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.

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.

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.

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.

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.

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.

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.

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.

Impact assessment of agricultural inputs into a Mediterranean coastal lagoon (Mar Menor, SE Spain) on transplanted clams (Ruditapes decussatus) by biochemical and physiological responses.

The Mar Menor is a coastal lagoon threatened by the development of intensive agriculture in the surrounding areas. Large amounts of pesticides from these areas are discharged into El Albujón, a permanent watercourse flowing into the lagoon. We have used a multi-biomarker approach to assess the biological effects of agricultural pollution on a bivalve species. Biomarkers indicative of neurotoxicity (acetylcholinesterase, AChE), oxidative stress (catalase, CAT; glutathione reductase, GR and lipid peroxidation, LPO), phase II biotransformation of xenobiotics (glutathione S-transferase, GST) and physiological stress (scope for growth, SFG) were measured in clams transplanted to four sites of the lagoon (two reference sites and two sites affected by the dispersion of the effluent of the El Albujón), for exposure periods of 7 and 22 days. The hazards of this effluent were also examined by simultaneously measuring up to 83 contaminants (pesticides, PCBs, PAHs and others) in samples of fresh water from the watercourse mouth and seawater from the deployed sites, as well as the bioaccumulation of organochlorinated compounds and PAHs in the transplanted animals. Biomarker responses showed marked differences between reference and affected sites after 7 and 22 days. However it was only after 22 days that principal component analysis (PCA) of the biomarker responses distinguished between clams deployed in sites affected by the dispersion of the effluent of the watercourse and those from the reference sites. The chemical analysis of water showed high concentrations of pesticides close to El Albujón watercourse mouth, with the greatest input flux corresponding to the organophosphate chlorpyrifos, followed by pendimethalin and naphthalene, and at lower levels acenaphthene, terbuthylazine-desethyl and chlorpyrifos-methyl. In this regard, PCA analysis showed that the biological effects of the mixture of pesticides in caged clams after 22 days were reduced levels of AchE and SFG and increased levels of GR and phase II GST activity. An integrated biomarker response index was calculated from the combination of these biomarkers, proving useful for the assessment of the impact of agricultural pollution in caged clams.