4-sec-butyl-2,6-di-tert-butylphenol

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:

24 May 2011 - 25 Jul 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

2004

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

1992

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 440/2008 C.2, Acute Toxicity for Daphnia

Version / remarks:

2008

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures

Version / remarks:

2000

Deviations:

not specified

Test guideline 5

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Version / remarks:

1996

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Remarks:

Swiss Federal Office of Public Health, Consumer protection directorate, Bern, 11 January 2011.

Test material

Test material information

Constituent 1

Reference substance name:

4-sec-butyl-2,6-di-tert-butylphenol

EC Number:

241-533-0

EC Name:

4-sec-butyl-2,6-di-tert-butylphenol

Cas Number:

17540-75-9

Molecular formula:

C18H30O

IUPAC Name:

4-(butan-2-yl)-2,6-di-tert-butylphenol

Test material form:

liquid: volatile

Details on test material:

Storage conditions: At room temperature at about 20 °C, under nitrogen in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: solvent control, 10, 22, 46, 100 and 220 μg/L (10 μg/L sampled but not analyzed).
- Sampling method: Duplicate samples, at start and end of test. Additionally, one sample was taken from the highest concentrated application solution at the start of the test.
- For the 48-hour stability samples, the contents of the respective replicates were combined prior to sampling.
- Sample storage conditions before analysis: Immediately after sampling, acetonitrile in the ratio of 1:1 was added to each sample from the test media of all test concentrations and the solvent control to stabilize the latter during the storage period. Thereafter, all samples were stored deep-frozen (at about -20 °C) until analysis.

Test solutions

Vehicle:

yes

Remarks:

N,N-Dimethylformamide p.a. (DMF)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The application solution for the highest test concentration (2200 mg/L) was freshly prepared by dissolving 88.0 mg of the test item in 40 mL of DMF. Adequate volumes of this application solution were diluted with DMF to obtain the application solutions for the lower test concentrations.
Initially, the test vessels were preconditioned. Therefore, the test vessels were filled with test water and treatment replicates were applied individually by adding and mixing adequate volumes of the respective application solution or DMF (for the solvent control) directly into the test water. A solvent concentration of 100 μL/L was finally reached in all treatments. Thereafter, the test vessels were stoppered with glass stoppers and left for about 1 hour. After the preconditioning period, the test media were discarded, the test flasks rinsed with test water (without test item), and remaining test water was drained off.
Subsequently, the test vessels were used for the test and the application solutions respectively DMF (for the solvent control) were applied again to the test water as described above. For the control, test water without addition of test item or solvent was used.
- Controls: test water without addition of test item or solvent; solvent control.
- Chemical name of vehicle: N,N-Dimethylformamide p.a. (DMF)
- Concentration of vehicle in test medium: 100 μL/L
- Evidence of undissolved material: no.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Strain/clone: Straus, clone 5.
- Source: in-house laboratory culture, original supplier University of Sheffield / UK (1992).
- Age of organisms at start of test: 6-24 h, not first brood progeny.
- Feeding during test: no

BREEDING
- Medium: reconstituted water of the quality identical to the water quality used in the tests (in respect to pH, main ions, and total hardness) and under temperature and light conditions identical to those of the tests.
- Feeding: three times a week with an algal suspension of the green algae Desmodesmus subspicatus CHODAT, Strain No. 86.81 SAG, or a mixture of this algal suspension and a commercial fish diet (Tetra Min® Hauptfutter).

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

Test water: 2.5 mmol/L (250 mg/L as CaCO3)

Test temperature:

21-22 °C

pH:

Start: 7.9
End: 7.7

Dissolved oxygen:

Start: 8.3-8.6 mg/L
End: 8.0-8.1 mg/L

Nominal and measured concentrations:

Nominal: 10, 22, 46, 100, 220 μg/L
Measured (four highest test concentrations only): 19.8, 44.1, 99.1, 218 μg/L (fresh, i.e. 90-99% of nominal); 18.5, 39.9, 88.7, 200 μg/L (old, i.e. 84-91% of nominal). Effect parameters are expressed as nominal test item concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flasks, completely filled, tightly closed with glass stoppers
- Preconditioning of vessels: one hour with respective test medium prior to star of the test, see 'Details on test solution'.
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water according to ISO 6341.
- Culture medium different from test medium: no
- Intervals of water quality measurement: At the start and end of the test, the pH values, dissolved oxygen concentrations and watertemperature were determined at each treatment. The appearance of the test media was visually recorded at the start of the test and after 24 and 48 hours.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 8:16 dark:light cycle with a 30 min transistion period.
- Light intensity: approx. 485-640 Lux.

EFFECT PARAMETERS MEASURED
- The immobility of the daphnids was determined by visual inspection after 24 and 48 hours ofexposure.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- The selection of the test concentrations was based on the results of range-finding tests and of pre-experiments to the dosage and solubility of the test item (non-GLP); details on range-finding test not specified.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (performed April 2011)

Results and discussion

Effect concentrations

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

92 µg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% C.I. 57-188 ug/L

Details on results:

- Behavioural abnormalities: none
- Mortality of control: none
- Effect concentrations exceeding solubility of substance in test medium: no
- No immobility or adverse effects were observed in any treatment at 24 h. After 48 h, 5% immobility was observed at 46 ug/L, which was considered a toxic effect, and 65% immobility was observed at 100 ug/L.

Other effect parameters determined:
– 24-hour EC50: >220 μg/L
– 24-hour EC0: ≥220 μg/L
– 24-hour EC100: >220 μg/L
– 48-hour EC50: 92 μg/L
(95% confidence limits: 57 and 188 μg/L)
– 48-hour EC0 and
48-hour NOEC: 22 μg/L
– 48-hour EC100: >220 μg/L

Results with reference substance (positive control):

- Results with reference substance valid? Yes, the sensitivity of the test organisms was within the internal historical range (48-hour EC50 from 2000 to 2011: 0.43-1.1 mg/L).
- 48-hour EC50: 0.62 mg/L

Reported statistics and error estimates:

48-hour EC50 and the 95% confidence limits were calculated by Weibull Analysis

Any other information on results incl. tables

Table 1: Effect of 2,6-di-tert-butyl-4-sec-butylphenol on the Mobility of Daphnia magna

Nominal test item concentration (μg/L)	No. of daphnids tested	Immobilized daphnids after 24 hours		Immobilized daphnids after 48 hours
		No.	%	No.	%
Solvent control	20	0	0	0	0
Control	20	0	0	0	0
10	20	0	0	0	0
22	20	0	0	0	0
46	20	0	0	1	5
100	20	0	0	13	65
220	20	0	0	17	85

Table 5 Results for Test Samples and Application Solution Samples

Timepoint       [day]	Nominal concentration of test item Cnom [μg/L]	Measured concentration of test item x [μg/L]	Sample preparation factor F	Determined concentration of test item C [μg/L]	% of nominal concentration     [%]
0 (fresh)	Control	*	2	<LOQ	n.a.
	22	9.89	2	19.8	90
	46	22.0	2	44.1	96
	100	49.6	2	99.1	99
	220	109	2	218	99
	2200000	89.7	25000	2242491	102
2 (old)	Control	*	2	<LOQ	n.a.
	22	9.24	2	18.5	84
	46	20.0	2	39.9	87
	100	44.3	2	88.7	89
	220	100	2	200	91

*the measured concentration was below the lowest calibration concentration

LOQ = 2.58 μg test item/L

n.a. = not applicable

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See 'Overall remarks' for details on validity criteria.

Conclusions:

The 48-hour EC50 was calculated to be 92 μg/L with 95% confidence limits of 57 and 188 μg/L.

Executive summary:

In a 48 h acute toxicity test conducted according to OECD Guideline 202 and GLP principles, Daphnia magna were exposed to the test item at nominal loading rates of 10, 22, 46, 100 and 220 μg/L, and a control and a solvent control, under static conditions. The measured concentrations were between 84 and 99% of the nominal values. Therefore, the effect parameters are based on nominal concentrations. At 24 h, no immoblity or adverse effects were observed. At 48 h, 85% of the test organisms were immobile in the highest test concentration. The 48h-EC50 for Daphnia magna was 92 μg/L with 95% confidence limits of 57 and 188 μg/L. The study met all validity criteria and is considered to be reliable without restrictions.