2-ethoxy-4-(hydroxymethyl)phenol

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:

12 August 2016 - 01 October 2017

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:

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

Version / remarks:

At the time of signing the study plan, the only test item information included was the identification EVA. Additional test item information was provided, however, a study plan amendment incorporating this information to the study plan was not issued. The test item information has been included in the report and the omission of this information from the study plan was considered to have no impact on the outcome or integrity of the study.

Deviations:

yes

Remarks:

The deviation was considered to have no impact on the outcome or integrity of the study.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

At the time of signing the study plan, the only test item information included was the identification EVA. Additional test item information was provided, however, a study plan amendment incorporating this information to the study plan was not issued. The test item information has been included in the report and the omission of this information from the study plan was considered to have no impact on the outcome or integrity of the study.

Deviations:

yes

Remarks:

The deviation was considered to have no impact on the outcome or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-ethoxy-4-(hydroxymethyl)phenol

EC Number:

674-192-5

Cas Number:

4912-58-7

Molecular formula:

C9H12O3

IUPAC Name:

2-ethoxy-4-(hydroxymethyl)phenol

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Storage Conditions:
Room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

The test samples were thawed in water bath, and then analyzed directly.
To demonstrate the validity of the analytical procedure, volumes of test medium were spiked with the test item and the recovery was assessed. The test item (nominal 100 mg) was initially dissolved in methanol to prepare a stock solution with a concentration of 1000 mg/L. A defined volume of this stock solution was diluted with test medium to obtain spiked recovery samples at a concentration of 1.0 mg/L. Five replicates were prepared and subjected to the same treatment as the test samples. In addition, test medium without the addition of the test item (synthetic control) was also analyzed.

Test solutions

Vehicle:

no

Details on test solutions:

The test item (100 mg) was dissolved in culture medium with the aid of ultrasonication and vigorous shaking by hand for approximately 25 minutes.

Test organisms

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4.7.5
- Method of cultivation: under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ± 1 °C.

ACCLIMATION
- Culturing media and conditions: The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

24 ± 1 °C

pH:

7.5 - 8.3

Nominal and measured concentrations:

Nominal Test Concentration: 1.0, 3.2, 10, 32 and 100 mg/L
Geometric Mean Measured Test Concentration: 0.74, 2.6, 9.0, 29, 96 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass conical flasks
- Type: closed with polyurethane foam bungs
- Initial cells density: 5 x 10^3 cells per mL
- No. of vessels per concentration (replicates): three flasks
- No. of vessels per control (replicates): six flasks

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: continuous illumination
- Light intensity and quality: approximately 7000 lux, warm white lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: counting chamber

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.010, 0.10, 1.0, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: Based on the results of the third range-finding test the following test concentrations were assigned to the initial experiments: 6.25, 12.5, 25, 50 and 100 mg/L. However, significant inhibition occurred at 6.25 mg/L and as such a test concentration range of 1.0, 3.2, 10, 32 and 100 mg/L was selected for use in the definitive test.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 95 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Remarks on result:

other: It was not possible to calculate an EC50 value as no concentration tested resulted in greater than 50% inhibition.

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

30 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

95 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat. (total fraction)

Basis for effect:

growth rate

Details on results:

The detection system was found to have acceptable linearity. The analytical procedure was found to have acceptable recoveries of test item in test medium. The method of analysis was validated and proven to be suitable for use.
Inspection of the growth rate data obtained from all tests showed that less than 50% inhibition occurred at the maximum test concentration employed, resulting in an ErC50 value of greater than 100 mg/L (nominal concentration). It is therefore considered that whilst variability in the response to exposure occurred, the end point of each and every test remained the same and thus gave a reliable indication of the toxicity of the test item to Pseudokirchneriella subcapitata.

Results with reference substance (positive control):

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-Hour period and based on the geometric mean measured test concentrations gave the following results (confirmatory test):
EC50 (mg/L) NOEC (mg/L) LOEC (mg/L)
Growth Rate: >95* 30 95
Yield >95* 30 95
* It was not possible to calculate an EC50 value as no concentration tested resulted in greater than 50% inhibition.