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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 30 March 2004 to 26 July 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Screening study according to the OECD TG209
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Two stock solutions at 2 g/L (expressed as triflic acid) were prepared by dissolving the test item in dechlorinated water (pH: 7.62). The pH of both stock solutions was 2.38.
One of these stock solutions was neutralized by addition of 267 mg of pure (100%) NaOH per g of triflic acid. The pH of this stock solution was 7.22 after neutralization.
Test solutions were prepared by further dilution of the appropriate stock solution with dechlorinated water.
The pH of the test solutions at 1000 mg/L were 6.17 when prepared from test item as is (without neutralization) and 7.65 when prepared from neutralized test item.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Type: inoculum was collected from a water treatment works containing effluent from a predominantly domestic origin.
Reason for this choice: this inoculum is recommended in the OECD guideline.
Origin: the water treatment plant of Evreux (27000 Evreux, France) or (at additional cost) the water treatment plant Emeraude (SIARR) (76141 Petit-Quevilly, France).
Preparation: the inoculum will be left to settle and the supematant rejected. It will be filtered and washed with dechlorinated water if necessary. The dry matter weight was adjusted to 4 g/L ± 10% with dechlorinated water, when required.

Conditions of culture:
The inoculum may be retained for up to 4 days in the laboratory before use in the test provided that it is topped up with 50 mL/L of synthetic sewage feed, daily. The culture room conditions were as follows:
Temperature: 20°C ± 2°C; the temperature in the culture room were recorded continuously and records retained.
Synthetic sewage feed: is prepared using dechlorinated water and analytical grade reagents following OECD recommendations.
Aeration: clean air were bubbled through the inoculum at the rate of 0.5 to 1 L/min until the start of the test.
pH: is checked before use and buffered, if necessary, to pH 6.0 to 8.0 using NaHCO3 or HCL
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
280 ± 20 mg/L as CaCO3.
Test temperature:
20°C ± 2°C throughout the test.
pH:
6.17 to 7.65
Nominal and measured concentrations:
Nominal: 0, 10, 100 and 1000 mg/L with and without pH adjustment.
Details on test conditions:
Conditions of test
Temperature: 20°C ± 2°C throughout the test; the temperature in the culture room was recorded continuously and records retained.
Illumination: no special precautions was taken during the test, although for inoculum stocked for up to four days, the stock vessel was kept away from light.
Dilution water: dechlorinated tap water was used, with a hardness of 280 ± 20 mg/L as CaC03.
Aeration: air was bubbled through each solution at the rate of 0.5 to 1 L/min until the start of the 02 concentration measurements.

Test system:
Groups of test and reference solutions and controls were up for the test:
- two control solutions containing inoculum at 1.6 g/L,
- three test concentrations containing the test item with pH adjustment (i.e. test item neutralization) at 10, 100 and 1000 mg/L and inoculum at 1.6 g/L,
- three test concentrations containing the test item without pH adjustment (i.e. test item as is) at 10, 100 and 1000 mg/L and inoculum at 1.6 g/L,
- three test concentrations containing the reference item (3,5-dichlorophenol) at 4, 12 and 36 mg/L and inoculum at 1.6 g/L.

Initially these solutions were prepared in test flasks and then immediately afterwards aerated for 3 hours before being transferred to an oxygen measuring apparatus.

Test method:
Bach concentration was prepared separately by adding 16 mL of sewage feed to each of the flasks which were made up to 300 mL with water only in the case of the control or a nominal mixture of water and test item (added by means of a stock solution) in the case of the test solutions. Each mixture was then made up to 500 mL by adding 200 mL of inoculum. As 200 mL of inoculum was added in a final volume of 500 mL, the organic material concentration from inoculum was 1.6 g/L (± 10%).
The same procedure was used to prepare the three solutions containing the reference item. The solutions are aerated at between 0.5 to 1 liter of air per minute using a Pasteur-pipette connected with a flexible tube to an air pump as an aeration device.

Solutions were prepared approximately every 15 minutes.
In order to verify the quality of the inoculum, the controls are prepared at the beginning (first control) and the end (second control) of the test.

After 3 hours of aeration/contact time, the contents of each test flask was poured into the measuring apparatus and the oxygen concentration determined for a period of approximately 10 minutes using a chart recorder.

If the respiration rates of the test solutions of highest concentration (1000 mg/L with and without pH adjustment) were equivalent to the respiration rate of the first control (i.e. these rates are within 15% of each other), the oxygen consumption rate of the four other test solutions (10 and 100 mg/L with and without pH adjustment) was not determined.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
Remarks on result:
other: neutralised or not
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Results with reference substance (positive control):
The 3h EC50 of the reference item was 11.7 mg/L.

Respiration rate:

 

Concentration (mg/L)

Respiratory rate

(mg O2.L-1.h-1)

Inhibition

(%)

Control 1

0

42

 

Control 2

0

42

 

Reference item

4

38

10

 

12

18

57

 

36

6

86

Triflic acid

With pH adjustment

10

ND

 

100

ND

 

1000

42

0

Triflic acid

without pH adjustment

10

ND

 

100

ND

 

1000

42

0

ND: Not determined. The respiration rate of the test solution of highest concentration (1000 mg/L) was equivalent to the respiration rate of the first control (i.e. these rates were within 15% of each other). Therefore, the oxygen consumption rate of the test solutions of lowest concentrations (10 and 100 mg/L) was not determined.

Validity criteria fulfilled:
yes
Remarks:
the two control respiration rates are within 15% of each other; the EC50 (3 hours) of 3,5-dichlorophenol is in the accepted range 5 to 30 mg/L.
Conclusions:
Trifluoromethanesulfonic acid is not harmful to microorganisms.
Executive summary:

The objective of the study was to assess the effect of the test item trifluoromethanesulfonic acid on the respiration of activated sewage sludge, over a period of 3 hours.

The criterion measured was the inhibition of the respiration rate of the activated sludge, exposed to different concentrations of the test item, and expressed as a percentage of the control.

From these values, the EC50 (concentration at which respiration rate is 50% of that in the control) was calculated.

The 3h EC50 of the reference item was 11.7 mg/L in this study. Therefore, the criterion for validation of the inoculum is fulfilled.

 

The highest tested concentration of the test item, i.e. 1000 mg/L, did not inhibited the respiration rate of the inoculum. This concentration can therefore be considered as a NOEC. 

The 3h EC50 of the test item, with test item as is or neutralized, was > 1000 mg/L.

In these conditions, trifluoromethanesulfonic acid is not harmful to microorganisms.


Description of key information

In an activated sludge respiration inhibition test, no effect was observed at test item concentration up to 1000 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

On reliable study is available for this endpoint. The objective of the study was to assess the effect of the test item trifluoromethanesulfonic acid on the respiration of activated sewage sludge, over a period of 3 hours.

The criterion measured was the inhibition of the respiration rate of the activated sludge, exposed to different concentrations of the test item, and expressed as a percentage of the control.

From these values, the EC50 (concentration at which respiration rate is 50% of that in the control) was calculated.

The 3h EC50 of the reference item was 11.7 mg/L in this study. Therefore, the criterion for validation of the inoculum is fulfilled.

 

The highest tested concentration of the test item, i.e. 1000 mg/L, did not inhibited the respiration rate of the inoculum. This concentration can therefore be considered as a NOEC. 

The 3h EC50 of the test item, with test item as is or neutralized, was > 1000 mg/L.

In these conditions, trifluoromethanesulfonic acid is not harmful to microorganisms.