Soil‐specific outcomes in the OECD 216 Nitrogen Transformation Test

The Organisation for Economic Co-operation and Development (OECD) 216 test guideline investigates the impact of agrochemicals on soil nitrogen transformation. After an evaluation of 465 OECD 216 studies, we describe two distinct yet contrasting outcomes in control nontreated samples that are possible in this testing framework, which we term the "rise" (consistent increases in nitrate concentrations throughout the test period) and "dip" (initial decline in nitrate concentration between Days 0-7, followed by a net-generation of nitrate across Days 7-28) responses. We raise significant concerns that control data from standardized, internationally recognized test guidelines can demonstrate such dissimilar patterns. We propose that, when present, the dip response undermines the intended functioning of the test system and removes the ability to draw appropriate ecotoxicological inferences from the data. In this work, we hypothesize the dip response is a product of conducting the study in low nitrogen content soils. Our results indicate that the dip response can be alleviated by using ammonium sulfate as an immediately available inorganic nitrogen source in place of the guideline-mandated complex, organic lucerne meal, demonstrating the influence of nitrogen availability and accessibility. However, not all low nitrogen soils exhibited the dip response, indicating the involvement of additional unidentified factors. Using our data and real-world regulatory examples, we advocate that datasets displaying the dip response should not be considered valid OECD 216 studies due to the influence of soil properties precluding an assessment of whether any impacts observed are driven solely by the test compound in question or are instead a product of the soil used. We propose methods to account for these soil-specific responses that could be integrated into the conduct and interpretation of OECD 216 studies. Such amendments will improve the reliability and robustness of the study system and enhance confidence in ecotoxicological conclusions derived from OECD 216 datasets. Integr Environ Assess Manag 2024;20:1611-1624. © 2024 SETAC.