ECOTOXICOLGICAL EFFECTS OF ATRAZINE AND ENDOSULFAN ON THE GROWTH AND BIOMARKER EXPRESSION OF THREE GREEN ALGAL SPECIES (CHLOROPHYTA) UNDER VARIOUS NUTRIENT CONDITIONS

Abstract

For the past decades, anthropogenic impacts on natural systems are of growing concern as the human population expands and global biological diversity declines. Agricultural practices often involve the use of fertilisers and pesticides to improve the yield of crops. Agricultural runoff with excess nitrogen (N) and phosphorus (P) could stimulate the excessive growth of algae in the aquatic ecosystems, including lakes and coastal regions, resulting in eutrophication. Besides fertilisers, pesticides are also used in large amounts for pest controls. Microalgae have been frequently used as bio-assay organisms to assess the toxicity of pesticides. However, most of the reports were on the effects of single or mixed pesticides without considering the possible influence of nutrients such as N and P on its toxicity. Therefore, our study aims to assess the changes of various biomarkers in microalgae after exposure to pesticides under nutrient-limited and nutrient-enriched conditions. In this study, two commonly used pesticides, atrazine and endosulfan were chosen. Atrazine, a herbicide of the triazine family, is one of the most frequently used herbicides worldwide while endosulfan is an organochlorine insecticide that causes acute neurotoxic to insects and mammals. A total of 10 isolates from Tasik Jaya and Cameron Highlands were selected to determine their sensitivities to atrazine and endosulfan. The EC50 for atrazine ranged from 43.07 µg/L to 1313.90 µ/L, while the EC50 for endosulfan ranged from 1.51 mg/L to > 50 mg/L after 96 h of exposure. Two of the microalgae, namely Scenedesmus arcuatus (highly sensitive) and Chlorella sp. (highly resistant) were selected for further studies, and compared with the model species, Pseudokirchneriella subcapitata. These three microalgae were also cultured in mediums with different levels of N (in the form of sodium nitrate, ammonium chloride and urea) and P (in the form of potassium phosphate) to determine the levels of N and P that limit/enhance the growth of the microalgae. The N-limited and N-excess levels were determined as 0.0117 mM and 3mM respectively while the P-limited and P-excess levels were 0.0002 mM and 1.7210mM respectively. To determine the effects of different nutrient conditions to the toxicity of atrazine and endosulfan, the three chosen microalgae were exposed to EC10 and EC50 of atrazine and endosulfan, being determined earlier in the dose-response assay in four different culture media: N- and P-limited (LNLP); N-limited, P-excess (LNHP); N-excess, P-limited (HNLP); as well as N- and P-excess (HNHP). In general,all three microalgae showed better tolerance to atrazine and endosulfan when they were cultured in nutrient-limited conditions as compared to nutrient-enriched conditions based on their growth response. In addition to growth response, the effects of different nutrient conditions to the toxicity of atrazine and endosulfan were also assessed using different biomarkers such as oxidative stress biomarkers, photosynthetic biomarkers and morphological biomarkers. The oxidative stress biomarkers of microalgae showed different changes when exposed to atrazine and endosulfan at different nutrient conditions. Reactive oxygen species (ROS) production and lipid peroxidation were induced significantly when microalgae were exposed to atrazine and endosulfan in nutrient-excess conditions, while superoxide dismutase (SOD) levels were generally higher in microalgae grown in nutrient-limited conditions. At N-limited conditions, exposure to atrazine and endosulfan has caused inhibition of SOD in P. subcapitata. The catalase (CAT) enzyme did not show many changes after exposure to atrazine and the response varied in different microalgae in different nutrient conditions after exposure to endosulfan. The photosynthetic biomarkers also showed different changes when microalgae were exposed to atrazine and endosulfan at different nutrient conditions. The maximum potential quantum efficiency of photosystem II (Fv/Fm) was significantly reduced with decreasing nutrient levels, while exposure to atrazine and endosulfan has stimulated the Fv/Fm regardless of the nutrient conditions. Similar trends were observed for the photosynthetic pigments. However, alpha and rETRmax were generally being reduced after exposure to atrazine and endosulfan in various nutrient conditions, except for Chlorella sp. 5 that showed significant elevation in alpha and rETRmax after exposure to endosulfan in nutrient-limited conditions. The presence of atrazine and endosulfan in nutrient-limited conditions has caused more severe damage to the algal cellular structure compared to those in nutrient-excess conditions, such as cell wall disruption and chloroplast damage. On the other hand, increased number of lipid bodies and starch granules were observed in nutrient-limited conditions, together with more severe damage to the cell wall and chloroplast of the microalgae compared to nutrient excess conditions. In conclusion, both atrazine and endosulfan caused significantly different adverse effects to different algal strains, based on their effects on various biomarkers assessed. In addition, nutrient conditions do affect the toxicity of pesticides.