IJERD – International Journal of Environmental and Rural Development (2010) 1–1

Research article
erd

Development of the Indigenous Chironomid Species as
Ecotoxicology Test: Tool for Water Quality Management inThailandATCHARAPORN SOMPARN
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand
E-mail: somparn_a@yahoo.com

CHULEEMAS BOONTHAI IWAI
Faculty of Agriculture, Khon Kaen University, Khon Kaen, Thailand

BARRY NOLLER
The University of Queensland, Centre for Mined Land Rehabilitation,
Brisbane, Australia
Received 21 December 2009

Accepted 5 March 2010

Abstract In Thailand, heavy metal contamination of the aquatic ecosystem is of concern
because of the effect on water quality and toxicity to aquatic biota. The aim of this study
was to develop ecotoxicology test protocols using a standard test organism the midge,
Chironomus striatipennis Kieffer for measuring acute and chronic toxicology.. This
species is an important indigenous freshwater invertebrate in Thailand. The acute effectsof cadmium using cadmium chloride and lead as lead nitrate on midge larvae were
investigated by static bioassay under laboratory conditions. The observed mortality data of
the acute test for median lethal concentration 48 h LC50 of cadmium chloride on first,
second, third and fourth instars of midge larvae were 25.1 (14.6-40.7), 201 (60.1-367), 579(537-620) and 641 (564-717) mg/L, respectively. The 48 h LC50 of lead on first, second,
third and fourth instars of midge larvae were 99.9 (14.7-209), 55.5 (266-897), 1,540(1,200-1,870) and 3790 (2,890-4,410) mg/L, respectively. The results showed that
cadmium chloride and lead nitrate were highly toxic to the first instar larvae followed bysecond, third, and fourth instar larvae, respectively. Sublethal studies of cadmium and leadon midge showed that both cadmium and lead had effects on the body length of larvae, thedevelopment time of larvae, dry weight and the number of female and male adults. The
results from this study would be useful for developing a biomonitoring tool for heavy
metal contamination assessment in freshwater ecosystem and water quality management inThailand.
Keywords ecotoxicology test, heavy metal, freshwater management

INTRODUCTION
Aquatic ecosystem contamination with heavy metals has been an increasing concern in Thailand,
due to their persistence and high toxicity to many living organisms when they exceed thresholdconcentrations (Chen et al., 2007; Be´chard et al., 2008). The impact of heavy metals on theecosystemare significant as they cannot be completely removed from water by natural self
purification in aquatic ecosystems and could accumulate in overlay water and in sediment and thus
enter the food chain (Loska and Wiechuła, 2003) and subsequently cause ecotoxicologicalproblems and then affect human health. Because of the great importance of this issue, there is anurgent need to have practical tools for water quality management and ecological risk assessment inThailand. However, the study of ecotoxicology is quite new and has many limitations, especiallythe availability of ecotoxicological data on aquatic organisms in Thailand. As with many
developing countries, we have relied upon overseas data (US.EPA, ATSM, OECD, etc.) for© ISERD

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developing ecotoxicology test guidelines. However, these guidelines may not be suitable for
Thailand. Because indigenous aquatic organisms in Thailand may be more or less sensitive tocontaminants than surrogate species, the characteristic of sediment and water in Thai river systems
may differ from other temperate locations.
Chironomid species have been adopted as test species for ecotoxicological assessments
because they are an important component of freshwater ecosystems and represent one of mostwidely distributed group of benthic organisms. They are adapted to almost every type of aquatic
ecosystem (Vos, 2000). These species are important as prey for juvenile and adult fish and aquaticbirds and can be easily cultured under laboratory conditions with a relatively short life cycle(Taenzler, 2007). Two species of Chironomus are typically used in ecotoxicity testing: these are C.
tentans and C. riparius (Watts and Pascoe, 2000). Unfortunately, these species are few in numberin Thailand freshwater ecosystems. Therefore, the use of indigenous species to assess contaminatedaquatic ecosystems should be investigated because the toxic response of such species represents
more accurately the adverse effect of pollution on the tropical invertebrate species in Thailand. In
addition, standardized protocols for acute and chronic aquatic toxicity testing should be developed
using indigenous species.
The aim of this paper is to study the ecotoxicology of cadmium and lead on the indigenous
freshwater invertebrate Chironomus striatipennis, a non-biting midge (Diptera; Chironomidae)
found in Thailand. The result would be a useful component in developing a biomonitoring tool
using an indigenous species test for evaluation heavy metal contamination in aquatic ecosystems
and provide useful information for pollution control authorities involved in water quality
management in Thailand.
MATERIALS AND METHODS
Test organism
Initially, a local chironomid species was found in Thailand, Chironomus striatipennis Kieffer,isolated from the field and cultured under control laboratory conditions at the Ecotoxicology andEnvironmental Sciences Laboratory, Faculty of Agriculture, Khon Kaen University, Thailand. C.
striatipennis were cultured in 3 L glass tanks of 15 x 30 x 30 cm3 in the aquarium and covered withnetting to trap the emerging C. striatipennis. Overlying water was replaced every three days. Eachegg mass was collected and placed in a 1 L beaker containing 500 mL of aerated water. When all
eggs hatched, larvae were transferred in to a clean beaker and cultured until the larvae reached asuitable stage and were ready for use in experiments. The test species was given fish food at a rateof 0.5 g/L for food and conditions were maintained at 23±2 oC, on a 16:8 h light: dark regime withpH of 7-8 and a dissolved oxygen (DO) concentration of 5-7 mg/L.
Test chemicals
The test heavy metals were cadmium chloride (CdCl2 99.5%) and lead nitrate ([Pb(NO3)2] 99%).
Stock solutions were prepared by dissolving the heavy metal directly in distilled water and test
solutions were prepared by appropriate dilution immediately before each experiment.
Acute toxicity test
Acute toxicity tests were conducted in order to calculate the Cd and Pb LC50 data of first, second,
third and fourth instars of midge larvae. All experiments were performed according to the US.EPA(1991) method for determining 48 h LC50 values for C. striatipennis. Five dilutions were prepared
for Cd: 200, 400, 600, 800 and 1,000 mg/L, and 6 for Pb: 100, 200, 300, 400 and 500 and 1,000
mg/L. Three replicates of 10 midge larvae per treatment plus a control were used. The larvae were
exposed in 20 mL glass beakers containing 10 mL for each test concentration and control and were
undertaken as static bioassay under laboratory conditions. Test organisms were not fed during the© ISERD

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testing period. Observations were made after 24 and 48 h, and results recorded. For water quality
purposes DO, pH, electrical conductivity (EC) and temperature were monitored at the beginningand end of the test. Mortality data were used to calculate 48 h LC50 values and 95% confidence
intervals by Probit analysis with the Statistical Analysis System (SPSS) Version 12 statisticalsoftware.
Chronic toxicity test
Chronic toxicity testing for heavy metal effects on midge larvae followed the procedure
recommended by USEPA document 6004-91/002 (1994). Based on the acute toxicity result, C.
striatipennis were exposed to Cd concentrations of 0, 0.5, 2.5 and 5.0 mg/L. Lead concentrations of0, 5, 25 and 50 mg/L were used.
The effects of Cd or Pb on the growth of midge larvae were evaluated by using 60 midge
larvae and 300 eggs to study the effect on emergence of adult, length, the development time anddry weight of larvae. These were randomly sampled from each treatment and control and were
exposed in 500 mL glass beakers for static bioassays containing 300 mL for each test
concentration. Test organisms were fed with fish food 0.5 g/L. The water quality at the beginningand end of the test on control and treatments were measured. Data from chronic tests were analyzed
using analysis of variance (ANOVA) to detect significant differences between treatment andcontrol. Statistical analysis was performed using SPSS Version 12 statistical software.
RESULTS AND DISCUSSION
Acute toxicity
Table 1 shows the estimated 48 h LC50 for Cd and Pb that were calculated from standard toxicity
tests on the first, second, third and fourth instars of C. striatipennis. The results of this investigation
demonstrated that the 48 h LC50 values of first to fourth instars of C. striatipennis for Cd
concentrations were between 25.1 - 641 mg/L and for Pb concentration between 99.9 - 3,720 mg/L,
respectively. The 48 h LC50 value of Cd on second, third and fourth instars of C. striatipennis were8, 23 and 25 greater than for first midge larvae, respectively. The 48 h LC50 value of Pb on second,
third and fourth instars of C. striatipennis were 6, 15 and 31 fold greater than for first midge larvae,
respectively.
On the basis of the experimental LC50 values from this study, C. striatipennis were moretolerant to the heavy metals Cd and Pb than some other Chironomid species (Bechard et al., 2008;
CCME, 2006; Milani et al., 2003; USEPA, 1996; Watts and Pascoe, 2000). Comparison of the LC50
values showed that C. striatipennis was more sensitive to Cd and Pb than other aquatic insects suchas stonefly and mayfly (William et al., 1985). Acute toxicity results from this study showed higher
Cd toxicity on C. striatipennis than found for Pb and LC50’s in similar reported findings (CCME,
2006; Suedel et al., 1997; US.EPA, 1996). The observed LC50 values of Cd and Pb showed hightoxicity to the the first instar larvae followed by the second, third, and fourth instar larvae,
respectively. The results were similar to other reported findings (Gills and Wood, 2007; Hooftman
et al., 1989). This study showed that the second instars of midge larvae were a suitable stageorganism to assess the ecotoxicology test because of its sensitivity as a test organism, suite size andbeing stronger than other instars of midge larvae. Williams et al. (1986) also reported that thesecond instar of midge was useful to assess the ecotoxicological test.
Chronic toxicity
Table 2 shows that the effect of sublethal Cd and Pb concentrations on emergence of the adult werereduced significantly (P<0.05) and that the number of females and males at 2.5 mg Cd/L and 50
and 5 mg Pb/L, respectively, were reduced. The LOEC of the number of emergent female and malewas 2.5 mg Cd/L and NOEC was 0.5 mg Cd/L. The LOECs of Pb concentration for the number of© ISERD

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IJERD – International Journal of Environmental and Rural Development (2010) 1–1

emergent females and males were 50 and 25mg Pb/L; a NOEC of 25 mg Pb/L was obtained for thenumber of emergent females.
Table 1 Acute toxicity (Medium lethal concentration [LC50]) of Cd and Pb on larvae of C.
striatipennis at 48 h
Larvae instar
First
SecondThird
Fourth

48h LC50 of Cd (95% C.I)
25.10 (14.61 - 40.67)
201.30 (60.05 - 367.03)
579.31 (536.74 - 620.07)
641.12 (564.30 -717.21)

48h LC50 of Pb (95% C.I)
99.89 (14.68 - 209.13)
555.49 (265.69 - 896.62)
1,539 (1,201- 1,874)
3,720 (2,890 - 4,405)

Table 2 Effect sublethal Cd and Pb concentrations on the number of female and male C.
striatipennis (n=300)
Chemical
Cd

Pb

Concentration(mg/L)
Control
0.5
0.6
5.0
Control
525
50

Number of emergence (number)
FemaleMale
6.83±0.75c
7.17±0.75b
6.50±1.05c
6.83±1.17bb5.50±0.55
5.33±0.82a
4.50±0.55a
5.50±0.84ab
6.83±0.75
7.17±0.75bc
6.83±1.47b
7.50±1.05cb6.67±0.82
6.33±0.82ab
4.83±0.75a
5.50±0.55a

Emergence (%)
Female
Male
2.20
2.39
2.17
2.28
1.83
1.78
1.50
1.83
2.28
2.39
2.28
2.50
2.22
2.11
1.61
1.83

Note: Values are mean ± standard deviation. Means with the same letter in the same column are not significantly
different (P>0.05)

The effect of sublethal Cd and Pb concentrations on length, development time and dry weightof C. striatipennis are shown in Table 3. Length of midge larvae in the control and 3 additional
concentrations of 0.5, 2.5 and 5 mg Cd/L and 5, 25 and 50 mg Pb/L showed no significant
reduction (P>0.05) in length of the first to fourth instars of C. striatipennis.
The development time of the second, third and fourth instars of C. striatipennis was reduced
significantly (P<0.05) at 0.5 mg Cd/L and higher concentrations. Lead concentrations at 0.5, 5 and25 mg/L significantly reduced (P<0.05) the development time of the first, third instars, and secondand fourth instars of C. striatipennis, respectively (Table 3). The development time is also an
important endpoint for the chironomid as it reflects larval growth to development emergence datathat were also similar to those found in other studies (Sikanchadra and Crane, 2000; Suedle et al.,1997). For the development time at each Cd concentration on the second, third and fourth instar of
C. striatipennis, a lowest observed concentration (LOEC) of 0.05 mg/L was obtained and for Pb
concentrations on the second and third instars, a LOEC of 5 mg and 50 mg/L was found while the
LOEC of the third instar was 50 mg/L and no observed effects of concentration (NOEC) for Pb at
25 mg/L were obtained.
No difference in reduction of the dry weight (P>0.05) of C. striatipennis was observed
between control and treatment with Cd concentration but in the fourth instars of C. striatipennis
there was a significant reduction on dry weight (P<0.05) with Pb at 5 and 25 mg/L (Table 3). The
LOEC of Pb concentration on fourth instar was 50 mg/L.
Table 3 shows that the effect of sub lethal Cd and Pb concentrations significantly reduced
emergence of the adults (P<0.05) and that the number of females and males at 2.5 mg Cd/L and 50
and 5 mg Pb/L, respectively, were reduced. The LOEC of the number of emergent females andmales was 2.5 mg Cd/L and NOEC was 0.5 mg Cd/L. The LOECs of Pb concentration for thenumber of emergent of female and male were 50 and 25mg Pb/L; a NOEC of 25 mg Pb/L was
obtained for number of emergent females.
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Table 3 Effect of sub lethal Cd and Pb concentrations (mg/L) on the body length (mm), thedevelopment time (days) and the dry weight (mm) of instar larvae of C. striatipennis(Effect)
InstarControl

Concentration of Cd (mg/L)
0.5
2.5(Length)
1st
2.21±0.46
2nd
3.88±1.17
3rd
8.60±1.83
4th
6.65±0.47(Development time)
1st
6.65±0.47
2nd
3.17±0.38a
3rd
4.33±0.48d
4th
8.67±0.75c(Dry weight)
1st
0.03±0.01
2nd
0.10±0.00
3rd
0.11±0.03
4th
0.30±0.01

5Control

Concentration of Pb (mg/L)
5
25

50

1.21±0.30
2.31±0.42
4.00±1.26
8.72±1.80

1.17±0.24
2.16±0.41
3.41±0.73
7.91±2.03

1.20±0.30
2.27±0.40
3.61±1.14
8.17±2.08

1.18±0.25
2.21±0.46
3.88±1.17
8.60±1.83

1.21±0.29
2.24±0.37
3.68±1.06
8.22±1.95

1.13±0.22
2.06±0.31
3.24±0.78
7.61±2.16

1.04±0.13
1.88±0.34
3.01±0.51
7.59±2.09

6.63±0.66
3.50±0.50b
4.00±0.22c
8.17±0.38b

6.62±0.51
3.50±0.50b
3.83±0.13b
7.50±0.97a

6.62±0.42
3.50±0.50b
3.67±0.48a
7.33±1.11a

7.00±0.47d
3.17±0.38b
4.33±0.48b
8.67±0.75c

6.50±0.50c
3.00±0.12a
4.33±0.75b
7.17±0.38b

6.00±0.23b
3.00±0.00a
4.17±0.91b
7.00±0.11a

5.00±0.12a
3.15±0.36b
3.67±0.48a
7.18±0.39b

0.03±0.00
0.07±0.02
0.16±0.00
0.26±0.02

0.04±0.00
0.15±0.06
0.12±0.05
0.24±0.02

0.04±0.01
0.11±0.05
0.10±0.00
0.21±0.10

0.03±0.01
0.10±0.01
0.11±0.03
0.30±0.01a

0.03±0.01
0.05±0.01
0.16±0.03
0.21±0.10b

0.04±0.00
0.08±0.01
0.26±0.01
0.31±0.00a

0.05±0.00
0.08±0.01
0.17±0.11
0.42±0.05b

Note: Values are mean ± standard deviation. Means with the same letter in the row are notsignificantly different to control (P>0.05)

CONCLUSION
The development of a method using an indigenous Chironomid species, C. striatipennis, as abioindicator and aquatic organism for ecotoxicology tests is reported as a biomonitoring tool forevaluation of heavy metal contamination in the aquatic ecosystem of Thailand. Compared to lead,
cadmium had higher acute toxicity to the first instar larvae followed by second, third, and fourth
instar larvae of C. striatipennis, respectively. Sub lethal effects of Cd and Pb concentration
significantly reduced the length of larvae, the development time for larvae, dry weight and thenumber of emergent females and males. These endpoints are also important for Chironomids
because they reflect larval growth and the development of the emergent Chironomid species and its
abundance in the aquatic ecosystem.
The results showed that C. striatipennis was sensitive to Cd and Pb concentrations. Thus thisspecies is suitable as a bioindicator and as a test organism for aquatic ecosystem assessment.
However, revision of water quality guidelines is required to provide more precise and conclusive
toxicology data for heavy metals on other indigenous aquatic organisms.
ACKNOWLEDGMENTS
The authors wish to express their sincere thanks to the research project participants: Graduate School,
Groundwater Research Center, Development Center for Integrated Water Resource Management inNortheast Thailand, Research Centre for Environmental and Hazardous Substance Management(EHsM), National Center of Excellence for Environmental hazardous Waste Management Khon Kaen
University (Thailand), Association of Environmental and Rural Development (AERD) and Prof. Dr.
Machito Mihara for special support.

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