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Endocrine disrupter testing in aquatic vertebrates – in vivo

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amphibian Xenopus laevis, eleutheroembryo: (sub)lethal effects  
Type of information:
experimental study
Adequacy of study:
weight of evidence
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
Urea is Necessary for the Culture of Embryos of the Marsupial Frog Gastrotheca riobambae, and is Tolerated by Embryos of the Aquatic Frog Xenopus laevis
del Pino EM; Alcocer I and H Grunz
Bibliographic source:
Develop. Growth & Differ., 36 (l), 73-80 (1994)

Materials and methods

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:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
It is assumed that the purity is 100%

Sampling and analysis

Analytical monitoring:

Test solutions


Test organisms

Aquatic vertebrate type:
Test organisms (species):
Xenopus laevis

Study design

Test type:
Water media type:
Remarks on exposure duration:
duration not explicitly provided, from stage 8 to stage 37.

Results and discussion

Effect concentrations
Dose descriptor:
Effect conc.:
>= 3 000 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
larval development
Remarks on result:
other: duration: from stage 8 to 37

Any other information on results incl. tables

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 such as 0.1MR. When embryos of Xeno-pusare placed in full strength physiological saline solutions, the epithelial layer is destabilized, resulting in abnormal ingression of surface cells during gastrulation and in exogastrulation.

Applicant's summary and conclusion

Validity criteria fulfilled:
not applicable

Executive summary:

The NOEC for Xenopus laevis (stage 8 to 37) was >= 3000 mg urea/L, the highest tested concentration. no developmental changes caused by urea were observed and hence it was proposed to add urea to the culture medium. No effects related to endocrine disruption were reported but assessment was reported only up to stage 37..