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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:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results. Reliability given as 2 due to results being obtained on a read-across substance.
Qualifier:
according to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
Sludge sampling were made at the following 10 places in Japan:
Fukogawa city sewage plant
Fukashiba industry sewage plant
Nakahama city sewage plant
Ochiai city sewage plant
Kitakami river
Shinano river
Yoshino river
Lake Biwa
Hisohima bay
Dookai bay

Sludge sampling method
City sewage - Returned sludge of sewage plants was collected.
Rivers, lake and sea -Surface water and surface soil which was in contact with the atmosphere was collected.

5L of the fitrate of the supernatant of an activated sludge in use was mixed with 500ml of the fitrate of the supernatant of a newly collected sludge and the mixture cultured at pH 7 ± 1 under aeration. 30 minutes after ceasing aeration to the sludge mixture, supernatant corresponding to approximately 1/3 of the whole volume was removed. An equal volume of dechlorinated water was added to the remaining portion and aerated followed by the addition of synthetic sewage* (concentration 0.1(w/v%). This procedure was repeated once everyday. The culturing was carried out at 25±2°C.

During cultivation the appearance of the supernatant, precipitability, formation of flock, pH, dissolved oxygen concentration, and temperature were checked and adjusted if necessary. The microflora in the activated sludge was observed microscopically and sludge with no abnormal symptoms used in the test.

*Synthetic sewage - Glucose, peptone and monopotassium phosphate were dissolved in dechlorinated water respectively. Each concentration of them was 5(w/v%) and the solution adjusted to pH 7 ± 1 with sodium hydroxide.

The concentration of activated seawge sludge used was 30mg/l ( as suspended solids)
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
other: BOD
Parameter followed for biodegradation estimation:
TOC removal
Parameter followed for biodegradation estimation:
test mat. analysis
Remarks:
For the test substance (4-Hydroxy-2,2,6,6-tetramethylpiperidine-1-oxy) and its converted product (4-Oxo-2,2,6,6-tetramethylpiperidine-1-oxy)
Details on study design:
Measurement of concentration of suspended solid -
Method in accordance with Japanese Industrial Standards (JIS) K 0102-1986-14.1. The concentration of suspended solid in the activated sludge was 5900mg/l

Preparation of mineral medium - Each 3 ml of solution A, solution B, solution C and solution D as prescribed in JIS K 0102-1986-21 were made upto 1000ml with purified water and then the pH of the solution was adjusted to 7.0

Preparation of test solutions
The following test solutions were prepared and cultured under the conditions described below -
a) Water and test substance. A test vessel containing 300 ml of purified water into which 30 mg of the test substance was added. the pH of the test solution was measured.
b) Sludge and test substance. In triplicate. A test vessel containing 300 ml of basal culture medium into which 30 mg of the test substance was added. The pH of the test solutions were measured.
c) Sludge and aniline. A test vessel containing 300ml of basal culture medium into which 30mg of aniline was added.
d) Control blank. A test vessel containing 300ml of basal culture medium into whcih neither the test substance nor aniline was added.
Activated sludge was added to each of the tets vessels in b, c, and d above at a concentration of 30 mg/l suspended solids.

Test conditions:
Concentration of the test substance :100mg/l
Concentration of activated sludge (as concentration of suspended solids) 30mg/l
Volume of test solution : 300ml
Cultivating temperature : 25 ± 1°C
Stirring : Test solutions were stirred by magnetic stirrer.
Cultivating duration : 28 days
Absorbent for carbon dioxide : Soda lime No. 1

Oxygen consumption was measured during the test period and after the termination of cultivation, total organic carbon, the tests substance and the converted product in the test solutions were determined. The pH of the test solutions were measured.

Measurement and analysis
Measurement of biochemical oxygen demamn (BOD) by closed system oxygen consumption measuring apparatus.
Determination of total organic carbon by total organic carbon analyser (TOC).
Determination of test substance by gas chromatography (GC)
Determination of converted product by gas chromatography (GC)
Reference substance:
other: aniline
Key result
Parameter:
other: BOD
Value:
0
Sampling time:
28 d
Key result
Parameter:
% degradation (TOC removal)
Value:
0
Sampling time:
28 d
Key result
Parameter:
% degradation (test mat. analysis)
Value:
20
Sampling time:
28 d
Details on results:
Please see attached background material
Results with reference substance:
Aniline attained 61%, 70% and 71% degradation after 7 days, 14 days and 28 days respectively, thereby confirming the suitability of the innoculum and the test conditions

Percentage biodegradation of the test substance after 28 days

 Method

          Percentage biodegradation (%)

   Vessel-3   Vessel-4   Vessel-5  Average
 BOD  0
 TOC  0
 GC  20 16  22  20 

Percentage residue of the test substance and percentage production of the converted product after 28 days

   Percentage residue of the test substance  Percentage production of the converted product  Total
 Vessel-2 103 103 
 Vessel-3 82 14  96 
 Vessel-4 86  14  100 
 Vessel-5 80  20  100 
       
       

 Vessel-2 - Water and test substance

 Vessel-3, 4, 5 - Sludge and test substance

Please see attached background material

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test substance showed 0% biodegradation after 28 days in a biodegradation test according to OECD 301C Ready biodegradability: Modified MITI test. It was also concluded that the test substance was converted in the presence of activated sludge, and the mixture of the test substance and the converted product remained in the test solutions.
Executive summary:

Introduction

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 301C, "Ready Biodegradability; modified MITI test.

Methods

The test item, at a concentration of 100 mg/l was exposed to activated sewage sludge micro-organisms with culture medium at approximately 25°C for 28 days.

The degradation of the test item was assessed by the determination of BOD, TOC and test material analysis. Control solutions with inoculum and the reference item, aniline were used for validation purposes.

Results.

The test substance showed 0% biodegradation after 28 days.

It was also concluded that the test substance was converted in the presence of activated sludge, and the mixture of the test substance and the converted product remained in the test solutions.

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Refer to read-across justification document in Section 13 of IUCLID
Reason / purpose:
read-across source
Key result
Parameter:
other: BOD
Value:
0
Sampling time:
28 d
Key result
Parameter:
% degradation (TOC removal)
Value:
0
Sampling time:
28 d
Key result
Parameter:
% degradation (test mat. analysis)
Value:
20
Sampling time:
28 d
Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
Based on a read-across approach, the test substance showed 0% biodegradation after 28 days in a biodegradation test according to OECD 301C Ready biodegradability: Modified MITI test. It was also concluded that the test substance was converted in the presence of activated sludge, and the mixture of the test substance and the converted product remained in the test solutions.

Description of key information

28d % biodegradation = 0 %; OECD 301C; Oyama (1992)

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

OECD 301C (Read-Across)

A study was performed to assess the ready biodegradability of the test item in an aerobic aqueous medium. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (1992) No 301C, "Ready Biodegradability; modified MITI test. The test item, at a concentration of 100 mg/l was exposed to activated sewage sludge micro-organisms with culture medium at approximately 25°C for 28 days. The degradation of the test item was assessed by the determination of BOD, TOC and test material analysis. Control solutions with inoculum and the reference item, aniline were used for validation purposes. The test substance showed 0% biodegradation after 28 days. It was also concluded that the test substance was converted in the presence of activated sludge, and the mixture of the test substance and the converted product remained in the test solutions.