1,3-Cyclohexanedimethanamine

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:

19 December 2008 to 12 March 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to GLP and internationally accepted test guidelines.

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:

- Concentrations: 1.94, 4.27, 9.39, 20.7, 45.5 and 100 mg/L.
- Sampling method: Samples were taken from control and test flasks at 24, 48 and 72 hours and the cell densities measured using a Coulter Z Series Particle Count and Size Analyser. The estimate of cell numbers in each sample was based on the mean of three consecutive counts, corrected for background counts of uninoculated dilution media. The presence of any abnormal cells was also noted during screening of each test level by microscopic examination.
- Sample storage conditions before analysis: dark, room temperature

Vehicle:

no

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: unicellular green alga
- Strain: CCAP 278/4
- Method of cultivation: The liquid slope cultures were stored in an illuminated refrigerator. Sterile algal nutrient medium was inoculated with cells aseptically removed from the slope culture; these primary liquid cultures (100 mL) were incubated for approximately three days in an orbital incubator under continuous illumination at nominal temperatures in the range 21 to 25 °C. These primary cultures provided an inoculum for use in the test (see protocol deviations) in the log phase of growth, characterised by a cell density of 2.19 x 106 cells/mL. Sterile algal nutrient medium as recommended in Official Journal No. L383A Part C.3 and OECD Procedure 201.

ACCLIMATION
- Acclimation period: 3 days

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

The test temperature in the non-adjusted media ranged from 22.4°C to 23.3°C.
In the pH-adjusted media, the temperature ranged from 22.0°C to 23.8°C.

pH:

In non-adjust media, the pH ranged from 7.62 up to 9.87 and varied among the test concentrations and over the duration of the test.
At the start of the test, the control pH was 7.44 and was 8.32 after 72h.
When the pH of the test concentration started below 8, it increased during the test period. For test substances whose initial pH was greater than 8, the pH decreased during the test period.

For pH-adjusted media, the range of starting pH was 7.4 to 7.58, with pH increasing in all concentrations over the duration of the test up to 9.18.

Nominal and measured concentrations:

At the start of the test, the measured levels of 1,3-bis(aminomethyl)cyclohexane in samples of the test cultures ranged between 71 and 108% of their nominal values in both non-adjusted and pH-adjusted media.
After 72 hours, the measured levels ranged between 34 and 111% of their nominal values (between 33 and 113% of their starting values), with losses evident at nominal concentrations of 1.94 to 9.39 mg/L in both non-adjusted and pH-adjusted media.

The overall mean measured levels of 1,3-bis(aminomethyl)cyclohexane were 1.27, 2.97, 5.63, 13.7, 39.2 and 104 mg/L in non-adjusted media and 1.44, 3.47, 5.59, 14.4, 45.8 and 100 mg/L in pH-adjusted media. These results indicate adjustment of pH had no influence on achieved 1,3-bis(aminomethyl)cyclohexane concentrations.

After 72 hours, analysis of samples of media containing 1,3-bis(aminomethyl)cyclohexane at nominal concentrations of 1.94 mg/L, which had been incubated without algal cells, indicated maintenance of test concentrations when compared to test media incubated in the presence of algal cells (83 and 100% of starting concentrations compared to 67 and 73%).
At nominal concentrations of 100 mg/L, results of analysis showed similar results to test media incubated in the presence of algal cells (102 and 115% of starting concentrations). These results indicate that the presence of algal cells affected the stability of the test substance in aquatic media.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type: closed
- Material, size, headspace, fill volume: 250mL conical flasks loosely stoppered with foam bungs, containing 100mL of test culture or control.
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): static
- Initial cells density: 0.930 x104/mL and 0.750x104
- Control end cells density: 1121406 and 1286178
- No. of vessels per concentration (replicates):3
- No. of vessels per control (replicates):6

GROWTH MEDIUM
- Standard medium used: yes
As recommended in Official Journal No. L383A Part C.3 and OECD Procedue 201

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

56.7 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL42.2 & 64.4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

29.7 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL, 25.0 & 37.4 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: pH Adjusted media; 95% CL, >100 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

58.4 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: pH Adjusted; 95% CL 43.2 & 75.8 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

25 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL 19.0 & 36.3mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

13.8 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% CL, 9.24 &31.2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

42.3 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: pH-adjusted media; 95% CL, 31.0& 54.4 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

16.5 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: pH adjusted; 95%CL 6.15 & 32.9 mg/L

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

39.2 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

39.2 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

45.8 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: pH-adjusted media

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

13.7 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

13.7 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

14.4 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: pH-Adjusted media

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): 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

Non-adjusted media
Area under the growth curve
EbC10 (72 h) : 13.8 mg/L (95% confidence limits, 9.24 & 31.2 mg/L)
EbC50 (72 h) : 29.7 mg/L (95% confidence limits, 25.0 & 37.4 mg/L)
No observed effect concentration (NOEC) : 13.7 mg/L
Lowest observed concentration (LOEC) : 39.2 mg/L
Average specific growth rate
ErC10 (0 - 72 h) : 25.0 mg/L (95% confidence limits, 19.0 & 36.3 mg/L)
ErC50 (0 - 72 h) : 56.7 mg/L (95% confidence limits, 42.2 & 64.4 mg/L)
No observed effect concentration (NOEC) : 13.7 mg/L
Lowest observed concentration (LOEC) : 39.2 mg/L
The mean coefficient of variation (CoV) for daily growth rates in non-adjusted control cultures ranged between 5.00 and 11.0% during the definitive test and the CoV for the average specific growth rates of control culture was 3.14% during the 72-hour exposure period.

pH-adjusted media
Area under the growth curve
EbC10 (72 h) : 16.5 mg/L (95% confidence limits, 6.15 & 32.9 mg/L)
EbC50 (72 h) : 58.4 mg/L (95% confidence limits, 43.2 & 75.8 mg/L)
No observed effect concentration (NOEC) : 14.4 mg/L
Lowest observed concentration (LOEC) : 45.8 mg/L
Average specific growth rate
ErC10 (0 - 72 h) : 42.3 mg/L (95% confidence limits, 31.0 & 54.4 mg/L)
ErC50 (0 - 72 h) : >100 mg/L (95% confidence limits, >100 mg/L)
No observed effect concentration (NOEC) : 14.4 mg/L
Lowest observed concentration (LOEC) : 45.8 mg/L
The mean coefficient of variation (CoV) for daily growth rates in pH-adjusted control cultures ranged between 1.78 and 16.3% during the definitive test and the CoV for the average specific growth rates of control culture was 4.24% during the 72-hour exposure period.

These results suggest that the increase in pH caused by the presence of 1,3-bis(aminomethyl)cyclohexane was partially responsible for the higher level of inhibition observed in the non-adjusted media when compared to the pH-adjusted media.

a) Cell densities - non-adjusted media

Exposure concentrations (mg 1,3-bis(aminomethyl)cyclohexane/L)		Replicate number	Cell densities (cells/mL)
Nominal	Measured		24 hours	48 hours	72 hours
Control	nd	R1	50200	274267	1170517
		R2	48100	248667	1202083
		R3	48067	283800	1248583
		R4	50600	263233	1258517
		R5	35200	218700	961317
		R6	36733	169800	887417
		Mean	44817	243078	1121406
1.94	1.27	R1	44533	242833	1082417
		R2	45167	243733	1068783
		R3	48667	271900	1195750
		Mean	46122	252822	1115650
4.27	2.97	R1	44267	252900	1108083
		R2	49767	270633	1183417
		R3	47267	235967	1098950
		Mean	47100	253167	1130150
9.39	5.63	R1a	30033	106233	502017
		R2	40367	240567	1131683
		R3	48333	266467	1082350
		Mean	44350	253517	1107017
20.7	13.7	R1	41400	212333	983483
		R2	45500	244833	1026983
		R3	46133	241867	949983
		Mean	44344	233011	986817
45.5	39.2	R1	18733	81000	296083
		R2	19733	104867	414517
		R3	20000	85333	312450
		Mean	19489	90400	341017
100	104	R1	5200	7567	8750
		R2	4800	6067	5483
		R3	6867	5933	4850
		Mean	5622	6522	6361

nd : none detected (less than the limit of detection of 0.5 mg/L).

R1-R6 : replicate number.

a : cell counts appear anomalous therefore vessel disregarded from analysis

Note : the initial cell density was estimated to be 0.930 x 104/mL

b) Cell densities - pH-adjusted media

Exposure concentrations (mg 1,3-bis(aminomethyl)cyclohexane/L)		Replicate number	Cell densities (cells/mL)
Nominal	Measured		24 hours	48 hours	72 hours
Control	nd	R1	50233	295067	1401517
		R2	48867	282633	1344483
		R3	50200	287000	1328817
		R4	27300	156200	835750
		R5	49767	292600	1427883
		R6	49867	269400	1378617
		Mean	46039	263817	1286178
1.94	1.44	R1	51467	279700	1292517
		R2	53933	282967	1414183
		R3	53933	290567	1366350
		Mean	53111	284411	1357683
4.27	3.47	R1	50900	266100	1189350
		R2	52467	274833	1078783
		R3	45800	263767	1165217
		Mean	49722	268233	1144450
9.39	5.59	R1	47667	263167	1268917
		R2	50400	276567	1409117
		R3	50467	283733	1454550
		Mean	49511	274489	1377528
20.7	14.4	R1	43033	238967	1094583
		R2	47533	244100	1139750
		R3	42633	232233	954350
		Mean	44400	238433	1062894
45.5	45.8	R1	41633	210367	937750
		R2	38133	194667	644950
		R3	43567	214033	736483
		Mean	41111	206356	773061
100	100	R1	25867	104867	268817
		R2	21167	92833	243917
		R3	25733	106433	263083
		Mean	24256	101378	258606

nd : none detected (less than the limit of detection of 0.5 mg/L).

R1-R6: replicate number.

Note : the initial cell density was estimated to be 0.750 x 104/mL

a) Inhibition of growth - non-adjusted media

Parameter	Concentration (mg/l)		Sample	Mean	% Inhibition	p
	Nominal	Measured	size
Area under curve to 72 hours	Control	nd	6	19.8	0.0	-
	1.94	1.27	3	20.0	-1.0	>0.999W
	4.27	2.97	3	20.2	-2.0	>0.999W
	9.39	5.63	2	19.8	-0.3	>0.999W
	20.7	13.7	3	17.9	9.5	0.221W
	45.5	39.2	3	6.1	69.0	<0.001***W
	100	104	3	-0.2	101.2	<0.001***W
Growth rate to 72 hours	Control	nd	6	0.065	0.0	-
	1.94	1.27	3	0.065	-0.1	>0.999W
	4.27	2.97	3	0.066	-0.3	>0.999W
	9.39	5.63	2	0.065	0.1	>0.999W
	20.7	13.7	3	0.064	2.5	0.383W
	45.5	39.2	3	0.049	25.3	<0.001***W
	100	104	3	-0.007	110.3	<0.001***W
Growth rate 0 - 24 hours	Control	nd	6	0.062	0.0	-
	1.94	1.27	3	0.064	-2.6	>0.999W
	4.27	2.97	3	0.065	-4.0	>0.999W
	9.39	5.63	2	0.062	0.2	>0.999W
	20.7	13.7	3	0.062	0.1	>0.999W
	45.5	39.2	3	0.028	55.2	<0.001***W
	100	104	3	-0.024	139.5	<0.001***W
Growth rate 24 - 48 hours	Control	nd	6	0.070	0.0	-
	1.94	1.27	3	0.071	-0.8	>0.999W
	4.27	2.97	3	0.070	0.3	>0.999W
	9.39	5.63	2	0.073	-3.5	>0.999W
	20.7	13.7	3	0.069	1.7	0.973W
	45.5	39.2	3	0.064	9.4	0.108W
	100	104	3	0.006	90.8	<0.001***W
Growth rate 48 - 72 hours	Control	nd	6	0.064	0.0	-
	1.94	1.27	3	0.062	3.3	0.590W
	4.27	2.97	3	0.062	2.4	0.590W
	9.39	5.63	2	0.061	3.9	0.538W
	20.7	13.7	3	0.060	5.8	0.220W
	45.5	39.2	3	0.055	13.8	0.004**W
	100	104	3	-0.002	103.4	<0.001***W

pvalues are for the comparison with Control using Williams' test (W)

**p< 0.01, ***p< 0.001

nd : none detected (less than the limit of detection of 0.5 mg/L).

Validity criteria fulfilled:

yes

Conclusions:

After 72 hours of exposure to 1,3-bis(aminomethyl)cyclohexane in non-adjusted media, the EbC50 and ErC50, respectively were 29.7 and 56.7 mg/L. In pH-adjusted media, the EbC50 and ErC50, respectively were 58.4 and >100 mg/L.

The following values were derived from the data.
Parameter (mg/L) Non-adjusted pH-adjusted
Area under the growth curve: EbC50 (72 h) 29.7 58.4
95% confidence limits 25.0 & 37.4 43.2 & 75.8
“No observed effect concentration” (NOEC) 13.7 14.4
Area under the growth curve: ErC50 (0 - 72 h) 56.7 > 100
95% confidence limits 42.2 & 64.4 > 100
“No observed effect concentration” (NOEC) 13.7 14.4

The “no observed effect concentration” (NOEC) for both area under the growth curve and growth rate in non-adjusted media was 13.7 mg/L. For, pH-adjusted media the NOEC for both area under the growth curve and growth rate was 14.4 mg/L.
These results suggest that the increase in pH caused by the presence of 1,3-bis(aminomethyl)cyclohexane was partially responsible for the higher level of inhibition observed in non-adjusted media.

Executive summary:

The effect of 1,3-bis(aminomethyl)cyclohexane on the growth of the unicellular green alga Pseudokirchneriella subcapitata was assessed under non-axenic conditions. The study was conducted in accordance with EC Methods for Determination of Ecotoxicity, Annex to Directive 92/69/EEC (O.J. No. L383A, 1992) Part C, Method 3 “Algal Inhibition Test” and Procedure 201 of the “Guidelines for Testing of Chemicals” of the Organisation for Economic Co-operation and Development: Freshwater Alga and Cyanobacteria, Growth Inhibition Test” (adopted 2006). Triplicate algal cultures, with an initial cell density of 1 x 10⁴/mL, were exposed to 1,3- bis(aminomethyl)cyclohexane dissolved in algal nutrient medium at nominal concentrations of 1.94, 4.27, 9.39, 20.7, 45.5 and 100 mg/L. The test media were prepared in OECD medium by serial dilution of an aqueous solution. During conduct of the rangefinding test, it was observed that addition of the test substance resulted in an increase in pH. In the definitive test, this effect was observed at nominal concentrations of 9.39 to 100 mg/L. Therefore, two batches of media were prepared. The first batch was tested without adjustment of pH; for the second batch of media, their pH was adjusted to within the range of 7.4 to 7.8, using 0.1M hydrochloric acid. The cultures were incubated in orbital incubators under continuous illumination at temperatures ranging from 21.7 to 23.2°C for 72 hours.

The measured levels of 1,3-bis(aminomethyl)cyclohexane in samples of test cultures ranged between 71 and 108% of their nominal values in freshly prepared non-adjusted and pH adjusted media. During the test, losses were observed at the lower test concentrations, with measured levels in expired (72 hour old) media decreasing to between 34 and 71% of nominal at 1.94 to 9.39 mg/L. At 20.7 to 100 mg/L, measured levels in expired media ranged between 62 and 111% of nominal. The overall mean measured levels of 1,3-bis(aminomethyl)cyclohexane were 1.27, 2.97, 5.63, 13.7, 39.2 and 104 mg/L in non-adjusted media and 1.44, 3.47, 5.59, 14.4, 45.8 and 100 mg/L in pH-adjusted media. These results indicate adjustment of pH had no influence on achieved 1,3-bis(aminomethyl)cyclohexane concentrations. Cell numbers were counted daily to monitor growth. The test results are expressed in terms of the area under the growth curve and growth rate.

The following values were derived from the data.

Parameter (g/L)	Non-adjusted	pH-adjusted
Area under the growth curve: EbC50(72h)	29.7	58.4
95% Confidence limits	25.0 & 37.4	43.2 & 75.8
“No observed effect concentration” (NOEC)	13.7	14.4
Area under the growth curve: ErC50(0-72h)	56.7	>100
95% Confidence limits	42.2 & 64.4	>100
“No observed effect concentration” (NOEC)	13.7	14.4

These results suggest that the increase in pH caused by the presence of 1,3- bis(aminomethyl)cyclohexane was partially responsible for the higher level of inhibition observed in the non-adjusted media when compared to the pH-adjusted media.

Description of key information

Algal growth inhibition, 72 hours; EbC50 = 29.7 mg/L, ErC50 = 56.7 mg/L.  In pH-adjusted media over the same period, EbC50 value was found to be 58.4 mg/L and ErC50 was >100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

56.7 mg/L

EC10 or NOEC for freshwater algae:

13.7 mg/L

Additional information

The effect of 1,3-bis(aminomethyl)cyclohexane on the growth of the unicellular green alga Pseudokirchneriella subcapitata was assessed under non-axenic conditions. The study was conducted in accordance with EC Methods for Determination of Ecotoxicity, Annex to Directive 92/69/EEC (O.J. No. L383A, 1992) Part C, Method 3 “Algal Inhibition Test” and Procedure 201 of the “Guidelines for Testing of Chemicals” of the Organisation for Economic Co-operation and Development: Freshwater Alga and Cyanobacteria, Growth Inhibition Test” (adopted 2006). Triplicate algal cultures, with an initial cell density of 1 x 10⁴/mL, were exposed to 1,3- bis(aminomethyl)cyclohexane dissolved in algal nutrient medium at nominal concentrations of 1.94, 4.27, 9.39, 20.7, 45.5 and 100 mg/L. The test media were prepared in OECD medium by serial dilution of an aqueous solution. During conduct of the rangefinding test, it was observed that addition of the test substance resulted in an increase in pH. In the definitive test, this effect was observed at nominal concentrations of 9.39 to 100 mg/L. Therefore, two batches of media were prepared. The first batch was tested without adjustment of pH; for the second batch of media, their pH was adjusted to within the range of 7.4 to 7.8, using 0.1M hydrochloric acid. The cultures were incubated in orbital incubators under continuous illumination at temperatures ranging from 21.7 to 23.2°C for 72 hours. The measured levels of 1,3-bis(aminomethyl)cyclohexane in samples of test cultures ranged between 71 and 108% of their nominal values in freshly prepared non-adjusted and pH adjusted media. During the test, losses were observed at the lower test concentrations, with measured levels in expired (72 hour old) media decreasing to between 34 and 71% of nominal at 1.94 to 9.39 mg/L. At 20.7 to 100 mg/L, measured levels in expired media ranged between 62 and 111% of nominal. The overall mean measured levels of 1,3-bis(aminomethyl)cyclohexane were 1.27, 2.97, 5.63, 13.7, 39.2 and 104 mg/L in non-adjusted media and 1.44, 3.47, 5.59, 14.4, 45.8 and 100 mg/L in pH-adjusted media. These results indicate adjustment of pH had no influence on achieved 1,3-bis(aminomethyl)cyclohexane concentrations. Cell numbers were counted daily to monitor growth. The test results are expressed in terms of the area under the growth curve and growth rate.

The following values were derived from the data.

Parameter (g/L)	Non-adjusted	pH-adjusted
Area under the growth curve: EbC50(72h)	29.7	58.4
95% Confidence limits	25.0 & 37.4	43.2 & 75.8
“No observed effect concentration” (NOEC)	13.7	14.4
Area under the growth curve: ErC50(0-72h)	56.7	>100
95% Confidence limits	42.2 & 64.4	>100
“No observed effect concentration” (NOEC)	13.7	14.4

These results suggest that the increase in pH caused by the presence of 1,3- bis(aminomethyl)cyclohexane was partially responsible for the higher level of inhibition observed in the non-adjusted media when compared to the pH-adjusted media.