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Ecotoxicological information

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
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:
Control, 2.2, 4.6, 10, 22, 46, 100 mg test item/L were sampled, since no biological effects were observed in any concentration, only the controls and teh 100 mg/L test solutions (freshly prepared and aged) were analysed.

- 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 sampling at the end of the test, the test medium of the treatment replicates were pooled. All samples were stored deep-frozen (at about -20 °C) immediately after sampling until analysis. In pre-experiments for investigation of the storage stability ot the samples (GLP), 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 concentration of 100 mg/L. From the control samples, one of the duplicate samples was analyzed per sampling time. Aliquots of 1 mL were diluted to 11 mL with test water giving a sample preparation factor of 11.

- Sample storage conditions before analysis:
The samples were stored deep frozen until analysis. Test samples and control samples were thawed at room temperature for 1.5 hours and shaken manually to obtain homogeneous sample solutions. The test and control samples from day 3 were centrifuged (3500 rpm, 5 min) due to the presence of algae. If necessary, the samples were further diluted into the calibration range with test water before they were analyzed.
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 100 mg/L was prepared by dissolving 50.18 mg of the test item completely in 500 mL of test water using ultrasonic treatment for 15 minutes and intense stirring for 10 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, analytical recoveries at the end of the test confirm that the test item was stable during the exposure period.
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 within 22-23 °C.
pH:
The pH of the test media was in the range of 7.8 to 9.1 during the test period
Nominal and measured concentrations:
nominal: control and 2.2, 4.6, 10. 22. 46 and 100 mg test item/L. measured concentrations were 91 and 92% of nominal concentration of 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: 735000 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: 6700 Lux (range: 6280 to 7180 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 fluorescence measurement (BIO-TEK Multi-Detection Microplate Reader, Model FLx800, wavelength: excitation 440 nm, emission 680 nm). The measurements were performed at least in duplicate.
- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- 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.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 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
- Other: No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the test period.
- 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, EC20and EC50 values of the test item could not be determined because of the absence of a significant inhibitory effect of the test item on the algal growth at the tested concentrations.

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 147 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 21%. 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 2.0%. 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 di(1.3-dimethylbutyl) sulfosuccinate 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 test concentrations were 0 (control), 2.2, 4.6, 10, 22, 46 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 at 100 mg/L. In order to determine the growth of the cultures, the cells were counted by fluorescence 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 > 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:
100 mg/L

Additional information

In the Klimisch 1 GLP study from Kimmel (2013) the toxicity ofSodium di(1.3-dimethylbutyl) sulfosuccinatetoDesmodesmus 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), 2.2, 4.6, 10, 22, 46 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 at 100 mg/L. In order to determine the growth of the cultures, the cells were counted byfluorescence 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 > 100 mg test item/L based on the nominal concentration.

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