Every step of microplastic analysis (collection, extraction and characterization) is time-consuming, representing an obstacle to the implementation of large scale monitoring. This study proposes a ...semi-automated Raman micro-spectroscopy method coupled to static image analysis that allows the screening of a large quantity of microplastic in a time-effective way with minimal machine operator intervention. The method was validated using 103 particles collected at the sea surface spiked with 7 standard plastics: morphological and chemical characterization of particles was performed in <3h. The method was then applied to a larger environmental sample (n=962 particles). The identification rate was 75% and significantly decreased as a function of particle size. Microplastics represented 71% of the identified particles and significant size differences were observed: polystyrene was mainly found in the 2–5mm range (59%), polyethylene in the 1–2mm range (40%) and polypropylene in the 0.335–1mm range (42%).
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•Semi-automated micro-spectroscopy Raman method coupled to static image analysis•Time-effective and reproducible method requiring minimum operator intervention•High particles identification rate (>70%)•Fast morphological and chemical characterization of environmental particles•Efficient method for microplastics monitoring in marine environment
Microplastics are present in marine habitats worldwide and may be ingested by low trophic organisms such as fish larvae, with uncertain physiological consequences. The present study aims at assessing ...the impact of polyethylene (PE 10–45 μM) microbeads ingestion in European sea bass (Dicentrarchus labrax) larvae. Fish were fed an inert diet including 0, 104 and 105 fluorescent microbeads per gram from 7 until 43 days post-hatching (dph). Microbeads were detected in the gastrointestinal tract in all fish fed diet incorporating PE. Our data revealed an efficient elimination of PE beads from the gut since no fluorescent was observed in the larvae after 48 h depuration. While the mortality rate increased significantly with the amount of microbeads scored per larvae at 14 and 20 dph, only ingestion of the highest concentration slightly impacted mortality rates. Larval growth and inflammatory response through Interleukine-1-beta (IL-1β) gene expression were not found to be affected while cytochrome-P450-1A1 (cyp1a1) expression level was significantly positively correlated with the number of microbeads scored per larva at 20 dph. Overall, these results suggest that ingestion of PE microbeads had limited impact on sea bass larvae possibly due to their high potential of egestion.
•First data on the effect of microplastic ingestion in marine fish larvae.•Fish larvae exhibit high potential of microplastic beads egestion.•Ingestion of microplastic beads has limited impact on traits linked to fitness.
The concentration and spatial distribution of microplastics in the Bay of Brest (Brittany, France) was investigated in two surveys. Surface water and sediment were sampled at nine locations in areas ...characterized by contrasting anthropic pressures, riverine influences or water mixing. Microplastics were categorized by their polymer type and size class. Microplastic contamination in surface water and sediment was dominated by polyethylene fragments (PE, 53–67%) followed by polypropylene (PP, 16–30%) and polystyrene (PS, 16–17%) microparticles. The presence of buoyant microplastics (PE, PP and PS) in sediment suggests the existence of physical and/or biological processes leading to vertical transfer of lightweight microplastics in the bay. In sediment (upper 5 cm), the percentage of particles identified by Raman micro-spectroscopy was lower (41%) than in surface water (79%) and may explain the apparent low concentration observed in this matrix (0.97 ± 2.08 MP kg−1 dry sediment). Mean microplastic concentration was 0.24 ± 0.35 MP m−3 in surface water. We suggest that the observed spatial MP distribution is related to proximity to urbanized areas and to hydrodynamics in the bay. A particle dispersal model was used to study the influence of hydrodynamics on surface microplastic distribution. The outputs of the model showed the presence of a transitional convergence zone in the centre of the bay during flood tide, where floating debris coming from the northern and southern parts of the bay tends to accumulate before being expelled from the bay. Further modelling work and observations integrating (i) the complex vertical motion of microplastics, and (ii) their point sources is required to better understand the fate of microplastics in such a complex coastal ecosystem.
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•Microplastics were detected throughout the Bay of Brest.•Dominance of polyethylene fragments both in surface water and sediment.•High spatial and temporal variations were observed.•Environmental pressures and hydrodynamics influenced microplastics distribution.•A convergence zone was observed despite no permanent accumulation structure.
First assessment of microplastic contamination in the Bay of Brest: buoyant polymer fragments dominated in both surface water and sediment; their distribution exhibited spatial and temporal variations related to hydrodynamic and anthropic factors.
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•Plastic particles crossing the intestinal barrier is debated.•PS-NP particles were tested in ex-vivo time series experiments using Ussing chambers.•Two PS-NP concentrations were ...tested on two locations of adult Seabass guts.•PS-NP directly crossed the intestinal barrier of adult Seabass within few minutes.•PS-NP translocation was confirmed by 3 complementary techniques.
Plastic pollution in marine ecosystems constitutes an important threat to marine life. For vertebrates, macro/microplastics can obstruct and/or transit into the airways and digestive tract whereas nanoplastics (NPs; < 1000 nm) have been observed in non-digestive tissues such as the liver and brain. Whether NPs cross the intestinal epithelium to gain access to the blood and internal organs remains controversial, however. Here, we show directly NP translocation across the intestinal barrier of a fish, the European seabass, Dicentrarchus labrax, ex vivo. The luminal side of median and distal segments of intestine were exposed to fluorescent polystyrene NPs (PS-NPs) of 50 nm diameter. PS-NPs that translocated to the serosal side were then detected quantitatively by fluorimetry, and qualitatively by scanning electron microscopy (SEM) and pyrolysis coupled to gas chromatography and high-resolution mass spectrometry (Py-GC-HRMS). Fluorescence intensity on the serosal side increased 15–90 min after PS-NP addition into the luminal side, suggesting that PS-NPs crossed the intestinal barrier; this was confirmed by both SEM and Py-GC-HRMS. This study thus evidenced conclusively that NPs beads translocate across the intestinal epithelium in this marine vertebrate.
The problematic of microplastics pollution in the marine environment is tightly linked to their colonization by a wide diversity of microorganisms, the so-called plastisphere. The composition of the ...plastisphere relies on a complex combination of multiple factors including the surrounding environment, the time of incubation along with the polymer type, making it difficult to understand how the biofilm evolves during the microplastic lifetime over the oceans. To better define bacterial community assembly processes on plastics, we performed a 5 months spatio-temporal survey of the plastisphere in an oyster farming area in the Bay of Brest (France). We deployed three types of plastic pellets in two positions in the foreshore and in the water column. Plastic-associated biofilm composition in all these conditions was monitored using 16 S rRNA metabarcoding and compared to free-living and attached bacterial members of seawater. We observed that bacterial families associated to plastic pellets were significantly distinct from the ones found in seawater, with a significant prevalence of filamentous Cyanobacteria on plastics. No convergence towards a unique plastisphere was detected between polymers exposed in the intertidal and subtidal area, emphasizing the central role of the surrounding environment on constantly shaping the plastisphere community diversity. However, we could define a bulk of early-colonizers of marine biofilms such as Alteromonas, Pseudoalteromonas or Vibrio. These early-colonizers could reach high abundances in floating microplastics collected in field-sampling studies, suggesting the plastic-associated biofilms could remain at early development stages across large oceanic scales. Our study raises the hypothesis that most members of the plastisphere, including putative pathogens, could result of opportunistic colonization processes and unlikely long-term transport.
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•Plastic pellets are enriched in Cyanobacteria as compared to seawater.•Plastisphere is constantly influenced by its surrounding environment.•Early-colonizers of plastisphere can thrive on floating microplastics.•Cyanobacteria are possibly inhibited by PVC associated chemical.•Members of Vibrio are more abundant on seawater large-particles.
•Paralytic shellfish toxins (PSTs) and bioactive extracellular compounds (BECs) of Alexandrium minutum have contrasted effects upon Crassostrea gigas oysters.•BECs affect nutrition and valve-activity ...behavior.•BECs induce hemocyte mobilization in gills.•PSTs disrupt biological rhythm.•PSTs provoke inflammation in the digestive gland.
Blooms of the dinoflagellate Alexandrium spp., known as producers of paralytic shellfish toxins (PSTs), are regularly detected on the French coastline. PSTs accumulate into harvested shellfish species, such as the Pacific oyster Crassostrea gigas, and can cause strong disorders to consumers at high doses. The impacts of Alexandrium minutum on C. gigas have often been attributed to its production of PSTs without testing separately the effects of the bioactive extracellular compounds (BECs) with allelopathic, hemolytic, cytotoxic or ichthyotoxic properties, which can also be produced by these algae. The BECs, still uncharacterized, are excreted within the environment thereby impacting not only phytoplankton, zooplankton but also marine invertebrates and fishes, without implicating any PST. The aim of this work was to compare the effects of three strains of A. minutum producing either only PSTs, only BECs, or both PSTs and BECs, on the oyster C. gigas. Behavioral and physiological responses of oysters exposed during 4 days were monitored and showed contrasted behavioral and physiological responses in oysters supposedly depending on produced bioactive substances. The non-PST extracellular-compound-producing strain primarily strongly modified valve-activity behavior of C. gigas and induced hemocyte mobilization within the gills, whereas the PST-producing strain caused inflammatory responses within the digestive gland and disrupted the daily biological rhythm of valve activity behavior. BECs may therefore have a significant harmful effect on the gills, which is one of the first organ in contact with the extracellular substances released in the water by A. minutum. Conversely, the PSTs impact the digestive gland, where they are released and mainly accumulated, after degradation of algal cells during digestion process of bivalves. This study provides a better understanding of the toxicity of A. minutum on oyster and highlights the significant role of BECs in this toxicity calling for further chemical characterization of these substances.
Oysters are keystone species that use external fertilization as a sexual mode. The gametes are planktonic and face a wide range of stressors, including plastic litter. Nanoplastics are of increasing ...concern because their size allows pronounced interactions with biological membranes, making them a potential hazard to marine life. In the present study, oyster spermatozoa were exposed for 1 h to various doses (from 0.1 to 25 µg mL
−1
) of 50-nm polystyrene beads with amine (50-NH
2
beads) or carboxyl (50-COOH beads) functions. Microscopy revealed adhesion of particles to the spermatozoa membranes, but no translocation of either particle type into cells. Nevertheless, the 50-NH
2
beads at 10 µg mL
−1
induced a high spermiotoxicity, characterized by a decrease in the percentage of motile spermatozoa (−79%) and in the velocity (−62%) compared to control spermatozoa, with an overall drop in embryogenesis success (−59%). This major reproduction failure could be linked to a homeostasis disruption in exposed spermatozoa. The 50-COOH beads hampered spermatozoa motility only when administered at 25 µg mL
−1
and caused a decrease in the percentage of motile spermatozoa (−66%) and in the velocity (−38%), but did not affect embryogenesis success. Microscopy analyses indicated these effects were probably due to physical blockages by microscale aggregates formed by the 50-COOH beads in seawater. This toxicological study emphasizes that oyster spermatozoa are a useful and sensitive model for (i) deciphering the fine interactions underpinning nanoplastic toxicity and (ii) evaluating adverse effects of plastic nanoparticles on marine biota while waiting for their concentration to be known in the environment.
Early life stages (ELS) of numerous marine invertebrates mustcope with man-made contaminants, including plastic debris, during their pelagic phase. Among the diversity of plastic particles, ...nano-sized debris, known as nanoplastics, can induce effects with severe outcomes in ELS of various biological models, including the Pacific oyster Crassostrea gigas. Here, we investigated the effects of a sub-lethal dose (0.1 µg mL
−1
) of 50 nm polystyrene nanobeads (nano-PS) with amine functions on oyster embryos (24 h exposure) and we assessed consequences on larval and adult performances over two generations of oysters. Only a few effects were observed. Lipid analyses revealed that first-generation (G1) embryos exposed to nano-PS displayed a relative increase in cardiolipin content (+9.7%), suggesting a potential modification of mitochondrial functioning. G1-larvae issued from exposed embryos showed decreases in larval growth (−9%) and lipid storage (−20%). No effect was observed at the G1 adult stage in terms of growth, ecophysiological parameters (clearance and respiration rates, absorption efficiency), or reproductive outputs (gonadic development, gamete quality). Second generation (G2) larvae issued from control G1 displayed a significant growth reduction after G2 embryonic exposure to nano-PS (−24%) compared to control (as observed at the first generation), while no intergenerational effect was detected on G2 larvae issued from G1 exposed embryos. Overall, the present experimental study suggests a low incidence of a short embryonic exposure to nano-PS on oyster phenotypes along the entire life cycle until the next larval generation.
Summer mortality associated with juveniles of the oyster
Crassostrea gigas is probably the result of a complex interaction between the host, pathogens and environmental factors. Genetic variability ...in the host appears to be a major determinant in its sensitivity to summer mortality. Previously, divergent selection criteria based on summer survival have been applied to produce oyster families with resistant and susceptible progeny. In this paper, we describe the use of suppression subtractive hybridization to generate 150
C. gigas clones that were differentially regulated between resistant and susceptible F2 progeny. The nucleotide sequence of these clones was determined. In 28%, the inferred amino sequence was found to match the products of known genes, 14% matched hypothetical proteins and a further 14% appeared to contain open reading frames (ORFs) whose product had no obvious homologue in the nucleotide databases. It has been hypothesized that differences exist in the level of energy generation and immune function between resistant and susceptible progeny. In light of this, clones encoding homologues of cavortin, cyclophilin, isocitrate dehydrogenase, sodium glucose cotransporter, fatty acid binding protein, ATPase H+ transporting lysosomal protein, precerebellin, and scavenger receptor were analyzed by real-time PCR. These transcripts were induced in resistant progeny when compared to their susceptible counterparts. A bacterial challenge of oysters resulted in the suppression of six of these transcripts in only those that were resistant to summer mortality. This study has identified potential candidates for further investigation into the functional basis of resistance and susceptibility to summer mortality.
Summer mortality of Pacific oysters is known in several countries. However no specific pathogen has been systematically associated with this phenomenon. A complex combination of environmental and ...biological parameters has been suggested as the cause and is now starting to be identified. A high genetic basis was found for survival in oysters when a first generation (G1) was tested in three sites during summer. This paper presents a synthesis on physiological characteristics of two selected groups (‘R’ and ‘S’, from families selected for resistance and susceptibility to summer mortality respectively), of the second and third generations. R and S showed improvement or reduction of survival compared with the control in both field and laboratory trials confirming the high heritability of survival of juveniles <
1 year old. Interestingly, no correlation was observed between growth and survival.
Comparison between the two selected groups showed that S oysters invested more energy in reproduction and stayed a longer time without spawning than R oysters which had high synchronous spawning. This was mainly shown with high rather than low dietary rations (respectively 12% and 4% DW algae/DW oyster) in a controlled experiment. Moreover, early partial spawning was detected in S oysters and not R ones in the high dietary ration. S showed a higher respiration rate and an earlier decrease in absorption efficiency than R during gametogenesis, but they were not significantly different in glycogen or ATP utilisation. Two months before a mortality episode, hemocytes from S oysters had a higher adhesive capacity than R hemocytes and significantly higher reactive oxygen species production capacity. One month before mortality, S oysters had the highest hyalinocyte concentration and their expression of genes coding for glucose metabolism enzymes (Hexokinase, GS, PGM, PEPCK) was significantly lower in the labial palps. After a thermal increase from 13 °C to 19 °C, during 8 days in normoxia, S oysters showed a large HSP70 increase under hypoxia contrary to R oysters, suggesting their high susceptibility to stress. Their catalase activity was lower than in R oysters and showed no further change to subsequent hypoxia and pesticide stresses, in contrast to R oysters.
These observations suggest possible links between higher reproductive effort in S oysters, their specific stress response to temperature and hypoxia, ROS production, partial spawning, hyalinocyte increase and the infection process. To compare R and S oysters in a more integrated way, a suppression subtractive hybridisation (SSH) library and a micro-array strategy are being undertaken.
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