Ethoxyresorufin-O-deethylase (EROD) and vitellogenin (VTG) in flounder (Platichthys flesus): system interaction, crosstalk and implications for monitoring.

The extent to which biological systems interact in fish from multi-contaminant areas needs to be understood for full interpretation of monitoring data. This study investigates the interaction between two biomarkers, ethoxyresorufin-O-deethylase (EROD) activity and plasma vitellogenin (VTG) in the European flounder (Platichthys flesus). Flounder were exposed to several waterborne EROD inducers and estrogenic chemicals on their own and in binary combinations. Each experimental exposure was for 10 days. The estrogenic chemicals suppressed PAH-mediated EROD induction. Ethynylestradiol (EE2) and nonylphenol (NP) had threshold concentrations of EROD inhibition similar to those at which they induced VTG production. Estradiol (E2), however, showed an ability to suppress EROD at a concentration much lower than that at which VTG was induced. This established that, although EE2 is a more potent VTG inducer than E2, it is less potent in its ability to inhibit EROD activity. The PAH, dibenz[a,h]anthracene (DbA), showed no effect on the VTG induction caused by EE2 and E2. A small effect was noted with NP at threshold concentrations for VTG induction. Archived data on flounder hepatic EROD activity collected during estuarine monitoring were reassessed in light of the project findings. It is hypothesised that published EROD monitoring data may be an underestimation of effects if it is assumed that estrogen-mediated MFO suppression is occurring in wild populations. A greater understanding of system interaction and other factors, including genetics, that influence biomarker response to contaminants would be required to interpret biomarker monitoring data.

Differential sensitivity of flounder (Platichthys flesus) in response to oestrogenic chemical exposure: an issue for design and interpretation of monitoring and research programmes.

This study was conducted as an initial investigation of 'differential response' in one of the main sentinel organisms used for monitoring programmes in United Kingdom estuaries, the flounder Platichthys flesus. It has been hypothesised that monitoring using species with a wide geographical spread and limited migration, such as flounder, might result in the comparison of different genetic stocks and certainly of populations with differing early life stage contaminant exposure histories. Furthermore, it is probable that these pre-exposure and genetic differences could manifest themselves in an ability to respond differently to contaminant exposure, so-called 'differential response'. It is important that the extent and nature of this response is understood, if we want to be able to fully interpret the monitoring data from such programmes. During this study, flounder were collected from four separate sources; wild caught fish from the estuaries of the Rivers Alde, Mersey and Tyne, and farmed flounder from Port Erin Farm, Isle of Man. Under controlled laboratory conditions, groups of fish from each source were exposed to water-borne concentrations of the synthetic oestrogen ethynylestradiol (EE2) at a nominal concentration of 50 ng/l. Plasma was taken from each male fish after 6 and 10 days exposure and analysed for the presence of vitellogenin (VTG) using an ELISA technique. Significant levels of VTG induction were evident in fish from all sources after both 6 and 10 days exposure. Flounder from the Mersey were the only fish with significantly elevated initial background levels of VTG (day 0) and this appeared to be reflected in that these specimens showed the highest induction response after day 6. However, after day 10, fish from all other sites had a slightly higher mean VTG than those from the Mersey which showed significantly (p < 0.05) lower mean plasma VTG. It is suggested that other differential responses may have been masked by the use of a high dose of EE2 which produced maximum induction in nearly all fish. The findings of the study are discussed in terms of implications for further research into the differential response issue and how the initial plasma VTG figures contribute to a time-series from the Mersey, Tyne and Alde estuaries.

Hepatic ethoxyresorufin O-deethylase (EROD) activity in flounder (Platichthys flesus) from contaminant impacted estuaries of the United Kingdom: continued monitoring 1999-2001.

The determination of hepatic ethoxyresorufin O-deethylase (EROD) has been used to assess the induction of the mixed function oxygenase system (MFO) of flounder (Platichthys flesus) in UK estuaries. Induction of the MFO system denotes possible exposure to certain organic contaminants (e.g. polycyclic aromatic hydrocarbons, polychlorinated biphenyls) and its measure has been incorporated in national monitoring programmes. This study presents EROD monitoring data from 5 UK estuaries taken between 1999 and 2001 and builds on data from previous years. The results reveal that for all sampled estuaries EROD values have been significantly (p < 0.05) elevated on the majority of occasions in comparison with the reference estuary, the Alde in Suffolk, UK. However, the limited temporal scale of the reported monitoring does not allow any conclusions to be drawn with respect to trends in the data. Possible factors influencing the data (size, gender, seasonality, reproductive status, etc.) are discussed and recommendations for continued monitoring are made.

32P-postlabelling analysis of DNA adducts and EROD induction as biomarkers of genotoxin exposure in dab (Limanda limanda) from British coastal waters.

Dab (Limanda limanda) were sampled from a number of polluted and unpolluted areas in British coastal waters. The 32P-postlabelling assay was used to analyse the level of aromatic/hydrophobic DNA adducts in pooled samples of liver tissue. The mean levels of DNA adducts detected from areas known to receive anthropogenic pollutants ranged from 4.0 to 26.8 adducts per 10(8) nucleotides, with all sites containing samples displaying DNA adduct profiles consisting of diagonal radioactive zones. In contrast no DNA adducts were detectable in samples from an unpolluted reference site. The ranking of polluted sites based on DNA adduct levels did not correspond with the ranking of sites based on sediment associated polycyclic aromatic hydrocarbon levels, highlighting the problem of linking the presence of contamination with detectable biological responses. No correlation could be found in this study between EROD activity and the level of DNA adducts.

The detection of biomarkers of genotoxin exposure in the European flounder (Platichthys flesus) collected from the River Tyne Estuary.

The Tyne Estuary (North East England) is known to contain elevated levels of polycyclic aromatic hydrocarbons (PAH), compared with other less industrialised English waterways. Previous studies suggest that such contamination is responsible for the toxicity detected in invertebrate bioassays conducted on water and sediment samples collected from the Tyne. Here we present data from a biomonitoring study using hepatic DNA adducts (32P-postlabelling assay) and bile metabolites (synchronous fluorescence spectrometry) to investigate genotoxic exposure in flounder (Platichthys flesus) collected from three sites (Scotswood, Newcastle and Redheugh) along the Tyne Estuary. Flounder were also collected from a clean reference site, the Alde Estuary. Levels of bile metabolites (microgram kg-1 wet weight 1-OH pyrene equivalents) were elevated in flounder caught from the Tyne (Scotswood = 22,247 +/- 3408; Newcastle = 14,572 +/- 1888; Redheugh = 21,872 +/- 2935) compared with those collected from the Alde (632 +/- 56). The levels of DNA adducts (adducted nucleotides per 10(8) normal nucleotides) were also elevated in Tyne flounder (Scotswood = 24.6 +/- 3.2; Newcastle = 34.4 +/- 3.7; Redheugh = 27.6 +/- 6.3) compared with fish collected from the Alde (10.1 +/- 4.8), suggesting that a proportion of the bioavailable PAH was being converted into genotoxic metabolites. All DNA adduct profiles in flounder collected from the Tyne consisted of diagonal radioactive zones of radiolabelled adducts, which were not present in fish sampled from the Alde. The in vivo dosing of flounder with benzo[a]pyrene (BaP) to produced DNA adducts in similar chromatographic positions to the diagonal radioactive zones in the Tyne caught flounder are also described.

EROD activity measured in flatfish from the area of the Sea Empress oil spill.

Dab (Limanda limanda) and plaice (Pleuronectes platessa) were collected at five stations near to the site of the Sea Empress oil spill within two weeks of the incident and a further fourteen stations three months after the spillage. Ethoxyresorufin-O-deethylase (EROD) activity was determined in the livers of the specimens to determine whether induction could be detected. Statistically significant inter-site differences in EROD levels in both species were demonstrated. Elevated levels of EROD activity in dab were found at the two stations nearest to the incident up to three months after the spill but no clear relationship to putative contaminant levels was determined. EROD levels in plaice showed a generally similar pattern of induction as in dab. Correlation of EROD levels with other variables showed that sexual maturity had the greatest influence on dab during the study period. The plaice specimens were sexually immature and, therefore, did not demonstrate a corresponding relationship. It was concluded that, for EROD monitoring purposes, fish should be sampled during their sexually inactive phase and that close attention needs to be paid to other variables (depth, temperature, GSI, length, influential contaminants etc.) when interpreting the results.