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Chemosphere 69 (2007) 48–54 A comparative study on characterization of textile
Chemosphere 69 (2007) 48–54 A comparative study on characterization of textile

Chemosphere 69 (2007) 48–54

Chemosphere 69 (2007) 48–54 A comparative study on characterization of textile

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

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

a Botany Department, University of Rajasthan, Jaipur 302004, India b 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


Toxicity of textile wastewaters (untreated and treated) and their ingredient chemicals was quantified in terms of their chemical char- acteristics, fish ( Gambusia affinis ) mortality and end point growth responses of duckweed ( Lemna aequinoctialis ) in short-term bioassays. Other parameters of fish bioassay were erythrocyte morphology and its counts. Despite of a definite correlation between data of biolog- ical tests (LC/EC 50 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, fish RBCs (morphology and counts) sensitivity to pollu- tants in the wastewaters was usually maximum and therefore, their study should be included in the routine fish 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 ingre- dient chemicals (major) contributing maximum toxicity to textile dye wastewater were, acids (HCl and H 2 SO 4 ), alkali (Na 2 O SiO 2 ), salt (NaNO 2 ) 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 phe- nomenon, which is more serious in the developing coun- tries on account of discharge of mostly untreated or partially treated municipal and industrial wastewaters into them. The relatively higher toxicity of industrial wastewa- ters to living organisms, especially plants, is of greater con- cern since they clean aquatic ecosystems. The industries of concern are; textile printing industry, tanneries, distilleries, sugar industry and paper and pulp industry, since they dis- charge large volume of wastewater in the environment.

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

0045-6535/$ - see front matter 2007 Elsevier Ltd. All rights reserved.


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 con- tributes 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 chemi- cal as well as biological oxygen demand. Thus, textile wastewater is chemically very complex in nature. Its dis- charge had led to complete disappearance of submerged and free-floating hydrophytes, and also selected marshy

K.P. Sharma et al. / Chemosphere 69 (2007) 48–54


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 flora 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 combina- tion with chemical analysis may however, be useful in deci- phering toxicity as they express living material response to the total effect 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 different trophic levels in the food chain may prove to be useful in forecast- ing 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 first 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, fish is favoured over other ani- mals 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 fish 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 waste- waters (untreated and treated) of Sanganer, Jaipur, includ- ing of ingredient chemicals, in relation to their toxicity to duckweed and fish (mortality and RBC), to quantify poten- tial threats in terms of discharge limits imposed by the reg- ulatory authority for conventional parameters.

2. Materials and methods

Textile wastewaters released at different steps of printing in Sanganer, Jaipur were collected separately in clean plas- tic cans and transported immediately to the laboratory. These were: bleach wastewater (chlorine bleaching using CaOCl 2 ), screen wash (released during washing of screen after manual printing) and wastewaters discharged at different steps of fixing dyes by diazotization and silicate processes. During diazotization process, indigo and rapid dyes are fixed 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 pro- cess. Thereafter, three washings of printed cloth remove bleeding dyes and chemicals. Textile wastewaters released at different steps of printing process are finally discharged (mostly untreated) in the pools and drains. Since unlike drains, pollutants discharged during a day in textile print- ing process get mixed in the pools, therefore their waters were collected in the morning (prior to initiation of print- ing processes on the next day) to quantify cumulative toxi- city of them. Also collected physico-chemically (FeSO 4 + slaked lime + polyelectrolyte) and biologically treated dye wastewaters from effluent treatment plants (ETP) in San- ganer, 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 (30 000–35 000 l/day) with slaked lime in the equalizer chambers followed by microbial degradation of dyes and other organic pollutants first in a bioreactor (I day Retention Time) and then in rhizosphere of con- structed wetland of Phragmites karka (2 day RT) and finally coagulation of suspended impurities by alum (100 mg/l) and polyelectrolyte in a clarifier and their removal through sand filter. Physico-chemical characteristics of wastewaters were analyzed within 24 h of collection using standard methods (APHA, 1989 ). We examined their toxicity to a freshwater fish Gambusia affinis (Baird and Gerard) as described ear- lier (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 fields (10 x · 100 x ) were observed to quantify morphological abnormality in a treatment (control/wastewater). Lemna aequinoctialis (Welwitsch) population main- tained in the unpolluted concrete tanks of University Botanical Garden was the source of plant material. Experi- ments were set in both green house (temperature = 28 ± 3.5 C) and growth chamber (temperature = 25 ± 5 C). Different dilutions (using tap water) of wastewater samples (both untreated and treated) were filled in (two-third) separately into 1.5 l sized plastic tubs (green- house 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 five replicates of a treatment. Toxicity of wastewaters was examined at three different occasions and data pre- sented 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) 48–54

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 toxi- city, 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, fish toxicity assay was made only for acidity (pH = 5.5 for HCl), copper and methyl red. LC (for fish mortality) and EC 50 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 stan- dard deviation) of data obtained independently at three dif- ferent 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 notice- able fish response in the textile wastewaters and ingredient chemicals. Dyes were deposited over the external (gills) and internal organs (lateral line and digestive system) of fish 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 fins. The dead fish had opened mouth and flared 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 com- pared with control fish (58.9 ± 3.3 · 10 4 mm 3 ). Morpho- logical abnormalities (poikilocytosis) in control fish RBCs (spherical and triangular) were relatively less (<5%), in

comparison to wastewater exposed fish, majority being tri- angular (30–40%) and quadrilateral (30–35%) 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 fish possibly because of maximum load of various pollutants and acidity ( Table 1 ). EC 50 values for poikilocy- tosis were generally minimum followed by values for counts and fish 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 (50–70%) in size and thickness of fronds, their chlorosis initiated first 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 fish and duckweed may be con- sidered 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) differed largely in their toxicity to fish, the former having higher residual chlorine (3 folds) was 10–14 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 LC 50 value, along with its pH (5.5) however, resulted in instant death of fish. Contrast to this, control fish survived for 96 h at pH 5.5. This means acidity increases residual chlorine toxicity. Thus chemical analysis forecasted lesser

chlorine toxicity. Thus chemical analysis forecasted lesser Fig. 1. Morphological abnormalities (Poikilocytosis) in the

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

K.P. Sharma et al. / Chemosphere 69 (2007) 48–54


Table 1 Physico-chemical characteristics of textile wastewaters of Sanganer and their toxicity to fish ( G. affinis) and Lemna


Wastewater characteristics


Fish bioassay (96 h)


Lemna bioassay 10th day (EC 50 : %)






EC (mS)

COD (ppm)

Cu (ppm)

LC 50 (%)

EC 50 (%)


Frond number

Dry weight

Bleach Acidic Neutral Screen wash Pass Raw Neutral I Wash Raw Neutral II Wash: Raw III Wash: Raw Silicate: I wash Pool waters




88.75 a 30.8 a 0.15 ± 0.2


2.38 (5.71)











8.6 ± 0.1

4.9 ± .06

896 ± 6

19.6 ± 1.1

41.3 ± 1.7


89.1 ± 71

58 ± 10

1.2 ± 0.3 7.6 ± 0.4

80.2 ± 41.8 11.2 ± 7.7

3963 ± 1115 3651 ± 1379

45.7 ± 34 6.0 ± 1.4

0.8 ± 0.6 3.2 ± 0.7

1.3 ± 0.9 (0.78)

1.0 ± 0.8 5.0 ± 0.3

0.7 ± 0.01 4.6 ± 0.3

35.3 ± 4.5


1.7 ± 0.4 7.5 ± 0.2 2.9 ± 0.5 7.2 ± 0.8 11.6 ± 0.1 6.9 ± 3.1

19.3 ± 19.6 5.2 ± 3.7 4.0 ± 1.6 2.6 ± 0.9 18.6 ± 4.8 3.9 ± 2.6

872 ± 116 716 ± 129 545 ± 132 291 ± 110 479 ± 29 255 ± 105

4.5 ± 1.6 1.2 ± 0.5 3.7 ± 0.4 2.8 ± 0.6 Nil 0.86 ± 0.4

4.9 ± 4.1 16.3 ± 5.0 12.1 ± 1.3 14.9 ± 0.6 4.2 ± 2.3 36 ± 18

0.75 ± 0.5


5.5 ± 4.4 34.6 ± 28.9 55.2 ± 3.4 107.4 ± 14 6.3 ± 2.6 53.3 ± 31

5.5 ± 2.8 14.8 ± 2.7 NA NA 8.4 ± 2.2 66 ± 23



NA NA 2.9 ± 1.0 (2.0) 39.7 ± 22.6

Wastewater after treatment in effluent treatment plants


Untreated wastewater Physico-chemical treatment Biological treatment After alum + sand filter treatment






NA (11.42) NA (25.1) 52.4 ± 14 (8.2)








NC 141.2 ± 38.5 NC


7.6 ± 1.0

4.4 ± 0.1

138 ± 109


39 ± 10

148 ± 64









a Residual chlorine content; NA = not available; NC = not calculable; Data in parenthesis are for EC 50 value for poikilocytosis.


K.P. Sharma et al. / Chemosphere 69 (2007) 48–54

toxicity than what actually assessed during fish bioassay. The comparatively higher sensitivity of fish (lower LC 50 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, EC 50 values for RBC counts and poikilocytosis were higher than LC 50 values of fish suggesting RBC to be lesser sensitive to bleach. The higher fish mortality may be ascribed to impairment of gaseous exchange through greater damage to gills. The toxicity order of colored wastewaters to fish (based on LC 50 value) was: pass > silicate water I wash > II wash > III wash > screen wash > pools ( Table 1 ). Further, their EC 50 values for poikilocytosis (in RBCs) were usually minimum indicating greater physiological distress in fish, unlike bleach wastewater ( Table 1 ). Acidic bleach water ranks almost similar to pass in terms of fish mortality while neutral one was closer to third wash, despite the fact that they differed largely in their physico-chemical characteris- tics. This fact also holds true for first wash and silicate wastewater. Contrast to this, first wash and screen wash almost similar in their COD values varied largely in their LC 50 values. Thus, physico-chemical characteristics of wastewaters alone are not sufficient to quantify their toxicity. With the exception of pass, although EC 50 values of dye wastewaters for Lemna were higher (low toxicity) than LC 50 values for fish, but general trend of their toxicity ranking was almost similar to fish: pass > I wash > silicate

water > pools > screen wash > II wash > III wash (Table 1 ). Thus, response of both the tested organisms to pollu- tants 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 effects 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 pro- cess 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 fish ( Tables 2, 3 ). In contrast to major ingredient chemicals, dyes such as C.I. Direct Blue 86 (EC 50 = 225 ppm), C.I. Direct Yel- low 44 (EC 50 = 1500 ppm) and Drimarene Yellow L2R (EC 50 = 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 alka- line, in comparison to acidic one. Its toxicity also increased in combination with copper (Table 4 ). In contrast to cop- per, nutrients had no role in reducing methyl red toxicity (at lower pH) to Lemna . Methyl red loses its azo character a bit in acidic solu- tions on account of protonation resulting in its planar structure (Park et al., 2005 ), which may increase its absorp- tion in fish in comparison to normal alkaline solution of

Table 2 Physico-chemical characteristics (range) of ingredient chemical treatments and their toxicity to Lemna (EC 50 )


Physico-chemical characteristics of varying dilutions (range)


Duckweed bioassay (EC 50 )



EC (mS)

TH (ppm)

Na (ppm)


Frond no.

Dry weight

Acids HCl H 2 SO 4 Salts NaNO 2 Na 2 OSiO 2 Tap water





0.0052 M

(pH = 3.8) (pH = 4.4)

0.0050 M (pH = 5.2) 0.0045 M (pH = 4.9)





0.0025 M





0.009 M


0.015 M






0.014 M


0.029 M






Data in parenthesis indicate EC 50 values of acids in terms of pH values, and of salts in percentage.


Table 3 LC and EC 50 values (ppm) of methyl red and copper for fish and Lemna at different nutrient and pH levels of the growing medium




Fish bioassay

Duckweed bioassay





Frond no.

Dry weight

Copper Nutrient poor medium Nutrient rich medium Methyl red Nutrient poor medium i. acidic ii. alkaline Nutrient rich medium



























NC = not calculable; NA = not available.

K.P. Sharma et al. / Chemosphere 69 (2007) 48–54


Table 4 Toxic effects 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)


% Reduction (counts)

Methyl red Copper Methyl red + copper




methyl red. The tested plant species may also respond sim- ilarly 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 pro- moted at lower concentrations of textile wastewaters of pools (<40–80%), pass (<0.5–0.25%), I (<3%), II (<10%) and III (<25%) wash of diazotization process, possibly on account of higher concentration of nutrients (filterable reactive phosphorus = 1.9–13.5 ppm; total phosphorus = 5.0–16.4 ppm; NH 3 –N = 1.4–266 ppm; NO x = 5.6– 67.2 ppm; organic nitrogen = 8.4–506.8 ppm; K = 2.8– 15.8 ppm) in comparison to control plants growing in the tap water (FRP and TP = traces; NH 3 –N = nil; NO x = 4.0 ppm; organic nitrogen = nil; K = traces). The reduc- tion in copper toxicity (higher EC 50 ) 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 LC 50 and EC 50 values, respectively, for fish (5–15 folds) and Lemna (5–32 folds) (Table 1 ). This has been ascribed pri- marily 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 evi- dent from LC/EC 50 values, treated wastewaters were more (3–4 folds) toxic to fish and poikilocytosis was the most sensitive parameter. No definite trend was recorded for EC 50 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 waste- waters 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 toxi- city of biologically treated wastewater was significantly higher after alum treatment, while their physico-chemical characteristics varied little ( Table 1 ). These findings signify importance of biological tests in characterizing textile wastewater toxicity.

3.4. Correlation between physico-chemical characteristics and LC/EC 50 values

LC and EC 50 values of both the test organisms showed significant negative correlation with conductivity (r = 0.53–0.59), COD (r = 0.55–0.71) and Cu ( r = 0.54) which was positive for pH (r = 0.59–0.97), to wastewaters of diazotization process (untreated and neutralized) and so, they are good physico-chemical parameters for character- izing toxicity. Pool water characteristics had significant positive corre- lations (pH: r = 0.82–0.93; EC: r = 0.78–0.90; COD: r = 0.98–0.99) with LC 50 and EC 50 values of fish, which were negative for number and dry weight of duckweed (pH:

r = 0.92–0.98; EC: r = 0.85–0.97; COD: r = 0.81–0.97). Thus, duckweed bioassay defined pool toxicity better when compared with fish. Only pH had significant positive correlation ( r = 0.58– 0.98) with LC and EC 50 values of fish and Lemna for bio- logically treated wastewaters, while their EC (r = 0.91) and COD ( r = 0.89) values had significant negative correla- tion 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 inju- ries noted during fish and duckweed bioassays may assist in rapid assessment of wastewaters toxicity in the natural eco- systems, whereas their EC/LC 50 values in quantifying toxi- city 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 fish RBCs to pollutants in dye waste- waters emphasizes inclusion of their study in routine fish bioassay. Present study documented increase in pollutants toxic- ity (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 rec- ommend discharge limit of textile wastewaters close to neutral. The considerations such as dilution factor and buffering capacity of the recipient water bodies are of little significance in monsoonic climate as that of India, where water flow (lotic system)/its volume (lentic system) remains low during most part of the year, except rainy season.


We are thankful to the Council of Scientific and Indus- trial Research, New Delhi for awarding Research Asso- ciateship 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, Univer- sity of Rajasthan, Jaipur for laboratory facilities.


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