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

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-05-28 to 2021-06-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

adopted 13 April 2004

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

May 30, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Version / remarks:

October 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling interval: sampled on day 0 and day 2

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution of 100 mg/L (nominal concentration) was first prepared by dissolving an amount of 0.055 g test item in 550 mL ISO medium using approx. 4 days stirring period (by magnetic stirrer). Non-dissolved material was removed by filtration through a membrane filter (pore size: 0.45 µm). The test solutions of subsequent lower concentrations were prepared by appropriate dilution of this stock solutions
- Controls: negative control, positive control
- Test concentration separation factor: 2
- Evidence of undissolved material: no

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Age at study initiation: less than 24 hours old at the start of exposure period
- Source: Daphnia were bred in the laboratory
- Animal Health: apparently healthy
- Food and Feeding: fed with concentrated algal suspension of Raphidocelis subcapitata during breeding; not fed during the exposure

ACCLIMATION
- Acclimation period: not necessary as test animals bred under same conditions as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

none

Hardness:

249 mg/L (as CaCO3)

Test temperature:

21.1 – 21.3 °C

pH:

7.41 – 8.13

Dissolved oxygen:

7.91 – 7.69 mg/L

Salinity:

not applicable

Conductivity:

not specified

Nominal and measured concentrations:

nominal: 6.25, 12.5, 25, 50 and 100 mg/L; measured: 5.27, 10.1, 20.2, 40.2 and 83.2 mg/L (geom. mean measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: beakers
- Type: open
- Material, size, fill volume: glass, 50 mL nominal volume, 40 mL fill volume
- Volume of solution: 40 mL
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration: 4
- No. of vessels per control: 4

TEST MEDIUM / WATER PARAMETERS: ISO medium

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light (artificial illumination) and 8 hours darkness
- Light intensity: 469 lux

EFFECT PARAMETERS MEASURED: immobilisation, mortality

RANGE-FINDING STUDY
- Test concentrations: Control 0.1, 1, 10, 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

16.2 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

other: EC20

Effect conc.:

17.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

18.9 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

10.1 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOEC

Effect conc.:

20.2 mg/L

Nominal / measured:

meas. (geom. mean)

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities:
- Observations on body length and weight: no
- Other biological observations: no
- Mortality of control: no
- Other adverse effects control: none
- Immobilisation of control: no
- Abnormal responses: no
- Any observations 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):

24-hour EC50: 0.94 mg/L

Reported statistics and error estimates:

To determine NOEC/LOEC values Step-down Cochran-Armitage test ( = 0.05, one-sided greater), to determine the ECx values Probit analysis was used by ToxRat Professional (Version 3.3.0) software.

Table 1: Immobilisation of the test animals (summary table)

Exposure Concentration [mg/L]	Number of Daphnia tested	Number of immobilised Daphnia		% of immobilised Daphnia
		24 h	48 h	24 h	48 h
Control	20	0	0	0	0
5.27	20	0	0	0	0
10.1	20	0	0	0	0
20.2	20	0	14	0	70
40.2	20	20	20	100	100
83.2	20	20	20	100	100

 

Table 2: Summary of biological endpoints

Endpoints	Concentration [mg/L]
48 h-EC10	16.2
95 % conf. limits	n.d.
48 h-EC20	17.1
95 % conf. limits	n.d.
48 h-EC50	18.9
95 % conf. limits	n.d.
48 h-NOEC	10.1
48 h-LOEC	20.2

n.d.: not determined due to mathematical reasons or inappropriate data

 

Table 3: Immobilization of the test animals (detailed table)

Exposure Concentration [mg/L]	Replicate	Number of treated animals	Number of immobilised animals
			24 h	48 h
Control	1	5	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0
5.27	1	5	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0
10.1	1	5	0	0
	2	5	0	0
	3	5	0	0
	4	5	0	0
20.2	1	5	0	4
	2	5	0	4
	3	5	0	3
	4	5	0	3
40.2	1	5	5	5
	2	5	5	5
	3	5	5	5
	4	5	5	5
83.2	1	5	5	5
	2	5	5	5
	3	5	5	5
	4	5	5	5

 

Table 4: Test Item concentration

Nominal concentration (mg/L)	Start of the test	End of the test
6.25	5.85	4.75
12.5	10.7	9.58
25	20.7	19.7
50	41.2	39.2
100	85.4	81.1

Validity criteria fulfilled:

yes

Conclusions:

In an immobilisation test with Daphnia magna according to OECD TG 202 the 48-hour EC50 of the test item was determined to be 18.9 mg/L.

Executive summary:

To evaluate the influence of the test item on the mobility respectively survival of Daphnia magna a 48-hour immobilisation test was carried out in accordance with OECD TG 202, EU Method C.2 and GLP-principles. Young Daphnia were exposed in an immobilisation test to aqueous test media containing the test item at a range of concentration for 48 hours. Twenty animals, divided into four groups (glass beaker; volume app. 50 mL; with ~40 mL test medium) of five animals each, were exposed to the test concentrations or ran as control (dilution water only) for 48 hours in a static test. The immobility or mortality of the Daphnia was determined by visual observation after 24 and 48 hours of exposure. Based on the results of preliminary experiment, nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg/L were investigated in the main study. Exposure concentrations were calculated as the geometric mean of the measured start and end concentrations and were the followings: 5.27, 10.1, 20.2, 40.2 and 83.2 mg/L. For determination of the test item concentrations, samples were taken from each concentration level and control at the start and at the end of the test. Concentrations were determined using HPLC-UV method. To determine NOEC/LOEC values Step-down Cochran-Armitage test (a = 0.05, one-sided greater), to determine the EC_x values Probit analysis was used. All validity criteria of the test guideline were fulfilled. After 48-h exposure no immobilisation was observed in the control and at the two lowest exposure concentrations of 5.27 and 10.1 mg/L. Significant immobilisation was observed at the exposure concentrations of 20.2, 40.2 and 83.2 mg/L (70 %, 100 %, 100 % immobility, respectively). As a result, the 48-h EC₅₀ value was determined to be 18.9 mg/L. The 48-h NOEC was determined to be 10.1 mg/L. The results are based on geometric measured test item concentrations. All validity criteria were met.

Description of key information

In an immobilisation test with Daphnia magna according to OECD TG 202 the 48-hour EC50 of the test item was determined to be 18.9 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

EC50

Effect concentration:

18.9 mg/L

Additional information

To evaluate the influence of the test item on the mobility respectively survival of Daphnia magna a 48-hour immobilisation test was carried out in accordance with OECD TG 202, EU Method C.2 and GLP-principles. Young Daphnia were exposed in an immobilisation test to aqueous test media containing the test item at a range of concentration for 48 hours. Twenty animals, divided into four groups (glass beaker; volume app. 50 mL; with ~40 mL test medium) of five animals each, were exposed to the test concentrations or ran as control (dilution water only) for 48 hours in a static test. The immobility or mortality of the Daphnia was determined by visual observation after 24 and 48 hours of exposure. Based on the results of preliminary experiment, nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg/L were investigated in the main study. Exposure concentrations were calculated as the geometric mean of the measured start and end concentrations and were the followings: 5.27, 10.1, 20.2, 40.2 and 83.2 mg/L. For determination of the test item concentrations, samples were taken from each concentration level and control at the start and at the end of the test. Concentrations were determined using HPLC-UV method. To determine NOEC/LOEC values Step-down Cochran-Armitage test (a = 0.05, one-sided greater), to determine the EC_x values Probit analysis was used. All validity criteria of the test guideline were fulfilled. After 48-h exposure no immobilisation was observed in the control and at the two lowest exposure concentrations of 5.27 and 10.1 mg/L. Significant immobilisation was observed at the exposure concentrations of 20.2, 40.2 and 83.2 mg/L (70 %, 100 %, 100 % immobility, respectively). As a result, the 48-h EC₅₀ value was determined to be 18.9 mg/L. The 48-h NOEC was determined to be 10.1 mg/L. The results are based on geometric measured test item concentrations. All validity criteria were met.