Cyclohexyl(ethyl)amine

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11.6.-13.7.2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

yes

Remarks:

see Overall remarks

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Cyclohexyl(ethyl)amine

EC Number:

226-733-8

EC Name:

Cyclohexyl(ethyl)amine

Cas Number:

5459-93-8

Molecular formula:

C8H17N

IUPAC Name:

N-ethylcyclohexanamine

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): N-ethylcyclohexylamine
- Physical state: clear colourless liquid
- Analytical purity: 99.5% (w/w)
- Impurities (identity and concentrations): cyclohexanol 0.22% (w/w), alifatic amines 0.23% (w/w), water 0.07% (w/w)
- Lot/batch No.: 89002001
- Expiration date of the lot/batch: 12/2012
- Storage condition of test material: in original packaging at the fridge

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: the highest (120 mg.L-1) and the lowest (10 mg.L-1) test concentration
- Sampling method: The samples for analysis (0 hours) were prepared at the beginning of the test and immediately delivered in transport box to analytical laboratory. The samples for analysis at the end of the test (48 hours) were delivered to analytical laboratory immediately after the end of testing.
- Sample storage conditions before analysis: All samples were stored at laboratory temperature.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Since the test substance is poorly soluble in dilution water at concentrations needed for the test, the saturated solutions of the test substance in dilution water were prepared for preliminary and full test.
The saturated solutions were prepared by adding measured amounts of the test substance to the dilution water. The saturated solutions were stirred for suitable period (3 hours) on a shaft stirrer and consequently filtered through 0.45 μm filter. These solutions were used for testing.
The suitable period for achieving equilibrated concentration of the test substance was determined in preliminary experiment. For preliminary experiment the saturated solutions were prepared as follows: 100 mg of the test substance in 1000 mL of dilution water was stirred for period 3, 24, 48 and 72 hours on a shaft stirrer, consequently filtered through 0.45 μm filter and the concentrations of the test substance in resulted solutions were determined.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain: Strauss
- Source: own laboratory breeding
- Age at study initiation (mean and range, SD): young daphnia born in 24 hours
- Method of breeding: aperiodic parthenogenesis
- Feeding during test: no

ACCLIMATION
- Acclimation period: The grow-up gravid females are separated into dilution water. Young daphnia born in 24 hours are segregated to the handling container from which they entered the experiment.
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: a mixture of algae

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

Content of (Ca+Mg) in dilution water: 2.59 mmol.L-1

Test temperature:

20 +/- 2°C, with a variation of +/- 1°C at each individual test

pH:

8

Dissolved oxygen:

8.2 mg.L-1

Nominal and measured concentrations:

Nominal concentrations: 120, 80, 53, 35, 23, 15, 10 mg.L-1

The analytical determination of the test substance concentrations was performed at the beginning and at the end of the test. The samples for analysis were taken from the highest (120 mg.L-1) and the lowest (10 mg.L-1) test concentration:
Measured concentration: 8.0 mg.L-1 and 126.1 mg.L-1 (0 hours), 9.41 mg.L-1 and 137.1 mg.L-1 (48 hours)

Details on test conditions:

TEST SYSTEM
- Test vessel: open beakers
- Aeration: no
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- Biomass loading rate: one daphnia per 5 mL of test solution (least 2 mL per individual)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Standard dilution water was prepared just before the test.
- The dilution water should be of pH 7.8 +/- 0.2. The sum of Ca + Mg ions should be approx. 2.5 mmol.L-1.
- Ca/Mg ratio: 2.59 mmol.L-1
- Conductivity: 0.183 mS/m

OTHER TEST CONDITIONS
- Photoperiod and Light intensity: natural variation of daily light and dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The daphnia immobilisation was observed after 24 and 48 hours. At the beginning and the end of the test the ambient temperature, the oxygen content and pH value were measured.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.5
- Range finding study
- Test concentrations: The test was performed in range nominal concentrations from 10 mg.L-1 to 100 mg.L-1.
- On the basic of the preliminary test results the full test was performed.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

66 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limit: 55.8 – 76.2 mg.L-1

Effect concentrations 3

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

< 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 5

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 6

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Results with reference substance (positive control):

- Results with reference substance valid? Yes.
The immobilisation of test organism by the reference substance (48h – EC50) obtained from last reference test meets the calculated range from the interlaboratory test.

Reference test:
48 hour – EC50 = 0.78 mg.L-1 (95% confidence limit: 0.64 – 0.93 mg.L-1)
Interlaboratory test:
48 hour – EC50 = 0.34 – 1.18 mg.L-1

Any other information on results incl. tables

EVALUATION OF RESULTS

The values of EC50 were calculated by means of software “Toxicita”(Water Research Institute, Ostrava, Czech Republic). The NOEC and EC100 values are identified by direct observation.

The nominal concentrations were used for all evaluations and results because, if evidence is available to demonstrate that the concentration of the test substance has been satisfactorily maintained within ± 20 percent of the nominal or measured initial concentration throughout the test, then the results can be based on nominal or measured initial values.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Executive summary:

The test substance, N-ethylcyclohexylamine, was tested in acute immobilisation test on Daphnia magna. The test was performed according to method C.2 – Daphnia sp. Acute Immobilisation Test, Council Regulation (EC) No. 440/2008, published in O.J.L 142, 2008.

 

Since the test substance is poorly soluble in dilution water at concentrations needed for the test, the saturated solutions of the test substance in dilution water were prepared for preliminary test. The saturated solutions were prepared by adding measured amounts of the test substance to the dilution water. The saturated solutions were stirred for period 72 hours on a shaft stirrer and consequently filtered through 0.45 μm filter.

 

The preliminary test was performed in range of test substance nominal concentrations 10 – 100 mg.L-1. Based on toxicity of the test substance found in preliminary test, the full test was performed in appropriate concentration range.

 

The analytical results showed that the test substance N-ethylcyclohexylamine was sufficiently stable in dilution water at conditions of the test, therefore the test was performed as static one. The full test was performed in range of test substance nominal concentrations

10 - 120 mg.L-1. Samples for analytical determination were taken at the beginning and at the end of the test.

The nominal concentrations were used for all evaluations and results because, if evidence is available to demonstrate that the concentration of the test substance has been satisfactorily maintained within ± 20 per cent of the nominal or measured initial concentration throughout the test, then the results can be based on nominal or measured initial values.

 

TEST RESULTS:

 

24 hour–EC50 > 120 mg.L-1 (nominal concentration)

48 hour–EC50 = 66.0 mg.L-1 (nominal concentration)

(95% confidence limit: 55.8 – 76.2 mg.L-1)

 

24 hour–NOEC = 10 mg.L-1 (nominal concentration)

48 hour–NOEC < 10 mg.L-1 (nominal concentration)

 

24 hour–EC100 > 120 mg.L-1 (nominal concentration)

48 hour–EC100 > 120 mg.L-1 (nominal concentration)