CONCLUSIONS AND RECOMMENDATIONS

Although there was insufficient time at the workshop to rank the recommendations, in the process of writing this report the authors have sought an indication of the priority for the work from the workshop participants. The following areas were considered to be of very high priority and should be recommended for further action:

Difficult substances and complex mixtures

It was clear that there were significant challenges in developing appropriate techniques to assess the persistence of complex substances. It was also clear that the higher tiered tests were developed for single substances and need to be tailored for complex substances. Approaches could be based on assessing the predicted properties of components, an assessment of the whole substance or a combination of both. Specific recommendations were as follows:

• Develop a non-target approach to look for both non-degrading components and metabolites. This would require advanced analytical procedures and composition would need to be linked to improved predictive models.
• Assess the potential role of microbial profiling to give an indication of the presence and growth of different types of degraders to provide weight of evidence that degradation of the substance (and relevant constituents) was occurring.
• Use chemical profiling to follow (ready) biodegradation as a pragmatic means to give more information than the usual secondary measures of ready biodegradation e.g. complex substance’s average molecular weight change (i.e. decline) over the course of a test using MS.
• Consider using an approach analogous to that recommended by OSPAR for whole effluent assessment (WEA) to not just assess the persistency of certain components but the persistence of potentially bioaccumulating and toxic substances. For example, solid-phase micro extraction (SPME) ran before and after degradation assessments can give information on the persistence of potentially bioaccumulating substances.
• Use sterile controls to help differentiate between physical and biodegradation losses.
• Monitor and assess value of RIFM research project on “Methods to improve the environmental assessment of poorly defined components of complex mixtures” and if appropriate provide to a wider spectrum of complex substances.

Screening level assessment of persistence

It was clear that the current OECD biodegradation screening tests have some limitations associated with the identification and relative prioritisation of chemicals that persist in the environment. There were also concerns expressed (i) about the extent to which many of these standardised tests had been subject to any rigorous validation and ring testing or inter-laboratory comparisons with a range of suitable test chemicals, and (ii) some of the standardised studies are being used in regulatory assessments in a manner for which  they were not designed or intended (e.g. the use of the OECD 308 study being used as a river simulation for down the drain chemicals). Specific recommendations were as follow:

• The OECD should consider convening an Expert Working Group to consolidate and update the RBTs to reflect (i) the availability of new instrumentation with increased analytical sensitivity (ii) the use of tests with combined analytes (e.g. BOD and carbon dioxide evolution), and (iii) the need to screen for biodegradability in water-sediment systems.
• A laboratory-based study to compare the performance of, and identify improvements to, the OECD 309 and relevant OECD 314 tests, using appropriate benchmark chemicals with known biodegradability / persistency is required to increase industrial and regulatory stakeholder confidence in these studies. It was also recommended that the enhanced biomass studies could be included as part of this exercise.
• Research is conducted to demonstrate the ecological significance of adaptation and validate an appropriate experimental approach for its inclusion within persistency assessments. This approach should also develop appropriate marine studies with a reference set of validation chemicals.
• A ring test for enhanced biomass studies to compare their performance against other current screening tests with a reference set of validation chemicals.
• Test durations should be made more flexible and be allowed to extend to beyond 60 days as marine biodegradation studies often have lengthy lag periods before the onset of degradation.
• It was recommended that the current REACH guidance allowing modified tests, to assess chemicals with (i) poor water solubility and/or (ii) toxicity, should be allowed as they overcame some of the limitations associated with the RBTs.

Interpretation of higher tiered studies

Whilst higher tiered biodegradation studies attempt to increase the realism and relevance of persistence assessment, the increased complexity of the test system poses several additional challenges and uncertainties associated with the interpretation of the test data. Specific recommendations were as follows:

• Clarification of terms and definitions associated with chemical-sediment and chemical-soil interactions (e.g. NER and bound residues, type 1-3 NER).
• Improved understanding of remobilisation of test substance or transformation products from NER and the resulting environmental risk.
• Guidance on the use of DegT 50 values, not DT 50 values for comparison against the persistence triggers and the role that inverse modelling and other model-based approaches can have in elucidating removal mechanisms.
• Development of guidance for concluding on a persistence assessment where multiple data exist.
• Improved understanding of the impact of temperature on degradation rates as opposed to variability of biomass and other possible influences (see research needs).
• Improved identification of transformation products and associated risks.
• Publish data on correlations between the phylogeny of STP microorganisms and those present in natural environmental waters.

Enhanced realism within persistence assessment

It is recognised that screening and higher tiered persistence assessments lack realism and often introduce laboratory artefacts. These tests are quite often conducted only once so the relevance of the rate of degradation measured and the inherent variability around that measured rate is unknown. Tests are also of very limited duration compared to the environmental exposure of the chemical and the persistence assessment is made using a small number of environmental microbes with no history of pre-exposure and limited or no time to adapt to the test chemical during the study period. Specific recommendations were as follows:

• OECD 314 tests involving activated sludge at high concentrations of biomass are considered valuable in assessing the environmental fate and behaviour for the purposes of risk assessment only. However, the tests would not be accepted (by regulatory agencies) as methods for assessing chemical persistence, even for chemicals discharged to sewer, because sewage treatment may not always be applied to all point source and diffuse discharges. The assessment of chemical transformation products is an important aspect of risk assessment. In most cases assessment of the fate of parent and transformation products can be made in a single study. Data from the OECD 314 activated sludge die-away studies could be used as part of a weight of evidence assessment at the screening stage for persistence if the criteria for the inherent biodegradation in the Zahn-Wellens test (OECD 302B) are fulfilled.
• A set of chemicals with defined persistence criteria, e.g. LRI ECO 12 chemicals, should be established for use in degradation studies to provide internal benchmarks. This is particularly important for aquatic studies where inocula tend to be more variable compared to those in soil systems. This would need to involve considering additional chemicals to the current reference set to include a wider set of chemical structures and properties. There would need to be consideration of the availability of 14 C labelled compounds when considering chemicals to include.
• It was considered that key reference chemicals should be assessed in multiple labs, focussing on the complete set of OECD 314 tests (with the exception of the anaerobic test). Ideally OECD 307/308/ 309 should also be included to cover all environmental compartments and to adhere to environmentally relevant conditions (e.g. temperature, suspended particle concentration, etc.). Practically there would need to be a focus around key chemicals and tests. An output of the project would be to improve guidance documents or to revisit persistence cut-off criteria e.g. to consider whether persistence assessment is better done as a relative measure compared to appropriate established benchmarks. It was considered important to standardise inocula collection / handling etc. to minimise variability in the tests, before the testing phase. There would need to be agreed criteria for inoculum quality to allow comparison and reproducibility.
• It was felt that there was a need for a Task Force or further workshop to consider the relevance of laboratory data for the field situation. The aim would be to compile existing data and compare the fit of laboratory enhanced / modified screening tests (not ready or simulation tests) and field data. There may be data available from key sectors e.g. the pesticide industry; there may be potential to use blind data. Some data will be publicly available e.g. FOCUS reports. Once data has been considered an expert workshop should be held. The findings could be used to determine the need to introduce further complexity into laboratory tests, such as light associated processes.
• Inclusion of an adaptation phase offers potential for relevant assessments of chemical biodegradation but test designs must be interpreted with care or superseded by simulation tests inoculated with adapted biomass. Test designs should take account of the rapid loss of inoculum activity in batch systems. Other modifications to laboratory studies can enhance field relevance, e.g. including phototrophic organisms when relevant.
• Microbial characterisation of inocula using next generation sequencing procedures, which are becoming cheap and accessible. There was debate about how to take this forward- a literature review was suggested although the use of next metagenome and metatranscriptome analysis could be suitably advanced now for application of techniques. Techniques could have application, for instance, to investigate adaptation processes; to identify differences and similarities of inocula and its consequences for test outcome; to compare the relevance of laboratory inocula compared to the field situation; to cross compare different communities (e.g. compartment, geographical region) and assess whether there is commonality in terms of diversity and composition; to investigate and predict generic processes which drive transformation e.g. cometabolism; to apply microbial ecology theory to interpret and understand test outcomes; and to improve and refine systems. There would need to be initial validation of next generation sequencing methods.
• Further work is needed to develop knowledge and predictive capability of covalent binding of chemicals in the environment.