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Environmental fate & pathways

Biodegradation in soil

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Reference
Endpoint:
biodegradation in soil: simulation testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Principles of method if other than guideline:
EPA Guideline, Subdivision N, Section 162-2
GLP compliance:
yes
Test type:
laboratory
Radiolabelling:
yes
Oxygen conditions:
anaerobic
Soil classification:
not specified
Soil type:
sandy loam
% Clay:
56
% Silt:
26
% Sand:
18
% Org. C:
1.6
pH:
6.8
CEC:
11.6 meq/100 g soil d.w.
Details on soil characteristics:
The particle size distribution of the Sandy Loam is constituted by: 56% w/w of sand, 26% w/w of silt and 18% w/w of clay
The percentage of organic matter is 1.6% w/w .
The cation exchange capacity is 11.6 meq/100 g.
The Bulk density is 1.51 gm/cm3.
The total hardness is 264
The Sandy Loam Contains the following exchangeable cations: K (227 ppm), Mg (202 ppm) and Ca (1221 ppm).
The pH of Sandy Loam is 6.8
The water content at 0.33 bar is 17.37% w/w
Soil No.:
#1
Duration:
70 d
Initial conc.:
10 mg/kg soil d.w.
Based on:
test mat.
Parameter followed for biodegradation estimation:
CO2 evolution
Temp.:
25 ± 1°C
Microbial biomass:
was conducted on day 0 and 10 following aerobic initiation and at day 15 and 60 following anaerobic initiation.
Details on experimental conditions:
MATERIALs & METHODS
An anaerobic soil metabolism study with 14C-Piperonyl Butoxide (purity = 99.1 %) applied to a sandy loam soil from Ashland, Nebraska dosed at a nominal 10 µg/g rate was conducted. The study lasted 70 days in the dark in an environmental chamber regulated at 25 ± 1°C. Following one half-life (10 days), the aerobically aged samples were flooded with well water and purged with nitrogen to induce anaerobic conditions. Samples were taken at days 0, 1, 3, 7 and 10 following aerobic initiation and at days 15, 30, 45 and 60 following anaerobic initiation. Day 0 samples were analysed in duplicate. The radioactivity level of aqueous soluble and extractable 14C-residues was determined by LSC. Soil extracts were characterized for parent compound by TLC. The level of bound 14C-residues was determined by combustion radioanalysis of the post-extracted samples. Radioactivity in the trapping solutions was also analysed by LSC. Possible degradation products were analysed by HPLC (detection limit of < 0.3 %) and EI-MS for identification. The half-life of Piperonyl Butoxide for the aerobic and anaerobic incubation period was calculated by means of linear regression analysis. Furthermore the microbial analysis was conducted on day 0 and 10 following aerobic initiation and at day 15 and 60 following anaerobic initiation.

APPLICATION OF TEST ITEM
In the preliminary study sandy loam soil was dosed at a nominal 10 µg/g rate of 14C-Piperonyl Butoxide and incubated in an environmentally controlled chamber at 25 ± 1°C in the dark.
To 35 of the silanized culture tubes containing the study soil and water, 83 µL of a 1214 mg/L solution of 14C-Piperonyl Butoxide was added. The initial measured dose was 10.1 µg Piperonyl Butoxide equivalents/g soil. The remaining 16 sample tubes were not dosed and served as control. The samples, excluding the day 0 and stability samples, were placed into metabolism vessels within the environmental chamber. 14 control samples were placed in the control vessel, 14 dosed samples were placed in the Replicate I test vessel and 11 dosed samples were placed in the Replicate II test vessel.
DURATION OF TEST
10 days aerobic and 60 days anaerobic incubation period

TEMPERATURE/LIGHT
All tubes were maintained in the dark and at a temperature of 25 ± 1°C.

SAMPLING
Preliminary study samples were collected and analysed at 0, 4 and 10 days after aerobic initiation and 1, 4 and 7 days after anaerobic initiation. Trapping solutions were analysed on days 4 and 10 following aerobic initiation and on days 1, 4 and 7 following anaerobic initiation.
Test samples were collected for analysis at days 0, 1, 3, 7 and 10 following aerobic initiation and at days 15, 30, 45 and 60 following anaerobic initiation. Day 0 samples were analysed in duplicate. Appropriate analysis was conducted on each Replicate I sample. Replicate II samples were placed in a freezer at approximately -22°C. Trapping solutions were collected on the days that samples were removed. Microbial analysis was conducted on day 0 and 10 following aerobic initiation and at day 15 and 60 following anaerobic initiation.
After the removal of the day 10 aerobic samples, all remaining sample tubes were removed from their respective metabolism vessel. A 30 mL aliquot of well water and a 0.1 g aliquot of glucose were added to each tube and vortexed to remove any trapped air. The sample tubes were returned to their appropriate metabolism vessels in the environmental chamber and connected to traps for 14C-volatile degradates. The vessels were then flushed with nitrogen to induce anaerobic conditions.
Extraction
During the analysis of the anaerobic samples, each sample tube containing the soil and water samples was centrifuged for approximately 10 min at 2000 rpm. The water layer was decanted and, if necessary, the volume was adjusted to 30 mL. Triplicate 1.0 mL aliquots of the test water were analysed by LSC.
The soil was triplicate extracted with 15 mL methanol. After 30 min shaking, 10 min centrifuging and decanting the extracts were combined and, if necessary, adjusted to 45 mL. Triplicate aliquots at 1.0 mL were analysed by LSC. The soil was then extracted with 25 mL 0.1 M NaOH:methanol (3:1, v:v). The sample was shaken for 4 hours, centrifuged for 10 min and the re-extract was decanted. Then the soil was rinsed twice with 10 mL aliquot of 0.1 M NaOH:methanol (3:1, v:v) and again centrifuged for 10 min. The rinses were combined with the re-extract and adjusted to 50 mL. Triplicate aliquots at 1.0 mL were analysed by LSC.
For analysing the organic phase a 25 mL aliquot of the re-extract was transferred to a separate 50 mL culture tube. The pH was adjusted to 2.0 with the dropwise addition of 6 N HCl. Then the re-extract was partitioned into 2 phases by adding a 10 mL aliquot of ethyl acetate. The final volumes of both the organic and the aqueous phase were measured and triplicate 1.0 mL aliquots of both phases were analysed by LSC.
For analysing the non extractable 14C-residues, triplicate aliquots of post-extracted soil were combusted and analysed by LSC. Each trapping solution was analysed by triplicate 1.0 mL LSC analysis.
DT50:
10 d
Type:
not specified
Remarks on result:
other: T not specified
Key result
DT50:
144 d
Type:
not specified
Remarks on result:
other: when flooding occurs
Transformation products:
yes
No.:
#1
Details on transformation products:
AEROBIC AGING
In addition to the evolution of 14CO2, 3 possible degradation products were observed during TLC analysis of water and soil extracts. The only degradate exceeding 10 % of the applied radioactivity was definitely identified and named as Metabolite F (origin). This non-mobile 14C-residue at the origin of the TLC plate obtained a maximum concentration of 28.5 % at day 10. The identities of the other two degradation products that accounted for <5 % of the applied radioactivity were theorised based on relative polarities. According to it, the second degradate with a maximum concentration of 1.16 % at day 3 was supposed to be Intermediate II (Rf = 0.05) and the third degradate with a concentration of 3.76 % at day 10 was most probably Intermediate I.

ANAEROBIC AGING
In addition to the evolution of 14CO2, 4 possible degradation products were observed during TLC analysis of water and soil extracts. The only degradate exceeding 10 % of the applied radioactivity was definitely identified and named as Metabolite F. This non-mobile 14C-residue at the origin of the TLC plate obtained a maximum concentration of 27.9 % at day 10. Two of the remaining 3 degradations products that accounted for <5 % of the applied radioactivity were theorised based on relative polarities. According to it, the second degradate with a maximum concentration of 2.91 % at day 3 was supposed to be Intermediate II (Rf = 0.05) and the third degradate with a concentration of 4.12 % at day 10 was most probably Intermediate I (Rf = 0.1). A degradate having a maximum concentration of 2.16 % of the applied radioactivity could not be identified (Rf = 0.2).
The proposed degradation pathway of 14C-Piperonyl Butoxide:
Parent compound --> Intermediate I --> Intermediate II --> Metabolite F --> 14CO2 + non-extractable 14C-residues.

All results are listed in the table "Degradation of Piperonyl Butoxide" attached in background material.
The proposed degradation pathway of 14C-piperonyl butoxide during the anaerobic soil metabolism study is reported in the scheme "Proposed degradation pathway " attached in background material
Evaporation of parent compound:
yes
Volatile metabolites:
yes
Residues:
yes
Details on results:
The results of the study have demonstrated that piperonyl butoxide undergoes microbial and/or chemical degradation under aerobic soil conditions to eventually mineralize to CO2 (t½ = 10.0 days). However, the degradation rate is reduced when flooding occurs and anaerobic conditions are induced (t½ = 144 days).

RECOVERY
The mean 14C-mass accountability for the study was 96.1 % based on the percent of initial 14C-residues measured following dosing.

MINRALIZATION
Just a small amount of 14C-volatiles evolved during the study and could be trapped (aerobic: 1.45 % at day 10, anaerobic: 1.57 % at day 60). The majority of 14C-volatiles generated (98-99 %) were trapped in the KOH traps and could be confirmed to be 14CO2.

IDENTIFICATION of RADIOACTVITY
Based on TLC analysis of the aerobic soil samples, parent compound accounted for 48.6 % of the applied radioactivity at day 10. Parent compound of the anaerobic soil samples accounted for 35.2 % of the applied radioactivity at day 60 confirmed by HPLC.
As non-extractable 14C-residues obtained concentrations of >10% of the applied radioactivity, an organic matter fractionation analysis of the day 10 (aerobic) and day 60 (anaerobic) samples was conducted. The majority were observed in the fulvic acid fraction.

DEGRADATION KINETICS
The half-life of Piperonyl Butoxide under aerobic and anaerobic conditions was calculated to be 10 days and 144 days, respectively. The kinetic constant for the degradation of the parent compound was obtained by means of a linear regression analysis.
aerobic conditions: y = 4.66-0.0693x, r = 0.966
anaerobic conditions: y = 3.81-0.00481x, r = -0.876

MICROBIAL VIABILITY
The bacterial and fungal plate count analysis indicated that the microbial viability was sufficient throughout the study.

Parent compound: Based on TLC analysis of the aerobic soil samples, parent compoundaccounted for 48.6 % of the applied radioactivity at day 10. Parent compound of the anaerobic soil samples accounted for 35.2 % of the applied radioactivity at day 60 confirmed by HPLC.

Non-extractable residues

As non-extractable 14C-residues obtained concentrations of >10% of the applied radioactivity, an organic matter fractionation analysis of the day 10 (aerobic) and day 60 (anaerobic) samples was conducted. The majority were observed in the fulvic acid fraction.

Conclusions:
The results of the study have demonstrated that Piperonyl Butoxide undergoes microbial and/or chemical degradation under aerobic soil conditions to eventually mineralize to CO2 (half-life: 10 days). The degradation rate is reduced when flooding occurs and anaerobic conditions are induced (half-life: 144 days).

Description of key information

Aerobic soil degradation: a normalised geometric mean value of 58.3 days should be considered for risk assessment purposes.

Anaerobic soil degradation: a DT50of 144 days has been calculated.

Key value for chemical safety assessment

Half-life in soil:
58.3 d

Additional information