Butanal, reaction products with aniline

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

est conducted with 100 mg/L activated sludge (dw/L) from three different sources; test item concentration = 15 mg/L

Principles of method if other than guideline:

As deviating from the guideline the test was conducted with 100 mg/L activated sludge (dw/L) from three different sources and a test item concentration of 15 mg/L.

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

other: Activated sludge from two different wastewater treatment plants treating predominantly domestic wastewater and activated sludge from a wastewater treatment plant treating predominantly wastewater of industrial origin

Details on inoculum:

- mixed population of aquatic microorganisms, origin from the aeration tank 3 different wastewater plant treating predominantly domestic or industrial waste water. (domstic WWTPS: Wupper area water authority, WWTP Odenthal, and WWTP Cologne-Stammheim, industrial: WWTP Leverkusen Bürrig
- The three sludge types were mixed taking 2 parts from each of the two domestic WWTPS plus 1 part from the industrial WWTP.
- Date of collection: 2013-03-19

Pre-treatment of the inoculum
− Before use, the inoculum was stored for three days at room temperature under continuous shaking with aeration.
− The sludges of the three different origins were mixed.
− The combined sludge was washed twice by adding mineral medium and centrifuging for 10 min at 2000 rpm and 20 °C and decanting off the supernatant.
− An aliquot of the wet sludge was dried in order to determine the wet weight / dry weight ratio of the sludge and to prepare a stock suspension with a defined concentration of suspended solids.
− The calculated amount of sludge, needed to achieve 300 mL of this stock suspension of 5 g dw/L, was dissolved in mineral medium and then filled up to a defined end volume.

Duration of test (contact time):

56 d

Initial conc.:

15 mg/L

Based on:

test mat.

Details on study design:

- Pre-treatment of the test item
3.75 mg of the test item were added to the test flasks, filled with 200 mL of mineral medium. Afterwards the volume was made up to 250 mL with mineral medium plus inoculum to give a final concentration of 15 mg test item/L and a final concentration of suspended solids of 100 mg/L.
- Pre-treatment of the reference compound
25 mg of the reference compound were added to the test flasks, filled with 200 mL of mineral medium. Afterwards the volume was made up to 250 mL with mineral medium plus inoculum to give a final concentration of 100 mg reference compound/L and a final concentration of suspended solids of 30 mg/L.
- Exposure conditions
Test volume : 250 mL, Test apparatus : OxiTop System (WTW), Mixing : 1 magnetic stirrer per test vessel, Incubation time : 56 days, Incubation temperature : 25 ± 2 °C
- CONTROL AND BLANK SYSTEM
- Inoculum blank
- The endogenous activity of the inoculum was checked running parallel blanks with inoculum but without test item
- A toxicity control (test item and reference compound mixed) was not run in parallel, because the chosen concentration of the test item was not inhibitory to microorganisms (see Biodegradation study Richter, 2011 report no.:2011/0032/06).

Reference substance:

benzoic acid, sodium salt

Remarks:

(Sigma-Aldrich, purity 99.7 %, Batch-no. 1438955)

Parameter:

% degradation (O2 consumption)

Value:

39

Sampling time:

56 d

Details on results:

Kinetic of test item ( % Degr.):
3 after 7 days
15 after 14 days
18 after 28 days
23 after 42 days
31 after 49days
39 after 56 days

Parameter:

COD

Value:

3.026 mg O2/g test mat.

Results with reference substance:

Kinetic of reference substance ( % Degr.):
73 after 7 days
78 after 14 days
90 after 50 days
82after 56 days

Validity criteria fulfilled:

yes

Remarks:

(-The percent degradation of the reference compound sodium benzoate reached the level of ≥ 40 % after 7 days and ≥ 65 % after 14 days..)

Interpretation of results:

not inherently biodegradable

Conclusions:

The test item is '' Not Inherently Biodegradable''. After 56 days, a degradation rate of 39 % was determined.

Executive summary:

The study was conducted in accordance with the OECD Guideline 302C “Inherent Biodegradability: Modified MITI Test (II)” with fresh inocula from different sources. A solution of the test item in a mineral medium (containing a known concentration of 15 mg/L of the test item was stirred in a closed flask at a constant temperature (25 ± 2 °C) under aerobic conditions in the dark)

was inoculated with 40 % sludge of each of two different municipal sewage treatment plants (STP) and 20 % sludge of an industrial STP and incubated under aerobic conditions. During this period, degradation was followed by continuous automated BOD determinations.

The consumption of oxygen (BOD) was determined by measuring the drop in pressure in the automated respirometer flasks. Evolved carbon dioxide was absorbed in sodium hydroxide. The amount of oxygen taken up by the test item (corrected for uptake by blank inoculum, run in parallel) was expressed as a percentage of the chemical oxygen demand (COD).

The test was prolonged until 56 days as the biodegradation curve showed that biodegradation had started but that the plateau had not been reached.

The endogenous activity of the inoculum was checked running parallel blanks with inoculum but without test item. A reference compound (sodium benzoate) was run in parallel to check the operation of the procedures.

A toxicity control (test item and reference compound mixed) was not run in parallel, because the chosen concentration of the test item was not inhibitory to microorganisms (see Biodegradation study Richter, 2011 report no.:2011/0032/06).

Because of the nature of biodegradation and of the mixed bacterial populations used as inoculum, determinations of test item were carried out in quadruplicate, for the inoculum blank in triplicate and for the reference compound in duplicate.

The oxygen uptake was calculated from the readings taken at regular and frequent intervals, using the method given by the manufacturer of the equipment. At the end of incubation, the pH was measured in the flasks.

The degradation rate of the test item was set in relation to its experimentally determined COD. As the COD implicitly covers the oxygen demand for the nitrification process, there was no need to take additional correction measures into account.

Reference 2

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Deviating from the methods a second test concentration (0.5 mg/L) was tested.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

Deviating from the methods a second test concentration (0.5 mg/L) was tested, the test runs for 40 days and was stirred during the test.

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Type: mixed population of aquatic microorganisms
- Origin: secondary effluent of a wastewater treatment plant treating predominantly domestic sewage (Wupper area water authority, WWTP Odenthal, Germany)
- Date of collection: 2014-02-10
- Pre-treatment: separation of coarse particles by filtration, aeration of the resulting inoculum for 2 days
- Effluent concentration of reaction mixture: 3.85 mL/L (1 mL/0.26 L)

Duration of test (contact time):

40 d

Initial conc.:

0.5 mg/L

Based on:

test mat.

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

PRE TREATMENT OF THE TEST ITEM:
- 0.13 respectively, and 0.52 mg of the test item were weighed out and added to the test flasks, filled with ca. 200 mL of mineral medium. Afterwards the inoculum was added and the flasks volume was made up to 0.26 L with mineral medium and the flasks were closed free from air bubbles with glass stoppers.
- Initial concentration of test item (2): 0.5 mg/L (0.13 mg/0.26 L)/2.0 mg/L (0.52 mg/0.26 L)

TEST SYSTEM
- Culturing apparatus: closed flasks with a test volume of 250 mL
- Number of culture flasks/concentration: 2 (test item & reference compound, inoculum blank)
- Test performed in closed vessels: Yes
- Test performed in open system: not applicable
- Incubation temperature : 22 ± 2 °C
- Continous darkness: yes
- Incubation time : 40 days
- pH value after 40 days: 7.0-7.1

CONTROL AND BLANK SYSTEM
- Inoculum blank: Blanks with inoculum were run in parallel
- Test substance: The concentrations of the test item were 0.5 and 2.0 mg/L and the test on degradation was run in parallel
- Reference substance: A reference compound (sodium benzoate) was run in parallel, to check the operations of the procedures. The concentration of the reference substance used in the test was 2.9 mg/L

SAMPLING:
- Sampling frequency: The BOD (in mg O2/L) was measured on day 0, 7, 14, 21, 28, 35 and 40

EXPOSURE CONDITIONS:
- Test volume : 0.26 L
- Incubation time : 40 days
- Incubation temperature : 22 ± 2 °C

Reference substance:

benzoic acid, sodium salt

Remarks:

(Purity: 99.7 %, Batch no.: 1438955 (Sigma-Aldrich) )

Parameter:

% degradation (O2 consumption)

Value:

37

Sampling time:

14 d

Remarks on result:

other: Test item concentration: 0.5 mg/L

Parameter:

% degradation (O2 consumption)

Value:

15

Sampling time:

28 d

Remarks on result:

other: Test item concentration: 0.5 mg/L

Parameter:

% degradation (O2 consumption)

Value:

16

Sampling time:

14 d

Remarks on result:

other: Test item concentration: 2 mg/L

Parameter:

% degradation (O2 consumption)

Value:

16

Sampling time:

28 d

Remarks on result:

other: Test item concentration: 2 mg/L

Details on results:

Degradation rates after 40 days:
15 % (O2 consumption/Test item concentration: 0.5 mg/L)
9 % (O2 consumption/Test item concentration: 2 mg/L)

Results with reference substance:

The reference compound sodium benzoate showed 73 % degradation after 14 days.

Validity criteria fulfilled:

yes

Remarks:

(biodegradation in parallels with test item did not differ by more than 20 percentage points; ref. compound >= 60 % for ready biodegradability within 14 days)

Interpretation of results:

not readily biodegradable

Conclusions:

Butanal, reaction products with aniline showed degradation rates of 15 respectively, and 9 % after 40 days depending on the particular test concentration of 2 and 7 mg/L. Therefore, butanal, reaction products with aniline is considered to be “Not Readily Biodegradable“. The reference compound sodium benzoate showed 73 % degradation after 14 days.

Executive summary:

The study was conducted in accordance with the Council Regulation (EC) 440/2008 Method C.4-E “Closed Bottle Test” (2008). This test method is in all essential parts identical with OECD Guideline 301 D (adopted July 1992).

The test item butanal, reaction products with aniline in a mineral medium was inoculated with secondary effluent to result in completely filled, closed flasks and incubated under aerobic conditions for 40 days. During this period, the biodegradation was followed by analysis of dissolved oxygen. The amount of oxygen taken up by the test chemical, corrected for uptake by the blank inoculum run in parallel, was expressed as a percentage of chemical oxygen demand (COD). Degradation rates between 15 and 9 % have been precisely measured depneding on the particular test concentration of 0.5 and 2 mg/L, which indicates that butanal, reaction products with anilin is not readily biodegradable under the conditions of the test.

Reference 3

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Deviating from the method a solubiliser was added to the test item.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

Deviating from the method a solubiliser was added to the test item, the test runs for 40 days and was stirred during the duration of the test.

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic (adaptation not specified)

Details on inoculum:

- Type: mixed population of aquatic microorganisms
- Origin: secondary effluent of a wastewater treatment plant treating predominantly domestic sewage (Wupper area water authority, WWTP Odenthal, Germany)
- Date of collection: 2014-02-10
- Pre-treatment: separation of coarse particles by filtration, aeration of the resulting inoculum for 3 days
- Effluent concentration of reaction mixture: 3.85 mL/L (1 mL/0.26 L)

Duration of test (contact time):

40 d

Initial conc.:

2 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

PRE TREATMENT OF THE TEST ITEM:
- 0.52 mg of the test item were weighed out and added to the test flasks, filled with ca. 200 mL of mineral medium. Additionally a solubiliser (Baywowet FT 248 [CAS: 29420-49-3, 51 % solution in water]) was added. Therefore a stock solution was prepared. 521.4 mg/ 50 mL were weighed in and added to each flask for a final concentration of 10.4 mg/ 0.26 mL. Afterwards the inoculum was added and the flasks volume was made up to 0.26 L with mineral medium and the flasks were closed free from air bubbles with glass stoppers.
- Initial concentration of test item : 2 mg/L (0.52 mg/0.26 L)

TEST SYSTEM
- Culturing apparatus: closed flasks with a test volume of 250 mL
- Number of culture flasks/concentration: 2 (test item & reference compound, inoculum blank)
- Test performed in closed vessels: Yes
- Test performed in open system: not applicable
- Incubation temperature : 22 ± 2 °C
- Continous darkness: yes
- Incubation time : 40 days
- pH value after 40 days: 6.8-7.1

CONTROL AND BLANK SYSTEM
- Inoculum blank: Blanks with inoculum were run in parallel
- Test substance: The concentration of the test item was 2.0 mg/L and the test on degradation was run in parallel
- Reference substance: A reference compound (sodium benzoate) was run in parallel, to check the operations of the procedures. The concentration of the reference substance used in the test was 2.9 mg/L

SAMPLING:
- Sampling frequency: The BOD (in mg O2/L) was measured on day 0, 7, 14, 21, 28, 35 and 40

EXPOSURE CONDITIONS:
- Test volume : 0.26 L
- Incubation time : 40 days
- Incubation temperature : 22 ± 2 °C

Reference substance:

benzoic acid, sodium salt

Remarks:

(Purity: 99.7 %, Batch no.: 1438955 (Sigma-Aldrich))

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

7 d

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

14 d

Parameter:

% degradation (O2 consumption)

Value:

36

Sampling time:

21 d

Parameter:

% degradation (O2 consumption)

Value:

17

Sampling time:

28 d

Details on results:

The incubation temperature exceeded the range of 22 +/- 2°C once on day 4. As the degradation values of the reference on day 7 showed expected and plausible results this deviation had no effects on the result.

Results with reference substance:

The reference compound sodium benzoate showed 83 % degradation after 14 days.

Validity criteria fulfilled:

yes

Remarks:

(O2-depletion in the blank control <= 1.5 mg dissolved O2/L after 28 days; biodegradation in parallels with test item did not differ by more than 20 percentage points; ref. compound >= 60 % for ready biodegradability within 14 days)

Interpretation of results:

not readily biodegradable

Conclusions:

Butanal, reaction products with aniline showed a degradation of 17 % after 28 days. Therefore, butanal, reaction products with aniline is considered to be “Not Readily Biodegradable“. The reference compound sodium benzoate showed 83 % degradation after 14 days.

Executive summary:

The study was conducted in accordance with the Council Regulation (EC) 440/2008 Method C.4-E “Closed Bottle Test” (2008). This test method is in all essential parts identical with OECD Guideline 301 D (adopted July 1992). The test item in a mineral medium was inoculated with secondary effluent to result in completely filled, closed flasks and incubated under aerobic conditions for 40 days. During this period, the biodegradation was followed by analysis of dissolved oxygen. The amount of oxygen taken up by the test chemical, corrected for uptake by the blank inoculum run in parallel, was expressed as a percentage of chemical oxygen demand (COD). Butanal, reaction products with aniline showed a degradation of 17 % after 28 days. Therefore, butanal, reaction products with aniline is considered to be “Not Readily Biodegradable“. The reference compound sodium benzoate showed 83 % degradation after 14 days.

Reference 4

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- mixed population of aquatic microorganisms, origin from the aeration tank of a wastewater plant treating predominantly domestic sewage
(Wupper area water authority, WWTP Odenthal, Germany)
- Date of collection: 2011-05-03
- activated sludge concentration in test flasks: 30 mg/L ss

Pretreatment
- the sludge was washed twice by adding mineral medium, centrifuging (10 min at 2000 rpm and 20 °C) and decanting off the supernatant
- wet sludge was dried in order to determine the wet weight / dry weight ratio of the sludge and to prepare a stock suspension (activated sludge) of 3 g dw/L.
- the calculated amount of sludge, needed to achieve 300 mL of this stock suspension, was dissolved in mineral medium and than filled up to a defined end volume
- the inoculum was stored for one day at room temperature under continuous shaking with aeration.

Duration of test (contact time):

28 d

Initial conc.:

100 mg/L

Based on:

test mat.

Details on study design:

INITIAL CONCENTRATION:
- Test substance: 100 mg/l
- Reference substance: 100 mg/l

TEST CONDITIONS
- Test temperature: (22 ± 1 °C)
- Incubation: 28d under aerobic conditions
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: OxiTopControl System (WTW)
- Flask: closed flask (automated respirometer flasks)
- Test volume: 250 mL
- Mixing: 1 magnetic stirrer per test vessel

SAMPLING
- Sampling frequency: dayly

CONTROL AND BLANK SYSTEM
- Inoculum blank
- The endogenous activity of the inoculum was checked running parallel blanks with inoculum but without test item
- A toxicity control (test item and reference compound mixed) was run in parallel, to ensure that the chosen concentration of the test item was not inhibitory to microorganisms

Reference substance:

benzoic acid, sodium salt

Remarks:

Sigma-Aldrich, purity 99.7 %, Batch-no. 1438955

Parameter:

% degradation (O2 consumption)

Value:

24

Sampling time:

28 d

Details on results:

Kinetic of test item ( % Degr.):
4 after 7 days
14 after 14 days
21 after 21 days
24 after 28 days

Parameter:

COD

Value:

3.026 other: mg O2/mg

Results with reference substance:

Kinetic of reference substance ( % Degr.):
84 after 7 days
92 after 14 days
93 after 21 days
93 after 28 days

Validity criteria fulfilled:

yes

Remarks:

(-Ready biodegradability of reference compound 60 percent within 14 days. -In the toxicity control degradation rates > 25 % within 14 days. -Replicates difference< 20% . - Oxygen uptake of blank inoculum=< 60mg/l. -No pH influence.)

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test item is '' Not Readily Biodegradable''. After 28 days, a degradation rate of 24 % was determined.

Executive summary:

According to OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test) a degradation of 24% was determined for the test item.

A suspension of 100 mg/L test item in a mineral medium, equalling to 50-100 mg COD/Litre as the nominal sole source of organic carbon, was stirred in a closed flask and inoculated at a constant temperature (22 ± 1 °C) for up to 28 days under aerobic conditions in the dark.

The consumption of oxygen (BOD) was determined by measuring the drop in pressure in the automated respirometer flasks. Evolved carbon dioxide was absorbed in sodium hydroxide. The amount of oxygen taken up by the test item was expressed as a percentage of chemical oxygen demand (COD).

Degradation was followed by the determination of oxygen uptake and measurements were taken at frequent intervals to allow the identification of the beginning and end of biodegradation and the slope of the biodegradation curve.

Because of the nature of biodegradation and of the mixed bacterial populations used as inoculum, determinations of test item were carried out in fourfold, inoculum blank in triplicate, reference compound in duplicate.

The oxygen uptake was calculated from the readings taken at regular and frequent intervals, using the method given by the manufacturer of the equipment. At the end of incubation, the pH was measured in the flasks.

The degradation rate of the test item was set in relation to its experimentally determined COD. As the COD implicitly covers the oxygen demand for the nitrification process, there was no need to take additional correction measures into account.

Because of the nature of biodegradation and of the mixed bacterial populations used as inoculum, determinations of test item were carried out in fourfold, inoculum blank in triplicate, reference compound in duplicate.

The oxygen uptake was calculated from the readings taken at regular and frequent intervals, using the method given by the manufacturer of the equipment. At the end of incubation, the pH was measured in the flasks.

The degradation rate of the test item was set in relation to its experimentally determined COD. As the COD implicitly covers the oxygen demand for the nitrification process, there was no need to take additional correction measures into account.

The 28-day degradation was 25% and for reference substance the biodegrdation was 92% after 14 days. In conclusion the substance is Not Readily Biodegradable.

Description of key information

Three studies  concerning the "Ready Biodegradabilty"  with different variations were conducted. In a ready biodegradation test according to OECD 301F a degradation of 24% was reported. Additionally two tests according to OECD 301D (closed bottle test) were performded. In one of the latter tests two test concentrations (0.5/2 mg/L) of butanal, reaction products with aniline were tested with a test period of 40 days. In another experiment a test concentration of 2 mg/L was examined by using a solubilizer. During the 28 day test period lasting the test substance was continuously stirred. However, only degradation rates up to 37%  were measured.  In another test to check the inherent biodegradability, a degradation rate of 39 % was determined  after 56 days. 

Key value for chemical safety assessment

Biodegradation in water:

under test conditions no biodegradation observed

Type of water:

freshwater

Additional information

Biodegradation tests according to the OECD 301 and 302 series require substance concentrations in the range of 30 to 100 mg/L. Poorly soluble substances with a solubility of 1 mg/L or lower, however, are not bioavailable for degradation via bacteria. Typically they are adsorbed to surfaces of the vessels or to particles. Butanal, reaction products with aniline is considered to be poorly soluble. Only after addition of 1% acetonitrile a solubility of 5.2 mg/L could be detected. However, biodegradation tests with lower concentration in the range of the water solubility according to OECD 301D (closed bottle) with concentrations of 0.5 to 2 mg/L did not show higher degradation rates.