Biomonitoring of Wastewater
Report No 1121/1/04
Access to an adequate supply of usable natural water is a basic
requirement for human life. At the present time, when the right to such
access is being written into law in some countries, there is a
diminishing likelihood of its being met for billions of people living
in Asia, Africa and Latin America.
Many different factors are involved, the most obvious being the
increased demand for water due to population growth and urbanisation,
without any matching increase in the supply of fresh water. In the
past, wastewater contained much biodegradable material, able to be
processed and removed by living organisms. Today's effluents also carry
other substances, some of them incompatible with any form of life.
An obvious solution is to deal with the problem at source, where
substance-based end-of-pipe methods of analysis are most
cost-effective, and regulatory controls can be enforced without
The current approach is to use effect-based monitoring systems,
including ecological ones, to detect the existence of a problem, and
then trace it back to its end-of-pipe source by means of whole organism
survival tests. These have their own limitations, and even the
simplified kit-type tests now available are not universally applicable.
The alternative to one-off testing is to use integrated batteries of
tests designed to meet the requirements of specific practical
situations, and for this purpose, human cell culture assays have both
advantages and shortcomings, which are discussed in detail in the
The original aim of the project was to develop a rapid, low-cost human
cell toxicity test that could be used for the universal monitoring of
complex effluents. This would be based upon earlier work listed in the References, and the
aims listed below were formulated as necessary stages in reaching this
Results and discussion
- Determining the range and sensitivity of the assay
- Simplifying the assay
- Controlling 'drift' in cellular responses to toxic agents.
The problem of drift (discussed in detail later in this report) is
essentially the changing responsiveness of the same cells to the same
amounts of the same toxic agents. Cells of higher organisms function by
means of biochemical pathways each of which consists of a train of
molecular reactions governed by mass action. The pathways are linked
together through source molecules and end products, and it is by
regulating the availability of these that cells are able to express
their different functions. Unused biochemical functions can become
attenuated during successive cell divisions and may eventually be lost
One solution to this problem is to use primary cell cultures at a fixed
passage number. This approach requires extensive back-up facilities in
the way of animal (or fish) colonies and holding cultures. Another
solution is to use established cell lines and select for their ability
to maintain alternative metabolic pathways by 'bouncing' them between
different cell culture environments. We find the following media:
go a long way towards solving this problem, apparently by stabilizing
glutamate metabolism prior to and during the assay.
- serum-free with glutamine,
- glutamine-free with serum,
- with glutamine and serum, and
- serum-free, glutamine-free (for the assay).
A major factor impacting on the cost and convenience of cell culture
assays is the need to maintain sterile working conditions. We have
introduced three simplifying modifications. The first is to work under
clean, non-sterile conditions up to the final stages of setting up an
assay. The entire plate is then sterilised by brief irradiation with UV
light before adding the cells and starting the incubation. The second
is to re-sterilise disposable equipment with 70% ethanol while it is
still in use and then flush it immediately afterwards with sterile
water. This saves time and reduces the consumption of disposable
plastic-ware. A third factor adding significantly to costs is the level
of staff training and time needed for interpreting results. We have
tackled this problem by putting appropriate controls into the test and
by using computer-controlled procedures for reading the assays and
Under our experimental conditions, each assay generates a number
between -30 and 100, which is indicative of the level of cytotoxicity
of the sample being tested. It is evident from our results that the
values we obtained for known toxic agents all occur at concentrations
well above their permissible limits in Class 2 drinking water.
The window between the lower limits of sensitivity of the assay and the
upper limits of safety for human consumption can often be closed by
concentrating individual samples 20-fold, a feasible approach when
working with the small volumes required for the assay. However, because
it adds to the costs in consumables and time, it would not be realistic
to treat large numbers of non-toxic samples in this way.
In practice, the opposite situation in which a significant percentage
of samples are off-scale with respect to toxicity is common in South
African mining and industrial effluents. Such samples are easily
assayed by sequential dilution.
A series of replicate assays carried out on unknown samples of
supposedly usable water from several environmental sources showed
characteristic cytotoxicity profiles for some of them.
During the course of the project, several minor changes were made in
the methodology with the aim of reducing the risk of variability due to
operator bias. These are noted in the report.
Cell culture-based toxicity assays, and specifically the human cell
assays described here have an obvious place in any battery of tests for
evaluating water quality. Their level of sensitivity covers the range
of toxicity found in many complex effluents and can be correlated with
clinical databases. In addition they integrate easily with currently
used methods of chemical analysis.
We conclude that effluents showing cytotoxicity in this assay should be
regarded as hazardous for human health until such time as the agents
causing the effects have been identified.