Fate and toxicity of azo dye metabolites under batch long-term anaerobic incubations

Enzyme and Microbial Technology40(2007)

934–939

Fate and toxicity of azo dye metabolites under batch

long-term anaerobic incubations

Mustafa Is¸ik a,∗,Delia Teresa Sponza b

a Aksaray University,Engineering Faculty,Environmental Engineering Department,68100Aksaray,Turkey

b Dokuz Eylul University,Engineering Faculty,Environmental Engineering Department,Buca Kaynaklar Campus,35160Izmir,Turkey

Received16November2005;received in revised form20July2006;accepted21July2006

Abstract

Anaerobic biodegradability and toxicity ofﬁve azo dye metabolites(Reactive Black5,RB5;Direct Red28,DR28;Direct Black38,DB38; Direct Brown2,DB2;Direct Yellow12,DY-12)at parent azo dyes co

ncentrations varying between200and1800mg l−1of were studied under long-term incubations such as72days.The metabolites of DB38and DB2azo dyes at a concentration of1800mg l−1,had low toxicity effect on methanogenic bacteria.TAA measurements in the beginning and last period of incubation period showed that the recovery percentages of aromatic amines for DR28,DB38,DB2and DY12azo dyes,were57.6,69.4,66.5,and77.1%,respectively.The low recovery of aromatic amines (57–77%)from the reduction of azo dyes was probably due to low level of cleveage of aromatic ring resulting in minor anaerobic transformation of azo dye structure.This study showed that azo dye metabolites(aromatic amines)are partly persistent under anaerobic conditions and had low toxicity under long-term incubations.

原子核物理学Keywords:Dye metabolites;Azo;Anaerobic;Aromatic;Batch

1.Introduction

Azo colorants are substances that have a coloring effect containing one or more azo groups(N N double bonds)in their chemical structure.They are synthesized by coupling one or more diazotized aromatic amines with other aromatic compounds[1].They are largest group of synthetic colorants (60

–70%)and can be used to color a large number of different substrates,such as synthetic and natural textileﬁbres,plastics, leather,paper,mineral oils,waxes,and even(with selected types) foodstuffs and cosmetics[2].The color of azo dyes is due to azo bonds,the associated auxochromes and a system of con-jugated double bonds.The apparently general reaction of this substance group,reductive cleavage of the azo linkage under anaerobic conditions,has prompted concerns about the poten-tial hazards associated with exposure to azo dyestuffs which could be metabolized with the release of known carcinogens, such as benzidine.Aniline and its derivatives are commonly ∗Corresponding author.Present address:Aksaray University,Engineering Faculty,Environmental Engineering Dep.,68100Aksaray,Turkey.

Tel.:+903822150953/132;fax:+903822150592.

E-mail address:mustafa.isik@(M.Is¸ik).produced as by-products of the petroleum,paper,coal and chem-ical industries.These aromatic amines are biorefractory and highly toxic because they can react easily in the blood to convert hemoglobin into methahemoglobin,thereby preventing oxygen uptake.Practical methods for an efﬁcient depollution of indus-trial wastewaters containing anilines then need to be established to avoid their dangerous environmental accumulation[3].

In the microbial degradation of azo dyes,the initial process is their decolorization.The highly electrophilic azo bond must be cleaved for azo decolorization to take place.Under aerobic conditions neither the activated sludge[4]nor aerobic bacterial isolates were able to degrade azo dyes.On the other hand, various azo dyes were shown to be decolorized by anaerobic sludge[5,6]anaerobic sediments[7]and by pure cultures of bacteria[8,9].Azo dye reduction leads to the formation of aromatic amines.Aromatic amines are generally not degraded and accumulate under anaerobic conditions[5,10],with the exception of a few aromatic amines characterized by the pres-ence of hydroxyl and/or carboxyl groups[11,12].Currently,as most of treatment plant has been conventional aerobic system, azo dyes in the wastewater has remained unchanged or has only slightly modiﬁed.Once discharged into receiving medium, possibly azo dyes will accumulate in sediments in where anaerobic conditions dominate.

M.I¸s ik,D.T.Sponza/Enzyme and Microbial Technology40(2007)934–939935 Table1

Properties of azo dyes used

Color index name No of C.I.a Formulas of dyes M.W.b(g mol−1)λmax(nm)1000mg dye(l)

COD(mg l−1)S.D.n Reactive Black5(RB5)20505C26H25N5O19S6Na4991.8597524283 Direct Red28c(DR28),22120C32H24N6O6S2Na2696.7497746223 Direct Black38,c(DB38)30235C34H25N9O7S2Na2781.7520370403 Direct Brown2c(DB2)22311C29H19N5Na2O7S627.5400103403 Direct Yellow12(DY12)24895C30H26N4O8S2Na2680.740049773

a Colour index.

b Molecular weight.

c Banne

d azo dyes which contain benzidine(MAK III A1amine).

Although several studies concerning the degradation and tox-icity of azo dyes in anaerobic condition[5,13–15]and aerobic conditions[16–18],little has been done to track the fate and tox-icity of these dye metabolites in sludge during long time.This subject is important because azo dyes discharged to receiving waters which they adsorbed to suspended material and accu-mulate in sedi城市社会学论文

ments.After short time,azo dyes will reduce to their metabolites resulting decolorization under anaerobic con-ditions.It is not well known yet the fate and toxicity effect of these metabolites in sediments or sludge under anaerobic con-ditions during long times anaerobic incubations.Therefore,the aim of this research is to study the long time anaerobic incu-bation of metabolites originated fromﬁve different azo dyes in order to understand the fate and toxicity of azo dye metabolites in sludge.

2.Material and methods东亚自贸区

2.1.Laboratory-scale batch anaerobic reactors,synthetic media, seed and dyes used throughout the experiments

The studies were performed in115ml dark glass serum bottles sealed with5mm rubber septum kept in place by a screw cap.Each serum bottle contained4.0mg l−1of partially granulated anaerobic sludge obtained from the UASB reactor treating the wastewaters of PAK MAY A yeast factory in Izmir,3000mg COD l−1of glucose and the necessary volume of macro and micro nutrients(Vanderbilt mineral medium).This mineral medium was used in all batch investigations and consisted of the following inorganic composi-tion(in mg l−1):NH4Cl,400;MgSO4·7H2O,400;KCl,400;Na2S·9H2O,300;

(NH4)2HPO4,80;CaCl2·2H2O,50;FeCl3·4H2O,40;CoCl2·6H2O,10;KI, 10;(NaPO3)6,10;l-cysteine,10;AlCl3·6H2O,0.5;MnCl2·4H2O,0.5;CuCl2,

0.5;ZnCl2,0.5;NH4VO3,0.5;NaMoO4·2H2O,0.5;H3BO3,0.5;NiCl2·6H2O, 0.5;NaWO4·2H2O,0.5;Na2SeO3,0.5[19].The anaerobic conditions were maintained by adding667mg l−1of sodium thioglycollate(0.067%)which is proposed between0.01and0.2%(w/w)for maintaining the conditions anaer-obic.The alkalinity and neutral pH were adjusted by addition of5000mg l−1 NaHCO3.Three thousand milligram per liter of glucose–COD was used as co-substrate providing reducing equivalents with electronﬁssion.All incubations were carried out in a temperature controlled incubator at35◦C.Firstly,sam-ples containing200–3200mg l−1of azo dyes were incubated under anaerobic condition during100h.Through this stage the azo dyes were decolorized.After that,the bioreduced and decolorized azo dyes were re-incubated under anaer-obic contidions.In other words,during this stage the azo ye metabolites were incubated through72–91days.The equivalent concentrations of intermediates of azo dyes were changed between0and1800mg l−1because of dilution of bioreduced samples with replenishment of sludge,mineral medium,alkalinity and co-substrate through72–91days.Azo dyes used in this study are summa-rized in Table1.These dyes were used in dissolved form.Open formulas of azo dyes used in this study are depicted in Fig.1.2.2.Experimental procedure

All substrate removal tests were conducted in duplicate in order to check the accuracy of the results.Throughout the batch tests,a control without dye and a seed blank sample were used to detect and compare the methane production in order to correct the gas productions in batch serum bottles containing the all dye concentrations.

2.3.Analytical procedure

桓仁东山中学TSS in granulated sludge was measured by theﬁltration technique using membraneﬁlters with pore sized0.45␮m[20].Gas productions were mea-sured with liquid displacement method[21].The total aromatic amines(TAA) were determined colorimetrically at440nm after reacting with4-dimethylamino benzaldehyde–HCl according to the method described by Oren et al.[22].TAA values are given as mg benzidine l−1with using equation benzidine concen-tration=0.0039×Absorbans at440nm−0.0005(R2=0.9976)obtained from benzidine versus absorbance straight line.The determination of TAA could not be carried out in samples containing RB5because the intermediates of RB 5re-colored immediately through auto-oxidation during sampling and analyz-ing.The accumulation of aromatic amines was determined to evaluate whether degradation of intermediates took place under anaerobic conditions.For this goal,samples containing azo dyes between200and3200mg l−1were incu-bated during100h,and accumulated TAAs were monitored with

diazotizated method as aforementioned.Measured TAAs values were accepted as the equiv-alent metabolites of each azo dye.The concentrations of samples containing azo dye metabolites originating from the incubation of parent azo dyes varied between200and1800mg l−1.

3.Result and discussions

3.1.The effect of increasing metabolite concentrations on methane gas production

Fig.2shows the cumulative methane gas productions during the incubation period in batch reactors containing3000mg l−1 glucose COD,and metabolites of the selected azo dyes sep-arately at concentrations varying between0and1800mg l−1. The quantities of cumulative methane gas showed that a quick initial methane production due to degradation of glucose on days 15–20through incubation period.Afterwards,the methane pro-duction became slower because of the decrease of substrate,and accumulation of aromatic amine and volatile fatty acids,which would progress the methanogenic stage less readily,was tak-ing place[21,23].For example,90ml of cumulative methane gas production was obtained on days20days,while150ml of cumulative methane was obtained on days72at a parent RB5azo dye concentration of1800mg l−1.In other words the methane

936M.I¸s ik,D.T.Sponza /Enzyme and Microbial Technology 40(2007)

934–939

Fig.1.Open formulas of azo dyes used in this study.

gas production is very high during the ﬁrst 10days while the gas

production decreased at the end of incubation period.In some samples,the cumulative methane decreased as the concentra-tions of azo dye metabolites increased.These products could inhibit the activity of anaerobic bacteria.Three thousand mil-ligram per liter of glucose–COD was completely converted to methane gas in metabolite-free batch reactors during incubation periods.Azo dye metabolites containing samples exhibited low methane gas production compared to metabolite-free samples due to inhibition of methanogenic activity via these metabolites.The effect of the azo dye metabolites towards methanogens was tabulated by measuring the cumulative methane yields in the presence of selected dye metabolites at the end of incubation periods.As shown in Table 2the cumulative methane gas mea-sured at 1800mg l −1of RB 5,DR 28,DB 38,DB 2and DY 12azo dyes metabolites were about 153,105,77,59and 128%,respectively,compared to metabolite-free samples containing batch reactor.The results of this study showed that the metabolites of DB 38and DB 2at concentrations of 1800,and 1600–1800mg l −1,respectively,had toxicity effect on methanogenic bacteria under anaerobic conditions.No inhibition was observed even at con-centrations as high as 1200mg l −1for all dyes.Methane ratios of dyes were 1.29,1.09,0.99,0.95,and 1.06for metabolites of

RB 5,DR 28,DB 38,DB 2and DY 12,respectively,at parent azo dye concentrations of 1200mg l −1as seen in Table 2.The metabo-lites of all azo dyes at concentrations lower than 1200mg l −1exhibited no toxicity on anaerobic microorganisms.This shows that high azo dye concentrations such as 1200mg l did not exhibited toxicity on the anaerobic microorganisms,which these microorganisms are responsible from the degradation of organic matter in sludge or in sediments in the environment.High ben-zidine concentrations originated from the azo dye Direct Black 38,at a concentration of 3200mg l −1,reduced the COD removal efﬁciencies,while no serious inhibition was observed for DB 38at concentration of 1600mg l −1through continuous anerobic

Table 2

Methane ratios of dye metabolites containing samples to dye metabolites free samples Parent azo dye concentration Ink.time (days)0200400800120016001800RB 572 1.00 1.35 1.34 1.20 1.29 1.51 1.53DR 2872 1.00 1.03 1.140.94 1.09 1.25 1.05DB 3874 1.00 1.03 1.09 1.020.99 1.060.77DB 290 1.00 1.08 1.09 1.100.950.700.59DY 12

1.00

1.06

1.05

1.06

1.26

1.28

M.I¸s ik,D.T.Sponza/Enzyme and Microbial Technology40(2007)934–939

937

Fig.2.Cumulative methane gas productions for:(a)RB5,(b)DR28,(c)DB38,(d)DB2and(e)DY12metabolites.

conditions[15].Anaerobic batch toxicity assays usually do not reveal severe inhibition through methanogenesis at dye concen-trations below100mg l−1[2,21],although nitro-substituted azo dyes may be more toxic[23].In this study it was reported that the toxicity of monosubstituted benzenes was observed in the following order:COOH<H<OH<NH2<NO2[23].All dyes used in this study do not contain NO2however contains NH2sub-stitutions.Therefore,the effect of toxicity of azo dye metabolites was very low at high azo dye concentrations.The data obtained in this study exhibits similar results with theﬁndings of Donlon et al.[23].

3.2.Fate of aromatic amines

TAA measurements were carried in the beginning and last period of incubation in order to determine the fate of aromatic amines splitting from the azo dyes.The benzidine equivalents for samples containing DR28azo dye metabolites,and recovery percentages of aromatics amines,which are calculated from the equations given in Fig.3,are tabulated in Table3for all samples. All the calculation

s are based on the azo dye concentration of 1800mg l−1.

Methane recovery percentages of azo dye metabolites of RB5showed that these metabolites(Fig.2)might be partly transformed to methane.Similarly,the study performed by Razo-Flores et al.showed a complete anaerobic mineralization of the azo dye azodisalicylate(ASA)under methanogenic conditions[11].This azo dye wasﬁrstly reduced to the aromatic amine5-aminosalicylic acid(5-ASA)and later 5-ASA was anaerobically mineralized[11].More evidence for anaerobic biodegradation he decrease of UV absorbance and the recovery of ammonium,was reported in another study performed under anaerobic conditions[12]. On the contrary,Libra et al.[24]reported that complete recovery of the expected amines from hydrolyzed Reactive Black5reduction(in the expected stoichiometric ratio of 2mol p-aminobenzene-2-hydroxylethylsulfonic acid and1mol 1,2,7-triamino-8-hydroxynaphthalene-3,6-sulfonic acid per mol hydrolyzed Reactive Black5)could be demonstrated by LC–MS analysis through the continuous operation of anaerobic rotating disc at a HRT of15h.The present study shows that long retention times could cause mineralization of metabolites of some azo dyes,such as RB5and DY12.2.2.Table3shows that the recovery percentages of57.6,69.4,66.5,and77.1% were obtained for aromatic amines of DR28,DB38,DB2and DY12azo dyes,respectively,through the anaerobic incubatio

n. The TAAs of DR28(1800mg l−1)was247.5mg benzidine l−1 at the beginning while it decreased to142.6through incubation period of72days.Low recovery percentages of aromatic amines could be attributed to further anaerobic transformations for all dyes,through the mineralization of DR28,DB38 and DB2,since the methane ratio of dye metabolites to dye free metabolites samples is greater than1for RB5and DY 12azo dyes.

938M.I¸s ik,D.T.Sponza /Enzyme and Microbial Technology 40(2007)

934–939

Fig.3.TAA values of samples at the beginning and last of incubation period for DR 28azo dye.

Table 3

Fate and recovery percentage aromatic amines for all dyes at a concentrations of 1800mg l −1Parent azo dyes

DR 28DB38DB 2DY 12Initial TAAs (mg benzidine l −1)247.5114.5172.9172.2Last TAAs (mg benzidine l −1)142.679.5115.1132.7Recovery percentage of TAA (%)

57.6

69.4

66.5

77.1

4.Conclusions

This study tested and assessed the anaerobic toxicity and fate of ﬁve kinds of dye metabolites under

long time incuba-tion periods.Results were based on measurements of cumulative methane production and TAA.Dye metabolites split from azo dyes at high concentration as high as 1200mg l −1had no toxicity effect on anaerobic microorganism,however the dye metabolites had lower toxicity at higher dye concentrations,especially,for DB 2and DB 38azo dye.As these concentrations are in excess of those expected in wastewater discharges,therefore,in prac-tice,the acute toxicity of these metabolites may be neglected on anaerobic microorganism in receiving water.However,chronic toxicity of dye metabolites should be investigated for anaerobic bacteria and other living species.The low recovery of aromatic amines (57–77%)from the reduction of azo dyes was proba-bly due to a minor anaerobic transformation resulting from the cleavage of aromatic ring from the structure of the dye.This study showed that azo dyes metabolites are partly persistent under anaerobic conditions and had low toxicity under long-term anaerobic conditions.Acknowledgments

内蒙古地勘十院This study was supported by the Turkish Scientiﬁc and Tech-nical Research Council (TUBITAK),and fund of Ni˘g de Univer-sity,TURKEY .The authors gratefully acknowledge the ﬁnancial support from these organizations.References

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