Determining the Ecological Impacts of Organic Contaminants in Biosolids Using a High-Throughput Colorimetric Denitrification Assay: A Case Study with Antimicrobial Agents

Land application accounts for ∼50% of wastewater solid disposal in the United States. Still, little is known regarding the ecological impacts of nonregulated contaminants found in biosolids. Because of the myriad of contaminants, there is a need for a rapid, high-throughput method to evaluate their ecotoxicity. Herein, we developed a novel assay that measures denitrification inhibition in a model denitrifier, Paracoccus denitrificans Pd1222. Two common (triclosan and triclocarban) and four emerging (2,4,5 trichlorophenol, 2-benzyl-4-chlorophenol, 2-chloro-4-phenylphenol, and bis(5-chloro-2-hydroxyphenyl)methane) antimicrobial agents found in biosolids were analyzed. Overall, the assay was reproducible and measured impacts on denitrification over 3 orders of magnitude exposure. The lowest observable adverse effect concentrations (LOAECs) were 1.04 μM for triclosan, 3.17 μM for triclocarban, 0.372 μM for bis-(5-chloro-2-hydroxyphenyl)methane, 4.89 μM for 2-chloro-4-phenyl phenol, 45.7 μM for 2-benzyl-4-chorophenol, and 50.6 μM for 2,4,5-trichlorophenol. Compared with gene expression and cell viability based methods, the denitrification assay was more sensitive and resulted in lower LOAECs. The increased sensitivity, low cost, and high-throughput adaptability make this method an attractive alternative for meeting the initial testing regulatory framework for the Federal Insecticide, Fungicide, and Rodenticide Act, and recommended for the Toxic Substances Control Act, in determining the ecotoxicity of biosolids-derived emerging contaminants.