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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-07-11 to 2014-08-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study in accordance with guidance with no deviations and test concentrations monitored by analytic measurement
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
2013-04-12
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
The dilutions 1:46, 1:22, 1:10, 1:4.6, 1:2.2 and the undiluted filtrate (with the loading rate of 100 mg/L) were used as test media. Additionally, a control was tested in parallel (test water without test item).

- Sampling method:
For measurement of the actual test item concentrations, 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 was pooled. Immediately after sampling, acetonitrile was added to each sample in the ratio of 9:1 to stabilize the latter during the storage period.

- Sample storage conditions before analysis:
All samples were stored deep-frozen at about -20 °C immediately after sampling.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
Due to the low solubility of the test item in test medium, dispersion with the loading rate of 100 mg/L was prepared at the start of the test by dispersing 107.28 mg of the test item in 1070 mL of test medium. This preparation was supported by ultrasonic treatment for 15 minutes and intense stirring on a magnetic stirrer over 3 hours in the dark, to dissolve a maximum amount of the test item in the dispersion. No auxiliary solvent or emulsifier was used.
After the 3-hour stirring period, the dispersion of the test item was filtered through a membrane filter (0.45 μm) after precondition of the filter with about 200 mL of the dispersion. The negative pressure of the filtration unit was reduced as far as possible to avoid losses of volatile components of the test item during filtration. The undiluted filtrate was used as the highest concentrated test medium and as a stock solution for the preparation of all test media. The test media were prepared just before the start of the test.
- Controls: Test water without test item
- Evidence of undissolved material: All test media were clear solutions throughout the entire test duration.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Strain: 61.81 SAG
- Source: Institute for Plant Physiology, University of Göttigen, 37073 Göttingen/Germany and cultivated at Harlan laboratory
- Method of cultivation: standardized conditions according to the test guidelines

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions: Same as test conditions
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
0.15 mmol/L (=15 mg/L as CaCO3)
Test temperature:
23-24 °C
pH:
pH in the control: 7.9-8.3
pH in the test media: 7.7-8.4
Nominal and measured concentrations:
The nominal concentrations were: dilution 1:46, 1:22, 1:10, 1:4.6; 1:2.2 and the undiluted filtrate.
Measured concentrations in the fresh test samples were: 0.546 mg/L (dilution 1:4.6), 1.04 mg/L (dilution 1:2.2) and 2.35 mg/L (undiluted filtrate).
Measured concentrations in the old test samples were: 0.365 mg/L (dilution 1:4.6), 0.764 mg/L (dilution 1:2.2) and 1.76 mg/L (undiluted filtrate).

Details on test conditions:
TEST SYSTEM
- Test vessel: 50 mL Erlenmeyer flasks with glass-stopper
- Type: closed
- Headspace: No head space
- Initial cells density: 5000 cells/mL
- No. of vessels per concentration (replicates): 3 replicates
- No. of vessels per control (replicates): 6 replicates

GROWTH MEDIUM
- Standard medium used: yes (AAP medium)

OTHER TEST CONDITIONS
- Adjustment of pH: 6 mmol/L HEPES-buffer (corresponding to 1430 mg/L)
- Light intensity and quality: 7200 Lux (range: 6450 to 8100 Lux) within +/-15% deviation
- Illumination: fluorescent tubes (Philips TLD 36W-1/840)
- Photoperiod: continuous

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: electronic particle counter
- Chlorophyll measurement: fluorescence measurement (wavelength: excitation 440 nm, emission 680 nm)

TEST CONCENTRATIONS
- Spacing factor for test concentrations of definitive study: 2.2
- Test concentrations for the range finding study: undiluted filtrate (with a loading rate 100 mg/L), dilution 1:10 and dilution 1:100
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
> 2 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.:
0.89 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 2 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.62 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95% CL:0.31-0.85 mg/L
Details on results:
- Exponential growth in the control: yes
- Observation of abnormalities: no difference between the algae growing at the highest concentration (undiluted filtrate) and Algal cells in the control.
- Unusual cell shape: not obviously affected by the test item
- Observation: all test media were clear solutions throughout the entire test duration
Results with reference substance (positive control):
- EC50 (for the growth rate): 1.1 mg/L (the range of EC50 (72 h) for the growth rate
Validity criteria fulfilled:
yes
Conclusions:
Based on growth rate the EC50 and EC10 were determined to be above the water solubility of the test substance.
Executive summary:

The toxicity of the test substance to the unicellular freshwater alga Pseudokirchneriella subcapitata was investigated under GLP according to the procedures of the OECD Guideline 201 (2011), as well as according to the EC-Guideline No. 761/2009 C.3 (2009).

Since the test item was determined to be volatile, a closed test system without headspace was used to avoid potential losses of the test item. The algae were exposed to saturated solution and five dilutions (three replicates were tested for each concentration) over a period of 72 hours at a temperature ranging from 23 to 24 °C. The test concentrations were monitored by analytic measurement. The measured concentrations of the test substance after 72 hours of the exposure were in the range of 67 - 75% of the initial measured values. Therefore, all effect values reported are based on mean measured test substance concentrations.

The ErC50 and ErC10 values were determined to be above the water solubility of the test substance (> 2 mg/L).

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1992-07-24 to 1992-08-07
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study according to official guideline, but no analytical confirmation of test concentrations. The test item was applied above the level of maximum water solubility.
Qualifier:
according to
Guideline:
other: DIN 38412, part 9
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: a stock solution was prepared at a concentration of 1 g/L by weighing of appropriate amounts, addition to water and vigorous stirring with an Ultraturrax
- Controls: three controls with no test substance
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no solvents, emulsifiers or dispersants were used
- Evidence of undissolved material (e.g. precipitate, surface film, etc): not reported
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
- Strain: SAG 8681
- Source (laboratory, culture collection): Pflanzenphysiologisches Institut der Universität Göttingen, Germany
- Age of inoculum (at test initiation): 1 mL of algal suspension was inoculated into test medium three days prior to the test
- Method of cultivation: in culture medium
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
not applicable
Hardness:
not reported
Test temperature:
22.5 to 23 °C
pH:
8.0 to 10.5
Dissolved oxygen:
not reported
Salinity:
not applicable
Nominal and measured concentrations:
10, 30 and 100 mg/L
Details on test conditions:
TEST SYSTEM
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 300 mL Erlenmeyer flasks, test volume was 100 mL
- Initial cells density: 10000 cells/mL
- Control end cells density: 3.9E06 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuous
- Light intensity and quality: 2000 lux (Li-Cor-Li-185 B lamp)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: cell growth
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
53 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC0
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
96 h
Dose descriptor:
EC10
Effect conc.:
23 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): none reported
- Unusual cell shape: none reported
- Colour differences: none reported
- Flocculation: none reported
- Adherence to test vessels: none reported
- Aggregation of algal cells: none reported
- Any stimulation of growth found in any treatment: no
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none reported
- Effect concentrations exceeding solubility of substance in test medium: Yes. Nominal test item concentrations were 10, 30, and 100 mg/L, whereas the study on water solubility revealed a solubility of 1.12 mg/L only.
Validity criteria fulfilled:
yes
Conclusions:
An ErC50 value of 53 mg/L over a period of 96 hours was determined in an algal growth inhibition study with Desmodesmus subspicatus. The test showed only effects above the water solubility of 1.12 mg/L.The inhibitory effect therefore presumably was caused by physical effects rather than systemic effects.
Executive summary:

The algal toxicity of the test substance was studied with the freshwater green alga Desmodesmus subspicatus under GLP in a valid test according to DIN 38412, part 9, which is essentially equivalent to EU Method C.3. Freshwater green algae (Desmodesmus subspicatus) were inoculated at 1 mL in standard test medium three days before the test to obtain an initial cell concentration of 10E04 cells/mL. The algae were then exposed to nominal test concentrations of 10, 30 or 100 mg/L (three replicates were tested for each concentration) over a period of 96 hours under continuous illumination (2000 lux) at a temperature ranging from 22.5 to 23 °C. The cell number was counted at defined intervals and the growth rate and biomass were determined and compared to those of the untreated controls (also three replicates treated in a similar way but not exposed to the test substance). The inhibition of the growth rate with the three nominal test concentrations was evaluated and the inhibition of the growth rate by 50% was calculated from the data: the ErC50 value was 53 mg/L over the period of 96 hours. No effects were seen at the lowest nominal test concentration of 10 mg/L and the 96-hour NOEC was 10 mg/L.

The observed growth inhibition effect (ErC50 = 53 mg/L) however is presumably due to physical effects rather than systemic effects. The test item was being applied far beyond the level of maximum water solubility, which was determined to be 1.12 mg/L in a valid study on water solubility. In the two other acute aquatic toxicity studies, slightly higher water solubility values were found in real test water (2.67 mg/L in the fish study, and 3.7 mg/L in the Daphnia study). Given the fact that the test item in this algal growth inhibition study was applied in nominal concentrations of 10, 30, and 100 mg/L, thus far above the level of maximum water solubility, the same (maximum) dissolved test item concentration was to be expected in all these saturated solutions. Subsequently at all the applied nominal test item concentrations the same level of maximum algal growth inhibition was to be expected if the effect was based on systemic toxicity caused by the fraction of dissolved (bioavailable) test item. As however no effect was observed at 10 mg/L test item concentration, but concentration-dependant algal growth inhibition was observed at 30 and 100 mg/L, it is safe to say that no effects were observed within the range of the water solubility of the test item. The growth inhibition effect therefore is very unlikely to be caused by systemic effects, but rather caused by physical effects which are coming from increased concentrations of non-dissolved test item inside the test batches. Adverse effects on algae therefore were caused by nominal test item concentrations that were at least one order of magnitude higher than the soluble concentration. Physical effects on the algae caused by the test item, such as adsorption and shading, are considered as likely causes for the observed algal growth inhibition effect, however such effects are not to be attributed to toxic properties of the substance.

Description of key information

The test substance is not toxic for aquatic algae. Two tests showed that the ErC50 and ErC10 are above the water solubility of the test item (> 2 mg/L). 

Key value for chemical safety assessment

Additional information

The toxicity of the test substance to algae was investigated in two valid tests showing that the ErC50 and ErC10 are above the water solubility of the test item.

In the key study (Symrise, 2014, RL1), the toxicity of the test substance to the unicellular freshwater alga Pseudokirchneriella subcapitata was investigated under GLP over 72 hours in a static test. The study was performed according to OECD Guideline 201. Since the test item was determined to be volatile, a closed test system without headspace was used to avoid potential losses of the test item. The algae were exposed to the saturated solution and five dilutions (1:2.2, 1:4.6, 1:10, 1:22 and 1:46). The test concentrations were monitored by analytic measurement at the beginning (t=0h) and at the end of the test (t=72h). The measured concentrations of the test substance after 72 hours of exposure were in the range of 67 - 75% of the initial measured values. Therefore, all effect values reported are based on mean measured test substance concentrations. The ErC50 and ErC10 values were determined to be above the water solubility of the test substance (> 2 mg/L).

In the supporting study the inhibitory effect of the substance to Desmodesmus subspicatus was studied under GLP over a period of 96 hours. The study was performed in accordance with DIN 38412, part 9, which is equivalent to EU Method C.3. Three nominal test concentrations of 10, 30 and 100 mg/L were tested. Since these concentrations were not confirmed analyticallyand far above the water solubility, the study should be regarded as reliable with restrictions(RL2). All three test concentrations are above the experimentally determined water solubility of the substance registered (1.1 mg/L) and there is the likelihood that the greatest portion of the test substance was present as non-dissolved substance. No effect on the algal growth rate was seen at the lowest test concentration of 10 mg/L.Due to the low solubility of the substance, the test medium should have been saturated with test substance at this nominal concentration.Therefore, itcanbe argued that no effects on algal growthwere observedwithin the water solubility of the test substance,which is supported by the key study.The observed growth inhibition effect (ErC50 = 53 mg/L) however is presumably due to physical effects rather than systemic effects. Given the fact that the test item in this algal growth inhibition study was applied in nominal concentrations of 10, 30, and 100 mg/L, thus far above the level of maximum water solubility, the same (maximum) dissolved test item concentration was to be expected in all these saturated solutions. Subsequently at all the applied nominal test item concentrations the same level of maximum algal growth inhibition was to be expected if the effect was based on systemic toxicity caused by the fraction of dissolved (bioavailable) test item. As however no effect was observed at 10 mg/L test item concentration, but concentration-dependant algal growth inhibition was observed at 30 and 100 mg/L, it is safe to say that no effects were observed within the range of the water solubility of the test item. The growth inhibition effect therefore is very unlikely to be caused by systemic effects, but rather caused by physical effects which are coming from increased concentrations of non-dissolved test item. Adverse effects on algae therefore were caused by nominal test item concentrations that were at least one order of magnitude higher than the soluble concentration. Physical effects on the algae caused by the test item, such as adsorption and shading, are considered as likely causes for the observed algal growth inhibition effect, however such effects are not to be attributed to toxic properties of the substance.