Size matters: ingestion of relatively large microplastics contaminated

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Ecotoxicology and Human Environmental Health

Size matters: ingestion of relatively large microplastics contaminated with environmental pollutants posed little risk for fish health and fillet quality Giedr# Ašmonait#, Karin Larsson, Ingrid Undeland, Joachim Sturve, and Bethanie M. Carney Almroth Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b04849 • Publication Date (Web): 19 Nov 2018 Downloaded from http://pubs.acs.org on November 20, 2018

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Environmental Science & Technology

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Size matters: ingestion of relatively large microplastics contaminated with environmental

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pollutants posed little risk for fish health and fillet quality

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Giedrė Ašmonaitė1*, Karin Larsson2, Ingrid Undeland2, Joachim Sturve1, Bethanie Carney

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Almroth1

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1Department

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Medicinaregatan 18A, 413 90 Göteborg, Sweden

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2Department

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University of Technology, Kemivägen 10, 412 96 Göteborg, Sweden

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*Corresponding author: [email protected]

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of Biological and Environmental Sciences, University of Gothenburg,

of Biology and Biological Engineering-Food and Nutrition Science, Chalmers

Graphic abstract

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1 ACS Paragon Plus Environment


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Abstract

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In this study, we investigated biological effects associated with ingestion of polystyrene (PS)

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microplastic (MPs) in fish. We examined whether ingestion of contaminated PS MPs (100-400

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µm) results in chemical stress in rainbow trout (Oncorhynchus mykiss) liver and we explored

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whether this exposure can affect the oxidative stability of the fillet during ice storage. Juvenile

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rainbow trout were fed for 4 weeks with four different experimental diets: control (1) and feeds

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containing virgin PS MPs (2) or PS MPs exposed to sewage (3) or harbor (4) effluent. A suite

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of ecotoxicological biomarkers for oxidative stress and xenobiotic-related pathways was

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investigated in the hepatic tissue, and included gene expression analyses and enzymatic

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measurements. The potential impact of MPs exposure on fillet quality was investigated in a

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storage trial where lipid hydroperoxides, loss of redness and development of rancid odor were

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assessed as indications of lipid peroxidation. Although, chemical analysis of PS MPs revealed

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that particles sorb environmental contaminants (e.g. PAHs, nonylphenol and alcohol

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ethoxylates and others), the ingestion of relatively high doses of these PS MPs did not induce

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adverse hepatic stress in fish liver. Apart from a small effect on redness loss in fillets, PS MPs

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ingestion did not affect lipid peroxidation or rancid odor development, thus did not affecting

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fillet’s quality.

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Keywords: Polystyrene, vector effects, rainbow trout, Oncorhynchus mykiss, oxidative stress,

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lipid, peroxidation, EDCs, biomarkers

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Introduction

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Polystyrene (PS) is a synthetic aromatic polymer, widely used in packaging, building and

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construction, and electric applications.1 It is one of the most commonly used polymers

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worldwide2 with an estimated annual production of several million tons.3 PS often ends up as

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plastic debris in the aquatic environment and has been identified in various environmental and

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biological matrices,4–12 often as low density expanded PS. PS is among the most studied

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polymers in the research field of microplastics (MP) in terms of uptake, localization and effects

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on various aquatic organisms.13,14,23,24,15–22 Exposure to PS MPs has documented negative

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effects on ecophysiological endpoints, such as reduced growth and survival,25 fecundity,22

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developmental impairment26,27 and changes in energy metabolism.28,29 As the majority of

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studies investigating effects of PS (and other types of MPs) have focused on lower micro size

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fraction (e.g. < 10 µm), the impacts of MPs at upper micro-size range (> 100 µm) at which

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particles are often detected in environmental samples30 or reported ingested by variety of

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aquatic animals,31–36 are much less known.37

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While the number of studies documenting the ingestion of MPs by fish has increased,38–42 fewer

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studies have addressed the toxicological effects of ingested MPs.43–46 As there could be many

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factors governing potential toxicity of MPs (size, shape, polymer type and additives), and it

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remains largely unknown if and how intrinsic polymer chemical attributes (e.g. additives or

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non-intentionally added substances (NIAS)) contribute to chemical stress. Chemical toxicity of

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synthetic polymers, plastic additives or NAIS has been associated with endocrine disruption

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and oxidative stress,15,47–50 having potential health implications not only for aquatic organisms,

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but also on human populations (e.g. BPA, DEHP51). In case of PS, it’s constituting monomer

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(styrene) has been shown to be genotoxic and potentially carcinogenic52–54 and oligomers have

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been shown to induce estrogenic effects,55,56 raising toxicological concerns of this plastic

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polymer. 3 ACS Paragon Plus Environment


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Furthermore, a controversial notion exists regarding whether MPs can act as vectors,57

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ab/adsorbing environmental contaminants from surrounding environment and further

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transferring them to aquatic organisms and causing adverse biological effects.43,58 While

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scientific discussions about overall importance and relevance of this phenomenon continues,

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societal, commercial, industrial and governmental concerns in regards to food quality and safety

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in this context arise.59–62 MPs have been identified in a number of marine species intended for

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human consumption.60 In regards to fish, accumulation of plastic particles per se in edible parts

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of the tissue may not be a considerable issue, as the gastrointestinal tract is normally removed

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prior the consumption.61 Thus, chemical toxicity and transfer of MP-associated chemicals to

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consumable parts of fish (e.g. fillet) may emerge as an issue of a greater concern. Upon

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ingestion, MPs can leach and/or release associated chemicals into fish, and these compounds

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could accumulate in edible parts of tissue.60,63 It is hypothesized that MPs may directly

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contribute to increased levels of trace contaminants in fish that could potentially have direct

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implications on human health. On the other hand, contaminants (e.g. phthalates64) released by

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MPs could also have indirect effects on food quality, for example, via induction of oxidative

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stress, which can further translate into lipid or protein peroxidation. Lipid peroxidation has been

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shown to be one of the key factors affecting the quality of commercial fish-products,65 since

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oxidation of unsaturated lipids induce formation of e.g. aldehydes, giving rise to development

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of a rancid odor and taste that reduce the attractiveness of fish commodities and diminish

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commercial value.66 It has also been documented that lipid peroxidation affects color and

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texture of fish, further reducing eating quality. Color changes are particularly characterized by

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redness loss caused by parallel oxidation of hemoglobin67 or loss of astaxanthin, the latter

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mainly in salmonid fish like rainbow trout.68 The decreased oxidative stability can affect the

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storage and processing of fish-products69 or, in extreme cases, may render the product harmful

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as some of the oxidation-derived aldehydes like 4-hydroxyhexenal (HHE) and 4-


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hydroxynonemal (HNE) can be e.g. cytotoxic and genotoxic.70 In this light, MP-mediated

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chemical transfer and subsequent oxidative stress could potentially play a role for food quality

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and safety of fish products.

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To address this, we designed a study that examined effects resulting from ingestion of virgin

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PS MPs (chemical toxicity of raw plastic material) and environmentally deployed MPs (vector

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effects) in fish. Using an effect-based approach we investigated the prevalence of chemical

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induced stress in fish liver, and explored whether MPs exposure could induce oxidative stress

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and subsequently have an impact on oxidative stability of fish fillet during ice storage, and

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affect its quality. A battery of well-established ecotoxicological biomarkers for oxidative stress

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and xenobiotic-related pathways was investigated in the hepatic tissue of rainbow trout exposed

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to PS MPs. Molecular markers specific for exposure to common environmental contaminants

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such as polycyclic aromatic hydrocarbons (PAHs), endocrine disrupting compounds (EDCs)

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and metals were included. Peroxide value, loss of redness and sensory analysis of rancid odor

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were utilized as markers during ice storage of fillets to screen of potential MP-mediated

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chemical effects in the muscle of commercial fish species. With complementary chemical

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analysis we determined the chemical constituents, associated with these particles.



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2

Materials and Methods

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2.1

Fish

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Juvenile rainbow trout (Oncorhynchus mykiss) were bought from a fish farm (Vänneåns

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Fiskodling AB, Knäred, SE). Fish were fed ad libitum three times a week using commercial

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feed. Animal husbandry and feeding experiments were conducted in compliance with ethical

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practices from Swedish Board of Agriculture (Ethical permit number: 220-2013).

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2.2

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2.2.1

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Polystyrene (PS) powder was purchased from Goodfellow Cambridge Ltd. (Huntington, UK).

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To characterize particle morphology, light microscopy images of PS MPs subsample were taken

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(LEICA M205C) (Supplementary Information (SI) Figure S1 A)) and to describe the surface

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topography of the particles scanning electron microscopy (SEM) (DSM 982 Gemini, Zeiss) was

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used (Figure S1 B-C). PS MPs had readily diverse morphology and ranged between 100 and

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400 µm in size. More detailed information on the morphology of the particles is presented in

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SI and Ašmonaitė et al, in preparation).

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2.2.2

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For in situ exposures, commercial PS powder was sieved with stainless steel net (> 100 µm).

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Particles were weighed and enclosed within handmade metallic mesh envelopes (20 x 10 cm)

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made from the same material (Swedaq, SE). Metallic packages (n = 5, 20 g) were deployed for

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3 weeks in two different aquatic matrices to allow sorption of pollutants. One set of packages

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was submerged in undiluted sewage effluent discharge in one of the largest sewage treatment

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facilities in Scandinavia (Ryaverket Gryaab AB, Göteborg, SE) with following water quality

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parameters: total nitrogen 32 mg L-1, total phosphorus 4.2 mg L-1, biochemical oxygen demand

Experimental design Plastic particles

Particle exposures in situ


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(BOD7) 206 mg L-1, total organic carbon (TOC4) 94 mg L-1. The average water flow in the

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deployment compartment was 3.7 m3 s-1 and temperature was 10-13 °C. The second set of

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packages was deployed in surface water (0.5-1 m depth; temperature: 4-8 °C), in the outer

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harbor of Gothenburg city, in location within close proximity to numerous industries (Hjuvik,

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Göteborg, SE) in April 2017.

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After the deployment period, packages were collected and briefly rinsed with water. Particles

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were allowed to dry in semi-closed glass containers for a week and thereafter used for

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preparation of experimental diets.

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2.2.3

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Qualitative assessment of chemical compounds associated with experimental PS particles was

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performed with Ultra Performance Liquid Chromatography (UPLC) system (Dionex Ultimate

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3000, Thermo Scientific) attached to Q Exactive™ Focus Hybrid Quadrupole-Orbitrap™ Mass

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Spectrometer (HRMS). The quantitative determination of PAHs in PS powder was carried out

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using high performance liquid chromatography (HPLC, Varian Prostar 240, M410, Agilent

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Technologies, USA) (for more detailed information see SI).

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2.2.4

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The experimental setup included 4 dietary treatments: 1) control (feed with no PS MPs); 2)

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diets containing virgin PS MPs (plastic control, PS-V), 3) diets, containing PS MPs exposed in

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sewage effluent (PS-SW) and 4) diets, containing PS MPs exposed in the outer harbor (PS-HB).

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Feed was administered daily in the form of hand-made pellets (n =5 pellets/fish/ day; all detailed

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information about feed preparation is provided in SI). Estimated intake of MPs in plastic-

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containing diets varied from 500-700 to 2226-2411 particles/fish/day (Ašmonaitė et al, in

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preparation), largely exceeding reported environmental thresholds. High exposures levels were

Chemical analysis of plastic particles

Dietary exposures



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selected with the intention of establishing high fugacity gradients between contaminated MPs

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and (uncontaminated) fish, leading to migration (exposure) of chemicals to the fish.71

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Exposures were conducted in glass aquaria (30 x 60 x 35; 48 L) subdivided in two

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compartments. In total, there were 12 individuals allocated to each treatment (114 ± 28.6 g;

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22.7 ± 1.3 cm; n = 48) and were placed in individual compartments. Fish were fed individually

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on daily basis for 28 days. Experiments were conducted under flow-through conditions at 12.1

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± 1.1 °C, under 12:12 h light: dark regime. The levels of ammonia (NH4+), nitrates (NO3-) and

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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

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± 0.2 mg L-1.

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2.2.5

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After 28 days of dietary exposure to PS MPs, the feeding experiment was terminated and fish

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were sampled. Fish were killed with an overdose Aquacalm™ (metomidate hydrochloride, 12.5

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mg L-1). The dissected liver was split into different pieces for gene expression and enzymatic

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analysis (n = 11-12 per treatment) and frozen in liquid nitrogen until analyzed. Fish fillet (right

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side; 19.68 ± 5.21 g) was sampled by removing the muscle along the spine. The skin attached

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to the sliced muscle was carefully removed and samples were immediately placed on dry ice

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and kept in -80 °C until analysis.

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2.3

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RNA extraction of hepatic tissue was performed using a commercial RNeasy® Plus mini kit

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(Qiagen, Hilden, DE) following manufacturer’s instructions. After extraction, RNA

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concentrations (ng µL-1) were determined spectrophotometrically using Nanodrop 2000 system

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(Thermo scientific, USA). Sample purity ratios (260/280 and 260/230 ratios) were also recorded

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and ranged 1.9-2.5 and 1.7-2.4, respectively. The RNA integrity was evaluated by automatic

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electrophoresis RNA ScreenTape System 2200 (Agilent Technologies, Inc., Waldbronn, DE).

Sampling

Hepatic gene expression analysis


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RNA integrity number (RINe) and rRNA (28/18S) ratio were 8.6-9.6 and 2.2-2.4, respectively.

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cDNA synthesis was performed using iScript™ cDNA synthesis kit (Bio-Rad Laboratories,

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Inc., USA) and the following thermal protocol: 1) 5 min at 25 °C; 2) 30 min at 42 °C and 3) 5

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min at 85 °C. In the synthesis reaction, 1 µg of mRNA was used and NoRT samples (no reverse

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transcriptase control) were synthesized and refrigerated at -20 °C until further analysis.

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Analyses of genes involved in antioxidant defense (NRF2, glutathione reductase (GR),

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glutathione S-transferase (GST), glutathione synthetase (GS), glutathione peroxidase (GP)

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catalase (CAT), glutamate cysteine ligase (GCLmodyfying

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dismutase (SODMn, SODZn_Cu) were used (Table S1). Also, genes involved in cytochrome P450

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(CYP1a) metabolism (aryl hydrocarbon receptors: AhRα, AhRβ and CYP1a), metal

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detoxification (metallothioneins: MTA, MTB) and endocrine regulation (ERα, ERβ, VTG and

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AR) were quantified. Reference genes (ELF1-α and β-actin) were used. Gene expression

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analyses were performed using real time polymerase chain reaction (RT-qPCR) in a CFX

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Connect system (Bio-Rad laboratories, Inc.). Thermal protocol included following steps: initial

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denaturation (1 min at 95 °C), followed by 39 cycles of denaturation (20 s at 94 °C) and

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annealing (30 s). In each RT-qPCR reaction (10 µL), 10 ng of cDNA was used. Each plate

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contained NoRT (no reverse transcription) and NTC (non-template) controls, to assess genomic

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DNA contamination and quality control, respectively.

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2.4 Hepatic enzymatic assays

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Assessment of the catalytic activities of antioxidant enzymes (GR, GPx, GST, CAT) in cytosol

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was performed spectrophotometrically (SpectraMax 190, Fisher Scientific) using following

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original methodologies,72–77 adapted for 96 well-microplate reader.78,79 Molecular glutathione

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(tGSH and GSSG) measurements were conducted according established methodologies.76,77

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Ethoxyresorufin O-deethylase (EROD) activity was measured in the microsomal fraction of

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hepatic tissue homogenates, using Rhodamine as a standard.

unit,

GCLcatalytic

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unit),

superoxide

Protein content in the 9

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microsomal and cytosolic fractions was determined by Lowry method81 using bovine serum

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albumin (BSA) as a standard. All hepatic tissue preparation for enzymatic activity

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measurements was performed according previously described procedures.78

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2. 5 Fish fillet quality assessment during ice storage

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2.5.1 Sample preparation

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Frozen fillets (n = 11-12 from each exposure group) were thawed in a plastic bag under running

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cold water and then homogenized using a food processor (Philips HR 2373) into a pooled mince

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which was fortified with streptomycin (200 ppm) to prevent microbial growth during the

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storage trial. Pooled mince samples (30 g, n=4) were flattened out in the bottom of 250 mL E-

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flasks (n = 4), so that the layer of mince was 6-8 mm thick, assuring full access to air. E-flasks

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were then stored on ice in darkness for up to 17 days to monitor lipid peroxidation. The pH of

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the pooled mince was determined at start of the storage with a Hamilton double pore electrode

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(Hamilton Double Pore, Bonaduz, CH).

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2.5.2 Determination of lipid peroxide value

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To determine primary lipid peroxidation products, i.e. lipid hydroperoxides, peroxide values

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(PV) were measured. Sample plugs (1 g; n =3) were withdrawn from the flasks with a hollow

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cylinder to obtain a constant surface-to-volume ratio of each sample. Thereafter plugs were

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wrapped in aluminum foil and stored at -80 °C until analysis. Lipids were extracted from the

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mince and lipid hydroperoxides were determined in the chloroform phase with the

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ferrothiocyanate method.82 A standard curve was prepared from cumene hydroperoxide and

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results were expressed as µmol hydroperoxides kg mince-1 (n = 3).

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2.5.3 Loss of redness measurement


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During the storage trial, colorimetric analysis was used to investigate the loss of redness in the

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minces. Redness (a* value in the CIE Laboratory color scale) was monitored from the bottom

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of the E-flask by a colorimeter (Minolta Chroma Meter CR-400, Minolta Corp., Ramsey, NJ).

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Five different spots were averaged from each E-flask (n = 4 for each exposure group) to

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determine the redness index.

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2.5.4 Sensory analysis of rancid odor

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To assess formation of rancid odor in mince, sensory analysis was deployed. The intensity of

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rancid odor in the headspace above the samples (n = 4 for each exposure treatment) was

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determined by smelling above the E-flask using the established methodology.83 This procedure

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was performed by 3 independent trained sensory panelists. To grade the intensity of rancid odor,

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a scale from 0 to 100 (where 100 represent strongest odor intensity) was used.

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2.6

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Prior to statistical analysis, data were investigated for normality using Shapiro-Wilk test and

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visual assessment of normal Q-Q plots. Levene’s test for homogeneity of variance was

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performed to confirm the equality of variances for the exposure groups for every measured

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variable. For comparative analysis of biomarker responses one-way ANOVA with post-hoc

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Dunnett’s test was employed. When assumption for homogeneity of variances was violated,

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Brown-Forsythe test was used. For lipid peroxidation data (odor sensory analysis, PV and loss

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of redness) repeated measures one-way ANOVA with Bonferonni corrections was used. For

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these statistical analyses SPSS (IBM® SPSS® Statistics, Version 22, 2013) was used.

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3

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To assess physiological effects relating to chemical toxicity of PS (and chemical additives),

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oxidative stress, cytochrome P450 (CYP1a) metabolism, metal detoxification and endocrine

Statistical analysis

Results and Discussion



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regulation were investigated. Our results showed that ingestion of virgin PS MPs did not induce

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adverse changes in hepatic biomarker responses, suggesting low chemical toxicity of

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commercial virgin PS MPs (100-400 µm) (Figure 1). Non-target chemical screening identified

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14 compounds associated with commercial PS powder (Table 1), presumably residuals or

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impurities remaining from the polymer synthesis. Decanoic acids are used as intermediates for

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industrial organic synthesis of various surfactants, detergents, fragrances, including plastic

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polymers.84 Long-chain fatty acids can be used as emulsifiers in polymerization processes,85

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lubricants or additives in plastic production.86 The use of vegetable oils as a base for production

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of environmentally-friendly and non-toxic alternatives to industrial lubricants87 has been

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suggested and argued as a means of producing less hazardous plastics. On the other hand, our

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chemical analyses also revealed the presence of DEHP, a phthalate of high toxicological

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concern,51 as well as different PAHs (Table 2), indicating that hazardous (also carcinogenic)

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compounds are retained in PS powder. While pure polystyrene is considered to be a chemically

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inert polymer, PS material can shed monomers that are present in the final polymer as a result

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of incomplete polymerization processes88 or can leach chemical residuals or NIAS.89 Although

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the virgin PS was shown to contain a variety of chemicals, we did not observe and statistically

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significant changes in hepatic xenobiotic biomarker responses both at mRNA (AhRβ, CYP1a,

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ERα, ERβ) and enzyme level (EROD) (p > 0.05 for all comparisons). PS can contain a mixture

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of aromatic compounds (e.g. PAHs) (Table 2), as well as styrene oligomers,90 prominent

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agonists for AhR receptor, activating cytochrome P450 metabolism, which also have been

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shown to have affinity to estrogen receptor (ER)55. On contrary to our initial hypothesis, we did

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not observe this here. Lack of responses to PS MPs exposure could be explained by limited

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bioavailability of the compounds or by the fact that identified chemicals were for the most part

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readily non-toxic and easily metabolized. In this light, the intrinsic polymer composition and

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presence (bioavailability) of additives or NIAS in raw plastics may be important factors


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determining potential biological effects (or lack of thereof) of exposure to plastics and

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highlights the importance of chemical characterization of plastic materials in (eco)toxicological

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studies. While our findings indicated no adverse impacts of these intermediate micro-sized

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particles on biomarkers related to oxidative stress and xenobiotic-related pathways, the results

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presented here are in line with recent publications.45,46,91,92 Nevertheless, with precautionary

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principle in mind, we take care to caution that these studies may not be generalized while

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addressing toxicological risks of PS (as polymer), nor of larger MPs (> 100 µm) in general.

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Current scientific literature suggests that polymer toxicity may greatly depend on the

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components of polymer formulation,93 thus the findings of this study may be polymer product-

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specific. It has been noted that raw plastic materials may contain less chemicals than those

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plastic products enriched with functional additives, thus intrincically possess lower

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toxicological potential.

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On the other hand, we did observe some trends in biomarker responses in the hepatic tissue that

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could potentially indicate limited MP-mediated chemical exposure by virgin PS MPs. For

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example, a tendency for low-level oxidative stress, mediated via the NRF2 pathway, could be

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observed in the liver of experimental fish exposed to virgin PS (Figure 1), as slight increase in

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gene expression of NRF2 was documented (p = 0.1; 0.29 fold-change). NRF2 is a transcription

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factor that acts as a cellular sensor, responding to oxidative and electrophilic stress and

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orchestrating expression and activation of multiple genes involved in detoxification and

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antioxidant responses.53 A growing number of studies indicate that particle contaminants can

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alter redox balance and induce oxidative stress.15,94,95 Exposure to MPs has been shown to

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mediate oxidative stress via NRF2 pathway, causing developmental delays and affecting

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fecundity in crustaceans (PS MPs; 100 µm) to induce

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oxidative stress. In line with our findings, previous animal studies using relatively large virgin

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MPs,92,95,97 including PS (40-150 µm98) demonstrate no or minor adverse effects related to

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oxidative stress.

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In regard to research question, addressing the propensity for MPs to act as vectors for

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environmental pollutants, our results showed that PS MPs can sorb xenobiotics from

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environment and act as environmental vectors.57 A number of compounds were found in

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environmentally exposed PS MPs but were not present in virgin PS microparticles (Table 1).

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Non-target chemical screening showed that during in situ exposures in sewage effluent, PS MPs

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obtained a more diverse chemical profile than particles exposed in a contaminated outer city

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harbor with 32 and 17 identified compounds, respectively (Table 1). Some chemicals (triclosan,

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nonylphenol ethoxylates, laurylsulphate, octylhydrogen sulphate) associated with sewage-

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contaminated PS MPs were ingredients of surfactants, detergents and fragrances commonly

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used in household applications, and they were also very characteristic to the deployment

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medium. Also, targeted chemical analysis revealed slightly increased amounts of certain PAHs

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in deployed MPs (Table 2). Harbor-exposed MPs did not show the chemical composition that

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we anticipated for this industrial environment, subjected to intense shipping and oil refinery

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activities99,100 (Table 1 and 2). Only, a surfactant component from personal care products,

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cocoamidopropylbetaine, was explicitly found bound to harbor-exposed particles. Moreover,

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we observed lower amounts of Ʃ16PAHs in harbor-exposed particles compared to virgin PS

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MPs (Table 2), suggesting loss of chemicals from the polymer matrix during in situ deployment.

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Over a course of a few weeks, 1 g of plastic particles could release from just a few up to hundred

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nanograms of certain PAHs (e.g. fenantrene, fluoranthene, naftalene). Once in the environment,

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PS degrades and shed oligomers in the surrounding environment96,97 and one month is ample 14 ACS Paragon Plus Environment

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time to allow for formation of oligomers on the polymer surface97 and could facilitate leaching

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of chemical additives. Also, we suggest that an initiation of (oxidative) degradation, or

317

weathering, could have occurred during in situ deployment (3 weeks) of particles in the

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environment. An increased relative abundance of carboxylic acids (compared to virgin PS

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powder) was documented in both in situ groups (Table 1). Also, yellowing of particles was

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observed following in situ exposure (Figure S2). Such color change has been associated with

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an increase of carbonyl bands and an increased oxygen content after artificial ageing of PS

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pellets101 and can be achieved via oxidation of phenolic additives.102 Formation of various

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functional groups (i.e. carboxylates103) can lead to increased polarity, further limiting sorption

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of hydrophobic organic pollutants104 and/or enhancing leaching of polymer additives. Overall,

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the greater number of compounds identified in PS particles exposed in situ to sewage effluent,

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compared to the PS-HB treatment (Table 1 and 2), is likely due to the higher concentration of

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chemicals and/or faster chemical exchange in undiluted effluent discharge versus the more

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dilute harbor water. Previous studies have highlighted differences in chemicals associated with

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MPs at different locations,89 potentially due to differring levels of background pollution. On the

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other hand, differences in the deployment medium (e.g. pH, temperature, salinity, etc.) could

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also play an important role for chemical partitioning onto plastic particulates,105 thus should be

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monitored and repoted during in situ deployment studies.

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In the line with these chemical analyses, our results suggested that the environmentally

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contaminated PS MPs could have greater toxicological potential compared to the virgin

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particles, as they yielded trends towards stronger biomarker responses, albeit with no statistical

336

significance (Figure 1A, p > 0.05 for all comparisons). For example, ingestion of PS MPs

337

exposed to sewage effluent showed more pronounced regulation of oxidative stress via NRF2

338

pathway (p= 0.1, 0.4 fold-change), which was followed by a consistent low-level upregulation

339

of multiple downstream NRF2-regulated antioxidant genes (GCLmod, GR, SODCu/Zn, GSTπ, 15 ACS Paragon Plus Environment


Page 16 of 41

340

GPx and GCLcat) and has led to marginal increases in activity of antioxidant enzymes (GSTπ,

341

GR, GPx). Although these findings were not statistically significant, they still could suggest a

342

coordinated biological response to reactive oxygen species (ROS) involving many components

343

of the antioxidant defense system, simultaneously acting to mitigate cellular stress in PS-SW

344

exposed fish. Simultaneous activation of oxidative stress genes may in fact be viewed as a

345

measure of homeostatic regulation, which is an indication of adaptive capacity of an organism

346

subjected to mild chemical exposure, or can be viewed as a general stress response.

347

Furthermore, despite observed differences in the chemical constitution of environmentally

348

contaminated PS treatments and virgin PS (Table 1 and 2), a PCA analysis demonstrated similar

349

grouping of biomarker responses of among different PS MPs exposures (Figure S3) and the

350

control group, suggesting negligible differences between PS MPs treatments and low

351

propensity for chemical toxicity of MPs-vector effects. Based on prevalent viewpoints,

352

presuming low incidence of ingestion of MPs by aquatic animals of commercial importance,

353

followed by low magnitude of PS MPs-mediated contaminant transfer,106–108 ingestion of MPs

354

by aquatic animals may not currently represent a direct toxicological threat. Hence, we could

355

argue MPs vector-mediated effects (in the aquatic environment) on fish health may be

356

negligible.

357

In this study, we also explored whether ingestion of PS MPs can have an indirect impact on

358

trout-based food commodities intended for human consumption, namely whether MPs induce

359

oxidative stress, leading to lipid peroxidation in the muscle, and subsequently reducing eating

360

quality of the fillet. Peroxide values (PV) (Figure 2A) and decrease in redness (a* value)

361

(Figure 2B) were measured in a storage trial using minces from each treatment group. The PV

362

is commonly used to investigate primary stages of lipid peroxidation,109 and the loss of redness

363

(decrease in a* value) is conventionally used to evaluate hemoglobin-mediated lipid

364

peroxidation in fish fillets67 or lipid peroxidation-induced loss of astaxanthin.68 The PV in 16 ACS Paragon Plus Environment

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365

minced fillets of MP-exposed fish was not discernable from controls during storage on ice.

366

Redness of the muscle tissue, on the other hand, was lower in MP-exposed fish (p < 0.05 for all

367

treatments after 3 days of storage, and for the duration of the experiment), and could indicate

368

slightly higher lipid peroxidation compared to the control group; or some direct effect of MP-

369

exposure on red pigments (heme-proteins and astaxanthin). Sensory analysis supported PV

370

data, it revealed slow development of rancid odor of all fillet minces that ranged from an

371

intensity of 0 to 30 (Figure 2C). This indicated slow overall intrinsic formation of volatile

372

compounds like aldehydes and ketones in the tissue over time. There were no clear differences

373

in rancid odor between fillet minces of fish exposed to PS MPs and control fish. Taken

374

altogether, while slightly higher loss of redness in fish fillet exposed to PS MPs (compared to

375

controls) could indicate more lipid peroxidation, this finding warrants further investigations,

376

before we can conclude that exposure to MPs has a potential to affect oxidative stability of

377

fillet, and its shelf-life and eating quality. With this current experimental evidence (based on

378

exposure scenarios with much higher levels of MPs than is reported in the wild fish), we suggest

379

that MPs-mediated chemical effects may be limited. In this scenario, MPs are not a cause for

380

concern to food quality, at least not to lipid quality (note: we did not measure chemical

381

contamination (bioaccumulation) in the fillets). According to United Nations Food and

382

Agriculture Organization, estimated chemical exposure (to humans) to perstsitent organic

383

pollutants and plastic additives, following consumption of seafood, containing MPs, is expected

384

to be neglibile (less than 0.1 %).61 Nevertheless, due to fragmented knowledge, food safety risk

385

analysis yet needs to be implemented to increase understanding of potential hazards and risks

386

associated with consumption of seafood, contaminated with MPs or nanoplastics.61,62 With

387

regards to the potential negative impacts of plastics per se on food safety, future studies could

388

address the leakage of plastic-related chemicals along the fish product cycle: from fisheries and

389

aquaculture, food processing110 and packaging,56,88 to provide relevant insights on plastic-



Page 18 of 41

390

mediated effects on the eating quality of fish, or for overall impacts on human food supply and

391

safety.


Page 19 of 41


392

393



Page 20 of 41

394 395

Figure 1 Hepatic gene expression (A) and enzymatic activities of proteins, involved in

396

antioxidant defense CYP450 metabolism and molecular glutathione (B) after dietary exposure

397

(28 days) to PS MPs. Data presented in a colorimetric scale as fold-change compared (gene

398

expression) and as relative activities (enzymatic biomarkers) compared to control treatment, n

399

= 11-12.

400


Page 21 of 41


401 402

Figure 2 Lipid hydroperoxides (peroxide value, A), redness (a* value, B) and development of

403

rancid odor (C) of pooled minced fish fillets in a storage trial on ice. Data are expressed as mean

404

± SD, n = 3-4. Significant differences from the control are denoted with (*), p > 0.05.



Page 22 of 41

405

Table 1 List of chemicals with a varying degree of hydrophobicity (LogKow = 4.76-7.5) were

406

found to be associated with virgin PS MPs and particles after in situ exposures in sewage

407

effluent (PS-SW) and harbor (PS-HB). The confidence level is expressed as the Schymanski’s

408

confidence criteria (1-5) where 1 indicates the highest confidence level and 5 – the lowest. The

409

relative abundance of a compound between the samples was calculated by comparison of the

410

peak areas.

411


Page 23 of 41


412

413



Page 24 of 41

414

Table 2 Quantitative analysis of PAHs detected on virgin PS MPs and particles after in situ

415

exposures in sewage effluent (PS-SW) and harbor (PS-HB), NR- not reported.

416 417

Supporting

information.

Complementary

information

about

methodologies

used

418

(experimental feed preparation, non-target chemcical analysis, analysis of PAHs and gene

419

expression analysis) and PCA analysis.

420 421

6

422

We are thankful to Gryaab AB sewage treatment facility for the opportunity to use facilities for

423

in situ particle deployment. The authors wish to thank the Centre for Cellular Imaging (CCI) of

424

the Sahlgrenska Academy at Gothenburg University for the opportunity to use instruments and

425

the kind support of its staff. Furthermore, Swedish Research Council (FORMAS), Helge

Acknowledgements


Page 25 of 41


426

Axelsson Johnsons and Adlerbertska scholarships’ foundations and SWEMARC (Swedish

427

Mariculture Research Centre) at Gothenburg University are acknowledged for financial support

428

for the project.



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Size matters: ingestion of relatively large microplastics contaminated

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