N-isopropylacrylamide

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 21 August 2017 and 12 December 2017.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)

Qualifier:

according to guideline

Guideline:

EU Method C.4-C (Determination of the "Ready" Biodegradability - Carbon Dioxide Evolution Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Batch: 2161004
Purity: 99.41%
Physical state/Appearance: white flakes
Expiry Date: 31 January 2018
Storage Conditions: room temperature in the dark

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

Inoculum
A mixed population of activated sewage sludge micro organisms was obtained on 13 November 2017 from the aeration stage of the Severn Trent Water Plc sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.

Preparation of Inoculum
The activated sewage sludge sample was washed twice by settlement and re suspension in mineral medium to remove any excessive amounts of dissolved organic carbon (DOC) that may have been present. The washed sample was then maintained on continuous aeration in the laboratory at a temperature of approximately 21 ºC and used on the day of collection. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 mL) of the washed activated sewage sludge by suction through pre-weighed GF/A filter paper* using a Buchner funnel. Filtration was then continued for a further 3 minutes after rinsing the filter three successive times with 10 mL of deionized reverse osmosis water. The filter paper was then dried in an oven at approximately 105 ºC for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 4.0 g/L prior to use.

Medium
The mineral medium used in this study was that recommended in the OECD Guidelines.
The deionized reverse osmosis water was used for the preparation of the mineral medium and the mineral medium used for the test contained less than 1 mg/L Total Organic Carbon (TOC).

Duration of test (contact time):

28 d

Initial conc.:

15.7 mg/L

Based on:

test mat.

Remarks:

equivalent to 10 mg carbon/L

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

Preliminary Work
In order to investigate whether the test item adsorbed to filter matrices and/or the activated sewage sludge, the following work was conducted and samples analyzed for Dissolved Organic Carbon (DOC) using a Shimadzu TOC VCPH TOC analyzer. During sample preparation, samples are either filtered or centrifuged to remove sewage sludge solids.
A nominal amount of test item (100 mg) was dissolved in mineral medium (1 liter) to give a 100 mg/L stock solution. Two samples were taken for DOC analysis; one untreated and one filtered through a 0.45 µm Gelman AcroCap filter (discarding the initial 5 mL to pre condition the filter). A further nominal amount of test item (100 mg) was dissolved in mineral medium and inoculated at a concentration of 30 mg suspended solids (ss)/L prior to adjusting to a final volume of 1 liter. Two samples were taken for DOC analysis; 1 after filtration through a 0.45 µm Gelman AcroCap filter (discarding the initial 5 mL to pre condition the filter) and the other after centrifugation at 4000 g for 15 minutes. Control samples were prepared by inoculating mineral medium (1000 mL) at a suspended solids level of 30 mg ss/L and then filtering or centrifuging as per the test item samples.

Test Item Preparation
The test item was dissolved directly in mineral medium.
A nominal amount of test item (300 mg) was dissolved in mineral medium with the aid of ultrasonication (approximately 5 minutes) and the volume adjusted to 1 liter to give a 300 mg/L stock solution. An aliquot (157 mL) of this stock solution was dispersed in inoculated mineral medium and the volume adjusted to 3 liters to give a final concentration of 15.7 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the test item was inverted several times to ensure homogeneity of the solution.
A test concentration of 10 mg carbon/L was employed in the test following the recommendations of the Test Guidelines.

Reference Item Preparation
A reference item, sodium benzoate (C6H5COONa), was used to prepare the procedure control vessels. An initial stock solution of 1000 mg/L was prepared by dissolving the reference item directly in mineral medium. An aliquot (51.4 mL) of this stock solution was added to the test vessel containing inoculated mineral medium and the volume adjusted to 3 liters to give a final test concentration of 17.1 mg/L, equivalent to 10 mg carbon/L. The volumetric flask containing the reference item was inverted several times to ensure homogeneity of the solution.

Toxicity Control
A toxicity control, containing the test item and sodium benzoate, was prepared in order to assess any toxic effect of the test item on the sewage sludge micro-organisms used in the test.
An aliquot (157 mL) of the test item stock solution was dispersed in inoculated mineral medium along with an aliquot (51.4 mL) of the sodium benzoate stock solution. The volume was adjusted to 3 liters to give a final concentration of 15.7 mg test item/L plus 17.1 mg sodium benzoate/L, equivalent to a total of 20 mg carbon/L.

Preparation of Test System
The following test preparations were prepared and inoculated in 5 liter test culture vessels each containing 3 liters of solution:
a) An inoculated control, in duplicate, consisting of inoculated mineral medium.
b) The procedure control containing the reference item (sodium benzoate), in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
c) The test item, in duplicate, in inoculated mineral medium to give a final concentration of 10 mg carbon/L.
d) The test item plus the reference item in inoculated mineral medium to give a final concentration of 20 mg carbon/L to act as a toxicity control (one vessel only).
Data from the inoculum control and procedure control vessels was shared with similar concurrent studies.
Each test vessel was inoculated with the prepared inoculum at a final concentration of 30 mg suspended solids (ss)/L. The test was carried out in a temperature controlled room at temperatures of between 20 and 24 °C, in darkness.
Approximately 24 hours prior to addition of the test and reference items the vessels were filled with 2400 mL of mineral medium and 22.5 mL of inoculum and aerated overnight. On Day 0, the test and reference items were added, and the pH of all vessels were measured using a Hach HQ40d Flexi handheld meter. The pH was adjusted to pH 7.4 ± 0.2 using diluted hydrochloric acid or sodium hydroxide solution prior to the volume in all the vessels being adjusted to 3 liters by the addition of mineral medium which had been purged overnight with CO2 free air.
The test vessels were sealed and CO2 free air bubbled through the solution at a rate of 30 to 100 mL/minute per vessel and stirred continuously by magnetic stirrer.
The CO2 free air was produced by passing compressed air through a glass column containing self-indicating soda lime (Carbosorb®) granules.
The CO2 produced by degradation was collected in two 500 mL Dreschel bottles containing 350 mL of 0.05 M NaOH. The CO2 absorbing solutions were prepared using purified water.

Reference substance:

benzoic acid, sodium salt

Preliminary study:

The results obtained from the samples taken for DOC analysis from the preliminary investigational work indicated that the test item did not adsorb to filter matrices or to activated sewage sludge. Therefore, for the purpose of the study, the samples taken for DOC analysis were filtered to remove the suspended solids present without the loss of any test item.

Test performance:

The total CO2 evolution in the inoculum control vessels on Day 28 was 33.52 mg/L and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC content of the test item suspension in the mineral medium at the start of the test was below 5% of the TC content and hence satisfied the validation criterion given in the OECD Test Guidelines.
The difference between the values for CO2 production at the end of the test for the replicate vessels was < 20 % and hence satisfied the validation criterion given in the OECD Test Guidelines.
The test item attained 3 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.
The OECD Test Guidelines requires the acidification of the test vessels on Day 28 prior to final analyses being performed on Day 29. This acidification effectively kills the micro-organisms present and drives of any dissolved CO2 present in the test vessels. Acidification of the test vessels was not performed during the study due to a concurrent study which shared the control and reference item vessels being extended past 28 days. The lack of acidification was considered not to have had an impact on this study as no significant biodegradation had been shown to have occurred, and past experience has shown that only limited amounts of dissolved CO2 are released by the acidification process in the test design employed.
The toxicity control attained 44 % biodegradation after 14 days and 52 % biodegradation after 28 days thereby confirming that the test item did not exhibit an inhibitory effect on the sewage treatment micro organisms used in the test.

Key result

Parameter:

% degradation (CO2 evolution)

Value:

3

Sampling time:

28 d

Results with reference substance:

Sodium benzoate attained 80 % biodegradation after 14 days and 100 % biodegradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Inorganic Carbon Values on Each Analysis Occasion

Day	Inorganic Carbon (mg IC)
	Inoculum Control				Procedure Control				Test Item				Toxicity Control
	R1		R2		R1		R2		R1		R2		R1
	Abs1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2	Abs 1	Abs 2
0	2.45	2.10	2.10	2.45	2.10	2.45	2.10	2.10	3.15	2.80	2.10	2.45	2.45	2.45
2	11.83	-	7.31	-	23.55	-	21.58	-	6.73	-	8.12	-	27.49	-
6	12.46	-	11.88	-	30.22	-	27.33	-	10.15	-	11.76	-	32.06	-
8	13.53	-	12.38	-	33.48	-	29.47	-	12.15	-	13.42	-	34.40	-
10	17.44	-	15.39	-	44.01	-	37.39	-	15.62	-	17.44	-	43.78	-
14	19.38	-	17.23	-	44.88	-	39.78	-	17.00	-	20.29	-	44.43	-
21	26.14	-	25.58	-	52.95	-	56.45	-	24.22	-	30.76	-	61.63	-
28	23.97/26.54*	-	22.51/28.33^*	-	55.33	-	59.25	-	28.22	-	28.22	-	58.57	-

 

R    =     Replicate

Abs=      CO2 absorber vessel

*     =     Results from re-analysis of original sample as original result deemed to be erroneous

Percentage Biodegradation Values

Day	% Biodegradation
	Procedure Control	Test Item	Toxicity Control
0	0	0	0
2	43	0	30
6	55	0	33
8	62	0	36
10	81	0	46
14	80	1	44
21	96	5	60
28	100	3	52

Total and Inorganic Carbon Values in the Culture Vessels on Day 0

Test vessel	Total Carbon* (mg/L)	Inorganic Carbon*(mg/L)	IC Content (% of TC)
Test Item 10 mg C/L R1	9.93	0.09	1
Test Item 10 mg C/L R2	9.86	0.02	0

R=   Replicate

*=   Corrected for control values

pH Values of the Test Preparations on Days 0 and 28

 

Test Vessel	pH
	Day 0 Pre-Adjustment	Day 0 Post-Adjustment	Day 28
Inoculum Control R1	7.7	7.6	7.6
Inoculum Control R2	7.7	7.6	7.6
Procedure Control R1	7.7	7.6	7.7
Procedure Control R2	7.7	7.6	7.6
Test Item R1	7.7	7.6	7.6
Test Item R2	7.7	7.6	7.6
Toxicity Control	7.7	7.5	7.6

R=   Replicate

 

Observations on the Test Preparations Throughout the Test Period

Test Vessel		Observations on Test Preparations
		Day 0	Day 6	Day 13	Day 20	Day 27
Inoculum Control	R1	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion
	R2	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion	Light brown dispersion
Procedure Control	R1	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible
	R2	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible	Light brown dispersion, no undissolved reference item visible
Test Item	R1	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible
	R2	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible	Light brown dispersion, no undissolved test item visible
Toxicity Control		Light brown dispersion, no undissolved test or reference item visible	Light brown dispersion, no undissolved test or reference item visible	Light brown dispersion, no undissolved test or reference item visible	Light brown dispersion, no undissolved test or reference item visible	Light brown dispersion, no undissolved test or reference item visible

R=   Replicate

Validity criteria fulfilled:

yes

Interpretation of results:

not readily biodegradable

Conclusions:

The test item attained 3 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Executive summary:

Introduction

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (m)).

Methods

The test item, at a concentration of 10 mg carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 20 and 24 °C for 28 days.

The biodegradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

Results

The test item attained 3 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Description of key information

Introduction

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No. 301B, "Ready Biodegradability; CO2 Evolution Test" referenced as Method C.4-C of Commission Regulation (EC) No. 440/2008 and US EPA Fate, Transport, and Transformation Test Guidelines OCSPP 835.3110 (Paragraph (m)).

Methods

The test item, at a concentration of 10 mg carbon/L, was exposed to activated sewage sludge micro-organisms with mineral medium in sealed culture vessels in the dark at temperatures of between 20 and 24 °C for 28 days.

The biodegradation of the test item was assessed by the determination of carbon dioxide produced. Control solutions with inoculum and the reference item, sodium benzoate, together with a toxicity control were used for validation purposes.

Results

The test item attained 3 % biodegradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No. 301B.

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater