Acute Toxicity of Chlorpyrifos on the Freshwater Bivalves ( Unio Tigridis ) and Effects on Bioindicators

: A freshwater bivalve plays a crucial function in aquatic habitats as the filtered water and burrowing mussels mix the sediment, thus increasing oxygen content and making the ecosystem healthier. The aim of the study is to see how chlorpyrifos affects biochemical markers in freshwater mussel Unio tigridis . About 180 individuals per taxon and water samples were collected from the Qandil water resource on the Greater Zab River, Erbil Province, Iraq. Once arrived at the lab, the individuals were kept in aquaria with river water and an air-conditioned room Temperature: 25±2 and Light: 12h/12h and acclimatized to laboratory conditions for seven days in aged tap water. The mussel's identification molecularly and the DNA sequence of the mussel includes U. tigridis supplied gene bank accession number ON872361, ON872362, ON872363, and ON872364 nucleotide sequencing. The 96-h toxicity of chlorpyrifos pesticide in the freshwater mussel U. tigridis was investigated using various nominal concentrations, including 50, 100, 200, 300 and 400 ppm. The water quality of the river and aquaria was tested for physicochemical parameters including water temperature, the potential of hydrogen ion pH, electrical conductivity EC, and total dissolved solids TDS, dissolved oxygen, total alkalinity, total hardness, calcium ion, magnesium ion. Water quality results of aquaria revealed that most tested variables were favorable for the breeding of mussels. The mortality of the mussels was observed daily and the 96 h LC 50 value for mussels was 157.99 ppm. Within the tissue of the gills, Acetylcholinesterase (AChE), Glutathione S-transferase (GST), Catalase (CAT), and Malondialdehyde (MDA) were determined. The chlorpyrifos exposures caused significant increases in GST, CAT, and MDA. The elevation of oxidative stress biomarkers was inversely related to the AChE inhibition in the examined species. In conclusion water pollution by chlorpyrifos lead to unsafe condition for aquatic taxa.


Introduction:
Bivalves are a significant animal that has made excellent bioindicators for evaluating water pollution and figuring out its level in bodies of water 1 . Stormwater runoff, untreated wastewater discharges, agriculture, and air deposition are just a few of the various ways that pollutants infiltrate water systems. Around the world, non-point source nutrient enrichment has taken over as the main cause of deteriorating water quality 2 . Pesticides and heavy metals are frequent freshwater pollutants that have a direct impact on aquatic life. Pesticides have a variety of negative consequences on public health, the ecosystem, the quality of food, and biodiversity. Pesticide poisoning of the environment ranks among the most significant issues facing the country due to its high persistence, potential for extreme toxicity, and slow breakdown. Pesticide employment is growing in farming, which causes environmental pollution. The majority of pesticides cause oxidative stress by disrupting the body's normal antioxidant system 3 . The sensitivity of freshwater species to organophosphates pesticides varies widely according to chemical compositions, duration of exposure, quality of water and taxa. In general, pesticide adversely affects non-target species, such as benthic invertebrates and fish, that live in the freshwater ecosystem due to their watery dispersal 4 . Pesticides are routinely identified in the aquatic ecosystem as artificial toxins; organophosphates and carbamates are new chemically synthesized insecticides that are strong neurotoxic chemicals. Chlorpyrifos is a common ingredient in organophosphates pesticides, and it's utilized on a vast range of products including wheat, fruit and vegetables 5 . Pesticide overuse has a variety of negative consequences on the environment and the creatures that are exposed to it, which has garnered interest in scientific fields. The inadequacy of a study based on empirical data on the opinions of farmers is one of Erbil's biggest issues. Typically, farmers are not well informed about the procedures for choosing and applying pesticides 6 .
Bivalves are generally long-lived between 15 to 40 years and have comparatively low mobility in their natural habitat. These creatures filter-feed on minute fine particles in the aquatic environment, but they have additionally been observed depositing food in the benthic habitats 7 . Mollusca, specifically bivalves, has played a significant influence in determining the rates of pollutants around the planet. This is due to the tactical advantages of collecting, global dispersion, generally quiescent behaviors, appropriate size, and, in many cases, environmental and socioeconomic significance 8 . A biomarker is a fundamental change that may be detected and/or assessed by bivalves at the genetic, metabolic, cell, physiological, or behavioral level and that discloses an organism present or the previous response to at least a single chemical in the ecosystem 9 .
Antioxidant enzymes are among the most popular markers utilized in pollution level monitoring. Among the first chemicals to also be utilized as a lipid peroxidation indicator was malondialdehyde (MDA) 10 . One of the important steps in oxidative stress is the modification of membrane phospholipids by lipid peroxidation, which is one of the indicators of stress. Chemically produced toxicity is frequently characterized by oxidative stress 11 . An enzyme called acetylcholinesterase (AchE) hydrolyzes acetylcholine and turns it into choline and acetic acid 12 . The enzyme regulates ionic currents in excitable membranes and is crucial for nerve conduction at the neuromuscular junction. The mechanism of the harmful effect of organophosphate insecticides is closely linked to the suppression of AchE. AChE has been and continues to be a widely utilized biomarker in freshwater ecotoxicity investigations, and it may also be employed as a neurotoxicity biomarker in species 10 . Glutathione S-transferase (GST) are detoxification enzymes that catalyze the attachment of glutathione (GSH) to a range of electrophilic molecules for the removal of potentially harmful xenobiotics. GST is found in the cytosol and microsomal fractions of cells 13 . Antioxidant enzymes like catalase (CAT) are part of the antioxidant defense system. By eliminating reactive oxygen species, this antioxidant enzyme helps to maintain cellular homeostasis and antioxidant defense 14 . The purpose of this research is to see how chlorpyrifos affects biochemical markers in freshwater mussel Unio tigridis gill tissue.

Water Quality
In September 2021, polyethylene bottles were used for the collection of river water at the study location. Water samples were examined for physical-chemical characteristics instantly such as water temperature °C, the potential of hydrogen ion pH, electrical conductivity EC (µS/cm), and total dissolved solids TDS (mg/l) at the sampling site. Dissolved oxygen (mg/l), total alkalinity (mg CaCO3/l), total hardness (mg CaCO3/l), calcium ion (mg/l), magnesium ion (mg/l), were measured as soon as arrived at the laboratory according to the procedures of 15,16 . The following water parameters analyzed in each glass aquarium were pH, EC, TDS, DO, total alkalinity, total hardness, Ca 2+ , and Mg 2+ utilizing the techniques listed above. Throughout the test, every 48 h, all parameters were measured.
One species of freshwater mussel's handcollected U. tigridis 7.5 cm long, 4.5 cm wide, and 26.2 g weight was found in the Greater Zab River's sediment area Fig. 1, branch in the Qandil area. About 180 individuals per taxon were sampled from the study area, cleaned, and transported to the laboratory with river water. Once arrived at the lab, the individuals were kept in aquaria with river water and an air-conditioned room Temperature: 25±2 and Light: 12h/12h and acclimatized to laboratory conditions for seven days in aged tap water. During animal rearing time, the water was changed every 24 h. Mussels were not fed during the experiment period. The sample of mussels was identified using the common keys 17,18 .

Molecular Study
Four replications of a Bivalvia U. tigridis were employed for DNA sequencing after morphological identification. Genomic DNA from adductor muscle was isolated and purified using the GeneAll® ExgeneTM for Clinic Cell SV small kit (Songpa-gu, Seoul, Korea). Agar gel electrophoresis was used to gauge the amount of genomic DNA that had been extracted before PCR 19 . The Nano Drop 1000 spectrophotometer is prepared to measure the optical density of a DNA sample as well as the concentration and purity of genomic DNA extraction to determine the amount and purity of DNA. The COI gene was successfully amplified using the primer pair LCO1490: 5'-GGTCAACAAATCATAAAGATATTGG-3' and HC02198: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3', which were ordered from Macrogen (Korea) Table  1 20 . The PCR experiment was conducted in a 50 µL reaction cocktail including 25 µL of 2× master mix (AMPLIQON, Denmark), 1.0 µL of each primer 10 pmol, and 3 µL of genome template. The amount reached 50 µL using PCR-grade water. To verify that the DNA templates were completely denaturized, DNA amplification for the COI gene was carried out in the thermal cycler for 5 minutes at 94 °C. The PCR was then carried out as follows: 94°C for denaturation for 50 sec, 50°C for annealing for 45 sec, and 72°C for an extension for 50 sec. These parts were repeated forty times, with a 7-minute extension at 72°C as the final cycle. 0.8% of agarose gel electrophoresis in 1× TAE buffer was used to examine the PCR products 21 . and were sent to (Macrogen/South Korea) for sequencing. MEGA software was used to analyze and alter the sequences that were acquired.

Experimental Design
The experiment consisted of 18 glass aquaria three controls and three replications of each other who received different exposure concentrations of chlorpyrifos 50, 100, 200, 300 and 400 mg/L. After applying chlorpyrifos immediately into the aquaria, they were left at 25 °C with a 12-h. darkness: 12 h. light cycle. The number of U. tigridis living and dead were recorded after 24, 48, 72, and 96 h by observing the mobility of individuals, the entry and escape of soft tissues from their shells in particular. The individual that couldn't enter their soft tissues and close their shells were considered as dead. Dead mussels were taken out of glass aquariums every day until the test was finished. Probit Analysis was used to measure the 96-h LC50 of chlorpyrifos following 22 . Additionally, the mortality percentage was calculated as a following Eq. 1: ℎ .

Biochemical Analysis
Mussels were dissected, and the gills of each U. tigridis were cleaned in an ice-cold saline fluid. A handheld glass homogenizer was used to homogenize the samples in a phosphate buffer. The homogenates were centrifuged at 4,000 rpm for 10 min at 4 °C. Before testing, the supernatants were taken and stored at -80°C 23 .
For Acetylcholinesterase AChE: BC 2020 and Glutathione S Transfer GST: BC0350, concentrations were measured by colorimetric method using commercial assay kits according to the manufacturer's protocol of Beijing Solarbio Science and Technology Co., Ltd. But, the tissue Catalase (CAT) activity was measured using the technique of 24 . Finally, the lipid peroxidation level in the tissue was quantified as malondialdehyde (MDA) according to the method of 25 .

Data Analysis
Data were analyzed by using the SPSS program version 25. Analysis of variance (ANOVA) is one way used to handle water quality and biochemical data. To look for significant variations between the treatments, Duncan's post hoc test was used. For statistical significance, a pvalue of 0.05 is used as the limit.

Results and Discussion:
It appears vital to assess the harmful effects of pesticides on aquatic creatures like freshwater mussels because there are numerous ways for environmental pollutants to infiltrate surface waters. The toxicity of the chlorpyrifos on U. tigridis biochemical markers is thus assessed in this work. The mussel identification molecularly by utilizing comprehensive or particular initial gene magnification was uniform with phenotypic assessment. Data for the molecular sample was provided by CDS nucleotide sequencing, which also provided isolation diagnostic and minute properties. The DNA sequence of the mussel includes U. tigridis supplied gene bank accession number ON872361, ON872362, ON872363, and ON872364 nucleotide sequencing Fig.  2.

Figure 2. Analysis of COI's molecular phylogeny using the Maximum Likelihood approach.
The values of water quality parameters in Qandil water resources during the study are represented in Fig.3. Environmental elements like physical and chemical variables have an impact on the ecology of freshwater mussels 26 .
While Table 2, summarizes the value of aquaria water quality parameters. During a laboratory experiment, these parameters must fall within the acceptable limits for aquatic species, especially for mussels' life. Water quality results of aquaria revealed that most tested variables were favorable for the breeding of mussels and were steady over the entire test. A control survival rate of 90% or more is typically required for short-term acute experiments with fish and invertebrates like bivalves 16 .  During the investigation, overproduction of mucus and a lengthening of shell closure was observed in all treated groups. These findings may help to explain why the investigated mussels had a defensive response, as other researchers found a similar pattern of behavior. Consequently, the findings of our investigation are consistent with 27 . As a typical response to stress, the mollusks secrete more mucus 28 [30][31][32] . The result of the mortality percentage illustrated at Table 3. The effect of chlorpyrifos on oxidative stress-related toxicity was studied including measurement of AChE, GST, CAT activities (U/mg protein), and MDA level (nmol/g protein) in gill tissue of freshwater mussels U. tigridis. Acetylcholinesterase (AChE) inhibition is the primary poisonous property of chlorpyrifos 33 . Table  4 shows how the organophosphorus pesticide chlorpyrifos affects AChE activity. The outcomes showed that the AChE activity in the gills of pesticide-exposed mussels had significantly decreased p ≤0.05 with increasing concentration of pesticide in aquaria. The AChE activity declined gradually from 24 h to 96 h of exposure. Indeed, the least activities were observed after 96 h of exposure with a value of 227.93±3.51 U/mg protein compared to control with a value of 492.81±0.54 U/mg protein. The findings of the present study are consistent with an earlier study of the inhibitory effect of this type of pesticide on mussels' AChE 34 . Glutathione S-transferase (GST) activity assessed in control and treated gill U. tigridis throughout the experiment period is displayed in Table 5. Indeed, pesticides raised GST activity in gills to 7.37±0.26 U/mg protein compared to the control value of 2.22±0.11 U/mg protein. A significant rise p ≤ 0.05 was noticed in most concentrations when compared with the gills of control mussels 35 . suggest that GST is crucial for detoxification and for preserving cells from oxidative damage. The detoxification of organophosphorus substances is facilitated by GST, which is essential for pesticide resistance. Similar results were observed by 36 . Additionally, variations in catalase (CAT) activity were seen as a result of chlorpyrifos exposure and its effects on the gills of mussels, U. tigridis Table 6. There is an obvious difference between the treatment and control groups. With an increase in chlorpyrifos dosage exposure, there were significant increases in CAT activity. At 400 ppm, the maximum CAT activity 45.90 U/mg protein was observed. The tendency in CAT activity is consistent with earlier studies that found that certain insecticides increased CAT activity. This increase in CAT activity in the gill's aids in the pesticide's detoxification 37 . Our results come to agree with those of 38 (3). Among the several aldehydes and ketones that are produced when monounsaturated and polyunsaturated fatty acids are per-oxidized is MDA. The maximum value of MDA 1.64±0.01 nmol/g protein was observed in an aquarium with a 400 ppm chlorpyrifos level during the 48 h of the test Table 7. A gradual increase in MDA levels was observed with increasing the concentration of pesticide during the test period. The mechanism for the rise in MDA may reveal that insecticide may enter the cell's lipid membrane and disrupt the orientation of the phospholipids, altering the fluidity of the membrane. Lipid peroxidation rises as a result of oxidative damage that tissue or cell is unable to prevent, as indicated by a rise in MDA levels 39 . Our results come to agree with that of 38 .

Conclusion:
The organophosphorus pesticide chlorpyrifos is toxic to U. tigridis at different concentrations. The biochemical impacts of sublethal concentrations of up to 400 ppm of chlorpyrifos pesticides were evaluated in U. tigridis under laboratory conditions. The chlorpyrifos exposures caused significant increases in GST, CAT, and MDA. The elevation of oxidative stress biomarkers was inversely related to the AChE inhibition in the examined species. Our findings suggest the value of using biochemical and oxidative biomarkers to identify the harmful outcomes and toxicological processes brought on by environmental contaminants. Water pollution by chlorpyrifos from human activities, especially agriculture leads to unsafe conditions for aquatic taxa.