Lethal and teratogenic effects of naringenin evaluated by means of an amphibian embryo toxicity test (AMPHITOX).

The effect of naringenin on the survival and morphogenesis of amphibian embryos was evaluated by means of the AMPHITOX test reporting early life stage and chronic toxicity effects. Lethality, malformation incidence and the degree of adverse effects were concentration-dependent. The Teratogenic Index (TI) for naringenin was 2 pointing out the high developmental hazard of this substance. For instance, 10 mg/l naringenin exerted 100% malformations while only 30% of the abnormal embryos died. Main abnormalities were reduced body size, axial curves, microcephaly, abdominal edema, underdeveloped gills and delayed development. Scanning electronic microscopy (SEM) showed alterations in epithelial cell shapes related to malformations. The results obtained by means of treatment of Bufo arenarum embryos with naringenin from complete operculum stage onwards show that at this final stage of development, the susceptibility of the embryos to this flavonoid is slightly lower for lethal effects but exerted sublethal adverse effects such as transient narcosis, abnormal contortions, loss of equilibrium, reduced motility and edema. Overall, amphibian early life stages appeared more susceptible to the embryotoxicity associated with exposure to naringenin, especially at concentrations greater than 5 mg/l. This increased susceptibility may result from the relatively high rates of cellular differentiation and morphogenesis that occur at this early stage of development.

Evaluation of nickel-zinc interactions by means of bioassays with amphibian embryos.

The nickel hazard was evaluated by means of a 7-day toxicity test with Bufo arenarum embryos. The LC(50) values for this metal from 24 to 168 h diminished from about 26 to 1.8 mg Ni(2+)/L, respectively, but from 96 h onward, the LC(50) varied very slightly. Although a noticeable difference among the LC(50) and LC(10) or LC(90) was observed at 24 h of exposure, these parameters tended to a similar value at 168 h of exposure while the confidence intervals of LC(50) overlapped all other confidence interval values. These results, plotted as toxicity profile curves, are useful for determining time and concentration thresholds for Ni. Nickel-zinc interactions on B. arenarum embryos were evaluated by means of simultaneous treatments with both cations (Ni: 5-35 mg Ni(2+)/L; Zn: 0.5-130 mg Zn(2+)/L). As a general pattern, low Zn concentrations (0.5 mg Zn(2+)/L) did not have a clear-cut effect on Ni toxicity, higher Zn concentrations (2-20 mg Zn(2+)/L) enhanced Ni toxicity, and concentrations of 30 mg Zn(2+)/L and higher had a beneficial effect in most cases. The metal interaction studies provide a scientific basis for the establishment of water quality criteria for wildlife protection purposes.

Metallothionein induction and cadmium uptake in Bufo arenarum embryos following an acclimation protocol.

Bufo arenarum embryos at the end of their embryonic development were acclimated to cadmium (Cd) by means of a 10-day treatment protocol. Embryos were processed for metallothionein (Mt) isolation and Cd and zinc (Zn) contents were measured. The results showed that: (1) the uptake of Cd in the experimental embryos was 7 microg/g embryo (wet weight) representing a bioaccumulation of Cd 255 times higher than in the maintaining medium; (2) a major Mt-like fraction was Cd-induced 7.8 times that in control embryos; two other protein fractions also bound Cd and Zn but were induced by Cd only about 2 and 1.4 times; (3) the Zn concentration was about 44 microg Zn/g embryo (wet weight) and did not change significantly (p>0.01) in the experimental embryos with respect to controls, but in acclimated embryos the essential metal was released from the Mts. The enhanced Mt synthesis and release of Zn from the native Mts are discussed in relation to the acclimation phenomenon.

Metallothioneins and cadmium uptake by the liver in Bufo arenarum.

Bufo arenarum females were treated daily with 0.5 mg Cd kg(-1) during 10 days to evaluate the uptake of this heavy metal and the induction of metallothionein synthesis in the liver. The liver incorporated 26% of the Cd administered, about 6.5 times higher than the average uptake of the other tissues of B. arenarum. Three protein fractions from the B. arenarum liver bound Cd, and were induced by this xenobiotic up to approx. 24 times above the basal level of these proteins.

Ecotoxicity in the Reconquista River, province of Buenos Aires, Argentina: a preliminary study.

The Reconquista River in Argentina is considered a "supercritical" river basin due to environmental degradation. Within its valley of 1.547 km2, there are more than 3 million inhabitants and 12,000 industries. Using early-life-stage toxicity tests with Bufo arenarum embryos (the most sensitive of three native species), we determined the water quality at six sampling stations of the river valley and expressed the results as acute and chronic toxicity units. Along most of the river, the toxicity was higher than the allowable level of whole industrial effluent toxicity recommended by U.S. EPA. In a tributary stream, Arroyo Moron, the water was about 10 times more toxic than the criteria maximum concentration (CMC) recommended by U.S. EPA for industrial effluents. Similar degradation of the water quality was found taking as a reference value an upstream sampling station. In all places where the water quality was worse than the CMC, no macroorganisms were found, and in one of these places, a large number of dead fishes was observed. Our study points out that amphibian early-life-stage toxicity tests could be appropriate for assessing water contamination and water quality, which is essential for diagnosis, protection of environmental services, monitoring, and restoration purposes.

Increased resistance against cadmium toxicity by means of pretreatment with low cadmium/zinc concentrations in Bufo arenarum embryos.

A significantly increased resistance against cadmium toxicity in Bufo arenarum embryos was obtained by means of pretreatments with low cadmium/zinc concentrations, allowing embryos to tolerate lethal cadmium concentrations. Slight variations in the pretreatment schedule could result in significant differences within this acclimation phenomenon. The probable mechanisms of action of this protective effect are discussed.

Antagonism and synergism between lead and zinc in amphibian larvae.

Lead and zinc effects on Bufo arenarum larval survival were studied in single and combined treatments. On a weight basis, lead is about twice as toxic as zinc. The antagonism or synergism between these heavy metals is dose-dependent.

Zinc protection against delayed development produced by cadmium.

The protective effect of zinc against slight teratogenical action, exerted by low cadmium concentrations, was evaluated in Bufo arenarum embryos treated simultaneously with both cations or preincubated with Zn before Cd treatment. Data on survival, malformations, and delay in development pointed out that Zn could prevent the deleterious effects of Cd in previous and simultaneous treatments with that heavy metal.

Stage dependent susceptibility to lead in Bufo arenarum embryos.

The stage dependent susceptibility to lead in amphibian development was studied by exposing Bufo arenarum embryos during neurulae, neuromuscular activity and gill circulation stages for twenty hours to 1 ppm Pb(2+). Survival, malformations and behavioral disorders were evaluated. The embryonic susceptibility to lead was markedly stage dependent. The survival at the neuromuscular activity stage was approximately half that of the other two periods; concerning malformations, the gill circulation stage was the least sensitive. The observed malformations consisted of failed closure of neural tube, hydropsy, small and cylindrical tails, reduced body size and incurvations in the body axis. Some alterations occurred in all experimental groups and therefore were considered non-dependent on the period of treatment. In all experimental embryos, neurological disorders such as trembles and loss of equilibrium were observed.

[Effects of cadmium on the development of an amphibian]. / Efectos del cadmio sobre el desarrollo de un anfibio.

The effects of continuous treatment with cadmium on Bufo arenarum embryos from the 2-cell stage onwards, were evaluated by light and scanning electron microscopy. Damages were concentration-dependent (between 0.03 and 4.00 mg Cd++/L in 10% Holtfreter's solution) and ranged between teratogenic effects and lethality. In the 0.50-4.00 mg Cd++/L range high mortality, delayed development and significant alterations in the gastrulation and neurulation processes were observed. In embryos maintained at concentrations between 0.03 and 0.25 mg Cd++/L, lethality was considerably lower and development proceeded to more advanced stages. In this group of embryos the toxicity signs were: retarded growth rate, reduced body size, behavioral disorders and a variety of malformations such as microcephaly, underdevelopment of gills and abnormal fins. The ectodermal tissue exhibited predominance of ciliated cells and/or atypical distribution of them. These findings were compared with the effect of cadmium and other heavy metals on embryos, larvae and adults of several amphibian, fish and mammalian species. The findings were interpreted on the basis of known biochemical effects of cadmium.