Sexual behavior in Japanese quail as a test end point for endocrine disruption: effects of in ovo exposure to ethinylestradiol and diethylstilbestrol.

Chemicals having a capacity to disturb the endocrine system have attracted considerable interest during recent years. There is a shortage of well-characterized in vivo tests with which to study such disturbances in different classes of vertebrates. In the present study, test end points related to reproduction in the Japanese quail were used to examine the estrogenic activity of chemicals. The synthetic estrogens ethinylestradiol (EE(2)) and diethylstilbestrol (DES), used as model compounds, were injected into the yolk of embryonated eggs. After the birds had been raised to sexual maturity, we examined sexual behavior, plasma testosterone concentrations, and testis morphology in adult males. The lowest doses resulting in a significantly depressed male sexual behavior were 6 ng/g egg for EE(2) and 19 ng/g egg for DES. Testis weight asymmetry was increased at 6 ng EE(2)/g egg, but DES had no effect at any treatment level. The area of the androgen-dependent cloacal gland was significantly reduced at 57 ng DES/g egg. No effects on plasma testosterone concentration or body weight following exposure to EE(2) or DES were observed at any dose level. Depressed male sexual behavior was the most sensitive of the end points studied, and we suggest that this ecologically relevant end point be included in avian in vivo testing for neuroendocrine disruptors.

Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife.

An expert meeting was organized by the World Health Organization (WHO) and held in Stockholm on 15-18 June 1997. The objective of this meeting was to derive consensus toxic equivalency factors (TEFs) for polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) and dioxinlike polychlorinated biphenyls (PCBs) for both human, fish, and wildlife risk assessment. Based on existing literature data, TEFs were (re)evaluated and either revised (mammals) or established (fish and birds). A few mammalian WHO-TEFs were revised, including 1,2,3,7,8-pentachlorinated DD, octachlorinated DD, octachlorinated DF, and PCB 77. These mammalian TEFs are also considered applicable for humans and wild mammalian species. Furthermore, it was concluded that there was insufficient in vivo evidence to continue the use of TEFs for some di-ortho PCBs, as suggested earlier by Ahlborg et al. [Chemosphere 28:1049-1067 (1994)]. In addition, TEFs for fish and birds were determined. The WHO working group attempted to harmonize TEFs across different taxa to the extent possible. However, total synchronization of TEFs was not feasible, as there were orders of a magnitude difference in TEFs between taxa for some compounds. In this respect, the absent or very low response of fish to mono-ortho PCBs is most noticeable compared to mammals and birds. Uncertainties that could compromise the TEF concept were also reviewed, including nonadditive interactions, differences in shape of the dose-response curve, and species responsiveness. In spite of these uncertainties, it was concluded that the TEF concept is still the most plausible and feasible approach for risk assessment of halogenated aromatic hydrocarbons with dioxinlike properties.

Toxicity and distribution in chick embryos of 3,3',4,4'-tetrachlorobiphenyl injected into the eggs.

3,3',4,4'-Tetrachlorobiphenyl (3TCB) was injected into the yolk of embryonated hens' eggs in doses of 4, 20 or 100 micrograms/kg egg. Twenty and 100 micrograms/kg resulted in the death of all the embryos. The hatching rate of the eggs treated with 4 micrograms/kg was 60% while that of the control eggs was 88%. This means that 3TCB is at least 10 000 times more toxic in chick embryos than a previously studied isomer, 2,2',4,5'-tetrachlorobiphenyl (2TCB). 3,3',4,4'-Tetrachlorobiphenyl might be teratogenic in chick embryos since eye and beak deformities were seen in 3 unhatched tetrachlorobiphenyl-treated embryos. In addition rump edema and excess fluid in the body cavity was observed in some embryos. The distribution of the 2 tetrachlorobiphenyls in chick embryos was studied by means of whole-body autoradiography. No differences in distribution, which could explain the extreme difference in toxicity, were detected. High amounts of radioactivity were found in the bile of the embryos, indicating a capacity of the embryos to metabolize and excrete the tetrachlorobiphenyls.

Embryolethality and induction of 7-ethoxyresorufin O-deethylase in chick embryos by polychlorinated biphenyls and polycyclic aromatic hydrocarbons having Ah receptor affinity.

The lethality and 7-ethoxyresorufin O-deethylase (EROD)-inducing potency of some individual polycyclic aromatic hydrocarbons (PAHs) and coplanar polychlorinated biphenyls (PCBs) in chick embryos were measured in order to compare the mechanisms of action of these compounds. In previous studies it was found that coplanar PCBs and certain PAHs have a high embryolethality in the chicken and that they induce embryonic EROD activity. Although the most potent PAHs were almost as embryolethal as the PCBs when injected into hens' eggs 72 h prior to measurement, they were considerably less potent EROD inducers. In the present study, three coplanar PCBs (3,3',4,4'-tetrachlorobiphenyl (TCB), 3,3',4,4',5-pentachlorobiphenyl (PeCB) and 3,3',4,4',5,5'-hexachlorobiphenyl (HCB)) and four of the most toxic PAHs (benzo[a]anthracene (BaA), benzo[k]fluoranthene (BkF), indeno[1,2,3-cd]pyrene (IP) and dibenzo[a, h]-anthracene (DBahA] were administered to chick embryos in different ways, including co-administration. Additive embryolethality was found when BkF and PeCB were co-administered as well as when BaA and DBahA were given simultaneously. The PAHs were more effective as EROD inducers when injected on day 9 (24 h prior to measurement) than when injected on day 7 (72 h prior to measurement). The opposite was found for PeCB and HCB, whereas no difference in potency was noted when comparing TCB injected 24 and 72 h before EROD determination. These substance-related differences were probably due, at least partly, to differences in biotransformation rates. EROD activities found after treatment with high doses of BkF, IP, or DBahA on day 9 were similar to those measured after treatment with PeCB in doses high enough to give maximal induction. Co-administration of high doses of BkF and PeCB did not further increase the activity, indicating that the PAHs and coplanar PCBs induce EROD to a common maximal value. To decrease the influence of metabolization of the PAHs on their EROD-inducing potency, EROD was determined early in development (day 8) and soon after treatment (24 h) in one experiment. In that experiment, the PAHs proved to be only a few times less potent EROD inducers in relation to their embryolethalities compared with the PCBs. The results of the present study, a previously observed similarity in pathology between chick embryos treated with PAHs and embryos treated with coplanar PCBs, and the fact that the most toxic PAHs also are the most avid Ah receptor binders suggest that the coplanar PCBs and the PAHs largely exert their toxicity in chick embryos via an Ah receptor-mediated mechanism. The differences between the compounds in their EROD-inducing potency/embryolethality ratios could probably be explained by their different rates of biotransformation.

3,3',4,4'-tetrachlorobiphenyl: metabolism by the chick embryo in ovo and toxicity of hydroxylated metabolites.

The metabolism of 3,3',4,4'-tetrachlorobiphenyl (TCB) has been studied in the chicken in ovo by analysis of bile from chick embryos. Four percent of the [14C]TCB dose injected into the air sac on day 13 of incubation was detected in the bile by day 19. An increase of more lipophilic TCB metabolites was observed by HPLC analysis after hydrolysis of the bile. TCB and three phenolic TCB metabolites were identified and quantified in the hydrolyzed bile: TCB (14 ng/gall bladder), 5-hydroxy-3,3',4,4'-tetrachlorobiphenyl (234 ng/gall bladder), 4-hydroxy-3,3',4',5-tetrachlorobiphenyl (45 ng/gall bladder) and 2-hydroxy-3,3',4,4'-tetrachlorobiphenyl (3 ng/gall bladder). The presence of two other TCB metabolites in the bile, a dihydroxy-tetrachlorobiphenyl and a dihydroxy-trichlorobiphenyl was also indicated. The method used in the present study is well suited for studies of metabolism in avian embryos in ovo. The three TCB metabolites identified all proved to be at least two orders of magnitude less toxic than TCB in a chick embryo test. These metabolites were also shown to bind with significantly lower affinity than TCB to the Ah receptor. TCB, 5-hydroxy-3,3',4,4'-tetrachlorobiphenyl, 4-hydroxy-3,3',4',5-tetrachlorobiphenyl and 2-hydroxy-3,3',4,4'-tetrachlorobiphenyl gave Kd values of 16, 33, 45 and 37 nM, respectively, in the Ah receptor test.

Ecotoxicological risk assessment of environmental pollutants in the Arctic.

Concentrations of such persistent organic pollutants (POPs) as polychlorinated biphenyls (PCBs) are high in certain Arctic animal species. The polar bear, Arctic fox, and glaucous gull may be exposed to PCB levels above lowest-observed-adverse-effect-level (LOAEL) values for adverse effects on reproduction in mammals and birds. However, the dioxin-like congeners seem to be major contributors to the reproductive effects of PCBs and the relative concentrations of these congeners are low in polar bears. Temporal trends for POPs in Arctic wildlife and the sensitivities of Arctic species to these compounds determine the risk for future adverse health effects.

Methods for studying xenoestrogenic effects in birds.

The embryonated bird egg provides a simple whole organism test system that allows examination of xenoestrogenic effects at different levels of biological organisation. Test compounds are injected into the yolk, the albumen or the air chamber at defined stages of embryonic development. Bioavailability and embryonic exposure may be determined by autoradiography and image analysis. Females represent the heterogametic sex (ZW) and estrogens determine differentiation into the female phenotype in birds. Xenoestrogenic effects can be examined by markers of gene expression and anatomical or histological characterization of the gonads and tubular sex organs. Chicks may be raised to sexual maturity and examination of sexual behaviour and reproductive physiology performed. The Japanese quail is a suitable test organism due to its small size and early sexual maturation.

Embryotoxicity of polycyclic aromatic hydrocarbons (PAHs) in three domestic avian species, and of PAHs and coplanar polychlorinated biphenyls (PCBs) in the common eider.

The embryotoxicity of an artificial mixture of 18 polycyclic aromatic hydrocarbons (PAHs) was tested by injection into the yolk sacs of eggs of four avian species: chicken Gallus domesticus, turkey Meleagris gallopavo, domestic duck Anas platyrhynchos and common eider Somateria mollissima. A dose of 2.0 mg kg egg(-1) of the PAH mixture increased the mortality among the embryos of all four species. In the domestic duck, but not in the three other species, there was a significantly increased embryonic mortality at a dose of 0.2 mg kg(-1) of this mixture. All 18 individual compounds in the mixture were tested for embryotoxicity in the chicken. The compound most toxic to chick embryos was benzo[k]fluoranthene. This substance also proved to be highly embryotoxic in the three other species. Previous studies have shown coplanar polychlorinated biphenyls (PCBs) to be much more embryotoxic in the chicken than in other avian species studied. In accordance with this, eider duck embryos proved to be considerably less sensitive to 3,3',4,4'-tetrachlorobiphenyl and 3,3',4,4',5-pentachlorobiphenyl than was previously found for chick embryos. For PAHs, however, chick embryos did not have a higher sensitivity than the other species tested.

The avian egg as a test system for endocrine disrupters: effects of diethylstilbestrol and ethynylestradiol on sex organ development.

Many environmental contaminants are known or suspected to interfere with hormonal function in animals. In vivo test methods to detect and characterize chemicals that disrupt the endocrine system are therefore urgently needed. In this study, we assessed the usefulness of abnormalities of the reproductive organs as test endpoints for estrogenic activity of xenobiotics in Japanese quail embryos. Two synthetic estrogens, diethylstilbestrol (DES) and ethynylestradiol (EE2), were injected into the yolks of embryonated eggs. At a dose as low as 2 ng EE2/g egg, all male embryos became feminized, containing ovary-like tissue in the left testis. The extent of feminization of the testes was determined by measuring the relative area of the ovary-like component. Persistent Müllerian ducts (oviducts) in male embryos, and malformations of the Müllerian ducts in females occurred at 2 ng EE2/g egg and higher doses. DES was approximately one-third to one-tenth as potent as EE2. The morphological changes studied were dose-dependent, indicating that they are useful as test endpoints for estrogenic activity. Feminization of the left testis in males proved to be the most sensitive endpoint. We propose the quail egg as a simple in vivo test system for estrogenic compounds.

EROD induction by environmental contaminants in avian embryo livers.

The CYP1A (EROD)-inducing potencies of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3',4,4',5-pentachlorobiphenyl (PCB126) and benzo(k)fluoranthene (B(k)F) were studied in avian embryo livers. TCDD and PCB126 proved to be much more potent as inducers in the chicken than in the other species examined. This finding is consistent with a considerably higher sensitivity of the chicken compared with a number of other avian species to the embryotoxic effects of these compounds. Furthermore, the relative potencies of the tested Ah receptor agonists as CYP1A inducers differed substantially between species. B(k)F and PCB126 showed similar induction potencies in domestic duck embryos, whereas PCB126 is much more potent than B(k)F in the chicken. Also, the potency of PCB126, relative to that of TCDD, was much lower in quail embryo liver in vitro than in chicken embryo liver. Thus, there are large interspecific differences in birds in the sensitivity to CYP1A inducers and furthermore, the relative potencies of these compounds may differ substantially between species.

Lethality and EROD-inducing potency of chlorinated chrysene in chick embryos.

Chrysene was non-specifically chlorinated and the toxic potency of the mixture formed was studied. The chlorinated mixture was considerably more potent than the parent compound in terms of embryolethality and EROD and AHH induction in a 2-week test in chick embryos. The chlorinated chrysene caused anomalies, including edema and beak defects, similar to those previously found after treatment of chick embryos with coplanar PCBs. Chlorinated chrysene was also much more potent than chrysene as an inducer of EROD in a 72-hour test in chick embryos in ovo and in chick embryo liver in vitro. It seems that the mechanism of toxicity of chlorinated chrysene in chick embryos is similar to that of the coplanar PCBs and other Ah receptor ligands. The effects of the chlorinated mixture were mainly accounted for by 6- chlorochrysene and 6,12-dichlorochrysene.

Levels of dioxin-like compounds in sewage sludge determined with a bioassay based on EROD induction in chicken embryo liver cultures.

A bioassay for the detection of dioxin-like compounds was used to estimate levels in sewage sludge from Swedish sewage treatment plants (STPs). The sludge extracts were HPLC-separated into three fractions containing a) monoaromatic/aliphatic, b) diaromatic (e.g. polychlorinated biphenyls [PCBs], polychlorinated dibenzodioxins and polychlorinated dibenzofurans [PCDDs/Fs]), and c) polyaromatic compounds (e.g. polycyclic aromatic hydrocarbons [PAHs]). The bioassay, which is based on EROD (7-ethoxyresorufin O-deethylase) induction in cultured chicken embryo livers detected dioxin-like activity in all unfractionated extracts and in the di- and polyaromatic fractions of all sludge extracts, but not in the monoaromatic/aliphatic fractions. The levels ranged between 6 and 109 pg bio-TEQ/g sludge (d.w.). In sediment samples from rural lakes in Sweden, levels of about 5 pg bio-TEQ/g (d.w.) have been found. The polyaromatic fractions of the sludge samples were potent in the bioassay, probably due to various PAHs and other polyaromatics in the sludge. The levels of six PAHs that are screened for in the sludge at Swedish STPs accounted for only 3-10% of the observed EROD-induction by the polyaromatic fractions. Consequently, many other polyaromatic EROD-inducing compounds were present in the sludge. Inclusion of a biological test like the chicken embryo liver bioassay in the screening of sludge would improve the ability to detect the presence of bioactive dioxin-like compounds. A theoretical estimation of bio-TEQ concentrations in farm-soil following long-term application of sludge with bio-TEQ concentrations similar to those observed in this investigation indicated that the bio-TEQ levels in soil would increase very slowly over time. The chicken embryo liver bioassay proved useful in assessing levels of dioxin-like compounds in sewage sludge and it gives valuable complementary information to chemical analysis data.

Effects of bisphenol A and tetrabromobisphenol A on sex organ development in quail and chicken embryos.

The plastic monomere bisphenol A (BPA) and the flame retardant tetrabromobisphenol A (TBBPA) were examined for estrogen-like developmental effects on the reproductive organs in avian embryos. The synthetic estrogen diethylstilbestrol (DES) was used as a positive control. The test compounds were injected into the yolk of quail and chicken eggs early during incubation and the embryos were examined 2 d before anticipated hatching. At 200 microgram/g egg, BPA induced Müllerian duct (embryonic oviduct) malformation in female quail embryos and feminization of the left testis (ovotestis) in male chicken embryos. The estrogenic potency of BPA compared with DES was species and endpoint specific. Müllerian duct malformation was the most sensitive endpoint in quail embryos, whereas ovotestis formation was the most sensitive response in chicken embryos. Tetrabromobisphenol A caused high embryo mortality at 45 microgram/g egg in both species, but no estrogen-like effects were observed. Bisphenol A caused mortality only in chicken embryos at 67 and 200 microgram/g egg. To our knowledge, this is the first report on estrogen-like or embryolethal effects of BPA and TBBPA in birds.

Reproductive toxicity in mink (Mustela vison) chronically exposed to environmentally relevant polychlorinated biphenyl concentrations.

Female mink were exposed to a technical polychlorinated biphenyl (PCB) preparation (Clophen A50 [A50]; 0.1 or 0.3 mg/animal/d), one fraction of A50 containing the non- and mono-ortho-chlorinated congeners (0-1-ortho-chlorobiphenyls [CBs]), another fraction of A50 containing the congeners with two to four ortho-chlorines (2-4-ortho-CBs), or an organic extract from Baltic gray seal blubber. The animals were exposed for 18 months, including two reproduction seasons. Among the animals given the highest dose of A50, the whelping frequency was reduced in the second reproductive season, and all kits died within 24 h of birth. Reproduction was also impaired by the lower dose of A50. Daily exposure to the 0-1-ortho-CBs separated from 0.3 mg A50 severely reduced kit survival. Reproduction was not significantly impaired by daily exposure to the 2-4-ortho-CBs separated from 0.3 mg A50 or by exposure to the blubber extract. We conclude that the reproductive toxicity in chronically PCB-exposed mink is caused by the aryl hydrocarbon (Ah) receptor agonists. The lowest-observed-effect level for reproductive impairment was 2.4 ng 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents (TEQs) per kilogram body weight and day (22 pg TEQs/g feed). Ethoxyresorufin-O-dealkylase (EROD) was strongly induced by the 0-1-ortho-CBs and pentoxyresorufin-O-dealkylase by the 2-4-ortho-CBs. High EROD activity was correlated with low kit production, and consequently EROD may serve as a marker for reproductive toxicity by Ah receptor agonists in mink.

Basal and induced EROD activity in the chorioallantoic membrane during chicken embryo development.

The chorioallantoic membrane (CAM) is a highly vascularized tissue that takes part in the respiratory exchange of gases through the eggshell. Although the CAM may be exposed to environmental contaminants, its response to pollutants has not been studied. We examined the cytochrome P4501A (CYP1A)-catalyzed deethylation of 7-ethoxyresorufin (EROD) in the CAM during chicken embryo development. EROD was constitutively present and was inducible by the aryl hydrocarbon (Ah) receptor agonist 3,3',4,4',5-pentachlorobiphenyl (PCB 126). Our results suggest the CAM as a first line of defence of the avian embryo against toxic compounds, but also as a target for CYP1A-activated chemicals.

Sensitivity of embryos from duck, goose, herring gull, and various chicken breeds to 3,3',4,4'-tetrachlorobiphenyl.

Yolks in embryonated eggs from duck, goose, herring gull, and various breeds of chicken were injected with 3,3',4,4'-tetrachlorobiphenyl (TCB). Hens' eggs were injected after 4 days of incubation and eggs from the other species were injected after 5 days of incubation. All breeds of chicken tested were very sensitive to TCB. At a dose of 20 micrograms/kg egg the death rate in chick embryos ranged from 70 to 100% at the end of the experiment by Day 18 of incubation. Liver lesions, hydropericardium, subcutaneous edema, shortened beak, and microphthalmia were found in both dead and living TCB-treated chick embryos. Embryos of the other species tested were considerably less sensitive than chick embryos to TCB. The highest dose administered to these species was 5,000 micrograms/kg egg for ducks and 1,000 micrograms/kg egg for geese and herring gulls. These doses did not affect the viability of the embryos and caused no gross abnormalities.

Defective reproductive organ morphology and function in domestic rooster embryonically exposed to o,p'-DDT or ethynylestradiol.

Environmental pollutants with estrogenic activity have a potential to disrupt estrogen-dependent developmental processes. The objective of this study was to investigate if embryonic exposure to the environmental estrogens o,p'-DDT (1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2,2-trichloroethane; 37, 75, 150 or 300 microg/g egg) and EE2 (17alpha-ethynyl estradiol; 60 ng/g egg) affects the reproductive system in domestic roosters. Following egg injection on Embryonic Day 4, the newly hatched chicks were sexed by cloacal inspection. A skewed phenotypic sex ratio with overrepresentation of chicks deemed as females was observed in the groups exposed to the three highest doses of o,p'-DDT but not in the EE2-exposed group. Normal sex ratios were observed in all groups at adulthood. However, a cloacal deformation seemed to remain in the adult roosters, causing an abnormal semen flow upon semen collection. Semen yield was significantly reduced in both o,p'-DDT-exposed and EE2- exposed birds, whereas semen quality was unaffected. When killed, deformations of the left testis were found in all treatment groups. Image analysis revealed a reduced seminiferous tubular area in the roosters exposed to the two highest doses of o,p'-DDT. Embryonic exposure to o,p'-DDT caused decreased comb weight and right-spur diameter, while EE2 only affected right-spur diameter. In conclusion, this study shows that embryonic exposure to estrogenic compounds can induce permanent effects in male birds. The effects of the two studied compounds were partly similar but o,p'-DDT also induced alterations not seen in the EE2-treated birds.

Mono-ortho-chlorinated chlorobiphenyls: toxicity and induction of 7-ethoxyresorufin O-deethylase (EROD) activity in chick embryos.

Six mono-ortho-chlorinated chlorobiphenyls were compared regarding their toxicity and 7-ethoxyresorufin O-deethylase (EROD)-inducing potency in chick embryos. Three of the tested chlorobiphenyls have a chloro substituent adjacent to the ortho-chlorine, and these congeners were about ten times more potent than the three having a meta-hydrogen adjacent to the ortho-chlorine. These more toxic mono-ortho-chlorinated congeners were, however, about three orders of magnitude less toxic and less potent as EROD inducers in chick embryos than 3,3',4,4',5-pentachlorobiphenyl in a previous similar study. Malformed eyes and beaks, degenerative hepatic lesions and subcutaneous as well as pericardial edema were detected in embryos exposed to the mono-ortho-chlorine-substituted congeners, as was previously found after exposure to the most toxic non-ortho-chlorinated, coplanar chlorobiphenyls. It is concluded that the mono-ortho-chlorinated chlorobiphenyls are considerably less toxic and less potent as EROD inducers than the most toxic coplanar ones. Owing to their relatively high concentrations in technical preparations of polychlorinated biphenyls (PCBs) the mono-ortho-chlorine-substituted congeners may, however, contribute to the overall toxicity of PCBs.

Activities in chick embryos of 7-ethoxycoumarin O-deethylase and aryl hydrocarbon (benzo[a]pyrene) hydroxylase and their induction by 3,3',4,4'-tetrachlorobiphenyl in early embryos.

Basal activities of 7-ethoxycoumarin O-deethylase and aryl hydrocarbon (benzo[a]pyrene) hydroxylase were determined in whole-liver homogenates from chick embryos of different ages, from newly hatched chicks and from chicks a few days old. The enzyme activities increased substantially in chick embryo livers between days five and 10, remained at a fairly constant level until day 19, and reached a peak in activity one day after hatching. The optimal pH value was lower than 7.0 for both enzyme activities. The total increase in activity from day five to one day after hatching was about 300-fold for 7-ethoxycoumarin O-deethylase and about 75-fold for aryl hydrocarbon (benzo[a]pyrene) hydroxylase. Treatment of eggs with 3,3',4,4'-tetrachlorobiphenyl resulted in increased metabolism of both substrates by five-day-old chick embryo livers. The increase in aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity was 14-fold while that of 7-ethoxycoumarin O-deethylase was approximately double.

Effects of chlorinated paraffins on liver weight, cytochrome P-450 concentration and microsomal enzyme activities in chick embryos.

Sublethal doses of three technical preparations of chlorinated paraffins (CPs) (Cereclor 42 (C22-26, 42% Cl w/w), Cereclor 50LV (C10-13, 49% Cl w/w) and Cereclor 70L (C10-13, 70% Cl w/w)) were injected into the yolks of hens' eggs after 4 days of incubation. The liver weight, the cytochrome P-450 concentration in the liver and the liver microsomal activity of aminopyrine N-demethylase (APND), aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) and 7-ethoxycoumarin O-deethylase (ECOD) were determined in chick embryos incubated for 20 days. The degree of chlorination and probably also the carbon chain length of the CPs were of importance for their effects. Cereclor 70L was the most potent in causing increases in liver weight, cytochrome P-450 concentration and APND activity. Cereclor 42 was the least potent in these respects, even causing reduced APND activity. A decrease in AHH activity occurred in chick embryos treated with Cereclor 50LV, and a reduction in ECOD activity was noted as a result of treatment with Cereclor 42 and Cereclor 50LV.