Herbicides are intentionally added to the environment, in contrast to other pollutants, with the aim of employing their deadly properties to reduce pests and insects that transmit disease. 99.9% of pesticides used on intended targets wind up in various environmental media, with less than 0.1% actually reaching the pests [1]. Pesticides in streams gradually disappear due to torrents, dilution, partitioning in water or air, attachment to sediment particles, accumulation in the tissues of aquatic animals, or burial in sediment [2]. Surface runoff from infrequent rainfall is the primary means of pesticide transmission from land to aquatic bodies [3], exposing non-target organisms like fish to pesticides on a pulse basis. Pesticide dissipation in lentic (still) systems may be the source of pulse exposure, but lotic (flowing) systems may have a connection to their hydrology (constant replenishing through water movement) [4]. Floods, dilution, partitioning in water or air, attachment to sediment particles, accumulation in aquatic animal tissues, and burial in the sediment are some of the ways that low pesticide concentrations can cause pulse exposure [5].

While there are many benefits to utilizing chemicals in farming, there are also major disadvantages that are directly tied to the use of chemicals, such as pollution and environmental damage [6]. Agrochemical pollution poses a severe threat to environmental safety and can have detrimental health impacts, including cancer and neurological system damage [7]. The use of agricultural chemicals has led to the presence of these chemicals in ecosystems becoming a major cause for concern that causes significant social and scientific anxiety worldwide. These chemicals pose a serious threat to the ecosystem and all living things, and they can easily contaminate water bodies, causing extensive harm to non-target species, including fish [8]. Globally, agrochemical pollution of the environment is becoming a serious threat to human health and wildlife conservation [9]. Intentional or inadvertent compounds applied directly to aquatic systems, spray drifts, air fallout from dust and rain, sewage, industrial effluent, and, very infrequently, spills can all contaminate water.

Biomarkers are sub-organism responses in organisms that can demonstrate exposure to or the effects of environmental pollutants [10]. Oxidative damage, haematological changes, biochemical and histological abnormalities, genotoxicity, and mutagenicity are a few of the indicators that are commonly evaluated in fish. Biomarkers can be used to monitor fish health and act as early warning systems for environmental threats [11]. After more than 20 years of studying aquatic organisms exposed to pesticides or other toxicants, nothing is known about the biochemical responses of S. melanotheron to herbicides. These creatures have a wide range of consequences on the economy and environment. The influence of butachlor on S. melanotheron metabolites is not well-documented in the literature. The metabolic responses of S. melanotheron exposed to varied lab-based butachlor concentrations were assessed in this work.

Experimental Location and Fish

The study was conducted at the African Regional Aquaculture Center in Buguma, Rivers State, Nigeria, which is a branch office of the Nigerian Institute for Oceanography and Marine Research. During low tide, a total of 180 S. melanotheron  used for the experiment were sourced from the recruitment ponds in the center. They were later sorted and grouped into three based on their sizes. Group 1(Juveniles) were of the size (mean length 12.45cm±1.98SD and mean weight 66.23g±3.04SD), Group 2 (Sub-Adults) were of the size (mean length 15.22cm±3.03SD and mean weight 100.34g±11.00SD) and Group 3 (Adults) were of the size (mean length 19.04cm±6.09SD and mean weight 142.05g±12.54SD). The fish were brought to the lab in six open, 50-liter plastic containers, where they acclimated for seven days.

Preparation of Test Solutions and Exposure of Fish

In the present study, Butachlor was used. Butachlor is an aromatic amide that is 2-choro-N-(2,6-diethylphenyl) acetamide in which the amide nitrogen has been replaced by a butoxymethyl group. It has a role as an herbicide, an environmental contaminant and a xenobiotic. It is an aromatic amide, an organochlorine compound and a tertiary carboxamide. It is functionally related to an N-phenylacetamide. Butachlor is a selective herbicide used worldwide in corn, soybean and other crop cultures. Elevated concentrations of these herbicides and their degradation products have been detected in surface and groundwater.  The herbicide was purchased from a commercial outlet in Port Harcourt, Nigeria. S. melanotheron were exposed to the chemical   at the concentrations of 0.00 (control), 0.05, 0.10, 0.15, and 0.20 mg/L in triplicates. Five fish were randomly distributed into each test tank. The experiment lasted for a period of 15 days. The water in the tanks was renewed daily. The fish were fed twice daily at 3% body weight with a commercial feed.

Determination of blood Plasma Metabolites

A 2ml sample of fresh blood was taken at the conclusion of each experimental period by puncturing the caudal artery with a tiny needle and pouring the sample into heparinized sample vials. Plasma was separated by centrifugation in a TG20-WS Tabletop High Speed Laboratory Centrifuge for 5-8 minutes at 10,000 rpm. Following the guidelines provided by APHA [12], the samples were examined for the metabolites creatinine, total bilirubin, total urea, and total protein. There were three copies of each test run. The methods APHA [12] were also used to determine water quality parameters.

Statistical Analysis

The mean and standard deviation of the mean were used to express all the data. The data analysis was done using SPSS Version 22, a statistical program. Using two-way ANOVA, the means were split, and the two means were deemed significant at 5% (P <0.05).

The parameters of water quality (Table 1) were all within the same range, with the exception of DO, where lower values were recorded at larger chemical concentrations. Table 2 shows how the chemical Butachlor affected the metabolites in the plasma of juvenile S. melanotheron. With rising Butachlor concentrations, it was seen that the values of creatinine, total protein, and total bilirubin levels dropped. When compared to the control values, while the values of urea considerably increased. Additionally, Table 3 shows how the chemical Butachlore affected the metabolites in the plasma of sub-adult sizes of S. melanotheron. With rising Butachlor concentrations, it was seen that creatinine, total protein, and total bilirubin levels dropped. When compared to the control values, however, the values of urea considerably increased. Moreover, the same trends were equally observed in adult of S. melanotheron exposed to the chemical (Table 4).

Table 1: Physico-chemical Parameters of Water in Experimental Tanks (Meant± SD).

Means within the row with different superscripts are significantly different (P<0.05).

Table2: Metabolites Activities in Juveniles of S. melanotheron Exposed to Butachlor in the Laboratory.

Means within the same column with different super scripts are significantly different (P<0.05).

Table3: Metabolites Activities in Sub-Adults of S. melanotheron Exposed to Butachlor in the Laboratory.

Means within the same column with different super scripts are significantly different (P<0.05).

Table 4: Metabolites Activities in Adults of S. melanotheron Exposed to Butachlor in the Laboratory.

Means within the same column with different super scripts are significantly different (P<0.05). 

Metabolite variations can generally be attributed to the influence of water quality parameters, especially in hazardous water. The water used in the experiment has no bearing on the changes and variations in metabolites that occur after the chemical is administered in this inquiry. This is because there was no appreciable difference (P>0.05) between the solution's various concentrations and the control in terms of temperature, salinity, pH, ammonia, and nitrite. The dissolved oxygen readings, however, revealed lower values. The pattern in the markers of water quality is similar to what other authors found when they subjected fish to varying chemical concentrations in a salinity chamber [21]. Blood or plasma's main job is to transport waste materials to various excretory organs for the body to eliminate them, as well as metabolites and nutrients (both organic and inorganic) that are absorbed throughout the body [22].The presence of metabolites in the blood, whether at high or low amounts, is an important clinical correlate. The measurements of total bilirubin dropped as the concentration of the chemical solution increased. A decrease in plasma total bilirubin indicates damage to the liver cells [23]. The investigation's lower bilirubin levels raise the possibility that the liver cells of S. melanotheron were harmed. A decrease in total bilirubin concentrations in the plasma may also be caused by the liver's incapacity to convert bilirubin into bile and urobilin, a disease that results in yellow urine in humans [24].

Urea and creatinine have been used as important indicators for the evaluation of the effects of stress on the kidney using a variety of in-vitro and in-vivo methods [25]. Over the duration of the exposure period, the experimental fish's urea levels increased in proportion to the concentration of the chemical solution, while its creatinine levels declined. The kidney's inability to remove these waste products was indicated by a decrease in glomerular filtration rate and an increase in urea level, according to Calbreath [26]. Fish health is fundamentally indicated by total protein concentration, a non-specific immunological parameter that is critical [27]. The serum protein levels of the experimental fishes dropped in the current study. Total protein levels can drop due to reduced amino transferase activity, changed liver structure, and inadequate fluid balance management. The results of Anyanwu study [28] about Sarotherodon melanotheron exposure to varying salinities are corroborated by this result. The protein content decline was most likely caused by a decrease in or disruption in the synthesis of microsomal proteins. Increased proteolytic activity and the potential application of the outcome for metabolic processes are possibilities brought about by the breakdown of proteins [29]. Because of an increase in the enzyme activity involved in protein synthesis, an increase in protein content may be associated with an increase in protein synthesis, whereas an occasional fall in protein content may result from an increase in proteolysis and a decrease in protein synthesis [30]. However, following stress, scientists have shown that fish protein content decreases.

The findings of this study have shown that different butachlor concentrations significantly affect S. melanotheron metabolites, with a particularly pronounced influence in juvenile fish. This is in line with past studies that demonstrate how stress can alter how fish organs, especially the liver, work inside. In conclusion, the study's reduced total protein levels point to either decreased protein synthesis or protein loss through excretion, both of which are signs of renal issues. In fish exposed to the toxin, a decrease in creatinine levels indicates stress. Elevated urea signifies a problem with the kidneys' ability to eliminate surplus waste. If fish exposed to the poison displayed changes in total bilirubin, it's probable that the liver was spared injury. According to the study's findings, total protein, total bilirubin, creatinine, and urea levels in the plasma of the probe organism may be helpful markers of butachlor's sublethal effects on aquatic life.

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