Comparative assessment of microplastics and microalgae as vectors of mercury and chlorpyrifos in the copepod Acartia tonsa.

Microplastics (MPs) raise concerns not only as pollutants themselves, but also due to their ability to act as vectors of pollutants adsorbed from seawater, transferring them to marine organisms. However, the relevance of MPs as carriers of pollutants compared to microalgae needs further exploration. This study compared the role of MPs (2-10 µm non-oxidized and 10-15 µm oxidized high-density polyethylene) and natural organic particles (Rhodomonas lens microalgae, MA) as carriers of mercury (Hg, 2.3 µg Hg/L) and chlorpyrifos (CPF, 1.0 µg CPF/L) to adult Acartia tonsa copepods, after 24-48 h exposure. Dose-response experiments were first performed with adult female copepods exposed to oxidized MPs (0.25-4.0 mg/L), waterborne Hg (0.01-10.0 µg/L) and Ox MPs + Hg (0.25-4.0 mg oxidized MPs/L + 0.50-8.0 µg Hg/L) for 48 h, to complement previous studies that focused on the pesticide CPF. Effects were evaluated with four replicates for physiological and reproductive responses (6 females/replicate), biochemical techniques (40 individuals/replicate) and Hg/CPF bioaccumulation measurements (1000 individuals/replicate). Copepods accumulated Hg/CPF similarly from dissolved pollutants (6204 ± 2265 ng Hg/g and 1251 ± 646 ng CPF/g) and loaded MPs (3125 ± 1389 ng Hg/g and 1156 ± 266 ng CPF/g), but significantly less from loaded MA (21 ± 8 ng Hg/g and 173 ± 80 ng CPF/g). After 24-48 h, copepods exposed to MPs + Hg/CPF showed generally greater biological effects than those exposed to dissolved Hg/CPF or to MA + Hg/CPF, although differences were not statistically significant. MA + CPF had significantly lower AChE inhibition (1073.4 nmol min-1 mg-1) and MA + Hg lower GRx induction (48.8 nmol min-1 mg-1) compared to MPs + Hg/CPF and dissolved Hg/CPF (182.8-236.4 nmol min-1 mg-1 of AChE and 74.1-101.7 nmol min-1 mg-1 of GRx). Principal component analysis suggested different modes of action for Hg and CPF.

Metal and metallothionein concentrations in mesozooplankton from an oligotrophic offshore area in the eastern Mediterranean Sea (Cretan Passage/Levantine Sea).

Spatial variability of Cd, Cu, Cr, Ni, Zn, Fe, Mn, Pb and metallothionein (MT) concentrations were determined in mesozooplankton samples along the west-east axis of the Cretan Passage in the western Levantine Sea (Eastern Mediterranean). Metal and MT values from the present study are proposed as background levels, due to the lack of substantial anthropogenic activities in the area, where only maritime traffic and atmospheric deposition could be potential sources. Higher concentrations, of both metals and MTs, were recorded mainly at the western stations indicating higher metal bioavailability than in the eastern part. An inverse relationship of the metal zooplankton levels with zooplankton biomass, abundance and vital rates (production, respiration and ingestion rates), as well as salinity, was evident. We discuss the hypothesis that physical and biological characteristics of the marine environment, affecting growth dynamics of phytoplankton and zooplankton communities, may also act on metal uptake in oligotrophic marine systems.

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.

Organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) occurrence in Sparus aurata exposed to microplastic enriched diets in aquaculture facilities.

Organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) are highly stable and bioaccumulative, and microplastics (MPs; plastics <5 mm) are ubiquitous in the marine environment. In this study we report the levels of a selection of pollutants in liver and muscle of juvenile gilthead seabream (Sparus aurata) exposed to virgin and weathered MP enriched diets during three months and followed by one month of MP depuration. In general, a major concentration of pollutants in liver was observed. According to OCPs, total DDTs was two or three times higher in liver. Levels in muscle suffer higher variability between treatments and sampling periods. The MP index was negatively correlated to HCB and positively to p,p'DDT in liver. Levels of pollutants are correlated to biological parameters such as total size and total weight. Our results suggest that the bioaccumulation is subjected to the molecular structure of the pollutants and that MPs are correlated with the detoxification system.

Sea snail (Hexaplex trunculus) and sea cucumber (Holothuria polii) as potential sentinel species for organic pollutants and trace metals in coastal ecosystems.

The seasonal bioaccumulation of trace metals, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and organochlorinated pesticides (OCPs) in sea snail (Hexaplex trunculus) and sea cucumber (Holothuria polii) from Mar Menor lagoon were characterised. The highest concentrations of p,p'-DDE were detected in the central and south part of Mar Menor lagoon. However, the highest concentrations of metals in sea snail and holothurians were detected in the influence area of El Beal wadi. Biomagnification factors (BMF) in sea snail from cockle (sea snail-cockle concentration ratio) were higher than 5 for metal and organochlorinated compounds. However, similar concentrations were observed in both species for PAHs due to gastropods capability of metabolising these pollutants. Consequently, sea snail is proposed as a sentinel for trace metals, PCBs and OCPs in the coastal lagoons, not only due to its bioaccumulation and biomagnification capacity but also the easy sampling and amply distribution in many coastal areas.

Metal and metallothionein levels in zooplankton in relation to environmental exposure: spatial and temporal variability (Saronikos Gulf, Greece).

Metal and metallothionein (MT) in mixed zooplankton were investigated as means of monitoring metal availability regarding environmental exposure. Spatial and temporal variability of Cd, Cu, Ni, Zn, Fe, Mn and Pb in zooplankton and seawater were studied in Saronikos Gulf (Aegean Sea, Eastern Mediterranean), once every second month during an annual cycle (2011-2012). Particulate organic carbon and chlorophyll α were also measured in seawater samples. Median zooplankton metal concentrations were 0.65, 32.4, 7.1, 864, 1420, 40.2 and 26.8 µg g-1 dw for Cd, Cu, Ni, Zn, Fe, Mn and Pb, respectively, and 109 µg g-1 ww for MTs. Metal levels in zooplankton and MTs were higher at sites influenced by human-derived pressures. Additionally, metal concentrations in pelagic fish flesh from the Greek MED-POL data base were used for bioconcentration and biomagnification factors calculation. Bioconcentration from water to zooplankton was higher than metal transfer from either seston to zooplankton or zooplankton to fish.

Combining an effect-based methodology with chemical analysis for antibiotics determination in wastewater and receiving freshwater and marine environment.

Two different methodologies were combined to evaluate the risks that antibiotics can pose in the environment; i) an effect-based methodology based on microbial growth inhibition and ii) an analytical method based on liquid-chromatography coupled to mass spectrometry (LC-MS). The first approach was adapted and validated for the screening of four antibiotic families, specifically macrolides/ß-lactams, quinolones, sulfonamides and tetracyclines. The LC-MS method was applied for the identification and quantification of target antibiotics; then, the obtained results were combined with ecotoxicological data from literature to determine the environmental risk. The two methodologies were used for the analysis of antibiotics in water samples (wastewater, river water and seawater) and biofluids (fish plasma and mollusk hemolymph) in two monitoring campaigns undertaken in the Ebro Delta and Mar Menor Lagoon (both in the Mediterranean coast of Spain). Both approaches highlighted macrolides (azithromycin) and quinolones (ciprofloxacin and ofloxacin) as the main antibiotics in wastewater treatment plant (WWTP) effluents with potential risk for the environment. However, no risk for the aquatic life was identified in the river, lagoon and seawater as antibiotic levels were much lower than those in WWTP effluents. Fish from Ebro River were the organisms presenting the highest antibiotic concentration when compared with bivalves (mussels) from the Mediterranean Sea and gastropods (marine snails) from the Mar Menor Lagoon. The effect-based methodology successfully determined antibiotic risk in wastewater, but its applicability was less clear in environmental waters such as seawater, due to its high detection limits. Improving sample preconcentration could increase the method sensibility. Overall, combination of both methodologies provides comprehensive insights in antibiotic occurrence and risk associated in areas under study.

The hydrological regime of a large Mediterranean river influences the availability of pollutants to mussels at the adjacent marine coastal area: Implications for temporal and spatial trends.

The present study reports the levels and trends (1993-2013) of DDTs, PCBs and metals in mussels collected at two coastal sites influenced by the River Ebro discharges. Results showed that levels of PCBs, DDTs, Cd and Hg were related to water inputs from the river, while these did not seem to be the main source for the Pb, Zn, Cu and As levels observed. Significant relationships were observed between PCBs and DDTs levels and the river flowrate in the fast-flowing months, suggesting that overflow periods promoted the mobilization of contaminants from sediments, and their subsequent discharge and spreading across the sea. Results indicated that PCBs were effectively southwestern transported, probably in relation to their soluble behavior in stream waters, while DDTs were limitedly spread in seawater, likely due to their particulate behavior. Significant declining trends observed for some metals, PCBs and p,p' DDE may reflect the reduction of pollutants inputs along the river basin. However, the results also showed significant relationships between the river's flowrate in the fast-flowing months and PCBs and DDTs levels, and significant decreases on the river flowrate in some fast-flowing months during the long-term series studies. Both facts suggested that the reduction on overflow periods influenced the decreasing pollutants trends observed in mussels. The present study advises that decreasing trends in contaminant levels in Mediterranean estuarine coastal areas may be related not only to successful remediation works/policies, but also to the fluctuations on the hydrological regime of the rivers, which are directly linked to current climatic trends.

Microplastic distribution in surface sediments along the Spanish Mediterranean continental shelf.

Microplastics (MPs) are widely recognised as a contaminant of emerging concern in the marine environment. This work provides original data of the presence of MPs in coastal sediments along the Spanish Mediterranean continental shelf. Ten surface sediment samples were collected in order to document baseline microplastic distribution from Algeciras to Barcelona. Microplastics were extracted from bulk sediments by density separation. The number of microplastics per kilogramme of dry weight ranged from 45.9 ± 23.9 MPs/kg d.w. observed at Palma de Mallorca to 280.3 ± 164.9 MPs/kg d.w. noted at Málaga, with an average value of 113.2 ± 88.9 MPs/kg d.w. The lower limit is defined by the pore filter size used (1.2 µm). For all analysed locations, the dominant microplastic type was fibres (82.9%), followed by fragments, and the main colours were transparent and blue. Microplastic size distribution was presented; in the case of fragments, 85% was lower than 0.5 mm, and in the case of fibres, the three studied intervals (0.5-1, 1-2, 2-5 mm) had similar distribution (35, 34 and 31%, respectively). Attending to all available data, no statistically significant relationship (Spearman's correlation) was found between microplastic average size and distance to the coast, the depth, density population and sediment grain size. Neither relationship was observed between these variables and microplastic concentration using the non-parametric Kruskal-Wallis H test. This study has confirmed the widespread distribution of MPs in surface sediments from the Spanish Mediterranean continental shelf, and these data are useful to define baselines for MPs in the Western Mediterranean region.

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.

Relationship between physiological measurements (SFG -scope for growth-) and the functionality of the digestive gland in Mytilus galloprovincialis.

The present study was aimed to establish the relationship between the functionality of the digestive gland and physiological rates including SFG (scope for growth) in wild mussels, Mytilus galloprovincilis. The experimental set-up consisted in the evaluation of changes in the morphology of the gland, as well as in the activity of some key digestive enzymes (amylase, laminarinase, cellulase and protease) within a broad range of SFG obtained through manipulation of food ration. The higher SFG values were correlated to an increase in both the size of the digestive gland and the activities of enzymes when expressed in relation to individual. In contrast, no clear relations were observed when the activity of enzymes was expressed in relation to soluble protein, with the exception to amylase. The higher protease activities measured in mussels showing lower SFG may reflect an initial stage of catabolic processes intended to compensate the energy deficit produced by food restriction. The potential use of parameters measured in digestive glands in studies of marine pollution was discussed.

Assessment of the mechanisms of detoxification of chemical compounds and antioxidant enzymes in the digestive gland of mussels, Mytilus galloprovincialis, from Mediterranean coastal sites.

In this study the effects of the main marine pollutants (metals, PAHs, PCBs and DDTs) were assessed in native mussels from the Mediterranean coast of Spain. For this purpose several biomarkers such as benzo[a]pyrene hydroxylase (BPH), DT-diaphorase (DTD), glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPs), glutathione reductase (GR), metallothionein (MT) and lipid peroxidation (LPO) were measured in the digestive gland. Results showed increased LPO levels in mussels which accumulated high loads of organic compounds and arsenic in their tissues. BPH levels correlated to the concentrations of organic compounds in mussel tissues, though the range of BPH response was low in relation to the high gradient of accumulation of organic pollutants. Increased BPH levels, concomitant to low DTD and GST activities, were detected in mussels which presented high levels of organic pollutants in their tissues. This suggests that signs of LPO present in these organisms are related to the imbalance between phase I and phase II biotransformation processes. Furthermore, the increased levels of MT and CAT detected in mussels which showed high levels of Cd in their tissues appear to reflect a coordinated response which protects against the toxicity of this metal. The application of these biomarkers in environmental assessment is discussed.

Micronuclei and other nuclear abnormalities in mussels (Mytilus galloprovincialis) as biomarkers of cyto-genotoxic pollution in mediterranean waters.

Environmental genotoxicity and cytotoxicity along the Spanish Mediterranean coast was investigated through the determination of levels of micronuclei (MN) and other nuclear abnormalities (NAs) such as nuclear buds (NB) and binucleated cells (BN) in gills of wild mussels, Mytilus galloprovincialis, from 17 study sites. The results obtained were studied in relation to the exposure to main pollutants (metals, PAHs, PCBs and DDTs), gill antioxidant enzyme activities and environmental variables (temperature and salinity). The highest MN and NB levels were found in mussels from metal-polluted sites, such as Cartagena (MN: 11.6‰, NB: 4.6‰) and Portman (MN: 8.0‰, NB: 3.5‰), where genotoxicity seemed to be related to the oxidative stress generated by exposure to metals. High frequencies of MN and NB in mussels from Algeciras 1 (MN: 8.6‰, NB: 4‰) and of MN (7.2‰) in mussels from Manilva also indicated genotoxicity, though not attributable to the pollutants analysed. In contrast, MN levels at sites highly polluted by organic contaminants such as Barcelona, Vallcarca, Tarragona and Valencia, were lower than expected (ranging from 3.8 to 5.8‰). On the other hand, evidences of cytotoxicity indicated by the high frequencies of BN found in mussels from Vallcarca, Barcelona, and Cartagena (3.9, 3.4, and 2.3‰, respectively) appeared to be specifically related to the accumulation of organic pollutants. Positive correlations found between MN and NB frequencies, and also between both NAs and water temperature, suggested that they were related genotoxic events, and that this environmental factor is able to influence their levels and has to be taken into account in the application of these biomarkers in field studies.

Western Mediterranean coastal waters--monitoring PCBs and pesticides accumulation in Mytilus galloprovincialis by active mussel watching: the Mytilos project.

In order to evaluate the contamination levels in the Western Mediterranean basin, the active mussel watch methodology has been applied. This methodology consists of mussel transplantation (Mytilus galloprovincialis) from non impacted areas to selected coastal areas, characterised by potential impact from the continent due to contaminating sources. The areas of interest were selected along the entire coastal development of the Western Mediterranean sea, 122 sites in total. The time of mussel caging exposure was 12 weeks. The project was co-financed in the frame of the Interreg IIIB Meddoc Programme, aimed at determining the overall chemical quality of the Mediterranean sea, consistent with the Water Framework Directive 2000/60. Several partners representative of the coastal Mediterranean Countries were involved in the Project, with the purpose of building up a common surveillance network, adopting shared methodologies. In this paper we present the results of three yearly monitoring campaigns (2004, 2005, 2006) carried out along the coasts of Italy, France, Spain, Morocco, Algeria and Tunisia, including the coastal environment of Baleares, Sicily, Sardinia and Corsica. The contamination levels of Pesticides (DDT and its metabolites, Hexachlorocyclohexane isomers alpha and gamma) and Polychlorinated biphenyls, are reported and discussed. Statistical elaborations performed on the original data set were mainly aimed at validating the raw sample distributions, by means of the Johnson method. Both DD and PCB species frequency distributions have been approximated to appropriate theoretical distributions, belonging to the Log-normal and Bounded families. By integrating the related Probability Density Functions (p.d.f.), different accumulation values for DDT, DDD and DDE and PCB species have been estimated, corresponding to fixed percentage points of the area under the respective curves. By choosing appropriate probability level boundaries (33rd and 66th percentile), different regional zones have been ranked in terms of low, medium and high accumulation for Pesticides and PCBs.

Integrated use of antioxidant enzymes in mussels, Mytilus galloprovincialis, for monitoring pollution in highly productive coastal areas of Galicia (NW Spain).

In the present work, we investigated the potential use of several antioxidant enzymes in wild mussels (Mytilus galloprovincialis) as biomarkers of marine pollution. The enzymatic activity levels of glutathione S-transferase (GST), glutathione peroxidase (GPx) and catalase (CAT) were measured in gills and digestive gland. Those enzymes participate in the cellular defense system that is involved in the adaptive response of organisms to chemical pollution. Adult mussels were collected at five sampling sites located at the Ría de Pontevedra and Ría de Vigo. Seasonal variations in the control site were observed for the CAT activity, but no significant variability was found for GST and GPx. Mussels from the most polluted sites exhibited a significantly greater GST activity compared to the control site (p<0.05) during the sampling period, whereas GPx and CAT activities have not such a marked pattern. Trace metals, PAHs, PCBs and DDT contents in mussels at sampling sites showed highly significant positive correlations with the GST activity. This study supports the use of GST as a useful biomarker for long-term pollution monitoring in marine coastal ecosystems, whilst more research is needed for GPx and CAT.

Application and evaluation of the neutral red retention (NRR) assay for lysosomal stability in mussel populations along the Iberian Mediterranean coast.

The neutral red retention (NRR) assay to determine lysosomal membrane stability (LMS) was applied to wild mussels (Mytilus galloprovincialis) sampled from fourteen sites with different degrees of chemical pollution along the Iberian Mediterranean coast in 2002 and 2003. Total body burdens for a range of contaminants were measured in pooled samples (n = 50) of whole soft tissues in order to explore possible causality. Mean LMS values (n = 15) were significantly greater in mussels from one of the selected two reference sites (San Diego) than in chemically well-characterized hotspot sites. Mean LMS values were inversely correlated with contaminant concentrations in mussel tissues (except for Zn). The multidimensional scaling ordination technique classified the sampling sites into three groups according to their chemical-physical-biological similarities and the results were statistically confirmed using ANOSIM analysis. The results show that lysosomal stability is an effective indicator of health status in mussels along the Iberian Mediterranean coast and that it is related to body burden of contaminants, particularly polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs).