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Environmental fate & pathways

Biodegradation in water: screening tests

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Administrative data

Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
4-chlorophthalic anhydride
EC Number:
204-251-9
EC Name:
4-chlorophthalic anhydride
Cas Number:
118-45-6
Molecular formula:
C8-H3-Cl-O3
IUPAC Name:
5-chloro-2-benzofuran-1,3-dione
Specific details on test material used for the study:
- Name of test material: 4-Chlorophthalic Anhydride (4-CLPA; CAS RN 118-45-6);
- Lot 22/02
- Purity: 93.8%
- Organic carbon content: 52.5%
- Appearance: chunky white powder
- Storage conditions: room temperature in the dark

Study design

Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
Fresh activated sludge was obtained from the municipal waste treatment facility at Newburyport, Massachusetts. This facility treats predominantly domestic waste. Viability of the microorganisms was confirmed after inoculation of the test solutions and activity was checked by means of the positive control. The sludge was used on the day it was collected and it was aerated for 4 hours prior to use. A subsample (approximately 1 L) of the activated sludge was homogenized for approximately 2 minutes with a mechanical blender, settled for approximately 30 minutes, and centrifuged in 6 tubes for approximately 30 minutes. The supernatant was decanted, pooled, and added to each test vessel to provide sufficient volume for a 1% inoculum. Carry-over of sludge solids, which would interfere with the measurement of CO2 production, was avoided. Viability counts were performed on the supernatant at the start of the test and the inoculum contained 1 x 10^6 colony forming units per mL and 0.030 g/L total suspended solids.
Duration of test (contact time):
28 d
Initial test substance concentration
Initial conc.:
38.1 mg/L
Parameter followed for biodegradation estimation
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
Basal Salts Medium (BSM) was used as dilution water. It was prepared immediately before use in deionized water. A representative sample of deionized water contained 1.0 mg/L total organic carbon. Basal Salts Medium (BSM) was prepared by mixing 10 mL of solution A with deionized water, then adding 1 mL each of solutions B, C, and D per 1,000 mL (the total volume prepared was 12 liters). Solution A, the phosphate solution, was prepared by dissolving the following materials in 1.0 liter of deionized water: KH2PO4 (potassium dihydrogen phosphate) = 8.5 g, K2HPO4 (dipotassium hydrogen phosphate) = 21.7 g, Na2HPO4 (anhydrous disodium monohydrogen phosphate) = 26.6 g, and NH4Cl (ammonium chloride) = 0.5 g. The pH of solution A was adjusted from 7.5 to 7.4 with 0.1 N hydrochloric acid. Solution B, the calcium chloride solution, was prepared by dissolving 2.75 g CaCl2 (calcium chloride, anhydrous) in 100 mL of deionized water. Solution C, the magnesium sulfate solution, was prepared by dissolving 2.25 g of MgSO4•7H2O (magnesium sulfate heptahydrate) in 100 mL of deionized water. Solution D, the ferric chloride solution, was prepared by dissolving 25 mg of FeCl3•6H2O [iron(III) chloride hexahydrate] in 100 mL of deionized water. The pH of the BSM was 7.4 at the start of the test. The pH of the BSM was 7.4 in the toxicity control and treatment vessels on the day of the test.

The definitive degradability test was performed using six test vessels (2.5 L capacity amber glass bottles); two for inoculated BSM (the inoculum blank), one for the inoculated positive control solution, one for the toxicity control, and two for the inoculated replicate test solution. Each test vessel was filled with 1,235 mL of BSM and 15 mL of the activated sludge supernatant inoculum. This mixture was aerated for approximately 24 hours to purge the system of carbon dioxide. After the aeration period, 100 mL of 0.0125 M Ba(OH)2 was introduced into each of three CO2 absorber bottles connected in series to the exit air line of each vessel. Test substance was added to the appropriate vessels to begin the test. The test substance was tested at 20 mg/L total carbon. The test concentration was prepared in duplicate by bringing 0.0572 g of test substance to a total volume of 250 mL with BSM and mixing on a magnetic stir plate for approximately 6 hours. The pH of the solutions was adjusted from an initial pH of 3.5 to 7.4 using 1N sodium hydroxide. After mixing, the solutions were added to each of two replicate test vessels. The inoculum blanks, which contained no test substance, received only 250 mL of CO2-free BSM. The positive control, sodium benzoate, was prepared by adding 0.0515 g to 250 mL of CO2 purged BSM. This stock solution was then added to the test vessel. The toxicity control was prepared by bringing 0.0572 g test substance and 0.0515 g sodium benzoate to a total volume of 250 mL with CO2 purged BSM. The solution was mixed on a magnetic stir plate for approximately 6 hours. The pH of the solution was adjusted from an initial pH of 3.5 to 7.4 using 1N sodium hydroxide. After mixing, the solution was added to the test vessel. The final volume in each test vessel was 1,500 mL.

CO2-free air was bubbled through the solutions at a rate of approximately 50 to 100 mL/minute. The CO2 produced in each test vessel reacted with the barium hydroxide and was precipitated as barium carbonate. Barium hydroxide was prepared by adding 4 g/L of Ba(OH)2•8H2O to up to 6 liters of deionized water and mixing until dissolved. The solution was then filtered (0.20 microns) and sealed in 1 liter plastic bottles prior to use. The amount of CO2 produced by each measurement day was determined by titrating the remaining Ba(OH)2 with 0.05 N (0.05 M) HCl. The CO2 absorber bottle nearest the test vessel was removed on each measurement day for titration. The remaining two absorber bottles were each moved one place closer to the test vessel, and a new absorber bottle containing 100 mL of fresh 0.0125 M Ba(OH)2 was placed at the far end of the series. The 100 mL Ba(OH)2 solutions were titrated with 0.05 N (0.05 M) HCl, using phenolphthalein as an indicator. Titrations were made every one to three days during the first 10 days, then at least every five days until day 28.

The test was performed at a target temperature range of 22 +/- 2°C (the temperature of a representative flask of water incubated among the test vessels was recorded daily during the test period). The pH of both BSM and stock solutions was measured at the start of the test. The pH was measured in all test vessels on day 27 and 1 mL of concentrated HCl was added to drive off inorganic carbonate. The test vessels were aerated overnight and final titrations were performed on day 28.
Reference substance
Reference substance:
benzoic acid, sodium salt
Remarks:
Sodium benzoate; C6H5COONa; FW 144.11; CAS No. 532-32-1; Lot No. X03H00; from J. T. Baker; a white flake; stored in the dark at room temperature

Results and discussion

Test performance:
The positive control, sodium benzoate, yielded 91% of the theoretical CO2 during the test, demonstrating the adequacy of the inoculum and the toxicity control allowed 38% biodegradation, indicating that 4-chlorophthalic anhydride was not toxic to the activated sludge at the tested concentration. The inoculum blanks evolved an acceptable amount of CO2/L (7.9 mg CO2/L). No unusual variation in pH was noted in any test vessel. Temperature remained within the acceptable range of 22 +/- 2°C throughout the test. Daily measured aeration flow rates were approximately 50 to 80 mL/minute in each test vessel.
% Degradationopen allclose all
Parameter:
% degradation (CO2 evolution)
Value:
4.2
Sampling time:
28 d
Remarks on result:
other: Replicate1
Parameter:
% degradation (CO2 evolution)
Value:
4.7
Sampling time:
28 d
Remarks on result:
other: Replicate 2
Parameter:
% degradation (CO2 evolution)
Value:
4.45
St. dev.:
0.25
Sampling time:
28 d
Remarks on result:
other: mean value of 2 replicates
Details on results:
The total amount of CO2 produced by the test substance was determined for the test period and calculated as a percentage of the total CO2 that the test substance, based on its carbon content, could have theoretically produced. The inoculum blank served as a control to correct for CO2 produced through endogenous respiration of bacteria. The amount of CO2 produced by the test substance was determined by the difference (in mL of titrant) between the test and inoculum blank Ba(OH)2 traps. As titrations were performed with 0.05 N (0.05 M) HCl, each 1.0 mL of HCl corresponds to 1.1 mg of CO2 produced.

Any other information on results incl. tables

Day

Mean

% Degradation of
4-CLPA

% Degradation of
Reference Substance

% Degradation of
Toxicity Control

2

0.6

43.4

11.1

3

0.7

70.8

23.5

4

1.2

81.3

27.3

6

1.5

85.8

28.6

8

1.5

87.7

28.9

11

1.6

88.6

28.9

14

2.1

89.8

28.9

18

2.4

90.6

30.5

22

3.6

91.1

34.9

25

3.9

91.1

36.7

27

4.3

91.4

37.7

28

4.5

91.0

38.3

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Interpretation of results:
under test conditions no biodegradation observed
Conclusions:
The test results (4.5% biodegradation) indicated that the test substance was not readily biodegradable under the test conditions.
Executive summary:

The biodegradation potential of the substance in water was determined in a screening study according to OECD TG 301B (CO2 -Evolution Test) and in compliance with GLP criteria. In this study 38.1 mg/L test substance (20 mg/L total carbon) was inoculated with activated sludge from predominantly domestic sewage for 28 days under aerobic conditions in the dark. During the incubation period degradation was followed by determining the carbon dioxide produced. After the 28 -day incubation period 4.5% of the test substance was biodegraded. Therefore, the results are interpreted as under these test conditions no biodegradation was observed.