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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Purity: 99.2%

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Samples of the test solutions were collected at approximately 0 and 72 hours to measure concentrations of the test substance. At test initiation, samples were collected from each test concentration and blank control solution prior to distribution into test chambers. At test termination, the biological replicates from each respective test concentration and blank control solution were pooled and then sampled and the single flask of the abiotic control was sampled. All samples were collected in glass vials.

Test solutions

Vehicle:
yes
Remarks:
freshwater algal medium
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
A primary stock solution was prepared by mixing 0.01 g of test substance in 1000 mL of freshwater algal medium for a nominal concentration of 10 mg/L. The stock was sonicated for 20 minutes, stirred for one hour, and inverted at least 20 times to mix. At the completion of mixing, the stock was cloudy with very slight particulates on the neck of the flask. A secondary stock was made by diluting 100 mL of the primary stock to a final volume of 1000 mL with freshwater algal medium for a nominal concentration of 1.0 mg/L and mixed by inversion. The secondary stock, which also served as the highest test concentration and the abiotic control, was proportionally diluted with freshwater algal medium to prepare the five additional test concentration solutions at nominal concentrations of 0.031, 0.063, 0.13, 0.25, and 0.50 mg/L. The secondary stock and all other test solutions appeared clear and colourless.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green alga
- Strain: Freshwater algae
- Source: University of Toronto Culture Collection (maintained in culture medium at Wildlife International, Ltd., Easton, Maryland)

ACCLIMATION
- Acclimation period: At least two weeks

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h

Test conditions

Test temperature:
Exposure phase: 23.4 to 24.2°C
Recovery phase: 23.5 to 24.9°C
pH:
At the beginning: 7.4
At termination: 7.5 to 7.9
Abiotic control: 7.4 (at the beginning and termination)
Nominal and measured concentrations:
Nominal: Blank control, 0.031, 0.063, 0.13, 0.25, 0.50, 1.0, and 1.0 mg/L (abiotic control)
Measured: Blank control, 0.025, 0.051, 0.10, 0.20, 0.40, 0.87, and 0.92 mg a.s/L (abiotic control)
Details on test conditions:
TEST SYSTEM
- Test vessel: 250-mL Erlenmeyer flasks plugged with foam stoppers and contained 100 mL of test or control solution
- Initial cells density: Approximately 10000 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per abiotic control (replicate): 1

GROWTH MEDIUM
- Medium: The algal cells were cultured and tested in freshwater (AAP) algal medium. Stock nutrient solutions were prepared by adding reagent-grade chemicals to purified test facility well water. The test medium was then prepared by adding appropriate volumes of the stock nutrient solutions to purified well water, adjusted to pH 7.5 with 0.1 N NaOH, and sterilized by filtration (0.22 μm) prior to use. A different lot of the same medium was used for the recovery phase.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Photoperiod: 24-hour photoperiod
- Light intensity and quality: 5100 to 6250 lux

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Electronic particle counter
- Other: Microscopic examination of the inoculum culture prior to initiation of the test and of a pooled sample from each test concentration and the controls at the end of the test was performed to assess whether the algae were normal in appearance. Each sample was examined for evidence of flocculation or aggregation of algal cells, or adherence of cells to the test chamber in any of the treatment or control groups.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
cell number
Remarks on result:
other: 95% C. I.: 0.0743 to 0.135 mg a.s./L
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.051 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: cell number and growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.025 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: cell number and growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.421 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% C. I.: 0.378 to 0.469 mg a.s./L
Details on results:
Inhibition of growth expressed as cell density of P. subcapitata exposed to geometric mean, measured concentrations of 0.025, 0.051, 0.10, 0.20, 0.40, and 0.87 mg a.s./L was 7, 38, 53, 63, 94, and 98%, respectively, relative to the blank control. Inhibition of growth expressed as growth rate was 2, 9, 15, 18, 53, and 74%, respectively, relative to the blank control.

At test initiation, algal cells in the inoculum appeared normal. Microscopic observations were made from samples of test solution collected from each treatment and the control group at approximately 72 hours. Cell morphology in the control and the 0.025, 0.051, and 0.10 mg a.s./L treatment concentrations were normal in size, shape, and color. Cells in the 0.20, 0.40, and 0.87 mg a.s./L treatment concentrations appeared to be enlarged relative to cells in the control group. There was no evidence of flocculation, aggregation or adherence to the test chambers in any group.

In the recovery phase it was demonstrated that the effects of test substance were algistatic (reversible) rather than algicidal, to P. subcapitata at concentrations less than or equal to the geometric mean, measured concentration of 0.87 mg a.s./L within three days.

Dunnett’s test indicated that mean cell density and growth rate of the 0.051, 0.10, 0.20, 0.40, and 0.87 mg a.s./L concentration were significantly reduced relative to the blank control. Consequently the NOEC and LOEC were determined to be 0.025 and 0.051 mg a.s./L, respectively, for cell density and growth rate. Cell density increased in the blank control by at least a factor of 16 in 72 hours. The coefficient of variation of average specific growth rates during the whole test period (0-72 hour) in replicate controls was 1.3%. The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2, and 2-3) in the controls was 17.6%. Therefore, the appropriate validity criteria were met.
Reported statistics and error estimates:
The EC50 values for cell density and growth rate were calculated by non-linear interpolation for the 72-hour exposure interval. The cell density (count) and growth rate data were evaluated for normality and homogeneity of variance (alpha = 0.01) using the Shapiro-Wilk’s and Levene’s tests, respectively. Since the data were normal with homogeneous variances, the treatment groups were compared to the untreated control using ANOVA and Dunnett’s test (alpha = 0.05). The results of the statistical analyses, as well as an evaluation of the concentration-response pattern, were used to determine the NOEC relative to each parameter at 72 hours.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Healthy Cell Count (density):
72-hr EbC50 (P. subcapitata): 0.100 mg a.s./L
72-hr NOEC (P. subcapitata): 0.025 mg a.s./L

Growth Rate:
0-72 hr ErC50 (P. subcapitata): 0.421 mg a.s./L
72-hr NOEC (P. subcapitata): 0.025 mg a.s./L
Executive summary:

The effect of the test substance on the green alga Pseudokirchneriella subcapitata was determined in a static 72-hour test without test medium renewal. The test was conducted according to OECD Guideline 201 and OPPTS Guideline 850.5400.

Treatments consisted of an untreated control, an abiotic (stability) control, and six nominal concentrations of 0.031, 0.063, 0.13, 0.25, 0.50, and 1.0 mg/L. The corresponding geometric mean, measured concentrations were 0.025, 0.051, 0.10, 0.20, 0.40, and 0.87 mg a.s./L.

The EC50 and NOEC for Pseudokirchneriella subcapitata were based on geometric mean, measured concentrations and cell count (density) and growth rate. The 72-hr EbC50 based on healthy cell count (density) was 0.100 mg a.s./L and the NOEC was 0.025 mg a.s./L. The 0-72 hr ErC50 based on growth rate was 0.421 mg a.s./L and the NOEC was 0.025 mg a.s./L. Recovery data indicated that the effects of test substance on Pseudokirchneriella subcapitata are expected to be reversible at concentrations less than or equal to 0.87 mg/L.