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Environmental fate & pathways

Biodegradation in soil

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Endpoint:
biodegradation in soil, other
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Measurement of the amount of 14CO2 produced from the degradation of MDA in anaerobic soil under a wide range of redox conditions.
GLP compliance:
no
Test type:
laboratory
Radiolabelling:
yes
Oxygen conditions:
anaerobic
Year:
2002
Details on soil characteristics:
see 'Other information on methods' for table of details on soil characteristics.
Duration:
365 d
Initial conc.:
2 other: mg/kg (dry weight) of [14C]-radiolabeled MDA
Parameter followed for biodegradation estimation:
radiochem. meas.
other: 14CO2 evolution
Details on experimental conditions:
The sorptive interactions and biodegradation of radiolabeled [14C]-4,4'-methylenedianiline (MDA) was studied in anaerobic soils under a wide range of redox conditions. Approximately 2 mg/kg (dry wt.) of [14C]-radiolabeled MDA were added to anaerobic laboratory batch microcosms (LBM) containing 25g soil and 10 mL of an aqueous mineral medium. LBMs were prepared using a sand soil, which had a history of contact with leachate from a municipal solid waste landfill. LBMs were also prepared from a loamy sand soil, which had a history of contamination by various aromatic amine compounds. The LBMs were poised under a wide range of redox conditions, which were maintained by stimulating the native soil microorganisms to use the reduction of nitrate, iron (III), sulfate, or carbon dioxide (i.e. methanogenesis) as terminal electron accepting processes in anaerobic respiration. Using these two soils and the wide range of established redox conditions, the effects of soil texture and redox potential on the covalent binding with organic matter, particle-associated adsorption processes, and especially biodegradation of MDA were examined.

Total radioactivity in the unextracted aqueous phase, liquid phase of extracted microcosms, caustic 14CO2 traps, and soil combustion trap solutions was determined using liquid scintillation counting (LSC) techniques. The filtered microcosm aqueous phase was counted by combining a single 200 µL aliquot with 5 mL Aquasol® scintillation cocktail (Perkin Elmer Life Sciences, Boston, MA). Filtered samples of the extracted microcosm liquid phase were counted in duplicate by combining 200 microlitre aliquots with 6 mL Aquasol in 7 mL glass scintillation vials. The 14CO2 trap solutions containing 1N NaOH were also counted in duplicate by combining 200 microlitre trap solution with 5 mL Aquasol and 500 microlitre MilliQ water. Trap solutions from soil combustion analyses were counted in single 20 mL scintillation vials by mixing the 15 mL of trap cocktail with 5 mL of Econofluor scintillation cocktail (Packard Instruments). All LSC analyses were performed using a Beckmann LS-6000 scintillation counter, which used either representative standard or quench-curve counting programs to correct for variation in counting efficiency due to matrix quenching effects.
Soil No.:
#1
% Degr.:
1.7
Parameter:
radiochem. meas.
Sampling time:
364 d
Soil No.:
#2
% Degr.:
0.2
Parameter:
radiochem. meas.
Sampling time:
364 d
Transformation products:
yes
No.:
#1
Details on transformation products:
Tentative identification, under denitrifying conditions.
Evaporation of parent compound:
not specified
Volatile metabolites:
not specified
Residues:
not specified
Details on results:
The anaerobic biodegradation of MDA in soil was evaluated under a series of different redox conditions. Detection of 14CO2 in the anaerobic LBMs indicated that slow biodegradation of [14C]-MDA occurred in the sand soil under denitrifying conditions, but not under Fe(III) reducing, sulfate reducing, or methanogenic conditions. Maximum yields of 14CO2 detected after 364 days were equivalent to 1.7% of the applied [14C]-MDA. The addition of a co-substrate, 2-aminobenzoic acid (6 mg/kg), to the reaction mixtures appeared to enhance the mineralization of MDA under denitrifying conditions. Yields of 14CO2 were increased to 2.7% of the applied [14C]-MDA in the presence of this co-substrate. Yields of 14CO2 under iron (III)-reducing, sulfate reducing, and methanogenic conditions remained < 1% of the applied radioactivity as [14C]-MDA after 374 days, thereby indicating that little or no biodegradation of the materials occurred under these anaerobic conditions. A similar lack of 14CO2 evolution indicated that no biodegradation of [14C]-MDA has occurred in the loamy sand soil when poised under denitrifying conditions.

MDA was shown to be slowly mineralized to CO2 under denitrifying conditions. Covalent binding of MDA to natural organic matter is the dominant process affecting the fate of this substance in soils.

Endpoint:
biodegradation in soil, other
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented and scientifically acceptable study.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Measuring the amount of 14CO2 produced form the aerobic biodegradation of MDA in soils.
GLP compliance:
no
Test type:
laboratory
Radiolabelling:
yes
Oxygen conditions:
aerobic
Soil classification:
other: Washington Silt Loam
Soil no.:
#1
Soil type:
silt loam
% Clay:
19
% Silt:
60
% Sand:
21
% Org. C:
1.3
pH:
5
CEC:
13.6 meq/100 g soil d.w.
Details on soil characteristics:
Surface soil obtained from a farm field in Pennsylvania.

Soil type: Washington silt loam
21% sand
60% silt
19% clay
Cation exchange capacity: 13.6 meq/100g
Ca: 7.59 meq/100g
Mg: 1.26 meq/100g
K: 0.68 meq/100g
pH: 5
Combustible content: 5.7 %
Field capacity at 1/3 Bar: 23.05%
TOC: 1.3%
Soil No.:
#1
Duration:
365 d
Parameter followed for biodegradation estimation:
CO2 evolution
radiochem. meas.
Soil No.:
#1
Temp.:
25°C
Details on experimental conditions:
Test substance added to the soil at 5 microg/g and at approx. 4-5*10(6) disintegrations per minute of 14C per flask (1.8 - 2.3 microCi per flask)

To 50g of air-dried, 2 mm sieved silt loam was added water to provide 75% of 1/3 bar moisture tension in the soil. The test substance was added to the soil at the 5 microg/g level and at approximately 4-5*10(6) disintegrations per minute of 14C per flask (1.8 - 2.3 microCi per flask). The flasks were incubated in darkness in a microbial incubator at 25°C for up to one year. Sodium hydroxide solution was used to trap the carbon dioxide formed and liquid scintillation counting was performed using a Beckman Model LS 3801 instrument.
Soil No.:
#1
% Degr.:
11.6
Parameter:
radiochem. meas.
Sampling time:
56 d
Transformation products:
not specified
Evaporation of parent compound:
not specified
Volatile metabolites:
not specified
Residues:
not specified
Details on results:
% Biodegradation

Time (days), % biodegradation

3d, 2.9%
7d, 9.1%
14d, 10.0%
28d, 11.2%
56d, 11.6%
210d, 30.5%
365d, 40.2%

The 14C labeled MDA started to biodegrade immediately with the aerobic soil. The biodegradation slowed later perhaps due to the increasing binding of the MDA with humic acids in the soil.

This study also demonstrated the strong binding of MDA to soil.

MDA apparently undergoes aerobic biodegradation in soil.

NB. Apparently some of the 14CO2 was lost during the latter period of incubation, so that the % biodegradation figures given above for 210d and 365d were calculated from the loss of C-14 from the soil plus the water extracts of the soil.

Endpoint:
biodegradation in soil, other
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Measurements of the amount of 14CH4 and 14CO2 produced from the anaerobic degradation of MDA in soil.
GLP compliance:
no
Test type:
laboratory
Radiolabelling:
yes
Oxygen conditions:
anaerobic
Soil classification:
other: Washington Silt Loam
Year:
1996
Soil no.:
#1
Soil type:
silt loam
% Clay:
19
% Silt:
60
% Sand:
21
% Org. C:
1.3
pH:
5
CEC:
13.6 meq/100 g soil d.w.
Details on soil characteristics:
Surface soil obtained from a farm field in Pennsylvania.

Soil type: Washington silt loam
21% sand
60% silt
19% clay
Cation exchange capacity: 13.6 meq/100g
Ca: 7.59 meq/100g
Mg: 1.26 meq/100g
K: 0.68 meq/100g
pH: 5
Combustible content: 5.7 %
Field capacity at 1/3 Bar: 23.05%
TOC: 1.3%
Soil No.:
#1
Duration:
73 d
Parameter followed for biodegradation estimation:
CH4 evolution
CO2 evolution
radiochem. meas.
Soil No.:
#1
Temp.:
30°C
Details on experimental conditions:
Anaerobic Biodegradation in Soil

Fresh silt loam soil was partially air-dried and sieved to pass a 2mm sieve, then 20g. of the soil was weighed into acid-washed, dried 16-mL serum bottles. Helium-stripped deionized water and 8 mL of 0.1M CaCl2, plus 30% CO2/70% N2 gas-stripped 0.1M sodium acetate (2 mL) and 0.1M glucose solution (2.1 mL) were added to give a final total volume of 80 mL minus the volumes of C-14 labeled MDA, or sodium acetate-2-C-14 to be added later after the bottles had attained an anaerobic condition.

The C-14 sodium acetate bottles served as a positive control, to ensure that methanogenic bacteria were present in the soil. Surrogate bottles containing soils, but with no labeled substrates were also set up to be opened for determining ORP conditions prior to spiking.

The contents of the bottles were stripped with 50 mL /min of 30% CO2/70% N2 for 4 min., then stoppered with gray butyl rubber caps sealed with aluminium-crimped seals. The bottles were allowed to incubate at 30°C for 6-8 days to allow the bottles to develop anaerobic conditions before adding labeled test compounds. After verifying that anaerobic conditions had been developed, the bottles were spiked with radioactive C-14 labeled compounds in a N2 atmosphere glove bag by syringe injection through the rubber cap. Duplicate bottles were set up to be analyzed for 14CO2 and 14CH4 gases after 0, 33, 45 and 73 days at 30°C.

After the initial venting of the excess pressure in the serum bottle to the gas analysis system, 1.4 mL of 1:1 H3PO4 in water was injected through the cap of the bottle in order to convert all of the C-14 carbonate and bicarbonate in the serum bottle to gaseous 14CO2, and a purge gas (N2) flow of 100 cc/min. was passed through the bottle via a 1/16-in, stainless steel tube through the cap and the vent needle connected to the CO2 traps. This flow was continued for four 4-min. oxidation cycles of the Harvey Model OX-500 Biological Oxidizer quartz tube furnace.

After the completed purge of the gases, the reactor was removed from the apparatus and the solids allowed to settle. An aliquot of the supernatant solution was filtered through a 0.2 micrometre nylon syringe filter for C-14 counting with 10 mL Ready Gel to determine the soluble C-14 fraction in the bottle. Each NaOH trap vial was capped and mixed, then a 1- mL aliquot was taken for LSC with a 4 mL water plus 10 mL Ready Gel to determine the fraction of C-14 present as C-14 carbonates or 14CO2 from the total in the two traps. The two liquid scintillation vials from the Harvey OX-500 were counted directly and totaled to determine the fraction of C-14 present as 14CH4 in the bottle. The supernatant solution not used for counting was poured off the soil or soil/sludge mixture, and the wet soilds were poured into an aluminium pan for air-drying and storage in the refrigerator for subsequent soil C-14 determination on the Harvey OX-500.

Anaerobic Biodegradation in Soil Plus Anaerobic Sewage Sludge
A set of bottles was also set up with soil + anaerobic digester sludge from the Lehigh County Sewage Pretreatment Plant in Trexlertown, Pennsylvania. The sludge solids were passed through a 2-mm sieve, then diluted by 1/5 with a sterile nutrient medium described by ASTM Method E 1196-87, but made up at 2X nutrient medium strength.

Forty mL of the sludge mixture was added to each bottle in addition to the 40 mL total volume soil/water plus C-14 labeled MDA, or sodium acetate plus the CaCl2, sodium acetate and glucose solutions described above. Surrogate bottles containing soils and soils + sludge but with no labeled substrates were also set up to be opened for determining ORP conditions prior to spiking. These sludge/soil bottles were incubated for 0, 33, 45 and 71 days at 30°C, and analyzed in the same manner as bottles containing only soil.
Soil No.:
#1
% Degr.:
ca. 0
Parameter:
radiochem. meas.
Sampling time:
73 d
Transformation products:
not specified
Evaporation of parent compound:
not specified
Volatile metabolites:
not specified
Residues:
not specified
Details on results:
Under methanogenic conditions no CH4 or CO2 were observed for MDA after up to 73 days.
Results with reference substance:
In experiments carried out with [2-14C]-acetic acid sodium salt and soil plus anaerobic sludge organisms, the production of 14CO2 and 14CH4 from the acetate was completed by 33 days, and a significant amount of degradation was observed on the first day.

Because of binding of MDA to soil, it was impossible to tell if any soluble metabolites were formed. Also, it is well known that many anaerobic biodegradation processes are very slow and an extended project, over a sufficient length or time, could show biodegradation.

Description of key information

4,4'-MDA is considered to be persistent in soil due to covalent binding to soils organic matter.

Key value for chemical safety assessment

Additional information

The microbial degradation of MDA in soil was investigated under aerobic and anaerobic conditions using carbon-14C labeled MDA. The results show, that biodegradation started immediately after mixing with the aerobic soil. With the binding of amine to soil the degradation rate decreased later. The test indicates biodegradation of 2.9% after 3 days,. 9.1% after 7 days and 11.6% after 56 days. During the latter period of the incubation some of the 14CO2 was lost, so results for 210 and 365 days must be rejected. The degradation rates after 7 and 56 days indicated that biodegradation is disrupted after MDA had formed covalent bounds with humic substances. From the remaining results it is not possible to calculate a half-life, but it can be assumed that MDA covalently bound to organic matter is degraded almost similar to the humic acids themselves.

Cowen et al (1996) showed that under anaerobic methanogenic conditions no 14CH4 or 14CO2 was recovered after 73 days of incubation. A further study (West et al 2002) was carried out over one year on the degradation of MDA in anaerobic soils under a range of redox conditions. Results indicated that MDA was slowly mineralized to carbon dioxide under denitrifying conditions.

Overall, covalent binding of MDA to natural organic materials present in soil is the dominant process affecting the fate of this substance in soils (see also IUCLID chapter 5.4.4).