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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
Study period:
20 January - 22 June 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
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
yes
Remarks:
No chemical analysis was conducted. The test substance is an UVCB , and no chemical analysis is available for the whole compound. Therefore, in agreement with the sponsor, no chemical analysis was performed.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
No chemical analysis was conducted. The test substance is an UVCB , and no chemical analysis is available for the whole compound. Therefore, in agreement with the sponsor, no chemical analysis was performed.
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Sulfite liquors and Cooking liquors, green
EC Number:
268-612-2
EC Name:
Sulfite liquors and Cooking liquors, green
Cas Number:
68131-30-6
Molecular formula:
HNa3OS
IUPAC Name:
Aqueous alkaline solution of sodium salts produced by dissolving melt from incineration of spent liquor from sodium based pulping processes.
Details on test material:
- Name of test material (as cited in study report): Green liquor 1
- Chemical name: Sulphite liquors and Cooking liquors, green.
- Molecular formula (if other than submission substance): UVCB
- Appearance: Yellowish liquid
- Solubility: In water: The substance is a water solution, The dry solids content is 17.7% dry solids/82.3% water by weight (at 20 deg. C)).
- pH: over 11
- Impurities (identity and concentrations): UVCB
- Lot/batch No.: Not stated
- Expiry date: Not available.
- Stability under test conditions: No data available
- Storage condition of test material: Room temperature. Storage in the dark but may be used under light.

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
- Method: Two stock solutions with nominal test substance concentrations of 6 658.0 and 79.4 mg/L respectively were prepared. Stock solution 1 was assembled by dissolving 3 329 mg test substance (wet weight) in 500 mL of nutrient medium, stock solution 2 was prepared by dissolving 39.7 mg test substance in 500 mL of nutrient medium. Both solutions were homogenised manually. Aliquots of this stock solution were diluted with nutrient medium to obtain lower concentrations, nominally spaced by a factor of about 3. The preparations were made freshly before the start of the exposure.
- 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:
21 °C
pH:
The pH was between 7.4 and 11.7 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 7.8 and 9.4 in the test cultures and it was 8.5 in the control cultures.
Dissolved oxygen:
Not reported.
Nominal and measured concentrations:
Nominal: 0, 0.8, 2.5, 8.1, 23.3, 71.5, 209.7, 649.2, 1997.4, and 5992.2 mg/L (substance wet weight, 82.3 % water included)
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 190.
- 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 4800 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: . In this preliminary study five test substance concentrations, nominal 10 000, 1 000, 100, 10, and 1 mg/L (wet weight), were tested.
- Results used to determine the conditions for the definitive study: Based on the inhibition of the algal yield and the growth rates, the range finding study revealed EC50 values between 125 and 2 000 mg/L (wet weight) (22 mg/L - 354 mg/L (dry weight)).
Reference substance (positive control):
yes
Remarks:
72h EC50 (K2Cr2O7): for growth rate 0.87mg/L and yield 0.65 mg/L

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
209.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
wet weight
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
71.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
wet weight
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1 380.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
wet weight
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
208.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
wet weight
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 190, corresponding to about 7.6 generations.
- Observation of abnormalities (for algal test): None
- Colour differences: No
- Any stimulation of growth found in any treatment: No.

Growth inhibition:
• Based on the yield and compared to the negative controls: In all concentrations tested, the influence on algal growth ranged from 1.4 % to 99.3 % inhibition.
• Based on the average growth rates and compared to the negative controls: In all concentrations tested, the influence on algal growth ranged from 0.3 % to 84.7 % inhibition.
Results with reference substance (positive control):
The last reference test with K2Cr2O7 was conducted from the 16th to the 19th of November
2009 with Pseudokirchneriella subcapitata to evaluate the reliability of the test conditions.
The 72 hour EC50 for growth rate and yield were 0.87 and 0.65 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

The pH was between 7.4 and 11.7 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 7.8 and 9.4 in the test cultures and it was 8.5 in the control cultures.

The pH in the control cultures changed by 1.2 units during the 72 hours of incubation, i.e. within the maximum change of 1.5 recommended by the guideline.

Marked inhibition of algal growth was mainly observed in the three highest concentrations tested which displayed basic pH values (11.7, 10.9, and 9.8 respectively). Therefore it is not unlikely that the algal growth inhibition was caused by pH. However, it should be noticed that after 72 hours of exposure the pH in the respective test cultures decreased to 8.0 - 9.1, which is in about the same pH range as the control culture.

Validity criteria:

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

·      During the 72 hours incubation period the cell density in the control cultures increased by a factor of about 190, corresponding to about 7.6 generations.

·      The mean coefficient of variation for section-by-section specific growth rates was 11.5 %.

·      The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures was 2.4 %.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
based on the yield NOEC = 71.5 mg/L (wet weight), 12,6 mg/L (dry weight)
based on the average growth rates NOEC = 209.7 mg/L (wet weight), 37 mg/L (dry weight)
based on the yield EC50 = 208.9 mg/L (wet weight), 37 mg/L (dry weight)
based on the average growth rates EC50 = 1380.4 mg/L (wet weight), 244 mg/L (dry weight)
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 "GREENLIQUOR 1" on the growth of a unicellular green algal species. Nine different concentrations of "GREEN LIQUOR 1" between nominal 0.8 and 5 992.2 mg per litre (substance wet weight, 82.3 % water included) nutrient medium, spaced by a factor of about 3.0, 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 beginning 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. The test substance is an UVCB (substance of unknown or variable composition, complex reaction products or biological material), and no chemical analysis is available for the whole compound. Therefore, in agreement with the sponsor, no chemical analysis was performed.

Results:

based on the yield NOEC = 71.5 mg/L (wet weight), 12,6 mg/L (dry weight)

based on the average growth rates NOEC = 209.7 mg/L (wet weight), 37 mg/L (dry weight)

based on the yield EC50 = 208.9 mg/L (wet weight), 37 mg/L (dry weight)

based on the average growth rates EC50 = 1380.4 mg/L (wet weight), 244 mg/L (dry weight)

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