TECHNOLOGICAL STATION

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1 RESTRICTED MATERIAL IN THIS REPORT IS NOT TO BE QUOTED WITHOUT EXPLICIT PERMISSION FISHERIES RESEARCH BOARD OF CANADA ANNUAL REPORT FOR OF THE TECHNOLOGICAL STATION HALIFAX N. S. AND THE TECHNOLOGICAL UNIT. ST. JOHNS NFLD.... I D. R.IDLER DIRECTOR ROC: R DUHAMEL,,,R.S.C. QUEEN'S f'njntt:r Al'fD COI'ITIMOltiR or STATlON'I:MV OTTAW A, 1!162 Ill

2 RESTRICTED MATERIAL IN THIS REPORT IS NOT TO BE QUOTED WITHOUT EXPLICIT PERMISSION FISHERIES RESEARCH BOARD OF CANADA ANNUAL REPORT FOR OF THE TECHNOLOGICAL STATION HALIFAX N.S. AND THE TECHNOLOGICAL UNIT ST. JOHNS NFLD. D. R.IDLER DIRECTOR

3 FISHERIES RESEARCH BOARD OF CANADA TECHNOLOGICAL STATIONn HA.LIFAXa N 2 S 9 TECHNOLOGICAL UNIT 8 S'r 2 JOHN 9 S 9 NFLD 9 INTRODUCTION The attention of members of the Eastern Advisory Committee is directed to Summary No o 40 which reports recent progress in a study on non-bacterial spoilage of ground fiflh 0 s:lngled out for special mention in recommendations by the Committee in Januaey o Mro No Dambergs 9 whose talents are particularly well dt:nre10jped in analytical chemistry 9 has been transferred from the product and proc~as group to the microbiology section in order to expedite this programo The attention of the Committee is also directed to SUmmary No, 13 where it will be noted that excellent progress has been made with the multi~ stage molecular stillo Four units are now in operation and the equipment is fun~tioning in a sat:isfacto:cy mannero The comprehensive bulletin on the pre~ servation of fresh fisb wi't..'». the antibiotic chlortetracycline (CTC) is ready for submission and ia discussed in Summary No, 4lo Certain investigations, including the prese~;ation of fillets with CTC 9 the heat-stable protein from cod muscle 9 the amino acid=pyrimidine compound from a cod parasite and the analyses of trop :)myos~n 9 have been taken to the publication stage ~d terminatec;t. Other :projects includiz!g nr'ln=bacterial spoilage of ground fishg fish protein, (flour) an.d the ut:i.lization :of marine oils have probably received increased ~-t= tentiono A majo:l:' new aree.,,f research at this Station has been concerned with studies on Newfoundland cod and to a much lesser extentp with steroid ho:..1.non;;3 and the culture of fish t.i.ssues o Studies on cod phospholipids and indeed all studiea on marine; lipids are continuing under new managemento The attention of the fishing industry is also directed to the studies on Newfoundland cod and particularly to Summaries Noo 22, and 36o Note thf;l.t in Summa.ry Nc, o 36 it is rep"jrted that the poor shelf life of the frozen fillets 0 prepared from water=thawed frozen round cod 9 correlated with an early change in the ultracentrifuge pattern of the muscle proteinso The potential application of this technique to the prediction of frozen storage quality of cod makes it worthy of further explorationo Time-temperature interrelationships as they affect the quality of frozen cod are discussed in Summary Noo 37o The im= portanoe of cooking procedures on the quality of cod 9 as assessed by taste panel, is under investigati z:m and a preliminary discussion is presented in Summaries Noo 28 and 38o Those interested primarily in fresh fish are directed also to Summary Noo 45 where the importance of temperature on rigor con~action and protein extractability is discussedo Those concerned with lobsters are directed to Summaries beginning with No. 59' and for those interested in "poundingtt it is noted that Uve=holding facilities will soon be completed at this Station which will permi"i;; such studies to proceed as indicated in Summary Noo 59o Those interested in special products or processing are referred to Summary Noo 71 co"'r;ern:i.ng fish protein (flour) 9 Summary Noo 78 describing a means of ut.11izing fish skins and fish skin residues v and Summaries No o 14 and 15 which are con= cerned with the utilization of marine oilso

4 In :re e.nt years fisheries scientists have come to realize that it is not :;;~eas~cnable fox one ~tj>up to concern themselves exclusively with the live animal and a.nothe:r" with the dead animal, Scientists at this Station are placing in= crea.s;'lng emphasis on the need for a better definition of the raw material ~ith wh:i,c:h th~y 'W"\Jl':k, Ow~r the years studies on fresh fish moved from the retail and wholesale outle~cs back i't:<e1 the plants and finally to the fishing vessels. At each stage the impor4~ance of handling or processing techniques were assessed" Aa improvements were realiz~d at each stage in this chain 9 it became increasingly apparent that the pictur'e could not be complete until more was known of the ef= feet of environmant and physiological processes on the subsequent qualit,y of the product whether it be freshp frozen 9 ealted 0 smoked 0 reduced to mealp freeze dried or otherwise preserved. Considering the variety of commercially important marine forms and the in~reasing :recognition of the importance of sexuai maturity, water temperature 0 effect of the ocean floor on bottom fish either directly or via the food supply 0 degree of struggle 0 condition of the fish in rel~tion to ri.gorv and other variables 0 it is essential that facilities be provided to permit the scientist to control these variableso At tne present time there r two re~ circulated sea~water units with a combined capacity of approximately 1400 gallons in use at the Station for holding live fisho These facilities are being ex- I panded. and within the next few months, four additional 700 &'allon live holding tank:s 0 one 12 x 6 x 4 deep rec:tangular tank with a capacity of 1700 gallons, 42 tanks for holding lobsters and other shellfish with a combined capacity of 2000 gallons 0 and a sea water line to supply a continuous flow of clarified sea water will be in operationo A new Dole Ho:t"izor.;tal Plate Freezer has been installed in an area adjacent to the livt!l fish holding facilities and is now in operation. It is a 6 station 0 Freon 22 Model with a freezing capacity of about 200 lb per ~ in l=lb packages 0 5 lb packages or 2=inch thick blockso A new Thermobank automatic defrost evaporator has been installed and is now operating in the standby com= pressor for the second floor ~entr.cold room and constant temperature boxes have been remodelled sufficiently to permit long term frozen storage studies on in= shore cod to be ~arried outo However 9 existing cold room facilities are inadequate and represent a major obstacle to the satisfactory pursuit of all studies requiring reliab~ controlled low temperatureso The needs of the Station for cold storage facilities have been assessed but thus far several attempts to interest any commercial refrigeration firm in assessing the existing facilities have been unsucc:essfulo A local engineering firm has carried out a preliminary in= spectio~ of existing facilities and may be prepared to undertake a more detailed assessmento In June of 1961 the technological aspects of the inshore cod project became a \:ollaborative investigation between scientists of the Newfoundland Technological Unit and this Stationo An experiment on freezingp thawing and refreezing trap cod was undertaken in July and has now been completedo Commencing September , the ptogram of the Newfoundland Unit was formulated by a programming group author= ized by Dr. Kask and composed of senior personnel selected from the two labora= torieso Sirtce January the programme of the Technological Unit has formed a. part of the Halifax Station programme and as a res\,llt the programming group was d:isbanded by a.::::t;:ion of the G'hairman of the Boardo

5 = 3 = For some years :it has not been possible for the limited engineering staff (2) of this. Static;m 1t0 carry on an effective research program because of i;ht"! ma:r"y ;Jth~r df!mands on their time (see the last Investigator 0 s Summary i"n 'thi.s repor~~). During the demands for technical assistance have de= ::r~;ased app:t.'eh~:,:_,abjyo Mr. Brian Meagher 9 Director 11 Department of Trade and Indastr;t frc;r the Pi:"cnr:Lnce of Ncnra Scotia 0 has agreed to having requests from No ga Scotia refenad to Mr. Morgan 9 an engineer with the Department. Mr. Morgan has worked closely with Mr. Wood for many years and this arrangement seems i;;o be working well 0 id th Mr. Wood acting in. an advisory capacity. In Mareh Mr. A. Fcc~alick of the Department of Fisheries 9 assumed respcnsibilit.y for the inspection of bait freezers 9 a duty previously as= signed to Mr. Wood. The exc:ellent cooperation of Mr. Loran Baker 9 Area D:trector of the Depa:r"bent of Fisheries 0 is gratefully acknowledged.: These d~velopments have made it possible for Mr. Wood to consider a reasontbly full time research prcjedo After due considerationp the freezecodry~ng of fishery products has been selected for study and.work will begin earlf in the new fis,~al year. Adequa te equipment is expensi'4e ($ = $ ) and it may be necessary 'l;o proceed on a limited basis for the immediate tuture. The matter is still und!sdr c:onsideration. ' Collaboratitr..1~1. bar made it possible for personnel to engage in1 projects ' that could not ~ther~lse have been considered. A collaborative proj~ct is underway with the Glcc,uc:heJster Technological Laboratory of the United. States RtU"eau of Cc:mmexocial Fisheries for the purpose of comparing microwav~ (diele~t:ric:) and water thawing of frozen fish. The relative merits of frying, ba.kl'lg and stream:l.'1g' <COd 0 as assessed by taste panel 0 are under investigation in : collaboration with personnel of the Home Economics Branch of the Department of Fisheriea 0 Ottawa.. This Sta.tion is not equipped to carry out nutrition studies and EH:d..entists of several laboratories are cooperating in order to ~rovide a thorough a.gsessment of the products produced by the fish protein ~flrour) p:rocess1es dev<t1loped here. Products are now being tested by Dr. w. w. Hawkins 9 National Research Council 9 Halifax~ Dr. Ao B. Morrison, Departmen t of National Health and Welfare~ Ottawa~ Dr. D. G. Snyder, United States Depart~ ment of the Interior 9 College Park 9 Maryland; Dr. Paul Gyorgy~ Philadelphia General Hospital9 and samples have been requested by 9 and sent to 9 representa~ ti ves of UNICEF\ FAO and the United States Food and Drug Administration. Te::::hnologi.s ts have continued to contribute to the education of persom1el concerned with the fishing industry. Two scientists lectured at Fishery In= specters Courses held a.t Lunenburg. Two scientists participated in a Con= ference of U.M.. F. Managers a.nd Extension Field Workers of St. Francis Xavier University held at the Fisheries Schoolp Pictou. Station personnel attended the Fisheries Exhibition at ~xnenburg and participated in a meeting on scallops. Two scientists contributed to two one=half hour television programmes produced by the Extension Department of Memorial University, Newfoundland, and other scientists parti~ipated in other television programmes 9 also of an educational nature. Several staff members have given lectures at Dalhousie University.

6 -4- During the past year there have been several changes both in senior scientific and administrative personnel. Mr. W.D. Cossaboom, the Station's dedicated Executive Assistant~ passed away on December 13, Miss I.J. Rattray, who has served since July~ 1961, as Administrative Assistant, was appointed acting Executive Assistant on the death of Mr. Cossaboom and her appointment as Executive Assistant was confirmed effective January 1, Mrs. G.A.M. Gordon was appointed to the vacancy created by Miss Rattray's promotion. Dr. E.G. Bligh left for Ottawa in March, 1962, to understudy Dr. N.M. Carter in preparation for assuming the duties of Special Assistant to the Chairman in Technology and Associate Editor of the Board's Journal. Dr. P.M. Townsleyp a graduate of the University of California and previous~ employed by the Canada Department of Agriculture, assumed his duties as leader of the process and product research group shortly after his arrival in August, Dr. W. J. Dyer assumed leadership in Octo bar, 1961, of the group concerned with studies on frozen and fresh fish with particular emphasis on protein$. On bie arrival in June~ 1961, Dr. D.R. Idler assumed temporary leadership of a marine natural products section 9 the nucleus of which is comprised of personnel previously associated with the fonner analytical chemistry section. Dr. R.G. Ackman has continued to serve as leader of the marine lipid section and Mr. C.H. Castell as assistant station director and leader of the microbiology group. Mr. W.A. MacCallum was confirmed as Chief of the Newfoundland Technological Unit in January Mr. E.J. Laishley, a graduate of MacDonald College, joined the staff of the Unit in September, 1961, and Dr. C.M. Blackwood resigned effective February 15, The chemistry and bacteriology laboratories on the main floor of the Cable Building at St. John s, Newfoundland, ha;'te been completely outfitted and equipped. Plans are now being formulated to provide a third laboratory on this floor. Freezing equipment used in freeze-thaw-refreeze studies during the year were installed at Job's Southside Plant, St. John's, in space generously provided by the management. Cold storage facilities of the plant were utilized and continue to be so used. During the year the Board acquired about two-thirds of the space available in a 26 x 37 one=story masonry building at the rear of the Cable Building with the intention that Unit freezing and storage faciiities would be installed at a later date. Recentlyp plans for refrigeration facilities were developed based on the Board obtaining occupancy of the whole of the building and on the assurance that a 14 ft. addition to the building could be provided. Information at time of writing would indicate that construction and renovations required will be completed in Aprilv A refrigerated sea water tank and ancillary equipment required for holding live fish were installed in the basement in June and were used throughout the summer and fall monthso Since January plans were made to increase holding capacity to 4 tanks and to provide stand-by refrigeration and equipment, At time of writingp delivery was being made on some materials and equipment. It is expected that the system will be ready for operation in May. D.R. Idler 9 Director.

7 GENERAL SUMMARY OF INVESTIGATIONS TABLE OF CONTENTS Page (s) Marine Lipids i-ii Marine Steroids Newfoundland Cod Quality of Frozen Cod Quality of Fresh Cod ii - viii iii Proteins of Fresh Fish Chemical Composition of Fish viii ~ ix = x i:x: Cod Lipids with Emphasis on Phospholipids Biochemical and Microbiological Studies Pertaining to Lobstere Studies on a Cod Parasite xi.. ~ :xii x:ii - xii xi.ii Products and Processing Cytology xii.i - xiv xiv

8 i Marine Lipidsg Utilization of Marine Oiis Conversion of the free f'attvacids of' cod oil to methyl esters"= Large I amounts of cod oil are available with free fatty acid content in the 10=1~ rang~~ and a lower free fatty acid content might improve the marketing situationo Ace= tone dimethyl acetal (2p2=dimethoxypropane) has been employed in the esterifica= tion of fatty acids~ primarily as a water sce.venger 2 but it also contributes methanol on hydrolysiso Adaption of this re-j3.cti,on to the methylation of the free fatty acids of cod oil.i!!, situ requires very simple a.pparatus in contrast to most other procedures for esterification in~o Hydrogen chloride in methanol was a moet satisfactory catalyst and facilitated refining of the producto In conjunction with definite amounts of reagent and catalyst and refining techniques it was found possible to obtain a stable product with any desired free fatty acid content down to ~o Blowing of marine oils o The air= blowing of marine oils bas be&n thoroughly explored and cod liver oil has been found t,~ yield a product whose properties approximate t~ specificatfons of oil industry a.dditivesv presently based on vegetable oilsv in all respects except iodine ~ralueo The more suit;:-~ble products do not~ howeverv appear to be susceptible to oxidation 9 and do not settle from mineral oil blends on standingo Samples have been submitted to a comme:rc:lal firm for testingo Marine Lipids ; Comoosi tion The flame ionization detector for gas chromatography was tested with various marine oils o Owing to the high sensi ti vi ty very small samples could be used 9 increasing the efficiency of the columnso The chromatograms obtained were too complex to permit immediate positive identification. of many of the peaksr since some of the shoulder peaks may be due to positionally isomeric fatty acids of the same chain length and number of double bondso

9 In contrast t.::~ the methyl este:r::;p 7olati:.e fatty acids are partially retained on the columna employed~ not necessa:d:ly in proportion to their original conc:entra tio:n.o It has been di;:e:-jvered t!:la t m 4~st of the absorbed In other developments the long«chain fat.ty alcohols may be total)$ absorbed on certain polyester columr..s 9 facilitating analyses of mixtures of carboxylic acids has been. prepared as: a qual:lta'.:;:i're sj~andard through spplication of a modified Varrentrapp I"ea.c:ti.r.~n t;c e:r'!_l.cic acd"d a:r.d subsequent ozonolysis., Four :stages of the projected ten sb,g;s,::> of the multistage moleculfl.:r still have been operated in series under conditizms of high reflux< ~lith a mixture of ethyl esters~ 16% paljjl-i..tate and 84% o=..ea:te 0 the enrighment :in the~ rtistillate was up to 85% palmi tateo Modific:atio:u.s are now being oarri.ed out to operate at lower reflux ratio to give greater output, Steroids Impaired hormone= metabolism:_k~n~;-n _J-&...;"i:22~~1_ng and_\i(i~t_h '~1L sg,_l= 1!!2!!" It is generally agreed t.nat all :fi.-v1~ spe:.d..es of Pac:Lfic: salmon dt:: after spawning while trout and Atlanti.-~ salmon fx'''?lqu<:ntl;y swtvive spawning" More than one investigator has suggested the:t a h,.vpex~secret:l;u of adrenocor,ti.coster= oids is responsible for the very hi.gh lev.els of these z-ub5't;ar1ces in the blood of sexually mature and spawned Pacific sal.mono Howe:'J!Sr v i-t has been demonstrated that the elevated blood hormone lenrels in moribm1d b:u:l1!&"1s arise from impaired hormone metabolism resulting in a decreased c;learanca of these substances from the bloodo The present studies are con.c0e>rned with t,he -dearance of intraarter= ially injected c 14 =adrenocorticosterdds from r;hj?. blood of spawned,atlrmhc sal= m.on and a comparison of these data with those pz'evi ) lsly obtained for se::-rnally

10 immatureg mature and spawned sockeye salloono The ~sul~;s show that there i.s an impaired hormone metabolism in sockeye salmon :Lmmedia tely before and ~te:t spawning while no such impairment is appa:r.en:t in 21pawned Atlantic salmon or sexually immature sockeye salmono It is ccx1clud~d tha+ {~he major impairment of ho~one metabolism is a sign of approaching death :rather than a result of spawn= ingo It is therefore unnecessary to po.st :llattj a b,;r})el"eecretion of c~ortic:-o " steroids from the adrenal gland in order to exp:.:.aln the el~nrated hormone levels found in sexually mature and spawned Paci.fir:" salmo::-.c 0 Q9njugated testosterone in blood a.:c1d 4arrt~,S,!~,t:,_ru~:~Lsalmonc;,!..ast yea:r testosterone was shown to occur in a water~soluble,:,011~ugated fo:rm in 'cht1 plasma of spawned female sockeye salmono Testoste,:rxme has now been.isolated frcm the conjugated steroid fractions of both plwma and testes of male sr,ckeye sal = mono The struc;ture of the steroid was confirmed by a<;:etylation" oxidat:l )n ar;.d infrared spectrao Glucuronide conjugation was indica ted by 1.::se of a sped fi ~ glucuronidose inhibitor P glucosaccharc ~l g4coladon<fl! o Conjugated cholesterol il1 ooi. co:rwscles ol~tanni:us, puscles of Stannius are located at the posterior end of the cod kidney and their function is in doubt although it has been suggested that they may secrete steroid ho:rmoneso A search for steroid hormones in a. r;:;latively large sample of c ::.:r = puscles of Stanniusp supplied by Dro Grace< Eo Pi~kford of Yale University, ~as not fruitful but in the course of this investigation H vras found that a very significant proportion of the cholesterol isolated f~~ t)m this source r.:ra:s water=, solubleo Cholesterol was liberated from the conjugate with ~glucuronidoseo To our knowledge cholesterol=glucuronide has not been prerlt<usly demonstrated from an animal source and the obvious significance of a fv.n:'m of cholesterol soluble in body fluids warrants further investigationo

11 Newfour1dland Cod fieezeo thaw'l refreeze e~iment4, As one p::;,ssible means cf extend= :ing the operating season for plants :handling tr.ap fish in Ne~Tfoundl.and 10 the feasability of freezing the headed, g<n;t~:jd ~:tap 'cod 0 followed later by thaw= ing and refreezing was studied. The main :pr Gb:iem 'i Rl,(-,: "',;ro deieruline if this treatment affected the storage iife of the tt:<i'lr.-;;~zen filleted product~' particularly when prepared from 'trap cod. Fish wb>r'e fr."c,zen and stored in Newfound= land in L';ooperation with the Newfoundland Un:!.tr ~i;hen tr..awed and refrozen at 2 month i.nter-rals and shipped to HaUfax by a:'lr wb.~:r~;; "rh.:o refrozen fillets were stored at c~l8 10 and at =23 Ce The ~amples of urj.tbaw,:~d round fish 0h.Bl[!!ged. l':>:i:y 1::;.tt1e up t:; 6 montt.s although taste panel scores were gom~lwbat below those for control offshore com= mero: ially frczen filletso The ref:!"ozen fillets s.:;ored immediately after re= freezing '#Eili." a unchanged from the rsund fish 0 io f'. 0 'tlre thawing (:ln \-mi;,:-r at 7 to 3 C) and rejfreezing treatment had no i:mmedlai:;t:;.: efft.<c,to After storage for 2 months o:r more at =23 and e.t =18 C 9 the re.:frt~:zen fillets deteriorateu mat'/"edly 0 at =18 to le'frels c'::msiderad )'bjectic::nable by the taste pmelo The de,,;:'saas oc~ curred in the first 2 months of storage 9 then the e(0:t<?js levelled off., T:!:Lh is probably an effec;t due to the temperature ri:'!lfi dur;lli,g tha\f:lngr whic:h allmm deteriorativ-e reactions to proceed at a faster I"atfl e':,en +.hough the temj:')&ratllre is quickly lowered again 9 as was d,emonst:rated :.n the aurefrigerator car" t~sts., In these 0 temperature rises from ~18 or ~23 t" abou ~ ~JL2 C re5ulted in marked later deterioration., Tests of fish sticks prepared by a commerdal plant from these refrozen fillets 9 while acceptable 9 showed d~f:jlnitely l;:;we:r tsxt:ure ratings when scored by industry and laboratory taste panelc'3 0 Samples of ro'tmd frozen trap fish 9 plate frozen postrigor 9 and brine frozen prerigor gr"aded similarly to the above plate froze'rl prerigor fish as did a sample of :::cldlllei' ::<~<.a1.1y frf:lzen trap cod fillets.

12 Onset and duration of, rigor a, Investigators working i.ndepende.v.t ly on freeze=thaw... refreeze studies on t:r.>ap cod and on the c:o:udi.tions of trap cod as euch at the time of capture and shor tly thejx'~safte<o:' ~:Oi."X'vborated 'Erwiden.ce ob= tained in 1960 that the ph of t:r'ap fish falls to a luw ;;.f 6o0 = 6.2 shortl.y after death 0 indicating that the g.lyr.;cgen I"e:!!IEJ!'1re <:.if d: t.~e fish is not e:x;pended to any great extent prior to kil:l.ing., Th.e ph,;_,f fj~i:ien;s f:t'"om nor.~.. =feedlng trap fish impounded in 37 F water for 30 daya dr\jpped from ' to 6o55 in one dby and rose again indicating that gly cog.en re~e;;ye had been e:z;pend.ed for surdvalo A somewhat similar o~currenc:e was noted :Ln f~.. 11Ee.~r:~ r~:.l'; f:':"om ;omme:t 'cd.a.::.~.y bf:.. a b:kl baited trawl fish landed in wl.ntero Data available to date, 'W\'JuJLd indicat~ i:l:.at su:mmer 9 fall and "rinter " caught cod go into rigor a few hours after" i,;aptur u Et~ren when ~~hilled in :.i.ce and in the ca~e of winter=eaught fish taken alive :fr,om wa!;er at.ice tempera tu..re ~ About half the iced cod examined in summer 9 fan a.:nd win.ter went into rigor with.in 5 to 10 hours of ~~apture and Btayed in rigor for about 3 dayso.m w-ould t.e t"n:,~ pected 9 the degree of rigor was found to depertd upon degree of struggl:ingo Since a. significant proportion of all c0d ayailable for f'reezl'lg in the unfilleted state within the first 3 days of ~Catching will be in incipient or in late stages of rigor~ r.esearch on the quality of f~~ozen blockt:s and fillei~!! p:r o= duced from this raw material is in pr0g:k"e9~5 e ph Measurement and sooilk~""~ Organolepti~::; examina.t:lu:m of fillet~ of rc;mn = parable age but of different ph le wels sho wed the oducr" ~.:.hreshold to be u.nrelated to pho Susceptibility of trap od t~~ elerwated te!j!~i!'s.a, Observations made on trap cod landed unic:ed after l t: 2 1/2 h:;mrs out 0f we/cer di.d not reveal softness designated as 11 ~Wggji 'u or :tlalqv o With cod gutted and iced at: tbe t!::'ap,s a..>1d fj. 1ld :L"l ijt:;e :tn the ~.ab :'>;,~ato:r.'jt until filleted at regular intenals 9 gaping an:i :soggi.les? a ::compar.cied c:v

13 vi age., On the other ha.nd 0 Grand Bank cod t:aught; about the aam~e' time wer~ firmer fit for filleting at''l;er a thawing p.er:li:;.d of BZ'\J. l~ ) ~ t"mrs was d,~ ~relopedo Oth.~ da t~~!'ijm..sqflj)oz;<.o{n j~n ~111. ~~B,iYeL~k~~_R:.\.a. te freezer ~ in b:;dmlo Stowing density wi thi:u ~)!0ck:2. c)f pn:. r::lg ;r= frr.;zen eviscerated~ headed When these blocks were thawed in water and filleted 9 ;:;he commercial yield in terms of gutted 0 headed weight was ll per cent., Small. eviscerated cod frozer. :nx. sa.turated NaC:l brine at =6 F had the fol,, lowing salt contents after 3 hours immersin:nt r,a k m?.-'.sc:;l"' 0., froz;en blocks whire:h had been in storage fo:ro 5 1/2 monthc? a.n.d were held frozen for the salt apparently had not cont:i'lbuted to a. :r -duction :L'l. ;'3~~orag~ life >:)V PT' that found in fish of the same season which had 'l:,f'en blo<k fh.z:8n ini ti.ally &

14 Collaborative experiment in m.icr<:n rave thawipg runong the ~ilix Station, the Newfoundland Unit and the Glouc:el!ter LaboratQtt_rvt U 11 S 9 F-lsh and Wild Life foundland Unit and the Glouceste.r La.borato:ry of the U. s. Fil!h and Wildlife Ser~ vice has been set up to compare the e:ffec;ts on the ;:;o: :f:r'dzen fillets of thawing the gain further!might into quali.ty r;;f (~od fx:ozqem in.:l tia11y ~m a plate freezer and Many panels were conducted on summer and fall=caught Ne:wfound1and cod in ord ~~: 1.:1 a.asess the influence of condition of rig\jr and free.ze: thaw 9 refreeze on quali t.;y. Samples and personnel were then exchanged between Halifax e.nd Newfoundland and the results now being obtained are ccnsis tent between the tw'j pa."lela, was similar in composition to the fle~h lipids of other ~cd previously i;rvest:!gate.:l. fuge pattern of the actomyosin system of pr<;:!tein wal!5 examined on flesh p:n.jtetms pre= iods of time., The poor shelf life of the f:r.:,zen fi.llet~l, prepared f'!'om th : w2ter~ thawed round frozen codp correlated with an early hange in the ult~acentrifuge pat= tern of the muscle proteinse Quality of Frozen Cod Time=temperature interrelationahi:ps a,~ they aff'e~t the frozen cod during subsequent storage were investigated over the tempe!r'a'cure range of ~~12~C to =26 C and for peri ode up to 35 months, Quality was a.~se8e,ed by ';:e.15te panel and extractable protein. A collaborative investigation is tmderway w:!l.'~h pereoxmel of the Home

15 viii Economics Section of the Department Of "~isheries 0 Ot+;a!iil'a, in order to e~tabli5h the effe~t of the method of cook..ing on the subsequent taste panel acceptabili.ty of variou.a quali'des of frozen codo eeesed by taste panel soon after ~:c\sa tmen~~ o pleted and a bulletirt was wri tteno isolated from spoiling cod flesh for the purpose of etudying the proteolytic enzymes of the organismo Black specks in e;od flesh were :identified aa blood., Proteinl! of Freah Fbh Studies on the composition of the muacle protein, trcpo~yosin, were concluded and a paper prepared for publicationo There is a substantial decrease in extractability of the actomyosin group of protei.rus when cod muscle goes into rigor at elevated temperaw.ree 9 whert? muscle contraction b very marked 9 but not when :r1g'n' O!"'curs at the temperature of iceo This observation may rtave important implicati'cm in the freezing of fish., Vertical etarch gel electrophoreei3 was applied to a study of c~. d muscle and blood proteins and to lobster blood proteim.lo Thin sli.cee of cod muscle were aleo subjected to eledrophore~.:l.s and two proteinsv not found in watex extracts~ were presento

16 tropomyosino A heat-stable protein in cod m-uscle has been identified a.s Studies on the structural proteins of cod DW.s cle were concerned Characterizationg The principal obje t:ii,ve o! thi.s work war the isolation of the muscle proteins and determiimtion vf their more important properties o TrlOptJmyosin and a~ tin he.,re bee;n i:sole.t;<f:<t~. and possibly also myosin., The ATPase activity of the latter h.ab bee:n in~:restigated as well as that of unfractionated extractae The effect of ATP 9 stc. 0 en the main components of salt extracts of muscle is being used in attempts to separate them, and to identify the substances responeible for the ul tracentrifuge patterns of extra ts lll8de aftex' various treatments of the fisho Certain of the ~omponents of th.e aqueous extracts of cod muscle~ and. to a lesser extent of other spe~ies 0 Interaction of componentsg have 'been isolated. A proposed model of the freezing of fish muscle suggests an environment.equi ;ralent to high ion concentratione Such a high ion concentration in a salt extract results in a dissociation of the larger protein moleculesv as is also found i.n extracts from frozen stored cc d muscleo This increasing dissociation precedes t~xture changes shown by Time= Temperature~Tolerance storage experiments. The dissociation reaction and the interaction of the four dispersed components and the gel fraction are being Chemical Composition of Fish Postmortem biochemical and physical ~es in cod muscle were in~ vestigated and the major changes occ:urring in f:r"::>zen fish on storage are ra= lated to the proteins and the lipid fractions but can also be.aff'~ted by post~ mortem cllanges.

17 Last year 9 in muscle from re,sted :n.r:~x,.feed.ing c;od 9 it was shown tha:i;, the higher temperat.~are of storage of the ~x:;i~;f)d filletsr the more rapid were the glycolytic p~':'odeesesp onset of rigo!'c dl'ipt )t~phu:rylatt.e;r, of high energy phosphat~s9 hydrolysis of glycogen to lact:j.c acid with e:t~ns~quent lowering of ph 9 etco Dea.minati<on of aden.i.ne nuc:ieotid~<.! to :inos:ln~ whh liberation of am= monia also oc:curred simultaneouslyo In :feeding f:ishr similar changes have now been fotmd 9 ex~cept that dephrosphorylation O:f ATP O(;c:mr:;:~1 much earlier and at a more rapid rate 9 although onse':;, f 1'ig::)r was n~:'lt a:ffe tedo The nucleotide. phosphates a~e being ~tudied by <chroma.tog:raphy w:b.i.c':n has f.:ihown the presence, of DPN 9 AMP 0 ADP and considerabli!> amoimts 'Of ATP and an ATP=like fract:icn. A fish with a very high glycogen value 0 Oo4s%:. reach~d an ultimate ph of ab:.mt 5o9v accompanied by marked drip formation and contracti:on e1ren at 9 C'c a mu.::h lower temperature for the occurr~nce of the,,'l'e phenomena than previously found with nonf'eeding fisho During 1960 quantitative analyses.;;f prote:in 9 lipid and water=soluble material in cod fillets were carried out on a monthly bas:tsa The results in~ dicated that these components varied consid!!re.bly with the spawning ycleoro The e~;periments were duplicated in 1961 on a magrdfied ~ampling scale with some qualitative explorations of the main <Components of the filleto The new results are in agreement with the results obtained in 1960 and indicate that besidee the spawning cycle 9 the nature and amo'w'i.t of foc d. avauable ma;y be another factor influencing fish compositiono The proximate composition and nitrogenoua extractives have been dete:rmined on samples of argentine 9.lrgentina si;lu~"9 and mackerel shark 9 ~~after frozen storage under defined c:ondi tions a."ld t;he g1yc:ogen content of la,rge snd small scallops was compared fresh and after stc,zage in i~::~ o

18 xi Cod Lipids With Emphasis on Phospholipids Free fatty acid precursors in frozen codg Studies have shown that quality deterioration in frozen cod is due in large measure to protein de=na~~ation which is accompanied by an in rease in the free fatt.y acid content of the tissuev and it was suggeeted that the two proce~ses may be related or that one may be the result of the othero Thus 0 more information on lipid de= composition was required in order to ass~ss ita poseible role in protein denaturationo Quantitative data have now been obtained confirming an earlier report that the increase in free fatty acid content of cod muscle during frozen storage is due to the hydrolysis of only two of ~he phospholipids present in the tissuep namely P phosphatidylcholine and phosphatidylethanolamine., This fulfils the objective of this part of the investigation which was to establish the precmrsors of the free fatty acids that develop in stored frozen cod. The specificity of the lipid hydrolysis supports the concept that it is enzym:ic. Lipids in cod fleshg It has been demonstrated that the lipid extra.ct.ed from the brown tissue of cod fillets has prac tj.cally the same composi= tion as that from the white mea to This is in contrast to previo~ concepts that the brown meat contains more trigylcer~ide 0 and does not support ar.y argument that lipid deterioration in cod may be localized in the brown layer. Ethylenediaminetetraactic did not inhibit the formation of free fatt.y acids in cod flesh homogenates stored at ~2 C for period.s up to 4 weeks. Cod phospholipid obtained by methanol=ehloroform extracu n and re=added to cod muscle homogenate was not hydrolyzed" A project was started to develop a procedure for analysis of phos~ pholipids of fish., The lipids are deacylated 9 and then separated by ion exchange chromatography.,

19 Li'\re holding fa~ilities are u:nder con,struction 1!1 prepara!;ion for a stereo Pla:sma ~ontaid.ed appr::>rl.mate:'y '5% p:to~"'.j.;.:, ru~d "" ~p ()f 85% hemocya.nin 0 8o5 = 9oafo fil>rinogsn and 4the J>: E!IfJcl.i:ndsc:" ('"n.,;>;:.;: i,\:>. i.l'.!l'!j.i.rl.den~;ified p::..~ctei::-1~.. and fibrinogen was deter~inedo when we;unded,~ven :"ilightly 0 bled t :; d.. :.stho 'I'hb ::,;o_,:,.::t:l.<:iu in blood ph 1 is ac= compani+-:ld by a large de ~rease in bl;3:;.,j pj:l.h&:l.y~.:;.e'l ~ 1.:3 (c5%) and since the that the animal suffocateao which suggest:!! that humans may be a so1ll.i'ce of the crganism wh).c:h infects lob= s ters o Several properties of the two micro,1rga."li:s:ms were,:;om pared and ga~~~ liquid chromatography was used to identify la,;;;ti~c' a,:;:j..d as the product of glueose metabolisms in both mic:roorganisllll3o Four phospholipid components rnake up 64% c.:f ~t.he total lipid )f 1ob~te;r

20 xiii The nematode from cod musculuture has 'been kept alive in synthetic in=vitro culture for three monthse During this time the nematode changed from the larva to a pre-adult atageo The full adult stage was not achieved jij. three months even though this stage may be achieved in 2 to 3 weeks under natural conditions. The fatty acid composition of the Ilematode differed markedly from the flesh of the host, Products and Processing The development of an improved process for producing fish prote.l..n (flour) is proceedinge The process~ originally developed on a laboratory scale, has been modified for limited pilot p1ant production (batchw~ae = 50 to 100 lbs. fresh cod fillets).. StabilityD colour and texture problems which were not apparent in the laboratory became noticeable in pilot plant productiono These latter problems have been solved and storage studies are in progress6 The nutritive value of various fish flours resulting from changes in processing precedure is being assessed in the laboratories of the Department of National Health and Welfare at Ottaw~P who have a paper ready for submissiont the National Research Council at Halifax and other laboratories. Preliminary results would indicate that the method of processing (solvent extraction, etc.) definitely influence the nutritive value of the flour. There is considerable and growing world=wide interest in the Canadian process. Taste panels have been carried out on commercially prepared freeze= dried fishery productso A survey has been made of available equipnent suitable for freeze=drying fishery products and a literature survey u..11.dertaken., Laboratory research will commence shortlye Studies were continued on the autolysis of herring and the preparation of meal from autolyzed herring preserved Ylth isopropancln

21 xiv Delicatessen products prepared at the Lond~n Unit were well re~ ceived by the trade in Sto Johnus 9 Newfoundland~ Spe~ial mild soaps were prepared from hydrolyz,rzd. fish skin and skin residues by combining the resulting peptides with fatty acids., ' Cytology: The culture of marine fish tissue c:ells is a relatively unex~ plored fieldo Tissue culture is being explorf:d a:s a t :HJl to obtain condi= tions to study such phenomena as mucous fvrma thm in fishp chi tin formation by lobster and hormoned response by marine foiwlo Cod epidermal tissue has been successfully culturedo Histological techniques hav-e been nsed to explore the changes iri fish muscle following starch gel electrophousis of thin layer tissue slices.

22 INDEX INVESTIGATORS 0 SUMMARIES SUMMARIES MARINE OII.S g GAS-LIQUID CHROMATOGRAPHY OF LIPIDS MARINE OILS g GENERAL LIPID RESEARCH 1 = 8 9 ~ 13 r-1a.rine OILS g UTILIZATION 14 = 15 MARINE STEROIDS NEWFOUNDLAND COD QUALITY OF FROZEN COD QUALITY OF FRESH COD PROTEINS OF FRESH FISH CHEMICAL COMPOSITION OF FISH COD LIPI DS WITH EMPHASIS ON PHOSPHOLI PIDS BIOCHEMICAL AND MICROBIOLOGICAL STUDIES 16 = = = = = = 54 55= = 67 PERTAINING TO LOBSTERS STUDIES ON A COD PARASITE PRODUCTS AND PROCESSING CYTOLOOY STORAGE QUALITY OF FROZEN SCALLOPS 68 = ~ = INFORr.~TION AND ASSISTANCE TO THE FISHING INDUSTRY 82

23 SUMMARY NQ2.. _1 MARINE OILS g IDEt~TIFICATION GAS= LIQUID CHROMATOGRAPHY OF LIPIDS~ OF P01rJNSA.TURATED FATTY ACIDS R. G. Ackman R. D. Burgher The separation of {~he xc.ethyl est~:;"s <;;f ti'j!f.fje r.oa tarij3.ls on polyester subs'trates with a minimum of ~werlappi~ {Appendix =61 Annual Report) is desirable but cannot be :readily a~hi~sve.d. with t:t>.e c:0mplex mixture of fatty acids of' marine oila. A uum.be;" of folyes~~l':'s we.re iz:.lr astigated to determine their potential applicationo DElGS diethylene g1:y::'jl =succinate) is a superior polyester with very good Z>esolving pr we:-: 0 but ser:im.:a.;s.overlapping of the more highly unsaturated fatty ac,ids D'CC~IT'!Si ~~:th those of lo:nger chain length but lesser degree of' unsatw:a ~hmo BDS (b;c:.~ ~a:::\e ii:jl=s':j:.cc:ina:te) aiso gives good separationp but fails tc r JE.olire two llw.,,cmps; n<emts 9 mt:r~h,yl eicosapentaenoate and methyl docosenoatep thils lim:tt~;jg allj.y Ur;ie w:tth mari.:ne oilso NPGS (neopentyl glycol=succinate) is an (ext;:remely h::~rt ~dt:abl'?i p,:;,lysl!ltt.or with only moderate resolving power., Its 1"ir~:c;:, is t:tw t each ewen=x1mbe:l':" t>'d. ehain length group of fatty acids is completely sepajt.ao~;ed :tn. )JIT,~ band o Thvs the o:nly overlapping is with certain branch=-eha:i:u. ru1d c ddc-o!liumbere:j~ fatty at.: ids wrlich ar e few in number and respectively total only 1% a:'ji.:l 2!'fo of' 1~he w:tco::.e o Th-e,:,dd =numbered fatty acids have been tentativ-ely id.e!nt::lic?.a ae; ~h:!.~;fl;}~ C15 run.d C27 gatu:rated acids, and Cl7 p Clg and c21 lll0ll(j'uji:.sb.'~l..1jr'at:ijd. I.?,;:;:Jic!;;,o T'}MS3~ app2.aj!' to be metabolic "mistakes" in the formati n.n (if' adjb.c;~ent r c:,la.~;<sd maj0))l: fa.tty acid components ( see.~: 'Ct' J.gs., ':tq ;J., 1. an d ~ 2 " 1,/V.. ~ ;:::r, ~" A' 9 J.:1'wv...:.L... u.ntli!l;. ll R e:p.c "'" ' ""' o Identificatio;n. of. Jt'a ;ty a.;dd~ depends?:ti a.n ;1,ytical techniques discussed below., The projejcit!=.: d is::;:~a.tivtjl ru:j<.d ciharacterbaticm of particular fa tty acids will be acllieved by 2s'tanut:e~1 :ooicr~, techniques c Th:L"'l. layer chromatography is also bging ~:~C:p~~o::red a2. a gitide t::j fatty acid structure. In this novel technique a mixtu:r~ 2Jf fa'':;ty flcid esters is tzeat:ed with mercuric acetate to form eth,yle:nic; do~ib~~"" h:.rd. 8/td;;,cts 9 and these are chromatographed in such a way that the saturated acid~:; (non~.dducted) are first' removed, and the addu::.:ts are then separated by n<.lmbe:r' e;.f dd;,'tle bcm.dso The recl()vered and reconstituted methyl esters may then be cc'n"ela'~ed with gas~liquid chromatographic peaks of differing cllain lengths b';~t the Bare.e dsgxo 'e of unsaturation. At preser~.t the :identifi.,~;a1~~- ':<ns aso:tg.1e: d to gas~uquid chromatography peaks rest on comparison with authe1r\t:l0 materials where availablep on published data of oil com]>osi tiona and on tabulat!'jd. da'\';a of retention times obtained by the few workers in. this field. The lf,tter- inf'ormat~ton has been carefully studied leading to the improvelllents outlined in Summaries 2 and 3.

24 2 SUMMARY NO" 2 MARINE OILS: GAS-LIQUID CHROMATOGRAPHY OF LIPIDS: IMPROVED RELATIVE RETENTION TIMES OP THE LONGER_. CHAIN HIGHLY UNSATURATED FATTY ACID mmrrl ES'fERS R. G. Aclaun The identificat.ion of gas-liquid.chroma tegraphy peakb prosm1u1d te be long-chain higbly tmsaturat~ fatty acid D!&~eyl eatem is idmjj;v: 1ll&da by iselation and characterization ef the material. A lesa safutactozy 1D!Itth&d i~ to repeat the analysis with the admixture ef authentic mterial where this is available, and the least accurate m thod is te c0111pare the retsn t;. on time;~ published data, although this procedure is oftm useful as a prel.im:i.na:'c'y ztl!p. The latter information is usually presmted in.t~elative form, with th.e ester of a commonly occurring fatty acid of convenient chain le.ngth assigned the value of 1.,00. Methyl tetradeca.noate and octadacsnoat.e ha7a bem wid.ely u&edt with the latter havidg the advantages of pore wig_espread ~ ar,j a loo.g-0r. retention time which should signi.ficantli reduce the e:crars.in measurem~nt~ In such tabulated data the relative retention times for matet'i.als with Ta:ry long retenuon times, such as nethyl docosahe:menoate, are found to vary widely (Table I-A). Semilog plots of relative retention time of both saturated and unsaturated fatty acid methyl esters, agaillst number of carbon atoms in the fatty acid chain, give linear results for the saturated aci.da.and usual~ for the monounsaturated acids as well. lru;iufficient points are available for plotting other unsaturated fatt,y acids systanatica~. It is noteworthy that the lines so drawn for the saturat~ and monounsaturated fatty acid esters are frequently nearly parallel, but in DBny cases are distinct)3 not parallel. Under the ll:t.tter circumstances it seems illogical to comp1re retention times for po~saturated fatty acid esters on the basis of relation to a saturated fatty acid ester. The use of the most commonly occurring monounsaturated fatty acid ester, methyl 9-octadecenoate, would appear to be more satisfactory, excepting tha. t in published data the retention times gi. van for this ester relative to methyl octadecanoate seldom differ and therefore no relative change is observed in the esters with longer retention times. The expedient was therefore adopted for the C2o and C22 series of methyl esters of calculating the relative retention times based on the monounsaturated fatty acid ester commonly occurring in each series. This has resulted (Table I-B) in a significant improvement in the correlation of relative retention times tabulated by various authors. In this table the revised relative retention times have been based on methyl 11-eicosenoate and methyl 13-docosenoate. Both occur naturally in many lipid systems, and the latter is readily available for addition to lipids if necessary. However. if one is lacking, tlle relative retention ti~ may be obtained by interpolat.ion or extrapolation, as employed in some casas in Table I, of a $emilog plot with methyl 9-octadecenoate as the other point. The use of methyl 9-hexadecenoat~ is not recommended since it frequently lies above such a line. It would appear that the use of such retention times, based on one chain length series of fatty acids, generally give greatly improved correla ti.on both in cases of data determined on differing polyesters and at differing temperatures on one polyester column, although not all the results presented are more satisfactory. w:l.. th

25 If this imprcyement and the i:iasis f<tll' it are arc:<cepted, sim:llaj:o reasoning :!.ndicates that the comparative appl:ll:cati e>lll of the carbon n"illll.ber OJr equivalent chain length identification sy~ri;en;;s r.rus i;, be Umited ex~ept in those ases of known parallel linearity in the semi log plots of both saturated and :fatty acid este::rs., In. the lattexo event such systems ot identification should be< satii3:\:~ac ~o:ry P.aven :lr:a c:11mpal'ing data from different polyesters 11 provided the tem:peratw.'es e:r~ app:rroximately ihr,:; sam " TABLE Ie Relative Retention Ti:mea;* of Som Highly U::x~atu:l"a.ted Fatty Ac:i.d Matb,yl E~ter:s on P0lyest~!r' Sub.st!"'ltes Reference Substrate Temperature Farquhab",James Hawke ~, Reoplex 400 IDA IDA IDA FJJA DEnS o5"' 184"' 19? 0 l8qo 200 ~~ LiiM!!I!!P':JII... Relative to Mateyl O:ctadfj ~anoat~ Liri.dgr"J1!1 DIDS Fatty Acid lo96 2ol6 2o (2o15) (2o04) 20:2 2o24 :2o48' 2o40 2.,32 20g3 2o68 :5o02 2., g4 2o90 3o32 3o 3o g5 3., o08 3o85 "''?'!I 0 7'3i...,» 22gl {3o4B) (4.,12) (3o90) (3.,65) 4., 1~5 22g o4Q 7o 9 7.,00 22g6 7., o59? o 7rs 9.,25:;1: 2.,065 2<761 ).,Ct() 3o '50 7.,01 Bo Relative to Corri!sponding M.o!:lotlllo':\a tuxa ted Ea c~r 1.,, 20g2 1.,14 l.,i5.:.0.i.:') 1"15 20g3 1.,37 1.,40 1.,38 1.,37 20:4 1.,48 1.,54 1.,:;1 los'!) 1.,55 lo53 20~5 lo lo95 lo90 2.,0:t; 22~5 lo92 2.,C4 lo97 1o92 22g 'i ""'~().t:...et.--=:u :2.,12 2o23+ -=---.-',_._ ,49 1., ,02 * Interpolated or extrapolated data i:n brackets" + Shortha.."ld nota tio:.j. after Farqu}:.ar" Strucd:;ta es identical except ::t (not specified).,

26 4 MARINE OII.Sg GAS=LIQUID CHROMATOGRAPHY OF LIPIDSg STRUCTURE AND RETENTION TIME OF UNSA.TURATED FATTY ACIDS ON POLYESTER SUBSTRATES R~ G. Ackman It is generally acc:epted that plots of log:j..o retention time in the gas= liquid C;hromatograph;y of t:h~ i:j>lc ge<x.:::lmiu n<:jrmal satu:t"at;ed fatty acid methyl esters against 'the number of ~arbol?l, atc;1::1l.z i;:j" the fatty acid chain g:bre a straight line with both non=pola~~ aud poiar au'bet:j:. ateso On the other hand, various simple plots of similar values obtained from unsaturated fatty ac-id esters wi t.il polar su"tlsb; a.teal) Jf');l"":r'l!'>la:l;l.:ag ctjja;r~ iengtj:1 or IJllwber of do1.1.ble bonds~ yield surprisingly little info:t."mb.ti.ono In the cowrse of gas= liquid c;:bromat~g:mphic examinati'i: D. :o;f tl-ae methyl este:r"s l)f the fatty acids of mar:f.ne oils the ll:'etention time data of a m1!l1be:r of' identified tmsaturai:ed fatty acid~ studied by different workersl) ali employing polyester substrates, were plotted on semilog paper against the number of arbon atqms in tl~ fatty acid chaino Despite the fact that a number of differing polyester substrates w ere employedp the rasultixlg plots showed a surprl.sixjg overall similaritya This s~jiggested that the key to the relation of the plotted poults lay in the structural position of the methylene=int~xtup',~ed double bond systems in (;he fatty acid rr:;ha:i.n;s in the ase of the polyethylenic fatty acids~ and.in the posi t::lon of the i.solated double bonds in the monoetbylenic: fatty a.cids 0 rather than :in the type r:jf polyestero The following hypothesi~:\ is theref',ore advanged~ "In plots of log10 retention time of ethyleni~;ally unsat'llrated fatty acids va, m.mbe12 of arbon atomsg where polyester phases\ are employed 9 parallel straight lines can be drawn between those points corresponding to fatty acids of' the same number ot meth,ylene~~i.nterrupted double bonds 0 or of single double bauds~ when the :nt.'mbe~ of carbc.m atoms beyond the double bond furthest removed from the carboxyl grr;~lp, including the terminal methyl group 0 is the sa.meo 11 A corollary to this hypothesis is that the 1.mes are parallel to tr..at derived from certain of the commonly occurring monoethylenirc fatty acids, namely octadec=9=enoic 0 eicos=ll~enoic, and dooos=l3=enoic 9 and not necessarily parallel to the plot of the saturated fatty acidso Since i.w or JIKl:re ryf these are normally :present in most natural lip:i.ds 0 the basis for plotti.ng >Other materials is readily availableo The gene:r'al observation may als.o be made that the precise nature of the polyeste:r is relatively unimportant 0 facilitating ~omparison of data from various authorso The data from Farquhar eu!:*g obtained on ethylene glyeol=adipa.te pc lyester at l97 C 9 provide the most comprehensive test of this hypothes.j.s, al thollgh similar data are available in pat';); from other authors o Certsin supplemental point~:! were also pj.otted 0 using an equivalent chain length systfml (ECL) based on the plotted mon eth,ylenic fatty acid li."leso These are :h:di(.lat;ed in. Table I ill the reference autht~:t:' (~olunm~ and are subject to sane.reser=.rati.ons as 't;r;;; accuraey as a result of the transfer., Of the 11arious groupa ~::f

27 Fig, 1 = Poin,t pl\ot f relative retentiq)l'l t:ime data fr::jlm Table II 9 with parall~jl lines correlating fatty ae;~.d stru~tm~~eo

28 6 TABLE I. Fatty Acid Structures and Retention Times Employed to Obtain Graphi.0 Linear Relationships Group Graph point:jj Retenti.::m time 0 relatbre to stearic acidt used by Author Reft;renc:e author* Fatty acid structr:re 0 Shorthand Double bond designation positions End chain+ I II III v VI VII VIII IX X XI XII l , , ,53 2.,76 3., o634 o o04 Jo o695' lo o75 4o32 lo ell ,02 [ECL 22.4] [ECL 19.~~] 2o c904 [1.04] o04 [ECL 22o6] [.68] L74J 1.,43 [1.,42] , (ECL 23.0] o ~38 18;;3 20~3 20g5 22g5 16:3 18g3 l6gl l6g3 l6g4 l8g4 20g4 22g2 16g2 16g2 18g2 16g2 l6g2 18~~ l8g2 18:2 20~2 20~4 22;6 22g2 (l8g3) 20~ (4) 20:(6) (22g2) ~12 5 J.l~ ~ !, , c. 7 pl0pl G 6p ,15 ~ r ::l 6~ 9 6 9~ v9 '' 9vl2 :? ~14 3 6~9 8 8pll 8 5v8vllvl rl3Pl6 9 l9 2 ( ) ( ) 8 or 5 (6v9rl2) 5 ( ?') 2 (2r rl7) 2 (l0vl3) ( ) 5 or 8 "' Points fallir...g very close togetl:1er may be assigned only one point c-;;;.d. numbero * Data of Farquhar et, alo P except as indicated by other references th:u~:> ( L 0 Parentheses ( ) indi ate hypotheti'cal st:ructureso + Number of ~arbon atoms after ultimate double bond including methyl grcm:po

29 '/',, fattj actda listed :i.n rabl~ 1 9 G::':JJ<.«.p~ I ~ 1'1 :t'r$j::re:se:-:,.t a(~:.u:rate correlation of ~;". + d d t. 4 '1" "*'"" " '" '. ~> ~ ""' ~ r,.-."' 'Y' rr:~,., t t., p, o;;.,lt.,..,., f G-:r"~'lP 17 P.!b. t '~ a'.a W'l.. " bile '-',}> Pv,,.ul!:c.~ :> u~ ~ ~..\., \i\.. - "" s - ~ " ~ d --.., '"C'" ' '.. agjtee a,o. well 9 btl!.t the> reten,tir,!!r tim,es 1.~~:::: t.:th~ s:ixteert.;a:c ho:n fati:;\r ac:ji,ds ~:r ~ pvssibly not <l'b'.r a.t:i..sfae:~o:ry a<.; c the-rl" s;,).(t::~ th.~ Jr'etenti,OJ.ti times t~:r.e ;shor<f:o:e;~ p.;r,, ~- -l:;h,e F'!"pa:<'at::cm?:'f i.s:jme:n" 1JrYJtr!.r~q,l1r.~:'::,t o T.n CL: ~;rp VI iil:'e list~d plotted point;;.; wll:i~~~.b sho~u1d not, f;;j J ::xjn a.'l1.y l!st.~:c»:t~,shed ~~,:L'<~~~ sillc ~ ~i t.her the nunfber' of do'i1,:ibl~ brjl-:tde or the end,~a.rbo~ :;ha::lll ~ d'jj!!'0+ w T.h~se in fa.:;ct di) not fall on an:p lfn,f! 9 (see 9 however 0 GJ:"io12'.p.IU f:lt' <, p?jsa:itle 1:1!'1ht'i~.enra:te; c'orrelation) although the sixteen carbon '-'hailt '3ata ax;,o, subjec;t t :. ~: ese::r'?;'fl.i:ior!.f o The 1ralidi ty of the ~1ee:es.si ty of the methylene ~~.!'.~ ~rc:r.~.:;:l'~~d system. :.:x1 P'~ly~tb~rlernil~ fatty ac~~ds h shown in Group VII 9 wher)e tb,~~ D f a d:ten_~ 11..ot :r.<~:sse.~--b!r;.g- th.is strtlt;i\u- e falls r~.ote fr om any d:ii~~ll.),~ li.lixi!l 0 Jll:'~ G;: ~<up YIII :a.:r~ :.:,:;ted three fa. tty a:::ids P the plotted PQ:ints of whi:ch -fall 111 a s':'::t'aigt:." :~iti~ 0.~HhoQ.g'tJ. they sho,~jtld n<o i,;o Hw we:n~::;;r in addi t!dr.: ~,;:, ;;;:;:!.~ (l,;:;,~:cy;;: affec 't.:.t: t';~ ~ :t('~i'2 c;;arhr.;n c;ha:~.i.:: a,:;:idb 9 the plottir;:g :Jf WJ.Y nw11be:c' of r~~: J::1ts wl J'tl ~~L'l,exr~. gi 'Y't SCJIDe fallh:g ~:.u a 'Straight l.:ilie th.:r"c:ljgh,;l/j.l)'r~eo W1t;t;, a. l':'~'f ~,se] r;.~~r be,s;~li '''::: ~ra"q;sf~rred da~\':.21) as, given :121 Group IXa tbji!'!!s~ jthi:.'il'il!: are ll'j 1'.\1ogetr 1-ii.ne.a:dy :r: e1at~d~ b':rg :in b th plots there is r!!>ag!o:a.s t.:_~,a.frt'.\"ji!lic\~n<~ ~),~/:r,?,~e'';, h!i!ixbli'~'':~,g ~ 12~"iiendJd.,:: B.:::id and oc:tadec=llgl4~ d.lenoic~ a.<~o::.ci. 0 'J:'he 11llpc;.fu ;an,t nega ;;.. ~.v~ cor:r't'!la ;.lj>;r., Of data ;.ri th theory is given in G:lloup X~ '!;lth.er~ 'tvf.l p:d~..ntr~ &;rht<:j1 sh:~uld b ~ fitted to a line o:a the basis of the g~~r'!'n "'l'"':r."tt:~t;~j:;::o~!'~:"~ ':a. J.ncc't -~~~ 1t:!;J.ld~d ~XJ a:n,y w&:::ro :rh~ ei~o:satetraeuoi:c and d,,,c;,gsa.her:ae;n<::i,':; e:::rii:~;:,u G:r:; :r;.p XI 0 a:r'e 't1jilique 9 u.nle?3s th8.., 1 t".~.~ t"' c '''-. F'... In. l'l.ppr,jx::una1ge e;rn:rfr; a.ton x ri).... '"~.,,\*.'a ~,e7 ;.;J: 'tfo. '.11~.;:J:i":l'-'WJ!.B, ~,.,.r:r'io"jr.p.ll. WJl.t.n. relat:nr~ ret;ention ;~ime!s ~- 0 1J a::::,:!. 4o.58 ::':!'? Jld ;',,;,;.d1:::1'::~ 4tl!< ~~ii,e p::teisen:ce of a.;,ry, eit:.,)s.ahe:r:aenoic aci.d of t:he ~,a;~ t;:vpe 0 although -trn:,;, ;;~, :im~;lz\)ba.blt<o The valid:.ty of ar;~il '3'\:'!':.~n:':ll!~.s.L d.ete:rn:dnalt;l,)r;, <il"!pemis upo:n the ntunl,er of bi is of ir.jfo:rma tit~~'!i po:i:c.tiy~g 't.(, 'i;ha.'jt, pa:::-i;ic./1~ a't :1txur ~ J.1'e o Thus if the retention ~J.llle i.~ lrn.::;wn 9 a.1;,d Jther,fa:t.a.tnd3 ca:~~ a twe:r.tty ~ t wo <G!J."bon d.i.ene at:d.d, tr.~.e assumpiion 'Of fen"' t=d;r'll,~ture e:f th~ a:i,d a,':! d:':':'\l>sp.c~o)/) 9 -~-}~ d.lercoic; a.c:i ::i. (Group XI!) :E:~eeiD.:S ind~~r:;.,a'q,~'.i by ich~ p1'""'it ctc:il:v:.::id1en(;:~ wi 'h a. :.C:t.<,e CJased J.n t'r.a.t die:r~e struoturre -~yp.eo R:::>,re,:."e':r: f) t pol-'~sid~.~ t<::t::t :::: '\:.. tl:':.sferri:ag ~he data a doc,osa~l3v 16~dien.:1:;; :s <;:o:'j..2:'::'!ll"~t i>:~ alst" :;'. aenrllj ble i.:f ~he structure assigned to point 21 i.r;; :.or:recto The :i.denti. :i~'o' ::,c.ggestecl for thi!i c,the:>.' unknowns of Group XII.!1.Xt5 h.:;,we~yex' 0 0 h.igl1ly spi6c:nlat1 ;:e 0 s:'..:f!{j~ in tw.:j; cae.~ei I'{'~ even the rel:>...ain le:o.gth :7~3. b'~-'t tb.~b~ Ht ~ pu.,; f uy~..v2:r1 R:5~ n.-a'te::r:i.a1:fi w~icj1 J:'':m.ld be 1Jb'3ed '111de:r <C<Ql] [:r.<~:ij.et,,;_ COriJJ:". ~;io;::s. : il~ :'k tl:j:i :iry-::.;,,.,yrt.\.,i-?::1.8 ttd7811g6d &..E' the basis of thi~ t:.j(bll'u<;:i S~!J~ c. 0

30 8 MAR:mE OILSg GAS=LIQUID CHROMATOGRAPHY OF LIPIDSg APPARATUS The convention~l gas=liquid chromatography apparatus available has proven inadequate for the d tailed sb1dies desir~ble not only with oils but also with various associated lipid system~o This is in part due to the extreme~ l~ng retention times of the methyl ester"s of the longer=chain highly unsaturated fatty acids character.istic of marine lipid systemso The limiting factgl" is th low sensitivi~ of the detecting elements of the catherometer typeo To offset thisv low gas flows must be uaed 9 in turn necessitating higher operating temperatures to give pew s~hsjr:p ttnough to be measurableo At these higher operating temperatures there is 9 during the ~lysis 9 a progressive interaction of the methyl ester with the po:iyest.r substrate 9 greatly reducing 1;he accuracy of quantitative work ara leading t~ rapid deterioration of the coluwno The hydrogen flame i~nization detector has largely been used for ether studies (see belo:w) but exploratl.\'l:ry work with the higher methyl esters (see Fig. 1) indicated that the high resolution possible through the employment of extremely small samples improved the separation of the numer~us individual tatt.r ac;:id$ described in Appendix 301) 1960=61 Annual Report. A number of other c~mponents indicatedg but incompletely separated 0 are believed to be fatty ~cids isomeric in the ~sition of the double bond aystems 9 as fo~d by other workers in the field. The auxiliar.y apparatus ne~essary for stabilizing the rigid cperat~~ conditions of such highly sensitive apparatus has been found to be of limit d utility at the necessary high operating temperatureso This ~pe of dinct rea.arch has been deferred pending acquisition of first-class argon f3,=ier.g\j.zation equipment sui ted to the nature of this and other studies 9 especia.lly il! the sterilllid fieldo Active investigations hawe h wever been carried out in a number, of fields based on application of gas~liquid chromatography to lipid research &ld these are described in detail belowo Fi.gure 1 =!r!b.lysis of ~od liver oil metl:zy-1 esters. Sample.0005 ml 9 c.ol\jllu: l/8 inch x 8 feet 0 pa~ked with 2Cf!, DE&S on 60-=80 mesh «Shromosorb. Operating o nd:itio:ne g temperature helium at 20 lbs pressure 9 attenuation 9 2 X. 2X. A) B) c) D) E) :f!") GJ ~~ Identity of principal peakag Methyl tet~~de~anoat~ (c ) 14 Methyl hen.d~ anoate< (c 16 ;J Metr~l h~xade~enoat~ (c 16 =2R) M~th,yl ':J~tadec:anoate (c~ ) Me:~cy~ o:'tt:adecenrciate., (c~ =2H) 8 Met,b,yl e:v;;osenrq\ate (««=~) 20 Met~~ d cosen~ate (c 2 =211) Metl~l ei osapentaenoate (c =loh) Met1t411 do~osa.hexaenoate (c 20 =12H) 22

31 --! ~- I ~I -=-~ ~ c--~ == 1 ~ '.. " '_,. ----~ ==--,:.:::J,, -. -=...-J i?"-, L~===::: -_----- j,-- ' I i

32 10 S'!JEMARY NOO 5 ===~- M.!\RINE OILSg QUANTITATIVE GAS=LIQUID CHROMATOGRAPHY R, G., Ackman Ro Do Burgher C~J,mparison -,f xoe la ti V\Eil p:rclpw:ti :ms of unsa tu:rated f'ai~ty ac:id methyl est;e.r~s am their hydrogenati,:jn produc'~s has shown serious discrepar,c:ies!) particularly in the C20 and C22 eer:kes 0 cha:racterized by their content of highly unsatwrated materials., I:n par t this an be ascribed to interact:l.;:;,r). cf,fhe ma te:rials with lojr!..ger rer'ten tton tim.es wi tb. the column subf:itra te (sac?: above) and :b1 par\g to relative ~'~response fa,~t.orstl! 0 known to be a characteristic of iir:jh fatty ar::id ester., The lattex' '!'i'a1ues are not available for most of the fatty Mids r:?'!1cou!i.'~erad in mar:ilne r;;:c,:.s 0 oo:;[; it is h{j,ped tc: determine thc-'jll through pr~pa:;;;at:bre thln=layel."' c:h!~'(:,matograph;yo A furthei" e;ompli~a:~lo!rr Jr:e(,~tly d.isc;:o1;ered is that ce:ctain. n0n= &:i&ponifiable ma~:gerial!31 c;cdlll.; ide fy;, gas=hq,i.l.b cr..r(omatog-.taph,y wi.th methyl este1~ ~a.k~o Thus squalene (C)Op=l2H) :o'rl'&r:llap;s the p~ c,f meth;;d dl(l1co~ati.exaendate tc23v=l2hl Squalene and oi:her isoprenoid hydrooarbonll';; occur particularly in mar:l>je Hvex oils 0 but it i~, belie~ e::l. tha t tll:ey mby alscj O((;,CUI' to a lesser extent in crther marine oilso Thus ::he rapid and widely used transesterification t~ghn1qu~ f<jx' the p:k:'epar&:don of metb,yl '3r:r.t:e::;.~s of oil fatty a(;ids may no longer be ur:h?d 9 since saponifioati:c:n.is ne\c't>abary to I'"emove non=saponifiable materials~ The recovery techniqt:e: f,,xr- the rna terials prepared by semi~micro hydrogenation is als'cl beir;g c.hgck ed for possi.ble pj;"eferential losses of the more vola tile rna terial<3" SUMMARY NO. 6 MARINE OilS~ GAS~LIQUID CHROMATOGRAPHY OF LIPIDSg ABSORPTION OF AlJJOHOlS APPLIED TO THE DETERMINATION OF NON=ALCOHOLIC IMPURITIES Ro Go Ackman R. Do Burgher The direct analysis (Q;f high molecular weight alc:/)lhcls o:n polyester gas~l:iquid chromatography substrates has been l."eported to :result in partial or co.m.plete absorption., Lcng=c:hain fatty alcohols have therefore been separated on pclyglyc,ol substrates 0 non polar substrates 0 or on polyestsr subs-i;rates after acetylation of the alcohole~e While carrj5:ng out a:n analysis of acetylated. long-chai.n fatty alcohols. prepared from marine oils 0 m1 a polyes"!:;ex ''olumn at high te-mperatures ( 220 ),. inadvertent inject:ton of a. sample cf the alc chols themsel'\lles gave vi:rtually no respon.beo Tlag &uggested the pi:l'se:i.bili ty of determining r,,~r~t:ai.}(l ::tmpur'i i:i.es ::mch aa hyd:t )t:::a:rbo:ns 9 aldehydes or es t;ersr which are known to pa~s through :p-:ll.yesier 00 tj;1jo'a~':' :)1!..EV 'i:i ~.9 'C:.vely v by complete abso:z.'ption of the ale:r::bo:.. This tee.~ '.l.qm:>..::-dld t;e ;..':1c. t1 ~ular2.y appli,~able ix~ c;ases where a mixture of a :number of ale ::t :;I? and their c :.r.::. espondirrg hy dr!'yja:cbc:;;lv aldehyde 0 or ester forms wculd render

33 The ird.tial obs~:r;ati vn ;. a.g mbl::;-, -~.::.. r:ol :CDIIl!ll9I"C'Ji&.lly :pj:(?p:l:r8d colunm (Wilk.ir:ts Irmtrtuna"lt and RS<S ba:r:::hn!d<:oo Wa.~.::,_ r <:'c <o, 7:1k~ C'El... f") p:od::e1 wit:h 2Cf//. ~--~.,.+'., ' 1,; '".,,.;:.., V (.Dt:~<"C.\,.,.,...,. t> "''. _C.I~\...,. '\, r.:;,' "ll''i"'"' t A '"~ b -~ a""~ ".ny.ler.~ g y-r::.:, J1 s,d,..: ~ :Ula. -"~, ~;;;>;.d~ --.;c~-., '"'" '-" >... a:"'--" --.~ : "'--' ''-" -1 -' rj...,l :<:;; e;,f DF;GS ~arupleb of d.i!-fej:.:'~:cg,,~ ajd.d. ~:.~-~_.,:n";', r~.>.f : ~.l'i'2>:. ~~l!-~'bstj':' tteb <~!PJ:{~~ te~d:ed w:i th va.:rying re~r.l'; ;;,o R:. werv.::~ x v r.ijj;,:.l '?,.i:' c: mp1e~'" of lgclg=cl'.."lin.f'atty alcohols at high! e"' ')6:l~p l:j.a:t?'<''' wa<-\ ':[...,.,,,,.,,,j r.ri ;f),_. nms p-~ l.)r,-f1~er r:.wnth4 - ;i~~d witho>u.t" e.ta:l.yl?l~;; {;;~; t(:,;y ~f D(,sctr:~..... r'-df.tg :-:, Inc"D Chicago, I:U.)~ ani cm.e prepared f:rom dj~e U:.yler.?. ~r ')3,,_,L: p.':.5."4'} ~ 9 :~atnol~:d.ge lnd,~ tries C,:_:," 9 Inc. ~ Cambridg~ 9 TABLE I" Area Resn ot:::.1'! 3: 9 Rc-;1~1'.::'1'13: ~J JVl-, 0f 00~ Oiey:'l Air.t}~'lr:.. 1.~ ll(ij;n,ld w;', M ~ " -.;;, ~' i'j. '-(a.-~-~~:~ ::.. 0 (VJ [t :-:<% 1' 1 :-.\!li,y:tl::ta ~""' <.'. ::> 1D.. s O.l.U"' o.. ~~~o* ()008 0 ~')3' Mas13.) 9 c;ontainijc',_g z% ' a:c:i:-:io?m ~;,':; :li'o.'' ~~ b' ~ ;:s9 t,?i,e abs:c~:r.-ptt.ij;)!l a~tivicy waa ~hort~jivscl 9 wheo:'t:ea.<=<'.,.,-; i-h ths <;;her ;, ~ ;:c;a~~ 2id.al:~l 1l-1.:p tr.t one h1;nd:ved a':lalysef:l,of' alc"'ho't::; cr, ::: 'lis "-t:l" -?<:rs w-<:~:r-:.1'.. '.ar.r:'.eori :YGJ; be>f ~ re i':r...a column ceased to absorb quanti ta.i.i:rf:'ly. To aases:s the se:rn!.t.~.vi.ty :-f the t'~ :;h':'.:1q~ t~ ruv1 l~-;c. r<rduce varietl:::ns i!:c instrument 'ii"'s~jx.(o.e'\9 d:ne t,:t -~emp-pn~. ~c,:r:t-, 3:~1 '.utx'':i8:; ga;;, ~:J(JW :?;:bnngesv Simples; of alcohol mixed with a pure IDE>thyi 3S'0~.I W S..ra t?-l::lp::t)yf..~o The a.!.oe:a.;,f the methyl ~ster peak was taken aa 1.00 ru:t.'l th-b< oce3,~jt t e a::o;~a ')T crh::;' i:..l..lcc1hol pe<ak wa.e exprt$ssed in prr::vp>:~rtic::n1 0 T:'ne res'.jllts.,)f t,":\"''t.'"' ai;?a:f:'ying c:oorperatu!"bb on "~o substrates< are pre~ented ir.. Ta.ble I ~.vnst' ~. a,,,;:gn:.;:,;:,;, :f S~~ alcohol (:pr<slpared by red:uctioti~ r;f highjy pll:.ti.f~l?r.::l. ~ ~>: a4: e> i<"' t;l:: ~...: U:>i'il.\11 ah 'J'1:il'.~).ill hydrid,:,) and 2r:J/o methyl :m;yx: is tate wao:< '*mpl:;;;.re:lo The a>0tl~e.tl :>:"E :::..ative x.e,:-:rp,:rase &~o m 3,,-, -:.hr:! u::;:,u.':{.ad a1et.::.h n1 p"~e1k Positi,r,n \.Jlrler c;gr..j.it~.m!~:> be:!j.e<>-~1 tc''.""_,jx-~ t-~-te.l i'l."j:~;.k~o:. ~.. :.,~. yrf 1.,.,,,..,~'i" d ~~,-,.,r.'l 3 tii") 6% of the methyl!:jst~n" :.:'<e'2jk11!1e'8 0 ~OT.t'BSpi: l'-"i~':.rg i;e) ai>..:.t 2.% ~f't~,.. :~yl ' ai~oholo T'b.is is b:elieved. b 'be an 9.C'tua1 ty i:n 'IJJ;;-.e :::r:n(l t:t;.i;;-; view :i~ s!1 PP'1Jl"'ted by!;he, fad: that ~ [1!'fif~.ll1~r ~~)"S tal:ll2 ' :! ::.a :Ami ty1 aj:j_5 t<.i::fl! C";,,ll alcohol~:: under the i:'i<s.z!'!>b' c:jndi 'tiox1s ge:~;f.,:, no ~:8-21Xir1S:fl 0 H,2Ji1JHJE./ 0 th,-, he:-~,.,:.:.=j tin,:: p riat P.nd limited sc lubili.i:y :rf '.-:1>.<0''"-' rr>a t:ft.t7cah "\... : :-:.1 ~~l10"5al ~!!.<, -:; ~~::l:us O$i:tig i!?!111pj th,e leyl a.l:c-ohol. 1,1Yld tw' th(1s~ c. Li'i~r;i.-o::"r~ >ta "'! 'L;,,,:, <:L c ~ il'p-:>x.3. r v '!ation tjn.at ttdto: ;;:E? spc:-nr>!' wfu; :::1.",, e:rj.tior, preliminary ~~x:pe:dment.s witt eli!"(y::. experiment 9 c;learji.;r ehowerl ~:.ha 1 t thi:t '3JY tr J.n ~ ~:f a.:j ~-,,;.t.o.i.7't'- 1 f:enti('~"l 'L'bs pcsstbi1tty d obsez ~ ;~r;.t t'8}i{jl} :,] ~J f:~..[:jj.. ~; ')b;:.;.'.?(< - i.me 0 7t:.s~c 20 ~ ~: a.~._~ ::crbed ir~ E~.rA?. a bs\\l' ~.,Btl :<s..fltcte- as~d. wi. td. th~

34 12 dis0'1unted 9 but in preliminary studies trace amounts of material with the same retention time as methyl oleate were oqsepved in preparations of oleyl alcohol other than the one employed in determining the results listed in Table Io The sharp transition in column activity 0 when exhausted 0 from complete absorption to decreasing partial absorption suggests that the absorption of the al ::ohol takes place at a limited number of reactive groups in the polyester, and not at the ester linkageso While free carboxyl grou;ps are a distinct possibility a~ active sitesg it is also possible that carboxylic acid ~drides would be present and would be very reactiveo E.x:perimental Th!i apparat\u'l employed '~a:e a Wilkins Aerograph Model A-110-Co All columns were l/4=inch in diameter 9 ajad lo feet in le:qgtho The f:i.lament current was maintained at 200 ma in all experimentso The flow rate of the carrier ga8, helium 9 was varied to keep the retention times (for the met~l ~istate) relatively constant at about 10 min at all temperatureso SUMMARY NO.. 1 MARINE OILS g GAS= LIQUID CHROMATOGRAPHY OF LIPIDS g OXIDATION OF HIGHLY UNSATURATED FATTY ACIDS Ro Go Acklnan R., D.. Burgher rr, UlSing the ~.rer'y small samples ( w=4 grai~b) necessary with the flame ionization detector it was noted that analyses on certain coluiillls showed gre<:~.tly reduced proportior~ of the fatty a~id methyl eaters with five and six double bondso This effect was traced to prior exposure of the copper columns to oxygen contailled in the standard grade nitrogen employed in the laboratory (up to 100 ppm oxygen) o Prolonged exposure of the columns to this gas at ca. 220 results in formation of a film of copper oxide. This may act directly as an oxidant or may be a catalyst promoting oxidation of the unsaturation by the oxygen in the nitrogen a After formation of this film partial oxidation may still be observed with helium, argon or nitrogen containing only 1 or 2 ppm oxygene The activity of the copper may however be neutralized by passing hydrogen through the column for aeveral hours under operating conditions. The column may then be used satisfactgr:uy with other gases of low oxygen contento Stainless eteel column~ appear tc be only slightly affected under similar circumstanceso The new apparatus contemplated can employ glass columns, whioh should be completely inert to this effecto Similar effects have not been noted with the conventional apparatus since the columns have not normally been exposed to carrier gas containing oxygenp and the sample is normally at least 100 times as large, reducing t:t>.e significance of any oxidationo

35 SUMMARY NO o 8 MA.RJCNE OILS~ GAS~L,IQUID CHHOMATOGRAPID OF LIPIDS~ VOLA.T!LE FATTY ACIDS R. :i o AcJ~7tkm R.D E' :Tg'b~ " The quali ta ti ve "tc::la1y~:i~ uf "lftllatlle fa tt;y ac.idl? :tn ajj,,xte aq11er.n.k~.;cu'!luti ~ns P important p:rodnc~r,'!'- of tt. "! ~'::!:r.ln~~2.y~j.!l p.r':t,c;e..i~tr~~ i.;e we:ry cre.nveniently ("Er'Iied out dix-ectly tb.rr 'J~sh tht!.. ~ '~ '-'f the f~.am.e 1oni>;:;a.~L: r1 di!'+l!':ctor~ which A.t' :insensitive i;:') wa.":;e:r and fc:: :rmi~ ac:~-~!. P~ly>"J;::;r:~r e;;ol~lm.nr..; CJTta:x.:Lng ;:.% H"".P04. ~. p t ' f t. "'.., +ex... r ~ i -:.,..-~.-...': ~~-" "?< i 4--, h~ ''!'~ h.,.. " _,.,.. ".,. ~t,,<> r,.: ~.. 11 e ;e,a...1s. a<c;.:or:y e~pare. ~d,\.u?i.. i;[,,.az. ' ~,a.,,'"~' e ' I!!SU.Ji..,s.>!;i... ~ ' l!':ert a.~sa.!t-'.~:l':i:!.,j. ;,lll'6 since. varying amc:unts of thes~ a c.:i.'ils (a.c~i\':t:ic; c:: ncrn&".\o:l;,:;) a 1.e ab&orbed by tlje cohann 0 pre,renting :.:eprodl;.~;ih..~.~ fj.na: 1.y~~s~ J, \.P~"i. be~i d:>.o~!rc:;j c~e!'ed ina i,; 1!" +.I~ {\Oll1lllll is fir~t,;;1ea~~d Of <ebje.~~t:ilr.nja'bl~ ll:.b.~~";;:ia~\tz~ l)y ln,jet:>':::axn of fo:x:m:ic ae:i.j.~ fcllowed By inj~ tiolm o f the 11Jil.k:rr \:rw.~ rc.:flxigur~~ ::her. ir;he abs<:.rbtiid materi.:ij!'i m1ijy be nearl;y q\:'c.9ltt:i.tatbrely di.~p:ie,c:,, i i::;v :G:.\!1: ;:;:; (11\'Cv ~d'l~~qe.~~:rd: mjecti0r~ cf f~:<:rmi~ a.c:id. 1'his requiret5 ~e veral in.:l!ll'"t:i.:.rn<.> fr J' ~ej.:.b e..naly'3:t:!io It i~ p~:st:l.bj.c' ~11</~ the re0ently disc.oyered r~'f.»ppet.' o:l'~:',(cte layer (Som;:,l~l"Jf Nr..,.~) ma.';r have s0n~ b"!larinr on thie eff eet and redmrel!r'i: i.ga. ti :))D. 5~;!i'. nl!'e<~:f!,~,a~y. MARINE Oll.Sg GENERAL LIPID RESEARCHg APPLICATION OF THE VARRENTRAPP REACTION 'I\0 'fhe PRE'PARATION 01'' LONG~CHAIN DICARBOXILIC ACIDS F o R. G. Arikman l~l Ao B;mner1l!l3:n M" E. RetRC n A. Vander... b.e ayel Aliphatic dbarb~>:cyli! ~ acid!s wi t!1.11.::::r rne. :cec :;arbon atom!! were required for ga8~l:l:p.llid C!hromatcgraphic :Jiden'tif:~~-a~~:L;n c~" :=tmilar material8 produced by czonoly5i.~ Jf ma.dne=oil fatty at:.i5~e A!S'ide f' r.jm ble'aseylic ac:id~ prepar~d. oy o::ddati we f:issio;::. ~Jf er;lc:ic :. arr;;id~ fhe5t! a"::'.1~ a:r'<e not C(Jmreniently availa.!:lle o As an altel"'.na ti "re t~'j jjnd.iv±.dua)~ S)lli.'thes~~ D mod.ifica ti.on of the Va::r::oentrapp reaction by 1i.miting =~ t'o red::~";t:::< i outi.;:r,l cf t:ne 13gl4 double bond i:n eru<g.il: f.l.~c:id intc1 adjac;ernt p:o,sititme 0 fol'l:rif~d by t xtd.ati v~ fi~sion aad re,c:ove~r :of the dic;arboxyli:a a ~ids 0 offe:"~ri,,~ r~.~ Vt~~ :-f 'J1:d;a:L:~r'..:t;"g a Dtllllbe:;:,:Jf thest! a ~idi5 in one pre~pajca ticne T~ prod~:.ll<:f~'; of alkali fu~i'j; a1; 320 C w3:': Bel~ ~ii f v cj'xidativa fis~::.on as the iodine: yalue of 6.,:) cl:ndj,.:;at;~d t:tja'f: t.be '.f;;u zt: ~.;a;,:p r."eae:tion pr0p~r~ productic:n of the Bat:urated a(;id +.wo ~a.rbcm atom~ f<:t.t:x c.,'!: c.bt:,n the or:tg:'.nal monounsatujtated ac;id, 1;.ait p:::o.~eeded t0 a.. itui.ed '"~:::: ~~':/;,, Sl.c1c~e d1:rr"b;:.e. bond~ migt'ati.ng to the 6 o:r :,f!, :vap:;d.i.y f'!."': te>; :i:< the fi.nal!!ltage:s of thi>"!. reacti.cj:n~ "~ t we,.~ ezi_l!'jc;ted d:.t:t1: '\;he li:'esidual doy!.ble l;on.ds :\.:".. the fueion prodvct WO':lld be iu p?si t!.z1y::;'!s m. Jre remote from th~ carboxyl gr "}'.'':P 6 The petrn:.eltm.. ether~:::d.l.j.it:;le ~.. :' J:l'r;du.:" c:mtaj.ned ch.>lefly mono:::ar bc;xyl.i,;: a.clda Sl!C'h F.t:5 palnrit:'..t:. ~t~&-ric" ~:::::-;d ax.s.cb:'.di~o with a.pp:r'~':::<..:.<.b,e amoun.ts of odd=nl..miberl'!'d :T-:iri 1~1El:t"b:c:rl!:y :Li,:;; a..;;id~,,f 11 or ::fewe-::r :::ar borc atoms.

36 14 Thi!',r.~tharw:t=~.oluble ):zr:molytda product contained approximately 6Cf% of roughly equa:: amounts of the di~arborylic; adds with 1 to 16 and 17 arbon atoms and 4o% of mom.)~arbor.rli.c. a!q:ids with 7 to 16 ~arbon a tom a Sin.ce double bonds are increasingly le::je susceptible to the Varrentrapp reac ::~~on when more re»1ote from the ~arbo:xyl group 0 a higher proportion of longrhain di~arbmcylire acid~ with 1.5 or mm:e c:a.rbon atom5 could be obtained by prolong~d fusion at lowez' temperature~o The over=all dicarboxylic acid yield wo1lld 0 however 9 be diminished as a. consequence of further completion of the Varrentrapp rearetiono ~xperimental A techni ~al erucic; ac:!.i 9 i0dine '\ra.lur.! 85r was employed in this reaction. Gas~liquid c.hromatography ild.dic:ated a composition of 72of:!/; erucic acid % ei.cosenoic. ac;id 0 6 o 7% oleic ac0:ircl 0 and mi.nor amounts of palmi tic 9 stearic, linolei~c 0 and arachidic ac:idt'!., Alk:aJ.i fue:lr.m~ w~re carried out in a covered iron vessel fitted with a nitrogen. inleft a.nd a stirrer of iron pipe.. Equal weightl!l of erucic acid 0 sodium hydroxid.e 0 ru:j~.d potassium hydro?cide were placed in this ve~sel 0 and it was then placed in a Woodus metal bath preheated 40 C above the re<aotio:n temperatureo The m_i.xi:;ure -was ~tirred continuou8ly for 1/2 hour and in :rermittently fox" a further half houro The cool~d r~action mass was dil!lsolved J.n wate!" 0 ac:idified 0 and the fatty acids extracted with petroleum ether. Weight recoveries were approximately so% 9 with iodine values of and 69 from fusion~ at 9 reepectivelyr and 300 C for 1 houro Gas~liquid chromatography indicated almost complete d.el!truction of the oleic 0 linoleic 9 and eicosep.oic acids 0 the productb thu5 consisting chiefly of saturated acids and d<e;<e:osenoic: acide o The alkali fusio:n _pr~duct of iodine value 60 (5 g) was ozonized in glacial a0etic acid and the ozonide decomposed with hydrogen peroxide., The final ~elution was dilut~ to 400 ml with water and extracted with diethyl ether (400 ml)o The ether solutil"jn weus washed with water (50 mll and the aqueous phaf!m discardedo The ether wae removed and the product (5o 7 g) was dissolved in 9t.Y/o methanol (200 ml) and extracted with petroleum ether (bopo C 9 2C~ ml)o The petroleum~et~er~soluble material (Oo9 g) and the methanol=soluble mat~rial (4o8 g) were converted to methyl esters with diazomethaneo Identifi= ~ation ot certain components was effected through mixed gas=liquid chromatograr~ with authentic materials employing both silicone grease and polyester ::substrate5o The identifitcation of the remainder wae cmlf'irmed by plote of loga:r"i trun ret en ti C n time vs o number of carbon a tome, SUMMARY NOo 10 MARINE OilS~ GENERAL, LIPID HESEAIWHg ELIMINATION OF EXCESS FORMIC ACID FROM OZONOLYSIS MONOCARBOXYLIC ACID PRODUCTS Ro Go Ackman 1 0 Ro Gallay M.. Lo Hughes The analy~it's of the mojra<o a:1'!:'bo:zylic acid products f'rom the high=yield oz(molyi'jil!l procedure (Append:JLx =60 Annual Report 0 Appendic:ee 33 and =61 Annual Report) may be c~nveniently arried out directly on the aqueous steam di.stilla.te by ga5 Ghromatography 'Dlsillg the flame ionization detector

37 ( g;.unrn: '"Y 8 9 tl'j.i.s ::- epo:::ct)" Hmf~iver it ~ de~:ira.ble to de:mon~ br'a'i;e the general appli ::xation of the procedure to xr;any C:CriilllO<lly Ufl<!id an.a1ytic:al sy~teim~ r usually basei on chromatography G The large excess of formi~ arc:i.j present (mere tha<l :100 t:cmes de5ired pr dtw c) would normally render the~e systems useleesf, &"l:i mean~ h~we been explm;ed to elim.inate th'ls Ev:dd wi thou.t serir usly affecti.:ng othez :e,.:;ids present v partlcularly aceti t.'. A number of t.eclmi.q\aes were studied (App"!adi-:,es 34 ru'11 35v 1960=61 A:..wual RepCJrt) 9 but aze0tropj~~~ die~ti~.laticn app~a:r~d ~RU1~iG\:.1arly promising Jwing t<oj it5 simplicity. Chlo:rcJfc:::rr~ {B.Po 6l C) wa~ :lj~;.~t:tally chosen since it :1 t'l sui table for extradlng the f.a tty acid~ from the a.qut";oue~ pha<l!!e ~- ru1d wal!,,,-,::ated to form an azeotrope; with frglmie; acid b:c-t not wi tr~ i'i!cet;.c a.::;ido Thi5 wa.t!l c.:n.f:irmed 9 but distillation o.!.both acids simultane rmsj~r indicated appreciablel lcl!'s~~ of a<.eti(:' acid~ r;_(~w k:n:y m t.c'' be i:~1e :,:. a '",e:r::j.ij,:l'jl sz~; t.j:<op~ -:;,:t :-h1orc fqrm~ac:etic acid~for-mic add~ 1nf'or:matiL>:n,~nly re' er~t.:.y pub1ish"!:d 0 Methylene :;hl,o:r:lde: (:e.p o 40o 1 C) ~a~! ner;t ~-~"';dt~d l!i::dh enc:ou.raging <:t!l:!!ult~. from the di~t1llat.~.r,i'1 p:-'i.:ryt,)f' 1r:l.ewp a::l.'~h.;j ugb j';'g t.;he~:.ia:ge F.illlOlmt~ mu.l!lt be distilled :eince thl! e,zet:-~!:'!'!-'~ C<Jnta.i:nf! only '1% fro:rmie ar.:n:j! 0 However a~ B ~v::>j!::~e;;1t for extral1tion o-f x:h~ a.oids fr;:m wate& i ; -~~a~ foun:i ~~ ) be l.m~ati.:!'if'actnry.! _ 0. ". ~ 0 '. 0 1 ~ '1. "'~ ~ t"" 0 d n 'l wa t~on l>l.zluu\1.~ e~te-8t' :.:J3.)JC~~. ::<!' p).~op~,;~:'!f.' 1 Jt(~ a~lf& X :.~./l:' ~J :;'~t<>~ ".l :CUL,Y ~i (JO r }.!....~~.e a~~l. ~8.!1 :removed 9 wherea.3 with d:lethyl ether 2 hvu.;'s e:x:tj'("etf.:t:te,rdc :Jt~moved 96% of the acid U."lder the ~ame cmltdi t.;'l..;:n~.o Ii;; wa~ "Jle:c efo~,e ne c~!>~~~/lcry tc1 introdv.tce a preliminary diethyl ethe!' l!ix1g:ca.ctiun 1(;;:, reml>v~; tf.eo?'j~ 1.<.1~ frgid. the aqueou~ :phaee. The bulk ~Jf the dieth;y!. ether e:ou.ld the:n be ~4~rli P~1.,off wi th,out lol!e of the ac:id~ 0 leaving a residue l':'f 5= 1.0 mj'. d.ie'th,yl 1\!:the:r., U;;~f,c,Jctunately th.e boiling pvint!'! of methyli!'j:ro.e ch:i:: Jd.de m1d dietb,.y:. ether a:n ; :_>;) ;;~!~c;se bgether that ttd.:r! ~:~ma.ll amount of the latter wal!t l!tll':fi::d.en'~ t:::,, l!f\l\pplt-~a>s thee f(c1rmation of the methylene C>hloride=formirc~ ac~id az~otr.ope 0!'.!<CJ attentton ~Ji'ae turned to highex boiling solventeo l=chlor,opropa&l~ (BoPo ft"'c) J.'hl'W a.ppear-~ t<; [,e a ~atisfactory material, sine& tne. azeotrope conta:i:rti'!i ff/; f<0:!tij~l\ ac,:j.d and the r~ddual diethyl ether i!'!l readily remo..,.edo DistillatioM perfor-med with a 30 x 1 inch (Vigreaux) column are listed m Table Ie Since it if'~ known tllat :formic ar.;id conta:r..ns ac~etie: acid and vice ver3a the pre3ence of rull acid re:!id,c,e frjlli tbefe:jrmic a;:;id and some acid dfstillation illx t:be am of the acetic a(d.d i1!1 not ~~llrprisingo A blauk distillation of diethyl ether ajll\d l~, :hlo:rtpr\';:pa;:be showed -~ery little background acidityo

38 16 <~.. - ~k:::j.g i ~ Amounts of Ool N NaOH Required to Neutralize 5 ml Lots f.)if Distillate from Distillation of 50 ml l~hloropn::»pane and 0.5 ml" each of Var~oue Acids Formi:~ Formic+ 10 ml dieteyl ether Acetlc Propionic F';action..!!. "' 18o30 ~ ~; 12o l0o69 o o8Q ,70 9 Disl!lontlinued 13o90 Residue 6o40 =- i ' o40 o40.41 i.37 i.24 I I I I.24 o28 o , SUMMARY NO. 11 MARINE OilS~ GENERAL LIPID RESEARCH g COMPOSITION OF THE FREE FATTY ACIDS R. G. Ack:mari R., D. Burgher Mo L.. Hughes The free fatty acids formed during the produytion of "sun rotted" cod.oil are varioualy stated to be releaee.d by liver lipaees or by lipases associated with ba~terial action on the liver proteins. A preliminary survey ~howe that there is a distinct possibility of e~pecific lipase activity involving a reduction in the amount of docoeahexaenoic 1 acid and an increase in the amount of ~ristic acid 0 in the free fatty acids relative to the neutral frac:tiono (Table IL If this assumption ie correct 9 bolation of l!luch an enzyme could yield a powerful agent for exploration of gl::yceride etructuree. Owing to uncertainties (Summariee and 7 9 thil!l report) involved in the quantitative gas=liquid chromatography this work will be repeated6

39 TABLE Ic ~~~--~- =---~ Oil eouzce Percentage fatty acid chain length compol!ition, (w/w) and pr'0perties :~a.c'..ous cod oil~r of the neutral and acid fractio:n~r and of fresh cod l:t'irer oil (ai;t;e:t I;;;rd.rC>genat:ion) - - Newfoundland Nova Scotia South~Ea~t Coa~t North~Ea~t Coaet Sambro Harbou:t Terence Bay I Terence Bay (April) (April) (January) % FFA IoYc* m~thy~~ of ~~tere 1008 lloo 2'i'o 9 23o o5 l72o ll55o ~ ';{Q., i ~ ~ ]- -- c,,.,. c:! 14"., ~ 4 ~ ~ c::2 ~ I ""',_ ~ Jl..~i_. o ~ ~... ~-4 1 +o,~ _,4,oJ.-iA1-.t 0,..1 '.)()<: )1.iS.) c ~-- ~,!,(0 0.!'-T ~ --~-~ {~---~-~-~~ ~F==a-c~;t:~ Whole Neut,!'FA 'Whole N ut, IT~ jllb:"_n_e~'.l-t~o FF_'A~,~- ~u_,t_o FFA j F!'esl? C.L.Oo ~~1~~ I I I -- 5o5 '5.4 4o8 6ol I "-o1 4o3 ';io c1s C2o C22 Otlw;c** ~ 20o~ 2::o:~ l~o~ ~ l~c~ I Oo9 \Jo6 J'.o.L j Je f 7~" I ~~~ * By h;ydrogenatipn :>""' " I 26,.L?, " I 2fl ''-' ''' 6 )" c" t '"'(' 8 --,;r o 0 ~ o 1' o- J, ~ o' - \_,. o ~, Ll: o ::;.A o c"~ ~ ~ -J o-.. 26o9 24ol 18 ':1, Oc< ** Chiefly fhs and C17 in roughly ~qual proportiona~o c! 2'71 <'' :>r:.: '7 '>n 81,- r: a I. o'-' ~ i oll -?o I C.:Jcl' i:!.: 0 ; LJ.c4 23o4 24oCl! 24o4 l J <1 ~ ") ' "'f "'~ h 1 0' >:) I ) '! "'. ~:: I <~:4 <~:; ~;:;I "~:; -~~~~~-,.. 3o o9 24o9 I 4o3 I,,.., I 2 0 ',,..,. I 3C'c 7 i..,, H, 0~ 0, I Jo? 21o9 28c2 ;~3o l 2 '4: 2 1-'~ _.-, ::n "" /o ( oo I 6:.:,dl.o-~ II 4J- j.., 9 ~---~ ~0

40 18 SUMMARY NO o 12 MARINE OILS~ GENERAL LIPID RESEARCH~ PREPARATIVE PARTITION CHROMATOGRAPHY Although this project has 1b>een su.ependedv a few f\jrther analyses were arried out to aesiet in publica:!;i,:.no These coneieted of _accurate determinations of the retention volumee of the even numbered eaturated methyl eeters with 6 to 16 carbon atom~sv run simultaneous~v with the 18 carbon eeters with lg 2 and 3 double bondeo The volumee for the ~=C12 esters could be read directly off the chromatogramsv but the overlapping of the C14 and C16 estere with those of the Cl8 group prevented any accurate determ:1nationo Accordingly the eluant from the unresolved peake was collected in 10 ml lotsv and extracted with petroleum ethero The extract was concentrated and analysed by gas chromatography, using the flame ionization detector and a polyester column., Table I shows the retention times obtained from this analyeiso TABLE I Methyl Eeter Retention Volume in mlo Hexanoate 56 Oc:tanoate 100 Decanoate 145 Dodecanoate 220 Octadecatrienoate 320 Tetradecanoate 330 Octadecadienoate 430 Hexadecanoate 550 Octadecenoate 620 Thil!'l data revea~ that a straight line plot can be drawn (Figure 1) between the number of carbon atoms in the fatty acid ~oiety of the saturated esters and the logarithm of the retention volume as mea~ured from the 5olvent front (indicated by a ~mall negative peak 0 (~ee Appendix 32p 1960=61 Annual Report) o This ie analagous to gal!l ~liqtti.d chromatography resultso A slight!shift in the position of the~e linee from run to run is probably due to varying solvent concentratio~o

41 19 -l Figo 1 = Semil gax'i thmi~ plot f :!C'e~ten<aor~, W«J l1\lmes vso mlmb: r ::!.~;IJ.l"ltiJ n t;.tom~. i!lc a~id baino

42 20 lwud OilBg GaERAL LIPID B&C':JU.Rt!R& XUL!IS'fAGI JIOLiCULAR.STILL J. e. Sipos... Ini~.1 ~(ti.es were came,d out with the oped". deaicn of atillllllit (ill:uat:rated U ~ 31.1, Annual.Report)..Uthough.IIOa~ 110lecalar. still. studin ijl tbe peat.ba"v:e :utilized oer't&u e.eten ot dicarboxtlic. ac14a tor baeic; etllcu.u, a!jlce tltei:r wapo~ prestllu"e Qharacteriatica were.,.u_ ubul&w; the -.teriala ~throughout the current.. uperi.md.ta were e111qrl.j*].id.tate ud etb;yl oleate, elected u representatives of the fatty acid chain leagtu libq to be enoo1m.te~ id practice add thw, peraitti.dg i edi te twv of ~tical operatb~& coddi. t.tou. ''... _lxpl9~t9i7 1"IJDII were earried out nth a ai.dcle llllit, operatiac at lgy ~ra~ Qd 111.a ~ :N.tio ot reet4ue to distillate.!his approxiatee ideal o.~at:i,dg.cojuu.t,iou.ill. a..u.l ataa- aolecular still~.!he reault... (!able I~A) i,j:idic,aw :tbat cu,stiu.timt. fl"qii a rot&17 418c.evaporator approaoud.. the aeparatioa ~ues.obt.b.lecl wtth ot)a.er ~ of' 110lecular still nell aa the f'al~ fila still earlie.r atu&ed U1 this laboratqr,y. ~ aucllllllita were thm"ef'ore,jset up, ope~tidg ool&~tl.t aa showa ill lipre 1.!o obtain pptirlua eparatioj! ~--utte were ope:rate4 at ditf'eri!jg t ptra:turea, d.ecreas~nc (l> 2 > 3>4). as ~ diet.ill&te 1IU progressiteq enriched. Speeds of retatiojl. ~J."e varied aa. dictated by eater lenla in the various 'Qllit reaervoira. J)upite this flexible ope:ration 1 t prqved difficult to maintain continuoua operation id all mrl.ta sia:.ult.al:l.eo~ly, and unfortunate4 acqurate t perature control depended on, qo.ntinuoua we Uinc of the_ th.erlaiat.or.. teaparature.probe.. lloreoy.er considerable loaeea.of eaters 'oc~ tm:ollth t ope. face of tbe unit. However the enrichaent in the final di8tillate_:bml the.four.»nita, 86.U' pal.dd.tate fro 16. ~ id tbe feed mixture; DJ5 yer,y encouracinc despite a rather illpractically high ref'lu ratio. The "open" clesign wu therefore abandoned, aud new uni ta aade, iacorpel"- ating tbe ;rotors p. other :~art.a o.f the previous design. 'fhe rotor ia now... co.ap~te,q encloe.td uqeptj for. a well";"baffled porl.,t the coolest poi.dt in the condenser shell. In ~Qctiqe pract~~all;y no loss, of 'estera.f'roa theee uuita baa occurred. 'fhe tt'!api!jr e control uaita were red ei&jled and repoeitioned a:a4 now ~1'11it accv,rate con.trol eve at Tery low rotor speeds. Other aodif'icatl.ona include automatic idl.et feed control and d.rop-countijjg transfer tips to pem:f.t.. comparison of proportions of distillate and residue at each unit. Operatioa of' a si.dgle unit ~der th.~~t condi tiona to which units 1, 2 and ; would nol!mllt be a~bject (distillate> residue) gave eatisf'actory- replta (Table I-B.J.. Po~ units hf.ve now.been Qperated succeestull.y u.co:t.mter-curreut flow,. with feed a~ 'the a~codd ~t.. ~er these conditiou the.ratio of' distillate to residue ~t lm,ita 1, 2 and ; mpt e~eed unity:, t.be actual exceaa dependij]g on tempe~ture md speed of rotation, am the reflux. ratio of the still aa a whole is govel'tl:ed bj' tl'j4t of ~t 4. With t!j,e two component qsta et~l palmitateetb 1 ol,ateij four lu,lita give VeJ:T good strippidc separatione in one -s of JB&terial. (Table!=C). The effect ot operation with a three component eyata, eth,yl palmi tate=et:tv'l oleate=ethyl el"'lcate, will be tested on four units, as a approximation of the distillation of' a marine oil concentrate, before proceedias to assemble the projected ten unite.

43 21 ~~~= ~--=-~ D=Dia. 'llatti'j R=Res U i I " l I I! 0~=c=c==- I '.::::====c'-=':::(~: \.J,J \, /" Residw~ V D~:t~~illate J R!iaeN{[l;il.i:' VaGJ'b~ R~S~erwoir L.~-~ ,. Figo 1 = Flow diagram illuatl"ating operation of fum;' atag~s, C»f' lilllllltistage moleaular Btillo

44 22 TABLE I. Operation wariable~ and results from warlm.t~ molecular distillation~ R.at.ie Distilla.te to Residue Feed mixtw:'e Distillate Reeidue Rotor Temp. C A ( O:ne: Unit) ~~~ 1 22 l ::.. l , 17.Is.l~ l6a 35o "I' 7 k,) 0 ~ , o B {One Unit) l C (F~ur Unite) 1 Unit No. 2* 3 4 ~-~--~---=~-=~---=--~ * Input Qo o SUMMARY NO. 14 MARnm OIJ..Sg UTILIZATION OF MARINE OII.Sg CONVERSION OF THE FREE FATTY ACIDS OF COD OIL TO METHYL ESTERS IN SITU R. G. Ackman L. R. Gallay P. M. Jangaard Ivt L., Hughee Th~ C (;ld o:u.:!. pr.jduced o:n the Atlantic coaet of Cana.daP particularly tho3e pre!d:uc~ed by ' 1 eu.1'l.=rott:ing" 9 may contain up to 3\f%, free fatty acid:! expreeeed a~.0lei: ac.:'.d" However,> la.rge quanti ties of oil are available v through Mgre= gati,oll f!;f nla.'t~r:iale pur:clla5etl at plants 0 with free fatty acid (FFA) conteui:;,:,,;~ 1'2% oz: l~~e o Refinir~g of the!!iej better qua.li ty cod oile through claeeical alka.h refining i~ f~asible 0 but the JL\QJ!IS<!il!! tend to be high and the recovered alkal:t foo{:!! of littl~ 'Walueo

45 &terif'i<eation of the FFA :f,_n ~1.':t'a w:mld. b~ ~ 1 atrra Jtive me.w.e of improv:ling the quali i;y a-r1d 111!U'kef:;abili ty of thee~ oil>5 o The use of gly erol in trds t;ype 0f rea:ction hm been the l!n::.bjeot of r-.rur:<.h tjn,tl!<:!:'~~t wi.th war:io'u!j type5 <" f roileo The u~e of gly'3~:f''(),:i and ~d.ll:d1ar!'mjjf!c".'il't~le. :Lri.~ ajlc: ;'IltWl~ requi:r-ee high 0 I, <l' c ~ *, t d 1 tempera~cijx>e~ ana. ~JY. 1~e;.iat1.ve 171 J.a.E\g e ajjlo,~mt!> :.:. ~ cx;n: lt? ~o.:.::_:.~. ~a:,.alyl!l :!"p an t';t!, u~ tively high ('c"ll!t piant!!'iq'!.1.i pm'!'nt o T.h(!. ecox:::r:d:'~,,;~. ajre l!l'gi:"k! t;b.~ t; thee~ p:roc~s!j~:5 have beer.<. applied only to high 1;:l)~t <OC~1'< 9 J::l p'!'!'x i.~r.:,rl.~ <Of' &.CD'~ ;~ai ~ho:rotage~o The C(inv ersion of FJ!"~A 'k, e~tere C'/f the ~l.?'ioi~j; alr;r;h )ls by conventional pro!:edure!5 ha~ al~o bee"1 App)Lied ~J J<l; p:n~t'rent~ <>cn~~dt!!jr'able diff1'::"'ltie~.ow'l"'lg t:: the competing reac:tiona 0!. e~5t'-':r.ifi:..atiov. (a) ard. t1'rul!<.~m'r.e:rif:l~'atizm (b). {a) (b) R~CO,={:E "j. 2 ~ ij R~,IJO., ~CH + 3 R 8 :OR ~ 7,c. ~ R..C0 2 =GH 2 Thlt'l wat«!:r l.ib.:!ra'~f:'i.~.'-: ;r;.t 1 y~ (.t.) prevent~!'! th~ rl!'lac..,tji.on f'ro;n p:l'\"h.:.eecn.r..g t0 corr.pleti:1fi, 9 ani if e~x ~>e<~!!' <iu :. t : ;~. iidded tc for:.:e the reaction toward completion the ove:ra:, L :JrCn'l1'et:'l!!~ ;Z!,~ e~n;en;>~ l.ncr~mtl!i!s d\:;1! to x:~actton (b). Th.e latter reaction 'i;ake~ :plaqe ~"t;epw.il~ttr ~~'j t:ha t Gtm~id!!!I( &).le amount~ of moxw~~ an'd di~lyc~:t5~de~ are fo:rmi!d a" wello T:he :remcval of the wa.1~tn' formed during el!ter= lfi(;;;ation i!! there.fvre th~ key fa~;;'t:;.:t iv. (!)st;~s:r<i.flr;at;i.un ::.. :f FFA etf c>ile i.n sit11j.. T'l"&.i~ d:t:f'f;l;..c:.lh~y lraa.~ t'm'-1 ~ VI!lrc:::m.e h1 :ne p:t'o'~~d:t:ee by c:onyerti:ag th~ F'iA \~CDde.r a< uzydr\)uei f;,;ndit:~. ;i:.~ t;:;, t:oe ;jr.,d~urb.~al t3 &:c:u o;r ~at:;,v~g the mixture ':Jf' csil an.d ~ ap with dimethyl ;'.~~~ d:teih?l Sr;,lpk.:e. "t" in th ~ ptt>j!ie::~c~ 0f a buffel":lng alkali whid1 a.becrb<s the ~u!ph~u;.i,r,~ a.<.jd ';L:~~:rat<!ld. The FFA '"f gy~!<a~~~ e::<:r tall,,;;,w~ tmij' th'~l~ 'De reduce~d from '22 t~'!) c ~5</oo Tt)e t'eac::.ti.oi1 i~,~~~::.~~. t:'. ;:e;..y l'!imple bu.+; require:s :s'tepw:i~e add:i ti;~jq Df ;,u,.~:\\'in:<.a~.~ lilnd. :ajr'~fl.il :A5ililfl~l:'&t1Are ':'/c,r~_':!~.'~) c The int:r~dugt'ti )n of acetone dimethyl acetal (:2r2~d.irn.e"ChoXJ propatu~) (DMP) for the pr epa_jt"at:fl.ol~ of methyl e1st~:r3 rf fa. tty acdt:i.l!l ~ :tgg~~ted that ~ problem of remmring i~he watei' :f.orid.ed during t:t~e e:o~i~erifi~,auon :;,; ~acti{.)jl\ cou:d b., e<olvedv ~L'fll('je< in thias l"eacticn ( ) the reagent may ~e:rve bot:t. >~.~ ~,;r,. ;;:;~ :;;e of metha.:o.":l.ol (MeOH) <1nd a wat~r ~~;:;a ~,enge:r:. Further advantag~e were c:ffer&!i'd ll'2 tl:&t. the l'~a.ction 1r'equ:J..;; ~~!i only simpie t1quipment and proceede at room t!!mperatu: teo h"~liruir.a:.r;s :~,;~eults ij;:di,;;~:l;;ed that thits rea,:tiw. could be ~uccef!f!fully applied to ~he f'lf!'terifi at:iou of th~; FFA in :::od o:i.l ana e:x:perimentf! were therefeire rried ~~ut tt; d,;iermi.:ne the :r:t~~ttfe <Lv.i al'l":ilillt,:;'{: ca.taly!lt nee<e~5~aey P the leaet e.cunt ot :L"eagerd; which I(;O'J.~d b~ employed~ pe:.c atl:ng condi tione 0 and the nejc~~~--rv purif:~r;~p.i:;iolu ;;d. ighe p.r;;d wi, 1;.( ensu:re!l)"pt.m'i:~m quahty and etabilityo Th~!i ~xpe:rim.em:~ Cla:c :ri"'d oui :Jct!l t:t.e pjre,!!l:e.:ool~ :5t.. ld,t" ~J(~!'e Ji;~ect cr. the pv~l!!ible W!l~ r )f ;;h:l~ )~~a<:;tion. :lin ~~il phnft;~ w:., the Jim:Ji.t.rre>d "ilq'ui.pment aj.tmi pe:reoimr.}:>. Consequently the ju~telr'i'i~s~d iptimi,lt1.ortl,gf' the :~f:sil.c'>:;i0n W;J\~:~d ;;aq<!a2re a covered. ves~el 0 m agitat1:1rr :Ji.:t.'~tle o:r!olo itenti>on during il:'ea.,ot:\ ay::d. a. l~ eact:ilor; period eui ted t:; Oin&co.~hift pla:.:jjt l:pl!l1t'a. \i.c:1iil. 0 Simila:;~ :;;:~J;JJ5.;'.. :l~~a tion:'! diota:ted thf.l

46 24 u~e c: ':il.e m.!r.t:\mt.~m of relll.gent ani ~imple ;ru.rification etep~ 9 employing equipment r~a{l~ Y availa.ble. C0d,)i.l wa~ qlyc:air~~d th:r'':ot1t','h the cm.u:'tmjt of F~hery Product~ Ltd., Sto J,:>l1l.\' Sv Newfoundland. 'J!he FFA ::;~,nl.tent wae ddermined (ae oleic acid) by the p1 ::c~d.ure ghren in the Cff5..::ial and Tentative Methode of the American Oil Chenn.istl". n Se,!ciety 9 C';)l";'I!Cted NovembeT 9 l947 v and wal! found to be 10.12%e The :!i!'~z!r c ~mtent ;,.ra:o. estin:u.o:i;ed b"j' azec1tr!)pic dif!tillation with toluene to lie 1. <H'<<een 0.3 an-i Clo4% w/w. Monoglyi;;~;:;:oide:s were determined ae 4o 76%. The reagent, ;~, 2LOd:,meth::xypr: pii..n>!'~ of 98% m7:::d1r.tn:- pu:'l:':i ty, wae eupplied through the courteey of Dr. N. Bo kre-l;t.ev D-;w Ch~;:;;::,;a:;.. Cr. o, F'reeport 9 Texae. MethJUlol, reagent grade, wa5 used thrcjughc ': ~ th::,."' '!\~'.udy P v?hi:!.e t:be acid ca talyete employed were Eastman reagent p,to}\;.enet~uj.pt.:m:'.t a;:::li~ '!;:e,:.:h.n::i Hal dimethyl sulphatep and dry hydrogen chloride=ga:.t~ pae::sec\ in1~;,:, :.b:ilh-3 1 metha:c.oj. 1mt1l sui table concentratione were reach~'do C':)mpari.son of var:l.(r;j.e a:::id. ccatalyl!t:5 and..~b:u.i ty o! ;c ea::-;t~ u:; m:ixtu...:,:et~ Cod o:tj. (100~g 1::-t;r:;r &pp:rc,x:ima.tely molee FFA, calculated a.e : :ieic:) was vf~igh~d l.n~r. 25Cbm::. Eh:Ienneye:r flaek~ (ground glass etoppers).: nt:c.:ining tef1m:.. -<Oa1;ed ma ;"T.<e-.ti ; stirring ~aj.. s" To each, while gently stirring, ' >'I added DMP {r;..oo mi :t n;xj:~e~) lmd cataly!5'c and MeOH a,:, indicated in 'l'able I. 'l'be ~tr-: ng~~ a.~id ;>'mct'mtr'a r,;-j :Ax m::lxtu.rel'j darkened :5.mmedi.ately, excepting in the C:<::U3e of th~ :P '""t: j.'le~~ E'~u1phoni.0 g;;,c;jd 9 which did not dil!leolve for eeveral mi.nut~s. Stirr::.~n.g w.._:!5~c:m 0 ~ l.yiji".ld fol' the balaw::~ of the day and eubsequently for a few mini.t~,s prior tc remm.,al G:f sample~5 :fr.j:r examinationo The FFA contente aftfllr?4 h(.~'.h'3 mel ~e :re:r"' "- ~!ayi"j <~re listed ::Ln 'I'able Io The three highe8t acid concen"" {;, ~''/.::; 1 rdx:i'.jt1 :'".':,3 y.;-~;:~e i"'y: mi.ne.i ;;..t l.:a.ter datees with the re!!lult~ indicated in }i'l'., c :.o G:.. ~ :;:Ll ('J>O~g 1.Jt::>J) wa.r!l +~:re;jted a~ deecribed above w ith DMP (3.0 ml,,-, u;<i_e;") ],T,j :::;,~+!:'2.' 1 )L~:~ H :-:' 1,3i)J' lt;:_ ~. E~ (l.3 ml, lo3 mlp lo5 ml and lo5 ml),,vlta.~ 'e:<!pec t.:-; ely IJ.(:\:>.22 9 :).C'{)5'7 0 Jo0102 and eq HClo The FFA ' :::J.t(~nt;:, :.;f th.e.::t a..<:.b 'H~\ m.ii:t'.j.:c'et>: at varioul'! ti.mel3 are illu!ltrated in Fig. 2. Exam:: r:.a t.:1 ':n.:.f ~ffe :: 't ::.;f leeser ~:Jr;,r::;&'.:: t~. ;;_:,_f ::'-11.,2.~:1!,met~ro~n Cc.d 1 (100-g! ~ts) wa3 treated a:s before with DMP (5o0~ 4o0 and 3 nl: r reep~..:. t:!.ve::ly Oa04l~Ci.033.a;nd mole:!!!) in the presemcfi of MeOH, w,cided ae mei;h~n::;li :~ HQ1 (1.3 ml, e5"dm&:ted at Oe03 melee MeOH and t;q H(:~ 1 o 'D>e ~ill:.,; LJ. the o:, :. w-a,-, t~~t~.mated not t o exceed Oo022 moles ar.d the >.r.~>d;e~r ~.:ber... ted dur'.ng ':;;he t'j~tet' 1,f:ii;ation a,~ mole:!!. The re3ult~ :..~ a fllt.n::y '-'X.Pe':~m~nt: the ~.ams p..t'oc d Ire was followed u~ing les~e;:' 'Jf D~l.P \C'o-,~:P ar::a. Clo00<'3 ro:"1.>!5 :re~p cthrely) with a higher conc<an- ~::;,;;~t.:'_ ~ J.l of H : ~ ~'~;"!l():i mlp ~t::.: u.ated t::. cnnta:tn 0.03 moles M OH and ~q HeaL The rl!ed ri"l a:t>e ~1J1ll111~H"ized in Tab:..e II and Figo 3o

47 Czod Ji:L (loo"~g lo~t~) wa.~ :vc~ated. '!.1 1 ::. r.q Df!P.~o::.q.~. ml)~arj) and dimetb.y 1 ~ui pha ~ts ( i). 60 m.l 0 q l o T,.,:. ''if ~. t ~ w ;.: a::id.c;:l MeOH \, 0. )2 ml and 0.64 i7.!l 0 :re.spectively ').!)1~1 ~i'~ i:, ri.: ~~!J '"~~L:At 1i. thj.::;j_ ~~;;,t re;.eivsd l:h MeOH. Aft;ex ::1 day~ t.t.e F.'Fk p'ti.:: e::': ~Ji\6<'S':' ~~' ~ :f C ~, r:< tct~ l«:~p~::: ~ive1y 1o69,, :: 71 and v and a.fter :u~ rl<i~j"''...:. '. 0 o ~- "' J:;. ~1. :1" A d3..rllethyll!l\i.~.pri.iii.'r.& l'f.l.i.<"t:c. :": :w'xt:::::"' 1 : ~(0 ~ g ''''!~!~.:;tj.7 '!1rl H:Ltb. Yary:ing lillliolt."ltl!l.')f di:ff~tcttil'i: i'ij. :~!'.' :i.jd3 i,;...: :::~:c;:_,,i~ >:;:f,n:: c.c "'. '.\Ci ~1'15, 0'7!!1:!:' ::;h:i.l!l time ~tirring wae for ~l:v. :::"'c, ;;.n<.e:~,:~.~: ~ez::. ;: :p r:c~,,~,s ~:u~.y. 8:.t::;;/.f.e::!l -~ ~~r~ Xt1i!1Y.r~rl aften: and 48 h<.1uj"~ 9 th~& fi ;~e: ~t~. :l "i'iil.!' :~~:;:T. 'trl. { 1.:: 0 li..':'.c' tbs 1 _!.Hrn.~ ci.l ~ 4 ;c r«~d irl 5C:tew~~ar 'Vh<l8 f c ~~ :ir:.rjlohll~\;(!1 li!nd ::..ii.y::er J!":ii 'A --1 er;,;,n;o,r~_ n,: : c The 1.'~t~ Jit;'l a.:'e presented in Table IIIo La~- ge~~hja 1~ :" ~~::::;m~ -~':.~L~E~:::Ei~:!~t ;:.:.z:"" ~: ±.! ~ ~ :: ;:: J ~.:':.~~~~. ~-0.c~,:; 2 l~ ~'-5 0 }1. ag, : ~ ")~.:: '.,:: d:. ; ~g"':_. ll~~~ W:.:.:1.e l.ll.(i'li~~ P=tolueneeulphonic at:.:.-ct V5~. gr. "' '~~O.Jr :mvd... ' IT.J 0.._,q. l!h_..ut!lj a.1d MeOH Lc. ml, 0.3 mt~1e~l JiJ'1ter ~3 bty ' ~:!.1 0,.,,-~;,'c:: ' rv.~ d:,_f::>:rntc: I.;;~~ ;;;.::::i... :~ ~.tt1:t 24 hcmr~ the FF'A had. fallen i ~ ~~J~ 3%. T1".~.: t. i~tr7e:: ~rp~c1 w-; offen<!l:i:re ~"'-~Phl:J.r:?Ut'!l odour on 1 tr atment with tent: ~'nir;~ " o '<%'., a::il <3r fuy~b'!c:" :ijc:r ;<ii:!p-:lg~i:;.;; w:c(ll were under":';~en. Ex pe r.1lreen it B o :'), _ 1 ~::: kg\ ~., 1'1 "',., ""' il.,.., ~ -,.r''"'"!'! ~~ 7 :1.. q o:; ga 1 ~ ~~-=-"'~'.,, <; 1.,.~ ~ijl...,~ _) 'j / <\: ~ ) ",_ -~ ~.- : ~ -,~-;~ f~.;-:. ;~~~~' l~'"~-'"-;, :::"- ~.';.'~ ",c-.j,.,_) O bctt.l.f.~ wtu.le decllltx.;;.l ~ll<t ' "l'" h- v ""0 '-" J.' "' '' ' D. u-.. '- '-'.-'o :> mole,_, were a.ddedo The m:txh:1"e va!'. ~-~:tcc r.>: ~~ c,.~mt1 w;,v 1 s :f:r:r 2'' b.)'ijy:"i~ FFA Ct'lntent then 3.19% 9 and ::1'i'C&.Inoncb ~ ::-e:>.: a fuxtbe::t :;.. iays ~rnt.b. a :f1.nal FFA :of 2o05%. Part '.Jf the pr )duct (5 kg),,~a~ :':"em.;-rre-1 ':ll'lci ~ i:ju'~:] 8 bn;.r.~ ~dt;h Johns~,ManYille Micro~"Cel (25 gr Oo5% w/>'ff). A.:f e.'t" a t ;;,:,a: of 24 h: :'.:co, t.;~-~;!< m:.:dure wae again l!tirred and filtered ~-~l c t;,.'no ~'he bulk ;,f t:t.e >i i ' aye,':" 1 J>',::t~ ~t:i.ned!d. th tap water (3 1) f D!' lo5 Jt~t'o A:ftl!l'r I'!: M'ttl~'~l- ;.:-erijrl. h.i:>u:!'5 9 part of the oil layer (4o3 kg) wa:! :::emcnr~d a c,d t::eated f cr: 24 l':al:r'l!l wi.th MJ.r:.ro~Cel ab de~!cribed aboveo Thi!i xern.a:'lnde:r- of the : :!.::..1aye.: wa..5 <::;"ars fi~d :::.n a Sharpie~ Super Centril~uge 9 and a portion (2 kg) tr'!>~::<"i:ed "''l.th M:.::.<':r,;,~~Cel He abov~o The vario'.l~ product~ were stored.tr~ g.~9.1!tl!!!,,,0:w.: H";r..e3:~ a11d "t.am1.n~"l fr,cm time tc 'time for FFAO The res"jllt:'! are preeent~.i :i.n. Table IVo Experiml!!nt C. Cod :1.J. (::4"8 1:.:g,:.,.a~ ~l..rn~ - _2.1: r;;:ea-::e.d whh DMP (700 ml 0 5~70 ;-;les)-;~d met.harjolie; HCl (., rr~:~o 0o614 e:.!, HC2'lo After 5tl.r:;cing 24 howr~ theffa wa!'s found t(' he ;_ o ""i'i%,, 8X!d a.f1:~1 a f\rr+,h~::r:- 2: day~ it had fallen to 2o04%. The further t:reabn~en.t; f~ llow!!id tb:<;:t,,m:l~.e"!! i ''l.b c:ve fox th.e dimethyl eulpha.te iciataly~ed rea0t').,:nr r~:x:e~ept that ~h.e :,ra~!'.. ed :'.. L~... ay~:c ;'!ampj;e< wa~ ~;reated with Mie:ro=Cel (0.5% w/w) fc_.r.?.~ b.;;;:'1r!l 9 fol.jwed by.f3.<.id~ ;::.on :f a. further 0.5% and treatment f0r en add:.t~0rca2 2:11. :'lr:m.r:!l :pr:l.:rr t.j f '.l"':-: ; ~>;:,o The :: e:sul+~ eu'e, ~ummarized ir. Table IV o Experiment D. Or::d ~L~ o "';::~ '~z~js'~'.:'.l. '-''!' El>::: e:..;ri t:~l T:l ~'--""l"!~e:r propo!'tion of DMP T?-5 ~111 0 OoG1 m,~,~e~) an:i IGt~rth.;.~; E,!~,. }Jt.:': ( 1.~ l!c.. 0 0,)06 eq) o After 2 day:!s the FFA ws.. ::S 1 0?:1% 9 axd a:fte::' ~.,;: o'?fj<f,o T part.f ~:hi!!! z~_ar:.t~_r: :: mixture ( 900 g) wm add~:'i 0. '5% M'i,- y,~,.ce: and af~ ~:r?4 b~y?';:; a :f ct.r ther 0 0 5%. After a total o:f 48 ho:.z1:"~ +}:.;.:'.. ~ m-:-~-v:+'x'te w3.~ r' ';: ~ i" 'l'h.p; a::an ::'!.' c f the reaction mixt\u e m~ :!t:l.:::;re:l wtth i;at 'ifate:r ' : ) ; ::Jr, :: R.:r;d. a12nwed tc ~ettle for 24 ho':.ji'>~o Pay1; Df tlle :1::, l.ay~ V8)~' -.~:e. :~~:a.n+~d a.::1.d ~im:u.ar.ly

48 t.reatec,\ with Mi.cro~Celo rr;,l!'l hal.anot"i wa!"l clarified by centrifugation the c 1a rif:ied rna terial wa:~ tr.~a ted as ab::ve with 1% Micro~Cel in two ~tages 0 wh.he the rema:index Wi3~ t,::: eated w:ltth. only 0.5% fror 48 hourso are 3U.m.:nari.zed in Table IV o and part of 24=hour The resultl!! Of th~ ad:l ~a"galy~t!i cho~en f{jjc' ~tudy 0 :hydrogen chlorid~ a.1ll.d r~ bluenel!iulphoni{; &A:<id [;,a lfej ueera employedp whi:e dimethyl >5ulphate wa~s Mlected a:~ a c'!:lnvenien,; meax1.~ c;::f :bh" Ddlwi.ng eu.lp,lu.u:ic acid into thl!l :reaction mixture~ The eydroly~5ie of the ::.~.:~;':ter.;;;ataly~t: may u:a.der ~hese cond.i tion~ not be complete, 1 j.:~c all the (;,ataly~t:5 u.ay uh:tj.llg.tl.'ly be partially c:ombinedv l!!ince the FFA valuel!! JJ;A.Ji be found to be of th.e.~cune order as thobe which would be given by the ":ate.iyed add itl!>e.!dp y~'c are nvt rew1uc"d oy ~a.shing 0 M would. be expected i.f they were all minere.l!1.clf,;.. Tht!i da."ta. :in Table I and Fig. 2 indi.cate that there i.~ a l:i.mi t:b~.g cataly~t c;.ty;:nc,'!7.1t.ratlon fe-r ea'l;;h t~ataly:5'g below which the eeterifi ~ (\at:ton rea ct:;:;n dg:e~ n'0t procf.'ed ve:ry rapidlyv nor J.~ the lowel!!t FFA level nec~~~ari.ly reached wi fu leiw con.<c.;ent:ratione of ataly~to The latter effect may be d,\le t'~ the competing al!;d.d~;;ai;a].y~ed reaction!~! of e:s terif:itcation and decompo= 5ition of thedmp ~ ieopr Jpen;~r:". methyl e.ther and Me0H 9 where the latter reaction deetroye tr.,e Jteagent before 1Che ~~Sterifie;ation l"eaotion ~a:n be!.';ompleted 0 or to lol5e of ac:j.d Gatalyet a~ de~:.;~~bed ab<r::nreo With a.u e.dd :::a:talye~t!l (Table l 9 Figo 3L, the l~ est FFA values a.re obtained w:l th the hi.ghe15'1,; c;o;n.e>et'ltration of.~.\0id c:ataly!st. Whl.l.E'! ~J=+c.::.u~ne~u..;.~pho:nic acid wal!l the mo~t aotive c:atalyst 9 effective even.tr:, lo11i C)ll<.;enb:ahon~ 9 i ;~ wa:5 l!!lvw to diesolve in the cod oilo Other die= advan~t;agel!l wtore t!j~s rapl.d irj.c:reae~.fm FFA content of the reaction mixture on etandi:ng (Figo l) 0 the d.~yelopment of cbjectionabl~ odour~ am the probable diffio.tl ty :iln removing 'Ghie material by wal!!hing proceduree 0 The dimethyl eulp:tmte did not Mem al!i act:bre a~ the other <Jatalye~t:s, and gave a eomewhat darker and le~e ~table produd;o The poel!!ibili ty of l!!ulphation of ethylenic double bonde m~t al!!o be co~ideredo The methanolic HCl solution was found to be particularly eatiesfa cto:cy as a catalyl!!t from the points of view of rapid reaet:lon 9 l~w free fatty 2'Ciid c.;:mtent at minimrun 0 l!!l!':lw increaee of free fatty ac;id c<qntent afte1~ m:::mim~llll 0 ~a.~e ~,f p>jtrtfi ation 0 and etabili ty of purified product a A Cl!)mparieon of Tabl~ I whh th<!i experimental details outlined above for la.rge=~ ale reaction~ :l..nd:ic.ate~ ~~hat even with contin1!lou~ agi ta tion the large=scale reaction~5 we:re ~mewhat glower than the emall=ecale reaction~o The :~'eal!!on for thie i!!! n:jt cl a4:' 9 although the fact that the la.:.:ge-ecale reactiom were not carried o~;rt 11:c!!~a led containers may ha->re some bearing o No c~ncl~i ve ev.tdenc:e wae ever obtarr'"ed that continuous stirring offered any advantagee over t;ht~r,~ugh mixing iqjnly a~l; the ~ta:r~ ~~f the :reaction. S:ince tht~ m:mimum FFA content,.:;;an be made fairly oon~i~ient ;v~ er a period of day~ flexibll!l plant operation would be pl\li:!!3ible. Heating of the l~~a<~tion mi:dure is not recommendedo Other ~u:rker'"' havt!!i ~ht:;'\\0! that MeOH pres~nt in even modest prf.t~,<ur'i:.~~o:r~ pr\)inllte~ 1;:ne ~a1:1y,~tagee of the X'eac:ti-t)nv but 1n the cal!!e of the eeterifi~at.l-n j,"jf a.rclda ;:nly ha~ li ttl.e.influetk~:;!l 1.m the ui tima te conversion. In the pre~ent ~tudy 0 where water wae ln:i.:tia:ily p:re:5ent to liberate some MeOH from the DMP P and the total MeOH l.jlbera:ted w"uld "-"X=>eea tr.a'l; necl!sl5a.ry for the el!lterifica.tion 0 :U;; wae e:li:pec:ted that lit'tl~ o:r: n~ 1>1eOH w1.:mld need to be addedo The experiln.ental

49 Jcnve~' 'gation c,f thi;,t.s poitrl:; (~e~ above) i:~d:t:;;a ~~J 0 ::'l.c\i'~wt>':.' 0 tc>:aa'c tsoll\i!'j benefit ;;;;oul:i ne e:q~c~~.i fnm i;hl! erd.d:i tlcd. "Jf :5I :all &llc.f!j::c r:~ ::r' MeOEL, and tlu i WA.15 done ~L~:; mo~'' t;xpl!rime?yts ex:'!~p~~ wh~re m~ t:nartoh.'" HC1 wtl~ ~:r;.~''ye3i ~-~ 'l;rje aciii cataly~t. e:!l~e.:t :Lf:c :;etl;:~j of tr~.glj,:~r';i~~- lil <1:.h M!!!GH 'ii!e\.!'l e:~r<>k~ -~~ :1 1G::<'.' r;;: ~he l.a:;,'ge ~l!'.cale ~ '-"'. im,-, 1()' ''t"' ;~.,.,,_...,,..,,. "''""' + '"',,.,. ~"'~'r ' -""" i:h,..e _,., O"lly!'l!!a.:v.:L :OrJ.~ '\AI'!..,),;..~.1, 0 A.).,,\,,)::'!1Agu ~,,,ll.\e ~'"''! ~<;ic.::. >A~. ;._, :;,;s<.,,v,... :\ij'i....;v > e.! -'-'" a 3hght :~:i~e, :.L:n the m.::;nogbrceride C''X'ite.n.t 9 :!.rli::'. ~;d::':.r;q ''"cia'~ tt~ e~t!!rifice;t:ion :r~a,,tion (a) proceeri~ tth:r~ :;:'a_pi.d.jy ~ban tne ~~-t-er~ete:r;j';,,;~<.t:icor~< xeact1,on (b)" The partial repls. ~!l'lm~;o"j: f:,f DMP ty MeOH : m.i,::.'c ', b :! (;.HJ-5:''-der~d U5ef:llp ~:i.nc-e the h;ydroly~i!!l of t:t~ DMP ~-:~~~lf p~~d..!.:;,.~ m :..:'J r1e0b. d:cau- 't.15 req_l~ired by 1Ghe reaction of eetex :i.f:\.t.a:t:\~5 -i~ ~rolle the are;>ulr'': : je DMP :::.~ -~;hl3r"' le~e than 'chair; ;).e~e;5~e,ry tc, 15~a1ren.g~ t~l.-e ~>:.r.i;a: ~ 'l.fst.e:t.~~::,.,",~ =o Jt. :rt,-.:,-e:r~ }:,.e~.e ~ r amt':.1r~.ta of DMP <~:;an be employed te glve ru;;j' de~:(.:e'ed. FFlt Ta::.,, o \of: :b. 'l. ;F_:~:"!<d. ;;.:::' <t :P Z rtion li)f acid cataj.y!!lt t;; tti:j. 9 qax~ring ::.r.e ant,;,jrt.:1 DMP ';!!''. e~; :; ' p:::..::allel C\U"Ve~ (Figo :J) 0 The e!lterifi(;a:~: ~T ;.:'Bi'l. :t :n:. l F"l~ al-;;e.,:rt'! '! ~ ~...ht~d '~:t t.h tne forma"cic;;l cf a black p<!:llymer wh.ich t<""c\.l'.'-i :.:: t ~n hi:d::.z,.. ;?.i\.fi.l. -:;;;,;:ci.a.::~!lv '.!i"er: d1.;.ri.ng agitat:jgn. Thi~ ma.t~rial wa.~ r;~ :. --::>: ~.\l~:'.>~y~it) (~td,:i ~:ij:.~:;t~~.. ~. -"~~.:~~ff~:... :-~~ :!nat :,,.-~ :.1.~, 't.r~ ~.J::,~ ~"~.\~r:f a<.~~~ to pe.::111ci t 4;:h.e el!ltexif:\.('!!:l. -rr,'l! t -~ ::.~.;::an'~ec't w7.th... ~.~;; t:,5 di.!5turt<ed 0 aj. thoug!~ it c:euld ea~!!:l!.y b~ re:,r :J''<t;!; t :, ~'"J.u.th~':g, Sbu'"" '!.::t>'il f!'il>cj1a!:.r ;)f i h:l~ mater:~_al forr ~ed. ii.d r.ot t=!pp~ay t:.. t.r :,c,.~ c: y '-~~,:.,, '.'. '"'15 t;~- ;<.1~e.'i 0 btd: tn~ quantity a.p:p!!~a:r ed to bt<!. ::~~h t ;: d~ pr r":c<:.i.-;; :~ ::1.. :; 3. p:ce~~nt., ~ugg~~t:~rtg -~hat.i 'i wa.:!\ d~-~--~ ~" -~-~~- ~'~.," ~~~~:'. -~,~-J.~ I>IVL'P :t '"!Jl:l~ t~~~~-r., :!Ome ma t;'!;i:'ia::. pre:i!i~n r;.:.:.:;, ~ 1 ::, c:~: is ' 8.15 llo:uh?gly(;;t,ridf!':~ 0 e:tr.o A.dl ~ 5 M.'",4 ~id.~~ :J:"'I'!;~;..:,~~i ~H: ~-:.~~t~~~ Al tl'l~'ij,g!l ~1crrm.'il a.:c~(:.-1.. ~~~: -~; ~i j~:jt:l...{;g ;rt~t-,~;~.,l:.lt~~~ t5h~:-lid ~Je ~~lj... tc.t.~.:i >1~~: a.~ ~: mean!! of purifying and ~:~~,;;t.b:l.. ::~:.ir..~.t:~ 1~}1~- J;Y:t'Gd.,_~.c ~~ 0 t.. t~ P~~'.'!:"!~Xl':-r.' o::~ t.e p::t.'l)~t~~ipi ta-~t;: ~ugge::!lted that a f:i.lt:ratio't' ::'!'t~p m.:lght be ad<'l~ab~.e~ ax1l t:rea.tmer:t: with t; f:u.ter aid wa:5 therefgre ad!'lpt~d. a~ a part of the p1,u. ::'.f:~::>o<t~ o.~\ ;pr >\eg.1jre 0 Ae~ illdl.cat('jd in Table IIID Micr<;;.-~Ce! a:pp~ared 7:.(< be tt~ me""~ ~.. i.<>hle m"ye;, ~:.a.:c fo:;; thi:! purp";~e 9 and e:1r:tend.ed l:t\!ar.me.nt t:1.m.e a t J:'(dJl! -(-~mpe: ato? -~ ~o?d: :re c;he: beet ~'ee~ults.~ 'fhe Teact:,i:rn~ mi:trtu:re ~:iarker.ed.t:l-ppre~~~alf.tb~~;:.-- ~.t: -~l~e:t"\;.1 ~tc:.d e;~ncent:rat.il:ml':\ 9 the d:i.m.e"ch:,rl l~ll:,}pbb.'i:-1!! :c.~,a.c;i~;,o-,;::c rrcre 8<i i:h.tl1 <;he 1 c t.h,'5r~, T:U~ purified e~terif:led c~-1 ~.~,1 f:::~m th~ H01 :~a ~~--"'y~.~!;d, d::n:-eri,o~rrt C d:\.d n.. t s1lffer from d:ll~c:.,leuratienr ~t;he GarlrL :>T :<,':.t.:hlll!i g ;:!lex ve.i te~c.: B',:,r~~g:inal c:~d Gil reacti,ola mixture ~~;fea~~e<d ~13;11 M:>.t::rr "':'el,;,a.~he,:;, 1 r:;~d tled a~,,;, h:'~sated with Kicl~<-=Gelp 13~ lv a~h~ :i 9 ~ettled azrl c1a.ri,fied~.~t!v'1d wael:t~dv i5t:;t; tled, <ClaJt ifted and trfjated with M:i.cso= CelD 13o It :it"i e-v~~c\~::: ~ tl:a i; ii:t~n~ 't.b.e preduc't nti!t t~eated with M:i'~H~{]el U! r~~ :ia:ckei~ thi:...ri. t!"''!!~' 1 r..:1 cx:.g :-:JJD' ':~:r.\.8.1,, the MtcN= Cel ha:! a ptwerf'!..ll bleaching!!lf'fec:t 1:.'!, a.dd:.ti~ll'), t;-:;, U:;:-!1 pc:-.. J:Ba.ty,:,llu;.:d;:i fhl,.,f ~'tab;h., izati'!)xl throiii.gh r'e!!i(j'\lli'el.. l iilf 8.'t.~ro llti:j.he'8.1 a.~ij :;,f \;tle f:' ~ e f.~t:ty a t].dl!,. The d~i5aget!i employed in t.beee ex~~ e:r-j.jnf'!!:atl!l,, (lo5 :~r ~.%D a:.:t::' m:~.ii!:i~-,~ ar:d ar:.. exp~!'!.ure time <Df 24 lii'r 48 h4<t:re ;,;t Y"fliO~lJ. -~emper~t-:='!l 1! :':!~ em:p:~~y-.d. : :.teu <Jtf the JvdustY."ia1 p1;acd;'fl,-. Qf br:1ef 'l.:roeatm ~:r:c- :s.t eltr.ra-i;e-:i ~;;ei:i:pe:r:: r _t.::e~ o l'ht'l ;p; i.i:~:r' J. Nwedure might have af.l:vanb.ge~.?.tt L'O!l'.:t'Vin.g ~r,} e.'~-1 ~~M "i;''~.j.d :render th~ e~terified ~ill! u:n~~n. i~able f!?<r som~ appli at:: "'"\1>: 0 On '!"~,e.,.'~'j $:1: ha."ld~ in mi!lrq ptd:ential ~e~ C 1 1!'l~r:.lj' a:nu tr:ar~~e~' ~f 1Yl) :'.~ ;:.U~'!' 11'\;J..J.Cl 0~ Of ;;;,; :411 :.c:;.cj.nc.~p a:;-:-,6_

50 ~ 28 ~.imp.""" t:rc'es:tment w1n1ld ~uffi'0e 0 prrtvided the product ie euffic:llently stable d,llrl.:ng shipping e.nd f!bt1rcage. The data :in Table IV indicate that stable pnducb f l. w f~ee fatt"j a~d-ai c:tntent ~an be pr.duced by the preceduree eutlined abeve.. TABLE Io,.f ac::id catalyst ctfn;:;;entratien n percentage FFA rtf 100 g ;';.ij;;i t~il t:r'eated with Oo041 melee DMP Influen,~e, -~ Cataly~t Hydregen r.~hl~::'1ld~ p=telueneeulphonic acid Dimethyl sulphate = Eq'li"'alente Oo Oo0106 Oo0054 Oo0025 =-"" MeOH added 0 ml lo3* Oo65* 0.32* Oo5 Oo5 Oo % FFA aftejt 24 heure lo41 6o lo o77 3o hture 0 u 0 v u 0 0 lo o58 3ol o90 4 day a lo ol0 0 0 c u 0 0 u day~ () o o40 4olb TABLE n. Influence ef the am tmt 0f DMP n percentage FFA of 100 g c d ~.il in the preeence ef MeOH DMPo mtllee Oo Me than~ lie HCl ~ luti(iu 9 m.l l o::j ~ HC:::' r eqcli val en te Oo !02 % FFA afte1r 24 htmrl!l o28 5o heure o htl!mre o h~~::,re 0 () 0 0 3o52 4o77 6o63

51 29 TABLE III. Influence f '!far:)'-il"lg runount 5 ef ~ome and f treatm.ent tiro.e~.-n ~'i'~;abi11:z:atign ~f a di,methyl :sulphate re.sct] on pr.rdac't filter aid:o~ Material (w/w) , ,,5 4f.1 ' "" ~\.o29 l.1.j I JL 1o it 1: '11~. 24 d~. 0 <--<..., l.l ~co26! r 1b24 ' 24 ::~ (100 I & e. t " l 0 0l 0., (~ofc ;:: Nene /

52 30 TABLE IVo Percentage FFA ef eeterified cod eile purified by varj"ou!i proced1.n e~r 1orhen freehly prepared 9 and after eterage Experiment and catalyet B(c~) 2 so 4 J C[HCl/MeOH] D[HCl/MeOH) Pr~Jduct treatment Origo Final Day~ Orig. Final Daye Orig. <=<O~,' --- I-1::\fa-oCel o:a.ly. lo ':il 3o Wa 5hed,. ~ettled and treated ' w:ith l'iicreq Celo lo Oo73 Oo o69 Wa~hedr Mttled and clarifiedo 2ol2 3o33 70 lo96 2o o29 Final 3o Day~ Wa~hed.r :!lettled 0 >e; larh i ed and t rea ted with (2.92* M1. :crcj~c!el lo31 lo51 70 lo38 lo34 70 ( (3.39** TABLE Vc Percentage menoglyceridee in eeterified cod eile purified by varicnm preceduree (original eil; 4. 76%) Experiment Product treatment c D Micro~JCel only ,19 Waehedr eettled and. treated with Micro=Celo 5o Wa.:shed 0 ~e-l:ued and. c:<; il.. ::i.fl,!!ie. 0 5o WMhed 0 :5etthd 9 ~,;la.r:i.f1ed and trmted wit.h M:lc:r ~-Cel. JL% Mi.cro=Cel - 5% M::.::n~C'el 5o 55 (5o74* ( (5,81** ~"'

53 ~ FREE () F' 1\i> \1.-~ FAT'T"l':" A0!:7J ~ \,_{i] 0!',~ '!)! \v.q, il- J ("""p -ll ~ ~ ~~ f!.l 1.!1 'IJ:J - >'~ 0 \ "\ \ \ \ \ \ ' \ \ \,~- d. ~ \ \ \ ' ~,., - r»; \ 1-l ~ \ \ \ \ \\ \ ' ' v'\, Figo 1 = Long=te>Jt:m of <:wid (P.Jl:ls) 9 d.i.~j<l~~tl',:r1!",3sp~cti~tr~ly,:~'~._.:;.,-~ ;z/"':-~_ :;:;\.~::--~~~ Wi' ~~:\ L:e=~t~.;I_~,l~~-J~;,~? c:~.2i>i :lll:.c~f;~;'~~.~~d_::'~:. r,_~ H(J'~-'-!l~ ~?.'Lid tta :;a:.;.r;;.. :.~

54 32 ~ ?.Jl-. "' t 0 I DAYS F~go ~. = Cou:r se Gf r~actirm;s of 100 g cod oil with moles of DMP and -wa.cying amounts (.~q) of HCl.

55 10 4'1""' ~~-~~~====ii ~ 8 ~P Qo ~002 1 ' ~- ~~ -~-~ = ~ ~ 1 16-~~~ ib-,_,.,.l;,~- -ll.~~~jj ~~~~~--=~o~=m.jh~ = ~ ~ 0 1 ;2 4 5 '1 8 9 DATS

56 1. ') I ' ~'

57 The codli ver I.Jtl wae f\tc.pplted by r.>t'j'u:n~e~:r '~'f Fi!.Jhex y P:roducte Limi bd. 0 Sto John 9 :!lr. Newflnmdland the s~a1 o.:tl wa~ rerhiereti fj r,m ;-r..acy seale and wa15 supplied by ctcuxteeiy of Ka~l Ka:rJ.~Jen and C ;:.np-an:~r I;:iJnit~d 0 HBJij.fax 9 Nova. sc~:jt:la 0 and the hetring cil was a 0ommercd.al 1):Ll p:rcdu :<!tcl in. B:r1 rl.~h Columbiao Th~ phy5i~:al and chemi.r~al con~tante o:f the~5e,;iiloe a1 ~ lil"'ted in Table Io Each bat,:;h con~5i5ted c:f \500 g ril and wa~ bljwn :u<t a heavy g1a~~ tube 9 7 em ineide di.ame't~r a::jisd '" : m da~:oo ~\'he e.i:rc wa~ taken f1 oru the b~ulding ;!!Upply of c:ompre:!ls~a ai?' g p~el!led t:'tj)>)l'l<gh 3 :~':'c!'.>her Ei.':t.d P:: r ter Flowrator,. s~:riee '700 rotameter 0 and wae C.i.!Spe~:: sed iu '[ be 'l the tot k»m of the tube trli'ou.gh a 3~cm diameter fritted gla<5~ ple! :(~ 0 The cu~ 'i"e ei hi!)ated by a fibregla5 hea;~i.;;::g tape wrapped arcund -~jbl~ ~il:j: e e.-~j.d :~> J>";tler c.'r!})l'<"i' "1?8!\ c;:,n c:c-'i:s J:.l<'.Ai by a Var :::~~~ t::t&'ll'!i= fermer o A therm ~<CJup:t~! '"~M~ :~mrn~,;~l's;.>;>d i:n th~ 1 r ac:d a. ec.nertant re.:.'t ri of the temperature wae kept Oil t~ li:.ie i.!!l.at1d N.t:r~:;hxup Sp.i!e1omax r.ecorder o Very U ttle heat was given c,ff dur,~ ~::~ ~:.lh!;,~~~xi.da:t,j~._jl1t:c 0 e,0 ' =\ ~ :=. h..lljg 'Wa;tt :cc t :nev:~eesar,y o lli~ illj.duetrial prae:ti ce 2.t W<.Gt'.J.d be n!1 :~l'!ll!i!t!':,:r 'C{, cu >..;.la i:e,-s :1ol.irJg w-a1[;er trmou.gh &he C n.ile during the ]per::b::ji :>:f; mof\ t: Ytg:;n 0'l~ rr.l!a~ti Gl1o DLil to >rariat:':r:zl L"l line voltage 0 a cex'tair. f i,.n tem.~-::at;:oc e ""a:'!! eu~periejnced 0 but. trj! average deviation i:n ll'!l\~ '~ X'J1\J :5 ')ll',~lf'l : J:;je :vax;,g ~ of ~.-3(t,'Q o The iodi.ne ""JS:!.ue.~,,]J_ FFA ier~m::.)b.::;icnl'.,;,,j, t,;j,e producte wei'~ rajr:'".rie:d cyu;t according t,,;-j ~:he Alna:;: ~ct'ax~ 0-:,:t Chemi~t~ S:<.l.ety f)ffi c:.al and ~rentativ~ Methodeo The vi.ec:ots".'t.v wa.~ d.~ t,m:ill.ill. IHi ',;;l!\\ ng.;;t Bubble Viecomete! n 0 V. _... S A ' T ~ d H B..... '' ~ r "'.. r axn:j..i:j,u, -oc d t. ~ erbe~ 'to 9 &t, ~a J" :>:tj..e11l W :t"j..es ~ ~,~ ~~,(: (._) -.ot::. a<{o:: t.::? an.r.. e :~~'ajj.. te~ obtained were oa:me:r c.e1 t;.j :l!!'~ok:et~o The CI:>L'UX"''5 {!i er~t< C Y!Lpa:c:eol wt th G aj.dner'"' Colour Standardeo Th~ 7."f:;;a::tiv e indlgee :..re:r~ :weal3m::~1 OIJ. a Bau~ch arkl. r~ ;mb prec~.. e:::.on refrllet.,\!!'jtd:e:o~ o The me...::all drie:rl!! manufactured wae in the icr'!l, ""'Q,.. ' '",., {.J<A.JJc.;} 'i' ''"'" U + '\.-;.7> "., "'" Q -d... 1""',Jo:""J~ ~. ~,. ~ f'l< <> 11 "'., "'"'' ''.. 71"3 0,.,'1;..4..'_;.,,,._ d\...~'1.", 'f''-- U ~,( ',.c:',, q6)." O.~s~.::..l "'''' r'~!" \' "' - :..t..-!_ ' cr. ( 1 f,oj'.,... ; Nt!:t'Yiex I'r ':ldue"r;l5 :: f Ca:ne.da 9 L.i:..mi~A:c';.o B:;roG t~:tfl ~'-<~ i l.~, :;:f t~be e ::ne:::"en:eo ~.n The f!ettlir..g te~.t:!l were.c;arried (;~xt ~1!'1 a Ivf, g,-:> l1.~tion of b::.cw::;,; l i Imperial Oil LCT 20 o The hydroxyl y a:rue~ "'lere de'~e:<:>ru:... tj.ed by phr h"!ijij'j.i:,t ::<n a.cc:ord:l1g t;:; Verbe0k~ and the near irrfrared!'lpeotra vn a. Be,;1JJ)an DK. -2 with the!'ul'np.!le~. die~ol;jed in ~arbon t;etrachlorideo Re:!!ultl!'l.!!nd Dil!'lcue.eion~ In Table II the ): e~ult~ of \?a:ri:. ui!'l rus:1!! a.:ce ta-.:>nja<tedo Theee re~"<.z.1~:.~ would be of only limited U:!le to indui!'ltry ~ ta:r d.ng ave upen:a-;::..:..)11 of thie type ::l:ue to wari.aticm.!t in bl.ow:ilng ve~sel 9 air distributit.'p. 0 heat t~: an~fer and other f actor.e o However 9 by plotting t~ ccon~ta:ntl!l :fr~.m a few cet~t rorl:!l again~5t b1<:ll1flng time or tempe:ra:bxre 0 the:!! dat:~;. ehr.>..~.l.d :prcn.ride a gu:ilde to th~ vi~e:jt'!i t;;y 9 iodine wa.l'.1e and colour of the pl"l~d \l:ct t.o "be ~xpecct~do Iodine Value~ 1..'1 Figo 1 the c:r.ar~.ge :':;a, i:)dine 'fyaj.l.l& 'Tf.l;o bl<twhlg time i!1 plott~d f: l;' eeveral temperatu.ree l!!"t o;n:~h.n'c ai:r.il.d.fl'llt ra.tea The :A owe~ ~; :i~j;dine >ra1:~.'!1 ~1<bitamed ueing thie ~.r-ti,'u~,i'ix" <C\ ~1dJ.rnn: o:.ll WM '79v but the product Yale! then

58 pra,; L;;il:.ly a ~: lid g~j. 0 One ~peci.fication for blown rapteeed oil ie an iodine value of 70 0 and addi ti:::n.al treatment would be neceeeary to bring the iodine va.lu.e down tc: thi!'l levelo Marine oile of lower iodine valuev eogo eeal oil and herring uil 9 beha~:re 5:i.milarly i:<n: e;odliver oil during blowing (Figo 2) 9 and ~'ari::j":.e ammmte (::'<>Juld be bj'.ended w.i th the codliver oil before or after blowinge AI!! <>)nwn :L':I. Figo 2 9 the dec1~e~.ee in :iodine v-alue when blowing eeal oil or herring ci1 parallele the d ::::rea:5e iu :.:odlicrer :::i.l 9 while the increal'!le in vii'!icoi'!ii ty ie almoet the!5aid.e in the thr.ee oileo The :inc;reaee in w"ie":;:oei ty varie~ with the tem~rature and the airflow. L"1 Figo l the viec<o~ity i~ plotted againl'!lt time at l'!leveral temperature!!! and at conetant airflow o It '"a::. l:e ~een tj::at ta::> blow codliver oil to a high viecoei ty g eogo higher than Gardl1e:r Z ~ 1S (99 :atokee) at an airflow of ml/min 0 would i~ake approximate.l,y ll h:r at :, hr at 130 v 19 hr at and 34 hr at 100. However 9 at higher airflg~w~ 0 a:s ~hmm in Fi.g. 3 9 this time could be ehortened considerablyo Sin.ce appr~ximately ml/min was the maximum the eintered gla~l'!l plate could handlfl 0 ~. levelling off in the rate of vil'!lcoei ty increase with increal'!led airflow wa!l not r~achf>do By plotting c:i1 vi~0o~i.ty Ro time on eemilog paper 9 a nearly linear relati.,o.nship wal'!l fou..."ld {Fig. 4L The pointe not falling on a straight line were irl moet aeee ~amplee taken when the temperature had fluctuated from the averageo Othere have also plotted these rel'!lultl'!l 9 obtained by blowing rapeseed on a logarithmic ~~t::a:;.e and fmmd a linear rela tiommip after the induction period and up to the poi:o:l; when gelation occurred. Whe:r~ blown oil!'! are to be ueed for lubricating purpoeel'!l 9 a FFA <L~m::tent higher than 3% i~ ccml'!lidered undesirable. In Fig. 5 9 the development t f FFA dt:1ring blow:ir>..g of codliver oil is plottedo In most ru.ne the FFA did not rise above 4o5 to 5'/o" Mc~t of thel'!le acide are formed ae a rel'!lult of peroxide decomp,)si tion to give cham!lcisl'!lion 0 eo alkali refining of the etarting ma ter ial only lower~ the final FFA by the ~ame number of uni tl'!l ae the original difference j~n FFA content of the two oile. The formation of acide eeeme to pro..::<~ted l,'!.t the ~ame rate in the alkali refined oi.l ae in the untreated oil. In a ~tudy of c:orrol'!lion in co~nnection with fatty oile in lubrication 9 it hal!! been p:c)i:nted out that attack on iron b not related to the total acidity of the oil but rather to the ""r'<lati.li t':'r and reactt;rity of the acids formed. At higher temperature~!~ ( ) the increal'!le in viecol'!lity ie relatively much more rapid than the increal'!le in free fatty acid!'!! since!'!lome lower molecular weight acide would blow off at thel'!le temperatures. Thie euggeet~ a method for lowering the FFA content cloeer to 3'/ov i.e. raieing tlw temperature and i:ncr eaeing the airflow toward the end of the procee~lingo Howe1.rer" thie ie apt to darken the ::Jil exceesivelyo Steam.=dietillation wal'!l cried with good re3u1t3o Ilt::. ~- T=hr run at 130 with an airflow of 9,500 ml/min, the FFA content WM 3o6%o By b~~jwip,g steam through for 20 minv the FFA dropped ';;:;. 3o7.% w:1th no effect c-n ":;he co1c;ur. According to!'!lome author!'!!, partial e!':!teril_.:~:.ation :J the a,~:id:!! wtt."'l hydroxyl group!'!! will occuro However 9 in one nm o 22) gly::.er, ~l w~l'l!iddod to poeeibly eeterify!'!lome of the free fatty acid~v bu.t the tempel"atur (1::50 ) wa~ too law for uncatalyzed el'!lterificationp and no reduction in FFA wal!! ob~ened.

59 3'7 Cataly~t~g A few experiment!! were abo carried out U8ing catalyets 9 e.g. bor,on tnfluoride and ;;ajcium and cobalt drier8. The cobalt drier in the t.m:nmt of % cobalt to oh wa~ found tc be moet effective in promoting polymerizatior. ae shown in Fig o 6 o However P the iodine value only dro ppsd to 99 wh:ic.h ':.~ ccme:'.dered too high for bj..r:rwn oils to be 1.H5ed in lubrication and the poly<~ merization ccmtj.. rrued on e'\:orager a tough film forming on the eurface. The u~ of 00025% boron trifluoride aleo accelerahd the polymerization 9 but Oo0042% <calcium produced a p:rcoduct only slightly better tha.'ll. the untreated oil. 'l'he use of theee catalyete wguld therefore not be considered to be ad ;rantageous i.n view of the high iodine valueo The catalyete would have to be neutrall.z~d OC:' r8moved 0 adding to the coet of the blown oilo Refractive Index~ The refractive index ine>l"'eaeed with blowing time ae ehown in ~"':'.go and repreeente the emieet and quickest analytical method to check on the progreee of the blowing once ~alib~ation has been achievedo c ) Colour: The colour wa~ net obje~tionably dark in most of the eample~, tr darkeet producte being from the rum! at higher temperature! and rusing certain catalysts o By >ll.eing high airflows to shorten the blowing time ae much ae poeeible 0 products with a Gardner colour of 11=12 eho~ld be attainableo Settlingg The only ~amplee that showed any appreciable eettling out from a lo% solution in LCT 20 after 1 month at room temperature 0 were samples from I'Uil8 No. 9v 14 and 15. Theee rune were catalyzed by driere or boron trifluor~.d r indicating that theee oile e;annot be used in lubrication without further t!'eat= mento Settling teets at other concentrations or temperatures were not attempted~ An attempt wae made to correlate the eettling out with hydroxyl content of the blown oils and aleo by comparison of the near infrared epectrao It wal! found that the hydroxyl values of a blown oil l!howing settling out (No., 14) v and oils blown at 100 and 120 (Noo 10 and 18) were nearly the same (lo2 to 1.5% OH). The near infrared spectra of the three samples were aleo nearly identical 0 all ehowing a broad maximum at mu. Summary: There is no doubt that a blown oil for lubricatior~ purpoees could b prepared from codliver oil or other marine oils o Some preliminary experimente would have to be carried out with the proposed plant as thl type of veeeelr blower 0 etco would eeriouely affect the producto The blown oil would probably have to be eteam-treated to remove odour and to lower the FFA contento The present ~tudy correlates temperature 0 time am airflo'iit wt rh ].(,din v-ah1.e 9 vi5co:"lity and FFA a~ a guide to large=~5cale air blowing operationg wtth certain marine 'C'~ili! o

60 0~~~,--~~~- TA_BLE L Phyei~al and chemical conetante r1f (marine) r;ji ~~. l'!tud~ed I Godliver.JU l SAa:J ):il ~ He:r:r:u:..e; ~-~~~~. +~~-~-~,~~~,---~ L:-.thne wa1ue 168 Fru fatty a,;idl!f. % Oc8 Uueapo-.Jnifiab.ls W!.t.rial % 1,5 Colour~ Gardx.. ~t:r 4 Vi8cl>s1ty, Gardl1er,. 25;l)C Viec:oeity 9 StokeeP 25 C Oc5 Saptmi.f:t.c":l.tion '1!'alue,, mg KOH/g 185 Refractive indexg 25 C A lo4'j744 Stearin~ Clear at; 2 C 1, _ i ' _:,4t ~ :1 ~ I! e.:::: '~ A OoS 192 lo47478 Almoet ~olid at 2 C I ~ l I.. " ~~~~-~--~--' ' o'.j 6 A l, i)1~<l Almo5t sol:td at 2 C i ~>t::.'. II &.RIC im3if&irmc$m 4tzsao.l: & ~l!*i~._,jaiii.!1!ii -i~.. -!._-~it!<p_j,~~ ~ ::» ~-'' ~ ~-'' p. 1:1 ~ ttl e._,, p.. c <:1 ;»o~ ~!"~ ~130rt j-'o i-f< f;:l" \)1 ~ ~. t+ j-'o ~.~:a 0 ('J f Otl --~ ~ ~0 tl tit S IF'" F!< '0...t ill 0 t-',_. ~. "< 1:1 c+ <Y"' m tl-' 1'1! t-0 G:.)~tl~~ 5 ~ ""i 0 r~ o..!\<oo [iii t~"'i n"pi :JJ ir'' <+ 0 t1 P,p,o!i1 e p.. &..0 O'l; -~~~'1 0 ~ Q ~ 1 P> IQJ cr' ~ P."'l "' {ill (AI'? ~ ~"'' rl- tw rei t't) "'. n "!» It. o.::: ~. '0 <:+ O'd 1-1 ""% ~!(,_~ j;li'..,, (>-Co <-? ~ 0 G~' ~ (j s 11,,... "< ~-"] Ill ::r 1\.'o ' ~ >o l::t rl' {.1 11 D-'<' fll ~, 0 II.:+ ii a: l:$' I= ~0~ ;I ~<4tafi ~ ~ i: tt~ J:t ~~1110 ~' c+ Ill ~ 01:1~0' f"t.p-11 1!!1!'1$1»~ ~i> "tl j.'l. 11 II> u, ~;. q c <i P r.t!» <+!-! o ~I~,_.,. j:;s c+ 1-" r.t ri' (llratllip. ~~

61 TABLE IIo Ph,ys:it"'a1 and chemh.al r~omrt<'!.n t~ of blown oil.~ Tim~o Run~ 1-!~Cidine noo. Type oil blrjvjn Cataly~t Ai!flow Tempo I valu hrc ml/mi~ "'C 6 CodUver None " a6 i\ fl 5 7lo5 " ~~ f1 i& 7 ' 73o lo4 II If o8!e ~~ II 10 4lo II. it!! 1""' ol 18 '!C i1l!i lo5 fi Cl II 21 23o o8 ~~ I! It c6 II II o5 ill % mj o5 ~ fi 8 " 49o5 Oo5% BF3 93o0 11 it n 26 Oo025% BF o5 ~ I! 13 29o5 Oo004% Ca " 90o8 ~ ll II % Ca 9loQ II II % Co " 98o8 16* Alkaline 30 None.6500 refined o5 codliver Seal 28 n II 79o? II ~ II 20 Herring 29 64o5 22 Codliver '!' IIH n a 130 l03o7 II 25 fi-l' 0 5 to if *** o8 n li 26-8 ii *** " 99o8 ' " * Th f:~ec :t~:!:ty acid of the alkajj refined oil WM Oo2% ** G ly(~en: added 0 *** Si:samed 0 - Refrac:ti ve 1 Vi~co3i ty r VisccJsi ty~"r Co:io11i~ FFA index Gardner Stoke igardnn! t" 25"' Io48498 Z-lotj 34-o 7 14 '7 lo48658 Z=~ 46o3 16 \~ 3c Z ~5 l33o or '1 lo48447 Z~o~ 88cQ 10 4c9 lc48518 >z-~6 )1ABc 13 4c9 i lo48442 Z~?o4 11Bo3 12 4ol I lo4850;f ;:;,Z=6.> o7 o c, >Z={S >148 c 0 3ol 1o48361 X+t"Jo5 15o3 14 3o6 1o48346 X+Oo6 22o7 12 5ol lo48558 )Z~6 > o9 lo48558 Z=5 98o5 16 3o9 lo48452 Z=4 63o4 12 4o8 lo48412 Z=4o3 73o o4 le48473 Z=5ol 103o5 13 2o )Z=ki ) o2 lo48372 Z=4 63o lo4805l z~~2o 7 47i '~... 0,.<- 7o6 lo47990 ~~4 65o4 11-3o5 lo48311 w ol lo48326 X+Oo5 15o3 I 12 3o2 lo4832l y l'foej 13 v.! <.{)

62 co 0 40 I 0 D I N E VALUE ~ ~.. ~ ~ ~ -~ 0 VISCOSITY 0 STOK&S Figo JL = Eff~K~r\f: (!J;f t~m~rature ( C) of blowing on iodine value and viscosity" Cu.rtit'iS readin.g upward to the right indicate changes in. viscosity 0 downward -':-: ::Le right c:hang~s in iodine walueo Air flow <Constant at ml/mino

63 IODINE VA.LUE Fig o 2 = l 1 'JJd~~<!l walu!!l md 'ri.s o~i ty of blown heh'ing and sal oil u igmpa:red 'bln.w,&. (»:ill liwelr' ~1:lULo ~~Si _ Jl:'ea.ding upward t tll.e right :imi ate J."'Aanges ;rr.;o~ viemoe~,s;ityf) dovnwud.. fg the rlght ~~s in i dine walu!!!o Tempt!IE'&h\re L):.'-~ ~ air fl~ ml/mino

64 42 IODINE VALUI 0 N 0 ~ VISCOSITY~' STOKES 3 = Ef'fe t of' air f'lmv on visco,si ty and iodine value. Curves reading upward to the rlght :~.xldicate changeb in vis:eosity, downward to the right changes in iodine va'!.utoo Temp~rature ~onstant at

65 J~ i I --"'-~~~._...: ~----~b--.~-~

66 44 FREE FATTY ACJil:IS 0 % l

67 ~,,~ T- l i I ' 1\ ~! IODINE VALUE, Q Fig-a 6 = E'tf:e~I'Jt f Cidalyets «:m viscosity and iodine walu.,o Cw:ves ~ead:ing ""'~a~~. t 'tt:h~ righ't~; hld.i ate hangesl in rls f>s:il:\t;y D d WWL.""d to th~ ri.gh'1 ~~tw~g:l':?j :irt i~chi~ walu~o Tempera:t1Ulre 100 CD au f:j!.(i)w 6o500 ml/miidlo

68 c,,-..l) ~"' <J r~- L ~r I i li ~ I

69 4? SUMMARY NOR 16 STEROID HORMONESg IMPAIRED HORMONE METABOLISM IN RELATION TO SPAWNING AND DEATH OF SAlMON g CLEARANCE OF c;l4~cortisone ACETATE FROM THE BWOD OF SPAWNED ATLANTIC SAlMON ~ SALAJ!) Beryl Truscott HoC., Freeman DoRo Idler These studies on steroid hormones were transferred from Vancouver to this Station when D:ro Idler moved in June 9 196L SL"l~~ this iis\ a new.investigation at this Stati.on a brief review may be in order and :has been :mserted following the abstrat0{;" Abstract I1~ is generally ag:ree1 that all five species of Pacific salmon die after spawning while trout and Atlantic ~:almon frequently survive spawningo More than one investigator has suggest:ed that a hypersecretion of adrenocorticosteroids is responsible for the very high levels,of these substances in the blood of se:rually mature and spawned Pacific salmono However,, it has been demonstra\~ed that the elevated blood hormone le~jels in moribund humans arise fr.om ijl1pairell ho:lmone metabolism resulting in a decreased clear&"l<ee of these substanc:~s from the bloodo The presern; etud:les a:rr!' con cerned wit:h the clearance ::>f ilti::r'e, arterially injected cl4"~adrenn:.;j::ctic:osteroids from the blood gf S~ifl~"!Ct Atlantic: salm:m and a c~omparis:m :)f these data with those previ.~ous:l y obtained for sexually immatur~u mature and spawned sockeye sai.rrk>no The results show that there is an impaired hormone metabcolism in sockeye sajmtm immediately before and after spawning while no such impairment ~cs app:n:ent in spawned Atlantic salmon c;r.sexually immature sockeye salld.' )Klo It is concluded that the major impairment of hormone metabolism is a sign of approaching death rather than a result of spaw.ningo It is therefore wnnee"essary to postulate a hypersecretion of corticosteroids fmm the adrenal gland in (,rder to explain the elevated hormone levels f ound in sexually mature and spawned Pacific salmono Sockeye salmon (~ ne~ka) is the most economica:~ly imra::rtb.rr~; (-.pec.ies on the Canadian Paci.hc coasto Between 1956 and the present ti.me s'tudies have been carried out at the Vancouver Technological Station on variou~.~ispe.c:b of the biochemistry of sockeye salmon during the spawing migrativno The freshwater phase of the spawning migration is extremely interesting from the physiological point of view;) in that the fish are in a state of starvation at a time of extreme exertion accompanied by a X"apid gonad de<relopme:nt" The longest :runp to Stuart Lake 9 British Columbiap involves a river mig.ration of nearly 700 mileso Steroid hormones related to cortisone regulate liver gly;eogen lenrels in several other species and might be expected to have a similar flm«.:,tion :Ln salmono The transition from ~a.lt to fresh water presents a problem in '!;h~

70 maj.ntenar. :~:, '(;:t i<.n Dai.ani'A" wh1.gh c,an, be!'egulated in other animal 9pe(;ies. by -:i1dosteron'?' 0 cortji~ost:erom> :)Jt ll~deoxycorticos;t<e;ro:ne0 Stickey:e salmon ha.v12 a e'c:.:c~ng t~c-)m:tng :Lnstj.ncc't!lnd. adhere ~10 a strict timo 50hedule; O(J>th beg:tmnlng ani d.u:::tng 1';1'1,Ch rn:',gxa *;:'.(m and this observahon s<uggests a p.:;,ssible 1 ''tr;ggrov" 1 t. r:.g 1he >Jl1J.i1l nceruer.\'~ ;,f the Ii'V<'!:lC' phaat> o:f +,,he SIJaW!11..iJ:Jg mig:rat,:~':jn 'l!..z\j. a;l l! i3! 1 Pl.r.l.gg'>~~'~.c:mB. be I): )rmonal JLn natureo k\rs.ilab::l~ tx1f(,r:rrjb.t):.. r" u1dl.:.1t8s :;:;.a.j: a:,:. L /?c :'ls ;yf P~ c2.fic salm,on d1.e after spa.,'ti.:t;lg whl.,. i.&jge pi:~r. t Xt.:.Ls.n~~jJ: ::;;aimcm 2nuviv~ E?:paw-.:d.rJg" Steroid ho:rmor -<'H:: ha fte: hc~r::m ~.llip1:'\cs.tsd.; z:;n '::he basis of :Jndirect e".ridenc~ 9 :in an a.ttempt by t?':'mf irrl'e&tl.gato:zc& tc ~xpla1n the dsath of Pal);ifi«J salmon after spawnin.go W1 th rh;:;.~.,. tl:..:;.uglrt8 <r:o, ;'\tud::les y,rere bl2!gu:n :in 1958 ~'" a:n atte:mlj' k.l- :;,sc,ia~';,-,: ad:t'eil1u<.. ~'r<;:_. : ~~,~~"~l' J:td3 f:::e:m th~~ olrv:.d o:f Pa.;,::i.fic salmono Blocxi. was c:~e.l,.r,,:-.te.d f-.,r i.jk1:"-s; fitudi.es because hc:;cmcnes are carried in thi.9 medium to targ191t >rgan~ t"j1a ::.:1-, waa te:h that \1.lood hormon~ levela would p!'\:yvld:e the grea:tes'c flc.:::-,;crr:ela'ia.o:n with obsf"i!"wad llioe;hemicaj. r:~p..angeso T.tJ.f~ :t 'ir ~t: ~tdy",~n.n<:,,)~~--tkt(a;sta! ~ i,~1., c~x~t.i-~vli' W-5 _,n1y ist1lated an.ti p:jsi. thrs,:i,y id.e::lt:i:':'18ct f:t Jm 1 1 'Jill.~n 'blc' )d in 1953 ' p:r "unarh,y be;c;.aj..<:~e it ;_,')Ctl:t'S in nomal man at a /":);:, ~";:;nt.)'f.:l.'ti irc r:f ::x.1~~y 10 ~/100 ml ~.:_:f plasma, The f:irst evidencc"3 of the; <;>;:;c;u:rr:en : 1; : f ad!" alvc ~::xvt,.'-c:st.rrv;.,ida in salm:m was an :obser~ratton that Y:hi?d~..,. '"~fa~ 8. Eubsta:n :<?l(s) ;,n the plasma of Atlantic 5almo:~ Is~~) wh1.c~. re,:rpond..::-d..: :~ e. :..: l:, :r:.l'n-'tr i.,.., proce<dure devised to determine cz:cz tis <t 1.n otha1 c:lpecl..;>":' c Thes"' :t\.~ew.j.t::3 we:;;-i'l ~mbetantiated when Fonta.in~ and Hn. shm..red t~.a J~ 1-":Ktxac,'~,.o: «!' th;o, bj,>d -~ f -~ ~~laxo prortded the T ).:'.~.ate e-n : :..;; t'is ;n,;,, l.io:--~hydroxyprogehtercme and i.tl:i 2(;f3~d.:.hydr Jspl.mer... ll~ket;,-i,<:m'-;;os+.erunf\ and.;;:o:rticosterc,ne,, ad:renc.stm:., me 9 t.estogtert ne ax~r:l.;:,)p bav& been :is.)llated and :td!f:;nb.fl.s d at the;!fan..:~ '.li"l<: ~ ec:ru:\):.. g. : "l-1 E<t~.d r-;h.own. tc occur in tha "<free~'..;:,;, x:...~n-~ :<::..v..j.;,ga:t~d Bt.at,,:~ in ~.-:.' f'~ -'.llll1 at~id. 0 Ei';ridenc;e W8.cl \}re.sentc>d B'J.ggeati.ng d-"u.r R<?~. ns-ts.l.n 1 B r;,j(',;-:,;::cn" "S''' ij.l deoxycoi"tisone) also ')cc;ur.;;: J~n the :non~ c-::;nj:ag:tle;'l. si::(-;rc,.td.f:ta:::.:l:'jn0.i\. subntru1(:<1 'ii' ith chromatographic propertiee~ c.;: nsist.emt w:l th tho~e: of ald::sr,erone 9 a potent mineral regulating hom.1me'" has belen i:2nlat6'.i f:.rom the plasma :.f pcst~sp&?med salmod. by Philipsp Holmes a:r.ld Bcnd:\' 1, (195q nut 8. s.un.ila7'' mater' :ial i.s:?jlated at Vanc:ou ve:<:" from pr"e~,spawned. l:k~ckey~ "ih1m :nc. :,:;t'rlwed. no m'..,nerajl<~"c.orticol.d activ-ity :in a. :r a:l;cobioasse.y" Ste:ryu;!!::... :rm:'n-:?s ar!f '.)iten ~-'~"<)J.ga.b?.d afs'. gluc-.uroni.des o:r: :sulpha tes and th9 sr:d.. y :flj>ggated r: terc.ii.d. th,.:h3 fcc :donthied f:,:,um e.almcn bl~:jd pla;s:ma "' 21 t~~;::~:-:,0ste:r J_n~ ~ o There i.!"i 1 : tth.l:l:tk, m (: :rm:;eming the physiological func:t:'!.i.:me< h1 s.<.tlmon O f thr,- ab,)\pa: n::~:n."cic ned stero11.d hormoneso ll~keb'ltestc!:n:er,cne has r~ot ye+ been X "porte1 in <ejther specei~.:=; but it bears an obvilro.a simila:kohy t o the me.)e -sex h':,r.nnone,. ~testo:::ter~on8 9 and Idler; Sehmidt and Bialy hay,& shown that it has 57% ( f th;:. hci.<olc.gi, :al _potency of tf:stosterone in pr ~omoti.r.g the gr owth,~,f the c hi.ck' s "7,}<mt;.. Th!'! s:&::tr~jaj.'l.y rna turirjg male sockeye sa.lnum is charat:t'?;:-:ze& by an &i.:,nga~:~.:ng srh:;ut, loss 0f pi.gmentation from the flesh~ inc;;reased pigrnentati.<clr'' ar..d :;htckrjes.s 'Jf the skin and a humped bac;ko Sexually imrnatur<::: se:ckey e' sajmu:e ra.pj.dly da :relop these secondary sex eharacter:\.sti.c~

71 49 wher. treated with ll~k:etotestosterone and Idler 0 Bitnera and Schmidt haye presented preliminary to.videnc:e for a stimulation of spermatogenesis by the hormoneo The plasma concentration of all hormones thus far isolated from salmon plasma have bee:n quantitatively determined by Schmidt and Idler at 'iariouel stage:il Clf the ~eri:lw.l de'welopment of the fisho The river ph..<is~ of the spawning migration coincided with an increase in ll~ketotestosterone and a decre8#3e i.n testosteron~ :t:n '~he plasma of the femaleo An opposite phenomena. oc:curred :Lu the male where the testosterone level increased with an a~;cm:npanying decrease in ll= ketotestosteroneo These rb>~ults give some support tro a hoxmone ~~trigger[~ theory for spawr.d.ng migrationo Evidenc:s has been presented by Scbmid'tt. and Idler which suggests a ti;)orrelation between liye:i" glycogen ccmtent and the blood levels of cortisol and cortisoneo The salmon pi tui ta:ry h,m been shmm by Rinfret and Hane to produt:!e a 9Ubstance with ACTH activity and this observation gave >.:'ise to Robertson s hypothesis that. t:he degentrative changes and death of the Pace<ific salmon after ~pawning war duill l;o th~1 production of intolerable c;ort,~ent!"ations of adrenocortical hol"mold.e!s U>.J.del' the influence of ACTHo Further st1.1d.iea hav<?i shr.nm that Pac;ific. :sa]mon undergo extensive degeneration of the pitui~ary gland at full ~e:rual maturity and spawning and this is ac.compan:tad by a marked h;yperplasia (and presumed increased secretory acti?'l.ty) of the adxenal glando In e:ontrasto matu.:re rainbow troutp which spawn repea:tedlyp general2y!';;how little P if axry pi tu:utary degeneration according to Robert son and hi a c~,o=wo:rkerso It has now also been established by Schmidt and Id:i.erg and.:::-:mfirmed by Hs.nep Robertson and Philips that cortisol and co:r:>tisone are presen~: at very r.r.igh levels in l;he blood of sexually m.ab.tre a.s well ~:2 spawned Pacific; salmono AHh:» ug:r. the cir<:rji!lstantial ev'ide~lc:e is ::omrin~ing~ tho~e who postulat"l a hyperfm.gtion of the adrenal in salmon appe:ial" to b;; overlooking an, important c:~bservation made on moribund humanso There j~. an impaired el::~mination cf infused cl4=cortisol from the blo::.,d of dy ing hu!ilfl'1c5.independent :of the \0!1fl,"1Se of dr~ath and the reb.ults were ir.terpn>.ted to indican~ that the f;leva~ed h:;nnone levels resulted from impaired me.tabojism in unthe p:resen:;e o:t coni:;i.nus.:l production and secretion of steroids by the> adrenal cortex at normal or reduced ratesiv. An impaired elimination of hormones has also been associated with late human pregnancy and occurs in the newborn infant o Six female slamon from River Philip 9 NOSO were a:rtinciall,.y spawnedv transferred to Grand Lake hate;he:ry 9 Wellingtonp No So 9 and held in the. Rawdon Ri.ver o Three days later 9 they were injected intraarterially with 2j.L.c; ot cl4~ortisohe acetatee Aft'i3r and ~ hours fallowing the injection 9 1 ml of' blood was withdrawn., transferred to a vial and frozen in dry ir:eo During this experiment one fish 9 Noo 6 9 died~ Six days after spawni11g 9 the experi= ment was repeated c)n the f:ive remaining salmon and blood ~amples were witbdraw at lp 39 5p and 11 hours after the injection of 2 j.v.:: cl4~~c:od:is.one acetat8o

72 All tb8 b.:.:-~, s~unp:::;,a. ~~~;;:~ a:c:l<:ilyeed for :rad:toactive at ;n."oid contento Th~:, to amr~es w ere r;h~;.;.,.,a.,. a..-:eu:ra'~ely we;.ghed 1 and extr'ac:ted w t th ch0hloro- mis r.hane., An 8.liq ;vjt of th"i.' extract wa.e :pl-st'~ed cut ajr;d COll.\'lted, The rate of cle:a:rance. :-f th~ C ).r t:.;,s~ rl8 aceh,te was "~ssentially the sa'lle for both s~ats of samples bnt, the s.. Y~!l':i~r Barnpl e f:tguj:;es we:r e U;3ed to ~al.;)u1ate the bi.o1ogicsl ha.:j.~hfe Hetoultr:<, : <, t..:<bn.. ojh d JII Tab:.~ L The rema~.xld.'sl:t :-)f the di<;::hlc r) ~ methan~ e.jctra.~tt:' ~"'re- dr- <id ani. ~hx., ri:!v~tc g;;:aphed vn pape::r.. Th.e areas of th ' pa.p!.'t :a:r rylng I~I)T't-t,:; : I r:c().f'luo'-1f1.1:\' a.ud. ('( rt.:is{'w? a:.;e ta+:e W9:{ cut cut~ elu>:ed. a.r1d al?b b81jj. -~ c..(.~jijl1't~=~~i 1'h-:; sax h~:<:r z "";:aranci'< l:.eud:.t':2 are :i.nc.lud>!'ld fo:r e:ompo.r-~.se:m {Ar.Jma1 Rep :T'L of the VHJ!(>~Il''''Y' T:?<.!tln.r).-.1.g; (a~ s r:at.i :m, Summar,;v- Nc' :.., ) 0 Cle:.,r::." "' ~ JV.:,i!:':. :-,. tr.:,, ~... \.],,:.~eng To:: aax tt.<i.. ~J..al InJu<"L.,.)n,):[ 4--"cl4.~~=id::_;.ntr:.JL!l~!lg.! FJ,sg6:c R:,:;:::r S(ck~y':'< :ntoi<: SR'-.He.:.i >f'f the west coast of VF..nc,:ru~ "',l'' lbla.nd n.eaxv p,,:.,.r~,, li'.f.; ~~f:tet...r udd!l-::1l.d,._; ~ -~~ ~ia.j' Ke-.~t:~~d tank ~: ;;~-'elnn.tgllt bsf--or \J tn.jecthm, to b8 -::sr~!:lij: that tl_'<': fish ilfi::xs not d.y:ing, On~1 m.i of a saline', 8olubon ;:>:m ~ai..rp.ng J I"" ~ 4~Gi.4~-ecH'tJ.son6 a o.:.;;::.te (.. 5 p.g of sternj 71) W"!S inje(,ted l.nt:r~3.3 x -+ -:-.r "'~a] :~.r.~."t:.. ~ to--'fu.:-j.:~ :f-_ ::)1 v 0~0~; W.. :. b..\.'c:od ~~~asnp.le;; were tal<~;\n. from ear,;c f 1 sh fr :.m tt"<f 'i,, r::.2 o<:a t eel b~:::h:..nd the t;hi:t'd gt ll arch~ at 5 P l P 1,5, 2.S ".md. 5J'i h:c, Tr:,,.-, -~1:up:0~:' w;.;-h,, fr~;zen :YJ. d:ry ic"eo An addith,nal group ( f fi r1h of tl ;e Cz.t~ ~x' Lak,: ~.'BL:t:: i<lbr'8 r m.<.ght w:lth a gil.lnet at Aj.bion nea.r the :mouth r:f d: 3 i''la=-:<::"r Ro.1 er and r-r:ant3:::'s-nt:,ri, under mild sedation with j.),s 222,, b:: the Var-',;,..., rs<l:,~~j~i...:.g1". a1 S'ra c.i 1:n where they ~'!ere helj in a 'd ".l"r~ ulnr l..,r.j. r, ''". "r'"''''"'""' t.. ''g......,,.,,.,.,1. s 'pp.ly Thr "'e day"' ""at'""" the f' 4.A. r" " :..,,f.,.j. \'!JJ '' "' ).J'.t..,.:~, "" ;,._ " - ~....,_.._. '. t4 1;1h appeared to Le 1.n (~X': e1j.:-,:nt,,,ondi ti.on and were.tnjected with 1 lj.c of 4~C.. - r r>y t':j c ol ;:,._..._,;;_j )..~, ~r' '-- < 1 "- J.L;.,A_',.,.,~, -.c,..,-->1-' ~,C.,~ ~ -.#..::1, p,,j ; Jl,_'/,.~.~t...,...,.~, (..;:.;;,}! "'"P"""'"''J..A.....A...,.,,,.~ely ~'1.>,.1 <-'"" -14' ''.g t" of co:r t sone).j...'o Sar.uples ( 1 rul) ( -~'' b ( S'Hl.,. :.-rt:; ': ermc >. ed. fr~m the same artery used for inje.:: tion at :i:c.tf:n'1ia.!.:~ of :5, 6. : rini c. h,":n '-~ f:.: om the t.irue :::f ::..njectiono The blood waf: h :pa:rwi.z8d and. f:r: :::.Rrt on d:r:t.ccs, F1.si1. werf.< also taken on the spawning grmmd. at c::h.l Uc:J Lake ::..nd j'tf>je~ ted w1 th l lj.c of.t~cl4-cortisol ( 46 j.j.g of l;.)j:'in>jne),, Blood samp:ee!1'(-:'!'":7 "(aken :.\11 o:he same manner as at the other 1ocation:3 after 1,, 2, 4 6 a.nd 8 hourso For analyses ca... 1 g of bloo:i was a<:::curately weighed a."ld diluted with 15 ml.:,f ('"9jb B!'i~l;;:-;0 G;rtJs:-;l M.d cortisone (20 j.j.g of each) were added hl the solution and tho::> p],amua e.a,uihbrated with 40 ml of methylene chloride with orc8s" )nt:d r:h.1:.1lcng o 8r a. one~r.talf hour interva.l" A 5 ml al:1quot of tr:.e methy u~r.ce < nj,xr:i de extract was taken and the rad.ioae:t2vi ty determined The ":;(;ta'i I'idioa.cti-a. ty.:.n tb.e m8thylene c.uj o:dde extract was then used to r::i'.1c'l19.te the ra:l.(;b.<:t:.;rity remaining ;in a unit weight of blood from '!?'3.ch 8u.mp:.. e Results and D1.scus'?I 011 ThHe 1e: a. shm p deo ea;:;e u, The radioactivt.ty of the hl;:,od from. all fish.lr.ltj.aj w (flgs" 1 ard 2), Th<2 shape of these curves is very similar to tjw8e obr:d::.n{;d fd:r c:he nc rt.aaj human... H:)WeYer 9 :,.n the case Jf S JC.keye 21.dmcm r: aptured :';mr.~dia oef.~:re., 1mmediately H.fter spawning, the' :ap.:d "J.!Ll'c\~J d.;::rrease u, ':he r.ad:j.:achv.it;y jn the p!.. asma levels cut and a f3)1j',j...:.a (!~;J :.:n.cl.t orrt<n:r~p.d for the clearance of c.. o:rtisal by

73 ". 51 moribund humans is obta1.nedo The half-lif~ of the stero:i.ds in salmon blood war determined from the latter half of the curveo The clearance Cc.A.r:.res of cl:lsteroid from the bl0od of spawned Atlantic salmon are sj.m.1la1' to those found fo:r s"lrj.ally immature sockeyeo Table I ::lu:tnma::r.:lzes the result::: obtained on the fish invest:igateo. to da:teo There appea.rs to be little difference in the clearance of cl4~ ccrtisonfi and :tt:-:;; acetate from male and female sockeye salmono 'rhe a'rerage half~~life fo:r the rad~.oactive stercids in aeraa.lly immature sockeye salmc:;-rl was 3a4 hro The ba.lf~life of the c14-steroids is considerably longer ":;hm the value of l" g:!: 02 hx found for normal humanso For pre~spawned and spawned sockeye salmon the average half=life of radioactiv:j.. ty in the p;,mrna w as 15d! hr" The clearance of corti.sol and cortisone is similar in h.umans arid :re;1u.l..ts vb ~;alned :i.n th Ls labm:a.tcry show that cort isone acetate ::s rapidly converted to cortisone in sockeye salmon (unpubeshed data). T(H9: :r,c,s.tllta si'.i(?w -~~r.a t t he:f', 5.is S.!'i impa.tred l:t(/.trn.orl~>:.rr.~ ;.:'~~ rll. i -.1 ~ ~f~n ~: sockey"' :'lelmo:r: ;trume:iiat"!jy bef,:;::: '' and. after spawning 0 In A)!e.:a. :5.....n ~ 1f hjcj1 fr~qc.:ce'::tt".:v s:xz:";"i'lrf;,~pa.wn.:i.ng;, the :rat~:5 of hormon& 6l:i.:tTt:c:::.tr~. ' :::. : day;;:t spa"lfming :ts '~omparabi<:~ t{j th8 c c bia:ined w.i'dl ~'<o:l:.a.l.t!!..jiu.i~.f"' scr.keye salmon,, ~;.~tnough s1i.gh;:ly sluwel""o It seernb :reb2 r;,e,'.2 '. :r: ::. J. +.hat th.e ma.j(';!c im:ps.t:r me;n:t.;yf tl O~"~n:~.:~nA:?! m.eta.bclism :i~s tl1.er-~~fo.~ B a. ~~ ~~i a.pp~'()~.~ob.i:':"lg ticj;h h.!"8 rhe:?' -~;'Jar; a,;;f spawnj..rg u Thig ; (.'!('; ;::. til:' > fur :t:~1e-ll'.:::;.ub::rtrur:.t!i 'e:.l ':Jf A~::.8J1t1.t: ~"almon No., 6 (Table I) wr:j.ct l ;:;ar ' :.~ in.j<f':'s: ~~d hormone a.t a s]c; ~t':rr' ra 1 :,_, ~:ttruj. djid the c)the:i.~!:'! 0nd d1 ~1 :, }~.:'J:,:: :> :o::: fh~f~ ~ -:,: i~_:l ~.,n. v T.h0,?8 e.xpe:r:i.jd.e.n.i.s coh:: w that :d.:..s unnec:sssary t: po.~~.. let..,,. : ~Gc:rnt:, ~n ;.~. : 1f.;\Cc,'1 ~At'r>~idfi ~::0m th.e adr~?nal gla.'1d :en : :r1en' tr: ~'~;, 1 : ' <'. 13lc>':'are:il h:.1"'dou>p. I~F els foutj. d. :w.; :senl.aily mature and ~.pa~e i Pa "'' rr:7.; S.:t(h may hs the -:A.M3C,. but :tt: ~ s e:qu.ahy pos.s:ible~ by ana.. L gy Y> m., j hu.m.a:nsn toot hr,nr:.m.<e of 'the adrenal gland l.'lk"ly t '- n ~-.:va:' ~., d.im:i.ll.shado Tbs AJJt;t,;.:JJ:""S Wl.sh to '/;;ha..'1k Dz o WoEo Vanstone a.l'1"i Mro Cu'l"'o ELl::~: f;hf.' F\.sh8':10;'; R&searr h Bc,ard. of Cax:v:d.a 9 B:i.o:logi.cal Sta.tc, (,C. N:?t.,':l ~a..,?.,, ;.~"". :: :~. Col.\:.n1-l1ia.,, ffi',j1 J 1:( o AoP o R": 1 nai:1 an.d. Miss Vo Cha.Ylg of the T6c-1"rr~ J t StR ~.,:_,; : n V.:?.ncouver, B:-: itish Columbia., for '!;heir assistance with m0ch ::f :h.:: "",~,.,.,, ~,.,

74 "=~«< R_..,._.,_,. ~----"-..,,, ~~.,.~,.,-.,,~~ -~ - ---,_,M~~T' -U--,..,...,..... _., ~--~-~-- "-.~ TABLE I Glearan.ce :,f Int!"aa.rtel'ially Admiiastered P..a.dJoa.ct v <' S.:ero~~ds from the Blood of Salmon.3pec1es Sample of.i'':;csh.no.~ Sex.AJ)IJ:t;)X o L_,.cat.-l~:',t1 State of Steroid -iveig:nt. ;:n:i j),'j.i:6 r::a tu::c:~ ty Injected Stert)id Half'--.u "f e('.n:rs) \ {\,J ~ Oo , Oo 0. 3 s S, 3. so s0 nerka lllb:, B.C. Irmno:.tture 4-~G 14 -co rt:i sone 4o6 Aug, 6(J nerka 2 lil... A 1'b:~orJ BvC Immature 4~C 1<Lcorti~>one 3A lt\.l{~" ~ nerk.a 3 F + p._:;rr uc,nf "~{ Immature 4-~ 1 4-cortisone :~' 0 t -,~ 1:~ 'c :. ~.:. /Lg,,.il.A.f;.,.D, ' acetate nerka 1 N + P :,:rt Rex~:: 0)~~ Irnmntu:re 4=C 14 ~co rti. sone 2,,, r) J:i (' An..., "~~~q '"(' > - L--. c... l.)~ J.',... acetate nerka? F + C:.t i l.k.j k-ake. Sc.awned 4~ Cl4.~cortisol 1608.t' {' ),_... 2'.c'Pv Jlc ;/59., I nerk11 7!ll + Ch1lk1 u:t.«:e,, Mature; 4-Cl4_cortisol 18,0 B,_,C 1 87:p0 ~.~ ;'7/59 pres pawned nerka 8 lvl + Cl:nlk,:. Lak::, Mab.re; 4-Cl4~cortisol 13,0 B c,,. "'".,.08p.,.,...;f'~ ~ 'i I" Cl P"-"espawned nerka 6 M G:hilkr; l,ake...l'tlature9 4--cl4~cort1sol 1206 B.C Sep() /59 pre:spawned salar l... 'fi' 1) lltve:c N s...p~r:d 1p; Spawned* 4=C14-cortisone 5v0 '.,Nov.21/6l acetate sai.ar 2 li' H~~_-te:r P.ln1ir,J. Spaw'!led 4-cl4_c:ortisone 4ol N,,u '~,,..,, /l 16 i l!.l'i\,1 "0 - " ~~ acetate 'Z salar,i F ) luver PhlhP. Spawned 4-Cl4-cortisone 4~7 " (''J l'j vu,, ~ Nc v, <.)... :."l/61 i aceta.te snlar 4 El 4 ihver Pin. lip, Spawned 4~C l4~cortimme 4 9 r s,, 'r,- ~" ~,,Nov",::.. 'J.l acetate salar 5 E' I) ' R.l\l'er Philip" Spawned 4-C 14 -cortisone 4,9 NoS.,,No,r,2l_/6l acetate t:; saiar 6 li' / River Fh:.L~) Spawned 4 ~cl4 -cortisone 7vl** LUS,.., N )i.t ' > ~2. i,/6j 11.cetate :..--:.~;-:.:=.:::.::::=:===..:::----~~=.;=.:~"':'!.::::..::..::.-====:~~-=~';':~.. ~.,...,.,...,~.,... * Spawned 1-iY't1f!.C1c.tl1y 6 day~> pr:jf..r' ;:;.; in;jed.:i-::m" The steroid half~1ife in -l:hre(:l female.s.~ ~~a :.a:c spuwr:,ed artif~.cially 3 days pn.cr to tnject.:'!. Jn wi.th 4-Cl Lc,:>rtieonf:' acetate was 4-,,'3,. 3d6 and 4"6 hoursu

75 Fig. 1 = Clearance of intraarterially injected c 14 = steroids trom the blood of O.nerka. (See Table I) fi ~ ~ ~ 0 ~ 0 f:i ~ ~ L_ f L I I I I I I I L..L..,,!....J-_-..! ~ ~-_--&.,_ <? 9 0 l '! 8 9 T I ME (:.ms) \.11 \J>l

76 ~ ~ A 0 s I:Q 0 ""' 0 c ~ ~ ~ ~ TIME (HRS) Figo 2 = Clearance of Intravenously injected c 14 = steroids from the blood of spawned S 2 Salar (see Table I)o

77 55 SUMMARY NOB 17 STEROID HORMONES g TESTOSTERONE IN THE BLOOD AND TESTES OF SPAWNED 9 FRASER RIVER SOCKEYE SALMON (ONCORHYNCHUS NERKA) Dov Grajcer* Do Ro Idler Abstraci Testosterone was isolated from the conjugated steroid fractions of both plasma and testes of spawning male sockeye salmono The structure of the steroid was confimed by several criteria including infrared spectrao Glucuronide conjugatjon was indi.c;ated by the use of a specific glucuronidase inhibitor 9 gluc;osaccn.. uu,, l ~ 4~~lactone P and the inability of the enzyme to hydrolyse testosteron6< sulphateo Testosterone ~Has J:: ecently shown to occur in a conjugated form in the plasma of spawned female sockeye salmono The steroid was recovered following t3~glucuronidaae treatmen!; of the solvent extracted 9 deproteinized plasrrw. 9 and no further testosterone was :E'eleased by acid hydrolysiso It therefore seemed likely that the testosterone was present in the form of a conjugate with glucuronic acido However 9 since crude p-glucuronidase was employed there was still a possibility that hydrolysis was effected either by the conditions of the reaction or a contaminant in the enzyme preparationo Glucosaccharo~l~4- lactone is a strong inhibitor for ~lucuronidase and this report shows that in its presence testosterone is not released from its conjugate. in male sockeye plasma by {3~ glucuronidase but is released after the removal of the inhibitor. It is also shown that testosterone is not released from its sulphate conjugate by the ~glucuronidase used in the experiments and thus indicates that testosteront: sulphate is not in:vohred in 1;Jle reactiono.a. search for th:?. origin of the testosterone conjugate in the plasma led to the discovery ~f a substantial amount of the conjugated steroid in the testes of spawning fish of t;he same speciesu An androgenic activity was recognized and described for fish testes for some time. It wa~ also shown that in spawning migration of Oncorhynchus ner~ the androgenic activity increased as the fish proceeded up river to the spawning groundso However 9 no characterization of the hormone(s) responsible was attempted 9 to our knowledgeo Plasma Blood was obtained from spawned Cul tus Ll;lke 9 male sockeye salmon in Novemberp Fish were bled by severing the caudal artery and precautions were taken to exclude slime a:c.d oaxcretao The heparinized blood wa~;j chilled on icer centrifuged promptly at 4000=5000 r"p"mo and the plasma stored at =35 Co The "free if steroids were extracted from the alkalized plasma three times with 2o5 volumes of dichlorometharte or ethyl acetate as previously described, * Mro Grajcer has spent the past few months in Halifax completing this work which was begun &t the Vancouver Technological Station (Annual Report for 1961=62;; SUmmary Noo 6L He will leave Halifax in MayP 19620

78 .U~lrorude~.o The plas::.1a :c esid:j.f:- fn)lll ~.he ext:.:actton r: f the free staro:!.ds was treated w;~th 2o5 vo~ijlme:s cf et.han,:;j t: rrt'6'::p:tta ~t~ the proteins and the prt.~c:ipcl.trte was reru{j'l(-.'d by 'ent.rifugat~.c.r. &.t 9'~1001\{L Th>.: l: recip1ta.te was washr,d w.::.th 'icjfs eth:.1.n :.~~- and thee waejtl.rgs were addii:<l '\" Gl1e m:.pero.at(o'lo Tho ent:.re p:r ocedure '<83 ea:r:r<h>d.:,ut at )r.co ~:Jw 'd!pe:nat;'-" wa.s ccn;:;entrated in vacuo at 'lli :50 0,;_:;lng '' flash 8'>1l.p~:r:, col' ths :~e,:,e;i ;-sr of \rh:u:::h was cooled in a dry ice=acetok.t8 ty(.}'.tj..?.r<~o '1 1 hf--:! rr;:.c: cj'... i~: ; w-as S~_ls_ p.elld~d "td. F.t. 1 r 101u:a:t.e -~f watex eq1.<ivalent to th1:j.t of t:h;.:; P.:vi ex~rf:.:.t ::>d three tirr.es with d~chloromethane to remove any.ster ::dd.s 1v:h'i ~"h m.2gh._ h':iye been :~~f.;lc~ased from the protein. DichloronH:.!thane wa~~ <se:i. al:: 21 PX"<Jc~ ::n:v::j.:,:;veo Ver:Jene (5 mg/100 ml) was added and the aql.h:'l,: \18 phase was ad,justed tc1 ph L 5 rilld. 'treatt>d w-1 th (3-~glucuronidase (pur ho:;<.'.l. fr~ail W': tl.,:,,g kid ~li~ -.:b:~m:.:. ::.:. C:::.:r!J:...rat;~.e>Lp F.:-eehold~ N., J") (,.., 1... cv). ' " ~.,."(' + ~s \ T. H '!,. c.lu mg_ J. " r:l.l) + t 3 5 d n 'l ' ' i,..,._ 'JT' ".tlou.jc'b o ' n1'j: p was tnhn 8.Q JUS,;ea 0 o an th"' hydro~tys..la was c(mt:.n:,ie::j. f.'jj:' J3.:i\ [ddj. t:;.'):rtal 24 hovr.s, Prtor to use 1, the i3 ~r:;j.uc: 1.lrom.daBe w.:ts Eu:.,f~ "'I:il.cd.:u. 1. wa':;;:a and extrac~ed tbr<?.e times w:t th 2 volumes of dj.chiol. metha!\f:'o rtw.s t.:;roi.ds f:tt:ed. by the gl;.wu.ronida.se treatment were removed by three e:d;j.,;ct:~c.:ns w:t th d:lchloro:11etha!1e" 'rhe d:i.chloromethcllle extract wa.s wa<hed three times w::..tn. (\o! vol;j.!?j,e -:;:f each of th.e follow1.ng: 5% sodh'lll bicarb x.ate 0 Oo 1. N acetj,., B.cicL Br\d wate>to The :xrganl:: solvent was removed in.xacuo as de:scribed above a.nd the rc.~sidue was JXl.rti tioned. between?ci/o methanol. ar:d h.e:t:ll.ylpo.rhe rnet:b.arw't was removod by flash eyaporation 9 the residue taken up in we; and the g:u.1euron:uie con.j'jgated ster,?ids extracted with djr;hjorc!!!!etta.neo 'rh"' p(asma Ext:l:acto to which :;;.nhib,tor ha1 D;::<:;n added 9 ivas takor, t ) dryness by ly;,;,ph:lll2.ation. 'rhe res2.due was suspended Jn ''s. te= and passed t hr r,.,atl,.,. "'""'e)l a i"q'j.,,.. 1'll1'"l """'...\).. (If ~~ Am'h.:J.ri.~'I;';. '--,:~.. f,-v "' (. (' Ll) IJ.J. J ~.J. u;"al ;rt' 'C'' ~ gy~ o11!"! i 'I~ ">""("h"11'l.<u:,.,..,ec l T1 1. ~!!...a.....~... c t.~ y.. ~ ~-. 1,... ~, -'~ c..." ;::;,J.,..,..,. (pu.r:j:... '1.t'ed from Roh!!l <::u:d P...aas Coc L:d.,), The resir:: bed m6af:;ly:ing lo8x 12 ern wc::.s washed. \Hth water t.;j remc'le thr g~'llc :;sacd:a.r::~1~4 -lar:tone, 'I'es bsit:<t"')zl S'.ll.pha te ;vas pr:3par<~':l. &s cte,scribed by Hold.Pn 21 JJ!.Jo lllopo 2l5 C with g:reen c:)l:m~~ ':;hanging t-:. brmm at 2.. t0 C c~ ;?J6 Co Neutral. Woe1m alununa (d5str:l.butod by Alu.pha:rm Chemicals~ P, 0, ')':' K 755,. New Orleans. Lao) er:t:rvity g:rade 1 (15 g) were p 111.red into a 18 mn1 i:n;;ernaj d.i.am&t<":::" colj.jlnc!ln.mal bnta.m:i. C,oo ml) was used to wa.sh the columu fc-1lowed by the :r.:,nr;:tldn Ft.::.xture :m. n r;utanol :5ochl.iln testosterone sulphate ras ejuted f:~.~om 'c,:: t=? absorbent with 2<)(J :m.1 of 1)}0 Etp,cer:.to" bu-'~arwl follow;;)d by 2CX.I ml of lc}b aq ueolh\ r.:~ tnrw J., Papei~ wa2 washf.'d in a S )xhlet apj:.18.ratus for long periods with acetic acid 9 ammonitilll hyd:rox::de : and methano1 as py eviously describedo The paper was then >va3ht.1d. w:t U. metha'lol. f:-::r 48 hottr"b hy d.sscending chromatography. Heptane~Bo% aquem:ts methane::\, (Hl\1=8\.J) and ~:oluens;, t~,bu+:anol.. acetic~ ar.::iddw-ater and hyd:co..:.,b1:):t'i..c: cv.:cd. J.n the _,~Elt:i of f:j0340o 30, "/\) a:nd 3 respect:tvely were used,

79 57 Selective Acetylatio]L The steroid (2(}~40!J ) was incubated overnight with O.,l ml of 20:1 mixture of a-:::~tic anhydride atld dey pyridineo The reaction was stopped rlth Oo4 ml of ethanol by :incubating for one hour at 37 C and the solvent removed under ni. trogen" T.bjs procedure a.cetylated testosterone but no.t l7cl=bydro:cyprogesteroneo Deacetylation of Testosteronil At~etate The steroid was dissolved in Oo5 ml of NaOH and lo5 ml of methanol and incubated for 45 mino at 37 Co Four volumes of water war& added and ~the steroid extracted with ethyl acetate in the usual mannero Chromic Trioxid~ Oxidation of 17~hydroxy Group_ The ste:n:ol.d wa5 dissolved in 20 ml glacial acetic acid and 2o0 mj. of lo2'% chxomic oxid~ lj.ji 6o/o acetit acid and sti:r:ted for about 30 mino Excess ch.romtc ox:id"' wa1:< d.estrjyg :l w:ith a few drops of saturated sodium bisu.lphite solut:to!2o Dichlor,omethana '..ra:s freshly distilled and stabilized with 1% methanolo Methano?. was added per:lod:...cally during the rej,iloval of dichloromethane from the steroid extra>c:tso All :OJthe:t' solvents were distilledo Glassware was washed with sulphuric ac1d<~ch:r:omie:,.'jxj~de cleal1:ing solution followed by water 9 versene in aqueous methp-nolu <'ll'~d d:i.stlll'id watero Infrarf'>d ~pe::r,xa wer~ rli'cord~~jd with a Beckman IR=4 doubl.e beam spectro:phctomet&::c: ~~ 'xlpf~d with a beam-condensing systemo Potassium bromide (5~10 mg :infrared gxade} was apr~ad over an area of. &o inch2 in a mortar warmed on a m.icro=heater to a temperature sufficient to volatilize dichloromethaneo The steroid (~o j.lg) was taken up in =0 0 1 ml of dichloromethane and dispersed on the potassium bromide which was then gently mixed with a pestleo The pellet waa pressed in the usual mannero Experimental and Results Plasma from ~:pa~~ed male sockeye salmon 9 taken at Cultus Lake 9 Bo Coo in November was processed to obtain the deproteinized plasmao Two portionsp each eguival~nt to 480 ml of plasma were takenr> one was treated with ~=glucuronidase \525 mg) and incubated at 37 C for 24 hourso The other portion was treated in an identical manner except that 45 mg of glucosaccharo=1:4~ lactone was addqd prior to the addition of' [3=glucuronidaseo The.ine<ubation mixtures were extracted with dichloromethane which was then washed with Oa05 N NaOH and Ool N acetic acid and the residue after removal of th:i solvent was partitioned bqtween hexane and?cif, aqueous meth.a.na~o The methanol=water phase was taken to dryness i~ a flash evaporator at room temperattira and the r~sidue suspended in acetone and filteredo The aceton~ soluble material wr~z c,:nromatographed on thoroughly washed Whatman Noo 1 filtii.l~

80 papiii<:r and Jw~ tu:.);;~'j:~-.t~cam ff!acj diov~j. i:p >,: w.th. a. HlVJ.~dO s:jlv!i>r.tt system for 16 b.r. '... ~~ 2) ~re~j t.~:j~~ --:.~ ~x: ""Hl:? - -~~-~~J-~-h:.:-rrlrox. 'lp~~:,:;~:~e: e. r.er(.t, e ru1d ll,:o.o1rut;.jt~s tvstero:tj.8 wilre J..ncl.u.ded. &.!0: refer uno:-~ ~ -::mda:rd :o:,. Ar;.d.t,p-c,..os.et (UV) --a.bsoybj.ng substance wi tb. <:h101 m.c:biut v of u':~' : :i~r-:'::ii! ;r,s.,:< <:;T :~ ;s:: : ::'-:-.:t::tf'gt"'d f::: Jm 'cr:.oa 1.ncubs.te prep9..1 ed wit:b.r.n,;:t -::h,.. B.dd t'l.~.- :' 'T.:, >Ot'.::-, t."jjs :..r. r,tlt? -- t.:tt :nn UV-abso:f"bing substance co.j.jd be :l ~"e:c" :d t ;.lit ~::; t; >vd:ich t'c,.,, ir..fc:j::: t.jr ~ra':'. add6da The GV ~ ab;:.:-r b~,:::1i; f..::.bsta.c.. ft );JJ t.:>s :.::1pex i:tnd GOLr:uoJ.':ed e. maxi!llulll. abnori} t ; o1~l.::,t 2~1()!llt!J :~ci_-~_;_\ ~': rd- ;.:.. r~~/,.~.f~(' xn}.., C _-p'a.a8tnn :.Yf ::-es~j-~. ter~:::-:je ':--alcnl.o.tro'd -: n tn!1 :;) o :n ~'4') a7;,a 260 u;.; and emp.i.. ;~yj11g.allen~ s corr ec tc.j;r; Tr.,; t'v!1 bs x b.:.rg ~;u:sta.nce was.::~cei;y L1ted and ('hroma.tographed ny desce.ndin.g :.., ~! /3C' :f ~Jr 2 tl-vll.t~so.truthenti~ :, ter:rt~)ste:r.or1e acet0t,;: and the TJV l:lb,so(l :~:v;;- E;:...t ::Hac:.:;:c.; LcoZ:illi :. c~~ pj..asma both had an R.f' of Oa80v The a: f'":v 1 -:n~c.:i rj\i.. a~,;s.::r',:.r_,,;-, :.=,,.. t<:.< '''.1.1E '' f'y,,~> th(~ p::..asma v.'iip :nh>:n po:ra.ted intz),.~, add eo. vaf:; ;, : r, ~tnu:jd f ( :~ a:n addj t.j..or:-91 t:est,)ste!ct~-9 CC"J1d. b,:;: d:) ;e: t.edo ~ 4't11 :.b no cnlub:i tol' ~a:::.::h1 i no 3:1.d. i t::l.ona1 q'ja.nt.::. ty of wrnch inbj_bj t:x haci t:.e:oe'i.~ 5% ac st'l' a:: l.d,c;.. f 'f''.,~."' i.l:o, s ~~c:. ~ - -~~rl'"'s{} ±:rntu -~:.b.e: p1.e.t::zna ~-:,art~p.ltsi to D~t E l:.lt:'.ng the A.mberlit.;,~400 (OH) column with.~: ~-~.- 1 b1 "i:; +.,_r lrif::ls w,a~~}:_t,:d "_,,ff ;~r:: t!.l -wa.-~~ S<~ 0 'I'be c'j{;, q\jf,.:",,..,.:, ;:.c'c ~v"j.'! e ',,atp m'' tak"'r' '"' dr{ll"'"''' on a fla8h ~, r,r--,.~~: '~',~~.. ~.; r: ~:.h.q.)' ~--~~ :: :;~ _u.~ ~~ : -~ ~:. ~-~~:~-~-;~-~~ --.. :~ : :.~... ~.)'" ~~ ~- 0 evap,rat.j, \~;::q.a:['.tj9.:' N.. Cu"' ".'."-e~t~ci'ot.t"l.b ~h.j... L_,_,,J...,.I..FJ.~I. er!,-..,.di. 20 Co The residue wa8 "ci=;k"<n lp.l\ '-' '1to<::J: and t:he ph adym :;(t t:, 4o5 w.".. tl;. sc c'uum hyd.roxtdt' 0 'rhs hyd:r'::> :..F p "..,.,'i Jr:i: HJ. tb!3~gluc,:.r:.n:;.li:ise was ~~epeate<l 'rhe sterojtd Yf.:~s:.d~IP. ~~ r~ i,;yt r:--,~/:;0. r.ry f~.i.i..::.~ n,ct.,.~.,n w ~j~tl1 m_?!th~,.jo.ne cl11c r.1.de:j -ia.~ ch.rtjlli.e!tc-- graphe i o:r.1 paper!i?> H~/ LC :c,~;-_:,e:;::y; :~ys"'e!i: f.';r h..-.jt<:1 0 The c:b..xcmatogram waej sprayed. w11;~;, 1.::1 Z 'm'll,:;1;:.;:.:'' :r eagen f. and a l:<jij.msrma.rl.n-"posi tive e-:.tbsta..."1cfj w1. th the chroma't:-gn=:j.;: ~ ' : m.)'b~.l..,y (f testos't8r=)!:.t? wa~! present from tb.e plasma sample" Testo.~ terou'!.. 8'l.Lpr.a.t8 (sod.j.um salt) (300 jlg) prepared as described by Holder:. e-r aj were 1lliL'l~l'sed :ln 400 ml of H20 and the ph was ad,ju:sted to 4" 5 with 1N H2S04,, Ac ~~;.at<' h:jff e:r at ph 4,,5 and. l1ersene,. a:~ w&ll as 440 mg of washed!3~-gl:j,.:u.r )nviase W<:Jc:'e added. an.d the preparation i.ncubated at 37 C for 48 bfc:}.rbo lt'y:.;,ed ster :,js +H're t'(jr t::r a '.ted ) tlm 'c'e' wi.th a. vo:ju.me of dichloro-~ met.harlo:>o T.he p:,o :ced d 1 ct1::.1:!':' methans:,:.;xt:r. a.c'ts were t::-eated S.H described for the lr:.:it.tj,on <jf t~st:~:-.st.e:u;r.f:l, tnt no Rte;r~)J:l wrth the cbrc~y.::lt,)gj:aph:i'"' m::, bility of l.,:-,.:;;"tostex<.ne couj_d t: 3 io::r:ter::.ted, The ac;''1.8yj,h phase wag &.dj\asted f.irst to ph 3a':l :,~~:.d then ph ll and ~:.. xts.a ~'ted E!ach (ucp vr' th n~butanclo Go :m.pounds were deter:to:;d 'th uv "C Vl.r. H :amp ajw i' 'OX1.\:~... c.!. ~.. <:.:; , ' f'' 0 ]J". ~., W..\ i.ug.. RT..,,._JL"',., I.e~...;.8c. "' (\ ~J." '""' 'h r. ) o ; s 0 d1't _, ID-~.. TweLre huwired g cd hjt:it;ef3 from.q,~~.~r ka ca1lgbt.:m. the ~pa.w:a:ing grounds at W&av"'-r G ~ t;t:. k, B. C... E. we.;: ;'J p:r-eserve:l '.n dry.l.ce at'l.d. :pub;rt=>.r - ized undf:!r 1_1Cfll;:J..d l"l~s-tl'l<.jgt ::"' :i.mrr~e d ~.att:> J)r:~..r:J.f, ~~,:~ e,o 1.. t.re~n.t.;xi~~l,actiotlo Th ~l powder \lias Lr":'a ted 1..:1 ttl 4 vo.lumea of 9"ifo ethano 1 a.t 4 C rtnd s.llaken. f<: r '? days after ~ h1.cn t;.he eh;s;.no) we.::: '1'":'-t:nve?!'!d t. y ce.:qtjl'ifug:tng ~.t ]860xgo 'llli..s ~~8:1 r'e p~.a ~-:Ei.i ttu t~l, r, ~.Ir1E~~4 :tnd t~~.~7~ t~.~r:.h:rnoi e~:t:'~'~act,r~ {t~j"sre l"ty01.edc-

81 Th';' p::r:jte~;r; -re.:iid.'::~g< (fraction 11 A 10 ) (:protei..."'l.=bound steroids) was reco.n,h1ltut:ed!o l2()() ml nth wa ;[;er 9 f,jtt.e ph wa<:~ adjut1ted to. 4o5 and lo5 g of [3=gl'J('i!lXOnidas~ addeda After 48 hc,j:rs in.cubati.on at 37 C the steroids were ext:r~h:oted 0 p:;~or.ess.<:ld and crrromatr: graphed as previously describedo No steroid with ttl ruobil.i t;y.~,f testrj8"t:e:r o:.!e could be deteded by UV light or Zimmermann sp:ra;yr :following ch.ron,mtogrbpt.cy with the HM=OO solvent system, The 95% eth.!:ln.ol extra::. r, r;as erwaporated in. ~ YQ. while the receiving flask was kbpt at "he tempercature of dry ice and ac:etoneo The residue was dissolved in 1500 ml wf water and ext,;: ac;ted three times with equal volumes of dichloromethaneo The C!".' rnbin!'. exg.rac1t (fjraction "B") (~~tfree" steroids) was -washed wtth Oo03 N NaOH and Oo I N W0<8'b.t' ae:id and thfl :r.es.idue after removal of solvent was pa:d:l t:or:.ed be,tweex'. hexrne and 7C'f/6 aqueous met;b.a.uol (this last P,.,, 't'' ('~~ "'l7'e' '~ ~ ~d-l,~ -... Iii'"' ' n~c.: """,)~>;::_. 11'''"'1'.'l''a+,~ 1.-~'\4. 1.., ;, """'" -, ~ - :.,,.\j 0 m i~.:> J..-.~ "" "" >~q " A... u;,..,.v\!-4.,.. ""'' m.e.!.:'l nnol.u.l: l..lj.c;l.u.... t'"'''"''~'-~r n A.~vi!.J.:&.w wo ' {~ ~ ak"'"' \11 ~.A. t~' ~... dryness at.. r:oom temr~<:1tnb1re in a flas!"j. t!f:;t'j'n ator and the :l:'esidue suspended in uceton6 an.-;. f1.it a:cnd, Th.& a.:.etone R Jluble mate::<1al -was chromatographed as twc pa.rallel s}:k;t~ re-presant.>-dg tcf,jt: t:md 90% )f +.:he ';;c>taj along w:i.th authentic testosterone ab a stnnda:rr:i.o N. :J mr~a.bsorb.ing S!lbstan.ce with th~ mobility of testosterone could bs d<::t,a:j:ted 1:r1 either the lo% r.r:: 9Cffo pc~rtionso Th~ area of the g(j}b port::u::n c.c:ct~'>~pofjd::.:ng.m m~bl.l:j. t;y tv, testosterone was c~.it out and eluted wtth meth:3.:r.tol f'1 :t :f,;=t;i,.ei:r sj~udyo The rest o the papsx was spra~yed with the Z.1..mme:rmmm reagent but notbj.ng was detecteda Th.;; ph of the a.qtl.eoua residue (fraction "Cw) (conjugated steroids) WM adj.ust<ed to 4o5 with :'./J N Sl"'lphuri~ acid, [3=glucuronidase was added 9 and the Ft5.. xture inc\!bated. a.t '3'':''0 ru1d extracted after 48 hours with dichloromethane and the extract +rea:tli'i E<s dc;;scr:tbed for fraction "B 11 o Chromatography.in the HMd30 sohrent system fn: 12 hours produced a DV -absorbing area. wl.th the mobility of tesh.l \te!o!wu Tbt< uv.~abs..:n:b.lng substa.rwe from fraction ncn was eluted from the paper and "''xr;.:uy:i t,ed. a ma:ximmd. abso!jlt.ion at 240 liljj. equivalent to 39 lj.g (Jf testosterone p.:~:r :;~00 g of r.estes calculated on the Oa Do reading at 220fl 240 and 260 II and o;;mp.i)y;;.ng AilenQB :;(;r.rec.tiono The elua.tes of the chromatograms,jf fraction "B' 1 a.ll:cd f.ra.cttrm '''C 11 corresponding to testosterone were concan= tzated under n:::c t:rngen 9 the.!' esid11es arc ;etylated with acetic anhydride=pyridinep chromatographed for 2 hours with the HM-80 solvent systema A UV-absorb.ing substance with the chromatographic mobility of testosterone acetate was detected in fraction "C" (conjugated) but not in fraction. "B" (freel Testos= terone acetate and tht"! conj:\agated steroid from testes were eluted from the paper with methanol and the i:nfra.red spectra determin~do The spe:ctra were identical with each ether and with the published spectra of teatosteron, acetate prepared from salmon plasmao The remainder of the chromatogram was sprayed with the Zimmermann reagent and no add1tio:na.1 substances vere detecteda The potac: slum bromide< pellets of testosterone acetate ru1d the acety= lated testo;s stercid were :Jlli!l~rsed :Ul H20 and the steroids ext:;::oacted wtth dichloromethane" The flolyt:mt was evaporated under N" the residues deacetylated and then oxidi-zed with chromic; oxido:;" 'fhe oxidized steroids were chromato~ graphed along w:i th atj.drost ~-4" ez:.e-j,,.r;~d::j:jne on Whatman No. 1 paper in the HM-00 sol vent system. foz: 2 hou.:rs, and E~ach produced a single UV=absorlling Z:unmermanrt.- pc\:~'3l.t.-:lve substance with a~?, Rf value of Oo3. (13o6 em;, 36.5 cml

82 Tl110re wer ii ~:So yg A' tef<t.t)e<'' ervn e r-eleased by P ~glucuronidas~ from 100 ml of ma:;.el s Jckey~ ~a:i.mox; I.. 6\sic.a. t:as~u. vn th>$ :: ecove:r;y :1f authen~,ic testostermu:l through tho 1211t}.re L:.>::~::a:i:iQr< pr.,~--:~gdu~re o In. a. p::;evicmn sh.<.dy s:pe xwd female; sdr;ln-;y~:. sa.l.cw:r;;; ~&lcen ;a;<; f:he cihllle \:J.li),~,,, con r:ail:l~d '7. 6 jig of testoste:r.on.e ra1~ast:'j P" gi..::/:j}xon~;das il f: c;l: loti ru:~ )f plasma. Thli\ fre(3 testos~erone conte:;.'i::,,f, tl:rj!i> male a:nd :fi<;j:ma.:ue. p:.r r:1.ma" xe9pec~:.ved.yp was lo7 and 'io8 pg/100 mlu rhe t 1 t::11 t;~s:,:>.:;;-~j.x:l~ ccn<,ertt A (t...e plasma ia therefore siin.;..j.a:r -;'cii t;h~:~ EH:lxes bnt t!j,tjo hcrrrl::p~> :1.s pres.~~r.t t,) a nf!.1(~h grtilatgr 111xtent in the conjuge.t;;;d fom u1 the m.aleu 'J1h.., p7'~l8gnt ert;;:.dy d)ii!s not E~~tablish the position thrj,ugh which the c:jt:j'.ghbon occ ;us., Prt# ;; 1ous J.nvastigat'K.ons i.n which injected ts<stoe'te:ron~ tj.as b"'sk!. :l''s i:o.;jvfiorad i;:l. the '!.\$!'Old {'.onjugat~ fraction cf ar.ir1e :-;f )t.h-~~~;~ i:.p~;:. : _, ;~_'::~ } _,=.. ;; ~-- f~.k~.{yr,t-'. : n.j :&TI. trl:;s~ ~~11s -~:a.n.rjtls ch.e conj'u.gb.. ~ ion i.s through tn~t~ 17 f)--.nyd:.r o::xj,' g:r).:p ~.<:t -het,~han t.h:r.ough th~ th«~oretica.lly possi.blw 3-~e:nol f:,rm Qf t.'.\o;,i',>'l. ::S. -Ae:;on<6< 0 In t11e pi 8t'~ ~ n:: 1..\b. i~,y ~-iii<jt::. it;e:r,::.ne J.B,::~lso sh :ti'm. to be a major androgfin ir"< thliil gl ~CUY:)t:cJ.de--::-: nj'>)-t';l'l.il;!!!d f:i:~lct:.on,,f the t~u;:te~ t)f the migrating sockeye sa1mi\ Ylu An androg,..r.r:.. ~" a. 'l;j_v:;, ~~.v wa:5 at;tr ibuted +;o <W1Ct:t acts of toleost f:l.sh test<:~s Rom time ag-,~, a.. hc.~ngh :~'::c& c.lww.l.s. n2.ture o:f th'il ht:~rmona was r.tot knoli:oo Hazelton and Go cjdc:;..c;h.,~;:;.~. ~.''\~'i :t;: ;, : an a2: ::b~<-ben:<hme ext:.r rwt Gf the test~?.s of Pac.ific ;)alm{.;l:i pr-c<1~.<1~ > i \.omn g.< -wtl~ i.u -~a.p :.cd.f3 0 Pr1t;t<t.r and II ; ar demnnst.ratild by chick b i(jae'sa;r tha:;; a l::eir~:rtvstei onfi - \:7.k<i> r,ct, ':-9r :,sd ~;c u ld bq< obta:tn.'!i!d fx"oro. the tes"~.es of chu.m s~i r,,f, islji._t~l'..:!) Dv U.J.( 7.(,;;_~ - $'itun exi:: act:..tdl!iqu.hai.emt to ~3 ~ of test<jstr.;;r or..'iil pr:-'i' Fxt:;, ' ~ P"'".K.:).. J ::,gn:.m "if Testli'ls taken from B]J~r~i~11ng fish.,!dle:r' and 'I'suy.;J.<::" s: : '"';.d. ax;.::.\: ~~'""'"' :!..r:, :~ne a.ndrogeru: ac ti v:l. ty of theil tas ty:3 rj' -- k y '' ~ \' ('..,.JJ. 8<.,C 8!,"" c 'laj.lflo,.. _ -!;;'::~:;;.;L;,J1~ ;:;,:;;l~;,-~:;:c~~-~;;:~: t, "'"'"''' ' -.,. k - \ pa_.,.. d -.1.~~-l..Lug 'i. '~: ' t:t is...,,:a.crll?ie!.,)iil. ~I "--- t B ~.""'a... u.~. matu.::ltyo Thus!;;:;;:, "" :Pliht1<1nt c:,:f!i.e. t.lg twsbstwt ':lfhio pr)p;.~.:na,t~ /kg 'GG:Hh~d i!\!18 f o'.'"'d,.~t.~j.. n~r: ~;~,,_ t'" ~I.J>.~< \..!..'I.;.:)C,.,. ~.,,-,., _..p' ":J.:. -...,.,,...,,., p"=- "''"'. i t:"'" 7'l..,.w:n~ng rn~g"a"' "on 'lo6 tw/t..,r,,i, :,;rj~,_)j., ~ "- '' ~ ' ~... _..o.q,y""-,. '\-....&..~,,').1""..._..,, = J.,....J.. \,,.._, o,.-oj.l"'5 """ ~ Km '!4J>-".r 1tEl1 an.j. 1-l{!, a~:: t~:.e o'p!i.w:t!.':.iig g;-:ot:.nd~:v It ls d.i.ffic.j.jt to eva:~uata jl.. ~st how much of th!i arj.d:c,gen~c a.ctivi ty c, th<? t~sr;es c-juld l.,., al:t.r::~.but~~<d ~.o th ~ 't!!lsr.osts;ron~ glucurontd~ a.e w'" k:r~ow of no A.'ail!.th~~ e c nl.\ioil's.ton factc:r to pi!y'(ll~ G::>W"fH'Si.on :,f its act:ivi ty tc- tho;. comb g:r: owth acb::r:j..ty of c.!if:;:;>:cs ;;;"':ron~p pjt :>,ptci:lare How~vlllr,,:f t!rle iffwct of w":.itlirific:at~lou by C;yc.J.c.p~n tylcar box.yu(~ a<..j. ds on '\tqstos-,r,fol:r.:;.n~ ~ s ar1 ind.1cat:or: ;;f thfil diig:rlilw of a(;'t.tvati:m 1ii1Xpii1;;tfid of ~J:~, glur~urontda,. s1xty".m~ p.g p-w:::- kg : L t,e,:,\'.gs of r.::mjugs.tlild tlilstostfirone :.alculat~wd from th\i! rwcovw::ry cf a.uthlimtl.c tlistost~::c:c""' throv,gh thlil proc.wdt..\rli ag:rees Wi!ll with l;ll,. qt'u.ntl tat.lv'ii androgillnic act:t~ti ty,:.;f fish a.t about th.- sar.uil stage of mjg:ratioj~l ( ~33 ~ per k.g),'.:!.nd wij..i S!il.iom to fjccowi.c f<:r most of thif achv1.tyo

83 SUMMARY NOR 18 STEROID HORMONES~ SEARCH FOR STEROID HORMONES IN CORPUSCLES OF STANNIUS AND ISOLATION OF CHOLESTEROL G LUCl 1 RONIDE Dov Grajcer Do R, Idler The ;;:j:rpjs.l~fl,::, St!lilJ.i.i'J.s 3:re located at tb.e poster.i;)j'." end of the cod k:1dney c:lr )se <;.:; ib.e f1.:rrs't he;;:xal arch; thei:t: s~ze" number and exact loca.tion are..lif.iz.iable and th.eb func:tton is.t.n. doubt'~ a1th,)ugh adrenal.cort:.cal as &i'el1 as.mt:ll2.j'.ox:,..:h~.ct b.bmr)logy ha ;re beer~. S\J.ggested 0 Racently the absence of adrenalcortj.. ~ :.s-'cfn'fji.de) w.::r ( 1:.,-se:r.ved. i;:: J.~u~pr flounder a:c.d Pacif::..c p:~nk salmon, wt1.~ e. i;ha y,::~:;}:;; t~.l :1.L ~~,~;.;x; t,(<:-p~;~;:~"., J.\.:.f~ ~ f ~p.:i.n:v~ sa la~ W&J."l)e.,:.. :.n tradi.,:;t~r:y > 'l'his :;,tudy ~Kt\5 ll~dgtrta\(en :in order' to de"<;errrd.ne the nature of ti"y;, si:eroid h :'!'fe )F!. 28 1:. t~j:pt. 'Jf th.i!':! endo,:;rine (h:istc::.>.l::gi("a.l evidence) bcdyo Mixed ma:.:.e c>.i.'l.d fld'ma:~.ei cod <.:':,rpusc~les cf Starm:b.is (?8 gl which were collected and f':rozen at Camprr:;i'Jell~; leland :i.n.ma;,y 9 195'3" by Dr" Grace Eu Pickfo::: do were made a'trai.l.~bl.e t0 tl.bo A almilar tf6ight Of kici.ney e;olle(;ted a+: the same p::ol~' E<. and Uilder the f:'bjn:;, c;m.d~~ r:ion ;<,, was '.lsed e.~~ a. Cl:.:ntrolo A systerr;.~:r: il' S'c&.:r\;b fc<r the C:( 1 rtic<0steroids 9 "conjugated" and "free 1 ',, Hs W?l.J. as a.ndrrjgen"l a:c.t.l r,.,,ij:;e :~: the oes crogens gave negative :results~ but in th( cou:rge r;,f investigat:\0;;. ch.::<lesir:;e.:..~'ol. was found.in the glu,c.uronide fxacti.ono Pre1:.minazy dal;a su.ggast ttie!. t the glucuronide form of :rholes't~rol in the c-:.lrpueh.;les r)f Stanm.. ;:B const:ttu.ted ~~'Yer 5o% of the total cholesterol of that G.rgan wheres.s i::h,,1 -r&tl:; :tn the k:ui."'ley vas very mui;h smallero Cod c~ lrp,.._w,~..:ls of Stanrnus, 78 g, were ground in a Waring blender WJ th 2 }5 vo..'..u:mes of 95% ethr3xj)1 and f>xtx'acted for an additional 3 hours at 4 C on a rotary shakex, Tt.e protein precipitate was centrifuged out at 9000 x g for 40 minutesv Ttn.s ptocedure was :repeated with 7Cffo aqueous ethanol and. the eh:ates wel"f..' c.ombined. L::.. te:r ed through glass woolv and concentrated in a flash~evaporat;or w.ith the ai.d ojt"' an acet.:.ne~dry :ice chilled rec:eivera 'rheo res:id'.u: was rec(jnsht:rted to, 300 ml w1. th water (fractl.on "A") and then e:;d::raci::ed 2h. w tth t~thyl acetateo The ethyl acetate ext:rac ~ (fraction "B") was combimd 0 washed w~th O.,o:m NaOH water 0 acetic: acid and again wi.th watera F:rse;ti;;n "B" Yif&/3 concentrated (dr~y ice and acetone:) and the residue parti tloned betwee:~',.,,;'f}b nq ue ~ UB methanol and hexane u Th<:'< 7Gfo,e.cpJ.e'c''1S metha.nol phase was concentra-ted an.d trj.<e' res:ld'..;.e e appl d ('> ') 114 _,_,... ;.1''" " ).'-.,, ~ T.J1... -!-.. l.e "'3 B ""~ i ~..c..~ 1 :xt... i l JLp, -r;,o ct nua.,.. <layl Noo l paper :t>:jr' Cfll'omatog.raphy in hexane~;& aqueous mettjb.r2o:. sc.~:~..vent systemo Testosteron.e, C C rtj.soned cortis~l N t, r"(;l""'<>w~, ~"JcA<': (uv j O'b<'<('J'b'l"IJJ '~ J.l... 'ti~!:"j..'(j.,..li..u_,.,_ li.a \~ }...,. ~---., '""'-..."'C mater:i.al was de tee it:.:: d. due to 1:h,,38.1Jlp1.H usedu With hexane-sv% aquec ::::* mgtt..a.zw_~ solvent.syst:em t:te f!ta:ndard ster<y'..de us~:;1 do nett al;l. separatea 'rherefo:::--e the chroruatog-.r am wns tt:c en~.;c.~.ura ti:je -l::olu(jl'v'3~7<$ aqueous methan,::d. Bt:Jhrent system,~ ths phase> WB.fl 1'11:-i.ceweai t) r&ach the bottzc:;m of the paper and a better

84 separa.tion of tlvs ;s-r;e.:..'lda:rds v;ae sffecteda Howe:wer., no IJV absorb1.ng a1'ea could bos ietected from the sample usedo The cljj:'.c!.f!b.bg:r'am Ka~ Ci.ivided :inf,.,; three areas co:rresponding Yo c )rtiscj1 9.:;.21rtiso:ne.;md c,n'tic.ostf!,rone: e,~:tch area waa el'lted with metha.n.)l" 'rhe- eluates w,e~:re con.::entrated 1.m.der a ~s:tream :}f.yjcj. c:::.;g~n and tb.er;, part:i tio.ne1 between benzene and watero The aq 1,:0u9 phases were ext;;, l.:lct;ed \V'ith ::U.c:h.lo.eomoth.ane and rc<~h:corr!b.tl:-g;:aph.ed in. t:.i.lj; r.c.:j.u m~~ -70% meth.mol solvent.systam as sepa:r.'a.te ::~pc, L:;o The ~:>c>l'\7r;:ynt front ~t<'t~"i again. :~1:-..owed to :reach bottom nf paper 0 N. uv a'bsdj:rb:j.r.<g mtl'>'1rl.ald z~;.~~2'iml.r..n pos:u::t ve c: 8 blue tetxaz ol'l:u:m pc'~s'l r.j r e rea:a',e-ria:", wac.; j,,;~ec:tt"<:io Ccmch:a~.iu!];.~ No ~~free"' fl..:ln~nalcoj:<t:ie.os ~erc:_d::: W>':...l~-:, de 'i;t<;:;;;tw1 in tbl~ corpuscles of Starm.iuB o FractJ'On ' 1 A 11 (aqne ~u8 :;:,r:,.:t;ut)n;. the ph adjusted. k 4o:i w \. tb :N su!.plrur:'i ~, ac'ld 9 a.c:eiate buffer and. T'eJ:sane add,t~d 0 ~S.Ec1 tl~&ated WJ. th ~~glucu:ron.;;.r.l.13,f>e BJi<':l incubated for 48 hours at 37 C.. S~saquently thl.s Ban.pl'"'' w.af:'.;,;rb;acted ')x \~~ th dic:hlor omethane 0 those exttacts were pooled,, wa~hed ~tnth QJ)5N s :-dim:;;. hyd:'r. li.. ~d~, water 0 acetic aci.d a..'1d again wt<-1\:jo!l, then i~ ;.ncent:r:ated (J.:: y ica and Cl.(eto:rk! The residue was :parti ti..:med b?r':ween bea! Ze:Ufs an,d wa :e.r o ':flh.oe \~Ya t8z pb.af'"' war extracted with d:tch.1,0romethane which. tt?. t.u:ru, was ccmc~?r. ':;l"fl.ted Hlld chr~jma t;o~ graphed 1.n the tcluan,;.~'::c'% aque(nls m~thanol. solvent system" No u"v absorbir:.g e:;r blue tetrazol1um pos~. tive ma.ter1..al ~as dets:yi;;edo The less pc.lar compounds in the benzene J:.h.ase w.:pre colulll!l c.hrmns:to= e,tamed (Al203 activity 3) and tabes 26A3 as ~rel~ a.s 44-~55 and 56 6.Ci. were poo1ed with methanol and dtchl.oromethaoe 0 Each group wa.e; G ij;n,;~~;~a tra '~ed 1.mdf'J: n:l c;:"ogen and each residue chromatogz arned as a spot ('\G WhatJLan No" l paper J.n the toluene~?~ aqu~ous methanol solvent system fc r 4 h'mrs 0 Testosterone~ l7a-.. hydrox:y proge:stel:,;.n::.9 a:n.d a.nd:r ;;,ate ron.~ were ch.l'oma tog:.~amf;d. t~onc~ur:rent.ly as standards" No UV absttrbj.. o.g :>Y: Zimm~r~tHX'in pc.sj..t:i.:;;;:e!illi'te.ria.l ccu}d be dstecte :io.con 1Us'J..:m.~ N0 ftee IJ! g1ll.:;1l!':m.::..de. cc:.n;)l,gated adrenalcc;r tiu;.l 17~ketosteroids w>:re det:e:~od :'lj~\ ';he c:rpnsc.les of Starmiuso 'rhe aquelo"as phase was made 2N with respect to sulphu:...~1(; a.::-:i.d P...CJ.d solvdlysis carried :Jut on an ethyl a,;,etate extract in the usual mtt.ni1 l" 0 1''il.e il;jxtji."act was washed consecut:'1vely with N sddium l'l,vdi.nxi.de, wate:t"n di:.\lt<:i acet:lc: acid and fi.nally wa.<~ero The tut:;;; act was c-::mcent:rated and the restdu"i'. partitioned between 7Cffo A.queous metha..rv :, and hexane; 0 'El1e mr1thanc: '-~~~" p;:,rt:i.on was concentx ated and ch:romatogramed em Wbatman Noo l fk:llk~!' tn ";;h8 toh.:.pne.~"cjfo aquecus methanol solvent; system f0r 9 hours" Allop:r S<gtiS.."lec dioi ~.3p J.l, 20~trione 11 cortisol and c,::::.~tisone were 1 clsed as standa::cd.~, N,.) UV s.bsorb.i.ng \;:r Zimmermann poai ti ve material could (.,;:: d~th.'\c i;e:d from the c.':l'imple, Conclusioq,~ N,) app;;:e:.:~la.ble amount o:f sulphai: ~ c;::.~1.ycgated adrenhl~ cortical or l7~ketost>'3roids were detg;.~tad in the corp''l.sc.les of i;;;ti'i.!ln:lus" The Oo05N sodium :hydrox:j.de wash of the g1ucur:n:'.de f:ract.ir.'!:! fxcn:; th.: corpuscles was neutralized and then ext:racted 3x with d:i.chl.:.:romethan.e Hn:.i another 3x with, ethyl acetate o Each extract was washed twj ce iv i th sod~my1.

85 b)j;a:rbonat'e (~.;%)" 'J'he it;;hi,:;.xom.e::tlane &:(Jd ethyl acetat& were1 combined a.vl.d ' i ) con-"... ~'="--~ ntratr.> i,!' d, ;,:,j. :.c~:: ' f\xl.:i ii 2E1Dne,. v (lesi:rogen estimation ii'bs tried on the reu:ldue 9 \;2.lov;ing a m:odifir:atton r~f t.h O< K:.~be!' f' reaction, The samples were read. 2 c 4t30, :;.:5 ar.d : ID.j.' and (;he:n:- ~ as no Kober p:;s:::.tiv.:;, present in the sample, Q<,;:ncJL~~~~!l?L. te;a"t,; :.g,.";;n wa~:; -~::e!:,:::; red al t;hough the) pr'j>i;;;ed.z.re would 1;; )~'; ha"i!." d~:':e<;;,ted a:j:" :. ;: :TA?;;.:::.::::.. r:n.,., 9~:i% E.n'OH <: xtx ~L t the : c :C'Jrc!~>C.J: s.c;: Stoa1m.i J.8 wars m;.bdhrided. :mto three pad:. tc:: h.:.:: his fx se stero::...de" ~-glueu.ronidas.r: '1ydro:i.ysad frac.tion,. and. 8" cr:.s :~:.d l' fz ae ~io!l, l:c. fj'i'l.gh!c;8f:.h-':j t;h~~ bti':ct:rldf! w.ere fieparbt6d fi:"ou, ~,h0' ~A;'l :. p.:la.~ &~;:etc~.is by the 1L"'f/o ::o,r.rue,;;-~!e' mt:d;hanol..~ h0:xam.' parti.t::.. on" A1:; th:,.~? uot tho:' J:i::;ran~< rm.-;.f e<tlt:"h ~t'at:; r..r n;::ent~~ated" 'f~hij residu.e :i:t;~.:~ol\; :::,:1 ~z:, Wb"fDl 8t.~~a.nc : ar:.,i rb.l3; c-:t ' rolf; a.ll<'r.~: ed :~o ~ry:s.talj.~ s.e ~_c'.t1t~.i.n the ~:,o ~.d" Whtt& ;,ty6t.9ll:..rj?< ;ci:'lei:.ss pr.;:.~;:q:rt~~attd fl in ':lle ~ r:r e.?l'f :t'.&."a.c,;:~ -n d/. well fw thtj. glil{, J.r~:,>u._,)n]'.-.;<' l ci:i f.r;cv T!M ma1;.erla:l.. w f;,e cy1~le( t.t.'"i,. dt"ieb. ill~'.d we::gb.e }u!fh.~ s,t_i~r.: > :/_eld.6 f~ -~~j~f:..::;, Gttl. ~G(: 1 ;: v 8,.r"1d tugl1lt/uxt.<tri 1 ~." CO!l.J'~~;.gEitt?d. 11 v\j 00::3;)g0 N~~~ st~~-~> ~ ~,:-\:_:.~<.i n~ :r ::t\::~~~.~s~~j. fj t :~lli ''(t~~? ~ 11.J")..lpr..._qt\8 cj>ljj\:~ gate11 f:ractiu;;v at 147' "'~;oc (hjjjhi_::,tj.t,2 -~-.. tt.r~l~:-~.. t~;.~ me-l t :.!~e; ro.in.t 't t~r.=:.\ :J~~~2'e J ~Le::.:. chole~:"<;(-:>!:t l, rl"' "",1. '"~.;;." ~ tne Il': e;::n2!a. wt ::h ~gtncu:nmidase both ruel':ed. K<:f lex ~t:icrc hot stag~)" Nec:l. tbs:r.',:;:,.:tsimli? ~.:1.2; m:;..xei w:d;h e:.;,_ttiern:1;:: An a. d.:<.t,;; ',cc;;.;, ]::-: sps.: s-d '::y rtt:<rl::'.;.1g ~.0 mg of ea::h sterol with 2: ml Of aceti,; ~:u:j:...vc:z ~ ic:.. e fc,,'is :f ir y pyx idineu and 2.J:.cu.bati.ng lywer.r.ight at 37' C" The ac<:yoate wa;; prt;.:;;.. p:>':a:,g&d the addition <'f' wa :~er a.yj.j. filteredo The prf,;;cipi ta t B wa~~ :r l..l.:".?;~e.d frr.~m fit~b..anol" The ~l;wo '"unknown.s" and choleste:ryl ac.str!ti::' :;.:.. - na. I!:~1 :1 aj, \?f: C o A modified. Liab.91."''llaf!.n-Bu:rchard re8a:otion showed that the ~unknown."'" produced a chromogel at a rate :-,.dentical "ir!ith cholestero::'..o CQ!""!QJLO?_i.,:>Jt;_; C':h,.:<l,tst"~:::'ol ccnjugatej. as a water-soluble glucuronid.s aceoum;:o; f n a sign.ifi.;:;ard; pc:.rtion rjf the tc.taj'. illlesterified ch,:jlesterol in the corpt:.scles of S~&.r..n:m~:>o A com.para.t1.~< a '! t:u::i:,",,f :;,-,d. kt±w:y :Cholesterol wnf; inl t.l~1ted 9 us trjg 70 g.of ki.dl:.ey and. fu:n:.wu1.g t:::; : saah? proce,dm'e a9 with the co:r P'..lscles of Stanniusv ThEBe r&jr.psximel;.te W>?-~'6 twt fill:ts.hed at this stage., but so far they seem to au'u.stant2at-a t:he prasenc.e of a ::ho:j e.~::i'i:erol glu.(i\.u'iyo.lde c;:onjuga'te although in case of the kidney th'9 C C.ll,Jugated r:'.hol ;.ste:r o:rt. 2.:.c-ccunts only fo:r a small fract:ion tjf the t.otal,::hol(::'ift"i;e;~x,l" A 1aJt:ge qua.r.::l;i":y :J f a wates' so't.uble fo:rm of cho:lesteroi S8ti:llS to be pree.ent in ~he c.:or:p'z'l.s::] ~ '" r)f Sta:rmi u-7, ~rr..i&'1. C.i!'lmpound st.:em~ to be a glu':tu"o:m~'- :18

86 conjugate~ and to our kn.owj~edgq thi:<:. io& ~:!1e first repor1; <O f tht1 e::xls tt:m:j.\<j: of thi 1 c'omprnmd in all ani.::r.9.1 br;,dy 0 Frarther inq\liry into th6 conftgu:;:at; :12"1. of thil c.omf <Jlt;::. i e.::~d ~ t, occurrence in "ra:r-:ious organs :'.s propos"d" Th"" :;;: s<'?.'i..tltf! m3,y <;:;;gg(~,at a fulj. th)n.for th.e e>rpu.sc:ie8 :Jf Sta;;:urv.1, a.wi a:::>u!ii.b.ka a _-,;::,t::::'}jutiu:r~ h.1 thro~ :~.r~;nratl problem cf cholestercl scjub-:.,h.7at:j\.. n a111j!.l~spere:t-~;r., Ui pb,y-:;<.ol Jgj -::;a.l syc:"'t~m.:-1 Ui h!i'alth and d:tseaseo STEROID HORMONES~ ISOLATION AND IDENTIFICATION FROM A'J.llk'lTIC SALMON ( ~--... S.AIMC SAl.!i~l:fl ~,'"""_. II" Freeman Berfi. rrri4sc:!)tt Do Ro Idler A large volum~.. :Jf :mal~ :<?.:Jed f&ma:a.~ r:<.as.:na ba::! b".i:en C )llefted fr J8 spawned Atlantic salmon primar:tj.l fer th!:' p-:ll'posta c.;;' ad.sw /<J.: U''..g i-;r~~rc. R.p~ci.L- ~ questionso lo Was the I:'Ub~rt-2<'1.:ii> w~.th ;.!;.;;. r})rrj:r.::.a.t ;g:cav~, :: rucb:i : t;t ~:f 11-~ketotestosterone pr&v!lou.sly :i.s olated f:rom th~ t.:~.c-vd. ;;f Gpaw:cu;;d me le A1:1aat:i ;-, salmon identie;-;al w:hh t:!tts ro, ze:;nnd? (V~:t.J'!.:;ou\?er 1'iD1'2.hno::...ogi;al Stst:cor An 1 :>.-:...H ~ R~pcrt for 1961~~62, S1J.iJillJ?.ry Noo 14) v 2 u F,CJxlta:iJo.~ ijr!d ~~{i R c~ ::=:~~-, 1 )r.k~rf:..t.d, ::: 9~~.4 ;>.>,ar-:' ~~ed...,,~j.t H ;::.l'"j.e:m.i ~~~al determ.j.nat:i'.:jn of tb.t> t<jtal ' - :b.yirmr::r::>!zt;.:;..c ;:;,:: Br.::.:7~-d.e, ix' x J..as:a..a. i; 8Illples tr: kii::'l from ~pawned ma]:.e an.d fiirrjaj.a SaJ.mv sala:r "l.nd es :ime.ted. -~ha.-:: thtrt'" ~,..<:,, dp~:r "!C\ mately 30 ~J.g/100 ml of ~~~~n=-al.entso In an<y~hex p;,;;;,;:.md. samp 'e tak-;,n from spawn~d fish!jf tmstat'itd s<rjr Yhey f'>~w.d appr:j,xj.iilb te :.:'' }(\_; f..ig,h.()o m::. r.;-:' :Qte :rtjl:sol ~qu.::tvalents ' Jn, the ba.sl :: ct' a roat lili'~r g2yc,:,ge:: d~-posit t:c>r ;;s~2...;:, Adrenal -c:;,!ri,tctjstez-o:td~l wh:i.,~t" ~1.re.l1~ ):tygenatil\i and :u;, aod;f,..-;} 1.>li P':l ses:.> t;)-.,,,a4-3keto and the ct~keto1 grcu:p:::cng at, C matntaill hf>!l i':t. ad:r~e;,1.r< 1 j;;.:t.: i~' Jt.z<id animal,s and :Ul add~ tic-n brb.g abuu.t tha.iep:;:;s it~.on f ::.: vet gly.;;.l)gwn i; m proteino lle~ketotestosterone :has alr ady 1:een test~d and shown tj havli' no liver glycogen deposition a~ti.v1 ty in a zat b:ioa:ssay ar:d l:erh~e trr:~s st:e:::-r:,~"-.1 cannot explain th~ discrepancy bi~tw Qen the :ehem:u:.al ai<'lld b t<:f.ibsei.ji d.~tarm.uj.c ations c;arried out on thri p.lasroa!q:f almo~lar-;"_., The chli.tji.j..ca:i. and bi.ologi ~a.l aesays will be ~arried,au-;: on the sam., bl'itt'h )f plas:m.dl since W9 now know that plasma leyiiills of adrenal cort:.v;;osterotds an; derpe:n.d.ent.::~n Sfflr, ~r.xnal matul\ ty Bn1 other factorso If' thine ie: still a d.l.sc"7"wpancy ;.,~'). t.tlgi :h~m:i.cal artd btological assay an i<xplanati.or. will b~ su.;.gh ~o If t:herf:o l.b nn rhscr~pa:ncy then the qugstion will ha.vq been l'esolvedo Salmon were caught i.u tr-aps in tbe Mi:tarrEchi lt,_voar. N" Bo,, du:r::lng th!fl month of October and werli h.ii!ld in ll'lfloats" by staff of the Depa:.::tmi>nt of Fisheries at South Esk 1 N.. Bo unt:,.l N<::'i!embe:~, 3o 1962,, when they were al'ti= ficially sr..awn~do On Nov.rmber 6p 1962,, fish W"i!Xf'.f "':ap:ped )!l thl'# head,, ffi'ld bled by severing the ca>.1dal a:t't~ry,, ta.ktng tb.e pret,llnrt:tons ne0!01ssa:r-y -~:: > prevent the collection of &xc:retao 'fhio blc od was hersrill:i:z$i ~\a::j."t':t':cfuggd., and 'thii pl.asma racove:l:"ed and f:r-nzen imm~di.a-r;611y Tn dz;y ir;~o It was ~torwd at th'e temp~raturll> untl.l ext:ractedv Male placma. (2315 m1) ar;.d femal.e p~\af!ma (2<'.32 ml) wa8

87 65 obtained from 76 and?5 fish 9 respectivelyo The male plasma was extracted with dichloro~ethane (stabilided with I% methanoll The ext::ract yas flash evaporated immediately and 6tc:red :in methanol at =12 C. The solvent was removed L11 vacu.o and the Tesidue partitioned between 7rffo aqueous metha.."lol and n~hex:ane" The=;;;Idt::e f:r'cid the?cf/o me'lthanol fraction was stored at =12 C in methanolo The dichlorcmethane extracted plasma wa,i, the;:~ extx-a.cted with ethyjt acetate in order to remove very P'.Jlar steroidsa The ot:b,yl a~;~tate extract: was removed in vacuo at 40 C 9 and tr.e residue stored for further investigation at =12 C in 7~hanol., In due course the methanol was removed with a flash evaporator and the residue par-titioned be"'tween benze:ce and. watero The very polar steroids were then extracted from the water phn.se with ethyl a.<::ertats which was then removed in~ at 40 C and the rasidue stored at "~12~C i.n methanolo All above fractions were saved 0 on dry ice" stored in methanol at ~l2 C,or fr:;zer! Extraction pr>t>cedures and methods used ar:e dis~ussed in detail ir... publications from the Vancouver Technological Station" All solvents were redistilled before useo The female plasma was t~ea ted in exactly the same manner as the mal 1 SUMMARY NO 9 20 STEROLS~ MARINE INVERTEBRATES T. Wai..nai. Do Ro Idler This work is being carried out under terms of a grant from the National Institutes of Health and is a continuation of the study carried out during 1960~1961 at the Vancouver Technolog:ical. Station (Annual Report for 1960=61~ Summary No. 39) and transferred to Halifax in October " Progress has been made towards the isolation of the rema1.m.ng sterols of scallops (24=methylenecholesterol was isolated previously) by employing adsorption and gas chromatographyo 24-Ketocholesterol has been prepared by ozon:l.z::~ng seaweed sterols and whl be used to synthesize cl4-24 methylenecholesterolo An atte t will then be made to establish if this sterol is the precursor of ~ 7,24\2t1Lergostadis>.Je~ 3~ol. previously isolated from a starfisho The synthesis of relatively large amounts,~"if 24-methylenecholeettHol for Dro Steinberg (at No Ia Ho) were completed at Vancomrero When aquaria facilities are available at this Station (.. {! May) an effort will be made to investi.ga~e the relative i.mpoi'tance of exogenous and endogenous sterols on the sterol composition of the tissues of invertebrat;;es.,

88 66 $ljmiyiary NO~ 2_! STEROlS~ GAS~ LIQUID CHROMATOGRAPHY OF SC.i\.LWP AND COD STER.OIS T. Wainai Ro Go Ackman R" Do B>.trgher D,, R, Idlex Recent devel~)pnents i.n the field ~ f glis ~:.d.cf.jid Ghromat;:,graphy have featu.red sterol analyseso PrelimJ_nar;:v work on BlllbsLr: ate~ and columns has been carried out using the flame ionizatton dete (;to;r 9 since :mry small samples and low substrate concentrat:f.om m~usi be employedo :rh:la d i~<te::tor is not entirely satisfactory for the purpof!b ~LVJ, :e en.~ l:,.igh. gas fl~ iili t"a.::er:-: uscessary 1ead to flame instability and h.igh baclr...gxolm.d ncise 0 The ~ :ray I o:nis:;at:i.on det.ect,1r, on the other ha.ndp is not ae:;:j,~yu.sly affeeted by gas f:~ow :rates.o Various combinati.ons of four diffe:'l:';.r.g subs4:;rate~ and tr.~.res suppcrts hawe been r:.cq;jcn~,i to determine optimum separ,at;:,ng ~ondltiims" Significant sepaxa.t:'.. on:s b.a'1re b~e:o. a: h' ~~ved h:;tb ~1'.\ polar (c.;~~ 1 did NPGS) and non~polar (SEo 30) p!,;,a,sesd based b<:th on m:<.:l~~mj:'.8!' weight and yarl, ations in st:r.uctureo Jmalyses o:f a n.aturai mi:&:i::tlre of scallop sterols (Figure 1) show a somewhat mcrt: e c,_,mp:;.e::r system them ::.s evident in analyses by other systemso In vi.ew of the pr«j!.a.ble thermal dec.)mpos:l tton of some sterol& on the columnsp and th~ p::ssibji.l:ity of copper column int~:ra:;;tion (see s~'mlllllb.iji?),, these analyses must be tl'.'i<aited w i ch t"ese::rvat1')rlt1 pending f''il.l.rth~s~r stud:lesc ft..n example of the appl.ijati.on of' th:ls W.rrk i.. ~ the analyses of a few millig'.rams of :sterol" isolated from cod flesh lipids ~;;y Dr" Bligh? ar..d found to be at least 95% chol~stercl 't Y gas~liquid chromat;ogr aphyo Figo 1 = S~all~p sterols 9,~.,,., ""'''J.lb~.:!>,.,,,...,,..,...,.., ~ / (.) ~. ~- t,.:;)l u,i\.,,'!.i!..='l;:r...',-,..._. ~~ --" IJfJ._} Gas Chromo iuztu o Operat:ii.ng I(';OndU:)[. C)nf: g.25 ne::i.i1!.1ilii 9 10 lbe hydrogoo C o ,;; 0 ::>1 'ra"'... ;[ '"'"'.. :OJ, ~~""'p' L, A ';'!'; ttl s~ :z~ ) Cl!~

89 67 UMMARY NO 2 l.f. FREEZE 0 THAW 9 Uf,REEZE EXPERIMENTS ON NEWFOUNDLAND TRAP COD" * W.,J 0!):y,n D,..I o Fraser DoGe Ellis E, La.ishley DoRo Idler A program to better &td more compbtely use the ;potentia1. supply cf Newfoundland trap c:od involves s nral aspec~tsg 1" Use of present knowledge, demonstration of proper hand:ting pro,edures 0 and provision of adequa'tg fa(jilities~, S;CJ that th fish now procass'*d will ybld the best possibl produc:t consistent with a rease:nable return ol'! industry investmento Indications are that the applic:ati:m of present k:n(;jwledge l.s no\:: adequate~ at least in some areas 0 as quanti ties of fr ozen b:jl x;;ks hawe hfld t~j be scrappedo 2o Increased size and mechanization of prssent plantsp so tb~t a larger volume of fish can be handled in the short txap fish seasono Thi!'l solution was adopted in one reoently built plant at La Scieo 3o Freezing of round fish followed by thawing and processing a:fter frozen storage until the glut is overo 4o Live holding of trap fish" The present work is involved with ths thi.rd ac;,pe.:t" freezing >:'mmd, thawing after storage and refreezingo There are two considerations which become evident immediate~9 first is it economically feasible 0 and second 9 can a first class prroduc:'l; result from this treatment? Economically labour is still required fer guttjng and hea.ding the r~xnd fish 9 and 2 to 3 times as much weight of fish ba~ to bs frozen and stored if the fish is not fille'i;ed prior to freezi:ngo On fac:toey shipap hrrw.e\ f.;!\ such a process may be more advantageous 0 ec:onomically 9 than in shore plant!\ where labour is usually more availableo Technioally 0 the freezing of prerigor fillets is not a problem since much of the worldvs production of frozen cod is now being processed in the prerigor stateo * It is difficult to assign authors since 'the project was fully co- operati~e between personnel at the Newfoundland Unit and the Halifax Station" Many personnel at Halifax played a role in planning these ~xpe~iments &ld their assistance is gratefully acknowledgedo

90 68 Llkewiser conside;rable e,mourrhi of whole (gutt.sd) cod ajr S ly2jjlng frozen in '\\Various om.mtries v pa:r~~icnlarly B:~:"i tain and R:us.fliB.o This ::..5 d<sf.ros ted pr:tor to distri.bution and most is sold as 11 fresh'~ fi:ll.ets., Some:, howev-e:r'r is refrozen but primarily for ~~.mmed:iate distrib'~'dono Tht:.s, here i"s our f'i.:r.::.;"r prcblem~ What ~ s th~j sn~:r.age. potential of' refxozen blocks.:ir f:::~1l,&t~?.no dat<~. are prgi~fmtly ava:uat ].oo What rdher. fac,-c:.t;:rs 1.':1.}'6 in''"(;'.1fl-3d.? Heac-:\:~y f;..l""d:i.r : : trap fish. are! '\;"BI'}l Sl1~jG8lJ"'~~blt-:; t:j sc:1.:c~;r:t.sbsy d.~~j:e p.r )l: a_t:~.ly ra th5 ~~n~-~x ee.bf_,.-:t proteolytic: enzymt; CJ:Jh'l.1.:en'i: of ihe :">'(:::.znach iif!d. p::>ssibiy of 'ths i"lesh as wc:l..;." coupled lrrl th the u,-;;,r:al pra..:::ticf? of i1~lkwing i\:::~d.g ;z.j.,~: :t :,sndi.ng, ('Tb.:L'> Station's Annual R8por fr~!'' l959p AppEmdix L The rate of free:-&ing may ~.J"3 e. p:r.e:,r lsm. ir: thf;':j.s th.1ck :f:i ;~h, ag :is the preferred method of f1:eez:tngo Vert:'tc~al plate: (.4 ), b:::'i.ne ar.d. t>.i:i' blast ma.y be usedo Ancthex pr ::blsm ::..A the method of r;h?."'i'~.ng c\b.-e u~t:hhi.' l:j_t'(':''c blo0k:e :1nvolvedo Air b: a8~ 9 wa::n:n a.j.d i..>wld wa M!d i~te:u:;.:.;t,r:i.x thaw..tng faa~.;;. been usedo Tha'!!i rigor 9 t\jugme:ss, e:xce<ssive d:r'i1\,.elt<r.,, may oc.c.~m: in imp:ropcr1; : handled prer.igor frozen f:.,sho Th;ir:; latt>?:r p t:olem tr~ v1'!.1l~.c in ;Yf~stigation H'L Halifax and elsewhere" A program l.fa.r~. <;;b.ex e:fw:: e. t;3e :: ::Jp to in:'ri!lst.:iga ro e-~~;(;,r al 0f thes~e a.spectso Technolngie:a:;.. :factujcs such as freez:lng and :;ha:wtng pro:::;edure.s wer.-~ to be undertaken by the Sl!"c hnn]~og.~c.a1 Um.t at St,.kbD. :Oo The question of k&e1p.ing qua::.:l ty rf re:fl: :~ z.an f:t~ih prepared f:n)m ttawed. fi"dzen round cod wal!i undertaken at Halifa:.: w~ t.h 08lll.plo::. <~o be suppli:~d witt. the G<Ctopera ti<lj.n :vf the Newfoundland Unjc t 0 S(:m.e t;;;s ~e< :m br:~ne fx ozen samples were also ean-iefl Ju: t 0 A cr:llabcrative e;:li:per<imen t: vd th the Gl Juc:i3~r~ex Lab;;ra ;ory 0 U "So Fish and Wildlife Sarvice,, :b.;:.1e be,)n <:1 '"t :,;cp,~,; c:m:pan' wa '::e1?:;::1.:1 w.iczuwa~ e thawi..:..g and the storage c:ha:ra::-: ~&~'7'istica.,,;f the'' :rcef:r:izen pr~j.uct~ 0 Pr21xigor an.d poi'5 ~;._, rigo r fish havt& l::jeen pr:~.c sssc,;:j at &l:'fax fi:_,:r" thi.~ p!lrp/ ;::<61 Studir:'>f:l c :l" yar:vous methods for.c: ::JokiP.g fish ;e<arnp.le:~ fer tast~ J.Xl.::MI testing a:re also b&ing undertaken in c:c:opera t.ion w:l th the; Hom\3 Ec,onom:ic.s Section of the Department of FiaheriP.e" Ottawaa In addition 1 11-onstderable taste panel cer:rdng of ~o:rumerv:::j..alj:.y L' ozt:'!l offshore cod wa~ und~:rtaken as c:<ontrol Ramples and S(tffie 0omparative Sf.illl.pl.:;a of' Newfoundland and F.alifa.x fji;ozers fish 9 inehoros and r.d'fbh:)re 0 were taste tes'hld by both baking and si;aamingo The work c:a.:rri.swd out i~. repc:c?:ed. :;.:n(t c X.cra:rtcus sf":,.:r:::t:ms f, ;,llt,wj.\j.gp partly under the Newfcund.land Unit a.nd tht'l remainder :frt;n th~ F'zozen Fish and Proteins Se c:tion :,f the Halifax Stah<C no The purpose :: '"" 'tj,i~'te:rmi:}e the 1!\tOJ:ag.s Iife.;f fju;,zen c:;d blvck~ prepa.red from thaw~~d f2:1nen r'i("und cnio The:refor""',:,nly bk'wti, p:ra;,t~lo:;a.j prto<e:eduree were tq be used in p:::-2\paj:a'hor.:. and assessmen~~ 1 :>f ~.mmpleso (s+uc:; of the many techn~cal :probjem;wj ccmld bes'\_c be l'lrlsw~?<red Jn the f'j.e]d or by '~h ' f,., il '+) ~e.:cu. un~". o

91 69 Initial experiments were c:onf:tned to Newfom1dJru.'1d trap cod.o Ths fish (medium size) well:"& to be landed. prefe:cably iced,, as ~oon as pv~sible after brailingr; and be immediately gutted and washed0 Freezing was to he by vertical plate 9 and storage at =10 to ~15 F in St" ~Tot::n''s;,, J.n a,::;c,mpe,~ct bl,pck, covered to nunim:tze temper~ti:lr:a fluctuation and de:!\'ijc:ccat:'.0no Thaw.ing in recirculating f:rel!lh water at F (!5 ) was MJ.ggested,, f\;j11 ~~wed by immediate filleting and ref:k"eev;ing 81!'1 l lb" Wlf~pped pi.f,kage!"l :tn a conventjona.l horizontal plate freezer, Quality deiiel'mina t:b::~n!s w~:re to b<e made on samp1~,~,.;f froz <e<n rt>tmd fish and on stored ref:r'ozen <'.amplee: prepared fr(;;m Jthn i:::'oi<:en l"(>und :Lil!5h thawed and proceesed at 2, 4 <'l."')jl 6 m':>nthsu using ta~l":.li panal5 aud ah:u protein analysiso The blocke se1ectea fox Ul!ie< in the R.!?41J.ie.:r ll!lurk were prepar:e d f1. >H fish caught July 21 and '22,, Thi3 wa~ d.u}."ing ':Che la1~t:ei" pa:r:t 1;:,f the Sto John s trap fish l!!eason, vh~n thbj supply of ftsh r.~ 3.s beginrring t :j d~":!crt=;c;,:o, ',o At t.his time they were still feeding on capalino The f15h,;:,arrte from '!;;., ;, trap;~ operated by one fishe:rmano Tb.E:y wez'a iced. when renewed. from the trapp gutt;ed and headed at sea~ ll\a~hed with salt watex and re=iced in metal fish boxe~o The fish were la."lded. iir S:re:tly a1t the i'i::r,h plan'(; whe:::"e freezing was done one half t on6 ~d one half ho1.ll'rs out of the 1,zap" On~ 1bat.ch of fi5h brought directly in f:rc om ih~ tra.p 9 ungutted and :in lc~,. ~ad an average fle"'h temperature of 51 Fo The gutted fish uaed. for block'!i :t>ar.~gad in temperat!:r::~ from 34 l/2 F io 40 F when loaded irrf.o ths fr.~lezf:f"o llll were prel'igor 0 firm and in excellent c<~.mdit:to:no The a7erage size x e,c:e1.1r:e1 was 8aid. to be larger than normal fol' eo late in the!ieason" Some land:tngs ~.owe'~j"er 9 'contained only emall fish" Freezing of the gutt9d 9 headed fi:!h was re::arrioo Jut 1n a threfl 5'.::etion Yertie;al plate freezer yielding blocke 36'V X re~c X 4 l/4i~ 0 The plate!!! wer s operated flooded 9 luling 8Jlllnonia from the fieh plant refrigeration :!Jystem" at a temperature of =28 F o The blocke were left in the freeze:t for a minimum of 4 to 5 houre 0 at which time the temperature wa~ bel0w 0 Fo Thay were dropped from the freezer by a :short hot gae defrost" Surface thawing of t.~e fbh block was minimized during thie operation by glazing the platee with ice pr:i\'>r to loading" The blocks r emoved from the freezer we:te ~e,aled in p:ilyethyle.n~ fum and placed in 'Cil)JLd!ltorage at =14 F,, After equ.h:ibtating at thi~ tf'jrnper~ ajture they were sto~d in insulated boxee 9 to even out te>jllp>:'lratu:r a variation3o The temperature range in one of the!!e boxee for a thrree week ~J(.'ivd Wai'J ~:~,0 F to ~140j'o Only one batch (if 250~275 lbs" of gutted,. haa.i:t~d f:.:"'!h could be frozen from eny one landing of fil!lho Aftez- 5 to 8 hou: '1!' i.n J.,:;e!. fish temperature 32=35op-v about 1/3 to l/2 the fish wa~ in o~lgoj: :o::r bn tbi1c.ll.g rigor" After 6 day!! st,orage, 3 bl J:;ke of the gu'tte,i a:ad headed f:ish WE<s<F.'l thawed in recirculated fre.sh watero The ini.tial tempe:r:e:t.rre of the watejt ~"""' 50 F 9 falling quickly to 45 F when th9 frozen blm:kl! w ~s::rcs inu!j.et!!edo Ai ":>ti~!

92 70 h.e.lf hour the water temperature had dropped to 40 F 0 c~"1d 8 1./2 h;j11.:rs i{~, had dropped to 36 F o At thi.s tim6l the fish were ramo;re,d fx1::m too water and EO rparated in metal fish buxeso The flesh was softo C,..:m ":~derable i.l.~bri.s was present in the water and ie;e was st.ill pr et2l n 1 t in the; c""nt.:er... f the f: sh along the backboneo The eot t fish suf:t\:me;i on g 4 :andi:ng a1g r.-oil!. t.&m.pert'lturf (65 F)" This appeared to llr& refr"eez:i.ng :rav;he:c :;.}:J.en th.o: <:<(,set l1f :: ig :':ll'" Some diff:"iaul\;y wa.n ~~fkpe::- ~i~~:,:t~~-<'i.2..!}. f~'~i~let1:r.-.g ~~ :,;,) '"}:8.11~$ r f ~~ ~~\ remaining in the tissue o The fj.lllets we:'!"b 80I\~~ bllt: ::: th.;,i"wis;,::,.,f' g~ <>i qualit;y-" '{ihe fillet temperatures ranged from 33 to 37 1!'0 They were pa::jraged.in l lb 1r1axed boxesv celli; w:rappedd refrozen i.n. a plat": freeze:r pa.3ked in 12 :~h cartm:s and sterad at ~l4 Fo Shipment ~-;~; Ha.~.t:faxD 'ii';wo r;_ays Iater 0 war~ J.n mau.lated boxes cc o1 vi with dry i~e. The au:.:"face temper.'ltu:re of th<">: 1 lb paekagas on ar rh al wt:w =7 l/2 F to ~14 F" The aaruples were dividt/1 :mt.:. :'>':C b;:;(;ches., "Jn6 equj.i.ih.ra"t-od. to ~ l0 F (;:!;: l/2 F) and the,,::-lhtn t;v o.of azd ps./'lced 1;1 ~onr.d~axr(; ~e.rnp-?.:tat'.o.l'8 boxes at these temperat:u.:;;:e,;:.o Additional sampl.ae :f '':: -:omme:ccial:ly pa.ch::ed fil:'b;e, ~B a:1:1 Jf fr.~ic;sr., gutted fish were also br Jt:ghY. t :l Hah.:fax f,n~ corup;...jij.son. The:se samples we:j. e trap fish caught and pro(:.eh~e;d the aame day the :fj_r st thawei fish were hlleted" ph valuea,.f tha freshly cut fillets W'O;J"i' <GBliY<'n '>U lanc1ing and up tc 4 1/2 days m ice ~x:vering prengm",, :LTJ. r:igc'l a.nd posh igor 0 small and medium fish. There was J:l_ ttle d.ii:fejrenctb in ph between JSampleB taken from the skin sid'3 and the cc:ut s.:i.de 0 (lr' 'tletwe<en r,;""' la;;j.ly flap and he:ad and 0enter of the fillets. HoweYe.r 0 ph values from tr1.e 'l!;a:~l :r4bgion were USlU\lJ.y 001 t~j 0.2 ph units and s:lm2timee. up 'to Oo5 ph u.n.vts ic wa:?.' than the rest Cff' the fishr Ts."ble I. The ph valuae; :;:apidly dropped t;, about 6.0 with c>nset of rigor. In full rigor values ranged from about 5A to 6.3p followed by a slight increase to about 6.0 to 6.2 for the postrigor fish iced 4 1/2 days. These values are rather lower than those usually observed in trawler caught offshore fish and may reflect; higher nu1t.y'i ti<onal state of these h~a1vily feedi::r.~.g fish. Higher initial muscle glycogen values could lead i-;o greater le.ctic add :formation and c;.m.. <>Jequently lower ph.as glycolysis pioceeded d1..u:~:ng post ~ mortem development of rigor mortis 0 It is planned t 1 'c~heck glycogen and high en2. rgy phosphate compounds this coming t::-ap seasono The values are in agreement with those rjbse.'t~ ;i by Ma.cOallum and Ebsa.ry~ Newfoundland Technologica.}. Uni"t Annual Report,. Append:i.x L

93 TABLE I ~ ph of trap fish Days in Ice Weight 1 (lbs) Condition H2 Cu't Side C T Fe Ft H s k i n S i d e C T Fe F;; 7 1/2 hrs 7.1/2 hrs 1/2 day 1/2 day 27 hrb 27 hrs 1.1/2 days l 1/2 2 days 2 days 2 1/2 days 2 1/2 days 2 l/2 days 2 1/2 days 3 days 3 days 3 l/2 days 3 1/2 days 4 1/2 days 4 1/2 days / l/2~2 5 5 r;; / = Prerigor 601 Prerigor 601 In rigor 602 In rigor 600 In rigor 5o9 In rigor 5o8 Starting out 6o4 slo relaxing Starting out 6c3 slo relaxing In rigor 601 In rigor 6c3 In rigor 601 In rigor 600 Coming out still slo stiff 6~0 Sl, rigor 5.9 Slo rigor 6.2 Slo rigor 6.2 Mostly out 6.1 Mostly out 602 Sl, stiffness 6c3 Slc stiffness 6cl o8 6J) ~ '),c; qol 5o9 5o8 5o9 ~;; 5 o I 5o'T 600 5o8 5o o9 5c9 5o9 5o ,0 6 ') c~ o o9 6 o"' () o c8 5o o9 5o a8 5o o o'-. " 5o9 6o o o8 5o o.b. " 6c c2 5o9 5o8 6 1 o~ o2 6o0 5o9 5o o o ,0 5o8 5o , o8 5o o c o7 5o7 5o c o8 5o ol 5o9 6ol ~ I-' 1 Weight of headed, gutted fishc 2 H9 C? T ~ Head 9 Center at"'ld Tail portion of f:u2,,~t Fe Ft ~ Belly flap at (::enter and tall sechor.~..s

94 This informati,on WM n8eded to a.u0w,;;alcu.:,at:i... m :f yields J-f f:i.sh useda The medium and SI!l.a ~.l gutted t"'lod used l J:s1; an tvrerage A" t::ci/c (O(e:gh;i; wh en beheaded" {Data. tiy Mr., H"Eo Po""ey ), The a.r"tu.al f:'igu:re was 20o1% with a standard devia'd.on f loz:, fc,r 6 samples" Taste p2nel a::;z,esscrjht>;; wa.c. carried C'Ut oj:; 'd1.<c o,arrrpl'sis w:i thout; thawin.g 9 using the standar i baki.r:<g pr;):.:~sd.'.:.:te t:n mse a d:.t:,. Stab.c:::1 fj:: se'~ eral ye.e.rs" Samples were also tested for fll:"'='"':;;in extlta,:;<ta:r.d.j..;, :f;orm.atir. n ~f fji."e:is fatty acidsr moisture and dlt'ipo Control sampleb foy the it;as'~e pal1rbl1 W<'il'~"".d:i; awn fr-<jm ''ia..mpl'i:-if~ tf (;ommerc:iatly prepared. fx ::lz.s,z: f12~:,ets frcm offshc: '.:a'.r~~;-;,r._ ;aught C<l(t IJI'-C ~~er-n:;a:i in Halifax and str0red 8.,: \;.'h2, la.h:itc;. S.s'? 3'1ID:r:tl.')!!, Gen,~:cs.:.:Jyj 3 to 4 samples were tes:-;ed 5,; c.i.'16 time 0 8IJ:J. t~a.;:,y,,;.. wn;> ~- 'l!.!a.n ':(;es'l;2:d :lr, a.1" ~<D'-:\(:';t; 3 separate p.:tnelsi! using 6 t,i 8 ~!2st;e.r's :f-er pg.ns&, ~e ""a:r e S':;c::red f)t texture~ 'Casts and ;)'IY'i r-:.1.1 g::-"jd.;o / Th "l +',... "'-' ' ;:,'"., -;;. A : /4u?..,_.., i ~lr " " '"' - "~.,-... J,.., e i' h SaTilp..L..i':'~8 Jb,t:,:L<?.n ~n ~LL~ ~,._;; '!-...L t,.:.oj~ ~. [LJ~.;,)\~.r.\..::J> W8.!~ W'.t~k- t.jpt:j,..i.."'!'... ~.," p:;~lysthylene sheet and store/! il~.8 ;;:f;.mm8.1:'': ::..a... s)-;o:-;:<1:'\g-.:.. ict:: m.pe;na lrui'e abc:_;c;:,~jq to =l5 Fo Large pi.et;es lifer~ G'Bwn ~;ff irtme.ltf..tre.,l:'i' ate-:"{<: f:rah?!z::]r,.g at 2'. 4 a:nd. 6 m\mths and shi.pp"i~d au' f:r:e;.ght 1r:o Ha:l.i:fa.JL wb.~~j:e y,-,~: ;,, =< ~~:.::ed >:-.. ;.. Jt.PF and analyzed w.i thin a faw liayr5 of r-e:;en.p ~o These resu~~ ts sho'w -.r;hat; 'til:~ trap f:l.sh ba't''8 n, :u'mal mo:lr~-+;u:c8 9 e~~..:lginiy higher total lipid, rrormal ::iah; "'x:tra- ':1table prote:i.r. e;.:(d 8.(:;i~"'myosln 0 Th<.::. 1 i.t;;,;, TF.iA -;ralue indicates no ta.:~~e:rj...al spcn.lage tut ~:hfj Ji.a. the~ high f.r ee :fa'~ty <: :':..is urrusual 9 and may indic:at,e important differences tn handling 0:r in :i.m.t:lal condition of the fish" Glycogen at 3 weeks is ra.the:r.h1.gner than US1X3.1 fc t' cod but more determinations are necessary" li~fi dis.appr">aran;:<~ on t'u:.%ih.:.y st o:rage is probably nomai but this phenomenon. v,f >:;, ::.rtt::tn c.tng gi}r(,c,:~:~l;~i~,;a at various temperatures :n;!'leds a.nd i.s b:ei.ng given further study,. The drip fi.gures,. 7' \::o %, are pr-obably abo<lt no:rmal f:~:~ cn:~-s fl~i)\:.sil f Lsh 9 though the ons 14% valu<& i.s r.igher fran nu r-ma:, 0 Taste pa..."1el g"~ade8 show '.?n1y a. p:_,ss :::Lble slight le>t~rease in 6 mcr:-thi::i atora.ge 9 in a.greemeni w ith prev:lous da"ca on orwe fr::r-i3:d., i _.~tnrel at _.,;his: temperatureo There :l..s a 0oneidera ble decrease i:o tasie panel s\co:. es bmr; little change in i~exture or gx:ade: n Hwwe nn:",, a~ 8(-l6E izl ~"ig 0 l: the g::ra.d'61:s assigned the :;:~ound trap fish samples by 'the t;aste }.1aiif"l Bre a.b.uut 15 if.') lf.f% brelow too oommercially frozen offshore.;: od M:~ c:r l sam_plo3<j" Th:A.R:I d::tfferem;;e is due chiefly to a..yj. inferi:.;:r texture score, signrf~c:a.ntly.lower (:!% l~sv,_.l) 0

95 TABLE II = Round 9 plate frozen trap codo Storage time in months J..ndica tii'ji b;r numbei' ~~~~-~~~~~~...!:RF=3.s../.L. RF 2 RF,L_.~L=6~ ~ Moisture % Drip% Lipid% FFAl % of lipid EPN2 % (Oa6 M NaCl) AM~N3% TMA.4 9 mg N per 100 g TMAO 9 mg N per 100 g Glycogen 9 mg per 100 g La.cti~ Acid 9 mg per 100 g Texture Taste Grade SoEo of mean Number of samples Oa al6 330 ~ o2 '+ 4 7Jl) ~- 0 ' -! 14 8lo4 8lo Oo84 Oo ,.-i2 la72 '..),0 lo~~ '0 4?o7 45o2 (+,. 6""~ -= 0 0 t~ t (! 4o50) FFA = free fatty acid 9 2 EPN = extraetable protein n:'b:x,-:.gen,. 3 AM~N = actomyosin ni tl"'gen 9 4 TMA = t:rimethy lalli::lrm BOo? 9 Oa6? 27 la93 l.60 Ooij, 9''.A.o..J; 1;:: o5 + 5o05) 19 Perhaps it should be ramphasized here that the taste IffilE!l u~ed was an analytic;al panel rather 'than a ~onsumer panelo The panel uaed j_~ t:rainto,d. -~.;.: p1.ck up traits in both texture and taste which may not b~ e.o obviou.s to an untrained person 9 who may actually like! his fish a shade off=flavow:' Ol' \\-"i th a bit of body to it 9 or sv smothered with condiments that ~;h-e t:tue texturee and flavour are lost a In other words 9 the panel is familia:rc with the characteristics of a good quality, fres.hly frozen cod fills{;; a.a :.~\egaj:ds texture and taste 9 and these trap fish were simply n0ot up to pa]" 0 Some panels lil'ere als!o' conducted at the Sta Joll>':l 8 s Urd<t; (SU!llill.9:ry Noo 26) where the differences were rep~rted to be much less between the h:up and offshore sampleso HoweverD steamj.ng was 122ed as the CC(:king proc:edure 9 together with a scoring system primari.ly dt=nreloped fvr detecting bacterial spoilageo It has also been found (Summa:cy NcJV 28) that steawir.g may not be as effechve a procedure for detecting hfferencr8s in qualit;r near the top or near the bottom of the scal:eo Thls. question cf cooki~'llg method as it affects assessmeni~ of quality is alae, under inves~~igat:tcn (Summary Noo 38L Since the trap fish are primarily used Jt~:::c the produ::-:"ion,of frozen blocks for later conversion to fish sticks,,~~r por'hone 9 it was of i.nterest to determine whether the poorer texture was detectable or objection= able in the final fish stick product (Summary Nco 23) c

96 There aze SE<'?'elfal poss.ibla t fa:s~~nz for ~;ru:< Icwerad texture bu:t the~ Ixt'f;sent data is insufficient tn def-rrntin.e t:h~: aq;;;1~ua:c ~c;a.use~ Yjfhathe:r e:ingle cr a combination of fa dors~ 'J.lhe relatiy<ely s.i.:~w f.:reezing of th~ 4 1/4 91 th;td!.:. blocks could result in some texture changt:?s and ajsc c«:mld albw :rigor to proceed further than,expe:ete<il '!:he :ti.hh may l:w:<r;:,. be ~:t Hl :p:~t::i' '.:::mdition 9 that is~ not '00mpleh ly :r~g~wf'/r' ild f::::om c:wawningu A1s n t<h~ C r:r;ap :fi.sh 8.1' ; caught in relatively 'Wa:rm wate:r and,;jtli.'uggle :;;on;;:~_;.~if'irabl.y c1n bli'ailing9 =~tc~ Even though _iced in this 'exp9rim""nt : 1 ien.rpera ~~nx ~~~ had r ')"i- reacht>v!i 32 F by the "time of prnces;:ctng thi?l prer:igo:::- nloclr.s 0 Prr.iss1rl1B :J:.ang:':!::J durl~g shipment ha~re largely been ru1e l:l om; sjn'c<:~ t:<"eatment sirrmla.'t;:lng r;hipm~rrt: ~r:f on.~ lot ~,f fish showed no signifit;ant <'?iffe(;i; &"ld al1'.k, a'tt lea.s:: 2 sampl~s of offshore Newfoundland fish shipped 'mjlder. thg same oondil;'fons ' 0 1-''\cred. normaliyo The refroz-en fill.egio' Wdi'F; ejta:u:tced a8 a.br;vc<..,. ithi:n a few days of p:roc,eszi:ng" Table III c TABLE III Re.f'::ozan Fll.leta Prt:h::essed f:' om Reo;. l.d FJ.fJh Stored a'h =io a.tj.d ~15 F.in lvkmtbb,, a:s E\1l'L'Yi!ff2 g Moistl.<re % Drip % LJLpid % FFA {% cf lipid) EPN (Oo6M NaCl) % AM % TMA Tl\'lAO Glycogen La.ct:1c Acid Te~tu.re Taste Grade So Eo of mean r n" 8: ~05 8lo6 8~~ 0 '7 820() l'i 18 ::CB looo t)o87 Oo95 Vo (Jo: "-"'! oC() lo 1 "-~ 1 9 lo74,, ~o la50 lo59 lo28 L lo09 Oo67 23 c::3 22 )::( T7 :T.Ol, i ' '59 68 ' ,,1 5lo9 50o4 4'-' "'+ :<, 0 o' 35o5 (! 4,64) (! 5o08) {+ 4o45) t+ 4 ao) \,.;;..:::. \ --~ (± 5o64) tJ The initial sample'2 were thawed L'l re'l':i::'c'.llat:mg water at about; 40=45 F 0 At 2 months and aft;er, the temperature of the th!lllling water \faa reduced gradually from 45 to ahr;;ut 37 to prevent wa:;;idj.ng e>f the tha.tmed partso MoistureD lipid and free fatty acidv TMA and TMAO appear not t>? be affe~ted by the thawing and :refr"eezingo Drip is atv:u fr; doubja,d al:' wa,s antij::;;ipatedo Extracta."ble protein and a,ctomyos.in show (;On:sidera:ble decreas~:, especially at 4 and 6 mon'i;hs, Glycog,sn J.s '7rariable, perhaps depending on ti.~& particular fish analysed 0

97 At and 4 months 0 the taste panel sc<eres are unchanged f:r:~-om the frozen round fisho At 6 months 0 value~, ar~ somoowhat l.rcjwerp about 35% as compared with 53%o Thus a'~ least up tcj 4 months" the thaw:ing and refreezing has not affected the quality of t,;hb's~ trap fisho Thl.s 5.s (Of course in line with pre:t_rious siudie;s in England and the TJoSaAo on haddock and c;odo There is ax'i> indicjatimj\ tna><r, tm l"ng8:r 1.!1-t..o:ra.ge "i';h(::ll'a may b'"' an. effec:t which is reached at about 5~6 m0n~~h'8 o These rceaul ta eh:cm.ld b~ checked with offshor~ fish sin,;;,5 on the pr~;)srar.t ~amples the l:o'!'rl illh:iq:'l quality scores may indi~a ~l':li!l d:if:'fe ren~ e"': f:rom ;;1oJ:ma1" Storage Quality of R.efrrc:Jzen Fi1~~~~ Part cf the z efrol!;en f':cllrets il!!ere ~tored at C>lC and ':t:he ::t<el'lt at 0 F 0 to simula.te storage Jl!D. f.lshiz1g pi:.j:-1t; f:j01ld e-to::.'~t,ge~ s:nd ::.n!i!>)tll\& inland warehousesr re.g,;mcti1'ts~:cya They '~~'~~#~ e:tam:l.:;;eq. a > 2 m.o:atq inteitalb" Sincle objections by thfl Jl.on~?h:\u::::'fer:l.D,g r:aste pall6l Wt:1<Y.8 ~~e;coming ti frequent 9 the exper~ment '!i>a.s d:l~l(f~n~:1.nued a'~ t:; r~nth~o Thrs 'f6:81~lts 9 Table IV and Tabl~:~ V a;.:yd. Figsa :Ji. ru~.d. 2 9 fl;h.)'" empr.a. 'trb.t refrozen fillets had been ad'v!e-orsely a~fferr;tedo Moisture value!& ;.U.d :t:..:d; change tbri::;ughout th!-1 1Sltpe1: Imen1~ and drip was unchanged flt'(.l.m che fre:sh.ly refrozen f...l.ire>~[lo I~~: was rather high for use as frozen fish-~stick blockso TMA.O ind"~c;a~;es that there was no appreciable leaching of solubl,e const:i tuents dwd:ng --::he thawing a;:;:< :l.t remains a t about tm initial le"iie1o Whil"'ll glyco.>g-en and lactate values are variable there appea:r s '~o be b. ra.p1.d 'c;on~.re:rsi' n t th;; 1La tter durl:og st)jr'agr!1lo There is a d.r,op in e:drac:t;able pr.:;tein which is c:oneiderable at 0 F sto:rageo At -l0 F 9 aatc,myos:..n 9 "i;he protei:.'l fract:1:m which becomes unext:ractable on s-torage>~ b~:comes progrreossiye1y- lower on 5tc-:cage being reduced to 50 to 6r::tf, at 4 to 6 mont!wo This :is E:V"'imt~whair; faster than norrnalo At 0 F the, d.ecn~ease ie more :~apido U1t:roacen~I'ifuga1 examinatinns,of the extractable pro";ein e_.~ 'B disc:ussed in SUIIll'llB.I'Y N -~ 0 )6. Fat hydrolysis pr<:.ceeds rather faster at; ~l0 P thw. would be expected at this temperatureo From the high initial -value of 22=23% free fatty acids increased t{l 29=36% in 2 to 6 monthe 9 t:he rate baing faster the longer the rc und fish were atored. prior to );hawing 0 At 0.1t proceeded much faster 9 va.lue,s c"lf 51% being reac:hsdo The :l:hnt :;f hyd!"olysis is indeed rema.r.ka.ble 0 The t;ast:e p.911el results are the most inte>:eshngo ThefJ:e iihr.nt that at =10 F ther1:< wae'i a drop in gra.de of some 18 t.:j 2(:1 p)in't~ in th~ first two month'3 after :r-ef1eezing (FigQ l)o The reffec.~; wae' aimjjar with pri.o:jr storage of the round fish either or 4 mon ~r,a be,fore thawing and reprocessingo After this i.ni tial decrease the rate of d~':ite:r i,rat:..'jn levelled off more or lea<s parall,sl to the c':l::cwe for ",t~he< tcmn:1 ox tj~ control fisho At 0 F (Figo 2) the magnitud~ of it:he d. ~l:;rebf~ 1.e g:-fe-aten"~, about 30 pointsv but again it levels off aftaz' 2 ffi.)~1tb.ao Th'.ls it;l$ st:;:rage potential of the refrozen trap cod t.iljl.e ts is p(l<cii", If these results are typical of those which would be obtained in c'olliillexcia.:, pra.c;tic;e 0 then H; appears that this method of handling is not likely to yie.1ld a g)r.jod :prod-uc" and thub some other solution may be more desirableo

98 - ~ TABLE IV - Storage quality of' refrozen fillets at c~l0 F Round Fish Stored-Months RFO RF2 Refrozen FllJets ~ Stored-r-1ontt.r.S 0 :..:_ RF4 0 2 RF6 0 - = - ~-~~-- Moisture 8] " Drip 1c:az l ; '<!::). "-I ~' '.../ Lipid lc.i,)q 0,~ Oo83 Oo95.~J"i FFA 23?9 29 '-' 22 EPN 2000 :t lo5q 774 AM 1 0 ~ 1 9 j_ 0 2? 0.93 )o04 1 7: -. ~- 0,,?F TMA lei) o.6l Trii.. AD 8L 15 8? Glycogen Laeti:: Ac1.d 541 {'>1?7'"'""!; t::r i Textu.r~ TastE'! 6'=' j?;4 43 2? 6~) ~ Gra ::J.e {r ~ ~ :: ~.-~! ;~2 0 ~ 33u0..!,-~ 0 ) 50 o-4 3cEo Ivlean (+A <, \ (t7 ;;;-z) (±7 '7'.>) ±:; (1 5~~) i+, F.. l ~,~ "',/.::...,r,_.. :.:, o:t:' c - l \ ' ""f'" o '.-,,J' ' Number ')"\ o~....,.l Oo84 "'!~,_; 1 06'1 lo :58 3'' <o "' t rt"' ;-::\ ~ )o>.,}f J ;21 Oo ':!;).,!!~ 1..!~ 0 10'?,...;;>..;: l'"t c1l6 ()0 :~;s 3i.) 0 ~,~) \ f t.f '\4 I -~,---tv.,..." 2/) 8L'i 18 Oo96 2) ~~ 0 ;\0 Oo~~,.~'"". -r,l,... r ~ :Jv 44.,:" {+4 (.._ ""-'---' Q C:,_J '~'") 21 8la )6 1o45 JLo (YJ (;o5:) ~~ C.o ft~ l~l \ ~o-j, l ()c?') 3I ~o50 _c<~o09 0.~67 l()j. 1 '" 'i;.'~~ "~'. ~~ ; (±5>iti) ":;'\ "'~', c ~1 Q'; ~C~-~-~~~-~~~~~~-~~~~-~~--~,~=-~~~~~~ ~-~~~---=~ = ~. ~~~~

99 TABLE V - Storage quality of refrozen fillets at 0 F Round Fish Stored Months Refrozen Fillets RFO RF2 BF4 Stored Months Moisture o9 8lo6 8lo L7 82ol Drip lo 11 l8 18 II=:.!._.! Lipid 1000 Oo84 Oo8l Oo72 Oo95 Oo73 Oo76 Oo96 Oo74 FFA EPN 2o00 lo63 lo l/74 lo48 Oo58 1o30 lo50 AM lo59 lol5 Oo92 Oo72 lo32 Oe95 Ool3 Oo88 lo30 7 TMl ~oj 3o3 100 TMAO S Glycogen lo lo 5 Lactic 347 Acid ~'jc "T I "j',~ Tenttu:re ':1 ll./-'' 1,. ~ 1~~ 4~. 2:2. rv -,- Taste c.:t _,;?" 5(> 29 Grade 47ol 2'7.,! 0..1.\. 30o5 9o; 50o l5o4 44o3 l4o3 SoEo Mean (±4o64) (±4o 11) (±3o9?) (±-z 56) (+~ (±.to45) 3,-,' (ljo33) (±4o23) '+~ 72) - Jc \=)o c..) ~. =.C.o Numbex ~-,;j =.11 --~~-~ --~~-~--~-

100 78 Fig. 1 = Taste panel scores of ro-undv frozen Nfld. trap cod and of the refrozen fillets stored at =23 C. t---qi=r""'=--1 I / I I I I / / I ~a/ / I I I ~~~! '! I

101 79 Figo 2 = Taste panel scores of round 0 frozen Newfoundland trap cod and of the refrozen fillets stored at =18 Co J )( / ~/ / I I i I 00 +> (1)0...-!CO li'"'ir-t ~u / / ~ ~ N't:l / ~ ~ ft.~ 0 / (i)~ ~ t1j +> ~ ~ OJ et (]) ~ ~ (I).p ~ om) ioc\!'<:1" ~! ~ ~ ~ oxe+<j (; rf1 i<rv'hf> 0

102 80 The decrea5e in grade found a.h e:r refreeztng is s:..m:lla&~ ~;r, -~hat r::>btal.ned by a tempo:rary r-ise in te>jn~ra(i;m:"!i :t.n the p::o~-;::...;yd.s1y :rep<>rtt~~j. refrigerator car experiments" It a.ppears tha'~ thie,!iff~ ~:t, is!!:lore ~~ ne::ral. and any appret"i~ble :r;.f.3e in t.. emperatnr~: esp~.::;ially ci.t 0:h~ h.!.gle.e::' te.c;.-~~}.;e;., 1<>:-,.:J.s to changes whi c::h. shew l p ;;:m :?!.it.~eq:j.~.nt 'J.t,)ra.g~. &"en '? -~ :> ~ ~ "> tempera tu:res, O:ne '~'"'m sugg ~c; t; tba + t~'le ~;;;_,'w::r~.g up a f ~.e.,: 1:: l'e>f! d.ng c:m.<1gb~: c:ell membra.."lea,, or i:n tzxfa.:'"js, and s<: 1.rw~ a :;e:r~~ai.n ::m ~UI:t uf BU:b:str att; Hnd enzyme ~r of cl"~acta.nt~ 't., -;:nf: J.n p:rox:lrrl.ity w:'. <n. 7~1-J(;. :, tj'1e:r, C j :l;>). a 1 with a slow r~actior. Ya:tr:e thie wcn~ld a.ljc.!':h th.' ::oe8<:.t~c:.:;j rs pr.:.: r3t'!;;[ ""'J'<H after the temperat:ur<s had. again i:'&sn kwe1ned b':jc\r m,t:u had again been Xe~a~:ohed" rui;i r:::~.tl 21:)'\"i or:.g::.:1:al diff~;_,zs~. :IJ..,.c,ntta J.c~lel Y\':8: T:J.,;:'. would proceed as b6:fgrso It wcn1ld. thbref,~rtl ;-y" ~:::.;;::.o:">',stj..ng,;;. <~zt:; "'rr1. ih ' ~h<::~ 1~ '"" a.'".:.d ch.a.rarc:te:: -cri"l the l'~'&a~:; li~.:t~rl~~ i~:r J~i~:, ~. ;, Bd o P:,.~:.~::.c. i~~ally ~, ~~:r.~. x1~~l~~,~. ig <::~f ~~t~ ra i.~~c~f during 1;he first 2 mcn"ths.:.t lo>~ '~ Bmps<cs.'cue,.,,~~.:.:~i 'i:rdl:.-a.t: 31 i;~h,-;. per'>.::. during which the X'&fl.i:'O%,en. p:rr0,.:bc:t sb :...;:i3.,..,,,,. CL:>'..iLffio:i" Li.kE:w:.,e.,: 9 S':lllib work should be underi~ak!?:n w-:1. tll ~:ffe:?~'1r.n~.,....:.-,,i. &~.:~ a:,;n c :.th<3l 51~'C"' :.:_.;:::;" FISH STICKS FROM TFJ\P Jll'J:U CON'IROL FISH Dolo F.r~f:HT w 0.J"' DyE:':' Wh<:m it bbg'>.me- ~"Cl.dE<nt tha:t. the stc:"i'id. :re:fj: Q"O~n tra:p :f~.<-jt. fi21..i:;t<fl deterio:r.a:'.~ed rather" rapid:y 0 "~he que12 tion az,,:.se a.-:; to whe.~,her thay wt'j.ld still mak e accep ~ablt:l fi 1h eti0kso There d0ea r.. c :' ::? :em t.:. be an,y :!.n:fo:rma.tj..on available on qua:.. i ty.:.,f :fii8h db;si:rtable fox fish 81t:i':Jrc:t 0 C':.n.suiDJ~r Rf:pC):f'tf> (February 1961) 5.. n a s::,v1xey,)n quality ~.n fish ati'f~kil :i.nd.l.ate,that.sel''ew.l:: brands wrare downgraded mainly be oause -0f :ran;:;.idity il1 the fish and bread.i.r.g 9 la\clk of clean sweet flavour &"ld toughness of t.'le fisho Several samples of the x~frozen fillets we,re.rbecrefor? stinm:l tt;<:;d to a fish stick p:~ant.. (where they were sawn i.nt~; sticks 9 and put thr r::ugr. t:ha regular fi.sh stick line) o They were bcr~telc'edp breead.ed,. :f:r:'.ed.c. c,;,.o1~1- pa.dced9 frozen and s~t:"red af~ co23't: fer th:rr16e hcu.r~o Cr.Jn h:"oj. samples cf freshly f:roz.en good q11al::lty trawle::r~ca.ught ::'" d :UPre cs. ar,.d a ~!DmpJ;~. t:f stored frozen f:i.ll">:,r;s,. Wb!'e< also in;:;lu.dedo They were d1en "taken out.. evoked :li.n an oven at 171 for abdu.'~ 20 nu.nutes and tasted by the plant quality (:ontr ol pax s.~;nn~).. 0 Oth-sr samplee were taken back t o the ]Jlbo:rat;ory "'i1e>r9 they were te<itr:.d r.y +~,:; regular panel who 9 howef!er" bad not had pre l,:nle e;xpe::rierice w1'th.sccz:i.?.:cg fish sti<~ks" Results are :r>:'po:rted ;,n Table L

103 81 TABLE I = Testing of Fish Sticks Sample Taste Panel Scores Texture Taste Grade Remarks by Plant Personnel lr oc RFOFF3=1/2 1R oc RFOF.F3~1./2 RF2FFl=l/2 =23 ~ :;..8 MT OT 57~78 98 ~~87 48 l ~ )77 90) " 80)84 87) 82)84 85) =~=-~"""~ Best 9 tho 0 accept- dcry t~ f'oo!'cer J.n Both these show rather able 9 tough cr ~exturr::; some coersenesh tasteless some stringy in thsn ~"10 0 and toughnesb some bitter te.l:ture<? approach.~ are quite dryness and cod not as ing acceptable (salt) good as 1R borderflavour and OC but line acceptable Trawler cod 0 commercially frozen fillet~: Oct~o 26/61) store.i three weeks at ~23o Similar samples proe;essed Mar_, 30/60 9 st.ored at -=23., Round frozen trap cod stored ;;:nly a day or tv.ro at ~23 p thawed a."l.d refrozen fillets stored 3=1/2 months at ~23 9 or at Round frozen trap cod stored lbw'o montruj at, "-23 t::> =26 " 2 lots of refrozen fillets stored l~l/2 months a-!: ~23 0 b1~ t In each e:ase the samples were identified only by oodenumberso 'J:ho processing plant personnel almost without exception noted a drynessv coarsenessp and usually indicated toughness in the refrozen sampleso Hc:w= everv they rated them as acceptable but certainly not comparable to their own product~ They also noted a trace of rancidity in the stie;ks from the control fillets stored 20 months at ~23 C 9 though they were still quite acceptableo The laboratory taste panel rated both control samples v-ery highly9 about 8Cifov whereas the refrozen fillets stored l=l/2 months &t c~23 C9 originating from round fish held two months at ~23 to ~26" grade:~d only about 4o%o Those stored 3-1/2 months from freshly :frozen and thawed :r.c>und fish graded slightly better when stored at =23 and poorer at ~180 Most members noted that while the -18 sample was edible,, they would not reorder sticl<..a of this qualityo

104 82 The conclusion of this experiment was that the factoi's" especially texi:u.re 9 which led to downgrading of the frozen fillets 9 were still a~ evident in the fish sti~ks prepared from themo The fish sticks prepared from the refrozen fish were d$fiui.tely not first quality 0 Surely 9 the aim of the modern fibhing :industry is to produce nothing less than a first cglass product, SlJMMAB.Y NO 2 24 EFFECT OF SHIPPING IN DRY ICE IN INSULATED BOXES EloJo Laishley WoJo Dyer To check whe~er there might be some e.ffect of shipping frozen bl,'jicks of fillets in close pro:dmi ty to dry ice 9 sampies of cdmmercially frozen cod fillets were packed as in the shipping ex~~rimants and left for 24 hourso They were brought to =26'tby sto:ri.ng 1n a rc,;;,m at this temperature for three days prior to packingo After 24 hours in t..he shipping boxes 9 which :containsd three cardboard cartons each containing 12 packages of: 1 lb :filleta 9 pa<cked with slabs of dry ~.ca which was separated from the ~art~ns by a shea ~ of ~a.rdboard 9 '\:he tempexatura of the fillets wa:r:ied between = and =30~ The c:a:i'igoy.\.8,gf fish WE'::'<!" then stored at ~23 along with the control which had been continuously at -23 o Samples were removed and t;estedo Analytice,l va.lues were practically identical and the taste panel sc:ores werg not statistically different (-5% level)o Effect of Shipping in Dry Ica Contr<ol Centre Carton End Cart '.:ln Taste Panel Grade EPN% Lipid% FFA % Lipid Mois-ture% Drip % 72o5 64o6 (±3o7)24* (±3o7) lo9 Oo69 Oo ol 82o4 8o5 llo (4.2) o9 * SoElo of the mean and number of testingso

105 83 FREEZING AND THAWING COD AND COD BLOCKS WoAo Mac;Ca11um DcGo Elli!!>i G,ao Windsox (a) General Freezing of cod blocks at ihe N<ewf\~undland TJ:n:i.'t to be used for tests (Summary Noo 22) wafl carried <.n~t in one of tw tltt'9ejc~stati.on sections of an ~ld model Torry=Hall vei."ti<c:al plat:&! freez<~:r o:n ::L:;ai~ frrom a commercial firmo In this device three h:~l)cks P ea~ch meast.ri:ng 36" lm1g x 20 1 v high x 4 l/4?1 thick 9 and each containing about $0 to 1.00 lb,.:,f heade;d gutted fish may be frozen on edge at Ol'li.<e timeo Af'tEJ:rc :f::c'1\f?l~ing 9 th~ blocks are released from the freezer plates and E!.::t e plau,;d :j.ij frozen er!:u ag!'< f'or subt=h:l<plent thawing and pro~esaing o Freezing in br ine 'flil'b.s.;;ar:r.'l~d {JUt ir;. the Unit:!S h;,cally designed freezer employing the tank=type.;,;;ooling methodo hj. ag.t ~ato:c circulates large quantities of saturated NaCl brine through the :t"efrigerated coila and between the :fish which are t'~ozen in baskets in a l~;{ S<B arrangement and in blccks l)f various p1.clr.ing densi ti,~so The bl.>:;ld.os frozen durlng the past summer vade~. in aize 9 the +arger:rt b~i.ng approximately 24't x 42?1 x?~ thick. The thawing de~"ic:e empl~y~:~d consist~:~ of a. rectangular tank :Ln which up to 4 blocks of the f'lplate freezer siz~" can be immersed in recireulatad tap wate? while standing on ~dgso IrMlividual blocks ar'i< separated by about 1 1/2 :incohe.so Ths wlit is pr(rdded with mr~an~ of recirculating the water used for ~rbawingo The i~empi'xf. ature,of the thawing water me:y be contrclled ovejc a range of abouic 35 tc, S:)l? and the velocity passing the surface of the fish ct;o er a range f'r"om zero to about 3 f~et per minute. (b) Packing in the vertical plate freezer Using fish either prer:igor or out of rigur ~ good sut.;cess wae achieved in producing uniform 0 well packed blocks meaeu~.ing 36~r long x 20f'l high x 4 1/4 19 thick with little c:;;: no twisting of 5.. ud::j.yidual fisho For various blocks 9 fish ranging in weight from 2 to ], and up to 10 lbs in the headed gutted state 9 were used su0c:eesf't~:i:c:v o_ (c) Removal of blocks f:t"om fl' eezer.!'3ta"t:::ton.s On a commercial scale there may be a problem in removing blocks from the plates of a vertical plate freezer without oo.using slight SUl'face thawing of the fisho Whether this~ would noti<:;~ably affe~t the quality of the twice frozen and stored product is not knowno We r<eq';.xire engineering data on the relationship of plate temperature with time; ~f app~lcation of hot gas and rl th degi"ee of a,?f'tening of the surface of '1~1:1.~ bl()cko On all blocks used in tests to date we haye avoided the possib:ui ty <r>f ~oftening by glazing the plates prior to paa;;king the fish inb the freezer s tations" This technique would not be acceptable to all :;:;o:mme:h'(:ia.l users 0

106 (d) FreezinE rates (i) Blocks frozen in vertical plate freezer" The longes.t t.i:rue required for fish llji. the: ~:rue t:entre.c:f a 4 l/4 1 v thick block to pass thr<mgh the c::r-iti<c>al zone,::f 30"~; F to 23 F was found to be about 1-3/4 hou:ts using ammc;nia rbfrigera:o.t at =20 tc; ~28~ 0 The total time fer the f':ish in this location to drop f:r.:om 370f to =5 F was about 5 hourso It is reported that 4Qj thic:lc ba.ocks ha'l:-e been frozen to a temperatui e of =5<>F in the new ate:r.-n t:t'awler ~vlord. Nelson" :'.n about 4 hours at a refrigerant temperature of =40 F 0 The fish which freeze slowest in the Unit us set~up are those located at the centre of the: block near the top sdge 0 The mu.':~<ele in fish loca:'bsd a.~~ chis point passes through the critical zone in 3 to 4 hourso This is about twice the time for fish in the true centre of tha block" Th~s P while it may be said thn t one obtains reasonably fast freezing using the verti::ja1 plate freezer th~~ rate over any vertical plane taken through the bloe:k dc:;es not appear~ to be constant for any given set f.)f physical condi tionso If this difference in rate were shown to be a factor in the quality of th,~ fj:::'ozen produ.:'tp desf&h changes in the freezer would be in ordero Wa have no knowledge whether newer types of plate freezers, such as those used in the "Lord Nelson~~ r provide uniform freezi:ng rates over a given vertical cross section of the blccko (ii) Cod and cod blocks frozen in brineo The fish in the c:entre of a 6" thick bl;:,ck were lowered in temperature to 0 F in three hours when held in recircula"c;ed brine maintained at about =6 F" (e) Water t awing bl.')cks frozen in the vertical J2!?.te freez!r, One of the objl3~ti;res of pilot plant thawing tests on frozen blocks was to provide condi tiona whereby individual fish could be separated from the blocks as soon as possible after the latter were introduoed to the thawing tanka Conditions employed in some tests (eogo water temperature 46<>F 9 water velocity 3 feet per minute) permitted periodic; separation of some of the fish 9 starting about 3 1/2 hours after the immersion of the block" Under these conditions even the slowest-to=thaw~fish were sufficiently thawed for the filleting line :in a total of 5 1/2 hours" Under the sam a conditions of water temperature and velocity 15 to 18 hours would elapse before all fish in an intact block (in which the fish were not separated by nand) would be tha~edo One test run was conducted to compare thawing ra'te:s of frozen fish which had been packed by two separate methodso In t:h:a first method the fish were packed in one continuous operationo In the aec:ond methtld polyethylene strips were placed hor.izontally between fish in t:he block every five or six inches of block heighto With wate1' at a6 F circuiated at 3 feet per minute9 the fish nth the polyethylene st:r~i.ps could be separated at the cleavage planes provided by the strips "it Rn e>arly stage

107 of tha.1j'ing"' Ind1v1d.ual fiah were aepa..t>ate~d ~m. thfl geueral lila~$ in the two blooks 'When posaible to increase the overa.ll thawing rates for the respective blooka....ibe initial advantage,given me fish~ oould 0. separated at the JOlevap planas Pe.e\Uted m mn np14 tbari.db f.ll a'u. fish in this blook than in the~ of the ot~ b~ A~ fin~ twice as many fish tlrom the fmm.er thf.m fioo tb'$ :Jl,a,t.ele bl~ ~ bt~ filletedo P!pre data a.re ~ui- before b a.jil~t:mt ad.v:an~~ of tht~ firs't tecbniqu.& ~ b8 ftp-\)~..ed 1n data.tlb.. Some e4wntaee ~ to b ~ by ~1.iiiwlatil:lg. at a velocity of 3 fe&t per minute versus a veloci\7 of.' Oo6 feet pe~ minuteo Additional data are required t}zl the effects of wate:t ~relocitiefslo Modest req~:lirtlments in r~gard t~ watei" velocit-y and the low temperatures sui table for tht :~: e;c:;:trc-c~ulated watetr i.ndi.< Jate minimal power costs arising from this method ~f thawingo While it is believed that tll-6 condil tion.~ wh:ich we <!mployed in ~ thawing should be conducive to the p!"oduc~tion ro:f f:tllet.~ r,e;ving an a<~cept.;;. able storage lifed t.~ere is n ed f r a compa.l"i&lion ryt th effegts of thawing at various water temperatureso This will :r~uin tasf.e panel evaluation of stored f'illetso ( 1) gutted., heads r;ff fish nuffibe~ of fish ~- 36 weight of frozen blocks ~ 189 l/2 1M averag<s weight of fiah ~ 5 1/4 lt:s 1rolume frozen blocks ~.3o65 ouv ft" density ~ 52 lb/cuo ftv (variation wi.thin 2 block~: 55 Jb/cu. ft. to 49 1/2 lb/cuo ft") (2) gutted, heads off fish number of fish = 28 weight of frozen blocks = 89 lb!z' average wefght of fish = 3 1/4 lbs volume frozen blotck = lo87 CUa f to density = 47 o 5 lbs/~~u. fto From the above it is appa::rr~nt that a. :;;;on~ido:>r:ib:.g ~1:::-cn ax:sts in t:ha method of determining volumes. It i:e equii.uy appar e,nt; that the stowing densityp which is importi!w.t from the standpoint of st.:j:rage capacity~ will probably be considerably less than. 50 lb/cuo fto gutted 9 heads i:i!ff fish number of fish = 36 weight of thawed fish l hour before filleting = 189 :t/2 1.bao (equal to the weight of the froz:en bloc:ks and sugg~sthr! of water absorption by the fish flesh during '~ha.wi.ng) 0

108 Yields = plate; fy'0~en f1sh..l_m;e:;~igo:r 1 average weight ;;,f fbh ~ 5 1/4 lba weight of processed fillets ~ 80 lf'?. l'ba ( 'com:rn.e::ccial yield 78 = 1 lb packs an,,jwixl.g 1/2 o~,, oveorweight per 1 lb pack) actual yield in % ;;Jf the g'l.rt t.ed 9 h.eadsjd weigm = 42o;~ commercial yield in % of th~ gu-t;!;e~d" headed weight o~ 4\ Salt absorbed _b;t: bl'il!~.1~~,j?n _'bl''b.i=;~ught cod." Small cod (heade> off w~ight lo5 tc:t:~ 2: 0 8 1bs) f~~o:z.en in saturated brine at =6 F in the gutted 0 heads on a."ld heads off',;;;,,:mditionsr absorbed a maximum of Oo35% salt in the ba.c:k m!lsc:l8 and a minimum ':lf ')o17%c Maximum an<8 minimum percentages sal i; :in the f1a.ps wen.1 1o25 and Oo5l% resper::tiv elyo Time of immersion 9 3 h;,ru:r'i1o When '~he fish were test,3d ~oon after freezing 9 the salt appearsd to erilia<"lc'e the tasteo Agai;u wh ~.n :fillets pr.;1pared from these fish were taated id. St;o.JohG..' s :ln Jarruar,y (afte;i"' bi.ocks had been 5 1/2 months in. fr.:: ~e.n atc:ra.gel no unusual am:n.h'"lt. of :Salt was noted by the panelistsv nor had the sal"\~ apparently e<(mtri~buied to a. :::edus;d;;io:n. in storage life' of th ; un:filleted block over that found in summer fish which ha.d been frozen in block.<j in the vertical plate freezex'o TASTE PANEL ASSESSMENT AT 8'I 1, JOHN S OF SUl\ll:MER AND FALL-CAUGHT NEWFOUNDLAND INSHORE FISK Sine~ WA' hat~;, <. :Lim."..tect lawwledge of the chemi,:;ajl and phys~.cai changes occurring in the ~..)111pc.nents of fish tissues duj:trjg 1;h9 fre.a:z~:;.=t.ha w ~ refreeze cycle 9 it is uecessar- y tc depend pr~marily or.< the senbory perceptible quality fai:;f;.rjrs as judged organol:?ptically tn.::,;:).:;; e7aluation of fish quality at an.y s i;age of processing o :r'or the ev-alu~:l-;ion reported below the technique of assessment by taste panel was employed,, The firs~ objective was to f'in1 C'lt the eff~;;-~ '-J>n quality resulting fr.:;m ;:mco and twice freezing Ne wfoundland.~n&1.ore c,od which ware i.n a :prerigo::t s ~a.<;:6 e.t time of first freezi.ng 0 and the ser;ond 0 t'c' diiferentiai;e, after oth.erwise comparable treatment 9 th.e qjality of sujr..mer~trf.tp.~caught cod which had been in frozen storage in the gut"~b<d.,; headed stat$ f'o:r e.bout 2 1/ ~ months from that of fall "hook a111 li.r1eq 1 caught; l(;od o For the la ~"ter comparison, once and twice frozen trap and fail~i:r.lght fish were ~:tsedo A tast~ panel wa~ not available in Newfoundland at t;he tim& when tr,r. summej: CcHi were ftozen,, hence these samples r.ad t c:: be held in fr,jzen Btorage for 2 1/2 m::n1ths bef;,:r-e being evaluated for qualityo * Personnel on the ta.s ::; s pan<16l were emplcyees cf ~be Federal Depa.rtmen t of Fisheries 9 Fish Inspection Laboratory 9 Sto Johr!;&." Newfoundland and of the New:f0un.d.l.s,nd Tcec;h!'wlogioa.l Unit o

109 The round 9 eviscerated fish were all in a prerigor c~onditiou when frozen (headed and gutted) in 90 to 100 lb bloc~ks using a vertical plate freezero The July-caught cod blocks were wzoapped in polyethylene and stored at -14 to -18 F for 2 1/2 monthao The October caught ~od blocks were in frozen storage for one day o The bloe;ks were thawed in l!>tat;er 9 filleted and refrozen in one pound packages i~ a horizontal plata ftaezero Fillets were cut from the prerigor tan fish before f~~eezing snd fjt:jzen i.n one pound packages to be used as controlso Before freezing thermoccup].es well:'il inserted at several locations in the thickest paxt r:lf the blocks9 the time required to lower the temperatures of the fal.l=caugh:t 4 1/2 to 5w b1cc:ks from 47 F to -13 F was 6 l/2 hours~ The CO!'responding thawing time a/-;; water bath temperature of 38 to 40'1' was 6 hourao Samples tested wers steam <OfJOkedo Por"t:~~.on::.~ of frozen fisb\ were arranged around the sid<e of a 600 ml bea.ke:r ;: the s 1 portions W&Jr-e kept upright by placing an inverted 60 ml beaksr w:i:~hin the 600 ml beaker which was then covered with aluminum foil and pla::';ed in a bcil::'.ng watex 'ba:tl1" No wa. ter or condiment was added; c;ooking time was 25 to ::,.'{) mi.nutes 0 Upcn removal of the 600 ml beaker fn1m the boiling watel'v the liquid fr:"lm the cooked fish was drawn up in'co the sma::uer inv-er led beake:r: o The mean scores of individual panelists far the various treai~ments 9 together with an analysis of this data may b : see;1 in Tables I 9 II and IIL A summary of the taste panel results is ghren in Fj.g" lo From the analysis of variance data there is convincing eviden ";8 of significant differences between the grades given the controls 1rs the :once. and twice frozen f:i.sho Similarly P there are significant differences among the once and twice frozen fall and aummer fisho There &re also significant differences between. panelistso Tuk:eyv s D-test may be used to "sort" out the differen~es between treatmentso Sx :-::: 8o98/7 = lo1:3y s2 = 8"98 belng th.61 E.st:imate of th& With 5 treatments and f = 24~ Q = 3o96o So, D = ~ = X = The experimental differences to be compared with D are shown in Table III" Of the 10 differences 0 5 exceed D = 4o2" The conclusions are thatlo Both the control and the fall fish frozen in t:b.e :rju.nd had a higher rat:b1g than fall fish twice frozen 9 and summer trap Hsh twice froz&no 2" The summer trap fish frozen in the round had a highe:..~ :eating than ',"he fall fish twice frozenc 3o There are no significant differences between :Btlmmer fish once and twice frozeno 4o There are no significant differences between once :~rozen summer and once frozen fall fish and between twice f't'ozen summer and twice frozen fall fish (in each instance the summer fi.sh (rouii.d) had been in f:zoozen storage for 2 l/2 months and the fall f:lsh had tnly r ecer.tly be><e<n frozen or frozen 0 thawed and ::ce,:frozen,. )

110 88 The hypothesi~ that there were no differencjes between pane.hstb was rejectedo Tukey 0 s D=test ~ also be used to vvaortw out the~e differ:ex1ceeo Foo- the pmelists 9 SJ: = 8o98/5 = lo34 and 4rv 24 = 4o54 D = 4o54 X lo34 = 6o08 The experimental differences to be compared with D may be se,e~kj. in Table IVo Of the 21 dif'fe;rences 9 5 exc6ed D = 6olo N.<l)JC,f'o :f the <Oth>8:t" differences are significant at 'the 5% levelo Panehs 1 c r grado::1d differently from panelists and lo Whew, the Ol'ganoleptic r-at;ix;_gs o:f panelist 7 were excluded from the data lmd );he analysis of the results for dif:fe:renees among the means repea.tedr ths ~onclusions ccon~geming trea:tm.enta wen WK~hangedo ~""'-~~- -"-=-~~. -=::c:-=..-~--==~,~~-~""' ""' "'' TABLE I =Mean score (!If aa<eh panelist loll >Eiammer a:ad fa1l eod after.:mot~< twice freezingo ii;d. ~~""" -=-C3l-== "'==-e~...,..._= -.:--".=-~--=--~"" ""'-" "1'"..::Q.::"--.,..,-,..,_~ PA.N E L I s T =-~ Treatment 1 2 : '1 Total Mean - Control 88o o8 88A o;2 89o4 605o0 86o4 Fall-Frozen o4 83o2 81o4 8 4-oO 92o8 59/o6 85o4 Round Fall=Twi<C~ 75o0 78o4 f34o2 74o0 73o2 77,b 80,8 543o2 Tlo6 Frozen SUDIDII6r (Trap} 82.o6 83o2 8?o8 80,9 83o0 84o '1'o5 83o9 Frozen Round Summer (Trap) 72o4 8001) o8 75o o Twice Frozen Total 405o2 404o4 433o o4 439o0 2894ol Mean Qo o5 79o5 82o3 B1o8 8207

111 89 TABLE II = Analysis of variance for data in Table I Sour~e of Variation Sum of Squares d,,f 0 Treatmentsg Control vs Freezl~v Tharlngv Refreezing" Among Freezing v Thawing~ 264o31 ~ Refreezing Between Panelists Error (Interaction and Discreptance)..,.. t Mean Square le Control vs FreezingD Thawing and R.efreezingg 2. Among Freezing[/ Thawing and Refreezing g F== 8 : 98 = Fo05=3.01 3o Between Panelists~ F = 2_!_ 4 1Q = ==-================================-====-=====~~~- TABLE III ~ Tukey 0 s ~test for treatments, ~~-=~-~.~-.. Treatment '.X: x-77.6 xoooso. l x=83.9 i~b5.4 Ccmt::ro Fall=fro~en ,5 round Summer (trap) Frozen round Summer (trap) Twice frozen Fall - twice 77.6 frozen

112 ~ 90 TABLE IV = Tr.lkey 0 s. D-test for panelists Panelist X l 2 4 5?9o5 104 la 6.,8 A Fall fish fr JZf.H\}. f! ~' l""i'"' (,. on -i"ro : ) ( f! dav " s'~ Y fi '"' '"') B Fall fish fl'~::' ~um l':;1~;i"('o'day~ ~t~r~a). - ' - c C Fall fish f:t'cj'z:gl< )"'Ull~L 1thawedo fille~~~d. and fill8ta Z'efr0zer. (0 days stoxag~) D T:t"ap fish f::oz sn :cl:':)wii.d (2 l/2 months.e:(:o~t ag!i!} E Trap fish f:c QzlS:o. ::o'ijyj,ii.. ;~hawed~ f:l.lleted &.nd : i1lr.,tb Y' Efr ozen ( ') 1 j 1,~...,''1 m y tj::.c... u., """,;.~ *r ' -'!b~ ~ Y """' '"''' ~. loo ~, Q ~ 10 re '-"' 60 ~ H ~ 50 ~ H 41"l ~ "' 0 30 ~ , Figo 1 = A B c -~----~------~-~-- Organolepti:;;; rating of steam c:ooked r;;od (ra:u. fish approach.ing :rigor and trap fish pre~ rigo:r before freezing)

113 91 SUMMARY NO 9 21 TASTE PANEL ASSESSMENT AT STo JOmPS OF PRERIGOR AND RIGOR FROZEN SUMMER=CAUGHT NEWFOUNDLA.ND INSHORE FISH" The state of rigor together with the m<bthod snd ra:~tl of freezing would be expected to affect the shelf=life of frozen fillets cut from fish which had been first frozen 111 in the round 111 and thawedo Preliminary experi= ments were carried out to test some of these ~ariabl~se Inshore cod taken by cod trap off Sto John n s in July were headed" gutted and immediately icedo Samples were taken after three hours and 48 b.ours in ice and frozen into blocks 1n vertical plate freezers aa drese;dbed in Sm.JWB.ey Noo 25" These samples represented prerigor and :rigcix' fif>ho Sum<e <of the prerigor cod were also frozen in brine as descx:fbed in Summa:ry Noo 25o All frozen samples were stored in poly~th.ylene bags at ~14 F for 5 1/2 months. They were then thawed 9 fille<{,ad am xoef'l"ozen as described in Summary No. 26 and the fillets were hsld a t =l4 F for approximately 2 weekso Samples of all treatments wex"e judged f<or quality by a taste panelo This panel was not available for e walua.ting the quality of the fish prior to 5 1/2 months storage of the once frozen sa.mpleso Inshore fall fish taken by 1 'hook and line' 1 w ere frozen prarigor and were used as controlso These controls were stored at ~14 F for two months. An analysis of the taste panel data is given in Table I" II and * Personnel on the taste panel were employees of the Federal Department of Fisheries" Fish Inspection La bora tory~ St o John~ s r Newfoundla.D.d and and of the Newfoundland Technological Unito

114 TABLE I ~ Organoleptic rating of inshore trap-cod~ comparisons among means, Treatment Paneli!:it A B C D E F G J:I I Total Mean =~ Control Prerigor> one~ frozen, plate.rigorp once frozen> plate Prerigor P one~ frozen" brine Prerigorg twice frozen, plate Rigor 0 twice fe'ozen., plate Prerigorp twice frozen 9 brine* Total Mean 8lo67 83~33 9QoQQ 85~33 79ol7 89o33 82o08 77o'f8 88o89 757c58 84ol8 77 coo 80S()() 82o22 79o03 75o56 89ol6 83,,3:3 7JJ;7 '73,33 fllo30 79o o75 93o33?loll 63o89 7lo67 70o67 74o44 83,33 662o52 73o61 74o04 75oOQ 77o55 7'lo55 77o41 88o86 76o42 70o0() o96 T'?o55 70o30 69o08 75~18 65o00 69o o60 74A4 74,:i4 656o06 72o89 64o23 77o50 75o83 62o22 6Qo94 79o00 72o50 7Jo'l5 75o23 639o2Q 7lo02 7loQQ 77 o50 78o33 74o16 76o04 79o99?Oc;66 76olJ?OJ)() 673/{'9?4o )28ol6 572o44 524AO 502, ,88 53lo A3 :t798,'-h n o45 8lo78 73o o89 73,74 78,06 76ol6 ~ ~ ~----~~~--~--~~----~=-~~ =-=--==-- * Round cod frozen in brine, t.hawed~ filleted and refrozen in plate freezerc 'IS

115 93 TABLE II = Analyses of variance for data in Table I Source of Variation S'um o.f Sq'lJB.res dofo M.s. Treatmentsg Control vs Freezing 9 Thawing and Refreezing 640., ,07 Among Freezing 9 Thawing and Refreezing Error (Interaction and Discreptance) HAg No difference for control vs freezing~ thawing aild refreezing< F = 640e07 = ~: No difference among freezing 9 thawing and refreezing. F = = Therefore HA is rejected. ~ is rejected. TABLE III = Treatment X x-11.o2 x x=73.61 i~74.86 x=77.55 x=79e03 Control Prerigor v once frozen 9 plate Prerigor? once frozenv brine Prerigor 9 twice frozen 9 brine* Rigor 9 once frozen 73o plate Prerigor v twice ,87 frozen 9 plate Rigor 9 twice 71.,02 frozen~ plate ~ *Round cod frozen in brine 9 thawed 9 filleted and refrozen in plate freezer.

116 94 In the analysis of variance. test there was c;.:mvincing eviden:r;e, of differences among the means a TukeyQ s ~test may be used to sort cruj~ these differences" For the treatments 9 :Lo )'[ =.?.2u12 9 "" 1 58 s 2-22o43 bel'lg '~:he e~:rb.mate of ir;he..<.o. p With 7' treatments and F = 48 0 Q "" 4o36o So" D = Q.~" "" 4._";t, ~:t the 5 percerd; significant;;;~ le~el the :f ll owir,g co:nclush,na may be drawn f:r'il)m the data show in Table III'~ t:n~a.tments ~ The control had a higher il)rgan<01eptic r.!'l:ttng than the fc,ll.;:iw.ing Rigor 'coa :fr.')z fil liiin. the :r. oundvr in ':Jertic;;al plat~ freezell>9 :rig.or 1JJd fr,ozen 11.i:!.1 the :~c:ucn.d" i.:n v-ertir:'ial p1ai:;e fx' ezexv t~hawod,, fi.lletcj and ref:r'ozen in horizoxrl;al plate freezer;; p:rerigo:t ;e,:jd fzoozen "in the rmu1c'f 11 ild. vertiaal platl!l freezer P tha.wedv filleted and reflt"oz.en in horizontal p\a't.s freezer' prarigor cod frozen 11 in "the round 11 i.n brine, tha,wed,, :filleted and refrozen in horizontal plate freezero None of the other differences for prerigm' and rigor fish war~ signifii:jan t at the 5 percent level o SUMMARY N TASTE PANEL GRADING AS AFFECTED :BY COOKING PROCEDURE EoJo Laishley WoJo Dyer Some preliminar,y results (see S~es NoD and 27) indi.caied that very different nsults were bf!iing obtain,&d 'by the Halifu Laboratory taste panel and one set up by the NewformdJ.~nd Unit and subsequent testing indicated that the discrepanc;y wa:b!."lot as great u " appeared at firsto However 9 'it was de ided ~o set up an experiment t'1:1 compare the procedures used in the two labo:ratorieso This was done in Halifax but 9 so far 9 rms been only pa.rtiahy completed by the Newfound1aJ'1d Unito The Halifax taste pmel uses samples baked in an cnen at 260 (500 FL without prior tha'llol:l.ng 0 and a scoring system wh:teh htii.~ been developed over the years for testing c:hanges in frozen fish occurring on storil.gt<o The procedure used by the Newfoundland Unit involved e:'c'oldr.g the sample~ by stemning and a sc:oring system de veloped primari.ly for degjr>ee CD'f freshness and bacterial spoilageo H must be realized 1(h.en t,ha'~ the h<fo

117 95 different cooking procedures and also different sc;ori.ng systems could not be expected to yield identical grades 0 though when the grades are related to 111 good if 9 "medium'~ and IWpoor" qu.alh:y fish 9 better agreement should resulto As well 9 the relative senaitivity cf the two procedures to quality differences is largely unkn.owno Three samples of c :Jm:IOOrdially prdcesaed orod fillets were selected, one freshly frozen 9 second 0 a m~dium quality ~ample etor'iid three years at =23 v and third 9 a py>or sample flfl:d<'.h ha,d been stored a m~onth at =12 9 followed by three weeks at =23 at time of shi.pnen:to The three samples were all good quality fish prier -to f;:,;>eezir.:~ o These sample!'il were teied;&d at Halifax in early January by both baking and steaming cooking pro~edures 0 along with the Nevfolmdland sampleso Part O>f s8.1~h l:ot was shipped by air freight in insulated boxes with d:ey 5"'1''1\:l t~o Sto John" s io.r testing there, lo Commereial off~hltl'x'~ tl"awlar"~~augh'!'; fishr :f:r-0zen December 12 after three days in iceo 5o Well handled fall inshore fish ~ October 9 l96lo The results of testing at Halifa.J: dll:r.ing the weeks of January 8 ~ and 15 are shown :LJ. the table and in Figs o 1 and 2 B..\1.d of baking tests ' undertaken at Sto JohnQs during t.he week of Febxuazy 200 Unfortunately 0 the original plan of the experiment was not tearried out and the ateam:i..'ng procedure has not yet been don~ in Newfoundland~ consequently ver,y little conclusions can be drawn from the worko Except for Sample Noo 1 0 there is very good agreement between ba.ld.ng and st~f!piing tests at Halifaxo The results of the baking tests in Newfoundland 9 which were probably st:ill preliminary 9 are however in excellent agreemento The four good samples are fairly closer and the other samples are all slightly higher than the Halifax scores but arrang&d in the same order o From the results it apperut"b that steaming ma.y not be as discriminatory in distinguishing differences in good quali.ty fisho Consideration of texture and taste scores separately indicates that the taste scores are not very d:i.scriminatory with fish in either the better quality or in the poor qual..i ty range 0 This is tl:"uei for 'both baking and steamingo Thus a scoring sy~tem which emphas.izes taste rather than texture would not be as effective in distingu.i.sh:i.rtg very g':;od fr(om fairly good fish 1, or similarly fish in the lower gradesa This at least partly explains the original differences in result~o

118 TABLE I = Comparative testing of baking and steaming Baking (Jano 8+) Steaming (Jan, 15+) Baking (Sto Jolul 1 s) (Febo 21) S~ple Texo Taste Grade So Eo Noo Texo Taste Grade So Eo Noo Tex, Taste Grade ScEo Noo Mean Mean Mean Halifax A , )5 5o c0 4o47 16 Halifax B o9 2o o7 2o o2 5o94 17 Halifax C o8 4o o6 3o o65 18 Nfldo } /3 3o o7 4o o6 4o80 17 \D Nfldo o9 3.; o4 2o o '\ Nfldo J o0 2o ol 2o78 22 Nfldo o7 4c o7 4o o26 17 Nfldo o o o02 19 Halifax A Dec" = 2 day icedf =26 B Novo = 1 month =12 9 then -26 c Jano 19" 1959 = 23 9 approxo 3 years Nfldo 1 Deco 9, iced 3 days '= commercial 2 Febo = frozen blocks.<: Ju&"le 22~ 1961 = trap cod,,j 4 Octo 24 - inshore 5 Octo ; inshore - well handled. All Brnnpl.t::s< ~<r,::;re (' :Jmmercially processed with the pos:r':~blf; E;.::r, eptinn ' / r.;<r::(." ~~).?

119 97 Fig o 1 = Texture and taste scores in baked and steamed samples of frozen cod fillets of various s~lected qualitieso cr, I * 'f, I \ I ' \ I \ I I I jf, / ' I/ ' "" ( 'o"' Texturr~ 'l'aste Baked s teamed SAMPLE NOO

120 98 90 ~ I 80 o o Baked 0 Nfld.,. Baked it =7\ Steamed 60 I J i ljq A < (l:j t.!l 50 = J I ~ SAMPLE NO" Figo 2 = Taste panel scores in baked and steamed frozen cod fillets.

121 99 SUDARY NO 2 29 BRINE FRQ&EN AND POST RIGOR PLATE FROZEN TRAP C!j'lD 9 AND COMMERCIALLY FROZEN TRAP COD FILLETS" D.I. Fraaer W.J. Dyer W.A. MacCallum While not originally a par G of tha f'reeze--"thaw=refreeze e:z:periment 9 some samples of round trap cod plate~frozen postrigor and some prerigor brine frozen samples prepared by tb.e Newfoundland U:ni t, were also tested. At the time of writing a few further samples along with some refrozen fillets prep1red from them are being exam3..ned. ThE< ::c'ebtu:'~;s to date are shown in Table I. The postrigor samples were trap fish ~aught J uly 21 and held on ice for six days after which they were frozen i.jo. the ;r>;~rt:lcal plate frea.z"""r (4 1/4" thick blocks) and stored at =23 and ~26 in a commercial storagso The brine frozen samples were prerigor trap cod frozen in b1 :ir,e jlj bloch,\ about thicko The results indicate that the po~trigor plate~frozen samples (Summa.ry No. 22). Similarlyr. the fish graded in the same rangev although drip was much lessp graded the same as the prerigor ones. brine~frozen only 6'J,a The data are too few to make f'irm. conclusions though they suggest that freezing the trap fish in the prerlgor or nea:r'-rigor state was net the cause of the somewhat low taste p&'lel ratings. Testing of commercially frozen trap fish samples was planned for the Newfoundland Uni to One sample only was obtained and examined in Halifaxg it graded the same as the experimentally prepared packs. More testing is necessary before one could suggest that this quality is typical of the whole trap fish production. Further data should bs obtained during the coming trap seasono

122 --~: TABLE I = Postrigor plate=frozen, prerigor brine=frozen trap cod and commercial fillets 0 Months Storage Postrigor BF3 Prerigor Brine~ Frozen BRF3 CommeJr.'dal T;r;.~ap CF 3/4 FiHdf' CF2* Moisture Drip Lipid FFA EPN AM NaCl Texture Taste Grade S.Eo Mean Number OG (!3.37) (±3.87) J,'J ol t 0 y (:!::4.74) (+Ll 76) * Stored at ~23 after receipt at 1;he laboratory ~========-====~============== -..,, _...,..~,.-_~,, ---= ~ST~"MMARY NOR 30 COLLABORATIVE EXPERIMENTS ON THAWING METHODS AND EF'FECTS OF MICROWAVE AND WATER THAWING ON REFR.!!.~ZING AND FROZEN STORAGE QUALITY OF PRE- AND POSTRIGOR COD FROZEN INITIALLY IN BRINE OR A PLATE FREEZER AS GUTTED p HEADED FISH E.J. Laish::.ey W.A. MacCallwn W.J. Dyer * C.M. Blackwood D.R. Id1er This collaborative experiment was set up among the u.s. Bureau of Commercial Fisheries 9 Technological Laboratoryr Gloucester, Massachusetts; the Technological Unit, St. John s, Newfoundland' and the Te:~hnc:lr.;g;.. c:al Station in Halifax at a meeting held in St. John s on December 1 and attended by J. Slavin 9 Director P and J 0 Peters of the Gloucester La. bora t;c;;:y, and DaR. Idlerp W.Jo Dyer~ W.A. MacCallum and C.Mc Blackwood. Phase I The process of microwave thawing was to ~e studied in Gloucestei" in preparation for Phase II 9 and water thawing by the Technological Un:l.tv St. JohnQs in preparation for the next trap season. * Left the Board before the March 14 samples were obtainedo

123 101 Phase II To measure the effect of the two thawing procedures~ o:q brine and plate frozen 9 pre and postrigor codo Samples of cod were co be omained in Sto Johnns on about February lr gutted; headed,, frvzen iu blocks and shipped by refrigerated boat to Gloucester. Freezing cu:rwes and other relevant data were to be carefully documen.tedo Fish as sillt};lar as :possible were to be processed in the prerigor condi'donp h.cdf being }:l:t i.ne f:r; A.m 0 the remainder being frozen in a plate freezer. A similar lo1~ ~ t,;,, b<'l ic::;,d for two to three days 9 until rigor had passed 9 and -;;w., 1x ta f;;: ozeu a.s aboye. Con.trol samples of similar prerigor f'ish were to b6 filleted~ packaged as l olb wrapped packagesp plate frozen (horizontal) and shipp~s'd to Gloucester whe::: e they would be repacked i:n cans under va:', uu.lli and ~to:;ced at =29 C. At Gloucester thb va:c::tour-. blocoks -~-ere to be tha-wed by the :c!lio methodsv micr"owave and water tr..aw:ingo Thf<tflafte:r ~:.hey we:c a to be filleted~ examined organoleptically r plate fxo.'~em. artd ::rcored at -l8 C (0 F) :i".qol4' 0 ;. The product: will oe f~s<8tad at two= month :i.ntenralsv from one day after refreezing to twelve mon.thso The Gl1.moeste.r La.borato:ry w7~11 org-anoleptically examine raw fillets 9 g;ade tr.e c;ookbd filiets by i;aste panels and determine thaw dripo Free fatty acids and protein disperaabili ty are to be determined at Halifax on samples shipped air freight under dry iceo Progress With the collaboration of the Newfoundland B:iological S"i;ation~ personnel of the Newfoundland Unit and the C:t"ew of the Ao To Cameron were able to bring in sufficient li'\:re c:od to permit completion of the pre and poetrigor plate freezing pha~e of the ezperimento However, in spita of another attempt to bring in fish w:i th the aid of the A. To Cameron and several attempts to use C<Ommerc;ial fisherme:a 9 i;g was not possible to obtain fish for the brine freezi:ng phase c f this i:o:-v<~st:igatj.,)no M.T'. Laishley came to Jtlhe Halifax Statiou in early March and wi. th the assistance of Halifa~ personnel brine freezing facilities were installed. A horizontal plate freezer had recently been installedo.artangements were made with staff of the Sta Andrews Biological StationQ and the crew of the A.T. Cameron were able to obtain sufficient li\re fish for the entire experiment (plate and bri.ne frozen pre and postrigor and controls) off the Western Bank during the vessel 0 s trip f'r,om Newfoundland to Halifax in the week of March 12. Mr o Laiahley 9 with the as sis tan.c e of Halifax per'sonnel 9 completed the experiment and arra.ngemen ts were made to ship the samples on a refrigerated vessel arriving in Halifax March 30o Mr. Slavin~ Director of the Gloucester Technological Laboratory 9 reports that the microwave thawing equipment is working well and they are ready to process the samples which will arrive in Gloucester on April 2o The contrt;l fis,h were processed in the postrigor condition rather than prer:igor as originally plann.eda This was done after mutual agreement,. to eliminate the :r:l.sk of the '-xm.t.rol samples going through r igor during processing or l.'!torage ~ it also served to reduce the requirements for live fisho

124 Crew t~f' the Ao To Ca.me:~n ca\ilght appr;:;x!mat~?.ly 2000 JLbe of!':lcjd~ in one drag lastilig 30 minutes 0 off tha fishlrj. b.anks of Sable IEilan.d on March 14v 1962o One thoa.~and 1bs of thio:~ o;;.;)d lh'l.s inm:j.:~:.>di.l:rte1y g11tted and iced while the remainder ~" lb~? :Lt'w~ 'tlla:tght) ~-as kept ali~re in fottr portable fish holding tanl...r;s aboa:rcvl. th<il Ao To Ce.mero~lo 0!1 ;rou' ;~ t r,om the fishing ground to ths &J.if~:U; Stat.io1n (7 b.:rs) ~ )0!~Dd ij.je-1.i];l the~e fish tankso They were!"amoved f:rom the tank 9 gutted. and :.rxli for u.ee as additional post!"igvr sampleo:<o On landing lb of live weight o.:x'l!'l (s\:.nd.) were lmmediat 'Jly guttedp headed and..teed fmr p~~erigor plat~ f:r ~e:tingo WHhin an!leu:.:." after the cod were S~i:>Xificed~ t...i.ery w~r<6 ps.cked int :: f. a1um:1.n.um pan~ C;tl\J :1.: 17 1 ' x 27" L Freezing rates -.rere re,;,.,_:.:r.d~d {Hc:~ne~eD. ts>:r!lpe:z abxre :r.~(>vr:ier) 'by inserting thermoc9u.ples into the nape am cd: ~~:Jt~"' d.eep dort-3al ID1J.t60le cf 5 cod in different pans 0 These }..le.!ls w~:re f;[n Bl:'t~J. 'bi t);!;:;.'l.r,~~~.1a111:'lnu.m sheeting in order to make con tad with!;he ~'Jd a r;n;;l:a;,;~ and 1Gf.'t" i;,;;:p p1att:~<!i of the Dole horizon.tal plate freezero Ea..:,h ahli!liiljjl.< p~.m held cf'jj!""'?:dmai;,ej.y 38 tc; 40 lbs of round gutted and headl5\i ~:;:rod ;-~cjd" The remaining live cod (~o 500 Ib Uv.~ weight) wer~ held in the Station~ s '37 F fish holding 1~a1:J.k f\:>r overnight k~~apir.g unti 1 tre orlne freezing unit was ready 0 By the nex1: monllingp it wa f~ared. that this dens.i ty of cod cc:rald not surrl'l!!e in the tar!k for any l.;,;ngth :Jf timt1 be\~ause of the filtration rate nec>a>ssaf.f i;;<q remo,: e the kji:>?.mendous am~imt nf i!l!ast'$ products being excretedo A jl:emp;;rary ar..~-~angement pr;y'r.:l.~ed Btme <:vicl~~.ttvtj.":'.1 filtering capacity o To further relieve \::hi~ cr.mditic"m., un t:u.. th._. br:tl,h freezing unit was operationa1, approximatsly : 150 1;::.,;f 1i.ve ::;od Wf>re trant1= ferred bac:k to a tank aboard t.he.ao To Came:rcmo Thea-c~ were k'~pi: a::~r~~ra by continuously pumping harbour water int,"':i the ta.nko A.rJ')t:h.ex' 50 Hlc of l:~t a cod were t:rffinaferred to the warm fish tank (45o:F) bu' : owi.'«< not tlse-d. in 1 th:is experimento The brine freezing unit was designed and bui.h by the Stat:l.. cr.:h~ technical staff and consisted of a 700 ga.ll n p2castic: fish holding tazxk (6 ft in diameter and 4 ft highl and evapor.ato:t 'Coil (ammonia u~ed a;:. refrigerant) and a mechanical stirrero The b1::-l11e sr.1lution ws 23% Na.Clo The cod (350 lb liwe weight of m:tx:ed. :~l~c:jt od and IDP..rkei) fox the prerigor brine freezing wer~ gutted. he.r;~,ded and il]oo.o They wer~s packed into twc; wire baskets (6 x 26 x 3?'vJ and pla ;;ed. intr) che brine {0 -t;o 1 F) to be frozenv within an hour af1t.e!' being sa(5rific:;:::do Each wire baskerii; was divided :Lnto three levels by 2 l?,"gj 8 ~f wil"e' sp9.cers fapp:::grlmat:ely 40 lb of cod per level) 9 to prevent the ~~od fr()m chl.mp;it~.g tc:lgi:yther intc a large solid mass whi<.~h would slow down the free2;ing Urn~ du.e to pno:r circulation of brine over th8 :round ccdo A f:;:'eezj.n.g.:mr;.re-< wa.s obtained by use of thermocou.ples plac!'sd i.nto a number r. f thds<.:c'd e.b PX'f.r:d0usly describedo

125 103 The postr:::..gor ple::~e and b:rine :3ampl ~s were frozen :.::1 the 8&.me quantities and manner ae< prerdously described f:rom the 1000 lb of g<:.tt.:'d cod which waa stored i.n ice for thras dayso The rema:lning '>Od wa' used for control samples in -thls ej::per:j.ment. The fish were ::~epa.:~ ~;i:e1d ~.. nto sc:::"c..d, market and steh.k.:;jj and.,.;e)(.:; pr:.v,;esca~d c::illill:sj:t"cia~.lly Lrt'J i lc:t.l;;crnen f:'.i3.~ts (200 lb) with '!;;he coope.r&.tiou of a cc:mnh';)x :::da)~ fi:n!!. The PX'd and. p)ertr.:- ~ '' samples fx ozen at the Ha1.::fax Sts.t.ion were w7apped i.n p).ty~ihy:l.s:>:h:j 8heet_\t.<;;' and sterad at ~15 F. Th~ ccy-:.rtrcl =ampj.~~,. cere aj.s') a i;:~r<.-: at -~I.5r'F afte:~ they were pa~:<k<;d by ;~h-9.';;:,m:jj.erc:<ia.l f_~j.'m" The f:t aezi:ng '~cr'!<e 1 '!Ire;~ ~ p1 0tt,~i fr":jill tt.e :r,sa~ :l 0f f:iyt8 th.e::: :n~ couple readings e:x,;ept f\:;,y "~..b.t-: prerigor brtne. 3:n :.:r~:.:,eh c;:;.x r ea.d:l.y.j Ei Wf.r;;~~ usedo From t.he. fr l:,t;;z.::;u_g-c,.r_,r~;e (F:ig'3o \ e.r;d ~~), 1J:.s :~/.d. E!.H.!!ipl,,,E' "''"?2tl frozen illl':t~e qi.:i.ich.:.;.y ;:;,y 'j;..»:~ne. ;tl1<m by p:u3:te f.r.'t.ib:;.;~' x:.g. T.nis wa.s d::;(.'l 1:,;:; '\~.'. better overall co:ntac:~ mad'!': by the ''l~'eulm;:ing :.:r"j,tje. a~ :>)mpa:n: d '': t:tw J)u it: freezer plates, The.'~r i'c:i~:a:~ f:ree~:;:';.;;j.g w:me c:f t.cu.~. :,:; :::.nges f:: :)m '(~ 23'='!? and was reached. in approxirnate:::.y 20 t,; 35 lll.~tx~i\.. ~: by 'brine f.,x ~"-'~Z.:'ng 9 w:o'. :.. : ~late freezing rgqu.~.z~e.j. abe.;t 4.5 T::' ::.;o mj.cx(;~,'-' ThB.fii:>r;tr ig:::.~ r;:.;ecia 8E-mp::., (.Figo 1) t.:,ok approx:ima1:8:l..y : " :tj2 lh.:tu:e :~.:::nge:t: ::c r.'v h O"F tl'-u:.t.n the ::;rre:: J.g.:~? samp1e 9 w'hieh was probably du.a t: J :pr:c~;," -:xnr~a. :t mad.c. :)ei;w~en th>&,,;:.cd ::r~.rfo.c:,~.' the thin aluminu!l1 sheeting &"Hi ~r.>b D.:;l<:~ f:.t-.'3e:r~i:n.g :pi,s\i;.::;s. St:.<z..:;d c:;;od aa:aq:..le.s were used in the plai9 tret>ze.. ~ as market :r: :i W<-!lre te:. ; ia~rge in d:i.aji!~ ~er ::o gi t be!twee!n the Dole f~ae:zi11g pld.teie< ( 3 : /~:31 1 ) o The samp=.el:.' ( =J5 F) we..: e. J-ackeJ :.a w~<lden -~ ""a tea :;ompl<.<ta~~;y insulated w"i th one :i.nr.h <Jf st:n\)foamo Tha c:este.f:' w~~re pj.ac..:d ::ll'i. the C<)l-:.t room for se ll"e.ral houxs bsf<)re the fish (wrapped. ~:r, p::11yethylene) w -"'"'"~ crated a:nd the shipmt=>..ni was l.::>aded Jast,. ee:;; ~ra A h<)urs after the ~oirlr.cj :n:,iaj. shipment was in P'.JSi tiono The trip from Hal:ifa:.&: :.) Gle<t1i2'ester ie, estima sed at 40 hours and the sr.g.pmen }; :i.a due to ar:d.'t~ on Ap:;: il 2. The refriger'atitm temperatm e :)n the rsssel il:3.orcated t ) be 0 F :: :t' c::..r.er. This experiment c~::,uli not ba 1re oeen ;;;ar::.<;.&j. t:lt witlwut tt.<: excellent cooperation of t:he building ma.intenan:,e staff and membe?."~z d' the process and produci~ resoa:t"ch g;rnup. Many work!:ld lor.lts hcn;;:rs ove:c a period of several days to install and opera'!;e> -:'-he ns~';essary fa.0ili tie~~.

126 ~ Postrigor plate samples 30 --{)--- Prerigor plate samples ~ Postrigor horizodtal Dole plate samples 20 ~10-30 TIME (MINUTES) =40 Figo 1 = Horizontal plate freezing curveao Prerigor cod O~dQ> postrigor cod 6-.6~.d 9 postrigor horizontal Dole plate CJ-D- D.

127 105 --o- Prerigor cod 30 ~ Postrigor cod ~ "Prerigorn brine -~- "Postrigor" brine TIME ( MINUTES) Figo 2 - Brine freezing curveso Prerigor cod postrigor cod Ll-4-L\-~ "prerigor" brine0-0-c- 9 "postrigor brine X-X-X.

128 106 NEWFOUNDLAND CODg RIGOR" ONSm' AND DURATION OF W o Ao &~C:alll:un H" Fougere Do G, KUis Co M:" :Blackwood Eo J o La.ishley E, Eo Coldwell Jo No Christie One of th~ obse:rvat:lons mad~ in r \elgard tg :t'.and.j~i:<1g Newfo~mdland cod is related to the onset and duration t;f rig~x " Summer=ea.ught (trap) fish ph readings 0 :eoupled with ivb\s Jft'e't~:ie>::;J.s (1n!'18\'~' were made on aevr,ral hund.loed pounds of {;Od held in i~~- st(:rag~ fn pe:rl;>'l.s of 9 to 98 hours~ :i.'h;l< fish were either iced at the 1l;;rap e.r.td the~af te.:.: (:w,,h~,., :"'n hour or two) gu:t;t.e-1" headed 9 washed and re=iced asho:t:,e 0 or were iceii E:,f; rl.~ -;~,~i' 8.r.:..d gj.tt~l'i; h~a~~~". washed and re=iced during the half hour long trip be rowecm. the trap and iht; stage, A small percentage was i:t1 1ni tial sr'tag'l5s of ::::?\.gt1r within 3 iz, 4 hon:t's of capture 9 temperatures of indhrid-uals being 33 F to 4"~ll'\, with most fi>!lh being at the lower end of the!'angeo In most mstant:~lil1 f':tf'ty :ij!i!l::~.srl.t or morl! of th!!; iced fish were in stiff rlgoi" at; 8 t:.j 10 h 1t.t:.: 'Bo Cm;d'i.m~: 1960 r~.snltei~ those of the present year indi ated that tbe ph of ic;ed tjrap :fish falls t.o e. Jew o:f about 6e0 to 6ol shortly after deatho This lr''fji wa.ln1'5 appears to ~J~ ;na:l~~---~ ;':i<'"'t up to 4 days or more caught=ageo From a cowille:jct~ial landing r::f se~re.&'al thousa:1.d pounds of tm:1ged. <:.ryil[j fish made at the Ste!'rs prem~.. ses a":r. t,he rea'~ 0f the C8h1e Bui1.ding 9 ll Jar,'~<!' number of evis erated.(;)od we:re examined one r.uur ot(~ :Jf wa t~!:' with tne fiii::j a:t a temperature of 45 F 0 the ambien~t tempe:;;>atu:re: being 65 F o No obsex-va.tioru:~ regarding rigor were ma.deo At 2 1/2!lours G at of wate!",, the splitting ope:rab..on being still in progre~ss 0 all fish whirc;h had yet t'j rec< t the cutters kn.:i.ves ~rer~ in rigor c A ae ond commercial landing of uni.t:~<'i fish was examine-d,~.t; a fl''i'!h.>h= frozen fi.sh plant, Ewiscerated fi.sh 9 abou-t:; 4 1/2 hnu~s {JUt «'Jf wate::l' ar;,d at a temperature of about 58 F 0 were j\ll.st being rem<:j'wed fi''()id the boat;o On.ly fl. :fe>'w of the fish appeared to be in firm :r.igoro Many more appeared tr1 b~ ir, a l~<l:j pronoun~ed ~tate of rigoro It was noted tnat some of th!! fish appeared to b., rubbery o Thr'ee frozen blocks wel<>e prepared fx" \llm thl.a landirii$o In a controlled experiment on~ lot xt' cod. l!!as~ ~,«.,;ed immediately at the trap and enother exposed to the sun for 2 ht;u~ i:n a;n a11!.l!ll:dnum boxp the ambient temperature being 65 F o Within 2 hrd;u:rs of :;.9'~;c:hing the second lo~i; was also icedo The first lot took approximately 72 J:"g;vurs and the se,~nd 36 houj::>s to enter and pass through rigoro

129 107 The curves (Fig. 1) show trends in ph values of fillets excised, from trap cod handled experimentally in various ways and from commercially-caught iced cod from the Grand Banks. The most noteworthy results are those related to fish gutted and iced at the trap and kept stored in ice, and to nonfeeding fish impounded for 30 days then stored in ice. The initial ph of 7 for fillets of fish gutted and iced at the trap and the subsequent drop to a ph of 6.2 is some indication that the glycogen reserve of these fish had not been expended to any great extent prior to killing. On the other hand, the ph of fillets from non-feeding fish impounded for 30 days dropped from 7 to 6.55 in one day and began to rise again indicating that the glycogen reserve in this case was expended for survival, This appears to be somewhat a similar sequence to that experienced with commercially boated trawl-caught fish which had been alive on the hooks for possibly 3 dafs - see below (Winter-caught (baited trawl) fish (a) Struggling magnified). Fall-caught fish (caught on lines with jiggers) Of about twelve fish obtained off Sugar Loaf and Cape Spear (near the entrance to St. Johnus Harbour) on November 8 and iced immediately, half were in rigor after 5 hours. Winter-caught (baited trawl) fish (a) Struggling magnified Baited trawl set on January 30 could not be reached before February 2. Forty-seven out of 55 cod taken were still alive. The live fish taken at a temperature of about 0 C from water of like temperatures were gutted and iced immediately. About 8 hours after capture, 7~ of these fish were in rigor. However, none of the fish went into stiff rigor throughout the period of observation amounting to 6 days post mortem age. The fish had apparently used up much of their glycogen reserve during the period on the hooks. There was no evidence of significant drop in ph after death. (see below). ph readings {p 9 m 9 = post mortem) Sample Weight 5-6 hrs. 7-8 hrs hrs hrs. 69 hrs. No. Kgms. p.m. age p.m. age p.m. age p.m. age p.m. age ~ a ,

130 100 -J 0 \ \ \ \ \ \ \ Fig. 1 =TRENDS IN ph VALUES OF FILLETS EXCISED FROM COD STORED IN ICE. A and C = Gutted and iced at trap. B ~ Gutted at trap but exposed 3 hours at 65 F then iced. D = Gutted at trap but exposed 1.5 hours at 65 then iced. E ~ Eastern Grand Bank cod iced on board vessel. F ~ Tank fishv 30 days non-feedingp iced when killed.

131 109 (b) Normal struggling Cod intended for ~e in the Gloucester experiments (Summgr.y No. 30) caught on February 7, 1962 under conditions given below: Count Count Depth Temp. Count Fish Fish Location Water Water Length Fish Caught Sacrificed of Catch (metres) oc Drag Caught Alive by Net 22 miles S.E. 150 surface 1/2 hr Cape Spear -0.1 bottom miles off 150 OQO 1/2 hr Bay BUlls 4 miles off 150 o.o baited approx. approx. St. John's trawl set 20 hrs were (a) gutted and iced soon after capture~ or were (b) held alive in seawater at 0 C, sacrificed and iced 7 hours after capture. As seen from the table immediately below, over half the representative samples from a total of 26 trawler-caught cod handled as in (a) above were in rigor 6 hours after capture. Feb. 7/62 Feb. 7/62 Feb. 9/62 Feb. 10/62 3:12 p.m. 9:30 p.m. 6:00 p.m. 1:30 p.m. Sample (time of catch) state state state No 11 ~th 7m) TemE 11 EH :e!! of rigor :e!! of r!gor :e!! of rigor J..t 1 72 o.oo rigor 6.5 rigor 6.8 sl. rl.g. o ~ 2 51 o.oo rigor 6.7 rigor 6.7 sl. rig. Cl>..., rigor 6.6 sl.rigor 6.8 sl. rl.g.:l rl 4 52 o.oo rigor 6.8 rigor 6.8 sl. rig o.oo rigor 6.7 rigor 6.8 sl. rig. 'i 6 54 o.oo prerig. ~ ~ 7 63 o.oo prerig o.oo prerig. ~.~ ~ t;j 9 61 o.oo prerig. Q)... Cl> :;Jet

132 110 The fish remained in rigor for about three days at which time they were frozeno The table immediately following gives data on 7 trawler-caught cod caught earlier in the same day and handled similarly to the 26 samples. Three of the seven fish were in firm rigor 9 1/2 hours after capture. Feb. 7/62 12~55 p.m. Feb. 7/62 Feb. 9/62 Feb. 10/62 (time of catch) 9g30 Pomo 6~00 Pomo 1(~0 p.m. Sample Tempo State State State No. ph of cod ph of rigor ph of rigor ph of rigor C 6.8 pre rigor rigor 6.7 rigor 6.9 sl. rigor rigor 6.6 sl. rigor 6.7 sl. rigor pre rigor rigor 6.6 sl. rigor 6.8 sl. rigor prerigor prerigor All fish were judged suitable for freezingo Conclusions~ All data available to date would indicate that summerp fall and winter-caught cod start into rigor a few hours after capture even when chilled in ice and this appears to be true even for winter-caught fishp when taken from water at ice temperature. About half the fish can be expected to be in rigor within 5 to 10 hours of deatho The duration of rigor can be expected to be about 3 days. The period of stiff rigor would be shorter but has not been defined. As would be expected 9 the degree of rigor was found to depend upon the amount of struggling the fish had undergone. Observation has shown that of the quanti ties of all cod avai.lable for freezing within the first 3 days of catching~ a significant proportion will be in incipient or in late stages of rigor. It is therefore important to determine the quality of frozen blocks and fillets produced from this raw material.

133 111 SUMMARY NOS 32 NEWFOUNDLAND COD: PH MEASUREMENT AND SPOILAGE He Fougere H. Wight The tendency of ph to seek lower levels as exemplified in curves D~ E 9 C (see Fig. 1, Summary No. 31) at the 6th and 8th day of fillet storage and a similar tendency though less obvious in curve Fat the 3rd dayp prompted further investigation to ascertain if this phenomenon is a common occurrence in spoiling fish muscle. Thereforep two hundred pounds of trap cod with guts in were landed at the laboratory. The history of this fish is little known but it is believed that it had been exposed to atmospheric temperature for at least three hourso They were eviscerated and stored in ice. Five fish were filleted 9 thus giving ten filletsp which were placed so they would not touch one another in a deep pan which was sunk in ice in an insulated ice boxo The temperature of these fillets was maintained at approximately 33 F except during the time when they were removed from the ice box with the pan, for ph readings. Each day 9 ph readings were taken~ always as much as poss:i!ble at the same spots on each fillet'- that is~ at both ends and at the middle section. During these operations the fillets were not removed from the pan and remained in their original order. In Figure 1 9 each point on curve A represents the average of 30 readingso Alsop each d~ six fillets were excised from three iced fish and ph readings made on them in much the same manner as above 9 then discarded. Each point on curve B represents 18 readings. Fu.rthermore 9 the quality of the fillets 9 rated by odour perception 9 was followed each day. Since the fillets of curve A were stored in the pre-cut state and were subjected to contamination during the filleting operation 9 evidently both curves A and B represent different spoiling rates. In curve A, the drop in ph after the 4th day is of short duration and together with the subsequent rapid rise is commensurate with a rapid spoiling rate. In curve B the drop in ph is more prolonged and the subsequent slow rise is in agreement with a slower spoiling rate. This phenomenon 9 of temporary drop in ph 9 may be linked, at a certain stage of spoilage 9 with the production of carboxylic acids resulting from bacterial deamination of amino acids to depress the ph level of the medium as long as the volatile bases are not generated faster. The rate at which these volatile bases are generated and the consequent ph levels depend on the concentration of spoilage bacteria and hence spoilage rate. It is at the point of rising ph that incipient odours begin to be noticeable. In curve A these odours were perceptible at about the 6th day and in curve Bat the loth to the 11th day. Fu.rthermore 9 the fillets excised from starved fish stored in ice 9 Fig. 1 9 curve F (Summary No. 31) 9 had perceptible seaweedy odours at the 6th day and strong seaweedy odours on the 7th day of storage" The fillets of the starved fish therefore spoiled at about

134 \} ~ 0'1 0'1 0'1 0'1 0'1 0'1 ~ \J1 0'1...;J tt P> t-< Cll I I I I I I I I d (\ 0\\ 00 Ji J ' ' ' ' \ b ~ ~\ ~\ ' ~ I \ \ b, ' ' f:g ' P>' 0 ' ' '0 ttl... 1-'~--~----~----._--~~--~----~--~-----L----L----L----~----L----- Fig. 1 = TRENDS IN ph VALUES OFg FILLETS STORED AT 33 F ~ -; FILLE"rS EXCISED FROM FISH STORED IN ICE:

135 the same rate as those of curve A~ indicating generally that the odour threshold ordinarily associated with s}ltluai;e is_}lot always related to the ph level of codfish muscle. The ph levelv in addition to its association with spoiling rates resulting from the manner in ~hich the fish have been handled from the time of catching to processing 9 such as in the case of the fish represented by curve A~ Fig. 1 9 could also be associated with starvation as occurring in pre-spawning and spawning timep a concept which seems to be in agreement with that of the difference in susceptibility to spoilage with the seasons of the year. SUMMARY NO 2 33 NEWFOUNDLAND COD~ SUSCEP1'Il3ILITY OF TRAP COD TO D:m'ERIORATION AT ELEVATED TEMPERATURES H. Fougere H. Wight (a) Less than Severe or Rough Handling Three hundred pounds of fish were eviscerated at the trap immediately upon being taken on board and divided into two lots. One lot was iced immediately on board the vessel 9 the other was packed in an aluminum box and left exposed on deck. They were landed within one hour. The iced fish were stored with further icing in an insulated box at the pilot planto The un-iced fish were given an additional one hour exposure in the sun at 65 F 9 then well packed in ice for storageo The fillets of both lots were generally of good physical consistency up to about the fifth day in icep but began showing gaping and sogginess on the sixth and seventh dayo On the seventh day none withstood water conveyance as practiced in some filleting plants. They broke up into pieces. Several similar experiments showed that trap fish varied in degrees of susceptibility to physical deteriorationo The fillets of some began to show signs of gaping and sogginess with appreciable drip formation on the fourth day while others showed similar behavior on the fifth or sixth day. While there was no external evidence of adverse effect of exposure of the fish to an ambient temperature of 65 F for two hours prior to icingp it should be noted that subtle proteolytic changes affecting the storage quality of fillets frozen from these fish might well have occurred in this intervalo In another experimentv 100 fish were picked at random from several thousand pounds of commercially-landed codo The fish had been out of water for about one hour and were at a temperature of 45 FP the ambient temperature being 65 F. All fish were firm as were the fillets cut from themo A one hundred pound lot from the above was then iced and inspected daily.

136 ll4 Up to and including the foufth day the fillets were firm with no perceptible odour. On the fifth day they showed signs of gaping and on the sixth day they were disintegrating with perceptible juiciness as if they had been slightly cookedp but had no spoilage odour. These fillets would not have withstood water conveyance as practiced in some filleting plants. On the seventh day they were slightly sour. One lot of trap fish with guts in was exposed outdoors at 65 F for eight hours. Every half hour or so the whole lot was transferred to another container to mix the fish to promote eveness in temperature. a) After three and one half hours the fillets were still firm - their temperature was 60 F. Their krinkled. surface after one half hour storage at 32 F indicated that they were still in the shrinking process and hence still going into rigor. b) The fillets excised after four and one half hours were of doubtful quality physicallyp although good organoleptically. The krinkling surface due to shrinking was less evidenc~, indicating that the rigor process was slowing down. c) At sb: hours the fillets were of poor physical quality. The belly flaps showed' yellow stain, gapingp and looked as if they had been slightly cooked. The shrinking process at this stage was terminated. d) At eight hours the fillets showed distinct changes. They were wet, soggy, flaky and tended to break up when lifted by one end. They were definitely not of good physical quality and the fillets had a muddy colour. Thus, evidence of deterioration of fish held at 65 F was found after about four hours storage at this temperature, whereas it had not been noted at two hours. Fifty medium size eviscerated fish were exposed at F and frequently transferred from one box by dumping them into another from a height of about four feet. After four hours of this treatment the fillets were soft, gaping, wet and of generally poor physical condition. One lot was left exposed at the same temperature and untouched for four hours. During the following half hour they were transferred several times from one box to another in the same manner as the first lot. The fillets of these fish were of the same general consistency of those of the first lot - soft 9 wet and gaping. (c) Effects of "Bagging" It often happens that fishermen will not land all the fish which may be found in a trap either because they haul their trap too late in the evening or because the filleting plant cannot process more on that particular day. In this case it is common practice to bag the fish. This consists of crowding them into a meshed bag,somewhat similar to a cod end

137 ll5 of an otter trawl,which is kept afloat near the trap for several hours or usually overnight or until such time as they can be disposed of. These fish usually die within a few minutes. Thus 9 twenty-five fish which had been bagged overnight, the exact history being unknown, were delivered to the laboratory for examination. The fillets from these were of the poorest condition yet seen. The fish were only good for the fish meal plant. (d) A Comparison of Trap Fish and Bank FiSh Caught During the Trap Season. c Cod fish caught by the A.T. Cameron on the eastern edge of the Grand Bank were gutted and iced fifteen minutes after being on board and landed three days after. They were stored in ice at the laboratory. The following is a brief summaxy of the observations on these fish. 3rd day - Fillets were firm and of prime qualityp both pbysically and organoleptically. 4th day - Fillets were firm and physically better than those from trap fish for the same period in ice. 5th ~ - Fillets firm, first grade quality physically and organoleptically. 6th day - Fillets firm, very good quality. 7th day - Fillets firm - otf odours. 8th day - Fillets slightly soggy, good sheen, good colour, off odour. loth ~ - Fillets soggy - sour odour, quite wet. General observations have indicated that the muscle of trap cod in Newfoundland is not soft at the trap. Under ideal conditions,and within limits short of these 9 the muscle of this fish will comply with the requirements of good filleting practice. However 9 the susceptibility of these fish to physical deterioration is greater than that of cod caught on the Grand Bank during the same season. Even under ideal conditions of eviscerating and icing it is not recommended to keep trap cod longer than four to five days in ice, whereas a Grand Bank fish will still produce rather relatively firm fillets,even though spoiling,after eight to ten days in ice. Trap fish should be eviscerated, landed and iced in the shortest possible time before being filleted. They should never be bagged 9 even for a few hours. If they must be transported to a distant plant 9 they should be well iced and packed in the same manner as on board a trawler with dividing boards in order to minimize excessive weight and crushing of the bottom fish. These fillets should never be water conveyedo

138 ll6 SUMMARY NO. 34 NEWFOUNDLAND COD: IN CAPTIVITY LOSS OF WEIGHT OF COD H. Fougere H. Wight On June 26, twenty live fish were put in a tank of 500 gallons of seawater maintained at 37 F in the Cable Building. Aerated water was constant~ recirculated through a charcoal filter and through a heat exchanger maintained at the desired temperature. After a short acclimatization period of two days,during which it was assumed that the food in their stomachs was digested, the fish were individual~ weighed and tagged. In the meantime, trap fish landed during commercial operations were eviscerated and gilled and each specimen weighed and its length determined. In addition Dr. Templeman of the Newfoundland Biological Station provided further data gathered during preceding years on weights of gutted and gilled trap cod versus their lengths. The purpose was to establish a statistical length-weight curve of trap caught fish as sold under current commercial conditions as a basis for comparing gutted w ights per lengths of non-feeding fish kept in captivity. After ten days, the impounded fish showed signed of a condition called "pop-eyed". After fifteen days they were totally blind. After 30 days the loss in weight was on the average a% of their initial weight ranging from one individual to another in the order of 5 to 1~. The fish were then eviscerated, gilled and their weights and lengths recorded. The gutted and gilled weights were plotted against lengths to ascertain if ths,y fitted within the curves plotted from weight-lengths obtained from commercial landings. The vertical lines in Fig. 1 represent the upper and lower weight limits per length of fish landed commercially. In general, the results as shown are based on too few specimens to permit statistical conclusions to be recorded; nevertheless, the trend seems to indicate that at 15 and 20 days the fish do not lose gutted weight and that at 30 days, at least under condi tiona simulating those of the present experiment, a. fisherman would sell on the aver&l'e the same gutted weight as he would ~e had he sold his fish gutted immediate~ after capture.

139 117 Fig. 1 - Gutted weights vs lengths of non-feeding Newfoundland traf codfish impounded at 37 F for 15, 20 or 30 days (0,.",.Q.) and the weight limits for given lengths of 150 commercially landed fish ( ) a g b 11 & 2.-A 00 o~o o 0 9oo LENGTH IN CENTIMFJrERS 65 70

140 118 SUMMARY NO, 3~ COD LIPIDS: LIPID IN NEWFOUNDLAND TRAP COD E.G. Bligh Margaret A. Whitman During investigations concerning the freezing, thawing 9 and refreezing of Newfoundland trap cod (Summary No. 22L it was found that the muscle tissue of these fish contained approximately 2o% more lipid than normal~ found in cod muscle. It was therefore considered that a study of the lipid composition would be desirable since the increased lipid content may be responsible for some of the characteristics of trap cod fillets. A sample of commercially prepared Newfoundland trap cod fillets which had been stored at -10 F for 3 months was extracted with chloroformmethanol-water and the composition of the extracted lipid determined by means of silicic acid chromatography and chemical analyses. This sample of trap cod contained 0.85 g lipid/100 g flesh, whereas cod generally contains about Oo7 g lipid/100 g muscle. The chromatograph for trap cod muscle is compared to that for normal fresh cod muscle in Fig. 1. The graphs clearly show that the on~ difference in the samples is that the trap cod contained more free fatty acid (component III) and sli~htly less phosphatidylethanolamine (component VI) and phosphatidylcholine (component VIII) as a result of frozen storage. Chemical data also showed that if both samples had been fresh, the lipid compositions would have been practically identical, Thus it is very doubtful that any of the atypical characteristics that Newfoundland trap cod may have are due to the lipids present.

141 j ~ H ~ ~ H E-t 0 ~ FRACTION NO. Fig. 1. The separation of the lipid components of fresh cod muscle (top graph) and Nfld. trap cod muscle (bottom graph) by silicic acid chromatographyo

142 120 SUMMARY NO. 3p ULTRACENTRIFUGE PATTERNS OF MUSCLE PROTEINS OF NEWFOUNDLAND TRAP COD D.G. Ellis J.T. Lauder D.I. Fraser A.s a result of the experiments on refreezing of Newfoundland trap cod, three series of samples were available in frozen storage at -10 F. 1. Prerigor round fish, frozen in a vertical plate freezer. 2. Fillets, prepared from sample No. 1, one week after freezing and immediately refrozen. 3. Commercially packed frozen fillets, frozen the same day as sample No. 2. The previous history of samples 1 and 2 was reasonably well known. Since there was little information available on the muscle proteins of trap cod, the ultracentrifuge patterns of the actomyosin system of proteins was determined for each series of samples. Unfortunately unfrozen prerigor samples were not available for comparison. The round fish was examined three weeks after freezing. The ultracentrifuge pattern of the protein was as expected for the storage time, with a normal amount of dispersed protein. There was slightly exhanced amount of the smaller protein components, indicating some dissociation of the actomyosin. After 2 months and 4 months storage these samples showed reduced dispersed protein and increasing dissociation. The refrozen fillets were examined 2 weeks, 2 months and 4 months after initial freezing of the original round fish. The 2 week sample showed slightly reduced dispersed protein and advanced dissociation of the actomyosin. The undissociated.protein was 37% of the actomyosin system as compared to 6CJI, for extracts of unfrozen prerigor muscle tissue. In the 2 month sample the dispersed protein was further reduced and the dissociation of the actomyosin almost complete. The frozen commercial:;fillets were intermediate between the first two samples with regard to ultr.acentrifuge patterns of the actomyosin proteins, after 3 weeks and 2 months storage. Previous studies of the ultracentrifuge patterns of the protein of frozen cod muscle have indicated that either the molecularly dispersed protein or the degree of dissociation of the actomyosin are more sensitive measures of the protein changes during frozen storage than the extractable protein. No attempt has been made to correlate either method with storage quality.

143 121 The patterns for the refrozen fillets suggested that even after two weeks storage the protein changes were well advanced. The commercial fillets, at three weeks, showed little evidence of these protein changes. After two months, however, there has been considerable protein denaturationo Even after four months storage, the round fish samples showed considerab~ less protein alteration. Ultracentrifuge patterns for the three samples under comparable conditions are shown below. The advanced protein changes in the refrozen fillet samples are indicated in FigureJl. ;;,',J. A B c Fig. 1 A - Round fish in frozen storage for 3 weeks. B - Refrozen fillets prepared from frozen round fieho time 2 weeks. Total storage C - Commercial~ frozen fillets in frozen storage for 3 weeks.

144 122 SUMMARY NO, 31 QUALITY OF FROZEN COD: l.. COMMERCIALLY FROZEN COD FILLETS AND CHANGES ON STORAGE W,J, Dyer E,J. La.ishley D.I. Fraser ~le many studies of the rate of quality change, as measured by taste panel, protein "denaturation", lipid hydrolysis, etc., have been reported previously on particular lots of frozen cod followed overf~a period of storage, no studies have been made of the initial quality of the commercial product at various seasons of the year. Similarly, the deterioration in quality on storage of samples taken at various seasons has not been carried out on a sufficient number of samples to allow correlation of the results. Taste panel testing of the number of samples required for such a project involves a tremendous amount of effort, not only in obtaining samples, preparation of the cooked products, and computation of results, but also in the time of the taste panel personnel participating in the tasting sessions. Consequently, the control samples used in taste panel testing of the freeze-thaw-refreeze experiment were selected to supply some of the required information. See also Summary No, 22, reported in the section on Newfoundland cod. Freshly frozen commercially prepared samples were obtained from a local processing plant and stored under various conditions at the Station, usually at either -23 C or at -12 C, Some ~amples stored up to three years were used. Taste panel testing was carried out by the procedure in use for several years. The still frozen samples were baked in an oven at 500~. Three to four samples were served at a time to a panel of eight testers. Each frozen sample was tested on three separate panels. One control, unknown, was usually included in each panel, Data on protein extractable in neutral 0,6 M salt solution were also obtained, though not necessarily at the same time intervals. Duplicate ext~actions were made and~ reported separately, It will be seen from the results that the variation in duplicates is much less than that sometimes reported in the literature. The results as related to storage time and temperature are shown in the Figures. During the first month after freezing, the taste panel scores on the samples stored at -23 ran from 70 to ~' with the exception of one August sample which scored only 56%. On further storage the grade gradually decreased, but even after three years, one sample graded 45%, the other was 24%. The storage life might be expected to depend on type of packaging, These were 1-lb consumer-type blocks, packed in waxed cartons and overwrapped with cellophane. The samples stored at -12 C deteriorated very rapidly, becoming unacceptable in a month or two.

145 123 The extractable protein values at -23 C decrease in a manner almost identical to the taste panel scores in contrast to some literature reports. At -12 the rate of decrease appears to be slight~ faster than taste scores. The agreement between duplicate extractions was very good. Since, as shown below, changes in the proteins during frozen storage occur in stages, and several other factors contribute 'to the taste panel scores, the latter agree remarkab~ well with protein extractability values. These results indicate that many different commercial samples yield quite consistent results.. As yetp insufficient samples have been tested to indicate any seasonal trend though it was noted that one August samples was rather poor. (Table I and graphs). Recently, the provision of ttme-temperature-tolerance data for various species of frozen fish has been brought to the fore by the appearance of a small time-temperature indicator on the market. These gadgets can integrate the time-temperature experience of a plrticular sample and give results more or less similar to the effect of those conditions on the product. Moreover, they have the distinct advantage of giving a visual indication. The use of these indicators along with packages of frozen product during storage, and especially during transportation and distribution, should bring home to the people handling the frozen product, the effect of poor handling on quality. At the same time, the argument shifts from actual quality of the product, a very difficult thing to measure accurately, to the more easily defined conditions during handling. Of course, the provision of reasonable time-temperature data then becomes necessary, so that the various frozen fish merchandisers can set reasonable limits of handling conditions.

146 124 \. TABLE I - Taste panel grades of stored cod fillets. (Standard error of the mean in brackets followed by number of tastings.) Date Date (Tested J Storage Storage Processed A.p.J>.rox_. TemJ>. Time Grade EPN Time-Temp Jan 15/62 Feb 15/62-26 C 1 month (:!:2.81)21 p 09 ) mos at ) 0 Jan 19/59 Dec. / mos. 23.8(:!:4.20~8 g:~~0.95' 35 mos at -23 Jan 19/59 Jan 8/ mos. 45.2(~ Feb 7/62 Feb 15/ week 78.0(±3.83)25 Feb.; 7/62 Mar 15/ weeks 1 7 ~~ wks at -26 Feb. 15/61 Oo35)o mos at ) Mar 10/61 Sep 10/ mos. 6o.o(:!:lo.7) 6 Mar 30/60 Aug 8/ mos. 42.5(4.53)8 Mar 30/60 Sep 26/ mos. 46.0(~.99)10 1o59~ mos at Oo23~o mos at Apr 17/61 Sep 19/ mos. 70.0(-6o83) lo85 8 mos at Sep 19/ mos (-5.85) mos at Nov 28/ mos. 26.8(-3.63)22 Apr 13/59 Dec 12/ mos. 5.0(:!:2.29) ) mos at ) 0 May 27/60 Sep ]8/61 ' mos. 11.7(:!:3.07)6 Aug 1/61 Aug 8/ week 55.8(4.74)24 Aug 1/61 Sep 20/ weeks 65.6(:4.91)16 Aug 1/61 Oct 4/ mos. 61.2(~o41)8 1.95) ) 4 1/2 moo -26 Oct 26/61 Oct 26/ day 77oo(:!: 7.1)6 0.45)0 44 OA2) 4 1/2 moo =12 Nov 3/61 Nov 8/ week 72~5(~3.71)24 Nov 30/ month 6~~ )7. + Nov 23/61 Jan 8/ month 25.9(-2.95)22 g:~i~ wks at wks at wks at -9 Dec 4/61 Dec 12/ week o7(:!:2.79) wks at Jan 8/ weeks 68.2(:4.19)22 Dec 12/61 Dec 12/61 - Live 80. 7(:!:3.23h4 pre rigor

147 Fig. 1 - Taste panel scores in commercially fo~en cod fillets stored at -23 and -12 C C X -12 A Prerigor, unfrozen Ol J>!l ~ \J1 ~ 0 0 Ol 0 l * I.J. I * I J.! 36

148 126 Fig. 2 ~ Extractable protein nitrogen in stpred commercial~ frozen cod fillets 0 0 ~ 0\ ' ' +c3... C\l... ' 0

149 127 SUMMARY NO. 38 QUALITY OF FROZEN COD: COLLABORATIVE TESTING OF COOKING PROCEDURES W.J. Dyer D.I. Fraser There are three main ways of cooking fish in general use, baking, frying and steaming, although 11 of course, there are many variations of each. Preliminary observations (see Summary No. 28) have suggested that these cooking procedures may not result in cooked dishes of the same level of taste appeal with a given quality of fish. For instance, a very good quality freshly frozen fish may score "extra good" when baked bu~ on~ "moderately good" when steamed. Differences may be even greater when poorer quality fish is cooked 9 and this is where t}!.e consumer and the fishing industry should be vitally concerned. Since there have been no studies in this regard, a collaborative experiment was set up with Miss Margaret ~er and Miss Ruth Macintosh of the Home Economics Section of the Department of Fisheries in Ottawa to study this question. Discussions on procedures for cooking, methods of scoring, training of taste panel personnelp etc., have been held both in Halifax lind in Ottawa. Two lots of preliminary samples have been sent to Ottawa and preliminary taste tests have been conducted. It is planned to test the three basic methods of cooking, steaming, baking and frying, using standardized procedures with various qualities of fish. The latter will be supplied by the Station and the cooking and tasting will be done in 4 0ttawa. Two types of deterioration will be assessed, that due to storage of the frozen products, and that caused by handling prior to freezing, primarily bacterial spoilage. Samples of first grade cod with good, medium and poor histories of frozen storage are presently being prepared. Samples of Grade II and III quality cod, each with similar frozen storage histories will follow. SUMMARY NO. 39 QUALITY OF FROZEN COD: TOO'ERATURE EFFECT OF VERY LOW D.G. Ellis D.I. Fraser W.J. Dyer A system of freezing, or of lowering the temperature of frozen fish to very low temperature for shipment in insulated packs rather than in refrigerated cars or ~rucks, has been introduced. It uses liquid nitrogen

150 128 which quickly drops the temperature to very low levels. The strains set up by such rapid lowering of temperature sometimes result in visible cracking in the product. It was not known whether this treatment affected the product, and consequently some experiments were set up to check this point. Freshly frozen packaged cod fillets, and also unpackaged fillets were submitted to rapid cooling by liquid nitrogen (supplied by a Montreal company). The temperature was lowered several times to below -730C and the samples were then stored at -26~. Taste panels were conducted a few days later, and more fish, along with untreated controls, will be examined after various periods of storage. The control sample scored 68, 65 and 65 units in texture, taste and overall grade respectively, while an unpackaged frozen fillet immersed in N2 for five minutes, then stored for three days at -26 C, scored 64, 67 and 64%. Thus there was no immediate effect on quality which could be detected by the taste panel. After 4 months' storage, the scores of the control were identical to those of the treated fish.

151 129 SUMMARY NO A 40 QUALITY OF FRESH FISH: METAL-INDUCED RANCIDITY NON-BACTERIAL SPOILAGE: C.H. Castell J. Dale N. Dambergs During this past year some studies were made on the effect of trace amounts of heavy metals on the preservative action of chlortetracycline when applied to fish muscle. Those experiments involving copper ions were invariably unsuccessful because the copper itself caused the fish to develop a series of rancid odours. To a lesser extent 9 iron had a similar but more transitory effect. Th~se odours ranged all the way from those suggesting the characteristic smell of algae-covered rocks at low tide, to that of very strong rancid cod liver oil. For several reaeons 9 it was decided to further investigate this phenomenon of metal-induced rancidity in the flesh of our common ground fish. It was planned to carry out the work in two directions: (l) To investigate the conditions under which these odours develop (i.e. the minimum concentration of metallic ions causing the rancid odours; the effect of flesh of different species of fishp and other environmental factors); (2) To determine the chemical changes involved. Many things suggested that this was probably a specific example of the classical oxidation of the unsaturated fats or lipids catalyzed by the metals. Mr. Lloyd Smith made a start on the chemical studiesp but left us when an opportunity opened up for him to continue graduate studies. His work has recently been taken over by Mr. Dambergs, an arrangement which is highly satisfactory to us all. For this reason 9 this preliminary report will deal only with the attempt to better define the conditions under which these rancid odours develop. Briefly, they are as follows: (1) Of the cations tested (Al, Ba, Ca, Co, Cu, Fe 2, Fe3, Li, Mg, Mn, Hg, Ni, Sn, and ZnL only copper and iron brought about the rancid odours, and copper was much more effective than iron. (2) Boiling or autoclaving the fish muscle increased rather than decreased the development of the rancid odours. This suggested that the reactions involved are independent of enzymes, unless they are very heat resistant (15 min at 120 C). (3) When added directly to a blended fish muscle 9 the minimum concentration of copper or iron ions required to induce rancidity in fish held one week or less at 0 C is 5 to 20 ppm (rarely at 5 and always at 20 ppm). (4) By adding hydrogen peroxide in addition to the metal ions, the rancid odours develop sooner and become much stronger. HYdrogen peroxide also lowers the minimum concentration of Cu required to produce rancidity. Hydrogen peroxide alone in the concentrations used (0.001 to 0.~) did not produce rancidity.

152 130 (5) In contrast to this, EDTA added to the fish muscle, either stops or retards the rancidity, depending upon the quantities added. For.example: 10 ppm eu++ added to blended haddock muscle caused the development of rancid odours in less than 24 hours at 0 C. EDTA (10 ppm) retarded the rancid odours for 5 days and then they were only slight compared to the others. EDTA (100 ppm) prevented development of the rancid odours completely during the test period of 10 days. ( 6) Tests were run in which copper and iron were added to the flesh of 4 species of fish - cod, haddock, flounder and redfish. The rancid odours were not identical but were quite similar with each species. For a given concentration of copper they were stronger in the redfish and flounder than in the cod or haddock. (7) In a series of tests~ both fillets and gutted whole fish were immersed in solutions containing various concentrations of copper ions. The object was to determine the effect of both immersion time and solution concentratiop. on the subsequent development of rancid odours. Again, an example will give some indication of the kind of results that were obtained: Haddock fillets were dipped for 10 and 30 seconds, 1 9 5, 10 and 30 minutes and 1, 2, 3 and 24 hours in solutions containing 5 and 20 ppm eu++. They were then stored at 0 C. Dipped in the 5 ppm solution,csliglilll:ljtgr~cwfl.odours developed on those fillets that were immersed for 2 hours or more. Di~ped in the solution containing 20 ppm, slightly rancid odours developed after 4 or 5 days when the immersion period was 1 to 10 minutes. When the immersion period was more than 30 minutes, the fillets became strongly rancid in less than 24 hours~ Those immersed for 24 hours were rancid on removal or shortly after. It was also possible to produce rancid odours in whole gutted fish by immersion in solutions containing 10 to 20 ppm copper ions. (9) Metal induced rancid odours developed more rapidly when the solutions were made from sea water or 3% solutions of solar salts. (10) Normal microbial spoilage failed to develop in rancid fish to which 15 to 20 ppm (or more) copper had been added. (11) Trace amounts of eu++ were added to muscle from which the fats had been extracted, and no rancid odours developed. Howeverv when 0.5% freshly extracted cod liver oil was added in addition to the copper typical rancid odours rapidly developed. Incidentally, it was found that pure cultures of many fish spoiling bacteria were inhibited or stopped growing altogether by these concentrations of copper. Ps. putrefaciens, isolated from spoiling fish,was one exception to this. It grew well in concentrations of copper that inhibited all other cultures. I As a matter of interest, it might be mentioned that odours similar or identical to those observed above are encountered in certain types of commercial fish spoilage - especially in frozen redfish, flounder and haddock. The metal content of the guts, undigested food and the flesh of these fish

153 131 is under investigation. It is anticipated that similar analyses of mud bottom sediments and various animals ~bat are used as food b,y our commercial groundfish will be carried out. SUMMARY NO, 41 QUALITY OF J'RESH FISH: PRF.SERVATION OF FILLETS WITH CHLOROTETRACYCLINE ( CTC) ] C,R, Castell During this past year, much of our time has been spent on problems related to the application of CTO to the preservation of fillets. This work has terminated and the results are included in a bulletin which has now been reviewed by Dr. M. C. Firman and his associates at American Cyanamid Company; Mr. M, Stansby, Director, u.s. Fish and Wildlife Laboratory, Seattle, Washington; Mr. R. R omans, Assistant Area Director, Maritime Area, Federal Department of Fisheries, and Mr. J. Morrow, Chief Engineer, National Sea Products Limited; and appropriate alterations have been incorporated into the manuscript. The proposed table of contents is listed below and the particular phases of the bulletin which have been under investigation during the past year are indicated with an asterisk. In the opinion of the senior author the bulletin is ready for submission to the Board's Editor and this will be done as soon as the observations of a reviewer 9 Dr. H. L, A, Tarr, Director, Vancouver Technological Station, have been received. OUTLINE OF CONTENTS This bulletin includes 19 tables and 23 figures. INTRODUCTION (1) Purpose of the bulletin~ To provide knowledge that is necessary for the most effective and economical use of antibiotic dips for fresh fillets. (2) Definition of an antibiotic. (3) Antibiotics as preservatives. (4) The wide spectrum antibiotics CTC and OTC. Page 1 3 4

154 132 PART ONE * SECTION ONE - A BRIEF OUTLINE OF OUR PRESENT KNOWLEDGE RmARDING THE COMMON CHARACTERISTICS AND MODE OF ACTION OF CHWRTETRACYCLINE (l) Chemical structure of CTC and some of its physical and chemical characteristics. 6a (2) Mode of act~on in food preservation 8 (3) Inhibition of CTC activity by metals 9 (4) Destruction of CTC by oxidizing agents and alkalinity 10 (5) Removal of CTC from solution by various agents 10 (6) Activation of enzymes by CTC 12 (7) The need to convert some of these findings into data that can be applied more directly to the problems of fish preservation by antibiotics 12 SECTION TWO - FACTORS AFFECTING THE STABILITY OF CTC IN SOLUTION * (1) ph - buffers - phosphates * (2) Metallic salts - metallic ions - metal surfaces * ( 3) Oxidizing agents - chlorine P iodine p permangana te 9 dichromate, periodate, perborate and hydrogen peroxide * (4) Metallic oxides and carbonates * (5) Water supplies - distilled, tap and seawaters. Hard and soft waters * (6) Brine solutions - reagent grade NaCl and fisheries salts 26 SECTION THREE - REMOVAL OF CT4 FROM SOLUTION BY VARIOUS FISHERY PRODUCTS * (1) Flesh of common species * (2) Cooked vs uncooked fish muscle * (3) Canned fishery products (4) Skin - scales - bones - fish meals etc. * (5) The adsorption of CTC on fish tissues and its subsequent disappearance 33

155 133 SECTION FOUR - ACTION OF CTC ON SOME FISH SPOILING BACTERIA * (1) Indirect destruction of CTC by bacteria (2) Comparative sensitivity of bacterial species to CTC (3) Possible build-up of a CTC resistant flora * (4) Effect of metallic ions on the bacteriostatic action of CTC on bacteria PART TWO EXPERIMENTS DEALING WITH THE MORE DIRECT APPLICATION OF CTA TO FISHERY PRODUCTS SECTION FIVE - FACTORS AFFECTING THE KEEPING TIME OF CTC-TREATED FILJ.E'l'S (1) Solution concentration - The most effective concentration for preserving fillets 43 (2) Storage temperature 45 (3) Quality of fillets at time of dipping 46 * (4) The presence of metallic ions 47 (5) Chlorine 49 SECTION SIX - FACTORS AFFECTING THE AMOUNT OF CTC TAKEN UP BY FILlETS FROM ACRONIZE SOLUTION (1) Size of fillet (2) Solution concentration (3) Immersion time (4) Fluming fillets in chlorinated water (5) Fluming in tap water before and after dipping SECTION SEVEN - FACTORS AFFECTING THE RATE DEPLETION OF CTC IN DIPPING SOLUTION (1) Aeration (2) Continuous dipping of fillets (3) Accumulation of organic matter in the solution

156 134 SECTION EIGHT - C!O STUDIES MADE IN COMMERCIAL FISH PLANTS (1) Keeping time of dry Acronize powder 67 (2) Weighing vs ladling out the required amounts 67 (3) Sanitation of tanks and conveyors 68 (4) Successive bacterial counts from dipping solution during commercial runs 69 (5) Microflora in high-count Acronize solutions 71 (6) Depletion of volume of solution by continuous dipping 72 (7) Depletion of CTC concentration in dipping solutions during commercial runs 7 4 (8) CTC content of commercially treated fillets 75 (9) The cost of treating fillets in a CTC dip 75 (10) The quantity of fillets that can be effectively dipped in a given volufue of Acronize solution 76 (11) Flavoursp off odours and discolouration attributed to CTC (12) Is there a health hazard in the use of antibiotics in food? SUMMARY AND CONCLUSIONS APPENDIX 1 - THE BIOLOGICAL ASSAY FOR CTC USED IN THESE EXPERIMENTS AND THE LIMITATIONS OF ITS ACCURACY BIBLIOGRAPHY SUMMARY NO. 42 QUALITY OF FRESH FISH: BACTERIAL SPOILAGE: PROTEOLYTIC ENZYMES FROM A BACTERIA FOUND IN COD MUSCLE. E.J.. Laishley J.E. Stewart The proteolytic enzyme from a Flavobacter species isolated from spoiling fish is embedded in lipid material when shake flask grown. Attempts to free the enzyme from the lipid were not successful. It was decided that still grown cultures, although involving longer growth periods~

157 ph ph 6.0 ph 7.0 -o- Enzyme activity --0--pH changes -L:r- - -l::r-- -x-- --x O'l +» g ~ ~ ~... ~ cd -~.... ~ ~ 0 cd! t'l a ' x-- --x ' ' ' ~""'- -:S:~ ~~~-"=~-"" -.e GROWTH TIME IN HOURS 5.0 Fig. 1 -Graph showing enzyme activity, ph changes vs time. Sample with 1.~ glucose.

158 136 would allow us to exclude much of the lipid material and markedly reduce our problems. Consequently this was tried and it was fol.uld that growth and enzyme production was poor in the medium used in themake flask studies. Several different media were examined and at the time of writing the medium which promises the most success contains: 50 g - Bacto Beef Heart for Infusion (Difco) 10 g - Proteose peptone (Difco) 2.5 g - NaCl 0.2 g - KCl and g - CaC ml Distilled Water Figure l shows the results obtained with the medium at ph 5, 6 SUMMARY NO. 43 QUALITY OF FRESH FISH: BLACK SPECKS IN COD FLESH Jacqueline Dale J.E. Stewart M. W. Mullins Examination of fillets with black specks throughout the muscle showed that the majority of the spots were on the surface of the fillet. Tiny black threads ledrl from these spots deeper into the muscle. Microscopic examination of these threads showed them to be tubules filled with black material. These tubules corresponded to blood vessels. These specks and black spots were carefully excised. The results of the subsequent ana~sis showed the black material to be blood. The positive test was the Benzedine Confirmatory Test. Since the black material was contained in tubules throughout the muscle and the large specks position coincided with breaks in these vessels it was concluded that this was fish blood. '

159 137 SUMMARY NO. 44 PROTEINS OP PRESR PISR: ANALYSIS OP TROPOMYOSIN Beryl Truscott P.R. Odense A.E. Waddell A study of enzyme digestion of tropomyosin for the preparation of a partial breakdown product of the protein suitable for amino acid sequence determinations was concluded, The action of trypsin on carbobenzoxy-substituted tropomyosin 9 reported last year, was shown to be reproducible. However, since less than 25~ of the substituted tropomyosin molecule seems to be hydrolyzed, (7~ by weight of the original CBZ-tropo~osin after trypsin digestion 9 is insoluble through the PH range ) trypsin does not seem to be a good choice of enzyme for the initial degradation of the protein. The action of o{-chymotrypsin on the oxidized protein was investigated. This enzyme is known to break a peptide bond involving an aromatic amino acid residue, i~e. phenylalanine 9 tyrosine and tryptophan. Some leucine and methionine bonds are also hydrolyzed by chymotrypsin. Theoretically, tropomyosin could yield a minimum of 10 and a maximum of 73 peptides. After digestion with chymotrypsin, 9~ by weight of the tropomyosin was soluble through the ph range 2.0 to 8.0. The peptides so obtained were separated by ion-exchange chromatography, using the Beckman/Spinco Amino Acid Analyzer. At least 34 peptides were shown to be present. This digestion is also reproducible. Of the three enzymes studied, lit-proteolytic, trypsin and 6{-ohym.otrypsin, the latter would seem to be the best choice of enzyme for the initial breakdown of tropomyosin for amino acid sequence determination. Further details of the experimental procedure follow. Cbymotryptic Digestion. Oxidized tropomyosin (1% solution) was suspended in water, 0.005M in ca++, and the preparation was adjusted to ph 8.0, with O.lN NaOR. In 1-2 hours with stirring and further additions of alkali to maintain the ph at 8.0, the protein dissolved to give a clear solution. Chymotrypsin (0.02% in water) was added, and the digestion allowed to proceed at room temperature with stirring, and maintaining the ph at 8.0 by the automatic addition of O.lN NaOH with a Radiometer ph stat. The uptake of NaOH was a measure of protein digestion; rapid for the first hour, then increasingly slower, until at the end of six hours, the digestion seemed complete. At this time, o.~ HCl was added to lower the ph of the solution to 3.5. A small amount of precipitate which formed was removed by centrifugation. The solution of peptides was freeze-dried.

160 138 Column Chromatography of Peptideso A column of Dowex 5G-X2 ion exchange resin, mesh, sodium form, 150 em x Oo9 em, water jacketed for temperature control, was connected to the Beckman/Spinco Amino Acid Analyzer o Fifteen mil11:grams of the chymotrytic digest of tropo,myosin was charged into this columno The peptides were separated by gradient elution with citrate buffers, ph 3o25 to ph 5o28o The results are being prepared for publication and it is Eplticipated that a manuscript will be submitted in Aprilo SUIJMARY NO 2 45 PROTEINS OF FRESH FISH~ THE DEUTICKE EFFECT IN MUSCLE FROM RESTED FISH W.,J. Dyer D.I. Fraser During rigor mortis, it has been found that the extractability of the acto,myosin group of muscle proteins sometimes becomes depressedo This is known as the Deuticke effecto However, there are many conflicting reports, some workers finding such an effect and others not 9 with no apparent consistency in conditions 9 etco Following the major effects observed on degree of contraction of cod muscle during rigor mortis as affected by temperaturep the extractability of the acto,myosin protein was determined in muscle from rested 9 non-feeding cod held at 0 and 24 C. The onset of rigor occurred at about 30 minutes and lasted for about 5 hours at 24 (Figo 1). Corresponding times for ice temperature were about 16 to 72 hours respectively. At 0 no decrease in extractable protein (EPN) was observed over this period. However, at 24, the extractable protein decreased from the initial value of 2.~, expressed as nitrogen % of wet weight in the tissue, to a value of about 1.3% during the period of maximum rigor. Subsequently, it returned to the initial value on resolution of rigor mortis. Thus 9 the Deuticke effect occurred only at the high temperature where contraction was very marked - about 3ctfo or more (Appendix No. 11, Halifax Annual Report 1960L This temperature dependence, then, may account for the conflicting reports in the literature. Further work is planned to determine the effect at intermediate temperatures. The level of glycogen may also influence the point at which the contraction and non-extractability of protein occurs. As found previously, the ultimate ph did not go below at either temperature. This effect of temperatu:ne may have some significance in the freezing of fish in the prerigor or near fitor condition, especially if they are taken from warm water or allowed to warm up prior to freezing. As found last year, handling may aggravate the effect.

161 Fig. 1 - Deuticke effect in the muscle of rested cod C-~======--RI~~G~O~R I I... 'vl 1..0 RIGOR J L 12 I I I ' HOURS DAYS

162 140 SUMMARY NO 2 46 PROTEINS OF FRESH FISH~ VERTICAL STARCH GEL ELECTROPHORESIS OF COD AND lobster MUSCLE AND BlOOD PROTEINS. P.H. ~ense C. \i. Shinners Many variables affect the optimum conditions for the resolution of fish muscle proteins by the technique of vertical starch gel electrophoresis. Among these factors are buffer composition~ buffer ionic strength, starch concentration of gel~ temperature of run, length of run~ current 9 sample size and concentration and staining method. :Numerous runs have been made in which these factors have been varied and the most successful system to date is described belovo The buffer used is a modification of the "high resolution" buffer employed by Aronsson and GrOnwall for paper electrophoresis. Its composition is: Trishydroxymethylaminomethane (TRIS) g/1 (O.l25M) Etbylenediaminotetraacetic acid (EDTA) 1.5 g/1 ( M) ~~~c acid lol5 g/1 (O.Ol875M) The ph is adjusted to 8.3. The current used is 50 milliamps. The vol*age varies in the 350 volt range. Run time is 6 hours. The Buchler Instruments, Inc., vertical starch gel apparatus and a similar apparatus 9 made at this Station after Smithie 9 s design, were employed. Samples subjected to electrophoresis included lobster serum~ lobster plasma and haemocyanin and fibrinogen isolated from lobster blood. Cod muscle albumins 9 blood serum and muscle tropomyosin were also run. The effect of the initial physiological condition of the fish 9 storage time and storage condition upon the cod muscle albumin patterns is being studied. Another run of special interest is the electrophoresis of tissue samples. Thin slices of cod muscle have been inserted in the starch gel sample slots and the normal electrophoresis run has been carried out. It was found that the pattern was identical to the electrophoresis pattern of the water extract of cod muscle albumins but two additional bands appear. These additional protein components are strong~ anionic. It has not yet been determined why they do not appear in the water extracts as well.

163 141 SUMMARY 10 R 47 PROTEINS OF FRESH FISH: STRUC!URAL PROTEINS OF COD MUSCLE: PROTEIN INTERACTIONS II THE ACTOMYOSIN SYSTEM D.G. Ellis J.T. Iauder Storage studies on frozen cod have indicated that texture changes are a major probl. A loss of dispersibility of the actodi\y'osin fraction of the muscle also occurs during storage. Protein studies on muscle extracts have shown the actojvosin eyst,m to include four coaponents, resolved in the ultracentrif'uce, and a gel fraction. Two large components (I and II) are assumed both to be actoayosins, the smaller components (III and IV) are assumed to be JVOSin and tropolii\y'osin respectively. The position of actin in this picture has not bean determined. It is assumed that free actin, at the ion concentration of the extracts, will appear in the gel fraction in the polymerized form. Dissociation of the actoayosins is produced by high ion concentrations, certain chalicals (ATP, pyrophosphate, potassium iodide, urea, etc.) and by prolonged frozen storage. Aggregation of JVOSin occurs readily in solutions of the isolated protein. Aggregation of component III of the muscle extract has been observed in the ultracentrifuge patterns. Based on studies of the muscle proteins and the storage behaviour of frozen fish muscle a simplita.ed. reaption has been proposed as a working hypothesis for the study of protein changes during frozeu storage: Dissociation I + II III + IV + G Actin l 1 Aggregation Po4nuer1zation I Ia F Actin ~~ Gel Fraction If we assume the identity of the components of the ultracentrifuge pattern this becomes: ActODI\Y'OSins I and II Dissociation Myosin + TropoiD1osin + G Actil'l 1 t Myosin Aggregated F Actin ~e--- Gel

164 142 A complete study of this reaction is essential to our understanding of acto~osin "denaturation" during frozen storage of cod muscle. Modification of the model will pro bably become necessary as the study continues. The present work is concerned primarily with the ~sociation step. There has been no evidence of reversibility. The variables of interest are time 9 temperature 9 protein concentration 9 solvent ion concentration 9 and ph. Time and temperature are the variables currently under study. Protein concentration has been held at approximately 0.6 mg protein H/ml 9 solvent ion concentration at 0~6 and ph at ibo. The extracts have been prepared using our standard procedure 9 with a solvent of 0.6 r/2 containing a 0.05 M phosphate buffer of ph 7 ooo The TSP (total soluble protein) extract has been used rather than a fractionated act~osin preparation. The presence of the albumen group did not appear to introduce problems. The precipitation technique (dilution with water to 0.05 Tr2 or less) for the preparation of acto~osin gave a more variable starting materialp with a higher initial dissociation. The actomyosin: dissociation pro4ucts ratio was approximately 45:55 as compared to an initial ratio of 60:40 for the TSP extract. Prerigor muscle has been used, generally obtained from rested non-feeding fish held in a tank at about 2 C. The tissue was held at 0 C until extraction, about 1/2 hour after death. The extracts were held at l0 C or 20 C (± l/4 C) during storage. The ultracentrifuge analyses were carried out at the temperature of storage. Component concentrations were determined from the areas of the corresponding peaks in the ultracentrifuge schlieren pattern. The accuracy of the method has been improved in the past year 9 but reproducibility of results is not as good as desired. The average of a number of experiments must be used. Components I and II 9 the undissociated protein 9 are measured together to give a single value. Similarly the dissociation products 9 III and IV, are measured together. This complicates the interpretation of results, but is necessary because of difficulty in separating the components. In additionp even at the relatively low protein concentrations used (about o.~ dispersed protein) the Johnston-Ogston effect would lead to considerable enhancement of component II and possibly of component IV. We are not in a position to correct for this error at the present time. The data now available also will permit gel fraction quantities to be calculated. This will require a reliable value for the concentration of albumen proteins present under these conditions. The data presented in Figs. 1, 2 and 3 are the average of several determinations at each temperature. The undissociated protein (I and II) and the dissociation products (III and IV) are plotted as a percentage of the total dispersed protein of the actomyosin system at zero time. This eliminates the effect of minor initial protein concen= tration variations and of the albumen and gel fractions 9 both of which are included in the biuret or total protein values.

165 143 Storage Time - hours Figure 1.

166 144 -g ~ Pt!l'l n A i 0 J.t '---~l,:------'-~:: Storage Time - hours ~ C Q) ~ ~ 80 i ~ 60 8.!l'l n A 40 ~ ~ 0 20 J.t 0 ll" Storage Time - hours Figure 2 Figure 3

167 145 The time of measurement has been extended somewhat over earlier data. The undissociated protein 9 as observed previously p drops rapidly at all three temperatures. The dissociation products (III and IV) have been found to go through a JD8%imum. The portion of the curves with a positive slope represents a period of buildup of dissociation products, when the dissociation rate exceeds the rate of loss due to aggregation. The negative slope region of the curves represents a loss of the dissociation products as the aggregation rate exceeds the dissociation, limited by the reduced concentration of undissociated protein. This is the form of the dissociation - aggregation curve predicted by the simple model of the denaturation reaction. At all three temperatures the actomyosin system has been found to be approximately 6Q% undissociated actomyosin and 4o% dissociation products at zero time. The slope of the dissociation curve is independent of temperature for the 0 C to 10 C range~!fhe aggregation curve has a steeper slope at 10 than at 0. The accuracy of the data at 20 C does not justify a comparison of rates at present. Because of the rapid changes involved at 20 C and the length of the ultracentrifuge analysisp very few points are available to determine the slope at each portion of the curve. At this temperature there is greater possibility of bacterial or enzymatic degradation of the protein. In all determinationsp however 9 the total protein, as measured by biuret, had not changed up to the time the dispersed protein concentration had approached zero. It is interesting to note the relatively high concentration of dissociation products reached at 0 C where aggregation is proceeding slowly. This m.ey be com}b':red with the results observed at "intermediate times" of frozen storage for cod muscle wheil the extractable protein is still high, but the ultracentrifuge analysis shows that the actomyosin has been completely dissociated, and the dispersed protein is the dissociation products. SUMMARY NO. 48 PROTEINS OF FRESH FISH: THE NATURE OF THE HEAT STABLE PROTEIN ISOLATED FROM COD MUSCLE P.H. Odense C.W. Shinners J.R. Dingle H. C. Freeman It had been reported earlier that the heat stable protein isolated by Bourdillon and Baker from bovine and human muscle had also been isolated from cod muscle. The method used by Bourdillon has been modified and the method of preparation from cod muscle is shown below in '!'able I.

168 g minced cod muscle 400 ml M KCl boil 1 minute filter Ppt. Filtrate Dialyze vs distilled water at 2 C overnight. Centrifuge Supernatant Ppt. Redissolve in Oo4M MgCl~ Dialyze vs dist1lled water at 2 C overnight centrifuge Supernatant Crystalline ppt. The crystallization step was sometimes repeated four times in attempts to purify the heat stable protein but the free boundary electrophoresis pattern always revealed the presence of several components. There was an additional difficulty in the electrophoresis of the protein. It was water insoluble and the electrophoresis had to be carried out at the relatively high ionic strength of The ultraviolet spectra of these solutions revealed the presence of large amounts of inosine in the extracts. On repeated crystallization the amount of nucleotide associated with the protein steadily decreased. The ultracentrifuge run on a four times crystallized fraction showed a main peak with a sedimentation constant of 2.8 x Io-13 and two very minor peaks with constants of 3.6 x lo-13 and 4.6 x lo-13. The protein concentration for this run was Oo72 mg N/ml and the protein was in molar NaCl solution, ph 7.o.. The amino acid analysis of the four times cr,rstallized fraction was smtrisingly similar to that of tropomyosin (Fig. 1). The similarity was at first discounted because tropomyosin, subjected to the treatment used for the extraction of the heat stable proteinp failed to produce a water insoluble product.

169 147 LYS HIS D ARG ASP GLU CIS MET THR SER PRO GIJ! ALA. VAL ILE LEU TYR PHED 0 25 Fig. 1 -Mole percent amino acid composition of heat stable protein.

170 LYS HIS ARG D ASP GID THR SER PRO GLY ALl V.AL ILl LEU TYR Pmi'D 0 25 Fig. 2 - MOle percent amino acid composition of water soluble fraction.

171 149 LYS HIS D ARG ASP GID CIS m TBR SER PRO GLY ALA. VAL ILE LEU TYR PHE 0..._I--J 0 25 Fig. 3 - Mole percent amino acid composition of tropom;rosin.

172 150 In a subsequent experiment four times crystallized heat stable protein was dialyzed 72 hours vs continuous changes of water. Instead of a yield of crystals a gelatinous precipitate formed and the entire contents of the dialysis sac formed a gel. The gel was broken by the addition of 0.05 M KCl solution and the precipitate was separated by centrifuging. A biuret on the supernatant revealed a high protein concentration mg 1/ml. Electrophoresis of the water soluble protein in the supernatant at an ionic strength of showed only one peako When the protein was subjected to electrophoresis under the same conditions used for tropo~osin (ionic strength 0.15) the characteristic descending and ascending peaks of tropo~osin appeared. The mobilities also corresponded to tropomyosin values. These values were 6.59 and 6.15 cm2volt-lsec-l for the ascending and descending peaks respectively.!he amino acid analysis of the water soluble protein in the supernatant after exhaustive dialysis of a four times crystallized heat stable protein preparation is shown in Fig. 2. It appears identical with the amino analysis of tropomyosin which is shown in Fig. 3. The final identification of the water soluble protein was achieved by dialyzing a solution of the protein vs the ammonium sulphate and acetate buffer solution used to crystallize tropomyosin. The typical bipyramidal crystals of tropomyosin formed. It has been concluded that the heat stable protein of Bourdillon and Baker is, to a large extent, tropomyosin. When initially extracted it might be associated with nucleotides or other proteins which render it water i.uo,luhle. Exhaustive dialysis liberates the characteristic water soluble tropojg"osin. It is a tribute to the protein's stability that it can be subjected to boiling in M 101 for 1 minute and still retain its ability to crystallize. This is also the first isolation of tropomyosin without the use of dehydrating organic solvents. SUJIMARY NO. 49 PROTEINS OF lresh lish: AQUEOUS EXTRAif.rs OF FISH MUSClE: FRACTIONATION WITH ZINC ACETATE. J.R.Dingle J.A. Hines Aqueous extracts ot fish muscle have been shown by electrophoretic analysis to contain several groupe of proteins, the relative proportions of which vary from one t,ype of fish to another. It may be that some of their biochemical oharaoteriatioa vary as well. Presumably moat of the enzymes aaaooiated with the metabolic processes of the muscle cell are to be found among these proteins. Relatively little progress has been reported in separating and identifying the various components 9 and most

173 151 of this has been achieved by fractionation with ammonium sulphate. Cohn in 1953 introduced the use of heavy metals as a means of fractionating the proteins of biological fluids, especially human blood, but this procedure appears to have been used rather sparingly since then. We have investigated the use of zinc acetate as a means of fractionating the dissolved components in aqueous extracts of cod and haddock muscle. Fillets cut from prerigor and postrigor cod, and from postrigor haddock, were minqed in a chilled meat grinder, and extracts made by stirring the minces with equal volumes of water at 2 for about 2 hours. The supernates obtained after centrif'uting at 1400 x g were used as the starting materials for fractionation. All operations were carried out at approximately 2o. Aliquots of' extracts were mixed with zinc acetate solution, water, and in some cases with IfaCl solution, to constant total volume and ph, the desired zinc acetate concentration, and total ionic strength. After standing for 20 minutes, soluble protein was determined in the supernates. The solubility curves so obtained were qualitatively reproducible, and there was generally a suggestion of a stepwise precipitation. An increase of' ionic strength by addition of IfaCl shifted the precipitation curve to higher zinc acetate concentrations. Under all conditions, approximately 35~ of the original protein remained soluble up to a zinc acetate concentration of 0.10 X. Precipitates formed between several zinc acetate concentrations were collected, and redissolved by stirring for several hours with 0.1 X EDTA. Electrophoresis of these fractions showed that some selective precipitation had occurred, but fractionation was not very cle~-cut. Since chromatography on DEAE cellulose columns will probably offer a b~tter method of fractionation, this work was not carried further. The protein that resisted precipitation by zinc seemed to warrant further study. Attempts to remove excess zinc ion by addition of EDTA or phosphate ion resulted in an immediate precipitation of further protein. Attempts to redissolve these precipitates were unsuccessful, and their natures remain uncertain. In subsequent work, the "zinc-acetate - soluble fraction" was routinely prepared by adding to a water extract of muscle one tenth volume of cold O.lM zinc acetate, centrifuging at 1200 x g, adding to the supernate one-tenth of its volume of cold 1.0 M KH2P04 buffer, ph 6.6, and centrifuging again as before. Approximately 18% of the original protein remained soluble after this treatment. After concentration by dialysis against Carbowax, fractions prepared from cod and haddock muscle gave the electrophoretic patterns shown in Fig. 1. The patterns for the whole extracts are also shown for comparison. The mobilities of the two main peaks in the cod fraction indicated that these were identical with components 3 and 4 of the original extract. Moreover, the mobilities of the two main components of the haddock fraction agreed closely with those of the cod fraction at comparable ph. In the analytical ultracentrifuge, the cod fraction showed two main components with ~edimentation constants of 2.5 and 1.1.

174 152 l3 D 4 3 Fig. 1 - Electrophoretic patterns of aqueous extracts of postrigor cod and haddock muscles, and of the zinc acetate-soluble fractions prepared from them. A: Cod~ whole extract; ph 6.5~ after 320 min. 13~ Cod 9 fraction; ph after 190 min. C: Haddock 9 whole extract; ph after 270 min. D: Haddock fraction; ph 7. 7, after 190 min. Migration left to right. In an attempt to separate the two main components 9 mercuric acetate was added to a concentration of M. A further precipitation of protein occurred 9 but electrophoresis of the concentrated supernate revealed that the composition had not been significantly changed. In another attempt 9 a concentrated fraction was Chromatographed on a cooled DEAE-cellulose column 9 eluting with KCl from 0 to 0.1 Mat ph 7.0. Seven sub-fractions were collected. The principal fraction was found by electrophoresis to consist almost entire~ of the original components 3 and 4 in the proportions of 58% and 4<>% 9 with 2:% impurity. Thus 9 while chromatography on DEAE cellulose did not give the desired separation of the two main components under the present conditions 9 the electrochemical properties of the proteins appeared to remain unaffected. Recovery from the column wasp however 9 only 7o% of the amount of protein added.

175 153 The ultraviolet absorption spectrum of this purified subtraction suggested the presence of an unusual amount of phenylalanine. This was confirmed by an amino acid analysis of a eydrolysate of the subfraction (by P.H. Odense), which gave the phenylalanine content as 11.~ by weight. Similar proteins have been isolated by others from aqueous extracts of carp and plaice, using ammonium sulphate fractionation. It appears, then, that this unusual protein may occur generally in fish muscle, but there are indications that its electrochemical properties differ from source to source. This work will be reported in greater detail in a paper to appear soon in the Board 0 s Journal. SUMMARY :NO a 50 PRO'l'EINS OF FRESH FISHg AQUEOUS EXTRACTS OF FISH MUSCLE: FRACTIONATION WITH AMMONIUM SULPHATE. In some further attempts to isolate the components of the sarcoplasmic protein system, aqueous extracts of postrigor cod muscle were treated with ammonium sulphate at various concentrations from 0 to 2.5 M, ph 7.3. Precipitates formed between different concentrations were collected, redissolved, and examined by free-boundary electrophoresis. In general, while there vas usually some preferential precipitation of one or two of the components in a given fraction~ most of the fractions contained many other components as well. The fraction obtained between 1.8 and 2.2 M ammonium sulphate, however 9 was particularly enr~qhed in component 6. For example, from an extract containipg about 33~ of this component (which may be complex), the M fraction contained 6~, with lesser amounts of several other componen~o A second fractionation of the redissolved precipitate between 1.8 and 2.0 M ammonium sulphate yielded a fraction of which 86~ was component 6. In several cases, the supernate at 2.0 M, after standing overnight at 2, yielded a crop of short rod-like crystals of which the largest observed was 52 x 12 microns. Crystallization was more rapid when the solution was warmed to room temperature. This precipitate was not qompletely soluble in waterv and the portion that did dissolve did not contain any greater proportion of component 6 than the original fraction. The nature of the crystals remains uncertain, but they did not appear to be inorganic. Attempts were made to separate component 6 from the other material in a primary M fraction by chromatography on a cooled DEAE cellulose column. It was necessary to collect the fractions at room temperature, howeverp and under these conditions, several of them precipitated irreversibly. The apparently more stable fractions, after concentration with Carbowax, were found by electrophoretic analysis to be complex.

176 154 A primary fraction obtained at lo8-2o2 M ammonium sulphate was examined by free-boundar,y electrophoresis at ph's from 5o2 to 6o6o The peak: due to component 6 remained undivided over this range~ indicating electrochemical homogeneityo The isoelectric point was found to be at ph 5o24~ at an ionic concentration of OolO in KCl'~'+ K-phosphateo The precipitates and supernates obtained in preparing purified component 6 by successive fractionations at lo8 to 2o0 M ammonium sulphate were assayed for aldolase and ATP-creatine kinase activitieso The aldolase activities became concentrated in the precipitated fraction 0 but the activity per unit of protein did not increase significantly beyond the first fractionationo The total activity from all portions of the preparation also fell off with successive fractionationsp presumably due to denaturation of the enzymep or possiblyp to the separation of a cofactoro The kinase activity was associated with the components remaining ~oluble in 2o0 M ammonium sulphate 9 but in this case also 9 the total accountable activity decreased with successive fractionationso PROTEINS OF.FRESH FISH~ AQUlOOUS EXTRACTS OF FISH MUSCLE: i,ariations IN WATER-EXTRACTABLE PROTEINS OF COD MUSCIS It was reported last year (Appendix 19) that several factors may affect the electrophoretic patterns of aqueous extracts of fish muscle that might reduce their value for identification of specieso Among these was a possible effect of starvationo To test this 9 fillets cut from tvo cod immediately after killing were minced and extracted with twice their weights of watero Both fish had been kept in holding tanks for at least 2 weeks~ one was held at 2o5 and refused to feed 9 while the other was held at 6 and was feeding freely o The electrophoretic patterns (free boundary) of the water extracts are shown Fig o 1, A and BP and the relatiife proportions of the several components determined by measurement of peak: areas are given in Table I 0 In our experience 9 the proportion of component 4 in aqueous extracts of cod muscle usually lay in the range 14~~P and of component 6 in the range 28 to ~o An enhanced amount of component 4 (greater than 2CJI,) hasp however 9 occasionally been found in found extracts of fish caught during the winter monthso As reported last year 9 a variation in the amount of this component has also been observed in extracts made from adjacent portions of the same filletp both prerigor and postrigor o All these differences appear to be real since duplicate electrophoretic analyses on one extract were indistinguishableo It is not known w~ther these variations in the patterns reflect a real difference in the amounts of components in the muscle 9 or whether they are due to differences in extractability caused by the condition of the fisho It has been foundp howeverp that the brownish muscle tissue of cod is much poorer in component 4 than is the much more abundant white tissueo This is illustrated in Figo lp D and Cp showing electrophoretic patterns of extracts made from the brown muscle and white muscle of the same prerigor codo The most rapid peak in the brown muscle extract probably represents ~oglobin.

177 155 A B 7 6 5a 5 4a a 5 4a c D 7 6 5a Pigo 1 - Variations in electrophoretic patterns of aqueous extracts of cod muscle. A. Feeding cod (prerigor) from 6 tank. B. Semi-starved cod (prerigor) from 2.5 tank. C. Extract of white muscle (post rigor) D. Extract of brown muscle (post rigor) Ionic concentration = 0.10; ph 6.6; migration left to right. TABLE I - Composition of aqueous extracts of starved and feeding cod. Percent of total electrophoretic pattern area Electrophoretic Starved Cod Feeding Cod Peak Number Ascendi~ DescendiEB: t.(scendi~ DescendinP: 1 7.1% o a a

178 156 SUMMARY NOR 52 CHEMICAL COMPOSITION OF FISH: POSTMORTEM BIOCHEMICAL CHANGES IN COD MUSCLE" Dolo Fraser So Punjamapirom WOJO Dyer Ao Effect of temperature on the biochemical processes occurring during rigor mortis in rested(, feeding cod muscleo The state of freshness of fish and especially the 'biochemical postmortem changes associated with rigor mortis play an important role in the subsequent storage life of the frozen producto In view of the increasing amounts of prerigor fish being processed industrially 0 it is essential that our knowledge of these char~es be increased 0 and so aid our understanding of some of the difficulties presently encountered in the processing of ver,y fresh fish such as shore-ca1~ht cod 9 and of the rather large changes in quality which sometimes occur under conditions of frozen storage o (See Summary No o 22 of this year 0 s report L Investigation of the rapid biochemical changes taking place in fish muscle immediately postmortem has shown that with fasting cod held in an aquarium at 2 C the rate of developnent of glycolysis and the accompanying dephosphorylation and deamination of the high energy phosphate compounds were markedly affected by increase in temperature from 0 C up to 25 Co These processes were complete in about 30 hr at 0 C~ 12 hr at 10 C and in just over 2 hr at 25 C (Appendix 10 of this Station s Annual Report for )0 With the installation of a second fish-holding tank at the laboratory these studies have been extended to include postmortem changes in feeding fish under various conditions, Rested cod were held live in seawater at a temperature of 7 C and maintained on a diet of frozen herring which had been cut up into bite-size pieces and allowed to thawo The cod were anaesthesized prior to filleting to minimize struggling and samples of muscle excised immediately for extraction of the metabolites with perchloric acido Changes in glycogen~ lactic acid 9 acid-soluble phosphorus compounds and ammonia were followed in the muscle at postmortem storage temperatures of and 25 Co The effect of temperature on the rate of glycogen depletion and of lactic acid formation in restedp feeding cod muscle is shown in Figo lo Initial glycogen values for rested cod fed thrice weekly for periods of from one to five weeks averaged 160 mg/100 g 9 and ranged from 108 mg/100 g to 220 mg/100 go One value of 480 mg/100 g was recordedo These levels are rather less than expected for feeding fish,, values up to Oo5% having been reported in the literaturev In contrast 9 as reported last year~ initial glycogen levels in non-feeding cod were usually less than 100 mg/100 go The rate of glycogen disappearance and lactic acid formation in the muscle from rested" feeding cod held at and 25 C was similar to that observed in muscle from rested 9 fasting fish at the same temperature? in both cases the rate of glycolysis was markedly

179 157 Fig. 1 - G~coger/ and lactic acid in the musb1e of rested, frding cod held at various temperatures. I I I ' \ \ \ \ \ ~ \ \ \ \ I I I I I I I 1 I I I I "' '.,., 0,...0~ ' ' o o 0 I.C\ 0

180 g. Tissue \ \ \ \ \ \ \ \ \ \ 0 \ s X \ ' ' \ \ \ \ ""' Fig. 2 - Dephosphorylation and deamination in the muscle of rested 9 feeding cod. \ \ \ \ \ \ \ \ '6 \ \ \ ' ' "' "\. ' ' ' ' \ 0 ~~ \ o""- """-... a.:::; ' ' '\ 0 1.1'\ C\1

181 159 increased with increase in temperature. Times to onset of rigor were also similar; in both feeding and non-feeding muscle, postmortem times at 0, 10 and 25 C were 12, 6 and 1/2 hour respectively. As was found with starved fish, the onset of rigor in feeding fish occurred much earlier at the higher temperatures. In the majority of these feeding cod, the ph was initial~ , falling to an ultimate level of during the resolution of rigor. Muscle with an initial glycogen level of close to 500 mg/100 g reached an ultimate ph level of The so-called "alkaline" rigor, characteristic of non-feeding cod, where the ultimate ph values during rigor remained near 7.0 was not encountered in feeding fish. The changes in ATP (acid-labile P) and in ammonia contents of rested, feeding cod muscle at temperatures of 0, 10 and 25 C are shown in Fig. 2. Initial ATP-P values averaged approximate~ 30 mg/100 g, corresponding to an ATP content of 5 x 10-4 moles per 100 g, similar to -.t found with non-feeding muscle. ATP disappeared very rapidly during the prerigor period; at the onset of rigor, on~ about 10!' of the level in the rested living Mimal remained. These results are in agreement with those reported for rested, feeding codling muscle by investigators at Torry and for trout muscle by Japanese workers. In contrast, ATP in fasting cod muscle had reached a level of on~ ~of its initial value at the onset of rigor. The results indicate an increased activity of the enzymes responsible for the postmortem breakdown of ATP in feeding fish. As illustrated in the Figure, the dephosphorylation of ATP was accompanied by deamination of adenine nucleotides to inosine nucleotides with the liberation of ammonia. This process also occurred much faster at the higher temperatures. The production of ammonia increased gradually during the resolution of rigor and reached a level of 3 times ita initial value during the poatrigor period. These results indicate that in feeding cod the enzymic breakdown of ATP through dephosphorylation to ADP and thence to AMP pr~cedea the conve~ion, by a specific deaminaae, of AMP to IMP with the release of ammonia. This is in contrast to fasting cod muscle, where both processes of dephosphorylation and deamination were found to occur simultaneously. It is expected that with increased fish-holding facilities at the Station, some of the difficulties which have been encountered in obtaining feeding fish will be overcome and t~t these studies will be extended to include postmortem changes in fish under conditions of exhaustion and others which more closely approximate those in commercial practice. The recent acquisition of a plate freezer will facilitate studies of the effects of freezing and frozen storage at various temperatures. B. The acid-soluble nucleotides of rested, feeding cod muscle. The breakdown of adenine nucleotidea and their conversion to inosine compounds have been followed in the postmortem muscle by determinations of acid-labile phosphorus and ammonia contents, and substantiated by corresponding changes in the ultraviolet absorption maxima of perchloric

182 160 IV I if o.a 0 \0 C\1..., ~ 0 A o.6 III 0, No.200 2N HCOOH 4N HCOOH 4N HCOOH + 0.2M NH4COOH Fig. 3 - Acid = soluble nucleotides in muscle of rested 9 feeding cod

183 161 v VI A ' I ' 250-c I ~0 400

184 162 extracts of the muscle from 258 ~ to 248 ~ 9 characteristic of adenine and inosine nucleotides respectively. These methods 9 however 9 give only an approximate picture of the nucleotide changes occurring in the muscle postmortem, and a more complete identification of the major constituents of the nucleotide fraction is considered necessary for increased understanding of these and related postmortem biochemical processes. Consequently 9 the acid-soluble nucleotides in the m~~cle of rested 9 feeding cod muscle immediately postmortem have been separated by a formate elution system on an ion-exchange resin (Dowex I). A typical separation 9 showing 6 main components 9 is illustrated in Fig, 3. The nucleotide fractions were located by reading their optical densities at 260 ~ in a Beckman spectrophotometer. The composition of each pooled peak 9 after removal of formate by absorption and elution of the nucleotide on charcoal 9 was examined by spectrophotometric and phosphate analyses. Peaks I to V were thus tentatively identified and estimated as DPN 9 AMP 9 IMP, ADP and ATP respectively 9 in the following amounts~ DP.N 9 0,094 lj.moles/g; AMP pmoles/g; DIP, 0.55 pmoles/g9 ADP pmoles/g~ and ATP pmoles/g. These results are in partial agreement with those reported for rested 9 feeding codling muscle by Jones and Murray (1960)P whose estimations of AMP, IMP and ATP were considera~ly higher , 1.26 and 5.34 ~oles/g respectively. However, the amount of acid-labile phosphorus in the perchloric acid extract used by us for chromatography corresponded to an ATP value of 5.92 ~ole/g. 9 and it is possible that the low recovery of 2.37 ~ole/g may be due to a loss of nucleotide during the charcoal absorption and elution procedure, The discrepancy in amount of ATP found by us and that reported by Jones and Murray (5.34 ~oles/g) may also lie in Peak VI 9 which contained as yet unidentified phosphorylated material, It closely resembled ATP 9 however 9 in its absorption spectrum 9 indicating adenine base, and in the amount of phosphate liberated by acid hydrolysis in IN H Cl for 7 minutes (69%). This substance may possibly be a decomposition product of ATP 9 since the latter is known to be unstable in acid solution at room temperature. The same double peak in the position of ATP in the elution pattern was found when a solution of standard nucleotides containing DP.N, AMP, IMP, ADP and ATP was chroma tographed under the same conditions. Subsequent investigations have indicated that Peak VI may be eliminated by lowering the operating temperature to 0 C by the use of a jacketed column through which refrigerant is circulated. Recently, a new Gilson fraction collector with attached automatic ultraviolet recorder has been set up in order that the changes in nucleotide concentration which are associated with other major biochemical changes in postmortem fish muscle may be more readily followed. C. Contraction in muscle with a high initial glycogen content. During the past year attempts have been made to obtain cod with high muscle glycogen levels. Cod have been kept in tanks at a temperature of 7 C where cod usually feed readily. When fed thawed frozen herring 9 3 times a week 9 there was only a slight increase in glycogen level as reported above, Section A 9 perhaps due to erratic feeding habits. However 0 one feeding cod was found with a very high initial muscle glycogen content 480 mg/100 go When the muscle was placed at 9 P rigor mortis set in at

185 163 about 6 hours, as with the non-feeding fish, but the ultimate ph reached 5.9 to 6.0 and considerable contraction occurred, about ~. Handling did not appreciab~ affect these values, unlike the effect reported last year with non-feeding fish where handling marked~ increased shortening and drip. It appears that with fish with very high muscle g~cogenp as might be expected in thebaavi~ feeding trap fish, contraction and drip formation occurs at considerab~ lower temperature levels. Unfortunate~ no data on the glycogen level of the trap fish have yet been obtainedg but the ultimate ph has been found to reach levels below ph 6.0, similar to the present case. Attempts will be made to obtain samples from heavily feeding fish to extend the above data and also to examine the lewfoundland trap fish. CHEMICAL COMPOSITION OF FISH: EXTRACTIVES OF COD: SEASONAL VARIATIONS IN THE CHEMICAL COMPOSITION OF COD FILLErS N.Dambergs After the optimum conditions for the frac;ionation of fish muscle by solvent extraction were established (J. Fish. Res. Bd. Canada. Vol. 16, p. 63, 1959), a new set of figures representing the composition of fillets of Nova Scotian inshore cod was obtained. (This Station's Annual Report for , Appendix 9). Then it also became possible to obtain an accurate picture of the distribution of fat, water-solubles, and protein throughout the edible part of the fish. The accumulation of these data partly carried out in 1960 and completed in 1961 was organized in a manner to pro,ide the first outline of the possible seasonal variations of these components. At that timep it was believed that the knowledge of the nature and of the extent of seasonal variations in the chemical composition of the muscle would provide a safe foundation for the exploration of the individual entities of the muscle and their role, thus leading to better understanding of the postmortem processes taking place in the muscle. The available data of this nature as a rule has been obtained without regard to the seasonal variations of these components nor knowledge of their local distribution in the tissue. The preliminary analyses showed that ver,y rapid chemical changes took place in fish muscle even during the prerigor period. To reduce the interference of the postmortem changes only live, freshly caught fish were used. Comparison of the composition of fillets obt~ed from large and from small specimens caught simultaneously on the same fishing grounds showed that the difference in composition of the fillet increased with the increasing difference of the size of the fish. The differences of

186 164 the concentrations of fat, water-solubles, protein and water in male and female specimens of the same size and caught in the same place and time were much less important. For this reason, the sampling was simplified by random selection of cod without regard to sex. On approximately the 15th of each month, five mature cod not shorter than 21 inchesp and not larger than 24 inches, were caught in Terrance Bay and delivered alive in a refrigerated sea-water tank to the laboratory. On arrival they were immediately killed and filleted. From the fillets, four samples were taken - one was prepared from the head-ends of the fillets, the second from the tail-endsp the third from the belly sections, and the fourth sample was prepared by mixing the remaining parts of the fillet. This last sample served as a control for the direct determination of water and total phosphorus. The samples were immediately solvent extracted at room temperature according to our previously published method. The extracts were then analysed for their content of fat and water-solubles. The conditions of extraction were adjusted so as not to extract any proteins. Thus the content of protein in the samples could be determined gravimetrically on the extracted residues. The method was accurate enough to provide a good agreement of the results obtained on separate parts of the sample and the direct determination of the total solids on the control sample. The results obtained can be summarized as follows~ The fat content of the fillet undergoes a considerable variation during the yearly cycle. The monthly plot of fat content shows a sharp minimum at the peak of the spawning period~ which for the cod at Terrance Bay is in April. Immediately after the end of spawning the fat content increases rapidly to reach the maximum level in July, and August and then very slowly decreases until February when it decreases rapidly towards the minimum in April. The seasonal variations of fat content in the head-end are more pronounced than in the belly section of the fillet, while the tailend is the least affected. The observed fluctuations from the maximum to minimum values for the three sections of the fillet are 24% decrease in the head-end, 15% in the middle section and lo% in the tail-end. The variations in the concentration of the water-solubles follow a rather opposite pattern. The maximum concentration of water solubles in the muscular tissue corresponds to the spawning peak and decreases symmetrically on both sides of the April value to reach the minimum level after the moat active feeding period is over, namely in September~ October and November. Then the water solubles begin to increase again towards the maximum value in April. These variations are similar in all three parts of the fillet and the increase from minimum to maximum values is 24% in all three sections. The variations in protein concentrations, due to the large amount of this component in the filletp are more difficult to detect 9 and they are not as obvious as the variations of the two previous components. However, there also seems to be a definite minimum which does not coincide with the spawning peak but occurs approximately a

187 165 month latero In other wordsp it seems that the recovery of protein begins short~ after the deficit of lipids has been partially compensatedo The results obtained for the monthly values of protein are scattered and would deserve further studies on strictly standardized sampleso Thelariations in water 9 the major component of the fillet 9 are symmetrical to the variations of the proteins and are little pronouncedo The extreme values registered over a two year period were 80o4% and 82o~ (only once) with an average value of 8lo~o The oft-stated rule for herring and some other fat fishes stipulating that the sum of fat and water in fish remain constftnt does not app~ to the lean cod fisho The decrease in the concentratlon of lipids in the tissue here is not compensated by the increase of water but b,y the increase of the water-soluble compoundso Also the distribution of fat and water in the fillet of cod is not complementary but both are at their maximum in the tail-end of the filleto Total phosphorus was determined on all the extracts as well as in the extracted protein residues and control sampleso The concentration of this element first follows the variations of the lipids and reaches a minimum at spawning time when fat is also minimalo Total phosphorus then increases ver,y rapid~ and reaches a maximum in June and fluctuates thereafter, probab~ with the lcind and abundance of the food taken by the codo The sulphur ana~ses are not yet completedo The head-end of ~ fillet is the richest in sulphur while the tail-end is the pooresto Eight percent of the total sulphur is extractable o In all three parts of the fillet, sulphur is at its minimum during the spawning periodo During the first nine months of the study when the main objective was the determination of the true values of protein 9 fat 9 water=solubles 9 and water in cod fillets as well as their distribution throughout the fillet only one mature cod monthly was usedo The monthly values for the components followed the same pattern as curves obtained in 1961 using five fish sampleso However 9 the points obtained using only one fish showed less deviation from the main curve than the points obtained from five fish sampleso This proves that the chemical seasonal changes in cod muscle can be followed even on a single fish if strictly standardized samples are usedo The scattering of results in the summer months of 1961 are explained by the difficulty to secure five full-size live fish when the large specimens in Terrance Bay were scarce and their capacity of survival considerably shortenedo To have at least five live fish for the samples 9 under-size specimens were usedo This fact may be responsible for the scattered results obtained in the summer and fall, but it is not excluded that at this time other populations of cod were entering Terrance Bay 0 During this study a collection of monthly extracts from standardized samples covering almost a full two year period were prepared and preserved for further analyseso With little additional effort these

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