The influence of microbial inocula on biodegradation outcome towards enhanced regulatory assessments

Abstract

Chemical compounds are ubiquitous in the aquatic environment, causing numerous negative impacts and raising concern over human health and the environment. The principal elimination process is microbial degradation, the prediction of which plays an important role in risk assessment. Current biodegradation tests are notoriously variable and not effective at characterising chemical persistence. Enhancements to existing biodegradation tests have been proposed to enable a more effective prioritisation on persistence. Several of the proposed enhancements are examined in this study, including increasing total cell numbers within tests and extending test duration. Activated sludge (AS) and marine inocula were incorporated in OCED 301B type studies with different inocula concentrations, test volumes and over extended study duration. Evolved ¹⁴CO² was captured as a measure of degradation, subsequently converted to a probability of degradation and used in the calculation of degradation descriptors. Culture-independent methods were employed to study the diversity within batch systems (PCR-DGGE and 454 sequencing). AS inocula exhibited faster rates of degradation and shorter lag phases when operated at higher cell concentrations, although the greatest impact of increased cell concentration was observed in decreased inter-replicate variation (P < 0.01). Test volume had less effect than concentration, with inter-replicate variation again the main beneficiary of the enhancement. Typically, band richness, used as a measure of diversity, increased with increasing biomass concentration. DGGE analysis also suggested greater similarity between higher cell concentration replicates. The ideal system proposed is 300 mg SS L-¹ at 0.5-1.0 L. Marine systems did not show a significant concentration or volume effect (P>0.05). A number of systems exhibited rapid degradation rates following lengthy lag phases suggesting the importance of increasing test duration in order to accurately predict environmental behaviour and fate. A major concern for a novel screening test would be to underestimate persistency and approve chemicals which could pose a significant threat to the environment. Chemical validation studies using a set of recognised reference compounds of varying persistence showed no false positives in the enhanced screening test.