TOXICITY OF CHLORPYRIPHOS CONTAINING FORMULATION AND HEAVY ELEMENTS (Cd, Pb) TO CHICKEN EMBRYOS.

The phytotoxic effect of cadmium sulphate, lead acetate and chlorpyriphos containing insecticide Pyrinex 48 EC were investigated on chicken embryos on late developmental stage (day 19 of incubation). The eggs were injected by 0.1 ml amount of lead acetate (0.1%), of cadmium sulphate (0.01%), of Pyrinex 48 EC (chlorpyriphos, 480 g/l; 1%) and as a control 0.1 ml of bird-physiologic saline solution (0.75%). The treatments were performed on day 0 of incubation, and the embryos were examined on day 19. Number of embryonic death, developmental abnormalities and body weight of embryos were recorded on day 19. The body weight of embryos reduced significantly, the rate of embryo mortality increased, but teratogenic effect was not realised by the single treatment of lead acetate and cadmium sulphate. By the single administration of PYRINEX 48 EC the average body weight of embryos resulted in a significant decrease and the rate of embryo mortality and developmental anomalies were increased and it was found to be embryo toxic and teratogenic in the embryos. The increased embryo toxic effect can occur by the additive effect of the heavy elements (cadmium sulphate and lead acetate) and the PYRINEX 48 EC.

Determination of carbendazim residues in the eggs, liver and pectoral muscle of Japanese quail (Coturnix coturnix japonica).

The effect of carbendazim, a widely applied cereal seed dressing agent, was studied in Japanese quail (Coturnix coturnix japonica) during an overall period of eight weeks, consisting of a four-week feeding phase and a subsequent four-week excretion period. Body mass and feed consumption of the birds were monitored and residues of the active ingredient were determined by an analytical chemical method. During the eight-week study period, changes (either decrease or increase) directly attributable to the toxic effects of carbendazim were not found either in body mass or in feed consumption. Active ingredient levels exceeding the limit of detection were found in the liver (average: 0.0262 mg/kg) and pectoral muscle (average: 0.0236 mg/kg) of the birds and also in the egg (0.0338 mg/kg) samples. From the results it can be concluded that through the consumption of cereal seeds dressed with carbendazim, this active ingredient can be incorporated into the tissues of animals. Via the food chain, carbendazim can also enter the human organism, where it may cause various pathological changes in interaction with other chemicals.

Toxicity of a pendimethalin containing herbicide formulation and three heavy metals in chicken embryos.

The toxic effects of a widely used herbicide (Stomp 330 containing 33% pendimethalin as active ingredient) applied alone or in combination with three heavy elements (copper sulphate, cadmium sulphate and lead acetate) modelling the heavy metal load of the environment were studied on chicken embryos with injection treatment. The treatment was done on day 0 of incubation. Solutions and emulsions of different concentrations were made from the test materials and injected in 0.1 ml volume into the air space of eggs. The macroscopical evaluations were done on day 19 of the incubation. Summarizing the findings, it can be established that the individual administration of the 33% pendimethalin containing herbicide formulation was less toxic compared to the control group than the simultaneous administration of the pesticide and heavy elements. As compared with each other the results from the combined administrations of the 33% pendimethalin containing herbicide formulation and heavy elements the simultaneous administration of cadmium and the herbicide caused the highest embryomortality while the incidence of developmental anomalies were the highest in the interaction study of the copper and the pesticide.

Teratogenicity testing of a 2,4-D containing herbicide formulation and three heavy metals in chicken embryos.

The toxic effects of a widely used herbicide (Dikamin D containing 72% 2,4-D-amine Na as active ingredient) applied alone or in combination with three heavy elements (copper sulphate, cadmium sulphate and lead acetate) modelling the heavy metal load of the environment were studied on chicken embryos with injection treatment. The treatment was done on day 0 of incubation. Solutions and emulsions of different concentrations were made from the test materials and injected in 0.1 ml volume into the air space of eggs. The macroscopical evaluations were done on day 19 of the incubation. Summarizing the findings, it can be established that the individual administration of the 72% 2,4-D containing herbicide formulation was less toxic compared to the control group than the simultaneous administration of the pesticide and heavy elements. As compared with each other the results from the combined administrations of the 72% 2,4-D containing herbicide formulation and heavy elements the simultaneous administration of cadmium and the herbicide caused the highest embryomortality while the incidence of developmental anomalies were the highest in the interaction study of the copper and the pesticide.

Early embryogenesis study on a dimethoate containing formulation and Cd-sulphate in chicken embryos.

The toxic effects of a widely used organophosphate insecticide (BI 58 EC containing 38% dimethoate as active ingredient) applied alone or in combination with cadmium sulphate modelling the heavy metal load of the environment were studied on chicken embryos in the early phase of development. Solutions and emulsions of different concentrations were made from the test materials and injected in 0.1 ml volume into the air space of eggs on the first day (day 0) of incubation. Subsequently, on days 2 and 3 of incubation permanent preparations were made from the embryos in order to study the early developmental stage. Embryos fixed on slides and stained with osmium tetroxide solution were studied under light microscope. Summarising the findings, it can be established that the embryotoxicity increased after the simultaneous administration of Cd-sulphate and 38% dimethoate containing insecticide formulation compared to the control or the individually treated groups.

Teratogenicity study of some pesticides in chicken embryos.

The use of pesticides involves the risk of poisoning on wild animals. Teratological tests carried out on avian embryos provide useful data for environmental protection and facilitate the development of environment-friendly chemical plant protection techniques. A 30% dimethoate containing insecticide formulation (BI 58 EC) and a 20% benfluralin containing herbicide formulation (Flubalex) and a 960 g/l S-metolachlor containing herbicide formulation (Dual Gold 960 EC) were studied in chicken embryos after single administration by immersion and injection technique. Treatment was done on day 0 of incubation. Applied concentration of pesticides were 0.1% (dimethoate) and 2.05% (S-metolachlor) and 0.375% (benfluralin) corresponding to that used in plant protection practice. Test materials were injected into the air chamber in a volume of 0.1 ml/egg, or eggs were treated by the immersion technique for 30 min. at 37 degrees C. Evaluation was done on day 19 of incubation. Injection treatment: the administration of S-metolachlor and benfluralin did not result a significant decrease in the average body weight of embryos. At the same time the body weight of embryos significantly decreased because of single administration of dimethoate. The embryomortality increased markedly after the administration of test materials (S-metolachlor, benfluralin, dimethoate). Immersion treatment: the administration of S-metolachlor and benfluralin and dimethoate did not result a significant decrease in the average body weight of embryos. The rate of embryomortality was low after the administration of S-metolachlor, benfluralin and dimethoate. After the immersion and the injection treatment the incidences of developmental anomalies were sporadic. In summary it can be established that the injection treatment was more toxic than immersion technique of the test materials in our study.

Irritative effects of some pesticides and a technical component on tissue structure of the chorioallantoic membrane.

The chorioallantoic membrane (CAM) is a complete tissue that responds to injury with a complete inflammatory reaction, this process similar to that induced by chemicals in the conjunctival tissue of the rabbit eye. During the study chemicals are placed directly onto the chorioallantoic membrane and the occurrence of vascular injury or coagulation in response to a compound is as an indication of the potential of a chemical to damage mucous membranes. In our study irritant pesticides (Fusilade S, Karathane LC) and a technical pesticide component (Trend) were tested and their effects on the tissue structures of CAM were examined. After treatment with the test materials, first lysis and then haemorrhage were observed macroscopically on CAM. In histological pictures stained with H-E the rupture of the blood vessel wall was seen and blood was observed around the blood vessels in the middle layer. The histological findings correlated well with the macroscopic appearance in this study. In general a good correlation was found between the HET-CAM results and reported data from Draize test. The subjective nature of the evaluation is reduced through the histological examination of treated CAM. The HET-CAM test can be a useful component of a battery of tests needed for replacing the Draize rabbit eye irritation test.

Teratogenicity test of dimethoate containing insecticide formulation and Cd-sulphate in chicken embryos after administration as a single compound or in combination.

Cd-sulphate and a 38% dimethoate containing insecticide formulation (BI 58 EC) were used as test material in a teratogenicity test in chicken after administration as a single compounds or in combination. The incubated chicken eggs were directly exposed to the applied test materials with injection into the air-chamber in a volume of 0.1 ml/egg before the starting of incubation. Applied concentration of Cd-sulphate was 0.01% and the concentration of pesticide was 0.1%. Evaluation was done on day 19. In test of individual toxicity after injection on day 0 of incubation Cd-sulphate did not cause a significant reduction in body mass of embryos. The rate of embryonic mortality was 26%. After the administration of dimethoate containing insecticide formulation on day 0 of incubation no decrease occurred in embryonic body mass. The rate of embryonic mortality was 31%. The developmental anomalies observed in the different treatment groups occurred sporadically. The simultaneous administration of Cd-sulphate and the dimethoate containing insecticide formulation on day 0 of incubation resulted in expressed embryonic mortality. The rate of embryonic mortality was 93%. In summary, it can be established that the simultaneous administration of Cd-sulphate and the dimethoate containing insecticide formulation on day 0 in studies of chemical interaction exerted an adverse effect on embryonic mortality.

Comparing two methods of examination in the interaction study of a pesticide and a heavy metal.

The use of pesticides in field application involves the risk of poisoning wild animals. The reproduction period of pheasant takes place at the same time as the spraying time of pesticides, which justifies, that we evaluate in a point of the ecotoxicologic view the influence of the pesticide on progressive avian embryo. The most frequent technical way is injecting the exam stuffs to the some part of the embryonated eggs under the bird teratological trials. The advantage of this method is that it can be injected in a correct measured dose into the optional part of eggs. The disadvantage of this method is that it can't model properly the influence on the environment. If adverse effect of the embraced chemical substance on the embryo is experienced under the study, it will be necessary to use an immersion treatment. This procedure shows only the possible indirect influence of the pesticide on the embryo but it can suitably model its influence in plant protection practice. Treatment was done on day 12 of incubation. Applied concentration of heavy element (Cd sulphate) was 0.01% and the concentration of pesticide (Dithane M-45) was 0.2%. Evaluation was done on day 19 of incubation. Injection treatment: the simultaneous administration of Cd sulphate and the 80% mancozeb containing fungicide formulation on day 12 of incubation did not result in a significant decrease in the average body weight of embryos compared to neither the control nor the pesticide individually treated group. At the same time the body weight of embryos significantly decreased because of combined administration as compared to the Cd sulphate treated group. The embryo mortality and the incidence of developmental anomalies markedly increased after the simultaneous administration. Immersion treatment: the combined administration of Cd sulphate and the mancozeb containing fungicide formulation on day 12 of incubation did not result in a significant decrease in the average body mass of embryos compared to neither the control nor the individually treated groups. The number of embryo mortality was very high after the simultaneous administration. The incidence of developmental anomalies was sporadic.

Embryonic toxicity of a mancozeb containing fungicide formulation and Cu-sulphate in pheasant after individual or combined administration.

Cu-sulphate and a 80% mancozeb containing fungicide formulation (Dithane M-45) were studied in pheasants after administration as single compounds or in combination. The test materials were injected directly into the air-chamber of eggs on day 12 of the hatching period and evaulation was carried out on day 23 of incubation. The pheasant embryos were examined for the following: rate of embryo mortality, body mass, type of developmental anomalies, light microscopic examination. After the administration of copper-sulphate on day 12 of incubation, the average body weight of pheasant embryos significantly did not decrease as compared to the control. The embryonic mortality was 68%. After the administration of a mancozeb containing fungicide formulation (Dithane M-45) on day 12 of incubation, the average body weight of embryos did not decrease as compared to the control. The embryonic mortality was 50%. After the individual administration of pesticide, the incidence of developmental anomalies was very high. After the combined administration of copper-sulphate and the 80% mancozeb containing fungicide formulation (Dithane M-45) on day 12 of incubation the embryonic mortality markedly increased. The rate of embryo mortality was 93%. We did not find any degenerative change in the liver tissue in either the control group or the treated groups by light microscopic examination. In summary, it can be established that the simultaneous administration of Cu-sulphate and mancozeb containing fungicide formulation caused high toxicity compared with the individual toxicity of test materials.

Toxicity and degradation of metolachlor (Dual Gold 960 EC) in chicken embryos.

The herbicide formulation Dual Gold 960 EC (960 g/l metolachlor) was applied, ROSS 308 embryonated hen eggs were treated on day 12 of incubation period. The pesticide was diluted in water to a concentration level 0.3% and the emulsion was injected into the air space in a volume of 0.1 ml/egg, or hen's eggs were treated by the immersion technique (30 min). Residues of metolachlor were measured by GC in 14 collected embryo samples on days 13, 15 and 19 of the incubation of chicken embryos, and macro- and microscopic morphological examinations of 49 embryos were performed simultaneously on day 19. Body mass of embryos was weighed on 13th, 15th and 19th day of incubation. After the both treatments the mortality rate of embryos was similar. The average data of body mass showed a significant decrease compared to the control in the immersion study on day 15 and 19 of the hatching period. This phenomenon may only be in connection with the presence of metolachlor over the limit of quantification (LOQ) on day 13 of incubation period. The macroscopic deformations were sporadic in the embryos. No histologically detected alterations were seen.

Toxicity of a mancozeb containing formulation and Cd-sulphate to chicken embryos after administration as single compounds or in combination.

Environmental pollution of metal modelled by cadmium-sulphate and a 80% mancozeb containing fungicide formulation (Dithane M-45) were studied on chicken embryos after administration as a single compounds or in combination. The test materials were injected into the air-chamber in a volume of 0.1 ml/egg on day 0 of incubation. The concentration of cadmium-sulphate was 0.01%. The applied concentration of Dithane M-45 fungicide was 0.2%. Evaluation was done on day 19 of the hatching period. The individual administration of cadmium-sulphate and the 80% mancozeb containing fungicide formulation did not cause a significant reduction in body weight as compared to the control data. Embryonic mortality increased at all individual treated groups and reached almost a 35% rate. After the individual administration of pesticide, the number of chicken embryos with developmental anomalies did not differ markedly from the control. After the combined administration of cadmium-sulphate and the 80% mancozeb containing fungicide formulation (Dithane M-45) on day 0 of the hatching period embryonic mortality markedly increased. 88% of the treated embryos were dead. Results from the combined administration of cadmium-sulphate and an 80% mancozeb containing fungicide formulation (Dithane M-45) caused higher embryomortality with respect to individual toxicity test of cadmium-sulphate and fungicide in our study.

One-generation reproduction toxicity study of Dithane M-45 (mancozeb) and lead acetate.

The reproductive toxicity of lead acetate and of a fungicide formulation (Dithane M-45) containing 80% mancozeb was studied on rats. Lead acetate was applied in the feed in the following dose groups: control, 1,000, 5,000 and 10,000 mg/kg of diet. The three treatment groups received, in addition to the above doses of lead acetate, 4,500 mg/kg Dithane M-45 in the diet. The method was based on the OECD Guideline for Testing of Chemicals No. 415 (1981). Clinical symptoms and mortality were not found in the parent generation. The body weight of female animals decreased significantly before the pregnancy period. This tendency was also seen in males after the combination treatment. At the two high dose levels a remarkable body weight increase was seen in the female animals during the lactation period. As a result of treatment, decreased body weight of offspring was measured during the lactation period. No gross pathological changes were seen. Histological examination showed general tubulonephrosis in the experimental animals. It can be established that the administration of Dithane M-45 did not enhance the reproductive toxicity of lead acetate.

Embryonic toxicity of a dimethoate containing insecticide formulation and Cu-sulphate in chicken after individual or combined administration.

The aim of this study was to determine the individual and combined toxic effects of a dimethoate containing insecticide formulation and Cu-sulphate on the development of chicken embryos. The test materials were injected directly into the air-chamber in a volume of 0.1 ml/egg, or eggs were treated by immersion technique on day 12. Applied concentration of Cu-sulphate was 0.01% and the concentration of insecticidE was 0.1%. After the injection treatment of a dimethoate containing insecticide on day 12 of incubation, the average body mass of embryos significantly decreased. The simultaneous injections of Cu-sulphate and a dimethoate containing insecticide a statistically significant reduction in embryonic body mass occurred. Embryonic mortality did not increase after the individual injection of test materials, while the combined injection of Cu-sulphate and a dimethoate containing insecticide killed 30% of embryos treated. After the individual and combined immersion treatment of Cu-sulphate and a dimethoate containing insecticide, the average body mass of embryos did not decrease significantly as compared to the control. After the combined immersion treatment the incidence of embryonic mortality and the number of embryos with developmental anomalies did not differ markedly from the control. In summary, it can be established that the combined injection treatment of Cu-sulphate and a dimethoate containing insecticide caused higher embryotoxicity with respect to the test of the combined immersion treatment of test materials.

Toxicity of a mancozeb containing fungicide formulation and CU-sulphate to chicken embryos after administration as single compounds or in combination.

Environmental pollution of metal modelled by copper-sulphate and a 80% mancozeb containing fungicide formulation (Dithane M-45) were studied on chicken embryos after administration as a single compounds or in combination. The test materials were injected into the air-chamber in a volume of 0.1 ml/egg on day 12 of incubation. The concentration of copper-sulphate was 0.01%. The applied concentration of Dithane M-45 fungicide formulation was 0.2%. Evaluation was done on day 19 of the hatching period. The combined administration of copper-sulphate and the fungicide formulation did not cause a significant reduction in body weight as compared to the control data and the results from individual toxicity study of the test materials. After the combined administration of copper-sulphate and the fungicide formulation the rate of embryomortality was 40%. The incidence of developmental anomalies were sporadic. Light microscopic findings exhibited a degenerative change in the liver tissue of combined administration group. Activities of GPT and GOT enzymes increased markedly in the combined administration group. In summary, it can be established that the interaction of copper-sulphate and an 80% mancozeb containing fungicide formulation (Dithane M-45) caused higher embryomortality with respect to the test of individual toxicity of copper-sulphate and fungicide in our study.

Toxicity and degradation of benefin in chicken embryos.

The herbicide formulation Flubalex (20% benefin) was applied, ROSS 308 embryonated hen eggs were treated on day 12 of incubation period. The pesticide was diluted in water to a concentration level of 3.0%, and the emulsion was injected into the air space in a volume of 0.1 ml/egg, or hen's eggs were treated by the immersion technique. Residues of benefin were measured in the samples on days 13, 15 and 19 of the incubation of chicken embryos, and morphological examinations were performed simultaneously. After the immersion treatment the mortality rate of embryos was remarkable compared to the injection treatment. Analytical chemistry data showed the concentration of the active ingredient which was 3.5 times higher on day 13 of incubation in the samples after immersion treatment than after the injection of benefin. This resulted an increased incidence rate of mortality. On day 19 of hatching period the benefin concentration was practically similar independently of treatment method. No macro- and microscopic alterations were seen.

Teratogenicity testing of BI 58 EC (38% dimethoate) in chicken embryos with special respect to degradation of the active ingredient.

The insecticide formulation BI 58 EC was tested for teratogenicity in chicken embryos, with particular reference to degradation of the active ingredient (dimethoate) after the treatment of embryonated eggs. The pesticide was diluted in water to a concentration level of 0.8%, and the emulsion was injected into the air space in a volume of 0.1 ml/egg, or hen's eggs were treated by the immersion technique. Residues of dimethoate were measured in the samples on days, 13, 15 and 19 of the incubation of chicken embryos, and morphological examinations were performed simultaneously. Analytical chemistry data indicated a slower degradation of dimethoate in embryos after the immersion of eggs, and cyllosis was remarkable in this group among the sporadic developmental anomalies. The liver tissues of both treated groups exhibited severe fatty infiltration.

Teratogenicity testing of dimethoate containing insecticide formulation (BI 58 EC) in chicken embryos.

BI 58 EC insecticide formulation was tested for teratogenicity in chicken embryos after the treatment of embryonated eggs. The pesticide was diluted in water to 0.8% concentration level, and the emulsion was injected into air space in a volume of 0.1 ml/egg or hen eggs were treated by the immersion technique. The morphological examinations were done on the days 13, 15 and 19 of incubation of chicken embryos. BI 58 EC produced an increased embryo mortality after the treatment which was the most expressive on day 15 of incubation. The trend of embryo weight showed similarity in the control and treated groups after both treatments. The developmental anomalies were sporadic and dose-effect dependency was not seen. Light microscopic findings exhibited a degenerative change in the liver tissue of both treated groups. In summary, the 38% dimethoate containing pesticide formulation (BI 58 EC) was toxic to the developing embryo at 0.8% concentration in our study.

One-generation reproduction toxicity study of mancozeb and lead acetate.

The reproduction toxicity of lead acetate and 80% mancozeb containing fungicide formulation (Dithane M-45) were studied on rats. The lead acetate was applied in diet at the following dose groups: Control-1,000-5,000-10,000 mg/kg. Three treatment and a control groups were applied, 4,500 mg/kg Dithane M-45 was administered in all the dose levels simultaneously in diet. The basis of the method was the OECD Guideline for Testing of Chemicals No. 415. Clinical symptoms and mortality were not found in the parent generation. The body weight of female animals diminished significantly before the pregnancy period. This tendency was also seen on males after the combination treatment. Remarkable body weight growth of female animals was observed during lactation period at the two high dose levels. Diminished body weight data of offsprings were measured after treatment at the end of the lactation period. The histological examination showed a general tubulonephrosis in the trial. Summing up, it can be established the administration of fungicide Dithane M-45 did not increase the toxicity of lead acetate.

Teratogenicity test of dimethoate containing insecticide formulation and heavy elements (Cu, Cd) in chicken embryos after administration as single compounds or in combination.

The teratogenic effects of heavy elements (Cu-sulphate, Cd-sulphate) and a 38% dimethoate containing insecticide formulation (Bi 58 EC) were studied on chicken after administration as a single compounds or in combination. The test materials were injected directly into the airchamber with an injector on day 12 of incubation. Applied concentrations of heavy elements (Cu-sulphate, Cd-sulphate) were: 0.01-0.001% and the concentration of pesticide was 0.1%. Final volume was 0.1 ml/egg. Evaluation was done on day 19 of incubation. After the administration of heavy elements (Cu-sulphate, Cd-sulphate) on day 12 of incubation, the average body mass of embryos significantly decreased at all the treated dose groups as compared to the control. Embryonic mortality did not increase at all the two dose levels of Cu-sulphate and at 0.001% conc. of Cd-sulphate, while the highest concentration of Cd-sulphate killed 20% of embryos treated. The number of embryos with developmental anomalies did not differ markedly from the control. The simultaneous administration of heavy elements (Cu-sulphate, Cd-sulphate) and dimethoate containing insecticide a statistically significant reduction in embryonic body mass occurred at all the treated dose groups. The incidence of developmental anomalies was markedly higher after the simultaneous use of heavy elements (Cu-sulphate, Cd-sulphate) and dimethoate containing formulation than in tests based on the separate use of heavy elements. Embryonic mortality increased at all the treated dose groups and reached almost 20% rate. In summary, it can be established that the simultaneous administration of heavy elements (Cu-sulphate, Cd-sulphate) and the dimethoate containing insecticide caused higher toxicity with respect to test of individual toxicity of heavy elements.