Evaluation of cataract formation in fish exposed to environmental radiation at Chernobyl and Fukushima.

Recent studies apparently finding deleterious effects of radiation exposure on cataract formation in birds and voles living near Chernobyl represent a major challenge to current radiation protection regulations. This study conducted an integrated assessment of radiation exposure on cataractogenesis using the most advanced technologies available to assess the cataract status of lenses extracted from fish caught at both Chernobyl in Ukraine and Fukushima in Japan. It was hypothesised that these novel data would reveal positive correlations between radiation dose and early indicators of cataract formation. The structure, function and optical properties of lenses were analysed from atomic to millimetre length scales. We measured the short-range order of the lens crystallin proteins using Small Angle X-Ray Scattering (SAXS) at both the SPring-8 and DIAMOND synchrotrons, the profile of the graded refractive index generated by these proteins, the epithelial cell density and organisation and finally the focal length of each lens. The results showed no evidence of a difference between the focal length, the epithelial cell densities, the refractive indices, the interference functions and the short-range order of crystallin proteins (X-ray diffraction patterns) in lens from fish exposed to different radiation doses. It could be argued that animals in the natural environment which developed cataract would be more likely, for example, to suffer predation leading to survivor bias. But the cross-length scale study presented here, by evaluating small scale molecular and cellular changes in the lens (pre-cataract formation) significantly mitigates against this issue.

Wide-scope screening of multi-class contaminants in seafood using a novel sample preparation (QuEChUP) procedure coupled with UHPLC-Q-TOF-MS: Application for semi-quantitation of real seafood samples.

A high-resolution mass spectrometry screening method was developed and validated based on EU SANTE/11312/2021 guidelines for the analysis of 850 multi-class contaminants in commercial seafood samples. Samples were extracted using a novel sequential QuEChUP preparation method that combines the QuEChERS and QuPPe procedures. The screening detection limits (SDLs) and limits of identification (LOIs) were equal to or lower than 0.01 mg·kg-1 for 92% and 78% of contaminants, respectively. This screening procedure was ultimately applied for a target screening analysis of 24 seafood samples. The concentrations of identified contaminants were assessed using semi-quantitative approach. Two identified contaminants, diuron and diclofenac, showed the highest estimated average concentrations: 0.076 and 0.068 mg·kg-1 respectively in mussel samples. Suspect screening was also performed. Target and suspect screening led to the identification of mixtures of contaminants (pesticides, veterinary products, industrial chemicals and personal care products) and the assessment of their frequencies of appearance (FoA).

Approaches to determine pesticides in marine bivalves.

Due to agricultural runoff, pesticides end up in aquatic ecosystems and some accumulate in marine bivalves. As filter feeders, bivalves can accumulate high concentrations of chemicals in their tissue representing a potential risk to the health of human and aquatic ecosystems. So far, most of the studies dealing with pesticide contamination in marine bivalves, for example, in the French Atlantic and English Channel coasts, have focused on the old generation of pesticides. Only a few investigated the newly emerging pesticides partly due to methodological challenges. A better understanding of the most sensitive and reliable methods is thus essential for accurately determining a wide variety of environmentally relevant pesticides in marine bivalves. The review highlighted the use of more environmentally friendly and efficient materials such as sorbents and the "quick easy cheap effective rugged safe" extraction procedure to extract pesticides from bivalve matrices, as they appeared to be the most efficient while being the safest. Moreover, this method combined with the high-resolution mass spectrometry (MS) technique offers promising perspectives by highlighting a wide range of pesticides including those that are not usually sought. Finally, recent developments in the field of ultra-high-performance liquid chromatography coupled to MS, such as two-dimensional chromatography and ion mobility spectrometry, will improve the analysis of pesticides in complex matrices.

Seasonal variations of low pesticides contamination and biomarker responses in marine bivalves from French estuaries.

Biomarkers involved in detoxification process (GST), oxidative stress (SOD and MDA), immune response (Laccase) and neurotoxic disorders (AChE) were analysed in Pacific oysters and blue mussels collected from 4 locations within the Pertuis sea (France). Seasonal variations of total pesticide mean concentrations were found in seawater with metolachlor being the main pesticide measured (up to 32 ng/L). The majority of pesticide concentrations in sediment were below the LOD. Seasonal contamination differences were evidenced for chlortoluron, especially in mussels where concentrations reached 16 ng/g (wet weight) during the winter, in the Charente estuary, but no relationships with any of the biomarkers selected arisen. Actually, low concentrations of alpha-, beta-BHC and alachlor were correlated to GST activity, and low levels of hexachlorobenzene were linked to the AChE activity and MDA content in oysters. In mussels, low concentrations of methylparathion, parathion and beta-BHC were correlated to laccase.

Spatio-temporal contamination of microplastics in shellfish farming regions: A case study.

The seasonal pattern of microplastics (MPs) contamination of the French littoral area of the Pertuis Charentais, one of the main French shellfish production regions, was assessed for the first time, between May 2019 and May 2020 at four different sites. The reference site was located at "Ile de Ré" and the other sites were located in the estuaries of the Sèvre Niortaise, Charente and Seudre rivers. Both blue mussels (Mytilus edulis) and Pacific oysters (Magallana gigas), that are considered sentinel species for the quality of the marine environment were analysed, along with sediment and seawater samples. MPs were extracted from each sample, counted, measured and sorted by colour and type. Micro-Raman spectroscopy was used to determine the proportion of confirmed MPs and the polymer types. The results showed that the contamination of mussels by fibres and fragments (1.9 ± 2.1 MPs/g ww) was significantly higher than for oysters (0.4 ± 0.4 MPs/g ww). Specifically, the contamination by fibres in both species was significantly greater than the contamination by fragments. Significant variations of MPs contamination were observed across the seasons and sites in bivalves, and depended on the species and the type of MPs (fibres or fragments). Mean concentrations of MPs measured in water and sediment were 0.007 MPs/L and 210 MPs/kg dw, respectively. Finally, blue was the dominant colour for fibres (79 %) and fragments (81 %). Blue fragments were mainly made of PS (70 %) followed by PC (18 %) and PP, PA or PLA (3 %) whereas blue fibres were mainly made of PA (80 %) followed by PET (13 %) or PP (7 %). This rare environmental case study of long-term chronic exposure of farming areas to MPs provides new knowledge on in situ variations of plastic fibres and fragments contamination throughout the seasons.

Development and validation according to the SANTE guidelines of a QuEChERS-UHPLC-QTOF-MS method for the screening of 204 pesticides in bivalves.

A qualitative screening high resolution mass spectrometry method was developed and validated according to the EU SANTE/12682/2019 guidelines for the analysis of 204 pesticides in seven commercial bivalve species spiked at three concentrations (0.01, 0.05 and, 0.1 mg.kg-1). Samples were extracted using QuEChERS and analysed using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. The QuEChERS method was optimised by the Taguchi Orthogonal Array approach. The best conditions were obtained with pure ACN, MgSO4/NaCl as extraction salts, MgSO4/PSA/C18 as clean-up, and the non-dilution of extracts. The impact of different HRMS acquisition modes on detection and identification rates were also evaluated. The screening detection limits were determined to be 0.01 mg.kg-1 and 0.1 for 66% and 87% of pesticides, respectively. These screening procedure was finally applied to different bivalve samples using target and suspect analysis. This allowed the identification of diuron and its metabolite 1-(3,4-dichlorophenyl)-3-methylurea in the investigated samples.

Pesticides, nonylphenols and polybrominated diphenyl ethers in marine bivalves from France: A pilot study.

The present pilot study aimed to provide an overview of organic contaminant concentration levels in the littoral ecosystems of the Pertuis seas. The study determined the concentrations of twenty-nine pesticides, six nonylphenols and seven polybrominated diphenyl ethers (PBDEs) in sediments, seawater, Pacific oysters and blue mussels. Oysters accumulated a higher number of pesticides than blue mussels. Indeed, alpha BHC (0.60-0.72 ng/g, ww), chlorfenvinphos (1.65-2.12 ng/g, ww), chlorpyrifos (0.79-0.93 ng/g, ww), chlortoluron (2.50-4.31 ng/g, ww), metolachlor (up to 0.38 ng/g, ww) and parathion (0.56-0.69 ng/g, ww) were quantified in oysters whereas only alpha BHC (0.24-0.31 ng/g, ww), was quantified in mussels. The present results also revealed that the POPs detected in water or sediments were not ultimately found accumulated in bivalves. Other molecules such as methylparathion and BDE47 were quantified in sediments. These molecules, BDE99 and one nonylphenol (OP2OE) were quantified in seawater. Finally, the comparison with the available environmental guidelines showed that the values measured were at concentrations not considered to cause adverse effects at the populations' level except for chlortoluron in seawater (15-50 ng/L).

Susceptibility of polar cod (Boreogadus saida) to a model carcinogen.

Studies that aim to characterise the susceptibility of the ecologically relevant and non-model fish polar cod (Boreogadus saida) to model carcinogens are required. Polar cod were exposed under laboratory conditions for six months to control, 0.03 µg BaP/g fish/week and 0.3 µg BaP/g fish/week dietary benzo(a)pyrene (BaP), a reference carcinogen. The concentrations of the 3-OH-BaP bile metabolite and transcriptional responses of genes involved in DNA adduct recognition (xpc), helicase activity (xpd), DNA repair (xpf, rad51) and tumour suppression (tp53) were assessed after 0, 1, 3 and 6 months of exposure, alongside body condition indexes (gonadosomatic index, hepatosomatic index and condition factor). Micronuclei and nuclear abnormalities in blood and spleen, and liver histopathological endpoints were assessed at the end of the experiment. Fish grew steadily over the whole experiment and no mortality was recorded. The concentrations of 3-OH-BaP increased significantly after 1 month of exposure to the highest BaP concentration and after 6 months of exposure to all BaP concentrations showing the biotransformation of the mother compound. Nevertheless, no significant induction of gene transcripts involved in DNA damage repair or tumour suppression were observed at the selected sampling times. These results together with the absence of chromosomal damage in blood and spleen cells, the subtle increase in nuclear abnormalities observed in spleen cells and the low occurrence of foci of cellular alteration suggested that the exposure was below the threshold of observable effects. Taken together, the results showed that polar cod was not susceptible to carcinogenesis using the BaP exposure regime employed herein.

Linking pollution and cancer in aquatic environments: A review.

Due to the interconnectedness of aquatic ecosystems through the highly effective marine and atmospheric transport routes, all aquatic ecosystems are potentially vulnerable to pollution. Whilst links between pollution and increased mortality of wild animals have now been firmly established, the next steps should be to focus on specific physiological pathways and pathologies that link pollution to wildlife health deterioration. One of the pollution-induced pathologies that should be at the centre of attention in ecological and evolutionary research is cancer, as anthropogenic contamination has resulted in a rapid increase of oncogenic substances in natural habitats. Whilst wildlife cancer research is an emerging research topic, systematic reviews of the many case studies published over the recent decades are scarce. This research direction would (1) provide a better understanding of the physiological mechanisms connecting anthropogenic pollution to oncogenic processes in non-model organisms (reducing the current bias towards human and lab-animal studies in cancer research), and (2) allow us to better predict the vulnerability of different wild populations to oncogenic contamination. This article combines the information available within the scientific literature about cancer occurrences in aquatic and semi-aquatic species. For the first aim, we use available knowledge from aquatic species to suggest physiological mechanisms that link pollution and cancer, including main metabolic detoxification pathways, oxidative damage effects, infections, and changes to the microbiome. For the second aim, we determine which types of aquatic animals are more vulnerable to pollution-induced cancer, which types of pollution are mainly associated with cancer in aquatic ecosystems, and which types of cancer pollution causes. We also discuss the role of migration in exposing aquatic and semi-aquatic animals to different oncogenic pollutants. Finally, we suggest novel research avenues, including experimental approaches, analysis of the effects of pollutant cocktails and long-term chronic exposure to lower levels of pollutants, and the use of already published databases of gene expression levels in animals from differently polluted habitats.

Transcriptional Changes in the Ovaries of Perch from Chernobyl.

Fish have been highly exposed to radiation in freshwater systems after the Chernobyl Nuclear Power Plant (NPP) accident in 1986 and in freshwater and marine systems after the more recent Fukushima NPP accident in 2011. In the years after the accident, the radioactivity levels rapidly declined due to radioactive decay and environmental processes, but chronic lower dose exposures persisted. To gain insights into the long-term effects of environmental low dose radiation on fish ovaries development, a high-throughput transcriptomic approach including a de novo assembly was applied to different gonad phenotypes of female perch: developed gonads from reference lakes, developed/irradiated from medium contaminated lake, and both developed/irradiated and undeveloped from more highly contaminated lakes. This is the most comprehensive analysis to date of the gene responses in wildlife reproductive system to radiation. Some gene responses that were modulated in irradiated gonads were found to be involved in biological processes including cell differentiation and proliferation (ggnb2, mod5, rergl), cytoskeleton organization (k1C18, mtpn), gonad development (nell2, tcp4), lipid metabolism (ldah, at11b, nltp), reproduction (cyb5, cyp17A, ovos), DNA damage repair (wdhd1, rad51, hus1), and epigenetic mechanisms (dmap1). Identification of these genes provides a better understanding of the underlying molecular mechanisms underpinning the development of the gonad phenotypes of wild perch and how fish may respond to chronic exposure to radiation in their natural environment, though causal attribution of gene responses remains unclear in the undeveloped gonads.

Subtle effects of radiation on embryo development of the 3-spined stickleback.

The Chernobyl and Fukushima nuclear power plant (NPP) accidents that occurred in 1986 and 2011 respectively have led to many years of chronic radiation exposure of wildlife. However, controversies remain on the dose threshold above which an impact on animal health occurs. Fish have been highly exposed immediately after both accidents in freshwater systems around Chernobyl and in freshwater and marine systems around Fukushima. The dose levels decreased during the years after the accidents, however, little is known about the effects of environmental low doses of radiation on fish health. The present laboratory study assesses the effects of an environmentally relevant dose range of radiation (0.1, 1 and 10 mGy/day) on early life stages of the 3-spined stickleback, Gasterosteus aculeatus. The cardiac physiology and developmental features (head width, diameter, area) of high exposed embryos (10 mGy/day) showed no significant change when compared to controls. Embryos exposed to the medium and high dose were slower to hatch than the controls (between 166 and 195 h post-fertilization). After 10 days of exposure (at 240 h post-fertilization), larvae exposed to the high dose displayed comparable growth to controls. High-throughput sequence analysis of transcriptional changes at this time point revealed no significant changes in gene regulation compared to controls regardless of exposure conditions. Our results suggest that exposure of fish embryos to environmental radiation elicits subtle delays in hatching times, but does not impair the overall growth and physiology, nor the gene expression patterns in the recently hatched larvae.

Chronic radiation exposure at Chernobyl shows no effect on genetic diversity in the freshwater crustacean, Asellus aquaticus thirty years on.

Analysis of genetic diversity represents a fundamental component of ecological risk assessments in contaminated environments. Many studies have assessed the genetic implications of chronic radiation exposure at Chernobyl, generally recording an elevated genetic diversity and mutation rate in rodents, plants, and birds inhabiting contaminated areas. Only limited studies have considered genetic diversity in aquatic biota at Chernobyl, despite the large number of freshwater systems where elevated dose rates will persist for many years. Consequently, the present study aimed to assess the effects of chronic radiation exposure on genetic diversity in the freshwater crustacean, Asellus aquaticus, using a genome-wide SNP approach (Genotyping-by-sequencing). It was hypothesized that genetic diversity in A. aquaticus would be positively correlated with dose rate. A. aquaticus was collected from six lakes in Belarus and the Ukraine ranging in dose rate from 0.064 to 27.1 µGy/hr. Genotyping-by-sequencing analysis was performed on 74 individuals. A significant relationship between geographical distance and genetic differentiation confirmed the Isolation-by-Distance model. Conversely, no significant relationship between dose rate and genetic differentiation suggested no effect of the contamination gradient on genetic differentiation between populations. No significant relationship between five measures of genetic diversity and dose rate was recorded, suggesting that radiation exposure has not significantly influenced genetic diversity in A. aquaticus at Chernobyl. This is the first study to adopt a genome-wide SNP approach to assess the impacts of environmental radiation exposure on biota. These findings are fundamental to understanding the long-term success of aquatic populations in contaminated environments at Chernobyl and Fukushima.

Impact of Environmental Radiation on the Health and Reproductive Status of Fish from Chernobyl.

Aquatic organisms at Chernobyl have now been chronically exposed to environmental radiation for three decades. The biological effects of acute exposure to radiation are relatively well documented, but much less is known about the long-term effects of chronic exposure of organisms in their natural environment. Highly exposed fish in freshwater systems at Chernobyl showed morphological changes in their reproductive system in the years after the accident. However, the relatively limited scope of past studies did not allow robust conclusions to be drawn. Moreover, the level of the radiation dose at which significant effects on wildlife occur is still under debate. In the most comprehensive evaluation of the effects of chronic radiation on wild fish populations to date, the present study measures specific activities of 137Cs, 90Sr, and transuranium elements (238Pu, 239,240Pu, and 241Am), index conditions, distribution and size of oocytes, as well as environmental and biological confounding factors in two fish species perch ( Perca fluviatilis) and roach ( Rutilus rutilus) from seven lakes. In addition, relative species abundance was examined. The results showed that both fish species are, perhaps surprisingly, in good general physiological and reproductive health. Perch, however, appeared to be more sensitive to radiation than roach: in the most contaminated lakes, a delay of the maturation of the gonads and the presence of several undeveloped phenotypes were evident only for perch and not for roach.

Hepatocellular adenoma in a European flatfish (Limanda limanda): Genetic alterations in laser-capture micro-dissected tissue and global transcriptomic approach.

Liver tumours in flatfish have been diagnosed using histopathology for decades to monitor the impacts of marine pollution. Here we describe the application of specific gene (retinoblastoma, Rb) profiling in laser capture micro-dissected samples, and a suppression subtractive hybridization (SSH) approach to isolate differentially expressed genes in hepatocellular adenoma (HCA) samples from dab, Limanda limanda. The Rb profiles from apparently normal and HCA micro-dissected samples of fish from the North Sea showed no significant difference, and genotypic heterogeneity within defined histological phenotypes was observed. In the SSH, sequences associated with cell signalling, cell cycle, gene expression regulation, protein transport and protein degradation were isolated. These included up-regulation of arrestin domain containing 3 (arrdc3), Rac-1 and tribbles, and down-regulation of ankyrin repeat/sterile alpha-motif domain-containing protein 1B-like (ANKS1B-like), c-fos, CDKN1B and RhoA-like sequences, previously implicated in mammalian HCA. This study offers new candidates involved in fish liver tumour development.

An optimized method for cryogenic storage of Xenopus sperm to maximise the effectiveness of research using genetically altered frogs.

Cryogenic storage of sperm from genetically altered Xenopus improves cost effectiveness and animal welfare associated with their use in research; currently it is routine for X. tropicalis but not reliable for X. laevis. Here we compare directly the three published protocols for Xenopus sperm freeze-thaw and determine whether sperm storage temperature, method of testes maceration and delays in the freezing protocols affect successful fertilisation and embryo development in X. laevis. We conclude that the protocol is robust and that the variability observed in fertilisation rates is due to differences between individuals. We show that the embryos made from the frozen-thawed sperm are normal and that the adults they develop into are reproductively indistinguishable from others in the colony. This opens the way for using cryopreserved sperm to distribute dominant genetically altered (GA) lines, potentially saving travel-induced stress to the male frogs, reducing their numbers used and making Xenopus experiments more cost effective.

Danio rerio ABC transporter genes abcb3 and abcb7 play a protecting role against metal contamination.

ATP-binding cassette (ABC) proteins are efflux transporters and some of them are involved in xenobiotic detoxification. The involvement of four zebrafish ABC transporters in cadmium, zinc and mercury detoxification was characterized in a metal hypersensitive mutant of Escherichia coli. The E. coli tolC mutant expressing ABCB3 or ABCB7 transporters exhibited higher survival ratios and lower metal accumulation under a metal exposure condition than the controls. For instance, in the presence of 8 and 10 µM of HgCl2 , the survival ratios of bacteria expressing ABCB3 were four and six-times higher than the control whereas the mercury concentrations were 2.5 and 2-times lower than in the control. This work provides new data on the function of zebrafish ABCB3 and ABCB7 transporters and highlights their significance in metal detoxification. Copyright © 2016 John Wiley & Sons, Ltd.

Molecular changes in skin pigmented lesions of the coral trout Plectropomus leopardus.

A high prevalence of skin pigmented lesions of 15% was recently reported in coral trout Plectropomus leopardus, a commercially important marine fish, inhabiting the Great Barrier Reef. Herein, fish were sampled at two offshore sites, characterised by high and low lesion prevalence. A transcriptomic approach using the suppressive subtractive hybridisation (SSH) method was used to analyse the differentially expressed genes between lesion and normal skin samples. Transcriptional changes of 14 genes were observed in lesion samples relative to normal skin samples. These targeted genes encoded for specific proteins which are involved in general cell function but also in different stages disrupted during the tumourigenesis process of other organisms, such as cell cycling, cell proliferation, skeletal organisation and cell migration. The results highlight transcripts that are associated with the lesion occurrence, contributing to a better understanding of the molecular aetiology of this coral trout skin disease.

Exposure to low pH induces molecular level changes in the marine worm, Platynereis dumerilii.

Fossil fuel emissions and changes in net land use lead to an increase in atmospheric CO2 concentration and a subsequent decrease of ocean pH. Noticeable effects on organisms' calcification rate, shell structure and energy metabolism have been reported in the literature. To date, little is known about the molecular mechanisms altered under low pH exposure, especially in non-calcifying organisms. We used a suppression subtractive hybridisation (SSH) approach to characterise differentially expressed genes isolated from Platynereis dumerilii, a non-calcifying marine polychaeta species, kept at normal and low pH conditions. Several gene sequences have been identified as differentially regulated. These are involved in processes previously considered as indicators of environment change, such as energy metabolism (NADH dehydrogenase, 2-oxoglutarate dehydrogenase, cytochrome c oxidase and ATP synthase subunit F), while others are involved in cytoskeleton function (paramyosin and calponin) and immune defence (fucolectin-1 and paneth cell-specific alpha-defensin) processes. This is the first study of differential gene expression in a non-calcifying, marine polychaete exposed to low pH seawater conditions and suggests that mechanisms of impact may include additional pathways not previously identified as impacted by low pH in other species.

The biological effects of ionising radiation on Crustaceans: A review.

Historic approaches to radiation protection are founded on the conjecture that measures to safeguard humans are adequate to protect non-human organisms. This view is disparate with other toxicants wherein well-developed frameworks exist to minimise exposure of biota. Significant data gaps for many organisms, coupled with high profile nuclear incidents such as Chernobyl and Fukushima, have prompted the re-evaluation of our approach toward environmental radioprotection. Elucidating the impacts of radiation on biota has been identified as priority area for future research within both scientific and regulatory communities. The crustaceans are ubiquitous in aquatic ecosystems, comprising greater than 66,000 species of ecological and commercial importance. This paper aims to assess the available literature of radiation-induced effects within this subphylum and identify knowledge gaps. A literature search was conducted pertaining to radiation effects on four endpoints as stipulated by a number of regulatory bodies: mortality, morbidity, reproduction and mutation. A major finding of this review was the paucity of data regarding the effects of environmentally relevant radiation doses on crustacean biology. Extremely few studies utilising chronic exposure durations or wild populations were found across all four endpoints. The dose levels at which effects occur was found to vary by orders of magnitude thus presenting difficulties in developing phyla-specific benchmark values and reference levels for radioprotection. Based on the limited data, mutation was found to be the most sensitive endpoint of radiation exposure, with mortality the least sensitive. Current phyla-specific dose levels and limits proposed by major regulatory bodies were found to be inadequate to protect species across a range of endpoints including morbidity, mutation and reproduction and examples are discussed within. These findings serve to prioritise areas for future research that will significantly advance understanding of radiation-induced effects in aquatic invertebrates and consequently enhance ability to predict the impacts of radioactive releases on the environment.

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.