Characterizing the marine mammal exposome by iceberg modeling, linking chemical analysis and in vitro bioassays.

The present study complements work on mixture effects measured with in vitro bioassays of passive equilibrium sampling extracts using the silicone polydimethylsiloxane (PDMS) in organs from marine mammals with chemical profiling. Blubber, liver, kidney and brain tissues of harbor porpoise (Phocoena phocoena), harbor seal (Phoca vitulina), ringed seal (Phoca hispida) and orca (Orcinus orca) from the North and Baltic Seas were investigated. We analyzed 117 chemicals including legacy and emerging contaminants using gas chromatography-high resolution mass spectrometry and quantified 70 of those chemicals in at least one sample. No systematic differences between the organs were found. Only for single compounds a clear distribution pattern was observed. For example, 4,4'-dichlorodiphenyltrichloroethane, enzacamene and etofenprox were mainly detected in blubber, whereas tonalide and the hexachlorocyclohexanes were more often found in liver. Furthermore, we compared the chemical profiling with the bioanalytical results using an iceberg mixture model, evaluating how much of the biological effect could be explained by the analyzed chemicals. The mixture effect predicted from the quantified chemical concentrations explained 0.014-83% of the aryl hydrocarbon receptor activating effect (AhR-CALUX), but less than 0.13% for the activation of the oxidative stress response (AREc32) and peroxisome-proliferator activated receptor (PPARγ). The quantified chemicals also explained between 0.044-45% of the cytotoxic effect measured with the AhR-CALUX. The largest fraction of the observed effect was explained for the orca, which was the individuum with the highest chemical burden. This study underlines that chemical analysis and bioassays are complementary to comprehensively characterize the mixture exposome of marine mammals.

Activation of the xenobiotic metabolism and oxidative stress response by mixtures of organic pollutants extracted with in-tissue passive sampling from liver, kidney, brain and blubber of marine mammals.

We used in-tissue passive equilibrium sampling using the silicone polydimethylsiloxane (PDMS) to transfer chemical mixtures present in organs from marine mammals with lipid contents between 2.3 and 99%into in vitro bioassays. Tissues from five harbor porpoises (Phocoena phocoena), one harbor seal (Phoca vitulina) and one orca (Orcinus orca) from the North and Baltic Seas were sampled until thermodynamic equilibrium was reached. Mixture effects were quantified with cellular reporter gene bioassays targeting the activation of the aryl hydrocarbon receptor (AhR-CALUX), the peroxisome proliferator-activated receptor gamma (PPARγ-bla) and the oxidative stress response (AREc32), with parallel cytotoxicity measurements in all assays. After removing co-extracted lipids and other matrix residues with a non-destructive cleanup method (freeze-out of acetonitrile extract followed by a primary secondary amine sorbent extraction), the activation of the PPARγ and AREc32 were reduced by factors of on average 4.3 ± 0.15 (n = 22) and 2.5 ± 0.23 (n = 18), respectively, whereas the activation of the AhR remained largely unaltered: 1.1 ± 0.075 (n = 6). The liver extracts showed the highest activation, followed by the corresponding kidney and brain extracts, and the blubber extracts of the animals were the least active ones. The activation of the PPARγ by the liver extracts was reduced after cleanup by a factor of 11 ± 0.26 (n = 7) and the AREc32 activity by a factor of 1.9 ± 0.32 (n = 4). The blubber extracts did not activate the AhR up to concentrations where cytotoxicity occurred or up to an acceptable lipid volume fraction of 0.27% as derived from earlier work, whereas all liver extracts that had undergone cleanup activated the AhR. The developed in-tissue passive sampling approach allows a direct comparison of the bioassay responses between different tissues without further normalization and serves as a quantitative method suitable for biomonitoring of environmental biota samples.

Influence of Co-Dosed Lipids from Biota Extracts on the Availability of Chemicals in In Vitro Cell-Based Bioassays.

Extraction of chemicals from biota leads to co-extraction of lipids. When dosing such extracts into in vitro bioassays, co-dosed lipids act as an additional phase that can reduce the bioavailability of the chemicals and the apparent sensitivity of the assay. Equilibrium partitioning between medium, cells, and co-dosed lipids was described with an existing equilibrium partitioning model for cell-based bioassays extended by an additional lipid phase. We experimentally investigated the influence of co-dosed lipids on the effects elicited by four test chemicals of different hydrophobicity in two bioassays, indicative of the aryl hydrocarbon receptor and oxidative stress response (AREc32). The partitioning model explained the effect of the test chemicals in the presence of spiked triolein within a factor of 0.33-5.83 between the measured and predicted effect concentration (EC). We applied the model to marine mammal blubber extracted with silicone. Extracts dosed in the AREc32 bioassay showed a linear increase of apparent EC with increasing lipid fraction. The partitioning model was used to interpret the role of the co-extracted lipid. A quantitative lipid correction of bioassay results in the presence of co-dosed lipids was possible for known compounds and defined mixtures, while we could only estimate a range for mixtures of unknown chemicals.