Multi-residue screening of non-polar hazardous chemicals in green turtle blood from different foraging regions of the Great Barrier Reef.

Green turtles spend a large part of their lifecycle foraging in nearshore seagrass habitats, which are often in close proximity to sources of anthropogenic contaminants. As most biomonitoring studies focus on a limited number of targeted chemical groups, this study was designed to screen for a wider range of hazardous chemicals that may not have been considered in prior studies. Whole blood of sub-adult green turtles (Chelonia mydas) were sampled from three different locations, a remote, offshore 'control' site; and two coastal 'case' sites influenced by urban and agricultural activities on the Great Barrier Reef in North Queensland, Australia. In order to screen blood samples for chemicals across a wide range of KOW's, a modified QuEChER's extraction method was used. The samples were analysed using a multi-residue gas chromatography with tandem mass spectrometry system (GC-MS/MS method that allowed simultaneous quantification of polychlorinated biphenyls (PCBs), polychlorinated diphenyl ethers (PBDES), organochlorine pesticides (OCPs) and polycyclic aromatic hydrocarbons (PAHs). While PBDEs, PCBs and OCPS were below the limits of quantification, PAHs were detected in all turtle blood samples. However, PAH levels were relatively low (maximum ΣPAH = 13 ng/mL ww) and comparable to or less than those reported from other green turtles globally. The present study provides the first baseline PAH levels in blood samples from green turtles from nearshore and offshore locations in the Southern Hemisphere.

Effect-based approach for screening of chemical mixtures in whole blood of green turtles from the Great Barrier Reef.

Organisms are exposed to mixtures of both known and unknown chemicals which are diverse and variable, and thus difficult and costly to characterise and monitor using traditional target analyses. The objective of this study was to validate and apply in vitro effect-based methods by which whole blood can be used to screen internal exposure to such complex chemical mixtures. For this study, we used whole blood of green sea turtles (Chelonia mydas). To ensure the chemical mixture in blood is transferred with minimal losses or bias, we tested a modified QuEChERS extraction method specifically developed for multi- and non-target instrument analysis. The extracts were dosed to a battery of in vitro bioassays (AhR-CAFLUX, AREc32, NFκB-bla, VM7Luc4E2, Microtox), each with a different mode of action (e.g., AhR receptor mediated xenobiotics, NrF2-mediated oxidative stress, NFκB mediated response to inflammation, estrogen activity and baseline toxicity oxidative stress, respectively) in order to cover a wide spectrum of chemicals. Results confirmed the absence of interferences of the blood extract with the responses of the different assays, thus indicating the methods' compatibility with effect-based screening approaches. To apply this approach, whole blood samples were collected from green turtles foraging in agricultural, urban and remote areas of the Australian Great Barrier Reef. The effect-based screening revealed significant differences in exposure, with higher induction of AhR-CAFLUX, AREc32 and Microtox assays in turtles from the agricultural foraging ground. Overall, these results corroborated with concurrent health, target and non-target analyses in the same animals performed as part of a larger program. This study provides evidence that the proposed effect-based approach is suitable for screening and evaluating internal exposure of organisms to chemical mixtures. The approach could be valuable for advancing understanding on multiple levels ranging from identification of priority chemicals in effect-directed investigations to exploring relationships between exposure and disease, not only in sea turtles, but in any organism.

Mechanisms of Action Point Towards Combined PBDE/NDL-PCB Risk Assessment.

At present, human risk assessment of the structurally similar non-dioxin-like (NDL) PCBs and polybrominated diphenylethers (PBDEs) is done independently for both groups of compounds. There are however obvious similarities between NDL-PCBs and PBDEs with regard to modulation of the intracellular calcium homeostasis (basal calcium levels, voltage-gated calcium channels, calcium uptake, ryanodine receptor) and thyroid hormone (TH) homeostasis (TH levels and transport). which are mechanisms of action related to neurobehavioral effects (spontaneous activity, habituation and learning ability). There also similarities in agonistic interactions with the hepatic nuclear receptors PXR and CAR. Several effects on developmental (reproductive) processes have also been observed, but results were more dispersed and insufficient to compare both groups of compounds. The available mechanistic information is sufficient to warrant a dose addition model for NDL-PCBs and PBDEs, including their hydroxylated metabolites.Although many of the observed effects are similar from a qualitative point of view for both groups, congener or tissue specific differences have also been found. As this is a source of uncertainty in the combined hazard and risk assessment of these compounds, molecular entities involved in the observed mechanisms and adverse outcomes associated with these compounds need to be identified. The systematical generation of (quantitative) structure-activity information for NDL-PCBs and PBDEs on these targets (including potential non-additive effects) will allow a more realistic risk estimation associated with combined exposure to both groups of compounds during early life. Additional validation studies are needed to quantify these uncertainties for risk assessment of NDL-PCBs and PBDEs.

Assessment of the neurotoxic potential of exposure to 50Hz extremely low frequency electromagnetic fields (ELF-EMF) in naïve and chemically stressed PC12 cells.

Increasing exposure to extremely low frequency electromagnetic fields (ELF-EMF), generated by power lines and electric appliances, raises concern about potential adverse health effects of ELF-EMF. The central nervous system is expected to be particularly vulnerable to ELF-EMF as its function strongly depends on electrical excitability. We therefore investigated effects of acute (30min) and sub-chronic (48h) exposure to 50Hz ELF-EMF on naïve and chemically stressed pheochromocytoma (PC12) cells. The latter have higher levels of iron and/or reactive oxygen species (ROS) and display increased vulnerability to environmental insults. Effects of ELF-EMF on Ca(2+)-homeostasis, ROS production and membrane integrity were assessed using Fura-2 single cell fluorescence microscopy, H2-DCFDA and CFDA assays, respectively. Our data demonstrate that acute exposure of naïve PC12 cells to 50Hz ELF-EMF up to 1000µT fails to affect basal or depolarization-evoked [Ca(2+)]i. Moreover, sub-chronic ELF-EMF exposure up to 1000µT has no consistent effects on Ca(2+)-homeostasis in naïve PC12 cells and does not affect ROS production and membrane integrity. Notably, in chemically stressed PC12 cells both acute and sub-chronic ELF-EMF exposure also failed to exert consistent effects on Ca(2+)-homeostasis, ROS production and membrane integrity. Our combined findings thus indicate that exposure to 50Hz ELF-EMF up to 1000µT, i.e. 10,000 times above background exposure, does not induce neurotoxic effects in vitro, neither in naïve nor in chemically stressed PC12 cells. Though our data require confirmation, e.g. in developing neuronal cells in vitro or (developing) animals, it appears that the neurotoxic risk of ELF-EMF exposure is limited.