AbstractThe EU directive 2013/39/EU has incorporated four biocidal compounds as priority substances: diuron, isoproturon, cybutryne, and terbutryn. The research was undertaken to determine the concentration of biocides in surface waters in three locations in southern Poland: the Wisła River in Kraków, the Wisłoka River in Mielec, and the drainage ditch draining water from arable fields located near Mielec. Environmental samples were taken in two series: winter (February) and spring (May and June). The analyses were carried out using gas chromatography with mass spectrometry. The seasonality of biocides in surface waters was observed. In winter samples, the concentrations were below MQL, while in spring, they ranged from a few to several dozen nanograms per liter. The highest concentrations of all analyzed compounds were recorded in water taken from the Wisła River. According to directive 2013/39/EU, the maximum allowable concentration was exceeded only in the case of cybutryne in water from the Wisła, both in May and in June. The assessment of the toxicity with the tested compounds was defined based on the Environmental Risk Assessment method. Low risk was estimated for diuron and isoproturon, while moderate risk for terbutryn and cybutryne.