thiourea; thiocarbamide

Biodegradation in soil

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

secondary literature

Remarks:

Secondary literature; original reference (Frederick et al., 1957.Decomposability of some organic sulfur compounds in soil. Soil Science Society Proceedings, p. 287-292) was not available for review.

Qualifier:

equivalent or similar to guideline

Guideline:

other: no guideline reported

Principles of method if other than guideline:

The biodegradation of thiourea in agricultural soil (sandy loam, pH 6.1) was followed in an experiment over 21 weeks at 28 °C and a field capacity of 40 %. The initial thiourea concentration applied was 10 g/kg soil dw.
After incubation for 21 weeks 0.5 % CaCO3 was added to the soil and the samples were again incubated for additional 21 weeks.
In addition bacterial and fungal populations were checked.
Besides, soil suspensions were incubated with 5 g/kg thiourea with mineral medium at 28 °C for one week, and 1g thiourea and 50 g soil were incubated with 150 mL perfusate for six weeks.

GLP compliance:

no

Test type:

laboratory

Radiolabelling:

no

Oxygen conditions:

not specified

Soil classification:

not specified

Soil no.:

#1

pH:

6.1

Soil No.:

#1

Duration:

>= 21 - <= 42 wk

Soil No.:

#1

Initial conc.:

10 g/kg soil d.w.

Based on:

test mat.

Soil No.:

#1

Temp.:

28 °C

Humidity:

40% field capacity

Microbial biomass:

no details reported

Key result

Soil No.:

#1

% Degr.:

9

Parameter:

other: formation of sulphate

Sampling time:

21 wk

Soil No.:

#1

% Degr.:

9

Parameter:

other: formation of sulphate

Sampling time:

42 wk

Transformation products:

yes

No.:

#1

Evaporation of parent compound:

not measured

Volatile metabolites:

not measured

Residues:

not measured

The biodegradation of thiourea in agricultural soil (sandy loam, pH 6.1) was followed in an experiment over 21 weeks at 28 °C, a field capacity of 40%, and an initial thiourea concentration of 10 g/kg soil dw. 9 % degradation (determined via formation of sulphate) was observed. Application of 0.5 % CaCO3 and additional incubation for 21 weeks did not alter the degradation results.

 

Bacterial and fungal populations were checked. The number of bacteria and actinomyces was reduced from several million per g untreated soil to several thousand per g treated soil. The number of fungus was reduced by > 99%. Incubation of soil suspensions with 5 g/kg thiourea with mineral medium at 28 °C for one week did not result in formation of sulphate. 1% of thiourea was degraded when 1g thiourea and 50 g soil were incubated with 150 mL perfusate for six weeks.

Conclusions:

Thiourea was only slightly degraded in the soil samples at 10 g/kg soil dw after incubation after 21 to 42 weeks.

Executive summary:

The biodegradation of thiourea in agricultural soil (sandy loam, pH 6.1) was followed in an experiment over 21 weeks at 28 °C, a field capacity of 40%, and an initial thiourea concentration of 10 g/kg soil dw. 9 % degradation (determined via formation of sulphate) was observed. Application of 0.5 % CaCO3 and additional incubation for 21 weeks did not alter the degradation results.

 

Incubation of soil suspensions with 5 g/kg thiourea with mineral medium at 28 °C for one week did not result in formation of sulphate. 1% of thiourea was degraded when 1g thiourea and 50 g soil were incubated with 150 mL perfusate for six weeks.

 

In addition, bacterial and fungal populations were checked. The number of bacteria and actinomyces was reduced from several million per g untreated soil to several thousand per g treated soil. The number of fungus was reduced by > 99%.

Reference 2

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

no data

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

Documentation insufficient for assessment (e.g. purity of the test substance as well as details on methods and results are not reported).

Qualifier:

equivalent or similar to guideline

Guideline:

other: no guideline reported

Principles of method if other than guideline:

The biodegradation of thiourea was studied in barnyard soil (200 g) at 0.02 and 0.1 M. 0.5 % Glucose was added to the soil samples with and without thiourea. The moisture content of the soil was brought to 50 % of field capacity. Water was added regularly to account for losses through evaporation. The soil samples were incubated at 28 °C for several weeks. Production of sulphate served as indicator for decomposition of thiourea.

GLP compliance:

not specified

Test type:

laboratory

Radiolabelling:

no

Oxygen conditions:

not specified

Soil classification:

not specified

Soil No.:

#1

Duration:

15 wk

Soil No.:

#1

Initial conc.:

0.02 other: M

Based on:

test mat.

Soil No.:

#1

Initial conc.:

0.1 other: M

Based on:

test mat.

Soil No.:

#1

Temp.:

28 °C

Humidity:

50 %

Microbial biomass:

no data

Soil No.:

#1

% Degr.:

96

Parameter:

other: % S recovered as sulphate; initial concentration: 0.02M

Sampling time:

15 wk

Key result

Soil No.:

#1

% Degr.:

28

Parameter:

other: % S recovered as sulphate; initial concentration: 0.1M

Sampling time:

15 wk

Transformation products:

yes

No.:

#1

Evaporation of parent compound:

not measured

Volatile metabolites:

not measured

Residues:

yes

Biodegradation of thiourea is inhibited at higher concentrations: at an initial concentration of 0.02 M thiourea 22% of the test substance is degradaed within one week, 27 % is degraded after 2 weeks. Within 15 weeks 96 % of the S in thiourea is recovered as sulphate. At an initial concentration of 0.1 M only 28 % of the thiourea-S is recovered as sulphate after 15 weeks.

Conclusions:

Biodegradation of thiourea is inhibited at higher concentrations: after 15 weeks thiourea is degraded to 96 % and 28 % at initial concentrations of 0.02 M and 0.1 M thiourea, respectively.

Executive summary:

The biodegradation of thiourea was studied in barnyard soil (200g) at 0.02 and 0.1 M. 0.5 % Glucose was added to the soil samples with and without thiourea.The moisture content of the soil was brought to 50 % of field capacity. Water was added regularly to account for losses through evaporation. The soil samples were incubated at 28 °C for up to 15 weeks. Production of sulphate served as indicator for decomposition of thiourea. Biodegradation of thiourea is inhibited at higher concentrations: at an initial concentration of 0.02 M thiourea 22% of the test substance is degraded within one week, 27 % is degraded after 2 weeks. Within 15 weeks 96 % of the S in thiourea is recovered as sulphate. At an initial concentration of 0.1 M only 28 % of the thiourea-S is recovered as sulphate after 15 weeks.

Reference 3

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-10-26 to 2013-06-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 307 (Aerobic and Anaerobic Transformation in Soil)

Version / remarks:

adopted 24t h April 2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Directive 98/8/EC of the European Parliament and of the Council of February 16, 1998, Annex IIIA, XII1.1

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

laboratory

Specific details on test material used for the study:

- Chemical name: [14C]Thiourea

Radiolabelling:

yes

Oxygen conditions:

aerobic

Soil classification:

USDA (US Department of Agriculture)

Year:

2013

Soil no.:

#1

Soil type:

sandy loam

% Clay:

1 215

% Silt:

15.47

% Sand:

72.38

% Org. C:

0.64

pH:

5.86

CEC:

11.82 other: mmol/100 g soil

Bulk density (g/cm³):

1.7

% Moisture content:

28

Soil no.:

#2

Soil type:

silt loam

% Clay:

22.61

% Silt:

63.82

% Sand:

1 357

% Org. C:

2.25

pH:

6.3

CEC:

24.7 other: mmol/100g soil

Bulk density (g/cm³):

1.7

% Moisture content:

29

Soil no.:

#3

Soil type:

loam

% Clay:

18.14

% Silt:

40.65

% Sand:

41.21

% Org. C:

2.39

pH:

7.01

CEC:

22.81 other: mmol/100 g soil

Bulk density (g/cm³):

1.7

% Moisture content:

27

Soil no.:

#4

Soil type:

loam

% Clay:

25.9

% Silt:

40.5

% Sand:

33.6

% Org. C:

2.26

pH:

7.2

CEC:

31.4 other: mmol/100 g soil

Bulk density (g/cm³):

1.7

% Moisture content:

43.6

Details on soil characteristics:

SOIL COLLECTION AND STORAGE
The soils were collected from different agricultural areas. Sampling and handling of the soils were performed under consideration of ISO 10381-6. The top plant cover was removed, thereafter, the soil top layer was added to containers with free access of air. A unique sample identification label was placed inside each container and the same information written with indelible pen on the outside of the containers.

Soil I (Mühlfeld) was freshly sampled from the top 30 cm layer from an uncultivated grassland in 85354 Freising (Bavaria, Germany; latitude 48° 24' 02''N, longitude 11° 42' 53''E) in August 2012. The soil had never been treated with the test item substance or its structural analogues.

Soil II (Bad Lauchstädt) was freshly sampled from the top 20 cm layer from an agricultural area in 06246 Bad Lauchstädt (Saxony-Anhalt, Germany, latitude 51° 23' 25''N, longitude 11° 52' 46''E) in August 2012. The soil had not been treated with the test item substance or its structural analogues during the past 4 years prior to sampling.

Soil III (Priesteblich) was freshly sampled from the top 20 cm layer from a grassland in Priesteblich (04420 Markranstädt, Saxony, Germany, latitude 51° 19' 59''N, longitude 12° 12'17''E) in October 2012. The soil had not been treated with the test item substance or its structural analogues during the past 4 years prior to sampling.

Soil IV (Speyer 2.4) was freshly sampled from the top 20 cm layer from a meadow in 76774 Leimersheim (Rhineland-Palatinate, Germany, latitude 49° 07'N, longitude 8° 20'E) in September 2012. The soil had not been treated with the test item substance or its structural analogues during the past 4 years prior to sampling.

Soil No.:

#1

Duration:

72 h

Soil No.:

#2

Duration:

72 h

Soil No.:

#3

Duration:

72 h

Soil No.:

#4

Duration:

72 h

Soil No.:

#1

Initial conc.:

10 other: mg/kg soil ww

Based on:

act. ingr.

Soil No.:

#2

Initial conc.:

10 other: mg/kg soil ww

Based on:

act. ingr.

Soil No.:

#3

Initial conc.:

10 other: mg/kg soil ww

Based on:

act. ingr.

Soil No.:

#4

Initial conc.:

10 other: mg/kg soil ww

Based on:

act. ingr.

Parameter followed for biodegradation estimation:

test mat. analysis

Soil No.:

#1

Temp.:

20.4 ± 0.1 °C

Humidity:

pF 2.0

Microbial biomass:

280 mg microbial C/kg dry soil

Soil No.:

#2

Temp.:

20.4 ± 0.1 °C

Humidity:

pF 2.0

Microbial biomass:

207 mg microbial C/kg dry soil

Soil No.:

#3

Temp.:

20.4 ± 0.1 °C

Humidity:

pF 2.0

Microbial biomass:

491 mg microbial C/kg dry soil

Soil No.:

#4

Temp.:

20.4 ± 0.1 °C

Humidity:

pF 2.0

Microbial biomass:

873 mg microbial C/kg dry soil

Details on experimental conditions:

1. PRELIMINARY EXPERIMENTS: non-GLP pre-experiment

2. EXPERIMENTAL DESIGN
- Soil incubation condition: fresh soil, incubated in environmental chamber, darkness, 20.4 ± 0.1 °C, open gas-flow system (all-glass flasks with a volume of approximately 1 litre), samples ventilated with moistened air (passed through a trapping system consisting of flasks of sodium hydroxide and ethylene glycol in series), soil moisture adjusted to pF 2.0 with water followed by mixing
- Soil (g/replicate): 100
- Control conditions: same as for treatments
- No. of replication controls: 2
- No. of replication treatments: 2
- Details of traps for CO2 and organic volatile: ethylene glycol or sodium hydroxide

- Test material application
Application of soil II (October 30, 2012): An amount of 28.13 mg of [ C]Thiourea (purity: 99.7%) was dissolved in 10 mL of purified water. Thereof, 9 mL were taken and spiked with 220 uL of the stock solution containing 0.364 mg [14C]Thiourea, making a total volume of 20 mL with purified water. The radioactivity determined by LSC in 3 aliquots of 2 uL was 485'690'000 dpm (or 0.321 mg) in 20 mL. Therefore, the overall concentration was 1.278 mg/mL and the new specific activity 0.32 MBq/mg. The application volume was 800 uL (1.022 mg) per 100 g wet soil.

Application of soils I and III (November 06, 2012): An amount of 56.06 mg of [ C]Thiourea (purity: 99.7%) was dissolved in 20 mL of purified water. Thereof, 18 mL were taken and spiked with 450 uL of the stock solution containing 0.746 mg [14C]Thiourea to a total volume of 25 mL with purified water. The radioactivity determined by LSC in 3 aliquots of 2 uL was 1'016'725'000 dpm in 25 mL. Thus, the overall concentration was 2.039 mg/mL and the new specific activity 0.33 MBq/mg. The application volume was 500 uL (1.019 mg) per 100 g wet soil.

Application of soil IV (November 20, 2012): An amount of 28.31 mg of [ C]Thiourea (purity: 99.7%) was dissolved in 10 mL of purified water. Thereof, 9 mL were taken and spiked with 220 uL of the stock solution containing 0.364 mg [14C]Thiourea. Then, the volume was filled up to 20 mL with purified water and the

Experimental conditions
- Moisture maintenance method: water added
- Continuous darkness: Yes

3. OXYGEN CONDITIONS
- Methods used to create the aerobic conditions: replicates ventilated with air

4. SUPPLEMENTARY EXPERIMENTS: none

5. SAMPLING DETAILS
- Sampling intervals: after treatment, and 2, 4, 6, 15, 24 and 72 hours of incubation
- Sampling method for soil samples: two replicates treated with the test item taken at each sampling event
- Method of collection of CO2 volatile organic compounds: traps
> Moisture content: pF 2.0

Parent/product:

parent

Key result

Soil No.:

#1

Sampling date:

2012

% Degr.:

77.5

Parameter:

CO2 evolution

Sampling time:

72 h

Parent/product:

parent

Key result

Soil No.:

#2

Sampling date:

2012

% Degr.:

75

Parameter:

CO2 evolution

Sampling time:

72 h

Parent/product:

parent

Key result

Soil No.:

#3

Sampling date:

2012

% Degr.:

72.6

Parameter:

CO2 evolution

Sampling time:

72 h

Parent/product:

parent

Key result

Soil No.:

#4

Sampling date:

2012

% Degr.:

71.2

Parameter:

CO2 evolution

Sampling time:

72 h

Key result

Soil No.:

#1

DT50:

7 h

Type:

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

Temp.:

20 °C

Key result

Soil No.:

#2

DT50:

14.8 h

Type:

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

Temp.:

20 °C

Key result

Soil No.:

#3

DT50:

18 h

Type:

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

Temp.:

20 °C

Key result

Soil No.:

#4

DT50:

18.2 h

Type:

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

Temp.:

20 °C

Transformation products:

yes

Remarks:

minor transient metabolites

Details on transformation products:

- Formation and decline of each transformation product during test: After representing between 78.1% and 90.3 % AR on Day 0, the concentration of [14C] Thiourea rapidly decreased in all soils to 2.8 % (soil I), 2.9 % (soil II), below limit of detection (soil III) and 3.7 % (soil VI) after 72 hours of being in contact with soil. Up to four minor transient metabolites were formed, one of them being urea (M1). The mean concentration of M1 was slightly above 5 % in two soils at one single time point (soil III and soil IV with 5.3 % at 2 hours of incubation). The mean concentration of all other metabolites was not > 4.3 % of applied at any time.

Evaporation of parent compound:

no

Volatile metabolites:

no

Remarks:

The mineralisation of [14C]Thiourea was rapid and significant in all four soils tested. 72 hours after application, 14CO2 reached maximum mean levels of 77.5%, 75.0%, 72.6% and 71.2% AR for soils I to IV, respectively.

Residues:

yes

Remarks:

On Day 0, the amount of bound residues was 2.1 %, 3.3 %, 2.6 % and 2.9 % for soils I to IV, respectively. Thereafter, the amount of bound residues increased to values of 8.8 %, 13.2 %, 16.0 % and 14.1 % for the respective soils. Harsh extractions using 0.

Details on results:

TEST CONDITIONS
- Aerobicity, moisture, temperature and other experimental conditions maintained throughout the study: Yes

MINOR TRANSFORMATION PRODUCTS
Up to four minor transient metabolites were formed, one of them being urea (M1). The mean concentration of M1 was slightly above 5% in two soils at one single time point (soil I I I and soil IV with 5.3% at 2 hours of incubation).The mean concentration of all other metabolites was not >4.3% of applied at any time.

NON-EXTRACTABLE RESIDUES
On Day 0, the amount of bound residues was 2.1 %, 3.3 %, 2.6 % and 2.9 % for soils I to IV, respectively. Thereafter, the amount of bound residues increased to values of 8.8 %, 13.2 %, 16.0 % and 14.1 % for the respective soils. Harsh extractions using 0.01N hydrochloric acid were performed for soil samples of soil II (Bad Lauchstädt), soil I I I (Priesteblich) and soil IV (Speyer 2.4), as the amount of nonextracted radioactivity exceeded 10% of the radioactivity applied after 72 hours of incubation. Maximum amounts of radioactivity of 1.9%, 2.6% and 2.5% of applied were released.

MINERALISATION
- % of applied radioactivity present as CO2 at end of study: After 72 hours of incubation, [14]CO2 reached mean levels of 71.2 to 77.5% AR.

VOLATILIZATION
- % of the applied radioactivity present as volatile organics at end of study: volatile organics other than CO2 were not detected

Results with reference substance:

not specified

Table: Summary of derived DT50 and DT90 values

Soil no.	Soil type	[^14C]thiourea
		DT50 [hours]	DT90 [hours]
Soil I	sandy loam	7.0 (0.3 days)	23.2 (1.0 day)
Soil II	silt loam	14.8 (0.6 days)	49.3 (2.1 days)
Soil III	loam	18.0 (0.8 days)	59.7 (2.5 days)
Soil IV	loam	18.2 (0.8 days)	60.3 (2.5 days)

 

Supplementary information is attached to this endpoint study record.

Conclusions:

In a GLP-compliant aerobic transformation test according to OECD 307 [14C]Thiourea degraded very fast with DT50 values between 7.0 and 18.2 hours. After 72 hours of incubation, [14]CO2 reached mean levels of 71.2 to 77.5% AR.

Executive summary:

The biodegradation of thiourea was additionally assessed in a GLP-compliant study on Aerobic Transformation in Soil according to OECD TG 307 (adopted April 24, 2002) and Directive 98/8/EC. The degradation of radiolabeled [¹⁴C]thiourea was followed in four soils of confirmed origin characterized as sandy loam (soil I), silt loam (soil II), loam (soils III and IV). The soils were treated with the test item at a rate of 10 mg/kg wet soil and incubated for 72 hours. This application rate corresponds to an application rate of 8.5 kg test item/ha when assuming an even distribution of the test item in the top 5 cm soil layer and a soil bulk density of 1.7 g/cm³. The treated soil samples were incubated at 20.4 ± 0.1 °C in the dark under continuous ventilation with moistened air. The exiting air was passed through a trapping system consisting of flasks of sodium hydroxide and ethylene glycol in series. Prior to treatment and at the end of the incubation period, the microbial biomass was determined for each soil, showing that the soils were viable during the study. Two replicates treated with the test item were taken immediately after treatment (Day 0) and after 2, 4, 6, 15, 24 and 72 hours of incubation. Each replicate was submitted to extraction with water at room temperature for two to four times followed by Soxhlet extraction using acetonitrile/water (4:1; v/v). The applied extraction method is a standard method of extraction from solid samples which has been proven for 150 years (ECHA, 2021). The individual extracts were combined, and the pooled extracts concentrated. The concentrated extracts were then analyzed for the test item and degradation products by HPLC. Selected samples were also submitted to TLC analysis. Immediately after treatment (Day 0), 95.0 %, 84.3 %, 91.3 % and 88.3 % AR could be extracted with water from soils I to IV, respectively, at room temperature. Thereafter, the total amount of extractable radioactivity rapidly decreased over time in all four soils. At the last sampling date (72 hours after application), the total extractable radioactivity amounted to 4.1 %, 3.6 %, 3.9 % and 4.7 % AR for soils I to IV, respectively. The amount of radioactivity recovered by Soxhlet extraction was low and reached maximum amounts of 2.1 % (soil I, 6 hours), 1.7 % (soil II, 72 hours), 2.7 % (soil III, 15 hours) and 1.3 % (soil VI, 24 and 72 hours) AR. On Day 0, the amount of bound residues was 2.1 %, 3.3 %, 2.6 % and 2.9 % for soils I to IV, respectively. Thereafter, the amount of bound residues increased to values of 8.8 %, 13.2 %, 16.0 % and 14.1 % for the respective soils.  After 72 hours of incubation, between 40.7 % and 58.1 % of non-extractable radioactivity was associated with the immobile fractions (humic acids and humin). The radioactivity associated with the fulvic acids was 53.9, 59.3, 52.3 % and 41.9 % of non-extractable radioactivity for soils I to IV, respectively. The humic acids and humin fractions recovered in total 4.1 %, 5.4 %, 7.6 % and 8.2 % AR for soils I to IV, respectively. The mineralization of [¹⁴C]thiourea was rapid and significant in all four soils tested. 72 hours after application, CO₂ reached maximum mean levels of 77.5 %, 75.0 %, 72.6 % and 71.2 % AR for soils I to IV, respectively. No other volatile products were detected. After representing between 78.1 % and 90.3 % AR on Day 0, the concentration of [¹⁴C]thiourea rapidly decreased in all soils to 2.8 % (soil I), 2.9 % (soil II), below limit of detection (soil III) and 3.7 % (soil VI) after 72 hours of being in contact with soil. Up to four minor transient metabolites were formed, one of them being urea (M1). The mean concentration of M1 was slightly above 5 % in two soils at one single time point (soil III and soil IV with 5.3 % at 2 hours of incubation). The mean amount of all other metabolites was never above 4.4 % of applied radioactivity at any time.

 

The calculated DT₅₀ (half-live) and DT₉₀ values for [¹⁴C]thiourea, based on single-first order kinetics (SFO), are shown in the table below:

 

Soil no.	Soil type	[^14C]thiourea
		DT50 [hours]	DT90 [hours]
Soil I	sandy loam	7.0 (0.3 days)	23.2 (1.0 day)
Soil II	silt loam	14.8 (0.6 days)	49.3 (2.1 days)
Soil III	loam	18.0 (0.8 days)	59.7 (2.5 days)
Soil IV	loam	18.2 (0.8 days)	60.3 (2.5 days)

 

In conclusion, [¹⁴C]thiourea degraded very fast in all four soils with DT₅₀ values between 7.0 and 18.2 hours. The main degradation pathway of [¹⁴C]thiourea in soil proceeded through formation of minor transient metabolites, mineralization, and formation of low amounts of bound residues. The mineralization of [¹⁴C]thiourea was extremely high in all four soils tested. After 72 hours of incubation, ¹⁴CO₂ reached mean levels of 77.5 %, 75.0 %, 72.6 % and 71.2 % AR for soils I to IV, respectively. No other volatile products were formed. The amount of bound residues increased during incubation to mean values of 8.8 %, 13.2 %, 16.0 % and 14.1 % AR for soils I to IV, respectively, at the last sampling date.

Reference 4

Endpoint:

biodegradation in soil: simulation testing

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

no data

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

equivalent or similar to guideline

Guideline:

other: no guideline reported

Principles of method if other than guideline:

Pure thiourea was added to two soils used in batch microcosm experiments to determine loss rates under controlled conditions.
Two soils (previously unexposed to industrial chemicals or waste) with different characteristics were used in the microcosm studies to obtain representative treatability data:
- Acid soil with low organic content;
- Basic soil with higher organic content and cation exchange capacity (CEC).
Soils were air dried, sieved, and stored at 4 °C in the dark. For the microcosm experiment both soils were used with a moisture content near saturation and the indigenous organisms to allow for the establishment of equilibrium concentrations.
Thiourea loss rates were determined by measurement of the test substance concentration over time. The specific chemical loss mechanism was not evaluated in this study. Therefore, the loss rates could have been due to: biodegradation, chemical degradation, hydrolysis, and volatilisation. Based on experience with these soils and the protocols used, the authors estimate that the major loss mechanism was biodegradation. However, abiotic controls to measure abiotic losses were part of the protocol. (Please note: it is assumed that the author's supposition is correct as thiourea is not volatile, does not hydrolyse and has a low potential for adsorption.)
The loss rate was determined by measuring the difference between the initial thiourea concentration added to the soil and that recovered after specified time intervals.

GLP compliance:

not specified

Test type:

laboratory

Radiolabelling:

no

Oxygen conditions:

aerobic

Soil classification:

not specified

Soil no.:

#1

Soil type:

sandy loam

% Clay:

8.6

% Silt:

23.4

% Sand:

68

% Org. C:

0.94

pH:

4.8

CEC:

6.35

Soil no.:

#2

Soil type:

other: Sandy silt loam

% Clay:

7.4

% Silt:

31.1

% Sand:

61.5

% Org. C:

3.25

pH:

7.8

CEC:

10.8

Details on soil characteristics:

SOIL COLLECTION AND STORAGE
- Geographic location:
* Sandy loam (acid soil): area near Wiggins, Mississippi
* Sandy silt loam: area near Austin, Texas
- Pesticide use history at the collection site: soils were not previously exposed to industrial chemicals or waste)
- Collection procedures:
* Sandy loam (acid soil): no data, soil was suppplied by researchers at Mississippi State University
* Sandy silt loam (basic soil): no data
- Sampling depth (cm):
* Sandy loam (acid soil): no data, soil was suppplied by researchers at Mississippi State University
* Sandy silt loam (basic soil): no data
- Storage conditions: 4 °C in the dark
- Storage length: no data
- Soil preparation: sieved and air dried

Soil No.:

#1

Duration:

64 d

Soil No.:

#2

Duration:

64 d

Soil No.:

#1

Initial conc.:

100 mg/kg soil d.w.

Based on:

test mat.

Soil No.:

#2

Initial conc.:

660 mg/kg soil d.w.

Based on:

test mat.

Parameter followed for biodegradation estimation:

test mat. analysis

Soil No.:

#1

Temp.:

20 °C

Humidity:

80 % of field capacity (= total moisture content of about 16%)

Soil No.:

#2

Temp.:

20 °C

Humidity:

80 % of field capacity (= total moisture content of about 12%)

Details on experimental conditions:

1. PRELIMINARY EXPERIMENTS:
1.1 Determination of the relative toxicity of thiourea:
- Microtox (Microbics Corp., Carlsbad, CA) experiment to determine the relative toxicity of thiourea in order to establish nontoxic chemical application concentrations (loading rates).
- Photobacterium phosphoreum was exposed to different concentrations of thiourea and the reduction of chemical luminescence after 5 min exposure was measured to determine the EC50 (Microtox Model 2055 Toxicity Analyzer System; proceudres as indicated in the Microtox System Operating Manual (Beckman Instruments, 1982)) .

1.2 Determination of chemical loading rates:
- Chemical-soil mixtures were prepared at different chemical concentrations.
- The water-soluble fraction (WSF) for each mixture was obtained using the following procedure:
* Deionized (DD) water (50 mL) was added to each chemical-soil (10 g d.w.) mixture in capped jars. Each jar was sealed with a gas tight cap, and placed on a shaker apparatus.
* The shaker apparatus was operated at 250 rpm for 1 h. Subsequently, the reactors were allowed to settle for 15-30 min.
* The supernatant was decanted from the reactors and re-centrifuged at 5000 rpm for 30 min.
* After that the supernatant was filtered through a 0.45-mm pore size filter. The filtrate was stored at 4 °C until analysis.
- The EC50 was determined for the WSF of thiourea.
- The relative toxicity units [TU] were calculated using the following equation: TU = 400/EC50.
- A log-log plot of TU values vs. loading rate was prepared; the interception point for 20 toxicity units (20 TU) is the desired loading limit for the soil. Because there can be a window of acceptable loading rates, a value of twice this amount was identified as the upper limit for the acceptable window.

2. EXPERIMENTAL DESIGN
- Soil pre-incubation conditions: soil samples were adjusted to 80 % field capacity and stored for 10 days at 20 °C in the dark to allow soil microorganisms to equilibrate to experimental conditions
- Soil condition: air dried
- Soil (g/replicate): 10 g
- Control conditions, if used: blank control
- Eight sample sets (one for each sampling point) with four beakers each.
- No. of replication controls, if used: 1 blank control per sample set
- No. of replication treatments: 3 replicates per sample set
- Test apparatus (Type/material/volume): 150 mL beakers
- Details of traps for CO2 and organic volatile, if any: Not applicable
- If no traps were used, is the system closed/open: Open (no closed system necessary due to monitoring by chemical analysis of the test substance)

Test material application
- Volume of test solution used/treatment: 100 µl
- Application method: 100 µl of test solution were added to the soil with a 20 µl pipette; the test solution was distributed to 5 points on the soil surface; subsequently soil and chemical solution were thoroughly mixed.
- Any indication of the test material adsorbing to the walls of the test apparatus: no

Experimental conditions
- Moisture maintenance method: yes, weekly adjustment to 80 % field capacity
- Continuous darkness: Yes

3. OXYGEN CONDITIONS
- Methods used to create the aerobic conditions: test vessels were only loosely covered with aluminium foil

4. SAMPLING DETAILS
- Sampling intervals: 0, 2, 4, 8, 16, 24, 32, and 64 days
- Sampling method for soil samples: whole sample sets were sacrificed at each sampling point
- The experiment ended when experimental data show ed that the thiourea concentration remaining in the soil is below the HPLC detection limit.

Key result

Soil No.:

#1

DT50:

18.7 d

Type:

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

Remarks on result:

other: r² = 0.45; 95 % C.I.: -8.3 to +76.2

Soil No.:

#2

DT50:

12.8 d

Type:

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

Remarks on result:

other: r² = 0.86; 95 % C.I.: 9.9-18.0

Transformation products:

not measured

Details on transformation products:

Not applicable as transformation products were not measured.

Evaporation of parent compound:

not measured

Volatile metabolites:

not measured

Residues:

not specified

Details on results:

No further details available.

Results with reference substance:

Not applicable.

Conclusions:

The half-life of thiourea biodegradation in acidic and basic soil under the conditions of the test was determined at 18.7 days and 12.8 days, respectively.

Executive summary:

The loss of pure thiourea in two soils was evaluated in microcosm experiments under controlled conditions (20 °C, moisture: 80 % of field capacity). The test soils were previously not exposed to industrial chemicals or waste and had different characteristics with regard to texture, organic content and pH:

- Acid soil with low organic content

- Basic soil with higher organic content and cation exchange capacity (CEC)

The initial test substance concentration in the acidic and basic soil was 100 mg/kg soil dw and 660 mg/kg soil dw, respectively. In a pre-experiment these concentrations were determined to have no toxic effects on the microorganisms present in the soil samples.

The soils were air dried, sieved, and stored at 4 °C in the dark. For the microcosm experiment both soils were moistened to near saturation and the indigenous organisms allowed establishing equilibrium concentrations. Thiourea loss rates were attributed to the biodegradation of the substance and were determined by measurement of the test substance concentration over time (sampling after 0, 2, 4, 8, 16, and 64 days). The loss rate was determined by measuring the difference between the initial thiourea concentration added to the soil and that recovered after specified time intervals.

First and zero-order rate constants were determined from a least-squares fit to the data. Standard deviation and Student's t-statistic were computed from the available data in order to estimate 95 % confidence intervals for the parameters of interest. The half-life of thiourea biodegradation in acidic and basic soil under the conditions of the test was determined to be 18.7 and 12.8 days, respectively.

Description of key information

In a GLP-compliant aerobic transformation test according to OECD 307 the degradation half-life of the substance was determined to be 0.6 days (13.6 hours, geometric mean, at 20 °C).

Key value for chemical safety assessment

Half-life in soil:

0.6 d

at the temperature of:

20 °C

Additional information

Experimental data on the biodegradation of thiourea in soil is available. Results from simulation studies were published by Frederick et al. (1957), Lashen and Starkey (1970) and Loehr and Matthews (1992). This information had been submitted in the EU in accordance with Regulation (EC) No 1907/2006 (REACH). However, considering current methods and criteria the quality of the data is not regarded acceptable for assessment anymore due to methodological deficiencies and insufficient documentation. Therefore, a new valid and GLP-compliant guideline study was carried out (Voelkel, 2013) to support the authorization of the product “ALZOGUR” under BPR. This study was not included in the dossiers previously submitted in the EU under Regulation (EC) 1907/2006 (REACH) and Regulation (EC) 528/2012 (BPR). A brief overview about the key information of the available studies is given in the table below.

 

Table: Available studies on degradation of thiourea in soil

Test guideline	GLP	Reliability	Initial test item concentration	Key result	Reference
non-guideline	no	Rel 3	0.02 and 0.1 M	22 % degradation after 1 week (0.02 M)   28 % degradation after 15 weeks (0.1 M)	Lashen and Starkey (1970)
non-guideline	no	Rel 4	10 g/kg soil dw	slight degradation	Frederick et al. (1957)
non-guideline	no	Rel 3	100 mg/kg soil dw and 660 mg/kg soil dw	DT50: 12.8 to 18.7 d	Loehr and Matthews (1992)
OECD 307	yes	Rel 1	10 mg /kg soil ww	DT50: 7.0 to 18.2 h	Voelkel (2013)

 

Key information

The biodegradation of thiourea was assessed in a new GLP-compliant study on Aerobic Transformation in Soil according to OECD TG 307 (adopted April 24, 2002) and Directive 98/8/EC. The degradation of radiolabeled [¹⁴C]thiourea was followed in four soils of confirmed origin characterized as sandy loam (soil I), silt loam (soil II), loam (soils III and IV). The soils were treated with the test item at a rate of 10 mg/kg wet soil and incubated for 72 hours. This application rate corresponds to an application rate of 8.5 kg test item/ha when assuming an even distribution of the test item in the top 5 cm soil layer and a soil bulk density of 1.7 g/cm³. The treated soil samples were incubated at 20.4 ± 0.1 °C in the dark under continuous ventilation with moistened air. The exiting air was passed through a trapping system consisting of flasks of sodium hydroxide and ethylene glycol in series. Prior to treatment and at the end of the incubation period, the microbial biomass was determined for each soil, showing that the soils were viable during the study. Two replicates treated with the test item were taken immediately after treatment (Day 0) and after 2, 4, 6, 15, 24 and 72 hours of incubation. Each replicate was submitted to extraction with water at room temperature for two to four times followed by Soxhlet extraction using acetonitrile/water (4:1; v/v). The applied extraction method is a standard method of extraction from solid samples which has been proven for 150 years (ECHA, 2021). The individual extracts were combined, and the pooled extracts concentrated. The concentrated extracts were then analyzed for the test item and degradation products by HPLC. Selected samples were also submitted to TLC analysis. Immediately after treatment (Day 0), 95.0 %, 84.3 %, 91.3 % and 88.3 % AR could be extracted with water from soils I to IV, respectively, at room temperature. Thereafter, the total amount of extractable radioactivity rapidly decreased over time in all four soils. At the last sampling date (72 hours after application), the total extractable radioactivity amounted to 4.1 %, 3.6 %, 3.9 % and 4.7 % AR for soils I to IV, respectively. The amount of radioactivity recovered by Soxhlet extraction was low and reached maximum amounts of 2.1 % (soil I, 6 hours), 1.7 % (soil II, 72 hours), 2.7 % (soil III, 15 hours) and 1.3 % (soil VI, 24 and 72 hours) AR.

On Day 0, the amount of bound residues was 2.1 %, 3.3 %, 2.6 % and 2.9 % for soils I to IV, respectively. Thereafter, the amount of bound residues increased to values of 8.8 %, 13.2 %, 16.0 % and 14.1 % for the respective soils.  After 72 hours of incubation, between 40.7 % and 58.1 % of non-extractable radioactivity was associated with the immobile fractions (humic acids and humin). The radioactivity associated with the fulvic acids was 53.9, 59.3, 52.3 % and 41.9 % of non-extractable radioactivity for soils I to IV, respectively. The humic acids and humin fractions recovered in total 4.1 %, 5.4 %, 7.6 % and 8.2 % AR for soils I to IV, respectively. The mineralization of [¹⁴C]thiourea was rapid and significant in all four soils tested. 72 hours after application, CO₂ reached maximum mean levels of 77.5 %, 75.0 %, 72.6 % and 71.2 % AR for soils I to IV, respectively. No other volatile products were detected. After representing between 78.1 % and 90.3 % AR on Day 0, the concentration of [¹⁴C]thiourea rapidly decreased in all soils to 2.8 % (soil I), 2.9 % (soil II), below limit of detection (soil III) and 3.7 % (soil VI) after 72 hours of being in contact with soil. Up to four minor transient metabolites were formed, one of them being urea (M1). The mean concentration of M1 was slightly above 5 % in two soils at one single time point (soil III and soil IV with 5.3 % at 2 hours of incubation). The mean amount of all other metabolites was never above 4.4 % of applied radioactivity at any time.

 

The calculated DT₅₀ (half-live) and DT₉₀ values for [¹⁴C]thiourea, based on single-first order kinetics (SFO), are shown in the table below:

 

Soil no.	Soil type	[^14C]thiourea
		DT50 [hours]	DT90 [hours]
Soil I	sandy loam	7.0 (0.3 days)	23.2 (1.0 day)
Soil II	silt loam	14.8 (0.6 days)	49.3 (2.1 days)
Soil III	loam	18.0 (0.8 days)	59.7 (2.5 days)
Soil IV	loam	18.2 (0.8 days)	60.3 (2.5 days)

 

In conclusion, [¹⁴C]thiourea degraded very fast in all four soils with DT₅₀ values between 7.0 and 18.2 hours. The main degradation pathway of [¹⁴C]thiourea in soil proceeded through formation of minor transient metabolites, mineralization, and formation of low amounts of bound residues. The mineralization of [¹⁴C]thiourea was extremely high in all four soils tested. After 72 hours of incubation, ¹⁴CO₂ reached mean levels of 77.5 %, 75.0 %, 72.6 % and 71.2 % AR for soils I to IV, respectively. No other volatile products were formed. The amount of bound residues increased during incubation to mean values of 8.8 %, 13.2 %, 16.0 % and 14.1 % AR for soils I to IV, respectively, at the last sampling date.

 

Other and disregarded information

The biodegradation of thiourea in soil was studied by Lashen and Starkey (1970) in a 15-weeks simulation test with barnyard soil (200 g) at initial test item concentrations of 0.02 and 0.1 M. The experiment was not carried out in accordance with an official test guideline and was not performed in compliance with the GLP principles. Little information is provided about the applied method. In this test an amount of 0.5 % glucose was added to the soil samples without (control) and with thiourea. The moisture content of the soil was brought to 50 % of field capacity. Water was added regularly to account for losses through evaporation. The soil samples were incubated at 28 °C for 15 weeks. The production of sulphate served as indicator for the decomposition of thiourea. For the analysis of sulphate production soil samples were extracted following a method described by Chesnin & Yien (1950) and Bartlett & Neller (1960). The sulphate content was measured colometrically. As a result, the biodegradation of thiourea was inhibited at higher concentrations. At an initial concentration of 0.02 M thiourea 22 % of the test substance were degraded within one week, and 27 % were degraded after 2 weeks. Within 15 weeks 96 % of the S in thiourea was recovered as sulphate. In contrast, at an initial concentration of 0.1 M only 28 % of the thiourea-S was recovered as sulphate after 15 weeks. Based on the outcome of this experimental study, it could be assumed that the biodegradation of thiourea is inhibited at higher concentrations. However, the information is not regarded reliable for several reasons: the study was not performed according to an official test guideline and the OECD GLP principles. The test principle and analytics significantly deviate from state-of-the-art methods as proposed in OECD TG 307. It is further not possible to quantify the observations of biodegradation in the form of kinetic rate expressions as requested under Regulation (EC) No 1907/2006 (REACH). Essential information such as the purity of the test substance as well as details on the applied methods and results are not reported. Therefore, the data is classified Klimisch 3 (not reliable).

 

The biodegradation of thiourea in agricultural soil (sandy loam, pH 6.1) was also followed in a simulation test over 21 weeks at 28 °C, a field capacity of 40 %, and an initial thiourea concentration of 10 g/kg soil dw (Frederick et al., 1957). The experiment was not performed in accordance with an accepted test guideline and GLP principles. The origin of the used soil and its properties such as the amount of clay, silt, sand, org. carbon, CEC and bulk density are not reported. As a result, 9 % degradation (determined via formation of sulphate) was observed. Application of 0.5 % CaCO₃ and additional incubation for 21 weeks did not alter the degradation results. Incubation of soil suspensions with 5 g/kg thiourea with mineral medium at 28 °C for one week did not result in formation of sulphate. 1 % of thiourea was degraded when 1 g thiourea and 50 g soil were incubated with 150 ml perfusate for six weeks. In addition, bacterial and fungal populations were checked. The number of bacteria and actinomyces was reduced from several million per g untreated soil to several thousand per g treated soil. The number of fungus was reduced by > 99 %. In conclusion, under the conditions of the test thiourea was only slightly degraded in the soil samples at 10 g/kg soil dw after incubation after 21 to 42 weeks. The reliability of this information is not assignable since the study did not follow an official test guideline and GLP principles. Further, the information is taken from secondary literature and the documentation of the study data is hence insufficient for assessment. Therefore, the information is classified Klimisch 4 (not assignable).

 

In a publication of Loehr and Matthews (1992) the biodegradation of thiourea in non-guideline microcosm experiments is reported. The loss of pure thiourea in two soils was evaluated under controlled conditions (20 °C, moisture: 80 % of field capacity). The test soils were previously not exposed to industrial chemicals or waste and had different characteristics regarding texture, organic content, and pH: an acidic soil (sandy loam) with low organic content and a basic soil (sandy silt loam) with higher organic content and cation exchange capacity (CEC) were used. The soils were air dried, sieved, and stored at 4 °C in the dark. For the microcosm experiment both soils were moistened to near saturation and the indigenous organisms allowed establishing equilibrium concentrations. The initial test substance concentration in the acidic and basic soil was 100 mg/kg soil dw and 660 mg/kg soil dw, respectively. 100 µl of test solution (test substance in methylene chloride) were added to the soil with a 20 µl pipette. The test vessels were only loosely covered with aluminum foil and CO₂ and organic volatile traps were not used. The test solution was distributed to 5 points on the soil surface; subsequently soil and chemical solution were thoroughly mixed. In a pre-experiment (Microtox test; Microbics Corp., Carlsbad, CA) these concentrations were determined to have no toxic effects on the microorganisms present in the soil samples. Thiourea loss rates were attributed to the biodegradation of the substance and were determined by measurement of the test substance concentration over time (sampling after 0, 2, 4, 8, 16, and 64 days). The loss rate was determined by measuring the difference between the initial thiourea concentration added to the soil and that recovered after specified time intervals. Soil samples were extracted with a Soxhlet apparatus for 16 h using methanol as solvent. The extracts were concentrated using a Kuderna-Danish extraction unit attached to a three-ball Snyder column (method 3540) (U.S. Environmental Protection Agency, 1986). Extracts were dried by passing through disposable sodium sulfate columns and then refrigerated at 4 °C until analysis. For the quantification of the parent compound a HPLC-UV method was applied. To determine the chemical concentration that actually remained in the soil, recovery efficiency data for thiourea were acquired. Recovery efficiency-corrected thiourea concentration was the basis for the calculation of the kinetic relationships and constants. Recovery efficiency for thiourea was 93 % for the acidic soil #1 and 88 % for the basic soil #2. First and zero-order rate constants were determined from a least-squares fit to the data. Standard deviation and Student's t-statistic were computed from the available data to estimate 95 % confidence intervals for the parameters of interest. The half-life of thiourea biodegradation in acidic and basic soil under the conditions of the test was determined to be 18.7 ((pseudo-)first order; r² = 0.45; 95 % C.I.: -8.3 to +76.2) and 12.8 days ((pseudo-)first order; r² = 0.86; 95 % C.I.: 9.9 to 18.0), respectively. Taken together, the experimental data is considered reliable and acceptable for assessment since the test procedure is in accordance with generally accepted scientific standards and described in sufficient detail. The information was classified Klimisch 1 (reliable without restriction) for the submission under Regulation (EC) No 1907/2006 (REACH). However, the specific chemical loss mechanism was not evaluated in this study. Therefore, the loss rates could have been due to biodegradation, chemical degradation, hydrolysis, and volatilization. Based on experience with these soils and the protocols used, the authors just estimated that the major loss mechanism was biodegradation. ditionally, the test substance was applied to the soil matrix using methylene chloride (= dichloromethane) as a solvent. In section 19 of the OECD Test Guideline 307 on test substance application it is explicitly stated that the use of vehicles which are known to inhibit the microbial activity, such as methylene chloride, “should be avoided”. Kanazawa and Filip (1986) determined the 28-day NOEC of methylene chloride for soil microorganisms to be 0.1 mg/kg soil dw. In the study of Loehr and Matthews (1992) the solvent was placed at a rate of 100 µL to 10 g of air-dried soil per treatment. Hence, the use of the solvent methylene chloride may have inhibited the microbial activity and thus the degradation of thiourea. Further, it was not specified whether the study was performed in accordance with the OECD GLP or comparable quality principles. Based on this information uncertainties remain regarding the biodegradation and the environmental fate of the substance. The study of Loehr and Matthews (1992) is thus regarded not reliable due to significant methodological deficiencies (Klimisch 3).

 

Conclusion:

For the assessment of the biodegradability of thiourea in soil under REACH Regulation, the information given in the publication of Loehr and Matthews (1992, reliability 3) was submitted as key information. Two supporting studies with reliability scores of 3 and 4 (Lashen and Starkey, 1970; BUA Stoffbericht 179 citing Frederick et al., 1957) are available but were not considered acceptable for assessment. Overall, the results obtained by Voelkel (2013) are most reliable, and a degradation half-life of 0.6 days (13.6 hours, geometric mean, at 20 °C) is considered relevant and conservative for the risk assessment. This conclusion is further expatiated in an Expert Statement on the biodegradation of thiurea in soil (SCC GmbH, 2021) presented in IUCLID section 5.2.4.