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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:
9 March - 3 August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): "3-HYDROXY-2-(HYDROXYMETHYL)-2-METHYLPROPIONALDEHYDE"
- Molecular formula (if other than submission substance):C5H10O3
- Lot No.: CH 138067/001.
- CAS No.: 185146-18-2.
- Appearance: uncolored and high viscous.
- Purity test date:
~ 82 % Dimethylpropanale
~ 10 % Hydroxymethanesulfonic acid, Na-salt (CAS No: 870-72-4)
~ 6 % water *)
~ 2 % Potassiumformiate (CAS No: 590-29-4).
- Solubility: In water: Soluble
- Conditions of storage: Room temperature.
- Stability at conditions of storage: Min. 1 year.
- Expiry date: 30 November 2010.

*) The results presented in the report refer to the dry weight of the test substance not including the 6 % water content.

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: Nominal: 0.0, 0.4, 1.0, 3.0, 7.0, 20.0, 60.0, 170.0, 500.0 mg/L
- Sampling method: At the start and at the end of the incubation period, samples of the test media were drawn and the concentrations of the test substance were determined in aliquots of the blank containing test substance, but no algae; of one replicate of each of the test and control cultures (after filtering through a 0.2 µm filter)
- Sample storage conditions before analysis: samples were immediately analysed by HPLC

Test solutions

Vehicle:
no
Details on test solutions:
- Method: A stock solution with a nominal test substance concentration of 555 mg/L (dry weight) was prepared by addition of 603 mg test substance (6 % water content) in 1 L of nutrient medium and stirring for 20 minutes.
- Controls: For the negative control group only nutrient medium was used.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehicle was used.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly Selenastrum capricornutum)
- Strain: ATCC (American Type Culture Collection) 22662.
- Source (laboratory, culture collection): LGC Promochem GmbH, Germany
- Method of cultivation: Stock cultures, initially cultivated from deep frozen algae, are continuously maintained in
250 mL conical flasks (Erlenmeyer), each containing 100 mL nutrient medium, at a temperature in the range of 21 to 24 (± 2) °C under permanent light with an intensity between 4400 and 8800 lux. In about weekly intervals 1 mL of the stock culture is
diluted 100-fold with nutrient medium for precultivation and incubation is continued.

ACCLIMATION
- Acclimation period: Precultures were prepared by incubating a new stock culture for four days.
- Culturing media and conditions (same as test or not): same as test.
- Any deformed or abnormal cells observed: no.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None.

Test conditions

Hardness:
not reported.
Test temperature:
22 °C
pH:
The pH was between 7.5 and 7.6 at the start of the incubation in the test cultures and it was 7.3 in the control cultures.
After 72 hours of incubation the pH was between 7.0 and 8.6 in the test cultures and it was 8.3 in the control cultures.

Dissolved oxygen:
Not reported.
Nominal and measured concentrations:
Nominal: 0.0, 0.4, 1.0, 3.0, 7.0, 20.0, 60.0, 170.0, 500.0 mg/L
Geometric mean of actual/calculated concentrations: 0.10 (calculated), 0.31 (calculated), 1.00 (calculated), 3.21 (calculated), 10.36 (calculated), 33.39, 118.86, 343.14 mg/L. For details see attached background material.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL closed Erlenmeyer flasks filled with 100 mL medium .
- Initial cells density: 10 000 cells/mL
- Control end cells density: the cell density in the control cultures increased by a factor of about 123.
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes.
- Sterile deionised water and sterile concentrated nutrient medium 9 + 1 was used.
- Conductivity of the deionised water: <5 µS/cm

OTHER TEST CONDITIONS
- Sterile test conditions: The test cultures are prepared under steril conditions.
- Adjustment of pH: no.
- Photoperiod: 24 h.
- Light intensity and quality: at least 4710 lux. Wavelength of 400 to 700 nm.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic cell counter with Casy Cell Counter after 24, 48, and 72 hours of incubation
- Chlorophyll measurement: no.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: < 3.2
- Justification for using less concentrations than requested by guideline: not applicable.
- Range finding study: yes .
- Test concentrations: . A preliminary range finding study not according to GLP using nominal concentrations of 10, 100, 1 000, and 10 000 mg/L (including 6 % water content) was conducted
- Results used to determine the conditions for the definitive study: The range finding study revealed EC50 values below 10 mg/L (based on the yield) and between 10 and 100 mg/L (based on the growth rates).
Reference substance (positive control):
yes
Remarks:
72h EC50 (K2Cr2O7): for growth rate 1.32 mg/L and yield 0.62 mg/L

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
67.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
16.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
- Exponential growth in the control (for algal test): During the 72 hours incubation period the cell density in the control cultures increased by
a factor of about 123, corresponding to about 7.0 generations.
- Observation of abnormalities (for algal test): None
- Colour differences: No
- Any stimulation of growth found in any treatment: Yes.
- Effect concentrations exceeding solubility of substance in test medium: No.

Growth Inhibition:
• Based on the yield and compared to the negative controls: In all concentrations tested, the influence on algal growth ranged from 98.2 % inhibition to 6.9 % enhancement.
• Based on the average growth rates and compared to the negative controls: In all concentrations tested, the influence on algal growth ranged from 76.9 % inhibition to 1.5 % enhancement.

Results with reference substance (positive control):
The last reference test with K2Cr2O7 was conducted from the 21st to the 24th of June 2010 with Pseudokirchneriella subcapitata to evaluate the reliability of the test conditions. The 72 hour EC50 for growth rate and yield were 1.32 and 0.62 mg K2Cr2O7/L, respectively. These results establish the reliability of the test procedures for this kind of study type.
Reported statistics and error estimates:
Based on the yield as well as on the average growth rates two "lowest observed effective
concentrations" (LOECs) are calculated by comparison of the data of the three replicates of
each test substance culture with the negative control (analysis of variance, followed by the
Scheffé-test, P = 0.05). The "no observed effect concentrations" (NOECs) are then derived
from these results (highest concentration with no statistically significant difference to the
control).

Any other information on results incl. tables

Validity criteria:

All acceptance criteria for controls given in the EC Regulation were met:

·      During the 72 hours incubation period the cell density in the control cultures increased by a factor of about 123, corresponding to about 7.0 generations.

·      The mean coefficient of variation for section-by-section specific growth rates was 10.4 %.

·      The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 2.7 %.

.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
based on the yield NOEC = 10.4 mg/L
based on the average growth rates NOEC = 10.4 mg/L
based on the yield EC50 = 16.8 mg/L
based on the average growth rates EC50 = 67.6 mg/L
Executive summary:

A Pseudokirchneriella subcapitata growth inhibition test according to the EC regulation 761/2009 Part C.3 and theOECD-Guideline 201 (adopted by the Council on 23rdMarch 2006)was performed to determine the possible effects of "3-HYDROXY-2-(HYDROXMETHYL)-2-METHYLPROPIONALDEHYDE"on the growth of a unicellular green algal species.

 Eight different concentrations of between nominal 0.4 and 500.0 mg per L nutrient medium, spaced by a factor of about 3, were tested against one concurrent negative control (nutrient medium only).

Three replicates were used for each test culture and six replicates for the control culture. The cell count of the algae was about 104cells/mL at the start of the exposure in each vessel.

In each vessel the cell density of the algae was determined 24, 48 and 72 hours after the onset of incubation and the pH was measured at the begin and at the end of the incubation period. Visual observations on the appearance of the algal cultures were made each time when the cell densities were determined.

Possible test substance effects were determined by comparison of the yield and of the growth rates.

At the start and at the end of the experiment, filtered samples of the cultures exposed to the test substance, of the control cultures and also of the blank with the highest concentration of the test substance, but no algae, were taken and immediately analysed in duplicate by HPLC.

Results:

based on the yield NOEC = 10.4 mg/L

based on the average growth rates NOEC = 10.4 mg/L

based on the yield EC50 = 16.8 mg/L

based on the average growth rates EC50 = 67.6 mg/L