Increased cell numbers improve marine biodegradation tests for persistence assessment

Currently available OECD biodegradation screening tests (BSTs) are not particularly suited for persistence screening. Their duration can be much less than international half-life thresholds for persistence and they are variable and stringent, therefore prone to false negatives. The present study extended test durations beyond 28 days and increased biomass concentrations for marine BSTs to better represent the microbial diversity inherent in the sampled environment. For this so-called environmentally relevant BST (erBST) marine cell concentrations were nominally increased 100-fold by tangential flow filtration. The marine erBST was validated against a standard BST using five ¹⁴C labeled reference compounds with a range of biodegradation potentials (aniline, 4-fluorophenol, 4-nitrophenol, 4-chloroaniline and pentachlorophenol) in a modified OECD 301B test. A full mass balance was collated to follow chemical fate in the tests. The erBST was more accurate and less variable than the comparator BST in assigning the reference compounds to their expected biodegradation classifications (non-persistent or potentially persistent). According to the REACH non-persistence criterion of ≥60% biodegradation over 60 days, the erBST correctly classified 60% of chemical replicates according to their expected biodegradation classification and had a coefficient of variation of 21% between replicates. In contrast, the BST correctly assessed 40% of reference chemicals in regards to their expected biodegradation classification with a coefficient of variation of 36%. All non-persistent chemicals showed increased degradation in the erBST, except for 4-chloroaniline, which did not degrade in either BST or erBST. Both tests showed no false positive results, correctly classifying the negative control pentachlorophenol as potentially persistent. Next, it is recommended to further validate the marine erBST in an inter-laboratory study incorporating different seawater sources to fully assess its variability and reliability.