Oxidative Stress Induced by Exposure of Microplastics in Labeo Rohita

The present study revealed the oxidative stress induced by the exposure of low density polyethylene microplastics (LDPE MPs) in Labeo rohita. Fingerlings were divided into four (control, T1, T2 and T3) groups. Fingerlings of T1, T2 and T3 groups were exposed respectively to 2, 20 and 200 mg/L of LDPE MPs for 45 days. A control was also maintained during experiment to which no MPs were added. Oxidative stress studies revealed a decrease in the activity of hepatic antioxidative enzymes i.e. superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and glutathione reductase (GR) in all treated groups in dose and time dependent manner. In gills, an increase in the activities of SOD, CAT and GPx but decrease in the activities of GR and GST was found in T1 group after 30 days of exposure. However, decrease in activities of these enzymes was observed in all treated groups in dose dependent manner after 45 days of exposure. Moreover, the activities of antioxidative enzymes were found to be decreased in time dependent manner. In kidneys, increase in the activities of antioxidative enzymes was observed in T1 group followed by their decrease in T2 and T3 groups in dose and time dependent manner. However, Lipid peroxidation was found to be increased in vital organs of all treated groups in dose and time dependent manner indicating MPs induced oxidative stress in these organs due to increased production of reactive oxygen species.


Introduction
Plastics are synthetic polymers with high molecular weights, made up of linked long chain of similar repeating units called monomers which are derived from petroleum or fossil fuels. These are cheap, strong, durable, water resistant materials thus making it a material of choice for manufacturers of several items of daily use including packaging, clothing and electrical appliances (Horton 2019).
For many years, extreme use and disposal, ineffective material recycling and dumping of plastics on land and sea lead to accumulation of mismanaged plastic litter in the environment. The properties of plastic materials such as durability, longevity and resistance to degradation due to presence of hydrocarbons allow their persistence in the natural environment. Thus, deposition and accumulation of plastics in the natural environment has become an emerging subject of environmental urgency (Asmonaite 2019).
Both marine and freshwater environments are contaminated with plastics all over the world. This huge presence of plastics in the water bodies have adverse effects on the aquatic ecosystems and related economic activities (Boucher and Friot 2017). Plastic waste has two forms: large plastic wastes and small plastic particles less than 5 mm in size termed as microplastics (Thevenon et al. 2014). Large sized plastic wastes are easily visible and studies of these large plastic wastes have revealed a number of undesirable social, ecological and economic impacts. These impacts vary from reduced food intake, intestinal injury, entanglement, intestinal blockage and death of animals. As MPs are not visible due to their small size so their harmful impacts on the animals are not easily identi ed (Boucher and Friot 2017). These ingested MPs may have many negative effects on marine organisms ranging from physical to physiological effects. These effects include internal abrasions and blockage of digestive tract which may cause starvation (Gall and Thompson 2015), histopathological changes in the intestine, behavioural changes and change in lipid metabolism (Jovanovic 2017) in sh. There is increasing concern about study of harmful effects of MPs on human health as they may move independently or may adsorb other toxic chemicals while moving in the food chain (Eriksen et al. 2014). Ingestion of these MPs is not only harmful to humans alone but also to ecological health due to accumulation of pathogens on surfaces of plastics.
On the basis of their origin, MPs can be classi ed into primary and secondary MPs. Primary MPs, manufactured in the form of microbeads, capsules, pellets or bers are directly released into the environment in the form of small particles. Examples include use of microbeads in personal care products such as in cosmetics, soaps, toothpaste and use of micro bers in textiles. Secondary MPs are produced by the degradation of larger plastic products into smaller plastic pieces through photodegradation and other weathering processes of improper management of wastes such as discarded plastic bags (Boucher and Friot 2017).
To date, several studies have been carried out on the contamination of marine environment by MPs and their effects on various marine organisms including marine shes. Although studies on MPs in freshwater environments are scarce than marine water studies, plastics have been reported in freshwater sh, insects and invertebrates also (Anderson 2019). Among aquatic organisms, sh are highly susceptible to MPs ingestion due to their buoyancy, attractive colouration and great resemblance to food (Jovanović et al. 2018). Thus, it becomes important to protect and preserve populations of freshwater sh from pollutants of appearing concern like MPs (Pinheiro et al. 2017).

Preparation of test chemical
Low Density Polyethylene (LDPE) in pellet form (CAS: 9002-88-4; melting point: 116ºC; density: 0.925g/ml at 25ºC) was purchased from SIGMA-ALDRICH Company, USA. 30 LDPE pellets were taken in each glass test tube. To it, 6 ml of toluene (used as solvent) was added. Then 200 µl of Tween-20 as surfactant was added by using a micropipette and mouth of each glass test tube was covered with aluminium foil/ para lm to prevent the evaporation of toluene on heating. The test tubes were kept in water bath shaker pre-heated at 95ºC until LDPE pellets were completely dissolved in toluene. After this process, 6 ml of double distilled water (DDW) pre-heated at 95ºC was added in each glass test tube containing the dissolved LDPE at 95ºC. The hot mixture was immediately homogenized at 10,000 rpm for about 30 seconds. The resultant mixture was rapidly spread on wide glass petriplate with the help of spatula and left undisturbed for few days or till the mixture was completely air dried. The obtained LDPE powder was sieved using siever to obtain ne particles of LDPE. The left over large particles were then crushed with the help of pestle and mortar. These crushed particles were then sieved to obtain ne LDPE powder. This formulation of LDPE powder was used as LDPE MPs in the present study.

Procurement and acclimatization of ngerlings
Healthy ngerlings of Labeo rohita (9 ± 2 cm in length) were procured from private sh farm, Ludhiana, Punjab, India. These were acclimatized to laboratory conditions for fortnight in plastic tanks of 100 litres capacity lled with 80 litres of de-chlorinated tap water with adequate values of temperature, pH, alkalinity, conductivity, hardness, dissolved oxygen and biochemical oxygen demand. A natural photoperiod of 12 hour light:dark cycle was maintained in the laboratory. Each tank was tted with electrically operated two water aerators (to ful l the oxygen demand) and two water lters (to avoid accumulation of excreta) during the acclimatization period. Fingerlings were fed with commercial sh food (Trade name: Tokyu oating type sh feed) once a day in afternoon during acclimatization period.
No feed was given to the ngerlings one day before the start of an experiment. The experimental protocol met the standard guidelines given by Organisation for Economic Co-operation and Development (OECD) (1992).

Exposure of ngerlings to LDPE MPs
In the present study, ngerlings were divided into four groups-1st group served as control and 2nd, 3rd and 4th served as MPs treated groups with three replicates per group. A total of nine ngerlings (n = 9) were kept in each tank (35 litres capacity) containing 25 litres of de-chlorinated tap water thus resulting in the use of total 108 ngerlings and 12 tanks. For exposure concentration selection, existing literature was thoroughly studied to nd an environmental relevant concentration of PE MPs in freshwater environments. Rochman et al (2017) stated the presence of maximum of 2.8 mg/L of PE MPs in the freshwater environments so accordingly, the present study denoted the concentration of 2 mg/L in water as environmental relevant concentration of PE MPs and this concentration is used in lowest treatment group (T1) in our study. Moreover, the other two concentrations used in the present study were taken as 20 and 200 mg/L in water (T2 and T3 respectively), which are 10 and 100 times higher than this environmental relevant concentration respectively and represented the worst-case scenario of contamination of MPs in the aquatic environment.
The experimental design used in the present study during chronic toxicity test is shown in the following During the whole experiment, water was completely changed after every 5th day to avoid the accumulation of excreta and the MPs were added to restore its concentration in each tank.

Assessment of oxidative stress
Fingerlings were sacri ced by putting them for 10-15 minutes in the beaker containing 2-3 drops of clove oil mixed with 1 litre of tap water. After sacri cing, ngerlings were carefully cut opened from the ventral side i.e. the anal n along the belly up to the operculum using small sharp surgical scissors (taking care not to cut into the ngerling's internal organs and prevent leakage of bile from gall bladder).
Various organs i.e. gills, liver, kidneys were dissected out with the help of scalpel, forceps and a pair of sharp surgical scissors for oxidative stress studies.
After sacri cing, 3 ngerlings (n = 3) from each tank were dissected at an interval of 30 days and 45 days to collect the required tissues. For preparing tissue (gills, liver, intestine, muscle and kidney) homogenate, 0.5 g of each tissue was taken in centrifuge tube containing 2 ml of freshly prepared PBS solution and homogenised for 2 minutes following centrifugation at 3,000 r.p.m. for 10 minutes. After centrifugation,

Statistical analysis
All values are expressed as mean ± S.E. Two way analysis of variance (ANOVA) and Tukey's post hoc test was used for determining signi cant differences among treatments i.e. control, T1, T2 and T3 groups using Statistical Package for Social Sciences (SPSS) version 16 for windows. Values with different alphabetical superscript(s) are considered signi cant at p < 0.05.

Results And Discussion
Results revealed a decrease in the activity of hepatic antioxidative enzymes i.e. SOD, CAT, GST, GR and GPx was observed in all treated groups in dose and time dependent manner (shown in tables). In gills, an increase in the activities of SOD, CAT and GPx but decrease in the activities of GR and GST was found in T1 group after 30 days of exposure. However, a signi cant decrease in activities of these enzymes was observed in all treated groups in dose dependent manner after 45 days of exposure. Moreover, the activities of antioxidative enzymes were found to be decreased in time dependent manner (shown in tables). In kidneys, increase in the activities of antioxidative enzymes was observed in T1 group followed by their decrease in T2 and T3 groups in dose and time dependent manner (shown in tables). However, LPO was found to be increased in vital organs of all treated groups in dose and time dependent manner (shown in table VI).
Oxidative stress can be de ned as a disturbed balance between the formation of damaging reactive species i.e. reactive oxygen species (ROS) and reactive nitrogen species (RNS) and a living organism's ability to deal with them. Increase in the production of ROS and RNS or decrease in The antioxidative system of organisms exposed to MPs revealed a variety of responses, ranging from induction and reduction to changes which are non-signi cant, depending on the size, type and concentrations of MPs and the studied tissues and organisms. The liver is the main organ which plays a vital role in the detoxi cation of pollutants as it is the site of storage of many substances, hence, exposure and accumulation of pollutants may affect its vital functions. Gills are paired organs in sh which possess osmoregulatory, respiratory and excretory functions. Their direct contact with the surrounding aquatic environment makes them more prone to exposure of various pollutants. Kidneys are the paired organs that are supposed to be exposed to various pollutants due to its excretory role.
Decrease in the activity of anti-oxidative enzymes in the liver with the increasing concentration of MPs and exposure period may be attributed to the fact that stress induced by the overproduction of ROS in the cells exceeds the self-clearing approach of the antioxidative defence system thereby inhibiting the antioxidative enzymes activity. The time dependent decrease in the GPx activity in the liver of all treated groups might be due to an increased production levels of hydroperoxides (Ajima et al. 2017), supposed tobe caused by the adverse effect of LDPE MPs (Iheanacho and Odo 2020).
However, at low concentrations of MPs, the activity of antioxidative enzymes is not always inhibited and consequently the ROS levels are not signi cantly increased. This suggests that the antioxidative system of sh is resistant to oxidative stress induced by low concentration of MPs, but once the concentration of MPs exposure is increased, the antioxidative capacity of sh is destroyed and sh could not resist the oxidative stress induced by the MPs (Yang et al. 2020). Moreover, increase in the exposure period of MPs to sh is thought to play an important role in decrease in the antioxidative capacity of sh due to continous contact of vital organs (like gills) with MPs. These facts could possibly explain the trends of antioxidative enzymes activities observed in gills and kidneys after exposure to MPs in the present study.
In the present study, LPO levels were found to be increased in all vital organs of ngerlings of all treated groups in a dose and time dependent manner in comparison to control group. This might be due to the increased levels of MDA, produced as a result of increased ROS, which causes damage to structure of cell membranes and cells hence causing oxidative stress in ngerlings (Umamaheswari et al. 2020).
There are enough studies reporting the decrease in the activities of antioxidative enzymes in the liver of MPs exposed sh but studies involving the changes in the activities of these enzymes in gills and kidneys after exposure to MPs are lacking.
Decrease in the SOD and CAT activity in the liver of European sea bass (Dicentrarchus labrax L.) was also reported by Espinosa et al. (2019) on exposure of sh to100 or 500 mg of virgin PE-MPs/ kg diet for 3 weeks. However, GR activity was not affected by MPs exposure. Moreover, no signi cant effects on the activities of these anti-oxidative enzymes were observed when sh were exposed to PVC MPs diets for 3 weeks.
Marine medaka were exposed to 2, 20 and 200 µg/L of PS MPs in a semistatic system for 60 days. After exposure period, the activities of various antioxidative enzymes and MDA content in gills and liver were recorded. In liver, the activity of GPx in liver was found to be signi cantly decreased in all exposure groups. But, sh groups exposed to high Similar to the ndings of the present study, adult male zebra sh exposed to 10 µg/L and 100 µg/L of polystyrene MPs in water for a period of 35 days resulted a dose and time dependent signi cant decrease in the activities of antioxidant enzymes (SOD, CAT and GPx) and signi cant increase in the LPO levels in the liver of sh. However, the activity of GST was found to be increased in the liver of MPs exposed groups in a dose and time dependent manner which may be due to increased e ciency of GST to detoxify the ROS produced as a result of MPs exposure (Umamaheswari et al. 2020).
Exposure of Clarias gariepinus juveniles to 0.5, 1.5 and 3.0% PVC MPs in their diet for 45 days resulted in a time dependent decrease in the activities of CAT and GPx in the liver of juveniles. However, the activity of SOD was found to be increased in all treated groups after 30 days followed by its decrease after 45 days of exposure. Moreover, the levels of LPO were found to be signi cantly increased progressively in all treated groups in a time dependent manner (Iheanacho and Odo 2020).
In contrary, there are several studies reporting the increase in the activities of antioxidative enzymes and consequently decline in the LPO levels in various organs of sh exposed to MPs. This could be due to the use of different species of sh and their life stages; different types, shapes, sizes of MPs; different concentrations and exposure.

Conclusion
Chronic exposure of Labeo rohita ngerlings to low density polyethylene microplastics caused oxidative stress in their vital organs (gills, liver and kidneys) as depicted by alterations in the activities of antioxidative enzymes and increase in the lipid peroxidation levels in these organs in dose and time dependent manner.

Declarations
Animal    Values with different alphabetical superscripts differ signi cantly at p<0.05. Values with different alphabetical superscripts differ signi cantly at p<0.05. Values with different alphabetical superscripts differ signi cantly at p<0.05.