Comprehensive target analysis and TOP assay of per- and polyfluoroalkyl substances (PFAS) in wild boar livers indicate contamination hot-spots in the environment.

The suitability of wild boar liver as a bioindicator of per- and polyfluoroalkyl substances (PFAS) in the terrestrial environment was investigated. Samples from 50 animals in three different areas associated with (1) contaminated paper sludges distributed on arable land (PS), (2) industrial emissions of PFAS (IE) and (3) background contamination (BC) were analyzed for 66 PFAS, including legacy PFAS, novel substitutes and precursors of perfluoroalkyl acids (PFAAs). Additionally, the Total Oxidizable Precursor (TOP) assay was performed to determine the formation potential of PFAAs from precursors. In total, 31 PFAS were detected with site-specific contamination profiles. PFAS concentrations in livers from area PS and IE (567 and 944 µg kg-1 wet weight, respectively) were multiple times higher than from area BC (120 µg kg-1). The dominating PFAS were the legacy compounds perfluorooctane sulfonic acid (PFOS) in areas PS and BC (426 and 82 µg kg-1, respectively) and perfluorooctanoic acid (PFOA) in area IE (650 µg kg-1). In area IE, the compounds 4,8-dioxa-3H-perfluorononanoic acid (DONA) and hexafluoropropylene oxide dimer acid (HFPO-DA) - which are used as substitutes for PFOA - were determined at 15 and 0.29 µg kg-1, respectively. The formation potential of PFAAs was highest in area PS, but generally lower than the contamination with PFAAs. The pattern of perfluoroalkyl carboxylic acids (PFCAs) in wild boar liver reflects the contamination of the local soil at the two hot-spot areas IE and PS. This first comparison of PFAS contamination between wild boars and soil suggests that wild boar livers are suitable bioindicators for PFAS contamination in the terrestrial environment. Moreover, in terrestrial samples from area IE, legacy PFAS were found to be retained for a longer period as compared to riverine samples (suspended particulate matter and chub filet).

Differences in the internal PFAS patterns of herbivores, omnivores and carnivores - lessons learned from target screening and the total oxidizable precursor assay.

Per- and polyfluorinated alkyl substances (PFAS) are a group of anthropogenic chemicals, which are not (fully) biodegradable and accumulate in different environmental compartments worldwide. A comprehensive, quantitative analysis - consisting of target analysis (66 different analytes, including e. g. ultrashort-chain perfluorinated carboxylic acids (PFCAs), precursor compounds and novel substitutes) and the Total Oxidisable Precursor (TOP) assay (including trifluoroacetic acid (TFA)) - were conducted to analyse the PFAS concentrations and patterns in 12 mammalian and two bird species from different areas of Germany and Denmark. The PFAS contamination was investigated in dependance of the trophic class (herbivores, omnivores, carnivores), ecological habitat (terrestrial, (semi-) aquatic) and body tissue (liver, musculature). PFAS concentrations were highest in carnivores, followed by omnivores and herbivores, with ∑PFAS concentration ranging from 1274 µg/kg (Eurasian otter liver) to 22 µg/kg (roe deer liver). TFA dominated in the herbivorous species, whereas perfluorooctanesulfonic acid (PFOS) and the long-chain PFCAs covered the majority of the PFAS contamination in carnivorous species. Besides trophic class, ecological habitat also affected the PFAS levels in the different species, with terrestrial herbivores and omnivores showing higher PFAS concentration than their aquatic counterparts, whereas for carnivores this relationship was reversed. The TOP assay analysis indicated similar trends, with the PFCA formation pattern differing significantly between the trophic classes. TFA was formed predominantly in herbivorous and omnivorous species, whereas in carnivorous species a broad spectrum of PFCAs (chain-length C2-C14) was formed. Musculature tissue of six species exhibited significantly lower PFAS concentrations than the respective liver tissue, but with similar PFAS patterns. The comprehensive approach applied in the present study showed, that primarily the trophic class is decisive for the PFAS concentration, as herbivores, omnivores and carnivores clearly differed in their PFAS concentrations and patterns. Additionally, the TOP assay gave novel insights in the PFCA formation potential in biota samples.

Assessment of contaminants of emerging concern in European apex predators and their prey by LC-QToF MS wide-scope target analysis.

Apex predators are good indicators of environmental pollution since they are relatively long-lived and their high trophic position and spatiotemporal exposure to chemicals provides insights into the persistent, bioaccumulative and toxic (PBT) properties of chemicals. Although monitoring data from apex predators can considerably support chemicals' management, there is a lack of pan-European studies, and longer-term monitoring of chemicals in organisms from higher trophic levels. The present study investigated the occurrence of contaminants of emerging concern (CECs) in 67 freshwater, marine and terrestrial apex predators and in freshwater and marine prey, gathered from four European countries. Generic sample preparation protocols for the extraction of CECs with a broad range of physicochemical properties and the purification of the extracts were used. The analysis was performed utilizing liquid (LC) chromatography coupled to high resolution mass spectrometry (HRMS), while the acquired chromatograms were screened for the presence of more than 2,200 CECs through wide-scope target analysis. In total, 145 CECs were determined in the apex predator and their prey samples belonging in different categories, such as pharmaceuticals, plant protection products, per- and polyfluoroalkyl substances, their metabolites and transformation products. Higher concentration levels were measured in predators compared to prey, suggesting that biomagnification of chemicals through the food chain occurs. The compounds were prioritized for further regulatory risk assessment based on their frequency of detection and their concentration levels. The majority of the prioritized CECs were lipophilic, although the presence of more polar contaminants should not be neglected. This indicates that holistic analytical approaches are required to fully characterize the chemical universe of biota samples. Therefore, the present survey is an attempt to systematically investigate the presence of thousands of chemicals at a European level, aiming to use these data for better chemicals management and contribute to EU Zero Pollution Ambition.

Evaluation of acute-to-chronic ratios of fish and Daphnia to predict acceptable no-effect levels.

BACKGROUND: Acute-to-chronic extrapolation is an important approach to predict acceptable no-effect levels from acute data which has some uncertainties, but is valuable for risk assessment of chemical substances. With regard to the still limited and heterogenic data of chronic fish tests, conclusions on aquatic hazard estimation need to be checked and the question arises whether the chronic toxicity to fish can be adequately derived from acute data. A comprehensive dataset including ecotoxicological studies of 203 substances was used to investigate acute-to-chronic ratios (ACR) for both fish and Daphnia. To address potential uncertainty parameters of the approach, the influence of the octanol-water partition coefficient Kow, the mode of action, and the acute toxicity levels on the ACR was evaluated. RESULTS: For industrial chemicals, median ACRs of 12.0 for fish and 8.8 for Daphnia and 90th percentiles of 68.0 and 50.2, respectively, were determined. The ACR for the most sensitive aquatic trophic level (ACRaqu) is derived by comparing the lowest acute and chronic effect value of Daphnia and fish. The median ACRaqu was 9.9, and the 90th percentile was determined to 58.5. The influence of the Kow on the ACR value was analysed and a correlation could not be confirmed. Non-polar narcosis was associated with a lower ACR, whereas polar narcosis was associated with an increased ACR. CONCLUSIONS: The result suggests that an acute-to-chronic extrapolation factor of 100 is protective for more than 90 % of the chemicals. Polar narcosis may represent a predictor for an increased ACR and an increased uncertainty of the approach. The result further suggests that a high Kow is probably not associated with increased ACRs and does not necessarily represent a determinant for chronic toxicity testing within this context.

Evaluation of the integrated testing strategy for PNEC derivation under REACH.

Species sensitivity evaluation represents an approach to avoid chronic toxicity testing of aquatic vertebrates in accordance with the animal welfare concept of the EU chemicals regulation. In this study a data set of chemicals is analysed for relative species sensitivity between Daphnia and fish in chronic testing to evaluate under what condition chronic fish tests can be waived without underestimating the environmental hazard. Chronic fish toxicity is covered in 84% of the evaluated substances by the chronic invertebrate test and an assessment factor of 50. Thus, animal testing can be avoided in environmental hazard assessment for many chemicals. Moreover, it is shown that species sensitivity in chronic testing is associated with species sensitivity in acute testing. The more sensitive species in chronic testing is predicted with a high probability if a species is >5x more sensitive in acute testing. If substances are comparable or more toxic to Daphnia in acute testing than to fish chronic fish toxicity is covered by the chronic Daphnia test and an assessment factor of 50 in about 95% of the evaluated cases. To provide decision support for the regulation of chemicals a categorization scheme on relative sensitivity comparison is presented.

The Dessau workshop on bioaccumulation: state of the art, challenges and regulatory implications.

Bioaccumulation plays a vital role in understanding the fate of a substance in the environment and is key to the regulation of chemicals in several jurisdictions. The current assessment approaches commonly use the octanol-water partition coefficient (log KOW) as an indicator for bioaccumulation and the bioconcentration factor (BCF) as a standard criterion to identify bioaccumulative substances show limitations. The log KOW does not take into account active transport phenomena or special structural properties (e.g., amphiphilic substances or dissociating substances) and therefore additional screening criteria are required. Regulatory BCF studies are so far restricted to fish and uptake through the gills. Studies on (terrestrial) air-breathing organisms are missing. Though there are alternative tests such as the dietary exposure bioaccumulation fish test described in the recently revised OECD test guideline 305, it still remains unclear how to deal with results of alternative tests in regulatory decision-making processes. A substantial number of bioaccumulation fish tests are required in regulation. The development of improved test systems following the 3R principles, namely to replace, reduce and refine animal testing, is thus required. All these aspects stress the importance to further develop the assessment of bioaccumulation. The Dessau Workshop on Bioaccumulation which was held from June 26th to 27th 2014, in Dessau, Germany, provided a comprehensive overview of the state of the art of bioaccumulation assessment, provided insights into the problems and challenges addressed by the regulatory authorities and described new research concepts and their regulatory implications. The event was organised by UBA (Dessau, Germany) and Fraunhofer IME (Schmallenberg, Germany). About 50 participants from industry, regulatory bodies and academia listened to 14 lectures on selected topics and joined the plenary discussions.

Problems faced when evaluating the bioaccumulation potential of substances under REACH.

The identification and regulation of substances that combine persistence, bioaccumulation potential, and toxicity ("PBT" substances) is one central aspect of the European chemical legislation REACH (Registration, Evaluation, Authorisation, and Restriction of Chemicals), because these substances may elicit adverse long-term effects after release to the environment. The determination of a substance that has persistence, bioaccumulation potential, and toxicity is based on a set of distinct cutoff criteria identified in Annex XIII of the REACH regulation. Regarding the bioaccumulation potential, the evaluation is focused on the substance's bioconcentration factor as single decisive criterion. In addition, the REACH guidelines provide a selection of standardized test procedures for measuring bioconcentration factor and guidance in appraising test results. However, alternative test results like bioaccumulation factors and biomagnification as well as additional indications for a bioaccumulation potential such as trophic magnification are only allowed for supporting evidence. The currently used test systems with aquatic exposure have been demonstrated to generate reliable results for the majority of neutral, lipophilic organic substances, which facilitate clear decision-making by means of the crucial bioconcentration factor cutoff criteria of Annex XIII. However, certain substance groups such as highly hydrophobic organic substances and amphiphilic and nonlipophilic compounds are difficult to evaluate with common test strategies due to inappropriate test systems or accumulation mechanisms not based on lipophilicity. Recent scientific progress has already been made to establish alternative test systems and to refine the bioaccumulation assessment by consideration of additive accumulation mechanisms and indications. This article gives an overview on actual shortcomings in the current bioaccumulation assessment under REACH and also provides suggestions for a refinement of evaluation.

Heavy metal toxicity to Lemna minor: studies on the time dependence of growth inhibition and the recovery after exposure.

Environmental concentrations of toxic substances are not necessarily constant but fluctuate over time. Periods of intense exposure might be followed by episodes with a relatively low or no exposure, in principle allowing exposed organisms to recover from toxic injury. The growth reproduction assay with the limnic vascular plant Lemna minor allows for convenient studies on the time dependence of the aquatic toxicity of chemicals. Here we report on a study with four priority metals (Zn, Cu, Ni, Cd). Aims of the study were to determine the impact of the exposure duration on the observed toxicities and to determine the potential for recovery. The bioconcentrations of the test metals were recorded during the exposure in order to analyse, whether changes in the internal concentrations are a governing factor for the dynamics of toxicity. After an exposure of 7 days, Cd and Cu showed the highest toxicity to Lemna (EC50's of 1.9 and 9.7 microM respectively), while Ni and Zn had a slightly lower toxicity (EC50's of 56.3 and 46.1 microM respectively). Additionally, Zn showed a severely delayed toxicity and the exposed plants did not recover even 7 days after the exposure had ended. This is in sharp contrast to the other test metals, for which a considerable recovery was observed. These results indicate the necessity to more thoroughly consider the dynamics of toxicity, instead of recording toxic effects only after a constant exposure over a fixed time.