1,3,3-trimethyl-N-(2-methylpropylidene)-5-[(2-methylpropylidene)amino]cyclohexanemethylamine

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:

2011-07-01 to 2011-07-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline Study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: samples taken from additional beakers without daphnia, kept under the same experimental conditions
- Sample storage conditions before analysis: samples were taken, immediately transferred to the analytical laboratory and analysed on the same day.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test substance concentrations were individually prepared in M4 medium, stirred for 24 h, and afterwards the preparations were filtered .
- Controls: control preparations without test substance but treated the same way.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: 86.81
- Source (laboratory, culture collection): Collection of Algae Cultures, Institute for Plant Physiology, University of Göttingen, Germany
- Age of inoculum (at test initiation): < 2 weeks
- Method of cultivation: A slant agar tube with Desmodesmus subspicatus is stored in the refrigerator. Two 300 mL conical flasks, filled with 100 mL of sterile culture medium were inoculated with algae from the slant agar. Twice a week, the algae stock culture is transferred: One mL of the stock culture was diluted in two 300 mL conical flasks with 100 mL of fresh sterile culture medium.

ACCLIMATION
- Acclimation period: none
- Culturing media and conditions (same as test or not): not
- Any deformed or abnormal cells observed: no such cells observed

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

25.1 - 25.2 °C

pH:

8.07 - 9.09

Nominal and measured concentrations:

Nominal: 6.25, 12.5, 25.0, 50.0, 100 mg/L
Measured (DOC at 72 h): 0.4 mg/L (control), 12.6mg/L (nominal 12.5 mg/L), 25.9 mg/L (nominal 50 mg/L), 52.4 mg/L (nominal 100 mg/L)

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glas, 300 mL volume, filled with 150 mL culture medium
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): static test
- Initial cells density: 5000 cells/mL
- No. of vessels per concentration (replicates): 3 with algal suspensions, 1 for analytical sampling
- No. of vessels per control (replicates): 6 with algal suspensions, 1 for analytical sampling
- No. of vessels per vehicle control (replicates): none

GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used: -

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: yes

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 16 h light /
- Light intensity and quality: lighting supplied by fluorescent tubes Osram Dulux L, “Cool White” was 0.72 x 1020 photons per m2s = 8000 +/- 1000 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: -
- Range finding study
- Test concentrations: 0.1 mg/L, 1.0 mg/L, 10 mg/L, 100 mg/L
- Results used to determine the conditions for the definitive study: growth inhibition observed in pre-test

Reference substance (positive control):

yes

Remarks:

potassium dichromate was tested in an independent experiment in October 2009 and adequate sensitivity of the algal culture was demonstrated

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 89.5-186.2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

17.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

26.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 27.3-30.2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

14.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

73.6 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

dissolved

Remarks:

(DOC)

Basis for effect:

growth rate

Remarks on result:

other: 49.9-251.2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

9.6 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

dissolved

Remarks:

(DOC)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

19.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

dissolved

Remarks:

(DOC)

Basis for effect:

biomass

Remarks on result:

other: 1.4-24.8

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

11 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

dissolved

Remarks:

(DOC)

Basis for effect:

biomass

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Other: no
- 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
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

- Results with reference substance valid? yes; test was conducted 5 November 2010
- Concentrations: 0.3, 0.6, 0.9, 1.2, 1.5 mg/L
- EC50 (72 h, growth rate): 0.9 mg/L

Reported statistics and error estimates:

Statistical analysis was done with ToxRat Professional (ToxRat Solutions GmbH, Alsfeld, Germany)

EC (probit analysis using linear max. likelihood regression), LOEC and NOEC values (Williams Multiple Sequential t-test)

 

	Effective concentrations (mg/L)
	Based on nominal conc.		Based on 0h DOC values
	Yield	Growth rate		Yield	Growth rate
EC10       (0-72h)	14.1	17.9		11.0	9.6
EC20       (0-72h	17.6	34.1		13.3	19.3
EC50       (0-72h)	26.7	>100*		19.2	73.6
95 % Confidence interval	23.7 – 30.2	89.5 – 186.2		1.4 – 24.8	49.9 – 251.2
LOEC    (0-72h)	25.0	25.0		-	-
NOEC    (0-72h)	12.5	12.5		7.6	7.6

*value was extrapolated by thestatistic software ToxRat® Professional

 

Extinction values

	0 hours	24 hours	48 hours	72 hours
control 1	0.002	0.012	0.066	0.219
control 2	0.001	0.012	0.069	0.216
control 3	0.001	0.010	0.054	0.221
control 4	0.000	0.011	0.055	0.208
control 5	0.002	0.011	0.047	0.173
control 6	0.001	0.011	0.047	0.185
blank value sample 7-9	0.000	0.000	0.000	0.001
6.25 mg/L -7	0.002	0.012	0.091	0.237
6.25 mg/L -8	0.002	0.012	0.096	0.212
6.25 mg/L -9	0.002	0.010	0.071	0.213
blank value sample 10-12	0.007	0.006	0.005	0.004
12.5 mg/L -10	0.009	0.023	0.084	0.219
12.5 mg/L -11	0.009	0.021	0.100	0.227
12.5 mg/L -12	0.009	0.021	0.089	0.229
blank value sample 13-15	0.000	0.000	0.018	0.010
25 mg/L -13	0.002	0.012	0.084	0.104
25 mg/L -14	0.002	0.015	0.102	0.146
25 mg/L -15	0.002	0.015	0.090	0.117
blank value sample 16-18	0.001	0.000	0.009	0.013
50 mg/L -16	0.002	0.013	0.040	0.046
50 mg/L -17	0.002	0.012	0.041	0.046
50 mg/L -18	0.002	0.014	0.046	0.049
blank value sample 19- 21	0.000	0.000	0.005	0.010
100 mg/L -19	0.002	0.010	0.037	0.045
100 mg/L -20	0.002	0.010	0.037	0.037
100 mg/L -21	0.002	0.014	0.038	0.045

 

 Inhibition in Yield and Growth rate after 72 h

mg/L	0 (Control)	6.25	12.5	25	50	100
Yield x 10^4 (mean cell number)	84.144	91.491	92.095	43.400	11.200	10.576
Inhibition (%)	0	-8.7	-9.5	48.4	86.7	87.4
Growth rate	1.701	1.730	1.732	1.479	1.043	1.022
Inhibition (%)	0	-1.7	-1.9	13.0	38.7	39.9

 

Analytical results

 

Time	Control (M4 medium)			12.5 mg/L
	TC (mg/L)	TIC (mg/L)	DOC^1 (mg/L)	TC (mg/L)	TIC (mg/L)	DOC (mg/L)^2	recovery in %
0 h	8.2	7.0	1.2	16.6	7.8	7.6
24 h	8.6	7.2	1.4	16.0	8.0	6.6	87
48 h	8.1	7.2	0.9	15.4	7.8	6.7	88
72h	7.4	7.0	0.4	20.8	7.8	12.6	**

   

 

Time	50 mg/L				100 mg/L
	TC(mg/L)	TIC (mg/L)	DOC (mg/L)^2	recovery in %	TC (mg/L)	TIC (mg/L)	DOC (mg/L)^2	recovery in %
0 h	41.4	9.7	30.5		73.6	11.0	61.4
24 h	37.7	10.5	25.8	85	70.4	14.1	54.9	89
48 h	35.0	10.4	23.7	78*	63.7	13.7	49.1	80
72h	36.5	10.2	25.9	85	66.4	13.6	52.4	85

 ¹DOC = TC – TIC

²DOC = DOC minus DOC Control

Recovery rate for DOC in % after 24h, 48h and 72h relating to 0h values.

Percent values were rounded.

* outlier

** outlier, percent value not calculated

Validity criteria fulfilled:

yes

Conclusions:

The ErC50 (72 h), based on nominal concentrations was determined as >100 mg/L , whereas the EbC50 (72 h) based on nominal concentrations was 27.7 mg/L. Based on DOC the ErC50 was 73.6 mg/L and the EyC50 19.2 mg/L. The present data indicate that the test substance may be harmful to aquatic algae.

Executive summary:

1,3,3-trimethyl-N-(2-methylpropylidene)-5-[(2-methylpropylidene)amino]cyclohexanemethylamine was tested in the Alga, Growth Inhibition Test according to the OECD Guideline for the Testing of Chemicals No. 201 (23 March 2006) using the single cell green algae Desmodesmus subspicatus as test organism. The experiment was carried out as a static test, i.e. the test substance preparations were not exchanged for the whole exposure period of 72 hours. Based on non-GLP pre-test results the main test was performed with test substance concentrations of nominal 12.5, 50.0 and 100 mg/L mg/L culture medium. During this study the DOC of the test substance concentrations of nominal 6.25, 25, and 100 mg/L culture medium and the control (culture medium) was determined with a TIC analyzer. The results of this study show that the test substance induced inhibition of algal growth after 72 hours of exposure. For the parameter yield, the EyC50 (72 h) value was 26.7 mg/L and the LOEC and NOEC values were 25 mg/L and 12.5 mg/L, respectively, based on nominal concentrations. Based on measured DOC values (0 h) the EyC50 value was 19.2 mg/L and the NOEC was7.6mg/L, respectively. For the parameter growth rate the ErC50 value (72 h) was >100 mg/L and the LOEC and NOEC values were 25 mg/L and 12.5 mg/L, respectively, based on nominal concentrations. Based on measured DOC values (0 h) the ErC50 value was 73.6 mg/L and the NOEC was 7.6mg/L

Description of key information

1,3,3-trimethyl-N-(2-methylpropylidene)-5-[(2-methylpropylidene)amino]cyclohexanemethylamine was tested in the Alga Growth Inhibition Test according to the OECD Guideline for the Testing of Chemicals No. 201 (23 March 2006) using the single cell green algae Desmodesmus subspicatus as test organism. The experiment was carried out as a static test, i.e. the test substance preparations were not exchanged for the whole exposure period of 72 hours. Based on non-GLP pre-test results the main test was performed with test substance concentrations of nominal 12.5, 50.0 and 100 mg/L mg/L culture medium. During this study the DOC of the test substance concentrations of nominal 6.25, 25, and 100 mg/L culture medium and the control (culture medium) was determined with a TIC analyzer. The results of this study show that the test substance induced inhibition of algal growth after 72 hours of exposure. For the parameter yield, the EyC50 (72 h) value was 26.7 mg/L and the LOEC and NOEC values were 25 mg/L and 12.5 mg/L, respectively, based on nominal concentrations. Based on measured DOC values (0 h) the EyC50 value was 19.2 mg/L and the NOEC was7.6mg/L, respectively. For the parameter growth rate the ErC50 value (72 h) was >100 mg/L and the LOEC and NOEC values were 25 mg/L and 12.5 mg/L, respectively, based on nominal concentrations. Based on measured DOC values (0 h) the ErC50 value was 73.6 mg/L and the NOEC was 7.6mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

73.6 mg/L

EC10 or NOEC for freshwater algae:

7.6 mg/L