N-[3-({[2,2-dimethyl-3-(morpholin-4-yl)propylidene]amino}methyl)-3,5,5-trimethylcyclohexyl]-2,2-dimethyl-3-(morpholin-4-yl)propan-1-imine

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2012-05-14 to 2012-05-25

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:

2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Version / remarks:

1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For determination of the test item concentration, samples were taken from each testing concentrations and from the controls at the start and at the end of the experiments.

Vehicle:

no

Details on test solutions:

- Dosage of Test Item: In order to select appropriate test concentrations for use in the definitive test, preliminary range-finding test was conducted to determine the approximate toxicity of the test item. A stock solution of 100 mg/L (nominal concentration) was first prepared by dissolving an amount of 0.05 g test item in 500 mL OECD medium using intensive shaking (until all of the test item was dissolved). The test solutions of the chosen test concentrations were prepared by appropriate diluting of this stock solution.
- Test Concentration: Concentrations of the test item were analytically determined at the start and at the end of the experiments. The measured concentrations deviated more than 20 % from the nominal at some cases, therefore, the exposure concentrations were calculated as geometric mean of the concentrations measured at the start and at the end of the tests. The measured concentrations of Sika Hardener MI varied between 75 and 104 % of the nominal during the experiments.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata (formerly: Selenastrum capricornutum) (Printz-Starr).
- Strain-Number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source (laboratory, culture collection): The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Nikolausberger Weg 18, D-37073 Göttingen, Germany
- Age of inoculum (at test initiation): 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.
- Method of cultivation: 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:

None

Hardness:

Not meausured

Test temperature:

measured in the flasks: 22.1 – 23.8 °C
measured in teh climate chamber: 22.3 - 23.0 °C

pH:

7.89 – 9.72

Dissolved oxygen:

Not measured

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentrations in Experiment I: 0.2, 0.5, 1.2, 3.1, 7.8 and 19.5 mg/L
Nominal concentrations in Experiment II: 29.6, 44.4 and 66.7 mg/L
The measured concentrations deviated more than 20 % from the nominal. The corresponding calculated geometric mean concentrations were the followings:
Experiment I: 0.2, 0.4, 1.0, 2.4, 5.9 and 15.5 mg/L
Experiment II: 27.7, 43.5 and 66.4 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks of 250 mL volume with 100 mL test medium. The test flasks were covered with air-permeable stoppers.
- Initial cells density: The test was started (0 hours) by inoculation of 0.1 mL algal biomass (107 algal cells per mL) to 100 mL test item solution. The initial cell density was about 104 cells/mL in each test flask. 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.
- Replicates: The test was performed with three replicates at each test concentration and six replicates were included in the untreated control.

GROWTH MEDIUM
- Standard medium was used: yes

OTHER TEST CONDITIONS
- Light Intensity and quality:

EFFECT PARAMETERS MEASURED:

- Determination of cell concentrations: The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscope with counting chamber.
- Other: The morphological changes of algal cells compared to the control were examined at 24, 48 and 72 hours after starting the test using a microscope in parallel.

TEST CONCENTRATIONS:
- Test concentrations were prepared by appropriate diluting of a stock solution.
Based upon the results from the preliminary experiment, nominal concentrations of 0.2, 0.5, 1.2, 3.1, 7.8 and 19.5 mg/L were investigated in the main study. The results obtained from these concentrations tested (i.e. Experiment I) did not allow an exact ErC50 calculation because the inhibition of growth rate was below 50 % at the highest concentration level. In order to obtain appropriate results for the exact EC50 calculation, the effect of the test item was further investigated in a complementary experiment (i.e. Experiment II) at the following nominal concentrations: 29.6, 44.4 and 66.7 mg/L.

The measured concentrations deviated more than 20 % from the nominal at some cases therefore the geometric mean of the measured concentrations were calculated in order to determine exposure concentrations. The corresponding calculated geometric mean concentrations were the followings:
Experiment I: 0.2, 0.4, 1.0, 2.4, 5.9 and 15.5 mg/L
Experiment II: 27.7, 43.5 and 66.4 mg/L

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (K2Cr2O7)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

89 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % conf. limits (59.0-134.3)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

3.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% conf. limits ( 2.6-5.4)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.4 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.:

0.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% conf.limits (0.4-0.9)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% conf. limits (7.6-11.7)

Results with reference substance (positive control):

The results for the reference item Potassium dichromate are as follows:
The 72h ErC50: 1.19 mg/L, (95 % confidence limits: 1.09 – 1.30 mg/L)
The 72h EyC50: 0.80 mg/L, (95 % confidence limits: 0.73 – 0.87 mg/L)

Reported statistics and error estimates:

- CELL DENSITY: cell density in the control cultures increased by a factor of more than 16 (by a factor of 64.00 at Experiment I and 73.50 at Experiment II) within 72 hours.
-pH: pH of the controls did not vary by more than 1.5 units.
THE MEAN COEFFICIENT: The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72 h-tests) in the control cultures did not exceed 35 %.
Experiment I
- CV for section-by-section growth rate day 0-1: 8.78 %
- CV for section-by-section growth rate day 1-2: 13.18 %
- CV for section-by-section growth rate day 2-3: 3.67 %
The mean coefficient of variation for section-by-section specific growth rates: 8.54 %.
Experiment II
- CV for section-by-section growth rate day 0-1: 16.75 %
- CV for section-by-section growth rate day 1-2: 14.17 %
- CV for section-by-section growth rate day 2-3: 10.53 %
The mean coefficient of variation for section-by-section specific growth rates: 13.82 %.
- COEFFICIENT OF VARIATION OF AVERAGE SPECIFIC GROWTH: the coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7 %.
Experiment I
- CV for average specific growth rate day 0-3: 1.56 %
Experiment I
- CV for average specific growth rate day 0-3: 1.08 %

Validity criteria fulfilled:

yes

Conclusions:

In a 72-h algal growth inhibition test with Pseudokirchneriella subcapitata, the 72-h EC50 value based on growth rate was determined as 89.0 mg/L and based on yield was determined as 9.4 mg/L.
The 72-h EC10 value based on growth rate was determined as 3.8 mg/L and based on yield was determined as 0.5 mg/L.
The 72-h NOEC related to growth rate was determined to be 2.4 mg/L and related to yield was determined to be 1.0 mg/L.
The results are based on the measured geometric mean concentrations.

Executive summary:

An acute growth inhibition test with Sika Hardener MI on Algae (Pseudokirchneriella subcapitata) over an exposure period of 72 hours in a static system was assessed according to OECD Guideline 201 and EU method C.3. Five different concentrations arranged in a geometric series and untreated control group were tested in the main study. The following nominal concentrations of test item were tested: In Experiment I: 0.2, 0.5, 1.2, 3.1, 7.8 and 19.5 mg/L and in the Experiment II: 29.6, 44.4 and 66.7 mg/L. The measured concentrations deviated more than 20 % from the nominal at some cases, therefore, the geometric mean of the measured concentrations were calculated in order to determine exposure concentrations. The corresponding calculated geometric mean concentrations were the followings: Experiment I: 0.2, 0.4, 1.0, 2.4, 5.9 and 15.5 mg/L and Experiment II: 27.7, 43.5 and 66.4 mg/L. Under the conditions of this acute algal growth inhibition test on Pseudokirchneriella subcapitata the observed endpoints on the growth rate were the followings: 72-h EC50 value: 89.0 mg/L, 72-h NOEC value: 2.4 mg/L and the 72-h EC10 3.8 mg/L. These results are based on the measured geometric mean test item concentrations.

Description of key information

In a 72-h algal growth inhibition test with Pseudokirchneriella subcapitata, the 72-h EC50 value based on growth rate was determined as 89.0 mg/L and based on yield was determined as 9.4 mg/L. The 72-h EC10 value based on growth rate was determined as 3.8 mg/L and based on yield was determined as 0.5 mg/L. The 72-h NOEC related to growth rate was determined to be 2.4 mg/L and related to yield was determined to be 1.0 mg/L. These results are based on the measured geometric mean concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:

89 mg/L

EC10 or NOEC for freshwater algae:

3.8 mg/L

Additional information

An acute growth inhibition test with Sika Hardener MI on Algae (Pseudokirchneriella subcapitata) over an exposure period of 72 hours in a static system was assessed according to OECD Guideline 201 and EU method C.3. Five different concentrations arranged in a geometric series and untreated control group were tested in the main study. The following nominal concentrations of test item were tested: In Experiment I: 0.2, 0.5, 1.2, 3.1, 7.8 and 19.5 mg/L and in the Experiment II: 29.6, 44.4 and 66.7 mg/L. The measured concentrations deviated more than 20 % from the nominal at some cases, therefore, the geometric mean of the measured concentrations were calculated in order to determine exposure concentrations. The corresponding calculated geometric mean concentrations were the followings: Experiment I: 0.2, 0.4, 1.0, 2.4, 5.9 and 15.5 mg/L and Experiment II: 27.7, 43.5 and 66.4 mg/L. Under the conditions of this acute algal growth inhibition test on Pseudokirchneriella subcapitata the observed endpoints on the growth rate were the followings: 72-h EC50 value: 89.0 mg/L, 72-h NOEC value: 2.4 mg/L and the 72-h EC10 3.8 mg/L. These results are based on the measured geometric mean test item concentrations.