Urea

Toxicity to other aquatic organisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to other aquatic vertebrates

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

Principles of method if other than guideline:

Materials and Methods
Eggs and embryos of G. riobambae were obtained from spontaneous matings that occur in the laboratory. Methods for the maintenance of tadpoles and adults of this species, as well as, for the handling of eggs and embryos have been described (9). Xenopus embryos were obtained by artificial insemination of eggs, after stimulation of male and female frogs with 500-1,000 units of human chorionic gonadotropin (HCG, Schering AG, Berlin). The jelly layers that surrounds the egg, in both species, were removed by treatment with 2.5% cysteine hydrocholoride pH 7.4-7.8 for 10 to 15 minutes with agitation. Gastrotheca tadpoles used in this study range from the recently born tadpoles (stage 33) to large premetamorphosing tadpoles (stage 40). Free-living tadpoles of G. riobambae were staged according to (ll), while the staging of G. riobambae embryos from the pouch, before birth, was done according to (5). Embryos of X. laevis were staged according to (20).

The capsular fluid of advanced embryos from the pouch of G. riobambae (stages 23 to 25) was collected in a capillary tube after puncturing the embryonic capsule with a pair of forceps. About 10 to 20 µl of fluid were obtained from each embryo. The fluid from several embryos was pooled and centrifuged to remove red blood cells, since the capsular fluid becomes contaminated with blood from the bell gills, at the time of opening the embryonic capsule. The supernatant was diluted with distilled water and frozen at -20°C until use. See (9) for the method used to remove embryos from the maternal pouch. Blood samples from adult frogs were taken in heparin treated capillary tubes from a vein of the jaw (21). To minimize the differences that may be due to the feeding conditions, the frogs were fasted for one week before obtaining a blood samples of 20 to 40 µl. The serum was separated from blood cells by centrifugation and was diluted with distilled water. Samples were frozen at -20°C until use. Urea-nitrogen and ammonia- nitrogen were measured by the Berthelot method (lo), using the colorimetric kits of Boehringer (Mannheim) and SIGMA. Absorbances were measured with a Beckman DU-64 spectrophotometer at 550 nm. Ammonia-nitrogen was determined by omitting urease from the reaction mixture. Urea- nitrogen was obtained by subtracting ammonia- nitrogen from the total-nitrogen determination (23). To test the tolerance of G. riobambae tadpoles to urea and other compounds, one or two free- living tadpoles (stages 33 to 40) were incubated in 45 ml of each solution at room temperature (21 "C). The incubation of tadpoles in 0.1 Modified Ringer's Solution (MR) (pH 6.5) served as control. All solutions were made in 0.1 MR and the tadpoles were maintained in the same solution for a 120- hour incubation period. In the case of sucrose, the solution was changed daily to reduce microbial growth. See Table2 for the composition of MR. The osmolarity of several amphibian saline solu- tions was measured in an osmometer (Fa Roebling, Berlin).

Animal cap explants of middle blastulae of X. laevis at stage 8, were isolated as described elsewhere (12), and treated with 50 mM urea in Steinberg's solution for 6 hours or with 250mM urea in Steinberg's solution for either 15 minutes or 3.25 hours. After these treatments, the explants were transferred to Steinberg's solution and cultured for 3 days at 20°C, fixed and analyzed by standard methods (13).

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Aquatic vertebrate type (other than fish):

frog

Test organisms (species):

Xenopus laevis

Details on test organisms:

Gastrotheca riobambae and Xenopus laevis

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

120 h

Duration:

120 h

Dose descriptor:

LC0

Effect conc.:

18 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Gastrotheca riobambae Tadpoles

Duration:

120 h

Dose descriptor:

LC0

Effect conc.:

300 mmol/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: Gastrotheca riobambae Tadpoles

Dose descriptor:

NOEC

Remarks:

In Steinberg´s medium

Effect conc.:

>= 50 mmol/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

morphology

Remarks on result:

other: X. laevis duration not explicitly provided, from stage 8 to stage 37.

Dose descriptor:

NOEC

Remarks:

in Steinberg´s medium

Effect conc.:

>= 3 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

morphology

Remarks on result:

other: X. laevis duration not explicitly provided, from stage 8 to stage 37.

Effects observed in Xenopus laevis were considered to be primarily caused by the high osmolarity of the solution.

 

Fertilized Xenopus eggs, whose jelly layers and vitelline envelope have been removed, were cultured in Steinberg's and GRS with and without the addition of urea. Up to stage 8, embryos were cultured with the addition of 30 mM urea. Afterwards, the concentration of urea was raised to 50 mM and embryos were cultured until stage37. Xenopus embryos acquired a characteristic U shape when cultured in GRS with urea (Fig.2a). Similar abnormalities were observed when embryos were cultured within the vitelline envelope in this solution (data not shown). In contrast, embryos cultured in GRS without urea developed normally (Fig.2b). Embryos cultured in Steinberg's solution with or without urea developed normally (Figs.2c,d). The altered morphology observed when Xenopus embryos were cultured in GRS plus urea (Fig.2a) may be due to the high osmolarity of this solution.

 

It has been reported that urea may influence the biological activity of proteinaceous inducing factors (16). In addition, there exists the possibility that urea may interact with extracellular matrix proteins or other proteins, including receptors for inducing factors, integrated into the plasma membrane. By change of the conformation or liberation of masked endogenous factors, mesodermal and neural structures maybe induced. However, we could show in the animal cap assay with Xenopus, that such an effect of urea can be excluded under our experimental conditions. We used not only 50mM urea, but raised the concentration to 250 mM urea in Steinberg's solution for the treatment of animal cap explants. Treatments with urea, including 250 mM urea for 3.25hours did not have mesodermal or neural inducing activity in animal explants of Xenopus.

The segmenting eggs and early embryos of X.laevis are routinely cultured in solutions of low ionic strength suchas0.1MR.WhenembryosofXeno-pusareplacedin full strength physiological saline solutions, the epithelial layer is destabilized, resulting in abnormal ingression of surface cells during gastrulation and in exogastrulation.

Conclusions:

120 h LC0: 18000 mg urea/L Gastrotheca riobambae
NOEC: 3000 mg urea/L Xenopus laevis (stage 8 to 37)

Executive summary:

The publication of del Pinto et al. 1994 state that "Until recently, embryos of Gastrotheca could be cultured in vitro only within the embryonic capsule (9). Based on the findings that urea occurs in the capsular fluid during incubation in the maternal pouch, and that free-living tadpoles of Gastrotheca tolerate urea, we designed a saline solution, that contains urea, which allows successful culture of early embryos of this frog. In addition, we found that urea is tolerated by embryos of X.laevis."

 

The 120 LC0 for Gastrotheca riobambae was 18000 mg urea/L.

The NOEC for X. laevis (stage 8 to 37) was >= 3000 mg urea/L.

 

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

Reference 2

Endpoint:

toxicity to other aquatic vertebrates

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:

according to guideline

Guideline:

other: ASTM 1997a, 1997b

Version / remarks:

American Society for Testing and Materials (ASTM) (1997a) Standard guide for
conducting the frog embryo teratogenesis assay--Xenopus (Fetax). ASTM E 1439-
91. Philadelphia, PA
American Society for Testing and Materials (ASTM) (1997b) Standard practice for
conducting acute toxicity tests with fishes, macroinvertebrates, and amphibians.
ASTM 729-96. Philadelphia, PA

Deviations:

not specified

GLP compliance:

no

Specific details on test material used for the study:

The purity of the urea was >99.9%.

Analytical monitoring:

no

Vehicle:

no

Aquatic vertebrate type (other than fish):

frog

Test organisms (species):

Xenopus laevis

Details on test organisms:

TEST ORGANISM
The Pacific treefrog, Pseudacris regilla (Baird and Girard), is the most common frog in the Pacific Northwest and is found in shallow wetland areas which are frequently dry prior to mid-summer (Corkran and Thoms 1996).

The African clawed frog, Xenopus laevis Daudin is a common test organism.

- Source: P. regilla tadpoles were raised from egg masses collected
in Corvallis, OR. X. laevis tadpoles were raised from eggs from an in-house breeding
colony.

- Age at study initiation (mean and range, SD): P. regilla tadpoles 12 d post hatch, X. laevis tadpoles 13 dph
- Weight at study initiation (mean and range, SD): 60 mg/ tadpole for P. regilla and 15 mg/tadpole for X. laevis

- Method of breeding: P. regilla tadpoles were raised from egg masses collected
in Corvallis, OR. X. laevis tadpoles were raised from eggs from an in-house breeding
colony.

- Feeding during test
P. regilla were fed ground pelleted rabbit food and X. laevis were fed dried Oregon Moist fish food pellet (3% of their body weight per day during testing).

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

10 d

Test temperature:

22 °C

Nominal and measured concentrations:

Contrrol, 330, 823, 2058, 5145, 12863 and 32157 mg urea/L

Details on test conditions:

TEST SYSTEM

- Test vessel fill volume: 1000 mL
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 5 fpr P. regilla and 15 for X.laevis
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Water quality parameters (mean ± SE) of well water drawn for the tests were: dissolved oxygen, 8.7 ± 0.2 mg/L; total hardness, 25.5 ± 1.7 mg/L as CaCO3, total alkalinity, 24.2 ± 1.6 mg/L as CaCO3; conductivity, 82.3 ± 3.7μS/cm; median pH, 6.8. Background ammoniumnitrogen concentrations in the well water ranged from 0.005 to 0.010 mg/L NH 4-N.
- Intervals of water quality measurement: daily

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16:8 light:dark cycle

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality, length weight

Reference substance (positive control):

no

Key result

Duration:

10 d

Dose descriptor:

LC50

Effect conc.:

19 526 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other:

Remarks:

X. laevis tadpoles

Key result

Duration:

10 d

Dose descriptor:

NOEC

Effect conc.:

5 145 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Remarks on result:

other: X. laevis

Remarks:

lowest NOEC

Duration:

10 d

Dose descriptor:

LC50

Effect conc.:

17 999 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks on result:

other: P. regilla tadpoles

Duration:

10 d

Dose descriptor:

NOEC

Effect conc.:

5 145 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Remarks:

and length

Remarks on result:

other: P. regilla tadpoles

Details on results:

- Behavioural abnormalities: not reported
- Observations on body length and weight: yes, NOEC based on these values
- Other biological observations: no
- Mortality of control: no mortality observed
- Abnormal responses: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none reported
- Effect concentrations exceeding solubility of substance in test medium: none reported

Validity criteria fulfilled:

yes

Conclusions:

10 day-LC50 for P. regilla of 17999 mg urea/L
10-day LC50 for X. laevis: 19526 mg urea/L

Executive summary:

In the study from Schuytema and Nebeker (1999) the effect of urea on tadpoles of the Pacific treefrog, Pseudacris regilla and the African clawed frog, Xenopus laevis was investigated according to ASTM 1997.

In its pure form urea affects tadpoles only at extremely high concentrations with LC50 for P. regilla of 17999 mg urea/L and 19526 mg urea/L for X. laevis. Growth effects were observed at 12,800 mg urea /L, the NOEC is 5145 mg urea/L for both species.

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

Reference 3

Endpoint:

toxicity to other aquatic vertebrates

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:

Two Asiatic toad egg masses were collected from a pond (104.0723E, 30.6309N) at the Chengdu Branch, Chinese Academy of Sciences, Chengdu, Sichuan Province, China, on March 15, 2016. The egg masses were returned to the laboratory to hatch and develop together in a plastic container (42 x 30 x 23 cm) containing 25 L of water that was filtered with activated carbon to remove nitrate, ammonium, and chloride until the tadpoles reached a stage equivalent to Gosner 26 (Gosner 1960).

Four different concentrations of urea treatments (200, 400, 600, and 1200 mg/L) and one control treatment (0 mg/L) were prepared using urea crystals (CAS#57-13-6; Aladdin, Shanghai, China) with water filtered with activated carbon. Each treatment was replicated three times. These concentrations were selected based on urea concentrations in farmland water bodies in China of approximately 2-800 mg/L (Wang et al. 2004; Ji et al. 2006). More importantly, our recent filed observations found that the highest concentrations in farmland water bodies could be up to 1200 mg/L.

Thirty mosquito fish individuals were sampled from a pond (104.0772 E, 30.6335 N) at Sichuan University, Chengdu, Sichuan Province, China, on March 3, 2016, to condition water with mosquito fish chemical cues. The fish were transported to the Chengdu Institute of Biology, Chinese Academy of Sciences. In the laboratory, we used three plastic tanks (31.5 9 24.5 9 9.5 cm) for fish husbandry and water conditioning. Specifically, these plastic tanks were filled with 7 L of filtered dechlorinated tap water separately, with one culture tank for feeding, one clearing tank for fish to empty the guts, and one conditioning tank for preparing water with mosquito fish chemical cues (Angelon and Petranka 2002). These fish were fed commercial fish food instead of natural prey in the culture tank, because tadpoles might sense predation risk from injured conspecifics (Ferrari and Chivers 2009). Two hours after feeding, fish were transferred into the clearing tank for 2 h to clear their guts before transfer to the conditioning tank. This process continued for more than 2 weeks before the experiments.

Experiment #1
Experiment #1 was composed of three steps, including tadpoles exposure in urea concentrations, behavior observation (with and without mosquito fish cues), and morphological traits measurement. Specifically, exposure was conducted in 1000-mL glass beakers, each of which contained 1 L of the solution. Tadpoles were randomly selected and separated to four trial groups and one control group. According to previous studies (Xu and Oldham 1997; Ortiz-Santaliestra et al. 2011), 10 tadpole individuals were placed in each glass beaker. Experiment #1 was conducted at an air temperature of 25.0 ± 0.5 C (mean ± SD; controlled by an air-conditioning system), and mean water temperature was 20.1 ± 0.5 C SD. We adjusted the pH to slightly above seven with baking soda in order to reduce tadpoles physical stress. pH, conductivity, and dissolved oxygen were measured using a portable multiparameter meter (WTW Multi 3400i, GmbH, Germany) before the experiment, and the data are presented in Table 2. Exposure lasted for 7 days, and all tadpoles were in stage 26. The mean body length and mean body mass of the tadpoles were 12.64 ± 1.18 mm (mean ± SD) and 0.037 ± 0.006 g (mean ± SD), respectively, before the experiment, based on the measurements of 15 randomly selected individuals. Test tadpoles were fed ad libitum with boiled lettuce on the first and fourth days of exposure. Excess food, feces and dead tadpoles were removed daily from the glass beakers. The solution was renewed entirely at day four of the experiment before feeding to maintain the same concentration. This was performed because previous studies demonstrated that no significant change occurred in the manipulated nitrogen concentrations within 7 days (Marco et al. 1999). Behavioral observations began after 7 days of exposure. Specifically, three tadpoles out of 10 per glass beaker were randomly selected and transferred into a plastic container (29 9 19.5 9 7 cm) containing 1 L of solution of the same concentration individually. This setup allowed us to have nine individuals from each concentration to conduct the behavioral observations (45 tadpoles in total). After acclimation for 10 min, we recorded the activities of the three tadpole individuals for 10 min. In order to reduce the disturbance of researchers, the observer stood motionless approximately 1 m away, and observations were dictated (Kruuk and Gilchrist 1997). Following Burgett et al. (2007), containers were divided into four quadrants and we recorded the number of times the tadpoles crossed the boundary of any two quadrants (as a measure of tadpole activity; Figure S1). After the first observation, 50 mL of water with mosquito fish chemical cues was added into the plastic container. Then, tadpoles were re-acclimated for 10 additional minutes, and we repeated the behavioral observation process as described above. This procedure is commonly used in many previous studies (Kruuk and Gilchrist 1997; Marquis et al. 2004; Burgett et al.2007) to observe the pre- and post-cue activity levels of tadpoles. At the end of Experiment #1 (7-day exposure), all surviving tadpoles were preserved in 5% formaldehyde. In total, 150 tadpoles were preserved in Experiment #1, and each individual was measured for body mass and morphological traits subsequently.

Experiment #2
Experiment #2 was conducted 10 days after the start of Experiment #1, which also had three steps as Experiment #1. During this period, tadpoles that were not exposed to solutions in Experiment #1 were fed ad libitum with boiled lettuce every day. With 10 days of development, these tadpoles were in stage 28 (Gosner 1960). Based on the measurements of 15 randomly selected individuals, the total length was 19.52 ± 1.70 mm (mean ± SD), and the body mass was 0.093 ± 0.017 g (mean ± SD). For combination with Experiment #1, Experiment #2 was conducted in 1000-mL glass beakers to identify whether urea and developmental stages affected tadpoles functional traits, and whether intraspecific trait variability was disturbed by urea at different concentrations. Therefore, we repeated the entire treatment process that was used in Experiment #1. All living tadpoles (150 individuals) were also preserved in 5% formaldehyde and measured for body mass and morphological traits after 7 days of exposure. The air temperature was 25.0 ± 0.5 C (mean ± SD), and the water temperature was 20.0 ± 0.5 C (mean ± SD). pH was also adjusted to around seven with baking soda, and data on pH, conductivity, and dissolved oxygen (WTW Multi 3400i, GmbH, Germany) are presented in Table 2.

Functional traits
Each preserved tadpole individual was measured for a set of morphological traits using the Mshot Image Analysis System (Mc50-N) on a stereomicroscope (JSZ8T; Jiang Nan Yong Xin, China) and a digital caliper to the nearest 0.01 mm in the laboratory. The morphological traits we measured include the following: body width (BMW), interocular distance (IO), body length (BL), and total length (TL). These morphological traits were used to estimate two of the three selected functional traits, the details of which are presented as follows: In our study, the body mass (M) of each individual was directly measured using an electronic scale to the nearest 0.001 g, and the logarithm was taken as log (M ? 1) for statistical analysis (Akin and Winemiller 2008). Eye distance values were estimated using IO/BMW, and the values of trunk bending shape were estimated as BL/TL (Table 1).


Statistical analyses
Tadpole activities (movements) and functional traits values were tested for normality using the Shapiro– Wilk test. We first used repeated measure ANOVAs (a = 0.05) to test the potential effects of different urea concentrations and mosquito fish chemical cues (defined as a repeated measure) on the activities of tadpoles, and determine whether tadpoles anti-predator behavior was altered by urea at different concentrations. Using the same test, we also examined the effects of urea concentrations and development stages (defined as a repeated measure) on each functional trait, and determined whether intraspecific trait variability was disturbed by different concentrations of urea. Mauchly’s sphericity tests were used to ensure that the sphericity assumption of the tests was met, and appropriate adjustments were made if the assumption of sphericity was violated. Then, Fisher’s LSD tests were used to compare (1) the difference in tadpole activities between different concentration groups in Experiment #1 and #2, separately, and (2) the difference of each functional trait between different concentration groups. All statistical analyses were conducted in R 3.3.2 (R development Core Team 2011).

GLP compliance:

no

Analytical monitoring:

no

Vehicle:

no

Aquatic vertebrate type (other than fish):

frog

Test organisms (species):

Bufo bufo gargarizans

Details on test organisms:

tadpoles

Test type:

static

Water media type:

freshwater

Remarks:

filtered dechlorinated tap water

Limit test:

no

Total exposure duration:

7 d

Test temperature:

25°C

pH:

7.05 to 7.14

Dissolved oxygen:

5.37 to 5.43 mg/L

Conductivity:

287 to 291 µS/cm

Nominal and measured concentrations:

Control, 200, 400, 600, 1200 mg urea/L

Details on test conditions:

see above

Reference substance (positive control):

no

Duration:

7 d

Dose descriptor:

LC0

Effect conc.:

>= 1 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

>= 1 200 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

behaviour

Remarks:

moves

Remarks on result:

other: At lower concentrations, some potential increase of activity was observed at the lower concentrations when compared to the control. This could not be confirmed in the high concentration (no difference to control) and neither in experiment #2.

Details on results:

Although no significant effects of developmental stages were detected, the interaction between urea concentrations and developmental stages on functional traits was significant (e.g., for trunk bending shape and eye position). This result indicated that functional traits of tadpoles in different developmental stages became similar with the exposure to different urea concentrations. (The functional similarity increased between developmental stages.)

Conclusions:

7-day LC0 tadpoles >= 1200 mg/L (mortality)
7-day NOEC tadpoles >= 1200 mg/L (development)

Executive summary:

Urea with concentrations up to 1200 mg urea/L had no effect on the mortality and the development of tadpoles of the Asian Toad Bufo gargarizans during a 7 day exposure (Zhao et al 2018). Some effects on e.g. the position of the eyes might be present (even if there might be a statistical significance, the results from the highest test concentration are within the range of the control animals.

Description of key information

Aquatic amphibians are not sensitive to urea

Additional information

Aquatic amphibians are not sensitive to urea:

Zhao et al. (2018) reported the effect of urea on Asiatic toad (Bufo gargarizans) tadpoles as 7 -day LC0 tadpoles >= 1200 mg/L (mortality) and the 7-day NOEC tadpoles >= 1200 mg/L (development).

del Pinto et al 1994 provided an 120 h LC0 of 18000 mg urea/L for Gastrotheca riobambae and a NOEC of >= 3000 mg urea/L Xenopus laevis (stage 8 to 37).

Schuytema and Nebeker 1999 determined the 10 day-LC50 for Pseudacris regilla 17999 mg urea/L and the 10-day LC50 for Xenopus laevis as 19526 mg urea/L The NOEC is 5145 mg urea/L for both species.