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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:
key study
Study period:
From 15 September 2005 to 14 October 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline 301 F and EU Method C.4-D with GLP statement. No deviation was observed and all validity criteria were fulfilled.
Qualifier:
according to
Guideline:
OECD Guideline 301 F (Ready Biodegradability: Manometric Respirometry Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.4-D (Determination of the "Ready" Biodegradability - Manometric Respirometry Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Specific details on test material used for the study:
No additional information
Oxygen conditions:
aerobic
Inoculum or test system:
activated sludge, domestic, non-adapted
Details on inoculum:
Fresh activated sludge from a biological waste water treatment plant treating predominantly domestic sewage (City of Geneva, Peney-Dessous) was used.

The sludge is collected in the morning, washed three times in the mineral medium (by centrifuging at 1000 g for 10 minutes, discarding the supernatant and resuspending in mineral medium) and kept aerobic until being used on the same day.
Duration of test (contact time):
29 d
Initial conc.:
107.4 mg/L
Based on:
test mat.
Initial conc.:
110.6 mg/L
Based on:
test mat.
Parameter followed for biodegradation estimation:
O2 consumption
Details on study design:
TEST CONDITIONS
- Composition of medium: Prepared by mixing 50 mL of solution A (KH2PO4, K2HPO4, Na2HPO4.2H2O and NH4Cl) and 2000 mL deionised water (containing less than 10 mg/L dissolved organic carbon), adding 5 mL of each of the solutions B (CaCl2), C (MgSO4.7H2O) and D (FeCl3.6H2O and HCl) and making up to 5 litres with deionised water. The pH is measured and if necessary adjusted to 7.4 +/- 0.2 with phosphoric acid or potassium hydroxide.
- Solubilising agent (type and concentration if used): none
- Test temperature: 22°C
- CEC (meq/100 g): no data
- Aeration of dilution water: no data
- Suspended solids concentration: 3.3 g/L (dry weight of suspended solids)
- Continuous darkness: no data
- Other: none

TEST SYSTEM
- Culturing apparatus: test flasks of the SAPROMAT
- Number of culture flasks/concentration: 2
- Method used to create aerobic conditions: no data
- Method used to create anaerobic conditions: not applicable
- Measuring equipment: The respirometers used during this study are SAPROMAT D 12, made by J. M. VOITH GmbH, Heidenheim, Germany (Serial Nos.: SAPROMAT 1: 9316, controller 1: 1921, SAPROMAT 2: 9511, controller 2: 2067).
- Test performed in closed vessels: yes
- Test performed in open system: no

CONTROL AND BLANK SYSTEM
- Inoculum blank: yes
- Procedure control: yes with the reference substance
- Toxicity control: yes

Principe of the test method:
The consumption of oxygen is determined by measuring the quantity of oxygen (produced electrolytically) required to maintain constant the gas volume in the respirometer flask. Evolved carbon, dioxide is absorbed in soda line pellets. The Biological Oxygen Demand (BOD), amount of oxygen taken up by the microbial population during biodegradation of the test chemical (corrected for uptake by blank inoculum, run in parallel) is expressed as a percentage of ThOD (Theoretical Oxygen Demand, calculated from the elemental composition, assuming that carbon is oxidized to carbon dioxide and hydrogen to water).
Reference substance:
benzoic acid, sodium salt
Preliminary study:
None
Test performance:
Every day the oxygen consumption of each flask is recorded and correct temperature and stirring are checked. At the end of the test period (29 days), the pH of each flask is measured again.
Key result
Parameter:
% degradation (O2 consumption)
Value:
71
Sampling time:
28 d
Remarks on result:
other: 10-day window criteria fulfilled
Details on results:
The curves obtained with the reference substance alone and with the test substance + reference substance show no toxic effect of the test item to the microorganisms at the test concentrations.
The test item undergoes 71% biodegradation after 28 days (and 71% after 29 days) in the test conditions. The 10-day window criterion is also fulfilled (14% biodegradation on day 12 and 63% on day 22).
See Tables 5.2.1/2 and 5.2.1/3 in "Any other information on results incl. tables" and "Attached background material".
Results with reference substance:
Degradation of sodium benzoate exceeds 40% after 7 days and 65% after 14 days: the activity of the inoculum is thus verified.

Table 5.2.1/2: Test substance - Biological Oxygen Demand (BOD, mg O2/L, adjusted to nominal concentrations)

Days :

7

12

14

22

28

29

BOD sludge

1

14.0

20.0

20.0

25.0

28.0

28.0

2

15.0

22.0

22.0

27.0

31.0

31.0

mean

14.5

21.0

21.0

26.0

29.5

29.5

BOD test substance

1

8.4

102.0

153.2

231.8

252.5

252.5

2

15.9

25.5

49.9

206.0

241.6

242.5

1 (corrected)

-6.1

81.0

132.2

205.8

223.0

223.0

2 (corrected)

1.4

4.5

28.9

180.0

212.1

213.0

% biodeg.

1

-2

27

43

67

73

73

2

0

1

9

59

70

70

mean

-1

14

26

63

71

71

Table 5.2.1/3: Reference substance (sodium benzoate) - Biological Oxygen Demand (BOD, mg O2/L, adjusted to nominal concentrations)

Days :

5

7

14

21

28

BOD sludge

1

12.0

14.0

20.0

23.0

28.0

2

11.0

15.0

22.0

27.0

31.0

mean

11.5

14.5

21.0

25.0

29.5

BOD reference substance

1

137.0

153.9

173.8

185.7

191.7

2

141.0

156.9

170.8

179.8

183.8

mean

139.0

155.4

172.3

182.7

187.7

% biodeg. Reference substance

1

75

84

92

96

97

2

78

85

90

93

93

mean

77

85

91

95

95

BOD refer. + test substance

1

129.2

149.6

213.8

295.2

346.5

2

135.3

151.2

184.7

305.4

346.4

mean

132.2

150.4

199.3

300.3

346.5

Validity criteria fulfilled:
yes
Interpretation of results:
readily biodegradable
Conclusions:
The test item undergoes 71% biodegradation after 28 days (and 71% after 29 days) in the test conditions. The 10-day window criterion is also fulfilled. Thus, the test item should be regarded as readily biodegradable according to this test. In addition, no toxic effects of the test substance was observed to microorganisms at the test concentrations.
Executive summary:

The ready biodegradability of the test item has been determined by the Manometric Respirometry Test according to the OECD Guideline No. 301F, EU Method C.4 -D with GLP compliance.

A measured volume of inoculated mineral medium, containing a known concentration of test substance (average 100 mg/L) as the nominal sole source of organic carbon, is stirred in a closed flask at a constant temperature (22 +/-1°C) for up to 29 days. The consumption of oxygen is determined by measuring the quantity of oxygen (produced electrolytically) required to maintain constant the gas volume in the respirometer flask. Evolved carbon, dioxide is absorbed in soda line pellets.

As suggested in the OECD 301F method, the toxicity of the test substance for the inoculum is checked. Therefore, a pair of flasks of the volumetric respirometer is filled with mineral medium + test substance + reference substance + inoculum and their respirations are recorded as for the other flasks. If they are lower than those of the flasks containing mineral medium + reference substance + inoculum, the test substance can be assumed to be inhibitory to the inoculum used.

The curves obtained with the reference substance alone and with the test substance + reference substance show no toxic effect of the test item to the microorganisms at the test concentrations. Degradation of sodium benzoate exceeds 40% after 7 days and 65% after 14 days: the activity of the inoculum is thus verified. The test item undergoes 71% biodegradation after 28 days (and 71% after 29 days) in the test conditions. The 10-day window criterion is also fulfilled (14% biodegradation on day 12 and 63% on day 22).

According to this test, the conclusion is that the test item should be regarded as readily biodegradable.

Endpoint:
biodegradation in water: screening tests
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
No data
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
documentation insufficient for assessment
Remarks:
This published study was not performed according to international guideline with GLP statement. Sufficient experimental details were not provided in this publications.
Principles of method if other than guideline:
The test item in DMSO-EtOH (5:1, 30 mL) was evenly distributed between 50 flasks of a 4-day-old shake culture of C. aphidicola. After a further 10 days, the broth was filtered and extracted with EtOAc (x2) and EtOAc-BuOH (1:1) (x1). The combined extracts were dried over Na2SO4, the solvent was evaporated and the residue was chromatographed on silica gel.
GLP compliance:
no
Specific details on test material used for the study:
No additional information
Oxygen conditions:
aerobic
Inoculum or test system:
other: Cephalosporium aphidicola
Details on inoculum:
No data
Duration of test (contact time):
10 d
Details on study design:
No data
Preliminary study:
Not applicable
Test performance:
No data
Details on results:
The major metabolites from the incubation of the test item with C. aphidicola for 10 days in shake culture were the 3beta-alcohol; the 3beta,12-diol (isolated as the butyl ether); the 3beta,6beta-diol and the 3beta,6beta-dihydroxylactone. The metabolism of the test item by C. aphidicola was not particularly clean and a number of mixtures were obtained from the chromatography, suggesting that epimers were being formed.
Results with reference substance:
Not applicable

The location of the additional oxygen gunctions at C-3, C-6 and C-12 followed from changed in the 13C NMR spectra. In particular, the methylene resonances associated with C-3 and C-6 were replaced by CH-O signals while the adjacent signals showed downfield shifts consistent with the insertion of a hydroxyl group. There were gama-upfield shifts on a number of carbon signals including C-1, C-18, C-19 and C-5 arising from the hydroxylation at C-3. As anticipated, the oxidation at C-12 affected the signal assigned to C-11. The stereochemistry of the additional hydroxyl groups followed from the multiplicity of the CH(Oh) signals in the 1H NMR spectrum. The formation of the butyl ether as an artefact arises because butanol was used in the extraction. The parent hemi-acetal was detected although it was not obtained in pure form.

Validity criteria fulfilled:
not applicable
Interpretation of results:
other: the pattern of hydroxylation is that of xenobiotic transformation.
Conclusions:
The test item was hydroxylated (by the fungus Cephalosporium aphidicola) at C-3beta, C-6beta and C-12 in a xenobiotic rather than a biosynthetically patterned fashion.
Executive summary:

This published study was performed, not according to international guideline without GLP statement, to assess the biotransformation of the test substance by the fungus Cephalosporium aphidicola.

General experimental details were not provided in this publications. However, the test item in DMSO-EtOH (5:1, 30 mL) was evenly distributed between 50 flasks of a 4-day-old shake culture of C. aphidicola. After a further 10 days, the broth was filtered and extracted with EtOAc (x2) and EtOAc-BuOH (1:1) (x1). The combined extracts were dried over Na2SO4, the solvent was evaporated and the residue was chromatographed on silica gel. The column was eluted with a gradient of EtOAc-petrol starting with 5% EtOAc and finishing with pure EtOAc.

The major metabolites from the incubation of the test item with C. aphidicola for 10 days in shake culture were the 3beta-alcohol; the 3beta,12-diol (isolated as the butyl ether); the 3beta,6beta-diol and the 3beta,6beta-dihydroxylactone. The metabolism of the test item by C. aphidicola was not particularly clean and a number of mixtures were obtained from the chromatography, suggesting that epimers were being formed.

In conclusion, although the fungus hydroxylated this substrate, the pattern of hydroxylation is that of xenobiotic transformation.

Description of key information

OECD Guideline 301F, GLP, key study, validity 1:

71% biodegradation after 28 days and satisfied the 10-day window validation criterion (14% biodegradation on Day 12, 63% biodegradation on Day 22).

The test substance is readily biodegradable.

Key value for chemical safety assessment

Biodegradation in water:
readily biodegradable

Additional information

A key study is available (Givaudan, 2005) to determine the biodegradation of the registered substance, (-)-(3aR,5aS,9aS,9bR)-3a,6,6,9a-Tetramethyldodecahydronaphtho[2,1-b]furan (Table 1).

Table 1: Data available

Biodegradation studies

References

Data owner

Test Guideline

Test type

Purpose flag - Reliability

Test substance

Results

Givaudan, 2005

GIVAUDAN

OECD Test Guideline 301F

Ready biodegradability

KS - 1

Registered substance

71% biodegradation after 28 days and satisfied the 10-day window validation criterion (14% biodegradation on Day 12, 63% on Day 22)

Biotransformation

Hanson, 1996

Data published

-

Biotransformation

WoE - 4

Registered substance

The test substance was hydroxylated (by the fungusCephalosporium aphidicola) at C-3β, C-6β and C-12 in a xenobiotic rather than a biosynthetically patterned fashion

According to the key study, performed on the registered substance according to OECD Test Guideline 301F, 71% of biodegradation of the substance was observed after 28 days of incubation and satisfied the 10 day window validation criterion. Therefore, the substance is readily biodegradable.

Furthermore, one published study is available to determine the biotransformation of the substance. This study was used as a weight of evidence approach. This data published shows that the substance was hydroxylated by the fungus Cephalosporium aphidicola at C-3β, C-6β and C-12 in a xenobiotic rather than a biosynthetically patterned fashion.