Registration Dossier

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:
2005-01-14 to 2005-03-31
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP compliant; Guideline Study (OECD 201); Deficiencies: The coefficient of analysis of the daily growth rate in the control replicates and the coefficient of analysis of the average specific growth rates in the control replicates were not reported according to the current guideline adopted 2006.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
see Sect. "Rationale for Reliability incl. deficiencies"
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
see Sect. "Rationale for liability incl. deficiencies"
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentration: For the spiked samples of the test item 103.71 mg test item was dissolved in about 90 mL purified water, ultrasonicated for five minutes and made up to the mark in a 100 mL volumetric fiask with purified water to prepare a stock solution of 1037 mg test item/L.
- Sampling method: For the analytical measurements the following samples were taken: fourfold samples (without algae) from each test medium and the control just before the test start and after 72 h.
- Sample storage conditions before analysis: The samples were stored deep-frozen and protected from light until analysis was performed.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: WAFs (Water Accommodated Fractions) were prepared according to the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures as described below:
Prior to the start of the test, six test media with loading rates of 0.32, 1.0, 3.2, 10, 32 and 100 mg/L were prepared.
The test media with loading rates 100, 32, 10, and 3.2 mg/L were prepared by mixing 150, 48.1, 20.0, and 6.4 mg test item into 1500, 1500, 2000, and 2000 mL of test water respectively. Ultrasonic treatment was applied for 15 minutes. No auxiliary solvent or emulsifler was used. The dispersions were then stirred on a magnetic stirrer at room temperature in the dark for 96 hours to dissolve a maximum amount of the test item in test water. The long stirring period of 96 hours was chosen according to the results of a pre-test (without GLP) which showed that during this time no signiflcant loss of the test item was to be expected.
After the stirring period of 96 hours, the saturated dispersions were filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 pm). The undiluted filtrates with dissolved test item were tested on algae as WAFs.
For practical reasons, the test media with loading rates 1.0 and 0.32 mg/L had to be prepared by diluting the WAF with the loading rate of 3.2 mg/L with test water. Additionally, a control was tested in parallel.

Test organisms

Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Scenedesmus subspicatus CHODAT,
- Strain: Strain No. 86.81 SAG
- Source (laboratory, culture collection): SAG, Institute for Plant Physiology, University of Göttingen, D-37073 Göttingen, Germany
- Age of inoculum (at test initiation): four days
- Method of cultivation: exponentially growing pre-culture

ACCLIMATION
- Acclimation period: one day
- Culturing media and conditions: same as test medium

Study design

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

Test conditions

Hardness:
24 mg/L as CaCO3
Test temperature:
22 - 23 °C
pH:
7.9 - 8.6
Nominal and measured concentrations:
Nominal WAF loading rates: 0.32, 1.0, 3.2, 10, 32, 100 mg/L
measured concentrations (main component): Since WAFs were tested, the reported biological results are based on the nominal loading rates.
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer glass flasks
- Type: closed with glass dishes
- Material, size, headspace, fill volume: Volumes of 15 mL algal suspension for each replicate were continuously stirred by magnetic stirrers in 50 mL Erienmeyer flasks.
- Type of flow-through: No
- Renewal rate of test solution (frequency/flow rate): No
- Initial cells density: 10E04 cells per mL;
- Control end cells density: 69 x 10E04 cells/mL
- No. of vessels per concentration (replicates): three
- No. of vessels per control (replicates): six


GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: synthetic test water, prepared according to the test guidelines. Analytical grade salts were dissolved in sterile purified water.
- Culture medium different from test medium: No
- Intervals of water quality measurement: At the beginning and at the end of the test period

OTHER TEST CONDITIONS
- Photoperiod: continuous illumination;
- Light intensity and quality: 8500 to 9500 lux;


Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
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:
EC10
Effect conc.:
12 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 3.5 - 28
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
91 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL: 32 - 2039
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
4.5
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL: 0.28 - 12
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
Reported statistics and error estimates:
The EbC50 and ErC50 values (the respective concentrations of the test item corresponding to 50% inhibition of algal biomass (b) and growth rate (r) compared to the control), and the corresponding EC10 value and their 95%-confidence limits were calculated by Probit Analysis.
For the determination of the LOEC and NOEC, the calculated mean biomass and the mean growth rate at the test concentrations were tested for significant differences when compared to the control values by a Dunnett-test

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
With high probability not acutely harmful to aquatic algae. No toxic effects within the range of solubility.