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Effects of different types of primary microplastics on early life stages of rainbow trout (<I>Oncorhynchus mykiss</I>),

Jakubowska Magdalena, Białowąs Marcin, Stankevičiūtė Milda, Chomiczewska Agnieszka, Jonko-Sobuś Karolina, Pažusienė Janina, Hallmann Anna, Bučaitė Agnė, Urban-Malinga Barbara

DOI wersji wydawcy / Published version

DOI http://dx.doi.org/10.1016/j.scitotenv.2021.151909

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Cytuj tę wersję / Cite this version

Jakubowska Magdalena, Białowąs Marcin, Stankevičiūtė Milda, Chomiczewska Agnieszka, Jonko-Sobuś Karolina, Pažusienė Janina, Hallmann Anna, Bučaitė

Effects of different types of primary Agnė, Urban-Malinga Barbara:

microplastics on early life stages of rainbow trout ( Oncorhynchus mykiss ),

Science of the Total Environment, vol. 808, no. art. ID 151909, 2022, pp. 1-11,

DOI:10.1016/j.scitotenv.2021.151909

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Effects of different types of primary microplastics on early life stages of rainbow trout (Oncorhynchus mykiss)

Magdalena Jakubowska

^a,

⁎ , Marcin Bia ł ow ą s

^a

, Milda Stankevi č i ū t ė

^b

, Agnieszka Chomiczewska

^c,d

, Karolina Jonko-Sobu ś

^a

, Janina Pa ž usien ė

^b

, Anna Hallmann

^c

, Agn ė Bu č ait ė

^b

, Barbara Urban-Malinga

^a

aNational Marine Fisheries Research Institute, Kołłątaja 1, 81-332 Gdynia, Poland

bLaboratory of Genotoxicology, Nature Research Centre, Akademijos st. 2, LT-08412 Vilnius, Lithuania

cDepartment of Pharmaceutical Biochemistry, Medical University of Gdańsk, Dębinki 1, 80-211 Gdańsk, Poland

dDepartment of Biochemistry, Medical University of Gdańsk, Dębinki 1, 80-211 Gdańsk, Poland

H I G H L I G H T S

• Effects of microplastic (MPs) on rainbow trout embryos and larvae were assessed.

• Exposure to MPs did not affect hatching success, rate and larvae survival.

• MPs had limited effect on growth and no effect on frequency of cytotoxic endpoints.

• Larvae exposed to polystyrene exhibited increased frequency of genotoxic endpoints.

• Elevated corticosterone concentrations were found in larvae exposed to polyethylene.

G R A P H I C A L A B S T R A C T

A B S T R A C T A R T I C L E I N F O

Article history:

Received 15 September 2021

Received in revised form 16 November 2021 Accepted 19 November 2021

Available online 25 November 2021 Editor: Henner Hollert

Plastic pollution is recognized as serious threat to aquatic organisms. The aim of this research was to determine the effect of environmentally realistic concentrations of various microplastics (MPs) on survival, growth, development and induction of endocrine, geno- and cytotoxic responses in the early life stages of rainbow troutOncorhynchus mykiss.

Fish were exposed for 69-days, from embryos at eyed-stage to mobile yolk-sac larvae, to pre-production pellets (3000μm; polystyrene - PS and polyethylene terephthalate - PET). Additionally, since salmonid larvae are particularly exposed to light polymers after swimming up from the bottom,ﬁsh were also treated with PE microspheres (150–180μm; polyethylene - PE) for both long (69-days, from embryos at eyed-stage) and short period (29 days, from larvae 3 weeks after hatching) to test the development stage-related effect on the growth parameters andﬁtness.

Hatching success, rate and the survival of larvae did not differ among treatments. Although some alterations were found in the length gain after the long-term exposure and in the yolk-sac exhaustion rate in all PE treatments, the final size of larvae did not differ from the respective controls. PE-treated larvae have shown elevated corticosterone concentrations being significantly higher infish exposed from the embryo stage. It was indicated for thefirst time that mobile yolk-sac larvae ingested MPs (up to 24% of larvae contained microspheres). No changes were recorded in cytotoxicity endpoints in any of the treatments, but exposure to PS pellets resulted in significantly higher frequencies of genotoxicity endpoints compared to the control treatment. This effect and aforementioned alterations in PE-treated larvae might result from the exposure to toxic MPs leaches. The fact that selected PAHs' levels reached the highest values in PS pellets and PE microspheres must be underlined.

Keywords:

Microplastics Fish larvae Growth rate Hatching Corticosteroids Cytogenetic damage

Science of the Total Environment 808 (2022) 151909

⁎Corresponding author at: Department of Fisheries Oceanography and Marine Ecology, National Marine Fisheries Research Institute, Kołłątaja 1, 81-332 Gdynia, Poland.

E-mail address:mjakubowska@mir.gdynia.pl(M. Jakubowska).

http://dx.doi.org/10.1016/j.scitotenv.2021.151909

Contents lists available atScienceDirect

Science of the Total Environment

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1. Introduction

Plastic is the most common component of marine litter (Galgani et al., 1995;Ramirez-Llodra et al., 2013). Microplastics (MPs) are usually deﬁned as particles orﬁbers within a size range of 1–5000μm (European Commis- sion, 2017;NOAA, 2020). They dominate in the plastic debris found in the marine (Van Cauwenberghe et al., 2015;Auta et al., 2017) and riverine systems (Lechner et al., 2014;Horton et al., 2017) where they pose a serious environmental threat (Auta et al., 2017;Worm et al., 2017). Microplastics are present in the water column (Lechner et al., 2014;Auta et al., 2017) as well as in sediments which constitute their ultimate sinks (Van Cauwenberghe et al., 2015;Horton et al., 2017).

The potential harmfulness of microplastics may be of both physical and chemical nature. MPs may cause harm to invertebrates and vertebrates upon ingestion (Pedà et al., 2016;Jin et al., 2018), accumulation in gills (Watts et al., 2014;Chen et al., 2020) or, in case of smaller nano- and microplastics, penetration through body membranes and into cells (Watts et al., 2014;Pitt et al., 2018;Hodkovicova et al., 2021). Chemical hazard related to the MPs exposure might be associated with their basic chemical composition i.e., unreacted residual monomers or polymerization impurities (Halden, 2010;Lithner et al., 2011), with the substances not bounded into the polymer matrix i.e., numerous additives used in production process like ﬂame retardants, stabilizers, antioxidants or plasticizers (Hermabessiere et al., 2017) or adsorbed pollutants from surrounding water (Rochman et al., 2013a). Potential transfer routes of pollutants to organisms include ingestion (Avio et al., 2015;Hollerová et al., 2021) and exposure to these chemicals present in the surrounding water due to plastic leaching (León et al., 2019).

Although an increasing number of studies is focused on MPs effects on biota, many of them use concentrations of MPs being much higher than those in the natural environment (Ogonowski et al., 2018). The investigation of the effects of MPs at environmentally relevant concentrations and under environmentally realistic exposure conditions is crucial for the un- derstanding of real impacts of MPs on aquatic ecosystems, proper risk assessment and thus further mitigation and prevention (Rochman and Boxall, 2014;de Sá et al., 2018).

Experimental studies conducted so far indicate that microplastics may pose a threat to aquatic organisms, irrespective of the exposure route. As the ingestion is the most frequent type of interaction between plastic debris and aquatic animals, an overwhelming majority of research is focused on the dietary exposure to MPs especially that contaminants associated with microplastics are more bioavailable to organisms upon ingestion due to desorption processes facilitated by gut surfactants (Bakir et al., 2014). The knowledge about non-dietary effects related to the chemical hazard associated with MPs, i.e. excluding the potential mechanical consequences of particle ingestion likely leading to the blockage of gastrointestinal tracts, false satiation and their subsequent physiological and toxicological consequences, is very limited. Negative effects of MPs leachates on the development of invertebrate embryos and early life stages of invertebrates andfish were documented even after short-term (24–72 h) exposure (Nobre et al., 2015;Gandara e Silva et al., 2016;Martínez-Gómez et al., 2017). Although fish are the most extensively studied group in terms of MPs effects (de Sá et al., 2018), the knowledge on their early life stages is exceptionally scarce, especially when related to embryos and endogenously feeding yolk-sac stage which are the most sensitive to environmental and anthropogenic pressures, and crucial for the population recruitment (Van Leeuwen et al., 1985). In addition, spawning, embryo development, hatching and the development of early larval stages of manyfish species such as salmonids, take place on the bottom where both embryos and larvae are exposed to plastic litter including its smallest size fractions (nano- and microplastics). Although embryos and newly hatched larvae are not vulnerable to MPs ingestion as they feed endogenously, they may be exposed to chemical substances leaching from the plastic.

Very recent research demonstrated for theﬁrst time that non-dietary exposure to polystyrene (PS) and polyethylene terephthalate (PET) pre- production pellets had signiﬁcant genotoxic effect on wild sea trout larvae

Salmo trutta(Jakubowska et al., 2020). However, since sea trout hatching and proper embryos and larvae development take place at comparatively low temperatures (approx. 5 °C), the duration of the embryo and larval stages is exceptionally long compared to many other commonﬁsh species.

The exposure of sea trout to MPs performed byJakubowska et al. (2020) from the fertilized eggs to yolk-sac swimming larvae was of a chronic nature and took 113 days. The question arises therefore about the effect of MPs on development of early life stages of many otherﬁsh species characterized by much shorter embryonic and larval development period.

The aim of the present study was toﬁnd out whether 69-day exposure to primary microplastics affects the survival, development parameters, leads to cellular damage and endocrine responses in early life stages of another salmonid - rainbow troutOncorhynchus mykisswhich is proposed to be a good model species over a wide range of research areas (Thorgaard et al., 2002;Finn, 2007). Since spawning, embryo development, hatching and development of the early larval stages of salmonids take place on the bottom, they are exposed to microplastic particles laying on the sediment in spawning grounds. Therefore, as experimental treatments we chose virgin pre-production pellets (3000μm) made of dense polymers - PS and PET.

Moreover, large MPs (1–5 mm), including pre-production pellets constitute a significant contribution to the plastic debris in marine and freshwater environments (Lechner et al., 2014;Van Cauwenberghe et al., 2015;Horton et al., 2017). We have hypothesized that exposure to large PS and PET pellets may induce toxic effects in larval rainbow trout. On the other hand, after swimming up from the bottom,fish larvae are likely to encounter less dense particles present in the water column or on the water surface, and as such are also potentially vulnerable to ingestion offloating MPs small enough to be swallowed. Despite being before the initiation of active feeding, the yolk-sac larvae are able for ingestion of exogenous particles as soon as their mouths open (Gerking, 1994). To investigate this effect both long-term (69 days, from eyed eggs) and short-term (29 days, from the stage of larvae few weeks after hatching) experiments were performed to assess whether duration of the exposure and two different PE microspheres (150–180μm) concentrations affect frequency of potential particle ingestion, larvae developmental parameters and induce toxic effects.

2. Materials and methods

2.1. Animal maintenance and experimental design

Embryos of rainbow trout in eyed stage 245 D° (degree-days i.e. °C day) were obtained from Dąbie Fish Hatchery (Dąbie, Poland) in January 2019.

After transportation to the laboratory and before the beginning of experiments, eggs were placed inflow-through system (freshwater, T = 5.5°, flow rate 5 L min⁻¹), described byJakubowska et al. (2020). Ten days before the start of exposure to MPs individuals were transferred to the experimental containers. The glass containers (V = 1.7 L) werefilled with 1.5 L of particle free (filtered through GF/Ffilters) water (freshwater,T= 5.5 °C) and contained 400 g of sediment (commercial gravel, diameter ~ 3 mm) on the bottom, to imitate the natural spawning ground of the species. The containers were placed in glass tanks connected to above mentioned recirculating system (V = 300 L,flow rate of 5 L min⁻¹; Fig. S1) equipped with a cooling unit (Titan 2000, Aqua Medic, Germany) to keep the constant water temperature at 5.5 °C. The water from containers did not mix with the surrounding water from the glass tanks which served as water baths. Two experiments were performed–long-, and short-term. For the long-term exposure (69 days) embryos were transferred to experimental containers (300 embryos per container) few hours after their transportation to the laboratory. For the short-term exposure (29 days) larvae being 3 weeks after mass hatching (i.e., 40 days after the embryos had been transported to laboratory) were moved to the experimental containers (200 larvae per container). Long-term and short-term experiment consisted of 5 and 3 treatments respectively, each treatment was performed in triplicate (i.e., consisted of 3 containers). Water in each container was vigorously aerated using diaphragm compressor (AT-40, Akwatech, Poland) with aer- ation stones. During the acclimation period as well as the exposure to MPs,

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the temperature in each glass tank was monitored continuously by Hobo loggers UA-001-08 (Onset, USA), with a resolution of 10 min. The oxygen concentration in each experimental container was measured every two days using HQ40d multi meter equipped with LDO oxygen sensor (Hach, Germany). The oxygen saturation did not fell below 83% (10.46 mg L), whereas mean saturation was 99.9% (12.6 mg L). Water in each container was exchanged once a week in a way to prevent removal of MP particles (ﬁltered through 100μm mesh). In case of evaporation the experimental water was supplemented to maintain the concentrations of MPs constant.

2.2. Microplastic exposure and data collection

Microplastics were added to each experimental container after the accli- matization period. Polyethylene (PE), polystyrene (PS) and polyethylene terephthalate (PET), belonging to most widely produced and most abun- dant polymers in aquatic environments (Plastics Europe, 2019) were used. Long-term experiment consisted of three MP treatments - commercial virgin pre-production pellets (particle diameter ~ 3000μm) made of PS and PET and commercial PE microspheres (150–180μm). In short-term experiment larvae were exposed only to PE microspheres (150–180μm) in different concentrations. The detailed information concerning treatments and used MPs are presented in theTable 1. In case of treatments with large microplastics (3000μm) the concentration equal to 0.1% of sediment dry weight (DW) was applied, as according toRedondo-Hasselerharm et al.

(2018), environmentally relevant microplastics concentrations in the sediment are within the range 0–1% plastic weight in sediment dry weight (DW). As PE microspheres due to their low density werefloating on the water surface their concentration was expressed per water volume. We chose the concentrations (0.33 and 1.33 mg L⁻¹, indicated as L (low) and H (high) respectively, thorough the manuscript) being within the environmental range and within the range of concentrations previously used in experiments onfish embryos and larvae (Karami et al., 2016, 2017;Chen et al., 2017;Pitt et al., 2018). Relevant (with similar density/buoyancy) natural particles (gravels ~3000μm and woodenflakes ~100–200μm) were used as controls to MPs, as according toOgonowski et al. (2018)similar effects may be also induced by natural particles, especially upon ingestion. Both microplastics and control particles had been kept in aerated water for 8 weeks before the start of the experiments.

Embryos were incubated in the dark, whilst the natural photoperiod (10:14–13:11 h light: dark) was applied after 100% hatching (on the 20th day). Asﬁsh were feeding endogenously, they were not fed during the experiments. During the exposure the number of dead embryos and larvae, as well as hatched larvae was recorded daily. Dead eggs and larvae were immediately removed. Approximately 30 larvae per replicate were randomly collected per week starting from 0 dph (days post hatching; long-term experiment) and 26 dph (short-term experiment) and preserved in ethanol for morphometric analyses. The day of mass hatching (0 dph) was considered to be the day when, on average, more than 95% of the larvae hatched in all treatments. Both experiments have been terminated close to the end of yolk-sack period. At the end of the experiments larvae from each variant (n= 3–11) were collected, immediately frozen in liquid nitrogen and

stored in−80 °C for the analysis of the concentrations of corticosteroid hormones in tissues. Blood samples (n= 15 per treatment) were also taken from larvae for determination of geno- and cytotoxicity responses by tail puncture. Additionally, in case ofﬁsh exposed to PE particles (n= 21 per treatment) were collected and frozen in−20 °C for the analysis of potential presence of MPs in their digestive tracts.

2.3. Survival, hatching, development and growth rate determination

Based on the number of dead eggs, dead larvae and hatched larvae recorded each day, the following parameters were calculated: time to hatch 50 and 90% of larvae, incubation period (time to hatch 100% of larvae), hatching period (the interval between hatching of theﬁrst and last egg), hatching rate (the percentage of larvae hatched each day) hatching success (the survival of embryos) and survival rate of larvae. The growth rate was determined based on measurements taken on larvae collected at 0, 24, and 52 dph (long-term experiment) and 26, 40 and 54 dph (short-term experiment). To this end the wet weight (wet wt.; ±0.0001 g) of each larva and its standard length (SL; ±0.01 mm) as well as length (L) and height (H) of yolk sac (±0.01 mm) were determined under stereoscopic microscope (SMZ 1500, Nikon Japan, total magniﬁcation 3.75x–270x). Since the yolk-sac in salmonids becomes elongated within a day after hatching (Groot, 1996), it was assumed to be an ellipsoidal mass (prolate spheroid) and its volume was calculated using the equation given byBagarinao (1986):

V¼0:1667πL H² (1)

The growth rates (daily weight and length gain) and the daily rate of yolk-sac absorption were calculated. All larvae collected during experiments were visually inspected for the occurrence of developmental malformations. Abnormally developed larvae were not taken into account in the calculations of growth rates.

2.4. Determination of whole-body steroid compounds concentration

Corticosteroids concentrations were determined according to Jakubowska et al. (2020). Each larva was homogenized separately using 5 mL of homogenization buffer (8.5 mM MgCl2, 3.13 mM KCl, 7.59 mM NaCl, 2.7 mM CaCl2, 50 mM Tris/HCl, pH 7.4) per gram of tissue. Next, MTBE (tert-butyl methyl ether) was added, the sample was mixed for 1 min and the mixture centrifuged at 3000 ×gfor 15 min at room temperature. The supernatant was transferred to a new glass vial and MTBE was allowed to evaporate to dryness in 45 °C. Upon evaporation, the dry residue of the eluate was dissolved in 100μL of methanol and 5μL of this solution was injected into the HPLC–ESI-MS/MS system. Calibration curves were prepared based on matrix and plotted as the peak area ratio (standard/IS) versus amount of analytes. All measurements in calibration curves were taken in triplicate. In the range of 0.03–100 ng g⁻¹regression coefﬁcients of all analytes were>0.99.

Table 1

Information on experimental treatments and characteristics of used particles.

Long-term experiment Short-term experiment

Treatment PS3000 PET3000 PE_150–180 PE_150–180L PE_150–180H

Material Polystyrene Polyethylene terephthalate Polyethylene Polyethylene Polyethylene

Polymer density (g L⁻¹) 1.04 1.38 0.96 0.96 0.96

Particle shape Pellets Pellets Spheres Spheres Spheres

Particle size (μm) 3000 3000 150–180 150–180 150–180

Concentration 0.1% of sediment DW 0.1% of sediment DW 0.33 mg L⁻¹ 0.33 mg L⁻¹ 1.33 mg L⁻¹

Amount of added particles 12–14^a 11–13^a ~221^b ~221^b ~885^b

Source BASF, Germany Polysciences, Inc., USA Cospheric LLC, United States Cospheric LLC, United States Cospheric LLC, United States a Range for three replications.

b Amount in experimental container (1.5 L), calculated based on the polymer density and mean particle volume (assuming mean diameters 0.165 and 0.275μm). Calcu- lations were veriﬁed by counting the particles in their known mass under stereomicroscope.

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2.5. Analysis of nuclear abnormalities (NAs) in an in vivo assay

After cutting the tail, whole-blood samples (~1–2μL) were dispersed directly on a microscope slide. Dried blood smears werefixed in ethanol for 10 min and stained with 10% Giemsa solution in phosphate buffer pH = 6.8 for 40 min. The stained slides were analyzed under bright-field Olym- pus BX51 microscopes (Tokyo, Japan) using an immersion objective (1000×). Two thousands erythrocytes with intact cellular and nuclear membranes perfish were evaluated by blind scoring. Thefinal results were expressed as the mean value (‰) of the sums of the analyzed individual lesions scored in 1000 cells per organism sampled from every study group. The formation of micronuclei (MN), nuclear buds (NB), nuclear buds onfilament (NBf) and blebbed nuclei (BL) erythrocytes were assessed as genotoxicity endpoints, and 8-shaped nuclei, fragmented apoptotic (FA) and binucleated (BN) cells as cytotoxicity endpoints. Due to the low frequencies of NB, NBf and BL cells, these parameters were summed up and expressed as total NB (NB + NBf + BL). Total genotoxicity (ΣGentox) levels were assessed as the sum of the frequencies of the detected genotoxicity (MN + NB + NBf + BL) endpoints. Morphological features of the nuclear abnormalities studied were identified using criteria described by Heddle et al. (1991)andFenech et al. (2003)(the detailed description of these criteria was presented in previous study (Baršienėet al., 2012).

2.6. Microplastics quantiﬁcation in larvae

Larvae recovered after the experiments were thawed and thoroughly rinsed withfiltered (GF/Ffilter Whatman 47 mm, 0.7μm), deionized water in order to remove any MPs potentially attached to the body. Each larva was placed into a separate glass vial and was digested with 2 mL of di- gestion solution (150 ml KOH (1120 g L⁻¹) and 150 ml NaClO (14% active chlorine) added to 700 mL water;Lenz et al., 2016). The vials were left overnight at the room temperature. The whole digested material from each individual wasfiltered throughfilters made of chromium‑nickel mesh (47 mm in diameter, 25μm mesh size) using vacuum pump. Filters were then inspected under the stereoscopic microscope (SMZ 18, Nikon Japan, total magnification 3.75x–270x) to assess the presence and the number of microplastic particles.

2.7. Chemical analyses of microplastics

The extraction method for chemical component of microplastics was based on theNguyen and Scales (2019)with modifications. In brief, 1 g per sample of each polymer was spiked with appropriate standard (isotope C13-labelled for PBDEs, PAHs' perdeuterated standards) and extracted according to the following procedure: a) PBDEs and PAHs were extracted two times with 10 mL of hexane and 10 mL of methanol using ultrasonic bath; b) PCBs were extracted using the same amount and type of solvents, placed on laboratory shaker for 8 h and set on the medium speed. Extracts were collected, combined and concentrated to 1 mL. Samples were purified on glass chromatographic columns packed with anhydrous sodium sulphate acidic and deactivated silica gel (for PBDEs, PCBs) or anhydrous sodium sulphate and deactivated silica gel (for PAHs). This step was brought to remove any remaining organic contamination or plastic particles which could affect thefinal results. Final steps were solvent exchange and eluates concentration to the eventual volume. The final volume was 0.2 mL in isooctane (PAHs) and 0.1 mL in nonan (PCBs and PBDEs). All analyses were performed using gas chromatography–mass spectrometry (GC–MS). Reagent blanks and replication of each sample were performed to ensure the quality control during the analysis. The laboratory glassware for PAHs analyses was pre-washed with dichloromethane directly before use.

2.8. Statistical analysis

The comparisons of the Kaplan-Meier survival curves were used to test for the signiﬁcance in the survival of embryos and larvae among

experimental treatments. The hatching rate (percentage distribution of the hatched individuals in time) in each treatment was compared to distribution in the relevant control group usingχ²-test. For all morphometric measurements, due to high inter-individual variability and lack of signiﬁ- cant differences between replicates, three replicates were pooled together for calculations and statistics to increase the sample size (to ~90 per treatment). For each experimental treatment, the linear regression lines were ﬁtted between measured wet weight, standard length or the yolk sac volume and the larvae age (dph). In order to compare the growth rates of larvae reared in each MP treatment and the control group, the homogeneity of regression slopes was compared using analysis of covariance (ANCOVA).

Before this analysis boxplots were created to identify and remove outliers (values>2 SD form the mean). The normal distribution of the data on wet weight, standard length and yolk sac volume on each day of larvae collection as well as geno- and cytotoxicity data and the concentrations of corticosteroids in larvae between various treatments were tested by the Shapiro-Wilk test. In case the data did not follow the normal distribution, the non-parametric Mann-WhitneyUtest or Kruskal-Wallis test at a confi- dence level ofα= 95% (with Dunn-Bonferroni post hoc) were used to test the significance of the differences among experimental treatments, depending on the number of treatments including relevant controls. One-way ANOVA with the Bonferroni post hoc or independent samplest-test was used in case the data met the assumption on normal distribution. In case of geno- and cytotoxic data for all sequential tests,p-values were corrected using FDR (Benjamini and Hochberg, 1995). The significance of differences in the proportion of individuals containing microspheres in the digestive tracts were compared among the treatments using the difference between two proportions test. All above mentioned analyses were performed using STATISTICA software (10.0 Software, Inc. PA, USA). Statistical analysis of the cytogenetic biomarkers was performed using R and RStudio (Versions 4.0.2 and 1.3.1073, respectively).

3. Results

3.1. Survival, hatching and growth rate

The hatching success (survival rate of rainbow trout embryos) was higher than 98% in all experimental as well as control treatments after long-term exposure (Table 2). No signiﬁcant differences in this parameter were recorded among the treatments (comparison of Kaplan-Meier survival curves,p>0.05). The survival rate of hatched larvae was not signiﬁcantly different among experimental variants (Table 2, comparison of Kaplan- Meier survival curves,p>0.05).

Similarly, in the short-term experiment no signiﬁcant changes were observed in the survival rate of larvae exposed to PE microspheres (Kaplan- Meier survival curves,p>0.05). After 29 days of exposure the mean survival rate of larvae was higher than 95% in all experimental treatments (Table 2).

The differences in hatching rate between individuals exposed to microplastics and the control ones were insigniﬁcant (χ²test,p>0.05;

Fig. S2). The incubation period (time to hatching = 100%) varied between 19 and 31 days depending on the treatment and replicate, however,>90%

of larvae were hatched after 16–17 days in all variants (Table 2).

In long-term experiment no changes in growth rate expressed in the weight gain were observed in any experimental treatment compared to relevant controls (ANCOVA slope,p>0.05;Fig. 1A;Table 2). The length gain was significantly higher in PE150–180treatment compared to the control (ANCOVA slope,p= 0.000, intercept,p>0.05;Fig. 2B;Table 2), whereas yolk sac absorption rate was significantly lower (ANCOVA slope,p= 0.014, intercept,p= 0.036). Significantly lower yolk sac absorption rate was also observed in rainbow trout exposed to PET (ANCOVA slope,p= 0.013, intercept,p= 0.033;Fig. 1C;Table 2). No changes in length gain and yolk sac absorption in larvae exposed to PS as well as in length gain of PET-exposedfish were noticed (ANCOVA slope,p>005). No significant changes in wet weight, standard length and yolk-sac volume between the treatments were noticed after 41-day (24 dph) or 69-day exposure (52

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dph) (Kruskal-Wallis/One-way ANOVA and Mann-Whitney U/t-tests p>0.05), whereas standard length and yolk-sac volume in larvae from PE_150–180treatment were signiﬁcantly lower (Mann-Whitney,p= 0.000;

t-test,p= 0.019, respectively) at hatch (after 17-day of embryo exposure) in PE150–180Ltreatment.

In short-term experiment no changes in growth rate of larvae expressed in weight or length gain were noticed (ANCOVA slope,p>0.05;Fig. 2A, B) in any experimental concentration. In larvae exposed to both PE concentrations PE_150–180Land PE_150–180Hyolk-sac absorption was signiﬁcantly higher from the control conditions (ANCOVA slope, intercept,p= 0.003;

ANCOVA slope,p= 0.004, intercept,p>0.05, respectively;Fig. 2C;

Table 2). There were no signiﬁcant changes in wet weight, standard length and yolk-sac volume between the treatments after 15-day (40 dph) or 29- day exposure (54 dph) (Kruskal-Wallis/One-way ANOVA and Mann- Whitney U/t-testsp>0.05).

We have observed one developmental abnormality in all experimental treatments including control - the accumulation ofﬂuid (edema) in the yolk (Fig. S3; Table S1). In long-term experiment, the highest frequency of edema occurrence (8.9%) was observed in 14 dph larvae exposed to PS and PET and in 24 dph larvae exposed to PE150–180(7.97%) while the low- est frequency was observed in the control treatments (2.4–4.4%). In the short-term experiments the edema occurrence after exposure to PE microspheres (both concentrations) was less evident and below 5% in all variants. The changes in frequency of edema occurrence were not signiﬁcant (difference between two proportions testp<0.05). The edema was not observed before 14 and after 45 dph. No other malformations were noticed.

3.2. Concentration of whole-body corticosteroids

The mean (±SE) and median values of whole-body corticosteroid hormones in rainbow trout after long- and short-term exposure to various microplastics are presented inTable 3. In both experiments there were no signiﬁcant differences in the whole-body concentrations of dehydrocorticosterone, cortisone and cortisol between MP-exposed larvae and relevant controls (Mann-Whitney U/Kruskal-Wallis tests,p>0.05).

Significantly higher concentrations of corticosterone were, however, observed in rainbow trout exposed to PE150–180Lfor 69 days (Mann-Whitney Utest,p= 0.014). High inter-individual variability in concentrations of cortisol was observed. Cortisol was not detected in larvae from the control treatment (control 3000) while its detectable concentrations were recorded in 60 and 30% offish exposed to PS and PET respectively. In case offish exposed to PE microspheres in long experiment, cortisol was detected in 40%

of exposedfish and in 50% in the relevant control. In the short experiment the percentage offish characterized by cortisol concentration higher than zero varied between 18% in the control and 54% infish exposed to PE150–180applied in lower concentration.

3.3. Geno- and cytotoxic effects

Frequencies of MN and NB did not show any significant (Kruskal-Wallis test,p>0.05) changes in the MPs treatment groups from the long-term experiment, except for NB (Kruskal-Wallis test,p= 0.001) in PS treatment group compared to the control value (Dunn test,p<0.001) (Fig. 3A). The exposure to PS induced significant formation of∑Gentox (Kruskal-Wallis test,p= 0.001) in rainbow trout erythrocytes compared to the control group (Dunn test,p<0.001) (Fig. 3B). Cytotoxicity endpoints were not significantly affected by long-term exposure to MPs (data not shown). 8- shaped nuclei erythrocytes was the only cytotoxicity endpoint detected in MPs exposed groups with frequencies varied from 0.17‰in PE group to 0.27‰in PS-exposed group (0.10–0.17‰in control groups, respectively).

PE microspheres were not found to induce a signiﬁcant (allp>0.05) changes in NAs frequencies in rainbow trout erythrocytes during 29-day exposure (Fig. S4), though frequencies of genotoxicity endpoints were slightly increased in PE-treated groups compared to control values. Signiﬁcant changes in cytotoxicity endpoints were not observed either (data not shown).

Table2 Parametersrelatedtohatchingandgrowthofrainbowtroutduringlong-(69days)andshort-term(29days)exposuretodifferentmicroplastics.Thevaluesaregivenasarangeforthreereplicationsexceptforthegrowthrateswhich werepooledforthereplicates.Asterisksindicatesigniﬁcantdifferencecomparedtorelevantcontrols. Long-termexperimentShort-termexperiment TreatmentControl3000PS3000PET3000Control150–180PE150–180Control150–180PE150–180LPE150–180H T(°C)5.4±0.2–5.5±0.35.4±0.2–5.5±0.35.4±0.2–5.5±0.35.4±0.2–5.5±0.35.4±0.2–5.5±0.35.3±0.4–5.4±0.55.3±0.4–5.4±0.55.3±0.4–5.4±0.5 O2(mgL−1)12.56±0.34–12.68 ±0.3212.45±0.51–12.67 ±0.3912.49±0.49–12.55 ±0.4812.53±0.42–12.69 ±0.2712.52±0.56–12.56 ±0.4312.50±0.21–12.70 ±0.2812.67±0.31–12.69 ±0.2712.49±0.49–12.55 ±0.48 Dayofﬁrsthatching9–127–117–1110–137–13 Timetohatch>50%(days)15–1615–1515–1515–1515–15 Timetohatch>90%(days)16–1716–1716–1616–1715–17 Incubationperiod(days)19–1919–1019–3119–2819–22 Hatchingperiod(days)8–139–1313–217–98–12 Hatchingsuccess(%)98.65–99.3398.65–99.6798.31–10098.31–10099.67–100 Larvaesurvival(%)88.13–95.7791.57–97.0089.24–96.9595.07–97.8992.84–94.2899.39–10097.53–99.2595.72–99.26 Growthrate(mgday−1)0.280.300.240.290.350.930.801.00 Growthrate(mmday−1)0.110.100.110.110.13⁎0.050.050.05 Yolk-sacexhaustion (mm3day−1)0.710.740.67⁎0.710.64⁎0.640.74⁎0.72⁎

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3.4. Presence of microplastics in larvae

After 69-day exposure to PE microspheres (0.33 mg L⁻¹) 19% of larvae contained MPs in their digestive tracts and the number of particles per one individual ranged from 0 to 2 (Fig. 4A). Both mean number of particles per larvae and percentage of larvae containing microplastics were similar in case of individuals from short-term experiment exposed for 29 days (Fig. 4). In short-term experiment the number of spheres per individual varied from 0 to 1 in PE150–180Land from 0 to 4 in the treatment with highest concentration of MPs (PE_150–180H). No significant differences in proportion offish containing MPs were observed among the treatments (difference between two proportions test.p>0.05). MPs were not identified infish from control treatment (n= 10) and in younger larvae (40 dph,n= 10).

3.5. Chemical analysis of microplastics

The concentrations of identiﬁed PAH congeners are presented in Table 4. None of the PBDE congeners nor PCBs were detected (<LOD) in

analyzed microplastics. Analyses of PBDEs in PE matrix did not succeed due to the unidentiﬁed residues on the sides of insert. After repetition of the clean-up stage the samples were eliminated from theﬁnal results be- cause of no improvement. Most likely small-sized particles (<300μm) begun to disintegrate during the extraction under the ultrasonic bath and released substances which are not well soluble in n-nonan were released.

4. Discussion

The hatching success of rainbow trout was high in all treatments and seemed to be unaffected by exposure to any polymer regardless MPs size.

Also the hatching rate was similar in all MPs treatments. The lack of observed effects of exposure to various MPs on various developmental parameters measured directly in the embryos is not entirely surprising since embryos are considered well protected from surrounding environment by the chorion (Van Leeuwen et al., 1985), and the migration rate of xenobi- otics through the chorion of embryo is lower compared to the transport across the gill epithelium of larvae (Petersen and Kristensen, 1998). It is un- likely that microplastics used in the present study penetrated the chorion since 150–180μm was the smallest MPs size fraction applied, while the Fig. 1.Changes in wet weight (A), standard length (B) and yolk-sac volume (C) of

larval rainbow trout during 69-day exposure to different microplastics. Open circles represent non-outlier data for all individuals, closed diamonds - means (n= 76–96). Asterisks indicate signiﬁcant difference compared to relevant controls.

Fig. 2.Changes in wet weight (A), standard length (B) and yolk-sac volume (C) of larval rainbow trout during 29-day exposure to different concentrations of PE microspheres. Open circles represent non-outlier data for all individuals, closed diamonds - means (n= 80–93).

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chorionic pores of salmonid embryos are 0.5–0.8μm in diameter (Groot and Alderdice, 1985). Small MPs may also accumulate on or adhere to the eggs and clog the chorionic pores, what was previously observed in ﬁsh embryos and was connected with decreased hatching success or rate (Li et al., 2020;Malafaia et al., 2020). In this study this mechanism was, however, hardly possible, especially in case of large PS and PET particles.

Similarly to ourfindings, the hatching success and hatching rate were unre- lated to MPs presence in otherfish: in another salmonid species sea trout S. truttaafter exposure to various pre-production pellets (Jakubowska et al., 2020) or in zebrafishDanio rerio(LeMoine et al., 2018;Pitt et al., 2018;Qiang and Cheng, 2019) and marine medakaOryzias melastigma (Beiras et al., 2018) exposed to nanoplastics and small MPs of various polymer types.

Also survival of yolk-sac larvae, which are considered the most sensitive fish life stage, was high and unaffected by any MP treatment what is similar to earlier results of the experiments with sea trout (Jakubowska et al., 2020) or zebrafish exposed to PS nano- and microplastics (Chen et al., 2017;Pitt et al., 2018). By contrast, increased mortality offish larvae, juveniles and/or adults was observed in variousfish species in response to MPs but only after exposure to high MPs (10 mg L⁻¹) concentrations (Lei et al., 2018) or after direct dietary exposure (Mazurais et al., 2015;Naidoo and Glassom, 2019;Pannetier et al., 2020).

In our study, large MPs had limited effect on rainbow trout growth expressed by both weight and length gain, and the yolk sac absorption rate, although the latter one was initially slower in PET-exposed larvae.

No changes in growth and absorption of yolk sacs were also observed in our previous study on larval sea trout exposed to PE, PS and PET pre- production pellets (Jakubowska et al., 2020) or in larval zebraﬁsh exposed to various MPs present in the water (Karami et al., 2017;LeMoine et al.,

2018;Qiang and Cheng, 2019) and in some adultﬁsh (European sea bass Dicentrarchus labraxand juvenile gilthead seabreamSparus aurata) fed with MPs (Mazurais et al., 2015;Jovanovićet al., 2018). Interestingly, Table 3

Mean (± SD) and median concentrations of whole-body corticosteroids in larval rainbow trout after long- (69 days) and short-term (29 days) exposure to different microplastics. Asterisk indicates signiﬁcant difference compared to relevant controls.

Concentration of corticosteroid Treatment Mean ± SD Median n Long-term experiment

Dehydrocorticosterone Control3000 3.66 ± 0.66 2.76 3

PS3000 2.13 ± 0.89 2.22 5

PET3000 4.26 ± 3.57 2.60 10

Control_150–180 4.20 ± 2.05 3.43 7

PE150–180 2.18 ± 0.72 2.36 5

Corticosterone Control3000 10.15 ± 3.67 9.46 3

PS3000 9.31 ± 2.05 9.21 5

PET3000 8.1 ± 2.93 7.21 10

Control_150–180 9.61 ± 2.96 10.74 7

PE_150–180 17.63 ± 4.47* 16.31 5

Cortisone Control3000 1.38 ± 1.09 1.22 3

PS3000 1.42 ± 1.11 1.08 5

PET3000 0.79 ± 0.99 0.41 10

Control150–180 1.44 ± 1.23 0.96 7

PE_150–180 0.92 ± 0.90 0.67 5

Cortisol Control3000 0.00 ± 0.00 0.00 3

PS3000 0.75 ± 0.68 1.22 5

PET3000 0.51 ± 1.37 0.00 10

Control150–180 1.76 ± 2.61 0.00 7

PE150–180 0.36 ± 0.71 0.00 5

Short-term experiment

Dehydrocorticosterone Control_150–180 2.97 ± 1.57 2.83 11

PE150–180L 3.22 ± 2.41 2.67 11

PE150–180H 3.11 ± 1.62 2.84 11

Corticosterone Control_150–180 5.14 ± 2.54 5.02 11

PE_150–180L 7.99 ± 3.87 8.40 11

PE_150–180H 10.98 ± 8.35 5.81 11

Cortisone Control_150–180 1.10 ± 0.73 1.33 11

PE_150–180L 2.33 ± 2.43 0.57 11

PE150–180H 3.05 ± 1.82 3.50 11

Cortisol Control150–180 0.08 ± 0.21 0.00 11

PE_150–180L 2.80 ± 3.86 0.18 11

PE_150–180H 2.18 ± 2.99 0.00 11

Fig. 3.Frequencies of genotoxicity endpoints (micronuclei (MN), nuclear buds (NB)) (A) and total genotoxicity (ΣGentox) levels induced in erythrocytes (B) of rainbow trout after 69-day exposure to microplastics of different polymer types.

Asterisks denote signiﬁcant differences from the control groups, mean ± SD (n= 15).

Fig. 4. (A) Mean + SD (n= 21) number of polyethylene microspheres found in rainbow trout larvae and (B) percentage of larvae containing the microspheres, after 69- and 29-day exposure.

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rainbow trout larvae exposed here to PE microspheres hatched smaller, but theirﬁnal length and yolk sac volume did not differ from the control group indicating some kind of growth compensation. Growth alterations observed earlier in variousﬁsh larvae resulted directly from the consumption of MPs (Naidoo and Glassom, 2019;Xia et al., 2020). Notwithstanding, in the present study, larvae were not able to intake MPs during most of the experimental period, while the number of PE particles potentially ingested by larvae at the end of exposure was generally very low.

Interestingly, we have observed higher frequency of a single measured developmental abnormality at the presence of all MPs types. Edema indicating the accumulation offluid in yolk-sac is considered an indicator of toxicity and a sublethal effect. It is usually connected with decreased bloodflow, increased energy expenditure, reducedfinfold surface area and spinal cur- vature which may impair the swimming performance, orientation and, in the further perspective, pray capture (Carls et al., 1999). Elevated frequency of yolk-sac edema has been previously observed after exposure of zebrafish to MPs (Malafaia et al., 2020) and nanoparticles (Duan et al., 2013), various chemical compounds including PAHs (Carls et al., 1999;Li et al., 2020). Edema can be observed during normalfish development but its frequency is usually very low (below 5%) (Carls et al., 1999;Li et al., 2020). Higher frequency of this malformation in our study (up to 9–10%

in some replicates) may, therefore, indicate some alterations in the osmotic processes leading to excessive water uptake at the presence of all MPs types.

In the present study the mean concentrations of cortisol–the most common bioindicator of stress in teleostﬁsh (Wendelaar Bonga, 1997), were close to zero and this hormone was detected only in some part of individuals in each treatment. High inter-individual variability of cortisol levels and of all other measured corticosteroids has been earlier recorded in larval sea trout exposed to similar set of microplastics (Jakubowska et al., 2020).

Interestingly, in the present study corticosterone concentrations were elevated in all PE treatments compared to the relevant controls being significant after 69 days what may be connected with the longer exposure or its beginning at embryo stage. Corticosterone is one of the intermediates in the steroid biosynthesis, and similarly to cortisol shows affinity to glucocor- ticoid receptors (Takahashi and Sakamoto, 2013), but its endocrine func- tion infish remains unclear (Sandor, 1979;Schreck and Tort, 2016). Its elevated plasma concentrations have been previously detected in Atlantic salmon (Salmo salar) during certain seasons and at the beginning of up- stream spawning migration (Idler and Truscott, 1972;Sangalang and Uthe, 1994). It is also considered primary stress hormone in many terres- trial animals (Wendelaar Bonga, 1997). Elevated corticosterone levels in PE treatment might be attributed to the microspheres ingestion. It was proposed that some well-known molecules and/or their receptors involved in stress response in larval stages are biologically active mainly in the gut (Pederzoli and Mola, 2016). It is, however not known if MPs may exert higher actions on larvae's endocrine axis by the presence in gastrointestinal tract, compering to the external MP exposure. It should be underlined

however, that due to limited number of analyzed samples from the long- term experiment, especially in the control treatment, these results should be interpreted with caution. In the context of extremely scarce literature concerning endocrine responses of larvalfish to MPs and the potential role of corticosteroids in their development, it is difficult to drawfirm conclusions. Further research concerning potential mechanisms involved in fish endocrine responses to MPs seem necessary to confirm if the changes in levels of whole-body corticosteroids are indicator of the effect of MP exposure in larvae.

Analyzed cytogenetic biomarkers indicate both clastogenicity and aneugenicity and allow evaluating the impact of genotoxic and cytotoxic substances even at low exposure concentrations as well as dose-response relationships of DNA reactive and non-DNA reactive genotoxins. The exposure to MPs resulted in slightly increased frequencies of all analyzed genotoxicity endpoints. A signiﬁcantly higher NB frequencies and∑Gentox level was observed only in the PS group compared to the control treatment.

Genotoxic, mutagenic, cytotoxic potential of nano-sized PS and impaired circulating erythrocytes morphometry in freshwaterfish–grass carp (Ctenopharyngodon idella) at early developmental stage was reported by Guimarães et al. (2021). The nano-sized PS was able to attack erythrocyte DNA, induce single- and double-strand DNA breaks, form DNA adducts and DNA-DNA and DNA-proteins cross-link resulting from interactions between contaminants or their metabolites and DNA. In vitro genotoxic effects of nano-sized PS were reported byPoma et al. (2019)andGiorgetti et al. (2020). Obtained results are similar to our previous study on early life stages of another salmonid - sea trout (Salmo trutta) (Jakubowska et al., 2020). Frequencies of DNA damage indicators levels have been shown to be significantly elevated after chronic (4 months) exposure of larvae to the same polymers as in present study, following the sequence PS>PET>PE. Significant genotoxic effect observed after much shorter exposure of rainbow trout in here to PS. Despite the similarities between these two experiments, which both were performed on salmonid species using similar plastic pellets, the experimental designs differed considerably (different water volumes/MPs concentrations;flow through system with large amount of water (250 L) vs. 1.5 L containers, lack of the water exchange vs. water partly exchanged once a week, and exposure from fertilized eggs vs. from eyed egg stage in the former and present study, respectively).

Genotoxicity in a non-dietary exposure can be related to the biological activity of various MPs co-contaminants (Jakubowska et al., 2020). The concentrations of selected parent-PAHs (PPAHs) measured in microplastics used in our experiment are in the range of their typical values recorded in blank pre-production plastic pellets (Rochman et al., 2013b;Rochman et al., 2013c;Nguyen and Scales, 2019). PAHs are associated with polystyrene via manufacture process as both benzene and styrene, which are PS components, are precursors of PAH formation (Rochman et al., 2013c). It was shown that PS used in the present study was characterized by the highest concentration of naphthalene (10 ng g⁻¹), phenanthrene and acenaphthene (4–5 ng g⁻¹), while PE microspheres contained considerably high levels of phenanthrene (36 ng g⁻¹), followed by fluoranthene (8 ng g⁻¹), naphthalene, anthracene, pyrene (~4 ng g⁻¹). Many PAHs and their metabolites are of particular concern and may possess genotoxic risk due to their toxic, mutagenic and carcinogenic properties (Baršienė et al., 2012,White, 2002). Inhibitory, synergistic or additive effects on genotoxicity responses, mutagenicity, carcinogenicity, and DNA binding activity of PAHs mixtures have been reported in aquatic biota (Meier et al., 2020;White, 2002).Rendell-Bhatti et al. (2021)demonstrated the exis- tence of PAHs and PCBs in leachates from plastic pellets after 24 and 72 h of exposure. Nonetheless, as in the present study, these compounds were only measured in MPs themselves, based on the available data concerning the leaching (Teuten et al., 2009) we may only speculate that they were present in experimental water in concentrations sufficient to induce observed changes in larvae. Since their toxicity is directly dependent on the duration of leaching, we can assess the risk of the toxic leachates as high in our study. The lack of genotoxic effect of PE in the present study might be connected with the fact that the distance betweenfish and MP particle Table 4

Levels (ng g⁻¹) of analyzed PAHs in microplastics used in the experiment.

PAH PE PET PS

Naphthalene 4.68 1.81 11.96

Acenaphthene 1.93 0.51 1.97

Acenaphthylene 1.29 0.53 4.25

Fluorene 2.73 0.45 2.36

Phenanthrene 36.28 2.15 5.59

Anthracene 3.95 0.44 1.21

Fluoranthene 7.57 <LOD <LOD

Pyrene 3.92 <LOD <LOD

Benz[a]anthracene <LOD <LOD <LOD

Chrysene <LOD <LOD <LOD

Benzo[b]ﬂuoranthene 0.19 <LOD <LOD

Benzo[k]ﬂuoranthene <LOD <LOD <LOD

Benzo[e]pyrene 0.43 0.12 0.53

Dibenzo[ah]anthracene <LOD <LOD 0.76

Benzo[ghi]perylene <LOD <LOD 0.68

Indeno[1,2,3]pyrene 0.19 0.19 2.74

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may modulate the impact of lipophilic contaminants. PE spheres, contrary to PS and PET pellets were not in the close proximity to embryos and larvae laying on the bottom. On the other hand, other effects of PE-exposure were observed in present study. Smaller plastic particles are characterized by higher capacity to release or bind compounds compared to larger particles (Teuten et al., 2009). Also, their different shape compared to the pellets might slightly modulate the surface/volume ratio and thus the rate of leaching of additives. Moreover, PE despite being considered as one of least hazardous polymers (Lithner et al., 2011), is characterized by low crystallinity, thus by higher desorption rates than, for example PET or PS (Teuten et al., 2009). It should be also emphasized that our analyses were limited to the congeners of PBDEs, PCBs and PAHs, whereas several thou- sand different additives exists for plastic polymers and that potential leachates from the pellets may also contain speciﬁc unpolymerized residual monomers or polymerization impurities (Lithner et al., 2011).

We have shown that PE microspheres were captured by rainbow trout larvae before the onset of exogenous feeding. Recent data suggest that fish larvae are not able to adaptively avoid MP ingestion and thus adapt to their presence in the environment (Huuskonen et al., 2020). The harmfulness of MPs intake or ingestion by aquatic organisms is debatable. The consumption of MPs may lead to gastrointestinal blockage, reduced feeding rates and energy intake. However, similar effects may be also caused by ingestion of other non-food particles (Ogonowski et al., 2018). Moreover, juvenile and adultfish as well as exogenously feeding larvae are able to easily egest ingested microplastic particles (Mazurais et al., 2015;Naidoo and Glassom, 2019). Recentfindings suggest that juvenilefish usually capture MPs visually similar to the food pellets, whereas other MPs (different colors) can be captured occasionally and often spit out (Ory et al., 2018).

It is however not known if similar mechanisms exist in larvae before the initiation of feeding. Yolk-sac larvae are able for ingestion of exogenous particles as soon as the mouth opens (Gerking, 1994). Moreover, rainbow trout atfirst feeding have mature digestive systems and they are capable of ingesting feed particles ~0.5 mm (FAO, 2021). Therefore, it is not surprising that studied larvae were capable for the intake of particles that were 150–180μm in diameter. However, as we identified whole-body MPs content, not the content in the particular elements of the digestive tracts, we have to be aware that the identified MPs might have originated both from the intestines and from the mouth. Nevertheless, the potential consumption of MPs byfish larvae during the transition from endogenous nutrition to exogenous feeding might pose a serious threat - even short period of food dep- rivation after yolk sac exhaustion can cause morphological and histological deformations, abnormal feeding behaviour and consequently reduce growth and survival (Yúfera et al., 1993). Also, the inadequate composition and quality of food may negatively affect the differentiation of the digestive tract (Yúfera et al., 1993). In addition, MPs-associated contaminants may desorb from microplastics, faster in the presence of gut surfactants becom- ing more bioavailable upon ingestion (Bakir et al., 2014).Karami et al.

(2016)concluded that bacteria able to biodegrade PE polymers to ethylene monomers (which can form toxic ethylene oxide and ethylene glycol) may be present in the digestive tracts of freshwaterfish and therefore might be responsible for observed toxicity of this polymer upon ingestion. Since our experiment was terminated upon 52–54 dph we were unable to investigate the potential consequences (fithess of juveniles) of occasional PE microspheres ingestion by larvae before the onset of exogenous feeding. This issue definitely deserves more attention in the future research.

5. Conclusions

Our results indicated that virgin pre-production pellets have limited impact on basic processes, such as survival, development and growth of rainbow trout early life stages. However, the signiﬁcant genototoxic effect expressed by elevated frequencies of genotoxicity endpoints (micronuclei and nuclear buds) detected at the end of exposure to PS pellets was observed. Similar response was earlier observed in the sea trout larvae after much longer exposure period. Interestingly, long-term (from the eyed eggs) exposure to PE microspheres resulted in slight but signiﬁcant changes

in growth, mainly in smaller size of hatched larvae, and in elevated whole- body corticosterone concentration. Shorter exposure (from larvae

~3 weeks after hatching) to the same or even higher concentrations of PE microspheres did not affect larvae growth (or affected in lesser extent) and steroid hormones concentrations. That may indicate time- or development stage-dependent rather than concentration-dependent effect of PE microspheres. Present study indicated that even low concentrations of both large pre-production pellets and small MPs may induce a negative response inﬁsh early life stages, therefore more research on the environmentally relevant concentrations is needed. It should be kept in mind, however, that the toxicity of MPs might be related to their basic composition or additives, also to compounds which were not investigated in the present study. Hence, studies on various polymers of different origin (i.e., pre-production pellets, pure polymers, MPs collected from the environment) connected with analyses of various chemical compounds which may be associated with MPs would be very useful in the assessment of the effects of microplastic pollution on biota. Moreover, during the last days of exposure larvae were able to ingest MPs with unknown consequences for further development. There- fore, more research on the effect of various microplastics on older, exogenously feeding larval stages of salmonids would be interesting. It would be also reasonable to investigate long-term growth and toxicity parameters in larvae which were exposed to various microplastics from fertilized or eyed embryos.

CRediT authorship contribution statement

Magdalena Jakubowska:Conceptualization, Methodology, Investiga- tion, Data curation, Visualization, Writing - original draft, Writing - Review

&Editing.

Marcin Białowąs:Conceptualization, Methodology, Investigation, Writing - Review&Editing .

Milda Stankevičiūtė:Investigation, Data curation, Visualization, Writ- ing - original draft.

Agnieszka Chomiczewska:Investigation, Validation.

Karolina Jonko-Sobuś:Methodology, Investigation, Data curation, Validation, Writing - original draft.

Janina Pažusienė:Investigation, Data curation.

AgnėBučaitė:Investigation, Data curation.

Anna Hallmann:Investigation, Writing - original draft.

Barbara Urban-Malinga:Conceptualization, Methodology, Investiga- tion, Writing - original draft, Writing - Review&Editing, Funding acquisi- tion, Project administration.

Declaration of competing interest

The authors declare that they have no known competingﬁnancial inter- ests or personal relationships that could have appeared to inﬂuence the work reported in this paper.

Acknowledgements

This research was funded by the BONUS MICROPOLL project supported by the joint Baltic Sea research and development programme BONUS (Art 185) funded jointly by the European Union and the National Centre for Re- search and Development. Poland (NCBR) (BONUS-BB/MICROPOLL/06/

2017). Determination of tissue steroid compounds concentration was performed under Medical University of Gdańsk grant number ST40. Genotox- icity and cytotoxicity studies were funded by the Research Council of Lithuania through the project MULTIS (S-MIP-21-10).

Mr. Thibault Pasquier from Dąbieﬁsh hatchery is gratefully acknowl- edged for providing us the embryos ofOncorhynchus mykiss. Ms. Agata Nowak from the National Marine Fisheries Research Institute and Ms.

MichalinaŚcibik from the University of Gdańsk are gratefully acknowl- edged for their assistance in the morphometric analyses of larvae. We wish to thank colleagues from the Leibniz Institute for Baltic Sea Research for providing us polystyrene pellets.