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Diss Factsheets

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 Feb 2018 to 16 Feb 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))
Deviations:
no
Principles of method if other than guideline:
Only minor SOP deviations that did not impact the quality or integrity of the study occurred during the course of the study.
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
4-(4-bromo-3-formylphenoxy)benzonitrile
EC Number:
822-547-1
Cas Number:
906673-54-9
Molecular formula:
C14H8BrNO2
IUPAC Name:
4-(4-bromo-3-formylphenoxy)benzonitrile
Test material form:
solid: particulate/powder
Details on test material:
Off-white powder
Specific details on test material used for the study:
Purity/Composition: 98.7% Test item storage: At room temperature protected from light Stable under storage conditions until: 20 November 2019 (retest date) (taken from label)

Sampling and analysis

Analytical monitoring:
not specified
Details on sampling:
For each batch (lot) of test item, a reserve sample (about 0.5 gram) was collected and maintained under the appropriate storage conditions by the Test Facility and destroyed after the expiration date.

Test item storage: At room temperature protected from light

Test solutions

Vehicle:
yes
Details on test solutions:
The batch of PF-06932437 tested was an off-white powder with a purity of 98.7%. A correction was made for the purity of the test item. A correction factor of 1.013 was used. All further concentrations reported are based on the pure test item. Preparation was as much as possible performed under low light conditions. 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 dark brown 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 and 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 and Table 2). Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring. Any residual volumes were discarded.

Reference Item – Preparation and Test Concentrations
The batch of activated sludge was checked for sensitivity by testing the reference item 3,5-dichlorophenol. A 1.0 g/L stock solution was prepared by dissolving 250.2 mg 3,5-dichlorophenol in Milli - RO water and making up to a total volume of 250 mL. The pH as used for the test was 8.0 in both the combined limit/range-finding and final test. The 3,5-dichlorophenol stock solution was stored in a freezer (≤-15℃) until use. The reference item solution was defrosted at room temperature and diluted to reach the test concentrations. Volumes of 0.5, 1.6, 5.0 and 16 mL from the stock solution were added to the test medium resulting in the final test concentrations of 1.0, 3.2, 10 and 32 mg/L, respectively.

Test organisms

Test organisms (species):
activated sludge, domestic
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. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test). 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.
Medium: Adjusted ISO-medium, formulated using RO water (tap water purified by reverse osmosis; GEON Waterbehandeling, BerkelEnschot, The Netherlands) 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:
other: continuous aeration and stirring
Water media type:
saltwater
Limit test:
yes
Total exposure duration:
3 h

Test conditions

Test temperature:
ranged between 18 and 22°C during the test
pH:
The pH in all test vessels, before addition of sludge was between 7.4 and 7.7. After the 3 hour exposure period the pH was between 7.7 and 8.4.
Dissolved oxygen:
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.
Nominal and measured concentrations:
Five concentrations were tested, ranging from 46 to 1000 mg/L and increasing with a factor 2.2.

Final Test – Test Concentrations: 46, 100, 220, 460 and 1000 mg/L.
Details on test conditions:
Test System
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. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test). 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
Medium: Adjusted ISO-medium, formulated using RO water (tap water purified by reverse osmosis; GEON Waterbehandeling, BerkelEnschot, The Netherlands) 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.
Combined Limit/Range-Finding Test: 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. In addition, a control (6 replicates) and an abiotic control (1 replicate) were tested. Test procedure and conditions were similar to those applied in the final test with the following exceptions: The pH of the sludge was 7.5 on the day of testing.
Final Test – Test Concentrations
PF-06932437: Five concentrations: 46, 100, 220, 460 and 1000 mg/L.
Replicates: 5 replicates per test group and 6 replicates for the control.
Control: Test medium without test item, treated in the same way as the test item solutions
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 16 g peptone (=sewage feed) 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 paragraph 4.7.2. Performance of the test 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 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-dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
ca. 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth inhibition
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
Reported statistics and error estimates:
ECx
For the reference item, calculation of the EC50 value 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 item.

NOEC determination
An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the control revealed significant inhibition of the respiration rate (Williams Multiple Sequential t-test Procedure, α=0.05, one-sided, smaller). Calculations were performed with ToxRat Professional v. 3.2.1 (ToxRat Solutions® GmbH, Germany).

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In conclusion, under the conditions of this present test PF-06932437 was not toxic to waste water bacteria (activated sludge) at or below a concentration of 100 mg/L (NOEC). The EC50 was above 1000 mg/L.
Executive summary:

The objective of the study was to evaluate PF-06932437 for its ability to adversely affect aerobic microbial treatment plants and, if possible, to determine the EC50 and/or the no - observed effect concentration (NOEC). The influence of PF-06932437 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-06932437 tested was an off-white powder with a purity of 98.7%.  A correction was made for the purity of the test item. A correction factor of 1.013 was used. All further concentrations reported are based on the pure test item. Preparation was as much as possible performed under low light conditions. 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 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. The final test was performed based on the result of a preceding combined limit/range-finding test.  Five concentrations were tested, ranging from 46 to 1000 mg/L and increasing with a factor 2.2.  Five replicates per concentration and six replicates for an untreated control group were tested. No statistically significant inhibition of the respiration rate of the sludge was recorded at 100 mg PF-06932437 per liter.  At higher concentrations the inhibitory effect of PF-06932437 on aerobic waste water (activated sludge) bacteria increased with increasing concentration, ranging from 9 % inhibition at 220 mg/L to 13% at 1000 mg/L. 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. PF-06932437 was not toxic to waste water (activated sludge) bacteria at a concentration of 100 mg/L (NOEC). The effect parameters for the inhibition of the respiration rate are presented below.

Parameter

PF-06932437 Concentration (mg/L)

NOEC 100

EC50 >1000