Quaternary ammonium compounds, C12-14-alkyl(hydroxyethyl)dimethyl, chlorides

Biodegradation in soil

Administrative data

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30-Sep-2009 to 21-Jan-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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)

Deviations:

no

Test guideline 2

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

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

[see concordance under 1.1. of the CSR]

IUPAC Name:

[see concordance under 1.1. of the CSR]

Details on test material:

14C-Labeled Test Item

Identification: [14C]HYEQS

Test Item name for Report: 14C-HYEQS

Structural Formula/ Pos. of Labeling(*): please see attached "Structural Formulas"

Batch Number: CFQ40638 Batch B1

Harlan Laboratories Internal Logistic No.:224388/A

Specific Radioactivity: 13.81 MBq/mg, 116 mCi/mmol, 4.29 GBq/mmol (310.7 g/mol at this specific radioactivity)

Radiochemical Purity: >95.26%; as determined by 1D-TLC analysis prior and after treatment (Figure 3).

Expiry Date: Not needed since the purity was determined before use.

Storage Conditions (Sponsor): -20 °C in the absence of moisture, light and air

Storage Conditions (Harlan Laboratories): -20 °C

Stability of Test Item in Vehicle: Stable as determined by analysing an aliquot of the application solution before and after application.

Safety Precautions: Routine hygienic procedures according to the Swiss Legislation on Radiological Protection (Switzerland, Ordinance of June 22, 1994)

Unlabeled Test Item
The unlabeled test item will be used for co-chromatography purposes.

Identification: HYEQS (Hydroxyethylquat Solid)

Chemical Name: Quaternary ammonium compounds, C12-18-alkyl (hydroxyethyl)dimethyl, chlorides

Test Item name for Report: HYEQS

CAS-No.: 85736-63-6

Structural Formula:(The percent values indicate the content of the main components in the test item) please see attached "Structural Formulas"


Physical Form: White, solid granules

Molecular Formula: C16H36NO Cl / C18H36NO Cl

Batch Number: DEG4052477
Harlan Laboratories Internal Logistic No.:223938/A

Molecular Weight: 265.92 g/mol (average of C12 and C14 homologs)
Purity: 98.8%

Solubility in Water: ca. 400 g/L

Expiry Date (Retest Date): 01-Mar-2011

Storage Conditions: Room temperature

Safety Precautions: Routine hygienic procedures are sufficient to assure personnel health and safety.

Radiolabelling:

yes

Study design

Oxygen conditions:

aerobic

Soil classification:

other: Three representative field soils I (Fislis, France; silty clay loam); II (Mechtildshausen, Germany; loam) and III (Speyer 2.3, Germany; sandy loam) were used in the study.

Year:

2009

Soil properties

Soil properties 1

Soil no.:

#1

Soil type:

silty clay loam

% Clay:

27.34

% Silt:

62.98

% Sand:

9.68

% Org. C:

2.19

pH:

6.85

CEC:

26.1 other: (mmol/100 g soil)

Soil properties 2

Soil no.:

#2

Soil type:

loam

% Clay:

20.29

% Silt:

41.04

% Sand:

38.67

% Org. C:

1.07

pH:

7.49

CEC:

12.8 other: (mmol/100 g soil)

Soil properties 3

Soil no.:

#3

Soil type:

sandy loam

% Clay:

8.8

% Silt:

29.3

% Sand:

61.9

% Org. C:

0.97

pH:

6.6

CEC:

9 other: (mmol/100 g soil)

Details on soil characteristics:

SOIL COLLECTION AND STORAGE

Soil Types
Three representative field soils I (Fislis, France; silty clay 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 February 2009 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 February 2009 and transported to Harlan Laboratories Ltd. shortly after sampling. It was sampled from a depth of 25-30 cm using a spade.

Soil III (Speyer 6S) was freshly sampled from a field in Speyer, Germany (49°12'N, 8°03'E) in February 2009 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 five years prior to sampling. There had also been no such treatment of the soils at Harlan Laboratories Ltd.

- Soil preparation (e.g., 2 mm sieved; air dried etc.):
Soil Preparation

After transportation to Harlan Laboratories Ltd. soil I was stored at 4±2 °C for about one month until use. Soil II and III were 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.

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), 19.8 g water per 100 g soil (soil II), and 12.9 g water per 100 g soil (soil III). These values correspond to a pF value of 2.5, as the soils agglomerated at higher water contents (lower pF values). 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.



PROPERTIES OF THE SOILS (in addition to defined fields)
Please see Table 1 Soil Types and their Characteristics

Duration of test (contact time)

Duration of test (contact time) 1

Soil No.:

#1

Duration:

62 d

Duration of test (contact time) 2

Soil No.:

#2

Duration:

62 d

Duration of test (contact time) 3

Soil No.:

#3

Duration:

62 d

Initial test substance concentration

Initial test substance concentration 1

Soil No.:

#1

Initial conc.:

0.45 other: mg a.i./kg dry soil

Based on:

test mat.

Initial test substance concentration 2

Soil No.:

#2

Initial conc.:

0.45 other: mg a.i./kg dry soil

Based on:

test mat.

Initial test substance concentration 3

Soil No.:

#3

Initial conc.:

0.45 other: mg a.i./kg dry soil

Based on:

test mat.

Parameter followed for biodegradation estimation:

radiochem. meas.

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 Füllinsdorf, Switzerland). The sludge moisture content was determined for triplicate sub-samples by oven drying and weighing. This determination revealed a water content of 69.3% (325.5 g 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 553 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 50 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 characteristics" , 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 18, 37, 45 and 49 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 14C-HYEQS at the rate of about 0.5 mg a.i./per kg dry soil
(= 0.05 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.


Preparation of the Application Solution and Treatment of the Test System
The solid test item was completely dissolved in 5 mL acetone. Prior to the application, a 60 µL aliquot was diluted in 10 mL acetone in order to determine the amount of radioactivity in the application solution. The radioactivity of the dilution measured by LSC was 59’446’200 dpm/10 mL. In order to reach the target concentration in the soil an aliquot of 40 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 37’156’500 dpm/50 mL, corresponding to 0.045 mg 14C HYEQS/100 g dry soil, given the specific activity of 13.81 MBq/mg. The latter dpm value was taken as 100% of the applied radioactivity.

The purity and stability of 14C- HYEQS 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 0.45 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" in section "Details on soil characteristics"). 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 40 µL of the application solution (or acetone 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 and Treatment of the Test System" ). 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" in section "Details on soil characteristics". Each replicate was attached immediately to the trapping system after treatment and mixing. The glass rod used was rinsed with acetone 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 0.02% 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 2, 4, 8, 15, 28 and 62 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 seven 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 4 to 4.5 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:

 Acetonitrile/0.1 M hydrochloric acid (4:1; v/v), up to four 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.


Characterisation of the Non-Extractable Radioactivity
The residues from the reflux extractions from day 62 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.

Results and discussion

Material (mass) balance

Material (mass) balance 1

Soil No.:

#1

% Recovery:

91.2

St. dev.:

3.7

Remarks on result:

other: The total mean recoveries in terms of percent of the applied radioactivity ± 3.7%

Material (mass) balance 2

Soil No.:

#2

% Recovery:

91.6

St. dev.:

5.3

Remarks on result:

other: The total mean recoveries in terms of percent of the applied radioactivity ± 3.4%

Material (mass) balance 3

Soil No.:

#3

% Recovery:

94.9

St. dev.:

1.4

Remarks on result:

other: The total mean recoveries in terms of percent of the applied radioactivity ± 1.4%

% Degradation

% Degradation 1

Soil No.:

#1

% Degr.:

6.4

Parameter:

radiochem. meas.

Sampling time:

62 d

% Degradation 2

Soil No.:

#2

% Degr.:

7.4

Parameter:

radiochem. meas.

Sampling time:

62 d

% Degradation 3

Soil No.:

#3

% Degr.:

13.7

Parameter:

radiochem. meas.

Sampling time:

62 d

Half-life / dissipation time of parent compound

Half-life / dissipation time of parent compound 1

Soil No.:

#1

DT50:

6.2

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: DT50 (d) at 20 °C

Half-life / dissipation time of parent compound 2

Soil No.:

#1

DT50:

20.7

Type:

other: DT90 (d)

Remarks on result:

other: DT90 (d) at 20 °C

Half-life / dissipation time of parent compound 3

Soil No.:

#2

DT50:

6

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: DT50 (d) at 20 °C

Half-life / dissipation time of parent compound 4

Soil No.:

#2

DT50:

18.6

Type:

other: DT90 (d)

Remarks on result:

other: DT90 (d) at 20 °C

Half-life / dissipation time of parent compound 5

Soil No.:

#3

DT50:

13.6

Type:

(pseudo-)first order (= half-life)

Remarks on result:

other: DT50 (d) at 20 °C

Half-life / dissipation time of parent compound 6

Soil No.:

#3

DT50:

45.3

Type:

other: DT90 (d)

Remarks on result:

other: DT 90 (d) at 20 °C

Transformation products:

no

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 14C-HYEQS in the application solution before and after treatment was determined by 1D-TLC to be >95.26% (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 summarised in Table 2 to Table 4 in terms of percent of the applied radioactivity and in Appendix II in mg test item equivalents/kg dry soil. Radiocarbon material balances for all soils are shown in Figure 1 to Figure 2.

The total mean recoveries in terms of percent of the applied radioactivity were 91.2 ± 3.7% (soil I), 91.6 ± 3.4% (soil II), and 94.9 ± 1.4% (soil III).

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


Characterisation of Radioactivity.
Extractable Radioactivity.
Immediately after treatment (day 0), 92.9-94.1% of the applied radioactivity could be extracted from the soils (Table 2 to Table 4 and Figure 1 and Figure 2). The amount of extractable radioactivity decreased rapidly to 35.7% (soil I), 33.8% (soil II) and 55.3% (soil III) of the applied radioactivity (day 8, mean values). By the end of the study (day 62), extractables accounted for between 6.4-13.7% of applied.


Non-extractable Radioactivity.
The amount of bound residues was high, increasing from levels of 1.4-3.50% on day 0 to values of 34.8% (soil I) and 35.0% (soil II) of the applied radioactivity on day 15. The non-extractables in soil III reached its maximum on day 28 with 31.7% of applied. By the end of the study (day 62), the level of bound residues had declined to 26.8% (soil I), 28.1% (soil II) and 27.2% (soil III).

Harsh extractions under reflux conditions with acetonitrile/0.1M hydrochloric acid (v/v), conducted at each incubation interval, released between 1.7% and 17.6% 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 17.8-18.7% of the applied radioactivity for all three soils (Table 5). The corresponding range for the fulvic acids was 8.5-10.2% of the applied radioactivity.


Volatiles.
The formation of 14CO2 was significant, increasing to maximum mean levels of 57.5%, 58.8% and 54.8% of the applied radioactivity for soils I to III, respectively, on day 62 (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 0.1% of the applied radioactivity.


Microbial Biomass.
The microbial biomass determined prior to the start of incubation was 51.0 mg, 35.1 mg and 20.8 mg microbial C/100 g dry soil for soils I, II and III, respectively (Table 1). At the end of incubation, the corresponding values were 40.6 mg, 27.4 mg and 14.7 mg microbial C/100 g dry soil, respectively.


Rate of Degradation of HYEQS in Soil.
The rate of disappearance of 14C-HYEQS from soil was described using simple first-order kinetics (described in Section "Calculations" in section "Calculations of DT50 and DT90 values"). The calculated fitted curves are presented in 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 14C-HYEQS 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 14C-HYEQS
(°C) DT50 (d) DT90 (d)
Soil I 20 6.2 20.7
Soil II 20 6.0 18.6
Soil III 20 13.6 45.3

In soil III, a sandy loam, the test item was degraded at slower rate.

Results with reference substance:

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

Any other information on results incl. tables

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:

Tables 1 to 6.

Schemes 1 to 3.

Figures 1 to 6.

Appendices 1 and 2.

Applicant's summary and conclusion

Conclusions:

14C-HYEQS degraded rapidly in all three soils with DT50 values of 6.2, 6.0 and 13.6 days in soils I, II and III, respectively. In soil III, a sandy loam, the test item was degraded at a slower rate.

The formation of radioactive carbon dioxide was high in all soils, reaching levels of 57.5%, 58.8% and 54.8% 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 values of 31.7% to 35.0% of the applied radioactivity during the 62-day incubation period. Organic matter fractionation indicated that the majority of the non-extractable radioactivity was bound to the immobile humic acids and humins (17.8-18.7% of the applied radioactivity). Only minor amounts of radioactivity (8.5-10.2% of applied) were detected in the more mobile fulvic acid fraction.

Degradation of 14C-HYEQS 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 of¹⁴C-HYEQS(Quaternary ammonium compounds, C12-18-alkyl (hydroxyethyl)dimethyl, chlorides)was investigated in three soils incubated under aerobic conditions for 62 days.The following soils were used for the study: soil I (,; silty clay 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 with¹⁴C-HYEQSat the concentration of 0.45 mg a.i./kg dry soil(= 0.045 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 2.5 was adjusted, since the soils agglomerated at water contents corresponding to pF values below 2.5. 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 2, 4, 8, 15, 28 and 62 days.The study was terminated after 62 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 62 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 four 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 91.2 ± 3.7% (soil I), 91.6 ± 5.3% (soil II), and 94.9 ± 1.4% (soil III).

 

Immediately after treatment (day 0), 92.9-94.1% of the applied radioactivity could be extracted from the soils. The amount of extractable radioactivity decreased rapidly to 35.7% (soil I), 33.8% (soil II) and 55.3% (soil III) of the applied radioactivity (day 8, mean values). By the end of the study (day 62), extractables accounted for between 6.4-13.7% of applied.Harsh extractions under reflux conditions with acetonitrile/0.1M hydrochloric acid (v/v), conducted at each incubation interval, released between 1.7% and 17.6% of the applied radioactivity.

 

The amount of bound residues was high, increasing from levels of 1.4-3.50% on day 0 to values of 34.8% (soil I) and 35.0% (soil II) of the applied radioactivity on day 15. The non-extractables in soil III reached its maximum on day 28 with 31.7% of applied. By the end of the study (day 62), the level of bound residues had declined to 26.8% (soil I), 28.1% (soil II) and 27.2% (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 17.8-18.7% of the applied radioactivity for all three soils. The corresponding range for the fulvic acids was 8.5-10.2% of the applied radioactivity.

 

Formation of¹⁴CO₂steadily increased with time, reaching maximum levels of 57.5%, 58.8% and 54.8%of the applied radioactivity for soils I to III, respectively, on day 62. Other volatile products were low, not exceeding 0.1% of the applied radioactivity during the entire incubation period.

 

The calculated DT₅₀and DT₉₀values for¹⁴C-HYEQS based on first-order kineticsare presented below:

 

Soil	Temperature	^14C-HYEQS
	(°C)	DT50(d)	DT90(d)
Soil I	20	6.2	20.7
Soil II	20	6.0	18.6
Soil	20	13.6	45.3

 

In soil III, a sandy loam, the test item was degraded at a slower rate.

 

 

Degradation of¹⁴C-HYEQSin soil incubated under aerobic conditions at 20 °C proceeds via the formation significant amounts of radioactive carbon dioxide and bound residues.