Size Matters: Ingestion of Relatively Large Microplastics Contaminated

Article Cite This: Environ. Sci. Technol. 2018, 52, 14381−14391

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Size Matters: Ingestion of Relatively Large Microplastics Contaminated with Environmental Pollutants Posed Little Risk for Fish Health and Fillet Quality Giedrė Ašmonaitė,*,† Karin Larsson,‡ Ingrid Undeland,‡ Joachim Sturve,† and Bethanie Carney Almroth† †

Department of Biological and Environmental Sciences, University of Gothenburg, Medicinaregatan 18A, 413 90 Göteborg, Sweden Department of Biology and Biological Engineering-Food and Nutrition Science, Chalmers University of Technology, Kemivägen 10, 412 96 Göteborg, Sweden

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‡

S Supporting Information *

ABSTRACT: In this study, we investigated biological effects associated with ingestion of polystyrene (PS) microplastic (MPs) in fish. We examined whether ingestion of contaminated PS MPs (100−400 μm) results in chemical stress in rainbow trout (Oncorhynchus mykiss) liver and we explored whether this exposure can affect the oxidative stability of the fillet during ice storage. Juvenile rainbow trout were fed for 4 weeks with four different experimental diets: control (1) and feeds containing virgin PS MPs (2) or PS MPs exposed to sewage (3) or harbor (4) effluent. A suite of ecotoxicological biomarkers for oxidative stress and xenobiotic-related pathways was investigated in the hepatic tissue, and included gene expression analyses and enzymatic measurements. The potential impact of MPs exposure on fillet quality was investigated in a storage trial where lipid hydroperoxides, loss of redness and development of rancid odor were assessed as indications of lipid peroxidation. Although, chemical analysis of PS MPs revealed that particles sorb environmental contaminants (e.g., PAHs, nonylphenol and alcohol ethoxylates and others), the ingestion of relatively high doses of these PS MPs did not induce adverse hepatic stress in fish liver. Apart from small effect on redness loss in fillets of fish exposed to PS MPs, the ingestion of these particles did not affect lipid peroxidation or rancid odor development, thus did not affect fillet’s quality.

1. INTRODUCTION

While the number of studies documenting the ingestion of MPs by fish has increased,38−42 fewer studies have addressed the toxicological effects of ingested MPs.43−46 As there could be many factors governing potential toxicity of MPs (size, shape, polymer type, and additives), it remains largely unknown if and how intrinsic polymer chemical attributes (e.g., additives or nonintentionally added substances (NIAS)) contribute to chemical stress. Chemical toxicity of synthetic polymers, plastic additives, or NIAS has been associated with endocrine disruption and oxidative stress,15,47−50 having potential health implications not only for aquatic organisms, but also on human populations (e.g., BPA, DEHP51). In the case of PS, the constituting monomer (styrene) has been shown to be genotoxic and potentially carcinogenic52−54 and oligomers have been shown to induce estrogenic effects,55,56 raising toxicological concerns of this plastic polymer.

Polystyrene (PS) is a synthetic aromatic polymer, widely used in packaging, building and construction, and electric applications.1 It is one of the most commonly used polymers worldwide2 with an estimated annual production of several million tons.3 PS often ends up as plastic debris in the aquatic environment and has been identified in various environmental and biological matrices,4−12 often as low density expanded PS. PS is among the most studied polymers in the research field of microplastics (MP) in terms of uptake, localization, and effects on various aquatic organisms.13,14,23,24,15−22 Exposure to PS MPs has documented negative effects on ecophysiological endpoints, such as reduced growth and survival,25 fecundity,22 developmental impairment,26,27 and changes in energy metabolism.28,29 As the majority of studies investigating effects of PS (and other types of MPs) have focused on lower micro size fraction (e.g., 100 μm) at which particles are often detected in environmental samples30 or reported ingested by variety of aquatic animals,31−36 are much less known.37 © 2018 American Chemical Society

Received: Revised: Accepted: Published: 14381

August 29, 2018 November 14, 2018 November 19, 2018 November 19, 2018 DOI: 10.1021/acs.est.8b04849 Environ. Sci. Technol. 2018, 52, 14381−14391

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Environmental Science & Technology

2. MATERIALS AND METHODS 2.1. Fish. Juvenile rainbow trout (Oncorhynchus mykiss) were bought from a fish farm (Vänneåns Fiskodling AB, Knäred, SE). Fish were fed ad libitum three times a week using commercial feed. Animal husbandry and feeding experiments were conducted in compliance with ethical practices from Swedish Board of Agriculture (Ethical permit number: 2202013). 2.2. Experimental Design. 2.2.1. Plastic Particles. Polystyrene (PS) powder was purchased from Goodfellow Cambridge Ltd. (Huntington, UK). To characterize particle morphology, light microscopy images of PS MPs subsample were taken (LEICA M205C) (Supporting Information (SI) Figure S1A) and to describe the surface topography of the particles scanning electron microscopy (SEM) (DSM 982 Gemini, Zeiss) was used (SI Figure S1B−C). PS MPs had readily diverse morphology and ranged between 100 and 400 μm in size. More detailed information on the morphology of the particles is presented in SI and Ref 71. 2.2.2. Particle Exposures in Situ. For in situ exposures, commercial PS powder was sieved with stainless steel net (>100 μm). Particles were weighed and enclosed within handmade metallic mesh envelopes (20 × 10 cm) made from the same material (Swedaq, SE). Metallic packages (n = 5, 20 g) were deployed in two different aquatic matrices to allow sorption of pollutants for 3 weeks. One set of packages was submerged in undiluted sewage effluent discharge in one of the largest sewage treatment facilities in Scandinavia (Ryaverket Gryaab AB, Göteborg, SE) with following water quality parameters: total nitrogen 32 mg L−1, total phosphorus 4.2 mg L−1, biochemical oxygen demand (BOD7) 206 mg L−1, total organic carbon (TOC4) 94 mg L−1. The average water flow in the deployment compartment was 3.7 m3 s−1 and temperature was 10−13 °C. The second set of packages was deployed in surface water (0.5−1 m depth; temperature: 4−8 °C), in the outer harbor of Gothenburg city, in location within close proximity to numerous industries (Hjuvik, Göteborg, SE) in April 2017. After the deployment period, packages were collected and briefly rinsed with water. Particles were allowed to dry in semiclosed glass containers for a week and thereafter used for preparation of experimental diets. 2.2.3. Chemical Analysis of Plastic Particles. Qualitative assessment of chemical compounds associated with experimental PS particles was performed with Ultra Performance Liquid Chromatography (UPLC) system (Dionex Ultimate 3000, Thermo Scientific) attached to Q Exactive Focus Hybrid Quadrupole-Orbitrap Mass Spectrometer (HRMS). The quantitative determination of PAHs in PS powder was carried out using high performance liquid chromatography (HPLC, Varian Prostar 240, M410, Agilent Technologies, Santa Clara, CA) (for more detailed information see SI). 2.2.4. Dietary Exposures. The experimental setup included four dietary treatments: (1) control (feed with no PS MPs); (2) diets containing virgin PS MPs (plastic control, PS), (3) diets, containing PS MPs exposed in sewage effluent (PSSewage), and (4) diets, containing PS MPs exposed in the outer harbor (PS-Harbor). Feed was administered daily in the form of handmade pellets (n = 5 pellets/fish/day; all detailed information about feed preparation is provided in SI). Estimated intake of MPs in plastic-containing diets varied from 500−700 to 2226−2411 particles/fish/day,71 largely

Furthermore, a controversial notion exists regarding whether MPs can act as vectors,57 ab/adsorbing environmental contaminants from surrounding environment and further transferring them to aquatic organisms and causing adverse biological effects.43,58 While scientific discussions about overall importance and relevance of this phenomenon continues, societal, commercial, industrial, and governmental concerns in regards to food quality and safety in this context arise.59−62 MPs have been identified in a number of marine species intended for human consumption.60 In regards to fish, accumulation of plastic particles in edible parts of the tissue may not be a considerable issue, as the gastrointestinal tract is removed prior to consumption.61 Thus, chemical toxicity and transfer of MP-associated chemicals to consumable parts of fish (e.g., fillet) may emerge as an issue of a greater concern. Upon ingestion, MPs can release associated chemicals into fish, and these compounds could accumulate in edible parts of tissue.60,63 It is hypothesized that MPs may directly contribute to increased levels of trace contaminants in fish that could potentially have direct implications on human health. On the other hand, contaminants (e.g., phthalates64) released by MPs could also have indirect effects on food quality, for example, via induction of oxidative stress, which can further translate into lipid or protein peroxidation. Lipid peroxidation has been shown to be one of the key factors affecting the quality of commercial fish-products,65 since oxidation of unsaturated lipids induce formation of, for example, aldehydes, giving rise to development of a rancid odor and taste that reduce the attractiveness of fish commodities and diminish its commercial value.66 It has also been documented that lipid peroxidation affects color and texture of fish, further reducing eating quality. Color changes are particularly characterized by redness loss caused by parallel oxidation of hemoglobin67 or loss of astaxanthin, the latter mainly in salmonid fish like rainbow trout.68 The decreased oxidative stability can affect the storage and processing of fish-products69 or, in extreme cases, may render the product harmful as some of the oxidation-derived aldehydes like 4-hydroxyhexenal (HHE) and 4-hydroxynonemal (HNE) can be, for example, cytotoxic and genotoxic.70 In this light, MP-mediated chemical transfer and subsequent oxidative stress could potentially play a role for food quality and safety of fish products. To address this, we designed a study that examined effects resulting from ingestion of virgin PS MPs (chemical toxicity of raw plastic material) and environmentally deployed MPs (vector effects) in fish. Using an effect-based approach we investigated the prevalence of chemical induced stress in fish liver, and explored whether MPs exposure could induce oxidative stress and subsequently have an impact on oxidative stability of fish fillet during ice storage, and affect its quality. A battery of well-established ecotoxicological biomarkers for oxidative stress and xenobiotic-related pathways was investigated in the hepatic tissue of rainbow trout exposed to PS MPs. Molecular markers specific for exposure to common environmental contaminants such as polycyclic aromatic hydrocarbons (PAHs), endocrine disrupting compounds (EDCs) and metals were included. Peroxide value, loss of redness and sensory analysis of rancid odor were utilized as markers during ice storage of fillets to screen of potential MPmediated chemical effects in the muscle of commercial fish species. With complementary chemical analysis we determined the chemical constituents, associated with these particles. 14382

DOI: 10.1021/acs.est.8b04849 Environ. Sci. Technol. 2018, 52, 14381−14391

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2.4. Hepatic Enzymatic Assays. Assessment of the catalytic activities of antioxidant enzymes (GR, GPx, GST, CAT) in cytosol was performed spectrophotometrically using the following original methodologies,73−78 adapted for 96 wellmicroplate reader (SpectraMax 190, Fisher Scientific).79,80 Molecular glutathione (tGSH and GSSG) measurements were conducted according established methodologies.77,78 Ethoxyresorufin O-deethylase (EROD) activity was measured in the microsomal fraction of hepatic tissue homogenates, using Rhodamine as a standard.81 Protein content in the microsomal and cytosolic fractions was determined by Lowry method82 using bovine serum albumin (BSA) as a standard. All hepatic tissue preparation for enzymatic activity measurements was performed according previously described procedures.79 2.5. Fish Fillet Quality Assessment during Ice Storage. 2.5.1. Sample Preparation. Frozen fillets (n = 11−12 from each exposure group) were thawed in a plastic bag under running cold water and then homogenized using a food processor (Philips HR 2373) into a pooled mince which was fortified with streptomycin (200 ppm) to prevent microbial growth during the storage trial. Pooled mince samples (30 g, n = 4) were flattened out in the bottom of 250 mL E-flasks (n = 4), so that the layer of mince was 6−8 mm thick, assuring full access to air. E-flasks were then stored on ice in darkness for up to 17 days to monitor lipid peroxidation. The pH of the pooled mince was determined at start of the storage with a Hamilton double pore electrode (Hamilton Double Pore, Bonaduz, CH). 2.5.2. Determination of Lipid Peroxide Value. To determine primary lipid peroxidation products, that is, lipid hydroperoxides, peroxide values (PV) were measured. Sample plugs (1 g; n = 3) were withdrawn from the flasks with a hollow cylinder to obtain a constant surface-to-volume ratio of each sample. Thereafter plugs were wrapped in aluminum foil and stored at −80 °C until analysis. Lipids were extracted from the mince and lipid hydroperoxides were determined in the chloroform phase with the ferrothiocyanate method.83 A standard curve was prepared from cumene hydroperoxide and results were expressed as μmol hydroperoxides kg mince−1 (n = 3). 2.5.3. Loss of Redness Measurement. During the storage trial, colorimetric analysis was used to investigate the loss of redness in the minces. Redness (a* value in the CIE Laboratory color scale) was monitored from the bottom of the E-flask by a colorimeter (Minolta Chroma Meter CR-400, Minolta Corp., Ramsey, NJ). Five different spots were averaged from each E-flask (n = 4 for each exposure group) to determine the redness index. 2.5.4. Sensory Analysis of Rancid Odor. To assess formation of rancid odor in mince, sensory analysis was deployed. The intensity of rancid odor in the headspace above the samples (n = 4 for each exposure treatment) was determined by smelling above the E-flask using the established methodology.84 This procedure was performed by three independent trained sensory panelists. To grade the intensity of rancid odor, a scale from 0 to 100 (where 100 represent strongest odor intensity) was used. 2.6. Statistical Analysis. Prior to statistical analysis, data were investigated for normality using Shapiro-Wilk test and visual assessment of normal Q-Q plots. Levene’s test for homogeneity of variance was performed to confirm the equality of variances for the exposure groups for every measured variable. For comparative analysis of biomarker responses one-way ANOVA with posthoc Dunnett’s test was

exceeding reported environmental thresholds. High exposures levels were selected with the intention of establishing high fugacity gradient between contaminated MPs and (uncontaminated) fish, leading to migration of chemicals into the fish.72 Exposures were conducted in glass aquaria (30 × 60 × 35; 48 L) subdivided in two compartments. In total, there were 12 individuals allocated to each treatment (114 ± 28.6 g; 22.7 ± 1.3 cm; n = 48) and were placed in individual compartments. Fish were fed individually on daily basis for 28 days. Experiments were conducted under flow-through conditions at 12.1 ± 1.1 °C, under 12:12 h light:dark regime. The levels of ammonia (NH4+), nitrates (NO3−) and nitrites (NO2−) in the water were as follows: 0.02 ± 0.01 mg L−1, 0.004 ± 0.002 mg L−1, and 0.9 ± 0.2 mg L−1. 2.2.5. Sampling. After 28 days of dietary exposure to PS MPs, the feeding experiment was terminated and fish were sampled. Fish were killed with an overdose Aquacalm (metomidate hydrochloride, 12.5 mg L−1). The dissected liver was split into different pieces for gene expression and enzymatic analysis (n = 11−12 per treatment) and frozen in liquid nitrogen until analyzed. Fish fillet (right side; 19.68 ± 5.21 g) was sampled by removing the muscle along the spine. The skin attached to the sliced muscle was carefully removed and samples were immediately placed on dry ice and kept in −80 °C until analysis. 2.3. Hepatic Gene Expression Analysis. RNA extraction of hepatic tissue was performed using a commercial RNeasy Plus mini kit (Qiagen, Hilden, DE) following manufacturer’s instructions. After extraction, RNA concentrations (ng μL−1) were determined spectrophotometrically using Nanodrop 2000 system (Thermo scientific, Waltham, MA). Sample purity ratios (260/280 and 260/230 ratios) were also recorded and ranged 1.9−2.5 and 1.7−2.4, respectively. The RNA integrity was evaluated by automatic electrophoresis RNA ScreenTape System 2200 (Agilent Technologies, Inc., Waldbronn, DE). RNA integrity number (RINe) and rRNA (28/18S) ratio were 8.6−9.6 and 2.2−2.4, respectively. cDNA synthesis was performed using iScript cDNA synthesis kit (Bio-Rad Laboratories, Inc., Hercules, CA) and the following thermal protocol: (1) 5 min at 25 °C, (2) 30 min at 42 °C, and (3) 5 min at 85 °C. In the synthesis reaction, 1 μg of mRNA was used and NoRT samples (no reverse transcriptase control) were synthesized and refrigerated at −20 °C until further analysis. Analyses of genes involved in antioxidant defense (NRF2, glutathione reductase (GR), glutathione S-transferase (GST), glutathione synthetase (GS), glutathione peroxidase (GP) catalase (CAT), glutamate cysteine ligase (GCL modyfying unit , GCL catalytic unit), superoxide dismutase (SODMn, SODZn_Cu) were used (SI Table S1). Also, genes involved in cytochrome P450 (CYP1a) metabolism (aryl hydrocarbon receptors: AhRα, AhRβ, and CYP1a), metal detoxification (metallothioneins: MTA, MTB) and endocrine regulation (ERα, ERβ, VTG, and AR) were quantified. Reference genes (ELF1-α and β-actin) were used. Gene expression analyses were performed using real time polymerase chain reaction (RT-qPCR) in a CFX Connect system (BioRad laboratories, Inc.). Thermal protocol included following steps: initial denaturation (1 min at 95 °C), followed by 39 cycles of denaturation (20 s at 94 °C) and annealing (30 s). In each RT-qPCR reaction (10 μL), 10 ng of cDNA was used. Each plate contained NoRT (no reverse transcription) and NTC (nontemplate) controls, to assess genomic DNA contamination and quality control, respectively. 14383


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Figure 1. Hepatic gene expression (A) and enzymatic activities of proteins, involved in antioxidant defense, CYP450 metabolism and molecular glutathione (B) after dietary exposure (28 days) to PS MPs. Data presented in a colorimetric scale as fold-change compared (gene expression) and as relative activities (enzymatic biomarkers) compared to control treatment, n = 11−12.

intermediates for industrial organic synthesis of various surfactants, detergents, and fragrances, including plastic polymers.85 Long-chain fatty acids can be used as emulsifiers in polymerization processes,86 lubricants or additives in plastic production.87 The use of vegetable oils as a base for production of environmentally friendly and nontoxic alternatives to industrial lubricants88 has been suggested and argued as a means of producing less hazardous plastics. On the other hand, our chemical analyses also revealed the presence of DEHP, a phthalate of high toxicological concern,51 as well as different PAHs (Table 2), indicating that hazardous (also carcinogenic) compounds are retained in PS powder. While pure polystyrene is considered to be a chemically inert polymer, PS material can shed monomers that are present in the final polymer as a result of incomplete polymerization processes89 or can leach chemical residuals or NIAS.90 Although the virgin PS was shown to contain a variety of chemicals, we did not observe any statistically significant changes in hepatic xenobiotic biomarker responses both at

employed. When assumption for homogeneity of variances was violated, Brown-Forsythe test was used. For lipid peroxidation data (odor sensory analysis, PV and loss of redness) repeated measures one-way ANOVA with Bonferonni corrections was used. Statistical analyses were conducted in SPSS (IBM SPSS Statistics, 319 Version 22, 2013).

3. RESULTS AND DISCUSSION To assess physiological effects relating to chemical toxicity of PS (and chemical additives), biomarkers related to oxidative stress, cytochrome P450 (CYP1a) metabolism, metal detoxification and endocrine regulation were investigated. Our results showed that ingestion of virgin PS MPs did not induce adverse changes in hepatic biomarker responses, suggesting low chemical toxicity of commercial virgin PS MPs (100−400 μm) (Figure 1). Nontarget chemical screening identified 14 compounds associated with commercial PS powder (Table 1), presumably residuals or impurities remaining from the polymer synthesis. For instance, decanoic acids are used as 14384


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Table 1. List of Chemicals with a Varying Degree of Hydrophobicity (LogKow = 4.76-7.5) Were Found to Be Associated with Virgin PS MPs and Particles after in Situ Exposures in Sewage Effluent (PS-Sewage) and Harbor (PS-Harbor)a

a The confidence level is expressed as the Schymanski’s confidence criteria (1−5) where 1 indicates the highest confidence level and 5 the lowest. The relative abundance of a compound between the samples was calculated by comparison of the peak areas.

mRNA (AhRβ, CYP1a, ERα, ERβ) and enzyme level (EROD) (p > 0.05 for all comparisons). PS can contain a mixture of aromatic compounds (e.g., PAHs) (Table 2), as well as styrene oligomers,91 prominent agonists for AhR receptor, activating cytochrome P450 metabolism, which also have been shown to have affinity to estrogen receptor (ER).55 On the contrary to our initial hypothesis, we did not observe this here. Lack of responses to PS MPs exposure could be explained by limited bioavailability of the compounds or by the fact that identified chemicals were for the most part readily nontoxic and easily

metabolized. In this light, the intrinsic polymer composition and presence (bioavailability) of additives or NIAS in raw plastics may be important factors determining potential biological effects (or lack of thereof) of exposure to plastics and highlights the importance of chemical characterization of plastic materials in (eco)toxicological studies. While our findings indicated no adverse impacts of these intermediate microsized particles on biomarkers related to oxidative stress and xenobiotic-related pathways, the results presented here are in line with recent publications.45,46,92,93 Nevertheless, with 14385


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Table 2. Quantitative Analysis of PAHs Detected on Virgin PS MPs and Particles after in Situ Exposures in Sewage Effluent (PS-Sewage) and Harbor (PS-Harbor); NR, Not Reported

In regard to research question, addressing the propensity for MPs to act as vectors for environmental pollutants, our results showed that PS MPs can sorb xenobiotics from environment and act as environmental vectors.57 A number of compounds were found in environmentally exposed PS MPs but were not present in virgin PS microparticles (Table 1). Nontarget chemical screening showed that during in situ deployment in sewage effluent, PS MPs obtained a more diverse chemical profile than particles exposed in a contaminated outer city harbor with 32 and 17 identified compounds, respectively (Table 1). Some chemicals (triclosan, nonylphenol ethoxylates, laurylsulfate, octylhydrogen sulfate) associated with sewagecontaminated PS MPs were ingredients of surfactants, detergents and fragrances commonly used in household applications, and they were also very characteristic to the deployment medium. Also, targeted chemical analysis revealed slightly increased amounts of certain PAHs in deployed MPs (Table 2). Harbor-exposed MPs did not show the chemical composition that we anticipated for this industrial environment, subjected to intense shipping and oil refinery activities100,101 (Tables 1 and 2). Only, a surfactant component from personal care products, cocoamidopropylbetaine, was explicitly found bound to harbor-exposed particles. Moreover, we observed lower amounts of Σ16PAHs in harbor-exposed particles compared to virgin PS MPs (Table 2), suggesting loss of chemicals from the polymer matrix during in situ deployment. Over a course of a few weeks, 1 g of plastic particles could release from just a few up to hundred nanograms of certain PAHs (e.g., fenantrene, fluoranthene, naftalene). Once in the environment, PS degrades and shed oligomers in the surrounding environment97,98 and one month is ample time to allow for formation of oligomers on the polymer surface98 and could facilitate leaching of chemical additives. Also, we suggested that an initiation of (oxidative) degradation, or weathering, could have occurred during in situ deployment (3 weeks) of particles in the environment. An increased relative abundance of carboxylic acids (compared to virgin PS powder) was documented in both in situ groups

precautionary principle in mind, we take care to caution that these studies may not be generalized while addressing toxicological risks of PS (as polymer), nor of larger MPs (>100 μm) in general. Current scientific literature suggests that polymer toxicity may greatly depend on the components of polymer formulation,94 thus the findings of this study may be polymer product-specific. It has been noted that raw plastic materials may contain less chemicals than those plastic products enriched with functional additives, thus intrincically possess lower toxicological potential. On the other hand, we did observe some trends in biomarker responses in the hepatic tissue that could potentially indicate limited MP-mediated chemical exposure by virgin PS MPs. For example, a tendency for low-level oxidative stress, mediated via the NRF2 pathway, could be observed in the liver of experimental fish exposed to virgin PS (Figure 1), as slight increase in gene expression of NRF2 was documented (p = 0.1; 0.29 fold-change). NRF2 is a transcription factor that acts as a cellular sensor, responding to oxidative and electrophilic stress and orchestrating expression and activation of multiple genes involved in detoxification and antioxidant responses.53 A growing number of studies indicate that particle contaminants can alter redox balance and induce oxidative stress.15,95,96 Exposure to MPs has been shown to mediate oxidative stress via NRF2 pathway, causing developmental delays and affecting fecundity in crustaceans (PS MPs; 0.05 for all comparisons). For example, ingestion of PS MPs exposed to sewage effluent showed more pronounced regulation of oxidative stress via NRF2 pathway (p = 0.1, 0.4 fold-change), which was followed by a consistent low-level upregulation of multiple downstream NRF2-regulated antioxidant genes (GCLmod, GR, SODCu/Zn, GSTπ, GPx, and GCLcat) and has led to marginal increases in activity of antioxidant enzymes (GSTπ, GR, GPx). Although these findings were not statistically significant, they still could suggest a coordinated biological response to reactive oxygen species (ROS) involving many components of the antioxidant defense system, simultaneously acting to mitigate cellular stress in PS-Sewage exposed fish. Simultaneous activation of oxidative stress genes may, in fact, be viewed as a measure of homeostatic regulation, which is an indication of adaptive capacity of an organism subjected to mild chemical exposure, or can be related to a general stress response. Furthermore, despite observed differences in the chemical constitution of environmentally contaminated PS treatments and virgin PS (Tables 1 and 2), a PCA analysis demonstrated similar grouping of biomarker responses of among different PS MPs exposures (SI Figure S3) and the control group, suggesting negligible differences between PS MPs treatments and low propensity for chemical toxicity of MPs-vector effects. Based on prevalent viewpoints, presuming low incidence of ingestion of MPs by aquatic animals of commercial importance, followed by low magnitude of PS MPs-mediated contaminant transfer,107−109 ingestion of MPs by aquatic animals may not currently represent a direct toxicological threat. Hence, we could argue MPs vectormediated effects (in the aquatic environment) on fish health may be negligible. In this study, we also explored whether ingestion of PS MPs can have an indirect impact on trout-based food commodities intended for human consumption, namely whether MPs induce oxidative stress, leading to lipid peroxidation in the muscle, and subsequently reducing eating quality of the fillet. Peroxide values (PV) (Figure 2A) and decrease in redness (a*

Figure 2. Lipid hydroperoxides (peroxide value, A), redness (a* value, B) and development of rancid odor (C) of pooled minced fish fillets in a storage trial on ice. Data are expressed as mean ± SD, n = 3−4. Significant differences from the control are denoted with (*), p < 0.05. 14387


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Environmental Science & Technology value) (Figure 2B) were measured in a storage trial using minces from each treatment group. The PV is commonly used to investigate primary stages of lipid peroxidation,110 and the loss of redness (decrease in a* value) is conventionally used to evaluate hemoglobin-mediated lipid peroxidation in fish fillets67 or lipid peroxidation-induced loss of astaxanthin.68 The PV in minced fillets of MP-exposed fish was not discernible from controls during storage on ice. Redness of the muscle tissue, on the other hand, was lower in MP-exposed fish (p < 0.05 for all treatments after 3 days of storage, and for the duration of the experiment), and could indicate slightly higher lipid peroxidation compared to the control group; or some direct effect of MP-exposure on red pigments (hemeproteins and astaxanthin). Sensory analysis supported PV data, it revealed slow development of rancid odor of all fillet minces that ranged from an intensity of 0 to 30 (Figure 2C). This indicated slow overall intrinsic formation of volatile compounds, like aldehydes and ketones, in the tissue over time. There were no clear differences in rancid odor between fillet minces of fish exposed to PS MPs and control fish. Taken altogether, while slightly higher loss of redness in fish fillet exposed to PS MPs (compared to controls) could indicate more lipid peroxidation, this finding warrants further investigations, before we can conclude that exposure to MPs has a potential to affect oxidative stability of fillet and its shelf life. With this current experimental evidence (based on exposure scenarios with much higher levels of MPs than is reported in the wild fish), we suggest that MPs-mediated chemical effects may be limited. In this scenario, MPs are not a cause for concern to food quality, at least not to lipid quality (note: we did not measure chemical contamination (bioaccumulation) in the fillets). According to United Nations Food and Agriculture Organization, estimated chemical exposure (to humans) to perstsitent organic pollutants and plastic additives, following consumption of seafood, containing MPs, is expected to be neglibile (less than 0.1%). 61 Nevertheless, due to fragmented knowledge, food safety risk analysis yet needs to be implemented to increase understanding of potential hazards and risks associated with consumption of seafood, contaminated with MPs or nanoplastics.61,62 With regards to the potential negative impacts of plastics per se on food safety, future studies could address the leakage of plastic-related chemicals along the fish product cycle: from fisheries and aquaculture, food processing111 and packaging,56,89 to provide relevant insights on plastic-mediated effects on the eating quality of fish, or for overall impacts on human food supply and safety.

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Bethanie Carney Almroth: 0000-0002-5037-4612 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are thankful to Gryaab AB sewage treatment facility for the opportunity to use facilities for in situ particle deployment. We thank the Centre for Cellular Imaging (CCI) of the Sahlgrenska Academy at Gothenburg University for the opportunity to use instruments and the kind support of its staff. Furthermore, Swedish Research Council (FORMAS), Helge Axelsson Johnsons and Adlerbertska scholarships’ foundations and SWEMARC (Swedish Mariculture Research Centre) at Gothenburg University are acknowledged for financial support for the project.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.est.8b04849. Complementary information about methodologies used (experimental feed preparation, nontarget chemcical analysis, analysis of PAHs and gene expression analysis) and PCA analysis (PDF)

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REFERENCES

AUTHOR INFORMATION

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*E-mail: [email protected]. ORCID

Giedrė Ašmonaitė: 0000-0002-4227-0792 14388


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