4,6-bis(octylthiomethyl)-o-cresol

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:

08.Nov. - 01.Dec.2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Principles of method if other than guideline:

The temperature in the reference study was higher than stated in the guideline. Because the controls showed a normal exponential growth, this can be stated as uncritical.
The light intensity in the main study was slightly higher than stated in the guideline. As the controls showed normal exponential growth, this can be also stated as uncritical.
In OECD Guideline 201 and EU Method C.3, different compositions of stock solution II are stated. The study was conducted in accordance with OECD Guideline 201, therefore, the composition of stock solution II must be stated as deviation from EU Method C.3.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4,6-bis(octylthiomethyl)-o-cresol

EC Number:

402-860-6

EC Name:

4,6-bis(octylthiomethyl)-o-cresol

Cas Number:

110553-27-0

Molecular formula:

C25H44OS2

IUPAC Name:

4,6-bis(octylthiomethyl)-o-cresol

Constituent 2

Reference substance name:

402-360-6

IUPAC Name:

402-360-6

Constituent 3

Reference substance name:

4,6-Bis(octylthtomethyl)-o-cresol

IUPAC Name:

4,6-Bis(octylthtomethyl)-o-cresol

Details on test material:

Name : Songnox 1520 LQ
Batch no. : 9AL5203802
Appearance : Slightly yellowish Liquid
Composition : 4,6-Bis(octylthtomethyl)-o-cresol (110553-27-0): Min.96.0
Molecular weight : 425 g/mol
Purity : 99.3% (LC method)
Vapour pressure : 2x10e-7 hPa at 25 °C
Stability : H2O; EtOH; acetone; CH3CN; DMSO: unknown
Solubility : H20; <0,1g/L; EtOH; acetone; Ch3CN; DMSO: >1g/L
Storage : Room Temperature: (20 ± 5 °C); Keep away from light; Keep away from humidity; Keep under nitrogene

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable, since neither a surrogate nor an analogue material was used in the test.

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

- Concentrations: As solubility lies below 100 mg/L, a saturated solution was prepared for the test. This was done by weighing of each nominal load, adding the corresponding amount of deionised water and shaking vigorously for 24 hours. The resulting solution was tested unfiltered.
- Sampling method: One day before the start of the test, a saturated solution was prepared. This was done by weighing the nominal load, adding the appropriate amount of deionised water and stirring slowly on a magnetic stirrer for 24 hours. The resulting solution was tested unfiltered.
- Sample storage conditions before analysis: The test item was stored in a closed vessel dark and dry at room temperature.
- Performance of the Study: For the treatment, 800 mL test solution was prepared and mixed with 200 mL of the test culture. In this mixture, the pH-value was measured. A sample for the analytical determination was taken.
The test vessels were filled with 250 mL of the mixture and were incubated open for 72 hours shaken on an orbital shaker in the light incubator. Before the start of incubation and every 24 hours, the cell number was determined and the values of the absorption were recorded. After the test, the pH value in treatment and control was measured once more.
At the end of the test, the treatments were observed microscopically in order to assess the appearance of the alga and detect abnormalities (e.g. caused by the exposure to the test item).
The content of the test item in the test vessels was measured at the start and every 24 hours during the test. The content of the test item in the control was measured at the start and at the end of the test

Test solutions

Vehicle:

no

Details on test solutions:

All solutions were sterilised before use. Composition of the Solutions:

1. Stock Solution I
NH4CI: 1500 mg
MgCI2*6H2O: 1200 mg
CaCI2*2H2O: 1800 mg
MgSO4*7H2O: 1500 mg
KH2PO4: 160 mg
H2O deionised: ad 1000 mL

2. Stock Solution II
FeCI3*6H2O: 64 mg
Na2EDTA*2H2O: 100 mg
H2O deionised: ad 1000 mL
The composition of stock solution II is taken from OECD Guideline 201; As in EU-Method C.3, a different composition {80 mg FeCI3*6H2O/L) is stated, this is stated as a deviation from the EU Method.

3. Stock Solution III
H3BO3: 135 mg
MnCI2*4H2O: 415 mg
ZnCI2: 3 mg
CoCI2*6H2O: 1.5 mg
CuCI2*2H2O: 0.01 mg
Na2MoO4*2H2O: 7 mg
H2O deionised: ad 1000mL

4. Stock Solution IV
NaHCO3: 50 g
H2O deionised: ad 1000 mL

5. Pre-culture-medium
Stock solution I: 10.0 mL
Stock solution II: 1.0 mL
Stock solution III: 1.0 mL
Stock solution IV: 1.0 mL
H2O deionised: ad 1000 mL

6. Nutrient-medium (10-fold concentrated)
Stock solution I: 100 mL
Stock solution II: 10 mL
Stock solution III: 10 mL
Stock solution IV: 10 mL
H2O deionised: ad 1000 mL

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

Specification
Unicellular freshwater green alga.
Genus, Species: Desmodesmus subspicatus
Strain: CHODAT
Family: Chlorophyceae
Order: Chloracoccalos

Origin and Culture
The culture of Desmodesmus subspicatus was obtained in Sep. 2010 by MBM Science-bridge GmbH (Institut fur Pfianzenphysiologie of Universitat Gtittingen). The algae are kept as stock culture on solid agar at 8 °C. From an aliquot of this stock culture the pre-culture was prepared.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

none

Test conditions

Hardness:

Data not reported

Test temperature:

Main Study
22°C

Reference Study (positive control)
24-27°C

pH:

Main Study
7.5-8

Reference Study (positive control)
7.6-9.2

Dissolved oxygen:

Data not reported

Salinity:

Data not reported

Nominal and measured concentrations:

Main Study
nominal: 0 and 100 mg/L
measured:
Reference study (positive control)
nominal: 0, 0.046, 0.063, 0.1, 0.16, 0.25, 0.4, 0.63, 1 and 1.6 mg/L

Details on test conditions:

Replicates:
six vessels, each containing 250 mL test solution for photometric measurement;
twelve vessels, each containing 250 mL test solution for analytical determination

Control:
six vessels, each containing 250 mL control soFution (deionised water with test culture)

Vessels:
glass flasks, nominal volume 250 ml

Lightning: 9000 Lux (main study), 6000 Lux (reference study)

Reference substance (positive control):

yes

Remarks:

Potassium dichromate K2Cr2O7 (CAS No. 7778-50-9)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: also on the basis of AUC and yield

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: also on the basis of AUC and yield

Effect concentrations 3

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: also on the basis of AUC and yield

Details on results:

No algal cell growth inhibition was recorded throughout the test.

Results with reference substance (positive control):

The EC50s of potassium dichromate were determined in a current reference test. For the estimation of the EC50s of the positive control, the fits showed sufficient statistical correspondence of die data with Ihe dose-response-equation The values were within the normal range of the laboratory.

NOEC 72h: 0.4 mg/L (95%-confidence-intervall not determinable)
LOEC 72h: 0.63 mg/L (95%-confidence-intervall not determinable)
72h ErC50: 2.0 mg/L (95%-confidence-intervall: 1.0 - 8.1 mg/L)
72h EbC50: 0.49 mg/L (95%-confidence-intervall: 0.28 - 0.86 mg/L)
72h EyC50: 0.35 mg/L (95%-confidence-intervall: 0.22 - 0.58 mg/L)

Reported statistics and error estimates:

Determination of the NOEC
For the endpoints µ, AUC and Yield, the differences between the treatment 100 mg/L nominal concentration (0.0075 mg/L measured concentration) and the control can be considered as not significant (level of significance: 97.5 %) as the calculated t-values were smaller than the limit of significance. Therefore, the concentration 0.0075 mg/L is stated as NOEC for these endpoints.

Determination of the LOEC
As for the endpoints µ, AUC and Yield, the differences between the treatment 100 mg/L nominal concentration (0.0075 mg/L measured concentration) and the control must be considered as not significant (level of significance: 97.5 %), no LOEC can be stated for these endpoints.

Any other information on results incl. tables

Findings

Main Study

The cell numbers were determined by photometric measurement of optical density. The means and standard deviations of the cell numbers of the control and the treatments are presented in the following table:

Table 1 - Cell number/mL

Nominal Concentration in mg/L	Parameter	Cell Number/mL
		0 h	24 h	48 h	72 h
0	Mean	7354	17159	91925	421629
0	SD	0	3798	4028	30735
100	Mean	7354	18385	77217	420404
100	SD	0	6153	9007	40658

SD = Standard Deviation

In the following table the appearance of the alga at the end of the test were stated:

Table 2 - Microscopical Observation

Nom. Conc. in mg/L	Observation
0	normal growth
100	normal growth

From the cell numbers, the Growth Rate µ, the Area under the Curve AUC, and the Yield were calculated. The means and standard deviations at the end of the test are given in the following table:

Table 3 - Growth Rate µ, Area under the Curve AUC, Yield

Nom. Conc. in mg/L	Meas. Conc. in mg/L		Growth Rate [day^-1]	AUC [Cell Concentration/mL*day]	Yield [Cell Concentration/mL]
0	<LOQ	Mean	1.35	301514	414275
		SD	0.02	12738	30735
100	0.0075	Mean	1.35	287419	413050
		SD	0.03	27947	40658

SD = Standard Deviation

The following mean inhibition values were calculated for the Growth Rate µ, the Area under the Curve AUC, and the Yield:

Table 4 - Inhibition Values

Nom. Conc. in mg/L	Meas. Conc. in mg/L	% Inhibition
		Growth Rate µ	Area under the Curve AUC	Yield
0	< LOQ	0!	0!	0!
100	0.0075	0.11	4.67	0.30

Table 5 - Biological Results of the Test Item

Parameter	Value (measured)	95%-Confidence-Interval
NOEC (Growth Rate) 72 h	0.0075 mg/L	not determinable
NOEC (AUC) 72 h	0.0075 mg/L	not determinable
NOEC (Yield) 72 h	0.0075 mg/L	not determinable
LOEC (Growth Rate) 72 h	> 0.0075 mg/L	not determinable
LOEC (AUC) 72 h	> 0.0075 mg/L	not determinable
LOEC (Yield) 72 h	> 0.0075 mg/L	not determinable
72h ErC50	> 0.0075 mg/L	not determinable
72h EbC50	> 0.0075 mg/L	not determinable
72h EyC50	> 0.0075 mg/L	not determinable
72h ErC100	> 0.0075 mg/L	not determinable
72h EbC100	> 0.0075 mg/L	not determinable
72h EyC100	> 0.0075 mg/L	not determinable

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The test substance '4,6-Bis(octylthtomethyl)-o-cresol' is non-toxic to Desmodesmus subspicatus [NOEC = 100 mg/L, nominal; EC50 > 100 mg/L, nominal].

Executive summary:

The acute toxicity of '4,6-Bis(octylthtomethyl)-o-cresol' to Desmodesmus subspicatus was determined according to OECD guideline 201 and EU Method C3 (duration 72 hours). The study demonstrated that the substance was non-toxic (NOEC = 100 mg/L, nominal) to Desmodesmus subspicatus.