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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:
From September 19th to October 17th, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Version / remarks:
July 17th, 1992
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
(3S,6E)-3,7,11-trimethyldodeca-6,10-dienal
EC Number:
810-298-1
Cas Number:
194934-66-2
Molecular formula:
C15H26O
IUPAC Name:
(3S,6E)-3,7,11-trimethyldodeca-6,10-dienal
Test material form:
liquid

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge (adaptation not specified)
Details on inoculum:
- Activated sludge: on-site sampling was carried out at 10 locations in Japan (samples were from surface water and surface soil of rivers, lakes and inland sea; return sludge from sewage plants).
- Method of cultivation: cultivated for 18 hours after the synthetic sewage addition. The synthetic sewage was prepared according to the following method: glucose, peptone and potassium dihydrogenphosphate were dissolved in purified water and the pH of the solution was adjusted to 7.0 ± 1.0.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
test mat. analysis
Details on study design:
TEST CONDITIONS
- Composition of medium: additive amount of activated sludge into the vessel was 2.28 ml on the basis of the concentration of suspended soil in the activated sludge.
- Preparation of the basal culture medium: the basal culture medium (5 l) was prepared adding purified water to each 3 ml aliquot of solution A, B, c and D, which are described in JKS K 0102-2008 Section 21, in order to prepare 1 litre of solution.
- Volume of the test solution: 300 mg/l
- Incubation temperature: 25 ± 1 °C
- pH adjusted: yes; adjusted to 7.0.
- Suspended solids concentration: 30 mg/l.
- Continuous darkness: yes.
- Stirring method: stirring with a stirrer.

REPLICATES
- Test solution (test item + water): one vessel; 30 mg of the test item were added to 300 ml of purified water.
- Test solution (test item + sludge): three vessels; 30 mg of the test item were added to the basal culture medium, thus the concentration reached was 100 mg/l.
- Reference solution (aniline + sludge): one vessel; 29.5 μl (30 mg) of aniline were added to the basal culture medium, thus the concentration reached was 100 mg/l.

TEST SYSTEM
- Culturing apparatus: closed system oxygen consumption measuring apparatus.
- Vessel: test solutions and control blank were placed in glass vessels with lid for volatile substance. Reference solution was placed in glass vessel.
- Adsorbed for carbon dioxide: soda lime.

OBSERVATION AND MEASUREMENT
- Observation of test solution: during the incubation period, the appearance of the test solution was observed once a day.
- Measurement of BOD: during the incubation period, BOD was measured continuously by a closed system oxygen consumption measuring apparatus.
- Temperature: the incubation temperature was measured and recorded once a day.

CONTROL AND BLANK SYSTEM
- Inoculum blank: one vessel; nothing was added to the basal culture medium.
Reference substance
Reference substance:
aniline

Results and discussion

% Degradation
Key result
Parameter:
% degradation (test mat. analysis)
Value:
86
Sampling time:
28 d
Details on results:
In the test solutions (sludge + test item) the average percentage residue of the test item was 0 %, the average percentage biodegradation of BOD was 86 %, growth of sludge was observed at the end of incubation. Additionally, no peaks of converted products were detected on total ion current (TIC) chromatogram of the qualitative analysis of converted products by LC-MS. Therefore, the test item was biodegraded under the test conditions of the experiment.
In the test solution (water + test item), the percentage residue of the test item was 6 %, the peaks of converted products were detected on HPLC chromatogram. The result suggests that most of the test item was converted by an abiotic reaction in water. Additionally, nine peaks of converted products were detected at 3.5 - 15.4 minutes on TIC chromatogram of the qualitative analysis of converted products by LC-MS using a ODS column. The retention times of the converted products (3.5 - 15.4 min) were shorter than that of the test item (about 3.5 min) on the chromatograms obtained under the analytical conditions of the experiment. Therefore, the polarities of converted products are thought to be higher than that of the test item. The converted products were likely to be produced by oxidation of the test item and addition of one water molecule to the test item. However, their structures were hardly presumed.

APPEARANCE OF THE TEST SOLUTION
Start of incubation water + test item: test item was not dissolved; the test solution was colourless.
Start of incubation sludge + test item: test item was not dissolved; the test solution was colourless.
End of incubation water + test item: insoluble compound was observed; the test solution was colourless.
End of incubation sludge + test item: insoluble compound except the sludge was not observed; Growth of the sludge was observed. The test solution was colourless.

BOD5 / COD results

BOD5 / COD
Key result
Parameter:
ThOD
Value:
90.6 mg O2/g test mat.
Results with reference substance:
Aniline degradation after 28 days: 91 %
TOD aniline: 72.3 mg

Any other information on results incl. tables

CALCULATION TABLE FOR PERCENTAGE BIODEGRADATION BY BOD

Vessel N. 7th day 14th day 21st day 28th day Average
BOD (mg) Deg. (%) BOD (mg) Deg. (%) BOD (mg) Deg. (%) BOD (mg) Deg. (%) Deg. (%)
1 (water + test item) 1.2 - 2.2 - 2.2 - 2.3 - -
2 (sludge + test item) 51.9 53 76.3 76 84.4 83 83 84 86
3 (sludge + test item) 50.0 50 75.6 76 83.6 82 82 83
4 (sludge + test item) 52.9 54 82.5 83 90.9 90 93.2 92
5 (control blank) 4.3 - 7.1 - 9.1 - 10.1 - -
6 (sludge + aniline) 60.1 77 72.1 90 75.1 91 75.7 91 -

ANALYTICAL RESULTS OF THE TEST SOLUTION

Analytical results of the test solution after 28 days


Water + test item Sludge + test item Theoretical amount
Vessel 1 Vessel 2 Vessel 3 Vessel 4
BOD*2 mg 2.3 76.0 75.5 83.1 90.6
Residual amount and percentage residue of test item (HPLC) mg 1.8 0 0 0 30.0
% 6 0 0 0 -
Detection of converted products (HPLC) - Detected Not detected -

*2: the value of the test solution (control blank) was subtracted from the values of the test solutions (sludge + test item)

Percentage of biodegradation after 28 days.

The percentage decrease of the test item, the ratio of decrease amount to the theoretical amount, was calculated instead of the percentage biodegradation by the chemical analysis because the percentage residue of the percentage biodegradation by the chemical analysis because the percentage residue of the test item in the test solution (water + test item) (6 %) was less than 90 %.


Sludge + test item
Vessel 2 Vessel 3 Vessel 4 Average
Percentage biodegradation by BOD % 84 83 92 86
Percentage decrease of test item (HPLC) % 100 100 100 100

RESULT OF QUALITATIVE ANALYSIS OF CONVERTED PRODUCTS

In the qualitative analysis of the test item, converted products were detected. Therefore, the qualitative analysis of converted products by LC-MS was performed. As the result of the test solution (water + test item), nine peaks of converted products were detected in 3.5 - 15.4 minutes on total ion current (TIC) chromatogram of positive ion and no peaks of converted products were detected on TIC chromatogram of negative ion. In the test solutions (sludge + test item) no peaks of converted products were detected on TIC chromatogram of positive and negative ion. The converted products were treated as Converted product A to I in elution order. The results of the mass spectral analysis of them showed that the presumed structural formula of all converted products was unknown, although the molecular weight of some converted products (A, C, D, E, H, and I) was presumed as reported below.

Table of converted products detected in the test solution

Converted product Retention time (min) Detection ion (m/z) and attribution (presumption) Presumed molecular weight Presumed structure
A 3.5 221.2 [M-2H2O+H]+
256.3 [M-H
2O-NH4]+
274.3 [M+NH
4]+
256 unknown
B 4.2 239.2 unknown unknown unknown
C 4.9 257.3 [M-H2O+H]+
275.3 [M+H]
+
274 unknown
D 6.5 243.2 [M-H2O+H]+
278.3 [M-NH
4]+
283.3 [M+Na]
+
260 unknown
E 7.7 221.2 [M-H2O+H]+
239.2 [M+H]
+
238 unknown
F 9.2 256.3 unknown unknown unknown
G 10.1 257.3 unknown unknown unknown
H 11.1 239.2 [M-H2O+H]+
257.3 [M+H]
+
256 unknown
I 15.4 205.2 [M-2H2O+H]+
223.2 [M-H
2O-H]+
263.3 [M+Na]
+
240 unknown

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test item was biodegradable under the test conditions.
Executive summary:

The substance was tested for biodegradation potential according to the OECD guideline 301C (July 17th, 1992) test method. The initial test concentration was 100 mg/l, while the sludge concentration was 30 mg/l; three vessels were prepared. Furthermore, one vessel was fulfilled with 30 mg of the test item and 300 ml of purified water, one vessel was used as control blank and another one as reference, testing aniline with the sludge. Converted products were detected on total ion current (TIC) chromatogram.

In the test solutions the average percentage biodegradation of BOD was 86 %, growth of sludge was observed at the end of incubation. Additionally, no peaks of converted products were detected. Therefore, the test item was biodegraded under the test conditions of the experiment. In the test solution (water + test item), the percentage residue of the test item was 6 %; the result suggests that most of the test item was converted by an abiotic reaction in water. Additionally, nine peaks of converted products were detected: the retention time of the converted products were shorter than that of the test item, therefore, the polarities of converted products are thought to be higher than that of the test item. The converted products were likely to be produced by oxidation of the test item and addition of one water molecule to the test item. However, their structures were hardly presumed.