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

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:

26 Aug 2011 - 20 Sep 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 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Deviations:

no

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, all test concentrations
- Sampling method: duplicate samples at start of the test (without algae) and at the end of the test (containing algae). For the 72-hour stability samples, the contents of the respective replicates were combined prior to sampling
- Sample storage conditions before analysis: Immediately after sampling, acetonitrile (1 mL acetonitrile per mL sample volume) was added to each sample in order to stabilize the latter during the storage period. Thereafter, all samples were stored deep-frozen (at about -20 °C).

Test solutions

Vehicle:

yes

Remarks:

N,N-dimethylformamide (DMF)

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The application solution for the dosage of the highest test concentration was prepared by dissolving 92.1 mg of the test item in 20 mL of DMF. This application solution was diluted with DMF to prepare the application solutions for the dosage of 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 28.5 μL of the respective application solution or DMF (for the solvent control) directly into 285 mL of the test water. A solvent concentration of 100 μL/L was finally reached in all treatments. Thereafter, the test vessels were stirred for 5 minutes and stoppered with glass stoppers and left for 55 minutes. After the preconditioning period, the test media were discarded, the test flasks rinsed with purified water, 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.
- Controls: test water without addition of test item or solvent; solvent control
- Chemical name of vehicle: N,N-dimethylformamide (DMF)
- Concentration of vehicle in test medium: 100 μL/L
- Evidence of undissolved material: no

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: Strain No. 61.81 SAG
- Source: Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany).
- Age of inoculum: 4 days
- Method of cultivation: An inoculum culture was set up four days before the start of the exposure. The algae were cultivated under the test conditions. The inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.
- Cultivation and test medium: Reconstituted test water (OECD medium), buffered with 6 mmol/L HEPES-buffer (corresponding to 1430 mg/L)

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 d

Test conditions

Hardness:

0.24 mmol/L (= 24 mg/L as CaCO3)

Test temperature:

22 °C

pH:

Start: 7.8-8.0
End: 10.7-11.1

Nominal and measured concentrations:

Nominal: 22, 46, 100, 220, 460 μg/L
Measured: 14, 22, 64, 145, 320 μg/L (geometric means of measured concentration at start and end of the test). The effect levels were expressed as mean measured test item concentrations. See table 5 in 'Any other information on results incl. tables' for the measured concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel: 285 mL glass Erlenmeyer flasks, completely filled, tightly closed with glass stoppers
- Aeration: no
- Initial cells density: 10000 cells/mL, 0.99 x 10^3 relative fluorescence units.
- Control end cells density: 306 x 10^3 relative fluorescence units (solvent control).
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): 6
- The test flasks were incubated in a temperature-controlled water bath, positioned randomly and repositioned daily.

GROWTH MEDIUM
- Standard medium used: yes, reconstituted water according to OECD Guideline.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water (OECD medium), made with sterile purified water.
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH: in each treatment at start and end of the test; temperature: daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: continuously
- Light intensity and quality: fluorescent tubes (Philips TLD 36W-1/840), approximately 6630 Lux (range: 6200 to 6930 Lux).

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: pre-culture: electronic particle counter (Coulter Counter, Model ZM); test samples: fluorescence measurement (BIO-TEK Multi-Detection Microplate Reader, Model FLx800)

TEST CONCENTRATIONS
- Range finding study performed, not further specified.
- Results used to determine the conditions for the definitive study: yes.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate (performed April 2011)

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 320 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 320 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

22 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

64 µg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none observed
- The algal growth (biomass) in the solvent control was not statistically significantly different from the control after 72 hours.
- The test item had a statistically significant inhibitory effect on the growth of the algae of the algae after the test period of 72 hours at the mean measured concentration of 64 μg/L and at all higher test concentrations.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- 72-hour EC50 for the growth rate: 1.3 mg/L; within the historical range (2000-2011) of 0.71-1.7 mg/L.

Reported statistics and error estimates:

Student-t test, α = 0.05, two-sided (to compare control and solvent-control). Williams t-test, onesided smaller, α = 0.05 (to determine NOEC). Probit Analysis.

Any other information on results incl. tables

Table 1     Biomass of Algae

Nominal test item concentration [µg/L]	Mean measured concentration [µg/L]	Rep. no.	Biomass of algae* 24 hours             48 hours            72 hours
Solvent Control	--	1 2 3 4 5	9.7 9.2 8.9 9.8 9.5	77.6 79.0 79.6 70.8 84.3	299.8 303.0 304.1 273.8 333.8
		6	9.6	82.2	321.8
		Mean	9.4	78.9	306.0
		SD	0.3	4.6	20.5
Control	--	1 2 3	9.7 9.4 9.3	74.0 85.6 83.4	262.1 305.9 287.8
		Mean	9.5	81.0	285.3
		SD	0.2	6.1	22.0
22	14	1 2 3	9.2 8.6 9.4	76.4 82.4 87.0	265.8 327.1 290.6
		Mean	9.1	81.9	294.5
		SD	0.4	5.4	30.8
46	22	1 2 3	9.4 9.3 9.4	82.0 83.1 93.0	248.4 311.6 267.8
		Mean	9.4	86.0	275.9
		SD	0.0	6.1	32.4

100	64	1 2 3	9.6 9.2 9.9	84.2 83.4 90.6	225.3 248.1 288.7
		Mean	9.6	86.1	254.0
		SD	0.3	3.9	32.1
220	145	1 2 3	9.0 9.1 8.8	72.5 78.0 74.3	249.5 253.0 256.3
		Mean	9.0	74.9	252.9
		SD	0.1	2.8	3.4
460	320	1 2 3	8.5 7.9 8.2	65.6 66.3 66.4	250.3 226.2 260.1
		Mean	8.2	66.1	245.5
		SD	0.3	0.4	17.5

SD:        Standard deviation

*: The biomass was determined by fluorescence measurement (at least duplicate measurements per replicate) and is given as relative fluorescence units (E+03). At the start of the test, the initial cell density was 10000 algal cells/mL, corresponding to 0.99E+03 relative fluorescence units.

Table 2: Average Growth Rates ( μ)

Nominal test item concentration (µg/L)	Mean measured concentration (µg/L)	Average growth rate µ (day-1) and inhibition of µ (Ir)
		0-24 h		0-48 h		0-72 h
		µ	Ir (%)	µ	Ir (%)	µ	Ir (%)
Solvent Control	---	2.26	0.0	2.19	0.0	1.91	0.0
Control	---	2.26	-0.3	2.20	-0.6	1.89	1.2
22	14	2.21	1.8	2.21	-0.9	1.90	0.7
46	22	2.25	0.4	2.23	-2.0	1.88	1.9
100	64	2.27	-0.7	2.23	-2.0	1.85*	3.3
220	145	2.20*	2.3	2.16	1.2	1.85*	3.3
460	320	2.12*	6.2	2.10*	4.0	1.84*	3.8

*: mean value statistically significantly lower than in the solvent control (according to Williams t-test, one-sided smaller, α = 0.05)

Table 5     Results for Test Samples

Timepoint	Nominal Concentration of  Test Item cnom	Measured Concentration of Test Item	Sample Preparation Factor F	Determined Concentration of Test Item  c	% of Nominal Concentration
[day]	[µg/L]	[µg/L]		[µg/L]	[%]
0	Control	*	2	<LOQ	n.a.
(fresh)	22	9.10	2	18.2	83
	46	18.0	2	36.0	78
	100	41.3	2	82.7	83
	220	88.7	2	177	81
	460	193	2	386	84
3	Control	*	2	<LOQ	n.a.
(old)	22	5.15	2	10.3	47
	46	7.00	2	14.0	30
	100	25.1	2	50.2	50
	220	59.1	2	118	54
	460	132	2	265	58

*the measured concentration was below the lowest calibration concentration

LOQ = 10.1 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 biological results of the study were as follows (based on mean measured test item concentrations):
EC50 (μg/L): >320
EC20 (μg/L): >320
EC10 (μg/L): >320
NOEC (μg/L): 22
LOEC (μg/L): 64

Executive summary:

In a 72 h toxicity study conducted according to OECD guideline 201 and GLP principles, freshwater algae (Pseudokirchneriella subcapitata) were exposed to the test substance at nominal concentrations of 22, 46, 100, 220, 460 μg/L. Geometric mean measured concentrations were 14, 22, 64, 145 and 320 μg/L, and thus the effect parameters were based on mean measured concentrations. Growth rate was statistically significantly reduced at and above 64 μg/L. The EC50 for growth rate inhibition (72h-ERC50) was >320 μg/L. The 72h-NOEC for growth rate inhibition was 22 μg/L, the 72 -h LOEC was 64 μg/L. The study met all validity criteria and is considered to be reliable without restrictions.