Karina Petersen

Environmental risk assessment of combined effects in aquatic ecotoxicology: a discussion paper.

Environmental regulatory edicts within the EU, such as the regulatory framework for chemicals REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals), the Water Framework Directive (WFD), and the Marine Strategy Framework Directive (MSFD) focus mainly on toxicity assessment of individual chemicals although the effect of contaminant mixtures is a matter of increasing concern. This discussion paper provides an overview of the field of combined effects in aquatic ecotoxicology and addresses some of the major challenges related to assessment of combined effects in connection with environmental risk assessment (ERA) and regulation. Potentials and obstacles related to different experimental, modelling and predictive ERA approaches are described. On-going ERA guideline and manual developments in Europe aiming to incorporate combined effects of contaminants, the use of different experimental approaches for providing combined effect data, the involvement of biomarkers to characterize Mode of Action and toxicity pathways and efforts to identify relevant risk scenarios related to combined effects are discussed.

Toxicity of Synthetic Naphthenic Acids and Mixtures of These to Fish Liver Cells

Environmental concerns have been raised over the toxicity of crude naphthenic acids (NA) originating from oil exploration activities offshore, oil sands exploitation onshore, and use of refined NA as wood preservatives, tire additives, and in various other applications. The NA exist in highly complex mixtures, so the toxic effects of the individual acids are rarely known. The present study investigated the relationships between the chemical structures of a range of synthetic alicyclic and aromatic acids and their acute toxicities both as single chemicals and as complex mixtures in a primary culture of rainbow trout (Oncorhynchus mykiss) hepatocytes. The combined toxicity of multicomponent mixtures of these NA was assessed using the concept of concentration addition (CA) and independent action (IA) prediction. All of the acids tested were moderately toxic, with EC(50) values in the range 108-405 μM (24-89 mg L(-1)) and 188-656 μM (43-148 mg L(-1)) when assessed by effects on metabolic inhibition or loss of membrane integrity, respectively. Binary and 6-compound mixture of NA caused combined toxicity according to the concept of additivity, although slight deviations from additivity were observed at a few mixture concentrations. Single NA and mixtures of NA with similar structures to those tested herein probably contribute to the toxicity of complex natural mixtures of NA. Toxicity tests on three commercial NA mixtures showed that these exhibited highly variable toxicities themselves probably reflecting their chemical heterogeneity.

Early stress responses in Atlantic salmon (Salmo salar) exposed to environmentally relevant concentrations of uranium

Uranium (U) is a naturally occurring heavy metal widely used in many military and civil applications. Uranium contamination and the associated potential adverse effects of U on the aquatic environment have been debated during recent years. In order to understand the effect and mode of action (MoA) of U in vivo, juvenile Atlantic salmon (Salmo salar) were exposed to 0.25 mg/L, 0.5 mg/L and 1.0mg/L waterborne depleted uranyl acetate, respectively, in a static system for 48 h. The U concentrations in the gill and liver were analyzed by inductively coupled plasma mass spectrometry (ICP-MS) and the resulting biological effects were determined by a combination of analysis of gene expression and micronuclei formation. The hepatic transcriptional level of 12 biomarker genes from four stress-response categories, including oxidative stress (γ-glutamyl cysteine synthetase (GCS), glutathione reductase (GR), glutathione peroxidase (GPx)), DNA damage and repair (P53, cyclin-dependent kinase inhibitor 1 (P21), growth arrest and DNA damage-inducible gene gamma (Gadd45G), proliferating cell nuclear antigen (PCNA), Rad51), apoptosis (Bcl2-associated X protein (BAX), Bcl-x, Caspase 6A,) and protein degradation (Ubiquitin) were evaluated by quantitative real-time polymerase chain reaction (q-rtPCR). The results clearly showed accumulation of U in the gill and liver with increasing concentrations of U in the exposure water. The effects of U on differential hepatic gene expression also occurred in a concentration-dependent manner, although deviations from ideal concentration-response relationships were observed at the highest U concentration (1.0 mg/L). All the genes tested were found to be up-regulated by U while no significant micronuclei formation was identified. The results suggest that U may cause oxidative stress in fish liver at concentrations greater than 0.25 mg/L, giving rise to clear induction of several toxicologically relevant biomarker genes, although no significant adverse effects were observed after the relatively short exposure period.

Combined effects of pharmaceuticals, personal care products, biocides and organic contaminants on the growth of Skeletonema pseudocostatum

Organisms in the environment are exposed to a number of pollutants from different compound groups. In addition to the classic pollutants like the polychlorinated biphenyls, polyaromatic hydrocarbons (PAHs), alkylphenols, biocides, etc. other compound groups of concern are constantly emerging. Pharmaceuticals and personal care products (PPCPs) can be expected to co-occur with other organic contaminants like biocides, PAHs and alkylphenols in areas affected by wastewater, industrial effluents and intensive recreational activity. In this study, representatives from these four different compound groups were tested individually and in mixtures in a growth inhibition assay with the marine algae Skeletonema pseudocostatum (formerly Skeletonema costatum) to determine whether the combined effects could be predicted by models for additive effects; the concentration addition (CA) and independent action (IA) prediction model. The eleven tested compounds reduced the growth of S. pseudocostatum in the microplate test in a concentration-dependent manner. The order of toxicity of these chemicals were irgarol>fluoxetine>diuron>benzo(a)pyrene>thioguanine>triclosan>propranolol>benzophenone 3>cetrimonium bromide>4-tert-octylphenol>endosulfan. Several binary mixtures and a mixture of eight compounds from the four different compound groups were tested. All tested mixtures were additive as model deviation ratios, the deviation between experimental and predicted effect concentrations, were within a factor of 2 from one or both prediction models (e.g. CA and IA). Interestingly, a concentration dependent shift from IA to CA, potentially due to activation of similar toxicity pathways at higher concentrations, was observed for the mixture of eight compounds. The combined effects of the multi-compound mixture were clearly additive and it should therefore be expected that PPCPs, biocides, PAHs and alkylphenols will collectively contribute to the risk in areas contaminated by such complex mixtures.

PAH related effects on fish in sedimentation ponds for road runoff and potential transfer of PAHs from sediment to biota

Road runoff is an important source of pollution to the aquatic environment, and sedimentation ponds have been installed to mitigate effects on the aquatic environment. The purpose of this study was to investigate if a) fish from sedimentation ponds were affected by road pollution and; b) the transfer of PAHs from road runoff material to aquatic organisms was substantial. Minnow from a sedimentation pond (Skullerud) near Oslo (Norway) had higher levels of CYP1A enzyme and DNA stand breaks than minnow from the nearby river, but high concentrations of PAH-metabolites in bile revealed that both populations were highly exposed. Principal component analysis revealed that CYP1A and age of fish were correlated, while levels of PAH-metabolites were not correlated to CYP1A or DNA damage. Minnow from a lake un-affected by traffic had much lower levels of PAH-metabolites than the exposed fish, and also an improved condition. The latter results indicate that fish health was affected by road runoff. A closer investigation of PAH levels of the ecosystems of two sedimentation ponds (Skullerud and Vassum) and nearby environments were conducted. The concentration of the 16 EPA PAHs in sediments of the sedimentation ponds were high (1900-4200ngg(-1)), and even higher levels were observed in plants. Principal component analysis of selected ion chromatograms of PAHs showed a clear separation of plants vs. sediments. The plants preferentially accumulated the high molecular PAHs, both from sedimentation ponds with a petrogenic PAH isomer ratio in sediments; and from a lake with pyrogenic PAH isomer ratio in sediments.

Mixture toxicity of five biocides with dissimilar modes of action on the growth and photosystem II efficiency of Chlamydomonas reinhardtii

ABSTRACT Biocides are extensively used and universally distributed. Some are highly toxic to algae, including antifoulants, herbicides, and fungicides. The inhibition of algal growth is an important regulatory endpoint for toxicity assessment of single compounds. However, in the aquatic environment, mixtures of compounds with unknown toxicities and mode of action (MoA) co-exist, making single toxicity assessment inadequate to ensure protection of the aquatic environment. This study aimed to characterize the combined toxicity of five environmentally relevant biocides—aclonifen, bifenox, dichlofluanid, metribuzin, and triclosan—with different MoA on growth and photosystem (PS) II efficiency of Chlamydomonas reinhardtii. For growth inhibition, herbicides bifenox and metribuzin were the most toxic, whereas triclosan was least. Only aclonifen and metribuzin exerted a significant effect on PSII, which was also correlated with reduced algal growth. The combined effect of the five biocides on growth inhibition was predominantly additive and presumed to act by independent MoA with potential antagonism observed only at low concentrations and at shorter duration of exposure. The binary mixture of metribuzin and aclonifen exhibited additive effects on diminished PSII efficiency, and effects were apparently induced by an independent MoA. Potential synergy of this mixture on growth inhibition was identified at the highest concentrations. Growth inhibition was found to be a more valuable endpoint for regulatory studies than PSII inhibition due to its environmental relevance, integration of multiple MoA and sensitivity.

Primary hepatocytes from Arctic char (Salvelinus alpinus) as a relevant Arctic in vitro model for screening contaminants and environmental extracts

Contaminants find their way to the Arctic through long-range atmospheric transport, transport via ocean currents, and through increased anthropogenic activity. Some of the typical pollutants reaching the Arctic (PAHs, PCBs) are known to induce cytochrome P450 1a (CYP1A) protein expression and ethoxyresorufin-O-deethylase (EROD) activity through the aryl hydrocarbon receptor (AhR). In addition, some endocrine disrupting chemicals (EDCs) such as estrogen mimics (xenoestrogens) have been documented in Arctic areas and they may interfere with natural sexual development and reproduction. In vitro assays that are capable of detecting effects of such pollutants, covering multiple endpoints, are generally based on mammalian or temperate species and there are currently no well-characterized cell-based in vitro assays for effect assessment from Arctic fish species. The present study aimed to develop a high-throughput and multi-endpoint in vitro assay from Arctic char (Salvelinus alpinus) to provide a non-animal (alternative) testing method for an ecologically relevant Arctic species. A method for isolation and exposure of primary hepatocytes from Arctic char for studying the toxic effects and mode of action (MoA) of pollutants was applied and validated. The multi-versatility of the bioassay was assessed by classical biomarker responses such as cell viability (membrane integrity and metabolic activity), phase I detoxification (CYP1A protein expression, EROD activity) and estrogen receptor (ER) mediated vitellogenin (Vtg) protein expression using a selection of model compounds, environmental pollutants and an environmental extract containing a complex mixture of pollutants. Primary hepatocytes from Arctic char were successfully isolated and culture conditions optimized to identify the most optimal assay conditions for covering multiple endpoints. The hepatocytes responded with concentration-dependent responses to all of the model compounds, most of the environmental pollutants and the environmental sample tested. The bioassay response and sensitivity of the hepatocytes from Arctic char differed slightly from closely related salmonid species, thus highlighting the need for developing in vitro assays relevant for Arctic species. The present multi-endpoint in vitro assay offer a highly versatile tool to screen potential effects of pollutants and complex samples relevant for Arctic exposure scenarios.

Characterizing cytotoxic and estrogenic activity of Arctic char tissue extracts in primary Arctic char hepatocytes

ABSTRACT Contaminants from various anthropogenic activities are detected in the Arctic due to long-range atmospheric transport, ocean currents, and living organisms such as migrating fish or seabirds. Although levels of persistent organic pollutants (POPs) in Arctic fish are generally low, local hot spots of contamination were found in freshwater systems such as Lake Ellasjøen at Bjørnøya (Bear Island, Norway). Higher concentrations of organic halogenated compounds (OHC), and higher levels of cytochrome P450 and DNA-double strand breaks were reported in Arctic char (Salvelinus alpinus) from this lake compared to fish from other lakes on Bjørnøya. Although several of the measured contaminants are potential endocrine disrupters, few studies have investigated potential endocrine disruptive effects of the contaminant cocktail in this fish population. The aim of this study was to compare acutely toxic and estrogenic potency of the cocktail of pollutants as evidenced by cytotoxic and/or estrogenic effects in vitro using extracts of Arctic char livers from contaminated Lake Ellasjøen with those from less contaminated Lake Laksvatn at Bjørnøya. This was performed by in situ sampling and contaminant extraction from liver tissue, followed by chemical analysis and in vitro testing of the following contaminated tissue extracts: F1-nonpolar OHC, F2-polar pesticides and metabolites of OHC, and F3-polar OHC. Contaminant levels were highest in extracts from Ellasjøen fish. The F2 and F3 extracts from Lake Laksvatn and Lake Ellasjøen fish reduced in vitro cell viability at a concentration ratio of 0.03–1 relative to tissue concentration in Arctic char. Only the F3 liver extract from Ellasjøen fish increased in vitro vitellogenin protein expression. Although compounds such as estrogenic OH-PCBs were quantified in Ellasjøen F3 extracts, it remains to be determined which compounds were inducing estrogenic effects.