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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
22 - 25 Aug 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
no chemical analysis
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test item (100 mg) and medium (1000 mL) were mixed to prepare the nominal concentration of 100 mg/L. The solution was stirred for 48 h.
- Eluate: no
- Differential loading: no
- Controls: yes, test medium control
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: ATCC 22662
- Source (laboratory, culture collection): American Type Culture Collection (supplied: 30 Jun 1995)
- Method of cultivation: Algae were cultured under sterile conditions.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Test temperature:
22.9 - 23.0 °C
pH:
7.9 - 8.5
Nominal and measured concentrations:
nominal: control, 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Sterilized 300 mL Erlenmeyer flask (with gas-permeable stopper); fill volume: 100 mL
- Aeration: constantly shaken (approx. 100 rpm)
- Initial cells density: 0.75 E+04 cells/mL (equivalent to 2.8 Chlorophyll fluorescence values)
- Control end cells density: Cell density not given; 260 Chrlorophyl lfluorescence value
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: purified water according to guideline
- Culture medium different from test medium: same as test

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous illumination
- Light intensity and quality: 88-91 µmol*m-2*s-1 (wavelength: 400-700 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable)
- Chlorophyll measurement: Chlorophyll was measured using a spectrophotometer every 24 h.

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: control, 0.1, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: No concentration dependant inhibition of growth was recorded. At 100 mg/L a maximum inhibition of growth of 2.8% was measured.
Reference substance (positive control):
yes
Remarks:
Reference item was tested periodically (potassium dichromate)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 100 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
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No precipitation of test substance was recorded.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 0.78 mg/L
- Other: EC50 is within the normal range of the reference substance in the laboratory [mean ± S.D.: 1.0 ± 0.19 mg/L (n=29)].
Reported statistics and error estimates:
F test was done to determine homogeneity of variances. Aspich-Welch t-test was used to estimate the significant difference in comparison with the control.

Table 1: Validity criteria

Criterion from the guideline

Outcome

Validity criterion fulfilled

The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.

 > 79

 yes

The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%

 13%

 yes

The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.

 2.6%

 yes

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Mitsubishi-Kagaku Foods Corporation, Lot # 55033111

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient conditions in the dark.
- Solubility and stability of the test substance in the solvent/vehicle: Analysis of test solutions showed a maximum of 2-7% of nominal test sustance in solution.


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was weighed , and then ground into a fine powder prior to dissolving in water. The test substance was then added to a 30 mL beaker, and rinsed into a 500 mL aspirator bottle containing freshwater medium. The test solution was then stirred for almost 47 hrs, and then allowed to settle for 1 hr.

FORM AS APPLIED IN THE TEST (if different from that of starting material): solution
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All
- Sampling method: Samples were taken from mid-depth.
- Sample storage conditions before analysis: Refrigerated in glass vials.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test substance was weighed , and then ground into a fine powder prior to dissolving in water. The test substance was then added to a 30 mL beaker, and rinsed into a 500 mL aspirator bottle containing freshwater medium. The test solution was then stirred for almost 47 hrs, and then allowed to settle for 1 hr.
- Controls: Negative control.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): None
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0, 6.3, 13, 25, 50, and 100 mg/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): Particulates were observed at the top of the 6.3 mg/L solution. The 25, 50, and 100 mg/L solutions were cloudy with particulates on the surface of the solution.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Source (laboratory, culture collection): University of Toronto Culture Collection
- Age of inoculum (at test initiation): At least 2 weeks.
- Method of cultivation: Algal cells were transferred to fresh media 4 days before the start of the test.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
No
Test temperature:
21.61-24.42 °C
pH:
7.5-10.1
Nominal and measured concentrations:
Nominal: 0, 6.3, 13, 25, 50, 100 mg/L
Measured: 0,
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): closed with foam stoppers
- Material, size, headspace, fill volume: filled with 100 mL
- Initial cells density: 10,000 cells/mL
- Control end cells density: 2,808,333 cells/mL
- No. of organisms per vessel: 1 x 10^6
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Wildlife International Well Water
- Total organic carbon: Below limit of detection
- Metals: Below limit of detection
- Pesticides: Below limit of detection

OTHER TEST CONDITIONS
- Adjustment of pH: yes
- Photoperiod: continuous
- Light intensity and quality: 6000 lux cool-white fluorescent lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: hemacytometer and microscope at 24 hour intervals

TEST CONCENTRATIONS
- Range finding study: 0, 1.0, 10, and 100 mg/L
- Test concentrations: Nominal: 0, 6.3, 13, 25, 50, 100 mg/L
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
WAF
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Particulates were noted at the top of the solution in most of the test concentrations.
- Effect concentrations exceeding solubility of substance in test medium: yes
Reported statistics and error estimates:
Dunnett's Test was used to deterimine if any of the test groups had significantly reduced growth as compared to controls. None of the test groups had significantly reduced growth as compared to controls.
Validity criteria fulfilled:
yes
Conclusions:
The 72-hr algal EC50 for the test substance is > 100 mg/L (WAF).
Executive summary:

The test substance Sucrose Palmitate Stearate MDT Grade was tested for toxicity to algae. Three replicates each at concentrations of 6.3, 13, 25, 50, and 100 mg/L of test substance were tested. Negative controls were also used. After 72 hrs of exposure, none of the test concentrations showed reduced growth as compared to controls. The 72 -hr EL50 is therefore > 100 mg/L water accomodated fraction. The 72 -hr NOELR is >= 100 mg/L.

Description of key information

ErL50 (72 h) > 100 mg/L (nominal, OECD 201)
NOErLR (72 h) ≥ 100 mg/L (nominal, OECD 201)

Key value for chemical safety assessment

Additional information

Two experimental studies are available investigating the toxicity of the test item to aquatic freshwater algae.
The first study was performed according to OECD 201 (Mitsubishi, 2016). Pseudokirchneriella subcapitata was exposed to nominal loading rates of 6.3, 13, 25, 50 and 100 mg/L for 72 h. The Water Accommodated Fractions (WAF) were prepared by adding an appropriate amount of test item to test medium, followed by intensive stirring (47 h) and a settling period (1 h). Particulates were observed at the top of the 6.3 mg/L solution. The 25, 50, and 100 mg/L solutions were cloudy with particulates on the surface of the solution. HPLC/MS analysis revealed only a low recovery of the test item. In the 25 and 100 mg/L loading rate concentrations of 1.69 and 2.56 mg/L was recorded, respectively. In the remaining loading rates the recovery was below the LOQ (< 1 mg/L). This result reflects the very low water solubility of the substance and indicates that the concentration of the test item is in line with the water solubility (< 1.51 mg/L). At the end of exposure after 72 h no effects of the test item on growth was recorded at any of the loading rates tested resulting in an ErL50 (72 h) > 100 mg/L and a NOELR (72 h) ≥ 100 mg/L.
The result from the key study was confirmed in one supporting study (DKS, 2016). This study was performed according to OECD 201 (GLP). Compared to the key study no chemical analysis of test concentrations was performed and no loading rate was tested. In this study a test solution of 100 mg/L was prepared and stirred for 48 h. This solution was used for testing without further processing (e.g. filtration, siphoning). In contrast to the key study these test solutions were clear and colorless and no precipitate was recorded. No effects on growth rate were observed after 72 h resulting in an EC50 (72 h) > 100 mg/L and a NOEC (72 h) ≥ 100 mg/L.