Microplastics increase the toxicity of mercury, chlorpyrifos and fluoranthene to mussel and sea urchin embryos.

The objective of this study was to determine whether and to what extent microplastics (MPs) enhance the toxicity of pollutants as well as whether pollutant-loaded MPs act as relevant vectors of chemical pollutants. With this aim, the toxicity for mussel and sea urchin embryos of: 1) three dissolved pollutants (Pol): chlorpyrifos (CPF), fluoranthene (FLT) and mercury (Hg); 2) their mixture with Microplastics (MP + Pol); and 3) pollutant-loaded MPs (MPPol), was assessed. Analyses of CPF, FLT and Hg were also performed to evaluate the transfer among dissolved and particulate phases. In general, the 'MP + Pol' treatments were more toxic as 48-h EC50 (µg/L) than the 'Pol' treatments for sea urchin or mussel. The 48-h and 120-h EC50s (µg/L) for sea urchin showed little variation for CPF and MP + CPF, and no clear pattern was found for FLT and MP + FLT. The performed chemical analysis in the MPPol tests indicated that desorption was the main route to explain the observed toxicity of Hg and a relevant route for CPF and FLT. This study contributes to improve the knowledge about the interactions between MPs and chemical pollutants, which is fundamental for a more realistic ecological risk assessment in aquatic ecosystems.

Interactive effects of palladium (Pd) and microplastics (MPs) on metal bioaccumulation and biological responses in the Mediterranean mussel, Mytilus galloprovincialis.

This study investigates the potential of MPs as carriers of pollutants as they can strengthen bioaccumulation of toxic metals on marine organisms. For the first time, the interaction of the metal palladium (Pd) with the widespread MPs, both with increasing concentrations in water environments from anthropogenic sources, was tested. Mytilus galloprovincialis, an important seafood product, was exposed to Pd (24 h) in two ways: water-dissolved and MPs-adsorbed, with depuration followed for 144 h. Quantification of Pd in tissues shown an accumulation 2-3 times higher (59 % of initial Pd) for mussels exposed to MPs-adsorbed Pd and higher in digestive gland than when exposed to water-dissolved Pd (25 %; higher in gills). Additionally, it was demonstrated that Pd induced oxidative stress and altered the feeding behavior of mussels. Therefore, this work support MPs as being vectors of metals (i.e. Pd) to enhance their bioaccumulation on marine organisms which highlights ecological risk of these emerging pollutants.

Occurrence and size distribution study of microplastics in household water from different cities in continental Spain and the Canary Islands.

The purpose of this study was to investigate the occurrence of microplastics (MPs) in drinking water in Spain by comparing tap water from different locations using common sampling and identification procedures. We sampled tap water from 24 points in 8 different locations from continental Spain and the Canary Islands by means of 25 µm opening size steel filters coupled to household connections. All particles were measured and spectroscopically characterized including not only MPs but also particles consisting of natural materials with evidence of industrial processing, such as dyed natural fibres, referred insofar as artificial particles (APs). The average concentration of MPs was 12.5 ± 4.9 MPs/m3 and that of anthropogenic particles 32.2 ± 12.5 APs/m3. The main synthetic polymers detected were polyamide, polyester, and polypropylene, with lower counts of other polymers including the biopolymer poly(lactic acid). Particle size and mass distributions were parameterized by means of power law distributions, which allowed performing estimations of the concentration of smaller particles provided the same scaling parameter of the power law applies. The calculated total mass concentration of the identified MPs was 45.5 ng/L. The observed size distribution of MPs allowed an estimation for the concentration of nanoplastics (< 1 µm) well below the ng/L range; higher concentrations are not consistent with scale invariant fractal fragmentation. Our findings showed that MPs in the drinking water sampled in this work do not represent a significant way of exposure to MPs and would probably pose a negligible risk for human health.

Comparative role of microplastics and microalgae as vectors for chlorpyrifos bioacumulation and related physiological and immune effects in mussels.

Microplastics (MP) are contaminants of concern per se, and also by their capacity to sorb dissolved chemicals from seawater, acting as vehicles for their transfer into marine organisms. Still, the role of MP as vehicles for contaminants and their associated toxicological effects have been poorly investigated. In this work we have compared the role of MP (high density polyethylene, HDPE, ≤22 µm) and of natural organic particles (microalgae, MA) as vehicle for chlorpyrifos (CPF), one of the most common pesticides found in river and coastal waters. We have compared the capacity of MP and MA to carry CPF. Then, the mussel Mytilus galloprovincialis has been exposed for 21 days to dissolved CPF, and to the same amount of CPF loaded onto MP and MA. The concentration of CPF in mussel' tissues and several physiological, energetics and immune parameters have been analyzed after 7 and 21 days of exposure. Results showed similar CPF accumulation in mussel exposed to MP and to MA spiked with CPF. This revealed that MP acted as vector for CPF in a similar way (or even to a lesser extent) than MA. After 21 days of exposure mussels exposed to MP spiked with CPF displayed similar or more pronounced biological effects than mussels exposed to dissolved CPF or to MA loaded with CPF. This suggested that the combined "particle" and "organic contaminant" effect produced an alteration on the biological responses greater than that produced by each stressor alone, although this was evident only after 3 weeks of exposure.

Trace Metal Residues in Marine Mussels: A Global Survey.

Pressures from anthropogenic activities are causing degradation of estuarine and coastal ecosystems around the world. Trace metals are key pollutants that are released and can partition in a range of environmental compartments, to be ultimately accumulated in exposed biota. The level of pressure varies with locations and the range and intensity of anthropogenic activities. The present study measured residues of trace metals in Mytilus mussel species collected from a range of locations around the world in areas experiencing a gradient of anthropogenic pressures that we classified as low, moderate, or high impact. The data showed no grouping/impact level when sampling sites in all countries were incorporated in the analysis, but there was significant clustering/impact level for most countries. Overall, high-impact areas were characterized by elevated concentrations of zinc, lead, nickel, and arsenic, whereas copper and silver were detected at higher concentrations in medium-impact areas. Finally, whereas most metals were found at lower concentrations in areas classified as low impact, cadmium was typically elevated in these areas. The present study provides a unique snapshot of worldwide levels of coastal metal contamination through the use of Mytilus species, a well-established marine biomonitoring tool. Environ Toxicol Chem 2021;40:3434-3440. © 2021 SETAC.

Mercury interactions with algal and plastic microparticles: Comparative role as vectors of metals for the mussel, Mytilus galloprovincialis.

The adsorption and desorption of Hg onto and from microplastics (MP) and microalgae (MA) were investigated, and fitted using pseudo-first-order and pseudo-second order kinetics models. Then, the potential role of MP as vector for the entrance and accumulation of Hg (MP-Hg) in comparison to natural pathways (via MA -MA-Hg-, and dissolved -WB-Hg-) was investigated in mussel. Mussels were exposed to a single dose of Hg (2375 ng ind-1) for 4 h. Although the clearance of MP-Hg was relevant (82 %), it was lower than that of MA (95 %) and MA-Hg (94 %). The amount of the Hg accumulated and eliminated was higher in mussels exposed to MP-Hg (1417 ng Hg) than in those exposed to MA-Hg (882 ng Hg) and WB-Hg (1074 ng Hg). However, Hg accumulation was similar in the three mussel groups (≈800 ng Hg). This was related to the fast elimination of Hg still attached to MP by MP-Hg mussels. Hg was mainly accumulated in digestive gland in MA-Hg and MP-Hg mussels, and in gills in WB-Hg mussels. Overall, the results indicated that MP facilitated the entrance of Hg in mussel but also promoted Hg elimination, which could limit the toxicological risk of Hg adsorbed onto MP.

Microplastics do not affect standard ecotoxicological endpoints in marine unicellular organisms.

In this study, the acute toxicity of microplastics (MPs) on unicellular organisms as marine decomposers and microalgae was assessed, by evaluating standards endpoints included in International Standard Organization (ISO) protocols. The bacteria Vibrio fischeri and the diatom Phaeodactylum tricornutum were exposed to different sizes (1-500 µm) of polyethylene MPs in order to evaluate bioluminescence inhibition and microalgal growth. No acute toxicity was found on bacteria or microalgae in an order of magnitude above environmentally relevant concentrations, suggesting that tested MPs did not affect the investigated biological processes. In conclusion, standard ecotoxicological endpoints are not sufficiently sensitive to assess the potential effects of MPs on decomposers and primary producers, conversely to nanoplastics. These findings highlight that the current approach for MP risk assessment in unicellular species should be revised, by providing alternative endpoints to be included in standardized protocols, able to monitor the fate and biological effects of MPs.

Dynamic of small polyethylene microplastics (≤10 µm) in mussel's tissues.

MPs' uptake and tissue accumulation were investigated in mussel exposed to a single dose (2.85 mg ind-1, 3 mg l-1) of a heterogeneous mixture of irregularly shaped particles of HDPE (mainly ≤10 µm), followed by a 7 days depuration period. The results showed that mussels efficiently cleared MPs from water during exposure, and that MPs were accumulated in digestive gland and gills during depuration. In digestive gland, the amount and size of the MPs accumulated decreased with time, indicating a slower processing and elimination of small MPs than of larger ones. In gills, MPs' burdens increased with time, the MPs accumulated were the smallest ones, suggesting the translocation of small MPs from the digestive system to the gills. The hazardous potential of the smaller fraction of MPs (≤4 µm) underlined that more focus should be directed towards the accumulation and effects of this fraction of MPs in the marine environment.

Biodynamics of mercury in mussel tissues as a function of exposure pathway: natural vs microplastic routes.

In the marine environment, metals can be present dissolved or adsorbed to suspended particles. In the last decades a new type of particle has been introduced, microplastics (MPs). The exposure route of pollutants influences their accumulation and distribution into tissues. A pulse-chase experiment was conducted in which mussels were exposed to Hg: adsorbed onto MPs and microalgae (MA) and dissolved (WB). Mussels accumulated the same amount of Hg independently of particle, due to the Hg loading in both particles and their acceptability were similar. The highest Hg accumulation occurred in gill when the Hg exposure was through water and in digestive gland when Hg was adsorbed to particles. More than 70% of the Hg uptake through MPs was quickly eliminated due to: i) part of the cleared MPs might not really be ingested but adhered to body surfaces of mussels, ii) MPs ingested were eliminated through faeces as they are non-nutritive particles which may be rejected in stomach preventing their entry into digestive gland and iii) high affinity of Hg on surface of MPs which meant that Hg was mainly eliminated jointly to MPs. The organic nature of MA facilitates the entry of Hg into digestive gland where MA are intracellularly digested releasing the Hg adsorbed onto their surfaces. In this case, Hg may reach deeper levels by translocation of the Hg incorporated into gland towards foot and remaining tissues, a process that might occur through haemolymph. All of the Hg accumulated in WB during the exposure was internally absorbed into tissues, and later translocated from gill to gland. Although Hg elimination rate in MPs mussels was greater than in the other exposure pathways, an important amount of Hg was maintained through the depuration period, thus we cannot and should not neglect the risk of MPs as vectors for mercury.

Insights into the uptake, elimination and accumulation of microplastics in mussel.

The majority of plastics present in the marine environment are microplastics (MPs, <5 mm). Suspension filter feeders are susceptible species to MPs ingestion. Once ingested MPs can be eliminated packed in fecal pellets, or they can be accumulated within tissues, and likely be transferred along the food web. The research on MPs is hampered by the difficulty on their quantification and the lack of standardized methodologies. Indeed, limited information exits about the capacity of marine organisms to ingest, accumulate and eliminate MPs. In this work we investigated the uptake, elimination and accumulation of MPs (irregularly shaped particles of high density polyethylene, ≤22 µm) in mussel. Mussels were exposed to two concentrations of MPs (2 and 4 mm3 l-1), and their uptake, elimination and accumulation in digestive gland was investigated. The results showed that the uptake of MPs increased at the high concentration tested, and that mussels cleared MPs at the same extent than a food item (microalgae) of similar size. Small MPs (2-4 µm) were less efficiently cleared than the larger ones. Large MPs (>10 µm) were faster eliminated than the smaller ones. The global balance showed that after 6 days of depuration mussels eliminated ≈85% of the MPs cleared, and that ≈2-6% of the MPs cleared remained in the digestive gland, essentially those <6 µm. We recorded a long retention time for MPs, contrasting with the lower times assumed to be necessary to empty mussel's gut before quantifying MPs. Our study emphasized the gap of knowledge on the feeding behaviour of mussels in relation to MPs, and the necessity to investigate it in different marine species, and under different exposure scenarios.

Effect of microplastics on the toxicity of chlorpyrifos to the microalgae Isochrysis galbana, clone t-ISO.

It is highly likely that phytoplanktonic organisms will interact with MPs in the ocean, and consequently with the pollutants sorbed onto their surfaces. Microalgae play an essential role in maintaining the balance of the marine ecosystem due to the fact that they are a primary producer and the base of marine trophic chains. Therefore, their fitness represents an important index in the assessment of water quality. The objectives of this study were i) to assess the toxicity of MPs and the pesticide chlorpyrifos (CPF) to the microalgae, Isochrysis galbana, clone t-ISO and ii) to ascertain whether the presence of MPs affects the toxicity of CPF. Microalgae growth rate was selected as the endpoint and a commercial virgin PE micronized powder was chosen as a micro-plastic model, with mean size ranging from 2 to 6 µm, assayed until 25 mg L-1. CPF was tested at concentrations ranging from 0 to 4 mg L-1. A constant concentration of MPs (5 mg L-1) was loaded with increasing doses of CPF (0-3 mg L-1) with a 2 h incubation period. Bioassays were performed at 20 °C, in glass tubes of 50 ml, with air and constant light and an exposure time of 72 h. Cell counts were performed using a Coulter Counter Multisizer III and HPLC was used to quantify the partition of this pollutant among MPs and water. Although microalgae growth was not impacted by MPs, growth was clearly affected by exposure to CPF from 2 mg L-1 and above, with a total growth inhibition at concentrations over 3 mg L-1. Subsequent to incubation, 80% of CPF was sorbed onto MP surfaces. Two different dose-response curves resulted from CPF bioassays depending on the presence of MP, with lower percentages of inhibition when CPF was presented through MP. Thus, the adsorption of CPF onto MP surfaces modulates the toxicity of CPF on I. galbana growth through a reduction in its toxicity, as CPF is adsorbed onto MP surfaces which are less bio-available to the algal cells.

Metabolomic responses of mussel Mytilus galloprovincialis to fluoranthene exposure under different nutritive conditions.

Biomarkers are useful tools to assess biological effects of pollutants that are extensively used in monitoring programs to assess ecosystem health. However, they are strongly affected by mussel physiological state, especially nutritive status, which has led to the search of new biological indicators of chemical pollutants exposition. Environmental metabolomics is an approach for examining the metabolic responses (measurement of low molecular weight endogenous metabolites) of an organism to both natural and anthropogenic stressors that can occur in its environment. The aim of the present work was to assess the effect of the polycyclic aromatic hydrocarbon fluoranthene (FLU) exposure on the metabolomic profiles of mussel digestive glands under different nutritive conditions. To achieve this objective, mussels were reared, for a period of 56 days, under three different food rations in order to obtain a gradient of nutritive status (negative, zero and positive energy balance), and after that, they were exposed, during 3 weeks, to a nominal concentration of 3 µg FLU L-1. A total of 43 metabolites, including aminoacids (Ala, Val, Leu, Ile, etc.), energy metabolism related metabolites (ATP, AMP, etc.), organic osmolytes (taurine, etc.), redox metabolism (GSH, NADP+) and nucleotides, were identified and quantified in the digestive glands of the mussels. Principal Component Analysis (PCA) defined two principal components (PC1 and PC2) that explained 55.6% of the total variance, although the first component explains more than 80% of this variance, this being related to the mussel nutritive condition. The effect of the toxicant, explained by the PC2, is similar to that produced under conditions of food restriction, which masks the effect of the toxicant under these conditions. As the feeding conditions are more favorable, the toxic effect becomes more apparent. Therefore, the great influence of nutritive condition on mussel metabolome implies a handicap for the use of metabolomic biomarkers, as previously demonstrated for biochemical and other molecular biomarkers, in large-scale monitoring programs in which several food conditions coexist with pollution levels.

Interactive effects of nutrition, reproductive state and pollution on molecular stress responses of mussels, Mytilus galloprovincialis Lamarck, 1819.

Marine bivalves including mussels Mytilus galloprovincialis are commonly used as sentinels for pollution monitoring and ecosystem health assessment in the coastal zones. Use of biomarkers to assess the pollution effects assumes that the effects of pollutants on the biomarkers exceed the natural background variability; yet this assumption has rarely been tested. We exposed mussels at different reproductive stages and nutritive states to two concentrations of a polycyclic aromatic hydrocarbon (fluoranthene, 3 and 60 µg L-1) for three weeks. Expression levels of the molecular biomarkers related to the detoxification and general stress response [cytochrome P450 oxidase (CYP450), glutathione S-transferases (GST-α; GST-S1; GST-S2), the multixenobiotic resistance protein P-glycoprotein (PgP), metallothioneins (MT10 and MT20), heat shock proteins (HSP22, HSP70-2; HSP70-3; HSP70-4), as well as mRNA expression of two reproduction-related genes, vitellogenin (Vitel) and vitelline coat lysin M7 (VCLM7)] were measured. The mussels' nutrition and reproductive state affected the baseline mRNA levels of molecular biomarkers and modulated the transcriptional responses of biomarker genes to the pollutant exposure. Thus, mussel physiological state could act as a confounding factor in the evaluation of the response of pollution through molecular biomarkers. The biomarker baseline levels must be determined across a range of physiological states to enable the use of biomarkers in monitoring programs.

Biomarkers of general stress in mussels as common indicators for marine biomonitoring programmes in Europe: The ICON experience.

This study investigated whether general stress biomarkers in mussels can be applied as common first-tier biomarkers in regional biomonitoring programmes in the North Sea (including Iceland) and western Mediterranean Sea. Stress on Stress (SoS) and lysosomal membrane stability (LMS) biomarkers were analysed in resident mussels (Mytilus sp.) from 8 coastal sites and in transplanted mussels (Mytilus galloprovincialis) from two Spanish Mediterranean coastal sites. The assessment of results, as input to pollution monitoring strategies, was performed jointly for LMS and SoS data from the two regions. Contaminant body burden of the mussels was compared with biomarker results. The results demonstrated that these two general and non-expensive stress biomarkers in mussel can be applied throughout European waters, providing a cost-effective and harmonised approach to screen contaminant-related biological effects within the framework of wide-scale pollution biomonitoring programmes, such as that proposed by the European Union, i.e. the Marine Strategy Framework Directive.

Effect of mussel reproductive status on biomarker responses to PAHs: Implications for large-scale monitoring programs.

Biomarkers are useful tools to assess biological effects of pollutants and have been extensively used in monitoring programs to determine ecosystem health. In these programs, a wide range of environmental conditions are covered and sometimes, obtained data are difficult to interpret because of natural variables are affecting biomarker responses. Among these variables, mussels reproductive status has been considered one of the most changing variables between sites in a monitoring survey. Thus, the main aim of this work was to identify the effect that mussel reproductive status has on biomarker responses. For that purpose, mussels sampled at two periods in the reproductive cycle (reproductive and resting stages) were conditioned to the same laboratory conditions and exposed to fluoranthene (FLU) for three weeks. Studied biomarkers covering a wide range of organism responses were included: bioaccumulation, physiological rates (clearance rate -CR-, absorption efficiency -AE-, respiration rate -RR- and their integration in the scope for growth -SFG-), antioxidant enzyme activities (superoxide-dismutase -SOD-, catalase -CAT-, glutathione reductase -GR-, glutathione peroxidase -GPx-, glutathione-S-tranferase -GST-) and biochemical damage responses (lipid membrane peroxidation -LPO-). The results obtained evidenced that the levels of the biomarkers studied (RR, SOD, CAT and GPx) were higher at reproductive than at resting stage. On the other hand, the effect of toxicant was observed in SFG, CAT and GPx but this effect was only detected during the resting period. Moreover, there was a deterioration of mussel gonadal tissue with FLU exposure during reproductive stage. FLU accumulation in mussel tissues was also dependent of the reproductive status with higher internal concentrations during resting than reproductive period. In conclusion, there was a strong effect of reproductive status on studied biomarkers which seems to mask the effect of FLU at reproductive stage. The present study evidences the need to include the measurement of mussel biological status in marine pollution monitoring programs for a correct interpretation of biomarker data.

Exposure of marine mussels Mytilus spp. to polystyrene microplastics: Toxicity and influence on fluoranthene bioaccumulation.

The effects of polystyrene microbeads (micro-PS; mix of 2 and 6 µm; final concentration: 32 µg L(-1)) alone or in combination with fluoranthene (30 µg L(-1)) on marine mussels Mytilus spp. were investigated after 7 days of exposure and 7 days of depuration under controlled laboratory conditions. Overall, fluoranthene was mostly associated to algae Chaetoceros muelleri (partition coefficient Log Kp = 4.8) used as a food source for mussels during the experiment. When micro-PS were added in the system, a fraction of FLU transferred from the algae to the microbeads as suggested by the higher partition coefficient of micro-PS (Log Kp = 6.6), which confirmed a high affinity of fluoranthene for polystyrene microparticles. However, this did not lead to a modification of fluoranthene bioaccumulation in exposed individuals, suggesting that micro-PS had a minor role in transferring fluoranthene to mussels tissues in comparison with waterborne and foodborne exposures. After depuration, a higher fluoranthene concentration was detected in mussels exposed to micro-PS and fluoranthene, as compared to mussels exposed to fluoranthene alone. This may be related to direct effect of micro-PS on detoxification mechanisms, as suggested by a down regulation of a P-glycoprotein involved in pollutant excretion, but other factors such as an impairment of the filtration activity or presence of remaining beads in the gut cannot be excluded. Micro-PS alone led to an increase in hemocyte mortality and triggered substantial modulation of cellular oxidative balance: increase in reactive oxygen species production in hemocytes and enhancement of anti-oxidant and glutathione-related enzymes in mussel tissues. Highest histopathological damages and levels of anti-oxidant markers were observed in mussels exposed to micro-PS together with fluoranthene. Overall these results suggest that under the experimental conditions of our study micro-PS led to direct toxic effects at tissue, cellular and molecular levels, and modulated fluoranthene kinetics and toxicity in marine mussels.

Effect of diet quality on mussel biomarker responses to pollutants.

The effect of the quality of two microalgal species on select biological and biochemical responses used as indicators of pollution were assessed. Mussels were conditioned for 6 weeks with the diatom Chaetoceros neogracile and the dinoflagellate Heterocapsa triquetra, chosen for being two clearly different types of primary production quality that differ in both biometric and biochemical characteristics. After dietary conditioning, the mussels were exposed to a polycyclic aromatic hydrocarbon, fluoranthene (FLU), for 1 week followed by 1 week of depuration. Results showed higher FLU accumulation in mussels fed on C. neogracile compared to those fed on H. triquetra. Concomitantly, a greater impact of this toxicant was observed in the biomarker responses of mussels fed on C. neogracile. These mussels showed an increase in the percentage of dead hemocytes, an activation of phagocytosis and ROS production of hemocytes after exposure. Some enzymatic activities also increased upon FLU exposure (superoxide dismutase -SOD-, catalase -CAT-, and glutathione reductases -GR-) and after depuration (glutathione-s-transferase -GST-). Results suggest that FLU exposure as well as food quality influence biomarker responses, with higher values of SOD, CAT and GR in non-exposed mussels fed on C. neogracile. In addition, upon exposure to the same FLU concentration, GR response varied according to dietary conditioning, suggesting that diet could act as a confounding factor in biomarker responses to pollution. Consequently, trophic conditions should be considered in marine pollution monitoring programs for a better interpretation of biomarker responses.

Effect of nutritive status on Mytilus galloprovincialis pollution biomarkers: Implications for large-scale monitoring programs.

Biomarkers have been extensively used in monitoring programs with the aim of assessing the biological effects of pollutants on marine organisms and determining environmental status. Data obtained from these programs are sometimes difficult to interpret due to the large amount of natural variables affecting biological processes, which could act as confounding factors on biomarker responses. The main aim of this work was to identify the effect of one of these variables, the food availability, and consequently, the mussel nutritive status, on biomarker responses. For that purpose, mussels (Mytilus galloprovincialis) were conditioned to three different food rations for 2 months in order to create three mussel nutritive statuses and afterwards, each status was exposed to three nominal concentrations of fluoranthene (FLU) for 3 weeks. A battery of biomarkers was considered in this study to cover a wide range of organism responses, both physiological (scope for growth - SFG) and biochemical (superoxide dismutase - SOD, catalase - CAT, glutathione reductase - GR, glutathione peroxidase - GPx, glutathione-S-transferase - GST and phenoloxidase - PO activities, and lipid membrane peroxidation - LPO). The results obtained, evidenced that most of the studied biomarkers (SFG, SOD, CAT, GPx, and PO) were strongly affected by mussel nutritive status, showing higher values at lower status, whereas the effect of toxicant was not always evident, masked by the nutritive status effect. This paper demonstrates that toxicants are not the only source of variability modulating pollution biomarkers, and confirms nutritive status as a major factor altering biochemical and physiological biomarkers.

Metabolomic responses in caged clams, Ruditapes decussatus, exposed to agricultural and urban inputs in a Mediterranean coastal lagoon (Mar Menor, SE Spain).

The Mar Menor is a coastal lagoon affected by the growth of intensive agriculture and urban development in the surrounding area. Large amounts of chemical pollutants from these areas are discharged into El Albujón, a permanent water-course flowing into the lagoon. Biomarkers such as the activity of acetylcholinesterase or antioxidant enzymes have been previously tested in this lagoon demonstrating the presence of neurotoxicity and oxidative stress in clams transplanted in sites affected by the dispersion of the effluent from El Albujón. To complete this traditional toxicology work, a metabolomic profiling of these transplanted organisms has been carried out for the detection of metabolic biomarkers induced by agricultural/urban pollutants. More than 70 metabolites have been quantified using a targeting metabolomics platform based on HPLC-MS. The intracellular metabolic pattern was analyzed by PCA from the digestive gland of clams after 7 and 22 days of transplantation. Results showed a different profile of metabolite between organisms collected from control and exposed sites. At the shorter exposure time, there was an increase in several metabolites in the latter when compared with those from control sites, whereas metabolic profiling at 22 days showed that those metabolites were drastically diminished, with even lower levels than at control sites. These metabolites included: (i) 12 amino acids from the 21 proteogenic and HomoSer, (ii) osmotic protectants such as γ-butyrobetaine and taurine and (iii) nucleotides such as ITP. Regarding sulfur-containing molecules, taurine could be highlighted as a potential biomarker since its concentration was reduced by more than 30 times after 22 days of exposure, whereas the antioxidant glutathione remained constant in the organisms from both control and exposed sites. Although targeted metabolomics has been shown as an early technique of pollutant effect detection, the two-phase pattern could highlight a more complicated metabolite response to pollutants than classical biomarkers.

Influence of mussel biological variability on pollution biomarkers.

This study deals with the identification and characterization of biological variables that may affect some of the biological responses used as pollution biomarkers. With this aim, during the 2012 mussel survey of the Spanish Marine Pollution monitoring program (SMP), at the North-Atlantic coast, several quantitative and qualitative biological variables were measured (corporal and shell indices, gonadal development and reserves composition). Studied biomarkers were antioxidant enzymatic activities (CAT, GST, GR), lipid peroxidation (LPO) and the physiological rates integrated in the SFG biomarker (CR, AE, RR). Site pollution was considered as the chemical concentration in the whole tissues of mussels. A great geographical variability was observed for the biological variables, which was mainly linked to the differences in food availability along the studied region. An inverse relationship between antioxidant enzymes and the nutritional status of the organism was evidenced, whereas LPO was positively related to nutritional status and, therefore, with higher metabolic costs, with their associated ROS generation. Mussel condition was also inversely related to CR, and therefore to SFG, suggesting that mussels keep an "ecological memory" from the habitat where they have been collected. No overall relationship was observed between pollution and biomarkers, but a significant overall effect of biological variables on both biochemical and physiological biomarkers was evidenced. It was concluded that when a wide range of certain environmental factors, as food availability, coexist in the same monitoring program, it determines a great variability in mussel populations which mask the effect of contaminants on biomarkers.