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

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 March - 08 May 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study is conducted according to EPA OPPTS 850.1010 and OECD TG 202 in compliance with GLP, without deviations that influence the quality of the results. However, since the measured concentrations in fresh media at t =0 and old media at t = 48h were higher than the calibration range and had to be determined by extrapolation of the calibration curve and the highest concentration for which the procedural recovery has been checked was 10 mg/L whereas the concentration in the test media were much higher, the study is considered to be reliable with restrictions (KC2). It has still been demonstrated that the concentrations to which the Daphnia have been exposed has was higher than the max. solubility and therefore the conclusion that there are no effects below the max. solubility in test medium remains valid.

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:

EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)

Version / remarks:

'Public Draft', EPA 712-C-96-114, April 1996

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Version / remarks:

(2004)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Version / remarks:

(2008)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))

Version / remarks:

(Third Edition, 1996)

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000

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) in the dark, purged with nitrogen.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Test concentrations were verified by chemical analysis. Water samples (3 mL, in duplicate) were taken from the approximate centre of the test vessels of the control and each exposure level at the start and end of both 24-h (renewal) periods.
At the end of each refreshment period, the replicates 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 at the highest loading rate of 1000 mg/L by applying one day of magnetic stirring to ensure reaching maximum dissolution in test medium. The resulting emulsion was left to settle for approximately 2 hours after which the clear and colourless Water Soluble Fraction (WSF) was collected by means of siphoning. In the 2nd full study the WSF was additionally centrifuged for 30 minutes at 10,000G to remove any remaining undissolved material that might still be present in the WSF after settlement. The lower test concentrations were prepared by subsequent dilutions of the WSF in test medium. Test solutions were renewed after 24 hours in all three experiments.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (Straus 1820)
- Source: in-house laboratory culture with a known history (at least third generation, obtained by acyclical parthenogenesis under specified breeding conditions)
- Age at study initiation: young daphnids with an age of < 24 hours, from parental daphnids of more than two weeks old
- Method of breeding: start of each batch with newborn daphnids, i.e. less than 3 days old, by placing about 250 of them into 5 litres of medium in an all-glass culture vessel. Renewal of the medium: after 7 days of cultivation half of the medium twice a week.
- Feeding during test: no

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Test conditions

Hardness:

180 mg/L (CaCO3)

Test temperature:

19.9 - 20.5°C

pH:

7.7-8.0

Dissolved oxygen:

8.7 - 9.1 mg O2/L
test end: 8.8 - 8.9 mg O2/L

Nominal and measured concentrations:

Based on the results of the combined limit/range-finding test the following dilutions of the WSF prepared at a loading rate of 1000 mg/L were assigned to the final tests: 4.6, 10, 22,46 and 100%
Measured test concentrations: see tables 1 and 2 in field 'Any other information on results'

Details on test conditions:

TEST SYSTEM
The tests were performed in all-glass vessels (100 mL) containing 80 mL of test solution.

* Combined limit/ range-finding test (semi-static):
- Four test concentrations (0.1, 1.0, 10 and 100% of WSF prepared at 1000 mg/L) plus one control.
- 0.1, 1.0 and 10% of WSF prepared at 1000 mg/L in duplicate with 5 animals/test vessel (i.e. 10 animals/concentration)
- Control and 100% of WSF prepared at 1000 mg/L in 4-fold (with 5 animals/test vessel (i.e. 20 animals/concentration)

* Final tests (semi-static):
- Five test concentrations (4.6, 10, 22,46 and 100% of WSF prepared at 1000 mg/L, "settled only" in first full test and "settled and centrifuged" in second full test) plus one control each in 4-fold with 5 animals/test vessel (i.e. 20 animals/concentration).
- Loading rate: 16 mL test solution per organism
- Test vessels were maintained at 20 °C under a photoperiod of 16 hours light : 8 hours dark and without supplementary aeration or feeding during the 48 hour exposure period.
- The temperature was measured continuously and the pH and dissolved oxygen levels were recorded at the start and at the end of the study.

TEST MEDIUM / WATER PARAMETERS
- Test medium: adjusted ISO
- Culture medium different from test medium: yes (M7)

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED: Immobility: Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation of the test vessel.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (March 2015)

Results and discussion

Effect concentrations

Duration:

48 h

Dose descriptor:

EC50

Basis for effect:

mobility

Remarks on result:

other: The EC50 was > maximum soluble concentration of test substance in medium (390 µg/L)

Details on results:

* Range-finding test:
Samples taken from 10 and 100% of the WSF were analysed. Analyses of the samples taken from the undiluted WSF showed that measured concentrations in the freshly prepared solutions ranged between 1128 and 601 µg/L. The concentrations measured in the ten-fold dilution (10% WSF) were at 116 and 71 µg/L in the freshly prepared solutions. The concentrations measured in the 24-hour old solutions all decreased, except the concentration measured after 48h at 100% of the WSF which was 119% of initial.

* First final test:
Measured concentrations ranged from 62 - 140 % of inital at the end of the first renewal period (t=0-24 hours) and 59-97 % of initial at the end of the second renewal period (t=24-48 hours), See Table 1.
Considering the fact that the expected maximum solubility was much lower, and considering the observed effects (see Table 3) it was decided to repeat the experiment with extra attention for removal of undissolved material (additional centrifugation) and extra attention for homogenisation before each dilution step.

* Second final test:
Samples were only analysed from the control and the highest test concentration considering the result obtained in this final study. The measured concentration was 44 % of inital at the end of the first renewal period (t=0-24 hours) and 81 % of initial at the end of the second renewal period (t=24-48 hours). See Table 2.
The geometric mean measured concentration was calculated to correspond with 390 µg/L.

Individual pH, temperature and dissolved oxygen values remained within acceptable limits throughout the duration of the study.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- 48-h EC50: 0.32 mg/L

Any other information on results incl. tables

Measured concentrations

Table 1: Concentrations of the test substance in test medium - first final test

Time of sampling [hours]	Percentage of WSF^1 [%]	Analysed concentration^2 [µg/L]	Relative to initial [%]
0	0	n.d.
(fresh)	4.6	42.0
	10	66.7
	22	404^3
	46	629^3
	100	1013^3
24	0	n.d.	n.a.
(old)	4.6	34.6	82
	10	93.1	140
	22	280^3	69
	46	675^3	107
	100	623^3	62
24	0	n.d.
(fresh)	4.6	604^3
	10	1358^3
	22	1963^3
	46	2212^3
	100	1950^3
48	0	n.d.	n.a.
(old)	4.6	356^3	59
	10	1198^3	88
	22	1498^3	76
	46	1936^3	88
	100	1897^3	97

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

²          Correction was made for the difference in purity of the test substance and the analytical standard used for calibration.

³           Estimated value, calculated by extrapolation of the calibration curve.

n.d.     Small response found at the retention time of the test substance. Since the response was similar as the analytical blanks it was considered as not detected.

n.a.     Not applicable.

Table 2:       Concentrations of the test substance in test medium−second final test

Time of sampling [hours]	Percentage of WSF^1 [%]	Analysed concentration^2 [µg/L]	Relative to initial [%]
0	0	n.d
(fresh)	100	295
24	0	n.d.	n.a.
(old)	100	129	44
24	0	^3
(fresh)	100	649^4
48	0	^3	n.a.
(old)	100	529^4	81

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

²          Correction was made for the difference in purity of the test substance and the analytical standard used for calibration.

³          Response found in blank samples. The maximum contribution to the test samples was 0.03%.

⁴         Estimated value, calculated by extrapolation of the calibration curve.

n.d.    Not detected.

n.a.     Not applicable.

Range-finding test

No or no significant immobility was observed up to and including concentrations containing 10% of the WSF during the test period. A total of 15 of the 20 daphnids exposed to the highest test group containing the undiluted WSF were immobilised at the end of the test period. Based on this result it was expected that the EC₅₀ranged between 10 and 100% of the WSF.

 

Final tests

First final test:

Table 3 shows the responses recorded during the first full test. No significant effects were observed after 24 hours, whereas most of the daphnids were immobilised after 24 hours at 10% WSF and higher. This was unexpected based on the result of the combined limit/range-finding test and considered related to the unexpected high measured concentrations obtained in the freshly prepared solutions after 24 hours. It was expected that testing was performed above the solubility limit and that this caused immobility by physical instead of toxic processes. It was decided to repeat the study with the same range of WSF dilutions but with introduction of centrifugation to ensure better removal of undissolved material before testing. In conclusion; results of the 1^stfull study were not considered reliable.

Biological data

Table 3: Cumulative immobilisation data for Daphnia magna exposed for 48 hours to the test substance (first final test)

 

Test group NS-1000 (% WSF)	Vessel number	Number Daphnia exposed	Response at 24 h		Response at 48 h
			number	Total %	number	Total %
Control	A  B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0
4.6	A  B C D	5 5 5 5	0 0 0 0	0	0 0 1 0	5
10	A  B C D	5 5 6* 5	0 1 0 0	5	3 (3) 4 (1) 6 (2) 2 (3)	71
22	A  B C D	5 5 5 6*	0 0 0 0	0	5 (3) 5 (4) 4 (2) 6 (6)	95
46	A  B C D	5 5 4* 5	0 0 0 0	0	5 (4) 5 (5) 4 (4) 5 (5)	100
100	A  B C D	5 5 5 5	0 1 0 0	5	5 5 5 5	100

* Accidentally an incorrect number of Daphnia was introduced in these vessels (protocol deviation). The study results are not affected.

( ) between brackets: number of daphnia observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

 A, B, C, D = replicate 1, 2, 3, 4

Second final test:

Table 4 shows the responses recorded during the second final test. No immobility was observed in any of the concentrations tested.

Table 4: Cumulative immobilisation data for Daphnia magna exposed for 48 hours to the test substance (second final test)

Test group NS-1000 (% WSF)	Vessel number	Number Daphnia exposed	Response at 24 h		Response at 48 h
			number	Total %	number	Total %
Control	A  B C D	5 5 5 6*	0 0 0 0	0	0 0 0 0	0
4.6	A  B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0
10	A  B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0
22	A  B C D	5 5 5 5	0 0 0 0	0	0 0 0 0	0
46	A  B C D	5 5 5 5	0 0 (2) 0 (1) 0	0	0 0 (1) 0 (1) 0	0
100 [0.39 mg/L]	A  B C D	5 5 5 5	0 0 (2) 0 0 (1)	0	0 (3) 0 (3) 0 (4) 0 (2)	0

* Accidentally, an incorrect number of Daphnia was introduced in these vessels (protocol deviation). The study results are not affected.

( ) between brackets: number of daphnia observed trapped at the surface of the test solutions. These organisms were reimmersed into the respective solutions before recording of mobility.

[ ] Average measured concentration

A, B, C, D = replicate 1, 2, 3, 4

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

<10% immobilization in control group, O2 conc. at end is >=3 mg/L in control and test vessels

Conclusions:

The 48h-EC50 exceeded a WSF prepared at a loading rate of 1000 mg/L that corresponds with a geometric mean measured concentration of 390 µg/L. It can consequently be stated that NS-1000 is not acutely toxic up to and including the limit of solubility in test medium under the conditions of the present semi-static test.

Executive summary:

A study was performed to assess the acute toxicity of the substance to Daphnia magna under semi-static conditions (renewal period: 24 hours). The study was conducted in accordance with EPA OPPTS 850.1010, OECD Guideline for Testing of Chemicals No. 202 and GLP.

Groups of twenty daphnids (less than 24 hours old) were exposed for 48 hours to fiveconcentrations of the substance dispersed in test water (4.6 - 100 % of a WSF prepared at a loading rate of 1000 mg/L, settled and centrifuged) and a control. The incidence of immobilisation was recorded for each test and control group at 24 hours and at 48 hours. The measured concentration was 44 % of inital at the end of the first renewal period (t=0-24 hours) and 81 % of initial at the end of the second renewal period (t=24-48 hours). The geometric mean measured concentration in the highest test group was calculated to correspond with 390 µg/L. The 48h-EC50 exceeded a WSF prepared at a loading rate of 1000 mg/L. It can consequently be stated that NS-1000 is not acutely toxic up to and including the limit of solubility in test medium under the conditions of the present semi-static test. The study is conducted according to EPA OPPTS 850.1010 and OECD TG 202 in compliance with GLP, without deviations that influence the quality of the results. However, since the measured concentrations in fresh media at t =0 and old media t = 48h were higher than the calibration range and had to be determined by extrapolation of the calibration curve and the highest concentration for which the procedural recovery has been checked was 10 mg/L whereas the concentration in the test media were much higher, the study is considered to be reliable with restrictions (KC2). It has still been demonstrated that the concentrations to which the Daphnia have been exposed has was higher than the max. solubility and therefore the conclusion that there are no effects below the max. solubility in test medium remains valid.