Cyclohex-1,2-ylenediamine

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:

Experimental phase: 2019-08-09 to 2019-09-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

Commission Regulation (EC) 761/2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2006, Annex 5 corrected: 28 July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for immediate quantitative analysis. A set of duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Vehicle:

no

Details on test solutions:

A nominal amount of test item (100 mg) was dissolved in culture medium and the volume adjusted to 1 liter to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 1.0, 3.2, 10 and 32 mg/L. The pH of the 100 mg/L stock solution (test item is a strong base) was checked and adjusted to pH 7.6. The pH of all subsequent stock solutions was checked and did not require adjustment. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (1.5 mL) to give an initial nominal cell density of 5.00 x 10^3 cells/mL.
The stock solutions and prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

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

Details on test organisms:

Pseudokirchneriella subcapitata strain CCAP 278/4
Origin: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland;

Inoculum culture:
Approximately 3 to 4 days before the start of the test, inoculum culture of algae was set up at an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 10^5 to 10^6 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

Calcium & Magnesium: 0.09 mM

Test temperature:

24 ±1 °C

pH:

Start of the test (t= 0 h):
Control pH 7.7
Treatments pH 7.5-7.7

End of the study (t= 72 h):
Control pH 8.4
Treatmens pH 7.8 (100 mg/L), 8.3 (32 mg/L) and 8.5 (1.0, 3.2, and 10 mg/L)

Dissolved oxygen:

Not applicable

Salinity:

n.a.

Conductivity:

not reported

Nominal and measured concentrations:

Nominal concentrations:
1.0, 3.2, 10 mg/L, 32 mg/L, and 100 mg/L

Measured concentrations:
t= 0 h [mg/L]: 1.04, 3.18, 8.71, 30.7, 98.7;
t= 72 h [mg/L]: 1.11, 2.96, 9.26, 32.4, 104;

Details on test conditions:

See section "Any other information on materials and methods incl. tables".

Reference substance (positive control):

yes

Remarks:

A positive control test using potassium dichromate as the reference item was performed twice in a 12-month period to demonstrate satisfactory conditions of the test.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

As analytically confirmed concentrations (0 h; 72 h) were very close to nominal, toxicity evaluation was based on nominal values.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

76 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

As analytically confirmed concentrations (0 h; 72 h) were very close to nominal, toxicity evaluation was based on nominal values.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 70 to 82 mg/L

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

35 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Remarks:

As analytically confirmed concentrations (0 h; 72 h) were very close to nominal, toxicity evaluation was based on nominal values.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 28 to 41 mg/L

Details on results:

All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures at 1.0, 3.2, 10 and 32 mg/L, however some cell debris and some healthy cells were observed to be present in the test cultures at 100 mg/L, with very few cells present overall.
At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control and test cultures at 1.0, 3.2, 10 and 32 mg/L were green dispersions (due to the presence of algal cells) and the 100 mg/L test culture replicates were clear colorless solutions.
For further information, see section "Any other information on results incl. tables".

Results with reference substance (positive control):

A positive control (Covance Study Number TP88DT) used potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L. The positive control was conducted between 16 July 2019 and 19 July 2019.
Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
Exposure of Pseudokirchneriella subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 to 72 hour) : 1.3 mg/L; 95% confidence limits 1.2 to 1.4 mg/L
EyC50 (0 to 72 hour) : 0.55 mg/L; 95% confidence limits 0.49 to 0.63 mg/L
No Observed Effect Concentration based on growth rate: 0.25 mg/L
No Observed Effect Concentration based on yield: 0.25 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.50 mg/L
Lowest Observed Effect Concentration based on yield: 0.50 mg/L
The results from the positive control with potassium dichromate were within the normal ranges for this reference item:
According to in-house data from the last 5 years the mean ErC50 value is 1.4 mg/L (standard deviation = 0.22) and the mean EyC50 value is 0.67 mg/L (standard deviation = 0.12).

Reported statistics and error estimates:

Statistical analysis of the growth rate and yield data was carried out for the control and all test concentrations using Shapiro-Wilk’s Test on Normal Distribution followed by Levene’s Test on Variance Homogeneity and Dunnetts Multiple t-test Procedure. Normality check (growth rate and yield) with p> 0.01.

Range-finding Test
No effect on growth was observed at the test concentrations of 0.010, 0.10 and 1.0 mg/L. However, growth was observed to be reduced at 10 and 100 mg/L.
Based on this information test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.

Definitive Test
Verification of Test Concentrations
Analysis of the test preparations at 0 hours showed measured test concentrations to range from 87 to 104% of nominal. There was no significant change in the measured concentrations at 72 hours and so the results are based on the nominal concentrations.
The following analytical results at 0 and 72 hours were obtained:

Analytical Identification	Sample Description	Time Point (Hours)	DCH Detected (mg/L)	Percentage of Nominal Concentration Detected (%)
19/3767	Control	0	ND	-
19/3768	1.0 mg/L		1.04	104
19/3769	3.2 mg/L		3.18	99
19/3770	10 mg/L		8.71	87
19/3771	32 mg/L		30.7	96
19/2772	100 mg/L		98.7	99
19/4089	Control	72	ND	-
19/4090	1.0 mg/L		1.11	111
19/4091	3.2 mg/L		2.96	93
19/4092	10 mg/L		9.26	93
19/4093	32 mg/L		32.4	101
19/4094	100 mg/L		104	104

ND – none detected less than the limit of detection of 0.1 mg/L

Growth Data
Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in following Table. Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks (R = Replicate).

Nominal Concentration (mg/L)		pH	Cell Densities (cells per mL)			pH
		0 Hours	25 Hours	52 Hours	72 Hours	72 Hours
Control	R1	7.7	3.29E+04	3.48E+05	1.82E+06	8.4
	R2		3.30E+04	3.74E+05	1.88E+06
	R3		3.18E+04	3.87E+05	1.94E+06
	R4		3.26E+04	3.99E+05	1.88E+06
	R5		3.54E+04	3.70E+05	1.88E+06
	R6		3.58E+04	3.83E+05	1.93E+06
	Mean		3.36E+04	3.77E+05	1.89E+06
1.0	R1	7.7	3.26E+04	3.48E+05	1.71E+06	8.5
	R2		3.18E+04	3.18E+05	1.68E+06
	R3		3.38E+04	3.75E+05	1.92E+06
	Mean		3.27E+04	3.47E+05	1.77E+06
3.2	R1	7.6	3.04E+04	2.25E+05	1.17E+06	8.5
	R2		3.10E+04	3.36E+05	1.77E+06
	R3		3.44E+04	4.16E+05	2.02E+06
	Mean		3.19E+04	3.26E+05	1.65E+06
10	R1	7.6	2.94E+04	3.31E+05	1.66E+06	8.5
	R2		3.13E+04	2.89E+05	1.48E+06
	R3		3.24E+04	3.13E+05	1.59E+06
	Mean		3.10E+04	3.11E+05	1.58E+06
32	R1	7.5	3.05E+04	2.69E+05	1.17E+06	8.3
	R2		3.14E+04	2.85E+05	1.27E+06
	R3		3.16E+04	2.51E+05	1.18E+06
	Mean		3.12E+04	2.68E+05	1.21E+06
100	R1	7.6	1.33E+04	2.54E+04	5.56E+04	7.8
	R2		1.13E+04	1.87E+04	2.30E+04
	R3		1.26E+04	2.20E+04	3.14E+04
	Mean		1.24E+04	2.20E+04	3.67E+04

Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in the following table (R = Replicate; SD = Standard Deviation; [ ] = Increase in growth compared to controls; - = Not applicable).

Nominal Concentration (mg/L)		Growth Rate (cells/mL/hour)		Yield (cells/mL)
		0 to 72 Hour	% Inhibition	0 to 72 Hour	% Inhibition
Control	R1	0 082	-	1.82E+06	-
	R2	0 082		1.87E+06
	R3	0 083		1.94E+06
	R4	0 082		1.87E+06
	R5	0 082		1.87E+06
	R6	0 083		1.93E+06
	Mean	0 082		1.88E+06
	SD	0 001		4.27E+04
1.0	R1	0 081	1	1.71E+06	9
	R2	0 081	1	1.67E+06	11
	R3	0 083	[1]	1.91E+06	[2]
	Mean	0 082	0	1.76E+06	6
	SD	0 001		1.28E+05
3.2	R1	0 076	7	1.16E+06	38
	R2	0 082	0	1.77E+06	6
	R3	0 083	[1]	2.02E+06	[7]
	Mean	0 080	2	1.65E+06	12
	SD	0 004		4.41E+05
10	R1	0 081	1	1.66E+06	12
	R2	0 079	4	1.48E+06	21
	R3	0 080	2	1.58E+06	16
	Mean	0 080	2	1.57E+06	16
	SD	0 001		9.12E+04
32	R1	0 076	7	1.16E+06	38
	R2	0 077	6	1.27E+06	32
	R3	0 076	7	1.17E+06	38
	Mean	0 076	7	1.20E+06	36
	SD	0 001		6.00E+04
100	R1	0 033	60	5.06E+04	97
	R2	0 021	74	1.80E+04	99
	R3	0 026	68	2.64E+04	99
	Mean	0 027	67	3.17E+04	98
	SD	0 006		1.69E+04

The mean cell densities versus time for the definitive test are presented in Figure 1 (attached illustration). Percentage inhibition values are plotted against test concentration in Figure 2 and Figure 3 (attached illustration).
From the data given above, it is obvious that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.
Accordingly, the following results were determined from the data:
Inhibition of Growth Rate
ErC10 (0 to 72 hour) : 35 mg/L; 95% confidence limits 28 to 41 mg/L
ErC20 (0 to 72 hour) : 46 mg/L; 95% confidence limits 39 to 51 mg/L
ErC50 (0 to 72 hour) : 76 mg/L; 95% confidence limits 70 to 82 mg/L
where ErCx is the test concentration that reduced growth rate by x%. Goodness of fit was calculated to be 1.000 whilst the significance of the dose-response relationship was <0.001.

There were no statistically significant differences (P≥0.05), between the control and the 1.0, 3.2 and 10 mg/L test concentrations; however, all other test concentrations were significantly different (P<0.05) and, therefore the
No Observed Effect Concentration (NOEC) based on growth rate was 10 mg/L.
Correspondingly the
Lowest Observed Effect Concentration (LOEC) based on growth rate was 32 mg/L.

Inhibition of Yield
EyC10 (0 to 72 hour) : 15 mg/L; 95% confidence limits 8.2 to 20 mg/L
EyC20 (0 to 72 hour) : 21 mg/L; 95% confidence limits 13 to 26 mg/L
EyC50 (0 to 72 hour) : 38 mg/L; 95% confidence limits 31 to 46 mg/L
Where EyCx is the test concentration that reduced yield by x%. Goodness of fit was calculated to be 1.000 whilst the significance of the dose-response relationship was 0.007.

There were no statistically significant differences (P≥0.05), between the control and the 1.0 and 3.2 mg/L test concentrations; however, all other test concentrations were significantly different (P<0.05) and, therefore the
NOEC based on yield was 3.2 mg/L.
Correspondingly the
LOEC based on yield was 10 mg/L.

 

 

Validity criteria fulfilled:

yes

Conclusions:

The following results in relation to growth rate were obtained from an algal growth inhibition test (OECD TG 201, GLP) based on analytically confirmed nominal test concentrations:
ErC10 (0 to 72 hour) : 35 mg/L; 95% confidence limits 28 to 41 mg/L;
ErC20 (0 to 72 hour) : 46 mg/L; 95% confidence limits 39 to 51 mg/L;
ErC50 (0 to 72 hour) : 76 mg/L; 95% confidence limits 70 to 82 mg/L;
NOEC (growth rate; 0 to 72 hour): 10 mg/L.

Executive summary:

A study on green algae growth inhibition was performed compliant with GLP according to OECD TG 201 (2006) and EU method C.3 (2009) using Pseudokirchneriella subcapitata.
Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to aqueous solutions of the test item at concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C. Due to the caustic nature of the test item, the pH of the stock solution was adjusted to pH 7.6 with HCl before use in the test.
Samples of the algal populations were removed daily, and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.
Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to be near nominal and so the results are based on nominal test concentrations only.
All validity criteria of the testing guidelines were met.

The following results were obtained for the period of 0h to 72 hours based on the analytically confirmed nominal test concentrations:
Inhibition of Growth Rate
ErC10 (0 to 72 hour) : 35 mg/L; 95% confidence limits 28 to 41 mg/L
ErC20 (0 to 72 hour) : 46 mg/L; 95% confidence limits 39 to 51 mg/L
ErC50 (0 to 72 hour) : 76 mg/L; 95% confidence limits 70 to 82 mg/L
NOEC (growth rate; 0 to 72 hour): 10 mg/L

Inhibition of Yield
EyC10 (0 to 72 hour) : 15 mg/L; 95% confidence limits 8.2 to 20 mg/L
EyC20 (0 to 72 hour) : 21 mg/L; 95% confidence limits 13 to 26 mg/L
EyC50 (0 to 72 hour) : 38 mg/L; 95% confidence limits 31 to 46 mg/L
NOEC (yield; 0 to 72 hour): 3.2 mg/L

Description of key information

The following results in relation to growth rate were obtained from an algal growth inhibition test (OECD TG 201, GLP) based on analytically confirmed nominal test concentrations:
ErC10 (0 to 72 hour) : 35 mg/L; 95% confidence limits 28 to 41 mg/L
ErC20 (0 to 72 hour) : 46 mg/L; 95% confidence limits 39 to 51 mg/L
ErC50 (0 to 72 hour) : 76 mg/L; 95% confidence limits 70 to 82 mg/L
NOEC (growth rate; 0 to 72 hour): 10 mg/L

Key value for chemical safety assessment

EC50 for freshwater algae:

76 mg/L

EC10 or NOEC for freshwater algae:

35 mg/L

Additional information

A study on green algae growth inhibition with the test item DCH (1,2-Cyclohexanediamine) was performed compliant with GLP according to OECD TG 201 (2006) and EU method C.3 (2009) using Pseudokirchneriella subcapitata.
Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to aqueous solutions of the test item at concentrations of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ±1 °C. Due to the caustic nature of the test item, the pH of the stock solution was adjusted to pH 7.6 with HCl before use in the test.
Samples of the algal populations were removed daily, and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.
Analysis of the test preparations at 0 and 72 hours showed measured test concentrations to be near nominal and so the results are based on nominal test item concentrations.
All validity criteria of the testing guidelines were met.

The following results were obtained for the period of 0h to 72 hours based on the analytically confirmed nominal test concentrations:
Inhibition of Growth Rate
ErC10 (0 to 72 hour) : 35 mg/L; 95% confidence limits 28 to 41 mg/L
ErC20 (0 to 72 hour) : 46 mg/L; 95% confidence limits 39 to 51 mg/L
ErC50 (0 to 72 hour) : 76 mg/L; 95% confidence limits 70 to 82 mg/L
NOEC (growth rate; 0 to 72 hour): 10 mg/L

Inhibition of Yield
EyC10 (0 to 72 hour) : 15 mg/L; 95% confidence limits 8.2 to 20 mg/L
EyC20 (0 to 72 hour) : 21 mg/L; 95% confidence limits 13 to 26 mg/L
EyC50 (0 to 72 hour) : 38 mg/L; 95% confidence limits 31 to 46 mg/L
NOEC (yield; 0 to 72 hour): 3.2 mg/L

Conclusions:
The following results in relation to growth rate were obtained from an algal growth inhibition test (OECD TG 201, GLP) based on analytically confirmed nominal test concentrations:
ErC10 (0 to 72 hour) : 35 mg/L; 95% confidence limits 28 to 41 mg/L
ErC20 (0 to 72 hour) : 46 mg/L; 95% confidence limits 39 to 51 mg/L
ErC50 (0 to 72 hour) : 76 mg/L; 95% confidence limits 70 to 82 mg/L
NOEC (growth rate; 0 to 72 hour): 10 mg/L