Urea

Toxicity to terrestrial plants

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to terrestrial plants: short-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 208 (Terrestrial Plants Test: Seedling Emergence and Seedling Growth Test)

Deviations:

not applicable

Principles of method if other than guideline:

The seed germination and seedling growth studies reported were performed with seeds of wheat (Triticum aestivum L.), sorghum [Sorghum bicolor (L.) Moench], alfalfa (Medicago sativa L.), rye (Secale cereale L.), oats (Avena sativa L.), and barley (Hordeum vulgare L.). The early plant growth studies were performed with plants grown from wheat and sorghum seeds. Wheat seeds were obtained from Merschman Seed & Fertilizer, West Point, IA. The other seeds used were obtained from May Seed & Nursery, Shenandoah, IA. The soils used were surface (0-15 cm) samples of a Typic Hapludoll of the Dickinson series and of Entic Hapludolls of the Buckney and Sparta series. Before use, each sample was air-dried and crushed to pass through a 2-mm screen. The soil analyses reported in Table 1 were performed as described by Zantua and Bremner (16). NBPT, N-(diaminophosphinyl)benzamide, and phosphoric triamide were obtained from Allied, Solvay, NY. PPD was obtained from K & K Labs Division, ICN Pharmaceuticals, Plainview, NJ. 4-Fluoro-N-(diaminophosphinyl)benzamide and N-phenylphosphoric triamide were obtained from Norwich Eaton Pharmaceuticals, Norwich, NY. All other chemicals used were obtained from Fisher Scientific, Itasca, IL. The following procedure, based on the rules for seed testing published by the Association of Official Seed Analysts, was used in the germination tests reported.

Air-dried soil (40 g) was placed in a 15 mm x 100 mm Petri dish (Fisher Scientific, Pittsburgh, PA) and moistened with 10 ml of water, 10 ml of water containing 100 mg of urea (control), or 10 ml of water containing 100 mg of urea plus 1-100 µg of the inhibitor under study. One hundred seeds of the plant under study were placed on the soil, and the Petri dish was covered with a lid and kept in the dark for 7 days in an incubator maintained at 20°C. The number of germinated seeds was then counted and calculated as a percentage of the number of seeds sown. The criterion for germination was the emergence of a root and coleoptile that were longer than the seed. All germination tests reported were performed in quadruplicate. The procedure used to study the effects of urease inhibitors on seedling growth in soils. treated with urea was similar to that used for the germination tests, but 15 seeds were used instead of 100 seeds. Seedling growth was determined by measuring shoot length after 7 days.
The following procedure was used to compare the effectiveness of NBPT and PPD for elimination of the adverse effect on early plant growth of urea granules placed in contact with seeds in soil. A 25-mg granule of urea containing PPD or NBPT was made by grinding urea or urea containing a small amount of PPD or NBPT to a powder with a mortar and pestle and then compressing 25-mg samples of these powders into 32-mm (o.d.) granules with a Parr pellet press. Three seeds of the plant under study were placed 2 cm below the surface of soil in a "Ray Leach" container (Polycast Plastics, Beaverton, OR). These containers contained 200 g of air-dried soil that had been moistened with 10 ml of water containing 10 mg of K2SO4, 10 mg of NaH2PO4, and 50 mg of (NH4)2SO4.

After placement of the seeds and the urea or urea granules containing PPD or NBPT, the containers were treated with 20 ml of water and placed in a growth chamber that was maintained at 22°C and illuminated for 12 hr/day. The containers were watered daily to replace water lost by evaporation as determined by weighing the containers. After 21 days, the plants were removed from the containers, rinsed with water to remove soil particles, dried for 3 days at 65°C, and weighed. All plant growth tests reported were performed in quadruplicate.

GLP compliance:

no

Species:

Triticum aestivum

Plant group:

Monocotyledonae (monocots)

Species:

Sorghum bicolor

Plant group:

Monocotyledonae (monocots)

Species:

other: Medicago sativa

Plant group:

Dicotyledonae (dicots)

Species:

Secale cereale

Plant group:

Monocotyledonae (monocots)

Species:

Avena sativa

Plant group:

Monocotyledonae (monocots)

Species:

Hordeum vulgare

Plant group:

Monocotyledonae (monocots)

Test type:

seedling emergence toxicity test

Study type:

laboratory study

Substrate type:

natural soil

Limit test:

yes

Total exposure duration:

7 d

Test temperature:

20°C

Nominal and measured concentrations:

2500 mg/kg dw

Species:

Triticum aestivum

Duration:

7 d

Dose descriptor:

ER50

Effect conc.:

< 2 500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

germination

Species:

Sorghum bicolor

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

< 2 500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

germination

Species:

other: Medicago sativa

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

< 2 500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

germination

Species:

Secale cereale

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

< 2 500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

germination

Species:

Avena sativa

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

< 2 500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

germination

Species:

Hordeum vulgare

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

< 2 500 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

germination

Details on results:

In none of urea only concentrations (2500 mg/kg dw; without urease), germination was observed.
After 7 days, at 2500 mg/kg dw the germination rate was 0% for all tested species. In controls without urea (and without urease inhibitor), the germination was 85 to 96%. Addition of different urease inhibitors increased the germination rate up to the blank control level.

Comparison of the effects of various amounts of 10 urease inhibitors on germination of seeds in Dickinson soil treated with urea showed that PPD and NBPT were considerably more effective than the other inhibitors tested for their ability to eliminate the adverse effect of urea solutions on seed germination (Table 2). Similar results were obtained when the same comparison was performed with the Buckney and Sparta soils. NBPT was found to be superior to PPD for elimination of the adverse effect of urea fertilizer on seed germination (Table 3) and on seedling growth (Table 4) in soils. Table 3 shows that it completely eliminated the adverse effect of urea on germination of barley, oats, sorghum, and wheat seeds when it represented only 0.005% (wt/wt) of the urea added to the Buckney soil and only 0.001% (wt/wt) of the urea added to the Dickinson and Sparta soils. Table 4 shows that NBPT also completely eliminated the adverse effect of urea on barley, oats, sorghum, and wheat seedling growth when it represented only 0.01% (wt/wt) of the urea added to the Buckney soil and only 0.005% (wt/wt) of the urea added to the Sparta soil. Table 5 shows results obtained in a study of growth in 21 days from sorghum and wheat seeds placed in Buckney and Sparta soil in contact with granules of urea or urea containing 0.001, 0.010, or 0.100% (wt/wt) PPD or NBPT. The data reported show that NBPT was superior to PPD for reducing the adverse effect of urea granules on early plant growth in both soils and that, in contrast to PPD, it markedly reduced the adverse effect of urea granules on early plant growth even when it represented only 0.01% (wt/wt) of the urea granule. The superiority of NBPT over PPD for reducing the adverse effect of urea on early plant growth is further illustrated by Fig. 1, which shows the growth in 21 days from sorghum seeds placed in Buckney soil in contact with granules of urea and with granules of urea containing 0.1% PPD or NBPT. The soils used in the work reported were selected because the adverse effects of urea fertilizer on seed germination and seedling growth were considerably greater with these soils than with other soils studied. Also, the soil concentrations of urea in the work reported exceeded those likely to occur in soils under field conditions. It seems reasonable, therefore, to conclude from the data reported that the adverse effects of urea fertilizers on seed germination, seedling growth, and early plant growth in soil could be eliminated or markedly reduced by amending these fertilizers with as little as 0.01% (wt/wt) NBPT.
Because Eskew et al. (18) observed with soybeans that a reduction in leaf urease activity can lead to leaflet-tip necrosis and concluded that this necrosis resulted from the accumulation of toxic concentrations of urea, it is possible that amendment of urea fertilizer with urease inhibitors may induce leaf-tip necrosis in some plants.

Validity criteria fulfilled:

not applicable

Conclusions:

In none of urea only concentrations (2500 mg/kg dw; without urease), germination was observed.
After 7 days, at 2500 mg/kg dw the germination rate was 0% for all tested species. In controls without urea (and without urease inhibitor), the germination was 85 to 96%. Addition of different urease inhibitors increased the germination rate up to the blank control level.

Executive summary:

Bremner and Krogmeier (1988) demonstrated that the adverse effect of urea on germination of various plant seeds can be reduced by adding urease inhibitors to the soil.

In none of the "urea only" concentrations (2500 mg/kg dw; without urease), germination was observed.

After 7 days, at 2500 mg/kg dw the germination rate was 0% for all tested species. In controls without urea (and without urease inhibitor), the germination was 85 to 96%. Addition of different urease inhibitors increased the germination rate up to the blank control level.

Reference 2

Endpoint:

toxicity to terrestrial plants: short-term

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: Association of Official Seed Analysts

Deviations:

not specified

Principles of method if other than guideline:

Two 85-mm discs of germination paper (Steel Blue Anchor seed germination blotter, Anchor Paper, St. Paul, MN) or 40 g of air-dried soil were placed in a 15 mm x 100 mm Petri dish (Fisher Scientific) and moistened with 10 ml of water (control) or with 10 ml of water containing the compound(s) under study. Seeds (25 with corn, 100 with others) were placed between the discs of germination paper or on the soil, and the Petri dish was covered with a lid and kept in the dark for 7 days in an incubator maintained at 20'C. The number of germinated seeds was then counted and calculated as a percentage of the number of seeds sown. The criterion for germination was the emergence of a radicle and a coleoptile that were longer than the seed.

GLP compliance:

no

Specific details on test material used for the study:

Urea from various sources were tested.
Urea fertilizers were obtained from C. F. Industries (Long Grove, IL), Cominco American (Spokane, WA), and Farmland Industries (Kansas City, MO). Purified urea was obtained from Fisher Scientific. PPD was obtained from ICN, and jack bean urease (urea amidohydrolase, EC 3.5.1.5) was obtained from Sigma. All other chemicals used were obtained from Fisher Scientific.

It is not clear which urea material has been used for this test.

Analytical monitoring:

no

Vehicle:

no

Species:

Hordeum vulgare

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

seeds used were obtained from May Seed and Nursery (Shenandoah, IA).

Species:

Zea mays

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

corn seeds were obtained from Mike Brayton Seeds (Ames, IA).

Species:

Secale cereale

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

seeds used were obtained from May Seed and Nursery (Shenandoah, IA).

Species:

Triticum aestivum

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

Wheat seeds were obtained from Merschman Seed and Fertilizer (West Point, IA)

Test type:

seed germination/root elongation toxicity test

Study type:

laboratory study

Substrate type:

filter paper

Limit test:

yes

Total exposure duration:

7 d

Test temperature:

20°C

Nominal and measured concentrations:

0 (control) and 10 mg urea/mL

Reference substance (positive control):

no

Species:

Hordeum vulgare

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

10 other: mg/mL

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

germination

Remarks on result:

other: no statistics performed, but no obvious difference between control and 10 mg/mL

Species:

Zea mays

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

10 other: mg/mL

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

germination

Remarks on result:

other: no statistics performed, but no obvious difference between control and 10 mg/mL

Species:

Secale cereale

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

10 other: mg/mL

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

germination

Remarks on result:

other: no statistics performed, but no obvious difference between control and 10 mg/mL

Species:

Triticum aestivum

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

10 other: mg/mL

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

germination

Remarks on result:

other: no statistics performed, but no obvious difference between control and 10 mg/mL

Details on results:

Details are provided in Table 4 of the publication.

Validity criteria fulfilled:

not applicable

Conclusions:

On filter paper 10 mg urea/mL had no adverse effect on the germination of barley (Hordeum vulgare L.), corn (Zea mays L.), rye (Secale cereale L.), and wheat (Triticum aestivum L.).

Executive summary:

Bremner and Krogmeier (1989) tested the gemination of barley (Hordeum vulgare L.), corn (Zea mays L.), rye (Secale cereale L.), and wheat (Triticum aestivum L.) on filter paper with a contriol and 10 mg urea/mL. 10 mg urea/mL had no relevant adverse effect on the germination of  these seeds.

Reference 3

Endpoint:

toxicity to terrestrial plants: short-term

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

Two 85-mm discs of germination paper (Steel Blue Anchor seed germination blotter, Anchor Paper, St. Paul, MN) or 40 g of air-dried soil were placed in a 15 mm x 100 mm Petri dish (Fisher Scientific) and moistened with 10 ml of water (control) or with 10 ml of water containing the compound(s) under study. Seeds (25 with corn, 100 with others) were placed between the discs of germination paper or on the soil, and the Petri dish was covered with a lid and kept in the dark for 7 days in an incubator maintained at 20'C. The number of germinated seeds was then counted and calculated as a percentage of the number of seeds sown. The criterion for germination was the emergence of a radicle and a coleoptile that were longer than the seed.

The soils used (Table 1) were surface (0-15 cm) samples of Entic Hapludolls of the Buckney and Sparta series and Typic Hapludolls of the Dickinson and Clarion series. Before use, each sample was air-dried and crushed to pass through a 2-mm screen. The analyses reported in Table 1 were performed as described by Zantua and Bremner.

GLP compliance:

no

Specific details on test material used for the study:

Urea from various sources were tested.
Urea fertilizers were obtained from C. F. Industries (Long Grove, IL), Cominco American (Spokane, WA), and Farmland Industries (Kansas City, MO). Purified urea was obtained from Fisher Scientific. PPD was obtained from ICN, and jack bean urease (urea amidohydrolase, EC 3.5.1.5) was obtained from Sigma. All other chemicals used were obtained from Fisher Scientific.

It is not clear which urea material has been used for this test.

Analytical monitoring:

no

Vehicle:

no

Species:

Hordeum vulgare

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

seeds used were obtained from May Seed and Nursery (Shenandoah, IA).

Species:

Zea mays

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

corn seeds were obtained from Mike Brayton Seeds (Ames, IA).

Species:

Secale cereale

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

seeds used were obtained from May Seed and Nursery (Shenandoah, IA).

Species:

Triticum aestivum

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

Wheat seeds were obtained from Merschman Seed and Fertilizer (West Point, IA)

Test type:

seed germination/root elongation toxicity test

Study type:

laboratory study

Substrate type:

filter paper

Limit test:

yes

Total exposure duration:

7 d

Test temperature:

20°C

Nominal and measured concentrations:

0 (control) and 10 mg urea/mL

Reference substance (positive control):

no

Species:

Hordeum vulgare

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

germination

Remarks on result:

other: Estimated from the dose response curve

Species:

Zea mays

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

germination

Remarks on result:

other: Estimated from the dose response curve

Species:

Secale cereale

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

germination

Remarks on result:

other: Estimated from the dose response curve

Species:

Triticum aestivum

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

germination

Remarks on result:

other: Estimated from the dose response curve

Details on results:

Details are provided in Table 2 of the publication.

In this germination test from Bremner and Krogmeier (1989) corn was the lest sensitive species, barley the second most sensitive, rye and wheate were more sensitive than the others.
The EC10 and EC50 was clearly > 1000 mg urea/kg dw for all species tested .

Comparison of the effects of different amounts of purified urea and urea fertilizers on seed germination in Dickinson soil showed that the adverse effects of different amounts of urea fertilizers were not significantly different from those of the corresponding amounts of purified urea (Table 2). Similar results were obtained with Buckney soil. The deduction from this observation that the adverse effect of urea fertilizers on seed germination is not due to fertilizer impurities was supported by our finding that biuret and cyanuric acid, compounds formed during manufacture of urea fertilizers, had little, if any, effect on seed germination in soil when compared with urea (Table 2). Urea fertilizers usually contain only -small amounts of biuret and other impurities, and the concentrations of biuret and cyanuric acid after application of urea fertilizers to soil are much smaller than those tested in the work reported in Table 2.

Additional experiments with autoclaved soils and addition of urease show that the adverse effect of urea on seed germination in soil is due to ammonia formed through hydrolysis of urea by soil urease and is not due to urea itself, to urea fertilizer impurities such as biuret, or to nitrite formed through nitrification of urea N by soil microorganisms.

Validity criteria fulfilled:

not applicable

Conclusions:

The EC10 and EC50 for germination was clearly > 1000 mg urea/kg dw for barley (Hordeum vulgare L.), corn (Zea mays L.), rye (Secale cereale L.), and wheat (Triticum aestivum L.).

Executive summary:

In the seed germination test from Bremner and Krogmeier (1989) corn was the lest sensitive species, barley the second most sensitive, rye and wheate were more sensitive than the others.

The EC10 and EC50 for germination was clearly > 1000 mg urea/kg dw for all species tested .

Comparison of the effects of different amounts of purified urea and urea fertilizers on seed germination in Dickinson soil showed that the adverse effects of different amounts of urea fertilizers were not significantly different from those of the corresponding amounts of purified urea (Table 2). Similar results were obtained with Buckney soil. The deduction from this observation that the adverse effect of urea fertilizers on seed germination is not due to fertilizer impurities was supported by our finding that biuret and cyanuric acid, compounds formed during manufacture of urea fertilizers, had little, if any, effect on seed germination in soil when compared with urea (Table 2). Urea fertilizers usually contain only small amounts of biuret and other impurities, and the concentrations of biuret and cyanuric acid after application of urea fertilizers to soil are much smaller than those tested in the work reported in Table 2.

Additional experiments with autoclaved soils and addition of urease show that the adverse effect of urea on seed germination in soil is due to ammonia formed through hydrolysis of urea by soil urease and is not due to urea itself, to urea fertilizer impurities such as biuret, or to nitrite formed through nitrification of urea N by soil microorganisms.

Reference 4

Endpoint:

toxicity to terrestrial plants: short-term

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

no guideline followed

Principles of method if other than guideline:

Petri dish Incubation Experiment. Soil ammonia volatilization from urea application and its effects on seed germination and early seedling growth of dry direct-seeded rice were examined in two Petri dish incubation experiments. Soils for Petri dish incubation experiments were collected from the top 25 cm of three fields, where tea plant (Soil I), wheat (Soil II), and vegetables (Soil III) were grown, respectively, in Hubei province. Physical and chemical properties of the three soil samples are listed in Table 1. Before filling the Petri dish, the soil was air-dried, pulverized, crushed to pass through a 2mm sieve, and well mixed. Two sizes of plastic Petri dishes with a diameter of 5.5 and 14.5 cm were used. A 150 g of air-dried soil from each soil sample was placed in a larger plastic Petri dish. In Petri dish incubation experiments, the N rate was 0.15 gN per Petri dish and NBPT was applied at 1.00% on a urea basis. Urea, ammonium sulfate, and NBPT were applied to the soil in each Petri dish in solution, and the total volume of solution was kept as 50mL. To simulate soil water content suitable for direct-seeded rice, soil inside the Petri dishes was kept under aerobic conditions.

A smaller uncovered Petri dish containing 10 presoaked seeds of Apo (an upland rice variety) with moistened filter paper was placed in the center of a larger Petri dish. Inside the sealed Petri dish, the presoaked seeds were exposed to ammonia volatilized, but without any contact with the soil.

Petri dishes were placed inside a darkened growth chamber for 4 d at 30◦C and 70% humidity using a completely randomized design. Each treatment was replicated four times with four Petri dishes.

The fresh shoot and root were weighed and recorded, and root tip number was counted using a WinRHIZO scanner (Reagent Instrument, Quebec, Canada).

GLP compliance:

not specified

Remarks:

No information on GLP compliance provided.

Analytical monitoring:

no

Vehicle:

no

Species:

Oryza sativa

Plant group:

Monocotyledonae (monocots)

Details on test organisms:

Apo (an upland rice variety)

Test type:

seedling emergence and seedling growth test

Study type:

laboratory study

Substrate type:

natural soil

Limit test:

yes

Total exposure duration:

4 d

Test temperature:

30°C

Moisture:

70%

Details on test conditions:

Petri dishes were placed inside a darkened growth chamber for 4 d at 30°C and 70% humidity using a completely randomized design. Each treatment was replicated four times with four Petri dishes.

Nominal and measured concentrations:

Control, 0.15 g N/150 g dw , equivalent to 1 g N/kg dw (=2144 mg urea/kg dw)

Reference substance (positive control):

no

Species:

Oryza sativa

Duration:

4 d

Dose descriptor:

NOEC

Effect conc.:

<= 2 144 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Remarks on result:

other: root growth; see details

Remarks:

soil I (pH 4.8, 7.3, 7.9)

Species:

Oryza sativa

Duration:

4 d

Dose descriptor:

NOEC

Effect conc.:

>= 2 144 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

germination

Remarks on result:

other: see details

Remarks:

Soil II (ph 7.3)

Species:

Oryza sativa

Duration:

4 d

Dose descriptor:

NOEC

Effect conc.:

>= 2 144 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

germination

Remarks:

shoot growth was increased

Remarks on result:

other: see details

Remarks:

Soil III (pH 7.9)

Details on results:

Application of urea to soils I–III at 0.15 gN per Petri dish (equivalent to 2144 mg urea/kg dw) significantly decreased parameters of root growth compared with zero N input control (Table 3). In soils I and II, the shoot growth was not inhibited by urea application, while shoot growth was increased by urea application in soil III.

Reported statistics and error estimates:

Data were analyzed following analysis of variance, and mean comparison between treatments was performed based on the Least Significant Difference (LSD) test at the 0.05 probability level.

Validity criteria fulfilled:

not applicable

Conclusions:

In natural soils with pH values of 4.8, 7.3 and 7.9, the root length of Oryza sativa was reduced at 2144 mg urea/kg dw, the only concentration tested. However, the shoot growth was not adversely affected at 2144 mg urea/kg dw.

Executive summary:

Qi et al. (2012) performed a laboratory test on germination and early growth ot upland rice (variety Apo) with 3 natural soils with pH values of 4.8, 7.3 and 7.9. Application of urea to soils I–III at 0.15 gN per Petri dish (equivalent to 2144 mg urea/kg dw) significantly decreased parameters of root growth compared with zero N input control. In soils I and II, the shoot growth was not inhibited by urea application, while shoot growth was increased by urea application in soil III.

Reference 5

Endpoint:

toxicity to terrestrial plants: 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:

no guideline followed

Principles of method if other than guideline:

The examinations were aimed at determining the dependence between form and rate of applied nitrogen fertilizer vs. yield and chemical composition of leaves of Swiss chard cultivated in greenhouse in spring. Nitrogen was applied in a form of urea, potassium nitrate, and ammonium nitrate at three amounts: 0.2; 0.4; 0.6 g N·dm-3 of substrate. Following items were assessed: yield, nutrients contents, as well as chemical analyses of substrate after plant harvest were made.

GLP compliance:

no

Vehicle:

no

Species:

Beta vulgaris

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

- Common name: Swiss chard
- Plant family: Chenopodiaceae
- Variety: Beta vulgaris L. var. cicla L.

Test type:

early seedling growth toxicity test

Study type:

semi-field study

Remarks:

greenhouse

Substrate type:

natural soil

Limit test:

no

Total exposure duration:

7 wk

pH:

5.8

Details on test conditions:

TEST SYSTEM
- Test container (type, material, size): cylinders of 5 dm3 capacity (one plant was grown in one cylinder).
- Amount of soil: 5 dm3
- No. of seeds per container: 1
- No. of replicates per treatment group: 6
- No. of replicates per control: 6

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Soil texture (if natural soil) peat substrate of pH 5.8

Nominal and measured concentrations:

0.2, 0.4, 0.6 g N/dm³ corresponding to 225, 450 and 675 mg urea/kg dw (assuming a soil ww of 1700 kg/m³ and a conversion factor of 1.13 kg ww/kg dw)

Reference substance (positive control):

no

Species:

Beta vulgaris

Duration:

7 wk

Dose descriptor:

NOEC

Remarks:

NOAEL

Effect conc.:

>= 0.6 other: g/dm³

Nominal / measured:

nominal

Conc. based on:

element

Basis for effect:

growth

Species:

Beta vulgaris

Duration:

7 wk

Dose descriptor:

NOEC

Remarks:

NOAEL

Effect conc.:

>= 1.3 other: g/dm³

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Species:

Beta vulgaris

Duration:

7 wk

Dose descriptor:

NOEC

Remarks:

NOAEL

Effect conc.:

>= 765 mg/kg soil ww

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Remarks on result:

other: assuming a wet weight of 1.7 kg/dm³

Species:

Beta vulgaris

Duration:

7 wk

Dose descriptor:

NOEC

Remarks:

NOAEL

Effect conc.:

>= 675 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Remarks on result:

other: assuming a conversion factor of 1.133 kg ww/kg dw. (see Technical guidance document)

Yield at 225, 450 and 675 mg/kg dw: 290, 289 and 289 g fresh weight /plot, respectively.

Validity criteria fulfilled:

not applicable

Conclusions:

NOAEL for Swiss Chard (Beta vulgaris L. var. cicla L.) >= 675 mg/kg dw

Executive summary:

In a 7 week greenhouse experiment Dzida and Pitura (2008) determined the NOAEL for Swiss Chard (Beta vulgaris L. var. cicla L.) to be >= 675 mg/kg dw. Under the conditions tested, the addition of urea alone had no effect on the growth of Swiss Chard.

Reference 6

Endpoint:

toxicity to terrestrial plants: 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:

no guideline followed

Principles of method if other than guideline:

The field trial was conducted on Research Farm, Faculty of Agriculture, Gomal University Dera Ismail Khan (GPS: 31° 42'48.80'' N, 70° 50' 09.53'' E) K.P.K, Pakistan during 2010-2011. Forty-five field-plots, each 5x3 m2 and separated by a 1 m buffer zone, were established in five rows. Prior to treatment application, three composite soil samples from 0-10 cm depth were taken for chemical analyses. The soil pH was 7.63. It contained NO3-N at 0.36 mg kg-1, total P at 2.98 mg kg-1, K at 244 mg kg-1, and ECe was at 1.42 d Sm-1. All plots received a basal rate of 150 kg P ha-1 and 200 kg K ha-1 using triple superphosphate and sulphate of potash respectively. Tubers were obtained from Hazara Research station, Abbotabad. Cut pieces of potato tubers, each about 40-50 g with at least two eyes, were planted with plant-to-plant spacing of 30 cm and row-to-row of 75 cm on 24 December 2010.

Nine treatments were tested: control (no N or Cytozyme), urea at 200 kg N ha-1, urea at 300 kg N ha-1, Agrotain treated-urea at 200 kg N ha-1, Agrotain treated-urea at 300 kg N ha-1, urea at 200 kg N ha-1+Cytozyme, urea at 300 kg N ha-1+Cytozyme, Agrotain treated-urea at 200 kg N ha-1+Cytozyme, and Agrotain treated-urea at 300 kg N ha-1+Cytozyme. Each treatment was replicated five times. The Cytozyme tested products included Seed+, Crop+, Bio-Nutrient Combination, and Bio-Nutrient Potassium. Seed+ was applied to the seeds at 2 kg/ton seed at the time of planting, while Crop+, Bio-Nutrient Combination and Bio-Nutrient Potassium were applied at 2 L/ha each at timing of fertilizer application. Urea with or without Agrotain was applied in three split doses, first dose at 30 days after planting, second at 20 days after first application and third at 30 days after the second application. The Cytozyme products (Crop+, Bio-Nutrient Combination, and Bio-Nutrient Potassium) were also applied with the three split applications of N. The experiment was conducted in a randomized complete block design. Common farming practices in the region like spraying herbicides and insecticides and irrigation were also followed. Growth and yield parameters included survival percentage, plant height, plant spread or canopy area, number of stems per plant, total yield, and yield and number of large, medium and small tubers. Potato crops were harvested in the first week of May after reaching maturity. Tubers from each plot were dug out by hand, brushed to remove loose soil, counted, separated into large (>55 mm), medium (35-55 mm) and small (<35 mm) grades according to their sizes. Each grade tubers were weighed for calculating tuber yield.

GLP compliance:

not specified

Remarks:

No information on GLP compliance provided.

Analytical monitoring:

no

Vehicle:

no

Species:

other: Solanum tuberosum L.

Plant group:

Dicotyledonae (dicots)

Details on test organisms:

Tubers were obtained from Hazara Research station, Abbotabad.

Test type:

other: Growth test

Study type:

field study

Substrate type:

natural soil

Limit test:

no

Total exposure duration:

19 wk

Remarks:

Cut pieces of potato tubers were planted on 24 December 2010. Potato crops were harvested in the first week of May after reaching maturity.

pH:

7.63

Details on test conditions:

TEST SYSTEM
- Testing facility: Research Farm, Faculty of Agriculture, Gomal University Dera Ismail Khan (GPS: 31° 42'48.80'' N, 70° 50' 09.53'' E) K.P.K, Pakistan
- Cut pieces of potato tubers, each about 40-50 g with at least two eyes, were planted with plant-to-plant spacing of 30 cm and row-to-row of 75 cm on 24 December 2010.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographic location: Research Farm, Faculty of Agriculture, Gomal University Dera Ismail Khan (GPS: 31° 42'48.80'' N, 70° 50' 09.53'' E) K.P.K, Pakistan
- Pesticide use history at the collection site: not provided
- Pretreatment of soil: Forty-five field-plots, each 5x3 m2 and separated by a 1 m buffer zone, were established in five rows. Prior to treatment application, three composite soil samples from 0-10 cm depth were taken for chemical analyses. The soil pH was 7.63. It contained NO3-N at 0.36 mg kg-1, total P at 2.98 mg kg-1, K at 244 mg kg-1, and ECe was at 1.42 d Sm-1. All plots received a basal rate of 150 kg P ha-1 and 200 kg K ha-1 using triple superphosphate and sulphate of potash respectively.


GROWTH CONDITIONS
- Field study performed from 24th December 2010 to first week of May 2011,
- Urea was applied in three split doses, first dose at 30 days after planting, second at 20 days after first application and third at 30 days after the second application.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Plant survival, growth (height, spread, stem/plant), tuber yield

Nominal and measured concentrations:

Control, urea at 200 kg N ha-1, urea at 300 kg N ha-1; corresponding to 429 and 643 kg urea/ha
- Urea was applied in three split doses, first dose at 30 days after planting, second at 20 days after first application and third at 30 days after the second application.

Reference substance (positive control):

no

Species:

other: Solanum tuberosum L.

Duration:

19 wk

Dose descriptor:

NOEC

Remarks:

NOAEC, no observed adverse effect concentration

Effect conc.:

>= 643 kg/ha

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Remarks on result:

other: Plots treated with urea resulted in better growth of the potato plants and higher yield of tubers when compared to the control (see Tables 1 and 2 of the publication) .

Details on results:

In this field study, urea was used as fertilizer and applied with 429 and 643 kg urea/ha. At both application rates, urea had a stimulating effect on potato survival, growth, and tuber yield when compared to the control plots (see Table 1 of the publication). Urea was applied in three split doses, first dose at 30 days after planting, second at 20 days after first application and third at 30 days after the second application, i.e., 143 and 214 kg urea per application, respectively.

The positive effective of applying urea with Cytozyme are related to alleviating the abiotic stress and better N nutrition as already discussed above. The benefits achieved by using Agrotain-treated urea compared to urea only in term of total tuber yield improvements were the same for both lower (200 kg N ha-1) (46%) and higher rate (300 kg N ha-1) probably because Agrotain only delays urea hydrolysis by 1 to 2 weeks during which time plants have access to N take in urea form. It is possible that at 200 kg N ha-1, N uptake is maximized in that 1 to 2 weeks period so there is no extra advantage over urea achieved by applying more Agrotain treated urea at the 300 kg N ha-1 application rate. When applied with urea, Cytozyme increased the tuber yield by 61% and 62% compared to the equivalent treatments with urea at 200 kg N ha-1 and urea at 300 kg N ha-1, respectively.

Reported statistics and error estimates:

Analysis of variance (ANOVA) was performed to compare fertilizer treatments with respect to various measured parameters. When significant effects of treatment were observed, these were further explored using the Tukey adjusted LSD value to make specific comparison among the different treatments. All the analyses were performed using Minitab (version 12).

Validity criteria fulfilled:

not applicable

Conclusions:

In this 19 week field study with potatoes, urea was used as fertilizer and applied with 429 and 643 kg urea/ha. At both application rates, urea had a stimulating effect on potato survival, growth, and tuber yield when compared to the control plots. Urea was applied in three split doses, first dose at 30 days after planting, second at 20 days after first application and third at 30 days after the second application, i.e., 143 and 214 kg urea per application, respectively.

Executive summary:

Khan et al. (2014) report a fied study performed at Research Farm, Faculty of Agriculture, Gomal University Dera Ismail Khan (GPS: 31° 42'48.80'' N, 70° 50' 09.53'' E) K.P.K, Pakistan.

In this 19 week field study with potatoes, urea was used as fertilizer and applied with 429 and 643 kg urea/ha. At both application rates, urea had a stimulating effect on potato survival, growth, and tuber yield when compared to the control plots. Urea was applied in three split doses, first dose at 30 days after planting, second at 20 days after first application and third at 30 days after the second application, i.e., 143 and 214 kg urea per application, respectively.

The results demonstrate that urea applied at a rate of up to 643 kg urea/ha (applied in three applications) had no adverse effects on potato growth and tuber yield. Hence, this application rate is considered to be the NOAEC in this study.

Description of key information

EC10 terrestrial plants >= 1000 mg urea/kg dw

Key value for chemical safety assessment

Long-term EC10, LC10 or NOEC for terrestrial plants:

1 000 mg/kg soil dw

Additional information

 

For the typical plant species as described in Annex 2 of the OECD 208, test data or fertilizer recommendations are available and provided in this document. Most of the data are germination tests, field tests or recommendations for the use of urea as fertilizer. Field tests and fertilizer recommendations were performed/made in order to allow maximum yield with a minimum of fertilizer used. Hence, the information obtained from such studies is typically an application amount (concentration) at which the optimum (economic) benefit is obtained and not a concentration where toxic effects start.

 

Laboratory test data:

In germination tests with various grain species, the EC10 was determined to be > 1000 mg/kg dw (Bremner and Krogmeier, 1988). In a rice germination and early growth limit test, Qi et al. (2012) found that the NOEC for Oryza sativa is >= 2144 mg urea/kg dw. On filter paper 10 mg urea/mL had no adverse effect on the germination of barley (Hordeum vulgare L.), corn (Zea mays L.), rye (Secale cereale L.), and wheat (Triticum aestivum L.) (Bremner and Krogmeier, 1989). Assuming that 133 mL pore water can be found per kg wet soil (conversion factor 1.1333 wet soil/dry soil according to guidance) this corresponds to 1333 mg urea/kg dw.

For the risk assessment, the EC10 of 1000 mg urea/kg dw will be used.

 

Fertilizer recommendations:

Fertilizer recommendations were all below application rates which correspond to 1000 mg/kg dw. Maximum application rates recommended in these fertilizer recommendations were, e.g.,  675 mg urea /kg dw (Dzida and Pitura, 2008, swiss chard Beta vulgaris L. var. cicla). Different varieties of cultured plants may have different nutrient requirement which is then reflected in the fertilizer recommendations. For example, recommendations for potatoes range from 120 to 240 kg urea/ha corresponding to 40 to 80 mg/kg dw for different varieties of potatoes. However, the field study from Khan et al. (2014) demonstrated that an application of 643 kg urea/ha (corresponding to 214 mg/kg dw) had not adverse effect on potato growth and yield and the NOAEC is hence higher than the fertilizer recommendations – at least for potatoes. It can be concluded that the fertilizer recommendations are mainly made based on economic considerations and not on toxic findings and need to be considered to be significantly below concentrations where adverse effects can be expected.

 

Mode of action:

The test data reported Bremner and Krogmeier (1988) indicate that the adverse effects of urea fertilizer on seed germination, seedling growth, and early plant growth in soil could be eliminated or markedly reduced by amending urease inhibitors. This supports the hypothesis that effects on plants are caused largely, if not entirely, by ammonia produced through hydrolysis of urea fertilizer by soil urease. Bremner and Krogmeier (1988) reported EC50 of 2500 mg/kg dw without urease inhibitor while addition of urease inhibitor eliminated or reduced the effect of urea significantly.

All relevant data for terrestrial plants demonstrate that terrestrial plants are less sensitive to urea (or its degradation products) than earthworms and therefore the terrestrial plants are less relevant for the risk assessment of urea than earthworms.