1,3-Cyclohexanedimethanamine, N1,N3-bis(2-methylpropylidene)-

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:

2010-11-12 to 2011-10-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study, in accordance to OECD, EU guidelines

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted 23rd March, 2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

dated July 23, 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling preparation:
At the start and end of the experiment three parallel samples were taken from each concentration level and from the control. Representative samples (in duplicate series) were taken at the start and at the end of the experiment. One series was stored frozen in the testing laboratory, one series was sent for analysis, and was analysed immediately after sampling.

- Storage of the Samples: The second series of samples were stored in a refrigerator immediately after sampling, and were kept stored until delivery of the final report to enable additional analyses on request of the sponsor. After delivery of the final report, all samples will be discarded.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Formulation: A solution of the test item with a concentration of 30 mg/L in Algal Mineral Salts Test Medium (“OECD Medium”) was prepared, thereafter sonicated for 30 minutes in ultrasonic bath (under cooling). The resulted “Algal Mineral Salts Test Medium (“OECD Medium”) stock solution” was centrifuged at 4000 rpm for 10 min. (20 °C) and filtrated using a 0.45 μm filter (VWR Vacuum Filtration System) and further diluted accordingly.

- Untreated Control: Algal Mineral Salts Test Medium was inoculated (without test item) and examined in parallel.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Source: SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Nikolausberger Weg 18, D-37073 Göttingen, Germany
- 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 standardized 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-cultured algal cultures were healthy and did not contain deformed or abnormal cells.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.2 – 22.4 °C

pH:

pH 7.85 to 8.72 at test start
pH 8.32 to 9.17 at test end

Nominal and measured concentrations:

Nominal: 1.875; 3.75; 7.5; 15, and 30 mg/L
Mean measured concentration with 95% confidence interval: 1.44 ± 0.00, 2.95 ± 0.07, 5.89 ± 0.30, 12.2 ± 0.89, 25.8 ± 0.12

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: Approximately 10.000 algal cells per mL test medium
- Replicates: 3 replicates per test concentration and six replicates in the control group. Volumes of 100 mL algal suspension per replicate were continuously shaken by a laboratory orbital shaker.

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Light intensity and quality: Approximately 8871 lux, which was ensured with fluorescent lamps (with a spectral range of 400-700 nm).

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Manual cell counting using a microscope with a counting chamber
- Other: The cell morphology was examined in parallel

TEST CONCENTRATIONS
- Range finding study: Two non-GLP preliminary range-finding tests were performed. In the first range-finding test the examined nominal concentrations were: 3.125, 6.25, 12.5, 25, 50 and 100 mg test item/L. In the second range-finding test the same concentration range was investigated.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

14.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 13.11 - 16.71 mg/L

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

12.22 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The INCOREZ 397 in aquatic formulation decomposes. The decomposition product of INCOREZ 397 was analysed after derivatisation using 9-Fluorenylmethyl-chlorformiat (FMOC), by HPLC method with UV detection on a BDS Hypersil C18, Thermo Scientific column. Three replicates samples were taken from each test concentration and from the control at the start and at the end of the study. The diluted samples were analysed. The decomposition product of INCOREZ 397 concentrations in the samples varied in the range from 74 % to 104 % in comparison to the nominal values. As the measured concentration levels did not maintain within the ± 20 % of the nominal range during the study, the analysis of the results was based on the calculated geometric mean of the measured test item concentration values. Therefore, all reported biological results are related to the geometric mean of measured concentrations of the test item.

Results with reference substance (positive control):

The ErC 50 of Potassium dichromate: 0.82 mg/L, (95 % confidence limits: 0.77 – 0.61 mg/L). The growth rate, 72h value is in accordance with the accepted 1.10 ± 0.48 mg/L range for Pseudokirchneriella subcapitata.

Reported statistics and error estimates:

The EC50 values of the test item and their confidence limits were calculated using Probit analysis. The analysis was done using the TOXSTAT statistical software program.
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 (TOXSTAT software). The data were checked for normality by Chi-Square Test, for homogeneity of variance by Levene’s Test.

The shape of the algal cells growing was obviously not affected at the test item concentration of 1.875 (measured 1.64) mg/L while thin and fragmented cells were observed in the concentration range of 3.75 (measured 3.22) – 30 (measured 24.81) mg/L.

Cell Density in Control: In the control the cell density has increased from nominal N = 1E+04 cells/mL at the start of the test (0 hours) to N = 91.50E+04 cells/mL (mean value) after 72 hours by a factor of 91.50. Thus, the algal growth in the control was high enough to pass the validity criteria in this assay.

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this acute algal growth inhibition test on Pseudokirchneriella subcapitata the observed endpoints for the effect of Incorez 397 based on the growth rate were the followings: 72h EC50 value: 14.80 mg test item/L; 48h NOEC value: 6.13 mg test item/L and 96h LOEC value: 12.22 mg test item/L. All calculations were based on the geometric mean of measured concentrations.

Executive summary:

An acute growth inhibition test of Incorez 397 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 an untreated control group were tested in the main study. The following concentrations were used in the main study: 1.875; 3.75; 7.5; 15, and 30 mg/L. The shape of the algal cells growing was obviously not affected at the test item concentration of 1.875 (measured 1.64) mg/L while thin and fragmented cells were observed in the concentration range of 3.75 (measured 3.22) – 30 (measured 24.81) mg/L. Under the conditions of this acute algal growth inhibition test on Pseudokirchneriella subcapitata the observed endpoints for the effect of Incorez 397 based on the growth rate were the followings: 72h EC50 value: 14.80 mg test item/L; 48h NOEC value: 6.13 mg test item/L and 96h LOEC value: 12.22 mg test item/L. All calculations were based on the geometric mean of measured concentrations.

Description of key information

Under the conditions of this acute algal growth inhibition test on Pseudokirchneriella subcapitata the observed endpoints for the effect of Incorez 397 based on the growth rate were the followings: 72h EC50 value: 14.80 mg test item/L; 48h NOEC value: 6.13 mg test item/L and 96h LOEC value: 12.22 mg test item/L. All calculations were based on the geometric mean of measured concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:

14.8 mg/L

EC10 or NOEC for freshwater algae:

6.13 mg/L

Additional information

An acute growth inhibition test of Incorez 397 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 an untreated control group were tested in the main study. The following concentrations were used in the main study: 1.875; 3.75; 7.5; 15, and 30 mg/L. The shape of the algal cells growing was obviously not affected at the test item concentration of 1.875 (measured 1.64) mg/L while thin and fragmented cells were observed in the concentration range of 3.75 (measured 3.22) – 30 (measured 24.81) mg/L. Under the conditions of this acute algal growth inhibition test on Pseudokirchneriella subcapitata the observed endpoints for the effect of Incorez 397 based on the growth rate were the followings: 72h EC50 value: 14.80 mg test item/L; 48h NOEC value: 6.13 mg test item/L and 96h LOEC value: 12.22 mg test item/L. All calculations were based on the geometric mean of measured concentrations.