The biotransformation of molinate (Ordram) in the striped bass (Morone saxatilis)

Aquatic Toxicology, 9 (1987) 305-317

305

Elsevier

AQT 00228 THE

BIOTRANSFORMATION

STRIPED

BASS (MORONE

OF MOLINATE

(ORDRAM)

IN THE

SAXA TILIS)

RONALD S. TJEERDEMA and DONALD G. CROSBY

Department of Environmental Toxicology, University of California, Davis, California, U.S.A. (Received 27 June 1986; revised version received 10 October 1986; accepted 14 October 1986)

Bioconcentration, depuration, and biotransformation of molinate in striped bass (Morone saxatilis) were studied in a flow-through metabolism system. When compared to static conditions, flowing water improved oxygenation, decreased volatilization and remetabolism and, run through a macroreticular resin, improved waste-product collection. Metabolite identification and quantitation employed highpressure liquid chromatography; gradient elution optimized peak resolution and minimized analysis time. Exposure of juvenile fish to 100 #g. 1-1 [ring-~4C]molinate for 24 h resulted in a bioconcentration factor of 25.3 and a 14C total concentration factor of 30.9 (in molinate molar equivalents). After 24-h depuration, 90.47% of the absorbed t4c had been excreted; metabolites accounted for 19.20% of the depurated and 7.92% of the retained '4C. The identified metabolites included molinate sulfoxide, carboxymolinate, 4-hydroxymolinate, molinate mercapturic acid, 4-ketomolinate, and hexahydroazepine. While molinate is highly toxic to Japanese carp (Cyprinus carpio var. Yamato Koi), sulfoxidation, followed by glutathione conjugation and/or rapid depuration, may effectively reduce the toxicity of molinate to striped bass. Key words: Molinate; Morone saxatilis; Bioconcentration; Xenobiotic biotransformation

INTRODUCTION

Almost two million pounds of the two principal thiocarbamate herbicides, molinate (Ordram) and thiobencarb (Bolero), were applied to California rice fields in 1984 (California Department of Food and Agriculture, 1985), representing a 30O7o increase over the amount used in 1980 (California Department of Food and Agriculture, 1981). Both herbicides control barnyard grass (Echinochloa spp.) (Weed Science Society of America, 1983), possibly by uncoupling plant oxidative phosphorylation (Wakamori, 1973; Fang, 1975). Inevitably, a small proportion of both herbicides reaches the contiguous drainage canals and rivers; during May and June, levels of 100 to 200 ttg. 1-1 once were not uncommon (California State Water Resources Control Board, 1984). Herbicide application coincides with the spawning Correspondence to: R.S. Tjeerdema, Department of Environmental Toxicology, University of California, Davis, CA 95616, U.S.A. 0166-445X/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

306

of several important game fish species, including striped bass (Morone saxatilis) and white sturgeon (Acipenser transmontanus) (Moyle, 1976; Fry, 1979). Each spring since 1965, thousands of fish, particularly common carp (Cyprinus carpio) have died in the Colusa Basin and adjacent drains (California State Water Resources Control Board, 1984). Although traces of other toxic agents also have been found in the drains, including methyl and ethyl parathion and MCPA (California State Water Resources Control Board, 1984), molinate has been singled out as the most likely cause of mortality (California Department of Fish and Game, 1982). While molinate has a low toxicity to rats (oral LDso = 720 mg. kg -1) and is rapidly metabolized to its sulfoxide and other products (Hubbell and Casida, 1977; DeBaun et al., 1978), it is highly toxic to common carp (28-day LCs0 = 0.21 mg. 1-~), particularly when compared to the less sensitive striped bass (96-h LCs0 = 12.10 mg.l-~; Finlayson and Faggella, 1986). Several investigations of molinate metabolism have been reported, but only two have involved an aquatic organism, the Japanese carp (C. carpio var. Yamato Koi) (Lay et al., 1979; Lay and Menn, 1979). In the Japanese carp, molinate is both very toxic (20-day LCs0 = 0.18 mg-l-~; Kawatsu, 1977) and extensively metabolized (Lay et al., 1979). An understanding of the bioconcentration, depuration, and metabolism of molinate in a less sensitive species may help to explain its toxic action in carp. The objectives of this investigation were to develop a dynamic, single-pass metabolism chamber for fish; to develop high-pressure liquid chromatographic (HPLC) methods, sensitive to /zg.1-1 levels, for the identification and quantitation of molinate and its metabolites; to measure bioconcentration and depuration of molinate in striped bass; and to identify and quantitate the principal molinate metabolites in this species. MATERIALS AND METHODS

Chemicals [Ring-14C]molinate, S-ethyl hexahydro-[2-14C]azepine-l-carbothioate (10.5 mCi. mmol-1), reference standard molinate (I; Fig. 5), 4-hydroxymolinate (II), and 4-ketomolinate (III) were provided by Stauffer Chemical Co., Richmond, CA. Identities were confirmed by gas chromatography-mass spectrometry (GC-MS) on a Finnegan Model 3200 instrument (70 eV) with a 25 m × 0.25 mm i.d. DB-5-fused silica column, helium flow rate 1.0 ml. min -1, as well as by published mass spectra (DeBaun et al., 1978; Lay et al., 1979). [Ring-14C]molinate was purified to a final radiopurity of 98~/0 by preparative thin-layer chromatography (TLC) on silica gel with acetone-hexane (1:1; Casida et al., 1975). Hexahydroazepine (hexamethyleneimine, HMI, IV) was purchased from Aldrich Chemical Co., Milwaukee, WI. Molinate sulfoxide (V) was prepared by oxidation of I with equimolar m-chloroperoxybenzoic acid in chloroform, while molinate sulfone (VI) required a five-molar

307

excess of the oxidant (Casida et al., 1975). Both products were purified by preparative TLC (acetone-hexane 1:1) and their identities confirmed with authentic standards by both TLC (acetone-hexane 1:1) and HPLC (water-methanol 40:60, isocratic). Carboxymolinate (VII) was prepared by reacting VI with a five-molar excess of mercaptoacetic acid in methanol and triethylamine (Hubbell and Casida, 1977) and purified by preparative TLC (ethyl acetate-formic acid 30:1; DeBaun et al., 1978). Molinate mercapturic acid (VIII) was prepared by reacting VI with equimolar N-acetyl-L-cysteine in ethanol, triethylamine, and water, and purified by preparative TLC (butanol-ethanol-water 4:1:1; DeBaun et al., 1978). Following methylation with ethereal diazomethane, the identities of VII and VIII were confirmed via either solid-probe MS or GC-MS and reference to published mass spectra (DeBaun et al., 1978; Lay et al., 1979). Both preparative and one-dimensional TLC utilized Silica Gel 60 F-254 plates (E. Merck, Darmstadt, F.R.G.) with 2 or 0.2 mm layers. Animals Striped bass, average age 14 months, were provided by the California Department of Fish and Game Central Valleys Hatchery in Elk Grove, CA. They were housed in a continuous-flow Fiberglas tank, fed BioDiet Grower (BioProducts Inc., Worrenton, OR), and acclimated for 30 days at 18°C prior to the study. Metabolism system A flow-through metabolism chamber developed and utilized in this laboratory has been described previously (Garnas and Crosby, 1979). Flow-through conditions reduced problems associated with poor oxygenation, chemical volatilization, and remetabolism. To further minimize volatilization losses, the previous chamber was modified (Fig. 1). Fresh wellwater entered a stripper (A) which reduced NE gas content and provided aeration; the 58 x 7.8 cm (i.d.) length of PVC pipe was filled with river rock and mounted in the center of an 80-1 bucket with water introduced at the top (B), an air pump connected to an airstone at the base provided aeration (C), and an overflow maintained water level (D). Chemical was infused by syringe pump (E) and, to minimize volatilization, water subsequently was isolated from atmospheric contact. Chemical and water were mixed in a tube filled with borosilicate glass beads (F) to create a homogeneous aqueous solution, and a thermometer (G) measured water temperature. The fish, shaded to minimize stress, was exposed so that its head always pointed into the influent (H), following which organic solutes were trapped on Amberlite XAD-4 resin (Rohm and Haas, Philadelphia, PA) (I). The waste water was discarded (J). Silicone tubing (8-mm i.d.) and glass chambers and columns of the following dimensions were used to minimize adsorption: mixing chamber, 21 x 2.5 cm (i.d.), 75 ml; metabolism chamber, 27 x 3.5 cm (i.d.), 310 ml; and resin column, 13 x 1.4 cm (i.d.), 15 ml.

308 A

)

c

E

I

B

l

D

J

II

F

Fig. 1. The flow-throughmetabolism system: (A) water stripper, (B) water inlet, (C) air pump, (D) water overflow, (E) metered syringe, (F) mixing chamber, (G) thermometer, (H) metabolism chamber, (1) XAD-4 resin column, and (J) water outlet.

Exposure Water composition was adjusted prior to the start of each exposure. To help alleviate osmotic shock and improve metabolite removal by the resin, Instant Ocean artificial seawater (Aquarium Systems, Eastlake, OH) was continuously added to maintain a solute level o f 3 g. 1-l (J.J. Cech, Jr., pers. comm., 1985). Temperature was maintained at 18°C, and flow was gravity-driven at 14 m l - m i n -1. Three striped bass (mean fork length, 14.7 ___ 1.2 cm; mean weight, 19.1 + 1.6 g) were exposed separately to I after fasting 24 h to decrease fecal accumulation in the metabolism chamber. Each 50-h exposure was divided into three phases: acclimation, 2 h; absorption, 24 h; and depuration, 24 h. During the acclimation phase, an individual was placed in the chamber and observed for signs of stress. During the uptake phase, 7 ml of I in methanol (0.29 mg. ml-t) was introduced into the water flow from a 10 ml-Hamilton gas-tight syringe, mounted on a Sage Model 255-2 syringe pump, to provide a water concentration of 100 ___ 5/~g. 1-1. During the depuration phase, I exposure was discontinued and only untreated water flowed through the system. The resin columns were removed at the end of each of the last

309 two phases and, upon termination, the fish was immediately placed in a freezer. Three controls were run parallel to the metabolism study: a vehicle control with methanol only, a system control without a fish, and a microbial breakdown control in which the X A D - 4 resin was preloaded with I. It determined transformation resulting from any waterborne bacteria (i.e., Escherichia coli) originating from the fish. The resin extracts were later analyzed.

Metabolite collection and isolation XAD-4 resin Fresh X A D - 4 resin, found to contain unacceptable levels o f organic contaminants, was subjected to cleanup by sequential 24-h solvent extractions with methylene chloride and then methanol in a Soxhlet extractor. The clean resin was transferred with methanol into an amber glass bottle for storage. Used resin was regenerated by the same procedure. Fig. 2 presents the analytical procedure. Following collection, the resin columns were flushed with distilled water; metabolites were extracted by vacuum aspiration o f excess water followed by sequential elution with 100 ml o f methylene chloride and Depurate Animal

I

Collect Metabolites on XAD-4 Resin Flush with Distilled Water

I

Vacuum Aspirate (A)

(B)

I

Elute with Methylene Chloride - I00 ml

I

Elute with Methanol - 100 ml

Dry with Anhydrous

CaSO4, Na2SO4

I

Reduce Volume

Reduce Volume

I

Combine

I

Quantitate by LSC (i)

(2)

I J

Confirm by TLC Identify Metabolites by HRLC Cochromatography

I

Remove Silica Gel

I

Elute with Methylene Chloride - 30 ml

Fraction Col lect

Reduce Volume

I

Quantitate Metabolites by LSC

Fig. 2. Analytical procedure for X A D - 4 resin.

Confirm by HPLC Cochromatography

310

then 100 ml of methanol (8-12 ml. mini). The methylene chloride eluates were dried through anhydrated granular sodium sulfate topped with anhydrated calcium sulfate (Drierite); both eluates were reduced, first by rotary evaporation and then under a stream of N2-gas, to 100 ~1. After combining the two, aliquots were quantitated for 14C by liquid scintillation counting (LSC); uptake phase extracts were used only to help quantitate total 14Crecovery. Remaining extracts were refrigerated for later analysis. Table I presents the reference standard elution efficiencies. Tissue Fig. 3 presents the analytical procedure developed for whole fish tissue. Each fish was homogenized whole in acetonitrile with a Polytron tissue homogenizer (Brinkmann Instruments, Westbury, NY). Tissue was removed by filtration, airdried for 24 h, and oxidized with a Packard Tri-Carb Model B-0306 sample oxidizer. An aliquot of the acetonitrile filtrate was removed for LSC, and the remainder was diluted to 1 liter with saturated aqueous sodium chloride and passed through an XAD-4 resin column (8-12 ml. min-‘) to collect metabolites. Column extraction was as previously described. Metabolite identification

and quantitation

Metabolites were identified by HPLC cochromatography with unlabeled reference standards on a Waters Associates integrated HPLC system equipped with an ISCO Model V4 variable wavelength detector and an Eldex Model 1044 fraction collector. The best wavelength for determination of molinate was 220 nm (Cabras et al., 1982). An Alltech reverse-phase Cl8 column (250 x 4.6 mm i.d., 10 pm) and a 40-min gradient elution (Waters Curve 9) with a mobile phase of water-methanol (initial, 70:30; final, 30:70), 1.O ml. min-‘, were used. Fractions were collected for

TABLE

I

Reference

standard

recoveries

from

Amberlite

XAD-4

resin and TLC RF values.

XAD-4

Component

recovery Molinate

(I)

4-Hydroxymolinate 4-Ketomolinate

(II) (III)

Hexahydroazepine Molinate

sulfoxide

Molinate

sulfone

Carboxymolinate Molinate

(V) (VII)

mercapturic

a Average, N = 2. b (A) acetone-hexane, acid-water, 13:3:3:1.

acid (VIII)

1:l; (B) ethyl acetate-formic

6: 1: 1; (E)

butanol-ethanol-water,

(%)”

TLC RF value? A

B

C

D

E

F

100.9

0.62

0.73

0.73

0.69

0.67

0.71

101.0

0.33

0.42

0.13

0.61

0.60

0.61

92.8

0.43

0.53

0.28

0.56

0.54

0.63

0.12

0.15

0.09

0.09

0.12

0.19

(IV) (VI)

resin

98.8

0.17

0.14

0.05

0.31

0.39

0.35

87.4

0.53

0.69

0.64

0.65

0.56

0.67

95.1

0.49

0.65

0.53

0.63

0.63

0.65

0.41

0.59

0.33

0.57

0.45

0.25

acid, 3O:l; (C) toluene-ether, 2:3; (D) butanol-acetic 4: 1: 1; (F) ethyl acetate-methanol-pyridine-water,

311 Defrost Animal

Weigh

Homogenize with Acetonitrile

Separate by Filtration

Aqueous

Solid

Quantitate by LSC

AirlDry Dilute with Distilled Water - i L Weigh Saturate with NaCI

Collect Metabolites on XAD-4 Resin

Repeat XAD-4 Resin Protocol

I

Solubilize, Deeolorize by Oxidation

quantitate by LSC

Fig. 3. Analytical procedure for whole striped bass tissue.

later 14C quantitation in Liquiscint scintillation cocktail (National Diagnostics, Somerville, N J) with a Packard Tri-Carb 2425 scintillation spectrometer, optimized for the cocktail. The samples were corrected for background, quenching, and efficiency. Amounts as small as 0.3 nmol could be detected accurately and reproducibly. Identities were confirmed by TLC on silica gel in at least three of six solvent systems: acetone-hexane (1:1), ethyl acetate-formic acid (30:1), toluene-ether (2:3), butanol-acetic acid-water (6:1:1), butanol-ethanol-water (4:1:1), and ethyl acetate-methanol-pyridine-water (13:3:3:1; Casida et al., 1975; Hubbell and Casida, 1977; DeBaun et al., 1978; Lay et al., 1979; Lay and Menn, 1979). Developed plates were scanned with a Packard Model 7230 two-dimensional radiochromatogram scanner, and the RF values were compared to those of standards (Table I); silica gel was removed, extracted with methylene chloride, volume reduced to 100/zl, and the metabolites re-analyzed by HPLC cochromatography and LSC. RESULTS

No toxic signs were detected in the vehicle control. Total ~4C recovery in the system control was 92.2°70, molinate radiopurity remained approximately 98070, no breakdown products were detected, and losses due to wall adsorption and other causes were insignificant. No breakdown products were detected in the microbial

312

,,,vii

VIII Ill

VlH

Vl VII

I

l 20

4'3

6'o

8'o

,,;o TIME

,fo

,,;o

~'o

do

6'0

8'0

(mlr3)

Fig. 4. Chromatograms of a reference standard mixture (see Table I for key to Roman numerals). Mobile phases: (A) isocratic water-methanol (60:40), 1.5 ml.min-l; (B) gradient water-methanol (70:30 to 30:70), 40 min, Waters Curve 9, 1.0 ml. min-~. Column: reverse-phase C~8. Detector: UV at 220 nm. control, and molinate radiopurity again was approximately 98%. We conclude that chemical changes were due to striped bass rather than to other factors. Fig. 4 illustrates metabolite separation by H P L C under isocratic conditions (A, w a t e r - m e t h a n o l 60:40, 1.5 ml. min-1) and gradient elution (B). While both systems provided adequate peak resolution, gradient elution minimized peak broadening, tailing, and analysis time. Average 14C recovery (three replicates) was 87.0 +_ 1.4%; a recovery in excess of 85% was considered acceptable. A whole fish bioconcentration factor (BCF) and ~4C total concentration factor (TCF) were measured for striped bass after the 24-h uptake phase. The BCF (ratio of the concentration in tissue, Ct, to that in water, Cw) was calculated as follows (Neely et al., 1974; Spacie and Hamelink, 1982): BCF = Ct" Cw 1. The TCF, based on molinate molar equivalents, is the analogous ratio involving total t4C concentration, and because the 24-h allowance for steady state was an estimation, both values may be approximations. The BCF was 25.3 _+ 3.7, based upon a whole body wet tissue concentration of 2.53 #g. g - l , and the T C F was 30.9 +_ 3.9, based on a concentration of 3.09/zg. g-1. Table II presents the disposition of 14C following 24-h depuration; striped bass eliminated 90.47%, while 9.53% was retained. Metabolites accounted for 19.20% o f the depurated and 7.92% of the retained t4c. Table III shows the identities and concentration profiles o f metabolites f r o m the acetonitrile filtrate and XAD-4 resin; due to lack of UV absorption, IV was identified by T L C and quantitated by H P L C and LSC. Although most of the retained 14C represented unchanged molinate, significant amounts of II, V, and VII were present. Most of the depurated 14C also represented molinate, but significant amounts o f II, III, IV, V, VII and VIII were found. Overall, V accounted for most of the metabolized 14C. Fig. 5 presents the structures of I and the six identified metabolites.

313 TABLE II ~4C disposition following 24-h depuration by striped bass. Fraction

J4C disposition nmol. g- la.b

070

XAD-4 resin Acetonitrile filtrate Dry tissue residue

14.91 (1.93) 1.01 (0.20) 0.56 (0.16)

90.47 6.13 3.40

Totals

16.48 (2.07)

100.00

Mean (SD), N = 3. b Wet weight. a

TABLE Ill Metabolite profiles following 24-h depurat~on by striped bass. Component

14C distribution Acetonitrile filtrate

Depurated water

nmol.g -la,b

nmol.g -la,b

Molinate (1) 4-Hydroxymolinate (11) 4-Ketomolinate (1II) Hexahydroazepine (IV) Molinate sulfoxide (V) Carboxymolinate (VII) Molinate mercapturic acid (VIII)

0.93 0.013 ND c ND c 0.017 0.050 ND c

(0.23) (0.006)

Totals

1.01 (0.20)

(0.006) (0.036) -

O7o 92.08 1.29 1.68 4.95 -

12.05 0.10 0.020 0.020 2.07 0.61 0.043

Total %

(1.69) (0.05) (0.010) (0.002) (1.54) (0.18) (0.006)

80.80 0.67 0.13 0.13 13.89 4.09 0.29

100.00 14.91 (1.93)

100.00

nmol. g -aa,b 12.98 0.11 0.020 0.020 2.09 0.66 0.043

%

(1.89) (0.05) (0.010) (0.002) (1.54) (0.21) (0.006)

81.51 0,69 0.13 0.13 13.13 4.14 0.27

15.92 (2.13)

100.00

Mean (SD), N = 3. b Wet weight. c None detected. a

DISCUSSION X e n o b i o t i c m e t a b o l i s m b y a q u a t i c o r g a n i s m s is u s u a l l y s t u d i e d in a n y o f f o u r d i f f e r e n t t y p e s o f e x p e r i m e n t a l s y s t e m s : static a q u a r i a ; static ' m o d e l e c o s y s t e m s ' ; static o u t d o o r p o n d s ; o r f l o w - t h r o u g h c h a m b e r s ( M e t c a l f , 1974; C r o s b y et al., 1979). S t a t i c a q u a r i a are t h e easiest to m a i n t a i n b u t p r o v i d e less c o n t r o l o f o x y g e n a t i o n a n d chemical volatilization, and time-dependent remetabolism of primary metabolites m a y o c c u r . F l o w - t h r o u g h c h a m b e r s a r e m o s t a n a l o g o u s to t e r r e s t r i a l m e t a b o l i s m c h a m b e r s a n d , w h i l e m o r e c o m p l e x t h a n static a q u a r i a , t h e y m a x i m i z e o x y g e n a t i o n and waste-product collection and minimize volatilization and remetabolism (Crosby et al., 1979). O u r i n v e s t i g a t i o n e m p l o y e d a f l o w - t h r o u g h s y s t e m . A s m o l i n a t e has a r e l a t i v e l y

314

O

O -~-S

-CH2-CH

3

I O

~

..~

O

- C - S -CH2-CH

3

V

HO.~ _O_s

- CH 2- CH 3

II 0

0 ,,

-S

- CH 2- C -OH

VII

-

-S-CH2-CH

3

III

O

VIII

O

HN-C

-CH

3

0

O

H IV

Fig. 5. Molinate and the metabolites from striped bass (see Table I for key to R o m a n numerals).

high vapor pressure (5.6 × 10 -3 Torr at 25°C; Weed Science Society of America, 1983), flow-through conditions minimized vaporization loss. Also, because several molinate metabolites are unstable at high temperatures, H P L C provided a nondestructive alternative to gas-liquid chromatography (GLC) with greater sensitivity than TLC. Along with variations in toxic action mechanisms and cellular receptor sites, toxicological differences between aquatic species may result from differences in bioconcentration factors, depuration rates, or metabolism rates and profiles. Molinate may be most toxic in either the parent form or as a metabolite. If the parent is most toxic, then organisms readily able to metabolize (detoxify) and depurate it would be least sensitive. If a metabolite is most toxic, then organisms least able to metabolize but most able to depurate the parent would again be least sensitive. Molinate bioconcentration in striped bass was significant but not particularly high; the BCF of 25.3 corresponded closely to the 26.0 reported for topmouth gudgeon (Pseudorasbora parva) (Kanazawa, 1981). The 24-h depuration of

315

molinate and metabolites was rapid and extensive, similar to that (90%) by channel catfish (Ictaluruspunctatus) (Stauffer Chemical Co., 1981). Striped bass readily oxidized molinate to a number of products, particularly the sulfoxide, and with subsequent hydrolysis, to hexahydroazepine (IV); they also produced a molinate conjugate with glutathione (GSH) as evidenced by the mercapturic acid (VIII). Oxidative xenobiotic metabolism of other compounds previously has been demonstrated in many fish species, including rainbow trout (Salmo gairdneri) (Melancon and Lech, 1976), coho salmon (Oncorhynchus kisutch) (Collier et al., 1978), and bluegill (Lepomis macrochirus) (Gingerich and Rach, 1985); hydrolysis similarly has been shown to occur in species such as catfish and bluegill (Rodgers and Stalling, 1972), and rainbow trout (Melancon and Lech, 1978); and GSH conjugation has been demonstrated in Japanese carp (Lay et al., 1979; Lay and Menn, 1979). No other conjugated products were observed in the striped bass, although they have been formed from other compounds in other species, including winter flounder (Pseudopleuronectes americanus) (acetate, glycine, glucuronide; Huang and Collins, 1962), rainbow trout (glucuronide; Lech, 1973), goldfish (Carassius auratus) (sulfate; Akitake and Kobayashi, 1975; Kobayashi et al., 1975), and dogfish shark (Squalus acanthias) (taurine; James and Bend, 1976). Metabolites in the striped bass appeared less diverse than those in Japanese carp in static aquaria (Lay et al., 1979). The increased diversity in Japanese carp, if not due to remetabolism, could indicate that one or more of the metabolites is the toxic agent. Also, less sulfoxide was observed in Japanese carp than in striped bass, which may be important; upon i.p. injection in mice, sulfoxides of the thiocarbamates EPTC and pebulate showed only one-third of the parents' toxicity (Casida et al., 1975). Thus, sulfoxidation may be considered to represent a mammalian detoxication pathway for molinate (Casida et al., 1974; Casida et al., 1975). Compared to the thiocarbamates, sulfoxides are better carbamylating agents and so are more toxic to plants (Casida et al., 1974) as well as more susceptible to GSH inactivation in mammals (Casida et al., 1975). Therefore, striped bass may effectively avoid intoxication by sulfoxidation, followed by GSH conjugation and rapid depuration. This investigation measured molinate bioconcentration, depuration, and biotransformation in a relatively insensitive species. We plan to compare molinate metabolism in the sensitive common carp and the relatively insensitive white sturgeon, and metabolic differences among the three species may indicate the reasons for the observed differential toxicity. ACKNOWLEDGEMENTS

We thank John Cornacchia (California State Water Resources Control Board) for advice; Jeffrey Miller (Stauffer Chemical Company) for samples of molinate, 4-hydroxymolinate, and 4-ketomolinate; and the California Department of Fish and Game Central Valleys Hatchery for striped bass. This research was supported in

316

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