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 February 2011 to 11 February 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to EU, OECD & EPA test guidance in compliance with GLP and reported with a valid GLP certificate.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Deviations:

no

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

Details on test material:

Name: THANOX 1520
Batch No.: 031006013
Purity: 97.0 %
Description: pale yellow to colourless liquid
Expiry date: 25 June 2011
Storage: room temperature, protect from light and humidity, under N2
Safety Precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials will be applied to assure personnel health and safety.

The test item of a suitable chemical purity was supplied by the Sponsor. All precautions required in the handling and disposal of the test item were outlined by the Sponsor. These documents are part of the raw data. Identification of the test item was performed in the Central Dispensary of LAB Research Ltd. on the basis of the information provided by the Sponsor.

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

All of the days of the test three parallel samples were taken from the test solution. One sample was taken from the control solution at the start of the study. The samples were analysed by an HPLC-UV method.

Test solutions

Vehicle:

no

Details on test solutions:

Because the test item is poorly soluble in water, in order to achieve the maximum solubility level of the test item (limit test concentration), a test solution was prepared using a saturated solution method. A supersaturated test item stock solution (nominally 100 mg/L) was prepared by dispersing/dissolving the amount of test item into the test medium (OECD Medium) two days before the start of the study. This solution was stirred for about 48 hours and then the non-dissolved test material was removed by appropriate method (decantation) to give the saturated solution.

Untreated Control

Algal growth medium was inoculated with algal cells (without test item) and was examined in parallel to the test item concentrations.

Reference Control

For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate (Batch Number: 0769128) is tested at least twice a year to demonstrate satisfactory test conditions.

The date of the last study (Study Code: 10/332-022AL) with the reference item Potassium dichromate is: 07 - 10 December 2010.
The 72h ErC 50 : 1.40 mg/L, (95 % confidence limits: 1.29 – 1.53 mg/L)
The 72h EbC 50 : 0.92 mg/L, (95 % confidence limits: 0.85 – 0.99 mg/L)
The 72h EyC 50 : 0.92 mg/L, (95 % confidence limits: 0.85 – 1.00 mg/L)

Dilution Water

Reconstituted algal growth medium (OECD medium, according to OECD 201) was used as dilution water for both the range finding and definitive tests.

Test organisms

Test organisms (species):

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

Details on test organisms:

Species: Pseudokirchneriella subcapitata (formerly known as Selenastrum capricornutum)
Strain number: 61.81 SAG (identical strains: CCAP 278/4; UTEX 1648; ATCC 22662)
Source: The algae were supplied by the SAG: Collection of Algal Cultures, Inst. Plant Physiology, University of Göttingen, Nikolausberger Weg 18, D-37073 Göttingen, GERMANY. Cultured under standardised conditions (see OECD 201) in the Ecotoxicological Laboratory of LAB Research Ltd.
Justification of species: The species of Pseudokirchneriella subcapitata used, being a fast-growing species, is convenient for culturing and testing and is a recommended species by relevant guidelines.
Initial cell number: The initial cell number in the test cultures was 104 cells/mL.
Pre-culturing: The pre-culture was intended to give an amount of alga suspension suitable for the inoculation of test cultures. The pre-culture was incubated under the conditions of the study in an aerated Algal Growth Medium and used when still exponentially growing (after an incubation period of 3 days). The cell count of above culture was determined by microscopic method and this cell suspension was diluted with Algal Growth Medium to 107 cells/mL.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No post exposure observation period

Test conditions

Hardness:

No data

Test temperature:

The temperature was in the range of 22.0 – 22.7 °C measured in the flask and between 21.5 and 23.0 °C measured within the climate chamber.

pH:

The range of the pH was 7.77 – 7.98 at the start and 8.68 – 8.82 at the end of the study.

Dissolved oxygen:

No data

Salinity:

Not applicable.

Nominal and measured concentrations:

The measured concentration of saturated test solution was 0.28 mg/L at the start of the experiment.

Details on test conditions:

Test conditions

Temperature
Culture temperature was checked at the beginning of the study and each day thereafter in a flask filled with water, in the climatic chamber. In addition, water temperature was continuously measured (with a min/max thermometer) within the climate chamber. The temperature was in the range of 22.0 – 22.7 °C measured in the flask and between 21.5 and 23.0 °C measured within the climate chamber.

pH
The pH was checked at the beginning and at the end of the study, in the control and each concentration. The pH of the control medium was not increased by more than 1.5 units during the test. The range of the pH was 7.77 – 7.98 at the start and 8.68 – 8.82 at the end of the study.

Light Intensity
The algal culture flasks were continuously illuminated. The light intensity at the position occupied by algal culture flasks during the test was about 110 uE/m2/s, which was ensured with fluorescent lamps (with a spectral range of 400-700 nm) and it is checked periodically.

Description of the Test Procedure
The exposure time was 72 hours. The test was started (0 hours) by inoculation of a biomass of approximately 104 algal cells per mL test medium.
The test was performed with six replicates per test concentration and six replicates in the control group. Volumes of 100 mL algal suspension per replicate in 250 mL Erlenmeyer flasks were continuously shaken by a laboratory orbital shaker to keep algae in suspension. The flasks were covered with air-permeable stoppers.

Preliminary Range Finding Test
A concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations can be selected for use in the definitive test. Algal cells were exposed to each concentration of the test item plus a control, for 72 hours. The test was performed with two replicates per each test concentration and three in the control group.
During the formulation procedure the test solutions were prepared by the method described above. Test solutions were prepared by appropriate diluting of the stock solution.

Concentration Levels Investigated in the Main Test
Because no toxic effect was observed in the preliminary range-finding test, only one test concentration at the solubility level of the test item in the test medium (100 % v/v saturated solution) and one control group were tested in a limit test.

Observations
The cell numbers were determined at 24, 48 and 72 hours after starting the test by manual cell counting using a microscopic method with a counting chamber.
Microscopic observation of the algal cells in each concentration and in the control was performed (at 24h, 48h and 72h) to detect any abnormal appearance of the algae.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

> 0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Concentrations of the test item
The measured concentration of saturated test solution was 0.28 mg/L at the start of the experiment. Concentration was below the Limit of Quantification (LOQ) at day 1 and could not be detected at day 2 and 3.
In order to calculate the mean exposure concentration, the concentrations below the limit of quantification (LOQ) at day 1, 2 and 3 were taken as the half of LOQ.

The corresponding calculated geometric mean concentration was 0.1 mg/L

Average Specific Growth Rates
The results of the statistical evaluation (based on 2 Sample t-Test; a=0.05) show that the 0-72 h average specific growth rate was not statistically significantly different from the untreated control value, accordingly the No Observed Effect Concentration (NOEC) was determined as 0.1 mg/L.

Areas under the Growth Curves
The results of the statistical evaluation (based on 2 Sample t-Test; a=0.05) show that the 0-72 h areas were not statistically significantly different from the untreated control value, accordingly the No Observed Effect Concentration (NOEC) was determined as 0.1 mg/L.

Yield
The results of the statistical evaluation (based on 2 Sample t-Test; a=0.05) show that the 0-72 h yield was not statistically significantly different from the untreated control value, accordingly the No Observed Effect Concentration (NOEC) was determined as 0.1 mg/L.

Based on the results of this study, the test item THANOX 1520 had no toxic effect at saturation; the EC50 results and the LOEC are higher than the solubility level of the test item in the test medium.

Results with reference substance (positive control):

For the evaluation of the quality of the algae and validation of the experimental conditions, Potassium dichromate (Batch Number: 0769128) is tested at least twice a year to demonstrate satisfactory test conditions.

The date of the last study (Study Code: 10/332-022AL) with the reference item Potassium dichromate is: 07 - 10 December 2010.
The 72h ErC 50 : 1.40 mg/L, (95 % confidence limits: 1.29 – 1.53 mg/L)
The 72h EbC 50 : 0.92 mg/L, (95 % confidence limits: 0.85 – 0.99 mg/L)
The 72h EyC 50 : 0.92 mg/L, (95 % confidence limits: 0.85 – 1.00 mg/L)

Reported statistics and error estimates:

The section-by-section specific growth rates in the control cultures were assessed (calculated as the specific growth rates for each day during the course of the test (days 0-1, 1-2 and 2-3) and to demonstrate exponential growth for the entire study period.

The inhibition of alga growth was determined from the biomass (area under the growth curves, A), the average specific growth rate (r) and from the yield (y). Mean values and standard deviations were calculated for each concentration at the start, and at the end of the test using Excel for Windows software (Microsoft Co./One Microsoft Way/Redmond, WA 98052-6399).

The ErC50, EbC50 and EyC50 values of the test item and their confidence limits were determined from the raw data.

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (a = 0.05) by TOXSTAT software.
For the determination of the LOEC and NOEC, the calculated mean biomass, growth rates and yield at the test concentrations were tested on significant differences to the control values by 2 Sample t-Test (a = 0.05) by TOXSTAT software.

Any other information on results incl. tables

Growth Rates (μ) and Percentage Inhibition of μ during the Test Period

Test group	Growth rate μ and % inhibition of μ
	0 – 24 h		0 – 48 h		0 – 72 h
	μ	%	μ	%	μ	%
Control	0.0573	0.0	0.0611	0.0	0.0629	0.0
100% v/v saturated solution (0.1 mg/L calculated)	0.0553	3.5	0.0609	0.3	0.0624+	0.8

⁺            at this value the rounding of the EXCEL and TOXSTAT software was different. The table contains the value calculated with EXCEL. The TOXSTAT rounding: 0.0625

 

Area under the Growth Curves (A) and Percentage Inhibition of A during the Test Period

Test group	Areas under the Growth Curves (A) and % inhibition of A
	0 – 24 h		0 – 48 h		0 – 72 h
	A	%	A	%	A	%
Control	36.0	0.0	286.0	0.0	1602.0	0.0
100% v/v saturated solution  (0.1 mg/L calculated)	34.0	5.6	280.0	2.1	1556.0	2.9

 

Yield (Y) and Percentage Inhibition of Y during the Test Period

Test group	Yield (Y) and % inhibition of Y (0-72 h)
	Y	%
Control	91.8	0.0
100% v/v saturated solution (0.1 mg/L calculated)	88.7	3.4

 

Cell Number (x 10⁴cell/mL) determined in the Main Experiment

Test group	Number of cells
		0 h	24 h	48 h	72 h
Control		1	4	19	88
		1	5	21	96
		1	4	20	94
		1	4	18	92
		1	3	17	97
		1	4	18	90
	Mean	1.00	4.00	18.83	92.83
	SD	0.0	0.6	1.5	3.5
100% v/v saturated solution  (0.1 mg/L calculated)		1	3	17	87
		1	3	18	92
		1	4	19	93
		1	5	21	86
		1	4	1	90
		1	4	18	90
	Mean	1.00	3.83	18.67	89.67
	SD	0.0	0.8	1.4	2.7

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of THANOX 1520 test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours.

Under the conditions of this algal growth inhibition test the observed endpoints for the effect of THANOX 1520 were the following:

The 72h EbC50 value (biomass): > 0.1 mg/L
The 72h ErC50 value (growth rate): > 0.1 mg/L
The 72h EyC50 value (yield): > 0.1 mg/L

The No-Observed Effect Concentration (NOEC): 0.1 mg/L
The Lowest Observed Effect Concentration (LOEC): > 0.1 mg/L

Based on the results of this study, the test item THANOX 1520 had no toxic effect at saturation; the EC50 results and the LOEC are higher than the solubility level of the test item in the test medium.

Executive summary:

This study has been performed in accordance with the study plan the OECD Guidelines for Testing of Chemicals (No. 201, 23 March, 2006) and Directive 92/69/EEC, Annex Part C, C.3 and the EPA Health Effects Test Guidelines (OPPTS 850.5400) and the Principles of Good Laboratory Practice (GLP) and reported with a valid GLP certificate.

 

The effect ofTHANOX 1520test item was assessed on algal growth using the unicellular green alga Pseudokirchneriella subcapitata (Selenastrum capricornutum), over an exposure period of 72 hours.

Because no significant inhibition of algal growth was observed during the preliminary range-finding test, a limit test was carried out using only one concentration at the solubility level of the test substance in the test medium (100 % v/v saturated solution) and one control group.

 

The measured concentration of saturated test solution was 0.28 mg/L at the start of the experiment. Concentration was below the Limit of Quantification (LOQ) at day 1 and could not be detected at day 2 and 3. The corresponding calculated geometric mean concentration was 0.1 mg/L

 

The test design included six replicates at test concentration and six replicates for the untreated control.

 

Statistical comparisons of biomass, average specific growth rates and yield in control and in treated groups were carried out using 2 Sample t-Test (a= 0.05) by TOXSTAT software.

TheE_rC_50,E_bC₅₀and E_yC₅₀values of the test item were determined directly from the raw data.

  

Under the conditions of this algal growth inhibition testthe observed endpoints for the effect o f THANOX 1520 were the following:

 

The 72h E_bC₅₀value (biomass): >0.1 mg/L

The 72h E_rC₅₀value (growth rate): >0.1 mg/L

The 72h E_yC₅₀value(yield): >0.1 mg/L

 

The No-Observed Effect Concentration (NOEC): 0.1 mg/L

The Lowest Observed Effect Concentration (LOEC): >0.1 mg/L

 

Based on the results of this study, the test itemTHANOX 1520 had no toxic effect at saturation; the EC₅₀results and the LOEC are higher than the solubility level of the test item in the test medium.