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Environmental fate & pathways

Biodegradation in water: screening tests

Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Study initiation date: November 8, 2007. Study completion date: December 21, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Appearance: Yellow powder
Stability: Stable
Storage: Dark storage place at room temperature.

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, adapted
Details on inoculum:
Sampling sites:
On-site sludge sampling was carried out at 10 locations in Japan, including sewage plants, rivers, lakes and the sea.

Preparation method of activated sludge:
The filtrate (5 L) of the supernatant of the activated sludge* cultivated about for 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* after the pH value of the mixture was adjusted to 7.0 ± l.0.

Cultivation:
Approximately 30 minutes after ceasing aeration of the sludge mixture, supernatant corresponding to about 1/3 of the whole volume was removed. Dechlorinated water was added to the remaining portion so that the total volume reached 10L. This mixture was aerated for 30 minutes or more, and then a predetermined amount of synthetic sewage was added to the mixture so that the concentration of the synthetic sewage was 0.1 % in the volume of dechlorinated water added. This procedure was repeated once every day. Cultivation was carried out at 25 ± 2°C.

Synthetic sewage:
Glucose, peptone and potassium dihydrogenphosphate were dissolved in purified water to obtain 50 g/L of the solution for each component. The pH of the solution was adjusted to 7. 0± 1.0 with sodium hydroxide.

Control and use:
During cultivation, the appearance of the supernatant, sedimentation of the sludge, formation of flock, pH, dissolved oxygen concentration in the solution and temperature were checked to maintain a normal state of sludge. It was confirmed that these were within the scope of the control standard stipulated in the "Testing Methods for New Chemical Substances". Microflora in the activated sludge was microscopically observed and sludge with no abnormal symptoms was used for the test. The activated sludge, which was cultivated for 19 hours after it had been added the synthetic sewage, was used.







Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimationopen allclose all
Parameter followed for biodegradation estimation:
other: Percenrage biodegradation by biological oxygen demand (BOD)
Parameter followed for biodegradation estimation:
other: Percentage biodegradation by dissolved organic carbon (DOC)
Parameter followed for biodegradation estimation:
other: Percentage biodegradation of test item (HPLC)
Details on study design:
Preparations for test:
1) Measurement of concentration of suspended solid in activated sludge
The concentration of suspended solid was measured to determine the amount of activated sludge to add.

2) Preparation of basal culture medium
Each 3 mL of solutions A, B, C and D, were made up to 1000 mL with purified water and then the pH of this solution was adjusted to 7.0.

Preparation of test solutions:
The following test solutions were prepared and cultivated.

(1) Addition of test or aniline
(a) Test solution (water + test item) (n=1, Vessel No.1)
In one test vessel, 3 mL of 10.0 g/L of the test item in water was added to 297 mL of purified water, so that the concentration of the test item reached 100 mg/L and then the pH of the solution was measured. 10.0 g/L of the test item in water was pretreated as follows. The test sample was accurately weighed and dissolved in purified water to obtain it.

(b) Test solution (sludge + test item) (n=3, Vessel No. 2, 3 and 4).
In each test vessel, 3 mL of 10.0 g/L of the test item in water was added to the basal culture medium [the volume was less than 297 mL by the volume (2.30 mL) of activated sludge inoculated], so that the concentration of the test item reached 100 mg/L and then the pH of the solution was measured. 10.0 g/L of the test item in water was pretreated as follows. The test sample was accurately weighed and dissolved in purified water to obtain it.

(c) Test solution (sludge + aniline) (n=1, Vessel No.6)
In one test vessel, 29.5 µL [30 mg = 29.5 µL x 1.022 g/cm3 (density)] of aniline was taken out by microsyringe and added to [the volume was less than 300 mL by the volume (2.30 mL) of activated sludge inoculated], so that the concentration of aniline reached 100 mg/L.

(d) Test solution (control blank) (n=1, Vessel No.5)
In one test vessel, nothing was added to the basal culture medium [the volume was less than 300 mL by the volume (2.30 mL) of activated sludge inoculated].

(2) Inoculation of activated sludge:
The activated sludge cultivated was added to each test vessel, b, c and d, so that the concentration of the suspended solid reached 30 mg/L.

Instruments and conditions for cultivation:
1) Instruments for cultivation:
Closed system oxygen consumption measuring apparatus.
Vessel: 300 mL in volume
Absorbent for carbon dioxide: Soda lime No.1

2) Conditions of cultivation:
Cultivation temperature: 25 ± 1°C
Cultivation duration: 28 days (in darkness).
Stirring method: Each test solution was stirred by a magnetic stirrer.

Observation and measurement of test conditions:
1) Observations of test solution:
During the cilltivation, the appearance ofthe test solution was observed once a day and conditions ofthe instruments were checked properly.

2) Measurement of biochemical oxygen demand (BOD):
During the cultivation period, the change in BOD of the test solutions was measured by autorecording using a data sampler. Cultivation temperature was measured and recorded once a day.





























Reference substance
Reference substance:
aniline

Results and discussion

Preliminary study:
Not applicable.
Test performance:
The validity criteria of the test and the values in this test are shown (see any other information on results section). The test was valid as all of the values met the criteria.
No adverse effects on the reliability of this test were noted.
% Degradationopen allclose all
Parameter:
other: Percentage biodegradation by BOD
Value:
0
Sampling time:
28 d
Remarks on result:
other: Average from 3 vessels.
Parameter:
other: Percentage biodegradation by DOC
Value:
3
Sampling time:
28 d
Remarks on result:
other: Average from 3 vessels
Parameter:
other: Percentage biodegradation of test item (HPLC)
Value:
1
Sampling time:
28 d
Remarks on result:
other: Average from 3 vessels
Details on results:
See 'any other information on results incl.tables' section

BOD5 / COD results

Results with reference substance:
Percentage biodegradation of aniline after 28 days: 76%

Any other information on results incl. tables

Validity of test conditions:

 

Value in test

Value of criterion

Differences of extremes of replicate values of % biodegradation

% biodegradation by BOD

2%

<20%

% biodegradation by DOC

4%

% biodegradation of test item

1%

Percentage biodegradation of aniline by BOD

After 7 days

63%

>40%

After 14 days

73%

>65%

BOD value of control blank

After 28 days

8.3 mg

<18 mg (<60 mg/L)

Analytical results of the test solutions after 28 days:

In the test solution (water + test item) and the test solutions (sludge + test item), approximately theoretical amount of the test item remained and no peak in addition to the test item was detected on the HPLC chromatogram. Therefore, any converted products

were judged not to be produced and then not analyzed.

Percentage biodegradation:

Percentage biodegradations after 28 days were as follows:

 

Sludge + test item

Table

Vessel 2

Vessel 3

Vessel 4

Average

% biodegradation by BOD

1

1

-1

0

1

% biodegradation by DOC

5

1

2

3

2

% biodegradation of test item (HPLC)

0

1

1

1

3

Tables 1, 2 and 3 attached as background material.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
other: not readily biodegradable
Conclusions:
The test item was not biodegraded by microorganisms under the conditions of this test.
Executive summary:

Biodegradation test of CY-9 by microorganisms

Conditions of cultivation:

1) Concentration of test item: 100 mg/L

2) Concentration of activated sludge (as the concentration of suspended solid): 30 mg/L

3) Volume of test solution: 300 mL

4) Cultivation temperature: 25 ± 1 °C

5) Cultivation duration: 28 days

Measurement and analysis:

1) Measurement of biochemical oxygen demand (BOD) by means of a closed system oxygen consumption measuring apparatus.

2) Determination of dissolved organic carbon (DOC) by a total organic carbon analysis (TOC)

3) Determination of test item by HPLC

Results:

1) Percentage biodegradation by BOD (1%, 1%, -1%): Average 0%

2) Percentage biodegradation by DOC (5%, 1%, 2%): Average 3%

3) Percentage biodegradation by HPLC (0%, 1%, 1%): Average 1%

Conclusion:

The test substance was not biodegraded by microorganisms under the test conditions.