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Department of Life Sciences, The Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, UK
Department of Civil Engineering, Loughborough University of Technology, Loughborough LE11 3TU, UK
Received 11 December 2001; received in revised form 10 September 2002; accepted 24 September 2002
Abstract
The toxicity of C.I. Reactive Black 5 and three Procion dyes, as found in textile effluents, was determined using the
bioluminescent bacterium Vibrio fischeri . Hydrolysed Reactive Black had a slightly greater toxicity than the parent
form (EC50 11.49/3.68 and 27.59/4.01 mg l1, respectively). A baffled bioreactor with anaerobic and aerobic
compartments was used to decolourise hydrolysed Reactive Black 5 in a synthetic effluent. Decolourisation of
hydrolysed Reactive Black resulted in an increased toxicity (EC50 0.29/0.03 mg l 1). Toxicity was not detectable when
decolourised Reactive Black 5 was metabolised under aerobic conditions. No genotoxicity was detected after the
decolourisation of either the parent or the hydrolysed reactive dyes, either in vitro or in the bioreactor. The toxicity and
genotoxicity of decolourised C.I. Acid Orange 7 was due to the production of 1-amino-2-naphthol (EC50 0.19/0.03 mg
l 1).
# 2002 Elsevier Science B.V. All rights reserved.
Keywords: Textile; Azo; Dyes; Decolourisation; Toxicity; Genotoxicity
1. Introduction
Reactive azo dyes occur in textile dyehouse
wastewater in concentrations ranging from 5 to
1500 mg l 1 due to their poor fixation to fabrics
(Pierce, 1994). Moreover, reactive azo dyes are not
degraded by conventional aerobic sewage treatment plants as they are resistant to biological
oxidative degradation (Easton, 1995). Decolourisation can be achieved using either anaerobic
digestion, microbial generation of oxygen radicals,
i.e. laccase activity of white-rot fungi (Schliephake
et al., 2000) or expensive physico-chemical treatments (ONeill et al., 2000). Although reactive
textile dyes can be decolourised under anaerobic
conditions due to reduction of the azo bond, the
resultant aromatic amines resist further degradation and may be toxic or genotoxic (Sweeney et al.,
1994). Toxicity could be eliminated through bacterial fission of the aromatic ring structure, a
process that requires oxygen. Therefore, secondary
0168-1656/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 1 6 5 6 ( 0 2 ) 0 0 3 0 2 - 4
50
Two bacterial strains were used for the decolourisation assays Enterococcus faecalis as previously used by Sweeney et al. (1994) and
Clostridium butyricum which was isolated from
the effluent stream of a textile dyehouse. Both
organisms had initially been screened for azo dye
decolourisation (as opposed to adsorption) by
plating onto tryptone soya agar plates and Schaedler anaerobe agar plates, respectively, containing
1% (w/v) Reactive Black 5 followed by anaerobic
incubation at 37 8C for 2 days. Decolourisation of
the agar without colony colouration indicated
azoreductase activity which was confirmed using
the following quantitative assay.
E. faecalis and C. butyricum were grown
anaerobically in tryptone soya broth and Schaedler anaerobe broth at 37 8C, respectively. They
51
52
Fig. 1. The structure of azo dyes and related compounds used in this study. The underlined region of Reactive Black 5 indicates the
portion of the vinyl sulphone group altered in the hydrolysed dye form, with OSO3 replaced by OH .
Table 1
Toxicity and genotoxicity of non-textile azo dyes and related
naphthol compounds
Compound
Azo dyes
Acid Orange 7
Acid Orange 7 (E. faecalis )d
Food Yellow
Food Yellow (E. faecalis )
EC50
(mg l 1)a
15.7
0.2
22.1
32.5
(2.68)c
(0.0)
(2.47)
(1.53)
CS valueb
1.1 (0.04)
0.04 (0.05)
0.9 (0.07)
0.4 (0.18)
53
54
Table 2
Toxicity and genotoxicity of textile dyes
Compound
Azo dyes
Reactive Black 5
Reactive Black 5 (E. faecalis )d
Reactive Black 5 (C. butyricum )
Reactive Black 5 (sodium dithionite)
Procion Navy
Procion Navy (E. faecalis )
Procion Navy (C. butyricum )
Procion Yellow
Procion Yellow (E. faecalis )
Procion Yellow (C. butyricum )
Procion Crimson
Procion Crimson (E. faecalis )
Procion Crimson (C. butyricum )
27.5
0.7
2.2
4.9
18.9
0.5
9.1
71.0
0.8
9.1
34.7
19.9
8.1
(4.01)c
(0.09)
(0.71)
(1.45)
(5.65)
(0.17)
(0.53)
(6.00)
(0.17)
(1.04)
(0.27)
(0.10)
(0.66)
CS valueb
Hydrolysede
Parent
Hydrolysed
11.4
0.2
0.3
0.2
27.9
0.5
8.6
66.4
0.5
5.6
37.7
18.2
9.1
1.5
1.4
0.6
0.6
/
/
/
/
/
/
/
/
/
0.7
1.3
0.8
0.6
/
/
/
/
/
/
/
/
/
(3.68)
(0.03)
(0.01)
(0.09)
(3.28)
(0.07)
(1.19)
(0.93)
(0.28)
(0.89)
(1.72)
(2.3)
(1.04)
(0.33)
(0.16)
(0.26)
(0.37)
(0.05)
(0.27)
(0.34)
(0.23)
55
Fig. 3. Toxicity of effluents from the laboratory scale baffled reactor for three consecutive periods; day 1 /76 anaerobic operation (A),
day 77 /154 anaerobic /aerobic operation without additional biomass (B), day 155 /205, anaerobic /aerobic operation with additional
biomass (C). -"-, Control, synthetic wastewater without hydrolysed Reactive Black 5; -j-, synthetic wastewater with hydrolysed
Reactive Black 5 (500 mg l 1).
56
Acknowledgements
The authors wish to express their gratitude to
the EPSRC (GR/L54738/01) for funding of this
project, Vanessa Kennerley and Cynthia Carliell
for their assistance and Dr Michael RaynerBrandes (Merck Speciality Chemicals Ltd) for his
constructive criticism of the V. fischeri work.
References
Easton, J.R., 1995. The dye-makers view. In: Cooper, P. (Ed.),
Colour in Dyehouse Effluent. Society of Dyers and Colourists, pp. 9 /21.