Stephan Fischer
Division Head Water Quality
stephan.fischer@aquatox-solutions.ch
Phone: +41 (0)71 571 09 72
ACUTE FISH TOXICITY ASSESSMENT
CHEMICAL ANALYSIS
Stephan Fischer
Division Head Water Quality
stephan.fischer@aquatox-solutions.ch
Phone: +41 (0)71 571 09 72
ACUTE FISH TOXICITY ASSESSMENT
CHRONIC (SUB-LETHAL) TOXICITY
CHEMICAL ANALYSIS
ACUTE FISH TOXICITY ASSESSMENT
ACUTE FISH TOXICITY ASSESSMENT
ISO 21115 - Determination of acute toxicity of water samples and chemicals to a fish gill cell line (RTgill-W1)
The RTgill-W1 (rainbow trout (Oncorhynchus mykiss) gill) cell line assay allows the prediction of acute fish toxicity based on three fluorescent indicator dyes. The assay is performed in 24-well plates in which the water sample is tested in a series of six dilutions containing 100%, 80%, 60%, 40%, 20% and 10% of the water sample. The results are expressed as % cell viability compared to an untreated control and the LID (lowest ineffective dilution) value is calculated.
Furthermore, a second 24-well plate is exposed with 100% of the water sample and spiked with the standard concentration range of the positive control 3,4-DCA. This additional exposure is used to test for potential interferences of the matrix of the water sample with chemicals.
ISO 21115 - Determination of acute toxicity of water samples and chemicals to a fish gill cell line (RTgill-W1)
The RTgill-W1 (rainbow trout (Oncorhynchus mykiss) gill) cell line assay allows the prediction of acute fish toxicity based on three fluorescent indicator dyes. The assay is performed in 24-well plates in which the water sample is tested in a series of six dilutions containing 100%, 80%, 60%, 40%, 20% and 10% of the water sample. The results are expressed as % cell viability compared to an untreated control and the LID (lowest ineffective dilution) value is calculated.
Furthermore, a second 24-well plate is exposed with 100% of the water sample and spiked with the standard concentration range of the positive control 3,4-DCA. This additional exposure is used to test for potential interferences of the matrix of the water sample with chemicals.
Extended OECD TG236 –Fish Embryo Acute Toxicity (FET) Test
Freshly fertilized zebrafish eggs are exposed to a dilution series of the water sample for a period of 120 h. Every 24 h, up to five apical observations are recorded as indicators of lethality: coagulation of fertilized eggs, lack of somite formation, lack of detachment of the tail-bud from the yolk sac, and lack of heartbeat. At the end of the exposure period, acute toxicity is determined based on a positive outcome in any of the five apical observations recorded, and the % mortality, LC50, LID (lowest ineffective dilution) value are calculated.
Scoring of apical observations resembling sub-lethal effects, such as occurrence of edema, malformations, deformations or uncontrolled movements are routinely included in the test performance by aQuaTox-Solutions, with the outcome being % sub-lethal effects, EC50 and LID value.
aQuaTox-Solutions GmbH has adapted the FET test specifically for testing water / wastewater samples and extended the test duration to 120 h. As a result, sub-lethal effects and indication for neurotoxic effects (altered behavior such as trembling, uncontrolled movement or abnormal reaction to a trigger) that may occur can be better assessed compared to the standard 96 h test duration.
Extending exposure to 120 h is not legally considered an animal experiment in jurisdictions such as Europe, in which animal protection laws exclude early life stages until independent feeding.
Extended OECD TG236 –Fish Embryo Acute Toxicity (FET) Test
Freshly fertilized zebrafish eggs are exposed to a dilution series of the water sample for a period of 120 h. Every 24 h, up to five apical observations are recorded as indicators of lethality: coagulation of fertilized eggs, lack of somite formation, lack of detachment of the tail-bud from the yolk sac, and lack of heartbeat. At the end of the exposure period, acute toxicity is determined based on a positive outcome in any of the five apical observations recorded, and the % mortality, LC50, LID (lowest ineffective dilution) value are calculated.
Scoring of apical observations resembling sub-lethal effects, such as occurrence of edema, malformations, deformations or uncontrolled movements are routinely included in the test performance by aQuaTox-Solutions, with the outcome being % sub-lethal effects, EC50 and LID value.
aQuaTox-Solutions GmbH has adapted the FET test specifically for testing water / wastewater samples and extended the test duration to 120 h. As a result, sub-lethal effects and indication for neurotoxic effects (altered behavior such as trembling, uncontrolled movement or abnormal reaction to a trigger) that may occur can be better assessed compared to the standard 96 h test duration.
Extending exposure to 120 h is not legally considered an animal experiment in jurisdictions such as Europe, in which animal protection laws exclude early life stages until independent feeding.
ISO 15088 - Determination of the acute toxicity of waste water to zebrafish eggs (Danio rerio)
Freshly fertilised zebrafish eggs are exposed to the water/effluent samples for a period of 48h. After 24h and 48h, four apical observations are recorded as indicators of lethality: coagulation of fertilised eggs, lack of somite formation, lack of detachment of the tail-bud from the yolk sac, and lack of heartbeat. At the end of the exposure period, acute toxicity is determined based on a positive outcome in any of the four apical observations recorded and the lowest ineffective dilution (LID – dilution at which 90% of the fertilised eggs survive) is given as test result.
Comparison of water quality test options:
ISO 15088 - Determination of the acute toxicity of waste water to zebrafish eggs (Danio rerio)
Freshly fertilised zebrafish eggs are exposed to the water/effluent samples for a period of 48h. After 24h and 48h, four apical observations are recorded as indicators of lethality: coagulation of fertilised eggs, lack of somite formation, lack of detachment of the tail-bud from the yolk sac, and lack of heartbeat. At the end of the exposure period, acute toxicity is determined based on a positive outcome in any of the four apical observations recorded and the lowest ineffective dilution (LID – dilution at which 90% of the fertilised eggs survive) is given as test result.
Comparison of water quality test options:
CHRONIC (SUB-LETHAL) TOXICITY
CHRONIC (SUB-LETHAL) TOXICITY
Gene Expression
We have an ever expanding list of primers to analyse the expression of a wide variety of genes, such as related to biotransformation, metal detoxification, cell proliferation, oxidative stress response and immune regulation. We frequently add new primers based on clients’ needs.
Gene expression analysis can be performed in fish cell lines as well as zebrafish embryos, using a concentration-response, as well as time-resolved, experimental design. These tests are usually preceded by an acute fish toxicity test with either the cell line of choice or the embryos to ensure that gene expression is indeed studied at non-acutely toxic concentrations.
Concentration-dependent gene expression analysis allows the derivation of Points-Of-Departure (POD), which are increasingly used to derive estimated low effect levels for chronic toxicity assessments.
Gene Expression
We have an ever expanding list of primers to analyse the expression of a wide variety of genes, such as related to biotransformation, metal detoxification, cell proliferation, oxidative stress response and immune regulation. We frequently add new primers based on clients’ needs.
Gene expression analysis can be performed in fish cell lines as well as zebrafish embryos, using a concentration-response, as well as time-resolved, experimental design. These tests are usually preceded by an acute fish toxicity test with either the cell line of choice or the embryos to ensure that gene expression is indeed studied at non-acutely toxic concentrations.
Concentration-dependent gene expression analysis allows the derivation of Points-Of-Departure (POD), which are increasingly used to derive estimated low effect levels for chronic toxicity assessments.
Monitoring Water Quality via fish tissue samples
aQuaTox-Solutions can also use fish tissue to assess water quality. Fish tissue has to be provided, aQuaTox is not performing any experiments with fish.
Via Gene Expression Analysis the following points can be assessed:
- Effects of waste water treatment plant (WWTP) effluents, by analyzing fish from upstream and downstream of WWTP
- Effects of pollutant gradients in surface waterbodies, by analyzing fish caught from different distances from the contamination source
- Effects of pollutants in small surface waterbodies, such as lakes or ponds, by analyzing fish from the target site and a defined reference site or lab controls
Furthermore, gene expression analysis could serve as an additional end point if tissue samples are provided from tests, such as OECD 203: Fish Acute Toxicity Test, OECD 210: Fish Early-life Stage Toxicity Test, and OECD 212: Fish Short-term Toxicity Test on Embryo and Sac-fry Stages.
Monitoring Water Quality via fish tissue samples
aQuaTox-Solutions can also use fish tissue to assess water quality. Fish tissue has to be provided, aQuaTox is not performing any experiments with fish.
Via Gene Expression Analysis the following points can be assessed:
- Effects of waste water treatment plant (WWTP) effluents, by analyzing fish from upstream and downstream of WWTP
- Effects of pollutant gradients in surface waterbodies, by analyzing fish caught from different distances from the contamination source
- Effects of pollutants in small surface waterbodies, such as lakes or ponds, by analyzing fish from the target site and a defined reference site or lab controls
Furthermore, gene expression analysis could serve as an additional end point if tissue samples are provided from tests, such as OECD 203: Fish Acute Toxicity Test, OECD 210: Fish Early-life Stage Toxicity Test, and OECD 212: Fish Short-term Toxicity Test on Embryo and Sac-fry Stages.
CHEMICAL ANALYSIS
CHEMICAL ANALYSIS
Information
We collaborate with well established contract research organisations who specialize in chemical analysis, including under GLP. If chemical analysis is desired, e.g. to verify exposure concentrations or biotransformation products, we offer bundled packages, which include co-ordination for a streamlined workup and reporting.
Samples can also be shipped to a laboratory of the client’s choice or for in-house analysis.
Information
We collaborate with well established contract research organisations who specialize in chemical analysis, including under GLP. If chemical analysis is desired, e.g. to verify exposure concentrations or biotransformation products, we offer bundled packages, which include co-ordination for a streamlined workup and reporting.
Samples can also be shipped to a laboratory of the client’s choice or for in-house analysis.