United States
Environmental Protection
Agency
Environmental Research Laboratory
Corvallis OR 97333
Research and Development
EPA-600/S3-83-017 June 1983
Project Summary
Bacterial Bioassayfor
Level 1 Toxicity Assessment
Kenneth J. Williamson and Peter O. Nelson
Nitrifying bacteria were tested to
determine their applicability as Level 1
bioassay organisms. Level 1 testing
involves general bioassay and analysis
procedures that will identify the presence
of toxic material in a given waste
stream.
The toxicity of five metals and three
organic toxicants to the nitrifying bacte-
ria (Nitrobacter and Nitrosomonas)
were determined and compared to other
common bioassay organisms. In gener-
al, the bacteria exhibited comparable
sensitivity for toxicants with affected
substrate metabolism, but lower sensi-
tivity for specific toxicants such as
pesticides that affect the activity of
nerve cells.
The application of the bacterial bio-
assay was shown for two cases of Level
1 testing: a field study of a toxic
industrial waste and its pretreatment,
and an assessment study of the poten-
tial leachate problems for a flue-gas
scrubber solid waste.
This Project Summary was developed
by EPA's Environmental Research Lab-
oratory, Corvallis, OR, to announce key
findings of the research project that is
fully documented in a separate report of
the same title (see Project Report order-
ing information at back).
Introduction
Bioassay tests are used to detect biolog-
ically harmful chemicals whose effects
can be manifested as cellular, genetic,
behavioral, or metabolic damage. The
U.S. Environmental Protection Agency
(EPA) has developed a three-phased
bioassay approach to performing assess-
ment of the toxicity of aqueous solutions
(including solid waste leachates); this
approach is divided into Level 1.2, and 3
tests. Level 1 involves general bioassay
analysis procedures that will identify the
presence of toxicity in a given waste
stream. Level 2 tests attempt to identify
and quantify the specific compounds
associated with the toxicity found in the
Level 1 test. In Level 3 tests, more details
concerning chronic health and ecological
effects of the stream components are
sought.
The focus in the Level 1 tests is a
complementary series of bioassay tests
for acute toxicity, mutagehicity, cytotoxic-
ity, and soil microbiological inhibition.
These tests provide no specific identifica-
tion of the toxicant, but serve as signals
for a wide range of potentially toxic
responses.
The primary difficulty with the applica-
tion of these tests is the complexity that
results in high costs. The tests require
highly trained personnel, modern labora-
tories, and long time periods. An attrac-
tive alternative to reduce costs is the use
of lower level organisms, especially bacte-
ria. Such a bacterial bioassay would be
supplemental to the proposed Level 1
bioassay tests and other health and
toxicity tests and, hopefully, correlative.
The advantage of using bacteria as
compared to the other Level 1 bioassay
organisms would be greater simplicity,
shorter testing times, and lower cost.
Such a test could be accomplished within
a few hours by chemical technicians and
would involve minimal laboratory facili-
ties.
Project
This research was conducted to deter-
mine the potential applicability of using
mixed cultures of the autotrophic orga-
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nisms Nitrosomonas and Nitrobacter to
detect toxic compounds under Level 1
type testing. Toxicity was measured by
reducing nitrogen oxidation. In addition, it
was desired to know the relative sensitiv-
ity of these two bacterial genera to other
bioassay organisms. Specifically, the objec-
tives were to:
1. Determine the toxic concentrations
of a wide range of known toxicants to
Nitrobacter and Nitrosomonas and
compare these concentrations to
known toxic levels for other health
and ecological bioassays presently
used.
2. Demonstrate the use of nitrifying
bacteria to estimate toxicity and the
type of toxicant present in complex
wastewaters.
The Test Procedure
Enriched cultures of Nitrobacter or Nitro-
somonas—Nitrobacter were grown in a
downflow column packed with polyethyl-
ene beads. Bacteria were removed from
the column and freeze-dried. Rehydration
was done with a solution of approximately
5 mg NO2-N/I for Nitrobacter and 5 mg
NHj-N/l for Nitrosomonas. The bio-
assay procedure was:
1. Fifty ml of 15 mg/l of NaNO2-N or
(NH«)2SO.«-N was prepared with the
selected concentrations of waste-
water and was placed in a similar
flask as the control.
2. An equal volume of Nitrobacter or
Nitrosomonas suspension was placed
in each flask and the flasks were
shaken at a constant temperature for
several hours.
3. One ml of each solution was taken
out periodically and measured for
NO2 or NHi spectrophotometrically.
4. At the end of the experiment, dry-
weight of the biomass was deter-
mined by fUtration of the solution and
by drying at 105° for one hour.
NOi-N or NHj-N concentrations versus
time for each flask were plotted and the
slope determined by a best squares fit. A
comparison of the calculated slope of
each line with the slope obtained for the
control yielded the relative metabolism
rate of the test solution. These rates are
plotted versus concentration of the waste-
water or toxicant; a reduced rate of
metabolism confirms a toxic response
from the wastewater sample.
Sensitivity of
Nitrosomonas and
Nitrobacter
The 50 percent and 90 percent relative
metabolisms were extrapolated from dose
response curves to estimate the range of
maximum sensitivity (Figures 1 and 2).
The ranges varied from about 101 to 102
/ug/l for silver to 1 0s to 1 08 Aig/l for zinc,
and from 104to 106A
-
i-
5O%
xx
Xx:
90%:
50%
^
^o^
50%
XX;
:90%
5O%
• 9O%
90%
-
90%
-
!
10°
5O%
No
toxic
response
SB"*
Figure 1
10"
10s
10"
? JO3
Summary of relative metabolism of Nitrobacter for various toxicant levels.
50%
~ 50%
$n% XXx 50%
x^xx r yt/%.
H- *?%:
9O%
50%
;90% "
1X^
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Table 1. Comparison of Sensitivity of Nitrobacter and Nitrosomonas to Other Bioassay
Organisms*
Bioassay Organism
Fathead
Toxicant Daphnia Trout Minnow Algae
Lead 0 + +0
Cadmium - - 0 -
Copper - - - 0
Silver + + + +
Zinc - - - -
Endosulfan - - - *
Heptachlor - - - -
Parathion - - - -
*+, more sensitivity; 0, comparable sensitivity; -, less sensitivity; x, no data available.
and could be standardized using freeze- as heavy metals) compared to other
dried organisms. It may be useful in cases common bioassay organisms, but exhibits
where comparative toxic levels of a large dramatically lower sensitivity for specific
number of samples are required, or for toxicants (such as pesticides).
field application with limited equipment The bacterial bioassay can be used for
and personnel. Level 1 type testing as shown by its
The bacterial bioassay with either successes in studies to optimize pretreat-
Nitrobacter or Nitrosomonas exhibits ment of toxic wastewaters, and to assess
approximately somewhat less sensitiv- toxicity of solid waste leachates under
ities for general metabolic toxicants (such various leaching conditions.
K. J, Williamson and P. 0. Nelson are with Department of Civil Engineering,
Oregon State University, Corvallis, OR 97331.
David T. Tingey is the EPA Project Officer (see below).
The complete report, entitled "Bacterial Bioassay for Level 1 Toxicity Assessment,"
(Order No. PB 83-182 287; Cost: $11.50, 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
Corvallis, OR 97330
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