Potential and challenge : biomarker response analyses in environmental risk assessment - case studies with fish

  • Potenzial und Herausforderung: Biomarkerantworten in der Umweltrisikobewertung - Fallstudien mit Fischen

Deutschmann, Björn; Hollert, Henner (Thesis advisor); Schäffer, Andreas (Thesis advisor)

Aachen (2020)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2020


The Water Framework Directive (WFD) of the European Commission represents the legal framework for water quality management and is transposed into national law in all of the member states of the European Union. It aims to reach or maintain waters in quality and quantity for the use for future generations. Fundamentally, a good chemical as well as ecological status of European surface waters is required. Therefore, the WFD can be considered as a visionary piece of environmental legislation. The member states are urged to accomplish efforts in monitoring to assess the chemical and ecological status of their water bodies. However, the last two rounds of the European water monitoring under WFD indicated that European water bodies failed to achieve a good chemical and ecological status to a large extent. The methods used in these monitoring efforts are under discussion and widely deemed to be not suitable to indicate the main drivers of toxicity for aquatic organisms and to establish reliable cause-effect relationships of biological active substances to adverse effects on the level of populations and communities which is crucial to establish good water qualities. Chemical contaminants can enter the aquatic environment via several intake routes and mostly originate from anthropogenic activities. Pharmaceuticals and other daily care products, agricultural chemicals and, to a lesser extent, industrial chemicals can be considered as the most prominent chemicals present in the environment. The contaminant concentrations are often low and do not exceed the Environmental Quality Standards (EQS) for each single substance which represents the maximal allowable concentration in surface waters. However, when effect-based methods (EBM) are applied to assess the ecotoxicological potential of water samples, the toxic potential of these samples is often demonstrated - the so-called "something from nothing"-effect. Since the WFD is under review in 2019, several projects (e.g. the EU project SOLUTIONS) and the NORMAN Network aim to contribute towards methods which are more consistent and therefore more suitable to deal with the challenges of environmental water quality management and to achieve a more holistic and solution-based approach for water quality monitoring. To safeguard the aquatic environment against pollution-induced adverse effects, it is suggested that several different methods should be considered in an integrative approach which, strictly speaking, would result in a paradigm shift in European water quality assessment. For example, the EU project SOLUTIONS agreed on 10 recommendations for future monitoring under WFD. One crucial part is the implementation of EBM (bioassays and biomarker) in the WFD monitoring activities. A main drawback of the actual concept of EQS of single substances in chemical monitoring is the non-consideration of mixture effects. This is problematic since environmental research is confronted with mixtures of several contaminants present in the aquatic environments which often result in cumulative toxic potentials. Therefore, the assessment of effect-based trigger values (EBT) can serve as a corresponding tool for chemical and ecological monitoring to indicate toxic pressure of mixtures and to identify sites of interests for further investigations. According to that, in situ biomarker assessment in fish can help to verify if these toxic potentials can be also demonstrated for aquatic organisms under realistic exposure scenarios. In Environmental Risk Assessment (ERA) fish play a prominent role since they are exposed in the surrounding medium, have highly permeable skins for contaminant uptake, accumulate large amounts of chemicals, are sensitive to several biological endpoints, and last but not least are significant for economically and human health aspects. Therefore, fish are one of the four quality elements for the assessment of the ecological status under WFD. Biomarker analyses in fish has been traditionally used to assess the impact of point-sources but are more and more used to investigate toxic pressure in large-scale monitoring approaches. Biomarker can demonstrate that contaminants entered the organism, were distributed in tissues and caused effects at the target sites. Biomarker can be considered as early-warning tools at lower levels of biological organization before adverse effects on populations occur. Several biological endpoints in fish, ranging from molecular activities up to histopathological lesions in tissues, have been successfully investigated to demonstrate toxic pressure at the sites of interest. However, since the shift from point-source assessment to larger monitoring approaches, the drawbacks of biomarker response analyses came more into focus. The resulting data are often not easy to interpret or even contradictory and methods are required to overcome these issues and to promote biomarker analyses as a reliable tool in ERA. Indeed, biomarker in fish have a great potential and combined with the promising concept of Adverse Outcome Pathways (AOPs) responses in fish will be increasingly linked to adverse effects in the environment in the future. Moreover, already today biomarker can provide valuable information in a Weight of Evidence (WOE) approach to link data from both extreme ends. Biomarker analyses were successfully performed in several studies and with differing study-setups in the past. However, it is reported that several studies were not able to detect toxic pressure in fish and therefore the underlying reasons need to be investigated. Potential causes that hinder biomarker analysis are study setup, river size, fish species or selection of unsuitable biomarkers for the specific context. Unfortunately, although there are suggestions to implement biomarkers as EBM in the WFD there is still a lack of guidelines and suggestions for the proper use of biomarkers for that purpose. In this thesis, four case studies were used to investigate in situ biomarker responses in fish to prove their applicability in ERA. The study designs differed in terms of river size and fish species, and aimed to determine the impact of point-sources and diffuse sources on feral fish species. This work aimed to (1) evaluate the water quality of a major river using established biomarkers (2) identify potential knowledge gaps and bottlenecks in biomarker assessment and (3) provide possible solutions to overcome limiting factors in study designs that are based on biomarker evaluation. With respect to that, a large-scale assessment of the Danube was performed in the course of the 3rd Joint Danube Survey (JDS3) to identify hot spots of toxic pressure along the river course on the basis of two feral fish species. Overall, the results demonstrated that the biomarker responses were not indicative for toxic pressure at the assessed hot sites. This highlighted the need for advanced tools for the aggregation of multibiomarker responses that account for high within sample variations which have been shown to be a limiting factor in the statistical evaluation. The Danube is a huge river with high dilution capacities. Gene expression analyses and the use of enzymatic biomarkers seemed not to be suitable to assess the impact of contaminants. However, in terms of DNA damages assessed in erythrocytes of one fish species a hotspot of micronucleus formation was observed and was correlated to the discharge of wastewaters in one tributary. This indicated, that the suite of biomarker should be carefully selected to observe chronic exposure to low concentration contaminants. The results demonstrated that cumulative endpoints such as micronucleus formation or histopathological lesions constitute suitable biomarkers since fish cannot adapt to these responses. The impact of wastewater effluents and historically contaminated small rivers on local biota were investigated using feral and caged fish. The results demonstrated that the used biomarkers were a good indication for the local contamination levels. This holds true for feral and for caged fish. Potential and drawbacks of biomarker response analyses were identified and critically discussed. However, high within-sample variation in all case studies hampered the statistical of biomarker response. Therefore, the concept of an aggregated biomarker response (ABR) index has been emphasized and might be suitable for future biomarker assessment, even for non-specialists. For a more comprehensive biomarker assessment and the use of biomarker for ERA and to establish cause-effect relationships univariate and multivariate statistical techniques were used to correlate biomarker response pattern with chemical pressure at investigated sites. Although the toxic stress at the investigated sites was relatively low, the investigations demonstrated the potential of the combination of these methods, to identify substance groups as main drivers of toxicity. Moreover, the gene expression analysis identified several additional responses in wastewater impacted fish and seems to be very promising to identify relevant biological endpoints for selection of suitable biomarkers. With ongoing establishment of reliable AOPs biomarker responses can be linked to adverse effects on the population level and therefore strengthen the diagnostic power for ERA. The presented results from the three case studies demonstrated the challenges of biomarker response analyses and identified crucial needs in making biomarker response analyses more reliable and therefore more prominent for future ERA and water quality management. Nevertheless, biomarker analyses have not reached their maximum potential and will play an important role in future monitoring within the WFD.