Quantitative determination of de-conjugated chrysene metabolites in fish bile by HPLC-fluorescence and GC--MS

Two analytical methods have been evaluated for quantitative determination of de-conjugated chrysene metabolites in fish bile. High performance liquid chromatography-fluorescence (HPLC-F) and gas chromatography-mass spectrometry (GC--MS) were compared regarding instrumental and overall limits of detection (LOD) as well as recoveries for the following nine chrysene compounds: 1-, 2,- 3-, 4- and 6-hydroxychrysene (1-, 2-, 3-, 4- and 6-OH-chr), 1,2-dihydroxy-1,2-dihydrochrysene (1,2-DHD-chr), 3,4-dihydroxy-3,4-dihydrochrysene (3,4-DHD-chr), 5,6-dihydroxy-5,6-dihydrochrysene (5,6-DHD-chr) and chrysene. Instrumental LODs were comparable for the two methods whereas the overall LOD was better for HPLC-F. Recoveries varied per chrysene compound for both HPLC-F (62-107%) and GC-MS (48-124%). In vivo formed chrysene metabolites were studied in the bile of Atlantic cod (Gadus morhua) exposed to chrysene (1 mg/kg) via intra-peritoneal (i.p.) and inter-muscular (i.m.) injection. Total amounts of chrysene metabolites were three times higher in i.p. compared to i.m. exposed cod bile, but the relative distribution of determined metabolites was very similar. 1,2-DHD-chr was the most prominent metabolite in de-conjugated bile and constituted more than 88% of the total chrysene metabolites. Additional chrysene metabolites formed were 3,4-DHD-chr and 1-, 2-, 3- and 4-OH-chr. K-region chrysene metabolites (oxidation at carbons 5 and 6) were not detected and seem to be a less favoured biotransformation route. The two methods were applied and evaluated for analysis of chrysene metabolites in two bile reference materials (BCR 720 and 721) and a limited number of field exposed cods.     

Bioavailability of PAHs in the Biobio river (Chile): MFO activity and biliary fluorescence in juvenile Oncorhynchus mykiss.

Cytochrome P450-dependent monooxygenase (MFO) activity and levels of bile PAH metabolites were measured in fish (Oncorhynchus mykiss) exposed to Biobio river (Chile) water. Experiments were performed simultaneously in both the field and laboratory to investigate whether the river water contained notable bioavailable PAH fractions. The field experiment was conducted using fish caged for 41 days at La Mochita (Biobio river mouth) whereas the laboratory experiment involved a 21-day exposure treatment with water collected in the same area. Induction of hepatic MFO, assayed by benzo(a)pyrene monooxygenase activity (BaPMO), and levels of PAH metabolites in the bile were measured in groups of eight (field experiment) and four specimens (laboratory experiment) sampled after 5, 10, 21 and 41 days of exposure. BaPMO was induced by a factor of 23 and 25 in fish caged for 21 and 41 days, respectively. Likewise, a significant BaPMO induction was found in the fish group experimentally exposed to river water for 21 days. Biliary fluorescence measurements, recorded by fixed wavelength fluorescence (FF) and synchronous fluorescence spectroscopy (SFS), revealed significantly high levels of PAH metabolites in bile samples of fish caged for 21 and 41 days. A positive relationship was found between BaPMO activity and biliary PAH metabolites. This study provides evidences that (i) Biobio river water contains a significant bioavailable PAH fraction in terms of marked effects on fish and (ii) the biological indicators MFO activity and bile PAH metabolites represent an excellent screening methodology to assess PAH exposure.     

Increased formation of carcinogenic PAH metabolites in fish promoted by nitrite

Nitrite (NO(2)(-)), a highly reactive chemical species, accumulates in coastal waters as a result of pollution with nitrogenous waste and/or an imbalance in the bacterial processes of nitrification and denitrification. The present study probed the impact of nitrite (NO(2)(-)) on the metabolism of polycyclic aromatic hydrocarbons (PAHs) in fish. In a laboratory experiment, exposure of euryhaline fish, Oreochromis mossambicus to industrial effluents containing PAHs in the presence of NO(2)(-) enhanced the cytochrome P450-dependent biotransformation activity determined as 7-ethoxyresorufin-O-deethylase (EROD), by nearly 36% compared to the value observed in the absence of NO(2)(-) (50.2 +/- 6.74 pmol resorufin min(-1) g(-1) liver). Fixed wavelength fluorescence measurements in bile revealed maximum enhancement to have occurred in the metabolites of benzo[a]pyrene, a carcinogenic PAH. Lasting, sublethal physiological deterioration was apparent in fish exposed simultaneously to an oil refinery effluent and NO(2)(-), from the unremittingly decreasing liver somatic index, even after the withdrawal of the contaminants.     

13C NMR spectroscopy in the analysis of conjugate metabolites in the bile of fish exposed to petroleum

The first natural abundance ¹³C NMR investigation of a complex mixture of conjugate metabolites obtained from the gall bladder bile of fish exposed to hydrocarbons is presented. Cunners were exposed to water accommodated No. 2 fuel oil containing about 68% saturates and 22% aromatics. Spectral analysis indicated that the hydrocarbon derivatives were present predominantly as β-glucuronides, with the oxygen at carbon-1 of glucuronic acid preferentially attached to an aliphatic carbon. The conjugate metabolites were enriched in aromatic-type carbons when compared to the fuel oil or the aromatic fraction of oil.     

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.     

Potential application of synchronous fluorescence spectroscopy to determine benzo[a]pyrene in soil extracts

Benzo[a]pyrene (BaP) is a significant environmental pollutant and rapid, accurate methods to quantify this compound in soil for both research and environmental investigation purposes are required. In this work, solvent extracts from five contrasting soils spiked with four different polycyclic aromatic hydrocarbons (PAHs) were rapidly analysed by using a synchronous fluorescence spectroscopy (SFS) method. The SFS method was validated using HPLC with ultraviolet detection. A good correlation for the quantification of BaP in soil extracts by the two methods was observed. The detection limit of the SFS method was 1.6 x 10(-9) g/ml in CTAB micellar medium (7.8 mmol/l). The work demonstrates that SFS has potential as a sensitive, accurate, rapid, simple and economic methodology and an efficient alternative to HPLC for fast confirmation and quantification of BaP in complex soil extracts.     

Measurement of pyrene in the gills of exposed fish using synchronous fluorescence spectroscopy

A synchronous fluorescence spectroscopy (SFS) method was developed for determination of pyrene in the gills of exposed fish. The wavelength differences (Δλ) of 50 nm was maintained between excitation and emission wavelengths and it was found to be suitable for the effective determination of pyrene in fish gills; the peak were observed at λ_ex 334.5 nm. Linear relationships between SFS intensity and the concentration of pyrene in n-hexane solution were established. It was demonstrated that the SFS method was effective, simple, and less expensive, providing an attractive alternative for the rapid analysis of pyrene in fish gills.     

Polycyclic aromatic hydrocarbons and their hydroxylated metabolites in fish bile and sediments from coastal waters of Colombia

Sediments and fish bile collected from the Atlantic coastal waters of Colombia were analyzed for 16 parent polycyclic aromatic hydrocarbons (PAHs), and 23 hydroxylated PAHs (OH-PAHs), respectively. Sediments contained overall mean SigmaPAH concentrations of 2090, 234 and 170 ng/g, dry wt, for Cartagena Bay, Caimanera Marsh, and Totumo Marsh, respectively. The mean concentration of the summed OH-PAHs in fish bile was 1250, 180 and 64.1 ng/g bile wt for Cartagena Bay, Caimanera Marsh, and Totumo Marsh, respectively. The results suggest that Cartagena Bay is heavily polluted by PAHs, and that exposure to high concentrations of PAHs together with other factors could contribute to the decreased health of fish living in this ecosystem. This is one of the first studies to describe the analysis of 23 individual OH-PAHs in fish bile, using authentic standards.     

Assessment of bioavailable PAH, PCB and OCP concentrations in water, using semipermeable membrane devices (SPMDs), sediments and caged carp

Bioavailable water concentrations of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) were assessed at several freshwater sites in and around the city of Amsterdam. Carp (Cyprinus carpio) were caged for 4 weeks at 10 sites, together with semipermeable membrane devices (SPMDs). In addition, sediment samples were taken at each site. SPMDs and sediments were analysed for PAHs, PCBs and OCPs. Carp muscle tissues were analysed for PCBs and OCP, while PAH metabolites were assessed in fish bile. Contaminant concentrations in the water phase were estimated using three different methods: 1. Using fish tissue concentrations and literature bioconcentration factors (BCFs), 2. Using SPMD levels and a kinetic SPMD uptake model, and 3. Using sediment levels and literature sorption coefficients (K_ocs). Since PAH accumulation in fish is not considered an accurate indicator of PAH exposure, calculated aqueous PAH concentrations from SPMD data were compared with semiquantitatively determined biliary PAH metabolite levels. Contaminant concentrations in the water phase estimated with fish data (Cw_fish) and SPMD data (Cw_spmd) were more in line for compounds with lower K_ow than for compounds with higher K_ow values. This indicates that the assumption of fish–water sorption equilibrium was not valid. At most sites, sediment-based water levels (Cw_sed) were comparable with the Cw_spmd, although large differences were observed at certain sites. A significant correlation was observed between biliary PAH metabolite levels in fish and aqueous PAH concentrations estimated with SPMD data, suggesting that both methods may be accurate indicators of PAH exposure in aquatic ecosystems.     

Characterization of alkylphenol metabolites in fish bile by enzymatic treatment and HPLC-fluorescence analysis

Alkylphenol (AP) metabolites were characterized in the bile of Atlantic cod (Gadus morhua L.) after exposure to nine individual compounds (10mg/kg fish), 2-methylphenol (2-MP), 4-methylphenol (4-MP), 3,5-dimethylphenol (3,5-DMP), 2,4,6-trimethylphenol (2,4,6-TMP), 4-tert-butylphenol (4-t-BP), 4-tert-butyl-2-methylphenol (4-t-B-2-MP), 4-n-pentylphenol (4-n-PP), 4-n-hexylphenol (4-n-HexP) and 4-n-heptylphenol (4-n-HepP), and a mixture (total dose; 13.5 mg/kg fish) of the nine APs by inter-muscular injection. The degree of alkylation ranged from methyl (C1) to heptyl (C7) and represents the types of APs present in produced water. Fish bile was collected on day 4 and 16 (exposure groups 2-MP, 3,5-DMP, 2,4,6-TMP and 4-t-B-2-MP) following exposure. Characterization of major metabolites was accomplished by enzymatic de-conjugation and analysis by high performance liquid chromatography connected to a fluorescence detector (HPLC-F) acquiring at ex/em 222/306 nm. Two solid phase extraction (SPE) columns were evaluated for clean-up of samples prior to analysis. Independent of alkyl homologue, the glucuronide conjugated APs were the most abundant metabolites (73-100%), whereas sulfates, glucosides and unchanged compounds were excreted in amounts of 0-21%, 0-6.1% and 0-6.3%, respectively. The total concentration of measured metabolites in the bile, determined as their respective APs after de-conjugation, increased with increasing degree of alkylation (3.2+/-2.6 microg/g bile for 2-MP and 571+/-81 microg/g bile for 4-n-HepP) after exposure to an equal dose of AP. Comparison of metabolite concentrations in bile sampled 4 and 16 days after exposure, showed that the levels of 2-MP, 2,4,6-TMP and 4-t-B-2-MP were reduced by 55%, 30% and 45%, respectively whereas 3,5-DMP increased by 25% (not significant). This study suggests that analysis of de-conjugated metabolites in fish bile can be used to monitor AP exposure to fish, due to the relatively high and persistent level of these compounds. However, although HPLC-F is suitable for laboratory exposures, it might not be sufficient selective for field studies.     

Rapid quantification of polycyclic aromatic hydrocarbons in hydroxypropyl-beta-cyclodextrin (HPCD) soil extracts by synchronous fluorescence spectroscopy (SFS)

Synchronous fluorescence spectroscopy (SFS) was directly applied to rapidly quantify selected polycyclic aromatic hydrocarbons (PAHs: benzo[a]pyrene and pyrene) in aqueous hydroxypropyl-beta-cyclodextrin (HPCD) soil extract solutions from a variety of aged contaminated soils containing four different PAHs. The method was optimized and validated. The results show that SFS can be used to analyse benzo[a]pyrene and pyrene in HPCD based soil extracts with high sensitivity and selectivity. The linear calibration ranges were 4.0x10(-6)-1.0x10(-3)mM for benzo[a]pyrene and 6.0x10(-6)-1.2x10(-3)mM for pyrene in 10mM HPCD aqueous solution alone. The detection limits according to the error propagation theory for benzo[a]pyrene and pyrene were 3.9x10(-6) and 5.4x10(-6)mM, respectively. A good agreement between SFS and HPLC was reached for both determinations of PAHs in HPCD alone and in soil HPCD extracts. Hence, SFS is a potential means to simplify the present non-exhaustive hydroxypropyl-beta-cyclodextrin (HPCD)-based extraction technique for the evaluation of PAH bioavailability in soil.     

Detection of DNA damage in fish Oreochromis mossambicus induced by co-exposure to phenanthrene and nitrite by ESI-MS/MS

Background, aim, and scope

Mutagenic nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) have been known to arise in the environment through direct emissions from combustion sources and nitration of PAHs, primarily in the atmosphere. In the marine environment, PAHs are one of the classic anthropogenic organic pollutants, while nitrite (NO ₂ ^– ) is produced naturally via various biological processes like imbalance in nitrification/denitrification or eutrophication and subsequent oxygen depletion from an oversupply of nutrients. In this paper, we report the formation of PAH-DNA adducts in fish contaminated with PAHs and exposed to NO ₂ ^– in the ambient water. Electrospray ionization tandem mass spectrometric (ESI-MS/MS) analysis of the bile of the euryhaline fish Oreochromis mossambicus exposed simultaneously to field relevant sublethal concentrations of phenanthrene and NO ₂ ^– and collision-induced dissociation of selected ions revealed the presence of DNA-PAH adducts. The present study indicates that, although several high sensitivity techniques have been developed for the analysis of PAH derived DNA adducts, MS/MS has emerged as a powerful tool in the detection and structure elucidation of DNA adducts.

Materials and methods

Juvenile O. mossambicus from a local estuarine fish farm were used with increasing frequency for carcinogenicity testing and comparative cancer research. The fish were exposed to the alkylating agent phenanthrene in the presence of NO ₂ ^– . Composite untreated bile samples after dilution with methanol: water (1:1; v/v) were analyzed by ESI-MS.

Results

Several adducts could be evidenced in the bile by MS/MS. Deoxyadenosine/deoxyguanosine having a mass in the range of 450–650 amu is detected. In addition, a segment of modified dinucleotide with a mass that corresponds to a dimer consisting of a modified guanosine and a normal guanosine has also been identified in the bile.

Discussion

The formation of certain types of DNA adducts is a crucial step in the induction of cancer and a primary stage in mutagenesis. Phenanthrene injected by i.p. route led to the transformation of phenanthrene to N-formyl amino phenanthrene-N ⁶-deoxyadenosine adduct, whereas the fish co-exposed to phenanthrene and ambient nitrite metabolizes PAH to mono-, di- as well as trinitro derivatives, which then react with DNA leading to the formation of mainly modified guanosine and adenosine adducts. In the present investigation, dinitrophenanthrene diol epoxide (DNPDE) adduct with guanosine (m/z 587) seems to be the dominant adduct in the mixture, and its presence is shown first as a comparatively less stable adduct, which decomposes to give a more stable N² adduct (m/z 567).

Conclusions

MS/MS has proved to be useful in the rapid determination and discrimination of structurally different phenanthrene/derivatives DNA adducts in a complex mixture of fish bile co-exposed to phenanthrene and nitrite. However, the nature of metabolites formed is likely determined by the route of PAH administration, and there is a need to further define the early biochemical events of carcinogenesis in these species.

Recommendations and perspectives

DNA adduct analysis in fish bile offers a promising approach to study the risk of potentiation of anthropogenic chemicals into genotoxic compounds in the presence of nitrite in the marine environment. We believe this is the first report on the formation of DNA-phenanthrene adducts on co-exposure of the fish to PAH and nitrite.     

Contents and sources of polycyclic aromatic hydrocarbons and organochlorine pesticides in vegetable soils of Guangzhou, China

We investigated contents, distribution and possible sources of PAHs and organochlorine pesticides (Ops) in 43 surface and subsurface soils around the urban Guangzhou where variable kinds of vegetables are grown. The results indicate that the contents of PAHs (16 US EPA priority PAHs) range from 42 to 3077 microg/kg and the pollution extent is classified as a moderate level in comparison with other investigations and soil quality standards. The ratios of methylphenanthrenes to phenanthrene(MP/P), anthracene to anthracene plus phenanthrene (An/178), benz[a]anthracene to benz[a]anthracene plus chrysene (BaA/228), indeno[1,2,3-cd]pyrene to indeno[1,2,3-cd]pyrene plus benzo[ghi]perylene (In/In+BP) suggest that the sources of PAHs in the soil samples are mixed with a dominant contribution from petroleum and combustion of fossil fuel. The correlation analysis shows that the PAHs contents are significantly related to total organic carbon contents (TOC) (R2=0.75) and black carbon contents (BC) (R2=0.62) in the soil samples. Dichlorodiphenyltrichloroethane and metabolites (DDTs) and hexachlorocyclohexanes and metabolites (HCHs) account largely for the contaminants of OPs. The concentrations of DDTs range from 3.58 to 831 microg/kg and the ratios for DDT/(DDD+DDE) are higher than 2 in some soil samples, suggesting that DDT contamination still exists and may be caused by its persistence in soils and/or impurity in the pesticide dicofol. The concentrations of HCHs are 0.19-42.3 microg/kg.     

PAH Metabolites in Bile Fluids of Dab (Limanda limanda) and Flounder (Platichthys flesus): Spatial Distribution and Seasonal Changes (7 pp)

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 <0.7 to 838 ng/ml in bile of dab (Limanda limanda) and flounder (Platichthys flesus) caught between 1997 and 2004. The values for 1-hydroxyphenanthrene in fish bile were considerably lower (<0.4 – 87 ng/ml). Significant differences in the 1-hydroxypyrene levels were found between summer and winter surveys as well as between the sampling sites in the data set from 2004 (383 dabs and 62 flounders): Highest levels of PAH contamination were found in dab from the German Bight and in flounder from the Baltic Sea. Discussion: Spatial differences in 1-hydroxypyrene concentrations between North Sea and Baltic Sea were discussed, as well as differences in relation to season, sex and species. Three parameters of normalisation (biliary protein, biliverdin and bile pigments) were discussed. Biliverdin was identified as a suitable parameter for the normalisation of PAH metabolites in field samples. Conclusions: Spatial differences in 1-hydroxypyrene concentrations of dab demonstrate the usefulness of PAH metabolites in fish bile as a monitoring parameter in marine regions. Significant differences in 1-hydroxypyrene concentrations were found between summer and winter sampling campaigns. This may be linked to an annual cycle of 1-hydroxyprene in dab. It is also possible that bile synthesis/release in dab differs between the seasons. There is no indication for a time trend from 1997 to 2004. Recommendations and Perspectives: It is recommended to relate PAH metabolites in fish bile to biliverdin concentrations. Although the concentrations are low in offshore regions and bile volumes are small, the method presented here allows one to measure PAH metabolites on an individual level which is a crucial prerequisite for meaningful monitoring studies.     

Residual levels of DDTs and PAHs in freshwater and marine fish from Hong Kong markets and their health risk assessment

Axial and ventral muscle from 10 each species of freshwater and marine fish purchased from markets in Hong Kong were analyzed for dichlorodiphenyltrichloroethane (total DDTs including DDE, DDD and DDT) and polycyclic aromatic hydrocarbons (PAHs). Among the 10 freshwater fish species, rice field eel (Monopterus albus) showed significantly higher levels of DDTs in both ventral (125 ng/g wet wt) and axial muscle (127 ng/g wet wt) than the other species. The highest concentration of PAHs was detected in catfish (Clarias fuscus), with 24.8 ng/g in ventral muscle and 9.1 ng/g in axial muscle. As to marine fish, snubnose pompano (Trachinotus blochii) showed significantly higher levels of DDT and its metabolites (1018 ng/g in ventral and 409 ng/g wet wt in axial tissues) than all other marine fish species. The overall concentrations of PAHs in marine fish species were 15.5-57.0 ng/g (axial muscle) and 18.1-118 ng/g wet wt (ventral muscle) where yellow seafin (Acanthopeyrus latus) and golden threadfin bream (Nemipterus virgatus) exhibited the highest concentrations of PAHs in the axial and ventral muscles, respectively. In general, results showed that levels of PAHs in Hong Kong market fish was low and do not expect to cause any concern for human consumption. However, the levels of DDTs in fish samples ranged from 1.10 to 1018 ng/g wet wt, and based on a fish consumption rate of 142.2g/day to calculate the screening value of 14.4 ng/g wet wt for human consumption (USEPA, 2000. Guidance for assessing chemical contaminant, data for use in fish advisories, vol. 1: fish sampling and analysis, third ed. EPA 823-R-95-007. Office of Water, Washington, DC), there were 9 out of 20 (45%) muscle samples of freshwater fish species and 14 out of 20 (70%) muscle samples of marine fish species had elevated levels of DDTs exceeded the screening value. It was also suggested to use ventral muscle for detecting concentrations of persistent organic pollutants in fish.     

Bioaccumulation of butyltins and liver damage in the demersal fish Cathorops spixii (Siluriformes,Ariidae)

The toxicity of butyltin compounds (BTs), mainly tributyltin (TBT), has been reported in different organisms. However, such an analysis in fish after field exposure with reference to the related biomarkers has not been commonly observed in the literature. This study presents the uptake of BTs in the liver of a neotropical marine catfish Cathorops spixii in Paranagua Bay, an important estuarine system located in southern Brazil. Two different areas, close to and distant from the harbor, were used for chemical analysis evaluation of hepatotoxicity through genetic, enzymatic, and histopathological biomarkers. The presence of polycyclic aromatic hydrocarbons in bile was also considered as a biomarker. The results showed a significant relationship between TBT levels and the inhibition of biotransformation enzymes and high occurrence of melanomacrophages in fish collected close to the harbor site. These effects were linked to the absence of TBT metabolites in the liver. In the second site, the presence of DBT was associated with an increase in EROD and GST activity. The larger amount of DNA damage as well as the highest oxidative stress was noted in fish from the less TBT-polluted area, where DBT and bile PAHs occurred. These findings showed different impact levels due to or increased by the chronic exposure of biota to BTs.     

A comparative study on the uptake of polycyclic aromatic hydrocarbons by Anodonta californiensis

Uptake of polycyclic aromatic hydrocarbons (PAHs) by the freshwater bivalve mollusc Anodonta californiensis was examined in the presence and absence of surfactant in order to gain further insight into mixture toxicity and to predict whether certain mixtures have negative and/or positive effects on aquatic organisms. In the presence of surfactant, the uptake of anthracene or chrysene was higher than that of naphthalene, given the same concentration in the solution. In the absence of surfactant, the trend was similar, but the uptakes were increased by approximately 100% compared to those in the presence of surfactant. On the uptake of naphthalene, the presence of anthracene showed only minor influence. The uptake of anthracene was affected by both naphthalene and chrysene. The uptake of chrysene was influenced by neither naphthalene nor anthracene. There was no observable displacement of divalent cations from the surface of the gill membrane by any of the PAHs studied.     

PAH metabolites,GST and EROD in European eel (Anguilla anguilla) as possible indicators for eel habitat quality in German rivers

The stock of the European eel (Anguilla anguilla L.) continues to decline and has reached a new minimum in 2011. Poor health status of the spawners due to organic contaminants is one of the possible causes for this dramatic situation. Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants, which are rapidly metabolized in vertebrates. EROD (ethoxyresorufin-O-deethylase) and GST (glutathione-S-transferase) are two enzymes involved in PAH detoxification in fish. In this study, PAH metabolites as well as EROD and GST activity in a large, comprising dataset of more than 260 migratory and pre-migratory eels from five large German river basin districts were used to describe PAH exposure and its metabolism as possible indicators for the habitat quality for eels. Eel from the river Elbe appear to be moderately contaminated with PAH. Highest mean values of PAH metabolites were analysed in fish from the river Rhine. However, the results suggest that contaminants such as PAH are metabolized in the fish and may have contributed to EROD activity in eels caught from the Elbe estuary to 600 km upstream. Since the eel’s onset of cessation of feeding is closely linked to maturation and migration, we propose bile pigments as new indicators contributing to identify the proportion of migratory eel, which is crucial information for eel management plans. We showed that PAH metabolites normalized to bile pigments as well as EROD could be used to describe the habitat quality and might be suitable parameters in search for suitable stocking habitats.     

Identification and dose dependency of ibuprofen biliary metabolites in rainbow trout

The biotransformation of the anti-inflammatory drug ibuprofen (IBF) was studied by exposing rainbow trout (Oncorhynchus mykiss) to IBF via intraperitoneal (i.p.) injection, and via water at four (0.17, 1.9, 13 and 145 μg L⁻¹) exposure levels for 4 d. Following exposure, the bile was collected and analyzed by LC–MS/MS methods. The identification of the formed metabolites in i.p. injected fish bile was based on the exact mass determinations by a time-of-flight mass analyzer (Q–TOF–MS) and on the studies of fragments and fragmentation patterns of precursor ions by ion trap mass analyzer (IT-MS). In addition to unmetabolized IBF, several phase I and phase II metabolites were found in the bile. The main metabolites were acyl glucuronides and taurine conjugates of IBF and of hydroxylated IBFs. The bioconcentration factors (BCF_bile), defined as the ratio of the sum of IBF and its metabolites in fish bile to the concentration of IBF in water, was determined following enzymatic deconjugation and was found to range from 14 000 to 49 000. The highest BCF_bile was found at the lowest exposure concentration (0.17 μg L⁻¹). The results show that rainbow trout has a high capacity for biotransformation of IBF, and the exposure of fish to sub μg L⁻¹ concentrations of IBF can be determined by the analyses of the biliary metabolites of the compound.