| This paper studied the semi-lethal concentration (LC50) of copper ion (Cu2+) on Hoplobatrachus chinensis. On the basis of the semi-lethal concentration, we carried out an acute toxicity test to study the toxic eff of Cu2+ on tricarboxyl acid (TCA) cycle and free radical metabolism in liver mitochondria of Hoplobatrachus chinensis. Six various concentrations (0.0,2.0,4.0,6.0,8.0 and 10.0mg/L) and five different exposure duration (0,24,48,72 and 96h) were used for discussing the effects of Cu2+ on isocitrate dehydrogenase (ICDHm) activity, a-ketoglutarate dehydrogenase (a-KGDH) activity, anti-superoxide anion (anti-·O2-) activity, hydrogen peroxide (H2O2) content, inhibit-hydroxyl radical (inhibit-·OH) activity, nitric oxide (NO) content and nitric oxide synthetase (NOS) activity to explore the mechanism of heavy metal pollution resulting in a decline in amphibian populations, as well as to provide theoretical basis for the establishment of copper wastewater discharge standards. At the same time it also could provide important data support for the species of the wild protection.The result shows that:(1) In the test of the LC50 of Cu2+ on Hoplobatrachus chinensis, when the concentration was low mortality of Hoplobatrachus chinensis was low as well. With the increase of Cu2+ concentration and the extension of exposure time, the mortality of Hoplobatrachus chinensis showed a trend of rising. When the test condition of Cu2+ concentration was 8.0,10.0,20.0,40.0,60.0,80.0 and 100.0 mg/L, the mortality of Hoplobatrachus chinensis was 0,0,0,10.00%,46.67%,73.33% and 76.67% at 24h. When the test condition of Cu2+ concentration was 8.0,10.0,20.0,40.0 and 60.0mg/L, the mortality of Hoplobatrachus chinensis was 6.67%,20.00%,36.67%, 46.67 and 100.00% at 48h. When the test condition of Cu2+ concentration was 8.0, 10.0 and 20.0mg/L, the mortality of Hoplobatrachus chinensis was 40%,53.33% and 100.00% at 72h. When the test condition of Cu2+ concentration was 8.0,10.0 and 20.0mg/L, the mortality of Hoplobatrachus chinensis was 46.67%,80% and 100.00% at 96h.(2) In TCA cycle, with the increasing concentration of Cu2+and the extension of the exposed time, no significant correlation was showed by two-factor, exposed time and concentration, variance analysis on ICDHm activity (P> 0.05). The extension of the exposed time had no significant effect on ICDHm activity (P> 0.05), with the increase of concentration of Cu2+, ICDHm activity decreased. However, two-factor, exposed time and concentration, variance analysis showed that there was significant correlation on a-KGDH activity (P< 0.05). a-KGDH activity decreased after the test, and groups of 24h and 96h with 4.0 mg/L and 6.0 mg/L had the lowest activities.(3) In free radical metabolism, two-factor, exposed time and concentration, variance analysis showed that there were significant correlation on the activity of anti-·O2- and H2O2 content (P< 0.05) and no significant correlation on the activity of inhibit-·OH, NO content and NOS activity. With the increase of concentration in different exposed time, the activity of anti-·O2- presented a decreasing trend. H2O2 content increased after the test, and groups of 24h with 6.0mg/L had the highest activities. With the extension of the exposed time and the increase of concentration of Cu2+ the activity of inhibit-·OH both reduced gradually. The NO content and NOS activity had a same variation trend, namely with the increase of concentration of Cu2+ the value increased first and then decreased to the value of the group with O.Omg/L, and both reached the maximum at the group of 6.0 mg/L.The conclusions:(1) Hoplobatrachus chinensis under the stess of Cu2+ in 24h,48h,72h,96h, the LC50 respectively reached 62.5 mg/L,24.7mg/L,9.5mg/L,8.2mg/L. With extension of the exposed time, the LC50 of Hoplobatrachus chinensis decreased. The safe concentration of Cu2+ on Hoplobatrachus chinensis was 0.82mg/L.(2) In the acute toxicity test Cu2+ had an effect on activity of the key enzyme, ICDHm and a-KGDH, in TCA cycle. We could speculate that Cu2+ could reduce the survival rate of Hoplobatrachus chinensis through reducing the TCA cycle efficiency, inhebiting energy metabolism.(3) In the acute toxicity test Cu2+ destroyed the balance of free radical metabolism by reducing the ability to resist the formation of free radicals and increasing free radical content in mitochondria. Those would damage mitochondrial structure and function, effecting the living conditions. |
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