United States
Environmental Protection
Agency
Environmental Research
Laboratory
Athens GA 30613
Research and Development
EPA-600/S3-84-043 May 1984
&EPA Project Summary
Microbial Transformation
Kinetics of Xenobiotics in
Aquatic Environment
J. E. Rogers, S-m. W. Li, and L. J. Felice
Valid second-order rate equations will
permit the wider use of mathematical
models for predicting the persistence of
organic chemicals in fresh and marine
waters. In the laboratory study, the
microbiological transformations of the
butoxyethylester of 2,4-dichlorophe-
noxyacetic acid (2,4-DBE), p-cresol, a-
naphthol and quinoline were examined
to determine whether their degradation
rates are reasonably described by
second-order rate equations. Graphical
analysis of the data with first-order log
plots indicated that quinoline, p-cresol,
and a-naphthol were only transformed
following a lag phase. The lag period
was followed by a transformation phase
where the detectable decrease in com-
pound concentration could be described
by a pseudo first-order rate equation
and for which pseudo first-order rate
constants could be determined. The
transformation of 2,4-DBE, however,
occurred immediately upon addition of
the compound to sample waters.
Much of the variability in first-order
constants for the different compounds
could be accounted for in the range of
average bacterial populations,
measured during the transformation
phase, that were used to calculate
second-order rate constants. Second-
order rate constants were clustered into
groups that were statistically
different. Within all but two groups the
range in first-order rate constants was
greater than the range in second-order
constants.
Results of the study support the
usefulness of the second-order trans-
formation kinetics concept for
describing the microbial transformation
of organic chemicals in the aquatic
environment. The research points to a
continuing need for efforts in the area of
quantitative kinetic approaches to the
predictive modeling of microbiological
transformation in the environment.
This Project Summary was developed
by EPA's Environmental Research
Laboratory, Athens, GA, 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
Estimates of the degradation rates and
thus the persistence of organic
compounds in a wide range of aquatic
environments require an in-depth
understanding of the microbiological
transformation pathways (reaction
sequences) of major compound classes
and the effects of physical-chemical
(environmental) parameters as well as
microbiological population dynamics on
the rates of these reactions. Although not
complete, a wealth of information
describing the metabolism of major
compound classes, especially in the area
of aerobic degradation, is available in the
scientific literature. Anaerobic
metabolism has not been characterized to
the same degree, however
The microbiological degradation and
transformation rates of numerous
organics have been determined in
environmental samples. Unfortunately,
much of this data base is sample specific
and not transferable to other sites. Only
in recent years have attempts been made
to quantitate these rates so that they can
be used to estimate the persistence of
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specific organics in a range of
environments. These recent studies have
led to the development of mathematical
models to estimate persistence of
organics.
The continued development of viable
predictive models of the degradation of
organic compounds in the environment
requires the interactions of the following
two research areas:
• The development of environmentally
applicable mathematical representa-
tions of the microbiological pro-
cesses involved in the transforma-
tion and ultimate degradation of
organic compounds
• The design and development of pro-
cedures to produce laboratory data
compatible with the mathematical
representations
Previously, these areas have developed
independently in many respects In the
future, as microbiological degradation
models become more sophisticated, it is
essential that the two areas be
investigated in an interactive manner,
each process being directed, in turn, by
the other as new information and
methodologies are developed. This will
ensure the most rapid and complete
development of useful microbiological
models.
This research approach does not
preclude theoretical studies. Models,
however, are generally of little practical
value when no consideration is given to
the availability of laboratory data to
exercise the model, or worse, if no
consideration is given to the development
of laboratory techniques to provide the
data. Also of little value are degradation
studies in which the usefulness of the
data as input for available or developing
computer models has not been
considered.
Continued efforts to integrate these
two research areas will lead to better
microbiological models and a better
understanding of microbiological degra-
dation in the environment. An important
first step in this direction has been to use
second-order rate equations (i.e., the
product of a second-order rate constant, a
bJomass term and the compound
concentration) to describe the microbio-
logical transformation of organics in
fresh and marine waters.
The potential utility of second-order
equations arises from the aspect that,
once reliable second-order rate constants
are available, the transformation rates at
specific study sites can be estimated with
reasonable accuracy from measure-
ments of the biomass (bacteria). In most
cases, these estimates are markedly less
costly and labor intensive than the
experimental determination of the
transformation rate. Ultimately, the utility
of second-order rate constants and
equations in predicting the transforma-
tion rates of organics will depend on the
reproducibility of second-order rate
constants experimentally determined in
different laboratories and on the range of
compound types for which second-order
rate equations will describe their
transformation in aquatic environments.
In this study, the microbiological trans-
formation of a series of organic
substrates was examined to determine
whether their rates of degradation are
reasonably described by second-order
equations. The compounds were n-
naphthol, qumoline, p-cresol, and the
butoxyethylester of 2,4-dichloropheno-
xyacetic acid.
Discussion
The microbiological transformation
rates of the four organic compounds
added to natural water samples were
examined in the laboratory. Graphical
analysis of the data with first-order log
plots indicated that transformation of
these compounds occurred in two
phases. The initial phase consisted of a
lag period during which no decrease in
compound concentration could be
detected. Quinoline, p-cresol, and a-
naphthol, were only transformed
following a lag phase. The transformation
of 2,4-DBE occurred immediately upon
addition of the compound to sample
waters. The lag period was followed by a
transformation phase where the
detectable decrease in compound con-
centration could be described by a psuedo
first-order rate equation and for which
psuedo first-order constants could be
determined.
The variability in first-order constants
for the different compounds ranged from
a low of 13.6-fold for 2,4-DBE to a high of
185-fold for quinoline. Much of the
variability could be accounted for in the
range in average bacterial populations,
measured during the transformation
phase, that were used to calculate
second-order rate constants and from the
observation that second-order rate
constants could be clustered into groups
that were statistically different.
The variability of second-order con-
stants within these groups ranged from
1.18- to 36.14-fold, whereas the first-
order constants ranged from 1.24- to
184.71-fold. Within all but two groups,
the range in first-order rate constants
was greater than the range in second-
order constants.
Comparison of the second-order rate
constants for 2,4-DBE and p-cresol and
the second-order constants reported in
an independent study indicated that,
although the mean values were markedly
different, the standard deviations were
remarkably close in all cases. Either of
two possible explanations can account
for the difference in these values: there is
a systematic difference between labora-
tory procedures or there were significant
differences in the microbial populations
in waters sampled by each of the labora-
tories and the differences are real.
Before a detectable decrease in
compound concentration could be
measured for three of the four
compounds examined, a significant lag
period was observed. This lag could have
importance in the approach used in
computer modeling of compounds that
behave in this manner.
One of the primary interests in
developing site independent second-
order rate constants has been their use in
exposure assessment modeling. The
reproducibility of these constants for a
number of compounds observed in our
work and that of others emphasizes their
utility in exposure assessment. The role
of microbiological transformation or
degradation of xenobiotics, however,
cannot be limited only to second-order
constants. The observation that second-
order rate constants fall into groups
suggests that the rate of transformation
of xenobiotics in the environment may
require a probability distribution function
(i.e., transformation models may need to
have a random variable format). The
conservative alternative would be simply
to use the smallest second-order rate
constant determined for a given
compound.
Although more data are needed with
different sites and different compounds,
these results do offer significant
encouragement for prediction of microb-
ial transformation rates in natural
systems.
Recommendations
A continued research effort in this
area, which is a "kinetic approach" to the
predictive modeling of microbiological
transformations in the environment,
should address the areas of adaption
kinetics, reaction kinetics, and quantita-
tion of biomass. Biomass is important in
extrapolations from basic research
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models to widely applicable predictive
models that are normally constructed to
use readily available or easily attainable
data. For example, a compound-specific
biomass measure can now be obtained by
using a most-probable-number
technique for enumerating microorgan-
isms. The technique incorporates 14C-
labeled substrates. In time, this
procedure may displace the standard
pour-plate methods currently being used.
Where second-order rate equations are
applicable, the "kinetic approach" has
rested on the ability of laboratory studies
to determine an average second-order
rate constant, using a number of different
waters with different microbial
populations. The average rate constants
could then be used to predict degradation
rates at other sites if the microbial popu-
lation and organic substrate concentra-
tions are known.
This approach also can be used when
the degradation rate is described by alter-
native mathematical expressions. For
example, if the Michaelis-Menten
equation were used, one would simply
determine the average values for Km,
Vmax, and the bacterial population of a
number of waters and then use these
average values to estimate degradation
rates in other waters where the substrate
concentration and bacterial populations
are known. The bacterial population in
this case is used to set the value of Vmax
from an average specific activity term (sp.
act. = Vmax/bacterial population)
determined in the initial evaluation
studies. Similar examples can be derived
from other mathematical expressions of
the degradation rate.
John E. Rogers. Shu-mei W. Li, and Lawrence J. Felice are with Battelle, Pacific
Northwest Laboratories, Rich/and, WA 99352.
William C. Steen is the EPA Project Officer (see below).
The complete report, entitled "Microbial Transformation Kinetics of Xenobiotics in
Aquatic Environment," (Order No. PB 84-162 866; Cost: $13.00. subject to
change) will be available only from.
National Technical Information Service
5285 Port Royal Road
Springfield, VA22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Environmental Research Laboratory
U S. Environmental Protection Agency
College Station Road
Athens, GA 30613
U S GOVERNMENT PRINTING OFFICE. 1984 — 759-015/7695
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Environmental Protection
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