Glass reinforced plastic (GRP) boats and the impact on coastal environment - Evidence of fibreglass ingestion by marine bivalves from natural populations.

Classified as marine debris, man made materials are polluting the world's oceans. Recently, glass reinforced plastic (GRP) has been shown to degrade and contaminate the coasts. In this pioneering study, fibreglass particles have been detected in the soft parts of oysters and mussels collected from natural populations, in front of an active boatyard. The presence of particulate glass, with concentrations up to 11,220 particles/kg ww in Ostrea edulis and 2740 particles/kg ww in Mytilus edulis, was confirmed by micro Raman spectroscopy. The results showed higher accumulation during the winter months, when boat maintenance activities are peaking and, through repair work, the release of glass fibres in the environment is more likely. Bivalves are considered high risk species due to their sessile nature and extensive filter feeding behaviour. The microparticle inclusion may contribute to adverse impacts on physiological processes and eventually to a decline in the overall health and subsequent death of the animal. The high costs involved in the proper GRP disposal and the lack of recycling facilities worldwide lead to boat abandonement and further contamination of the coasts. For the first time this study presents the extensive fibreglass contamination of natural bivalve populations, in a popular South England sailing harbour, designated a biological and geological site of specific scientific interest (SSRI).

Gonadal Atresia, Estrogen-Responsive, and Apoptosis-Specific mRNA Expression in Marine Mussels from the East China Coast: A Preliminary Study.

This preliminary survey analysed mussel atresia incidences, estrogen-responsive and apoptotic-specific molecular end points, and aqueous and gonadal levels of selected estrogens from the East China coast. Estrogen levels were low (e.g. < LOD-28.36 ng/L, < LOD-3.88 ng/g wet weight of tissue for BPA) relative to worldwide freshwater environments, but high oocyte follicle atresia incidences (up to 26.6%) occurred at selected sites. Expression of estrogen-responsive ER2 was significantly increased in males relative to females at sites with high atresia incidences in females. A second estrogen-responsive gene, V9, was significantly increased at two sites in April in females relative to males; the opposite was true for the remaining two sites. Apoptosis-specific genes (Bcl-2, fas) showed elevated expression in males relative to females at the site with the highest atresia incidence. These results provide coastal estrogen levels and the utility of several estrogen-specific molecular-level markers for marine mussels.

Induction of apoptosis in the gonads of Mytilus edulis by metformin and increased temperature, via regulation of HSP70, CASP8, BCL2 and FAS.

Pharmaceutically active compounds have been considered contaminants of emerging concern, in response to evidence that these substances may adversely affect aquatic organisms. Here we expose mussels for 7 days to metformin, the most commonly prescribed anti-diabetes treatment, at a concentration of 40 µg/L and a high temperature of 20 °C. The apoptosis-related genes HSP70, CASP8, BCL2 and FAS showed variation in expression in gonadal tissue. The results suggest that complex interactions between these genes are modulating the onset of apoptotic changes such as atresia and follicle degeneration. The temperature induced apoptosis may be initiated by overexpression of CASP8. Conversely, metformin may induce apoptosis by suppressing the anti-apoptotic gene BCL2, thus promoting the process. Interestingly, apoptosis and follicle degeneration are likely FAS-mediated, following the synergistic effect of metformin and temperature. The potential of metformin to act as a non-traditional EDC, due to its impact on the reproductive system in mussels is discussed.

Chemical characterization of variably degraded fibre glass reinforced plastic from the marine environment.

Glass reinforced plastic (GRP) constitutes the commonest component of small sea going craft of all descriptions. This study provides a baseline molecular and elemental account of GRP's recovered from the marine environment. Fourteen samples of GRP sourced from scrapyards and one sample sourced from a GRP boat manufacturer were examined. Samples were analysed by x-ray fluorescence and mid infrared (MIR). The latter technique confirmed that all samples contained the same polyester resin, poly diallyl phthalate (PDP). The two techniques in combination indicate the presence of aluminium calcium borosilicate E-glass fibres (E denotes electrical) of variable origins. MIR results are consistent with hydrolysis of polyester, weakening of the glass fibre resin interface facilitating exposure of e-type fibres to water which accelerates fibre breakage. The implication being that aging of GRP in the marine environment represent sources for micro (<5 mm) and macro plastic release, plus fragmented asbestiform-like silicate fibres.

High concentrations of paracetamol in effluent dominated waters of Jakarta Bay, Indonesia.

The occurrence of several aquatic contaminants, including pharmaceuticals, were investigated in seawater samples collected from effluent-dominated sites in Indonesia: 4 sites in Jakarta Bay and one on the north coast of Central Java. The data presented in this preliminary study provide a snapshot of seawater quality in these areas. Results show that nutrient parameters exceeded the Indonesian Standard Quality of Seawater limits, and some metals were also present. Interestingly, high concentrations of paracetamol were detected at Angke (610 ng/L) and Ancol (420 ng/L), both in Jakarta Bay. To date, this is the first study to report the presence of paracetamol (acetaminophen) in the coastal waters around Indonesia. The high concentrations detected, compared to other levels reported in scientific literature, raise concerns about the environmental risks associated with long-term exposure and, especially, the impact on nearby shellfish farms. Given pharmaceuticals' consideration as emerging contaminants, these data suggest further investigations are needed.

Long-term exposure of marine mussels to paracetamol: is time a healer or a killer?

Pharmaceuticals pose a major threat to the marine environment, and several studies have recently described their negative effects on marine organisms. Pharmaceutical compounds are constantly being released into aquatic ecosystems, and chronic exposure, even at low concentrations, may have a major impact on marine organisms. The purpose of the present study is to evaluate the biological changes induced by one of the most widely used pharmaceuticals-paracetamol-in the blue mussel Mytilus edulis, after a long-term exposure at environmentally relevant concentrations. We present our data alongside and in comparison with results from a previous short-term exposure, to demonstrate the significance of exposure period on the effects of paracetamol in adult blue mussels. After 24 days of laboratory exposure, seven potential target genes were selected to examine toxicological effects in mussels' gonads and possible disruptive effects on reproductive processes. The results show the modulation of some important reproduction-related genes: estrogen receptor-2 (ER2), vitelline envelope zona pellucida domain-9 (V9), and vitellogenin (VTG). Variations in mRNA expression of four other genes involved in apoptosis (HSP70, CASP8, BCL2, and FAS) are also highlighted. Histopathological alterations caused by paracetamol, together with neutral red retention time response in mussels' hemocytes, are presented herein. Overall, this study highlights the exacerbated effects of low concentration of paracetamol after chronic exposure, similar to the damage induced by higher concentrations in a short exposure scenario, thus emphasizing the importance of length of exposure period when studying the effects of this substance. Additionally, this study also discusses the potential of paracetamol to inflict several major changes in the reproductive system of mussels and thus possibly affect the survival of populations.

Glass reinforced plastic (GRP) a new emerging contaminant - First evidence of GRP impact on aquatic organisms.

Plastics and synthetic materials are polluting the world's oceans. In this study we exposed juvenile mussels, Mytilus edulis, to glass reinforced plastic (GRP) dust, under laboratory conditions. The study ran for a period of 7 days, to test for the morphological and potential physiological impacts of GRP. Infrared spectroscopy has revealed that the GRP resin material is poly diallyl phthalate. In mussels, particulate glass and plastics were detected in the digestive tubules and gills, with a suite of inflammatory features observed in all examined organs. In parallel, we observed the effect of powdered GRP on swimming behaviour and survival of water fleas, Daphnia magna. Polymer particles and fibreglass adhered to the filament hairs on appendages, including the caudal spine, in exposed organisms. Most importantly, swimming impairment and sinking of the animals were recorded shortly after exposure. The potential implications for severe localized impact of GRP on aquatic environment are discussed.

Effects of short-term exposure of paracetamol in the gonads of blue mussels Mytilus edulis.

A growing body of literature suggests that pharmaceutical contamination poses an increasing risk to marine ecosystems. Paracetamol or acetaminophen is the most widely used medicine in the world and has recently been detected in seawater. Here, we present the results of 7 days' exposure of blue mussel adults to 40 ng/L, 250 ng/L and 100 µg/L of paracetamol. Histopathology shows that haemocytic infiltration is the most observed condition in the exposed mussels. The mRNA expression of VTG, V9, ER2, HSP70, CASP8, BCL2 and FAS in mussel gonads present different patterns of downregulation. VTG and CASP8 mRNA expression show downregulation in all exposed mussels, irrespective of sex. The V9, HSP70, BCL2 and FAS transcripts follow a concentration-dependent variation in gene expression and may therefore be considered good biomarker candidates. ER2 mRNA expression shows a downregulated trend, with a clearer dose-response relationship in males. In conclusion, this study suggests that paracetamol has the potential to alter the expression of several genes related to processes occurring in the reproductive system and may therefore impair reproduction in blue mussels.

Intersex related gene expression profiles in clams Scrobicularia plana: Molecular markers and environmental application.

Intersex, the appearance of female characteristics in male gonads, has been identified in several aquatic species. It is a widespread phenomenon in populations of the bivalve, Scrobicularia plana, from the southwest coast of the U.K. Genes previously identified as differentially expressed (ferritin, testicular haploid expressed gene, THEG, proliferating cell nuclear antigen, PCNA; receptor activated protein kinase C, RACK; cytochrome B, CYB; and cytochrome c oxidase 1, COX1) in intersex clams relative to normal male clams, were selected for characterisation and an environmental survey of the Channel region. Transcripts were significantly differentially expressed at sites with varying intersex incidence and contaminant burdens. Significant correlations between specific gene expressions, key contaminants and sampling locations have been identified, though no single gene was associated with intersex incidence. The results highlight the difficulty in understanding the intersex phenomenon in molluscs where there is still a lack of knowledge on the control of normal reproduction.

Intersex in Scrobicularia plana: transcriptomic analysis reveals novel genes involved in endocrine disruption.

Intersex, the appearance of female characteristics in male gonads, has been identified in a wide range of aquatic species worldwide, yet the underpinning molecular etiology remains uncharacterized. The presence of intersex has been shown to be a widespread phenomenon in bivalve, S. plana, populations from the southwest coast of the U.K., as well as inducible in an experimental exposure regime using endocrine disrupting compounds (EDCs). Herein, we use the suppressive subtractive hybridization approach to isolate differentially expressed transcripts in S. plana males exhibiting intersex. Transcripts involved in cell signaling, cell cycle control, energy production/metabolism, microtubule assembly, and sperm physiology are all highlighted as differentially expressed in intersex male clams. These provide both an insight into the molecular mechanisms of action involved in the development of intersex, as well as facilitating potential molecular-level "early warning" biomarkers of the condition.

Two CYP3A-like genes in the marine mussel Mytilus edulis: mRNA expression modulation following short-term exposure to endocrine disruptors.

Members of the vertebrate CYP3A subfamily are involved in the metabolism of steroids and a wide range of xenobiotics. In this study two CYP3A-like mRNAs have been isolated from the mussel (Mytilus edulis), and their seasonal expression profile and modulation by estrogens examined. Sexual dimorphism of CYP3A-like mRNA expression was not observed in mussel gonads of individuals collected throughout a year. Nevertheless, natural variation in gonadal CYP3A-like mRNA expression was observed, with highest levels of CYP3A isoform1 and lowest levels of CYP3A isoform2 mRNA during the maturation and spawning season. Exposure to a 10% sewage treatment works extract did not result in any significant changes in mRNA expression of CYP3A-like. In contrast, exposure to E2 (200 ng/L) and TBT (100 ng/L) significantly down-regulated the expression of CYP3A-like isoform1 but not CYP3A-like isoform2 suggesting differential regulation.

Identification of reproduction-specific genes associated with maturation and estrogen exposure in a marine bivalve Mytilus edulis.

BACKGROUND: While it is established that vertebrate-like steroids, particularly estrogens (estradiol, estrone) and androgens (testosterone), are present in various tissues of molluscs, it is still unclear what role these play in reproductive endocrinology in such organisms. This is despite the significant commercial shellfishery interest in several bivalve species and their decline. METHODOLOGY/PRINCIPAL FINDINGS: Using suppression subtraction hybridisation of mussel gonad samples at two stages (early and mature) of gametogenesis and (in parallel) following controlled laboratory estrogen exposure, we isolate several differentially regulated genes including testis-specific kinases, vitelline lysin and envelope sequences. CONCLUSIONS: The differentially expressed mRNAs isolated provide evidence that mussels may be impacted by exogenous estrogen exposure.

Reference gene selection for qPCR in mussel, Mytilus edulis, during gametogenesis and exogenous estrogen exposure.

PURPOSE: The aim of this study is to develop a normalization method for real-time PCR data by analyzing the most stably expressed control genes in mussel (Mytilus edulis) reproductive tissue. METHODS: To facilitate this, six candidate genes, including several commonly used in the literature, were investigated in mussels at different stages of gametogenesis and following experimental exposure to a model estrogen (17b-estradiol). GeNorm and NormFinder softwares were employed to assess the stability of the reference genes. RESULTS: Our results demonstrate that the most stable reference genes are not the same in mussels at different stages of gametogenesis and in experimentally E2-exposed mussels. Interestingly, HEL (helicase) and ACT (actin) mRNA expression levels were most affected by the stage of gametogenesis and yet, in molluscan studies, ACT is possibly the most frequently used reference gene. CONCLUSIONS: We demonstrate that the experimental results are highly dependent on the reference gene chosen and that statistically significant contrasting differences between sample groups are present or absent depending on the reference gene employed.

Effects of estrogen exposure in mussels, Mytilus edulis, at different stages of gametogenesis.

Mytilus edulis were exposed to 17beta-estradiol (E2) and the synthetic estrogens ethinyl estradiol (EE2) and estradiol benzoate (EB) for 10 days. Two exposures were performed to determine their effect on vitellogenin (VTG) and estrogen receptor 2 (ER2) mRNA expression at different stages of the reproductive cycle. Significant natural variation was not observed in VTG mRNA expression, though ER2 mRNA expression displayed significantly lower values during January, February and July compared with other times of the year. A significant increase in VTG and ER2 mRNA expression was observed in mussels exposed to estrogens at the early stage of gametogenesis. In contrast, mature mussels displayed no statistically significant change in the VTG or ER2 mRNA expression. The data presented suggests that the reproductive physiology of molluscs, in terms of VTG and ER2 mRNA expression, may be susceptible to damage by environmental estrogens at certain points in their gametogenesis process.

Estrogens disrupt serotonin receptor and cyclooxygenase mRNA expression in the gonads of mussels (Mytilus edulis).

Estrogenic contaminants in the aquatic environment are associated with feminisation of male fish, however their effects on some invertebrate species, such as bivalve molluscs, have yet to be characterised. Gametogenesis represents a critical step in the reproductive process and is subjected to hormonal control by serotonin (5-HT), prostaglandins (synthesised by cyclooxygenases-COX) and steroids such as 17beta-estradiol (E2). Here, we examine the responses of 5-HT receptor and COX mRNA expression in mussels, Mytilus edulis, exposed to estrogenic compounds during different stages of their reproductive cycle. In mature mussels, 5-HT receptor mRNA expression decreased following E2 exposure. The opposite trend was observed in mussels at early gametogenesis stages. COX mRNA expression levels at both stages were generally decreased by E2 exposure. Mussels at early gametogenesis stages were also exposed to ethynylestradiol (EE2) and estradiol benzoate (EB) and a significant increase in 5-HT receptor mRNA expression was observed with both xeno-estrogens. COX expression levels were increased with EB exposure but no significant effects were found with EE2 exposure. These results show that the natural estrogen, E2, as well as the synthetic estrogen, EE2, induce alterations, dependent on reproductive stage, in the mRNA expression levels of 5-HT receptor and/or COX in the marine bivalve M. edulis.

The role of ras gene in the development of haemic neoplasia in Mytilus trossulus.

Disseminated neoplasia has been reported in mussels (Mytilus spp) from numerous locations worldwide. This condition is progressive and fatal and the aetiology is unknown. In vertebrates, oncogenes such as ras, and tumour suppressor genes such as p53, play important roles in carcinogenesis. We have cloned a Mytilus trossulus homologue of the vertebrate ras gene, which shows conserved sequence in regions of functional importance. Neoplastic hemolymph samples derived from M. trossulus have been investigated for the presence of ras gene mutations and changes in expression.

Conservation of cancer genes in the marine invertebrate Mytilus edulis.

Mussels are susceptible to a wide range of environmental toxicants, including carcinogens, and thus are often employed as bioindicator species. To elucidate the molecular aetiology of such neoplastic damage, we have cloned Mytilus edulis homologues of the vertebrate ras proto-oncogene, and p53 tumor suppressor gene. The M. edulis ras cDNA encodes a predicted protein of 184 amino acids. The DNA sequence analysis with vertebrate ras sequences demonstrates that the M. edulis ras cDNA is highly conserved in regions of functional importance, including mutational hot spots. The partial p53 sequence also demonstrates that M. edulis p53 is highly conserved in two regions of functional importance and that these regions also include four of the five mutational hot spots for this gene. In contrast, the M. edulis p53 sequence shows little similarity to the other published invertebrate p53-like sequences. The cancer gene sequences characterized herein will allow development of specific biomarkers of genotoxic damage.

Disseminated neoplasia in blue mussels, Mytilus galloprovincialis, from the Black Sea, Romania.

Disseminated neoplasia, also called leukemia or hemic neoplasia, has been detected in 15 species of marine bivalve mollusks worldwide. The disease is characterized by the presence of single anaplastic cells with enlarged nuclei and sometimes frequent mitosis, in hemolymph vessels and sinuses. The neoplastic cells gradually replace normal hemocytes leading to the increased mortality of animals. The neoplasia reaches epizootic prevalences in blue mussels, Mytilus trossulus, in some areas, whereas prevalences in Mytilus edulis are generally very low. Mytilus galloprovincialis was suggested to be resistant to the disease although very low prevalences were documented from Spain in the Atlantic Ocean and Italy in the Mediterranean Sea. A case of disseminated neoplasia was discovered in M. galloprovincialis from among 200 specimens studied from the coast of the Romanian Black Sea. Histological preparation revealed the presence of large anaplastic cells with lobed nuclei. This observation extends the geographic range of marine bivalve mollusks with disseminated neoplasia to include the Black Sea.

Cadmium induction of metallothionein isoforms in juvenile and adult mussel (Mytilus edulis).

Isoforms of metallothionein in the digestive gland of control and experimentally Cd-exposed mussels (Mytilus edulis) (200 microg L(-1) Cd2+ and 400 microg L(-1) Cd2+; 20 days) were studied using the reverse transcriptase-polymerase chain reaction. In addition, glutathione S-transferase (GSTpi) primers were designed to evaluate the reduction in the antioxidant defense systems (glutathione) accompanying the aging process in the same organisms. Following experimental exposure, an accumulation of Cd was observed in the digestive gland of exposed mussels, both adults and juveniles, up to 500 times higher than in the control. An induction of the dimeric form MT20 II was detected in 400 microg L(-1) exposed mussels, as well as a visible inhibition of the monomeric form MT10 IV. After 20 days of exposure juveniles expressed increased GSTpi compared with adults. Results reveal individual variation of both metallothioneins and GSTpi expression among control and Cd2+-exposed mussels of different ages. The ecotoxicological significance of MT utilization in biomonitoring of seawater for trace metals has been considered in light of these results.