Reaction mass of decyl acrylate and octyl acrylate

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:

2016-11-10 to 2017-

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))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PRELIMINARY RANGE-FINDING TEST:
In the range-finding test, activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 10, 100 and 1000 mg/L. Two replicate vessels were prepared per treatment (including the controls). The test item was dispersed directly in water.

The reference item, 3,5-dichlorophenol, was included at concentrations of 3.2, 10 and 32 mg/L in order to confirm the suitability of the inoculum: a stock solution at 1 g/L was prepared by dissolving the reference item (100.3 mg) directly in previously heated water to aid dissolution, the volume was then made up to 100 mL with ultra-pure water. Aliquots of the stock solution were added to activated sewage sludge, synthetic sewage and water to achieve final concentrations of 3.2, 10 and 32 mg/L, each with a single replicate.

At the start of the test, 16 mL of synthetic sewage feed were made up to 250 mL with ultra-pure water and 250 mL activated sludge inoculum were added to the first control vessel (final suspended solid of nominally 1.5 g/L/dw). Thereafter, at time intervals of about fifteen minutes, the procedure was repeated with each of the test item and reference item test vessels, in which 16 mL of synthetic sewage were made up to 250 mL with the appropriate amount of reference or test item diluted in ultra-pure water. As for the control, 250 mL of activated sludge inoculum were then added. Fifteen minutes after the preparation of the last treatment, the second control vessel was prepared at last. The fifteen minutes interval was necessary so that the 10 min incubation period with O2 depletion monitoring could take place exactly 3 hours after start of incubation of each test vessel.

Upon addition of the microbial inoculum, the pH was measured in order to identify if there was any variation in pH caused by the addition of the test item. The pH was not adjusted.

DEFINITIVE TEST:
The definitive test was conducted using a range of concentrations deduced from the preliminary range-finding test: activated sewage sludge micro-organisms were exposed to a series of nominal test concentrations of 62.5, 125.0, 250.0, 500.0 and 1000.0 mg/L. In order to obtain both a NOEC and EC50, five treatment concentrations in a geometric series with 2 replicates were prepared. The test item was dispersed directly in ultra-pure water. The same inoculum as for the range-finding test was used.

The reference item, 3,5-dichlorophenol, was included at concentrations of 3.2, 10 and 32 mg/L in order to confirm again the suitability of the inoculum (test solutions were prepared with a single replicate from the same stock solution as the range-finding test).

The test protocol was identical to the one described for the range-finding test, except that more test item concentrations were tested.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

The inoculum was obtained on 14 November 2016 from the activated sludge of the biological wastewater treatment plant from Abidos (France, 64) which handles predominantly domestic sewage.

On arrival at the laboratory, the activated sludge (around 10L) was centrifuged (1000g for 10 minutes) and the solid sewage inoculum pellet was re-suspended in 10L deionised water. 500 mL synthetic sewage feed was then added. Sewage sludge was maintained under aerated conditions for a maximum of 4 days and fed daily with synthetic sewage feed.

In order to obtain the required sludge suspended solids concentration of 3 g/L in the stock solution to be used in the experiments, the solid content of the 10-L sludge solution was determined by removing a sub-sample of homogenised sludge and drying in an oven at approximately 105°C. The suspended solids concentration was subsequently adjusted with the source water until the recommended guideline value of 3 g/L ± 10% is achieved.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

Note: two temperature controlled rooms were necessary for this test which includes many devices. These room were controlled with similar conditions and temperature was recorded in both rooms.

The ambient temperature remained constant at 20°C within ± 2°C during the definitive test:
• Room 1: mean measured temperature: 21.5°C, min.: 20.8°C, max.: 21.9°C.
• Room 2: mean measured temperature: 21.7°C, min.: 21.6°C, max.: 21.7°C.

pH:

All measured pH were in the range 7 – 8.

Nominal and measured concentrations:

Nominal concentrations (mg/L): 0 (Control), 62.5, 125.0, 250.0, 500.0, 1000.0.

Details on test conditions:

The addition of the inoculum was considered as the start of the 3-hours incubation time period.

All solutions were incubated at 20 +/- 2°C in darkness for 3 hours in a temperature controlled room. Solutions were mixed by stirring and aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of 0.5 to 1.0 liter per minute during 3 hours. At the end of the incubation period, a well-mixed sample of each solution (following their order of preparation) were poured into a BOD bottle in which dissolved oxygen concentrations were measured at 1 min intervals for up to a maximum period of 10 min or until the oxygen concentration felt below 1 mg/L (during the definitive test, a rapid respiration rate was observed. Consequently, dissolved oxygen concentrations were measured at 30 seconds intervals).

The temperature was monitored throughout the test period.

Reference substance (positive control):

yes

Key result

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

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

Remarks on result:

not determinable

Remarks:

No significant inhibition in respiration up to the highest tested concentration.

Range-finding test

The oxygen uptake and the coefficient of variation of oxygen uptake rate in control replicates were as follows:

 

Oxygen uptake (Rs) (mg O2/g of activated sludge (dry weight of suspended solids)/h)				Coefficient of variation of oxygen uptake rate
Item	Control a	Control b	Mean
Reference item	18.76	20.44	19.6	6.1%
Test item	13.78	18.22	16.0	19.6%

 

This test met the validity criteria of the guideline:

• The coefficient of variation of oxygen uptake rate in the controls should be no more than 30% at the end of the definitive test: it was determined to be 6.1 and 19.6%
• The 3h-EC50 for 3,5-dichlorophenol (23.0 mg/L) was between 2 and 25 mg/L for total respiration

The following validity criterion of the test guideline was not respected:

• The blank controls oxygen uptake was lower than 20 mg oxygen/g of activated sludge (dry weight of suspended solids) in an hour: 19.6 and 16.0 mg O₂/g of activated sludge (dry weight of suspended solids)/h) F

For total respiration, negative inhibitions in respiration were observed at the 10 and 100 mg/L test concentrations. A slight inhibition (7.9%) was calculated at the highest tested concentration. Although the statistical analysis showed no significant inhibition and since this range-finding test could not be validated, it was decided to conduct the definitive test at the following test item nominal concentrations:62.5, 125.0, 250.0, 500.0 and 1000.0 mg/L.

Definitive test

The oxygen uptake and the coefficient of variation of oxygen uptake rate in control replicates were as follows:

Oxygen uptake (Rs) (mg O2/g of activated sludge (dry weight of suspended solids)/h)				Coefficient of variation of oxygen uptake rate
Item	Control a	Control b	Mean
Reference item	31.2	31.2	31.2	0.0
Test item	31.2	25.7	28.5	13.7

 

This test met the validity criteria of the guideline:

• The coefficient of variation of oxygen uptake rate in the controls should be no more than 30% at the end of the definitive test: it was determined to be 0 and 13.7%
• The 3h-EC50 for the reference item (23.3 mg/L) passed the validity criterion of the guideline set between 2 and 25 mg/L for total respiration
• The blank controls oxygen uptake was not less than 20 mg oxygen/g of activated sludge (dry weight of suspended solids) in an hour: 31.2 and 28.5 mg O₂/g of activated sludge (dry weight of suspended solids)/h)  

Total respiration was inhibited at each tested concentrations as described below:

Test item nominal concentration (mg/L)	Inhibition of total respiration (%)
62.5	-9.0
125.0	-5.4
250.0	-30.1
500.0	4.4
1000.0	3.7

No significant inhibition in respiration was observed at any tested concentrations as confirmed with the statistical analysis.

Validity criteria fulfilled:

yes

Conclusions:

This study was designed to determine the inhibition of respiration rates of an activated sludge by the test item following the procedure described in the corresponding OECD guideline 209.

No significant inhibition in respiration was observed at any tested concentrations as confirmed with the statistical analysis.

LOEC and NOEC values under the conditions of this test were thus calculated to be respectively >1000.0 mg/L and =1000.0 mg/L.

Executive summary:

This study was designed to determine the effects of the test item on sewage micro-organisms by measuring the respiration rate in 3 hours test according to the OECD Guideline 209 (July 2010).

 

At the request of the sponsor, the inhibition of oxygen uptake by ammonium oxidation (i.e. nitrification) was not determined, so that only total respiration inhibition was assessed in this study.

 

The definitive test was conducted at the following nominal concentrations: 62.5, 125.0, 250.0, 500.0 and 1000.0 mg/L.

 

A reference item (3, 5-dichlorophenol) was tested as a reference inhibitory substance at the following nominal concentrations: 3.2, 10.0 and 32.0 mg/L.

 

The prepared vessels were aerated for 3h, after which dissolved oxygen concentrations were measured at 1 min intervals for up to a maximum period of 10 min. The respiration rate for each vessel was calculated from the measured values of recorded oxygen concentrations versus time andexpressed as mg O₂/L/h for the linear part of the graphs (then converted in mg O₂/g of activated sludge dry weight of suspended solids/h).

 

The definitive test met the validity criteria of the guideline detailed as follows:

• The coefficient of variation of oxygen uptake rate in the controls should be no more than 30% at the end of the definitive test: it was determined to be 0 and 13.7%
• The 3h-EC50 for the reference item (23.3 mg/L) passed the validity criterion of the guideline set between 2 and 25 mg/L for total respiration
• The blank controls oxygen uptake was not less than 20 mg oxygen/g of activated sludge (dry weight of suspended solids) in an hour: 31.2 and 28.5 mg O₂/g of activated sludge (dry weight of suspended solids)/h)

No significant inhibition in respiration was observed at any tested concentrations as confirmed with the statistical analysis.

 

LOEC and NOEC values under the conditions of this test were thus calculated to be respectively >1000.0 mg/L and =1000.0 mg/L.

Description of key information

A test was performed according to the OECD Test Guideline 209 to assess respiration inhibition effects of Reaction mass of decyl acrylate and octyl acrylate (EC number 911-295-9) on an activated sludge from a sewage treatment plant which handles predominantly domestic sewage.

 

The 3h-EC50 was not determined due to the maximum observed effect being -30.1% (positive effect) respiration inhibition. It is therefore considered that the EC50 is greater than the highest tested concentration: 1000 mg/L.

 

Since no significant effects were observed, the NOEC is equal to or greater than the highest tested concentration: 1000 mg/L. This is the endpoint value used in chemical safety assessment.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L