United States
Environmental Protection
Agency
Water Engineering
Research Laboratory
Cincinnati OH 45268
/1 \
Research and Development
EPA/600/S2-85/141 Feb. 1986
Project Summary
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Protocol Development for the
Prediction of the Fate of
Organic Priority Pollutants in
Biological Wastewater
Treatment Systems
E. J. Kirsch, C. P. L. Grady, Jr., R. F. Wukasch, and H. H. Tabak
Many of the organic chemicals classi-
fied as priority pollutants can be
present in the wastewater entering mu-
nicipal and industrial biological waste-
water treatment plants. A biodegrad-
ability test protocol was developed and
tested to provide a scientific basis for
predicting the fate of organic priority
pollutants in typical activated sludge
treatment systems and anaerobic di-
gestion processes.
The biodegradability testing proce-
dure focuses on the study of: (1) sub-
strate toxicity to activated sludge sys-
tems and anaerobic digestion,
(2) partitioning and intermedia trans-
port of pollutants including volatiliza-
tion and sorption, and (3) substrate
biodegradability, including kinetics and
effluent quality as a function of solids
retention time.
This report describes the evolution of
the testing protocol, the development
of necessary laboratory procedures, the
results of the application of these tests
to the priority pollutant pentachloro-
phenol (PCP), and the utilization of
these data to evaluate and predict the
fate of PCP in biological wastewater
treatment systems.
This Project Summary was devel-
oped by EPA's Water Engineering Re-
search 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
Approximately 129 extensively manu-
factured and widely used organic chem-
icals are classified as priority pollutants.
Since many of these chemicals can be
detected in wastewater, questions have,
naturally, been raised as to their fate in
biological wastewater treatment sys-
tems. The degree of hazard posed by a
chemical depends greatly on its concen-
tration and exposure time. Thus, the ex-
tent of biodestruction in wastewater
treatment systems will probably be one
of the factors affecting regulatory deci-
sions on the continued manufacture
and use of these substances. This re-
search program was initiated to estab-
lish and test an experimental protocol
for predicting the fate of selected prior-
ity pollutants in aerobic and anaerobic
biological wastewater treatment sys-
tems.
The objectives of the program were
(1) to develop and test a biodegradabil-
ity protocol for determining the toxicity
of organic compounds to aerobic bio-
logical systems and anaerobic digestion
processes, (2) to quantitate the adsorp-
tion and stripping losses of the com-
pound, (3) to develop kinetic informa-
tion allowing prediction of effluent
quality as a function of the sludge reten-
tion time (SRT) of the biological treat-
ment system, and (4) to estimate the ex-
tent of biodegradation (mineralization)
of the compound.
The study evaluated the application
of the experimental biodegradability
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testing protocol to the testing of se-
lected priority pollutants. Pen-
tachlorophenol (PCP) was chosen as the
typical priority pollutant because it is
known to exhibit high resistance to un-
acclimated systems, but it is substan-
tially degraded in acclimated systems.
Furthermore, PCP shows significant
bioinhibitory properties even in sys-
tems that lead to its eventual decompo-
sition. These considerations implied
that the use of PCP would be a severe
test for the proposed methodology. The
proposed methodology is also being
evaluated with seven additional pollu-
tants that will be the subject of a future
report.
Experimental Approach
The study emphasized determining
the kinetic relationship between the
growth rate of organisms in the system
and the concentration of organic com-
pounds. This information can then be
used with an appropriate mass balance
equation to predict the fate of the sub-
strate for the full-scale system. Since
activated-sludge systems are basically
large chemostats, the growth rate of or-
ganisms in the system is controlled by
the SRT. Thus, knowing the SRT of a
given full-scale system and the kinetics
of biodegradation will permit an esti-
mate of the effluent concentration of the
substrate.
The biodegradability studies followed
the noninteractive kinetic approach;
this implies that the concentration of the
priority pollutant in a steady-state con-
tinuous feed reactor is governed solely
by the specific growth rate of the organ-
isms responsible for its degradation
and that the specific growth of these or-
ganisms is controlled by the SRT and
cell decay rate.
The relationship between specific
growth rate of microorganisms respon-
sible for substrate degradation and con-
centration of organic substrate can be
expressed by the equation M, = f(S), in
which the ^ (specific growth rate) is a
function of substrate (S) concentration.
At substrate concentration well below
the inhibitory threshold, the equilibrium
concentration of a growth-limiting sub-
strate can be related to the specific
growth rate of microbial population by
the Monod equation.
tion constant for the organic substrate)
are empirically determined parameters
describing the hyperbolic relationship
between ^ and S. When S is much lower
than Ks, as in the case of most treatment
systems, the Monod equation reduces
to a simple first order rate equation:
or
The relationship between specific
growth rate of microorganisms and SRT
of a reactor is expressed by the equa-
tions:
and
M- = (1/SRT) + ds
M- = (Ks/n,m)(1/SRT) +
* ~ Ks + S '
where ^m (maximum specific growth
rate of microorganisms) and KS (satura-
2
where ds is the decay rate constant for
the organisms degrading the substrate.
A plot of S versus 1/SRT should yield a
straight line with a slope equal to (Ks/
ixm), an ordinate intercept of (Ks/|im)ds,
and an abscissa ordinate as -d. Once M-m-
Ks and ds values are determined, the
concentration of organic pollutant in the
reactor may be predicted for a selected
SRT.
A multi-tiered approach was used to
obtain information needed to describe
the fate of a priority pollutant and is out-
lined briefly in Figures 1 and 2. A three-
level approach was used for aerobic
studies: Level I (acclimation and abiotic
removal). Level II (biodegradation kinet-
ics), and Level III (extent of biodegrada-
tion). Anaerobic studies involved a two-
level approach: Level I (acclimation and
substrate toxicity assay) and Level II
(growth kinetics and operational per-
formance of digester systems).
An effective way of establishing the
kinetic relationship between the specific
growth rate of a microbial culture and
the concentration of a growth-limiting
substrate is through continuous culture
in a continuous stirred-tank reactor
(CSTR). The specific growth rate may be
controlled by fixing the SRT of the reac-
tor, and under steady-state conditions,
the physiological state of the culture
reaches a stable condition reflecting the
concentration of the substrate in the re-
actor. Operating several reactors (each
at a different SRT) and measuring the
steady-state concentration of the com-
pound of interest provides the informa-
tion necessary to evaluate the kinetic
parameters for biodegradation of the
compound.
Experimental Results
The efficacy of the proposed protocol
was examined experimentally with four
priority pollutantspentachlorophenol,
dimethyl phthalate, monochloroben-
zene, and bis-(2-ethyl hexyl) phtha-
lateto determine whether needed in-
formation was provided on the fate of
these organics when subjected to the
outlined levels of testing.
Aerobic Testing
Level I: Acclimation Studies
Fiber-wall acclimation reactors were
operated for 2.5 months with an input
flow containing domestic wastewater
supplemented with dog food extract to
yield a soluble chemical oxygen de-
mand (SCOD) of 200 mg/L. Pen-
tachlorophenol (PCP) was added as the
sodium salt at specified concentrations,
which were increased periodically from
1 to 20 mg/L. Effluent PCP concentra-
tions were measured quite often in the
final month of acclimation and were
consistently 100 |xg/L or less.
Alternative removal mechanisms of
the compound, such as adsorption and
volatilization, were tested in Level I.
Consequently, a study was made of the
possibility that these abiotic removal
mechanisms were responsible for the
observed PCP removal. The adsorptive
capacity of biomass at equilibrium con-
ditions was measured with unaccli-
mated biomass from a control reactor in
a manner similar to an adsorption
isotherm. The specific adsorptive ca-
pacities at the various equilibrium PCP
concentrations were calculated, and
data were used to estimate the relative
importance of the sorptive mechanism
for PCP removal relative to the degrada-
tion. Sorption was shown to contribute
less than 0.1% to PCP removal in the
acclimation reactor. A long-term air-
stripping test demonstrated that PCP
concentration decreased very slightly. A
first-order rate constant for the gas
stripping (0.0076 day"1) was deter-
mined by plotting the natural log of the
PCP concentration versus time and
measuring the slope. This rate constant
indicated that gas stripping made a neg-
ligible contribution to PCP removal.
Less than 0.5% of PCP entering the
reactor left by means of air stripping
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and sorption. Conversely, 99.5% of the
PCP eliminated was the result of
biodegradation. Consequently, the re-
moval of PCP during Level I testing was
primarily attributed to biodegradation
and indicated that acclimation had been
achieved.
Level II: Biokinetic Studies
In the second-level aerobic testing to
determine the kinetics of biodegrada-
tion, continuous-flow reactors without
cell recycle (continuous stirred tank re-
actors, CSTR's) were operated on PCP-
spiked wastewater at SRT's of 3, 7, 11,
and 15 days. In addition, four control
reactors were fed wastewater without
PCP while operating at the same SRT's.
Only data from the last few months of
operation were used to determine the
steady-state values. Log normal proba-
bility plots of PCP and SCOD data were
established for the four reactors. Since
concentrations higher than 350 jig/L of
PCP were somewhat inhibitory to PCP-
degrading organisms, only data with a
PCP concentration of 350 mj/L or less
were used to calculate the mean values
for a given reactor and were used in the
kinetic evaluation.
Figure 3 shows a plot of effluent PCP
concentration as a function of (1/SRT)
for the four reactors. A straight line with
a slope equal to (Ks/n-m) and an ordinate
intercept of (Ks/|Am)ds was fitted to the
data points by linear regression, and an
excellent fit was obtained (correlation
coefficient = 0.998). The excellent de-
gree of correlation found indicates that
PCP biodegradation is a first-order reac-
tion at growth rates encountered in
these reactors. The slope of the line was
determined as 593 (n,g«day)/L, and the
ordinate intercept was 27 n-g/L. Using
these values, ds (decay rate of cells) was
calculated to be 0.046 day'1.
The use of the above kinetic relation-
ship to describe PCP biodegradation for
the purpose of estimating the steady-
state performance of a full-scale system
should give an accurate prediction for
reactors that approach the completely
mixed state. The reasons are that the
organisms in such reactors all have the
same average specific growth rate (de-
termined by the SRT and decay rate for
the system) and that the growth equa-
tion is based solely on the growth rate.
This relationship can be used to predict
effluent concentration of PCP from a re-
actor operation at a given SRT.
The aerobic Level II testing indicated
quite certainly that:
a) Substantial biodestruction of PCP
Levell
No Change in PP Cone.
Descrease in PP Cone.
1
1) Determine Gas Stripping
2) Determine Biomass Sorption
1 +
Partial Complete
PP PP
f Accountability Accountability
Non-Degradable proceed t7~l rNon-Degradable~~[
Non-Removable LeveU, s
Stop 1 ^-" J
Level II
Determine Steady State PP Cone.
Treatment Feasible
Treatment Not Feasible
C'roceed to \
Level III \
Clot Treatable \
Stop j
Level III
Test for Ultimate Biodegradation
Method 1 Method 2
4COS from 1 'C-PP Microbial Growth with PP as Sole C Source
I Prepare Statement of Fate of PP in Aerobic Treatment ]
Figure 1. Logic flow diagram for aerobic testing.
occurred at nominal SRT's of 3, 7,
11, and 15 days (true SRT's of 3.2,
7.8, 12.8, and 18.3 days), since in-
put PCP concentration of 20 mg/L
dropped to mean steady-state
concentrations of 214,105,68, and
65 n-g/L, respectively.
b) Stable steady-state operation im-
proved with the longer SRT's.
Clear evidence of substrate inhibi-
tion was exhibited by PCP at the
shorter SRT's.
c) The threshold inhibitory concen-
tration of PCP was determined to
be 350 |xg/L; reactor instability at
low SRT's may be associated with
transitory excursions into in-
hibitory PCP concentrations.
d) The first-order PCP degradation
rate constant (|xm/Ks) was shown
to be 0.0017 L/n-g per day.
e) The lower limit for PCP concentra-
tion in a single, completely mixed
reactor with infinitely long SRT
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Level I
Anaerobic Toxicity Assay
Five Concentrations of PP
Determine Maximum Tolerated Dose of PP
Triplicate Acclimation Reactors
Semi-Continuous Flow Reactors with
Sludge Recycle. 60 Day SRT Minimum
Operate 90 Days, Save Biomass
Operation Comparable to Control
[ Proceed to Phase II}
Level II
r
Semi-Continuous Flow Reactors
at 3 SRTs
Determine
Determine
Sorbed
PP
Soluble
PP
Gas Phase*
PP
Gas Production Volatile Suspended
and Quality Acids Solids
Extent of Primary Biodegradation
Compare with Controls
[ Prepare Statements of Fate and Effect of PP in Anaerobic Digestion ]
*Not required if gas stripping in aerobic experiments negligible.
Figure 2. Logic flow diagram for anaerobic testing.
was estimated to be 27 ^g/L at in-
fluent concentration of 20 mg/L of
PCP.
Level III: Extent of Biodegrada-
tion Studies
Two types of experiments were used
to demonstrate that PCP was mineral-
ized in the Level II reactors. Since ran-
domly labelled 14C-PCP was available,
the biologically mediated formation of
14C02 was demonstrated and used as
evidence for ultimate biodegradation.
Experiments also showed that when
PCP was supplied as the sole source of
carbon and energy in a continuous-flow
system, a new steady state was ob-
tained that could only have been main-
tained for extended periods of time by
the de novo growth of PCP-degrading
microorganisms. This result implied
that PCP acted as a carbon and energy
source undergoing ultimate degrada-
tion by means of aerobic respiration.
These data were interpreted to mean
that PCP undergoes ultimate biodegra-
dation and, to a large extent, the disap-
pearance of PCP in acclimated biologi-
cal reactors is well correlated with its
mineralization.
Aerobic Level III testing established
that PCP undergoes significant mineral-
ization in the complex reactor environ-
ment. A comparison of the radioiso-
topic method and the microbial growth
method indicated that either method
used alone would have established that
PCP is mineralized.
Anaerobic Testing
Level I: Anaerobic Toxicity As-
say and Acclimation Studies
Level I of the anaerobic protocol was
found to require modification. Acclima-
tion of digester biomass to PCP was
found to be quite sensitive to PCP con-
centration. Accordingly, a preacclima-
tion toxicity assay test was inserted to
determine the inhibitory levels of the
priority pollutant prior to acclimation.
The toxicity assay test suggested that
a soluble concentration of PCP in excess
of 200 |Jig/L initiates significant inhibi-
tion of gas production by unacclimated
organisms. Consequently, the acclima-
tion feeding schedule was adjusted to
minimize inhibition.
Since methanogenesis is a critical
final step in the complete digestion
process, toxicity to this microbial popu-
lation can result in decreased perform-
ance and progressive failure of the
system.
The time-related gas production at
various PCP concentrations is shown in
Figure 4. Slight inhibition was observed
at a concentration of 200 (jig/L. This re-
sult means that the concentration of
PCP added daily should not be high
enough to result in a digester concen-
tration much in excess of 150 to 200
jxg/L. The PCP dosing regime during ac-
climation was established to avoid toxic
concentration effects on digester
biomass.
Three acclimation digesters were
started at an SRT of 73 days. The operat-
ing parameters normally used to assess
the performance of anaerobic digesters
were not significantly affected by PCP in
these systems. Gas production in the
test reactors was slightly lower than in
the controls, but the difference was not
significant. Level I anaerobic reactors
were successfully acclimated to a PCP
input level of 7.6 jixj/L.
Significant biological removal of PCP
was observed during Level I anaerobic
testing and was accompanied by the ap-
pearance of PCP-related decomposition
products in gas chromatograms (GC).
Direct determination of PCP sorption to
digester sludge by solvent extraction
and GC analysis indicated that less than
2% of the applied PCP was bound to the
sludge.
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Level II: Growth Kinetics and
Operational Performance
Studies
The SRT values chosen for Level II
testing were 10, 20, and 40 days. Since
no sludge recycle was used, these val-
ues represent the hydraulic retention
time (HRT) as well. Reduction of the SRT
of an anaerobic digester increases the
organic loading rate and stresses the
system which, in itself, could cause re-
actor failure. Thus, a slow transition
from Level I to Level II testing is critical.
After the transition period, three reac-
tors with SRT's of approximately 10,20,
and 40 days were fed once daily with
raw sludge spiked with 5 mg/L of PCP
and compared with control reactors
supplied with sludge containing no
added PCP. As an indication of opera-
tional performance, the gas production
data for each of the three reactors are
shown in Figure 5 as ratios to the com-
parable control reactors. After some
early adjustment, the mean gas ratio
was shown to be close to a value of 1.0,
indicating no difference between the
controls and the test reactors.
This study indicates that PCP would
have no significant effect on gasifica-
tion if digesters were operated within
the SRT limits tested. But an anaerobic
digester would probably be susceptible
to PCP toxicity if shock loads of PCP
were encountered, as evidenced by the
experiences with the toxicity test.
Once steady state was achieved in
each reactor, the concentration of solu-
ble PCP was below the detectable limit
of 10 (Jig/I. This result indicated that the
desired kinetic relationship between
pollutant concentration and SRT was
not attainable. The data are no less im-
portant, however, because the concen-
tration of PCP in the anaerobic reactor
with a short SRT of 10 days is lower than
the concentration of the substrate in any
of the anaerobic reactors, even those
with longer SRT values. The data are
also interesting because they suggest
that PCP was undergoing very complete
removal under anaerobic conditions.
One can attribute this removal to
biodegradation only after evaluating
the alternative abiotic mechanisms.
When alternative abiotic tests were per-
formed, less than 2% of the PCP re-
moved could be accounted for by abi-
otic mechanisms. Thus, more than 95%
of the PCP removed anaerobically was
the result of biotic mechanisms.
Phase II anaerobic testing with reac-
tors at 10-, 20-, and 40-day SRT's and
200
150
I
roo
so
Slope = S93 lttg day/L)
Intercept = 27 ng/l
I
I
.05 .10 .15 .20 .25
1/SRT (day''1!
.30
.35
Figure 3.
Kinetic relationship between mean steady state PCP concentrations and solids
retention times.
input of PCP concentration 5 mg/L re-
sulted in normal digester performance
when compared with unspiked controls.
During the steady state. Level II anaer-
obic testing soluble PCP concentrations
at all SRT's testing were almost unde-
tectable (<10 (xg/L). However, oscilla-
tions in PCP concentration and gas pro-
duction were observed earlier in the
testing period.
It appears that PCP feed concentra-
tions of less than 5 mg/L would not sig-
nificantly effect anaerobic digestion if
digesters are operated within the SRT
limits tested. However, shock loads of
PCP would probably result in essentially
irreversible inhibition of digestion.
It should be made clear that only pri-
mary biodegradation was evaluated
and that there was evidence of PCP
metabolites accumulating in the reac-
tors. Acclimation was very important to
the success of anaerobic digestion and
PCP biodegradation. A second study of
PCP toxicity toward acclimated biomass
clearly revealed that this biomass could
tolerate three or more times the PCP
concentration than unacclimated
sludge could. The slow rate of acclima-
tion combined with the obvious sensi-
tivity of the biomass to PCP would sug-
gest that intermittent shock loads of
PCP would have a very deleterious ef-
fect on digester performance as well as
on PCP degradation.
Conclusions
An experimental protocol for evaluat-
ing the fate of priority pollutants in aero-
bic and anaerobic wastewater treat-
ment systems was developed. The
protocol was tested and refined by ap-
plying it to a study of the fate of PCP in
aerobic and anaerobic bioreactors.
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Pentachlorophenol
Concentration
Gas Production
Rate
ml/day
120
Figure 4. Toxicity of pentachlorophenol to biological methane production.
The aerobic test protocol allows one
to predict that PCP can be effectively de-
stroyed in an acclimated municipal
wastewater treatment system operated
at SRT'sof 3 to 15 days to produce efflu-
ent PCP levels below 100 jjig/L. How-
ever, biodegradation kinetics also imply
that biodestruction of PCP to levels be-
low 30 jjig/L would be extremely difficult
to achieve. Biodegradation with miner-
alization was shown to be the primary
mechanism of PCP removal in waste-
water treatment.
Neither sorption of PCP to biomass
nor gas stripping were significant fac-
tors in PCP removal. Sorption ac-
counted for less than 0.1% of the PCP
loss, and the gas stripping was respon-
sible for less than 0.17% loss of sub-
strate.
Neither soluble organic carbon nor
methylene blue active substance could
be correlated with PCP biodegradation.
Thus, the use of an internal standard
was discontinued.
Generally, the aerobic test protocol
was shown to be an effective approach
to evaluating the fate and kinetics of
PCP removal in aerobic waste treatment
systems.
Acclimated anaerobic systems were
also shown to biotransform PCP very
effectively to levels below 10 ^.g/L at
SRT's as short as 10 days. Here again,
biological activity was the primary re-
moval mechanism. PCP did not inter-
fere with normal digester performance
at any SRT; however, there were clear
indications that PCP toxicity could dis-
rupt performance when a PCP concen-
tration in the reactor exceeded approxi-
mately 200 M-9/L for unacclimated
biomass or 600 n.g/1 for acclimated
biomass. Since intermittent feeding is
the norm for digester operation, toxic
pulse loading of PCP would be expected
to pose a greater hazard to the anaero-
bic process than to the aerobic process.
Overall, the anaerobic test protocol
was shown to be an effective approach
in evaluating both the fate of PCP in
anaerobic digesters and the influence of
PCP on normal anaerobic digestion.
The full report was submitted in fulfill-
ment of Grant No. R805858-01-1 by Pur-
due University under the sponsorship
of the U.S. Environmental Protection
Agency.
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1.2
1.1
k.
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United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
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