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Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24/01/1991-07/03/1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Reliability 1 is assigned because the study conducted according to OECD TG 301C in compliance with GLP, without deviations that influence the quality of the results.
Qualifier:
according to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Deviations:
no
Qualifier:
according to
Guideline:
other: "Biodegradation test of chemical substance by microorganisms etc." stipulated in the Order Prescribing the Items of the Test Relating to the New Chemical Substances
Deviations:
no
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
mixture of sewage, soil and natural water
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure):
Sludge sampling were made at the following 10 places in Japan:
Fukogawa city sewage plant (Sapporo-shi Hokkaido)
Fukashiba industry sewage plant (Kashima-gun Ibaragi)
Nakahama city sewage plant (Osaka-shi Osaka)
Ochiai city sewage plant (Shinjuku-ku Tokyo)
Nitakami river (Ishinomaki-shi Miyagi)
Shinano river (Nishikanbara-gun Niigata)
Yoshino river (Tokushima-shi Tokushima)
Lake Biwa (Otsu-shi Shiga)
Hiroshima bay (Hiroshima-shi Hiroshima)
Dookal bay (Kitakyushu-shi Fukuoka)

Date: December 1990
Sludge sampling method:
- City sewage: Returned sludge of sewage plants were collected.
- Rivers, lake and sea: Surface water and surface soil which is in contact with atmosphere were collected.

Mixing of fresh and old activated sludge
5 L of the filtrate of the supernatant of an activated sludge in the present use was mixed with 500 mL of the filtrate of the supernatant of a newly collected sludge respectively and the mixture was cultured at pH 7.0 ± 1.0 under the sufficient aeration (prefiltered open air was used for aeration).

Culture
About 30 minutes after ceasing the aeration to the sludge mixture, supernatant corresponding to about 1/3 of the whole volume was removed.
Then the equal volume of dechlorization water was added to the remaining portion and aerated again, followed by addition of synthetic sewage (Glucose, peptone and monopotassium phosphate were dissolved in dechlorization water respectively. Each concentration of them was to be 5(W/V)% and the solution was adjusted to pH 7.0 ± 1.0 with sodium hydroxide), concentration of that portion was to be 0.1(W/V)%. This procedure was rapeated once every day. The culturing was carried out at 25±2°C.

- Concentration of sludge: Concentration of suspended solid in the activated sludge was 4700 mg/l.
The activated sludge cultivated under the conditions described was so added to each test vessel containing sludge, that the concentration of suspended solid reached to 30 mg/L in them.
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Each 3 mL of solution A, solution B, solution C and solution D, which are prescribed in JIS K 0102-1986-21, were made
up to 1000 mL with purified water (Takasugi Seiyaku Co.. Ltd.), and then pH of this solution was adjusted to 7.0.
- Test temperature: 25 ± 1°C
- Aeration of dilution water: Prefiltered open air was used for aeration.
- Suspended solids concentration: 30 mg/L
- Continuous darkness: yes

TEST SYSTEM
Instrument for cultivation
Closed system oxygen consumption measuring apparatus (Coulometer: Ohkura Electric Co.. Ltd.)
Data processor (Data sampler: Chemicals Inspection & Testing Institute)

Vessel: 300 mL in volume (vessel for volatile substance)
Absorbent for carbon dioxide: Soda lime No.1 (extra pure reagent. Wako Pure Chemical Industries, Ltd.)
Stirring method: Test solution was stirred by a magnetic stirrer.

Number of culture flasks/concentration: 3 (test substance)

Following test solutions were prepared:

(1) Addition of test substance or aniline
(a) (water + test substance) (n=1)
A test vessel containing 300 mL of purified water into which 31.5 μL by micro cylinder (30.2 mg) of the test substance was added.

(b) (sludge + test substance) (n=3)
Each test vessel containing 300 mL of basal culture medium into which 31.5 μL by micro cylinder (30.2mg) of the test substance was added, respectively.

(c) (sludge + aniline) (n=1)
A test vessel containing 300 mL of basal culture medium into which 30 mg of aniline was added.

(d) (control blank) (n=1)
A test vessel containing 300 mL of basal culture medium into which was not added test substance nor aniline.

(2) Inoculation of activated sludge
Activated sludge was so added to each test vessel (b), (c) and (d), that the concentration of suspended solid reached to 30 mg/L in them.
SAMPLING
After the termination of cultivation, residual amount of the test substance in the test solutions was subjected to analysis.


Reference substance:
aniline
Remarks:
guaranteed reagent, Showa Chemicals Inc. Lot No. SB-2638
Test performance:
Appearance of the test solutions:
At the initiation of cultivation it was observed in both solutions (water + test substance) and (sludge + test substance) that the test substance was not dissolved.
At the completion of cultivation, in both solutions no changes were observed.
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Remarks on result:
other: arithmetical mean of 3 vessels
Key result
Parameter:
% degradation (test mat. analysis)
Value:
11
Sampling time:
28 d
Remarks on result:
other: arithmetical mean of 3 vessels
Details on results:
For the percentages biodegradation in the individial test vessels see overall remarks below. More details on measured values at different times and the biodegradation curve based on BOD can be found in the attached background material.
Results with reference substance:
Percentage biodegradation of aniline calculated by BOD value were 62% and 92% on the 7th and 14th day respectively.

Percentage biodegradation after 28 days:

Method

Percentage biodegradation (%)

Refer to2

Vessel-1

Vessel-2

Vessel-3

Mean

BOD

0

0

1

0

Table-1

GC1

14

13

8

11

Table-2

1Please note the report states HPLC, but the analysis was done by GC so this is mentioned here

2See attached background material

Validity criteria fulfilled:
yes
Remarks:
oxygen uptake in inoculum blank < 60 mg/l in 28 days, differences between replicates <20%, percentage aniline degradation >40% and >65% after 7 days and 14 days respectively.
Interpretation of results:
other: Not readily biodegradable under the present test conditions
Conclusions:
The test substance was not readily biodegradable under the present test conditions.
Executive summary:

The biodegradability of OTNE was tested in a Modified MITI test (OECD TG 301C). The validity criteria for the test were met. The concentration of the test substance added was 100 mg/l, with an activated sludge concentration of 30 mg/l suspended solids. The mean percentage biodegradation found was 0% based on BOD and 11% based on analysed test material concentration. In conclusion, OTNE was not readily biodegradable under the conditions of this test.

Description of key information

The biodegradability of OTNE was tested in a Modified MITI test (OECD TG 301C). The validity criteria for the test were met. The concentration of the test substance added was 100 mg/l, with an activated sludge concentration of 30 mg/l suspended solids. The mean percentage biodegradation found was 0% based on BOD and 11% based on analysed test material concentration. In conclusion, OTNE was not readily biodegradable under the conditions of this test.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

Besides this key study, another ready biodegradability test according to OECD TG 301F is available.

The Ready Biodegradability of OTNE was determined by the Manometric Respirometry Test according to the OECD Guidelines for Testing of Chemicals, Method No. 301 F. The concentration tested was 100 mg/l OTNE, with an activated sludge concentration of 30 mg/l. In the conditions of the test, OTNE undergoes no biodegradation, even after 42 days. Thus, OTNE should be regarded as not readily biodegradable according to this test (Rudio, 1995a).

Also the inherent biodegradability of OTNE was determined by the Modified MITI Test (II) according to the OECD Guidelines for Testing of Chemicals, Method No. 302 C. The concentration tested was 30 mg/L OTNE, with an activated sludge concentration of 100 mg/L. OTNE undergoes no biodegradation and should be regarded as not inherently biodegradable according to this test (Rudio, 1995b).

Under the conditions of these studies OTNE is not readily biodegradable and not inherently biodegradable.