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Background In Morocco, fish is an important protein source especially, even not exclusively, for coastal communities and marketed fresh all along the coastline. One of the main targets of coastal artisanal fisheries is the European conger (Conger conger, Linnaeus 1758), a widely distributed benthic predatory species of a maximum weight of up to 50 kg. However, information on heavy metal contamination of conger is scarce. Therefore, concentrations of mercury, lead and cadmium were analysed in 108 European conger specimens from nine locations along the along the Atlantic and Mediterranean coasts of Morocco to describe the spatial distribution of heavy metal contamination. Results The average heavy metal concentration in all conger samples under investigation was 246.90 ± 216.83 µg mercury/kg wet mass, 74.14 ± 87.02 µg lead/kg wet mass and 255.12 ± 287.15 µg cadmium/kg wet mass respectively. Mercury and cadmium showed a clear site-specific bioaccumulation in European conger but lead does not. Hence, the effect of fish length bias on contamination was corrected through a generalized linear model (GLM) prior to any spatial comparison. Conclusions Different regional hotspots for the three analysed metals are identified and local sources are discussed. Mercury levels in big specimens of European conger exceeded the European threshold level for human consumption at some of the locations under investigation.

As a toxic and harmful global pollutant, mercury (Hg) enters the marine environment through natural sources, and human activities. It bioaccumulates through the food chain and therefore, Hg is of great importance for environmental monitoring. This study aims to answer the question if Hg contamination in fish and sediment from the German Bight follows temporal trends. Therefore, 496 individual female dab (Limanda limanda) were analyzed. The Hg concentrations in the muscle of dab from the German Bight showed significant increase in function of time with an annual percental change of 1.4%, leading to a 41% increase in Hg contamination level within 25 years of monitoring. At the same time, Hg concentrations in sediment—analyzed in 86 samples—significantly decreased in the nearby North Sea environment. This surprising contradiction is shown in the present study and possible causes are discussed. It could be clearly shown that contamination in sediment and biota can follow completely different time courses and therefore, different environmental matrices should be considered in future monitoring studies. Age of the fish turned out to be a biological factor of particular importance for temporal trend analysis.

For half a decade, the Atlantic salmon in the Baltic Sea has been facing severe health issues. Clinical signs like haemorrhage, erosions and ulcerative/necrotic skin conditions in returning adults have been reported from different Swedish rivers. These primary disease signs precede a secondary, terminal fungal infection. As initial investigations of the disease did not provide conclusive answers regarding the pathogenesis, this study was initiated to gain insight into a possible link between this so‐called Red Skin Disease and anthropogenic influences. Therefore, returning salmon were caught in rivers along the Swedish coast and different tissues were sampled. The focus was put on the measurements of a battery of biomarkers as well as biochemical and haematological parameters, which were analysed using multivariate statistics. The main findings were a severe osmotic haemodilution, an immune response and an alteration of the carbohydrate metabolism in diseased fish. Furthermore, oxidative stress does not seem to be a likely factor in the pathogenesis. Concluding, certain changes in physiological parameters were shown to be indicative for the disease patterns, while others were ruled out as significant factors. Thus, this study contributes to the understanding of the Red Skin Disease and may act as a hypothesis generator for future studies.

Microplastic fibers are frequent anthropogenic contaminants in most aquatic environments and have consequently been detected in the digestive tract of many fish species. Upon ingestion, microplastic fibers pose risks of interference with nutrient uptake, impaired intestinal health, and as a consequence may alter growth performance and fitness. In addition, foreign particles such as fibers might cause tissue irritations and stress, and thus interfere with immune parameters. In nature, fish regularly encounter microplastic fibers as well as fiber debris from natural sources and materials. Thus, we wanted to test the potential impact of microplastic fibers on growth, organosomatic indices, and immune parameters of subadult fish and compare these to possible effects caused by natural fibers. We administered sticklebacks diets, which were supplemented with either polyester or cotton fibers (each at concentrations of 0.2 mg/g and 2 mg/g feed) or a control diet without fiber supplementation for nine weeks. Mortalities did not occur and sticklebacks grew equally well across treatments. Neither organosomatic indices nor immune parameters revealed significant differences between treatments. While natural differences between males and females were observed for some parameters, no treatment-related gender-specific effects were detected. Our results suggest that the dietary uptake of polyester fibers does not affect growth, body condition, gonad development, and immunity of sticklebacks - even at fiber concentrations higher than what can be encountered in the wild. Furthermore, virgin microplastic fibers do not seem to affect fish differently than fibers from natural origin. The present study implies that at least some species are resilient towards pollution with (virgin) microplastic fibers even at high concentrations.

Dumped munitions contain various harmful substances which can affect marine biota like fish. One of them is mercury (Hg), included in the common explosive primer Hg fulminate. There is still a lack of knowledge whether dumped munitions impact the Hg concentrations in the Baltic Sea environment. This study aims to answer the question if dab caught at the dump site Kolberger Heide show higher Hg concentrations released from munition sources and whether Hg in fish is a usable marker for munition exposure. Therefore, a total of 251 individual dab (Limanda limanda) were analysed including 99 fish from the dump site. In fish from the Kolberger Heide, no elevated Hg concentrations were found compared to reference sites when age-dependent bioaccumulation of mercury was considered. Therefore we conclude that Hg in fish is no suitable indicator for exposure to munition dumping, e.g. in the frame of possible future monitoring studies as Hg exposure originating from dumped munition is only a small contributor to overall Hg exposure of fish.

Microplastic fibers are frequent contaminants of aquatic ecosystems. Early life stages of aquatic organisms are predicted to be especially vulnerable to microplastic pollution. We hypothesized that microplastic fibers in the water column might interfere with fertilization and embryonic development of fish. We tested this with an in vitro fertilization system with three-spined sticklebacks. Six egg clutches were divided and one half was fertilized and bread out in water with polyester fibers (PET fibers; mean diameter 9.7 ± 2.3 μm; mean length 245.6 ± 163.1 μm) at a concentration of 1 × 104 fibers/L while the other half served as control without fibers. Observation with a dissection microscope revealed that some polyester fibers stuck to the outside of the eggs in the fiber treatments. Yet, overall 67.4 ± 12.9% eggs were fertilized from which 97.2 ± 4.2% larvae hatched without any significant difference between treatments. Mortality and abnormal development of larvae was low and was not changed by microplastic fibers, as was the heart rate of developing embryos five days post fertilization. The present study illustrates that polyester fibers, even at concentrations three to four orders above levels reported from the environment, do not impair fertilization success, embryonic and early larval development of sticklebacks. Accordingly, concentrations of microplastic fibers currently observed in aquatic habitats do not appear to be harmful to early live stages of fish. First use of fish egg in vitro fertilization assay for microplastic fiber exposure Fertilization and hatching success of fish was not altered by microplastic fibers Fish early life stage development was unaffected by microplastic fiber presence

Marine environments are contaminated with enormous amounts of warfare agents due to military activity and exercise, and the disposal of unused ordnance. Due to corrosion of munition shells, substances are leaking from the warfare materials into the environment. It has to be expected that climate change will influence munition corrosion and distribution of their content. Although there is no doubt about the principle toxicity of many of the munition compounds, including their transformation/degradation products, the impact of munition compounds on marine environments, including their biota are yet only at the beginning to be understood. Recently the intake of munition compounds has been confirmed in mussels and fish collected from contaminated areas. It has become clear that dumped munitions are a continuous source of toxic substances leaking into the environment and that ongoing corrosion will worsen the problem. The present review intends to evaluate the available literature on how climate change might influence the contamination of marine environments and inhabiting biota with munition compounds. Direct testing (or modelling) of climate change scenarios in the context of the marine munition problems has yet not been undertaken. Nevertheless, it can be predicted that climate change effects such as rising temperature and higher frequencies of extreme weather events will accelerate the rates at which disposed ordnance corrodes and consequently accelerate the rate at which munition compounds are leaking out. Climate change will cause elevated stress to biota, ranging from temperature stress and lower availability of oxygen to shifts in salinity and pH. In combination, elevated release of munition related compounds and elevated environmental stress, will put biota under threat, in particular in areas with high munition contamination and limited water exchange, such as the Baltic Sea. On a positive side, biodegradation of organic munition compounds by biota and microorganisms is likely to be accelerated with rising temperature.

Different litter types accumulate in all marine environments. Plastics are of special interest because of their high abundance and possible threats to marine organisms. Polymer type is crucial for their distribution and fate in marine environments. Seafloor litter abundance and composition in the Baltic and North Sea were analysed based on three sampling campaigns according to the protocol of ICES International Bottom Trawl Survey. Polymers were identified via attenuated total reflection-Fourier transform infrared spectroscopy. General litter abundances differed significantly between the Baltic and North Sea with 9.6 items/km2 and 70.7 items/km2, respectively. Plastic built the dominating litter group in both seas (62.2% and 91.3%, respectively). Polymer identification revealed clear dominance of polyethylene, polypropylene and polyamide. Most polymers were positively buoyant in seawater (89.5%), thereby excluding polymer density as the main driver of vertical plastic litter transportation. Plastics at the seafloor basically reflected the entirety of polymers entering marine environments.

Corrosion and disintegration of munition shells from the World Wars increase the risk that explosives are released into the marine environment, exposing a variety of organisms. Only few studies investigated contamination of fish with explosives in the field under environmental conditions. Here we present a comprehensive study on the contamination status of dab (Limanda limanda) from a munition dumpsite and from reference sites in the Baltic Sea. Bile of 236 dab from four different study sites, including a dumpsite for conventional munitions, was investigated and explosive compounds were detected by high performance liquid chromatography-mass spectrometry. Five explosive compounds were identified, including 2,4,6-trinitrotoluene, 4-amino-2,6-dinitrolouene, and hexahydro-1,3,5-trinitro-1,3,5-triazine. 48% of the samples from the dumpsite contained at least one explosive compound. The results prove that toxic explosive compounds from a dumpsite in the Baltic Sea are accumulated by flatfish and may therefore pose a risk to fish health and human food safety.

The pollution of the aquatic environment is currently characterised by a large number of contaminants, especially by mixtures of micro-pollutants including neurotoxins. The ecotoxicological consequences of this burden are not yet assessable. Within the present study, a new test method was applied which evaluates behavioural changes in zebrafish (Danio rerio) embryos to quantify the neurotoxic effect of selected chemicals. Changes in the frequency of spontaneous tail movements – a parameter of locomotion – occurring during embryogenesis was assessed as parameter for neurotoxic effects. Embryos were exposed in 24-microwell plates to neurotoxic compounds. Behaviour was examined after 24 h of exposure by videotaping and quantifying spontaneous locomotion. Additionally, acute toxicity was determined after 48 h of exposure by utilising the fish embryo toxicity test. Abamectin, emamectin benzoate, chlorpyrifos-oxon and carbamazepine were analysed using both: the new and classic acute test methods. The results showed the neurotoxic effect of the substances. Furthermore, an increased sensitivity compared to acute toxicity data was shown. The aim of the present study, to illustrate the sensitivity and specificity of the established endpoint to reliably assess adverse, neurotoxic effects of compounds on the model organism Danio rerio and to apply a new test method was achieved. Therefore, the present study is a substantial contribution to an effect-based risk assessment of contaminants in aquatic ecosystems.

Awareness of microplastic pollution in aquatic environments increased strongly during the last decade. Environmental monitoring studies detected microplastic items in every tested water body and found them in various aquatic organisms. Yet, many studies conducted so far, refer to microplastic particles and spheres but not fibers. Microplastic fibers are often not considered due to methodological issues and high contamination risk during sampling and analysis. Only a few of the microplastic exposure studies with aquatic organisms were conducted with microplastic fibers. Recent effect studies demonstrated several negative impacts of microplastic fibers on aquatic organisms, which include tissue damage, reduced growth, and body condition and even mortality. Such negative effects were predominantly observed in taxa at the basis of the food chain. Higher taxa were less heavily affected in direct exposure experiments, but they presumably suffer from negative effects on organisms at lower food chain levels in the wild. Consequently, ongoing and future pollution with microplastic fibers may disturb the functioning of aquatic ecosystems. The present review outlines the current state of knowledge on microplastic fiber abundance in nature, bioavailability, and impacts on aquatic animals. Based on these findings, we recommend inclusion of microplastic fibers in prospective monitoring studies, discuss appropriate methods, and propose to conduct exposure studies with - as well as risk assessments of - these underestimated pollutants.

Lethal and sublethal effects of trinitrotoluene (TNT) and its degradation products 2-amino-4,6-dinitrotoluene (2-ADNT) and 4-amino-2,6-dinitrotoluene (4-ADNT) to zebrafish embryos (Danio rerio) were investigated in a 120 h exposure scenario. Lethal concentrations (LC50) were 4.5 mg/l for TNT, 13.4 mg/l for 2-ADNT and 14.4 mg/l for 4-ADNT. Embryos exposed to 2-ADNT or 4-ADNT revealed a high proportion of chorda deformations among the surviving individuals. Genotoxicity of the nitroaromatic compounds in zebrafish embryos was investigated by comet assay isolating cells from whole embryos after 48 h in vivo exposure. Significant genotoxicity was induced by all three compounds tested, in comparison to the corresponding controls at 0.1 mg/l and 1.0 mg/l as lowest tested concentrations. The genotoxicity caused by TNT was about three to four times higher than that of 2-ADNT and 4-ADNT. To our knowledge, this is the first study demonstrating the genotoxicity of TNT in fish embryos by in vivo exposure. The results are discussed in the context of dumped munition in the marine environment.

Large quantities of chemical warfare agents (CWAs), such as phenylarsenic chemicals, were disposed by sea-dumping after World War II. Nowadays, the release of these toxic chemicals from munitions poses a potential threat to living organisms. This study investigates the fate of these chemicals in fish by exposing selected CWA-related phenylarsenic chemicals and their oxidation products to cod (Gadus morhua) liver S9 fraction in vitro. Clark I (DA), Adamsite (DM) and their corresponding oxidation products as well as triphenylarsine oxide (TPA[ox]) and phenylarsonic acid (PDCA[ox]) were used as chemicals in in vitro experiments. Glutathione (GSH) conjugates of DA, DM and PDCA-related chemicals were found to be the most dominant metabolites, and methylated metabolites were detected as well, suggesting that these compounds are metabolised in the presence of cod liver enzymes. TPA[ox] was the only compound tested that did not form a GSH conjugate or methylated metabolite, indicating a different biotransformation pathway for this compound. Furthermore, hydroxylated metabolites were detected for each tested chemical. Due to their reactive nature, GSH conjugates may be difficult to detect in fish samples from CWA dumpsites. In contrast, both methylated and hydroxylated metabolites of phenylarsenic chemicals are promising target chemicals for the detection of CWA-related contamination in fish.

Fipronil is an insecticide which, based on its mode of action, is intended to be predominantly toxic towards insects. Fipronil bioaccumulates and some of its transformation products were reported to be similar or even more stable in the environment and to show an enhanced toxicity against non-target organisms compared to the parent compound. The current study investigated the occurrence of Fipronil and two of its transformation products, Fipronil-desulfinyl and Fipronil-sulfone, in water as well as muscle and liver samples of eels from the river Elbe (Germany). In water samples total concentrations of FIP, FIP-d and FIP-s ranged between 0.5-1.6ngL(-1) with FIP being the main component in all water samples followed by FIP-s and FIP-d. In contrast, FIP-s was the main component in muscle and liver tissues of eels with concentrations of 4.05±3.73ngg(-1) ww and 19.91±9.96ngg(-1) ww, respectively. Using a physiologically based toxicokinetic (PBTK) model for moderately hydrophobic organic chemicals, the different distributions of FIP, FIP-d and FIP-s in water and related tissue samples could be attributed to metabolic processes of eels. The measured concentrations in water of all analytes and their fractional distribution did not reflect the assumed seasonal application of FIP and it seems that the water was constantly contaminated with FIP, FIP-d and FIP-s.

Regarding cod as sea food for human consumption and as bio indicator of the marine eco system, this study is the first approach to combine the analysis of organic and inorganic contaminants and radionuclides in cod muscle as well as PCDD/Fs and dl-PCBs in its livers from the same fishing areas. Concentrations of 1-hydroxypyrene, PCDD/Fs, PCBs, cesium-137 (Cs-137), cadmium and lead were determined in individual or pooled samples over a wide geographic area, including Greenland Seas, Barents Sea, North and Baltic Sea. Highest concentrations were found in samples from the Baltic Sea, lowest in the pristine areas of the Barents Sea and Greenland. Levels of contaminants in cod muscle were found to be far below the established EU maximum levels (ML), regardless of which fishing grounds. In contrast to this, most cod liver samples from the North and Baltic Sea showed PCDD/F and PCB contents exceeding the ML. In addition, new background assessment criteria (BAC) for 1-hydroxypyrene in cod of 4.6 ng mL(-1) bile and for Cs-137 a BAC of 0.16 Bq kg(-1) wet weight are proposed to be included in the European Marine Strategy Framework Directive for cod from the Northeast Atlantic.

Livers from dab (Limanda limanda), plaice (Pleuronectes platessa) and flounder (Platichthys flesus) sampled from the Baltic Sea were used to determine the interaction of flatfish CYP1A enzymes with 2,4,6-trinitrotoluene (TNT) in vitro. Competitive inhibition of 7-ethoxyresorufin-O-deethylase (EROD) and 7-methoxyresorufin-O-deethylase (MROD) could be demonstrated for all three flatfish species. The highest inhibition of CYP1A activities was measured in liver samples of flounder resulting in a half maximal inhibitory concentration (IC50) of 28.1 μM TNT. Due to their lower inhibition (EROD IC50 65.2 μM TNT, MROD IC50 40.3 μM TNT), dab liver samples were used to conduct in vitro metabolization experiments with TNT. The metabolization of TNT in fish was investigated with post-mitochondrial fractions (PMF) of dab liver as a model system after adding different cofactors. Rapid and time-dependent enzymatic degradation of TNT was observed. The concentrations of 4-amino-2,6-dinitrotoluene and 2-amino-4,6-dinitrotoluene increased in the samples over time. Additionally, 2,2,6,6-tetranitro-4,4-azoxytoluene was detected in one sample. The results of this study indicate that in vitro experiments are useful to investigate the xenobiotic metabolism of fish under controlled conditions prior to field studies. The metabolites found can serve as target compounds for marine monitoring of TNT contamination in munition dumpsites.

Environmental pollution of fish with organic contaminants is a topic of rising attention in Morocco. Polycyclic aromatic hydrocarbons (PAH) are prominent organic contaminants which are rapidly metabolized in fish. Their metabolites are accumulated in the bile fluid and can be used to assess PAH exposure. The two PAH metabolites 1-hydroxypyrene and 1-hydroxyphenanthrene were quantified in European eels (Anguilla anguilla) from two Moroccan river systems by high-performance liquid chromatography with fluorescence detection. Mean values ranged from 52 to 210 ng/mL 1-hydroxypyrene and from 61 to 73 ng/mL 1-hydroxyphenanthrene. The overall concentrations of PAH metabolites in eel from Morocco appeared moderate compared to eel from European rivers and coastal sites. The present study provides first information on concentrations of PAH metabolites in fish from Morocco.

Litter is present in all marine waters around the globe. It consists of several compound classes of which plastic is of special interest because of its high abundance and possible threat to marine organisms. The regional distribution, composition and abundance of large litter items (LI) at the sea floor of the North Sea and the Baltic Sea were investigated based on 175 bottom trawls between 2013 and 2015. Different types of marine litter >2.5cm were classified according to the protocol of the ICES International Bottom Trawl Survey. The results showed considerable geographical variation: In the North Sea, a mean litter abundance of 16.8LI/km2 was found, whereas the litter abundance in the Baltic Sea was significantly lower (5.07LI/km2). In general, plastic represented 80% of the litter items. During the study, some methodical aspects with possible impact on the results were identified that need to be addressed in future sampling campaigns.

There is a consensus within the scientific community that sediments act as a long-term sink for a variety of organic and inorganic pollutants, which, however, can re-enter the water column upon resuspension of deposited material under certain hydraulic conditions such as flood events. Within the implementation of the European Water Framework Directive, it is important to understand the potential short- and long-term impact of suspended particulate matter (SPM)-associated contaminants on aquatic organisms as well as the related uptake mechanisms for a sound risk assessment. To elucidate the effects of sediment-bound organic pollutants, such as polycyclic aromatic hydrocarbons (PAHs), rainbow trout (Oncorhynchus mykiss) were exposed to three resuspended natural sediments with different contamination levels. Physicochemical parameters including dissolved oxygen concentration, pH and temperature, total PAH concentration in sediments and SPM as well as different biomarkers of exposure in fish (7-ethoxyresorufin O-deethylase activity, biliary PAH metabolites, micronuclei, and lipid peroxidation) were measured following seven days of exposure within an annular flume, a device to assess erosion and deposition processes of cohesive sediment. Concentrations of PAHs in SPM remained constant and represented the different contamination levels in the un-suspended sediments. Significant differences in bile metabolite concentrations as well as in 7-ethoxyresorufin O-deethylase induction compared to control experiments (untreated animals and animals that were exposed in the annular flume without sediment) were observed for all exposure scenarios. The ratio between 1-hydroxypyrene in bile from fish exposed to the three different contamination levels was 1.0:3.6:10.7 and correlated well with (1) the ratio of pyrene concentrations in corresponding sediments which was 1.0:3.1:12.7 and (2) with the ratio of particle-bound pyrene in SPM which was 1.0:2.7:11.7. In contrast, hepatic lipid peroxidation and micronuclei formation represented the different contamination levels less conclusive. The results of this study clearly demonstrate that firmly bound PAH from aged sediments can become bioaccessible upon resuspension under flood-like conditions and are readily absorbed by aquatic organisms such as rainbow trout. Associated short-term effects were clearly documented and possible adverse long-term impacts due to genotoxicity are likely to follow.

Increasing frequency and intensity of flood events are major concerns in the context of climate change. In addition to the direct hydrological implications of such events, potential ecotoxicological impacts are of increasing interest. It is vital to understand mechanisms of contaminant uptake from suspended particulate matter (SPM) and related effects in aquatic biota under realistic conditions. However, little is known about these processes. Due to recent changes in climate, during summer temperatures of German rivers frequently exceed 25°C. Effects of re-suspension of sediments on biota under elevated temperature regimes are likely to differ from those under lower temperature regimes. To elucidate this differential response of aquatic vertebrates, rainbow trout were exposed to suspensions of sediment from the Rhine River that was spiked with a mixture of polycyclic aromatic hydrocarbons (PAH). The experiments were conducted under two different temperature regimes (24°C or 12°C). Physicochemical parameters, including concentration of PAHs in SPM, and biomarkers in fish (biliary PAH metabolites, 7-ethoxyresorufin O-deethylase activity, lipid peroxidation (LPO), mRNA expression of some genes and micronuclei) were measured over the course of a 12d study. Concentrations of pyrene and phenanthrene decreased over time, while no decrease was observed for chrysene and benzo[a]pyrene. The biomarker cascades, more specifically the temporal dynamics of biomarker reactions, did not only show quantitative differences (i.e. different induction intensity or rate of biomarker responses) at the two temperatures but also qualitative differences, i.e. different biomarker responses were observed. A slight significant increase of biliary metabolites in fish was observed in un-spiked sediment at 24°C. In bile of fish exposed to PAH spiked sediment concentrations of 1-hydroxypyrene and 1-hydroxyphenanthrene increased significantly during the first two days, and then decreased. At 12°C uptake of PAHs was slower and maximum metabolite concentrations in bile were less than in fish exposed at 24°C. Following a latency of two days, concentrations of PAH metabolites in bile of fish exposed at 24°C were followed by a peak in LPO. PAHs spiked into sediments under laboratory conditions were significantly more bioavailable than the PAHs that were already present in un-spiked field-collected sediments.

Background Both frequency and intensity of flood events are expected to increase as a result of global climate change in the upcoming decades, potentially resulting in increased re-suspension of sediments in fluvial systems. Contamination of these re-suspended sediments with legacy contaminants, including dioxins and dioxin-like compounds (DLCs), as well as polycyclic aromatic hydrocarbons (PAHs) is of great ecotoxicological concern. DLCs, and to some extent also PAHs, exhibit their toxicity through activation of the aryl hydrocarbon receptor (AhR). However, interactions of DLCs with pathways other than those known to be mediated through the AhR are not fully understood to date. Methods This study aimed to investigate molecular and biochemical effects in roach (Rutilus rutilus) during a 10 days exposure to suspensions of three natural sediments that differed in the level of DLC contamination. Concentrations of biliary PAH metabolites and hepatic 7-ethoxyresorufin-O-deethylase activity were quantified in exposed fish. Furthermore, the abundance of transcripts of several genes related to energy metabolism, response to oxidative stress, and apoptosis, as well as cytochrome P450 1A (cyp1a) was quantified. Results Biliary PAH metabolites and activation of the AhR were confirmed as suitable early warning biomarkers of exposure to suspended sediments containing DLCs and PAHs that corresponded well with analytically determined concentrations of those contaminants. Although the abundances of transcripts of superoxide dismutase (sod), protein kinase c delta (pkcd), and ATP-binding cassette transporter c9 (abcc9) were altered by the treatment compared with unexposed control fish, none of these showed a time- or concentration-dependent response. The abundance of transcripts of pyruvate carboxylase (pc) and transferrin variant d (tfd) remained unaltered by the treatments. Conclusions We have shown that contaminated sediments can become a risk for fish during re-suspension events (e.g., flooding and dredging). We have also demonstrated that roach, which are native to most European freshwater systems, are suitable sentinel species due to their great sensitivity and ecological relevance. Roach may be particularly suitable in future field studies to assess the toxicological concerns associated with the release of DLCs and PAHs during sediment re-suspension.

To increase our understanding of possible chemical impacts on coastal fish populations in the Baltic Sea, Kattegat and Skagerrak, the viviparous eelpout (Zoarces viviparus) was used as sentinel species in two major sampling campaigns (spring and autumn) in 16 different coastal sites. Condition factor (CF), liver somatic index (LSI), gonad somatic index (GSI) were measured and the activity of the hepatic enzymes ethoxyresorufin-O-deethylase (EROD), glutathione reductase GR), glutathione S-transferase (GST), catalase (CAT) and muscular activity of acetylcholinesterase (AChE) were assessed. PAH metabolites in bile were also analyzed. The most notable finding in the data set was the low EROD activity in eelpouts collected at the relatively polluted region in Germany compared to the other regions, which could be due to an inhibition of the CYP1A-system or to adaptation to chronic exposure of pollutants in this area. Additionally, low AChE activity was noted in the German region in the autumn campaign and low AChE activity detected in the Danish region in the spring campaign. These differences suggest possible season-specific differences in the use and release of AChE-inhibiting chemicals in the Danish and German regions. Clustering of biomarkers on site level indicated a relationship between CF and GSI and suggested that sites with a high CF contained eelpout that put a larger effort into their larvae development. Clustering of the oxidative stress markers GR, GST and CAT on the individual level reflected a possible coordinated regulation of these enzymes. Overall, the results support the importance of taking into account general regional differences and seasonal variation in biomarker activity when monitoring and assessing the effects of pollution. Despite the expected seasonal variation for most of the measured endpoint, several markers (GSI, EROD and CF) vary similarly between all selected sites in both spring and autumn. This suggests that the differences between sites for these endpoints are independent of season.

Purpose Flood events are expected to increase both in intensity and frequency due to climate change in the near future. From an environmental toxicology perspective, there is concern that such flood events could lead to the remobilization of contaminated sediment layers in rivers. The aim of this pilot study was to establish a novel and interdisciplinary framework combining methods of hydrodynamic engineering and ecotoxicological assessment to enable investigation of the potential risks associated with such remobilization events.

One of the central issues related to global changes in weather is the increasing occurrence of flood events that can result in the re-suspension of contaminated sediments in rivers. Here, we report on a proof-of-concept study combining hydraulic engineering and ecotoxicology in a new interdisciplinary approach to assess the toxicity of re-suspended polluted sediments after a simulated flood event. Rainbow trout (Oncorhynchus mykiss) were exposed for 5 days under simulated flood conditions in an annular flume with artificial sediments that were spiked with a mixture of polycyclic aromatic hydrocarbons (PAH) at environmentally relevant concentrations. Specifically, the objective of this study was to bridge the gap between the physical re-suspension of pollutants and resulting toxicological impacts on aquatic organisms. A suite of different molecular, biochemical and histological markers was used to test the hypothesis that re-suspension of sediments can lead to re-mobilization of PAHs and subsequently to effects on aquatic organisms. The micronucleus frequency was significantly 4.3-fold elevated after exposure. There was no significant indication of Aryl hydrocarbon receptor signaling (no EROD induction or increased CYP1A protein content, only slight induction of CYP1A gene expression). Biliary metabolite concentration was the most sensitive marker of PAH exposure. Results for other biomarkers (glutathione-S-transferase, catalase and lipid peroxidation) were inconclusive. In combination with chemical analyses of suspended matter, the presented approach will be used to improve understanding of the re-mobilization of pollutants from sediments in support of environmental risk assessment.

Hintergrund und Ziel der Studie Die Fischbestande in der Donau sind seit Ende der 1980er-Jahre stark rucklaufig. Trotz intensiver bestandsstutzender Masnahmen und einer verbesserten Wasserqualitat entlang der Donau seit den 1970er-Jahren konnte diese Entwicklung nicht gestoppt werden. Ziel der umfassenden Weight-of-Evidence-Studie (WOE) mit verschiedenen Lines-of-Evidence war es, 1) mogliche kausale Verbindungen zwischen molekularen und zellbiologischen Endpunkten sowie okologisch relevanten Effekten zu analysieren, und 2) zu uberprufen, ob die okotoxikologischen Effekte fur den Ruckgang der Fische verantwortlich sein konnten. Dazu wurden Sedimente und Fische an verschiedenen Standorten der Donau entnommen und in einem integrierten Ansatz untersucht. Das Ziel dieses Beitrages ist es, die Untersuchungsstrategie und den Stand der seit 2003 laufenden Studie vorzustellen. Wie kurzlich von Chapman und Hollert (2006) vorgestellt, wurde als Untersuchungsstrategie eine WOE-Studie mit zahlreichen Lines-of-Evidence eingesetzt. 1) Mit einer Biotestbatterie wurde die akute und mechanismusspezifische Toxizitat untersucht, 2) histologische Untersuchungen und der Mikrokerntest mit Fischzellen wurden als In-situ-Studien durchgefuhrt. 3) Die Diversitat und Abundanz von Makroinvertebraten und Fischen sowie 4) die Konzentrationen von ausgewahlten persistenten organischen Verbindungen, endokrinen Disruptoren und Schwermetallen wurde bestimmt. 5) Um die Substanzen zu identifizieren, die fur die toxischen Effekte in den Sedimenten verantwortlich sind, wurden effektdirigierte Analysen durchgefuhrt.

In response to increasing concerns about the potential toxicological impacts of (extreme) flood events, scientists from several disciplines have joined to form the interdisciplinary research project named FLOODSEARCH. FLOODSEARCH is one of the recent Pathfinder Projects supported by the German Excellence Initiative via the Exploratory Research Space at RWTH Aachen (ERS). FLOODSEARCH aims to combine methodologies of hydraulic engineering and ecotoxicology in a new interdisciplinary approach to assess the risks associated with the re-mobilisation of particulate bound contaminants often observed after severe flood events. Impacts of extreme flood events and aspects of re-mobilisation of sediment-bound toxic compounds will be characterised and evaluated in controlled experiments fusing flood simulation technologies with biological effects assessment. The overall goal is to establish a novel and more realistic approach towards flood event testing that can be applied to a number of different questions and species. Specifically, model aquatic species such as rainbow trout (Onchorhynchus mykiss) will be exposed to particle-bound contaminants in flood-like conditions in a specifically designed annular flume that permits monitoring of both physical/chemical and biological parameter. Ultimately, this approach will assist to further our understanding of the potential biological risks associated with increasingly frequent extreme flood events, e.g., as a consequence of climate change, by bridging the gap between the physical (re-)mobilisation of contaminants and resulting toxicological impacts on aquatic organisms. Thus, it is the objective of the project to derive relationships between the hydrodynamic parameters such as velocities and turbulences, the parameters associated to sediment transport such as sediment concentration and grain sizes and the biological parameters.

As a consequence of flood events, runoff and remobilized sediments may cause an increase of ecotoxicologically relevant effects from contaminant reservoirs. Aquatic and terrestrial organisms as well as cattle and areas of settlement are exposed to dislocated contaminants during and after flood events. In this study, the impacts of two flood events triggered by intense rain at the rivers Neckar and Rhine (Southern Germany) were studied. Effects in correlation to flood flow were assessed at the river Neckar using samples collected at frequent intervals. River Rhine suspended particulate matter (SPM) was sampled over a longer period at normal flow and during a flood event. Three cell lines (H4L1.1c4, GPC.2D.Luc, RTL-W1) were used to compare Ah receptor agonist activity in different biotest systems. Multilayer fractionation was performed to identify causative compounds, focusing on persistent organic contaminants. Native water and SPM of flood events were collected at the river Neckar and at the monitoring station (Rheinguetestation, Worms, Germany) of the river Rhine. Water samples were XAD-extracted. SPM were freeze-dried and Soxhlet-extracted using acetone and finally dissolved in dimethyl sulfoxide. Resulting crude extracts were analyzed for cytotoxicity with the neutral red assay. Aryl hydrocarbon receptor (AhR) agonist activity was measured in a set of biological test systems (DR-CALUX, GPC.2D, and ethoxyresorufin-O-deethylase (EROD) assay) and different cell lines. In addition, crude extracts were fractionated using a combined method of multilayer (sequence of acidified silica layers) and carbon fractionation. Fractions from the multilayer fractionation contained persistent organic compounds (polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), polychlorinated biphenyls (PCBs), and some polycyclic aromatic hydrocarbon (PAHs)); fractions from the carbon fractionation were separated into a PCDD/F and a PCB fraction. Dioxin-like activity of multilayer and carbon fractions was determined in the EROD assay and expressed as biological toxicity equivalency concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin (bio-TEQs). The calculation of chemical equivalency concentrations (chem-TEQs) and comparison to bio-TEQ values allowed the determination of the contribution of the analyzed persistent compounds to the total biological effects measured. Soluble compounds in native and extracted water samples resulted in no or minor activity in the toxicity tests, respectively. Filter residues of native water caused increased AhR-mediated activity at the peak of the flood. Activities of SPM of the river Neckar correlated well with the flow rate indicating a flood-dependent increase of toxicity culminating at the peak of flow. River Rhine SPM showed a decrease of activity regarding an SPM sample of the flood event compared to a long-term sample. Excellent correlations with AhR agonistic activity were determined for DR-CALUX and EROD assay, while the GPC.2D assay did not correlate with both other biotests. The activity of persistent dioxin-like acting compounds in multilayer and carbon fractionated PCDD/F and PCB fractions was low if compared to corresponding crude extracts. The congener pattern of PCDD/F revealed that the contaminations mainly originated from products and productions of the chlorine and organochlorine industries. Native and extracted water samples could be shown to contain little or no cytotoxic or AhR agonistic compounds. In contrast, particle-bound compounds were shown to be the relevant effect-causing fraction, as indicated by the activities of filter residues of native water and SPM. Compounds other than fractionated persistent PCBs and PCDD/Fs were more relevant to explain AhR-mediated activities of crude flood SPM at both rivers assessed. Biologically detected activities could at least in part be traced back to chemically analyzed and quantified compounds. The calculation of the portion of persistent PCBs and PCDD/Fs in multilayer fractions causing the high inductions in the EROD assay in combination with chemical analysis provides a suitable tool to assess dioxin-like activity of persistent compounds in SPM sampled over the course of flood events. Depending on the catchment area and annual course of flood events, end points may either indicate an increase or a decrease of activity. In order to determine the ecological hazard potential of mobilized contaminants during flood events, the focus should be set on particle-bound pollutants. Furthermore, PCDD/Fs and PCBs, commonly expected to be the most relevant pollutants in river systems, could be shown to contribute only to a minor portion of the overall AhR-mediated activity. However, they might be most relevant for human exposure when considering persistence and bioaccumulation–biomagnification in the food chain. As a consequence of climate change, flood events will increase in frequency and intensity at least in some regions such as Central Europe. Thus, it is crucial to identify the potential hazard of (re-)mobilized contaminants from reservoirs dislocated via floods and threatening especially aquatic organisms and cattle grazing in flood plains. Since other less persistent compounds seem to be more relevant to explain AhR-mediated activities in flood SPM, nonconventional PAHs and more polar compounds also need to be considered for risk assessment. Effect-directed analysis using broad-range fractionation methods taking into account compounds from polar to nonpolar should be applied for identification of pollutants causing biological effects, thus integrating biological and chemical parameters.

Gene expression analyses with real-time (RT)-polymerase chain reaction (PCR) gains importance in marine monitoring. This new technique has to be compared to the classical approaches like the well known biomarker ethoxyresorufin-O-deethylase (EROD) to test their suitability for monitoring programmes. The goal of the present study is to compare EROD activity and CYP1A1 mRNA expression in the important monitoring fish species dab (Limanda limanda) and to answer the question of whether these parameters reflect the polycyclic aromatic hydrocarbon (PAH) contamination of the fish. Further on, glyceraldehyd-3-phosphate dehydrogenase (GAPDH) was investigated as a potential housekeeping gene. Female dab were caught in the summer of 2004 in the North Sea and in the Baltic. EROD activity was determined in liver samples by a kinetic fluorimetric assay according to a standard protocol. The gene expression of CYP1A (cytochrome P450 1A) and GAPDH were determined by means of RT-PCR. Results were compared to gonado somatic index and to the concentration of PAH metabolite 1OHPyr (1-hydroxypyrene) analysed in the bile fluids of the fish, respectively. Dab from all stations showed a considerable individual variation in the levels of both CYP1A mRNA and EROD. Highest mean values for CYP1A mRNA and EROD were detected in the northern part of the sampling area. In contrast, the PAH metabolite 1OHPyr was found at the highest concentration in fish caught near the German coast. CYP1A mRNA and EROD showed only a minor but significant correlation (r = 0.32, p

Heat shock proteins 70 (hsp70) are known to be induced by a great variety of chemical stressors. The effects of different environmental contaminants, which were identified in sediments of the North Sea and the Baltic Sea as main contaminants or as contaminants of special toxicological interest, on the extent of expression of hsp70 in a permanent cell line Epithelioma papulosum cyprini (EPC) were investigated. Concentrations of the single contaminants leading to elevated hsp70 levels were compared to the respective concentrations of these contaminants encountered in the sediment extracts thus allowing an assessment of their contribution to the toxicity of these sediments. Analysis of hsp70 was performed using western blotting techniques with subsequent comparative quantification by densitometry. Eleven of thirteen contaminants have shown a dose-dependent increase in hsp70 levels. An expression of hsp70 was observed in most cases between 0.1 and 10 microg/mL test solution. Some of the investigated stressors led to extremely high hsp70 contents even at low concentrations. However, in most cases contaminant concentrations in sediments were too low to cause an effect. For this reason, effects of the tested sediments are attributed to other contaminants or rather to mixtures of stressors.

Persistent organic pollutants (POPs) are monitored regularly in water, sediment, and biota in the Baltic Sea. Lipophilic substances are measured in remarkable concentrations especially in the fatty parts of fish, such as herring (Clupea harengus). However, less lipophilic POPs, e.g. perfluorinated compounds (PFCs), can also be detected. For the first time to our knowledge, this study provides a broad range of contaminant concentrations simultaneously measured in filet, liver, and gonads of both sexes of Baltic herring. We analysed organochlorines, polybrominated diphenyl ethers (PBDEs), and PFCs in mature autumn-spawning individuals and found distinct organ pollutant pattern for all POPs in both sexes. POP concentrations found in the gonads of both sexes indicate that not only females but also males tend to reduce contaminants via reproduction. However, sex-dependent differences could be identified for hexachlorobenzene, PBDEs, and were most remarkable for PFCs. This transfer of contaminants to the gonads in both male and female herring is being underestimated, as it may directly affect the general reproduction success as well as the healthy development of the next generation. Hence, the accumulation of contaminants in the gonads should be considered one possible threat to a healthy wildlife as its achievement is stated by the Baltic Sea Action Plan. Inclusion of a periodic monitoring of POP concentrations in gonads of fish may be an important bioeffect measure to assess the environmental status of biota in the Baltic Sea.

The European eel (Anguilla anguilla) is a facultatively catadromous fish species with a complex life cycle. Its current population status is alarming: recruitment has decreased drastically since the 1980s and its stock is still considered to be outside safe biological limits. Although there is no consensus on the reasons for this situation, it is currently thought to have resulted from a combination of different stressors, including anthropogenic contaminants. To deepen our understanding of the processes leading to the accumulation of lipophilic organic contaminants in yellow eels (i.e. the feeding, continental growth stage), we developed a physiologically based toxicokinetic model using our own data and values from the literature. Such models can predict the uptake and distribution of water-borne organic chemicals in the whole fish and in different tissues at any time during exposure. The predictive power of the model was tested against experimental data for six chemicals with n-octanol-water partitioning coefficient (log Kow) values ranging from 2.13–4.29. Model performance was excellent, with a root mean squared error of 0.28 log units. This model has the potential to help identify suitable habitats for restocking under eel management plans.

Sediments can act as long-term sinks for environmental pollutants. Within the past decades, dioxin-like compounds (DLCs) such as polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) have attracted significant attention in the scientific community. To investigate the time- and concentration-dependent uptake of DLCs and PAHs in rainbow trout (Oncorhynchus mykiss) and their associated toxicological effects, we conducted exposure experiments using suspensions of three field-collected sediments from the rivers Rhine and Elbe, which were chosen to represent different contamination levels. Five serial dilutions of contaminated sediments were tested; these originated from the Prossen and Zollelbe sampling sites (both in the river Elbe, Germany) and from Ehrenbreitstein (Rhine, Germany), with lower levels of contamination. Fish were exposed to suspensions of these dilutions under semi-static conditions for 90 days. Analysis of muscle tissue by high resolution gas chromatography and mass spectrometry and of bile liquid by high-performance liquid chromatography showed that particle-bound PCDD/Fs, PCBs and PAHs were readily bioavailable from re-suspended sediments. Uptake of these contaminants and the associated toxicological effects in fish were largely proportional to their sediment concentrations. The changes in the investigated biomarkers closely reflected the different sediment contamination levels: cytochrome P450 1A mRNA expression and 7-ethoxyresorufin-O-deethylase activity in fish livers responded immediately and with high sensitivity, while increased frequencies of micronuclei and other nuclear aberrations, as well as histopathological and gross pathological lesions, were strong indicators of the potential long-term effects of re-suspension events. Our study clearly demonstrates that sediment re-suspension can lead to accumulation of PCDD/Fs and PCBs in fish, resulting in potentially adverse toxicological effects. For a sound risk assessment within the implementation of the European Water Framework Directive and related legislation, we propose a strong emphasis on sediment-bound contaminants in the context of integrated river basin management plans.

The Three Gorges Reservoir (TGR), created in consequence of the Yangtze River's impoundment by the Three Gorges Dam, faces numerous anthropogenic impacts that challenge its unique ecosystem. Organic pollutants, particularly aryl hydrocarbon receptor (AhR) agonists, have been widely detected in the Yangtze River, but only little research was yet done on AhR-mediated activities. Hence, in order to assess effects of organic pollution, with particular focus on AhR-mediated activities, several sites in the TGR area were examined applying the "triad approach". It combines chemical analysis, in vitro, in vivo and in situ investigations to a holistic assessment. Sediments and the benthic fish species Pelteobagrus vachellii were sampled in 2011/2012, respectively, to identify relevant endpoints. Sediment was tested in vitro with the ethoxyresorufin-O-deethylase (EROD) induction assay, and in vivo with the Fish Embryo Toxicity Test and Sediment Contact Assay with Danio rerio. Activities of phase I (EROD) and phase II (glutathione-S-transferase) biotransformation enzymes, pollutant metabolites and histopathological alterations were studied in situ in P. vachellii. EROD induction was tested in vitro and in situ to evaluate possible relationships. Two sites, near Chongqing and Kaixian city, were identified as regional hot-spots and further investigated in 2013. The sediments induced in the in vitro/in vivo bioassays AhR-mediated activities and embryotoxic/teratogenic effects - particularly on the cardiovascular system. These endpoints could be significantly correlated to each other and respective chemical data. However, particle-bound pollutants showed only low bioavailability. The in situ investigations suggested a rather poor condition of P. vachellii, with histopathological alterations in liver and excretory kidney. Fish from Chongqing city exhibited significant hepatic EROD induction and obvious parasitic infestations. The polycyclic aromatic hydrocarbon (PAH) metabolite 1-hydroxypyrene was detected in bile of fish from all sites. All endpoints in combination with the chemical data suggest a pivotal role of PAHs in the observed ecotoxicological impacts.

The Water Framework Directive (WFD), 2000/60/EC, requires an integrated approach to the monitoring and assessment of the quality of surface water bodies. The chemical status assessment is based on compliance with legally binding Environmental Quality Standards (EQSs) for selected chemical pollutants (priority substances) of EU-wide concern. In the context of the mandate for the period 2010 to 2012 of the subgroup Chemical Monitoring and Emerging Pollutants (CMEP) under the Common Implementation Strategy (CIS) for the WFD, a specific task was established for the elaboration of a technical report on aquatic effect-based monitoring tools. The activity was chaired by Sweden and co-chaired by Italy and progressively involved several Member States and stakeholders in an EU-wide drafting group. The main aim of this technical report was to identify potential effect-based tools (e.g. biomarkers and bioassays) that could be used in the context of the different monitoring programmes (surveillance, operational and investigative) linking chemical and ecological status assessment. The present paper summarizes the major technical contents and findings of the report.

Migrants from can coatings consist of a complex mixture of a variety of substances that mostly are not toxicologically evaluated. 95% ethanolic migrates from a polyester and an epoxy coating were screened in different bioassays on mutagenicity (Ames II-Test), on aquatic toxicity (Fish Embryo Assay) and on cytotoxicity (BrdU-ELISA, WST-1-Assay, Neutral Red Assay (NRA), RNA-Synthesis-Inhibition). No mutagenic effect was detected in the Ames II-Test and no lethal effects were induced in the fish embryo test up to a concentration of 60 dm2/l. Strongly varying cytotoxicity effects have been determined depending on the type of the assay, the cell line in use (Hela-S3, Caco-2, HT-29, Hep-G2) as well as the incubation conditions.

Background, Aim and Scope Polycyclic aromatic hydrocarbons (PAH) are important environmental contaminants which may lead to increased levels of neoplastic aberrations or tumours in fish liver. Therefore, monitoring of PAH and their effects are part of several international environmental programmes. The aim of the present field study was to investigate the concentrations of the PAH metabolites in fish bile, to elucidate spatial, seasonal and species differences as well as to discuss different strategies of normalisation with regard to environmental monitoring. Materials and Methods: PAH metabolites were determined in the bile fluid of dab (Limanda limanda) and flounder (Platichthys flesus) caught in the North Sea and Baltic Sea between 1997 and 2004. After enzymatic deconjugation, two metabolites were determined by means of HPLC. The limit of detection and the limit of quantification were calculated. The accuracy of the method was tested with a standard reference material. Results were referred to bile volume as well as to biliverdin. Results: The main metabolite, 1-hydroxypyrene, was determined in concentrations from

Microinjection was tested as a potentially powerful tool to introduce natural and anthropogenic pollutants directly into fish eggs to determine their toxicological impact on fish. With this technique, parental transfer of lipophilic contaminants may be mimicked. Here, we investigated the applicability of pollutant injection into the yolk of early zebrafish (Danio rerio) eggs with special regard on survival after vehicle injection. Tested vehicles were autoclaved tap water, dimethyl sulfoxide (DMSO), methanol, and triolein. Highest mortality occurred after the injection of DMSO and methanol. The lethality rates were up to 40% higher than under control conditions. Best survival rates were obtained after triolein and water injections. However, the triolein droplet was not assimilated by the embryo within 96 h post fertilization suggesting an incomplete uptake of triolein-solubilized chemicals. Technical aspects concerning microinjection in zebrafish eggs are discussed with special emphasis on quantitative injection. Microinjection into the yolk cell of zebrafish eggs is feasible, but the application of exact volumes appears problematic. However, microinjection is a powerful tool for studies without the demand for high volume accuracy. Adopting microinjection for pollutant research requires further investigation.

Goal, Scope and Background Ethoxyresorufin-O-deethylase (EROD) is a well-accepted marker for biological effects in fish and is, therefore, part of numerous monitoring programmes. EROD activity was measured in livers of dab (Limanda limanda) from the German Bight (North Sea) from 1995 to 2003. The aim of the present long-term study was (a) time trend monitoring of EROD activity of dab from the German Bight and (b) to elucidate the needs for a successful application of EROD activity as an early warning system. Methods. EROD activities were determined fluorimetrically in dab liver microsomes, using resorufin as an external standard. The limit of detection (LD) and the limit of quantification (LQ) were calculated. Results were referred to protein concentrations. Results and Discussion. EROD activities of 610 female dab caught in different seasons between January 1995 and August 2003 were analyzed individually. Activities varied from 〈 LD to 1768 pmol/(min mg protein) and showed an annual cycle as well as significant differences between the years. Highest EROD activities were observed in early summer and lowest activities during the winter period. In autumn 2002, significantly elevated EROD activities were detected, possibly related to effects of the River Elbe flood event. Two scenarios with different EROD baseline data are presented to discuss the prerequisites for the use of EROD as a monitoring tool. The comparison of these scenarios underlines the importance of appropriate season-specific baseline data. Conclusion. The use of EROD as an early warning tool for contaminant effects in dab in the German Bight has different prospects during the year, because, due to the high background variability, elevated EROD activities are less easy to detect in spring/summer than during the remaining times of the year. Recommendation and Outlook. The availability of site-specific data on the EROD baseline level, its random variation and its annual cycle is a necessary prerequisite for monitoring. If monitoring is to be carried out only for a limited time period of the year, a season with low background variability in EROD activity (autumn) should be chosen to avoid the need for a compensation of the temperature-triggered shift in sexual cycles and the resulting changes in EROD activity.

Organic extracts of marine sediments from the North Sea and the Baltic Sea were investigated with two toxicity assays. The comet assay based on the fish cell line Epithelioma papulosum cyprini (EPC) was applied to determine the genotoxic potential; zebrafish embryos ( Danio rerio ) were used to quantify the teratogenic potential of the samples. EC 50 values were calculated from dose–response curves for both test systems. Highest teratogenic and genotoxic effects normalised to total organic carbon (TOC) content were detected in sediment samples of different origins. Polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) are not likely to be the causes of the observed effects, as demonstrated by a two-step fractionation procedure of selected extracts. The toxic potential was more pronounced in fractions having polarity higher than those possessed by PAHs and PCBs. The suitability of the two in vitro test systems for assessing genotoxic and teratogenic effects of marine sediment extracts could be demonstrated.

Part I: Determination and identification of organic pollutants Part II: Results of the biotest battery and development of a biotest index

Part I: Determination and identification of organic pollutants Part II: Results of the biotest battery and development of a biotest index Preamble. This series of two papers presents the results of an interdisciplinary research project (ISIS) dealing with bioassay-directed fractionation of marine sediment extracts. Part I presents the extraction and fractionation procedure as well as the results of chemical analysis, including non-target analysis of sediments. Part II describes the results of the biotest battery in relation to chemicals possibly causing parts of the observed effects. A biotest index is used to compare the toxicities of the samples. AUTHORS / AFFILIATIONS Ninja Reineke (3), Werner Wosniok (4), Dirk Danischewski (1), Heinrich Huhnerfuss (3), Angelika Kinder (5), Arne Sierts-Herrmann (5), Norbert Theobald (2), Hans-Heinrich Vahl (6), Michael Vobach (1), Johannes Westendorf (6) and Hans Steinhart (5). (1) Federal Research Centre for Fisheries, Institute for Fishery Ecology, Palmaille 9, 22767 Hamburg, Germany (2) Federal Maritime and Hydrographic Agency, Bernhard-Nochtstr. 78, 20359 Hamburg, Germany (3) University of Hamburg, Institute for Organic Chemistry, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany (4) University of Bremen, Institute of Statistics, Bibliothekstr. 1, 28334 Bremen, Germany (5) University of Hamburg, Institute for Food Chemistry, Grindelallee 117, 20146 Hamburg, Germany (6) University of Hamburg, University Hospital Hamburg-Eppendorf, Department for Toxicology, Vogt-Kolln-Str. 30, 22527 Hamburg, Germany (7) Eurofins Wiertz-Eggert-Jorissen, Stenzelring 14b, 21107 Hamburg, Germany The ecological relevance of contaminants in mixtures is difficult to assess, because of possible interactions and due to lacking toxicity data for many substances present in environmental samples. Marine sediment extracts, which contain a mixture of environmental contaminants in low concentrations, were the object of this study. The extracts were investigated with a set of different biotests in order to identify the compound or the substance class responsible for the toxicity. For this goal, a combination of biotests, biotest-directed fractionation and chemical analysis has been applied. Further on, a strategy for the development of a biotest index to describe the toxicity of the fractions without a prior ranking of the test results is proposed. This article (Part II) focuses on the biological results of the approach. The toxicological potential of organic extracts of sediments from the North Sea and the Baltic Sea was analyzed in a bioassay-directed fractionation procedure with a set of biotests: luciferase reporter gene assays on hormone receptor and Ah receptor, arabinose resistance test, fish embryo test (Danio rerio), comet assay, acetylcholinesterase inhibition test, heat-shock protein 70 induction, oxidative stress and luminescence inhibition test (Vibrio fischeri). The test results provided the basis for the calculation of a biotest index by factor analysis to compare the toxicity of the samples and fractions. Results of 11 biotests on different fractionation levels of the samples were described and discussed with regard to the occurrence of contaminants and their toxic potentials. Polychlorinated biphenyls, polycyclic aromatic hydrocarbons, quinones, brominated indoles and brominated phenols were in the focus of interest. A biotest index was constructed to compare the toxic responses in the samples and to group the biotest results. The procedure presented in this study is well suited for bioassay-directed fractionation of marine sediment extracts. However, in relatively low contaminated samples, high enrichment factors and sufficient fractionation is necessary to allow identification of low concentrations of contaminants which is required to link effects and possible causes. In the present case, the relation between substances and effects was difficult to uncover due to relatively low concentrations of pollutants compared to the biogenic matrix and to the remaining complexity of the fractions. The results, with respect to the brominated phenols and indoles in the samples, highlight the successful use of bioassay directed fractionation in the case of high concentrations and high toxicity. In general, it has been shown that a marine risk assessment requires focusing on the input of diffuse sources and taking into account the fact of mixture toxicity. Effects resulting from biogenic substances will make the assessment of the influence of anthropogenic substances even more difficult.

The present study was undertaken to investigate the influence of natural and anthropogenic stressors on the induction of apoptosis, metallothionein (MT) isoforms, heat shock proteins and DNA strand breaks in the marine flatfish dab (Limbanda limanda) Seasonal changes and possible physiological influences were evaluated over a 1-year period at a fixed location northwest of Helgoland in the German Bight. These results were compared with data from sampling sites in the North Sea and the Baltic Sea. Annual cycles could be observed for all parameters except for Cd. The data revealed that changes in biomarker are not only linked to physiological processes related to reproduction but also to factors like water temperature changes, lipid content and zinc content. Cd and organochlorines had no influence on biomarkers whereas an influence of Cd on MT levels revealed in the regional observations was possibly masked by the major changes in Zn content during the annual cycle. Due to different abiotic factors we supposed that the annual cycles at each sampling site in the North and the Baltic Sea might be shifted temporally and therefore measurements at different locations during a small time window of a few weeks may lead to misinterpretation in biomarker research.

As a consequence of global climate change, we will be likely facing an increasing frequency and intensity of flood events. Thus, the ecotoxicological relevance of sediment re-suspension is of growing concern. It is vital to understand contaminant uptake from suspended sediments and relate it to effects in aquatic biota. Here we report on a computational study that utilizes a physiologically based toxicokinetic model to predict uptake, metabolism and excretion of sediment-borne pyrene in rainbow trout (Oncorhynchus mykiss). To this end, data from two experimental studies were compared with the model predictions: (a) batch re-suspension experiments with constant concentration of suspended particulate matter at two different temperatures (12 and 24°C), and (b) simulated flood events in an annular flume. The model predicted both the final concentrations and the kinetics of 1-hydroxypyrene secretion into the gall bladder of exposed rainbow trout well. We were able to show that exhaustive exercise during exposure in simulated flood events can lead to increased levels of biliary metabolites and identified cardiac output and effective respiratory volume as the two most important factors for contaminant uptake. The results of our study clearly demonstrate the relevance and the necessity to investigate uptake of contaminants from suspended sediments under realistic exposure scenarios.

The alkaline comet assay is a method for detecting DNA strand breaks and alkali labile sites in individual cells. An in vitro system was used to investigate the genotoxic potential of complex mixtures such as organic extracts of marine sediments. DNA damage was induced in leukocytes isolated from carp (Cyprius carpio) by exposure to organic sediment extracts from the North Sea or hydrogen peroxide as positive control, respectively. The minimum concentration for significant effects ranged from 1 to 40 mg sediment dry weight per milliliter assay volume. The sensitivity of the method was enhanced by using the DNA repair inhibitor, 1-β- d -arabinofuranosylcytosine (ara C). From the results, it can be suggested that total organic carbon (TOC) as well as the different compositions of contaminants present in the sediment extracts may contribute to the genotoxic effects observed. The comet assay can be applied successfully as an in vitro bioassay for investigations on genotoxicity of marine sediment extracts.

Apoptosis, also known as programmed cell death, is a physiological and irreversible process in tissue homeostasis that leads to DNA fragmentation of multiples of 180–200 bp. Because apoptosis can be initiated not only by physiological stimuli but also by various chemical substances, the present paper investigates the suitability of apoptosis as a biomarker for biological effect monitoring in the marine environment. Aquarium experiments with dab (Limanda limanda) were carried out to examine the effects of exposure to cadmium, PCB 118, and PCB 77 (each 1 mg/kg fish wt) on apoptosis in dab liver. Determination of apoptosis was carried out by DNA gel electrophoresis and quantification of DNA fragments smaller than 1500 bp. In addition, accumulated amounts of cadmium, PCB 118, and PCB 77 in dab liver were analyzed. Quantification of the three xenobiotics resulted in an accumulation of about factor 102–104. Exposure to PCB 118 and cadmium resulted in an increase in apoptotic DNA fragmentation. Exposure to PCB 77 led mainly to cell death by necrosis.

Extreme hydrodynamic events such as flood events or dredging activities bear the risk of eroding sediments in rivers, reservoirs, harbour basins or estuaries. One of the key concerns associated with these erosion processes is the re-mobilisation of sediment-bound pollutants in highly contaminated sediments. To date, much research has been conducted to characterise flow and sediment processes associated with hydrological events such as floods. Furthermore, there is a large body of literature describing the interaction of contaminants associated with particulate matter to aquatic biota. However, there is little knowledge regarding interactions between hydro-sedimentological and ecotoxicological processes. Understanding of the ecotoxicological consequences and associated risks to aquatic wildlife associated with hydraulic events can provide critical information to regulatory bodies or managing authorities. Specifically, it will aid in assessing risks associated with current management practices and will aid in developing more sustainable future management practices for waterways or harbours. Therefore, a combined experimental methodology between hydraulic engineers and ecotoxicologists was developed to investigate the ecological and toxicological relevance of sediment re-suspension and transport during erosion. An overview of this methodology is given in the present paper.

The stock of the catadromous European eel ( Anguilla anguilla L.) continues to decline and there is growing evidence that poor health status due to contaminants might be a key element in this decrease. Organic contaminants such as polycyclic aromatic hydrocarbons (PAHs) belong to the major threats to yellow eel in their growth habitat and their metabolites are detectable in the bile. Starting the silvering process eels undergo physiological and morphological changes including cessation of feeding and downstream migration back to their spawning grounds. Reduced feed intake results in a diminishment of bile production and induces accumulation of e.g. PAH-metabolites in bile. Therefore, the aim of the present study was to demonstrate the impact of silvering on biliary PAH metabolite concentrations and to utilize normalization procedures to overcome silvering related accumulation effects of PAH-metabolites in eel bile. We investigated the hydroxyl-metabolites of pyrene (1-OH Pyr) and phenantrene (1-OH Phen) in the bile of different maturation stages of eels (silvering index I–V) from nine German rivers. We detected increasing absolute PAH metabolite levels in bile during the silvering process. The highest rise could be observed at the transition from pre migration stage III to the migrating stage IV, suggesting the onset of cessation of feeding at this stage. A cessation bias in PAH metabolite measurement could be diminished by normalization of absolute values against bile pigments (A 380 , biliverdin). In conclusion, we demonstrated the impact of silvering on PAH metabolite concentrations in eel bile and present suitable normalization procedures to overcome silvering related accumulation effects. Thus, for a future eel monitoring we recommend (1) to regularly monitor PAH metabolites in bile, (2) to determine silvering index of eel and (3) to normalize PAH metabolite values in bile based on maturation/silvering stages. The knowledge of the silvering stage is mandatory for an unbiased evaluation of PAH contamination of European eel towards an international harmonized eel monitoring program.

In the light of the alarming decline of the European eel (Anguilla anguilla L.) population, there is an urgent need to define ecological indicators for eel habitat quality. Due to an increasing shortage of glass eels available for local stock enhancement, the decision of whether restocking is a valuable management tool to increase high-quality silver eel escapement to the sea needs to be evaluated. Organic contaminants, such as polycyclic aromatic hydrocarbons (PAHs), are among the major threats to fish in their habitat. Therefore, the aim of the investigation presented here was to examine metabolites of PAHs in eel bile as one possible marker for habitat quality. In total, 170 yellow eels were collected in the rivers Rhine, Ems, Weser, Elbe, Havel, Schlei, Eider, Trave, Warnow, Peene, Uecker, and Oder in 2009. PAH metabolites in eel bile were analyzed using high-performance liquid chromatography with fluorescence detection. Metabolites of pyrene and phenanthrene were investigated. Concentrations of PAH metabolites in eel bile varied significantly between several rivers, with the highest mean concentrations of 1-hydroxypyrene and 1-hydroxyphenanthrene in eel bile from the river Trave (2421 and 632 ng/ml). Moreover, huge differences in the ratio of 1-hydroxypyrene to 1-hydroxyphenanthrene, with the highest mean value in eel bile from the river Ems (7.43) and the lowest mean value in eel bile from the river Uecker (0.70), indicate different sources of PAH contamination. A comparative analysis of PAH-metabolite contamination of eels in different river systems is seen as a first step toward a classification of freshwater habitats for restocking purposes.