Do polystyrene microplastics affect juvenile brown trout (Salmo trutta f. fario) and modulate effects of the pesticide methiocarb?


 Background During the last decade, there has been rising interest of the scientific community and the public in the environmental risk related to the abundance of microplastics in aquatic environments. Besides potential effects of the particles themselves, also their interaction with organic micropollutants is of particular concern. Up to now, however, scientific knowledge in this context is scarce and insufficient for a reliable risk assessment. This is especially true for data on microplastics in freshwater ecosystems.Results Against the background of this shortage, we investigated possible adverse effects of polystyrene particles (10 4 particles/L) and the pesticide methiocarb (1 mg/L) both alone as well as in combination in juvenile brown trout ( Salmo trutta f. fario ) after a 96 h laboratory exposure. PS beads (density 1.05 g/mL) were cryogenically milled and fractionated resulting in irregular shaped particles (<50 µm). Besides body weight of the animals, biomarkers for proteotoxicity (stress protein family Hsp70), oxidative stress (superoxide dismutase, lipid peroxidation) and neurotoxicity (acetylcholinesterase, carboxylesterases) were analysed. As an indicator of overall health histopathological effects were studied in liver and gills of exposed fish. Polystyrene particles alone did not influence any of the investigated biomarkers. In contrast, the exposure to methiocarb led to a significant reduction of the activity of acetylcholinesterase and the two carboxylesterases. Moreover, the tissue integrity of liver and gills was impaired by the pesticide. Body weight, the oxidative stress and the stress protein levels were not influenced by methiocarb. Effects caused by the mixture of polystyrene microplastics and methiocarb were the same as those caused by methiocarb alone.Conclusions Overall, methiocarb led to strong effects in juvenile brown trout. In contrast, polystyrene microplastics in the tested concentration did not negatively affect the health of juvenile brown trout and did not modulate the toxicity of methiocarb in this fish species.


Abstract
Background During the last decade, there has been rising interest of the scientific community and the public in the environmental risk related to the abundance of microplastics in aquatic environments. Besides potential effects of the particles themselves, also their interaction with organic micropollutants is of particular concern. Up to now, however, scientific knowledge in this context is scarce and insufficient for a reliable risk assessment. This is especially true for data on microplastics in freshwater ecosystems.

Results
Against the background of this shortage, we investigated possible adverse effects of polystyrene particles (10 4 particles/L) and the pesticide methiocarb (1 mg/L) both alone as well as in combination in juvenile brown trout ( Salmo trutta f. fario ) after a 96 h laboratory exposure. PS beads (density 1.05 g/mL) were cryogenically milled and fractionated resulting in irregular shaped particles (<50 µm). Besides body weight of the animals, biomarkers for proteotoxicity (stress protein family Hsp70), oxidative stress (superoxide dismutase, lipid peroxidation) and neurotoxicity (acetylcholinesterase, carboxylesterases) were analysed. As an indicator of overall health histopathological effects were studied in liver and gills of exposed fish.
Polystyrene particles alone did not influence any of the investigated biomarkers. In contrast, the exposure to methiocarb led to a significant reduction of the activity of acetylcholinesterase and the two carboxylesterases. Moreover, the tissue integrity of liver and gills was impaired by the pesticide. Body weight, the oxidative stress and the stress protein levels were not influenced by methiocarb. Effects caused by the mixture of polystyrene microplastics and methiocarb were the same as those caused by methiocarb alone.

Conclusions
Overall, methiocarb led to strong effects in juvenile brown trout. In contrast, polystyrene microplastics in the tested concentration did not negatively affect the health of juvenile brown trout and did not modulate the toxicity of methiocarb in this fish species. Despite most research focusses on the abundance and possible effects of MP in marine environments, the number of studies in freshwater ecosystems is also rapidly increasing (Horton et al. 2017). Nevertheless, many gaps in knowledge about abundance, toxicity and hazard of MP in freshwater systems still exist.
For an environmental risk assessment of MP, measured concentrations in the environment must be related to effect concentrations in exposed organisms.  Ding et al. 2018). In addition to this physical damage MP can also potentially affect organisms due to the leakage of hazardous substances like residual monomers, polymerization solvents or additives (Lithner et al. 2009, Schiavo et al. 2018. A third process that should be considered in this context is that MP might ad/absorb hydrophobic organic pollutants like PAHs, PCBs or pesticides (reviewed by Wang et al. 2018) and transport them into organisms. Sorption of hydrophobic organic pollutants to MP is especially important for freshwater, since the concentrations of these pollutants are generally higher than in marine ecosystems (Dris et al. 2015). The sorption of organic pollutants to MP may alter the bioavailability of the pollutants. The bioavailability (and thereby effects) of pollutants can be decreased due to sorption to the polymer ( Beckingham and Ghosh 2017). To assess the risk particle filter, activated charcoal filter) for almost three months. Trout originated from a commercial fish breeder (Forellenzucht Lohmühle, D-72275 Alpirsbach-Ehlenbogen, Germany). In regular controls the breeding establishment is categorized as category I, disease free (EU 2006). The fish can be considered as close to feral forms and robust since they are also used for fishery restocking campaigns in German streams. Subsequently, the particles were fractionated to a size < 50 µm. The number of PS particles in the pure suspension was determined with a particle counter (SVSS, PAMAS, Germany) by light extinction in a laser-diode sensor (type HCB-LD-50/50).

Test substances
The particle size distribution is provided in the supplement ( Figure S1). Methiocarb ( Fig. 1) was purchased from Sigma-Aldrich (product line: PESTANAL®; CAS number: 2032-65-7; purity 99.8%; molecular formula: C 11 H 15 NO 2 S). The melting point of methiocarb is 119 °C, the vapour pressure 0.015 mPa (20 °C) and the octanol/water partition coefficient as log Pow 3.08. According to literature the solubility in water (20 °C) is 27 mg/L (Worthing et al. 1991, ILO 2012. Nevertheless, in our experiment it was not possible to solve methiocarb without a solvent. Therefore, dimethyl sulfoxide (DMSO) was used to solve methiocarb in water.
2.3. Exposure of juvenile brown trout Fish were exposed in a static three-block design for 96 h (18.11. − 23.11.2017).
Each of the three blocks consisted of five tanks with the different treatment groups: a control group, a solvent control (0.01% DMSO), PS-MP (10 4 particles/L), methiocarb (1 mg/L) and a mixture (10 4 particles/L and 1 mg/L methiocarb). The test concentration of 1 mg/L methiocarb was selected due to published LC 50 values for rainbow trout (Onchorynchus mykiss): The reported LC 50 (96 h)  Exposure took place in a thermo-constant chamber set to 7 °C with a light/dark cycle of 10 h/14 h (tanks were shaded from direct light). Aeration of the tanks was ensured by glass pipettes, which were connected to compressed air via silicone tubes.

Chemical analyses
At the beginning and the end of the experiment, in both control groups, the methiocarb concentrations were below the detection limit (LOD) of 0.25 µg/L (Table 1). In the PS-MP exposure, no methiocarb could be detected (< LOD). In both exposure groups with methiocarb (methiocarb and mixture), the measured concentrations at the beginning of the experiment were 50% of the nominal concentrations. After 96 h, in the treatment group with solely methiocarb, the concentration was further reduced by approximately 50%, in the mixture treatment by 34%.

Mortality and weight
After 48 h, three fish exposed to methiocarb and one fish exposed to the mixture had to be euthanized due to their poor health conditions. Apart from that, no mortality occurred during the experiment. Weight (overall mean: 2.96 ± 1.01 g) did not differ between the treatment groups (

Oxidative Stress
To assess the oxidative stress level SOD activity and the degree of LPO were investigated.
No difference between SOD activity in the different treatment groups was found (

Neurotoxicity
No difference in the activity of AChE occurred between the solvent control and the treatment group with PS-MP (p = 0.8192). However, methiocarb and the mixture of methiocarb and PS-MP led to a significantly reduced activity of AChE in comparison to the solvent control and PS-MP alone (Fig. 1, DMSO-exposure groups: Nested-ANOVA: d.f. =3/101, F = 77.77, p < 0.0001). The activity of AChE in the methiocarb-exposed fish was 59% reduced compared to the solvent control (DMSO-methiocarb and PS-MP-methiocarb: p < 0.001). In fish exposed to the mixture of PS-MP and methiocarb the activity of AChE was reduced by 58% compared to the solvent control (DMSO-mixture and PS-MP-mixture: p < 0.001; Table 2). Between fish exposed to methiocarb alone and methiocarb plus PS-MP no differences were found (p = 0.9951).  4A and 4B). In fish exposed to methiocarb alone or the mixture treatment, a reduction of the glycogen stores became obvious (Fig. 4D) resulting in atrophic cells and enlarged intercellular spaces. Furthermore, in both groups containing methiocarb vacuolization and inflammations occurred cumulatively, and in some cases, even small zones with karyopycnosis and focal necrosis were found (Fig. 4C). Semi-quantitative analyses showed a very similar condition of fish from the control, solvent control and exposure with PS-MP, whereas livers of fish exposed to methiocarb or the mixture treatment showed significantly stronger reactions (Fig. 5

Gills
Observed pathological alterations in gills of brown trout included hyperplasia and hypertrophy of chloride and pillar cells resulting, in some cases, in lamellar fusion ( Figure   6). Furthermore, an increase of mucus secreting cells, lifting of epithelia, oedema and even necrosis in small areas occurred. Most of these symptoms occurred in the methiocarb and the mixture treatment groups. Semi-quantitative analysis revealed a significantly worse condition of the two treatment groups containing methiocarb compared to the solvent control and fish exposed solely to PS-MP (Figure 7; DMSO-exposure groups: No difference was found between the control groups and the group containing solely PS-MP.

Discussion
In the present study, the effects of PS-MP and the pesticide methiocarb either applied alone or as their binary mixture were investigated in juvenile brown trout. To the best of our knowledge, the present study is the first that investigated potential proteotoxic effects of MP. In the tested concentration, no effect of PS-MP on the stress protein level (Hsp70) was found in brown trout after 96 h exposure.
Also, the activity of AChE and two investigated CbE were not altered after exposure to PS- In the present study, also no alterations in the histopathological status of the liver and gills were found in fish exposed to PS-MP alone when compared to the solvent control Subsequently exposed clams were fed to white sturgeon (Aciper transmontanus). In clams, the only histological reactions were mild or moderate tubular dilation in digestive glands, while no effect of MP were found in liver and gastrointestinal tract of white sturgeon. In

Does methiocarb affect juvenile brown trout?
Three fish exposed to methiocarb as sole pollutant showed strong behavioral reactions after 24 h and had to be euthanized. After 96 h, also all other fish exposed to methiocarb exhibit behavioral abnormalities like slower swimming and reduced escape behavior. To the best of our knowledge, this is the first study that investigated the effects of methiocarb on brown trout. In the present study weight of methiocarb-exposed fish were comparable to the control group. However, 96 h is a relative short time to observe changes regarding this endpoint in brown trout.
The observed reactions of fish can be seen as a consequence of the AChE inhibition by methiocarb. Acetylcholine (ACh) accumulates in the synaptic cleft leading to a cholinergic crisis (Rosman et al. 2009). In the present study, methiocarb led to a reduction of the AChE activity by 59% in the tested juvenile brown trout. The mode of action of carbamate pesticides is based on carbamylation of AChE and, thereby, inhibition of its ability to hydrolyze acetylcholine (Fukuto 1990). Therefore, it could have been expected that methiocarb reduces the activity of AChE also in brown trout. To our knowledge no one has analyzed the effect of methiocarb on stress proteins so far, and rarely proteotoxic effects of carbamate pesticides were studied in general. Seleem   found no histopathological alterations in liver, kidney, brain and spleen of rainbow trout after exposure to 2.5 or 3.75 mg/L methiocarb for 21 days. However, in gills of rainbow trout lamellar lifting occurred when fish were exposed to 3.75 mg/L methiocarb for 21 days (Altinok and Capkin 2007). Brown trout seem to be more sensitive to methiocarb than rainbow trout as indicated by the more severe effects in the present study. This was also shown in the past for other environmental stressors When considering all investigated endpoints, fish exposed to the mixture of methiocarb and PS-MP showed the same reactions as fish exposed to methiocarb solely. Weight as well as the stress protein level and the level of oxidative stress remained unchanged.
However, the activity of AChE and CbE were significantly reduced in the mixture to almost the same extent as caused by methiocarb alone. Furthermore, the observed histopathological alterations in liver and gills in fish of the mixture treatment were comparable to those found in fish exposed solely to the pesticide. Thus, the toxicity of  to sorption to the plastic particles. In the present study, chemical analyses revealed that the concentration of methiocarb was higher in the mixture treatment than in the groups exposed to methiocarb alone after 97 h. Thus, it is probable, that methiocarb did not sorb in considerable amounts to the plastic microparticles. In contrast to reports on a reduction Compared to the uptake pathway via the water, PS-MP seemed to have a negligible effect on the uptake of methiocarb in juvenile brown trout.

Conclusion
Based on the results of our study, we conclude that methiocarb heavily impairs the health of brown trout whereas the studied PS-MP in a concentration of 10 4 The experiment was approved by the animal welfare committee of the regional council of Tübingen, Germany (authorisation number ZO 2/16).
The datasets used and analysed during the current study are available from the corresponding author on request.

Competing interests
The authors declare that they have no competing interests.

Funding
The

Chemical analyses
At the beginning and the end of the experiment, in both control groups, the methiocarb concentrations were below the detection limit (LOD) of 0.25 µg/L (Table 1). In the PS-MP exposure, no methiocarb could be detected (< LOD). In both exposure groups with methiocarb (methiocarb and mixture), the measured concentrations at the beginning of the experiment were 50% of the nominal concentrations. After 96 h, in the treatment group with solely methiocarb, the concentration was further reduced by approximately 50%, in the mixture treatment by 34%. Table 1 Nominal and measured concentrations of methiocarb in the different treatment groups. Displayed are the arithmetic means ± standard deviation of the three aquaria. The limit of detection was 0.25 µg/L methiocarb.

Mortality and weight
After 48 h, three fish exposed to methiocarb and one fish exposed to the mixture had to be euthanized due to their poor health conditions. Apart from that, no mortality occurred during the experiment. Weight (overall mean: 2.96 ± 1.01 g) did not differ between the treatment groups ( Table 2; DMSO-exposure groups: Nested-ANOVA: d.f.=3/104, F = 1.14, p = 0.3384).

Oxidative Stress
To assess the oxidative stress level SOD activity and the degree of LPO were investigated. No difference between SOD activity in the different treatment groups was found (Table 2;

Proteotoxicity
The analysis of the Hsp70 level did not reveal any differences between the control, solvent control and the exposure groups (Table 2;

Neurotoxicity
No difference in the activity of AChE occurred between the solvent control and the treatment group with PS-MP (p = 0.8192). However, methiocarb and the mixture of methiocarb and PS-MP led to a significantly reduced activity of AChE in comparison to the solvent control and PS-MP alone (Fig. 1, DMSO-exposure groups: Nested-ANOVA: d.f. =3/101, F = 77.77, p < 0.0001). The activity of AChE in the methiocarb-exposed fish was 59% reduced compared to the solvent control (DMSO-methiocarb and PS-MP-methiocarb: p < 0.001). In fish exposed to the mixture of PS-MP and methiocarb the activity of AChE was reduced by 58% compared to the solvent control (DMSO-mixture and PS-MP-mixture: p < 0.001; Table 2). Between fish exposed to methiocarb alone and methiocarb plus PS-MP no differences were found (p = 0.9951). Methiocarb alone reduced the activity compared to the solvent control by 42%, the mixture by 44%.
Even more pronounced was the inhibition of CbE with the substrate pnpv: Compared to the solvent control, the activity was reduced by 83% both in methiocarb exposed fish and in fish in the mixture treatment ( 3.6. Histopathology

Liver
Healthy liver tissue consists of large bright cells containing high amounts of glycogen ( Fig. 4A and 4B). In fish exposed to methiocarb alone or the mixture treatment, a reduction of the glycogen stores became obvious (Fig. 4D) resulting in atrophic cells and enlarged intercellular spaces. Furthermore, in both groups containing methiocarb vacuolization and inflammations occurred cumulatively, and in some cases, even small zones with karyopycnosis and focal necrosis were found (Fig. 4C). Semi-quantitative analyses showed a very similar condition of fish from the control, solvent control and exposure with PS-MP, whereas livers of fish exposed to methiocarb or the mixture treatment showed significantly stronger reactions (Fig. 5  To the best of our knowledge, the present study is the first that investigated potential proteotoxic effects of MP. In the tested concentration, no effect of PS-MP on the stress protein level (Hsp70) was found in brown trout after 96 h exposure.
Also, the activity of AChE and two investigated CbE were not altered after exposure to PS-MP.
In In the present study, also no alterations in the histopathological status of the liver and gills were found in fish exposed to PS-MP alone when compared to the solvent control group. Also 4.3. Does methiocarb affect juvenile brown trout? Three fish exposed to methiocarb as sole pollutant showed strong behavioral reactions after 24 h and had to be euthanized. After 96 h, also all other fish exposed to methiocarb exhibit behavioral abnormalities like slower swimming and reduced escape behavior. To the best of our knowledge, this is the first study that investigated the effects of methiocarb on brown trout. In the present study weight of methiocarb-exposed fish were comparable to the control group. However, 96 h is a relative short time to observe changes regarding this endpoint in brown trout.
The observed reactions of fish can be seen as a consequence of the AChE inhibition by methiocarb. Acetylcholine (ACh) accumulates in the synaptic cleft leading to a cholinergic crisis (Rosman et al. 2009). In the present study, methiocarb led to a reduction of the AChE activity by 59% in the tested juvenile brown trout. The mode of action of carbamate pesticides is based on carbamylation of AChE and, thereby, inhibition of its ability to hydrolyze acetylcholine (Fukuto 1990  ionic imbalance due to inhibition of AChE activity. In a follow up study,  found no histopathological alterations in liver, kidney, brain and spleen of rainbow trout after exposure to 2.5 or 3.75 mg/L methiocarb for 21 days. However, in gills of rainbow trout lamellar lifting occurred when fish were exposed to 3.75 mg/L methiocarb for 21 days . Brown trout seem to be more sensitive to methiocarb than rainbow trout as indicated by the more severe effects in the present study. This was also shown in the When considering all investigated endpoints, fish exposed to the mixture of methiocarb and PS-MP showed the same reactions as fish exposed to methiocarb solely. Weight as well as the stress protein level and the level of oxidative stress remained unchanged. However, the activity of AChE and CbE were significantly reduced in the mixture to almost the same extent as caused by methiocarb alone. Furthermore, the observed histopathological alterations in liver and gills in fish of the mixture treatment were comparable to those found in fish exposed

Conclusion
Based on the results of our study, we conclude that methiocarb heavily impairs the health of brown trout whereas the studied PS-MP in a concentration of 10 4 particles/L do not. It can also be excluded that the studied PS-MP modulate methiocarb-induced effects in brown trout. In general, literature provides a diverse and inconsistent image with respect to the capacity of MP to modulate the toxicity of environmental chemicals. Although our study does not speak for an environmental risk related to the investigated polystyrene particles and their interaction with the pesticide methiocarb, this study provides only a very small piece of knowledge for a defined type and size class of plastics and a single pesticide, and emphasizes the need of further research in this field.

Competing interests
The authors declare that they have no competing interests.

Funding
The experiment was conducted within the joint project MiWa (Microplastics in the water cycle -sampling, sample preparation, analytics, occurrence, removal, and assessment)        Supplement.docx