United States
Environmental Protection
Agency
Risk Reduction
Engineering Laboratory
Cincinnati, OH 45268
Research and Development
EPA/600/S2-89/051 Jan. 1990
Project Summary
Aerobic and Anaerobic
Treatment of C. I. Disperse
Blue 79
David A. Gardner, Thomas J. Holds worth, Glenn M. Shaul , Kenneth A.
Dostal, and L. Don Betowski
This study summarized here was
conducted to determine the fate of
C.I. Disperse Blue 79, one of the
largest production-volume dyes, and
select blodegradatlon products in a
conventionally operated activated
sludge process and in an anaerobic
sludge digestion system. To achieve
this objective, a pilot study was
conducted from "November 1987 to
February 1989. Two continuous-feed
pilot-scale waaftewater treatment
systems, one control and one
experimental, were operated during
the pilot study. Yfee experimental
treatment system was fed screened,
raw municipal wastewater dosed with
a target concentration of 5 mg/L of
active Ingredient in the commercial
formulation of C. I. Disperse Blue 79.
The control system was fed only the
screened, raw municipal wastewater.
After acclimation and after steady
state conditions were reached,
samples from each system were
analyzed for the dye and related
compounds. A bench-scale activated
sludge system was also operated to
assess the fate of dye degradation
products from the anaerobic digester
In an aerobic treatment system. This
system was operated to simulate the
recycle of digester supernatant to the
head-end of a typical wastewater
treatment system. The results of this
extensive research project are
presented In Volume I of the full
report. Findings are presented
regarding: (1) the development of an
analytical procedure to determine C.I.
Disperse Blue 79 in various sample
matrices; (2) the effect of C.I.
Disperse Blue 79 on the operation of
an activated sludge system and an
anaerobic digester; (3) the fate of the
dye in the treatment systems; and (4)
the detection of any degradation
products in the systems. Laboratory
and operating data collected during
the study are presented In Volume II.
This Project Summary was
developed by EPA's Risk Reduction
Engineering 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 fate of specific azo dyes in
wastewater treatment facilities is
unknown since few detailed studies have
been reported in the literature. A better
understanding of this large class of
organic compounds is necessary to
assess their effect on the environment
and on human health. Several azo dyes
and possible biodegradation products,
such as aromatic amines, have been
shown to be, or are suspected to be,
carcinogenic. C.I. Disperse Blue 79, one
of the largest production-volume dyes, is
a water insoluble bromodinitroaniline-
derived compound. The empirical formula
for the bromomethoxy form of C.I.
Disperse Blue 79 used in this study is
C23H25BrN6010, the molecular weight is
625.4, and the structural formula is shown
in Figure 1.
The purpose of this study was to
determine the fate of C.I. Disperse Blue
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OCH3
NH-CO-CH3
Figure 1. C. I. Disperse Blue 79
79 and select biodegradation products in
a conventionally operated activated
sludge process and in an anaerobic
sludge digestion system. Before testing,
an analytical procedure for measuring C.I.
Disperse Blue 79 concentrations was
developed. Two continuous-feed, pilot-
scale wastewater treatment systems (one
control [Unit 1] and one experimental
[Unit 2]) were operated at the Milwaukee
Metropolitan Sewerage District (MMSD)
South Shore Wastewater Treatment
Plant.
In addition to these pilot-scale
systems, a bench-scale activated sludge
system (Unit 3) was operated to assess
the fate of dye degradation products from
a digester in an aerobic treatment
system. This system was operated to
simulate the recycle of digester
supernatant to the head-end of a typical
wastewater treatment system.
Experimental Procedures
The two pilot-scale treatment systems
were operated for the entire study, from
November 1987 to February 1989. The
bench-scale system was operated from
November 1988 to February 1989.
C.I. Disperse Blue 79 Extraction
and Analysis
Because a reliable method for dye
analysis was needed to determine the
fate of C.I. Disperse Blue 79 in the
treatment system, an analytical
procedure was developed. Various
extraction methods and solvents were
investigated to develop a suitable
extraction procedure to prepare samples
for high-performance liquid chromatog-
raphy (HPLC) analysis.
Pilot-Scale Treatment Systems
Both pilot-scale activated sludge
systems included a contact tank, a
conical-shaped primary clarifier, an
aeration basin, and a conical-shaped
secondary clarifier. The contact tanks
were installed to ensure the dye was
mixed with the feed and to obtain a 30-
min contact time between the raw
wastewater and the dye. The primary and
secondary clarifiers were approximately
49L, and the aeration tanks were
approximately 185 L.
The activated sludge basins were
separated into three cells to operate as a
plug-flow system. Peristaltic pumps
supplied the screened, raw wastewater to
the contact tanks. Gravity moved the
wastewater from the contact tanks to the
primary clarifiers, then to the aeration
basins, and on to the secondary clarifiers.
Activated sludge was wasted from the
aeration basins via peristaltic pumps.
Primary sludge was wasted manually
once each day. The target hydraulic
retention time (HRT) was 5.5 hr and the
solids retention time (SRT), 7 days.
The anaerobic digesters were
cylindrical-shaped vessels constructed of
clear PVC. Each digester had a total
volume of 70 L with an operating volume
of 39 L. The digesters were completely
mixed and heated to maintain an
operating temperature of 35°C. Gas
production from the digesters was
monitored with gas meters.
Waste activated sludge and primary
sludge from each activated sludge unit
were mixed, thickened, and fed to the
respective anaerobic digesters. The
target SRT of the anaerobic digesters
was 15 days and the target loading was
1.2 kg total volatile solids (TVS)/m3»day.
The experimental treatment received
screened, raw wastewater dosed with a
target concentration of 5 mg/L of the
active ingredient in C.I. Disperse Blue 79.
The control system received only the
screened, raw wastewater. After
acclimation and steady state conditions
were reached, the following samples from
each system were analyzed for the dye
and related compounds: influent, primary
effluent, activated sludge effluent,
primary sludge, waste activated sludge,
digester feed, digester supernatant, and
digester effluent.
Bench-Scale Treatment System
The bench-scale system was an
activated sludge unit operated on a feed
mixture prepared from the experimental
system. The activated sludge unit
consisted of a 6-L conical reactor, which
served as the aeration basin; a 2-L inner
cone for solids recycle; and a 125-ml
clarifier tube for effluent clarification.
Peristaltic pumps were used to deliver
the feed and remove waste activated
sludge from the unit.
The feed mixture included primary
effluent from the experimental system,
supernatant from digester I
preparation (primary and waste acti\
sludge thickening), and centrate
centrifuging digested sludge from
anaerobic digester. The mixture
prepared to simulate the recycl
digester supernatant and primary
thickened waste activated sli
supernatant to the head-end i
treatment plant.
Results and Discussion
C.I. Disperse Blue 79 Extract/
and Analysis
Initial experiments with two lie
phase extractions did not yield
acceptable procedure for C.I. Disp
Blue 79; therefore, the approach
changed to dispersing the aqu<
sample in acetonitrile. A spectropt
meter monitored the extrac
procedure. C.I. Disperse Blue 79 cor
trations in the extracts were anal'
with the use of HPLC.
Analytical spike and analyl
duplicate analyses (part of the Qu
Assurance/Quality Control (QA/
program) monitored the accuracy
precision of the extraction procedure
HPLC analyses. For the 57 analy
duplicate analyses performed
spectrophotometry, the average rel
difference was 4.3% with a stan
deviation of 6.7%. For the 29 anal}
spike analyses performed
spectrophotometry, the average s
recovery was 105% with a stam
deviation of 13.8%. For the se
analytical duplicate measurem
performed on the HPLC, the ave
relative difference was 6.8% wil
standard deviation of 9.1%. Tl
replicate analyses performed on
same sample had an average rel,
difference of 4.8% and standard devi.
of 3.1%.
Field spike samples prepared 1
field duplicate samples ensured
proper sample collection proced
were used. The average rela
difference for HPLC analyses was 1C
with a standard deviation of 15.3%.
Pilot-Scale Systems Operat/oi
Operating data were collet
throughout the entire period the syst
were operated. Because of analy
capacity limitations, however, the f
scale activated sludge systems,
anaerobic digesters, and the bench-s
activated sludge system "Were'
sampled over the entire period for
analyses. Thus, all discussion:
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}uipment performance include only data
jring the time dye analyses were
>nducted.
Operating and analytical data for the
lot-scale activated sludge units are
mmarized in Table 1. The data for both
lits 1 and 2 were similar. The average
X)D value for Unit 2 was 73.5 mg/L and
t for Unit 1 was 59.2 mg/L. Although
3 slightly higher effluent TCOD value
• Unit 2 may have been caused by
ding dye to the unit, the data indicate
\ the overall performance of the
perimental activated sludge system
is not affected by this addition.
tile 1. Summary of the Pilot-Scale
Activated Sludge Systems'
Operational and Analytical Data for
the Dye Testing Period
Parameter
Unit 1
Unit 2
leddata
rSS (mg/L
TCOD (mg/L)
TBOD (mg/L)
NH3-N (mg/L)
perational data
HRT (hr)
SRT (days)
238
364
182
22.5
5.28
5.94
211
375
177
20.7
5.28
5.87
ixed liquor data
Temperature (°C) 20.0 20.0
pH (range) 6.8-8.0 6.8-7.6
DO, Cell 1 (mg/L) 2.4 2.8
DO, Cell 2 (mg/L) 3.6 3.5
DO, Cell 3 (mg/L) 3.6 3.9
TSS (mg/L) 3,030 3,060
02 Uptake rate 6.8-8.0 6.8-7.6
(mg/Lhr) 73.1 58.0
SSVI (ml/g)
rimary effluent data
rSS (mg/L) 134 139
NH3-N(mg/L) 22.7 21.7
nal effluent data
TCOD (mg/L)
TBOD (mg/L)
TSS (mg/L)
NH3-N (mg/L)
59.2
16
27
0.26
73.5
21
31
0.18
The anaerobic digester's operating
id analytical data are summarized in
ible 2. The feed, effluent, and
)erational data indicate no significant
ference between the two units. No
Jverse affect was detected on the
Deration of the experimental digester as
result of adding dye.
ye and Related Compounds
esults
The influent and effluent streams (feed
ream, primary clarifier effluent, primary
Table 2. Summary of the Anaerobic
Digesters' Operational and
Analytical Data for the Dye Testing
Period
Parameter
Unitl
Unit 2
Feed data
7SS (mg/L)
TS (%)
TVS (%)
21,300
2.42
1.81
21,400
2.42
1.82
Effluent data
pH (range) 6.6-7.0 6.6-7.0
Temperature (°C) 35.0 35.0
TSS (mg/L) 12,700 12,200
TS (%) 1.46 1.48
TVS (%) 0.94 0.97
Operational data
Alkalinity (mg/L) 2,930 2,820
Volatile acids < 51 < 50
(mg/L)
Loading 1.21 1.22
(kg TVS/m3 day)
TVS reduction (%) 47.8 46.4
Gas production 0.76 0.87
(m3/kg TVS
destroyed)
Percent CH4 in gas 58.9 57.9
sludge, waste activated sludge, and final
clarifier effluent) from the experimental
activated sludge systems were sampled
and analyzed for C.I. Disperse Blue 79
and any related compounds to determine
the fate of the dye in the treatment
system. The average dye and TSS
concentrations from the Unit 2 samples
are summarized in Table 3. Influent and
waste mixed liquor samples were
analyzed from Unit 1. The dye was not
detected in any of the control unit
samples analyzed (i.e., no background
concentration of dye was present in the
raw municipal wastewater feed).
The average dye concentration in the
Unit 2 feed to the primary clarifier was
4.40 mg/L and the average final effluent
concentration was < 0.93 mg/L, so that
the average dye removal was greater
than 79%. Although 5 of 19 analyzed
effluent samples were below the 0.25
mg/L detection limit, the effluent dye
concentration varied from < 0.25 mg/L to
3.70 mg/L. The variation in effluent dye
concentration may have been caused by
the variation in effluent TSS concen-
tration.
The correlation coefficient between
TSS and dye concentrations in the Unit 2
effluent was determined to be 0.78. In
addition, calculations performed on Table
3 data show that each gram of
suspended solids in the waste activated
sludge contained 30 mg of dye whereas
each gram of suspended solids in the
final effluent contained 33 mg of dye.
These data indicate that the dye has a
high affinity for the activated sludge
solids. Approximately 21 % of the dye fed
to the unit was in the final effluent;
however, most of the dye was probably in
the suspended solids in the effluent. The
average final effluent TSS concentration
was 28 mg/L. Lowering this TSS concen-
tration by improving solids removal in the
final clarifier could result in a lower dye
concentration in the final effluent.
Table 3. Average C. I. Disperse Blue 79 and
TSS Concentration in the Unit 2
Experimental Activated Sludge Unit
Samples
C.I. Disperse
Sample
Location
Feed
Primary effluent
Primary sludge
Waste activated
sludge
Blue 79
(mg/L)
4.40
4.71
31.8
93.5
TSS
(mg/L)
212
133
14,500
3,060
Final effluent
< 0.93
28
Mass balance calculations were
performed with the use of the measured
dye concentrations and measured
flowrates for each process stream. Mass
balance calculations across the entire
activated sludge system showed that an
average of 86.5% of the dye contained in
the feed stream was accounted for in the
effluent streams. The primary sludge
contained an average of 3.6% of the dye
fed to the system; waste activated
sludge, 62.3%; and final effluents, 20.4%
(the percentages of the three streams do
not equal 86.5% because of rounding off
the individual values). Since most of the
dye fed to the system was recovered and
no other related compounds were
detected, it can be concluded that no
significant biodegradation of C.I. Disperse
Blue 79 occurred in the activated sludge
system.
Feed sludge and effluent (digested
sludge) samples from both the control
and experimental digesters were
analyzed for dye content. Detectable
concentrations of dye were identified by
HPLC-UV in 5 of 10 control-unit feed
samples and in 4 of 10 effluent samples.
The average concentrations were low,
however, at < 1.45 mg/L for the feed
samples and < 1.22 mg/L for the effluent
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sample. The low level of dye in these
control unit samples is negligible when
compared with the much higher
concentrations of dye in the experimental
unit samples.
The average experimental unit feed
dye concentration was 443 mg/L, and the
average effluent concentration was 7.86
mg/L. On the average, 98.2% of the dye
contained in the feed sludge was
degraded in the anaerobic digester.
Thermospray ionization mass
spectrometry was used to identify
degradation products of C.I. Disperse
Blue 79 in the anaerobic digester effluent.
With this ionization technique, the parent
dye was observable, but because of the
electronegativity of many of the functional
groups on the molecule (e.g., N02, Br),
the sensitivity of the technique for this
compound was poor. Four major
degradation compounds were tentatively
identified and found in significant
amounts in the digester effluent. Their
exact identity and amounts have not been
verified because appropriate analytical
standards were not available. The
degradation products showed a better
response than the parent dye by the
ionization technique; the increased
sensitivity probably resulted from either
removal or reduction of the electro-
negativity that was suppressing the
sensitivity of C.I. Disperse Blue 79. The
relative amounts of these compounds (A,
B, C, and D having molecular weights of
283, 358, 400, and 478 daltons,
respectively) measured in one set of
samples are summarized in Table 4. The
relative amounts of these compounds
were greater in the digester effluent than
in the digester feed. (Information about
the structure of these compounds is
discussed in detail in Volume I of the full
report).
Some of the potential degradation
pathways of C.I. Disperse Blue 79 could
liberate bromide from the dye compound.
Anaerobic digester feeds and effluents
were analyzed to determine if bromide
was liberated in the digester. In one set
of data from the control unit, sample
analyses did not show any significant
difference in bromide concentration
between the feed and effluent (Table 5).
In both sets of data from the experimental
unit, however, the bromide concentration
was much lower in the feed than in the
effluent. The two feed samples averaged
2.50 mg/L, and the two effluents
averaged 39.0 mg/L. For the one set of
samples for which dye analyses are also
available, the dye concentration was
reduced from 250 mg/L to 9.46 mg/L.
These data indicate that bromide was
being liberated during dye degradation in
the anaerobic digester.
Bench-Scale Activated Sludge
System
During normal operation of a
wastewater treatment system, the super-
natant from sludge lagoons or other
digester sludge thickening operations is
returned to the head-end of the plant for
treatment. The bench-scale activated
sludge system (Unit 3) was operated to
study the fate of dye degradation
products from the anaerobic digester in
an activated sludge system. The
supernatant from the sludge thickening
operation used to prepare the digester
feed was mixed with centrate from
centrifuging digester effluent and primary
effluent to prepare feed for Unit 3. The
supernatant was added to simulate the
effluents produced from thickening waste
activated sludge in a typical plant.
The operating and analytical data from
Unit 3 are presented in Table 6. The
average HRT was 6.04 days, which was
slightly higher than the Unit 2 value of
5.28 days; the average SRT for Unit 3
was 4.83 days, which was lower than the
Unit 2 average of 5.87 days. The Unit 3
average SRT was lower than the target
value of 7 days because of a relatively
high average effluent TSS value of 40
mg/L. The bench-scale unit settling
performance was more variable than that
in the pilot units.
The average effluent TCOD and TBOD
values were also higher than the pilot unit
values. The higher effluent values
probably resulted from the higher TSS
levels in the final effluent. The
performance of Unit 3 with respect to
TSS, TBOD, and TCOD removal was not
as good as that of the pilot units but was
typical of a bench-scale unit.
Table 7 summarizes the dye data from
the bench-scale unit feed, waste
activated sludge, and final effluent
sample analyses. The average feed dye
concentration was 3.43 mg/L and the
average effluent concentration was 1.32
mg/L, for a removal efficiency of 62%.
The effluent concentration was probably
high because of the relatively high TSS
concentration in the final effluent.
Mass balance calculations of the dye
across Unit 3 showed that an average of
75.3% of the dye fed to the unit was
accounted for in the effluents from the
unit. The mass balance for Unit 2 showed
86.5% of the dye was recovered.
Although the recovery from Unit 3 was
slightly lower, it does not appear that
significant degradation of the d
occurred in the bench-scale activa
sludge system.
Degradation products of C.I. Dispe
Blue 79 were also monitored in
influent, effluent, and waste sludge fr
Unit 3. Because no positive identifical
was made of the by-products, quan
cation was not possible. Some gen<
observations can, however, be rm
concerning the degradation produ
based on relative amounts. The obser
trend indicated that the concentration
these compounds decreased across I
3. The final effluent samples alw;
contained the lowest concentrations
the degradation products, but because
limited data, further conclusions can
be drawn. Further evaluation of
degradation products and their fate
biological treatment systems may
subject for further project work.
Conclusions
1. The addition of C.I. Disperse Blue
did not adversely affect the opera'
of the pilot activated sludge unil
that of the anaerobic digester. E
the control and experimer
activated sludge units produi
effluents typical of munici
wastewater treatment systems.
anaerobic digesters achieved vol<
solids reductions within the nor
operating range for munici
digesters.
2. No evidence of C.I. Disperse Blue
degradation in the activated slu
systems was found. Mass bala
calculations showed that, on avers
86.5% of the dye contained in
feed to the system was presen
the effluent streams.
3. The majority of the C.I. Disp€
Blue 79 fed to the activated slu
system was removed in the wi
activated sludge. The average
mass balance obtained around
system was 86.5%; the dye
partitioned in the effluent stream;
follows: 3.6% in the primary slu<
62.3% in the waste activated slue
and 20.4% in the final effluent.
4. The C.I. Disperse Blue 79
degraded in the anaerobic digei
The dye concentration was redi
from an average feed value of
mg/L to an average effluent valu
15.0 mg/L, or a 97.4% reduction.
5. Possible degradation products ol
dye were detected in the dige
effluent. Although some prelimi
measurements were made to ide
the structure of these compounds
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Table 4. Mass Spectrometric Analysis of Anaerobic Digester Samples
Relative Sample Amounts (Arbitrary Units)
Sample Location
Control digester feed
Experimental digester feed
Experimental digester effluent
A(MW 283)
26,000
24,000
568,000
B(MW 358)
7,300
6,400
264,000
C(MW 400)
7,400
6,300
365,000
D(MW 478)
1,700
43,000
225,000
Table 5. Bromide Analyses of Anaerobic Digester Samples
Sample
Location
Experimental unit feed
Experimental unit effluent
Control unit feed
Control unit effluent
Experimental unit feed
Experimental unit effluent
Sample Date
H5/89
1/5/89
217189
2/7/89
2/7/89
2/7/89
Bromide
Concentration
(mg/L)
1.75
40.8
0.85
0.66
3.24
37.1
C.I. Disperse Blue 79
Concentration
(mglL)
A
*
*
*t
250
9.46
"Analysis not performed.
Table 6. Summary of Activated Sludge Unit
3's Operational and Analytical Data
Parameter
Average Value
Feed
rss (mg/L)
TCOD (mglL)
TBOD (mg/L)
Operation data
HRT (hr)
SRT (days)
Mixed liquor data
Temperature (°C)
pH (range)
DO (mg/L)
TSS (mg/L)
Final effluent data
TSS (mg/L)
TCOD (mg/L)
TBOD (mg/L)
130
336
158
6.04
4.83
21.5
6.8-8.1
5.6
T,650
40
116
31
Table 7. Bench-Scale Activated Sludge System's C. I. Disperse Blue 79
Analytical Results
C. I. Disperse Blue 79 TSS
Sample Location (mg/L) (mglL)
Feed
Waste activated sludge
Final effluent
3.43
37.6
1.32
14.5
2,150
53
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positive identification or quantification products from the anaerobic digester The full report was submitted i
of the compounds was made. were destroyed when treated in a fulfillment of Contract No. 68-03-3371 I
6. Based on limited semi-quantitative bench-scale activated sludge Radian Corporation under th
results, some of the dye degradation system. sponsorship of the U.S. Environment
Protection Agency.
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