Model study on chicken embryos to investigate potential teratogenic risk of wild birds due to pesticide and environmental contaminant

Single and simultaneous toxic effects of glyphosate (Amega Up, 360 g/L, 4%) and copper sulphate (0.01%) were studied on avian embryos treated with injection directly into the air chamber or by immersion application for 30 minutes on day 0 of incubation. Alterations of the chicken embryos were evaluated during the necropsy performed on day 19 of incubation including the mortality, the body weight, and the type of developmental abnormalities. Based on the results, the injection application was to be more toxic than the immersion method, induced increasing of mortality and decreasing of body weight, and the incidence of the congenital anomalies was more frequent. Supposedly, an additive-type toxicodynamic interaction was occurred between the copper sulphate and glyphosate that may result in reduced vitality of the embryos and thus the number of offspring of wild-life birds.


Introduction
Pesticides are used mainly in agriculture to control pest animals, fungi, and weeds. Application of pesticide in agriculture helps to increase the yield, improve the quality as well as extend the storage life of food crops. As plant protection products are biologically active, they have the potential to affect nontarget organisms, particularly in terrestrial environments where exposure may be continual. Excessive use of pesticides may lead to the destruction of biodiversity. Many birds, aquatic organisms and animals are under the threat of harmful pesticides for their survival (Mahmood et al. 2016).
Besides the adult birds, also embryos in the eggs are threatened, because these chemicals can penetrate the porous eggshell and increase the mortality or development anomalies of embryos as well (Kertész 2001).
An ecosystem can be contaminated by pesticides sprayed alone or applied together with other xenobiotics (e. g. heavy metals). Thus, the chemical load can generally appear in a complex way, therefore the simultaneous toxic effect and interaction of the chemical agents can be expected at the same time.
Over the past 30 years, there has been a signi cant increase in number of studies on the effects of pesticides including their accumulation and their impact of wildlife. In the 1960's and 1970's was a dramatic decline in wildlife numbers resulted from the use of pesticides based on organochlorine compounds, that can be traced back to the death of the birds of prey (Köhler and Triebskorn 2013). The components can modify each other's toxic effect. Therefore, the examination of the combination of heavy metals and other chemicals gained signi cant ground in both avian (Fejes et al. 2001;Kertész and Hlubik 2002) and mammalian (Institóris et al. 2001;Pecze et al. 2001) toxicological research studies. Furthermore, the interaction effects are examined not only in the eld of ecotoxicology, but also in all other areas dealing with health care and chemical safety issues (Oskarsson 1983;Danielsson et al. 1984;Speijers and Speijers 2004).
Pesticides have been used for centuries. Copper has been used in agriculture due to its fungicide and bactericide properties for over a century (La Torre et al. 2018). Copper, in trace amounts, is essential for the normal development of living organisms, as it is a cofactor in many enzymes responsible for important processes in cells. On the other hand, it can trigger the production of free radicals, which can damage proteins and DNA (Ogórek et al. 2017). Copper is strongly bioaccumulative. Biological activity is a major factor in determining the occurrence and distribution of copper in the ecosystem (Aaseth and Norseth 1986;TOXNET 1975TOXNET -1986. It can be found in agricultural soils in varying degrees originated from agrarian sources, e. g. manure, sewage sludge, fertilizers, and pesticides (Mantovi 2003). Copper is important in integrated pest management. In organic farming it is almost exclusively used to control many bacterial and fungal diseases. However, the long-term consequences of its accumulation in the soil cannot be ignored (La Torre et al. 2018) since it can be transferred easily to animals and humans as well through food chains. More than the essential amount can be toxic, but sensitivity to the toxicosis is species dependent. Generally, poultry resist chronic toxicosis better than most mammals (NRC 1977). The contamination of agricultural soil with copper is a concern for the state of our environment and food safety (Komárek et al. 2010) The usage of chemical pest control began around the time of World War II, because of the development of the synthetic organic chemical industry.
In the 1970s glyphosate came on the market what is the world's one of the most commonly used herbicidal active ingredient present day. It is a broad-spectrum herbicide, used in weed control in agriculture, and vegetation control in non-agricultural areas (Solomon et al. 2007.) Thus, based on the widespread use it may have negative effects on animal and human health due to possible residue of glyphosate in feed and foods (Blaylock 2015;Cox 1995;Faria 2015;Krüger et al. 2014).
The herbicide glyphosate disrupts the Shikimate pathway of plants and some microorganisms by inhibiting the enzyme EPSP (5-enolpyruvylshikimate-3-phosphate) synthase (Lipok et al. 2010). This process reduces the biosynthesis of aromatic amino acids in target organism, resulting in their death (Nielsen et al. 2018). This pathway is not found in animal cells, and thus, glyphosate is considered relatively non-toxic to animals and is also environmentally safe (Annett et al. 2014;Duke et al. 2012;Giesy et al. 2000;Helander et al. 2012).
Teratology test performed on bird embryo is quick and accurate and allows the chemical impact on the development of the foetus to be investigated. The further advantage of this method is its low cost, the sensitivity against various agents, as well as its high degree of similarity to the morphological development of mammals (Korhonen et al. 1982). The major disadvantage of this type of study is the lack of maternal-foetal relationship and the high sensitivity of the embryo (Wilson 1978).
Different application methods are recommended to investigate the toxic effect of chemical agents on the egg and avian embryo (Lutz and Oterag 1973;Hoffman and Gay 1981). The most frequently used administration is the injection of test item into different part of the egg. Injection into the air chamber is the best-known method because the physical injuries of the embryo can be avoided during the application (Lutz 1974;Meiniel 1977;Várnagy et al. 1982). Advantages of it are that exact dose can be applied, so the teratogenic alterations caused by the given concentration can be precisely calculated and the results of the study can be properly evaluated. However, the injection method does not re ect eld exposure, and the residue of the chemicals cannot appear at the same stage of embryonic development as after immersion treatment (Varga et al. 2002).
In case of immersion method, the amount of the chemicals that enter into the egg cannot be exactly calculated because its penetration can be in uenced by the permeability of the eggshell that is characteristic to a given species, and different environmental factors (Tullett and Deeming 1982;Tyler 1955).
Teratological tests carried out on avian embryos provide useful data for environmental protection and for protection of wild birds and their progeny and facilitate the development of environmental-friendly chemical plant protection techniques (Várnagy et al. 1996).
The current study was designed to examine the individual and combined embryotoxic effects of heavy metal (copper) modelling the heavy metal load of the environment and a glyphosate-based herbicide formulation (Amega Up) widely applied in the practice on the development of chicken embryos.

Test System
Three hundred and twenty, mixed-use Farm hen eggs (Goldavis Ltd., Hungary) with good fertility were used in the experiment (Table 1). They were randomized into eight groups (40 eggs/group) based on their size and weight. The eggs were incubated in Ragus type table incubator (Vienna, Austria) applying adequate temperature (37-38ºC), relative humidity (65-70%) and daily rotation of them during the incubation (Bogenfürst 2004). During the injection and immersion treatment the copper sulphate (Reanal-Ker Ltd., Budapest) was applied with a concentration of 0.01% using the results of a prior examination (Fejes 2005). The glyphosate-containing herbicide (Amega Up, 360 g/L, Nufarm Hungary Ltd.) was administered as 4% emulsion corresponding to that used in plant protection practice. They were used in the individual and simultaneous application with these concentrations.
Avian physiological saline solution (0.75% of sodium chloride) was used to dilute the test items and for the treatment of the control eggs.

Treatments
The study and the treatments were performed in accordance with methods described by Várnagy et al. (1981). The experimental protocol of the study was approved by the local Committee of Animal Welfare at Pannon University, Georgikon Faculty (permission No.: MÁB-10/2019).

Injection method
The test items applied individually and/or simultaneously, and the saline solution were administered as 0.1 ml directly into the air chamber of the egg on day 0 of the incubation depending on the treatment groups. The calciferous eggshell was drilled through before the injection, and it was closed with para n after treatment (Clegg 1964).

Immersion method
During the immersion method using the test items and/or saline solution the chicken eggs were dipped into the solution or emulsion for 30 minutes on day 0 of incubation. The applied concentration of the test items was the same as at injection method.

Investigated parameters
The eggs and the embryos were processed by necropsy on day 19 of incubation, and the following parameters were noted for evaluation: mortality, body weight, developmental abnormalities of the embryo. Skeletal preparations of the embryos were made using alizarin red staining to paint the bones and the cartilages and to detect any possible developmental changes in the skeletal system followed by stereo-microscopic evaluation (Dawson 1926).

Statistical Analysis
Statistical analysis of the body weight of the live embryos was performed by One-Way ANOVA after controlling of their distribution using Comparison-Quantile Plot. Comparative evaluation of the results of different groups was carried out by Tukey and Dunnett tests. The mortalities and the developmental abnormalities of embryos were analysed statistically using Fischer-type exact test (Baráth et al. 1996).

Injection method
Embryonic mortality The results of the embryonic mortality are presented in Table 2.
The mortality rate was 7.5% in the control group. The single administration of copper sulphate increased the mortality up to 20% in the Group II. The changes were not statistically different as compared to the control. Glyphosate caused an increase in mortality (35%) in Group III that was statistically signi cant as compared to the control (p < 0.05). Due to the simultaneous administration (Group IV) signi cant increase of embryonic mortality was induced up to 40% as compared to the control group (p < 0.001).

Developmental abnormalities
The rate of developmental abnormality was 2.7% in Group I ( Table 2). The single injection of copper sulphate induced leg deformation and open abdomen with 3.1% (Group II) without statistical difference as compared to the control group. Similarly, low malformation rate was observed in Group III. However, the simultaneous administration of copper sulphate and glyphosate (Group IV) produced the increase of developmental anomalies up to 12.5% including open abdomen and leg deformation (Fig. 1). The average body weight of the embryos was 20.87 ± 1.61 g in Group II., which was signi cantly lower, as compared the control group (21.96 ± 1.16 g, p < 0.05). Due to glyphosate treatment (Group III) no statistically signi cant change was observed in the body weight (21.04 ± 1.92 g). The simultaneous administration of copper sulphate and glyphosate resulted in signi cant reduction (p < 0.001) of average body weight (Group IV: 20.16 ± 1.96 g), as compared to the control ( Fig. 2A).

Skeletal staining preparation
The highest rate of development disorder of the skeletal system (leg deformation, growth retardation; Fig. 3.) were caused by the simultaneous use of copper sulphate and glyphosate, however, the anomalies were sporadic if the test items were applied individually (Table 2).

Immersion Method
Embryonic mortality The results of the mortality of embryos are presented in Table 3.
The rate of dead embryo was 5% in the control group. The single administration of copper sulphate increased the mortality up to 7.5% in Group VI. The changes were not statistically different as compared to the control (Group V). Application of glyphosate caused 10% mortality of the treated embryos in Group VII., which was also not signi cant as compared to the control. Due to the simultaneous administration of copper sulphate and glyphosate (Group VIII) not statistically signi cant increase of embryonic mortality was induced (12.5%). Generally, leg deformation and growth retardation were induced by glyphosate-containing herbicide applied alone or in combination (Group VIII).
Rate of developmental anomalies was similar in all groups treated with the investigated chemicals.

Body weight
The average body weight of the embryos was 20.26 ± 1.83 gin Group VI. It was not signi cantly reduced compared to the control group 20.97 ± 1.29 g. Due to the glyphosate treatment (Group VII) the body weight was 19.89 ± 1.28 g, which was statistically lower than in the control group (p < 0.05). The simultaneous administration of copper sulphate (0.01%) and glyphosate (Group VIII) was resulted in signi cant decrease (p < 0.001) of average body weight (19.61 ± 1.68 g) compared to the control (Fig. 2B).

Skeletal staining preparation
The development disorders of the skeletal system were sporadic due to the individual and simultaneous application of the test items resulted in incorrect posture of the feet (Table 3).

Discussion
The agricultural areas provide feedstuffs, living and hatching territory for wild birds thus the study of environmental effect of different chemical agents including pesticides is very important. Depending on the exposure level they can induce acute damage or even destruction of living organisms.
The results of the individual teratogenicity studies on copper sulphate in chicken are in accordance with results of toxicity studies in other species. Depending on the dose, copper has embryotoxic potential and may cause developmental anomalies (Ferm and Carpenter 1967;Várnagy and Budai 1995).
According to our ndings during the evaluation on day 19, copper sulphate signi cantly increased the rate of mortality and decreased the body mass of the embryos compared to the control group. This result is similar to the experiment performed by Fejes (2005), where the eggs were treated with injection of copper sulphate to the air chamber on the 1st and 12th day of incubation.
In the injection groups, adverse effects were more pronounced, the rate of mortality and developmental anomalies were higher than in the immersion groups. It is in line with study conducted on chicken embryos by Kertész (2001), indicating a slower onset of toxicity because the compounds must penetrate the eggshell and the shell membranes.
According to our ndings, the single administration of the herbicide caused signi cant increase in embryo mortality rate and developmental abnormalities.
Some studies have been labelled herbicides with glyphosate as a teratogen (Dallegrave et al. 2003), but it should not be overlooked that adverse effects occur in different animal species (Hued et al. 2012;Mottier et al. 2013;Tompa 2005), which are affected by duration and dose (Várnagy and Budai 1995). Roongruangchai et al. (2018) studied the effect of 0.1 ml of 0.01%, 0.05%, 0.3%, and 0.5% w/v glyphosate solution injected to yolk sacs of eggs at 21h of incubation and repeated at the volume of 0.05 ml on the 3rd day of incubation. They have found that glyphosate was toxic to chick embryos. It caused mortality, growth retardation and malformations on day 3 and skeletal alteration on day 6, which also indicates its teratogenic effect. Winnick (2013) investigated the toxic effect of glyphosate and Roundup (glyphosate-containing product) on developing chicken embryos applied with a concentration of 19.8 and 9.9 mg of active ingredient glyphosate/kg egg mass in both preparations. Survivability was signi cantly lower in chickens treated with Roundup than in case of glyphosate-treated embryos. The body weight of the embryos was signi cantly reduced due to the treatment of 9.9 mg/kg glyphosate in Roundup and 19.8 mg/kg glyphosate. Signi cant reduction in tibiotarsus and beak length was observed in embryos treated with Roundup (9.9 mg/kg). Ruuskanen et al. (2020) studied the effects of glyphosate-based herbicide on feather and body weight gain applied as contaminated food on Japanese quails (Coturnix japonica) to collect more data about the effects of glyphosate containing herbicides in avian taxa, because it is a poorly understood area. The results showed that the consumption of contaminated food caused delayed plumage development, and the residues of the herbicide were present in eggs, muscles, and liver.
The glyphosate (Roundup) induced reproductive changes (reduced level of testosterone and estradiol, morphological alteration of testis and epididymal region) in male duck (Anas platyrhynchos) (Oliveira et al. 2007).
Toxic interaction between glyphosate-based herbicide and copper sulphate on chicken embryos is a quite rarely investigated topic. However, the results of the combined administration in our experiment showed that in both methods, that the embryo mortality was increased, and the body weight was decreased signi cantly compared to the control groups.
Due to the individual and simultaneous application the developmental abnormalities (leg deformation, growth retardation, opened abdomen) were sporadically observed in our studies including both treatment methods. Our experience is in line with the scienti c literature that is the incidence of the gross developmental anomalies is sporadic and not characteristic, and the abnormalities are the most frequent on the skeletal system. It is not surprising as the skeletal system has outstanding importance in experimental teratological studies (Tompa et al. 1971). However, in some cases, developmental changes may be also developed on the soft tissues.
Toxic interaction, at least additive action, may be realised between two pesticides if they act together in the same organism at the same time. Especially, if the combination contains insecticidal component, this undesirable effect may be more severe, even synergistic with increasing toxicity up to 100-fold. However, these effects depend on the animal species, the exposure time, and the dose, and thus their routine prediction is di cult (Thompson 1996).

Conclusions
Based on the data presumably a slight addition effect was realised at the mortality due to the simultaneous injection and immersion application of copper sulphate and glyphosate. The body weight of the embryos was reduced due to the both experimental exposure without any combined relation. The injection method induced synergistic effect at developmental abnormalities between the test items; however, any toxic interaction was not occurred due to the immersion treatment.
Comparing the result of the two treatments, we can conclude, that the more provoking injection treatment proved to be more toxic than the immersion treatment in the case of both single and combined treatments, which appeared in the increase of embryo mortality, more frequent occurrence of developmental disorders, and decrease of body weight.
The advantage of injection method is that the experimental material can be directly introduced into the required part of the egg with an exact dose, while immersion application of the chemical into the egg represents better the exposure realised in the environment. Body weight (average±SD; g) of the embryos after single and simultaneous administration of glyphosate and copper sulphate by injection method (Fig. 2A) and by immersion method (Fig. 2B). a=signi cant decrease as compared to the control data (p<0.05) b=signi cant decrease as compared to the control data (p<0.001)

Figure 3
Skeletal staining preparation: Normal development of control embryo (A) and Growth retardation of embryo (B) caused by the simultaneous use of copper sulphate and glyphosate