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Journal of Stress Physiology & Biochemistry, Vol. 18, No. 2, 2022, pp. 84-92 ISSN 1997-0838 Original Text Copyright © 2021 by Joseph and John
ORIGINAL ARTICLE
Stress Induced Biochemical Changes in the Liver of Oreochromis mossambicus Exposed to Pulp and
Papermill Effluent
Kiran Joseph1 and Elizabeth John2
1 Department of Zoology, Catholicate College, Pathanamthitta-689 645, Kerala, India
2 Department of Zoology, St. Gregorios College, Kottarakkara-691 531, Kollam (Dist.), Kerala, India
*E-Mail: kirankj007@gmail. com
Received January 11, 2022
The stress induced effect of pulp and papermill effluent in the fish, Oreochromis mossambicus was investigated in the present study. Pulp and papermill effluent discharged from Hindustan News Print LTD, Peruva, Kottayam (Dist), Kerala was collected from the discharging point and used for the study.
The adult Oreochromis mossambicus of size 15 ± 2 g were exposed to a sublethal concentrations (1/5th and 1 /10th LC50 value) of the effluent for a period of 10, 20 and 30 days. biochemical parameters in the liver such as carbohydrate, protein, lipid and enzymes were analysed. Energy yielding nutrients such as carbohydrates, proteins and lipids were decreased tremendously for the study as the concentration and duration of the effluent exposure increased. Increased activity of enzymes such as GOT, GPT and LDH showed that the fish was under stress when exposed to pulp and papermill effluent. Fish exposed to papermill effluent showed reduced activity of ACP and ALP as the duration of exposure and concentration increased.
Key words: biochemical studies, liver, Oreochromis mossambicus, pulp and papermill effluent
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The rapid industrial growth throughout the world is common and in India, particularly due to alarming rise in human population caused tremendous environmental pollution. The aquatic environment is severely affected by different types of chemicals which are toxic to the aquatic organisms (Kopecka et al., 2006). The freshwater organisms particularly fishes are more susceptible to these pollutants, since, their habitats are confined and escape from such polluted habitats is impossible (Pathan et al., 2009)
Fishes are widely used to evaluate the health of aquatic systems and their biochemical and physiological changes serve as biomarkers of environmental pollution (Lavado et al., 2006). Pollutants are entering into fish through five main routes: via food or non-food particles, gills, oral consumption of water and the skin. On absorption, the pollutant is carried in blood stream to either a storage point or to the liver for transformation. Pollutants reached in the liver may be stored or excreted through bile or transported to other excretory organs such as gills or kidneys for elimination (Nussev et al., 2000).
Biochemical studies are good parameters which help to see the effect of pollutants on biochemical composition of vital tissue of fish. For the determination of both exposure and effect of pollutant on an organism biochemical alteration can serve as markers (Anel Joubert, 2000). The liver is considered to be a principal organ of detoxification in vertebrates and particularly in fish. It is also the potential site for lipid deposition in these animals (Freeman et al., 1983). Fish liver is a good indicator of aquatic environmental pollution. One of the important functions of the liver is to clean any poisons or pollutants from the blood coming from the intestine (Saleh, 1982).
Keeping the information in view, the present study has been undertaken with the following objectives such as to study the effect of pulp and paper mill effluent on estimation of Carbohydrate, Protein, Lipid, Glutamate Oxaloacetate Transaminase (GOT), Glutamate Pyruvate Transaminase (GPT), Lactate Dehydrogenase (LDH), Acid Phosphatase (ACP) and Alkaline Phosphatase
(ALP).
MATERIALS AND METHODS
For the analysis of biochemical and enzymatic parameters, fishes from control and sublethal conditions, were sacrificed after the exact periods (10, 20 and 30 days). For biochemical analysis, liver tissues was dissected out from the control and experimental fish. The tissues were weighed and centrifuged at 4,000 rpm using double distilled water. The supernatant was stored at 20C for analytical purpose. Colorimetric determination was performed using BTR - BIOTRONE 810(German) semi auto analyzer. Estimation of Carbohydrate
Carbohydrate was analysed using carbohydrate test kit (AGAPPE Diagnostics, India) based on GOD - PAP methodology (Trinder, 1969). Blank is prepared by taking 1000|l working reagent. Standard is prepared by adding 1000|l working reagent and 10|l standard. Sample is prepared by mixing 1000|l working reagent and 10|l sample. Incubate at 370C for 10 minutes and measure the absorbance of the sample and standard against the reagent blank at 505nm. Calculation:
Carbohydrate concentration (mg/dl) = Absorbance of sample x 100
Estimation of Protein
Total protein was analysed using protein test kit (AGAPPE Diagnostics, India) based on direct biuret method (Gomall, 1949).1000|l total protein reagent serves as the blank. Standard is prepared by adding 1000|l total protein reagent and 20|l standard. Sample is prepared by mixing 1000|l reagent and 20|l sample. Incubate for 10 minutes at 370C. Measure the absorbance of standard and sample against reagent blank.
Calculation:
Total protein Concentration (gm/dl) = Absorbance of sample x 6
Estimation of lipid
Total Lipid was analysed using lipid test kit
(AGAPPE Diagnostics, India) based on CHOD - PAP methodology (Allain, 1974). Blank is prepared by taking 1000|l working reagent. Standard is prepared by adding 1000|l working reagent and 10|l standard. Sample is prepared by mixing 1000|l working reagent and 10|l sample. Incubate at 370C for 5 minutes and measure the absorbance of he sample and standard against the reagent blank at 630nm.
Calculation:
Lipid concentration (mg/dl) = Absorbance of sample x 200
Estimation of Glutamate Oxaloacetate Transaminase (GOT)
Glutamate Oxalo Transaminase was analysed with GOT test kit (AGAPPE Diagnostics, India) based on IFCC methodology (Anonymous, 1976). Working reagent is prepared by mixing GOT reagent 1 and GOT reagent 2 (1000|l) and mixed with 100|l sample. Incubate at 370C for 1 minute. Measure the change in absorbance/minute (AOD/min.) during 3 minutes at 340nm.
Calculation:
GOT activity (U/L) = (AOD/min.) x 1768. Estimation of Glutamate Pyruvate Transaminase (GPT)
Glutamate Phosphate Transaminase was analysed in liver, kidney, gill, gonads and muscle with GPT test kit (AGAPPE Diagnostics, India) based on IFCC methodology (Anonymous, 1976). Working reagent is prepared by mixing GPT reagent 1 and GPT reagent 2 (1000|l) and mix with 100|l sample. Incubate at 370C for 1 minute. Measure the change in absorbance/minute (AOD/min.) during 3 minutes at 340nm.
Calculation:
GPT activity (U/L) = (AOD/min.) x 1768. Estimation of Acid Phosphatase (ACP)
Acid Phosphatase was analysed in liver, kidney, gill, gonads and muscle with ACP test kit (AGAPPE Diagnostics, India) based on alpha - naphthylphosphate method (Hillman, 1971). Working reagent is prepared by dissolving one tablet citrate buffer with 2ml naphthylphosphate reagent. Add 1000 |l working
reagent to 100 ц1 sample to analyse Total Acid Phosphatase. Incubate for 5 minutes at 370C. Measure the change in absorbance per minute (AOD/min.) during 5 minutes.
Calculation:
Total acid phosphatase (U/L) = 750 x (AOD/min.) Estimation of Alkaline Phosphatase (ALP)
Alkaline Phosphatase was analysed in liver, kidney, gill, gonads and muscle with ALP test kit (AGAPPE Diagnostics, India) based on DGKC - SCE recommended procedure with Diethanolamine buffer (Klin,1972). Working reagent is prepared by reconstituting nitrophenyl phosphate reagent with the volume of diethanolamine reagent. Add 1000 ц1 working reagent to 20 ц1 sample to analyse. Incubate for 1 minute at 370C. Measure the change in absorbance per minute (AOD/min.) during 3 minutes.
Calculation:
ALP activity (U/L) = 2750 x (AOD/min.) Estimation of Lactate Dehydrogenase (LDH)
LDH was analysed using LDH test kit (AGAPPE Diagnostics, India) based on SCE recommended method (Wei Bhaar, 1975). Add 1000ц1 working reagent with 10ц1 sample, mix and incubate at 370C for 1 minute. Measure the change in absorbance per minute (AOD/min.) during 3 minutes at 340nm.
Calculation:
LDH activity (U/L) = (AOD/min.) x 16030.
RESULTS
The effect of pulp and papermill effluent on biochemical constituents in the liver, of Oreochromis mossambicus was studied at two sublethal concentrations for long term exposure (10, 20 and 30 days). 1/5th LC50 of the effluent was 0.546% whereas 1/10th of LC50 of the effluent was 0.273%. Carbohydrate
The carbohydrate level was found to be 57.86, 56.41 and 54.65mg/g during exposure period. Where as in sub lethal concentration 0.546% (1/5th LC50 of 96 hrs) the values were noted as 51.82, 49.61 and 47.71mg/g. When compared to control fishes the percentage of
decrease in the concentration of 0.273% was ranged between -6.28% to -10.14%, whereas in 0.546% concentration it was ranged between -16.07% to -21.56%. The carbohydrate levels in the liver of exposed fishes were decreased when compared to control. (Table- 1). Proteins
The level of protein in the liver of the fish groups exposed to 0.273% (1/10th LC50 concentration) of effluent for 10, 20 and 30 days was found to contain 270.77, 256.28 and 241.36 mg/g. In the case of 0.546% (1/5tl LC50 concentration) of effluent the values were noted as 246.82, 221.63 and 203.42 mg/g when exposed to 10, 20 and 30 days respectively. When compared to control, the percentage of decrease noticed in 0.273% (1/10th LC50 concentration) was -2.02% to -10.70%, whereas for 0.546% (1/5th LC50 concentration) it was from -10.68% to -24.73%. (Table-2). Lipid
The lipid level in the liver of the fish groups exposed to 0.273% (1/10th LC50 concentration) of effluent for 10, 20 and 30 days was found to contain 9.990, 9.740 and 9.410 mg/g. In the case of 0.546% (1/5th LC50 concentration) of effluent the values were 9.580, 9.215 and 8.800 mg/g when exposed to 10, 20 and 30 days respectively. When compared to control, the percentage of decrease noticed in 0.273% (1/10th LC50 concentration) was -5.04% to -10.11%, whereas for 0.546% (1/5th LC50 concentration) it was from -8.94% to -15.93%. (Table- 3).
Glutamate oxaloacetate transaminase (GOT)
The fish groups exposed to 0.273% (1/10th LC50 concentration) of effluent for 10, 20 and 30 days the level of GOT in the liver was recorded as 13.26, 14.32 and 15.86 IU/min/g. In the case of 0.546% (1/5th LC50 concentration) of effluent the GOT values were 16.31, 18.26 and 20.24 IU/min/g on exposure to 10, 20 and 30 days respectively. When compared to control, the percentage of increase of GOT noticed in 0.273% (1/10th LC50 concentration) was 29.87% to 46.18%, whereas in 0.546% (1/5th LC50 concentration) it was from 59.75% to 86.54%. (Table-4).
Glutamate pyruvate transaminase (GPT)
The fish groups exposed to 0.273% (1/10th LC50 concentration) of effluent for 10, 20 and 30 days the GPT level in the liver was recorded as 10.99, 12.56 and 15.54 IU/min/g. In 0.546% (1/5th LC50 concentration) of effluent the values were 11.44, 18.21 and 24.53 IU/min/g on exposure to 10, 20 and 30 days respectively. Percentage increase over control noticed in 0.273% (1/10th LC50 concentration) was 9.46% to 52.50%, whereas for 0.546% (1/5th LC50 concentration) it was from 13.94% to 140.73%. (Table-5). Lactate Dehydrogenase (LDH)
The LDH level in the liver of the fish groups exposed to 0.273% (1/10th LC50 concentration) of effluent for 10, 20 and 30 days was recorded as 129.20, 139.50 and 143.76 IU/min/g. In the case of 0.546% (1/5th LC50 concentration) of effluent the values were 131.64, 152.83 and 169.53 IU/min/g on exposure to 10, 20 and 30 days respectively. Percentage of increase noticed in 0.273% (1/10th LC50 concentration) was 3.96% to 15.43%, whereas in 0.546% (1/5th LC50 concentration) it was from 5.92% to 36.12%. (Table- 6). Acid Phosphatase (ACP)
The ACP level in the liver of the fish groups exposed to 0.273% (1/10th LC50 concentration) of effluent for 10, 20 and 30 days was recorded as 4.86, 3.48 and 3.21 IU/min/g. In 0.546% (1/5th LC50 concentration) of effluent the values were 3.89, 2.34 and 2.00 IU/min/g on exposure to 10, 20 and 30 days respectively. When compared to control, the percentage of decrease noticed in 0.273% (1/10th LC50 concentration) was -4.89% to -36.44%, whereas for 0.546% (1/5th LC50 concentration) it was from -23.87% to -60.40%. (Table-7). Alkaline Phosphatase (ALP)
The ALP level in the liver of the fish groups exposed to 0.273% (1/10th LC50 concentration) of effluent for 10, 20 and 30 days was recorded as 37.94, 35.36 and 28.58 IU/min/g. In 0.546% (1/5th LC50 concentration) of effluent the values were 34.47, 31.00 and 27.05 IU/min/g on exposure to 10, 20 and 30 days respectively. When compared to control, the percentage of decrease noticed in 0.273% (1/10th LC50 concentration) was -3.31% to -27.86%, whereas in 0.546% (1/5th LC50 concentration) it
was from -12.16% to -31.74%. (Table-8).
Table 1. The level of Carbohydrate (mg/g) in the Liver of Oreochromis mossambicus exposed to two sublethal concentrations for 10, 20 and 30 days (long term).
Duration (Days) Control (mg/g) Concentration of Effluent
0.546% (1/5th of LC5q ) 0.273% (1/10th of LC5q )
Experiment (mg/g) %difference over control Experiment (mg/g) % difference over control
10 61.74±0.18 51.82±0.51 -16.07% 57.86±0.64 -6.28%
20 61.25±0.21 49.61±0.34 -19.00% 56.41±0.52 -7.90%
30 60.82±0.42 47.71±0.38 -21.56 54.65±0.48 -10.14%
Table.2-The level of Protein in the Liver (mg/g) of Oreochromis mossambicus exposed to two sublethal concentrations for 10, 20 and 30 days (long term)
Duration (Days) Control (mg/g) Concentration of Effluent
0.546% (1/5th of LCsq ) 0.273% (1/10th of LCsq )
Experiment (mg/g) %difference over control Experiment (mg/g) %difference over control
10 276.34±0.55 246.82±0.75 -10.68% 270.77±0.35 -2.02%
20 267.91±0.45 221.63±0.80 -17.27% 256.28±0.60 -4.34%
30 270.27±0.52 203.42±0.50 -24.73% 241.36±0.30 -10.70
Table.3-The level of Lipid (mg/g) in the Liver of Oreochromis mossambicus exposed to two sublethal concentrations for 10, 20 and 30 days (long term)
Concentration of Effluent
Duration (Days) Control (mg/g) 0.546% (1/5th of LCsg ) 0.273% (1/10th of LC50 )
Experiment (mg/g) %difference over control Experiment (mg/g) %difference over control
10 10.520±0.35 9.580±0.52 -8.94% 9.990±0.48 -5.04%
20 10.501±0.45 9.215±0.32 -12.25% 9.740±0.25 -7.25%
30 10.468±0.43 8.800±0.28 -15.93% 9.410±0.17 -10.11%
Table.4-The level of GOT (IU/min/g) in the Liver of Oreochromis mossambicus exposed to two sublethal concentrations for 10, 20 and 30 days (long term)
Duration (Days) Control (IU/min/g) Concentration of Effluent
0.546% (1/5th of LC5g ) 0.273% (1/10th of LC50 )
Experiment (IU/min/g) %difference over control Experiment (IU/min/g) %difference over control
10 10.21±0.15 16.31±0.18 59.75% 13.26±0.12 29.87%
20 10.52±0.14 18.26±0.20 73.54% 14.32±0.14 36.12%
30 10.85±0.10 20.24±0.15 86.54% 15.86±0.17 46.18%
Table.5-The level of GPT (IU/min/g) in the Liver of Oreochromis mossambicus exposed to two sublethal concentrations for 10, 20 and 30 days (long term)
Concentration of Effluent
Duration (Days) Control 0.546% (1/5th of LC5q) 0.273% (1/10th of LC50 )
(IU/min/g) Experiment (IU/min/g) %difference over control Experiment (IU/min/g) %difference over control
10 10.04±0.25 11.44±0.43 13.94% 10.99±0.29 9.46%
20 10.20±0.31 18.21±0.48 78.53% 12.56±0.36 23.14%
30 10.19±0.37 24.53±0.51 140.73% 15.54±0.44 52.50%
Table.6-The level of LDH (IU/min/g) in the Liver of Oreochromis mossambicus exposed to two sublethal concentrations for 10, 20 and 30 days (long term)
Duration (Days) Control (IU/min/g) Concentration of Effluent
0.546% (1/5th of LCsq ) 0.273% (1/10th of LC50)
Experiment (IU/min/g) %difference over control Experiment (IU/min/g) %difference over control
10 124.28±0.54 131.64±0.68 5.92% 129.20±1.02 3.96%
20 122.13±0.61 152.83±0.71 25.14% 139.50±1.07 14.22%
30 124.54±0.57 169.53±0.92 36.12% 143.76±1.12 15.43%
Table.7-The level of ACP (IU/min/g) in the Liver of Oreochromis mossambicus exposed to two sublethal concentrations for 10, 20 and 30 days (long term)
Concentration of Effluent
Duration (Days) Control (IU/min/g) 0.546% (1/5th of LC50) 0.273% (1/10th of LC50)
Experiment (IU/min/g) %difference over control Experiment (IU/min/g) %difference over control
10 5.11±0.12 3.89±0.20 -23.87% 4.86±0.14 -4.89%
20 5.09±0.16 2.34±0.31 -54.03% 3.48±0.17 -31.63%
30 5.05±0.19 2.00±0.25 -60.40% 3.21±0.22 -36.44%
Table.8-The level of ALP (IU/min/g) in the Liver of Oreochromis mossambicus exposed to two sublethal concentrations for 10, 20 and 30 days (long term)
Duration (Days) Control (IU/min/g) Concentration of Effluent
0.546% (1/5th of LC50) 0.273% (1/10th of LC50)
Experiment (IU/min/g) %difference over control Experiment (IU/min/g) %difference over control
10 39.24±0.22 34.47±0.23 -12.16% 37.94±0.25 -3.31%
20 39.59±0.18 31.00±0.28 -21.70% 35.36±0.33 -10.68%
30 39.62±0.15 27.05±0.11 -31.74% 28.58±0.40 -27.86%
DISCUSSION
Fishes were exposed to different sub lethal concentrations for long term exposure, the biochemical parameters like Carbohydrate, Protein, Lipid, ACP and ALP showed significant decrease in relation to control. Similarly, enzymes like GOT, GPT and LDH showed
acceleration and inhibition of their activities increase in effluent concentration and exposure duration. Carbohydrate
Carbohydrates are important components of biological system. During stress condition, fish need more energy to detoxify the toxicants and to overcome
stress. In the present investigation, significant decrease in the carbohydrate content has been noticed in the liver when the exposure duration of effluent is increased. The percentage of decrease was more pronounced in fish groups exposed to higher exposure period of 1/5th concentration. The decrease in carbohydrate is mainly due to the hepatic synthesis of detoxifying enzymes requires high energy levels (Hori et al., 2006). Protein
Protein is one of the main sources of energy. The quantitative determination of total protein reflects the liver capacity of protein synthesis and denotes the osmolarity of the blood and the renal impairments. The data showed that the protein content was decreased in the liver, of the fish when fishes were exposed to long term in two sub lethal concentrations of the effluent. The decrease in protein content was dose dependent as well. The depletion in the proteins may be due to impaired or low rate of protein synthesis, their utilization in cell repair and organization and the decrease in uptake of amino acids into the polypeptide chain (Rajamanickam and Muthuswamy, 2008). Lipid
The direction of change in the lipid parameter seems to be dependent on types of contaminants, the concentration, mode of its action, duration of exposure and fish species (Heath, 1991). The results of the present study showed a significant decrease in the lipid content of liver in both concentrations. As the concentration and exposure duration was increased, the lipid content was decreased. This may be due to increased utilization of lipid to meet additional energy requirements under stress of low oxygen take up. Similar findings are also observed by Kulkarni and Suniti Dharawadkar (1998).
Glutamate oxaloacetate transaminase (GOT)
GOT is an enzyme involved in transamination reactions and present in most of the tissues which shift metabolic cycle from amino acids to alpha keto acids which enter into Kreb's cycle and hence cause the acceleration or deceleration of the intermediary metabolism. The results of the present study showed increase in the GOT activity in the liver tissues. This
may be due to decreased metabolic activity, destruption of enzyme system by blocking the active sites and tissue damage (Bhatanagar and Tyagi, 1995). Glutamate pyruvate transaminase (GPT)
GPT is another transaminase enzyme involved in transamination reactions. GPT activity is high in liver tissues and hence the level increases markedly in acute liver disorders when the liver cells are damaged. In the present study on exposure to pulp and papermill effluent, the GPT level was increased in liver. The increase in GPT activity may indicate anaerobic nature of carbohydrate metabolism in fish, possibly to meet the increased energy demands under sustained and prolonged toxic stress as supported by Ramana Rao et al., (1990).
Lactate Dehydrogenase (LDH)
LDH is an active enzyme of glucose metabolism and any effect on its activity directly affects the energy production of the cell.
The LDH activity in the fish groups exposed to the effluent showed remarkable increase in the liver tissues. As the time of exposure and concentration of effluent increased, the activity also increased. Increased activity of LDH in the tissues could be because of increased secretion by the specialized cells or by changes in the permeability of their cell membranes to stop leach out of the enzymes and keep stored in the cells as stated by Abdel-Hameid (2009). Acid Phosphatase (ACP)
Acid phosphatases are hydrolytic lysosomal enzymes and are released by the lysosomes for the hydrolysis of foreign material and hence it has a role in certain detoxification functions (Jaroli and Sharma, 2005). The fishes were exposed to pulp and paper mill effluent showed remarkable decrease in the ACP activity as the days of exposure and concentration of effluent increased. This pattern of decrease in ACP activity was noticed in liver tissues. The decreased ACP activity indicates the disturbance in the structure of cell organelles, like endoplasmic reticulum and membrane transport system. Such damage to cell organelles has been reported by (Nchumbeni et al., 2007).
Alkaline phosphatase (ALP)
ALP is composed of several isoenzymes that are present in practically all tissues of the body, especially in cell membranes. In the present study, the liver tissues of the effluent exposed fish, the ALP level was found to be reduced. This reduction was time dependent. The ACP level was decreased in 1/5th concentration when compaired to 1/10th concentration of LC50. The decrease in the ALP level in the present study may be attributed to the accumulation of toxicants in the tissues of the fish (Humtsoe et al., 2007), which interfere the synthesis of enzyme protein and increased metabolism due to an increase of toxic substances and the production of toxic metabolic products destructive to the enzymes.
ACKNOWLEDGMENTS
It gives me immense pleasure to express my deep sense of gratitude and indebtedness to Dr. K. Shanthi, Associate Professor (Rtd.) department of Zoology, Kongunadu Arts and Science College, Coimbatore and Fr. G. P. Mathen, Lecturer (Rtd.), Department of Zoology, St. Stephen's College, Pathanapuram, Kerala for the help, encouragement and moral support rendered by him throughout the study period.
CONFLICTS OF INTEREST
The authors declare that they have no potential conflicts of interest.
REFERENCES
Abdel-Hameid, N.A.H. (2009). A Protective Effect of Calcium Carbonate against Arsenic Toxicity of the Nile Catfish, Clarias gariepinus. Turk. J. Fish. Aquat. Sci., 9: 191-200. Allain,C.C. (1974). Enzymatic determination of total
serum cholesterol. Clin. Chem., 20: 470-475. Anel Joubert, (2000). Evaluation of Biomarkers for Pollution In Orechromis mossambicus. Master of Science Thesis, Faculty of Science, Rand Afrikaans University, South Africa. Anonymous, (1976). Expert panel on enzyme of the
IFCC. Clin.Chem. Acta., 70, F19. Bhatnagar, M.C. and Meenakshi Tyagi, (1995). Pyrethroid induced alterations in Aransaminases in
liver and muscles of Clarias batrachus (Linn). Proc. Acad. Environ. Biol., 5: 251 - 253.
Freeman, H.C., Sangalang G.B., Uthe J.F, Garside E.T. and Daye P.G. (1983). A Histopathological examination and analysis for polychlorinated hydrocarbons in shore Atlantic Cod (Gadus morhua). Arch. Environ. Contam. Toxicol., 12: 627632.
Gornall, A. G., Bardawill, C. J. & David, M. M. (1949). Determination Of Serum Proteins By Means Of The Biuret Reaction. Biol. Chem., 177, 751-766
Heath, A.G. (1991). Water Pollution and Fish Physiology. Lewis Publishers, Boca Raton, Florida, USA., ISBN: 0873716329, pp: 359.
Hillman GZ (1971) Fortlaufende photometrische Messung del. Säuren Phosphatase Aktivität. Klin Chem Klin Biochem. 9:273-274.
Hori, T.S.F., Avilez, T.N. Inoue, L.K. and Moraes, G.
(2006). Metabolical changes induced by chronic phenol exposure in Matrinxa bryconcephalus (Teleostei: Characidae) juveniles. Comp. Biochem., Physiol, 143: 67 - 72,
Humtsoe, N., Dawoodi R., Kulkarni B. and Chavan B.
(2007). Effect of arsenic on the enzymes of the rohu carp, Labeo rohita (Hamilton, 1822). Raffles Bull. Zool., 14: 17 - 19.
Jaroli, D.P. and Sharma B. L. (2005). Effect of Organophosphate Insecticide on the Organic Constituents in Liver of Channa punctatus . Asian J. Exp. Sci., 19: 121-129.
Klin, Z. (1972). Alkaline phosphatase activity is determined by measuring the rate of conversion p-nitrophenyl phosphate (pNPP) to p-nitrophenol (pNP). Chemklin Biochem, 182-192.
Kopecka, J., Lehtonem K.K., Barsiene J., Broeg K., Vuorien P.J., Gercken J. and Pempkowiak J. (2006). Measurement of biomarker level in flounder (Platichthys flesus) and blue mussel (Mytilus trossulus) from the Gulf of Gdansk (Southern Baltic). Marine Poll. Bull., 53: 406- 421.
Kulkarani, G.P.P. and Suniti Dharwadkar, M. (1998). Effect of dairy effluent on biochemical parameters
of wheat seeds and fish. Proc. Acad. Environ. Biol. 7: 57 - 60.
Lavado, R., Urena R., Martin-Skilton R., Torreblanca A., Del Ramo J., Raldua D. and Porte C. (2006). The combined use of chemical and biochemical markers to assess water quality along the Ebro River. Environmental Pollution, 139: 330-339.
Nchumbeni Humtsoe, Reza Davoodi, Kulkarni B.G. and Bhavita Chavan. (2007). Effect of arsenic on the enzymes of the rohu carp, Labeo rohita (Hamilton, 1822). Raffles Bull.Zool., 14: 17 - 19.
Nussev G., Van Vuren J.H.J. and Du Preez H.H. (2000). Bioaccumulation of chromium, manganese, nickel and lead in the tissues of the moggel, labeo umbratus (Cyprinidae), from Witbank Dam, Mpumalanga. Water SA, 26: 269-284.
Pathan, T.S., Sonawane D.L. and Khillare Y.K. (2009). Toxicity and Behavioural Changes in Freshwater Fish Rasbora daniconius Exposed to Paper Mill Effluent. Bot. Res. Intl., 2: 263-266.
Rajamanicckam, V. and Muthuswamy N. (2008). Effect of heavy metals induced toxicity on metabolic biomarkers in common carp (Cyprinus carpio L.). Maejo Int.J.Sci.Technol., 2: 192 - 200.
Ramana Rao, S. V., Padmavathy B., Sathyavelu Reddy K. and Indira K. (1990). Carbon tetrachloride induced hepato toxicity in a fresh water fish Sarotherodon mossambicus. Poll. Res., 15: 385387.
Saleh, H.H. (1982). Study on the entrance and accumulation of pollutants through the different organs and tissues of Tilapia zillii Gerv. Living in and fresh and saline water. Bull. Inst. Ocean. Fish. A.R.E. 8: 81-86.
Trinder, P. (1969). Enzymatic methods for glucose determination. Ann Clin Biochem, 6, 24-26.
Weibhaar D, Grossau E, Faderal B. (1975) Normal ranges of alphaHBDH, LDH, AP and LAP as measured with substrateoptimated test charges. Med Welt 26, 387-392.
