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1. The acute oral LD50 and chronic LC50 toxicity values for ethylene dibromide (EDB) were estimated for japanese quail. 2. Single sub-acute oral and intraperitoneal doses of EDB (1/2 LD50) and chronic oral doses of EDB (1/3 LC50) were administered to quail in order to characterise the sub-lethal effects of EDB residues. 3. At 24 h after sub-acute dosing, relative liver weight, plasma aspartate aminotransferase (AT) [EC 2.6.1.1] and L-iditol (sorbitol) dehydrogenase (SDH) [EC 1.1.1.14] were elevated and decreases were found in hepatic total lipid, total protein, AT and glutamic dehydrogenase (NAD (P)+) (GDH) and plasma cholinesterase (ChE) [EC 3.1.1.8] and total lipid. 4. Following chronic administration, elevations in relative liver weight, plasma ChE and total lipid, haemoglobin and haematocrit were found and hepatic AT, GDH and total lipid were decreased. 5. The changes in hepatic and plasma enzymes and constituents are discussed in relation to possible biphasic effects resulting from EDB exposure.

The opportunity presented by the introduction of a coal-based granular formulation of the carbamate nematocide, aldicarb for use on sugar beet and potatoes in the United Kingdom has been used to evaluate a number of new methods for the assessment of the environmental hazard presented by pesticides. In an intensive field trial with sugar beet, well established biological procedures for recording changes in animal populations and behaviour were combined with a planned programme involving the sampling of animals, plants and soil. Emphasis was placed on sampling and observing mammals in addition to birds which have been more extensively studied in the past. The samples were submitted to a variety of chemical and biochemical analyses designed to reveal the extent to which the pesticide was distributed temporally and spatially and the effects it produced on individuals. The results indicate that although the pesticide is not persistent, it is mobile especially in wet soil, systemic in plants and can be found widely in the local vertebrate fauna for up to 90 days after application. Specific hazards were identified as arising from the ingestion of uncovered granules and from worms containing residues coming to the surface following application in wet conditions. Subsequently a surveillance exercise was mounted on the commercial use of the pesticide at eight sites where sugar beet and potatoes were grown. The hazards identified in the field trial were confirmed and their practical importance assessed, resulting in modifications to the application method in order to eliminate them. The value of the various methods employed in the trials and the significance of the results are discussed. It is concluded that the approach used will find wider and more general use in assessing the environmental hazard posed by new pesticides.

The wood mouse ( Apodemus sylvaticus ) has been used as an indicator species to assess, both in the field and the laboratory, the effects of carbophenothion and, in the laboratory, the effects of chlorfenvinphos as seed dressings on winter wheat. Wood mice were fed in the laboratory with wheat treated with carbophenothion and chlorfenvinphos and the effects on esterase levels were determined for periods of up to 7 days and on returning the mice to untreated wheat. The significant inhibition of plasma acetylcholinesterase and cholinesterase and brain and liver nitrophenyl acetate esterase during the 6-month period after carbophenothion-treated grain was drilled was found to reflect the carbophenothion residue in the gut contents of the mice. The degree of esterase inhibition differentiated mice trapped on the field from those caught in adjacent woodland. Inhibition of the plasma, brain, and liver esterases in wood mice fed wheat treated with the recommended rate of carbophenothion confirmed that the continued esterase inhibition obtained in the field-trapped mice during the field trial was due to chronic pesticide exposure. The pattern of esterase inhibition in the wood mice reflected the persistence of the two organophosphorus pesticides.

Individual di-, tetra-, hexa-, and decachlorobiphenyl isomers have been compared with commercial mixtures of polychlorinated biphenyls by examining the residue levels and the effects on hepatic microsomal enzymes which arise following administration at low levels in the diet of Japanese quail. In a preliminary experiment, 4,4′-dichlorobiphenyl was shown to be rapidly eliminated even at 500 ppm in diet but to affect enzyme activity to a variable extent depending upon the duration and level of feeding. In the major experiment animals were maintained on diets containing 50 ppm of chlorinated biphenyl for 20 days. The residues and effects observed generally increased with the degree of chlorination although individual variations appeared to depend on isomeric structure. The commercial mixtures, Aroclor 1016 and Aroclor 1242, were similar to the di- or less persistent tetrachlorobiphenyl isomers in causing minimal changes in enzyme activity. Aroclor 1254 and 2,4,6,4′-tetrachlorobiphenyl were both persistent and appeared to belong to a new class of microsomal enzyme inducer which produce a spectral shift in the observed cytochrome P -450 spectrum to 448 nm. Most of the other highly chlorinated isomers fed gave rise to large residues and generally to elevated enzyme activities. 3,4,5,3′,4′,5′-Hexachlorobiphenyl appeared to be particularly toxic. Some general conclusions are drawn, which underline the difficulty of treating chlorinated biphenyl isomers as a group of compounds with identical effects on biological systems.

The acute single dose oral toxicity of carbophenothion (S-4-chlorophenylthiomethyl OO-diethyl phosphorodithioate) has been determined in Canada geese (Branta canadensis), pigeons (Columba livia) and Japanese quail (Coturnix coturnix japonica). At post mortem examination gross pathological changes were observed in Canada geese and pigeons and esterase levels were determined by conventional and electrophoretic methods on extracts of liver and brain from these two species. Carbophenothion residue levels were determined in liver, brain and gizzard contents from the geese and pigeons. The overall pattern of results suggests that esterase inhibition may not be the dominant factor in carbophenothion poisoning in geese. It is suggested that a brain carbophenothion residue level of 1 part/106 is indicative of death by poisoning in geese.

Organic pesticides have been used for more than 50 years, and have made a major contribution to the profound alteration of agricultural practice which has occurred during this period. Among the many effects arising from these developments in agriculture, various environmental changes have been identified, which have been the subject of considerable public debate and disagreement. Attention has focussed particularly on the popularly assumed adverse influence of pesticides on the natural flora and fauna. This review examines the position in the United Kingdom where interest in wildlife has always been extensive and where many amateur observations and professional scientific studies have been brought to bear on the problem. It differentiates between ‘direct’ effects which may be attributed to particular uses of certain compounds and ‘indirect’ effects normally arising from changes in habitat or food supply brought about by pesticides. The approach adopted has been to assess the effects of pesticide use on populations rather than attempting to place a financial estimate on any adverse environmental effect attributed to pesticides. Systems of surveillance are described and their ability to detect adverse effects is assessed. The difficulty of correlating such effects with pesticide use is examined. In general, it is concluded that other than for insects the levels of surveillance have been adequate to detect adverse effects on fauna and flora arising from pesticide use. Where direct effects have been discerned in the past, action has, where possible, been taken to rectify the position and it is suggested that this will continue. Overall there is little evidence that the survival of individual species is threatened by the direct effects of pesticides. Indirect effects are neither simple to delineate nor readily corrected and it is recommended that more research be carried out to develop the ability to assess their influence.

It was established in the 1950s that the use of pesticides could present a hazard to nontarget species but it was not until later that the investigation of environmental incidents revealed that other chemicals produced as a result of the recent rapid growth of the chemical industry could also present hazards to wildlife. The identification of problems in the field together with extensive laboratory studies on pollutants has shown that there are some common mechanisms by which many pollutants exert their sublethal or lethal effects on wildlife. The recognition of these mechanisms and thus the identification of hazardous features of novel chemicals forms the basis of the hazard assessment undertaken by chemical registration authorities. This review discusses current knowledge of the mechanisms of action of environmental pollutants on wild vertebrates and attempts to assess the significance of pollutants in the light of this knowledge. It lays emphasis on the primary mechanisms by which chemicals cause the observed change in the wildlife species while recognizing that pollutants often also cause secondary or general responses which are difficult to differentiate from the primary mechanisms. The review briefly considers the mechanisms of action of environmentally significant acute poisons and then reviews in more depth the toxic mechanisms which are responsible for the effects observed after chronic exposure to persistent chemicals. Important biochemical, physiological, developmental, morphological, and behavior mechanisms are presented and discussed with examples.

Following the deaths of blackbirds in 1971 and 1972 an investigation was mounted into the comparative levels of DDT and its metabolites in soil, earthworms, moles, birds and their eggs in a small orchard in East Anglia. The build up of residues in the various organisms was demonstrated. It is suggested that blackbirds and thrushes store DDT in fat through the year. Due to increased food intake or stored fat mobilisation in the spring, there is a large and often lethal build-up of DDE residues in the birds due to a metabolic block. Pheasants appear to be affected directly byt he spraying operation. There appears to be no serious effect on the bird population in the orchards as a result of high residues and individual mortalities. Moles, although apparently not affected by the high DDT residues they carry, could be a hazard to certain predatory birds.

DDT and to a lesser extent DDD were until recently used extensively in agriculture and horticulture as insecticides. The metabolism of DDT was first investigated in the rabbit (WHITE, 1945) but it was only in 1964 that a complete metabolic pathway was proposed for the conversion of DDT to DDA in the rat (PETERSON and ROBISON 1964). This involved the initial conversion of DDT to DDE or DDD and the subsequent conversion of DDD to DDA by a series of alternate dehydrohalogenation and hydrogenation stages with a final oxidation step to DDA.

Techniques are described for the demonstration of animal poisoning arising from the misuse of agricultural chemicals. Data are presented on incidents investigated during the last five years and the implications are discussed.

Fumigation of wheat with methyl bromide causes a change in the polyacrylamide gel, isoelectric focusing profile of esterase proteins from aqueous extracts of wheat. The significance of this effect is discussed in the light of known reactions of methyl bromide in stored products.

Changes in the liver resulting from the low level dietary administration of 1,1-di(p-chlorophenyl)-2-chloroethylene (DDMU),p,p'-DDT, o,p'-DDT, p,p'-DDD and p,p'-DDE to Japanese Quail have been monitored. DDMU was exceptional in causing substantial increases in relative liver wt. and hepatic glucose-6-phosphatase after feeding at 100 ppm for 28 days. The time course of liver enzyme induction by DDMU has also been studied in Japanese Quail after periods of dietary administration ranging from 1--28 days with particular reference to changes in hepatic cytochrome P-450 and relative liver wt. Structural changes in the liver have been followed by reference to protein and lipid components. The hepatic response to DDMU appears to be biphasic. Initially there are substantial increases in hepatic cytochrome P-450 and relative liver wt., but the latter is largely due to accumulation of triglycerides. After approximately 20 days the level of hepatic cytochrome P-450 remain at a high 'plateau' level. This secondary phase of liver induction probably involves cell proliferation. It is concluded that DDMU causes major changes in the avian liver and either directly or through a metabolite causes pronounced microsomal enzyme induction.

Pigeons have been fed diets containing 1000 ppm of the halogenated hydrocarbons, 1,1-di(p-chlorophenyl)-2,2,2-dichloroethylene (DDE) and 1,1-di(p-chlorophenyl)-2,2-dichloroethylene (DDMU) for 21 days and 14 or 28 days, respectively. Some birds died both during and after the period of feeding of DDE. Three birds in the group exposed for 14 days to DDMU-treated food died of starvation, and evidence is presented to show that this food was unpalatable. No deaths occurred in the group of birds fed DDMU-treated food for 28 days. Residues of the fed compounds were measured at intervals after the cessation of feeding, and the results from seven tissues were treated statistically to ascertain the rate of elimination. DDE was shown to be eliminated slowly from all tissues (half-life 250 days), and no metabolites were found. Comparison of the residues from birds which survived to sacrifice with those that died following the cessation of feeding suggest that about 200 ppm in representative tissue such as muscle is indicative of death from chronic DDE poisoning. It is suggested that DDE is also the toxic agent following ingestion of DDT in pigeons. DDMU was shown to be eliminated from all tissues at a rate (half-life 27 days) comparable to that of DDT and DDD. Small residues of 1,1-bis-(p-chlorophenyl)ethane were found. No 1,1-di(p-chlorophenyl)-2-chloroethane (DDMS) was found, and it is suggested that the metabolic pathway of DDT in the pigeon differs from that postulated in the rat. Large increases in average liver weight were found in birds fed DDE or DDMU.

An investigation into the poisoning of birds with 1,1,1-trichloro-2, 2-di (4-chlorophenyl) ethane (DDT) on a fruit farm in South-East England is described. Pesticide levels in wild birds were sufficient to be held responsible for deaths. It is suggested that levels in samples of soil, earthworms, sludge, eggs, etc. did not indicate the source of the poisoning. Examination of caged birds following exposure to normal spraying indicated little hazard from this type of operation. Some discussion of the assessment of levels of intoxication is included.

Hepatic glucose-6-phosphate (G-6-P) dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate (6-P-G) dehydrogenase (EC 1.1.1.44) levels have been investigated in Japanese Quail (Coturnix coturnix japonica) fed diets containing various low levels of p,p'-DDT (1,1-di(4-chlorophenyl)-2,2,2-trichloroethane] or a number of saturated or unsaturated analogues of p,p'-DDT. In general the unsaturated compounds lowered G-6-P dehydrogenase levels and increased 6-P-G dehydrogenase levels. The saturated analogues caused a smaller decrease in G-6-P dehydrogenase levels and had little effect on 6-P-G dehydrogenase activity at this dietary level. p,p'-DDMU [1,1-di(4-chlorophenyl)-2-chloroethylene] exhibited anomalous behaviour in causing elevated G-6-P dehydrogenase levels. These effects may be due to interference in protein metabolism primarily by the unsaturated analogues and metabolites of p,p'-DDT.

The acute oral toxicity of chlorfenvinphos [2-chloro-1-(2′, 4′-dichlorophenyl)vinyl diethyl phosphate] was measured in pigeon (Columba livia), pheasant (Phasianus colchicus), and quail (Coturnix coturnix japonica) and the compound was shown to be particularly toxic to pigeons. Additionally, all three species were fed chlorfenvinphos at 100 ppm in their diet for two or four weeks and esterase measurements were made by conventional and electrophoretic methods on extracts of liver, kidney and brain. The conventionally measured esterase inhibition correlated well with the acute oral toxicity figures. A more detailed study of the histochemically stained electrophoregams showed some discrepancies compared with conventional methods but offered a possible explanation of the inter-species toxicity difference in that it revealed differential inhibition of some brain iso-esterases in pigeon but not in pheasant or quail.

The effects of dietary administration of 1,1-di(p-chlorophenyl)-2-chloroethylene (DDMU) to Japanese quail at a concentration of 100 ppm were investigated for periods of up to 32 days. Hepatic morphology was studied by light microscopy. Histologic changes observed included cytoplasmic and nuclear degeneration in the hepatocytes followed by severe lipid accumulation and hepatocellular hypertrophy. There was a progressive increase in cytoplasmic vacuoles containing lipid up to Day 24 followed by a decrease by Day 32 when the numbers of vacuoles remained greater than those in untreated quail livers. The vacuoles showed a distribution which followed the functional acinar units of the liver. Increased numbers and hypertrophy were observed in Kupffer cells and fibrocytes. There was an occasional necrotic hepatocyte observed but this lesion was not a prominent feature. Hepatocellular hyperplasia occurred as the lipid accumulation decreased. The histologic findings are compared with the biphasic response previously described.

The response of various species of Anser and Branta geese and other avian species to the ingestion of carbophenothion (S-[[(4-chlorophenyl)thio]methyl] O,O-diethyl phosphorodithioate) has been investigated. Optimum assay conditions for measurement of glutamate oxaloacetate transaminase, glutamate dehydrogenase, sorbitol dehydrogenase, and cholinesterase in avian plasma were developed for the study. The administration of acutely toxic doses of carbophenothion to Japanese quail, pigeons, and chickens, and to Greylag, Pink-footed, Greenland White-fronted, and Canada geese led to species-dependent responses for both plasma glutamate oxaloacetate transaminase and cholinesterase levels. Carbophenothion administered to Japanese quail at several dose levels produced changes in plasma enzyme levels which were dependent on dose and time. The level of plasma glutamate oxaloacetate transaminase after dosing in the Anser family of geese rose more rapidly than in the Branta species but no change was found in this enzyme in either chickens or pigeons. In contrast to geese and pigeons, chickens exhibited no plasma cholinesterase inhibition for 3 hr after dosing. These enzyme changes demonstrate a species variation in the toxicological response of birds to a pesticide and indicate the desirability of using more than one avian species for pesticide toxicity testing.

1. Hepatocytes have been isolated from adult Japanese quail in high yields (26-40 X 10(6) cells/g) with 95% viability. Variation in the collagenase and hyaluronidase levels and incubation time used in the isolation procedure affected the yield and viability of the hepatocytes. Quail hepatocytes were more stable in a standard nutrient medium than those of the rat. 2 1,1-Di-(4-chlorophenyl)-2-chloroethylene (DDMU), a metabolite of DDT, depleted quail hepatic GSH levels both in vitro and in vivo, but had no effect on rat hepatocytes. DDMU (0.1 mM) depleted GSH levels in the quail to the same extent as diethyl maleate (a known GSH-depleting agent) (0.04 mM) but the latter acted more rapidly. 3. Pretreatment of quail in vivo with DDMU or phenobarbitone, known inducers of the hepatic mixed-function oxidases, resulted in faster depletion of GSH when the hepatocytes were incubated subsequently with DDMU in vitro. 4. In contrast, 1,1-di-(4-chlorophenyl)-2,2-dichloroethylene (DDE), another metabolite of DDT, did not deplete GSH levels in the quail but did cause some reduction in the rat. Phenobarbitone pretreatment had no effect on GSH depletion by DDE in vitro in quail hepatocytes but enhanced GSH depletion in rat hepatocytes.

The metabolism and excretion of 14C-DDT, 14C-DDD, 14C-DDE and 14C-DDMU were compared in male rats and male Japanese quail after i.p. injection. The rate of excretion of radioactivity was greater in the rat than in the quail for all compounds except DDMU. Skin and fat were major sites of residual radioactivity in both species, the compound administered and DDE accounting for most of the radioactivity except in the case of DDMU. The four radiolabelled compounds were all present in significant quantities in excreta of both species in unchanged forms. The metabolic patterns for DDT and DDD were similar in rat and quail, except that the rat formed 2,2-di(4-chlorophenyl)ethanol (DDOH) from DDT while the quail did not. Rat and quail metabolized DDE and DDMU differently. Ring-hydroxylated derivatives of DDE were formed only in the rat and analogous metabolites of DDMU were produced only by quail. Both species produced di(4-chlorophenyl)acetic acid as a metabolite of all four compounds administered, although the formation was generally less and slower in quail than rat. Comparative metabolism of DDMU with the other compounds indicated that this compound is not a metabolic intermediate in the metabolism of DDT in either rat or quail.

A procedure is described for the determination of low levels of organochlorine residues in avian tissues collected for environmental monitoring. The analytical scheme combines published and modified methods for the identification, confirmation and quantitation of these residues. A hexane extract is cleaned up using chromatography on alumina and silica gel followed by reaction with chromium (VI) oxide before measurement of residues using electron-capture gas chromatography. Identities of a range of compounds are confirmed by chemical derivatisation.

A study in progress to examine the metabolic fate of DDT in birds and mammals is discussed. The first phase of the study, which is reported in this article, has been to establish the rate of excretion of ratioactivity following the intraperitoneal administrations of /sup 14/C-DDT, /sup 14/C-DDE, /sup 14/C-DDD, and /sup 14/C-DDMU to male rats and male Japanese quail. The mean values from the three animals in each experimental group for the amount of radioactivity excreted daily are given, and it was found that the rats excreted the radioactivity administered as DDT, DDD, and DDE substantially faster than did the quail. DDMU was excreted relatively rapidly and at similar rates. This finding suggests that apparent differences in the rates of excretion of DDT by birds and mammals probably arise from differences in the conversion of DDT to DDD or DDE or in the degradation of these metabolites to DDMU. The Japanese quail differ from the rats in excreting substantial amounts of unchanged DDT, DDE, and DDD, which probably reflects the inability of the Japanese quail to readily metabolise these compounds.

The use of the wood mouse ( Apodemus sylvaticus ) as an indicator species to assess the effects of chlorfenvinphos and an organomercurial fungicide used as seed dressings on winter wheat have been investigated. Optimum assay conditions for measurement of glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, glutamate dehydrogenase, and sorbitol dehydrogenase in rodent plasma were developed for this study. The amount of surface grain and the chlorfen vinphos and total mercury residues on the surface grain declined with time after drilling. Wood mice were captured using a grid of small mammal traps placed over the surface of a field sown with treated winter wheat and also in adjacent woodland. No trend in mouse body total mercury was discernible after sowing unlike mouse gut chlorfenvinphos which declined with time. Mice trapped in the wood contained very low levels of chlorfenvinphos and total mercury unlike those mice trapped on the field. Plasma acetylcholinesterase and cholinesterase showed significant inhibition 10 days after drilling and liver nitrophenyl acetate esterase showed significant inhibition during the first 3 days after drilling in mice trapped on the field. This inhibition of esterase activities reflected the chlorfenvinphos residue in the gut contents of the mice. The enzyme changes further differentiated mice trapped on the field from those caught in the wood.

Pigeons have been fed diets containing 1000 ppm of the halogenated hydrocarbons, 1,1-di( p -chlorophenyl)-2,2,2-trichloroethane (DDT) and 1,1-di( p -chlorophenyl)-2,2-dichloroethane (DDD) for 24 days. None of the birds fed either chemical died during the course of the experiment. Residues of these compounds and their metabolites were measured at intervals after the cessation of feeding and the results were treated statistically to determine rates of elimination or change from seven tissues. DDT was shown to be eliminated from all tissues at the same rate (half-life 28 days) and gave rise to DDE and DDD by separate pathways. DDD was shown to be eliminated from all tissues at a similar rate (half-life 24 days) and gave rise mainly to DDMU together with a very small amount of DDE. Large increases in average liver weights were noted in birds fed DDT. With the DDD-fed birds the increase in liver weight was very slight.

Hepatic microsomal protein, cytochrome P-450, aniline hydroxylase, aromatic nitroreductase, phenyl benzoate esterase and total vitamin C, as well as tissue residue levels were measured following the feeding of low levels of 1,1-di( p -chlorophenyl)-2,2-dichloroethylene (DDE) or 1,1-di( p -chlorophenyl)-2,2,2-trichloroethane (DDT) to rats and Japanese quail. Changes in the factors measured were more readily related to the tissue residue levels than to the dietary feeding levels. It is suggested that DDE is a more potent inducer of microsomal protein, cytochrome P-450 and aniline hydroxylase in both species, than DDT fed at the same level, and the effects of DDT were best expressed in terms of the resulting DDE residue levels. Aromatic nitroreductase was not induced by feeding either DDT or DDE, and phenyl benzoate esterase levels showed a biphasic effect following DDE feeding. Vitamin C levels were raised more by DDT than DDE in both species. Residue levels were considerably higher in the quail than in the rat fed at the same rate. Basal levels of all the factors measured were higher in the rat and induction generally occurred to a greater extent and at a lower tissue residue level than in the quail.