Propan-2-yl-oxocyclobutane carboxylate

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

20-12-2018 to 21-12-2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

propan-2-yl 3-oxocyclobutane-1-carboxylate

EC Number:

834-970-9

Cas Number:

130111-95-4

Molecular formula:

C8H12O3

IUPAC Name:

propan-2-yl 3-oxocyclobutane-1-carboxylate

Test material form:

liquid

Details on test material:

Batch (Lot) Number: GR13224
Expiry date: 31 January 2020 (expiry date)
Physical Description: Colourless to pale yellow liquid
Purity/Composition: 99.6%
Storage Conditions: At room temperature

Sampling and analysis

Analytical monitoring:

no

Test solutions

Vehicle:

yes

Remarks:

milli-RO water

Details on test solutions:

The batch of PF-06238566 tested was a colourless to pale yellow liquid with a purity of
99.6%. No correction was made for the purity/composition of the test item.
The test item was not sufficiently soluble to allow preparation of an aqueous solution at a
concentration of 10 g/L. Therefore, aliquots of test item corresponding with the final test
concentration were pipetted directly into the amber test bottles 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, 16 mL synthetic medium made up to 50 mL with Milli - RO water and 250 mL
sludge were added resulting in the required concentrations (see Table 1). Optimal contact
between the test item and test organisms was ensured by applying continuous aeration and
stirring. Any residual volumes were discarded.

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Test system: Micro-organisms in activated sludge.
Source: Municipal sewage treatment plant: 'Waterschap Aa en Maas',
's-Hertogenbosch, The Netherlands, receiving predominantly
domestic sewage.
Preparation of the sludge:
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 of sludge, as used for the test). The pH was 7.2 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.
Medium: Adjusted ISO-medium, formulated using RO-water (tap water
purified by reverse osmosis; GEON Waterbehandeling, BerkelEnschot,
TheNetherlands)with the following composition:

CaCl2.2H2O 211.5 mg/L
MgSO4.7H2O 88.8 mg/L
NaHCO3 46.7 mg/L
KCl 4.2 mg/L

Rationale: Recognized by international guidelines as the recommended
test system.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Test conditions

pH:

The pH was within 7.5 ± 0.5.

Details on test conditions:

Test Procedure and Conditions
Contact time: 3 hours, during which aeration and stirring took place.
Vessels: All glass open bottles/vessels.
Milli-RO / Milli-RO water: Tap water purified by reverse osmosis (Millipore Corp., Bedford, Mass., USA).
Synthetic medium (=sewage feed):
16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
Dissolved in Milli-RO water, made up to 1 litre and filtered.
The pH was within 7.5 ± 0.5.
Air supply Clean, oil-free air.
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/L at 20°C) and to maintain the
sludge flocs in suspension.
Test set up See Table 1

Performance of the test:
The synthetic medium (= sewage feed, 16 mL) and an
appropriate amount of the test item stock were mixed and made
up to 250 mL with Milli-RO water. 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 consumption was
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 all test/reference item
concentrations and controls.
The medium temperature was recorded continuously in
temperature control vessels. The temperature control vessels
were identically prepared compared to the control vessels. A
temperature control vessel with a REES sensor was placed in
each fume cupboard of the climate room.

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

Reference substance (positive control):

yes

Remarks:

3,5-Dinitrophenol

Results and discussion

Effect concentrations

Effect concentrations 1

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

Effect concentrations 2

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:

The mean control oxygen uptake rate exceeded 20 mg oxygen per gram of activated
sludge (dry weight of suspended solids) per hour (21 mg oxygen per gram of activated
sludge).
The coefficient of variation of oxygen uptake in control replicates did not exceed 30%
at the end of the test (15%).

Results with reference substance (positive control):

The EC50 of 3,5-dichlorophenol was in the accepted range of 2 to 25 mg/L for total
respiration (5.9 mg/L)

Any other information on results incl. tables

Experimental Conditions

The pH in all test vessels, before addition of sludge was 7.1. After the 3 hour exposure period

the pH was between 7.0 and 7.9.

The temperature continuously measured in the temperature control vessels ranged between

20.0 and 23.3°C during the test, and was outside the range prescribed by the study plan (20 ±

2°C).

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of this present test PF-06238566 was not toxic to waste water bacteria
(activated sludge) at 1000 mg/L (NOEC).
The EC50 was above 1000 mg/L.

Executive summary:

The objective of the study was to evaluate  PF-06238566 for its ability to adversely affect

aerobic microbial treatment plants and, if possible, to determine the no - observed effect

concentration (NOEC).

The influence of  PF-06238566 on the respiration rate of activated sludge was investigated

after a contact time of 3 hours.

The study procedures described in this report were based on the OECD guideline No. 209,

2010.

The batch of  PF-06238566 tested was a colourless to pale yellow liquid with a purity of

99.6%. No correction was made for the purity/composition of the test item.

The test item was not sufficiently soluble to allow preparation of an aqueous solution at a

concentration of 10 g/L. Therefore, aliquots of test item corresponding with the final test

concentration were pipetted directly into the test-bottles containing Milli-RO  water. The test

item – Milli-RO  water mixtures were magnetically stirred for a short period and subsequently,

synthetic medium, sludge and Milli-RO  water were added resulting in the required

concentrations. Optimal contact between the test item and test medium was ensured by

applying continuous aeration and stirring during the 3 hour exposure period. Thereafter,

oxygen consumption was recorded for approximately 10 minutes.

In a combined limit/range-finding test concentrations of 10, 100 and 1000 mg/L were tested.

The highest concentration was tested in triplicate, lower concentrations consisted of one

replicate. Furthermore, at 1000 mg/L an abiotic control (1 replicate) was tested. Responses

were compared to the controls.

No statistically significant inhibition of the respiration rate of the sludge was recorded at a

concentration of 1000 mg  PF-06238566 per litre. Therefore, the EC50 was above the highest

concentration tested (1000 mg/L).

There was no oxygen uptake from abiotic processes.

The batch of activated sludge was tested for sensitivity with the reference item

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 present test  PF-06238566 was not toxic to waste water (activated

sludge) bacteria at a concentration of 1000 mg/L (NOEC).

The EC50 was above 1000 mg/L.