AUTEX Research Journal, Vol. 7, No3, September 2007 © AUTEXThe mean IC50 value for the blue dye was 278µg/ml and the mean IC20 value was 112µg/ml(Figure 3).120100y = 0,0001x 2 - 0,2357x + 107,73R 2 = 0,9454prot, %80604020IC20 IC5000 112 200 278 400 600 800the blue dye, µg/mlFigure 3. The IC20 and IC50 values for the blue dye after 72 hours exposure (n=4 and n=2)The IC20 and the IC50 values were lowest for the red dye and highest for the blue dye (Figure 4).300250IC20, IC502001501005001 2 3red yellow blueFigure 4. The IC20 and the IC50 values for three reactive dyes (red, yellow, blue)All the fabric extracts had protein contents of more than 80% of the total protein contents of theunexposed samples, showing that none of the fabric extracts were toxic to skin cells (Figure 5).prot, %1201008060402001 2 3yellow red blueFigure 5. Values of protein content of the fabric extractsThe non-exposed cells had good viability. Using the 2,4-dinitrophenol samples as positive controlsshowed that, at low concentrations, the mean value of the protein content was 80% of the total proteincontent of the non-exposed samples. The lower control concentration of 0,05 mg/ml resulted in a 60%protein content, while the highest control concentration resulted in a 10% protein content.For the red and blue dye samples, for all non-toxic concentrations, the coefficient of variation (C of V)values were all less than 10%. For toxic concentrations, the C of V values ranged between 7-15%. Forhttp://www.autexrj.org/No3-2007/0231.pdf 220
AUTEX Research Journal, Vol. 7, No3, September 2007 © AUTEXthe yellow dye, the C of V values for the non- toxic samples were between 2-14%, and for the toxicsamples were between 17-21%. For fabric extracts, the C of V values were between 9-13%.DiscussionThis study has shown that the reactive dyes were toxic in concentrations which were lower than thoseused in commercial dyeing processes. Reactive dyes react with cellulose under alkaline conditions[24]. The reactive dyes have studied pH values between 4,5-6,5; however, the dyes are very reactivein human keratinocyte cells. The textile dyes when used commercially contain many other chemicalsin addition to the dye molecules. There are different salts, including calcium stearate, CMC and otherchemicals. This means that the discussion of the results of this work relates to the toxicity of a mixtureof chemicals and not just to the pure molecules: the effect of the pure dye alone cannot be evaluatedand it is also not possible to know the concentration of the dye itself in the powder used as thecommercial dyestuff. However, since these dyes are part of a mixture when they are used in industry,it is more useful to know the toxicity of this mixture than that of the pure dye when assessing thesafety of commercial dye formulations.The results show that the dyes are toxic, but the dyed fabrics are not. This can be explained by thefact that the pure reactive dye in powder form is very active, but after the dyeing process many of thereactive sites on the dye molecules have taken part in the formation of covalent bonds with fibremolecules [8]. These bonds are very stable and this can explain why the dyed fabric material is nottoxic.The red dye produced adverse effects under the lowest concentration of the dye: the IC20 value was28µg/ml: the IC50 value (155µg/ml) was the lowest compared to the values for the other dyes. Theblue dye and the yellow dye had IC20 values between 78-112µg/ml: IC50 values were between 237-278µg/ml, showing clear toxicity. When 80% of the cells are dead IC20 values indicate low toxicity andthe results show that the red dye is very toxic even at low concentrations.Although the dyes were toxic, extracts from the dyed fabrics were not toxic. Although the extractscontained dye (they were coloured), the results showed that they were not toxic and it can beassumed that the dye in solution had been hydrolyzed. The protein contents of all fabric samples wereover 80% compared to the non-exposed cells. For some samples the C of V values showed theprotein content to be under 80%. However, the mean percentage values for the protein contents of thefabric extracts showed clearly that these were non-toxic.This study therefore supports earlier studies which have shown pure reactive dyes to have adverseeffects. Allergic reactions are commonly known to cause allergic diseases for workers in industry [5,7].Park et al. detected workers in the textile industry as having changes in the amounts ofimmunoglobulins in their blood [20]. Keneklis noticed some textile dyes are mutagenic [12]. HaCaTcells were used by Wollin et al. [28] to show that several azo are genotoxic. Birhanli et al haveestablished that some reactive dyes are teratogenic [2].The Chemical Safety Data Sheets [3] show the LD50 value for the yellow dye to be 5000mg/kg, higherthan the 2000mg/kg values for the red and blue dyes. According to toxicity tests using activatedsludge, the toxicity of the blue dye measured as EC50 (the molar concentration of an agonist whichproduces 50% of the maximum possible response for that agonist) was higher than 100mg/l. Using theOECD 209 method, 1984, the red and yellow dyes had IC50 values higher than 1000mg/l. Using theOECD 203 method (an acute fish toxicity test using Salmo gairneri and Oncorhynchus mykiss) thevalues of LC50 (the concentration of a chemical which kills 50% of a sample population) for the redand yellow dyes were found to be higher than 100mg/l.[3]. In this study, the IC50 values for all threedyes showed the limit value of toxicity to be over 100µg/ml as in the tests using activated sludge andfish. The cytotoxicity test using HaCaT cells is very sensitive and the IC20 values indicated toxicity inconcentrations lower than 100mg/l.Kopponen et al [13] have used hepa-1 cells to test the toxicity of textile dyes and dyed fabrics. Theymeasured IC50 values but not IC20 values. The dyes that they used were not the same as the onesused in this study, but the results showed similar levels of dye concentration which resulted in toxicity.Klemola et al have used the spermatozoa motility test for studying the same textile dyes as in thiswork (paper submitted to AUTEX Research Journal). In this test the IC50 values were calculated afterhttp://www.autexrj.org/No3-2007/0231.pdf 221
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