The accumulation, elimination and effect of heavy metals (as, cd, pb) on cortisol levels in oreochromis sp

study purposes. 5 3.1.2. Validated of Cd , Pb and As analytical method - The linear range of Cd analysis method was from 2 µg/L to 200 mg/L and linear regression was y = 1864.9x + 44.675, with correlation coefficient 0.9999. The limit of detection and limit of quantification were 0.52 and 1.73 µg/L, respectively. Recovery yield of Cd in the spiked Oreochromis sp. samples was almost 94.4%. A method using ICP-OES is proposed that was shown to be sensitive and specific for the determination of Cd in fish. - The treatment sample proceed and the determination of lead using ICP-OES was shown suitable to be sensitive and specific. LOD and LOQ of the method were found to be 1.2 𝜇g/L and 3.9 𝜇g/L, respectively. The recoveries was hight at 92.2%, the precision was less than 5%. The ICP-OES was suitable to determination of trace level of lead in fish. - The treatment sample proceed and the determination of arsenic using ICP-MS was shown suitable to be sensitive and specific. LOD and LOQ of the method were found to be 0.076 𝜇g/L and 0.253 𝜇g/L, respectively. Intra-assay precision levels was between 0.41 and 2.69%. Inter-assay precision levels was between 2.11 and 4.17%. Recovery of arsenic from fish muscle was found to be from 96.0 to 98.6%. The ICP-MS was suitable to determination of trace level of arsenic in fish. 3.2. Arsenic 3.2.1. The 96 h LC50 value of As Fig. 3.2 Mortality rate of Oreochromis sp. exposure to As Fig. 3.5 The accumulation of arsenic in fish gill No Oreochromis sp. fish died in the first 96 h for the control treatment and the group exposed to 20 mgCd/L. In the case of the As 6 concentrations in the waterborne were about 20-40 mg/L, 13.3% to 100% of the fish are died for 96 hours. In this study, the 96 h LC50 value of As for Oreochromis sp. was found to be high level which was approximately 29.26 mg/L. This results demonstrated that inorganic arsenic forms were able to be biotransformed to the less toxic organic asenic forms. The previous studies demonstrated that As was biotransformed to the less toxic monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), and arsenobetaine (AsB) in the Tilapia mossambica fish in the inorganic arsenic (III) waterbone. 3.2.2. Sub-chronic toxicity studies of As to Oreochromis sp. 3.2.2.1. Accumulation of As (exposure phase) Observation of the growth of the control fish showed that fish was not tired, which eat normally while the treatment fish was tired and grew slower than. On day 20 of the exposure time, the weight of the fish from the group exposed to 1-3 mgAs/L only increased by 2.7- 5.7% compared to the beginning of the experiment while the weight of the control fish increased significantly by 19.3% compared to the beginning of the experiment. Accumulation of As in fish gill: As accumulation in the gill of fish was dependent upon the exposure time and exposure dose. As concentrations in the gill of the fish was significantly higher than in the control group (Fig. 3.5). In the group exposed to 1-3 mgAs/L for 4 day, As accumulation in the gill was 0.18 – 0.60 mg/kg. After 20 day of exposured As, the As concentrations in the gill increased from 0.89 to 1.29 mg/kg. Fig. 3.6 The accumulation of arsenic in fish liver Fig. 3.7 The accumulation of arsenic in fish muscle 7 Accumulation of As in fish liver: As accumulation in the liver of fish was dependent upon the exposure time and exposure dose (Fig. 3.6). As accumulation in the liver of treatment fish was increased significantly than control fish. Short-term exposure (4 days) of fish to 1.0-3.0 mgAs/L, As concentration in fish liver was 0.38-0.88 mg/kg. Long-term exposure (20 days) of fish to 1.0-3.0 mgAs/L, As concentration in liver was 1.15-1.80 mg/kg. Accumulation of As in fish muscle: Short-term exposure (4 days) of fish to 1.0-3.0 mgAs/L, As concentration in fish muscle was 0.57- 1.21 mg/kg. Long-term exposure (20 days) of fish to 1.0-3.0 mgAs/L, As concentration in muscle was 2.27 đến 3.01 mg/kg dry weigh. As accumulation in the muscle of fish was dependent upon the exposure time and exposure dose (Fig. 3.7). As accumulation in the Oreochromis sp. tissues were dependent upon the exposure dose and its increased significantly higher than control group. The distribution patterns of As concentrations presented the sequence: muscle > liver > gill. Gill surfaces are the first target for waterborne metals. The gill surfaces are negatively charged and, thus, provide a potential site for positively charged metal accumulation. However, in the present study, a lower concentration of As was found in the gills of Oreochromis sp. compared to liver and muscle. The result might be because the arsenic compounds were in the third oxidation state (AsO2-), which is negatively charged, and thereby have a lower afinity to gill. The As concentrations in the gills of Mystus gulio, Catla catla and Mystus seenghara have also been found to be lower than in muscles. Metal enters to the cells via binding to intracellular ligands (metallothioneins, metallochaperones or metal-binding proteins), or through metal efflux across the basolateral membranes. Lipophilic metal compounds (i.e., metals complexes with hydrophobic ligands) enter fish cells by passive diffusion through the cell membrane. Fig. 3.9 shows that, the ratio 𝐴𝑠𝑓𝑖𝑠ℎ 𝑜𝑟𝑔𝑎𝑛𝑠 𝐴𝑠𝐻2𝑂⁄ is low when the concentration of As in water is high. It might be due to fish exposed to low metals concentration have failed to recognize toxicity, metals enter to the cell via membrane protein transporters and passive diffusion through the cell membrane. Fish exposed to high metals concentration have recognized toxicity, metals enter to the cell mainly 8 via passive diffusion through the cell membrane. Although the concentration of As in fish from the group exposed to high concentration of As in water was significantly higher than that of fish from the group exposed to low concentration of As in water. However, the increases in As concentration in fish organs were not significantly in comparison with the increases in As concentration in the water. Therefore, ratio of 𝐴𝑠𝑓𝑖𝑠ℎ 𝑜𝑟𝑔𝑎𝑛𝑠 𝐴𝑠𝐻2𝑂⁄ decreased as increasing of As concentration in the water. Fig. 3.9 The ratio of As concentration betwen As treatment groups tissue and waterbone Fig. 3.15 The suppression of plasma cortisol levels between As treatment groups and the control group 3.2.2.2. Elimination of As (recovery phase) Observation of the growth of the control fish showed that fish was not tired, which eat normally but the treatment fish was tired and less grew in fresh water. There was no significant difference in weight of fish exposed to As between day 20 of exposure and day 10 of recovery time. On day 10 since fish started recovery time, the body weight of the control fish increased about 12.3% while the weight of fish exposed to As increased less than 1.9%. Table 3.8. The elimination rate of accumulated As from fish organs at day 10 of recovery time Experiment The elimination rate (%) Muscle Liver Gill Treament 1.0As 16.7 24.6 19.1 1.5As 19.3 29.4 18.2 3.0As 12.3 14.7 18.7 9 Concentration of As in all organs of fish exposed to As at day 10 of recovery time significantly decreased. No significant differences in As concentration in the control fish were observed. The weight of treatment fish was not significantly increased. These results also demonstrated that accumulated As was eliminated from Oreochromis sp. fish. The order elimination of accumulated As in fish was liver > gill ≈ muscle. 3.2.2.3. Mechanism of detoxification of As in Oreochromis sp The DMA, AsB and unknown species was measured in liver and muscle of Oreochromis sp. fish. As (III) and As (V) did not present in liver and muscle. These results also demonstrated that, As biotransformation from the inorganic forms to the organic forms in liver and muscle tissues of Oreochromis sp. fish. Fig. 3.10. The chromatography of standard of As species Fig. 3.11. The chromatography of As species in CMR sample Fig. 3.12. The chromatography of As species: a) in muscle and b) liver 3.2.2.4. The effect of As on plasma cortisol levels in Oreochromis sp. Plasma cortisol levels in Oreochromis sp in exposure phase: The plasma cortisol levels in Oreochromis sp. varied widely with increases in As concentration in the water or as time proceeded (Fig. 3.15). On day 4 of the exposure phase, the plasma cortisol levels in Oreochromis sp. were elevated in response to all water As concentrations compared to the control treatment. Exposure to high As concentrations induced 10 a significant rise in the level of plasma cortisol. The plasma cortisol level increased by 21.5%, 36.5% and 51% for the groups exposed to As at 1.0, 1.5 and 3.0 mg/L compared to the control treatment. On day 12 of the exposure phase, the plasma cortisol level in fish exposed to As at 1.0 mg/L still increased compared to day 4 of the exposure phase. In the case of the groups exposed to As at 1.5 and 3.0 mg/L, the plasma cortisol levels declined in comparison to day 4; however, the cortisol levels were still higher than those in the control group. On day 20 of the exposure phase, plasma cortisol levels in Oreochromis sp. in all As treatment groups were significantly reduced in comparison to day 4 levels and lower than those in the control group. The reduction in plasma cortisol levels in Oreochromis sp. between day 20 and day 4 were 14.8%, 25.1% and 33.9%, corresponding to the groups exposed to As at 1.0, 1.5 and 3.0 mg/L. In comparison to the control group, the plasma cortisol level in Oreochromis sp. on day 20 of the exposure phase declined by 11.7%, 16.5% and 18.5% for the groups exposed to As at 1.0, 1.5 and 3.0 mg/L, respectively. Plasma cortisol levels in Oreochromis sp. fish in recovery phase: On day 10 of the recovery phase, plasma cortisol levels in Oreochromis sp. decreased in the group exposed to As at 3.0 mg/L, while they did not change much in the groups exposed to As at 1.0 and 1.5 mg/L. This result indicated that high arsenic concentrations in water had a significant effect on the endocrine systems and could impair the endocrine system. 3.3. Cadmium 3.3.1. The 96 h LC50 value of Cd Fig. 3.17 Mortality rate of Oreo - chromis sp. exposure to Cd Fig. 3.20 The accumulation of Cd in fish gill 11 Fig. 3.17 shows that fish died in all treatment groups within 96 h. Fish mortality increased with increasing Cd concentration in water. No fish died in the first 72 h for the group exposed to 2 mgCd/L, which lasted 96 h, about 7% of fish died. When the Cd concentration in the water is about 5-45 mg/L, 27 to 100% of the fish are died. No control fish died within 96 h. The 96 h LC50 value of Cd for Oreochromis sp. was found to be approximately 19.63 mg/L. 3.3.2. Sub-chronic toxicity studies of Cd to Oreochromis sp. 3.3.2.1. Accumulation of Cd (exposure phase) Cadmium has deleterious effected to Oreochromis sp. fish. The control fish was not tired, which eat normally while the treatment fish was tired and grew slower than. On day 20 of the exposure time, the weight of the fish from the group exposed to 0.66-2.00 mgCd/L only increased by 4.0-6.3% compared to the beginning of the experiment while the weight of the control fish increased significantly by 19.3% compared to the beginning of the experiment. These findings were compatible with observations reported by previous studies, Oncorhynchus mykiss exposed to higher Cd concentrations grew slower than fish exposed to the lower Cd concentrations and the control fish. Fig. 3.21 The accumulation of Cd in fish liver Fig. 3.22 The accumulation of Cd in fish muscle Accumulation of Cd in fish gill: The fish gill acts as an interface between the environment and the blood, especially for continuous diffusion of oxygen, maintaining acid-base and osmotic and ionic regulation. Due to the large surface area, the gills are assumed major sites of heavy metals uptake. During 20 days of exposure to 0.66-2.0 mgCd/L, gill Cd accumulation were about 0.82-1.44 mg/kg dried 12 weight, these values were approximately 10-18 times higher than the control group. Accumulation of Cd in fish liver: Concentration of Cd in Oreochromis sp. liver increased with increases in Cd concentration in water or as time proceeds. Long-term exposure (20 days) of fish to 0.66-2.0 mgCd/L, Cd accumulation in fish liver was 1.84 ± 0.17; 2.06 ± 0.04 và 2.53 ± 0.05 mg/kg. Due to the chemical similarity of Cd, Ca and Zn, Cd easily enters to the cells through the calcium channels or zinc transporter protein. Cd concentration in the liver of Oreochromis sp. was significantly higher than other organs. Accumulation of Cd in fish muscle: Concentration of Cd in Oreochromis sp. organs increased with increases in Cd concentration in water or as time proceeds. After 4, 12 and 20 days exposures to Cd at 0.66, 1.0 and 2.0 mg/L, concentration of Cd in fish muscle reached 0.14-0.19 mg/kg, 0.15-0.24 mg/kg and 0.29-0.39 mg/kg, respectively. Cd accumulation in the Oreochromis sp. tissues were dependent upon the exposure dose and time proceeds. The groups exposed to Cd had significantly higher accumulation of Cd in tissues than the control group. The distribution patterns of Cd concentrations presented the sequence: liver > gill > muscle. Similarly, Kah Hin Low et al. (2011), studies documented that metal accumulation in the liver tissue of Oreochromis sp. in Jelebu, Malaysia was higher than in muscle and gill. Metal enters to the cells via binding to intracellular ligands (metallothioneins, metallochaperones or metal-binding proteins), or through metal efflux across the basolateral membranes. Lipophilic metal compounds (i.e., metals complexes with hydrophobic ligands) enter fish cells by passive diffusion through the cell membrane. Fig. 3.25 shows that, the ratio 𝐶𝑑𝑓𝑖𝑠ℎ 𝑜𝑟𝑔𝑎𝑛𝑠 𝐶𝑑𝐻2𝑂⁄ is low when the concentration of Cd in water is high. It might be due to fish exposed to low metals concentration have failed to recognize toxicity, metals enter to the cell via membrane protein transporters and passive diffusion through the cell membrane. Fish exposed to high metals concentration have recognized toxicity, metals enter to the cell mainly via passive diffusion through the cell membrane. Although the concentration of Cd in fish from the group exposed to high concentration of Cd in water was significantly higher than that of fish 13 from the group exposed to low concentration of Cd in water. However, the increases in Cd concentration in fish organs were not significantly in comparison with the increases in Cd concentration in the water. Therefore, ratio of 𝐶𝑑𝑓𝑖𝑠ℎ 𝑜𝑟𝑔𝑎𝑛𝑠 𝐶𝑑𝐻2𝑂⁄ decreased as increasing of Cd concentration in the water. 3.3.2.2. Elimination of Cd (Recovery phase) On day 10 since fish started recovery time, the body weight of the control fish increased about 12.3% while the weight of fish exposed to Cd increased from 1.9 to 4.7% compared to the beginning of the experiment. Fish exposed to Cd tired, ate, swam slower than control fish. Fish exposed to higher Cd concentrations (1-2 mgCd/L) grew slower than fish exposed to lower Cd concentration (0.66 mgCd/L) and the control fish. This shows that the growth of test fish was dose- dependent. These findings were compatible with observations reported by previous studies; Oncorhynchus mykiss exposed to higher Cd concentrations grew slower than fish exposed to the lower Cd concentrations and the control fish. Fig. 3.25 The ratio of Cd concentration betwen Cd treatment groups tissue and waterbone Fig. 3.29 The suppression of plasma cortisol levels between Cd treatment groups and the control group Table 3.13. The elimination rate of accumulated Cd from fish organs at day 10 of recovery time Experiments The elimination rate (%) Gill Liver Muscle Treatment 0.66Cd 54.3 34.2 48.9 1.0Cd 33.8 26.4 27.8 2.0Cd 46.0 28.1 30.5 14 At day 10 of recovery time, Cd accumulation in the Oreochromis sp. tissues decreased from 26 to 54%, meanwhile the weight of the exposed fish increased less than 4%. The decrease of Cd in fish organs caused the Cd elimination in fish. The order elimination of accumulated Cd in fish was gill > muscle > liver. The elimination of accumulated Cd from fish organs depended mainly on function of organs. Quick decrease of Cd was observed in the gill (33.7- 54.3%) and muscle (27.8 to 48.8%). Cd elimination from liver was slightly slower (26.4 to 34.3%), probably due to their role in the removal of this element from body. 3.3.2.3. Mechanism of detoxification of Cd in Oreochromis sp The Cd concentration in Cd-MT complex in the liver and muscle fish was about 59.8% and 85.3% in comparison with the total Cd concentration. In fish, the liver is an organ where there is continuous accumulation, biotransformation and detoxification of metals through the induction of metal-binding proteins such as metallothioneins (MTs). Cd is reabsorbed by active transport mechanism in the cells of proximal convoluted tubules that are rich in a metal binding protein (MT). It seems that liver is the first organ for detoxification. Cd–MT complexes are transported to the kidney. Then, metallothionein occurs in the kidney as a response of reabsorbing circulatory Cd–MT complex and biosynthesis of renal MT for Cd storage. Table 3.10. The comparison of Cd concentration in MT - Cd complex and total cadmium concentration in tissues treatment fish. Tissue Total of Cd conc. (mg/kg) Cd conc. in MT complex (mg/kg) The ratio of 𝐶𝑑𝑀𝑇−𝑐𝑜𝑚𝑝𝑙𝑒𝑥 𝐶𝑑𝑇𝑜𝑡𝑎𝑙⁄ % Liver 2.536 ± 0.053 1.517 ± 0.108 59.8 Muscle 0.394 ± 0.022 0.336 ± 0.019 85.3 3.3.2.4. The effect of Cd on plasma cortisol levels in Oreochromis sp. Plasma cortisol levels in Oreochromis sp in exposure phase: Plasma cortisol levels in Oreochromis sp. decreased with increasing concentration of Cd in water and increasing number of exposure days (Fig. 3.29). For the long-term exposure (20 days) to Cd, the suppression of cortisol release decreased steadily from 78% to 91% in all exposure groups compared to the control group. This result 15 indicated that Cd had a significant effect on the endocrine system of Oreochromis sp. until the end of the exposure period (20 days). The exposure to chemicals may directly compromise the stress response by interfering with specific neuroendocrine control mechanisms. Some chemicals affect metabolic pathways, which eventually influence neural and internal tissue functions. Cortisol secretion has been found to be affected by waterborne contaminants because they are toxins that target multiple sites along the Hypothalamus Pituitary Interrenal (HPI) axis, resulting in the decreased secretion of adrenocorticotrophic hormone, which in turn has been shown to promote minor cortisol release from interrenal tissue. Similar observations reported that serum cortisol activities of Anguilla rostrata lesueur and Oreochromis mossombicus increased compared to control treatment, meanwhile its decreased in Oncorhynchus mykiss. Plasma cortisol levels in Oreochromis sp.in recovery phase: Within the 10-day recovery period, the plasma cortisol levels elevated by approximately 21.0-64.4% in the Cd treatment groups. This indicated that the affected Oreochromis sp. had recovered from the stress response. 3.4. Lead 3.4.1. The 96 h LC50 value of Pb Fig. 3.31 Mortality rate of Oreochromis sp. exposure to Pb Fig. 3.34 The accumulation of Pb in fish gill Fig. 3.31 shows that fish died in all treatment groups within 96 h. Fish mortality increased with increasing Pb concentration in water and increasing number of exposure days. Gills of fish exposed to lead 16 presented a higher occurrence of histopathological lesions such as epithelial lifting, hyperplasia, and lamellar aneurism which caused death of fish. The 96 h LC50 value of Pb for Oreochromis sp. was found to be approximately 3.24 mg/L. The 96 h LC50 value of Pb for Capoeta fusca and Goldfish was 7.58 mg/L and 5.02 mg/L, respectively. 3.4.2. Sub-chronic toxicity studies of Pb to Oreochromis sp. 3.4.2.1. Accumulation of Pb (Exposure phase) Observation of the growth of the control fish showed that fish was not tired, which eat normally while the treatment fish was tired and grew slower than. The high Pb concentration in waterborne was the less the growth of fish. On day 20 of the exposure time, the weight of the fish from the group exposed to 0.12-0.33 mgPb/L only increased by 5.0-8.7% compared to the beginning of the experiment while the weight of the control fish increased significantly by 19.3% compared to the beginning of the experiment. Accumulation of Pb in fish gill: Concentration of Pb in Oreochromis sp. organs increased with increases in Pb concentration in water or as time proceeds. Pb concentrations in the liver, gill and muscle of the fish were significantly higher than in the control group. On day 20, accumulation of Pb in fish gill from the group exposed to 0.12-0.33 mgPb/L reached to 8.63-9.03 mg/kg dry weight and was about 10.6-11.1 times higher in comparison with the control group. Accumulation of Pb in fish liver: Concentration of Pb in Oreochromis sp. organs increased with increases in Pb concentration in water or as time proceeds. The concentration of Pb in fish liver from the group exposed to 0.12-0.33 mgPb/L on day 20 of exposure time reached to 3.86-5.99 mg/kg dry weight and was about 6.0-9.4 times higher than the control group. Accumulation of Pb in fish muscle: Pb concentrations in muscle of the fish was significantly higher than in the control group. On day 4, the concentration of Pb in fish muscle from the group exposed to 0.12-0.33 mgPb/L reached to 0.13-0.24 mg/kg dry weight. On day 20 of the exposure time, Pb concentration in fish muscle reached from 0.43 to 0.95 mg/kg dry weight, these values were about 6.1-13.6 times higher than the control group. 17 Fig. 3.35 The accumulation of Pb in fish liver Fig. 3.36 The accumulation of Pb in fish muscle Pb accumulation in the Oreochromis sp. tissues were dependent upon the exposure dose and time proceeds. The groups exposed to Pb had significantly higher accumulation of Pb in tissues than the control group. The distribution patterns of Pb concentrations presented the sequence: gill > liver > muscle. Similarly, Kah Hin Low et al. (2011), studies documented that Pb accumulation in the gill tissue of Oreochromis sp. in Jelebu, Malaysia was higher than in muscle and liver. High concentration of Pb in fish gill may be associated with ionic exchange and fish gill can produce mucus, which can serve as a binding site to capture metals. In fish, the liver is an organ of continuous accumulation, biotransformation and detoxification of metals through the induction of metal-binding proteins such as metallothionein (MT). In this study, the accumulation of Pb in Oreochromis sp. muscle was significantly lower than in the liver. This may be due to metabolic activity in fish. Organs with higher metabolic activity, such as the liver, accumulate more metals than those with lower metabolic activity, such as muscle. Metal enters to the cells via binding to intracellular ligands (metallothioneins, metallochaperones or metal-binding proteins), or through metal efflux across the basolateral membranes. Lipophilic metal compounds (i.e., metals complexes with hydrophobic ligands) enter fish cells by passive diffusion through the cell membrane. Fig. 3.38 shows that, the ratio 𝑃𝑏𝑓𝑖𝑠ℎ 𝑜𝑟𝑔𝑎𝑛𝑠 𝑃𝑏𝐻2𝑂⁄ is low when the concentration of Pb in water is high. It might be due to fish exposed to low metals concentration have failed to recognize toxicity, metals enter to the cell via membrane protein transporters and passive 18 diffusion through the cell membrane. Fish exposed to high metals concentration have recognized toxicity, metals enter to the cell mainly via passive diffusion through the cell membrane. Although the concentration of Pb in fish from the group exposed to high concentration of Pb in water was significantly higher than that of fish from the group exposed to low concentration of Pb in water. However, the increases in Pb conc