1-Octanethiol, 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluoro-

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:

09 - 13 March 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Reliability 1 is assigned because the study conducted according to EPA OCSPP 850.4500 and OECD TG 201 in compliance with GLP, without deviations that influence the quality of the results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: EPA Ecological Effects Test Guidelines, OCSPP 850.4500, Algal toxicity, EPA 712-C-006, January 2012.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

(2006; Annex 5 corrected 28 July 2011)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctane-1-thiol

EC Number:

628-448-8

Cas Number:

34451-26-8

Molecular formula:

C8H5F13S

IUPAC Name:

3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctane-1-thiol

Test material form:

other: Liquid

Details on test material:

- Name of test material (as cited in study report): NS-1000
- Appearance: Colorless clear liquid
- Storage condition of test material: Refrigerated (2ºC to 8ºC), purged with nitrogen.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Test concentrations were verified by chemical analysis. Water samples (3 mL, duplicate) were taken from the control and each exposure level at the start of the test and after 24 and 96 hours.
At the end of the exposure period, the replicates with algae were pooled at each concentration before sampling.
Samples were stored in the freezer until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
Preparation of test solutions started with the highest concentration of 1000 mg/L applying 22 hours of magnetic stirring to ensure reaching maximum dissolution in test medium. The resulting emulsion was left to settle for 2.5 hours after which the clear and colourless Water Soluble Fraction (WSF) was collected by means of siphoning. The WSF was used to prepare three lower test concentrations by subsequent dilutions in test medium.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: NIVA CHL 1
- Source: in-house laboratory culture

ACCLIMATION
- Pre-culture: 3 days before the start of the test, cells from the algal stock culture were inoculated in culture medium (M2) at a cell density of 1 x 10^4 cells/mL. The pre-culture was maintained under the same conditions as used in the test. The cell density was measured immediately before use.

After preparation, volumes of 50 mL test solution were added to each replicate vessel of the respective test concentration. Subsequently, 1 mL of an algal suspension was added to each replicate providing a cell density of 10^4 cells/mL.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test conditions

Hardness:

24 mg CaCO3/L

Test temperature:

21.3 - 22.9 °C

pH:

7.5 - 8.3

Nominal and measured concentrations:

Test concentrations: 0.1, 1.0, 10 and 100% of WSF prepared at a loading rate of 1000 mg/L.
Only samples from the the 100% WSF were analysed. The Time Weight Average (TWA) measured concentration was calculated to correspond with 122 µg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel:
100 mL all-glass capped vessels were used, each containing 50 mL of test solution for the control and each treatment group.
All test vessels were maintained under identical conditions.

- Initial cells density:
Pre-culture conditions gave an algal suspension in log phase growth which was diluted to a cell density of 1 x 10^4 cells per mL prior to use.

- No. of vessels per concentration (replicates): 3, except for 100% of WSF prepared at a loading rate of 1000 mg/L for which 6 replicate vessels were tested
- No. of vessels per control (replicates): 6
- No. of vessels without algae per concentration (replicates): 1-2
- 1 extra replicate of each test concentration and the control for sampling purposes

GROWTH MEDIUM
- Stock culture medium: M1 prepared according to the NPR 6505 (“Nederlandse Praktijk Richtlijn no. 6505”) formulated using Milli-RO water (tap-water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA)

- Pre-culture and test medium: M2 according to OECD 201. The medium was prepared using reverse osmosis purified deionised water (Milli-RO, Millipore)

- Illumination: continuously using TLD-lamps with a light intensity within the range of 83-84 µE.m-2.s-1 while constantly shaking.

- Determination of cell concentrations: Samples were taken at at t = 0, 24, 48, 72 and 96 hours. At the beginning of the test, cells were counted using a microscope and a counting chamber. Thereafter, cell densities were determined by spectrophotometric measurement of samples at 720 nm using a spectrophotometer with immersion probe (pathlength =20 mm). Algal medium was used as blank.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (March 2015)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Basis for effect:

growth rate

Remarks on result:

other: Determined by reviewer. The 72h value is key for REACH and CLP. The EC50 was > maximum soluble concentration of test substance in medium (TWA measured concentration 122 µg/L)

Effect concentrations 2

Duration:

72 h

Dose descriptor:

NOEC

Basis for effect:

growth rate

Remarks on result:

other: Determined by reviewer. The 72h value is key for REACH and CLP. The NOEC was > maximum soluble concentration of test substance in medium (TWA measured concentration 122 µg/L)

Effect concentrations 3

Duration:

96 h

Dose descriptor:

EC50

Basis for effect:

growth rate

Remarks on result:

other: The EC50 was > maximum soluble concentration of test substance in medium (TWA measured concentration 122 µg/L)

Effect concentrations 4

Duration:

96 h

Dose descriptor:

NOEC

Basis for effect:

growth rate

Remarks on result:

other: the NOEC was > maximum soluble concentration of test substance in medium (TWA measured concentration 122 µg/L)

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no

Results with reference substance (positive control):

- Results with reference substance valid? yes
- EC50: growth rate: 1.3 mg/L (95% CI: 1.1-1.5 mg/L)
The 72h-ErC50 for the algal culture tested falls within the historical range at the test facility (0.82-2.3 mg/L).

Reported statistics and error estimates:

For determination of the NOEC and the EC50 the approaches recommended in the EPA OCSPP 850.4000 guideline (referred to in EPA OCSPP 850.4500 guideline) and the OECD guideline 201 were used. An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the negative control revealed significant inhibition of growth rate, inhibition of yield or biomass (Student-t test for homogenous variances, α=0.05, one-sided, smaller).

No EC50-values could be calculated because the test substance proved to be non-toxic (EC50 > maximum soluble concentration).

The calculations were performed with ToxRat Professional v. 2.10.05 (ToxRat Solutions® GmbH, Germany).

Any other information on results incl. tables

Measured test substance concentrations

The actual measured concentration at the start of the test was 2034 µg/l in the WSF prepared at a loading rate of 1000 mg/L. The concentration decreased during the test period to 67 and 23 µg/L after respectively 24 and 96 hours of exposure (see Table1). The Time Weight Average (TWA) exposure concentration was calculated to correspond with 122 µg/L. The same concentration incubated without algae showed a similar decrease, thus indicative that adsorption to or by algae was not an issue.

Table 1: Concentrations of the test item in test medium

Time of sampling [hours]	Percentage of WSF^1 [%]	Analysed concentration^4 [µg/L]	Relative to initial [%]
0	0	^3
	100	2034
	100^2	1967
24	0	^3	n.a
	100	67.2	3.3
	100^2	87.7	4.5
96	0	^3	n.a
	100	23.0	1.1
	100^2	29.4	1.5

¹          Percentage of a water soluble fraction (WSF) prepared at a loading rate of 1000 mg/L.

²          Without algae.

³          Response found in blank samples. The maximum contribution to the test samples was 1.5%.

⁴          Correction was made for the difference in purity of the test substance and the analytical standard used for calibration.

n.d.    Not detected.

n.a.     Not applicable.

Table 2: TWA concentrations

Test group NS-1000 (% WSF)	Measured concentration (µg/L)			TWA (µg/L)
	t=0h	t=24h	t=96 h
100	2034	67.2	23.0	122

Inhibition results

Table 3: Percentage inhibition of growth rate (total test period) during the final test

 

Treatment (% WSF of 1000 mg/L)		Mean	Std. Dev.	n	%Inhibition
Control	1.512		0.0063	6	0.0
0.10	1.510		0.0087	3	0.1
1.0	1.513		0.0191	3	0.0
10	1.505		0.0075	3	0.5
100	1.504		0.0136	6	0.6

Microscopic observations

Microscopic observations at the end of the test in the highest concentration revealed a normal and healthy appearance of the exposed cells when compared to the control.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

See 'Remarks on results including tables and figures'

Conclusions:

No significant effects were observed at all test concentrations. The ErC50 and NOErC were > maximum soluble concentration of test substance in medium (TWA measured concentration 122 µg/L)

Executive summary:

A study was performed to assess the effect of the test material on the growth of the green alga Pseudokirchneriella subcapitata, strain: N1. The method followed that described in EPA OCSPP 850.4500 and OECD TG No 201. Pseudokirchneriella subcapitata was exposed to a Water Soluble Fraction (WSF) prepared at a loading rate of 1000 mg/L (six replicate flasks), 3 dilutions of this WSF (three replicate flasks per concentration) and a control (six replicate flasks) for 96 hours, under constant illumination and shaking at a temperature between 21.3 and 22.9°C.

Preparation of test solutions started with the highest concentration of 1000 mg/L applying 22 hours of magnetic stirring to ensure reaching maximum dissolution in test medium. The resulting emulsion was left to settle for 2.5 hours after which the clear and colourless Water Soluble Fraction (WSF) was collected by means of siphoning. The WSF was used to prepare three lower test concentrations by subsequent dilutions in test medium.

The actual measured concentration at the start of the test was 2034 µg/L in the WSF prepared at a loading rate of 1000 mg/L. The concentration decreased during the test period to 67 and 23 µg/L after respectively 24 and 96 hours of exposure. The Time Weight Average (TWA) exposure concentration was calculated to correspond with 122 µg/L. The same concentration incubated without algae showed a similar decrease, thus indicative that adsorption to or by algae was not an issue.

No significant effects were observed at all test concentrations. The ErC50 and NOErC were > maximum soluble concentration of test substance in medium (TWA measured concentration 122 µg/L)