Increasing amounts of manufactured nanomaterials (MNMs) are produced for their industrial use and released to the environment by the usage or disposal of the products. As depending on their annual production rate, substances are subjected to PBT assessment, the availability of reliable methods to evaluate these endpoints for (corresponding) nanoforms/MNMs becomes relevant. The classical method to elucidate the bioaccumulation potential of chemicals has been the flow-through study with fish, which has limitations as regards meeting the requirements of MNMs. Most MNMs tend to sediment in the aquatic environment. Thus, maintenance of stable exposure conditions for bioaccumulation testing with fish is nearly impossible to achieve when using MNMs. Corbicula fluminea, a freshwater filter-feeding bivalve distributed worldwide, has been previously shown to ingest and accumulate MNMs present in the water phase. To investigate the suitability of C. fluminea for bioaccumulation testing we developed a new flowthrough system to expose mussels under constant exposure conditions. Two nanoparticles (NPs), the AgNP NM 300K and the TiO 2 NP NM 105, were applied. In addition, C. fluminea was exposed to AgNO 3 as a source of dissolved Ag + to compare the bioaccumulation of Ag in dissolved and nanoparticulate forms. For each MNM exposure scenario we were able to determine steady-state bioaccumulation factors. BAF ss values of 31 and 128 for two NM 300K concentrations (0.624 and 6.177 μg Ag per L) and 6150 and 9022for TiO 2 (0.099 and 0.589 μg TiO 2 per L) showed the exposure dependence of the BAF ss estimates. The progression of metal uptake and elimination in the soft tissue provided clear indications that the uptake and thus accumulation is mainly driven by the uptake of NPs and less of dissolved ions.Filter-feeding organisms such as bivalves represent a major target for the bioaccumulation of nanomaterials in the aquatic environment. Therefore, bivalves should be considered as test organisms for the bioaccumulation assessment of nanomaterials. A new flow-through system to expose the freshwater bivalves under constant exposure conditions was developed. Bioaccumulation studies with the freshwater bivalve C. fluminea on two nanoparticles, the AgNP NM 300K and the TiO 2 NP NM 105, demonstrated the suitability of the new test system. The results obtained with this test system can be used to generate useful endpoints required for regulatory purposes and could be included in a tiered bioaccumulation testing strategy for manufactured nanomaterials.

Background The high production volume of engineered nanomaterials (ENMs) may lead to high pressure on the environment, and a scientific assessment of ENMs that bioaccumulate in organisms and biomagnify in the food web is necessary. Within the regulation of chemicals in several jurisdictions, such as the European regulation REACH, the bioconcentration factor is the standard endpoint. The bioconcentration factor is mostly determined by flow-through fish tests. However, nanomaterials tend to agglomerate, which may lead to sedimentation in aquatic environments. The bioavailability of the tested nanomaterials may be thus impaired for pelagic species, including fish, in comparison to benthic or filtrating species. Several risk assessment regulations allow the usage of data gained during tests using invertebrates and such data may allow a waiver of further tests using vertebrates. The aim of this study was to elucidate the potential of different freshwater invertebrate species to be used in laboratory bioaccumulation studies on ENMs and to give some guidance for the use of bioaccumulation endpoints derived from studies using aquatic invertebrate species in the risk assessment process for ENMs. Results The existing literature related to the testing of nanomaterial bioaccumulation with freshwater invertebrates was screened and reviewed to find suitable test species with regard to their ecology and physiology, as well as laboratory test systems allowing to investigate the bioavailability/bioaccumulation of nanomaterials with the respective species. Bivalvia, gastropoda, isopoda, amphipoda, and branchiopoda were reviewed and their suitability for bioaccumulation testing was assessed. Amphipods and bivalves represent worst-case scenarios and show clear advantages to be used as test organisms. However, only amphipods allow the examination of two clearly independent exposure pathways (water and diet). Conclusion Amphipods are suitable test organisms for bioaccumulation testing of ENMs. The results from amphipod bioconcentration and biomagnification tests can be included in a tiered assessment suggested at the end of this study allowing a clear grading of the tested nanomaterials as “bioaccumulative” or “non bioaccumulative.” Due to the worst-case scenario of the amphipod test, this approach may allow a waiver of further vertebrate tests.

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