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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:
key study
Study period:
From 2018-03-13 to 2018-03-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
Lot No.: 20170707;
Purity: 99.13%
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control), 0.95, 3.1, 9.8, 31, and 100 mg/L
- Sampling method: At initiation (0 hour) and termination (72 hours), a 5-mL sample was collected from the control and each test substance treatment using a serological pipette, and the samples were placed in separate 10-mL culture tubes.
- Sample storage conditions before analysis: analysed immediately
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A 0.10 mg/mL primary standard was prepared by transferring 0.1009 g of test item to a 1-L glass volumetric flask, and bringing the flask to volume with test medium. This primary standard was inverted, stirred for approximately 5 minutes on a stir plate, and sonicated for approximately 5 minutes following preparation. Appropriate aliquots of the primary standard solution were diluted with test medium to a volume of 0.5 L to prepare the test substance treatments at concentrations of 0.95, 3.1, 9.8, and 31 mg/L .
- Controls: consisted of test medium only
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Selenastrum capricornutum
- Strain: unicellular green alga
- Source: Department of Botany, Culture Collection of Algae, University of Texas at Austin
- Method of cultivation: The prepared cultures were maintained in a temperature-controlled environmental chamber under continuous light. Periodically, new cultures were cloned from an existing culture derived from the parent stock. All cultures were maintained under the same conditions as those used for testing. The algal culture used for this test was four days old and the biomass had increased exponentially (i.e., specific growth rate of 1.4 day-1) during the culture period.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
22.2 to 23.4°C
pH:
7.4 to 7.5 at initiation and from 8.2 to 8.7 in biological replicates at 72 hours
Nominal and measured concentrations:
Nominal: 0 (control), 0.95, 3.1, 9.8, 31, and 100 mg/L
Measured:
Details on test conditions:
TEST SYSTEM
- Test vessel: 250-mL Erlenmeyer flask
- Type: closed, with foam stoppers
- Fill volume: 100 mL
- Initial cells density: 5.0 E+ 03 cells/mL
- Control end cells density: 7.95 E+05 cells/mL
- 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: prepared by the addition of appropriate reagent grade salts to autoclaved reagent water

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: 7,321 to 7,366 lux, cool-white fluorescent lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: direct microscopic counting with a hemacytometer

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0 (control), 0.10, 1.0, 10, and 100 mg/L
- Results used to determine the conditions for the definitive study: The control mean cell density at termination was 74.2 E+04 cells/mL, respectively. The mean cell density at 72 hours was 74.3, 74.5, 63.6 and 38.9 E+04 cells/mL in the 0.10, 1.0, 10, and 100 mg/L treatments, respectively.
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
31 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Analytical Results: Concentrations of test item in the test substance treatment solutions at initiation were 0.976, 3.07, 9.43, 30.9, and 97.0 mg test item/L. Measured concentrations of test item at 72 hours were 1.13, 3.19, 10.1, 31.5, and 102 mg/L. The overall mean measured concentrations in the test substance treatment solutions during the 72-hour exposure were 1.05, 3.13, 9.77, 31.2, and 99.5 mg/L, which represented recoveries of 99 to 111% of the nominal concentrations.
- Biological Results:
Percent inhibition in algal growth at 72 hours, as compared to the control, ranged from -1% at the concentration of 3.1 mg /L to 40% at the concentration of 100 mg/L.
Percent inhibition in growth rate at 72 hours as compared to the control ranged from 0% at the concentrations of 0.95, 3.1, and 9.8 mg/L to 11% at the concentration of 100 mg /L.
The percent inhibition in yield at 72 hours, as compared to the control, ranged from -1% at the concentration of 3.1 mg/L to 41% at the concentration of 100 mg /L.
Reported statistics and error estimates:
The NOEC values, based on growth rate and yield, were estimated using a one-way analysis of variance (ANOVA) procedure and a one-tailed Dunnett’s test (p = 0.05) where the alternate hypothesis was the mean for the growth parameter was reduced in comparison to the control. Prior to the Dunnett’s test, a Shapiro-Wilk’s test and a Levene’s test were conducted to test for normality and homogeneity of variance, respectively, over treatments at each time point. If the results from the Shapiro-Wilk’s and Levene’s tests indicated normality and insignificant heterogeneity (i.e., p > 0.01), the analysis was performed on the non-transformed raw data. In instances of non-normality or heterogeneity (i.e., p < 0.01), a square root transformation was performed. If both the non-transformed raw data and the transformed data exhibited non-normality or inequality of variance, a non-parametric analysis of variance was performed on the ranks of the raw data values. Non-parametric analyses were performed on the 24-hour cell density, growth rate, and yield data. Parametric analyses were performed on the 48- and 72-hour cell density, growth rate, and yield data.
The ErC50 and EyC50 estimates were calculated using a logistic (sigmoid-shaped) model fit to the data with percent inhibition as the dependent variable and concentration as the independent variable.

Results Based on Nominal Concentrations:

Hour

EC Type

EC Value/ mg/L

95% Confidence Limits 

/ mg/L

NOEC 

/ mg/L

24 Hours

ErC50

99.4

Not statistically sound

31

EyC50

67.6

53.4 – 81.8

31

48 Hours

ErC50

>100

Not calculated

31

EyC50

>100

Not calculated

31

72 Hours

ErC50

>100

Not calculated

31

EyC50

>100

Not statistically sound

31

Validity criteria fulfilled:
yes
Conclusions:
Based on growth rate and nominal concentrations, the estimated 72 hour ErC50 was >100 mg a.i./L, the highest concentration tested, the NOEC at 72 hours is 31 mg/L.
Based on yield and nominal concentrations, the estimated 72 hour EyC50 value was >100 mg/L, the NOEC at 72 hours is 31 mg/L.
Executive summary:

A 72-hour growth inhibition test was performed with the unicellular green alga, Pseudokirchneriella subcapitata, exposed to test item according to OECD 201.

A definitive test with a nominal concentration range of 0 (control), 0.95, 3.1, 9.8, 31, and 100 mg/L was conducted. The control was replicated six times and each test substance treatment was replicated three times. At 24, 48, and 72 hours (±1 hour), cell density was measured in all replicates of the control, as well as each test substance treatment by direct microscopic counting with a hemacytometer.

Based on growth rate and nominal concentrations, the estimated 72 hour ErC50 was >100 mg a.i./L, the highest concentration tested, the NOEC at 72 hours is 31 mg/L.

Based on yield and nominal concentrations, the estimated 72 hour EyC50 value was >100 mg/L, the NOEC at 72 hours is 31 mg/L.

Description of key information

A 72-hour growth inhibition test was performed with the unicellular green alga, Pseudokirchneriella subcapitata, exposed to test item according to OECD 201.

Based on growth rate and nominal concentrations, the estimated 72 hour ErC50 was >100 mg a.i./L, the highest concentration tested, the NOEC at 72 hours is 31 mg/L.

Based on yield and nominal concentrations, the estimated 72 hour EyC50 value was >100 mg/L, the NOEC at 72 hours is 31 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
31 mg/L

Additional information

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