Biomonitoring of Wastewater
Report No 1121/1/04
Dec 2004



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 difficulty.

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 following pages.


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 final objective:
Results and discussion

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 altogether.

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.

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 calculating results.

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.

Conclusions and recommendations

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.