A call to update reproduction test criteria for Enchytraeus crypticus: why a 50% CV threshold no longer fits

The ISO 16387 and OECD 220 guidelines provide standardized protocols for assessing the effects of contaminants on reproduction and survival in Enchytraeus species, key soil invertebrates involved in nutrient cycling and soil functioning. These guidelines include a validation criterion requiring the coefficient of variation (CV) for reproduction in control groups to not exceed 50%. This threshold was originally derived from tests using Enchytraeus albidus, a species with a longer test duration (42 days) and historically higher variability in reproductive outputs. However, the currently preferred species in ecotoxicological testing is Enchytraeus crypticus, which has a shorter test duration (varying between 21 and 28 days), higher reproductive consistency, and a markedly lower CV in empirical studies. Data compiled from 2006 to 2025, including recent studies across a range of artificial and natural soils, consistently show that E. crypticus control CVs remain below 30%, with many values between 6 and 15%. Rare outliers above 30% occurred under specific conditions. In contrast, other OECD and ISO guidelines for soil invertebrates (e.g., collembolans, earthworms, mites) already adopt stricter CV thresholds (≤ 30%), ensuring higher test sensitivity and comparability. We propose revising the CV acceptance threshold for E. crypticus reproduction tests to ≤ 30%, aligning validation criteria with demonstrated species performance and broader international standards. This adjustment would improve data precision, reduce false positives, and enhance reproducibility across laboratories, contributing to more robust Environmental Risk Assessments (ERAs) for plant protection products and other chemicals. We further call on the scientific community to contribute data and support harmonized efforts to refine guideline criteria for enchytraeids, strengthening their role in soil ecotoxicology and regulatory science.