Investigation of heavy metals and lipid peroxidation level in the muscles of Clarias gariepinus from IBI, Gindin-Dorowa and Donga community rivers in Taraba State, Nigeria

Heavy metals have harmful effects on human health, and exposure to these metals has been increased by industrial and anthropogenic activities and modern industrialization. Heavy metals content of the liver tissues was determined using Atomic Absorption Spectrophotometer method, while lipid peroxidation was carried out. Heavy metals analyzed include; lead (Pb), cadmium (Cd), zinc (Zn), Arsenic (As), and Mercury (Hg). The findings revealed that the heavy metal Zinc (Zn) has high concentrations in the muscles of the fish species, the concentration of this heavy metal Zinc is high in River Gindin Dorowa than in River Ibi and River Donga shows less concentration of this heavy metal though it’s above WHO permissible limits. Results revealed that only Zn and Cd were present in the muscle from the three rivers. Pb was found only in the liver from Gindin-Dorowa at the concentration of 0.017 mg/kg, which is not significant ( P < 0.05) when compared with other locations, while Hg and As were absent in all the muscle samples. The highest concentration of Zn was found in the muscle sample from Gindin-Dorowa (7.450 mg/kg) followed by Ibi (6.16 mg/kg) and the least being Donga (4.365 mg/kg) which are significantly ( P < 0.05) different from one another. However, there was no significant ( P < 0.05) difference among the Cd composition of muscle from Gindin-Dorowa (0.025 mg/kg), Donga (0.024 mg/kg) and Ibi (0.015 mg/kg), respectively. The TBA was found in the hepatic tissue sample from Gidin-Dorowa, which has the highest Zn, Cd and no Pb content, followed by Ibi and then the Donga sample. This suggests that there is a positive relationship between heavy metals and the effect of TBA on the hepatic tissues, justifying the fact that heavy metals affect the hepatic tissues of fish, while on the cerebral tissue. In conclusion, it revealed that there is a negative relation between heavy metals and the effect of TBA on the cerebral tissues to protect or save aquatic habitats of fish quality and aquatic life.


Introduction
Heavy metal exposure has increased as a result of anthropogenic, industrial, and agricultural activity as well as modern industrialization, all of which have negative impacts on human health. This is taken into consideration when assessing the concentration of heavy metals in water resources, air, and food [1]. Therefore, it is inevitable for humans to come into contact with metals, and some research has indicated that the toxicity of metals varies depending on gender [2]. According to several studies by Fernandes Azevedo et al. [3], cumulative effects from concurrent exposure to two or more metals are possible. Continuous exposure to heavy metals, especially mercury and lead, can result in serious side effects like kidney failure, bloody diarrhea, and abdominal cramping [4]. On the other hand, low-dose exposure is a covert and undetectable danger unless it is repeated frequently, at which point its complications, such as neuropsychiatric disorders like fatigue and anxiety as well as negative effects on children's IQ and intellectual function, may be recognized [5]. Arsenic, cadmium, and chromium are carcinogenic metals that may interfere with DNA synthesis and repair [6]. Heavy metal toxicity and carcinogenicity are dose-dependent. According to Gorini et al. [7], high-dose exposure induces severe reactions in both humans and animals that result in increased DNA damage and neuropsychiatric problems.
Lipid peroxidation is thought to be one of the key molecular processes responsible for cell death and oxidative damage to cell structures. Lipid peroxidation results from hazardous metabolites that damage cells rupture intracellular membranes and produce highly reactive species. However, little is known about the presence of heavy metals in the fish from the aforementioned region in Taraba State and whether they are dangerous to people. Lipid peroxidation is thought to be one of the primary molecular processes responsible for cell death by toxicity and oxidative damage to cell structures. Lipid peroxidation results from hazardous metabolites that damage cells, rupture intracellular membranes and produced highly reactive species. According to studies, several heavy metals that are present in high concentrations cause lipid peroxidation processes to start in order to manifest their toxicity. A wide variety of reactive intermediates, including F2-IsoPs and MDA, are produced by the endoperoxides. According to Grimsrud, et al. [8], aldehyde toxicity is caused by changes in a number of cellular processes, which primarily depend on the creation of covalent adducts with cellular proteins. By means of a Michael addition to either a thiol (-SH) or Cys, His, or Lys residues, HNE can create adducts with three distinct amino acyl side chains. According to reports by Slatter et al. [9] and Lamore et al.

Fish preparation
The liver, brain, and muscle tissues of the fish were utilized in this investigation. According to Tuzen [11], fish tissues were chopped and oven-dried at 110°C to a consistent weight, sufficiently dissecting the fish to remove its liver, brain, and muscle, as well as other necessary organs. Based on analytical techniques for atomic absorbance spectrometry, the digestion was completed.

Ashing
Each organ sample weighed 2 grams and was placed in a platinum dish before being placed in a muffle furnace. The temperature was raised to roughly 550 C for 4-5 hours, and once the sample had completely turned to ash, it was removed and allowed to cool in a desiccator.

Samples digestion
25 mL of digesting acid (Aqua regia HCL: HNO3, 3:1) was applied to 2.00 g of the ash-prepared sample material (muscles). Swirl and heat slowly at first to stop frothing, then more vigorously to obtain a clear, pale yellow solution. The mixture was allowed to cool before the digest was placed into a volumetric flask measuring 100 mL, which was then filled to the proper level with distilled water and filtered using Whatman No. 1 filter paper. The filtrate was brought to the AAS (Bulk Scientific, VPG 20, La Vegas, USA), where each desired metal's hollow cathode lamp was installed, and the wavelength characteristics of each heavy metal were set for the measurements using the air acetylene integrated flame mode (all heavy metals). According to Perkin Elmer [12], the calibration curve of the standard was extrapolated to provide the standard for each metal.

Heavy metals analysis
The heavy metals in the muscle tissues was assessed using the AOAC (2019) method.

Preparation of tissue homogenate
Animals were given mild ether anesthesia before being decapitated to end their lives. As a result, the liver and brain were separated, put on ice right away, washed in a suitable buffer solution including protease inhibitors, and then homogenized (1:5 in the liver and 1:8 in the brain) in cold 50 mM Tris-HCl buffer (pH 7.4) at 40 C. A low-speed supernatant (S1) was produced by centrifuging the homogenates at 4,000 rpm for 10 minutes. Both the enzymatic assay and the antioxidant assay utilized the homogenate.

Thiobarbituric acid Reactive Species (TBARS) assay
The modified method of Ohkawa et al. [13] was used to measure TBARS (thiobarbituric acid reactive species), which is a marker of lipid peroxidation. A reaction system containing 50 mM Tris-HCl buffer (pH 7.4), 100 µL of tissue homogenate, and an aliquot of 100 L of S1 were incubated for 1 hour at 370 C in the presence of ebselen (final concentrations range of 0-200 µM). After adding 200 µL of 8.1% SDS (Sodium Dodecyl Sulphate) to the reaction mixture containing S1, the colour reaction was created. Next, 500 µL of acetate buffer, pH 3.4, and 500 µL of 0.8% TBA were added. For 30 minutes, the mixture was incubated at 1000 C. In a UV-visible spectrophotometer, the amount of TBARS generated was measured at 532 nm.

Analysis
After digestion, the concentrations of the heavy metals (Cd, Zn, As, Hg, and Pb) were examined using an atomic absorption spectrometer (Shimazu-AA-t300). The AAS values were given as µg/g, which were then converted to mg/kg in the final results. Analytical-grade reagents were utilized throughout. Results were presented as Mean ± SEM. The proper ANOVA was used to evaluate the data, and then, where necessary, Duncan's multiple range tests. P <0.05, represents a significant difference from the control.

Heavy metals composition of fish muscle samples
The levels of heavy metals found in the muscle of catfish (C. gariepinus) from the rivers Donga, Ibi, and Gindin-Dorowa are displayed in Table 1. Zn and Cd were identified in the muscle from all three rivers, but Pb was only found in the muscle of fish from Gindin-Dorowa at the concentration of 0.017 mg/kg, which is significantly (P <0.05) different from the Pb-free Ibi and Donga muscles. The muscle samples collected from all areas lacked Hg and As. The Zn concentrations in the liver samples varied significantly (P < 0.05), with Gindin-Dorowa having the greatest amount (7.450 mg/kg), followed by Ibi (6.165 mg/kg), and Donga having the lowest level (4.365 mg/kg). Moreso, the Cd compositions of muscle from Gindin-Dorowa (0.025 mg/kg) and Donga (0.024 mg/kg) were significant (P <0.05) compared to Ibi (0.015).

Thiobarbituric acid reactive substances in the fish liver and brain samples
The results of TBARS levels in the hepatic and cerebral tissues of fishes from the three locations are displayed in Figure 1 and 2, respectively. TBARS level in the liver tissue from Gindin-Dorowa is significantly (P < 0.05) higher when compared to Ibi, with Donga showing the least amount ( Figure 1). In addition, there is no significant (P < 0.05) difference in the amount of lipid peroxidation adducts formed in the cerebral tissues from Gindin-Dorowa and Donga with Ibi showing the least amount (Figure 2). Table 1 shows the concentrations of heavy metals in the muscle of catfish (C. gariepinus) from river Donga, Ibi and Gindin-Dorowa. Zn and Cd were present in fish muscles from the three rivers, Pb was found only in the muscle from Gindin-Dorowa at the concentration of 0.017 mg/kg which is not significantly (P < 0.05) different from Ibi and Donga with no Pb. This value is below the permissible limits of 0.02 mg/kg and 0.2 mg/kg reported by the European Union (EU) [14], respectively. Pb is a harmful environmental pollutant that has high toxic effects on many body organs. Exposure to high lead levels can severely damage the brain, and kidney and ultimately cause death Pb can produce alteration in physiological functions of the body and is associated with many diseases [15][16][17]. Pb is highly toxic and has adverse effects on the neurological, biological, and cognitive functions in the body. Hg and As were found to be absent in all the muscle samples from all locations. There was a significant (P < 0.05) difference among the Zn concentrations of the liver samples with the highest level found in Gindin-Dorowa (7.450 mg/kg) followed by Ibi (6.165 mg/kg) and the least being Donga (4.365 mg/kg). This value is lower than the permissible limits (40 mg/kg) according to World Health Organization [18]. Zn accumulates in the gills of fish and creates adverse effects on fish by causing structural damage that affects growth, development, and survival. It also alters fish behavior, hematological parameters, and swimming ability [19]. Moreso, the Cd compositions of muscle from Gindin-Dorowa (0.025 mg/kg) and Donga (0.024 mg/kg) were more significant (P < 0.05) than that of Ibi (0.015 mg/kg) respectively. This value of Cd is below the permissible limits of 0.2 mg/kg [18]. Exposure to Cd, especially chronic, can cause renal dysfunction, calcium metabolism disorders, and increased incidence of some forms of cancer [20]. The present study observed the concentration of heavy metals (Pb, Cd, As, Hg, and Zn) and lipid peroxidation in the fish tissues (liver, brain, and muscles) of Clarias gariepinus from the three freshwater sources in Taraba State. This is in line with Siong et al. [21] that the mobility and availability in aquatic environments are primarily controlled by water quality parameters, including pH, dissolved oxygen, and organic matter content. Likewise, Ani et al. [22] opined that environmental factors such as pH, turbidity, dissolved oxygen, temperature, and conductivity influence the reaction rate of the pollutants entering the water or the lethal effects on the aquatic organisms.

Discussion
Lipid peroxidation mediated by free radicals is considered to be primarily responsible for cell membrane destruction and cell damage leading to tissue injury and failure of the antioxidant defense mechanism [23]. The results of TBARS levels in the hepatic and cerebral tissues of fishes from the three locations are displayed in Figure 1 and 2 respectively. TBARS level in the liver tissue from Gindin-Dorowa is significant (P < 0.05) higher, when compared to Ibi with Donga showing the least amount ( Figure 1). In addition, there is no significantly (P < 0.05) difference in the amount of lipid peroxidation adducts formed in the cerebral tissues from Gindin-Dorowa and Donga with Ibi showing the least amount. Generally, study has revealed that heavy metals can generate free radicals which attack membrane lipids and consequently lead to cell damage [24]. It is, therefore, rational to suggest that the presence of Pb in a sample from Gindin-Dorowa could be responsible for the high-level lipid peroxidation observed.

Conclusion
The present study revealed the presence of Zn and Cd in muscles from the three rivers, Pb only in muscle from Gindin-Dorowa only limits and therefore safe for human consumption Hg and as were found to be absent in all the muscle samples from all locations. Zn was significantly (P < 0.05) higher in Gindin-Dorowa and Ibi (6.165 mg/kg) when compared with Donga, while Cd compositions of muscle from Gindin-Dorowa and Donga were significantly (P < 0.05) higher than that of Ibi. TBARS level in the liver tissue from Gindin-Dorowa is significantly (P < 0.05) higher, when compared to Ibi and Donga, while the TBARS levels in the cerebral tissues from Gindin-Dorowa and Donga were significantly (P < 0.05) higher than Ibi. We suggest that the cumulative effect of heavy metals reported from Gindin-Dorowa sample might be responsible for the high-level lipid peroxidation observed in both the cerebral and hepatic tissue. However, heavy metal concentration from all river samples did not transcend the permissible limits, hence safe for human consumption. The present conditions of the river should therefore be monitored to maintain better fish quality.