1-[([[(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl]oxy]carbonyl)oxy]pyrrolidine-2,5-dione

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:

19-12-2018 to 21-12-2019

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

Qualifier:

according to guideline

Guideline:

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

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I18IB2522
- Purity: 100.1%
- Purity test date: 19 March 2019 (retest date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8°C)
- Solubility and stability of the test substance in the solvent/vehicle: Not stable in water

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)

The test item was not sufficiently soluble to allow preparation of an aqueous solution at a concentration of 10 g/L. Therefore,weighed amounts were added to the ambertestbottles containing 200 mL Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). The test item –Milli-RO water mixtures were magnetically stirred for a short period. Subsequently, 16mL synthetic medium,made up to 50 mL with Milli-RO water,and 250 mL sludge were added,resulting in the required concentrations. Optimal contact between the test item 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 sewage treatment plant where inoculum was collected: Municipal sewage treatment plant: '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. The concentration of the suspended solids was determined (3 g/L, as used for the test).The pH was 7.2on the dayof 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.

Medium: Adjusted ISO-medium, formulated using RO-water (tapwater purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands) with the following composition:
CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO 346.7 mg/L
KCl 4.2 mg/L

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Hardness:

not measured

Test temperature:

20-23°C

pH:

5.7 - 7.8

Dissolved oxygen:

not measured

Salinity:

not applicable

Conductivity:

not applicable

Nominal and measured concentrations:

Nominal concentrations: 10, 100 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: All glass open bottles/vessels.
- Type (delete if not applicable): open
- Material, size, headspace, fill volume:

- Aeration: The aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/Lat 20°C) and to maintain the sludge flocs in suspension. Air supply: Clean, oil-free air.

- No. of vessels per concentration (replicates): 1000 mg/L: 3 replicates, 100 mg/L: 1 replicate, 10 mg/L: 1 replicate
- No. of vessels per control (replicates): 6 replicates
- No. of vessels per abiotic control (replicates): 1 replicate

- Sludge concentration (weight of dry solids per volume): 3 g/L of suspended solids

- Weight of dry solids per volume of reaction mixture per unit of time:
- Nutrients provided for bacteria: 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.
Sewage feed:
16 g peptone
11 g meat extract
3 g urea
0.7 g NaC
l0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO / Milli-RO water- Tapwater purified by reverse osmosis (Millipore Corp., Bedford, Mass., USA).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Respiration rate, defined as the oxygen consumption of aerobic activated sludge or waste water micro-organisms expressed generally as mg O2per litre per hour.

The synthetic medium (16 mL) made up to 50 mL with Milli-RO and 200 mL test item solution were mixed (total volume 250 mL). The pH was determined. Thereafter,250 ml activated sludge was added. This was the start of the test. After the 3 hour contact time the oxygen consumptionwas recorded for a period of approximately 10 minutes. During measurement, the sample was not aerated but continuously stirred on a magnetic stirrer.The pH was determined in the remaining part of the reaction mixture. This procedure was repeated for alltest/reference item concentrations and controls.The medium temperature was recorded continuously in temperature control vessels. The temperature control vessels wereidentically prepared compared to the control vessels. A temperature control vessel with a REES sensor was placed in each fume cupboard of the climate room.

Determination of oxygen was performed with multiple oxygen probes connected to a BlueBox (GO-Systemelektronik GmbH, Germany), a multichannel measuring and controlling system.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Test concentrations: 10, 100 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

Details on results:

- Blank controls oxygen uptake rate: There was no significant oxygen uptake from abiotic processes.

The combined limit/range-finding test showed no inhibition, 14%, and an average of 41% inhibition of the respiration rate at 10, 100 and 1000 mg/L, respectively. The EC50was above the highest concentration tested (1000 mg/L).

The second replicate of 1000 mg/L could be considered as an outlier. The value was not removed from the statistical calculation. The outcome of the effect parameters were not different. The average ofthe two other replicates was 28% and therefore significant different from the blank control. Consequently, a NOEC could not be determined

The temperature was outside the range prescribed by the study plan (20 ± 2°C) during the exposure phase, the temperature was 20-23°C. The validity criteria for the controls and reference item were met and therefore this deviation is considered not to have influenced the sludge.The study integrity was not adversely affected by the deviations.

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: The EC50of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/Lfor total respiration(5.9mg/L)

Reported statistics and error estimates:

ECx
For the reference item, calculation of the EC50 value was based on a 3-parameter logisticcumulative distribution function (CDF) using non-linear regression analysis, with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference item. For T002632, no EC50 value could be calculated because effects were below 50% (EC50> 1000mg/L)

NOEC determination
In the present test T002632 was toxic to waste water (activated sludge) bacteria at 1000 mg/L(Two-sample t-test Procedure, α=0.05, one-sided, smaller). Therefore, a NOEC could not be determined.

The calculations were performed with ToxRat Professional v. 3.2.1.(ToxRat Solutions®GmbH, Germany).

Validity criteria fulfilled:

yes

Conclusions:

A 3-h microorganisms respiration inhibitation study was performed with the test substance T002632 according to the OECD guideline 209. The determined EC50 was above 1000 mg/L. The results of the test can be considered reliable without restrictions.

Description of key information

A 3-h microorganisms respiration inhibitation study was performed with the test substance T002632 according to the OECD guideline 209. The determined EC50 was above 1000 mg/L. The results of the test can be considered reliable without restrictions.