The great strength with effect-based methods is that they detect the effects of all hazardous compounds in a sample, instead of focusing on a few selected compounds, which is done with traditional chemical analyses. The generated knowledge contributes to our understanding of how human health and the environment are affected by all the chemicals we are exposed to simultaneously, the so-called cocktail effect. This new strategy to monitor chemical hazards in water has been developed and applied in multiple research projects, where we have collaborated with drinking water and wastewater treatment plants.
A battery of cell-based tests
We use a battery of cell-based tests to measure toxic effects that are relevant for human and environmental health, such as endocrine disruption and DNA damage.
Mammalian cells, which are cultured in the laboratory, have been modified to respond to specific toxic effects. When the cells are exposed to a sample that contains chemical substances, causing for example estrogenic effects, the cells produce a signal protein that is easily detected by luminescence. By treating the cells with a water sample and then measuring the amount of produced signal protein, we investigate whether, and to which extent, the sample is contaminated with hazardous chemicals. The methods are suitable to monitor drinking water quality and efficiency in wastewater treatment, to perform environmental monitoring and to evaluate new technical solutions in the water industry.
The whole iceberg, not just the tip
Tens of thousands of chemicals can potentially contaminate our waters. Despite that, chemical hazards in water is currently monitored mainly by target chemical analysis, a methodology that only detects a very limited number of all the chemical substances that can contaminate water.
Many international research studies have shown that up to 99% of the effects that can be detected with effect-based methods cannot be explained by the chemicals identified by chemical analysis. A vast majority of the toxicity is caused by unknown chemicals or cocktail effect. Thus, focusing only on the concentrations of known chemicals resembles looking only on the tip of an iceberg. With such a strategy, there is a great risk that chemical hazards from unknown compounds or cocktail effects pass undiscovered. Effect-based methods allow us to gain a holistic view of chemical hazards in water, by looking on the entire iceberg of effects.
We need 1 L water sample sent to our lab in Uppsala.
When the samples arrives a solid phase extraction is performed – just like for chemical analysis of organic micropollutants.
Solid phase extraction
Cells that are sensitive to different effects are exposed to water samples and reference substances in dilution series.
The water extract is usually concentrated 5000 times but is then diluted in the medium the cells grow in.
The occurrence of toxic chemicals in the sample is analyzed with cultured cells
Concentrated water sample
After 24 hours exposure the amount of signal protein is measured which the cells produce in relation to the amounts and potencies of bioactive substances in the sample.
Results from the reference substances are used to express the effect in a sample as a concentration of the reference substance in a biological equivalent concentration – BEQ.
The amount of signal protein is measured
Results are compiled and analyzed
1. Water sample
We need 1 L water sample which is sent to our laboratory in Uppsala. The sampling bottles have been carefully tested to assure that no compounds we measure effects from are leached from the bottles (or bound to the bottle material).
3. Concentrated water sample
The water extract is usually concentrated 5000 times but is then diluted in the cell growth medium. We measure effects in dilution series commonly at a relative enrichment factor (REF) of 1-50.
4. The occurrence of toxic chemicals in the sample is analyzed with cultured cells
The concentrated sample is added to different cell lines, usually human cells. The cells have been modified to detect specific effects such as endocrine disuption or oxidative stress in what is called reporter gene assays. When the cells are exposed to a sample that contains compounds causing the specific effects the reporter gene is activated and the cells produce a signal protein.
5. The amount of signal protein is measured
The amount of signal protein is measured both in samples and for the reference substances that are specific for each assay. Results for the reference substances are used to translate effects in samples to biological equivalent concentrations (BEQ) of the reference substances. For example, estrogenic effects are expressed as estradiol equivalents per litre water sample where the effect may come from several different compounds or mixtures with estrogenic activity. BEQ values can be used to compare effects between different studies.
6. Results are compiled and analyzed
BEQ values are compiled for active samples and can thereafter be compared with values from other studies or historical data. Average or median values and percentiles demonstrate reference values and define a ”normal” interval. Measured effects can be compared to this interval and deviating values indicate that a water should be further investigated. Some of the assays have suggested benchmark values (so called effect-based trigger values – EBT) relevant for human health or the environment.
Effects on sex hormone receptors, endocrine disruptors (EDCs)
Substances can activate or inhibit the estrogen and androgen receptors. Estrogens and androgens have many important physiological functions not only for reproduction but also for the cardiovascular, immune, muscular, and nervous systems. Examples of chemical contaminants in water that affect sex hormone receptors are natural sex hormones, birth control pills, pharmaceuticals used to treat breast and prostate cancer, as well as isoflavones (so-called phytoestrogens) and certain chemicals used in plastic products.
Oxidative stress occurs from excess reactive oxygen radicals and imbalance in the antioxidant defense system. It is a common mechanism behind various types of toxicity, such as teratogenicity and carcinogenicity. Many toxic substances, e.g., organic pollutants, certain pesticides, and natural substances can cause oxidative stress. Oxidative stress is also induced by disinfectant by-products (DBPs), which can be formed during water disinfection. Nearly 700 DBPs have been identified.
AhR activity (aryl hydrocarbon receptor)
When the Ah receptor is activated, metabolizing enzymes are induced and the effect of AhR activation is often called metabolic activation. The Ah receptor has many physiological functions, including chemical and microbial defense, development, and in the regulation of inflammatory reactions. Many toxic substances activate the Ah receptor, such as halogenated organic compounds, polycyclic aromatic hydrocarbons (PAHs), certain pesticides and pharmaceuticals, and naturally occurring substances such as indoles and stilbenes.
Genotoxicity or DNA damage is a serious effect, which requires extensive testing and investigation for registration of chemicals such as pesticides, food additives and flavorings. DNA damage in body cells can lead to cancer and to reproductive disorders when affecting germ cells.
Sampling and results
Water samples are collected by the customer in sampling bottles supplied by us. The samples are then sent to our laboratory, where they are extracted and analyzed. The results are compiled in a report that is sent to the customer. We are happy to contribute with our knowledge in interpretations and discussions of the results.
You are welcome to contact us for further discussions and advice on how our effect-based analysis can be used!