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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Occurrence and identification of microplastics in beach sediments from the Hauts-de-France region.

The present work was carried out to quantify microplastics (MP) from three sandy beaches along the Côte d'Opale coastline located in the Hauts-de-France region of northern France. Three different study sites located along the English Channel were investigated due to different levels of anthropopression and hydrodynamic conditions. Sediments were collected at three different tide lines: high tide line (HTL), middle of the intertidal zone (IZ), and low tide line (LTL), to investigate the effects of tide line on microplastic contamination. Particles and fibers were counted and colors were recorded; polymer identification was then performed using pyrolysis-gas chromatography-mass spectrometry (Py-GC/MS). Particle and fiber abundances ranged from 23.4 ± 18.9 to 69.3 ± 30.6 items kg-1 dry weight sediment, with a trend towards fiber predominance, were observed. No difference in particle and fiber abundance was found between the different beaches and tide lines, except for Boulogne-sur-Mer, where the particle number was significantly different between tide lines. Major polymers identified were polyethylene (36.6%) and polypropylene (10.7%). This citizen science project provided preliminary data about the abundance and polymeric nature of MP along the Côte d'Opale coastline.

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.

Microplastic contamination and pollutant levels in mussels and cockles collected along the channel coasts.

Nowadays, environmental pollution by microplastics (<5 mm; MP) is a major issue. MP are contaminating marine organisms consumed by humans. This work studied MP contamination in two bivalve species of commercial interest: blue mussel (Mytilus edulis) and common cockle (Cerastoderma edule) sampled on the Channel coastlines (France). In parallel, 13 plastic additives and 27 hydrophobic organic compounds (HOC) were quantified in bivalves flesh using SBSE-TD-GS-MS/MS to explore a possible relationship between their concentrations and MP contamination levels. MP were extracted using a 10% potassium hydroxide digestion method then identified by µ-Raman spectroscopy. The proportion of contaminated bivalves by MP ranged from 34 to 58%. Blue mussels and common cockles exhibited 0.76 ±â€¯0.40 and 2.46 ±â€¯1.16 MP/individual and between 0.15 ±â€¯0.06 and 0.74 ±â€¯0.35 MP/g of tissue wet weight. Some HOC and plastic additives were detected in bivalves. However, no significant Pearson or Spearman correlation was found between MP loads and plastic additives or HOC concentrations in bivalve tissues for the two species.

Mitochondrial activity as an indicator of fish freshness.

The current methods used to routinely assess freshness in the fishing industry reflect more a state of spoilage than a state of freshness. Mitochondria, the seat of cellular respiration, undergo profound changes in post mortem tissues. The objective of this study was to demonstrate that mitochondrial activity constitutes a putative early fish freshness marker. The structure of gilthead sea bream (Sparus aurata) muscle tissue was evaluated over time by transmission electron microscopy. Respiration was assessed in mitochondria isolated from sea bream fillets using oxygraphy. Membrane potential (ΔΨm) was determined by fluorescence (Rhodamine 123). Mitochondrial activity of fillets stored at +4 °C was studied for 6 days. Changes in mitochondrial cristae structure appeared from Day 3 highlighting the presence of dense granules. ΔΨm and mitochondrial activity were significantly disrupted in sea bream fillets after 96 h of storage at +4 °C. Mitochondrial activity constituted a reliable and early indicator of fish freshness.

LF 1H NMR T2 relaxation rate as affected by water addition, NaCl and pH in fresh, frozen and cooked minced hake.

Low-Field Nuclear Magnetic Resonance of proton transverse relaxation signal (T2) was monitored in hake (Merluccius merluccius) mince with different thermal histories (fresh, frozen, cooked) as affected by pH, water and NaCl addition, and it was related to water holding capacity (WHC). Modifications of T2 signals were found in terms of changes in relaxation times and relative abundance of the relaxation components. The relaxation rate of the major component (1/T21) increased significantly upon frozen storage or pH increase, whereas water or NaCl addition had the opposite effect. WHC decreased with freezing or water addition and increased with NaCl or basic pH; thus, T2 and WHC best correlated when each factor was analysed separately. Linearity found for pH and protein concentration with 1/T21 was consistent with chemical exchange being responsible for these changes. The significance of these results for technological situations where compositional and biochemical changes are occurring is discussed.

Validation of standard method EN ISO 19343 for the detection and quantification of histamine in fish and fishery products using high-performance liquid chromatography.

In contaminated fish, bacterial decarboxylases produce histamine from histidine, thereby causing scombroid fish poisoning. European Regulation (EC) No 2073/2005 on microbiological criteria for foodstuffs requires using a fully validated, standardized reference HPLC method for detecting and quantifying histamine. After optimizing this reference method for the quantification of histamine in fish muscle, we organized an inter-laboratory study in 2013 across nine laboratories from seven European countries using defined criteria of method performance. The optimized, validated method was standardized (Standard EN ISO19343) as part of Mandate M381 from the European Commission to the European Committee for Standardization (CEN), signed in December 2010. The standard method was validated for three types of foodstuffs (fish with enzymatic maturation, fish without enzymatic maturation and fish sauce).

Use of Ratiometric Probes with a Spectrofluorometer for Bacterial Viability Measurement.

Assessment of microorganism viability is useful in many industrial fields. A large number of methods associated with the use of fluorescent probes have been developed, including fluorimetry, fluorescence microscopy, and cytometry. In this study, a microvolume spectrofluorometer was used to measure the membrane potential variations of Escherichia coli. In order to estimate the sensitivity of the device, the membrane potential of E. coli was artificially disrupted using an ionophore agent: carbonyl cyanide 3-chlorophenylhydrazone. The membrane potential was evaluated using two ratiometric methods: a Rhodamine 123/4',6-diamidino-2-phenylindole combination and a JC-10 ratiometric probe. These methods were used to study the impact of freezing on E. coli, and were compared with the conventional enumeration method. The results showed that it was beneficial to use this compact, easy-to-use, and inexpensive spectrofluorometer to assess the viability of bacterial cells via their membrane potential.

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.