Suspended micro-sized PVC particles impair the performance and decrease survival in the Asian green mussel Perna viridis

MPB-07880; No of Pages 8 Marine Pollution Bulletin xxx (2016) xxx–xxx

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Suspended micro-sized PVC particles impair the performance and decrease survival in the Asian green mussel Perna viridis Sinja Elena Rist a,⁎, Khoirunnisa Assidqi b, Neviaty Putri Zamani b, Daniel Appel c, Myriam Perschke d, Mareike Huhn b,e, Mark Lenz e a

Department of Biology, Faculty of Mathematics and Natural Sciences, Dresden University of Technology, 01062 Dresden, Germany Department of Marine Science and Technology, Faculty of Fisheries and Marine Sciences, Bogor Agricultural University, Jl. Agatis No.1, Bogor 16680, West Java, Indonesia Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Campus Kiel, Brunswiker Str. 10, 24105 Kiel, Germany d Institute for Chemistry and Biology of the Marine Environment, Carl-von-Ossietzky-Str. 9-11, 26111 Oldenburg, Germany e GEOMAR Helmholtz Centre for Ocean Research Kiel, Düsternbrooker Weg 20, 24105 Kiel, Germany b c

a r t i c l e

i n f o

Article history: Received 4 April 2016 Received in revised form 2 July 2016 Accepted 4 July 2016 Available online xxxx Keywords: Microplastics Perna viridis Clearance rate Respiration rate Byssus production Jakarta Bay

a b s t r a c t Marine bivalves are known to ingest microplastics, but information on the consequences for their physiological performance is limited. To investigate a potential exposure pathway that has not yet been addressed, we mimicked the resuspension of microplastics from the sediment in a laboratory exposure experiment. For this, we exposed the Asian green mussel Perna viridis to 4 concentrations (0 mg/l, 21.6 mg/l, 216 mg/l, 2160 mg/l) of suspended polyvinylchloride (PVC) particles (1–50 μm) for two 2-hour-time-periods per day. After 44 days, mussel filtration and respiration rates as well as byssus production were found to be a negative function of particle concentration. Furthermore, within 91 days of exposure, mussel survival declined with increasing PVC abundance. These negative effects presumably go back to prolonged periods of valve closure as a reaction to particle presence. We suggest that microplastics constitute a new seston component that exerts a stress comparable to natural suspended solids. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction It was estimated that in 2010 a total of 4.8 to 12.7 million metric tons of plastics entered the oceans worldwide and the yearly input is predicted to increase by an order of magnitude until 2025 (Jambeck et al., 2015). Plastic particles are therefore accumulating in all parts of the oceans: at the sea surface, in the water column and in sediments (Moore et al., 2001; Thompson et al., 2004; Barnes, 2005). Their fragmentation at sea is the most important source of marine microplastics (i.e. plastic particles b5 mm) (Thompson et al., 2004; Claessens et al., 2011). In addition to this, micro-sized plastic particles enter the oceans as pre-production pellets, textile fibers and as scrubbers from cosmetics or from abrasives used in air-blasting machines (Gregory, 1996; Fendall and Sewell, 2009). Microplastics are widely distributed in the water column (Thompson et al., 2004; Lima et al., 2014; Song et al., 2014) as well as in sediments (Claessens et al., 2011; Vianello et al., 2013; Nor and Obbard, 2014) and have also reached pristine habitats like the deep sea (Van Cauwenberghe et al., 2013) and the Arctic ocean (Obbard et al., 2014).

⁎ Corresponding author. E-mail address: [email protected] (S.E. Rist).

Due to their small size, microplastics can be taken up by a wide range of animals. This includes large predators like fish (Davison and Asch, 2011), seabirds (Colabuono et al., 2009) and mammals (Besseling et al., 2015), but also benthic invertebrates (Thompson et al., 2004; Graham and Thompson, 2009, Goldstein and Goodwin, 2013, Watts et al., 2014) and even zooplankton (Cole et al., 2013; Besseling et al., 2014). However, since the majority of all plastic fragments will finally sink to the seafloor (Woodall et al., 2014), benthic invertebrates are presumably the group of organisms that is most exposed to microplastic pollution. This is particularly true for benthic filter feeders, such as mussels, which ingest organic material that is suspended in near-bottom waters. It originates either directly from vertical transport or from the wave- or current-induced resuspension of deposited material. Several studies have documented the ingestion of microplastics by mussels and observed effects on their performance at the physiological, cellular and humoral level. Inflammatory responses followed by the accumulation of particles in the lysosomal system of the blue mussel Mytilus edulis occurred after the animals were exposed to polyethylene particles (1–80 μm) for a few hours (Von Moos et al., 2012). Furthermore, microplastics were found to be translocated to the cells of M. edulis 3 days after the ingestion of polystyrene beads (3 and 9.6 μm) (Browne et al., 2008) and reduced filtration rates were observed in individuals of M. edulis that were exposed to polystyrene particles (0.03 μm)

http://dx.doi.org/10.1016/j.marpolbul.2016.07.006 0025-326X/© 2016 Elsevier Ltd. All rights reserved.

Please cite this article as: Rist, S.E., et al., Suspended micro-sized PVC particles impair the performance and decrease survival in the Asian green mussel Perna viridis, Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.07.006

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S.E. Rist et al. / Marine Pollution Bulletin xxx (2016) xxx–xxx

for 8 h (Wegner et al., 2012). Many factors are assumed to influence the impact of microplastics on marine organisms, including particle size, shape, concentration, composition, weathering and erosional status as well as exposure time (Cole et al., 2011; Wright et al., 2013b), but the role of most of them remain to be investigated. Exposure time is of high ecological relevance, since marine invertebrates in many habitats most likely face microplastic pollution throughout their entire lifespan, which can reach from b 1 year to N 10 years, and this fact limits the interpretability of short-term tests. However, so far only one study on marine bivalves realized an exposure time that was longer than a few days: Sussarellu et al. (2016) exposed oysters to polystyrene microspheres (2 and 6 μm) for 2 months to cover a whole reproductive cycle and after this time they found that reproduction was impaired as they observed decreased sperm velocity, oocyte number and diameter as well as lower larval yield and growth. This was caused by a shift in energy allocation from reproduction to structural growth. Although many of these experiments clearly documented negative effects of microplastics on animal performance, their interpretation is limited. This is because all but one were run for timespans that were by far shorter than the exposure times marine invertebrates very likely experience in polluted environments. However, exposure scenarios, which are realistic with regard to the length of exposure as well as to the concentration of particles, are central for assessing the ecological consequences of microplastic pollution. Therefore, more studies that cover the time scale of months to even years are currently needed to come to a comprehensive understanding of what the consequences of the enrichment of microplastics in marine systems can be. A further aspect of microplastic pollution is the interaction of these materials with organic pollutants. In the marine environment plastic particles accumulate and transport persistent organic pollutants (POPs) like polychlorinates biphenyls (PCBs) or polycyclic aromatic hydrocarbons (PAHs) (Rios et al., 2007) and it is widely assumed that by this they can serve as a vector for pollutants to organisms (Teuten et al., 2007). The microplastics-mediated transfer and subsequent accumulation of POPs in tissues of Arenicola marina have already been documented for 19 different polychlorinated biphenyls (PCBs) (Besseling et al., 2013). Furthermore, increased genotoxic effects were observed in Mytilus galloprovincialis as a response to a 7-day exposure to pyrenecontaminated polyethylene and polystyrene (b 100 μm) in comparison to virgin plastic. Although the authors found an accumulation of pyrene in the tissue, the majority of the observed biological responses (immunological, lysosomal, cholinesterasic and antioxidant effects) were particle-induced and not influenced by the pollutant (Avio et al., 2015). These studies show that a microplastics-mediated transfer of POPs is possible, but it is still controversially debated whether this actually represent an ecologically relevant pathway for organic pollutants to marine organisms (Koelmans et al., 2013; Ziccardi et al., 2016). In a laboratory study, we simulated the resuspension of microplastics from coastal sediments and implemented different pollution levels. The particle loads covered several orders of magnitude to determine the threshold concentration that leads to a measurable impairment in physiological performance. Respiration and clearance rates were measured because they have widely been recognized as sensitive and reliable measures of stress in mussels (Chandurvelan et al., 2013; Zhao et al., 2014). Furthermore, we recorded byssus production as a response variable to assess physiological activity and to detect the impact of unfavorable conditions that may trigger stress responses that, in turn, consume energy which is then not available for the formation of byssus threads (Paul, 1980; Wang et al., 2012). Since microplastics can constitute a stress for mussels, we hypothesized the physiological performance to decrease with increasing amounts of microplastics. We used PVC particles (1– 50 μm) that were contaminated with an organic pollutant to mimic the situation in the field. The influence of microplastics on the performance of the Asian green mussel Perna viridis was then assessed over the course of 91 days.

2. Methods 2.1. Study site and mussel collection Individuals of the Asian green mussel Perna viridis used in this study were collected in Jakarta Bay, which is under the influence of various anthropogenic impacts. The waters of the bay are eutrophic and polluted with heavy metals and organic pollutants (Damar, 2003; Arifin, 2004). Concentrations of PAHs in Jakarta Bay sediments were found to range between 257 and 1511 ng/g due to petrogenic pollutants that presumably originate from the use of fossil fuels (Rinawati et al., 2012), while a constant import of suspended particulate matter into the bay leads to enhanced sedimentation rates (Arifin, 2004). Furthermore, water turbidity is high due to the complete mixing of the water column during each tidal change as Jakarta Bay is a shallow sea area with a mean depth of 8.6 m (Damar, 2003). We collected mussel individuals with a shell length of 3.5–4.0 cm from bamboo constructions near Muara Kamal ( 6° 4′ S, 106° 43′ E). The racks were deployed by fishermen as a settlement substratum for the bivalves, which represent an important protein source for the coastal population of the region (Arifin, 2004). During the 2 h transport to the marine laboratory in Bogor, we kept the mussels in cooled insulation boxes without seawater. After arrival, we randomly formed 6 groups of 30 mussels each, which all had the same shell length. Each group was transferred into a glass aquarium with 20 l of seawater and provided constant aeration. For the experiment, 2000 l of clean seawater were transported from the coast to the laboratory in Bogor, where they were distributed to two separate water cycles, of which both contained a storage tank and a filter unit. Mussel acclimatization to laboratory conditions lasted for 2 weeks, during which half of the water in the aquaria was exchanged daily and the mussels were fed with one million cells of an Isochrysis galbana culture per aquarium twice per day. Prior to the exposure experiment, we tested whether P. viridis takes up the PVC particles we chose for this study (see below). Ingestion of particles was confirmed by microscopic inspection of the faeces. Animals that were used in this pilot study were not included in the main experiment. 2.2. Exposure to microplastic particles 2.2.1. Plastic material We chose PVC particles due to their negative buoyancy in seawater, which makes them available for benthic invertebrates even if they are devoid of fouling. Furthermore, PVC makes up 19% of the global plastic production and is therefore a common component of marine microplastic waste (Andrady, 2011; Browne et al., 2010; Nor and Obbard, 2014). We purchased the material in a size range of 1–50 μm from PyroPowders (www.pyropowders.de). Prior to the experiment, the PVC material was submersed in fluoranthene-contaminated seawater for 24 days. This was done to mimic the conditions in the Bay of Jakarta, where plastic material presumably gets quickly contaminated due to the omnipresence of PAHs of which fluoranthene is a common component (Rinawati et al., 2012). Fluoranthene is a PAH that is released through the combustion of fossil fuels and has already been detected on microplastic particles in the ocean (Rios et al., 2007). For contaminating the particles in the laboratory, 100 g of PVC were mixed into 500 ml of seawater that contained 2 μg fluoranthene per l. We chose this concentration as a rather high but still ecologically relevant level since similar amounts of PAHs can occur in seawater close to oil spills and petroleum installations (Jensen et al., 2012; Neff et al., 2006; Reddy and Quinn, 1999). As fluoranthene is a hydrophobic solid, it was first dissolved in acetone and then aliquots of a stock solution with 100 μg fluoranthene per 1 ml of acetone were mixed into the seawater. This water came from Jakarta Bay and therefore surely contained organic material, including bacteria and pollutants, which are likely to have interacted with the PVC particles as

Please cite this article as: Rist, S.E., et al., Suspended micro-sized PVC particles impair the performance and decrease survival in the Asian green mussel Perna viridis, Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.07.006

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well. But since this was the same for all treatment groups and could therefore not have introduced a bias, this aspect was not further investigated. A small pump ensured constant mixing of the PVC/seawater suspension and every four days the seawater-fluoranthene solution was renewed to maximize the loading of the particles. On the last day, particles were washed with clean seawater for 1 h to remove excessive fluoranthene. This was done by letting the particles settle, decanting the old water and replacing it by clean seawater. Additionally, one part of the PVC material was stored in clean seawater for the same time span (24 days) to serve as a non-contaminated control. 2.2.2. Experimental design We ran a laboratory experiment for 91 days, in which mussels were exposed to microplastics that were regularly resuspended from the sediment into the water column, mimicking a near-coastal habitat under tidal influence. Several pollution scenarios were implemented that differed with regard to the amount of PVC particles present (‘particle concentration’): 0 mg PVC/l (‘particle control’), 21.6 mg PVC/l, 216 mg PVC/l and 2160 mg PVC/l. In terms of particle numbers the 3 levels chosen were equivalent to 1.2 ∗ 107 particles/l, 1.2 ∗ 108 particles/l and 1.2 ∗ 109 particles/l, respectively. These values were reached when the PVC material was completely suspended, which was only given during the resuspension events (see below). We implemented one further control group (‘pollutant control’) with clean PVC particles that had been kept in non-contaminated seawater prior to the experiment. This was done to check whether there are any signs that the contamination state of the particles can influence the measured responses. It was, however, not the intention of this study to disentangle possible single effects of the PVC particles and/or of the pollutant on the animals. Therefore we also did not include an experimental group exposed to fluoranthene only. This would only have been meaningful if we had been able to ensure that fluoranthene uptake rates are the same between treatment levels that had fluoranthene on particles and fluoranthene dissolved in seawater. Otherwise the difference in the doses would have biased the comparison. Furthermore, we were not able to quantify the exact amounts of fluoranthene that dissolved from the particles into the water due to technical limitations. Since we expected the most pronounced impairment of mussel performance at the highest particle concentration (2160 mg PVC/l), we realized the ‘pollutant control’ at this level (Fig. 1). Each treatment group consisted of 15 replicates, i.e. single mussels, which were kept in individual containers with 1.2 l of seawater. Half of the water in each container was exchanged manually every day prior to the first of two resuspension events (see below). Twice a day, immediately before resuspension, each mussel was fed with 2 ∗ 106 I. galbana cells. We added 0.86 g of sieved and dried sand from Rambut Island/Jakarta Bay (− 5° 58′ N, 106° 41′ O) to each container since not only

Fig. 1. Experimental design of the laboratory exposure assays that were conducted with the Asian green mussel Perna viridis to assess the effects of microplastic pollution on mussel performance and survival. Fluoranthene-contaminated PVC particles were applied at 4 concentration levels (in black), while clean particles occurred only in one concentration (in gray).

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Table 1 Abundance of suspended PVC particles during resuspension events. Samples were taken from 2 containers per treatment level on 3 consecutive days immediately after the PVC material was renewed. The latter was a weekly routine. Group

21.6 mg/l 21.6 mg/l 216 mg/l 216 mg/l 2160 mg/l 2160 mg/l

Particles per ml Day 1

Day 2

Day 3

198,900 6000 381,000 736,000 29,480,000 13,600,000

167,400 209,200 302,000 420,000 6,760,000 7,080,000

72,900 30,700 529,000 270,000 2,320,000 Data missing

microplastics but also sediments are transported into the water column during resuspension events. The resuspension of the sediment/PVC particle mix was achieved by aeration. We aerated the containers continuously for 2 h in the morning and then, after a break of 6 h, again for 2 h in the evening. This was meant to simulate two tidal currents, one flood and one ebb tide, during which high current velocities can lead to a strongly increased resuspension of seston (Fegley et al., 1992; Mantovanelli et al., 2004). Thus, we did not apply a constant but a regularly reoccurring particle exposure. The sediment and the PVC material in the experimental containers was replaced by new sediment and freshly contaminated or clean particles, respectively, once per week. Due to the formation of aggregates and because mussels ingested particles or embedded them in pseudofaeces, their concentration in the experimental containers declined over the course of the week until the system was reset again. In a pilot study we measured this by counting the particles in the water column in two test containers from every experimental group within 3 days after the renewal of the PVC material (Table 1). 2.3. Response variables Physiological response variables were measured for all replicates once at the beginning of the experiment and again after 40–44 days of exposure to the PVC particles. A further measurement at the end of the total exposure phase of 91 days, as it was initially planned, was not possible due to high mortality in all groups. 2.3.1. Clearance rates To quantify mussel filtration activity we determined a clearance rate, i.e. the uptake of plankton cells per individual per unit time. For this we placed the mussels separately in a container with 500 ml seawater that was devoid of PVC particles. After 2 h of acclimatization, 2.7 ∗ 107 I. galbana cells were added and the water in the container was carefully mixed. With a syringe a water sample of 2 ml was taken from the water body in front of the mussel's siphon shortly after the cells were added. This was repeated after 30 min. We verified that the mussels were still filtering after this time span. Then Lugol solution was added to all samples and they were stored at 4 °C until the cells were counted using a Sedgewick rafter. While assuming that the mussels retained 100% of the algal cells they ingested, the change in cell numbers over the course of 30 min was used to determine clearance rates. 2.3.2. Respiration rates To assess oxygen consumption in P. viridis in response to the exposure to different concentrations of microplastics, we measured the decrease of dissolved oxygen per unit time per mussel individual in a closed system (Lampert, 1984). For this, mussels were transferred to a glass container that was completely filled with 200 ml of clean seawater and sealed with a rubber-stopper. An oxygen sensor (Oxi 2305, WTW) was inserted through a hole in the rubber-stopper into the measurement chamber that was then placed on a magnetic stirrer to assure a constant mixing of the water inside. Oxygen concentration was measured in the moment when the mussel opened its valves and then

Please cite this article as: Rist, S.E., et al., Suspended micro-sized PVC particles impair the performance and decrease survival in the Asian green mussel Perna viridis, Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.07.006

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again after 15 min. We calculated the rate of respiration as the difference between the two measurements. These values were corrected for microbial oxygen consumption, which was assessed by measuring the change in oxygen concentration in the empty chamber. The latter measurement was repeated after every fourth mussel individual. 2.3.3. Byssus production For measuring the production of byssus threads in P. viridis, we transferred the mussels to clean containers, after having carefully cut all existing threads. After 24 h, the number of newly formed threads was determined by counting the byssus discs that were attached to the container walls. 2.3.4. Survival time Mortality among mussels was monitored twice per day throughout the whole experiment by inspecting every single mussel. They were considered dead when their shell was wide open, the gills were not extended and the mussels did not react to careful pricking with a wooden stick. Dead individuals were removed and immediately frozen at −20 ° C. Despite the high frequency of checks, in most cases the soft body of dead individuals had already started to dissolve by the time they were removed. Due to this, we were not able to quantify the dry weight of these individuals and could not calculate their body condition indices (BCI). 2.4. Fluoranthene concentrations on PVC particles and in mussel tissue Measuring fluoranthene concentrations on PVC particles or in mussel tissue was not possible during the exposure experiment in Indonesia due to technical constraints. Therefore, samples were transported to Germany to be analyzed by high performance liquid chromatography (HPLC) at the Institute for Toxicology and Pharmacology in Kiel. However, the material was discarded because the samples got damaged during the transport. The same protocol for PVC particle contamination, as it was described for this study, was also applied in a parallel experiment that was run in Akkeshi, Japan. For this experiment, the accumulation of fluoranthene on PVC particles was confirmed by HPLC. We provide the Japan data here to document that the method we employed is generally useful for the contamination of the plastic material that was used in our experiments. Since our contamination procedure was identical with the one in Akkeshi, we believe that the method was also successful in Bogor. The biggest difference between both locations was the ambient temperature in the laboratory, which was on average 15 °C higher in Bogor. However, due to this the accumulation rate of fluoranthene on the PVC particles should have been even higher in the Indonesia experiment. For the plastic samples, we determined the concentration of fluoranthene that was reached on the particles after 4, 12, 16 and 24 days of contamination using HPLC with fluorescence detection. In September 2015, the samples were transported cooled to the Institute of Toxicology and Pharmacology in Kiel. Here each portion of the PVC material was mixed into 6 ml of hexane (Merck, Darmstadt, Germany) and carefully shaken for 1 min. The supernatant was then transferred to a tube and the hexane was evaporated using a nitrogen stream. Afterwards, the residue was dissolved in 500 μl acetonitrile (J.T. Baker, Center Valley, USA) of which 20 μl were then used for HPLC analysis. HPLC was equipped with a Supelcosil™ LC-PAH column (15 cm × 4.6 mm, Sigma–Aldrich Chemie GmbH, Steinheim, Germany) and we used a water:acetonitrile eluent (40%:60%) as a mobile phase with a flow rate of 1.5 ml/min. Fluoranthene standard solution was obtained from Sigma–Aldrich (St. Louis, MO). All solvents used for the extractions were of HPLC gradient grade. The fluorescence detector was set to an excitation wavelength of 260 nm and an emission wavelength of 460 nm. Fluoranthene retention time was 10.2 min. Fluoranthene content was quantified by measuring the area of signal peaks from the

sample extracts in relation to those of a fluoranthene standard (Sigma–Aldrich, Steinheim, Germany) with a detection limit of 0.01 ng. 2.5. Data analysis Physiological data were analyzed using analysis of variance (ANOVA) and simple linear regression. All statistical analyses and graphs were made with the free statistical computing software R (version 3.0.2 (2013-09-25) “Frisbee Sailing”) for Mac OS X (R Core Team, 2014). The assumptions of homogeneous variances and of normal errors were checked graphically by the use of residual plots and histograms. In addition to this, homogeneity of variances was tested with the Fligner-Killeen Test and normality of residuals with the ShapiroWilks-W-Test. Due to inhomogeneous variances in the data of byssus production and clearance rate, an ANOVA with Welch-adjustment was calculated for these responses. Respiration rates did not show normality of residuals, which was, however, overcome by square root transforming the data. Possible differences between the group of mussels that was not exposed to microplastic particles (‘particle control’) and all other groups were tested by Welch adjusted t-tests. This was preferred over a usual post hoc test (e.g. Tukey's HSD), because of the higher test power. Mortality rates between groups were compared by Cox-regression procedures and differences between single groups were tested for significance with pairwise Peto-Wilcoxon tests. The significance thresholds of the Welch's t-test and the Peto-Wilcoxon tests were bonferroni-corrected to avoid the inflation of the Type I error rate. 3. Results 3.1. Physiological response variables After 40–44 days of exposure to microplastics, clearance and respiration rates as well as byssus production in Perna viridis were found to decrease with increasing PVC particle loads (Figs. 2–4). In mussels that experienced the severest pollution level (2160 mg contaminated PVC/ l), clearance rates were on average by 79% (Fig. 2) and respiration rates by 64% (Fig. 3) lower than in conspecifics that were kept in the absence of plastics (‘particle control’). The production of byssus threads even showed a decrease by 97%, since, with the exception of one individual, it completely ceased in the 2160 mg contaminated PVC/l group

Fig. 2. Influence of various concentrations of suspended PVC particles on clearance rates (means +95% CI) in Perna viridis after 40 days of exposure. Groups shown in black had fluoranthene-contaminated PVC, the group in gray had clean PVC (the particle concentration of this group was also 2160 mg/l but for better readability it is shifted to the right). The horizontal line indicates the mean clearance rate that was measured immediately before the start of the experiment. Asterisks indicate significant differences between the respective groups and the group without PVC based on Welch's t-tests. Results from linear regression: R2 = 0.30, F = 15.64, p = 3.4 ∗ 10−4.

Please cite this article as: Rist, S.E., et al., Suspended micro-sized PVC particles impair the performance and decrease survival in the Asian green mussel Perna viridis, Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.07.006

S.E. Rist et al. / Marine Pollution Bulletin xxx (2016) xxx–xxx

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Table 2 Effects of PVC particle exposure on physiological responses (ANOVA) and survival (Cox regression) in Perna viridis after 40–44 days and 91 days, respectively. Response

F-value

df

p-Value

Clearance rate Respiration rate Byssus production Survival

25.07 6.45 13.24 30.56

4 4 4 4

2.79 ∗ 10–7 3.42 ∗ 10–4 2.67 ∗ 10–4 3.77 ∗ 10–6

almost the same response as the group of mussels that was confronted with the same amount of contaminated PVC (Figs. 2–4). Mean clearance and respiration rates in the ‘particle control’ group were very close to the baseline measurements that we did at the start of the experiment. Byssus production rates, however, were too variable between individuals to see such a trend (Fig. 4). Fig. 3. Influence of various concentrations of suspended PVC particles on respiration rates (means +95% CI) in Perna viridis after 40 days of exposure. Groups shown in black had fluoranthene-contaminated PVC, the group in gray had clean PVC (the particle concentration of this group was also 2160 mg/l but for better readability it is shifted to the right). The horizontal line indicates the mean respiration rate that was measured immediately before the start of the experiment. Asterisks indicate significant differences between the respective groups and the group without PVC based on Welch's t-tests. Results from linear regression: R2 = 0.14, F = 6.36, p = 0.02.

(Fig. 4). In mussels that were exposed to the second highest pollution level, i.e. 216 mg PVC/l, the difference to the ‘particle control’ group was still 41% for both clearance and respiration rates and 76% for byssus production. ANOVA revealed highly significant differences between the groups for all response variables (Table 2). Single comparisons showed that the means of the groups with 2160 mg and 216 mg PVC/l were significantly different from the mean of the control group in all three responses. For clearance rates this was also true for the group that experienced the lowest pollution scenario of 21.6 mg PVC/l, while in the other two responses there was a clear but non-significant trend for a reduction in respiration rates by 32% and in byssus production by 41% at the 21.6 mg PVC/l level. The ‘pollutant control’ group showed

Fig. 4. Influence of various concentrations of suspended PVC particles on byssus production (means +95% CI) in Perna viridis after 44 days of exposure. Groups shown in black had fluoranthene-contaminated PVC, the group in gray had clean PVC (the particle concentration of this group was also 2160 mg/l but for better readability it is shifted to the right). The horizontal line indicates the mean byssus production that was measured immediately before the start of the experiment. Asterisks indicate significant differences between the respective groups and the group without PVC based on Welch's t-tests. Results from linear regression: R2 = 0.15, F = 5.12, p = 0.03.

3.2. Survival time During the 91 days of the experiment, we observed substantial mortality in all experimental groups - even in the group without microplastic particles (‘particle control’), but this was presumably caused by a sudden decline in water quality or the occurrence of a pathogen that only affected this group (Fig. 5). However, median survival times decreased clearly with increasing pollution by PVC particles (Fig. 6). In both groups with 2160 mg PVC/l, i.e. with and without fluoranthene contamination, the animals only survived half as long as in the control group. Statistical analysis of the mortality data with a global testing procedure (Cox regression) indicated highly significant differences between at least some of the experimental groups (Table 2), but we could not find any significant difference between the ‘particle control’ group and the others when we applied pairwise comparisons.

3.3. Fluoranthene contamination of PVC particles During 24 days of particle contamination in a fluoranthene-seawater solution the pollutant accumulated on the PVC material. The concentration increased from 6 ng fluoranthene per g PVC on day 4 to a maximum of 20 ng per g PVC at the end of the contamination phase after 24 days (Table 3). Only between day 12 and day 16 no increase was observed.

Fig. 5. Influence of various concentrations of suspended PVC particles on the survival of Perna viridis during 91 days of exposure. The group marked with an asterisk (*) had uncontaminated PVC particles, while the plastic material of all other groups was contaminated with fluoranthene.

Please cite this article as: Rist, S.E., et al., Suspended micro-sized PVC particles impair the performance and decrease survival in the Asian green mussel Perna viridis, Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.07.006

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Fig. 6. Median survival time of Perna viridis in the 91 days exposure to various concentrations of PVC particles. The group in gray had uncontaminated PVC particles, while the plastic material of all other groups was contaminated with fluoranthene.

Table 3 Fluoranthene concentration on the PVC particles during the 24 days contamination phase measured by HPLC. Day of particle contamination 4 12 16 24

Fluoranthene concentration on PVC particles (ng/g) 6.02 14.33 14.05 20.22

4. Discussion We investigated the effects of suspended microscopic PVC particles on the performance and survival of the Asian green mussel Perna viridis over the course of 91 days. To our knowledge this is the first study that simulated microplastic exposure that originates from particle resuspension from the sediment. To identify the pollution threshold, beyond which an effect on the physiological performance of the mussels can be detected, we chose a wide gradient of particle concentrations along a logarithmic scale (0–2160 mg/l). We were aware that the highest concentrations we chose (i.e. 216 and 2160 mg/l, equivalent to 1.2 ∗ 108 and 1.2 ∗ 109 particles/l) exceed microplastic particle loads that have so far been reported from the water column, with maximum values of 9.2 particles per l (Desforges et al., 2014). However, since sediments are the ultimate sink for microplastics, particle concentrations on the sea floor and - in case resuspension occurs - also in near-bottom waters should exceed those in the water column. Lee et al. (2013), for example, found up to 27,606 microplastic particles per m2 on the sea floor in a Korean estuary. This number presumably still underestimates the total plastic abundance, since they only took particles in a size range of 1– 5 mm into account and ignored smaller size fractions. Resuspension of deposited microplastics is a process that has not been investigated yet, but it is likely that microplastics show a behavior that is similar to natural seston, which is known to be resuspended by high current velocities (Fegley et al., 1992; Mantovanelli et al., 2004). Therefore events like strong tides should locally lead to high particle concentrations in near-bottom waters that may exceed the abundance of microplastics in the upper water column by far. In our study, mussel clearance rates, respiration rates and byssus production were found to decrease with increasing particle abundance after 40–44 days of exposure to different amounts of PVC particles. In

case of the filtering activity, already the lowest particle concentration led to a significant reduction in mussel performance, while for respiration and byssus production, a significant change emerged at the second highest pollution level. Furthermore, during the 91 days of exposure, mussel survival was a negative function of particle abundance. All in all, we observed a strong impact of the PVC particles on P. viridis, which very likely went along with metabolic depression and a depletion of energy reserves. The substantial decrease in clearance as well as in respiration rates can presumably be explained by particle-induced closing of the valves. This behavior obviously limits the uptake of food as well as gas exchange in bivalves. It is known that Mytilids adjust the opening width of their valves to the amount of food particles in the water column and also to the prevailing seston quality. An increase in the abundance of inorganic particles, which can potentially harm the animals by injuring epithelia or by causing blockages, leads to a rapid reduction of valve opening (Madon et al., 1998; Riisgard et al., 2003; Wright et al., 2013b). An impairment of the filtering activity and of oxygen uptake by suspended inorganic particles has also been described for several other bivalve species, including the scallop Placopecten magellanicus, the clam Mya arenaria, the turkey wing Arca zebra, the Atlantic pearloyster Pinctada imbricata and the zebra mussel Dreissena polymorpha (Bacon et al., 1998; Ward and Macdonald, 1996; Grant and Thorpe, 1991; Alexander et al., 1994). Shin et al. (2002) reported that P. viridis showed a significant decrease in filtration rates within 96 h of exposure to 1200 mg suspended material per l. Our highest pollution scenario exceeded this amount and should therefore have constituted a severe stress for the mussels, which was, however, not immediately lethal. This was presumably due to the fact that we did not apply it continuously but restricted it to the resuspension events of 2 × 2 h per day and short periods thereafter (until all particles deposited). Therefore, mussels were able to recover between the events. Nevertheless, they showed a strong decrease in clearance and respiration rates as a consequence of this stress. A similar but less pronounced decline was observed when mussels experienced a particle load of 216 mg/l and even in the group of animals that faced only 21.6 mg PVC/l clearance rates were significantly lowered in comparison to the control group without PVC. A similar trend emerged for respiration rates. Unfortunately, we were not able to monitor valve opening and closing in our test animals to verify whether mussel gaping differed significantly between the experimental groups. This can be done by the use of Hall sensors (Wilson et al., 2005; Robson et al., 2006), but we were not able to employ such a system in the laboratory in Indonesia. Reduced feeding, caused by a particle-induced decrease in filtration rates, should restrict the energy budget and the metabolism of the affected mussels. Although we were not able to quantify this in our test animals, the drastic decrease in clearance rates we observed with increasing particle density must have been associated with a reduction in food uptake and this should have, in turn, impaired the mussels' energy reserves. Such a depletion of energy reserves has already been shown for Arenicola marina during a 28 days exposure to a PVC particle pollution equivalent to 1% and 5%, respectively, volume share of the surrounding sediment (Wright et al., 2013a). Similar to our system, reduced food uptake was also discussed as a possible cause by the authors, although the mechanism that led to a reduction in food uptake presumably differs between the filter feeder (reduced filtration rates, prolonged periods of valve closure) and the deposit feeder, for which it was speculated that the surface properties of the clean PVC weakened particle adhesion to the worms' feeding apparatus, thus reducing uptake efficiency. A further factor that could have burdened the mussels' metabolism is the enhanced production of pseudofaeces, which allow the mussels to eliminate rejected particles from their gills and ultimately from their mantle cavity (Garrido et al., 2012). This behavior was observed in all groups of animals that were exposed to PVC particles (Rist, pers. obs.). The excretion of pseudofaeces requires the production of mucus and therefore consumes energy.

Please cite this article as: Rist, S.E., et al., Suspended micro-sized PVC particles impair the performance and decrease survival in the Asian green mussel Perna viridis, Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.07.006

S.E. Rist et al. / Marine Pollution Bulletin xxx (2016) xxx–xxx

An impairment of the mussels' energy budget should also have restricted their capacity to form byssus threads. This process is energy-intensive: For P. viridis it was found that 10% of the animal's total energy budget that is available for the production of biomass is consumed by the synthesis of the collagen threads (Cheung, 1991). In accordance with this, we observed a substantial drop in byssus formation rates during exposure to PVC particles. A perpetual production of byssus is not ultimately essential for the survival of mussels and under experimental conditions a reduction in byssus formation does not lead to mortality among the test animals. However, in the field the threads anchor the mussels and allow them to orient their siphons towards the water column (Gosling, 2008). They are therefore important for the viability and the fitness of the animals and in their habitats mortality in mussels is considerably increased if they are not able to attach themselves to the substrate (Cheung et al., 2006). Beside sublethal signs of energy depletion, such as the reduction in byssus thread formation, we also observed substantial mortality in all treatment groups during the 91 days of the experiment. Furthermore, survival time in the test animals was a negative function of PVC particle loads. The increased mortality presumably goes back to the fact that the depletion of energy reserves also affected essential life functions, such as ATP synthesis. This finding was statistically significant, although we observed a not understood increase in mortality among individuals of the control group between day 28 and 35. This incident had the potential to cause a type II error (overlooking an existing effect) by masking treatment-induced differences between groups, but this was not the case. So far, we discussed the observed negative effects on P. viridis as a consequence of exposure to suspended solids, but did not consider the potential role of the fluoranthene that was absorbed to the PVC particles in most of our experimental groups. However, it was not the aim of this study to assess the role of microplastics as vectors for POPs. The fluoranthene-contamination of the PVC particles rather served to simulate the conditions in anthropogenically impacted coastal habitats, in which plastic particles do not occur in their pristine form but are loaded with organic pollutants (Rios et al., 2007; Frias et al., 2010; Rochman et al., 2013). The method we used for particle contamination proved to be successful in the parallel experiment in Japan, since the PVC particles there were loaded with up to 20 ng fluoranthene per g PVC within 24 days. We therefore assume that the loading was also successful in the laboratory in Bogor, although we could not analyze the sample material (PVC and mussel tissue) from there. In order to have an indication of whether the presence of the pollutant can modify the effect of the plastic particles on the mussels, we had an additional experimental group with uncontaminated PVC (‘pollutant control’ with 2160 mg PVC/l). Since animals in this group did not differ in their performance from their conspecifics in the sibling group with fluoranthene-contaminated PVC (2160 mg/l), we believe that the pollutant did not influence P. viridis. At least not in a way that could be detected within the time span of the experiment and with the methods we applied. This is in line with the resumes of a recent review by Ziccardi et al. (2016), which summarized that effects of microplastic-transported POPs were so far only reported from the cellular and the subcellular level, whereas effects on physiological and reproductive performance have not been documented. In fact, the majority of studies that were reviewed reported particle-induced rather than pollutant-induced effects of microplastic contamination (Ziccardi et al., 2016). These findings are in accordance with our results that also document a particle effect rather than an influence of the PAH that was absorbed to the plastic. When summarizing the existing literature about the relevance of microplastics as a vector for POPs, it is concluded that microplastics do not enhance their bioavailability for aquatic animals since natural sources like prey items outweigh microplastics in terms of transfer of pollutants by several orders of magnitude (Koelmans et al., 2016; Ziccardi et al., 2016). The results of this study show that microplastic particles can exert a serious stress on the tropical bivalve P. viridis, which can lead to a

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fundamental impairment of the mussels' physiological performance. This was the case although exposure to microplastic particles in this study was not continuous but restricted to a few hours per day, since we mimicked a scenario in which particles got resuspended at regular intervals. When considering our findings for the assessment of possible environmental implications of the pollution of marine benthic habitats with microplastics, it must be noted that the highest chosen particle density is only representative for extremely polluted sites, such as Camillo Beach in the Hawaii archipelago (Carson et al., 2011). It certainly exceeds current pollution levels in most coastal habitats by far and also the second highest pollution level, although by one magnitude lower than the highest, is, luckily, not representative of the presentday situation in the majority of coastal sites on the planet. Here it is important to emphasize that all this is said with regard to pollution levels at the surface of sediments, where microplastic particles accumulate, and it does not at all apply to the water column. Here particle concentrations are again much lower, since it has been shown that microplastics only transit through this part of the oceans and finally sink to the seafloors (Woodall et al., 2014). However, this aspect of our work is new, since so far the resuspension of microplastics from sediments has been neglected as a possible exposure pathway. Although we cannot provide any field data that could document how common this process is in coastal habitats and whether it substantially enhances the bioavailability of microplastics, we documented that negative effects can be caused by a discontinuous exposure that is limited to a few hours per day – at least if the frequency of resuspension events is high. More field as well as laboratory studies are needed to evaluate the prevalence of this process in coastal benthic systems and its ecological relevance. Acknowledgments We are grateful to all technicians and lab assistants who, with their support and expertise, enabled us to realize the comprehensive experiments in the Marine Habitat Laboratory in Bogor. Furthermore, we would like to thank Carsten Thoms and Martin Wahl for their advice and indispensable support in Bogor and Kiel. Sinja Rist and Khoirunnisa Assidqi received stipends that were generously granted by Briese Schiffahrt and Kieler Nachrichten. References Alexander, J.E., Thorp, J.H., Fell, R.D., 1994. Turbidity and temperature effects on oxygenconsumption in the zebra mussel (Dreissena polymorpha). Can. J. Fish. Aquat. Sci. 51, 179–184. Andrady, A.L., 2011. Microplastics in the marine environment. Mar. Pollut. Bull. 62, 1596–1605. Arifin, Z., 2004. Local Millennium Ecosystem Assessment: Condition and Trend of the Greater Jakarta Bay Ecosystem. The Ministry of Environment, Indonesia. Avio, C.G., Gorbi, S., Milan, M., Benedetti, M., Fattorini, D., D'errico, G., Pauletto, M., Bargelloni, L., Regoli, F., 2015. Pollutants bioavailability and toxicological risk from microplastics to marine mussels. Environ. Pollut. 198, 211–222. Bacon, G.S., Macdonald, B.A., Ward, J.E., 1998. Physiological responses of infaunal (Mya arenaria) and epifaunal (Placopecten magellanicus) bivalves to variations in the concentration and quality of suspended particles: I. Feeding activity and selection. J. Exp. Mar. Biol. Ecol. 219, 105–125. Barnes, D.K., 2005. Remote islands reveal rapid rise of southern hemisphere, sea debris. Sci. World J. 5, 915–921. Besseling, E., Wegner, A., Foekema, E.M., Van Den Heuvel-Greve, M.J., Koelmans, A.A., 2013. Effects of microplastic on fitness and PCB bioaccumulation by the lugworm Arenicola marina (L.). Environ. Sci. Technol. 47, 593–600. Besseling, E., Wang, B., Lurling, M., Koelmans, A.A., 2014. Nanoplastic affects growth of S. obliquus and reproduction of D. magna. Environ. Sci. Technol. 48, 12336–12343. Besseling, E., Foekema, E.M., Van Franeker, J.A., Leopold, M.F., Kühn, S., Bravo Rebolledo, E.L., Heße, E., Mielke, L., Ijzer, J., Kamminga, P., Koelmans, A.A., 2015. Microplastic in a macro filter feeder: humpback whale Megaptera novaeangliae. Mar. Pollut. Bull. 95, 248–252. Browne, M.A., Dissanayake, A., Galloway, T.S., Lowe, D.M., Thompson, R.C., 2008. Ingested microscopic plastic translocates to the circulatory system of the mussel, Mytilus edulis (L.). Environ. Sci. Technol. 42, 5026–5031. Browne, M.A., Galloway, T.S., Thompson, R.C., 2010. Spatial patterns of plastic debris along estuarine shorelines. Environ. Sci. Technol. 44, 3404–3409. Carson, H.S., Colbert, S.L., Kaylor, M.J., Mcdermid, K.J., 2011. Small plastic debris changes water movement and heat transfer through beach sediments. Mar. Pollut. Bull. 62, 1708–1713.

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Please cite this article as: Rist, S.E., et al., Suspended micro-sized PVC particles impair the performance and decrease survival in the Asian green mussel Perna viridis, Marine Pollution Bulletin (2016), http://dx.doi.org/10.1016/j.marpolbul.2016.07.006