TOMILAC 214

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:

18 April 2008 - 11 June 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Conducted to recognised testing methodology to GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Test method relating to the new chemical substances ” (Yakushokuhatsu No. 1121002 on November 21, 2003, Heisei 15.11.17 Seikyoku No. 2, Kanpokihatsu No. 031121002

Deviations:

no

Principles of method if other than guideline:

The biochemical oxygen demand (BOD) generated by degradation of the test substance under the presence of activated sludge was measured during 28 days by a closed system oxygen consumption measuring apparatus. After the completion of measurement of BOD, dissolved organic carbon (DOC) and residual amount of the test substance were also measured. The degradability was calculated based on these measured values to evaluate biodegradability of the test substance.

Measurement of Biochemical Oxygen Demand (BOD) by a closed system oxygen consumption measuring apparatus (for 28 days)
Measurement of Dissolved Organic Carbon (DOC) by total organic carbon measuring apparatus (at day 28)
Measurement of residual amount of test substance by High-Performance Liquid Chromatography (HPLC) (at day 28)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): JKY-214
- Molecular formula (if other than submission substance): Not applicable
- Molecular weight (if other than submission substance): Not applicable
- Smiles notation (if other than submission substance): Not applicable
- InChl (if other than submission substance): Not applicable
- Structural formula attached as image file (if other tha n submission substance): Not applicable
- Substance type: Organic
- Physical state: White crystal
- Analytical purity: 99.5% w/w
- Impurities (identity and concentrations): Not specified
- Composition of test material, percentage of components:
- Isomers composition: Not applicable
- Purity test date: Not specified
- Lot/batch No.: RM05-58
- Expiration date of the lot/batch: Not specified
- Radiochemical purity (if radiolabelling): Not applicable
- Specific activity (if radiolabelling): Not applicable
- Locations of the label (if radiolabelling): Not applicable
- Expiration date of radiochemical substance (if radiolabelling): Not applicable
- Stability under test conditions: Stable. After the completion of the study, infrared absorption spectrum of the test substance stored in a desiccator for test material storage. The spectrum obtained was consistent with that measured before the initiation of the study. Therefore it was determined that the test substance was stable during storage
- Storage condition of test material: Room temperature, dark, tightly sealed
- Other: Not specified

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
Not applicable.

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge (adaptation not specified)

Details on inoculum:

MLSS: 3700mg/L
Date of receipt: April 17, 2008

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Chemicals Evaluation and Research Institute, Japan
- Laboratory culture: Not specified
- Method of cultivation: Not specified
- Storage conditions: Not specified
- Storage length: Not specified
- Preparation of inoculum for exposure: Not specified
- Pretreatment: Not specified
- Concentration of sludge: 30 mg/l
- Initial cell/biomass concentration: Not specified
- Water filtered: Not specified
- Type and size of filter used, if any: Not specified

Duration of test (contact time):

28 d

Initial test substance concentration

Initial conc.:

30 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

other: The biochemical oxygen demand (BOD) generated by degradation of the test substance under the presence of activated sludge was measured during 28 days by a closed system oxygen consumption measuring apparatus. After the completion of measurement of BOD, di

Details on study design:

Test method

This study was performed in accordance with “Test method relating to the new chemical substances ” (Yakushokuhatsu No. 1121002 on November 21, 2003, Heisei 15.11.17 Seikyoku No. 2, Kanpokihatsu No. 031121002).
The biochemical oxygen demand (BOD) generated by degradation of the test substance under the presence of activated sludge was measured during 28 days by a closed system oxygen consumption measuring apparatus. After the completion of measurement of BOD, dissolved organic carbon (DOC) and residual amount of the test substance were also measured. The degradability was calculated based on these measured values to evaluate biodegradability of the test substance.

Degradability test conditions

Degradability test was performed under the following conditions.

(Standard activated sludge)
1) MLSS: 3700mg/L
2) Source: Chemicals Evaluation and Research Institute, Japan
3) Date of receipt: April 17, 2008

(Conditions)
1) Temperature: 25±1oC
2) Period: 28 days (BOD measurement)
3) Test volume: 300mL
4) Concentration: Test substance and aniline* (control material): 100mg/L
Standard activated sludge: 30mg/L
* Kanto Chemical Co., Inc., special grade reagent, Lot No. 907X1751

(Test system and addition of the test material)

No. 1: Confirmation of degradation activity (aniline + sludge + basal medium)
Basal medium*1 was placed in a cultivation bottle, 29.5µL (30.0mg) of aniline was added with microsyringe and mixed, then sludge was added.

No. 2: Basal respiration of sludge (sludge + basal medium)
Basal medium*1 was placed in a cultivation bottle, then sludge was added.

No. 3, 4, 5: Degradation of test substance-1, 2, 3 (test substance + sludge + basal medium)
Basal medium*1 was placed in a cultivation bottle, the test substance was weighed in a glass-cup for 30.0mg and added, then sludge was added.
No. 6: Stable in water (test substance + purified water)
300mL of purified water*2 was poured into the cultivation bottle, the test substance was weighed in a glass-cup for 30.omg and added.

*1: Added amount of sludge suspension for 2.4mL was deducted from 300mL.
*2: JIS K0557 A4 grade.

Calibration curve
Test substance of 30.0mg was weighed and dissolved in acetone to make constant volume of 100mL. 300mg/L of standard solution was prepared and diluted in acetone. Acetone was used as 0mg/L of standard solution, and 0, 75.0, 150 and 300mg/L of the test substance standard solution was analyzed by HPLC to obtain peak area. Calibration curve was prepared with the concentration on the horizontal axis and peak area on the vertical axis.
The least-squares analysis was used to obtain the correlation coefficient of linear regression. The linearity was good as the correlation coefficient was 1.00 and it was accounted that a straight line would pass through the origin. Therefore the amount of the test substance in test solution was calculated by comparing to peak area that was obtained with standard solution of 300mg/L.

Detection limit
The minimum detectable peak area was established to be 10000 mAbs•sec. Based on the amount of the test substance that corresponded to this, the detection limit of the test substance was determined as 0.1mg.

Recovery test
The test solution of Degradation of test substance (n=2) and Basal respiration of sludge (n=1) was prepared in accordance with the preparation method shown in section 2.1, stirred for 30 minutes in closed-sysmtem oxygen consumption measuring apparatus.
Pre-treatment was held in accordance with the procedures in Measurement of residual amount of the test substance in flow sheet of pre-analysis treatment, the residual amount of the test substance was measured, and recovery rate of the test substance was calculated from the value of Degradation of test substance. As the result of two measurements, the recovery rate were 102 and 103 % respectively. The measurement result of the residual amount of test substance was calculated by correcting with the mean recovery rate of 103%. It was confirmed that the residual amount of the test substance was below detection limit in Basal respiration of sludge.

Evaluation of the test results

Formula to calculate degradability

The formula to calculate degradability from the measured value of BOD, DOC and the residual amount of the test substance are shown below.

1) BOD degradability
Degradability (%) = (BODs – BODb) / ThOD x 100
BODs: Oxygen consumption in Confirmation of degradation activity or Degradation of test substance (mg)
BODb: Oxygen consumption in Basal respiration of sludge (mg)
ThOD: Theoretical oxygen demand of aniline or the test substance (mg)
Calculation of theoretical oxygen demand (ThOD)
Aniline: 90.2mgO2/ 30.0mg
Aniline was assumed to be mineralized as below.
C6H7N + 35/4O2 → 6CO2 + 7/2H2O + NO2
The test substance: 59.5mgO2/ 30.0mg
The test substance was assumed to be mineralized as below.
C31H32O8S2 + 37O2 = 31CO2 + 16H2O + 2SO2

2) DOC degradability
Degradability (%) = [1- (DOCs – DOCb) / DOCc] x 100
DOCs: DOC in Degradation of test substance (mg)
DOCb: DOC in Basal respiration of sludge (mg)
DOCc: DOC in Stable in water (mg)
If the DOC in Stable in water is below 90% of initial theoretical amount, DOC degradability was not calculated.

3) Degradability from the residual amount of the test substance
Degradability (%) = (1- Cs/Cc) x 100
Cs: Residual amount of the test substance in Degradation of test substance (mg)
Cc: Residual amount of the test substance in Stable in water (mg)

Judgement of degradability

The degradability of the test substance is judged comprehensively based on BOD, DOC, measured residual amount of the test substance and calculated degradability.

Validity of the study

The study is considered to be valid when all of the following items are satisfied;
1) BOD degradability of reference substance at day 14 is greater than 60%.
2) The difference of the maximum value and the minimum value of the degradability at day 28 does not exceed 20%.
3) BOD in Basal respiration of sludge at day 28 does not exceed 18mgO2.

Reference substance

Reference substance:

aniline

Results and discussion

Preliminary study:

None conducted; main study only.

Test performance:

The study was judged as valid because the measured results satisfied all the items of the validity of the study.

The study is considered to be valid when all of the following items are satisfied;
1) BOD degradability of reference substance at day 14 is greater than 60%.
2) The difference of the maximum value and the minimum value of the degradability at day 28 does not exceed 20%.
3) BOD in Basal respiration of sludge at day 28 does not exceed 18mgO2.

% Degradation

Parameter:

other: The biochemical oxygen demand (BOD) and Dissolved Organic Carbon (DOC) where monitored as an assessment of ready biodegradability.

Value:

0

Sampling time:

28 d

Details on results:

Environmental factor which might have affected the reliability of the study results:

There was no factor which is applicable to the captioned.

The observation results of the contents in cultivation bottle after BOD measurement:

The solution was cloudy in Confirmation of degradation activity (No. 1), Degradation of test substance-1, 2 (No. 3, 4) and Stable in water (No. 6), slightly cloudy in Degradation of test substance-3 (No. 5).
Growth of sludge was noted in Confirmation of degradation activity compared to Basal respiration of sludge, no growth was noted in Degradation of test substance.

The result of pH measurement:

After the BOD measurement, pH was 7.2 respectively in Degradation of test substance, 8.4 in Stable in water.

BOD degradability:
BOD at day 28 was -0.3, -0.3, -0.2 mg in Degradation of test substance (the values after correction by BOD of Basal respiration of sludge, in the order of No. 3, 4, 5, the same order hereafter) against theoretical value of 59.5mg, 0.5mg in Stable in water. BOD degradability at day 28 was all calculated to be 0% (the calculated values were -1, -1, 0%).

DOC degradability:
DOC at day 28 was 0.1, 0.0, -0.1 mg in Degradation of test substance (the values after correction by DOC of Basal respiration of sludge) against initial theoretical value of 18.7mg, 0.3mg was detected in Stable in water. The test substance was hardly soluble in water and DOC in Stable in water was below 90% of initial theoretical value. Therefore DOC degradability was not calculated.

Degradability from the residual amount of the test substance:

The test substance was detected for 29.7, 30.1, 29.6mg in Degradation of test substance, 29.7mg in Stable in water against initial amount of 30.0mg. The degradability from the residual amount of the test substance was calculated as 0% respectively (calculated values were 0, -1, 0%).

Validity of results:
The study was judged as valid because the measured results satisfied all the items of the validity of the study.

BOD5 / COD results

Results with reference substance:

The reference substance fulfilled the validity requirements of the study.

Any other information on results incl. tables

Results (measured values at day 28)

Measured item	Degradation of test substance			Stable in water	Theoretical value
	1	2	3
BOD, mg^*1	-0.3	-0.3	-0.2	0.5	59.5
DOC, mg^*1	0.1	0.0	-0.1	0.3	18.7
Test substance, mg	29.7	30.1	29.6	29.7	30.0

*1 Values of Degradation of test substance is shown after deducting the values of Basal respiration of sludge.

 

Degradability at day 28

Degradability	Degradation of test substance			Average
	1	2	3
Degradability of BOD, %	0 (-1)^*2	0 (-1)^*2	0	0
Degradability of DOC, %	Not calculated because the test substance was hardly soluble in water.
Degradability of residual amount of test substance, %	0	0 (-1)^*2	0	0

*2 Calculated degradability is shown in the parentheses because degradability was calculated to be negative value.

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

Discussion
It is judged that the test substance was not readily biodegradable and not transformed because degradability of BOD at day 28 and degradability of residual amount of test substance were 0% at average.

Executive summary:

Discussion

It is judged that the test substance was not readily biodegradable and not transformed because degradability of BOD at day 28 and degradability of residual amount of test substance were 0% at average.