Time-of-flight secondary ion mass spectrometry (ToF

Science of the Total Environment 563–564 (2016) 261–266

Contents lists available at ScienceDirect

Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Time-of-flight secondary ion mass spectrometry (ToF-SIMS)-based analysis and imaging of polyethylene microplastics formation during sea surf simulation H. Jungnickel a,⁎, R. Pund b,c, J. Tentschert a, P. Reichardt a, P. Laux a, H. Harbach c, A. Luch a a b c

German Federal Institute for Risk Assessment (BfR), Department for Chemical and Product Safety, Max-Dohrn-Strasse 8-10, 10589 Berlin, Germany Lower Saxony State Office for Consumer Protection and Food Safety, Institute for Fishery Products Cuxhaven (LAVES), Schleusenstrasse 1, 27472 Cuxhaven, Germany German Federal Institute for Risk Assessment (BfR), Department of Experimental Toxicology and ZEBET, Max-Dohrn-Strasse 8-10, 10589 Berlin, Germany

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Pre-production microplastic pellets were used to evaluate the methodology • Secondary microplastic b than 10 μm can be detected directly in sea sand • Surface Ions of microplastic particles were used to identify them in sea sand matrix • Chemical images gave size distribution patterns of microplastic particles in matrix • Larger microplastic particles were exposed to a sea surf simulation • Particle number (b 10 μm) increased from 14 to 31 d exposure

a r t i c l e

i n f o

Article history: Received 30 October 2015 Received in revised form 4 April 2016 Accepted 5 April 2016 Available online 30 April 2016 Editor: D. Barcelo Keywords: Microplastics Particles Polyethylene Sea surf simulation

a b s t r a c t Plastic particles smaller than 5 mm, so called microplastics have the capability to accumulate in rivers, lakes and the marine environment and therefore have begun to be considered in eco-toxicology and human health risk assessment. Environmental microplastic contaminants may originate from consumer products like body wash, tooth pastes and cosmetic products, but also from degradation of plastic waste; they represent a potential but unpredictable threat to aquatic organisms and possibly also to humans. We investigated exemplarily for polyethylene (PE), the most abundant constituent of microplastic particles in the environment, whether such fragments could be produced from larger pellets (2 mm × 6 mm). So far only few analytical methods exist to identify microplastic particles smaller than 10 μm, especially no imaging mass spectrometry technique. We used at first time-of-flight secondary ion mass spectrometry (ToF-SIMS) for analysis and imaging of small PE-microplastic particles directly in the model system Ottawa sand during exposure to sea surf simulation. As a prerequisite, a method for identification of PE was established by identification of characteristic ions for PE out of an analysis of grinded polymer samples. The method was applied onto Ottawa sand in order to investigate the influence

Abbreviations: DDT, dichlorodiphenyltrichloroethane; m/z, mass-to-charge ratio; SIMS, secondary ion mass spectrometry; ToF-SIMS, time-of-flight secondary ion mass spectrometry; PE, polyethylene. ⁎ Corresponding author. E-mail address: [email protected] (H. Jungnickel).

http://dx.doi.org/10.1016/j.scitotenv.2016.04.025 0048-9697/© 2016 Elsevier B.V. All rights reserved.

262 Degradation Time-of-flight secondary ion mass spectrometry (ToF-SIMS)

H. Jungnickel et al. / Science of the Total Environment 563–564 (2016) 261–266

of simulated environmental conditions on particle transformation. A severe degradation of the primary PE pellet surface, associated with the transformation of larger particles into smaller ones already after 14 days of sea surf simulation, was observed. Within the subsequent period of 14 days to 1 month of exposure the number of detected smallest-sized particles increased significantly (50%) while the second smallest fraction increased even further to 350%. Results were verified using artificially degraded PE pellets and Ottawa sand. © 2016 Elsevier B.V. All rights reserved.

1. Introduction The contamination of the environment with all kinds of plastic debris, resulting from an ever increasing worldwide production of carbon based polymer products, is known since the 1970s (Carpenter et al., 1972; Colton et al., 1974). In the mid-1980s national and international concern started to increase. For example, the National Oceanic and Atmospheric Administration (NOAA) of the USA initiated the first of a series of international conferences on marine debris. Since then the accumulation and distribution of plastic litter in marine and terrestrial ecosystems increased dramatically on a worldwide scale (Ministry of Environment and Food of Denmark, Environmental Protection Agency, 2015). For smaller plastic particles (e.g. particles smaller than 5 mm in one dimension (NOAA definition) the term “microplastics” was coined in the last decade (Thompson et al., 2004, NOAA). Various sources for the introduction of such materials into the aquatic environment are discussed. This includes microplastic particles originating from personal care products (Fendall and Sewell, 2009) such as facial cleansers (Eriksen et al., 2013). However, degradation of originally larger sized plastic debris, e.g. condoms (Lambert et al., 2014), under environmental conditions contributes to the increased occurrence of microplastics as well. Due to unpredictable human littering behavior and different sources depending on the geographical location, release of microplastics is difficult to monitor and affects aquatic ecosystems (Lambert et al., 2014; McCormick et al., 2014; Free et al., 2014; Wagner et al., 2014) in particular since floating particles of a size of 0.5 mm or smaller are not completely retained by wastewater treatment facilities (Eriksen et al., 2013). On the other hand, the particle fraction retained in sewage sludge may contaminate agricultural land when used as a fertilizer (Saruhan et al., 2010). For the eastern Pacific Ocean N20.000 tons of floating microplastics was estimated based on an 11-year data set (Law et al., 2014). Microplastic particles were not only detected in the surface region of the water column, but also in sub-surface water layers (Kukulka et al., 2012; Desforges et al., 2014). The predominant distribution mechanism for plastic particles and especially microplastics in aquatic environments is current circulation either in surface or sub-surface water regions (Kukulka et al., 2012). Accumulation of plastic particles and fragments was not only observed in sea birds (Franeker et al., 2011; Kenyon and Kridler, 1969), but also in a variety of marine organisms. Besides sea turtles (Tomas and Guitart, 2002), whales (Lusher et al., 2015), seals (Bravo-Rebolledo et al., 2013) and mussels (Cauwenberghe et al., 2015) the records comprise lugworms (Cauwenberghe et al., 2015) and sea urchins (Nobre et al., 2015). Even within scleractinian corals (Hall et al., 2015) microplastic particles were already detected. Due to its capability to accumulate in rivers, lakes and the marine environment (Wagner et al., 2014), the issue of microplastics in the environment became of interest not only in (eco-) toxicology, but also with regard to potential effects on human health. In addition to the uncertain effects of pristine plastic particles as such, they may accumulate and translocate environmental toxicants along the food chain. Uptake and release capabilities depend on physico-chemical parameters like pH, temperature (Bakir et al., 2014) and polymer composition (Teuten et al., 2007) and so the surface properties of microplastic particles become a key factor for the understanding of surface uptake and release

capabilities with respect to various environmental toxicants and heavy metals. Thus, persistent organic contaminants like polychlorinated biphenyls (Rios et al., 2007), polycyclic hydrocarbons (Teuten et al., 2007) or polybrominated diphenyl ethers (Chua et al., 2014) may adsorb to the predominantly hydrophobic particles under environmental conditions and may be released again from the particle within the gastrointestinal tract following to ingestion by animals or humans. Such a putative “Trojan horse” function is discussed with regard to unidentified health risks of microplastic particles (Endo et al., 2005; Mato et al., 2001; Rios et al., 2007; Teuten et al., 2007). A study of microplastic particles collected from Hawaiian, Mexican and Californian beaches showed significant levels of persistent organic pollutants being adsorbed (Rios et al., 2007). Especially DDT (22–7100 ng/g), polychlorinated biphenyls (27–980 ng/g) and polycyclic aromatic hydrocarbons (39–1200 ng/g) were detected as organic contaminants of microplastic particles in this study (Rios et al., 2007). In addition to invertebrates (Cole et al., 2015) such as zooplankton (Cole et al., 2013) especially filter feeders (Fossi et al., 2014) may be affected by uptake of organic contaminant-loaded microplastic particles. Furthermore, heavy metals like cadmium, lead, nickel, or cobalt (Holmes et al., 2014) may translocate together with plastic particles. The finding that epithelial enterocytes of the rat gut are able to phagocytize 415 nm and 14 nm plastic microspheres (Hussain et al., 2001) illustrates the need for an assessment of possible human health risks arising from microplastics. This is further underpinned by the recently discovered degradation processes for plastic polymers that are commonly considered most biopersistent (Barnes et al., 2009; Sivan, 2011). Degradation of larger plastic constituents into smaller particles in water can occur over decades (Hidalgo-Ruz et al., 2012; Muthukumar et al., 2011). Mechanisms like biodegradation by microorganisms (Kathiresan, 2003; Sen and Raut, 2015; Yang et al., 2014), photodegradation by sunlight (Lambert et al., 2014) and thermo-oxidative degradation at low temperatures (Sen and Raut, 2015) are known to contribute to a slow but permanent degradation; even the hydrolysis with water (Andrady, 2011) was shown. As a result, larger plastic pieces (e.g. larger than 5 mm) are transformed into smaller pieces and finally into microplastic particles (Andrady, 2011). In addition to the routes of microplastic contamination, the capability of the aquatic environment to produce “secondary microplastics” from larger precursors under sea surf conditions requires further attention in order to predict long term impacts of marine plastic contamination. Especially weathering and degradation at coast-lines is assumed a major reason for embrittlement of polymers which may finally yield microplastics. We therefore analyzed exemplarily the degradation of PE, the material of which most microplastic particles in the environment consist (Ivar do Sul and Costa, 2014; Thompson et al., 2004), during a 30 day sea surf simulation. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) was employed to characterize emerging fragments in composition and size. 2. Materials and methods 2.1. Materials Ottawa sand and PE pellets (high density PE pellets) (6 mm × 2 mm, see Fig. S1a) were bought from Sigma-Aldrich. A synthetic sea water mixture was acquired from Tropic Marin GmbH, Wartenberg, Germany (Standard Tropic Marin).


263

2.2. Sea surf simulation

2.4.3. Degraded samples

A laboratory size sea surf simulation in a custom made instrument (130 L water basin, 1 m × 40 cm × 40 cm, equipped with an axial flow pump (Tunze Turbelle Stream, Tunze Aquarientechnik GmbH, Penzberg, Germany) and two sea wave simulator pumps (Waverbox 6215, Tunze Aquarientechnik GmbH, Penzberg, Germany) were used at full power for the wave simulation process. A “Standard Marin” sea water solution with a salinity of 15 ppm was generated from Tropic Marin® sea salt CLASSIC (Tropic Marin, St. Niklausen Switzerland) and osmotic water with an electric conductivity of 4 μS/cm using a two stage reverse osmosis apparatus RWS 50P (Julius Wassertechnik, Oschersleben, Germany). All experiments were performed at room temperature and a pH of 7.5 ± 0.1. The sea surf tank was exposed to natural daylight for 10 h a day.

2.4.3.1. Fresh water samples. 100 mg of the artificially grinded PE pellets and 900 mg Ottawa sand were submitted for 14 days to a fresh water pond at approximately 50 cm depth.

2.3. ToF-SIMS analysis Ion images and spectra measurements were performed using a ToFSIMS V instrument (ION-TOF GmbH, Münster, Germany) with a 30 keV nano-bismuth primary ion beam source (Biy+ x -cluster ion source with a BiMn emitter of the latest generation; lateral resolution in imaging mode 70 nm, depth resolution 9 nm). The ion currents were measured to be 0.5 pA at 5 kHz using a Faraday cup. A pulse of 0.7 ns from the bunching system resulted in a mass resolution that usually exceeded 6000 (full width at half-maximum) at m/z b 500 in positive mode. The primary ion dose was controlled below 1012 ions cm−2 to ensure static SIMS conditions. Charge compensation on the sample was obtained by pulsed electron flood gun (20 eV electrons). During operation the primary ion gun typically scans a field of view of 500 × 500 μm2 applying a 2048 × 2048 pixel measurement raster. Once the primary ion gun was aligned, a ToF-SIMS mass spectrum was generated by summing the detected secondary ion intensities and plotting them against the mass channels. The mass peak integration limit was set to the peak centre plus/minus half the dead time of τ = 65.4 ns. Since the primary ion peak width was adjusted to 50 ns FWHM, the 50 ns of the main secondary ion mass peak as well as 7.7 ns before and after this main peak were considered in the integration. Collection times were 120 s for each sample spot. In order to enhance the ion count per pixel, pixel binning was used. The 2048 × 2048 pixel image array was reduced to 512 × 512 for the mint samples and 128 × 128 for the environmental samples respectively by summing the spectral data for each pixel into larger pixels. Normalization of the mass spectrum in each pixel to the total intensity minus hydrogen peak in that pixel and autoscaling were obtained. To facilitate the comparison of the compositions of the different samples a common peak list (positive mode) was applied to each analysis. Analyses were carried out on cross-sections and surfaces of six samples of mint pellets, mint Ottawa sand, mint mixed PE/Ottawa sand samples and degraded mixed PE/Ottawa sand in fresh water samples (14 days) and three degraded mixed PE/Ottawa sand in seawater samples (14 days) and nine samples exposed for one month (Tab. S1).

2.4.3.2. Sea water samples. 1 kg Ottawa sand and 500 g PE pellets were exposed for 14 days and 1 month to a sea surf simulation. 2.4.4. Sample introduction Entire and grinded PE pellets as well as Ottawa sand and the respective artificial mixtures were analyzed following to exposure in a frozen or frozen-hydrated form “as is” without further sample pre-treatment. The samples were frozen via cooled propane gas and introduced into the vacuum chamber of the instrument. 2.5. Particle detection For particle detection ion images of the following fragment ions deriving from PE were considered: m/z: 109, 111, 113, 137, 139, 141, 151, 153 and 155. The fragment ions follow in positive mode the series [C3H3(CH2)n]+, [CnH2n − 1]+ and [CnH2n + 1]+. Only the fragment ion m/z 113 demonstrated a sufficient discriminating power for all matrices investigated and therefore was considered suitable for PE identification in experiments with fresh and sea water samples. Particles were detected using the freeware spot detection program “Icy” version 1.6.1.1. For the Ottawa sand the following characteristic ions were selected: 29 Si, 30Si, 28SiO+, 29SiO+ and 30SiO+. 2.6. Statistical analysis Using the respective peak lists as described above the raw data sets were further processed using Principal Components Analysis (PCA) using ION-TOF Surface Lab 6.3. 3. Results For the subsequent comparison of the generated mass spectra and their corresponding images representative data were gathered for the mint samples of PE, Ottawa sand as well as the water samples by analyzing six independent samples each.

2.4. Sample preparation 2.4.1. Artificial grinding of PE pellets 1 g PE pellets was used for forced degradation (10 min at amplitude 2) using a cryo-mill (Analysette 3 with a Pulverisette Cryobox, Fa. Fritsch, Idar-Oberstein, Germany) with liquid nitrogen. 2.4.2. Artificial mixture of grinded PE pellets and Ottawa sand 100 mg of the artificially grinded PE pellets and 900 mg Ottawa sand were mixed thoroughly together in a glass test tube on a vortex mixer for 15 min.

Fig. 1. ToF-SIMS image (ca. 500 × 500 μm) of total ions from a sample consisting of PE (blue circle) and Ottawa sand grains on top (green circles, indicated by green arrows).

264


Fig. 2. ToF-SIMS image (ca. 500 × 500 μm) of total ions from a sample consisting of PE (blue circle), Ottawa sand grain on top (green circle) and organic matter from fresh water environment (white circles).

For PE the prevalent fragment ions obtained are, as expected, the [C3H3(CH2)n]+, [CnH2n − 1]+ and [CnH2n + 1]+ series when the analyses were conducted in positive mode. Additionally, in positive mode adsorbed ionic species on the surfaces of the samples include sodium, potassium, and calcium. The Ottawa sand samples contained also traces of aluminum. In the sea water samples significantly higher amounts of sodium were present compared to the fresh water samples. As a comparison to the sample exposed to sea surf simulation milled PE flakes were mixed with Ottawa sand. A representative image of the surface examinations is shown in Fig. 1. In this portion of the mixture a PE fragment is present with two small sand grains on top of a PE flake (green circles). The subsequent

PCA analyses were calculated with three kinds of processing: pixel binning, normalization and autoscaling. Pixel binning was necessary to enhance the ion yield per pixel. The PCA data associated with Fig. 1 are depicted in Figure S2. The PCA for PE with its associated loading values results in an image that is in good compliance with the originally one where the sand grains are slightly darker (Fig. S2a). The inverse image (Fig. S2b) illustrates the loadings for the Ottawa sand grains, creating the bright yellow spots in the darker brown PE matrix. In a further step a more realistic situation was created, when a mixture of PE and Ottawa sand was analyzed after it was submitted for 14 days to the environmental impacts of a fresh water pond (Fig. 2). Figure S3 shows the associated PCA images and the corresponding loadings. Here, we could clearly discriminate between PE, Ottawa sand grains and the organic matter of the environment. The fragment ion m/z 113 demonstrated for all of these statistical analyses good discriminating power, which is indicated by the high absolute loading values for this fragment ion. In the presence of additional organic matter from the fresh water pond m/z values between 46 and 109 lost their significance for either PE or Ottawa sand and thus lack the necessary discrimination power. The m/z values of 44, 45 and 46 can be attributed to silicon and silicon oxide. These data sets were the baseline for the following investigations whether micro-sized particles could be formed from larger PE pellets (2 mm × 6 mm), when exposed to a sea surf simulation. The results showed significant disintegration of the surface of the PE pellets, when exposed for 1 month to a sea surf simulation set-up (Fig. S1d) in contrast to pristine PE pellets (Fig. S1b). Surface imaging of PE pellets revealed m/z 113 (C8H+ 17) as a major surface ion (Fig. S1c). Micrometer-sized plastic particles could be identified already after 14 days of exposure in the Ottawa sand fraction (Fig. 3a and Fig. 4b for a size distribution pattern of the observed plastic particles). ToF-SIMS imaging could be used to demonstrate a significant increase in the smaller-sized particle fraction from 14 days to 1 month of exposure (see Fig. 3a and 3b). The smallest fraction (category I, 1–1.4 μm2) increased by approximately 50%, while category II (1.5–2.4 μm2) increased N3.5-fold.

Fig. 3. ToF-SIMS overlay images (ca. 100 × 200 μm) of total ions (greyscale) and ion m/z 113 (red) from the PE pellets (C8H+ 17). The red circles depict areas with high PE particle densities. The pictures were acquired from the sea sand fraction in which PE pellets were exposed for 14 days (a) or 1 month (b) to a sea surf simulation.


Conversely, the largest particle group, group IV (larger than 3.5 μm2), kept numbers constant from 14 days to 1 month of exposure time. This indicates a degradation process, where larger particles slowly degrade from the PE pellets into smaller particles over time by a dynamic constant degradation process. This causes the smaller plastic particle fractions to increase significantly over time. Forced degradation experiments using the PE pellets showed the presence of ion m/z 113 on the surface of the generated particles (see Fig. S6a), that were subsequently mixed with Ottawa sand. ToF-SIMS imaging could be successfully used to visualize the microplastic particles within the mixtures (see Fig. S6b). This proves that ion m/z 113 derived from the PE matter is suitable to detect and visualize microplastic particles directly within Ottawa sand with no prior sample preparation.

4. Discussion and conclusion Microplastics in the aquatic environment comprise common polymers used in daily life such as the most prominent PE as well as polypropylene, polyvinylchloride or polyethylene terephthalate. While Fouriertransform infrared microscopy was proven suitable for identification of such plastic particles down to a size of 50 μm (Chae et al., 2015), the characterization of smaller objects remains challenging. This is of particular relevance in light of the current uncertainty on adverse effects of nanosized plastic particles (Bouwmeester et al., 2015). Such a fraction may possess a higher potential for the translocation of toxicants due to a “Trojan horse” function compared to larger particles, and the better penetration of biological barriers such as the intestinal epithelium. In general, more information is needed on time span, quantity and mechanism for the generation of particles in the lower micrometer and nanometer scale from larger precursor materials, especially under the highly relevant conditions of sea surf. In order to record such small particles an analytical methodology is needed that possesses a sufficient lateral resolution alongside with the capability for material identification. Using imaging mass spectrometry (ToF-SIMS) we could identify microplastics deposited on Ottawa sand when exposed to natural matter in freshwater environment for 14 days. As there is a significant overlap of fragment ions characteristic for Ottawa sand and PE (e.g. m/ z = 29 stands for silicon or CH3–CH+ 2 ) a statistical analysis (PCA) was performed to deduct the discriminating fragment ions. While several fragment ions were found suitable for discriminating PE from sand in the presence of organic matter from the environment—fresh or sea water—only fragment ion with m/z = 113 remained with sufficient discriminating power for polyethylene. In this environment the m/z values between 46 and 109 lost their significance, most likely due to the influence of organic matter. Only fragment ion m/z = 111 had further discriminating power, however its influence remained negligible in the presence of m/z = 113. In subsequent experiments using artificial sea water subjected to a laboratory sea surf simulation we could observe a severe surface damage of the primary PE pellets simultaneously with the formation of secondary microplastic particles. ToF-SIMS has been already used in the past for the identification of the polymer directionality (Karar and Gupta, 2015) and the occurrence of nanoclay in polymer plates (Tentschert et al., 2014). In the study presented here, ToF-SIMS imaging without prior clean-up steps was successfully used to identify microplastic particles in the model system of Ottawa sand already after 14 days of exposure to sea surf simulation. After 1 month the smallest size microplastic fraction (1–1.4 μm2) increased up to 50% and the second smallest size fraction (1.5–2.4 μm2) even further up to 350%, while the largest size fraction remained at the same numbers. To verify these results an artificial degradation of the PE beads was performed using a cryo-mill. The resulting microplastic particles were successfully imaged by application of the established methodology. When mixed with reference Ottawa sand, the microplastic particles could also be identified in the resulting artificial mixture of Ottawa sand and microplastic particles.

265

With the presented method polyethylene particles down to ca. 1– 5 μm in diameter were identified for the first time directly within the model system of Ottawa sand. Furthermore ToF-SIMS analysis revealed a detailed mass spectral pattern of the polymer assigned to specific ion fragments of the particle surface. Using the recent generation of SIMS instruments equipped with the latest ion cluster sources this was possible even in the presence of silicon oxide. Here, the suitability of the method for an investigation of the aging process of PE-microplastics under controlled conditions is demonstrated. This is providing a basis for further studies on representative environmental samples in which ToF-SIMS could provide a complementation of other methods such as scanning electron microscopy. Besides of a size measurement of particles by different principles, such an approach would provide mass spectrometric identification of the material investigated. This could help to create a chemical fingerprint of polymers and to understand their environmental breakdown which remains also in case of PE still uncertain (Prasun et al., 2011). Furthermore the confirmation of crack formation in the polymer particles in this study might also contribute to the elucidation and understanding of comparable environmental breakdown processes.

Fig. 4. a) Fragment ion m/z 113 (C8H+ 17 ) originating from PE that resulted from three control samples of Ottawa sand without exposure to PE pellets and from three Ottawa sand fractions after 14 days of exposure of PE pellets to a sea surf simulation. Secondary PE microplastic counts from the Ottawa sand fraction after b) 14 days (n = 9) and c) 1 month exposure (n = 9) of PE pellets to a sea surf simulation (ToF-SIMS images from a 0.25 mm2 area). The secondary PE microplastics were categorized into 4 groups. Group I: 1–1.4 μm2; Group II: 1.5–2.4 μm2; Group III: 2.5–3.4 μm2; Group IV: N3.5 μm2 particle area.

266


All this could be of special interest, if detailed information about the possible origin of microplastic litter is required to assess different entry pathways and pollution sources, including both secondary microplastics formed by degradation of larger pieces of debris and primary microplastics originating from consumer products. Ultimately the methodology could contribute to establish a specific monitoring program for microplastic contaminations of the aquatic environment. Such measures, especially with regard to smaller sized particles, are urgently needed over longer time periods to allow a full understanding of the total exposure of the environment to microplastic contaminants. Competing financial interest declaration The authors declare they have no actual or potential competing financial interests. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2016.04.025. References Andrady, A.L., 2011. Microplastics in the marine environment. Mar. Pollut. Bull. 62, 1596–1605. Bakir, A., et al., 2014. Enhanced desorption of persistent organic pollutants from microplastics under simulated physiological conditions. Environ. Pollut. 185, 16–23. Barnes, D.K.A., et al., 2009. Accumulation and fragmentation of plastic debris in global environments. Philos. Trans. R. Soc., B 364, 1985–1998. Bouwmeester, H., et al., 2015. Potential health impact of environmentally released micro- and nanoplastics in the human food production chain: experiences from nanotoxicology. Environ. Sci. Technol. 49, 8932–8947. Bravo-Rebolledo, E.L., et al., 2013. Plastic ingestion by harbour seals (Phoca vitulina) in The Netherlands. Mar. Pollut. Bull. 67, 200–202. Carpenter, E.J., et al., 1972. Polystyrene spherules in coastal waters. Science 178, 749–750. Cauwenberghe, L.V., et al., 2015. Microplastics are taken up by mussels Mytilus edulis and lugworms Arenicola marina living in natural habitats. Environ. Pollut. 199, 10–17. Chae, D.H., et al., 2015. Abundance and distribution characteristics of microplastics in surface seawaters of the Incheon/Kyeonggi coastal region. Arch. Environ. Contam. Toxicol. 69, 269–278. Chua, E.M., et al., 2014. Assimilation of polybrominated diphenyl ethers from microplastics by the marine amphipod, Allorchestes compressa. Environ. Sci. Technol. 48, 8127–8134. Cole, M., et al., 2013. Microplastic Ingestion by Zooplankton. Environ. Sci. Technol. 47, 6646–6655. Cole, M., et al., 2015. The impact of polystyrene microplastics on feeding, function and fecundity in the marine copepod Calanus helgolandicus. Environ. Sci. Technol. 49, 1130–1137. Colton, J.B., et al., 1974. Plastic particles in surface waters of the northwestern atlantic. Science 185, 491–497. Desforges, J.P.W., et al., 2014. Widespread distribution of microplastics in subsurface seawater in the NE Pacific Ocean. Mar. Pollut. Bull. 79, 94–99. Endo, S., et al., 2005. Concentration of polychlorinated biphenyls (PCBs) in beached resin pellets: variability among individual particles and regional differences. Mar. Pollut. Bull. 50, 1103–1114. Eriksen, M., et al., 2013. Microplastic pollution in the surface waters of the Laurentian Great Lakes. Mar. Pollut. Bull. 77, 177–182. Fendall, L.S., Sewell, M.A., 2009. Contributing to marine pollution by washing your face: microplastics in facial cleansers. Mar. Pollut. Bull. 58, 1225–1228. Fossi, M.C., et al., 2014. Large filter feeding marine organisms as indicators of microplastic in the pelagic environment: the case studies of the Mediterranean basking shark

(Cetorhinus maximus) and fin whale (Balaenoptera physalus). Mar. Environ. Res. 100, 17–24. Franeker, J.A., et al., 2011. Monitoring plastic ingestion by the northern fulmar Fulmarus glacialis in the North Sea. Environ. Pollut. 159, 2609–2615. Free, C.M., et al., 2014. High-levels of microplastic pollution in a large, remote, mountain lake. Mar. Pollut. Bull. 85, 156–163. Hall, N.M., et al., 2015. Microplastic ingestion by scleractinian corals. Mar. Biol. 162, 725–732. Hidalgo-Ruz, V., et al., 2012. Microplastics in the marine environment: a review of the methods used for identification and quantification. Environ. Sci. Technol. 46, 3060–3075. Holmes, L.A., et al., 2014. Interactions between trace metals and plastic production pellets under estuarine conditions. Mar. Chem. 167, 25–32. Hussain, N., et al., 2001. Recent advances in the understanding of uptake of microparticulates across the gastrointestinal lymphatics. Adv. Drug Deliv. Rev. 50, 107–142. Ivar do Sul, J.A., Costa, M.F., 2014. The present and future of microplastic pollution in the marine environment. Environ. Pollut. 185, 352–364. Karar, N., Gupta, T.K., 2015. Study of polymers and their blends using TOF-SIMS ion imaging. Vacuum 111, 119–123. Kathiresan, K., 2003. Polythene and plastics-degrading microbes from the mangrove soil. Rev. Biol. Trop. 51, 629–634. Kenyon, K.W., Kridler, E., 1969. Laysan albatrossess swallow indigestible matter. Auk 25, 339–343. Kukulka, T., et al., 2012. The effect of wind mixing on the vertical distribution of buoyant plastic debris. Geophys. Res. Lett. 39, L07601. Lambert, S., et al., 2014. Occurrence, degradation, and effect of polymer-based materials in the environment. Rev. Environ. Contam. Toxicol. 227, 1–53. Law, K.L., et al., 2014. Distribution of surface plastic debris in the eastern pacific ocean from an 11-year data set. Environ. Sci. Technol. 48, 4732–4738. Lusher, A.L., et al., 2015. Microplastic and macroplastic ingestion by a deep diving, oceanic cetacean: the True's beaked whale Mesoplodon mirus. Environ. Pollut. 199, 185–191. Mato, Y., et al., 2001. Plastic resin pellets as a transport medium of toxic chemicals in the marine environment. Environ. Sci. Technol. 35, 318–324. McCormick, A., et al., 2014. Microplastic is an abundant and distinct microbial habitat in an urban river. Environ. Sci. Technol. 48, 11863–11871. Ministry of Environment and Food of Denmark, Environmental Protection Agency, 2015l. Microplastics, occurrence, effects and sources of releases to the environment in Denmark. Environmental project No. 1793. Muthukumar, T., et al., 2011. Fouling and stability of polymers and composites in marine environment. Int. Biodeterior. Biodegrad. 65, 276–284. Nobre, C.R., et al., 2015. Assessment of microplastic toxicity to embryonic development of the sea urchin Lytechinus variegatus (Echinodermata: Echinoidea). Mar. Pollut. Bull. 92, 99–104. Prasun, K.R., et al., 2011. Degradable polyethylene: fantasy or reality. Environ. Sci. Technol. 45, 4217–4227. Rios, L.M., et al., 2007. Persistent organic pollutants carried by synthetic polymers in the ocean environment. Mar. Pollut. Bull. 54, 1230–1237. Saruhan, V., et al., 2010. The effects of sewage sludge used as fertilizer on argonomic and chemical features of bird's foot trefoil and soil pollution. Sci. Res. Essays 5, 2567–2573. Sen, S.K., Raut, S.J., 2015. Microbial degradation of low density polyethylene (LDPE): a review. Environ. Chem. Eng. 3, 462–473. Sivan, A., 2011. New perspectives in plastic biodegradation. Curr. Opin. Biotechnol. 22, 422–426. Tentschert, J., et al., 2014. Identification of nano clay in composite polymers. Surf. Interface Anal. 46, 334–336. Teuten, E.L., et al., 2007. Potential for plastics to transport hydrophobic contaminants. Environ. Sci. Technol. 41, 7759–7764. Thompson, R.C., et al., 2004. Lost at sea: where is all the plastic? Science 304, 838. Tomas, J., Guitart, R., 2002. Marine debris ingestion in loggerhead sea turtles, Caretta caretta, from the Western Mediterranean. Mar. Pollut. Bull. 44, 211–216. Wagner, M., et al., 2014. Microplastics in freshwater ecosystems: what we know and what we need to know. Environ. Sci. Eur. 26, 12–20. Yang, J., et al., 2014. Evidence of polyethylene biodegradation by bacterial strains from the guts of plastic-eating waxworms. Environ. Sci. Technol. 48, 13776–13784.