THE TOXICITY OF PARATHION TO ORCONECTES RUSTICUS AND VIVIPARUS MALLEATUS

Parathion is an organophosphate pesticide used in great quantities in the United States and around the world. The mechanism of toxicity for parathion in mammals has been attributed to its enzymatic desulfuration to its oxygen analog paraoxon which subsequently fonns a covalent bond with acetylcholinesterase (AChE), inhibiting the activity of that enzyme and precipitating cholinergic toxicity. The mechanism by which parathion produces its toxic effects in insects has not been completely determined, but it is believed to be due to the same mechanism. The effect parathion exposure has on fresh water invertebrates has not been investigated to any great extent, and a goal of this project was to determine the effects of parathion exposure and the relationship of this toxicity to the metabolism of parathion by the fresh water snail Viviparus malleatus and the crayfish Orconectes rusticus. The determination of the toxicity of parathion in Orconectes and Viviparus was made by exposing the organisms to different concentrations of parathion or by the direct injection of parathion. The possibility that any toxicity exhibited by parathion was produced by paraoxon was determined by observing the effect the oxygen analog of parathion would have when exposed to the species, and determining the metabolism of parath ion by the two species . The metabolism of parathion was determined in vitro and ..iI!. vivo. The efficient and specific separation and identification of parathion and its metabolites were accomplished through the use of thin layer chromatography. Parathion has been shown to be metabolized by different species by a multitude of pathways. Three of the important pathways produce £_-nitrophenol as an end product. A spectrophotometric assay was used to measure the in vitro production of £_-nitrophenol from homogenates of crayfish and snail tissues incubated with parathion. The excretion of parathion metabolites by Orconectes and Viviparus was determined by identifying the compounds extracted from water samples that had contained crayfish or snails exposed to parathion. The accumulation of parathion or metabolites in the species was determined by extracting the parathion exposed tissues of crayffsh or snails and identifying and quantifying the parathion and metabolites present. The excretation and accumulation experiments were accomplished with the use of 14c labeled parathion which was labeled either in the ring or ethyl position. The possible metabolism of parathion was also investigated by the direct determination of the formation of paraoxon, £_-nitrophenol, diethyl phosphate and diethyl phosphorothionate by the homogenates of crayfish or snail tissues incubated with parathion and appropriate cofactors. This determination was also aided by the use of labeled parathion. The snail Viviparus malleatus did not metabolize parathion or

. 10 The organophosphates are a very large class of compounds as there are over 50,000 compounds in that category today. Originally developed in Germany as nerve gases, the most important ones being tabun, sarin, and soman, they have since been found to be of great use to mankind as pesticides.
Parathion (0,0-diethyl 0-Q_-nitrophenyl phosphorothionate) was introduced in 1944 by Gerhard Schrader (Neal 1971), it is an ester of the acid (H0) 3 P=S with two molecules of ethanol and one molecule of the weakly acidic alcohol Q_-nitrophenol.
Unquestionably the organophosphates elicit their pharmacological activity in vertebrates predominatly via an inhibition of the activity of esterase enzymes, especially acetyl cholinesterase (AChE) (EC 3.3.3.7) (Neal 1971). The extent to which enzyme inhibition is the mechanism of toxicity to other susceptible species is still being investigated, and it is not established that AChE inhibition is the 1 2 only factor of its toxicity~ O' Brian (1967) and Matsumura (1975).
Parathion requires enzymatic activiation to its oxygen analog to exhibit significant anti-esterase activity. This is supported by the work of Alary and Brodeur (1970), Bartels and Nachmansohn (1965), Neal (1971), Ptashne and Neal (1972), Roth and Neal (1972). The anti-cholinesterase effect of parathion is, therefore, dependent on the amount of free paraoxon generated. Thus the conversion of parathion to its oxygen analog paraoxon is of great toxicological importance. The relative degree of formation of alternative products, Q_-nitrophenol, paraoxon, diethyl phosphate and diethyl phosphorothionate produced by reactions with parathion are interesting because of their divergent toxicities, environmental contamination potentials. and use as a marker for parathion contamination.
The possible accumulation of parathion and/or its metabolites in species has been investigated. Miller et al. (1966) showed that some fish and the freshwater mussel accumulate parathion. No accumulation of parathion in crayfish or lobster tissues has been reported and Yu and Sanborn (1975) could not show any accumulation of parathion in the snail used in their investigations.
Parathion has been shown to be toxic to many different species of animals. However, those species differ in their degree of susceptibility . The specifics of parathion toxicity in each individual species is considered in the literature review section as parathion has been shown to be toxic in the hamster, guinea pig~ mouse, rat, rabbit, cow, dog, pig, cat (Whitehouse and Ecobichon 1975) sunfish (Benke and M urphy 1974) mosquito fish (Chambers and Yarbough 1973) fathead minnow (Faust 1964) pink shrimp (Coppage and Mathews 1974) and lobster (Carlson 1973). 3 Not only is there a difference in species susceptibility to parathion but the age and sex of the animal also plays a role in the susceptibility of the species (Neal and Du Bois 1965) . The factor relating the differential toxicities produced by parathion exposure is the differential rate of metabolism of parathion to toxic or nontoxic compounds and the detoxification of the toxic metabolite paraoxon (Benke and Murphy 1975).
The chemical reactions and metabolism of this dialkyl phosphoric acid triester, parathion, are thus the essence of its toxicological importance. The two most important of the chemical reactions of parathion are hydrolysis and isomeriztion .
Little research into the toxicity and metabolism of parathion in crayfish and snails has been done. However, some interesting data have been gathered. Albaugh (1972) and Muncy and Oliver (1963) both have shown that crayfish are very sensitive to parathion exposure , perishing at concentrations below 1 ppm. However, when Carlson (1973 ) and Elmamlouk and Gessner (1976)  nitrophenol moiety . Acid environments would lead to a rupture of the OCH2CH3 moiety as the initial step (Faust et al. 5 6 1972). The hydrolysis of parathion or paraoxon is dependent on pH and temperature, and the hydrolysis of both compounds is faster at higher pH and temperature  However, aging of the compound occurs within hours when one of the alkyl groups is cleaved off the complex. The resulting aged organophosphate enzyme complex is stable and the only source of viable enzyme after aging is new synthesis . When sufficient AChE is inhi.bited by this mechanism the animal will exhibit signs of cholinergic toxicity as endogenously released ACh is not inactivated.

Metabolism 8
The metabolism of parathion is the essence of its toxicological importance, as it is the conversion of parathion to its oxygen analog paraoxon that produces the toxic AChE inhibitor.
The details of the pathways by which parathion is metabolized have been researched extensively in the past decade. Figure 1 has been produced by combining all the routes of metabolism referred to in this section of the review. Neal and DuBois (1965) reported that parathion was more toxic to female rats than male rats but that the highly toxic desulfurated metabolite of parathion, paraoxon did not exhibit this sex difference. The conversion of parathion to paraoxon by desulfuration was then tested in vitro in livers of both male and female rats, and it was discovered that male rats converted parathion to paraoxon faster than females did. These data set the stage for the future work on parathion metabolism by establishing that not only must parathion be metabolized to paraoxon to exhibit toxic effects in mammals, but there must also be some kind of detoxification mechanism for both parathion and paraoxon.
The experiments of Neal (1967) showed that the metabolism 9 of parathion by rat liver microsomes was typical of a mixed function oxidase (MFO) system as inhibitors of MFO microsomal system~ reduced the metabolism of parathion. Neal (1967) also showed that Q_-chloromercuribenzoate, cu2+ and 8-hydroxyquinoline inhibited the conversion of parathion to paraoxon more than the conversion of parathion to nontoxic diethyl phosphorothionate and Q_-nitrophenol.
This type of metabolism was also stimulated by reduced sulfur, EDTA, and ca2+. These results led Neal to the conclusion that the metabolism of parathion to both paraoxon and diethyl phosphorothionate was being catalyzed by an enzyme or enzymes in the liver microsomes by a MFO system.
Experiments by Neal (1971) established that reduced nicotinamide adenine dinucleotide phosphate (NADPH) and o 2 were needed for the metabolism of parathion to the oxygen analog paraoxon (pathway 1, figure 1) or to diethyl phosphorothionate and £_-nitrophenol (pathway 3, figure 1). and that this metabolism was inhibited by carbon monoxide. These results further strengthened the contention that parathion was metabolized to these substances by a MFO system associated with cytochrome P-450. The enzymatic mechanism for the metabolism of parathion was also found in lung, kidney, and brain tissues ( N eal 1971), but the greatest enzyme source was liver. Oil + The metabolism of parathion by soluble liver fractions (pathway 1, figure 1) was investigated by Dauterman (1971, Ku andKahm 1973), Lichtenstein et al. (1973), andNeskovic et al. (1973).
The procedure used by Neal and DuBois (1964) to demonstrate the relationship between the MFO and parathion metabolism was to measure the production of .e_-nitrophenol by incubation mixtures of differentially contrifuged liver homogenates with parathion and appropriate cofactors. The production of colored .e_-nitrophenol was measured spectrophotometrically. Neal continued his investigation of parathion metabolism (Neal 1967) evaluating his contention, at that time, that the metabolism of parathion to either paraoxon or diethyl phosphorothionate and Q_-nitrophenol employed two different MFO enzymes. The .e_-nitrophenol determination, assay has been used by many investigators to follow the metabolism of parathion and to show that liver microsomal enzymes of the MFO system are involved with parathion metabolism via these two pathways in the vertebrate. Benke and Murphy (1975) used the .e_-nitrophenol assay in their investigation on the influence of age and sex on the toxicity of parathion and methyl parathion in the rat. Their work also showed that the toxicity of parathion varied depending on the age and sex of the rat. Whitehouse and Ecobichon (1975) reported data analogous to that of many investigators, that there is a great disparity among species concerning the rate of metabolism of parathion.
They found that the desulfurating ability of the hamster, guinea pig, mouse, rat, rabbit, cow, pig, and cat declined in that order. Differential reaction rates shown by Benke and Murphy (1975) for the metabolism of parathion were shown to be the cause of the sex and age toxicity differences. The work also supported the previous finding that liver microsomal MFO was important in the metabolism of parathion. Similarly use of the Q_-nitrophenol method for parathion metabolism detection in vertebrate microsomal MFO P-450 systems was previously done by Ptashne et al. (1971) who showed the same association for MFO catalyzed desulfuration of parathion (pathway l, figure 1) or oxidation to diethyl phosphorothionate plus Q_-nitrophenol (pathway 3, figure 1) and also a dearylation reaction. Hollingworth (1969) demonstrated that the rate of dealkylation of organophosphates (pathway 4, figure 1) was dependent on the R groups of the molecule. He showed that the dealkylation by mouse hepatic enzymes of diethyl organophosphates preceded much faster than the dealkylation of diethyl compounds. He also showed that these 0-dealkylation reactions ..i!!_yivo proceeded to a large extent through alkylation of reduced glutathione. Cramer and Hollingworth (1976) investigated the dearylation reaction of a series of paraoxon analogs by mouse liver homogenates (pathway 2, figure 1) and determined that the reaction was N ADP H independent , and preceeded via an A-esterase. They showed that t he determining factor as to whether the reaction would proceed by a MFO enzyme or by the A-esterase pathway was the specific length of the alkyl side chain. The ethyl side chain of the paraoxon molecule favored the A-esterase reaction.
14 Villeneuve et al. (1970) used the .e_-nitrophenol assay to investigate the MFO role in parathion metabolism by liver microsomes.
They evaluated plasma esterase activity, and liver carboxylesterase (A-esterase) activity to releate the effect of hepatic microsomal enzyme induction and inhibition on altering the toxicity and metabolism of parathion. They used SKF-525A, 3, 4-benzpyrene, DDT and phenobarbital for the pretreatment of the rats. They determined that DDT decreased the acute toxicity of parathion but increased carboxylesterase activity and parathion metabolism while plasma esterase levels were not affected. SKF-525A pretreatment caused an increase in carboxylesterase activity but no change in the LD 50 of parathion, plasma esterase activity or parathion metabolism. Benze[a]pyrene decreased the toxicity of parathion and increased plasma esterase carboxylesterase and metabolism of parathion. Alary and Broudeur (1970) also investigated the association between microsomal metabolism of parathion and toxicity. They determined that the ability of microsomal fractions to metabolize parathion to diethyl phosphoric acid and diethyl phosphorothioinic acid \vas a good index for the estimation of the~ vivo toxicity of parathion in adult rats. They concluded that phenobarbital stimulates only the direct degradation of parathion to diethyl phosphorothioic acid and £_-nitrophenol.
fraction had a high rate of activity for converting paraoxon to 2._-nitrophenol and diethyl phosphate (pathway 2, figure 1). Lichtenstein et al. (1973) also showed that the majority of the paraoxon produced from parathion was from the soluble liver fraction, a basic difference from the work of Neal's group. Lichtenstein, et al. (1973) did not use the E_-nitrophenol spectrophotometric assay technique but employed gas liquid chromatography, thin layer chromatography, colorimetric assays and radioactive parathion and paraoxon to quantify their results. These methods will be discussed in detail in the detection section of the literature review. Whitehouse and Ecobichon (1975) investigated the metabolism of parathion adopting techniques comparable to those of Lichtenstein et al. (1973) analyzing extracted products from incubation mixtures of liver and parathion. However, they also used the spectrophotometric .2_-nitrophenol assay similar to the one previously discussed. Kamataki et al. (1976b) investigated whether parathion oxidation to paraoxon and diethyl phosphorothionate plus E_-nitrophenol was catalyzed by two different MFO enzymes or utilized different cytochrome P-450 systems or a combination of the both. Previous work by Neal (1967) had contended that various inhibitors of hepatic MFO enzymes had a differential effect on the products of parathion metabolism Pretreatment of animals with inducers of hepatic MFO indicated that the metabolism of parathion to paraoxon and diethyl phosphorothionate and Q_-nitrophenol were cata1yzed by separate MFO enzymes (Nea1 1967, Norman et al. 1974. Utilizing a homogenous preparation of rabbit liver cytochrome P-450 Kawataki et al. (1976b) presented evidence that parathion could be metabolized to not only both paraoxon (pathway l, figure   1) and diethyl phosphorothionate plus p-nitrophenol {pathway 3, Glutathione dependent metabolism of parathion has been investigated by Hollingworth (1969), Benke and Murphy (1975), Lichtenstein et al. (1973), and Whitehouse and Ecobichon (1975).
The method used by Hollingworth, and Benke and Murphy employed the soluble fraction of liver homogenate and radioactive (35s) glutathione (GSH). Incubation of this mixture with parathion was followed by the identification of the metabolities by thin layer chromatography and quantification by scintillation counting. Benke and Murphy (1975) determined that parathion did not undergo glutathiond dependent dearylation (pathway 6, figure 1) in either male or female rats as had been shown for mice (Hollingworth 1969). Glutathione dependent dealkylation of paraoxon did not occur, but GSH dependent dearylation of paraoxon (pathway 7, figure 1) did occur, and that reaction varied with the age and sex of the rat.
17 Hollingworth (1973) also found that the GSH dependent dearylation of paraoxon in rat livers (pathway 7, figure 1) was more rapid than deethylation. His previous work showed GSH dependent 0-dealkylation of methyl organophosphates but little or no 0-dealkylation of ethyl organophosphates, (pathways 4, 5, figure 1) (Dauterman 1971, Hollingworth 1973. Ku and Dahm (1973) demonstrated that 0-dealkylation by liver microsomal enzymes was either not effected or only slightly effected by reduced glutatione, while Lichtenstein et al. (1973) reported that even though liver microsomal MFO was the major route for arylphosphate cleavage of parathion there was some glutatione dependent reaction. Whitehouse and Ecobichon (1975) concluded that paraoxon detoxification by liver homogenates involves hydrolytic dearylation by arylesterase glutathione mediated dealkylation, oxodative dealkylation and dearylation of nonspecific binding to tissue proteins. They believe the role of GSH dependent detoxification is slight in organophosphates that contain other then methyl side groups, but Benke and Murphy (1975) concluded that GSH dependent detoxification of parathion in rats may be m ore i m portant than previously thou ght.
Studies by Roth and Neal (1972) established that parathion bound to cytochrome P-450 creating both type I and II difference spectra. Their experiments also showed that there were at least three separate type I spectral binding sites for parathion and hexobarbital on cytochrome P-450 as evidenced by a hexobarbital and parathion. Stevens (1974) investigated the binding of parathion to cytochrome P-450 and found that both parathion and paraoxon would bind to reduced P-450 from rats and mice.
A question that had been unanswered for years was the fate of the sulfur released when parathion was converted to paraoxon. Norman et al. (1974) used radioactively labeled sulfur (35s) and showed that the sulfur would covalently bind to the macromolecules of the microsomal membrane and would do so maximally in the presence of NADPH. This binding led to a decrease in cytochrome P-450 in the microsomes.  showed that the sulfur released from the metabolism of parathion to paraoxon became covalently bound predominately to cytochrome P-450. They showed that 50% of the bound sulfur attached to the side chain of the cysteine in the P-450 apoenzyme to form hydrodisulfide.
The detoxification of paraoxon to p-nitrophenol plus diethyl phosphate is a critical reaction in controlling the toxicity of parathion in the species discussed. Unlike the reaction of parathion to paraoxon or to p-nitrophenol and diethyl phosphorothionate which is catalyzed very rapidly by the MFO system, requires MADPH and 02, and is inhibited by CO, this reaction proceeds rapidly in particulate liver fractions or serum without NADPH and is carried out via esteratic enzymes (pathway 2, figure 1) (Neal 1971). The activity of the esterase enzyme that catalyzes paraoxon to Q_nitrophenol and diethyl phosphate was further investigated by Neal (1967) showing that this esteratic enzyme required ca++ 19 for optimal activity. Benke and Murphy (1975) showed the toxicological importance of the esterase (paraoxonase) reaction converting paraoxon to diethyl phosphate plus Q_-nitrophenol by demonstrating that the LD 50 of parathion in rats correlated with the different rates of the reaction associated with age and sex in the rat rather than to the activation reaction or detoxification of parathion directly.
The association between compounds that effect liver microsomal enzyme induction such as phenobarbital, 3-methylcholanthrene, DDT, dieldrin or endrin and organophosphate metabol ism has been investigated by Alary and Brodeur (1969), Harbison (1975), Ku andDahm (1973)~ andVilleneuve et al. (1970). Alary and Brodeur (1969) showed that phenobarbital pretreatment increased liver paraoxonase activity 1.5 fold but had little effect on serum paraoxonase activity, both of which convert paraoxon to diethyl phosphate plus Q_-nitrophenol. They also showed that phenobarbital pretreatment led to a greater increase in the reaction rate for parathion conversion to diethyl phosphorothionate plus Q_-nitrophenol (pathway 3, figure 1) than to paraoxon (pathway l, figure 1), although the reaction to paraoxon was increased. Villeneuve et al. (1970) demonstrated that DDT, phenobarbital and benzo [a] pyrene decreased the toxicity of parathion in rats three fold. Harbison (1975) investigated the phenomena of phenobarbital induced protection against parathion toxicity in mice, and showed that phenobarbital protected mice fetuses against parathion. 20 Ku and Dahm (1973) showed that phenobarbital, DDT, dieldrin and endrin, all compounds that induce liver microsomal enzymes, led to an increase in the NADPH-independent paraoxonase (pathway 2, figure   1). Ku and Dahm (1973) also noted that the 0-dealkylation detoxification of paraoxon (pathway 10, figure 1) is dependent on NADPH and o 2 and inhibited by CO.
Work designed to specifically investigate liver microsomal paraoxonase activity was done by Neal and DuBois (1965) and more recently by Whitehouse and Ecobichon (1975) who showed that only rats exhibited a sex difference in the hydrolysis of paraoxon, males possessing higher activity then females, and that various species did exhibit different rates of paraoxon hydrolytic activity. The highest activity was found in mice followed by cow, rat, guinea pig, rabbit, hamster, cat, dog, and pig in decreasing order. They showed that the hydrolysis of paraoxon could be by paraoxonases from various body tissues including liver or by a MFO system requiring NADPH and o 2 , but the hepatic paraoxonase was the major route for paraoxon detoxification.
The nature of the serum enzyme capable of catalyzing the hydrolysis of paraoxon to diethyl phosphate and Q_-nitrophenol in mammals was reviewed and investigated by Lenz (1973). Lenz found that the reaction was not susceptible to substrate or p~o duct inhibition and that the enzyme contained one active group which was hydrophobic and required an electron withdrawing group in the substracte for binding.

21
In summary the most important discovery brought about by this type of research was that the phosphorothionate organophosphates must be metabolized by desulfuration to an active toxic compound (paraaxon in the instance of parathion) to exhibit antiacetylcholinesterase activity. Mammals evaluated in the preceeding papers predominatly accomplish this metabolism by a liver microsomal MFO cytochrome P-450 dependent system but this desulfuratiorr can also be produced by soluble fraction enzymes. The detoxification of paraoxon has been shown to be associated with liver microsomal paraoxonase, and the rates of production of the different metabo-1 ites of parathion are species, and for rats, sex dependent. There is also direct detoxification of parathion by a microsomal MFO system to diethyl phosphorothionate plus E_-nitrophenol and the rate of this reaction does not respond to enzymatic inhibition or induction the same as the microsomal MFO reaction that converts parathion to paraoxon.

Toxicity
As stated above, the toxicity of parathion in mammals has been shown to be due to its metabolite paraoxon. Parathion is very toxic to adult rats, Faust and Gomaa (1972) reported LOSO values in the rat of 3-30 mg/kg orally and 4-200 mg/kg dermally. The results for the oral LOSO agree with those of Villeneuve et al. (1970) who reported oral LOSO values for parathion of 6.3 to 12.0 mg/kg. Villeneuve et al. (1970) also showed that DDT, benzo[a]pyrene and phenobarbital pretreatment increased that LOSO value to greater than 20 mg/kg. DuBois et al. (1968) showed that male rats were more resistant to organophosphate toxicity through different rates of liver enzyme activity when compared to female rats. Benke and Murphy (197S) showed that male rats were more resistant to parathion than females and that an LOSO in adults (greater than S6 days old) was 2-6 mg/kg i .p. Benke and Murphy (197S) noted that the toxicity steadily declined as the rats aged, reporting LOSO values of 2-4 mg/kg i.p. for rats 23-24 days old and only 1 mg/kg for rats 12 days old. Single lethal dose ranges for parathion via i .p. administration in mice were reported by  to be 13-lS mg/kg. The work of Alary and Brodeur (1970) agrees with the preceding data concerning acute LOSO values in rats and the effects of phenobarbital pretreatment. Parathion toxicity in dogs has been reported (Faust and Gamaa 1972) Research concerning the toxicity of parathion in nonmammals and invertebrate species in particular is scarce compared to the data available concerning mammals (Carlson 1973). Research has been done in the area and work by Benke and Murphy (1974, 197S) showed that sunfish were much more resistant to parathion on a mg/kg basis than the mammals studied. The sunfish LOSO values of 10-200 mg/kg was determined, but they also concluded that the rate of onset of AChE inhibition did not explain all of their toxicological findings.  continued their investigation of parathion toxicity in sunfish (Lepomis gibbosus) and calculated an LD 50 of 110 mg/kg. They showed that fish cholinesteras~ was inhibited by paraoxon, but that the fish had a lower sensitivity to paraoxon than mice did. Glutathione dependent metabolism of methyl parathion and methyl paraoxon could be detected in liver homogenates but not for paraoxon or parathion. Other investigators that have delved into the toxicity of parathion in fish include, Faust (1964), Yarbough (1973, 1974). Chambers and Yarbough investigated the toxicity of parathion in the mosquito fish (Gambusia affinis). They found that methyl parathion was more toxic in the fish than parathion. They also found that resistant fish had a higher level of microsomal MFO. Their data indicate that the toxicity of parathion varied depending on whether the fish were in the susceptable or resistant group and the ti me of year, but in all cases the compound was toxic in water at concentrations of 1.0 ppm or less. The toxicity of parathion in fathead minnows was investigated by Faust (1964) who recorded that parathion was acutely toxic to the fatheads at 1 .4 ppm in 96 hours. Miller et al. (1966) showed that the estuarine fish (Fundulus heteroclitus) would accumulate parathion from a model ecosystem experimental apparatus. Experi ments have thus shown that parathion is toxic at low concentr ations in fish, and that the tox i n can accumulate i n f ish. The involvement of the cholinesterase inhibiting action of parathion in its toxic manifestations has been shown by Potter and O'Brian (1964), , and  who reported cholinesterase inhibition in exposed animals.

24
The toxicity of parathion in nonvertebrates other than insects has received the least attention. Research in this area has been done by Miller et al. (1966) who showed that using a model ecosystem developed to si~ulate a cranberry bog they could demonstrate the accumulation of parathion in the freshwater mussel (Elliptio complanatus). They concluded that since it has been shown that oysters and mussels accumulate pesticides, there may be an environmental hazard. They also concluded that the mussel had a much slower rate of parathion metabolism as measured by the TLC identification of radioactive metabolites of parathion than fish or mammals. Yu and Sanborn (1975) also used a model ecosystem to study parathion toxicity and metabolism. Employing radioactive parathion and utilizing extraction techniques, they concluded that parathion diq not accumulate in the snail used in their study.
The toxicity of parathion to crustaceans has been investigated to a limited extent. The anti-AChE mechanism of toxicity being credited for the toxic effects of parathion has been supported by Coppage and Matthews (1974) who showed a reduction of AChE activity following organophosphate exposure to pink shrimp (Penaeus duorarum). Carlson (1973) investigated the toxicity and metabolism of parathion in lobsters and obtained very interesting data. Pointing out that phosphorothionates must be metabolized to their oxygen analog to be toxic, and with the knowledge that Brodie and Maickel had shown that the lobster hepatopancreas metabolized many drugs, he attempted to demonstrate the correlation between the lobster hepatopancreas 1 ability to metabolize or detoxify parathion and the susceptability of the lobster to parathion. Elmamlouk et al. (1974) have since shown that the lobster hepatopancreas does contain cytochrome P-450. Carlson determined that after injecting parathion into the cheliped sinus of the lobsters an acute dose LD 50 of 0.3 mg/kg could be calculated, a value considerably lower than that for any of the mammals as yet studied. Carlson investigated the ability of the lobster hepatopancreas to desulfurate the parathion to paraoxon. Measuring the ability of incubated samples of hepatopancreas homogenate or microsomes with parathion to inhibit AChE activity he was not able to demonstrate that the lobster hepatopancreas formed paraoxon.
Carlson also used the spectrophotometric 2_-nitrophenol assay to investigate the effect lobster heptopancreas had on parathion metabolism to paraoxon and subsequent ly to diethylphosphate plus Q_-nitrophenol or directly to Q_-nitrophenol plus diethyl phosphorothionate. The lobster did demonst r ate a temperature dependent production of Q_-nitrophenol from parathion. Carl son concluded that since the lobsters were susceptible to parathion but no paraoxon co uld be detected i n the .i..!!_ vitro experi ments then , the i n vitro experi men t s di d not r efl ect the j_Q_ s i t u rea ct ion r ates or that other organs in the lobster were responsible for parathion conversion to paraoxon.

26
The toxicity of organophosphates to crayfish has been investigated by Muncy and Oliver (1963) and Albaugh (1972). Albaugh (1972) showed that there was a difference in the susceptibility of two different crayfish populations to methyl parathion. The crayfish from an enviromental area that was exposed to insecticides capable of causing microsomal enzyme induction produced a toxicity value of 3.4 parts per billion whi 'le those from an area not exposed to microsomal inducing pesticides produced a value of 2.4 parts per billion . Muncy and Oliver (1963) used a median tolerance limit technique (Tlm) to establish the toxicity in red crawfish (Procambarus clarki) by ten insecticides. They determined a 24 hour Tlm value of 0.05 ppm and 48 and 72 hour Tlm value, of 0.04 ppm for methyl parathion.
The perplexing problem of potent parathion toxicity to crustaceans but their apparent lack of ability to produce paraoxon was further investigated by Elmamlouk and G~ssner (1976). They . tested the ability of lobster hepatopancreas derived microsomes to convert parathion to paraoxon and compared that with the ability of mouse liver microsomes to carry out the same reaction. They concluded that no metabolism of parathion to paraoxon or to £_-nitrophenol occured in hepatopancreas preparation. They do mention that it is possible for the conversion to have ta ken place below the l evel of t he ir detection capabilities.

Detection 27
The determination of metabolites of parathion in snails and crayfish would be impossible without using effective extraction techniques for separating parathion and its metabolites from both tissue and water samples. The extraction, concentration, and clean up techniques that can be used for organophosphates in water environments have been throughly investigated by Appleton and Nakatsugawa (1972), Burchfield and Storrs (1975), Burkhard and Voss (1972), Coburn and Chau (1974), Faust and Gomaa (1972) Faust . and Suffer (1969, 1972 , Gomaa and Faust (1972), Faust (1964), Nakatsugawa (1972), Ripley et al. (1974), Suffet and Faust (1972a) The direct measurment of parathion in water samples is impossible due to sensitivity and specificity limitations (Faust and Suffet 1969, 1972and Suffet and Faust 1972a.

Carbon/Absorption
The carbon absorption method is applicable to large samples of water from natural sources in the field or for the preliminary cleanup of water samples in the labroatory. This method of organophosphate contaminated water sample through activated charcoal and then the extraction of the organophosphate that has bound to the activated charcoal with solvents (Faust andSuffet 1969, Nicholson et al. 1962). Nicholson et al. (1962) reported that this technique was inadequate for the extraction of parathion from a farm pond.

28
Liquid-liquid extraction has replaced carbon absorbtion (Faust and Suffet 1969) for most organophosphate extraction requirements. The choice of solvent to be used in liquid-liquid extraction is critical, and there are a large number to choose from.
The re are many important general criteria when choosing the proper liquid-liquid extraction solvents. They include, the pH of the water to be extracted and whether that will effect the organophosphate and/or the extraction procedure; if the compound being extracted is volatile, if it undergoes spontaneous reactions under the conditions which it is being extracted; and the ultimate goal of the extraction procedure. There are also important specific criteria that must be evaluated when determining liquid-liquid extraction solvents and procedures, such as: the solubility of the solvent in water; whether the solvent is polar or nonpolar, aromatic or aliphatic ; its ability to be used with the eventual detection system; the availability of pure solvents that are pesticide quality and the ease of solvent handling, toxicity and flamability .
The pH of the water in which the organophosphate is dissolved is important. Parathion will hydrolyze in alkali environments to _E-nitrophenol and diethyl phosphorothionate (Faust and Gomaa 1972 ).
Therefore, it is important that the pH of the water be kn own or adjusted, and any extraction procedure that would greatly increase the pH of the sample be avoided. Faust and Suffet (1969) determined that neutral pH values are the best for maintaining the integrity of parathion samples as the sample will also hydrolyze under extremely acid conditions. Volatilization of parathion samples may occur under some condition (Faust and Suffet 1969). It has been suggested (E.P.A. Training Manual 1974) that when drying parathion samples on thin layer chromatography (tlc) plates, the use of hot air be avoided.
One of the problems of extracting parathion from water samples is that parathion itself is not indefinately stable in the water environment and undergoes hydrolysis and desulfuration (Faust andSuffet 1969. 1972a). To determine the extent to which parathion is being metabolized to paraoxon or other compounds, it is mandatory to recover all of the compounds. This necessitates that the pH and solvent be evaluated critically.

29
Experiments by Faust and Gomaa (1972) show that both parathion and paraoxon are more resistant to hydrolysis under acid condition than alkali conditions. They reported rate constants for hydrolysis and half lives for the two compounds under different pH conditions (table 1). 10.4 1. 15 x 10-l 6.0 aFaust and Gomaa (1972) Different methods for the determination of the extent to which the organophosphates will partition between the water and solvent phase of the extraction fluid have been tried. A thermodynamic partition coefficient determination system that measures the fraction of solute that partitions into the nonpolar solvent is the best system. That system is called the p value determination approach. 31 Coburn and Chau (1974) showed that benzene could be used as a solvent for extracting parathion and metabolites. Benzene extraction was also done by Ripley et al. (1974) who reported 95 to 100% recovery of pesticide from water samples using that method. Kliger and Varon (1975) showed that by extracting water samples with a mixture of water and hexane 5:2 the parathion could be completely partitioned into the solvent phase while £_-nitrophenol and diethylphosphoric acid would remain in the aqueous phase.
White et al. (1973) showed that in extracting parathion from bean plants 5% ethyl ether in benzene was an acceptable solvent, but to determine the solvent that would yield the best return the investigator should use the p value approach.
The p value determination approach demonstrated that parathion and paraoxon in an acid (pH 3.1) environment are effectively extracted by hexane benzene, ethyl acetate, and ether, and that ether is the superior solvent due to it's having the highest p value (Faust and Suffet 1972a). W hen Q_-nitrophenol was to be determined, ether with a p value of 0.98 was again the best solvent, especially whe n compared to he xane wh ic h only had a p value of 0.20 or benz ene wi t h a va lue of 0.60 (Suffet and Faust 1972b).

Extraction from Tissue
The extraction of parathion and metabolites from samples containing animal cells or cell fragments may require different so l vents or procedures than those used in the extraction of these compounds from water samples. Lichtenstein et al. (1973) found it necessary to extract parathion and metabolites from homogenate or microsomal samples. and their metabolites (Joiner andBaetcke 1973, Stahl 1965).
Several stationary phases have been evaluated in TLC systems.
Silica gels H, G, and G-HR are used to a great extent as are neutral .
aluminum oxide G and adsorbosil g-1 (Burchfield and Storrs 1975), Silca gel is the most popular stationary phase and it was used by Gunther et al. (1970), Norman et al. (1974), Joiner and Baetcke (1973), Lichtenstein et al. (1973). However, there are some unusual phases such as cellulose MN300 that have been used (Hollingworth 1969

36
There have been many other mobile phases used for organophosphate analysis. Burchfield and Storrs (1975)  .07 and E_-aminophenol at 0.00. This system was also used by Elmamlouk and Gessner (1976).
Autoradiography was one technique used by Hollingworth (1969), White et al. (1973) and Lichtenstein et al. (1973) to quantify the amounts of compound that were present on the plates. Gas liquid chromatography was also a very popular technique used to quantify the amounts of compounds present, and Gunther (1970)  1he procedure of scraping the coating from the TLC plate that contained the compound in question or cutting out that spot from paper strips used in TLC was a technique used with great success by Benke and Murphy (1975), Elmamlouk and Gessner (1976), and MacNeil and Frei (1975. A technique for recovering the coating by using an eye dropper plugged with glass wool connected to a vacuum was presented in the EPA manual (1974) and was reported to be extremely effective. The possible interferenc.e that could be caused by the coating in the counting process was evaluated by MacNeil and Frei (1975)   Corrections were made for nonenzymatic hydrolysis by incorporating appropriate control {heat deactivated homogenate) and blank samples.
The results were expressed as µg of 2_-nitrophenol formed/hour/gram of tissue.

Procedure for Spotting, Scraping, and Eluting Samples off Thin Layer Chromatography Plates
Thin layer chromatography was used to separate, identify and quantify parathion and its metabolic products.
Silica Gel-G coated TLC plates 250 microns in thickness were used. The plates were spotted with microliter quantities of parathion, paraoxon, 2_-nitrophenol, diethyl phosphate, diethyl phosphorothionate or unknown above the level of the solvent bath present in the chromatography tank. The solvent system employed consisted of hexane, chloroform, and methanol 7:2:1, all pesticide grade. One hundred ml of solvent mixture was used with this amount forming a 1 cm deep pool of solvent in the · tank. A large square of filter paper was placed in the tank to assure saturation.
The solvent front was allowed to develop to 10 cm before the plate was removed from the tank to dry. Identification of the separated spots was aided by the use of Rhodamine B 0.1 mg/ml in ethanol which was sprayed on the plate until a light pink color covered the plate.
The plate was then viewed under ul traviolent light where the parathion 44 and metabolite spots were plainly visible against a light background.
When radioactive compounds were to be recovered, the sprayed plates were marked to isolate the desired area of the plate to be scraped by tracing around the area with a dissecting needle. That area of the coating was scraped from the plate with a small spatula. The silia gell coating was then retrieved with the use of a glass wool plugged eye dropped attached to a vacuum hose. Care was taken to be sure no significant amount of coating was lost in this process and controls were run to determine the effectiveness and reliability of this process. The glass wool containing the Silica Gel G and compounds was then deposited into a scintillation vial containing Hydromix Packard Liquid Scintillation Counter. Blanks and controls were included to determine the effect of the glass wool, Silica Gel G and Rhodamine B spray. Raw counts were used for all subsequent data calculations.

Elution Procedure
Parathion and metabolites were recovered from the TLC plates in liquid form for subsequent gas liquid chromatograph characterization or as a comparison to the above procedure by scraping the area of the plate and then using the eye dropper technique to transfer the coating along with glass wool to a vial containing 5 ml of anhydrous ethyl ether. This mixture was shaken and poured into a funnel with filter paper. The filtering funnel was rinsed with 5 ml more of ether, and the liquid containing the parathion or metabolites was evaporated with 45 suction down to less than 4 ml. The ether was then placed into a scintillation counting vial containing 10 ml of Hydromix and counted or analyzed with the gas chromatograph.

Extraction of Parathion and Metabolites from Water Samples
The extraction of parathion, paraoxon, .P_-nitrophenol, diethyl phosphate and diethyl phosphorothionate from 500 ml samples of water was done using anhydrous ethyl ether as the sole extracting solvent.
The water sample was placed in a large (1000 ml) separatory funnel and extracted with 40 ml of ether. The sample was shaken for 2 minutes. The ether layers from three consecutive extractions were pooled and passed through a glass column containing 3 inches of anhydrous sodium sulfate to remove any water. Ether layers that were in a semigel state had additional 20 ml ether portions added to re-establish the more liquid state before passage through the column.
The dried sample was then placed in rotary evaporator flasks and the volume reduced under suction but without heat. The sample was then either spotted on a thin layer chromatography plate or injected into the gas chromatograph.

Extraction of Parathion and Metabolites Excreted into Water Samples by Crayfish
Four crayfish were individually placed into 1 liter beakers contain i ng 500 ml of glass distilled water. The water in two of the beakers then had 14 c parathion labeled in the ethyl group added while the other two beakers had 14 c parathion ring labeled added. The amounts of parathion added to all of the beakers were such that a concentration of 100 ppb was attained. One and one-half hours after exposure, the crayfish were removed from the water and frozen for 46 subsequent analysis of their tissues for parathion and metabolites.
The water in which they were exposed was immediately extracted employing the water extraction procedure to determine if any metabolites could be detected.

Extraction of Parathion and Metabolites from Crayfish and Snail Tissue
The recovery of parathion and metabolites from the tissues of crayfish following exposure to parathion in water was accomplished using the crayfish that had been exposed to 100 ppb parathion and then frozen. The same procedure for extraction from tissue was performed on the whole snail tissue from the snails that were exposed to radioactivity labeled parathion at a concentration of 320 ppm for 48 hours.
The day following the exposure, the hepatopancreas and tail muscles of the 14 c-ethyl parathion exposed crayfish tissue were pooled as were the tissues from the crayfish exposed to the ring labeled compound. The tissues were homogenized with a blade homogenizer in 9 ml of ether for 30 seconds. The homogenate was then scraped into centrifuge tubes containing 9 ml of ether. The tissue attached to the homogenizer blades was rinsed into the tube with additional ether. The centrifuge tube containing the tissue and ether was shaken for 2 minutes and then centrifuged at low speed to separate the tissues from ether layer.
Following the centrifugation the ether layer was removed with 47 a Pasteur pipet and transferred to a rotary evaporator flask. The sample was then evaporated without heat to a volume small enough to be spotted onto TLC plates. The plates were developed and sprayed as described along with standards for identification of the products. The spots were removed and counted in the liquid scintillation counter to quantify the amount of product recovered.

Production of 2_-Nitrophenol, Paraoxon, Diethyl Phosphate or Diethyl Phosphorothionate from Parathion via in vitro Metabolism by Orconectes or Viviparus Tissues
The procedure for this assay was almost identical to that for the 2_-nitrophenol spectrophotometric assay.
Tissues from snail or crayfish were removed and homogenized in NaCl, MgS0 4 , nicotinamide in the same way. The tissue was incubated in the same way with the parathion added to initiated the reaction containing a known amount of radioactive parathion. Ring labeled 14 c parathion was used to follow the fate of parathion, paraoxon and 2_-nitrophenol while 14 c ethyl label was employed to investigate the possible production of diethyl phosphate and diethyl phosphorothionate.

A. Parathion Toxicity
The fresh water snail Viviparus malleatus was not sensitive to parathion in the dose range to which the animal was exposed in these experiments. A level of 1000 ppm failed to exert any observable toxic effects on the snail (table 3). The solubility of parathion in water is only 20 ppm so the attempt to create a 1000 ppm concentration was to be certain that saturation was reached.
The crayfish Orconectes rusticus demonstrated extreme sensitivity to parathion exposure in its water environment. Concentrations as low as 1 .0 parts per billion (ppb) produced death in 100% of the crayfish exposed in less than 24 hours (table 2). The toxicity was evidenced by twitching movements and exaggerated muscle contractions in response to provocation immediately before death.  aNumber of animals individually exposed. aNumber of animals exposed .
bParathion solubility in water is 20 ppm. The resistance of the snails to parathion was further studied by injecting the snail directly with parathion. The two snails that were each injected with 5.0 mg (approximately 250 mg/kg) of parathion showed no toxic sign in 96 hours.

B. Paraoxon Toxicity
The susceptibility of Viviparus malleatus or Orconectes rusticus was determined for the oxygen analog of parathion, paraoxon.
These data show no snail mortability due to paraoxon exposure at 10 ppm exposure in 96 hours (table 5).
All of the animals exposed to concentrations ranging from 0.1 to 20 ppb died in less than 24 hours and exhibited the same toxic signs (e.g., twitching movements and exaggerated muscle contractions in response to provocation) as those that dies in the parathion exposure experiment.
The resistance of the snails to paraoxon was studied by injecting paraoxon directly into two snails. The snails that were each injected with 1.0 mg (approximately 50 mg/kg) of paraoxon showed no toxic signs in 96 hours.
crayfish gill, snail whole intestine and rat liver homogenates .
The results of these experiments are presented in table 6.
These data show that rat liver homogenates produce .E_-nitrophenol from parathion at a rate of 32.40 µg/hour/gram tissue when determined over a 1 hour incubation period. The male rat is a species known to be susceptible to parathion toxicity at a dose of 7.0 mg/kg ip (Benke and Murphy, 1975) .
The homogenate of whole snail intestine did not produce .E_-nitrophenol at a level that could be measured by this technique. The possibility that the reaction that could produce .E_-nitrophenol by these tissues was NADPH dependent and that the NADPH generating system employed in the assay procedure was ineffective in the incubation mixture was tested by the addition of NADPH in place of NADP. No .E_-nitrophenol was detected. Dithiothreitol at 10-4 M was also tested for its effect on .E_-nitrophenol production. However, no .E_-nitrophenol production was discernible.
The hepatopancreas of crayfish Orconectes rusticus was evaluated using the .E_-nitrophenol assay for its ability to metabolize parathion. The data of table 6 show that no conversion of parathion to .E_-nitrophenol by Orconectes hepatopancreas homogenates could be detected.
The effect the addition of NADPH and/or OTT would have on the production of .E_-nitrophenol was also tested. No convers i on of parathion to .E_-nitrophenol could be detected using the spectrophotometric assay.

55
The lungs of mammals have the ability to metabolize parathion, are in ready contact with the material and are capable of lipid absorption, so the gills of Orconectes were tested for parathion metabolizing ability as a comparison. The data in table 6 show that no .e_-nitrophenol production could be detected using that tissue and the spectrophotometric assay.

Thin Layer Chromatography
The use of Rhodamine B spray (0.1 mg/ml ethanol) made it possible to visually identify parathion, .e_-nitrophenol, paraoxon, and diethyl phosphorothionate spots under ultraviolet light. Initial experiments determined that 1 .0 µg of parathion spotted on a TLC plate and subsequently developed with the hexane; chloroform, methanol, solvent mixture (7:2:1) could be visualized. Amounts of parathion below 1 .0 µg were occasionally difficult to visualize so that identification was inconsistent.
The efficiency and reliability of the techniques used in this study that required TLC were evaluated. The Rf values for .e_-nitrophenol, paraoxon, parathion and diethyl phosphorothionate were 0.17, 0.42, 0.69 and 0.75 respectively, using the hexane, chloroform, methanol (7:2:1) system with the 250 µSilica Gel-G plate, but because the Rf value of a compound may change with slight alterations in the solvent mixture, standards were run concurrently with the unknowns to facilitate accurate identification.
Radioactive parathion and its metabolites were separated, 56 identified and quantitatively recovered by TLC using techniques that required knowledge of the effect glass wool, Silica Gel-G and Rhodamine B spray would have on recovery and counting efficiency. Preliminary tests determined that glass wool, scraped Silica Gel-G coating and Rhodamine B spray in the amounts used in these experiments did not interfere with the liquid scintillation counting procedure.
The efficiency of the procedure used to recover parathion and its metabolites from TLC plates is presented in table 7. These data show that TLC followed by liquid scintillation counting could be effectively used as a procedure for the separation, identification, recovery and quantification of parathion and its metabolites.

E. Extraction of Parathion and Metabolites From Water Samples
The extraction of parathion and metabolites from water samples was necessary to evaluate the excretion of parathion metabolites from crayfish and snails exposed to the pesticide. Prior to experiments to determine if parathion and/or metabolites could be recovered, the efficiency and reproducibility of the extraction process was determined (table 8). The ether extraction procedure of water samples extracted 77% of the parathion in the sample with less than 10% standard error.
The ability of crayfish to metabolize parathion and then to excrete those metabolites into their water environment was tested by exposing the crayfish for 1-1/2 hours to water samples containing 100 ppb parathion (labeled in the ring or ethyl position) and then determining the metabolites present in the water samples. The ether  x 387 ± 12.0 = 2.5 µg parathion x 298 ± 24.0 = 1 .9 µg parathion = 3.85 ppb aStandards were the radioactive compounds placed directly into scintillation vials. The amount of radioactive parathion was equivalent to 2.5 µg or the amount of parathion in 500 ml of water to equal 5 ppb. bsamples were 2.5 µg of radioactive parathion dissolved in 500 ml of water and then extracted using the water extraction procedure. 58 59 extraction, thin layer chromatographic separation and scintillation counter quantification technique was used for that determination. The data in table 9 show that unquestionably the vast majority of the total amount of compound recovered from the exposure was in the form of parathion.
The snails were exposed to 320 ppm of ring labeled parathion for 48 hours with subsequent extraction of the aquarium water as with the crayfish (table 10). The results presented in tables 9 and 10 show that for both the crayfish and the snail there was no excretion of parathion metabolite into the water environment. There was no production of 2_-nitrophenol with either species. However high values for 2_-nitrophenol were obtained which was shown to be due to spontaneous hydrolysis.

F. Evaluation of Parathion and Metabolites Accumulation in
Crayfish and Snail Tissues Following Parathion Exposure in a Water Environment To study the accumulation of parathion by tissues of the species animals were exposed to 100 ppb 14 c labeled parathion for one-half hour and were then sacrificed for subsequent analysis. The extraction of labeled 14 c parathion and metabolites from crayfish tissues showed that there was no indication of any accumulation of these compounds in hepatopancreas or muscle tissues. The data (table 11) show that when hepatopancreas or muscle samples from animals exposed to ring or ethyl labeled 14 c parathion were pooled, no accumulation could be detected. bValue not above background (does not indicate compound production-test sensitivity 4.7 µg ethyl and 9.3 µg ring).
cHigh value due to spontaneous hydrolysis.   The results (table 13) show that there was no production of .e..-nitrophenol, paraoxon or diethyl phosphorothionate from snail tissues that were active or deactivated by boiling. That there was 120 µg of radioactive parathion present in the incubation mixture (4.0 cpm/µg for ring label and 3.0 cpm/µg for ethyl label) and a large percentage of that radioactivity was effectively extracted, separated and recovered is illustrated by the CPM values for parathion itself.
The resu l ts of the experiments with crayfish hepatopanc reas

H. In Vitro Metabolism of Paraoxon by Orconectes and Viviparus
The possibility that both crayfish and snails might be able to metabolize the oxygen analog of parathion, paraoxon, to diethyl phosphate and E_-nitrophenol was evaluated by using the E_-nitrophenol spectrophotometric assay with paraoxon added to initiate the reaction in place of parathion. The results from this experiment (table 15) show that in no instance did crayfish or snail tissue demonstrate any significant production of E_-nitrophenol that could be measured by this method. dAbsorbance measured at 410 nm ± standard error.

V. Discussion
The investigation of the metabolism of parathion by fresh water invertebrates is more than an academic exercise in pesticide metabolism and detection. The problems that followed the use of the chlorinated hydrocarbon pesticides were in part due to the lack in understanding of the environmental consequences of their use and ignorance as to the effects the compounds would have on organisms other than the target species and man.
The increase in use of the organophosphates along with a limited understanding of their toxicity and metabolism in nonmammals is a situation analogous to the one that led to problems with chlori:;rated hydrocarbon pesticides. Nicholson et al. (1962) did investigate the environmental exposure consequences of parathion and showed that parathion would accumulate in a farm pond, and that it would persist in the environment for at least nine months. These times are similar to those that were reported by Faust (1964). Nicholson et al. (1962)   transformations. These pathways are all presented in Figure 1. The important point is that these reactions occur and that the rates at which they compete for parathion and paraoxon influences the toxicity of parathion exhibited in that particular species (Benke and Murphy, 1975).
The parathion exposure experiments showed that Viviparus was not sensitive to parathion in its water environment even when the solubility of parathion in the water was exceeded while Orconectes was sensitive to parathion in its water at 1 ppb. The question that presented itself was why there was such a species difference in 72 susceptibility. Refering to Figure 1 and with an understanding of the data from other investigators the possible mechanisms for these results were envisioned. The snail could be resistant to parathion exposure due to the following reasons: 1. Parathion was not entering the shell of the snail.
2. Parathion was entering the shell but not being metabolized to paraoxon, which if produced would cause toxicity.
3. The parathion was being metabolized extremely rapidly and efficiently via pathways 3,4,6,9 or 11 (Fig. 1) to the nontoxic metabolites or being bound to tissues where no metabolism could occur.
5. The snail may be converting parathion to paraoxon but be insensitive to paraoxon.
The goal of the investigation of the resistance of Viviparus malleatus to parathion was then to design experiments to determine which of these mechanisms was responsible for the lack of parathion toxicity in the snail.
The possibility that the parathion was not entering the shell of the snail was evaluated by injecting snails with 5.0 mg/kg. The results show that there were no toxic signs demonstrated by the snails.
Paraoxon was also injected directly into snails. Two snails were exposed to 50 mg/kg of paraoxon by direct injection with no toxic 73 signs demonstrated in 96 hours. The results of these two experiments establish that the ability of the compound to enter the shell of the snail was not a limiting factor in its lack of toxicity. The results also show that the snail is resistant to paraoxon as well as parathion so that the absence of pathway 1 of Figure 1 would not be the mechanism responsible for the animals' resistance to the organophosphate.
The 2_-nitrophenol spectrophotometric assay was used with whole snail homogenates to determine if the snail was capable of metabolizing parathion via pathways 2, 3 or 9 of Figure 1. Since these pathways are the most important detoxification pathways for parathion resistance, the demonstration of measurable 2_-nitrophenol would show that the parathion or paraoxon was being detoxified and could possibly be the cause for the animals' resistance to parathion and paraoxon exposure.
The 2_-nitrophenol spectrophotometric assay with whole snail intestine did not produce Q_-nitrophenol at a level that could be measured by this technique. The reason for the lack of production was not due to deficiency of NADPH for dependent enzyme reactions or the destruction of essential sulfhydryl groups of the membranes or enzymes, as NADPH and dithiothreitol were added. The same spectrophotometric assay incorporating paraoxon in the place of parathion was also done to determine if pathway 2 of Figure 1 was possible but not detected with parathion in the incubation due to the absence of the conversion of parathion to paraoxon (pathway 1, Fig. 1). Again, no 2_-nitrophenol was produced indicating the lack of pathway 2 route of metabolism.
The Q_-nitrophenol assay is designed to demonstrate t he presence of metabolism occurring but because the measured compound can be produced by numerous pathways if the compound is detected the exact pathway followed cannot be determined. A method to determine if specific pathways were used in the metabolism of parathion would be to determine the presence of the specific compounds paraoxon and diethyl phosphorthionate, along with E_-nitrophenol and parathion.
The extraction of parathion and metabolites from water samples measured the excretion of metabolites of parathion into the water environments of snails. The results of that experiment ( AChE if present has no effect on the organism.
The crayfish demonstrated susceptibility to acute parathion toxicity, and the muscle twitching signs were consistent with AChE inhibition. The mechanism by which this toxicity was produced was assumed to follow the desulfuration of parathion to paraoxon via pathway 1 of Figure 1. This conversion would have to be at a rate high enough to account for a paraoxon concentration that would cause toxicity. The reaction rates for the toxic conversion reaction and detoxification reactions has been shown to be an important factor in the toxicity exhibited by species to parathion exposure does not establish the existence or absence of detoxifying reactions by Orconectes, only the existence of a toxic conversion reaction 75 The goal, then, for the investigation of the Orconectes susceptibility to parathion was to demonstrate the presence of the reaction coverting parathion to paraoxon and any other metabolic pathways present as depicted in Figure 1. This is closely related to the question of the accumulation ~f parathion and/or its metabolites in crayfish tissues. The results presented in table 11 show that there was no accumulation of these compounds in the gill, muscle or hepatopancreas of crayfish following exposure to parathion.
The possibility that the crayfish toxicity was due to concentrations of paraoxon too low to detect could not be overlooked in this investigation considering the research of Carlson (1973) andElmanlouk andGessner (1976). They reported that the hepatopancreas of the lobster is the organ responsible for drug metabolism, but the hepatopancreas of the lobster had very little, if any, observable ability to convert parathion to paraoxon.
Establishing if paraoxon was capable of producing the toxicity in crayfish was done by exposing the crayfish to paraoxon in the aquarium water as had been done with parathion. The experimental results showed that the crayfish was sensitive to paraoxon and that the toxic signs exhibited by the crayfish following paraoxon exposure were the same as those following parathion exposure. The concentration range that produced the toxicity in the crayfish due to paraoxon (table 4) was consistent with the theory that the parathion was exhibiting its toxicity through conversion to paraoxon and subsequent AChE inhibition.
The toxicity determination experiments did little to increase the understanding of the metabolism of parathion by Orconectes.
Experimental procedures similar to the ones used to determine the metabolism of parathion in snails needed to be done to determine the pathways of metabolism of parathion and paraoxon in crayfish. 76 The .e_-nitrophenol spectrophotometric assay was used to ascertain if any metabolism of parathion or paraoxon could be determined with that technique. Rats are susceptible to parathion toxicity at 7.0 mg/kg i.p. (Benke and Murphy, 1975) so they were used as controls in these experiments to compare their metabolism of parathion to that in the crayfish. The .e_-nitrophenol assay was perfonned on rat liver homogenates as well as homogenates of crayfish hepatopancreas and gill (t able 6). The results show t hat the rat liver homogenate caused the production of 32.4 µg/hour/gram of tissue when determined over a l hour incubation period, but the crayfish hepatopancreas and gill homogenates which would be expected to be the organs capable of metaboliz i ng parathion produced no .e_-nitropheno l that could be m easured spectrophotometrically. These results were not due to lack of N ADPH for dependent enzyme processes or the destruction of vital enzymes since the addition both NADPH and dithiothreitol (l0-4 M ) added to some incubation samples as had been done in the snail tissue experiments, were not effective. 77 Since the E_-nitrophenol assay was done using both parathion and paraoxon in the incubation flasks, the assay had the capability of monitoring pathways 2, 3 and 9 of Figure 1 all of which cause the production of E_-nitrophenol. No E_-nitrophenol could be detected by this technique. However, because the crayfish were sensitive to the parathion and paraoxon exposures other experimental designs were used in an attempt to monitor some metabolism of parathion or paraoxon by the crayfish or its tissues.
The possibility that the crayfish was able to absorb parathion from its water environment and then to excrete metabolites of parathion back into the water was evaluated. 14 c Parathion labeled either in the ethyl or ring positions was used for these exposure-excretion experiments. Following the exposure of crayfish to labeled parathion at a concentration of 100 ppb for 1-1/2 hours, the water in which the crayfish were kept was extracted with ether, the samples separated by thin layer chromatography and the appropriate spots scraped and counted by liquid scintillation counting. The results showed that after the spontaneous hydrolysis of stock parathion is considered there was no excretion of parathion metabolites into the water that would be indicative of parathion metabolism.
The ability to produce even minute amounts of parathion metabolites by crayfish tissues was tested by the use of an incubation technique similar to the E_-nitrophenol assay. Homogenates of crayfish The question that rises from these results is how is the parathion producing its toxicity if no metabolism can be detected.

VI. Conclusions
The goal of determining the toxicity and metabolism of parathion in the fresh water invertebrates Orconectes rusticus and Viviparus malleatus made the extraction, detection and quantification of minute quantities of organophosphate and its metabolites necessary.
Groups of the crayfish Orconectes rusticus when exposed to The ability of the crayfish and the snail to metabolize parathion was determined through the use of the £_-nitrophenol spectrophotometric assay, the investigation of excreted metabolites that were extractable from water samples, the determination of the presence of metabolites accumulating in tissue, detection of radioactive metabolites from tissue incubation experiments and the toxicity experiments.
All of the experimental data support the conclusion that there was no metabolism of parathion by the snail and that this lack of metabolism and the insensitivity of Viviparus to paraoxon form the basis for the lack of toxicity exhibited by those compounds to the snail.
The data indicate no metabolism or accumulation of parathion or its metabolites by the crayfish, but the conclusion that no metabolism was taking place cannot be made as the toxicity experiments do not support that contention.
The data from the £_-nitrophenol spectrophotometric assays with parathion and paraoxon using either crayfish or snail tissue revealed no production of £_-nitrophenol by either the snail or the crayfish indicating that pathways 1, 2, 3 and 9 of Figure 1 could not be demonstrated by that technique.
The extraction experiments show that there was no detectable accumulation of parathion or metabolites in Orconectes or Viviparus and no detectable excretion of parathion metabolites into the water environment.

81
The experiments performed with radioactive parathion incubated with either crayfish or snail tissues were unable to demonstrate any metabolism of parathion, paraoxon, p_-nitrophenol, diethyl phosphorothionate and diethyl phosphate by crayfish or snails.
The resistance to parathion and paraoxon demonstrated by the snail does not stem from enzyme systems capable of detoxifying the compounds. The snail is biologically resistant to the inhibition of AChE.
The mechanism by which parathion exhibits toxicity in Orconectes cannot be determined from the results obtained by these experiments. The extreme sensitivity to parathion and the clinical signs demonstrated by the crayfish following parathion exposure are consistent with the production of effective concentrations of paraoxon that can inhibit the AChE at synapse and neuromuscular junctions.
However, until the production of some metabolism of parathion by some crayfish tissue can be demonstrated more definitive conclusion cannot be made.