3-chloro-2-[3-(chloroacetyl)-4,5-dihydro-1,2-oxazol-5-yl]phenyl methanesulfonate

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:

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:

adopted: 22 July 2010 and considered the Question-and-Answer Document by the German Federal Environment Agency (Version 2012-03-02).

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Pre-treatment of test item: Direct weighings were prepared to give the different test item concentrations. The test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water and was stirred before testing (equilibration phase) overnight for 17 hours. The pH was measured to be pH 5.8-6.3. The pH was adjusted to pH 7.2-7.4 with NaOH.
Pre-treatment of test item with ATU: Direct weighings were prepared to give the different test item concentrations. The test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water and was stirred before testing (equilibration phase) overnight for 17 hours. The pH was measured to be pH 6.0-6.4 The pH was adjusted to pH 7.2-7.3 with NaOH.
For the ATU solution 2.32 g/L N-allylthiourea were weighed out and diluted with deionized water to 1 litre. 1.25 mL of the solution was given to all replicates for the determination of the heterotrophic oxidation before start of the incubation period.
- Controls: Physico-chemical oxygen consumption control: 100 mg/L test item.
- As nearly the same inhibition was observed for the total oxygen consumption and the heterotrophic oxygen consumption, no nitrification was calculated and the main test was carried out only with the test set without ATU.

Test organisms (species):

activated sludge, domestic

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: aeration tank of a domestic waste water treatment plant (Municipal WWTP Cologne-Stammheim)
- Method of cultivation: aeration of the activated sludge at 20 ± 2 °C, daily fed with synthetic medium
- Preparation of inoculum for exposure: The sludge was settled and the supernatant was decanted. After centrifuging the sludge (15 min at 3500 rpm and 20°C) the supernatant was decanted again. Approximately 1 g of the wet sludge was dried in order to calculate the amount of wet sludge to achieve a concentration of activated sludge of 3 g/L (dry weight) suspended solids. The calculated amount of sludge was dissolved in synthetic medium and then filled up to a defined end volume with deionised water.
- Initial biomass concentration: 800 mg/L suspended solids
- pH of the suspension before application: 7.7 (range finding test), 7.7 (main test)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

no data

Test temperature:

Test without ATU: 19.6 °C
Test with ATU: 19.7 °C

pH:

Test without ATU: 8.4
Test with ATU: 8.4

Dissolved oxygen:

no data

Salinity:

no data

Conductivity:

no data

Nominal and measured concentrations:

- 10, 32, 100, 320 and 1000 mg/L (without ATU), 100 mg/L with ATU
- Test concentrations are given as nominal concentrations as they were not confirmed by analytical methods.

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL glass Erlenmeyer flasks
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume:
- Aeration: permanent aeration
- No. of vessels per concentration (replicates): 3 replicates, 2 replicates with ATU
- No. of vessels per control (replicates): 6 replicates, 4 replicates with ATU
- Sludge concentration (weight of dry solids per volume): 800 mg/L suspended solids
- Nitrification inhibitor used (delete if not applicable): N-allylthiourea (ATU)

OTHER TEST CONDITIONS
-To determine the heterotrophic oxidation a second set of control vessels, test item vessels, reference substance vessels and vessels to determine the physic-chemical oxygen consumption was prepared. Before they were filled up with deonised water to 250 mL end volume, to each vessel 1.25 mL of ATU-solution (N-allylthiourea), which equals to a final concentration of 11.6 mg ATU/L was added. The mixture was filled up with deionised water to 250 mL and aerated at 20 ± 2 °C.
- Since some substances may consume oxygen by chemical reactivity, a physico-chemical oxygen consumption control was carried out additionally for both sets. In order to be able to differentiate between physico-chemical oxygen consumption and biological oxygen consumption (respiration), at least the maximum concentration of the test item was tested without activated sludge.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Oxygen consumption, temperature and ph were measured after an aeration time of 3 hours

TEST CONCENTRATIONS
- Range finding study: Direct weighing was prepared to give test item concentrations of 100 mg/L. The test item was added into Erlenmeyer flasks (incubation vessels) to about 130 mL deionised water and was stirred before testing (equilibration phase) overnight for 17 hours. The pH was measured to be pH 6.2-6.5. The pH was adjusted to pH 7.2-7.3 with NaOH.
For the ATU-solution 2.32 g N-allylthiourea were weighed out and diluted with deionized water to 1 litre. 1.25 mL of the solution were given to all replicates for the determination of the heterotrophic oxidation immediately before start of the incubation period
- Test concentrations: 100 mg/L, 3 replicates; 100 mg/L with ATU, 2 replicates
-Control: 6 replicates, 4 replicates with ATU
- Results used to determine the conditions for the definitive study: respiration rate 15.734 mg/L, 42.433 % inhibition (Test with ATU: respiration rate 15.644 mg/L, 44.281 % inhibition)

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol , concentration (2.5, 5, 10, 20, 40 mg/L)

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

0.052 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 899 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

< 0.052 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

- As the NOEC was below the lowest tested concentration, the EC10 value was used as a surrogate.
- 3-chloro-2-[3-(chloroacetyl)-4,5-dihydro-1,2-oxazol-5-yl]phenylmethanesulfonate showed 48.253 % respiration inhibition of activated sludge at the highest test item concentration of 1000 mg/L.
- The oxygen consumption in the presence of N-allylthiourea was determined in four controls without test item and in two replicates of the test item concentration 100 mg/L.As nearly the same inhibition was observed for the total oxygen consumption and the heterotrophic oxygen consumption, no nitrification was calculated and the main test was carried out only with the test set without ATU.

The respiration rate for each concentration was determined from the linear part of the curve of the oxygen content versus time. The inhibitory effect of the test item at a particular concentration is expressed as a percentage of the mean of the respiration rates of the six controls.

ECx values for the test item and the reference substance were calculated from the respiration rates at different test item concentrations using the statistics programme ToxRatPro Version 2.10 (released 2010-09-10).

Validity criteria fulfilled:

yes

Remarks:

(oxygen uptake rate blank controls differ not less than 20 mg oxygen -coefficient of variation of oxygen uptake in control not more than 30 % at end of test - EC50 reference compound in the range of 2–25 mg/L total respiration,5-40 mg/L heterotrophic)

Conclusions:

3-chloro-2-[3-(chloroacetyl)-4,5-dihydro-1,2-oxazol-5-yl]phenylmethanesulfonate showed an EC50 = 899 mg/L and a NOEC of < 10 mg/L (0.052 mg/L).. As the NOEC was below the lowest tested concentration, the EC10 value was used as a surrogate (EC10 = 0.052mg/L).

Executive summary:

The study was conducted in accordance with OECD Guideline 209 and considered the Question-and-Answer Document by the German Federal Environment Agency (Version 2012-03-02).. The respiration rate is classified into two processes of oxidation. The oxidation of organic carbon and the ammonium oxidation (nitrification). The use of the specific nitrification inhibitor, ATU (N-allylthiourea), enables the direct assessment of the inhibitory effects of test substances on heterotrophic oxidation, and by subtracting the oxygen uptake rate in the presence of ATU from the total uptake rate, the effects on the rate of nitrification may be calculated.

The activated sludge was exposed to 3-chloro-2-[3-(chloroacetyl)-4,5-dihydro-1,2-oxazol-5-yl]phenylmethanesulfonate at different concentrations ( 10, 32, 100, 320 and 1000 mg/L without ATU, 100 mg/L with ATU).

Before use the wet weight/dry weight relationship of the activated sludge was determined by drying 10 mL of sludge suspension. Subsequently, a sludge suspension of 2 g (dry weight)/L was prepared. The pH of this suspension was measured and adjusted to 6-8. 8 mL of the synthetic medium and 100 mL of activated sludge were added to the dissolved test item. The mixture was filled up with deionised water to 250 mL and aerated at 20 ± 2 °C. The exposure medium with the reference substance was prepared by adding 8 mL of the synthetic medium, 100 mL of activated sludge and a defined amount of the stock solution to achieve the test concentrations, and was filled up with deionised water to 250 mL and aerated at 20 ± 2°C. Control vessels (inoculated sample without test item) were prepared the same way. Additional vessels to determine the physico-chemical oxygen consumption were prepared containing the test item, and the synthetic medium but no activated sludge.

To determine the heterotrophic oxidation a second set of control vessels, test item vessels, reference substance vessels and vessels to determine the physic-chemical oxygen consumption was prepared. Before they were filled up with deonised water to 250 mL end volume, to each vessel 1.25 mL of ATU-solution (N-allylthiourea), which equals to a final concentration of 11.6 mg ATU/L was added. The mixture was filled up with deionised water to 250 mL and aerated at 20 ± 2 °C.

Oxygen consumption, temperature and pH were measured and recorded after an aeration time of 3 hours.

For the test substance an EC50 = 899 mg/L and a NOEC < 10 (0.052) mg/L were determined. As the NOEC was below the lowest tested concentration, the EC10 value was used as a surrogate (EC10 = 0.052mg/L).

The test substance showed 48.253 % respiration inhibition of activated sludge at the highest test item concentration of 1000 mg/L. The oxygen consumption in the presence of N-allylthiourea was determined in four controls without test item and in two replicates of the test item concentration 100 mg/L.As nearly the same inhibition was observed for the total oxygen consumption and the heterotrophic oxygen consumption, no nitrification was calculated and the main test was carried out only with the test set without ATU.

This toxicity study is classified as acceptable and satisfies the guideline requirements for the toxicity study to microoragnisms.

Description of key information

3-chloro-2-[3-(chloroacetyl)-4,5-dihydro-1,2-oxazol-5-yl]phenylmethanesulfonate showed an EC50 = 899 mg/L and a NOEC of < 10 mg/L (0.052 mg/L). As the NOEC was below the lowest tested concentration, the EC10 value was used as a surrogate (EC10 = 0.052mg/L).

Key value for chemical safety assessment

EC50 for microorganisms:

899 mg/L

EC10 or NOEC for microorganisms:

10 mg/L

Additional information

should read < 10.0