United States
Environmental Protection
Agency
Health Effects
Research Laboratory
Research Triangle Park NC 27711
•<*:
Research and Development
EPA/600/S1-88/003 Sept. 1988
SEPA Project Summary
Health Hazard Evaluation of
Waste Water Using Bioassays:
Preliminary Concepts
C. E. Easterly, L. R. Glass, T. D. Jones, B. A. Owen, R. L. Schenley,
P.J.Walsh, andL C. Waters
Effluents from waste water treatment
facilities are discharged as a complex
mixture of numerous chemical sub-
stances, which may include cytotoxic,
ca-xsinogenic and mutagenic compounds.
Historically, Federal and State agencies
have relied upon chemical-based
analyses to set and enforce regulatory
limits for these effluents. One problem
with this approach is that many potential-
ly hazardous chemicals may not be quan-
tifiable in complex chemical effluents but
are none the less discharged into the
environment.
The U.S. Environmental Protection
Agency has recently established a re-
search program to determine if a
bioassay approach for evaluating the
potential adverse human health effects
from exposure to complex mixtures might
supplement conventional chemical
analysis for setting regulatory limits for
waste waters. The full report summarizes
a bioassay testing strategy for charac-
terizing cytotoxic and mutagenic activity
of various waste water effluents. Use of a
relative potency framework for assessing
complex mixtures for potential health
hazards is addressed.
This Project Summary was developed by
EPA's Health Effects Research Laboratory,
Research Triangle Park, NC, 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
The assessment of potential human health
hazards from exposure to chemicals present
in waste effluents is more difficult than the
determination of toxic effects on the biota of
receiving waters. The difficulties rest in
several areas: the possibility of indirect,
chronic exposure with low effective dose
rates, the inability to test humans directly, and
the difficulty in extrapolating between actual
exposure and potential effects. The evalua-
tion of potential human health effects involves
an investigation of chemical specific toxicrties
or bioassays of whole effluents, a determina-
tion of the potential exposure to those agents,
and a risk assessment based on the data
generated.
Chemical specific analysis, which has
traditionally been done in human health
evaluations, requires the specific identifica-
tion of pollutants and their concentrations in
the effluent. Once identified these chemicals
can be evaluated by consulting existing ex-
perimental and epidemiological data or by
performing toxicological tests on surrogate
organisms. This process has been applied
to chemicals which are known to be present
in an effluent which can be measured by
standard chemical analyses, but the evalua-
tion becomes more difficult for complex mix-
tures found in uncharacterized effluents.
Synergistic or antagonistic interactions be-
tween chemicals can occur in the complex
effluents. The process of screening for in-
dividual chemicals in complex waste ef-
fluents, even when limited to a list of priority
chemicals can be tedious and expensive.
Toxicological assessment of the whole ef-
fluent may correct some of the disadvantages
of chemical specific testing. Bioassays are
utilized in this approach to estimate relative
hazards from exposure to the chemicals in
waste effluents. The use of the health-related
bioassays as an approach to evaluating the
hazards of chemical contamination in an un-
characterized waste effluent is discussed in
the full report.
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Biotesting Waste Water Effluents
Although it is recommended that all waste
water samples be initially biotested directly
without concentration, in practice a majority
of samples will require some concentration
before a positive response can be detected
or a sample is declared negative. A major
problem is that no currently available concen-
tration procedure(s) can produce a concen-
trate from a water sample that contains all
the original chemical constituents in an
unaltered state and in the original relative
concentrations. Moderately volatile and less
volatile constituents may be tested as con-
centrates Concentration methods may be
divided into the following two major groups:
Concentration: These methods refer to
techniques in which water is removed and
the dissolved substances are left behind. Ex-
amples of such methods are freeze concen-
tration, lyophilization, vacuum distillation, and
membrane processes such as reverse
osmosis and ultrafiltration. A common disad-
vantage of these methods is that inorganic
species such as sodium and chloride ions
are concentrated along with the toxic consti-
tuents High concentrations of such salt
species that are normally non—toxic can
disrupt cell membranes through hyperos-
motic effects.
Isolation: This group consists of processes
in which the chemicals are removed from the
water. Examples are solvent extraction and
adsorption onto resins. Advantages of isola-
tion methods include the fact that inorganic
species are generally not concentrated. A
disadvantage is that the isolation is selective
in that neither solvent nor resin extraction will
qualitatively and quantitatively yield all the
organics present in a waste water sample.
After comparing the available methods
against the optimal criteria for a sample pro-
cessing procedure, the full report recom-
mends that an EPA method using sorbent
resins followed by organic elution and con-
centration would probably come closest to
the ideal system for routine waste water pro-
cessing for mutagenicity testing. A styrene
divinylbenzene copolymer (XAD), was the
recommended resin for concentration pur-
poses. XAD resin can efficiently remove a
large variety of model organic compounds
from water.
"Blue cotton," a copper phthalocyanine
derivative covalently attached to cotton, has
been shown to be an effective adsorbent for
mutagens with three or more fused aromatic
rings in their structure. Although its limited
adsorption properties prohibit the use of blue
cotton in a comprehensive toxicological
assessment of waste waters, it has features
that could make it useful as a qualitative
"spot" screen of potentially hazardous waste
water sites. Blue cotton is simple to use and
is amenable to batchwise extraction of water
samples. Blue cotton can be contained in a
mesh bag, suspended in the test water for
the desired period of time, transported to the
laboratory, rinsed with water, dried, extracted
with ammoniacal methanol, concentrated
and biotested.
Even when comprehensive testing is war-
ranted, on-site concentration by collecting the
sample through XAD columns has advan-
tages. Problems associated with transporting
and storing large volumes are avoided. The
XAD columns and the small volumes re-
quired for sample characterization and for
direct biotesting are relatively easy to handle.
In most cases toxic biological effects will
not be detected in the unconcentrated water.
However, the extent of concentration that
should be done before a sample is judged
to be safe is a difficult question to answer;
these points are discussed in the report. In
practice, toxicity sets upper limits on concen-
tration. If the in vitro preparation is killed, it
cannot be used for mutation of other assays.
The volumes required for adequate biotesting
plus the concentration factor set lower limits
on the volume of sample that is required. For
example, if a 1000-fold concentration factor
is desired and a 10 ml volume of concentrate
is required for the test procedures, then at
least 10 L of sample must be concentrated.
Once the waste water effluents are proper-
ly collected and stored, chemically analyzed
and concentrated if required, they are then
subjected to bioassay procedures. Two pro-
cedures that have been successfully
employed in directly biotesting waste water
samples have been the Salmonella
mutagenicity assay and the Chinese ham-
ster ovary cell/HGPRT assay (CHO/HGPRT).
Since environmental samples generally do
not contain sufficient levels of contaminants
to produce an effect in these two bioassays,
the sensitivity of the two assays can be im-
proved by increasing the size of sample
volume or by concentrating the sample as
described in the report.
In order to accommodate larger sample
volumes and thereby biotest waste water
samples directly, five times (5X) concentrated
top agar and treatment medium have been
used in the Salmonella and CHO/HGPRT
assays, respectively. With this modification
aqueous test sample volumes can be in-
creased to 2.5 ml in the Salmonella assay
and to 3.2 ml in the CHO/HGPRT assay, ef-
fecting a 17- to 25-fold concentration in situ
relative to the standard (1X) assays. Control
studies have shown the 5X and the standard
assays to be equivalent when direct or
indirect-acting standard mutagens are tested.
Salmonella and CHO/HGPRT assays are
outlined in the report.
When large volumes of environmental
samples are assayed, certain properties of
the samples become important. Microorgan-
isms, when present, must be removed by
filter sterilization. Particulates might also
have adsorbed toxic chemicals. It is recom-
mended that particulates be removed by cen-
trifugation and/or filtration. In order to assess
the biological activity of substances adsorb-
ed to the particulates, the pellets and/or filters
can be dried by lyophilization, extracted with
dimethyl sulfoxide and assayed in the stand-
ard systems. The pH and ionic strength are
also important properties to be considered
when assaying large sample volumes. To
avoid potential pH effects, the samples
should be neutralized prior to being assayed,
Hypertonicity considerations may limit the
degree to which a specific sample may be
concentrated.
Bioassay results obtained for a variety of
unconcentrated waste water samples are
described. The mutagenic responses ob-
served to date have been greater in one
Salmonella strain and do not require
metabolic activation. Cytotoxicity to CHO cells
was observed with waste effluents, but no
mutagenic activity was observed in this cell
line. One general observation of the data in-
dicates that mutagenic chemicals may not be
adequately removed by a municipal waste
treatment facility.
Relative Toxicity Evaluation
Samples of complex mixtures can be
evaluated by comparing toxicological
responses to reference chemicals in a bat-
tery of biological test systems. Given equal
biological response in a biological test system
relative doses necessary to produce that
degree of biological response may be used
to evaluate the hazard represented by com-
plex mixtures. Responses would reflect
characteristics of both the sample being
assayed and the test system used in the
analysis
A relative potency framework could be
used to evaluate complex mixtures as
unknown test samples in comparison to
reference chemicals. Relative potency (RP)
is defined as
RP =
dose of reference material _ Dr
dose of test material Dt
In a given biological system, responses to the
test and reference materials must be com-
pared in an equivalent test system. Reference
materials are those chemicals that have been
well characterized in terms of biological test
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data. In such cases, it is suggested that
biological results for test material can be in-
directly related to health risk based guidelines
or standards (Sr) of the reference material as
follows:
RP, • St = RPr • Sr (but RPr = 1), so
where St is the inferred guideline of the test
material. The "leap of faith" implied in the
use of such indirect methods for assessing
potential risk results from the belief that short-
term tests can be used as a scientific basis
for decision making. Whether such a
presumption is true has not been fully
demonstrated at the present time and is a
subject for further research.
Efforts to date have demonstrated that, in
principle, a battery of short-term bioassays
can be used to rank the relative hazard
represented by chemicals which may be
human carcinogens. The number of assays
and the specific tests comprising a practical
battery are not yet completely defined. Short-
term test system results for a variety of
materials should be incorporated into a data
base that includes results for an inventory of
chemicals and mixtures. Ultimately, it is en-
visioned that new results would be assess-
ed by their position on a "relative toxicity
scale." However, because of the small
number of test results generally available
from bioassays of waste water samples,
variability due to experimental design and
noise from random error, categorical
assignments are currently preferred. For ex-
ample, potential exposure via a water
pathway will be excessive if the hazard in-
dex (HI) is
HI = - >1
St
where St is a criterion or guidance value in
units of concentration, and R is the measured
or calculated concentration of the pollutant
(compound or mixture) in the water sample
at the point of consumption in the same units
as St.
When the water sample is taken at a point
of human consumption as opposed to a point
of contaminant release, then dilution occurs
between the release point and human ex-
posure. Thus, R may be expressed as Cw/D
where D is a generic dilution factor taken
from EPA recommendations, and Cw is the
concentration of a pollutant in the waste sam-
ple; thus, an unacceptable exposure could
be present if
HI = R/St<(Cw • RPt)/(D • Sr) >1
D is the generic dilution factor, Sr is an EPA
criterion for the reference chemical, and RPt
is the potency of the contaminant of concern
relative to the reference chemical. The
denominator (i.e. D • Sr) models the change
in concentration of the reference agent be-
tween points of release and consumption.
The numerator (i.e. Cw • RPt) scales the
measured concentration of the (tested) sam-
ple into an effective dose of the reference
agent.
Since no definitive battery of assays has
been identified as being predictive of human
response, specific guidance for the composi-
tion of a battery cannot be offered at this time.
However, a combination of bacterial mutation
assays coupled with mammalian cell assays
has been suggested by a variety of authors.
A variety of different bioassays yielding
positive results should be used. Constraints
of time and money will probably limit the bat-
tery size to between three and six bioas-
says for most applications. Future work will
hopefully identify the most useful battery of
assays for the assessment of waste waters.
Conclusions
Regulation of waste water effluents on the
basis of chemical analysis requires that all
the chemicals which are present in a sam-
ple be identified and quantified and that the
biological effects of those chemicals are
known. The impracticality of meeting these
requirements is evident. Short-term biotests
can be useful and are probably necessary
adjuncts to chemical analysis for waste water
evaluation. Further work is necessary to iden-
tify the "best" short-term assay or battery of
assays for biotesting waste water. Abundant
evidence exists to indicate that short-term
tests represent useful methods which need
to be better validated for the biological
assessment of the hazard posed by waste
waters. It is recommended that research in
this area be expanded, including the
development of a data base devoted to
results from the analysis of waste waters in
a variety of short-term tests.
This Project Summary was submitted in
fullfillment of Interagency Agreement
(DW89931236) between the U.S. En-
vironmental Protection Agency and the
Department of Energy. This paper has been
reviewed by the Health Effects Research
Laboratory, U.S. Environmental Protection
Agency and approved for publication. Ap-
proval does not signify that the contents
necessarily reflect the views and policies of
the Agency.
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C. E. Easterly, L R. Glass, T. D. Jones, B. A. Owen, R. L. Schenley. P. J.
Walsh, and L. C. Waters are with Oak Ridge National Laboratory, Oak Ridge,
TN 37831-6101.
L. W. Condie is the EPA Project Officer (see below).
The complete report, entitled "Health Hazard Evaluation of Waste Water Using
Bioassays: Preliminary Concepts," (Order No. PB 88-243 860/AS; Cost:
$19.95, 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:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, NC 27711
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Official Business
Penalty for Private Use $300
EPA/600/S1-88/003
'0000329 PS
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