IJERD ± International Journal of Environmental and Rural Development (2018) 9-2

Research article
erd

Determination of Lethal Concentration of Copper
Compounds on Nile Tilapia (Oreochromi nilo ic
Linnaeus, 1758) Larvae
NATSIMA TOKHUN*
Environmental Science Program, Faculty of Science and Technology, Valaya Alongkorn
Rajabhat University under the Royal Patronage, Pathum Thani, Thailand
Email: natsima@vru.ac.th

KANITTHA PAMONPOL
Environmental Science Program, Faculty of Science and Technology, Valaya Alongkorn
Rajabhat University under the Royal Patronage, Pathum Thani, Thailand

CHULEEMAS BOONTHAI IWAI
Integrated Water Resource Management Research and Development Center in Northeast
Thailand, Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand

ATCHARAPORN SOMPARN
Environment Division, Office of General Education, Udon Thani Rajabhat University, Udon
Thani, Thailand
Email: somparn_a@yahoo.com
Received 8 December 2017

Accepted 20 October 2018(*Corresponding Author)

Abstract Copper is an essential element for cell organisms. However, irregularity copperlevel could have effect on the growth of organism. In this research, we aim at considerationof copper compounds toxicity against larvae of Nile tilapia (Oreochromis niloticus
Linnaeus, 1758) by statistic assays. Four days old of test fish had an average 7.10+0.03 mmof length and 9.72+0.04 mg of fresh weight. All experiments were carried out for a periodof 48 to 96 hours at 27 30oC. The number of dead fish was counted every 24 hours and themortality rate was determined after 96 hours. The statistical data was eval a ed b Finne
probit analysis method. The fish died 50% (LC50 95% confidence limits) after 48, 72, 96
hours of concentration copper chloride (CuCl2) 595.75, 231.11, 124.64 µg/L, respectively,
and copper nitrate (Cu (NO3)2) 1,025.40, 588.79, 456.50 µg/L, respectively. The LC50
results clearly show that copper compounds toxicity for fish increased with increasing
concentration and exposure time. The percentage of survival decreased with increasing ofboth copper compound concentrations and it was significantly different from control(p<0.01). This study showed that Nile tilapia larvae were more sensitive to copper chloride
than copper nitrate. Further study needs the processes by which these chemicals affectbiochemical changes of the fish.
Keywords copper, lethal concentration, LC50, tilapia, toxicity

INTRODUCTION
The copper-gold (Cu-Au) has been consumed for a large quantity in Thailand. The internal main
supply could be found in Loei province (Yang et al., 2014). Due to high demand, it was imported invarious forms i.e. pure copper, copper matt, copper anode, unwrought and copper products in alarge amount for 422,619 metric tons in 2012 (Office of Industrial Economics, 2012). Copper andits compounds has been used in various industries such as electrical products, building construction,
chemical and pharmaceutical manufacturing (National Pollutant Inventory, 2014). They are directly
discharged from industrial effluents and also from polluted runoff in urban and agricultural areas(FAO, 1992; Yirgu, 2011). Therefore, surface water and groundwater are being polluted from theseⒸ ISERD

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IJERD ± International Journal of Environmental and Rural Development (2018) 9-2

effluents and runoff that contains copper and its compounds. Copper has been detected in water
range from 1 to 5 mg/L, which is classified as small amounts or not generally considered to be
toxic. However, gradually accumulation of copper in body could become large doses enough tocause sickness, and could lead to liver damage in extreme cases. The distribution of copper ions has
been determined in the aquatic system to assess the influence of biological processes on the copper
estuarine behavior. The copper chloride prominently affects the fish muscles as some amino acids(Histidine, Proline, Glycine, Alanine, Methionine and Valine) are found to be reduced or evencompletely missing (Alkesh, 2016). Aquatic life is more sensitive 10 100 times to the hazardous
effects of copper than mammals (Shah and Vyas, 2015).
Lethal concentration test measure the susceptibility and survival potential of organisms to aparticular toxic substances such as heavy metals. Pollutants with higher median lethal
concentration (LC50) values are required to induce mortality in organisms (Eaton et al., 2005;
Zahedi et al., 2012). Nile tilapia is one of the most common freshwater fish used in toxicologicalstudies (Figueiredo-Fernandes et al., 2006a, b, 2007; Garcia-Santos et al., 2006) because it is easy
handling, culture and maintenance in laboratory (Alkobaby and El-Wahed, 2017). Moreover, it
promptly responds to environmental alterations so this species is also a well-established model for
toxicological research (Almeida et al., 2002; Figueiredo-Fernandes et al., 2006a, 2007). Hence, theaim of this study is to determine the value of copper as chloride and nitrate lethal concentration for
Nile tilapia (Oreochromis niloticus Linnaeus, 1758) larvae in order to determine specific times.
MATERIALS AND METHODSThe experiment was conducted at Environmental Science Laboratory, Faculty of Science andTechnology, Valaya Alongkorn Rajabhat University under the Royal Patronage, Pathum Thani
province, Thailand. The acute toxicity of copper compounds for Nile tilapia larvae was determinedin terms of 48, 72 and 96 hours LC50 and lethal concentrations. The determination of lethal
concentration of copper compounds on Nile tilapia larvae was the following.
Test Organism
Two days old of Nile tilapia (Oreochromis nileticus Linnaeus, 1758) fish larvae were obtained fromthe Pathum Thani Inland Aquaculture Fisheries Research and Development Center, Thailand for
toxicity testing in an oxygenated plastic bag (Fig. 1). The test was conducted at the Environmental
Science Laboratory, Valaya Alongkorn Rajabhat University under the Royal Patronage in January,
2017. The tested fish were acclimatized in distilled water. Each holding tray was aerated by using a
portable pump and air stone for a period of 48 hours at a room temperature before experiment use. If
survival rate was <10%, the entire stock was discarded (Boonsomboone, 2004). Test fish fused or
toxicity tests had an average 7.10+0.03 mm of length and 9.72+0.04 mg of wet weight.

Fig. 1 The test organism, Nile tilapia (Oreochromis nileticus Linnaeus, 1758) larvaeⒸ ISERD

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IJERD ± International Journal of Environmental and Rural Development (2018) 9-2

Test Media
Analytical grade copper chloride (CuCl2·2H2O) and copper nitrate (Cu(NO3)2·3H2O) form SigmaAldrich were used for preparation of stock solutions that were diluted as desired concentrations.
Fish were exposed for 24 to 96 hours, separately, against different concentrations of two copper
compounds at 0, 10, 50, 100, 300 and 500 µg/L. The investigation was performed in laboratory
conditions at dissolved oxygen (8.06+0.05), temperature (27.77+0.76oC) and pH (6.50+0.50). All
physico-chemical parameters were performed by following APHA-AWWA-WEF (1998) on daily
basic.
Determination of Lethal Concentration
Four-day static acute toxicity test was performed at five different concentrations (10, 50, 100, 300
and 500 µg/L of copper chloride and nitrate) were used in the test series (10 fish for each). A
control group (10 fish) was maintained in deionized water container throughout entire experimental
period, the fish were not fed. Fish mortalities were record at 48, 72 and 96 hours after starting oftests, and dead fish were removed immediately from test media. Mortality was assessed in the
different copper concentrations along with the control group to determine the 96 hours medianlethal concentration (LC50) al e b he e of Finne p obi anal i me hod (Finne , 1971).
Statistical Analysis
The entire experiment was analyzed using a completely randomized design (CRD) method.
Mortality data for 48, 72 and 96 hours was calculated for LC50 values (95% confidence limits) byFinne
p obi analysis (Finney 1971) using the Statistical Analysis System (SPSS) version 11.5
program. Standard error of mean value (SEM) of replication sampling (n=12) was taken for eachanalysis. A significantly different result was established by a one-way ANOVA, and mean
comparisons of different treatments were carried out by least significant difference (LSD) on
Duncan multiple test of Statistix 8 Software (Analytical Software, Tallahassee, FL). The
acceptance level of significance was a probability value less than 0.05 (p<0.05).
RESULTS AND DISCUSSIONMedian lethal concentration (LC50, 95% confidence limits) for copper chloride and copper nitrateon the larvae of Nile tilapia (Oreochromis nileticus Linnaeus, 1758) calc la ed b Finnep obi
analysis method and SPSS Statistical Software at 48, 72 and 96 hours of exposure was shown in
Table 1 and Fig. 2.
Table 1 LC50 value with 95% confidence limit of copper chloride and copper nitrate on fish
Exposure times(hour)
48
72
96

Lethal concentration (µg Cu/L, lower-upper values)
Copper chloride (CuCl2)
Copper nitrate (Cu (NO3)2)595.754
1,025.395(391.083 1,118.644)(668.913 2,207.740)
231.110588.785(171.199 335.477)(418.447 988.509)
124.637456.504(94.508 167.266)(322.974 755.933)

Note: LC50 is concentration of copper compounds that caused dead 50% of the exposed fish at a specific time ofobservation (e.g. 48 hours LC50), (expressed as µg/L of copper concentrations)

Results showed that the 48 hours LC50 (lower upper values) for copper chloride was 595.754(391.083 1,118.644) µg/L which sharply declined to 231.110 (171.199 335.477) and 124.637(94.508 167.266) µg/L at 72 and 96 hours, respectively. LC50 value for copper chloride was lower
than copper nitrate at exposure times. At 48, 72 and 96 hours LC50 values for copper nitrate on fish
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IJERD ± International Journal of Environmental and Rural Development (2018) 9-2

were 1,025.395 (668.913 2,207.740), 588.785 (418.447 988.509) and 456.504 (322.974 755.933)µg/L, respectively. This study showed that copper chloride was more toxic than copper nitrate on
Nile tilapia larvae.

Fig. 2 Shown LC50 value of both copper compounds on fish
The 96 hours LC50 value for copper chloride in Nile tilapia larvae was found 124.637 µg/L in
the present work, and here we report copper chloride to be highly toxic to fish. The Material Safety
Data Sheet (MSDS) reports copper chloride acute toxicity to carp fish (Cyprinus carpio) inlaboratory tests, in the average range LC50 value of 120 230 µg/L (Sigma-Aldrich Corporation,2014). Our result is in good agreement with this report. These values are considerably lower than
96 hour LC50 (900 µg/L) of Bluegill (Lepomis macrochirus), indicating higher vulnerability tocopper chloride in the former two species (Sigma-Aldrich Corporation, 2014; NTU chemistry
Deportment, 2017). Conversely, Warrin et al. (2009) found that copper chloride in sediment media
had higher toxic on Chironomus tentans than copper sulfate and copper nitrate. The results wereobtained by Taweel et al. (2013) reported that mean of 48, 72 and 96 hours LC 50 value for copper
sulfate in fingerlings of Nile tilapia (Oreochromis nileticus) were 1,863 (1,031 5,194), 1,368 (741
2,802) and 1,093 (581 1,613) µg/L, respectively. According to Iwai et al. (2016) estimates of 48,
72 and 96 hour LC50 value for copper sulfate to Nile tilapia (Oreochromis nileticus) larvae were1,869 (1,157 5,235), 1,740 ( ) and 1,383 (859 2,718) µg/L, respectively, these results are higher
than present study (Table 1). On the other hand, LC50 for copper oxide on Barnacle (Balanus
improvises) was 350, 140 and 20 µg/L for 48, 72 and 96 hours, respectively (U.S.EPA, 2009a). For
copper sulfate, the larva was the most sensitive stage of development of cobia (Rachycentron
canadum): eggs, larvae (one-day-old fish), juveniles (20 days old) and young fish (40 days old) in
toxicity tests, with an 96 hours LC50 of 91 (56 134), 60 (29 96), 87 (49 133) and 240 (119 378)
µg/L, respectively as the early stages were more sensitive than the later stages, (Le et al., 2005). In
addition, copper was most toxic to fingerling tilapia fish and the toxicity ranking of four heavy
metals was copper > lead > cadmium > zinc (Taweel et al., 2013). Also, copper was more toxic
than cadmium for common carp (Cyprius carpio L.) embryos and larvae (Jezierska et al., 2009).
The observed percentage of Nile tilapia mortality for copper in static tests continuous fordifferent hours and different concentrations were shown in Table 2. There was positive relationshipbetween the mortality and concentration levels; when concentration increased, the mortality rateincreased as well. Similar, the percentage of survival decreased with increasing of both copper
compound concentrations and exposure times. The result showed significant decreased percentage
of fish survival in copper chloride and copper nitrate when compared with control (p<0.01). The
fish tests significant responded to both copper concentrations (50 µg/L) in percent survival (72.0
85.6%) at specific times in Table 2. The copper concentrations that caused 50% of the exposed fishat 96 hour were found 100 µg/L of copper chloride and 300 µg/L of copper nitrate. Similar to
Howell et al. (1984) reported that copper (94 µg/L) reduced the filtration rate of mussels (Mytilus
edulis) by half, probably through reducing the ciliary beation via the bronchial nerves. Chen et al.
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IJERD ± International Journal of Environmental and Rural Development (2018) 9-2(2012) reported that even short-term pulsed exposure to low levels of copper (1.6 2.0 µg/L)
reduced growth rate of tilapia (Oreochromis mossambicus) larvae. Shah and Vyas (2015) reported
that at 2 mg/L of copper chloride concentration caused damage to cell membrane and vaculation ofLabeo rohita on 15 day, nucleus was affected on 30 days and on 45 days the cell membrane waswrinkled and damaged.
Table 2 Percent survival of exposed Nile tilapia larvae at specific times
Concentrations(µg /L)
0
10
50
100
300
500
LSD
CV (%)% Survival for Nile tilapia larvae (n=120)
Copper chloride (CuCl2)
Copper nitrate (Cu (NO3)2)
48 h
72 h
96 h
48 h
72 h
96 h
94.7 + 4.2a 90.2 + 3.5a87.4 + 4.2a
100.0 + 0.0a 100.0 + 0.0a
98.9 + 3.3a
90.1 + 5.2ab 84.7 + 3.8a 79.0 + 6.3b
98.9 + 3.3ab 95.6 + 5.3ab 92.2 + 6.7ab
85.6 + 5.1b 77.3 + 7.2b 72.0 + 5.0c
92.2 + 6.7bc 85.6 + 7.3b 81.1 + 9.2bc
79.0 + 6.1c 68.6 + 8.5c 56.6 + 9.9d
92.2 + 8.3bc 88.9 + 7.8b 82.2 + 10.9c
61.2 + 7.0d 47.3 + 6.1d 39.4 + 8.7e
88.9 + 7.8c 73.3 + 13.2c 66.7 + 14.1d
47.1 + 9.8e 31.8 + 9.7e
21.8 + 3.9f
57.8 + 13.0d 43.3 + 19.4d 37.8 + 17.2e
**
**
**
**
**
**
8.56
11.11
11.40
8.71
13.24
14.67

Note: ** showed significantly different from control (p<0.01)

CONCLUSION
Based on the acute toxicity tests performed in this study, Nile tilapia larvae was shown to be moresensitive to copper chloride than copper nitrate which reducing LC50 value and survival rate of fish
tests in copper chloride concentrations. The LC50 determination of copper compounds on fishlarvae was copper toxicity increased with increasing concentration and exposure time. The copper
concentrations that caused 50% of the exposed fish at 96 hours were found 100 µg/L of copper
chloride and 300 µg/L of copper nitrate.
ACKNOWLEDGEMENTS
The authors would like to express their gratitude to Environmental Science Program, Faculty of
Science and Technology, and Research and Development Institute, Valaya Alongkorn Rajabhat
University under the Royal Patronage, Environment Division, Office of General Education, Udon
Thani Rajabhat University, and Integrated Water Resource Management Research andDevelopment Center in Northeast Thailand, Faculty of Agriculture, Khon Kaen University,
Thailand for financial support.
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