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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 Mar - 26 Apr 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2011
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
solid: crystalline
Details on test material:
Appearance: white crystalline powder
Storage conditions: at room temperature protected from light
Specific details on test material used for the study:
Water solubility at 20°C: 347 g/L

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (Control), 0.1, 1.0, 10, 100 mg/L
- Frequency: at t=0 h, t=24 h, and t=72 h
- Sampling volume: 3.6 mL; at the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
- Sample storage conditions before analysis: Samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preparation of test solutions started with the highest concentration of 100 mg/L. No other treatment than vigorous shaking was needed to completely dissolve the test item in medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. Because the test item is sensitive to light, during preparation procedure and light was dimmed and glassware was covered with aluminum foil.
- Controls: Test medium without test item or other additives

- Evidence of undissolved material: All test solutions were clear and colorless at the end of the preparation procedure.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: In-house laboratory culture
- Age of inoculum (at test initiation): 4 d (pre-culture)
- Method of cultivation: Algae stock cultures were started by inoculating growth medium (M1; according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)) with algal cells from a pure culture on agar. The suspensions were continuously aerated and exposed to light in a climate room at a temperature of 21-24°C, until the pre-culture started.

PRE-CULTURE:
- Pre-culture method: 4 days before the start of the test, cells from the algal stock culture were inoculated in culture medium (M2; according to the OECD 201 Guideline, formulated using Milli-RO water) at a cell density of 1 x 10^4 cells/mL.
- Culturing media and conditions (same as test or not): Yes

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h

Test conditions

Hardness:
Hardness of M2 medium: 0.24 mmol/L (24 mg CaCO2/L)
Test temperature:
The temperature measured in the incubator was maintained between 22 and 24°C. Temperature remained within the limits prescribed by the study plan (21-24°C, constant within 2°C)
pH:
Control: 8.0 - 8.3
100 mg/L: 7.7-7.8
Dissolved oxygen:
Not measured
Nominal and measured concentrations:
Control nominal: 0 mg/L, measured 0 mg/L at t=0 h and 0.016 mg/L at t=72 h.
Limit concentration nominal: 100 mg/L, measured 105 mg/L at t=0 h and 108 mg/L at t=72 h (103-112 mg/L without algae).
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL, all-glass
- Type (delete if not applicable): closed (capped vessels)
- Test solution volume: 50 mL
- Aeration: No
- Initial cells density: 1x10^4 cells/mL
- Control end cells density: 103.1x10^4 (stdev: 5.5x10^4)
- No. of vessels per concentration (replicates): 6 for 100 mg/L test item concentration, 3 for other test item concentrations.
- No. of vessels per control (replicates): 6
- 1 or 2 replicates of each concentration without algae


GROWTH MEDIUM
- Standard medium used: yes (M2, according to OECD 201 Guideline)


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M2 test medium, according to the OECD 201 Guideline, formulated using Milli-RO water.

- Culture medium different from test medium: Stock culture medium: M1, pre-culture medium: M2.
- Intervals of water quality measurement: pH at the beginning and at the end of the test; Temperature of medium continuously in a temperature control vessel.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: TLD-lamps with a light intensity within the range of 82 to 89 µE.m^2.s^1.


EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: At the beginning of the test, cells were counted using a microscope and acounting chamber; thereafter cell densities were determined by spectrophotometric measurement of samples at 680 nm using a spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank and the extra replicates as background for the treated solutions.Cell densities were recorded at 24-hour intervals in the control and the limit concentration. Intermediate concentrations were measured only at the end of the exposure period.
- Appearance of the cells: At the end of the test microscopic observations were performed in the control and the limit concentration to observe any abnormal appearance of the algae.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10x
Reference substance (positive control):
yes
Remarks:
K2Cr2O7 (Potassium dichromate)

Results and discussion

Effect concentrationsopen allclose all
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:
EC10
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
Remarks on result:
other:
Remarks:
Based on biological relevance
Details on results:
Actual concentrations measured in the limit concentration were at the level of nominal during the entire exposure period (103-112%) in solutions incubated with and without algae. Therefore, the effect parameters were expressed in terms of analytically confirmed nominal concentrations.

The inhibition of growth ranged between 0.7 to 1.7% at the end of the exposure period. The effect observed at the limit concentration was statistically significant and therefore, no NOEC can be given based on statistical significance. As this effect was biologically insignificant (<10%), the NOEC based on biological relevance was set at 100 mg/L.

- Observation of abnormalities (for algal test): Microscopic observations at the end of the test revealed a normal and healthy appearance of the algal cells exposed to the limit concentration when compared to the control.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50:
The EC50 for growth rate inhibition (72h-ERC50) was 1.2 mg/L with a 95% confidence interval ranging from 1.1 to 1.2 mg/L.

Reported statistics and error estimates:
Growth rate: Shapiro-Wilk's test on normal distribution and Levene's test on variance-homogeneity (normal-distribution and variance-homogeneity requirements are fulfilled); Two-sample t-test.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study with Pseudokirchneriella subcapitata, no biologically relevant inhibition of growth rate was recorded at any of the concentrations of the substance tested.
The EC50 for growth rate inhibition (72h-ERC50) was beyond the range tested, i.e. exceeded an analytically confirmed nominal concentration of 100 mg/L.
The 72h-NOEC for growth rate inhibition was 100 mg/L based on the biological relevance. The EC10 for growth rate inhibition (alternative to NOEC) was also beyond the range tested, i.e. exceeded an analytically confirmed nominal concentration of 100 mg/L.