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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
26 August to 08 October 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
This study was performed according to OECD Guideline 301B with GLP statement. All validity criteria were fulfilled.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Specific details on test material used for the study:
- Storage condition of test material: Keep in the dark, at 4 °C, under N2
- Stability under test conditions: Stable in container after opening/in light
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge: Aeration tank of Liede Sewage Treatment Plant of Guangzhou
- Batch No. IAs20140820-1
- Treatment: The sludge was removed any coarse particles and impurities on the surface, then was washed with the test medium (1100 g, 10 min, repeated for 6 times), the supernatant was decanted and the suspended solids were re-suspended in the test medium. The dry weight of the suspended solids was determined as 4.2 g/L. Before the test, 143 mL of the above inoculum was taken to triangular flask and suspended in 7 mL of the test medium to obtain a concentration equivalent to 4.0 g suspended solids per liter. Kept the sludge aerobic until required. 15.0 mL of the above inoculum was added into each test vessel to give a final concentration of 30 mg suspended solids per liter.
Duration of test (contact time):
28 d
Initial conc.:
17.6 mg/L
Based on:
TOC
Remarks:
12.3 mg/L
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
PREPARATION OF THE TEST SUBSTANCE STOCK SOLUTION
178 mg of the test substance was weighted into triangular flask, added 700 mL of the test medium and mixed for 2 h, then transferred to 1000 mL volumetric flask and where was diluted with test medium to 1000 mL, and the stock solution with the effective concentration of 176 mg/L was obtained (the purity of the test substance is 98.9%).

TEST CONDITIONS
- Composition of medium: Stock solution a (in water): 1 L contains 8.50 g KH2PO4, 28.50 g K2HPO4. 3H2O, 67.15 g Na2HPO4.12H2O and 0.5 g NH4CI
Stock solution b (in water): 0.5 L contains 13.75 g CaCl2
Stock solution c (in water): 0.5 L contains 11.25 g MgSO4.7H2O
Stock solution d (in water): 0.5 L contains 0.125 g FeCl3.6H2O
180mL of solution (a) was mixed with about 16 L of deionized water, then 18 mL of solutions (b), (c) and (d) were added respectively and then made up to 18 L with deionized water.
- The test was started by bubbling CO2-free air through the suspensions at a rate of 30-100 mL/min at 22 ± 2 °C (20.9-22.5 °C) in the dark.
- Suspended solids concentration: 4.2 g/L
- Concentration of the inoculum: 30 mg/L
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: 3 L flasks, each fitted with an aeration tube reaching nearly to the bottom of the vessel and an outlet
- Number of culture flasks/concentration: 2 (Flasks 1 & 2 (test suspension) containing test substance, inoculum and test medium)
- Addition of the test medium and the inoculum: 1500 mL of the test medium were added into flask and the inoculum 1, 2, 3, 4, 5, 6 and A, and then 15.0 mL of the 4.0 g/L inoculum were added respectively. These mixtures were aerated with CO2-free air overnight.
- Addition of the test substance stock solution: 200 mL of the test substance stock solution were added directly into flasks 1, 2 and 6 respectively.
- Addition of the test medium: The final volumes of the suspensions in flasks 1, 2, 3, 4, 5, 6 and A were made up to 2000 mL by the addition of the test medium previously aerated with N2.

ANALYSIS AND DETERMINATION
Determination of the content of TIC: before the study, 25.0 mL of the solution from flask A was filtered through 0.45 μm membrane and the first 5 mL of the filtrate was discarded, then the residual filtrate was used for analysis.
Determination of the amount of CO2 produced: analyses of CO2 were made on Days 2, 4, 7, 10, 14, 17, 21, 24 and 29.
On the days of CO2 measurement, the barium hydroxide absorber closest to the test vessel was disconnected and the hydroxide solution was titrated with 0.05098 mol/L and 0.05085mol/L HCl using phenolphthalein as the indicator. The remaining absorbers were moved one place closer to the test vessel and a new absorber containing 100 mL of 0.0125 mol/L fresh barium hydroxide was placed at the far end of the series.
On day 28, 1 mL of concentrated hydrochloric acid were added into each test vessel and aerated them overnight to drive off the carbon dioxide present in the test suspensions. On day 29, the last analyses of evolved carbon dioxide were made.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Flasks 3, 4 & A (inoculum blank) containing only inoculum and test medium
- Procedure control: Flask 5 (procedure control) containing reference substance, inoculum and test medium
- Toxicity control: Flask 6 (toxicity control) containing test substance, reference substance, inoculum and test medium
Reference substance:
benzoic acid, sodium salt
Remarks:
21.1 mg/L; TOC concentration 12.3 mg/L
Key result
Parameter:
% degradation (CO2 evolution)
Value:
52.3
Sampling time:
28 d
Details on results:
Biodegradation of the toxicity control after 14 and 28 days were 38.6 and 61.6%, respectively, which showed that the test substance was considered not to have a toxic effect on the sewage sludge micro-organisms used in the study.
At the end of the test, the percentage biodegradation of the two replicates of the test substance were 52.6% and 52.0%, with an average of 52.3%.
Results with reference substance:
Degradation of sodium benzoate after 14 and 28 days were 77.9 and 88.7%, respectively: the activity of the inoculum was thus verified (validity criterion).

Table 5.2.1/2: TIC concentration of the test medium and the ratio of TIC and TC

 

Test groups

Test substance

Reference substance

Test medium

TC concentration

of the test system

mg/L)

TIC/TC%

TIC concentration

mg/L)

TOC concentration (mg/L)

TIC concentrationmg/L)

TOC

concentration

mg/L)

TIC

concentration

mg/L)

TOC

concentration

mg/L)

Test

suspension

0

12.3

-

-

<0.5

0.6

13.4

3.7

Procedure

control

-

-

0

12.3

<0.5

0.6

13.4

3.7

Toxicity

control

0

12.3

0

12.3

<0.5

0.6

25.7

1.9

1. As the purity of the test substance is 98.9%, so the TIC concentration is negligible.

2. The purity of the reference substance is regarded as 100%

3. It was regarded as 0.5mg/L when the TIC concentration was <0.5mg/L.

 

Table 5.2.1/3: Percentage biodegradation during the test(%)

 

Time (days)

Test suspension

Procedure

control

Toxicity control

Flask 1

Flask 2

Mean

Flask 5

Flask 6

2

0.6

-0.2

0.3

25.8

11.5

4

2.4

0.5

1.5

45.2

22.1

7

3.2

1.2

2.2

62.5

30.4

10

5.8

2.6

4.2

72.6

35.1

14

18.2

15.2

16.7

77.9

38.6

17

32.3

29.6

31.0

85.8

42.7

21

40.2

36.8

38.5

82.2

45.9

24

46.0

43.1

44.6

84.5

51.4

28

52.6

52.0

52.3

88.8

61.6

 

Note: It was regarded as zero when the percentage biodegradation was negative.

 

Validity of the test

At the beginning of the test, TIC concentration of the test medium was < 0.5 mg/L which was within 1.9- 3.7% of TC in the test solution, less than 5%.

The total CO2 evolution in the inoculum blank at the end of the test was 65.1 mg/L which was not greater than 70 mg/L.

The percentage degradation of the procedure control and the toxicity control were 77.9% and 38.6% which were greater than 60% and 25% respectively (based on the total ThCO2) within 14 days.

The difference of extremes of replicate values of the removal of the test substance at the plateau, at the end of the test or at the end of 10 day window was less than 20%.

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the test conditions, the percentage biodegradation of the test item did not reach 60% in 10 day-window, so the test item can’t be considered to be readily biodegradable.
Executive summary:

A ready biodegradability test was performed according to the OECD Guideline 301B, to determine the biodegradability of the test item in the CO2 Evolution Test.

 

Effective concentration of the test item: 17.6 mg/L (TOC concentration from the test item 12.3 mg/L). Activated sludge was used as inoculum (concentration of the inoculum 30 mg/L). As a reference item sodium benzoate (21.1 mg/L; TOC concentration from the reference item 12.3 mg/L) was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. The ready biodegradability of the test item was determined in the dark at 20.9-22.5 °C over 28 days.

 

Biodegradation of the toxicity control after 14 and 28 days were 38.6 and 61.6%, respectively, which showed that the test substance was considered not to have a toxic effect on the sewage sludge micro-organisms used in the study.

 

Degradation of sodium benzoate 14 and 28 days were 77.9 and 88.7%, respectively: the activity of the inoculum was thus verified (validity criterion).

 

At the end of the test, the percentage biodegradation of the two replicates of the test substance were 52.6% and 52.0%, with an average of 52.3%.

 

Under the test conditions, the percentage biodegradation of the test item did not reach 60% in 10 day-window, so the test substance can’t be considered to be readily biodegradable.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
November 30th 2016 to January 30th 2017
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
Comparable to guideline, performed after 2008 but non-GLP . In addition, the test concentration used (ca. 100 mg/L) is greater than the EC20 value obtained in the ASRIT study.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
GLP compliance:
no
Remarks:
Comparable to guideline, performed after 2008 but non-GLP
Specific details on test material used for the study:
Storage conditions: stored at 4°C (refrigerator) in the dark under N2 conditions.
Oxygen conditions:
aerobic
Inoculum or test system:
sewage, predominantly domestic, non-adapted
Details on inoculum:
A mixed population of activated sewage sludge micro-organisms (Test System) was obtained on November 30th, 2016 from the secondary treatment stage of the sewage treatment plant at Villette (STEP Villette, avenue de Thônex 105, 1226 THONEX (Geneva, Switzerland)), which treats predominantly domestic sewage.
The sample of activated sewage sludge was maintained on continuous aeration upon receipt. A sample of the activated sewage sludge was washed three times by settlement (centrifuge: Heittich rotenta 460 RS) and suspension in culture medium. To remove any excessive amounts of Dissolved Organic Carbon (DOC) that may have been present, the solution was stirred and maintained on with pure oxygen at room temperature. Determination of dry weight is made to inoculate final solution with 30mg/L dry weight activated sludge.
Duration of test (contact time):
60 d
Initial conc.:
98 mg/L
Based on:
test mat.
Initial conc.:
104.5 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
Deionised water was prepared using water purification system Millipore milliQ. The high purity of the test water is necessary in order to eliminate high blank values.

Experimental Preparation:
The test item (test 1) was dispersed directly in culture medium. An amount of test item (19.6mg) was dispersed in 200mL of culture medium inoculated to give the test concentration of 98.0mg/L.
The test item (test 2) was dispersed directly in culture medium. An amount of test item (20.9mg) was dispersed in 200mL of culture medium inoculated to give the test concentration of 104.5mg/L.
Analysis of the concentration, homogeneity and stability of the test item in the test solutions were not appropriate to the test guideline.

Reference Material:
The reference material (Sodium Benzoate) was dispersed directly in culture medium (See preparation in Appendix 1). An amount of reference material (20.0mg) was dispersed in 200mL of culture medium inoculated to give the test concentration of 100.0mg/L.

Toxicity Control:
The reference material (Sodium Benzoate) was dispersed directly in culture medium. An amount of reference material (20.0mg) was dispersed in 200mL of culture medium inoculated to give the test concentration of 100.0mg/L.
The test item was dispersed directly in culture medium. An amount of test item (19.6mg) was dispersed in 200mL of culture medium inoculated to give the test concentration of 98.0mg/L.

Abiotic Control:
The test item was dispersed directly in culture medium. An amount of test item (19.6mg) was dispersed in 200mL of culture medium inoculated to give the test concentration of 98.0 mg/L with abiotic agent (NaN3).

METHOD:
Culture medium
The culture medium used in this study (see Appendix 1) was that recommended in the OECD Guideline N° 301F.

Preparation of the experiment
On Day 0, the following solutions were inoculated in 1000mL glass culture vessels (total volume when full 1145mL) each containing 200mL of solution:
- A control, in duplicate, consisting of inoculated culture medium.
- The test item (ST 14 C12), in duplicate, in inoculated culture medium.
- The reference material (Sodium Benzoate) in inoculated culture medium.
- The test item and the reference material in inoculated culture medium as a toxicity control.

The system consists of a sample flask sealed by a sensor head/CO2 trap in a temperature controlled incubator. The samples were stirred for the duration of the study with a magnetically coupled stirrer.
As biodegradation progresses, the micro-organisms convert oxygen to carbon dioxide which is absorbed into NaOH causing a net reduction in gas pressure within the sample flask.

Sampling and analysis
WTW oxitopC calculate automatically the consumption of oxygen.
The daily Biochemical Oxygen Demand (BOD) values for the control, test and reference materials and the toxicity control are given below. These were calculated
The BOD was adjusted automatically at the temperature measurement.
On day 28, all vessels were sampled for pH measurements. The pH was measured using a pHmeter Mettler Toledo SevenEasy. The test was conducted in diffuse light at a temperature of 22°C +/- 1°C in thermostatic incubator cabinet (WTW TS606).

Evaluation of Data
Calculation of Theoretical Oxygen Demand (ThOD)
The total amount of oxygen required to oxidise a chemical completely is calculated from the molecular formula (assuming no nitrification occurs) and expressed as mg oxygen required per mg test item as described in the OECD Guideline N° 301F.

Calculation of Biological Oxygen Demand (BOD) and Percentage degradation
The biological oxygen demand (BOD) mg O2/ mg of the test item and of the reference material, sodium benzoate was calculated as:
BOD=(mgO2/L uptake by test substance - mgO2/l uptake by blank)/ mg test substance/l in vessel= mg 02/mg test substance

The percentage biodegradation of the test item and of the reference material, sodium benzoate was calculated as:
% biodegradation = BOD(mgO2/mg test substance)/ ThOD (mg O2/mg test substance) x 100


Validation criteria
A test is considered valid if the difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the test or at the end of the 10-d window, as appropriate, is less than 20%, and if the percentage degradation of the reference compound has reached the pass levels by day 14. If either of these conditions is not met, the test should be repeated. Because of the stringency of the methods, low values do not necessarily mean that the test item is not biodegradable under environmental conditions, but indicates that more work will be necessary to establish ready biodegradability.
The BOD of the inoculated culture control medium (control) is normally 20 to 30mgO2/L and should not exceed 60mgO2/L after 28 days. Value higher than 60mgO2/L requires critical examination of the data and experimental technique. If the pH value is outside the range 6-8.5 and the oxygen consumption by the test item is less than 60%, the test should be repeated with a lower concentration of test item.
The results of the degradation test are considered valid if in the same test the reference material yields >= 60% degradation by Day 14.
The test item may be considered to be readily biodegradable if >= 60% degradation is attained after 28 days. This level of degradation must be reached within 10 days of biodegradation exceeding 10%.

The toxicity control should attain 25% degradation by Day 14 for the test item to be considered as non-inhibitory.
Reference substance:
benzoic acid, sodium salt
Remarks:
Appearance: white crystals Batch number:1290372 21806P02 / Purity>99%/ Storage conditions:at ambient temperature
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
Test 1
Value:
48
Sampling time:
28 d
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
Test 1
Value:
70
Sampling time:
60 d
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
Test 2
Value:
20
Sampling time:
28 d
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
Test 2
Value:
76
Sampling time:
60 d
Details on results:
The test item (test 1) attained 48% degradation after 28 days and attained 70% degradation after 60 days.
The test item (test 2) attained 20% degradation after 28 days and attained 76 % degradation after 60 days.
Therefore the test item cannot be considered to be readily biodegradable under the strict terms and conditions of OECD Guideline N° 301F.
The toxicity test attained 33% degradation after 14 days thereby confirming that the test item was not toxic to the sewage treatment micro-organisms used in the study.


Results with reference substance:
Sodium Benzoate attained 74% degradation after 14 days thereby confirming the suitability of the inoculum and test conditions.
Validity criteria fulfilled:
yes
Interpretation of results:
inherently biodegradable
Conclusions:
The test item (test 1 and 2) attained respectively 48% and 20% degradation after 28 days.
The test item (test 1 and 2) attained respectively 70% and 76% degradation after 60 days.
Under the test conditions, the test item is classified as not readily biodegradable within 28days.
Under the extended study period of 60 days, a biodegradation of min 70% was reached. This indicated that the test item is inherently and ultimately biodegradable. The test item can be considered to be non-persistent.

Executive summary:

This study was performed to assess the ready biodegradability of the test item in an aerobic aqueous media. The method followed that described in the OECD Guideline for the testing of Chemicals (1992) N°301 F, “Ready Biodegradability; Manometric Respirometry Test”.

In addition, the procedures were designed to meet the test method of the Council Regulation (EC) No. 440/2008 of 30 May 2008, Publication No. L142, Part C.4-D laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

The test item, at a concentration of 98.0 mg/L in test 1 and at a concentration of 104.5 mg/L in test 2 was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in diffused light at 22°C ± 1°C for 28 days.

The degradation of the test item was assessed by the measurement of daily oxygen consumption between days 0 and 60. Control solutions with inoculum and the reference material, sodium benzoate, together with a toxicity control were used for validation purposes.

The test item (test 1 and 2) attained respectively 48% and 20% degradation after 28 days. The test item (test 1 and 2) attained respectively 70% and 76% degradation after 60 days. Under the test conditions, the test item is classified as not readily biodegradable within 28 days.

Under the extended study period of 60 days, a biodegradation of min 70% was reached. This indicated that the test item is inherently and ultimately biodegradable.The test item can be considered to be non-persistent.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
October 21st 2014 to December 22nd, 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The test was performed according to OECD 301 C guideline (1992) and Japanese guideline (2011). However, the test concentration used (100 mg/L) is greater than the EC20 value obtained in the ASRIT study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Version / remarks:
OECD guidelines for testing of chemicals No. 301 C, July 1992, Ready biodegradability : Modified MITI test (I)
Deviations:
not specified
GLP compliance:
yes
Remarks:
GLP statement (December 22nd, 2014)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Firmenich SA, MHR2079

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test sample was sealed with nitrogen gas in cold and dark storage place.
- Stability under storage conditions: The infrared IR spectrum of the test item measured at this laboratory was confirmed to be identical to that provided by the sponsor. The stability of the test item was confirmed by comparing the IR spectrum of the test item after the completion of the experiement with that before the start of the experiement.
Oxygen conditions:
aerobic
Inoculum or test system:
mixture of sewage, soil and natural water
Remarks:
On-site sludge sampling at 10 locations in Japan : from surface water and surface soil of rivers lakes and inland sea, return sludge from sewage plants. Synthetic sewage : glucose peptone potassium dihydrogenphosphate, dissolved in purified water.
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): On-site sampling was carried out at 10 locations in Japan (samples were from surface water and surface soil of rivers, lakes and inland sea; return sludge from sewage plants).
- Laboratory culture: Activated sludge, which was prepared and controlled in this laboratory according to the test method described in the Japanse guideline, was used in this study (sampling period: September, 2014, inititation date of use: October 22, 2014).
- Method of cultivation: The activated sludge, which was cultivated for 18 hours after synthetic sewage was added, was used. The synthetic sewage was prepared according to the following method; glucose, peptone, and potassium dihydrogenphosphate were dissolved in purified water, and the pH of the solution was adjusted to 7.0 +-1.0.
- Concentration of sludge: 30 mg/L of suspended solids
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Remarks:
a) Measurement of BOD with a closed system oxygen consumption measuring apparatus b) Determination of DOC by a TOC analysis c) Determination of test item by HPLC
Details on study design:
TEST CONDITIONS
- Composition of medium: The b asal culture medium (5L) was prepared at the same proportion as the following method; purified water (The Japanese Pharmacopoeia, Takasugi Pharmaceutical) was added to each 3 mL aliquot of solutions A, B, C and D, which are described in JIS K 0102-2008 Section 21, in order to prepare 1L of solution. The pH of this solution was then adjusted to 7.0.

- Test temperature: 25 +- 1°C
- pH: 7 +/- 1
- pH adjusted: yes
- Aeration of dilution water: not specified
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes

TEST SYSTEM
- Number of culture flasks/concentration:
Vessel No. 1 (n=1): water + test item, test item concentration = 100 mg/L
Vessels No. 2, 3, 4 (n=3) : in each test vessel, 30 mg of test sample was added to basal culture medium, test item concenteation = 100 mg/L. The activated sludge was added to each test vessel described in 2), 3), 4) so that the concentration of the suspended solid reached 30 mg/L.
Vessel No. 5 : nothing was added to basal culture medium
Vessel No. 6 29.5 µL (30 mg) of aniline was taken out by microsyringe and added to the basal culture medium, aniline concentration = 100 mg/L.
- Measuring equipment: Closed system oxygen consumption measuring apparatus was used
- Details of trap for CO2 and volatile organics if used: Absorbent for carbon dioxide : Soda lime No. 1

SAMPLING
- Sampling frequency: During the incubation period, the appearance of the test solution was observed once a day. BOD of the test solutions was measured continuously by a closed system oxygen consumption measuring apparatus. The incubation temperature was measured and recorded once a day.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Vessel No. 5 : nothing was added to basal culture medium
Reference substance:
aniline
Preliminary study:
None
Test performance:
No data
Parameter:
% degradation (O2 consumption)
Remarks:
Biological Oxygen Demand
Value:
33
Sampling time:
28 d
Parameter:
% degradation (DOC removal)
Value:
48
Sampling time:
28 d
Key result
Parameter:
% degradation (test mat. analysis)
Remarks:
HPLC
Value:
98
Sampling time:
28 d
Details on results:
In the qualitative analysis with LC-MS, two converted products (A and B) were detected in the test solutions (sludge + test item). Presumed molecular weights of the converted product A and B were 222 and 208 respectively, according to the results of mass spectrum analysis of the converted products. From the presumed molecular weights and the chemical structure of the test item, the converted product A may have a carbonyl group and a hydroxyl group. The converted product B may be produced by partial degradation of alkyl chain of the test item by microorganisms or by transformation from carbonyl group of the test item to hydroxyl group.
Results with reference substance:
Aniline showed 92 % biodegradation after 28 days. The calculation is described in "Any other information on meterials and methods".

Results:

Table 5.2.1 / 1 :

Duration of incubation: 28 days

Vessel No.

7thday

14thday

21thday

28thday

Average

BOD (mg)

Deg (%)

BOD (mg)

Deg (%)

BOD (mg)

Deg (%)

BOD (mg)

Deg (%)

Deg (%)

[1]

0.0

-

0.0

0.1

0.1

-

0.1

-

-

[2]

2.2

0

4.1

-3

17.7

14

32.0

35

33

[3]

2.5

0

4.4

-2

9.3

1

31.1

34

[4]

2.1

0

4.1

-3

8.7

0

28.1

29

[5]

2.2

-

6.0

-

8.9

-

8.9

-

-

[6]

53.3

71

70.1

89

75.1

91

75.3

92

-

Vessel No. 1: Water + test item

Vessel No. 2, 3, 4: Sludge + test item

Vessel No. 5: Control blank

Vessel No. 6: Sludge + aniline

Table 5.2.1 / 2 :

 

Sludge + test item

Vessel No.2

Vessel No.3

Vessel No.4

Average

Percentage biodegradation by BOD

%

35

34

29

33

Percentage biodegradation by DOC

%

50

50

45

48

Percentage biodegradation of test item by (HPLC)

%

100

100

95

98

Table 5.2.1 / 3 : Analytical results of test solutions after 28 days

 

Water + test item

Slude + test item

Theoretical amount

Vessel No.1

Vessel No.2

Vessel No.3

Vessel No.4

BOD *3

mg

 0.1

 23.1

 22.2

 19.2

65.1

Residual amount and percentage residue of DOC *3

mgC

 21.2

 10.7

 10.6

 11.8

21.0

%

 101

 51

 51

 56

-

Residual amount and percentage residue of test item (by HPLC)

mg

 29.9

 0

 0

 1.6

30.0

%

 100

 0

 0

 5

-

Detection of converted products (by LC-MS) *4

-

Not detected

Detected (two components)

-

*3 The value of the test solution (control blank) was substracted from the values of the test solutions (sludge + test item).

*4 The quantitative analysis of the converted products could not be performed because their authentic samples were not available.

Table 5.2.1 / 4 : Validity of test conditions

 

Value in this test

Value of criterion

Difference between extremes of replicate values of percentage biodegradation

Percentage biodegradation by BOD

6%

20 %

Percentage biodegradation by DOC

5%

Percentage biodegradation of test item

5%

Percentage biodegradation of aniline by BOD

After 7 days

71%

>40%

After 14 days

89%

>65%

BOD value of control blank

After 28 days

30 mg*²

=< 60 mg/L

*² Measurement value of BOD (8.9 mg) / [test volume (300 mL) / 1000] = 30 mg/L

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
Under the test conditions of this study, almost all of the test item disappeared by biodegradation and two water-soluble converted products were produced. One of the converted products may have a carbonyl group and a hydroxyl group. The other converted product may be produced by partial degradation of alkyl chain of the test item by microorganisms or by transformation from carbonyl group of the test item to hydroxyl group.
Executive summary:

This study was performed to assess the ready biodegradability of the test item in an aerobic aqueous media.

The method followed is described in the OECD Guideline for the testing of Chemicals (1992) N°301 C, “Ready Biodegradability; Modified MITI Test I”. In addition, the procedures were designed to meet the test method of the "Method for testing the biodegradability of chemical substances by microorganisms" stipulated in the "Testing Methods for New Chemical Substances (March 31, 2011, No. 0331-7, Pharmaceutical anf Food Safety Bureau, Ministry of Health, Labour and Welfare; March 29 2011, No. 5, Manufacturing Industries bureau, ministry of economy, trade and industry; No. 110331009, Environmental policy bureau, Ministry of the environement, Japan; April 2, 2012 partial revision, No. 0402-1, Pharmaceutical and Food safety bureau, Ministry of Health, Labour and Welfare; March 28, 2012, No. 2, Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry; No. 120402001, Environmental Policy Bureau, Ministry of the environment, Japan.)

The test item, at a concentration of 100.0 mg/L was exposed to a mixture of sludge coming from 10 different locations in Japan, from surface water and surface soil of rivers lakes and inland sea, return sludge from sewage plants, with culture medium, in sealed culture vessels, in the dark, at 25°C ± 1°C for 28 days (n=3). The activated sludge was prepared and controlled in the laboratory according to OECD guideline for Modified Miti test (I). The activated sludge which was cultivated for 18 hours after the synthetic sewage was added, was used.

Percentage biodegradation was calculated thanks to measurement and analysis:

a) measurement of biochemical oxygen demand (BOD) with a closed system oxygen consumption measuring apparatus

b) determiantion of dissolved organic carbon (DOC) by a total organic carbon analysis (TOC)

c) determination of test item by high-performance liquid chromatography (HPLC)

Aniline was used as a reference iten in order to confirm that the sludge was sufficiently active. Aniline showed 92 % biodegradation after 28 days.

The test item attained:

a) 33% biodegradation, using BOD

b) 48 %, using DOC

c) 98 % using HPLC.

Under the test conditions of this study, the test substance should not be regarded as readily biodegradable but disappears almost totally (av. 98%, n=3). Almost all of the test item disappeared by biodegradation and two water-soluble converted products were produced.

In the qualitative analysis with LC-MS, two converted products (A and B) were detected in the test solutions (sludge + test item). Presumed molecular weights of the converted product A and B were 222 and 208 respectively, according to the results of mass spectrum analysis of the converted products. From the presumed molecular weights and the chemical structure of the test item, the converted product A may have a carbonyl group and a hydroxyl group. The converted product B may be produced by partial degradation of alkyl chain of the test item by microorganisms or by transformation from carbonyl group of the test item to hydroxyl group.

Description of key information

Weight of evidence approach, OECD Guidelines 301B/F/C:

The registered substance does degrade significantly (up to 52%) within 28 days but no enough to be classified as readily biodegradable.

The substance will continue to degrade if exposed to micro-organisms for a longer period and reaches criteria for non-persistency within 60 days.

According to the HPLC measurement at 28 days in the OECD 301C, almost all the substance disappear to form two transformation products.

Key value for chemical safety assessment

Biodegradation in water:
inherently biodegradable
Type of water:
freshwater

Additional information

To assess the biodegradation potential of the registered substance, a weight of evidence approach was performed using three experimental studies.

 

The first study (Guangdong Detection Center of Microbiology, 2014) was performed on the registered substance according to the OECD Guideline 301B and GLP. The test substance was exposed to activated sludge at an effective concentration of 17.6 mg/L. As a reference item, sodium benzoate (21.1 mg/L) was tested simultaneously under the same conditions as the test item, and functioned as a procedure control. The ready biodegradability of the test item was determined in the dark at 20.9 -22.5 °C over 28 days. Biodegradation of the toxicity control after 14 and 28 days were 38.6 and 61.6%, respectively, which showed that the test substance was considered not to have a toxic effect on the sewage sludge micro-organisms used in the study. Degradation of sodium benzoate 14 and 28 days were 77.9 and 88.7%, respectively, so the activity of the inoculum was thus verified (validity criterion). At the end of the test, the percentage biodegradation of the two replicates of the test substance were 52.6% and 52.0%, with an average of 52.3%. Under the test conditions, the percentage biodegradation of the test item did not reach 60% in 28 days, so the test substance can’t be considered to be readily biodegradable, acccording to CLP Regulation No 1272/2008.

 

The second study (FIR, 2018) was performed on the registered substance in a non-GLP test according to a method similar to OECD Guideline 301F. The test substance, at a concentration of 98.0 mg/L in test 1 and 104.5 mg/L in test 2, was exposed to activated sewage sludge micro-organisms with culture medium in sealed culture vessels in diffused light at 22°C ± 1°C for 28 days. The degradation of the test substance was assessed by the measurement of daily oxygen consumption between days 0 and 60. Control solutions with inoculum and the reference material, sodium benzoate, together with a toxicity control were used for validation purposes. The test item (test 1 and 2) attained respectively 48% and 20% degradation after 28 days. The test item (test 1 and 2) attained respectively 70% and 76% degradation after 60 days. Under the test conditions, the test substance is classified as not readily biodegradable within 28 days. However, under the extended study period of 60 days, a biodegradation of min 70% was reached. This indicated that the test substance is inherently and ultimately biodegradable. The registered substance can be considered to be non-persistent. This study was performed after 2008 but not according to GLP, and is therefore not normally accepted as valid to fulfil the endpoint. However, this study followed OECD Guidelines and due to the similarity of the results with the other biodegradation studies, the result of this OECD 301F study can be considered as valid and sufficient evidence that the substance is non-persistent.

 

The third study (CERI, 2014) was performed on the registered substance according to the OECD Guideline 301C and GLP. The test substance was exposed to a mixture of sludge at a concentration of 100 mg/L, with culture medium, in sealed culture vessels, in the dark, at 25°C ± 1°C for 28 days. Percentage biodegradation was calculated thanks to measurement and analysis: measurement of biochemical oxygen demand (BOD) with a closed system oxygen consumption measuring apparatus; determiantion of dissolved organic carbon (DOC) by a total organic carbon analysis (TOC); and determination of test item by high-performance liquid chromatography (HPLC). Aniline was used as a reference iten in order to confirm that the sludge was sufficiently active. Aniline showed 92 % biodegradation after 28 days. The test item attained 33% biodegradation after 28 days using BOD, 48 % biodegradation after 28 days using DOC, and 98 % biodegradation after 28 days using HPLC. Therefore, under the test conditions of this study, the test substance should not be regarded as readily biodegradable but disappears almost totally (av. 98%, n=3). Almost all of the test substance disappeared by biodegradation and two water-soluble converted products were produced. In the qualitative analysis with LC-MS, two converted products (A and B) were detected in the test solutions. Presumed molecular weights of the converted product A and B were 222 and 208 respectively, according to the results of mass spectrum analysis of the converted products. From the presumed molecular weights and the chemical structure of the test item, the converted product A may have a carbonyl group and a hydroxyl group. The converted product B may be produced by partial degradation of alkyl chain of the test item by microorganisms or by transformation from carbonyl group of the test item to hydroxyl group.

 

The three studies, taken together support the hypothesis that the registered substance does degrade significantly (up to 52%) within 28 days but no enough to be classified as readily biodegradable, under strict terms of the OECD Guideline, but will continue to degrade if exposed to micro-organisms for a longer period and reaches criteria for non-persistency within 60 days. A long lag time is observed in all studies (>= 12 days) and biodegradation values in the OECD 301F and OECD 301C are negative during these lag times which suggests an inhibition of bacterial during this period (concentration tested (ca. 100 mg/L) greater than the EC20 value obtained in the ASRIT study). Biodegradation is slow down, no "plateau" phase is observed in the curves. However, according to the HPLC measurement at 28 days in the OECD 301C, almost all the substance disappear to form two transformation products. In conclusion, the registered substance is not persistent in the environment.