Cyclohexyl(ethyl)amine

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11.6.-19.7.2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was carried out in accordance with internationally valid GLP principles.

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.3 (Algal Inhibition test)

Deviations:

no

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 during the study

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: the highest (86 mg.L-1) and the lowest (5 mg.L-1) test concentration and from the highest (86 mg.L-1) concentration without algae.
- 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 (72 hours) were delivered to analytical laboratory immediately after the end of testing. The samples were analyzed on the day of delivery.
- 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 test medium at concentrations needed for the test, the saturated solutions of the test substance in test medium were prepared for preliminary and full test. The saturated solutions were prepared by adding measured amounts of the test substance to the test medium. 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 the test medium 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):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Strain: Brinkmann 1953/SAG 86.81
- Source (laboratory, culture collection): The collection of autotrophic organisms of The Botanic Institute of the Czech Academy of Science, Třeboň
- Age of inoculum (at test initiation): 3 days
- Method of cultivation: The strain culture was preinoculated from the stock solution and cultivated in flasks with the test medium on indirect daylight at laboratory temperature.

ACCLIMATION
- Acclimation period: 3 days
- Culturing media and conditions (same as test or not): same as test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

23°C

pH:

7.6

Nominal and measured concentrations:

86, 54, 34, 21, 13, 8, 5 mg.L-1 (nominal concentration)

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 (86 mg.L-1) and the lowest (5 mg.L-1) test concentration and from the highest (86 mg.L-1) concentration without algae (w.a.):
Measured concentration: 5.34 mg.L-1, 80.9 mg.L-1 and 81.6 mg.L-1 w.a. (0 hours), 5.27 mg.L-1, 79.5 mg.L-1 and 81.4 mg.L-1 w.a. (72 hours)

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks with stoppers venting air
- Aeration: no
- Initial cells density: 5000 cells in a 1 mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Test medium: The water with conductivity smaller than 5 μS.cm-1 was used for the preparation of solutions.
- According to Method C.3 Algal Inhibition Test were prepared four basic stock solutions. The test medium was prepared by admixing of basic stock solutions.

OTHER TEST CONDITIONS
- Lighting: continuous white light, 4 440 lux to 8 880 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- The density of algae culture was evaluated microscopically at 24, 48 and 72 hours.
- At the beginning and the end of the test the pH of the test mixtures was measured. The light intensity and temperature were measured every 24 hours.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.6

- Range finding study
- Test concentrations: 100, 60, 40, 20 and 10 mg.L-1 (nominal concentration)
- On the basis of 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:

72 h

Dose descriptor:

other: ErC50

Effect conc.:

53.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% confidence limit: 48.4 – 58.6 mg.L-1

Effect concentrations 2

Duration:

72 h

Dose descriptor:

other: EyC50

Effect conc.:

8.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95% confidence limit: 7.5 – 9.3 mg.L-1

Effect concentrations 3

Duration:

72 h

Dose descriptor:

other: NOECr

Effect conc.:

5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

other: NOECy

Effect conc.:

< 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Results with reference substance (positive control):

- Results with reference substance valid? Yes.
The inhibition of test organisms by the reference substance (72h – ErC50) obtained from last reference test meets the calculated range from the interlaboratory test.

-Reference test: 72 hour – ErC50 = 0.99 mg.L-1 (95% confidence limit: 0.88 – 1.09 mg.L-1)

- Interlaboratory test: 72 hour – ErC50 = 0.38 – 1.30 mg.L-1

Any other information on results incl. tables

TEST EVALUATION

The values of ErC50 and EyC50 were calculated using the software “Toxicita”(product of Water Research Institute, Ostrava, Czech Republic).

The determination of NOEC values was done by ANOVA (Analysis of Variance) analysis. Used ANOVA method is the part of statistical software QC.Expert 2.5 © 1998-2000 (TriloByte, Czech Republic).

For the evaluation EC and NOEC values the nominal concentrations were used.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Executive summary:

The test substance, N-ethylcyclohexylamine, was tested for growth inhibition on algae Desmodesmus subspicatus. The test was performed according to method C.3. - Freshwater Algae and Cyanobacteria, Growth Inhibition Test, Commission Regulation (EC) No. 761/2009, Annex IV.

 

Since the test substance is poorly soluble in test medium at concentrations needed for the test, the saturated solutions of the test substance in test medium were prepared for preliminary and full test. The saturated solutions were prepared by adding measured amounts of the test substance to the test medium. The saturated solutions were stirred for period 3 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. The analytical results showed, that the test substance

N-ethylcyclohexylamine was sufficiently stable in the test medium at conditions of the test. Based on the toxicity of the test substance found in preliminary test, the full test was performed in appropriate concentration range.

 

The full test was performed in range of test substance nominal concentrations 5 – 86 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 percent of the nominal or measured initial concentration throughout the test, then the results can be based on nominal or measured initial values.

 

 

Test results:

72 hour – ErC50 = 53.5 mg.L-1 (nominal concentration)

(95% confidence limit: 48.4 – 58.6 mg.L-1)

 

72 hour – EyC50 = 8.4 mg.L-1 (nominal concentration)

(95% confidence limit: 7.5 – 9.3 mg.L-1)

 

72 hour – NOECr = 5 mg.L-1 (nominal concentration)

72 hour – NOECy < 5 mg.L-1 (nominal concentration)