2,4,6-tri-sec-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:

From 2017-06-26 to 2017-07-07, with the definitive exposure phase from 2017-06-27 to 2017-06-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

2,4,6-tri-sec-butylphenol

EC Number:

227-572-6

EC Name:

2,4,6-tri-sec-butylphenol

Cas Number:

5892-47-7

Molecular formula:

C18H30O

IUPAC Name:

2,4,6-tris(butan-2-yl)phenol

Test material form:

liquid

Details on test material:

Test item 2,4,6-Tri-sec-Butylphenol
Batch number DEG4355819
CAS No. 5892-47-7
Chemical name 2,4,6-tri-sec-butylphenol
Composition
94.7 [GC area-%] Tri-sec-butylphenol;
2.57 [GC area-%] Di-sec-butylphenol;
1.82 [GC area-%] Tetra-sec-butylphenol
Water solubility 0.79 mg/L
Appearance yellowish slightly viscous clear liquid
Density 0.91 g/cm³ (20 °C)
Stability under test conditions not specified
Expiry date 2017-07-07
Recommended storage tightly closed in a cool, well-ventilated place.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
All concentration levels and the control were analytically verified via LC-MS/MS after 0 hours with algae (start of exposure) and 72 hours (end of the exposure) with and without algae.

Test solutions

Vehicle:

no

Details on test solutions:

Stock solution, dispersion treatment
A stock solution with a nominal loading of 8.79 µL/L of the test item, which corresponds to a nominal concentration of 8.00 mg test item/L (density of 0.91 g/cm³ taken into account), was prepared with demineralized water one day prior to the start of the exposure in a glass flask.
The glass flask was filled with an appropriate amount of demineralized water. The test item (8 µL/L) was introduced into the dilution water and stirred on a magnetic stirrer at approximately 1100 rpm for 24 hours at room temperature. After completion of the stirring period and a separation phase of 1 hour, the stock solution was removed from the approximate center of the aqueous phase. The stock solution was used as the highest concentration level. The stock solution was checked after stirring via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion), which was negative. Then the components of the dilution water were added to the stock solution.

Test concentrations (nominal)
The stock solution and four dilution levels out of the stock solution were tested in a geometrical series with a dilution factor of √10: 1.00 - 3.16 - 10.0 - 31.6 - 100 % of the stock solution, corresponding to the geometric mean measured test item concentrations 0.00180 - 0.0242 - 0.0675 - 0.221 - 1.86 mg/L. The dilution levels are based on the results of a preliminary range finding test (non-GLP, flasks with headspace).

Control
Six replicates (without test item) were tested under the same test conditions as the test replicates.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus CHODAT
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG, Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2590 - 5180 lux corresponding to 35 - 70 µE ∙ m-2 ∙ s-1 for 24 h per day.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Remarks on exposure duration:

No

Post exposure observation period:

No

Test conditions

Hardness:

Not specified

Test temperature:

min.: 22.0 max.: 23.5 mean value: 22.8

pH:

Geometric mean measured test item concentration pH-values
[mg/L] Start; 0 hours End; 72 hours
1.86 7.73 7.90
0.221 7.75 8.03
0.0675 7.74 8.12
0.0242 7.66 8.18
0.00180 7.64 8.26
Control 7.52 8.30

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

nominal: 1.00 - 3.16 - 10.0 - 31.6 - 100% of the stock solution
geometric mean measured test item concentrations 0.00180 - 0.0242 - 0.0675 - 0.221 - 1.86 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x 10^3 cells/mL, Actual: mean 6312 cells/mL
- Control end cells density: Mean 833970 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6


GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064
Na2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 x 10^-3
Na2MoO4 x 2 H2O 7 x 10^-3
CoCl2 x 6 H2O 1.5 x 10^-3
CuCl2 x 2 H2O 1 x 10^-5
NaHCO3 50
pH 8.1 ± 0.2



OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120
µE ∙ m-2 ∙ s-1, mean value: 5800 lux,



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-The cell density was measured daily via Chlorophyll-a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.No self-fluorescence was found in the preliminary range finding test at the concentration of 8 mg/L.

Reference substance (positive control):

yes

Remarks:

(Potassium dichromate)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.068 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.221 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.258 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: 0.0748 - 1.41

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.301 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: 0.221 - 1.44

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.391 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: CI: 0.223 - 1.55

Effect concentrations 6

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.068 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 7

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.221 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.192 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI: 0.00180 - 0.874

Effect concentrations 9

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.22 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI: 0.0702 - 0.889

Effect concentrations 10

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.268 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: CI: 0.0807 - 0.954

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) to the unicellular freshwater green alga Desmodesmus subspicatus was determined over a period of 72 hours from 2017-04-10 to 2017-04-13. The reference item toxicity is in the valid range following test facility SOPS.

EC50-Values of the Reference Item
based on nominal concentrations mg/L, (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate Inhibition
ErC50 0.697 0.668 ± 0.375
95% confidence interval 0.647 – 0.755
Yield Inhibition
EyC50 0.325 0.318 ± 0.130
95% confidence interval 0.308 – 0.343
SD = Standard deviation

Reported statistics and error estimates:

ECx-values and statistical Analyses
ECx-values with confidence intervals of growth rate inhibition and for yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.

NOEC, LOEC and statistical analyses
NOEC/LOEC was determined by calculation of statistical significance of growth rates and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s test was used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05. For results, please refer to section 13. The Equal Variance test and Normality test failed for growth rate. Therefore, the data were transformed (Y squared).

Software
The data for the tables in this report were computer-generated and have been rounded for presentation from the full derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
• Excel, MICROSOFT CORPORATION
• SigmaPlot, SPSS INC.
• GraphPad Prism, GRAPHPAD SOFTWARE, INC.
• ToxRat Professional, Version 3.2.1, TOXRAT SOLUTIONS GMBH

Any other information on results incl. tables

Cell Densities

Geometric mean measured test item concentration	Replicate	Cell density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
1.86	1	6312	10582	7390	3719
	2	6312	11683	7530	8874
	3	6312	13091	14374	14431
	Mean	6312	11785	9765	9008
0.221	1	6312	22858	117402	539407
	2	6312	20216	113420	761169
	3	6312	23037	132392	691664
	Mean	6312	22037	121071	664080
0.0675	1	6312	23451	158569	919804
	2	6312	20759	164582	821184
	3	6312	26123	154614	917298
	Mean	6312	23444	159255	886095
0.0242	1	6312	23879	142300	712808
	2	6312	22722	135983	739722
	3	6312	22031	146733	716262
	Mean	6312	22877	141672	722931
0.00180	1	6312	23686	127290	813402
	2	6312	22623	137342	798218
	3	6312	22798	160649	809682
	Mean	6312	23036	141760	807101
Control	1	6312	24157	173199	810251
	2	6312	23044	151364	829839
	3	6312	25223	166814	861611
	4	6312	26935	140948	891486
	5	6312	22927	166161	874973
	6	6312	25853	139369	735660
	Mean	6312	24690	156309	833970

 Evaluation after 72 hours

                  Statistically significant differences of growth rates and yield compared to

                  control values are marked (+), not significant differences are marked (-).

 

Geometric mean measured test item concentration	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
1.86	1		-0.176	100		-2593	100
	2		0.114	93		2562	100
	3		0.276	83		8119	99
	Mean	(+)	0.071	92	(+)	2696	100
0.221	1		1.48	9		533095	36
	2		1.60	2		754857	9
	3		1.57	4		685352	17
	Mean	(+)	1.55	5	(+)	657768	21
0.0675	1		1.66	-2		913492	-10
	2		1.62	0		814872	2
	3		1.66	-2		910986	-10
	Mean	(-)	1.65	-1	(-)	879783	-6
0.0242	1		1.58	3		706496	15
	2		1.59	2		733410	11
	3		1.58	3		709950	14
	Mean	(-)	1.58	3	(-)	716619	13
0.00180	1		1.62	0		807090	2
	2		1.61	1		791906	4
	3		1.62	1		803370	3
	Mean	(-)	1.62	1	(-)	800789	3
Control	1		1.62			803939
	2		1.63			823527
	3		1.64			855299
	4		1.65			885174
	5		1.64			868661
	6		1.59			729348
	Mean		1.63			827658

 

 

 Section-by-Section and Average Specific Growth Ratesof the Control Group (0 – 72 hours)

 

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.34	1.97	1.54	1.62	0.321	19.8	16.0
	2	1.30	1.88	1.70	1.63	0.301	18.5
	3	1.39	1.89	1.64	1.64	0.252	15.4
	4	1.45	1.66	1.84	1.65	0.197	11.9
	5	1.29	1.98	1.66	1.64	0.346	21.0
	6	1.41	1.69	1.66	1.59	0.153	9.64
				Mean	1.63
				SD ±	0.02
				CV [%]	1.43

CV= Coefficient of variation

SD= Standard deviation

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, 2,4,6-Tri-sec-Butylphenol was found to inhibit the growth rate of the freshwater green alga Desmodesmus subspicatus after 72 hours with the following values: The EC50-values with 95 % confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 0.391 (0.223 – 1.55) and 0.268 (0.0807 – 0.954 mg/L, respectively. The NOEC-values for both inhibition of growth rate and yield after 72 hours were 0.0675 mg/L, respectively.
All effect levels are given based on the geometric mean measured test item concentration of 2,4,6-Tri-sec-Butylphenol.

Executive summary:

The toxicity of 2,4,6-Tri-sec-Butylphenol (batch number:DEG4355819) to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 (2011)and Council Regulation (EC) No. 266/2016/Method C.3 (2016) at the test facility from 2017-06-26 to 2017-07-07, with the definitive exposure phase from 2017-06-27 to
2017-06-30. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours.

 

The study was conducted under static conditions with an initial cell density of 6312 cells/mL. A stock solution with a nominal loading of 8.79 µL/L, which corresponds to a nominal concentration of 8.00 mg test item/L was prepared in demineralized water one day prior to the start of exposure in a glass flask. The glass flask was filled up with an appropriate amount of demineralized water. The test item (8 mg/L) was introduced into the dilution water and stirred on a magnetic stirrer at approximately 1100 rpm for 24 hours at room temperature. After completion of the stirring period and a separation phase of 1 hour, the stock solution wastaken from the approximate center of the aqueous phase of the flasks.

The stock solution was checked after stirring via laser beam (Tyndall effect) for undissolved test item (formation of an emulsion), which was negative. Then the components of the dilution water were added to the stock solution.The stock solution and fourdilution levels of the stock solution were tested in a geometrical series with a dilution factor of √10:1.00 - 3.16 - 10.0 - 31.6 - 100% of the stock solution, corresponding to the geometric mean measured test item concentrations 0.00180 - 0.0242 - 0.0675 - 0.221 - 1.86 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits.

 

The test media were visually clear throughout the exposure period. The concentrations of the test item 2,4,6-Tri-sec-Butylphenol were determined at the start of the exposure (0 hours) and at the end of the exposure (72 hours) in all test media and the control via LC-MS/MS. The measured initial concentrations of 2,4,6-Tri-sec-Butylphenol were 0.0108 – 0.110 – 0.236 – 0.855 – 2.18 mg/L. The measured concentrations at the end of the exposure (72 hours) were in the range of < LOQ to 73% of the initially measured concentrations.

 

All effect values of 2,4,6-Tri-sec-Butylphenol are given based on the geometric mean measured test item concentrations.

 

 NOEC, LOEC, EC - values of 2,4,6-Tri-sec-Butylphenol (0-72 hours)

                  based on geometric mean measured test item concentration [mg/L]

                               

	Growth Rate Inhibition
NOEC	0.0675
LOEC	0.221
ErC10	0.258 (0.0748 – 1.41)
ErC20	0.301 (0.221 – 1.44)
ErC50	0.391 (0.223 – 1.55)
	Inhibition of Yield
NOEC	0.0675
LOEC	0.221
EyC10	0.192 (0.00180 – 0.874)
EyC20	0.220 (0.0702 – 0.889)
EyC50	0.268 (0.0807 – 0.954)