Aquatic Test Systems
Bioassays for assessing the toxicity of water samples are mainly conducted in the Dübendorf lab. In vitro assays for detecting compounds with specific modes of action are used as well as in vivo assays with whole organisms.
Hormonal effect
Yeast Estrogen and Androgen Screens
Yeast Estrogen and Androgen Screens
Yeast Estrogen Screen = YES, Yeast Androgen Screen = YAS
Test organism
- Baker’s yeast (Saccharomyces cerevisiae)
Detectable effects (impact)
Activation or inhibition of the human estrogen receptor (YES, (anti-)estrogenic effect)
Activation or inhibition of the human androgen receptor (YAS, (anti-)androgenic effect)
Test principle, taking YES as an example
- In the Yeast Estrogen Screen, genetically modified yeast cells (Saccharomyces cerevisiae), which contain the gene for the human estrogen receptor coupled with a so-called reporter gene (LacZ), indicate estrogenic effects.
- If an estrogenically active substance binds to the estrogen receptor in the cell, the corresponding gene and then the reporter gene are read. The latter gene encodes for an enzyme (beta-galactosidase), which converts a dye (yellow to red) and thus induces a colour reaction, which is directly correlated to the existence of estrogenically active substances.
- After 18-hour exposure to chemicals or environmental samples, the estrogeneity of the analysed samples can be measured based on colour induction.
The same assay principle applies to the detection of anti-estrogenic substances in YES, as well as androgenic and anti-androgenic substances in YAS.
Flow Chart
Video tutorial
Macro alias: Video
Test duration
- 3 days (exposure time: 18 hours)
Relevance
- Suitable for detecting numerous natural and synthetic hormonally active substances such as environmental toxins from everyday products, e.g. birth control pill ingredients (17α-ethinylestradiol), synthetic materials (bisphenol A, phthalates), pesticides (methoxychlorine) and non-ionic surfactants (alkylphenoles).
- Substances with an activating or inhibitory effect on the estrogen and/or androgen receptor of organisms may interfere with reproduction, affect the metabolism and immune system and induce the formation of tumours.
Guidelines and literature
- ISO 19040-1:2018 Water quality — Determination of the estrogenic potential of water and waste water — Part 1: Yeast estrogen screen (Saccharomyces cerevisiae)
- Routledge EJ and Sumpter JP (1996). Estrogenic activity of surfactants and some of their degradation products assessed using a recombinant yeast screen. Environ. Toxicol. Chem. 15(3): 241-248.
H295R Steroidogenesis Assay (assigned to external lab)
H295R Steroidogenesis Assay (assigned to external lab)
Test organism
- Human adrenocortical carcinoma cells
Detectable effects (impact)
- Changes in steroid hormone concentration (impact on steroidogenesis)
Test principle
- In vitro assay system for determining changes in the formation of steroid hormones. A human cell line capable of expressing all enzymes of the steroidogenic metabolic pathway is used. Moreover, the cell line has the potential of producing all steroid hormones of the fetal and adult adrenal cortex.
- The hormone concentration (e.g. testosterone, estradiol) is determined with commercially available kits (e.g. Elisa).
Test duration
- At least 3 d (exposure time: 48 h)
Relevance
- In vitro assay system for the identification of receptor-dependent and non-receptor-dependent effects of hormonally active substances.
- Suitable for determining whether steroidogenesis can be affected by chemicals and environmental samples
- Tool for investigating the correlations between chemicals and the molecular and biochemical processes of steroidosynthetic pathways
Guidelines and literature
- ISO 19040-1:2018 Water quality — Determination of the estrogenic potential of water and waste water — Part 3: In vitro human cell-based reporter gene assay
- Gracia T, Hilscherova K, Jones PD, Newsted JL, Zhang X, Hecker M, Higley EB, Sanderson JT, Yu RMK, Wu RSS, Giesy JP (2006). The H295R system for evaluation of endocrine-disrupting effects. Ecotoxicol. Environ. Saf. 65:293-305.
- Hecker M, Newsted JL, Murphy MB, Higley EB, Jones PD, Wu R, Giesy JP (2006). Human adrenocarcinoma (H295R) cells for rapid in vitro determination of effects on steroidogenesis: Hormone production. Toxicol. Appl. Pharmacol. 217:114-124.
- Hecker M, Giesy JP (2008). Novel trends in endocrine disruptor testing: The H295R Steroidogenesis Assay for identification of inducers and inhibitors of hormone production. Anal. Bioanal. Chem. 390:287-291.
ER- and AR-Calux
ER- and AR-Calux
and Anti-ER- / Anti-AR-Calux®
Test organism
- Human cell line U2OS-ERα /U2OS-AR
Detectable effects (impact)
- Activation or inhibition of the human estrogen receptor α (estrogenic effect)
- Activation or inhibition of the human androgen receptor (androgenic effect)
Test principle
- A human cell line containing the gene for the human estrogen/androgen receptor coupled with a so-called reporter gene (gene for the enzyme luciferase) is used for this test.
- If an estrogenically/androgenically active substance binds to the estrogen/androgen receptor in the cell, the corresponding gene and then the reporter gene are read. The latter gene encodes for an enzyme (luciferase), which oxidises luciferin to generate light.
- The luminescence intensity is directly correlated to the amount of the substance bound to the receptor.
- The reaction is measured after 24 h exposure to chemicals or extracts from environmental samples.
- The same test principle is used for the detection of anti-estrogenic substances in the anti-ER-Calux® and the detection of anti-androgenic substances in the anti-AR-Calux®
Test duration
- 3 d (exposure time: 24 h)
Relevance
- Suitable for detecting numerous environmental toxins from everyday products, e.g. birth control pill ingredients (17α-ethinylestradiol), synthetic materials (bisphenol A, phthalates), pesticides (alachlor,methoxychlorine) and non-ionic surfactants (alkylphenoles)
- Usually more sensitive than the YES and YAS tests
Guidelines and literature
- No international standard available
- Van Der Linden SC, Heringa MB, Man HY, Sonneveld E, Puijker LM, Brouwer A, Van Der Burg B (2008). Detection of multiple hormonal activities in wastewater effluents and surface water, using a panel of steroid receptor CALUX bioassays. Environ. Sci. Technol. 42: 5814-5820.
Chronic Reproduction Test with Gastropods (assigned to external lab)
Chronic Reproduction Test with Gastropods (assigned to external lab)
Test organism
- New Zealand Spiresnail (Potamopyrgus antipodarum)
Test principle
- In Europe, the New Zealand Spiresnail (Potamopyrgus antipodarum) reproduces through parthenogenesis and is viviparous.
- The reproduction rate of the animals is determined based on the number of offspring.
- An increased or decreased number of embryos can be determined by comparing the number of offspring in controls with that of exposed animals.
Analysed parameters (impact)
- Number of offspring (number of shelled and unshelled embryos in the brood pouch of adult animals) (reproduction toxicity)
- Mortality of parent animals
Test duration
- 28 days and/or 56 days
Relevance
- The reproduction rate is controlled by sexual hormones and influenced by endocrine substances.
- Generally toxic, androgenic and anti-estrogenic substances cause a decrease in the number of embryos.
- Estrogenic substances specifically cause an increase in the number of embryos.
Guidelines and literature
- No international standard available
- Duft M, Tillmann M, Schulte-Oehlmann U, Markert B, Oehlmann J (2002). Entwicklung eines Sedimentbiotests mit der Zwergdeckelschnecke Potamopyrgus antipodarum (Gastropoda: Prosobranchia). Umweltwissenschaften und Schadstoff-Forschung 14: 12-17.
Herbicidal Effect
Combined Algae Test with Unicellular Green Algae
Combined Algae Test with Unicellular Green Algae
Test organism
- Unicellular freshwater green algae (Raphidocelis subcapitata)
Detectable effects (impact)
- Inhibition of photosynthesis (herbicidal effect)
- Inhibition of growth
Test principle
- In this test, the herbicide-induced, specific effects of chemicals or environmental samples on the photosynthesis of algae after 2 h and 24 h exposure time are recorded and the unspecific effects on the growth of the algae after 24 h exposure time are measured.
- The test combines the assessment of photosynthesis and growth inhibition.
- Growth inhibition stands for unspecific toxicity and is determined by means of the cell density through absorption at 685 nm.
- Photosynthesis inhibition is based on the inhibition of Photosystem II (PSII) and indicates herbicide-specific toxicity.
Test procedure
- Download flow chart combined algae test
- SOP available on request from Andrea Schifferli
Video tutorial
Macro alias: Video
Test duration
- 24 h
Relevance
- Substitute organism for primary producers
- Standard test organism for water quality evaluation (OECD, US EPA, ISO)
Guidelines and literature
- Escher BI, Bramaz N, Mueller JF, Quayle P, Rutishauser S (2008). Toxic equivalent concentrations (TEQs) for baseline toxicity and specific modes of action as a tool to improve evaluation of ecotoxicity tests on environmental samples J. Environ. Monit. 10, 612-621.
- Escher BI, Rutishauser S (2007). The combined algae test- a new routine 96-well-plate biotest for simultaneously assessing the photosynthesis inhibition and effect on growth in green algae. Internal Report, Eawag, Dübendorf, Switzerland
- International Organization for Standardization (2004). Water quality -- Freshwater algal growth inhibition test with unicellular green algae. ISO 8692: 15 p.
- Schreiber U, Quayle P, Schmidt S, Escher BI, Mueller J (2007). Methodology and evaluation of a highly sensitive algae toxicity test based on multiwell chlorophyll fluorescence imaging. Biosens. Bioelectron. 22, 2554-2563.
Neurotoxicity
Acetylcholinesterase Inhibition
Acetylcholinesterase Inhibition
Test organism
- Enzyme acetylcholinesterase (e.g. from the eel)
Detectable effects (impact)
- Inihibtion of the enzyme acetylcholinesterase (neurotoxic effect due to organophosphates or carbamates = insecticides)
Test principle
- Environmental toxins that inhibit the enzyme acetycholinesterase cause the accumulation of the transmitter substance acetylcholine in organisms. This leads to permanent muscle and nerve excitation and thus to damage of the organisms.
- Inhibition of the enzyme is determined after exposure to the substances or environmental samples to be analysed.
- Inhibition can be measured both in entire organisms and the isolated enzyme.
Test duration
- 1 day (exposure time: 10 min)
Relevance
- Suitable for example for detecting specific pesticides (organophosphates, carbamates) that specifically inhibit the enzyme acetylcholinesterase.
Guidelines and literature
- Deutsches Institut für Normung (1995). Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammuntersuchung - Suborganismische Testverfahren (Gruppe T) - Teil 1 : Bestimmung von Cholinesterase-hemmenden Organophosphat und Carbamat-Pestiziden (Cholinesterase-Hemmtest) (T 1). DIN 38415-1.
- Ellman GL, Courtney KD, Andres jr V, Featherstone RM (1961). A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7: 88-90.
- Hamers T, Molin KRJ, Koeman JH, Murk AJ (2000). A small-volume bioassay for quantification of the esterase inhibiting potency of mixtures of organophosphate and carbamate insecticides in rainwater: Development and optimization. Toxicol. Sci. 58:60-67.
Unspecific Toxicity
Test with Luminescent Bacteria
Test with Luminescent Bacteria
Test organism
- Marine luminescent bacterium (Vibrio fischeri)
Detectable effects (impact)
- Inhibition of bioluminescence (unspecific toxicity)
Test principle
- Based on the inhibition of the enzyme luciferase, which oxidises luciferin to generate light. Positive results indicate that a substance interferes with the energy metabolism of the cell.
- Effects of chemicals or environmental samples on the bioluminescence of the bacteria are measured after an exposure time of 30 min.
Flow Chart
Test duration
- approx. 0.5 days (exposure time: 30 min)
Relevance
- Screening test for the toxicity of unknown samples
- Standard test organism for water quality evaluation (e.g. OECD, ISO, US EPA)
- Common in wastewater analyses and partly stipulated in ordinances (e.g. German Wastewater Ordinance)
Guidelines and literature
- International Organization for Standardization (2007). Water quality -- Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test) -- Part 3: Method using freeze-dried bacteria. EN ISO 11348-3.
- Escher BI, Bramaz N, Mueller JF, Quayle P, Rutishauser S, Vermeirssen ELM (2008). Toxic equivalent concentrations (TEQs) for baseline toxicity and specific modes of action as a tool to improve interpretation of ecotoxicity testing of environmental samples. J. Environ. Monit. 10, 612-621.
In situ feeding assay with gammarids
In situ feeding assay with gammarids
Test organism
- Gammarus fossarum
Detectable effects (impact)
- Inhibition or stimulation of the feeding activity of gammarids
Test principle
- Gammarids are individually deployed in cages in the stream above and below a point source. Preconditioned leaf discs serve as food.
- After a week, the cages are removed and the gammarids and leaf discs are dried.
- The feeding rate is then determined from the dry weight of the gammarids and leaves before and after exposure.
Test duration
- Min. 7 days
Relevance
- Gammarids are detritivores that primarily feed on coarse particulate organic matter, but also have other food sources such as algae or animals (MacNeil et al. 1997). They play an important role in detritus processing in streams and serve as important prey for fish (Andersen et al. 1993).
- The inhibition of the feeding activity is a general stress response of organisms and can be induced by a variety of stressors in gammarids (Maltby et al. 2002).
- A reduced feeding activity can effect the development, growth and reproduction of animals (Naylor et al. 1989). In addition, effects on the level of communities and ecosystems are possible such as such as effects on the detritus processing or the total leaf decomposition.
- Intrinsic and extrinsic factors can influence the feeding activity of gammarids: examples include parasites, origin of the population and body size (intrinsic) as well as temperature, dissolved oxygen concentration and pH (extrinsic).
- To be able to evaluate effects of wastewater discharges, these parameters should be well characterized. A comparison of measurements upstream and downstream of the discharge should be well possible because of the similar conditions.
- In earlier studies, a reduction of the feeding activity of gammarids was measured downstream of wastewater treatment plants (Bundschuh et al. 2011).
Literature
- Andersen TH, Friberg N, Hansen HO, Iversen TM, Jacobsen D, Krojgaard L. 1993. The effects of introduction of brown trout (Salmo trutta L.) on Gammarus pulex L. drift density in two fishless Danish streams. Arch Hydrobiol 126:361–371.
- Bundschuh M, Pierstorf R, Schreiber WH, Schulz R, 2011. Positive effects of wastewater ozonation displayed by in situ bioassays in the receiving stream. Environmental Science and Technology 45:3774-3780.
- Bundschuh M, Zubrod JP, Kosol S, Maltby L, Stang C, Duester L, Schulz R, 2011. Fungal composition on leaves explains pollutant-mediated indirect effects on amphipod feeding. Aquatic Toxicology 104:32-37.
- MacNeil C, Dick JTA, Elwood RW (1997): The trophic ecology of freshwater Gammarus spp. (Crustacea:Amphipoda): Problems and Perspectives concerning the functional feeding group concept. Biological Reviews 72, 349-364
- Maltby L, Clayton SA, Wood RM, McLoughlin N, 2002. Evaluation of the Gammarus pulex in situ feeding assay as a biomonitor of water quality: robustness, responsiveness, and relevance. Environmental Toxicology and Chemistry 21:361-368.
- Naylor C, Maltby L, Calow P, 1989. Scope for growth in Gammarus pulex, a freshwater benthic detritivore. Hydrobiologia 188-189:517-523.
Acute Toxicity Test with Daphnids (assigned to external lab)
Acute Toxicity Test with Daphnids (assigned to external lab)
Test organism
- Water Flea (Daphnia magna)
Test principle
- This test records the effects of chemicals and environmental samples on the mobility of water fleas after 24 h and/or 48 h.
Analysed parameters
- Mobility inhibition (= mortality)
Test duration
- 24 h and/or 48 h
Relevance
- Component of zooplankton in standing water bodies feeding on algae and representing the basic food resource for fish
- Standard test organism for determining the acute ecotoxicity of chemicals and water samples
Guidelines and literature
- OECD (2004). Guideline for the testing of chemicals 202, Daphnia sp., Acute Immobilisation Test.
Chronic Reproduction Test with Daphnids (assigned to external lab)
Chronic Reproduction Test with Daphnids (assigned to external lab)
Test organism
- Ceriodaphnia dubia
- Water Flea (Daphnia magna)
Test principle
- Pollutants can have a detrimental effect on the offspring production of daphnids.
- The effects of environmental pollutants on the reproduction of daphnids are investigated in a chronic test during 7/8 and/or 21 days.
- For this purpose, adult daphnids are exposed to a medium contaminated with environmental pollutants or to environmental samples and the number of offspring after a defined period of time is determined.
- An increased or reduced reproduction rate can be determined by comparing the number of offspring in controls with that of exposed animals.
Analysed parameters (impact)
- Effects on the population growth/number of offspring (reproduction toxicity)
- Mortality of parent animals
Test duration
- 7 days and/or 8 days (C. dubia)
- 21 days (D. magna)
Relevance
- Component of zooplankton in standing water bodies feeding on algae and representing the basic food resource for fish
- Standard test organism for determining the chronic ecotoxicity of chemicals and water samples
Guidelines and literature
- International Organization for Standardization (2005) Water quality -- Determination of chronic toxicity to Ceriodaphnia dubia. ISO 20665. 21 p.
- Association Française de Normalisation (2000). AFNOR NF T 90-376, Water quality—determination of chronic toxicity to Ceriodaphnia dubia in 7 days. Population growth inhibition test.
- OECD (2004). Guideline for the testing of chemicals 211, Daphnia magna Reproduction Test.
Cell Toxicity
Cytotoxicity Test Kit PAN 1 on Mammalian Cells (assigned to external lab)
Cytotoxicity Test Kit PAN 1 on Mammalian Cells (assigned to external lab)
Test organism
- Mammalian cells: connective tissue cells (fibroblasts) of mice
Test principle
- After exposure to environmental pollutants, several cell toxicity parameters are examined on a cell culture sample to record the effects of environmental pollutants on cell vitality, i.e. cytotoxicity.
Detectable effects (measured parameters)
- Membrane integrity (extracellular lactate dehydrogenase, LDHe)
- Cell respiration (tetrazolium salt, XTT)
- Protein content (Sulforhodamine staining, SRB)
- Lysosome integrity (Neutral Red, NR)
Test duration
- 3 d (exposure time: 48 h)
Relevance
- Suitable for detecting substances that may have a damaging effect on important cell functions, which may be relevant to the entire organism.
Mutagenicity
Ames Test (assigned to external lab)
Ames Test (assigned to external lab)
Test organism
- Salmonellae (Salmonella typhimurium)
Detectable effects (impact)
- Heritable changes in the genetic material (mutagenic effect)
Test duration
- 3 d (exposure time: 48 h)
Test principle
- Mutants of the bacterium Salmonella typhimurium, which themselves are not capable of producing the amino acid histidine (his-deficient mutants), cannot grow on histidine-free culture media under controlled conditions.
- Upon contact with a mutagenic substance, the bacteria can mutate back and thus regain the histidine-producing capability.
- Such bacteria can now reproduce on the histidine-free culture medium
- The higher the number of mutated colonies of bacteria exposed to environmental pollutants, the higher the mutagenic activity of these pollutants.
Relevance
- Indicates Relatively quickly a potential mutagenic effect of substances or mixtures of substances (short waiting time for the occurrence of mutations due to fast bacterial reproduction)
- Easy detectability of a small number of mutated cells besides numerous undamaged cells.
However:
- In-vitro assay system: the behaviour of substances in the body cannot be exactly simulated (partial simulation by adding S9 mixture (liver homogenate with enzymes of the xenobiotic metabolism) is possible)
- Mutagen for bacteria ≠ mutagen for mammals
- Different complexitiy of bacterial and mammalian cells (mammalian cells activate a cytostatic or apoptotic mechanism when DNA defects are detected; the bacterium is a unicellular organism)
Guidelines and literature
- Deutsches Institut für Normung (1999). Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammuntersuchung - Suborganismische Testverfahren (Gruppe T) - Teil 4: Bestimmung des erbgutverändernden Potentials mit dem Salmonella-Mikrosomen-Test (Ames Test) (T 4). DIN 38415-4.
- International Organization for Standardization (2005). Water quality -- Determination of the genotoxicity of water and waste water -- Salmonella/microsome test (Ames test). ISO 16240, 20 p.
Gentoxicity
umuC Test (assigned to external lab)
umuC Test (assigned to external lab)
Test organism
- Salmonellae (Salmonella typhimurium)
Detectable effects (impact)
- Activation of the SOS repair system of a cell (genotoxicity)
Test principle
- The umuC test is based on a genetically modified strain of the bacterium Salmonella typhimurium.
- A genotoxic substance induces the so-called umuC gene in the bacterial cell as part of the SOS repair system of the cell to counteract DNA damage. A reporter gene producing an enzyme (beta-Galactosidase) is coupled to this umuC gene. The enzyme converts a dye and thus induces a colour reaction that indicates the existence of genotoxic substances.
Test duration
- 1.5 d (exposure time: 2 h)
Relevance
- For the detection of DNA damage (genotoxicity)
- Gentoxicity stands for any damage to the genetic apparatus, the genome. Genotoxic substances acting on the cell may cause chromosome breakage, the insertion or deletion of bases as well as DNA frameshifts. Most of such changes in the genetic substance are detected and repaired by the SOS repair system. These effects can be demonstrated with the umuC test.
- Any unrepairable damage to the genetic substance will be passed on to the daughter cells during cell division. This is called mutagenity, the heritable, irreparable results of genotoxicity. These effects are demonstrated in the Ames test.
Guidelines and literature
- International Standard Organisation (1996/2000) Water quality - Determination of the genotoxicity of water and waste water using the umu-test, EN ISO 13829 (2000) and 38415-3 (1996).
- Escher BI, Bramaz N, Quayle P, Rutishauser S (2008). Monitoring of the ecotoxicological hazard potential by polar organic micropollutants in sewage treatment plants and surface waters using a mode-of-action based test battery, J. Environ. Monit. 10, 622-631.