Cyclohex-1,2-ylenediamine

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental phase: 2019-08-09 to 2019-11-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Version / remarks:

Commission Regulation (EC) No. 440/2008, amended by Commission Regulation (EU) 2017/735 of 14 February 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Water samples were taken from the control and each surviving test group (replicates pooled) for immediate quantitative analysis. Samples of the fresh test preparations were taken on Days 0, 5, 12 and 19 and of the expired test preparations on Days 3, 7, 14 and 21. All samples were analyzed on the day of sampling. Duplicate samples were taken 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 test water and the volume adjusted to 1 liter to give a 100 mg/L stock solution. A series of dilutions was made from this stock solution to give test solutions of 0.32, 1.0, 3.2, 10 and 32 mg/L. The pH of the 100 mg/L stock solution was checked and adjusted to 7.9 ± 0.3. The pH of all subsequent stock solutions was checked prior to test organism addition.
The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.

Test organisms (species):

Daphnia magna

Details on test organisms:

The test was carried out using 1st instar Daphnia magna derived from in-house laboratory cultures.
Adult Daphnia were maintained in 150 mL glass vessels containing Elendt M4 medium (according to OECD TG 211, Annex 2) in a temperature controlled room at approximately 20 °C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension Raphidocelis subcapitata (formally known as Pseudokirchneriella subcapitata) and GEMMA Micro 300 fish food suspension. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated the day before initiation of the test, such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing. The diet and diluent water are considered not to contain any contaminant that would affect the integrity or outcome of the study.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

no

Hardness:

Water hardness measured as CaCO3 was determined in the control and the highest treatment group (32 mg/L) in fresh and old media with the following results:
Control: 240-252 mg/L
DCH (32 mg/L): 244-254 mg/L

Test temperature:

The temperature was maintained at 20 °C to 21 °C throughout the test

pH:

Control group: pH 7.9-8.4
Treatment groups (over all treatment concentrations and media preparations): pH 7.7-8.6

Dissolved oxygen:

= 8.3 mg O2/L throughout the study

Salinity:

n.a.

Conductivity:

not reported

Nominal and measured concentrations:

Nominal concentrations [mg/L]: 0.32, 1.0, 3.2, 10, 32.


Geometric mean measured concentrations [mg/L]: 0.22, 0.89, 3.0, 10, 32

Details on test conditions:

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

Reference substance (positive control):

no

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 32 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: parental immobilization not observed, only one inadvertent death in the control

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

32 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: reproduction & parental length

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: reproduction & parental length

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

32 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% CI: 21 – 49 mg/L

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 95% CI: 9.4 – 18 mg/L

Details on results:

At the start and throughout the test all control and test solutions were observed to be clear colorless solutions.
Effects on the Filial Generation (F1)
Information on the effects of the test item on the F1 generation is limited, since, by study design, the young are removed soon after liberation from the brood pouch. However, an assessment made at each media renewal showed the "filial" daphnids produced by all the test groups were in the same general condition as the young produced by the controls over the duration of the test. This was with the exception of young produced on Day 17 in the 32 mg/L test group, some of which were observed to be small.
Young were first produced in the control test group on Day 8 of the test.
For further information, see section "Any other information on results incl. tables".

Results with reference substance (positive control):

n.a.

Reported statistics and error estimates:

Analysis of Numbers of Live Young Produced per Adult:
Results from the control and each test group were compared using Shapiro Wilks test on Normal Distribution, Levenes test on Variance Homogeneity followed by the Multiple Sequentially-rejective Welch-t-test After Bonferroni-Holm as the normality check was passed however the variance homogeneity check failed.

Analysis of the Daphnia Length Data:
Results from the control and each test group were compared using Shapiro Wilk’s Test on Normal Distribution, Levene’s Test on Variance Homogeneity and Dunnett’s Multiple t-test Procedure. Both the normality check and the variance homogeneity check were passed (p> 0.010).

Range-finding Test
No immobilization was observed at the test concentrations of 0.10 and 1.0 mg/L, however, a single immobilized daphnia was observed at 10 mg/L and all 5 daphnids were observed to be immobilized at 100 mg/L. Sub-lethal effects of exposure were observed at the test concentration of 100 mg/L. These responses were a small size and reduced mobility.
Based on this information test concentrations of 0.32, 1.0, 3.2, 10 and 32 mg/L were selected for the definitive test.
Chemical analysis of the test preparations on Days 0 and 3 showed that near nominal measured concentrations were obtained indicating that the test item was stable under test conditions.

 

Definitive Test
Based on the results of the preliminary range-finding test, test concentrations of 0.32, 1.0, 3.2, 10 and 32 mg/L were selected for the definitive test.
Verification of Test Concentrations
Analysis of the fresh test preparations on Days 0, 5, 12 and 19 showed measured test concentrations to range from 81 to 119% of nominal. A decline in a proportion of the aged test groups was observed on Days 3, 7, 14 and 21 to give measured test concentrations of between 36 and 123% of nominal and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentration only in order to give a “worst case” analysis of the data.
The following table reports the measured concentrations in detail:

Sample Description	Time point (days)	DCH Detected (mg/L)	Percentage of Nominal Concentration Detected (%)
Control	0 (fresh)	ND	-
0.32 mg/L		0.277	87
1.0 mg/L		0.925	93
3.2 mg/L		3.090	97
10 mg/L		9.670	97
32 mg/L		31.300	98
Control	3 (old)	ND
0.32 mg/L		0.198	62
0.32 mg/L		0.189	59
1.0 mg/L		0.798	80
3.2 mg/L		2.660	83
10 mg/L		9.590	96
32 mg/L		30.000	94
Conlrol	5 (fresh)	ND	-
0.32 mg/L		0.259	81
1.0 mg/L		0.865	87
3.2 mg/L		2.950	92
10 mg/L		10.400	104
32 mg/L		30.500	95
Control	7 (old)	ND	-
0.32 mg/L		0.232	73
0.32 mg/L		0.201	63
1.0 mg/L		0.901	90
3.2 mg/L		2.720	85
10 mg/L		10.100	101
32 mg/L		29.600	93
Control	12 (fresh)	ND	-
0.32 mg/L		0.301	94
1.0 mg/L		1.010	101
3.2 mg/L		3.410	107
10 mg/L		11.900	119
32 mg/L		35.400	111
Control	14 (old)	ND	-
0.32 mg/L		0.163	51
0.32 mg/L		0.133	42
1.0 mg/L		0.676	68
1.0 mg/L		0.622	62
3.2 mg/L		2.500	78
3.2 mg/L		2.510	78
10 mg/L		9.770	98
32 mg/L		31.100	97
Control	19 (fresh)	ND	-
0.32 mg/L		0.246	77
0.32 mg/L		0.297	93
1.0 mg/L		1.010	101
3.2 mg/L		3.080	96
10 mg/L		10.900	109
32 mg/L		34.500	108
Control	21 (old)	ND	-
032 mg/L		0.112	35
032 mg/L		0.117	37
1.0 mg/L		0.980	98
3.2 mg/L		3.360	105
10 mg/L		12.800	128
10 mg/L		11.700	117
32 mg/L		30.700	96

Accordingly, the geometric mean measured test concentrations were determined to be [mg/L]: 0.22, 0.89, 3.0, 10, 32.

 

Validity criteria
All validity criteria of OECD TG 211 were fulfilled:
Control mortality (<= 20%): 10%
Mean number of live young per surviving adult (control group; >=60 after 21 days): 157
Coefficient of variation for control group* (<=25%): 4%
No ephippia produced: true
Dissolved oxygen (>3 mg O2/L): ≥8.3 mg O2/L
pH (control group; 6 to 9, variation <=1.5): 7.9 to 8.4, 0.5

 

Lethal Effects on the Parental Generation (P1)
No mortalities occurred in the test item test groups throughout the test. Accordingly, the following ECx (immobilization) values based on the geometric mean measured test item concentrations were estimated by inspection of the data:
EC10 (parental immobilization; 21 days) > 32 mg/L
NOEC (parental immobilization; 21 days) >= 32 mg/L

 

Sub-lethal Effects on the Parental Generation (P1)
No effect on the parental generation was observed in the control, 0.22, 0.89, 3.0 and 10 mg/L test groups, however, on Days 4 to 7, all adults in the 32 mg/L test group were observed to be small.
After 21 days the length of each surviving adult was determined. The results showed that there were no statistically significant differences (P>= 0.05) between the control and the 0.22, 0.89, 3.0 and 10 mg/L test groups in terms of length of the daphnids after 21 days exposure to the test item. However, a significant difference was found between the control and the 32 mg/L test group:
NOEC (parental length; day 21) = 10 mg/L
LOEC (parental length; day 21) = 32 mg/L
Body length data (day 21) for adults are given in the attached illustration.

 

Effects on Reproduction
After 21 days there were no statistically significant differences between the control and the 0.22, 0.89, 3.0 and 10 mg/L test groups in terms of the number of live young produced per adult. The 32 mg/L test group showed a statistically significant difference from the control after 21 days in terms of producing fewer numbers of live young per adult.
Given that all parental mortality was determined to be accidental/inadvertent, the results are the same in terms of young produced per adult surviving to the end of the test versus young produced per adult which did not die accidentally/inadvertently.
Details on reproduction data and evaluation are given in the attached illustration.
The following ECx (reproduction) values based on geometric mean measured test concentrations were calculated via fitting a 3-param. normal CDF to the data (no significant lack of fit):
EC10 (reproduction; 21 days) = 13 mg/L (95% CI: 9.4 – 18);
EC50 (reproduction; 21 days) = 32 mg/L (95% CI: 21 – 49);
NOEC (reproduction; 21 days) = 10 mg/L;
LOEC (reproduction; 21 days) = 32 mg/L.

Validity criteria fulfilled:

yes

Conclusions:

The following results were obtained for the test item DCH (1,2-Cyclohexanediamine) from a Daphnia magna reproduction test (OECD TG 211; GLP) based on geometric mean measured concentrations:

EC10 / NOEC (parental immobilization; 21 days) > 32 mg/L / >= 32 mg/L;

EC10 (reproduction; 21 days) = 13 mg/L (95% CI: 9.4 – 18);
EC50 (reproduction; 21 days) = 32 mg/L (95% CI: 21 – 49);
NOEC (reproduction & parental length; 21 days) = 10 mg/L;
LOEC (reproduction & parental length; 21 days) = 32 mg/L.

Executive summary:

A study on Daphnia magna reproduction with the test item DCH (1,2-Cyclohexanediamine) was performed compliant with GLP according to OECD TG 211 (2012) and EU method C.20 (2017) to assess the chronic toxicity of the test item to aquatic invertebrates.
Based on the results of a preliminary range-finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to solutions of the test item at nominal concentrations of 0.32, 1.0, 3.2, 10 and 32 mg/L for a period of 21 days in a semi-static test design (renewal 3 times per week). Due to the caustic nature of the test item, the pH of the stock solution was adjusted to pH 7.9 with HCl before use in the test. Test item concentrations in fresh and used media were analytically verified.
The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The Daphnia were fed daily with algal suspension and on Days 0-3 with GEMMA Micro 300 fish food suspension.

Analysis of the fresh test preparations on Days 0, 5, 12 and 19 showed measured test concentrations to range from 81 to 119% of nominal. A decline in a proportion of the aged test groups was observed on Days 3, 7, 14 and 21 to give measured test concentrations of between 36 and 123% of nominal and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations:
0.22, 0.89, 3.0, 10 and 32 mg/L.
All the validity criteria of the testing guideline were met.

Exposure of Daphnia magna to the test item gave the following results based on geometric mean measured test item concentrations:

EC10 / NOEC (parental immobilization; 21 days) > 32 mg/L / >= 32 mg/L

EC10 (reproduction; 21 days) = 13 mg/L (95% CI: 9.4 – 18);
EC50 (reproduction; 21 days) = 32 mg/L (95% CI: 21 – 49);
NOEC (reproduction & parental length; 21 days) = 10 mg/L;
LOEC (reproduction & parental length; 21 days) = 32 mg/L.

Although it cannot be inferred directly, it can be noted that the reduction in young production in the highest concentration of 32 mg/L could be linked to the significantly smaller size of the parental daphnia in this test group.

Description of key information

The following results were obtained for the test item DCH (1,2-Cyclohexanediamine) from a Daphnia magna reproduction test (OECD TG 211; GLP) based on geometric mean measured concentrations:

EC10 / NOEC (parental immobilization; 21 days) > 32 mg/L / >= 32 mg/L;

EC10 (reproduction; 21 days) = 13 mg/L (95% CI: 9.4 – 18);
EC50 (reproduction; 21 days) = 32 mg/L (95% CI: 21 – 49);
NOEC (reproduction & parental length; 21 days) = 10 mg/L;
LOEC (reproduction & parental length; 21 days) = 32 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC10

Effect concentration:

13 mg/L

Additional information

A study on Daphnia magna reproduction with the test item DCH (1,2-Cyclohexanediamine) was performed compliant with GLP according to OECD TG 211 (2012) and EU method C.20 (2017) to assess the chronic toxicity of the test item to aquatic invertebrates.
Based on the results of a preliminary range-finding test, Daphnia magna were exposed (10 replicates of a single daphnid per group) to solutions of the test item at nominal concentrations of 0.32, 1.0, 3.2, 10 and 32 mg/L for a period of 21 days in a semi-static test design (renewal 3 times per week). Due to the caustic nature of the test item, the pH of the stock solution was adjusted to pH 7.9 with HCl before use in the test. Test item concentrations in fresh and used media were analytically verified.
The numbers of live and dead adult Daphnia and young daphnids (live and dead) were determined daily. The Daphnia were fed daily with algal suspension and on Days 0-3 with GEMMA Micro 300 fish food suspension.

Analysis of the fresh test preparations on Days 0, 5, 12 and 19 showed measured test concentrations to range from 81 to 119% of nominal. A decline in a proportion of the aged test groups was observed on Days 3, 7, 14 and 21 to give measured test concentrations of between 36 and 123% of nominal and hence it was considered appropriate to calculate the results based on the geometric mean measured test concentrations:
0.22, 0.89, 3.0, 10 and 32 mg/L.
Exposure of Daphnia magna to the test item gave the following results based on geometric mean measured test item concentrations:

 

EC10 / NOEC (parental immobilization; 21 days) > 32 mg/L / >= 32 mg/L

 

EC10 (reproduction; 21 days) = 13 mg/L (95% CI: 9.4 – 18);
EC50 (reproduction; 21 days) = 32 mg/L (95% CI: 21 – 49);
NOEC (reproduction & parental length; 21 days) = 10 mg/L;
LOEC (reproduction & parental length; 21 days) = 32 mg/L.

 

Although it cannot be inferred directly, it can be noted that the reduction in young production in the highest concentration of 32 mg/L could be linked to the significantly smaller size of the parental daphnia in this test group.