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The effluents released from different agricultural fields into the local water bodies leads to pollution of the aquatic environment and it is one of the major concern to the present human civilisation. Pesticides, fertilisers and other components of agricultural runoff may adversely affect the metabolism of aquatic organisms by alternating the enzyme activities. The main aim of the present study is to assess the comparative behavioural and physiological responses of Catla catla and Clarias magur exposed to agricultural runoff and also evaluating the toxicity of the agricultural runoff of the Burdwan district, West Bengal. The fingerlings were exposed to agricultural runoff of different concentrations (i.e. 0.0%, 10%, 20%, 30%, 40%, 50%) for 72 hours and their behavioural and physiological changes were observed. Both fish species exhibited abnormal behavioural effects such as random surfacing and erratic movement as well as physiological responses such as increased opercular movement, particularly at higher concentration. However, the adverse effects were more pronounced in Catla compared to Clarias. The present findings indicate that the agricultural runoff is highly toxic to the living organisms and the results could be used for better agricultural practices and in environmental legislation, respectively.