Evaluating acute toxicity in enriched nitrifying cultures: Lessons learned

Toxicological batch assays are essential to assess a compound's acute effect on microorganisms. This methodology is frequently employed to evaluate the effect of contaminants in sensitive microbial communities from wastewater treatment plants (WWTPs), such as autotrophic nitrifying populations. However, despite nitrifying batch assays being commonly mentioned in the literature, their experimental design criteria are rarely reported or overlooked. Here, we found that slight deviations in culture preparations and conditions impacted bacterial community performance and could skew assay results. From pre-experimental trials and experience, we determined how mishandling and treatment of cultures could affect nitrification activity. While media and biomass preparations are needed to establish baseline conditions (e. g., biomass washing), we found extensive centrifugation selectively destabilised nitrification activities. Further, it is paramount that the air supply is adjusted to minimise nitrite build-up in the culture and maintain suitable aeration levels without sparging ammonia. DMSO and acetone up to 0.03% (v/v) were suitable organic solvents with minimal impact on nitrification activity. In the nitrification assays with allylthiourea (ATU), dilute cultures exhibited more significant inhibition than concentrated cultures. So there were biomass-related effects; however, these differences minimally impacted the EC50 values. Using different nutrient-media compositions had a minimal effect; however, switching mineral media for the toxicity test from the original cultivation media is not recommended because it reduced the original biomass nitrification capacity. Our results demonstrated that these factors substantially impact the performance of the nitrifying inoculum used in acute bioassays, and consequently, affect the response of AOB-NOB populations during the toxicant exposure. These are not highlighted in operation standards, and unfortunately, they can have significant consequential impacts on the determinations of toxicological endpoints. Moreover, the practical procedures tested here could support other authors in developing testing methodologies, adding quality checks in the experimental framework with minimal waste of time and resources.

Automated summary

Toxicological batch assays are essential for assessing the acute effects of a compound on microorganisms. However, the experimental design criteria for nitrifying batch assays are often overlooked in the literature. This study found that slight deviations in culture preparations and conditions can significantly impact bacterial community performance and skew assay results. Factors such as media and biomass preparations, centrifugation, air supply, and organic solvents can affect nitrification activity. These factors can have substantial consequences on the determinations of toxicological endpoints and should be considered in testing methodologies.