DEVELOPMENT OF TOXICITY IDENTIFICATION EVALUATION PROCEDURES FOR PYRETHROID DETECTION USING ESTERASE ACTIVITY

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1 Environmentl Toxiology nd Chemistry, Vol. 23, No. 11, pp , SETAC Printed in the USA /4 $12.. DEVELOPMENT OF TOXICITY IDENTIFICATION EVALUATION PROCEDURES FOR PYRETHROID DETECTION USING ESTERASE ACTIVITY CRAIG E. WHEELOCK, JEFF L. MILLER, MIKE J. MILLER, SHIRLEY J. GEE, GUOMIN SHAN, nd BRUCE D. HAMMOCK,* Deprtment of Entomology nd Cner Reserh Center, University of Cliforni t Dvis, Dvis, Cliforni 95616, USA AQUA-Siene, Dvis, Cliforni 95616, USA (Reeived 7 Otoer 23; Aepted 25 Mrh 24) Astrt Reent grohemil usge ptterns suggest tht the use of orgnophosphte (OP) pestiides will derese, resulting in onomitnt inrese in pyrethroid usge. Pyrethroids re known for their potentil toxiity to quti invertertes nd mny fish speies. Current toxiity identifition evlution (TIE) tehniques re le to detet OPs, ut hve not een optimized for pyrethroids. Orgnophosphte identifition methods depend upon the use of piperonyl utoxide (PBO) to identify OP-indued toxiity. However, the use of PBO in TIE ssys will e onfounded y the o-ourrene of OPs nd pyrethroids in reeiving wters. It is neessry, therefore, to develop new TIE proedures for pyrethroids. This study evluted the use of pyrethroidspeifi ntiody, PBO, nd roxylesterse tivity to identify pyrethroid toxiity in quti toxiity testing with Ceriodphni dui. The ntiody used signifint mortlity to the C. dui. Piperonyl utoxide synergized pyrethroid-ssoited toxiity, ut this effet my e diffiult to interpret in the presene of OPs nd pyrethroids. Croxylesterse tivity removed pyrethroidssoited toxiity in dose-dependent mnner nd did not ompromise OP toxiity, suggesting tht roxylesterse tretment will not interfere with TIE OP detetion methods. These results indite tht the ddition of roxylesterse to TIE proedures n e used to detet pyrethroids in quti smples. Keywords Esterse Toxiity identifition evlution Ceriodphni dui Pyrethroid Orgnophosphte INTRODUCTION In reent yers, inresed emphsis hs een pled on the use of routine toxiity tests using quti orgnisms to mesure wter qulity [1 5]. Methods hve een developed for determining the impts of dishrges of muniipl [1] nd industril effluent [6], griulturl runoff [4,5,7] nd storm-wter dishrges [8,9] on reeiving wter qulity. Conomitntly, inresed ttention hs foused on methods for identifying the hemil(s) tht re responsile for the toxiity so tht pproprite ontrol mesures n e tken. The U.S. Environmentl Protetion Ageny (U.S. EPA) hs pulished series of toxiity identifition evlution (TIE) methods tht n e used to identify the uses of toxiity in queous smples using hemil hrteriztion, identifition, nd onfirmtion proedures [1 12]. The TIE methods hve een pplied rodly to identify the uses of toxiity in multiple quti mtries s well s sediment. Toxiity identifition evlution testing routinely hs identified orgnophosphte (OP) insetiides, inluding dizinon nd hlorpyrifos, s uses of toxiity in muniipl effluents nd surfe wters in northern Cliforni, USA [3 5]. However, the use of these OP insetiides is deresing in Cliforni, with susequent oserved inrese in pyrethroid usge [13]. Pyrethroids re diffiult to identify using stndrd TIE methods euse of their physil hemil properties [14,15] nd the lk of sensitive nd seletive nlytil methods tht detet these insetiides t iologilly relevnt onentrtions [16,17]. New TIE tehniques re needed to identify pyrethroid- * To whom orrespondene my e ddressed (dhmmok@udvis.edu). used toxiity. To hieve widespred ppliility in TIEs, the proedures must e rpid, reltively inexpensive, nd not require high level of expertise or expensive equipment. We hve exmined numer of methods for their ility to remove, redue, or detet pyrethroid-ssoited toxiity. This work investigted the use of esterse tivity, pyrethroid-seletive ntiody, or piperonyl utoxide (PBO) s possile tretments. Croxylesterse is n enzyme tht rpidly degrdes oth type I nd type II pyrethroids (Fig. 1). This lss of enzymes hs een demonstrted to e effetive in reduing pyrethroid-ssoited toxiity in oth mmmls nd insets [18] nd is sensile trget for removing pyrethroid toxiity in TIE smples. Previous work hs identified this enzyme s good trget for identifying pyrethroid-ssoited toxiity in quti smples [19]. We determined previously tht ntiodies seletive for dizinon nd hlorpyrifos ould e used to identify toxiity in surfe wters used y these OP insetiides [2]. Therefore, we evluted these proedures using ntiodies developed y our group tht ind oth type I nd type II pyrethroids [21,22]. To our knowledge the use of ntiody or enzymti proedures to identify nd onfirm toxiity due to pyrethroids hs not yet een reported. Certin ompounds (e.g., OPs) must e tivted metolilly y the test orgnism efore they n exert their toxi effet. Mny of these tivtion retions onsist of oxidtive metolism y group of enzymes olletively known s mixed-funtion oxidses (MFOs), of whih the heme-protein ytohrome P45s re suset [23]. Compounds suh s PBO, syntheti methylenedioxy phenyl derivtive, ind to nd lok the tlyti tivity of some MFOs, preventing the toxiity of metolilly tivted OP insetiides inluding dizinon, hlorpyrifos, mlthion, prthion, nd fenthion 2699

2 27 Environ. Toxiol. Chem. 23, 24 C.E. Wheelok et l. Fig. 1. Both type I nd type II pyrethroids re hydrolyzed y esterses to the orresponding lohol nd id. The enzyli ron of the ester moiety in type II pyrethroids hs yno group tthed (forming seondry ester), wheres the type I pyrethroids ontin hydrogen tom in this position (forming primry ester). Cynohydrins quikly rerrnge to the orresponding ldehyde t si ph. [24]. Thus, when nontoxi level of PBO is dded to test smples ontining one or more of these OPs, the toxiity is gretly redued or ompletely loked. The use of PBO to identify toxiity used y metolilly tivted OP insetiides hs een desried previously [25] nd inorported in pulished U.S. EPA TIE mnuls [1,11]. Conversely, PBO synergizes the toxiity of pyrethroid insetiides y loking MFO-medited metolism of these hemils [26]. Thus, PBO ddition to smples ontining one or more pyrethroids inreses nd/or prolongs the toxi effet [27]. This dul tion of PBO ould led to onfounding signl in TIE testing of smples tht ontin oth OPs nd pyrethroids. Alterntively, the tion of PBO my e useful tool to identify the presene/ sene of pyrethroids nd metolilly tivted OPs in queous smples. The pyrethroids permethrin, ifenthrin, ypermethrin, yfluthrin, nd esfenvlerte were identified s trget ompounds of interest sed on quntities pplied in Cliforni ( nd vrition in struture (Tle 1). In ddition, -yhlothrin ws identified s pyrethroid of interest in ntiiption of its inresed usge in ontrolling mosquitoes rrying West Nile virus [28]. Using these pyrethroids, we hve tken three pprohes to develop method for identifying pyrethroid-used toxiity. In the first pproh, n ntiody tht seletively inds pyrethroids ws exmined for its ility to derese toxiity. The seond pproh employed PBO s known pyrethroid synergist in n effort to identify individul pyrethroids sed upon the level of oserved synergism. Lst, ommeril preprtion of roxylesterse ws used to medite pyrethroid toxiity through hydrolysis of the prent ompound to nontoxi produts. These pprohes my serve s ost-effetive, rpid, mehnistilly sed methods for identifying pyrethroid-ssoited toxiity in quti smples. MATERIALS AND METHODS Chemils nd instrumenttion All hemils were purhsed from Aldrih Chemil (Milwukee, WI, USA) unless otherwise noted nd were used without further purifition. Pyrethroid nd OP stndrds were purhsed from either Chem Servies (West Chester, PA, USA) or Riedel de Hen (Seelze, Germny). Struturl hrteriztion nd purity were provided y 1 H nd 13 C nuler mgneti resonne (NMR) nd gs hromtogrphy oupled to mss spetrometry (GC/MS) with eletron impt (EI) ioniztion detetion. The NMR spetr were quired on Merury 3 spetrometer (Vrin, Plo Alto, CA, USA). Chemil shift vlues were reorded in prts per million (ppm) using tetrmethylsilne (TMS) s the internl referene. For GC nlysis, smples were nlyzed on HP 689 GC (Agilent Tehnologies, Engelwood, CO, USA) equipped with.25 mm i.d. 3 m,.25- m film DB-5MS olumn (J&W Sientifi, Folsom, CA, USA) with onstnt He flow rte of.8 ml/ min. The injetor temperture ws 25 C. The initil olumn temperture of 5 C ws held for 5. min nd then rmped t 15 C/min to 32 C nd held for 2. min. The GC ws interfed with HP 5973 MS run in full sn mode from 5 to 55 m/z with qudrupole temperture of 186 C nd soure temperture of 24 C. The GC/MS eletron impt frgmenttion ptterns nd NMR spetr supported ll reported strutures. Compound purity ws 97% s determined y NMR nd GC/MS. Toxiity testing The test orgnism ws the ldoern, Ceriodphni dui, freshwter inverterte widely used for ute toxiity tests [29], short-term hroni studies [3], nd TIEs [31]. Ceriodphni dui neontes ( 24-h-old) were otined from ultures mintined t AQUA-Siene (Dvis, CA, USA) in reverse osmosis treted well wter mended with dry slts to U.S. EPA modertely hrd speifitions (ph, 7.8; hrdness, 8 mg/l; lklinity, 6 7 mg/l). Cultures were mintined t 25 1 C with photoperiod of 16:8-h light: drk nd were fed mixture of the green lg, Selenstrum priornutum (University of Texs Alge Type Colletion, Austin, TX, USA) nd lended trout how (Silver Cup, Murry, UT, USA). The 48-h toxiity test proedures followed those outlined y the U.S. EPA [29]. Neontes used for testing were 24-hold, olleted within 8 h from dults t lest 7-d-old. All tests were onduted in 2-ml glss sintilltion vils ontining 18 ml of test wter. A modertely hrd wter ontrol, methnol ontrol, nd five to seven onentrtions of the test mteril with two or four replites ontining five neontes eh were used for eh test series. The neontes were pipetted into the vils using strtified rndom ssortment. Test solutions were not renewed nd the orgnisms were not fed during the exposures. Test temperture nd photoperiod were identil to ulture onditions. Mortlities were monitored dily. Tests were onsidered invlid if ontrol survivl ws 9%. The onentrtions required to use n effet in 5% of the popultion (EC5) were lulted from the mortlity dt using omputer progrm (ToxCl, Tidepool Sientifi, M- Kinnleyville, CA, USA). Insetiide stndrds (99%.i., g/l in methnol) were otined from AuStndrd (New Hven, CT, USA). Piperonyl utoxide (9%.i.) ws otined from Aldrih Chemil

3 Development of toxiity identifition evlution Environ. Toxiol. Chem. 23, Tle 1. Struture nd physil onstnts of pyrethroids used in this study Dt re from Lskowski [39]. Chemil nmes of eh pestiide re s follows: Permethrin, ([3-phenoxyphenyl]methyl 3-[2,2-dihloroethenyl]-2,2-dimethylylopropne roxylte, CAS ); ifenthrin, ([2-methyl(1,1 -iphenyl)-3-yl]methyl [1R,3R]-3-[(1Z)-2-hloro-3,3,3-trifluoro-1-propenyl]-2,2-dimethylylopropneroxylte, CAS ); ypermethrin, ([ ] -yno[3-phenoxyphenyl]methyl 3-[2,2-dihloroethenyl]-2,2-dimethylylopropneroxylte, CAS ); esfenvlerte, ([S]- -yno[3- phenoxyphenyl]methyl [ S]-4-hloro- -[1-methylethyl]enzeneette, CAS ); yfluthrin, ( -yno[4-fluoro-3-phenoxyphenyl]methyl 3-[2,2-dihloroethenyl]-2,2-dimethylylopropneroxylte, CAS ); -yhlothrin, ([S/R]- -yno[3-phenoxyphenyl]methyl [1S,3S]-3-[(1Z)-2- hloro-3,3,3-trifluoro-1-propenyl]-2,2-dimethylylopropneroxylte, CAS ). Type II pyrethroids ontin n -yno group on the enzyli ron (Fig. 1). d Vlues re the log of the prtitioning onstnt etween otnol nd wter (K OW ). ClogP refers to the lulted log P. e Units re g/l. f Units re tm m 3 mol 1. (Milwukee, WI, USA). Working stndrds were prepred y diluting the hemils in high-performne liquid hromtogrphy (HPLC) grde methnol (Fisher Sientifi, Firlwn, NJ, USA). Aliquots of the working solutions were then used to prepre the test solutions in modertely hrd dilution wter. Methnol onentrtion in ll test solutions ws.1%. All stok nd working solutions were stored in the drk t 4 C. Dissolved oxygen, ph, ondutivity, lklinity, hrdness, nd temperture were mesured t the initition of exposure. Dissolved oxygen, temperture, nd ondutivity were determined with Yellow Springs Instrument proes nd meters (Yellow Springs, OH, USA), nd ph ws mesured with Hh ph meter (Lovelnd, CO, USA). Alklinity nd hrdness were determined with Hh titrnt kits. Temperture ws mesured ontinuously in the temperture-ontrolled room throughout the exposure period. Dissolved oxygen, temperture, nd ph lso were mesured t the onlusion of eh test. All instrumenttion were lened thoroughly etween nlyses using hot wter nd sop followed y rinsing with methnol. Pyrethroid removl methods All three of the methods desried elow were performed using the toxiity testing proedures desried in the previous setion. Any method devitions re listed; otherwise, ssys were ll performed identilly. The ntiody used in this study ws polylonl permethrin seletive ntiody developed y our lortory, whih hd high level of ross-retivity for ypermethrin (65%) [21]. The stok solution ws prepred in sodium phosphte uffer (.1 M, ph ) y diluting rude serum 5-fold. Working ntiody solutions of - nd 1,-fold dilutions were prepred in the sme uffer. Different mounts of these working solutions (1 L) were dded into the test ontiners ontining the pyrethroids 1 to 3 h prior to ddition of C. dui. Tehnil grde (9%.i.) PBO ws diluted in HPLC-grde methnol (.1 mg/ml) nd dded to test ontiners ontining the pyrethroids for finl onentrtion of g/l. Smples were inuted for 1 to 3 h prior to the ddition of C. dui. Porine esterse ws otined from Sigm Chemil (tlog E-2884, lot 17H716, 25 U/ml, 15 mg/ml; St. Louis, MO, USA). Stok enzyme solution ws prepred in sodium phosphte uffer (.1 M, ph ) nd diluted ppropritely for eh ssy. Enzyme ws dded to test ontiners ontining the pyrethroid for finl tivity of units/ml (U/ ml) unless otherwise noted (one unit of tivity will hydrolyze

4 272 Environ. Toxiol. Chem. 23, 24 C.E. Wheelok et l. 1. mole of ethyl utyrte to utyri id/min t 25 C s defined y the supplier). Test solutions were inuted with the enzyme for 1 h prior to ddition of C. dui. Esterse tivity ssys Esterse ssys with p-nitrophenyl ette (PNPA) were performed using sodium phosphte uffer (.1 M, ph ) ording to methods of Wheelok et l. [32] s dpted from Ljungquist nd Augustinsson [33]. Assys were preformed in 96-well mirotiter styrene flt ottom pltes (Dynex Tehnologies, Chntilly, VA, USA) nd nlyzed on Spetrmx 34PC plte reder (Moleulr Devies, Sunnyvle, CA, USA). Porine esterse ws used t finl onentrtion of.1 g/ml ( 1.8 mu/ml; tlog no. E-2884, lot no. 12K762, 184 U/ml, 1 mg/ml). All ssys were designed suh tht no more thn 1% of the sustrte ws hydrolyzed over the length of the ssy nd solvent ontent never exeeded 1% of the totl ssy volume. Reported results re ll orreted for kground hydrolysis of the sustrte. Ativity ws monitored using 2.-min kineti red t 45 nm. Pyrethroid hydrolysis ssys were performed s desried in Wheelok et l. using porine esterse [34]. Briefly, ssys were onduted in 2 ml of sodium phosphte uffer (.1 M, ph or 8.) t either 25 or C. Protein onentrtion vried depending upon pyrethroid nd ssy onditions, ut rnged from low of 2.6 g/ml (.5 U/ml; lot no. 12K762) for permethrin t C nd ph 8. to high of 2.8 g/ml ( 3.8 U/ml; lot no. 12K762) for ifenthrin t 25 C nd ph. Assys were liner over the life of the ssy nd were onfigured suh tht no more thn 1% of the sustrte ws onsumed. Pyrethroid sustrte ws dded nd the mixture inuted for 5 min t the indited temperture, followed y ddition of 1 ml of rine nd 1 ml of ethyl ette (EtOA). Smples were vortexed for 3 s, entrifuged for 5 min, nd then 25- L liquot of the EtOA ws nlyzed y GC/MS. Upon hydrolysis, pyrethroids form the orresponding ynohydrin s shown in Figure 1, whih spontneously rerrnges to the ldehyde t si ph [34]. The rte of pyrethroid hydrolysis ws mesured y quntifying the resultnt ldehydes. Quntifition stndrds were: 3-phenoxy-enzldehyde for ypermethrin, esfenvlerte, nd -yhlothrin; 3-phenoxyenzyllohol for permethrin; 2-methyl-3-iphenyl methnol for ifenthrin; nd 4-fluoro-3-phenoxy-enzldehyde for yfluthrin. All stndrds were purhsed from Aldrih Chemil exept for 4-fluoro-3-phenoxy-enzldehyde, whih ws synthesized s desried elow. All ssys to determine the onentrtion of inhiitor tht redued enzyme veloity y 5% (IC5) were designed suh tht there were t lest two dtum points ove nd elow the IC5 vlue in the liner rnge. Inhiitor solutions were prepred in ethnol nd diluted s required for eh ssy. The IC5 determintions with permethrin or ypermethrin s the sustrte were onduted s desried ove using porine esterse t 5.2 g/ml ( 1. U/ml; lot no. 12K762). Assys with PNPA were performed s desried ove using.2 g/ml ( 3.7 U/ml; lot no. 12K762) porine esterse. Solvent never exeeded 1% of the ssy volume nd no solvent effets were oserved. Synthesis of 4-fluoro-3-phenoxy-enzldehyde Cyfluthrin (16.9 mg) ws dissolved in 2-ml tetrhydrofurn nd 3 L of 1M NOH ws dded. The solution ws stirred slowly t room temperture for 96 h, fter whih the Tle 2. Effet of pyrethroid ntiody (A) on the toxiity of ypermethrin to Ceriodphni dui Smple tretment L ontrol A ontrol A ontrol Antiody 48-h % pmole IgG/ml mortlity.1 1 Cypermethrin Intive A d 1 4 Antiody onentrtion in immunogloin G (IgG) per ml of test solution. Men mortlity in two replites of 5 neonte C. dui. Cypermethrin ws spiked t 8 ng/l nominl wter onentrtions ( 1 toxi unit). d Antiody ws heted t 75 C for 1 h nd smple ontined 8 ng/l ypermethrin. mixture ws wshed with 3 2 ml of wter nd 1 ml of EtOA ws dded. The orgni frtion ws wshed with 1 25 ml of rine nd dried over MgSO 4, filtered, nd stripped to give visous yellow oil. The oil ws extrted with hexne nd stripped to give visous ple yellow oil. The oil ws then dissolved in miniml EtOA nd loded onto preprtive thin-lyer hromtogrphy plte (PK6F sili gel 6 Å, 2 2 m, 1,- M thik; Whtmn, Clifton, NJ, USA), whih ws developed in 7:3 hexnes:etoa. The produt ws extrted from the sili gel using EtOA, whih ws stripped to give drk ornge oil (4.5 mg, 8.5% yield). The 1 H NMR (CDCl 3 ) showed ppm 7.3 (2, J 7.8 Hz, 2H), 7.18 (t, J 7.2 Hz, 1H), 7.38 (m, 2H), 7.52 (dd, J 7.5, 1.8 Hz, 1H), 7.64 (m, 1H), 9.87 (s, 1H). GC/eletron impt-ms (m/z) 216 (M, %), 187 (38%), 159 (35%), 139 (2%), 133 (24%), 77 (56%). RESULTS Effet of tretment upon pyrethroid toxiity A series of 48-h toxiity tests were onduted with pyrethroid-seletive ntiody developed in our lortory [21]. Antiody onentrtions of.1 to 1 pmole IgG/ml were effetive in reduing ypermethrin toxiity to C. dui (Tle 2). However, oth ntiody ontrols produed detetle toxiity to C. dui. To exmine nonspeifi inding effets, the ntiody ws detivted y inution for 1 h t 75 C. The dentured ntiody redued pyrethroid-ssoited mortlity y pproximtely 5%, eing nerly s effetive in reduing ypermethrin toxiity s the highest tive ntiody onentrtion tested. The effet of PBO on the toxiity of suite of type I nd type II pyrethroids to C. dui ws investigted. Solutions ontining PBO-synergized individul pyrethroid toxiity to C. dui y 9- to 1-fold ompred to solutions without PBO (Tle 3). The lrgest PBO toxiity rtio (1-fold) ws oserved with ypermethrin; n intermedite toxiity rtio (4- fold) ws oserved with -yhlothrin; nd lower toxiity rtios (9- to 17-fold) were oserved with ifenthrin, yfluthrin, esfenvlerte, nd permethrin. No distint ptterns etween pyrethroid type nd level of synergism were oserved mong the different pyrethroids. Permethrin, ifenthrin, nd yfluthrin ll hd pproximtely the sme rtio, yet re mixture of type

5 Development of toxiity identifition evlution Environ. Toxiol. Chem. 23, Tle 3. Effet of piperonyl utoxide (PBO) on pyrethroid toxiity to Ceriodphni dui Pyrethroid (type) Permethrin (I) Bifenthrin (I) Cypermethrin (II) Esfenvlerte (II) Cyfluthrin (II) -Cyhlothrin (II) PBO ( ) EC5 (ng/l) PBO ( ) Rtio d Men EC5 (onentrtion t whih 5% of the popultion exhiits n effet) of 3 to 4 pired toxiity tests the stndrd devition. All vlues re sed on nominl wter onentrtions. Indites type I or type II pyrethroid s determined y the presene of the -yno group. PBO ws spiked t g/l in ll PBO ( ) smples. d Rtio of PBO( )/PBO( ). I nd type II pyrethroids. The differenes etween ypermethrin (type II) nd permethrin (type I) were extremely lrge nd these two ompounds differ only y the presene of the -yno group, suggesting effets upon ester hydrolysis. No struture-tivity reltionships were oserved mong the different pyrethroids exmined. A 48-h toxiity study ws onduted to determine if the porine roxylesterse ould degrde high onentrtions of the pyrethroid nd OP insetiides to nontoxi levels to C. dui. Exposure onentrtions were pproximtely 2 to 3 times the estimted EC5 for eh ompound. In ll ses, the roxylesterse hydrolyzed the six pyrethroids to nontoxi levels, wheres smples tht ontined no esterse hd % mortlity (Tle 4). The enzyme hd no detetle effet on the toxiity of the two OP insetiides (dizinon nd hlorpyrifos) to C. dui, with ll smples exhiiting % mortlity in the presene or sene of esterse. No seletivity towrds type I versus type II pyrethroids ws oserved, with the enzyme removing 2 to 3 toxi units (TUs) of ll pyrethroids Tle 4. Effet of esterse ddition on pyrethroid-ssoited toxiity to Ceriodphni dui Tretment Control Permethrin Bifenthrin Cypermethrin Esfenvlerte Cyfluthrin -Cyhlothrin Dizinon Chlorpyrifos Conn. (ng/l) (TUs) 6 (2) 66 (4) 1,45 (2) 7 (3) 56 (2) 6 (3) 76 (2) 16 (2) 48-h % mortlity Esterse ( ) Esterse ( ) Conentrtion t whih smples were spiked. All vlues re nominl wter onentrtions. Vlues in prentheses re toxi units (TUs) defined s /EC5 (the onentrtion t whih 5% of the popultion exhiits n effet). Men mortlity in two replites of five neonte C. dui. Stndrd devitions for ll smples re zero. In ll smples, either or % mortlity ws oserved. Esterse ( ) smples ontin enzyme spiked t U/ml. Esterse ( ) smples ontin no dded enzyme. Controls were performed with nd without the ddition of the esterse. Tle 5. Hydrolysis of seleted pyrethroids y porine liver esterse Sustrte Permethrin 25 Temp. ( C) ph Ativity Bifenthrin 25 Cypermethrin 25 Esfenvlerte 25 Cyfluthrin 25 -Cyhlothrin 25 PNPA ND ND ND ND Ativity is in units of nmol/min/mg protein. All ssys were performed in triplite nd dt re the verge the stndrd devition. Bkground hydrolysis t 25 C nd ph ws.7 nmol/min for ifenthrin,.11 nmol/min for esfenvlerte,.38 nmol/min for ypermethrin, nd.39 nmol/min for permethrin. Not deteted. The method detetion limit ws.1 M, whih orresponds to.2 nmol/min/mg protein. p-nitrophenyl ette. Ativity is in units of mol/min/mg protein. exmined. Studies with ifenthrin used four TUs, whih were remedited suessfully y the enzyme. A TU is defined s /EC5 (the onentrtion t whih 5% of the popultion demonstrtes the desired effet, in this se mortlity). The enzyme did not hve ny oservle toxi effets upon C. dui, with enzyme ontrols showing % mortlity. Esterse hrteriztion Initil studies suggested tht the porine liver esterse ws the most promising tehnique for removing pyrethroid-ssoited toxiity from TIE smples. The esterse preprtion, therefore, ws hrterized further to understnd its performne under potentil TIE onditions. The esterse hydrolysis of ll six pyrethroids exmined in this study s well s the stndrd esterse sustrte PNPA ws mesured t rnge of ssy onditions (Tle 5). The porine esterse hydrolyzed ll of the pyrethroids to some extent, exept for -yhlothrin, with hydrolysis inresing with temperture nd ph. Of prtiulr interest ws the lk of oservle hydrolysis of yhlothrin even though toxiity ssys with -yhlothrin showed tht the esterse ws ple of removing ll pyrethroid-ssoited toxiity (Tle 4). The rte of pyrethroid hydrolysis oserved ws pproximtely 1,-fold slower thn tht of PNPA under ll onditions exmined. To test the effiy of the esterse over rnge of pyrethroid onentrtions, the enzyme ws hllenged with 1 to 5 TUs of ifenthrin (35 1,75 ng/l) or ypermethrin (75 3,75

6 274 Environ. Toxiol. Chem. 23, 24 C.E. Wheelok et l. Tle 6. Effet of vrying esterse onentrtion upon pyrethroid toxiity to Ceriodphni dui 48-h % mortlity Smple Pyrethroid onn. Enzyme tivity Control d e Intive enzyme f Cypermethrin g (type II) Bifenthrin h (type I) 75 1,5 2,25 3, 3, ,5 1,4 1,75 8 Men mortlity in two replites of five neonte C. dui. Conentrtions, whih re ll nominl wter onentrtions in ng/l. Enzyme tivity is in units of 1 3 U/ml (, 75, 15, nd 3 ng/ml protein, respetively). These numers give the finl onentrtion of enzyme in eh 2-ml sintilltion vil. One unit of esterse will hydrolyze 1. mole of ethyl utyrte to utyri id nd ethnol per min t ph 8. t 25 C. d Controls were performed oth with nd without the ddition of enzyme. e indites tht the test ws not performed. f Enzyme ws heted t 8 C for 1 h. g Conentrtions of ypermethrin rnge from 1 toxi unit (TU) t 75 ng/l to 5 TU t 3,75 ng/l. A toxi unit is defined s /EC5 (the onentrtion t whih 5% of the popultion exhiits n effet). h Conentrtions of ifenthrin rnge from 1 TU t 35 ng/l to 5 TU t 1,75 ng/l ng/l). Additionlly, three different onentrtions of enzyme were used to exmine the dose-response reltionship. Dt provided dose-response reltionship with inresing levels of enzyme ple of mediting more pyrethroid TUs (Tle 6). All smples with no enzyme used % mortlity (exept for the lowest onentrtion of ypermethrin, whih used 8% mortlity). At the highest onentrtion of enzyme exmined in this study ( U/ml), ll ypermethrin toxiity ws removed. Bifenthrin smples exhiited some toxiity t the highest level of enzyme used; however signifint mount (7 9%) of the toxiity ws removed. No toxiity to C. dui ws oserved with either the enzyme ontrol or the intivted enzyme. The mount of ompound required to redue esterse veloity y 5% (IC5) ws mesured for oth the prent nd oxon-forms of hlorpyrifos nd dizinon. Results showed tht the oxon form of oth dizinon nd hlorpyrifos re extremely potent inhiitors of the esterse-medited hydrolysis of permethrin (type I) nd ypermethrin (type II) pyrethroids, s well s PNPA (Tle 7). However, the prent OPs re poor inhiitors, with IC5 vlues M for ll sustrtes exmined. DISCUSSION In the phse I TIE proess, the toxi environmentl smple is sujeted to numer of tretments tht identify tioni metls, oxidnts, mmoni, nd polr nd nonpolr orgni toxints [1 12,29]. The TIE proedures inlude ph djustment, ddition of helting nd oxidizing regents, seprtion on solid-phse extrtion (SPE) olumns, nd frtiontion of the SPE olumn elutes using HPLC, followed y nlysis of the toxi HPLC frtions y dvned instrumenttion (GC/ MS nd/or HPLC/MS) s shown in Figure 2. One the toxint(s) is identified, toxiity tests re onduted to determine if the onentrtions of suspeted toxint(s) n ount for the mount of toxiity mesured in the smple. Two TIE tehniques re useful in identifition of OP- nd pyrethroid-used toxiity. Use of SPE olumns removes toxiity due to nonpolr orgni toxints, inluding OP nd pyrethroid insetiides. Alterntively, tretment with PBO loks tivtion of OP insetiides to their toxi form [25] nd inreses the toxiity of pyrethroid insetiides [26]. Thus, if PBO dereses the toxiity, metolilly tivted OP insetiides (e.g., dizinon nd/or hlorpyrifos) re suspeted. If PBO inreses the smple toxiity, pyrethroid insetiides re suspeted. However, if there is mixture of ompounds, the gents using the toxiity re more diffiult to pinpoint nd the signl is onvoluted. Therefore, we propose intervening in the TIE proess t this juntion with n dditionl step to either medite or identify pyrethroid-ssoited toxiity s shown in Figure 2. Tle 7. Inhiition of esterse-medited pyrethroid hydrolysis y orgnophosphtes Sustrte Permethrin Cypermethrin PNPA Permethrin Cypermethrin PNPA Dizinon-oxon Dizinon d Inhiitor IC5 Chlorpyrifos-oxon Chlorpyrifos d IC5 Conentrtion of inhiitor required to redue enzyme veloity y 5%. All ssys were performed in triplite nd eh IC5 vlue is the verge of three different experiments the stndrd devition. IC5 vlues re in nm. p-nitrophenyl ette. d IC5 vlues re in M.

7 Development of toxiity identifition evlution Environ. Toxiol. Chem. 23, Fig. 2. Applition of esterse proedures to the phse I toxiity identifition evlution (TIE) proess. Arevitions used in figure re piperonyl utoxide (PBO), orgnophosphte (OP), nd onentrtion to use 5% effet (EC5). Effet of tretment upon pyrethroid toxiity The first tretment exmined ws pyrethroid-seletive ntiody tht ws hypothesized to remove pyrethroids from solution, therey reduing toxiity. At higher levels of IgG, this phenomenon ws in ft oserved. However, signifint mortlity ws ssoited with the ntiody ontrols. In ddition, ntiody intivted y heting redued toxiity lmost s effetively s the highest onentrtion of ntive ntiody used in this study. Heted ntiody should e dentured nd therefore unle to tively ind ypermethrin, suggesting tht the oserved effets upon toxiity were nonspeifi. Due to their extreme hydrophoiity, pyrethroids will dsor to lipophili surfes [14]. Protein ddition, therefore, ould e suffiient to remove some of the pyrethroid from solution. It is unler wht perentge of this ound pyrethroid would remin ioville. This tehnique might e useful method for pyrethroid removl through the ddition of suffiiently hydrophoi mteril to the test solution. However, it would most likely remove OPs s well, thus eliminting ny seletivity for deteting OPs versus pyrethroids. Additionl studies utilizing wider rnge of ntiody onentrtions will e needed to estlish if diret ntiody ddition n e used to prevent ypermethrin or other pyrethroid toxiity. To determine the extent of the nonspeifi protein dsorption, susequent studies should utilize n ntiody tht does not ind pyrethroids rther thn dentured ntiody. Further studies ould exmine rnge of pyrethroid ntiodies, inluding lss-speifi (e.g., type I or type II) ntiodies. In ddition, purifying the ntiody nd tthing it to solid mtrix (suh s glss eds) my improve the inding pity while eliminting the toxiity of the ntiody itself [2]. The inresed mortlity oserved likely is due to ftors in the serum other thn the ntiody. Future work thus will need to e performed with enrihed ntiody frtions or monolonl ntiodies. The resulting lower protein onentrtion should derese the redution in toxiity seen with intive ntiody (Tle 2). Further reserh will e neessry to develop these ntiody tehniques for use in TIE proedures. The use of PBO in quti toxiity testing hs een reported y severl reserhers [2,4,25,35] nd reommended y the U.S. EPA [29]. However, with the o-ourrene of OPs nd pyrethroids in TIE smples, the use of PBO to identify speifilly the ompounds responsile for the toxiity ould result in onvoluted test results. It lso is possile tht the synergisti effets of PBO upon pyrethroid toxiity ould e used s pyrethroid signture in TIE protools. We therefore exmined the ility of PBO toxiity rtios to identify individul pyrethroids (vs the entire lss s whole) in the sene of OPs. It ws hypothesized tht eh pyrethroid ould e identified y its individul PBO toxiity signture in the presene nd sene of PBO. However, the lk of struture-tivity reltionships nd differentil toxiity etween mny of the pyrethroids mkes it unlikely tht this tehnique will e useful (Tle 3), with the possile exeption of ypermethrin identifition. The PBO tretment did synergize pyrethroid toxiity in ll ses nd, therefore, my e useful generl identifier for the presene of pyrethroids. None of these studies exmined the effets of PBO tretment on smples ontining oth OPs nd pyrethroids. Additionl reserh will e needed to determine if PBO toxiity rtios n e used to identify toxiity due to OPs nd pyrethroids when oth lsses of toxints oour in smples. The esterse tretment provided the most promising results s the enzyme eliminted ll pyrethroid-ssoited toxiity. An dvntge of working with the esterse is tht the mehnism of toxiity redution is well understood [18] s shown in Figure 1. The hydrolysis of pyrethroids y esterses hs een hrterized y severl reserhers [15,18,36]. The esterse preprtion used for these studies is from porine liver nd ontins mixture of esterse isozymes [] with vrying ffinity for pyrethroids (dt not shown). The mount of esterse used ws suffiiently high to hydrolyze quikly ll pyrethroids nd no pyrethroid-speifi effets were oserved. It might e possile to detet differenes etween type I nd type II pyrethroids t lower onentrtions of esterse. The esterse ws inuted with the pyrethroids for 1 h efore the ddition of C. dui to llow for pyrethroid hydrolysis. We did not ttempt to optimize the length of the inution nd it is possile tht shorter inution time would e eptle. If the enzyme is ssumed to e operting under V mx onditions (n ssumption tht most likely is invlid), the theoretil mount of pyrethroid hydrolyzed over 1 h n e lulted using the tivity numers in Tle 5 nd the mount of enzyme dded per ssy ( 2.7 g). Porine esterse theoretilly n hydrolyze pproximtely 2, TUs

8 276 Environ. Toxiol. Chem. 23, 24 C.E. Wheelok et l. of permethrin (1.5 mg/l) nd 3 TUs of ifenthrin (1.5 g/l) in 1 h. However, s sustrte onentrtions derese, the enzyme will no longer funtion t V mx (i.e., non Mihelis- Menton onditions) due to deresed sustrte onentrtion nd tlyti tivity. Thus the tul mount of pyrethroid hydrolyzed most likely will e sustntilly less thn the theoretil mx lulted here. However, given tht most smples will ontin signifintly less pyrethroid, the esterse should e ple of hydrolyzing signifint mount of the pyrethroid s shown in Tle 6. These numers represent idel onditions nd it is likely tht omplited mtries, suh s effluent or pore wter, will redue the tlyti effiieny nd/ or lifetime of the enzyme. The presene of esterse hd no oservle ffet upon the toxiity of the OPs dizinon nd hlorpyrifos, inditing speifiity in the esterse mehnism of tion (Tle 4). Results from these studies suggest tht the enzyme ould e used to seletively degrde pyrethroid toxiity in OP-ontining smples. It should then e possile to perform the PBO tretment fterwrds to test for n OP signture. It lso is possile tht esterses of vrying speifiity ould e used to distinguish etween individul pyrethroids. Even if it is not possile to identify speifi pyrethroids, it should e hievle to find n esterse tht n distinguish etween type I nd type II pyrethroids. Numerous esterses re ville ommerilly s well s in different reserh groups. Further work should exmine severl esterses to determine whih one hs superior performne in TIE formts. One ould envision TIE proedure where ttery of esterses ws used to identify quikly the pyrethroids responsile for the oserved toxiity. Esterse hrteriztion Initil studies showed tht, out of the three methods exmined in this study, esterse ws the most promising tretment for deteting or removing pyrethroid toxiity nd therefore ws hosen for further hrteriztion for use in TIE ssys. Esterse ssys with mmmlin enzymes generlly re performed t ph 8. t C [38]. However, most TIE ssys re performed t 25 C or ooler t ph [29]. The effiieny of the enzyme t hydrolyzing pyrethroids therefore ws exmined under multiple onditions. The oserved responses were preditle in tht higher temperture nd higher ph resulted in inresed rtes of pyrethroid hydrolysis. The hydrolysis rtes of the pyrethroids exmined vried y pproximtely three orders of mgnitude, demonstrting tht there re strong struturl effets upon pyrethroid hydrolysis. Sterilly unhindered type I pyrethroids re hydrolyzed quiker thn type II pyrethroids. This effet n e seen most drmtilly in the differene in hydrolysis rtes for permethrin versus ypermethrin shown in Tle 5 (16 vs 64 nmol/min/mg protein, respetively t 25 C t ph ). However, the type I pyrethroid ifenthrin exhiited slower hydrolysis rte reltive to ypermethrin or yfluthrin (type II pyrethroids). This oservtion is most likely due to the ortho methyl sustituent tht sterilly hinders the ester, ffeting the hydrolysis rte nlogously to the -yno moiety of the type II pyrethroids. Hydrolysis tivity for -yhlothrin ould not e mesured with these methods, yet ll -yhlothrin ssoited toxiity ould e removed y the ddition of esterse s shown in Tle 4. This oservtion onfirms tht the enzyme still n serve its funtion even on pyrethroids tht re relitrnt to estersemedited hydrolysis. Dose-response studies with the enzyme demonstrted tht it is ple of reduing multiple TUs of oth ifenthrin nd ypermethrin. Tle 5 shows tht of ll the pyrethroids for whih hydrolysis ould e deteted, ifenthrin hd the slowest hydrolysis rte. However, t the highest onentrtion of esterse used in Tle 6, 9% of the mortlity of five TUs of ifenthrin ws eliminted. It will e neessry to quntify this effet for multiple pyrethroids in vriety of mtries. Mtrix effets (suh s orgnis, other toxints, ph, et.) ould e very lrge for this system, possily resulting in derese of esterse tivity. At the highest onentrtion of enzyme used in Tle 6, no enzyme-ssoited toxiity ws oserved. However, it is possile tht the enzyme ould exert toxi effets t inresed levels. Further studies should exmine rnge of enzyme onentrtions to determine the mximum onentrtion of enzyme tht does not use signifint toxiity. In order to hieve optiml effiieny for removl of pyrethroids, it is desirle to use the highest onentrtion of esterse possile. The lk of toxiity of het-intivted enzyme ws very promising nd suggests tht it is n pproprite ontrol for nonspeifi inding effets, unlike the het-intivted ntiody. As it is likely tht OPs nd pyrethroids will o-our in TIE smples [9], the inhiition of the esterse y OPs ws exmined using dizinon nd hlorpyrifos s model ompounds. The effets of OPs upon esterse tivity were exmined y mesuring the onentrtion of OP (inhiitor) required to redue enzyme veloity y 5% (IC5). The tive form of OP inhiitor is the oxon metolite following ytohrome P45 oxidtion [23]. Therefore, oth prent nd oxon OP were exmined for their ility to inhiit porine liver esterse. The prent OPs s thiones (P S) did not inhiit the enzyme s would e expeted. However, the oxon forms (P O) were potent inhiitors of oth permethrin nd ypermethrin hydrolysis s shown in Tle 7. This oserved inhiition most likely is not onern for use of the esterse in TIE ssys for numer of resons. First, formtion of the oxon form of the OPs usully requires metoli tivtion y MFOs, whih n our only inside of the test orgnism. For the oxon metolite to inhiit the esterse, it would hve to diffuse out of the orgnism into the test wter, whih is unlikely to our in signifintly high enough onentrtions to use inhiition of the esterse. Seondly, the mount of oxon formed reltive to the totl mount of esterse dded to the test solutions is very smll. Additionlly, the esterse ws dded to the test solutions 1 h efore the ddition of the C. dui, llowing the enzyme mple opportunity to hydrolyze pyrethroids. Therefore, even if the esterse ws inhiited y oxon formed in vivo in the test orgnism, it should not influene the ility of esterses to detoxify the test smples prior to orgnism ddition. The ility of the esterse preprtion to funtion with dditionl orgnisms should e exmined. These studies were limited to C. dui, ut it is likely tht the tehnique will e pplile to wide rnge of quti testing orgnisms. One potentil limittion will e enzyme use in sediment-sed ssys. It is possile tht high levels of sediment or orgni mteril will redue signifintly enzyme tivity. However, in ssys tht re sed upon prtitioning of pyrethroid from sediment to solution phse, it is likely tht the esterse n e used suessfully to remove pyrethroid toxiity. An interesting oservtion in this study ws the pprent redution in pyrethroid levels in the queous phse over the life of the toxiity test (dt not shown). A time-dependent effet of pyrethroid dsorption to the ontiner ws oserved

9 Development of toxiity identifition evlution Environ. Toxiol. Chem. 23, nd is utionry point for the formtting of quti toxiity testing. These oservtions hve een noted y other reserhers s well nd re known diffiulty in working with pyrethroids [14,17,39]. Further work should ttempt to quntify pyrethroid dsorption to smpling nd test ontiners s well s determine its effets upon the outomes of toxiity testing. Given the extreme hydrophoiity of mny pyrethroids, it is possile tht sorption to test ontiners signifintly ffets test outome. CONCLUSION Three different tehniques were ompred for their ility to identify or remedite pyrethroid toxiity in quti toxiity tests, with esterse tivity proving to e the most promising. An dvntge to the use of esterse tivity to remedite pyrethroid toxiity is its ommeril vilility nd reltively low ost. It will e neessry to hrterize the enzyme preprtion fully nd exmine the vriility in tivity nd stility of different ommeril lots. Additionl studies will e needed to onfirm tht the enzyme proedures re effetive in reduing or eliminting pyrethroid-used toxiity. Speifilly, work should fous on mtrix effets upon enzyme tivity. If suh studies demonstrte the effetiveness of the proedures, Figure 2 shows how they n e used either singly or in omintion in the TIE proess. If the smple toxiity ws deresed y the enzyme tretment, pyrethroid nlyses would e onduted on the originl test smple nd/or the SPE olumn elutes nd toxi HPLC frtions. Use of the esterse preprtion is n inexpensive, simple, mehnistilly sed method for removing pyrethroid toxiity from quti smples. Addition of esterse to the TIE proedure llows for the seletive detetion of the presene of pyrethroids, nd does not interfere with susequent detetion of OP toxiity with stndrd TIE proedures. This esterse preprtion should serve s new tool in the development of TIE proedures for hrterizing the ontriution of pyrethroid insetiides to the toxiity of wter smples. Aknowledgement C.E. Wheelok ws supported y Ntionl Institute of Helth (NIH) post dotorl trining grnt T32 DK nd University of Cliforni Toxi Sustnes Reserh nd Tehing Progrm Grdute Fellowship. This work ws supported in prt y Stte Wter Resoures Control Bord Contrt , Ntionl Institute of Environmentl Helth Sienes (NIEHS) grnt R ES271, NIEHS Superfund grnt P42 ES4699, NIEHS Center for Environmentl Helth Sienes grnt P3 ES577, nd NIH/Ntionl Institute of Allergy nd Infetious Diseses grnt U1 AI The uthors thnk Jim Snorn nd Ås Krlsson for ritil reding of the mnusript. REFERENCES 1. Biley HC, Miller JL, Miller MJ, Dhliwl BS Applition of toxiity identifition proedures to the ehinoderm fertiliztion ssy to identify toxiity in muniipl effluent. Environ Toxiol Chem 14: Biley HC, DiGiorgio C, Kroll K, Miller JL, Hinton DE, Strrett G Development of proedures for identifying pestiide toxiity in mient wters: Crofurn, dizinon, hlorpyrifos. 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