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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