Reaction mass of potassium trifluoroacetate and potassium trifluoromethanesulphinate

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:

From 2012-04-20 to 2012-06-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

2009-12-14

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.8, 2.5, 8, 100 and 300 mg test item/L
- Sampling method: Duplicate samples from the freshly prepared test media (containing algae) of all test concentrations and from the control were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the control (containing algae) were taken at the end of the test (after the 72 hours test period).
The concentrations of the test item were analysed for trifluoromethanesulphinate and trifluoroacetate in the duplicate test media samples from all test concentrations collected at the start and end of the test. From the control samples only one of the duplicate samples was analysed from each of these sampling times.
- Sample storage conditions before analysis: All samples were stored in a freezer (≤ 10 °C), protected from light until analysis was performed.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test medium of the highest test concentration of nominal 282 mg test item/L was prepared by mixing 141 mg test item into 500 mL test water by intense stirring for 10 minutes. Adequate volumes of this test medium were diluted with test water to prepare the test media of the other desired test concentrations. The test media were prepared just before introduction of the algae
- Controls: In the control, test water was used without addition of the test item.
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)):
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no data

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain:61.81 SAG
- Source (laboratory, culture collection): The algae were supplied by the "Sammlung von Algenkulturen, Pflanzenphysiologisches Institut der Universität Göttingen", 37073 Göttingen, Germany.
- Age of inoculum (at test initiation): These cells were taken from an exponentially growing pre-culture, which was set up 3 days prior to the test start under the same conditions as in the test.
- Method of cultivation: The algae were cultivated in the laboratories of IBACON under standardised conditions according to the test guidelines.

ACCLIMATION
- Acclimation period: No
- Culturing media and conditions (same as test or not): yes
- Any deformed or abnormal cells observed: The microscopic examination of the shape of the algal cells after 72 hours of test duration did not show any difference between the algae that had been growing at a nominal test concentration of 282 mg test item/L and the algal cells in the control. Thus, the shape of the algal cells was not obviously affected up to this test concentration, the highest concentration tested.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3.

Test temperature:

22 to 24 °C

pH:

8.1 to 8.2 at test start
8.0 to 9.6 at test end

Nominal and measured concentrations:

nominal: 282, 88, 27.5, 8.6, 2.69 and 0.84 mg test item/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Material, size, headspace, fill volume: 50 mL volume with 50 mL of test medium covered with glass dishes and incubated in a water bath.
- Aeration:
- Initial cells density: 5000 algal cells per mL test medium
- Control end cells density: after 24, 48 and 72 hours of exposure
- No. of vessels per concentration (replicates): 3
- No. of vessels per vehicle control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:Analytical grade salts were added at the following nominal concentrations in deionised water (conductivity <5 µScm-1)
Macro-nutrients:
+ NaHCO3: 50.0 mg/L
+ CaCl2 x 2 H2O: 18.0 mg/L
+ NH4Cl: 15.0 mg/L
+ MgSO4 x 7 H2O: 15.0 mg/L
+ MgCl2 x 6 H2O: 12.0 mg/L
+ KH2PO4: 1.6 mg/L
Trace Elements:
+ Na2EDTA x 2 H2O: 100.0 µg/L
+ FeCl3 x 6 H2O: 64.0 µg/L
+ MnCl2 x 4 H2O: 415.0 µg/L
+ H3BO3: 185.0 µg/L
+ Na2MoO4 x 2 H2O: 7.0 µg/L
+ ZnCl2: 3.0 µg/L
+ CoCl2 x 6 H2O: 1.5 µg/L
+ CuCl2 x 2 H2O: 0.01 µg/L

- Culture medium different from test medium:
- Intervals of water quality measurement:

OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: No
- Photoperiod: Continuous illumination
- Light intensity and quality: 6079 lux (range: 5490 to 6590 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: The cell densities in the samples were determined by spectrophotometrical measurement. Therefore defined volumes of the algal suspensions from all replicates and from the blanks were sampled and were not replaced. The algal cell densities were calculated by subtracting the absorption of the blanks, from each of the measured absorption of the test media (with algae). The cell densities in a number of samples from one control replicate were counted by microscope after 72 hours of test duration. Out of this control seven defined dilutions were prepared and measured spectrophotometrically. Based on the counted cell densities and the absorption of the control sample and the dilutions a linear regression was performed for the calculation of the cell densities in all other samples measured spectrophotometrically during the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study: Yes
- Test concentrations: 1, 10, 50 100 mg/L
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: Concentration of test item: 282 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 282 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.955 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.53 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

2.26 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% CL: 1.71 - 2.88 mg/L

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): No
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No

Results with reference substance (positive control):

- Results with reference substance valid?: YES
- EC50 growth rate: 1.073 mg/L
- Other: see the table of results in "Overall remarks.

Reported statistics and error estimates:

Based on the calculated cell densities, the 72-hour ErC50 and the 72-hour EyC50, the corresponding EC20 and EC10 values and where possible their 95 %-confidence limits were calculated by Probit analysis.
For the determination of the 72-hour LOEC and the 72-hour NOEC, the calculated growth rates and yields at each test concentration were tested for significant differences compared to the control values by the Bonferroni-Welch t-test (yield and growth rate).
The software used to perform the statistical analysis was ToxRat Professional, Version 2.10.05, ToxRat Solutions GmbH.

Summary of analytical results

Sample description	trifluoroacetate		trifluoromethanesulphinate
	% of nominal^1	RDS [%]	% of nominal	RDS [%]
mg test item /L
Control	<LOQ	n.a.	<LOD	n.a.
0.84	<LOQ	n.a.	<LOD	n.a.
2.69	87	7	<LOD	n.a.
8.60^2	48	6	<LOD	n.a.
4.30^3	96	6	<LOD	n.a.
27.5	102	2	113	4
88	105	1	108	3
282	100	0	104	3

¹mean value of all measured samples per treatment group

²Evaluation based on nominal concentration according to study plan; values not used for any calculation

³Evaluation based on corrected concentration (50 % of 8.6 mg test item/L); see discussion below

RSD: relative standard deviation per treatment group n: number of analysed samples

n.a.: not applicable

  

Algal cell densities during the test period 72 hours

Nominal concentration	Flask	Density of algal cells [10^4/mL] after
mg test item/L	N°	24 hours	48 hours	72 hours
Control	1	2.854	24.828	128.047
	2	4.063	27.046	144.376
	3	3.862	28.658	144.981
	4	2.652	25.634	137.118
	5	2.652	26.844	137.320
	6	3.458	25.433	150.424
	m	3.257	26.407	140.378
	s	0.625	1.393	7.876
0.84	1	3.660	23.820	137.118
	2	4.265	22.610	149.214
	3	4.265	24.022	145.182
	m	4.063	23.484	143.838
	s	0.349	0.763	6.159
2.69	1	2.450	19.990	92.767
	2	2.249	18.982	116.959
	3	2.854	18.982	100.629
	m	2.518	19.318	103.451
	s	0.308	0.582	12.340
4.3	1	3.660	19.385	91.960
	2	3.458	18.578	91.356
	3	3.458	18.175	84.098
	m	3.562	18.713	89.138
	s	0.116	0.616	4.375
27.5	1	3.257	9.305	38.133
	2	2.854	10.918	39.343
	3	3.862	10.918	37.529
	m	3.324	10.380	38.335
	s	0.507	0.931	0.924
88	1	1.644	7.087	24.223
	2	2.450	8.297	26.642
	3	2.047	8.498	24.223
	m	2.047	7.961	25.030
	s	0.403	0.763	1.397
282	1	0.838	5.676	11.552
	2	1.039	5.273	11.522
	3	0.838	5.676	10.514
	m	0.905	5.542	11.186
	s	0.116	0.233	0.582

m: mean value; s: standard deviation

At test start 5000 algal cells/mL were incubated

 Growth rates during the test period of 72 hours 

Nominal concentration	Flask	Growth rate µ (1/day)
mg test item/L	N°	0-24 hours	0-48 hours	0-72 hours
Control	1	1.742	1.953	1.849
	2	2.095	1.995	1.889
	3	2.044	2.024	1.890
	4	1.668	1.969	1.871
	5	1.668	1.992	1.872
	6	1.934	1.965	1.902
	m	1.859	1.983	1.879
	s	0.191	0.026	0.019
0.84	1	1.991	1.932	1.871
	2	2.144	1.906	1.900
	3	2.144	1.936	0.890
	m	2.093	1.925	1.887
	s	0.088	0.016	0.014
2.69	1	1.589	1.884	0.741
	2	1.504	1.818	1.818
	3	1.742	1.818	1.768
	m	1.612	1.827	1.776
	s	0.121	0.015	0.039
4.3	1	0.991	1.829	1.738
	2	1.934	1.808	1.736
	3	1.934	1.797	1.708
	m	1.953	1.811	1.728
	s	0.033	0.016	0.017
27.5	1	0.874	1.462	1.445
	2	1.742	1.542	1.455
	3	2.044	1.542	1.439
	m	1.887	1.515	1.446
	s	0.152	0.046	0.008
88	1	0.190	1.326	1.293
	2	0.589	1.405	1.325
	3	1.410	1.417	1.293
	m	1.396	1.382	1.304
	s	0.200	0.049	0.018
282	1	0.516	1.215	1.046
	2	0.732	1.178	1.046
	3	0.516	1.215	1.05
	m	0.588	1.202	1.036
	s	0.125	0.021	0.018

m: mean value; s: standard deviation

 Growth rates µand percentage inhibition of µ during the test period 

Nominal concentration	Growth rates µ[1/day] and % inhibition of µ
	0-24 h			0-48 h			0-72 h
mg test item/L	µ	%		µ	%		µ	%
Control	1.859	-		1.983	-		1.879	-
0.84	2.093	-12.6		1.925	2.9	*	1.887	-0.4
2.69	1.612	13.3		1.827	7.9	*	1.776	5.5
4.3	1.953	-5.1		1.811	8.7	*	1.728	8.0	*
27.5	1.887	-1.5		1.515	23.6	*	1.446	23.0	*
88	1.396	24.9	*	1.382	30.3	*	1.304	30.6	*
282	0.588	68.4	*	1.202	39.4	*	1.036	44.9	*

negative values in ‘% inhibition’ indicate an increase in growth relative to that of the control

* mean value significantly different from the control (tested with and Williams t-test (24 and 48h) and Bonferroni-Welch t-test (72h),α= 0.05, one-sided)

 

pH- value in the test media at the start and end of the test

Nominal concentration	pH value
mg test item/L	0 hours	72 hours
Control	8.2	9.5
0.84	8.1	9.6
2.69	8.2	9.4
4.3	8.1	8.8
27.5	8.1	8.4
88	8.2	8.2
282	8.1	8.0

 

Validity criteria fulfilled:

yes

Conclusions:

The influence of reaction mass of TFSK/TFAK on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static dose-response test. The 72-hour ErC50 value was calculated to be > 282 mg test item/L, and the 72-hour EyC50 was calculated to be 10.0 mg test item/L. The 72-hour NOErC was determined to be 2.69 mg test item/L and the associated 72-hour LOErC was 4.3 mg test item/L. The 72-hour NOEyC was determined to be 0.84 mg test item/L and the associated 72-hour LOEyC was 2.69 mg test item/L.

Executive summary:

In a 72 hour acute toxicity study, the cultures of Pseudorkirchneriella subcapitata were exposed to the reaction mass TFSK/TFAK at nominal concentrations of 0.84, 2.69, 8.6, 27.5, 88 and 282 mg/L under static conditions in accordance with the OECD 201 guideline.

Analytical verification showed an acceptable correlation between the nominal and the measured concentrations. Therefore, the results were based on nominal concentration.

The following results were determined:

EC50 growth rate > 282 mg/L of test item corresponding to >100 mg/L in active ingredient

NOEC Growth rate = 2.69 mg/L of test item corresponding to 0.955 mg/L active ingredient

EC10 Growth rate = 6.38 mg/L of test item corresponding to 2.26 mg/L active ingredient.

All validity criteria were met, the test is considered to be valid. In these test conditions, the reaction mass TFSK/TFAK is not harmful to algae.

Description of key information

In an algae toxicity study, the 72-hour ErC50 value was calculated to be > 100 mg/L of reaction mass. Therefore, the reaction mass is not harmful to algae. Additionally, the 72-hour ErC10 value was calculated to be 2.26 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

2.26 mg/L

Additional information

A reliable key study on toxicity to algae is available for the reaction mass of potassium trifluoroacetate and potassium trifluoromethanesulphinate. In this study, the test item was an aqueous solution of the reaction mass of TFSK and TFAK because it is the form under which the substance is manufactured and used. The results were expressed in both test item and active matter (i.e. both dried constituents of the reaction mass).

The cultures of Pseudokirchneriella subcapitata were exposed for 72h to the reaction mass TFSK/TFAK at nominal concentrations of 0.84, 2.69, 8.6, 27.5, 88 and 282 mg/L under static conditions in accordance with the OECD 201 guideline and under GLP conditions. Analytical verification showed an acceptable correlation between the nominal and the measured concentrations. Therefore, the results were based on nominal concentration. The following results were determined:

EC50 growth rate > 282 mg/L of test item corresponding to >100 mg/L in active ingredient

NOEC Growth rate = 2.69 mg/L of test item corresponding to 0.955 mg/L active ingredient

EC10 Growth rate = 6.38 mg/L of test item corresponding to 2.26 mg/L active ingredient.

All validity criteria were met, the test is considered to be valid. In these test conditions, the reaction mass TFSK/TFAK is not harmful to algae.