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Biodegradation in water: screening tests

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Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
according to
Guideline:
other: ISO 7827 (Water quality -- Evaluation in an aqueous medium of the "ultimate" aerobic biodegradability of organic compounds -- Method by analysis of dissolved organic carbon (DOC))
Version / remarks:
1994
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
other: activated sludge and real waste water
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Raw and treated sewage samples were collected in different days during April and May 2010, at three different urban WWTPs. These plants receive the discharges from small and medium size cities (18,000, 15,000 and 125,000 inhabitants, respectively) located in the same metropolitan area of the northwest of Spain. All of them comprise a primary and a secondary (activated sludge) treatment.
- Preparation of inoculum for exposure: Samples were collected in amber glass bottles previously rinsed with methanol and ultrapure water and extracted (SPE) in less than 6 h after sampling. Prior to extraction, particulate matter was removed using a combination of glass fibre prefilters and 0.45 µm nitrocellulose filters (Millipore, Bedford, USA).
Duration of test (contact time):
6 d
Initial conc.:
5 mg/L
Based on:
not specified
Parameter followed for biodegradation estimation:
DOC removal
Test performance:
No concentration changes were observed neither in the control nor in the inhibited media in the course of the whole study, proving that neither adsorption nor degradation due to abiotic processes occurred and that any loss in the test solutions had to be attributed to biological routes.
Parameter:
% degradation (DOC removal)
Value:
99
Sampling time:
5 d
Details on results:
In the test with activated sludge the test item was readily biodegraded, presenting half-lives lower than 3 days and reaching a 99% degradation within 3.7 to 4.5 days. In the test with real waste water more than 50% degradation was reached after 12 h and the half-life was 9.6 h.
Validity criteria fulfilled:
not specified
Interpretation of results:
readily biodegradable
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 Sep - 22 Oct 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 301 B (Ready Biodegradability: CO2 Evolution Test)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Niedersächsisches Umweltministerium, Hannover, Germany
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): municipal plant at 31137 Hildesheim, Germany; lnoculum of the aqueous phase of non adapted activated sludge
- Pretreatment: The activated sludge was maintained in an aerobic condition by aeration for four hours and then homogenized with a mixer. The sludge was filtered and the filtrate (30 mL) was subsequently used to initiate inoculation.
- Colony forming units of the inoculum: 10E+07 - 10E+08 CFU/L
- Colony forming units in the test vessels: 10E+07 - 10E+06 CFU/L
Duration of test (contact time):
28 d
Initial conc.:
15 mg/L
Based on:
test mat.
Initial conc.:
10.2 mg/L
Based on:
TOC
Parameter followed for biodegradation estimation:
CO2 evolution
Details on study design:
TEST CONDITIONS
- Composition of medium: Mineral nutrient solution acc. to OECD 301 B
- Test temperature: 20 - 24 °C
- pH: 7.57 - 7.88
- pH adjusted: no

TEST SYSTEM
- Culturing apparatus: 5000 mL, brown glass; filled with 3000 mL
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: The activated sludge was maintained in an aerobic condition by aeration for four hours and then homogenized with a mixer
- Measuring equipment: pH-Meter, Multilab 340i, VV-rw; Thermohygrograph, type 3.015/3 K, fabr.-no. 9003146; Flow meter, KROHNE DUISBURG TYP DK 800 PV
- Details of trap for CO2 and volatile organics if used: Determination of CO2 was carried out by titration subsequent to complete adsorption of the released CO2 in a basic solution

SAMPLING
- Sampling frequency: Backtitration of the residual Ba(OH)2 with 0.05 N HCI was carried out three times a week during the first ten days and thereafter twice weekly (altogether 12 measurements). On the 28th day the pH of all solutions were measured prior to acidification.

CONTROL AND BLANK SYSTEM
- Inoculum blank: Nutrient solution and inoculum in duplicates
- Toxicity control: single
- Fuctional control: single
Reference substance:
acetic acid, sodium salt
Parameter:
% degradation (CO2 evolution)
Value:
64
Sampling time:
18 d
Remarks on result:
other: mean of two replicates
Details on results:
The percentage degradation of the functional control reached the pass level of 60% after 8 days
In the toxicity control containing both test and reference item a biodegradation rate of 77% occurred within 14 days and came to a maximum of 100 % after 28 days.
The biodegradation of the reference item was not inhibited by the test item in the toxicity control.
For the test item the 10% level (beginning of biodegradation) was reached after an adaptation phase of 3 days and in the 10d window a mean biodegradation of 61% was reached. The mean maximum biodegradation came to 64% after 18 days. At this point of time the plateau was reached.
Results with reference substance:
Degradation with sodium acetate reached 77% and 95% after 14 and 28 days, respectively.

Table 1: CO2-Production and Biodegradation after 28 Days

CO2-production after 28 d

control (mean)

functional control (35 mg/L)

test item (15 mg/L)

toxicity control (35 + 15 mg/L)

No.1

No. 2

gross [mg/3 L]

157.9

264.9

230.5

217.4

391.1

[mg/L]

52.6

88.3

76.8

72.5

130.4

net [mg/3 L]

-

107.0

72.6

59.5

233.2

[mg/L]

-

35.7

24.2

19.8

77.7

theor. [mg/3 L]

-

112.4

112.5

112.5

224.9

[mg/L]

-

37.5

37.5

37.5

75.0

Degradation [%] after 28 d

-

95

65

53

100

Table 2: CO2-Production and Biodegradation for all Determination Points in the Control, Functional Control and Toxicity Control Samples

study day

date

control [mg CO2/3L] [mean]

functional control (35 mg/L)

toxicity control (35 mg/L reference item + 15 mg/L test item)

[mg CO2/3L]

degr.

[mg CO2/3L]

degr.

gross

net

[%]

gross

net

[%]

1

24.09.

4.4

11.6

7.2

6

5.4

1.0

0

4

27.09.

19.0

65.1

46.1

41

50.2

31.2

14

6

29.09.

28.9

95.0

66.1

59

104.8

75.9

34

8

01.10.

39.9

116.1

76.2

68

159.0

119.1

53

11

04.10.

52.9

134.5

81.6

73

202.0

149.1

66

14

07.10.

68.8

155.1

86.3

77

242.6

173.8

77

18

11.10.

87.6

177.8

90.2

80

277.1

189.5

84

21

14.10.

104.1

196.7

92.6

82

303.4

199.3

89

25

18.10.

126.1

219.4

93.3

83

333.3

207.2

92

28

21.10.

145.6

244.4

98.8

88

361.7

216.1

96

29*

22.10.

157.9

264.9

107.0

95

391.1

233.2

100

* results of the last two gas wash bottles (degr. = degradation)

Table 3: CO2-Production and Biodegradation for all Determination Points in the Control and Test Item Samples Samples

study day

date

control [mg CO2/3L] [mean]

test item (15 mg/L)

replicate 1

test item (15 mg/L)

replicate 2

[mg CO2/3L]

degr.

[mg CO2/3L]

degr.

gross

net

[%]

gross

net

[%]

1

24.09.

4.4

3.6

-0.8

0

3.9

-0.5

0

4

27.09.

19.0

34.3

15.3

14

47.2

28.2

25

6

29.09.

28.9

68.5

39.6

35

77.8

48.9

43

8

01.10.

39.9

92.9

53.0

47

101.9

62.0

55

11

04.10.

52.9

113.6

60.7

54

122.3

69.4

62

14

07.10.

68.8

135.6

66.8

59

142.5

73.7

66

18

11.10.

87.6

156.5

68.9

61

162.9

75.3

67

21

14.10.

104.1

169.4

65.3

58

179.1

75.0

67

25

18.10.

126.1

196.9

70.8

63

194.8

68.7

61

28

21.10.

145.6

218.4

72.8

65

207.3

61.7

55

29*

22.10.

157.9

230.5

72.6

65

217.4

59.5

53

* results of the last two gas wash bottles (degr. = degradation)

Table 4: Validity criteria of OECD guideline 301B.

Criterion from the guideline

Outcome

Validity criterion fulfilled

Difference of extremes of replicate values of the removal of the test chemical at the plateau, at the end of the test or at the end of the 10-d window, as appropriate, is less than 20%.

The difference of extremes of duplicate values at the end of the 10-day window and at the end of the test was less than 20%.

Yes

Percentage degradation of the reference compound has reached the pass levels by day 14.

The reference item sodium acetate was degraded to 77% after 14 days of incubation.

Yes

The toxicity control should degrade to at least 35% (based on DOC) or at least 25% (based on ThOD or ThCO2) within 14 d.

In the toxicity control degradation reached 77% after 14 days of incubation.

Yes

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to "Any other information on results incl. tables".
Interpretation of results:
readily biodegradable
Endpoint:
biodegradation in water: ready biodegradability
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Qualifier:
according to
Guideline:
OECD Guideline 301 A (Ready Biodegradability: DOC Die Away Test)
GLP compliance:
no
Oxygen conditions:
aerobic
Inoculum or test system:
natural water: freshwater
Details on inoculum:
- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): Tamiya River (October 2006) and Tsumeta River (September and November 2007) of Tokushima City
- Preparation of inoculum for exposure: filtered through 3 µm pore size membrane filter (Millipore)
Initial conc.:
100 µg/L
Based on:
not specified
Parameter followed for biodegradation estimation:
DOC removal
Details on study design:
TEST SYSTEM
- Culturing apparatus: 100 mL Erlenmeyer flasks

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes (prepared from sterilized milli Q water)

For the degradation of the test item the following half-lives were determined:

9.5 h (sample from Oct 2006),

16 h (sample from Sep 2007),

15 h (sample from Nov 2007).

Degradation is presented as C/C0, whereby C is the aqueous concentration at sampling time and C0 the initial concentration. C/C0 was zero, and thus the test item degraded at about 21 h, 22 h, and 28 h for the samples from Oct 2006, Sep 2007, and Nov 2007, respectively.

Data demonstrate the potential biodegradability of the test item.

Validity criteria fulfilled:
not specified
Interpretation of results:
other: No conclusion can be reached since the interpretation of results is not according to standards necessary for regulatory purposes.

Description of key information

Readily biodegradable: 64% (CO2 evolution) in 18 days (OECD 301B)

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

The key study was conducted according to OECD 301B and GLP standards (Noack, 2004). Non adapted activated sludge was used as inoculum. The concentration of the test item and the theoretical CO2 production (ThCO2) were calculated based on the determined carbon content of the test item. The percentage degradation of the functional control reached the pass level of 60% after 8 days. In the toxicity control containing both test and reference item a biodegradation rate of 77% occurred within 14 days and came to a maximum of 100 % after 28 days. The biodegradation of the reference item was not inhibited by the test item in the toxicity control, thus the test substance can be considered to be not toxic to microorganisms. For the test item the 10% level (beginning of biodegradation) was reached after an adaptation phase of 3 days and in the 10 d window a mean biodegradation of 61% was reached. The mean maximum biodegradation came to 64% after 18 days. At this point of time the plateau was reached. Thus, the test item is considered readily biodegradable according to the stringent requirements of the OECD 301B guideline.

In addition public data is available supporting this result. In the test with activated sludge (Gonzales-Marino et al., 2011) the test item was readily biodegraded, presenting half-lives lower than 3 days and reaching a 99% degradation within 3.7 to 4.5 days. In the test with real waste water more than 50% degradation was reached after 12 h and the half-life was 9.6 h. In the study by Yamamoto et al. (2007b) for the degradation of the test item half-lives in the range of 9.5 h to 15 h were determined. Degradation is presented as C/C0, whereby C is the aqueous concentration at sampling time and C0 the initial concentration. C/C0 was zero, and thus the test item degraded at about 21 to 28 h, demonstrating the biodegradation potential of the test item.

[Type of water: freshwater]