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.
4.1. General considerations concerning the conducted experiment
The measured methiocarb concentrations corresponded to approximately half of the nominal concentration in the methiocarb and mixture groups at the beginning of the experiment and 24% (methiocarb group) and 31% (mixture group) at the end. Methiocarb has a comparable low persistence in laboratory aerobic soil-water systems (Arena et al. 2018). The degradation of methiocarb is pH dependent and higher under alkaline conditions. The main metabolites are methiocarb phenol and methiocarb sulfoxide phenol. The dissipation half-life (DT50) of methiocarb in water at pH 7 is 24 days and 0.21 days at pH 9 (EFSA 2006). Thus, a degradation of methiocarb in this experiment was likely, but occurred to a greater extend as it could have been assumed in an environment with a pH of 7.3. During the experiment, beside the abiotic degradation, a metabolization by the fish may have contributed to the decreased methiocarb concentration after 96 h. In humans, mainly cytochrome p450 and flavin-containing monooxygenases in liver and kidney contribute to the metabolism of methiocarb (Usmani et al. 2004, Furnes and Schlenk 2005). For longer exposure to methiocarb regular exchange of the water with freshly prepared methiocarb solutions is recommended.
4.2. Do PS-MP affect juvenile brown trout?
In juvenile brown trout none of the investigated parameters were affected by 104 polystyrene particles/L. In the past, MP were found to cause oxidative stress in different organisms like in monogonont rotifers (Jeong et al. 2016), nematodes (Lei et al. 2018), mussels (Avio et al. 2015, Magni et al. 2018) or fish (Lu et al. 2016, Paul-Pont et al. 2016, Qiao et al. 2019). In contrast to these results, other studies found either no effects (Oliveira et al. 2013, Luís et al. 2015, Fonte et al. 2016) or effects in only some of the investigated endpoints for oxidative stress (Avio et al. 2015, Ferreira et al. 2016, Ding et al. 2018, Magni et al. 2018). The oxidative defense system is complex and consists of enzymatic (e.g. SOD, catalase (CAT), glutathione reductase) and non-enzymatic compounds (Lushchak 2016). In addition, the types of the investigated polymers, their concentrations, their potential additives as well as the size of the used MP highly vary in the different studies. This complexity is reflected by the literature: as for example, the activity of CAT was found to be increased (Lu et al. 2016, Qiao et al. 2019) as well as decreased (Paul-Pont et al. 2016) or remained unchanged (Chen et al. 2017, Guilhermino et al. 2018) after exposure to MP. Jeong et al. (2016) analyzed the influence of the particle size on oxidative stress responses and showed that there is a clear connection to this parameter. In general, however, the database is still far too limited to decide on whether or not MP cause oxidative stress and, in case of any influence, which pathways are affected.
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 the past several studies reported significant reductions about 20% of AChE activity in common goby (Pomatoschistus microps) after exposure to 184 µg/L polyethylene (PE; 1–5 µm) for 96 h (Oliveira et al. 2013, Luís et al. 2015, Fonte et al. 2016). Under the same test conditions, Ferreira et al. (2016) only found a reduction of AChE activity by 13% in common goby. In red tilapia (Oreochromis niloticus) an even higher inhibition of AChE by 37.7% was found after exposure to PS nanoplastics (0.1 µm) in concentrations of circa 1.8 × 106, 1.8 × 107, 1.8 × 108 particles/L for 14 days (Ding et al. 2018). Chen et al. (2017) did not find any neurotoxic effect of PS-MP (45 µm, 20 particles/mL) in zebrafish (Danio rerio) but reported a significant reduction of AChE activity by 40% after exposure to PS nanoplastics (50 nm, 1.5 × 1010 particles/mL). Furthermore, a decrease of AChE activity in Amazonian discus fish Symphysodon aequifasciatus was found after exposure to 200 µg/L fluorescent PE (70–88 µm) for 30 days (Wen et al. 2018). Reduction of AChE was not only observed in fish: in the crab Eriocheir sinensis exposed to 40 µg/L PS microspheres (5 µm) for 21 days AChE activity was significantly reduced (Yu et al. 2018). An exposure of freshwater clam Corbicula fluminea to 0.2 mg/L caused an inhibition of 31% AChE activity, but a concentration of 0.7 mg/L only led to a non-significant reduction of 19% (Guilhermino et al. 2018). Avio et al. (2015) found no effect in the heamolymph of Mytilus galloprovincialis on AChE activity after exposure to PE and PS (< 100 µm 7 days 1.5 g/L) but an AChE activity decrease in gills. In peppery furrow shell Scrobicularia plana a reduction of the AChE activity in response to PS-MP exposure (20 µm, 1 mg/L) was not only found after 14 d of exposure but also still after a depuration period of 7 days (Ribeiro et al. 2017). In contrast, a study of Magni et al. (2018) on the neurotoxic effects of a mixture of PS microbeads (with 5 × 105 particles/L − 10 µm and 5 × 105 particles/L − 1 µm) on the zebra mussel (Dreissena polymorpha) showed no neurotoxic effect in 6 days despite of an increase in the dopamine amount. Other investigated parameters were serotonin, glutamate, AChE and monoamine oxidase. Magni et al. (2018) suggested that the increased dopamine level reduces the intake of MP or improves their elimination. In general, the mode of action how MP might cause neurotoxicity remains unclear. Besides the type of the polymer (including different additives) also the particle size seems to be of importance in this context, since neurotoxic effects were mainly found in studies using very small micro- or even nanoplastics. Furthermore, Ding et al. (2018) reported accumulation of nanoplastics in the brain of red tilapia. These findings are supported by a study of Mattsson et al. (2017) who demonstrated that polystyrene nanoplastics are capable to penetrate the blood-brain barrier of Crucian carp (Carassius carassius). Since the plastic particles we used in the present study were < 50 µm with an unknown number of very small particles, possibly the larger particles were dominant in our experiment which might explain the lack of influences in neurotoxicity.
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 Lei et al. (2018), did not find any effect of PS-MP (0.1, 1.0 and 5 µm up to 10 mg/L) on tissue integrity of intestine, gills, kidney and liver of zebrafish after 10 days of exposure. Furthermore, besides effects on the intestine Lei et al. (2018) did not find any alterations in gills, liver and kidney of zebrafish exposed to polyamide (PA), polypropylene (PP), polyvinyl chloride (PVC) and PE (each ~ 70 µm in a concentration up to 10 mg/L) for 10 days. Moreover, low-density PE (125–250 µm) caused no alterations in liver of zebrafish after 3 weeks of exposure (Rainieri et al. 2018). In silver barb fry (Barbodes gonionotus) no histopathological reactions were caused by PVC (0.2, 0.5 and 1.0 mg/L; 0.1–1000 µm) fragments besides a slight thickening of the intestinal mucosal epithelium (Romano et al. 2018). Rochman et al. (2017) exposed Asian clams (Corbicula fluminea) to polyethylene terephthalate (PET), PVC, PE and PS MP for 28 days. 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 contrast, Lu et al. (2016) observed early inflammation responses as well as lipid droplets in the liver of zebrafish after exposure to 5 µm polystyrene particles in a concentration of 2.9 × 104 particles/L for three weeks. In Japanese medaka (Oryzias latipes) severe glycogen depletion and fatty vacuolization in the liver occurred, but no alterations were found in gonads after exposure to PE for 2 months (Rochman et al. 2013, Rochman et al. 2014). Karami et al. (2016) exposed African catfish (Clarias gariepinus) to low-density PE (< 60 µm) for 96 h. They observed hyperplasia and sloughing and even necrosis in gills at a concentration of 50 µg/L and even more severe reactions like desquamation of cells at a concentration of 500 µg/L. Additionally, the degree of tissue damage in the liver of the fish was increased after exposure to low-density PE in a concentration of 500 µg/L (Karami et al. 2016). In general, there is no evidence for a uniform pattern under which conditions histopathological changes may occur after exposure to MP.
Reports of the environmental concentration of MP in surface waters of a size < 50 µm are rather scarce possible due to difficulties with the sampling and detection methodology (de Sá et al. 2018). Nevertheless, reported results of MP indicate higher concentrations of smaller MP compared to larger ones (Triebskorn et al. 2019). Karlsson et al. (2017) found a concentration of MP particles of 27 particles/L (> 30 µm) at the coast of the North Sea (Netherlands). In canals of Amsterdam even higher concentrations of MP (> 10 µm) between 48 and 187 particles are reported (Leslie et al. 2017). In the Chinese river Yangtze MP concentrations between 0.5 and 3.1 particles/L (> 20 µm) were found (Su et al. 2018). In the German river Elbe concentrations of MP particles as high as 1000 and 9000 particles/L were detected (Triebskorn et al. 2019). In the present study the concentration of PS particles was 104 and therefore higher than the environmental concentration. Our study does not indicate a risk for brown trout at environmental concentrations. Nevertheless, to exclude potential negative effects of MP on brown trout other experiments with longer exposure time and other polymer types are necessary.
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). Therefore, it could have been expected that methiocarb reduces the activity of AChE also in brown trout. Comparable effects were observed by Essawy et al. (2008) in the land snail Eobania vermiculata in which AChE activity was reduced up to 69.3% by methiocarb. Carboxylesterases play an important role in pesticide detoxification (Wheelock et al. 2005). Sanchez-Hernandez et al. (2009) suggested that CbE might act as biochemical barrier for organophosphate pesticides in Lumbricus terrestris. Maymó et al. (2006) found that esterase activity is higher in western flower thrips (Frankliniella occidentalis) with increased resistance against methiocarb. To our knowledge no studies about the effect of methiocarb on AChE and CbE activities in fish were performed up to date. However, in the present study, CbE as well as AChE was inhibited by methiocarb and no protective effect became obvious. This might possibly be related to the fact that the methiocarb concentration was rather high and all three enzymes were inhibited and the protective effect of CbE ceased.
Samples of brown trout showed neither an increase of SOD nor an altered amount of LPO exposure to methiocarb. An increase of LPO and an alteration of reduced glutathione level was found in male Wistar rats fed with 2, 10 and 25 mg/kg methiocarb (Ozden and Alpertunga 2010). Ozden and Alpertunga (2010) found the highest malondialdehyde level in the brain and explained their finding by the comparably large amount of fatty acids in this organ. In another study, Ozden et al. (2012) found also an increase of glutathione as well as of the activities of SOD, CAT and glutathione peroxidase in male Wistar rats after administration of 25 mg/kg methiocarb.
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 (2019) found an increase of Hsp70 in Arabian toad (Bufo arabicus) after exposure of tadpoles to the carbamate pesticide methomyl. Bierkens et al. (1998) investigated the effect of carbaryl on the Hsp70 level of the microalga Raphidocelis subcapitata. Hsp70 responded in a dose-dependent manner to carbaryl, with a lowest observed effect concentration of 10.3 µM. Moreover, the carbamate pesticides formetanate, methomyl and pirimicarb induced an overexpression of the chaperone GRP78 and reduced Hsp27, Hsp72/73 and Hsp90 levels in human cell cultures (Skandrani et al. 2006). However, in the present study, methiocarb did not cause any alteration of Hsp70 level of juvenile brown trout.
After exposure to methiocarb, prominent histopathological alterations became evident in livers and gills of the exposed fish. Altinok et al. (2006) observed similar effects in gills of rainbow trout exposed to 3.75 and 7.5 mg/L methiocarb. After 96 h, symptoms like lamellar edema, lifting of epithelia, telangiectasis, increased cytoplasmic granularity and lamellar fusion occurred. Effects were reversible in concentrations below 3.75 mg/L. In the fish livers Altinok et al. (2006) found necrosis. Altinok et al. (2006) assumed that the alterations are caused by ionic imbalance due to inhibition of AChE activity. In a follow up study, Altinok and Capkin (2007) 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 (Schmidt-Posthaus et al. 2001).
The tested methiocarb concentration (1 mg/L) was considerably higher than the average surface water concentration of 6–40 ng/L in an EU wide monitoring campaign (Loos et al. 2018). The study was designed to investigate acute effects of methiocarb and a potential modulation of these effects by PS-MP. Nevertheless, the strong acute effects of methiocarb in brown trout elucidate the need for experiments in which brown trout are exposed to methiocarb at environmentally relevant concentrations for a longer exposure time. Such experiments would allow to assess the current risk of methiocarb exposure for brown trout in the environment.
4.4. Do PS-MP modulate the effects of methiocarb?
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 methiocarb on brown trout was not modulated by PS-MP. To the best of our knowledge, the interaction of carbamate pesticides and MP have not been investigated before. In their review de Sá et al. (2018) identified only one out of 59 studies where no interaction between MP and another tested contaminant was found. Ferreira et al. (2016) reported, that the effects of gold nanoparticles were not modulated by PE particles (1–5 µm). Of course, de Sá et al. (2018) could not account in their meta-analysis for the probable bias against publishing negative results. Multiple studies show that effects of different pollutants were decreased in combination with MP. For example, PS-MP alleviated the effects of 17 α-ethinylestradiol (EE2) on locomotion in zebrafish (Chen et al. 2017). Similar results were found after exposure of zebrafish larvae to EE2, phenanthrene and a mixture of both with PVC particles. In the presence of PVC particles, the expression of cytochrome P4501A and vitellogenin was reduced up to 48% for EE2 and by 33% for phenanthrene (Sleight et al. 2017). Furthermore, in Daphnia magna PA particles reduced the immobilization caused by bisphenol A (BPA) compared to an exposure of BPA alone. BPA in water appeared to be the most bioavailable fraction for daphnids compared to the BPA adsorbed to MP (Rehse et al. 2018). In general, a reduced toxicity of co-contaminants in the presence of MP might be explained by a decreased bioavailability of the chemicals due 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 of chemical-induced effects by MP, other studies conducted with MP in combination with chemicals found an intensification of such effects. For example, in common carp (Cyprinus caprio), MP increased the effects of paraquat on biochemical blood parameters. Thereby, higher concentrations of MP increased the toxicity (Nematdoost Haghi and Banaee 2017). Moreover, Fonte et al. (2016) found a significant interaction between PE-MP and cefalexin. In the mixture of both, the effect of cefalexin on the predatory performance of common goby was increased at 20 °C, but reduced at 25 °C. In the freshwater clam (Corbicula fluminea) the oxidative stress level and the AChE inhibition of the antimicrobial florfenicol was increased in a mixture with MP. Furthermore, the mixture led to a feeding inhibition and reduction of isocitrate dehydrogenase suggesting interactions of florfenicol and MP since a simple additive approach could not explain the observed effects (Guilhermino et al. 2018). In the present study the toxicity of methiocarb was not enhanced by MP. 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.