Journal of Hazardous Materials Advances 19 (2025) 100834
Contents lists available at ScienceDirect
Journal of Hazardous Materials Advances
journal homepage: www.elsevier.com/locate/hazadv
A comprehensive review on biodegradation of azo dye mixtures, metabolite
profiling with health implications and removal strategies
Bhavana Balachandran a, P.C. Sabumon b,*
a
b
School of Civil Engineering, Vellore Institute of Technology, Chennai Campus, Chennai 600127, India
Centre for Climate Change and Environment, Vellore Institute of Technology, Chennai Campus, Chennai 600127, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
Azo dye mixture
Biodegradation
Textile dyes
Azo dye metabolites
Health effects
Circular economy
Azo dye mixtures present in industrial effluents cause significant threats to both environmental integrity and
human health. Biological treatment methods of azo dyes offer an affordable and nature-friendly solution;
however, the metabolites resulting from biodegradation require further investigation. Different microbial groups
including bacteria, yeast, and fungi, have shown promise in the degradation of azo dye mixtures, yet there re­
mains a substantial gap in research concerning algal-based methods. The present work comprehensively high­
lights microorganisms that possess the ability to degrade azo dye mixtures. This review further serves to guide
readers in the identification of azo dye metabolites and its potential health risks and toxic effects including
responses within the human body. Finally, azo dye metabolites removal methods reported after biodegradation is
summarized. Therefore, this review would ultimately encourage the adoption of microbial mediated azo dye
degradation along with addressing future challenges and opportunities for research advancement in this field.
Future investigations should prioritize (i) the recovery of valuable azo dye metabolites, such as aniline and its
derivatives; to foster a circular economy, (ii) the mineralization of harmful metabolites generated during
biodegradation and (iii) developing advanced computational methods to expedite the identification of effective
biodegradation pathways and enhance process efficiency for the removal of azo dyes from industrial effluents.
1. Introduction
Mani et al., 2021). Moreover, azo dyes have been associated with cancer
in organs such as the liver, bladder, and spleen, as well as chromosomal
abnormalities in mammalian cells (Ben Younes et al., 2016; Zouar­
i-Mechichi et al., 2006). Considering the significant environmental and
health risks associated with dye-contaminated wastewater, it is essential
for industries that utilize dyes to effectively treat these complex pol­
lutants before discharging them into water bodies.
Multiple treatment options encompassing physical, chemical
(including Advanced Oxidation Process, AOPs) and biological processes
are being used by industries to eliminate dyes from effluent (Selvaraj
et al., 2021). However, physical and chemical methods often encounter
challenges such as high operational costs, the creation of concentrated
sludge that needs disposal, and the potential generation of further toxic
byproducts due to chemical usage. In contrast, biological
methods-particularly the biodegradation of azo dyes have received
considerable attention for being more cost-effective and environmen­
tally sustainable. Still, a significant drawback of biodegradation is the
production of aromatic amines as intermediate or end products, which
necessitates further treatment because of their toxicity. Additionally, the
Each year, about 7 × 108 kg of synthetic dyes are manufactured, with
azo dyes containing the –N = N– (azo) bond constituting nearly 70% of
the overall dye production (El Awady et al., 2024). The contamination of
water by these dyes can negatively impact the aquatic ecosystem,
resulting in reduced light penetration that adversely influences aquatic
organisms. This disruption, in turn, interferes with the essential process
of photosynthesis. Additionally, the toxic and carcinogenic character­
istics of azo dyes present dangers not only to humans but also to various
other living beings (Aounallah et al., 2024). The parent compound of azo
dye benzidine is known for causing carcinogenicity (Lellis et al., 2019).
Dye contaminated water can result in irritation of the eyes and the skin
through inhalation or contact, and it is connected to allergic reactions
including conjunctivitis, contact dermatitis, asthma, and rhinitis (Ghosh
et al., 2019). Studies have demonstrated that azo dyes can disrupt
ovulation and spermatogenesis, while reactive dyes may bind to human
serum albumin, which triggers the production of immunoglobulin E and
the release of histamine (Hemashenpagam and Selvajeyanthi, 2023;
* Corresponding author.
E-mail address: pcsabumon@vit.ac.in (P.C. Sabumon).
https://doi.org/10.1016/j.hazadv.2025.100834
Received 24 May 2025; Received in revised form 10 July 2025; Accepted 20 July 2025
Available online 21 July 2025
2772-4166/© 2025 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
B. Balachandran and P.C. Sabumon
Journal of Hazardous Materials Advances 19 (2025) 100834
efficiency of microbial decolorization is contingent upon several factors,
including the structural complexity of the dyes, environmental condi­
tions, the presence of redox mediators, and the specific enzymes
involved (Ngo and Tischler, 2022). The degradation pathways utilized
by these microorganisms are yet to be investigated. The chemical
structure of dyes, microbial systems, enzymes present and other factors
can lead to distinct pathways for bacteria-mediated biodegradation
(Ambika et al., 2022). Finding the degradation pathways becomes far
more complex and challenging when azo dye mixtures are treated (Zhu
et al., 2021). This is because the dye mixtures typically include a range
of chemical structures, each possessing unique substituents and bonding
arrangements. This diversity in structure indicates that no single enzyme
or pathway is capable of efficiently degrading all components, thus
requiring a consortium of enzymes with diverse substrate specificities
(Saratale et al., 2011b).
Bacterial degradation based on anaerobic conditions utilizes the
azoreductase enzyme to commence the process by catalyzing the
reductive cleavage of azo bonds (–N=N–). This results in the generation
of aromatic amine intermediates. These enzymes exhibit a high degree
of substrate specificity and necessitate electron donors such as NADH or
NADPH for their activity. The aromatic amines produced are toxic and
require further degradation. Typically, these aromatic amines are
eliminated under aerobic conditions. Oxidative enzymes, including
laccases, lignin peroxidases, and manganese peroxidases, predomi­
nantly found in fungi, are involved in breaking down aromatic rings and
aiding in the mineralization of these intermediates into simpler, less
harmful compounds (Mishra et al., 2022; Qiu et al., 2022). In the case of
mixed microbial consortia, this synergistic action results in a more
thorough degradation than that achieved by individual strains. But
sometimes, the complexity of this process increases due to the necessity
for different enzymes to degrade various dyes and their breakdown
products. Additionally, environmental parameters including pH,
salinity, oxygen levels, and the types of microbes involved significantly
impact the performance of these enzymes. When multiple dyes coexist in
a mixture, they can vie for the same enzymes or create new intricate
by-products. All these considerations complicate the goal of achieving
complete and efficient biodegradation (Kamal et al., 2022; Qiu et al.,
2022). A simple diagram showing how a bacterial-fungal consortium
degrades azo dye into less toxic compounds is illustrated in Fig. 1.
Taking each dye and studying their toxicity and biodegradation
pathways are tedious and not economic. In this case incorporating
computational tools in the biodegradation process makes a huge change
and needs to be pursued.
This review presents a distinctive viewpoint on the biodegradation of
azo dyes by identifying several gaps that have not been addressed in
earlier research. While Senthil Rathi and Senthil Kumar (2022) exam­
ines various microbial agents, including algae, in the context of textile
azo dye biodegradation, it does not provide a thorough analysis of the
metabolites produced during this process and their associated health
risks, which this review thoroughly investigates. Furthermore, while Shi
et al. (2021) highlights the significance of microbial consortia, biore­
actor advancements, and the physicochemical factors affecting dye
biodegradation, it fails to consider the recovery of metabolites or the
potential for a circular economy involving these metabolites, such as
aniline derivatives.
In the above context, this review focuses on mixed azo dyes by mi­
croorganisms and aims to gather information about the degradation
products of various azo dyes, including their specific details. The
biodegradation process reviewed includes microorganisms which are
capable of degrading the azo dye mixture, the enzymes which takes part
in the decolorization process, the experimental conditions employed in
the decolorization process, the metabolites formed during the experi­
ment and their health effects, toxicity, reaction/response inside the
human body and how these metabolites are treated or mineralized. By
doing so, this review streamlines the identification of azo dye degraded
compounds and its health impacts and facilitates the identification of
suitable subsequent treatment options to mineralize such compounds to
make biological treatment attractive. Finally, the future challenges and
scope of further research are identified including the recovery of
metabolites.
2. Biological methods of decolorization
Bioremediation is a boon for mitigating current environmental
pollution. Pollutant degradation via biological means is environmen­
tally favourable, as it results in minimal sludge production. Decolor­
ization and biodegradation of dye can be done either aerobically or
anaerobically (Yaseen and Scholz, 2018). A range of living organisms,
including bacteria, fungi, plants, algae, and yeast, are employed in the
processes of decolorization and mineralization of dye mixtures. Dyes can
Fig. 1. Degradation of azo dye by bacterial-fungal consortium.
2
B. Balachandran and P.C. Sabumon
Journal of Hazardous Materials Advances 19 (2025) 100834
be decolorized either by biosorption or by mineralization using micro­
organisms (Dissanayake et al., 2021). During biosorption, the dyes are
entrapped in both living and dead cells without undergoing any modi­
fications to their chemical structures. Consequently, biosorption is not
always a good choice. Biosorption is used only when no other biological
methods are available. But in some cases, biosorption act as a recovery
method (Fu and Viraraghavan, 2001). On the other hand, during
biodegradation, dye’s chromophore will break and it becomes colorless
(Kaushik and Malik, 2009). Most of the time the degraded products
would be aromatic amines and, in certain situations, the hazardous
molecules can be completely mineralized, or changed into harmless
substances like carbon dioxide, water, and even some inorganic salts
(Ali, 2010).
The decolorization of dye molecules occurs more swiftly and
comprehensively in mixed bacterial cultures than in pure cultures, due
to the presence of multiple enzymes that act on various locations of the
dye molecule. Dye degradation by microbes occurs by the action of
different enzymes. Laccase, peroxidase, azoreductase, tyrosinases,
veratryl alcohol oxidase are some of the enzymes found in these mi­
crobes. Among which azoreductase is the common one in dye degrading
microbes such as Pseudomonas sp., Bascillus sp. etc. (Kamal et al., 2022).
Anaerobic degradation has become a relatively straightforward and
promising approach for the treatment of wastewater containing colours.
The non-specific reduction of azo dyes is the main step in this procedure.
One method of accomplishing this reduction is by direct enzymatic
catalysis, in which specific microbes assist in catalysing the azo dye
reduction process. Even with the use of these microbes, the particular
enzyme that is in charge of the reduction remains unidentified and
unverified. This absence of clear confirmation begs the question of what
specific biological processes lead to the disintegration of azo dyes.
Additionally, cofactors and redox intermediates produced during the
degradation process plays a crucial role in facilitating reduction process.
Flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucle­
otide phosphate [NAD(P)H] are currently known to be redox active
coenzyme intermediates. They have the ability to receive electrons from
an azo reductase and subsequently donate electrons to azo dye molecule.
This leads to the breakdown of azo-bonds in the dyes, producing aro­
matic amines as a result (Selvaraj et al., 2021).
Mono-oxygenases, dioxygenases, and hydrolases are a few of the
several enzymes those helps the microorganisms to break the aromatic
rings. When aromatic rings are broken up into smaller components,
those that remain can go through additional reactions like conjugation
with other substances or functional group modification. Enzymes that
assist in the attachment or removal of functional groups, such as hy­
drolases and transferases, may be involved in these processes. Biodeg­
radation by-products of azo dyes can be further metabolised or
mineralized depending on microbial metabolism and environmental
factors. The transformation of the by-products into carbon dioxide,
water, and inorganic ions is referred to as complete mineralization. On
the other hand, microbes could take up the byproducts of degradation
and employ them as their carbon and energy source, thereby promoting
their growth and reproduction. Based on the enzyme involved in the
biodegradation process, the pathways differ in microorganisms. Fungi,
especially those classified as white-rot fungi like Phanerochaete chrys­
osporium, initiate the degradation of azo dyes via extracellular, nonspecific oxidative enzymes such as laccases, manganese peroxidases
(MnP), and lignin peroxidases (LiP). The by-products produced are
mainly less toxic in this case. On the other hand, bacterial consortia
typically begin with the reductive cleavage of the azo bond under
anaerobic or microaerophilic conditions, which is mediated by mainly
azoreductase enzymes. This reduction process results in colorless but
often toxic aromatic amines. In both instances, the end products will
vary. A detailed representation of the overall mineralization process of
azo dye in the context of a microbial consortium involving predomi­
nantly either bacteria or fungi and their major enzymes involved are
shown in Fig. 2.
2.1. Bacterial degradation of azo dye mixtures
It is been already proved by research that bacteria can be used to
treat wastewater containing single dye. But, when two or more dyes
presents in wastewater, the complexity increases. The bacteria could
grow and destroy a wide range of synthetic dyes in low concentration in
a short amount of time once they acclimatized, but they were more
resistant in harsh environment such as high concentration of dyes
(Yadav et al., 2019). Since bacteria are easy to culture, most of the cases
bacteria are used to treat textile effluent by genetically modifying those
bacteria (Yadav et al., 2019).
Azo dye degradation through anaerobic processes is commonly
perceived as a relatively straightforward and non-selective
Fig. 2. (a) Azo dye degradation by anaerobic-aerobic bacterial consortium and (b) Azo dye degradation by fungal strain.
3
Journal of Hazardous Materials Advances 19 (2025) 100834
B. Balachandran and P.C. Sabumon
phenomenon. However, the redox potential between the azo dye and
electron transporter determines whether this extracellular process will
take place (Shi et al., 2021). The following two mechanisms have re­
ported to prove this. During the catabolism ATP will be generated.
During this step electrons also generated. These electrons will transfer to
azo dyes; thereby breaking the azo bonds by accepting these electrons.
The reduction of azo dyes would be aided in the alternate route by en­
ergy, NADH, and FADH2 entering the electron transport chain from the
TCA cycle (Chen et al., 2021). Some anaerobic microbes can partially
biodegrade dyes with the help of azoreductase, which has the undesir­
able consequence of generating aromatic amines. Additionally, reverse
colorization may occur when anaerobic breakdown products are
exposed to oxygen. These issues restrict the use of anaerobic bacterial
decolorization on a broad scale. This happens due to the presence of lone
pairs in the amines formed. When these amines with lone pair electrons
exposed to air, they will get oxidized and forms stable colored long chain
compounds (Jafari et al., 2014a).
It is challenging to degrade azo dyes aerobically, because azo dyes
are weak electron acceptors than oxygen. Several microbial strains, such
as Aeromonas hydrophila and Lysinibacillus sphaericus, are capable of
effectively decolorize azo dyes in aerobic environments. These micro­
organisms employ the dye as a substrate, resulting in its degradation
(Srinivasan and Sadasivam, 2018). However, some studies reported that
the aerobic treatment can remove the aromatic amines generated during
the anaerobic process. Therefore, it can be inferred that the azo dyes can
be fully mineralized through an anaerobic-aerobic sequential process.
Anaerobic microbial consortium can eliminate color from wastewater,
according to a dye decolorization study conducted in the later 1990s.
The study used nine structurally distinct dyes, and the consortia were
able to significantly decolorize the mixture (Nigam et al., 1996).
The co-substrate has an essential function in the decolorization
process facilitated by bacteria. It promotes cell proliferation and sup­
ports the primary functions of bacterial activity. The co-substrates that
are most frequently utilized in the bacteria mediated decolorization
process is overpriced and increase the nitrogen and carbon loads in the
treated water (Mishra et al., 2019). Bacteria use the nutrients as an
energy source for their survival and to cleave the azo and anthraquinone
bonds in synthetic dyes during decolorization operation (Mishra et al.,
2019; Popli and Patel, 2015). Glucose, yeast extract, ethanol, lactose,
sucrose, acetate, starch etc. are the commonly used co-substrate by the
researchers. The most often utilized co-substrate among these is yeast
extract, which is used in bacterially mediated dye decolorization pro­
cesses (Imran et al., 2016; Mishra et al., 2019).
Table 1 shows the comprehensive information regarding decolor­
ization of dye mixtures using various bacterial species. It is observed that
the microbial species Alishewanella sp. strain KMK6, Clostridium bifer­
mentans, Sphingobacterium species, Bacillus odyssey and Pseudomonas
desmolyticum proved to working in mixed dye conditions. Studies using
the Pseudomonas bacteria have generated a lot of interest in the degra­
dation of dyes, Pseudomonas aeruginosa 23N1 revealed a notable decol­
orization efficiency of 79.38% for a synthetic azo dye mixture that
included Reactive Red 21 and Reactive Orange 16, both at an optimized
Table 1
Azo dye mixture decolorization studies using bacterial species.
Species / Consortia
Enzyme(s)
Concentration of dye
mixture
Experimental
conditions
C, N sources
Decolorization
(%)
References
Bacillus spp. (6 strains)
Not reported
40◦ C, pH ~7
Arabinose,
peptone
88.5
(Saha and Rao, 2024)
Same Bacillus spp.
Not reported
40◦ C, pH ~7
Arabinose,
peptone
61.4
(Saha and Rao, 2024)
Nesterenkonia lacusekhoensis
Not reported
Mixture A: RR195, RO16,
RB5, RB221, RB250 (120
mg/L)
Mixture B: Mixture A +
RY145, RO107, etc. (90
mg/L)
11 azo dyes (110 mg/L)
pH 11, static
95.6
(Prabhakar et al., 2022)
Thermophilic consortia
Immobilized sludge
reactor (species not
reported)
Immobilized alkalophilic
consortia
Aerobic bacterial
consortium
(Pseudomonas, Bacillus)
Alcaligenes aquatilis
Not reported
RB221, RY145, RR195 (50
mg/L)
RB221, RY145, RR195 (50
mg/L)
RB221, RY145, RR195 (50
mg/L)
AB113, DR81, DG26 (100
mg/L)
37◦ C, pH 10
37◦ C, pH 10
37◦ C, pH 10
Peptone, yeast
extract
Citrate,
ammonium sulfate
Citrate,
ammonium sulfate
Citrate,
ammonium sulfate
Glucose, yeast
extract
71.3
71.9
53
(Iqbal et al., 2022)
52.9
(Joshi et al., 2022)
37◦ C, static
(Ajaz et al., 2019)
Azoreductase
20-96
(Kolekar et al., 2013)
Bacillus subtilis
Not reported
28 C, pH 7, shaking
Sawdust, yeast
extract
Corn flour, starch,
ammonium sulfate
Yeast extract
86
Alishewanella sp. KMK6
71.8-100
(El-Rahim et al., 2021)
Pseudomonas aeruginosa
23N1
Shewanella sp. IFN4
Not reported
20◦ C, pH 5, salt 6 g/L
Yeast
79.4
28◦ C, pH 7.2
Yeast extract
80
(Mishra and Maiti,
2019)
(Imran et al., 2016)
Anaerobic bacterial
consortium (Clostridium,
Pseudomonas, etc.)
Bacterial consortium
(Arthrobacter,
Sphingomonas, etc.)
Bacillus subtilis
Not reported
CR, MR, PR, BB, NB (50
mg/L each)
Orange M2R etc. (0.5 g/L
each)
Mix of 15 dyes (20 mg/L
each)
RR21, RO16 (50 mg/L
each)
RB5, DR81, AR88 (200
mg/L)
Mixture of 18 dyes (360
mg/L)
30 C, pH 7, MFC
Molasses, cellulose
90
(Fernando et al., 2013)
Not reported
AO7, AR88 (50 mg/L each)
28◦ C, shaking
Dyes
~100
Azoreductase
pH 8.5
Not reported
Dyes, ammonium
chloride
Reactive dyes
87.4
Proteus vulgaris NCIM-2027
78-90
(Saratale et al., 2011b)
Mixed bacterial consortia
Azoreductase,
NADH–DCIP
reductase
RR, RB, RBrown (200 mg/
L)
RB172, RR2, RO4, RY84A,
RY17, RV5, RB25 (50 mg/
L each)
RR2, RR198, RR120,
RB160, RB13, RB172 (50
mg/L each)
(de los
Cobos-Vasconcelos
et al., 2012)
(Krithika et al., 2021)
Glucose,
ammonium sulfate
99.5
(Balapure et al., 2015)
Not reported
Not reported
Azoreductase
37◦ C, static
37◦ C, pH 7
◦
◦
37 C, fixed bed
reactor
◦
37◦ C, pH 7,
microaerophilic
reactor
4
B. Balachandran and P.C. Sabumon
Journal of Hazardous Materials Advances 19 (2025) 100834
concentration of 50 mg/L (using Response Surface Methodology - Cen­
tral Composite Design (RSM-CCD)), even in the presence of hexavalent
chromium at 50 mg/L (Mishra and Maiti, 2019). In addition, this strain
illustrated the capacity to utilize alternative carbon sources, like jack­
fruit powder, for effective dye decolorization (Mishra et al., 2019).
Pseudomonas bacteria demonstrate significant efficacy in the degrada­
tion of azo dyes, attributed to their advanced enzymatic systems,
versatility in various environmental settings, and capacity to convert
dyes into less harmful substances. These traits render them particularly
useful for bioremediation efforts aimed at addressing dye-contaminated
wastewater. Notable species such as Pseudomonas aeruginosa and Pseu­
domonas stutzeri possess substantial azoreductase activity, which is an
important factor for the breakdown of azo bond in dyes. Pseudomonas
species are effective in slightly alkaline conditions, specifically at a pH of
8 to 9 and within a temperature range of 30 to 40 ◦ C. They are also
capable of degrading azo dyes into less toxic byproducts. A notable
example is Pseudomonas aeruginosa, which converted methyl red into
benzoic acid and o-xylene (Bera and Tank, 2021; Ikram et al., 2022).
Studies involving bacteria typically favor a neutral or slightly alka­
line pH due to several reasons, such as enzyme activity and microbial
growth. The majority of enzymes exhibit optimal activities at neutral to
slightly alkaline pH levels, as their active sites are most stable within this
range. Although some bacteria can tolerate a wide range of pH levels,
most perform best in neutral to slightly alkaline conditions. In contrast,
Iqbal et al. (2022) in his study used two different pH; when the exper­
iments took place in the neutral pH the decolorization efficiency was
above 70% but when the pH changed to 10 the decolorization efficiency
reduced to 53%.
Research with bacteria often requires an external energy source, as
the degradation process involves complex enzymatic reactions that de­
mand energy. Bacteria are unable to utilize azo dyes as an energy source
for degradation, necessitating an external energy source to boost the
metabolic rate within the bacteria. In contrary, some rare cases are there
where azo dye itself used as an energy source. A microbial consortium
consists of Arthrobacter, Variovorax, Agrococcus, Sphingomonas, Sphingo­
pyxis, Methylobacterium, Mesorhizobium, and Microbacterium can use azo
dye as a carbon (de los Cobos-Vasconcelos et al., 2012). Additionally,
studies on mesophilic temperatures have not been thoroughly
investigated.
Nesterenkonia lacusekhoensis has demonstrated remarkable results,
achieving a 90% degradation rate for a dye mixture containing 10 dyes
under static conditions. The dyes contained distinct functional groups
that might contribute to a reduction in degradation rates. However, a
considerable level of decolorization was recorded in static environ­
ments. This phenomenon may be attributed to reduced disturbances and
environmental fluctuations. Such conditions likely facilitated optimal
enzyme activity.
Saha and Rao (2024) conducted research on two different dye mix­
tures using a microbial consortium. Despite the fact that all the dyes they
tested had the same functional group and a similar range of molecular
weight, they observed varying removal efficiency. Prior to the mixed dye
studies, they carried out individual dye degradation studies and selected
dyes for the mixtures based on the results. The first set of dye mixtures,
consisting of 5 dyes, exhibited good color removal efficiency, while the
second set, which included additional dyes, showed less color removal
efficiency. However, they were unable to identify a specific reason for
the lower removal efficiency based on factors such as molecular weight,
functional group, number of azo bonds, or structure (Liu et al., 2022).
This could be attributed to the absence of an enzyme necessary for
degrading these dyes, or the metabolites formed by the dyes may have
hindered bacterial growth. Prabhakar et al. (2022) studied 11 different
azo dyes among which Evans blue showed difficulty in degradation.
When comparing with the other dyes in the study, Evans Blue has the
highest molecular weight and highest number of functional groups
attached to it. This may the reason for the lesser degradation capacity of
this dye. Joshi et al. (2022) used three different dyes for the
decolorization study but the removal efficiency was very low. The Direct
Green 26 in the dye mixture has a complex structure consisting of 9 rings
and around 10 functional groups were attached to it. It is reported that if
the molecular weight and complexity of dye increases the dye molecules
cannot transfer through the microbial membrane and cannot get
degraded easily. The findings presented in Table 1 indicate that the
majority of studies demonstrate that single strains, when subjected to
anaerobic conditions, yield favorable results with reduced incubation
times. Conversely, consortia operating under aerobic conditions exhibit
enhanced color removal efficiency.
2.2. Azo dye mixture decolorization studies using fungal species
For the biodegradation of dyes, fungi are excellent choices. Using an
extracellular enzymatic system, they can change aromatic substances
like lignin, PAHs, or pesticides. The capacity of various fungal species to
remediate wastewater discharged by the textile industries has been
found and tested. But most of the studies are focused on white rot fungi.
In comparison to bacteria, white rot fungi develop slowly. If there are
bacteria in the reactor, they will take the food from the white rot fungus,
preventing it from growing. As a result, the process by which white rot
fungi degrade and discolour dye gradually comes to an end. Fungi can
synthesize various enzymes include lignin peroxidase (LiP), manganese
peroxidase (MnP), and laccase. These enzymes efficiently oxidise
different aromatic compounds (Przystas et al., 2015). Fungi possess a
high surface-to-cell ratio, which enables their mycelia to navigate effi­
ciently around dye molecules, resulting in substantial enzymatic reac­
tion. So, many studies of decolorization were conducted on viable and
non-viable cells of fungi. Fungi use different mechanisms to enable
complete mineralization of azo dyes (Gajera et al., 2015). Other than
biodegradation fungi adsorb the compounds to their hyphae. This bio­
sorption capacity of fungi depends on cell surface characteristics, cell
surface area and the functional groups of dye molecules. Azo dyes are
mobilised by fungi’s hyphae and spores, which then attach to the roots
of the organisms and create a wide surface area for biosorption (Sen
et al., 2016).
By removing some trace elements like iron, lowering pH (4-6),
creating a nitrogen-limited environment, temperature (25◦ C-35◦ C),
regularly adding fresh fungal biomass, and managing the development
of bacteria, it is possible to increase the activity of fungi that degrade
synthetic dyes. Also, the morphology of fungi shows different traits. It
can vary the enzyme produced, alter the growth rate and the metabolite
they produced (Sen et al., 2016). Fungal related azo dye mixture studies
are less explored compared to bacterial mediated studies.
Table 2 shows the comprehensive information regarding azo dye
mixture studies using fungal species. Most of the fungi examined for
degradation fall under the basidiomycota category. These fungi are fila­
mentous in nature. Within the basidiomycota category, the polyporales
order is commonly utilized in dye mixture studies. Examples of fungi in
this category include Pycnoporus sanguineus, Phlebia tremellosa, Phaner­
ochaete, and Lentinus squarrosulus. These organisms are known for their
rapid adaptability. Filamentous fungi show good dye mineralization
capacity, preventing the production of amines in the final effluent. For
example, a dye mixture used in the study by Saroj et al. (2015) is having
a complex dye structure and high molecular weight; when given suffi­
cient amount of time it decolorized upto 92%. Most of the article did not
explain the mechanism behind this. This area needs studies to find out
the reason. The oxidation of azo dyes in filamentous fungi is predomi­
nantly facilitated by the enzyme peroxidases and phenoloxidases. They
thrive in aerobic conditions for azo dye degradation, with agitation
enhancing the efficiency of the decolorization process. Agitation facili­
tates oxygen transfer to the system, improving the decolorization pro­
cess. Even under nitrogen-limited conditions, fungi can effectively
decolorize azo dye mixtures and achieve high removal efficiency
(Harazono and Nakamura, 2005).
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Journal of Hazardous Materials Advances 19 (2025) 100834
B. Balachandran and P.C. Sabumon
Table 2
Azo dye mixture degradation using fungal species.
Fungal species / consortia
Enzyme(s)
Concentration of dye mixture
Experimental conditions
Decolorization (%)
References
Lentinus squarrosulus AF5
MnP, LiP,
laccase
Laccase
Amido Black 10B, RB5, RB160
(500 mg/L total)
Allura Red, Tartrazine (40 mg/
L)
30 C, pH 7, shaking 200 rpm,
48 h
Solid-state fermentation, 25◦ C
20 days
~50
(Mathur et al.,
2023)
(Jiménez et al.,
2019)
Trametes versicolor, Pleurotus
pulmonarius, and consortium
◦
96 (T. versicolor); 93
(P. pulmonarius); 63
(consortium)
92
Aspergillus flavus SAB-3,
Penicillium oxalicum SAR-3,
A. niger SAR
Cyathus bulleri
MnP
Direct Blue 15, Acid Red 183,
Direct Red 75 (100 mg/L)
30◦ C, alkaline pH, shaking 200
rpm, 96 h
Laccase
MnP
Thelephora sp.
Phlebia tremellosa
LiP, MnP,
laccase
Laccase
30◦ C, pH 4.5, shaking 100 rpm,
3 h, with ABTS or vanillin
30◦ C, pH 4.5, shaking 150 rpm,
N-limited media, 48 h
39◦ C, pH 4.5, batch, 3 days
80 (ABTS); 69 (vanillin)
Phanerochaete sordida
RO1, RB5, RO7, RR198, AV17
(200 μM total)
Four reactive dye mixtures (200
mg/L each)
Dyeing unit effluent
Not
reported
28◦ C, stationary, 14 days,
glucose 5 g/L
28◦ C, shaking 130 rpm, malt
extract broth
~96
Trametes villosa, Pycnoporus
sanguineus
Remazol & Cibacron dyes (200
mg/L total)
Levafix, Drimaren, Remazol,
Procion dyes (10 mg/L total)
(Chhabra et al.,
2008)
(Harazono and
Nakamura, 2005)
(Selvam et al.,
2003)
(Kirby et al., 2000)
90 (T. villosa, 21 d); 80
(P. sanguineus, 10 d)
(Machado et al.,
2006)
2.3. Azo dye mixture decolorization studies using yeast species
90
61
(Saroj et al., 2015)
yeast can effectively decolorize unless bio-removal is based on bio­
sorption process with non-viable yeast biomass. Yeast related studies on
azo dye mixture are rare. But species like Pichia occidentalis, Meyerozyma
guilliermondii, Candida tropicalis, Scheffersomyces spartinae are shown
good decolorization efficiency above 90% within small amount of time
(Ali et al., 2021; Song et al., 2017; Tan et al., 2016; Wang et al., 2021).
Since the 1990s, there has been a growing focus on studying the
degradation of azo dyes through yeast-related processes. Notably, en­
zymes derived from diverse yeast varieties have shown remarkable
effectiveness in breaking down these dyes (Jafari et al., 2014b). But
yeast related study on dye degradation got only lesser attention compare
to other biological methods. Yeasts are advantageous for a variety of
reasons, including their quick development and capacity to withstand
harsh environments. Additionally, it has been learned that yeasts are
particularly effective at treating high-strength organic wastewaters
(Martorell et al., 2012).
Bioremediation by yeasts typically happens via three processes:
biosorption, bioaccumulation, and biodegradation (Jafari et al., 2014b).
Yeast can mediate the decolorization of azo dyes through two primary
processes: reductive and oxidative reactions. The effectiveness of
adsorption on yeast biomass is notably enhanced at lower pH values.
That may be due to the positive charge of yeast surface. If sulfonated
group present in a dye that will show a negative charge and this negative
charge will get attracted to the positive charge of the yeast surface. The
growth of yeast and its main metabolism are closely related to dye
degradation. A readily accessible carbon supply is always required for
yeast cells to reproduce. Additionally, a carbon supply is necessary for
the decolorization process (Song et al., 2017).
Table 3 shows the comprehensive information regarding azo dye
mixture studies using yeast species. The yeast related studies are con­
ducted in mesophilic condition. The ideal growth temperature and the
ideal temperature for dye decolorization utilising yeast cells are
frequently connected. Temperature variations can impact yeast cells’
biological responses, particularly those related to enzyme activity. But at
high temperatures, a decolorization drop was observed, which may have
been caused by the enzyme denature or due to non-viable cells. Acidic or
neutral media cause yeast species to exhibit more biodegradation be­
haviours. Additionally, cell viability is impacted by pH. When dyes are
the only carbon source used in biodegradation processes, no strain of
2.4. Azo dye decolorization studies using algal species
Algae demonstrate an impressive ability to engage with dyes present
in wastewater. They employ dye substances as substrates via three main
mechanisms as follows. (i) Biosorption, which takes place via two
methods: physisorption and chemisorption. The composition of the cell
wall significantly affects algal biosorption, as it is crucial for electro­
static attraction and complexation/chelation. Functional groups present
on the surface of algal cells, including phosphate, carboxylate, hydroxyl,
and amino groups, are responsible for the elimination of various con­
taminants from effluents. (ii) Bio coagulation, which is defined as the
coagulation of dye molecules onto the surfaces of extracellular bio­
polymers released by algae during the metabolic conversion of dyes.
These long-chain biopolymers possess surface functional groups and
demonstrate excellent coagulation capabilities. As a result, dye mole­
cules in the aqueous phase tend to adhere to these polymers and settle.
(iii) Biodegradation - algae are capable of breaking down azo dyes via an
induced azoreductase, which promotes the reductive cleavage of azo
bonds, leading to the release of aromatic amines (Ayele et al., 2021).
Then these aromatic amines are further degraded by the algae by
peroxidase enzymes. The rate of degradation depends on the chemical
composition of the dye and algal species involved. The main enzymes
documented are azoreductase, laccase and polyphenol oxidase (Sarkar
and Dey, 2021). Several investigations have reported the use of algae for
the degradation of single azo dyes (Aravindhan et al., 2007; Kousha
et al., 2012; Moradi et al., 2024; Vasanth Kumar et al., 2006). But
limited studies are focused on azo dye mixture degradation by algae.
Brar et al. (2019) studied the ability of Anabaena ambigua, Scenedesmus
Table 3
Azo dye mixture decolorization studies using yeast species.
Yeast species / consortia
Concentration of dye mixture
Experimental conditions
Decolorization (%)
References
Oleaginous yeast consortium (Yarrowia sp., Barnettozyma
californica, Sterigmatomyces halophilus)
Sterigmatomyces halophilus SSA1575
Mixtures I-VI (100 mg/L each dye)
18◦ C, static, 72 h, Bushnell
Haas Medium
18◦ C, static, 72 h
80-94
(Ali et al., 2020)
81-94
Candida sp. MM 403
Vilmafix Yellow, Green, Blue, Red
(50 mg/L each)
25◦ C, pH 4, shaking
Decolorization
observed
(Al-Tohamy et al.,
2020)
(Martorell et al.,
2012)
Sets I-VI (100 mg/L each dye)
6
B. Balachandran and P.C. Sabumon
Journal of Hazardous Materials Advances 19 (2025) 100834
abundans and Anabaena ambigua in removing nutrients and dyes from
real textile wastewater. The study confirmed the degradation of dyes in
the wastewater by analyzing the peaks in the UV-Vis spectra obtained
during the treatment process. Tarbajova et al. (2023) studied azo dye
degradation in the case of azo dye mixture using Chlorella sorokiniana
species. He obtained a remarkable decolorization efficiency of 70%.
In contrast to bacteria and fungi, which necessitate the incorporation
of carbon and other resources to eliminate dyes, algae do not need extra
nutrients. As photosynthetic entities, they flourish in polluted settings
without being hindered by dyes, rendering them a viable option for
bioremediation. Considering their renewable biomass capabilities and
self-sustaining characteristics, algae offer an environmentally friendly
substitute for traditional dye removal methods (Ayub et al., 2025).
Some studies related to algal degradation of azo dyes showed com­
plete mineralization. For example, Chlorella and Oscillatoria are crucial
for breaking down azo dyes into amine intermediates and subsequently
mineralizing them entirely into simple compounds or CO2 (Acuner and
Dilek, 2004). It has been observed that in excess of 30 azo compounds
can be biodegraded and decolorized by Chlorella pyrenoidosa, Chlorella
vulgaris, and Oscillatoria tenuis, leading to the breakdown of azo dyes into
simpler aromatic amines. Thus, the earlier results imply that algae can
significantly aid in the removal of azo dyes and aromatic amines.
Moreover, biosorption technique could be embraced as a cost-efficient
and effective solution for the decolorization of effluents, possibly
serving as a viable option compared to more expensive alternatives
(Saratale et al., 2011a). However, a scaled-up study is essential to prove
the practical applications involving algae and coloured effluents.
3.1. Molecular perspective on azo dye degradation
Different oxido-reductive enzymes act on the azo dye to get a com­
plete mineralization. The breakdown of azo dyes happens when the
microbial enzymes act on the structural characteristics of the dye mol­
ecules. It happens through three principal phenomena; (i) the cleavage
of azo bonds, (ii) transformation of aromatic rings, and (iii) and alter­
ation of functional group. The azoreductase enzyme is the key factor for
the breakage of azo bonds in anaerobic conditions. The enzymes first
acts on the azo bond, since it is the most electrophilic location in the
molecules. This connection shows significant resonance stabilization
between the nitrogen atoms and the conjugated aromatic rings. Azor­
eductase utilizes this electrophilic characteristic by transferring elec­
trons through cofactors such as NADH or NADPH (Chen, 2006). The
enzyme interacts with the azo dye, positioning the azo bond in align­
ment with the electron-donating groups of the active site. This disrupts
the π-electron delocalization, resulting in bond cleavage; leads to the
production of aromatic amines.
3.2. Enzyme perspective on azo dye degradation
Enzymes such as laccase and peroxidases can induce oxidative or
reductive stress, which leads to the destabilization of benzene rings.
Laccase acts by introducing hydroxyl groups to the aromatic structures,
resulting in the formation of intermediates like quinones or poly­
hydroxylated derivatives. These newly formed functional groups in­
crease the susceptibility of the rings to cleavage. On the other hand,
peroxidases generate radical species that can cause the spontaneous
breakdown of the aromatic system into smaller, less stable compounds
such as catechol or muconic acid (Chen, 2006). Functional groups such
as sulfonic acids (-SO3H) or chlorine (-Cl), attached to the rings, can
significantly affect the efficacy of the enzymes. Enzymes like quinone
reductase remove these groups, transforming the dye into forms that are
less soluble yet more amenable to degradation (Chen, 2006). Addi­
tionally, electron-withdrawing groups (for instance, -NO2 and -Cl) pro­
mote the activation of the rings for cleavage, while electron-donating
groups (-OH and -NH2) provide stabilization to the intermediates,
thereby requiring additional enzymatic processes.
2.4.1. Bacterial-algal consortium for azo dye degradation
Bacterial-microalgal consortia (BMC) have been utilized for the
treatment of industrial effluent for many years and have proven to be
more innovative and less time-intensive than engineered systems. BMC
represents a symbiotic relationship between bacteria and algae, where
both organisms positively influence each other’s growth in a photo­
bioreactor. In these symbiotic interactions, algae assist in the absorption
of contaminants due to their extensive surface area and serve as hosts to
create conducive conditions for bacteria to thrive in challenging envi­
ronments. Conversely, bacteria enhance algal growth by breaking down
toxic pollutants and releasing phytohormones. Moreover, prior to
employing such consortia in reactor systems, it is essential to select
strains that are compatible with one another in terms of size, growth
rate, and genetic stability (Rathour et al., 2024).
Mubashar et al. (2020) studied a real textile wastewater diluted to
concentrations of 5%, 10%, and 20%, and subsequently treated with a
consortium of Chlorella vulgaris and Enterobacter sp. MN17. This culture
demonstrated superior decolorization rates of 71.5%, 56%, and 49%,
respectively, across all dilutions when compared to the introduction of
these species individually (Mubashar et al., 2020). A synthetic textile
wastewater consisting of disperse blue 1 and disperse orange 3 was
treated by anaerobic-aerobic algal-bacterial photobioreactor and suc­
cessfully removed 82% disperse orange and 75% disperse blue
(Dhaouefi et al., 2022). This indicates, algal bacterial consortium can be
employed for azo dye mixture. There are several studies successfully
treated single azo dyes by bacterial-algal consortium (Dhaouefi et al.,
2018, 2019; Sun et al., 2015).
3.3. Fate of functional groups during azo dye degradation
After the ring cleavage, the functional group modification happens
through sulfonation and desulfonation, hydroxylation, introduction of
ether group, addition of -OH group, aliphatic chain formation etc. Based
on the functional group; the presence of hydroxyl groups may enhance
the reactivity of metabolites, potentially resulting in further breakdown
or interactions with other components. It can facilitate transformations
such as polymerization or oxidation. Conversely, sulfonate groups are
highly soluble and resistant to removal, contributing to the environ­
mental persistence of sulfonated aromatic amines. Azo dyes containing
sulfonate group may produce sulfanilic acid as metabolites. Further this
sulfanilic acid degraded to produce aniline and other metabolites.
Amino groups may undergo deamination, yielding phenolic compounds
that can be removed under aerobic conditions. In the degradation of
Disperse orange 2RL produced dillapiole; it is a methoxy-phenyl deriv­
ative (El-Sheekh et al., 2018). Dillapiole can be seen as a secondary
metabolite that arises from the degradation of substituted aromatic
rings, demonstrating the modification and stabilization of intermediates
resembling phenolic or catechol structures. The detection of dillapiole
points to the active pathways of methylation and ring modification
involved in the degradation of azo dyes. Additionally, nitro groups can
convert into amino groups in the metabolites, which are known to be
carcinogenic (Khan et al., 2021).
Hydroxyl groups are incorporated into aromatic compounds,
enhancing their polarity and diminishing toxicity. Phenol is oxidized to
catechol, which serves as a precursor for ring-opening processes. Amines
3. Azo dyes and their metabolites during biodegradation
The formation of azo dye metabolites during the biodegradation
relies on so many factors, including the parent dye structure, functional
group attached to the ring, surrounding environment, enzymes inter­
acting with the dye, and various other variables (Ambika et al., 2022).
As a result, several intermediates and products can appear during azo
dye degradation, and each azo dye may have its own distinct degrada­
tion pathway.
7
B. Balachandran and P.C. Sabumon
Journal of Hazardous Materials Advances 19 (2025) 100834
generated by azo bond cleavage (e.g., aniline) undergo subsequent
oxidation or deamination, resulting in metabolites such as quinones or
acids (e.g., benzoic acid). In the case of reactive black 5 addition of
amine (-NH2) (eg: aniline) and hydroxyl group (-OH) (eg: 2-naphthanol)
happened. In reactive red 239 addition of Nitroso (-NO) and Hydroxyl
(-OH) groups formed 2-Nitroso Naphthol and 5-Amino-1-Naphthol. In
Disperse orange 2RL addition of Hydroxyl (-OH) and Ether (-O-) groups
leads to the formation of 2-Cyclohexen-1-one, 2-methyl-5-(1-methyl­
ethenyl) and Dillapiole. In Synazol Red 6HBN addition of Aldehyde
(-CHO) and Acid (-COOH) groups leads to the formation of Pentadecanal
and [Z]-Hexadec-9-enoic Acid. In Acid Scarlet 3R the addition of Hy­
droxyl (-OH) and Polyhydroxylation leads to the formation of 7,8-Dihy­
droxynaphthalene-1,3-disulfonic Acid.
Malik, 2016). But when the same dye was degraded with Candida boidinii
MM 4035 no aromatic amines were produced (Martorell et al., 2017). It
is evident from Table 4 that one azo dye can have distinct biodegrada­
tion routes and degradation metabolites. For example, when methyl
orange reacts with laccase, three metabolites are produced, while
azoreductase produces a separate set of metabolites.
At the same time from the metabolites analysis, it was found that the
metabolites of reactive yellow have some valuable uses in some of the
industry. The metabolite 5-Nitroso-2, 4, 6-triaminopyrimidine can be
employed in drug production; cyclohexane can be used as solvent in
many industries. Tetramethyl-2-hexadecen-1-ol can acts as an antimi­
crobial and antiduretic substances, and 9-Octadecanoic acid, methyl
ester and hexadecanoic acid, methyl ester can act as an antioxidant
(Singh et al., 2022). o-Toluidine, also known as 2-methylaniline, serves
as an important intermediate in the production of dyes, rubber, and
pharmaceutical products. Aniline plays an essential role in the
manufacturing processes of polyurethane, diphenylmethane-3,
4-diisocyanate, rubber, dyes, pesticides, fibers, and pharmaceuticals
(Chung, 2016a). This is the area in the biodegradation of these dyes
lacking attention. Depending upon the nature of metabolites; their re­
covery or proper disposal is to be planned according to their chemical
characteristics.
3.4. Mechanistic role of molecular structure in azo dye degradation
The mechanistic importance of molecular structure in degradation
encompasses three key aspects: electron density and reactivity, steric
effects, and resonance effects. The arrangement of electron density
within dye molecules significantly impacts enzyme activity. Dyes char­
acterized by electron-rich aromatic rings, particularly those modified
with hydroxyl or amino groups, demonstrate a heightened susceptibility
to enzymatic degradation. Conversely, the presence of large substituents
or complex dye structures can hinder enzyme binding, resulting in
reduced degradation rates (Zhu et al., 2021). For instance, dyes like
Reactive Red 239, which are heavily substituted, require multiple
enzymatic processes for thorough degradation. Additionally, the inter­
action between azo bonds and aromatic rings contributes to resonance
stability, which enzymes can disrupt through electron transfer, thereby
increasing the reactivity of the molecules and facilitating their
degradation.
The cleavage of rings results in the formation of smaller aliphatic
compounds, including acetic acid and succinic acid. These products are
then further processed by microbial enzymes, leading to their conver­
sion into carbon dioxide and water, which ensures the complete
degradation of dye molecules.
3.6. Toxicity of metabolites
Some azo dyes are categorized as toxic and non-toxic azo dyes based
on certain scientific investigations. However, it has been noted that
several non-toxic azo dyes have hazardous metabolites. As the process of
analysing the toxicity of each dye is hampered by the structural differ­
ences and abundance of azo dyes on the market. Therefore, a system for
forecasting the ecological effects of dyes while taking into account their
structure and chemical properties needs to be developed (Rawat et al.,
2016).
Several studies were conducted related to the metabolism of azo dyes
in mammals. When azo dyes are ingested, intestinal microbiota or liver
azoreductase convert them to aromatic amines. Mammalian microsomal
enzymes metabolise aromatic amines into genotoxic chemicals. While
conducting Ames Salmonella/microsomal test technique, a large num­
ber of these aromatic amines are found to be mutagenic. The compounds
that contain benzidine and p-phenylenediamine moieties are primarily
the only ones that are biologically active dyes.
3.5. Fate of ring structure during biodegradation
Based on the ring structure specifically, the presence of benzene rings
can result in the formation of simple aromatic amines such as aniline and
its derivatives. In contrast, naphthalene rings give rise to naphthyl­
amines, which possess a more complex structure and exhibit persistence
in the environment. Congo Red consists of two benzidine moieties linked
via azo bonds to a naphthalene ring. The naphthalene moieties in the
dye formed 1-Naphthylamine as a result of biodegradation (Gul et al.,
2023). Heteroaromatic rings contribute to the development of resilient
compounds that exhibit recalcitrance, primarily due to the incorpora­
tion of nitrogen or sulfur atoms in their molecular framework.
Understanding the particular biodegradation mechanisms for each
azo dye requires thorough research and characterization. A range of
mechanisms have been identified in the degradation of azo dyes. These
encompass non-specific reduction facilitated by electron transporters
originating from metabolic pathways, reduction facilitated by external
redox mediators, enzymatic reduction specific to the dye, and chemical
reduction through sulfide generated during the microbial sulfate
reduction process (Zafar et al., 2022).
When comparing two similar azo dyes Acid Red 18 and Acid Red 88
we will get to know that these two azo dyes will produce two different
categories of metabolites. When Acid Red 18 was treated with treated
with Shewanella sp. RQs-106 the main metabolites identified comes
under aniline and its derivatives (Zhou et al., 2018). While Acid Red 88
was treated with Achaetomium strumarium the metabolites formed did
not contain any aniline or aniline derivatives (Bankole et al., 2018). RB5
dye was degraded with Pseudomonas entomophila and produced
naphthalene-1,2-diamine and 4-(methylsulfonyl) aniline (Khan and
3.6.1. Toxicity of metabolites in human body
Some aromatic amines such as biphenylamines, benzidine can un­
dergo biomagnification in the food chain and reported to be a problem
for human health and ecosystem (Choudhary, 1996). Benzidine is
recognized as a carcinogenic substance that poses a risk for the devel­
opment of urinary bladder cancer in humans. Additionally, it has been
associated with various other cancers, including those of the genito­
urinary tract, pancreas, liver, gallbladder, bile duct, lung, large intes­
tine, stomach, lymphatic system, and renal cells, as well as
non-Hodgkin’s lymphoma. The International Agency for Research on
Cancer (IARC) has classified all azo dyes that can be metabolized into
benzidine as category 1 carcinogens. Currently, most of these azo dyes
are no longer available in the market (Chung, 2016b). The derivatives of
benzidine, including 4-Aminobiphenyl [(1, 1’-biphenyl)-amine] and 3,
3′-dimethoxy-benzidine, as well as their salts, are categorized as human
carcinogens.
The use of p-phenylenediamine has been linked to an increased risk
of non-Hodgkin’s lymphoma, multiple myeloma, acute leukemia, and
bladder cancer (Chung, 2016a). The carcinogenic properties of aniline
are attributed to β-naphthylamine. Additionally, p-nitroaniline has been
identified as a potential human carcinogen. Research indicates that 2,
4-Dimethylaniline exhibits both mutagenic and carcinogenic effects.
Numerous research articles have established that o-toluidine can lead to
urinary bladder cancer in both animals and humans. In 2010, the IARC
8
Journal of Hazardous Materials Advances 19 (2025) 100834
B. Balachandran and P.C. Sabumon
Table 4
Microbial systems, enzymes, degradation products and associated health effects during azo dye mixture decolorization.
Azo dye
Species/consortium
involved
Enzymes involved
Main metabolites formed
Molecular formula
Health effects
Toxicity
index
References
Reactive
Black 5
Oleaginous yeast
consortium
Lignin
peroxidase,
Manganese
peroxidase,
Laccase, Azo
reductase, NADHDCIP reductase
2,7,8-triaminonaphthalen1-ol
2-chloro-4,6-diamino-1,3,5trizine
2-amino-5-chlorotriazine
Aniline
2-naphthol
Lauric anhydride
C10H11N3O
C3H4ClN5
C4H4ClN3
C6H7N
C10H8O
C24H46O3
Ingestion can produce renal
damage, vomiting,
diarrhoea, abdominal pain,
syncope, convulsions, and
haemolytic anaemia (2naphthol) Skin and eye
irritant (2-amino-5chlorotriazine, Lauric
anhydride)
(Ali et al.,
2020)
Disperse
Orange 2RL
Chlorella vulgaris
algae
Azoreductase
N- methyl-1- adamantane
acetamide
Tris (Tertbutyldimethylsilyoxy)
arsane
Dillapiole
2-cyclohexen-1-one,2methyl-5-(1-methylethenyl)
6- Aza-5,7,12,14 –
Tetrathiapentacene
Cyclotetra siloxane,
decamethyl
2,5 – Dihydroxyac
etophenone,bis
(trimethylsilyl) ether
C13H21NO
C18H45AsO3Si3
C12H14O4
C10H16O
C17H9NS4
C8H24O4Si4
C14H24O3Si2
Harmful if swallowed
(Dillapiole)
Methyl
Orange
Periphyton
Not reported
Phenol
Ethyl acetate
Acetyl acetate
C6H6O
C4H8O2
C4H5O+
3
Probable oral lethal dose to
humans of 50-500 mg/kg
(phenol) Causes skin, eye,
throat irritation (ethyl
acetate)
Synazol Red
6HBN
Alcaligenes aquatilis
3c
Not reported
Pentadecanal
2-acetyl-3methylhexahydropyrrolo
[1,2-a]pyrazine-1,4-dione
[Z]-hexadec-9-enoic acid
palmitic acid
C15H30O
C10H14N2O3
C16H30O2
C16H32O2
C11H18N2O2
Not mentioned
Skin and eye irritant
(Pentadecanal, palmitic acid)
NA
Oral LD50
(rat) 1,320
mg/kg
NA
LD50 (rat)
250–442
mg/kg
LC50 (rat)
1.86–3.3
mg/L in 4
h
LD50 (rat)
13001320 mg/
kg
LC50 (rat)
2.2 mg/L
in 4 h
LD50 (rat)
>5000
mg/kg
LC50 (rat)
0.162 mg/
L in 4 h
LD50
(mouse)
175 mg/
kg
Not
reported
LD50
(mouse)
1000 mg/
kg
Not
reported
Not
reported
LD50 (rat)
>4800
mg/kg
LC50 (rat)
36 mg/L
in 4 h
Not
reported
LD50 (rat)
317-400
mg/kg
LC50 (rat)
0.32 mg/L
in 4 h
LD50 (rat)
5620 mg/
kg LC50
(rat)
16000
mg/L in 6
h
LD50 (rat)
570-760
mg/kg
LC50 (rat)
16000
mg/L in 6
h
Not
reported
Not
reported
Not
reported
(El-Sheekh
et al., 2018)
(Shabbir
et al., 2017)
(Ajaz et al.,
2019)
(continued on next page)
9
Journal of Hazardous Materials Advances 19 (2025) 100834
B. Balachandran and P.C. Sabumon
Table 4 (continued )
Azo dye
Species/consortium
involved
Enzymes involved
Main metabolites formed
Molecular formula
3-isobutylhexahydropyrrolo
[1,2-a]pyrazine-1,4-dione
3-benzylhexahydropyrrolo
[1,2-a]pyrazine-1,4-dione,
bis(6 methylheptyl)
phthalate
Chlorobenzene
N′-(3,6-dichloro-2,7-bis(2(ethyl(methyl)amino)
ethoxy-9H-fluoren-9
ylidene) pivalohydrazide
C6H5Cl
Not mentioned
Health effects
Reactive red
141
Reactive
red 239
Halotolerant and
thermo-alkaliphilic
bacterial consortia
Not reported
2-nitroso naphthol
p-dinitrobenzene
1,3, 5-triazine 2,4-diol
Napththalene diazonium
Aniline
5-amino-1-naphthol
1,3,5-triazine 2,4- diol
C10H7NO2
C6H4N2O4
C3H4N4O2
C10H7N+
2
C6H7NO3
C10H9NO
C3H4N4O2
Methemoglobinemia,
Mutation in organisms
Acid Scarlet
3R
Scheffersomyces
spartinae TLHS-SF1
Not reported
4-aminonaphthalene-1sulfonic acid
7,8-dihydroxynaphthalene1,3-disulfonic acid
3,4-dihydroxynaphthalene1-sulfonic acid
Naphthalene-1,2,6,8-tetraol
Naphthalene-1,2,4-triol
C10H9NO3S
C10H8O8S2
C10H10O3S
C10H8O4
C10H8O3
Skin and eye irritation (4aminonaphthalene-1sulfonic acid)
Reactive
black 5
Trichoderma
atroviride
Laccase
2,4-ditertbutylphenol
1,2,4-trimethylbenzene
Benzoic acid
C14H22O
C9H12
C7H6O2
Cutaneous depigmentation
(2,4-ditertbutylphenol)
Bronchitis, hypochromic
anemia (1,2,4trimethylbenzene)
Corneal damage (Benzoic
acid)
Reactive Red
2, Reactive
Red 198,
Reactive
Bacterial
consortium
Azoreductase,
NADH–DCIP
reductase
1-phenylazo-2-napthol 5,8
disulfonic acid
1-amino triazine 3
pyridine sulfonic acid
Not mentioned
Not mentioned
C5H5NO3S
C10H10NNaO8S2
Toxic Pneumonitis (pyridine
sulfonic acid)
Toxicity
index
LD50 (fish)
>5000
mg/kg
LC50 (rat)
150 mg/L
in 96 h
Not
reported
Not
reported
LD50 (rat)
11002290 mg/
kg LC50
(rat) 12.2
mg/L in 6
h
Not
reported
LD50
(mouse)
42.30 mg/
kg LC50
(fish) 0.4
mg/L in
48 h
LDL0 (rat)
50 mg/kg
LC50 (fish)
0.603-2
mg/L in
96 h
Not
reported
Not
reported
Not
reported
Not
reported
LD50 (rat)
>7500
mg/kg
Not
reported
Not
reported
Not
reported
Not
reported
LD50 (rat)
~2000
mg/kg
LC50 (rat)
128.2 mg/
L in 4 h
LD50 (rat)
6000 mg/
kg
LC50 (rat)
11000
mg/m3 in
4h
LD50 (rat)
1700 mg/
kg
LC50 (rat)
>12.2
mg/L in 4
h
Not
reported
Not
reported
References
(Tizazu et al.,
2023)
(Tan et al.,
2016)
(Adnan et al.,
2015)
(Balapure
et al., 2015)
(continued on next page)
10
B. Balachandran and P.C. Sabumon
Journal of Hazardous Materials Advances 19 (2025) 100834
Table 4 (continued )
Azo dye
Species/consortium
involved
Enzymes involved
Red 120,
Reactive
Blue 160,
Reactive
Blue 13 and
Reactive
Blue 172
Main metabolites formed
Molecular formula
8-amino-naphthol 3,6
disulfonic acid
vinyl sulfone phenyl
diazene
2-diazonium 8nitronaphthoxide
8-hydroxynaphthalene
sulfonic acid
1-phenylmethanediamine
2-aminobenzene sulfonic
acid
8-aminonaphthalene1,2,3,6-tetraol
C8H8O2S
Not mentioned
Not mentioned
Not mentioned
C7H12N2
Health effects
Reactive Red
195,
Reactive
Orange 16,
Reactive
black 5,
Reactive
blue 221,
Reactive
blue 250
Bacillus firmus
VITEPB1, Bacillus
flexus VITEPB2,
Bacillus aryabhattai
VITEPB3, B. flexus
VITSP6, Bacillus
paraflexus VITSPB7,
Bacillus megaterium
VITSPB9
Not reported
Delta-3,4,5,6Tetrachlorocyclohexene
Sulfuric acid
1,2-dichloroethane
Hydroxyphenoxyethyl
aminohydroxypropanol
C6H6Cl4
H2SO4
C2H4Cl2
C11H17NO4
Pulmonary edema,
hepatocellular damage and
liver atrophy and necrosis
(1,2-Dichloroethane)
Trypan Blue
Vibrio sp. JM-17
Azo reductase
and laccase
Benzoic acid
Octanoic acid
Dodecane
1,3-Benzenedicarboxylic
acid
Bis(2-ethylhexyl)ester
Tris(2,4-di-tertbutylphenyl)phosphate
n-Hexadecanoic acid
5-Octadecene
5-Tetradecene
C7H6O2
C8H16O
C12H26
C8H6O4
C24H38O4
C42H63O4P
C16H32O2
C18H36
C14H28
These are nontoxic chemicals
and easily get degraded
Toxicity
index
LD50 (rat)
>2000
mg/kg
LC50 (rat)
>1 mg/L
in 4 h
Not
reported
Not
reported
Not
reported
LD50 (rat)
1127 mg/
kg
LC50 (rat)
0.65 mg/L
in 3 h
Not
reported
Not
reported
Not
reported
LD50
(human)
135 mg/
kg
LC50 (rat)
510 mg/
m3 in 2 h
LD50 (rat)
400-1000
mg/kg
LC50 (rat)
7.2-7.8
mg/L in 4
h
Not
reported
LD50 (rat)
10001700 mg/
kg
LC50 (rat)
>12.2
mg/L in 4
h
LD50 (rat)
200010080
mg/kg
LC50 (fish)
22-110
mg/L in
96 h
LD50 (rat)
>5000
mg/kg
LC50 (rat)
5.6 mg/L
in 4 h
LD50 (rat)
>2000
mg/kg
LC50 (rat)
>2000
mg/m3 in
4h
LD50 (rat)
>20000
mg/kg
LC50 (rat)
10.62 mg/
L LD50
References
(Saha and
Rao, 2024)
(Khandare
et al., 2023)
(continued on next page)
11
B. Balachandran and P.C. Sabumon
Journal of Hazardous Materials Advances 19 (2025) 100834
Table 4 (continued )
Azo dye
Species/consortium
involved
Amaranth
Acinetobacter
calcoaceticus NCIM
2890
Reactive
Black 5
Marine Shewanella
strains
Reactive
yellow-145
Acid Blue 113
Enzymes involved
Main metabolites formed
Molecular formula
Health effects
Lignin
peroxidase,
laccase,
dichlorophenol
indophenol
reductase and
riboflavin
reductase
Oxidoreductase
Naphthalene sulfamide
Hydroxyl naphthalene
diazonium
Naphthyl diazonium ion
C10H9NO2S
Not mentioned
C10H7N+
2
Skin, aye irritant
(Naphthalene sulfamide)
(Z)-4-oxobut-2-enoic acid
4-aminophenol
Z)-4,6-dioxooct-2-enedioic
acid
5,7-dihydroxynaphthalene2,7-disulfonic acid
3,6-diamino-4hydroxynaphthalene-2,7disulfonic acid
2-(4aminobenzenesulphonyl)
ethanol
C4H4O3
C6H7NO
C8H8O6
Not mentioned
C10H10N2O7S2
Not mentioned
Mutation (4-aminophenol)
Bacterial
consortium
Azoreductase
2-cyclohexen-1-ol
5-Nitroso-2,4, 6triminopyrimidine
Octahydroquinoline-9hydroxyperoxide
Tetramethyl-2-hexadecen1-ol
9-Octadecanoic acid,methyl
ester
C6H10O
C4H6N6O
C9H15NO2
C20H40O
C19H34O2
Not reported
Bacterial
consortium
Azoreductase
Palmitic acid
Diethyl phthalate
C16H32O2
C12H14O4
Causes skin eye and
respiratory irritation.
Harmful to aquatic life with
long lasting effect (Palmitic
acid)
Endocrine disruptor
(Diethyl phthalate)
Toxicity
index
(rat) 6000
mg/kg
LC50 (fish)
66 mg/L
in 4 h
LD50 (rat)
>5000
mg/kg
LC50 (fish)
150 mg/L
in 96 h
LD50 (rat)
5000
-10000
mg/kg
LC50 (fish)
1000 mg/
L in 96 h
Not
reported
LD50 (rat)
2500 mg/
kg
Not
reported
Not
reported
LD50 (rat)
375-671
mg/kg
LC50 (fish)
>3.42
mg/L in 4
h
Not
reported
Not
reported
Not
reported
Not
reported
Not
reported
Not
reported
Not
reported
LD50 (rat)
>5000
mg/kg
LD50 (rat)
>2000
mg/kg
LC50 (fish)
>5 mg/L
in 4 h
LD50 (rat)
>5000
mg/kg
LC50 (fish)
150 mg/L
in 96 h
LD50 (rat)
8600 mg/
kg
LC50 (fish)
>4.64
mg/L in 6
h
References
(Ghodake
et al., 2011)
(Liu et al.,
2023)
(Singh et al.,
2022)
(Shanmugam
et al., 2017)
(continued on next page)
12
Journal of Hazardous Materials Advances 19 (2025) 100834
B. Balachandran and P.C. Sabumon
Table 4 (continued )
Azo dye
Species/consortium
involved
Enzymes involved
Main metabolites formed
Molecular formula
Health effects
Toxicity
index
References
Orange II
Fungal strains
Not reported
α-naphthol
Sulfanilic acid
Aniline
C18H12F3NO
C6H7NO3S
C6H7NO3
Potent neurotoxins, causing
excessive salivation and eyewatering in low doses,
followed by muscle spasms
and ultimately death
(α-naphthol)
Respiratory irritation,
reproductive and
development effects(aniline)
(Ali et al.,
2010)
Reactive
Black 5
Pseudomonas
entomophila
Azo reductase
Naphthalene- 1,2- diamine
4- (methylsulfonyl) aniline
C10H10N2
C7H9NO2S
Skin and eye irritation,
carcinogenity, reproductive
problem
Sudan I-IV
dyes
Methyl
Orange
Shewanella
oneidensis MR-1
Laccase-producing
bacteria
Not reported
1-amino-2-naphthol Aniline
C10H9NO
Not reported
Laccase
C7H7NO
C14H14N3NaO3S
Not mentioned
Color vision defects(4(methylimino) cyclohexa2,5-dien-1-one)
Mixed azo
dyes
Alkaliphilic
Bacillus subtilis
Azo reductase
4-(methylimino) cyclohexa2,5-dien-1-one
3-((4-(dimethylamino)
phenyl) diazenyl)
benzenesulfonate
Benzene sulfonate + 3-((4(dimethylamino)phenyl)
diazenyl) benzenesulfonate
complex
Aniline
Benzoic acid3-hydroxy-2Pyridinamine,4,6-dimethylCyclohexanecarboxylic acid
3-[4-Hydroxyisopent-2(Z)enyl]-4hydroxyacetophenone
Chromone, 5-hydroxy6,7,8-trimethoxy-2,3dimethyl
Catechin
1,8-Anthracenediol,
diacetate
LD50 (rat)
18701879 mg/
kg
LC50 (fish)
>3.57
mg/L in
96 h
LD50 (rat)
12300
mg/kg
LC50 (fish)
>100 mg/
L in 96 h
Not
reported
Not
reported
Not
reported
Not
reported
LD50
(rabbit) 60
mg/kg
Not
reported
C7H6O3
C7H10N2
C7H12O2
C13H16O3
C14H16O6
C15H14O6
C18H14O4
Diarrhoea predominant
irritable bowel syndrome,
Nonalcoholic fatty liver
disease
(Cyclohexanecarboxylic
acid)
Congo Red
Fungal species
Laccase
C12H13N
Not mentioned
Methyl
Orange
Serratia sp. WKD
Laccase, NADHDCIP reductase
and azo reductase
N-ethyl-1-naphthalenamine
sodium 3-[(4-ethoxyphenyl)
amino]-2methylbenzenesulfonate
N,N dimethylbenzene
sulfonamide
1,4 diamine
Skin eye irritant (N-ethyl-1naphthalenamine)
Methemoglobinemia (Nphenyl-1-naphthalenamine)
Not reported
Methyl Red
Bacterial and fungal
consortium
L. fusiformis
L. macrolides
B. subtilis
A. terreus, A. oryzae
and A. fumigatus
Azo reductase
Anthranilic acid
Benzamine
Benzene propionic acid 4[(2,4-dinitrophenyl) azo],1methylethyl ester
Benzene, 1,3- bis(1,1dimethylethyl
Hexane, 1,3,4 triol, 3,5,
dimethyl
Pentyl phenylacetate
4- isothiocyanato-N, Nmethyl benzamine
C10H16NO5PS2
C4H12N2
C7H7NO
C6H7N
C18H18N4O6
C14H22
C8H18O3
C13H18O2
Uremic toxins (Anthranilic
acid)
LD50
(mouse)
2000 mg/
kg
Not
reported
LD50
(mouse)
3265 mg/
kg
Not
reported
Not
reported
LD50 (rat)
10000
mg/kg
LC50 (fish)
0.4-4.2
mg/L in
96 h
Not
reported
Not
reported
Not
reported
Not
reported
Not
reported
LD50 (rat)
45495410 mg/
kg
LC50 (fish)
>5.3 mg/
L in 4 h
LD50
(mouse)
1160 mg/
kg
Not
reported
LD50 (rat)
2000 mg/
(Khan and
Malik, 2016)
(Ji et al.,
2012)
(Ambika
et al., 2022)
(Krithika
et al., 2021)
(Gul et al.,
2023)
(Du et al.,
2015)
(El-Rahim
et al., 2021)
(continued on next page)
13
Journal of Hazardous Materials Advances 19 (2025) 100834
B. Balachandran and P.C. Sabumon
Table 4 (continued )
Azo dye
Species/consortium
involved
Enzymes involved
Main metabolites formed
Molecular formula
Health effects
Direct Violet
Bacterial strains
B. coagulans and
L. macrolides
Azo reductase
Ethanol, 2(2-butyxyethoxy)
4-methyl benzoic acid
Phenol 2,4-bis (1,1dimethyl ethyl)
C8H18O3
C8H8O2
C17H30OSi
Central nervous depression,
no hypocalcemic tetany or
metabolic acidosis (ethanol,
2(2-butyxyethoxy)
Shock, delirium, coma,
pulmonary distress, phenolic
breath, scanty/dark urine,
and death (Phenol 2,4-bis
(1,1-dimethyl ethyl))
Reactive
Yellow 145
Enterobacter
hormaechei
Azoreductase
C9H9ClN6
C9H11N5O
C3H5N5
C3H4N4
C8H10N4O
C9H12N4O
C8H10N2
Not reported
Reactive red
180
Enterobacter
hormaechei
Azoreductase
N2 –(4-aminophenyl)6chloro-1,3,5triazine-2− 4
diamine methylhydrazine
1-(3-(formamido)phenyl)
urea
1,2,3,5-triazin-2,4-diamine
Triazin-4-amine
6-(1,4,5,6tetrahydropyrimidin-2-yl)1H-pyrazin-2-one
1-(3-(formamidophenyl)
urea
N-methyl-Nphenylformamidine
Naphthalen-2-amine
N-(3-hydroxybut-2-enyl)
benzamide
N-allyl-2-methylenebut-3enamide
2-methylenebut-3-enamide
N-allylformamide
Methacrylamide
C10H9N
C11H13NO2
C8H11NO
C5H7NO
C4H7NO
C4H7NO
Carcinogen (Naphthalen-2amine)
Neurotoxin & reproductive
toxin (Methacrylamide)
Red HE7B
Bacillus sp.
Azo reductase,
lignin peroxidase,
laccase
6 aminonaphthalene 1-sul­
fonic acid
8 nitroso 1 naphthol
2 diazonium naphthalene
1,3-dihydroxy 8-amino
naphthalene 6-sulphonic
acid
C10H9NO3S
C10H7NO2
C10H7N+
2
C10H9NO5S
Dermatitis, phototoxic (8
nitroso 1 naphthol)
Brown 703
Pseudomonas
aeruginosa
Azoreductase
Toluene
p-Xylene
Benzene,1,2,3-triMethyl
Phenol,2,5-bis(1,1diMethylethyl)
Benzenepropanoic acid,
butyl ester
1,2Benzenedicarboxylicacid,
diisooctyl ester
C7H8
C8H10
C9H12
C14H22.O
C13H18O2
C24H38O4
Sinus tachycardia,
bradycardia (Toluene)
Toxicity
index
kg
LC50 (rat)
>20 mg/L
in 4 h
Not
reported
Not
reported
Not
reported
Not
reported
LD50 (rat)
2000 mg/
kg
LC50 (fish)
63.9 mg/L
in 96 h
Not
reported
Not
reported
Not
reported
Not
reported
Not
reported
Not
reported
Not
reported
Not
reported
LD50 (rat)
8730 mg/
kg
Not
reported
Not
reported
Not
reported
Not
reported
LD50 (rat)
1815 mg/
kg
LC50 (fish)
100 mg/L
in 96 h
Not
reported
Not
reported
Not
reported
Not
reported
LD50 (rat)
>2000
mg/kg
LC50 (fish)
49000
mg/m3 in
4h
LD₅₀ (rat)
~4,0005,000 mg/
kg
LC50 (rat)
4500 mg/
L in 4 h
Not
reported
References
(El-Rahim
et al., 2021)
(Thangaraj
et al., 2021)
(Thangaraj
et al., 2021)
(Thakur
et al., 2014)
(Ullah Khan
et al., 2023)
(continued on next page)
14
B. Balachandran and P.C. Sabumon
Journal of Hazardous Materials Advances 19 (2025) 100834
Table 4 (continued )
Azo dye
Species/consortium
involved
Enzymes involved
Main metabolites formed
Molecular formula
Health effects
Reactive
orange 16
Aspergillus flavus
A5P1
Lignin
peroxidase,
quinone
reductase, laccase
Metanilic acid
2-Diethylamino-5-nitro-1,4naphthoquinone
Naphthalene, 2-methylBenzenamine, N-hydroxy1,2-Benzenedicarboxylic
acid, monobutyl ester
0-Diacetylbenzene
C6H7NO3S
C14H14N2O4.
C11H10.
C6H7NO
C12H14O4
Not mentioned
Corrosive to tissue (Metanilic
acid)
Destroys red blood cells,
causes cancer in stomach,
kidney and respiratory tract
(Naphthalene, 2-methyl-)
Methemoglobinemia Autism
spectrum disorder,
aneuploidy, endometriosis
Motor disorder, memory
disorder, gait disorder
(Benzenamine, N-hydroxy-)
Amido black
10B,
Reactive
Black 5,
Reactive
blue 160
L. squarrosulus AF5
Manganese
peroxidase, lignin
peroxidase,
laccase
C22H14N6Na2O9S2.
Not mentioned
Not mentioned
Not mentioned
Not mentioned
C10H11N3O7S2
C8H11NO6S2.
Not mentioned
Not mentioned
Pulmonary edema,
hypovolemia, arrhythmias
Acid Red B
Candida tropicalis
Not reported
4-amino-5-hydroxy-3-((E)(4-nitrophenyl)diazenyl)-6((E)phenyldiazenyl)
naphthalene-2,7disulfonate
(E)-3,5-diamino-4hydroxy6-((4-nitrophenyl)diazenyl)
naphthalene-2,7disulfonate
(E)-4,6-diamino-5-hydroxy3-((4-nitrophenyl)diazenyl)
naphthalene-2-sulfonate
3,4,6-triamino-5hydroxynaphthalene-2,7disulfonate
2-((4-aminophenyl)
sulfonyl)ethyl sulfate
2-amino-5-((4-chloro-1,3,5triazin-2-yl) amino)
benzene-1,4-disulfonic acid
2-amino-5-((4-chloro-1,3,5triazin-2-yl)amino)
benzenesulfonic acid
3,5-diamino-4hydroxybenzenesulfonic
acid
3-amino-4hydroxybenzenesulfonic
acid
4-hydrazinylnaphthalene
-1-sulfonic acid
4-hydroxynaphthalene-1sulfonic acid
4-aminonaphthalene-1sulfonic acid
3,4-dihydroxynaphthalene1- sulfonic acid
-hydroxynapththalene-1,2dione
C10H10N2O3S
C10H8O4S
C10H9NO3S
C10H8O5S
Not mentioned
Not reported
Acid Red B
Pichia occidentalis
G1
NADH-DCIP
reductase
4-amino-naphthalene-1sulfonic acid
3,4-dihydroxynapththalene-1-sulfonic
acid
Naphthalene-1,24-triol
C10H9NO3S
Not mentioned
C10H8O3
Not reported
Toxicity
index
Not
reported
LD50 (rat)
735 mg/
kg
LC50 (rat)
15.246
mg/L in 4
h
LD50 (rat)
22000
mg/kg
LD50 (rat)
>5000
mg/kg
LC50 (rat)
1.6 mg/L
in 4 h
Not
reported
Not
reported
Not
reported
LD50 (rat)
1000 mg/
kg
Not
reported
Not
reported
Not
reported
Not
reported
Not
reported
LD50 (rat)
>5000
mg/kg
Not
reported
Not
reported
Not
reported
Not
reported
Not
reported
Not
reported
LD50 (rat)
>7500
mg/kg
Not
reported
Not
reported
Not
reported
Not
reported
References
(Qin et al.,
2024)
(Mathur
et al., 2023)
(Tan et al.,
2019)
(Song et al.,
2017)
(continued on next page)
15
B. Balachandran and P.C. Sabumon
Journal of Hazardous Materials Advances 19 (2025) 100834
Table 4 (continued )
Azo dye
Species/consortium
involved
Enzymes involved
Main metabolites formed
Molecular formula
Health effects
Toxicity
index
References
Sudan I-IV
Shewanella
oneidensis MR-1
Shewanella sp. RQs106
Azoreductase
1-amino-2-naphthol
Aniline
7-hydroxy-8-amimo-1,3naphthalene disulfonic acid
1-naphthylamine-4-sulfonic
acid
C10H9NO
Not reported
Not
reported
Not
reported
LD50 (rat)
>7500
mg/kg
Not
reported
Not
reported
​
(Ji et al.,
2012)
(Zhou et al.,
2018)
LD50 (rat)
>750 mg/
kg
LC50 (rat)
47.5 mg/L
(Xu et al.,
2010)
Acid Red 18
Acid Orange
G
Candida tropicalis
TL-F1
Reactive
Orange 16
Pichia kudriavzevii
CR-Y103
Para Red
29 human intestinal
bacteria
​
​
​
NADH-DCIP
reductase and azo
reductase
1-Aminonaphthylene-2hydroxy-3,6-disulfonic acid
naphthalene-1,2,6,8-tetraol
aniline
Aromatic amines
4-nitroaniline
1-amino-2-naphthol
skin irritation, serious eye
irritation
eye damage, skin burn
​
​
Skin irritation
​
​
C6H6N2O2
C10H9NO
elevated o-toluidine’s classification from Group 2A to Group 1, affirming
its carcinogenic potential in humans. Numerous monocyclic aromatic
amines (MAAs) exhibit genotoxic properties and pose significant risks to
human health. Notable examples include 2-nitro-p-phenylenediamine
(4-nitro-1,4-diaminobenzene),
4-nitro-2-amino-6β-hydroxyethylani­
line, 4-nitro-2-amino-6β-hydroxypropylaniline, 4-amino-3-nitro-6-­
fluoroaniline, 4-amino-3-nitro-6-chloroaniline, 2,4-Diaminotoluene,
and m-phenylenediamine (1,3-diaminobenzene) (Chung, 2016a). The
main metabolite of Sudan dye is 1-amino-2-naphthol. When this toxin
was administered at a 0.2 mol/m3 dosage to human intestinal bacteria,
there was a noticeable suppression of the bacterial strains occurred. The
results demonstrated that the common 1-amino-2-naphthol metabolites
of Sudan dye are harmful to the majority of human gut flora. Therefore,
long-term exposure to this toxin causes serious digestive issues in
humans (Pan et al., 2012). Metabolites such as aniline, triazine,
benzene-1,4-diamine, 1-amino-2-napthol are reported to be genotoxic
(Rawat et al., 2016).
The Centers for Disease Control and Prevention (CDC) in the United
States identifies p-PDA as a contact allergen. Exposure to p-PDA can lead
to throat irritation affecting the pharynx and larynx, bronchial asthma,
and sensitization dermatitis. Additionally, o-phenylenediamine is
known to cause eye irritation, skin irritation, dermatitis, and allergic
reactions. A serious condition associated with these substances is
methemoglobinemia, which presents symptoms such as dizziness,
drowsiness, headaches, shortness of breath, cyanosis, rapid heart rate,
chocolate-brown blood, and potential liver damage. Furthermore, mPDA may trigger sensitization reactions, eye irritation and injury, skin
irritation, dermatitis, blackened skin, and bronchial asthma. Other
associated symptoms include allergic skin reactions, mucous membrane
irritation, coughing, burning sensations, runny nose, sore throat,
methemoglobinemia, cyanosis, headaches, dizziness, drowsiness,
mental confusion, pulmonary edema, kidney and liver damage, central
nervous system effects, and conjunctivitis. Contact with the eyes may
result in discomfort, tearing, blurred vision, reddening, partial corneal
clouding, and swelling of the eye and surrounding tissues (Chung,
2016a).
Benzidine poses a significant acute toxicity risk to humans when
ingested. The associated symptoms encompass cyanosis, headaches,
mental disorientation, nausea, and dizziness. Exposure through the skin
can lead to rashes and irritation, in addition to potential bladder dam­
age. o-Aminoazotoluene has been linked to the development of eczema
on the hands and arms. Furthermore, 1-Amino-2-naphthol-4-sulfonic
acid may result in irritation of the eyes and skin, along with gastroin­
testinal disturbances (Agency for Toxic Substances and Disease Registry,
ATSDR, 2025).
​
(Tan et al.,
2014)
(Rosu et al.,
2018)
3.6.2. Response of the metabolites in the human body
Aromatic amines, including aniline, are enzymatically activated in
the liver, primarily by cytochrome P450 enzymes, resulting in the for­
mation of reactive metabolites. These metabolites can covalently bond
with DNA, leading to the creation of adducts that may induce mutations
and trigger carcinogenesis. Additionally, amines can oxidize hemoglo­
bin to methemoglobin, which diminishes the oxygen-carrying capacity
of the blood, potentially resulting in symptoms such as cyanosis. The
metabolites also produce reactive oxygen species (ROS), which can
inflict damage on lipids, proteins, and nucleic acids. Phenolic metabo­
lites, including hydroquinone and catechol, generate ROS through redox
cycling, contributing to lipid peroxidation and damage to cell mem­
branes. Furthermore, phenols can form adducts with cellular proteins,
hindering enzymatic functions and disrupting metabolic processes. The
ROS produced by phenolic compounds can oxidize DNA bases, leading
to strand breaks and mutations. Quinones are capable of accepting and
donating electrons, which generates ROS that contribute to oxidative
stress and cellular injury. They can also covalently modify proteins,
particularly those containing thiol groups, thereby impairing essential
metabolic functions. The ROS can adversely affect mitochondrial func­
tion, resulting in decreased ATP production and triggering apoptosis.
Chlorinated metabolites can mimic or disrupt hormonal activity by
binding to hormone receptors, thereby interfering with signalling
pathways. These compounds tend to accumulate in fatty tissues over
time, leading to chronic exposure. Chlorinated compounds can also
generate electrophilic metabolites that alkylate DNA, resulting in
mutagenesis. Nitroso compounds, such as 2-nitroso-naphthol, can
alkylate DNA bases, creating bulky lesions that interfere with replication
and transcription. The release of reactive nitrogen species (RNS) can
induce nitrosative stress, damaging various cellular components. RNS
can alter protein structure and function, thereby impairing cellular
repair mechanisms. In contrast, aliphatic acids like acetic acid are
rapidly metabolized through cellular pathways, such as the citric acid
cycle, and do not produce harmful metabolites, allowing for easy
excretion (Patnaik, 2007). Fig. 3 depicts the severity indexes of different
metabolic compounds on human health. The scoring system, which
ranges from 0 to 5, along with the comparative graphical representation,
was created by modifying existing toxicity scoring frameworks, such as
the Poisoning Severity Score (PSS) and the NCI Common Toxicity
Criteria. Scores for each class and endpoint were obtained through a
comprehensive literature review to guarantee consistency with docu­
mented toxicological effects and severity classifications.
Table 4 provides comprehensive information about the microor­
ganisms that can break down the azo dyes, species/consortium and
enzymes involved in the process, metabolites of azo dyes from the
16
B. Balachandran and P.C. Sabumon
Journal of Hazardous Materials Advances 19 (2025) 100834
Fig. 3. Comparative severity index scores (0–5) for six major chemical classes across four toxicological endpoints: endocrine disruption, toxicity, mutagenicity, and
carcinogenicity.
(Sources: 11th Report on Carcinogens, 2021; Alva-Gallegos et al., 2024; Ansari et al., 2023; Bolt, 2023; Edenharder and Tang, 1997; Gonsioroski et al., 2020; Huang
et al., 1992; Komulainen, 2004; Lin and Horng, 1986; O’Bryan and Ross, 1988; Persson et al., 1998;Preussmann, 1984; Schwarz et al., 2017; Sui et al., 2022).
process and their potential health effects.
reduction, particularly in anaerobic settings such as those found in the
gut, resulting in the formation of reactive intermediates like hydroxyl­
amines and nitroso derivatives that readily create DNA adducts and
induce mutations (Chung and Cerniglia, 1992). For example, metabo­
lites such as 1-(4-nitrophenylazo)-2-naphthol and other nitro-containing
aromatic amines have exhibited mutagenic and carcinogenic effects in
both in vitro and in vivo investigations, including the induction of chro­
mosomal aberrations and organ-specific cancers. Thus, the presence of a
nitro group in azo dye metabolites does not diminish, and may even
sustain or enhance, their toxicological risk due to their metabolic acti­
vation and DNA-damaging properties (Feng, 2012; Zafar et al., 2022).
Metabolites that possess a nitro group, such as derivatives of nitroaniline
or chloro-nitroaniline, are identified for their genotoxic and mutagenic
risks. For example, the action of E. coli azoreductase on Disperse Red 73
and Disperse Red 78 leads to the production of 4-nitroaniline and
2-chloro-4-nitroaniline, both of which are harmful and present health
and environmental dangers. Metabolites containing nitro groups can
interact with DNA, which may result in mutagenicity and carcinoge­
nicity (Franco et al., 2018).
The existence of a carboxyl group (–COOH) in azo dye metabolites is
significantly associated with decreased toxicity, as this functional group
enhances water solubility and polarity, thus promoting swift excretion
via the kidneys and restricting systemic absorption. Carboxylated me­
tabolites are less prone to cellular uptake or metabolic activation into
genotoxic intermediates, leading to a reduced mutagenic and carcino­
genic potential when compared to their non-carboxylated equivalents.
For example, carboxylated aromatic amines exhibit a lower capacity to
bind to DNA, and their increased hydrophilicity facilitates effective
conjugation (such as glucuronidation) in the liver, further aiding in
detoxification and elimination (Chung and Cerniglia, 1992).
Chloro groups enhance metabolic activation by elevating lip­
ophilicity, which facilitates bioaccumulation and chronic toxicity (Sun
et al., 2017). Amines that are ortho-substituted, for example, o-amino­
azotoluene, are found to be more carcinogenic than their para coun­
terparts, resulting in the induction of tumors in the bladder and liver.
The accumulation of hydrophobic metabolites, like eicosenoic
acid-coupled Sudan III, in tissues results in a prolonged exposure period
(Feng, 2012; Sun et al., 2017); however its toxicity is not reported in the
work.
Electron-donating groups (EDGs) such as amino (–NH2), methyl
(–CH3), and hydroxyl (–OH) in azo dye metabolites considerably in­
crease toxicity by elevating electron density on aromatic rings and
3.6.3. Correlation between azo dye metabolites toxicity and its structural
properties
The toxicity associated with azo dye metabolites is significantly
related to the presence of electron-withdrawing groups, electrondonating groups, and overall molecular complexity. These properties
impact the way metabolites interact with biological systems, their
persistence, and their potential to lead to mutagenic, carcinogenic, and
other toxic consequences (Brown and De Vito, 1993). Metabolites that
contain unsubstituted aromatic amines (like benzidine and phenyl­
enediamine) are characterized by high genotoxicity due to their ability
to generate DNA adducts. For example, 4-aminoazobenzene, resulting
from the metabolism of Sudan III, is known to cause liver tumors in
rodents. The methylation or halogenation of amine groups does not
mitigate mutagenicity, whereas sulfonation or carboxylation of amine
groups lead to a reduction in toxicity by enhancing the excretion process
(Bienstock et al., 2022; Chung and Cerniglia, 1992; Feng, 2012). Planar
aromatic metabolites, such 1-amino-2-naphthol, intercalate into DNA to
cause helical distortions and replication errors. As an example, Sudan I
metabolites bind to deoxyguanosine at the C8 or N2 sites, destabilizing
the DNA and impeding the repair process.
Electron withdrawing groups containing metabolites; sulfonated
metabolites, such as 1-amino-2-naphthol-6-sulphonate derived from
Sunset Yellow, exhibit decreased toxicity as a result of diminished
membrane permeability and accelerated excretion via urine and bile
(Feng, 2012). In a similar manner, incorporating sulfonic acid groups
into benzidine, which is recognized as a human carcinogen, significantly
diminishes its mutagenic and carcinogenic properties. After the meta­
bolic degradation of the azo bond, the resulting sulfonated aromatic
amines are quickly handled by the liver and expelled, which further
lessens the risk of systemic toxicity (Feng, 2012). In contrast, unsulfo­
nated analogues, like 1-amino-2-naphthol, demonstrate mutagenic
properties in Salmonella assays (de Almeida et al., 2021; Feng, 2012).
Metabolites of azo dyes that include nitro groups (–NO2) are significant
for their persistent genotoxic and carcinogenic potential, even though
the nitro group serves as a strong electron-withdrawing substituent. In
contrast to sulfonation or carboxylation, which typically diminish
toxicity by enhancing water solubility and facilitating excretion, the
addition of a nitro group does not reduce mutagenicity; rather,
nitro-substituted metabolites often remain highly mutagenic. This is due
to the fact that nitroaromatic compounds can undergo enzymatic
17
B. Balachandran and P.C. Sabumon
Journal of Hazardous Materials Advances 19 (2025) 100834
encouraging metabolic activation to genotoxic intermediates. These
groups aid in enzymatic oxidation, resulting in the production of DNAbinding electrophiles like nitrenium ions, which can create covalent
adducts with DNA and lead to mutations. Amino groups undergo
enzymatic oxidation (such as by cytochrome P450), resulting in the
formation of nitrenium ions that covalently bond to DNA, which can
lead to mutations and chromosomal anomalies. Metabolites including
benzidine (from benzidine-based dyes) and 4-aminoazobenzene are
powerful hepatocarcinogens, triggering liver tumors in rodent models.
2,5-diaminotoluene (a metabolite of certain dyes) shows genotoxicity in
bacterial tests and is associated with organ-specific carcinogenic effects.
Unsubstituted amino groups (for example, in p-phenylenediamine) can
cause acute toxicity, which includes renal failure and hemolysis, along
with minimal genotoxicity. Di-amino substitutions (like 1,2,4-triamino­
benzene from the reduction of chrysoidin) significantly boost mutage­
nicity due to their enhanced reactivity. The positioning of substituents is
critical: Ortho-amino substituents (as found in o-aminoazotoluene) in­
crease carcinogenicity, leading to tumor formation in the bladder, liver,
and lungs across various species (Colin Garner and Nutman, 1977; Feng,
2012). Electrophilic nitrenium ions, originating from metabolites such
as 4-aminobiphenyl, preferentially attack guanine bases, which results
in mutations (Bienstock et al., 2022). On the other hand, modifications
to the structure, such as sulfonation (–SO3H), can offset the impacts of
EDGs by boosting hydrophilicity and encouraging excretion, thereby
lessening mutagenicity. Methyl groups in substances such as o-amino­
azotoluene and 2,5-diaminotoluene are correlated with pronounced
mutagenic and carcinogenic properties, which include tumor induction
in the bladder, liver, and other organs. Hydroxyl groups, as illustrated by
1-amino-2-naphthol, can augment mutagenicity and carcinogenicity via
redox cycling and the creation of reactive oxygen species. Alkoxy
groups, though not as frequently highlighted, also enhance electron
density and may lead to similar toxic consequences. The presence of
several EDGs, or their strategic arrangement (such as ortho or para to the
azo bond), further magnifies these effects by stabilizing reactive in­
termediates that can interact with DNA or disrupt cellular mechanisms
(Feng, 2012).
overall reliability. Utilizing advanced analytical techniques to observe
biotransformation products during microbial degradation further re­
inforces these assessments. Together, these methodologies deepen our
understanding of the health and ecological hazards related to azo dye
metabolites (Funar-Timofei and Ilia, 2020).
Despite their potential, QSAR models aimed at predicting the toxicity
of azo dye metabolites encounter several significant challenges and
indicate clear pathways for future investigation. A primary limitation is
the lack and inconsistency of high-quality experimental toxicity data,
particularly for complex or less-explored metabolites such as haloge­
nated, carboxylated, or mixture-derived compounds, which hinders the
precision and reliability of model predictions (Huang et al., 2021;
Keşkek Karabulut and Yalçin Gürkan, 2023). Moreover, existing QSAR
methodologies frequently find it difficult to address the intricacies of
metabolic activation pathways such as those facilitated by cytochrome
P450 enzymes which can transform relatively inert parent dyes into
highly reactive and toxic intermediates, complicating the prediction of
chronic or long-term effects (Huang et al., 2021; Rawat et al., 2016). The
toxicity of mixtures and the possibility of synergistic interactions among
various metabolites also remain inadequately addressed, as the majority
of models are tailored for single-compound predictions and lack de­
scriptors specific to mixtures (Frindt et al., 2017). In addition, conven­
tional 2D molecular descriptors may not sufficiently capture 3D steric
and electronic influences or distinguish between mono and poly­
sulfonated metabolites, thereby limiting their capacity to represent
real-world structural diversity. To tackle these issues, future initiatives
should prioritize the expansion of curated experimental datasets, the
development of advanced descriptors (including 3D and
mixture-specific features), and the integration of QSAR with machine
learning and in silico metabolic simulation tools (Funar-Timofei and Ilia,
2020; Reddy Ramireddy et al., 2023). The incorporation of multi-omics
data and the validation of models through multispecies microcosm
studies will also improve ecological relevance. Ultimately, enhancing
collaboration between regulatory agencies and industry will be essential
for establishing QSAR-guided safety-by-design frameworks and ensuring
the effective application of these models in risk assessment and regu­
latory decision-making concerning azo dye metabolites (Brown and De
Vito, 1993).
3.6.4. Role and relevance of QSAR approaches in predicting the toxicity of
metabolites
QSAR models function as computational tools that can anticipate the
toxicity of azo dye metabolites by evaluating the relationship between
molecular attributes and biological activity. These models utilize mo­
lecular descriptors (for example, electronic properties, hydrophobicity,
and functional groups) to predict toxicity endpoints such as mutage­
nicity, carcinogenicity, and aquatic toxicity, eliminating the necessity
for laboratory experiments. QSAR models (ProTox-II and the OECD
QSAR Toolbox) assess the degradation products of azo dyes, like Reac­
tive Orange 16 and Direct Red 80, through ozonolytic or microbial
processes. The findings from these models indicate that certain metab­
olites are carcinogenic, immunotoxic, and cytotoxic, with a notable
impact on androgen receptors and mitochondrial function (Reddy
Ramireddy et al., 2023). To illustrate, QSAR models have been suc­
cessfully employed to predict the acute aquatic toxicity of azo dyes and
their degradation products, identifying substances such as Disperse Blue
291 as significantly toxic to fish (LC₅₀ = 0.0675 mg/L) and highlighting
aromatic amines like 2-bromo-4,6-dinitroaniline as particularly harmful
to aquatic invertebrates. Additionally, QSAR approaches have been
utilized to assess the mutagenic and carcinogenic potential of azo dye
metabolites, indicating that electron-donating groups (for example,
amino or methyl) tend to elevate mutagenicity, while sulfonation typi­
cally mitigates toxicity by enhancing excretion and reducing bioavail­
ability (Enslein and Borgstedt, 1989). Additionally, QSAR models are
vital for environmental risk assessment. They facilitate the screening of a
vast array of dye structures and highlight compounds that pose signifi­
cant risks. This process supports the prioritization of regulatory actions.
Merging QSAR predictions with experimental toxicity data improves the
4. Treatment of azo dye metabolites formed after
biodegradation
Most of the metabolites come under aniline category but only few
studies reported how to control or remove these products from waste­
water. Therefore, when considering the environmental risk of these
metabolites it is important to focus on how it is transformed, how to
increase degradation, and how to reduce its production. For example,
aniline can be biodegraded under aerobic condition. Finally, it will enter
into a TCA cycle (Zhang et al., 2021). This session is focussing on the
treatment of metabolites which is formed after the biodegradation
process. There are very few studies focussed on this case. Therefore,
recovery of metabolites or proper secondary treatment of azo dye me­
tabolites requires additional attention.
These metabolites (amines) can be broken down by different
methods such as AOP, biological, chemical, physical, and photochemical
processes, each of which has pros and cons of their own. A variety of
products can be produced by amines during the chemical oxidation
process, depending on the oxidant used, the amines’ structure, and the
conditions applied during the reaction. But sometimes instead of
mineralization polymerisation also happens. In physical process
adsorption gets more attention. Activated carbon, copper treated chi­
tosan, alumina nano particles, carbon black and montmorillonite are
found to be good in adsorbing specific type of amines.
Ekici et al. (2001) selected 8 different azo dye metabolites which are
commonly seen in textile wastewater for further biodegradation studies.
He established that these metabolites maintain relative stability in
18
Journal of Hazardous Materials Advances 19 (2025) 100834
B. Balachandran and P.C. Sabumon
aquatic ecosystems and are not effectively degraded by typical waste­
water treatment processes. The degradation of these particular metab­
olites under aerobic sludge conditions is facilitated by the oxidation of
substituent groups located on the aromatic ring or within the side chain.
But in the anaerobic condition reductive cleavage of the azo bond
happens. This leads only a partial degradation. Ekici and Lahaniatis
(1998) examined the photo induced reaction for the breakdown of
specific azo dye metabolites. The outcomes demonstrated an effective
and quick way to remove these metabolites from industrial effluent.
A very few studies were reported on the treatment of metabolites
formed from the biodegradation of azo dyes. Most of the studies focussed
on the anaerobic-aerobic combinations of treatment to reduce the
toxicity of the metabolites. But by the aerobic treatment of the in­
termediates of Ramazol Yellow RR; the color reappeared (Jonstrup et al.,
2011). Also, the aeration treatment was not successful for treating the
metabolites of Ramazol Red RR (Jonstrup et al., 2011). This is due to the
auto oxidation of the metabolites upon exposure to the aerobic condi­
tion. These results showed that some recalcitrant amines are unable to
degrade using aerobic means. So, aeration methods are not always a
reliable choice for the treatment of metabolites formed during the
biodegradation of azo dyes.
A combination of biological process followed by a short post ozon­
ation process treats recalcitrant metabolites. A real textile effluent
treated with same biological process followed by a 6 min post ozonation
process reduced the toxicity 20 times lower than the raw effluent (Punzi
et al., 2015b). Among all the treatment methods reported, photo­
catalytic method is better. It can degrade complex molecules into
simpler one under optimal condition. In the case of adsorption, the used
adsorbent and its regeneration will be a problem and also the disposal of
adsorbed compound. Even though aeration enhances the degradation
process, but it may fail when the complexity of the effluent increases.
Ozonation also has drawbacks such as partial degradation of the com­
pound and need of an optimal condition. Although there are few studies
focused on the treatment of metabolites produced from the biological
degradation of azo dyes, both ozonation and adsorption have consis­
tently shown greater effectiveness compared to other available methods.
Future research should focus on this area to find a complete solution in
terms of complete mineralization, energy efficiency and nontoxic sec­
ondary metabolites. The reported removal methods of formed azo dye
cleaved metabolites after the biological process are shown in Table 5.
5. Future challenges and scope
The biodegradation of azo dyes has been recognised as a practical
and effective strategy, but it also requires further study of its difficulties
in order to obtain better results for actual applications.
• Studies should concentrate on fully mineralizing synthetic dyes using
sustainable practises. Azo dyes are difficult to fully mineralize
because of their intricate structure. In order to ensure that the
complicated dye structure breaks down into a colourless liquid that is
safe, suitable biotechnological processes must be designed and
tested.
• The biodegradation pathways and the metabolites formed need
additional attention. It is essential to comprehend and reduce the
possible toxicity of these metabolites. Depending on the type of
metabolites generated, the proper polishing steps such as ozonation,
adsorption, photocatalytic degradation or aerobic biodegradation
are used. Identification of the metabolites and biodegradation routes
is required for that. If the metabolites can be recovered and used in
other domains, research should concentrate on them. Pharmaceu­
tical firms use metabolites like aniline and its derivatives as their
basic materials. If it can be gathered and put to use, it would promote
the circular economy concept in dye effluent management.
• In order to give researchers a fresh perspective on biodegradation
mechanisms, modern molecular biology approaches should be used
to identify microbial species and to better examine the genes and
enzymes involved for azo dye treatment.
• In upcoming research, computational methods like molecular dock­
ing and quantum mechanics/molecular mechanics (QM/MM) simu­
lations may be utilized to forecast enzyme–dye interactions.
Additionally, machine learning strategies-including graph neural
networks for predicting metabolites, reinforcement learning for
enhancing bioreactor conditions, and deep learning frameworks for
enzyme engineering-ought to be employed to expedite the identifi­
cation of effective biodegradation pathways and enhance process
efficiency for the removal of azo dyes.
6. Conclusions
Industrial effluent containing azo dyes poses a significant threat to
both the environment and human health if not properly treated. Based
on this review, biological approaches offer a more environment friendly
and cost-efficient solution with long-term benefits. Nevertheless, the
Table 5
Removal methods of formed azo dye cleaved metabolites after the biological process.
Dye
Metabolites / Intermediates
Removal Method
Key experimental
conditions
Main Outcome / Final Products
References
Fast violet B
Biodegradable aldehydes & acids
Ozonation
Not reported directly
Ozonation
Methyl Red
Phenolic intermediates & diamines
Reactive Black
Aniline
Photocatalytic ZnO
treatment
Biochar adsorption
Complete decolorization; formation
of small acids & aldehydes
COD reduction 59% & TOC
reduction ~31%
Formation of 1,4-diaminobenzene
(Lopez et al., 1998)
Ramazol Red
Alkaline pH, short
contact (10-90 min)
Short treatment (4 min)
Complete removal of aniline
(Mahmood et al., 2015)
Acid Orange 7
& AR88
Acid Red 14
Aminonaphthalene sulfonic acid,
aminobenzenesulfonic acid
Aminonaphthalene sulfonic acid
High surface area
biochar, 24 h
Aeration, moderate
organic load
Not detailed
No accumulation of intermediates
(de los Cobos-Vasconcelos
et al., 2012)
(Franca et al., 2020)
Methyl Red
2-aminobenzoic acid, N,N’-dimethylp-phenylenediamine
Not reported
DO 6-7.5 mg/L
89% metabolite removal; final
nitrobenzene & benzoic acid
COD reduction 92%
(Jayapal et al., 2018)
Partial peak reduction in HPLC;
recoloration observed
(1, 4-diaminobenzene) and one
unidentified compound
(Jonstrup et al., 2011)
Remazol Red
Remazol Red
& Yellow
Methyl red
Not reported
2-{[4(methylamino)phenyl]
diazenyl}benzoic acid
Packed bed
bioreactor
Aerobic
biodegradation
Aerobic
biodegradation
Photo-Fenton
oxidation
Aerobic
biodegradation
Photocatalytic
treatment-ZnO
Mild pH, 2 h
H2O2 20 mM
DO ~5-6 mg/L; HRT 7
days
30 ◦ C, pH 7.0 for 2 h
19
63% metabolite reduction
(Punzi et al., 2015b)
(Waghmode et al., 2019)
(Punzi et al., 2015a)
(Waghmode et al., 2019)
Journal of Hazardous Materials Advances 19 (2025) 100834
B. Balachandran and P.C. Sabumon
metabolites formed during the biodegradation of azo dyes require
careful consideration and additional treatment methods. Microbial or­
ganisms like bacteria, yeast, and fungi have shown promising results in
degrading azo dye mixtures, with most studies focusing on bacterial
degradation. However, research on algal-based studies on azo dye
mixture is still limited, despite their proven ability to act as an excellent
bio-sorbents and thrive in extreme saline conditions. Furthermore, there
is a lack of research on the metabolites formed during the degradation
process, particularly those falling under aniline and its derivatives,
which have potential value-added applications. The recovery of these
metabolites has not received sufficient attention, and finding an
economical and environmentally friendly approach to treat other toxic
metabolites remains a challenge. Although physicochemical and
advanced oxidation processes are commonly employed to handle the
metabolites, some biological studies have shown complete mineraliza­
tion in the case of single azo dye degradation. Therefore, there is still
immense potential to explore the capabilities of microorganisms, as well
as the numerous enzymes they possess, which could be further studied
using biotechnology and molecular biology techniques. Enhancing the
biodegradation process, especially in the case of azo dye mixtures, re­
mains an exciting avenue for research. Ultimately, harnessing the power
of microorganisms can lead to a safe and sustainable conversion of azo
dyes and other harmful substances.
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Declaration of generative AI and AI-assisted technologies in the
writing process
During the development of this work, the first author made use of
Grammarly to examine the text for spelling and grammatical mistakes,
in addition to ensuring clarity. Subsequent to the application of this tool,
the authors carefully reviewed and modified the content as necessary,
taking full accountability for the publication’s content.
Funding information
We (authors) gratefully acknowledge Department of Science and
Technology (DST), Government of India, for supporting this work
through the research grant DST/TM/WIC/WTI/2K17/82(G4).
CRediT authorship contribution statement
Bhavana Balachandran: Writing – original draft, Visualization,
Software, Resources, Methodology, Investigation, Formal analysis, Data
curation, Conceptualization. P.C. Sabumon: Writing – review & editing,
Validation, Supervision, Resources, Project administration, Funding
acquisition, Data curation, Conceptualization.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgment
We gratefully acknowledge Department of Science and Technology
(DST), Government of India, for supporting this work through the
research grant DST/TM/WIC/WTI/2K17/82(G4).
Data availability
Data will be made available on request.
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