2-[7-(diethylamino)-2-oxo-2H-1-benzopyran-3-yl]benzoxazole-5-sulphonamide

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:

01 February 2017 to 03 February 2017

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:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

ISO 8192 (Water quality - Test for inhibition of oxygen consumption by activated sludge for carbonaceous and ammonium oxidation)

Version / remarks:

2007

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The test material was not sufficiently soluble to allow preparation of a 10 g/L stock solution in water. Therefore, 1-litre test bottles were filled with 200 mL of test material mixtures in Milli-RO water (tap water purified by reverse osmosis; Millipore Corp), with initial loading rates of 2.5 times the final loading rate. These mixtures were stirred in closed dark brown bottles for approximately 24 hours. Subsequently, 16 mL synthetic medium made up to 50 mL with Milli-RO water and 250 mL sludge were added resulting in the required loading rates. Optimal contact between the test material and test organisms was ensured by applying continuous aeration and stirring.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of municipal sewage treatment plant where inoculum was collected: 'Waterschap Aa en Maas', 's- Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ca. 105 °C to determine the amount of suspended solids. The pH was 7.9 on the day of testing. The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium (sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.
- Initial biomass concentration: Suspended solids 3.0 g/L of sludge

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test temperature:

19 to 22 °C

pH:

7.1 to 8.1

Nominal and measured concentrations:

- Nominal concentration: 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: Bottles/vessels.
- Type: Open
- Material, size, headspace, fill volume: All glass, filled with 500 mL. The synthetic medium (16 mL) made up to 50 mL with Milli- RO and 200 mL test material solution were mixed (total volume 250 mL). Thereafter, 250 mL activated sludge was added.
- Aeration: Yes, with clean, oil-free air. The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60 to 70 % saturation (60 % of air saturation is > 5 mg/L at 20 °C) and to maintain the sludge flocs in suspension.
- No. of vessels per concentration (replicates): Triplicate vessels
- No. of vessels per control (replicates): 6 vessels
- No. of vessels per abiotic control (replicates): 1 vessel
- Sludge concentration: Suspended solids 3.0 g/L
- Nutrients provided for bacteria: Synthetic medium (sewage feed) consisted of 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.4 g CaCl2.2H2O, 0.2 g MgSO4.7H2O and 2.8 g K2HPO4 dissolved in Milli-RO water, made up to 1 litre and filtered. The pH was within 7.5 ± 0.5.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water; tap water purified by reverse osmosis (Millipore Corp., Bedford, Mass., USA).
- Composition of medium: Adjusted ISO-medium, formulated using RO-water (tap water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition: 211.5 mg/L CaCl2.2H2O, 88.8 mg/L MgSO4.7H2O, 46.7 mg/L NaHCO3 and 4.2 mg/L KCl.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: Not specified
- Details on termination of incubation: After the 3-hour contact time, the oxygen consumption was recorded. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After the 3-hour contact time, the oxygen consumption was recorded for a period of approximately 10 minutes. Determination of oxygen was performed with multiple oxygen probes connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.
The pH was determined in the remaining part of the reaction mixture after oxygen consumption measurements. The medium temperature was recorded continuously in temperature control vessels.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: one, limit test
- Range finding study: Yes. The study was carried out as a combined limit/range-finding test. The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate.
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: Not necessary; the highest concentration was suitable to use as a limit value.

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:

other: NOELR

Effect conc.:

1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

In the combined limit/range-finding test no statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg per litre. Therefore, the ELR50 was above the highest loading rate tested (1000 mg/L). There was no significant oxygen uptake from abiotic processes.

Results with reference substance (positive control):

The EC50 for 3,5-dichlorophenol was determined to be 5.6 mg/L (95 % CL 4.2 to 7.1 mg/L).

Reported statistics and error estimates:

Evaluation was based on the inhibition of the total respiration.
The calculation of the EC50 value of the reference material was based on a 3- parameter logistic cumulative distribution function (CDF) using non-linear regression analysis with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference material. For the test material no ELR50-value could be calculated because the test material proved to be non-toxic (ELR50 > 1000 mg/L).
An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the blank control revealed significant inhibition of the respiration rate (Two-sample t-test Procedure, α = 0.05, one-sided, smaller).
No inhibition of the respiration rate was observed at any of the tested loading rates (for 1000 mg/L the mean value was considered). Therefore, the NOELR was considered to be the highest test concentration.
The calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this study, the test material was not toxic to waste water (activated sludge) bacteria at a loading rate of 1000 mg/L (NOELR). The ELR50 was above 1000 mg/L.

Executive summary:

The influence of the test material on the respiration rate of activated sludge was investigated after a contact time of 3 hours in accordance with the standardised guidelines OECD 209, EU Method C.11 and ISO Standard 8192 under GLP conditions.

In a combined limit/range-finding test, loading rates of 10, 100 and 1000 mg/L were tested. The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate. Furthermore, a blank control (6 replicates) and an abiotic control at 1000 mg/L (1 replicate) were tested. Responses were compared to the controls.

The test material was not sufficiently soluble to allow preparation of a 10 g/L stock solution in water. Therefore, test material-Milli-RO water mixtures were magnetically stirred for a period of approximately 24 hours. Subsequently, synthetic medium, sludge and Milli-RO water were added resulting in the required loading rates. Optimal contact between the test material and test organisms was ensured by applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

No statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg per litre.

The batch of activated sludge was tested for sensitivity with the reference material 3,5- dichlorophenol, and showed normal sensitivity.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Under the conditions of this study, the test material was not toxic to waste water (activated sludge) bacteria at a loading rate of 1000 mg/L (NOELR). The ELR50 was above 1000 mg/L.

Description of key information

Under the conditions of this study, the test material was not toxic to waste water (activated sludge) bacteria at a loading rate of 1000 mg/L (NOELR). The ELR50 was above 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

Additional information

The influence of the test material on the respiration rate of activated sludge was investigated after a contact time of 3 hours in accordance with the standardised guidelines OECD 209, EU Method C.11 and ISO Standard 8192 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 a combined limit/range-finding test, loading rates of 10, 100 and 1000 mg/L were tested. The highest loading rate was tested in triplicate, lower loading rates consisted of one replicate. Furthermore, a blank control (6 replicates) and an abiotic control at 1000 mg/L (1 replicate) were tested. Responses were compared to the controls.

The test material was not sufficiently soluble to allow preparation of a 10 g/L stock solution in water. Therefore, test material-Milli-RO water mixtures were magnetically stirred for a period of approximately 24 hours. Subsequently, synthetic medium, sludge and Milli-RO water were added resulting in the required loading rates. Optimal contact between the test material and test organisms was ensured by applying continuous aeration and stirring during the 3-hour exposure period. Thereafter, oxygen consumption was recorded for approximately 10 minutes.

No statistically significant inhibition of the respiration rate of the sludge was recorded at a loading rate of 1000 mg per litre.

The batch of activated sludge was tested for sensitivity with the reference material 3,5- dichlorophenol, and showed normal sensitivity.

The study met the acceptability criteria prescribed by the study plan and was considered valid.

Under the conditions of this study, the test material was not toxic to waste water (activated sludge) bacteria at a loading rate of 1000 mg/L (NOELR). The ELR50 was above 1000 mg/L.