United States
Environmental Protection
Agency
Water Engineering
Research Laboratory
Cincinnati, OH 45268
Research and Development
EPA/600/S2-88/030 July 1988
&EPA Project Summary
Fate of Water Soluble Azo
Dyes in the Activated Sludge
Process
Glenn M. Shaul, Clyde R. Dempsey, and Kenneth A. Dostal
The objective of this study was to
determine the partitioning of water
soluble azo dyes in the activated
sludge process (ASP). Azo dyes are
of concern because some of the
dyes, dye precurors, and/or their
degradation products such as
aromatic amines (which are also dye
precurors) have been shown to be,
or are suspected to be, carcinogenic.
Specific azo dyes were spiked at 1
and 5 mg/L to pilot-scale treatment
systems with both liquid and sludge
samples collected. Samples were
analyzed by high performance liquid
chromatography (HPLC) with an
ultraviolet-visible detector.
Mass balance calculations were
made to determine the amount of the
dye compound in the waste activated
sludge (WAS) and in the activated
sludge effluent (ASE). Of the 18 dyes
studied, 11 compounds were found
to pass through the ASP
substantially untreated, 4 were
significantly adsorbed onto the WAS,
and 3 were apparently biodegraded.
This Project Summary was
developed by EPA's Water Engineering
Research Laboratory, Cincinnati, OH,
to announce key findings of the
research project that is fully
documented in a separate report of
the same title (see Project Report
ordering information at back).
Introduction
The U.S. Environmental Protection
Aqency's (EPA) Office of Toxic
Substances evaluates Premanufacture
Notification (PMN) submissions under
Section 5 of the Toxic Substances
Control Act. Azo dyes constitute a
significant portion of these submissions.
Generally, azo dyes contain between one
and three azo linkages (-N = N-),
linking phenyl and naphthyl radicals that
are usually substituted with some
combination of functional groups
including: amino (-NHa); chloro (-CI);
hydroxyl (-OH); methyl (-CH3); nitro
(-NOg); and sulfonic acid, sodium salt
(-S03Na).
One aspect of the PMN review
process is to estimate the release of a
new chemical. The industrial
manufacturing and processing of azo
dyes will generate a wastewater
contaminated with azo dyes, which is
typically treated in a conventional
wastewater treatment system. The
effectiveness of this treatment must be
known in order to estimate the release
from this source. Therefore, EPA's Water
Engineering Research Laboratory, Office
of Research and Development undertook
a study to determine the fate of specific
water soluble azo dye compounds in the
ASP.
The study was approached by
dosing the feed to the pilot ASP systems
with various water soluble azo dyes and
by monitoring each dye compound
through the system, analyzing both liquid
and sludge samples. The fate of the
parent dye compound was assessed via
mass balance calculations. These data
could determine if the compound was
removed by adsorption, apparent
biodegradation, or not removed at all.
The report presents results for 18 dye
compounds tested from June 1985
through August 1987. The study was
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conducted at EPA's Test and Evaluation
Facility in Cincinnati, OH.
Experimental Program
Screened raw wastewater from the
Greater Cincinnati Mill Creek Sewage
Treatment Plant was used as the influent
(INF) to three pilot-scale activated
sludge biological treatment systems (two
experimental and one control) operated
in parallel. Each system consisted of a
primary clarifier (33 L), complete-mix
aeration basin (200 L), and a secondary
clarifier (32 L).
Each water soluble dye was dosed
as commercial product to the screened
raw wastewater for the two experimental
systems operated in parallel at targeted
active ingredient doses of 1 and 5 mg/L
of influent flow (low and high spike
systems, respectively). The principal
focus of this work was on the ASP, and,
as such, the primary sludge was not
sampled. Table 1 presents a summary of
the average operating conditions of the
pilot-plant systems.
Before each data collection phase,
dye analytical recovery studies were
conducted using organic-free water,
influent wastewater, and mixed liquor.
These studies were run in duplicate and
each recovery study was repeated at
least once to ensure that the compound
could be extracted from these samples.
Purified dye standards were analytically
prepared from the commercial dye
product by repeated recrystallization.
The INF, primary effluent (PE), and
ASE were filtered, and the filtrate was
passed through a column packed with
resin. The filter paper and resin were
soaked in an ammonia-acetonitrile
solution and then Soxhlet extracted with
ammonia-acetonitrile. The extract was
concentrated and brought up to 50 mL
volume with a methanol/dimethyl-
formamide solution. The mixed liquor
(ML) samples were separated into two
components, the filtrate or soluble (SOL)
fraction and the residue (RES) fraction.
The SOL fraction was processed similar
to the INF, PE, and ASE samples. The
RES fraction and the filter paper were
processed similar to these samples but
the resin adsorption step was omitted. All
extracted samples were analyzed by
HPLC with an ultraviolet-visible
detector. Total suspended solids (TSS)
analyses were also performed on the
INF, PE, ML, and ASE samples.
All systems were operated for at
least three times the solids retention time
to ensure acclimation prior to initiation of
data collection. All samples were 24 hr
composites made up of 6 grab samples
collected every 4 hr and stored at 4°C.
The 18 water soluble, acid and direct azo
dyes studied in pilot-scale ASP
systems are listed below in Table 2 by
Colour Index name and number. Figure 1
presents the chemical structure for each.
Results and Discussions
Before a compound was judged
acceptable for spiking into the pilot-
scale treatment systems, spike recovery
studies were conducted for each dye.
These tests were conducted using
laboratory, organic-free water samples
and several wastewater and sludge
samples from the control ASP. All
samples were spiked and held at 4°C for
24 hr before recovery was assessed. The
possible removal mechanisms for a dye
compound in the ASP system include
adsorption, biodegradation, chemical
transformation, photodegradation, and air
stripping. Table 3 presents the results
from these determinations. Recovery for
most dyes was within the targeted range
of 80% to 120%; thus, it appeared that
little or no chemical transformation
occurred for these dyes because of
contact with" the variable wastewater
and/or sludge matrix under these
conditions. Some recoveries from
wastewater and/or sludge samples for
four of the dyes were outside the
targeted range, but these dyes were
accepted for sampling because such
recoveries were considered acceptable
to the general project guidelines. As the
recoveries for all 18 dyes were generally
very good and with relatively low
standard deviations, all values in Tables
3-5 are presented as measured and no
correction made for recovery. In addition,
no photodegradation of the dyes was
found in laboratory studies. Moreover,
the estimated Henry's law constant for
each dye tested was less than 10'15
atm-m3/mol, and, as such, air stripping
was very unlikely. Therefore, adsorption
and/or biodegradation appeared to be the
only removal mechanisms.
Table 4 presents the mean
concentrations for each of the dyes
tested. Four dyes have two runs reported
whereas all other dyes have just one.
Additional runs were conducted for
quality assurance/quality control reasons.
From the results in Table 4 and TSS
data, mass balance calculations can be
made (see Table 5). If a compound in
Table 5 was recovered near the targeted
range of 80% to 120%, then it was
assumed that this compound was not
biodegraded since most of the
compound was recovered. Conversely, if
the recovery was less than 20% to 30°
then it was assumed the compound w;
apparently biodegraded. This assumpti<
was valid only because prelimina
recoveries (Table 3) indicated little or r
problems in recovering the compoun<
from the various sample matrices. Lastl
if the compound was recovered near tt
targeted range of 80% to 120%, then or
must investigate the percentac
adsorbed data. If these data indicate
less than 20% adsorbed, then it w;
assumed that the compound w<
substantially untreated by the AS
However, if these data indicated that tt
amount adsorbed was greater than 30°
then it was concluded that such
compound was removed by appare
adsorption.
Eleven of the 18 azo dyes studied
Table 5 passed through the AS
substantially untreated with the data fro
the low and high spike systems
excellent agreement for these dye
These were:
C.I. Acid Black 1
C.I. Acid Orange 10
C.I. Acid Red 1
C.I. Acid Red 14
C.I. Acid Red 18
C.I. Acid Red 337
C.I. Acid Yellow 17
C.I. Acid Yellow 23
C.I. Acid Yellow 49
Cl. Acid Yellow 151
C.I. Direct Yellow 4
The relatively high sulfonic ac
substitution of these dyes may expla
why they were not removed. If the a;
dye has high sulfonic acid substitutio
then little or no adsorption of the dye t
the microbial cell or cell byproduc
would occur, thus limiting the chance
aerobic biodegradation. Ten of the 1
above dyes have at least two sulfon
acid functional groups, C I. Acid Red 3Z
has one.
The positioning of the sulfonic ac
functional group(s) and the molecul.
weight of the compound also appeared
have an affect on how the compour
partitions. Note in Table 5 that foi
compounds were adsorbed onto tr
WAS and apparently not biodegradei
These were
Cl. Acid Blue 113
Cl Acid Red 151
C.I. Direct Violet 9
C I. Direct Yellow 28
C.I. Acid Blue 113, C I. Acid Re
151, and C I. Direct Violet 9 represe
three of the four disazo (two azo bond
structures Although these dyes ai
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Table 1. Summary of Operating Conditions
Parameter
Value
Influent flow rate, Ud
Primary sludge flow rate, Ud
Primary effluent flow rate, Ud
Mixed liquor wastage flow rate, Ud
Secondary effluent flow rate, Ud
Solids retention time, days
Hydraulic retention time, days
Dissolved oxygen, mgIL
Target influent spike dosages, mg/L
Low
High
Influent pH, pH units
Aeration basin temperature, °C
720
6
714
67
647
2.7
0.28
2.0-4.0
1
5
7.0-8.0
21-25
Table 2. Dye Compounds Spiked to
the Activated Sludge
Process
Colour Index Name
C.I. Acid Black 1
C.I. Acid Blue 113
C.I. Acid Orange 7
C.I. Acid Orange 8
C.I. Acid Orange 10
C.I. Acid Red 1
C.I. Acid Red 14
C.I. Acid Red 18
C.I. Acid Red 88
C.I. Acid Red 151
C.I. Acid Red 337
C.I. Acid Yellow 17
C.I. Acid Yellow 23
C.I. Acid Yellow 49
C.I. Acid Yellow 151
C.I. Direct Violet 9
C.I. Direct Yellow 4
C.I. Direct Yellow 28
Colour Index
Number
20470
26360
15510
15575
16230
18050
14720
16255
15620
26900
18965
19140
18640
13906
27885
24890
19555
"Not assigned as of 12/87. Chemical
Abstracts Number 67786-14-5.
sulfonated compounds with two of the
three having two sulfonic acid functional
groups, they also have a greater
molecular weight than the other
compounds. Further investigations into
the affect of sulfonation (both in number
of groups and position) versus molecular
weight are necessary before a
relationship, if any exists, could be
developed.
Note also in Table 5 that three
compounds appeared to be biodegraded.
These were:
C.I. Acid Orange 7
C.I. Acid Orange 8
C.I. Acid Red 88
The conclusion that these
compounds were apparently
biodegraded comes from an inspection
of the mass balance data; for each
compound, very little of the dye was
recovered during sampling. However, the
preliminary recovery studies showed that
the compound could be recovered
without difficulty from wastewater and
sludge matrices (see Table 3). Since the
compounds were not found in the ASE or
ML samples and chemical transformation
appeared not to be occurring, then
biodegradation would account for the
loss of the parent compound.
In addition to the 18 dyes thus far
discussed, 11 other azo dyes were
investigated during this study but the
analytical recovery methodology did not
produce satisfactory recoveries from the
various matrices for these dyes. Table 6
identifies these dyes.
Conclusions
1. A total of 18 water soluble azo dyes
were successfully monitored in
wastewater and sludge samples
collected from pilot-scale ASP
treatment systems. The study of 11
additional dyes was attempted but
could not be accomplished because
of poor analytical recovery from
wastewater and/or sludge samples.
2. Based on the compounds tested in
this study, high water solubility, as
judged by the degree of sulfonation,
seemed to be a major factor in
preventing an azo dye compound
from being either apparently
adsorbed or biodegraded by the
ASP.
3. Of the 18 dyes studied, 11
compounds were found to pass
through the ASP substantially
untreated, 4 were significantly
adsorbed onto the WAS and 3 were
apparently biodegraded.
Recommendations
1. Since several azo dyes passed
through the ASP relatively untreated,
further investigations into how to
remove these compounds, and
others like them, may be necessary
2. Investigations into the degradation
products resulting from the aerobic
biodegradation of azo dyes may be
necessary to determine if the
degradation products, such as
aromatic amines, persist in the water
3. For those compounds that strongly
adsorb onto WAS, investigations into
their fate in anaerobic environments
(e.g., anaerobic digesters or landfills)
would be of value.
4. Additional testing of structurally
related compounds to those tested in
this study may allow structure
activity relationships to be
developed.
-------
02/V(
\NH(
C.I. Acid Black 1
C.I. Acid Blue 113
HO
CH3 HO
C./. Acid Orange 7
C.I. Acid Orange 8
HO
-0*0
WO NH-CO-CH3
C./. Acid Orange 10
C.I. Acid Red 1
HO
'OaS
WO
C.l. Acid Red 14
C.I. Ac id Red 18
HO
HO
-03S(
C/ Xlc/dRec/88
C.I. Acid Red 151
Figure 1. Chemical structures of test dyes.
-------
Cf3
'SOa'
C/
HOC -
it I
> /V=/V-C N
V
CH3
)S03"
C/
C.I. Acid Red 337
C.I. Acid Yellow 17
"03S(C ))/V=/V-C% N
%c*
COO"
C/
SI HOC - /V^)S03"
' /V t/
x«*
"C/ c
CH3
C.I. Acid Yellow 23
C.I. Acid Yellow 49
CH3
COH
N=NCCOHNI{
H3C
C.I. Acid Yellow 151
C.I. Direct Violet 9
H0
>OH
S03"
C./. D/recf Yellow 4
HaCr
C./. Direct Yellow 28
Figure 1. (continued).
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Table 3. Percent Recovery of Test Dyes from Sample Matrices
Sample Matrix
Dye Compound Name
1
C.I. Acid Black 1, Run 1
C.I. Acid Black 1, Run 2
C.I. Acid Blue 113
C.I. Acid Orange 7,
2
C.I. Acid Orange 8
Runs 1
and
Org. Free
mg/L
90
69
61
101
115
Water
5 mg/L
93
78
89
97
111
C.I. Acid Orange 10 ~
C.I. Acid Red 1
C.I. Acid Red 14
C.I. Acid Red 18
C.I. Acid Red 88
C.I. Acid Red 151,
C.I. Acid Red 151,
C.I. Acid Red 337
C.I. Acid Yellow 17
C.I. Acid Yellow 23
C.I. Acid Yellow 49
Run 1
Run 2
M
707
98
93
74
**
89
707
708
90
«.
90
98
706
85
•*
88
98
702
93
C.I. Acid Yellow 151
C.I. Direct Violet 9,
C.I. Direct Violet 9,
C.I. Direct Yellow 4
Run 1
Run 2
C.I. Direct Yellow 28
"nof tested.
Table 4. Dye Concentrations Data
Summary
-
**
86
96
„
~
90
98
Wastewater
1 mg/L 5 mg/L
87
79
95
97
703
97
705
98
94
92
97
703
95
92
703
99
776
720
703
87
94
72
90
88
97
707
80
95
97
88
82
83
83
88
97
703
700
98
92
98
92
87
Low Spike +
Dye Compound Name
Acid Black 1, Run 1
Acid Black 1, Run 2
Acid Blue 113
Acid Orange 7, Run 1
Acid Orange 7, Run 2
Acid Orange 8
Acid Orange 10
Acid Red 1
Acid Red 14
Acid Red 18
Acid Red 88
Acid Red 151, Run 1
Acid Red (57, Run 2
Acid Red 337
Acid Yellow 17
Acid Yellow 23
Acid Yellow 49
Acid Yellow 151
Direct Violet 9, Run 1
Direct Violet 9, Run 2
Direct Yellow 4
Direct Yellow 28
INF
0.53
0.43
1.00
0.99
1.12
0.80
1.17
1.01
0.90
1.21
"
"
0.96
1.20
0.97
1.33
1.14
1.29
0.95
0.98
0.84
0.93
PE
0.44
<0.11
0.84
0.95
1.04
0.82
0.96
0.90
0.66
1.23
0.68
0.56
0.71
7.73
0.95
7.23
7.74
0.67
0.78
0.83
0.76
0.87
ASE
0.41
0.28
0.07
0.19
0.31
<0.03
7 07
0.89
0.77
7.33
0.04
0.77
0.24
0.93
0.92
/.32
0.84
0.49
0.47
0.67
0.76
0.78
SOL
0.40
0.79
0.04
<0.08
0.25
<0.02
0.88
0.83
0.74
7.72
0.02
0.08
0.09
0.68
0.93
7.30
0.86
0.26
0.27
0.32
0.58
0.77
RES"
0.13
<0.04
3.98
<0.03
<0.03
<0.03
<0.03
<0.07
<0.03
<0.05
0.77
2.90
2.67
7.75
<0.06
<0.03
0.74
0.46
2.93
7.74
0.08
5.78
INF
2.21
2.59
5.27
4.96
6.18
4.39
5.44
4.71
4.61
5.11
~
*•
4.87
5.46
4.58
5.08
5.77
6.44
5.30
5.22
3.90
3.74
Mixed
1 mg/L
87
77
85
98
86
74
65
88
87
109
94
101
95
99
102
99
109
87
101
93
101
Liquor
5 mg/L
80
83
76
90
94
73
92
92
90
96
90
84
85
97
103
99
113
89
95
94
97
High Spike +
PE
2.20
<0.75
4.55
5.34
5.53
4.02
5.55
4.43
2.87
4.54
3.96
3.64
4.37
5.06
4.57
5.25
5.42
4.05
4.77
4.72
3.37
3.69
ASE
2.29
2.02
0.84
0.24
0.77
<0.70
5.49
4.48
4.76
4.77
<0.07
0.44
0.54
4.40
4.55
5.39
3.59
4.08
0.99
7.32
3.77
0.63
SOL
2.07
1.41
0.44
0.15
0.58
<0.04
4.94
4.43
3.98
4.76
<0.07
0.77
0.36
3.67
4.45
5.35
3.79
2.66
038
0.59
2.97
0.25
RES*
0.47
0.13
19.86
<0.03
<0.03
<003
<0.04
0.05
<0 11
<0.04
0.07
79.86
78.85
4.08
<0.05
<0.03
0.44
4.37
23.38
23.44
0.27
22.83
+ Concentration in mg/L.
" Mass in mg of dye adsorbed/gm of MLSS.
"Not sampled.
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Table 5. Mass Balance Data Summary
Low Spike
High Spike
Dye Compound Name
Acid Black 1, Run t
Acid Black 1, Run 2
Acid Blue 113
Acid Orange 7, Run 1
Acid Orange 7, Run 2
Acid Orange 8
Acid Orange 10
Acid Red 1
Acid Red 14
Acid Red 18
Acid Red 88
Acid Red 151, Run 1
Acid Red 151, Run 2
Acid Red 337
Acid Yellow 17
Acid Yellow 23
Acid Yellow 49
Acid Yellow 151
Direct Violet 9, Run 1
Direct Violet 9, Run 2
Direct Yellow 4
Direct Yellow 28
%
Recovered
96
244
74
19
30
4
104
98
116
107
7
73
82
95
98
107
75
89
93
100
99
78
%
Adsorbed
3
6
66
<1
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Glenn M. Shaul, Clyde R Dempsey, and Kenneth A. Dostal are with the Water
Engineering Research Laboratory, U.S. Environmental Protection Agency,
Cincinnati, OH 45268.
The complete report, entitled "Fate of Water Soluble Azo Dyes in the Activated
Sludge Process," (Order No. PB 88-208 251; Cost: $14.95, subject to
change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA authors can be contacted at:
Water Engineering Research Laboratory
U.S. Environmental Protection Agency
Cincinnati, OH 45268
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
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