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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:
supporting study
Study period:
01-Jul-2010 to 08-Oct-2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission Directive 95/36/EC of July 14, 1995; amending Council Directive 91/414/EEC, Annex I, 7.1.1.2 rate of degradation; 7.1.1.2.1 laboratory studies - aerobic degradation.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: SETAC (Europe): Procedures for assessing the environmental fate and ecotoxicity of pesticides, March 1995, Part 1 - Aerobic degradation.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
(Date of inspection: 05th to 09th and 26th to 30th November 2007 Date of decision: 30th April 2008)
Test type:
laboratory
Radiolabelling:
yes
Oxygen conditions:
aerobic
Soil classification:
other: Three representative field soils I (Fislis, France; silt loam); II (Mechtildshausen, Germany; loam) and III (Speyer 2.3, Germany; sandy loam) were used in the study.
Year:
2010
Soil no.:
#1
Soil type:
silt loam
% Clay:
26.24
% Silt:
64.15
% Sand:
9.62
% Org. C:
2.05
pH:
6.43
CEC:
26.23 other: (mmol/100 g soil)
Soil no.:
#2
Soil type:
loam
% Clay:
29.28
% Silt:
42.31
% Sand:
38.41
% Org. C:
1.25
pH:
7.41
CEC:
14.7 other: (mmol/100 g soil)
Soil no.:
#3
Soil type:
sandy loam
% Clay:
10.19
% Silt:
20.98
% Sand:
68.83
% Org. C:
0.83
pH:
7.04
CEC:
9.77 other: (mmol/100 g soil)
Details on soil characteristics:
SOIL COLLECTION AND STORAGE

Soil Types
Three representative field soils I (Fislis, France; silt loam); II (Mechtildshausen, Germany; loam) and III (Speyer 2.3, Germany; sandy loam) were used in the study. Sampling and handling of the soils was performed in accordance with ISO 10381-6 (“Soil quality-Sampling-Guidance on the collection, handling and storage of soil for the assessment of microbial processes in the laboratory”).

Soil Collection

Soil I (Fislis) was freshly sampled from a field in Fislis, France (47°30'N, 7°21'E) in June 2010 and transported to Harlan Laboratories Ltd. shortly after sampling. It was sampled from a depth of 0-20 cm using a spade.

Soil II (Mechtildshausen) was freshly sampled from a field in Neukirchen, Germany (50°02'N, 8°19'E) in June 2010 and transported to Harlan Laboratories Ltd. shortly after sampling. It was sampled from a depth of 20 cm using a spade.

Soil III (Speyer 2.3) was freshly sampled from a field in Speyer, Germany (49°11'N, 8°11'E) in July 2010 and transported to Harlan Laboratories Ltd. shortly after sampling. It was sampled from a depth of 0-20 cm using a spade.

For all soils, the top plant cover was removed during sampling and soil was put in containers with free access to air. A unique sample identification label was placed inside the container and the same information written with indelible pen on the outside of the container.

All three soils had not been subjected to any pesticide, organic or inorganic fertiliser treatment for at least the last four years prior to sampling. There had also been no such treatment of the soils at Harlan Laboratories Ltd.

Soil Preparation

After transportation to Harlan Laboratories Ltd. soil I was stored for about three weeks and soil II for about two weeks at 4 ± 2 °C until use. Soil III was used immediately after arrival without storage. The soils were sieved through a 2-mm screen prior to testing, and characterized for particle size distribution, percent moisture at water holding capacity, pH, % organic carbon and matter, nitrogen content, carbonate, cation exchange capacity and microbial biomass. The soil parameters are shown in Table 1.

About four days before the start of the study, the soils were conditioned to room temperature. The soils were finger-crumbled and turned over frequently to avoid excessive surface drying. The soils were watered if needed. The soil moisture content was determined for triplicate sub-samples by oven drying and weighing. An adequate water content was obtained by adding purified water to reach final water contents of 27.9 g water per 100 g soil (soil I), 15.7 g water per 100 g soil (soil II), and 12.0 g water per 100 g soil (soil III). These values correspond to a pF value of below 2.5, as the soils agglomerated at higher water contents.

In addition, the water content had to be kept low enough to allow for thorough mixing of the soils with the anoxic digested sludge. Nevertheless, the soil moisture was sufficient to support microbial degradation.
Soil No.:
#1
Duration:
37 d
Soil No.:
#2
Duration:
37 d
Soil No.:
#3
Duration:
37 d
Soil No.:
#1
Initial conc.:
0.46 other: mg a.i./kg dry soil
Based on:
test mat.
Soil No.:
#2
Initial conc.:
0.46 other: mg a.i./kg dry soil
Based on:
test mat.
Soil No.:
#3
Initial conc.:
0.46 other: mg a.i./kg dry soil
Based on:
test mat.
Parameter followed for biodegradation estimation:
radiochem. meas.
Soil No.:
#1
Temp.:
20+-2°C
Humidity:
27.9g water per 100g soil
Microbial biomass:
start: 40.5, end: 64.1mg org. C/100g dry soil
Soil No.:
#2
Temp.:
20+-2°C
Humidity:
15.7g water per 100g soil
Microbial biomass:
start: 37.3, end: 49.7mg org. C/100g dry soil
Soil No.:
#3
Temp.:
20+-2°C
Humidity:
12.0g water per 100g soil
Microbial biomass:
start: 19.3, end: 38.9mg org. C/100g dry soil
Details on experimental conditions:
Test System
Rationale for the Selection of the Test System
Anthropogenic chemicals are frequently detected in the environment and wastewater and sewage sludge can be important emission pathways for them. Sludge from municipal wastewater treatment plant is sometimes used as an organic fertiliser, resulting in the transfer of any chemicals contained within it to the soil. Aerobic and anaerobic conditions occur in soils. The use of aerobic and anaerobic soil test systems enables the study of the fate of chemicals under laboratory conditions, while providing relevant information about natural systems.

Three fresh soil types were selected to evaluate the rate of degradation of the test item in the environment.

Anoxic Digested Sludge

The anoxic digested sludge (dewatered) was sampled from a municipal wastewater treatment plant (ARA Sissach, Switzerland). The sludge moisture content was determined for triplicate sub-samples by oven drying and weighing. This determination revealed a water content of 65.7% (496 mg wet sludge corresponded to 100 g dry sludge).

The amount of sludge added to the soil reflected normal sludge loading to agricultural soils according to REACH Guidance (5000 kg/ha) and should correspond to about 0.17 g dry sludge/ 100 g dry soil. Therefore, an amount of 496 mg sludge per sample was mixed with the soil.

Determination of Soil Microbial Biomass
The microbial biomass of each soil was determined for all soils prior to treatment and at the end of the incubation period. A modification of the respiratory method described by Anderson and Domsch [see References (1)] was used. For this purpose, sub-samples of the sieved soil were amended with increasing amounts of glucose and submitted to respiratory measurements to determine the microbial biomass of the soil. The determination of the microbial biomass was performed at 20 ± 2 °C in the dark.

The soil aliquots were packed into all-glass columns and measured semi-continuously for about 24 hours by means of an IR-gas-analyser (X STREAM R, Emerson Process Management) until CO2 decreased after the peak phase. The total volume of CO2 evolved during approximately one hour was calculated. For the calculations, the values which corresponded to a maximum initial and constant CO2-production, were used (i.e. the so-called plateau values). The value obtained was extrapolated to 100 g dry soil (for details see Section "Determination of the MIcrobial Biomass").


Experimental Conditions
Test System

Samples of 100 g soil based on the dry weight were incubated under aerobic conditions in all-glass metabolism flasks (inner diameter: about 10.6 cm, volume: ca. 1 litre, see attached Scheme 1) in the dark at 20 ± 2 °C. The flasks were equipped with an air inlet and outlet. The system was continuously ventilated with moistened air. For each flask, the exiting air was passed through a trapping system, equipped with two absorption traps containing 50 mL of ethylene glycol and 60 mL 2N sodium hydroxide to trap organic volatiles and 14CO2, respectively.

For the time 0 samples, no absorption traps were set up.

Each test system was uniquely identified according to Harlan Laboratories identification system to assure unmistakable identification.

Temperature

The soil samples were incubated in an air-conditioned room at 20 ± 2 °C.

Moisture Content

The soil moisture content of each soil, described in Section "Soil Types" in section Details on soil characteristic, was held throughout the study. Soil water losses were kept to a minimum by circulating moistened air at a minimum volumetric flow rate sufficient enough to pass through the trapping system. Samples were weighed during the incubation period on day 21 in order to determine the amount of water lost by evaporation. To compensate for this loss, an amount of water equal to that lost by evaporation was added, followed by mixing.

Treatment and Sampling
Rationale for the Application Rate
The soil samples were treated with [1-14C]-HEXADECANAMINE hydrochloride at the rate of about 0.46 mg a.i./per kg dry soil (= 0.046 mg a.i./100 g dry soil), sufficient to determine its degradation rate. The application rate was based on an exposure modeling using realistic use rates.


Preparation of the Application Solution
An unlabeled stock solution was prepared by diluting 41.02 mg of unlabeled 1 HEXADECANAMINE in 100 mL ethanol, corresponding to a concentration of 0.41 mg/mL.

The radioactive test item was delivered as a stock solution in acetone. Its radioactive content was measured to be 11.6 MBq/mL. From this solution, an aliquot of 20 MBq was transferred to an empty vial and the organic solvent evaporated under a gentle stream of nitrogen. From the unlabeled stock solution, an aliquot of 4 mL was added and the dried radioactive test item re-dissolved by ultrasonic treatment.

The radioactive content of this application solution was determined by diluting 50 µL in 50 mL ethanol and measuring 100 µL aliquots in triplicate. In order to reach the target concentration in the soil an aliquot of 50 µL of the application solution was calculated to be applied to each sludge sample. The radioactivity of the dilution in 50 mL acetone measured by LSC was 12’844’233 dpm/50 µL, corresponding to 0.046 mg [1-14C]-HEXADECANAMINE hydrochloride/100 g dry soil, given the specific activity of 4.66 MBq/mg. The latter dpm value was taken as 100% of the applied radioactivity.

The purity and stability of [1-14C]-HEXADECANAMINE hydrochloride in the application solution was determined before and after treatment (see Section Results and Discussion and attached Figure 3).


Treatment of the Test System
Samples containing 100 g soil were treated with the test item at the concentration of 0.46 mg test item/kg dry soil. In order to achieve a realistic exposure regime the test item was added to the soil via sewage sludge (see Section soil types). The amount of sludge added to the soil reflected normal sludge loading to agricultural soils according to REACH Guidance (5000 kg/ha) and corresponds to about 0.17 g dry sludge / 100 g dry soil.

Due to the low amount of sludge which was used per sample, the soil was first transferred to the metabolism flask. Prior to application a small amount of the soil from the metabolism flask was placed on a weighing boat and the solid sludge was added on top. Thereafter, a defined aliquot of 50 µL of the application solution (or ethanol for the control samples) was applied to each solid sludge sample to reach the target concentration in the soil (see Section Preparation of the application solution). Care was taken to absorb the complete application volume into the sludge using a Hamilton syringe. After the application procedure, the soil and sludge were put back into the metabolism flask and then mixed with the soil using a glass rod. Afterwards, the soil moisture content was adjusted as described in Section Soil types. Each replicate was attached immediately to the trapping system after treatment and mixing. The glass rod used was rinsed with ethanol and the radioactivity content measured by LSC, to ensure that no test item residues were removed from the metabolism flasks. The LSC measurements revealed that no significant amounts (not exceeding the maximum of 0.028% of the applied radioactivity) were adsorbed by the glass rod.


Sampling
Soil Samples

Duplicate samples from all three soils were taken for extraction and analysis immediately after treatment (day 0) and after 3, 7, 14, 28 and 37 days of incubation.

The untreated samples were taken for the determination of the microbial biomass at the start and end of incubation.

Trapping Solutions

Radioactivity in the traps was periodically monitored by liquid scintillation counting (LSC). The traps were exchanged several times during the study. Prior to the determination of radioactivity, the volume of the liquid in each ethylene glycol and sodium hydroxide trap was recorded. In order to confirm the presence of 14CO2, the radioactivity contained in the sodium hydroxide traps was precipitated with barium hydroxide on pool samples (pooled aliquots of each soil from twelve intervals). For this purpose, 0.5 mL of the alkaline solutions were diluted with 3 mL of bi distilled water and the precipitations were induced by addition of 3 mL of a saturated barium hydroxide solution. The suspensions were centrifuged and the supernatants were tested for quantitative precipitation by adding another one to two drops of the saturated Ba(OH)2 solution. If no turbidity developed upon the second addition of Ba(OH)2, the supernatants were counted by LSC measurement. If turbidity was observed, LSC measurements were performed after another precipitation step. The absence of radioactivity in the supernatants after precipitation was taken as proof that only 14CO2 was present in the sodium hydroxide solutions.


Extraction and Isolation of Radioactivity from Soil
A flow-chart of the sample work-up is shown in attached Scheme 2.

Extractable and non-extractable radioactivity was determined by liquid scintillation counting the extract and combusting the extracted soil. The total amount of soil contained in each flask (about 100 g dry weight) was exhaustively extracted using the following work-up procedure:

 Acetone/0.1 M hydrochloric acid (4:1; v/v), up to five times.
 Acidic reflux extraction for four hours using Acetonitrile/0.1 M hydrochloric acid (1:1; v/v)

The amount of solvent used for each extraction step was about 1 mL per g soil. Each room temperature extraction was performed in a shaker at about 250 vibrations per minute for about 30 minutes. The individual extracts were quantified by LSC and then combined. Acidic reflux extraction was conduced subsequent to the ambient extractions.

The residual radioactivity remaining in soil after the extraction procedure was quantified by LSC after combustion of aliquots of the air-dried and homogenised soil.


Characterization of the Non-Extractable Radioactivity
The residues from the reflux extractions from day 37 were submitted to organic matter fractionation in order to measure the radioactivity bound to the humic and fulvic acids as well as to the humin fraction of the soil. A procedure based on Stevenson [see Reference (3)] was used. It is shown in attached Scheme 3.

Soil No.:
#1
% Recovery:
83.9
St. dev.:
3.4
Remarks on result:
other: The total mean recoveries in terms of percent of the applied radioactivity ± 3.4% Note: The low recoveries obtained are derived from losses of 14CO2.
Soil No.:
#2
% Recovery:
86
St. dev.:
1.5
Remarks on result:
other: The total mean recoveries in terms of percent of the applied radioactivity ± 1.5% Note: The low recoveries obtained are derived from losses of 14CO2.
Soil No.:
#3
% Recovery:
87.2
St. dev.:
2.7
Remarks on result:
other: The total mean recoveries in terms of percent of the applied radioactivity ± 2.7% Note: The low recoveries obtained are derived from losses of 14CO2.
Soil No.:
#1
% Degr.:
11.9
Parameter:
radiochem. meas.
Sampling time:
37 d
Soil No.:
#2
% Degr.:
11.3
Parameter:
radiochem. meas.
Sampling time:
37 d
Soil No.:
#3
% Degr.:
16.6
Parameter:
radiochem. meas.
Sampling time:
37 d
Soil No.:
#1
DT50:
9.6
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: DT50 (d) at 20 °C
Soil No.:
#1
DT50:
31.8
Type:
other: DT90 (d)
Remarks on result:
other: DT90 (d) at 20 °C
Soil No.:
#2
DT50:
8.7
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: DT50 (d) at 20 °C
Soil No.:
#2
DT50:
29
Type:
other: DT90 (d)
Remarks on result:
other: DT90 (d) at 20 °C
Soil No.:
#3
DT50:
9.9
Type:
(pseudo-)first order (= half-life)
Remarks on result:
other: DT50 (d) at 20 °C
Soil No.:
#3
DT50:
33
Type:
other: DT90 (d)
Remarks on result:
other: DT 90 (d) at 20 °C
Transformation products:
no
Details on transformation products:
Hexadecylamine is completely metabolized into biomass, CO2, H2O and NO3
Evaporation of parent compound:
not measured
Volatile metabolites:
yes
Residues:
yes
Details on results:
Radiochemical Purity and Stability of the Test Item
The purity of [1-14C]-HEXADECANAMINE hydrochloride in the application solution before and after treatment was determined by 1D-TLC to be >99% (Figure 3). Stability tests before and after the treatment procedure indicated that the test item was stable during application.


Material Balance (Overall Recovery of Radioactivity)
The results are summarized in attached Table 2 to Table 4 in terms of percent of the applied radioactivity and in attached Appendix II in mg test item equivalents/kg dry soil. Radiocarbon material balances for all soils are shown inattached Figure 1 to Figure 2.

The total mean recoveries in terms of percent of the applied radioactivity were 83.9 ± 3.4% (soil I), 86.0 ± 1.5% (soil II), and 87.2 ± 2.7% (soil III).

On the whole, duplicate samples gave similar results, therefore the results in the following sections are expressed as the mean value.


Characterization of Radioactivity
Extractable Radioactivity
Immediately after treatment (day 0), 81.7-86.0% of the applied radioactivity could be extracted from the soils (attached Table 2 to Table 4 and attached Figure 1 and Figure 2). The amount of extractable radioactivity decreased to 24.3% (soil I), 23.2% (soil II) and 26.5% (soil III) of the applied radioactivity (day 14, mean values). By the end of the study (day 37), extractables accounted for between 11.3-16.6% of applied.


Non-Extractable Radioactivity
The amount of bound residues was high, increasing from levels of 2.3-5.3% on day 0 to values of 22.8% (soil I), 24.0% (soil II) and 19.7% (soil III) of the applied radioactivity on day 28. By the end of the study (day 37), the level of bound residues had declined to 21.5% (soil I) and 23.1% (soil II). In soil III it increased to 21.1% (soil III).

Harsh extractions under reflux conditions with acetonitrile/0.1M hydrochloric acid (v/v), conducted at each incubation interval, released between 2.6% and 9.6% of the applied radioactivity. Subsequent organic matter fractionation of the non-extractable residues indicated that the majority of the non-extractable radioactivity was bound to the immobile humic acids and humins amounting to 19.5%, 20.8% and 18.6% of the applied radioactivity for Soils I, II and III, respectively.
The corresponding values for the fulvic acids were 2.0%, 2.3% and 2.5% of the applied radioactivity.


Volatiles
The formation of 14CO2 was significant, increasing to maximum mean levels of 46.3%, 48.2% and 50.7% of the applied radioactivity for soils I to III, respectively, on day 37 (Table 2 to Table 4 and Figure 1 and Figure 2). The identity of 14CO2 was confirmed by precipitation with barium hydroxide.

Volatile products other than 14CO2 did not exceed the maximum of 2.5% of the applied radioactivity.


Microbial Biomass
The microbial biomass determined prior to the start of incubation was 40.5 mg, 37.3 mg and 19.3 mg microbial C/100 g dry soil for soils I, II and III, respectively (attached Table 1). At the end of incubation, the corresponding values were 64.1 mg, 49.7 mg and 38.9 mg microbial C/100 g dry soil, respectively.


Rate of Degradation of [1-14C]-HEXADECANAMINE hydrochloride in Soil
The rate of disappearance of [1-14C]-HEXADECANAMINE hydrochloride from soil was described using simple first-order kinetics (Section Calculation of DT50 and DT90 values). The calculated fitted curves are presented in attached Figure 4 to Figure 5. These calculations were performed using the data of the total extractables. The total extractables decreased during incubation due to degradation of
[1-14C]-HEXADECANAMINE hydrochloride to radioactive carbon dioxide and the formation of bound residues. The total extractables were considered to contain the test item exclusively.

The calculated DT50 and DT90 values are presented below:

Soil Temperature [1-14C]-HEXADECANAMINE hydrochloride
(°C) DT50 (d) DT90 (d)
Soil I 20 9.6 31.8
Soil II 20 8.7 29.0
Soil III 20 9.9 33.0

Results with reference substance:
Reference Item
Only the unlabeled test item was used as reference item to confirm the identity and radiochemical purity of the test item by 1D-TLC.

Due to the required amount of tables etc it is not possible to input all of them in this section, also it would render the information less understandable to split this information between this section and attachments section. Therefore please see "Results" in the overall attachment section for:

Results Structural Formulas

Conclusions:
[1-14C]-HEXADECANAMINE hydrochloride degraded rapidly in all three soils with DT50 values of 9.6, 8.7 and 9.9 days in soils I, II and III, respectively.

The formation of radioactive carbon dioxide was high in all soils, reaching levels of 43.5%, 47.1% and 47.9% of the applied radioactivity in soils I to III, respectively, after 62 days of incubation.

The amount of non-extractable radioactivity was also significant, amounting to maximum mean values of 19.7% to 24% of the applied radioactivity during the 37-day incubation period. Organic matter fractionation indicated that the majority of the non-extractable radioactivity was bound to the immobile humic acids and humins (18.6-20.8% of the applied radioactivity). Only minor amounts of radioactivity (2.0-2.5% of applied) were detected in the more mobile fulvic acid fraction.

Degradation of [1-14C]-HEXADECANAMINE hydrochloride in soil incubated under aerobic conditions at 20 °C proceeds via the formation significant amounts of radioactive carbon dioxide and bound residues.
Executive summary:

 SUMMARY

The rate of degradation of [1-14C]-HEXADECANAMINE hydrochloridewas investigated in three soils incubated under aerobic conditions for 37 days.The following soils were used for the study: soil I (,; silt loam), soil II (,; loam) and soil III (2.3,; sandy loam).

 

The freshly collected soils were passed through a 2 mm sieve.Samples containing 100 g dry soil were treated with[1-14C]-HEXADECANAMINE hydrochlorideat the concentration of 0.459 mg a.i./kg dry soil(= 0.0459 mg a.i./100 g dry soil), sufficient to determine its degradation rate. The application rate was based on an exposure modelling using realistic use rates.In order to achieve a realistic exposure regime the test item was added to the soil via sewage sludge.The amount of sludge added to the soil reflected normal sludge loading to agricultural soils according to REACH Guidance (5000 kg/ha) and corresponds to about 0.17 g dry sludge / 100 g dry soil.

 

Adequate moisture contents were adjusted with purified water. A pF value of below 2.5 was adjusted, since the soils agglomerated at higher water contents. Nevertheless, the soil moisture was sufficient to support microbial degradation. In addition, the water content had to be kept low enough to allow for thorough mixing of the soils with the sewage sludge.The treated soil samples were incubated at 20 ± 2 °C in the dark under continuous ventilation with moistened air. The exiting air was passed through a trapping system consisting of flasks of ethylene glycol and sodium hydroxide in series. Prior to treatment and at the end of the incubation period, the microbial biomass was determined for each soil. The results showed that the soils were viable during the study.

 

Duplicate samples treated with the test item were taken immediately from each soil after treatment (day 0) and after 3, 7, 14, 28 and 37 days.The study was terminated after 37 rather than the standard 120 days, since a significant amount of the radioactivity was detected as radioactive carbon dioxide or was bound rapidly to the soils within 37 days.Each sample was submitted to the following extraction procedure: room temperature extraction using acetone/0.1 M HCl (4:1; v/v) for up to five times, followed by acidic reflux extraction using acetonitrile/0.1 M HCl (1:1; v/v) for 4 hours. The individual extracts were measured by LSC and combined.

 

The total mean recoveries in terms of percent of the applied radioactivity were 83.9 ± 3.4% (soil I), 86.0 ± 1.5% (soil II), and 87.2 ± 2.7% (soil III).

 

Immediately after treatment (day 0), 81.7-86.0% of the applied radioactivity could be extracted from the soils. The amount of extractable radioactivity decreased rapidly to 48.1% (soil I), 40.9% (soil II) and 46.5% (soil III) of the applied radioactivity (day 7, mean values). By the end of the study (day 37), extractables accounted for 11.9% (soil I), 11.3% (soil II) and 16.6% (soil III) of applied.Harsh extractions under reflux conditions with acetonitrile/0.1M hydrochloric acid (v/v), conducted at each incubation interval, released between 2.2% and 9.6% of the applied radioactivity.

 

The amount of bound residues was high, increasing from levels of 2.3-5.3% on day 0 to values of 17.3% (soil I), 21.9% (soil II) and 17.1% (soil III) of the applied radioactivity on day 7. By the end of the study (day 37), the level of bound residues had declined to 21.5% (soil I), 23.1% (soil II) and 21.1% (soil III).

 

Subsequent organic matter fractionation of the non-extractable residues indicated that the majority of the non-extractable radioactivity was bound to the immobile humic acids and humins amounting to 19.5%, 20.8% and 18.6% of the applied radioactivity for Soils I, II and III, respectively. The corresponding values for the fulvic acids were 2.0%, 2.3% and 2.5% of the applied radioactivity.

 

Formation of14CO2steadily increased with time, reaching maximum levels of 46.3%, 48.2% and 50.7%of the applied radioactivity for soils I to III, respectively, on day 37. Other volatile products were low, not exceeding 2.5% of the applied radioactivity during the entire incubation period.

 

The calculated DT50and DT90values for [1-14C]-HEXADECANAMINE hydrochloridebased on first-order kineticsare presented below:

 

Soil

Temperature

[1-14C]-HEXADECANAMINE hydrochloride

(°C)

DT50(d)

DT90(d)

Soil I

20

9.6

31.8

Soil II

20

8.7

29.0

Soil

20

9.9

33.0

 

 

Degradation of [1-14C]-HEXADECANAMINE hydrochloridein soil incubated under aerobic conditions at 20 °C proceeds via the formation significant amounts of radioactive carbon dioxide and bound residues.

Description of key information

Based on the available studies on biodegradation, the substances are estimated to be readily biodegradable. The 10-days-window criterion should not be evaluated for a UVCB thus there is no requirement for performing a soil biodegradation test (Annex IX. 9.2.1.3). No simulation tests for degradation rates under environmental conditions are available for primary fatty amine ethoxylates but for a very similar substance (hexadecyl amine) and these results will be used for read-across to primary fatty amine ethoxylates (2EO). For soil and aerobic sediment this is a half-life value of 17 days at 12 °C.

Key value for chemical safety assessment

Half-life in soil:
17 d
at the temperature of:
12 °C

Additional information

Default half-life’s

For the derivation of the default half-life’s the bioavailability is taken into account via the sorption properties of the substance. This is realistic for soluble non-toxic substances. For poorly soluble/strongly sorbing substances however, the degradation rate in a standard ready test is limited by the dissolution rate and in many cases not 60% degradation is achieved within 28 days but in a slightly longer time frame. Such a substance is in fact completely degraded to CO2and H2O and thus completely biodegradable. The bioavailable fraction is readily biodegradable but the due to the stringency of the test setup the substance this cannot be observed.

The Kpsoil was determined as 4526 L/kg. The suggested maximum half-life for a readily degradable substance with a Kpsoil in the range >1000 and < 10000 L/kg is 3000 days for soil (at 12 ºC). These values are considered as extremely conservative but in the absence of measured data can be used in the exposure assessment as a worst-case. The half-life of the bioavailable fraction of primary fatty amine ethoxylates in the water phase of soils is expected to be in the order of a few days, which is based on experiments with dialkyldimethylammonium salts (van Ginkel et al, 2003).

Read across from primary alkyl amines

For hexadecylamine[1-14C] a substance which isvery similar to 2,2'-(C12-18 evennumbered alkyl imino) diethanol (CAS no 71786-60-2) there is an OECD 307 aerobic soil transformation study available and the results of this study will be used for read across to the primary alkyl amine ethoxylates for both the soil and sediment compartment. Although this C16 amine is strongly sorbing to soil (median Kp soil of 3875 L/kg at lowest measured concentration) the following half-life’s at 20 °C were determined for three soils: Soil 1 t1/2 = 9.0 d; Soil 2 t1/2 = 8.1 d; Soil 3 t1/2 = 8.9 d.

The median Half-life of 8.9 d at 20 °C corresponds to a median Half-life of 16.9 d at an environmental temperature of 12 °C (see REACH Guidance). This study demonstrates that 1-Hexadecanamine (C16 amine) is rapidly degraded in various soils and that the assumption of low degradation rates for strongly sorbing substances could be unjustified.