1-butylpyrrolidin-2-one

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-10-13 to 2012-10-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material: Not applicable

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Effluent from a biologic sewage treatment plant was used. The chosen plant is treating mostly household sewage. The effluent was taken from the effluent channel of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, NW-Lachen-Speyerdorf, Germany.
- Storage conditions: The effluent was kept aerobic during transport and storage.
- Storage length: Date of collection: 10. Oct 2012, batch no: 20121010.
- Preparation of inoculum for exposure: Before usage, the effluent was left to settle for one hour.
- Pretreatment: The effluent was aerated for six days at room temperature in order to lower the oxygen consumption of the blanks.
- Concentration of sludge: 0.5 mL/L test medium were used.
- Initial cell/biomass concentration: 0.5 mL/L test medium were used.
- Water filtered: No
- Type and size of filter used, if any: Not applicable

Duration of test (contact time):

28 d

Initial conc.:

2.08 mg/L

Based on:

act. ingr.

Initial conc.:

5.9 mg/L

Based on:

ThOD/L

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Stock solutions
I. Solution a:
Potassium dihydrogenephosphate (KH2PO4): 8.5 g,
Di-potassium hydrogenephosphate (K2HPO4): 21.75 g,
Sodium di-hydrogenephosphate hydrate (NaH2P04*H20): 33.4 g,
Ammonia chloride (NH4CI): 0.5 g,
H2O demin. ad.: 1000 mL. The pH was adjusted to 7,4 ± 0,1.

II. Solution b:
Calcium chloride dihydrate (CaCI2*2H20): 36.4 g,
H2O demin. ad.: 1000 mL.

III. Solution с:
Magnesium sulfate heptahydrate (MgS04*7H20): 22.5 g,
H2O demin. ad.: 1000 mL.

IV. Solution d:
Iron(lll) chloride hexahydrate (FeCI3*6H20): 0.25 g,
Di-sodium-ethylendiamintetraacetate dihydrate (Na2EDTA*2H20): 0.4 g,
H2O demin.: ad 1000 mL.

Test medium
The medium was freshly prepared. Composition:
Solution a: 1 mL,
Solution b: 1 mL,
Solution с: 1 mL,
Solution d: 1 mL,
H2O demin. ad.: 1000 mL

Test Vessels
Incubation and measurement were performed in 250 mL narrow neck bottles with glass stoppers
All glassware was cleaned with the laboratory cleaning agent Mucasol® and then rinsed with tap water (thrice), diluted HCL (once), tap water (thrice) and diluted water (thrice).

Instruments and Devices:
The following instruments and devices were used in the performance of the study:
• Data logger for temperature
• Analytical Scales Mettler Toledo XS 205 LAUS No. 2
• Precision Scales Mettler XS 6001S
• Adjustable pipettes with one-way tips Rainin®; LAUS-No. 29
• Glass measuring cylinders and flasks
• Glass aspirator bottles (10 L)
• Oximeter 538 wtw
• Magnetic stirrers
• Photometer Specord 205 Analytik Jena

Preparations:
The effluent was taken from the sewage treatment plant and was aerated for six days in order to lower the oxygen demand of the effluent.
Two days before the start of the test, the medium was prepared from the stock solutions and the effluent and was then aerated for at least 24 hours. On the day of the start of the test, the stock solutions of the test and the positive control were prepared.

Reference substance:

benzoic acid, sodium salt

Remarks:

Sodium benzoate, С7Н5NaО2, p.A. was used as positive control.

Test performance:

Pre-Treatment:
A stock solution containing 111.1 mg N-Butylpyrrolidone/L was prepared. 18.75 mL of the stock solution were added to each litre of the test solution. The content of the test item in the test vessels was 2.08 mg/L (corresponding to 5.90 mg/L ThOD).

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

28 d

Remarks on result:

other: No biodegradation was observed after 28 days neither with nor without consideration of nitrification

Details on results:

No biodegradation was observed after 28 days neither with nor without consideration of nitrification. A 10-day-window could not be detected. The positive control reached the pass level (>60% degradation) on day 8. As degradation in the toxicity flask was more than 25 % at the end of the test, the test item can be stated as not toxic towards the inoculum in a concentration of 1.11 mg/L.

Results with reference substance:

The theoretical oxygen demand (ThOD) of the positive control was calculated. With the formula of the compound given as C7H5Na02, and the molecular weight MR of 144 g/mol, a ThOD of 1.665 mg O2 /mg sodium benzoate was calculated. A stock solution containing 180.0 mg sodium benzoate/L in deionised water was freshly prepared. The content of the positive control in the test vessels was 3.60 mg/L (corresponding to 5.99 mg/L ThOD).
Result: Degradation >60% within 8 days.

For the determination of the biological results, the data were evaluated with and without consideration of nitrification. The amount to be added to the test flasks was based on the THOD (theoretical oxygen demand) of the test item. THOD differentiates between O2-uptake caused by nitrification and O2-uptake caused by carbon oxidising. Therefore, the O2-uptake caused by nitrification was considered for the determination of the degradation.

As degradation in the toxicity flask was more than 25 % at the end of the test, the test item can be stated as not toxic towards the inoculum in a concentration of 1.11 mg/L.

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Based on the results of the Closed Bottle Test, N-Butylpyrrolidone is not readily biodegradable.

Executive summary:

The ready biodegradability study (Closed bottle test) was performed according to OECD Guideline 301D (1992) and EU Method C.4-E (2008). This study was performed in order to evaluate aerobic elimination and degradation potential of N-Butylpyrrolidone in a test for ready biodegradability, using a test item concentration of 2.08 mg/L (corresponding to a theoretical oxygen consumption of 5.90 mg/L). The solution of the test item in mineral medium was inoculated with a relatively small number of micro-organisms from a mixed population and kept in completely full, closed bottles in the dark at constant temperature. Degradation was followed by analysis of dissolved oxygen over a 28-d period. The amount of oxygen taken up by the microbial population during biodegradation of the test substance, corrected for uptake by the blank inoculum run in parallel, is expressed as a percentage of COD.

No biodegradation was observed after 28 days neither with nor without consideration of nitrification. A 10-day-window could not be detected. The positive control reached 67% degradation on day 8. As degradation in the toxicity flask was more than 25 % at the end of the test, the test item can be stated as not toxic towards the inoculum in a concentration of 1.11 mg/L.

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-04-02 to 2013-07-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline Study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 B (Inherent biodegradability: Zahn-Wellens/EMPA Test)

Qualifier:

according to guideline

Guideline:

EU Method C.9 (Biodegradation: Zahn-Wellens Test)

Principles of method if other than guideline:

The test was left running for 112 days (sponsor's intent).

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material:
Not applicable

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, predominantly domestic (adaptation not specified)

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Effluent from a biologic sewage treatment plant was used. The chosen plant is treating mostly household sewage. The effluent was taken from the effluent channel of the ESN (Stadtentsorgung Neustadt) sewage treatment plant, Im Altenschemel, NW-Lachen-Speyerdorf, Germany.
- Storage conditions: The effluent was kept aerobic during transport and storage.
- Storage length: Date of collection: 03. April 2013, batch no: 20121010.
- Pretreatment: The sludge was washed with tap water twice, then filtrated through a cloth and washed and resuspended in test medium. It was then aerated. The dry matter was determined as 4100 mg suspended solids/L.
- Concentration of sludge: concentration 601 mg dry matter/L
- Initial cell/biomass concentration: Inoculum was added in order to reach a content of 600 mg dry matter/L in the final volume
- Water filtered: Yes
- Type and size of filter used, if any: Not reported

Duration of test (contact time):

112 d

Initial conc.:

306.6 mg/L

Based on:

test mat.

Initial conc.:

200 mg/L

Based on:

other: organic carbon/L

Initial conc.:

288.42 - 329.85 mg/L

Based on:

DOC

Parameter followed for biodegradation estimation:

DOC removal

Details on study design:

Stock solutions
I. Solution a:
Potassium dihydrogenephosphate (KH2PO4): 8.5 g,
Di-potassium hydrogenephosphate (K2HPO4): 21.75 g,
Sodium di-hydrogenephosphate hydrate (NaH2P04*H20): 33.4 g,
Ammonia chloride (NH4CI): 0.5 g,
H2O demin. ad.: 1000 mL. The pH was adjusted to 7,4 ± 0,1.

II. Solution b:
Calcium chloride dihydrate (CaCI2*2H20): 36.4 g,
H2O demin. ad.: 1000 mL.

III. Solution с:
Magnesium sulfate heptahydrate (MgS04*7H20): 22.5 g,
H2O demin. ad.: 1000 mL.

IV. Solution d:
Iron(lll) chloride hexahydrate (FeCI3*6H20): 0.25 g,
Di-sodium-ethylendiamintetraacetate dihydrate (Na2EDTA*2H20): 0.4 g,
H2O demin.: ad 1000 mL.

Test medium
The medium was freshly prepared. Composition:
Solution a: 1 mL,
Solution b: 1 mL,
Solution с: 1 mL,
Solution d: 1 mL,
H20 demin. ad.: 1000 mL

Test Vessels and Apparatus:
Erlenmeyer flasks (nominal volume 2000 mL) were used. For aeration, glass tubes which reach to 2 cm above the bottom of the respective flask, were used. The air was purified by activated charcoal and moistened. An orbital shaker was used for shaking.

Instruments and Devices:
The following instruments and devices were used in the performance of the study:
- Data logger for temperature
- Analytical scales Mettler Toledo XS 205 DU LAUS No. 2
- Precision scales Mettler Toledo XS 6001S
- Adjustable pipettes with one-way tips Rainin®; LAUS-Nos. 29; 40
- Carbon analyser TOC multi N/C 2100S, Analytik Jena
- pH-meter3310 wtw
- Heating chamber Memmert LAUS No. 4
- Orbital shaker GFL3019 LAUS No. 11
- Fridge LAUS No. 9 and 11
- Standard laboratory glassware
- Syringe filters nylon 0.45 pm

Reference substance:

aniline

Remarks:

A stock solution containing 2.0005 g/L was prepared and TOC was measured in filtrated and unfiltrated solution. The TOC of the unfiltrated solution was 1483.5 mg/L, corresponding to 74.2 % organic carbon.

Test performance:

The test was left running for 112 days (sponsor's intent).
To measure the degradation of test item and positive control, the content of dissolved organic carbon (DOC) in the test vessels was measured twenty times during the test.
All validity criteria were met. Degradation of the positive control was 98% after 8 days.

Parameter:

% degradation (DOC removal)

Value:

2.9

Sampling time:

14 d

Parameter:

% degradation (DOC removal)

Value:

7.1

Sampling time:

28 d

Parameter:

% degradation (DOC removal)

Value:

11.2

Sampling time:

42 d

Parameter:

% degradation (DOC removal)

Value:

51.7

Sampling time:

84 d

Parameter:

% degradation (DOC removal)

Value:

81

Sampling time:

112 d

Remarks on result:

other: End of experiment after 112 days

Details on results:

Experiment was performed over a period of 112 days. After a lag phase (day 0 to 35), degradation of the test compound was observed (degradation phase day 36 to 112).

Results with reference substance:

Degradation of the positive control was 98% after 8 days.

Normally, duration of the test is 28 days. On request of the sponsor, the test duration was prolonged to 112 days. After adaption of the inoculum, on day 49, a rapid increase in degradation could be observed. The test item showed good correlation within the test replicates up to day 84. Afterwards, the values differ extremely. It may be possible that the sludge lost its activity in one replicate, due to the fact that the test duration was much longer than described in the guideline. So the test item can't be stated as inherent biodegradable after 28 days but as biodegradable after adaption of the inoculum. One replicate reached 100 % degradation at the end of the test. The mean degradation at the end of the test was 81 %. In a non-GLP pre-test, it was tested whether the test item is volatile. Because the test item was not volatile under the test conditions, a biodegradation test according to OECD 302B / EU C.9 was conducted. In the abiotic control and in the adsorption control, a marginal increase in degradation (marginally lower DOC concentrations) was observed starting at day 92. No explanation could be found.

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable, not fulfilling specific criteria

Conclusions:

The degree of biodegradation reached 7 % after 28 days. Mean degradation at the end of the test 81 % (lag-phase day 0 - 35, degradation phase day 36 -112).

Executive summary:

A Zahn-Wellens Test was performed with N-Butylpyrrolidone to characterise inherent biodegradation potential of the compound. The test item was tested using a concentration of 200 mg organic carbon/L (nominal) in test medium following OECD 302B/EU-Guideline C.9-C. Aniline was chosen as positive control. Activated sludge from a sewage treating plant was used as inoculum (concentration 601 mg dry matter/L). The test was left running for 112 days (sponsor's intent). To measure the degradation of test item and positive control, the content of dissolved organic carbon (DOC) in the test vessels was measured twenty times during the test. All validity criteria were met. Degradation of the positive control was 98% after 8 days. The following data could be determined for the test item N-Butylpyrrolidone: The degree of biodegradation reached 7 % after 28 days. Mean degradation at the end of the test (112 days) was 81 % (lag-phase day 0 - 35, degradation phase day 36 -112). As degradation in the toxicity flask was 50 % on day 14, the test item N-Butylpyrrolidone can be stated as "not toxic towards the inoculum in a concentration of 306.2 mg/L".

Reference 3

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-09-12 to 2014-11-24 (experimental phase of study)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))

Qualifier:

according to guideline

Guideline:

other: Commission Directive 1999/11/EC of 8 March 1999 amending Council Directive 87/18/EEC, Official Journal of the European Communities No L 77: pages 8 – 21

Principles of method if other than guideline:

According to the guideline OECD 302 C, the inoculum should be sampled in Japan at not less than 10 places throughout the country (waste water treatment plants, rivers, lakes, sea). The inoculum has to be prepared from the different samples during a one month lasting procedure. For this study, activated sludge from a well working municipal waste water treatment plant in Bensheim, Germany was used immediately after sampling.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material: Not relevant

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): For this study, activated sludge from a well working municipal waste water treatment plant in Bensheim, Germany was used immediately after sampling.
- Laboratory culture: Not relevant
- Method of cultivation: Not relevant
- Storage conditions: No storage, activated sludge was used immediately after sampling.
- Storage length: No storage, activated sludge was used immediately after sampling.
- Preparation of inoculum for exposure: The aerobic activated sludge used for this study was washed by centrifugation and the supernatant liquid phase was decanted. The solid material was re-suspended in tap water and centrifuged again. This procedure was done three times.
- Pretreatment: An aliquot of the final sludge suspension was weighed, dried and the ratio of wet sludge to its dry weight was determined. Based on this ratio, calculated aliquots of washed sludge suspension, corresponding to 2 g dry material per litre were mixed with test water. This suspension was used for the experiment.
- Concentration of sludge: 2 g dry material per litre.
- Initial cell/biomass concentration: No details reported.
- Water filtered: No.

Duration of test (contact time):

56 d

Initial conc.:

9 other: mg/300 mL test solution

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Remarks:

Biodegradability (% BOD = mg O2 per mg test item) exerted after each period was calculated

Details on study design:

TEST CONDITIONS
- Composition of medium:
In deionised water analytical grade salts were added to prepare the following stock solutions (basal culture medium):
a) 4.25 g KH2PO4, 10.875 g K2HPO4, 11.1 g Na2HPO4 x 2 H2O, 0.85 g NH4Cl filled up with deionised water to 500 mL volume. The pH was 7.2.
b) 11.25 g MgSO4 x 7 H2O filled up with deionised water to 500 mL volume.
c) 13.75 g CaCl2 filled up with deionised water to 500 mL volume.
d) 0.125 g FeCl3 x 6 H2O filled up with deionised water to 500 ML volume.
15mL of each of stock solutions a) to d) were combined and filled up to a final volume of 5000 mL with deionised water.

Preparation of test solutions:
Test Item (3 replicates): basal culture medium (285 mL) + activated sludge (30 mg, dry basis) + test item 9.0 mg.
(activated sludge to reach 30 mg (dry base) concentration in the test vessel (15 mL of a stock suspension of 2 g/L suspended solids); the final volume was 300 mL).

- Additional substrate: No
- Solubilising agent (type and concentration if used): None.
- Test temperature: 25 ± 1 °C (climatised room).
- pH: 7.2-7.3 (measured at the start of the test), 6.2–7.5 (measured at the end of the test).
- pH adjusted: No.
- CEC (meq/100 g): Not reported.
- Aeration of dilution water: Not reported.
- Suspended solids concentration: 2 g dry material per litre.
- Continuous darkness: Yes.
- Other: --

TEST SYSTEM
- Culturing apparatus: BSB/BOD-Sensor-System, Aqualytic (Dortmund, Germany). Manometric test system with test flasks of 504 mL volume containing 300 mL test liquid. The test flasks were closed gas-tight by a measuring head.
- Number of culture flasks/concentration: 3 replicates
- Method used to create aerobic conditions: Permanent stirring
- Measuring equipment: The change of pressure in the test flasks was measured automatically by means of a manometric method (BSB/BODSensor-System, Aqualytic Dortmund, Germany) about 4 to 6 times a day for a 56 day incubation period. The device consists of reaction vessels with CO2-absorber, measuring heads and a controller (infrared communication).
- Test performed in closed vessels due to significant volatility of test substance: The closed BOD-meter test flasks were incubated in a climatised chamber under continuous stirring. The consumption of oxygen was determined by measuring the change of pressure in the flasks.
- Test performed in open system: No, flasks were closed.
- Details of trap for CO2 and volatile organics if used: Evolved carbon dioxide was absorbed in an aqueous solution (45%) of potassium hydroxide.
- Other: --

SAMPLING
- Sampling frequency: After 0, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52 and 56 days.
- Sampling method: No details reported.
- Sterility check if applicable: Not applicable.
- Sample storage before analysis: No details reported.
- Other: --

CONTROL AND BLANK SYSTEM
- Inoculum blank: Yes
- Abiotic sterile control:Yes
- Toxicity control: Yes
- Other: --

STATISTICAL METHODS: No specific statistical procedures applied.

Reference substance:

benzoic acid, sodium salt

Remarks:

purity: 100.4%, ThODNH4: 1.666 mg oxygen per mg sodium benzoate (calculated)

Preliminary study:

Before test start, a pre-test was conducted with the test item and activated sludge in the same concentrations as in the main test to eliminate the possibility that the test item has an influence on the activated sludge. The pre-test showed no influence of the test item on the activated sludge.

Test performance:

The test flasks prepared were incubated at 25 ± 1 °C. The pressure decrease in the reaction vessels was measured over complete experimental phase of 56 days. The test flasks were closed gas-tight by a measuring head. Potassium hydroxide solution (45%) was used for trapping the produced carbon dioxide. The amount of O2 consumed by the activated sludge was calculated from the decrease of pressure in the reaction vessel.

Parameter:

% degradation (O2 consumption)

Remarks:

Result for ThOD(NO3) and ThOD(NH4)

Value:

0

Sampling time:

24 d

Remarks on result:

other: ThODNH4 of N-Butylpyrrolidone: 2.379 mg O2/mg test item, ThODNO3 of N-Butylpyrrolidone: 2.833 mg O2/mg test item

Parameter:

% degradation (O2 consumption)

Remarks:

Results for ThOD(NO3) and ThOD(NH4), respectively

Value:

13.7 - 16.3

Sampling time:

28 d

Remarks on result:

other: ThODNH4 of N-Butylpyrrolidone: 2.379 mg O2/mg test item, ThODNO3 of N-Butylpyrrolidone: 2.833 mg O2/mg test item

Parameter:

% degradation (O2 consumption)

Remarks:

Results for ThOD(NO3) and ThOD(NH4), respectively

Value:

83 - 98.9

Sampling time:

36 d

Remarks on result:

other: ThODNH4 of N-Butylpyrrolidone: 2.379 mg O2/mg test item, ThODNO3 of N-Butylpyrrolidone: 2.833 mg O2/mg test item

Parameter:

% degradation (O2 consumption)

Remarks:

Results for ThOD(NO3) and ThOD(NH4), respectively

Value:

112.2 - 133.6

Sampling time:

56 d

Remarks on result:

other: ThODNH4 of N-Butylpyrrolidone: 2.379 mg O2/mg test item, ThODNO3 of N-Butylpyrrolidone: 2.833 mg O2/mg test item

Details on results:

N-Butylpyrrolidone had an extended lag phase in this biodegradation test of more than 24 days. Between days 24 and 28, the biodegradation started and was at about 99% on day 36.The time between more than 10% degradation (around day 25) and 60% degradation (around day 33) was about 8 days. Complete degradation (>99%) was reached on day 36, about 12 days after the 10% limit was exceeded. After 56 days of incubation, the test sample was found to be biodegradable to an excess of 100% (ThODNH4, ThODNO3) based on BOD-measurement. This additional oxygen consumption can be attributed to the release of most of the organic carbon ‘trapped’ in the sludge, e.g. lysis of sludge microorganisms.

Results with reference substance:

The reference item sodium benzoate was sufficiently degraded to 87.2% after 4 days and to 97.8% after 12 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used. The degradation rate of sodium benzoate was clearly within the pass level for validity (>65% degradation within 14 days).

Table 1. Cumulative Biochemical Oxygen Demand (mg O2/L) in Test Flasks during the Test Period of 56 days

Cumulative O2-Consumption [mg/TestVessel]

Days		Test item		Control		Sodium Benzoate	Toxicity Control	Abiotic Control
Flask	1	2	3	4	5	6	8	7
0	0.000	0.000	0.000	0.000	0.000	0.000	0.000	0.000
4	9.054	9.418	8.693	8.697	9.059	52.166	55.428	-3.262
8	14.846	14.850	14.123	13.768	14.853	61.582	65.211	-2.538
12	18.104	17.384	17.381	17.026	17.750	65.926	68.108	-3.986
16	20.638	19.556	19.915	19.922	20.285	65.564	67.746	-4.710
20	22.086	21.728	21.363	21.732	22.459	65.202	67.384	-2.537
24	23.172	23.176	23.173	23.542	23.547	65.202	66.660	-1.451
28	27.518	27.159	29.690	24.628	24.636	65.202	67.022	-0.365
32	38.386	38.750	36.570	26.076	25.363	65.202	66.660	-0.727
36	48.893	47.084	47.436	26.800	26.448	64.840	67.022	-1.089
40	52.151	49.620	52.867	27.524	27.174	64.840	67.022	-3.262
44	54.686	52.517	56.487	28.610	27.537	64.840	67.022	-3.623
48	56.496	54.327	58.297	29.334	28.623	64.840	67.022	-2.174
52	58.306	55.775	59.383	30.058	28.986	64.840	67.022	-0.364
56	59.754	56.862	60.469	30.782	30.074	65.202	66.660	-0.002

 

Table 2. Corrected Cumulative Biochemical Oxygen Demand (mg O2/L) in Test Flasks During the Test Period of 56 Days Cumulative O2-Consumption [mg/Test Vessel] Mean Values

Days	TS 28 d	Control	Sodium Benzoate	Tox.Control	Abiotic Control
0	0.000	0.000	0.000	0.000	0.000
4	9.055	8.878	52.166	55.428	-3.262
8	14.606	14.311	61.582	65.211	-2.538
12	17.623	17.388	65.926	68.108	-3.986
16	20.036	20.104	65.564	67.746	-4.710
20	21.726	22.096	65.202	67.384	-2.537
24	23.174	23.545	65.202	66.660	-1.451
28	28.122	24.632	65.202	67.022	-0.365
32	37.902	25.720	65.202	66.660	-0.727
36	47.804	26.624	64.840	67.022	-1.089
40	51.546	27.349	64.840	67.022	-3.262
44	54.563	28.074	64.840	67.022	-3.623
48	56.373	28.979	64.840	67.022	-2.174
52	57.821	29.522	64.840	67.022	-0.364
56	59.028	30.428	65.202	66.660	-0.002

 

Table 3. Percentage Biodegradation of Test Item, of Sodium Benzoate and of the Toxicity Control based on ThODNH4 and ThODNO3

% degradation based on ThODNH4                            % degradation based on ThODNO3          

Days	Test Item^1	Sodium Benzoate	Tox.Control	Days	Test Item^1	Sodium Benzoate	Tox.Control
0	0.0	0.0	0.0	0	0.0	0.0	0.0
4	1.1	87.2	65.4	4	0.9	87.2	61.8
8	1.7	95.2	71.5	8	1.4	95.2	67.6
12	1.1	97.8	71.2	12	0.9	97.8	67.3
16	0.8	91.6	66.9	16	0.7	91.6	63.3
20	0.0	86.8	63.6	20	0.0	86.8	60.1
24	0.0	83.9	60.5	24	0.0	83.9	57.3
28	16.3	81.7	59.5	28	13.7	81.7	56.3
32	56.9	79.5	57.5	32	47.8	79.5	54.4
36	98.9	77.0	56.7	36	83.0	77.0	53.6
40	113.0	75.5	55.7	40	94.9	75.5	52.7
44	123.7	74.1	54.7	44	103.9	74.1	51.7
48	127.9	72.2	53.4	48	107.4	72.2	50.5
52	132.2	71.1	52.7	52	111.0	71.1	49.8
56	133.6	70.0	50.9	56	112.2	70.0	48.1

ThODNH4 of N-Butylpyrrolidone: 2.379 O2/mg test item

ThODNO3 of N-Butylpyrrolidone: 2.833 mg O2/mg test item

ThODNH4 of sodium benzoate: 1.666 mg O2/mg reference item

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable, not fulfilling specific criteria

Conclusions:

It can be concluded that the test item N-Butylpyrrolidone is completely primary biodegradable after a lag phase of four weeks resulting in 100% degradation after 56 days of incubation. 55% of the test item could be detected 28 days after test start; at the end of the test at day 56, the concentration of the test item was below the limit of detection. The absence of detectable degradation products confirmed that the test item was completely degraded.

Executive summary:

The test item N-Butylpyrrolidone was investigated for its inherent biodegradability in a modified MITI Test (II) over a period of 56 days. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. For the assessment of primary degradation, the test item concentration was determined in aqueous phase as well as the adsorption on the sludge. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. The grade of primary degradation was assessed by measurement of the test item concentration in the test vessels by a substance-specific analysis. This study is recognized by the OECD guideline (302C) and should provide a rational basis to assess the inherent biodegradation properties of the test item when incubated with activated sludge.

Results:

ThODNH4: Based on the BOD measurement, the biodegradation of the test sample was above 100% after 56 days of incubation. ThODNO3: Based on the BOD measurement, the biodegradation of the test sample was above 100% after 56 days of incubation. The primary degradation, measured by a test item specific GC-method, resulted in 100% degradation after 56 days, the concentration of test item was below the limit of detection. It can be concluded that the test item N-Butylpyrrolidone is completely primary biodegradable after a lag phase of four weeks resulting in 100% degradation after 56 days of incubation.

The reference item sodium benzoate was sufficiently degraded to 87.2% after 4 days and to 97.8% after 12 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

Description of key information

Based on the results of the Closed Bottle Test, N-Butylpyrrolidone is not readily biodegradable.

In a further study to characterise inherent biodegradability (Zahn-Wellens-Test), the degree of biodegradation reached 7 % after 28 days. Mean degradation at the end of the test (after 112 days) revealed 81 % (lag-phase day 0 - 35, degradation phase day 36 -112).  Hence the substance can be assigned as ultimately biodegradable.

In the second study on the substance’s inherent biodegradability (modified MITI Test (II)), N-Butylpyrrolidone was found to be completely primary biodegradable after a lag phase of four weeks resulting in 100% degradation after 56 days of incubation. This test results are further confirmed by the results of a modified MITI Test (II) over a period of 28 days, which showed no biodegradation.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable, not fulfilling specific criteria

Additional information

Regarding biodegradability, four studies are available for N-Butylpyrrolidone: Ready biodegradability was investigated in a Closed Bottle Test (OECD 301D), whereas in a consecutive test (OECD 302B), possible inherent biodegradation of the compound was characterised. In addition, another study (OECD 302C) to further investigate the inherent biodegradability of the test substance was performed.

The ready biodegradability study (Closed bottle test; Muckle, 2013) was performed according to OECD Guideline 301D (1992) and EU Method C.4-E (2008). This study was performed in order to evaluate aerobic elimination and degradation potential of N-Butylpyrrolidone in a test for ready biodegradability, using a test item concentration of 2.08 mg/L (corresponding to a theoretical oxygen consumption of 5.90 mg/L). The solution of the test item in mineral medium was inoculated with a relatively small number of micro-organisms from a mixed population and kept in completely full, closed bottles in the dark at constant temperature. Degradation was followed by analysis of dissolved oxygen over a 28-d period. The amount of oxygen taken up by the microbial population during biodegradation of the test substance, corrected for uptake by the blank inoculum run in parallel, is expressed as a percentage of COD. As final result, no biodegradation was observed after 28 days neither with nor without consideration of nitrification. A 10-day-window could not be detected. The positive control reached 67 % degradation on day 8.

A Zahn-Wellens Test was performed with N-Butylpyrrolidone to characterise inherent biodegradation potential of the compound (Muckle, 2013). The test item was tested using a concentration of 200 mg organic carbon/L (nominal) in test medium following OECD 302B/EU-Guideline C.9-C. Aniline was chosen as positive control. Activated sludge from a sewage treating plant was used as inoculum (concentration 601 mg dry matter/L). The test was left running for 112 days (sponsor's intent). To measure the degradation of test item and positive control, the content of dissolved organic carbon (DOC) in the test vessels was measured twenty times during the test. All validity criteria were met. Degradation of the positive control was 98 % after 8 days. The following results could be determined for the test item N-Butylpyrrolidone: The degree of biodegradation reached 7 % after 28 days. Mean degradation at the end of the test (112 days) was 81 % (lag-phase day 0 - 35, degradation phase day 36 -112). As degradation in the toxicity flask was 50 % on day 14, the test item N-Butylpyrrolidone can be stated as "not toxic towards the inoculum in a concentration of 306.2 mg/L".

 

The test item N-Butylpyrrolidone was further investigated for its inherent biodegradability in a modified MITI Test (II) over a period of 56 days (Hammerfahr and Münz, 2015). The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. For the assessment of primary degradation, the test item concentration was determined in aqueous phase as well as the adsorption on the sludge. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. The grade of primary degradation was assessed by measurement of the test item concentration in the test vessels by a substance-specific analysis. This study is recognized by the OECD guideline (302C) and should provide a rational basis to assess the inherent biodegradation properties of the test item when incubated with activated sludge. The following results were obtained:

- ThODNH4: Based on the BOD measurement, the biodegradation of the test sample was above 100% after 56 days of incubation.

-ThODNO3: Based on the BOD measurement, the biodegradation of the test sample was above 100% after 56 days of incubation. The primary degradation, measured by a test item specific GC-method, resulted in 100% degradation after 56 days, the concentration of test item was below the limit of detection.

It can be concluded that the test item N-Butylpyrrolidone is completely primary biodegradable after a lag phase of four weeks resulting in 100% degradation after 56 days of incubation. The reference item sodium benzoate was sufficiently degraded to 87.2% after 4 days and to 97.8% after 12 days of incubation, thus confirming the suitability of the aerobic activated sludge inoculum used.

The test item N-Butylpyrrolidone was investigated in a further supporting study for its inherent biodegradability in a modified MITI Test (II) over a period of 28 days. The purpose of the study was to determine the Inherent Biodegradability of the test substance in the dark at 25°C ± 2°C. The biodegradation was followed by the oxygen uptake of the microorganisms during exposure. As a reference item sodium benzoate was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. This study is recognized by the OECD guideline (302C) and should provide a rational basis to assess the inherent biodegradation properties of the test item when incubated with activated sludge.

Results:

Under the conditions of the study, percentage biodegradation rate of the test substance on day 28 which was calculated with BOD was 3.0%, DOC removal rate was 11.4%, primary biodegradation rate was -0.8%.

Sodium benzoate was used for reference substance, the percentage biodegradation of the procedure control was 78.5% (>40%) on 7d and was 85.5% on 14d (>65%) of the test. At the end of the test, test substance residual concentration in abiotic control was 28.1mg/L, 93.7% of initial concentration, which was greater than 10%. The above results revealed that the test results were valid.

The results of this study are in line with the results of the inherent biodegradation Key study described above, as they confirm the existence of a lag phase of about 4 weeks (28 days).