Characterization of complex environmental contaminant mixtures in common toad breeding ponds in Flanders, Belgium

The European common toad (Bufo bufo) is undergoing widespread population declines, potentially influenced by multiple environmental stressors, including chemical pollution. This study aimed to link the toad population status and contaminant levels in 20 breeding ponds in Flanders (Belgium). A multi-contaminant analytical approach was applied, using solid-phase extraction (SPE) combined with advanced instrumental methods: ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for pesticides and mycotoxins, UHPLC-Orbitrap-high resolution MS for pharmaceuticals, inductively coupled plasma-optical emission spectrometry (ICP-OES) and ICP-MS for high and low-concentrated metals, respectively, and ion chromatography (IC) for nutrients. Across ponds, 25 pesticides, 11 mycotoxins, 13 antimicrobial drugs, 5 antiparasitic drugs, and 11 metals were detected, reaching concentrations up to 114 ng/L (pesticides), 88.2 ng/L (mycotoxins), 837 ng/L (antimicrobials), 26.8 ng/L (antiparasitic drugs), and 7382 μg/L (metals). Temporal variability was pronounced, with episodic increases in pesticide and metal levels, declining nitrate and sulphate concentrations, and increasing enniatin levels from March to June. Despite frequent co-contamination, no direct relationship was observed between individual contaminant levels and toad population status. These findings suggest that population declines are unlikely to be driven by a single substance, but rather by combined pressures associated with complex contaminant mixtures and other environmental stressors.