IJERD – International Journal of Environmental and Rural Development (2015) 6-1

Research article
erd

Ecotoxicology of Copper on Local Freshwater Organisms
in Mekong River Cambodia
SOEUNG LYLY
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand
Email: lylysoeung@gmail.com

CHULEEMAS BOONTHAI IWAI*
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand
Email: chulee_b@kku.ac.th; chuleemas1@gmail.com

THAM HOANG
Loyola University Chicago, Chicago, IL, USA
Received 23 February 2015

Accepted 1 April 2015(*Corresponding Author)

Abstract The protection of aquatic habitat from damage and understanding of bothsensitivity of aquatic organisms to contaminant and ecological effects. Mekong Riverquality criteria of aquatic life for metals are largely driven by the extremely sensitive smallorganisms toxicity which are the Mekong native species. In this study we assessed thetoxicity of Copper in the Mekong river water with Chironomids Species (Chironomusjavanus) and Nile tilapia (Oreochromis niloticus). Acute toxicity effect of copper
concentration to freshwater animals occur after the exposure at tested with Mekong water
was studied by observing mortality and LC50 over a 24 hours test period. The LC50 with
95% confidence limit of the 96-hours toxicity was performed to contrast responses ofChironomids Species (C. javanus) and Nile tilapia (O. niloticus). The result showed that theLC50 with 95% confidence limit obtained were 742 µg/L in Chironomids Species (C.
javanus) and 853µg/L in Nile tilapia (O. niloticus). Copper (Cu) is a big concern forenvironment, human and aquatic organisms because it can accumulate in to plant andanimals via food web. The out coming of this series of laboratory experiment will provides
a worst-case scenario and useful for determine the risk assessment of copper on local
freshwater organisms in Mekong River Cambodia as well as Mekong River Basin.
Keywords ecotoxicology, acute toxicity, copper, local freshwater animals, Mekong River

INTRODUCTION
The resources of the Mekong Basin, such as fishes, other aquatic resources and plants are playmain role in food security, income and livelihood for many people across the Lower Mekong Basin.
Around 60 million people live in the lower Mekong Basin and most of people in the basin areclosely linked to the Mekong and resources of it to support their livelihood (MRC, 2007; Ferguson
et al., 2011). The development activities during the past decade and up to now, including mining,agriculture, deforestation, grazing and urbanization have caused of extensive soil erosion andcontribute increasingly to environmental levels of heavy metals especially copper (Cu) into water
body (Ti and Facon, 2004; Coates et al., 2006)
Copper mining was known as the important sector for economics. Therefore, it represents anessential in all living organisms that is required in small amounts (Heath, 1995). Humans, otheranimals, fish and shellfish require 5-20 micrograms per gram (µg/g) for carbohydrate metabolism
and the function of more than 30 enzymes. However, too much of copper concentration which
exceed 20 microgram per gram (µg/g) will be toxic, was explained by Wright and Welbourn (2002)
and Bradl (2005). Copper has been documented as one of the most toxic metals to aquatic organismand ecosystem (Scudder et al., 1998; Bradl, 2005; Carreau and Plye, 2005). Heavy metalⒸ ISERD22

IJERD – International Journal of Environmental and Rural Development (2015) 6-1

contamination of copper (Cu) is a big concern for environment, human and aquatic organisms.
Copper (Cu) does not break down in the environment because of that it can accumulate in to plantand animals.
The United States Environmental Protection Agency (US.EPA) has issued a guideline for
conducting early-life-stage toxicity test suitable for acute and chronic toxicity tests used formeasuring the aggregate toxic chemicals in an effluent or receiving water to freshwater and marineorganisms (US. EPA, 2002). In Cambodia, the information on the impact of toxicity effects ofsoluble copper on the tropical aquatic biota is limited. Therefore, many research papers weredesigned and conducted on ecotoxicology on copper everywhere in the world, but most them were
the different species and different from local species in the Mekong River. The out coming of
toxicity tests for copper will contribute data to aquatic environments and ecotoxicologicalfreshwater system for environmental quality standard in order to help and protect the MekongRiver in Cambodia as well as Mekong River Basin.
OBJECTIVES
The objective of studying is focus on ecotoxicology of copper on freshwater biota of Nile tilapia(Oreochromis niloticus) and Chironomids species (Chironomus javanus) with Mekong River inCambodia.
METHODOLOGY
Sampling
This study was conducted in the Lower Mekong Basin of Cambodia which located in the Kampong
Cham Province (Fig. 1).

Fig. 1 Map showing the sampling location
Organisms
Native species of Mekong River fish Nile tilapia (O. nileticus) larvae were obtained from theDepartment of Fisheries, Khon Kaen, Thailand. The tested fish larva was immediately collected
after hatching in oxygenate bags to the laboratory and handle properly to minimize injury andstress physiological in order to reduce the number of dead organisms. The test was conducted at
Ecotoxicology laboratory in Khon Kaen University. Average weight of Nile tilapia wasⒸ ISERD

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IJERD – International Journal of Environmental and Rural Development (2015) 6-1

9.717±0.040 mg which used for acute toxicity testing. Young organisms are often more sensitive
than adults. For this reason, the use of early life stages such as fish fingerling is required for alltests. In a given test, all organisms should be taken from the same source in order to minimize theorganisms diversity of response to experimental materials (US.EPA, 2002).
Chironomids Species (C. javanus) midge larvae were cultured at ecotoxicology laboratory ofKhon Kaen University, Thailand. Organisms were cultured in a glass container, covered with a net
to trap emerging adults. Since the aquarium already contains male and female species, mating andproduction of eggs is possible. To produce eggs of similar age, each egg mass collected was placedon the beaker containing 25 mL of tap water that was aged overnight. After two days, when alleggs were hatched, larvae were transferred on a 14 inches x 10 inches x 6 inches aquarium andgiven fish flakes for food. This was used as substrate. Overlying water was being replaced every
three days. The second instar organisms were placed individually in micro test tubes file with test
solutions. Tube was added, just enough for the organisms to create their own tubes.
Chemical and Test Procedure
The standard stock solution (100 mg/L) for studied metals was freshly prepared by dissolving ofcopper sulfate CuSO4 5H2O. The test organisms were subjected to different concentrations (450,500,600 1000, 1500 µg/L) for the fish and (500, 800, 1000, 3000, 5000 µg/L) for C. javanus of thestock copper solution in each container. The control was kept in experimental water without adding
copper. Water quality parameters (temperature, DO, alkalinity, hardness and pH) used in containers
were periodically determined before toxicity test (table 2). In addition, the experimental medium
was aerated in order to keep the amount of DO not less than 6 mg/L (Ezeonyejiaku et al., 2011).
Acute Toxicity Test
Acute copper toxicity experiments were performed for a 4-d period (96h) using small fishes at 5days old and the second instar larva of (C. javanus). The number of dead organisms were counted
every 24 hours and removed from aquarium as soon as possible. During the toxicity test, organisms
were not fed. The experimental were performed at room temperature of 25± 1 °C, with aPhotoperiod of 12h light: 12 darkness. All control result in lower mortality, less than 10% which
revealed the acceptability of the test (US. EPA, 2002).
Water Quality
The water quality parameters measured during the test were pH 7.77 ± 0.02, Conductivity 191±1.53 µS/cm, TDS 45±005mg/L, dissolve oxygen 10.46±0.05mg/L, and total hardness (mg2+ andCa2+) 88±4 mg/L as CaCO3. The mean value of other water quality parameters such as DOC, BOD
and alkalinity were 5.74 ±0 mg/L, 1.33±0.20 mg/L and 104±0 mg/L, respectively (Table 1).
Table 1 Water quality parameter of Mekong River for acute toxicity of copper on Nile tilapiaand C. javanus
Parameters
pH
Temperature (°C)
DO (mg/L)
EC (µS/cm)
TDS (mg/L)
Alkalinity (mg/L)
Hardness (mg/L)
BOD (mg/L)
DOC (mg/L)

ResultWater quality of Mekong River
7.77±0.02
28±0.0310.46±0.05
191±1.53
45±0
104±0
88±4
1.33±0.20
5.74± 0Ⓒ ISERD24

US. EPA, 2002
6.8-8.4
25±1
Above 80%-

IJERD – International Journal of Environmental and Rural Development (2015) 6-1

Statistical Analysis
All the results were carried out by analysis of variance (ANOVA) with Duncan’s Multiple Rang Test(DMRT) by Statistic 8 software (version 8, USA) on the Factorail experiment in Completely
Randomized Design (CRD). The significance was reported at p<0.05 levels. Toxicological doseresponse data involving quintal response (mortality) following toxicity of copper on the test species,
Nile tilapia and C. javanus larvae were determined by use of Probit Analysis LC50 Determination
Method (SPSS, version 19 software). The rate response determined at the end of the 96th hour.
Significance in 95% confidence interval (95%CI) of detected 96 hours LC50 value was determinedusing the Chi-Square technique (Ezeonyejiaku et al., 2011).
RESULTS AND DISCUSSION
The data from acute toxicity test of copper for Nile tilapia (O. niloticus) and C. javanus larvae
released that the mortality of organisms increased with increasing copper concentration andexposure time (Table 2). Copper was found toxic to Nile tilapia responding higher than C. javanus.
The Mean value 24, 48, 72 and 96 hr LC50 for CuSO4 5H2O in the Nile tilapia were 1228, 1052,
939 and 742 µg/L. Hence, the acute test of 96, 72, 48 and 24 hr showed an opposite relationshipbetween LC50 and exposure time, increase in the concentration reduces the time to kill 50% of thetilapia fish. Previous studies (Taweel et al., 2013) showed the LC50 96 hr value for copper was
1093 µg/L on the fingerline Tilapia fish (O. niloticus). However, this result is higher than presentstudy. Various authors in different parts of the world including tropical (Shuhaimi-Othman et al.,2013; Mastin and Rodgers, 2000, Taylor et al., 1991) have observed same and recorded differential
toxicity of heavy metal compounds against different test animals. Cusimano et al., (1986) andSolbe (1984) also reported that the acting metal (copper) might be due to the physicochemical
characteristics of the test medium, species and ages of fishes used and their susceptibility rates tothe test chemical.
Mean 24, 48, 72 and 96 hr LC50 values for Cu as CuSO4 5H2O in the C. javanus are presented
in the table 2. The 24 hr LC50 for CuSO4 was 8237 µg/L which sharply declined to 5033, 2206 and853 µg/L at 48, 72 and 96 hr, respectively. Also, it could be noted that Copper effect to the C.
javanus in the long time exposure. The 48 hr LC50 for CuSO4 was 1073 µg/L in C. ramousus third
instar larvae (Majumdar and Gupta, 2012) and the 96 hr LC50 in C. tentans and C. ramousus 170
and 183µg/L, respectively (Shuhaimi-Othman et al., 2013; Majumdar and Gupta, 2012). Thesevalues are considerably lower than responding value in C. javanus, indicating higher vulnerability
to Cu in the former two species. Mastin and Rodgers (2000) reported that 48 hr LC50 of midge
larvae C. tentans exposed to Clearigate and Cutrine-Plus were 373.5 and 460.9 µg/L, respectively.
On the other hand, Copper sulfate was an order of magnitude less toxic to C. tentans than
Clearigate and Cutrine-Plus with a mean 48 hr LC50 1,136.5 µg/L (Mastin and Rodgers, 2000).
Percentage mortality of Nile tilapia (O. niloticus) and C. javanus larvae to copper solution
with Mekong River water for 96 hours are presented in Fig. 2. From the concentration- response
obtained, it could be seen that C. javanus was more sensitive than Nile tilapia. Lower thresholdresponses of C. javanus and Nile tilapia were 538 and 462.5 µg/L, respectively. And upper
thresholds of response of CuSO4 with 100% mortality were observed for C. javanus and Nile tilapia
at 5095 and 1595 µg/L, respectively.
Table 2 LC50 Value of CuSO4 5H2O, for Nile tilapia and C. javanus
SpeciesNile tilapia
C. javanus

24h
1228
(1138-1340)
8237(5471-32105)

LC50 with 95% CL (µg/L)
48h
72h
1052
939
(890-1296)(771-1185)
5033
2206
(3035-88359)(-)

96h742(562-981)
853(-)

CL= Confidence limit, LC50= Median lethal concentrations, (-) = 95% Confidence limit (lower-upper value) exposure
at 96 hoursⒸ ISERD

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IJERD – International Journal of Environmental and Rural Development (2015) 6-1% Mortality

Nile tilapia
100
9080
70
60
50
40
30
20
1000500

1000

1500

2000

Copper concentrations (µg/L)

Fig. 2 Percentage mortality of Nile tilapia and C. javanus larvae to copper solution with
Mekong River water for 96 hours
CONCLUSION
Present study indicated that mortalities for both species increase with increasing copper but LC50
value decreased as more toxic on Nile tilapia and C. javanus. The 96 hr LC50 for CuSO4 5H2O in
the Nile tilapia and C. javanus were 742 and 853 µg/L, respectively. C. javanus was found moresensitive than Nile tilapia to copper.
Copper sulphate is still commonly used in the study area and in many developing countries tocontrol the water pollution. It is also used as fungicide in agricultural fields study area where fromit could be transported into the freshwater ecosystems via surface run off. Therefore, the hightoxicity of CuSO4 to non- target organisms like aquatic organisms needs to be taken into carefulconsideration before applying it indiscriminately in freshwater system. Due to the lack of the dataon ecotoxicology of copper on the local species of Mekong River, The further studies on theconcentration of heavy metals in other tropical freshwater should be studied.
ACKNOWLEDGEMENTS
I would like to thank to Integrated Water Resource Management Research and Development
Center in Northeast Thailand, Khon Kaen University and the research collaboration project
between Loyola University Chicago and Khon Kean University supported by International Copper
Association, Ltd. (ICA).
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