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This study investigated the use of enzymes and histology as potential biomarkers in Sand and Blood Rivers in Limpopo Province, South Africa. Physico-chemical parameters were analyzed at sites S1, S2, S3, S4, and S5 of the Sand River and sites B1 and B2 of the Blood River. Sites S1 and B1 were selected as the reference sites and located upstream of the Polokwane and Seshego wastewater treatment plants (WWTPs) effluent discharge points, respectively. Water quality parameters assessed were water temperature, dissolved oxygen (DO), biologica