ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY ENVIRONMENTAL RESEARCH SECTION B ARTICLE NO.

39, 2126 (1998)

ES971586

Cadmium Uptake and Bioaccumulation in Xenopus laevis Embryos at Different Developmental Stages
Jorge Herkovits,* Pietro Cardellini,- Cristina Pavanati,- and Cristina S. Perez-Coll*
- Dipartimento di Biologia, Universita degli Studi di Padova Via Trieste, 75-35121 Trieste, Italy; and *Programa de Seguridad Qumica, & Instituto de Ciencias Ambientales y Salud, Fundacion PROSAMA, Nicasio Orono 710, 1405 Buenos Aires, Argentina & J Received April 21, 1997

The uptake of cadmium in Xenopus laevis embryos was studied by exposing them to solutions containing cadmium concentrations ranging from 0.1 to 2 mg Cd2 /L at seven developmental stages for 72 h. The uptake values were from 0.0027 g Cd2 /embryo (two blastomeres stage) to 0.081 g Cd2 /embryo (hindlimb bud distinct stage). During early developmental stages, a limited permeability to cadium could play a signicant role for the survival of embryos. At the last developmental stage evaluated there was a signicantly higher uptake of cadmium simultaneously with a very signicant increase in the resistance against cadmium toxicity. The cadmium bioaccumulation factor (BF) ranged from 5 to 460. As a general pattern at all developmental stages the BF was higher in embryos exposed to the lower cadmium concentration, and as develop1998 Academic Press ment advanced the BF increased.

INTRODUCTION

The worldwide decline of amphibian populations may be related to their high susceptibility to environmental pollution (Simms, 1969; Baringa, 1990; Herkovits et al., 1996), particularly in certain early stages of their development (Herkovits and Perez-Coll, 1990, 1993). Amphibian em bryos are increasingly taken into account for hazard assessment of chemicals (Cook, 1981; Bantle et al., 1989; Herkovits and Perez-Coll, 1990; Vismara et al., 1993; Bernardini et al., 1994), water quality for wildlife protection purposes (Bohmer and Rahmann, 1990; Boyer and Grue, 1995), and in toxicity test of environmental samples (Dawson et al., 1985, Vismara et al., 1993; Herkovits, 1995; Herkovits et al., 1996). FETAX is the best known rapid test to identify developmental toxicants with amphibian embryos starting from midblastula to early gastrula stage onward for 96 h (ASTM, 1994). The results (mortality, teratogenicity, and growth inhibition) within this test involve stages which exhibit very
 To whom correspondence should be addressed. Fax: 54 1 431 4206. 21

dierent susceptibility to xenobiotics (Bustuoabad et al., 1977, Perez-Coll et al., 1986; Perez-Coll and Herkovits, 1990; Beattie et al., 1992; Herkovits and Perez-Coll, 1993). Although the stage-dependent susceptibility could be related in some extent to a dierential uptake of the xenobiotic, there are unexpected situations, e.g., during the last embryonic stages of Bufo arenarum, in which a 25 times increase in the resistance to the lethal eect exerted by cadmium occurs while the uptake of this heavy metal only increases about 15 times (Perez-Coll and Herkovits, 1996). The stage-dependent changes in the uptake of xenobiotics also could be relevant for bioavailability studies and seem to oer protable information in hazard and risk assessment of environmental stressors. Moreover, acute exposure-uptake data could be meaningful for water quality criteria and water quality-based toxics control (USEPA, 1991). Cadmium is released to the air, water, and land by human activities. It exerts a large number of adverse eects on humans and animals (IPCS, 1992) including developmental and reproductive impairments (Belmonte et al., 1989; Rivera et al., 1990). In Xenopus laevis embryos, based on lethality data, susceptibility to cadmium seems to increase gradually from 2 blastomeres stage up to stage 40 (open mouth) while an increase in resistance occurs (Herkovits et al., 1997) at stage 47 (the end of embryonic development). The main aim of this study was to evaluate the cadmium uptake and bioaccumulation in X. laevis embryos at 7 dierent developmental stages from the 2 blastomeres up to stage 47 (Niewkoop and Faber, 1985) and to correlate this result to the embryonic susceptibility to this heavy metal. For the record of intraspecic variation of the biological material, the coefcients of variation (CV) for susceptibility to cadmium among embryos obtained from the dierent couples of parents, at each of the 7 developmental stages evaluated, will also be reported.
MATERIALS AND METHODS

Ovulation of X. laevis females was induced by means of ip injection of 500 IU of chorionic gonadotrophin (Profasi,

0147-6513/98 $25.00 Copyright 1998 by Academic Press All rights of reproduction in any form reserved.

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HERKOVITS ET AL.

Serono, Italy) 24 h before eggs were needed. Squeezed eggs were fertilized in vitro with a sperm suspension made in 10% Holtfreters solution (HS). Jelly coat was dissolved by means of 3-min treatment with 2% L-cysteine at pH 8.1. The development of embryos was staged according to the table of Niewkoop and Faber (1985). Embryos were treated with cadmium during 72 h beginning at the following stages: two blastomeres stage (S.2), midcleavage (S.8), neural plate (S.13), tail bud (S.27S.28), heart beat (S.33), blood circulation in gills and mouth broken throughout (S.40S.41), and hindlimb bud distinct (S.47). For uptake experiments, in each case 20 individuals were placed in glass petri dishes containing 20 ml of cadmium solutions at the following nominal concentrations: 0.1, 0.2, 0.5, 1.0, and 2.0 ppm. The uptake/bioaccumulation experiments were repeated four times with embryos obtained from four couples of parents. For toxicity data the range of cadmium concentrations was extended up to 4 mg Cd>/L (nominal concentration) and LC , LC , and LC for 72 h were obtained by means of    PROBIT analysis for the dierent developmental stages. The controls were groups of 20 individuals at each evaluated stage incubated in HS without additions and maintained simultaneously with treated embryos. Experiments were carried out at a controlled temperature of 20$0.5C. Cadmium solutions were prepared from a standard solution of 1 g Cd>/L (CdClH O, Merck, Germany) and diluted  with HS. The lethal eect of cadmium on embryonic survival was evaluated daily and reported as LC , LC , and   LC for 72 h of exposure. At the end of the treatments,  embryos were twice washed with HS, dehydrated to constant weight, and digested with 1 ml of nitrosulfuric acid (BDHD Aristar, 70.5%) until complete mineralization.

Cadmium contents from the surviving embryos were quantied with a PerkinElmer 4000 atomic absorption spectrophotometer at the wavelength of 228.8 nm, detection limit 0.005 mg Cd>/L. Calibrations were performed using a standard solution (Sigma) for atomic absorption spectrophotometry. The accuracy of the determination was checked by analyzing three replicates of a solution containing 0.1 mg Cd>/L. The bioaccumulation factor (BF) for cadmium was reported as the ratio of its concentration in tissue versus its concentration in the experimental maintaining media. Results were statistically analyzed with the nonparametric KruskalWallis test with multiple contrasts using the STATIGRAPHICS program, version 4.0.
RESULTS

A summary of the stage-dependent susceptibility to cadmium on X. laevis embryos based on LC , LC , and LC    data can be observed in Fig. 1. As a general pattern there was an increase in the susceptibility to cadmium from the early developmental stages onward up to S.40, with a LC /72 h ranging from about 1 to 0.2 mg Cd>/L, respec tively, while at S.47 the resistance to cadmium increased up to 1.6 mg Cd>/L as the LC /72 h.  Figure 2 demonstrates the cadmium uptake by X. laevis embryos based on the above mentioned stage-dependent susceptibility protocol. In order to compare cadmium uptake results during developmental stages with dierent susceptibility, the concentrations evaluated in the present study ranged between values which allow, in most of the cases, the survival of a signicant number of embryos at least during 72 h of exposure (Figs. 3a, 3b, and 3c). Cadmium uptake

FIG. 1. Stage development susceptibility of Xenopus laevis embryos based on LC , LC , and LC values for 72 h of exposure.   

CADMIUM UPTAKE IN AMPHIBIAN EMBRYOS

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FIG. 2. Cadmium uptake in Xenopus laevis at dierent developmental stages after 72 h of treatment. All the values are signicantly dierent (P(0.05) from those corresponding to S.2 except the data noted by an asterisk.

increased as developmental stages advanced (e.g., for exposure to 0.1 mg Cd>/L at S.47 it was about 6 times higher than at S.2, while for 2 mg Cd>/L at the same developmental stages it was about 20 times higher). Up to stage 33 the cadmium uptake appeared not to be very related to the amount of cadmium in the maintaining media, while in later stages, especially at stage 47, it could be observed that the uptake was clearly related to the external cadmium concentration (Fig. 2). Moreover, at early developmental stages (S.2S.33) a slightly lower uptake of cadmium was registered in the embryos treated with the higher cadmium concentrations (Fig. 2), while on the other hand, there was an increase in the resistance to cadmium at the last embryonic stage evaluated in the present study (S.47) in spite of the simultaneous increase in the uptake of cadmium (Figs. 1 and 2). The mean embryonic (dry) weight was 0.44 mg$ 0.05 mg and no signicant dierence (P(0.05) was found among the developmental stages evaluated. The BF for cadmium ranged from 5 to 460 (Fig. 4). As a general pattern, at all developmental stages the BF was higher in embryos exposed to the lower cadmium concentration and as development advanced the BF increased. The CV for cadmium uptake at early developmental stages (S.2S.27/S.28) ranged between 5 and 44%, indicating more constant behavior than at the last stages in which the CV ranged between 45 and 82%.
DISCUSSION

The results of the present study reveal that cadmium is highly toxic to X. laevis embryos because a concentration of about 0.2 mg Cd>/L arrested the development of 50% of the embryos at stage 40. Regarding the stage-dependent

susceptibility, there is an increase in the susceptibility to cadmium from the early developmental stages onward up to S.40, while at S.47 the resistance to cadmium increases. On the other hand, Fig. 1 also indicates that the slope of the curve is more abrupt in the most susceptible stages, providing information on the doseresponse features (for additional information on stage-dependent susceptibility to cadmium in Xenopus embryos, see Herkovits et al., 1997). As a general pattern, the cadmium uptake increases as development advances. The low cadmium uptake obtained at early developmental stages could be related to the protective coat of the vitelline membrane and the cortex, preventing the high toxicity exerted by cadmium. The high increase in the uptake values at the last developmental stages could be related to the gradual increase in the external surface of the embryo, the development of gills, and the blood transporting cadmium from the surface to inner organs. At early developmental stages (S.2, S.8, S.13, S.27/28, S.33) a slightly lower uptake of cadmium was registered in the embryos treated with the higher cadmium concentrations (Fig. 2), which could be related to the possibility that only those embryos with a lower uptake of cadmium can survive in spite of the high cadmium concentration in the maintaining media (Figs. 3a and 3b). Thus, at early developmental stages, reduced permeability could represent a main protective mechanism against this heavy metal and probably also against other environmental stressors. At more advanced developmental stages (S.47) it can be observed that the uptake is directly related to the external cadmium concentration (Figs. 2, 3a, and 3b). As a whole, these results are in agreement with cadmium uptake data obtained with B. arenarum embryos (Perez-Coll and Herkovits, 1996) and suggest a sharp stage-dependent phenomenon with

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HERKOVITS ET AL.

FIG. 3. (a) Xenopus laevis embryo survival and cadmium uptake with 0.2 mg Cd>/L after 72 h of exposure. (b) X. laevis survival and cadmium uptake with 0.5 mg Cd>/L after 72 h of exposure. (c) X. laevis survival and cadmium uptake with 1.0 mg Cd>/L after 72 h of exposure.

saturation of the cadmium transport system at early developmental stages and not saturation at the end of embryonic development in amphibian embryos. It is noteworthy that this fact was recorded even at 4 h of exposure in the case of B. arenarum embryos (Perez-Coll and Herkovits, 1996). Regarding the uptake mechanism for cadmium, in Mytilus edulis there was no energy, temperature, aerobic dependency, or competition with other cations such as zinc, demonstrating that cadmium uptake was by diusion (Carpene and George, 1981). However, in other experimental systems using simultaneous incubations of human erythrocytes with Zn or Cd, a decrease in the cadmium uptake rate and transport constant was observed, suggesting that the cadmium uptake occurred by a mediated transport via cations chemically similar to it (Nguyen and Chien, 1988). The increase in the resistance to cadmium at the last embryonic stage evaluated in the present study (S.47) in spite of the simultaneous increase in the uptake of cadmium

(Figs. 1 and 2), could be related to an eventual enhancement of another defense mechanism, e.g., induction of metallothioneins (Mt). A relationship between Mt-bound cadmium and tissuecadmium concentration (Noel-Lambot et al., 1980) is generally accepted, while on the other hand acclimation experiments to cadmium enhance metallothioneins synthesis in B. arenarum embryos (unpublished data obtained in this laboratory). Other features such as cytosolic molecules, membrane-limited vesicles, and the increase in the extracellular compartment that occur as development advances could be involved as mechanisms against cadmium toxicity toward the end of embryonic development. At all developmental stages the BF is higher in embryos exposed to the lower cadmium concentration and, as a general pattern, as developments advance the BF increases. This fact points out the role that amphibian embryos could play in the biomagnication process even when they are exposed to very low cadmium concentrations in the environment.

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FIG. 4. Bioaccumulation of cadmium in Xenopus laevis embryos at dierent developmental stages. All the data are signicantly dierent (P(0.01) in respect to the cadmium concentration in the maintaining media as well as among the dierent developmental stages. Mean embryonic weight, 0.44 mg$0.05 mg.

The intraspecic variation of amphibian embryos to xenobiotics has been evaluated to some extent (Pierce and Harvey, 1987; Perez-Coll et al., 1988; Herkovits et al., 1997). On the other hand, it has been reported that the intralaboratory precision for embryonic survival and teratogenicity test methods have coecients of variation ranging from about 10 to 50% (USEPA, 1991). In the present study the cadmium uptake for each developmental stage was quantied with embryos obtained from four independent mating pairs for each developmental stage. It is noteworthy that all the animals belong to the same amphibian facility and therefore they are maintained continuously under similar laboratory conditions. The CV for cadmium uptake at early developmental stages (S.2S.27/S.28) indicated more constant behavior than at the last stages. The high variability obtained toward the end of embryonic development could be related to the expression of genetic dierences in embryos provided from dierent parents, that is, an inherent variability phenomenon.
CONCLUSION

for the surviving of the embryos, while at the last developmental stage evaluated in this study, high uptake values do not exert lethal eect; (iv) the BF for cadmium is inversely proportional to the concentration of cadmium in the maintaining media and as a general pattern its value increases as development advances.
ACKNOWLEDGMENTS
The work was supported by a grant from CNR, No. 9200668. J.H. is member of the Consejo Nacional de Investigaciones Cienticaas y Tecnicas, Argentina (CONICET), and C.S.P.C. has a CONICET contract.

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The following conclusions may be drawn from the results obtained by exposing amphibian embryos for 72 h to a range of cadmium concentration solutions: (i) exposure to low concentrations of cadmium, e.g., 0.20.5 mg Cd>/L results in an increased uptake of this heavy metal as development advances; (ii) in the most susceptible developmental stage of this heavy metal (stage 40), low cadmium concentrations in the maintaining media (e.g., 0.2 mg Cd>/L) result in an uptake of about 0.015 g Cd>/embryo which is lethal to most of the embryos; (iii) at early developmental stages a reduced uptake of cadmium could play a signicant role

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