PhD Graduation

Given the need for continued quality control of surface waters used for the production of drinking water by state-of-the-art bioassays and biological early warning systems, the objective of the present thesis was to validate and improve some of the bioassays and biological early warning systems used for quality control of surface water.

Due to the lower (geno)toxicity, bioassays with increased sensitivity are needed because of i) the increasing number of chemicals present in surface water and ii) the fact that concentrations of chemicals of concern may be toxicologically relevant but below the level of detection of the currently applied protocols for bioassays or biological early warning systems. The River Rhine was selected as a model river for our studies.

In vitro bioassays for several extracts of Rhine water were used and evaluated in relation to sensitivity, selectivity and suitability for future routine monitoring programs. An ecological risk model evaluation based on the bioassay data obtained is included to judge the ability of the in vitro bioassays to detect effects on the organisms in the ecosystem as a whole. It is concluded that an optimal bioassay battery of in vitro tests for water quality monitoring of the River Rhine includes the Daphnia IQ assay, the Raphidocelis sp. PAM test and the Microtox® test as general toxicity tests, together with the Ames TA98 test (with metabolic activation) to monitor genotoxicity. The results also reveal that the water quality of the River Rhine has improved over the years. The potentially affected fraction of organisms which is predicted to experience adverse effects of chemicals present in the surface water is lower than 5%. All together it was concluded that the results obtained measuring overall toxicity with bioassays as described above provide essential information about the quality of surface water that is not obtained by chemical analysis.

Next to the use of bioassays in the laboratory, the online biological early warning systems have proven useful in the monitoring of water quality of surface waters. Solid Phase Extraction (SPE) as an online concentration step was developed and validated in the bbe Algaetoximeter and Microlan TOXcontrol biological early warning systems. The results obtained provide a proof of principle for online bioassays with increased sensitivity, providing a suitable method for surface water quality control now indicating that water quality has been improved over the past decades, but still requires adequate online monitoring. The use of SPE connected to a biological early warning system will also prove a way to judge whether pollutants, chemically detected with ever increasing sensitivity, will still result in toxicity.

Although in vitro mutagenicity studies of the RIWA showed that the genotoxicity of the River Rhine steadily decreased during the last decades, there is still concern about the presence of some residual mutagenicity. In order to be able to make a comparison with the water quality 27 years ago, a study was performed with the same experimental design as before in order to measure the effect of Rhinewater on the induction of Sister Chromatide Exchange (SCE) in the Eastern mudminnow (Umbra pygmaea). As a new endpoint the single cell gel electrophoresis assay (Comet assay) was performed. Fish exposed for 11 days to Rhine water had a significantly higher number of SCE and an increased comet tail-length compared with control fish exposed to groundwater. After exposure for three days to Rhine water there was no difference in SCE and a slightly increased comet tail-length compared with the control. It was concluded that genotoxins are still present in the river Rhine, but that the genotoxic potential has markedly decreased compared with 27 years ago. In the next study the objectives were to investigate i) whether further prolonged exposure results in a further increase in in vivo genotoxicity, ii) whether new data corroborate that in vivo genotoxicity of Rhine water is at present lower than in 1978, and iii) whether the Comet assay is a suitable alternative to the SCE assay. Prolonging the exposure time of Eastern mudminnows to River Rhine water from 11 days to 42 days, did not give a significant increase in SCEs and DNA damage (Comet assay) in gill cells. The new data corroborate that in vivo genotoxicity of River Rhine water is at present lower than in 1978. The Comet assay is a useful addition but does not provide a substitute for the SCE endpoint in these in vivo genotoxicity studies.

In addition to (geno)toxicity measurements to monitor the quality of the surface water that is used as a source for the production of drinking water, the quality of water retrieved from different processes during the drinking water treatment, can also be monitored by bioassays. UV/H₂O₂ treatment can be part of the process converting surface water to drinking water, but would pose a potential problem when resulting in genotoxicity. The genotoxicity of samples collected from a water treatment plant , applying UV/H₂O₂ treatment was investigated. All samples showed negative results in in vitro Ames and Comet assay. Samples were also tested in in vivo SCE and Comet genotoxicity tests in Eastern mudminnow (Umbra pygmaea).  No significant increase in SCEs was observed, but gill cells isolated from fish exposed to water obtained immediately after UV/H₂O₂ treatment and to Lake IJsselmeer water showed significantly increased DNA damage in the Comet assay. All other samples tested were negative in this Comet assay. This indicates that DNA damaging compounds may result from the UV/ H₂O₂ treatment, but also that these compounds are efficiently eliminated by granular activated carbon (GAC) treatment of the water before distribution. It is concluded that when combined with this subsequent GAC treatment, UV/H₂O₂ treatment for the production of drinking water from surface water is not of concern with respect to genotoxicity.

Overall, it was demonstrated that the use and further optimisation of bioassays will strengthen the current state of the art in water quality assessment.

Promoter: prof. dr ir IMCM Rietjens
(Co)Promoter: dr. GM Alink, dr W Hoogenboezem