Uptake and Effects of Microplastics on Cells and Tissue of the Blue

Sep 10, 2012 - University of Basel, Programme Man-Society-Environment, Department of Environmental Sciences, Vesalgasse 1, CH-4051 Basel,...
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Uptake and Effects of Microplastics on Cells and Tissue of the Blue Mussel Mytilus edulis L. after an Experimental Exposure Nadia von Moos,*,†,∥ Patricia Burkhardt-Holm,‡ and Angela Köhler*,§

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University of Basel, Department of Environmental Sciences, Vesalgasse 1, CH-4051 Basel, Switzerland and Alfred Wegener Institute for Polar and Marine Research, Am Handelshafen 12, 27570 Bremerhaven, Germany ‡ University of Basel, Programme Man-Society-Environment, Department of Environmental Sciences, Vesalgasse 1, CH-4051 Basel, Switzerland § Alfred Wegener Institute for Polar and Marine Research, Cell Biology and Toxicology, Chemical Ecology in Biosciences, Am Handelshafen 12, 27570 Bremerhaven, Germany S Supporting Information *

ABSTRACT: In this study, we investigated if industrial high-density polyethylene (HDPE) particles, a model microplastic free of additives, ranging > 0− 80 μm are ingested and taken up into the cells and tissue of the blue mussel Mytilus edulis L. The effects of exposure (up to 96 h) and plastic ingestion were observed at the cellular and subcellular level. Microplastic uptake into the gills and digestive gland was analyzed by a new method using polarized light microscopy. Mussel health status was investigated incorporating histological assessment and cytochemical biomarkers of toxic effects and early warning. In addition to being drawn into the gills, HDPE particles were taken up into the stomach and transported into the digestive gland where they accumulated in the lysosomal system after 3 h of exposure. Our results show notable histological changes upon uptake and a strong inflammatory response demonstrated by the formation of granulocytomas after 6 h and lysosomal membrane destabilization, which significantly increased with longer exposure times. We provide proof of principle that microplastics are taken up into cells and cause significant effects on the tissue and cellular level, which can be assessed with standard cytochemical biomarkers and polarized light microscopy for microplastic tracking in tissue.

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manufacture, are released during transport and handling.2,4 Other sources of micro sized particles and smaller are increasing amounts of consumer products (e.g., cosmetics) that contain exfoliants as well as industrial abrasives from sandblasting cleaning media (acrylic or polyester beads), which are commonly applied in the maritime industry.16−19 With decreasing size, the plastic fragments are potentially available to an increasing number of marine species, especially to indiscriminate feeders at the base of the food web.3,4 Microplastics are known to be ingested both by invertebrates such as polychaetes, crustaceans, echinoderms, bryozoans, and bivalves14,20−22 and by vertebrates such as fish and seabirds.2,4,23−25 Microplastics have also been detected in planktonic organisms such as zooplankton, chaetognatha, larval fish, copepods, and salps.9,19,26 However, neither the extent of microplastic ingestion, nor the possible pathways of transition into cells or the toxicological effects at the cellular and tissue level are known.4 Therefore, the goal of the present exposure study was to evaluate whether the chosen model microplastic is

INTRODUCTION The marine environment is polluted by large amounts of anthropogenic marine debris, which principally consists of synthetic polymers (plastics) of all shapes, colors, and sizes that originate from industrial applications and consumer products.1−5 The mostly petroleum derived plastics exhibit yet unknown, but long degradation times in the marine environment and can therefore persist for years to decades.4,6 Once in the oceans, the long-lived, often buoyant plastics are dispersed by winds and currents over long distances across the globe. Plastics have become ubiquitous in the marine environment and are present even in the remotest areas.2,5,7,8 With time plastics at the water surface accumulate in gyres,9,10 or sink to the sea bed11,12 due to waterlogging or surface fouling followed by colonization13 and accumulate in sediments14,15 or are washed ashore and litter coastlines.2,4,5 Under the influence of wave action and UV, large plastic debris items gradually degrade into smaller fractions, thus giving rise to fragments generally categorized as microplastics ( 0−80 μm in size, as provided by manufacturers as the raw material for various industrial applications. Since polyethylene and polypropylene together account for about 50% of all plastics consumed in Europe,27 polyethylene is likely to be fairly abundant in the marine environment.16 High-density microplastics in the environment can temporarily be suspended in the watercolumn by turbulence and even remain suspended, especially if they enter the sea through estuaries, when high flow rates predominate or if they possess a high surface area.15 Microplastics on the seafloor can also be resuspended after storms that cause sufficient turbulence in the water column.28 By virtue of its broad geographical distribution, abundance, basal position in the food web, easy accessibility and cultivation as well as its well understood biology, the blue mussel (Mytilus edulis L.) was chosen as the model organism. Being a sessile suspension feeder, the blue mussel effectively reflects ambient environmental contamination. It is therefore an internationally accepted sentinel species of early warning for monitoring marine pollution and is applied in the U.S. Mussel Watch, Assessment and Control of Pollution in the Mediterranean region (MEDPOL) and the North East Atlantic Oslo and Paris Commission (OSPAR) monitoring programmes.29,30 The digestive gland is a well-known target organ of xenobiotic effects in the blue mussel.31−33 Cellular alterations and physiological responses due to contaminant exposure are readily detected within it using an array of standardized procedures.34,35 To monitor mussel health we selected a set of established cytochemical biomarkers, which were supplemented with histological observations of response. The cytochemical tests included lysosomal membrane stability (LMS), neutral lipid content and lipofuscin accumulation in the mussel digestive gland. Lysosomal membrane destabilization is associated with general stress, but also with specific toxicological, physiological and pathological responses. The LMS test visualizes alterations of lysosomal membrane integrity and enables the evaluation of a mussel’s current health status. It is a sensitive bioindicator for monitoring and predicting ecosystem health.36 Lipid storage disorders are a common response to xenobiotic exposure, which can lead to an excess or deficiency of unsaturated neutral lipids in the lysosomal vacuolar system of digestive cells which can be visualized by the Oil Red O (ORO) technique.33,37,38 The accumulation of lipofuscin in the lysosomal vacuolar system of the digestive cells of molluscs is an indicator of oxidative stress in cells and is related to the level of membrane lipid peroxidation.39 Histological analyses involved the observational study of particle uptake into intact gills as well as the semiquantitative assessment of particle uptake and host response (granulocytoma formation) in hematoxylin and eosin (H&E) stained tissue sections of the digestive gland. To achieve this we developed a method based on polarized light microscopy (PLM) that allowed us to microscopically track plastic particles in intact gills and cryostat tissue sections of the mussel digestive gland.

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MATERIALS AND METHODS

The Model Organism. Blue mussels were sampled on July 22, 2009 at the offshore terminal of Helgoland (54°10′13.80″ N; 7°53′28.58″ E). A stock of approximately 500 mussels were acclimatized for 12 weeks in three large flow-through maintenance tanks, each containing ca. 150 L of filtered North Sea water (31‰ salinity) at 15 °C, which was entirely turned over about once a day. The closed seawater circulation system at the Alfred Wegener Institute is supplied with seawater from a designated reference area in the North Sea. The water quality of the circulation system is regularly monitored for all relevant parameters such as oxygen, nitrite, nitrate and ammonium as well as for chemical pollutants. The mussels were fed the recommended product concentrations (1 drop per 4−5 L) of “Liquifry Marine” (Interpet House, Vincent Lane, Dorking, Surrey, RH4 3YX, United Kingdom) twice a week at 9 a.m. Water inflow was stopped for 1−2 h during feeding. The maintenance tanks were cleaned weekly and their water renewed by ca. 30%. All mussels used in the experiment were carefully inspected during analysis and were free of parasites. The Model Microplastic. The synthetic polymer powder used as a model microplastic was a high-density polyethylene (HDPE) fluff (abifor 1300/20, Abifor Zürich, Switzerland) with nonuniformly shaped grains ranging > 0−80 μm in size (Gaussian distribution). The grain size distribution according to EN-ISO 4610 standards is: < 50 μm = 35−45%, < 63 μm = 60− 80%, < 80 μm = 98−100%. According to Abifor AG, the agranular fluff provided for this exposure experiment was free of any additives (Pers. comm. Marco Hilhorst, Abifor AG). A nominal concentration of 2.5 g HDPE-fluff/L was added to the treatment tanks as powder. Exposure concentrations were chosen on the basis of trial experiments. Each exposure beaker was equipped with two aeration stones, one at the top and one at the bottom, to ensure constant aeration and to attempt an even distribution of HDPE particles in suspension. Mortality of exposed mussels was zero during the experiments. Experimental Setup and Sampling Procedure. The exposure experiments were conducted at a constant temperature of 15 °C and a 12 h light-dark illumination regime. Exposure times were 3, 6, 12, 24, 48, and 96 h. In total, six independent exposure experiments were performed, one for every exposure time. The basic experimental setup consisted of six glass beakers containing 2 L of filtered North Sea water. Each glass beaker contained three mussels, which were randomly selected from the main mussel maintenance tank, amounting to a total of 18 mussels per experiment. Three beakers received the HDPE treatment (i.e., nine mussels) and three beakers served as unexposed negative controls (i.e., nine mussels). This basic setup was repeated the same way for every exposure time (i.e., six times). The water in exposure beakers was exchanged every 12 h and replenished with HDPE where necessary. Liquifry Marine was administered in accordance with the regular feeding schedule of the main mussel maintenance tanks. Experiments were planned such that feeding during the exposure experiments was avoided. However, this was not possible for the 96 h exposure experiment, which received food once after 48 h (half a drop of LiquifryMarine per beaker, i.e. 0.5 L of a 1 L solution containing 1 drop). Sampling was always carried out at 10 a.m. Total body weight (g) was measured with a precision balance (0.01 g precision). Shell length (cm), shell width (cm), and shell height (cm) were determined with a 11328

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Figure 1. H&E stained tissue sections showing particles in the intestine (arrows) after 3 h of exposure, in bright field microscopy (A) and bright field overlaid with polarized light (B). Microplastic particles (arrows) appear glowing blue in polarized light (B).

regions of interest in which the stained lysosomes are measured with respect to the unstained tissue background in area percent. For analysis, three measurements were performed for every destabilization time and mussel. The results obtained were averaged for statistical evaluation. All samples were assessed blindly. Lipofuscin. Cryostat sections were stained using the Schmorl’s reaction32 and observed under the microscope (Zeiss Axio Scope). Lipofuscin content was quantified by image analysis analogously to the quantification of the LMS staining intensity. Neutral Lipids. Following post fixation in Baker’s calcium formol and staining with ORO, frozen sections were mounted in Kaiser’s glycerine gelatin. Neutral lipid content was semiquantitatively assessed with the microscope (Zeiss Axio Scope, 100× magnification). Histological Analyses. We analyzed intact gills for the presence of plastic particles and signs of uptake by PLM. The gill data obtained were strictly descriptive and observational. Microplastic uptake into the digestive system and host response (granulocytoma formation) were semiquantitatively assessed with H&E stained cryostat sections using bright field and polarized light microscopy (Zeiss Axio Scope, 100× and 400× magnification). Semiquantitative Assessments. In a first step, all cryosections were quickly screened under the microscope to assess the range of the response of interest. The sections with the least and greatest responses were searched and assigned the values 0 and 3, forming the lower (minimum response) and upper end (maximum response) delineations of the custom scale. In a second step, several representative intermediate sections in steps of 0.5 were searched to complete the scale (PubMed (SI) Table S1). For the evaluation of neutral lipid a different approach was tested using a visual scoring chart (PubMed SI Figure S1). All scoring scales had an equally finely graduated scale of seven grades. Finally, to assess the responses all cryosections were assigned a value according to the custom scale developed in this way. For every mussel, one slide with two intact cryosections was screened and the assigned score represented the overall condition of both sections. Every slide was screened blindly in three independent rounds. The distance between sections was not monitored. However, special consideration was given to eliminating peripheral sections and selecting sections nearest to the center of the digestive gland, those closest to the optimal cross section. To guarantee

vernier caliper (0.1 mm precision) and softbody weight (g) and shell weight (g) were weighed (0.01 g precision). Shell length varied between 5 and 6 cm. Five digestive tracts were mounted onto a single precooled metal chuck at a time. The loaded chucks were briefly immersed in a hexane bath previously cooled to −70 °C in liquid nitrogen. The chucks holding the super cooled tissue samples were wrapped in parafilm and aluminum foil and stored at −80 °C for further histological and histochemical processing. Condition Index (CI). The CI was calculated for each mussel. It is an indicator of starvation and is defined as the quotient of wet meat weight (g)] and shell weight (g) multiplied by 100 (1):40 CI = (wet meat weight[g]/shell weight[g]) × 100

(1)

Preparation of Gills. Entire gills were removed with sharp scissors, cut into streaks and fixed in Baker’s calcium formol (4%). For analysis, the branchiae were separated and all gill surfaces as well as all frontal faces of ciliated filaments were screened for the presence of microplastic particles using polarized light microscopy at 100×, 400×, and 600× magnification. The epithelium of the gills and blood vessels were also screened to verify whether smallest particles possibly entered the gills directly. Cryotomy. Tissue sections were prepared with a cryostat (MICROM HM 500 0M) equipped with a motorized cutting device at a chamber temperature of −25 °C and object temperature of −22 °C. The cryostat tissue sections of 10 μm thickness were collected onto glass slides kept at room temperature causing tissue sections to flash-dry immediately upon contact. Two tissue sections were collected onto every glass slide. The serial tissue sections for the measurement of lysosomal membrane stability were stored at −20 °C for no longer than 24 h before further histochemical processing whereas the rest of the tissue sections were stored at −80 °C until needed. Lysosomal Membrane Stability (LMS). The LMS assay was performed on serial sections (14 per mussel) of the sampled mussel digestive glands prepared according to Moore et al.41 Further details on the testing procedure are provided in the PubMed Supporting Information (SI). The reaction product in the serial sections was quantified with a light microscope (Zeiss, Axio Scope) using 400× magnification in combination with computer-assisted image analysis software (Axiovision, Zeiss). The software allows the designation of 11329

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the smallest particles or dust. We also detected particles in the blood lacunae of the gills and in the areas of lamellar junctions (PubMed SI Figure S2, D, small arrow). Larger particles were seen on the gill surface (PubMed SI Figure S2, B). The second pathway occurred via ciliae movement, which transferred the particles to the stomach, intestine and primary as well as secondary digestive tubules. Histologically, microplastic particles were found in the intestine (Figure 1) and numerous particle aggregates were observed in the lumina of primary and secondary ducts and in tubules of the digestive gland (Figure 2). In the digestive tubules, microplastic particles were taken up and accumulated in the lysosomal system of ductular and tubular epithelial cells as early as from 3 h on (Figure 2). The median values in exposed mussels were significantly higher than those of controls (0−0.25) and steadily increased from 1.0 after 3 h to nearly 2.0 after 96 h of exposure (p-value ≤ 0.01, PubMed SI Table S1). Numerous particle aggregates were observed in the lumina of primary and secondary ducts and in the tubules of the digestive gland, which clearly displayed lysosomal characteristics after fusion with lysosomes (Figure 3C). We also observed particles in the connective tissue (Figure 1B, top right), which were likely eliminated by epithelial cells of the ducts and tubuli and subsequently phagocytosed by the attracted eosinophilic granulocytes that migrated into the tissue and formed the observed granulocytomas. Effect of Exposure to Microplastics (Main Effect 1). Exposure to microplastics induced a significant increase in the end point granulocytoma formation and a significant decrease in lysosomal membrane stability (LMS) (main effect 1, p-value ≤ 0.01, PubMed SI Table S1). The onset of granulocytoma formation in exposed mussels occurred after 6 h of exposure and remained significantly higher than in control mussels during all subsequent exposure periods (Figure 4). Granulocytomas were comprised of eosinophilic granulocytes containing vacuoles with accumulated microplastic particles (Figure 5) and were mainly observed in the connective tissue of the digestive gland and in the direct periphery of the ducts and tubules. Compared to the formation of granulocytomas, the LMS test resulted in less pronounced differences between control and exposed mussels until 48 h of exposure and exhibited much higher fluctuations in the response patterns (Figure 6). After 96 h of exposure a dramatic and highly significant disruption of the lysosomal membrane integrity was observed for all three peaks. No effects were observed for the biomarkers of oxyradical damage (lipofuscin accumulation), disturbance in lipid metabolism (neutral lipid content) and in the condition index (data not shown). Effect of Exposure Duration (Main Effect 2). The duration of exposure (main effect 2) affected all response variables significantly except the variable “particle uptake” (p = 0.2247, PubMed SI Table S1) and was therefore retained in the model. Effect of Exposure and Duration (Interaction Term). The results of the fitted linear mixed-effects model, as summarized in SI Table S1, indicate that the interaction term of exposure and duration of exposure significantly affects the response variables LMS peak 2 (p-value = 0.01, Chisq = 6.38, Df = 6), LMS peak 3 (p-value = 0.01, Chisq = 5.93, Df = 6), and the degree of granulocytoma formation (p-value < 0.001, Chisq = 23.71, Df = 7). The degree of particles accumulated in vacuoles is just not significant (p-value = 0.05, Chisq = 3.83, Df

representative scoring of samples, the inhomogeneous fringes of cryosections were not considered to exclude possible artifacts from preparation. Statistics. A linear mixed-effects model was fitted for the experimental data with the lmer function in R42 to characterize the dependence of the response variables of an individual mussel on HDPE exposure (main effect one), on the time under treatment (main effect two) and on the interaction between the two main effects, all included as fixed-effects term. For the response variable “host response” (granulocytoma formation) the term I(Ttime∧2) was inserted into the model to correct for the nonlinear relationship between response variable and exposure time. Tank affiliation was incorporated as a random-effects term to correct for pseudoreplication. The main predictions for the linear model are that controls do not exhibit a slope in the linear model while treatments do. The main predictions were tested by running the lmer function on the maximum model containing all three factors of interest and comparing the AIC, BIC, Chi square, and p-values. Lastly, goodness of model fit was visually judged by plotting the residuals against the fitted values with the qqnorm function. Data analysis was performed with R 2.11.1 (R Development Core Team, 2010).

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RESULTS We generally observed the presence of the model microplastic HDPE particles on gills and inside the digestive system (Figure 1). Our approach using polarized light microscopy (PLM) facilitated the tracking and localization of the ingested microplastic particles on and inside the gills (PubMed SI Figure S2). PLM also allowed us to semiquantitatively determine both the amount of particles that had accumulated inside phagocytotic vacuoles of the lysosomal system of digestive tubules (Figure 2) and the subsequent host response

Figure 2. Median ± SE of particles accumulated in the lysosomal system of ductular and tubular epithelial cells in the digestive gland (scale ranging from 0 to 3, in 0.5 steps) at different exposure times for control (filled dots ●) and exposed (empty dots ○) mussels.

demonstrated by the formation of granulocytoma in the connective tissue surrounding the ducts and tubules after an exposure of up to 96 h (Figure 4). Uptake of HDPE Particles. HDPE particles were seen on the gills, indicating that they were trapped from the water column (PubMed SI Figure S2). We observed two pathways for the uptake of the model microplastic particles. One uptake pathway was mediated via the gill surface and presumably by microvilli, which directly transported the particles into the gills by endocytosis. This pathway is most likely mainly accessible to 11330

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Figure 3. H&E stained tissue sections of the mussel digestive gland tubuli. Image (A) shows a section of an unexposed mussel. Image (B) shows tubules of an exposed mussel (48 h) with vacuoles (heterophagosomes) that adopted lysosomal properties by fusion with the endolysosomal system, such as illustrated in image (C). Image (C) is a black and white image of an unstained section under PLM. Note the uptake of the plastic particles into the lysosomes by fusion events (arrows). LU stands for lumen and EC for epithelial cell.

the first time, they have been tracked inside epithelial cells of the primary and secondary ducts as well as in tubules of the digestive gland using polarized light microscopy. In the digestive gland microplastics caused the formation of granulocytomas (after 3 h) and a steady decrease in lysosomal stability (after 6 h). It was found that all observed biological responses, save for LMS peak 1, increased significantly with longer exposure times. No effects were observed in the end points lipofuscin accumulation, neutral lipid content (ORO) and condition index. The results suggest a clear sequence of response: particle ingestion (as of 3 h) is followed by granulocytoma formation (after 6 h) at the tissue level and finally by lysosomal destabilization at the cellular and subcellular level. Effects at the cellular and tissue level of biological organization generally occur rapidly and can serve as a good early warning system to assess the potential toxicological effects at higher levels of biological organization.43 Histologically, microplastic particles were found in the intestine, in the lumina of the primary and secondary ducts of the digestive gland and in endocytotic vacuoles of digestive epithelial cells. This suggests that particles were taken up via mouth, transported to the gastrointestinal tract and internalized into cells of the digestive system by endocytosis. The formation of granulocytomas is a non-neoplastic inflammatory cellular response, which is often associated with environmental pollution.44−46 It is an accepted indicator of hemocytic response to aquatic contamination44 as well as of a general loss of health in bivalves.31 The main cells forming granulocytomas around the accumulated HDPE particles are eosinophilic granulocytes, which are readily identifiable in H&E stained sections by their bright red coloration. As neither particle accumulation nor granulocytoma formation was observed in control mussels, the emergence and rapid increase of granulocytomas suggests a considerable cellular host response to microplastic exposure. As opposed to particle uptake, which quickly rose after 3 h to remain high, the formation of granulocytomas steadily increased as a function of exposure time (highly significant). The results on the granulocytoma condition are strongly supported by the observations on the lysosomal membrane stability (LMS), which is a well-established and very sensitive biomarker for xenobiotic-induced pathological changes.31,32,47 Numerous studies show that damage to the lysosomal membrane correlates with stress, toxicological responses and associated pathological reactions in social amoebae, molluscs and fish.36 Lysosomes are cell organelles that are involved in

Figure 4. Median ± SE of the degree of granulocytoma formation in the digestive gland (scale ranging from 0 to 3, in 0.5 steps) at different exposure times for control (filled dots ●) and exposed (empty dots ○) mussels as depicted in the corresponding image (in bright field overlaid with polarized light) of an exposed mussel (48 h) with accumulated plastic particles engulfed by granulocytomas (arrows).

Figure 5. H&E stained tissue section (in polarized light) of the digestive gland showing granulocytoma formation around accumulated HDPE particles (blue) in a vacuole, which was finally encapsulated by connective tissue (red arrow). The inset (bright field overlaid with polarized light) shows HDPE particles (blue) with a single eosinophilic granulocyte attached (red, black arrow), suggesting a possible migration of active phagocytotic blood cells attracted by the microplastic deposits in the connective tissue.

= 6) and no significant effects of the interaction term resulted in lysosomal peak 1 (p = 0.17, Chisq =1.87, Df = 6) (PubMed SI Table S1).

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DISCUSSION Our study confirms that the industrial HDPE particles (>0−80 μm) enter the digestive system of Mytilus edulis L. where, for 11331

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Figure 6. Means ± SE of lysosomal destabilization time for (A) peak 1, (B) peak 2, and (C) peak 3 at different exposure times for control (filled dots ●) and exposed (empty dots ○) mussels.

Euparal, Kaiser’s Glycerine or Fluorescence Mounting Medium (Dako, S3023) as well as in samples of intact gills. PLM may be a useful new tool for histological analyses of microplastic uptake in ecotoxicological studies as it enables the detection and localization of plastic particles in organisms. However, a drawback of PLM is the inability to differentiate particle sizes. What is more, the ingested particles often displayed a nonuniform distribution within the tissue of the digestive gland and also had the tendency to cluster within vacuoles. We therefore mainly observed aggregates of particles rather than single particles. When viewed with PLM, the accumulated particle aggregates appeared as large, grainy blue spots. It was neither possible to differentiate and quantify the number of particles constituting an aggregate, nor to determine their sizes. However, particle size is a crucial factor determining particle interactions with biota and lastly toxicity.53,54 The proper characterization of particle size but also other material properties such as shape, surface properties and chemical composition is of toxicological importance and a fundamental issue for future ecotoxicological research.55,56 No studies have ever addressed these issues in the context of microplastic ecotoxicity up to now. Apart from size, the particle dispersion state is another pivotal factor in understanding the toxicity of colloidal systems such as dispersed microplastics in aquatic environments. Experimentally, an even suspension was difficult to achieve due to particle behavior (electrostatic adherence and agglomeration). Once introduced into the exposure beakers, the particles aggregated and collected on exposed surfaces of the beaker, the mussels and tubing of the aeration stones. The aeration stones used in this study provided a reasonable and pragmatic approach to meet this challenge. For future work in this field solutions must be found to these methodological issues and standardized material characterization and toxicological test methodologies must be implemented so as to ensure the validity and comparability of studies on the toxicity of microplastics. To date, little or no data exist on the real concentrations of microplastic particles (and smaller) in the marine environment. In consequence, we consciously settled for a relatively high exposure concentration for a proof of principle regarding the putative pathways of uptake and hazards in cells and tissue connected to the chosen material. Clearly, concentration levels and experimental design must be refined in future experimental studies to better approximate the real world situation in which not only low concentrations most probably predominate, but also a mix of different plastic types and plastic types in

numerous vital cellular functions such as turnover of macromolecules, digestion of cell organelles as well as enclosure of toxic substances and particles.48 There is substantial evidence that xenobiotics are incorporated into lysosomes to prevent toxic injury.33 An overloading of lysosomes with xenobiotics and/or direct damage to their membranes can cause their destabilization, which leads to increased autophagy and the release of hydrolases into the surrounding tissue that finally results in necrotic processes.48,49 Neutral red and cytochemical techniques for assessing lysosomal destabilization have successfully been used in fish and invertebrate taxa41,50−52 and are being incorporated into major European monitoring programs (BIOMAR, Black Sea Mussel Watch, OSPAR, MEDPOL). Thus, LMS tests provide valuable information on the physiological stress an individual mussel experienced. We showed that endocytotic vacuoles containing microplastic particles fuse with lysosomes in the digestive tissue of exposed mussels. The lysosomes exhibited shorter destabilization times that, in an overall trend, significantly decreased with longer exposure times. Often three peaks were observed in the destabilization time series of a single mussel, which can be ascribed to different development stages of lysosomal populations present in cells (old vs young lysosomes). Peak 1 represents late lysosomes (residual bodies with decreasing enzyme contents) that exhibit relatively low membrane stability of less than 10 min. It is likely that this aged lysosomal population accumulated large amounts of microplastic particles during their maturation process involving various fusion events. Interestingly, we observed that this lysosomal population only responded after 96 h of exposure but then by a complete breakdown of LMS in less than 3 min. This indicates that enlarged, late lysosomes have a high storage capacity for microplastic particles. Peaks 2 and 3 usually represent newly formed, “young” and rather small lysosomes with relatively high lysosomal membrane stability of over 35 min in normal conditions. These primary lysosomes usually receive digestive enzymes from the Golgi apparatus and fuse with secondary lysosomes. Yet, these two younger populations responded more sensitively and in a time dependent manner to microplastic exposure. This may be due to their comparatively limited storage capacity, which made them more susceptible to microplastics than the larger, aged lysosomal population. Polarized light microscopy (PLM) proved to be a useful tool for the detection of plastic particles in intact organs and tissue sections. It is a contrast-enhancing technique that is generally used for imaging doubly refracting materials. The HDPE particles were equally detectable in tissue sections mounted in 11332

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Environmental Science & Technology combination with other pollutants. In future studies it would therefore be interesting to investigate the effects of long-term chronic exposure (≥30 days) to low doses. Finally, it was hardly possible to discern whether the observed effects of microplastic exposure were solely induced by the physical ingestion of the particles and the potentially caused mechanical injuries or by the chemical toxicity of the microplastic particles themselves. In future, an additional control with particulate matter of similar shape and size, but of naturally occurring particulate material such as mineral grains would be appropriate.20 Regardless of the relatively high exposure concentration of HDPE particles used in the present study, which are undoubtedly unparalleled in the environment, we have shown that microplastic particles, as an emerging pollutant, can potentially be taken up by marine filter feeders at the base of the food chain and negatively affect their health condition. Upon ingestion, microplastics can potentially cause physical injury in the intestinal tract.23 They also have the potential to cause chemical harm by leaching inherent toxic monomers, chemical additives incorporated during manufacture or adsorbed persistent organic pollutants (POPs, such as polycyclic hydrocarbons (PAHS), polychlorinated hydrocarbons (PCBs) and alkylbenzenes). POPs have recently been traced on the surface of marine resin pellets and postconsumer plastic fragments which have been found to adsorb and concentrate lipophilic contaminants from ambient seawater by several orders of magnitude.57−60 Synthetic polymers in the aquatic environment must therefore also be considered as a potential contaminant reservoir and as potential vectors for the global dispersal of hydrophobic contaminants across the oceans and between pelagic and benthic oceanic compartments, and likewise, once ingested, as potential vectors for the transmission of adsorbed environmental chemicals or inherent additives to pelagic and benthic organisms (the “Trojan horse effect”). This has important ecotoxicological implications. The results obtained in this study provide a first proof of principle that microplastics are taken up into digestive cells of Mytilus edulis L. where they induce distinct adverse effects and that polarized light microscopy can be used to track and localize microplastic particles within organisms and organs.

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ACKNOWLEDGMENTS

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REFERENCES

N.v.M. is very grateful for the scholarships from Kanton AG, Switzerland and the financial contribution of the Usitawi Club, Basel, Switzerland. We are also much obliged to Katja Broeg, Tobias Roth, and to all collaborators at AWI and University of Basel for their contributions. We thank Abifor AG, Switzerland for providing the model microplastic and Schaetti Switzerland for material samples.

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ASSOCIATED CONTENT

* Supporting Information S

Supporting information includes details on the LMS test; Figure S1 showing the scoring chart for the accumulation of unsaturated neutral lipids; Figure S2 depicting transport and uptake of plastic particles by gills; Table S1 showing the results of the fitted mixed-effects model. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (N.v.M.); Angela.Koehler@ awi.de (A.K.). Present Address ∥

University of Geneva, Institute F.A. Forel, 10, route de Suisse, 1290 Versoix, Switzerland.

Notes

The authors declare no competing financial interest. 11333

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