Single and combined effects of dimethoate and malathion on oxidative stress biomarkers in the non-target freshwater mussel Dreissena polymorpha

Pesticides enter aquatic ecosystems through a variety of processes, including direct application, industrial and urban wastewater discharges, and runoff from some other non-point sources (Sharma 1990). The intensive use of organophosphorus pesticides in many fields such as agriculture, medicine and industry can cause many problems in humans and wildlife (Garcia et al. 2003, Kalender et al. 2010, Banaee et al. 2019a). MA is one of the most heavily used organophosphate pesticides against various pests in agriculture, as this insecticide is also considered to be of low toxic value for domestic use (Lasram et al. 2008). Exposure of MA on experimental animals, however, has shown that it can change biochemical and hematological parameters as well as antioxidant enzyme activities in different tissues (Uzun and Kalendar 2011). DI is a dithiophosphate insecticide with wide application on various fruits and vegetables and some other crops (Hassall 1990).

Exposure to chemicals often causes oxidative stress. Oxidative stress induced by overproduction of reactive oxygen species (ROS) is a precursor to many of the problems associated with this organophosphorus exposure and many findings have reported the enhancement of oxidative stress in animals (Hu et al. 1994, Franco et al. 2009). Mixed pesticides can cause significant synergistic effects of toxicity on target species, although they are also effective on non-pest species compared to single pesticides (Bonansea et al. 2017).

In aquatic ecosystems, the organophosphate insecticide dimethoate could increase the environmental risk to nontarget organisms (Acquaroni et al. 2021). Understanding the relationship between pesticides and their toxic effects and accumulation in D. polymorpha can provide better insight into the ecological risks from pesticide contamination and is crucial for accurately monitoring and predicting the effects of these pollutants in the aquatic environment (Bashnin et al. 2019). Biomarkers can provide information about the health status of organisms and, therefore, these markers can be used as early warning signals of general or specific stress (Korte et al. 2000). Biochemical and oxidative biomarkers are very useful to detect harmful effects and toxicological mechanisms caused by environmental pollutants (Banaee et al. 2019a). Therefore, the enzymes such as SOD, CAT and GSH can be used as biomarkers in the case of oxidative stress caused by pollutants (Rocher et al. 2006, Acquaroni et al. 2021).

The SOD catalyzes the superoxide dismutation into O₂ and H₂O₂, which in turn is further reduced by CAT to H₂O and O₂. GSH is a vital antioxidant that acts as a direct scavenger of oxidants as well as being a substrate for antioxidant enzymes (Ferrari et al. 2007). Pesticides, metals and other xenobiotics cause lipid peroxidation, which is considered as an important indicator of oxidative damage of cell membrane components, known as the first step in cell membrane damage (Gamble et al. 1995, Serdar et al. 2018). The zebra mussel D. polymorpha is considered an invasive species found from the Caspian Sea to European and North American rivers and lakes. Due to its moderate sensitivity against anthropogenic pollutants, D. polymorpha established sustainable populations also in urban watercourses (Horgan et al. 1997).

This study was conducted to determine the sublethal effects of exposure, both single and combined, of the two insecticides DI and MA on oxidative stress in D. polymorpha and to evaluate its underlying mechanism.

Pesticides are chemicals that are widely used, especially to increase food production efficiency and to control disease vectors. These chemicals are released into the environment from many sources and contaminate water, soil and foodstuffs. In this study, the toxic potential of DI and MA, currently used in agriculture, was investigated at field realistic exposure levels in D. polymorpha. The capacity of D. polymorpha to cope with the environmental pollution stress was clearly evidenced. The selected biomarkers including SOD, CAT, GSH and TBARS displayed significant changes in activities and levels at different times of exposure.

The acute toxicity value (LC50) in G. pulex of the DI pesticide was found to be 167 μg l⁻¹ (Serdar 2019). Pandey et al. (2009) carried out a 96 hr static acute toxicity test to determine the LC50 value of DI on the freshwater airbreathing catfish Heteropneustes fossilis (Bloch). The LC50 values for DI for 24, 48, 72 and 96 h were 3.38, 3.23, 3.08 and 2.98 mg l⁻¹, respectively. MA is highly toxic to many organisms, although there is a wide variation from one species to another. The 96 h LC50 of MA was estimated for rohu in a semi static system and was found to be 5 μg l⁻¹ (Ullah et al. 2016). There was a wide variation in the 48 to 96 h LC50 for 5 test Freshwater molluscs species. Thiara (two species) had figures of 31 to 37 μg l⁻¹; Viviparus bengalensis (96 hours) had 1510 to 2340 μg l⁻¹; Pila globosa; 10,000 μg l⁻¹ to 15,490 μg l⁻¹; and Biomphalaria havanensis, 126,270 μg l⁻¹ (Anzecc&Armcanz 2000). In this study, the average of the LC50 value of D. polymorpha, exposed to three repetitive DI pesticides, was found to be 120.14 ± 6.26 mg l⁻¹. The LC50 value of the MA was determined to be 52.06 ± 5.33 mg l⁻¹. The LC50 value of the MA and DI was determined to be 39.91 ± 2.47 mg l⁻¹.

At low or non-high concentrations, ROS are considered a result of normal oxidative metabolism, but when they reach high levels, they cause many problems, including DNA damage, lipids, protein oxidation, and enzyme inactivation (Dal-pizzol et al. 2001). During the stress reactions caused by pollution, the antioxidant system increases its activities to scavenge harmful substances, reducing their toxicity and increasing their excretion. Pesticides may cause oxidative stress by increasing the formation of free radicals, resulting in changes in protective enzymatic and non-enzymatic antioxidants and lipid peroxidation (Abdollahi et al. 2004). Banaee et al. (2019b) investigated acute and subacute toxicity tests for chlorpyrifos and glyphosate performed on the crayfish Astacus leptodactylus. They found subacute concentrations of chlorpyrifos and glyphosate could lead to a collapse of cellular homeostasis and changes in the hemolymph biochemical parameters in the crayfish. Gunderson et al. (2018) indicate that the biomarkers are sensitive to the pollutants tested and that animals exposed in the wild are potentially compromised in their ability to detoxify environmental contaminants.

Lipid peroxidation is a degenerative process that affects polyunsaturated fatty acids in membrane phospholipids, resulting in toxic aldehydes that react with protein and non-protein substances causing diffuse changes in cell membranes. It is also suggested that organisms exposed to methamphetamine (MA) form covalent adducts between proteins and the carbonyl groups of malondialdehyde. Additionally, covalent adducts between proteins and the carbonyl groups of malondialdehyde are proposed to be generated in organisms exposed to MA (Chitra 2013). Lipid peroxidation and its end product, malondialdehyde (MDA), can occur when antioxidant defenses are not sufficient to neutralize excess ROS, presumably produced during the biotransformation process (Modesto & Martinez 2010). TBARs are used to measure the lipid peroxidation products in cells, tissues and bodily fluids (Frost et al. 2019). Previous studies have shown that DI intoxication causes oxidative stress by producing free radicals, resulting in cellular damage and lipid peroxidation (Singh et al. 2004). Wankhade (2012) demonstrated that sustained and prolonged exposure of mice to a non-lethal dose of MA induced lipid peroxidation.

Exposure to dimethoate has been reported to result in increased MDA in freshwater fish such as Channa punctatus (Ali et al. 2014), Oncorhynchus mykiss (Dogan & Can 2011), Cyprinus carpiove (Shadegan et al. 2018) and Danio rerio (Maharajan et al. 2018). Akhgari et al. (2003) found that MA exposure (100, 316, 1000, 1500 ppm) for 4 weeks resulted in increases in MDA levels in red blood cells (RBC) and the liver. Aksoy and Alper (2019) suggested that lipid peroxidation occurred in rats exposed to MA. Due to its lipophilic nature, MA was thought to penetrate the membrane and initiate lipid peroxidation. Similarly, in our study, TBARS levels were found to increase in all the exposure groups compared to the control after 24 and 96 h.

SOD is an important enzyme involved in removing oxyradicals and scavenges superoxide into hydrogen peroxide (H₂O₂) and oxygen (Su et al. 2014). The H₂O₂ is then converted to H₂O and O₂ by CAT, a type of enzyme containing Fe-protoporphyrin (Koivula et al. 2011). CAT activity in contaminated environments may be increased or suppressed depending on the type of contaminant (Sobjak et al. 2017). The reaction mechanism of SOD and CAT enzymes after exposure to pesticides differs according to the structure of the pesticides, the type of organism and the targeted part of the organism (Oruc&Usta 2007). In a study conducted by Serdar et al. (2021), D. polymorpha was exposed to non-lethal concentrations (1/20, 1/10 and 1/5 of the LC₅₀) of cyfluthrin for 24 and 96 hours. These researchers found that the enzyme activities of SOD were increased, but the CAT activity was inhibited compared to the control. In another study, Pala et al. (2019) found a statistically significant reduction in CAT activity and GSH levels when exposed to non-lethal concentrations of the glyphosate-based herbicide for 24 and 96 hours. Barski et al. (2012) showed that subchronic exposure of rats to DI produced a strong oxidative stress resulting in a significant increase in CAT and SOD activity in erythrocytes. In the study conducted by Serdar et al. (2021) on D. polymorpha exposed to Cyfluthrin, the SOD activity increased but CAT was inhibited compared to the control. Serdar et al. (2019) found that SOD activity has been decreased after DI exposure in G. pulex. However, John et al. (2001) found increased erythrocyte CAT and SOD activities in rats exposed to DI and/or MA compared to the control. They stated that SOD and CAT activities in erythrocytes increased in rats exposed to DI and/or MA, possibly to degrade superoxide anions (O₂^.−) and decompose H₂O₂. Banaee et al. (2022) investigated chlorpyrifos-induced oxidative stress in Cyprinus carpio, They determined the activity of SOD, and total antioxidant levels, significantly decreased in the hepatocytes of C. carpio exposed to chlorpyrifos, while malondialdehyde (MDA) increased.

The increase in the activity of these enzymes likely occurred as a response to increased ROS formation due to organophosphate toxicity. Shadegan et al. (2018) found that a significant increase in MDA and CAT activity occurred in fish liver and kidneys exposed to dimethoate alone or in combination with Bacilar. Demirci et al. (2018) investigated the toxic effects of the combined exposure of the herbicide atrazine and the insecticides endosulfan, indoxacarb and thiamethoxam using oxidative stress biomarkers found in Gammarus kischineffensis. They determined higher CAT and SOD activities when atrazine was combined with either endosulfan or indoxacarb compared with atrazine alone. In the present study, an increase was detected in DI+ MA exposure groups after 96 h compared to the control group (p < 0.05). CAT activity increased in all the DI exposure groups after 24 h compared to the control (p < 0.05). Compared to the single exposure, the CAT activity was reduced in the combined exposure at 24 h (p < 0.05). The increase in activity of the CAT enzyme could be due to the engagement of CAT enzyme to convert hydrogen peroxide into water molecules, whereas the decrease in activity detected could be due to the saturation of hydrogen peroxide leading the deactivation or suppression of CAT enzymes for a duration of time (Pawar 2020). Aksoy and Alper (2019) found that SOD and CAT concentrations in rats exposed to MA decreased significantly. In the present study, It was observed that SOD activity decreased in all groups after 24 h compared to the control group (p < 0.05). The constant level of increase in the ROS generation and at the same time inadequate supply of SOD to detoxify these ROS’, results into a decrease of the level of SOD enzymes in organisms (Pawar 2020).

GSH, which is an important tripeptide in the detoxification system, scavenges organic and metallic xenobiotics with its reduced form (Vasseur and Leguille 2004). It also plays an important role as a reducing agent in preventing the damaging effect of ROS (Gismondi et al. 2012). An increase in GSH levels was observed for diazinon and diuron pesticides in the study conducted by (Velki et al. 2019). They also suggested that the increased GSH levels indicate the occurrence of oxidative stress. Aksoy & Alper (2019) investigated the effects of royal jelly on toxicity and biochemical changes in rats exposed to MA. They determined that the GSH concentrations increased in the brain and decreased in the erythrocyte, liver and kidneys in the group exposed to MA compared to the control groups. They suggested that MA administration increased toxicity in the erythrocyte, liver and kidney tissues and that GSH was utilized as an antioxidant defence agent. Taherdehi et al. (2019) investigated the effects of MA on the GSH level in the testis of male rats. They showed that MA decreased the GSH level compared with the control group. Serdar et al. (2019) determined the response of some biochemical biomarkers in G. pulex exposed to DI pesticide. They found that GSH levels in G. pulex exposed to the DI pesticide were reduced at both 24 and 96 hours compared to the control group. Smilarly, in the present study GSH levels were decreased in all the exposure groups after 24 h and 96 h compared to the control. The decrease in the GSH levels may be due to the fact that it is the substrate of GST, which takes part in the detoxification reaction (Serdar et al. 2019). This decrease in GSH levels with exposure to pesticides may be related to the increased use of GSH converted to oxidized glutathione and insufficient GSH production.

The present study investigated the toxic potential of insecticides DI and MA in D. polymorpha at field-realistic exposure levels. The results show that the toxicity of the combination of DI and MA was dependent on their concentrations. The exposed higher concentrations of DI and MA induced significant changes in some biochemical parameters. Sub-lethal concentrations of DI and/or MA caused oxidative damages in D. polymorpha.

Exposure times also had an effect on some biomarkers. Therefore, different combinations of pesticides can cause changes in their toxicity. To determine the combined effect of pesticides, it is necessary to evaluate the responses of the right biomarkers, and this is quite difficult. According to the results of our study, it was concluded that the multiple biomarkers we used are suitable for determining the single and combined toxicity of DI and MA.