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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:
weight of evidence
Study period:
2009-05-29 to 2009-10-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2006
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Details on sampling:
The test was started (0 hours) by inoculation of a biomass of approximately 1E04 algal cells per mL test medium. These cells were taken from an exponentially growing pre-culture, set up three days prior to the test start using the same conditions as in the test.
Vehicle:
no
Details on test solutions:
A supersaturated test item solution (Stock solution = S.S.) was prepared by dispersing/dissolving the test item amount without the use of any organic solvent into the Test Medium (OECD Medium) two days before the start of the definitive study (on day -2). This solution was shaken for 24 hours at app. 30 °C, then was left settling for 24 hours at app. 20 °C and thereafter filtrated through a 0.45 μm filter.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The algae were supplied by the Georg-August-Universität Göttingen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften Experimentelle Phykologie und Sammlung von Algenkulturen (SAG).
The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardised conditions according to the test guidelines.
The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with Algal Mineral Salts Culture Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about three days. (The pre-culture was incubated for three days at this test.)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No post exposure observation period.
pH:
pH 7.50 to 8.02 at test start
pH 7.60 to 9.31 at test end
Nominal and measured concentrations:
The following nominal concentrations were used in the main study: S.S./1; S.S./2; S.S./4; S.S./8 and S.S./16 (S.S.: supersaturated Stock Solution >> limit of solubility of the test item in the media used). The corresponding calculated test item concentrations were 6.25; 12.5; 25.0; 50.0 and 100.0 mg/L as the percentage of the limit of solubility.
Details on test conditions:
- Breeding conditions:
The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardised conditions according to the test guidelines. The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with Algal Mineral Salts Culture Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about three days. (The pre-culture was incubated for three days at this test.)
- Temperature:
The cultures were maintained at a temperature in the range of 21 – 24 °C ± 2 °C, which was checked and recorded at the beginning of the study and every 24 hours in a flask filled with water. In addition, the temperature was continuously measured (with a min/max thermometer) within the climate chamber.
- pH:
The pH was checked at the beginning and at the end of the study in the controls and every concentration. The pH was not adjusted throughout the test period and the deviation was less than 1.5 units in each test.
- Light intensity:
The light intensity at the position occupied by algal culture flasks during the test was about 8127 lux, which was ensured with fluorescent lamps (with a spectral range of 400-700 nm), and checked periodically.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
37.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95 % conf. limits: 34.6 – 41.3 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
51.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % conf. limits: 48.7 – 55.2 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
38.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95 % conf. limits: 35.9 – 41.2 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
The test item had a statistically significant inhibitory effect on the growth based on the average specific growth rate, yield and areas under the growth curves of Pseudokirchneriella subcapitata after the exposure period of 72 hours in the concentration range of 50 – 100 mg/L of the test item. The test concentration of 50 mg/L was determined as the 72-hour LOEC. The test concentration of 25 mg/L was determined as the 72-hour NOEC.
The EC-values were calculated for the algal biomass b, for the growth rate r and for the yield y after 72-hour test duration and they are detailed in the results section above.
Shape of the algal cells: The shape of the algal cells growing was obviously not affected.
Cell density on control: In the control the cell density has increased from nominal N = 1E04 cells/mL at the start of the test (0 hours) to N = 70.50E04 cells/mL (mean value) after 72 hours by a factor of 70.50. Thus, the algal growth in the control was high enough to pass the validity criteria in this assay.
Results with reference substance (positive control):
Results of the reference substance proved that the test is valid.
Reported statistics and error estimates:
Mean values and standard deviations of cell concentrations were calculated for each treatment at the start, after 0 h, 24 h, 48 h and at the end of the test (72 hours after the start of the test) using Excel for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399). Percentage inhibition of biomass (area under the growth curves, A), growth rate r and yield Y were calculated using EXCEL for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399). The EC50 values of the test item and their confidence limits were calculated using Probit analysis. The analysis was done using the statistical software program “TOXSTAT 3.5”. For the determination of the LOEC and NOEC, the calculated mean biomass b (area under the growth curve), growth rates r and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test by TOXSTAT software.
Validity criteria fulfilled:
yes
Conclusions:
In this 72-h algal growth inhibition test on Pseudokirchneriella subcapitata with Sika Hardener LG, the 72-h EC50 based on growth rate was determined as 51.9 mg/mL. The 72-h EC50 based on yield was 38.5 mg/mL and the overall NOEC was determined to be 25.0 mg/mL of the limit of solubility.
Executive summary:

The purpose of this study was to determine the effect of the test item Sika Hardener LG on the growth of an unicellular green algal species Pseudokirchneriella subcapitata (formerly: Selenastrum capricornutum) according to EU method C.3 and OECD guideline no. 201. Exponentially growing cultures of Pseudokirchneriella subcapitata were exposed to various concentrations of the test item (6.25; 12.5; 25.0; 50.0 and 100.0 mg/L.) over several generations under defined conditions. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and thus, over several algal generations. A supersaturated test item solution was prepared by dispersing the test item amount (nominal load of 100 mg/L) into the test medium two days before the start of the renewal periods. This solution was shaken for 24 hours at app. 30 °C, then was left settling for 24 hours at app. 20 °C and thereafter filtrated through a 0.45 μm filter.

The shape of the algal cells growing was obviously not affected. The test item had a statistically significant inhibitory effect on the growth based on the average specific growth rate, yield and areas under the growth curves of Pseudokirchneriella subcapitata after the 72 exposure period in the concentration range tested. A test concentration of 50.0 mg/mL of the was determined as the 72-hour LOEC. A test concentration of 25.0 mg/mL was determined as the 72-hour NOEC. The 72- hour EC50 based on growth rate was determined as 51.9 mg/mL, a EC50 based on yield of 38.5 mg/mL.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-08-18 to 2005-08-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study, OECD guideline, Read-across to hydrolysis product.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
adopted 2002
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
adopted 1992
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Sampling preparation:
Aqueous samples (10.0 mL) were membrane filtrated (Nylon, 0.45 μm). The filtrate was acidified with 2M-HCl to pH 2 – 3. 10.0 mL of the filtrate were given on the top of a column with 5.0 g Extrelute. After 30 minutes, elution was slowly performed with ethyl acetate (50 mL, 30 minutes), the eluate was rotated down to dryness and 500 μL ISTD-solution were added. At last, 400 μL of the solution were mixed with 50 resp. 100 μL SIL -Mix in vials, and silylation was performed at 80 °C for 60 minutes.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
1. Experiment:
- Method: As solubility lies below 100 mg/L, the water-accommodated fraction was prepared for the test. This was done by weighing the nominal load of 100 mg/L, adding the corresponding amount of deionized water and shaking vigorously for 24 hours. The resulting solution was filtrated through 0.45 μm filters.
- Differential loading: 100 mg/L
- Controls: six replicates (deionized water with nutrient medium and algae)
- Replicates: six replicates
2. Experiment:
- Method: The water-accommodated fraction was prepared for the test. This was done by weighing the nominal load of 100 mg/L, adding the corresponding amount of deionized water and stirring slowly for 2 hours. The solution was left to stand for 15 minutes. Then the solution was filtrated.
- Differential loading: 100 mg/L
- Controls: six replicates (deionized water with nutrient medium and algae)
- Replicates: three replicates
3. Experiment:
- Method: A stock solution containing 1 g/L in acetone was prepared. This solution was used to prepare the treatment. 100 μL of the acetonic stock solution per litre were used.
- Differential loading: 1 mg/L
- Controls: six replicates (deionized water with 100 μL/L acetone, nutrient medium and algae)
- Replicates: six replicates for the treatment, additional three replicates of control and treatment for analytical determination after 4 hours
4. Experiment:
- Method: A stock solution containing 1 g/L in acetone was prepared. This solution was used to prepare the treatments to be tested. 100 μL of the acetonic stock solution per litre were used at the most.
- Differential loading: 1 mg/L
- Controls: six replicates (deionized water with 100 μL/L acetone, nutrient medium and algae)
- Replicates: three replicates for each treatment, additional three replicates of control and each treatment for analytical determination after 4 hours
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: CHODAT
- Source: Institut für Pflanzenphysiologie of Universität Göttingen
- Method of cultivation: Four days before the start of each test, an aliquot of the stock culture containing a few cells was brought into 50 mL pre-culture medium and incubated for 96 hours. The resulting culture is growing exponentially. After adjustment to a cell concentration of about 5E04 cells/mL by photometric measurement and addition of pre-culture medium, the culture was usable for the test.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
1. Experiment: 24 - 25 °C
2. Experiment: 23 - 25 °C
3. Experiment: 24 °C
pH:
1. Experiment: 8.9 to 9.6
2. Experiment: 8.5 to 9.4
3. Experiment: 8.6 to 9.4
Nominal and measured concentrations:
1. Experiment
100 mg/L
2. Experiment
100 mg/L
3. Experiment
1 mg/L
4. Experiment
0.1 mg/L; 0.18 mg/L; 0.32 mg/L, 0.56 mg/L and 1 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: screw cap cuvettes d = 6mm
- Initial cells density: 5E04 cells/mL
- No. of vessels per concentration (replicates): six replicates
- No. of vessels per control (replicates): three to six replicates

GROWTH MEDIUM
- in accordance to the guideline

TEST MEDIUM / WATER PARAMETERS
- in accordance to the guideline

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light intensity and quality: 8000 ± 2000 Lux

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: photometer
Reference substance (positive control):
no
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
1. Experiment: The test solution went flocculent during the test, probably caused by undissolved parts of the test item. Therefore the WAF for the second experiment was prepared by stirring instead of shaking.
2. Experiment: Because of the test item’s physico-chemical properties causing film-formation at the top of the water phase, the preparation of the WAF is not very reproducible. Therefore for the third experiment, a stock solution in acetone was prepared and the test solutions were prepared by spiking the necessary amount of deionized water with this stock solution.
3. Experiment: As the algal growth was inhibited, a further experiment using five concentrations of the test item in a geometric series was performed.
4. Experiment: Only a slight inhibition could be observed. As a dose-response correlation is visible, this test was used to determine the results.
Reported statistics and error estimates:
Determination of the NOEC
The difference between the treatment 0.18 mg/L and the control can be considered as not significant (level of significance: 97.5 %) as all calculated t-values were smaller than the limit of significance. Therefore the concentration 0.18 mg/L is stated as NOEC.
Determination of the LOEC
As the difference between the treatment 0.32 mg/L and the control can be considered as significant, the treatment 0.32 mg/L can be stated as LOEC.

3. Experiment:

At the beginning and 4 hours after the start of the test, the content of the test item in each test solution was determined using GC. The measured concentration of the fresh treatment at the start of the test was 32 % of the nominal concentration. Sample preparation and clean-up requires about one hour. During this time, the test item already undergoes hydrolysis which probably caused this low recovery. The measured concentration after 4 hours was below the limit of quantification. The test item is not stable in water (as has been demonstrated in the respective study “determination of water solubility”). Due to the rapid degradation, referring to determined values is not meaningful and only the nominal values were used for the determination of the biological results.

4. Experiment:

At the begin and 4 hours after the start of the test, the content of the test item in each test solution was determined using GC. The measured concentrations of the freshly prepared test solutions (at the start of the test) were between 27 and 30 % of the nominal concentrations. The three lower treatments lay below the limit of quantification. Sample preparation and clean-up requires about one hour. During this time, the test item already undergoes hydrolysis which probably caused these low recoveries. After 4 hours, the test item could be detected only in the highest treatment. The measured concentration was 8 % of the nominal concentration. The test item is not stable in water (as has been demonstrated in the respective study “water solubility determination”). Due to the fast degradation referring to determined values is not meaningful and only the nominal values were used.

Validity criteria fulfilled:
yes
Conclusions:
The 72-h EC50 was determined to be > 1 mg/L and the 72-h NOEC was 0.18 mg/L.
Executive summary:

The second study was performed in order to evaluate the toxic potential of the hydrolysis product 2,2 -Dimethyl-3 -lauroyloxy-propanal (aldehyde component) the toxic potential of test item towards Desmodesmus subspicatus CHODAT, an unicellular freshwater green alga. The incubation period of 72 hours corresponds to a subchronic test, as during this period, about five cell divisions take place. Four experiments were performed. As the test item is poorly water soluble, the “water-accommodated fraction” was tested in the first experiment. This was done by weighing the nominal load 100 mg/L, adding the corresponding amount of dilution water and shaking vigorously for 24 hours. The resulting solution was filtrated through 0.45 μm filters. The treatment was used to incubate the unicellular freshwater green alga Desmodesmus subspicatus for a period of 72 hours. The cell concentration of each replicate was determined by measuring the absorption of the cuvettes at 440 nm every 24 hours with a spectral photometer. With these measured values, the number of cells was calculated (linear correlation between cell concentration and absorption given). Then the growth rate, the area under the growth curve (AUC1) and the yield2 were determined. The test solution went flocculent during the test probably caused by undissolved parts of the test item. The second experiment was performed likewise, but with stirring over 2 hours instead of shaking. The resulting solution was left to stand for about 15 minutes, and then the solution was filtrated. No inhibition was observed. The test item forms a surface film, thus the preparation of the WAF is not very reproducible. Therefore for the third experiment, a stock solution in acetone was prepared. The treatment concentration of 1 mg/L was prepared by spiking the corresponding amount of deionized water with this stock solution. The treatment concentration of 1 mg/L is considered to be well above maximal water solubility of 0.227 mg/L. 25 % inhibition of growth rate, 72 % inhibition of AUC, and 73 % inhibition of yield were observed. At the beginning and after 4 hours of the test, the content of the test item and its oxidation product (carbonic acid) in each test solution was determined using GC. Based on these experiments, the fourth experiment was performed using five concentrations between 0.1 and 1 mg/L. The test solutions were prepared by spiking with a stock solution in acetone. No inhibition occurred in the two lower concentrations. The three higher concentrations inhibited the algal growth only slightly. At the beginning and 4 hours after the start of the test, the content of the test item in each test solution was determined using GC. The measured concentrations of the freshly prepared test solutions (at the start of the test) were between 27 and 30 % of the nominal concentrations. The three lower treatments were below the limit of quantification. Sample preparation and clean-up requires about one hour. During this time, the test item already undergoes hydrolysis which caused these low recoveries. After 4 hours remaining test item was only detected in the highest concentration. The measured concentration was 8 % of the nominal concentration. The test item is not stable in water (as has been demonstrated in the respective study “water solubility determination”). Due to the fast degradation referring to determined values is not meaningful and only the nominal values were used. The 72-h EC50 was determined to be > 1 mg/L and the 72-h NOEC was 0.18 mg/L. These data were already submitted in a NONS dossier under Directive 92/32/EEC (notification number 05 -04 -1922 -00 from 2005 -10 -25) and considered valid and uncritical from the German Competent Authority (BAUA).

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2009-06-12 to 2009-06-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Read-across to supporting substance
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
2006
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Details on sampling:
The test was started (0 hours) by inoculation of a biomass of approximately 1E4 algal cells per mL test medium. These cells were taken from an exponentially growing pre-culture, set up three days prior to the test start using the same conditions as in the test.
Vehicle:
no
Details on test solutions:
The test item solution was prepared according to the WAF method (Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, section 3.1.2 Media preparation methods, Direct addition. OECD Series Testing and Assessment No.23, Paris September 2000).
A supersaturated test item solution (Stock solution = S.S.) was prepared by dispersing/dissolving the test item amount (nominal load of 100 mg/L) without the use of any organic solvent into the Test Medium (OECD Medium) two days before the start of the definitive study (on day -2). This solution was shaken for 24 hours at app. 30 °C, then was left settling for 24 hours at app. 20 °C and thereafter filtrated through a 0.45 μm filter.
The test solutions were prepared by appropriate dilution of the stock solution on day 0, before the start of the definitive test (= introduction of algae).
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
The algae were supplied by the Georg-August-Universität Göttingen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften Experimentelle Phykologie und Sammlung von Algenkulturen (SAG).
The stock cultures are small algal cultures that are planted on agar regularly. These are transferred to fresh medium at least once every two months under standardised conditions according to the test guidelines.
The pre-culture is intended to give an amount of algae suitable for the inoculation of test cultures. The pre-culture was prepared with Algal Mineral Salts Culture Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of about three days. (The pre-culture was incubated for three days at this test.)
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Test temperature:
23.3 – 23.5 °C
pH:
pH 7.58 to 8.00 at test start
pH 7.88 to 9.36 at test end
Nominal and measured concentrations:
The following nominal concentrations were used in the main study: 6.25; 12.5; 25.0; 50.0 and 100.0 mg/L.
Details on test conditions:
Test Units
- Type and Size:
Erlenmeyer flasks of 250 mL volume with 100 mL test medium
- Identification:
Each test unit was uniquely identified with study code, test item concentration and replicate number.

Test Conditions
- Temperature:
The cultures were maintained at a temperature in the range of 21 – 24 °C ± 2 °C, which was checked and recorded at the beginning of the study and every 24 hours in a flask filled with water. In addition, the temperature was continuously measured (with a min/max thermometer) within the climate chamber.
- pH:
The pH was checked at the beginning and at the end of the study in the controls and every concentration. The pH was not adjusted throughout the test period and the deviation was less than 1.5 units in each test.
- Light Intensity:
The light intensity at the position occupied by algal culture flasks during the test was about 8287 lux, which was ensured with fluorescent lamps (with a spectral range of 400 - 700 nm), and checked periodically.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
104.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95 % conf. limits: 86.4 – 125.8 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
180.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % conf. limits: 122.8 – 265.1 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
102.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95 % conf. limits: 86.3 – 122.2 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Details on results:
The test item had a statistically significant inhibitory effect on the growth based on the average specific growth rate, yield and areas under the growth curves of Pseudokirchneriella subcapitata after the exposure period of 72 hours in the concentration range of 50 – 100 mg/L of the test item.
The test concentration of 50 mg/L was determined as the 72-hour LOEC. The test concentration of 25 mg/L was determined as the 72-hour NOEC.
Results with reference substance (positive control):
ErC50 : 0.98 mg/L, (95 % confidence limits: 0.90 – 1.06 mg/L)
EbC50 : 0.67 mg/L, (95 % confidence limits: 0.62 – 0.73 mg/L)
EyC50 : 0.63 mg/L, (95 % confidence limits: 0.58 – 0.69 mg/L)
Reported statistics and error estimates:
Mean values and standard deviations of cell concentrations were calculated for each treatment at the start, after 0 h, 24 h, 48 h and at the end of the test (72 hours after the start of the test) using Excel for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399). Percentage inhibition of biomass (area under the growth curves, A), growth rate r and yield Y were calculated using EXCEL for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399). The EC50 values of the test item and their confidence limits were calculated using Probit analysis. The analysis was done using the statistical software program “TOXSTAT 3.5”. For the determination of the LOEC and NOEC, the calculated mean biomass b (area under the growth curve), growth rates r and yield at the test concentrations were tested on significant differences to the control values by Bonferroni t-Test by TOXSTAT software.
Validity criteria fulfilled:
yes
Conclusions:
A water accommodated fraction (WAF) of 100 mg/L was prepared with Sika Hardener LI. In this 72-h algal growth inhibition test with Pseudokirchneriella subcapitata the 72-h EC50 based on growth rate was determined as 180.4 mg/L. The 72-h EC50 based on yield was 102.7 mg/L. The NOEC based on growth rate was determined to be 50.0 mg/L. The results are based on the nominal concentrations.
Executive summary:

The effect of the test item Sika Hardener LI on the growth of an unicellular green algal species Pseudokirchneriella subcapitata (formerly: Selenastrum capricornutum) was assessed according to EU-method C.3 and OECD guideline 201. Exponentially growing cultures of Pseudokirchneriella subcapitata were exposed to various concentrations of the test item over several generations under defined conditions. The algal growth in relation to a concurrent control culture was determined over a fixed test period of 72 hours and thus, over several algal generations.

A supersaturated test item solution was prepared by dispersing the test item amount (nominal load of 100 mg/L) into the test medium two days before the start of the renewal periods. This solution was shaken for 24 hours at app. 30 °C, then was left settling for 24 hours at app. 20 °C and thereafter filtrated through a 0.45 μm filter. The following nominal concentrations were used: 6.25; 12.5; 25.0; 50.0 and 100.0 mg/L.

In this 72-h algal growth inhibition test with Pseudokirchneriella subcapitata the 72-h EC50 based on growth rate was determined at 180.4 mg/L. The 72-h EC50 based on yield was 102.7 mg/L. The NOEC based on growth rate was determined to be 50 mg/L. The overall NOEC was determined to be 25.0 mg/L. All results were based on the nominal concentrations.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
For justification for type of information please refer to the Read-Across Justification attached to IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.18 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
0.32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
For justification for type of information please refer to the Read-Across Justification attached to IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
104.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95 % conf. limits: 86.4 – 125.8 mg/L
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
180.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95 % conf. limits: 122.8 – 265.1 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
102.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield
Remarks on result:
other: 95 % conf. limits: 86.3 – 122.2 mg/L

Description of key information

Sika Hardener LG was assessed in an aquatic toxicty test to unicellular green algal (Pseudokirchneriella subcapitata) according EU-method C.3 and OECD guideline 201. A water accommodated fraction (WAF) was prepared and nominal concentrations of up to 100 mg/L tested. The 72-h EC50 based on growth rate was determined as 51.9 mg/L. The 72-h EC50 based on yield was 38.5 mg/L and the overall NOEC was determined to be 25.0 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:
51.9 mg/L
EC10 or NOEC for freshwater algae:
25 mg/L

Additional information

Due to the high reactivity of the substance in water, a weight of evidence approach using data form the parent molecule, its two primary hydrolysis products and from an analogous substance were assessed:

 

In the study with the parent compound Sika Hardener LG, the effect of the test item on the growth of an unicellular green algal species Pseudokirchneriella subcapitata (formerly: Selenastrum capricornutum) according to EU method C.3 and OECD guideline no. 201 were determined. Exponentially growing cultures of Pseudokirchneriella subcapitata were exposed to various concentrations of the test item (6.25; 12.5; 25.0; 50.0 and 100.0 mg/L.) over several generations under defined conditions. The algal growth in relation to a control culture was determined over a fixed test period of 72 hours and thus, over several algal generations. A supersaturated test item solution was prepared by dispersing the test item amount (nominal load of 100 mg/L) into the test medium two days before the start of the renewal periods. This solution was shaken for 24 hours at app. 30 °C, then was left settling for 24 hours at app. 20 °C and thereafter filtrated through a 0.45 μm filter. The shape of the algal cells growing was obviously not affected. The test item had a statistically significant inhibitory effect on the growth based on the average specific growth rate, yield and areas under the growth curves of Pseudokirchneriella subcapitata after the 72 exposure period in the concentration range tested. A test concentration of 50.0 mg/L was determined as the 72-hour LOEC. A test concentration of 25.0 mg/L was determined as the 72-hour NOEC. The 72- hour EC50 based on growth rate was determined as 51.9 mg/L, a EC50 based on yield of 38.5 mg/L.

 

The second study was performed in order to evaluate the toxic potential of the hydrolysis product 2,2-Dimethyl-3-lauroyloxy-propanal (aldehyde component) the toxic potential of test item towards Desmodesmus subspicatus CHODAT, an unicellular freshwater green alga. The incubation period of 72 hours corresponds to a subchronic test, as during this period, about five cell divisions take place. Four experiments were performed. As the test item is poorly water soluble, the “water-accommodated fraction” was tested in the first experiment. This was done by weighing the nominal load 100 mg/L, adding the corresponding amount of dilution water and shaking vigorously for 24 hours. The resulting solution was filtrated through 0.45 μm filters. The treatment was used to incubate the unicellular freshwater green alga Desmodesmus subspicatus for a period of 72 hours. The cell concentration of each replicate was determined by measuring the absorption of the cuvettes at 440 nm every 24 hours with a spectral photometer. With these measured values, the number of cells was calculated (linear correlation between cell concentration and absorption given). Then the growth rate, the area under the growth curve (AUC1) and the yield were determined. The test solution went flocculent during the test probably caused by undissolved parts of the test item. The second experiment was performed likewise, but with stirring over 2 hours instead of shaking. The resulting solution was left to stand for about 15 minutes, and then the solution was filtrated. No inhibition was observed. The test item forms a surface film, thus the preparation of the WAF is not very reproducible. Therefore for the third experiment, a stock solution in acetone was prepared. The treatment concentration of 1 mg/L was prepared by spiking the corresponding amount of deionized water with this stock solution. The treatment concentration of 1 mg/L is considered to be well above maximal water solubility of 0.227 mg/L. 25 % inhibition of growth rate, 72 % inhibition of AUC, and 73 % inhibition of yield were observed. At the beginning and after 4 hours of the test, the content of the test item and its oxidation product (carbonic acid) in each test solution was determined using GC. Based on these experiments, the fourth experiment was performed using five concentrations between 0.1 and 1 mg/L. The test solutions were prepared by spiking with a stock solution in acetone. No inhibition occurred in the two lower concentrations. The three higher concentrations inhibited the algal growth only slightly. At the beginning and 4 hours after the start of the test, the content of the test item in each test solution was determined using GC. The measured concentrations of the freshly prepared test solutions (at the start of the test) were between 27 and 30 % of the nominal concentrations. The three lower treatments were below the limit of quantification. Sample preparation and clean-up requires about one hour. During this time, the test item already undergoes hydrolysis which caused these low recoveries. After 4 hours remaining test item was only detected in the highest concentration. The measured concentration was 8 % of the nominal concentration. The test item is not stable in water (as has been demonstrated in the respective study “water solubility determination”). Due to the fast degradation referring to determined values is not meaningful and only the nominal values were used. The 72 h EC50 was determined to be > 1 mg/L and the 72 h NOEC was 0.18 mg/L. These data were already submitted in a NONS dossier under Directive 92/32/EEC (notification number 05 -04 -1922 -00 from 2005 -10 -25) and considered valid and uncritical from the German Competent Authority (BAUA).

 

The toxicity of the second hydrolysis product 2-(2-aminoethoxy)ethanol to Algae was evaluated according to German Industrial Standard DIN 38412 part 9. In a 72 hour toxicity study, cultures of Scenedesmus subspicatus were exposed to 2-(2-aminoethoxy)ethanol at nominal concentrations of 0 (control), 3.9, 7.8, 15.625, 31.25, 62.5, 125, 250, 500 mg/L under static conditions. The 72 h-EC50 value based on growth rate was 202 mg/L. The 72-h NOEC based on growth rate was 62.5 mg/L (see ECHA dissemination homepage).

 

In addition data from the analogues substance Sika Hardener LI (Dodecanoic acid , 3-[[3-[[[2,2-dimethyl-3-[(1-oxododecylo)oxy]propylidene]amino]methyl]-3,5,5-trimethylcyclohexyl]imino]-2,2-dimethylpropyl ester, CAS 932742-30-8) is reported. Sika Hardener LG and Sika Hardener LI both have the same aldehyde component (2,2-Dimethyl-3-lauroyloxy-propanal), but different amine components. Data from the OECD toolbox support the use of data from Sika Hardener LI also as read across fro Sika Hardener LG (see attachment in section 13). In this study, exponentially growing cultures of Pseudokirchneriella subcapitata were exposed to various concentrations of the test item over several generations under defined conditions. The algal growth in relation to a concurrent control culture was determined over a fixed test period of 72 hours and thus, over several algal generations. A supersaturated test item solution was prepared by dispersing the test item amount (nominal load of 100 mg/L) into the test medium two days before the start of the renewal periods. This solution was shaken for 24 hours at app. 30 °C, then was left settling for 24 hours at app. 20 °C and thereafter filtrated through a 0.45 μm filter. The following nominal concentrations were used: 6.25; 12.5; 25.0; 50.0 and 100.0 mg/L. In this 72-h algal growth inhibition test with Pseudokirchneriella subcapitata the 72-h EC50 based on growth rate was determined at 180.4 mg/L. The 72-h EC50 based on yield was 102.7 mg/L. The overall NOEC was determined to be 25.0 mg/L. All results were based on the nominal concentrations.

 

Conclusion:

Due to the physical-chemical properties of Sika Hardener LG (fast hydrolysis) it is not possible to perform standard ecotoxicology tests. It was decided to do the test by preparing a WAF and base the EC50 and NOEC calculations on nominal concentrations (justification see study record). A value of 51.9 mg/L was derived. The data presented for the supporting substance Sika Hardener LG with a LC50 of 180 mg/L show a lower toxicity as compared to Sika Hardener LG. Overall, taking all ecotoxicological data into account, the same classification and labelling results (chronic aq. toxicity cat.3) was derived. Results from the two hydrolysis products support the findings and lead to the same environmental classification and labelling (aq. chronic cat.3). For 2,2-Dimethyl-3-lauroyloxy-propanal (aldehyde component) different tests were done to assess the ecotoxicology towards algae. In the end it was also not possible to use measured data for EC50 calculations (details see above). As the substance is readily biodegradable, the LC50 of > 1 mg/L and the NOEC of 0.18 mg/L together lead to a classification as chronic aq. cat.3 according to the 14. ATP of the CLP Regulation. The data for 2-(2-aminoethoxy)ethanol with an LC50 of 202 mg/L show that this hydrolysis product is not severely toxic to the environment. These data confirm that the classification and labelling and the hazard assessment of the ecotoxicology of Sika Hardener LG can be based on the main study with the product itself.