(Received 19 May 2005; Accepted 12 August 2005)

Transcription

1 Environmentl Toxiology n Chemistry, Vol. 25, No. 4, pp , SETAC Printe in the USA /6 $12.. USE OF CARBOXYLESTERASE ACTIVITY TO REMOVE PYRETHROID-ASSOCIATED TOXICITY TO CERIODAPHNIA DUBIA A HYALELLA AZTECA IN TOXICITY IDENTIFICATION EVALUATIONS CRAIG E. WHEELOCK, JEFF L. MILLER, MIKE J. MILLER, BRYN M. PHILLIPS, SARAH A. HUNTLEY, SHIRLEY J. GEE, RONALD S. TJEERDEMA, n BRUCE D. HAMMOCK* Deprtment of Entomology n Cner Reserh Center, University of Cliforni t Dvis, Dvis, Cliforni 95616, USA AQUA-Siene, Dvis, Cliforni 95616, USA Deprtment of Environmentl Toxiology, University of Cliforni t Dvis Mrine Pollution Stuies Lortory, Monterey, Cliforni 9394, USA (Reeive 19 My 25; Aepte 12 August 25) Astrt Inreses in the use n pplition of pyrethroi insetiies hve resulte in onern regring potentil effets on quti eosystems. Methos for the etetion of pyrethrois in reeiving wters re require to monitor environmentl levels of these insetiies. One metho employe for the ientifition of uses of toxiity in quti smples is the toxiity ientifition evlution (TIE); however, urrent TIE protools o not inlue speifi methos for pyrethroi etetion. Reent work ientifie roxylesterse tretment s useful metho for removing/eteting pyrethroi-ssoite toxiity. The present stuy hs extene this erlier work n exmine the ility of roxylesterse tivity to remove permethrin- n ifenthrin-ssoite toxiity to Ceriophni ui n Hylell zte in vriety of mtries, inluing lortory wter, Srmento River (CA, USA) wter, n Slins River (CA, USA) interstitil wter. Esterse tivity suessfully remove 1, ng/l of permethrin-ssoite toxiity n 6 ng/l of ifenthrin-ssoite toxiity to C. ui in Srmento River wter. In interstitil wter, 2 ng/l of permethrinssoite toxiity n 6 ng/l of ifenthrin-ssoite toxiity to H. zte were remove. The seletivity of the metho ws vlite using het-intivte enzyme n ovine serum lumin, emonstrting tht tlytilly tive esterse is require. Further stuies showe tht the enzyme is not signifintly inhiite y metls. Mtrix effets on esterse tivity were exmine with muniipl effluent n sewter in ition to the mtries isusse ove. Results onfirme tht the esterse retins tlyti funtion in iverse rry of mtries, suggesting tht this tehnique n e pte to vriety of quti smples. These t emonstrte the utility of roxylesterse tretment s vile step to etet the presene of pyrethrois in reeiving wters. Keywors Croxylesterse Pyrethroi Toxiity ientifition evlution Ceriophni ui Hylell zte INTRODUCTION The use of pyrethroi insetiies is inresing, whih oul result in onomitnt rise in their ourrene in reeiving wters n seiments [1]. Pyrethrois re potentilly toxi to numer of quti orgnisms, inluing mny fish speies s well s quti invertertes [2 7]. Reent stuies hve ientifie pyrethroi-ssoite toxiity to quti orgnisms in seiments [4,5], interstitil wters [8 1], n storm-wter runoff [3,11]. The effets of inresing pyrethroi usge on quti eosystems therefore oul e importnt [12] ( n reeiving wters n seiments shoul e monitore for the presene of pyrethrois. However, urrent nlytil tehniques for pyrethroi etetion re lor-intensive, time-onsuming, n expensive [4,5,13]. The use of toxiity ientifition evlution (TIE) methos is n effiient, routine tehnique to etet n ientify mny toxints in environmentl smples [14 16]. However, pulishe U.S. Environmentl Protetion Ageny (U.S. EPA) TIE protools o not ress the issue of pyrethroi etetion speifilly. Previous work ientifie roxylesterse tivity s useful tool for the etetion of pyrethrois in reeiving wters * To whom orresponene my e resse (hmmok@uvis.eu). The urrnet ress of C. E. Wheelok is Bioinformtis Center, Institute for Chemil Reserh, Kyoto University, Uji, Kyoto , Jpn. tht ppere to e pproprite for use in TIE testing with Ceriophni ui [13]. These stuies showe tht the introution of roxylesterse to wter smple efore the ition of test orgnisms ws suffiient to remove pyrethroissoite toxiity. Croxylesterses re enzymes tht hyrolyze ester-ontining ompouns (e.g., pyrethrois) to their orresponing i n lohol, whih re usully etoxifition prouts [17] (Fig. 1). Croxylesterse tivity is importnt for the metolism n susequent etoxifition of mny exogenous ester-ontining ompouns, inluing rmtes [18], orgnophosphtes (OPs) [19], n pyrethrois [2]. The present stuy ws esigne to further vlite the use of esterse tivity in TIE testing in multiple systems. Ielly, ny metho evelope for pyrethroi etetion woul e funtionl for multiple orgnisms in vriety of mtries. This stuy extene erlier work to new mtries, Srmento River (CA, USA) wter n Slins River (CA, USA) interstitil wter, s well s to new test speies, Hylell zte. Two ifferent pyrethrois were use, permethrin n ifenthrin, whih were hosen se on their usge ptterns [5] n rtes of esterse-meite hyrolysis [13]. Permethrin is one of the most ommonly use pyrethrois in Cliforni (USA), n s suh, the likelihoo tht this pyrethroi woul e oserve in quti smpling is inrese ( Bifenthrin is of onern to mny groups performing seiment tox- 973

2 974 Environ. Toxiol. Chem. 25, 26 C.E. Wheelok et l. Fig. 1. Esterse-meite hyrolysis of the pyrethroi ifenthrin. iity stuies, n work hs shown tht ifenthrin toxiity n our in interstitil wter toxiity stuies [5,21]. Previous work ientifie permethrin s eing hyrolyze reltively quikly y the porine roxylesterse employe in these stuies, wheres ifenthrin ws relitrnt to esterse-meite hyrolysis [13]. These two pyrethrois therefore represent the extremes of hyrolyti tivity tht the esterse most likely will enounter uring toxiity stuy. This projet provies further eviene for the utility of roxylesterse tivity in eteting the presene of pyrethroi-ssoite toxiity in TIE testing systems. MATERIALS A METHODS Chemils All hemils were purhse from Alrih Chemil (St. Louis, MO, USA) or Fisher Sientifi (Pittsurgh, PA, USA) unless otherwise note. Pestiie stnrs were quire from Austnr (New Hven, CT, USA). The esterse-inhiitor 1,1,1-trifluoro-3-otylthiol-propn-2-one (OTFP) ws synthesize oring to the methos esrie y Wheelok et l. [22]. Porine liver esterse n ovine serum lumin (BSA) were purhse from Sigm Chemil (St. Louis, MO, USA). Esterse onentrtion is reporte in terms of units of enzyme tivity per milliliter of wter, with n enzyme level of units of esterse tivity per milliliter of wter efine s 1x onentrtion for the purposes of this projet. A unit of tivity is the mount of enzyme tht hyrolyzes 1. mol of ethyl utyrte to utyri i n ethnol per minute t ph 8. t 25 C (s efine y Sigm Chemil). Different preprtions of enzyme n vry in their tivity level, ut the nomenlture of 1x ws kept onstnt for ll enzyme stuies. Two ifferent esterse preprtions were use in the present stuy, n esterse solution from porine liver in n mmonium sulfte suspension (3.2 M, ph 8., tlog no. E-2884; Sigm Chemil) n rue lyophilize power ( 5% uffer slts, tlog no. E-319; Sigm Chemil). Esterse tivity n protein onentrtion vry with lot numer n shoul e reporte in ll stuies. Toxiity testing Stuies with the loern C. ui, freshwter inverterte, were performe s etile y Wheelok et l. [13] n re riefly esrie here. Ceriophni ui neontes (ge, 24 h) were otine from ultures mintine t AQUA-Siene (Dvis, CA, USA). Cultures were mintine t 25 1 C, with photoperio of 16:8-h light:rk, n were fe mixture of the green lg Selenstrum priornutum (University of Texs Alge Type Colletion, Austin, TX, USA) n lene trout how (Silver Cup, Murry, UT, USA). Lortory wter onsiste of reverse osmosis trete well wter mene with ry slts to U.S. EPA moertely hr speifitions. Srmento River wter ws ollete from the Srmento River t Freeport Mrin (Srmento, CA, USA). The 48-h toxiity tests were onute in 2-ml glss sintilltion vils ontining 18 ml of moertely hr ilution wter. Test solutions were not renewe, n the orgnisms were not fe uring the exposures. Mortlities were monitore ily. The onentrtions require to use n effet in 5% of the popultion (EC5) were lulte from the mortlity t using omputer progrm (ToxCl TM, Ver 5..23; Tiepool Sientifi, MKinnleyville, CA, USA). Insetiie working stnrs (1 g/l) were prepre y issolving the hemils in high-performne liqui hromtogrphy gre methnol. Permethrin exposures were performe t 5, 75, n 1, ng/l, n ifenthrin exposures were onute t 3, 45, n 6 ng/l. Methnol onentrtion in ll test solutions ws less thn.1%. Initil toxiity stuies were performe with C. ui using two ifferent esterse preprtions, n esterse solution in n mmonium sulfte suspension (tivity 1,84 U/ml [184 U/ mg protein, 1 mg protein/ml], lot 12K762; Sigm Chemil) n lyophilize power (tivity 2 U/mg, lot 39H75; Sigm Chemil). Tolerne of C. ui to esterse exposure ws exmine t x, 1x, 5x, 1x, 1x, 5x, n 1,x. Susequent exposures to the liqui preprtion were performe t x, 1x, 5x, 1x, n 1x. Stuies with the hetintivte enzyme n with the nontlyti protein BSA were prepre extly s esrie for the esterse, with onentrtions reporte in terms of x. In these ses, x refers to the equivlent level of protein tht woul e present in 1x of the esterse preprtion. Het-intivte enzyme ws prepre y heting the enzyme t 1 C for 4 min. The liqui enzyme stok solutions were prepre y iluting the net enzyme 1:1 in ultrpure wter ( 1 S/m s prepre y ion-exhnge tretment; Cole Prmer, Vernon Hills, IL, USA), giving finl enzyme ilution of 184 U/ml. The lyophilize enzyme ws reonstitute in ultrpure wter (1 mg/ml, 2 U/ ml). Enzyme stok solutions were liquotte into the test ontiners using preision pipette (Rinin, Wourn, MA, USA) to hieve the esire enzyme onentrtion. Solutions were llowe to stn for 1 h efore ition of the test orgnisms. Hylell zte exposures were onute t the University of Cliforni t Dvis Mrine Pollution Stuies Lortory. Amphipos were otine from Chespeke Cultures (Hyes, VA, USA) 48 h efore test initition, mintine t 23 C, n fe yest erophyl trout how [23]. Exposures were onute in 2-ml glss sintilltion vils (three replites) tht h een prelehe with freshwter for minimum of 2 h n rinse thoroughly. Test ontiners hel 15 ml of trete smple n five 7- to 14--ol mphipos, whih were loe in.5 ml of ulture wter. Exposures were onute for 96 ht23 C following U.S. EPA guielines [24]. Wter-qulity prmeters of issolve oxygen, ph, n onutivity (unorrete; i.e., not speifi onutne) were mesure using Hh SensION seletive ion meter (Hh, Loveln, CO, USA) with pproprite eletroes t test initition, n temperture ws mesure using ontinuously reoring thermogrph n thermometer. Ammoni ws mesure with olorimetri metho (sliylte metho) using Hh 231

3 Use of roxylesterse tivity in TIEs Environ. Toxiol. Chem. 25, spetrophotometer. Totl mmoni ws mesure in terms of mg/l, n un-ionize mmoni ws lulte from the totl onentrtion using ph, temperture, n slinity (whih ws zero for these stuies). Wter hrness ws pproximtely 8 mg/l s CCO 3, ph vrie from 7.8 to 8., n un-ionize mmoni ws 5.2 mg/l. Slins River interstitil wter ws ollete from referene site ner the ity of Chulr ( 2 km south of Slins, CA, USA). No OPs were etete in this smple using enzyme-linke immunosorent ssys (t not shown). Conentrtions of ifenthrin n permethrin were prepre from 1 mg/l stok solutions. Superstok solutions were ilute to 1 g/l seonry stok solutions in methnol, then ilute further to test onentrtions using Mrine Pollution Stuies Lortory well wter. Permethrin exposures were onute t 1, 15, n 2 ng/l n ifenthrin exposures t 2, 6, n 1 ng/l. A tretment lnk ws use to monitor for methnol-ssoite toxiity. Initil experiments use liqui preprtion of enzyme (tivity 1,84 U/ml, lot 12K762; Sigm Chemil) tht ws ilute to 1.3 U/ml. Tolerne of H. zte to esterse ws etermine y exposing the mphipos to rnge of esterse onentrtions (x, 1x, 5x, 1,x, n 5,x). Esterse stok solutions were prepre in glss volumetri flsks (545 l of enzyme per 1 ml of Milli-Q wter, filtere t 18.2 M t 25 C with 25- m filter; Millipore, Billeri, MA, USA). Susequent experiments use lyophilize power enzyme preprtion (tivity 2 U/mg soli, lot 123K733; Sigm Chemil) tht ws use to rete stok solution of 1 mg/ml. Tolerne of H. zte to exposure to the lyophilize enzyme ws exmine t x, 1x, 5x, 1,x, n 5,x. Aitionl experiments exmining the ility of the esterse to remove pyrethroi-ssoite toxiity were onute with the lyophilize esterse t x, 1x, 5x, 1x, n 5x. Net liqui enzyme or enzyme stok solutions were trnsferre to volumetri flsks of spike wter n liquotte to test ontiners y pouring ( 1 ml per test ontiner). The esterse ws e to the wter smple t lest 1 h efore introution of the orgnisms. Mortlity ws then monitore ily for the urtion of the stuy. Enzyme ssys All esterse tivity ssys were onute oring to the methos esrie y Wheelok et l. [22] s pte from those esrie y Ljungquist n Augustinsson [25] using the stnr esterse sustrte p-nitrophenyl ette (PNPA). Assys were initite y ing esterse to flt-ottom, 96- well polystyrene mirotiter plte (Dynex Tehnologies, Chntilly, VA, USA) in Tris uffer (.1 M, ph 7.4) t 25 C, followe y ition of the sustrte n nlysis t 45 nm for 2. min in kineti moe on Spetrmx 34PC plte reer (Moleulr Devies, Sunnyvle, CA, USA). For inhiition ssys, the esterse ws inute with the inhiitor for 1 min efore the ition of sustrte. Mtrix effets on esterse tivity were exmine over 48-h perio. Stuies were initite y ing 2 l of esterse (1,84 U/ml, lot 12K762; Sigm Chemil) to 1 ml of mtrix in 15-ml Flon Blue Mx polypropylene onil tue (1 mm; Beton Dikinson Lwre, Frnklin Lkes, NJ, USA). Ativity ssys were onute y removing 1- l liquots of eh mtrix n mesuring esterse tivity s esrie ove. Finl esterse onentrtion in ll ssys ws.1 g/ml (1.84 mu/ml). Severl ifferent mtries were exmine, inluing soium phosphte uffer (.1 M, ph 7.4), Milli-Q wter (Millipore), nturl sewter (ollete from Boeg By, CA, USA, n filtere to.45 m), muniipl effluent (ollete from the Srmento Regionl Wstewter Tretment Plnt, Srmento, CA, USA), Srmento River wter (ollete from the Srmento River t Freeport Mrin, Srmento, CA, USA), n interstitil wter extrte from two seiments from the lower Snt Mri River wtershe (ner Gulupe, CA, USA). The hrteristis of the seiment smples, Snt Mri n Orutt Creek, s well s the preise lotion of smpling hve een esrie y Anerson et l. [7]. Totl orgni ron of the Orutt Creek seiment ws.95%, n tht of the Snt Mri seiment ws 1.1%. The Orutt Creek seiment ontine.322 g/l of hlorpyrifos n the Snt Mri.589 g/l. The effet of metls on esterse tivity ws exmine using 19 ifferent metl tions tht were purhse s the hlorie slt. Stok solutions were prepre in Milli-Q wter (.1 M). Assys were initite y ing esterse to 96-well mirotiter plte in Tris uffer (.1 M, ph 7.4), followe y 2 l of eh metl (finl metl onentrtion, 1 mm). Inhiite esterse ws generte for stuies exmining the nee for tlytilly tive enzyme to remove pyrethroissoite toxiity. To 2.8-ml ottle of esterse (184 U/ml, 1 mg/ml, lot 12K762; Sigm Chemil), 28 l of 5 mm stok solution of hlorpyrifos-oxon (in ethnol) were e to give finl onentrtion of.5 mm. An equivlent ontrol ottle ws prepre with 28 l of ethnol. Following ition of the inhiitor, no esterse tivity ws etete y PNPA ssy. The exess hlorpyrifos-oxon (not oun to the esterse) ws then remove y filtering the enzyme mixture through 3-ml, 5K utoff Centrion olumn (Millipore) t 6, rpm on Sorvll entrifuge (Kenro Lortory, Asheville, NC, USA) for 2 min. After entrifugtion, 2 ml of hille phosphte uffer (.1 M, ph 7.4) were e to the olumn, whih ws entrifuge gin. This metho ws repete eight times, until ll hlorpyrifos-oxon ws remove. Removl ws verifie y ition of 1 l of the inhiite enzyme preprtion to untrete enzyme n then ssying for enzyme tivity with PNPA. Suessful removl of the exess hlorpyrifos-oxon ourre when no esterse inhiition ws oserve. Control enzyme ws mnipulte ientilly, n no signifint loss in tivity ws oserve uring the entrifugtion proess. The inhiite enzyme preprtion ws then store t 4 C for pproximtely 12 h efore use in toxiity ssys. Stuies were performe with n itionl esterse-inhiitor OTFP ientilly s esrie for hlorpyrifos-oxon. RESULTS Toxiity testing with C. ui Results emonstrte tht the liqui enzyme preprtion ws not overtly toxi t levels up to 1x (Tle 1). However, t inrese onentrtions, signifint toxiity ws oserve. The lyophilize power i not exhiit ny signifint toxiity t enzyme levels up to 1,x. Neither the het-intivte enzyme nor the protein BSA exhiite toxi effets t ny of the onentrtions teste. Permethrin-ssoite toxiity in lortory wter ws prtilly remove t 5x n ompletely remove t 1x n 1x (Tle 2). No onentrtion response reltionship ws oserve for the three ifferent onentrtions of permethrin exmine. Similr effets were oserve with the Srmento River wter; however, the enzyme t suggeste inrese

4 976 Environ. Toxiol. Chem. 25, 26 C.E. Wheelok et l. Tle 1. Aute esterse toxiity to Ceriophni ui Enzyme onentrtion x 1x 5x 1x 1x 5x 1,x 48-h mortlity (%) Liqui Lyophilize BSA e Dt re the verge of four replites with five neontes stnr evition. lx U/ml in lortory wter. Ammonium sulfte suspension esterse preprtion (lot 12K762; Sigm Chemil, St. Louis, MO, USA). Lyophilize esterse preprtion (lot 39H75; Sigm Chemil). e BSA ovine serum lumin. effiy in Srmento River wter versus lortory wter, emonstrting mtrix-speifi effiieny. A onentrtion response reltionship ws oserve t the 5x level, with inresing onentrtions of permethrin using inrese mortlity. Bifenthrin-ssoite toxiity ws remove similrly to permethrin-ssoite toxiity, with 1x removing ll toxiity in lortory wter (Tle 2). However, 1x ws neessry to remove ll toxiity in Srmento River wter. Esterse removl of pyrethroi-ssoite toxiity ws more effiient for permethrin in lortory wter ut less effiient in Srmento River wter. Conentrtion response reltionships were oserve for oth mtries, with higher levels of ifenthrin eliiting inrese toxiity. Two ifferent ontrols were use to verify tht the oserve toxiity removl ws use y tlytilly tive enzyme. Het-intivte enzyme i not remove ny permethrin- or ifenthrin-ssoite toxiity t n esterse onentrtion of 1x (Tle 2). At 1x, slight reution in permethrin-ssoite toxiity ws oserve t the lowest onentrtion of permethrin exmine (5 ng/l). However, no reution in ifenthrin-ssoite toxiity ws oserve t 1x (Tle 2). Stuies performe with BSA i not signifintly remove permethrin- or ifenthrin-ssoite toxiity t 1x or 1x in either mtrix exmine (Tle 2). The lyophilize esterse preprtion ws exmine for its ility to remove ifenthrin-ssoite toxiity (Tle 3). At 5x or greter, ll ifenthrin-ssoite toxiity ws remove. Equivlent stuies performe with BSA showe tht no reution in toxiity ourre up to 25x. However, t 25x, signifint reution ws foun in toxiity (3% mortlity) t the lowest onentrtion of ifenthrin exmine (3 ng/l). Toxiity testing with H. zte Signifint ifferenes were oserve in the ute toxiity of the two ifferent esterse preprtions to H. zte (Tle 4). The liqui preprtion use 37% mortlity t 5x, Tle 2. Effet of liqui esterse on pyrethroi toxiity to Ceriophni ui 48-h mortlity (%) Pyrethroi (ng/l) Ative enzyme x f 1x 5x 1x 1x Intive enzyme 1x 1x 1x BSA e 1x Permethrin Lortory wter g , Srmento River wter h , Bifenthrin Lortory wter g Srmento River wter h Dt re the verge of four replites with five neontes stnr evition. Pyrethroi onentrtions re nominl wter onentrtions. Esterse ws prepre s esrie in Mterils n Methos. Esterse ws hete t 1 C for 4 min. e Bovine serum lumin (BSA) ws e t protein onentrtions equivlent to those use in the esterse preprtions. f 1x ilution U/ml. A unit of tivity is the mount of enzyme tht hyrolyzes 1. mol of utyrte to utyri i n ethnol per minute t ph 8. t 25 C (s efine y Sigm Chemil, St. Louis, MO, USA). g Wter mene to U.S. Environmentl Protetion Ageny moertely hr speifitions. h Srmento River wter ws ollete t Gri Ben, ner Srmento (CA, USA).

5 Use of roxylesterse tivity in TIEs Environ. Toxiol. Chem. 25, Tle 3. Effiy of lyophilize esterse on removl of ifenthrin-ssoite toxiity to Ceriophni ui 48-h Mortlity (%) Bifenthrin (ng/l) Esterse x e 1x 5x 1x 25x Bovine serum lumin 1x 5x 1x 25x Dt re the verge of four replites with five neontes stnr evition. Stuies were performe in lortory wter mene to U.S. Environmentl Protetion Ageny moertely hr speifitions. Bifenthrin onentrtions re nominl wter onentrtions. Esterse ws prepre s esrie in Mterils n Methos using lyophilize esterse (lot 39H75; Sigm Chemil, St. Louis, MO, USA). Bovine serum lumin (BSA) ws e t protein onentrtions equivlent to those use in the esterse preprtions (25x BSA mg protein/ml). e 1x ilution U/ml. A unit of tivity is the mount of enzyme tht hyrolyzes 1. mol of utyrte to utyri i n ethnol per minute t ph 8. t 25 C (s efine y Sigm Chemil). wheres the lyophilize preprtion exhiite % mortlity. The mximum esterse onentrtion exmine ws 5,x, whih use 1% mortlity with the liqui preprtion ut only 53% with the lyophilize preprtion. Initil stuies with the liqui esterse showe signifint mortlity in the ontrols t enzyme onentrtions greter thn 1x (t not shown). However, t n esterse onentrtion of 1x in lortory wter, 67 n 2% mortlity ws still oserve in exposures to 1 ng/l of ifenthrin n of permethrin, respetively. The eision therefore ws me to use higher enzyme onentrtions for the H. zte stuies. However, there ws onern regring the esterse-ssoite toxiity following exposure to the liqui enzyme preprtion. This toxiity ws ttriute to elevte mmoni levels, whih were etete in ll smples n orrelte iretly with the onentrtion of enzyme e to the smple (Tle 5). The lyophilize esterse preprtion therefore ws use for further stuies with H. zte. The lyophilize esterse remove essentilly ll permethrin-ssoite toxiity in lortory wter, with no oserve onentrtion response reltionship (Tle 6). The interstitil wter stuies showe tht onentrtion of 5x ws neessry to remove ll permethrin-ssoite toxiity. No signifint enzyme-ssoite toxiity ws oserve in ny of the stuies. Esterse tivity ws not s effiient t removing ifenthrin-ssoite toxiity in lortory wter (Tle 6). A signifint reution in ifenthrin-ssoite toxiity ws foun t the 2 ng/l ose, ut only for the higher enzyme onentrtions. Enzyme onentrtions s high s 5x i not signifintly reue ifenthrin-ssoite toxiity Enzyme onentrtion x 1x 5x 1,x 5,x Tle 4. Aute esterse toxiity to Hylell zte Liqui h mortlity (%) Lyophilize Dt re the verge of three replites with five mphipos stnr evition. 1x U/ml in lortory wter. Ammonium sulfte suspension esterse preprtion (lot 12K762; Sigm Chemil, St. Louis, MO, USA). Lyophilize esterse preprtion (lot 123K733; Sigm Chemil). t 6 n 1 ng/l. However, signifint reution in toxiity ws oserve in the interstitil wter ose with 6 ng/l n 5x esterse. No signifint mortlity ws oserve in the ontrols. Esterse hrteriztion The time epenene of esterse tivity ws exmine in severl ifferent mtries (Tle 7). A phosphte uffer ws most effiient t mintining esterse tivity over 48 h, with 24% reution in tivity oserve. Milli-Q wter ws essentilly s effiient s phosphte uffer, with 29% reution in tivity uring the monitoring perio. A signifint reution in tivity ws oserve in sewter, with no etetle tivity y 48 h. The other exmine mtries performe similrly, with loss of ll tivity y 12 to 24 h. However, ll exmine mtries h greter thn 5% remining tivity up to 2 h fter test initition. Esterse inhiition stuies performe with permethrin n two inhiitors showe tht 1x tlytilly tive esterse remove ll toxiity t three ifferent pyrethroi onentrtions (Tle 8). Inution of the enzyme with hlorpyrifosoxon i not ffet the ility of the esterse to reue toxiity. Mesure prmeter Tle 5. Ammoni n ph omprison of liqui versus lyophilize esterse Liqui esterse ph Totl mmoni (mg/l) Un-ionize mmoni (mg/l) Lyophilize esterse ph Totl mmoni (mg/l) Un-ionize mmoni (mg/l) Enzyme onentrtion x 1x 5x Vlues were mesure uring toxiity stuies with Hylell zte. 1x ilution U/ml. A unit of tivity is the mount of enzyme tht hyrolyzes 1. mol of utyrte to utyri i n ethnol per minute t ph 8. t 25 C (s efine y Sigm Chemil, St. Louis, MO, USA). Enzyme ws from n mmonium sulfte suspension from porine liver (3.2 M, ph 8., lot 12K762; Sigm Chemil). Enzyme ws from rue lyophilize power (lot 123K733; Sigm Chemil).

6 978 Environ. Toxiol. Chem. 25, 26 C.E. Wheelok et l. Tle 6. Effet of lyophilize esterse on pyrethroi toxiity to Hylell zte 96-h mortlity (%) per enzyme onentrtion Pyrethroi (ng/l) x 1x 5x 1x 5x Permethrin Lortory wter Chulr interstitil wter e Bifenthrin Lortory wter Chulr interstitil wter e Dt re the verge of three replites with five mphipos stnr evition. Esterse ws prepre from rue lyophilize power s esrie in Mterils n Methos (lot numer 123K733; Sigm Chemil, St. Louis, MO, USA). Pyrethroi onentrtions re nominl wter onentrtions. 1x ilution U/ml. A unit of tivity is the mount of enzyme tht hyrolyzes 1. mol of utyrte to utyri i n ethnol per minute t ph 8. t 25 C (s efine y Sigm Chemil). Grnite Cnyon well wter (Grnite Cnyon, CA, USA). e Chulr pore wter ws ollete from referene site on the Slins River ner the ity of Chulr (CA, USA). However, use of the esterse-inhiitor OTFP resulte in oseepenent inrese in permethrin-ssoite toxiity. Inution of the esterse-inhiitor OTFP with the enzyme efore test initition reue the enzyme s pility to reue pyrethroi toxiity in ose-epenent mnner. A series of 19 ifferent metls were sreene for their ility to inhiit esterse tivity (Tle 9). Only iron(iii), zin, ruiium, nikel, iron(ii), n opper(ii) signifintly inhiite tlyti tivity (p.5). The gretest level of inhiition ws oserve using opper(ii), with 31% inhiition. A positive Tle 7. Time epenene of esterse tivity in vrious mtries Time Muniipl (h) Buffer Wter effluent Srmento River wter Interstitil wter Snt Mri Orutt Creek e Sewter f All vlues re shown s the perentge of tivity reltive to initil uffer ontrol (1 mm, ph 7.4, phosphte uffer) n re the verge of three replites stnr evition. Enzyme (.1 g/ml, 18.4 mu/ml) ws inute in eh mtrix t 25 C, n tivity ws mesure using the sustrte p-nitrophenyl ette. A unit of tivity is the mount of enzyme tht hyrolyzes 1. mol of utyrte to utyri i n ethnol per minute t ph 8. t 25 C (s efine y Sigm Chemil, St. Louis, MO, USA). Soures of ll wter smples re esrie in Mterils n Methos. Soium phosphte uffer (1 mm, ph 7.4). Milli-Q wter (Millipore, Billeri, MA, USA). Collete from the Snt Mri River ner Gulupe (CA, USA). e Collete from Orutt Creek ner Gulupe (CA, USA). f not etete.

7 Use of roxylesterse tivity in TIEs Environ. Toxiol. Chem. 25, Tle 8. Effet of enzyme inhiition on esterse effiy in removing permethrin-ssoite toxiity to Ceriophni ui Esterse inhiitor Permethrin (ng/l) No enzyme Ative enzyme Inhiite enzyme e Chlorpyrifos-oxon f , OTFP g , Dt re shown s the men perentge mortlity stnr evition for 48-h C. ui toxiity using five neontes with four replites. Permethrin onentrtions re nominl wter onentrtions. Toxiity ssys were performe in the sene of esterse, or x, where 1x ilution U/ ml. A unit of tivity is the mount of enzyme tht hyrolyzes 1. mol of utyrte to utyri i n ethnol per minute t ph 8. t 25 C (s efine y Sigm Chemil, St. Louis, MO, USA). Uninhiite enzyme (1x) ws e to eh ontiner (liqui preprtion, lot numer 12K762; Sigm Chemil). e The inite inhiitor (1x) ws e to the esterse t finl onentrtion of.5 mm. f The speifi tivity of the porine esterse preprtion efore inhiition ws mol/min/mg s etermine y p-nitrophenyl ette (PNPA) hyrolysis. Following inhiition with.5 mm hlorpyrifosoxon n susequent filtrtion-se inhiitor removl, no PNPA hyrolysis tivity ws etete. However, 72 h fter toxiity ssy initition, the esterse speifi tivity h inrese to 81.2 mol/ min/mg, initing retivtion of the enzyme. g OTFP 1,1,1-trifluoro-3-otylthiol-propn-2-one. ontrol using izinon-oxon, known inhiitor of porine esterse, showe 1% inhiition. DISCUSSION Esterse toxiity to C. ui n H. zte The first issue exmine ws overt toxiity of the enzyme preprtion to the testing orgnisms. Currently, two ommeril preprtions of the porine esterse re ville. Erlier stuies with the enzyme reporte use of n mmonium sulfte suspension [13]. The seon enzyme preprtion is ville s rue lyophilize power, whih hs the vntge of erese mmonium slts, reue ost, n inrese homogeneity. The hyrolyti tivities of the two preprtions iffer y pproximtely one orer of mgnitue (lyophilize power, 2 U/mg protein; liqui preprtion, 184 U/mg protein). The min vntges of the liqui preprtion inlue inrese speifi tivity, ese of hnling for ition to testing ontiners, n existing t regring enzyme effiy from previous stuies (one lortory involve in the present stuy, however, reporte tht hnling of the lyophilize power ws esier). We evlute oth preprtions for toxiity to the testing orgnisms. Initil stuies eviene no signifint ifferenes in toxiity up to 1x for oth speies. This level of enzyme ws shown to e more thn suffiient in previous stuies for omplete removl of pyrethroi-ssoite toxiity [13]. The liqui preprtion therefore ws use in further stuies with C. ui, minly to enle omprisons with preexisting t. However, susequent stuies with H. zte using omplite mtries, suh s interstitil wter, emonstrte nee to inrese the enzyme onentrtion ove 1x to hieve removl of pyrethroi toxiity (t not shown). Toxiity stuies showe tht for enzyme onentrtions greter thn 1x, signifintly greter mortlity ws ssoite with the liqui preprtion versus the power (Tle 4). Therefore, stuies with H. zte were performe with the lyophilize power preprtion. The oserve enzyme-ssoite toxiity t higher esterse onentrtions ws ttriute to the presene of mmoni. Hylell zte hve higher mmoni tolerne thn C. ui, with 96-h mein lethl onentrtion (LC5) vlues of 5.2 mg/l (B.M. Phillips, unpulishe t) n.78 mg/l [15] of un-ionize mmoni, respetively. The 96-h Metl Control Brium Cerium(III) Cesium Clium Lithium Copper(I) Mngnese Cmium Colt Le(II) Merury Iron(III) Potssium Zin Ruiium Soium Nikel Iron(II) Copper(II) Dizinon-oxon Tle 9. Effet of metls on esterse tivity Speifi tivity ( mol/min/mg) % Control * 9 88* 88* 84 74* 72* 69* Assys were performe in quruplite using the liqui esterse preprtion (lot 12K762; Sigm Chemil, St. Louis, MO, USA). Ativity ssys were performe with the sustrte p-nitrophenyl ette. An sterisk inites tht the vlue is signifintly lower thn ontrol using Stuent s t test (p.5). All metls were purhse s the hlorie slt n were present t finl onentrtion of 1 mm. Control is efine s the tivity mesure in soium phosphte uffer (.1 M, ph 7.4, 25 C) in the sene of ny metls.

8 98 Environ. Toxiol. Chem. 25, 26 C.E. Wheelok et l. LC5 vlues for H. zte ompre well to pulishe vlues of 2.14 mg/l t ph 7.5 n 5.38 mg/l t ph 8.5 for hr wter [26]. These t show the importne of vliting the esterse tretment for eh new orgnism n mtrix, euse sensitivity to esterse ute toxiity s well s enzyme effiy likely will vry. Toxiity stuies with C. ui The esterse generlly ws very effetive t removing oth permethrin- n ifenthrin-ssoite toxiity. Reutions in permethrin-ssoite toxiity were greter thn reutions in ifenthrin-ssoite toxiity. This fining most likely resulte from the inrese hyrolyti rte of permethrin over ifenthrin [13]. Mtrix effets were oserve, in tht permethrin removl in Srmento River wter ws greter thn tht in lortory wter, possily euse of the sorption of permethrin y orgni mteril in the river wter. The effets oserve with ifenthrin were the opposite, in tht toxiity removl ws lower in river wter. These results re more iffiult to explin. Permethrin hs signifintly greter wter soluility thn ifenthrin (5.5 vs.14 g/l [27]); therefore, it woul e expete tht ifenthrin woul e remove through hyrophoi intertions to greter extent thn permethrin. Given tht essentilly equivlent toxi units of eh pyrethroi were use in the ssys, the results were unexpete. Although vriility ws foun t low enzyme tivities, ll pyrethroissoite toxiity in oth mtries ws remove t n enzyme level of 1x. It is importnt to point out tht the onentrtions of pyrethrois use in this stuy were quite high n not neessrily environmentlly relevnt [5]. It therefore is possile tht signifintly lower levels of esterse oul e employe in future ssys. For pplying this TIE metho to n environmentl smple, it will e esirle to use the gretest level of esterse possile while still mintining seletive removl of pyrethroi toxiity. This pplition will require lne of esterse level with potentil mmoni toxiity s well s overll protein level. As emonstrte in Tle 3, higher levels of protein n result in nonseletive removl of pyrethroi toxiity s oserve with the 25x BSA preprtion. The nonseletive removl of pyrethroi toxiity is not neessrily eterrent, euse the ssy results remin vli. The prolem rises in the presene of other hyrophoi ontminnts tht oul e remove from solution in ition to the pyrethrois, therey onvoluting the signifine n mening of the oserve rop in toxiity. The issue of ssy seletivity in the presene of high onentrtions of esterse oul e resse, in prt, y using other TIE testing protools, suh s employing piperonyl utoxie to etet the presene of OPs [14,15,28]. Toxiity stuies with H. zte Oservtions with H. zte were similr to those with C. ui; however, it ws neessry to inrese the enzyme onentrtion to 5x to remove ifenthrin-ssoite toxiity. The lk of strong onentrtion response reltionship etween inrese enzyme levels n ifenthrin toxiity ws interesting. The 1x n 5x preprtions were essentilly equivlent in lortory wter, n the 5x preprtion ws slightly more effiient in interstitil wter. The erese effiy of the esterse tretment with ifenthrin oul, potentilly, e resse with moifitions of the urrent testing methos. Currently, the esterse is e to the smple n inute for pproximtely 1 h efore the ition of the orgnisms. The length of inution oul e inrese signifintly in n ttempt to hyrolyze greter mount of pyrethrois. However, inresing the length of inution to 3 h with the H. zte stuies i not mke signifint ifferene in toxiity reution (t not shown). Therefore, it my e neessry to inute the esterse with the wter smple for severl hours or overnight. One oul formt the ssy suh tht esterse ws e to the smple either 12 or 24 h efore the initition of toxiity testing to provie the esterse with itionl time to hyrolyze pyrethrois in the wter smple. Our t show tht even in the most hllenging mtrix exmine uring the present stuy, the Snt Mri interstitil wter, signifint esterse tivity ws present more thn 8 h fter enzyme ition (Tle 7). The enzyme onsistently remove 2 ng/l of ifenthrin n up to 2 ng/l of permethrin in interstitil wter. Given the hllenging nture of the mtrix, this oservtion is useful. Interstitil wter n e omplite mtrix, ontining fine prtiles, ollois, orgni ron, n so on [29]. These omponents n erese pyrethroi iovilility n e expete to result in reue toxiity in interstitil wter [5,29,3]. In the ontext of TIE, ny reution in toxiity is enefiil n n e use in weight-of-eviene pproh to implite use of toxiity. Even in the most omplex system exmine in the present stuy, whih use pyrethroi tht is relitrnt to esterse-inue hyrolysis n omplite mtrix, we were le to oserve signifint reutions in pyrethroi-ssoite toxiity. These oservtions suggest tht this metho will e useful for eteting the presene of pyrethrois in quti testing. Esterse hrteriztion One of the vitl points to estlish in vliting the pplition of esterse tretment for use in TIEs is the speifiity of the metho. In prtiulr, it is neessry to show unequivolly tht the esterse is tlytilly removing the pyrethrois (i.e., through enzymti hyrolysis) n not through nonspeifi effets (i.e., sorption to hyrophoi surfes). Removl of pyrethroi toxiity through nontlyti mehnisms is still useful. However, it elimintes the speifiity of the metho, euse other hyrophoi ontminnts most likely will e remove s well. Erly work h shown tht esterse tretment i not ffet OP toxiity [13]. However, to emonstrte onlusively tht the oserve reution in toxiity following enzyme ition ws use y tlytilly tive esterse, we performe numer of stuies. Exposures onute with het-intivte enzyme n BSA h essentilly no effet on pyrethroi-ssoite toxiity. However, slight reution in toxiity ws oserve with the permethrin exposures, ut not in the ifenthrin exposures. It woul e expete tht the inrese hyrophoiity of ifenthrin woul result in greter sorption to the protein thn for permethrin. Tht the opposite results were oserve suggests tht the reutions were use y the presene of smll mount of tlytilly tive enzyme. It is possile tht following het intivtion, some of the protein ws ple of refoling to regin some tlyti tivity. This theory ws supporte further y the oservtion tht BSA i not hve ny signifint effets on either permethrin- or ifenthrin-ssoite toxiity. These t strongly inite tht tlytilly tive enzyme is essentil for the reution in toxiity. These oservtions were verifie further y inhiiting the enzyme with two known porine esterse inhiitors, hlorpyrifos-oxon [13] n OTFP [22]. Interestingly, hlorpyrifos-

9 Use of roxylesterse tivity in TIEs Environ. Toxiol. Chem. 25, oxon prove to e ineffetive t inhiiting the esterse for extene intervls. Following ition of the inhiitor to the enzyme preprtion, tivity ssys performe efore initition of the toxiity test inite no remining tlyti tivity. However, tivity ssys performe t the en of the toxiity stuy (48 h lter) showe tht sustntil tlyti tivity h een restore (72% of ontrol vlue) (Tle 8). These t suggest tht esterse inhiite with hlorpyrifos-oxon n e retivte. These results my e expline y greter stility of OTFP in our system ompre to tht of hlorpyrifosoxon n/or tht retivtion of the slow, tight-ining OTFP inhiition ours more slowly thn the retivtion of esterse phosphorylte y hlorpyrifos-oxon. A ution is tht thione insetiies in the smple oul generte oxon over perio of time. Uner some onitions, running simple olorimetri esterse ssy efore n fter the iossy oul test if the esterse tivity hs een reue y some ontminnt in the smple wter. Previous stuies h reporte tht hlorpyrifosoxon ws potent inhiitor of porine esterse, with vlues for the onentrtion of inhiitor require to reue enzyme veloity y 5% in the low nm rnge, epening on the sustrte [13]. In tht stuy, the inhiitor ws inute with the enzyme for only 1 min, s oppose to more thn 48 h in the present work. This oservtion further shows the utility of using roxylesterse tivity to etet pyrethroi toxiity in reeiving wters, euse the presene of OPs my not inhiit the enzyme signifintly over extene intervls. Inhiition of the esterse with OTFP signifintly reue the ility of the enzyme to remove permethrin-ssoite toxiity. These t suggest tht OTFP forms tighter ssoition with the enzyme thn with hlorpyrifos-oxon. Dt for other roxylesterses (e.g., juvenile hormone esterse) hve shown tht OTFP is reversile inhiitor, with retivtion times on the orer of ys to weeks [31]. These results further support the hypothesis tht tlytilly tive esterse is require to hieve removl of pyrethroi-ssoite toxiity. A numer of ifferent mtries were exmine for their effets on esterse tivity. Strong mtrix effets were oserve, showing tht eh new mtrix nees to e vlite efore the esterse n e use in routine toxiity testing. However, the key point is tht urrent methos ll for only 1-h inution of the esterse efore ition of the test orgnisms. In every mtrix exmine, signifint tlyti tivity ws present to t lest 8 h fter test initition (Tle 7). Therefore, in ll mtries exmine in the present stuy, loss of esterse tivity most likely is not signifint issue. As expete, oth phosphte uffer n Milli-Q wter were the most effiient t preserving esterse tivity. Interstitil wter ws the most hllenging mtrix euse of the presene of orgni mteril, ut the esterse ws still ple of reuing signifint pyrethroi-ssoite toxiity. The reution of esterse tlyti effiieny in interstitil wter suggests tht pplying these methos in TIEs with ulk seiment will e more hllenging, euse exessive prtiles most likely will reue the enzyme s effiieny. However, enzyme oul e e to wter overlying seiment in stti exposure to hyrolyze (i.e., etoxify) pyrethrois tht iffuse out of the seiments. Orgnisms woul then e e fter n pproprite intervl. This metho my not e useful for stritly enthi orgnisms, ut it oul hve ppliility to epienthi orgnisms. It lso oul e useful for nektoni or plnktoni orgnisms to etermine if ioville frtion of pyrethroi ws iffusing from the seiments. For exmple, enzyme ition woul work well in the seiment wter interfe exposure system evelope y Anerson et l. [32,33]. In this metho, orgnisms re hel ove the seiment surfe in sreene ontiner, ensuring tht only fluxe ontminnts ffet the orgnism. It is importnt tht this metho e evelope for use with seiment orgnisms. Beuse of the hyrophoi nture of pyrethrois, these hemils most likely will e etete in seiments n interstitil wters rther thn in the wter olumn [5,27]. University of Cliforni t Dvis reserhers hve experimente with ing the enzyme to overlying wter in H. zte soli-phse tests n oserve reutions in toxiity [8 1]. Currently, stuies re unerwy to refine the metho y testing it with pyrethroi-spike seiments. Very little metl-inue esterse inhiition ws oserve, with only opper(ii), iron(ii), n nikel using signifint ereses in esterse tivity (p.5). Suh high levels of metls re unlikely to e oserve in environmentl smples (1 mm). In ition, muh of the inhiitory tivity ws use y soluility issues, euse the limit of soluility of mny of the metls exmine in the present stuy ws exeee t 1 mm. Therefore, the presene of metls most likely will not e mjor inhiitor of esterse tivity in TIE testing. In ition, TIE protools ll for the use of ethyleneiminetetr-eti i s helting ftor to etet the presene of ivlent metl tions. Thus, the presene of mny metls oul e verifie inepenently in TIE protool vi the ethyleneiminetetr-eti i metho or use of tion soli-phse extrtion olumn if metls were suspete (e.g., se on ln-use prties where the smple ws ollete). In ition, the gretest level of inhiition oserve ws pproximtely 31% with opper(ii), whih mens tht 69% of esterse tivity remine to hyrolyze pyrethrois. Tken together, these results strongly suggest tht metls will not interfere with the use of esterse in TIE protools. Reprouiility of the esterse solution An importnt issue in the use of esterse tivity uring routine toxiity testing is the homogeneity n reprouiility of the esterse preprtion. Beuse the esterse use in this projet is ommeril prout, it is wiely ville to the reserh n toxiity testing ommunities. The mnufturer provies qulity-ssurne informtion for the prout on the ompny wesite ( No expirtion te for either esterse preprtion hs een estlishe y the ompny. The tivity of the enzyme hs een ressye every two to three yers fter the mnufturing te n reporte to e essentilly onstnt. However, survey of the ville esterse preprtions from Sigm Chemil shows tht enzyme tivity of the ommeril prout hs erese steily sine the ineption of this projet. The urrently ville lot of the mmonium sulfte preprtion hs reporte tivity of 13 U/mg, s oppose to 184 U/mg for the enzyme use in the present stuy. Our experiments hve shown slight loss in tivity of the liqui preprtion over six months when store t 4 C (t not shown). However, n importnt issue is tht the liqui preprtion is heterogeneous suspension. Therefore, trnsferring smll mounts of the enzyme hs inrese vriility. Experiments in whih 2 l of esterse were liquotte to smple ontiner le to reltive stnr evitions (RSDs) s high s 21% (n 3) s oppose to the trnsfer of 1 l, whih never exeee 1% RSD (n 3) (t not shown). Initil stuies with H. zte ttempte to

10 982 Environ. Toxiol. Chem. 25, 26 C.E. Wheelok et l. trnsfer smll quntities of esterse iretly from the vil to the testing ontiner (t not shown). This metho resulte in extremely high RSDs, n it ws estlishe tht single stok solution shoul e generte n lrger volumes trnsferre to the testing ontiners. These tehniques gretly reue vrition. To ompre vil-to-vil vriility, we exmine the esterse tivity in five ifferent ottles from the sme lot numer, ll purhse t the sme time, n foun n pproximtely 11% RSD mong the five ottles (t not shown). This evition n esily e ttriute to experimentl vriility, initing tht vil-to-vil reprouiility is eptle. Esterse tivity in these sme five ottles ws monitore weekly for six months, n no hnge in verge perentge RSD ws oserve. Another importnt issue is the vriility in tivity etween enzyme lots. Beuse these preprtions ome from ifferent thes of porine liver tissue, onern exists tht isozyme unne n tivity my vry on th-to-th sis. Inee, some of our initil experiments oserve ifferenes in the ility to remove pyrethroi-ssoite toxiity (t not shown). Therefore, toxiity testing lortories most likely will nee to purhse given mount of esterse n hrterize its tivity n pyrethroi-removl ility in their testing system. This hrteriztion woul remin onstnt s long s they h the sme th of enzyme, ut it oul e verifie every few months for qulity-ontrol purposes with either stnr tivity ssy using n esterse sustrte (e.g., PNPA) or in omintion with orgnism exposure to known onentrtion of pyrethroi. Aoring to t supplie y the mnufturer s well s tht generte in our lortory, the tivity of the enzyme shoul not erese signifintly uring storge (uner pproprite onitions). These proeures woul enle the pproprite qulity ontrols require in toxiity testing lortory. Both enzyme preprtions were effetive t removing pyrethroi-ssoite toxiity, ut elevte levels of mmoni ssoite with the liqui preprtion re of some onern if high onentrtions of esterse re require. The mmoni n e remove with quik uffer-exhnge step. An vntge of len-up step woul e the ility to onentrte the enzyme further n to inrese the speifi tivity. This step woul enle the ition of higher levels of tive esterse while mintining reltively low levels of overll protein. Filtering of the enzyme preprtion through 5K Centrion filter serve to inrese the speifi tivity (t not shown). The enzyme lso oul e ilyze efore use or ethnol-preipitte to remove mmonium sulfte. A numer of other mnipultions re possile tht oul inrese the effiy of the urrent ssy, ut tre-off exists etween effiieny n effiy. An vntge of the urrent metho is the ese of pplition, ut some lortories might not hve the equipment neessry to implement these itionl proeures. Bse on the present results, ition of len-up step oes not pper to e neessry, ut it oul eome useful in the presene of more omplite mtries. In the future, the use of sol gel enpsulte esterse or of polyethylene glyol or glutrlehye-stilize esterse oul e employe to inrese the roustness of the proeure. Other esterses lso re ville ommerilly, inluing rit liver esterse tht hs inrese tivity ompre to the lyophilize porine preprtion (75 U/mg vs 2 U/mg for the preprtions use in the present stuy; Sigm Chemil). The min isvntge of the rit enzyme is the ost, whih is pproximtely 8-fol greter thn tht of the porine enzyme. Another possiility for improving the effiy of the metho woul e to move wy from rue enzyme preprtion n use purifie roxylesterse. The ientifition n isoltion of pyrethroi-hyrolyzing esterse hs een reporte in the literture [34]. If this type of esterse oul e expresse n purifie on lrge sle n me ommerilly ville, it oul e n iel solution for use in TIE. The esterse lso oul e prepre in low-mmonium-slt uffer to prevent the omplitions ssoite with mmoni toxiity. This solution is the most ttrtive in terms of prouing the optiml ssy. However, numer of sientifi n ommeril hurles nee to e overome efore it oul e prtil. Applition of esterse tivity in TIE stuies The present stuy hs emonstrte tht the inlusion of esterse tivity in phse I TIE protools hs the promise of eing useful ssy. These erly stuies were performe with pyrethroi onentrtions tht most likely re muh greter thn the levels tht woul e experiene in environmentl settings. It ws eeme to e vntgeous to use elevte pyrethroi onentrtions for numer of resons. First n foremost, it ws neessry to hieve essentilly 1% mortlity in the testing orgnisms to emonstrte tht the orgnisms oul e resue from toxiity. Seon, euse the min effort of this work ws to emonstrte the effiy of the esterse preprtion, it ws thought tht performing the stuies t greter pyrethroi onentrtions woul show etter proof of onept. If the esterse is effetive t removing multiple toxi units (efine s 1/EC5) of pyrethroi toxiity, it lso shoul e effetive t reuing toxiity t lower pyrethroi onentrtions. In ll ses uring the present stuy, the esterse emonstrte the gretest effiy t reuing toxiity from lower pyrethroi onentrtions reltive to higher onentrtions (Tles 2, 3, n 6). Therefore, this tren is expete to ontinue t even lower, more environmentlly relisti pyrethroi onentrtions. It is vitl tht future stuies vlite the speifiity of the esterse tretment for the reution of pyrethroi toxiity. Results of previous stuies with OPs (izinon n hlorpyrifos) suggeste tht the metho ws seletive [13]. However, these were isolte stuies n were not performe within the ontext of full phse I TIE. Therefore, it is vise tht vlition stuies work on estlishing the speifiity of the metho. This ojetive oul e omplishe using rel-worl smples tht hve een shown (vi nlytil onfirmtion) to ontin pyrethrois n/or spiking stuies. The stuy shoul e onute with rnge of prtilly to very toxi onentrtions of hemils other thn pyrethrois, to whih the roxylesterse n e e to sertin whether the enzyme/protein removes toxiity to the test orgnisms in nonspeifi mnner. These experiments shoul e performe with rnge of ommonly ourring toxints in queous smples tht possess ifferent physiohemil properties n ifferent moes/ mehnisms of tion. For exmple, the inlusion of metls (e.g., opper n zin), OPs, rmtes, n other physil stressors long with the pyrethrois woul e useful for estlishing the speifiity of the esterse tretment. By performing these stuies with multiple orgnisms in vriety of mtries, suh s interstitil wter n griulturl or stormwter runoff, the utility n speifiity of the metho oul e estlishe. This metho proly will e most pplile t the Phse