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
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
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
The
The living
DI was obtained from the local market, in the form of Polygor EC (purity 25%, dissolved in 75% acetone). MA was obtained from the local market, in the form of 65% malathion EC. Each pesticide was prepared from a stock solution weighed in a volumetric flask containing distilled water. Dilutions of the defined stock solution were used for the tests described below. The stock solutions were renewed every 12 h. The lethal concentration-50 (LC50) value of the DI and MA was determined after 96 hours of exposure. For this, first of all the death range was determined and then the static test was conducted (APHA, 1998). The LC50 values of the DI and MA were measured as 120.14 ± 6.26 and 52.06 ± 5.33 mg l−1 respectively. The LC50 value of the combined exposure of the two insecticides was determined to be 39.91 ± 2.47 mg l−1. Three subletal doses of the DI, MA and combined exposure of the two insecticides (1/16, 1/8 and 1/4 ratio of the LC50 value) were applied to the
The test organism individuals were opened with a scalpel and the dissection process was performed on each of them. 0.5 g of the organism was weighed and homogenized using a homogenizer with ice, adding a PBS buffer (phosphate buffered salt solution) at a ratio of 1/5 w/v. These homogenized samples were then centrifuged in a refrigerated centrifuge at 17.000 rpm for 15 minutes and the supernatants obtained were stored in a -86°C deep freezer until the measurement process was completed (Serdar 2021).
In this study, the activities of the SOD, CAT and the levels of GSH, TBARS were measured to determine the oxidative biomarker response of the
SPSS version PASW Statistics 18 was used for the statistical analysis. A one-way ANOVA and Duncan’s multiple range tests were applied to determine the statistical differences in the control and all exposure groups (A, B, C, D, E, F, G, H and I) at the same exposure time (abc
In our study, the average of the LC50 value of
The LC50 value of the combined exposure of the two insecticides was measured as 39.91 ± 2.47 mg l−1.
The activities of the SOD, CAT and the levels of GSH, TBARS are given in Table 1.
Biochemical response parameters
Parameters | Groups | Exposure Time | |
---|---|---|---|
24 h | 96 h | ||
GSH (μत) | Control | 2.66 ± 0.61 | 2.95 ± 0.04 |
A | 1.50 ± 0.62 | 1.70 ± 0.20 | |
B | 1.37 ± 0.14 | 1.32 ± 0.36 | |
C | 1.55 ± 0.21 | 1.24 ± 0.49 | |
D | 1.05 ± 0.32 | 1.04 ± 0.10 | |
E | 1.43 ± 0.24 | 0.94 ± 0.01 | |
F | 0.64 ± 0.10 | 0.80 ± 0.009 | |
G | 1.34 ± 0.46 | 1.16 ± 0.16 | |
H | 0.68 ± 0.05 | 0.55 ± 0.05 | |
I | 1.05 ± 0.07 | 0.5 ± 0.01 | |
CAT (nmol/min/ml) | Control | 3.01 ± 0.21 | 3.64 ± 0.09 |
A | 6.79 ± 0.42 | 2.83 ± 0.12 | |
B | 5.49 ± 1.09 | 7.66 ± 0.73 | |
C | 3.13 ± 0.39 | 6.57 ± 0.15 | |
D | 3.24 ± 0.90 | 5.08 ± 0.90 | |
E | 2.36 ± 0.18 | 2.56 ± 0.21 | |
F | 2.04 ± 0.02 | 2.22 ± 0.009 | |
G | 2.56 ± 0.04 | 9.18 ± 0.05 | |
H | 2.68 ± 0.13 | 5.19 ± 0.006 | |
I | 2.64 ± 0.07 | 2.47 ± 0.07 | |
SOD (U/ml) | Control | 0.034 ± 0.0005 | 0.0245 ± 0.001 |
A | 0.0260 ± 0.006 | 0.0266 ± 0.003 | |
B | 0.0206 ± 0.002 | 0.0377 ± 0.002 | |
C | 0.0233 ± 0.003 | 0.0307 ± 0.001 | |
D | 0.0328 ± 0.002 | 0.0326 ± 0.001 | |
E | 0.0247 ± 0.002 | 0.0367 ± 0.002 | |
F | 0.0254 ± 0.001 | 0.0187 ± 0.0006 | |
G | 0.0283 ± 0.004 | 0.0272 ± 0.0003 | |
H | 0.0309 ± 0.003 | 0.0287 ± 0.002 | |
I | 0.0317 ± 0.001 | 0.0220 ± 0.003 | |
TBARS (μत) | Control | 4.78 ± 0.59 | 2.93 ± 0.24 |
A | 5.43 ± 0.13 | 6.58 ± 0.26 | |
B | 9.46 ± 0.11 | 8.85 ± 0.45 | |
C | 7.09 ± 0.70 | 7.34 ± 0.40 | |
D | 5.83 ± 0.15 | 7.01 ± 0.23 | |
E | 6.50 ± 0.53 | 8.49 ± 0.19 | |
F | 14.41 ± 1.73 | 11.43 ± 1.05 | |
G | 6.13 ± 1.73 | 12.36 ± 0.08 | |
H | 6.55 ± 0.26 | 14.40 ± 0.05 | |
I | 15.07 ± 0.65 | 47.71 ± 0.76 |
It was determined that the SOD activity decreased in all the groups after 24 h compared to the control (
The CAT activity increased in all the DI exposure groups after 24 h compared to the control (
The GSH levels were decreased in all exposure groups after 24 and 96 h compared to the control (
The TBARS levels were increased in all exposure groups after 24 h and 96 h compared to the control group (
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
The acute toxicity value (LC50) in
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
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
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),
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
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
The present study investigated the toxic potential of insecticides DI and MA in
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.