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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:
29 Nov - 27 Dec 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Version / remarks:
17 Jul 1992
Qualifier:
according to guideline
Guideline:
other: "Method for Testing the Biodegradability of Chemical Substances by Microorganisms", Testing Methods for New Chemical Substances, No. 0331-7, Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare
Version / remarks:
31 Mar 2011
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of 2-ethyl-4-methyl-1H-imidazole-1-propiononitrile and 2-ethyl-5-methyl-1H-imidazole-1-propiononitrile
EC Number:
947-727-8
Cas Number:
568591-00-4
Molecular formula:
C9H13N3
IUPAC Name:
Reaction mass of 2-ethyl-4-methyl-1H-imidazole-1-propiononitrile and 2-ethyl-5-methyl-1H-imidazole-1-propiononitrile

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
mixture of sewage, soil and natural water
Remarks:
Sampled from surface water, surface soil of rivers, lake and inland sea and from return sludge of sewage plants
Details on inoculum:
- Source of inoculum/activated sludge: Activated sludge was sampled at 10 locations in Japan from surface water and surface soil of rivers, lake and inland sea and from return sludge of sewage plants.
- Storage length: Sampling period: September 2017; Initiation of use: 20 Oct 2017. The activated sludge was cultivated for 19.5 h after feeding with synthetic sewage.
- Pretreatment: Activated sludge was fed with synthetic sewage, which contained glucose, peptone and potassium dihydrogenphosphate dissolved in purified water (pH 7.0 ± 1.0)
- Concentration of sludge: 3830 mg/L
- Initial cell/biomass concentration: 30 mg suspended solids/L (in test vessels)
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
Parameter followed for biodegradation estimation:
DOC removal
Parameter followed for biodegradation estimation:
test mat. analysis
Details on study design:
TEST CONDITIONS
- Composition of medium: Basal culture medium
- Test temperature: 25 ± 1 °C
- pH: 7.0 - 9.0
- pH adjusted: Yes, the pH of the basal culture medium was adjusted to pH 7.0 and the pH of the test solution (sludge + test item in basal culture medium) was adjusted to pH 7.0.
- Suspended solids concentration: 30 mg suspended solids/L
- Continuous darkness: Yes
- Other: Each test solution was stirred with a stirrer.

TEST SYSTEM
- Culturing apparatus: Glass vessels and temperature controlled bath with measuring unit
- Number of culture flasks/concentration: 3
- Measuring equipment: Closed system oxygen consumption measuring apparatus (No. CM-068)
- Details of CO2 trap: Soda lime No. 1 for absorption of CO2 (Wako Pure Chemical Industries)

SAMPLING
- Sampling frequency: During the incubation period, the appearance of the test solutions was observed once a day. BOD was measured continuously. The incubation temperature was measured once a day. At the end of incubation, dissolved organic carbon (DOC) and test item concentrations were determined.
- Sampling method: 40 mL were sampled from the test solutions, which were then centrifuged to obtain supernatants for analytics.

CONTROL AND BLANK SYSTEM
- Negative control: 1 replicate: 100 mg/L test item in water (3 mL of 10.0 g/L aqueous solution of test item was added to 297 mL purified water)
- Test solution: 3 replicates: 100 mg/L test item in basal culture medium + activated sludge (3 mL of 10.0 g/L aqueous solution of test item + 2.35 mL activated sludge + 294.65 mL basal culture medium)
- Procedure/Positive control: 1 replicate: 100 mg/L aniline in basal culture medium + activated sludge (29.5 µL/30 mg aniline + 2.35 mL activated sludge + 297.65 mL basal culture medium)
- Blank control: 1 replicate: 2.35 mL activated sludge in 297.65 mL basal culture medium
Reference substance
Reference substance:
aniline

Results and discussion

% Degradationopen allclose all
Parameter:
% degradation (O2 consumption)
Value:
0
Sampling time:
28 d
Remarks on result:
other:
Remarks:
The calculated average value was -4%
Parameter:
% degradation (DOC removal)
Value:
0
Sampling time:
28 d
Remarks on result:
other:
Remarks:
The calculated average value was -4%
Parameter:
% degradation (test mat. analysis)
Value:
0
Sampling time:
28 d
Details on results:
The average percent biodegradation after 28 d was 0% by BOD, DOC and by HPLC.
The growth of the sludge was not observed at the end of cultivation.

Any other information on results incl. tables

VALIDITY CRITERIA

The test fulfilled the validity criteria of the guideline (Table 1).

 

Table 1. Validity criteria for OECD 301C

Criterion from the guideline

Outcome

Validity criterion fulfilled

The oxygen uptake of the inoculum blank is normally 20-30 mg O2/L and should not be greater than 60 mg O2/L in 28 days.

BOD (blank control) after 28 d: 32 mg/L

Yes

A test is considered valid if the difference of extremes of replicate values of the removal of the test substance at the plateau or at the end of the test, as appropriate is less than 20%.

Difference between extremes of replicate values of % biodegradation

by BOD: 2%

by DOC: 3%

by HPLC: 1%

Yes

Percentage degradation of aniline calculated from the oxygen consumption exceeds 40% after 7 days and 65% after 14 days.

 

After 7 d: 68%

After 14 d: 86%

Yes

 

RESULTS

Visual inspection of the test vessels, summarized in Table 2.

Table 2. Apperance of test solutions

 

Test solution

Visual appearance

 

At the start of incubation

water + test item

The test item was dissolved.

The test solution was colorless.

sludge + test item

The test item was dissolved.

The test solution was colorless.

blank control

Insoluble compound except the sludge was not observed.

The test solution was colorless.

 

At the end of incubation

water + test item

Insoluble compound was not observed.

The test solution was colorless.

sludge + test ite,

Insoluble compound was not observed.

Growth of sludge was not observed.

Test solution was colorless.

blank control

Insoluble compound except the sludge was not observed.

Test solution was colorless.

 

ANALTICAL RESULTS

Analytical results of the test solutions after 28 d are summarized in Table 3.

Table 3. Analytical results.

 

water + test item

sludge + test item

theoretical amount

replicate 1

replicate 2

replicate 3

BOD*

mg

0.0

-2.3

-4.2

-2.5

89.7

Residual amount and % residue of DOC

mgC

19.8

20.6

20.8

20.2

19.9

%

100

104

105

102

--

Residual amount** and % residue of test item (HPLC)

mg

29.2

29.0

29.1

29.2

30.0

%

97

97

97

97

--

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

**         Calculated based on the total peak area of the chromatograms

 

BIOLOGICAL RESULTS

Percent biodegradation is summarized in Table 4. The % degradation of the reference control (sludge + aniline) was 68% after 7 d, 86% after 14 d, 89% after 21 d and 88% after 28 d.

Table 4. % biodegradation after 28 d.

 

sludge + test item

replicate 1

replicate 2

replicate 3

average

Percent biodegradation by BOD

-3

-5

-3

0 (-4)*

Percent biodegradation by DOC

-4

-5

-2

0 (-4)*

Percent biodegradation of test item by HPLC

1

0

0

0

* The average % biodegradation was regarded as 0% because the calculated average value, indicated in parentheses, was negative.

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:
under test conditions no biodegradation observed
Conclusions:
The substance is not readily biodegradable according to OECD criteria.