Material-specific binding peptides empower sustainable innovations in plant health, biocatalysis, medicine and microplastic quantification.

Material-binding peptides (MBPs) have emerged as a diverse and innovation-enabling class of peptides in applications such as plant-/human health, immobilization of catalysts, bioactive coatings, accelerated polymer degradation and analytics for micro-/nanoplastics quantification. Progress has been fuelled by recent advancements in protein engineering methodologies and advances in computational and analytical methodologies, which allow the design of, for instance, material-specific MBPs with fine-tuned binding strength for numerous demands in material science applications. A genetic or chemical conjugation of second (biological, chemical or physical property-changing) functionality to MBPs empowers the design of advanced (hybrid) materials, bioactive coatings and analytical tools. In this review, we provide a comprehensive overview comprising naturally occurring MBPs and their function in nature, binding properties of short man-made MBPs (<20 amino acids) mainly obtained from phage-display libraries, and medium-sized binding peptides (20-100 amino acids) that have been reported to bind to metals, polymers or other industrially produced materials. The goal of this review is to provide an in-depth understanding of molecular interactions between materials and material-specific binding peptides, and thereby empower the use of MBPs in material science applications. Protein engineering methodologies and selected examples to tailor MBPs toward applications in agriculture with a focus on plant health, biocatalysis, medicine and environmental monitoring serve as examples of the transformative power of MBPs for various industrial applications. An emphasis will be given to MBPs' role in detecting and quantifying microplastics in high throughput, distinguishing microplastics from other environmental particles, and thereby assisting to close an analytical gap in food safety and monitoring of environmental plastic pollution. In essence, this review aims to provide an overview among researchers from diverse disciplines in respect to material-(specific) binding of MBPs, protein engineering methodologies to tailor their properties to application demands, re-engineering for material science applications using MBPs, and thereby inspire researchers to employ MBPs in their research.

Towards a reference material for microplastics' number concentration-case study of PET in water using Raman microspectroscopy.

Increasing demand for size-resolved identification and quantification of microplastic particles in drinking water and environmental samples requires the adequate validation of methods and techniques that can be used for this purpose. In turn, the feasibility of such validation depends on the existence of suitable certified reference materials (CRM). A new candidate reference material (RM), consisting of polyethylene terephthalate (PET) particles and a water matrix, has been developed. Here, we examine its suitability with respect to a homogeneous and stable microplastic particle number concentration across its individual units. A measurement series employing tailor-made software for automated counting and analysis of particles (TUM-ParticleTyper 2) coupled with Raman microspectroscopy showed evidence of the candidate RM homogeneity with a relative standard deviation of 12% of PET particle counts involving particle sizes >30 µm. Both the total particle count and the respective sums within distinct size classes were comparable in all selected candidate RM units. We demonstrate the feasibility of production of a reference material that is sufficiently homogeneous and stable with respect to the particle number concentration.

Sublethal effects induced by different plastic nano-sized particles in Daphnia magna at environmentally relevant concentrations.

A growing number of studies have reported the toxic effects of nanoplastics (NPs) on organisms. However, the focus of these studies has almost exclusively been on the use of polystyrene (PS) nanospheres. Herein, we aim to evaluate the sublethal effects on Daphnia magna juveniles of three different NP polymers: PS-NPs with an average size of 200 nm, polyethylene [PE] NPs and polyvinyl chloride [PVC] NPs with a size distribution between 50 and 350 nm and a comparable mean size. For each polymer, five environmentally relevant concentrations were tested (from 2.5 to 250 µg/L) for an exposure time of 48 h. NP effects were assessed at the biochemical level by investigating the amount of reactive oxygen species (ROS) and the activity of the antioxidant enzyme catalase (CAT) and at the behavioral level by evaluating the swimming behavior (distance moved). Our results highlight that exposure to PVC-NPs can have sublethal effects on Daphnia magna at the biochemical and behavioral levels. The potential role of particle size on the measured effects cannot be excluded as PVC and PE showed a wider size range distribution than PS, with particles displaying sizes from 50 to 350 nm. However, we infer that the chemical structure of PVC, which differs from that of PE of the same range size, concurs to explain the observed effects. Consequently, as PS seems not to be the most hazardous polymer, we suggest that the use of data on PS toxicity alone can lead to an underestimation of NP hazards.

Microplastics in the bogue, Boops boops: A snapshot of the past from the southern Tyrrhenian Sea.

The present investigation focuses on Boops boops specimens gathered in the Gulf of Patti in 2010. Providing a snapshot from the past, this paper represents, chronologically, the first record of microplastic ingestion in the Mediterranean bogue. The plastic abundance and composition in gastrointestinal tracts of the bogue was assess, in order to improve the knowledge on spatial-temporal variability of microplastics pollution in the Mediterranean basin and in particular, in the southern Tyrrhenian Sea. In a total of 65 specimens, 180 particles of plastic (2.8 items/specimens), mainly belonging to microplastics class, were found. Fragments (63%) and fibres (30%) were the predominant shape categories. Eleven polymers were identified: polypropylene and polyethylene were the most abundant. Several synthetic polymers belonging to the class of elastomers were also observed. The study area is strongly influenced by the absence of trawl fishing activities and a low mixing level of the seabed that, together with the confluence of different watercourses and the presence of different kind of anthropic impact, including motorway, could make it a 'waste disposal site'. Finally, our results suggest the usefulness to retrieve older samples to better understand spatial-temporal changes in marine litter pollution over time.

Screening of suspected micro(nano)plastics in the Ebro Delta (Mediterranean Sea).

This is the first work reporting the use of a double suspect-screening to assess most common polymers and additives in micro(nano)plastics (NPLs/MPLs) found in environmental waters. The method consisted of water filtration followed by ultrasonic-assisted extraction with toluene and analysis employing size exclusion chromatography using an advanced polymer chromatography column coupled to high-resolution mass spectrometry with an atmospheric pressure photoionisation source by negative ionisation conditions (LC(APC)-APPI(-)-HRMS). The identification of NPL/MPLs polymers has been based on increasing confirmation level, including the monomers characterisation by the Kendrick Mass Defect and confirmation and quantification when standards were available. In parallel, the identification of main additives in NPL/MPLs composition, as well organic contaminants adsorbed onto the plastic particles were carried out by analysis of the extracts by LC(C18)-APPI (+/-)-HRMS. To assess the impact of plastic pollution it is necessary to assess the composition in terms of polymers but also the additives. This screening approach has been employed to study composition of NPL/MPLs in the Ebro Delta. Two sampling campaigns including freshwater and seawater samples have been investigated to assess plastic composition in the top 5 cm. Polystyrene (PS), polyethylene (PE), polyisoprene (PI), polybutadiene (PBD), polypropylene (PP) and polysiloxanes were the most detected polymers and PP and PE, sizing between < 1000 and 2000 Da, were found at concentrations reaching up to 7000 ng/L in some areas. The pentadecanoic acid, 1,2,3-benzotriazoles, 2-ethylhexanoic acid (2-EHA), and phthalates such as dimethyl phthalate, mono(2-ethylhexyl) phthalate (MEHP) and the phthalimide were more frequently detected plastic additives. Finally, series of organic contaminants were as well detected in the particulate fraction. These organic contaminants cannot be associated to plastic compositions but can be associated to their adsorption to the particulate matter, in particular to NPL/MPLs, due to their non-polar character. Among these organic contaminants, the more frequently detected were pharmaceutical compounds, food additives and pesticides.

A new digestion approach for the extraction of microplastics from gastrointestinal tracts (GITs) of the common dolphinfish (Coryphaena hippurus) from the western Mediterranean Sea.

Plastic ingestion is one of the main impacts of marine litter on organisms. The occurrence of microplastics (MPs < 5 mm) in the stomachs of Mediterranean species was already reported in several studies. In this context, the present study aims to develop a new approach of digestion for the identification of MPs in the gastrointestinal tracts (GITs) of marine organisms. The new approach combines two digestion protocols, including potassium hydroxide (KOH) and nitric acid (HNO3), to remove most organic and inorganic materials. This digestion allows recording small MPs that are difficult to find via routinely stomach content analysis and also to minimize the overestimation of the phenomenon trough the control of airborne contamination. The new approach was tested on a voracious pelagic opportunistic predator, the common dolphinfish, a fishery resource exploited in several Mediterranean areas. The results showed that a large amount of ingested meso- and microplastics, such as fragments or sheets, was recorded in GITs (F = 65.5 %). The FTIR analysis on litter samples allowed to identify polyethylene, polypropylene and polystyrene as dominant constituent polymers of microplastics. These results confirmed that our novel combined digestion protocol represents a reliable approach to detect MPs in opportunistic pelagic predators.

Riverine anthropogenic litter load to the Mediterranean Sea near the metropolitan area of Barcelona, Spain.

Every year >4 million tonnes of plastic are estimated to enter the oceans and much of it comes from land-based sources through rivers and estuaries. To fill the lack of information related to plastic inputs from rivers, a harmonized approach based on visual observations for monitoring floating macro litter was followed in this work. We provide the results of one-year monitoring (October 2016-September 2017) in the Llobregat and El Besòs rivers, which are flowing through an industrialized and populated area nearby the city of Barcelona (Catalonia, NE Spain). Floating litter items categories were influenced by urban centres located along the rivers. Overall, similar litter composition was observed in both rivers with a prevalence of plastics, mainly related to the food and beverage sectors. Seasonal variability showed significant correlations with natural factors such as wind and rainfall. Approximately 0.4-0.6 tonnes of plastic per year were estimated to be loaded into the sea by these two Catalan rivers. This study contributes to enlarge our knowledge on anthropogenic riverine litter entering the NW Mediterranean Sea, providing a starting point for the development of further mitigation strategies.

Trace analysis of polystyrene microplastics in natural waters.

The development of quantitative and qualitative analytical methods to assess micro-plastics (MPLs) and nano-plastics (NPLs) content in the environment is a central issue for realistic risk assessment studies. However, the quantitative analysis continues being a critical issue, in particular for MPLs from 100 µm down to the nano-sized range in complex environmental samples. This paper evaluates the potential of mass spectrometry for the analysis of MPLs and NPLs. The performance of different techniques including matrix-assisted laser desorption ionisation (MALDI) coupled to time-of-flight mass spectrometry (TOF-MS), liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS), and the ambient ionisation approaches as desorption electrospray ionisation (DESI) and direct analysis real-time (DART), were assessed for the study of polystyrene (PS) MPLs and NPLs in natural waters. A method based on LC-HRMS, equipped with an atmospheric pressure photoionisation source (APPI), operated in negative conditions for the quantitative analysis of PS MPLs and NPLs in natural waters, was developed. The chromatographic separation was achieved using an advanced polymer chromatographic (APC) column using toluene isocratic as the mobile phase. The optimal analytical method showed an instrumental limit of detection (ILOD) of 20 pg and methods limits of detection and quantification around 30 pg L-1 and 100 pg L-1, respectively. And, recoveries of 60 and 70% in samples from rivers and the marine coast, respectively. The performance of the new method was proved by the analysis of fortified samples and natural seawater samples.

Adsorption of perfluoroalkyl substances on microplastics under environmental conditions.

Plastic debris has become an environmental problem during recent years. Among the plastic debris, microplastics (<5 mm; MPLs) imply an extra problem due to their capacity to enter into the fauna through ingestion. In this work, we study the capacity of three MPLs, that include high-density polyethylene (HDPE), polystyrene (PS) and polystyrene carboxylate (PS-COOH), to sorb 18 perfluoroalkyl substances (PFASs; including carboxylic acids, sulphonates and one sulphonamide) from the surrounding waters (freshwater and seawater). Conclusions drawn from the results are that perfluoro sulphonates and sulphonamides have more tendency to be sorbed onto MPLs. In addition, PS and PS-COOH have more affinity for PFASs than HDPE. Finally, the increment of conductivity and pH of the water decreases the exposure time that is necessary to reach equilibrium. However, the presence of salts decreases the tendency of PFASs to be sorbed onto plastic surfaces. These results highlight the problem associated with the presence of MPLs in inland and marine waters since toxic compounds can be sorbed onto surrounding plastics that could be ingested by aquatic fauna.

Metabolic Responses of Mytilus galloprovincialis to Fullerenes in Mesocosm Exposure Experiments.

In this study, Mediterranean mussels (Mytilus galloprovincialis) were exposed through the diet to fullerene soot at three concentrations in parallel to a control group. Their metabolomics response was assessed by high-performance liquid chromatography coupled to high-resolution mass spectrometry (HPLC-HRMS). The experiments were conducted in marine mesocosms, during 35 days (7 days of acclimatization, 21 days of exposure, and 7 days of depuration). Real conditions were emulated in terms of physicochemical conditions of the habitat. Results confirmed the bioaccumulation of fullerenes, and the metabolome of the exposed organisms revealed significant differences in the concentrations of seven free amino acids in comparison to the control group. An increase in small nonpolar amino acids (e.g., alanine) and branched chain amino acids (leucine and isoleucine) were observed. Also, glutamine concentrations decreased significantly, suggesting the activation of facultative anaerobic energy metabolism. Branched chain amino acids, such as leucine and isoleucine, followed the opposite trend after the highest level of exposure, which can imply hormesis effects. Other significant differences were observed on lipids content, such as the general increase of free fatty acids, i.e., long-chain fatty acids (lauric, myristic, and palmitic acids) when the concentration of exposure was increased. These results were consistent with hypoxia and oxidative stress.

Cytotoxic effects of commonly used nanomaterials and microplastics on cerebral and epithelial human cells.

Plastic wastes are among the major inputs of detritus into aquatic ecosystems. Also, during recent years the increasing use of new materials such as nanomaterials (NMs) in industrial and household applications has contributed to the complexity of waste mixtures in aquatic systems. The current effects and the synergism and antagonisms of mixtures of microplastics (MPLs), NMs and organic compounds on the environment and in human health have, to date, not been well understood but instead they are a cause for general concern. The aim of this work is to contribute to a better understanding of the cytotoxicity of NMs and microplastics/nanoplastics (MPLs/NPLs), at cell level in terms of oxidative stress (evaluating Reactive Oxygen Species effect) and cell viability. Firstly, the individual cytotoxicity of metal nanoparticles (NPs) (AgNPs and AuNPs), of metal oxide NPs (ZrO2NPs, CeO2NPs, TiO2NPs, and Al2O3NPs), carbon nanomaterials (C60fullerene, graphene), and MPLs of polyethylene (PE) and polystyrene (PS) has been evaluated in vitro. Two different cellular lines T98G and HeLa, cerebral and epithelial human cells, respectively, were employed. The cells were exposed during 24-48h to different levels of contaminants, from 10ng/mL to 10µg/mL, under the same conditions. Secondly, the synergistic and antagonistic relationships between fullerenes and other organic contaminants, including an organophosphate insecticide (malathion), a surfactant (sodium dodecylbenzenesulfonate) and a plasticiser (diethyl phthalate) were assessed. The obtained results confirm that oxidative stress is one of the mechanisms of cytotoxicity at cell level, as has been observed for both cell lines and contributes to the current knowledge of the effects of NMs and MPLs-NPLs.