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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:
guideline study
Remarks:
Guideline compliant GLP study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
Control, 0.32, 1.0, 3.2, 10, 32 and 100 mg test item/L were sampled. At the lowest test concentration of 0.32 mg/L the measured concentration of the test item was lower than LOQ (LOQ=0.981 mg/L) at the start and end of the test. .

- 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 characterization of the WAFs, duplicate samples were taken from the test media of all test concentrations at the start of the test (without algae, 100 and 7.5 mL volume) and at the end of the test, with algae (100 mL and 7.5 mL volume). At the same sampling times, duplicate samples were also taken from the control. One sample without algae (7.5 mL volume) of all test concentrations and control was also taken at the end of the test. For all 72-hour stability samples, additional flasks containing the test media with and without algae were incubated for each treatment under the test conditions. All 7.5 mL samples were diluted with acetonitrile. Immediately after sampling, acetonitrile (2.5 mL 85% phosphoric acid per 7.5 mL sample volume) was added to each 7.5 mL sample in order to stabilize the latter during the storage period. The 100 mL samples were not diluted with acetonitrile.

- Sample storage conditions before analysis:
Thereafter, all samples were stored deep-frozen (at about -20 °C). 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 Sodium bis(C11-14-isoalkyl, C13-rich) sulfosuccinate were determined in one of the duplicate test media samples from each sampling time
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For preparation of the WAFs, individual dispersions of the test item with the loading rates as mentioned above were prepared using ultrasonic treatment for 30 minutes. The WAFs of 3.2 to 100 mg/L were prepared by diuting the appropriate amounts in 3000 mL dilution water. The dispersions were stirred for 24 hours at room temperature in the dark to dissolve a maximum amount of the different compounds of the test item. The dispersions were not filtered since the test item was uniformly distributed in the test media and the dispersions were directly tested after stirring. Due to technical reasons, the dispersions with the lowest concentrations of 0.32 and 1.0 mg/L were prepared as dilutions of the WAF with the concentration of 3.2 mg/L.
- Controls: dilution water only
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The test media with the test concentrations of 0.32 and 1.0 mg/L were clear solutions throughout the test period. The test media with the test concentrations of 3.2 to 100 mg/L were turbid and homogeneous dispersions in the test water on Day 0. Thereafter the test media were clear (3.2 and 10 mg/L) or homogeneous dispersions (32 and 100 mg/L) until the end of the test.
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 23 °C.
pH:
The pH of the test media was in the range of 7.9 to 8.4 during the test period
Nominal and measured concentrations:
nominal: control and 0.32, 1.0, 3.2, 10, 32 and 100 mg/L. measured concentrations were 84 and 114% of nominal concentrations in the test solutions of 1.0 to 100 mg/L of the freshly prepared and aged test solutions, respectively. Hence the data were reported based on nominal concentrations.
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: 143000 cells/mL (average)
- 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: 7900 Lux (range: 7190 to 8460 Lux; fluorescent tubes (Philips TLD 36W-1/840)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations:The algal biomass in the samples was determined by measurement of the algal cell density using an electronic particle counter (Coulter Counter, Model Z2). The measurements were performed at least in duplicate.
- 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.:
8.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
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
- 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 100 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
- 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, no difference found
- 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 29 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) during 72 hours was 34.5%. 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 3.0%. 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 Sodium bis(C11-14-isoalkyl, C13-rich) sulfosuccinate toDesmodesmus subspicatuswas 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 test concentrations were 0 (control), 0.32, 1.0, 3.2, 10, 32 and 100 mg test item/L. Six control replicates and 3 replicates from each test solution were set up. Dose verification analysis was performed and confirmed the correct dosage and stability of the test item throughout the test period. In order to determine the growth of the cultures, the cells were counted by coulter counter measurement. 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 both 8.7 and > 100 mg test item/L based on the nominal concentration.

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

Description of key information

Key value for chemical safety assessment

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

Additional information

In the Klimisch 1 GLP study from Kimmel (2013) the toxicity of Sodium bis(C11-14-isoalkyl, C13-rich) sulfosuccinate toDesmodesmus subspicatuswas 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 test concentrations were 0 (control), 0.32, 1.0, 3.2, 10, 32 and 100 mg test item/L. Six control replicates and 3 replicates from each test solution were set up. Dose verification analysis was performed and confirmed the correct dosage and stability of the test item throughout the test period. In order to determine the growth of the cultures, the cells were counted by coulter counter measurement. 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 both 8.7 and > 100 mg test item/L based on the nominal concentration.

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