- Email: [email protected]
Comparative metabolism of fenitrothion in aquatic organisms
ECOTOXICOLOCY
AND
Comparative
ENVIRONMENTAL
SAFETY
Metabolism
13,118-l 25 ( 1987)
of Fenitrothion
in Aquatic Organisms
II. Metabolism in the Freshwater Snails, Cipangopaludinajaponica and Physa acuta YOSHIYUKITAKIMOTO,MASAKOOHSHIMA,ANDJUNSHIMIYAMOTO Takarazuka
Research
Center,
Sumitomo
Chemical
Received
Co., Ltd.,
September
I-2-1,
Takarazuka.
Hyogo
665, Japan
2, 1985
When freshwater snails, Cipangopaludina japonica and Physa acuta. were exposed to 0.1 ppm [“‘Clfenitrothion in a dynamic flow system. the concentrations of fenitrothion and “‘C in the body reached equilibrium on Day I of exposure. The maximum bioaccumulation ratios of fenitrothion were 18 and 53 in C. japonica and P. acuta, respectively. These snails metabolized the compound primarily by demethylation, hydrolysis, and reduction. The liberated phenol moiety was found to be conjugated with sulfate in C. japonica and mainly with glucose in P. acuta. When the snails were transferred to a freshwater stream, fenitrothion and its metabolites were rapidly excreted, and the half-life of the parent compound was less than 0.5 day in both snails. Fenitrothion and its decomposition products were mainly distributed in liver ofP. acuta. as evidenced by whole-body radioautography. o 1987 Academic press, I~C.
INTRODUCTION To establish a biochemical background for the use of aquatic organisms in ecotoxicology testings, previous reports have dealt with metabolism studies of fenitrothion in fish including rainbow trout (Salvo gairdneri) (Takimoto and Miyamoto, 1976) killifish (Oryzias latipes) at five different developmental stages (Takimoto et al., 1984), and mullet (Mugil cephalus) (Takimoto et al., 1987a), and it turns out that fish metabolize the compound through hydrolysis, demethylation, oxidation of P=S to P=O, and conjugation ofthe liberated phenol with glucuronic acid. Although only a few data on accumulation of the compound and other organophosphorus pesticides in mollusca are available (McLeese et al., 1979; Kanazawa, 1978) the metabolism has not been elucidated. Therefore, the metabolism study was conducted in two species of freshwater snails, Cipangopaludina japonica and Physa acuta, in which the gill and lung, respectively, are used as the respiration system. 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 activities of the preparations were 20.6 mCi/mmol for C. japonica and 39.1 mCi/mmol for P. acuta. Radiochemical purity of both [‘4C]fenitrothion was more than 99%. The nonradioactive authentic compounds were prepared and used for identification for 14C-labeled compounds (Takimoto et al., 1987a). Limpet arylsulfatase Type III and /3glucosidase Type II were purchased from Sigma Chemical Co. (St. Louis, MO). 0147-6513187 $3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
118
FENITROTHION
METABOLISM
IN
FRESHWATER
SNAILS
119
Snails. Pond snails (C. japonica) were obtained from the Biwa Lake and acclimated to the laboratory conditions at 25 ? 2°C in dechlorinated tap water for at least 2 weeks. Physa snails were bred in our laboratory and acclimated under the same conditions until about 3 months after hatching. The average body weights used in the tests were 2.6 g (5.5 g including shell) and 0.040 g (0.084 g including shell) for the pond snail and Physa snail, respectively. Uptake and elimination study with [‘4Clfenitrothion. Acute toxicity (96-hr LCsO) of fenitrot hion was found to be more than 10 ppm to both snails. The fate of fenitrothion in two species of the snails was examined in a flow system as follows: [‘4C]fenitrothion was dissolved in an aliquot of methanol and diluted with dechlorinated water of pH 7.2-7.8 and a hardness of 60-80 ppm as CaC03 to prepare a 0.1 -ppm fenitrothion aqueous solution. Twenty-nine pond snails in 3 liters and 144 Physa snanls were maintained in 2 liters of the solution, and the fresh 14C solution was passed into a glass aquarium with a micropump (Shibata Kagaku Kiki Co., Tokyo) at the renewal rate of 2.5 times per day. At specified intervals during the exposure period, 14C concentrations were monitored by taking 0.2 ml of the exposure water. After a .3-day exposure, 20 pond snails and 104 Physa snails were transferred to 2 and 1 liter 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 held below 3°C to prevent the degradation of 14C excreta. The overflow water and the aquarium water (recovery water) were mixed and the total volume was measured. 14C concentration was analyzed by taking 1 or 10 ml of the solution. Phvsa snails were kept in a glass vessel which had some holes at the bottom and a glass cove:r with some holes, and the entirety was immersed in a glass beaker to prevent snails from escaping from the solution. Physa snails were allowed to breathe air two times per day. The aquaria were maintained in a water bath controlled at 25 f 0.5”C. During the test period, snails were fed no bait. Extraction of radioactivity from the snail. The snails exposed to [ “C]fenitrothion were rand’omly sampled at specified intervals and processed for solvent extraction as follows: The snails were shucked, excess water was drained off, and then the body weights were measured. The body portion of the snail was cut into small pieces and processed for methanol extraction according to the previous report (Takimoto et al., 1987a). Extraction of radioactivityfrom the exposure water. Extraction of 14C-labeled compounds from 20 ml of water was performed according to the previous report (Takimot0 et al’., 1987a). Extract,ion of radioactive compounds from the recovery water. After determination of radioactivity of the recovery water, an appropriate volume of the water was sampled and extracted (Takimoto et al., 1987a). Analysi,s and measurement of radioactivity. Quantitative and qualitative analyses of radioactive compounds in the organic extracts of snails and water were carried out according to the foregoing study (Takimoto et al., 1987a). 3-Methyl-4-nitrophenyl+?-glucoside was hydrolyzed by incubation with &glucosidase in 0. I: Macetate buffer (pH 5) at 37°C for 5 hr. Whole-body radioautography. After a 3-day exposure and a 6-hr recovery period, Physa snails were taken up and excess water around them was removed by filter
120
TAKIMOTO, 10:
t.
OHSHIMA.
AND MIYAMOTO
C.joponica -~
1..
.i
1 2 3.0 1 2 -Exp.-Rec.-
3 -Exp.*Rec.
FIG. 1. Uptake and elimination of [‘4C]fenitrothion in mollusca. 14C,0; fenitrothion. 0; demethylfenitrothion, A; 3-methyl-4-nitrophenyl sulfate, A; 3-methyl-4-nitrophenyl-&glucoside, n . Exp.: exposure: Rec.: recovery.
paper. Thereafter, whole-body radioautography was conducted according to the foregoing study (Takimoto et al., 1987a). Calculation of bioaccumulation ratio and biological half-life and recovery ratio. Accumulation ratio, half-life, and recovery ratio were calculated according to the previous report (Takimoto et al., 1987a). RESULTS C. japonica During the 3-day exposure, average concentration and standard deviation of fenitrothion and 14C in the water were 73.8 f 10.8 and 93.0 k 9.9 ppb, respectively, by seven analyses, as shown on the left in Fig. 1. Fenitrothion contents were more than 72% of the r4C in the water, and the major decomposition product was 3-methyl-4-nitrophenol, amounting to 7- 10% of the 14C. In addition to the above compounds, demethylfenitrothion (accounting for 1S-4.3% of the 14C), fenitrooxon (0.4-0.9%), demethylfenitrooxon (0.4- 1.2%) aminofenitrothion (0.6-0.9%), and 3-methyl-4-nitrophenyl sulfate (0.3-l. 1%) were detected in the water. In the pond snail, fenitrothion concentration increased to 1.13 ppm on Day I of exposure and was constant during the exposure period, as shown on the left in Fig. 1.
FENITROTHION
METABOLISM
TABLE
IN FRESHWATER
121
SNAILS
1
MAXIPJUM BIOACCUMULATION RATIO(MBR), BIOLOGICALHALF-LIFE(BHL),AND RECOVERYRATIO(RR)OF['~C]FENITROTHION MBR Species Physa acuta Cipangopaludina
japonica
BHL (day)
RR
14C
Fen.
14c
Fen.
Fen.
79 35
53 18
0.75 1.05
0.35 0.40
0.820 0.517
As the concentration of 14Cincreased gradually with time, the content of fenitrothion decreased from 52.4% on Day 1 to 38.9% of the 14C on Day 3 of exposure. Major :metabolites in the snail were demethylfenitrothion and 3-methyl-4-nitrophenyl sulfate, and the concentrations increased gradually with time (the left of Fig. 1). The contents accounted for 19.2-23.2 and 8.1-9.9% of the 14C, respectively. 3-Methyl-4-nitrophenol, aminofenitrothion, demethylaminofenitrothion, and demethylfenitrooxon were also produced and the contents were 3.7-8.2, 2.2-2.3, 1.6-5.5, and 0.2-0.4% of the 14C during the exposure period, respectively. No fenitrooxon was detected (~0.1%). Based on these concentrations in the snail and the water, bioaccumulation ratios were 18 and 35 for fenitrothion and 14C, respectively (Table 1). When the snails were transferred to fresh water after attainment of an equilibrium of fenitrothion, the concentrations of fenitrothion and 14C decreased rapidly, with half-lives of 0.40 and 1.05 days, respectively (Table 1). Analyses of 14Cin the snail and the water showed that 54.9,76.7, and 88.9% of the 14C originally contained in the snail were excreted into the surrounding water on Days 1, 2, and 3 of recovery, respectively. In the 3-day recovery water, the contents of fenitrothion and 3-methyl-4-nitrophenol as major 14C compounds accounted for 20.0 and 20.6%, respectively (the left of Fig. 2). Therelore, 5 1.7% of fenitrothion originally contained in the snail was excreted directly into the recovery water by transference; that is, the recovery ratio was 0.5 17 (Table 1). Besides the above excreted metabolites, demethylfenitrothion (4. I%), demethylfenitrooxon (6.5%) 3-methyl-4-nitrophenyl sulfate (6.9%), fenitrooxon (O.S%), and aminofenitrothion (0.7%) were also found in the water, and the latter two compounds are included as “others” in the column of Fig. 2. Physa ac;mta As Physa snail is an air-breathing animal, the container was lifted above the water surface two times per day. In the exposure water, fenitrothion and 14C concentrations were 92.9 k 6.2 and 101 + 4.9 ppb, respectively, by seven analyses, as shown on the right in Fig. 1. The fenitrothion content in the water was more than 88.8% that of the 14C. During the exposure period, 3-methyl-4-nitrophenol (2.5-5.6% of the i4C), fenitrooxon (0.82.1%), demethylfenitrothion (0.2-0.9%), demethylfenitrooxon (0.4- 1.3%), and aminofenitrothion (0.2-0.8%) were detected.
122
TAKIMOTO,
OHSHIMA,
AND MIYAMOTO
FIG. 2. Distribution of “‘C during recovery period. 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&lucoside.
In Physa snails, fenitrothion concentration reached equilibrium on Day 1 of exposure (4.53 ppm), as shown on the right in Fig. 1, and its content amounted to 6 1.566.0% of the i4C. As major metabolites, demethylfenitrothion and 3-methyl-4-nitrophenyl+glucoside accounted for 18.3-2 1.4 and 5.3-6.8% of the 14C, and their concentrations were also constant during the exposure period (the right of Fig. 1). Other metabolites detected were 3-methyl-4-nitrophenol ( 1.7-3.0% of the 14C), its sulfate (0.3-0.9%) fenitrooxon (0.3-0.4%), demethylfenitrooxon (0.3-0.4%) aminofenitrothion (0.5-l .4%), and demethylaminofenitrothion (0.2-0.8%). Maximum bioaccumulation ratios were 53 and 79 for fenitrothion and 14C,respectively (Table 1). When the animals were transferred to a freshwater stream, fenitrothion and its metabolites diminished from the snails with time (the right of Fig. 1). The half-lives of fenitrothion and 14Cin the Physa snail were 0.35 and 0.75 day, respectively (Table 1). During the 6- and 24-hr recovery periods, 65.4 and 87.5% of the 14C contained in the snail were excreted into the recovery water, respectively. In the 24-hr recovery water, the contents of fenitrothion and 3-methyl-4-nitrophenol amounted to 49.7 and 14.7% as major 14C compounds (the right of Fig. 2). This result showed that the recovery ratio of fenitrothion was 0.820 (Table 1). Other excreted compounds were
FENITROTHION
METABOLISM
IN FRESHWATER
SNAILS
123
fenitrooxon (3.4%), aminofenitrothion ( 1. l%), demethylfenitrothion (2.3%), demethylfenitrooxon (3.5%), demethylaminofenitrothion (0.2%), 3-methyl-4-nitrophenyl sulfate (0.3%), and 3-methyl-4-nitrophenyl-P-glucoside (0.4%). Distribution
of 14C in Physa acuta
To clarify the distribution of 14C-labeled compounds in the Physa snail, a wholebody radioautographic technique was employed. As shown in Fig. 3, almost all the 14C was located in the liver and some of 14C was detected in mantle after the 3-day exposure. IDuring the 6-hr recovery period, some of the 14C remained in the liver and most of the radioactivity disappeared from the other tissues. DISCUSSION Few data on accumulation and metabolism of pesticides in mollusca are available. McLeese et al. ( 1979) reported that accumulation ratios of fenitrothion were independent of the exposure levels, being 19-35,78- 130, and 9 in marine clam (Mya arenaria), mussel (Mytilus edutis), and freshwater clam (Anodonta cataractae), respectively. From the present study, maximum accumulation ratios of fenitrothion in the pond snail and Physa snail were 18 and 53, respectively, and these values are in accordancle with the above results. However, the chemical uptake mechanism is considered to be clearly different; in the pond snail, like fish, the uptake is through the gill, while in the Physa snail uptake may be through the body surface, due to lack of gill filaments. Kanazawa (1978) also observed a higher ratio in red snail (Indoplanorbis exustus), an air-breathing animal, than in pond snail (Cipangopaludina malleata), with average bioaccumulation ratios of diazinon of 17.0 and 5.9, respectively. The accumulation ratio is, rather, related more closely to the fat content of the snails (0.55% in the pond snail and 1.84% in Physa snail) than to the respiration system. Because snails have low fat contents, they usually show a lower accumulation potential than do fish species, as observed with fenitrothion (Takimoto et al., 1984) and diazinon (Kanazawa, 1978). The 14C absorbed in the Physa snail was located mainly in the liver, and some was detected in the mantle, but was not found in the intestine. These findings are different from fish in which 14Cderived from fenitrothion was located primarily in gall bladder and intestine (Takimoto and Miyamoto, 1976; Takimoto et al., 1987a), indicating enterohepatic circulation as with other chemicals (Lech and Bend, 1980). When transferred to fresh water, an appreciable amount of 14C remained in the liver, and 14C concentration decreased with the biological half-lives of 0.8- 1.1 days. These values are similar to those in the killifish (1.2-1.3 days without feeding) (Takimoto et al., 1984). Furthermore, the half-life (0.4 day) of fentitrothion in the snails was comparable to that in the fish (0.3-0.4 day) (Takimoto et al.. 1984, 1987a). Recovery ratios of fenitrothion were 0.52 and 0.82 from the pond snail and Physa snail, respectively. These values were comparable to those obtained from the killifish (0.58-0.95) (Takimoto et al., 1984, 1987a) and higher than the mullet (0.1 g-0.47) (Takimoto et al., 1987a). In both snails fenitrothion is metabolized primarily through demethylation of P-0-alkyl and hydrolysis of P-0-aryl linkage, but the conjugation system of the phenol was species-different; the pond snail produced sulfate, but the Physa snail formed mainly glucoside together with minor amounts of sulfate. The contents of the de-
124
TAKIMOTO,
OHSHIMA.
section
AND
MIYAMOTO
distribution
of 14C
distribution
of 14C
3 day exposure
section
6 hr in fresh water after 3 days exposure FIG.
3. Radioautograms
of Physa acuta exposed
to [ ‘%]fenitrothion.
methylated metabolites (sum of demethylfenitrothion, -aminofenitrothion, and -fenitrooxon) were about 27 and 23% in the pond snail and Physa snail at maximum, respectively, and the values were much higher than in the fish: 1% in rainbow trout
FENITROTHION
METABOLISM
IN FRESHWATER
SNAILS
125
(S&W guirdneri) (Takimoto and Miyamoto, 1976) and 3-6% from the postlarva to adult stages of killifish. However, the contents were comparable to those obtained from the yolk sac fry (28%) of killifish (Takimoto et al., 1984) at maximum and mullet (70%) (Takimoto et al., 1987a). Hydrolytic metabolism of fenitrothion is common in aquatic organisms such as fish (Takimoto and Miyamoto, 1976; Takimoto et al., 1984, 1987a), algae (Kikuchi et al., 1984), and crustaceans (Takimoto et al., 1987b). Unique to the mollusk is reduction of a nitro group to an amino derivative, the content being about 2%. Although the oxon analogs were detected in very low amounts (lower than 1%) in the body, they were found in the recovery water from both snails at about 7% of the 14C originally contained in the snails. Therefore, snails have excretory oxidation activity, but the compounds are not retained in the body. CONCLUSION These results demonstrate that because of their low fat contents, mollusca accumulate relatively low amounts of foreign compounds and metabolize them through demethylation, hydrolysis, reduction, and oxidation. REFERENCES KANAZAWA, J. (1978). Bioconcentration ratio of diazinon by freshwater fish and snail. Bull. Environ. Contam. Toxicol. 20,6 13-6 17. KIKUCHI, R.. YASUTANIYA T., TAKIMOTO, Y., AND MIYAMOTO, J. (1984). Accumulation and metabolism of fenitrothion in 3 species of algae. J. Pest. Sci. 9,33 l-337. LECH, J. J., AND BEND, J. R. (1980). Relationship between biotransformation and the toxicity and fate of xenobiotic chemicals in fish. Environ. Health Perspect. 34, 115- I3 1. MCLEESE, D. W., ZITKO. V., AND SERGEANT, D. B. (1979). Uptake and excretion offenitrothion by clams and mussels. Bull. Environ. Contam. Toxicol. 22,800-806. TAKIMOTO, Y., AND MIYAMOTO, J. (1976). Studies on Accumulation and metabolism of Sumithion in fish. J. Pest. Sci. 1,26 I-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. EcotrMcol. Environ. Saf 13, 104-l 17. TAKIMOTO, Y., OHSHIMA, M., AND MIYAMOTO, J. (198713). Comparative metabolism of fenitrothion in aquatic organisms. III. Metabolism in the crustaceans, Daphniapulexand Palaemonpaucidens. Ecotoricol. Environ. Saf 13, 126- 134. TAKIMOTO, Y., OHSHIMA, M., YAMADA, H., AND MIYAMOTO, J. (1984). Fate of fenitrothion in several developmental stages of kiliifish (Oryzias t’atipes). Arch. Environ. Contam. To.uicoi. 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 I.
AND
Comparative
ENVIRONMENTAL
SAFETY
Metabolism
13,118-l 25 ( 1987)
of Fenitrothion
in Aquatic Organisms
II. Metabolism in the Freshwater Snails, Cipangopaludinajaponica and Physa acuta YOSHIYUKITAKIMOTO,MASAKOOHSHIMA,ANDJUNSHIMIYAMOTO Takarazuka
Research
Center,
Sumitomo
Chemical
Received
Co., Ltd.,
September
I-2-1,
Takarazuka.
Hyogo
665, Japan
2, 1985
When freshwater snails, Cipangopaludina japonica and Physa acuta. were exposed to 0.1 ppm [“‘Clfenitrothion in a dynamic flow system. the concentrations of fenitrothion and “‘C in the body reached equilibrium on Day I of exposure. The maximum bioaccumulation ratios of fenitrothion were 18 and 53 in C. japonica and P. acuta, respectively. These snails metabolized the compound primarily by demethylation, hydrolysis, and reduction. The liberated phenol moiety was found to be conjugated with sulfate in C. japonica and mainly with glucose in P. acuta. When the snails were transferred to a freshwater stream, fenitrothion and its metabolites were rapidly excreted, and the half-life of the parent compound was less than 0.5 day in both snails. Fenitrothion and its decomposition products were mainly distributed in liver ofP. acuta. as evidenced by whole-body radioautography. o 1987 Academic press, I~C.
INTRODUCTION To establish a biochemical background for the use of aquatic organisms in ecotoxicology testings, previous reports have dealt with metabolism studies of fenitrothion in fish including rainbow trout (Salvo gairdneri) (Takimoto and Miyamoto, 1976) killifish (Oryzias latipes) at five different developmental stages (Takimoto et al., 1984), and mullet (Mugil cephalus) (Takimoto et al., 1987a), and it turns out that fish metabolize the compound through hydrolysis, demethylation, oxidation of P=S to P=O, and conjugation ofthe liberated phenol with glucuronic acid. Although only a few data on accumulation of the compound and other organophosphorus pesticides in mollusca are available (McLeese et al., 1979; Kanazawa, 1978) the metabolism has not been elucidated. Therefore, the metabolism study was conducted in two species of freshwater snails, Cipangopaludina japonica and Physa acuta, in which the gill and lung, respectively, are used as the respiration system. 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 activities of the preparations were 20.6 mCi/mmol for C. japonica and 39.1 mCi/mmol for P. acuta. Radiochemical purity of both [‘4C]fenitrothion was more than 99%. The nonradioactive authentic compounds were prepared and used for identification for 14C-labeled compounds (Takimoto et al., 1987a). Limpet arylsulfatase Type III and /3glucosidase Type II were purchased from Sigma Chemical Co. (St. Louis, MO). 0147-6513187 $3.00 Copyright 0 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
118
FENITROTHION
METABOLISM
IN
FRESHWATER
SNAILS
119
Snails. Pond snails (C. japonica) were obtained from the Biwa Lake and acclimated to the laboratory conditions at 25 ? 2°C in dechlorinated tap water for at least 2 weeks. Physa snails were bred in our laboratory and acclimated under the same conditions until about 3 months after hatching. The average body weights used in the tests were 2.6 g (5.5 g including shell) and 0.040 g (0.084 g including shell) for the pond snail and Physa snail, respectively. Uptake and elimination study with [‘4Clfenitrothion. Acute toxicity (96-hr LCsO) of fenitrot hion was found to be more than 10 ppm to both snails. The fate of fenitrothion in two species of the snails was examined in a flow system as follows: [‘4C]fenitrothion was dissolved in an aliquot of methanol and diluted with dechlorinated water of pH 7.2-7.8 and a hardness of 60-80 ppm as CaC03 to prepare a 0.1 -ppm fenitrothion aqueous solution. Twenty-nine pond snails in 3 liters and 144 Physa snanls were maintained in 2 liters of the solution, and the fresh 14C solution was passed into a glass aquarium with a micropump (Shibata Kagaku Kiki Co., Tokyo) at the renewal rate of 2.5 times per day. At specified intervals during the exposure period, 14C concentrations were monitored by taking 0.2 ml of the exposure water. After a .3-day exposure, 20 pond snails and 104 Physa snails were transferred to 2 and 1 liter 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 held below 3°C to prevent the degradation of 14C excreta. The overflow water and the aquarium water (recovery water) were mixed and the total volume was measured. 14C concentration was analyzed by taking 1 or 10 ml of the solution. Phvsa snails were kept in a glass vessel which had some holes at the bottom and a glass cove:r with some holes, and the entirety was immersed in a glass beaker to prevent snails from escaping from the solution. Physa snails were allowed to breathe air two times per day. The aquaria were maintained in a water bath controlled at 25 f 0.5”C. During the test period, snails were fed no bait. Extraction of radioactivity from the snail. The snails exposed to [ “C]fenitrothion were rand’omly sampled at specified intervals and processed for solvent extraction as follows: The snails were shucked, excess water was drained off, and then the body weights were measured. The body portion of the snail was cut into small pieces and processed for methanol extraction according to the previous report (Takimoto et al., 1987a). Extraction of radioactivityfrom the exposure water. Extraction of 14C-labeled compounds from 20 ml of water was performed according to the previous report (Takimot0 et al’., 1987a). Extract,ion of radioactive compounds from the recovery water. After determination of radioactivity of the recovery water, an appropriate volume of the water was sampled and extracted (Takimoto et al., 1987a). Analysi,s and measurement of radioactivity. Quantitative and qualitative analyses of radioactive compounds in the organic extracts of snails and water were carried out according to the foregoing study (Takimoto et al., 1987a). 3-Methyl-4-nitrophenyl+?-glucoside was hydrolyzed by incubation with &glucosidase in 0. I: Macetate buffer (pH 5) at 37°C for 5 hr. Whole-body radioautography. After a 3-day exposure and a 6-hr recovery period, Physa snails were taken up and excess water around them was removed by filter
120
TAKIMOTO, 10:
t.
OHSHIMA.
AND MIYAMOTO
C.joponica -~
1..
.i
1 2 3.0 1 2 -Exp.-Rec.-
3 -Exp.*Rec.
FIG. 1. Uptake and elimination of [‘4C]fenitrothion in mollusca. 14C,0; fenitrothion. 0; demethylfenitrothion, A; 3-methyl-4-nitrophenyl sulfate, A; 3-methyl-4-nitrophenyl-&glucoside, n . Exp.: exposure: Rec.: recovery.
paper. Thereafter, whole-body radioautography was conducted according to the foregoing study (Takimoto et al., 1987a). Calculation of bioaccumulation ratio and biological half-life and recovery ratio. Accumulation ratio, half-life, and recovery ratio were calculated according to the previous report (Takimoto et al., 1987a). RESULTS C. japonica During the 3-day exposure, average concentration and standard deviation of fenitrothion and 14C in the water were 73.8 f 10.8 and 93.0 k 9.9 ppb, respectively, by seven analyses, as shown on the left in Fig. 1. Fenitrothion contents were more than 72% of the r4C in the water, and the major decomposition product was 3-methyl-4-nitrophenol, amounting to 7- 10% of the 14C. In addition to the above compounds, demethylfenitrothion (accounting for 1S-4.3% of the 14C), fenitrooxon (0.4-0.9%), demethylfenitrooxon (0.4- 1.2%) aminofenitrothion (0.6-0.9%), and 3-methyl-4-nitrophenyl sulfate (0.3-l. 1%) were detected in the water. In the pond snail, fenitrothion concentration increased to 1.13 ppm on Day I of exposure and was constant during the exposure period, as shown on the left in Fig. 1.
FENITROTHION
METABOLISM
TABLE
IN FRESHWATER
121
SNAILS
1
MAXIPJUM BIOACCUMULATION RATIO(MBR), BIOLOGICALHALF-LIFE(BHL),AND RECOVERYRATIO(RR)OF['~C]FENITROTHION MBR Species Physa acuta Cipangopaludina
japonica
BHL (day)
RR
14C
Fen.
14c
Fen.
Fen.
79 35
53 18
0.75 1.05
0.35 0.40
0.820 0.517
As the concentration of 14Cincreased gradually with time, the content of fenitrothion decreased from 52.4% on Day 1 to 38.9% of the 14C on Day 3 of exposure. Major :metabolites in the snail were demethylfenitrothion and 3-methyl-4-nitrophenyl sulfate, and the concentrations increased gradually with time (the left of Fig. 1). The contents accounted for 19.2-23.2 and 8.1-9.9% of the 14C, respectively. 3-Methyl-4-nitrophenol, aminofenitrothion, demethylaminofenitrothion, and demethylfenitrooxon were also produced and the contents were 3.7-8.2, 2.2-2.3, 1.6-5.5, and 0.2-0.4% of the 14C during the exposure period, respectively. No fenitrooxon was detected (~0.1%). Based on these concentrations in the snail and the water, bioaccumulation ratios were 18 and 35 for fenitrothion and 14C, respectively (Table 1). When the snails were transferred to fresh water after attainment of an equilibrium of fenitrothion, the concentrations of fenitrothion and 14C decreased rapidly, with half-lives of 0.40 and 1.05 days, respectively (Table 1). Analyses of 14Cin the snail and the water showed that 54.9,76.7, and 88.9% of the 14C originally contained in the snail were excreted into the surrounding water on Days 1, 2, and 3 of recovery, respectively. In the 3-day recovery water, the contents of fenitrothion and 3-methyl-4-nitrophenol as major 14C compounds accounted for 20.0 and 20.6%, respectively (the left of Fig. 2). Therelore, 5 1.7% of fenitrothion originally contained in the snail was excreted directly into the recovery water by transference; that is, the recovery ratio was 0.5 17 (Table 1). Besides the above excreted metabolites, demethylfenitrothion (4. I%), demethylfenitrooxon (6.5%) 3-methyl-4-nitrophenyl sulfate (6.9%), fenitrooxon (O.S%), and aminofenitrothion (0.7%) were also found in the water, and the latter two compounds are included as “others” in the column of Fig. 2. Physa ac;mta As Physa snail is an air-breathing animal, the container was lifted above the water surface two times per day. In the exposure water, fenitrothion and 14C concentrations were 92.9 k 6.2 and 101 + 4.9 ppb, respectively, by seven analyses, as shown on the right in Fig. 1. The fenitrothion content in the water was more than 88.8% that of the 14C. During the exposure period, 3-methyl-4-nitrophenol (2.5-5.6% of the i4C), fenitrooxon (0.82.1%), demethylfenitrothion (0.2-0.9%), demethylfenitrooxon (0.4- 1.3%), and aminofenitrothion (0.2-0.8%) were detected.
122
TAKIMOTO,
OHSHIMA,
AND MIYAMOTO
FIG. 2. Distribution of “‘C during recovery period. 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&lucoside.
In Physa snails, fenitrothion concentration reached equilibrium on Day 1 of exposure (4.53 ppm), as shown on the right in Fig. 1, and its content amounted to 6 1.566.0% of the i4C. As major metabolites, demethylfenitrothion and 3-methyl-4-nitrophenyl+glucoside accounted for 18.3-2 1.4 and 5.3-6.8% of the 14C, and their concentrations were also constant during the exposure period (the right of Fig. 1). Other metabolites detected were 3-methyl-4-nitrophenol ( 1.7-3.0% of the 14C), its sulfate (0.3-0.9%) fenitrooxon (0.3-0.4%), demethylfenitrooxon (0.3-0.4%) aminofenitrothion (0.5-l .4%), and demethylaminofenitrothion (0.2-0.8%). Maximum bioaccumulation ratios were 53 and 79 for fenitrothion and 14C,respectively (Table 1). When the animals were transferred to a freshwater stream, fenitrothion and its metabolites diminished from the snails with time (the right of Fig. 1). The half-lives of fenitrothion and 14Cin the Physa snail were 0.35 and 0.75 day, respectively (Table 1). During the 6- and 24-hr recovery periods, 65.4 and 87.5% of the 14C contained in the snail were excreted into the recovery water, respectively. In the 24-hr recovery water, the contents of fenitrothion and 3-methyl-4-nitrophenol amounted to 49.7 and 14.7% as major 14C compounds (the right of Fig. 2). This result showed that the recovery ratio of fenitrothion was 0.820 (Table 1). Other excreted compounds were
FENITROTHION
METABOLISM
IN FRESHWATER
SNAILS
123
fenitrooxon (3.4%), aminofenitrothion ( 1. l%), demethylfenitrothion (2.3%), demethylfenitrooxon (3.5%), demethylaminofenitrothion (0.2%), 3-methyl-4-nitrophenyl sulfate (0.3%), and 3-methyl-4-nitrophenyl-P-glucoside (0.4%). Distribution
of 14C in Physa acuta
To clarify the distribution of 14C-labeled compounds in the Physa snail, a wholebody radioautographic technique was employed. As shown in Fig. 3, almost all the 14C was located in the liver and some of 14C was detected in mantle after the 3-day exposure. IDuring the 6-hr recovery period, some of the 14C remained in the liver and most of the radioactivity disappeared from the other tissues. DISCUSSION Few data on accumulation and metabolism of pesticides in mollusca are available. McLeese et al. ( 1979) reported that accumulation ratios of fenitrothion were independent of the exposure levels, being 19-35,78- 130, and 9 in marine clam (Mya arenaria), mussel (Mytilus edutis), and freshwater clam (Anodonta cataractae), respectively. From the present study, maximum accumulation ratios of fenitrothion in the pond snail and Physa snail were 18 and 53, respectively, and these values are in accordancle with the above results. However, the chemical uptake mechanism is considered to be clearly different; in the pond snail, like fish, the uptake is through the gill, while in the Physa snail uptake may be through the body surface, due to lack of gill filaments. Kanazawa (1978) also observed a higher ratio in red snail (Indoplanorbis exustus), an air-breathing animal, than in pond snail (Cipangopaludina malleata), with average bioaccumulation ratios of diazinon of 17.0 and 5.9, respectively. The accumulation ratio is, rather, related more closely to the fat content of the snails (0.55% in the pond snail and 1.84% in Physa snail) than to the respiration system. Because snails have low fat contents, they usually show a lower accumulation potential than do fish species, as observed with fenitrothion (Takimoto et al., 1984) and diazinon (Kanazawa, 1978). The 14C absorbed in the Physa snail was located mainly in the liver, and some was detected in the mantle, but was not found in the intestine. These findings are different from fish in which 14Cderived from fenitrothion was located primarily in gall bladder and intestine (Takimoto and Miyamoto, 1976; Takimoto et al., 1987a), indicating enterohepatic circulation as with other chemicals (Lech and Bend, 1980). When transferred to fresh water, an appreciable amount of 14C remained in the liver, and 14C concentration decreased with the biological half-lives of 0.8- 1.1 days. These values are similar to those in the killifish (1.2-1.3 days without feeding) (Takimoto et al., 1984). Furthermore, the half-life (0.4 day) of fentitrothion in the snails was comparable to that in the fish (0.3-0.4 day) (Takimoto et al.. 1984, 1987a). Recovery ratios of fenitrothion were 0.52 and 0.82 from the pond snail and Physa snail, respectively. These values were comparable to those obtained from the killifish (0.58-0.95) (Takimoto et al., 1984, 1987a) and higher than the mullet (0.1 g-0.47) (Takimoto et al., 1987a). In both snails fenitrothion is metabolized primarily through demethylation of P-0-alkyl and hydrolysis of P-0-aryl linkage, but the conjugation system of the phenol was species-different; the pond snail produced sulfate, but the Physa snail formed mainly glucoside together with minor amounts of sulfate. The contents of the de-
124
TAKIMOTO,
OHSHIMA.
section
AND
MIYAMOTO
distribution
of 14C
distribution
of 14C
3 day exposure
section
6 hr in fresh water after 3 days exposure FIG.
3. Radioautograms
of Physa acuta exposed
to [ ‘%]fenitrothion.
methylated metabolites (sum of demethylfenitrothion, -aminofenitrothion, and -fenitrooxon) were about 27 and 23% in the pond snail and Physa snail at maximum, respectively, and the values were much higher than in the fish: 1% in rainbow trout
FENITROTHION
METABOLISM
IN FRESHWATER
SNAILS
125
(S&W guirdneri) (Takimoto and Miyamoto, 1976) and 3-6% from the postlarva to adult stages of killifish. However, the contents were comparable to those obtained from the yolk sac fry (28%) of killifish (Takimoto et al., 1984) at maximum and mullet (70%) (Takimoto et al., 1987a). Hydrolytic metabolism of fenitrothion is common in aquatic organisms such as fish (Takimoto and Miyamoto, 1976; Takimoto et al., 1984, 1987a), algae (Kikuchi et al., 1984), and crustaceans (Takimoto et al., 1987b). Unique to the mollusk is reduction of a nitro group to an amino derivative, the content being about 2%. Although the oxon analogs were detected in very low amounts (lower than 1%) in the body, they were found in the recovery water from both snails at about 7% of the 14C originally contained in the snails. Therefore, snails have excretory oxidation activity, but the compounds are not retained in the body. CONCLUSION These results demonstrate that because of their low fat contents, mollusca accumulate relatively low amounts of foreign compounds and metabolize them through demethylation, hydrolysis, reduction, and oxidation. REFERENCES KANAZAWA, J. (1978). Bioconcentration ratio of diazinon by freshwater fish and snail. Bull. Environ. Contam. Toxicol. 20,6 13-6 17. KIKUCHI, R.. YASUTANIYA T., TAKIMOTO, Y., AND MIYAMOTO, J. (1984). Accumulation and metabolism of fenitrothion in 3 species of algae. J. Pest. Sci. 9,33 l-337. LECH, J. J., AND BEND, J. R. (1980). Relationship between biotransformation and the toxicity and fate of xenobiotic chemicals in fish. Environ. Health Perspect. 34, 115- I3 1. MCLEESE, D. W., ZITKO. V., AND SERGEANT, D. B. (1979). Uptake and excretion offenitrothion by clams and mussels. Bull. Environ. Contam. Toxicol. 22,800-806. TAKIMOTO, Y., AND MIYAMOTO, J. (1976). Studies on Accumulation and metabolism of Sumithion in fish. J. Pest. Sci. 1,26 I-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. EcotrMcol. Environ. Saf 13, 104-l 17. TAKIMOTO, Y., OHSHIMA, M., AND MIYAMOTO, J. (198713). Comparative metabolism of fenitrothion in aquatic organisms. III. Metabolism in the crustaceans, Daphniapulexand Palaemonpaucidens. Ecotoricol. Environ. Saf 13, 126- 134. TAKIMOTO, Y., OHSHIMA, M., YAMADA, H., AND MIYAMOTO, J. (1984). Fate of fenitrothion in several developmental stages of kiliifish (Oryzias t’atipes). Arch. Environ. Contam. To.uicoi. 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 I.