IJERD – International Journal of Environmental and Rural Development (2015) 6-2

Research articleerd

Ecotoxicology of Copper on Freshwater Fish with Different
Water Hardness on the Mekong River, Lao PDR
NAKSAYFONG KHOUNNAVONGSA
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand
Email: nakstudent@gmail.com

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

THAM C. HOANG
Institute of Environmental Sustainability, Loyola University Chicago, Chicago, USA
Received 23 February 2015

Accepted 11 September 2015(*Corresponding Author)

Abstract The aquatic resources of the Mekong River are important to support the livelihoods ofa large percentage of 60 million or more people living in the Lower Mekong Basin. A study on
the impact of heavy metals on tropical freshwater fish in the Mekong River is needed.
Ecotoxicology of copper on freshwater fish was studied using field-collected water from two
local sites along the Lower Mekong Basin in Lao PDR, which focused on two different water
hardness. In this study, US EPA method was used for the acute toxicity test to larvae Labeorohita with 7 different copper concentrations (0, 0.02, 0.09, 0.16, 0.23, 0.3, and 0.37 mg/L) in
moderately hardness of water (108±0.00 mg/L as CaCO3) and (0, 0.02, 0.03, 0.04, 0.05, 0.09,
and 0.13 mg/L) in soft hardness of water (20±2.83 mg/L as CaCO3). The mortalities of fish
were observed at 24, 48, 72, and 96 hr. The results clearly showed that amount of mortalities
were increased from low to high copper concentrations. The 96-hr LC50 values of copper onlarvae L. rohita with moderately hardness and soft hardness of water were 0.106 and 0.038
mg/L, respectively.
Keywords acute toxicity, copper, Labeo rohita, the lower Mekong basin

INTRODUCTION
The Mekong River is the 12th longest river in the world in terms of volume of water. The river runs forapproximately 4,350 km from China through five countries, namely Myanmar, Lao PDR, Thailand,Cambodia, and Viet Nam. The millions of people living in different parts of the Lower Mekong Basinrely on some extent on the water resources of the Mekong River Basin for food supply andsustainability of their livelihoods (MRC, 2010a), due to the fact that the resources have potential tocontribute to economic development of the countries. If not properly planned, managed, and monitored,
it could also exert tremendous pressure on the basin’s ecological health, livelihoods, and water quality.
Copper in the aquatic environment is usually related to anthropogenic sources rather than naturalsources (McNeely et al., 1979). Industrial sources of copper include mining, electroplating, petroleum
refining, metal works, and foundries. Copper is a micronutrient for both plants and animals at low
concentrations and is recognized as all plants and animals (Kapustka et al., 2004). Concentrations ofcopper have been reported from 0.03 to 0.23 µg/L in surface seawaters and from 0.20 to 30 µg/L in
freshwater systems (Bowen, 1985). However, it may become toxic to some forms of aquatic life atelevated concentrations.
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Water hardness is one of the water qualities, usually expressed as mg/L CaCO3. It substantiallyaffects metal toxicity but seemingly has little effect on the toxicity of organic chemicals (Sprague,1985; Mayer, 1988; Inglis, 1972). The relation of water hardness to metal toxicity concerning the lethal
tolerance of freshwater fish to copper, is mainly dependent to the hardness of the water; as theincreasing hardness reduces the uptake rate of copper by gill tissue. One hypothesis suggested thatcalcium-magnesium hardness may act intrinsically upon cell-membrane permeability at the gills (Spearand Pierce, 1979). Therefore, the objective of this study was carried out on the tropical fish species
Cyprinidae, Labeo rohita to evaluate acute toxicity of copper with different water hardness tocontribute the ecotoxicology data for the management of aquatic environment towards environmental
quality standard revision in Lao PDR.
METERIALS AND METHOD
Location and sampling: The study sites were conducted at two different locations along the Lower
Mekong Basin in Lao PDR by focusing on different water hardness. The first study site was Vientiane,
the capital city (Lao PDR), located at 17°58´1.18´´N/ 102°35´1.66´´E. The city is around 692,900
inhabitants with a density of 176 people per km2 (ADB, 2000). Municipal sewage is normallydischarged into That Luang wetland and is consequently discharged into the Mekong Riverdownstream. The second study site was Pakxan district, located at 18°22´23.29´´N /103°39´43.85´´E.
Sources of pollution of this district included domestic, industrial waste, agricultural runoff, and mining
activities. The sites were chosen based on proximity and activities that potentially polluted the MekongRiver.
Reagents: All laboratory glassware, polyethylene, and polypropylene were soaked in 10% HNO3 acidfor at least 48 hr and rinsed with distilled water more than 3 times prior using. Deionized (DI) waterfrom a Millipore Milli-QTM ultra-pure (<18.2 MΩ/cm) water system was used throughout the study.
Water hardness was measured by EDTA titration method. A standard solution of copper 1000 mg/L
(Spectrosol grade, Merk) was prepared and diluted 100 mg/L with DI water for acute toxicity test.
Test specimen: Tropical freshwater fish, juvenile Cyprinidae Labeo rohita used in this investigation
were obtained from the hatchery maintained by Khon Kaen Department of Fisheries, Thailand. The test
specimen was conducted at Ecotoxicology Laboratory in Khon Kaen University. Juvenile L. rohita
were acclimated with tap water by a portable pump for a period of 48 hr at 25⁰C±1⁰C and arewere
more sensitive to toxicants than adults, appeared healthy, behaved normally, fed well, and had low
mortality in cultures12a. Therefore, juvenile L. rohita 1-14 day(s) of age were used for acute toxicity
test. At the beginning of bioassay, 25ºC±1ºC at a temperature control room with 16-hr light, 8-hr
darkness photoperiod. Moreover, copper concentrations for each test chambers were determined basedon the factorial experiment in Completely Randomized Design (CRD). One fish was randomly selected
at a time from the aquaria to complete the experiment of 7 treatments and 4 replications of 20 fish at
200 mL of test solution volume.
Data analysis: The acute toxicity of copper was determined as the median lethal concentration (LC50)
for 96 hr in each exposure time, with 95% confidence intervals. The analysis used the Probit Analysis
function of SPSS Version 17, statistical software.
RESULTS AND DISCUSION
Percent mortality of larvae Labeo rohita was evaluated at soft hardness and moderately hardness ofwater (20±2.83 and 108±0.00 mg/L as CaCO3). After being exposed in different periods of time,copper concentrations are resulted in highly significant difference (P<0.01), as presented in (Tables 1ⒸISERD

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IJERD – International Journal of Environmental and Rural Development (2015) 6-2and 2). The LC50 values of copper to larvae L. rohita at 24, 48, 72 and 96-hr for 20±2.83 mg/L as
CaCO3 were 0.083 (0.069-0.106), 0.065 (0.06-0.071), 0.055 (0.045-0.069), and 0.038 (0.021-0.077)
mg/L; and for 108±0.00 mg/L as CaCO3 were 0.271, 0.157, 0.129, and 0.106 mg/L, respectively. The
result of LC50 values with 95% confidence limits of copper are summarized in (Table 3) which clearly
indicates that at 20±2.83 mg/L as CaCO3 soft hardness of water was more toxic than 108±0.00 mg/L as
CaCO3 moderate hardness to larvae L. rohita.
Table 1 Percent mortality of larvae Labeo rohita at 20±2.83 mg/L as CaCO3 soft hardness at
exposure time with different copper concentrations% mortality on exposure time

Exposure concentration, (mg/L Cu)

24hr
48hr
72hr
96hr
00±0f
0±0e
4±3e
10±4e
0.02
1±3ef
3±3e
10±6e
18±10de
0.03
6±3df
14±3d
20±4d
24±9d
cd
cd
0.04
11±521±324±329±5dccc
0.05
14±8
25±4
39±6
54±10c
0.09
46±3b
69±5b
75±9b
80±9baaa
0.13
86±591±398±3100±0a
F-test
***
***
***
***
C.V. %
17.63
9.71
13.717.08
Note:Values
Value
mean
SD (n=4).
Difference
littersaindicate
a significant
difference(p<0.01)
byNote:arearemean± SD±(n=4).
Difference
litters indicate
significant
difference (p<0.01)
by one-way
ANOVA
analysiswith lsdanalysis
comparison
one-way
ANOVA
with lsd comparison.

Table 2 Percent mortality of larvae Labeo rohita at 108±0.00 mg/L as CaCO3 moderately
hardness at exposure time with different copper concentrations% mortality on exposure timeExposure concentration, (mg/L Cu)
00.02
0.09
0.16
0.23
0.3
0.37F-test
C.V. %

24hr
3±3d4±8d5±0d
13±3d40±8c
59±10b76±9a***
24.38

48hr
4±5d10±11cd
14±3c19±3c63±9b95±4a96±5a***14.472hr
6±6e15±14de
21±3cd26±3c66±9b98±3a99±3a***14.7296hr
9±9e20±17de
30±0cd34±6c
68±9b
99±3a100±0a***
16.11

Note: Values are mean ± SD (n=4). Difference litters indicate a significant difference (p<0.01) by one-way ANOVA
analysis with lsd comparison

Acute toxicity of copper in soft water hardness with copper concentration 0.13 mg/L, themortality at 96 hours of exposure was 100%. On the other hand, 100% mortality at 96 hours inmoderately water hardness was observed at copper concentration 0.37 mg/L, due to the toxic effect ofheavy metals on freshwater organisms related to hardness (Kim et al., 2001; Plye et al., 2002; Rathorand Khangarot, 2003; Markich et al., 2006).

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Comparing the effect of copper at two different water hardness on larvae L. rohita, it wasobserved that copper at the soft water had high toxic to the fish with LC50 value of 0.038 (0.021-0.077)
mg/L, whereas copper at the moderately hardness of water was less toxic with LC50 value of 0.106
mg/L. It could be seen that the tropical freshwater fish was potentially sensititive to toxic elements.
Fish can have a quick response to various copper concentrations in the form of increased surface
movements were observed in the first few hours of the experiment, which normalized within 24hr ofexposure, thereafter fish settled on the bottom of the chamber. The response of fish mortality withexposure time is shown in Fig. 1. In freshwater, as water hardness increased, heavy metal toxicity
decreased due to competition between metal ions and calcium and magnesium ions for the uptake sitesof organisms (Javid et al., 2007; Kim et al., 2001; Pyle et al., 2002; Ebrahimpour et al., 2010).
According to Penttinen et al., (1998), the uptake of calcium and magnesium ions by the cell membranecaused it to stabilize, and this reduced its permeability to metal ions. Water hardness reduced metal
toxicity by saturating gill surface binding sites with Ca2+ to the exclusion of metal cations (Pyle et al.,2002).
Table 3 Lethal concentration (LC50 to 95% confidence limits) of copper to larvae Labeo rohita
at exposure time
LC50 (Medium Lethal Concentration) mg/L
95% Confidence Limits for conc.
24hr
48hr

72hr

96hr

20±2.83 mg/L as CaCO3
soft hardness

0.083(0.069-0.106)

0.065(0.06-0.071)

0.055(0.045-0.069)

0.038(0.021-0.077)

108±0.00 mg/L as CaCO3
moderately hardness

0.271-0.157-0.129-0.106-

Note: (-) =95% Confidence limit (lower-upper value exposure timeFig. 1 Relation of percent mortality of larvae Labeo rohita to exposure copper concentrations
with 2 different water hardness.

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CONCLUSION
The toxicity of copper on larvae Labeo rohita under low water hardness was higher than in high water
hardness. The results of this toxicity tests for copper contribute to ecotoxicology data to themanagement of aquatic environment towards environmental quality standard revision in Lao PDR.
ACKNOWLEDGEMENTS
This research project was funded by Integrated Water Resource Management Research andDevelopment Center in northeast Thailand, Khon Kaen University; and the research collaboration
project between Loyola University Chicago and Khon Kaen University supported by International
Copper Association, Ltd. (ICA).
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