Reaction mass of potassium trifluoroacetate and potassium trifluoromethanesulphinate

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2012-03-19 to 2013-06-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

december 2012

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

A reference control stock solution of 3,5-dichlorophenol, a known inhibitor of microbial respiration, was prepared as follows: Dissolved 0.5024 g of 3,5-dichlorophenol in 10 mL of 1 mol/L NaOH; Diluted to approximately 30 mL with 20 mL distilled water; Added, while stirring, 0.5 mol/L H2SO4 to the point of incipient precipitation-approximately 5 mL of 0.5 mol/L H2SO4 was required; Finally, diluted the mixture to one liter with laboratory water. The pH was 7.89 and adjusted with 0.5 mol/L H2SO4.
The test substance (Reaction mass of potassium trifluoromethanesulphinate and potassium trifluoroacetate) was directly added to test bottles after appropriate amounts of Reaction mass of potassium trifluoromethanesulphinate and potassium trifluoroacetate was weighed. The ingredient concentration of the TFAK and TFSK was used and expressed as mg a.i./L, because the test substance was a water solution of TFAK and TFSK.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Source: Activated sludge microorganisms were obtained from a chengdong waste water treatment plant receiving predominantly domestic sewage, Lot: AS20130428.

- Method of cultivation: On arrival at the laboratory, a small amount of the fresh sludge was weighed, oven-dried, and reweighed. A calculation was made from these results to determine the amount of wet sludge that must be suspended in deionised water in order to obtain an activated sludge with a mixed-liquor suspended solids (MLSS) level of approximately 3 g/L.

- Preparation of inoculum for exposure: The sludge was washed three times with deionised water using the following method: after centrifuging at approximately 10000 rpm for 10 minutes, the supernatant was decanted and the remaining sludge solids re-suspended in deionised water and mixed well with mechanical stirrer. After the third spin, the wet solids were homogenized for 2 minutes in a blender. The 90 g wet solids were suspended in 3 L of deionised water and 150 mL of the synthetic feed stock solutions. The sludge suspension was aerated overnight at (20±2) ºC. The pH of the overnight suspension was 7.48. Viability of the microorganisms was checked by means of a reference control. In studies of this sort no attempt was made to identify the large variety of organisms present in the samples or the degree of absorption of the test and control substances.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

20 ± 2°C

pH:

7.54 - 8.19 in the definitive test.

Dissolved oxygen:

0.30 mg/L

Nominal and measured concentrations:

2124 mg a.i./L(TFAK: 1002 mg/L, TFSK: 1122 mg/L)

Details on test conditions:

Range-finding Test
At time “0”, 16 mL of the synthetic sewage feed and 250 mL of microbial inoculum were added to a first vessel (first blank control RC1), then fixed final volume of 500 mL with deionised water. Whereafter aerate the mixture at 0.8 litre per minute using a Pasteur-pipette by an aeration device.
At time “15 min” the above was repeated, except that appropriate amount of the test substance was added to the 16 mL of synthetic sewage. Then 250 mL microbial inoculum was added to make a total volume of 500 mL. This mixture was then be poured into a second vessel and aerated as above. This process was repeated at 15-minute intervals with different amounts of the test substance to give a series of vessels containing widely-spaced concentrations of the test substance.
In the range-finding test, the test solutions of the test substance with nominal concentration 10, 100, 1000 and 2000 mg a.i./L were established, with no replicates for each concentration, but triplicates at the highest tested concentration 2000 a.i.mg/L.

DEFINITIVE LIMIT TEST
As the test substance is a reaction mass containing two constituents in water, the concentration of test item was selected to ensure at least 1000 mg/L of each constituent. Therefore, the limit test of the test substance was conducted at the concentration of 2124 mg a.i./L(TFAK: 1002 mg/L, TFSK: 1122 mg/L), and triplicates.

The reference control was tested on the microbial inoculums in the same way. Appropriate volumes stock solution of reference substance was added to test bottle with exposure solutions at 3 mg/L, 10 mg/L, and 30 mg/L.

Finally, a second Negative Control (RC2) was prepared. Oxygen consumption was measured and recorded after an aeration time of 3 hours. Air temperature was 20.3 ºC at test start. The pH of all test solutions were measured at test start and test end.

After 3 hours the respiration rate was measured directly in the vessel between oxygen concentrations between 2.0 mg/L and 7.0 mg/L over a period of up to 10 minutes.
This determination was repeated on the contents of each vessel at 15-minute intervals, in such a way that the contact time in each vessel was three hours.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 2 124 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Remarks:

TFAK: 1002 mg/L, TFSK: 1122 mg/L

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

2 124 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

At the tested concentration of 2124 mg a.i./L, from 11 to 15% of inhibition of the respiration of the activated sludge were observed. The 3 h-EC50 value of the test substance (Reaction mass of potassium trifluoromethanesulphinate and potassium trifluoroacetate) was therefore >2124 mg a.i./L(TFAK: 1002 mg/L, TFSK: 1122 mg/L). 3 h-NOEC of the test substance (Reaction mass of potassium trifluoromethanesulphinate and potassium trifluoroacetate) was 2124 mg a.i./L (total concentration of TFAK and TFSK), no statistical significance of this effect with p=0.225 (p>0.05)

Results with reference substance (positive control):

The 3h-EC50 value of the reference substance (i.e. 3,5-dichlorophenol) was calculated by regression analysis to be 19.3 mg/L, and the 95% confidence limit ranged from 16.3 mg/L to 22.8 mg/L. The 3-hour EC50 of 3, 5-dichlorophenol was therefore in the validity range of 2 mg/L-25 mg/L.

Table1: Results of Respiration Inhibitionduring Range-finding Test

#Beakers	Conc. (mg a.i./L)	O2 Concentration		Time(min)2	Respiration ratemg O2/g.h	%Inhibition
		Upper value	Lower value
1 Neg. Control	0	7.00	2.50	6.59	27.3	-
2 Test	10	7.00	2.50	6.59	27.3	-2.24
3 Test	100	7.00	2.50	6.63	27.1	-1.35
4 Test	1000	7.00	2.50	6.63	27.2	-1.35
5 Test	1000	7.00	2.50	6.63	27.1	-1.35
6 Test	1000	7.00	2.00	7.32	27.3	-2.24
7 Test	2000	7.00	2.00	8.33	24.0	10.3
8 Test	2000	7.00	2.00	8.25	24.3	9.42
9 Test	2000	7.00	2.00	8.33	24.0	10.3
10 Pro. Control	3	7.00	2.50	7.50	24.0	10.3
11 Pro. Control	10	7.00	2.50	12.1	14.9	44.4
12 Pro. Control	30	7.00	4.00	12.9	9.30	65.0
13 Neg. Control	0	7.00	2.50	6.89	26.1	-

 

EC50value of Test	>2000mg a.i./L	95% confidence limit	-
EC50value of Reference	15.5mg/L	95% confidence limit	12.4 ~ 19.3 mg/L

 

1=mg of oxygen from dissolved oxygen meter test readings (as mg/L)

2=Time for Resp. Rate calculations

3=Respiration rate as mg of oxygen per liter hour,(Upper value – Lower value)/Time in min. × 60

Note: the Negative Controlswas4.48%(within30%).

(Relative Percent Difference between reps ÷MEAN × 100)

Table 2: Results of Respiration Inhibition during Limit Test

#Beakers	Con. (mg a.i./L)	O2Concentration		Time (min)^2	Respiration rate mg O2/g·h		% Inhibition
		UpperValue^1	LowerValue^1
1 Neg. Control	0	7.00	2.50	7.35	24.5		-
2 Test	2124 (TFAK:1002 mg/L,TFSK:1122 mg/L)	7.00	3.00	7.49	21.3		11.0
3 Test	2124 (TFAK:1002 mg/L,TFSK:1122 mg/L)	7.00	3.00	7.84	20.4		15.0
4 Test	2124 (TFAK:1002 mg/L,TFSK:1122 mg/L)	7.00	3.00	7.66	20.9		13.0
5 Pro. Control	3	7.00	2.50	7.65	23.5		2.00
6 Pro. Control	10	7.00	2.50	11.5	15.6		35.0
7 Pro. Control	30	7.00	4.00	13.2	9.13		62.0
8 Neg. Control	0	7.00	2.50	7.65	23.5		-
EC50 value of Test	>2124 mg a.i./L		95% confidence limit			-
NOEC	2124 mg A;I./L
EC50 value of Reference	19.3 mg/L		95% confidence limit			16.3 ~ 22.8mg/L

1=mg of oxygen from dissolved oxygen meter test readings (as mg/L)

2=Time for Resp. Rate calculations

3=Respiration rate as mg of oxygen per liter hour,(Upper value – Lower value)/Time in min. × 60

Note: the Negative Controlswas4.17%(within30%).

(Relative Percent Difference between reps ÷MEAN × 100)

Table 3: Parameters of Test Solutionsduring Range-finding Test

Nominal Concentration(mg/L)	Duration(h)	pH	Temperature(°C)
1 Neg. Control	0	7.55	20.3
	3	8.17	21.0
2 Test	0	7.56	20.3
	3	8.16	20.8
3 Test	0	7.58	20.2
	3	8.17	20.9
4 Test	0	7.54	20.0
	3	8.16	20.5
5 Test	0	7.57	20.3
	3	8.18	20.7
6 Test	0	7.55	20.4
	3	8.17	20.6
7 Test	0	7.56	20.1
	3	8.18	20.5
8 Test	0	7.57	20.3
	3	8.17	20.8
9 Test	0	7.54	20.2
	3	8.19	21.0
10 Pro. Control	0	7.61	20.0
	3	8.18	20.4
11 Pro. Control	0	7.62	20.4
	3	8.18	20.7
12 Pro. Control	0	7.63	20.3
	3	8.18	20.8
10 Neg. Control	0	7.57	20.5
	3	8.18	21.1

Validity criteria fulfilled:

yes

Conclusions:

EC50>2124 mg a.i./L
NOEC=2124 mg a.i./L

Executive summary:

A 3-hour activated sludge respiration inhibition test was performed using activated sludge from a domestic waste treatment plant according to “The guidelines for the testing of chemical (HJ/T 153-2004)” and with reference to OECD TG 209.

The sludge microorganisms were exposed to three concentrations of the test substance (Reaction mass of potassium trifluoromethanesulphinate and potassium trifluoroacetate) at nominal concentration of 10, 100, 1000 and 2000 mg a.i./Lin range-finding test and 2124 mg a.i./L in limit test. These concentrations were selected to ensure a high concentration of each constituent of the reaction mass.The respiration rate, expressed as oxygen consumption by the microbes in mg O2 per liter per hour, was measured under defined conditions following the 3-hour exposure period. Inhibition values were calculated by comparing test respiration rates to negative control rates.

EC50 values were calculated by regression analysis. The result indicated that under valid test conditions the 3 h-EC50 value of 3,5-dichlorophenol was 19.3mg/L. Therefore, the 3-hour EC50 of 3,5-dichlorophenol was in the range 2 - 25 mg/L. Additionally, the two negative control respiration rates were within 30% of each other and the blank controls oxygen uptake rates were greater than 20 mg per one gram of activated sludge (dry wet of suspended solids) in an hour. Therefore, the validity criteria were fulfilled.

The 3 h-EC50 value of the test substance (Reaction mass of potassium trifluoromethanesulfinate and potassium trifluoroacetate) was higher than 2124 mg a.i./L (TFAK: 1002 mg/L,TFSK:1122 mg/L);

3 h-NOECof the test substance (Reaction mass of potassium trifluoromethanesulphinate and potassium trifluoroacetate) was 2124 mg a.i./L (total concentration of TFAK and TFSK), no statistical significance of this effect with p>0.05.

 

Description of key information

In a study on microorganisms with the reaction mass, the EC50 (3h) was found to be higher than 2124 mg a.i./L (corresponding to TFAK: 1002 mg/L, TFSK: 1122 mg/L actice ingredient).
The NOEC is 2124 mg a.i./L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

2 124 mg/L

Additional information

One reliable toxicity study on microorganisms is available for the reaction mass of potassium trifluoroacetate and potassium trifluoromethanesulphinate and is selected as key study. This study is a 3-hour activated sludge respiration inhibition test performed using activated sludge from a domestic waste treatment plant according to the OECD Testing Guideline 209.

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 ingredient (i.e. both dried constituents of the reaction mass). The ecotoxicity profile of the substance has been determined based on the results expressed as active matter and therefore are applicable to the substance described in the section 1.2 of the iuclid.

The sludge microorganisms were exposed to three concentrations of the test substance (Reaction mass of potassium trifluoromethanesulphinate and potassium trifluoroacetate) at nominal concentration of 10, 100, 1000 and 2000 mg a.i./L in range-finding test and 2124 mga.i./L in limit test. These concentrations were selected to ensure a high concentration of each constituent of the reaction mass.The respiration rate, expressed as oxygen consumption by the microbes in mg O₂ per liter per hour, was measured under defined conditions following the 3-hour exposure period. Inhibition values were calculated by comparing test respiration rates to negative control rates.

EC50 values were calculated by regression analysis.The result indicated that under valid test conditions the 3 h-EC₅₀ value of 3,5-dichlorophenol was19.3mg/L. Therefore, the 3-hour EC₅₀ of 3,5 -dichlorophenol was in the range 2 mg/L-25 mg/L. Additionally, the two negative control respiration rates were within 30% of each other and the blank controls oxygen uptake rates were greater than 20 mg per one gram of activated sludge (dry wet of suspended solids) in an hour. Therefore, the validity criteria were fulfilled.

The 3 h-EC50 value of the test substance (Reaction mass of potassium trifluoromethanesulfinate and potassium trifluoroacetate) was higher than 2124 mg a.i./L (TFAK: 1002mg/L,TFSK:1122mg/L); The 3 h-NOEC of the test substance (Reaction mass of potassium trifluoromethanesulphinate and potassium trifluoroacetate) was 2124 mga.i./L (total concentration of TFAK and TFSK).