1,2-Bis[2-(1,1,2-trifluoro-2-heptafluoropropyloxy-ethylsulfany)-ethoxycarbonyl]-ethanesulfonate sodium salt

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2018 - April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Details on sampling:

At the start of the exposure (0 hours), samples of the fresh media were taken after preparation of each concentration and analysed. At the end of the exposure (72 hours), samples of the old media were taken from the test vessels for analyses.

Vehicle:

no

Details on test solutions:

A saturated solution with a nominal loading of 116 mg/L was prepared once 24 ± 1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out and transferred with an appropriate amount of the dilution water into a glass flask. This dispersion was stirred on a magnetic stirrer at approx. 1100 rpm for 24 ± 1 hour at room temperature. After completion of stirring, the solution was centrifuged for 15 minutes with 14000 rpm at 20 °C, to remove undissolved particles, and used for testing. After centrifugation, the supernatant was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Dunstaffnage Marine Laboratory, Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK
- Age of inoculum: A four days old preculture, prepared in dilution water and incubated under test conditions, was used as inoculum.
- Method of cultivation: Nutrient medium Z according to LUTTGE et al. (1994)

ACCLIMATION
- Acclimation period: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22 - 24 °C

pH:

7.85 - 8.81

Nominal and measured concentrations:

Nominal concentrations: 0.316, 1.00, 3.16, 10.0, 31.6, 100% of the saturated solution
Geometric mean measured test item concentrations: 0.0181, 0.0448, 0.148, 0.588, 2.32, 8.48 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile glass Erlenmeyer flasks
- Type: closed (sealed with cotton wool plugs)
- Material, size, fill volume: glass, 25 mL, 10 mL
- Aeration: no
- Initial cells density: 6442 cells/mL
- Control end cells density: 1984933 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
- Preparation of dilution water: Dilution water (OECD TG 201 medium)

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 24 hours/day light
- Light intensity and quality: 5484 Lux

EFFECT PARAMETERS MEASURED
The cell density was measured daily via Chlorophyll a- fluorescence, excitation at 436 nm, emission at 685 nm.
Microscopic evaluation of the cells at the start and the end of the incubation period was carried out. The cells were checked for unusual cell shapes, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and agglutination of algae cells.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: √10
- Range finding study
- Test concentrations: 1, 10, 100 % saturated solution
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 8.48 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.32 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

8.48 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and biomass

Details on results:

- Exponential growth in the control: yes
- Observation of abnormalities: no
- Unusual cell shape: no
- Colour differences: no
- Flocculation: no
- Adherence to test vessels: no
- Aggregation of algal cells: no
- Any stimulation of growth found in any treatment: no
- Any observations that might cause a difference between measured and nominal values: 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 Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2018-09-25 to 2018-09-28. The reference item toxicity is in the valid range following test facility SOPs. EC50 (growth rate): 0.799 (0.707-1.3), EC50 (biomass): 0.607 (0.404-0.694)

Table 1: Cell Densities

Geometric mean measured test item concentration [mg/L]	Replicate No.	Cell density [cells/mL]
		0 hours	24 hours	48 hours	72 hours
8.48	1	6442	16332	104017	1303653
	2	6442	16970	110290	1342131
	3	6442	17765	118472	1414918
	Mean	6442	17022	110926	1353567
2.32	1	6442	25222	206514	1920263
	2	6442	27571	216161	1828312
	3	6442	23082	176277	1845143
	Mean	6442	25292	199651	1864573
0.588	1	6442	29673	237369	1875377
	2	6442	28514	228129	1922098
	3	6442	29601	234822	1671117
	Mean	6442	29263	233440	1822864
0.148	1	6442	27484	231394	1960487
	2	6442	28730	216692	1877373
	3	6442	30284	215198	1909397
	Mean	6442	28833	221095	1915752
0.0448	1	6442	26925	229800	1992000
	2	6442	16782	228514	1886295
	3	6442	29514	235326	1873535
	Mean	6442	24407	231213	1917277
0.0181	1	6442	27595	221265	1987087
	2	6442	28499	206072	1943246
	3	6442	29097	219215	1824335
	Mean	6442	28397	215517	1918223
Control	1	6442	27034	244191	1919710
	2	6442	28861	237369	1910822
	3	6442	30123	225389	2056744
	4	6442	32118	221079	2039466
	5	6442	30887	218899	2015435
	6	6442	33772	248853	1967423
	Mean	6442	30466	232630	1984933

 

Table 2: Inhibition of Growth rate and Yield: 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 [mg/L]	Replicate No.	Growth Rate [d-1]	Inhibition of Growth Rate [%]	Yield [cells/mL]	Inhibition of Yield [%]
8.48	1	1.77	7	1297211	34
	2	1.78	7	1335689	32
	3	1.80	6	1408476	29
	Mean	(+) 1.78	7	(+) 1347125	32
2.32	1	1.90	1	1913821	3
	2	1.88	1	1821870	8
	3	1.89	1	1838701	7
	Mean	(-) 1.89	1	(-) 1858131	6
0.588	1	1.89	1	1868935	6
	2	1.90	1	1915656	3
	3	1.85	3	1664675	16
	Mean	(+)^11.88	2	(+)^21816422	8
0.148	1	1.91	0	1954045	1
	2	1.89	1	1870931	5
	3	1.90	1	1902955	4
	Mean	(-) 1.90	1	1909310	4
0.0448	1	1.91	0	(-) 1985558	0
	2	1.89	1	1879853	5
	3	1.89	1	1867093	6
	Mean	(-) 1.90	1	(-) 1910835	3
0.0181	1	1.91	0	1980645	0
	2	1.90	0	1936804	2
	3	1.88	1	1817893	8
	Mean	(-) 1.90	1	(-) 1911781	3
Control	1	1.90		1913268
	2	1.92		1904380
	3	1.92		2050302
	4	1.92		2033024
	5	1.90		2008993
	6	1.91		1960981
	Mean	1.91		1978491

1: Since inihibtion values were <5% and showed no concentration dependent correlation, growth rate inhibition is considered to be not significant

2: statistically significant, but with a higher concentration level showing no harmful effect/statistical significance compared to control (as definition of NOEC/LOEC).

Table 3: Measured Concentration of the test item in the Definitive Test with Algae

Sampling date	Fresh Media, 0 hours	Old Media, 72 hours
Dilution of a saturated Solution [%]	Test item
	Meas. conc. [mg/L]	Meas. conc. [mg/L]	%	Geometric mean measured concentration* [mg/L]
100	9.19	7.83	85	8.48
31.6	2.68	2.00	75	2.32
10.0	0.897	0.386	43	0.588
3.16	0.251	0.0867	35	0.148
1.00	0.0845	0.0237	28	0.0448
0.316	0.0274	0.0119	43	0.0181
Control	< LOQ	< LOQ

Meas. conc.= measured concentration of the test item, dilution factors taken into account

% = percent of the initial measured concentration of the test item

LOQ = limit of quantification of the analytical method (0.0200 mg/L of the test item)

*= formula for the calculation of geometric mean measured concentration (GM =\jyl * y2)

GM = Geometric mean ; y1 = Meas. cone, of the fresh media ; y2 = Meas. cone, of the old media

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201. The results based on the geometric mean measured test item concentrations were: NOEC: 2.32 mg/L, EC50 > 8.48 mg/L (for growth rate and biomass).

Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3. The aim of the study was the determination of 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 6442 cells/mL. A saturated solution with a nominal loading of 116 mg/L, corresponding to 100 mg active substance/L was prepared once 24 ± 1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out and transferred with an appropriate amount of the dilution water into a glass flask. This dispersion was stirred on a magnetic stirrer at approx. 1100 rpm for 24 ± 1 hour at room temperature. After completion of stirring, the solution was centrifuged for 15 minutes with 14000 rpm at 20 °C, to remove undissolved particles, and used for testing. After centrifugation, the supernatant was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative. Six concentration levels were tested in a geometrical series with a spacing factor of: 0.316 - 1.00 - 3.16 - 10.0 - 31.6 - 100 % of the saturated solution, corresponding to the geometric mean measured test item concentrations: 0.0181 - 0.0448 - 0.148 - 0.588 - 2.32 - 8.48 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. At the end of exposure after 72 hours, the measured concentrations were in the range of 28 to 85% of the initially measured concentrations. All effect values given are based on the geometric mean measured test item concentrations of the test item. The results based on the geometric mean measured test item concentrations were: NOEC: 2.32 mg/L, LOEC: 8.48 mg/L, EC50 > 8.48 mg/L (for growth rate and biomass).

Description of key information

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201. The results based on the geometric mean measured test item concentrations were: NOEC: 2.32 mg/L, EC50 > 8.48 mg/L (for growth rate and biomass).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:

2.32 mg/L

Additional information

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 (reference 6.1.5 -1). The aim of the study was the determination of 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 6442 cells/mL. A saturated solution with a nominal loading of 116 mg/L, corresponding to 100 mg active substance/L was prepared once 24 ± 1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out and transferred with an appropriate amount of the dilution water into a glass flask. This dispersion was stirred on a magnetic stirrer at approx. 1100 rpm for 24 ± 1 hour at room temperature. After completion of stirring, the solution was centrifuged for 15 minutes with 14000 rpm at 20 °C, to remove undissolved particles, and used for testing. After centrifugation, the supernatant was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was negative. Six concentration levels were tested in a geometrical series with a spacing factor of: 0.316 - 1.00 - 3.16 - 10.0 - 31.6 - 100 % of the saturated solution, corresponding to the geometric mean measured test item concentrations: 0.0181 - 0.0448 - 0.148 - 0.588 - 2.32 - 8.48 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. At the end of exposure after 72 hours, the measured concentrations were in the range of 28 to 85 % of the initially measured concentrations. All effect values given are based on the geometric mean measured test item concentrations ofthe test item. The resultsbased on the geometric mean measured test item concentrations were: NOEC: 2.32 mg/L, LOEC: 8.48 mg/L, EC50 > 8.48 mg/L (for growth rate and biomass).