N-isopropylmethacrylamide

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 16 October 2007 and 11 December 2007.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 835.3110 (Ready Biodegradability)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

Test Species:
A mixed population of activated sewage sludge micro-organisms was obtained on 5 November 2007 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 three times by settlement and resuspension in culture 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°C 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 funnel three successive times with 10 ml of deionised reverse osmosis water. The filter paper was then dried in an oven at approximately 105°C 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 2.6 g/l prior to use.

Culture medium:
The culture medium used in this study was that recommended in the OECD Guidelines (defined below)

Solution a
KH2PO4: 8.50 g/l
K2HPO4: 21.75 g/l
Na2HPO4.2H2O: 33.40 g/l
NH4Cl: 0.50 g/l

pH = 7.4

Solution b: CaCl2: 27.50 g/l
Solution c: MgSO4.7H2O: 22.50 g/l
Solution d: FeCl3.6H2O: 0.25 g/l

To 1 litre (final volume) of purified water was added the following volumes of solutions a – d.
10 ml of Solution a
1 ml of Solution b
1 ml of Solution c
1 ml of Solution d

Duration of test (contact time):

28 d

Initial conc.:

10 other: mg Carbon/l

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Details on study design:

EXPERIMENTAL PREPARATION:
For the purpose ofthe test the test material was dissolved directly in culture medium.

An amount of test material (300 mg) was dissolved in culture medium with the aid of ultrasonication (approximately 5 minutes) and the volume adjusted to 1 litre to give a 300 mg/l stock solution. An aliquot (189 ml) of this stock solution was dispersed in inoculated culture medium and the volume adjusted to 3 litres to give a final concentration of 18.9 mg/l, equivalent to 10 mg carbon/l. The volumetric flask containing the test material 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 Guideline.

STANDARD MATERIAL:
For the purposes of the test a standard material, sodium benzoate (C6H5COONa), was used. An initial stock solution of 1000 mg/l was prepared by dissolving the standard material directly in culture medium with the aid of ultrasonication for approximately 1 minute, and a 51.4 ml aliquot added to the test vessel to give a final test concentration of 17.1 mg/l, equivalent to 10 mg carbon/l. The volumetric flask containing the standard material was inverted several times to ensure homogeneity of the solution.

TOXICITY CONTROL:
For the purposes of the test a toxicity control, containing the test material and sodium benzoate, was prepared in order to assess any toxic effect of the test material on the sewage sludge microorganisms used in the test.
An aliquot (189 ml) of the test material stock solution was dispersed in inoculated culture medium along with an aliquot (51.4 ml) of the sodium benzoate stock solution. The volume was adjusted to 3 litres to give a final concentration of 18.9 mg test material/l plus 17.1 mg sodium benzoate/l, equivalent to a total of 20 mg carbon/l.

PRELIMINARY INVESTIGATIONAL WORK:
During the study, samples are taken for Dissolved Organic Carbon (DOC) analysis and as part of the sample preparation the samples are either filtered or centrifuged to remove the sewage sludge solids. Thus the following work was conducted and samples analysed for Dissolved Organic Carbon (DOC) using a Shimadzu TOC analyser (see Appendix 1 – attached background material).

An amount of test material (l00 mg) was dissolved in reverse osmosis purified water (1000 ml) with the aid of ultrasonication for approximately 5 minutes to give a 100 mg/l stock solution. Two samples were taken for DOC analysis; one neat and one filtered though a Gelman 0.45 µm Acrocap filter (discarding the initial 5 ml to pre-condition the filter). A further amount of test material (100 mg) was dissolved in reverse osmosis purified water with the aid of ultrasonication for approximately 5 minutes and inoculated at a concentration of 30 mg suspended solids (ss)/l prior to adjusting to a final volume of 1000 ml. Two samples were taken for DOC analysis; one filtered though a Gelman 0.45 µm Acrocap filter (discarding the initial 5 ml to pre-condition the filter) and the other centrifuged at 3500 rpm for 15 minutes. Control samples were prepared by inoculating reverse osmosis water (1000 ml) at a suspended solids level of 30 mg ss/l and then filtering or centrifuging as per the test material samples.

PREPARATION OF TEST SYSTEM:
The following test preparations were prepared and inoculated in 5 litre glass culture vessels each containing 3 litres of solution:
a) A control, in duplicate, consisting of inoculated culture medium.
b) The standard material (sodium benzoate), in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/l.
c) The test material, in duplicate, in inoculated culture medium to give a final concentration of 10 mg carbon/l.
d) The test material plus the standard material in inoculated culture medium to give a final concentration of 20 mg carbon/l to act as a toxicity control (one vessel only).

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 approximately 21°C, in darkness.

Approximately 24 hours prior to addition of the test and standard materials the vessels were filled with 2400 ml of culture medium and 34.6 ml of inoculum and aerated overnight. On Day 0 the test and standard materials were added and the volume in all the vessels adjusted to 3 litres by the addition of culture medium.

The culture vessels were sealed and CO2-free air bubbled through the solution at a rate of approximately 40 ml/minute 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 de-gassed water.

SAMPLING AND ANALYSIS:
CO2 ANALYSIS:
Samples (2 ml) were taken from the first C02 absorber vessel on Days 0, 1, 2, 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 27, 28 and 29. The second absorber vessel was sampled on Days 0 and 29.

The samples taken on Days 0, 1, 2, 3, 6, 8, 10, 14, 16, 20, 22, 24, 27,2 8 and 29 were analysed for CO2 immediately. The samples taken on Days 12 and 18 were stored at approximately -20°C. However, these samples (except Day 12 control and standard material samples which were analysed for a concurrent study) were not analysed for CO2 as the results obtained from previous and subsequent analyses showed that the level of degradation of the test material did not significantly increase during this time and therefore additional analyses were considered to be unnecessary.

On Day 28, 1 ml of concentrated hydrochloric acid was added to each vessel to drive off any inorganic carbonates formed. The vessels were resealed, aerated overnight and the final samples taken from both absorber vessels on Day 29.

The samples were analysed for CO2 using a Tekmar-Dohrmann Apollo 9000 TOC analyser and a Shimadzu TOC-Vcsh TOC analyser. Samples (300 or 50 µl) were injected into the IC (Inorganic Carbon) channel of the TOC analyser. Inorganic carbon analysis occurs by means of the conversion of an aqueous sample to CO2 by orthophosphoric acid using zero grade air as the carrier gas. Calibration was by standard solutions of sodium carbonate (Na2CO3) Each analysis was carried out in triplicate.

DISSOLVED ORGANIC CARBON (DOC) ANALYSIS:
On Days 0 and 28 samples (20 ml) were removed from all culture vessels and filtered through Gelman 0.45 µm Acrocap filters (approximately 5 ml discarded) prior to DOC analysis.

The samples were analysed for DOC using a Shimadzu TOC-5050A TOC analyser. Samples (27 or 13 µl) were injected into the Total Carbon (TC) and Inorganic Carbon (IC) channels of the TOC analyser. Total carbon analysis is carried out at 680°C using a platinum based catalyst and zero grade air as the carrier gas. Inorganic carbon analysis involves conversion by orthophosphoric acid at ambient temperature. Calibration was performed using standard solutions of potassium hydrogen phthalate (CC8H5KO4) and sodium carbonate (Na2C03) in deionised water. Each analysis was carried out in triplicate.

pH MEASUREMENTS:
The pH of the test preparations was determined on Day 28, prior to acidification with hydrochloric acid, using a WTW pH/Oxi 3401 pH and dissolved oxygen meter.

Reference substance:

benzoic acid, sodium salt

Preliminary study:

Preliminary Investigational Work:
The results obtained from the samples taken for DOC analysis from the preliminary investigational work indicated that the test material did not absorb to filter matrices or to activated sewage sludge (see Appendix 1 – attached background material). 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 material.

Test performance:

The total CO2 evolution in the control vessels on Day 28 was 25.93 mg/l and therefore satisfied the validation criterion given in the OECD Test Guidelines.
The IC content of the test material suspension in the mineral medium at the start of the test (see Table 3 – attached background material) 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 C02 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.

Parameter:

% degradation (CO2 evolution)

Value:

14

Sampling time:

28 d

Details on results:

Inorganic carbon values for the test material, standard material, toxicity control and control vessels at each analysis occasion are given in Table 1. Percentage biodegradation values of the test and standard materials and the toxicity control are given in Table 2 and the biodegradation curves are presented in Figure 1. Total and Inorganic Carbon values in the culture vessels on Day 0 are given in Table 3, and the results of the Dissolved Organic Carbon analyses performed on Days 0 and 28 are given in Table 4. The pH values of the test preparations on Day 28 are given in Table 5 (see attached background material for Tables 1-5 and Figure 1).

Acidification of the test vessels on Day 28 followed by the final analyses on Day 29 was conducted according to the methods specified in the Test Guidelines. This acidification effectively kills the micro-organisms present and drives off any dissolved CO2 present in the test vessels. Therefore any additional CO2 detected in the Day 29 samples originated from dissolved CO2 that was present in the test vessels on Day 28 and hence the biodegradation value calculated from the Day 29 analyses is taken as being the final biodegradation value for the test material.

The results of the inorganic carbon analysis of samples from the first absorber vessels on Day 29 showed an increase in all replicate vessels with the exception of control replicate R2. Inorganic carbon analysis of the samples from the second absorber vessels on Day 29 confirmed that no significant carry-over of CO2 into the second absorber vessels occurred.

The test material attained 14% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

The toxicity control attained 44% degradation after 14 days and 43% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the test. The slight decrease in degradation obtained for the toxicity control between Day 14 and Day 28 was considered to be due to a slightly greater increase in CO2 evolution values in the control inorganic carbon values compared to the increase in inorganic carbon within the toxicity control vessel during this time period.

Analysis of the test media from the test material culture vessels on Days 0 and 28 for Dissolved Organic Carbon (DOC), see Table 4 (attached background material), gave percentage degradation values of 11% and 3% respectively for the test material Replicates R1 and R2 and 51% for the toxicity control. These results were in line with those calculated from the IC analyses.

Observations made throughout the test period (see Table 6 – attached background material) showed the contents of the control vessels to be light brown dispersions and the contents of the standard material vessels were light brown dispersions with no undissolved standard material visible. The test material vessels were observed to contain light brown dispersions with no undissolved test material visible. The toxicity control vessel contained a light brown dispersion with no undissolved test or standard material visible.

Results with reference substance:

Sodium benzoate attained 85% degradation after 14 days and 88% degradation after 28 days thereby confirming the suitability of the inoculum and test conditions.

Sodium benzoate attained 100% degradation for both Replicates R1 and R2 calculated from the results of the DOC analyses. The degradation rates calculated from the results of the DOC analyses were higher than those calculated from inorganic carbon analysis. This was considered to be due to incorporation of sodium benzoate into the microbial biomass prior to degradation, and hence CO2 evolution occurring.

Validity criteria fulfilled:

yes

Interpretation of results:

readily biodegradable

Conclusions:

The test material attained 14% degradation 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 material in an aerobic aqueous medium. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; C02 Evolution Test" referenced as Method C.4-C of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC), and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 Paragraph (m).

Methods.

The test material, at a concentration of 10 mg Carbon/l, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at approximately 21°C for 28 days.

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

Results.

The test material attained 14% degradation 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

The test material attained 14% degradation after 28 days and therefore cannot be considered to be readily biodegradable

Key value for chemical safety assessment

Biodegradation in water:

not biodegradable

Type of water:

freshwater

Additional information

Introduction.

A study was performed to assess the ready biodegradability of the test material in an aerobic aqueous medium. The method followed that described in the OECD Guidelines for Testing of Chemicals (1992) No 301B, "Ready Biodegradability; C02 Evolution Test" referenced as Method C.4-C of Commission Directive 92/69/EEC (which constitutes Annex V of Council Directive 67/548/EEC), and US EPA Fate, Transport, and Transformation Test Guidelines OPPTS 835.3110 Paragraph (m).

Methods.

The test material, at a concentration of 10 mg Carbon/l, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in the dark at approximately 21°C for 28 days.

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

Results.

The test material attained 14% degradation after 28 days and therefore cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline No 301B.

The toxicity control attained 44% degradation after 14 days and 43% degradation after 28 days thereby confirming that the test material was not toxic to the sewage treatment micro-organisms used in the test.