This paper examines the interaction between dissolved natural organic matter and pesticide residues, both of which are found in raw water sources, using three dimensional excitation–emission matrix (3DEEM) fluorescence spectroscopy. It was observed that pesticide residue at 0.1 mg L−1 formed a complex with humic-like fluorophores that are commonly found in raw water samples. Applying 3DEEM fluorescence to investigate the humic fractions, it was found that identification of changes in water sources was possible, and, importantly, the presence of a number of pesticides was able to be determined. In addition, the formation of this complex, and the influence of soluble cations and anions upon it, was shown to impact the efficiency of analytical extraction procedures for pesticides; however, 3DEEM fluorescence could be an approach to account for such losses.

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