Aquatic Test Systems

Test organism

• Human cell line U2OS

Detectable effects (impact)

• Inihibition of the expression of the enzyme luciferase 

Test principle

• The Cytotox CALUX® consists of a human osteosarcoma cell line (U2OS) that constitutively expresses a high level of luciferase.
• By addition of the appropriate substrate for luciferase, light is emitted.
• If the cells are exposed to cytotoxic compounds as a result the amount of luciferase expressed will decrease. This can be measured as a decrease in the light signal.
• The line is also used as a generic control in CALUX assay panels.

Test duration

• Exposure time: 24 h

Relevance

• The Cytotox CALUX reports whether a sample is cytotoxic, and at which concentration or dilution factor the cytotoxicity occurs.

Guidelines and literature

• ECVAM method DB-ALM Protocol n° 197.: Automated CALUX reporter gene assay procedure.

• Fish gill cell line RTgill-W1 from rainbow trout (Oncorhynchus mykiss)

Detectable effects (impact)

• Cell viability is assessed based on three fluorescent indicator dyes

Test principle

• The RTgill-W1 cell line assay comprises a 24-well plate format acute toxicity test based on the permanent cell line from rainbow trout (Oncorhynchus mykiss) gill.

• After 24 h of exposure to a test chemical or product at six concentrations, cell viability is assessed based on three fluorescent indicator dyes.

• Data are expressed as percent cell viability of unexposed controls versus the test chemical concentration.

• The resulting concentration-response curves serve to determine the effective concentrations causing 50% loss in cell viability (EC50 values). The lowest observed EC50 value is taken as proxy to predict the fish acute toxicity (LC50 value).

Test duration

• Exposure time: 24 h

Relevance

• Aside from being an animal-free alternative to the high severity traditional fish test (e.g. according to OECD TG203), the gill cell line assay requires about three orders of magnitude less testing material, making it the perfect choice for screening, e.g. in new chemical synthesis and product development.

Guidelines and literature

• OECD (2021), Test No. 249: Fish Cell Line Acute Toxicity - The RTgill-W1 cell line assay, OECD Guidelines for the Testing of Chemicals, Section 2, OECD Publishing, Paris, https://doi.org/10.1787/c66d5190-en.

Test organism

• Human cell line U2OS

Detectable effects (impact)

• The expression of the luciferase gene shows the activation of the Nrf2 pathway which is activated by oxidative stress and antioxidants. 

Test principle

• The Nrf2 Responsive (Nrf2) CALUX® is composed of a human cell line (U2OS)
  containing the firefly luciferase gene under control of four Electrophile Responsive
  Elements (EpREs).
• The luciferase serves as a reporter gene for activation of the Nrf2
  pathway. This pathway is activated by oxidative stress and antioxidants. Activation of
  the pathway will lead to luciferase expression, and by addition of the appropriate
  substrate for luciferase, this can be detected as light.
• The amount of light produced is proportional to the concentration of the Nrf2-pathway activating compounds.
• The pathway activation caused by the sample is compared to the activation elicited by the positive control, curcumine. Nrf2 CALUX bioassays report total curcumine equivalents

Test duration

Exposure time: 24 h

Relevance

• Numerous pollutants lead to oxidative stress through the increased formation of free radicals.

Guidelines and literature

• Van der Linden, SC, von Bergh A, Van Vugt-Lussenburg B, Jonker L, Brouwer A, Teunis M, Krul C and Van der Burg B. (2014) Development of a panel of high throughput reporter gene assays to detect genotoxicity and oxidative stress, Mutation Res.760,23-32

Test organism

• Rat cell line (H4IIE)

Detectable effects (impact)

• The test indicates the presence of dioxins (PCDDs), dioxin-like compounds (e.g. furans (PCDFs) and dioxin-like PCBs (dl-PCBs)) via their binding to the aryl hydrocarbon receptor (AhR).

Test principle

• In the cell line, the firefly luciferase gene is coupled to Dioxin Responsive Elements
  (DREs) as a reporter gene for the presence of dioxins (PCDDs) and dioxin-like compounds (e.g., furans (PCDFs) and dioxin-like PCBs (dl-PCBs)).
• Following binding of dioxins and/or dioxin-like compounds to the cytosolic Aryl-hydrocarbon receptor (AhR), the ligand-receptor complex binds the DRE.
• Cells that are exposed to dioxins or dioxin-like compounds not only express proteins that are under normal circumstances associated to DRE, but also luciferase. By addition of the appropriate substrate for luciferase, light is emitted.
• The amount of light produced is proportional to the amount of ligand-specific receptor binding, which is benchmarked against the relevant reference compounds (2, 3, 7, 8-TCDD). DR CALUX bioassays report total 2,3,7,8-TCDD TEQs for environmental matrices and total BEQs for food/feed matrices.
  
  

Test duration

• Exposure time 24 h

Relevance

• The DR CALUX indicates whether a sample contains dioxins or dioxin-like compounds.

Guidelines and literature

• Van Vugt-Lussenburg, B., Van der Burg, B., Besselink, H.,  Brouwer, A. (2013) DR CALUX^®, A high-throughput screening assay for the detection of dioxin and dioxin-like compounds in food and feed. In “High throughput screening methods in toxicity testing” (P. Steinberg, ed). John Wiley and Sons, Inc. New York. ISBN 9781118065631. Pp 553-546.

Test organism

• Human cell line U2OS

Detectable effects (impact)

• The test indicates the presence of various pollutants via their binding to the pregnane X receptor.

Test principle

• The pregnane X receptor PXR is a nuclear receptor with the main task of detecting toxic exogenous substances and activating the expression of proteins responsible for detoxification and clearance.
• In the cell line, the firefly luciferase gene as a reporter gene is coupled to the PXR to indicate the presence of pollutants.
• Following binding of these compounds to the PXR, the corresponding genes are read and also the reporter gene. This gene codes for an enzyme (luciferase) that converts luciferin to produce light.
• The amount of light produced is proportional to the amount of ligand-specific receptor activation, which is benchmarked against the relevant reference compound nicardipine, and expressed as toxic equivalents (TEQs), or bioanalytical equivalents (BEQs).

Test duration

• Exposure time 24 h

Relevance

• The PXR CALUX indicates the presence of numerous contaminants such as steroids, antibiotics, antimycotika, pharmaceuticals and non-dioxin-like PCBs.

Guidelines and literature

• Piersma AH, Schulpen SHW, Uibel F, Van Vugt-Lussenburg, B, Bosgra S, Hermsen SAB,
  Roelofs MJE, Man, H., Jonker, L., Van der Linden, S, Van Duursen MBM, Wolterbeek APM, ,
  Schwarz M, Kroese ED, Van der Burg B. (2013) Evaluation of an alternative in vitro test
  battery for detecting reproductive toxicants. Reprod. Toxicol. 38,53-64

Test organism

• Rat cell line (H4IIE)

Detectable effects (impact)

• The test indicates the presence of polycyclic aromatic hydrocarbons (PAH) via their binding to the aryl hydrocarbon receptor (AhR).

Test principle

• In the cell line, the firefly luciferase gene as a reporter is coupled to dioxin-reactive elements (DREs), which also indicate the presence of polycyclic aromatic hydrocarbons (PAH). Due to the specific work-up method and short incubation time, these typically only bind PAH.
• After PAH binds to the cytosolic aryl hydrocarbon receptor (AhR), the ligand-receptor complex binds to the DRE.
• Cells exposed to PAH express not only proteins that are associated with the DRE under normal circumstances, but also luciferase. By adding the appropriate substrate for luciferase, light is emitted.
  The amount of light produced is proportional to the amount of ligand-specific receptor binding, which is compared to the corresponding reference compound (benzo(a)pyrene). The concentration is expressed as toxic equivalents (TEQ) or bioanalytical equivalents (BEQ).

Test duration

• Exposure time 6 h

Relevance

• The PAH CALUX indicates the presence of polycyclic aromatic hydrocarbons (PAH).

Guidelines and literature

• Pieterse B, Felzel E, Winter R, van der Burg B, Brouwer A (2013) PAH-CALUX, an
  optimized bioassay for carcinogenic hazard identification of polycyclic aromatic
  hydrocarbons (PAHs) as individual compounds and in complex mixtures. Environ Sci
  Technol, 47, 11651-11659.

Test organism

• Human cell line 

Detectable effects (impact)

• The transport of the thyroid hormone thyroxine (T4) by the transporter protein transthyretin (TTR) is disrupted by per- and polyfluorinated alkyl compounds (PFAS).

Test principle

• PFAS interfere with the transport of the natural ligand thyroxine (T4) by the transporter protein transthyretin (TTR). The PFAS CALUX® uses this to measure PFAS compounds independently of their structure.
• The PFAS CALUX consists of a TTR-binding assay in combination with the TRbeta CALUX bioassay. The disruption of TTR binding by T4 is compared to perfluorooctanoic acid (PFOA) as a reference compound and expressed as PFOA equivalents/g processed sample.
• Sample preparation and purification are simple and allow successful application to a wide range of matrices.

Test duration

• 42 h

Relevance

• The PFAS CALUX shows the presence of numerous per- and polyfluorinated alkyl compounds (PFAS).

Guidelines and literature

• Behnisch P, Besselink H, Weber R, Willand W, Huang J, Brouwer A. (2021) Developing
  potency factors for thyroid hormone disruption by PFASs using TTR-TRβ CALUX®
  bioassay and assessment of PFASs mixtures in technical products. Environment
  International 157, 106791

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.
• By means of lyticase digestion of the yeast cells (L-YES) after exposure, the sensitivity can be increased by about one order of magnitude.
  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

• Download flow chart YES
• Download flow chart L-YES

Video tutorial

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.

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

• 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

• 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.

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.

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.

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.

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

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.

Test organism

• Various microorganisms (bacteria, yeast, see information on Yeast estrogen screen, umuC, bacterial luminescence inhibition test)

Detectable effects (impact)

• Several endpoints can be combined with high performance thin-layer chromatography (HPTLC). The Ecotox Centre routinely evaluates inhibition of bacterial luminescence, activation of estrogen receptor (yeast estrogen screen), activation of DNA repair pathways (umuC SOS response test)
• The effects of multiple separated chemicals are detectable simultaneously, linking to individual chemicals or giving a profile of bioactivity

Test principle

• Chemicals in a sample are first chromatographically separated from each other on a HPTLC plate.
• Microorganisms of a desired test system are applied to the plate containing the separated chemicals.
• The plate is sprayed with the substrate, which, in the bioassay, leads to a color change or luminescence.
• The test organisms respond to bioactive chemicals by reduced luminescence or induction of a color change, thereby indicating the location of the chemicals on the HPTLC plate.
• The chromatographic behavior of chemicals can be compared between samples, and to standard chemicals.
• The bioactive bands are extracted from the plate and used for further biological or chemical analyses. For example, the identity of the bioactive substances can be clarified using high-resolution mass spectrometry (LC-HRMS/MS).

Test duration

• 1-3 d (exposure time: 0.5 - 3 h)

Relevance

• Can detect multiple, separated, chemicals simultaneously.
• Can reduce impact of matrix on the test’s specific effect (for example, masking from cytotoxicity).
• HPTLC assays are usually more sensitive than the microtiter based counterparts.

Guidelines and literature

• Bergmann, A. J., Simon, E., Schifferli, A., Schönborn, A., & Vermeirssen, E. L. M. (2020). Estrogenic activity of food contact materials - evaluation of 20 chemicals using a yeast estrogen screen on HPTLC or 96-well plates. Analytical and Bioanalytical Chemistry, 412, 4527-4536. doi.org/10.1007/s00216-020-02701-w 
• Bergmann, A. J., Breitenbach, M., Muñoz, C., Simon, E., McCombie, G., Biedermann, M., Schönborn, A., Vermeirssen, E. L. M. (2023). Towards detecting genotoxic chemicals in food packaging at thresholds of toxicological concern using bioassays with high-performance thin-layer chromatography. Food Packaging and Shelf Life, 36, 101052 (11 pp.). doi.org/10.1016/j.fpsl.2023.101052 
• Bergmann, A. J., Masset, T., Breider, F., Dudefoi, W., Schirmer, K., Ferrari, B. J. D., & Vermeirssen, E. L. M. (2024). Estrogenic, genotoxic, and antibacterial effects of chemicals from cryogenically milled tire tread. Environmental Toxicology and Chemistry. doi.org/10.1002/etc.5934 

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

• Download flow chart test

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.

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.

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.

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 (2008) Water quality -- Determination of chronic toxicity to Ceriodaphnia dubia. ISO 20665:2008. 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.

Test organism

• Zebrafish embryos (Danio rerio) aged 0-5 days

Detectable effects (impact)

• The mortality of the embryos is assessed.

Test principle

• Freshly fertilised zebrafish eggs are exposed to the test chemical for 96 hours.
• Four parameters are recorded every 24 hours as indicators of mortality: the coagulation of the fertilised eggs, the absence of somite formation, the absence of tail detachment from the yolk sac and the absence of a heartbeat.
• At the end of the exposure period, acute toxicity is determined based on at least one of the four mortality parameters and the LC50 is calculated.

Test duration

• Exposure time 96 h

Relevance

• The use of zebrafish (Danio rerio) embryos for the prediction of acute fish toxicity is a valuable and increasingly accepted alternative to animal testing with juvenile and adult fish. Numerous studies have demonstrated excellent comparability between zebrafish embryo toxicity data and acute toxicity in fish (OECD 203) has been demonstrated.

Literature

• OECD (2013), Test No. 236: Fish Embryo Acute Toxicity (FET) Test, OECD Guidelines for the Testing of Chemicals, Section 2, OECD Publishing, Paris, https://doi.org/10.1787/9789264203709-en.