Experts Review Advances in Bioaccumulation Science and Identify Future Priorities
Thomas Parkerton, Exxon Mobil Biomedical Sciences, Caren Rauert, German Federal Environment Agency, Duane Huggett, University of North Texas, Kent Woodburn, Dow Corning, John Nichols, U.S. Environmental Protection Agency, Adam Lillicrap, Norwegian Institute for Water Research, Michelle Embry, ILSI Health and Environmental Sciences Institute
An expert workshop on in vivo methods for bioaccumulation assessment was held prior to the SETAC Berlin World Congress on 18 May 2012. The workshop was hosted by Umweltbundesamt (German Federal Environment Agency) and sponsored by the ILSI Health and Environmental Sciences Institute’s (HESI) Bioaccumulation Project Committee. The workshop had three objectives:
- Share recent advances in bioaccumulation assessment of organic chemicals
- Prioritize critical research needs to inform current and future planning efforts
- Promote incorporation of new data and methods into regulatory decision-making
The focus was on in vivo (laboratory-based) bioaccumulation endpoints involving whole organisms, and linkages of these endpoints with in silico (computer modeling), in vitro (sub-organismal) and in situ (field) approaches. Further background information about HESI and the Bioaccumulation Project can be found at www.hesiglobal.org.
Twenty-four experts from government, academia and private sectors participated in the workshop. Participants came from eight countries: Canada, Denmark, Germany, Japan, Norway, Sweden, the United Kingdom and the United States. The morning session featured 12 invited presentations highlighting recent work and suggesting short and longer-term needs in this field. Abstracts and presentations from the workshop are available at www.hesiglobal.org/i4a/pages/index.cfm?pageid=3604. During the afternoon session, participants provided information about new developments and data and ideas for future work that were not captured in the morning session. Participants then spent the remaining time prioritizing a consolidated list of future work recommendations.
A number of general themes emerged regarding recent developments in this field.
- Considerable effort has focused on standardization and refinement of in vivo methods for bioaccumulation assessment. Several participants highlighted the role of recent revisions to the OECD 305 test guideline that now includes an abbreviated bioconcentration test as well as a new dietary exposure method (OECD 2012). A comprehensive ring test involving ten laboratories has recently been completed for this latter test procedure (OECD 2011). The ring test involved simultaneous dietary exposure of rainbow trout to five test substances. Test substances were selected to provide similar hydrophobicity but different susceptibility to fish biotransformation. Increasingly, new in vivo bioaccumulation data are being generated using this updated guideline and one recent publication has shown an excellent correlation between measured aqueous (BCF) and dietary (BMF) test endpoints for a training set of nine chemicals with log Kow values ranging from 4.3 to 9.0 (Inoue et al. 2012).
- Additional efforts to improve in vivo methods have focused on increasing test precision and reducing cost, time and animal use. One advance involves the use of passive dosing techniques to deliver constant, better-defined aqueous exposures for poorly water soluble test substances that are prone to abiotic or biotic losses (Adolfsson-Erici et al. 2012a). A second advance is solvent-free passive sampling methods that quantify chemical concentrations in tissue and exposure media (Jahke et al 2011; Togunde et al. 2012).
- Several recent studies have employed experimental designs that involve testing multiple compounds simultaneously. As a result, the concept of including a conserved (i.e., non-metabolized) compound as an internal benchmark in these studies for subsequent use in data analysis and improved interpretation of test results has emerged as an innovative approach in bioaccumulation testing. Recent work suggests this approach can account for potential growth dilution and reduce variation in tissue measurements that are attributable to variation between individual fish or test substance analyses between samples (Adolfsson-Erici et al. 2012b).
- While testing multiple compounds simultaneously is attractive, concern was raised about potential metabolic interactions between compounds that may alter observed bioaccumulation potential. One study discussed at the workshop investigated this issue by determining steady-state BCFs for three compounds tested independently and simultaneously as a mixture. Results from this study indicated that BCFs derived from the mixture test were the same or higher than those determined in corresponding single exposure tests (Huggett and Springer 2012). While further research is needed, this case study suggests that bioaccumulation test designs involving multiple test substances may provide a conservative basis for characterizing substance-specific bioaccumulation potential.
- Recognizing the importance of biotransformation in modulating chemical bioaccumulation, considerable research has been devoted to developing methods and predictive models that quantitatively estimate this process. For example, back-calculation procedures were used to derive apparent whole-body biotransformation rates from in vivo aqueous and dietary bioaccumulation data. The resulting in vivo biotransformation rates were then employed to develop a quantitative model that predicts fish biotransformation rates based on chemical structure. This algorithm has subsequently been incorporated into the USEPA’s EPISuite software for in silico BCF and BAF prediction (Costanza et al. 2012).
- Several participants highlighted progress in the development of in vitro methods involving fish liver S9 fractions or hepatocytes and subsequent progress in the development of models for extrapolating in vitro measurements to in vivo biotransformation rates. Passive dosing techniques that can facilitate reliable exposure delivery are well suited for in vitro test designs with hydrophobic test substances (Lee et al. 2012). However, a number of challenges were described including lack of standardized in vitro test protocols and limited comparative in vivo data on the same chemicals and test species (e.g., same strain, life stages) that are needed to adequately validate in vitro to in vivo extrapolation procedures.
- A final theme discussed at the workshop was progress in developing integrated assessment approaches to support bioaccumulation assessment. One recent development that was outlined is the conversion of lab (e.g., BCF, BMF) and field (BAF, BMF, TMFs) bioaccumulation metrics into a common, fugacity ratio scale (Burkhard et al. 2011). This normalization is particularly useful in providing a weight-of-evidence framework for identifying chemicals that biomagnify in the environment.
Workshop participants identified five strategic priorities for future consideration, summarized below.
Compile, analyze and communicate new, reliable in vivo data
Participants highlighted the general need to expand the range of chemical classes for which we can predict biotransformation rates in fish. They also identified the need for comparative in vivo biotransformation data. The need for models to extrapolate across invertebrate and vertebrate species, particularly for chemicals that are known to be susceptible to biotransformation, was another identified need under the first strategic priority. They also identified the need for further work to reliably quantify and model dietary uptake and biotransformation rates in the gut, for the purpose of improving in silico prediction of in vivo dietary bioaccumulation endpoints.
Recent and ongoing chemicals management regulations such as REACH (Registration, Evaluation, Authorization and Restriction of Chemical Substances) have spurred the generation of in vivo bioaccumulation data. The availability of these new data provides a unique opportunity to build on earlier work by refining and expanding the applicability domain of mechanistic models for predicting in vivo endpoints. A key element of this effort is to use new in vivo data to derive apparent tissue biotransformation rate estimates for a broader range of chemicals and expand this database to include gut biotransformation rates. Such databases, in turn, can be used for validation of in silico or in vitro models that predict in vivo biotransformation rates.
A recent HESI project that is focused on compilation and analysis of in vivo bioaccumulation data for fish has been initiated to advance this recommendation. Individuals possessing fish BCF or BMF data that have been generated following OECD test guidelines and can be shared publicly are welcomed to contribute to this project by contacting Jon Arnot. Extension of this project to exploit existing in vivo lab data on species other than fish would provide a logical next project phase. To support linkages to other bioaccumulation assessment approaches, data, calculation methods and resulting apparent biotransformation rates should be made publicly available. One way to do that is to disseminate the data via the HESI website.
Continue in vivo method improvement
The abbreviated aqueous test method was developed by using subsets of available bioconcentration data to determine whether or not a simplified test design could yield reliable BCF values. To assess if a streamlined dietary test method is possible, a similar analysis of the existing data developed from a recently completed dietary round-robin study was recommended. As previously highlighted, passive dosing and sampling techniques offer promise for improving in vivo bioaccumulation testing. It was agreed that guidance should be published that describes principles for determining when and how these methods can be consistently applied.
Participants recognized that “back calculation” of apparent biotransformation rates from in vivo bioaccumulation data was a significant step forward. Nonetheless, they identified development of an experimental test protocol specifically designed to estimate tissue and gut biotransformation rates as a priority research need. Such guidance is particularly needed for test substances whose bioaccumulation behavior is not well described by existing models. Examples include substances with high protein-binding affinity and ionic chemicals. The incorporation of conserved chemical benchmarks in this protocol was suggested as a potential path forward. Once developed, this protocol also can be used to better evaluate fish size effects (allometry), temperature impacts, life-stage and species differences, as well as potential mixture interactions on biotransformation rates derived in lab tests.
While standardized laboratory bioaccumulation test methods such as OECD 315 are available for sediment-dwelling organisms using synthetic sediments, several participants raised concern about extrapolation to natural field sediments. Further work was recommended to better understand and possibly reduce the uncertainty in extrapolating lab-derived bioaccumulation endpoints using synthetic soils or sediments to field bioaccumulation endpoints in benthic and soil organisms.
Standardize and validate in vitro to in vivo extrapolation procedures
In vitro methods for determining biotransformation rates offer promise for reducing animal use and testing costs associated with bioaccumulation assessment. These methods, though, are still in an early phase of development. To advance their use in decision-making, standardized test guidelines and extrapolation procedures must be agreed upon and published. These methods then need to be applied to a wide range of chemicals and the results compared to reliable in vivo biotransformation rates for the same chemicals and test species in order to assess extrapolation performance.
Evaluate assumption that in vivo tests can be used to predict in situ biotransformation rates
Biotransformation estimates derived from laboratory studies currently are used to calibrate food chain models that are used to predict bioaccumulation in the field. Further research is needed to determine whether laboratory results are consistent with apparent field biotransformation rates.
To support an improved understanding of the linkage between laboratory and field biotransformation, guidance on field sampling methods and analysis (e.g., benchmarking) for deriving apparent biotransformation rates from field studies is needed. This guidance could then be used to target collection of field bioaccumulation data on chemical classes with varying susceptibility to biotransformation based on in silico, in vitro or in vivo data. Further compilation and sharing of field datasets is needed to better evaluate the performance of food chain models across different trophic levels and ecosystems for a broader range of chemicals.
Provide guidance to support bioaccumulation assessment that integrates multiple lines of evidence
The use of fugacity ratios was viewed as a promising starting point for interpreting laboratory and field data in chemical management. Workshop participants agreed that further practical experience with applying fugacity ratios is needed. In particular, defining the domain of applicability of this approach across chemicals is essential. For chemical classes that exhibit unusual behavior such as high-affinity binding to specific proteins, alternative normalization procedures are probably needed. Further integration of in silico and in vitro endpoints was viewed as a logical next step in updating guidance for assessing bioaccumulation based on weight-of-evidence approaches.
In closing, a cross-cutting element that is pertinent to all strategic priorities identified is the need for effective collaboration, communication and leveraging of funding resources across boundaries of traditional expertise. It is intended that the recommendations from this workshop will help to guide such collaborative work within academic centers and organizations such as HESI, the SETAC Bioaccumulation Science Advisory Group (BSAG), and European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC), as they set strategic priorities. The HESI Bioaccumulation Project Committee is working to prioritize these recommendations and develop a strategic plan, which could influence research funding allocation in key areas. Communication among research scientists and the regulatory community is a critical need, and this workshop was intended to facilitate constructive dialog.
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