Effects of in ovo injection of carbamates on chick embryo hatchability, esterase enzyme activity and locomotion of chicks

Carbaryl and aldicarb, two carbamate pesticides used extensively throughout the United States, are known to act as acetylcholinesterase inhibitors. We have demonstrated previously that exposure to carbaryl and aldicarb in young chicks caused persistent locomotion alterations with no correlation to esterase inhibition. In this study, we investigated the effects of these carbamates when injected in ovo to chick embryos, at two time periods (days 5 and 15) during incubation. Carbaryl dosed at 45 mg kg-1 egg weight was extremely toxic to the embryos on day 5 of incubation. Hatchability was reduced to 0% as compared to 80% when carbaryl was injected on day 15 of incubation. Aldicarb at 1.5 mg kg-1 egg weight had no major effect on hatchability when injected either on day 5 or day 15 of incubation (hatchability = 90 and 100%, respectively). Plasma, liver and brain esterases were measured in the chick at different time points during incubation and after hatching. Brain acetylcholinesterase (AChE) and liver cholinesterase (ChE) were inhibited significantly during incubation in embryos dosed on day 15 with both carbaryl and aldicarb. Liver carboxylesterase was inhibited significantly during incubation with only the carbaryl treatment. All esterase enzyme activities returned to normal after hatching. Plasma ChE and carboxylesterase levels were not affected with either carbaryl or aldicarb treatment from 8 until 47 days after hatching. Neither carbamate had any effect on brain neuropathy target esterase (NTE) activity either during incubation or after hatching. The locomotion of chicks was affected in both treatment groups until 47 days after hatching. This study indicates that carbaryl and aldicarb may cause long-term delayed alterations in the chicks.     

Toxicity and distribution of 2-methyl-4-chlorophenoxyacetic acid (MCPA) in developing chick embryos

The toxicity of the herbicide Erbitox E30, a commercial formulation of 2-methyl-4-chlorophenoxyacetic acid (MCPA) containing 28% MCPA as sodium-potassium salt and 72% of unknown ingredients, was tested on chick embryos. Sterile aqueous solutions of MCPA were injected into the air chamber at doses of 0, 1.5, 3.0, 6.0, 9.0, or 10.5 mg/egg on day 0 or on day 4 of incubation. The mortality rate for the embryos treated on day 0 of incubation was high in the first 5 days, low from 5-12 days and again increased by 15 days. The 15-day LD50 was 4.4 mg/egg (95% C.I. 3.7-5.3 mg/egg). HPLC analysis of albumen and yolk showed that concentrations of MCPA in the albumen were detectable at 5 min, highest at 7 days and markedly diminished by 14 days of incubation; a significantly lower concentration of MCPA was found in the yolk throughout the incubation period, except at 14 days when the yolk concentration was 4 times higher than the albumen concentration. At 15 days of incubation, MCPA was evenly distributed in the tissues of the embryo. MCPA was more toxic to 4-day embryos; concentrations above 6.0 mg/egg were lethal to all embryos within the first week of incubation. The 15-day LD50 for treatment on day 4 of incubation was 2.8 mg/egg (95% C.I. 2.5-3.2 mg/egg). The liver was affected by treatment with MCPA, being green in treated embryos. However, histological examination revealed few changes in the liver parenchyma.     

Development of opiate tolerance in the chick embyro

Tolerance to morphine was produced in the chick embryo. Eggs were injected with morphine sulfate (MS) (20 mg/kg egg) or H₂O daily starting on incubation day 12. On day 16, embryo activities were recorded and eggs were injected with either MS or naloxone. Activity of H₂O-pretreated controls decreased after both MS and naloxone. Embryos treated with MS from incubation days 12–15 showed no activity change after morphine and responded to naloxone with increases in activity. Baseline rates of distress vocalizations (DV) of 1–2 day old chicks were not affected by MS pretreatment during incubation days 12–19. However, 1 mg/kg MS decreased the rate of DV of control chicks by 90% whereas MS-pretreated chicks were unaffected. At age 4–5 days, the baseline rate of DV and rate after MS were higher in MS-pretreated chicks. However, all chicks showed significant decreases in rate of DV after MS injection. Naloxone increased the rate of DV of paired 1–2 day old chicks, but response of MS-pretreated chicks was significantly greater than controls.     

Effects of fenthion, fenitrothion and desbromoleptophos on gait, acetylcholinesterase, and neurotoxic esterase in young chicks after in ovo exposure

Previous studies have demonstrated that gait is affected in chicks exposed to organophosphorus esters (OPs) that induce delayed neurotoxicity (OPIDN) in adult hens. To investigate the developmental relationship between such functional deficits and OPIDN, chicks were exposed to 3 OPs with different OPIDN potential. Desbromoleptophos (DBL) induces OPIDN in adult hens; fenthion (FEN) has uncertain OPIDN potential; fenitrothion (FTR) does not induce OPIDN. Chicks were treated by injection into the egg on day 15 of incubation, after the presumed period of OP-induced structural teratogenesis. AChE and neurotoxic esterase (NTE) were assayed during incubation and in parallel with post-hatching evaluations of gait. DBL, 125 mg/kg in ovo, caused paralysis in 70% of chicks after hatching. The gait of surviving chicks was affected for at least 6 weeks and marked by toes curling under. NTE was inhibited until 10 days post-hatching and AChE until hatching. FEN did not inhibit NTE significantly, but AChE was significantly inhibited until hatching. Chicks exposed as embryos to FEN were hyperactive and aggressive. Gait was still affected 6 weeks after treatment with 3 mg/kg FEN. FTR at 125 mg/kg inhibited AChE until day 10 post-hatching, but neither inhibited NTE nor affected gait. The growth of OP-exposed chicks was not significantly decreased, so the decreased length and increased width of the stride could not be ascribed to stunted growth. We conclude that OPs cause irreversible effects on gait that are not related to their defined neurotoxic effects, since altered gait (1) occurs below the age of sensitivity to OPIDN, (2) is seen in the absence of NTE inhibition and (3) does not invariably accompany AChE inhibition.     

Drug withdrawal prior to hatch in the morphine tolerant chick embryo

Morphine tolerance and dependence were produced in chick embryos by injecting 20 mg/kg into the air space of the egg daily from incubation day 12. Starting on day 16, two groups of eggs were withdrawn from morphine by either substituting water or by treating with naloxone. Chick embryo activities were monitored on incubation day 18. Activities of the embryos withdrawn from morphine did not differ from control, but naloxone injection produced a substantial increase in activity. Neither regimen of morphine withdrawal affected the hatchability of the eggs. When these chicks were 1 day old, the effect of 1 mg morphine/kg on the rate of distress vocalization was measured. The withdrawn chicks vocalized at a rate that was intermediate between that of controls and that of nonwithdrawn chicks indicating that tolerance was still present one week after the last morphine injection.     

Developmental and behavioral effects of medetomidine following in ovo injection in chicks

Developmental and behavioral effects of medetomidine were assessed in chicks following in ovo exposure on incubation day 4. Medetomidine at 25 and 50 μg/egg injected once into the air cell on incubation day 4, dose-dependently decreased the number of viable chick embryos starting on day 10 of the incubation. The percentages of successful hatching in the control and medetomidine treated groups were 93, 60 and 47%, respectively. Embryo lethalities in these groups were 7, 40 and 53%, respectively. In ovo exposure of the chicks to medetomidine at 25 and 50 μg/egg did not significantly affect the body weight of the chicks as well as their morphometric measurements. In another experiment, 3- and 8-day old chicks exposed to medetomidine in ovo (25 μg/egg) were monitored in the open-field for 5 min. Medetomidine suppressed the open-field activity of both 3- and 8-day old chicks. This was manifested by a significant increase in the latency to move from the central square of the open-field arena and a decrease in the number of lines crossed (ambulation) with an additional decrease in vocalizations of the 3-day old chicks when compared with respective age-matched control values. In the same medetomidine-exposed chicks the duration of tonic immobility significantly increased in comparison with respective control values. Pharmacological challenge of the medetomidine-exposed chicks (8-day old) with medetomidine at 25 μg/kg, intramuscularly significantly increased the latencies to onset of sedation and loss of righting reflex and decreased the duration of sleep when compared with the saline-control group challenged in the same manner. The data suggest that medetomidine could be a behavioral teratogen in chicks following in ovo exposure.     

Toxicity of styrene and styrene oxide on chick embryos

Styrene and styrene oxide were injected into the air space of fertilized chicken eggs at different times during an incubation period of 14 days. The toxicity of styrene and styrene oxide when injected on the fourth day of incubation revealed an LD50 of 40 mumol/egg and 1.5 mumol/egg, respectively. Malformations were found in 0-20% of the embryos, but never in the controls. The results obtained point to a need for further experimental, and possibly epidemiologic, studies on the consequences of styrene exposure.     

Time dependent effects produced in chicks after prenatal injection of methylmercury

Methylmercury dicyandiamide (0.05 to 10 mg/kg egg) injected into the yolk sac of fertilized chicken eggs prior to incubation produced a dose related decrease in the percentage of chicks hatched (90-57% of control). With dosage fixed at 0.5 or 5.0 mg/kg egg and injections made on Days 0, 7 or 14 of incubation, hatches were 90, 68 and 75%, respectively, for the low dose and 63, 13 and 18% for the high dose. In contrast to results obtained from chicks hatched from eggs injected on Day 0 of incubation, chicks hatched from eggs injected with 0.5 or 5.0 mg MMD/kg on Day 7 or 14 were not different from controls in a detour learning situation. Administration of 14-C methylmercury revealed maximal brain radiolabel in embryos injected on Day 0 to be 10% that seen with eggs injected on Day 7 but twice that seen with eggs injected on Day 14. We tentatively conclude that a period of maximal sensitivity to the behavior effects exists prior to Day 7 and that the mechanisms of embryolethality is different from that producing the functional deficits.     

Effects of in ovo exposure to PCBs 126 and 77 on mortality, deformities and post-hatch immune function in chickens

In laboratory experiments, planar PCBs produce immune organ atrophy in chicken embryos. To study the immunotoxic effects of PCBs in birds, the coplanar congeners 3,3',4,4',5-pentachlorobiphenyl (PCB 126) and 3,3',4,4'-tetrachlorobiphenyl (PCB 77) were injected into the air cell of fertile white leghorn chicken eggs before incubation at doses of 0.25 and 0.5 ng/g egg PCB 126 and 0.64 ng/g egg PCB 77. Mortality and deformities were assessed during incubation of the eggs, and immune function was analyzed post-hatch using phytohemagglutinin (PHA) skin test for T-cell mediated immunity, antibody titers to sheep red blood cells (SRBC), mitogenesis of peripheral blood lymphocytes, and immune organ mass and cellularity. Exposure to 0.25 ng/g PCB 126 elevated mortality (61% and 69%) and deformities (31% and 32%), three or more times higher than controls. Two-fold suppression of antibody titers was observed in 28 day old chicks exposed to PCB 126 or PCB 77. No consistent alterations in PHA skin response or lymphocyte proliferation were observed. In 14 day old chicks in experiment two, PCB 126 decreased thymus and bursa cellularity by 33% and 35%, respectively. Immune organ atrophy was transient, recovering to control levels by 42 days of age. These experiments demonstrate that PCB 126 and 77 suppress antibody responses in juvenile chickens following an in ovo PCB exposure. Results reinforce the need for measuring multiple immune endpoints to detect immunotoxicity.     

Abnormal embryogenesis induced by thiopental

The effects of thiopental on chick embryos were analyzed in the present study. Thiopental was dissolved in saline and injected into embryonating chicken eggs at doses ranging from 0.2 to 4.0 mg per egg. The injections were made into the air sacs of eggs after two to four days of incubation. Control eggs were injected with an equivalent volume of saline (0.1 ml per egg). In all 1080 chicken eggs were used for this study. All embryos were examined on day 7. The LD50 for eggs injected on days 2, 3 and 4 was 2.1, 1.9, and 4.1 mg per egg, respectively. The principal malformations observed were exencephaly, anencephaly, twisted limbs, twisted neck, microphthalmia, everted viscera, and hemorrhage above the left eye and in both cerebral hemispheres. The results of the present study indicate that thiopental has a tendency to cause malformations in the chick embryos tested.     

Oxidative Stress Induced by Diclofenac Alone and under the Influence of Certain Variables in Broilers

Diclofenac toxicity was studied in male broiler chicks (Cobb strain) of a day old age. The chicks were randomly divided into eight groups consisting of 10 in each group. Group 1 was kept as basal diet control (1-32 days), group 2 on basal diet for 32 days + diclofenac (0.8 mg/kg body weight I/M) on day 24, 26, 28, 30, and 32, group 3 on basal diet for 32 days + cyclophosphamide (50 mg/kg body weight I/M once daily) from day 20 to 23, group 4 on high-protein, high-calcium, low vitamin A (HPHC) diet (1-32 days), group 5 on basal diet + diclofenac + cyclophosphamide (as per the schedule), group 6 on HPHC + diclofenac (as per the schedule), group 7 on HPHC + cyclophosphamide (as per the schedule), and group 8 on HPHC + diclofenac + cyclophosphamide (as per the schedule). The activity of TBARS, SOD, and catalase revealed a significant (P< 0.05) increase, while there was a significant (P < 0.05) decrease in the concentration of GSH in liver in the groups given diclofenac either alone or in combination with other variables. There was no significant difference in the diclofenac residue levels. The electron microscopy of liver revealed lesions of mild to marked severity in different combinations. It can be concluded that diclofenac has the toxic potential in poultry at subtherapeutic doses and further the toxic effects were more pronounced under the influence of immunosuppressants and HPHC diet.     

Effects of injected methylmercury on the hatching of common loon (Gavia immer) eggs

To determine the level of in ovo methylmercury (MeHg) exposure that results in detrimental effects on fitness and survival of loon embryos and hatched chicks, we conducted a field study in which we injected eggs with various doses of MeHg on day 4 of incubation. Eggs were collected following about 23 days of natural incubation and artificially incubated to observe hatching. Reduced embryo survival was evident in eggs injected at a rate of ≥1.3 μg Hg/g wet-mass. When maternally deposited Hg and injected Hg were considered together, the median lethal concentration of Hg (LC₅₀) was estimated to be 1.78 μg Hg/g wet-mass. Organ mass patterns from eggs of chicks injected at a rate of 2.9 μg Hg/g differed from that of controls and chicks from the 0.5 μg Hg/g treatment, largely related to a negative relation between yolk sac mass and egg mercury concentration. Chicks from eggs in the 2.9 μg Hg/g treatment were also less responsive to a frightening stimulus than controls and chicks from the 0.5 μg Hg/g treatment. We also found that the length of incubation period increased with increasing egg mercury concentration. Tissue Hg concentrations were strongly associated (r ² ≥ 0.80) with egg Hg concentration.     

Permanent chondrification in the pelvis and occurrence of hernias in mice treated neonatally with tamoxifen

Male and female C57BL/Tw mice were given 5 daily subcutaneous injections of 100 micrograms tamoxifen (Tx), starting on the day of birth (Tx mice). In untreated fetal mice on day 18 of gestation, the greater part of the pubic and ischial bones were cartilaginous. At more than 30 days of age, however, untreated mice showed completely calcified pelvic bone, whereas in age-matched Tx mice the greater part of the junctional regions in the pelvis remained cartilaginous. Treatment with Tx starting within 5 days of age caused bladder hernia with or without cecum hernia. The pubic ligament in Tx mice at ages of 30-540 days was markedly expanded as compared with that in age-matched controls. The permanent chondrification in the pelvis was found in all mice given Tx starting within 10 days of age. By contrast, neonatal treatments of mice with other antiestrogens, clomiphene and nafoxidine (100 micrograms/day), induced neither permanent chondrification in the pelvis nor expansion of the pubic ligament nor hernia. These findings suggest that Tx has a specific effect on the symphysis pubis and some junctional regions of the developing pelvis in mice when given neonatally.     

Gait analysis of chicks following treatment with tri-ortho-cresyl phosphate in ovo

Chick embryos were treated during late embryonic development with tri-ortho-cresyl phosphate (TOCP), an organophosphate compound that causes delayed neurotoxicity in humans and some other species. Embryos were treated on incubation d 14 with either 62 or 250 microliters TOCP/kg egg. The higher dose reduced the number hatched, and signs of cholinergic toxicity were apparent in the newly hatched chick. All chicks that survived this dose were unable to stand. Recovery from the cholinergic effects occurred within a few days after hatching, but the chicks remained severely ataxic through 3 wk of observation. The mortality of embryos treated with 62 microliters TOCP/kg egg was not higher than that of controls, and young chicks showed no overt signs of cholinergic toxicity or ataxia. Motor impairment was detected by measuring gait parameters. These chicks had a short stride and walked with a more open angle of foot placement. These are adjustments in gait that provide a more steady base of support. The change in gait developed over a 3-wk period after hatching. The hindlimb motor impairment detected at both doses is consistent with neuropathy such as is seen in the adult chicken. The value of gait analysis is the ability to quantify effects that are not apparent by simple observation.     

Evidence for embryotoxicity of gossypol in mice and chicks with no evidence of mutagenic activity in the Ames test

Effects of gossypol treatment were studied in pregnant mice and chick embryos. Pregnant Balb C mice were treated orally with 60 or 120 mg/kg of gossypol acetic acid on days 6-13 of pregnancy and killed on day 18. The uteri were removed, the number of resorptions and late fetal deaths were recorded, and the fetuses were weighed and assessed for malformations. Fertilized hen eggs were injected with 0.25 mg gossypol/egg at 24, 48, 72, or 96 h of incubation. The embryos were examined at day 9 of incubation. The mutagenic potential of gossypol was determined by the Ames test. Treatment of mice with gossypol produced significant adverse effects on the dam and offspring including decreased pregnancy weight gain of the dam and growth retardation of the offspring. There were increased resorptions and late fetal deaths in mice and high mortality in chick embryos. Exencephalic fetuses were observed in one of four litters exposed to the higher dose of gossypol, micromelia was observed in one of 26 chick embryos treated at 24 h, and gastroschisis was observed in one of 21 chick embryos treated at 72 h. No malformations were observed in the controls. The number of revertants per plate in the gossypol treatment group (100 or 500 mg gossypol/plate) did not differ significantly from that of control. This study provides evidence that gossypol has embryotoxic and possibly teratogenic activity in mouse and chick embryos but no mutagenic activity according to the Ames assay.     

Development of esterase activities in the chicken before and after hatching

The embryonic chick has long been a model for developmental biology and has often been recommended as a model system in developmental toxicology. More recently, several investigators have shown that the chick embryo also provides a good model for identifying the neurotoxic effects of environmental pollutants, especially cholinesterase-inhibiting pesticides. Although numerous studies detail the structural development of chick embryos, few describe embryonic levels of enzyme synthesis and their changes during development. In this study, the development of esterase activity in chick embryos was measured from day 9 of incubation until 46 days after hatching. Brain acetylcholinesterase (AChE) activity was detected on day 9 of incubation at a concentration of 0.364 mumoles/min/g tissue. An increase between AChE activity and age of the embryos was observed. In the liver, the nonspecific cholinesterases (ChE) and carboxylesterase activities during incubation were not different from activities after the chicks had hatched. Plasma ChE and carboxylesterase activities did not change with age after hatching. Brain neuropathy target esterase (NTE) activity was not detected on day 9 of incubation and was extremely low (6.12 nmoles/15 min/mg protein) the next day, but increased rapidly with increasing age. This study demonstrates that chick embryos have developed esterase activities in the brain and liver by day 10 of incubation and again confirms that the insensitivity of chick embryos and young chicks to organophosphorus ester-induced delayed neurotoxicity is not due to absence of NTE. In addition, the results provide baseline data for evaluating the response of embryonic and immature chicks to neurotoxicants and teratogens.     

The teratogenic effect of phenobarbital on the embryonic chick heart

Phenobarbital was observed to produce cardiovascular malformations in embryonic chicks. Malformations included simple ventricular septal defect, ventricular septal defect associated with dextroposition of the aorta, double outlet right ventricle, and several types of aortic arch anomalies. Embryos were exposed to phenobarbital at doses of 1-25 mumol on day 4 of incubation (Hamburger-Hamilton developmental stage 24). Doses equal to and greater than 5 mumol phenobarbital (26 mg/kg egg) significantly increased the frequency of embryos with cardiovascular malformations compared with lesser doses and with saline. A significant reduction in heart rate and abnormal rhythm of the heart were observed in embryos treated with teratogenic doses of phenobarbital. No arrhythmia nor significant changes in heart rate were observed in embryos exposed to subteratogenic doses of phenobarbital or to saline.     

Penetration studies and residue determinations of 2,4-dichlorophenoxyacetic acid butyl ester in fertile hen eggs and chicks hatched from treated eggs

Fertilized hen eggs were topically applied with 2,4-Dichlorophenoxyacetic acid butyl ester (3.1 mg/egg) before starting incubation. During the incubation time 20% of the compound applied on eggs was lost by volatilization. At different incubation times (0, 1, 5, 10 and 15 days) the wash-off technique was applied to fertile hen eggs and residue analyses of 2,4-D ester were determined and quantified by gas chromatography with an electron capture detector. We found that 2,4-D ester began to be detectable at the embryo from the 5th incubation day and the amount of the compound progressively increased during chick embryonic development. Furthermore chicks hatched from treated eggs showed the presence of the compound in all studied tissues and the highest levels were found in organs such as brain and kidney.     

Chick embryo exposure to carbamates alters neurochemical parameters and behavior.

Recent evidence has shown that exposure to pesticides can lead to long-term neurophysiological and functional deficits. We have demonstrated previously that locomotion in chicks exposed to some organophosphates and carbamates could be altered persistently without concomitant central or peripheral esterase inhibition. Furthermore, histopathology of the ataxic chickens showed no lesions in either the central or peripheral systems. In this study, we examined whether locomotion alterations seen in chicks exposed in ovo to carbaryl and aldicarb are accompanied by perturbations in particular central neurotransmitter systems. Carbaryl and aldicarb were injected in ovo on day 15 of incubation at 6, 16 and 65 mg kg-1 egg weight and at 0.2, 0.4 and 35 mg kg-1 egg weight, respectively. Neurotransmitter levels (assayed by high performance liquid chromatography with electrochemical detection) and locomotion were measured at various times (1-43 days) after dosing. At the lower doses of both carbaryl and aldicarb, a trend towards prolonged decreases in cerebral dopamine and homovanillic acid was seen. The high dose of carbaryl significantly reduced dopamine and the high dose of both compounds significantly decreased homovanillic acid and 5-hydroxyindoleacetic acid. Persistent locomotion alterations were observed only at the higher doses of both carbaryl and aldicarb when the specific neurotransmitters measured returned to normal levels.     

Visual deficits and retinotoxicity caused by the naturally occurring anthelmintics,Embelia ribes and Hagenia abyssinica

The naturally occurring anthelmintics, Embelia ribes (Enkoko) and Hagenia abyssinica (Kosso), have been reported to possibly cause optic atrophy among the Ethiopian population. In this study we found retinal pathology and defects in visual behavior in chicks treated with Enkoko, Kosso, or embelin, a crystalline extract of E. ribes. The chicks were fed orally with a high dose of 0.25 g (5 g/kg) or a low dose of 0.025 g (0.5 g/kg) per day of Enkoko or Kosso, beginning on Day 2 of life. The high dose for Enkoko was administered for 1 or 5 days, while that for Kosso was administered for 1 or 9 days. For the low dose of both Enkoko and Kosso, the dosing regime was for a period of 1, 4, or 9 days. Embelin was administered at a dose of 0.001 g (0.02 g/kg) per day for 9 days. Control chicks were force fed an equivalent amount of chick feed. Treatment with Enkoko or Kosso significantly reduced the ability of chicks to detect a moving bead introduced into the peripheral field of vision. The degree of constriction of the visual field for detection was dependent upon the total amount of drug administered. Performance on a visual discrimination task, which required discrimination of feed grains from pebbles, was also impaired in chicks treated with total doses of 0.200 and 0.250 g of Enkoko or Kosso. Thus, the extent of deficit in visually guided tasks was found to be dose dependent. The visual deficits observed in Enkoko-treated chicks were mimicked by embelin, which suggests that embelin may be responsible for the visual defects. Anatomical evidence of degeneration of ganglion cells was found in retinae exposed to high doses of Enkoko (1.25 g) and Kosso (2.25 g). However, no retinal lesions were detected in chicks following treatment with cumulative doses of less than 0.25 g of Enkoko or Kosso. Similarly, retinal pathology was not observed in embelin-exposed retinae.