Summary of Chemistry doctoral thesis: The accumulation, elimination and effect of heavy metals (As, Cd, Pb) on cortisol levels in Oreochromis sp.

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Research scope and content: Study on Lethal Concentration 50 (LC50 - 96 hours) of As and Cd and Pb in Oreochromis sp.; the accumulation, elimination of heavy metals (As, Cd, Pb) in Oreochromis sp. in exposure phase and elimination phase; the effects of heavy metals (As, Cd, Pb) on plasma cortisol levels in Oreochromis sp.

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MINISTRY OF EDUCATION VIETNAM ACADEMY AND TRAINING OF SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY SCIENCE AND TECHNOLOGY ---------------------------- NGUYEN QUOC THANG THE ACCUMULATION, ELIMINATION AND EFFECT OF HEAVY METALS (As, Cd, Pb) ON CORTISOL LEVELS IN OREOCHROMIS SP. Major: Inorganic Chemistry Code: 9.44.01.13 SUMMARY OF CHEMISTRY DOCTORAL THESIS Ho Chi Minh City - 2018
The dissertation was completed at: Graduate University of Sciences and Technology, Vietnam Academy of Science and Technology; Institute of Applied Materials Science; Industrial University of Ho Chi Minh City, Korea Instite of Toxicology – Gajeong-ro, Yuseong-gu, Daejeon; The Department of Biology and Chemistry - Changwon National University; HoChiMinh City Institute of Resources Geography. Scientific Supervisors: 1. Assoc. Prof. Dr. Le Van Tan 2. Assoc. Prof. Dr. Nguyen Thi Kim Phuong 1 st Reviewer: ........................................................................... .................................................................................................... 2 nd Reviewer: .......................................................................... ................................................................................................... 3 rd Reviewer:........................................................................... ................................................................................. …………….. The dissertation will be defended at Institute of Applied Materials Science, Graduate University of Science And Technology, Vietnam Academy of Science and Technology, 01A, Thanh Loc 29, Thanh Loc ward, 12 District, Ho Chi Minh City. At ….. hour….. date….. month …..2018. The dissertation can be found in National Library of Vietnam and the library of Graduate University of Science And Technology, Vietnam Academy of Science and Technology.
1 INTRODUCTION 1. The necessity of the thesis Heavy metals are particularly significant in ecotoxicology because of their toxicity, persistence, and bioaccumulation in the food chain. Among aquatic organisms, fish are particularly sensitive to many toxicants and are valuable bioindicators for an estimation of the metal pollution level. Fish typically occupy higher positions in the aquatic food chain and may accumulate metals and pass them to human beings through food, which can cause chronic or acute diseases. It is well known that heavy metals easily accumulate in bones, gills, kidneys, and the liver of fish and can cause various detrimental eﬀects. Heavy metals can induce histological changes and can cause changes in enzymatic activity, carbohydrate metabolism and electrolyte levels; they can also introduce metallothionein and reduce glycogen content, leading to disturbances in neuromuscular transmission and contraction, and can alter metabolic functions and impair detoxification mechanisms. The routes of metal uptake in fish are through the gills, the digestive system and the skin. Therefore, the importance of studies of heavy metal bioaccumulation in fish, which represent a valuable source of food for humans, in the context of environmental pollution, seems unquestionable. 2. Objectives Study on the accumulation, elimination of heavy metals (As, Cd, Pb) and its effects on plasma cortisol levels in Oreochromis. sp in waterborne. 3. Research scope and content - Study on Lethal Concentration 50 (LC50 - 96 hours) of As and Cd and Pb in Oreochromis sp. -The accumulation, elimination of heavy metals (As, Cd, Pb) in Oreochromis sp. in exposure phase and elimination phase. - The effects of heavy metals (As, Cd, Pb) on plasma cortisol levels in Oreochromis sp. 4. Structure of the thesis The dissertation has 112 pages, including the Preface, Chapter 1: Overview, Chapter 2: Experiment, Chapter 3: Results and discussions,
2 Conclusions, Publications, with 43 images, 17 tables and 194 references. Chapter 1. OVERVIEW Cadmium belongs to a group of toxic metals which have no function in the physiological processes of human, thus its bioaccumulation in different organs and tissues of living organisms exposed to Cd is a common occurrence. Lead can enter the human and cause several unwanted effects, such as: Disruption the biosynthesis of haemoglobin and anaemia, a rise in blood pressure, kidney damage and subtle abortions, disruption of nervous systems, brain damage. The LC50 values of Pb was vary from species to species and organism age. Main effect of As were coagulation of protein, reaction coenzym to complex, destroys the phosphate. Arsenic combinate with sulfhydryl groups of mitochondrial enzymes and prevents cellular respiration. Inorganic As (III) was the most toxicity. The major routes of toxic chemistry uptake in organism are through the respiratory system, the digestive system and the skin. Metals enter to the cell via membrane protein transporters and passive diffusion through the cell membrane. The accumulation of heavy metals in fish tissue is dependent on numerous of factors including water concentrations of metals, exposure period, water temperature, oxygen concentration, pH, hardness, salinity, alkalinity and dissolved organic carbon, …The elimination of metals from the tissues depends on time, age, metabolic activity, interacting agents, the biological half- life of the metals and aquatic species. Toxic chemistry got into the other organs from the blood, in here, it was bio-transformed, eliminated or accumulated. The rate of elimination depend on toxic chemistry concentration, metabolic activities in fish and depended on metal species. Cortisol is the most potent and abundant glucocorticoid secreted by the outer cortex of the adrenal gland. Cortisol plays an important role in mobilizing fuels such as glucose, lipids, and fatty acids, for the maintenance of homeostasis. Additionally, it exerts direct and indirect effects on an organism’s intermediary metabolism, particularly in response to stress. In some cases, stress can lead to
3 disease and/or death. Factors such as water pollution, grading, handling, transportation, and vaccination cause stress for fish. Cortisol is one of the most common stress indicators in fish. In recent years, accumulation of heavy metals in aquatic organism have attracted the intention of many research groups. Mohammed Aldoghachi Jasim Aldoghachi (2016), Kah Hin Low (2011), Abdulali Taweel (2011) Mohammed Aldoghachi Jasim Aldoghachi et al. (2016) reported on heavy metal accumulation in fish living in the natural environment. Several previous studies documented that metal accumulation in the liver tissue was higher than in muscle (Elzbieta Kondera, 2014, El-Moselhy KM, 2014, Canli M, 2003). Many authors have reported that gill have a high tendency to accumulate heavy metals (Wong et al. 2001, Altındağ and Yiğit, 2005, Karadede-Akin and Ünlü, 2007). In Vietnam, several studies have been conducted on the accumulation of heavy metals in plants and bivalve mollusk. No research has been done to study heavy metals accumulation and biochemical changes in fish. In general, until now, no work has been done to study on the accumulation, elimination and eﬀects of heavy metals (As, Cd, Pb) on the biochemical composition of Oreochromis sp. Chapter 2. EXPERIMENT 2.1. LC50 test Oreochromis sp. were exposed to waterborne arsenic or cadmium or lead at each concentration for 96 h. Thirty fish were randomly assigned to each treatment, and each treatment consisted of three tanks with ten fish per tank. Fish mortality was recorded after 0, 24, 48, 72, and 96 h of exposure to heavy metal solution. LC50 value was calculated by simple graphic method. 2.2. Sub-chronic exposure Oreochromis sp. were divided into seven treatments, which were exposed to control water (control treatment) and water contaminated with diﬀerent concentrations of arsenic or cadmium or lead (3.5%, 5%, and 10% of 96 h LC50): - As concentrations: 1.00 mg/L; 1.50 mg/L and 3.00 mg/L. - Cd concentrations: 0.66 mg/L; 1.00 mg/L and 2.00 mg/L. - Pb concentrations: 0.12 mg/L; 0.18 mg/L and 0.33 mg/L.
4 Seventy-five fish were randomly assigned to each treatment; each treatment consisted of three tanks with twenty-five fish per tank (n = 3). Water samples for total metal analysis were collected twice a week and analyzed by atomic spectrometry. Fish were sampled between 8:00 AM to 9:00 AM in all treatments to minimize variations in the hormonal responses caused by diel endocrine cycles. On days 4, 12, and 20, fish were washed with bi-distilled water before blood and tissue sampling. Blood samples were taken from the caudal vasculature. Whole blood samples were centrifuged at 5500 rpm for 5 min to obtain plasma samples before they were analyzed for their cortisol concentrations. Muscle, gill and liver tissues were collected from Oreochromis sp. fish randomly selected from each group. The tissues were dried in an oven at 105 °C for 10 h before analysis of As or Cd or Pb concentration. As analyses were carried out using ICP- MS 7700 (JP 13052271, Agilent, USA). Cd and Pb analyses were carried out using ICP-OES (Optima 2100 DV, Perkin Elmer). Cortisol was carried out using HPLC-DAD. HPLC-UV-HG-AAS was using to determination of the species of arsenic. 2.3. Statistical analysis Statistical analyses were performed using the Statistical Package for Social Sciences (SPSS) version 22.0. Data were expressed by mean ± SD. Diﬀerences between means were evaluated by one-way analysis of variance (ANOVA). Statistical signifcance of the diﬀerences between the means was assessed by Duncan’s test, and p < 0.05 was considered signifcance. Chapter 3. RESULTS AND DISCUSSIONS 3.1. Validated of analytical methods 3.1.1. Validated of cortisol analytical method Cortisol elutes as a sharp symmetrical peak at about 7.442 min. The limit of detection (LOD) and limit of quantification (LOQ) of the method were found to be 0.87 µg/L và 2.90 µg/L, respectively. The recovery was from 91.2% to 94.5%. The RSD precision ranged 1.1 to 2.6% respectively. A method using RP-HPLC/UV is proposed that was shown to be sensitive and specific for the determination of plasma cortisol for environmental toxicology 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 Fig. 3.5 The accumulation of Oreochromis sp. exposure to As 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 Fig. 3.7 The accumulation of arsenic in fish liver 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 Fig. 3.15 The suppression of plasma concentration betwen As cortisol levels between As treatment treatment groups tissue and groups and the control group waterbone 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 The elimination rate (%) Experiment Muscle Liver Gill 1.0As 16.7 24.6 19.1 Treament 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 Fig. 3.11. The chromatography standard of As species 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 - Fig. 3.20 The accumulation of chromis sp. exposure to Cd 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 Fig. 3.22 The accumulation of fish liver 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 Fig. 3.29 The suppression of concentration betwen Cd treatment plasma cortisol levels between groups tissue and waterbone 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 The elimination rate (%) Experiments Gill Liver Muscle 0.66Cd 54.3 34.2 48.9 Treatment 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. Cd conc. in The ratio of Total of Cd Tissue MT complex 𝐶𝑑𝑀𝑇−𝑐𝑜𝑚𝑝𝑙𝑒𝑥 ⁄𝐶𝑑 𝑇𝑜𝑡𝑎𝑙 conc. (mg/kg) (mg/kg) % 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 Fig. 3.34 The accumulation of Oreochromis sp. exposure to Pb 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 Fig. 3.36 The accumulation of fish liver 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 concentration in fish organs were not significantly in comparison with the increases in Pb concentration in the water. Therefore, ratio of Pb𝑓𝑖𝑠ℎ 𝑜𝑟𝑔𝑎𝑛𝑠 ⁄Pb𝐻2𝑂 decreased as increasing of Pb concentration in the water. Fig. 3.38 The ratio of Pb Fig. 3.40 Plasma cortisol levels concentration betwen Pb treatment in Oreochromis sp. groups tissue and waterbone 3.4.2.2. Elimination of Pb (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 Pb increased from 2.9 to 4.0% compared to the beginning of the experiment. Fish exposed to Pb were tired, ate, swam slower than control fish. Fish exposed to higher Pb concentrations grew slower than fish exposed to lower Pb concentration and the control fish. The decreased of Pb concentration in tissues of treatment fish was greater than the increased of fish weight. This result indicated that Oreochromis sp. had elimination of lead in tissues. Depuration of accumulated Pb from organs during exposure of 20 days depended mainly on tissue. The order of Pb elimination in the tissues during depuration was gills > livers > muscles. Quick decrease of Pb was observed in the gills (14.18% - 39.26%) and livers (14.64% - 26.71%). Pb elimination from muscles was slightly slower (14.68% - 18.00%),