Registration Dossier

Environmental fate & pathways

Biodegradation in water: screening tests

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
GLP compliance:
yes
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic (adaptation not specified)
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Elkton, Maryland (U.S.A.) Publically Owned Treatment Works (POTW)
- Storage conditions: glass containers under aerobic conditions in the period between sampling and application on return to the laboratory
To each of the test vessels needed to conduct the study, a total volume of 3 L was added as follows: 1) Test water: 2861 mL; 2) Solution A: 30 mL; 3) Solution B, C, and D: 3 mL; 4) Inoculum: 17.00 mL. This mixture was aerated with CO2-free air for one day to purge the system of carbon dioxide. After the aeration period, test and reference substances were added using the final 83.0 mL of test water for quantitative transfer of the test and reference substances to bring the final volume to 3 litres. The 83.0 mL of test water was also added to the inoculum control blanks. Three CO2 gas diffusion bottles were filled with 100 mL of 0.0125 M Ba(OH)2 solution and connected in series to the exit air line of each 4 L vessel. The pH of each test vessel was measured and recorded.
Duration of test (contact time):
28 d
Initial conc.:
20 other: mg C/L
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: 20 mg C/L
- pH: measured on days 0 and 25

TEST SYSTEM
- Reference Substance Name: Benzoic acid, sodium salt, purity: 99.9%
- Mineral Media Stock Solutions: 1) Mineral Medium Stock Solution A: 4.25 g KH2PO4 + 10.88 g K2HPO4 + 16.70 g Na2HPO4•2H2O + 0.25 g NH4Cl added to 0.5L-1 of water, 2) Mineral Medium Stock Solution B: 13.75g CaCl2 added to 0.5 L-1 of water, 3) Mineral Medium Stock Solution C: 11.25 g MgSO4.7H2O added to 0.5 L-1 of water, 4) Mineral Medium Stock Solution D: 0.25 g FeCl3•6H2O added to 1.0 L-1 of water.

The biological system used was secondary activated sludge from the Elkton, Maryland (U.S.A) Publicly-Owned Treatment Works (POTW). The biodegradation process was monitored on days 1, 4, 6, 8, 12, 18 and 25 when carbon dioxide (CO2) trapped in barium hydroxide was measured by titration of the residual hydroxide. Additionally, traps were titrated on day 26 to quantify any remaining CO2 after acidifying the test systems on day 25. The amount of CO2 produced from the test substance (corrected for that from the Inoculum Control Blank) is expressed as a percentage of the total CO2 that the test substance could have theoretically produced (ThCO2) based on the measured total carbon (TC) content of the test substance.
Reference substance:
benzoic acid, sodium salt
Parameter:
% degradation (CO2 evolution)
Value:
72.5
Sampling time:
25 d
Parameter:
% degradation (CO2 evolution)
Value:
60
Sampling time:
12 d
Details on results:
The ThCO2 produced by the test and reference substances was determined to be 2.55 and 2.14 mg CO2/mg substance, respectively (see Table 1). The value for the test substance was determined by measuring its total carbon content. The value for the reference substance was determined using its molecular formula and the purity because the composition was known.

In the test, the following results (see Table 2) were observed: 1) the test substance reached a final biodegradability of 72.5% on day 25. Since biodegradability was >60% by day 12 and reached this level within 10 days of reaching 10%, this chemical is considered “Ready Biodegradable”, and 2) Sodium benzoate (the reference substance) was greater than 60% biodegradable within 14 days confirming that the inoculum was viable.

In the Toxicity Control, which included the test and reference substances in the same vessel, biodegradation was greater than 25% within 14 days. Therefore the test substance was not inhibitory to microorganisms in the inoculum.

The lag phase of the test substance was from the start of the test until about day two. The lag phase is the period from inoculation until the degradation percentage has increased to 10%. There was a well-defined degradation phase from day 2 to day 12. The degradation phase is from 10% biodegradation until biodegradation reaches greater than 60% degradation. The pH of the solutions with the test and reference substances and the Inoculum control blank solutions on day 25 varied by 0.5 pH units or less from that on day 0 (see Table 3). The total CO2 evolution in the inoculum control blanks at the end of the test averaged 42.9 mg CO2, which is equivalent to 14.3 mg CO2/L (Table 2). The difference of extremes of replicate values of the test substance at the end of the test was less than 20% (Table 2).
Results with reference substance:
Sodium benzoate (the reference substance) was greater than 60% biodegradable within 14 days confirming that the inoculum was viable.

Table 1. Descriptions of Inoculum Control Blank and Test

and Reference Substances

Substance Test Systems

Initial Amount

of Substance (mg)

ThCO2 (mg THCO2/

mg substance)

Inoculum Control Blank Rep 1

NA*

NA

Inoculum Control Blank Rep 2

NA

NA

Inoculum Control Blank Rep Avg

NA

NA

Test Substance Rep 1

90.3

218

Test Substance Rep 2

91.6

221

Test Substance Avg

90.95

219.5

Test Substance TOX

91.1mg test substance +102.0 mg

reference substance

102 mg Ref. sub.

438

Reference Substance,

Substance Benzoate

102.9

220

*Not Applicable

 

Table 2. Cumulative CO2 Production in the Inoculum Control Blank and Test Substance, Toxicity Control and Reference Substance Biodegradation

Days

Inoculum Control Blank (mg CO2)

Test Substance

Avg Biodegradation(%)

Toxicity

Control (%)

Reference

Substance (%)

0

0.0

0

0.0

0.0

1

2.9

1.9

2.2

0.4

4

17.9

40.5

32.4

49.5

6

19.8

45.2

37.7

54.7

8

23.5

50.3

43.6

59.6

12

28.7

62.2

54.2

68.6

18

35.2

64.9

55.5

70.1

25

42.0

68.4

58.0

72.5

26

42.9

72.5

60.6

75.9

 

Table 3. pH Values on Days 2 and 25

Substance

Day 0, Rep 1

Day 0, Rep 2

Day 2, Rep 1

Day 25, Rep 2

Test Substance

7.07

7.09

6.80

6.81

Toxicity Control

7.11

NA*

6.93

NA

Inoculum Control Blank

7.18

7.13

7.02

6.94

Reference Substance

7.12

NA*

6.97

NA

*Not Applicable – these two substances were not replicated.

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test substance met the criteria for “Ready Biodegradable” under conditions of this test and was not inhibitory to microorganisms in the inoculum. The test was valid.
Executive summary:

The test substance was tested for ready biodegradability using the 28-day COEvolution test for “Ready Biodegradation” according to OECD Guideline 301B in the version dated July 17, 1992. This test is also known as the Modified Sturm Test. The biological system used was secondary activated sludge from the Elkton, Maryland (U.S.A) Publicly-Owned Treatment Works (POTW). The biodegradation process was monitored on days 1, 4, 6, 8, 12, 18 and 25 when carbon dioxide (CO2) trapped in barium hydroxide was measured by titration of the residual hydroxide. Additionally, traps were titrated on day 26 to quantify any remaining COafter acidifying the test systems on day 25. The amount of COproduced from the test substance (corrected for that from the Inoculum Control Blank) is expressed as a percentage of the total COthat the test substance could have theoretically produced (ThCO2) based on the measured total carbon (TC) content of the test substance.

 

Findings:

- the test substance reached a maximum biodegradability of 72.5 % by Day 26;

- greater than 60% biodegradability was reached by day 12 and was within 10 days of exceeding 10% biodegradation.

- the toxicity control, which included both the test substance and the reference substance in the same vessel, attained greater than 25% biodegradation within 14 days.

- the reference substance attained a biodegradation level of greater than 60% within 14 days.

Description of key information

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

The test substance was found to fulfil the criteria for readily biodegradable in a GLP-compliant OECD 301B guideline test.