The Organisation of Economic Co-operation and Development (OECD) 216 nitrogen transformation test is used to understand the impacts of plant protection products (PPPs) on the soil microbiome. However, there is significant interest in developing the European PPP risk assessment to include new technologies such as amplicon sequencing to assess impacts on soil microbial community composition and diversity. We have little understanding of how to generate endpoints from amplicon sequencing data sets, their robustness, and whether they provide an appropriate level of protection to the soil microbiome. Our study addresses this key knowledge gap. We conducted a dose-response OECD 216 study with two chemicals, nitrapyrin and streptomycin, and calculated traditional functional endpoints, in accordance with the OECD 216 guideline, and used amplicon sequencing techniques to generate a range of endpoints based on soil bacterial diversity, richness, dissimilarity from the control, species sensitivity distributions, and threshold indicator analysis. We show it is possible to generate a range of endpoints from amplicon sequencing data sets
however, these endpoints varied significantly based on the calculation method, with up to a 101-fold difference between the least and most sensitive endpoints. Additionally, the relative sensitivity of these endpoints compared to the currently used functional OECD 216 metrics was compound dependent, with many endpoint calculation methods unable to detect the impacts of nitrapyrin on the soil microbiome at concentrations deemed ecotoxicologically relevant by OECD 216. Our study shows amplicon sequencing methods to study soil microbial ecotoxicology did not perform consistently and reliably when considering both nitrapyrin and streptomycin treatments and in many cases did not consistently provide an enhanced degree of protection over the functional OECD 216 assessments already integrated into the PPP risk assessment.