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The National Academies
NATIONAL RESEARCH COUNCIL
1998
Health-Effect Studies of Reuse Systems
P. 174
The water reclamation projects reviewed here very rarely took the approach of screening with bioassays and then
applying more detailed toxicological evaluations. In fact, this approach was applied only to one aspect of the
health-effect study conducted by Orange and Los Angeles Counties (OLAC) (Baird et al., 1980, 1987; Jacks et al.,
1983). The bulk of this OLAC work was performed using the Salmonella/microsome assay. The first efforts were
directed at simply identifying the relative mutagenic activity of chemicals that could be isolated from differing source
waters. Certain of the more mutagenic waters were then fractionated and studied in more detail. While the approach
met with some modest success, the mutagenic activity of the fractions was greater than could be accounted for by
known mutagens. Part of the discrepancy was probably caused by the reliance on gas chromatography-mass
spectrometry (GCMS) as the analytical tool, because most of the chemicals in water, including some that are potent
mutagens, cannot be measured with GC-MS.
The OLAC study also attempted to use derivatization methods (which use a second chemical to react with and thus
detect the target chemical) to detect certain chemical targets (Baird et al., 1987; Jacks et al., 1983). While this test
produced some significant modifications of mutagenic activity in some water samples, it revealed no consistent pattern
of mutagen reduction. This suggests that the chemicals present in different waters may be of different characters.
It is of interest to contrast the inconclusive nature of the OLAC study's results with the progress that has been made in
drinking water chlorination. The initial studies of mutagenicity in drinking waters around the world first traced
mutagenicity to the process of chlorination; the major mutagenic activity was found to be associated with a very potent
mutagen referred to as MX (Meier et al., 1987), which is produced in chlorination. The whole-animal carcinogenesis
testing of this chemical recently ended and concluded that MX induces tumors (Tuomisto et al., 1995). However, a
recent workshop (ILSI, 1995) focused on the toxicology data for MX and concluded that MX is probably not a major
contributor to cancer via drinking water because it is found at extremely low levels in chlorinated drinking waters.
P. 175
In the Tampa project, positive mutagenic activity was associated with both the river water and the AWT effluents after
filtration, ultrafiltration, and reverse osmosis. In most of these cases the addition of chlorine tended to increase
mutagenic activity in the Ames test, whereas ozonation produced inconsistent effects.
The interpretation of the results of the Tampa study is subject to the same ambiguity identified with other studies that
have depended prima-rily on in vitro test systems. While screening assays showed some mutagenic activity, there was
no attempt to collect further data that would allow a comparison of the relative actual risks of disease posed by these
two water sources.
P.176
A more serious drawback of relying only on mutagenic testing is that such testing provides no "toxicological
characterization" of potential hazards. As pointed out in Chapter 4, negative results in such tests do not guarantee
safety. This uncertainty has three sources. First, many adverse health effects of chemicals do not require a chemically
induced mutation. Second, research has shown virtually no correlation between the genotoxic potency and the
carcinogenic potency of chemicals. Third, subsequent work has shown that the collective false negative rate for
carcinogenic chemicals in mutagenesis assays is much higher than initially believed (Ashby and Tennant, 1988),
leaving more carcinogenic chemicals undetected than previously thought.
P. 177
In spite of these excellent efforts at conducting well-conceived, long-term toxicological studies, there are technical
problems with the testing of organic concentrates. The preparation of representative concentrates of organics in water
is not simple. While recoveries of organic material can approach 70 percent in some systems (Jenkins et al., 1983),
they are often significantly lower in many studies. In the studies reviewed here, recovery levels were not always
reported. Tampa reported 20.9 percent recovery from ozone-disinfected Hillsborough River water and 9.1 percent
from ozone-disinfected GAC product water (CH2M Hill, 1993). Recovery in various OLAC studies ranged from less
than 50 percent (Baird et al., 1980) to 70 percent (Jenkins et al., 1983).
A second issue is the degradation of samples over time. While some effort was made to stabilize the samples, it is
impossible to know that no changes occurred that could have affected the result. In a situation where major portions of
the material cannot be identified, obtaining objective measures of stability is difficult.
A third commonly voiced criticism of using concentrates is that it is impossible to know whether the concentration
procedure itself produces or destroys some products by accelerating chemical reactions. Each of these criticisms is
relevant to the confidence of negative results.
Other criticisms deal with the completeness of the testing. While the tests applied had elements of currently accepted
protocols for assessing the safety of commercial products (FDA, 1982; U.S. EPA, 1979), pragmatic concerns forced
some potentially significant departures from conventional practice. For example, conventional practice in safety testing
dictates that materials should be tested at the maximum tolerated dose (MTD). But in the Denver and Tampa studies,
the cost of concentrate preparation limited the amount of concentrate that could be prepared, and the MTD was not
approximated. The MTD may not have been approached even if the concentration factors had been increased by
another order of magnitude. Nevertheless, in the opinion of the committee, these two studies approach the practical
limit of the type of study that could be performed using organic concentrates of reclaimed wastewater.
An issue in any retrospective evaluation such as this is that public health concerns evolve over time. The focus of
safety testing in the 1990s has moved beyond where it was in the 1970s and 1980s when most of these studies were
performed. For example, there is now considerable concern about chemicals loosely referred to as endocrine
disrupters (Kavlock et al., 1996). Much of the controversy over this type of chemical concerns estrogen-like chemicals
such as dioxin and polychlorinated biphenyls (PCBs) and their potential relationships with diseases like breast cancer.
The concern has recently broadened to include chemicals, such as alkylphenol ethoxylate, that have produced
apparent estrogen effects in fish (see Chapter 2). Endocrine disrupters are specifically identified for evaluation of
health impacts in the latest reauthorization of the Safe Drinking Water Act. A potentially important issue for potable
use of reclaimed water is that chemicals producing endocrine disruption have been associated with municipal
wastewater effluents (Sumpter, 1995).
P. 180
One critical problem is with the preparation of the water sample to be tested. As long as the sample can be considered
less than fully representative of the chemical constituents in the source water, the testing can be criticized as
incomplete. As discussed in Chapter 4, organics are concentrated both to increase the effective dose for testing
purposes and to separate inorganics that might dehydrate the test organisms. But the processes that concentrate the
organics may create reactions that remove or add chemical compounds, thus changing the mixture of chemicals. So
far we have no reliable way to verify how well a sample represents the water from which it is derived. As explained in
Chapter 2, certain chemical characteristics can be used to describe the nature of the organic chemicals in water. It is
possible that a confirmatory procedure could be developed that would (1) verify the consistency of the chemical
characteristics of samples produced and (2) verify that the process of concentration did not cause chemical
components of the mixture to react and change. Developing such a procedure would require a very significant
research effort.
Another major issue is the expense of completing an adequate safety evaluation. Cost estimates should consider not
only the investment for original testing at the pilot stage but also for ongoing measures to monitor and ensure the
safety of the product water over time. Such ongoing efforts must address not only potential changes in the quality of
the water but also changing priorities regarding what health risks should be addressed.
P. 181
These difficulties suggest that alternative strategies for testing reclaimed water must be sought. One option is to
employ conventional safety testing protocols used to evaluate new food additives and drugs. However, there are
problems with this approach. One is that the decision logic used in conventional safety testing calls for testing at or
approaching the MTD. The lack of substantive effects in the Denver and Tampa studies at 500- to 1000-fold
concentration factors suggests that testing at the MTD is impractical, if not impossible.
A final problem is the timing of results. For potable reuse projects, continuous toxicity testing is desirable to provide
project operations with an additional "warning system" in the event of unanticipated changes in product water quality.
Conventional methods of toxicity testing do not allow for such continuous monitoring and the production of rapid
results.
Another problem in applying the logic of safety testing to reclaimed water is that, unlike product developers, water
utilities cannot simply drop their product lines. If a commercial product is shown to be mutagenic by simple in vitro
tests, the producer can avoid the costs of further testing by terminating its production. This is not a reasonable option
for drinking water, which always requires further testing. For example, most drinking waters in the United States would
show a mutagenic response associated with disinfection (Cheh et al., 1980; Meier et al., 1987) if so tested; yet this
does not mean the water is unsafe, since mutagenesis does not necessarily indicate carcinogenicity or other health
threats. And in view of the well-established contribution that disinfection makes to public health, it would be foolish to
discard either the water or the disinfection process simply because disinfection increases mutagenic activity. Thus, the
question should not be whether a chemical is mutagenic in a bacterial system but whether it presents a carcinogenic
hazard to humans and at what levels of exposure. And determining carcinogenicity requires tests of intact animals
regardless of whether the in vitro mutagenesis test is positive or negative.
Final problems with trying to apply conventional safety testing to reclaimed water are timing of the results and
determining what action is required if a positive response is detected in live animals. For example, what should be
done if a chronic rodent study (which typically takes two years to run and another year to analyze) finds a marginally
significant increase in the incidence of tumor-bearing animals exposed to the test water? It is highly unlikely that a
specific chemical agent could be identified within a reasonable time frame. In this situation, one is caught in the
dilemma of deciding whether this test (1) was a statistical fluke, (2) reflected some transient changes in water quality
that occurred during the sample, or (3) represented a true hazard. The only way to answer the
P. 182
question might be to rerun the study. Consistent results from a second study would be clear cause for concern. But
what if the second test was negative? Would a tiebreaker be needed? While the testing scheme proposed by the NCR
(1982) was the proper approach to be taken from a toxicologist's point of view, the approach is exceedingly difficult to
implement for testing reclaimed water projects. It also does not provide results rapidly enough to respond to important
changes in water quality.
Potable reuse projects need a new approach to toxicity testing. Future toxicological characterizations of wastewaters
intended for potable reuse or water derived for potable use from wastewaters should focus specifically on data
needed for risk assessment. Because the substance being tested is essentially unknown, it is important that whole
animals be used for testing to allow the concurrent evaluation of multiple end points. If only in vitro tests are
conducted, the test system becomes a potentially large collection of independent tests that frequently cannot be
integrated into a realistic estimate of human health risk. Also essential is a toxicity testing system that can allow
continuous monitoring and produce timely results.
P. 202
Of the toxicological studies, only the Denver and Tampa studies addressed a broad range of toxicological concerns.
Those studies suggested that no adverse health effects should be anticipated from the use of Denver's or Tampa's
reclaimed water as a source of potable drinking water. However, these studies, drawn from two discrete points in time
and conducted only at a pilot plant level of effort, provide a very limited database from which to extrapolate to other
locations and times.
Because of the high cost and methodological problems inherent in the testing of concentrated samples on rats and
mice and because of the difficulty in applying the logic of safety testing to reclaimed water, the strategy set forth by the
1982 National Research Council panel is potentially too costly to implement and will not resolve health-effect questions
in a timely manner for an operational potable reuse system.
P. 203
Numerous epidemiologic studies (ecological, case-control, cohort, and outbreak investigations) have examined the
relationship between various microbial and chemical contaminants in drinking water and a wide range of acute and
chronic health outcomes in populations exposed either to a specific contaminated water supply or to specific types of
source waters and treatment processes. However, only three such studies apply to potable reuse of reclaimed water,
and only one set of epidemiological studies (Los Angeles County) evaluating the health effects associated with the
consumption of reclaimed water has been conducted in a setting that is useful for assessing possible health effects in
other parts of the United States or other industrialized countries. These studies have used an ecologic approach,
which is appropriate as an initial step when the health risks are unknown or poorly documented, but negative results
from such studies do not necessarily prove the safety of reclaimed water
P. 204
for human consumption. These studies can only be considered as preliminary examinations of the risks of exposure to
reclaimed water.
The committee recommends that alternative epidemiologic study designs and more sophisticated methods of exposure
assessment and outcome measurement be undertaken at a national level to evaluate the potential health risks
associated with reclaimed water. Ecologic studies should be conducted in a variety of water reuse situations (e.g.,
ground water, surface water) in areas with low population mobility. Case-control studies or retrospective cohort studies
should be undertaken to provide information on health outcomes and exposure for an individual level while controlling
for other important risk factors. Although cohort studies are the most difficult and expensive to perform, this is the only
study design that can examine the temporal relationship between exposure to reclaimed water and the development of
adverse health effects. Increasing interest in and need for potable water reuse may justify such efforts