Histopathological observations on liver,kidney and gonad of plaice (Pleuronectes platessa) taken from the Mersey estuary

The Mersey estuary is the most contaminated estuary in British waters. Detailed studies are underway on the pathology of flounder (Platichthys flesus) from the Mersey and on flounder from the nearby, but less contaminated, Dee estuary. Flounder breed offshore but spend a lot of time in the estuaries, penetrating into freshwater. These flatfish are in close contact with sediments and will be exposed to exenobiotic stored in the sediments. Plaice (Pleuronectes platessa) are flatfish which enter the Mersey with the tidal flow but spend less time in the estuary than flounder and are less likely to show evidence of pollutant exposure. Over 20 plaice were collected from the Mersey. Samples of the liver, kidney and gonad were fixed in 10% formal saline, processed into paraffin wax and 5 microm sections cut and stained with haematoxylin and eosin. Liver changes consisted of variable amounts of glycogen/lipid storage product, minor perivenular and perivascular fibrosis, and helminth parasitisation. No tumours or foci of cellular alteration, necrosis and inflammation were seen. In the gonad only changes consistent with normal gonadal cycling was seen. These studies provide evidence that plaice show little or no evidence of toxic pathological damage resulting from their stay in the estuary.     

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.     

Histopathological biomarkers in estuarine fish species for the assessment of biological effects of contaminants

The increasing emphasis on the assessment and monitoring of estuarine ecosystems has highlighted the need to deploy appropriate biological indices for these locations. Fish diseases and histopathology, with a broad range of causes, are increasingly being used as indicators of environmental stress since they provide a definite biological end-point of historical exposure. This study reports on the histopathological alterations observed in selected organs and tissues of three species of estuarine fish (Platichthys flesus, Pomatoschistus minutus and Zoarces viviparus), captured from four British estuaries (the Tyne, Tees, Mersey and Alde), differently impacted by contaminants, including PAHs. A biannual sampling regime was used to identify the important seasonal variations that occur in terms of the observed biological effects. Inflammatory lesions and hepatocellular fibrillar inclusions attained their highest prevalence in P. flesus captured from the Tyne, Tees and Mersey. The presence of pre-neoplastic and neoplastic toxicopathic lesions was highest in P. flesus captured from these sites, when compared to fish from the Aide reference site. In particular, the prevalence of hepatic foci of cellular alteration (up to 43.3%) and hepatocellular adenoma (up to 10%) were highest in P. flesus captured from the Mersey estuary. Intersex (ovotestis) was only recorded in male P.flesus captured from the Mersey estuary (up to 8.3%) and from male Z. viviparous captured from the Tyne estuary (25%). Pathologies associated with the gill and the kidney were also most prevalent in fish captured from the Tyne, Tees and Mersey estuaries. This study has successfully applied histopathology to an estuarine monitoring program, both for the recording of toxicopathic lesions in the liver and other organs, and for the detection of the endpoint of endocrine disruption, intersex. As such, it provides a powerful integrative tool for the assessment of biological effects of contaminants in these environments.     

The viviparous blenny (Zoarces viviparus) as a bioindicator of contaminant exposure: application of biomarkers of apoptosis and DNA damage

The viviparous blenny (Zoarces viviparus), also known as eelpout, is considered a suitable biomonitor for use in European estuarine and coastal waters. We have previously shown that Z. viviparus from the industrialised Tyne estuary (UK) display certain histopathologies (e.g., ovotestis and hepatic nuclear and cellular pleomorphism), which are associated with contaminant exposure [Mar. Environ. Res. 55 (2003) 137]. Furthermore, the prevalence of these pathologies was higher than in fish collected from a less contaminated reference site (the Alde estuary, UK). Here, tissue samples were collected from Z. viviparus from the Tyne and Alde estuaries (in the spring and autumn) and analysed for DNA adducts using the (32)P-postlabelling assay and for histopathology. Z. viviparus caught from the Tyne during the spring (56.4+/-18.5 adducts per 10(8) nucleotides) and autumn (63.1+/-7.5 adducts per 10(8) nucleotides) exhibited patterns of DNA adducts which indicated exposure to a complex mixture of genotoxins. In contrast, levels of DNA adducts in Z. viviparus from Alde estuary were significantly lower during both sampling periods. Histopathological changes to the gonads included intersex (ovotestis) and germ cell apoptosis (detected via the TUNEL assay) in male fish captured from the Tyne estuary. Further studies on the use of germ cell apoptotic rate at different estuarine sites are required if it is to be used as a reproductive biomarker of contaminant exposure in male fish.     

Kinetics of vitellogenin protein and mRNA induction and depuration in fish following laboratory and environmental exposure to oestrogens

Plaice, flounder and sand goby were exposed to ethynylestradiol (EE2) for 21 days and then followed for up to 31 days after removal of the oestrogen. Plasma vitellogenin (VTG) and hepatic VTG mRNA were determined in groups of fish sampled during the induction and post-exposure phases. VTG mRNA increased slightly earlier than plasma protein, but both reached maxima by 21 days. In contrast, VTG mRNA decayed much more rapidly than protein after EE2 exposure was terminated (typical values t(1/2) mRNA 3 days, protein 15-30 days). Vitellogenin and VTG mRNA thus measure different temporal events and this is illustrated by field data of male flounder in which both parameters have been determined. Few fish show co-ordinate increased VTG mRNA and vitellogenin but rather more fish have increased vitellogenin. Low level increases of VTG mRNA (5 X) is observed in some fish without increased vitellogenin and this may represent polymorphic differences between individual fish.     

The impact of oestrogenic and androgenic contamination on marine organisms in the United Kingdom--summary of the EDMAR programme. Endocrine Disruption in the Marine Environment

This paper summarises results of the EDMAR programme which is investigating oestrogenic and androgenic endocrine disruption in UK coastal waters. Most of the data concern fish. Four species (flounder, viviparous blenny and two sand gobies) are experiencing feminisation in industrialised estuaries. In males this includes vitellogenin (VTG) synthesis, ovotestis induction and/or feminised sexual characteristics. Although reproductive success may be impaired in some cases, implications for fish populations are still unclear. Suspected causative contaminants include natural oestrogenic substances and synthetic oestrogen mimics. The majority of the oestrogenic activity is adsorbed to sediments, and routes of exposure may include benthic food chain transfer. Some natural androgenic substances are also being discharged to estuaries, but their activity appears low.     

Assessing food web dynamics and relative importance of organic matter sources for fish species in two Portuguese estuaries: a stable isotope approach

Stable carbon and nitrogen isotopes (δ13C, δ1?N) were used to analyse food web dynamics of two of the main estuaries of the Portuguese coast: Tejo and Mira. The ultimate sources of organic matter supporting production of some of the most abundant and commercially important fish species were determined; and seasonal, inter- and intra- estuarine differences in the trophic relations among producers and consumers were identified. Stable isotope analysis was performed in different producers, primary consumers (main prey items for fish) and fish species (Solea solea, Solea senegalensis, Pomatoschistus microps, Dicentrarchus labrax, Liza ramada, Diplodus vulgaris and Atherina presbyter) of two areas in each estuary, in July and October 2009. Model calculations showed that the main prey for the fish species in the Tejo estuary used mostly salt marsh-derived organic matter as nutritional sources, with no marked differences between the sampled months. Trophic levels of fish species from the same estuary differed at multiple scales: inter-species, seasonally and spatially (both between and within estuaries). Significant differences in isotopic composition of fish species were more pronounced spatially (between the two sampled areas in the estuary) than seasonally (between sampled months). Trophic relationships in both estuaries demonstrated that organic matter is transferred to higher trophic positions mainly through benthic pathways. This shows the flexibility of these species to share resources and to exploit temporary peaks in prey populations. The present results showed that extensive disturbance in intertidal habitats from both estuaries may potentially change the balance of organic matter in the base of these complex food webs.     

Habitat selection of stone and starry flounders in an estuary in relation to feeding and survival

Juveniles of both stone flounder Platichthys bicoloratus and starry flounder Platichthys stellatus utilize estuaries as nursery grounds. To understand their habitat selection and the functions of habitats such as food supply, we defined the seasonal distribution of recently settled fish of these species in shallow nursery areas and investigated their feeding habits in the Natori River estuary, Japan. Distribution of stone flounder was limited to the lower estuary (<3 km upriver from the mouth) and stone flounder were most abundant near the mouth. Recently settled starry flounder were first detected further upstream in areas characterized by low salinity <10 and by the absence of the predatory sand shrimp Crangon uritai. Early juvenile stone and starry flounders consumed mainly siphons of the bivalve Nuttallia olivacea and the mysid Neomysis awatschensis, respectively; however, 1- and 2-yr-old fish of both stone and starry flounders fed mainly on the bivalve siphons. These results indicate that habitat selections of juvenile stone and starry flounders enable utilization of preferred prey and predator avoidance, respectively, and that non-overlap of these species' habitats results in avoidance of inter-specific competition for food between these two species.     

4-tert-octylphenol and 17beta-estradiol applied by feeding to flounder Platichthys flesus: induction of vitellogenin and accumulation in tissues

In order to simulate a possible natural administration route of xenoestrogens male flounder Platichthys flesus were exposed via the diet to the alkylphenol 4-tert-octylphenol. Treatment with 4-tert-octylphenol (10, 50 and 100 mg OP kg(-1) BW) or 17beta-estradiol (0.05 mg kg(-1) BW) every second day during a period of 11 days resulted in a significant increase in plasma vitellogenin (Vtg) concentrations. The induction of Vtg was greatest in the fish receiving 50 mg OP kg-' BW. A significant accumulation of 4-tert-octylphenol was found in liver and muscle tissue of the OP treated groups. The tissue concentrations of 4-tert-octylphenol and the plasma vitellogenin concentration were positively correlated. The results show that 4-tert-octylphenol accumulates in liver and muscles of flounder P. flesus, and exerts estrogenic effects such as vitellogenin induction.     

Occurrence of juvenile Japanese flounder Paralichthys olivaceus in brackish estuaries

Juvenile Japanese flounder Paralichthys olivaceus usually inhabit high salinity inshore sandy areas. In June–August 1997, 25 individuals of juvenile Japanese flounder (33–75 mm total length) were collected in the Natori and Nanakita River estuaries in Sendai Bay, Japan. This is the first record of this species being collected in brackish estuaries in which salinities fluctuate from 0 to 30 over a spring tidal cycle. Factors of rainfall, river flow, or year class strength of Japanese flounder were unable to explain the unusual occurrence of this species in the estuaries. The collected juvenile Japanese flounder were considered to have migrated into the estuaries around the time of the passage of an unusually early typhoon that affected this area, indicating the possibility that this species utilizes estuaries for refuge from strong disturbance in its usual habitats. The juveniles fed mainly upon mysids both in the estuaries and the sandy beach area, indicating that estuaries can be a substitute nursery habitat for this species.     

Uptake of estradiol from sediment by hornyhead turbot (Pleuronichthys verticalis) and effects on oxidative DNA damage in male gonads

Male and female hornyhead turbot (Pleuronichthys verticalis) were exposed to four concentrations (0, 0.75, 14.7 and 46.5 ng/g dry weight) of E2-amended sediment for 7 days. Sediment-derived E2 was bioavailable to the flatfish, though the route of uptake was unclear. A concentration of 46.5 ng/g E2 in sediment led to a significant increase in vitellogenin concentrations in the plasma in both sexes after 7 days of exposure. Though plasma E2 concentrations increased significantly in males at sediment E2 concentrations of 0.75 ng/g dry weight and above, a dose-dependent increase was not observed. There was also no correlation between sediment E2 concentrations, plasma E2 concentrations, and oxidative DNA damage in male gonads. The results suggest that the DNA damage previously seen in the gonads of feral hornyhead turbot at a sewage outfall is likely not caused by acute exposure to exogenous E2 from sediments.     

Variations of the Nutrients Loads to the Mainland U.K. Estuaries: Correlation with Catchment Areas, Urbanization and Coastal Eutrophication

The annual loads of nutrients (TO_xN equal to nitrate+nitrite; ammonium, phosphate, silicate) to all the estuaries on the mainland of the United Kingdom were estimated from data on water flow through gauging stations at the tidal limits of estuaries, and from concentration measurements under the Harmonised Monitoring Scheme of nutrient concentrations in water samples from these stations. The annual loads of nutrients showed distinct regional variations, with estuaries along the west coast of Wales and northern Scotland having much smaller loads than those along the east coast of England. The largest nitrogen loads were of TO_xN, and ammonium loads were usually small in comparison. The Severn, Mersey, Humber and Thames had the highest loads, although these were small in relation to the larger continental European estuaries. Loads of TO_xN per unit of catchment area were surprisingly constant (about 10⁵ moles N km⁻² y⁻¹). The nutrient loads showed that most U.K. catchments were influenced by human activity, the majority being in the ‘ moderately influenced ’ category. Nutrient loads were also normalized for the area of each estuary, as a measure of the relative influence of nutrients on the receiving estuaries. The ratios of N:P, N:Si and P:Si in the annual loads suggested that most estuaries were likely to be, if anything, P limited rather than N or Si limited. However, crude annual loads may conceal significant seasonal variations.The spring maximum chlorophylla concentrations in coastal waters adjacent to each estuary were significantly correlated with the log total annual loads of TO_xN, ammonium and phosphate (but not silicate) for each estuary, providing a direct link between a measure of the degree of biological response in coastal waters and the nutrient load through the estuaries. There were no significant correlations between spring maximum chlorophyll a concentrations and either catchment-normalized or estuary-normalized nutrient loads. There was significant correlation between catchment area-normalized loads of phosphate and an urbanization index for the catchments, but not with the catchment area-normalized loads of the other nutrients.     

Influence of intermittent estuary outflow on coastal sediments of adjacent sandy beaches

Outflows from estuaries potentially contribute to the productivity of adjacent coastal waters, although most previous work has been on estuaries with considerable river discharge. We investigated the influence of estuary outflow on aspects of coastal sediments adjacent to two seasonally intermittent estuaries, the Curdies and Anglesea Rivers, in southwest Victoria, Australia. For each estuary, we measured sediment organic matter, microphytobenthic chlorophyll a and microbial utilization of carbon sources at three locations associated with each estuary: (1) inside estuary mouth, (2) estuary swash and (3) control swash (an open beach distant from any estuarine influences). Sampling occurred one week before and at one and nine weeks after both an artificial mouth opening and a separate natural flood at both estuaries. Significant temporal changes were detected for all three variables at the estuary mouth and estuary swash but the direction of change was inconsistent across the two estuaries and between the artificial mouth opening and natural flood. Organic matter in both estuaries showed no difference after the artificial mouth openings. Only Anglesea showed an increase in organic matter in the estuary mouth and estuary swash after the floods. Microphytobenthic chlorophyll a concentrations were highest when the estuary mouths were closed. Concentrations decreased at all locations at Curdies after the mouth was artificially opened. The estuary mouth at Anglesea sustained high chlorophyll concentrations and the estuary swash increased one week post artificial opening. The flood event resulted in an increase in chlorophyll a at the estuary mouth and swash at both estuaries, one week post flood. At Curdies, the microbial utilization of different carbon sources changed after both mouth events; estuary mouth and estuary swash showed similar patterns at one and nine weeks post opening. At Anglesea, the bacteria utilized different carbon sources between locations and the only significant interaction between location and time was post flood with change in carbon sources utilized by bacteria in the estuary mouth and estuary swash for one and nine weeks post flood. The southern coastline of Australia is characterized by estuaries with small catchments. This study highlights the spatial and temporal variability in the effects of the output of relatively small, intermittent estuaries on coastal sediment of adjacent beaches, particularly during prolonged periods of drought.     

Evidence for estrogenic endocrine disruption in an offshore flatfish, the dab (Limanda limanda L.)

Dab (Limanda limanda) caught in UK offshore waters show evidence of being exposed to estrogenic endocrine disrupters at a relatively low level. Two of 449 males caught between June and July 2005 had markedly elevated levels of vitellogenin (VTG; 21 and 750 microg/ml) and the remainder ranged from <0.01 to 8.6 microg/ml. Omitting the two outliers, there was a very significant positive relationship with the mass of individual males (a feature noted in previous studies on cod). Mean VTG concentrations in males differed significantly between sites. The site with the highest mean (1.6 microg/ml) was North East of the Dogger Bank and the site with the lowest (0.04 microg/ml) was in Cardigan Bay. Mean VTG concentrations in all North Sea fish were significantly higher than English Channel and Irish Sea fish, but this difference disappeared when fish mass was taken into account. VTG concentrations showed no relationship to water depth, stage of sexual maturity or age of the males. Sixty selected male plasmas were assayed for 17beta-estradiol but only two had measurable amounts (assay limit 0.04 ng/ml). Despite being the start of summer, the gonads of many of the males and females (especially those caught in the North Sea) showed signs of sexual maturity (presence of sperm in males and vitellogenic eggs in females). Many females had high VTG concentrations (up to 14 mg/ml) and 78 out of 80 had measurable concentrations of 17beta-estradiol. The cause of elevated VTG levels in male dab is unknown. As seen in cod, the presence of affected males does not appear to be linked to proximity to land or to known point sources of endocrine disrupters. However, our data, showing that larger fish are more likely to have elevated VTG concentrations, suggests a gradual accumulation by marine fish, probably through feeding, of persistent (probably relatively weak) estrogenic compounds.     

Effects of artificial openings of intermittently opening estuaries on macroinvertebrate assemblages of the entrance barrier

Intermittently opening estuaries are artificially opened to manage flood risk, water quality, recreational amenity, and fisheries; however, the ecological impacts of this management technique are incompletely understood. During 2001 and 2004, this study assessed the impacts of artificial openings on the macroinvertebrates of entrance barriers of intermittently opening estuaries in New South Wales (Australia). In 2001 macroinvertebrates were sampled once before artificial opening and 9 and 25 d after re-formation of the entrance barrier. A multiple before–after-control-impact analysis found that, although entrance barriers were destroyed by the artificial openings and then re-formed naturally by wave action, significant interactions for taxonomic richness, density of the amphipod Paracalliope australis (Gammaridae) and density of the gastropod mollusc Aschoris victoriae (Hydrobiidae) meant that the effects of this disturbance could not be distinguished from the natural variations that occurred in unopened estuaries. Multivariate analyses found that assemblages at both opened and unopened estuaries changed from before to after the openings, and the magnitude of the dissimilarity between times varied between estuaries. In 2004, macroinvertebrates were sampled on three randomly selected days within each of three periods (before, 3 d and 42 d after) at one opened and three unopened estuaries. Asymmetrical analysis of this modified before–after-control-impact study found that the change in taxonomic richness at the opened estuary from before to after opening did not differ from temporal changes that occurred in unopened estuaries. Short-term variation (i.e. between days) in total density of macroinvertebrates and density of P. australis in the re-formed entrance barrier of the opened estuary also did not differ from the variation in the control estuaries. Additionally, assemblage structure was not significantly changed by the opening and assemblages at two control estuaries were also unchanged over the same time. Individual taxa and assemblages of macroinvertebrates in entrance barriers of these intermittently open estuarine systems appear to be resilient to the habitat disturbance caused by artificial openings.     

Introduced Pacific oysters Crassostrea gigas in South Africa: demographic change,genetic diversity and body condition

Successful aquaculture species are often chosen for their fast growth rates and fecundity, which are also characteristics of invasive species. The Pacific oyster Crassostrea gigas, which constitutes 80% of global oyster trade, has been confirmed as invasive in 17 of the 66 countries where it is cultured. The single study of its status in South Africa reported populations in six South Coast estuaries in 2001, dropping to three sites in 2003. We resurveyed these estuaries, visited others in the Eastern and Western Cape provinces, and sampled oyster tissue for molecular analyses of population structure. Pacific oysters have disappeared from Knysna and, following our collections, possibly also from the GouKou Estuary. Between 2003 and 2012, the Breede Estuary population decreased by 87%, from an estimated 184 206 to 23 760 individuals. Within this estuary, oysters 12 km upriver had denser shells and higher body condition indices than did those within 1.4 km of the river mouth, presumably reflecting higher availability of suspended organic matter. However, low salinity over most of the species’ range in the estuary probably inhibits recruitment. New populations of Pacific oysters in the Swartkops and Kaaimans estuaries urgently require monitoring and eradication. Haplotype (h) and nucleotide (π) diversities across all oyster populations sampled (h = 0.2300 [SD 0.0595], π = 0.0006 [SD 0.0007]) were lower than those of co-occurring indigenous Cape rock oysters Striostrea margaritacea from the GouKou and Breede estuaries (h = 0.9076 [SD 0.0386], π = 0.00589 [SD 0.00347]). Pacific oysters either have been introduced to South African estuaries infrequently, or have experienced genetic bottlenecks following river floods or human exploitation, or both. Populations growing outside culture infrastructure are restricted to estuaries in the Eastern and Western Cape provinces, with no evidence of occurrence in fully marine shelf environments. Given the species’ considerable socio-economic importance, estuarine and coastal surveillance coupled with aquaculture zoning are required to integrate biodiversity and food security considerations.     

Elevated serum vitellogenin levels and gonadal abnormalities in wild male flounder (Pleuronectes yokohamae) from Tokyo Bay, Japan.

Concentrations of vitellogenin (VTG) in serum of wild male flounder (Pleuronectes yokohamae) were investigated, as a biomarker for environmental estrogens in marine waters, from January 1997 to May 1998 in Tokyo Bay, Japan (n = 130) and from a reference site in Hokkaido, Japan, far from urban areas (about 40 km from Hakodate city) from March 1997 to June 1998 (n = 62). Statistically higher concentrations of serum VTG were detected in wild male flounder collected in Tokyo Bay (range: 25-2200 ng ml-1) compared to fish from Hokkaido (range: 31-91 ng ml-1) throughout the entire sampling period. Three out of 20 males collected off Haneda, in the inner part of Tokyo Bay, showed testes containing small numbers of oocytes. This intersex condition was not found among males collected from the reference site. This study suggests that the abnormally high levels of VTG and the presence of oocytes in the testis of male flounder collected from Tokyo Bay could be the effect of environmental estrogens.