Polyethylene (PE; order no. MPP-635 G) with a size range of 11–13 μm and a density of 0.96 kg/m³ purchased from Micro Powders (New York, USA) was kindly provided within the JPI Oceans project EPHEMARE as a dry powder by Marina Albentosa (Instituto Español de Oceanografía, Centro Oceanografico de Murcia, Spain). Benzo[a]pyrene (BaP; CAS 50-32-8; purity ≥ 96%), benzophenone-3 [(2-hydroxy-4-methoxyphenyl)-phenylmethanone, BP3; CAS 131-57-7; purity ≥ 98%] and PFOS (1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-heptadecafluorooctane-1-sulfonic acid; CAS 2785-37-3; purity ≥ 98%) were purchased from Sigma Aldrich (Stockholm, SE). All other chemicals and reagents were purchased at the highest purity available from Sigma-Aldrich, unless stated otherwise.

Setups for the spiking, e.g., MPs and chemical concentrations were previously determined with preliminary experiments in order to determine all parameters to reach aimed concentrations, based on fish toxicity data, e.g., BP3 (Blüthgen et al., 2012; Paredes et al., 2014); BaP (Weigt et al., 2011) and PFOS (Yamashita et al., 2005). Preliminary studies demonstrated that to minimize loss of chemicals, PFOS exposure has to be prepared in polypropylene bottles (Lamaplast; Sesto San Giovanni, Italy), while for BaP and BP3, glass bottles (Thermo Scientific, Lund, SE) were the most efficient to avoid loss of compound.

According to the pretests, for the sorption of BP3 and BaP to PE, 125 g/L PE were added in 400 mL septa bottles (Thermo Scientific, Lund, SE) and bottles were filled to the maximum with double-deionized water, to avoid gas headspace in the bottle, and so avoid volatilization of compound. BP3 and BaP concentrations in the solutions were 20 and 2,500 μg/L, respectively. For the sorption of PFOS on PE, 50 g/L of plastic were weighed into 1 L polypropylene bottles (Lamaplast; Sesto San Giovanni, Italy) filled with double-deionized water and PFOS was added to a final concentration of 20 mg/L. All bottles were placed on a rotary shaker for 48 h (BP3 and BaP) or 7 days (PFOS) at 20 rpm and at room temperature (20 ± 1°C). Based on preliminary experiments, after 48 h, concentrations of BaP and BP3 adsorbed on MPs were higher than samples shaken for more than 48 h due to volatilization of compound, and an increase of the adsorption to glass bottle. While, for PFOS, the sorption time of 7 days was maintained because of the lowest standard deviation of the results and the highest concentration of PFOS adsorbed on particles, compared to other time points.

Concentrations of chemicals (BP3, BaP, and PFOS) were measured in corresponding spiked MPs as well as in virgin MPs to determine background contamination of each chemical.

After filtration of the MP preparations with a funnel equipped with a 1 μm Whatman^® glass microfiber filter (GE Healthcare Life Sciences; Uppsala, SE), the filters were rinsed with double deionized water and dried by vacuum evaporation on a ceramic funnel.

The analysis of BP3 was performed by Eurofins Germany (Pforzheim, DE) by using an 0.3 g aliquot of the sample spiked with internal standards (i.e., 3,4,5,6-pentadeuteriobenzoic acid, 1-bromo-4-phenylbenzene, 1,2,3,4,5,6,7,8-octadeuterionaphthalene, 1,2,3,4,5-pentachloro-6-(2,3,4,5,6-pentachlorophenyl)benzene, and 1,2,3,4,5,6,7,8,9,10-decadeuteriophenanthrene) and concentrated with a factor of 10. The samples were extracted with a 1:1 solvent mixture of ethyl acetate and cyclohexane. Additionally, an external calibration standard and a blank sample were prepared. The extracts were measured with GC/MS/MS, electron impact ionization (GC: Agilent7890 A, MS/MS: Agilent 7000 multiple-reaction monitoring mode, Agilent Technologies, Kista, SE) using a split-less injection of 2 μl (column DB5-MS, 30 m × 0.25 mm × 0.25 μm). Identification and quantification were performed against the retention time and the external calibration using the BP3- specific selected reaction monitoring (one quantifier and one qualifier).

BaP analysis has been conducted according to Larsson et al. (2013): MPs were extracted in hexane (≥98%, SupraSolv; Merck, Darmstadt, DE) after addition of an internal standard (500 ng BaP D12 in toluene; Chiron, Tuttlingen, DE; prepared standard: 10 ng/μl in toluene) by sonication and centrifugation at 2,000 rpm. Extracts were filtered through fiberglass and transferred to toluene (purity 96%, SOLVECO; Rosersberg, SE). GC vials for analysis were filled with 100 ng recovery standard perylene D12 (Chiron; 2 ng/μl in toluene, 50 μl added) and 500 μl of extract. The sample volume was reduced to 500 μl using a nitrogen stream. Concentrations of BaP were quantified using a Micromass Autospec Ultima high-resolution GC/MS system, separation on a 30 meters (0.25 mm internal diameter, 25 μm film thickness) DB-5MS column (J&W Scientific, Folsom, USA). Procedure blanks were included in all batches. Target BaP was quantified by using a five-point calibration curve. Relative standard deviation of the relative response factor values was <15% for PAHs. Quantification standard replicates were analyzed after every tenth sample. Concentrations of BaP were calculated by use of the internal standard method. The limit of detection (LOD) was defined as mean concentration in blanks plus three times the standard deviations and was 5 pg/g. Samples which had concentrations exceeding the range of the calibration curve were diluted and reanalyzed. Procedure blanks were included in all batches.

Chemical analysis of PFOS has been conducted according to Eriksson et al. (2016): PFOS (linear and isomers) adsorbed to MPs were extracted in methanol (>99.9% purity, Fisher Scientific) by ultra-sonication following by centrifugation (7,000 rpm). Extracts were filtered using a 5 mL PE syringe (Norm-Ject^® Henke Sass Wolf, Tuttlingen, DE) with a filter of 0.2 μm (13 mm, 0.2 μm AcrodiscGHP; Pall, Dreieich, DE). LC vials were prepared with 2 ng of recovery standard (mass-labeled PFOS). Samples were diluted 100 times using 40% methanol (v/v) and 60% ammonium acetate (v/v). Analysis was performed on an Acquity UPLC system coupled to a Xevo TQ-S quadrupole MS (Waters, Milford, USA). A guard column (PFC isolator, Waters) was inserted between the pump and the injector to prevent contamination from the system. PFOS were separated on 100 mm Acquity BEH C18 column (2.1, 1.7 mm) using methanol and water as mobile phases, both with 2 mM ammonium acetate. Quantification of total PFOS was performed by summarization of linear PFOS and individual branched PFOS calculated against an external calibration curve using technical PFOS as standard. Recoveries were assessed using labeled internal standards. The mean recoveries of labeled PFOS internal standards was 83–96%. The LOD was calculated as above was 10 pg/g. Procedure blanks were included in all batches.

Fish husbandry in the three participating laboratories conditions fully complied with OECD TG 236 (Westerfield, 2007; OECD, 2013), but maintenance of adult zebrafish (Danio rerio) differed slightly regarding dark/light cycles, size of the rearing tanks, size of heterosexual groups, and water conditions including temperature, pH and conductivity (Table 1). In each laboratory, feeding was twice daily ad libitum with commercially available artificial diets and freshly hatched brine Artemia nauplii. Constant filtering or permanent flow-through conditions guaranteed that ammonia, nitrite, and nitrate were kept below detection limits (5, 1, and 140 mg/L, respectively). Zebrafish eggs were collected according to OECD TG 236 (OECD, 2013).

For both the FET and the in vivo EROD assay, three independent runs were performed, and data were tested for normality with the Shapiro-Wilk and Kolmogorov-Smirnov tests. If data were normally distributed, one-way analysis of variance (ANOVA) was run in combination with a post-hoc Dunnett's test; otherwise, a non-parametric Kruskal-Wallis, Mann-Whitney U or Wilcoxon's matched-pairs tests were used for statistical comparison.

Data were first analyzed for differences between runs (biological replicates). Since there were no significant differences between independent runs, single data sets were merged for each laboratory, and tests on different exposure groups were performed.

In the case of LPMR, a repeated-measure ANOVA was performed to take into account the three successive periods of the test.

A p-value of 0.05 was considered statistically significant for all analyses. Graphical illustrations and statistical tests were performed with Sigma Plot 12.5 (Jandel-Systat, Erkrath, Germany).

Background contamination by all three pollutants was measured on virgin microplastics. Results showed no presence of BP3 (<0.05 ng/g, LOD) and BaP (<5 pg/g, LOD). However, PFOS was found on the PE particles at a concentration of 12.1 ± 3.5 pg/g. This background concentration was taken into consideration when analyzing concentrations of PFOS adsorbed to MPs particles.

Chemical quantification of the three pollutants on PE showed that each pollutant has specific sorption efficiencies: 51% (BP3), 84% (BaP), and 18% (PFOS) of the total amount of the pollutants in the solution adsorbed to the PE particles, respectively. Concentrations of BP3, BaP and PFOS on MPs are reported in Table 4.

Embryotoxicity and teratogenicity of the selected pollutants diluted in water and DMSO without MPs were evaluated using a FET supplemented by various sublethal endpoints. The results did not show any significant effect (lethal, sublethal effects as malformations and hatching rate) between the negative control and the exposed individuals (p > 0.05).

The spiked MPs particles with BP3, BaP, and PFOS caused neither acute toxicity nor any teratogenic effects in the embryos using particle concentrations of 10 and 100 mg/L (p > 0.05; Figure 1). The examination of embryos exposed to the different MPs particles showed minor changes of the hatching rate at 72 hpf. Results were, however, statistically not different at any time point measured (p > 0.05; Figure 2). Moreover, measurements of the larvae standard length at 5 dpf showed no significant differences between all conditions tested (p > 0.05; Figure S1).

EROD activity was measured in 96 hpf embryos incubated to 100 mg/L of the different MPs for either a 3 h pulse or 96 h continuously. After the 3 h pulse (Figure 3A), only MPs spiked with BP3 (MP + BP3) induced a significant increase in EROD activity (p < 0.05), whereas after 96 h of continuous exposure to MP + BP3 there was no induction of EROD activity (Figure 3B). In contrast, 96 h of continuous exposure to MPs spiked with BaP (MP + BaP) produced a significant increase in EROD activity (p < 0.05). MPs spiked with PFOS did not show any change in EROD activity whatever condition tested.

Gene transcription of cyp1a was measured in zebrafish embryos after 3 h of exposure starting at 96 hpf. Figure 4 shows normalized fold-change in expression of cyp1a relative to negative controls. Virgin MPs particles did not show any significant change of cyp1a transcription (p = 0.569). In contrast, exposure to MP + BaP and MP + PFOS induced a mild, but significant increase in cyp1a transcription (p = 0.013 and p = 0.039, respectively), while in the case of MP + BP3 the significance was just above the threshold (p = 0.059).

The larval photomotor response (LPMR) was performed at 5 dpf to monitor early behavioral disruption. Independent of the treatment, LPMR showed an increase in distance traveled during the dark phase (LOFF), if compared to both lights-on phases (LON1 and LON2; ANOVA F_(2,321) = 38.07, p < 0.001). Between treatments, however, there was no difference [repeated-measures ANOVA F_{(15, 281.98)} = 0.96, p = 0.496; Figure 5].

Embryotoxic and teratogenic effects were investigated for the single pollutants without MPs assuming that 100% of the pollutants desorb from 10 mg/L of spiked MPs. Therefore, concentrations tested for BP3, BaP, and PFOS were assumed to be 0.0008, 0.17, and 0.7 μg/L, respectively. For none of the compounds, any embryotoxic effects were observed, which is in agreement with the fact that the concentrations tested were several magnitudes lower than concentrations inducing toxicity reported in the literature for different aquatic organisms. For instance, immobilization tests with Daphnia magna at 48 h showed an EC₅₀ for BP3 between 1.67 and 1.9 mg/L (Fent et al., 2010; Sieratowicz et al., 2011). Sublethal effects of BP3 in zebrafish, rainbow trout (Oncorhynchus mykiss) and Japanese medaka (Oryzias latipes) were observed from 132 μg/L (Coronado et al., 2008; Blüthgen et al., 2012; Kim and Choi, 2014). A previous study showed that a 72 h exposure to BaP induced teratogenic and lethal effects zebrafish embryos at 63 and 126 μg/L, respectively (Weigt et al., 2011). PFOS produced sublethal and lethal effects in zebrafish embryos at 10 mg/L (Shi et al., 2008).

Likewise, PE particles up to 100 mg/L (with a diameter between 11 and 13 μm) without pollutants did not show any embryotoxic or teratogenic effects after 96 h of exposure. Similar results were found after exposure of marine medaka (Oryzias melastigma) using 10 mg/L of 11–13 μm PE particles (Beiras et al., 2018). Lithner et al. (2011) ranked polymers based on monomer hazard classification, and PE was classified as one of the less hazardous polymers for humans and the environment.

Regarding MPs spiked with pollutants, in the only comparable experiment so far, embryonic stages of marine medaka were exposed to 10 mg/L PE spiked with BP3 at the same concentration as in present study. Exposure to MP + BP3 induced premature hatching and a significant decrease in hatching rate (Beiras et al., 2018). The same authors also demonstrated a direct contact of MPs to villi at the surface of the marine medaka chorion, which may facilitate the uptake of toxicants. Although villi or similar structures do not exist on the zebrafish chorion, Batel et al. (2018) have shown that small MPs (1–5 μm) were able to adhere to the outer surface of the zebrafish chorion and to transfer BaP to embryos across the chorion. In order to force contact of the embryos with MPs, the exposure protocol by Ifremer used a smaller water volume than the other laboratories; yet this exposure protocol failed to induce toxic effects.

Differences have been observed between zebrafish and medaka, and beside the difference in egg surface (Hart and Donovan, 1983; Hart et al., 1984), which may interfere with MPs contact, additional factors such as exposure time might also account for the different responses observed between marine medaka and zebrafish. Indeed, different sensitivities in lethal and sublethal endpoints have been demonstrated between early life stages of Japanese medaka and zebrafish exposed to sediments spiked with two models PAHs (Perrichon et al., 2014). In the frame of FET test, one major difference between these two species is the duration of embryonic stage. For Japanese medaka, the embryonic stage lasts until days 10–11 dpf (Padilla et al., 2009), whereas this period is restricted to 48–72 hpf in zebrafish, depending on temperature (Kimmel et al., 1995). Similarly to Japanese medaka, mean hatching time for marine medaka is 11 dpf (Beiras et al., 2018). Therefore, future investigations on MPs effects in zebrafish might consider prolonged or even chronic exposure; however, this would be an animal experiment clearly requiring authorization under Directive 2010/63/EU.

CYP1A is involved in phase I of xenobiotic metabolism and is often used as a biomarker of exposure. For the investigation of sublethal effects due to MP exposures, changes in CYP1A expression was evaluated by analyzing (a) EROD activity in 96 h old zebrafish embryos exposed to 100 mg/L of MPs for 3 h (93–96 hpf) or 96 h (0–96 hpf) and (b) cyp1a gene transcription. Since previous studies documented short–term (3 h pulse) exposure to reference molecules to cause a more prominent EROD induction than 72 h continuous exposure (Kais et al., 2017, 2018) and has been shown to be long enough to induce cyp1 transcription (Larcher et al., 2014), the present study tested cyp1a transcription levels after short-term exposure to 10 mg/L MPs for 3 h only. Independent of MP concentrations, MPs alone did not induce EROD activity nor cyp1a transcription.

For BP3, only the short-term exposure (93–96 hpf) to 100 mg/L MPs + BP3 significantly induced EROD activity (p < 0.05), while cyp1a transcription was slightly upregulated (p < 0.1) after exposure to MP + BP3 at 10 mg/L. To our knowledge, BP3 induction of EROD activity has never been shown before in embryonic stages of aquatic organisms. In adult zebrafish, Blüthgen et al. (2012) documented an increase in cyp1a transcription in the brain of adult zebrafish after 14 days exposure to 312 μg/L BP3.

BaP is well-known to modulate EROD activity and to induce cyp1a transcription in various species (Jönsson et al., 2009; Rochman et al., 2013b; Batel et al., 2016). In the present study, MPs spiked with BaP caused an induction of EROD activity after 96 h of exposure. Meanwhile, a significant induction (p < 0.05) of cyp1a transcription was also shown after 3 h of continuous exposure to BaP-spiked MPs.

MPs spiked with PFOS did not modify EROD activity, but induced cyp1a transcription upregulation. Both in adult marine medaka and zebrafish, previous studies showed that PFOS modifies cyp1a transcription after exposure to 1–16 mg/L PFOS (Fang et al., 2012; Jantzen et al., 2016).

Albeit with some differences in response with respect to exposure duration—which may reflect the need for translation or enzyme accumulation—an overall triggering of CYP1A (either as EROD activities and/or as cyp1a transcription) was observed after exposure to MPs spiked with pollutants, whereas virgin MPs failed to do so. Results indicate that pollutants spiked on MPs are able to desorb and reach concentrations sufficient to induce biotransformation.

An increasing number of studies uses behavioral responses to monitor sublethal effects of pollutants such as PFOS or BPA on aquatic organisms (Huang et al., 2010; Wang J. et al., 2015), and changes in, e.g., the photomotor response are commonly interpreted as evidence of potential neurotoxicity (Le Bihanic et al., 2015; Legradi et al., 2018). Yet, our results did not show any alteration of the LPMR by any of the different MPs preparations. Similar results were shown after exposure of zebrafish embryos to pure polystyrene MPs particles by Chen et al. (2017), who spiked polystyrene MPs with EE2 (17α-ethinylestradiol) and found hypoactivity at elevated concentrations only. However, previous studies showed that microplastics may reduce other behavioral parameters such as predatory performance of fish depending on the shape of plastic particles (de Sá et al., 2015; Choi et al., 2018).

As water-borne pollutants, both PFOS (Huang et al., 2010) and BaP (Knecht et al., 2017) were shown to be able to modify the behavior of zebrafish. However, concentrations applied in these studies were several magnitudes higher than those used in this study and most likely lacked any environmental relevance. Therefore, further investigations are required to assess the toxicity of microplastics on behavioral aspects considering different endpoints and different kind of particles.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.