Registration Dossier

Environmental fate & pathways

Biodegradation in soil

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
biodegradation in soil
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
January - June 1996
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Highly relevant study. Protocol for this study is developed and detailed data available. However, study not performed by GLP laboratory.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: Protocol for soil screening, isolation of pure cultures, organic matter determination in soil and analysis of the biodegradation of test material (Tonalid).
Principles of method if other than guideline:
Biodegradation is tested in various soil samples. Samples were analysed by Thin Layer Chromatography.
GLP compliance:
no
Test type:
laboratory
Radiolabelling:
no
Oxygen conditions:
aerobic
Soil classification:
other: Different soil types (sand, clay, peat and loam)
Year:
1995
Soil No.:
#1
% Degr.:
> 80
Parameter:
test mat. analysis
Remarks:
in soil with fungus A. pollulans
Sampling time:
3 wk
Soil No.:
#2
% Degr.:
100
Parameter:
test mat. analysis
Remarks:
in soil with P. chrysosporium
Sampling time:
3 d
Remarks on result:
not measured/tested
Transformation products:
not specified
Evaporation of parent compound:
not measured
Volatile metabolites:
not measured
Residues:
yes
Details on results:
Soil: Samples taken from soil showed a 53% chance of biodegradation that increases to 60% in soil samples from forests. In clay having an average organic carbon concentrationof 3%, only 1 out of 14 showed positive degradation. These results were expected from the theory that high microbial activity is expected in soils rich in organic matter.

A. Pollulans: No abiotic degradation was found. AHTN biodegraded and only one peak emerged as can be seen in figure 1 (attached). The degradation product was identified by GC-MS and indicated the reduction of the acetyl group resulting in the formation of the AHTN-alcohol (see figure 2 attached). A. Pollulans was also incubated with AHTN-alcohol during 4 weeks, but no further intermediates were detected.

P. Chrysosporium: Samples were analysed by GC. No abiotic degardation was observed.AHTN completely disappeared after 10 days with the appearance of two intermediates identified by GC-MS as AHTN+O and AHTN+2O. The formed degradation products were further analysed by GC-IR and the proposed structures can be found in figure 3 (attached). The concentrations of AHTN and degradation products as function of time can be found in figure 4 (attached). Degradation product AHTN+O shows a typical intermediate profile. The increase of AHTN+2O is slower than the increase of AHTN+O suggesting that AHTN+O is (partly) metabolised to AHTN+2O, but this can not be proven.
In addition, AHTN-alcohol was also studied and a significant decrease was observed (figure 5 attached). Small amounts of oxydation products, AHTN-alcohol+O and AHTN-alcohol+2O were detectyed by GC-MS, but the concentrations were too low to carry out further GC-IR analyses.

With P. chrysosporium there was complete disappearance of test material within 10 days; two intermediates were detected, one with one oxygen atom added and the other with two oxygen atoms added; no abiotic degradation was found. With A. pullulans the test material decreased over a period of 5 weeks. An intermediate with the acetyl group reduced to an alcohol was detected; no abiotic degradation was found. 

 

Figure 1 shows the biodegradation curve of AHTN incubated with A. Pollulans. No degradation was observed in the first week, but then the curve shows a distinct decrease over a two week period and levels with time. The AHTN-alcohol concentration gradually increases over the 5 week period. The proposed structure of AHTN-alcohol can be found in figure 2. The suggested or possible structures of the several degradation products are displayed in figure 3.

The biodegradation curve of AHTN incubated with P. Chrysosporium and the concentrations of two degradation products as function of time are shown in figure 4. The AHTN concentration rapidly decreases, while a single oxydated degradation product (AHTN+O) emerges, reaches a concentration plateau followed by decline due to the unavailability of the AHTN. This is a typical behaviour of an intermediate. The double oxydated degradation product (AHTN+2O) gradually increases.

The figures are attached.

Conclusions:
A. Pollulans and P. chrysosporium have the ability to degrade Tonalid (AHTN). Oxidation degradation products have been identified.
Executive summary:

In a screening experiment, 18 of 64 samples indicated biodegradation of Tonalid. Most of the 18 positive samples were sandy soils taken in forests and moors and at the PFW industrial site. The number of positive samples for Tonalid increases with an increasing carbon content.

Incubations of the fungus A. pollulans with Tonalid showed a reduction of the acetyl group to an alcohol. 80% of the AHTN disappeared within 3 weeks. No further transformation was observed.

Incubations with P. chrysosporium showed complete disappearance within three days. Degradation products were analysed and proposed structures were oxidised forms of AHTN. (AHTN+O and AHTN+2O).

A separate incubation of P. chrysosporium with Tonalid-alcohol showed a significant decrease in concentration over time and further oxidation products (AHTN-alcohol+O and AHTN-alcohol+2O). Structures further in the biodegration process were too polar to be extracted by ethyl acetate.

Endpoint:
biodegradation in soil
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
May - December 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Highly relevant screening study. Protocol for this study is developed and detailed data available. However, study not performed by GLP laboratory.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: Protocol for soil screening and tests of pure cultures (Tonalid).
Principles of method if other than guideline:
Biodegradation is tested in various soil samples. Samples were analysed by determining the low Rf values on Thin Layer Chromatography (TLC) plates.
GLP compliance:
no
Test type:
laboratory
Radiolabelling:
no
Oxygen conditions:
aerobic
Soil classification:
other: Different soil types
Year:
1995
Details on soil characteristics:
origin of soil samples:
1. Forests and moors
2. Rivers and meadows
3. Agricultural fields
4. Grassland
5. Ditch
6. Industrial area
7. verge/covered area
8. Recreation area
Duration:
9 wk
Parameter followed for biodegradation estimation:
test mat. analysis
Details on experimental conditions:
Soil screening: 64 soil samples were screened fro Tonalid. The soil was shaken with water and the water filtered. Flasks containing a sterile medium was inoculated with with soil filtrate and Tonalid. Incubation at 25 degrees Celsius. Samples were weekly taken and analysed.
Pure cultures: Three strains of Aureobasidium pullulans (ATCC-60435, ATCC-66657, ATCC-48433) and a pure culture isolated from a soil sample #HP1D were tested for biodegradation. The fungus was grown on a petri plate with a saline-tween solution. A Tonalid stock solution (50g/l in ethanol) was added and the mixture incubated at 25 degrees Celsius. Samples were weekly taken and analysed with TLC
Remarks on result:
not measured/tested
Remarks on result:
not measured/tested
Transformation products:
not specified
Evaporation of parent compound:
not measured
Volatile metabolites:
not measured
Residues:
not measured
Details on results:
15 of the 64 screened soil samples indicated biodegradation.
In the pure cultures, the A. pullulans and the #HP1D showed several intermediates in time.
The biodegradation of Tonalid by ATCC 60435 has been studied in more detail. Five intermediates were identified in week 3. One intermediate is replaced by a new one in week 8 and another new intermediate forms in week 5.

Biodegradation of test material was detected in 18 of 64 soil samples; a majority of positive samples were found in sandy soils taken in forests and moors and at an industrial site; the number of positive soil samples increased with an increasing organic carbon content.

No abiotic degradation was found

Conclusions:
Tonalid indicated biodegradation in 18 of 64 soil samples.
A. pulllulans showed biodegradation of Tonalid.
Executive summary:

A screening test for microbial degradation of Tonalid has been conducted in soil samples and in three strains of A. pullulans.

64 soil samples were drawn from various locations in the Netherlands and 18 samples showed biodegradation.

All strains of the A. pullulans indicated biodegradation with a number of intermediates identified by TLC.

Endpoint:
biodegradation in soil
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Thesis and article.
Principles of method if other than guideline:
The dissipation of AHTN in sludge- amended soils was studied in a 1-year die-away experiment which involved four different soil types, with and without spiking. The samples were put in a tray and subjected to environmental conditions and the AHTN analysed by GC/MS SIM.
GLP compliance:
not specified
Test type:
laboratory
Radiolabelling:
no
Oxygen conditions:
aerobic
Soil classification:
not specified
Soil no.:
#1
Soil type:
sand
% Org. C:
1.35 - 1.63
pH:
>= 6.5 - <= 6.6
Bulk density (g/cm³):
>= 1.38 - <= 1.42
Soil no.:
#2
Soil type:
Silt
% Org. C:
2.72 - 3.39
pH:
>= 5.7 - <= 5.8
Bulk density (g/cm³):
>= 1.19 - <= 1.21
Soil no.:
#3
Soil type:
clay
% Org. C:
5.17 - 6.93
pH:
>= 6.8 - <= 7.4
Bulk density (g/cm³):
>= 0.99 - <= 1.01
Soil no.:
#4
Soil type:
other: Highly-weathered, oxide rich
% Org. C:
0.72 - 1.25
pH:
>= 5.8 - <= 6.3
Bulk density (g/cm³):
>= 1.35 - <= 1.39
Soil No.:
#1
Duration:
1 yr
Soil No.:
#2
Duration:
1 yr
Soil No.:
#3
Duration:
1 yr
Soil No.:
#4
Duration:
1 yr
Soil No.:
#1
Initial conc.:
13 mg/kg soil d.w.
Based on:
other: test material in spiked soil from Georgetown amended with sludge from Georgetown.
Soil No.:
#1
Initial conc.:
0.12 mg/kg soil d.w.
Based on:
other: test material in unspiked soil from Georgetown amended with sludge from Georgetown.
Soil No.:
#2
Initial conc.:
7.5 mg/kg soil d.w.
Based on:
other: test material in spiked soil from Midwest amended with sludge from Georgetown.
Soil No.:
#3
Initial conc.:
ca. 10 mg/kg soil d.w.
Based on:
other: test material in spiked soil from Newark amended with sludge from Georgetown.
Soil No.:
#3
Initial conc.:
0.27 mg/kg soil d.w.
Based on:
other: test material in unspiked soil from Newark amended with sludge from Georgetown.
Soil No.:
#4
Initial conc.:
6.3 mg/kg soil d.w.
Based on:
other: test material in spiked soil from Aiken (South Carolina) amended with sludge from Georgetown.
Details on experimental conditions:
A one-year study was conducted to assess the dissipation of fragrance material from four different soils amended with digested sludge with and without spiking of fragrance materials, and to evaluate the fate process involved. The spiking cocktails were made by dissolving weighed amounts of test materials in ethanol, storing at 5° Celsius in clear glass bottles capped with Teflon-lined closures and sealing with low-permeability vinyl tape. Four soils were chosen to give a range of texture and organic matter content: a sandy agricultural soil from Georgetown, DE, a silty Midwestern agricultural soil from Illinois, a clayey, high-organic carbon soil from Newark, DE, and a highly weathered, oxide-rich soil from Aiken, SC. Anaerobically digested and dewatered sludge was obtained from two activated sludge plants: Georgetown WWTP and Wilmington WWTP, both in Delaware. The sludge was collected fresh from the conveyor belts. A total of 20 trays were set up, which included 16 trays containing the four soils amended with either raw (unspiked) or test material-spiked sludge from one of the two WWTPs, plus four duplicate trays. Each tray was given a name describing the sludge-soil mixture, as follows: sludge type is G for Georgetown sludge, W for Wilmington sludge; soil type is G for Georgetown, M for Midwestern, N for Newark, and S for South Carolina; spiked and unspiked is designated as S or U, respectively. The name is followed by I or II if the tray is a duplicate. Each tray had 24 L of soil and 1 L of sludge. They were made of stainless steel and exposed to environmental weather conditions. A wooden frame helped to support the trays and to lift the trays to allow leachate collection. For trays with spiked sludge, 1 L of sludge was spiked with a cocktail of the test materials using a wall-coating method, as referenced. Soil and sludge were mixed in a cement mixer for 30 minutes then transferred to trays. Soil samples were taken from the trays at predetermined times over 1 year, based on the assumption of annual land application of sludge. Soil cores were taken from each tray at 1 and 2 weeks and 1, 3, 6 and 12 months, using a brass soil corer. Holes were plugged immediately following sampling to minimize soil disturbance. Leachate samples were collected at the end of each rain event for the first 3-5 months of the experiment. To minimize biological activity, leachate samples were treated with a formaldehyde solution in methanol (3% v/v). When volumes of leachate exceeded 4 L during periods of heavy rain, a 10% subsample of leachate was collected from each tray and subsamples from the same tray were composited for extraction. Soil core and sludge samples were extracted by accelerated solvent extraction. All extracts of solid and liquid samples were analyzed for test materials by GC/MS using selected ion monitoring.
Remarks on result:
not measured/tested
Remarks on result:
not measured/tested
Transformation products:
not measured
Evaporation of parent compound:
not measured
Volatile metabolites:
not specified
Residues:
not specified

The sludge to soil ratio in the field was representative of the field conditions.

Rates of degradation varied with the type of soil. The test item was not measured in agricultural soil in Georgetown prior to a subsequent sludge application. It was concluded that it already had dissipated below detection limits (0.05 mg/kg) before new sludge was applied.

Comparison between the trays and field showed that the test item dissipated slower in the trays than in the field. A possible explanation is that field soil is more subjective to flora and fauna activities.

Conclusions:
AHTN concentrations rapidly decreased during the first month in spiked soil, which was not observed in unspiked soil. After one year the concentrations of AHTN in unspiked soils ranged from 42 to 61%. Leaching was negiglible.
Executive summary:

The dissipation of fragrance materials in sludge-amended soils was studied in a 1-year dieaway experiment with four different soils, with and without spiking of the test materials. The four different soils were characterised as: sandy agricultural soil from various locations in the USA.

Anaerobically digested and dewatered sludge was obtained from two activated sludge plants. The test was carried out in trays containing 24 liter mixed with 1 liter of sludge, simulating sludge applications of 7000 wet gallons per acre and a 15 cm plow depth (or 0.6 to 1.1 kg sludge per m2). Each of the four soils was incubated with digested sludge, unspiked as well as spiked with a mixture of fragrance ingredients. The spiking of sludge was performed by the wall coating method and the soil and sludge were mixed during 30 minutes in a cement mixer to ensure uniform mixing. Four combinations were duplicated, so a total of 20 trays were set up outdoors.

 

Leachate samples were collected at the end of each rain event. Soil samples were extracted by accelerated solvent extraction, the leachate was extracted using C-18 speed disks. All extracts were analysed by GC/MS. The initial concentrations in spiked soil were 6 and 13 mg/kg soil, whereas the levels in unspiked soil, simulating real practice were between 0.1 and 0.27 mg/kg.

In the spiked sludge experiments, the concentrations in soil rapidly decreased during the first month and then decreased steadily in time. In the non-spiked soils this phenomenon was not observed. After 3 months, the concentrations were 65 to 80% of the initial concentrations.

During the next three months period the soil was frozen and the concentrations of all test materials remained stable.

After one year the concentrations of AHTN in unspiked soils ranged from 42 to 61% of the initial concentration. In spiked soils the concentrations were slightly more variable. Loss processes may include volatilisation, leaching and biotransformation. Leachate was collected during 3 to 5 months. The leached amount was 0.04 to 0.18% of the initial amount in the spiked soils. A relation with the organic material content was absent. In the leachate from the unspiked soils AHTN was not detected.

(source: EU Risk Assessment Report AHTN, ECB, May 2008)

Description of key information

The test item is inherently biodegradable, not fulfilling criteria.

Key value for chemical safety assessment

Additional information

In a monitoring study, in 18 of 64 soil samples, biodegradation of the test item was observed. There was a positive relation between biodegradation and the organic carbon content.

In a subsequent study, two fungi have been identified capable of biodegrading of the test item i.e. A. pollulans and P. chrysosporium. These fungi are ubiquitous in soil. Especially the latter is capable of quickly degrading the test item. The results indicate that the test item in soil can be transformed into polar metabolites.

Outdoor experiments on sludge amended soils (Difrancesco 2004) were conducted showing a slow degradation. However, in the same study, the authors also mention that dissipation in the environment was faster, but they failed to quantify this process.

For the environmental risk assessment, the test item may be considered as inherently biodegradable', not fulfilling criteria'.

Source: EU Risk Assessement Report, AHTN, ECB, May 2008