ASSESSMENT OF THE TOXICITY OF PESTICIDES IN MICROALGAE AND ANIMAL CELLS

Abstract

Pesticides are widely used in agricultural activities in Malaysia. Some of the common pesticides used include organophosphate compounds such as chlorpyrifos and diazinon, and organochlorine chemicals such as lindane, heptachlor, endosulfan, DDT, chlordane, aldrin, dieldrin and endrin. Pesticides can cause adverse effects on the ecosystems due to their long half-life, and ultimately may end up in the sea affecting the marine ecosystems. Humans are inevitably exposed to pesticides through environmental contamination or occupational use. The primary aim of this study was to assess the toxicity of five pesticides, namely chlorpyrifos, lindane, endosulfan, dichlorvos and malathion using an integrated approach based on microalgae and animal cells. In addition, the mutagenicity of the pesticides was assessed by Ames test using Salmonella typhimurium. Results showed that lindane was the only pesticide that could cause mutation after liver enzyme activation. The toxic effect of the pesticides on normal human liver (THLE-2) and mouse embryo fibroblast (3T3) cells was assessed based on 72 h MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazoliumbromide) assay. Of the pesticides tested, endosulfan was most toxic (EC50 = 20.4 μg/mL) against THLE-2 cells while chlorpyrifos was most toxic (EC50 = 2.5 μg/mL) against 3T3 cells. Toxicity testing of the pesticides on three marine microalgae, namely Pavlova gyrans, Isochrysis sp. (clone T-ISO), and Dunaliella tertiolecta was conducted based on their five-day growth response assessed by optical density at 620 nm (OD620). Of the five pesticides tested, endosulfan was most toxic against Pavlova gyrans (EC50 = 0.17 μg/mL) and Dunaliella tertiolecta (EC50 = 0.35 μg/mL). In comparison, Dunaliella tertiolecta was most resistant to all the pesticides tested (EC50 = 0.35 - 28.09 μg/mL). Random Amplified Polymorphic DNA (RAPD) technique was used to detect DNA damage caused by the pesticides in animal and algae cells. A total of 31 sets of primers were used and appearance/disappearance of bands in the pesticides-treated cells were compared with the control. The change in RAPD band patterns was more obvious in animal cells compared to microalgae. Most of the changes in PCR banding patterns were random, but the frequencies were higher when the DNA was amplified with primer OPB 5 series, OPB 7 series and OPB 8. Based on the RAPD analysis, Pavlova gyrans showed the least changes in terms of DNA band pattern compared to the other species of microalgae. The induction of apoptosis by the pesticides in animal and algae cells was assessed based on oxidative DNA damage, nucleus condensation, and caspase-3 activity. Endosulfan and dichlorvos caused oxidative DNA damage in both mouse embryo fibroblast and human liver cells, while chlorpyrifos only caused oxidative DNA damage in mouse embryo fibroblast cells. In nucleus and DNA condensation study by Hoechst stain, apoptosis was detected in malathion-treated mouse embryo fibroblast cells with obvious DNA condensation after exposure to the pesticides for 4 h. Results from the caspase-3 activation study showed that human liver cells were more susceptible to caspase-3 induction by pesticides than mouse embryo fibroblast cells. Endosulfan and lindane showed the ability to increase the caspase-3 activity in liver cells. The findings suggest that the pesticides tested could adversely affect the growth, and may cause DNA damage in both marine microalgae and animal cells. The pesticides may impact on the marine ecosystem as well as human health.