2-Propenamide, 3-(1,3-benzodioxol-5-yl)-N,N-diphenyl-, (2E)-

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:

19 March to 20 July 2020

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:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: MEP, P.R. China, the Guidelines for the Testing of Chemicals, Effects on Biotic Systems (the Second Edition), No. 209 "Activated Sludge, Respiration Inhibition Test". (2013.8)

Version / remarks:

2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Details on sampling:

Not applicable

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test item was directly weighed into the test vessels and destilled water was added. The following test concentrations were tested: 10, 100, 300 and 1000 mg/L.
- Eluate: no
- Controls: blank control, abiotic control, reference control
- Evidence of undissolved material: no

Test organisms (species):

activated sludge

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: The activated sludge was derived from a treatment plant receiving predominantly demestic sewage in Shenyang North Sewage Treatment Plant (one day before the test was started).
- Preparation of inoculum for exposure: On the day of activated sludge collection, coarse particles were removed by settling for 15 minutes and by decanting the upper layer of finer solids. The sludge was wached as follows: The sludge was first centrifuged for 5 minutes at ca. 2500 rpm. The supernatant was discarded and the sludge was re-suspended in chlorine-free tap water by shaking. The wash-water was removed by re-centrifugation and discarding the supernatant. After re-suspension of the sludge pellet in chlorine-free tap water, the solids dry weight was determined by drying 3 replicates of 10 ml suspended sludge at 105 °C for 1 h and 30 min. From the measured results, the sludge was diluted in chlorine-free tap water to obtain the required sludge solid concentration of 3 g/L.
- Cultivation and preparation of the sludge: The activated sludge was fed with the synthetic seage feed (50 mL synthetic sewage feed/L activated sludge) and continuously aerated at the test temperature (19.9 - 21.8 °C) overnight prior to use in the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

20 ± 2 °C (actual values: 20.7 - 21.8 °C)

pH:

7.98 - 8.15 (test item treatments, 8.01 - 8.22 (blank controls)

Dissolved oxygen:

2.76 - 8.78 mg/L

Salinity:

Not applicable

Conductivity:

Not specified

Nominal and measured concentrations:

10, 100, 300 and 1000 mg/L (nominal), no measured concentrations

Details on test conditions:

TEST SYSTEM
- Test vessel: not specified
- Fill volume: 500 mL
- Aeration: 0.5 - 1 L air/min
- No. of vessels per concentration: 1 (each for 10, 100 and 300 mg/L) and 3 (for 1000 mg/L), respectively
- No. of vessels per blank control: 4
- No. of vessels per abiotic control: 3
- No. of vessels per reference item concentration: 1
- Sludge concentration: 1.5 g/L
- Weight of dry solids per volume of reaction mixture per unit of time: 1.5 g/L
- Nutrients provided for bacteria: 16 mL synthetic sewage feed per replicate (500 mL total volume of mixture)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: destilled water

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: darkness

EFFECT PARAMETERS MEASURED: respiration inhibition

TEST CONCENTRATIONS
- Range finding study/definitive study: Triplicates were examined at the highest tested concentration of 1000 mg/L. Additionally, three lower concentration levels of 10, 100 and 300 mg/L were examined with one vessel each. Blank (4 replicates), abiotic (three abiotic controls with the highest concentration of the test item) and reference controls were included.
- Test concentrations: 10, 100, 300 and 1000 mg/L

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:

inhibition of total respiration

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

Details on results:

- Any observations that might cause a difference between measured and nominal values: no
- Adsorption: no
- Blank controls oxygen uptake rate: 21.5 mg/(g.h ss) oxygen
- Coefficient of variation of oxygen uptake rate in control replicates: 3.8 % at the end of the test

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: EC50 (3 hours) = 6.2 mg/L (95 % confidence limits 4.1 - 9.4 mg/L

Reported statistics and error estimates:

The EC50 value of the reference item was calculated using Probit analysis.

Validity criteria fulfilled:

yes

Conclusions:

In a test according to OECD 209, the 3-hour EC50 value of the test item was greater than 1000 mg/L. The NOEC was determined to be 1000 mg/L, the highest concentration tested.

Executive summary:

The purpose of the test was to evaluate the influence of the test item on the activity of an activated sludge by measuring the respiration rate under defined conditions. The respiration rates of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. This pre-test was used serve as stand-alone (limit test) according to OECD 209. The test item was investigated in this study at the nominal concentrations of 10, 100, 300 and 1000 mg/L. Defined amounts of the test item were added directly into the test vessels. Triplicate vessels were prepared and investigated at the highest examined test item concentration. In parallel with the test item treatments, 3,5-Dichlorophenol as positive reference control at concentrations of 0.5, 1.3, 3.5, 9.4 and 25 mg/L; furthermore, blank (inoculum) control and abiotic controls were investigated. Abiotic controls (investigated in three parallels) were prepared containing the test item at 1000 mg/L, synthetics sewage feed, but without inoculum. The experiment was performed without pH adjustment. All validity criteria of the study were met. The average specific respiration rate of the blank was 21.5 mg O2 /gh activated sludge (based on dry weight) in an hour with a coefficient of variation of 3.8 % (clearly below 30 % as required in the validity criteria). The 3-hour EC50 of the reference item 3,5-Dichlorophenol was 6.2 mg/L being will within the range of 2 mg/L to 25 mg/L as required for total respiration. The observed oxygen consumption rates and consequently the specific respiration rates in all examined test item concentrations remained in the range of the blank controls (the average specific respiration rate at 1000 mg/L was 22.4 mg O2/gh). This corresponds to -4.4 % inhibition which is well within the biological variability range of the applied test system. Consequently, no inhibitory effect of the test item up to 1000 mg/L (the limit concentration according to OECD 209) was observed. Based on measured oxygen consumption values and calculated specific respiration rates it can be stated that the 3-hour EC10 and EC50 values of the test item are greater than 1000 mg/L. The NOEC was determined to be 1000 mg/L, the highest concentration tested.

Description of key information

In a test according to OECD 209, the 3-hour EC₅₀ value of the test item was greater than 1000 mg/L. The NOEC was determined to be 1000 mg/L, the highest concentration tested.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

The purpose of the test was to evaluate the influence of the test item on the activity of an activated sludge by measuring the respiration rate under defined conditions. The respiration rates of samples of activated sludge fed with synthetic sewage were measured in an enclosed cell containing an oxygen electrode after a contact time of 3 hours. This pre-test was used serve as stand-alone (limit test) according to OECD 209. The test item was investigated in this study at the nominal concentrations of 10, 100, 300 and 1000 mg/L. Defined amounts of the test item were added directly into the test vessels. Triplicate vessels were prepared and investigated at the highest examined test item concentration. In parallel with the test item treatments, 3,5-Dichlorophenol as positive reference control at concentrations of 0.5, 1.3, 3.5, 9.4 and 25 mg/L; furthermore, blank (inoculum) control and abiotic controls were investigated. Abiotic controls (investigated in three parallels) were prepared containing the test item at 1000 mg/L, synthetics sewage feed, but without inoculum. The experiment was performed without pH adjustment. All validity criteria of the study were met. The average specific respiration rate of the blank was 21.5 mg O2 /g activated sludge (based on dry weight) in an hour with a coefficient of variation of 3.8 % (clearly below 30 % as required in the validity criteria). The 3-hour EC50 of the reference item 3,5-Dichlorophenol was 6.2 mg/L being will within the range of 2 mg/L to 25 mg/L as required for total respiration. The observed oxygen consumption rates and consequently the specific respiration rates in all examined test item concentrations remained in the range of the blank controls (the average specific respiration rate at 1000 mg/L was 22.4 mg O2/gh). This corresponds to -4.4 % inhibition which is well within the biological variability range of the applied test system. Consequently, no inhibitory effect of the test item up to 1000 mg/L (the limit concentration according to OECD 209) was observed. Based on measured oxygen consumption values and calculated specific respiration rates it can be stated that the 3-hour EC10 and EC50 values of the test item are greater than 1000 mg/L. The NOEC was determined to be 1000 mg/L, the highest concentration tested.