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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From November 11, 1999 to January 07, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
The concentration and stability of the test substance and its oxidation product Diammonium Dithiodilactak in the test solutions were verified by chemical analysis at 0 and 72 h.
Vehicle:
no
Details on test solutions:
For the purpose of the definitive study, the test substance was prepared by a direct solution in culture medium.
400 mg of the test substance 70 % was dissolved in culture medium and the volume was adjusted to 1 L to give a 400 mg/L stock solution from which serial dilutions were made to give 200, 100, 50 and 25 mg/L stock solutions. An aliquot (500 mL) of each of these stock solutions was separately inoculated with algal suspension (500 mL) to give the required test concentrations of 12.5, 25, 50, 100 and 200 mg/L.
Test organisms (species):
Scenedesmus sp.
Details on test organisms:
The test was carried out using Scenedesmus subspicatus strain CCAP 276/20. Liquid cultures of Scenedesmus subspicatus were obtained from the Culture Centre for Algae and Protozoa (CCAP), Institute of Freshwater Ecology, Ferry House, Ambleside, Cumbria. Cultures were maintained in the laboratory by the periodic replenishment of culture medium. The culture was maintained in the laboratory at a temperature of 21 ± 1 °C under continuous illumination (intensity approximately 7000 lux) and constant aeration.

Test type:
static
Water media type:
other: deionised water Elga Optima 15+
Limit test:
no
Total exposure duration:
72 h
Test temperature:
24 ± 1 °C
pH:
The pH values of the control cultures were observed to increase from pH 7.4 at 0 hours to pH 8.2 at 72 h.
The pH of the test substance vessels at 0 hours decreased with increasing test substance concentration, this is considered to be due to the acidic nature of the test substance.
Nominal and measured concentrations:
Range finding study: 0.010, 0.10, 1.0, 10 and 100 mg/L;
Main study: 12.5, 25, 50, 100 and 200 mg/L.
Details on test conditions:
Range-finding study:

Nominal test concentrations: 0.010, 0.10, 1.0, 10 and 100 mg/L.
Design:
- The study was conducted in 250 ml glass conical flasks loosely covered with aluminium foil to reduce evaporation. Two replicate flasks were prepared for each control and test concentration. The test substance was dissolved directly in culture medium. The control group was maintained under identical conditions but not exposed to the test substance.
- At the start of the range-finding study a sample of each test and control culture was removed and the cell density determined using a Coulter Multisizer II Particle Counter. The flasks were then covered with aluminium foil and incubated (GaHenkamp INR - 401-0IOW) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) and constantly shaken at 100 rpm for 72 hours.
- After 72 hours the cell density of each flask was determined using a Coulter® Multisizer II Particle Counter.

Main study:

Nominal test concentrations: 12.5, 25, 50, 100 and 200 mg/L.
Design:
- As in the range-finding study 250 mL glass conical flasks were used. Three flasks each containing 100 mL of solution were prepared for the control, and each treatment group. The control group was maintained under identical conditions but not exposed to the test substance.
- Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 1.25+E6 cells per mL. This suspension was diluted to a cell density of 2.77+E4 cells per mL prior to use. At initiation of the study the culture contained a nominal cell density of 10+E4 cells per mL.
- The flasks were plugged with polyurethane foam bungs and incubated (Gallenkamp INR-401-01 OW) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) and constantly shaken at 100 rpm for 72 h. Polyurethane foam bungs were used to plug the flasks in the definitive study rather than aluminium foil in order to enhance the transfer of CO2 from the atmosphere into the test vessels.
- Samples were taken at 0, 24, 48 and 72 h and the cell densities determined by using a Coulter Multisizer II Particle Counter.

Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
150 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
12.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
Range finding study:

The results showed no effect on growth at the test concentrations of 0.010, 0.10, 1.0 and 10 mg/L. However, inhibition of growth (28 %) was observed at 100 mg/L. Based on this information test concentrations of: 12.5, 25, 50, 100, and 200 mg/L were selected for the definitive study.

Definitive study:

Both the growth (r) and the biomass (b) of Scenedesmus subspicatus (CCAP 276/20) were affected by the presence of the test substance over the 72 h exposure period.
- The EC50 value with respect to biomass, Eb50 (72 h), was determined by inspection of are under the growth curve data after 72 h.
- The EC50 value with respect to growth rate, ErC50 (0 - 72 h), was determined by inspection of the growth rates for the period 0 - 72 hours.

Statistical analysis of the area under the growth curve data was carried out for the control and all test concentrations using one way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control (1 and 2). There were no statistically significant differences between the control and 12.5 mg/L test concentration (P<0.05), however all other test concentrations were significantly different (P<0.05) and, therefore the "No Observed Effect Concentration" (NOEC) is 12.5 mg/L.
The following data show that the cell concentration of the control cultures increased by a factor of 21 during the test in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 h.

Mean cell density of control at O hours Mean cell density of control at 72 h:
Control after 0 h: 1.32 x 104 cells per mL
Control after 72 h: 2.74 x 105 cells per mL
All test and control cultures were inspected microscopically at 72 h. There were no abnormalities detected in any of the control or test cultures.
Reported statistics and error estimates:
One way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple· comparison procedure for comparing several treatments with a control (1955) (1 and 2) was carried out on the area under the growth curve data at 72 h for the control, and all test concentrations to determine any statistically significant differences between the test and control groups.

Analysis of the test solutions at 0 hours showed the measured test concentrations of the parent test substance range from 75 % to 99 % of nominal. The range of measured test concentrations followed a concentration dependent pattern with the lower measured test concentrations being determined from the lower nominal test concentrations. This effect is considered to be due to the unstable nature of the test substance in algal culture medium as indicated by the pre-study stability analyses performed. No significant levels of the oxidation product, DADTL, were detected the 0 hour test samples.

Analysis of the 72 hour test samples showed the concentration of DADTL to range from 6.45 mg/L to 98.5 mg/L thereby confirming that approximately 50 % of the parent test substance oxidised over the study period. However due to chromatographic interference from an unknown biological source which 'masked the test material peak on the chromatography, it was not possible to calculate the concentration of parent test substance remaining in the 72-hour test samples.

Given that the test substance was shown to oxidise over the study period, the toxicity cannot be attributed to the parent test substance alone but to the test substance/oxidation product mixture as a whole. As such, EC50 values cannot be calculated based on the measured test concentrations of the parent test substance or oxidation product and therefore the EC50 values given have been calculated based on nominal test concentrations alone.

Validity criteria fulfilled:
yes
Conclusions:
Under the study conditions, EC50 values could be calculated based on the measured test concentrations of the parent test substance or oxidation product and therefore the EC50 values given have been calculated based on nominal test concentrations alone. Exposure ofScenedesmus subspicatusto the test substance gave a nominal 72 h EbC50 value of 150 mg/L and a 72 h ErC50 value greater than 200 mg/L. The NOEC was 12.5 mg/L.
Executive summary:

A study was conducted to determine the toxicity of the test substance (Ammonium 2-mercaptopropionate) against Scenedesmus subspicatus according to OECD Guideline 201 and EU Method A.C. Following a preliminary range-finding study, Scenedesmus subspicatus was exposed to an aqueous solution of the test substance at concentrations of 12.5, 25, 50, 100 and 200 mg/L (three replicate flasks per concentration) for 72 h, under constant illumination and shaking at a temperature of 24 ± 1°C. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter Multisizer II Particle Counter. The test substance oxidises in water to form diammonium dithiodilactate, therefore the samples taken from the definitive study were analysed for both test substance and oxidation product concentration at 0 and 72 h. Analysis of the test solutions at 0 h showed the measured test concentrations of the parent test substance to range from 75 to 99% of nominal. No significant levels of the oxidation product, DADTL, were detected the 0 h test samples. Analysis of the 72 h test samples showed the concentration of DADTL to range from 6.45 to 98.5 mg/L thereby confirming that approximately 50% of the parent test substance oxidised over the study period. However due to chromatographic interference from an unknown biological source which ‘masked’ the test substance peak on the chromatography, it was not possible to calculate the concentration of parent test substance remaining in the 72 h test samples. Under the study conditions, EC50 values could be calculated based on the measured test concentrations of the parent test substance or oxidation product and therefore the EC50 values given have been calculated based on nominal test concentrations alone. Exposure ofScenedesmus subspicatusto the test substance gave a nominal 72 h EbC50 value of 150 mg/L and a 72 h ErC50 value greater than 200 mg/L. The NOEC was 12.5 mg/L (Mead, 2000).

Description of key information

Key value for chemical safety assessment

EC50 for freshwater algae:
200 mg/L
EC10 or NOEC for freshwater algae:
12.5 mg/L

Additional information

A study was conducted to determine the toxicity of the test substance (Ammonium 2-mercaptopropionate) against Scenedesmus subspicatus according to OECD Guideline 201 and EU Method A.C. Following a preliminary range-finding study, Scenedesmus subspicatus was exposed to an aqueous solution of the test substance at concentrations of 12.5, 25, 50, 100 and 200 mg/L (three replicate flasks per concentration) for 72 h, under constant illumination and shaking at a temperature of 24 ± 1°C. Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group, using a Coulter Multisizer II Particle Counter. The test substance oxidises in water to form diammonium dithiodilactate, therefore the samples taken from the definitive study were analysed for both test substance and oxidation product concentration at 0 and 72 h. Analysis of the test solutions at 0 h showed the measured test concentrations of the parent test substance to range from 75 to 99% of nominal. No significant levels of the oxidation product, DADTL, were detected the 0 h test samples. Analysis of the 72 h test samples showed the concentration of DADTL to range from 6.45 to 98.5 mg/L thereby confirming that approximately 50% of the parent test substance oxidised over the study period. However due to chromatographic interference from an unknown biological source which ‘masked’ the test substance peak on the chromatography, it was not possible to calculate the concentration of parent test substance remaining in the 72 h test samples. Under the study conditions, EC50 values could be calculated based on the measured test concentrations of the parent test substance or oxidation product and therefore the EC50 values given have been calculated based on nominal test concentrations alone. Exposure ofScenedesmus subspicatusto the test substance gave a nominal 72 h EbC50 value of 150 mg/L and a 72 h ErC50 value greater than 200 mg/L. The NOEC was 12.5 mg/L (Mead, 2000).