3,5-di-tert-butyl-4-hydroxybenzyl alcohol

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:

23 Jun - 12 Jul 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

2011

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: SANCO/3029/99 rev. 4 11/07/00: Residues: Guidance for generating and reporting methods of analysis in support of pre-registration data requirements for Annex II (part A; Section 4) and Annex III (part A; Section 5) of directive 91/414

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Germany

Test material

Test material information

Constituent 1

Reference substance name:

3,5-di-tert-butyl-4-hydroxybenzyl alcohol

EC Number:

201-815-6

EC Name:

3,5-di-tert-butyl-4-hydroxybenzyl alcohol

Cas Number:

88-26-6

Molecular formula:

C15H24O2

IUPAC Name:

2,6-Di-tert-butyl-4-(hydroxymethyl)phenol

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations and method: duplicate samples from the freshly prepared test media filtrates (containing algae) of all concentrations and from the control were taken at the start of the test.
Duplicate samples from the test media filtrates of all test concentrations and the control (containing algae) were taken also at the end of the test by pooling the replicates. Additional samples of the control blank and the dilution solvent were taken at test start and test end without any sample treatment.
- Sample storage conditions before analysis: all samples were diluted by a factor of 2 with acetonitrile and stored at ≤ - 20 °C, protected from light

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: a stock solution was prepared with a concentration of the test substance as close as possible to the saturation level by dissolving 100 mg test item into 1000 mL test water. Solutions was mixed for 49 h. Then, non-dissolved fractions of the test item were separated from the test medium by membrane filtration (0.45 μm cellulose acetate filter). Then dilutions of the stock solution were prepared to obtain the remaining concentrations.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: 61.81 SAG
- Source: laboratory culture (original source: Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen, Germany)
- Age of inoculum (at test initiation): 4 d old exponentially growing pre-culture
- Method of cultivation: under standardised conditions according to the test guidelines

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

0.24 mmol/L as CaCO3

Test temperature:

21.1 - 23.1 °C

pH:

8.0 at test start
8.0 to 9.9 at test end

Dissolved oxygen:

-

Salinity:

-

Conductivity:

-

Nominal and measured concentrations:

Nominal test concentrations: 0 (control), 1.2 mg/L, 3.7 mg/L, 11.1 mg/L, 33.3 mg/L and 100 mg/L
Mean measured concentrations: 0 (control), 0.286 mg/L, 0.9 mg/L, 2.65 mg/L, 8.44 mg/L and 25.4 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks (covered with glass dishes)
- Fill volume: 50 mL
- Initial cells density: 5000 algal cells per mL test medium
- Control end cells density: 121.935 * 10^4 cells /mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per blank control (replicates): 1 for each test concentration (blank for the spectrophotometric measurements)
- Other: continuously stirred with magnetic stirrers

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Intervals of water quality measurement: temperature was measured daily in a flask filled with water and incubated under the same conditions as the test flasks, pH was measured in all concentrations and the control at the start and the end of the test

OTHER TEST CONDITIONS
- Adjustment of pH in dilution water: yes with 1M HCl to pH 8.1
- Photoperiod: continuous
- Light intensity: 5248 lux (mean)

EFFECT PARAMETERS MEASURED : samples were taken after 24, 48 and 72 h
- Determination of cell concentrations: spectrophotometrical measurement

TEST CONCENTRATIONS
- Range finding study: yes, non GLP (not included in final report)

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.:

> 25.4 mg/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.:

4.81 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % conf. interval: 4.14 - 5.59 mg/L

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): at 8.44 and 25.4 mg test item/L, cells appeared to be smaller and roundish compared to control cells (microscopic observations)

Results with reference substance (positive control):

- Results with reference substance valid: yes
- ErC50 (72 h) = 1.5 mg/L
- Other: ErC10 (72 h) = 0.502 mg/L

Reported statistics and error estimates:

72 h ErC50 and EyC50 as well as the corresponding EC20 and EC10 values and where possible their 95%-confidence limits were calculated by Probit analysis.
For the determination of 72 h LOEC and 72 h NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by Williams t-test (yield and growth rate).
ToxRat Professional, Version 3.2.1, was used to perform the statistical analysis.

Any other information on results incl. tables

Table 1: Biological Results (mean measured concentrations).

Parameter	Yield	Growth rate
	[mg test item/L]	[mg test item/L]
72-hour EC50	5.76	> 25.4
	95 % CI: 4.65 - 7.13	-
72-hour EC20	2.29	9.06
	95 % CI: 1.64 - 3.18	95 % CI: 8.25 - 9.96
72-hour EC10	1.41	4.81
	95 % CI: 0.908 - 2.19	95 % CI: 4.14 - 5.59
72-hour NOEC	0.9	0.9
72-hour LOEC	2.65	2.65

Table 2: Algal Cell Densities, Yield and Growth rate as well as the inhibition percentage after 72 h of exposure.

Mean measured concentration [mg/L]	Density of algal cells [1000/mL] after 72 h	Yields y [10000 cells/mL]	% inhibition of y	Growth rates μ [1/day]	% inhibition of μ
Control	121.935	121.435	-	1.831	-
0.286	130.714	130.214	-7.2	1.855	-1.3
0.9	118.481	117.981	2.8	1.818	0.7
2.65	96.097	95.597	21.3	1.752	4.3
8.44	44.004	43.504	64.2	1.492	18.5
25.4	10.219	9.719	92	1.006	45.1

At the start of the test 26% of the nominal test concentrations were recovered (average of all test concentrations).

After 72 h exposure, 23% of the nominal values was determined (average of all test concentrations).

Therefore, during the test algae were exposed to a mean of 25% of nominal.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

Increase in Control Cultures: 243.9-fold increase within 72 h, Coefficient of Variation of Sectional (Daily) Growth Rates in Control Cultures: 10.9 %, Coefficient of Variation of Average Growth between Control Replicates: 2%