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Comparative metabolism of fenitrothion in aquatic organisms
ECOTOXICOLOGY
AND
Comparative
ENVIRONMENTAL
SAFETY
Metabolism
13,126-l 34 ( 1987)
of Fenitrothion
III. Metabolism in the Crustaceans, Daphnia
in Aquatic Organisms
pulex
and Palaemon
paucidens
YOSHIYUIU TAKIMOTO, MASAKO OHSHIMA, AND JUNSHI MIYAMOTO Takarazuka
Research
Center,
Sumitomo
Chemical
Received
Co., Ltd., 4-2-1,
September
Takarazuka,
H.yogo
665, Japan
2. 1986
When the waterflea Daphnia pulex and the shrimp Palaemon paucidens were exposed to 1.O ppb [“‘C]fenitrothion in a flowthrough system, the concentrations of fenitrothion and 14Cin the body reached equilibrium, and the maximum bioaccumulation ratios of fenitrothion were 7 1 and 6 in the daphnia and shrimp, respectively. These crustaceans primarily metabolized the compound by oxidation of P=S to P=O. hydrolysis of P-0-aryl linkage, and demethylation. The liberated phenol was found to be conjugated with sulfate in the daphnia and with glucose in the shrimp. Whentheorganisms weretransferred to a freshwater stream,fenitrothionandits metabolites were rapidly excreted from their bodies, and the half-life of the parent compound was less than 0.2 day in both species. o 1987 Academic PIW, IX
INTRODUCTION To establish a biochemical background for the use of aquatic organisms in ecotoxicology testings, studies of fenitrothion metabolism have been carried out on killifish (Oryzias Zatipes) at five different developmental stages (Takimoto et al., 1984), mullet (Mugil cephalus) (Takimoto et al., 1987a), carp (Cyprinus carpio) (Takimoto et al., 1985), algae (Kikuchi et al., 1984), and mollusca (Takimoto et al., 1987b), and the results demonstrate that metabolic activities do not vary greatly among species of the same phylum but clearly depend on phyla systematics. Other than the test organisms described above, crustacean species are also key organisms in ecotoxicology. However, the metabolic activities have not been fully elucidated in crustaceans. Therefore, to compare fenitrothion metabolism in other aquatic organisms, in vivo metabolism studies have been undertaken for the waterflea (Daphnia pulex) and the shrimp (Palaemon paucidens). This paper also deals with summarized comparative metabolism in several aquatic organisms in relation to ecotoxicology testings. MATERIALS
AND
METHODS
Special reagents. [14C]Fenitrothion labeled at the m-methyl group of the phenyl moiety was prepared in this laboratory (Yoshitake et al., 1977). The specific activity of the preparation was 39.1 mCi/mmol. Radiochemical purity of [ “C]fenitrothion was 399%. The nonradioactive authentic compounds were prepared and used for identification (Takimoto et al., 1987a). 0147-6513/87 $3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
126
FENITROTHION
METABOLISM
IN CRUSTACEANS
127
Limpet arylsulfatase Type III and P-glucosidase Type II were purchased from Sigma Chemical Co. (St. Louis, MO). Test animals. D. pulex were reproduced at 18°C in a solution containing soil, manure, and pond water (Bar&, 1921) in this laboratory. They were fed dry chlorella and acclimated to dechlorinated tap water for 1 day. Freshwater shrimp (P. paucidens) were purchased from Nihon Youshoku Co. (Nara Prefecture), and acclimated to laboratory conditions at 25 * 2°C in dechlorinated water for at least 2 weeks. The shrimp used for the test had an average body weight of 0.375 g and a fat content of 1.64% after extraction with methanol/chloroform (l/2) (Folch et al., 1957). Uptake and elimination study with [‘“Cl fenitrothion. Acute toxicities of fcnitrothion were 1.6 ppb for D. pulex (in terms of 24-hr ECSo) and 2.2 ppb for the shrimp (96-hr L&). The fate of fenitrothion in the crustaceans was examined in a flow system as follows: [‘4C]fenitrothion was dissolved in an aliquot of methanol and diluted in dechlorinated tap water with a pH of 7.2-7.8 and a hardness of 60-80 ppm as CaC03 to prepare a 1-ppb fenitrothion aqueous solution. In 10 liters of the solution 7.87 g of daphnia or in 40 liters of the solution 137 shrimp were maintained, and then the fresh r4C solution was passed into the glass aquarium with a micropump (Shibata Kagaku Kiki Co., Tokyo) at a renewal rate of 2.5 times (daphnia) or 1 time (shrimp). At specified intervals during the exposure period, 14Cconcentrations were monitored by taking 10 ml of the exposure water. After 1 or 3 days of exposure, 2.9 g daphnia or 64 shrimp were transferred to 5 and 10 liters of fresh water, respectively. Additional fresh water was run into the vessels at the renewal rate of one time per day. The overflow water was trapped in a glass vessel held1 below 3°C to prevent the degradation of 14C excreta. The overflow water and the aquarium water were mixed (recovery water), and the total volume was measured. The 14C concentration was analyzed by taking 10 ml of the solution. Organisms were randomly sampled at specified intervals, and excess water around the organisms was removed by filter paper and the body weights were measured. The aquaria were maintained in a water bath controlled at 18 f 0.5”C (daphnia) or 25 + 0.5”C (shrimp), and the dissolved oxygen concentrations were 6-7 ppm in the tests. Daphnids and shrimp were fed dry chlorella and carp bait (Kyorin Co.), respectively, twice a day. Extraction of radioactivity from the organisms. The daphnia and shrimp exposed to [‘4C]ferdtrothion were processed for solvent extraction according to the previous report (Takimoto et al., 1987a). Extraction of radioactivity from the exposure water. For determination of 14C-labeled compounds in the water, 500 ml of the exposure water or all the recovery water was extracted according to the previous report (Takimoto et a/., 1987a). Analysis and measurement of radioactivity. Quantitative and qualitative analyses of radioactive compounds in the organic extracts of the organisms and water were carried out according to the foregoing study (Takimoto et al., 1987a). Enzymatic hydrolysis of 3-methyl-4-nitrophenyl sulfate and glucoside was examined according to the previous study (Takimoto et al., 1987b). Calcula,tion of bioaccumulation ratio, biological half-life, and recovery ratio. Accumulation ratio, half-life. and recovery ratio were calculated according to the previous report (Takimoto et al., 1987a).
128
TAKIMOTO.
OHSHIMA,
FIG. 1. Uptake and elimination of [“‘Clfenitrothion sure; Rec., recovery.
AND MIYAMOTO
in crustaceans. ‘%2, 0; fenitrothion, 0; Exp., expo-
RESULTS During the exposure and recovery period, neither abnormal was observed.
behavior nor death
Daphnia pulex During the 24-hr exposure, average concentrations of fenitrothion and 14C in the water were 0.99 k 0.09 and 1.06 -+ 0.10 ppb, respectively (the right of Fig. 1). Fenitrothion contents were more than 90% of the 14C in the water, and the major decomposition product was 3-methyl-4-nitrophenol, amounting to 2.4% of the 14C at its maximum. Besides the above compounds, fenitrooxon (accounting for 0.20.8% of the 14C), demethylfenitrooxon (0. I-0.3%), demethylfenitrothion (O.l%), and 3-methyl-4-nitrophenyl sulfate (0.4-0.6%) were detected in the water. In the daphnids, fenitrothion concentration increased to 68 ppb at 8-hr exposure, and then became constant (right side of Fig. 1). The 14C concentration also reached equilibrium (about 90 ppb) after 8 hr exposure. The contents of fenitrothion were 72.9-80.4% ofthe i4C. Metabolites found in the daphnids were demethylfenitrooxon, 3-methyl-4-nitrophenyl sulfate, demethylfenitrothion, 3-methyl-4-nitrophenol, and fenitrooxon, amounting to 2.4-8.2, 3.4-4.9, 0.6-4.0, 3.4-4.0, and 0.8-I .7% of the 14C during the exposure period, respectively (Fig. 2). Based on these concentrations in the daphnids and the water, bioaccumulation ratios (BR) were 88 and 71 for 14C and fenitrothion, respectively (the right of Fig. 1 and Table 1). When the organisms were transferred to fresh water after attainment of an equilibrium of fenitrothion, the concentrations of 14C and fenitrothion decreased rapidly, with half-lives of about 6 and 5 hr, respectively (Table 1).
FENITROTHION
METABOLISM
ou3
129
IN CRUSTACEANS
01
FIG.2. Dtstribution of “‘C during recovery period. Fen, fenitrothion; Oxon, fenitrooxon; DM-fen, demethylfenitrothion; DM-oxon, demethylfenitrooxon; NMC, 3-methyl-4-nitrophnol; 3-methyl-4 -nitrophnol; NMC-sul. 3-methyl4-nitrophenyl-sulfate: NMC-gluco. 3-methyl-4-nitrophenyl-&lucoside.
Analysis of the 14C in the daphnids and recovery water revealed that 93.8% of the 14Coriginally contained in the daphnids was excreted into the recovery water on Day 1 of recovery (right side of Fig. 2). In the water, the content of fenitrothion accounted for 64.2% of the original 14C. Therefore, the recovery ratio was 0.9 10 (Table 1). Besides fenitrothion, 3-methyl-4nitrophenyl sulfate (8.2%) 3-methyl-4-nitrophenol (7.6%), fenitrooxon (4.5%), demethylfenitrooxon (2. I%), and demethylfenitrothion (0.6%) were also found in the water. Palaemon paucidens In the exposure water, I4C and fenitrothion concentrations were 1.OO + 0.04 and 0.89 k 0.10 ppb, respectively, as shown on the left of Fig. 1. The content of fenitrothion in the water was more than 84.6% of the 14C. During the 72-hr exposure period,
TABLE
I
MAXIMUMBIOACCUMULATIONRATIO(MBR),BIOLOGICALHALF-LIFE(BHL),AND RECOVERYRATIO OF['~C]FENITROTHION MBR Species Daphnia pulex Palaemon paucidens
BHL (hr)
RR
14C
Fen.
14C
Fen.
Fen.
88 11
71 6
6 2.5
5 1.5
0.913 0.197
130
TAKIMOTO.
OHSHIMA,
AND
MlYAMOTO
3-methyl-4-nitrophenol(2.5-9.5% ofthe 14C), fenitrooxon (0.3-0.8%) demethylfenitrothion (0.2-OS%), demethylfenitrooxon (O.l-0.2%) and 3-methyl-4-nitrophenylP-glucoside (0.2-0.3%) were detected. In the shrimp, fenitrothion concentration reached equilibrium on Day 1 of exposure (about 5 ppb), as shown on the left of Fig. 1, and its content amounted to 4453% of the 14C. As major metabolites, 3-methyl-4-nitrophenol and its glucoside accounted for 8.3- 10.2 and 8.1-10.8% of the 14C, respectively. Other metabolites detected were demethylfenitrothion (3.0-4.7% of the 14C), demethylfenitrooxon (3.44.6%), and fenitrooxon (1.7-3.9%) (left side of Fig. 2). The maximum bioaccumulation ratios were 11 and 6 for 14C and fenitrothion, respectively (Table 1). When the shrimp were transferred to fresh water, 14C and fenitrothion decreased rapidly, with biological half-lives of 2.5 and 1.5 hr, respectively (Table 1). During the 8-hr recovery period, 79.8% of the 14Coriginally contained in the shrimp was excreted into the recovery water. Fenitrothion, 3-methyl-4-nitrophenol, 3-methyl-4-nitrophenyl-@-glucoside, demethylfenitrooxon, fenitrooxon, and demethylfenitrothion accounted for 8.3, 38.4, 22.3, 0.7, 0.3, and O.l%, respectively, of the 14C in the water (Fig. 2). From these data, the recovery ratio of fenitrothion was 0.197 (Table 1). DISCUSSION Although in vitro metabolism studies of parathion, O,O-dimethyl o-4-nitrophenyl phosphorothioate, resulted in no conversion to paraoxon by hepatopancreas microsomes of lobsters (Homarus americanus) (Carlson, 1973; Elmamlouk and Gessner, 1976), Bend et al. (198 1) reported that the microsomes from marine crustacean species (H. americanus and Punulirus urgus) contained relatively high amounts of cytochrome P-450 (up to 1 nmol/mg protein), but NADPH-dependent monooxogenase activity was very low or undetectable due to the apparent absence of NADPH-cytochrome P-450 reductase activity and the presence of inhibitors. Therefore, they concluded that metabolism studies in microsomal fractions of crustacean hepatopancress do not accurately represent the in vivo metabolism. However, few data are available on in vivo metabolism of xenobiotics in crustaceans. Benzo[u]pyrene injected into the pericardial sinus of lobsters (H. americanus) was metabolized oxidatively to 3-hydroxybenzo[u]pyrene (Bend et al., 198 1). When biphenyl was injected into stomach of Cirolunu borealis, the compound was converted to 2-hydroxy derivative in the tissue and excreted as 4-hydroxy and 4,4’-dihydroxy-biphenyl as well as the above metabolite into water (Meyer and Bakke, 1977). Our studies also demonstrate that both crustacean species exposed to fenitrothion metabolized it oxidatively to the oxon analogs. It is toxicologically interesting that relatively high residues of fenitrooxon, known as an inhibitor of cholinesterase (O’Brien, 1960) are found in the body, and this retention may be related to high toxicity to the crustaceans. Other than oxidation, crustaceans metabolize in vivo the compound through demethylation of P-0-alkyl and hydrolysis of P-0-aryl linkage, and the liberated phenol is conjugated with sulfuric acid in the daphnia and glucose in the shrimp. Based on the recovery ratio of fenitrothion obtained from organisms transferred to fresh water, shrimp actively metabolized and excreted fenitrothion (RR of 0.197) while D. pulex was inactive (RR of 0.9 10). Higher amounts of the conjugates were
FENITROTHION
METABOLISM
IN CRUSTACEANS
131
detected in the recovery water than in the body, perhaps implying that crustaceans have little ability to hydrolyze the conjugates or have excretion routes different from those of the fish (Takimoto et al., 1987a). Bioaccumulation ratios of fenitrothion are relatively low in the crustaceans, at 6 and 7 1, an.d biological half-lives are very short, less than 0.25 day. Reinert (1972) reported that the BR of dieldrin was highest in guppy (Poecilia reticulata) followed by Daphnia magna and alga (Scenedesmus obliguus), with values of 49,307, 13,954, and 1282, respectively. However, accumulation of chlordane was highest in D. magna (BR of 24,000) followed by the alga Ankistrodesmus amalloides (5560) and the goldfish Curassius auratus (162) (Moore et al., 1977). Comparative bioaccumulation ratios are discussed below. In conclusion of our studies on comparative metabolism. the following points are clarified in relation to ecotoxicology testings. (1) Bioaccumulation
Ratio and Biological
Half-life
(i) Bioaccumulation ratio. When aquatic organisms were exposed to fenitrothion solution, fenitrothion concentrations reached equilibrium after short exposure time (usually in l-3 days), and bioaccumulation ratios (BR) of fenitrothion are in the range of 6-540, as shown in Table 2. This table demonstrates that among four phyla, fish show the highest value, followed by algae, mollusca, and crustaceans, although one order of magnitude difference is observed among species in the same phylum. Although there is good correlation between fat content and BR values of fenitrothion in the developmental stages of the killifish (Takimoto et al., 1984) and of 1,2,4trichlorobenzene in eight fish species (Geyer et al., 1985) fat content does not explain the ratios of fenitrothion among phyla. Water temperature and salinity do not give much different BR values (Takimoto et al., 1987a). (ii) Biological half-life. Biological half-lives of fenitrothion are very short in any organism of less than 1 day except for the killifish at the embryo stage (1.4 days), whereas those of 14C are somewhat longer (0.1-3.8 days), depending on feeding due to enterohepatic circulation of metabolites. Both fish and mollusca have the longest fenitrothion half-lives, followed by crustaceans and algae. Therefore, fish are thought to be the most suitable organism for accumulation test. (2) Metabolism Metabolites found during the exposure period are shown in Fig. 3 and proposed metabolic pathways of fenitrothion are given in Fig. 4. Metabolism is not dependent on species in the same phylum and surrounding conditions so much, but on the phyla of systemics, as described below. (i) Hydrolysis and conjugation. All organisms have hydrolyzing activity to produce 3-methyl-4-nitrophenol, but conjugation is dependent on the species. In fish, glucuronide conjugate is predominant, and a small amount of the sulfate is found only in the killifish at the adult stage. Mollusca produce sulfate (pond snail) and glucose conjugate (P/zysa snail), and crustaceans are similar to the mollusca. On the other hand, such conjugations are not detected in algae. Furthermore, the proportion of the conjugate to the free form is highest in fish, followed by mollusca, crustaceans, and algae.
132
TAKIMOTO,
OHSHIMA,
AND
TABLE
MAXIMUM BIOACCUMULATION RECOVERY RATIO (RR)
MIYAMOTO
2
RATIO (MBR), BIOLOGICAL HALF-LIFE (BHL), OF FENITROTHION IN AQUATIC ORGANISMS MBR
Organism Fish Rainbow trout (Salmogairdneri) Topmouth gudgeon (Pseudorasbora Killifish (Oryzias latipes) Embryo Yolk sac fry Postlarva Juvenile Adult (Male) (Female) (Ed Postlarva Postlarva Juvenile Mullet (Mugil cephalus)
Carp (Cyprinus carpio) Crustaceans Daphnia pulex Shrimp (Palaemon paucidens) Mollusca Pond snail (Cipangopaludina japonica) Physa acuta Algae Chlorella vulgaris Nitzschia closteriurn Anabaena flos-aquae
parva)
(day)
RR
Salinity (%o)
Temp PJ
“‘C
Fen
‘%I
0 0
1s 23
-h -’
249 203
-h -h
0 0 8 0 0 0 0 23 0 0
25 25 25 25 25 25 25 25 25 15 25
163 287 146 610 559 544 207 299 377 421 210
155 173 88 441 520 540 224 235 303 339 30
23 0
25 25
343 176
179 96
2.33 1.28 0.27 1.17 0.63 0.52 0.62 0.21 0.41 0.36 2.36 0.50’ 3.75 1.27
1.42 0.27 0.28 0.31 0.38 0.40 0.56 0.24 0.25 0.26 0.26 0.24’ 0.36 0.28
0.842 0.900 0.945 0.437 0.466’ 0.177 0.78 1
18 25
88 11
71 0.25 6 0.10
0.21 0.06
0.913 0.197
25 25
35 79
18 53
1.05 0.75
0.40 0.35
0.517 0.520
23 23 23
51 102 98
44 105 53
0.13 0.04 0.61
0.04 0.04 0.11
-d -d 0.919
a References: (1) Takimoto and Miyamoto. 1976; (2) Takimoto 1985: (5) this report; (6) Takimoto et al., 1985b: (7) Kikuchi b Nonradioactive fenitrothion was used. ‘Fish were fed bait during the recovery period. dNot determined.
etul..
BHL
AND
ef ul.. 1984; (3) Takimoto 1984.
etal..
Fen
0.4 0.3
etal..
Fen
Ref.”
-’ -h 0.821 0.971 0.641 0.629 0.581 3 3 3 3 4
1987a; (4) Takimoto
These characteristic conjugation reactions and proportions are thought to be related to systematic development of the organisms. (ii) Demethylation. Among four phyla, demethyl derivatives including demethylfenitrothion, fenitrooxon, and aminofenitrothion are produced by mollusca in relatively high amounts, followed by algae, crustaceans, and fish. However, killifish at the yolk sac fry stage produce the compound at an amount comparable to mollusca. Furthermore, mullet forms the highest amount of demethylfenitrothion among the species tested, and the activity was independent of the salinity. (iii) Oxidation and reduction. Oxidation of P=S to P=O is predominantly found in the body of crustaceans and A. Jos-aquae of blue-green alga. Oxon analogs of phosphorothioate insecticides are known to inhibit cholinesterase activity. Therefore, the high toxicity of fenitrothion to crustaceans can be explained by their higher production of the compound. It is interesting that fenitrooxon is also produced in the other organisms, as proved by analysis of the recovery water, but the compound is
FENITROTHION
Fenitrothion
METABOLISM
Hydrolysis
CKM
and Conjugation
SnP
ShD
Demethylation
ChNA
m DM-aminofen
10 ”
CKM
%P
YID
ChNA
C KM
SnP
ShD
133
IN CRUSTACEANS
ChN A
CKM C: K : M: Sn: P : sh: D : Ch: N : A :
%P
ShD
ChNA
Car Killi Pish Mullet Snail Physa acuta Ylrimp oophnia pulex Cvulgaris Nxlosterium A.flos-aquae
1
Fishes M4usca
3
Crustaceans
1
Algae
FIG. 3. Comparative metabolism of fenitrothion in aquatic organisms. Fen. fenitrothion: Oxon, fenitrooxon; Aminofen, aminofenitrothion; DM-fen, demethylfenitrothion; DM-oxon. demethylfenitrooxon: DM-aminofen. demethylaminofenitrothion; NMC: 3-methyl-4-nitrophnol: NMC-sul. 3-methyl-4-nitrophenyl-sulfate; NMC-gluco, 3-methyl-4-nitrophenyl-P-glucoside: NMC-glu, 3-methyl-4-nitrophenyl-flglucuronide.
not retamed in their bodies, in contrast to the crustaceans, probably due to rapid hydrolysis and/or rapid excretion. Reduction of the nitro group to amino group is specifically observed in mollusca. (iv) Excretion offenitrothion. Based on the recovery ratio of fenitrothion (RR), fenitrothion itself is excreted directly into fresh water, due to the higher RR values of 0.52-0.98’ except in the mullet (0.18-0.47) and shrimp (0.20) as shown in Table 2. The values are related to developmental stages of killifish, and this indicates that as the stage developed, the metabolism proceeded more rapidly.
F Fishes M Mollusca
FIG. 4. Proposed metabolic pathways for fenitrothion in aquatic organisms.
134
TAKIMOTO,
OHSHIMA.
AND
MIYAMOTO
CONCLUSIONS From the above comparative metabolism studies, it can be concluded that metabolism is dependent on phyla systematics and generally differences among species in the same phylum are relatively small. Therefore, this information will afford a sound basis for the selection of the organisms in ecotoxicology testings, and if combined with appropriate model ecosystems (Miyamoto et al., 1985), the environmental impact of chemicals, including their eventual fate, can be assessed fairly accurately during laboratory trials. REFERENCES BANTA, A. M. (1921). A convenient culture medium for daphnidaes. Science53,557-558. BEND, J. R., JAMES, M. 0.. LITTLE, P. J., AND FOUREMAN, G. L. (198 1). In vitro and in viva metabolism of benzo[a]pyrene by selected marine crustacean species. In Phyletic Approaches to Cancer (C. J. Daive. J. C. Harshbarger, S. Kondo, T. Sugimura, and S. Takayama. Eds.), pp. 179-194. Japan Sci. Sot. Press, Tokyo. CARLSON, G. P. (1973). Comparison of the metabolism of parathion by lobsters and rats. Bull. Environ.
Contam. Toxicol. 9,296-300. ELMAMLOUK, T. H., AND GESSNER, T. (1976). Species difference in metabolism of parathion. Apparent inability of hepatopancreas fractions to produce paraoxon. Comp. Biochem. Physiol. C: Comp. Pharmacol. 53, 19-24. FOLCH, J., LEES, M., AND STANLEY, G. H. S. (1957). A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226,497-509. GEYER, H., SCHEUNERT, I., AND KORTE, F. (1985). Relationship between the lipid content of fish and their bioconcentration potential of 1.2.4~trichlorobenzene. Chemosphere 14,545-555. KIKUCHI, R.. YASUTANIYA, T., TAKIMOTO, Y.. AND MIYAMOTO, J. (1984). Accumulation and metabolism of fenitrothion in 3 species ofalgae. J. Pest. Sci. 9,33 l-337. MEYER, T., AND BAKKE, T. (1977). The metabolism of biphenyl. V. Phenolic metabolites in some marine organisms. Acta Pharmacol. Toxicol. 40,20 l-208. MIYAMOTO, J.. KLEIN, W.. TAKIMOTO, Y., AND ROBERTS, T. R. (1985). Critical evaluation of model ecosystems. Pure Appl. Chem., 57, 1523- 1536. MOORE. R., TORO, E., STANTON. M., AND KHAN, M. A. Q. (1977). Absorption and elimination of 14C-aand y-chlordane by a freshwater alga, daphnid. and goldfish. Arch. Environ. Contam. Tosicol. 6,4 I l420. O’BRIEN, R. D. (1960). To.xic Phosphorus Esters, p. 73. Academic Press. New York. REINERT. R. E. (1972). Accumulation ofdieldrin in an alga (Scenede.ymus obhquus). Daphnia mugna, and the guppy (Poecilia reticuluta). J. Fish Res. Board Canad. 29, 14 13- 14 18. TAKIMOTO. Y., AND MIYAMOTO, J. (1976). Studies on Accumulation and metabolism of Sumithion in fish. J. Pest. Sci. 1,26 l-27 1. TAKIMOTO, Y., OHSHIMA, M., AND MIYAMOTO, J. (1987a). Comparative metabolism of fenitrothion in aquatic organisms. I. Metabolism in the euryhaline fish, Oryzias latipes and Mugil cephalus. Ecotoxicol. Environ. .Saf 13, 104-I 17. TAKIMOTO, Y., OHSHIMA. M., AND MIYAMOTO, J. (1985). Fate of fenitrothion in carp, C.vprinus carpio by oral administration and exposure. Submitted for publication. TAIUMOTO, Y., OHSHIMA, M., AND MIYAMOTO, J. (1987b). Comparative metabolism of fenitrothion in aquatic organisms. II. Metabolism in freshwater snails, Cipnngopnludina juponica and Physa Acura. Ecotoxicol. Environ. Saf: 13, 11 E- 125. TAKIMOTO, Y., OHSHIMA, M., YAMADA, H.. AND MIYAMOTO, J. (1984). Fate of fenitrothion in several developmental stages of killifish (Oryzias latipes). Arch. Environ. Contam. Toxicol. 13,579-587. YOSHITAKE, A., KAWAHARA, K., KAMADA, T., AND ENDO. M. (1977). Labelled organophosphorus pesticides. I. Synthesis of carbon 14 labelled O,O-dimethyl 0-(3-methyl-4-nitrophenyl) phosphorothioate (Sumithion@). J. Labelled Compd. 13,323-33 1.
AND
Comparative
ENVIRONMENTAL
SAFETY
Metabolism
13,126-l 34 ( 1987)
of Fenitrothion
III. Metabolism in the Crustaceans, Daphnia
in Aquatic Organisms
pulex
and Palaemon
paucidens
YOSHIYUIU TAKIMOTO, MASAKO OHSHIMA, AND JUNSHI MIYAMOTO Takarazuka
Research
Center,
Sumitomo
Chemical
Received
Co., Ltd., 4-2-1,
September
Takarazuka,
H.yogo
665, Japan
2. 1986
When the waterflea Daphnia pulex and the shrimp Palaemon paucidens were exposed to 1.O ppb [“‘C]fenitrothion in a flowthrough system, the concentrations of fenitrothion and 14Cin the body reached equilibrium, and the maximum bioaccumulation ratios of fenitrothion were 7 1 and 6 in the daphnia and shrimp, respectively. These crustaceans primarily metabolized the compound by oxidation of P=S to P=O. hydrolysis of P-0-aryl linkage, and demethylation. The liberated phenol was found to be conjugated with sulfate in the daphnia and with glucose in the shrimp. Whentheorganisms weretransferred to a freshwater stream,fenitrothionandits metabolites were rapidly excreted from their bodies, and the half-life of the parent compound was less than 0.2 day in both species. o 1987 Academic PIW, IX
INTRODUCTION To establish a biochemical background for the use of aquatic organisms in ecotoxicology testings, studies of fenitrothion metabolism have been carried out on killifish (Oryzias Zatipes) at five different developmental stages (Takimoto et al., 1984), mullet (Mugil cephalus) (Takimoto et al., 1987a), carp (Cyprinus carpio) (Takimoto et al., 1985), algae (Kikuchi et al., 1984), and mollusca (Takimoto et al., 1987b), and the results demonstrate that metabolic activities do not vary greatly among species of the same phylum but clearly depend on phyla systematics. Other than the test organisms described above, crustacean species are also key organisms in ecotoxicology. However, the metabolic activities have not been fully elucidated in crustaceans. Therefore, to compare fenitrothion metabolism in other aquatic organisms, in vivo metabolism studies have been undertaken for the waterflea (Daphnia pulex) and the shrimp (Palaemon paucidens). This paper also deals with summarized comparative metabolism in several aquatic organisms in relation to ecotoxicology testings. MATERIALS
AND
METHODS
Special reagents. [14C]Fenitrothion labeled at the m-methyl group of the phenyl moiety was prepared in this laboratory (Yoshitake et al., 1977). The specific activity of the preparation was 39.1 mCi/mmol. Radiochemical purity of [ “C]fenitrothion was 399%. The nonradioactive authentic compounds were prepared and used for identification (Takimoto et al., 1987a). 0147-6513/87 $3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
126
FENITROTHION
METABOLISM
IN CRUSTACEANS
127
Limpet arylsulfatase Type III and P-glucosidase Type II were purchased from Sigma Chemical Co. (St. Louis, MO). Test animals. D. pulex were reproduced at 18°C in a solution containing soil, manure, and pond water (Bar&, 1921) in this laboratory. They were fed dry chlorella and acclimated to dechlorinated tap water for 1 day. Freshwater shrimp (P. paucidens) were purchased from Nihon Youshoku Co. (Nara Prefecture), and acclimated to laboratory conditions at 25 * 2°C in dechlorinated water for at least 2 weeks. The shrimp used for the test had an average body weight of 0.375 g and a fat content of 1.64% after extraction with methanol/chloroform (l/2) (Folch et al., 1957). Uptake and elimination study with [‘“Cl fenitrothion. Acute toxicities of fcnitrothion were 1.6 ppb for D. pulex (in terms of 24-hr ECSo) and 2.2 ppb for the shrimp (96-hr L&). The fate of fenitrothion in the crustaceans was examined in a flow system as follows: [‘4C]fenitrothion was dissolved in an aliquot of methanol and diluted in dechlorinated tap water with a pH of 7.2-7.8 and a hardness of 60-80 ppm as CaC03 to prepare a 1-ppb fenitrothion aqueous solution. In 10 liters of the solution 7.87 g of daphnia or in 40 liters of the solution 137 shrimp were maintained, and then the fresh r4C solution was passed into the glass aquarium with a micropump (Shibata Kagaku Kiki Co., Tokyo) at a renewal rate of 2.5 times (daphnia) or 1 time (shrimp). At specified intervals during the exposure period, 14Cconcentrations were monitored by taking 10 ml of the exposure water. After 1 or 3 days of exposure, 2.9 g daphnia or 64 shrimp were transferred to 5 and 10 liters of fresh water, respectively. Additional fresh water was run into the vessels at the renewal rate of one time per day. The overflow water was trapped in a glass vessel held1 below 3°C to prevent the degradation of 14C excreta. The overflow water and the aquarium water were mixed (recovery water), and the total volume was measured. The 14C concentration was analyzed by taking 10 ml of the solution. Organisms were randomly sampled at specified intervals, and excess water around the organisms was removed by filter paper and the body weights were measured. The aquaria were maintained in a water bath controlled at 18 f 0.5”C (daphnia) or 25 + 0.5”C (shrimp), and the dissolved oxygen concentrations were 6-7 ppm in the tests. Daphnids and shrimp were fed dry chlorella and carp bait (Kyorin Co.), respectively, twice a day. Extraction of radioactivity from the organisms. The daphnia and shrimp exposed to [‘4C]ferdtrothion were processed for solvent extraction according to the previous report (Takimoto et al., 1987a). Extraction of radioactivity from the exposure water. For determination of 14C-labeled compounds in the water, 500 ml of the exposure water or all the recovery water was extracted according to the previous report (Takimoto et a/., 1987a). Analysis and measurement of radioactivity. Quantitative and qualitative analyses of radioactive compounds in the organic extracts of the organisms and water were carried out according to the foregoing study (Takimoto et al., 1987a). Enzymatic hydrolysis of 3-methyl-4-nitrophenyl sulfate and glucoside was examined according to the previous study (Takimoto et al., 1987b). Calcula,tion of bioaccumulation ratio, biological half-life, and recovery ratio. Accumulation ratio, half-life. and recovery ratio were calculated according to the previous report (Takimoto et al., 1987a).
128
TAKIMOTO.
OHSHIMA,
FIG. 1. Uptake and elimination of [“‘Clfenitrothion sure; Rec., recovery.
AND MIYAMOTO
in crustaceans. ‘%2, 0; fenitrothion, 0; Exp., expo-
RESULTS During the exposure and recovery period, neither abnormal was observed.
behavior nor death
Daphnia pulex During the 24-hr exposure, average concentrations of fenitrothion and 14C in the water were 0.99 k 0.09 and 1.06 -+ 0.10 ppb, respectively (the right of Fig. 1). Fenitrothion contents were more than 90% of the 14C in the water, and the major decomposition product was 3-methyl-4-nitrophenol, amounting to 2.4% of the 14C at its maximum. Besides the above compounds, fenitrooxon (accounting for 0.20.8% of the 14C), demethylfenitrooxon (0. I-0.3%), demethylfenitrothion (O.l%), and 3-methyl-4-nitrophenyl sulfate (0.4-0.6%) were detected in the water. In the daphnids, fenitrothion concentration increased to 68 ppb at 8-hr exposure, and then became constant (right side of Fig. 1). The 14C concentration also reached equilibrium (about 90 ppb) after 8 hr exposure. The contents of fenitrothion were 72.9-80.4% ofthe i4C. Metabolites found in the daphnids were demethylfenitrooxon, 3-methyl-4-nitrophenyl sulfate, demethylfenitrothion, 3-methyl-4-nitrophenol, and fenitrooxon, amounting to 2.4-8.2, 3.4-4.9, 0.6-4.0, 3.4-4.0, and 0.8-I .7% of the 14C during the exposure period, respectively (Fig. 2). Based on these concentrations in the daphnids and the water, bioaccumulation ratios (BR) were 88 and 71 for 14C and fenitrothion, respectively (the right of Fig. 1 and Table 1). When the organisms were transferred to fresh water after attainment of an equilibrium of fenitrothion, the concentrations of 14C and fenitrothion decreased rapidly, with half-lives of about 6 and 5 hr, respectively (Table 1).
FENITROTHION
METABOLISM
ou3
129
IN CRUSTACEANS
01
FIG.2. Dtstribution of “‘C during recovery period. Fen, fenitrothion; Oxon, fenitrooxon; DM-fen, demethylfenitrothion; DM-oxon, demethylfenitrooxon; NMC, 3-methyl-4-nitrophnol; 3-methyl-4 -nitrophnol; NMC-sul. 3-methyl4-nitrophenyl-sulfate: NMC-gluco. 3-methyl-4-nitrophenyl-&lucoside.
Analysis of the 14C in the daphnids and recovery water revealed that 93.8% of the 14Coriginally contained in the daphnids was excreted into the recovery water on Day 1 of recovery (right side of Fig. 2). In the water, the content of fenitrothion accounted for 64.2% of the original 14C. Therefore, the recovery ratio was 0.9 10 (Table 1). Besides fenitrothion, 3-methyl-4nitrophenyl sulfate (8.2%) 3-methyl-4-nitrophenol (7.6%), fenitrooxon (4.5%), demethylfenitrooxon (2. I%), and demethylfenitrothion (0.6%) were also found in the water. Palaemon paucidens In the exposure water, I4C and fenitrothion concentrations were 1.OO + 0.04 and 0.89 k 0.10 ppb, respectively, as shown on the left of Fig. 1. The content of fenitrothion in the water was more than 84.6% of the 14C. During the 72-hr exposure period,
TABLE
I
MAXIMUMBIOACCUMULATIONRATIO(MBR),BIOLOGICALHALF-LIFE(BHL),AND RECOVERYRATIO OF['~C]FENITROTHION MBR Species Daphnia pulex Palaemon paucidens
BHL (hr)
RR
14C
Fen.
14C
Fen.
Fen.
88 11
71 6
6 2.5
5 1.5
0.913 0.197
130
TAKIMOTO.
OHSHIMA,
AND
MlYAMOTO
3-methyl-4-nitrophenol(2.5-9.5% ofthe 14C), fenitrooxon (0.3-0.8%) demethylfenitrothion (0.2-OS%), demethylfenitrooxon (O.l-0.2%) and 3-methyl-4-nitrophenylP-glucoside (0.2-0.3%) were detected. In the shrimp, fenitrothion concentration reached equilibrium on Day 1 of exposure (about 5 ppb), as shown on the left of Fig. 1, and its content amounted to 4453% of the 14C. As major metabolites, 3-methyl-4-nitrophenol and its glucoside accounted for 8.3- 10.2 and 8.1-10.8% of the 14C, respectively. Other metabolites detected were demethylfenitrothion (3.0-4.7% of the 14C), demethylfenitrooxon (3.44.6%), and fenitrooxon (1.7-3.9%) (left side of Fig. 2). The maximum bioaccumulation ratios were 11 and 6 for 14C and fenitrothion, respectively (Table 1). When the shrimp were transferred to fresh water, 14C and fenitrothion decreased rapidly, with biological half-lives of 2.5 and 1.5 hr, respectively (Table 1). During the 8-hr recovery period, 79.8% of the 14Coriginally contained in the shrimp was excreted into the recovery water. Fenitrothion, 3-methyl-4-nitrophenol, 3-methyl-4-nitrophenyl-@-glucoside, demethylfenitrooxon, fenitrooxon, and demethylfenitrothion accounted for 8.3, 38.4, 22.3, 0.7, 0.3, and O.l%, respectively, of the 14C in the water (Fig. 2). From these data, the recovery ratio of fenitrothion was 0.197 (Table 1). DISCUSSION Although in vitro metabolism studies of parathion, O,O-dimethyl o-4-nitrophenyl phosphorothioate, resulted in no conversion to paraoxon by hepatopancreas microsomes of lobsters (Homarus americanus) (Carlson, 1973; Elmamlouk and Gessner, 1976), Bend et al. (198 1) reported that the microsomes from marine crustacean species (H. americanus and Punulirus urgus) contained relatively high amounts of cytochrome P-450 (up to 1 nmol/mg protein), but NADPH-dependent monooxogenase activity was very low or undetectable due to the apparent absence of NADPH-cytochrome P-450 reductase activity and the presence of inhibitors. Therefore, they concluded that metabolism studies in microsomal fractions of crustacean hepatopancress do not accurately represent the in vivo metabolism. However, few data are available on in vivo metabolism of xenobiotics in crustaceans. Benzo[u]pyrene injected into the pericardial sinus of lobsters (H. americanus) was metabolized oxidatively to 3-hydroxybenzo[u]pyrene (Bend et al., 198 1). When biphenyl was injected into stomach of Cirolunu borealis, the compound was converted to 2-hydroxy derivative in the tissue and excreted as 4-hydroxy and 4,4’-dihydroxy-biphenyl as well as the above metabolite into water (Meyer and Bakke, 1977). Our studies also demonstrate that both crustacean species exposed to fenitrothion metabolized it oxidatively to the oxon analogs. It is toxicologically interesting that relatively high residues of fenitrooxon, known as an inhibitor of cholinesterase (O’Brien, 1960) are found in the body, and this retention may be related to high toxicity to the crustaceans. Other than oxidation, crustaceans metabolize in vivo the compound through demethylation of P-0-alkyl and hydrolysis of P-0-aryl linkage, and the liberated phenol is conjugated with sulfuric acid in the daphnia and glucose in the shrimp. Based on the recovery ratio of fenitrothion obtained from organisms transferred to fresh water, shrimp actively metabolized and excreted fenitrothion (RR of 0.197) while D. pulex was inactive (RR of 0.9 10). Higher amounts of the conjugates were
FENITROTHION
METABOLISM
IN CRUSTACEANS
131
detected in the recovery water than in the body, perhaps implying that crustaceans have little ability to hydrolyze the conjugates or have excretion routes different from those of the fish (Takimoto et al., 1987a). Bioaccumulation ratios of fenitrothion are relatively low in the crustaceans, at 6 and 7 1, an.d biological half-lives are very short, less than 0.25 day. Reinert (1972) reported that the BR of dieldrin was highest in guppy (Poecilia reticulata) followed by Daphnia magna and alga (Scenedesmus obliguus), with values of 49,307, 13,954, and 1282, respectively. However, accumulation of chlordane was highest in D. magna (BR of 24,000) followed by the alga Ankistrodesmus amalloides (5560) and the goldfish Curassius auratus (162) (Moore et al., 1977). Comparative bioaccumulation ratios are discussed below. In conclusion of our studies on comparative metabolism. the following points are clarified in relation to ecotoxicology testings. (1) Bioaccumulation
Ratio and Biological
Half-life
(i) Bioaccumulation ratio. When aquatic organisms were exposed to fenitrothion solution, fenitrothion concentrations reached equilibrium after short exposure time (usually in l-3 days), and bioaccumulation ratios (BR) of fenitrothion are in the range of 6-540, as shown in Table 2. This table demonstrates that among four phyla, fish show the highest value, followed by algae, mollusca, and crustaceans, although one order of magnitude difference is observed among species in the same phylum. Although there is good correlation between fat content and BR values of fenitrothion in the developmental stages of the killifish (Takimoto et al., 1984) and of 1,2,4trichlorobenzene in eight fish species (Geyer et al., 1985) fat content does not explain the ratios of fenitrothion among phyla. Water temperature and salinity do not give much different BR values (Takimoto et al., 1987a). (ii) Biological half-life. Biological half-lives of fenitrothion are very short in any organism of less than 1 day except for the killifish at the embryo stage (1.4 days), whereas those of 14C are somewhat longer (0.1-3.8 days), depending on feeding due to enterohepatic circulation of metabolites. Both fish and mollusca have the longest fenitrothion half-lives, followed by crustaceans and algae. Therefore, fish are thought to be the most suitable organism for accumulation test. (2) Metabolism Metabolites found during the exposure period are shown in Fig. 3 and proposed metabolic pathways of fenitrothion are given in Fig. 4. Metabolism is not dependent on species in the same phylum and surrounding conditions so much, but on the phyla of systemics, as described below. (i) Hydrolysis and conjugation. All organisms have hydrolyzing activity to produce 3-methyl-4-nitrophenol, but conjugation is dependent on the species. In fish, glucuronide conjugate is predominant, and a small amount of the sulfate is found only in the killifish at the adult stage. Mollusca produce sulfate (pond snail) and glucose conjugate (P/zysa snail), and crustaceans are similar to the mollusca. On the other hand, such conjugations are not detected in algae. Furthermore, the proportion of the conjugate to the free form is highest in fish, followed by mollusca, crustaceans, and algae.
132
TAKIMOTO,
OHSHIMA,
AND
TABLE
MAXIMUM BIOACCUMULATION RECOVERY RATIO (RR)
MIYAMOTO
2
RATIO (MBR), BIOLOGICAL HALF-LIFE (BHL), OF FENITROTHION IN AQUATIC ORGANISMS MBR
Organism Fish Rainbow trout (Salmogairdneri) Topmouth gudgeon (Pseudorasbora Killifish (Oryzias latipes) Embryo Yolk sac fry Postlarva Juvenile Adult (Male) (Female) (Ed Postlarva Postlarva Juvenile Mullet (Mugil cephalus)
Carp (Cyprinus carpio) Crustaceans Daphnia pulex Shrimp (Palaemon paucidens) Mollusca Pond snail (Cipangopaludina japonica) Physa acuta Algae Chlorella vulgaris Nitzschia closteriurn Anabaena flos-aquae
parva)
(day)
RR
Salinity (%o)
Temp PJ
“‘C
Fen
‘%I
0 0
1s 23
-h -’
249 203
-h -h
0 0 8 0 0 0 0 23 0 0
25 25 25 25 25 25 25 25 25 15 25
163 287 146 610 559 544 207 299 377 421 210
155 173 88 441 520 540 224 235 303 339 30
23 0
25 25
343 176
179 96
2.33 1.28 0.27 1.17 0.63 0.52 0.62 0.21 0.41 0.36 2.36 0.50’ 3.75 1.27
1.42 0.27 0.28 0.31 0.38 0.40 0.56 0.24 0.25 0.26 0.26 0.24’ 0.36 0.28
0.842 0.900 0.945 0.437 0.466’ 0.177 0.78 1
18 25
88 11
71 0.25 6 0.10
0.21 0.06
0.913 0.197
25 25
35 79
18 53
1.05 0.75
0.40 0.35
0.517 0.520
23 23 23
51 102 98
44 105 53
0.13 0.04 0.61
0.04 0.04 0.11
-d -d 0.919
a References: (1) Takimoto and Miyamoto. 1976; (2) Takimoto 1985: (5) this report; (6) Takimoto et al., 1985b: (7) Kikuchi b Nonradioactive fenitrothion was used. ‘Fish were fed bait during the recovery period. dNot determined.
etul..
BHL
AND
ef ul.. 1984; (3) Takimoto 1984.
etal..
Fen
0.4 0.3
etal..
Fen
Ref.”
-’ -h 0.821 0.971 0.641 0.629 0.581 3 3 3 3 4
1987a; (4) Takimoto
These characteristic conjugation reactions and proportions are thought to be related to systematic development of the organisms. (ii) Demethylation. Among four phyla, demethyl derivatives including demethylfenitrothion, fenitrooxon, and aminofenitrothion are produced by mollusca in relatively high amounts, followed by algae, crustaceans, and fish. However, killifish at the yolk sac fry stage produce the compound at an amount comparable to mollusca. Furthermore, mullet forms the highest amount of demethylfenitrothion among the species tested, and the activity was independent of the salinity. (iii) Oxidation and reduction. Oxidation of P=S to P=O is predominantly found in the body of crustaceans and A. Jos-aquae of blue-green alga. Oxon analogs of phosphorothioate insecticides are known to inhibit cholinesterase activity. Therefore, the high toxicity of fenitrothion to crustaceans can be explained by their higher production of the compound. It is interesting that fenitrooxon is also produced in the other organisms, as proved by analysis of the recovery water, but the compound is
FENITROTHION
Fenitrothion
METABOLISM
Hydrolysis
CKM
and Conjugation
SnP
ShD
Demethylation
ChNA
m DM-aminofen
10 ”
CKM
%P
YID
ChNA
C KM
SnP
ShD
133
IN CRUSTACEANS
ChN A
CKM C: K : M: Sn: P : sh: D : Ch: N : A :
%P
ShD
ChNA
Car Killi Pish Mullet Snail Physa acuta Ylrimp oophnia pulex Cvulgaris Nxlosterium A.flos-aquae
1
Fishes M4usca
3
Crustaceans
1
Algae
FIG. 3. Comparative metabolism of fenitrothion in aquatic organisms. Fen. fenitrothion: Oxon, fenitrooxon; Aminofen, aminofenitrothion; DM-fen, demethylfenitrothion; DM-oxon. demethylfenitrooxon: DM-aminofen. demethylaminofenitrothion; NMC: 3-methyl-4-nitrophnol: NMC-sul. 3-methyl-4-nitrophenyl-sulfate; NMC-gluco, 3-methyl-4-nitrophenyl-P-glucoside: NMC-glu, 3-methyl-4-nitrophenyl-flglucuronide.
not retamed in their bodies, in contrast to the crustaceans, probably due to rapid hydrolysis and/or rapid excretion. Reduction of the nitro group to amino group is specifically observed in mollusca. (iv) Excretion offenitrothion. Based on the recovery ratio of fenitrothion (RR), fenitrothion itself is excreted directly into fresh water, due to the higher RR values of 0.52-0.98’ except in the mullet (0.18-0.47) and shrimp (0.20) as shown in Table 2. The values are related to developmental stages of killifish, and this indicates that as the stage developed, the metabolism proceeded more rapidly.
F Fishes M Mollusca
FIG. 4. Proposed metabolic pathways for fenitrothion in aquatic organisms.
134
TAKIMOTO,
OHSHIMA.
AND
MIYAMOTO
CONCLUSIONS From the above comparative metabolism studies, it can be concluded that metabolism is dependent on phyla systematics and generally differences among species in the same phylum are relatively small. Therefore, this information will afford a sound basis for the selection of the organisms in ecotoxicology testings, and if combined with appropriate model ecosystems (Miyamoto et al., 1985), the environmental impact of chemicals, including their eventual fate, can be assessed fairly accurately during laboratory trials. REFERENCES BANTA, A. M. (1921). A convenient culture medium for daphnidaes. Science53,557-558. BEND, J. R., JAMES, M. 0.. LITTLE, P. J., AND FOUREMAN, G. L. (198 1). In vitro and in viva metabolism of benzo[a]pyrene by selected marine crustacean species. In Phyletic Approaches to Cancer (C. J. Daive. J. C. Harshbarger, S. Kondo, T. Sugimura, and S. Takayama. Eds.), pp. 179-194. Japan Sci. Sot. Press, Tokyo. CARLSON, G. P. (1973). Comparison of the metabolism of parathion by lobsters and rats. Bull. Environ.
Contam. Toxicol. 9,296-300. ELMAMLOUK, T. H., AND GESSNER, T. (1976). Species difference in metabolism of parathion. Apparent inability of hepatopancreas fractions to produce paraoxon. Comp. Biochem. Physiol. C: Comp. Pharmacol. 53, 19-24. FOLCH, J., LEES, M., AND STANLEY, G. H. S. (1957). A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226,497-509. GEYER, H., SCHEUNERT, I., AND KORTE, F. (1985). Relationship between the lipid content of fish and their bioconcentration potential of 1.2.4~trichlorobenzene. Chemosphere 14,545-555. KIKUCHI, R.. YASUTANIYA, T., TAKIMOTO, Y.. AND MIYAMOTO, J. (1984). Accumulation and metabolism of fenitrothion in 3 species ofalgae. J. Pest. Sci. 9,33 l-337. MEYER, T., AND BAKKE, T. (1977). The metabolism of biphenyl. V. Phenolic metabolites in some marine organisms. Acta Pharmacol. Toxicol. 40,20 l-208. MIYAMOTO, J.. KLEIN, W.. TAKIMOTO, Y., AND ROBERTS, T. R. (1985). Critical evaluation of model ecosystems. Pure Appl. Chem., 57, 1523- 1536. MOORE. R., TORO, E., STANTON. M., AND KHAN, M. A. Q. (1977). Absorption and elimination of 14C-aand y-chlordane by a freshwater alga, daphnid. and goldfish. Arch. Environ. Contam. Tosicol. 6,4 I l420. O’BRIEN, R. D. (1960). To.xic Phosphorus Esters, p. 73. Academic Press. New York. REINERT. R. E. (1972). Accumulation ofdieldrin in an alga (Scenede.ymus obhquus). Daphnia mugna, and the guppy (Poecilia reticuluta). J. Fish Res. Board Canad. 29, 14 13- 14 18. TAKIMOTO. Y., AND MIYAMOTO, J. (1976). Studies on Accumulation and metabolism of Sumithion in fish. J. Pest. Sci. 1,26 l-27 1. TAKIMOTO, Y., OHSHIMA, M., AND MIYAMOTO, J. (1987a). Comparative metabolism of fenitrothion in aquatic organisms. I. Metabolism in the euryhaline fish, Oryzias latipes and Mugil cephalus. Ecotoxicol. Environ. .Saf 13, 104-I 17. TAKIMOTO, Y., OHSHIMA. M., AND MIYAMOTO, J. (1985). Fate of fenitrothion in carp, C.vprinus carpio by oral administration and exposure. Submitted for publication. TAIUMOTO, Y., OHSHIMA, M., AND MIYAMOTO, J. (1987b). Comparative metabolism of fenitrothion in aquatic organisms. II. Metabolism in freshwater snails, Cipnngopnludina juponica and Physa Acura. Ecotoxicol. Environ. Saf: 13, 11 E- 125. TAKIMOTO, Y., OHSHIMA, M., YAMADA, H.. AND MIYAMOTO, J. (1984). Fate of fenitrothion in several developmental stages of killifish (Oryzias latipes). Arch. Environ. Contam. Toxicol. 13,579-587. YOSHITAKE, A., KAWAHARA, K., KAMADA, T., AND ENDO. M. (1977). Labelled organophosphorus pesticides. I. Synthesis of carbon 14 labelled O,O-dimethyl 0-(3-methyl-4-nitrophenyl) phosphorothioate (Sumithion@). J. Labelled Compd. 13,323-33 1.