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Biodegradation in water: screening tests

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Reference
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-07-25 - 2005-09-25
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to
Guideline:
OECD Guideline 301 C (Ready Biodegradability: Modified MITI Test (I))
Version / remarks:
1992
Qualifier:
according to
Guideline:
other: Biodegradation Test of Chemical Substance by Microorganisms specified in the “Methods of Testing New Chemical Substances."
Version / remarks:
2003
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, non-adapted
Details on inoculum:
Collected sludge
(1) Sewage treatment plant: Return sludge
(2) River, lake, and sea: Topsoil on the shore in contact with the atmosphere and surface water

Preparation of activated sludge
To secure uniform activated sludge, 5 L of filtrate of sludge mixture collected from each region as described above was mixed with 5 L of filtrate of activated sludge(*2) cultured for about three months to prepare a 10 L sludge mixture, the pH was adjusted to 7.0 ± 1.0, and aeration(*3) was carried out in a culture tank.

*2 :10 L of filtrate of the sludge mixture solution collected from the region as described above was the activated sludge cultured according to section 2.4 below.

*3:Outdoor air was allowed to pass through a pre-filter and used in the aeration.

Culture
After the aeration in the culture tank was stopped for about 30 minutes, about 1/3 of the entire amount of the supernatant liquid was removed. This was added with dechlorinated tap water so as to be 10 L in total volume, aeration was performed again (for 30 minutes or longer), and 50 g/L of synthetic sewage(*4) was added so that the concentration of the synthetic sewage in the added dechlorinated tap water became 0.1 wt%. This operation was repeated once a day, and culture was performed to prepare activated sludge. The culture temperature was set to 25 ± 2ºC.

*4: Glucose, peptone, and potassium dihydrogen phosphate were dissolved in purified water so that each became 50 g/L, and the pH was adjusted to 7.0 ± 1.0 with sodium hydroxide.
Duration of test (contact time):
28 d
Initial conc.:
100 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Remarks:
BOD
Parameter followed for biodegradation estimation:
TOC removal
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
Test preparation

(1) Measurement of the concentration of the suspended substance of the activated sludge
The concentration of the suspended substance was measured to determine the addition amount of the activated sludge.
Measurement method: Measurement was carried out in accordance with the “Testing Methods for Industrial Wastewater, Suspended Substance” (14.1 of JIS K 0102-1998).
Measurement date: July 25, 2005
Measurement results: The concentration of the suspended substance of the activated sludge was 4300 mg/L.

(2) Preparation of the basic culture medium
Each of 3 mL of Solution A, solution B, solution C, and solution D of the composition specified in the “Testing Methods for Industrial Wastewater, Biochemical Oxygen Consumption” (section 21 in JIS K 0102-1998) was added with purified water (conforming to the Japanese Pharmacopoeia, product of Takasugi Pharmaceutical Co., Ltd.) so as to prepare a 1-L solution, and the pH was adjusted to 7.0.

(3) Control substance
Aniline (reagent grade, lot no. SP-3442Z, product of Showa Chemical Industry Co., Ltd.) was used as a control substance to confirm that the activated sludge has a sufficient degree of activity in the implementation of this test.

Preparation of test solutions
Six test containers were used to prepare test solutions according to the following methods. These test solutions were cultured under the conditions listed in section 3.3.

(1) Test substance and aniline addition
(a) System containing (water + test substance) (one, test container 1)
300 mL of purified water and 30 mg of the test substance were poured into a test container so that the concentration of the test substance became 100 mg/L. The test substance was accurately weighed by an electronic balance.

(b) System containing (sludge + test substance) (three, test container 2 3 4)
A basic culture medium [the amount obtained by deducting the addition amount of activated sludge (2.09 mL) from 300 mL] and 30 mg of test substance were placed in a test container so that the concentration of the test substance became 100 mg/L. The test substance was accurately weighed on an electronic balance.

(c) System containing (sludge + aniline) (one, test container 5)
A basic culture medium [the amount obtained by deducting the addition amount of activated sludge (2.09 mL) from 300 mL] and 29.5 μL of aniline [30 mg in addition amount = 29.5 μL x 1.022 g/cm3 (density)] were placed in a test container so that the concentration of aniline became 100 mg/L. Aniline was sorted and added using a micro syringe.

d) System containing a sludge blank (one, test container 6)
A basic culture medium [the amount obtained by deducting the addition amount of activated sludge (2.09 mL) from 300 mL] was placed in a test container.

(2) Inoculation of activated sludge
The activated sludge prepared under the conditions described in section 2 was inoculated in solutions (b), (c), and (d) so that the concentration of the suspended substance became 30 mg/L.

Test solution culture device and environmental conditions

(1) Test solution culture device
Closed-type oxygen consumption measurement device
Thermostat and measurement unit: Asahi Techneion Co., Ltd.
Data processor : Asahi Techneion Co., Ltd.
Test container: Culture bottle for 300 mL (improved culture bottle)
Carbon dioxide gas absorbent: Soda Lime, No. 1
(For carbon dioxide absorption, product of Wako Pure Chemical Industries, Ltd.)

(2) Environmental conditions
Test solution culture temperature: 25 ± 1ºC
Test solution culture period: 28 days (under shielding of light)
Stirring method: Rotating stirring by a magnetic stirrer

(3) Place of implementation: Low temperature-controlled room

Calculation method of the degree of degradation
The degradation was calculated based on the following equation, and the value was rounded at the first digit after the decimal point and displayed at an integer position.

(1) BOD degradation

Degradation (%) = (BOD – B / TOD*6) x 100

BOD: Biochemical oxygen consumption in the system containing (sludge + test substance) (Measured value) (mg)
B: Biochemical oxygen consumption in a sludge blank (Measured value) (mg)
TOD(*6): Theoretical oxygen consumption required in the event of a complete oxidation of the test substance (Measured value) (mg)

*6: Calculated as 100% pure

(2) DOC degradation

Degradation (%) = (DOCw - DOCs / DOCw) x 100

DOCs: Residual amount of dissolved organic carbon in the system containing (sludge + test substance)
(Measured value) (mgC)
DOCw: Residual amount of dissolved organic carbon in the system containing (water + test substance)
(Measured value) (mgC)

(3) Degradation of the test substance

degradation (%) = (Sw - Ss / Sw) x 100

Ss: Residual amount of test substance in the system containing (sludge + test substance)
(Measured value) (mgC)
Sw: Residual amount of test substance in the system containing (water + test substance)
(Measured value) (mgC)


Reference substance:
aniline
Key result
Parameter:
% degradation (DOC removal)
Value:
2
St. dev.:
1
Sampling time:
28 d
Key result
Parameter:
% degradation (O2 consumption)
Remarks:
BOD
Value:
0
St. dev.:
1
Sampling time:
28 d
Results with reference substance:
valid
BOD aniline = 71.2 mg after 28 days
59 % after 7 days and 66 % after 14 days
Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test substance was not degraded by microorganisms under the conditions of this test.
Executive summary:

The test substance 2,5-xylenol was assessed in an GLP-biodegaradtion study according to OECD Guildeine 301C (modified MITI Test (I)).

The test substance was incubated in activated sludge for 28 days at a concentration of 100 mg/L with a liquid volume of 300 mL at 25 +/- 1°C. The measurement of the biochemical oxygen demand (BOD) was performed by a closed oxgen consumption measurment device and the quantitative analysis of dissolved organic carbon (DOC) was performed by the analysis method of total organic carbon (TOC). Quantitative analysis of the test substance was done by high-preformance liquid chromatography (HPLC).

The test results were as follows:

BOD degradation rate: 0%, -1%, 0%, average 0%

DOC degradation rate: 2%, 3%, 1% average 2%

Test substance degradation (HPLC): 3%, 2%, 1%, average 2%

The tes substance was not degraded by microorganisms under the conditions of the test.

Description of key information

Biodegradation test (OECD 301C), GLP; activated sludge, 28 days: not biodegradable

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed

Additional information

The test substance 2,5-xylenol was assessed in an GLP-biodegaradtion study (KS_Biodegradation in water: screening tests_2005)) according to OECD Guildeine 301C (modified MITI Test (I)).

The test substance was incubated in activated sludge for 28 days at a concentration of 100 mg/L with a liquid volume of 300 mL at 25 +/- 1°C. The measurement of the biochemical oxygen demand (BOD) was performed by a closed oxgen consumption measurement device and the quantitative analysis of dissolved organic carbon (DOC) was performed by the analysis method of total organic carbon (TOC). Quantitative analysis of the test substance was done by high-preformance liquid chromatography (HPLC).

The test results were as follows:

BOD degradation rate: 0%, -1%, 0%, average 0%

DOC degradation rate: 2%, 3%, 1% average 2%

Test substance degradation (HPLC): 3%, 2%, 1%, average 2%

The test substance was not degraded by microorganisms under the conditions of the test.