United States
Environmental Protection
Agency
Environmental
Research Laboratory
Athens. GA 30613
-
Research and Development
EPA/600/S3-88/007 Apr. 1988
f/EPA Project Summary
Interim Protocol for Measuring
Microbial Transformation Rate
Constants for Suspended
Bacterial Populations in Aquatic
Systems
William C. Steen
An interim protocol for performing
research to measure microblal
degradation rates of organic
chemicals in freshwaters Is
presented. Microblal degradation is a
major transformation pathway
influencing the environmental fate of
chemicals. The interim protocol
presented provides a basis for
measurement of microblal
degradation rates such that reliable,
comparable and consistent data can
be obtained by different laboratories
and research investigations. As
additional research and information
Is gathered on the environmental
factors affecting microblal
degradation by suspended bacterial
populations In freshwater, the
outlined protocol will be modified.
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
Under the Toxic Substances Control
Act of 1976 (PL 94-469), EPA's Office
of Toxic Substances is required to review
the potential risk to human health and the
environment posed by new chemicals
before manufacture and use are
permitted. For many chemicals, microbial
degradation is a major transformation
pathway that influences their
environmental fate. Therefore, in
assessing risk, it is necessary to have
some estimate of the microbial
transformation rate of each chemical.
The Office of Toxic Substances
estimates microbial transformation rates
of chemicals proposed for manufacture
by comparing each new chemical's
organic structure (or other known
properties) with those of chemicals
whose microbial transformation rates
have been established. Chemicals with
similar structures/properties are expected
to have similar microbial transformation
rates. Unfortunately, microbial
transformation rates and rate constants
have been measured for only a few
chemicals. Investigations are being
expanded, however, as a better
understanding of the many environmental
factors that influence microbial
degradation is achieved. Much remains
to be learned about ways in which
population density and diversity,
accessibility of chemical substrate within
microcosms, and other factors influence
transformation rates.
While these investigations continue, it
is necessary to provide measured
microbial transformation rates for
chemicals based on current knowledge.
These microbial rates must be measured
in a manner that is reproducible and that
assures the results of measurement of
one chemical can be compared to those
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of another with confidence, even though
the influence of some environmental
factors is not fully understood.
This report represents a sampling and
measurement protocol that has been
applied over several years at the
Environmental Research Laboratory,
Athens, GA. The methodology has been
found to provide reproducible second-
order rate constants using suspended
natural populations in aerobic aquatic
systems. Transformation rate is based on
the rate of disappearance of the test
chemical.
The interim protocol provides a
stepwise description of methods for
establishing aerobic biodegradation
investigations. In a typical biodegradation
study, natural aquatic sites within the
vicinity of the investigator's laboratory
are sampled. Water temperature is
recorded, and the samples are
transported to the laboratory. After
receipt at the laboratory, samples are
filtered to remove coarse debris.
Population densities should be
measured by standard plate counts
(heterotrophic plate counts). This
characterization serves to establish the
baseline population density of the
ambient water and to determine the need
for a concentration procedure for
increasing population densities in order
to observe measurable rates in the
transformation of test chemicals.
The time lapse between sampling and
initiation of the rate constant
measurement phases should be no more
than 12 hours. Once the microbial
samples have been returned to the
laboratory, handling prior to experimental
rate constant measurement can take two
courses.
In one procedure, the sample is used
at the natural population density when
sampled. The test chemical is added and
the chemical's transformation rate is
measured.
In the alternative procedure, the
natural microbial population is enhanced
through a concentration step. This 10:1
concentration step requires larger
volumes of source water.
Bacterial populations in water samples
are concentrated 10-fold by filtering 10
liters through a membrane filter (0.2 pm
pore diameter, Nucleopore or equivalent)
that has been prewashed with distilled
water. After filtration, filters are placed in
a 2-liter, wide-mouth, cotton-plugged
Erlenmeyer flask containing 1 liter of the
original source sample.
Sterile, aqueous, concentrated stock
solutions of nutrients are prepared to
yield concentrations (g/L) of each of the
following: NH4CI (0.5), (NH4)2SO4 (0.5),
Na2HP04 (0.5), KH2P04 (0.5), MgSO4
(0.001), and FeCIa (0.001). No more than
1 ml of each nutrient is added to the
concentrated bacterial population. These
bacterial suspensions are then incubated
for 48 hours at 22 °C in a temperature-
controlled shaker (150 to 200 rpm)
before each experiment is initiated. This
procedure enhances the bacterial
population 10- to 100-fold.
Bacterial concentrations are
determined by pour plating techniques
using Tryptone Glucose Extract Agar
(TGE) from serial dilutions of each
reaction flask. Each dilution is plated in
duplicate or triplicate. Pour plates are
then incubated for 48 hours at 22±1°C
in the temperature-controlled
incubator/shaker. Following the 48-hour
incubation, plates are removed and
bacteria are counted (using a suitable
counting instrument), tabulated, and
arranged.
Following preparation of abiotic and
biotic treatment flasks, test chemical
disappearance is measured within each
reaction flask by either gas
chromatographic or high performance
liquid chromatographic methods. Raw
data on the test chemicals are obtained
by measuring the concentration of
chemical remaining in the reaction flask
at specific time intervals. The measured
concentration at time zero serves as the
reference point for the remaining points.
From these data, the first-order slope or
rate constant (k, hr1) is determined
either through standard laboratory
computer programs or manually through
graphical manipulations using semi-log
paper.
Using the mean of bacterial
concentration determined from plate
counts and first-order slopes (k, hr1)
for chemical disappearance, the
second-order rate constant (kb, L org-1
hr1) is calculated. The applicable form
of the second-order rate expression
used throughout is:
-ds/dt = kb[BT][ST]
where:
kb = Second-order rate con-
stant (L org-1 hr-1)
[BT] = Measured bacterial
concentration (CFU or org
per liter)
[87] = Measured substrate
(chemical) concentration
(mg per liter)
The utility of this protocol has been
demonstrated by comparative
determinations of the second-order
microbial rate constants of standard
reference chemicals. Two standar
reference chemicals, methyl ester <
2,4-dichloro phenoxy acetic acid (2,'
OME) and propanil, were investigate
using natural pond water over a period <
1 year. Second-order rate constant
ranged from 7.8 to 9.2 X 10-9 | org
hr1 for nine determinations with th
methyl ester of 2,4-D. Propanil yielde
rate constants ranging from 1.1 to 6.4
10"11 I org-1 hr1 for six determir
ations. Coefficients of variation were 3
to 72% for the methyl ester of 2,4-
and 42 to 69% for propanil. No season;
effects were observed. Use of a standar
reference chemical directly aids th
investigator in establishing his confidenc
in the protocol measurements and allow
for interlaboratory comparativ
investigations in application of th
protocol.
Several basic assumptions serve a
the foundation for measurements c
microbial transformation rate constant
under this protocol. Use of total viabl
plate count as a measure of microbii
population concentrations provides
measurement that is assumed to b
proportional to the number c
microorganisms participating in th
biodegradation process. For chemical
for which the concept was developed an
tested, many of the culturabl
populations indigenous to aquati
systems support the necessar
constitutive enzymes for microbiall
mediated hydrolysis and oxidatio
reactions. Moreover, the transform
ation/biodegradation process is pseudc
first-order with respect to bacterie
concentration and, therefore, i
proportional to the density of total viabl
bacteria in the system plated on TGE.
is further assumed that the substrate (tes
chemical) concentration is much les
than the theoretical Ks half-saturatio
concentration and that reaction kinetic
are first-order with respect to substrat
concentration. It also is assumed the
carbon and energy contributions from th
chemical under investigation are nc
sufficient to cause measurable growth t
the constitutive populations. Th
second-order mathematical descriptio
of biodegradation serves as
reproducible and reliable measurement c
microbial transformation rate constant
for organic chemicals in aquatic system;
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The EPA Author, William C. Steen, (also the Project Officer) is with the
Environmental Research Laboratory, Athens GA 30613.
The compieie report, entitled "Interim Protocol for Measuring Microbial
Transformation Rate Constants for Suspended Bacterial Populations in
Aquatic Systems," (Order No. PB 88-165 7091 AS; Cost: $12.95, subject
to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Environmental Research Laboratory
U.S. Environmental Protection Agency
Athens, GA 30613
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Official Business
Penalty for Private Use $300
EPA/600/S3-88/007
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CHICAGO
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