ToxTracker included in KWR’s water quality assessment
The Dutch Water Research Institute KWR is dedicated to ensuring that the surface water quality in Europe improves, or at least not deteriorates. Considering that the water quality in the Netherlands is the worst of all EU member states (only 1% of Dutch waters are rated ‘good’ according to the current KWR methodologies), continuous monitoring of (drinking) water quality is crucial both for health and the safety of the environment. Especially when alternative sources are used, such as rainwater or brackish water, potential risks of chemicals and other pollutants in the water must be assessed.
In vitro bioassays can be effective tools to detect toxic pollutants in water. These bioassays can assess toxicological endpoints such as genotoxicity, reproductive toxicity and endocrine disruption. Selecting the right battery of assays to cover different toxicological effects in an efficient and cost-effective manner requires careful evaluation of each candidate assay.
Earlier evaluation of ToxTracker as a candidate assay
In KWR’s evaluation in 2017, below set of criteria was used to construct a scoring matrix.
| Criteria | ToxTracker Score | Maximum Scorea |
| Assay applicability and ease of use | ||
| Applied to environmental samples | 1 | 3 |
| Validated to water samples | 1 | 3 |
| Standardized protocol available | 2 | 3 |
| Service and support available | 3 | 3 |
| Costs | 1 | 3 |
| Ease of use · non-GMOc · no specialized equipment or skill required · automation possible · non-licensed in vitro (cell) model · commercial kit available · training available | 3 | 6b |
| Assay performance | ||
| Selectivity | ? | 3 |
| Accuracy | ? | 3 |
| Reproducibility | 3 | 3 |
| Robustness | 3 | 3 |
| Sensitivity | ? | 3 |
| Specificity | 3 | 3 |
| Limit of detection | ? | 3 |
| Cytotoxicity control | 2 | 3 |
| Speed | 2 | 3 |
| Clear/straightforward read-out | 3 | 3 |
| High-throughput capacity | 3 | 3 |
aNumber of points represent poor (1 point), good (2 points) or excellent (3 points) score. bScore based on sub criteria (1 point each). cGenetically Modified Organism
Based on the combined score in the scoring matrix, ToxTracker was selected as a candidate in vitro bioassay as an alternative for the Ames fluctuation assay for gene mutations and the micronucleus test for chromosomal aberrations.
Since ToxTracker was developed primarily for predicting genotoxicity of new drugs, chemicals, personal care products and food ingredients, its applicability in water quality testing would require some additional validation.
ToxTracker received high scores for assay performance for the criteria available in 2017. At the time, information on selectivity, accuracy, sensitivity, and limit of detection were missing. Extensive validation confirms the high accuracy of ToxTracker to correctly predict in vivo genotoxicity.
We recently classified 68 test chemicals with ToxTracker and compared the results to data from in vitro and in vivo micronucleus tests. ToxTracker correctly identified all tested genotoxicants and there was a very high accuracy (92% sensitivity, 93 specificity) to predict in vivo clastogenicity.
Relevant for water quality monitoring?
A recently published comparison between different bioassays indicated that not every bioassay gives the same result for a specific water sample based on their mechanism of action, sensitivity, and specificity. Because the comet assay, micronucleus test, p53-CALUX and ToxTracker are performed with human or mammalian cells, they were considered more relevant for the assessment of human health effects than bioassays using bacteria. However, the Ames test was mentioned as an effective gene mutation assay when the starting point of a test strategy is to cover different mechanisms of DNA damage.
And when it comes to ToxTracker, the conclusion was: “The test can be seen as a far-reaching test in water quality monitoring due to its thorough protocol (it measures effects on six different mechanisms).” This means that ToxTracker may be a consideration in the future, in case it can replace other assays, providing a resource-effective alternative.
