N-[[3,5-dichloro-2-fluoro-4-(1,1,2,3,3,3-hexafluoropropoxy)phenyl]carbamoyl]-2,6-difluorobenzamide

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:

2 October 2003 - 25 November 2003

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

Analytical monitoring:

no

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
A stock solution containing 37,300 mg/L of the test material (corrected for purity) and a 1:10 dilution of the test material stock (3730 mg/L) were prepared in acetone due to the low water solubility of the test material. The same volume of acetone (0.025 mL) was added to all of the reaction mixtures.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Activated sludge was obtained from the Midland Municipal Wastewater Treatment Plant on 13 October 2003. Once returned to the laboratory, the activated sludge was washed three times with tap water. The washing step was accomplished by allowing the sludge to settle and replacing the supernatant aqueous phase with tap water. The mixed liquor suspended solids (MLSS) were determined to be 1740 ± 163 mg/L (n = 3) using a standard procedure. The activated sludge was removed from aeration and the MLSS were allowed to settle out. The MLSS were increased to an estimated 4000 mg/L by removing 4.5 L of clarified supernatant liquid, effectively reducing the activated sludge mixed liquor volume from 8.0 to 3.5 L. The pH of the concentrated activated sludge was 7.5.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 ± 2 °C

Nominal and measured concentrations:

Nominal concentrations: 0.02, 0.06, 0.19, 0.60 and 1.9 mg/mL

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flask
- Material, size, headspace, fill volume: 16 mL of the synthetic sewage feed, 284 mL water, 0.025 mL acetone, and 200 mL of microbial inoculum were mixed (final volume 500 mL). To this was added the appropriate amount of test material stock solution.
- Aeration: The reaction mixture was vigorously aerated with compressed air passed through a gas purification cartridge containing a moisture indicator and molecular sieve pellets. The air was introduced into the reaction mixture using a Pasteur pipette.
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Loading rate: The MLSS were 1600 mg/L in the test system

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Water used to prepare stock solutions was purified using the Milli-Q® water treatment system.
- Synthetic sewage feed: A synthetic sewage feed was made by dissolving the following in 1 L of tap water: 16 g peptone; 11 g meat extract; 3 g urea; 0.7 g NaCl; 0.4 g CaCl₂.2H₂O; 0.2 g MgSO₄.7H₂O; 2.8 g K₂HPO₄. The synthetic sewage was stored under refrigeration (4 °C) until used.

EFFECT PARAMETERS MEASURED
After three hours of aeration, the contents of the reaction flask were poured into a 300 mL BOD bottle, an oxygen electrode was inserted, and the dissolved oxygen concentration of the stirred reaction mixture was recorded over a period of up to 10 minutes. These measurements were performed at 15 minute intervals until all control, reference material, and test material mixtures were analysed.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: approximately 3
- Range finding study: No

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: The test material did not inhibit the respiration rate of the activated sludge compared to the control mixtures; thus, an EC50 value could not be determined

Details on results:

The results are summarised in Table 1. The test material, at nominal concentrations that ranged from 1.9 mg/L (10-fold greater higher than the reported water solubility) to 0.02 mg/L did not inhibit the respiration rate of the activated sludge compared to the control mixtures (no test material added). Thus, an EC50 value could not be determined under the conditions of this test guideline.

The measurement of the respiration rates of control mixtures C1 and C2 at the beginning and end of the experiment averaged 34.5 mg O2 per litre per hour ± 2.7 percent. The average consumption rate for the five test reaction mixtures (± 1 standard deviation) was 35.2 ± 0.7 mg O2 per litre per hour. There was negligible difference in respiration rates between the control mixtures and the five test mixtures, indicating that the test material did not inhibit the respiration rate of the activated sludge at concentrations up to 10-fold greater than the water solubility limit.

The validity of the test results was confirmed. The average respiration rates of the control mixtures at the beginning and conclusion of the experiment, 34.9 and 34.0 mg O2 per litre per hour, were within the accepted range of 15 percent of each other. The recorded temperatures of the aerated reaction mixtures ranged from 20.2 to 20.3 °C during the experiment, and were therefore maintained within the required range of 20 ± 2 °C.

Results with reference substance (positive control):

The 3 h EC50 of the reference material, 3,5- dichlorophenol, was determined to be 8.3 mg/L. This results falls within the accepted range of 5 to 30 mg/L.

Table 1: Results of the Activated Sludge Respiration Inhibition test

Concentration (mg/L)	Time¹ (minutes)	O₂ Start (mg/L)	O₂ End (mg/L)	O₂ Change	Respiration² (mg O₂/L/hr)	% Inhibition³
Control 1	2.8	6.45	4.82	1.63	34.9	-
3,5-DCP 2.98	2.4	4.95	3.80	1.15	28.8	16.6
3,5-DCP 9.94	3.2	6.70	5.99	0.71	13.3	61.4
3,5-DCP 29.8	1.3	8.19	8.04	0.15	6.9	79.9
1.90	2.1	5.11	3.82	1.26	36.9	0
0.60	2.4	5.45	4.02	1.34	35.8	0
0.19	2.6	5.25	3.73	1.52	35.1	0
0.06	2.5	6.43	4.97	1.46	35.0	0
0.02	1.6	6.17	5.22	0.95	35.6	0
Control 2	1.2	6.70	6.05	0.68	34.0	-

¹ Time period used in calculation of respiration rate, designated “measurement period”

² Respiration rate = Change in O₂ x (60 / Time)

³ Percent inhibition = [1 - ((2 x Respiration) / (34.9+34.0))] x 100

Validity criteria fulfilled:

yes

Conclusions:

The test material, at nominal concentrations that ranged from 1.9 mg/L (10-fold greater higher than the reported water solubility) to 0.02 mg/L did not inhibit the respiration rate of the activated sludge compared to the control. Thus, an EC50 value could not be determined.

Executive summary:

The potential for the test material to inhibit the respiration of municipal activated sludge was evaluated in accordance with the standardised guideline OECD 209 under GLP conditions.

During the study, activated sewage sludge was exposed to the test material for 3 hours at nominal concentrations of 0.02, 0.06, 0.19, 0.60 and 1.9 mg/mL at a temperature of 20 ± 2 °C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 3 hours of contact time and compared to data for the control and the reference material 3,5-dichlorophenol.

At a nominal concentration of 1.9 mg/L (approximately 10-fold higher than the reported water solubility of 0.19 mg/L) the test material did not inhibit the rate of the activated sludge respiration compared to untreated activated sludge controls. An EC50 value could therefore not be determined.

While an EC50 value could not be derived from this test, the material appears to have a low potential for inhibiting activated sludge respiration at concentrations of up to 10-fold higher than its water solubility limit.

Description of key information

At a nominal concentration of 1.9 mg/L (approximately 10-fold higher than the reported water solubility of 0.19 mg/L) the test material did not inhibit the rate of the activated sludge respiration compared to untreated activated sludge controls. An EC50 value could therefore not be determined.

Additional information

The potential for the test material to inhibit the respiration of municipal activated sludge was evaluated in accordance with the standardised guideline OECD 209 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study, activated sewage sludge was exposed to the test material for 3 hours at nominal concentrations of 0.02, 0.06, 0.19, 0.60 and 1.9 mg/mL at a temperature of 20 ± 2 °C with the addition of a synthetic sewage as a respiratory substrate. The rate of respiration was determined after 3 hours of contact time and compared to data for the control and the reference material 3,5-dichlorophenol.

At a nominal concentration of 1.9 mg/L (approximately 10-fold higher than the reported water solubility of 0.19 mg/L) the test material did not inhibit the rate of the activated sludge respiration compared to untreated activated sludge controls. An EC50 value could therefore not be determined.

While an EC50 value could not be derived from this test, the material appears to have a low potential for inhibiting activated sludge respiration at concentrations of up to 10-fold higher than its water solubility limit.