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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
standard activated sludge 30 mg/L
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:
DOC removal
Details on study design:
Initially, the biological oxygen demand (BOD) associated with the degradation of the test substance in the presence of standard activated sludge was measured over time using a closed oxygen consumption measurement device over 28 days, and after BOD measurement was complete, the dissolved organic carbon concentration (DOC) and the residual test substance concentration were measured.

- Culture: Once a day, approximately 1/3 of the entire volume of supernatant liquid was removed, and an equivalent volume of 0.1% sewage was added, and the mixture was cultured.
- MLSS: Experiment 1: 4700 mg/L, Experiment 3: 4250 mg/L

- Temperature: 25 ± 1°C (actual measurement values in experiment 1: 25.0°C, in experiment 3: 24.9 - 25.0°C)
- Period: 28 days (BOD measurement)
- Volume: 300 mL
- Concentration: Test substance and aniline (reference substance): 100 mg/L
- Standard activated sludge: 30 mg/L

Test composition and addition of test substance
No.1: Degradation activity confirmation system (aniline + standard activated sludge** + basic culture medium***)
The basic culture medium was added to a culture bottle, 30 mg of aniline was weighed in a glass cup, added and mixed to the medium, then standard activated sludge was added to the culture bottle.
The weights were as follows - Experiment 1: 30.2 mg, Experiment 3: 30.0 mg.
No. 2: Basic sludge respiration system (standard activated sludge** + basic culture medium***)
The basal medium was placed in a culture bottle and standard activated sludge was added.
No. 3 to No. 5: Test substance degradation system (test substance + standard activated sludge** + basic culture medium***)
The basic culture medium was inserted into a culture bottle, then 30.0 mg of the test substance was weighed in a glass cup, added to the medium and mixed, then standard activated sludge was added to each culture bottle.
No. 6: Aqueous stability system (test substance + Millipore water****)
300 mL of Millipore water was inserted into a culture bottle and 30.0 mg of the test substance, which was weighed in a glass cup, was added.

**) The amount of standard activated sludge to be added is determined by measuring the MLSS (suspended matter concentration) with a portable sludge concentration meter before the start of the experiment, and based on the concentration, the test solution volume and test sludge concentration were calculated as follows.

Added volume
Experiment 1: 30(mg/L)/4700(mg/L:MLSS)×300(mL)=1.9(mL)
Experiment 3: : 30(mg/L)/4250(mg/L:MLSS)×300(mL)=2.1(mL)
***) In the basic culture medium, 6 mL of Millipore water was added to each of the lots of solution A, solution B, solution C and solution D (created by adding 100 mL of distilled water to 0.025g of FeCl3.6H2O to a constant volume during use), then mixed to prepare a volume of 2L.

The pH of the basic culture medium was adjusted to pH 7 using 1N H3PO4, then the volume of added standard activated sludge (experiment 1: 1.9mL, experiment 3: 2.1 mL) was subtracted from 300 mL. ****) Water was deionized water, purified using a Milli-Q (Merck Millipore) deionized water, resistivity experiment 1: 17.0 and experiment 3: 18.2 MΩcm.

BOD measurement
The BOD was measured for 28 days using the following device, and during the measurement period, observations were made to determine the presence of abnormal temperatures in the thermostatic chamber of the device, the presence of activated sludge proliferation, the degree of test substance dissolution and changes in color.

Formula To Calculate Degradation
The formula for calculating the degradation from BOD, DOC and residual test substance concentration is shown below.
The original elimination rate should be calculated, since the residual test substance concentration in experiment 1 was ≤90%, but the degradation was calculated to perform a comprehensive analysis with experiment 3.
1) Degradation from BOD (after 7, 14, 21 and 28 days)
Degradation (%)=[1—(BODs-BODb)/ThOD]x100
BODs: Oxygen consumption (mg) in the test substance degradation system or degradation activity confirmation system
BODb: Oxygen demand (mg) in the sludge basic absorption system
ThOD: The theoretical oxygen demand (mg) of aniline or test substance
Theoretical oxygen demand (ThOD) calculation
aniline: 90.2mg-O2/30mg
The mineralized aniline was calculated as follows.
C6H7N+35/4Ch->6CO2+7/2H2O+NO2
Test substance: 59.0 mg-O2/30mg
Calculated assuming that the test substance is mineralized as described below.
C7H6O2+15/2O2->7CO2+3H2O
2) Degradation from DOC (after 28 days)
Degradation (%)=[1—(DOCs—DOCb)/DOCc]X100
DOCs: DOC (mg/L) in the test substance degradation system
DOCb: DOC (mg/L) in the sludge basic absorption system
DOCc: DOC (mg/L) in the aqueous stability system
3) Degradation from the residual test substance concentration (after 28 days)
Degradation (%)=(1—Cs/Cc) x 100
Cs: Test substance concentration (mg/L) in the test substance degradation system
Cc: Test substance concentration (mg/L) in the aqueous stability system


Reference substance
Reference substance:
aniline
Remarks:
100 mg/L

Results and discussion

% Degradation
Key result
Parameter:
% degradation (DOC removal)
Value:
100
Sampling time:
28 d
Details on results:
1. Observations of the culture flask after BOD measurement
The solution in the degradation activity confirmation system (no. 1) is very light pale yellow, while the sludge basic absorption system (no. 2), test substance decomposition system -2, 3 (no. 4, 5) and aqueous stability system (no. 6) were colorless. The degradation activity confirmation system showed sludge proliferation, when compared to the sludge basic absorption system, sludge proliferation was also observed in the test substance degradation system, and the test substance dissolved.

2. pH measurement results
There was no change in the pH before and after the BOD measurement both in the test substance degradation system and the aqueous stability system.

3. Degradation Activity of the Standard Activated Sludge
The BOD degradation of aniline after 7 days was 56% in experiment 1 and 59% in experiment 3, and the activity of standard activated sludge was favorable in both cases.

4. BOD measurement results
The BOD after 28 days is the theoretical oxygen demand (59.0 mg), compared to those of the test substance degradation system, which were 78.6, 56.0, and 54.4 mg (value after correction with values measured in the sludge basic absorption system, and the same applies below) for experiment 1 and 34.6, 55.7 and 55.0 mg for experiment 3. Despite an absence of device and operational abnormalities, the values from test substance degradation system-1 (no. 3) in experiment 1 were abnormally high. The values were abnormally low in test substance degradation system-1 (no. 3) during experiment 3, and these values were considered to be abnormal, so they were rejected as abnormal values during the Grubb’s test using a level of significance of 5%. The non-rejected values from experiment 1 and experiment 3 were then used, and the degradation from BOD after 28 days was 95% and 92% in experiment 1. 94% and 93% in experiment 3, and a mean value of 94%.
The degradation is considered to be favorable.

5. DOC Measurement Results
Compared to the theoretical organic carbon concentration (68.8 mg/L), the DOC after 28 days was 0.0, 0.0mg/L in the test substance degradation system in experiment 1, and 0.0, 0.0 mg/L for experiment 3 (value after correction using the values measured for the sludge basic absorption system), 64.2 mg/L in the aqueous stability system in experiment 1, and 72.0 mg/L in experiment 3, therefore the DOC after 28 days was 100% in all cases.

6. Residual Test Substance Concentration Measurement Results
Compared to the prepared test substance concentration (100.0 mg/L) after 28 days, the values for the test substance degradation system were 0.0 mg/L in experiment 1 and 0.0 mg/L for experiment 3, and 72.6 mg/L for experiment 1 and 95.9 mg/L in experiment 3 in the aqueous stability system. The degradation from the test substance degradation system after 28 days was therefore 100% in all cases.
A new peak appeared after 28 days during an HPLC retention time of approximately 3 minutes in the aqueous stability system. This suggests that structurally modified substances were generated. When these structurally modified substances were analysed under the same HPLC conditions as the degradation test, the peaks that appeared suggested the presence of p-hydroxybenzoic acid.

Any other information on results incl. tables

Measured values (after 28 days)

 Measurement items  Test substance degradation system*           Aqueous stability     Preparation

 Test no.:

    1     3  1  3  Theoretical
Bottle no.  4  5  4  5      
 BOD, mg 56.0   54.4  55.7 55.0   8.0  0.0  59.0
 DOC, mg/L (TOC)  0.0  0.0  0.0  0.0  64.2  72.0  68.8
 Test substance, mg/L (HPLC)  0  0  72.6 95.9   100.0

* Values after measured values were corrected using the sludge basic absorption system

Degradation after 28 days

 Degradation              Test substance degradation system  Mean
   Test no.     1     3  
   Bottle no.  4  5  4  5  
 Degradation from BOD, %     95  92  94  93  94
 Degradation from DOC, %     100  100  100  100  100
 Degradation from residual test substance concentration, %     100  100  100  100  100

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
Since the mean decomposition from BOD is 94%, the mean decomposition from DOC is 100%, and the decomposition from the remaining concentration of the test substance is 100%, the test substance is judged to exhibit favorable degradability.