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.

TOXIC INTERACTION OF CHLORPYRIPHOS AND COPPER SULPHATE ON CHICKEN EMBRYO.

The single and simultaneous phytotoxic effect of copper sulphate and Pyrinex 48 EC insecticide were investigated on chicken embryos. The eggs were injected by 0.1 ml of copper sulphate solution (0.05%) and/or by 0.1 ml of Pyrinex 48 EC (chlorpyriphos, 480 g/l; 1%). The treatments were performed on day 0 of incubation, and the embryos were examined on day 3 and 19 of it. Germinal disc was prepared to study the early stage of development. Number of embryonic death, developmental abnormalities and body weight of embryos were recorded on day 19. Liver samples were taken for histology and the skeleton was stained by Dawson method. The embryo mortality was not influenced by single treatment of copper sulphate, however, Pyrinex 48 EC and the combination of the test items significantly increased it on day 3. Same tendency was observed in the case of developmental aberration. Single administration of both test items and their combination significantly increased the embryo mortality on day 19. Frequency of abnormalities was not influenced by copper sulphate but single and simultaneous application of insecticide increased it significantly. Pyrinex 48 EC alone and in combination with copper sulphate significantly reduced the body weight, however, the copper sulphate alone did not influence it. Developmental abnormalities were observed sporadically in all cases. There were no findings of drug-induced hepatopathy, however, the ratio of the mitotic cells were markedly reduced. Based on the results, addition and synergistic toxic interaction may be between the copper sulphate and Pyrinex 48 EC that can highly reduce the viability of the embryos.

Toxicity of pendimethalin containing formulation and copper sulphate to chicken embryos.

The aim of this study was to determine the individual and combined toxic effects of STOMP 330 EC herbicide (33% pendimethalin) and copper sulphate on the development of chicken embryos. On the first day of incubation chicken eggs were dipped in the solution or emulsion of the test materials for 30 minutes. Applied concentration of copper sulphate was 0.01% and of herbicide STOMP 330 EC was 1.25%. The chicken embryos were examined for the followings: rate of embryo mortality, body weight, type of developmental anomalies, macroscopic examination. Our teratogenicity study revealed that, the individual toxic effect of copper sulphate and pendimethalin containing herbicide formulation (STOMP 330 EC) were embryotoxic but not teratogenic in chicken. The combined administration of STOMP 330 EC and copper sulphate did not increase the embryotoxic effect.

In vitro eye corrosion study of agrochemicals on isolated chicken eye.

Agrochemicals must undergo numberless toxicological tests before marketing. The eye irritation test is part of this test packet. Nowadays, OECD 405 can be used to classify the irritation potential of substances, the base of the OECD 405 guideline is the Draize test, which is one of the most criticized in vivo methods because of the injuries of the test animals and subjective nature of the test in recording the results. Therefore, several in vitro tests have been developed to replace totally or partly the in vivo eye irritation testing. The isolated chicken eye test method (OECD 438), which was used, is one of these alternative methods. Five different agrochemicals were examined in the following way: All test compounds were applied in a single dose onto the cornea of isolated chicken eyes in order to potentially classify the test compounds as ocular corrosive and/or severe irritant. The damages caused by the test substances were assessed by the determination of corneal swelling, opacity, fluorescein retention and morphological effects. These parameters were evaluated pre-treatment and starting at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test substance exposure) were determined at each of the above time points. Positive and negative controls were used and they showed the expected results in each study. In these in vitro eye corrosives and severe irritants studies, using the Isolated Chicken Eye model with five different products, no ocular corrosion or severe irritation potential were observed. These results correspond to the available information about the tested agrochemicals, so these studies with isolated chicken eye are considered to be successful.

Toxicity of chlorothalonil containing formulation and Cu-sulphate to chicken.

The agrochemicals are being used increasing amounts in plant protection. The plant protection must pay close attention to the decrease or complete elimination of the damage to the environment which affects wildlife, because the cultivated plants provided not only feedstuffs for wild birds but a living and hatching territory as well. The pesticides sprayed during the plant protection activities might have an influence not only on mature birds but also on the embryos in the eggs. The eggs may be exposed to pesticides, the toxic effect of which may appear as embryo lethality and development anomalies. The aim of this study was to determine the individual and combined toxic effects of a chlorothalonil containing fungicide formulation (BRAVO 500) and copper 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 on the first day of incubation. Applied concentration of copper sulphate was 0.01% and the concentrations of fungicide BRAVO 500 were 1% and 10%. The chicken embryos were examined for the followings: rate of embryo mortality, body weight, type of developmental anomalies, light microscopic examination. Our teratogenicity study revealed that, the combined administration of injected copper sulphate and chlorothalonil containing fungicide formulation (BRAVO 500) caused a significant reduction in the body weight of embryos, markedly increased the rate of embryonic mortality and the incidence of developmental anomalies. The joint toxic effect of copper sulphate and chlorothalonil containing fungicide formulation (BRAVO 500) is an additive effect compared to the individual toxicity of the test materials.

Toxicity of S-metolachlor containing formulation and heavy metals to chicken embryos.

Environmental pollution of metal modelled by copper sulphate, cadmium sulphate and a 960 g/l S-metolachlor containing herbicide formulation (Dual Gold 960 EC) 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 copper, and cadmium sulphate was 0.01%. The applied concentration of Dual Gold 960 EC was 0.375%. Evaluation was done on day 19 of the hatching period. In comparison with the values of the control group, the rate of embryomortality also increased significantly in the groups which were treated by herbicide and heavy metals individually. When the herbicide (Dual Gold 960 EC) was used individually, one quarter of the treated animals died in comparison with the values measured in the control group. In the groups which were treated with cadmium sulphate and Dual Gold 960 EC embryomortality increased significantly in comparison with both the control and individually treated groups. In the groups, which were treated together, the occurrence of development disorders remained as low level as in the case individual treatments. Looking at the types of development disorders, the most frequent problems were the oedema, the shortening of the beak mandible, and the incorrect posture of the feet and the neck. As the collective result of the treatment with cadmium sulphate and Dual Gold 960 EC, the body mass of the treated embryos decreased significantly in comparison with the control and individually treated groups, therefore we can conclude that the combined treatment resulted in increased embryo toxic effect in comparison with the individual embryo damaging effect of the used components.

Study of the toxic effects of an insecticide and copper sulphate on chicken embryos.

The toxic effects of the BI 58 EC insecticide (38% dimethoate) applied alone or in combination with copper sulphate were studied on chicken embryo in the early phase of development. The test materials were injected in 0.1-0.1 ml volume into the air chamber of eggs on the first day of incubation. Subsequently, on days 2 and 3 of incubation permanent preparations were made from the embryo in order to study the early developmental stage. Embryos fixed on slides and stained with osmium tetroxide solution were studied under light microscope. According to the result of the statistical evaluation, to sum up, we can say that the simultaneous administration of the test materials did not result in a significant increase in the embryo mortality, but after the combined administration the rate of embryonic mortality markedly increased. As a result of combined administrations the developmental anomalies included the apperance of a blood ring, poor development or absence of somites, the retarted development of the vascular system, the head and the body, irregular differentiation of the brain vesicles. Summarising the findings, it can be established that the insecticide treatment combined with heavy metal resulted in enhanced embryotoxicity in the case of both combinations, which was primarily manifested in an increased embryonic mortality rate.

Study on weevil pests of oilseed rape at Keszthely, Hungary.

One of the most important pests of rape in early spring are weevils (Ceutorhynchus spp.). The aim of our studies was to identify the occurring weevil species and to study their emergence, swarming, mating and damage. Our observations were performed on an experimental plot at Keszthely (Hungary, Zala County) in early spring of four consecutive years. Following species were collected and identified: Ceutorhynchus pallidactylus MARSHAM, Ceutorhynchus obstrictus MARSHAM, Ceutorhynchus napi GYLLENHAL, Ceutorhynchus pleurostigma MARSHAM. The obvious dominance of C. pallidactylus and C. obstrictus was detected. Their frequency of occurrence was 88-90% among the identified adults. We found that C. obstrictus was the last species to settle. Complete developmental period was 68 +/- 7 days in case of C. pallidactylus and 70 +/- 7 days in case of C. obstrictus. Average number of eggs laid by one female was 176 +/- 23 in case of C. obstrictus and 21 +/- 6 in case of C. pallidactylus.

Investigation on possible ecotoxicological risk of carbofuran insecticides.

Carbofuran-containing insecticides are widely used agents in plant protection. Their use may pose considerable environmental risk for both the protected and non-protected predator and plantivorous birds. For defence of wild birds a model experiment was carried out on broiler chickens. In the study, eight animals were treated orally by gastric tube with a carbofuran-containing insecticide at a single dose of 2.5 mg/kg b.w. One animal served as untreated control specimen. Forage and drinking water were provided ad libitum. After the treatment, the possible clinical signs were observed carefully, blood samples were obtained from each bird and after exsanguinations liver, breast and leg muscle samples and stomach content were taken. The carbofuran concentration in blood, tissues and stomach content was determined by gas chromatographic method. Thirty minutes after poisoning, the average carbofuran concentration in breast muscle of chickens exceeded the maximum level of 0.1 mg/kg permitted in edible tissues, whereas ninety minutes after poisoning the concentration of one sample was still above the limit value. In the liver, leg-muscle and blood samples, the measured carbofuran concentration was lower than the permitted maximum value, except in the blood of two animals. The carbofuran concentration of the stomach content markedly exceeded the limit value. The sublethal concentration of the pesticides can reduce the capable of living of wild animals. Due to the sub toxic dose the poisoned birds can survive; however, the residue of insecticides can lead to secondary toxicosis of other animals.

Eye irritation study of some pesticides on chorioallantoic membrane of the egg.

The chorioallantoic membrane of chick embryo has been used extensively for many years in various fields of biological research, including virology, bacteriology and toxicology. 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. A possible model for assessing the irritation potential of a chemical or product to such a vascularised tissue is the choriallantoic membrane of the embryonated hen's egg, as this is a highly vascular, thin membrane with relatively easy access for both treatment and assessment. In recent years various in vitro methods have been developed to replace the heavily criticized Draize rabbit eye test for irritation testing. One of the most studied alternative methods is the Hen's Egg Test - Chorioallantoic membrane (HET-CAM). In our studies a comparative screening was done with a set of pesticides to establish parallel data on in vitro (HET-CAM) and in vivo (Draize) results. In most cases good correlation was found between the HET-CAM assessment and results from the Draize rabbit eye test. The actual form of the HET-CAM test is a valuable pre-screen for predicting ocular irritation potential of chemicals, and can be used to reduce the number of experimental animals. The HET-CAM test is useful as a part of a battery of tests to replace the Draize rabbit eye test.

An alternative test battery in detecting ocular irritancy of agrochemicals.

Using animals in toxicological screening is a controversial issue. To get knowledge about eye irritation, recently only the in vivo Draize-test is accepted, which is one of the most criticized methods because of the injuries inflicted on the test animals. In recent years various in vitro methods have been developed to replace the primary irritation test. Most of these tests are in process of validation. The Draize rabbit eye test, or some modification of this test is essentially the only method for determining ocular irritation that is accepted by authorities. Several in vitro methods have been used to investigate the toxicity of potential eye irritants with the aim of replacing in vitro eye irritation testing. This study reports the results of an alternative approach for predicting irritation potential of agrochemicals. The approach was a two-stage test battery in vitro. The first stage was a cytotoxicity test, the MTT assay. The second stage was the HET-CAM test. The chick chorioallantoic membrane (CAM), being a connective tissue sheet with a visible blood supply, has been proposed as a substrate to identify the eye irritation potential of chemicals. During the HET-CAM test the chemicals are placed directly onto the chorioallantoic membrane. The changes of the vascular injury (haemorrhage, lysis or coagulation) are indications of the potential of the chemical to damage mucous membranes in vivo. MTT assay is a simple method to determine the viability of cells in the presence of a chemical. Cells are cultured with several concentrations of a substance or product, then the ratio of cell destruction is determined. LCC50 (concentration lethal for the 50 % of the cells) is assessed. This is in correlation of the eye irrigative potential of the chemical. In our studies comparative screening was performed with 6 agrochemicals to establish parallel data on alternative test battery (HET-CAM, MTT) and in vivo (Draize) results. In most cases, this study showed a good correlation between in vitro and in vivo data. By these results the present form of an alternative test battery (HET-CAM and MTT) can be proposed as a prescreen method of eye irritation tests.

Estimation of nutrient load from urban diffuse sources: experiments with runoff sampling at pilot catchments of Lake Balaton, Hungary.

About a quarter of the total nutrient loading of Lake Balaton (Hungary) originates from urban diffuse sources, mostly from direct shoreline watersheds. This load cannot be measured directly. Sampling of urban runoff can help improving load estimations. The dynamic processes characterizing the accumulation and washoff of contaminants suggest that randomly observed concentrations are likely under- or overestimated. The results of two recent pilot programs aimed towards achieving continuous measurement of nutrient load carried by urban runoff are introduced. Stations were implemented in two pilot catchments located on the shore of Lake Balaton. Storm event runoff was sampled automatically and manually. Discharge, precipitation and rainfall intensities were also recorded. Results proved that the more a specific pollutant is associated with solid particles, the more of its load comes from a few but large storm events, nevertheless the cumulative effect of small rainfall events is not negligible, either. Event mean concentrations of solid-related pollutants were found to be dependent on rainfall intensity. The derived empirical relationships for SS, TP and TN event mean concentrations were indeed found to be applicable for reducing the uncertainty of load estimations of these pollutants significantly, as compared to using long-time average (i.e. annual mean) concentration values.

The use of HET-CAM test in detecting the ocular irritation.

Agrochemicals must undergo numerous toxicological tests before registration. One of these experiments is the examination of eye irritation potential. To get knowledge about eye irritation, recently only the in vivo Draize-test is accepted, which is one of the most criticized methods because of the injuries inflicted on the test animals. Several in vitro methods have been used to investigate the toxicity of potential eye irritants with a view to replacing in vivo eye irritation testing. In the HET-CAM test chemicals are placed in direct contact with chorioallantoic membrane of the hen's egg. The occurrence of vascular injury or coagulation in response to a compound is the basis for employing this technique as an indication of the likelihood that a chemical would damage mucous membranes (especially the eye) in vivo. In our studies comparative screening was performed with a set of agrochemicals to establish paralell data on in vitro (HET-CAM) and in vivo (Draize) results in case of 6 agrochemicals. The solutions to be tested are added to the membrane and left in contact for 5 minutes and the membrane is examined for vascular damage at set time periods. Irritancy is scored according to the severity and speed at which damage occurs providing an indication of the likely irritant effect of the compound. Our study showed good correlation between results obtained by the HET-CAM test and those of the Draize rabbit eye test most cases. The present form of the HET-CAM test can be proposed as a pre-screen method of eye irritation tests.

Toxicity study of agrochemicals on chorioallantoic membrane of the egg.

Using animals in toxicological screening is a controversial issue. The Draize eye irritation test is one of the most criticized methods because of the injuries inflicted on the test animals. That's why several various tests have been developed to replace the rabbits in detecting the irritation potential of chemicals. The potential irritancy of compounds may be detected by observing adverse changes which occur in the chorioallantoic membrane (CAM) of the egg after to test chemicals. During the test the chemicals are placed directly onto the chorioallantoic membrane. The changes of the vascular injury (haemorrhage, lysis) or coagulation in response to a compound is an indication of the potential of the chemical to damage mucous membranes. The CAM is a complete tissue including arteries, capillaries and veins, and is technically easy to study. It responds to injury with a complete inflammatory reaction, similar to the tissue of the rabbit eye. In our experiment 6 agrochemicals were tested on the chorioallantoic membrane of the hen's egg. In most cases good correlation was found between the HET-CAM results and reported data based on the Draize eye test. The present form of the HET-CAM test can be proposed as a pre-screen method of eye irritation tests, therefore the number of test animals can be reduced.

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.

Histological examination of CAM treated with irritative pesticides.

The potential irritancy of compounds may be detected by observing adverse changes which occur in the chorioallantoic membrane (CAM) of the egg after exposure to tested chemicals. In our experiment irritating pesticides (Fusilade S, Karathene LC) and a technical component of pesticide (Trend) are tested and their effects are examined on tissue structure of CAM. After 10-11 days of incubation of chicken embryos the chorioallantoic membrane becomes trilaminar. The outer layer is chorionic epithelium between the two rows of cuboidal cells, thin-walled vascular sinuses can be found. In the intermediate layer, the mesenchyme, well-developed blood vessels can be seen. The inner layer is formed by allantoic epithelium. After the treatment with Fusilade S the wall of the blood vessels was damaged, and blood diffused from the blood vessels. After the treatment with Karathane LC rupture of the wall of the blood vessels could be seen and blood appeared around the blood vessels. Blood vessels of the yolk sac were frequently damaged under CAM.

In vitro ocular irritation toxicity study of some pesticides.

The use of animals in toxicological screening is a controversial issue. The Draize eye irritation test receives particular criticism because of the injuries inflicted on the test animals. In recent years various in vitro methods have been developed to replace the heavily criticised Draize rabbit eye test for irritation testing. One of the best-studied alternative methods is the Hen's Egg Test-Chorioallantoic Membrane (HET-CAM). In the present studies comparative screening was performed with a set of pesticides to establish parallel data on in vitro (HET-CAM) and in vivo (Draize) results. The tested pesticides included Arelon 500 FW (isoproturon), Banvel 480 (dicamba), Dikamin D (2.4 D), Karathane LC (dinocap), Ronstar (oxadiazon) and Modown 4 F (bifenox). In most cases a good correlation was found between the HET-CAM assessment and results of the Draize rabbit eye test. Although the current form of the HET-CAM test is a valuable prescreen method for predicting the ocular irritation potential of chemicals, and can be used for reducing the number of experimental animals, a number of technical problems must still be addressed before these systems can replace whole animal tests. The HET-CAM test can be a useful component of a battery of tests needed for replacing the Draize rabbit eye test.

Effect of immersion fluid temperature on the chicken embryo in teratogenicity tests: short communication.

The influence of immersion fluid temperature on the development of the chicken embryo was studied on the day most commonly used for treating incubated eggs in teratological trials. Embryonated eggs were immersed in tap water for 30 min on the 12th day of incubation at 22-25 degrees C or at incubation temperature without a waiting time or after 30 min. The incubation was then continued and the eggs were processed on day 19 of the incubation period. Treatment of eggs at 22-25 degrees C caused a significant increase in embryonic mortality, while the 30-min waiting time did not exert an influence on embryogenesis.