The geographical and seasonal effects on the composition of marine microplastic and its microbial communities: The case study of Israel and Portugal.

Introduction: Floating microplastic debris are found in most marine environments around the world. Due to their low density and high durability, plastic polymers such as polyethylene, polypropylene, and polystyrene serve as stable floating substrates for the colonization of diverse communities of marine organisms. Despite the high abundance of microplastic debris in the oceans, it is not clear how the geographical location and season affect the composition of marine microplastic and its bacterial microbiome in the natural environment. Methods: To address this question, microplastic debris were collected from the sea surface near estuaries in the Mediterranean Sea (Israel) and in the Atlantic Ocean (Portugal) during summer and winter of 2021. The microplastic physical characteristics, including shape, color, and polymer composition, were analyzed and the taxonomic structure of the microplastic bacterial microbiome was characterized using a high-resolution metabarcoding pipeline. Results: Our results, supported by previously published data, suggest that the plastisphere is a highly diverse ecosystem which is strongly shaped by spatial and temporal environmental factors. The geographical location had the highest impact on the plastisphere physical characteristics and its microbiome composition, followed by the season. Our metabarcoding analysis showed great variability between the different marine environments with a very limited microbiome "core." Discussion: This notion further emphasizes the importance of plastisphere studies in different geographical locations and/or seasons for the characterization of the plastisphere and the identification of plastic-associated species.

Interaction of Polycyclic Aromatic Hydrocarbon compounds in fish primary hepatocytes: From molecular mechanisms to genotoxic effects.

Polycyclic Aromatic Hydrocarbons (PAHs) are persistent pollutants normally found in the environment as complex mixtures. Although several individual PAHs are classified as mutagenic and carcinogenic pollutants, the interaction effects between compounds in a mixture may trigger different toxicological mechanisms and, consequently, yield different effects to organisms which are not accounted for in risk assessment guidelines. Given the ubiquity of PAHs, understanding the mechanistic features of their mixtures is a pressing research need. Therefore, the present work aimed to disclose the interaction effects of three PAHs with different carcinogenic potential and chemical structure, in primary hepatocyte cells of gilt-headed seabreams (Sparus aurata). Hepatocytes were exposed to Phenanthrene (Phe), Benzo[a]pyrene (B[a]P) and Benzo[b]fluoranthene (B[b]F) and their mixtures at different proportions and several cellular responses were analyzed: cellular viability, CYP1A1 activity (EROD assay) and protein expression level (Western blot); transcript (mRNA) levels of CYP1A1, EPXH1 and GST-3 (qRT-PCR); genotoxic effects (DNA strand breakage) by the Comet assay. Results show that B[a]P induced CYP1A1 gene and protein expression increasing its activity and, therefore, increasing the production of metabolites that trigger genotoxic DNA damage (%). Most importantly, mixtures containing Phe and B[a]P increased even further CYP1A1 mRNA levels and DNA damage (up to 70 %) which suggests that, although Phe is considered a non-carcinogenic PAH, it potentiates CYP1A1 synthesis induced by B[a]P, increasing its genotoxicity. These findings indicate that the upregulation of CYP1A1 by carcinogenic PAHs will not weaken even when in mixtures with non-carcinogenic PAHs. On contrary, non-carcinogenic PAHs may potentiate the genotoxic effect of carcinogenic PAH and therefore mixture composition should be taken in account when assessing PAH toxicity. In fact, our results point to the need of redefining Environmental Risk Assessment protocols for mixtures of carcinogenic pollutants.

Implication of microplastic toxicity on functioning of microalgae in aquatic system.

Microplastics (MPs) released from both primary and secondary sources affect the functioning of aquatic system. These MPs and components leached, can interact with aquatic organisms of all trophic levels, including the primary producers, such as microalgae. Considering the ecological value of microalgae and the toxicological effects of MPs towards them, this review provides: (1) a detailed understanding of the interactions between MPs and microalgae in the complex natural environment; (2) a discussion about the toxic effects of single type and mixtures of plastic particles on the microalgae cells, and (3) a discussion about the impacts of MPs on various features of microalgae -based bioremediation technology. For this purpose, toxic effects of MPs on various microalgal species were compiled and plastic components of MPs were ranked on the basis of their toxic effects. Based on available data, ranking for various plastic components was found to be: Polystyrene (PS) (rank 1) > Polyvinyl Chloride (PVC) > Polypropylene (PP) > Polyethylene (PE) (rank 4). Furthermore, the review suggested the need to understand joint toxicity of MPs along with co-contaminants on microalgae as the presence of other pollutants along with MPs might affect microalgae differently. In-depth investigations are required to check the impact of MPs on microalgae-based wastewater treatment technology and controlling factors.

Marine Fish Primary Hepatocyte Isolation and Culture: New Insights to Enzymatic Dissociation Pancreatin Digestion.

Primary cell cultures from wild organisms have been gaining relevance in ecotoxicology as they are considered more sensitive than immortalized cell lines and retain the biochemical pathways found in vivo. In this study, the efficacy of two methods for primary hepatocyte cell isolation was compared using liver from two marine fish (Sparus aurata and Psetta maxima): (i) two-step collagenase perfusion and (ii) pancreatin digestion with modifications. Cell cultures were incubated in L-15 medium at 17 ± 1 °C and monitored for up to six days for cell viability and function using the trypan blue exclusion test, MTT test, lactate dehydrogenase (LDH) activity, and ethoxyresorufin O-deethylase (EROD) activity after Benzo[a]Pyrene exposure. The results showed significant differences between the number of viable cells (p < 0.05), the highest number being obtained for the pancreatin digestion method (average = 4.5 ± 1.9 × 107 cells). Moreover, the hepatocytes showed solid adherence to the culture plate and the rounded shape, changing into a triangular/polygonal shape. The cell viability and function obtained by pancreatin digestion were maintained for five days, and the EROD induction after exposure to the B[a]P showed that cells were metabolically active. This study shows that the optimized pancreatin digestion method is a valid, cost-effective, and simple alternative to the standard perfusion method for the isolation of primary hepatocytes from fish and is suitable for ecotoxicological studies involving marine pollutants, such as PAHs.

Evidence of Mercury Methylation and Demethylation by the Estuarine Microbial Communities Obtained in Stable Hg Isotope Studies.

Microbial activity is a critical factor controlling methylmercury formation in aquatic environments. Microbial communities were isolated from sediments of two highly mercury-polluted areas of the Tagus Estuary (Barreiro and Cala do Norte) and differentiated according to their dependence on oxygen into three groups: aerobic, anaerobic, and sulphate-reducing microbial communities. Their potential to methylate mercury and demethylate methylmercury was evaluated through incubation with isotope-enriched Hg species (199HgCl and CH3201HgCl). The results showed that the isolated microbial communities are actively involved in methylation and demethylation processes. The production of CH3199Hg was positively correlated with sulphate-reducing microbial communities, methylating up to 0.07% of the added 199Hg within 48 h of incubation. A high rate of CH3201Hg degradation was observed and >20% of CH3201Hg was transformed. Mercury removal of inorganic forms was also observed. The results prove the simultaneous occurrence of microbial methylation and demethylation processes and indicate that microorganisms are mainly responsible for methylmercury formation and accumulation in the polluted Tagus Estuary.

Effects of microplastics and mercury in the freshwater bivalve Corbicula fluminea (Müller, 1774): Filtration rate, biochemical biomarkers and mercury bioconcentration.

The main objectives of this study were to investigate the effects of a mixture of microplastics and mercury on Corbicula fluminea, the post-exposure recovery, and the potential of microplastics to influence the bioconcentration of mercury by this species. Bivalves were collected in the field and acclimated to laboratory conditions for 14 days. Then, a 14-day bioassay was carried out. Bivalves were exposed for 8 days to clean medium (control), microplastics (0.13 mg/L), mercury (30 µg/L) and to a mixture (same concentrations) of both substances. The post-exposure recovery was investigated through 6 additional days in clean medium. After 8 and 14 days, the following endpoints were analysed: the post-exposure filtration rate (FR); the activity of cholinesterase enzymes (ChE), NADP-dependent isocitrate dehydrogenase (IDH), octopine dehydrogenase, catalase, glutathione reductase, glutathione peroxidase and glutathione S-transferases (GST), and the levels of lipid peroxidation (LPO). After 8 days of exposure to mercury, the bioconcentration factors (BCF) were 55 in bivalves exposed to the metal alone and 25 in bivalves exposed to the mixture. Thus, microplastics reduced the bioconcentration of mercury by C. fluminea. Bivalves exposed to microplastics, mercury or to the mixture had significantly (p ≤ 0.05) decreased FR and increased LPO levels, indicating fitness reduction and lipid oxidative damage. In addition, bivalves exposed to microplastics alone had significant (p ≤ 0.05) reduction of adductor muscle ChE activity, indicating neurotoxicity. Moreover, bivalves exposed to mercury alone had significantly (p ≤ 0.05) inhibited IDH activity, suggesting alterations in cellular energy production. Antagonism between microplastics and mercury in FR, ChE activity, GST activity and LPO levels was found. Six days of post-exposure recovery in clean medium was not enough to totally reverse the toxic effects induced by the substances nor to eliminate completely the mercury from the bivalve's body. These findings have implications to animal, ecosystem and human health.

Microplastics cause neurotoxicity, oxidative damage and energy-related changes and interact with the bioaccumulation of mercury in the European seabass, Dicentrarchus labrax (Linnaeus, 1758).

Microplastics pollution is a global paradigm that raises concern in relation to environmental and human health. This study investigated toxic effects of microplastics and mercury in the European seabass (Dicentrarchus labrax), a marine fish widely used as food for humans. A short-term (96 h) laboratory bioassay was done by exposing juvenile fish to microplastics (0.26 and 0.69 mg/L), mercury (0.010 and 0.016 mg/L) and binary mixtures of the two substances using the same concentrations, through test media. Microplastics alone and mercury alone caused neurotoxicity through acetylcholinesterase (AChE) inhibition, increased lipid oxidation (LPO) in brain and muscle, and changed the activities of the energy-related enzymes lactate dehydrogenase (LDH) and isocitrate dehydrogenase (IDH). All the mixtures caused significant inhibition of brain AChE activity (64-76%), and significant increase of LPO levels in brain (2.9-3.4 fold) and muscle (2.2-2.9 fold) but not in a concentration-dependent manner; mixtures containing low and high concentrations of microplastics caused different effects on IDH and LDH activity. Mercury was found to accumulate in the brain and muscle, with bioaccumulation factors of 4-7 and 25-40, respectively. Moreover, in the analysis of mercury concentrations in both tissues, a significant interaction between mercury and microplastics was found. The decay of mercury in the water increased with microplastics concentration, and was higher in the presence of fish than in their absence. Overall, these results indicate that: microplastics influence the bioaccumulation of mercury by D. labrax juveniles; microplastics, mercury and their mixtures (ppb range concentrations) cause neurotoxicity, oxidative stress and damage, and changes in the activities of energy-related enzymes in juveniles of this species; mixtures with the lowest and highest concentrations of their components induced different effects on some biomarkers. These findings and other published in the literature raise concern regarding high level predators and humans consuming fish being exposed to microplastics and heavy metals, and highlight the need of more research on the topic.

Optimization of microbial detoxification for an aquatic mercury-contaminated environment.

Mercury (Hg) reduction performed by microorganisms is well recognized as a biological means for remediation of contaminated environment. Recently, studies demonstrated that Hg-resistant microorganisms of Tagus Estuary are involved in metal reduction processes. In the present study, aerobic microbial community isolated from a highly Hg-contaminated area of Tagus Estuary was used to determine the optimization of the reduction process in conditions such as the contaminated ecosystem. Factorial design methodology was employed to examine the influence of glucose, sulfate, iron, and chloride on Hg reduction. In the presence of several concentrations of these elements, microbial community reduced Hg in a range of 37-61% of the initial 0.1 mg/ml Hg2+ levels. The response prediction through central composite design showed that the increase of sulfate concentration led to an optimal response in Hg reduction by microbial community, while the rise in chloride levels markedly decreased metal reduction. Iron may exert antagonistic effects depending upon the media composition. These results are useful in understanding the persistence of Hg contamination in Tagus Estuary after inactivation of critical industrial units, as well as data might also be beneficial for development of new bioremediation strategies either in Tagus Estuary and/or in other Hg-contaminated aquatic environments.

Aerobic Mercury-resistant bacteria alter Mercury speciation and retention in the Tagus Estuary (Portugal).

Aerobic mercury-resistant bacteria were isolated from the sediments of two highly mercury-polluted areas of the Tagus Estuary (Barreiro and Cala do Norte) and one natural reserve area (Alcochete) in order to test their capacity to transform mercury. Bacterial species were identified using 16S rRNA amplification and sequencing techniques and the results indicate the prevalence of Bacillus sp. Resistance patterns to mercurial compounds were established by the determination of minimal inhibitory concentrations. Representative Hg-resistant bacteria were further tested for transformation pathways (reduction, volatilization and methylation) in cultures containing mercury chloride. Bacterial Hg-methylation was carried out by Vibrio fluvialis, Bacillus megaterium and Serratia marcescens that transformed 2-8% of total mercury into methylmercury in 48h. In addition, most of the HgR bacterial isolates showed Hg(2+)-reduction andHg(0)-volatilization resulting 6-50% mercury loss from the culture media. In summary, the results obtained under controlled laboratory conditions indicate that aerobic Hg-resistant bacteria from the Tagus Estuary significantly affect both the methylation and reduction of mercury and may have a dual face by providing a pathway for pollution dispersion while forming methylmercury, which is highly toxic for living organisms.

Mercury-resistant bacteria from salt marsh of Tagus Estuary: the influence of plants presence and mercury contamination levels.

Mercury (Hg) contamination of aquatic systems has been recognized as a global, serious problem affecting both wildlife and humans. High levels of Hg, in particular methylmercury (MeHg), were detected in surface sediments of Tagus Estuary. MeHg is neurotoxic and its concentration in aquatic systems is dependent upon the relative efficiency of reduction, methylation, and demethylation processes, which are mediated predominantly by the microbial community, in particular mercury-resistant (HgR) bacteria. Plants in contaminated ecosystems are known to take up Hg via plant roots. Therefore, the aims of this study were to (1) isolate and characterize HgR bacteria from a salt marsh of Tagus Estuary (Rosário) and (2) determine HgR bacteria levels in the rhizosphere and, consequently, their influence in metal cycling. To accomplish this objective, sediments samples were collected during the spring season in an area colonized by Sacocornia fruticosa and Spartina maritima and compared with sediments without plants. From these samples, 13 aerobic HgR bacteria were isolated and characterized morphologically, biochemically, and genetically, and susceptibility to Hg compounds, Hg(2+), and MeHg was assessed by determination of minimal inhibitory concentration (MIC). Genetically, the mer operon was searched by polymerase chain reaction (PCR) and 16S rRNA sequencing was used for bacterial identification. Results showed that the isolates were capable of growing in the presence of high Hg concentration with MIC values for HgCl2 and MeHgCl in the ranges of 1.7-4.2 µg/ml and 0.1-0.9 µg/ml, respectively. The isolates from sediments colonized with Sacocornia fruticosa displayed higher resistance levels compared to ones colonized with Spartina maritima. Bacteria isolates showed different capacity of Hg accumulation but all displayed Hg volatilization capabilities (20-50%). Mer operon was found in two isolates, which genetically confirmed their capability to convert Hg compounds by reducing them to Hg(0). Thus, these results are the first evidence of the relevance of interaction between bacteria and plants in Hg cycling in Tagus Estuary.

Isolation and characterization of mercury-resistant bacteria from sediments of Tagus Estuary (Portugal): implications for environmental and human health risk assessment.

Mercury (Hg) contamination of aquatic systems has been recognized as a global and serious problem affecting both human and environmental health. In the aquatic ecosystems, mercurial compounds are microbiologically transformed with methylation responsible for generation of methylmercury (MeHg) and subsequent biomagnification in food chain, consequently increasing the risk of poisoning for humans and wildlife. High levels of Hg, especially MeHg, are known to exist in Tagus Estuary as a result of past industrial activities. The aim of this study was to isolate and characterize Hg-resistant bacteria from Tagus Estuary. Mercury-resistant (Hg-R) bacteria were isolated from sediments of two hotspots (Barreiro and North Channel) and one reserve area (Alcochete). Mercury contamination in these areas was examined and bacterial susceptibility to Hg compounds evaluated by determination of minimal inhibitory concentrations (MIC). The isolates characterization was based on morphological observation and biochemical testing. Bacteria characteristics, distribution, and Hg resistance levels were compared with metal levels. Barreiro and North Channel were highly contaminated with Hg, containing 126 and 18 µg/g total Hg, respectively, and in Alcochete, contamination was lower at 0.87 µg/g total Hg. Among the isolates there were aerobic and anaerobic bacteria, namely, sulfate-reducing bacteria, and Hg resistance levels ranged from 0.16 to 140 µg/ml for Hg(2+) and from 0.02 to 50.1 µg/ml for MeHg. The distribution of these bacteria and the resistance levels were consistent with Hg contamination along the depth of the sediments. Overall, results show the importance of the characterization of Tagus Estuary bacteria for ecological and human health risk assessment.