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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The analysis has been run on one of the "Rape oil, sulfated, sodium salt" representative sample.
Please, see section 13.2 for further details justifing the read-across approach from this supporting substance.
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Nominal loadings: 0, 1.0, 3.16, 10.0, 31.6 and 100.0 mg/mL
- Sampling method: Samples taken from each test vessel at test start (0 hours) and test end (after 72 hours)
- Sample storage conditions before analysis: in original contyainer, sat room temperature, protected from light.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: In view of the difficulties associated with poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed, exposing the organisms to a Water Accommodated Fraction (WAF) of the test item, as endorsed by several regulatory authorities in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted solvents. Aqueous media were prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. At the completion of mixing and following a 1-hour settlement period, the test item phase was separated by siphon and the test organisms exposed to the aqueous phase, the WAF (which may contain dissolved and/or suspended and/or emulsified fractions of the test item mixture). Exposures were expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of the test item in the WAF.

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicata CHODAT SAG 86.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): 4-days
- Method of cultivation at test facility: Fresh stocks are prepared every month on Z-Agar. Light intensity amounts to 35-70 µE · m-2 · s-1 for 24 h per day.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): yes, OECD medium
- Any deformed or abnormal cells observed: No
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No
Hardness:
Nominal hardness of 0.24 mmol Ca+Mg/L
Test temperature:
min. 21 deg C; max 23 deg C
pH:
The pH-value at the beginning of the test was measured in one additional replicate per loading level and the control. At the end of the exposure, it was measured in pooled replicates per loading and the control.
At test-start the values were between 7.89 and 7.91 for the test concentrations and 8.00 for the control.
At test-end ther values were between 8.10 to 8.29 for the test concentrations and 8.28 for the control.
Dissolved oxygen:
No data
Salinity:
According to the guideline - OECD medium used
Nominal and measured concentrations:
Nominal concentrations : 0, 1.0, 3.16, 10.0, 31.6 and 100.0 mg/L

Measured concentrations : At start of the test (0 hr), ; at end of the test (72 hr), mg/L respectively
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile 250 mL Erlenmeyer flask, sealed with cotton wool plugs, allowing ventilation.
- Material, size, headspace, fill volume: 100 mL OECD medium in a 250 mL Erlenmeyer flask
- Aeration: Rotary shaker at approximately 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A; static
- Renewal rate of test solution (frequency/flow rate): N/A
- Initial cells density: Nominal: 2E3-5E3 cells/mL; Actual: 4332 cells/mL
- Control end cells density: Control mean cell density increased 196-fold after 72 h
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes - OECD medium
- Detailed composition if non-standard medium was used: N/A

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium
- Intervals of water quality measurement: 0 & 72h

OTHER TEST CONDITIONS
- Sterile test conditions: yes - filtered before use
- Adjustment of pH: yes. All test concentrations were acidifed with 2 N HCl (150 µL/30mL)
- Photoperiod: 24 hours/day light
- Light intensity and quality: Nominal 60 - 120 µE · m-2 · s-1; Actual min. 60.2 µE · m-2 · s-1; max 72.2 µE · m-2 · s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Growth inhibition at 0, 24, 48 & 72h,
- Determination of cell concentrations: Fluorescence measurements at 0, 24, 48 and 72 hours. Chlorophyll-a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal

TEST CONCENTRATIONS
- Spacing factor for test concentrations: geometric series with a factor of square-root of 10 (3.16)
- Justification for using less concentrations than requested by guideline: N/A
- Range finding study: Yes
- Test concentrations: 1.0, 10.0 and 100.0 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
17.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 13.7-22.6mg/L
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
3.16 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
70.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 66.1 - 75.1 mg/L
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
3.16 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: WAF approach used
- Effect concentrations exceeding solubility of substance in test medium: Yes
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 0.59 mg/L (0.564-0.623 mg/L) - based on growth rate inhibitrion, 72h, nominal concentration
- EC50: 0.299 mg/L (0.278-0.324 mg/L) - based on yield inhibition, 72h, nominal concentration
Reported statistics and error estimates:
The percentage inhibitions of the mean growth rate or yield based on the mean growth rate or yield of the control were calculated for each loading rate. The values which are close to x% were used for the determination of the ELx via linear interpolation using a plot with a logarithmic scale for the concentration versus the percentage of inhibition.
Dunnett’s test (one-sided) was carried out at a 95 % significance level, based on the assumption that each higher tested concentration must have at least the same or a stronger effect than the LOELR.

 

 

 

 

 

 

 

 

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions in which this study was conducted, using the WAF approach endorsed for complex substances with a low solubility, the EL50 values for inhibition of growth rate (ErL50) and yield (EyL50) resulted to be 70.4 and 17.4mg/L, respectively. The NOELR for inhibition of growth rate (ErC0) and yield (EyC0) was 3.16 mg/L, the LOELR 10.0 mg/L.
Executive summary:

Algal growth inhibition on an analogue of the substance (Rape oil, sulfated, sodium salt) has been investigated in a 72-hour test according to OECD/EU test method (OECD 201, 2011), with Desmodesmus subspicatus. The WAF apporach for complex substances with a low solubility has been followed. The EL50 values for inhibition of growth rate (ErC50) and yield (EyC50) were 70.4 and 17.4 mg/L, respectively. The NOELR for inhibition of growth rate (ErC0) and yield (EyC0) was 3.16 mg/L, the LOELR 10.0 mg/L.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Nominal loadings: 0, 1.0, 3.16, 10.0, 31.6 and 100.0 mg/mL
- Sampling method: Samples taken from each test vessel at test start (0 hours) and test end (after 72 hours)
- Sample storage conditions before analysis: in original contyainer, sat room temperature, protected from light.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: In view of the difficulties associated with poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed, exposing the organisms to a Water Accommodated Fraction (WAF) of the test item, as endorsed by several regulatory authorities in cases where the test item is a complex mixture and is poorly soluble in water and in the permitted solvents. Aqueous media were prepared by mixing the test item with water for a prolonged period sufficient to ensure equilibration between the test item and the water phase. At the completion of mixing and following a 1-hour settlement period, the test item phase was separated by siphon and the test organisms exposed to the aqueous phase, the WAF (which may contain dissolved and/or suspended and/or emulsified fractions of the test item mixture). Exposures were expressed in terms of the original concentration of the test item in water at the start of the mixing period (loading rate) irrespective of the actual concentration of the test item in the WAF.

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicata CHODAT SAG 86.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): 4-days
- Method of cultivation at test facility: Fresh stocks are prepared every month on Z-Agar. Light intensity amounts to 35-70 µE · m-2 · s-1 for 24 h per day.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): yes, OECD medium
- Any deformed or abnormal cells observed: No
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No
Hardness:
Nominal hardness of 0.24 mmol Ca+Mg/L
Test temperature:
min. 21 deg C; max 23 deg C
pH:
The pH-value at the beginning of the test was measured in one additional replicate per loading level and the control. At the end of the exposure, it was measured in pooled replicates per loading and the control.
At test-start the values were between 7.89 and 7.91 for the test concentrations and 8.00 for the control.
At test-end ther values were between 8.10 to 8.29 for the test concentrations and 8.28 for the control.
Dissolved oxygen:
No data
Salinity:
According to the guideline - OECD medium used
Nominal and measured concentrations:
Nominal concentrations : 0, 1.0, 3.16, 10.0, 31.6 and 100.0 mg/L

Measured concentrations : At start of the test (0 hr), ; at end of the test (72 hr), mg/L respectively
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterile 250 mL Erlenmeyer flask, sealed with cotton wool plugs, allowing ventilation.
- Material, size, headspace, fill volume: 100 mL OECD medium in a 250 mL Erlenmeyer flask
- Aeration: Rotary shaker at approximately 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A; static
- Renewal rate of test solution (frequency/flow rate): N/A
- Initial cells density: Nominal: 2E3-5E3 cells/mL; Actual: 4332 cells/mL
- Control end cells density: Control mean cell density increased 196-fold after 72 h
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes - OECD medium
- Detailed composition if non-standard medium was used: N/A

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: OECD medium
- Intervals of water quality measurement: 0 & 72h

OTHER TEST CONDITIONS
- Sterile test conditions: yes - filtered before use
- Adjustment of pH: yes. All test concentrations were acidifed with 2 N HCl (150 µL/30mL)
- Photoperiod: 24 hours/day light
- Light intensity and quality: Nominal 60 - 120 µE · m-2 · s-1; Actual min. 60.2 µE · m-2 · s-1; max 72.2 µE · m-2 · s-1

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Growth inhibition at 0, 24, 48 & 72h,
- Determination of cell concentrations: Fluorescence measurements at 0, 24, 48 and 72 hours. Chlorophyll-a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal

TEST CONCENTRATIONS
- Spacing factor for test concentrations: geometric series with a factor of square-root of 10 (3.16)
- Justification for using less concentrations than requested by guideline: N/A
- Range finding study: Yes
- Test concentrations: 1.0, 10.0 and 100.0 mg/L
- Results used to determine the conditions for the definitive study: yes
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
17.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 13.7-22.6mg/L
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
3.16 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
70.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 66.1 - 75.1 mg/L
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
3.16 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: WAF approach used
- Effect concentrations exceeding solubility of substance in test medium: Yes
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 0.59 mg/L (0.564-0.623 mg/L) - based on growth rate inhibitrion, 72h, nominal concentration
- EC50: 0.299 mg/L (0.278-0.324 mg/L) - based on yield inhibition, 72h, nominal concentration
Reported statistics and error estimates:
The percentage inhibitions of the mean growth rate or yield based on the mean growth rate or yield of the control were calculated for each loading rate. The values which are close to x% were used for the determination of the ELx via linear interpolation using a plot with a logarithmic scale for the concentration versus the percentage of inhibition.
Dunnett’s test (one-sided) was carried out at a 95 % significance level, based on the assumption that each higher tested concentration must have at least the same or a stronger effect than the LOELR.

 

 

 

 

 

 

 

 

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions in which this study was conducted, using the WAF approach endorsed for complex substances with a low solubility, the EL50 values for inhibition of growth rate (ErL50) and yield (EyL50) resulted to be 70.4 and 17.4mg/L, respectively. The NOELR for inhibition of growth rate (ErC0) and yield (EyC0) was 3.16 mg/L, the LOELR 10.0 mg/L.
Executive summary:

Algal growth inhibition has been investigated in a 72-hour test according to OECD/EU test method (OECD 201, 2011), with Desmodesmus subspicatus. The WAF apporach for complex substances with a low solubility has been followed. The EL50 values for inhibition of growth rate (ErC50) and yield (EyC50) were 70.4 and 17.4 mg/L, respectively. The NOELR for inhibition of growth rate (ErC0) and yield (EyC0) was 3.16 mg/L, the LOELR 10.0 mg/L.

Description of key information

Toxicity to aquatic algae: EC50 -100 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
70 mg/L

Additional information

Algal growth inhibition has been investigated in a 72-hour test according to OECD/EU test method (OECD 201, 2011), with Desmodesmus subspicatus, on Rape oil, sulfated, sodium salt - an analogue of the substance. The WAF approach for complex substances with a low solubility has been followed. The EL50 values for inhibition of growth rate (ErC50) and yield (EyC50) were 70.4 and 17.4 mg/L, respectively. The NOELR for inhibition of growth rate (ErC0) and yield (EyC0) was 3.16 mg/L, the LOELR 10.0 mg/L.