Registration Dossier

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:
22 Jun - 20 Jul 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: "Method for Testing the Biodegradability of Chemical Substances by Microorganisms", Testing Methdos for New Chemical Substances
Version / remarks:
No. 1121002 (Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare); No. 2 (Manufacturing Industries Bureau, Ministry of Economy, Trade and Industry); No. 031121002 (Environmental Policy Bureau, Ministry of the Environment, Japan)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
mixture of sewage, soil and natural water
Details on inoculum:
- Source of inoculum/activated sludge: Return sludge from sewage plants and surface water and surface soil which was in contact with the atmosphere were collected from 10 locations in Japan: Fushikogawa city sewage plant (Sapporo-shi, Hokkaido), Fukashiba industrial sewage plant (Kashima-gun, Ibaraki), Nakahama city sewage plant (Osaka-shi, Osaka), Ochiai city sewage plant (Shinjuku-ku, Tokyo), Kitakami River (Ishinomaki-shi, Miyagi), Shinano River (Niigata-shi, Niigata), Yoshino River (Tokushima-shi, Tokushima), Lake Biwa (Otsu-shi, Shiga), Hiroshima Bay (Hiroshima-shi, Hiroshima), Dokai Bay (Kitakyushu-shi, Fukuoka)
- Preparation of inoculum for exposure: Activated sludge was prepared by mixing a filtrate of activated sludge cultivated for about 3 months (5 L) and the filtrate (5 L) of newly collected sludge from each loacation. The mixed filtrate (10 L) was aerated (pre-filtered open air) after the pH value of the mixture was adjusted to 7.0 ± 1.0.
- Cultivation: Roughly 30 min 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 10 L. This mixture was aerated for 30 min 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 wt% in the volume of dechlorinated water added. This procedure was repeated once every day. Cultivation was carried out at 25 ± 2 °C. Composition of 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.
- Concentration of sludge: 3850 mg/L suspended solids
- Concentration of activated sludge: 30 mg/L suspended solids
- Water filtered: Yes
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:
O2 consumption
Remarks:
Biochemical oxygen demand (BOD)
Parameter followed for biodegradation estimation:
DOC removal
Remarks:
Dissolved organic carbon (DOC) determined by analysis of total organic carbon (TOC)
Parameter followed for biodegradation estimation:
test mat. analysis
Remarks:
by high-performance liquid chromatography (HPLC)
Details on study design:
TEST CONDITIONS
- Composition of medium: Basal culture medium (prescriebed in JIS K 0102-1998, Section 21) prepared with purified water (Takasugi Pharmaceutical, The Japanese Pharmacopoeia)
- pH: The pH of the basel culture medium was adjusted to 7.0
- Test temperature: 25 ± 1 °C
- Suspended solids concentration at test start: 30 mg/L
- Continuous darkness: Yes

TEST SYSTEM
- Culturing apparatus: Closed system oxygen consumption measuring apparatus (300 mL)
- Number of culture flasks/concentration: 3
- Measuring equipment: Asahi Techneion
- Details of trap for CO2: Soda lime No. 1 (Wako Pure Chemical Industries)
- Other: Each test solution was stirred with a magnetic stirrer.

SAMPLING
- Sampling frequency: DOC, test item concentration and concentration of methacrylic acid, 3,5-dimethylpyrazole and 2-aminoethanol in the test solutions (transformation products): After 28 d. Measurement of biochemical oxygen demand (BOD): Continuous
- Other: During the cultivation period, the appearance of the test solutions and the instruments were checked once a day.

CONTROL AND BLANK SYSTEM
- Test suspension (3 replicates): 297.66 mL basal culture medium + 2.34 mL activated sludge (30 mg/L) + 30 mg test item (100 mg/L)
- Control blank (1 replicate): 300 mL purified water + 30 mg test item (100 mg/L) (1 replicate)
- Inoculum blank (1 replicate): 297.66 mL basal culture medium + 2.34 mL activated sludge (30 mg/L)
- Toxicity control (1 replicate): 297.66 mL basal culture medium + 2.34 mL activated sludge (30 mg/L) + 29.5 µL aniline (30 mg, corresponding to 100 mg/L)
Reference substance
Reference substance:
aniline
Remarks:
Showa Chemicals, reagent grade, Lot No. SP-3442Z

Results and discussion

% Degradationopen allclose all
Parameter:
% degradation (O2 consumption)
Value:
32
Sampling time:
28 d
Remarks on result:
other:
Remarks:
average
Parameter:
% degradation (DOC removal)
Value:
35
Sampling time:
28 d
Remarks on result:
other:
Remarks:
average
Parameter:
% degradation (test mat. analysis)
Value:
100
Sampling time:
28 d
Remarks on result:
other:
Remarks:
average

BOD5 / COD results

Results with reference substance:
74.2% degradation in 28 d.

Any other information on results incl. tables

VALIDITY CRITERIA

The study met the validity criteria prescribed by the guideline and is thus considered valid and reliable (Table 1).

 

Table 1. Validity criteria biodegradation study.

 

 

Value in this test

Criterion

Criterion met

Difference between extremes of replicate values of percentage biodegradation or decrease

% biodegradation by BOD

5%

 

 

 

< 20%

Yes

% biodegradation by DOC

7%

Yes

% decrease of test item

0%

Yes

% biodegradation of aniline by BOD

after 14 d

75%

≥ 60%

Yes

 

 

ANALYTICAL RESULTS

CONTROL SAMPLE (purified water + test item without sludge)

The percentage residue of the test item by HPLC analysis was 42% and six peaks were identified, indicating that the parent compound degraded to several conversion products, including 2,5-Dimethylpyrazole, Methacrylic acid and 2-Aminoethanol in the test solution (test medium + test item without sludge) (Table 2). The low mass balance of fragments A (65%) and B (54%) are assumed to be due to the production of unknown converted products containing these fragments.

The percentage DOC residue was 93%, which is explained by the likely decarboxylation reaction of the carbonyl group of the amide bond of the test item. When 58% of the additive amount of the test item is decarboxylated considering the remaining test item residue (42%), the calculated DOC is 16.4 mg C, which is close to the measured value of 15.9 mg C.

 

TEST SOLUTION (test medium + test item and sludge)

The test item was not detected by HPLC in any test solution containing sludge and test item (Table 2). The percentage residue was 57 – 63% in terms of DOC, indicating that water-soluble transformation products were produced. Methacrylic acid, 2-Aminoethanol and 3,5-Dimethylpyrazole were expected to be produced but only 3,5-Dimethylpyrazole was detected. Since biodegradation was 29 – 34% and growth of sludge was observed, Methacrylic acid and 2-Aminoethanol were likely biodegraded by microorganisms.

Three or four peaks (peaks A – D), corresponding to transformation products, were detected in the HPLC chromatograms, of which the 3 containing structural fragment C were qualitatively analyzed by LC-MS.

 

Table 2. Analytical results of the test solutions after 28 d.

 

 

Water + test item

Sludge + test item

Theoretical amount

 

 

vessel 1

vessel 2

vessel 3

vessel 4

 

BOD

mg

0.0

19.9

22.8

22.1

67.8

Residual amount and % residue of DOC*10

mgC

15.9

10.4

10.8

9.7

17.2

%

93

61

63

57

-

Residual amount and % residue of test item (HPLC)

mg

12.6

0

0

0

30.0

% (1)

42

0

0

0

-

Produced amount and % production of Methacrylic acid (HPLC)

mg

2.4

0

0

0

10.3

% (2)

23

0

0

0

-

Produced amount and % production of 3,5-Dimethylpyrazole (HPLC)

mg

6.4

7.5

8.5

8.3

11.5

% (3)

55

65

74

72

-

Produced amount and % production of 2-Aminoethanol (LC-MS)

mg

0.9

0

0

0

7.3

% (4)

12

0

0

0

-

Production of other converted products (HPLC)*11

 

 -

 

detected

 

detected

 

-

Mass balance of fragment A (1+2)*12

%

65

0

0

0

-

Mass balance of fragment B (1+4)*12

%

54

0

0

0

-

Mass balance of fragment C (1 + 3)*12

%

97

65

74

72

-

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

*11       In the analysis of the test item, converted products except methacrylci acid, 3,5-dimethylpirazole and 2-aminoethanol were detected in the test solution (water + test item) and the test solutions (sludge + test item).

*12       The test item was divided into 3 structural fragments: Methacrylic acid (A), 2-Aminoethanol (B) and 3,5-Dimethylpyrazole (C)

 

CONCLUSION

The test item was entirely degraded into six transformation products, including Methacrylic acid (fragment A), 2-Aminoethanol (fragment B), 3,5-Dimethylpyrazole (fragment C), 2-[(3,5-dimethylpyrazolyl)carbonylamino]acetaldehyde, 2-[3,5-Dimethylpyrazolyl)carbonylamino]ethanol and 2-[3,5-Dimethylpyrazolyl)carbonylamino]acetic acid. Methacrylic acid and 2-Aminoethanol underwent complete biodegradation. On the other hand, the other conversion products were not biodegraded and remained.

 

BIOLOGICAL RESULTS

The percentage decrease of the test item was calculated instead of the percentage biodegradation of the test item (percentage biodegradation by the chemical analysis of the test item) because the percentage residue of the test item in the test solution (test medium + test item without sludge) was less than 90% (42%) (Table 3).

 

Table 3. Percentage biodegradation after 28 d.

 

 

sludge + test item

vessel 2

vessel 3

vessel 4

average

biodegradation by BOD

%

29

34

33

32

biodegradation by DOC

%

34

32

39

35

decrease of test item (HPLC)

%

100

100

100

100

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.
Interpretation of results:
not readily biodegradable