4-cyclohexylphenol

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

2008

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-cyclohexylphenol

EC Number:

214-465-4

EC Name:

4-cyclohexylphenol

Cas Number:

1131-60-8

Molecular formula:

C12H16O

IUPAC Name:

4-cyclohexylphenol

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 mg/L
- Sample storage conditions before analysis: Routinely, the samples were analysed immediately. Only in exceptional cases, they were stored overnight deep frozen and protected from light.

Test solutions

Vehicle:

no

Remarks:

medium was used

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution was prepared to give the desired series of test concentrations. 101 mg of the test item were added to 1 litre of dilution water, treated for 1 h in an ultrasonic bath and stirred for 24 h on a magnetic stirrer. Undissolved particles of the test item were removed by filtration using a folded filter with a pore size of 7 - 12 μm. The pH was measured to be 7.9.
To produce the different test item concentrations appropriate amounts of the stock solution were diluted with dilution water to a volume of 500 mL. 50 mL of the prepared solutions were taken per replicate and 0.5 mL of dilution water containing 5 daphnids was given to all replicates resulting in the final nominal concentrations. For each test item concentration and the control 4 replicates were prepared.
- Controls: medium (composition see attachment to OECD 202) without any test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none stated

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Source: Strain of Bundesgesundheitsamt Berlin
- Maintenance and Acclimatisation : A population of parthenogenetic females of synchronized age structure has been maintained for more than 20 years in the test facility under constant temperature conditions (20 +/- 2 °C) at a 16 : 8 hour light-dark photoperiod (light intensity: < 20 μE x m-2 x s-1). The culture water (so-called 'M4 medium') was partly renewed once a week. The Daphnia were exclusively fed unicellular green algae (Desmodesmus subspicatus) 'ad libitum'. Mortalities of parent Daphnia during the culture period were recorded daily. The neonates were separated from their parent Daphnia by filtration prior to the acute test.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

none

Test conditions

Hardness:

The hardness of the dilution water used was 14.7 °dH (= 262 mg/L CaCO3).

Test temperature:

19.8 - 20.8 °C

pH:

7.9-8.0 at the start of the test, 8.5-8.7 at the end of the test

Dissolved oxygen:

8.5 - 8.8 mg O2/L

Nominal and measured concentrations:

Nominal: 0, 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 mg/L
Measured: --, 0.255, 0.505, 1.015, 1.960, 4.084, 8.183 and 16.662 mg/L after 0 h; <0.024, 0.250, 0.484, 0.990, 1.926, 3.967, 8.041* and 16.403* mg/L after 48 h (*: values were measured after 24 hours of exposure because of early 100 % immobilisation rates of the daphnids)

Details on test conditions:

TEST SYSTEM
- Test vessel, Type: 100 mL glass beakers covered with watch glasses holding 5 neonates in 50 mL of test medium
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): None.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (so-called 'M4 medium' according to OECD 202 and EC Method C.2, see Attachment to OECD 202) was used for the maintenance of the test animals and the preparation of stock and test solutions of the test item.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light : 8 h dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The criterion of adverse effects used in this study was the item-induced alteration of the normal mobility behaviour and the loss of locomotory actions of the neonates, observed at 24 and 48 hours.

VEHICLE CONTROL PERFORMED: yes, medium only

RANGE-FINDING STUDY
- Test concentrations: A range finding test preceded the main test. It provided information about the range of concentrations which were used in the main test. The following nominal concentrations of the test item were tested in the range finding test: 0.1, 1, 10 and 100 mg/L.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC0

Effect conc.:

1.96 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

3.82 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

8.18 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 4

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

0.51 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 5

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

1.56 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 6

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

4.08 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to EU method C.2, which is equivalent to OECD guideline 202, on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the toxicity of the test substance towards daphnids.
The toxicity against Daphnia magna was tested in a static test at nominal test concentrations of 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 mg/L, the EC 50 (48h) has been determined to be 1.56 mg/L (measured initial concentrations).
Based on these results, the test item does not need to be classified as acute toxic to the aquatic environment. With regard to chronic toxicity, taking into account the fact that the test item is not readily biodegradable, the test item should be classified as aquatic chronic Cat. 2.

Executive summary:

A GLP study was performed to assess the acute toxicity of p-Cyclohexylphenol to Daphnia magna STRAUS under static conditions.

The study was conducted in accordance with the Council Regulation (EC) No 440/2008, Method C.2 ‘Acute toxicity for Daphnia’ (2008) which is equivalent to OECD Guideline for Testing of Chemicals No. 202 'Daphnia sp., Acute Immobilisation Test' (adopted April 13, 2004).

The Daphnia were exposed to a range of concentrations, nominally 1.56, 3.13, 6.25, 12.5, 25, 50 and 100 mg/L of p-Cyclohexylphenol dissolved in dilution water. Auxiliaries used to prepare the test media were an ultrasonic bath, a magnetic stirrer and a folded filter.

Observations were made on the swimming ability and the immobilisation rate, respectively, after 24 and 48 hours of exposure. The following values were determined:

Time [h]	EC50 [mg/L]
24	3.82
48	1.56

The results are expressed in terms of measured initial concentrations at 0 and 48 h. Effective concentrations ranged from 15.7 % to 16.7 % of nominal values at 0 hours and from 15.4 % to 16.0 % of nominal values at 48 hours. The hardness of the dilution water used was 14.7 °dH (= 262 mg/L CaCO3).