Vertical distribution of microplastics in a river water column using an innovative sampling method.

Due to limitations of sampling methods, subsurface water is usually a less well-investigated compartment of the water column when scientists assess microplastic contamination. In this study, microplastic (MP) contamination was assessed in a freshwater river both in surface and subsurface using an innovative sampling method. Microplastic contamination in the lower part of the water column, i.e., near-bottom water and in sediments, was also studied. Three sampling campaigns were carried out during different weather conditions: stormy, rainy, and dry in order to observe their influence on the microplastics vertical distribution. No significant difference was observed between the abundance and types of MPs in surface and subsurface water. The proportion of polymer with theoretical density < 1 (polypropylene d = 0.9, polyethylene d = 0.91-0.95) and polystyrene (d = 0.1-1.06) in the surface and subsurface samples was 73.5%, and this proportion drops to 40.8% for the samples located in the near-bottom water and the sediments. Our results indicate that the MP concentration of the different compartments analyzed can be significantly influenced by rainfall during and prior to the sampling day. This study highlights that in shallow rivers, surface water sampling is representative of the water column MP contamination, but that sampling without taking environmental conditions into account may lead to erroneous estimation of MPs concentration and flux entering the marine environment.

Oral exposure to polyethylene microplastics alters gut morphology, immune response, and microbiota composition in mice.

The ubiquitous and growing presence of microplastics (MPs) in all compartments of the environment raises concerns about their possible harmful effects on human health. Human exposure to MPs occurs largely through ingestion. Polyethylene (PE) is widely employed for reusable bags and food packaging and found to be present in drinking water and food. It is also one of the major polymers detected in human stool. The aim of this study was to characterize the effects of intestinal exposure to PE MPs on gut homeostasis. Mice were orally exposed for 6 weeks to PE microbeads of 2 different sizes, 36 and 116 µm, that correspond to those found in human stool. They were administrated either individually or as a mixture at a dose of 100 µg/g of food. Both PE microbead sizes were detected in mouse stool. Different parameters related to major intestinal functions were compared between control mice, mice exposed to each type of microbead, or co-exposed to the 2 types of microbeads. Intestinal disturbances were observed after individual exposure to each size of PE microbead, and the most marked deleterious effects were found in co-exposed mice. At the histomorphological level, crypt depth was increased throughout the intestinal tissues. Significant variations of gene expression related to epithelial, permeability, and inflammatory biomarkers were quantified. Defective recruitment of some intestinal immune cells was observed from the proximal portion of the small intestine to the colon. Several bacterial taxa at the order level were found to be affected by exposure to the MPs by metagenomic analysis of cecal microbiota. These results show that ingestion of PE microbeads induces significant alterations of crucial intestinal markers in mice and underscores the need to further study the health impact of MP exposure in humans.

Histamine food poisoning: a sudden, large outbreak linked to fresh yellowfin tuna from Reunion Island, France, April 2017.

On 20 April 2017, an outbreak of histamine food poisoning occurred in a French military unit located near Paris. A total of 40 cases were identified (attack rate: 16.6%). We conducted a case-control study on 31 cases and 63 controls. Multivariate analysis pointed to cooked yellowfin tuna fillet as the very likely source of food poisoning (odds ratio = 156.8; 95% confidence interval: 18.4-1,338.4). The fresh yellowfin tuna was from Reunion Island and was supplied vacuum-sealed and packed with ice at the principal food market of Paris. No cold chain issues could be established in the upstream and downstream supply chains. Histamine concentration was found to be 1,720 mg/kg in leftover raw tuna, and 3,720 mg/kg in control cooked tuna, well above the threshold limit values defined by European regulations (200 mg/kg). The presence of Klebsiella variicola and Pantoea agglomerans, microorganisms of the Enterobacterales order that have been reported to produce histamine, was confirmed in the leftover raw tuna. This type of food poisoning is rarely recognised and confirmed. We describe the outbreak to highlight the specific key points of this type of investigation.

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.

Development of an SPME-GC-MS method for the specific quantification of dimethylamine and trimethylamine: use of a new ratio for the freshness monitoring of cod fillets.

BACKGROUND: Fish is a highly perishable food, so it is important to be able to estimate its freshness to ensure optimum quality for consumers. The present study describes the development of an SPME-GC-MS technique capable of quantifying both trimethylamine (TMA) and dimethylamine (DMA), components of what has been defined as partial volatile basic nitrogen (PVB-N). This method was used, together with other reference methods, to monitor the storage of cod fillets (Gadus morhua) conserved under melting ice. RESULTS: Careful optimisation enabled definition of the best parameters for extracting and separating targeted amines and an internal standard. The study of cod spoilage by sensory analysis and TVB-N assay led to the conclusion that the shelf-life of cod fillet was between 6 and 7 days. Throughout the study, TMA and DMA were specifically quantified by SPME-GC-MS; the first was found to be highly correlated with the values returned by steam distillation assays. Neither TMA-N nor DMA-N were able to successfully characterise the decrease in early freshness, unlike dimethylamine/trimethylamine ratio (DTR), whose evolution is closely related to the results of sensory analysis until the stage where fillets need to be rejected. CONCLUSION: DTR was proposed as a reliable indicator for the early decrease of freshness until fish rejection. © 2015 Society of Chemical Industry.

Monitoring the freshness of fish: development of a qPCR method applied to MAP chilled whiting.

BACKGROUND: Monitoring of early stages of freshness decay is a major issue for the fishery industry to guarantee the best quality for this highly perishable food matrix. Numerous techniques have been developed, but most of them have the disadvantage of being reliable only either in the last stages of fish freshness or for the analysis of whole fish. This study describes the development of a qPCR method targeting the torA gene harboured by fish spoilage microorganisms. torA encodes an enzyme that leads to the production of trimethylamine responsible for the characteristic spoiled-fish odour. RESULTS: A degenerate primer pair was designed. It amplified torA gene of both Vibrio and Photobacterium with good efficiencies on 7-log DNA dilutions. The primer pair was used during a shelf-life monitoring study achieved on modified atmosphere packed, chilled, whiting (Merlangius merlangus) fillets. The qPCR approach allows the detection of an increase of torA copies throughout the storage of fillets in correlation with the evolution of both total volatile basic nitrogen (-0.86) and trimethylamine concentrations (-0.81), known as spoilage markers. CONCLUSION: This study described a very promising, sensitive, reliable, time-effective, technique in the field of freshness characterisation of processed fish.

Microplastic leachates inhibit small-scale self-organization in mussel beds.

Self-organized spatial patterns are increasingly recognized for their contribution to ecosystem functioning. They can improve the ecosystem's ability to respond to perturbation and thus increase its resilience to environmental stress. Plastic pollution has now emerged as major threat to aquatic and terrestrial biota. Under laboratory conditions, we tested whether plastic leachates from pellets collected in the intertidal can impair small-scale, spatial self-organization and byssal threads production of intertidal mussels and whether the effect varied depending on where the pellets come from. Specifically, leachates originating from plastic pellets collected from relatively pristine and polluted areas respectively impaired and inhibited the ability of mussels to self-organize at small-scale and to produce byssal threads compared to control conditions (i.e., seawater without leaching solution). Limitations to natural self-organizing processes and threads formation may translate to a declined capacity of natural ecosystems to avoid tipping points and to a reduced restoration success of disturbed ecosystems.

Think positive: Proposal of a simple method to create reference materials in the frame of microplastics research.

In the context of the harmonization of methodologies employed to isolate and count microplastics in samples or to organize ring trials tests, the use of reference materials, i.e. samples with controlled amount of particles is required. The method proposed here uses transparent, sealed capsules containing in-house generated microplastics as a convenient way to generate microplastic reference materials. This method is a simple approach for adding particles to samples without risk of loss during particle extraction or transporting/handling.•Low-cost and easy-to-use preparation of heterogeneous mix of microplastic reference particles•Possibility to control microplastic size, shape, and polymeric composition•Applicable to many protocols and wide range of applications on water, sediments and biota.

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.

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.

WEFTA interlaboratory comparison on total lipid determination in fishery products using the Smedes method.

Lipid determination by the Smedes method was tested in an interlaboratory trial performed by nine laboratories from seven countries belonging to the West European Fish Technologists Association Analytical Methods Working Group. Five samples of fish and fishery products with different lipid contents, including two blind duplicates, were distributed among the participants. All laboratories applied a slightly modified Smedes method, which included extraction of lipids by cyclohexane and isopropanol, transfer of lipids to the cyclohexane phase by addition of water, phase separation by centrifugation, and gravimetric lipid determination. The results indicate that the RSD for reproducibility (RSD(R)) was between 4.11 and 6.31% for samples with moderate (7%) and high (14%) lipid content, depending on the sample. Larger SDs among the laboratories were obtained for a cod sample with low lipid content of 0.5%. The method is judged to be suitable as a routine method for lipid determination in fish and fishery products.

Differentiation of fresh and frozen/thawed fish, European sea bass (Dicentrarchus labrax), gilthead seabream (Sparus aurata), cod (Gadus morhua) and salmon (Salmo salar), using volatile compounds by SPME/GC/MS.

BACKGROUND: A simple method based on solid phase microextraction/gas chromatography/mass spectrometry (SPME/GC/MS) was applied for studying the volatile profiles of whole fish samples of European sea bass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata) and fillets of cod (Gadus morhua) and salmon (Salmo salar) during frozen storage in order to be able to differentiate a fresh product from one that has been frozen. Analysis of volatile compounds was performed on these two product types, fresh and after freezing/thawing following storage at - 20 °C for 30 and 90 days. RESULTS: More than a hundred volatile compounds were found by SPME/GC/MS. Statistical processing by principal component analysis and ascending hierarchical classification was used to classify the samples into categories and verify the possibility of separating fresh samples from those that had been frozen and thawed. The compounds to be used as differentiators were identified. Four compounds were common to all species: dimethyl sulfide, 3-methylbutanal, ethyl acetate and 2-methylbutanal. Not only were they found in larger quantities after thawing but they also increased with the duration of storage at - 20 °C. CONCLUSION: These four compounds can therefore be considered as potential markers of differentiation between a fresh product and one that has been frozen.

Microplastic leachates disrupt the chemotactic and chemokinetic behaviours of an ecosystem engineer (Mytilus edulis).

The massive contamination of the environment by plastics is an increasing global scientific and societal concern. Knowing whether and how these pollutants affect the behaviour of keystone species is essential to identify environmental risks effectively. Here, we focus on the effect of plastic leachates on the behavioural response of the common blue mussel Mytilus edulis, an ecosystem engineer responsible for the creation of biogenic structures that modify the environment and provide numerous ecosystem functions and services. Specifically, we assess the effect of virgin polypropylene beads on mussels' chemotactic (i.e. a directional movement in response to a chemical stimulus) and chemokinetic (i.e. a non-directional change in movement properties such as speed, distance travelled or turning frequency in response to a chemical stimulus) responses to different chemical cues (i.e. conspecifics, injured conspecifics and a predator, the crab Hemigrapsus sanguineus). In the presence of predator cues, individual mussels reduced both their gross distance and speed, changes interpreted here as an avoidance behaviour. When exposed to polypropylene leachates, mussels moved less compared to control conditions, regardless of the cues tested. Additionally, in presence of crab cues with plastic leachates, mussels significantly changed the direction of movement suggesting a leachate-induced loss of their negative chemotaxis response. Taken together, our results indicate that the behavioural response of M. edulis is cue-specific and that its anti-predator behaviour as well as its mobility are impaired when exposed to microplastic leachates, potentially affecting the functioning of the ecosystem that the species supports.

Identification and quantification of plastic additives using pyrolysis-GC/MS: A review.

Analysis of organic plastic additives (OPAs) associated to plastic polymers is growing. The current review outlines the characteristics and the development of (multi-step) pyrolysis coupled with a gas chromatography mass spectrometer (Py-GC/MS) for the identification and semi-quantification of OPAs. Compared to traditional methods, Py-GC/MS offers advantages like suppressing extensive steps of preparation, limiting contamination due to solvents and the possibility to analyse minute particles. Its key advantage is the successive analysis of OPAs and the polymeric matrix of the same sample. Based on the studied articles, numerous methods have been described allowing identification and, in some case, semi-quantification of OPAs. There is nevertheless no gold standard method, especially given the huge diversity of OPAs and the risks of interferences with polymers or other additives, but, among other parameters, a consensus temperature seems to arise from studies. More broadly, this review also explores many aspects on the sample preparation like weight and size of particles and calibration strategies. After studying the various works, some development prospects emerge and it appears that methodological developments should focus on better characterizing the limits of the methods in order to consider which OPAs can be quantified and in which polymers this is feasible.

Quantitative PCR method for evaluating freshness of whiting (Merlangius merlangus) and plaice (Pleuronectes platessa).

We have developed a method for rapid quantification of fish spoilage bacteria based on quantitative PCR with degenerated oligonucleotides that hybridize on the torA gene coding for trimethylamine N-oxide reductase, one of the major bacterial enzymes in fish spoilage. To show the utility of this gene, we used a regular PCR with DNA extracts from whiting (Merlangius merlangus) and plaice (Pleuronectes platessa) stored in ice. Quantitative PCR showed that the number of copies of the torA gene, i.e., the number of spoilage bacteria, increases with length of storage. This approach can therefore be used to evaluate freshness for the two fish species studied (whiting and plaice).

Freshness characterisation of whiting (Merlangius merlangus) using an SPME/GC/MS method and a statistical multivariate approach.

BACKGROUND: The freshness of whiting was studied at five stages of ice storage by comparing the analysis of volatile compounds obtained through solid phase microextraction/gas chromatography/mass spectrometry (SPME/GC/MS) with two sensory methods. RESULTS: Of the volatile compounds identified, 38 were analysed using a statistical multivariate approach and classified according to their role in the estimation of freshness during storage as markers of freshness or spoilage. Regarding the evolution of the presence or absence of individual compounds, three categories were defined. For example, the volatile compounds propanal, hexanal, 1-penten-3-ol, pentanal, 2,3-pentanedione, 1-penten-3-one, heptanal, (E)-2-pentenal, 2,3-octanedione, (Z)-2-penten-1-ol, 1-pentanol, butanal, octanal, 3,5,5-trimethyl-2-hexene, 1-hexanol and 4,4-dimethyl-1,3-dioxane appeared highly relevant, because they are found throughout storage and can be divided into several categories that are directly related to the quality of fish. CONCLUSION: SPME/GC/MS combined with a statistical multivariate approach may be a useful method to identify volatile compounds and characterise fish freshness during storage.

Impacts of microplastics exposure on mussel (Mytilus edulis) gut microbiota.

Microplastics (MPs), plastics with particles smaller than 5 mm, have been found almost in every corner of the world, especially in the ocean. Due to the small size, MPs can be ingested by animals and enter the marine trophic chain. MPs can affect animal health by physically causing damage to the digestive tract, leaking plastic chemical components, and carrying environmental pollutants and pathogens into animals. In this study, impacts of MPs ingestion on gut microbiota were investigated. Filter feeding mussels were exposed to "virgin" and "weathered" MPs at relatively realistic concentration 0.2 mg L-1 ("low") and exaggerated concentration 20 mg L-1 ("high") for 6 weeks. Influence in mussel gut microbiota was investigated with 16S rRNA gene high-throughput sequencing. As compared with non-exposed mussels, alteration of gut microbiota was observed after mussels were exposed to MPs for 1 week, 3 weeks, 6 weeks, and even after 8-day post-exposure depuration. Potential human pathogens were found among operational taxonomic units (OTUs) with increased abundance induced by MP-exposure. Faecal pellets containing microorganisms from altered gut microbiota and MPs might further influence microbiota of surrounding environment. Our results have demonstrated impacts of MP-exposure on mussel gut microbiota and suggested possible consequent effects on food quality, food safety, and the well-being of marine food web in the ecosystem for future studies.

An Irgafos® 168 story: When the ubiquity of an additive prevents studying its leaching from plastics.

Plastic pollution is a source of chemical to the environment and wildlife. Despite the ubiquity of plastic pollution and thus plastic additive in the environment, plastic additives have been studied to a limited extend. As a prerequisite to a study aiming to evaluate the leaching of a common additive used as an antioxidant (Irgafos® 168) from polyethylene microparticles, an inventory of the potential background contamination of the laboratory workplace was done. In this study, Irgafos® 168 (tris(2,4-ditert-butylphenyl) phosphite) and its oxidized form (tris (2,4-ditert-butylphenyl) phosphate) were quantified in different laboratory reagents, including the plastic packaging and the powders, using Pyrolysis-GC/MS. At least one form of Irgafos® 168 was detected in all tested laboratory reagents with higher concentrations in caps and bottles as compared to the powders. Additionally, oxidized Irgafos® 168 was also found in the reverse osmosed and deionized water container used in the laboratory. The same profile of contamination, i.e. higher concentration of the oxidized form and higher concentrations in acidic reagents, was observed when comparing the reagent and their respective containers suggesting that the additive is leaching from the container into the powder. Overall, this study demonstrates that the antioxidant additive Irgafos® 168 is ubiquitous in the laboratory workplace. Plastic additives such as Irgafos® 168 can therefore largely interfere and biased ecotoxicological and toxicological studies especially using environmentally relevant concentrations of microplastics. The source, fate and effects of plastic additive from plastic debris should be carefully considered in future studies that require setting up methods to overcome these contaminations.

Juvenile fish caging as a tool for assessing microplastics contamination in estuarine fish nursery grounds.

Estuaries serve as nursery grounds for many marine fish species. However, increasing human activities within estuaries and surrounding areas lead to significant habitat quality degradation for the juveniles. In recent years, plastic pollution has become a global environmental issue as plastic debris are found in all aquatic environments with potential adverse impacts on marine biota. Given the important ecological role of estuaries and implications of microplastics (MP) in ecosystems, here we assess the occurrence, number, size, and polymer types of MP ingested by wild and caged juvenile European flounder (Platichthys flesus). We deployed caged fish for 1 month at five sites in three estuaries in the eastern English Channel. The Seine estuary, heavily impacted by manmade modifications and one of the most contaminated estuaries in Europe, was compared to two smaller estuaries (Canche and Liane) less impacted by industrial activities. We found that juvenile flounders (7-9 cm) were vulnerable to plastic ingestion. Seventy-five percent of caged fish and 58% of wild caught fish had the presence of MP items in their digestive tract. Fibers (69%) dominated in the fish's digestive tract at all sites. An average of 2.04 ± 1.93 MP items were ingested by feral juvenile flounder and 1.67 ± 1.43 by caged juvenile flounder. For the caged fish, the three sites impacted by wastewater treatment plant (Liane, Le Havre harbor, and Rouen) were those with the highest percentage of individuals that has ingested MP items. Most of the isolated items were fibers and blue in color. Polymers identified by micro Raman spectroscopy were polycaprolactam, polyethylene terephthalate, and polyurethane. Although other environmental factors may have affected caged fish condition and mortality, we found no significant correlation with the number of ingested MP. However, the high occurrence of MP ingested by juvenile fish on nursery grounds raises concerns on their potential negative effects for fish recruitment success and population renewal. Finally, this study describes, for the first time, the feasibility of using caged juvenile fish as an assessing tool of MP contamination in estuarine nursery grounds.