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Chemosphere 69 (2007) 4854 www.elsevier.


A comparative study on characterization of textile wastewaters (untreated and treated) toxicity by chemical and biological tests
K.P. Sharma

, S. Sharma b, Subhasini Sharma b, P.K. Singh a, S. Kumar a, R. Grover a, P.K. Sharma a

a b

Botany Department, University of Rajasthan, Jaipur 302004, India Zoology Department, University of Rajasthan, Jaipur 302004, India

Received 10 November 2006; received in revised form 14 April 2007; accepted 25 April 2007 Available online 20 June 2007

Abstract Toxicity of textile wastewaters (untreated and treated) and their ingredient chemicals was quantied in terms of their chemical characteristics, sh (Gambusia anis) mortality and end point growth responses of duckweed (Lemna aequinoctialis) in short-term bioassays. Other parameters of sh bioassay were erythrocyte morphology and its counts. Despite of a denite correlation between data of biological tests (LC/EC50 values) with that of chemical tests, biological tests were found to be relatively more sensitive to both wastewaters and ingredient chemicals. Amongst all the examined parameters of test organisms, sh RBCs (morphology and counts) sensitivity to pollutants in the wastewaters was usually maximum and therefore, their study should be included in the routine sh bioassay. Other advantage of biological test such as on Lemna is even detection of eutrophic potential of wastewaters, as noted at their higher dilutions. The ingredient chemicals (major) contributing maximum toxicity to textile dye wastewater were, acids (HCl and H2SO4), alkali (Na2O SiO2), salt (NaNO2) and heavy metal (Cu), whereas dyes (4) were relatively less toxic. 2007 Elsevier Ltd. All rights reserved.
Keywords: Textile wastewaters; Fish bioassay; Duckweed bioassay; Dyes; Copper

1. Introduction Today, pollution of aquatic habitats is a universal phenomenon, which is more serious in the developing countries on account of discharge of mostly untreated or partially treated municipal and industrial wastewaters into them. The relatively higher toxicity of industrial wastewaters to living organisms, especially plants, is of greater concern since they clean aquatic ecosystems. The industries of concern are; textile printing industry, tanneries, distilleries, sugar industry and paper and pulp industry, since they discharge large volume of wastewater in the environment.

Corresponding author. Tel.: +91 9828418664; fax: +91 01412711654. E-mail address: sharmakp_in@yahoo.com (K.P. Sharma).

Amongst them, the textile printing industry which grew at much faster pace in the developing countries due to cheaper labour and less stringent waste disposal norms is of greater concern. For example, the magnitude of this industry in India can be adjudged from the fact that it contributes almost one-third of the total export and employ 35 million people directly (Chavan, 2001). Textile wastewater, which is a by-product of textile industry, is a mixture of colorants (dyes and pigments) and various organic compounds used as cleaning solvents, plasticizers etc. It also contains high concentrations of heavy metals, total dissolved solids, and has higher chemical as well as biological oxygen demand. Thus, textile wastewater is chemically very complex in nature. Its discharge had led to complete disappearance of submerged and free-oating hydrophytes, and also selected marshy

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K.P. Sharma et al. / Chemosphere 69 (2007) 4854


species in the pools and a wastewater drain in the industrial area of Sanganer town, Jaipur. Algal species richness also decreased markedly in them. These water bodies were also devoid of fauna (Chaturvedi et al., 1999; Sharma et al., 2001). Thus, textile wastewaters are highly toxic to both ora and fauna. The highly complex nature of textile wastewaters may prove to be a limitation factor for complete assessment of toxicity with chemical analysis. Biological tests in combination with chemical analysis may however, be useful in deciphering toxicity as they express living material response to the total eect of actual and potential disruption (Chen et al., 1999). Besides, such tests also assist in developing precautionary measures and strategy for environmental management (Slabbert, 1996). Biological tests made on organism at dierent trophic levels in the food chain may prove to be useful in forecasting impacts at ecosystem level. Phytoplankton, duck weeds (Blinova, 2000) and submerged macrophytes (Lee et al., 1998; Rai et al., 2003; Kumar and Prasad, 2004) are the test materials for monitoring toxicity of man-made chemicals on primary producers in the aquatic ecosystems, which have an additional advantage of detecting even stimulatory role of pollutants. However, duckweed such as Lemna is the rst choice of ecotoxicologists because it is wide spread, fast growing and reproduce faster. It is sensitive to many pollutants, which are assimilated from the growing medium (or aquatic environment) through the underside of the leaf (Greenberg et al., 1992; Becker et al., 2002; Sharma et al., 2006). Among aquatic fauna, sh is favoured over other animals and their mortality is the sole criteria for quantifying pollutant toxicity (Sharma et al., 2005a,b). Recently, Sharma et al. (2006) reported higher sensitivity of sh RBCs (their counts and morphological abnormalities) to an azo dye methyl red in comparison to their mortality, and hence they may also be explored for quantifying textile wastewaters toxicity. With this background, we have detailed physico-chemical characteristics of textile wastewaters (untreated and treated) of Sanganer, Jaipur, including of ingredient chemicals, in relation to their toxicity to duckweed and sh (mortality and RBC), to quantify potential threats in terms of discharge limits imposed by the regulatory authority for conventional parameters. 2. Materials and methods Textile wastewaters released at dierent steps of printing in Sanganer, Jaipur were collected separately in clean plastic cans and transported immediately to the laboratory. These were: bleach wastewater (chlorine bleaching using CaOCl2), screen wash (released during washing of screen after manual printing) and wastewaters discharged at dierent steps of xing dyes by diazotization and silicate processes. During diazotization process, indigo and rapid dyes are xed on the printed cloth by soaking them in a mixture of acid (hydrochloric/sulphuric acid) and sodium

nitrite (commonly referred to as Pass) for about 15 min. Whereas soaking printed cloth for 12 h in the concentrate solution of sodium silicate bind reactive dyes in silicate process. Thereafter, three washings of printed cloth remove bleeding dyes and chemicals. Textile wastewaters released at dierent steps of printing process are nally discharged (mostly untreated) in the pools and drains. Since unlike drains, pollutants discharged during a day in textile printing process get mixed in the pools, therefore their waters were collected in the morning (prior to initiation of printing processes on the next day) to quantify cumulative toxicity of them. Also collected physico-chemically (FeSO4 + slaked lime + polyelectrolyte) and biologically treated dye wastewaters from euent treatment plants (ETP) in Sanganer, Jaipur, to quantify toxicity reduction. The biological treatment process which has been patented by Sharma and Sharma (2006) comprises of neutralization of textile dye wastewaters (30 00035 000 l/day) with slaked lime in the equalizer chambers followed by microbial degradation of dyes and other organic pollutants rst in a bioreactor (I day Retention Time) and then in rhizosphere of constructed wetland of Phragmites karka (2 day RT) and nally coagulation of suspended impurities by alum (100 mg/l) and polyelectrolyte in a clarier and their removal through sand lter. Physico-chemical characteristics of wastewaters were analyzed within 24 h of collection using standard methods (APHA, 1989). We examined their toxicity to a freshwater sh Gambusia anis (Baird and Gerard) as described earlier (Sharma et al., 2003). After 96 h exposure, autopsy was done for RBC counts, blood smear preparation (Lee et al., 1993) and examination of their internal organs. Almost 200 RBCs in 20 microscopic elds (10x 100x) were observed to quantify morphological abnormality in a treatment (control/wastewater). Lemna aequinoctialis (Welwitsch) population maintained in the unpolluted concrete tanks of University Botanical Garden was the source of plant material. Experiments were set in both green house (temperature = 28 3.5 C) and growth chamber (temperature = 25 5 C). Dierent dilutions (using tap water) of wastewater samples (both untreated and treated) were lled in (two-third) separately into 1.5 l sized plastic tubs (greenhouse study)/200 ml sized plastic glasses (growth chamber study), while tap water in the control sets. Fifty Lemna plants (100 fronds) were added in a tub, while 20 plants (40 fronds) in a glass. Frond number and oven dry weight of plants (at 60 C for 7 days) were measured after 10 days in ve replicates of a treatment. Toxicity of wastewaters was examined at three dierent occasions and data presented are their mean values. We also performed Lemna assay on ingredient chemicals (analar grade) of textile wastewaters of Sanganer viz. hydrochloric acid, sulphuric acid, sodium nitrite, copper (present in wastewaters of diazotization process), sodium silicate (silicate process) and few selected dyes such as C.I. Direct Blue 86, C.I. Direct Yellow 44, C.I. Drimarene


K.P. Sharma et al. / Chemosphere 69 (2007) 4854

Yellow L2R and Methyl Red (C.I. 13020). Their stock solutions prepared in distilled water were diluted with tap water for experiment. To analyze role of nutrients on toxicity, stock solutions of methyl red and copper sulphate were diluted with Hoagland medium and the controls were also run in Hoagland medium. This treatment is referred to as nutrient rich hereafter in the text. A similar study made using tap water as a diluent of stock solution of these two chemicals is referred to as nutrient poor hereafter in the text. Among chemical ingredients, sh toxicity assay was made only for acidity (pH = 5.5 for HCl), copper and methyl red. LC (for sh mortality) and EC50 values (for RBC count, poikilocytosis, frond number and dry weight of Lemna) were calculated using COMPAQ personal computer BASICA version 1.13. They are mean values (with standard deviation) of data obtained independently at three different occasions for most of the textile wastewaters. 3. Results and discussion 3.1. External and internal injuries 3.1.1. Fish The darkening of body and pronounced secretion of mucous (a defensive mechanism) were the two most noticeable sh response in the textile wastewaters and ingredient chemicals. Dyes were deposited over the external (gills) and internal organs (lateral line and digestive system) of sh only in methyl red, and screen and silicate wastewaters. The gills were hemorrhaged at higher concentrations in all treatments. Additional injuries in the pass (wastewater of diazotization process) were whitening of eye-balls, and erosion of scales from the skin and of tissue from pelvic and caudal ns. The dead sh had opened mouth and ared operculum possibly due to asphyxia on account of damaged gills. RBC counts decreased with an increase in concentration of copper, methyl red and textile wastewaters, when compared with control sh (58.9 3.3 104 mm3). Morphological abnormalities (poikilocytosis) in control sh RBCs (spherical and triangular) were relatively less (<5%), in

comparison to wastewater exposed sh, majority being triangular (3040%) and quadrilateral (3035%) while the rest were spherical, beaked, kidney, pentagonal and dumble shape (Fig. 1). Other abnormalities exclusively found in pass were their hypochromic nature and some even had vacuoles and damaged cell membrane. Thus, pass was most toxic to sh possibly because of maximum load of various pollutants and acidity (Table 1). EC50 values for poikilocytosis were generally minimum followed by values for counts and sh mortality. 3.1.2. Duckweed The immediate plant responses to higher concentrations of wastewaters (not in bleach) and ingredient chemicals were their fragmentation into singlets (paired frond into unpaired frond) and loss of roots, while fronds turned white in bleach waste. Other injuries were however, dose and time dependent, and were reduction (5070%) in size and thickness of fronds, their chlorosis initiated rst in mature one, but younger one in silicate wastewater and sodium silicate. Dyes were deposited on the frond (lower surface) and root in screen wash, silicate wastewater and dye treatments. The aforesaid injuries to sh and duckweed may be considered as biomarkers of textile wastewater toxicity, as they were also noted during bioassays of ingredient chemicals. 3.2. Relationship between physico-chemical characteristics, and toxicity of ingredient chemicals and raw wastewaters Acidic and neutral bleach having almost similar values for chemical oxygen demand (COD) diered largely in their toxicity to sh, the former having higher residual chlorine (3 folds) was 1014 folds more toxic in comparison to neutral one, whereas their toxicity to Lemna was almost similar, possibly due to their wide ecological amplitude (Table 1). The lowering of residual chlorine content (4.2 ppm) of neutral bleach wastewater equal to its LC50 value, along with its pH (5.5) however, resulted in instant death of sh. Contrast to this, control sh survived for 96 h at pH 5.5. This means acidity increases residual chlorine toxicity. Thus chemical analysis forecasted lesser

Fig. 1. Morphological abnormalities (Poikilocytosis) in the RBCs of wastewater exposed sh comparison to control after 96 h of the study (N = nucleus; R = RBC membrane).

Table 1 Physico-chemical characteristics of textile wastewaters of Sanganer and their toxicity to sh (G. anis) and Lemna Wastewaters Wastewater characteristics Fish bioassay (96 h) Mortality pH Bleach Acidic Neutral Screen wash Pass Raw Neutral I Wash Raw Neutral II Wash: Raw III Wash: Raw Silicate: I wash Pool waters 5.3 7.3 8.6 0.1 1.2 0.3 7.6 0.4 1.7 0.4 7.5 0.2 2.9 0.5 7.2 0.8 11.6 0.1 6.9 3.1 EC (mS) 1.55 10.74 4.9 .06 80.2 41.8 11.2 7.7 19.3 19.6 5.2 3.7 4.0 1.6 2.6 0.9 18.6 4.8 3.9 2.6 4.5 4.5 4.4 0.1 4.45 COD (ppm) 1200 1430 896 6 3963 1115 3651 1379 872 116 716 129 545 132 291 110 479 29 255 105 900 520 138 109 140.5 Cu (ppm) 88.75a 30.8a 0.15 0.2 45.7 34 6.0 1.4 4.5 1.6 1.2 0.5 3.7 0.4 2.8 0.6 Nil 0.86 0.4 0.3 Nil Traces Nil LC50 (%) 1.13 15.71 19.6 1.1 0.8 0.6 3.2 0.7 4.9 4.1 16.3 5.0 12.1 1.3 14.9 0.6 4.2 2.3 36 18 18.8 47.5 39 10 165.2 RBC K.P. Sharma et al. / Chemosphere 69 (2007) 4854 EC50 (%) 2.38 (5.71) 25.0 41.3 1.7 1.3 0.9 (0.78) 35.3 4.5 (0.8) 0.75 0.5 (0.8) NA (1.1) NA NA 2.9 1.0 (2.0) 39.7 22.6 NA (11.42) NA (25.1) 52.4 14 (8.2) 153.8 Frond number 23.6 25.7 89.1 71 1.0 0.8 5.0 0.3 5.5 4.4 34.6 28.9 55.2 3.4 107.4 14 6.3 2.6 53.3 31 139.4 NC 141.2 38.5 NC Dry weight 28.8 32.0 58 10 0.7 0.01 4.6 0.3 5.5 2.8 14.8 2.7 NA NA 8.4 2.2 66 23 55.3 119.5 148 64 170 Lemna bioassay 10th day (EC50: %)

Wastewater after treatment in euent treatment plants Untreated wastewater 8.7 Physico-chemical treatment 10.5 Biological treatment 7.6 1.0 After alum + sand lter treatment 8.54

Residual chlorine content; NA = not available; NC = not calculable; Data in parenthesis are for EC50 value for poikilocytosis.



K.P. Sharma et al. / Chemosphere 69 (2007) 4854

toxicity than what actually assessed during sh bioassay. The comparatively higher sensitivity of sh (lower LC50 values) may be attributed to rapid penetration of residual chlorine in to their gills, which may be slow in Lemna (hence low sensitivity), possibly due to presence of cell wall. Further, EC50 values for RBC counts and poikilocytosis were higher than LC50 values of sh suggesting RBC to be lesser sensitive to bleach. The higher sh mortality may be ascribed to impairment of gaseous exchange through greater damage to gills. The toxicity order of colored wastewaters to sh (based on LC50 value) was: pass > silicate water % I wash > II wash > III wash > screen wash > pools (Table 1). Further, their EC50 values for poikilocytosis (in RBCs) were usually minimum indicating greater physiological distress in sh, unlike bleach wastewater (Table 1). Acidic bleach water ranks almost similar to pass in terms of sh mortality while neutral one was closer to third wash, despite the fact that they diered largely in their physico-chemical characteristics. This fact also holds true for rst wash and silicate wastewater. Contrast to this, rst wash and screen wash almost similar in their COD values varied largely in their LC50 values. Thus, physico-chemical characteristics of wastewaters alone are not sucient to quantify their toxicity. With the exception of pass, although EC50 values of dye wastewaters for Lemna were higher (low toxicity) than LC50 values for sh, but general trend of their toxicity ranking was almost similar to sh: pass > I wash > silicate

water > pools > screen wash > II wash > III wash (Table 1). Thus, response of both the tested organisms to pollutants in wastewaters was almost identical, and therefore, they are ideal for quantifying their toxicity. During duckweed bioassay, frond number was found to be a more sensitive parameter in comparison to dry weight; suggesting that adverse eects on vegetative propagation of plants were more severe than possibly on their photosynthesis responsible for an increase in their dry weight. The higher toxicity of wastewaters of diazotization process may be ascribed to its major ingredients such as acids, sodium nitrite and copper, whereas of silicate wastewater to sodium silicate, as noted in Lemna and sh (Tables 2, 3). In contrast to major ingredient chemicals, dyes such as C.I. Direct Blue 86 (EC50 = 225 ppm), C.I. Direct Yellow 44 (EC50 = 1500 ppm) and Drimarene Yellow L2R (EC50 = 2150 ppm) were less toxic to Lemna whereas methyl red toxicity was at par with pool waters. Similar to bleach wastewater, methyl red was less toxic to the test organisms at pH ranging between neutral to slightly alkaline, in comparison to acidic one. Its toxicity also increased in combination with copper (Table 4). In contrast to copper, nutrients had no role in reducing methyl red toxicity (at lower pH) to Lemna. Methyl red loses its azo character a bit in acidic solutions on account of protonation resulting in its planar structure (Park et al., 2005), which may increase its absorption in sh in comparison to normal alkaline solution of

Table 2 Physico-chemical characteristics (range) of ingredient chemical treatments and their toxicity to Lemna (EC50) Chemicals Physico-chemical characteristics of varying dilutions (range) pH Acids HCl H2SO4 Salts NaNO2 Na2OSiO2 Tap water 2.86.5 2.86.4 8.18.3 9.910.1 7.0 EC (mS) 0.681.05 0.681.12 2.58.42 1.593.09 0.58 TH (ppm) 208210 207209 188236 5368 206 Na (ppm) 27 25 1452900 1251250 26 Duckweed bioassay (EC50) Frond no. 0.0052 M (pH = 3.8) 0.0025 M (pH = 4.4) 0.009 M (0.062%) 0.014 M (0.17%) Dry weight 0.0050 M (pH = 5.2) 0.0045 M (pH = 4.9) 0.015 M (0.096%) 0.029 M (0.36%)

Data in parenthesis indicate EC50 values of acids in terms of pH values, and of salts in percentage.

Table 3 LC and EC50 values (ppm) of methyl red and copper for sh and Lemna at dierent nutrient and pH levels of the growing medium Chemicals pH Fish bioassay Mortality Copper Nutrient poor medium Nutrient rich medium Methyl red Nutrient poor medium i. acidic ii. alkaline Nutrient rich medium 8.5 5.956.2 0.25 NA RBC 0.19 NA Duckweed bioassay Frond no. 0.47 5.48 Dry weight NC 3.13

6.0 7.48.7 5.45.9

7.3 54.8 NA

4.98 41.4 NA

12.1 23.2 33.9

8.5 56.4 3.2

NC = not calculable; NA = not available.

K.P. Sharma et al. / Chemosphere 69 (2007) 4854 Table 4 Toxic eects of methyl red (10 ppm), copper (3.0 ppm) and their combination at sub-lethal concentrations on Lemna counts (on 7th day), in comparison to control (nutrient rich medium) Treatments Methyl red Copper Methyl red + copper % Reduction (counts) 33 18 68


3.4. Correlation between physico-chemical characteristics and LC/EC50 values LC and EC50 values of both the test organisms showed signicant negative correlation with conductivity (r = 0.530.59), COD (r = 0.550.71) and Cu (r = 0.54) which was positive for pH (r = 0.590.97), to wastewaters of diazotization process (untreated and neutralized) and so, they are good physico-chemical parameters for characterizing toxicity. Pool water characteristics had signicant positive correlations (pH: r = 0.820.93; EC: r = 0.780.90; COD: r = 0.980.99) with LC50 and EC50 values of sh, which were negative for number and dry weight of duckweed (pH: r = 0.920.98; EC: r = 0.850.97; COD: r = 0.810.97). Thus, duckweed bioassay dened pool toxicity better when compared with sh. Only pH had signicant positive correlation (r = 0.58 0.98) with LC and EC50 values of sh and Lemna for biologically treated wastewaters, while their EC (r = 0.91) and COD (r = 0.89) values had signicant negative correlation with frond number. Present study thus established superiority of biological tests in deciphering wastewaters toxicity as compared to their chemical analysis. Various external and internal injuries noted during sh and duckweed bioassays may assist in rapid assessment of wastewaters toxicity in the natural ecosystems, whereas their EC/LC50 values in quantifying toxicity more accurately along with chemical attributes in the laboratory. Duckweed bioassay has an added advantage of detecting eutrophication potential of wastewaters. The greater sensitivity of sh RBCs to pollutants in dye wastewaters emphasizes inclusion of their study in routine sh bioassay. Present study documented increase in pollutants toxicity (residual chlorine, copper and methyl red) in the acidic medium. The higher alkalinity of silicate wastewater may similarly be toxic to Lemna, since it decreased after its neutralization (Soni, 2004). In view of these facts, we recommend discharge limit of textile wastewaters close to neutral. The considerations such as dilution factor and buering capacity of the recipient water bodies are of little signicance in monsoonic climate as that of India, where water ow (lotic system)/its volume (lentic system) remains low during most part of the year, except rainy season.

methyl red. The tested plant species may also respond similarly since Zhou (2001) has reported absorption of organic dyes in vegetables cultivated using textile dye wastewaters. This explains greater toxicity of methyl red in acidic solution. It is important to note that Lemna growth was promoted at lower concentrations of textile wastewaters of pools (<4080%), pass (<0.50.25%), I (<3%), II (<10%) and III (<25%) wash of diazotization process, possibly on account of higher concentration of nutrients (lterable reactive phosphorus = 1.913.5 ppm; total phosphorus = 5.016.4 ppm; NH3N = 1.4266 ppm; NOx = 5.6 67.2 ppm; organic nitrogen = 8.4506.8 ppm; K = 2.8 15.8 ppm) in comparison to control plants growing in the tap water (FRP and TP = traces; NH3N = nil; NOx = 4.0 ppm; organic nitrogen = nil; K = traces). The reduction in copper toxicity (higher EC50) in nutrient rich medium supports this inference. 3.3. Treated wastewaters Neutralization of pass and 1st wash with lime decreased their toxicity markedly, as evident by an increase in LC50 and EC50 values, respectively, for sh (515 folds) and Lemna (532 folds) (Table 1). This has been ascribed primarily to reduction in acidity and copper content which are found to be very toxic to the test organisms (Table 2). Physico-chemical and biological treatments decreased toxicity of wastewaters to the tested organisms, as also noted for their chemical characteristics (Table 1). As evident from LC/EC50 values, treated wastewaters were more (34 folds) toxic to sh and poikilocytosis was the most sensitive parameter. No denite trend was recorded for EC50 values of Lemna for frond number and dry weight. The biologically treated wastewater (after alum treatment) also stimulated Lemna growth at 25% dilution. Thus, Lemna bioassay detected toxicity and stimulation of wastewaters simultaneously, as also noted for untreated wastewaters. It is important to note that silicate wastewater (untreated) almost similar to physico-chemically treated wastewater in chemical characteristics were however, more toxic to the tested organisms. Similarly, reduction in toxicity of biologically treated wastewater was signicantly higher after alum treatment, while their physico-chemical characteristics varied little (Table 1). These ndings signify importance of biological tests in characterizing textile wastewater toxicity.

Acknowledgements We are thankful to the Council of Scientic and Industrial Research, New Delhi for awarding Research Associateship to Shweta Sharma, Ministry of Science and Technology, Department of Biotechnology, New Delhi for research grants to K.P. Sharma and Subhasini Sharma, and the Head, Zoology and Botany Departments, University of Rajasthan, Jaipur for laboratory facilities.


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