Urea

Toxicity to other above-ground organisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to other above-ground organisms

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

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

Remarks:

Species investigated not relevant for Europe

Qualifier:

no guideline available

Principles of method if other than guideline:

Review article, hence no detailed information on the methods.

GLP compliance:

no

Analytical monitoring:

not specified

Vehicle:

not specified

Test organisms (species):

other: Amphibians, affected amphibians were from North America

Study type:

other: various

Remarks:

including temperate and boreal forests

Duration:

5 d

Dose descriptor:

other: significant mortality

Effect conc.:

ca. 107 000 g/ha

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Validity criteria fulfilled:

not applicable

Executive summary:

The effects on terrestrial amphibians are not clear. Some species react already at 107 kg urea/ha with significant mortality while other species are unaffected.

It is unclear as to the mechanism of urea toxicity in amphibians and why some species seem relatively unaffected.

The natural habitat of the affected species is in North America. Therefore, the relevance of this finding is limited for the risk assessment for Europe.

Reference 2

Endpoint:

toxicity to other above-ground organisms

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 available

Principles of method if other than guideline:

Review article, hence no detailed information on the methods.

GLP compliance:

no

Analytical monitoring:

not specified

Vehicle:

not specified

Test organisms (species):

other: various mammals (39 different groups/species) from mouse to moose

Details on test organisms:

see text below
various mammals (39 different groups/species) ranging from mouse to moose

Study type:

field study

Remarks:

temperate and boreal forests

Dose descriptor:

NOEC

Effect conc.:

> 1 600 000 g/ha

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Population growth

Remarks on result:

other: multiple year studies

Validity criteria fulfilled:

not applicable

Executive summary:

The majority of mammalians responded positively to the fertilization of forest with urea (concentrations up to 750 kg N/ha; 1600 kg urea/ha). This effect might be secondary due to improved availability of food. Few mammals showed a decline with is likely also due to secondary effects.

Reference 3

Endpoint:

toxicity to other above-ground organisms

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 available

Principles of method if other than guideline:

Review article, hence no detailed information on the methods.

GLP compliance:

no

Analytical monitoring:

not specified

Vehicle:

not specified

Test organisms (species):

other: Ruminants

Study type:

other: various

Remarks:

including temperate and boreal forests

Dose descriptor:

LD0

Effect conc.:

ca. 500 mg/kg bw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: no information on duration provided

Validity criteria fulfilled:

not applicable

Executive summary:

It may be reasonable to conclude that urea toxicity in native ungulates could be similar to cattle, goats, or sheep and might be in the vicinity of 500 mg/kg of body weight.

In Sweden, nitrate concentrations appearing in plants soon after fertilization with 150 kg N/ha (ammonium nitrate, corresponding to 321 kg urea/ha) were not toxic to reindeer (Nordkvist and Erne 1983). However, urea-fertilized areas were avoided by reindeer for winter grazing which takes place through snow cover (Eriksson and Raunistola 1993).

Reference 4

Endpoint:

toxicity to other above-ground organisms

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:

Study Species
The red-backed salamander Plethodon vehiculum, roughskin newt Taricha granulosa, and Southern torrent salamander Rhyacotriton variegatus inhabit humid coastal forests west of the Cascade range crest of northern California, Oregon, and Washington (Nussbaum et al. 1983).
Amphibians were collected in areas when they were abundant. We collected red-backed and Southern torrent salamanders from Fall Creek in Lincoln County, OR, and the Corvallis watershed in Benton County, OR, in October 1997. We collected roughskin newts from the Corvallis watershed in Benton County, OR, in February 1998. In the laboratory, animals were maintained in 40-L glass tanks with a damp paper towel substrate and wet moss. They were kept on a 14:10 light:dark photoperiod at 15°C and were periodically fed crickets. Animals were in captivity for at least 4 days before we began our experiments. Each test animal was used in only one experiment.

Avoidance Experiments
In avoidance experiments, we tested if amphibians could discriminate between a substrate sprayed with urea versus a control substrate with no urea. The experiments were conducted in the laboratory at 15°C under artificial incandescent light with a natural photoperiod. For each trial, we used a rectangular plastic container (32 3 18 3 8 cm) as a testing chamber. Each half of the floor of a container was lined with paper towels. We sprayed pilled urea in the treatment side of the container at a dose of 225 kg N/ha. The spraying of urea on the left or right side of the container was determined randomly. Paper towels were placed 2 cm apart to minimize diffusion of urea from the treatment side to the control side (no urea). The walls of the containers were not covered by the paper towels and thus had no urea. Before each test, paper towels were sprayed with dechlorinated water to dissolve the granular urea and to control for potential differences in moisture level between the sides.
For each trial, we introduced a single salamander into the center of each container and recorded the side of the container occupied by each salamander every 2 min for 1 h. If the test animal was located in the middle of the enclosure, we used the position of the salamander’s snout to assign location. We also recorded if the animal was in the bottom or on the walls of the container. All containers were carefully rotated 180° every 15 min to avoid any bias of animal orientation in the room.

Toxicity Experiments
In static experiments, we tested whether adults of three amphibian species were sensitive to urea at recommended fertilization doses. The experiments were conducted in the laboratory at 15°C under artificial incandescent light with a natural photoperiod. Forty-five individuals of each species were used in every experiment (3 treatments and 15 replicates per treatment). Fifteen animals were exposed to urea at a dose of 450 kg N/ha, 15 were exposed to urea at a dose of 225 kg N/ha, and finally 15 control animals were not exposed to urea. Tests were conducted in rectangular plastic containers (32 3 18 3 8 cm) for 96 h. The 45 containers for each experiment were randomly assigned to one of three experimental conditions. The floor of each container was lined with four layers of paper towels. We spread the urea on the floor of the treatment containers. Then paper towels of all containers were sprayed with dechlorinated tap water to dissolve the granular urea and to humidify the area. We used dechlorinated tap water that was chemically treated to remove ammonia, chlorine, chloramine, and heavy metals and to buffer pH. When the urea was totally dissolved, we introduced a single salamander into the center of each container. All the experiments were conducted for 96 h. Mortality and the presence of abnormalities were monitored and dead animals were removed every 12 h.

Analysis of Data
In the avoidance experiment and for each trial, we calculated the number of observations out of 30 (1 observation every 2 min for 60 min) that the test animal spent on each side of the testing container. We then used a Wilcoxon signed-ranks test (two-tailed) to determine whether the salamanders spent significantly more time on one side compared to the expected 50% (15 observations). We also used a Wilcoxon signed-ranks test (two-tailed) to determine whether the test animals spent significantly more time in the floor of the container (with urea in the treatment containers) when they were in the treatment or the control side. To determine if mortality differed among treatments, or among species, we used Chi-square tests, where the variable was the absolute frequency of dead animals at different times (12–96 h).

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

other: three species of forest-dwelling amphibians (Plethodon vehiculum, Rhyacotriton variegatus, and Taricha granulosa)

Details on test organisms:

The red-backed salamander Plethodon vehiculum, roughskin newt Taricha granulosa, and Southern torrent salamander Rhyacotriton variegatus inhabit humid coastal forests west of the Cascade range crest of northern California, Oregon, and Washington (Nussbaum et al. 1983).
Amphibians were collected in areas when they were abundant. We collected red-backed and Southern torrent salamanders from Fall Creek in Lincoln County, OR, and the Corvallis watershed in Benton County, OR, in October 1997. We collected roughskin newts from the Corvallis watershed in Benton County, OR, in February 1998. In the laboratory, animals were maintained in 40-L glass tanks with a damp paper towel substrate and wet moss. They were kept on a 14:10 light:dark photoperiod at 15°C and were periodically fed crickets. Animals were in captivity for at least 4 days before we began our experiments. Each test animal was used in only one experiment.

Study type:

laboratory study

Limit test:

no

Total exposure duration:

96 h

Test temperature:

15 °C

Photoperiod and lighting:

14:10 light:dark

Nominal and measured concentrations:

Control, 225 and 450 kg N/ha equivalent to Control, 482.35 and 964.7 kg urea/ha

Reference substance (positive control):

no

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 482 350 g/ha

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

morphology

Remarks on result:

other: P. vehiculum and R. variegatus

Duration:

96

Dose descriptor:

LC50

Effect conc.:

>= 964 700 g/ha

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

morphology

Remarks on result:

other: T. granulosa

Avoidance Experiments

Nine of 20 P. vehiculum tested were more often found on the side of the container with urea. The mean number of observations spent on the side without urea was not significantly greater than 50% (Wilcoxon signed-ranks test, Z = 0.348, p =0.728, n = 20; Figure 1). However, when salamanders were in the urea side of the container, they were preferentially found on the walls of the container (Wilcoxon signed-ranks test, Z = 2.929, p = 0.003, n = 20, Figure 1). When salamanders were in the control side of the container, they were found with similar frequency either on the floor or on the walls (Wilcoxon signed-ranks test, Z = 0.284, p = 0.776, n = 20; Figure 1). Seventeen of 20 R. variegatus tested were more often found on the control side of the container. The mean number of observations spent on the side without urea was significantly greater than 50% (Wilcoxon signed-ranks test, Z = 2.688, p = 0.01, n = 20, Figure 1). This species was found with low frequency on the walls of the container (Wilcoxon signed-ranks test, Z = 2.260, p = 0.036, n = 20). Sixteen of 20 T. granulosa tested were more often found on the control side of the container. The mean number of observations spent on the side with urea was significantly lower than 50% (Wilcoxon signed-ranks test, Z = 3.435, p = 0.001, n = 20, Figure 1). This species was found with very low frequency on the walls of the container (Wilcoxon signed-ranks test, Z = 3.800, p = 0.001, n = 20).

Toxicity Experiments

No mortality occurred in control tanks. The observed effects increased with both concentration and time, and there were significant differences in sensitivity among species (Figures 2 and 3). Both doses of urea had an acute effect on survival of P. vehiculum and R. variegatus. For the urea doses of 225 kg N/ha and at 48 h, P. vehiculum and R. variegatus had a mortality of 47%. At 12 h, mortality at the highest doses of urea was 60% for P. vehiculum and 40% for R. variegatus. At 48 h and at 450 kg N/ha, 14 of 15 P. vehiculum and 12 of 15 P. vehiculum died. T. granulosa had no mortality at both urea concentrations after 96 h of exposure. Sensitivity to urea increased with concentration in P. vehiculum (Chi-square test, x = 46.64, p = 0.001, df = 7) and in R. variegatus (Chi-square test, x = 19.10, p = 0.01, df = 7). Sensitivity to urea at 450 kg N/ha was similar between the two terrestrial salamanders (P. vehiculum and R. variegatus)(Chi-square test, x = 4.02, p = 0.78, df = 7, Figure 3), but sensitivity to urea showed differences between both species at the concentration of 225 kg N/ha (Chi-square test, x = 17.91, p = 0.012, df = 7). P. vehiculum was more sensitive during the first hours of exposure, but after 48 h of exposure R. variegatus was more sensitive (Figure 2).

Validity criteria fulfilled:

not applicable

Conclusions:

96 h LC50 < 482.35 kg/ha (the lowest tested concentration). Salamander Plethodon vehiculum, Rhyacotriton variegatus
96 h LC50 > 964.7 kg/ha (the highest tested concentration). Newt Taricha granulosa

Executive summary:

These results from (Marco et al 2001) suggest that urea-based fertilizers at 482.35 kg urea /ha could be affecting the survival of two amphibian species Salamander Plethodon vehiculum, Rhyacotriton variegatusi in fertilized forests within 96 hours of exposure in laboratory experiments. The roughskin newt Taricha granulosa showed no mortality at 964.7 kg urea/ha. Avoidance experiments did not correlate between toxicity and avoidance behaviour.

The results are considered to be relevant and reliable for the risk assessment.

Reference 5

Endpoint:

toxicity to other above-ground organisms

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:

Investigation of the effects of diet on urea toxicity in cattle.

The work was designed to determine if Starea would have a similar effect in vivo. Also, the value of Starea in preventing urea toxicity was tested.


Different types of administration.

Experiment I
Two sets of rumen-fistulated identical-twin cattle were used. One pair was Shorthorn steers (04-05) weighing about 500 kg and the other pair was Holstein cows (18-19) weighing about 510 kg. During a preliminary period of 3 weeks all animals were fed equal quantities of alfalfa hay and a grain ration containing 3% urea. Following the preliminary period, 4 different rations were fed to each animal. The rations were changed each week so that after 4 weeks the individual animal had been fed all 4 rations. That sequence was repeated once.

Four different methods of processing sorghum grain were tested. The same batch of sorghum grain was used for all four rations. The first ration (cracked) was produced by cold rolling the sorghum grain, adding 5% urea and mixing. For a uniform mix the ration was remixed and divided into daily individual portions. The second ration (fine grind) was grain finely ground (to produce flour) through Miag 3 Mutlimat experiment mill. A dry sieve analysis (6) of the ground grain gave a Fineness Modulus of 1.155 and a Modulus of Uniformity of 0:2:8. It was then nfixed with 5% urea and pelleted through a 0.76 cm die. The third ration (expanded) consisted of expanded sorghum grain. The grain was ground through a 0.16 cm hammermill screen and processed by a Wenger 4 X-50 continuous extruder cooker. The expanded grain was dried, cold rolled and mixed with 5% urea. The fourth ration (Starea) was also sorghmn grain, with 5% urea added before it was processed by a Wenger X-50 continuous extruder cooker, as described by ttelmer et al. (7). Degree of gelatinization was estimated by microscopic examination (11). The urea content and relative degree of gelatinization of the four rations are shown in Table 2.

Each animal received daily a total of 3.6 kg grain ration and 3.6 kg of long alfalfa hay in 2 equal feedings. Due to difficulty experienced in getting the cattle to eat the cracked, finely ground and expanded rations, all grain rations were placed in the rumen by way of the fistula at both feedings. Rumen fluid samples for ammonia determinations were obtained at the end of each 7-day feeding period. The samples were taken before the morning feeding and at 1, 2, 3, 4, 6 and 8 hours after feeding. Samples were also obtained the second day after the rations were changed before and at 1, 2, 3 and 4 hours after the morning feeding. Ammonia was determined by the method of Conway (5).

Experiment II
In this experiment the rumen fluid was analysed in detail and is not relevant for the risk assessment of urea since no "pure" urea was used in this experiment.

Experiment III. Procedures
Two sets of mature rumen-fistulated identical twin cattle were used to study urea toxicity. One pair was Holstein females (14-15) weighing 680 kg and the other pair was Shorthorn steers (04-05) weighing 500 kg. The Holsteins had not been fed urea for 250 days previous to the trial while the Shorthorns had been fed 180 to 200 g of urea daily up to 10 days previous to the trial. All animals were fed 2.8 kg of rolled sorghum grain and 2.8 kg of alfalfa hay daily. On the day of each trial all animals were fed 1.4 kg of sorghum grain and 1.4 kg of alfalfa hay at 8 A~. At 1 P~ they were fed 1.4 kg of alfalfa hay and a quantity of grain mixture containing 20% urea. The quantity of grain mixture fed was based on the weight of the animal on the trial day. Due to poor acceptability, the grain mixture containing urea was placed in the rumen by way of the fstula after the first day in amounts to supply the quantities of urea shown in Table 8. The alfalfa hay was fed at the time grain was administered to help promote normal actions resulting from the ingestion of feed. Acetic acid (5% aqueous solution) was used to attempt to alleviate toxicity when it occurred.

Rumen fluid samples for ammonia determinations were obtained before feeding and at 30-min intervals post feeding.

GLP compliance:

no

Specific details on test material used for the study:

urea pure (within the study denoted as "Control")
expansion-processed mixture of grain starch and urea (Starea)
cracked grain plus urea
further feeds admninistered with urea.

Analytical monitoring:

yes

Test organisms (species):

other: Cattle

Study type:

laboratory study

Limit test:

no

Total exposure duration:

56 d

Nominal and measured concentrations:

Animals were fed quantities of grain containing 20% urea at dose levels equivalent to 26-118 g/100 kg bw (260-1180 mg/kg bw).

Dose descriptor:

other: 4 animals tested one died at 600 mg/kg bw/day, another at 1080 mg urea/kg bw / day

Effect conc.:

ca. 600 mg/kg bw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: for details see text and Table 8 in publication

Experiment I

The Starea ration was the only ration readily consumed. Palatability problems were encountered with the other 3 rations so all grain rations were placed in the rumen through the fistula. Urea is an unpalatable feed ingredient (8,15). Urea often segregates in rations containing ground ingredients. Considerable variation existed in nitrogen contents of the cracked and expanded rations (Table 2) suggesting some urea segregation. The most uniform ration was Starea.

The content of the nitrogenous constituents of the feeds are in Table 2. The nonprotein nitrogen content of the grain mixtures consisted primarily of urea nitrogen. Since the intake of the nonprotein nitrogen fraction was more variable among rations than the protein nitrogen intake, the average rumen ammonia concentration was adjusted to a common nonprotein nitrogen content of 2.25% (5.0% urea equivalent) by multiplying the ammonia concentration by 2.25 divided by the urea nitrogen content of the grain mixture. A composite of daily feed samples was analyzed each week so it was possible to adjust the ammonia data for a given week by an analysis value for the feed used during the week.

The adjusted and unadjusted average rumen ammonia concentrations by rations for the second and seventh days after the rations were changed are shown in Table 3. By the second day the rumen ammonia concentration was lowest (P < .01) for the Starea ration. The finely ground pelleted and expanded rations had intermediate effects which were not significantly different from that of the control ration. By the seventh day the rumen ammonia concentration was lowest for the Starea ration, but the ammonia concentration for the finely ground and expanded grain rations approached that of the Starea ration. The Staten, expanded and finely ground rations were significantly different (P < .01) from the control ration but not different frmn one another. The diurnal pattern was similar for both pairs of twins (data not shown). The fine grinding of grain to produce a flour apparently results in sufficient damage to starch to provide carbohydrate in a form that assists urea nitrogen utilization

The results with expanded grain and Starea are similar to those obtained in vitro by Helmer et al. (7). They observed that Starea or a mixture of expanded grain plus urea lowered rumen ammonia concentration and increased microbial protein synthesis in vitro when compared with a mixture of ground grain plus urea. Starea, when tested in vitro, was slightly superior to the expanded grain and urea ration suggesting that the reacting of starch and urea may result in a product that affects the rate of ammonia release from urea to make its conversion to microbial protein more efficient. If expansion processed grain is valuable in urea utilization, it would be more logical to process the grain with urea (Starea) than to feed expended grain and urea separately. Starea provides enhanced palatability and reduced segregation of urea in mixed feed. The recovery of urea nitrogen in the Starea supplements processed was 98%, indicating little if any loss of nitrogen during processing.

Experiment II

Results not reported here since not relevant for hte risk assessment of urea.

Experiment III

The Holstein twins (14-15) which had not been fed urea for 250 days previous to the trial appeared to be more susceptible to ammonia toxicity than did the Shorthorn steers (04-05) which had been adapted to urea (Table 8). The Holsteins exhibited toxic symptoms when they were fed 60 g of urea (control ration) per 100 kg body weight. However, that level of urea in Stared did not produce toxic symptoms.

The steers (04-05) which had been adapted to urea were less sensitive to high urea. It required 108 g of urea per 100 kg weight to produce toxic symptoms. As was the case with the Holsteins, urea in combination with rolled grain was toxic whereas greater amounts of urea in Stared were not. In one trial a steer (05) was given 616 g of urea in the form of Stared, but toxicity did not occur.

Toxicity developed in Animal 15 on the second day while she was receiving the control ration. This animal responded favorably when given 18 liters of 5% acetic acid by way of the tureen fistula. Similarly Animal 04 (control) was treated on the eleventh day and responded. However, Animals ~4 (control - 14th day) and 05 (control - 25th day) were treated with acetic but failed to respond. Perhaps that failure was due to a delay in administering acetic acid after first signs of toxicity were observed. To be effective it appears that acetic acid should be administered within 30 rain of the first signs of toxicity. The symptoms of toxicity were: dullness, muscle tremors, eyes rolling back into the head, frequent urination, excessive salivation, muscular incoordination, labored breathing and prostration at the approach of the terminal stage. In the time preceding the death of animal 14, the venous circulatory system was in a state of collapse (determined by venous puncture). The first toxic symptoms were usually noticed 20 min after urea administration.

The results of the experiment indicate that urea in Starea is not so toxic to animals as is urea mixed with rolled sorghum grain. An adaptation to high levels of urea was also illustrated because animals recently fed urea ingested almost twice as much urea before toxicity occurred as that ingested by nonadapted animals.

The concentration of ammonia in the 30-min sample appeared to have a greater bearing on whether toxicity would result than the concentration in samples taken later. If the ammonia concentration approached 100 mg/100 mL of rumen fluid in the 30 min sample, toxicity usually resulted. Ammonia concentration often exceeded the 100 mg concentration at later times without evidence of toxicity. On one occasion when Starea was fed, the ammonia concentration rose to 216 mg at 120 min without producing toxicity.

Validity criteria fulfilled:

not applicable

Conclusions:

Urea is an unpalatable feed ingredient for cattle so that the experiments were performed in rumen fistulated cattle.

Deaths occurred in animals administered urea at dose levels equivalent to 600 and 1080 mg/kg bw. Adaptation was demonstrated as lower toxicity seen in animals previously administered urea.

Executive summary:

In the study from Stiles et al. (1970) it was shown that urea is an unpalatable feed ingredient for cattle so that the experiments were performed in rumen fistulated cattle. The Starea ration (expansion-processed mixture of grain starch and urea) was the only ration readily consumed.

For the toxicity tests, the animals were "fed" with 1.4 kg of alfalfa hay and a quantity of grain mixture containing 20% urea (i.e., 200 g urea per kg feed).

Deaths occurred in animals administered urea at dose levels equivalent to 600 and 1080 mg/kg bw. Adaptation was demonstrated as lower toxicity seen in animals previously administered urea.

Since this formulation of urea is not relveant for the use of urea other then for feeding purposes of lifestock this formulation is not relevant for the environmental risk assessment. However, the finding that urea in feed other than Starea was unpaptable for cattle can be considered relevant for the risk assessment of wildlife. Hence, the results of thsi study are considered to have limited relevance for the environmental risk assessment of urea.

Reference 6

Endpoint:

toxicity to other above-ground organisms

Type of information:

other: summary of clinical signs in online handbook for veterinarians

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:

Description of urea poisoning in ruminants.

GLP compliance:

no

Test organisms (species):

other: Ruminants like cattle, horses, sheep

Duration:

24 h

Dose descriptor:

LC0

Effect conc.:

1 000 mg/kg bw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: For safety reasons 500 mg urea/ kg bw/day are recommended

Poisoning by ingestion of excess urea or other sources of nonprotein nitrogen (NPN) is usually acute, rapidly progressive, and highly fatal. NPN is any source of nitrogen not present in a polypeptide (precipitable protein) form. Sources of NPN have different toxicities in various species, but mature ruminants are affected most commonly. After ingestion, NPN undergoes hydrolysis and releases excess ammonia (NH₃) into the GI tract, which is absorbed and leads to hyperammonemia.

Etiology:

Ruminants use NPN by converting it via the ruminal microflora to ammonia, which is then combined with carbohydrate-derived keto acids to form amino acids. The most common sources of NPN in feeds are urea, urea phosphate, ammonia (anhydrous), and salts such as monoammonium and diammonium phosphate. Because feed-grade urea is unstable, it is formulated (usually pelleted) to prevent degradation to NH₃. Biuret, a less toxic source of NPN, is being used less frequently than in the past. Natural protein sources such as rice hulls, beet or citrus pulp, cottonseed meal, and straw or other low-quality forages may be treated with anhydrous ammonia to increase available nitrogen in supplemented livestock diets. Fermentation byproducts from alcohol (ethanol) manufacture are a source of NPN that comes from incomplete proteins, and these products are commonly used in liquid or feed supplements. Most sources of NPN are provided to ruminants by direct addition of dry supplement to a complete mixed or blended diet, by free-choice access to NPN-containing range blocks or cubes, or by lick tank systems combined with molasses as a supplement. Ammonia or NPN poisoning is a common sequela of abrupt change to urea or other NPN in the diet when only natural protein was previously fed; animals have to be gradually acclimated to NPN so that rumen microflora can increase in numbers to use the NH₃produced. Also, farm animals sometimes drink liquid fertilizers or ingest dry granular fertilizers that contain ammonium salts or urea.

Ruminants are most sensitive, because urease is normally present in the functional rumen after 50 days of age. Dietary exposure of unacclimated ruminants to 0.3–0.5 g of urea/kg body wt may cause adverse effects; dosages of 1–1.5 g/kg are usually lethal. Urease activity in the equine cecum is ~25% that of the rumen, and horses may receive NPN as a feed additive; however, horses are more sensitive to urea than other monogastrics, and dosages ≥4 g/kg can be lethal. Ammonium salts at 0.3–0.5 g/kg may be toxic in all species and ages of farm animals; dosages ≥1.5 g/kg usually are fatal. Pigs and neonatal calves are generally unaffected by ingestion of urea except for a transient diuresis. Wild birds (silver gulls) reportedly have been poisoned after consuming water contaminated with urea fertilizer spillage.

Livestock may require days or weeks for total adaptation before rumen microflora can utilize the gradually increasing amounts of urea or other NPN in the diets; however, adaptation is lost relatively quickly (1–3 days) once NPN is removed from the diet.

Diets low in energy and high in fiber are more commonly associated with NPN toxicosis, even in acclimated animals. Highly palatable supplements (such as liquid molasses or large protein blocks crumbled by precipitation), range cubes, or improperly maintained lick tanks may lead to consumption of lethal amounts of NPN.

A related CNS disorder in cattle fed ammoniated high-quality hay, silage, molasses, and protein blocks is thought to be caused by formation of 4-methylimidazole (4-MI) through the action of NH₃on soluble carbohydrates (reducing sugars) in these feedstuffs. Cattle fed dietary components containing 4-MI develop a syndrome known as the “bovine bonkers syndrome,” named for the wildly aberrant behavior exhibited. Signs relate to CNS effects, with stampeding, ear twitching, trembling, champing, salivating, and convulsions. Because nursing calves are affected, the toxic principle apparently is excreted in milk. Ammoniated low-quality forages do not have sufficient concentrations of reducing sugars to form 4-MI, and thus serve as a relatively safe nitrogen source for acclimated animals.

Another related disorder involves accidental excessive exposure of ruminants (cattle and sheep) to raw soybeans. Soybeans have high concentrations of both carbohydrates and proteins, as well as urease. Overconsumption can cause acute carbohydrate fermentation and excessive ammonia release, resulting in ammonia toxicosis and lactic acidosis. Affected animals have engorged rumens with a gray, amorphous mass inside.

Clinical Findings:

The period from urea ingestion to onset of clinical signs is 20–60 min in cattle, 30–90 min in sheep, and longer in horses. Early signs include muscle tremors (especially of face and ears), exophthalmia, abdominal pain, frothy salivation, polyuria, and bruxism. Tremors progress to incoordination and weakness. Pulmonary edema leads to marked salivation, dyspnea, and gasping.

Horses may exhibit head pressing; cattle are often agitated, hyperirritable, aggressive, and belligerent as toxicosis progresses; sheep usually appear depressed. An early sign in cattle is ruminal atony; as toxicosis progresses, ruminal tympany is usually evident, and violent struggling and bellowing, a marked jugular pulse, severe twitching, tetanic spasms, and convulsions may be seen. Affected cattle with belligerent aberrant behavior may have produced some 4-MI in vivo through reaction of excessive NH₃, released from NPN, with carbohydrates and reducing sugars in the rumen. The PCV and serum concentrations of NH₃, glucose, lactate, potassium, phosphorus, AST, ALT, and BUN usually are significantly increased.

As death nears, animals become cyanotic, dyspneic, anuric, and hyperthermic, and blood pH decreases from 7.4 to 7.0. Regurgitation may occur, especially in sheep. Death related to excess NPN usually occurs within 2 hr in cattle, 4 hr in sheep, and 3–12 hr in horses. Survivors recover in 12–24 hr with no sequelae.

Lesions:

Carcasses of animals dying of NPN poisoning appear to bloat and decompose rapidly, with no specific characteristic lesions. Gross brain lesions are not usually reported in NPN-induced ammonia toxicosis, but histopathologic lesions may include neuronal degeneration, spongy degeneration of the neuropil, and congestion and hemorrhage in the pia mater. Frequently, pulmonary edema, congestion, and petechial hemorrhages may be seen. Mild bronchitis and catarrhal gastroenteritis are often reported. Regurgitated and inhaled rumen contents are commonly found in the trachea and bronchi, especially in sheep. The odor of NH₃may or may not be apparent in ingesta from a freshly opened rumen or cecum. A ruminal or cecal pH ≥7.5 from a recently dead animal is highly suggestive of NPN poisoning. The ruminal pH remains stable for several hours after death under most circumstances but continues to rise in NPN toxicosis.

Diagnosis:

Ammonia or NPN poisoning is suggested by signs, lesions, history of acute illness, and dietary exposure. Exposure to excess NPN may be evaluated through laboratory analysis for the ammonia nitrogen (NH₃-N) in both antemortem and postmortem specimens and for urea or other NPN in suspected feeds and other dietary sources. Specimens for NH₃-N analysis include ruminal-reticular fluid, serum, whole blood, and urine. All specimens should be frozen immediately after collection and thawed only for analysis; alternatively, ruminal-reticular fluid may be preserved with a few drops of saturated mercuric chloride solution added to each 100 mL of specimen.

Animals dead more than a few hours in hot ambient temperatures or 12 hr in moderate climates probably have undergone too much autolysis to be of diagnostic value.

The amount of urea or the equivalent NPN in biologic specimens is meaningless; however, urea and NPN should be determined in representative feeds and other dietary sources. Values for urea and NPN in feed permit calculation of the protein equivalent (1 part protein = 0.36 parts urea; 1 part urea = 2.92 parts protein) in feed as well as the total estimated dose of NPN ingested.

NH₃-N concentrations of ≥2 mg/100 mL in blood, serum, or vitreous humor indicate excess NPN exposure. Clinical signs usually appear at ~1 mg/100 mL. The concentration of NH₃-N in ruminal-reticular fluid is >80 mg/100 mL in most cases of NPN poisoning and may be >200 mg/100 mL. Acclimated ruminants fed diets high in legume hay, soybean meal, cottonseed meal, linseed meal, fish meal, or milk byproducts may have NH₃-N concentrations in rumen fluid approaching 60 mg/100 mL with no apparent toxicity. The pH of ruminal-reticular fluid should also be determined; a pH of 7.5–8 (at time of death) is indicative of NPN toxicity.

Differential diagnoses include poisonings by nitrate/nitrite, cyanide, organophosphate/carbamate pesticides, raw soybean overload, 4-methylimidazole, lead, chlorinated hydrocarbon pesticides, and toxic gases (carbon monoxide, hydrogen sulfide, nitrogen dioxide); acute infectious diseases; and noninfectious diseases such as encephalopathies (eg, leukoencephalomalacia, hepatic encephalopathy, polioencephalomalacia), enterotoxemia or rumen autointoxication, protein engorgement, grain engorgement, ruminal tympany, and pulmonary adenomatosis. Nutritional and metabolic disorders related to hypocalcemia, hypomagnesemia, and other elemental aberrations should also be considered.

Treatment:

Examination and treatment may be difficult because of sudden and violent behavior. Animals that are recumbent and moribund usually do not respond favorably to treatment.

If possible, affected animals should be treated by ruminal infusion of 5% acetic acid (vinegar, 0.5–2 L in sheep and goats and 2–8 L in cattle). Ruminal-reticular fluid specimens for analysis should be taken before acetic acid therapy. Concomitant infusion of iced (0–4°C) water (up to 40 L in adult cattle, proportionally less in sheep and goats) is also recommended. Acetic acid lowers rumen pH and prevents further absorption of NH₃by converting uncharged NH₃to the charged ammonium ion (NH₄⁺); administration may have to be repeated if affected animals again show clinical signs. Acetic acid inactivates existing NH₃in the GI tract and rapidly forms ammonium acetate, which can be used by rumen microflora but does not release NH₃. Cold water lowers the rumen temperature and dilutes the reacting media, which slows urease activity. In severely affected valuable animals, removed rumen contents should be replaced with a hay slurry, and a transfer of some rumen contents from a healthy animal may serve as an inoculum to restore normal function. Ruminal tympany should be corrected if indicated, and a trocar may be installed to prevent recurrence.

Supportive therapy is indicated and includes IV isotonic saline solutions to correct dehydration, and IVcalcium gluconateand magnesium solutions to relieve tetanic seizures. Convulsions may also be controlled with sodiumpentobarbitalor other injectable anesthestic agents.

Prevention and Control:

Urea should not be fed at a rate exceeding 2%–3% of the concentrate or grain portion of ruminant diets and should be limited to ≤1% of the total diet. Additionally, NPN should constitute no more than one-third of the total nitrogen in the ruminant diet. Once the decision is made to feed NPN, animals must be slowly adapted to, and maintained on, a consistent dietary NPN content with no significant deviation; cows fed range cubes with NPN must receive the cubes daily with no interruptions. Temporary absences of NPN from the diet should be avoided at all costs. Overconsumption of palatable liquid supplements can be controlled by the addition of phosphoric acid; 1% phosphorus from phosphoric acid should restrict consumption of liquid supplement to ~2 lb/animal/day. Although properly adapted adult cattle can tolerate urea at a rate of up to 1 g/kg body wt/day, a safer feeding rate is no more than half that amount.

Validity criteria fulfilled:

not applicable

Executive summary:

Clinical Findings:

The period from urea ingestion to onset of clinical signs is 20–60 min in cattle, 30–90 min in sheep, and longer in horses. Early signs include muscle tremors (especially of face and ears), exophthalmia, abdominal pain, frothy salivation, polyuria, and bruxism. Tremors progress to incoordination and weakness. Pulmonary edema leads to marked salivation, dyspnea, and gasping.

Horses may exhibit head pressing; cattle are often agitated, hyperirritable, aggressive, and belligerent as toxicosis progresses; sheep usually appear depressed. An early sign in cattle is ruminal atony; as toxicosis progresses, ruminal tympany is usually evident, and violent struggling and bellowing, a marked jugular pulse, severe twitching, tetanic spasms, and convulsions may be seen. Affected cattle with belligerent aberrant behavior may have produced some 4-MI in vivo through reaction of excessive NH₃, released from NPN, with carbohydrates and reducing sugars in the rumen. The PCV and serum concentrations of NH₃, glucose, lactate, potassium, phosphorus, AST, ALT, and BUN usually are significantly increased.

As death nears, animals become cyanotic, dyspneic, anuric, and hyperthermic, and blood pH decreases from 7.4 to 7.0. Regurgitation may occur, especially in sheep. Death related to excess NPN usually occurs within 2 hr in cattle, 4 hr in sheep, and 3–12 hr in horses. Survivors recover in 12–24 hr with no sequelae.

Prevention and Control:

Urea should not be fed at a rate exceeding 2%–3% of the concentrate or grain portion of ruminant diets and should be limited to ≤1% of the total diet. Additionally, NPN should constitute no more than one-third of the total nitrogen in the ruminant diet. ... Although properly adapted adult cattle can tolerate urea at a rate of up to 1 g/kg body wt/day, a safer feeding rate is no more than half that amount.

Reference 7

Endpoint:

toxicity to other above-ground organisms

Type of information:

calculation (if not (Q)SAR)

Adequacy of study:

weight of evidence

Reliability:

3 (not reliable)

Principles of method if other than guideline:

Deer and moose are ruminants. No toxicity data specific to them was found. Given that they are ruminants, it seems reasonable to conclude that urea toxicity is similar to other ruminants, therefore in the vicinity of 500mg/kg of body weight.

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Test organisms (species):

deer

Study type:

other:

Dose descriptor:

LD0

Effect conc.:

500 mg/kg bw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: estimated from toxicity to cows

Remarks:

Duration not provided

The publication from Scott 2008 contains following statement: "Deer and moose are ruminants. No toxicity data specific to them was found. Given that they are ruminants, it seems reasonable to conclude that urea toxicity is similar to other ruminants, therefore in the vicinity of 500mg/kg of body weight."

Validity criteria fulfilled:

not applicable

Executive summary:

The publication from Scott 2008 contains following statement: "Deer and moose are ruminants. No toxicity data specific to them was found. Given that they are ruminants, it seems reasonable to conclude that urea toxicity is similar to other ruminants, therefore in the vicinity of 500mg/kg of body weight."

Description of key information

In the monitoring of field studies no direct toxic effects were found for mammalians (Sullivan and Sullivan, 2017). However, urea-fertilized areas were avoided by reindeer for winter grazing (Sullivan and Sullivan, 2017).

 

Avoidance of urea in feed was observed for cattle ("Urea is an unpalatable feed ingredient" Stiles et al. 1970). This and the relative low toxicity of about 500 mg urea/kg/day in cattle (Thompson, 2015) might be the reason why no toxic effects were observed on mammals in field trials (Sullivan and Sullivan 2017).

 

Some amphibians react when exposed to urea while others are relatively insensitive (Marco et al. 2001, Sullivan and Sullivan 2017). It is unclear as to the mechanism of urea toxicity in amphibians and why some species seem relatively unaffected. Sensitive species were reported predominantly for North American forest dwelling amphibians and hence this finding might have limited relevance for risk assessment for European ecosystems. For some of these North American forest dwelling amphibians avoidance behaviour has been described (Hatch et al. 2001).

Key value for chemical safety assessment

Short-term EC50 or LC50 for mammals:

106 g/kg food

Long-term EC10, LC10 or NOEC for mammals:

30 g/kg food

Additional information

Calculation of the EC50:

In the study reported by Stiles et al. (1970) the rumen-fistulated Holstein cows (680 kg) received 60 g urea per 100 kg=> 408 g urea.

On the day of each trial all animals were fed 1.4 kg of sorghum grain and 1.4 kg of alfalfa hay at 8 AM. At 1 PM they were fed 1.4 kg of alfalfa hay and a quantity of grain mixture containing 20% urea. The quantity of grain mixture fed was based on the weight of the animal determined that day.

The exact % of urea in the feed was not provided in the study report. However, when 408 g are considered to be 20 % in the grain, this results in the amount of 2040 g grain for the application. Adding the this amounts to 3440 g of feed plus 408 g of urea this summarizes to total 3848 g. Hence, the share of urea is 408/3848 =10.6 % or 106 g urea /kg feed.

Note: Due to poor acceptability, the grain mixture containing urea was placed in the rumen by way of the fistula after the first day in amounts to supply the quantities of urea. Hence, the amount was added within very short time and not in a normal feeding process. Hence, it represents the worst case.

Information for the EC10/NOAEC for mammals:

"Use of low concentrations of urea (up to 3%) as a nitrogen supplement to ruminant feeds serves as an inexpensive low-toxicity source of protein for domestic animal production (Stanton and Whittier 2006)", cited from Sullivan and Sullivan (2017).