IJERD – International Journal of Environmental and Rural Development (2013) 4-1

Research article
erd

Acute Toxicity of Textile Metal Complex Dark Green Azo
Acid Dye (53) and Anionic Surfactant Oil on Nile Tilapia,
Oreochromis niloticus and Bioconcentration of Total
Chromium and Copper in Gill Tissues
HILMA RANTILLA AMWELE
Khon Kaen University, Khon Kaen, Thailand

RAKPONG PETKAM*
Khon Kaen University, Khon Kaen, Thailand
Email: rakpong@kku.ac.th

FREDERICK WILLIAM HENRY BEAMISH
Burapha University, Bangsaen, Chonburi, Thailand

KANIT CHUKANHOM
Khon Kaen University, Khon Kaen, Thailand
Received 13 December 2012

Accepted 30 January 2013(*Corresponding Author)

Abstract The experiment was conducted on Nile tilapia Oreochromis niloticus todetermine the lethal concentration of textile metal complex dark green azo acid dye in thepresence and absence of anionic surfactant oil during an exposure period of 96 hr. The
design consisted of four treatments: control, metal complex dark green azo dye, a mixtureof metal complex dark green azo acid dye with anionic surfactant oil and anionicsurfactant oil; and each was applied at four concentrations (1%, 5%, 10% and 15% ofstock solution). Data were analyzed by using Probit analysis. The 96 hr LC50 was 7.25%(95% CL = 6.55-7.96) of stock solution for metal complex dark green azo acid dye, 2.85%(95% CL 2.14-3.90) for the mixture of metal complex dark green azo acid dye withanionic surfactant oil and 7.27% (95% CL = 6.45-8.43) stock anionic surfactant oil. Total
chromium concentration in the gill filaments of tilapia increased linearly with dyeconcentration and was independent of anionic surfactant oil. Contrary, total copper
concentration in the gill tissue increased linearly in with the present of anionic surfactant
oil. The lethal effect observed during the trial was dose depended, the mortality rateincreased with the increase in treatments concentration.
Keywords mortality rate, LC50, dyebath, surfactant oil, aquatic animal

INTRODUCTION
Synthetic dyes and anionic surfactant oil contain substances potentially harmful to the environment
and are important in the production of textiles (Christie, 2007). In Northeast of Thailand, there isevidence that the silk textile community discharge dye effluent without treatment, raising
environmental concern (Thailand Institute of Science and Technological Research, 2004). One of
the synthetic dye that cause environmental concern is a metal complex azo acid dye, commonly
called azo dye and classified as acid dye that contain the potentially toxic metals copper, cobalt,
aluminum, iron, nickel and chromium (Clark, 2011; Jo et al., 2010; Christie 2001; Hunger, 2003).
These metals bind with dye ligands such as sodium sulfate and provide colour stability and firm
attachment to the yarn (Clark, 2011; Adachi, 2004; Christie, 2001).
Metal complex azo acid dyes are commonly used to dye silk and cotton yarn and are appliedin neutral to weakly acidic baths (Christie, 2007). Surfactant oil is used for removing the sericin orsilk gum, improves the sheen, colour and texture of the silk yarn. In addition, it is also used forⒸ ISERD

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IJERD – International Journal of Environmental and Rural Development (2013) 4-1

colour uniform and accelerates dye firm attachment to the yarn (Chen et al., 2010). Azo dyes havebeen associated with skin and eye irritations in humans and carcinogenic, mutagenic and lethal
effects in other organisms (Christie, 2007; Bae et al., 2006; Popma and Masser, 1999). Potentiallyharmful effect of metals, particularly the heavy metals, copper, and chromium, to aquaticorganisms include impaired growth, reproduction (Segner, 2011; Hayat, 2007) and in extreme
cause death (Eisler, 1998). This is explicit in water quality criteria (Eneji, 2007).
Acute lethal concentrations vary among metals, their chemical forms and other ambient
factors. In Thailand, maximum allowable concentrations of total copper (0.1 mg/L) and chromium(0.05 mg/L) for surface water (Pollution Control Department Ministry of Natural Resources andEnvironment, 2007). Some metals rarely, if ever, occur in water at lethal concentrations but even atlower concentrations may, impose chronic deleterious effects particularly in the presence of othersubstances (Eisler, 1998).
Nile tilapia, Oreochromis niloticus, is a freshwater and tolerant to harsh condition such as
water relatively low in dissolved oxygen and high pH (Delong et al., 2009 and Popma and Masser,1999).
The objective of this study was to determine the lethal concentration (96 hr LC50) of textile
metal complex dark green azo acid dye (53), surfactant oil and a mixture of dye with surfactant oil
to Oreochromis niloticus and bioconcentration of total chromium and copper from the dye andanionic surfactant oil in gill tissues.
MATERIALS AND METHODS
Nile tilapia (n= 630) of 6.4 to 7.9 g live weight and 7.5 to 8.3 cm total length, were obtained from
Khon Kaen University, fisheries farm. Fish were held in static dechlorinated water (50 L) that wascontinuously aerated for 7 days. Ambient conditions included Dissolved oxygen (≥4  mg/l), watertemperature (26.5 - 28.1 °C), pH (6.5 - 7.2), Total dissolved solids (89.4 mg/l) and salinity (0.09ppt), Alkalinity (47 mg/l), NH3 (0.01 mg/l) and NH4+ (0.74) (APHA, 2005). Fish were fed ad
libitum daily with commercial feed (Gorbest Cooperation Co. Company). No mortality wasrecorded during this period. After 7 days, fish were exposed to experimental concentrations ofsurfactant oil, dark green azo acid dye and a mixture of dark green azo acid dye with surfactant oil.
Metal complex dark green azo acid dye (53) and surfactant oil used in this study were obtained
from Chonnabot dye shop, (Khon Kaen Province, Thailand) and analyzed (EPA, 1996) for total
copper (1.09 mg/l) and chromium (5314 mg/l). Water solubility of the azo dye was 64.3 g/l wereanalyzed using methods EN 14362-1 described in (British Standard, 2003). Surfactant oil analyzed
for total copper was present at 0.002 mg/l and total chromium was present at 0.049 mg/l, sulfate (asSO42-) was present at 553.82 mg/l, alkyl benzene sulfonate was present at 3.57 mg/l (APHA, 2005)
and solubility of surfactant oil in water was > 1 kg/l.
A completely randomized design was applied and consisted of 3 replicates of each of 13
treatments. Fish were held in 39 plastic tanks, each containing 50 L of dechlorinated andcontinuously aerated water. Stocking density was 15 fish / tank (5 kg/m3).
The stock solution was prepared according to dyebath procedure used by community members
in Chonabot. Dye stock solution contained 65 g of metal complex dark green azo acid dye,
dissolved in 1 L of hot water and boiled for 5 minutes, allowed to cool at a room temperature andthe solution was made up to 30 L with tap water. Stock solution with mixture contained 65 g ofmetal complex dark green azo acid dye with 20 ml of anionic surfactant oil, while a stock solution
for anionic surfactant oil only contained 20 ml of oil. Experimental concentrations for each of thethree stock solutions (100%) were 15, 10, 5 and 1%. Control treatment was normal tape water.
Fish were not fed for 4 days during the trial. Number of dead fish were recorded and removed
at 24, 48, 72 and 96 hr. The 96 hr LC50 values were determined by Probit analysis (SPSS software
11.5 program).
Total chromium and copper were measured in gill tissue of fish from each of the threetreatments and four concentrations (1%, 5%, 10% and 15% of stock solution) and of control
treatment. After 96 hrs two fish that did not survived and two live fish that survived were collectedⒸ ISERD

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from each tank of three replicates, from which gill tissue were collected, from six of the 45 dead orlive fish. Ground gill tissues of 1 g was digested in a solution of 7 ml nitric acid (65%) and 1 mlhydrogen peroxide (30%), heated at 105 °C in a microwave, cooled, filtered (Whatman no. 1) andanalyzed for total copper and chromium using Inductively Coupled Plasma – Optical Emission
Spectroscopy (ICP-OES) (EPA, 1996; AOAC, 2005). Total chromium and copper
bioaconcentration factor was calculated according to the standard method of fish recommended by
OECD (1996) and USEPA, (1996): Bioconcentration factor (BCF) is a ratio of concentration of
metal in fish gills (µg/kg) / Concentration of metal in water (µg/l).
The LC50 value was determined by probit analysis method by SPSS software 14 program(Finnery,   1971).   The   percentages   for   corrected   fish   mortality   rate   were   determined   by   Abbot’s  formula (Abbott, 1925). Corrected mortality (%) = (X – Y / X) 100; Where, X = percentages of live
fish in control sample and Y = percentages of live fish in treatment sample.
RESULTS AND DISCUSSION
Lethal toxicity
No mortalities occurred in any of the control tanks over the 96 hr experimental period. Exposure tometal complex dark green azo acid dye in the absence of anionic surfactant oil produced no
mortality rate at a concentration of 1 % and 36% fish mortality rate at 5 % concentration, while themortality rate was 91% at 10% concentration and 96% at 15% concentration within 96 hr. The fishmortality rate of fish exposed to a mixture of metal complex dark green dye with anionic surfactant
oil was 2% at 1% concentration and 100% mortality rate occurred at 5, 10 and 15 % concentrationsin 24 hr. No fish mortality rate occurred at the lowest concentration of 1% anionic surfactant oil
only, 2% mortality rate occurred at 5% concentration and 100% fish mortality occurred at 10 and
15 % concentrations within 24 hr. Clearly, the lethal effects of all treatments of metal complex darkgreen azo acid dye, the mixture of metal complex dark green azo acid dye with anionic surfactant
oil and anionic surfactant oil reflected a concentration rather than exposure time effect.
The 96 hr LC50 for dye without anionic surfactant oil was 4.71 g metal complex dark green
azo acid dye per liter, equivalent to 7.25% ( 95% CL = 6.56 - 7.96) of that in the stock solution.
The lethal toxicity of the mixture of dye and surfactant oil was calculated to contain 1.85 g metal
complex dark green azo acid dye per liter, equivalent to 2.85 % ( 95% CL = 2.14 - 3.90) of that in
the stock solution, much less than that in the absence of oil. The mixture of oil and dye wascalculated to contain 0.57 ml/l of oil at the lethal toxicity, equivalent to 2.85% of that in the stock
solution. The LC50 of anionic surfactant oil alone was 1.45 ml/l, equivalent to 7.27 % (95% CL =6.45-8.43) of that in the stock solution and well above that calculated at the lethal toxicity of the oiland dye mixture.
Acute toxicity of metal complex dark green azo acid dye increased in the presence of
surfactant oil. It is interesting that for each of metal complex dark green azo acid dye and anionicsurfactant oil the lethal toxicities represented almost equal proportions of their respective stock
solutions. This indicates the mixture to be proportionately more toxic than the additive toxicities ofmetal complex dark green azo acid dye and surfactant oil.
Water quality parameters in experimental tanks were in the range recommended for tilapia
survival rate and production (DeLong et al. 2009), where, dissolved oxygen 4.6±0.4 mg/l - 6.7±0.3
mg/l, pH 6.7 ± 0.1 – 7.1 ± 0.2, temperature 27.8±0.3 ºC – 28. 3 ± 0.0 ºC. Total dissolved solids
were the higher when dye was present and increased directly with concentration from 95 ± 0.3 to
165 ± 0.2 mg/l and similar pattern occurred with the concentrations of mixture of metal complex
dark green azo acid dye with anionic surfactant oil were 96 ± 0.2 to 161 ± 0.2 mg/l. The anionicsurfactant oil was expected to increases overall solubility of dye in solution. However, total
dissolved solids increased with dye concentration with values being almost identical to those for
mixtures of metal complex dark green azo acid dye and anionic surfactant oil at comparable
concentrations. Total dissolved solids associated with surfactant oil alone increased slightly from91 ±0.1 to 96 ± 2.4 mg/l with ambient concentration. This implies the toxic qualities of metalⒸ ISERD

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complex dark green azo acid dye may not relate to its solubility but instead to total concentration.
Bioaccumulation of total chromium and copper in O. niloticus gills in 96 hr exposure periods
Table 1 Mean total chromium and copper concentration in pooled gill samples
from O. niloticus after 96 hr exposure to concentrations of metal complex dark green azo acid dye (D),a mixture of metal complex dark green azo acid dye with anionic surfactant oil (SOD) and anionic surfactant
oil (SO)

Total Cr concentration (µg/kg)
Concentration

D

SOD

SO

Cr

Cu

Cr

Cu

Cr

Cu

0

72

900

72

900

72

900

1%

354

809

300

862

191

704

5%

508

760

530

953

116

771

10%

534

713

600

999

92

1109

15%

757

994

750

1222

125

1042

Total chromium concentration in gill tissue increased with dye concentration between 0 and 15 %of stock solution in each of the two treatments containing metal complex dark green azo acid dye(Table 1). Some chromium was present in the gills of fish from untreated water (controls). Total
chromium concentration in gill tissue correlated significantly (P < 0.05) with dye concentration
when exposed only to metal complex dark green azo acid dye (n = 5, r = 0.91) and equally on
exposure to a mixture of metal complex dye and oil (n = 5, r = 0.93). These relationships aredescribed by the regressions: Metal complex dark green azo acid dye only: Cr (µg/kg) = 36.49
stock solution (% Total) + 218.79 (R = 0.82). The mixture of metal complex dark green azo aciddye with anionic surfactant oil: Cr (µg/kg) = 39.28 stock solution (% Total) + 206.87 (R = 0.86).
Thus, from the equations the rate of total chromium concentration is similar for the two treatments,on the contrary, in gill tissue exposed to anionic surfactant oil only (n = 5, r = 0.11) did notcorrelated significantly (P > 0.05) as expressed by regressions as Cr (µg/kg) = -0.81 stock solution(% Total) + 124.24 (R = 0.01).
Total copper was detected in the gills of fish exposed to normal condition. Total copper
accumulation in gill tissue correlated significantly (P < 0.05) in fish gills exposed to a mixture ofmetal complex dye with anionic surfactant oil (n = 5, r = 0.94) and those exposed to anionicsurfactant oil (n = 5, r = 0.74). These relationships are described by the regressions, where, amixture of metal complex dark green azo acid dye with anionic surfactant oil: Cu (µg/kg) = 21.06
stock solution (% Total) + 856.65 (R = 0.88) and the anionic surfactant oil: Cu (µg/kg) = 20.26
stock solution (% Total) + 779.57 (R = 0.55). Therefore, from the equations the rate of total copper
accumulation is similar to the treatment with a present of anionic surfactant oil. However, in gilltissue exposed only to metal complex dark green azo acid dye (n = 5, r = 0.26) did not correlated
significantly (P > 0.05) as described by regressions, where, Cu (µg/kg) = 4.77 stock solution (%Total) + 805.61 (R = 0.07).
Total copper and chromium concentrated in O.niloticus gills exposed to normal condition(Table 1) might come from the water as copper was detected at 0.4 µg/l which is below
recommended 50 µg/l for water consumption and 20 µg/l for aquatic animal production while
chromium was below detection level 50 µg/l recommended for water consumption in Thailand(Pollution Control Department, 2007). Generally metal occurs in naturally water from theweathering of rocks, industrial discharges and leakage (Eneji, 2011; Yilmaz et al. 2011). Fishuptake the metal across body surfaces and thin epithelium of gill filaments become concentrated ingills as their outer layer is negatively charged and attracts positively charged metallic ions (Heath,1995). Prior to the experiment fish were fed with commercial feed. Normally fish are supplemented
with copper and chromium in feed because essential elements for animal health at the low
concentration (Abdel-Baki et al., 2011; Watanabe, 1997) but can inflict deleterious effects at higherⒸ ISERD

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concentrations (Eisler, 1998).
Chromium concentration in the gill filaments of tilapia increased linearly with dyeconcentration and was independent of anionic surfactant oil. Total chromium bioconcentration
factor (BCF) in a present of metal complex dark green dye concentrations was 6.71 (1%), 2.03(5%), 1.22 (10%), 1.54 (15%) and in control was zero. Similar pattern occurred in fish exposed to
mixture of metal complex dark green dye with anionic surfactant oil concentrations were 5.18 (1%),
2.43 (5%), 1.55 (10%), 1.41 (15%), and in the present of anionic surfactant was zero. Hence, theconcentration of total chromium was higher in fish gills than in water. Despite similar chromium
accumulations in the gills at comparable dye concentrations the presence of anionic surfactant oil
increased lethality in a synergistic manner. It is interesting that Hickey (1990) found some heavy
metal toxic in the presence of SO42-. As noted earlier the surfactant oil used in this study contained
a considerable amount of SO42- which could relate to synergistic response of the mixture of metal
complex dark green azo acid dye and surfactant oil.
Contrary, total copper concentration in the gill tissue increased linearly in the present ofanionic surfactant oil and also increased lethality in a synergistic manner. In addition, the BCF for
total copper in the present of mixture of metal complex dark green dye with anionic surfactant oilconcentration at were 57 (15%), 38 (10%), 20 (5%) in a control and at 1% concentration was zero,in the present of anionic surfactant oil was 329 (15%), 38 (10%) but in control, 5%, and 1%
concentration was zero, while in a present of metal complex dark green dye at 15% was 147 and at
10%, 5%, 1% and 0% stock solution was zero. Similarly, the concentration of copper was higher infish gills than in water.
Generally, it is found that surfactant action activates Cu permeability through Na + uptake
pathway in gills (Grosell et al., 2007). In a present study lethal toxicity was supported by Sharma etal. (2009) finding that methyl red composed of Cd, Cu, Ni less toxic to fish. Sharma et al. (2006)
also found that the LC50 of methyl red dye to be 27.2 mg/l to Poecilia reticulate. Fish mortality rateoccurred during the exposure of period was not based only on copper and chromium but from theinteraction of chemical used during the study.
In Thailand the estimated contaminant from dyeing 1 kg silk yarn in Chonnabot community
was COD was 6315 mg, SS 121 470 mg and DSS 248 180 mg (Thailand Institute of Science andTechnological Research, 2004). The study indicated that waste from silk dyeing are harmful andcan accumulate in tissues to O. niloticus, the accumulated chemical can be transferred to humanthrough fish consumption that potentially caused negative effect on human health in a long run.
CONCLUSION
The metal complex azo acid dye and surfactant oil used by the community for silk dyeing areharmful to O. niloticus. Therefore, results of the present study strongly suggest that unmanaged
discharge of effluent from silk dyeing, especially those that employ azo dyes is likely to impose
harmful effects to aquatic ecosystems and recommend the wastewater to be handle with care.
Therefore, this warrants further research.
ACKNOWLEDGEMENTS
We express our sense of gratitude to Khon Kaen University, Thailand International Development
Cooperation Agency (TICA), Namibia National Council for High Education for financial support.
We also express our sincere appreciation to Dr. Peerapol Sukon for facilitation and guidance on
acute toxicity. Sincere thanks to Siriporn Tola for guidance and support.
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