The sea urchin test system has been determined in our laboratory to be an extremely informative bioassay for pollutants, occurring as individual or as mixtures of xenobiotics. Harmful agents may be identified and characterized biologically to give an integrated set of data dealing with several key events, such as fertilization, mitotic activity, and embryogenesis. Therefore this bioassay may contribute to the assessment of the environmental impact of pollutants, by focusing on several important toxicological aspects. This study was carried out on some inorganic pollutants [Cd(II), Cu(II), Zn(II), and Se(IV)] and their mixtures. Sublethal pH changes were taken into account as an additional stressor, capable of influencing the toxicities of tested agents. The experiments were performed on Paracentrotus lividus sperm and embryos, and on Echinus esculentus sperm. The results showed distinct patterns of embryotoxicity for the agents tested, which resulted in developmental defects as relatively high levels (>10−5 kmol/m3) for Cd(II) and Se(IV), whereas the embryotoxic effects of Cu(II) and Zn(II) were exerted at lower concentrations (5 × 10−7 kmol/m3) close to their natural seawater levels. Cytogenetic analysis of exposed embryos failed to reveal any morphological or quantitative changes in mitotic activity, at embryotoxic levels of all tested contaminants. The fertilization success of exposed sperm showed peculiar dose-response changes, as a result of the specific contaminant levels and of the simultaneous presence of other contaminants. Specifically, slight increases in Cd(II) or Cu(II) levels enhanced fertilization success, up to critical levels (10−7 to 10−6 kmol/m3); however, further increases in these metal ion concentrations led to a drop in fertilization success. The simultaneous presence of Zn(II) or Se(IV) dramatically changed the dose-response patterns, depending on the different mixtures and the test species. The results may represent an example of the need to account for the simultaneous presence of several pollutants, whose combined impacts may be substantially different from those of the individual toxins. Moreover, the results attest to the high sensitivity of the sea urchin bioassay, which responds to very slight increases in microelements or contaminant levels above their natural concentrations.
The effects of different pH conditions have been investigated on sea urchin larval development following exposure of embryos to controlled, though changing, decreases or increases of seawater pH. The pH of filtered natural seawater was initially adjusted with 1 N HCl of 1 N KOH and then was altered back to its normal values (8.0-8.2) by the exchange with atmospheric CO2 and subsequent carbonic acid equilibrium. During cultures, pH was regularly monitored. When developing embryos were reared in different pH conditions, larval differentiation was sharply affected by an apparently moderate pH decrease, such as 0.5 pH units. However, even pH decreases as small as 0.2 pH units from the normal value showed reproducible damage to embryogenesis. This damage appeared to be early and irreversible, since the exposure of cleaving embryos resulted in more severe developmental defects than exposure of posthatching blastulae. Moreover, mitotic abnormalities were observed following early exposure of embryos to decreased pH. Increased pH, up to 8.6 (approximately 0.5 pH units above normal value), failed to exert any adverse effect on subsequent development. Moreover, an initial pH increase (8.5-8.7) resulted in the final adjustment of culture pH to 8.1-8.2, thus providing optimal conditions for rearing embryos. Two attempts to stabilize culture pH were performed by decreasing gaseous exchanges or by using Tris as a buffering agent. Both approaches appeared to be impractical, thus ruling out any further attempts. The results point out the hazards of acid contamination in restricted bodies of seawater, leading to apparently "moderate" decreases in pH, which can result in severe damage to some marine organisms, both adult and larval forms.
pH decrease and increase were tested for their ability to affect the fertilizing capacity of sea urchin sperm, as well as to induce developmental defects and mitotic abnormalities in the embryos generated by pretreated sperm. Seawater (sw) at different pH values was obtained by mixing acidified (HCl) sw and alkalinized (KOH) sw. Thereafter sperm were exposed to different pHs for a defined time interval, or subjected to inactivation, while suspended in sw at defined pHs, as long as their fertilizing capacity was maintained. The study was carried out over the pH range 5-9; sperm showed optimal fertilizing capacity between pH 6 and 7. Below pH 6 and above pH 8 a drop in the fertilizing capacity was observed, whereas at normal sw pH (8.0-8.2) sperm showed intermediate values. pH decrease in sperm suspension induced a reproducible increase in developmental defects and mitotic abnormalities in the offspring of pretreated sperm. This effect displayed a dose-response relationship, which was most evident for pH ranging from 7 to 8. The effects of decreased pH were detected as well in terms of quantitative changes in mitotic activity, which decreased in embryos generated by sperm exposed to low pH (5-6). The results are consistent with the induction of genetic damage following exposure of sperm to low pHs. This observation suggests a genotoxic action of hydronium ions per se. Another possible explanation for the observed genotoxicity of decreased pH might be based on the pH-dependent activity of several genotoxins present in sw at trace levels.
Further evidence is reported here of genetic and developmental damage that can be induced by a sublethal pH decrease. The effects of three inorganic acids (HCl, H2SO4, and H3PO4) on embryos and sperm from the sea urchins Sphaerechinus granularis and Paracentrotus lividus were evaluated. In addition, acidification of the medium was tested for spontaneous reversion to His+ prototrophy in Salmonella typhimurium (strains TA97, TA98, TA100, TA102, TA1535) up to toxic levels, by both liquid incubation and agar plate incorporation. The induction of developmental and mitotic abnormalities in S. granularis confirmed our previous observations on P. lividus. Embryotoxicity was exerted in S. granularis more severely by H3PO4 than by HCl or H2SO4 (pH 7 to 6), while the induction of mitotic abnormalities appeared at a pH of less than or equal to 6.5 irrespective of the acids used. By suspending S. granularis or P. lividus sperm in acidified filtered seawater (fsw) and then inseminating the eggs in natural fsw (pH = 8.0), the offspring showed developmental and mitotic abnormalities. Low-pH-induced spermiotoxicity was ruled out in our experiments, since fertilization success of acid-exposed sperm was actually enhanced, as compared to sperm suspended in untreated fsw. The exposure of S. typhimurium to different pH's (ranging from 4 to 9) invariably failed to induce any changes in reversion rates, regardless of the acids (or alkali) being used. These results suggest that extracellular acidification may cause sublethal damage that in turn leads to an impairment of mitotic activity and cell differentiation.
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