In vivo Acute Toxicity Tests of Some Heavy Metals to Tilapia Fish (Oreochromis niloticus)
A. K. Ahmad
Fingerlings tilapia fish (Oreochromis niloticus) were exposed in laboratory conditions to a range of Copper (Cu), Cadmium (Cd), Lead (Pb) and Zinc (Zn) concentrations separately and the mortality rate were registered after 24, 48, 72 and 96 h. Median Lethal Concentrations (LC50s) and Median Lethal Times (LT50s) values were calculated by static bioassays for 24, 48, 72 and 96 h with the 95% fiducial limits through Probit analysis. LC50 and LT50 increased with the decrease in mean exposure times and concentrations, respectively. The LC50s for 96 h for Cu, Cd, Zn and Pb were 1093, 3751, 16177 and 1494 μg L-1, respectively and the LT50s for maximum concentration used for Cu, Cd, Zn and Pb were 20.25, 11.48, 20.70 and 24.66 h, respectively. Metals bioconcentration in tilapia fish increases with exposure to increasing concentrations and Cu was the most toxic to tilapia fish, The toxicity ranking of the four heavy metals was Cu>Pb>Cd>Zn. Comparison of LC50 values for studied heavy metals for this species with those for other fishes reveals that tilapia fish (Oreochromis niloticus) is equally or less sensitive to heavy metals than most other tested fishes.
Received: March 15, 2013;
Accepted: April 01, 2013;
Published: July 12, 2013
Aquatic organisms have been considered to concentrate metals several times
greater than environmental levels. Fishes have been used for many decades to
evaluate the pollution status of water and are thus considered as excellent
biological indicator of heavy metals in aquatic environments (Hedayati
et al., 2010). Fishes are extensively distributed creatures in the
marine and freshwater environments (Taweel et al.,
2012). Tilapia fish Oreochromis niloticus have some features such as the
ability to persist in poor water quality and quick growth (Ayotunde
and Ofem, 2008). Trace metals occur naturally in the environment and found
in varying levels and are considered the main ecosystem pollutants to the aquatic
life (Azmat et al., 2012). Anthropogenic activities
cause an increased discharge of these metals into the aquatic ecosystem; fish
are relatively sensitive to these changes in their environment (Abdel-Warith
et al., 2011). Toxicity tests are conducted to assess the effects
of single or complex mixture of contaminants on the organisms. In freshwater,
the existence or absence of fish has been extensively used as a biological indicator
of the level of pollution. In acute tests experiments, one of the commonly used
measures is the LC50s. In other words, the lethal median concentration
that causes mortality in 50% of the test organisms (Boateng
et al., 2006).
The main purpose of ecotoxicologyy is the estimation of risk for an ecosystem
exposed to environmental pressure, including contamination. Although physical-chemical
parameters are essential for risk evaluation, the results of biological response
to chemical stress have been used as reference to determine the expected biological
damage (Ferrer et al., 2006). Lead is postulated
as one of the most vital pollutants in the environment which builds up in the
body of the human due to its low of elimination; cadmium is very toxic to the
aquatic organisms, commonly found in industrial area and ninety percent of cadmium
derived from human activities (Omer et al., 2012).
The test fish chosen for the current study, tilapia is available throughout
the year, cheaper and protein rich food. Tilapia are presently known as aquatic
chicken due to their quick growth, adaptability to a broad range of environmental
stresses, disease confrontation, high flesh quality. Thus, tilapia becomes a
chosen fish for aquaculture, mainly in tropical and semitropical countries (Nguyen,
2008). The tilapia fish was selected as an experimental organism on the
basis of being representative of an ecologically significant animal, widely
edible, moreover, amenable to laboratory testing (Al-Attar,
2005). Undertaking the acute tests experiments of heavy metals using aquatic
organisms such as fish will assist to improve the environmental observing process.
Some heavy metals (Cu and Zn) are essential and some nonessential (Pb and Cd).
They intensively spread due to anthropogenic activities and natural sources,
however, metals accumulate in fish and transfer through food chain to human
body (Kamaruzzaman et al. 2010; Kamaruzzaman
et al., 2008).
The results of toxicity tests for copper, cadmium, zinc and lead will contribute
data to environmental quality standards in Malaysia. In addition, many laboratory
experiments have been carried out to assess the toxicity of heavy metals on
different test organisms (Verma et al., 2012;
Mebane et al., 2012; Benjamin
and Thatheyus, 2012; Shuhaimi-Othman et al., 2011;
Bhamre et al., 2010; Shuhaimi-Othman
et al., 2010; Ferrer et al., 2006;
Straus, 2003; Zyadah and Abdel-Baky,
2000). In the present study, the effect of median lethal concentrations,
time and bioconcentration of four selected heavy metals (Cu, Cd, Zn and Pb)
on the tilapia fish Oreochromis niloticus has been investigated.
MATERIALS AND METHODS
Apparatus: An Oven for drying the samples, Electronic balance-AB 204-S METTLER TOLEDO, Block thermostat-SASTEC model No ST-DBMK2004, Malaysia, Water parameter meter-Quanta and Inductively-coupled plasma Mass spectrometry (ICP-MS) (Model ELAN 9000 Perkin Elmer ICP-MS, USA).
Reagents and quality assurance and quality control: All reagents were
of analytical reagent grade. Ultra high purity deionised water was used for
all dilutions throughout the whole experimental work done in this study including:
preparation of working standards, dilution of digested samples and cleaning
of digestion vessels and other equipment that was used. Nitric acid, HNO3
(65%) and hydrogen peroxide, H2O2 (30%) were of ultrapure
quality (Merck, Darmstadt, Germany). The element standard solutions from Merck
Company to make the calibration were prepared by diluting the stock solutions
of 1000 mg L-1 of each element. For validation of the analytical
method, a recovery study for each run, duplicate samples and blanks were carried
out through the digestion reaction. The certified standard reference materials
of marine biota sample (SRM2976, freeze-dried mussel tissue, National Institute
of Standards and Technology, USA) was used, the results of measurements of CRMs
are summarized in Table 1. All the glassware and plastics
were soaked overnight in 10% (v/v) nitric acid and rinsed with distilled and
deionised water and dried before being used (Khansari et
Blank and drift standards were run after fifty determinations to preserve instrument
||Trace metal concentrations in certified reference material
with microwave digestion method, n = 6
||Operational parameter settings were used in ELAN 9000 Perkin
The concentrations of heavy metal were expressed on a dry weight basis (μg
g-1 dry wt.). The operational parameter settings were used in ELAN
9000 Perkin Elmer (Table 2).
Studied fish review: Fingerlings of tilapia fish Oreochromis niloticus
(mean total length, TL, 3.3±0.3 cm; mean weight, 1.5±0.2 g). Fingerlings
of tilapia were purchased from an aquarium commercial shop in Bangi, Selangor,
Malaysia. Young organisms are often more sensitive to toxicants than is adults
viz, the early stages are more sensitive than the later stages (Dung
et al., 2005). For this reason, the use of early life stages, such
as fish fingerlings is required for all tests. In a given test, all organisms
should be approximately the same age and should be taken from the same source
in order to minimize the diversity of response to experimental materials (US
EPA, 2002). Fish fingerlings were transported in oxygenated bags to the
laboratory and handled properly to minimize injury and stress physiology in
order to reduce the number of dead organisms.
Sample preparation procedures: The purpose of the acute test with fish
species is to facilitate in the measurement of possible risk to similar species
in natural ecosystems. Acute testing must be performed for a minimum of 96 h
(USEPA, 1999). Fish were acclimatized in aquarium under
laboratory conditions for two weeks in the laboratory at a temperature of 27-30°C,
Light: dark cycle was maintained at 12 h light: 12 h darkness, using fluorescent
lights (334-376 Lux) in 150 L stocking tanks using dechlorinated tap water with
adequate filtration system (USEPA, 1999). Throughout
the acclimatization process, chemical water parameters such as pH, dissolved
oxygen concentration and temperature of water in the tanks were monitored daily.
In addition, the fish were fed with commercial fish food Aquadene®
four times a day. The standard stock solutions (100 mg L-1) for studied
metals were freshly prepared by dissolving of copper sulfate CuSO4.5H2O,
cadmium chloride CdCl2.2.5H2O, lead nitrate
(Pb (NO3)2 and zinc sulfate ZnSO4.7H2O
obtained from Merck (Germany) with deionized water . A control with dechlorinated
tap water only also was used (Shuhaimi-Othman et al.,
2010). All toxicity test concentrations were made from the stock solution
using appropriate calibrated analytical pipettes and graduated cylinders. Preliminary
tests were conducted prior to the definitive test to establish the actual five
concentrations for each studied heavy metal. Following a range finding test,
five Cu (100,560, 1000, 1800 and 3200 μg L-1), Cd (1000, 5600,
10000, 14000 and 18000 μg L-1), Pb (100, 870, 3200, 5600 and
10000 μg L-1), Zn (5600, 10000, 18000, 32000 and 56000 μg
L-1) concentrations were chosen. In the definitive test, the concentrations
used in each test were chosen to give from 0 to 100% mortality of test animals
within 96 h based on the results of preliminary studies. Tilapia fingerlings
were exposed for a four-day period with a range of copper, cadmium, lead and
zinc concentrations. The tests were carried out under static conditions with
the renewal of the exposure solution every two days by siphoning out the old
solution and adding the freshly prepared solutions with the designated studied
metal levels. (APHA, 1998). Two replicates of control
group with five fish for each group and also for each metal-treated groups consisted
of five replicates of ten randomly allocated fish in a 1000 mL glass beaker
(Schott Duran®) containing 800 mL of the appropriate solution.
Based on the progressive bisection of intervals on a logarithmic scale, log
concentrations were selected as the experimental concentrations (APHA,
1975). A total of 10 animals per treatment were used in the experiment and
a total of 210 animals were employed in the investigation (APHA,
1992). Samples of water for metal analysis taken before and immediately
after each solution renewal were acidified to 1% with ARISTAR®
nitric acid (65%) before metal analysis using inductively-coupled plasma mass
spectrometry (ICP-MS) (model ELAN 9000 Perkin Elmer ICP-MS, USA) depending on
Analysis of the water physico-chemical variables: For each concentration
of the prepared solution, water parameters such as temperature, pH, conductivity,
total dissolved solids and dissolved oxygen readings were measured before the
tests carried out and every two days by using a Multiparameter hydrolab Quanta®
which was recalibrated before each measurement. (The pH must be monitored in
low, medium and high test concentrations and must remain >6.0 and <8.0
for freshwater testing (USEPA, 1999).
The mean values for tested water quality parameters were presented in Table
3. Freshwater samples are adjusted to pH 7.0, by adding 1N NaOH or 1N HCl
dropwise, as required, being careful to avoid over adjustment (US
EPA, 2002, 1998).
The experiment was monitored from the beginning and mortality was recorded at the time intervals (each 3 h onward till 96 h). The criteria used to determine the death the absence of motion of the tail and gills. The fish were considered dead when there was a lack of response to gentle prodding with a glass rod. The dead fish were removed immediately from exposure beakers. At the end of exposure for four days (96 h), a live fish used to determine the bioconcentration of metals in fish tissues.
Chemical analysis: Samples of water from the control and test metal beakers were taken at intervals, when the experiment was commenced at the beginning, before and after 48 h and at 96 h when the experiment finished for metal analysis. Samples were acidified to 1% with ultra pure nitric acid (70%) and saved for metal analysis by inductively coupled plasma-mass spectrometry (ICP-MS Model Perkin Elmer Elan 9000).
Accumulation-fish tissue digestion: A fish tissue digestion process
was conducted to measure the concentrations of studied heavy metals. After the
acute test experiment has been finished the rest of tested fish which still
alive and fish from the control were taken to perform accumulation tests for
all studied metals. Fish were rinsed with deionized water and left for dead
before admission to the oven. Each sample comprised two replicates in acid-washed
Petri dishes and dried at 80°C for 48 h. Fish were crushed into a fine powder
by using a porcelain mortar and pestle. The fish were placed in test tubes in
replicates. Each replicate was digested (whole fish) in 2.0 mL nitric acid (Aristar
65%) and left overnight and heated on a block thermostat at a temperature of
100°C for 2 h for the purpose of fish tissue digestion. Heating process
was carried out in the fume chamber with a block thermostat-SASTEC model No
ST-DBMK 200-4 (Malaysia). Solutions were heated to turn to yellow color and
allowed to cool at room temperature. Upon cooling, 0.8 mL of hydrogen peroxide
(30%) was added to the solutions. The test tubes were put back on the block
thermostat for an hour until the solutions became clear. The solution was then
allowed to cool and put in a 25 mL volumetric flask and added with deionized
water, make up to 25 mL. The solutions were then transferred into polyethylene
bottles and sent to analyzed metals concentrations in fish tissues by Using
An Inductively-Coupled Plasma Mass Spectrometer (ICP-MS) for the determinate
the values of Cu, Cd, Pb and Zn (Kamaruzzaman et al.,
2010). The accuracy was also examined by analyzing, in duplicate standard
reference materials (SRM) 2976 Mussel Tissue (Trace Elements and Methylmercury)
Freeze-dried tissue, National Institute of Standards and Technology (NIST),
(USA) and the results matched with the certified values, (Stephenson
and Mackie, 1988).
Statistical analysis: Results were analyzed by one-way ANOVA with Tukeys
multiple comparison depending on the experimental design. The significance was
reported at p = 0.05 levels. The acute toxic effects of studied heavy metals
on Nile tilapia were determined by the use of Probit Analysis LC50
and LT50 determination method. The computer model (Probit Program
Version 1.5 software) was developed by the Environmental Protection Agency (USEPA,
RESULTS AND DISCUSSION
In all data analyses, the actual, rather than nominal, Cu Pb, Zn and Cd concentrations
were used. The LC50s values for tilapia fingerlings obtained for
the four metals at different periods of exposure were summarized in Table
4. The data obtained from the acute toxicity test of Cu, Cd, Zn and Pb for
Nile tilapia revealed that the fish mortality increased with increasing concentration
and/or exposure time. All control results in lower mortality, less than 10%
which revealed the acceptability of the tests. The LC50-96 h values
for zinc was the highest among all studied metals 16177 μg L-1
and copper was the lowest 1093 μg L-1, whereas, for 24 h, it
was 64897 μg L-1 for Zn and 3286 μg L-1 for
Cu. Ferrer et al. (2006) has reported the LC50-96
h values 1093.40 μg L-1 for Pb, 219.20 for Cu and 172.10 μg
L-1 for Zn in the early life stages of the carb Chasmagnathus
granulata. Hence, the acute tests of 96, 72, 48 and 24 h showed an opposite
relationship between LC50s and exposure time, increase in the concentration
reduces the time to kill 50% of the tilapia fish. Straus,
(2003) estimated the LC50-96 values for Cu to blue tilapia in
different values of alkalinities (43060, 6061, 690 and 180 mg L-1
Cu) in water having total alkalinities of 225, 112, 57 and 16 mg L-1
||Median lethal concentrations (LC50) for tilapia
fish fingerlings at different exposure times for Cu, Cd, Pb and Zn
||Median lethal times (LT50) for tilapia fish fingerlings
exposed to different concentrations of Cu, Cd, Pb and Zn
Cu was the most toxic to tilapia fish and the toxicity ranking of the four
heavy metals was Cu>Pb>Cd>Zn Shuhaimi-Othman et
al. (2010) reported that LC50s for 24 and 96 h for Cu were
54.2 and 5.6 μg L-1 and for Cd 1440.2 and 101.6 μg L-1,
respectively for R. sumatrana. For P. reticulata, LC50s
for 24 and 96 h for Cu were 348.9 and 37.9 μg L-1 and for Cd
8205.6 and 168.1 μg L-1, respectively, the Cu was more toxic
than Cd to both fishes (Cu>Cd), these result are higher than present study.
Zyadah and Abdel-Baky (2000) recorded that the LC50-24
h 15300, 8700 and 102200 μg L-1; LC50-96 h 13100,
4500 and 91200 μg L-1 for Cd, Cu and Zn, respectively on T.
In view of median lethal times (LT50s), the median lethal times
(LT50) increased with a decrease in mean exposure concentrations
for all studied metals (Table 5) the lowest metals was Cd
with 11.48 h in measured concentration of 17612.11 μg L-1 and
with 266.46 h in measured concentration 1205.95 μg L-1.
||Relationship between LC50 and exposure times for
tilapia fish fingerlings
Whereas, the highest metals was Zn with 25.70 h in concentration of 53412.91
μg L-1 and with 185 h in measured concentration 5280.20 μg
L-1. Shuhaimi-Othman et al. (2009)
reported that LT50 of Cu for freshwater prawn M. lanchesteri with
concentration of 6.43 μg L-1 was 151.1 h and LT50
for 97.65 μg L-1 was 49.7 h. Results were obtained by Shuhaimi-Othman
et al. (2011) revealed that LT50 for measured Zn concentration
779, 1010, 2456, 3213 and 4822 μg L-1 were 260.33, 100.63, 52.52,
17.59 and 13.17 h, respectively. On the other hand, Pb LT50 for 475,
1160, 3410, 4829 and 8973 μg L-1 were 110.26, 72.39, 55.66,
43.55 and 9.15, respectively.
The relationship between LC50s and exposure times for tilapia fish fingerlings were shown in Fig. 1. It is very clear that the zinc is the highest concentration among all metals, this is reflect the less effect of zinc compared with copper, cadmium and lead, whereas, the strongest metal effect on tilapia is the copper.
Bioconcentration results: Bioconcentration of Cu, Cd, Pb and Zn in surviving
tilapia fish were as shown in Fig. 2 and 3.
Data for life fish were obtained from four Cu concentrations (68.95, 460.95,
964.07 and 2144.12 μg L-1); four Cd concentrations (1205.29,
5205.24, 9554.71 and 12800 μg L-1); four Pb concentrations (128.13,
710.48, 3365.50 and 5460.12 μg L-1; four Zn concentrations (5280.20,
9860.80, 17600 and 30200.21 μg L-1). Bioconcentration of metals
in tilapia fingerlings increases with increasing concentration exposure. Statistical
analyses show that there are significant differences (ANOVA, p<0.05) in all
studied metals bioconcentration compared with control water at the end of four
days. The bioconcentrations of heavy metal increase with additions of metal
concentrations in water (Rao and Patnaik, 1997).
||Bioconcentration of Cu and Cd in tilapia fish fingerlings
after four day exposure to different concentrations of Cu and Cd
||Bioconcentration of Pb and Zn in tilapia fish fingerlings
after four day exposure to different concentrations of Pb and Zn
A comparison of bioaccumulation of heavy metals in tilapia fish showed that
among the four metals, Cu was the least accumulated and Zn and Pb were the most
accumulated. Shuhaimi-Othman et al. (2011) have
reported that Cu and Cd were the most accumulated and Ni was the least accumulated
to fresh water ostracod Stenocypris major. The rate of bioaccumulation
of studied metals by the tilapia fish appeared within a wide range, the bioaccumulation
factor of Cu, Cd, Pb and Zn by tilapia was 79, 774, 374 and 26 times more than
control concentration respectively after 96 h exposure for maximum metals concentration
Present study indicated that copper highly toxic to tilapia fish. The tested
organism was shown to be most sensitive to Cu and most resistant to Cd. The
toxicity of the studied metals followed the order: Cu>Pb>Cd>Zn. Large
differences appeared in sensitive of tilapia fish fingerlings to Cu, Cd, Zn
and Pb, these results indicate that tilapia is comparatively tolerant to metals
in comparison to other fish species. In terms of accumulation, Zn was the least
accumulated and Cd was the most accumulated followd by Pb.
The authors would like to express their sincere appreciation and gratefulness to the Ministry of High Education, Libya for financial support and to University Kebangsaan Malaysia (UKM) for providing facilities. Our gratitude to laboratory assistants for their help during the study.
1: Abdel-Warith, A.A., E.M. Younis, N.A. Al-Asgah and O.M. Wahbi, 2011. Effect of zinc toxicity on liver histology of nile tilapia, Oreochromis niloticus. Sci. Res. Essays, 6: 3760-3769.
Direct Link |
2: Al-Attar, A.M., 2005. Biochemical effects of short-term cadmium exposure on the freshwater fish, Oreochromis niloticus. J. Biol. Sci., 5: 260-265.
CrossRef | Direct Link |
3: APHA, 1975. Standard Methods for the Examination of Water and Wastewater. 14th Edn., American Public Health Association, Washington, DC., USA.
4: APHA, 1992. Standard Methods for the Examination of Water and Wastewater. APHA, Washington, DC., USA.
5: APHA, 1998. Standard Methods for the Examination of Water and Wastewater. 20th Edn., American Public Health Association, Washington, DC., USA., ISBN: 0-309-09122-5.
6: Ayotunde, E.O. and B.O. Ofem, 2008. Acute and chronic toxicity of pawpaw (Carica papaya) seed powder to adult Nile tilapia (Oreochromis niloticus Linne 1757). Afr. J. Biotechnol., 7: 2267-2274.
Direct Link |
7: Azmat, H., M. Javed and G. Jabeen, 2012. Acute toxicity of aluminium to the fish (Catla catla, Labeo rohita and Cirrhina mrigala). Pak. Vet. J., 32: 85-87.
Direct Link |
8: Benjamin, D. and A.J. Thatheyus, 2012. Acute toxicity of nickel and cadmium to the cichlid fish, Oreochromis mossambicus (Peters). Res. Zool., 2: 19-22.
9: Bhamre, P.R., S.P. Thorat, A.E. Desai and B.M. Deoray, 2010. Evaluation of acute toxicity of mercury, cadmium and zinc to a freshwater mussel Lamellidens consobrinus. Our Nat., 8: 180-184.
Direct Link |
10: Boateng, J.O., F.K. Nunoo, E.H.R. Dankwa and M.H. Ocran, 2006. Acute toxic effects of deltamethrin on tilapia, Oreochromis niloticus (Linnaeus, 1758). West Afr. J. Applied Ecol., 9: 1-5.
Direct Link |
11: Dung, L.Q., N.M. Cuong, N.T. Huyen and N.D. Cu, 2005. Acute toxicity test to determine the effects of copper, zinc and cyanide on cobia (Rachycentron canadum) resources in North Vietnam. Aust. J. Ecotoxicol., 11: 163-166.
Direct Link |
12: Ferrer, L., S. Andrade, R. Asteasuain and J. Marcovecchio, 2006. Acute toxicities of four metals on the early life stages of the crab Chasmagnathus granulata from Bahia Blanca estuary, Argentina. Ecotoxicol. Environ. Saf., 65: 209-217.
13: Hedayati, A., A. Safahieh, A. Savari and J.G. Marammazi, 2010. Detection of range finding test of mercury chloride inyellowfin sea bream (Acanthopagrus latus). Iran. J. Energy Environ., 1: 228-233.
Direct Link |
14: Kamaruzzaman, B.Y., M.C. Ong, K. Zaleha and S. Shahbudin, 2008. Levels of heavy metals in green-lipped mussel Perna veridis (Linnaeus) from Muar Estuary, Johore, Malaysia. Pak. J. Biol. Sci., 11: 2249-2253.
CrossRef | PubMed | Direct Link |
15: Kamaruzzaman, B.Y., B. Akbar, K.C.A. Jalal and S. Shahbudin, 2010. Accumulation of metals in the gills of Tilapia fingerlings (Oreochromis niloticus) from In vitro toxicology study. J. Fish. Aquat. Sci., 5: 503-509.
CrossRef | Direct Link |
16: Khansari, F.E., M. Ghazi-Khansari and M. Abdollahi, 2005. Heavy metals content of canned tuna fish Food Chem., 93: 293-296.
17: Mebane, C.A., F.S. Dillon and D.P. Hennessy, 2012. Acute toxicity of cadmium, lead, zinc and their mixtures to stream-resident fish and invertebrates. Environ. Toxicol. Chem., 31: 1334-1348.
18: Nguyen, T.N., 2008. The utilization of soybean products in tilapia feed-A review. Proceedings of the 8th International Symposium on Tilapia in Aquaculture, October 12-14, 2008, Nong Lam University, pp: 53-65.
19: OECD, 2006. OECD Guideline for the testing of chemicals: Draft proposal for a new guideline; Fish Embryo Toxicity (FET) test. Draft Guideline, May 30, 2006 (1st Version). http://www.oecd.org/env/ehs/testing/36817070.pdf.
20: Omer, S.A., M.A. Elobeid, D. Fouad, M.H. Daghestani and E.M. Al-Olayan et al., 2012. Cadmium bioaccumulation and toxicity in tilapia fish (Oreochromis niloticus). J. Anim. Vet. Adv., 11: 1601-1606.
CrossRef | Direct Link |
21: Rao, L.M. and R.M.S. Patnaik, 1997. Acute toxicity of Zn, Pb and Cd in the freshwater catfish Mystus vittatus (Bloch). Indian J. Fish., 44: 405-408.
Direct Link |
22: Stephenson, M. and G.L. Mackie, 1988. Total cadmium concentrations in the water and littoral sediments of central Ontario lakes. Water Air Soil Pollut., 38: 121-136.
23: Shuhaimi-Othman, M., Y. Nadzifah, R. Nur-Amalina and A. Ahmad, 2009. Toxicity of copper and nickel to freshwater prawn macrobarchium lanchesteri. Sains Malaysiana, 38: 353-358.
Direct Link |
24: Shuhaimi-Othman, M., Y. Nadzifah and A.K. Ahmad, 2010. Toxicity of copper and cadmium to freshwater fishes. World Acad. Sci. Eng. Tech., 65: 869-871.
Direct Link |
25: Shuhaimi-Othman, M., N. Yakub, N.A. Ramle and A. Abas, 2011. Toxicity of metals to a freshwater ostracod: Stenocypris major. J. Toxicol., 10.1155/2011/136104
26: Straus, D.L., 2003. The acute toxicity of copper to blue tilapia in dilutions of settled pond water. Aquaculture, 219: 233-240.
27: US EPA, 1998. Ecological effects test guidelines fish acute toxicity test: Freshwater and marine. http://www.epa.gov/opptsfrs/publications/OPPTS_Harmonized/850_Ecological_Effects_Test_Guidelines/Drafts/850-1075.pdf.
28: USEPA, 1999. LC50 software program, version 1.50. Ecological Monitoring Research Division, Environmental Monitoring Systems Laboratory, EPA, Cincinnati, OH., USA.
29: USEPA., 2002. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms EPA-821-R-02-012. 5th Edn., U.S. Environmental Protection Agency, Washington DC., USA.
30: Verma, S.R., P. Chaudhari and S. Satyanaranyan, 2012. Acute toxicity of copper and cadmium, to the freshwater fish Lebistesreticulatus (Peter). J. Ind. Pollut. Control, 28: 119-124.
Direct Link |
31: Zyadah, M.A. and T.E. Abdel-Baky, 2000. Toxicity and bioaccumulation of copper, zinc and cadmium in some aquatic organisms. Bull. Environ. Contamin. Toxicol., 64: 740-747.
32: Taweel, A.K.A., M. Shuhaimi-Othman and A.K. Ahmad, 2012. Analysis of heavy metal concentrations in Tilapia fish (Oreochromis niloticus) from four selected markets in Selangor, Peninsular Malaysia. J. Biol. Sci., 12: 138-145.