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:
23 June - 25 July, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: all test concentrations and control
- Sampling method: 2 ml at t=0, t=24 h and t=96 h
- Sample storage conditions before analysis: freezer

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Preparation of test solutions for the combined limit/range-finding test (0.1, 1.0, 10 and 100 % of water soluble fraction) started with a loading rate of 100 mg/l. A 20-minute period with ultrasonic waves was followed by 16 minutes of magnetic stirring before a stabilisation period of 1 hour. A hazy solution with a floating layer was obtained. The hazy solution was then siphoned off over glass wool before use as highest test concentration.
For the final test a highest test concentration was prepared starting with a loading rate of 100 mg/l that was magnetically stirred for 1 hour and subsequently stabilised for 1 hour. The resulting solution was clear and colourless with a floating layer. The clear and colourless Water Soluble Fraction (WSF) was collected and used for testing. The lower test concentrations for both tests (1.0, 3.2, 10 and 32 % of water soluble fraction) were prepared by subsequent dilution of the highest test concentration in test medium. All were clear solutions except for the two highest solutions tested in the combined limit/range-finding test. Note that formulation of the test solutions was performed under dimmed light conditions.
After preparation, volumes of 50 ml were added to each replicate of the respective test concentration. Subsequently, 1 ml of an algal suspension was added to each replicate providing a cell density of 10^4 cells/ml.

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 (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): not indicated
- Method of cultivation: Algae stock cultures were started by inoculating growth medium 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.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): stock culture medium is M1 (according to the NPR 6505, formulated using Milli-Ro water); test medium and pre-culture medium is M2 (according to the OECD 201 Guideline, formulated using Milli-Q water (tap water purified by reverse osmosis and subsequently passed over activated carbon and ion-exchange cartridges) preventing precipitation.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
No post exposure observations.

Test conditions

Hardness:
0.24 mmol/l (24 mg CaCO3/l)
Test temperature:
22.7 - 23.4 °C
pH:
7.7 - 8.3
Nominal and measured concentrations:
Nominal concentrations final test: 0, 1.0, 3.2, 10, 32 and 100 % of a water soluble fraction (WSF) prepared at 100 mg/l.
Analyses of samples taken from the WSF prepared at 100 mg/l showed a measured concentration of 0.8 mg/l, which was in agreement with what was expected based on the water solubility limit. The measured concentration decreased to 0.09 mg/l after 24 hours, while the concentration measured after 96 hours was 0.14 mg/l. Hence, the actual concentration appeared to remain at a relatively stable level of approximately 0.1 mg/l during the 72-hour test period in light. Based on these results, the TWA concentration was calculated to correspond to 0.15 mg/l.
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 ml, all-glass, containing 50 ml of test solution
- Initial cells density: 10000 cells/ml
- Control end cells density: 2947000 cells/ml after 96h
- No. of vessels per concentration (replicates): 3 replicates of each test concentration, 1 extra replicate of each test group for sampling after 24 hours, 1 or 2 replicates of each test concentration without algae.
- No. of vessels per control (replicates): 6 replicates of the control

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 82 to 94 µE.m-2.s-1 using TLD-lamps of the type ‘Cool-white’ of 30 Watt.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : 0, 24, 48, 72 and 96h
- Determination of cell concentrations: spectrophotometer

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.1 in the final test (1-100% of WSF prepared at 100 mg/l), 10 in the range finding study (0.1-100 % of WSF prepared at 100 mg/l)
- Range finding study: the study is a combined limit/range finding study
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 0.15 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
> 0.8 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
growth rate
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities observed
- Any stimulation of growth found in any treatment: no
Results with reference substance (positive control):
- Results with reference substance valid: Yes
- EC50: The EC50 for growth rate reduction (ERC50: 0-72h) was 1.7 mg/l with a 95 % confidence interval ranging from 1.1 to 2.7 mg/l.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present study with Pseudokirchneriella subcapitata, no significant reduction of growth rate or inhibition of yield was recorded at any of the concentrations of the substance tested. The study included a highest concentration prepared at a loading rate of 100 mg/l, with an initial measured concentration of 0.8 mg/l that equalled the limit of solubility (NOEC).
Due to the low solubility of LE 5288 in water, concentration levels that might be toxic for algae could not be reached.
Executive summary:

The toxicity of the substance to algae was determined according to OECD Guideline 201 using a static test design for 72 hours. The water accomodated fraction of the substance was used. Under the conditions of the present study with Pseudokirchneriella subcapitata, no significant reduction of growth rate or inhibition of yield was recorded at any of the concentrations of the substance tested. The study included a highest concentration prepared at a loading rate of 100 mg/l, with an initial measured concentration of 0.8 mg/l that equalled the limit of solubility (NOEC).