2-ethylhexyl 2-([1,1'-biphenyl]-4-ylcarbonyl)benzoate

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:

20 November 2019- 27 April 2020

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:

22 July 2010

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Identification: C991
Appearance: Colourless to pale yellow liquid
Expiry date: 14 June 2020

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The batch of C991 tested was a colourless to pale yellow liquid with a purity of 96.27%. 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, weighed amounts were added to the amber coloured 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. Optimal contact between the test item and test organisms was ensured by applying continuous aeration and stirring.

Combined limit/Range-Finding Test - Weighed Amounts of C991

Treatment Concentration (mg/L) Weighed amount (mg) of C991
Replicate A Replicate B Replicate C
T1 10 5.19 - -
T2 100 49.57 - -
T3 1000 500.71 499.25 500.08
TA (abiotic) 1000 500.8 - -
- = Not applicable

Final Test - Weighed Amounts of C991

Treatment Concentration (mg/L) Weighed amount (mg) of C991
Replicate A Replicate Replicate C Replicate D Replicate E
T1 10 5.04 5.07 5.08 5.08 4.9
T2 32 15.9 16.07 16.17 16.05 15.97
T3 100 50.09 50.19 50.07 50.11 50.01
T4 320 160.26 160.24 160.4 160.04 160.65
T5 1000 500.9 500.04 501.04 499.27 501.05

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, as used for the test). The pH was 7.3 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, Berkel-Enschot, 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.

Test type:

static

Water media type:

freshwater

Total exposure duration:

3 h

Test temperature:

Temperature control vessels ranged between 19 and 22°C during the test and complied with the requirements as laid down in the Study Plan (20 ± 2°C).

pH:

The pH was within 7.5 ± 0.5

Nominal and measured concentrations:

Five concentrations: 10, 32, 100, 320 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.

Details on test 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.

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 vessel. The temperature control vessel was 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

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/L

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Combined Limit/Range-Finding Test
The combined limit/range-finding showed 23% inhibition, no inhibition and an average of 25% inhibition of the respiration rate at a concentration of 10, 100 and 1000 mg/L, respectively. The inhibition of the respiration rate at 1000 mg/L was statistically significant. Therefore, the NOEC could not be determined.
The EC50 was above the highest concentration tested (1000 mg/L). There was no significant oxygen release from abiotic processes.
All test conditions and acceptability criteria prescribed by the Study Plan were met.

Final Test
The effects observed were smaller than what was expected based on the results of the combined limit/range-finding test. The results of this test allowed for reliable determination of a NOEC and EC50 value. No inhibition of the respiration rate was observed at any of the tested concentrations. Therefore, the NOEC was considered to be the highest concentration tested. The EC50 was above the highest concentration tested (1000 mg/L).

Calculations
Blank and reference item concentrations were shared with parallel studies. All information pertaining to shared vessels is archived in the raw data. All results presented in the tables of the report are calculated using values as per the raw data rounding procedures and may not be exactly reproduced from the individual data presented.

Calculation of oxygen uptakes
The respiration rate (R) from each vessel, in mg O2/L.h was calculated or interpolated from the linear part of the respiration curve, which was generally between 2 and 7 mg O2/L.
R was calculated by the BlueBox software as (V1 – V2)/Δt * 60
Where:
V1=Value 1: the oxygen concentration at the start of the selected section of the linear phase (mg O2/L),
V2=Value 2: the oxygen concentration at the end of the selected section of the linear phase (mg O2/L),
Δt is the time interval between these two measurements.
Negative R values were expressed as 0 mg O2/L.h (V1
Furthermore, the respiration rate was expressed as the amount of oxygen consumed per g dry weight of sludge per hour (Rs in mg O2/g.h).
Rs = R / SS
Where SS is the concentration of suspended solids in the test mixture (g/L).

Calculation of the inhibition of the respiration rate
The percentage inhibition, IT, of total oxygen consumption is given below:
IT = [1- (RT/RC)] x 100%
Where:
RT is the respiration rate of the test item (T) (mg O2/L.h or mg O2/g.h).
RC is the respiration rate of the control (C) (mg O2/L.h or mg O2/g.h).
In case abiotic uptake was observed (>5% of control respiration) a correction for this oxygen uptake was made.

Final Test - Overivew of Results
Treatment	Concentration (mg/L)	Mean respiration rate		% inhibition of the respiration rate (mean value)
		(mg O2/L h)	(mgO2/g h)1
Control		58.91	39.27
T1	10	62.78	41.85	-6.57
T2	32	61.58	41.05	-4.53
T3	100	65.4	43.06	-11.02
T4	320	64.12	42.75	-8.86
T5	1000	61.7	41.13	-4.74
1) The amount of suspended solids in the final test mixture was 1.5g/L

Interpretation of results

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.

For C991, no EC50 value could be calculated because the test item proved to be non-toxic (EC50 > 1000 mg/L)

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).

COMPUTERIZED SYSTEMS

Critical computerized systems used in the study are listed below. All computerized systems used in the conduct of this study have been validated; when a particular system has not satisfied all requirements, appropriate administrative and procedural controls were implemented to assure the quality and integrity of data.

Critical Computerised Systems
System name	Version No.	Description of data collected and/or analysed
Deviation Information Library	2.1	Deviations
Share Document Management System	1	Reporting
DocuSign	19	Collection of part 11 compliant signature
REES Centron	SQL 2.0	Temperature, relative humidty, light cycle and/or atmospheric pressure monitoring
BlueBox Software	3.4.2.0	System control, data acquisition and processing

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present test C991 was not toxic to waste water bacteria (activated sludge) at or below a concentration of 1000 mg/L (NOEC).
The EC50 was >1000 mg/L.

Executive summary:

The objective of the study was to evaluate C991 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 C991 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 C991 tested was a colourless to pale yellow liquidwith a purity of 96.27%.  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, 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. 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 and in total two full tests were performed due to the different effect of the test item on the pH of the test solution. The final study was used to determine the NOEC and EC50 value.. In this final test, five concentrations were tested, ranging from 10- 1000 mg/L and increasing with a factor ≤ 3.2. Five replicates per concentration and six replicates of an untreated control group were tested.

 

No inhibition of the respiration rate was observed at any of the tested concentrations. Therefore, the NOEC was considered to be the highest concentration tested.

  

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 the present test C991 was not toxic to waste water bacteria (activated sludge) at or below a concentration of 1000 mg/L (NOEC).

Description of key information

Toxicity to microorganisms: Key study CRL 2020

OECD 209

Under the conditions of the present test C991 was not toxic to waste water bacteria (activated sludge) at or below a concentration of 1000 mg/L (NOEC).

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

Study conducted to recognised testing guidelines with GLP certification.