Ecotoxicology of phenylphosphonothioates.

The phenylphosphonothioate insecticides EPN and leptophos, and several analogs, were evaluated with respect to their delayed neurotoxic effects in hens and their environmental behavior in a terrestrial-aquatic model ecosystem. Acute toxicity to insects was highly correlated with sigma sigma of the substituted phenyl group (regression coefficient r = -0.91) while acute toxicity to mammals was slightly less well correlated (regression coefficient r = -0.71), and neurotoxicity was poorly correlated with sigma sigma (regression coefficient r = -0.35). Both EPN and leptophos were markedly more persistent and bioaccumulative in the model ecosystem than parathion. Desbromoleptophos, a contaminant and metabolite of leptophos, was seen to be a highly stable and persistent terminal residue of leptophos.


Introduction
The first reference to the chronic toxicity of phenylphosphonates was the evocation of delayed neurotoxicity in hens fed diets containing EPN [0-(4-nitrophenyl) 0-ethyl phenylphosphonothioate] (1). EPN in 1956 was a minor pesticide, and since there was no indication of human danger, this early warning was ignored. Ten years later, as the phenylphosphonothioates began to complement the alkyl phosphates in replacing the extremely persistent organochlorine pesticides, the neurotoxicity of leptophos [0-(2,5-dichloro-4bromophenyl) 0-methyl phenylphosphonothioate] forced reconsideration of the real hazards of organophosphate-induced neurotoxicity (2). The present study compares the toxicity of nine phenylphosphonothioates in terms of the traditional parameters of their acetylcholinesterase inhibition in flies and their acute toxicity in rats, and also in terms of their neurotoxic activity in hens. In addition, the behavior of leptophos and EPN in an aquatic-terrestrial ecosystem (3)(4)(5) is assessed in order to evaluate environmental effects and food chain transfers.
In this manner, the toxicological profiles of leptophos and EPN have been characterized with respect to their acute toxicity in target and nontarget organisms, their environmental degradation and persistence, and their chronic toxicity. Comparisons with the other phenylphosphonothioates permit some generalizations about the relationship between delayed neurotoxicity and the classical organophosphorus ester toxicity due to acetylcholinesterase inhibition. Such comparisons in turn permit realistic evaluation of the hazards which must be set against the benefits of using a particular pesticide or class of pesticides.

Ecosystem
In the terrestrial-aquatic model ecosystem studies, 5 mg of the pure "4C-labeled EPN, leptophos or desbromoleptophos, was applied from acetone solution to sorghum leaves in the terrestrial phase of the ecosystem, simulating application of these pesticides to crops. The contaminated sorghum leaves were eaten by salt marsh catepillars (Estigmene acrea), dispersing the ra-diolabeled products into the seven liters of standard reference water (6) containing plankton, alga (Oedogonium cardiacum), daphnia (Daphnia magna), and snails (Physa sp.). Subsequently mosquito larvae (Culex pipiens) and fish (Gambusia affinis) were added to complete the foodchain interactions. The entire ecosystem unit in a 10-gal aquarium was kept in a programmed environmental growth chamber for 12 hr daylight cycles of 5000 ft-candles light intensity and with constant temperature of 26 + 1°C.
At the termination of the 33-day experimental period, the organisms and water samples were collected separately and weighed. The organisms were extracted with acetone, and the water extracted with diethyl ether. The total bioaccumulation and the nature of the degradation products was determined by liquid scintillation counting and thin layer chromatography. The environmental properties of the parent compound and major metabolites were expressed quantitatively as ecological magnification (EM = concentration of parent compound in the organism/concentration of parent compound in the water) and biodegradability index (BI = concentration of polar metabolites/concentration nonpolar metabolites).
The unextractable radioactivities (water-partitioning metabolites) were assayed by the Schoniger oxygen flask technique (7). Quenching was corrected by channels ratio methods (8). The radioassay technology, separation and identification were carried out as previously described (5).

Fly Toxicity
The insecticidal activity of the chemicals, due to the acute inhibition of acetylcholinesterase, was Ml OMS 160 EPBP Parathion measured by using standard weight/volume solutions in acetone and applying microliter droplets to adult houseflies (Musca domestica NAIDM) as has been described previously (9).
Chemicals. The organophosphorus esters discussed in the text are listed in Table 1 with their chemical structures and trade or generic names. The structural relationships among tested compounds and other phenylphosphonothioates are shown in Table 2. Technical grade chemicals were used for neurotoxicity tests and fly toxicity tests, but highly purified radiolabeled compounds were used for ecosystem studies.
Treatment. Several dosing schedules were used, but compounds were always administered in gelatin capsules. Hens were not protected from   acute cholinergic effects with atropine; instead, doses were manipulated to fall below acutely lethal levels. After initial attempts at using single or multiple doses at maximum tolerated levels, two treatment schedules were adopted. Acute doses were begun at 50 mg/kg and chronic dosing was begun at 10 mg/kg/day and continued for 90 days. Treatment levels in subsequent hens were decreased until a nonlethal, non-neurotoxic dose was established and/or increased until a neurotoxic or lethal dose was established. In several cases testing was limited by the small quantities of chemical available. Thus negative results do not necessarily demonstrate nonneurotoxicity but only inactivity at the levels tested.
Observation. Hens were weighed on arrival and as necessary to calibrate dosages. In chronic dosing studies, hens were weighed every second week. All hens were observed daily in their cages and evaluated at intervals for their ability to stand, walk and fly. Because of the variability in posture, gait and activity between hens and between observations for the same hen, only gross ataxia (+2), paraplegia (+3), paralysis (+4), and quadriplegia (+5) were used to define neurotoxicity. Increased nervousness or minor disturbances of gait (+1) were noted but not used to define neurotoxic chemicals or levels of treatment.

Rho-Sigma Evaluation
The acute toxicity of organophosphorus insecticides is well known to result from their irreversible inhibition of acetylcholinesterase. This occurs in phosphonothioates (P=S) following microsomal oxidation to the corresponding phosphonates (P=O) which then undergo a bimolecular reaction with a serine hydroxyl group in the enzyme to irreversibly inactivate it (13). The relative rates of inactivation of acetylcholinesterase by phosphonates are governed by the electrophilic properties of the phosphorus atom and these are a function of the electron-withdrawing properties of the groups attached to the phenoxy group as well as the electronic and steric properties of the November 1980 phosphonate group (14,15). These effects can be quantified by the Hammett p-a (rho-sigma) analysis, and by the Taft steric parameter E. (16).
The concept of a neurotoxic esterase that is inactivated by interaction with organophosphorus ester neurotoxins (17) suggests that the reactivity, i.e., electrophilic character of the P atom, should relate quantitatively to the magnitude of neurotoxic effect in a manner similar to acute toxicity, i.e., phosphorylation of acetylcholinesterase. We have explored this possibility by plotting the summation of a values for the aromatic substituents of the 0-phenyl 0-alkyl phenylphosphonothioates vs. acute topical LD,, to the female house fly Musca domestica as determined in this laboratory and versus the rat oral LD. using values from the literature (10)(11)(12). The Taft a* values were used for the ortho substituents (16) after correcting them to H=O rather than CH3=O. The general validity of the a values was also confirmed by relating them to the Ka values for the ionization of the various phenols. The resulting plots ( Fig. 1) leave no doubt of the validity of this concept of phosphorylation in regard to acute tox- icity for the phosphonothioates. The correlation coefficient for a vs. fly LD5o was r = -0.91 and for rat LD. was r = -0.71. These indicated significance at p <0.001 and p <0.05, respectively, and also show the relative efficiency of the microsomal oxidation of P=S to P=O which does not appear to be significantly affected by the electronic and/or steric character of the remainder of the molecule.
In contrast is the corresponding plot of a versus minimum neurotoxic dose (Table 3 and Fig. 2). The correlation coefficient was r = -0.35, significance level p >0.20. Since these data include ethoxyphenyl-phosphonothioates (EPN, ethoxyleptophos) as well as methoxy phenylphosphonothioates, some heterogeneity would be expected. But within the class of methoxyphenylphosphonothioates, the neurotoxicity of desbromoleptophos (la = 0.573) is sixfold that of leptophos (la = 0.805). The effect of the 0-alkyl chain can be seen in the absence of overt neurotoxicity when ethoxyleptophos is given at 1000 mg/kg (18). Other studies have shown that the nature of the group involved in the direct phosphorus-carbon bond is also critical; for example, the methylphosphonothioate analog of compound 160 [i.e., 0-(2,4,6,-trichlorophenyl) O-ethyl methyl-phosphonothioate] is neurotoxic at a single oral dose of 20 mg/kg, while OMS 160 did not induce paralysis at 300 mg/kg (unpublished data).
The neurotoxic esterase theory of Johnson (17,(19)(20)(21) requires the neurotoxin to phosphorylate an esterase of unknown specificity within the nervous system. It is therefore necessary for a would-be neurotoxin to enter the nervous system in sufficient quantity, and to persist a sufficiently long time, to reach this esterase. A minimum lipophilicity and stability are required. Subsequently the neurotoxin must "fit" the enzyme sufficiently well to inhibit it, and finally the neurotoxin must possess the necessary electron-withdrawing/donating characteristics to phosphorylate the enzyme.
Thus it is not surprising that the correlation between neurotoxic activity and a is not as pronounced as the correlation between a and the acetylcholinesterase inhibition of the peripheral nervous system. It is even plausible that in some cases the requirements of persistence and lipophilicity counteract the electronegativity requirements sufficiently well that the observed results are an increase in neurotoxicity with decreasing a values, as is seen for leptophos and its desbromo analog. Precisely which aspects of molecular structure relate to fit must await either a better understanding of the natural substrate of the neurotoxic esterase or of its function within the nervous system. In addition to treating hens with single oral doses of phenylphosphonothioates, we tested the effects of low level chronic treatment with EPN, cyanofenphos, and leptophos, the three commercial phenylphosphonothioates available to us. All three were neurotoxic at 10 mg/kg/day (Table 4), but EPN was so acutely toxic that hens had to be treated at lower doses for the first week. Even so, EPN treated hens became ataxic 24 days after the start of treatment; ataxia progressed to irreversible paralysis (+4) and in one case to quadriplegia (+5). For leptophos, ataxia (+2) resulted from 90 days of 10 mg/kg/day. Cyanofenphos was intermediate, with ataxia (+2) after 34 days treatment at 10 mg/kg/day. The precise ordering of neurotoxic potential at low levels of chronic dosing is unfortunately irrelevant, since it is already established that the least potent compound, leptophos, caused nerve damage in factory workers and in farm animals. The critical factor in these studies is that paralysis can occur in the absence of symptoms of acute poisoning (leptophos, cyanofenphos) or with sublethal doses which the animal could survive even without atropine (EPN). Inasmuch.-as human acute poisoning with organophosphorus esters is aggressively treated with atropine and acetylcholinesterase regenerators, the levels of ex-  posure we used are conservative. The cumulative corresponds to a farm pond surrounded by a wanature of the neurotoxic potential must be considtershed under cultivation which has been treated ered in determining the risk involved in manufac-with 1 lb/acre (1.12 kg/hectare) of the test comturing, formulating and using organophosphorus pound. Data for EPN, leptophos, and desbromopesticides.

leptophos (Tables 5-7) can be compared with the
The aquatic-terrestrial model ecosystem pro-data for parathion [0-(4-nitrophenyl 0-ethyl vides a valid method for predicting the behavior phosphorothioate], which is generally regarded as of pollutants in the environment. It is particularly a nonpersistent insecticide. Parathion in the valuable, because over 150 chemicals have been model ecosystem was found in algae, snails, and compared under standardized conditions, allowing fish as well as in water ( Table 8). The highest levassessment of relative persistence, degradation els were found in snails, which accumulated paraand bioaccumulation. The design of the system thion 746-fold over the levels found in water. Environmental Health Perspectives Some bioaccumulation was seen in algae and fish as well, but relatively little. The Biodegradability Index (BI) for snails was greater than one, demonstrating the metabolism of parathion to polar, water soluble products. The presence of aminoparathion, p-nitrophenol and p-aminophenol in most components of the ecosystem is in accord with the well documented degradation pattern of parathion (5,22).
The behavior of EPN ini the model ecosystem is similar to that of parathion, but EPN is clearly more persistent. The values for bioaccumulation or ecological magnification (EM) of EPN in snail and fish were 12,561 and 346 vs. 747 and 87 for parathion (Tables 5,8,and 9). Moreover, the EPN metabolites were relatively less polar than those of parathion, so that biodegradability index val-ues (BI) of EPN for snail and fish were 0.15 and 0 vs. 1.23 and 0.24 for parathion. Leptophos in turn, was more bioaccumulative than EPN (Table  9) with EM values of snail 21,232 and fish 972; and was correspondingly more recalcitrant to biodegradation with BI values of snail 0.85 and fish 0.090. These values from the present study using 14C-phenyl ring-labeled leptophos are in excellent agreement with EM values of 48,358 in snail and 1,443 in fish previously obtained with 14CH30-labeled leptophos (23).
Desbromoleptophos, a persistent metabolite of leptophos with enhanced neurotoxic properties (24) was found in all components of the model ecosystem treated with leptophos, at levels ranging from 2% of leptophos concentration in snails, 9% in alga, 19.5% in fish, and 19.6% in mosquito   (Tables 7 and 9), the EM values were snail 34,164 and fish 3,477. The BI values were snail 0.006 and fish 0.030. Thus desbromoleptophos appears to represent a highly stable and persistent terminal residue of leptophos metabolism; its greater persistence in other systems has also been established (24). The five-fold greater neurotoxicity of desbromoleptophos, its role as a photodecomposition product of leptophos, and its substantially greater persistence and decreased biodegradability all argue for its suggested role as the active heurotoxin in agricultural poisonings by leptophos (24). The environmental persistence of this highly active delayed neurotoxin emphasizes the importance of evaluation of environmental residues of potentially neurotoxic compounds for neurotoxic action.

Summary
The evaluation of phenylphosphonothioates in terms of their acute, chronic, and environmental hazards illustrates the complexity of risk determination in pesticide use. The use of a-coefficients to establish relative acetylcholinesterase inhibition within a class of closely related organophosphorus esters is an eminently reliable predictor of insecticidal activity, and a fairly reliable indicator of mammalian toxicity. To what extent the heterogeneity of the data used for assessment of mammalian acetylcholinesterase inhibition is responsible for the lower correlation between a and LD. in rodents and to what extent the complexities of oral administration (e.g., digestion, transport, hepatic metabolism) affect the lower correlation is uncertain.
Delayed neurotoxicity, in sharp contrast to acute toxicity, is not well correlated with a. The marked effect of the ethoxy-group in reducing neurotoxic potential was far more significant than alterations in a among 0-methyl phenylphosphonothioates. Since a similar benign effect of ethoxy moieties-or malignant effect of methoxy moieties-is nQt seen in methyl phosphonothioates (unpublished data), the role of the P-C bond is also of considerable importance.
The ecosystem data demonstrate the variability of persistence of organophosphorus pesticides and suggest that their degradability is relative. Recent studies on the persistence of para-oxon on citrus foliage (25) illustrate the absence of absolutes in real world residue studies, but under the standardized conditions of the terrestrial aquatic model ecosystem, both EPN and leptophos are clearly more persistent than parathion. Their potential for inducing permanent paralysis after Environmental Health Perspectives low level chronic exposure is therefore an extremely significant datum in the evaluation of their hazards to man and animals. In a more general fashion, the multifaceted evaluation of closely related phenylphosphonothioates illustrates the inadequacy of a single determinant of toxicity. Acute toxicity, chronic toxicity, and knowledge of environmental behavior are required to assess the safety of environmental chemicals. This work was supported by NIEHS grant 5 ROI-ES-01492