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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline compliant GLP study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
9.19, 18.4, 36.8, 73.5, 147 and 294 mg test item/L (corresponding to 3.125, 6.25, 12.5, 25, 50 and 100 mg solid content/L).

- Sampling method:
For measurement of the actual concentrations of the test item, duplicate samples were taken from the test media of all test concentrations at the start of the test (without algae) and at the end of the test (containing algae). At the same sampling times, duplicate samples were also taken from the control. For the 72-hour stability samples, additional flasks containing the test medium with algae were incubated for each treatment under the test conditions. This was necessary as the volume of test solution of the treatment replicates (3 x 15 mL) was too small to perform the analyses. All samples were stored deep-frozen (at about -20 °C) immediately after sampling until analysis. In pre-experiments for investigation of the storage stability of the samples, the test item proved to be stable under these storage conditions. The concentrations of the test item were determined in one of the duplicate test medium samples from the nominal test concentrations of 36.8 to 294 mg test item/L. The samples from the nominal test concentrations of 9.19 and 18.4 mg test item/L were not analyzed, since these concentrations were below the NOEC determined in this test. From the control samples, one of the duplicate samples was analyzed per sampling time.

- Sample storage conditions before analysis:
All samples were stored deep-frozen (at about -20 °C) immediately after sampling until analysis. In pre-experiments for investigation of the storage stability of the samples (non-GLP), the test item proved to be stable under these storage conditions.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test medium of the highest nominal concentration of 294 mg test item/L was prepared by dissolving 294.03 mg of the test item completely in 1000 mL of test water using intense stirring for 15 minutes at room temperature. The test medium of the highest test concentration was used in a series of dilution with test water to prepare the test media of the lower test concentrations. The test media were prepared just before the start of the test.
- Controls: dilution water only
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None. The test item was stable in the test media over the test period of 72 hours.
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus CHODAT
- Strain:Strain No. 86.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany)
- Age of inoculum (at test initiation): An inoculum culture was set up four days before the start of the exposure.
- Method of cultivation: The algae were cultivated under the test conditions and were kept in the exponential growth phase until inoculation of the test solutions.

ACCLIMATION
- Culturing media and conditions (same as test or not): yes
- Any deformed or abnormal cells observed: not reported
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
The water temperature during the test was maintained at 21 °C.
pH:
The pH of the test media was in the range of 7.6 to 7.7 during the test period
Nominal and measured concentrations:
nominal: control and 9.19, 18.4, 36.8, 73.5, 147 and 294 mg test item/L (corresponding to 3.125, 6.25, 12.5, 25, 50 and 100 mg solid content/L). The measured concentrations of Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18 unsaturated alkyl) tetrasodium salts in the test media of the test concentrations of 36.8 to 294 mg test item/L were between 87 and 105% of the nominal values at the start of the test and between 85 and 97% at the end of the test. Thus, the correct dosing of the test item was confirmed. The test item was stable in the test media over the test period of 72 hours. The biological results were related to the nominal concentrations of the test item.
Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 50 mL Erlenmeyer flasks were used per replicate containing 15 mL of test solution.
- Aeration: no
- Initial cells density: 5000 cells/mL
- Control end cells density: not provided
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes AAP medium

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: AAP medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: start and end of test

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: approximately 5200 Lux (range: 4550 to 5640 Lux, measured at nine places in the experimental area)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations:fluorescent measurement
- 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: The selection of the test concentrations was based on the results of a range-finding test and on results of pre-experiment to determine the solubility of the test item.
- Results used to determine the conditions for the definitive study: not provided
Reference substance (positive control):
yes
Remarks:
performed about every half year
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
48 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
79 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: corresponds to nominal > 100 mg a.i./L
Details on results:
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no
- Unusual cell shape: The microscopic examination of the algal cells at the end of the test showed no difference between the algae growing at the nominal test concentration of 294 mg test item/L and the algal cells in the control. The shape and size of the algal cells were obviously not affected by the test item up to at least this concentration.
- Colour differences: not reported
- Flocculation: not reported
- Adherence to test vessels: not reported
- Aggregation of algal cells:no
- Other: No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period (foamy solutions were observed on Day 0 at the test concentrations of 147 and 294 mg test item/L).
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no, measured concentrations within 80-120% of nominal
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
For evaluation of the algal quality and experimental conditions, potassium dichromate is tested as a positive control twice a year to demonstrate satisfactory test conditions. The result of the latest positive control test performed in September 2012 showed that the sensitivity of the test system was within the internal historical range (72-hour EC50 for the growth rate: 0.69 mg test item/L (Harlan Laboratories Study D64300), range of the 72-hour EC50 for the growth rate from 2000 to 2012: 0.64-1.1 mg test item/L).
Reported statistics and error estimates:
The 72-hour EC10, EC20 and EC50 values for the inhibition of average growth rate and yield and their 95% confidence intervals were calculated as far as possible by Probit Analysis using linear maximum likelihood regression.

For the determination of the LOEC and NOEC, the average growth rate and yield at the test concentrations were compared to the control values by Williams t test.

In the control, the biomass increased by a factor of 64 over 72 hours. The validity criterion of increase of biomass by at least a factor of 16 within three days was fulfilled. The mean coefficient of variation of the daily growth rates in the control (section-by-section growth rates, Table 4) during 72 hours was 23%. According to the OECD test guideline, the mean coefficient of variation must not be higher than 35%. Thus, the validity criterion was fulfilled. The coefficient of variation of the average specific growth rates in the replicates of the control after 72 hours was 3.8%. According to the OECD test guideline, the coefficient of variation must not be higher than 7%. Thus, the validity criterion was fulfilled.

Validity criteria fulfilled:
yes
Executive summary:

In the Klimisch 1 GLP study from Kimmel (2013) the toxicity of Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18 unsaturated alkyl) tetrasodium salts to Desmodesmus subspicatus was determined in a static 72-Hour Algal Growth Inhibition Test. The test was performed according to OECD 201 and EU Method C.3. The nominal concentrations of the test item of 9.19, 18.4, 36.8, 73.5, 147 and 294 mg test item/L(corresponding to 3.125, 6.25, 12.5, 25, 50 and 100 mg solid content/L) were tested in parallel with a control. Dose verification analysis was performed . The measured concentrations of the test item in the biologically relevant test media of the nominal test concentrations of 36.8 to 294 mg test item/L were between 87 and 105% of the nominal values at the start of the test and between 85 and 97% at the end of the test. In order to determine the growth of the cultures, the cultures were measured with the fluorescent method . The cell density was determined at 0, 24, 48 and 72 hours. The growth of the control cultures fulfilled the validity criteria from OECD 201 (2006). The 72 -hour ErC10 and ErC50 are 48 and > 100 mg solid content/L based on the mean measured concentration.

This information is considered to be relevant and reliable for the further risk assessment.

Description of key information

72 h ErC10: 48 mg/L
72 h ErC50: > 100 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:
100 mg/L
EC10 or NOEC for freshwater algae:
48 mg/L

Additional information

In the Klimisch 1 GLP study from Kimmel (2013) the toxicity of Aspartic acid, N-(3-carboxy-1-oxo-sulfopropyl)-N-(C16-C18 (even numbered), C18 unsaturated alkyl) tetrasodium salts to Desmodesmus subspicatus was determined in a static 72-Hour Algal Growth Inhibition Test. The test was performed according to OECD 201 and EU Method C.3. The nominal concentrations of the test item of 9.19, 18.4, 36.8, 73.5, 147 and 294 mg test item/L(corresponding to 3.125, 6.25, 12.5, 25, 50 and 100 mg solid content/L) were tested in parallel with a control. Dose verification analysis was performed . The measured concentrations of the test item in the biologically relevant test media of the nominal test concentrations of 36.8 to 294 mg test item/L were between 87 and 105% of the nominal values at the start of the test and between 85 and 97% at the end of the test. In order to determine the growth of the cultures, the cultures were measured with the fluorescent method . The cell density was determined at 0, 24, 48 and 72 hours. The growth of the control cultures fulfilled the validity criteria from OECD 201 (2006). The 72 -hour ErC10 and ErC50 are 48 and > 100 mg solid content/L based on the mean measured concentration.

This information is considered to be relevant and reliable for the further risk assessment.

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