Biliary Polycyclic Aromatic Hydrocarbons Metabolites Research Articles

Spatiotemporal patterns in the prevalence of microscopic hepatic changes in Gulf of Mexico Tilefish (Lopholatilus chamaeleonticeps) and associations with hepatic PAHs

Following the 2010 Deepwater Horizon blowout, demersal longline surveys were conducted across the Gulf of Mexico (GoM) continental shelf to evaluate polycyclic aromatic hydrocarbon (PAH) exposure, tissue accumulation, and health indices in demersal fishes. Tilefish (Lopholatilus chamaeleonticeps), a target species due to Gulf-wide distribution with documented high exposure to PAHs, were collected in the north central GoM at repeat stations 2012 to 2015, and from the northwest GoM, Bay of Campeche, and Yucatán Shelf in 2015 and 2016. Liver samples (n = 239) were analyzed for microscopic hepatic changes (MHCs) by a board-certified veterinary pathologist. Histological analyzes identified 14 MHCs. Prevalence of MHCs was generally uniform throughout the GoM, except for low prevalence on the Yucatán Shelf. Inflammatory and vacuolar changes were most prevalent, while pre-neoplasia and neoplasia were rare. Tilefish sampled annually in the north central GoM showed increases in inflammatory MHCs and glycogen-type vacuolar change over time, while lipid-type vacuolar change decreased over time. Short-term exposure to PAHs was assessed by measuring PAH metabolites in bile (n = 100) using high performance liquid chromatography with fluorescence detection. Longer-term accumulation of PAHs in tissue was assessed by analyzing liver (n = 111) for PAHs and alkylated homologs using QuEChERS extractions and gas chromatography tandem mass spectrometry. Six MHCs including glycogen-type vacuolar change, biliary fibrosis, foci of cellular alteration, parasites, hepatocellular atrophy, and necrosis were significantly associated with hepatic PAH accumulation in Tilefish from the northern central GoM; however, no MHCs were associated with biliary PAH metabolites. Combined with previous studies of PAH exposure and health indices in north central GoM Tilefish post-Deepwater Horizon, which also identified decreases in hepatic lipid storage and Fulton's condition factor that were correlated to increasing PAH exposure, these data indicate concerning temporal trends and changes in hepatic energy storage.

The EU Interreg Project “GEREMIA” on waste management for the improvement of port waters: results on monitoring the health status of fish as bioindicator

Highly anthropized areas as ports represent complex scenarios that require accurate monitoring plans aimed to address the environmental status. In this context, the activities of the EU Interreg Project “GEstione dei REflui per il MIglioramento delle Acque portuali (GEREMIA)” were focused on comparing sites differently affected by human presence, as the Port of Genoa and the natural area of the S’Ena Arrubia fishpond: a panel of analyses was carried out on Mugilidae fish sampled in these two areas, aimed to address trace metal accumulation in the liver, gills, and muscle, as well as cytochrome P450 (CYP450) induction in liver and biliary polycyclic aromatic hydrocarbon (PAH) metabolites, and histopathological alterations in the liver and gills. Chemical analyses in the liver, gills, and muscle of specimens collected in the port area showed an overall higher degree of trace metal contamination compared to the natural fishpond, and similar results were obtained in terms of CYP450 induction and biliary PAH metabolites, suggesting a higher exposure to organic compounds. In addition, histopathological analyses revealed a significant alteration and then a loss of functionality of liver and gill tissue in individuals from the port. Overall, this study describes the complex environmental pollution scenario in the Port of Genoa, confirming the importance of using multidisciplinary approaches and different types of analyses to address both the presence and the effects of contaminants in marine environments.

BILIARY PAH METABOLITES IN FISH FROM THE HIGHLY IMPACTED GUANABARA BAY, IN SOUTHEASTERN BRAZIL, DETERMINED BY FIXED AND SYNCHRONOUS FLUORESCENCE SPECTROSCOPY

Guanabara Bay (GB) covers the metropolitan region of the state of Rio de Janeiro in Brazil. GB is subject to heavy contamination by polycyclic aromatic hydrocarbons (PAHs), from intense oil activities, which pose an ecotoxicological threat. The aim of this study is to implement and optimize a fluorescence methodology for the determination of biliary PAH metabolites in fish species (burrfish and whitemouth croaker), in order to evaluate biliary PAH metabolites as a biomarker of exposure. Fish were sampled from GB and a control region. Naphthalene, pyrene and benzo(a)pyrene metabolites were determined by Fixed Fluorescence Method (FF), while 1-hydroxypyrene was assessed by Synchronous Fluorescence Spectroscopy (SFS). The implementation and optimization of the FF and SFS methods allowed the determination and evaluation of the exposure of these species to PAHs of pyrogenic and petrogenic origin. Biliary PAH metabolite determinations was proven to be a useful tool for environmental monitoring contamination assessments.

A First Comprehensive Baseline of Hydrocarbon Pollution in Gulf of Mexico Fishes

Despite over seven decades of production and hundreds of oil spills per year, there were no comprehensive baselines for petroleum contamination in the Gulf of Mexico (GoM) prior to this study. Subsequent to the 2010 Deepwater Horizon (DWH) spill, we implemented Gulf-wide fish surveys extending over seven years (2011–2018). A total of 2,503 fishes, comprised of 91 species, were sampled from 359 locations and evaluated for biliary polycyclic aromatic hydrocarbon (PAH) concentrations. The northern GoM had significantly higher total biliary PAH concentrations than the West Florida Shelf, and coastal regions off Mexico and Cuba. The highest concentrations of biliary PAH metabolites occurred in Yellowfin Tuna (Thunnus albacares), Golden Tilefish (Lopholatilus chamaeleonticeps), and Red Drum (Sciaenops ocellatus). Conversely, biliary PAH concentrations were relatively low for most other species including economically important snappers and groupers. While oil contamination in most demersal species in the north central GoM declined in the first few years following DWH, more recent increases in exposure to PAHs in some species suggest a complex interaction between multiple input sources and possible re-suspension or bioturbation of oil-contaminated sediments. This study provides the most comprehensive baselines of PAH exposure in fishes ever conducted for a large marine ecosystem.

Spatial contrasts in hepatic and biliary PAHs in Tilefish (Lopholatilus chamaeleonticeps) throughout the Gulf of Mexico, with comparison to the Northwest Atlantic

A multinational demersal longline survey was conducted on the Gulf of Mexico continental shelf over the years 2015 and 2016 to generate a Gulf-wide baseline of polycyclic aromatic hydrocarbon (PAH) concentrations in demersal fishes. Tilefish (Lopholatilus chamaeleonticeps) were sampled in all regions of the Gulf of Mexico for biometrics, bile, and liver. Tilefish liver was also obtained from surveys in the northwest Atlantic Ocean for comparison. Liver tissues (n = 305) were analyzed for PAHs and select alkylated homologs using QuEChERS extractions and gas chromatography tandem mass spectrometry. Bile samples (n = 225) were analyzed for biliary PAH metabolites using high-performance liquid chromatography with fluorescence detection. Spatial comparisons indicate the highest levels of PAH exposure and hepatic accumulation in the north central Gulf of Mexico, with decreasing concentrations moving from the north central Gulf counterclockwise, and an increase on the Yucatán Shelf. Hepatic PAH concentrations were similar between the Gulf of Mexico and the northwest Atlantic, however, Tilefish from the northwest Atlantic had higher concentrations and more frequent detection of carcinogenic high molecular weight PAHs. Overall, results demonstrate that PAH pollution was ubiquitous within the study regions, with recent exposure and hepatic accumulation observed in Tilefish from both the Gulf of Mexico and northwest Atlantic.

Hepatobiliary Analyses Suggest Chronic PAH Exposure in Hakes (Urophycis spp.) Following the Deepwater Horizon Oil Spill

Prior to the Deepwater Horizon oil spill, we lacked a comprehensive baseline of oil contamination in the Gulf of Mexico's sediments, water column, and biota. Gaps in prespill knowledge limit our ability to determine the aftereffects of the Deepwater Horizon blowout or prepare to mitigate similar impacts during future oil spill disasters. We examined spatiotemporal differences in exposure to and metabolism of polycyclic aromatic hydrocarbons (PAHs) in 2 hake species (Urophycis spp.) to establish a current baseline for these ecologically important, abundant, and at‐risk demersal fishes. Gulf hake (Urophycis cirrata) and southern hake (Urophycis floridana) were collected throughout the Gulf of Mexico during extensive longline surveys from 2012 to 2015. Analyses of biliary PAH metabolites and liver PAH concentrations provided evidence of exposures to di‐ and tricyclic compounds, with the highest concentrations measured in the northern Gulf of Mexico. Species‐specific differences were not detected, but temporal trends observed in biliary PAHs suggest a decrease in acute exposures, whereas increasing liver PAHs suggest chronic exposures marked by greater assimilation than metabolism rates. To our knowledge, the present study provides the first multitissue contaminant analyses, as well as the most exhaustive biometric analyses, for both gulf and southern hakes. Though sources of exposure are complex because of multiple natural and anthropogenic PAH inputs, these results will facilitate the development of much needed health metrics for Gulf of Mexico benthos. Environ Toxicol Chem 2019;38:2740–2749. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Naturally occurring hydrocarbon content and baseline condition of deep-sea benthic fauna from the Great Australian Bight

The deep sea (> 500 m) of the Great Australian Bight (GAB) has not been well studied even though it is believed to have high conservation values as evidenced by large marine reserves, and where adjacent (continental shelf) waters support important commercial fisheries and large populations of marine mammals. Recent interest in developing the region's deep-sea hydrocarbon resources has drawn attention to this sparsity of data and the need to characterise the region's benthic ecology in order to detect and understand any potential impacts linked to a developing oil and gas industry. To that end, we measured the naturally occurring hydrocarbon content and baseline condition of a variety of benthic fishes and invertebrates collected during two surveys of the continental slope and seabed geological features. A total of 225 individuals, representing 15 species (7 invertebrates and 8 fishes) were analysed for (i) polycyclic aromatic hydrocarbons (PAH) body burdens or biliary PAH metabolites; (ii) 7-Ethoxyresorufin O-deethylase (EROD), a commonly used oil exposure biomarker; and (iii) lipid profiles and histology. Hydrocarbon concentrations, although measurable at some sites, did not indicate any anthropogenic contamination or unequivocal hydrocarbon seepage. PAH body burdens in the invertebrate species were below the levels of detection, and phenanthrene metabolites showed differences between species of fish surveyed, but were consistently low relative to oil exposed fish. EROD levels varied between species but also did not indicate exposure to oil. Lipid levels were highly variable between individuals and sites, suggesting that a lipid-related metric would not be suitable for monitoring organism health. Histological analysis indicated no gross deformity or infection in either the liver, gill, kidney or spleen. Pathologies identified were of minimal pathological important and likely to be recoverable. Liver exhibited varying levels of lipid accumulation across all fish species. These results, obtained before the commencement of oil and gas industry activity, can be used as a part of a baseline benthic ecosystem characterisation to guide future ecological monitoring programs.

Potential of Periophthalmus sobrinus and Siganus sutor as bioindicator fish species for PAH pollution in tropical waters

Polycyclic Aromatic Hydrocarbons (PAHs) are ubiquitous class of toxic organic compounds in coastal waters. The aim of this study was to assess the potential of goby fish (Periophthalmus sobrinus) and rabbit fish (Siganus sutor) as bioindicators for PAH pollution in Zanzibar coastal waters. The fish samples were collected from four sites along the coasts of Zanzibar between February to July, 2016. The analysis of PAH metabolites from bile was conducted using Synchronous Fluorescence Scanning (SFS) spectroscopy. The parent PAH compounds (naphthalene; fluorene; phenanthrene; pyrene; fluoranthene; anthracene; benzo (a) pyrene) in the fish muscle tissues were analyzed using Gas Chromatography with Mass Spectrometric detection (GC/MS). The levels of PAH metabolites in bile of goby fish (P. sobrinus) ranged from 100 ng mL−1 to 690 ng mL−1. The PAH contents in goby tissues fish ranged from 0.028 ± 0.008 ng g−1 ww to 0.037 ± 0.005 ng g−1 ww with the average of 0.032 ± 0.005 ng g−1 ww. PAH metabolites were not detected in the bile samples from rabbit fish (S. sutor) in any sampled areas. The results suggest that the goby fish (P. sobrinus) could be promising PAH biondicator in coastal waters of tropical regions including Zanzibar islands. This is the first study applying biliary PAH metabolites in bioindicator fish species to trace PAH pollution conducted in West Indian Ocean region that can serve as a baseline information of biliary PAH metabolites as biomarkers of fish PAH pollution in the regional waters.

Antioxidant defenses in polar cod (Boreogadus saida) and responsiveness toward dietary crude oil exposure

Increasing anthropogenic activities in the Arctic pose the risk for accidental oil spills but our knowledge of ecotoxicological effects is still limited for Arctic organisms. This study aimed to provide insights in baseline levels of antioxidants in polar cod (Boreogadus saida) from different Arctic locations and investigate the susceptibility of this species to oxidative stress during a 32 day dietary crude oil exposure. Baseline levels of individual antioxidants and total oxyradical scavenging capacity (TOSC) varied among different Arctic fjords. Upon crude oil exposure, dose- and time-dependent polycyclic aromatic hydrocarbons (PAHs) biotransformation was evident through hepatic ethoxyresorufine-O-deethylase activity, glutathione S-transferase mRNA expression and biliary PAH metabolites. Changes in the activity of individual antioxidants and TOSC was, however, slight and transient upon exposure. In conclusion, biotransformation was clearly induced by the sub-lethal levels of crude oil in polar cod, while the antioxidant defense responded with high variability.

Toward understanding the impacts of sediment contamination on a native fish species: transcriptional effects, EROD activity, and biliary PAH metabolites.

BackgroundBoth 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.MethodsThis 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.ResultsBiliary 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.ConclusionsWe 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.

Integrating Multiple Biomarkers of Fish Health: A Case Study of Fish Health in Ports.

Biomarkers of fish health are recognised as valuable biomonitoring tools that inform on the impact of pollution on biota. The integration of a suite of biomarkers in a statistical analysis that better illustrates the effects of exposure to xenobiotics on living organisms is most informative; however, most published ecotoxicological studies base the interpretation of results on individual biomarkers rather than on the information they carry as a set. To compare the interpretation of results from individual biomarkers with an interpretation based on multivariate analysis, a case study was selected where fish health was examined in two species of fish captured in two ports located in Western Australia. The suite of variables selected included chemical analysis of white muscle, body condition index, liver somatic index (LSI), hepatic ethoxyresorufin-O-deethylase activity, serum sorbitol dehydrogenase activity, biliary polycyclic aromatic hydrocarbon metabolites, oxidative DNA damage as measured by serum 8-oxo-dG, and stress protein HSP70 measured on gill tissue. Statistical analysis of individual biomarkers suggested little consistent evidence of the effects of contaminants on fish health. However, when biomarkers were integrated as a set by principal component analysis, there was evidence that the health status of fish in Fremantle port was compromised mainly due to increased LSI and greater oxidative DNA damage in fish captured within the port area relative to fish captured at a remote site. The conclusions achieved using the integrated set of biomarkers show the importance of viewing biomarkers of fish health as a set of variables rather than as isolated biomarkers of fish health.

PAH Exposure in Gulf of Mexico Demersal Fishes, Post-Deepwater Horizon

Following the 2010 Deepwater Horizon (DWH) blowout, we surveyed offshore demersal fishes in the northern Gulf of Mexico (GoM) in 2011-2013, to assess polycyclic aromatic hydrocarbon (PAH) exposure. Biliary PAH metabolites were estimated in 271 samples of golden tilefish (Lopholatilus chamaeleonticeps), king snake eel (Ophichthus rex), and red snapper (Lutjanus campechanus), using high performance liquid chromatography with fluorescence detection. Mean concentration of naphthalene metabolites in golden tilefish (240 μg g(-1)) was significantly higher (p = 0.001) than in red snapper (61 μg g(-1)) or king snake eel (38 μg g(-1)). Biliary naphthalene metabolite concentration decreased over the study period in red snapper (58%) and king snake eel (37%), indicating likely episodic exposure, while concentrations were persistently high in golden tilefish. Naphthalene metabolite levels measured in golden tilefish are among the highest concentrations measured in fishes globally, while concentrations for red snapper and king snake eel are similar to pre-DWH levels measured in GoM species. In contrast, concentrations of benzo[a]pyrene metabolites were similar for all three species (p = 0.265, mean 220 ng g(-1)) and relatively low when compared to GoM, global data and previous oil spills. These data support previous findings that fish life history and physiology play significant roles in exposure and uptake of PAH pollution.

PAH metabolites in European eels (Anguilla anguilla) as indicators of PAH exposure: Different methodological approaches

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants of aquatic environments derived from pyrogenic and petrogenic sources. In fish, as in other vertebrates, PAHs are rapidly metabolized. However, the metabolites have been proven to induce multiple deleterious effects in fish. The concentrations of biliary polycyclic aromatic hydrocarbon metabolites in eels (Anguilla anguilla) caught in Polish waters were measured. The main objectives of the study were to provide information on the levels of PAH metabolites in eels inhabiting Polish waters and to discuss which methodological approach is appropriate for assessing PAH exposure in aquatic ecosystems. The non-normalized median concentration of 1-OH Pyr and 1-OH Phe measured in eels from Polish waters ranged from 11 to 1642ngml−1 bile and from 83 to 929ngml−1 bile, respectively, depending on the sampling site. Data normalization in relation to bile pigment content reduced inter-site variation, and the normalized median concentrations of 1-OH Pyr and 1-OH Phe ranged from 0.44 to 20.24ngA−1380 and from 1.58 to 11.11ngA−1380, respectively.Our study indicated that results were more consistent for the two species sampled in the same area (eel examined in the current study and flounder examined in our previous study) when the fluorescence response of diluted bile samples was compared than when concentrations of 1-OH Pyr determined with the mean of HPLC were compared.

An attempt to assess the relevance of flood events—biomarker response of rainbow trout exposed to resuspended natural sediments in an annular flume

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.

Comparative study of different exposure routes on the biotransformation and genotoxicity of PAHs in the flatfish species, Scophthalmus maximus

In this study, laboratory experiments were carried out in order to come to a better understanding of the fate of polycyclic aromatic hydrocarbons (PAHs) in the marine environment and especially on their bioaccumulation, biotransformation and genotoxic effects in fish. Juveniles of turbot (Scophthalmus maximus) were exposed to PAHs through different routes via (1) a mixture of dissolved PAHs, (2) a PAH-polluted sediment and (3) an oil fuel elutriate. Fish were exposed 4 days followed by a 6-day depuration period. In each experiment, PAH concentrations in the seawater of the tanks were analysed regularly by gas chromatography coupled with mass spectrometry. Muscle and liver samples were also analysed for parent PAH levels and PAH bioconcentration factors were calculated. Biotransformation was evaluated by measuring the levels of PAH metabolites in fish bile. Genotoxicity was assessed by the alkaline comet assay. Regardless of exposure route, the parent PAH concentrations in the liver and muscle showed a peak level 1 day after the beginning of the exposure, followed by a decrease up to the background level towards the end of the experiment, except for the exposure to dissolved PAHs for which levels were relatively low throughout the study. As a consequence, no bioaccumulation was observed in fish tissues at the end of the experiment. In contrast, regardless of exposure routes, a rapid production of biliary metabolites was observed throughout the whole exposure experiment. This was especially true for 1-hydroxypyrene, the major metabolite of pyrene. After 6 days of recovery in clean water, a significant decrease in the total metabolite concentrations occurred in bile. Fish exposed through either route displayed a significant increase in DNA strand breaks after 4 days of exposure, and significant correlations were observed between the level of biliary PAH metabolites and the level of DNA lesions in fish erythrocytes. Overall results indicate that exposure to either a mixture of dissolved PAHs, a PAH-contaminated sediment or a dispersed oil fuel elutriate leads to biotransformation and increase in DNA damage in fish. The quantification of PAH metabolites in bile and DNA damage in erythrocytes appear to be suitable for environmental monitoring of marine pollution either in the case of accidental oil spills or sediment contamination.

Biliary PAH metabolites, EROD activity and DNA damage in dab (Limanda limanda) from Seine Estuary (France)

The Seine Estuary is well known to be widely contaminated by organic pollutants and especially by polycyclic aromatic hydrocarbons (PAHs). Fish are known to metabolize PAHs, leading to different toxic effects at both cellular and sub-cellular levels. In this work, we studied the relationships between the 7-ethoxyresorufin-O-deethylase (EROD) activity in the liver, the level of DNA strand breaks in blood cells and the concentration of PAH metabolites in the bile of the sentinel flatfish species Limanda limanda. Muscle and liver samples were analysed for parent PAH levels. Female and male dabs of two size classes (juveniles and adults) were collected by trawling in two sites with different degrees of pollution during March and September 2005 and 2006. Significant effects of sex, age, site and season were demonstrated on EROD activity and the level of strand breaks. Parent PAH concentrations in dabs did not allow discriminating of the two sampling sites. However, for PAH metabolites, significant differences were observed with sites and seasons. Dabs collected at the mouth of the estuary appeared to be the most impacted when looking at the results obtained with the three selected markers. The significant correlations observed between the level of PAH metabolites and the level of DNA lesions showed the importance of a combined analysis of chemical and biochemical markers to correctly assess the contribution of chemical contamination to the toxic effects measured in situ in fish.

How flood events affect rainbow trout: Evidence of a biomarker cascade in rainbow trout after exposure to PAH contaminated sediment suspensions

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.

The silvering process of European eel (Anguilla anguilla) influences PAH metabolite concentrations in bile fluid: Consequences for monitoring

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 (A380, 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.

PAH biomarkers in common eelpout (Zoarces viviparus) from Danish waters

Eelpouts (Zoarces viviparus) sampled at surveillance stations during the fall of 2007 and spring 2008 in different Danish coastal areas, were studied for biomarkers of polycyclic aromatic hydrocarbons (PAHs) exposure and effects. Two analytical techniques, synchronous fluorescence spectrometry (SFS) and high-performance liquid chromatography with fluorescence detection (HPLC/F), were applied for detecting PAH metabolites in bile and urine. CYP1A activity, in this study regarded as potential biomarker of effect, was measured as 7-ethoxyresorufin-O-deethylase (EROD) activity in liver of eelpouts from different stations. Biliary PAH metabolite measurements were used for monitoring the environmental PAH load at the surveillance stations. There was found significant difference in biliary PAH metabolite content between sexes with male fish containing higher concentrations of PAH metabolites than females. The urinary PAH metabolite content did not show the same spatial trends as biliary PAH metabolites. However, fish from Aarhus Bight and Vejle Fjord had significantly higher levels of PAH metabolites in both urine and bile compared to the reference station Agersø. Normalisation methods applied for bile and urine matrices did not have any effect or only slightly reduced the coefficients of variation in data sets. The CYP1A activity in eelpout liver did not show the same spatial distribution trends between sampling sites as did biliary or urinary PAH metabolite contents. Male eelpouts showed significantly higher CYP1A activity than females in fall sampling period but there were no differences found in the spring period. General comparison between both seasons showed that eelpouts sampled in the fall had significantly higher CYP1A activity than fish sampled during spring season. Overall, the results of this study describe selected biomarker responses in eelpouts to environmental PAH load at the different areas along Danish coasts.

Assessment of polycyclic aromatic hydrocarbon contamination of the Bílina River (Czech Republic) using passive water samplers and fish biliary metabolites

The aim of the study was to assess polycyclic aromatic hydrocarbon (PAH) contamination on the Bílina River (the Czech Republic) by measurement of three PAH biliary metabolites in fish and 16 PAHs in passive samplers. A total of sixty-one fish were collected; the indicator species were chub (Leuciscus cephalus L.; n = 25), roach (Rutilus rutilus L.; n = 17) and brown trout (Salmo trutta m. fario L.; n = 19). Three biliary PAH metabolites were measured: 1-hydroxypyrene (1-OHP), 2-naphtol, and 1-hydroxyphenanthrene, but only 1-OHP was detected in all fish. The highest median value of 32.3 ng·mg-1of 1-OHP was found at Ústí nad Labem, while the lowest median value of 27.6 ng·mg-1was found in the control site, Březenec. No significant differences (P < 0.05) among the sites were found. The highest concentration of PAH was detected in Litvínov-Záluží (172.0 ng·l-1) and the lowest concentration (7.9 ng·l-1) was detected in Březenec. A positive, but non-significant correlation (rs = 0.8) was confirmed between biliary 1-OHP and total PAH in passive samplers. These results indicate the highest pollution in the middle stretches of the Bílina River, especially at Litvínov-Záluží. We confirmed 1-OHP as the most prevalent PAH biliary metabolite in fish that could be used as a biomarker for assessment of PAH pollution of the aquatic ecosystem. The main importance of the present study is in the combination of biochemical and chemical monitoring that provides complex evaluation of aquatic environment contamination. It was the first study on the Bílina River, in which the assessment of PAH contamination in the aquatic ecosystem was realized using combination of biotic and abiotic monitoring.