United States
Environmental Protection
Water Engineering
Research Laboratory
Cincinnati OH 45268
/1 \
Research and Development
EPA/600/S2-85/141 Feb. 1986
Project  Summary

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
  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-
  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

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

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
  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:
  The  relationship between specific
growth rate of microorganisms and SRT
of a reactor is expressed by the equa-
          M- = (1/SRT) + ds
     M- = (Ks/n,m)(1/SRT) +
            •* ~ Ks + S  '

where ^m (maximum specific growth
rate of microorganisms) and KS (satura-
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
  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

Experimental Results
  The efficacy of the proposed protocol
was examined experimentally with four
priority pollutants—pentachlorophenol,
dimethyl phthalate,  monochloroben-
zene, and  bis-(2-ethyl hexyl)  phtha-
late—to 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

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

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
              No Change in PP Cone.
                                             Descrease in PP Cone.

1) Determine Gas Stripping
2) Determine Biomass Sorption
1 +
Partial Complete
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

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
                        Semi-Continuous Flow Reactors
                                at 3 SRTs
Gas Phase*
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

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
                                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
  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
  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

Level II:  Growth  Kinetics and
Operational Performance
  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
                          Slope = S93 lttg • day/L)
                    Intercept = 27 ng/l
                   .05     .10     .15     .20     .25

                                    1/SRT (day''1!
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.

                              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.

Gas Production
 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-
  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
  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



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Environmental Protection
Center for Environmental Research
Cincinnati OH 45268
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