A weight-of-evidence approach to assessing the ecological impact of organotin pollution in Dutch marine and brackish waters; combining risk prognosis and field monitoring using common periwinkles (Littorina littorea)

In the present study an integrated ecological risk assessment based on multiple lines of evidence (LOEs) was evaluated in order to better assess the risk from TBT in Dutch harbours and open coastal waters. On the basis of spatial distributions of measured tributyltin (TBT) concentrations in sediments and suspended matter, predictions of the intersex index (ISI) in Littorina littorea and the ecological risk expressed as the Potentially Affected Fraction (PAF) of species were made. The results were compared to actual ISI measurements and presence of L. littorea in the field. The PAF calculated on the basis of TBT levels for open coastal waters ranged from 4.2% to 15.3%; for harbours it ranged from 3.5% to 26.9%. Significant intersex levels were observed only in waters where the risk was calculated above 10% PAF. This study suggests that the absence of L. littorea from some harbours with high ecological risk values can be explained by high TBT concentrations. A call is made for the use of integrated approaches like weight-of-evidence (WOE) to help practitioners improve ecological risk assessment.     

Esterase variation in the periwinkle Littorina littorea, along the western and eastern Scheldt estuarium.

Variation in esterase expression (EST) has been investigated in 360 specimens of the periwinkle Littorina littorea collected at nine sites along the polluted western Scheldt and the relatively clean eastern Scheldt estuary. Mean number of EST bands differed among the sites, while a Dice similarity based multi-dimensional scaling along with a minimum spanning tree procedure, revealed an estuary based structuring, clustering the most polluted and least saline like sites together. The underlying factors and mechanisms (i.e. selection, regulation) which are responsible for this structuring remain to be determined.     

Endocrine disruption in a marine amphipod? Field observations of intersexuality and de-masculinisation

Intersexuality has been found in both males and females of the marine/estuarine amphipod, Echinogammarus marinus, at polluted and reference sites in East Scotland. Polluted sites had significantly more intersex specimens than reference sites, however the cause of intersexuality is unclear. Discriminant analysis of morphometric data showed that normal male specimens from the most polluted site resembled pooled intersex males, suggesting that subtle endocrine disruption (ED) maybe occurring in these otherwise apparently normal males. The main discriminating character was gnathopod size, recognised to be under androgenic gland control. The association of distinctive morphometry with intersexuality may provide a new approach to biomarkers of ED in crustaceans. The opportunities for other novel biomarkers, for example biochemical or behavioural markers, may also be explored through study of intersex animals.     

Intersex in feral marine and freshwater fish from northeastern Germany

A histopathological assessment of the gonad of male fish was performed as part of biological field studies carried out in coastal waters and small rivers in Mecklenburg-Western Pomerania, northeastern Germany. In the marine environment the eelpout (Zoarces viviparus) was selected as sentinel species. The three spined stickleback (Gasterosteus aculeatus) and perch (Perca fluviatilis) were chosen at freshwater locations. Histopathology of the testis revealed the presence of intersexuality in specimens of all three species. The intersex condition was defined by the simultaneous presence of primary oocytes within the apparently normal testis tissue. In comparison to stickleback and perch the eelpout exhibited the highest intersex prevalence and the most severe histological alterations. Fish with intersex were found at contaminated marine and freshwater stations as well as at sites with apparently little pollution. The findings suggest that feminised male fish were exposed to endocrine disrupting substances in the aquatic environment.     

Intersexuality in crustaceans: genetic, individual and population level effects

Scientists investigating toxicants such as endocrine disrupting chemicals (EDCs) at the cellular at the sub-cellular level are often faced with criticisms as to how these effects can be extrapolated to the level of individuals and their populations. This report aims to provide an overview of the studies undertaken on crustacean model, Echinogammarus marinus LEACH (AMPHIPODA), and intersex phenotypes, at the individual and population levels, and provide additional emergent data at the genomic level. These, normal and intersex, males and females have been investigated by cross-hybridisation microarray analysis and specific sexually dimorphic genes and corresponding properties identified between each sexual phenotype. The morphology, physiology and histology of these intersexes have been investigated in detail and a number of reproductive costs have been identified including reduced fecundity and fertility. These costs have been incorporated into a population model and simulated over a ten-year period to ascertain how different levels of intersexuality affect the stability of populations. Based on the information gained through study of intersex models (with known endocrine dysfunction) together with the substantial quantity of historical data relating to effects of chemicals on amphipod fecundity, growth and mortality, the development of appropriate biomarkers is nearer to being assessed from the level of genes to that of the population.     

Intersexuality incidence, sex ratio fluctuations and intersex reproductive output as factors affecting the temporal variation of intersexed populations of the marine amphipod Echinogammarus marinus

The occurrence of intersexuality in populations of the marine amphipod Echinogammarus marinus has been related to several causes, including anthropogenic contamination, microsporidian parasites and environmental sex determination (ESD). At the individual-level, the costs of intersexuality in E. marinus are reflected in reduced reproductive output of intersexes compared to normal specimens. The impacts of intersexuality and other reproductive disorders in nature will only be fully understood when individual-level effects are upscale to the population-level, which can be achieved through ecological models. The aim of the present work is to simulate the density variation of real populations of E. marinus with different intersexuality incidence, proportions of phenotypes and intersex female reproductive rates. To achieve that, we have incorporated empirical data obtained at the individual-level from three intersexed populations of E. marinus into a population dynamic model. The original model was calibrated and validated for a long-term stable population without intersexuality and sex ratio 1. In the present version, the model includes an extra population group, intersex females, but the total sum of flow rates from juveniles to adults was kept the same as in the original model to respect the previous validation. In these circumstances, results indicate that the male-biased population is very sensitive to intersexuality, even at low incidence levels, and exhibits significant density decrease if conditions do not change. On the contrary, the two populations with a preponderance of female-biased sex ratios, low or moderate intersexuality incidence (up to an average value of 9% of the population) and with intersex reproductive rates corresponding to 80% the value of normal specimens, exhibit long-term persistence.The model is highly sensitive to adult female parameters (e.g. mortality), indicating that small variations on its values can significantly change population responses.Overall the present results suggest that, in spite the costs of intersexuality in E. marinus, the temporal variation of intersexed populations depend on intersex incidence level, sex ratio fluctuations and reproductive output of intersexes. Simulations also suggest that other processes (e.g. mortality rates of normal females) may play important roles in the maintenance of populations with intersexuality. In the future, it will be crucial to improve and validate this type of models with long-term observations and monitoring of intersexed populations.     

Determining the sensitive developmental stages of intersex induction in medaka (Oryzias latipes) exposed to 17 beta-estradiol or testosterone.

Certain environmentally persistent compounds can adversely affect reproduction by acting as steroid hormone agonists or antagonists. The goal of the present study was to determine the developmental stage most susceptible to exogenous hormone (estradiol and testosterone) exposure using a small teleost model. In the first (pilot study) of two experiments, medaka (Oryzias latipes), at varying developmental stages, were bath-exposed to 5 micrograms/l 17 beta-estradiol for 24 h. At 5 months of age, fecundity, fertility and embryo and larval viability (reproductive success) were investigated in control and exposed groups. Fish at 1, 1.5, 2 and 5.5 months of age were also sampled, processed and examined histologically for gonadal alteration. No significant differences in mortality, gonadal morphology, body weight, sex-ratio or time to maturity were seen between control and exposed fish. At 5 months, however, when exposure groups were compared to controls, significant differences were seen in reproductive success and viability of offspring. A second experiment exposed embryo stage 10, and 1-, 7- and 21-day-old larvae for 6 days to 15 micrograms/l 17 beta-estradiol or 100 micrograms/l testosterone. No significant differences were seen at 5 months in mortality, body weight, or time to sexual maturity. However, sex-ratios were significantly biased toward female in the stage 10, 1- and 7-day post-hatch estradiol exposure groups. No significant changes in sex-ratio were associated with testosterone exposure at any developmental stage. Further, intersex gonads were observed in fish from all groups exposed to 15 micrograms/l estradiol. Only those fish exposed as newly hatched fry or at 1 week post-hatch displayed intersex gonads following 100 micrograms/l testosterone exposure. Data from these experiments show that newly hatched fry are that life stage most sensitive to hormone exposure and the most appropriate to use in determining effects of known endocrine-disrupting compounds.     

Flounder health status in the Seine Bay. A multibiomarker study.

The Seine Bay is used as a pilot area to assess the usefulness of monitoring programmes using a suite of biological measurements. These biomarkers included ethoxyresorfin-O-deethylase (EROD) and acetylcholinesterase (AChE) activities, multixenobiotic resistance (MXR) protein expression level assessment and gonad histopathology. Samples of European flounder collected in three sites close to the Seine Estuary in late September 1998 showed that 8% of the males were intersex, i.e. had gonads with both male and female tissues. Another 10% of individuals, identified as male by morphological observation during sampling, showed only female tissues on histological sections. These dramatic changes were associated with different patterns of EROD activity, MXR expression or AChE activity inhibition that might reflect shorter time effects of xenobiotics and constitute a starting point to integrate biological responses for the assessment of the health status of flounder in the Seine Bay.     

波纹巴非蛤性逆转时生殖腺的组织学变化

波纹巴非蛤由雌性逆转为雄性时，其生殖腺雌性滤泡内卵细胞败育，逐渐退化分解吸收。同时，分布于滤泡壁上的原始精原细胞开始生长发育。残留的雌性生殖细胞与发育的雄性生殖细胞共同存在于滤泡腔内，即为波纹巴非蛤的雌雄间性过渡阶段。随着滤泡内卵细胞的退化吸收，精细胞与雄性泡逐渐形成。波纹巴非蛤雄性个体是由雌性个体通过性反转发育而来，这种性别的转化是双方向的。此外，波纹巴非蛤雌雄同体存在两种滤泡结构类型。     

A study of Cu turnover in proteins of the visceral complex of Littorina littorea by stable isotopic analysis using coupled HPLC-ICP-MS

A two-dimensional HPLC system, tandemly coupled to an ICP-MS, has been used to study copper accumulation and turnover in the visceral complex cytosol of the gastropod, Littorina littorea. Animals were exposed for 8 weeks to NTA-buffered seawater containing stable isotopic 65Cu and then transferred to media containing stable isotopic 63Cu. The free ion activity of each isotope was maintained at 10(-11) M. Size exclusion (SE) HPLC showed Cu associated with haemocyanin (HC) and metallothionein-like (MT) proteins in two ligand pools with apparent molecular weights of >300 kDa and approximately 17 kDa, respectively. The MT pool was inducible by Cu, could assimilate the metal from both intrinsic and extrinsic sources and showed a higher rate of Cu accumulation and turnover than the HC pool. The induction of this pool also caused the sequestration and cytosolic redistribution of Zn, Cd, Pb, Mn and Co. Further fractionation of the MT pool by ion-exchange (IE) HPLC revealed that the Cu was associated with a single, major isoform of the protein that was Cu inducible and also bound trace quantities of Zn and Pb. A number of additional metal containing proteins were also resolved by IE. the most prominent of which also bound Pb, Mn and minor quantities of Zn. The significance of these findings in metal homeostasis and detoxification is discussed.     

Effect of animal orientation on acoustic estimates of zooplanktonproperties

It is well known that the behavior of zooplankton and, in particular, their orientation distribution dramatically affects the level of backscattered acoustic energy. As a result, interpretation of acoustic survey data in the ocean is subject to error. In order to quantify these effects, laboratory data from two important classes of animals were collected. The data involved broad-band (350-650 kHz) acoustic signals insonifying individual animals whose orientation was varied over the range 0°-360° in 1° increments. The animals were from two major anatomical groups: fluid-like (decapod shrimp; Palaemonetes vulgaris) and elastic-shelled (periwinkles; Littorina littorea). The data were analyzed both in the time domain (with pulse compression processing) and the frequency domain. Averages of the laboratory data over different orientation distributions illustrate the variability in average target strength that can be expected in the ocean environment. The average target strength of the shrimp varied by 3 dB when averaged over orientation distributions centered around broadside and end-on incidence. In addition, size estimates from pulse compression processing of the broad-band echoes were made for various orientation distributions for both the shrimp and periwinkles. These results show the necessity of animal orientation information for the proper interpretation of acoustic backscatter data     

Pathology of the liver, kidney and gonad of flounder (Platichthys flesus) from a UK estuary impacted by endocrine disrupting chemicals.

The Mersey estuary is highly contaminated with xenobiotics compared to the nearby Dee estuary. Male flounder, a migratory flatfish caught in the Mersey frequently contains high blood concentrations of the oestrogen controlled, female protein vitellogenin, suggesting that Mersey flounder have been exposed to endocrine disrupting contaminants. Males caught from the Dee contain lower blood vitellogenin levels. Preliminary histopathological examination of 410 flounder from both estuaries, focuses on the liver, kidney and gonads. Hepatic tubular vacuolation, foci of cellular alteration, and hepatocellular tumours were seen. Renal and gonadal pathology included the presence of two Mersey fish with enlarged and abnormal glomeruli, three phenotypic male Mersey flounder with unilateral intersex gonads and one male with bilateral, abnormal ovarian follicular components in the testis. These initial results provide pathological evidence of xenobiotic exposure in flounder sampled from both estuaries. Preliminary findings from flounder sampled from Millport, an offshore site in Southwest Scotland, showed no histopathological evidence of xenobiotic exposure.     

Food Preferences of Dominant Salt Marsh Herbivores and Detritivores

Abstract. Food preferences of six dominant salt marsh invertebrates were studied to identify detritivores and to assess differences in their diets. Animals fed on agar suspensions of dead and live foods in petri dishes with four compartments. Only two foods were included in each dish. Relative palatability was assessed by measuring the amount of suspension removed or by counting the number of feeding marks left on the surface of the suspensions.
Feeding marks reflected differences in mouth parts and feeding mechanisms of the six invertebrates. Melampus bidentatus, Orchestia grillus , and Philoscia vittata preferred dead litter over live tissues of marsh graminoids, blue-green algae, and sulfur bacteria. No preference for detritus from different graminoids was shown by M. bidentatus. Orchestia grillus fed preferentially on Spartina patens detritus. Philoscia vittata preferred detritus from S. alterniflora and S. patens. Blue-green algae and sulfur bacteria were preferred over detritus by Littorina saxatalis , but detritus was preferred over live graminoids. Littorina saxatalis fed preferentially on Juncus gerardi detritus. Blue-green algae, sulfur bacteria, and live graminoids were preferred over detritus by L. littorea. Talorchestia longicornis also preferred blue-green algae.
On the basis of their food preferences, Littorina saxatalis, Melampus bidentatus, Orchestia grillus , and Philoscia vittata were classified as detritivores. Feeding on detritus from different plant species could result in a partitioning of this food resource in the detritus-based food chains of the salt marsh ecosystem.     

Surflan and oryzalin impair reproduction in the teleost medaka (Oryzias latipes)

We conducted studies to determine if the xenoestrogens Surflan and its active ingredient oryzalin, affect indices of reproductive fitness in medaka (Oryzias latipes). Oryzalin (0.5, 0.25mg/l) or Surflan (2.0 microl/l) and oryzalin (0.5mg/l) significantly increased the mean number of non-fertilized eggs produced by treated females paired with untreated males, or by untreated females paired with treated males. Oryzalin (1.0, 0.5, 0.25mg/l) and Surflan (3.8, 2.0, 1.0 microl/l) significantly affected the time to hatch of eggs from treated females paired with untreated males, and from untreated females paired with treated males. Surflan (3.8, 2.0, 1.0 microl/l) induced intersex lesions in 80-100% of males. Oryzalin-exposed males exhibited a significant increase in the incidence of necrotic spermatids and necrotic spermatogonia, while oryzalin-exposed females had significantly fewer immature oocytes and an increase in the occurrence of hyperplastic ovaries. Our results indicate that Surflan and oryzalin affect both reproduction and gonadal histology in male and female medaka.     

Pathological reactions and recovery of hepatopancreatic digestive cells from the marine snail Littorina littorea following exposure to a polycyclic aromatic hydrocarbon

The aim of this study was to investigate the cellular pathological responses of hepatopancreatic digestive cells from the periwinkle Littorina littorea exposed to the polycyclic aromatic hydrocarbon (PAH) fluoranthene and to ascertain whether any injurious effects were reversible within the experimental time scale. A secondary objective was to establish the relationship of the various reactions to animal health status, using lysosomal stability as an index of well-being. Exposure of snails to a concentration of 335 microgl(-1) (1.7 microM) fluoranthene (seawater renewed and spiked daily with fluoranthene) for 5 days resulted in a reduction in lysosomal stability (neutral red retention) and endocytosis; and an increase in smooth endoplasmic reticulum (ER) and 7-ethoxycoumarin-o-deethylase (ECOD; measured as cyano-ECOD) activity measured in isolated live digestive cells. Exposed snails treated with clean seawater for a further 8 days resulted in a return to control levels of lysosomal stability, ECOD and ER; endocytosis showed only a partial recovery. Multi-variate and uni-variate analysis showed that there were strong correlations between the various cellular biomarker responses. These findings are interpretable within the current framework of molluscan biomarker responses to PAHs. Principal component analysis was used to derive the first principal component for endocytosis, ER and ECOD reactions and these were plotted against lysosomal stability as a measure of cellular well-being. The resulting significant regression represents the mapping of the individual biomarkers within health status space for a gradient of fluoranthene toxicity. From this analysis, we concluded that endocytosis is an indicator of healthy snails while proliferation of ER and to a lesser extent induced ECOD are indicative of dysfunction and reduced health. Finally, the results indicate that stress induced by chronic exposure to a PAH is reversible.     

The reproductive style of Diplodus capensis (Sparidae) in southern Angola: rudimentary hermaphroditism or partial protandry?

The reproductive style of Diplodus capensis was examined in an unexploited coastal area in southern Angola. The mean length of females was significantly greater than that of males and, although not significant, females dominated the older age classes. The overall sex ratio was 1 male:4.7 females and female dominance increased with size. Histological analysis of gonads revealed that this species is a rudimentary hermaphrodite, with individuals possessing a non-functional bipotential ovotes-tis in the juvenile phase. Histological examination of the gonads of four macroscopically staged ‘intersex’ individuals suggested that they were functional males with a persistent ovarian portion in their gonads from the juvenile ovotestis. The heavily female-biased sex ratio and female dominance in the larger length classes are thought to be an alternative to sex change that is more resilient to exploitation when compared with the protandrous strategy displayed by the South African population of D. capensis.     

The Effects of Hermit Crabs on Hydractiniid Hydroids The effects of hermit crabs on hydractiniid hydroids

Abstract. Hermit crab shells are often encrusted with diverse communities of epibionts. To explore the effects of hermit crabs on the interactions of these encrusting species, two species of hydractiniid hydroids, Hydractinia symbiolongicarpus and Podocoryna carnea , were examined in the presence and absence of Pagurus longicarpus hermit crabs. Colonies growing on Littorina littorea snail shells with hermit crab hosts were compared to those growing on shells without crabs. These experiments suggest that hermit crabs variably affect colony polyp number, colony morphology and the outcome of interspecific competition, according to the size and number of crabs; other factors include water temperature, food availability and hydroid clonal genotype. In order to allow image analysis of perturbations of colony morphology, hydroids were grown on glass surfaces both exposed and unexposed to hermit crabs. Relative to colonies growing on unexposed surfaces, colonies growing on exposed surfaces exhibited larger within-colony patches, thus decreasing the total encrusted surface area and polyp number of colonies. Further, on glass surfaces, hermit crabs accelerated the time to the resolution of interspecific competition ( i.e. , the overgrowth of one colony by the other). While a number of factors may contribute to the relative abundance of these encrusting species, under most circumstances P. longicarpus have large effects on hydractiniid hydroids. Mechanisms underlying these effects likely include hydrodynamics and mechanical disturbance.     

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.     

An investigation into intersex amphipods and a possible association with aquaculture

The amphipod, Echinogammarus marinus, is common in sheltered coastal inlets, such as estuaries and sea lochs, in Scotland and exhibits increased levels of intersex in some contaminated areas. Sea lochs are commonly the location for coastal aquaculture development, and some chemicals used in fish-farming are specifically designed to target the crustacean nervous system. Therefore it was hypothesised that these chemicals may also affect neuro-endocrine pathways, causing morphological and reproductive abnormalities in non-target Crustacea. Based upon this hypothesis, Echinogammarus marinus amphipods from two different Scottish sea lochs containing salmon farms were investigated. Morphology, intersexuality, and the incidence of microsporidian parasites were recorded at sites close and at distance from fish-farms. Results suggest a higher incidence of intersexuality at sites within sea lochs, comparable to that observed in industrially contaminated sites elsewhere in Scotland. The data suggest that fish farming activity may influence the observed distributions of intersexuality within lochs. Intersex specimens were more likely to be infected by microsporidian parasites than non-intersex specimens. Normal females were found more likely to be infected by microsporidian parasites at sites associated with high intersexuality, suggesting the parasite as the probable feminiser. The cause(s) for the observed patterns of intersexuality are unclear, although suggestions relating to discharges from fish farms are discussed.     

Intersexes in Mississippi River shovelnose sturgeon sampled below Saint Louis, Missouri, USA.

The Missouri Department of Conservation detected high organochlorine levels in tissues of fish from the Mississippi River south of Saint Louis. Chlordane, which is both a carcinogen and an endocrine disrupter, reached 2960 micrograms/kg in fillets of shovelnose sturgeon, Scaphirhynchus platyorynchus, and 1926 micrograms/kg in their roe, wet weight basis. Polychlorinated biphenyls (PCBs) and p,p'-dichlorodiphenyldichloroethylene (DDE) in roe have reached 5810 and 780 micrograms/kg, respectively. The purpose of this study was to determine if endocrine disrupters and carcinogens are a potential cause of the significantly diminished reproduction capacity and population declines in the endangered pallid sturgeon, Scaphirhynchus albus. Twenty-four surrogate shovelnose sturgeons were sampled north of Saint Louis where contaminant levels were lower compared with 17 sampled south of Saint Louis where contaminant levels were high. Liver and gonad tissues were formalin-fixed, histologically processed and examined. Neoplasms were not found. The principal finding was that the testes of two of the seven downstream male fish contained ovigerous lamellae, in addition to mature sperm. Since sturgeons are gonochoristic and intersexes are rare and unusual, the occurrence of intersex in 29% of the male Mississippi River shovelnose sturgeon examined from a population contaminated with chlordane and other chemicals raise concern for human health, species biodiversity and preservation of waterway quality. These findings, however, need to be corroborated by expanded studies with a higher sample size.