Improved reliability of ready biodegradability assessments in the Closed Bottle test (OECD 301D) by omission of ammonium chloride

Ammonium-nitrogen is supplemented with the medium of the Closed Bottle test (OECD 301D) as macronutrient for growth. Nitrification of this ammonium-nitrogen results in an increased background respiration and a higher chance of generating invalid test results. The omission of ammonium chloride from the Closed Bottle test medium raises regulatory concerns, as this modification is assumed to lead to less stringent test conditions. To address this concern and demonstrate that the removal of ammonium chloride does not result in unduly favorable conditions, the biodegradability of seven test substances was assessed in OECD 301D tests using standard and ammonium-free medium and results were compared. Nitrate and nitrite analyses were included to determine the extent of nitrification. Biodegradation results of the OECD 301D tests performed with standard medium and ammonium-free test medium were comparable and most of the ammonium-nitrogen dosed with the medium was recovered after 28 days in the form of nitrate-nitrogen. These results indicate that sufficient nitrogen for biomass growth of the heterotrophic microorganisms is introduced with the inoculum of the OECD 301D test. The stringency in the OECD 301D tests was demonstrated by the low biomass-to-test substance ratio and the test accuracy was demonstrated by oxygen consumption in the inoculum blank control bottles of ≤ 1.5 mg L−1. OECD 301D tests performed with standard test medium are more likely to exceed the validity criterion for the oxygen consumption in inoculum blank control bottles because of nitrification-caused respiration. Hence, the omission of ammonium chloride from the test medium will improve the reliability of the OECD 301D test by reducing the likelihood ofs generating invalid test results. Scientific evidence demonstrates that the omission of ammonium chloride from the OECD 301D test medium is justified for regulatory acceptance, as empirical data confirms this modification enhances test reliability while maintaining both stringency and accuracy of biodegradability assessment.