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Environmental fate & pathways

Biodegradation in soil

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Administrative data

Endpoint:
biodegradation in soil
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: - Guideline IV, 4-1 of the Biologische Bundesanstalt (BBA), Braunschweig (FRG)
GLP compliance:
not specified
Test type:
laboratory

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
R,S-[benzene ring-U-14C] WARFARIN
Radiochemical purity by analytical HPLC : 96.5%

Total activity : 80 MBq, 2.16mCi
Specific activity : 470 MBq/mmol, 12.7mCi/mmol
(determined by gravimetric analysis) : 1.53 MBq/mg, 41 µCi/mg
Molecular weight : 308 (at this specific activity)
Radiolabelling:
yes

Study design

Soil classification:
not specified
Details on soil characteristics:
Soil: Marsh Soil (Kalkmansch, silt loam)
Origin: Finkhaushalligkoog, southwest of Husum (FRG), close to the North Sea cost.
Vegetation: pasture, sheep grazing


Soil Analysis (Lufa Speyer)

Particle Size Distribution
Size Range in mm [%]
<0.002 mm [16]
0.002 to 0.006 mm [3.6]
0.006 to 0.020 mm [6.6]
0.020 to 0.063 mm [59.7]
0.063 to 0.200 mm [12.5]
0.200 to 0.630 mm [1.4]
0.630 to 2.000 mm [0.2]

Content of Organic Carbon: 1.8%
pH (CaCl2): 6.5
max. Water Capacity: 51.6%

Microbial Biomass (determined by LUPA Augustenburg): mg C/100g soil (dry weight)
start of the experiment: 50%
end of the experiment: 28%
Duration of test (contact time)
Soil No.:
#1
Duration:
134 d
Initial test substance concentration
Soil No.:
#1
Initial conc.:
550 mg/kg soil d.w.
Based on:
test mat.
Parameter followed for biodegradation estimation:
radiochem. meas.
Experimental conditions
Soil No.:
#1
Temp.:
20 ± 2 ºC
Humidity:
90%
Details on experimental conditions:
Soil Preparation: 15 Erlenmeyer flasks were prepared each containing 50 g (dry weight) soil, being adjusted to 40% of the maximum water capacity. The flasks were allowed to equilibrate for 2 weeks at 20 ± 2 ºC in the dark before the test substance was applied.

Unlabeled Warfarin and [14C]-Warfarin were dissolved in acetone, resulting in a concentration of [14C]-Warfarin at 26.1 mg/ml or 33 mg/g of solution. To 14 flasks an exactly weighed amount of ca. 0.8 g of the [14C]-Warfarin solution was added by pipette corresponding to a rate of 27 mg [14C]-Warfarin per 50 g (dry) soil or 550 mg/kg.

After application of the test substance each flask was equipped with a trap for volatile compounds including carbon dioxide. The trap consisted of a glass tube filled with paraffin oil impregnated quartz wool and soda lime. The flasks were stored in the dark at 20 ± 2 ºC and 90% relative humidity until analysis. The loss in water was checked by weighing from time to time and replaced by distilled water as appropriate.

Sampling occurred at days 0, 1.8, 6.7, 22, 52, 84, and 134. At each sampling, the volatile compounds were driven from the headspace of the flask into the trap by a stream of air. The soil was extracted with ultrasonication at room temperature for 10 min each with acetone (3 times) followed by soxhlett extraction with methanol for 12 h. The radioactivity in the different extract solutions was determined via LSC. The extract solutions were concentrated using rotary evaporation and analyzed by radio-LSC. After drying, the radioactivity retained in the soil samples was determined by means of combustion/LSC.

Analysis of the traps: Traps were extracted with ethyl acetate. For determination of the amounts of 14C-labelled carbonate, the soda lime was suspended in aqueous HCl (pH 1) in a closed system and during stirring and heating (70 ºC) a slight stream of nitrogen was driven over this solution and passed through an absorption liquid for carbon dioxide. The radioactivity in this solution was determined.

Radioactivity was measured in all liquid samples, in the combusted dried soil samples and for all samples analyzed by TLC.


Methylation of Warfarin for Comparative Analysis of Metabolites. Two experiments were run to obtain reference materials.
1. Methanol. Warfarin was refluxed with methanol and some hydrochloric acid for 15 hours. The solution was analyzed using the TLC method.
2. Diazomethane. Warfarin, dissolved in diethyl ether was treated with an etheral solution of diazomethane at room temperature. After evaporation of the solvent in a stream of nitrogen the residue was redissolved in methanol for TLC investigation.

Results and discussion

% Degradation
Soil No.:
#1
% Degr.:
53.6
Parameter:
radiochem. meas.
Sampling time:
134 d
Half-life / dissipation time of parent compound
Soil No.:
#1
DT50:
150 d
Type:
(pseudo-)first order (= half-life)
Transformation products:
yes
Identity of transformation productsopen allclose all
No.:
#1
Reference
Reference substance name:
Unnamed
Inventory number:
InventoryMultipleMappingImpl [inventoryEntryValue=EC 204-696-9]
IUPAC name:
dioxomethane
Identity:
Carbon dioxide
CAS number:
124-38-9
Molecular formula:
CO2
Molecular weight:
44.01
SMILES notation:
O=C=O
InChl:
InChI=1/CO2/c2-1-3
No.:
#2
Reference
Reference substance name:
Unnamed
IUPAC name:
hexahydro-coumarin
Identifier:
common name
Identity:
hexahydro-coumarin
No.:
#3
Reference
Reference substance name:
Unnamed
IUPAC name:
3-(alpha-hydroxy-benzyl)-4-hydroxycoumarin
Identifier:
common name
Identity:
3-(alpha-hydroxy-benzyl)-4-hydroxycoumarin
Evaporation of parent compound:
yes
Volatile metabolites:
yes
Residues:
yes
Details on results:
The amount of radioactivity extractable from the soil in organic solvents (acetone and methanol) decreased during the study from 102% on day 0 to 64% on day 134, whereas the radioactivity retained in the soil increased to 14%. Within the same period, the amount of 14C-labelled carbon dioxide being the main metabolite increased to 16%.

The extractable radioactivity consisted mainly of parent compound, thus the amount of [14C]-Warfarin in the soil decreased from 98% at day 0 to 54% at day 134. From these data, it is concluded that the degradation of Warfarin follows a first order kinetics and a disappearance time DT50 of 150 days was calculated.

The main metabolite was found to be carbon dioxide which after 134 days storage of the soil comprised 16% of the radioactivity applied. Another 14% of the radioactivity were found to be retained in the soil after extraction with acetone and methanol.

Some minor amounts of radioactivity were found in three different peaks after separation of the extracted radioactivity using a TLC system with further purification by HPLC. The individual components were analysed by MS. A comparison is also made with experimentally derived metabolites from the methylation of unlabeled Warfarin. The report explains in detail the interpretation of the data leading to the identification and source of these minor transformation products.

Any other information on results incl. tables

Percent Degradation of [14C]-Warfarin in Soil

Sampling Day

Extractable

Residual

Volatiles, CO2

Volatiles, other

Σ (Balance)

Warfarin

0

101.9

0.4

-

-

102.3

98

1.8

100.6

1.1

0.1

<0.01

101.8

95.9

6.7

98.6

2.1

0.5

<0.01

101.2

93.9

22

93

4.5

4.1

<0.01

101.6

86

52

82.6

8.6

9.5

<0.01

100.7

77

84

75.8

10.1

11.9

<0.01

97.7

66.8

134

64.3

13.6

16.4

<0.01

94.3

53.6

Applicant's summary and conclusion

Conclusions:
The amount of [14C]-labeled test substance in the soil decreased from 98% at day 0 to 54% at day 134. From these data, it was concluded that the degradation of the test substance follows a first order kinetics and a disappearance time, DT50, of 150 days was calculated.
Executive summary:

The degradation of [14C]-labeled test substance in soil was investigated at an application rate of 550 mg/kg using radio-analytical techniques and mass spectrometry. The disappearance time, DT50, was found to be approximately 150 days. The main metabolite was found to be carbon dioxide, after 134 days comprising 16% of the radioactivity applied. Furthermore, two minor metabolites were separated, comprising at most 5% each. From mass-spectrometric investigations, the following structures were derived: hexahydro-coumarin and 3-(alpha-hydroxy-benzyl)-4-hydroxycoumarin.