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EC number: - | CAS number: -
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Toxicity to aquatic algae and cyanobacteria
Administrative data
Link to relevant study record(s)
- Endpoint:
- toxicity to aquatic algae and cyanobacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 08-06-2020 to 06-10-2020
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Guideline study performed under GLP. All relevant validity criteria were met.
- Reason / purpose for cross-reference:
- other:
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 201 (Alga, Growth Inhibition Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method C.3 (Algal Inhibition test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- inspected: July 2016 ; signature: January 2017
- Analytical monitoring:
- yes
- Details on sampling:
- - Concentrations: All relevant concentration levels and the control were analytically verified via GC-MS/MS at the start (0 hours) and at the end of the exposure (72 hours). A saturated solution with a nominal loading rate of 100 mg test item/L was prepared was prepared once at room temperature 48 ± 1 hour prior to the start of the exposure. 5 test item concentrations in a geometric series with a separation factor of 2, were prepared by diluting the stock solution with dilution water as follows: 6.25%, 12.5%, 25.0%, 50.0% and 100% of the saturated solution. For the definitive test: equivalent geometric mean measured concentrations were: 0 (control), 0.0179, 0.0431, 0.0481, 0.0597 and 0.221 µg/L (or 17.9, 43.1, 48.1, 59.7 and 221 ng/L) which were based on analysis during the definitive test period.
- Sampling method: Analytical evaluation of the concentrations of the test item were carried out via GC-MS/MS from freshly prepared media after 0 hours (with algae) and old test media after 72 hours (with algae) of exposure. Three replicates per concentration and six for the control (without test item) were prepared. Separate replicates for each measuring time were prepared. All samples for the test item analysis were taken from additionally prepared replicates with algae. The method was validated prior to this study according to SANCO 3029/99 rev.4 (2000).
- Sample storage conditions before analysis: All original samples were stored at room temperature (ca. 20 ± 2 °C) before preparation. Prepared samples were stored in an autosampler at room temperature until analysis. - Vehicle:
- no
- Details on test solutions:
- PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Test item exposures was prepared from stock solution. A saturated solution with a nominal loading rate of 100 mg test item/L was prepared was prepared once at room temperature 48 ± 1 hour prior to the start of the exposure. A glass bottle was filled with an appropriate amount of the dilution water. The headspace in the glass flask was minimized. An appropriate volume of the test item was weighed out and placed by pipette onto the water surface and the bottle was closed with the screw cap. A slow stirring procedure was applied. Gentle stirring (to avoid formation of emulsion) was carried out for 48 ± 1 hour with a magnetic stirrer at room temperature. After completion of stirring, the dispersion was allowed to stand for at least 1 hour at room temperature for separation of undissolved test item. Thereafter, the clear water phase was removed by siphoning from the bottom of the glass flask. The test item solution was checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion). The Tyndall effect was negative. The saturated solution was used as highest concentration level and as a stock solution for the preparation of further dilution levels by diluting with dilution water. 5 test item concentrations in a geometric series with a separation factor of 2, were prepared by diluting the stock solution with dilution water as follows: 6.25%, 12.5%, 25.0%, 50.0% and 100% of the saturated solution. For the definitive test: equivalent geometric mean measured concentrations were: 0 (control), 0.0179, 0.0431, 0.0481, 0.0597 and 0.221 µg/L (or 17.9, 43.1, 48.1, 59.7 and 221 ng/L) which were based on analysis during the definitive test period.
- Eluate: Not applicable.
- Differential loading: Not applicable.
- Controls: For positive control - reference item: potassium dichromate were prepared in a separately conducted reference test (documented in the full study report). A negative/blank control without test item or reference item was also included.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Prior to start of the exposure, the test solutions were checked for undissolved test item. Presence of undissolved test item during preparation and during the test was not observed. - Test organisms (species):
- Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
- Details on test organisms:
- TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: HINDÁK, CCAP 278/4 (axenic)
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Dunstaffnage Marine Laboratory, Dunbeg, Oban, Argyll, Scotland, United Kingdom, PA37 1QA
- Method of cultivation: Fresh stocks are prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux for 24 hours per day.
ACCLIMATION
- Acclimation period: No. However, a three days old preculture, prepared in dilution water, was used as inoculum.
- Culturing media and conditions (same as test or not): No. Culture Medium: Nutrient medium Z according to LÜTTGE et al. (1994) Botanica Acta, Journal of the German Botanical Society, No. 3 Volume 107 page 111-186 (June 1994), THIEME-VERLAG.
Dilution water: OECD TG 201: (mg/L) - NH4Cl 15 ; MgCl2.6 H2O: 12 ; CaCl2.2 H2O: 18 ; MgSO4.7H2O: 15 ; KH2PO4: 1.6 ; FeCl3.6H2O: 0.064 ; Na2EDTA.2H2O: 0.1 ; H3BO3: 0.185 ; MnCl2.4H2O: 0.415 ; ZnCl2: 3x10-3 ; Na2MoO4.2H2O: 7x10-3 ; CoCl2.6H2O: 1.5x10-3 ; CuCl2.2H2O: 1x10-5 ; NaHCO3: 50 ; NaHCO3* : 250 ; MES monohydrate*: 2665.6 ; pH 8.1 +/- 0.2. This medium has a nominal hardness of 0.24 mmol Ca+Mg/L.
* additional compounds were added to enable sufficient growth under conditions without headspace.
- Any deformed or abnormal cells observed: None reported. - Test type:
- static
- Water media type:
- freshwater
- Limit test:
- yes
- Total exposure duration:
- 72 h
- Remarks on exposure duration:
- In accordance with the OECD TG 201 guideline.
- Test temperature:
- Nominal range: 21 - 24 °C, controlled at ± 2°C - measured room temperature values were min: 21.5 ; max 23.5 and mean 22.5 °C
- pH:
- 0 hours: pH 8.1 ± 0.1 (and 8.22 in control); 72 hours: pH 9.21-9.42 (definitive test concentrations) and pH 9.46 (controls). pH did not vary more than 1.5 units.
- Nominal and measured concentrations:
- - Preliminary test: Not applicable. See below
- First definitive test: 0 (control), and 100% of the saturated solution ; the results in the first definitive limit test showed a higher inhibition as expected. Therefore, the study had to be repeated as dose response test (Second definitive test)
- Second definitive test (dose-response test) : 0 (control), 6.25%, 12.5%, 25.0%, 50.0% and 100% of the saturated solution
- Equivalent geometric mean measured concentrations (with algae): 0 (control), 0.0179, 0.0431, 0.0481, 0.0597 and 0.221 µg/L (or 17.9, 43.1, 48.1, 59.7 and 221 ng/L) which were based on analysis during the definitive test period.
- Measured concentrations of the test item were in the samples with algae in the range of 0.0322 to 0.897 µg/L at the start of exposure (0 h) and in the range of: < LOQ (Limit of Quantification) to 0.0545 µg/L (and/or 0 to 6% and 20% of the initial test item concentrations) at the end of exposure (72 h). All effect values given were based on geometric mean measured concentrations after 72 hours for samples. - Details on test conditions:
- TEST SYSTEM
- Test vessel: Glass.
- Type: Closed - Static. Sterile headspace flasks
- Material, size, headspace, fill volume: , volume: 59 mL, with aluminium tops with PFTE seals. Minimum headspace.
- Aeration: Vessel shaken continuously. Test containers were placed on a rotary shaker and oscillated at approximately 70 rpm.
- Initial cells density: nominal: 5 x 10^3 - 10^4 and current: 5375 cells/mL
- Control end cells density: First definitive (limit test): Mean (of replicates after 72 hours) 354000 cells/ml (or ca. x66 increase in cell density) ; Second definitive (dose-response test): Mean (of replicates after 72 hours) 390680 cells/ml (or ca. x73 increase in cell density)
- No. of vessels per concentration (replicates): 3 replicates of each test concentration; 1 extra replicate of each test group for sampling purposes
- No. of vessels per control (replicates): 6 replicates of the control
- No. of vessels per vehicle control (replicates): Not applicable.
GROWTH MEDIUM
- Standard medium used: Yes. OECD TG 201 medium. Additionally, compounds were added to enable sufficient growth under conditions without headspace. In accordance to OECD Guidance Document No. 23.
TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Prepared according to guidelines (see 'Details on test organisms' field for more details on composition). Ultrapure water was used to prepare the dilution water (conductivity max. = 0.1 μS/cm)
- Culture medium different from test medium: Yes. (see 'Details on test organisms' field)
- Intervals of water quality measurement: Start and end of the test period.
OTHER TEST CONDITIONS
- Sterile test conditions: No.
- Adjustment of pH: No.
- Photoperiod: 24 hours ; continuous
- Light intensity and quality: 60 - 120 µE/m2/s ; within ± 15 % over incubation area (or 4440 to 8880 lux)
- Salinity (for marine algae): Not applicable.
EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal. No self-fluorescence was found at the highest preliminary test concentration level of 100% saturated solution.
- Other: Initial cell density: Microscopic evaluation of the cells was carried out at the start and end of the exposure. The cells were checked for any unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation and adherence of algae to test containers or aggregation of algae cells.
TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2 In second definitive test justified from the results of the first definitive test (limit test : 0 (control), and 100% of the saturated solution) ; the results in the first definitive limit test showed a higher inhibition as expected. Therefore, the study had to be repeated as dose response test (Second definitive test).
- Justification for using less concentrations than requested by guideline: Not applicable.
- Range finding study: No, however first definitive limit test could be considered a range-finder.
- Test concentrations: limit test : 0 (control), and 100% of the saturated solution
- Results used to determine the conditions for the definitive study: Yes. Second definitive test (dose-response test) : 0 (control), 6.25%, 12.5%, 25.0%, 50.0% and 100% of the saturated solution - Reference substance (positive control):
- yes
- Remarks:
- potassium dichromate
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- > 0.221 µg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Remarks on result:
- other: 95% C.I.: - µg/L ; based on geometric mean measured concentrations ; > 100 mg/L nominal concentration
- Remarks:
- i.e. ErC50 is above the solubility limit
- Key result
- Duration:
- 72 h
- Dose descriptor:
- EC10
- Effect conc.:
- > 0.221 µg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Remarks on result:
- other: 95% C.I.: - µg/L ; based on geometric mean measured concentrations ; > 100 mg/L nominal concentration
- Remarks:
- i.e. ErC10 is above the solubility limit
- Key result
- Duration:
- 72 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 0.221 µg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Remarks on result:
- other: or 100 mg/L nominal concentration
- Remarks:
- i.e. no effects were observed below the solubility limit (set as the NOEC)
- Duration:
- 72 h
- Dose descriptor:
- LOEC
- Effect conc.:
- > 0.221 µg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- growth rate
- Remarks on result:
- other: or 100 mg/L nominal concentration
- Duration:
- 72 h
- Dose descriptor:
- EC50
- Effect conc.:
- > 0.221 µg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- other: yield
- Remarks on result:
- other: 95% C.I.: - µg/L ; based on geometric mean measured concentrations ; > 100 mg/L nominal concentration
- Remarks:
- i.e. EyC50 is above the solubility limit
- Duration:
- 72 h
- Dose descriptor:
- EC10
- Effect conc.:
- 0.1 µg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- other: yield
- Remarks on result:
- other: 95% C.I.: > 0.0597 - < 0.221 µg/L ; based on geometric mean measured concentrations ; 72.4 (C.I. > 50 - < 100) mg/L nominal concentration
- Duration:
- 72 h
- Dose descriptor:
- NOEC
- Effect conc.:
- 0.06 µg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- other: yield
- Remarks on result:
- other: or 50 mg/L nominal concentration
- Duration:
- 72 h
- Dose descriptor:
- LOEC
- Effect conc.:
- 0.221 µg/L
- Nominal / measured:
- meas. (geom. mean)
- Conc. based on:
- test mat.
- Basis for effect:
- other: yield
- Remarks on result:
- other: or 100 mg/L nominal concentration
- Details on results:
- - Exponential growth in the control (for algal test): Yes.
- Observation of abnormalities (for algal test): Microscopic evaluation of the cells at the start of the incubation period and at test end revealed no morphological abnormalities in the test item concentration or control.
- Unusual cell shape: No.
- Colour differences: None.
- Flocculation: Not reported.
- Adherence to test vessels: None reported.
- Aggregation of algal cells: No.
- Other:
- Any stimulation of growth found in any treatment: No.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No precipitate or similar observations reported. At the start of the exposure the measured concentrations were in the range of 0.0322 to 0.897 μg/L. At the end of exposure after 72 hours, the measured concentrations were in the range of < LOQ to 0.0545 μg/L. The analytical values indicate that the sampling points were not completely homogeneous, which can be caused by the low water solubility of the test item, which is in accordance with the results of related studies (Water Solubility and/or Acute Daphnia Toxicity – cited in the full study report). The analysis of additional replicates (not shown) confirmed the inhomogeneous results but also the tendency of declining analytical values at higher diluted test item solutions. Therefore, the values of replicate 1 were used for the evaluation.
- Effect concentrations exceeding solubility of substance in test medium: Yes. See above and below. - Results with reference substance (positive control):
- - Results with reference substance valid?: Yes.
- EC50: The EC50 for growth rate reduction (ERC50: 0-72h) was 1.12 mg/L with a 95% confidence interval ranging from 1.08 to 1.15 mg/L with headspace and 0.949 mg/L with a 95% confidence interval ranging from 0.901 to 0.995 mg/L without headspace. The EC50 for yield inhibition (EYC50: 0-72h) was 0.594 mg/L with a 95% confidence interval ranging from 0.518 to 0.664mg/L with headspace and 0.551 mg/L with a 95% confidence interval ranging from 0.472 to 0.604 mg/L without headspace.
The results with headspace and without headspace were within the test facility SOPs (historic values).
- Other: The sensitivity of the test system was in agreement with the historical data. - Reported statistics and error estimates:
- EC10-, EC20- and EC50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression. The NOEC / LOEC was determined by calculation of statistically significant differences of growth rate and yield using: As a standard, One Way Analysis of Variance (ANOVA) and Dunnett’s test. When running a One Way Analysis of Variance, a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The alpha-value (acceptable probability of incorrectly concluding that there is a difference) is alpha = 0.05.
- Validity criteria fulfilled:
- yes
- Conclusions:
- The test item 72h-ErC50 (growth rate reduction) was > 0.221 µg/L based on geometric mean measured concentrations. The corresponding ErC10 was > 0.271 µg/L and the NOErC was considered to be 0.271 µg/L. No effects on growth rate were seen below the limit of solubility.
- Executive summary:
The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines under GLP. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 5375 cells/mL. A saturated solution with a nominal loading rate of 100 mg test item/L was prepared was prepared once at room temperature 48 ± 1 hour prior to the start of the exposure. A glass bottle was filled with an appropriate amount of the dilution water. The headspace in the glass flask was minimized. An appropriate volume of the test item was weighed out and placed by pipette onto the water surface and the bottle was closed with the screw cap. A slow stirring procedure was applied. Gentle stirring (to avoid formation of emulsion) was carried out for 48 ± 1 hour with a magnetic stirrer at room temperature. After completion of stirring, the dispersion was allowed to stand for at least 1 hour at room temperature for separation of undissolved test item. Thereafter, the clear water phase was removed by siphoning from the bottom of the glass flask.The test item solution was checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion).The Tyndall effect was negative. The saturated solution was used as highest concentration level and as a stock solution for the preparation of further dilution levels by diluting with dilution water. In the definitive, dose-response test: five test item concentrations in a geometric series with a separation factor of 2, were prepared by diluting the stock solution with dilution water as follows: 6.25%, 12.5%, 25.0%, 50.0% and 100% of the saturated solution. For the definitive test: equivalent geometric mean measured concentrations were: 0 (control), 0.0179, 0.0431, 0.0481, 0.0597 and 0.221 µg/L (or 17.9, 43.1, 48.1, 59.7 and 221 ng/L) which were based on analysis during the definitive test period. Glass flasks without headspace were used to reduce losses of the test item. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. The test media were clear throughout the test period. Concentrations were analytically verified via GC-MS/MS at test start and the end of exposure. At the start of the exposure, the measured concentrations were in the range of 0.0322 to 0.897 μg/L. At the end of exposure after 72 hours, the measured concentrations were in the range of < LOQ (0.02 μg/L) to 0.0545 μg/L. Effect concentrations were calculated based on geometric mean measured concentrations. All the relevant validity criteria of the test guideline were fulfilled. The test item 72h-EyC50 (yield) was > > 0.221 µg/L based on geometric mean measured concentrations. The corresponding EyC10 was 0.0998 (C.I. > 0.0597- < 0.221) µg/L. The 72h-ErC50 (growth rate reduction) was > 0.221 µg/L based on geometric mean measured concentrations. The corresponding ErC10 was > 0.271 µg/L and the NOErC was considered to be 0.271 µg/L. Under the conditions of this study, no effects on growth rate were seen below the limit of solubility.
Reference
Table 1. Results of the First Definitive (Limit) Test (0 - 72 hours)
Saturated solution |
Nominal test item loading rate [mg/L] |
Geometric mean measured test item concentration [µg/L] |
Growth Rate Inhibition |
Yield Inhibition |
100 |
100 |
6.92 |
28 |
70 |
Control |
0 |
< LOQ |
- |
- |
|
|
|
|
|
All biological observations are documented in the full study report and all validity criteria were considered to be met.
The results in the first definitive limit test showed an inhibition, therefore requiring a repetition as a dose response test.
Table 2. Percentage reduction in growth rate and inhibition of yield in the Second Definitive (dose response) Test (at 72 hours)
Concentration based on Saturated Solution |
Nominal test item loading rate |
Geometric mean measured test item concentration |
Replicate |
Growth rate |
Growth rate inhibition |
Yield |
Yield Inhibition |
||
[%] |
[mg/L] |
[µg/L] |
No. |
[d-1] |
[%] |
[d-1] |
[%] |
||
100.0 |
100.0 |
0.221 |
1 |
1.38 |
3 |
333621 |
13 |
||
2 |
1.39 |
2 |
345472 |
10 |
|||||
3 |
1.36 |
5 |
308502 |
20 |
|||||
Mean |
(+)* |
1.38 |
4 |
(+) |
329198 |
15 |
|||
50.0 |
50.0 |
0.0597 |
1 |
1.39 |
19 |
343175 |
11 |
||
2 |
1.39 |
20 |
344030 |
11 |
|||||
3 |
1.42 |
18 |
|
376871 |
2 |
||||
Mean |
(-) |
1.40 |
19 |
(-) |
354692 |
8 |
|||
25.0 |
25.0 |
0.0481 |
1 |
1.41 |
5 |
364995 |
5 |
||
2 |
1.40 |
6 |
354097 |
8 |
|||||
3 |
1.39 |
8 |
|
342662 |
11 |
||||
Mean |
(-) |
1.40 |
6 |
(-) |
353918 |
8 |
|||
12.5 |
12.5 |
0.0431 |
1 |
1.41 |
2 |
359180 |
7 |
||
2 |
1.40 |
1 |
357298 |
7 |
|||||
3 |
|
1.40 |
1 |
|
352411 |
9 |
|||
Mean |
(-) |
1.10 |
1 |
(-) |
356296 |
8 |
|||
6.25 |
6.25 |
0.0179 |
1 |
1.39 |
2 |
342833 |
11 |
||
2 |
1.42 |
2 |
373254 |
3 |
|||||
3 |
1.41 |
3 |
360573 |
6 |
|||||
Mean |
(-) |
1.41 |
2 |
(-) |
358887 |
7 |
|||
0% (Control) |
0 (Control) |
0 (Control) |
1 |
1.42 |
369662 |
|
|||
2 |
1.42 |
370567 |
|
||||||
3 |
1.42 |
374501 |
|
||||||
4 |
1.44 |
404385 |
|
||||||
5 |
1.41 |
359107 |
|
||||||
6 |
1.47 |
433609 |
|
||||||
Mean |
1.43 |
385305 |
|
Negative inhibition = increase in growth
n.a. = data not determinable
Statistically significant differences of growth rates and yield compared to control values are marked (+) and non-statistically significant are marked (-)
* = inhibition < 5%, therefore considered not biologically relevant
Table 3. Measured Concentration of test item in the Second Definitive (dose response) Test with Algae
Sampling |
Fresh Media, |
Old Media, |
Geometric mean measured test item concentration |
|
Saturated test item solution |
Meas. conc. [µg/L] |
Meas. conc. [µg/L] |
% |
Meas. conc. [µg/L] |
100 |
0.897 |
0.0545 |
6 |
0.221 |
50 |
0.134 |
0.0266 |
20 |
0.0597 |
25 |
0.231 |
< LOQ |
- |
0.0481 |
12.5 |
0.186 |
< LOQ |
- |
0.0431 |
6.25 |
0.0322 |
< LOQ |
- |
0.0179 |
Control |
< LOQ |
< LOQ |
|
|
PC |
75% / 72% 83% / 80%* |
86% / 83% |
|
Meas. conc. = measured concentration of the test item, dilution factors taken into account
% = percent of the initially measured concentration of the test item
LOQ = limit of quantification of the analytical method (0.02 µg/L of the test item).
For Geometric mean measured test item concentration, 1/2 LOQ was used for the calculation.
PC = Procedural recovery, % recovery calculated to:
weighed amount of the test item / mean value of the method validation.
* Value for 50% saturated solution, since it had to be separately
reanalysed
Table 4. Section-by-Section and Average Specific Growth Rates of the Control Group (0 - 72 hours) in the Second Definitive (dose response) Test
|
Replicate No. |
Specific growth rate [d-1] |
Mean (0 - 72 hours) |
SD ± |
CV |
Mean CV [%] |
||
section-by-section |
||||||||
0 - 24 hours |
24 - 48 hours |
48 - 72 hours |
||||||
Control |
1 |
1.49 |
1.63 |
1.13 |
1.42 |
0.256 |
18.1 |
|
2 |
1.47 |
1.65 |
1.13 |
1.42 |
0.267 |
18.9 |
|
|
3 |
1.52 |
1.68 |
1.06 |
1.42 |
0.321 |
22.6 |
|
|
4 |
1.60 |
1.49 |
1.24 |
1.45 |
0.183 |
12.7 |
|
|
5 |
1.58 |
1.63 |
1.01 |
1.41 |
0.342 |
24.3 |
|
|
6 |
1.54 |
1.58 |
1.28 |
1.47 |
0.165 |
11.3 |
17.9 |
|
|
|
|
Mean |
1.43 |
|
|
||
|
|
|
SD ± |
0.02 |
||||
|
|
|
CV [%] |
1.64 |
SD = Standard deviation
CV = Coefficient of variation
Description of key information
ErC50 (algae; growth rate) = > 0.221 µg/L based on geometric mean measured concentrations, or > 100 mg/L based on nominal loading concentration, 72-hour, freshwater, OECD TG 201, 2020
ErC10 (algae; growth rate) = > 0.221 µg/L based on geometric mean measured concentrations, or > 100 mg/L based on nominal loading concentration, 72-hour, freshwater, OECD TG 201, 2020
NOEC (algae; growth rate) = 0.221 µg/L based on geometric mean measured concentrations, or 100 mg/L based on nominal loading concentration, 72-hour, freshwater, OECD TG 201, 2020
Conclusion : The substance has apparent lower solubility in algal medium OECD TG 201 (2011), than in pure water within as measured in the flask method to EU Method A.6 (2020). This is potentially a result of differences in ionic strength and other parameters between the exposure matrix and that of pure water. The conclusion of the experimental study was that no effects were observed on algal growth rate at up to 100% saturation, with geometric mean measured concentration of 0.221 µg/L. Consequently, it can be concluded the test item has no effects on growth rate below the water solubility limit, observed experimentally.
Key value for chemical safety assessment
Additional information
Key study : OECD TG 202, 2020 : The algal growth inhibition to Pseudokirchneriella subcapitata, was carried out according to OECD TG 201 Freshwater Alga and Cyanobacteria, Growth Inhibition Test and EU Method C.3 guidelines under GLP. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 5375 cells/mL. A saturated solution with a nominal loading rate of 100 mg test item/L was prepared was prepared once at room temperature 48 ± 1 hour prior to the start of the exposure. A glass bottle was filled with an appropriate amount of the dilution water. The headspace in the glass flask was minimized. An appropriate volume of the test item was weighed out and placed by pipette onto the water surface and the bottle was closed with the screw cap. A slow stirring procedure was applied. Gentle stirring (to avoid formation of emulsion) was carried out for 48 ± 1 hour with a magnetic stirrer at room temperature. After completion of stirring, the dispersion was allowed to stand for at least 1 hour at room temperature for separation of undissolved test item. Thereafter, the clear water phase was removed by siphoning from the bottom of the glass flask.The test item solution was checked via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion).The Tyndall effect was negative. The saturated solution was used as highest concentration level and as a stock solution for the preparation of further dilution levels by diluting with dilution water. In the definitive, dose-response test: five test item concentrations in a geometric series with a separation factor of 2, were prepared by diluting the stock solution with dilution water as follows: 6.25%, 12.5%, 25.0%, 50.0% and 100% of the saturated solution. For the definitive test: equivalent geometric mean measured concentrations were: 0 (control), 0.0179, 0.0431, 0.0481, 0.0597 and 0.221 µg/L (or 17.9, 43.1, 48.1, 59.7 and 221 ng/L) which were based on analysis during the definitive test period. Glass flasks without headspace were used to reduce losses of the test item. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. The test media were clear throughout the test period. Concentrations were analytically verified via GC-MS/MS at test start and the end of exposure. At the start of the exposure, the measured concentrations were in the range of 0.0322 to 0.897 μg/L. At the end of exposure after 72 hours, the measured concentrations were in the range of < LOQ (0.02 μg/L) to 0.0545 μg/L. Effect concentrations were calculated based on geometric mean measured concentrations. All the relevant validity criteria of the test guideline were fulfilled. The test item 72h-EyC50 (yield) was > > 0.221 µg/L based on geometric mean measured concentrations. The corresponding EyC10 was 0.0998 (C.I. > 0.0597- < 0.221) µg/L. The 72h-ErC50 (growth rate reduction) was > 0.221 µg/L based on geometric mean measured concentrations. The corresponding ErC10 was > 0.271 µg/L and the NOErC was considered to be 0.271 µg/L. Under the conditions of this study, no effects on growth rate were seen below the limit of solubility.
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