Cyclohexanamine, 4,4’-methylenebis[N-(2,2-dimethyl-3-(4-morpholinyl)propylidene)]-

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-06-01 to 2021-06-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

adopted 23 March 2006

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

01 March 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling procedure: Three samples were analysed from each test concentration and six samples were analysed from the control solutions on both analytical occasions.
- Sampling interval: sampled on day 0 and and day 3

Vehicle:

no

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
- Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Untere Karspüle 2, D-37073 Göttingen, Germany
- Stage: exponential growth
- Method of cultivation: The stock cultures are small algal cultures that are planted on agar. These are transferred to fresh medium at least once every two months under standardised conditions according to the test guidelines. The pre-culture was prepared with OECD Medium, incubated under the conditions of the test and used when still exponentially growing, normally after an incubation period of 2-4 days. (The pre-culture was incubated for four days at this test.)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

post exposure observation not performed

Hardness:

not specified

Test temperature:

22.4 – 22.8 °C

pH:

7.72 – 9.21

Dissolved oxygen:

not specified

Salinity:

not applicable

Conductivity:

not specified

Nominal and measured concentrations:

Nominal: 0 (control), 0.30, 0.95, 3.1, 9.8, 31.3 and 100 mg/L; measured:

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type: closed
- Material, size, fill volume: glass, 250 mL volume, filled up with 100 mL test medium.
- Aeration: not actively aerated
- Initial cells density: 1 × 104 cells/mL
- Control end cells density: 104.0 × 104 cells at the end of the test (72 h)
- No. of vessels per concentration: 3
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ultrapure water prepared by Direct-Q 5 system, Millipore

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: counting chamber

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: performed
- Test concentrations in range finding study: 0.1, 1, 10, 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study: yes

CULTURING APPARATUS
-Details on culturing apparatus used: climate chamber

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.89 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

3.08 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.8 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.84 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

25.22 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

6.75 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

8.99 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

15.55 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

4.84 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.:

25.22 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: Some swollen cells were observed together with the normal cells in the exposure concentration of 4.84 mg/L, some swollen cells and thin cells were observed together with the normal cells in the exposure concentration of 25.22 mg/L. In the highest exposure concentration (88.93 mg/L) thin and swollen cells were observed together with the normal cells, amongst the very few number of algal cells found in the test solution.
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no
- Any observations that might cause a difference between measured and nominal values:
- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

72h ErC50: 0.62 mg/L, (95 % confidence limits: 0.59 – 0.65 mg/L); 72h EyC50: 0.33 mg/L, (95 % confidence limits: 0.32 – 0.34 mg/L)

Reported statistics and error estimates:

The ECx values and their 95 % confidence limits for growth rates and yield were calculated by Probit-analysis using linear max. likelihood regression. For the determination of the NOEC/LOEC values, Step-down Jonckheere-Terpstra test (alpha = 0.05, 2-sided) was performed with growth rates and yield. Pretesting on normal distribution was performed by Shapiro Wilk’s test and on variance homogeneity by Levene’s test.

Table 1: Test Item concentration

Nominal concentration (mg/L)	Start of the test	End of the test
0.30	0.283	0.130
0.95	0.97	not detected
3.1	3.0	BQL
9.8	9.0	2.6
31.3	27.9	22.8
100	91	86.9

BQL: below quantitation limit

 

Table 2: Growth Rates and Yield calculated for the entire test duration (day 0-3)

Exposure Concentration [mg/L]		Growth Rates	% inhibition	Yield	% inhibition
		day 0-3		day 0-3
Control		1.48		85
		1.51		91
		1.51		91
		1.67		147
		1.56		108
		1.52		96
	Mean	1.54		103.0
	SD	0.1		22.9
	CV	4.3		22.2
0.19		1.57	-2.0	111	-7.8
		1.48	4.0	84	18.4
		1.50	2.5	90	12.6
	Mean	1.52	1.5	95.0	7.8
	SD	0.0		14.2
	CV	3.2		14.9
0.49		1.55	-0.8	105	-1.9
		1.55	-0.8	105	-1.9
		1.52	1.6	94	8.7
	Mean	1.54	0.0	101.3	1.6
	SD	0.0		6.4
	CV	1.4		6.3
0.87		1.44	6.4	75	27.2
		1.51	2.0	92	10.7
		1.45	6.1	76	26.2
	Mean	1.47	4.9	81.0	21.4
	SD	0.0		9.5
	CV	2.6		11.8
4.84		1.46	5.3	79	23.3
		1.51	1.8	93	9.7
		1.48	4.0	84	18.4
	Mean	1.49	3.7	85.3	17.2
	SD	0.0		7.1
	CV	1.8		8.3
25.22		0.23	85.0	1	99.0
		0.37	76.3	2	98.1
		0.46	70.0	3	97.1
	Mean	0.35	77.1	2.0	98.1
	SD	0.1		1.0
	CV	32.9		50.0
88.93		0.00	100.0	0	100.0
		0.00	100.0	0	100.0
		0.00	100.0	0	100.0
	Mean	0.00	100.0	0.0	100.0
	SD	0.0		0.0
	CV	NA		NA

 

Table 3: The Section by section growth rates (days 0-1, 1-2 and 2-3)

Exposure Concentration [mg/L]		Section by section growth rates (µ)
		µ (day 0-1)	% inhibition	µ (day 1-2)	% inhibition	µ (day 2-3)	% inhibition
Control		1.39		1.50		1.56
		1.10		1.79		1.63
		1.10		1.79		1.63
		1.39		1.66		1.95
		1.10		1.67		1.92
		1.79		1.10		1.68
	Mean	1.31		1.59		1.73
	SD	0.3		0.3		0.2
	CV	21.0		16.5		9.5
0.19		1.39	-5.8	1.50	5.2	1.83	-5.6
		1.39	-5.8	1.39	12.6	1.67	3.5
		1.10	16.1	1.47	7.6	1.95	-12.5
	Mean	1.29	1.5	1.45	8.5	1.81	-4.9
	SD	0.2		0.1		0.1
	CV	12.9		4.1		7.6
0.49		1.39	-5.8	1.25	21.0	2.02	-17.0
		1.39	-5.8	1.50	5.2	1.77	-2.5
		1.39	-5.8	1.50	5.2	1.66	3.9
	Mean	1.39	-5.8	1.42	10.5	1.82	-5.2
	SD	0.0		0.1		0.2
	CV	0.0		10.2		10.2
0.87		1.61	-22.9	1.16	26.7	1.56	10.0
		1.61	-22.9	1.44	9.5	1.49	14.0
		1.10	16.1	1.79	-12.9	1.45	16.0
	Mean	1.44	-9.9	1.46	7.8	1.50	13.3
	SD	0.3		0.3		0.1
	CV	20.5		21.5		3.6
4.84		0.69	47.1	2.30	-45.1	1.39	19.9
		1.39	-5.8	1.61	-1.5	1.55	10.6
		1.39	-5.8	1.45	8.8	1.61	7.0
	Mean	1.16	11.8	1.79	-12.6	1.51	12.5
	SD	0.4		0.5		0.1
	CV	34.6		25.4		7.6
25.22		1.10	16.1	0.51	67.8	-0.92	153.0
		0.00	100.0	1.39	12.6	-0.29	116.6
		0.69	47.1	0.92	42.2	-0.22	112.9
	Mean	0.60	54.4	0.94	40.9	-0.48	127.5
	SD	0.6		0.4		0.4
	CV	93.0		46.7		-80.5
88.93		0.00	100.0	0.00	100.0	0.00	100.0
		0.00	100.0	0.00	100.0	0.00	100.0
		0.00	100.0	0.69	56.3	-0.69	140.1
	Mean	0.00	100.0	0.23	85.4	-0.23	113.4
	SD	0.0		0.4		0.4
	CV	NA		173.2		-173.2

 

Table 4: Biomass (algal cell concentrations) during the test

Exposure Concentration [mg/L]		Number of Cells (× 10^4/mL)
		0 h	24 h	48 h	72 h
Control		1	4	18	86
		1	3	18	92
		1	3	18	92
		1	4	21	148
		1	3	16	109
		1	6	18	97
	Mean	1.0	3.8	18.2	104.0
	SD	0.0	1.2	1.6	22.9
	CV	0.0	30.5	8.8	22.0
0.19		1	4	18	112
		1	4	16	85
		1	3	13	91
	Mean	1.0	3.7	15.7	96.0
	SD	0.0	0.6	2.5	14.2
	CV	0.0	15.7	16.1	14.8
0.49		1	4	14	106
		1	4	18	106
		1	4	18	95
	Mean	1.0	4.0	16.7	102.3
	SD	0.0	0.0	2.3	6.4
	CV	0.0	0.0	13.9	6.2
0.87		1	5	16	76
		1	5	21	93
		1	3	18	77
	Mean	1.0	4.3	18.3	82.0
	SD	0.0	1.2	2.5	9.5
	CV	0.0	26.6	13.7	11.6
4.84		1	2	20	80
		1	4	20	94
		1	4	17	85
	Mean	1.0	3.3	19.0	86.3
	SD	0.0	1.2	1.7	7.1
	CV	0.0	34.6	9.1	8.2
25.22		1	3	5	2
		1	1	4	3
		1	2	5	4
	Mean	1.0	2.0	4.7	3.0
	SD	0.0	1.0	0.6	1.0
	CV	0.0	50.0	12.4	33.3
88.93		1	1	1	1
		1	1	1	1
		1	1	2	1
	Mean	1.0	1.0	1.3	1.0
	SD	0.0	0.0	0.6	0.0
	CV	0.0	0.0	43.3	0.0

 

Table 6: Summary of biological results

Endpoints (0-72 h)	Growth rate [mg/L]	Yield [mg/L]
EC10	6.75	1.89
95 % conf. limits	4.59 – 8.62	0.71 – 3.15
EC20	8.99	3.08
95 % conf. limits	6.64 – 10.93	1.49 – 4.74
EC50	15.55	7.80
95 % conf. limits	13.28 – 17.42	5.11 – 12.33
NOEC	4.84	4.84
LOEC	25.22	25.22

 

VALIDITY OF TEST

Cell density in the control cultures increased by a factor of more than 16 (by a factor of 104.0) within 72 hours. This corresponds to a specific growth rate of 1.54 day-1.

The mean coefficient of variation (CV) for section-by-section specific growth rates (days 0-1, 1-2 and 2-3) in the control cultures did not exceed 35 %.

The mean CV for section-by-section specific growth rates: 20.4 %. The coefficient of variation (CV) of average specific growth rates during the whole test period in replicate control cultures did not exceed 7 % in test.

The CV for average specific growth rate (day 0-3): 4.3 %

All validity criteria were met, therefore the study can be considered as valid.

Validity criteria fulfilled:

yes

Conclusions:

In a Growth Inhibition Test with Raphidocelis subcapitata according to OECD 201 the 72-h EC10 and EC50 of the test item for growth rate were determined to be 6.75 and 15.55 mg/L, respectively. The 72-h EC10 and EC50 for yield were determined to be 1.89 and 7.80 mg/L, respectively. The 72-h NOEC for growth rate and yield was determined to be 4.84 mg/L. All biological results are based on geometric mean measured concentrations.

Executive summary:

To determine the effect of the test item on the growth of a unicellular green algal species a Growth Inhibition Test according to OECD 201 and EU Method C.3 was carried out in compliance with the GLP principles. Exponentially growing cultures of Raphidocelis subcapitata (previously known as Pseudokirchneriella subcapitata) were exposed for a duration of 72 hours to nominal test item concentrations of 0.30, 0.95, 3.1, 9.8, 31.3 and 100 mg/L. For determination of the test item concentrations, samples were taken from each concentration level and from the control at the start and at the end of the test. Using a HPLC-UV the geometric mean test item concentrations of the measured start and end concentrations were calculated to be 0.19, 0.49, 0.87, 4.84, 25.22 and 88.93 mg/L. Biological results and endpoints are thus based on the measured concentrations. The test design included three replicates at each test concentration and six replicates for the untreated control. The alga cell concentration was approximately 10⁴ cells/mL at the start of the test in all of the test cultures. Glass flasks with total capacity of 250 mL were used as test vessels. The algal cell concentration was determined by manual cell counting by microscope in each testing flask during the 72-hour test, in 24-hour intervals. The results of the statistical evaluation showed that the 0-72 h average specific growth rate and yield was not statistically significantly different from the control in the exposure concentrations of 0.19, 0.49, 0.87 and 4.84 mg/L and significantly different in the exposure concentrations of 25.22 and 88.93 mg/L. The 72-h EC10 and EC50 of the test item for growth rate were determined to be 6.75 and 15.55 mg/L, respectively. The 72-h EC10 and EC50 for yield were determined to be 1.89 and 7.80 mg/L, respectively.  The 72-h NOEC for growth rate and yield was determined to be 4.84 mg/L. All validity criteria of the guidelines were met; the study is hence considered valid.

Description of key information

In a Growth Inhibition Test with Raphidocelis subcapitata according to OECD 201 the 72-h EC10 and EC50 of the test item for growth rate were determined to be 6.75 and 15.55 mg/L, respectively. The 72-h EC10 and EC50 for yield were determined to be 1.89 and 7.80 mg/L, respectively. The 72-h NOEC for growth rate and yield was determined to be 4.84 mg/L. All biological results are based on geometric mean measured concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:

15.55 mg/L

EC10 or NOEC for freshwater algae:

6.75 mg/L

Additional information

To determine the effect of the test item on the growth of a unicellular green algal species a Growth Inhibition Test according to OECD 201 and EU Method C.3 was carried out in compliance with the GLP principles. Exponentially growing cultures of Raphidocelis subcapitata (previously known as Pseudokirchneriella subcapitata) were exposed for a duration of 72 hours to nominal test item concentrations of 0.30, 0.95, 3.1, 9.8, 31.3 and 100 mg/L. For determination of the test item concentrations, samples were taken from each concentration level and from the control at the start and at the end of the test. Using a HPLC-UV the geometric mean test item concentrations of the measured start and end concentrations were calculated to be 0.19, 0.49, 0.87, 4.84, 25.22 and 88.93 mg/L. Biological results and endpoints are thus based on the measured concentrations. The test design included three replicates at each test concentration and six replicates for the untreated control. The alga cell concentration was approximately 10⁴ cells/mL at the start of the test in all of the test cultures. Glass flasks with total capacity of 250 mL were used as test vessels. The algal cell concentration was determined by manual cell counting by microscope in each testing flask during the 72-hour test, in 24-hour intervals. The results of the statistical evaluation showed that the 0-72 h average specific growth rate and yield was not statistically significantly different from the control in the exposure concentrations of 0.19, 0.49, 0.87 and 4.84 mg/L and significantly different in the exposure concentrations of 25.22 and 88.93 mg/L. The 72-h EC10 and EC50 of the test item for growth rate were determined to be 6.75 and 15.55 mg/L, respectively. The 72-h EC10 and EC50 for yield were determined to be 1.89 and 7.80 mg/L, respectively.  The 72-h NOEC for growth rate and yield was determined to be 4.84 mg/L. All validity criteria of the guidelines were met; the study is hence considered valid.

Categories Display