Urea

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Deviations:

not applicable

Remarks:

2 instead of 4 replicates used, adult worms left in the containers after day 30 until the end of the test on day 60.

GLP compliance:

not specified

Remarks:

No information on GLP compliance provided.

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

Earthworms (E. foetida) were procured from the vermicomposting unit of Rajasthan College of Agriculture, Udaipur. They were maintained in the laboratory conditions and acclimatized for 15 days. The worms used in the experiment were approximately same body weight and body length.

Study type:

laboratory study

Substrate type:

natural soil

Limit test:

no

Total exposure duration:

60 d

Moisture:

70 percent moisture level water

Details on test conditions:

Plastic tubs were used for preparations of soil beds. Dried soil (from nearby farmland) was crushed and filtered through a fine mesh sieve. One kg of fine soil was then poured in each plastic tub and then water was added to moistened the soil then 250gm dried powdered (3 week old) cow dung was also added to each plastic tub to avoid starvation.

TEST SYSTEM
- Test container (material, size): plastic tub
- Amount of soil or substrate: 1 kg of soil and cow dung mixture made in the ratio of 1:1.
- No. of organisms per container (treatment): 20
- No. of replicates per treatment group: 3
- No. of replicates per control: 3
- No. of replicates per vehicle control: NA


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Biomass and mortality on days 0, 15, 30, 45 and 60.

VEHICLE CONTROL PERFORMED: no

TEST CONCENTRATIONS
The Urea dose being practically applied in the local agricultural lands for the Kharif crop was found to be 174 kg/ hectare of land area. Here, in our experimental set up the soil bed contained 1 kg of soil and cow dung mixture made in the ratio of 1:1. Therefore, the calculated value of Urea for the soil bed was 3.48gm/ kg of soil. In addition to the dose being practiced by the farmers i.e. 3.48gm/ kg, three more doses of Urea were set viz. 0.75gm/ kg, 1.5gm/ kg, and 2.25gm/ kg.

Nominal and measured concentrations:

Control, 750, 1500, 2250 and 3480 mg/kg

Reference substance (positive control):

no

Duration:

30 d

Dose descriptor:

LC50

Effect conc.:

3 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: calculated with the results from 1500 and 2250 mg/kg dw since the 750 and 3480 mg /kg dw resulted in 0 and 100 % mortality respectively.

Duration:

30 d

Dose descriptor:

EC10

Effect conc.:

520 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Remarks on result:

other: calculated with the results from the 750, 1500 and 2250 mg/kg dw groups, the 3480 mg/kg dw group was not used since there was 100 % mortality within 24 hours.

Duration:

30 d

Dose descriptor:

EC50

Effect conc.:

1 437 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Remarks on result:

other: calculated with the results from the 750, 1500 and 2250 mg/kg dw groups, the 3480 mg/kg dw group was not used since there was 100 % mortality within 24 hours.

Key result

Duration:

60 d

Dose descriptor:

EC10

Effect conc.:

160 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: calculated with the results from the 750, 1500 and 2250 mg/kg dw groups, the 3480 mg/kg dw group was not used since there was no reproduction.

Duration:

60 d

Dose descriptor:

EC50

Effect conc.:

921 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: calculated with the results from the 750, 1500 and 2250 mg/kg dw groups, the 3480 mg/kg dw group was not used since there was no reproduction.

Details on results:

- Mortality at end of exposure period:
0, 0, 15, 30 and 100% after 30 days in the control and 750, 1500, 2250 and 3480 mg/kg dw
0, 0, 15, 35 and 100% after 60 days in the control and 750, 1500, 2250 and 3480 mg/kg dw

- Total mass of adults at beginning of test: 3.96 g at start in the control
- Changes in body weight of live adults (% of initial weight) at end of exposure period:
95, 73, 57, 22 % after 30 days in the control and 750, 1500, 2250 mg/kg dw. At 3480 mg/kg dw all worms were dead and hence no biomass was available.

- No. of offspring produced:
57, 32, 21, 14 and 0 juveniles after 60 days in the control and 750, 1500, 2250 and 3480 mg/kg dw

- No. of unhatched cocoons:
not reported
The number of cocoons was highest in the control set and least in urea dose of 2250 mg/kg dw.(the 3480 mg/kg dw was not considered here since all adult worms were dead. See fig. 2 of the publication)

- Morphological abnormalities:
At the urea dose of 3480 mg/kg dw, the entire worms were found dead within 24hrs which is shown in the photograph. Shrinking of earthworm body and rupturing of the epidermis and cuticle with the secretion of yellowish fluid was observed in this dose of urea. Least number of earthworms i.e. 13 were left in the soil bed treated with urea dose of 2250 mg/kg dw at the end of experiment. Although, these number of worms were alive in this experimental set up but the worm‘s body was found weakened with less body weight.

- Behavioural abnormalities:
At 2250 mg/kg dw the worms tried to escape out from the tub; (see fig.-3(c) of the publication).

Reported statistics and error estimates:

The EC10 and EC 50 values were calculated using an excel spreadsheet (attached) with log transformed concentrations and probit transformed % responses relative to the control.
Test groups with 0 and 100% responses were not used for the endpoint calculation.

Validity criteria fulfilled:

yes

Conclusions:

LC50 (30 day mortality): 3000 mg urea/kg dw
EC50 (30 day growth inhibition): 1437 mg/kg dw
EC10 (30 day growth inhibition): 520 mg/kg dw
EC50 (60 day reproduction): 921 mg urea/kg dw
EC10 (60 day reproduction): 160 mg urea/kg dw

Executive summary:

In the study from Rai et al (2014), the toxicity of urea on Eisenia fetida was investigated with a test similar to the OECD 222. The study is well described and results are provided in sufficient detail. Major deviations to OECD 222 were that 2 instead of 4 replicates were used and adult worms were left in the containers after day 30 until the end of the test on day 60.

The study was performed with a control, 750, 1500, 2250 and 3480 mg urea/kg dw.

In the study, the endpoints were not calculated. However, based on the provided data, the following endpoints could be derived from the data.

LC50 (30 day mortality): 3000 mg urea/kg dw

EC50 (30 day growth inhibition): 1437 mg/kg dw

EC10 (30 day growth inhibition): 520 mg/kg dw

EC50 (60 day reproduction): 921 mg urea/kg dw

EC10 (60 day reproduction): 160 mg urea/kg dw

The results from this study are considered relevant and sufficient reliable for the risk assessment.

Reference 2

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

2 instead of 4 replicates

Qualifier:

according to guideline

Guideline:

OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

Deviations:

yes

Remarks:

2 instead of 4 repicates

Principles of method if other than guideline:

This study was carried out in laboratory of Zoology Department - DDE Wing, Annamalai University. In this study two different artificial soils had been identified for laboratory tests. One is the standard OECD artificial soil and the other is the modified Tropical Artificial Soil (TAS) incorporating coir pith as organic matter. The preparation of the main test substrate for toxicity studies, artificial soil, was based on an international guideline published by OECD (1984a) and the modified Tropical Artificial Soil (TAS) was prepared by following the same guideline substituting the coir pith instead of sphagnum peat. The components of OECD artificial soil and modified TAS are depicted in Tables 1 & 2 of the publication (see attached document).

GLP compliance:

not specified

Remarks:

No information on GLP compliance provided.

Specific details on test material used for the study:

The test chemical urea was obtained from the Preliminary Agricultural Co-operative Bank, Annamalai Nagar, Tamilnadu, India.

Vehicle:

yes

Remarks:

Deionized water

Details on preparation and application of test substrate:

Urea was mixed in different concentrations in the soil substrate and was applied only once at the beginning of the test. The preparation of the test solutions was done for each concentration by weighing and dissolving an amount of urea in deionized water. Deionized water was obtained using a Millipore Super-Q water purification system and was used throughout this study. All glass wares were washed with phosphate-free detergent, rinsed with acetone, and acid-washed before a final and thorough rinse with deionized water.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
Adult earthworms (with clitellum) with a fresh weight between 300 and 600 mg were used.

ACCLIMATION
In this work, mass cultures of E. foetida were established in laboratory from samples provided from the vermicomposting unit of Annamalai University for temperate and tropical artificial soils in plastic boxes (25 x 36 cm area, 12 cm height) (Figure 2). The temperate culture of Eisenia foetida was kept in a mixture of OECD soil at room temperature of 15 - 30 °C (mean: 22 °C). The tropical culture was kept in a mixture of tropical artificial soil (TAS) at room temperature 23 - 30 ºC (mean = 26 ºC), relative air humidity near 90%. The animals were maintained in a shaded place with a natural light cycle (12h light/12h dark). The breeding substrates were maintained
with a pH-value set to 6 ± 0.5. The moisture was adjusted to 50 ± 10% dry weight using deionized water. The earthworms were fed according to demand, usually once a week, with finely ground cattle manure free of any chemical contamination.
According to the calculations based on our results (Table 5), the LC50 of the tested earthworms after a 14-day exposure to urea was 1107.59 mg/kg. Similar LC50 values were recorded in Maroni et al. work (2002) (i.e. >1000mg). An acute 14 day study in the same laboratory resulted in an LC50 of 1080 mg/kg dw.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Test temperature:

20 °C (OECD), 28°C (tropical artificial soil, TAS)

pH:

The soil pH was adjusted to 6.0 ± 0.5 with calcium carbonate.

Moisture:

The moisture was adjusted to 50 ± 10% dry weight using deionized water.

Details on test conditions:

TEST SYSTEM
- Test container (material, size): glass vessels (12.5 cm diameter,
16 cm height)
- Amount of soil or substrate: filled with 500g (dw) soil
- No. of organisms per container (treatment): not explicitly provided in the publication (but since the OECD 222 was followed, and the validity criterion of 30 juveniles is cited, it can be assumed that 10 worms were used per test container.
- No. of replicates per treatment group: 2
- No. of replicates per control: 2
- No. of replicates per vehicle control: NA

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: artificial soils (OECD and TAS)
- Composition (if artificial substrate): see Table 3 and 4 of the original publication
- Organic carbon (%): 47.2% and 43.6 % for OECD and TAS soil, respectively.
- Maximum water holding capacity (in % dry weight): 56.1% and 48.5 % for OECD and TAS soil, respectively.
The breeding substrates were maintained with a pH-value set to 6 ± 0.5. The moisture was adjusted to 50 ± 10% dry weight using deionized water. The earthworms
were fed according to demand, usually once a week, with finely ground cattle manure free of any chemical contamination.

OTHER TEST CONDITIONS
The animals were maintained in a shaded place with a natural light cycle (12h light/12h dark).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mortality

VEHICLE CONTROL PERFORMED: no

Nominal and measured concentrations:

control, 500, 1000, 1500 and 2000 mg urea/kg dw

Reference substance (positive control):

no

Key result

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

417 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: OECD test (20°C)

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

1 018 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: OECD test (20°C)

Duration:

56 d

Dose descriptor:

EC10

Effect conc.:

409 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: Tropical artificial soil (28°)

Duration:

56 d

Dose descriptor:

EC50

Effect conc.:

1 177 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: Tropical artificial soil (26°C)

Details on results:

- Changes in body weight of live adults (% of initial weight) at end of exposure period: not reported
- No. of offspring produced:
OECD-test day 28: 118.4, 97.9, 78.0, 43.4 and 28.8 juveniles at control, 500, 1000, 1500 and 2000 mg urea/kg dw
OECD-test day 56: 103.1, 81.8, 69.5, 29.4 and 12.1 juveniles at control, 500, 1000, 1500 and 2000 mg urea/kg dw
TAS-test day 28: 122.6, 117.3, 87.6, 54.9 and 48.3 juveniles at control, 500, 1000, 1500 and 2000 mg urea/kg dw
TAS-test day 56: 122.3, 101.2, 81.2, 42.7 and 31.0 juveniles at control, 500, 1000, 1500 and 2000 mg urea/kg dw

The ECx values were calculated using log transformed concentrations and probit transformed % reproduction (relative to the corresponding controls). The calculation file is attached as background information.

In all of the experiments carried out in this study, there was a consistent and significant decrease in fecundity (number of surviving juveniles) at sublethal concentrations of urea in the spiked soil samples.

Since only the mean number of juveniles per concentration was reported, the EC10 and EC50 were calculated for OECD and TAS.

The mortality was reported for day 7 and day 14, but not for day 28 (Table 8 of the publication). Hence, the mortality was not considered for this evaluation

Validity criteria fulfilled:

yes

Conclusions:

EC10 Eisenia fetida 56 day reproduction (OECD 222): 417 mg/kg dw

Executive summary:

Abbiramy et al. (2012) investigated the acute toxicity of urea on Eisenia fetida according to OECD 222 (artificial soil). Since only 2 instead of 4 replicates were used, the study is considered to be Klimisch 2. All validity criteria were met. The 56-day EC10 for reproduction is 417 mg/kg dry weight. Mortality data of adults were not provided for day 28.

In addition to the OECD parameters, Abbiramy et al. (2012) investigated the toxicity of urea under increased temperature (28°C) with a slightly modified soil. The corresponding EC10 for reproduction was 409 mg/kg dw.

Beside the above mentioned shortcomings the results of the OECD study are considered relevant and reliable for the risk assessment. The results from the OECD test design are supported by the findings from the TAS test.

Hence, the EC10 of 417 mg/kg dw will be used for the risk assessment.

Reference 3

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

1 instead of 4 replicates

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Deviations:

yes

Remarks:

1 instead of 4 replicates

Principles of method if other than guideline:

This study was carried out in laboratory of Zoology Department - DDE Wing, Annamalai University. In this study two different artificial soils had been identified for laboratory tests. One is the standard OECD artificial soil and the other is the modified Tropical Artificial Soil (TAS) incorporating coir pith as organic matter. The preparation of the main test substrate for toxicity studies, artificial soil, was based on an international guideline published by OECD (1984a) and the modified Tropical Artificial Soil (TAS) was prepared by following the same guideline substituting the coir pith instead of sphagnum peat. The components of OECD artificial soil and modified TAS are depicted in Tables 1 & 2 of the publication (see attached document).

GLP compliance:

not specified

Remarks:

No information on GLP compliance provided.

Specific details on test material used for the study:

The test chemical urea was obtained from the Preliminary Agricultural Co-operative Bank, Annamalai Nagar, Tamilnadu, India.

Vehicle:

yes

Remarks:

Deionized water

Details on preparation and application of test substrate:

The preparation of the test solutions was done for each concentration by weighing and dissolving an amount of urea in deionized water. Deionized water was obtained using a Millipore Super-Q water purification system and was used throughout this study. All glass wares were washed with phosphate-free detergent, rinsed with acetone, and acid-washed before a final and thorough rinse with deionized water.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
Adult earthworms (with clitellum) with a fresh weight between 300 and 600 mg were used. The selected test animals were acclimatized in untreated soil substrates at least 24 hours prior to the start of the urea test.

ACCLIMATION
In this work, mass cultures of E. foetida were established in laboratory from samples provided from the vermicomposting unit of Annamalai University for temperate and tropical artificial soils in plastic boxes (25 x 36 cm area, 12 cm height) (Figure 2). The temperate culture of Eisenia foetida was kept in a mixture of OECD soil at room temperature of 15 - 30 °C (mean: 22 °C). The tropical culture was kept in a mixture of tropical artificial soil (TAS) at room temperature 23 - 30 ºC (mean = 26 ºC), relative air humidity near 90%. The animals were maintained in a shaded place with a natural light cycle (12h light/12h dark). The breeding substrates were maintained
with a pH-value set to 6 ± 0.5. The moisture was adjusted to 50 ± 10% dry weight using deionized water. The earthworms were fed according to demand, usually once a week, with finely ground cattle manure free of any chemical contamination.
According to the calculations based on our results (Table 5), the LC50 of the tested earthworms after a 14-day exposure to urea was 1107.59 mg/kg. Similar LC50 values were recorded in Maroni et al. work (2002) (i.e. >1000mg).

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

14 d

Test temperature:

20 °C (OECD), 28°C (tropical artificial soil, TAS)

pH:

The soil pH was adjusted to 6.0 ± 0.5 with calcium carbonate.

Moisture:

The moisture was adjusted to 50 ± 10% dry weight using deionized water.

Details on test conditions:

TEST SYSTEM
- Test container (material, size): glass vessels (12.5 cm diameter,
16 cm height)
- Amount of soil or substrate: filled with 500g (dw) soil
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 1
- No. of replicates per control: 1
- No. of replicates per vehicle control: NA

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: artificial soils (OECD and TAS)
- Composition (if artificial substrate): see Table 3 and 4 of the original publication
- Organic carbon (%): 47.2% and 43.6 % for OECD and TAS soil, respectively.
- Maximum water holding capacity (in % dry weight): 56.1% and 48.5 % for OECD and TAS soil, respectively.
The breeding substrates were maintained with a pH-value set to 6 ± 0.5. The moisture was adjusted to 50 ± 10% dry weight using deionized water. The earthworms
were fed according to demand, usually once a week, with finely ground cattle manure free of any chemical contamination.

OTHER TEST CONDITIONS
The animals were maintained in a shaded place with a natural light cycle (12h light/12h dark).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mortality

VEHICLE CONTROL PERFORMED: no

TEST CONCENTRATIONS
- Range finding study: yes, see below
- Test concentrations: 1000; 100; 10; 1; 0.1 mg of urea per kilogram of test substrate (dry weight)

Nominal and measured concentrations:

1000; 100; 10; 1; 0.1 mg of urea per kilogram of test substrate (dry weight)

Reference substance (positive control):

no

Key result

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

1 080 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: OECD test (20°C)

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

1 135 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Tropical artificial soil (28°C)

Validity criteria fulfilled:

yes

Conclusions:

LC50 Eisenia fetida 14 day: 1080 mg/kg dw

Executive summary:

Abbiramy et al. (2012) investigated the acute toxicity of urea on Eisenia fetida according to OECD 207 (artificial soil). Since only 1 instead of 4 replicates were used in this range finding test. The study is considered to be Klimisch 2. According to the authors all validity criteria were met. The LC50 determined during this test is 1080 mg/kg dry weight. Biomass decrease was reported.

In addition to the OECD parameters, Abbiramy et al. (2012) investigated the toxicity of urea under increased temperature (28°C) with a slightly modified soil. The corresponding LC50 was 1135 mg/kg dw.

The results of this study are considered relevant and reliable for the risk assessment. Since the OECD test design is relevant for the risk assessment, this value will be used. Furthermore, it is more conservative.

Reference 4

Endpoint:

toxicity to soil macroorganisms except arthropods: short-term

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

3 instead of 4 replicates

Qualifier:

according to guideline

Guideline:

OECD Guideline 207 (Earthworm, Acute Toxicity Tests)

Deviations:

yes

Remarks:

3 instead of 4 replicates

Principles of method if other than guideline:

This study was carried out in laboratory of Zoology Department - DDE Wing, Annamalai University. In this study two different artificial soils had been identified for laboratory tests. One is the standard OECD artificial soil and the other is the modified Tropical Artificial Soil (TAS) incorporating coir pith as organic matter. The preparation of the main test substrate for toxicity studies, artificial soil, was based on an international guideline published by OECD (1984a) and the modified Tropical Artificial Soil (TAS) was prepared by following the same guideline substituting the coir pith instead of sphagnum peat. The components of OECD artificial soil and modified TAS are depicted in Tables 1 & 2 of the publication (see attached document).

GLP compliance:

not specified

Remarks:

No information on GLP compliance provided.

Specific details on test material used for the study:

The test chemical urea was obtained from the Preliminary Agricultural Co-operative Bank, Annamalai Nagar, Tamilnadu, India.

Vehicle:

yes

Remarks:

Deionized water

Details on preparation and application of test substrate:

The preparation of the test solutions was done for each concentration by weighing and dissolving an amount of urea in deionized water. Deionized water was obtained using a Millipore Super-Q water purification system and was used throughout this study. All glass wares were washed with phosphate-free detergent, rinsed with acetone, and acid-washed before a final and thorough rinse with deionized water.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

TEST ORGANISM
Adult earthworms (with clitellum) with a fresh weight between 300 and 600 mg were used. The selected test animals were acclimatized in untreated soil substrates at least 24 hours prior to the start of the urea test.

ACCLIMATION
In this work, mass cultures of E. foetida were established in laboratory from samples provided from the vermicomposting unit of Annamalai University for temperate and tropical artificial soils in plastic boxes (25 x 36 cm area, 12 cm height) (Figure 2). The temperate culture of Eisenia foetida was kept in a mixture of OECD soil at room temperature of 15 - 30 °C (mean: 22 °C). The tropical culture was kept in a mixture of tropical artificial soil (TAS) at room temperature 23 - 30 ºC (mean = 26 ºC), relative air humidity near 90%. The animals were maintained in a shaded place with a natural light cycle (12h light/12h dark). The breeding substrates were maintained
with a pH-value set to 6 ± 0.5. The moisture was adjusted to 50 ± 10% dry weight using deionized water. The earthworms were fed according to demand, usually once a week, with finely ground cattle manure free of any chemical contamination.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

14 d

Test temperature:

20 °C (OECD), 28°C (tropical artificial soil, TAS)

pH:

The soil pH was adjusted to 6.0 ± 0.5 with calcium carbonate.

Moisture:

The moisture was adjusted to 50 ± 10% dry weight using deionized water.

Details on test conditions:

TEST SYSTEM
- Test container (material, size): glass vessels (12.5 cm diameter,
16 cm height)
- Amount of soil or substrate: filled with 500g (dw) soil
- No. of organisms per container (treatment): 10
- No. of replicates per treatment group: 1
- No. of replicates per control: 1
- No. of replicates per vehicle control: NA

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: artificial soils (OECD and TAS)
- Composition (if artificial substrate): see Table 3 and 4 of the original publication
- Organic carbon (%): 47.2% and 43.6 % for OECD and TAS soil, respectively.
- Maximum water holding capacity (in % dry weight): 56.1% and 48.5 % for OECD and TAS soil, respectively.
The breeding substrates were maintained with a pH-value set to 6 ± 0.5. The moisture was adjusted to 50 ± 10% dry weight using deionized water. The earthworms
were fed according to demand, usually once a week, with finely ground cattle manure free of any chemical contamination.

OTHER TEST CONDITIONS
The animals were maintained in a shaded place with a natural light cycle (12h light/12h dark).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Mortality

VEHICLE CONTROL PERFORMED: no

TEST CONCENTRATIONS
- Range finding study: yes, see below
- Test concentrations: 1000; 100; 10; 1; 0.1 mg of urea per kilogram of test substrate (dry weight)

Nominal and measured concentrations:

Control, 500, 1000, 1500 and 2000 mg of urea per kilogram of test substrate (dry weight)

Reference substance (positive control):

no

Key result

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

> 2 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: OECD test (20°C)

Duration:

14 d

Dose descriptor:

LC50

Effect conc.:

> 2 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Tropical artificial soil (28°C)

Details on results:

- Changes in body weight of live adults (% of initial weight) at end of exposure period: not reported
- No. of offspring produced:
Mortality OECD-test day 14: 2.5, 2.5, 10.5, 12.5 and 15.5 % mortality at control, 500, 1000, 1500 and 2000 mg urea/kg dw
Biomass OECD-test day 14: 11.1, 12.5, 13.8, 25.4 and 33.7 % biomass reduction at control, 500, 1000, 1500 and 2000 mg urea/kg dw
Mortality TAS-test day 14: 0.0, 1.5, 5.0, 5.0, 7.5 % mortality at control, 500, 1000, 1500 and 2000 mg urea/kg dw
Biomass TAS-test day 14: -16.1, -9.9, -7.1, 11.4 and 16.6 % biomass reduction at control, 500, 1000, 1500 and 2000 mg urea/kg dw (note, negative values indicate biomass increase)

Since both the mortality and the biomass reduction relative to the starting biomass were < 50%, the LC50 and EC50 were not calculated, but assessed to be greater than the highest test concentration, i.e., higher than 2000 mg/kg dw.

Validity criteria fulfilled:

yes

Conclusions:

LC50 Eisenia fetida 14 day: > 2000 mg/kg dw

Executive summary:

Abbiramy et al. (2012) investigated the acute toxicity of urea on Eisenia fetida according to OECD 207 (artificial soil). Since only 3 instead of 4 replicates were used, the study is considered to be Klimisch 2. All validity criteria were met. The LC50 determined during this test is greater than the highest test concentration, i.e., >2000 mg/kg dry weight. Biomass decrease was reported and the corresponding biomass reduction when compared to the initial biomass did not exceed 50%. Hence that EC50 is also > 2000 mg/kg dw.

In addition to the OECD parameters, Abbiramy et al. (2012) investigated the toxicity of urea under increased temperature (28°C) with a slightly modified soil. The corresponding LC50 was > 2000 mg/kg dw as well. Biomass decrease was reported and the corresponding biomass reduction when compared to the initial biomass did not exceed 50%. Hence that EC50 is also > 2000 mg/kg dw.

The results of this study are considered relevant and reliable for the risk assessment. Since the OECD test design resulting in an LC50 of > 2000 mg/kg dw is relevant for the risk assessment, this value will be used.

Description of key information

14 day acute LC50: > 1000 mg urea/kg dw, i.e., the limit concentration for that test type, the most relevant acute study resulted in > 2000 mg/L

30 day long-term LC50: > 3000 mg urea/kg dw

30 day long-term EC10 growth: 520 mg/kg dw

60 day long-term EC10 reproduction: within 160 to 409 mg/kg dw (3 studies)

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

2 000 mg/kg soil dw

Long-term EC10, LC10 or NOEC for soil macroorganisms:

160 mg/kg soil dw

Additional information

For the assessment of the toxicity of urea to earthworms (Eisenia fetida) , six studies with a Klimisch score of 2 are available. In addition, 2 studies with a Klimisch score of 3 or 4 are referred to. All except one study was performed with Eisena fetida. The second species tested is Eudrilus eugeniae. Data are available for filter, artificial and natural soil. The studies cover short-term and long-term endpoints.

 

The Klimisch 2 studies indicate that the acute toxicity of urea when applied to the soil is low, i.e., greater than the maximum concentration of 1000 mg/kg dw as provided in the OECD 207. The most reliable study Abbriarmy et al. (2012) indicated that the 14-day LC50 is > 2000 mg urea/kg dw. When urea is tested in a filter test (OECD 207), the toxicity is "very high" (LC50: 0.028 mg/cm²). Rai et al. (2012) reported that at higher concentrations (2250 mg urea/kg dw) the worms tried to escape from the test vessels. This might be an indication that urea causes local effects on the skin of the earthworms. Shruthi et al. (2017) investigated the effect of urea on Eudrilus eugeniae and found out that the 10-day LC50 for this species is < 5000 mg urea/kg (94 % mortality at 5000 mg urea/kg, lowest concentration tested).

In a long-term study Rai et al. (2014) showed that in natural soil the 30-day LC50 is > 3000 mg urea/kg dw. From the data provided, the EC10 for growth was calculated to be 520 mg urea/kg dw. The 60-day EC10 for reproduction was calculated to be 160 mg urea/kg dw.

Abbiramy et al. (2012) investigated the long-term toxicity of urea at 20°C (according to OECD 222) and 28°C in artificial soils. The worms were adapted to the temperatures prior to start of the study. At 20°C the 56 day EC10 for reproduction was 417 mg urea/kg dw. At 28° the 56 day EC10 for reproduction was 409 mg urea/kg dw. Hence, there was no relevant temperature effect observed in this study.

One field study with nematodes (reliable with restrictions) indicated that the number of free living nematodes increased with increasing urea concentration up to 707 kg/ha which was the highest tested concentration. This corresponds to a concentration of 236 mg urea/kg dw. Hence free living nematodes are less sensitive than earthworms.

For the risk assessment the acute 14-day LC50 value of 2000 mg/kg dw as reported by Abbriarmy et al. (2012) will be used (> 2000 mg/kg dw cannot be entered). Furthermore, the EC10 of 160 mg urea/kg dw for reproduction (Rai et al. 2014) will be used as most conservative value will be used as long-term value for the risk assessment. These endpoints are considered sufficiently robust and reliable for the risk assessment of urea.

"Generally, it was observed that management practices that encourage earthworm abundance (like mineral fertilization with nitrogen and phosphorous) are the same as those assuring a good support for a large crop productivity necessary for sustainable agroecosystems." cited from M. Iordache, I. Borza 2010 Relation between chemical indices of soil and earthworm abundance under chemical fertilization ( PLANT SOIL ENVIRON., 56, 2010 (9): 401–407)