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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:
23 July 2015 to 18 September 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
other: Chemical Registration Center of MEP. The Guidelines for the Testing of Chemicals Degradation and Accumulation, 301D Closed Bottle Test [M]. Second Edition. Beijing: China Environmental Science Press. 2013: 44-50.
Deviations:
no
Qualifier:
according to
Guideline:
other: The national hazardous chemical management standardization technical committee. GBff 21831-2008 Chemicals-Ready Biodegradability: Closed Bottle Test (S]. Beijing: China Standards Press. 2008.
Deviations:
no
Qualifier:
according to
Guideline:
other: State Environmental Protection Administration of China. HJ/T 153-2004 The Guidelines for the test of chemical [S]. Beijing: China Environmental Science Press. 2004.
Deviations:
no
Qualifier:
according to
Guideline:
other: State Environmental Protection Administration of China. HJ/T 84-2001 Water quality­Determination of inorganic anions-Ion charomatography method [S]. Beijing: China Environmental Science Press. 2001.
Deviations:
no
Qualifier:
according to
Guideline:
other: Urban construction industry standard of the People's Republic of China. CJ/T 221-2005 Examination methods for sludge of Urban Sewage Treatment Plant [S]. Beijing: China Standards Press. 2006.
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
31.6 mg of the test material was weighted into the beaker and dissolved with some test medium. The solution was further be transferred to 250 mL volumetric flask and diluted with test medium to 250 mL, and the stock solution with the concentration of 120 mg/L was obtained (as the purity of 95 %).
Oxygen conditions:
not specified
Inoculum or test system:
other: Secondary effluent
Details on inoculum:
- Source: Liede Sewage Treatment Plant of Guangzhou
- The inoculum is recommended by "The Guidelines for the Testing of Chemicals".
- The inoculum was through a coarse filter paper to removed any coarse particles and impurities on the surface, then kept the filtrate aerobic until required. 1.0 mL of inoculum per litre of test medium was added into each test system when testing.
- The total number of cell presented in secondary effluent was determined as 6.9 x 10^4 cfu per millilitre using nutrient agar.
Duration of test (contact time):
28 d
Initial conc.:
2 mg/L
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium:
Stock solution (a): Potassium dihydrogen phosphate 8.50 g KH2PO4, Dipotassium hydrogen phosphate trihydrate 28.50 g K2HPO4.3H2O, Sodium phosphate di basic dodecahydrate 67.15 g Na2HPO4.12H2O, Ammonium chloride, NH4CI 0.50 g. Dissolved in water and made up to 1 L, the pH value is 7.43.
Stock solution (b): Anhydrous calcium chloride CaCl2 13.75 g. Dissolved in water and made up to 0.5 L.
Stock solution (c): Magnesium sulphate heptahydrate MgSO4.7H2O 11.25 g. Dissolved in water and made up to 0.5 L.
Stock solution (d): Iron (W) chloride hexahydrate FeCl3.6H2O 0.125 g. Dissolved in water and made up to 0.5 L.
20 mL of solution (a), (b), (c) and (d) were mixed with 16 L of deionised water and made up to 20 L with deionised water. Prepare 2 bottles in the same way. Then 14 mL of solution (a), (b), (c) and (d) were mixed with 12 L of deionised water and made up to 14 L with deionised water. Prepared 54 L test medium in all.
- The test medium was allowed to fully aerate.
- Test temperature: 20 ± 1 °C
- pH: 6.60 - 7.19
- pH adjusted: no
- Suspended solids concentration: 1.0 mL filtrate/L medium
- Continuous darkness: yes

TEST SYSTEM
- Culturing apparatus: BOD bottles (about 300ml, with glass stoppers)
- Number of culture flasks/concentration: 16
- Prepration of the test system: 5800, 5900, 5900 and 5800 mL of test medium which had fully-aerated were added to flask I, flask 2, flask 3 and flask 4, respectively. 100 mL of the test material stock solution were added into flask 1 and flask 4, respectively. 12.0 mL of the reference material stock solution were added into flask 3 and flask 4, respectively. 6.0mL of the secondary effluent filtrate was added into each flask, respectively. The final volume of the solutions in each flask was made up to 6000 mL with the fully-aerated test medium, respectively. Then these solutions were mixed uniformly. The prepared solutions were dispensed immediately into the respective group of BOD bottles by hose from the lower quarter of the large flasks, so that all the BOD bottles were completely filled. When dispensing, ensured that the contents of the large flasks were well mixed by stirring and tapped gently to remove any air bubbles.
- Measuring equipment:
The temperature and humidity recorder was set to record the temperature automatically during the experiment period.
During the 28 days incubation, duplicate bottles of each series were withdrawn for the determination of dissolved oxygen and pH values at time intervals (0, 5, 10, 14, 19, 24 and 28 days).
During the 28 days incubation, after determination of dissolved oxygen concentrations and pH values in BOD bottles of the test suspensions and the inoculum blanks, a sample from each bottle were analysed for NO2--N and NO3--N concentrations after 0.45 µm filtered membrane filtration.

CONTROL AND BLANK SYSTEM
- Inoculum blank: 16 BOD bottles containing only inoculum
- Procedure control: 16 BOD bottles containing reference material and inoculum. 100 mg of the sodium benzoate was weighted into the beaker and dissolved with some test medium, then transferred to 100 mL volumetric flask and diluted with test medium to 100 mL, and the stock solution with the concentration of 1000 mg/L was obtained.
- Toxicity control: 16 BOD bottles containing test material, reference material and inoculum

DATA ANALYSIS
Theoretical Oxygen Demand (ThOD)
The theoretical oxygen demand (ThOD) may be calculated if the elemental composition is determined or known. For the compound: CcHhClclNnNanaOoPpSs, the ThOD without nitrification would be:
ThOD NH4 = (16[2c + 0.5(h – cl – 3n) +3s + (5/2)p + 0.5na – o]) / MW
with nitrification: ThOD NO3 = (16[2c + 0.5(h – cl) + (5/2)n +3s + (5/2)p + 0.5na – o]) / MW
Where MW= molecular weight
ThODNH4 and ThODNO3 of the test material are calculated as 2.24 and 3.36 mg/mg.
ThODNH4 of the reference material is calculated as 1.67 mg/mg.

Biochemical oxygen demand (BOD)
From the results of ‘NO2--N and NO3--N contents of the test suspensions during the test", there were no nitrifications occurred in the test material during the test. BOD exerted was obtained from:
BOD = (m1 – m2) / m3
m1 = oxygen uptake of each test group, mg/L
m2 = oxygen uptake of the inoculum blank, mg/L
m3 = the concentration of the test material or the reference material (or test material + reference material), mg/L.
Percentage biodegradation
The percentage biodegradation of each test group was obtained from:
D = (BOD / ThOD NH4) x 100
D= percentage biodegradation
Reference substance:
benzoic acid, sodium salt
Test performance:
The validity criteria of the test were fulfilled.
Key result
Parameter:
% degradation (O2 consumption)
Value:
-9.9
Sampling time:
28 d
Details on results:
NO2--N and NO3--N concentrations:
The results revealed that there were no nitrifications occurred in the test material during the test.

Consumed oxygen of each test group:
- Inoculum blank (28 d): 1.14 mg/L
- Procedure control (28 d): 3.48 mg/L and 3.34 mg/L (2 replicates)
- Toxicity control (28 d): 3.39 mg/L and 3.47 mg/L (2 replicates)
- Test suspension (28 d): 0.70 mg/L and 0.69 mg/L (2 replicates)

Percentage biodegradation:
- Procedure control (14 d): 65.1 %
- Procedure control (28 d): 68.0 %
- Toxicity control (14 d): 31.7 %
- Toxicity control (28 d): 29.2 %
- Test suspension (28 d): -9.8 and -10.0 % (2 replicates), -9.9 % (average)
- On day 14, the percentage biodegradation of the procedure control and the toxicity control were 65.1 % and 31.7 %, respectively, which showed that the activity of the inoculum met the requirement of the test and the test material was considered not to have a toxic effect on the sewage sludge micro-organisms used in the study.

Validity of the test
- Oxygen depletion in the inoculum blank was 1.14 mg/L, which less than 1.5 mg dissolved oxygen/L after 28 days.
- The minimum concentration of dissolved oxygen during the test was measured as 4.89 mg/L, which met that the residual concentration of oxygen in the test bottles should not fall below 0.5 mg/L at any time.
- The percentage biodegradation of the procedure control and the toxicity control were 65.1 and 31.7 % respectively within 14 days, which had reached the pass level (60 and 25 % of ThOD).
- The difference of extremes of replicate values of the removal of the test material at the plateau, at the end of the test or at the end of the 10-d window were less than 20 %.
Results with reference substance:
- Procedure control (14 d): 65.1 %
- Procedure control (28 d): 68.0 %

Table 1: Summary of Percentage Biodegradation During The Test

Time (Days)

Test Material Suspension (Mean)

Procedure Control (Mean)

Toxicity Control (Mean)

5

-0.6

57.9

29.1

10

0.8

65.4

31.2

14

0.1

65.1

31.7

19

-2.1

70.8

32.0

24

-7.9

70.4

30.0

28

-9.9

68.0

29.2

Validity criteria fulfilled:
yes
Interpretation of results:
not readily biodegradable
Conclusions:
Under the conditions of this study, the test material is not readily biodegradable.
Executive summary:

The ready biodegradability of the test material was investigated in accordance with the standardised guideline OECD 301D and other Chinese guidelines.

Secondary effluent was exposed to the test material for 28 days, during the 28 days incubation, duplicate bottles of each series were withdrawn for the determination of dissolved oxygen and pH values at time intervals (0, 5, 10, 14, 19, 24 and 28 days), a sample from each bottle were also analysed for NO2--N and NO3--N concentrations after 0.45 µm filtered membrane filtration.

The results revealed that there were no nitrifications occurred in the test material during the test. On day 14, the percentage biodegradation of the procedure control and the toxicity control were 65.1 % and 31.7 %, respectively, which showed that the activity of the inoculum met the requirement of the test and the test material was considered not to have a toxic effect on the sewage sludge micro-organisms used in the study. The validity criteria of the test were met.

The percentage biodegradation of the test material was 0 % at the end of the test.

Under the conditions of this study, the test material is not readily biodegradable.

Description of key information

Under the conditions of this study, the test material is not readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
under test conditions no biodegradation observed
Type of water:
freshwater

Additional information

The ready biodegradability of the test material was investigated in accordance with the standardised guideline OECD 301D and other Chinese guidelines. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Secondary effluent was exposed to the test material for 28 days, during the 28 days incubation, duplicate bottles of each series were withdrawn for the determination of dissolved oxygen and pH values at time intervals (0, 5, 10, 14, 19, 24 and 28 days), a sample from each bottle were also analysed for NO2--N and NO3--N concentrations after 0.45 µm filtered membrane filtration.

The results revealed that there were no nitrifications occurred in the test material during the test. On day 14, the percentage biodegradation of the procedure control and the toxicity control were 65.1 % and 31.7 %, respectively, which showed that the activity of the inoculum met the requirement of the test and the test material was considered not to have a toxic effect on the sewage sludge micro-organisms used in the study. The validity criteria of the test were met.

The percentage biodegradation of the test material was 0 % at the end of the test.

Under the conditions of this study, the test material is not readily biodegradable.