Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline

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 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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Appearance: Light orange liquid
Purity/Composition: UVCB
Storage Conditions: At room temperature protected from light
Substance Name: Reaction products of 2-ethyl-2-[[(1-oxoallyl)oxy]methyl]-1,3-propanediyl diacrylate and N-methylaniline
Chemical name (IUPAC): 2,2-bis[({3-[methyl(phenyl)amino]propanoyl}oxy)methyl]butyl 3-[methyl(phenyl)amino]propanoate

Vehicle:

no

Details on test solutions:

Test Item – Preparation and Test Concentrations
The batch of C894 tested was a light orange liquid (UVCB). No correction was made for the purity/composition of the test item. Preparation was as much as possible performed under yellow light and/or 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 amber test bottles containing 200 mL Milli-RO water (tap water purified by reverse osmosis; Millipore Corp., Bedford, Mass., USA). To this end, small watch glasses were used to transfer weighed amounts of test item to respective test bottles. 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. Any residual volumes were discarded.

Combined limit/Range -finding test - weighed amounts of C894

Treatment Concentration (mg/L) Weighted amount (mg) of C894
Replicate A Replicate B Replicate C
T1 10 5.12 - -
T2 100 50.37 - -
T3 1000 499.45 500.36 500.01
TA (abiotic) 1000 500.3 - -
-=: Not applicable


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 g/L stock solution was prepared by dissolving 500.8 mg 3,5-dichlorophenol in Milli-RO water and making up to a total volume of 500 mL. The pH as used for the test was 7.9. 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 (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 7.8 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

Limit test:

yes

Total exposure duration:

3 h

Hardness:

Not reported

Test temperature:

The temperature continuously measured in the temperature control vessel ranged between 19.7 and 19.9°C during the test and complied with the requirements as laid down in the Study Plan (20 ± 2°C)

pH:

The pH in all test vessels, before addition of sludge was between 7.7 and 7.8. After the 3-hour exposure period the pH was between 7.6 and 8.4.

Dissolved oxygen:

Not reported

Salinity:

0.7g NaCl in synthetic medium

Conductivity:

Not reported - freshwater study

Nominal and measured concentrations:

Test concentration(s): 10, 100 and 1000 mg/L
Control(s): Blank control
Number of replicates: 3 replicates at 1000 mg/L; 1 replicate at 10 and 100 mg/L; 6 replicates of an untreated control group

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: 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 a 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

Nominal / measured:

meas. (initial)

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

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Details on results:

Inhibition of the Respiration Rate

The combined limit/range-finding test showed 17%, 8% and an average of 23% inhibition of the respiration rate at 10, 100 and 1000 mg/L, respectively. The observed inhibition of the respiration rate at a concentration of 1000 mg/L was not statistically significant. Therefore, the NOEC was considered to be the highest test concentration. The EC50 was above 1000 mg/L. There was no significant oxygen release from abiotic processes.

Detailed study results are presented in Table 4 of Appendix 1.

Results with reference substance (positive control):

Results with the reference substance were valid. Full results are presented in Table 4 of Appendix 1.

Reported statistics and error estimates:

Statistics are reported in Appendix 2, appended to attached background material

COMPUTERIZED SYSTEMS

Critical computerized systems used in the study are listed below. All computerized systemsused 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.

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 humidity, 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 this present test, the test item 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 the test item 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 the test item 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 the test tiem tested was a light orange liquid (UVCB).  No correction was made for the purity/composition of the test item. Preparation was as much as possible performed under yellow light and/or 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. 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. In addition, a control (6 replicates) and an abiotic control (1 replicate) were tested.

 

The combined limit/range-finding test showed 17%, 8% and an average of 23% inhibition of the respiration rate at 10, 100 and 1000 mg/L, respectively. The observed inhibition of the respiration rate at a concentration of 1000 mg/L was not statistically significant. Therefore, the NOEC was considered to be the highest test concentration

 

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

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

EC50 for microorganisms:

1 000 mg/L

EC10 or NOEC for microorganisms:

1 000 mg/L