Investigation of toxic effects of novaluron on daphnia magna at the level of development, morphology, physiology and gene expressi̇on

Pesticides are chemicals used to control harmful organisms and are widely used in agriculture to increase productivity. With the increase in human population, it has become very important to increase productivity in agricultural activities in order to meet the demand for food. In this direction, the use of pesticides has become widespread and their environmental impacts have become a subject of debate. They are classified as insecticides, herbicides, fungicides and rodenticides against pests such as insects, weeds, fungi and rodents. Apart from agriculture, they are also used in homes, workplaces and garden maintenance. Novaluron is an insect growth regulator insecticide and is used especially against agricultural pests. Novaluron, chemically known as a chitin synthesis inhibitor, inhibits the formation of chitin in target organisms, stops the development of the exoskeleton and prevents the growth of larvae. However, the spread of these pesticides used in agricultural areas into the environment and their effects on non-target organisms pose a serious risk, especially in aquatic ecosystems. Pesticides such as Novaluron can be transported to aquatic ecosystems by various means such as surface runoff, soil erosion, groundwater infiltration and atmospheric transport, and can have toxic effects on organisms there. There is limited information on the effects of Novaluron especially in aquatic ecosystems. Aquatic organisms such as Daphnia magna have an important place in ecotoxicological research with their sensitivity to environmental pollutants. These organisms are used as biomarkers to evaluate the effects of pollutants. These biomarkers can be morphological such as secondary antennae, caudal abdominal flagella, length and heart diameter; physiological such as heart rate, post abdominal cavity, thorax activity or genetic such as changes in gene expression. Heart rate, as a parameter that responds rapidly to environmental stressors, provides critical information on the overall health status of the organism. Thoracic limb activity serves as an important biomarker for early detection of responses to environmental toxins as well as vital functions such as nutrient collection and oxygen transport. The abdominal flagellum generates the post-abdominal pincer movement. Abdominal flagella regulate the movement of water and are involved in essential functions such as nutrient collection and oxygen transport. Environmental pollutants, such as pesticides, distort the shape of this structure, directly affecting the speed and rhythm of movements, and are therefore an important marker in toxicological studies. Secondary antennae, with their functions of movement and adaptation to environmental changes, are a valuable parameter in monitoring both physical and behavioural effects. In addition, tail structure is an important biomarker for balance and mobility, and may show significant changes such as deformation when exposed to environmental stressors. Stature is a basic parameter that provides information about the growth rate and general health status of the organism. Cardiophysiological indicators such as heart diameter are considered as an important tool to understand the metabolic and circulatory effects of environmental pollutants on the organism. Finally, pericardial oedema is characterised by the accumulation of fluid in the region of the heart, which interferes with the efficient functioning of the organism's cardiovascular system, negatively affecting cardiac function. Fluid accumulation can significantly weaken the general state of health of the organism by disrupting the regular functioning of the circulatory system. The presence of pericardial oedema can be considered an indicator of cardiovascular damage caused by exposure to environmental pollutants In this study, to determine the toxicity of Novaluron in Daphnia magna, daphnids less than 24 hours old were treated at concentrations of 100 mg/L, 10 mg/L, 1 mg/L, 0,1 mg/L, 0,01 mg/L, 0.001 mg/L and 0.0001 mg/L. The 48-hour LC50 concentration of novaluron in Daphnia magna was determined as 61 μg/L. In our study, 4 groups were formed in total, one control and 3 experimental groups, and 10 daphnids younger than 24 hours were used in each group. While no treatment was applied to the control group, sublethal concentrations of 3.75 μg/L 7.5 μg/L and 15 μg/L Novaluron were applied to the experimental groups for 72 hours. During the experiments, survival rates, length, developmental deformities, heart size, heart rate, thoracic and abdominal cavity movements, as well as heart size, heart rate, thorax and abdominal cavity movements of the organisms were examined by inverted microscope. Thus, data on the sublethal effects of the pesticide in Daphnia magna were obtained. In addition, in this study, in order to investigate the expression levels of genes related to detoxification system (cyp314, cyp360A8, gst and p-gp) in Daphnia magna juveniles exposed to sublethal concentrations of Novaluron pesticide for 24 hours, sublethal concentrations of Novaluron pesticide (3.75 μg/L, 7.5 μg/L and 15 μg/L) were applied to Daphnia magna for 24 hours. Expression profiles of genes were determined using Real Time PCR. The distribution of the obtained data was analysed by Shapiro-Wilk test using SPSS programme. One-way ANOVA, which is a parametric test, was used for statistical significance for groups with normal distribution, while the non-parametric Kruskal-Wallis test was used for groups without normal distribution. Then, post-hoc tests were applied for significance between groups. The analyses were performed using 95% confidence level; 0.05, 0,01 and 0,001 significance value. The statistical analyses used in the experiments confirmed the significance of the observed differences and increased the reliability of the results. Data from both acute and chronic toxicity tests provide strong evidence for the ecological risks of Novaluron in aquatic ecosystems. When the data obtained were evaluated, the highest toxic effect was found in the group exposed to 15 μg/L Novaluron. While an increase in heart rate was observed in all experimental groups compared to the control group, a decrease in other physiological activities such as thorax and abdominal cavity was detected. In particular, it was observed that abdominal cavity activity came to a halt in the group exposed to 15 μg/L concentration at 72 hours. Changes in biological parameters such as heart rate, thoracic activity and post-abdominal pincer movements were considered as valuable biomarkers for early detection of toxic effects of pesticide. Moreover, these changes clearly reveal the harmful effects of the pesticide on the organism in response to environmental stress factors. When Novaluron-treated individuals were compared with the control, significant changes such as tail breakage, pericardial oedema, secondary antennal deformations, abdominal clefts and decreased heart diameter were observed at 72 hours of exposure. In addition, it was determined that there was an increase in the length of the experimental groups after Novaluron exposure, while there was a decrease in heart diameter. At the end of the study period, the expression levels of cyp314 and gst genes in pesticide-treated Daphnia fry showed a statistically significant increase compared to the control group (p<0.05, p<0.01 and p<0.001), while the expression level of cyp360A8 gene decreased statistically compared to the control group (p<0.05, p<0.01 and p<0.001). The expression of p-gp gene did not cause a statistically significant change compared to the control group. In summary, the findings revealed that Novaluron causes significant toxic effects on Daphnia magna even at low concentrations. It was observed that at high concentrations the survival rate of organisms was significantly reduced, while at low concentrations exposure caused changes in physiology, morphology and gene expression in the organism. In conclusion, this study comprehensively demonstrated the acute toxic effects of Novaluron on Daphnia magna. The findings indicate that this pesticide, which is frequently used in agriculture, can cause significant effects on physiology, morphology and gene expression in the ecosystem, especially in aquatic organisms, even at low concentrations. Since Daphnia magna is an important species in freshwater ecosystems and an important model organism for the assessment of aquatic toxicity, these findings are important for understanding the potential ecological risks posed by Novaluron. In conclusion, this study reveals the acute toxic effects of Novaluron on Daphnia magna. Therefore, it is recommended that regulations to reduce the environmental impacts of Novaluron should be implemented and the pesticide should be used in a controlled manner. Furthermore, the promotion of environmentally friendly agricultural practices is recognised as an important strategy to reduce the impacts of pesticides on aquatic ecosystems. Future studies should examine the acute and chronic consequences of Novaluron as well as other pesticides in the aquatic ecosystem. More detailed assessment of the effects of pesticides on non-target organisms can provide a stronger basis for environmental risk management. This study provides important information on the environmental effects of pesticides and constitutes a valuable reference for the protection of aquatic ecosystems and the promotion of sustainable agricultural practices. Keywords: Daphnia magna, Novaluron, acute toxicity, morphological deformations, physiological changes, gene expression