Technical BRIEF
INNOVATIVE RESEARCH FOR A SUSTAINABLE FUTURE
www.epa.gov/research
Bioassays for Evaluating Water Quality
Screening for total bioactivity to assess water safety
Background
Water quality assessment and characterization
techniques typically rely on analyses and data for
individual contaminants. Essentially, individual chemical
concentrations in water are measured or modeled.
These results are then compared to the known
concentrations at which the chemicals have been shown
to cause adverse health outcomes (usually mortality,
growth, or fecundity). This approach is helpful to
approximate the risk posed by known chemicals;
however, it may have limited usefulness.
Real-world exposures generally do not occur from
individual chemicals; they typically occur as mixtures of
different chemical compounds that can change over
time. This presents challenges in understanding the risks
posed to human and environmental health: (1) many
chemicals lack toxicity data even if measurable, which
means there is a lack of information on the effects to
exposed organisms; (2) although individual compounds
can accurately be measured at low concentrations,
information is lacking on the presence and
concentrations of other compounds; and (3) chemicals in
a mixture tend not to work alone, but together
additively, synergistically or antagonistically. For
example, many chemicals can act as estrogens and often
co-occur in environmental samples. If the risk estimate is
based on one estrogen, we would underestimate the risk
posed since it is the total estrogenic potency (or
potential) that is important. To better understand the
risk posed by these complex samples, we need methods
that can characterize potential cumulative effects on the
organisms without necessarily needing to know all the
components of the samples.
Bioassays are a potential solution for assessing complex
samples since they screen for total bioactivity for a given
pathway or mode of action (MOA), such as estrogen
receptor activation. Overall, they can account for the
three challenges listed above, and can simplify complex
samples by reducing them to a few activated biological
pathways or MOA. EPA has made considerable progress
in the development of these assays as effects-based
monitoring tools, and has applied them in a growing
number of water quality assessments.
What are Bioassays?
The term 'bioassay' represents a variety of assays.
Despite their variety, bioassays tend to have several
characteristics in common. Generally, bioassays target a
biological change that occurs in an organism rather than
a change that occurs on an organism. These endpoints
can be on the cellular level, such as cellular receptors
being activated or genes, proteins, or metabolites
changing in abundance in response to the water sample.
Bioassays can also target higher biological levels, such as
cell growth, tumor development, or changes in
morphology and function of organs. They can be in vitro
(outside a living organism), monitoring natural responses
of cells in culture or responses from genetically modified
cell lines. The former often look for changes within a
particular targeted cell type (e.g., kidney or bladder) as
an indicator of an adverse outcome (e.g., cancer);
whereas, the latter often target the activity of some class
U.S. Environmental Protection Agency
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March 2018

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of toxicant (e.g., activating an estrogen dependent
pathway). Alternatively, bioassays can utilize in vivo
systems (inside a living organism), such as exposure to
an embryo or a whole fish.
Advantages to using Bioassays
Both in vitro and in vivo systems have advantages and
disadvantages when using bioassays. One advantage of
bioassays is their ability to detect the cumulative toxicity
of mixtures of both known and unknown chemicals in a
sample. Many attributes of bioassays make them an
appealing option for a number of environmental
applications. For example, some in vitro assays are
amenable to high-throughput testing, suggesting their
potential to be used in rapid screening and monitoring
approaches. Whether the bioassays are high throughput
or not, they have the ability to detect toxicity and
provide needed biological context, providing some
measure of risk in terms of the potential for an adverse
effect.
Present and Future Applications
Currently, bioassays are used alone or are applied in
conjunction with other technologies, such as analytical
chemistry. When used with analytical chemistry, they
can direct analytical methods or give some biological
meaning to the concentrations of chemicals measured.
Bioassays have been very useful in characterizing
impacts to surface waters and determining potential
impacts of wastewater discharges. Additionally, they
have provided useful input to established adverse
outcome pathways (AOPs), connecting exposure to the
toxic effects. The combination of bioassays, chemical
analysis, and AOPs provides a powerful approach in a
host of water quality applications.
An area of active research is the development of suites
of bioassays that each target different biological
pathways. Alternatively, other bioassays are being
developed that focus on specific MOA, such as
neurotoxicity, developmental toxicity, or hepatotoxicity.
Applications are not limited to the biological activity of
chemicals in water, but also include the development of
salivary immunoassays that can evaluate human
exposure to some pathogens.
Environmental samples can be evaluated against suites
of assays to give an integrated measure of the toxicity of
the biologically active substances, identify potential
adverse effects, and to assign some degree of risk posed.
This is similar to the approach used in whole effluent
toxicity testing, where a complex sample has been
shown to be toxic using traditional means, yet the
drivers or the specifics of that toxicity remain unknown.
Bioassays can be used to identify the pathways leading
to toxicity, which can provide a means to prioritize
chemicals that work along that pathway in the
uncharacterized sample. They can also be used to target
a specific protection goal (e.g., evaluate removal of
hormonally active compounds prior to water reuse). This
concept can be applied to any unknown water sample in
many regulatory contexts, such as screening finished
drinking water for carcinogenic potential, reclaimed
water, or wastewater effluent for the potential to cause
adverse outcomes.
Comparative studies indicate that some of the more
well-established techniques respond in a predictable and
reproducible fashion. Progress has been made to
evaluate assay response in relation to the occurrence
and concentrations of both known and unknown
constituents in water samples. Additional work is
ongoing to provide guidance on the management and
interpretation of bioassay data to facilitate decision
making. As bioassays evolve and new applications are
identified, it is envisioned that they could increasingly be
used as a standalone technology, potentially even
replacing analytical methods in some applications.
Technical Contacts:
•	Vickie Wilson. wilson.vickie(5)epa.gov
•	Adam Biales, biales.adam (Sepa.gov
•	Jane Ellen Simmons, simmons.iane(5)epa.gov
•	Dan Villeneuve, villeneuve.dan(5)epa.gov
Communications Contact:
•	Michelle Latham, latham.michelle(5)epa.gov
Additional EPA Information
• Water Research
epa.gov/water-research
• Chemical Interaction with Biological Systems:
epa.gov/chemical-research/research-understanding-
chemicals-interactions-biological-svstems
• Research on Evaluating Chemicals for Adverse
Effects
eva
epa.gov/chemical-research/research-
evaluating-chemicals-adverse-effects
BIESfl epa.gov/chemical-research/
adverse-outcome-pathway-aop-research-brief
n
U.S. Environmental Protection Agency

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