3-[(phenylcarbamoyl)amino]phenyl 4-methylbenzenesulfonate

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:

9 May 2018

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 (Carbon and Ammonium Oxidation))

Version / remarks:

22 July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification and stability under storage conditions: The infrared (IR) spectrum of the test material measured at the test facility was confirmed to be identical to that provided by the sponsor. The stability of the test material was confirmed by comparing the IR spectrum of the test material after the completion of the experiment with that before the start of the experiment.

The test material was treated as 100% in purity.

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Test mixture: The test material (250 mg), the synthetic sewage (8 mL), purified water (117 mL) and the activated sludge (125 mL) were added to each test vessel. The test material was accurately weighed with an electric analytical balance and added. The pH of the test solution of the test material was not measured because the test material was not dissolved in test solutions.

- Reference mixture: 1000 mg/L stock solution of 3,5-dichlorophenol, the synthetic sewage (8 mL), activated sludge (125 mL) and purified water.

- Blank control: Synthetic sewage (8 mL), activated sludge (125 mL) and purified water.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: The activated sludge obtained from an aeration reactor of a sewage treatment plant receiving predominantly domestic sewage from Kurume central sewage treatment centre (Kurume-shi, Fukuoka, Japan)
- Method of cultivation: The activated sludge (5 L) was fed with the synthetic sewage (250 mL) and cultivated for 22 hours at 20 ± 2 °C under aerobic condition in order to acclimate the test temperature. The activated sludge was then used. Acclimation to the test material was not performed.
- Preparation of inoculum for exposure: The synthetic sewage (500 mL) was prepared at the same proportion as follows: the following reagents were dissolved in 1 L of purified water.
1) Peptone 16 g
2) Meat extract 11 g
3) Urea 3 g
4) Sodium chloride 0.7 g
5) Calcium chloride dihydrate 0.4 g
6) Magnesium sulfate heptahydrate 0.2 g
7) Dipotassium hydrogen phosphate 2.8 g
The pH of the prepared synthetic sewage was 7.2. The synthetic sewage was prepared the day before the test and was stored in a refrigerator until use.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

20 ± 2 °C

pH:

The pH of the reference mixture and blank control was measured and confirmed to be within 7.5 ± 0.5.

Nominal and measured concentrations:

1000 mg/L as nominal loading concentration

Details on test conditions:

TEST SYSTEM
- Test vessel: Approximately 300 mL glass vessel
- Fill volume: 250 mL
- Measurement bottle: 102 mL glass incubator bottle
- Aeration: 0.5–1 L/min (Air pump)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6 (3 replicates at the beginning and the end of the exposure period in the test, individually).
- Sludge concentration (concentration of suspended solids): 2760 mg/L

EFFECT PARAMETERS MEASURED
Measurement of dissolved oxygen concentration to calculate oxygen consumption rate: After 3 hours of exposure, the measurement bottle was filled with the test solution promptly and softly with attention to avoid foaming. Afterwards, the concentration of dissolved oxygen (DO) in the test solution was measured continuously for 10 minutes with stirring.

TEST CONCENTRATIONS
- Range finding study: In the preliminary test, three test concentrations of the test material at 10, 100 and 1000 mg/L were prepared, and their percentages of respiration inhibition were -6 %, -10 % and 22 %, respectively, after 3 hours of exposure. The results suggested that EC50 of the test material was higher than 1000 mg/L. Therefore, the test mixture was prepared as a limit test.
- Test concentrations: 1000 mg/L (nominal loading concentration)
- Results used to determine the conditions for the definitive study: Yes.

ESTIMATION OF EC50
- Calculation of percentage inhibition of respiration: solution, and a regression formula was derived from the linear part in the graph. The oxygen consumption rate (mgO2/L/h) was obtained from the absolute value of slope of the regression formula. The percentage inhibition of respiration was then calculated by the following equation:

Percentage inhibition of respiration (%) = (1-RS/RC ) × 100
RS : Oxygen consumption rate in test or reference mixture (mg O2/L/h)
RC : Average oxygen consumption rate in blank control (mg O2/L/h)

- Estimation of EC50: In the test mixture, the EC50 of the test material was estimated as " >1000 mg/L" because less than 50 % of inhibition rate was obtained within the exposure level. In the reference mixture, the percentage inhibition in each exposure level was plotted on a semilogarithmic graph against the corresponding concentration. The EC50 of the reference substance (3,5-dichlorophenol) was calculated from the regression formula obtained by probit analysis. The EC50 was determined using computer program (running on Microsoft software "Excel").

VALIDITY CRITERIA
- The EC50 of 3,5-dichlorophenol as reference item should be between 2–25 mg/L
- The coefficient of variation of the oxygen consumption rate in the blank controls between at the beginning and at the end of the exposure period should be less than 30%
- The oxygen consumption rate per gram (dry weight) of activated sludge per hour in the blank control should be ≥20 mg O2/g/h

Oxygen consumption rate (mg O2/g/h) = [(RC × V) / (SS × VS)] × 1000
RC : Average oxygen consumption rate in blank control (mg O2/L/h)
V : Volume of test solution (L)
SS : Concentration of suspended solid in activated sludge (mg/L)
VS : Additive amount of activated sludge (L)

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol, 4.0, 8.0, 16 and 32 mg/L (geometric series with a factor of 2), 1 replicate/exposure level.

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:

inhibition of total respiration

Details on results:

PRELIMINARY TEST
- Three test concentrations of the test material at 10, 100 and 1000 mg/L were prepared, and their percentages of respiration inhibition were -6 %, -10 % and 22 %, respectively, after 3 hours of exposure. The results suggested that EC50 of the test material was higher than 1000 mg/L, therefore the test mixture was prepared as a limit test.

MAIN TEST
- Average oxygen consumption rate in blank control: 47.9 mg O2/L/h.
- The average percentage inhibition of respiration of the test mixture at 1000 mg/L was 5 %.

VALIDITY OF TEST
The test was valid because the test results met the following criteria:
- The EC50 of 3,5-dichlorophenol as reference item was 11 mg/L this value met the criterion (2–25 mg/L).
- The coefficient of variation of the oxygen consumption rate in the blank controls between at the beginning and at the end of the exposure period was 4.8 %, and this value met the criterion (less than 30%).
- The oxygen consumption rate per gram (dry weight) of activated sludge per hour in the blank control was average 34.7 mgO2/g/h, and this value met the criterion (≥20 mg O2/g/h).

Results with reference substance (positive control):

A stock solution of the reference substance was prepared, 3,5-dichlorophenol (100 mg) was accurately weighed with an electronic analytical balance and dissolved in approximately 80 mL of purified water. The pH of this solution was adjusted to 7.5 with 1 mol/L sodium hydroxide solution. And then, this solution was filled up to 100 mL by purified water to obtain 1000 mg/L stock solution of 3,5-dichlorophenol.

The following concentrations of the reference mixture were tested: 4.0, 8.0, 16 and 32 mg/L (geometric series with a factor of 2) with 1 replicate per exposure level. At the nominal concentrations of 4.0, 8.0, 16 and 32 mg/L the oxygen consumption rate was 38.9, 34.6, 16.9 and 5.18 mg O2/L/h respectively. The EC50 was determined to be 11 mg/L.

Oxygen consumption rate and percentage inhibition of respiration after 3 hours of exposure

Test solution	Nominal loading concentration (mg/L)	Oxygen consumption rate (mg O2/L/h)	Percentage inhibition of respiration (%)	Average percentage inhibition of respiration (%)
Test mixture	1000 1000 1000	42.6 49.8 43.6	11 -4 9	5
Reference mixture	4.0 8.0 16 32	38.9 34.6 16.9 5.18	19 28 65 89	-
Blank control	-	At the beginning: 42.5, 47.9, 48.3 At the end: 45.1, 52.0, 51.4	-	-

Average oxygen consumption rate in blank control: 47.9 mg O2/L/h

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study the average percentage inhibition of respiration of the test mixture at 1000 mg/L was 5 %, therefore the EC50 of the test material was estimated as >1000 mg/mL (as nominal loading concentration). 

Executive summary:

To determine the 3-hour EC50 of the test material an activated sludge respiration inhibition test was conducted in accordance with the standardised guidelines OECD 209, under GLP conditions. 

In the preliminary test, three test concentrations of the test material at 10, 100 and 1000 mg/L were prepared, and their percentages of respiration inhibition were -6 %, -10 % and 22 %, respectively, after 3 hours of exposure. The results suggested that EC50 of the test material was higher than 1000 mg/L. Therefore, the test mixture was prepared as a limit test, and the test concentration of the test material was carried out at 1000 mg/L as nominal loading concentration. The reference mixture used was 3,5-Dichlorophenol. After 3 hours of exposure, the measurement bottle was filled with the test solution promptly and softly with attention to avoid foaming. Afterwards, the concentration of dissolved oxygen  DO) in the test solution was measured continuously for 10 minutes with stirring. The activated sludge was obtained from the aeration reactor of the sewage treatment plant receiving predominantly domestic sewage. This test was valid because all of the values in this test met the criteria. The EC50 of the reference substance was determined to be 11mg/L. 

Under the conditions of this study the average percentage inhibition of respiration of the test mixture at 1000 mg/L was 5 %, therefore the EC50 of the test material was estimated as >1000 mg/mL (as nominal loading concentration). 

Description of key information

The average percentage inhibition of respiration of the test mixture at 1000 mg/L was 5 %, therefore the EC50 of the test material was estimated as >1000 mg/mL (as nominal loading concentration). 

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

Additional information

To determine the 3-hour EC50 of the test material an activated sludge respiration inhibition test was conducted in accordance with the standardised guidelines 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).

In the preliminary test, three test concentrations of the test material at 10, 100 and 1000 mg/L were prepared, and their percentages of respiration inhibition were -6 %, -10 % and 22 %, respectively, after 3 hours of exposure. The results suggested that EC50 of the test material was higher than 1000 mg/L. Therefore, the test mixture was prepared as a limit test, and the test concentration of the test material was carried out at 1000 mg/L as nominal loading concentration. The reference mixture used was 3,5-Dichlorophenol. After 3 hours of exposure, the measurement bottle was filled with the test solution promptly and softly with attention to avoid foaming. Afterwards, the concentration of dissolved oxygen  DO) in the test solution was measured continuously for 10 minutes with stirring. The activated sludge was obtained from the aeration reactor of the sewage treatment plant receiving predominantly domestic sewage. This test was valid because all of the values in this test met the criteria. The EC50 of the reference substance was determined to be 11mg/L. 

Under the conditions of this study the average percentage inhibition of respiration of the test mixture at 1000 mg/L was 5 %, therefore the EC50 of the test material was estimated as >1000 mg/mL (as nominal loading concentration).