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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:
23 July 1998 to 25 September 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
other: Method for Testing the Biodegradability of Chemical Substances by Microorganisms
Version / remarks:
stipulated in the "Testing Methods for New Chemical Substances" (July 13, 1974, Kanpogyo No.5, Planning and Coordination Bureau, Environment Agency, Yakuhatu No.615, Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, and 49 Kikyoku No.392, Basic Industries Bureau, Ministry oflnternational Trade and Industry, Japan)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Version / remarks:
1992
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
Sampling sites: On-site sludge sampling was carried out at the following 10 locations in Japan :
- Fukogawa city sewage plant (Sapporo-shi Hokkaido)
- Kashima industry sewage plant (Kashima-gun Ibaragi)
- Nakahama city sewage plant (Osaka-shi Osaka)
- Ochiai city sewage plant (Shinjuku-ku Tokyo)
- Kitakami river (lshinomaki-shi Miyagi)
- Shinano river (Nishikanbara-gun Niigata)
- Yoshino river (Tokushima-shi Tokushima)
- Lake Biwa (Otsu-shi Shiga)
- Hiroshima bay (Hiroshima-shi Hiroshima)
- Dookai bay (Kitakyushu-shi Fukuoka)

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

Preparation of activated sludge
- Activated sludge was prepared as follows to maintain its uniformity: The filtrate (5 L) of the supernatant of the activated sludge (the activated sludge cultivated the mixed filtrate (10 L) of the supernatant of sludge collected at the ten locations) cultivated for ca. 3 months was mixed with the mixed filtrate (5 L) of the supernatant of a sludge collected newly at each location. The mixed filtrate (10 L) was aerated and the pH adjusted to 7.0 ± 1.0.

Cultivation
- Roughly 30 minutes after ceasing aeration of the sludge mixture, supernatant corresponding to about 1/3 of the whole volume was removed. Then an equal volume of de-chlorinated water was added to the remaining portion. This mixture was aerated, and then an amount of synthetic sewage (glucose, peptone and potassium dihydrogenphosphate were dissolved in de-chlorinated water to obtain 5 (W/V) % of the solution for each components. The pH of the solution was adjusted to 7.0 ± 1.0 with sodium hydroxide) was added to the mixture so that the concentration of the synthetic sewage was 0.1 (W/V) % in the volume of the de-chlorinated water added. This procedure was repeated once every day. Cultivation was carried out at 25 ± 2°C.
- During cultivating, the appearance of the supernatant, setting of the sludge, formation of flock, pH, dissolved oxygen concentration in the solution and temperature were checked and adjusted if necessary. Microflora in the activated sludge was microscopically observed and the sludge with no abnormal symptoms was used for the test.
Activity of the sludge was assessed using a reference substance and the relation between new and old activated sludge was taken into account.
- Concentration of sludge: 30 mg/L
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, B, C and D, which are prescribed in JIS K 0102-1993-21 , were made up to 1000 mL with purified water (Takasugi Seiyaku Co., Ltd.), and then the pH of this solution was adjusted to 7.0.
- Test temperature: 25 ± 1°C
- pH: 7
- Suspended solids concentration: 30 mg/L

TEST SYSTEM
- Culturing apparatus: 300 mL in volume (improved type vessel)
- Number of culture flasks/concentration: 3
- Measuring equipment: Closed system oxygen consumption measuring apparatus (Coulometer: Ohkura Electric Co., Ltd.) (Data sampler: Asahi Techneion Co., Ltd.)
- Details of trap for CO2 and volatile organics if used: Soda lime No. 1 (for absorption of carbon dioxide, Wako Pure Chemical Industries, Ltd.)

PREPARATION OF TEST SOLUTIONS
- Test solution (water + test material) (n=1, Vessel No.4): In one test vessel, 30 mg of the test material was accurately weighed and added to 300 mL of purified water, so that the concentration reached 100 mg/L.
- Test solution (sludge + test material) (n=3, Vessel No. 1, 2 and 3): In each test vessel, 30 mg of the test material was accurately weighed and added to the basal culture medium (300 mL - a volume of the activated sludge inoculated), so that the concentration reached 100 mg/L.
- Test solution (sludge + aniline) (n=1 , Vessel No.6): In one test vessel, 29.5 μL [30.0 mg = 29.5 μL x 1.022 g/cm³ (density)] of aniline was added into the basal culture medium (300 mL- a volume of the activated sludge inoculated), so that the concentration reached 100 mg/L.
- Test solution (control blank) (n=1, Vessel No.5): In one test vessel, nothing was added to the basal culture medium (300 mL – a volume of the activated sludge inoculated).
- The activated sludge was added to each test vessel (b), (c) and (d), so that the concentration of the suspended solid reached 30 mg/L.

CALCULATION OF PERCENTAGE BIODEGRADATION
The percentage biodegradation was calculated by the following equations and expressed in whole numbers:
- Percentage biodegradation by BOD
Percentage biodegradation (%) = [(BOD – B) / TOD] x 100
BOD = Biochemical oxygen demand in the test solution (sludge + test material) (experimental) (mg)
B = Biochemical oxygen demand in the control blank (experimental) (mg)
TOD = Theoretical oxygen demand required when the test material was completely oxidised (theoretical) (mg)

- Percentage biodegradation by HPLC
Percentage biodegradation (%) = [ (Sw- Ss) / Sw ] x 100
Ss = Residual amount of the test material in the test solution (sludge + test material) (experimental) (mg)
Sw = Residual amount of the test material in the test solution (water + test material) (experimental) (mg)
Reference substance:
aniline
Test performance:
Percentage biodegradations of aniline calculated by the BOD values was 71 and 76 % at the 7th and 14th day, respectively. It was concluded that the test conditions were valid.
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
BOD
Value:
1
Sampling time:
28 d
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
HPLC
Value:
0
Sampling time:
28 d
Details on results:
APPEARANCE OF TEST SOLUTIONS
- At the initiation of cultivation:
Water + test material: test material was not dissolved
Sludge + test material: test material was not dissolved

- At the completion of cultivation:
Water + test material: insoluble compound was observed
Sludge + test material: Insoluble compound except for the sludge was observed. Growth of the sludge was not observed.
Results with reference substance:
Percentage biodegradations of aniline calculated by the BOD values was 71 and 76% at the 7th and 14th day, respectively.

Table 1: Analytic results of the test solution after 28 days

 

Water + Test material

Sludge + Test material

Theoretical Amount

Vessel 4

Vessel 1

Vessel 2

Vessel 3

BOD

mg

0

0

1.4

1.2

74.4

Residual amount and percentage residue of test material

(HPLC)

mg

29.2

29.6

29.9

29.9

30.0

%

97

99

100

100

-

 

Table 2: Percentage biodegradation after 28 days

Method

Percentage biodegradation (%)

Vessel 1

Vessel 2

Vessel 3

Average

BOD

0

2

2

1

HPLC

0

0

0

0

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the conditions of this study, the test material was not biodegraded by microorganisms.
Executive summary:

The biodegradation of the test material was investigated in accordance with Japanese guidelines in a study similar in design to OECD 301C. The test was performed under GLP conditions.

The activated sludge at suspended solids concentration of 30 mg/L was exposed to the test material at 100 mg/L for 28 days at 25 ± 1 C. Biochemical oxygen demand (BOD) was measured by means of a closed system using oxygen consumption measuring apparatus. The test material was determined using high performance liquid chromatography (HPLC).

After 28 days the average percentage biodegradation by BOD was 1% and by HPLC was 0%.

Percentage biodegradations of aniline, the positive control used in the study, calculated by the BOD values was 71 and 76% at the 7th and 14th day, respectively. It was concluded that this test conditions were valid.

Under the conditions of this study, the test material was not biodegraded by microorganisms.

Description of key information

Under the conditions of the study, the test material was not biodegraded by microorganisms.

Key value for chemical safety assessment

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

Additional information

The biodegradation of the test material was investigated in accordance with Japanese guidelines in a study similar in design to OECD 301C. The test was performed under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The activated sludge at suspended solids concentration of 30 mg/L was exposed to the test material at 100 mg/L for 28 days at 25 ± 1°C. Biochemical oxygen demand (BOD) was measured by means of a closed system using oxygen consumption measuring apparatus. The test material was determined using high performance liquid chromatography (HPLC).

After 28 days the average percentage biodegradation by BOD was 1% and by HPLC was 0%.

Percentage biodegradations of aniline, the positive control used in the study, calculated by the BOD values was 71 and 76% at the 7th and 14th day, respectively. It was concluded that this test conditions were valid.

Under the conditions of the study, the test material was not biodegraded by microorganisms.