Pearl Farming Micro-Nanoplastics Affect Oyster Physiology and Pearl Quality.

Pearl farming is crucial for the economy of French Polynesia. However, rearing structures contribute significantly to plastic waste, and the widespread contamination of pearl farming lagoons by microplastics has raised concerns about risks to the pearl industry. This study aimed to evaluate the effects of micro-nanoplastics (MNPs, 0.4-200 µm) on the pearl oyster (Pinctada margaritifera) over a 5-month pearl production cycle by closely mimicking ecological scenarios. MNPs were produced from weathered plastic pearl farming gear and tested at environmentally relevant concentrations (0.025 and 1 µg L-1) to decipher biological and functional responses through integrative approaches. The significant findings highlighted the impacts of MNPs on oyster physiology and pearl quality, even at remarkably low concentrations. Exposure to MNPs induced changes in energy metabolism, predominantly driven by reduced assimilation efficiency of microalgae, leading to an alteration in gene expression patterns. A distinct gene expression module exhibited a strong correlation with physiological parameters affected by MNP conditions, identifying key genes as potential environmental indicators of nutritional-MNP stress in cultured oysters. The alteration in pearl biomineralization, evidenced by thinner aragonite crystals and the presence of abnormal biomineral concretions, known as keshi pearls, raises concerns about the potential long-term impact on the Polynesian pearl industry.

Detection of ostreid herpesvirus-1 in plankton and seawater samples at an estuary scale.

Ostreid herpesvirus-1 (OsHV-1) is known to associate with particles in seawater, leading to infection and disease in the Pacific oyster Crassostrea gigas. The estuarine environment is highly complex and changeable, and this needs to be considered when collecting environmental samples for pathogen detection. The aims of this study were to (1) compare different aspects of collecting natural seawater and plankton samples for detection of OsHV-1 DNA and (2) determine whether detection of OsHV-1 DNA in such environmental samples has merit for disease risk prediction. The results of one experiment suggest that sampling on the outgoing tide may improve the detection of OsHV-1 DNA in seawater and plankton tow samples (odds ratio 2.71). This statistical comparison was not possible in 2 other experiments. The method (plankton tow or beta bottle) and depth of collection (range: 250-1250 mm) had no effect on the likelihood of detection of OsHV-1. OsHV-1 DNA was found at low concentrations in plankton tow and seawater samples, and only when outbreaks of mortality associated with OsHV-1 were observed in nearby experimental or farmed populations of C. gigas. This suggests that single point in time environmental samples of seawater or plankton are not sufficient to rule out the presence of OsHV-1 in an estuary. The association of OsHV-1 with particles in seawater needs to be better understood in order to determine whether more selective and sensitive methods can be devised to detect it, before environmental samples can be reliably used in disease risk prediction.

Oyster reproduction is affected by exposure to polystyrene microplastics.

Plastics are persistent synthetic polymers that accumulate as waste in the marine environment. Microplastic (MP) particles are derived from the breakdown of larger debris or can enter the environment as microscopic fragments. Because filter-feeder organisms ingest MP while feeding, they are likely to be impacted by MP pollution. To assess the impact of polystyrene microspheres (micro-PS) on the physiology of the Pacific oyster, adult oysters were experimentally exposed to virgin micro-PS (2 and 6 µm in diameter; 0.023 mg·L(-1)) for 2 mo during a reproductive cycle. Effects were investigated on ecophysiological parameters; cellular, transcriptomic, and proteomic responses; fecundity; and offspring development. Oysters preferentially ingested the 6-µm micro-PS over the 2-µm-diameter particles. Consumption of microalgae and absorption efficiency were significantly higher in exposed oysters, suggesting compensatory and physical effects on both digestive parameters. After 2 mo, exposed oysters had significant decreases in oocyte number (-38%), diameter (-5%), and sperm velocity (-23%). The D-larval yield and larval development of offspring derived from exposed parents decreased by 41% and 18%, respectively, compared with control offspring. Dynamic energy budget modeling, supported by transcriptomic profiles, suggested a significant shift of energy allocation from reproduction to structural growth, and elevated maintenance costs in exposed oysters, which is thought to be caused by interference with energy uptake. Molecular signatures of endocrine disruption were also revealed, but no endocrine disruptors were found in the biological samples. This study provides evidence that micro-PS cause feeding modifications and reproductive disruption in oysters, with significant impacts on offspring.

Counting the dead to determine the source and transmission of the marine herpesvirus OsHV-1 in Crassostrea gigas.

Marine herpesviruses are responsible for epizootics in economically, ecologically and culturally significant taxa. The recent emergence of microvariants of Ostreid herpesvirus 1 (OsHV-1) in Pacific oysters Crassostrea gigas has resulted in socioeconomic losses in Europe, New Zealand and Australia however, there is no information on their origin or mode of transmission. These factors need to be understood because they influence the way the disease may be prevented and controlled. Mortality data obtained from experimental populations of C. gigas during natural epizootics of OsHV-1 disease in Australia were analysed qualitatively. In addition we compared actual mortality data with those from a Reed-Frost model of direct transmission and analysed incubation periods using Sartwell's method to test for the type of epizootic, point source or propagating. We concluded that outbreaks were initiated from an unknown environmental source which is unlikely to be farmed oysters in the same estuary. While direct oyster-to-oyster transmission may occur in larger oysters if they are in close proximity (< 40 cm), it did not explain the observed epizootics, point source exposure and indirect transmission being more common and important. A conceptual model is proposed for OsHV-1 index case source and transmission, leading to endemicity with recurrent seasonal outbreaks. The findings suggest that prevention and control of OsHV-1 in C. gigas will require multiple interventions. OsHV-1 in C. gigas, which is a sedentary animal once beyond the larval stage, is an informative model when considering marine host-herpesvirus relationships.

Optimization, performance, and application of a pyrolysis-GC/MS method for the identification of microplastics.

Plastics are found to be major debris composing marine litter; microplastics (MP, < 5 mm) are found in all marine compartments. The amount of MPs tends to increase with decreasing size leading to a potential misidentification when only visual identification is performed. These last years, pyrolysis coupled with gas chromatography/mass spectrometry (Py-GC/MS) has been used to get information on the composition of polymers with some applications on MP identification. The purpose of this work was to optimize and then validate a Py-GC/MS method, determine limit of detection (LOD) for eight common polymers, and apply this method on environmental MP. Optimization on multiple GC parameters was carried out using polyethylene (PE) and polystyrene (PS) microspheres. The optimized Py-GC/MS method require a pyrolysis temperature of 700 °C, a split ratio of 5 and 300 °C as injector temperature. Performance assessment was accomplished by performing repeatability and intermediate precision tests and calculating limit of detection (LOD) for common polymers. LODs were all below 1 µg. For performance assessment, identification remains accurate despite a decrease in signal over time. A comparison between identifications performed with Raman micro spectroscopy and with Py-GC/MS was assessed. Finally, the optimized method was applied to environmental samples, including plastics isolated from sea water surface, beach sediments, and organisms collected in the marine environment. The present method is complementary to µ-Raman spectroscopy as Py-GC/MS identified pigment containing particles as plastic. Moreover, some fibers and all particles from sediment and sea surface were identified as plastic. Graphical abstract ᅟ.

Detection of ostreid herpesvirus 1 microvariant DNA in aquatic invertebrate species, sediment and other samples collected from the Georges River estuary, New South Wales, Australia.

Ostreid herpesvirus 1 microvariants (OsHV-1) present a serious threat to the Australian Crassostrea gigas industry. Of great concern is the propensity for mortality due to the virus recurring each season in farmed oysters. However, the source of the virus in recurrent outbreaks remains unclear. Reference strain ostreid herpesvirus 1 (OsHV-1 ref) and other related variants have been detected in several aquatic invertebrate species other than C. gigas in Europe, Asia and the USA. The aim of this study was to confirm the presence or absence of OsHV-1 in a range of opportunistically sampled aquatic invertebrate species inhabiting specific locations within the Georges River estuary in New South Wales, Australia. OsHV-1 DNA was detected in samples of wild C. gigas, Saccostrea glomerata, Anadara trapezia, mussels (Mytilus spp., Trichomya hirsuta), whelks (Batillaria australis or Pyrazus ebeninus) and barnacles Balanus spp. collected from several sites between October 2012 and April 2013. Viral loads in non-ostreid species were consistently low, as was the prevalence of OsHV-1 DNA detection. Viral concentrations were highest in wild C. gigas and S. glomerata; the prevalence of detectable OsHV-1 DNA in these oysters reached approximately 68 and 43%, respectively, at least once during the study. These species may be important to the transmission and/or persistence of OsHV-1 in endemically infected Australian estuaries.

Colonization of Polystyrene Microparticles by Vibrio crassostreae: Light and Electron Microscopic Investigation.

Microplastics collected at sea harbor a high diversity of microorganisms, including some Vibrio genus members, raising questions about the role of microplastics as a novel ecological niche for potentially pathogenic microorganisms. In the present study, we investigated the adhesion dynamics of Vibrio crassostreae on polystyrene microparticles (micro-PS) using electronic and fluorescence microscopy techniques. Micro-PS were incubated with bacteria in different media (Zobell culture medium and artificial seawater) with or without natural marine aggregates. The highest percentage of colonized particles (38-100%) was observed in Zobell culture medium, which may be related to nutrient availability for production of pili and exopolysaccharide adhesion structures. A longer bacterial attachment (6 days) was observed on irregular micro-PS compared to smooth particles (<10 h), but complete decolonization of all particles eventually occurred. The presence of natural marine agreggates around micro-PS led to substantial and perennial colonization featuring monospecific biofilms at the surface of the aggregates. These exploratory results suggest that V. crassostreae may be a secondary colonizer of micro-PS, requiring a multispecies community to form a durable adhesion phenotype. Temporal assessment of microbial colonization on microplastics at sea using imaging and omics approaches are further indicated to better understand the microplastics colonization dynamics and species assemblages.

Resistance of Black-lip learl oyster, Pinctada margaritifera, to infection by Ostreid herpes virus 1µvar under experimental challenge may be mediated by humoral antiviral activity.

Ostreid herpesvirus 1 (OsHV-1) has induced mass mortalities of the larvae and spat of Pacific oysters, Crassostrea gigas, in Europe and, more recently, in Oceania. The production of pearls from the Black-lip pearl oyster, Pinctada margaritifera, represents the second largest source of income to the economies of French Polynesia and many Pacific Island nations that could be severely compromised in the event of a disease outbreak. Coincidentally with the occurrence of OsHV-1 in the southern hemisphere, C. gigas imported from New Zealand and France into French Polynesia tested positive for OsHV-1. Although interspecies viral transmission has been demonstrated, the transmissibility of OsHV-1 to P. margaritifera is unknown. We investigated the susceptibility of juvenile P. margaritifera to OsHV-1 µvar that were injected with tissue homogenates sourced from either naturally infected or healthy C. gigas. The infection challenge lasted 14 days post-injection (dpi) with sampling at 0, 1, 2, 3, 5, 7 and 14 days. Mortality rate, viral prevalence, and cellular immune responses in experimental animals were determined. Tissues were screened by light microscopy and TEM. Pacific oysters were also challenged and used as a positive control to validate the efficiency of OsHV-1 µvar infection. Viral particles and features such as marginated chromatin and highly electron dense nuclei were observed in C. gigas but not in P. margaritifera. Mortality rates and hemocyte immune parameters, including phagocytosis and respiratory burst, were similar between challenged and control P. margaritifera. Herpesvirus-inhibiting activity was demonstrated in the acellular fraction of the hemolymph from P. margaritifera, suggesting that the humoral immunity is critical in the defence against herpesvirus in pearl oysters. Overall, these results suggest that under the conditions of the experimental challenge, P. margaritifera was not sensitive to OsHV-1 µvar and was not an effective host/carrier. The nature and spectrum of activity of the humoral antiviral activity is worthy of further investigation.

Experimental infections of Pacific oyster Crassostrea gigas using the Australian ostreid herpesvirus-1 (OsHV-1) µVar strain.

In Australia, the spread of the ostreid herpesvirus-1 microvariant (OsHV-1 µVar) threatens the Pacific oyster industry. There is an urgent need to develop an experimental infection model in order to study the pathogenesis of the virus under controlled laboratory conditions. The present study constitutes the first attempt to use archived frozen oysters as a source of inoculum, based on the Australian OsHV-1 µVar strain. Experiments were conducted to test (1) virus infectivity, (2) the dose-response relationship for OsHV-1, and (3) the best conditions in which to store infective viral inoculum. Intramuscular injection of a viral inoculum consistently led to an onset of mortality 48 h post-injection and a final cumulative mortality exceeding 90%, in association with high viral loads (1 × 105 to 3 × 107 copies of virus mg-1) in dead individuals. For the first time, an infective inoculum was produced from frozen oysters (tissues stored at -80°C for 6 mo). Storage of purified viral inoculum at +4°C for 3 mo provided similar results to use of fresh inoculum, whereas storage at -20°C, -80°C and room temperature was detrimental to infectivity. A dose-response relationship for OsHV-1 was identified but further research is recommended to determine the most appropriate viral concentration for development of infection models that would be used for different purposes. Overall, this work highlights the best practices and potential issues that may occur in the development of a reproducible and transferable infection model for studying the pathogenicity of the Australian OsHV-1 strain in Crassostrea gigas under experimental conditions.

Spatial distribution of mortality in Pacific oysters Crassostrea gigas: reflection on mechanisms of OsHV-1 transmission.

The ostreid herpesvirus OsHV-1 has the potential to devastate Pacific oyster Crassostrea gigas culture in Australia as it has done in many other countries, highlighting the need for a better understanding of disease expression and transmission. The aim of this study was to assess the spatial distribution of OsHV-1-associated mortalities in one of only two infected areas in Australia, Woolooware Bay (Botany Bay, New South Wales). In October 2011, healthy sentinel Pacific oysters were placed in 3 different locations at 3 different tidal levels, and OsHV-1 associated mortalities were closely monitored over 7 mo. The outbreak started in November 2011, and the disease remained active until April 2012. Three major mortality events were detected. Rather than being a propagating epizootic, it appeared that most oysters were infected from a common environmental source. The distribution of OsHV-1-associated mortalities was spatially clustered, highly variable and clearly dependent on the age of oysters and their position in the water column. Non-random distribution of mortalities at macro scale (sites several km apart) and micro scale (within rearing trays), and vertical clustering patterns in the water column are discussed in regard to factors known to influence mechanism of disease transmission in aquatic environments (hydrodynamics, physical disturbances, host density/distribution, and variations of environmental parameters). A new hypothesis proposing that OsHV-1 may be carried through water by particles, possibly plankton, is also suggested to explain the patchy distribution of mortalities in Woolooware Bay.

Additives in polypropylene and polylactic acid food packaging: Chemical analysis and bioassays provide complementary tools for risk assessment.

Plastic food packaging represents 40 % of the plastic production worldwide and belongs to the 10 most commonly found items in aquatic environments. They are characterized by high additives contents with >4000 formulations available on the market. Thus they can release their constitutive chemicals (i.e. additives) into the surrounding environment, contributing to chemical pollution in aquatic systems and to contamination of marine organism up to the point of questioning the health of the consumer. In this context, the chemical and toxicological profiles of two types of polypropylene (PP) and polylactic acid (PLA) food packaging were investigated, using in vitro bioassays and target gas chromatography mass spectrometry analyses. Plastic additives quantification was performed both on the raw materials, and on the material leachates after 5 days of lixiviation in filtered natural seawater. The results showed that all samples (raw materials and leachates) contained additive compounds (e.g. phthalates plasticizers, phosphorous flame retardants, antioxidants and UV-stabilizers). Differences in the number and concentration of additives between polymers and suppliers were also pointed out, indicating that the chemical signature cannot be generalized to a polymer and is rather product dependent. Nevertheless, no significant toxic effects was observed upon exposure to the leachates in two short-term bioassays targeting baseline toxicity (Microtox® test) and Pacific oyster Crassostrea gigas fertilization success and embryo-larval development. Overall, this study demonstrates that both petrochemical and bio-based food containers contain harmful additives and that it is not possible to predict material toxicity solely based on chemical analysis. Additionally, it highlights the complexity to assess and comprehend the additive content of plastic packaging due to the variability of their composition, suggesting that more transparency in polymer formulations is required to properly address the risk associated with such materials during their use and end of life.

Diversity and molecular epidemiology of Ostreid herpesvirus 1 in farmed Crassostrea gigas in Australia: Geographic clusters and implications for "microvariants" in global mortality events.

Since 2010, mass mortality events known as Pacific oyster mortality syndrome (POMS) have occurred in Crassostrea gigas in Australia associated with Ostreid herpesvirus 1. The virus was thought to be an OsHV-1 µVar or "microvariant", i.e. one of the dominant variants associated with POMS in Europe, but there are few data to characterize the genotype in Australia. Consequently, the genetic identity and diversity of the virus was determined to understand the epidemiology of the disease in Australia. Samples were analysed from diseased C. gigas over five summer seasons between 2011 and 2016 in POMS-affected estuaries: Georges River in New South Wales (NSW), Hawkesbury River (NSW) and Pitt Water in Tasmania. Sequencing was attempted for six genomic regions. Numerous variants were identified among these regions (n = 100 isolates) while twelve variants were identified from concatenated nucleotide sequences (n = 61 isolates). Nucleotide diversity of the seven genotypes of C region among Australian isolates (Pi 0.99 × 10-3) was the lowest globally. All Australian isolates grouped in a cluster distinct from other OsHV-1 isolates worldwide. This is the first report that Australian outbreaks of POMS were associated with OsHV-1 distinct from OsHV-1 reference genotype, µVar and other microvariants from other countries. The findings illustrate that microvariants are not the only variants of OsHV-1 associated with mass mortality events in C. gigas. In addition, there was mutually exclusive spatial clustering of viral genomic and amino acid sequence variants between estuaries, and a possible association between genotype/amino acid sequence and the prevalence and severity of POMS, as this differed between these estuaries. The sequencing findings supported prior epidemiological evidence for environmental reservoirs of OsHV-1 for POMS outbreaks in Australia.

On the horns of a dilemma: Evaluation of synthetic and natural textile microfibre effects on the physiology of the pacific oyster Crassostrea gigas.

Fast fashion and our daily use of fibrous materials cause a massive release of microfibres (MF) into the oceans. Although MF pollution is commonly linked to plastics, the vast majority of collected MF are made from natural materials (e.g. cellulose). We investigated the effects of 96-h exposure to natural (wool, cotton, organic cotton) and synthetic (acrylic, nylon, polyester) textile MF and their associated chemical additives on the capacity of Pacific oysters Crassostrea gigas to ingest MF and the effects of MF and their leachates on key molecular and cellular endpoints. Digestive and glycolytic enzyme activities and immune and detoxification responses were determined at cellular (haemocyte viability, ROS production, ABC pump activity) and molecular (Ikb1, Ikb2, caspase 1 and EcSOD expression) levels, considering environmentally relevant (10 MF L-1) and worst-case scenarios (10 000 MF L-1). Ingestion of natural MF perturbed oyster digestive and immune functions, but synthetic MF had few effects, supposedly related with fibers weaving rather than the material itself. No concentration effects were found, suggesting that an environmental dose of MF is sufficient to trigger these responses. Leachate exposure had minimal effects on oyster physiology. These results suggest that the manufacture of the fibres and their characteristics could be the major factors of MF toxicity and stress the need to consider both natural and synthetic particles and their leachates to thoroughly evaluate the impact of anthropogenic debris. Environmental Implication. Microfibres (MF) are omnipresent in the world oceans with around 2 million tons released every year, resulting in their ingestion by a wide array of marine organisms. In the ocean, a domination of natural MF- representing more than 80% of collected fibres-over synthetic ones was observed. Despite MF pervasiveness, research on their impact on marine organisms, is still in its infancy. The current study aims to investigate the effects of environmental concentrations of both synthetic and natural textile MF and their associated leachates on a model filter feeder.

Discussion about suitable applications for biodegradable plastics regarding their sources, uses and end of life.

This opinion paper offers a scientific view on the current debate of the place of biodegradable plastics as part of the solution to deal with the growing plastic pollution in the world's soil, aquatic, and marine compartments. Based on the current scientific literature, we focus on the current limits to prove plastic biodegradability and to assess the toxicity of commercially used biobased and biodegradable plastics in natural environments. We also discuss the relevance of biodegradable plastics for selected applications with respect to their use and end of life. In particular, we underlined that there is no universal biodegradability of plastics in any ecosystem, that considering the environment as a waste treatment system is not acceptable, and that the use of compostable plastics requires adaptation of existing organic waste collection and treatment channels.

A critical review on the evaluation of toxicity and ecological risk assessment of plastics in the marine environment.

The increasing production of plastics together with the insufficient waste management has led to massive pollution by plastic debris in the marine environment. Contrary to other known pollutants, plastic has the potential to induce three types of toxic effects: physical (e.g intestinal injuries), chemical (e.g leaching of toxic additives) and biological (e.g transfer of pathogenic microorganisms). This critical review questions our capability to give an effective ecological risk assessment, based on an ever-growing number of scientific articles in the last two decades acknowledging toxic effects at all levels of biological integration, from the molecular to the population level. Numerous biases in terms of concentration, size, shape, composition and microbial colonization revealed how toxicity and ecotoxicity tests are still not adapted to this peculiar pollutant. Suggestions to improve the relevance of plastic toxicity studies and standards are disclosed with a view to support future appropriate legislation.

Mission Tara Microplastics: a holistic set of protocols and data resources for the field investigation of plastic pollution along the land-sea continuum in Europe.

The Tara Microplastics mission was conducted for 7 months to investigate plastic pollution along nine major rivers in Europe-Thames, Elbe, Rhine, Seine, Loire, Garonne, Ebro, Rhone, and Tiber. An extensive suite of sampling protocols was applied at four to five sites on each river along a salinity gradient from the sea and the outer estuary to downstream and upstream of the first heavily populated city. Biophysicochemical parameters including salinity, temperature, irradiance, particulate matter, large and small microplastics (MPs) concentration and composition, prokaryote and microeukaryote richness, and diversity on MPs and in the surrounding waters were routinely measured onboard the French research vessel Tara or from a semi-rigid boat in shallow waters. In addition, macroplastic and microplastic concentrations and composition were determined on river banks and beaches. Finally, cages containing either pristine pieces of plastics in the form of films or granules, and others containing mussels were immersed at each sampling site, 1 month prior to sampling in order to study the metabolic activity of the plastisphere by meta-OMICS and to run toxicity tests and pollutants analyses. Here, we fully described the holistic set of protocols designed for the Mission Tara Microplastics and promoted standard procedures to achieve its ambitious goals: (1) compare traits of plastic pollution among European rivers, (2) provide a baseline of the state of plastic pollution in the Anthropocene, (3) predict their evolution in the frame of the current European initiatives, (4) shed light on the toxicological effects of plastic on aquatic life, (5) model the transport of microplastics from land towards the sea, and (6) investigate the potential impact of pathogen or invasive species rafting on drifting plastics from the land to the sea through riverine systems.

Cloning, characterization and gene expression of a metallothionein isoform in the edible cockle Cerastoderma edule after cadmium or mercury exposure.

Metallothionein (MT) genes encode crucial metal-binding proteins ubiquitously expressed in living organisms and which play important roles in homeostasis of essential metals and detoxification processes. Here, the molecular organization of the first metallothionein gene of the edible cockle Cerastoderma edule and its expression after cadmium (Cd) or mercury (Hg) exposures were determined. The resulting sequence (Cemt1) exhibits unusual features. The full length cDNA encodes a protein of 73 amino acids with nine classical Cys-X((1-3))-Cys motifs, but also one Cys-Cys not generally found in molluscan MT. Moreover, characterization of the molecular organization of the Cemt1 gene revealed two different alleles (A1 and A2) with length differences due to large deletion events in their intronic sequences involving direct Short Interspersed repeated Elements (SINE), while their exonic sequences were identical. To our knowledge, such large excision mechanisms have never before been reported in a bivalve gene sequence. After 10 days of Cd exposure at environmentally relevant doses, quantitative real-time PCR revealed a strong induction of Cemt1 in gills of C. edule. Surprisingly, neither induction of the Cemt1 gene nor of MT protein was shown after Hg exposure, despite the fact that this organism is able to bioaccumulate a high amount of this trace metal which is theoretically one of the most powerful inducers of MT biosynthesis.

Chemical effects of different types of rubber-based products on early life stages of Pacific oyster, Crassostrea gigas.

Rubber products and debris with specific chemical signatures can release their constitutive compounds into the surrounding environment. We investigated the chemical toxicity of different types of new and used rubber products (tires, crumb rubber granulates, aquaculture rubber bands) on early life stages of a model marine organism, Pacific oyster Crassostrea gigas. Leachates obtained from used products were generally less toxic than those from new ones. Leachates from new products induced embryotoxicity at different concentrations: oyster-farming rubber bands (lowest observed effect concentration, LOEC = 1 g L-1) and crumb rubber granulates (LOEC = 1 g L-1) > tires (LOEC = 10 g L-1). Moreover, new oyster-farming rubber bands induced spermiotoxicity at 10 g L-1 (-29% survival) resulting in decreased oyster reproductive output (-17% fertilization yield). Targeted chemical analyses revealed some compounds (2 mineral contaminants, 15 PAHs, 2 PCBs) in leachates, which may have played a role. Rubber used in marine aquaculture (rubber bands) or present at sea as waste (tire, crumb rubber granulates) therefore release hazardous chemical molecules under realistic conditions, which may affect oyster development. Aquaculture development work is necessary to improve practices for eco-safety, as efforts to limit the contamination of marine environments by terrestrial rubber debris.

Cryogrinding and sieving techniques as challenges towards producing controlled size range microplastics for relevant ecotoxicological tests.

The impact of microplastics (MP) has attracted much attention from the scientific community and many laboratory assessments have been made of their effects on aquatic organisms. To produce MP from real environmental plastic waste, which would enable more realistic experiments, we used plastic pearl farming equipment from French Polynesian lagoons. Here, the pearl oyster Pinctada margaritifera could encounter MP coming from their breakdown in its surrounding environment. We tested an established method based on mechanical cryogenic grinding and liquid sieving. Our desired size range was 20-60 µm, corresponding to the optimal particle size ingested by P. margaritifera. The protocol was effective, generating MP particles of 20-60 µm (∼17,000-28,000 MP µg-1), but also produced too many smaller particles. The peak in the desired size range was thus flattened by the many small particles <3 µm (∼82,000-333,000 MP µg-1; 53-70% of total analysed particles), visible at the limit of Coulter counter analysis (cut-off point: 2 µm). Laser diffraction analysis (cut-off point: 0.4 µm) provided greater detail, showing that ∼80-90% of the total analysed particles were <1 µm. Diverging particle size distributions between those expected based on sieving range and those really observed, highlight the need to perform fine-scaled particle size distribution analyses to avoid underestimating the number of small micro- and nanoplastics (MNP) and to obtain an exact estimation of the fractions produced. Size and microstructure characterization by scanning electron microscopy suggested spontaneous particle self-assembly into crystal superstructures, which is the supposed cause of the divergence we observed. Overall, our results emphasize that particle self-assembly is a technical hurdle requiring further work and highlight the specific need to finely characterize the size distribution of MNP used in ecotoxicological experiments to avoid overestimating effects.