4-cyclohexylphenol

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

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.3 (Algal Inhibition test)

Version / remarks:

2009

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006

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 (nominally)
- 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. 100.8 mg of the test item were added to 2 litres 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 7.7.
To produce the different test item concentrations appropriate amounts of the stock solution were diluted with dilution water to a volume of 100 mL and 0.357 mL of the algal inoculum was added to each replicate resulting in a final cell density of 5000 cells/mL. For each test item concentration and the control 3 replicates were prepared. All flasks were sealed with cotton stoppers.
- Controls: medium without any test item
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): see attachment 1

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Source (laboratory, culture collection): Strain of the test species obtained from 'The Collection of Algal Cultures' of the Institute of Plant Physiology at the University of Göttingen (Germany).

ACCLIMATION
- Maintenance and Acclimatisation: Exponentially growing stock cultures were maintained in the test facility under constant temperature conditions (21 - 24 °C with a maximum fluctuation of +/- 2 °C) at a light intensity in the range 60 to 120 μE x m-2 x s-1 (measured in the range 400 to 700 nm using a spherical quantum flux meter). The growth medium (according to BRINGMANN & KÜHN (1977) was renewed once a week. Cell density measurements were made using a microcell counter, Particle Count and Size Analyzer Z2, Beckman Coulter.
- Culturing media and conditions (same as test or not): Pre cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different growth medium (attachment 1).
- Test cultures: The algal inocula for the test were taken from an exponentially growing pre culture and were mixed with the growth medium (attachment 1) to make up to a final cell density of about 5000 cells per millilitre in the test medium.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

Water hardness of the final nutrient medium was 1.3 °dH, corresponding to 22.5 mg/L CaCO3.

Test temperature:

21 - 24 °C

pH:

7.8 - 7.9 (start of the study); 7.8 - 8.1 (end of the study)

Nominal and measured concentrations:

Nominal: 0 (Control), 1.56, 3,13,6.25, 12.5, 25 and 50 (+ 50 mg/L without algae)
Measured: --, 0.345 mg/L, 0.731 mg/L, 1.426 mg/L, 2.824 mg/L, 5.626 mg/L and 11.262 mg/L (11.364 mg/L without algae) after 0 h; <0.024 mg/L, 0.302 mg/L, 0.637 mg/L, 1.293 mg/L, 2.788 mg/L, 5.524 mg/L and 11.370 mg/L (10.856 mg/L without algae) after 72 h

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks with cotton stoppers, test volume: 100 mL
- Initial cells density: approximately 5000 cells per millilitre
- No. of vessels per concentration (replicates): 3. In order to check whether or not significant amounts of the test item were incorporated into the algal biomass during the test period, at all measuring points (0, 24, 48 and 72 hours) a test flask without algae was run in parallel to the geometric series of test concentrations.
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: yes (OECD medium of OECD TG 201 was used for the growth of the algae in the pre cultures and the preparation of stock and test solutions of the test item; see attachment 1)

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: Pre cultures were set up three days before the start of a test. They were grown under identical exposure conditions as the stock cultures, except from the use of a different growth medium (attachment 1).

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Light intensity and quality: 60 to 120 μE x m-2 x s-1, or an equivalent range of 4000 to 8000 lux, was measured. The light intensity was checked before the start of the study.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The criteria of adverse effects used in this study were the item-induced inhibition of yield [y] and growth rate [r] of the algal population.
- Determination of cell concentrations: Cell densities were measured in a microcell counter (Z2, Beckman Coulter) by taking aliquots from each test flask, which were not replaced.

TEST CONCENTRATIONS
- Range finding study: No
- Test concentrations: nominally 0.1, 1.0, 10 and 100 mg/L

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.3 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.59 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.35 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.73 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The study was conducted under GLP according to EU method C.3, which is equivalent to OECD guideline 201, 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 algae.
The toxicity against planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) 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. Results based on growth rate after 72h:
EC50 = 1.3 mg/L          
EC10 = 0.59 mg/L
NOEC = 0.35 mg/L                      
LOEC = 0.73 mg/L
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 adverse effects of p-Cyclohexylphenol on the growth rate (= rate of increase in cell density with time) and the yield (= biomass at time t minus initial biomass) of the planktonic freshwater algal species Desmodesmus subspicatus (former name: Scenedesmus subspicatus) over several generations.

The study was conducted in accordance with Commission Regulation (EC) No 761/2009 amending Regulation No 440/2008, Method C.3 ‘Freshwater Alga and Cyanobacteria, Growth inhibition test’ (2009) which is equivalent to OECD Guideline for Testing of Chemicals No. 201 (2006).

Exponentially growing algal cells were exposed for a period of 72 hours 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.

The cell densities were measured at 24 hour intervals. Inhibition of the algal population was measured as reduction in growth rate (index r), relative to control cultures grown under identical conditions. Growth rates were also used to calculate a No Observed Effect Concentration (NOEC) and a Lowest Observed Effect Concentration (LOEC) according to Williams Multiple Sequential t-test Procedure. The following values were determined:

ErC 50* (0-72 h): 1.3

ErC 10* (0-72 h): 0.59

NOEC [r]* (tα 0.05): 0.35

LOEC [r]* (tα 0.05): 0.73

* Reduction of growth rate (ErCx, NOEC [r]) is the preferred endpoint according to OECD 201 and for regulatory purposes in the EU. Results relating to yield (EyCx, NOEC [y]) were calculated to fulfil regulatory requirements in some countries (but not in the EU) and are given in the results section of this report.

The results are expressed in terms of measured initial concentrations. Effective concentrations ranged from 22.1 % to 23.4 of nominal values at 0 hours, and from 19.4 % to 22.7 % of nominal values at 72 hours.