Ecotoxicological Effects Of Waterborne Cadmium Exposure On Spinibarbus Sinensis,associated With The Subcellular Partitioning Of Cadmium In Various Organs

To examine the ecotoxicological effects on survival,growth performance,and energy metabolism of waterborne cadmium(Cd)exposure in fish,and to explore mechanisms in response to heavy metal stress at the subcellular level in various organs of the fish,a series of experiments were conducted on Qingbo(Spinibarbus sinensis),a valuable and rare freshwater fish endemic to China.The juvenile fish(body weight:45.30±0.16 g)were exposed to three nominal Cd treatments at concentrations of 0(control),30(low-dose),and 90(high-dose)?g/L in artificial soft water with a hardness of 25 mg Ca CO₃/L at 27.5?.During the exposure period,the fish were fed to satiation daily,and the food intake and mortality of the fish were recorded.The exposure period was 28 d.The resting metabolic rate and the indices of growth performance were measured at the beginning of the experiment(day 0)and on days7,14,21,and 28.On each sampling date,the metabolic function of mitochondria in hepatopancreas and kidney,the antioxidant capacity,and the accumulated Cd concentrations at the tissue and subcellular levels in various organs were measured.The subcellular partitioning of Cd in various organs was compared for the surviving fish with the dead to investigate the subcellular lethal mechanism in fish caused by subacute Cd exposure.The main results obtained in this study were as follows:1?The survival rates for the low-and high-dose exposure groups were 71.67%and66.67%,respectively.The rate was 100%for the control group.2?The resting metabolic rates were significantly higher for the exposed groups than for those in the control group at the early stage of the exposure period(P<0.05).Then,the rates gradually recovered and finally reached a level comparable to the control group at the late stage of the exposure period.3?The daily feeding rate of the tested fish in the Cd-exposed groups was significantly lower than that in the control group under each exposure condition(P<0.05).The specific weight growth rates(SGR)in both Cd-exposed groups were negative and significantly lower than those in the control group at each sampling point(P<0.05).At the late stage of exposure,the SGR in the high-dose exposure group was significantly lower than that of the low-dose group(P<0.05).The condition factors for the low-and high-dose exposure groups were significantly lower than those in the control group,beginning on days 14 and 7,respectively(P<0.05).4?Cd concentrations in the gill,hepatopancreas,kidney,and brain increased with exposure concentration and duration,and were significantly higher than those in the control group at each sampling point(P<0.05).The lowest concentration of Cd was found in the brain under each treatment condition.At the early stage of exposure,Cd concentrations in gill were higher than those in the hepatopancreas and kidney.In contrast,at the late stage,the highest levels of Cd were observed in the kidney,followed by the gill and hepatopancreas.These results demonstrated obvious organ-specific Cd concentrations.5?Cd concentrations in all subcellular fractions of gill,hepatopancreas,kidney and brain increased with exposure concentration and duration.For the gill the distribution order of Cd accumulation in subcellular fractions was as follows:heat stable-protein fraction(HSP)>nuclei and cellular debris fraction(N-D)>mitochondria fraction(Mit)?heat-labile proteins fraction(HLP)>lysosomes and microsomes fraction(L-M)>metal-rich granules fraction(MRG).For the hepatopancreas,the order was HSP>Mit?N-D?HLP>L-M>MRG.The order for the kidney was HSP>N-D>Mit>HLP?MGR?L-M.The order for the brain was HSP?N-D>Mit>MRG>HLP?L-M.6?Compared with the control group,the proportion of Cd content in the metal-sensitive fraction(MSF)to the total Cd concentration in the gill and kidney in both Cd-exposed groups was relatively lower,while that in detoxified-metal fraction(MDF)was relatively higher.This was caused by a smaller increment of Cd accumulated in the HLP and a greater increment of Cd in HSP with Cd exposure.In the hepatopancreas,the proportion of Cd content in MSF in the Cd-exposed groups was significantly higher than that in control group under each treatment condition(P<0.05),while there was no significant difference in MDF between the Cd-exposed group and the control group(P>0.05).The proportion of Cd content in MSF to the total Cd content in the brain increased and that in MDF decreased significantly after Cd exposure(P<0.05).7?For hepatopancreas and kidney mitochondria,Cd concentrations in the two Cd-exposed groups were significantly higher than those in the control group at each sampling point(P<0.05).The high-dose exposure group had significantly higher values than the low-dose exposure group beginning on day 14(P<0.05).Regardless of the exposure conditions,Cd concentrations were higher in the kidney mitochondria than in the hepatopancreas.8?The respiratory control rates(RCR)in the hepatopancreas and kidney were more than4 in each experimental treatment.When hepatopancreas mitochondrial Cd concentrations in Cd-exposed groups were>5.5?g/g dwt,their state III respiration rates were significantly lower than the control(P<0.05).However,the values did not significantly decrease further with increasing exposure concentration and time(P>0.05).The activity of cytochrome c oxidase(CCO)in the high-dose exposure group was significantly lower than that in the control group from day 21(P<0.05).Dose and time did not significantly affect the state III respiration rate and CCO activity in the kidney,and there were no significant differences among the groups(P>0.05).The Cd concentration in the kidney HSP fraction was significantly higher than that in the hepatopancreas(P<0.05).There was a difference of 2?5times under each exposure condition,exhibiting organ-specificity.9?By Cd exposure,the contents of malondialdehyde(MDA)increased significantly in the hepatopancreas.Conversely,the exposure had no effect on those in gill and kidney.The activity of catalase(CAT)in the hepatopancreas was inhibited by Cd exposure.The CAT activity in the high-dose exposure group was significantly lower than that in the control group at each sampling time point(P<0.05).Dose and time did not significantly affect CAT activity in the gill and kidney.The activities of superoxide dismutase(SOD)in the gills of both Cd-exposed groups were significantly higher than that in the control group under each exposure condition(P<0.05).In the hepatopancreas,the SOD activity in the low-dose exposure group was significantly higher than that in the control group at the end of exposure(P<0.05).Dose and time did not significantly affect SOD activity in the kidney(P>0.05).10?The total Cd concentrations in various organs of surviving fish were higher than those of dead fish.Compared with the surviving fish,the Cd concentration in various subcellular fractions of the hepatopancreas of dead fish was relatively lower,and no significant difference was observed in the Cd accumulation rate between the two groups(P>0.05).Cd concentrations in the MSF(HLP and L-M)for gills between the dead and surviving fish were not significantly different(P>0.05),but the Cd accumulation rates in the two fractions of MSF in dead fish were higher significantly than those of surviving fish(P<0.05).For the kidney of dead fish both the Cd concentration and Cd accumulation rates in MSF were significantly lower than those of surviving fish(P<0.05).The conclusions suggested in this study were as follows:1?The resting metabolic rate of S.sinensis increased by Cd exposure and its feeding intake decreased,resulting in insufficient energy supply for the growth.2?Waterborne Cd exposure could induce metallothionein(MT)synthesis in both gill and kidney and transform Cd in the cells to MT-Cd that should be nontoxic,the mechanism could effectively reduce Cd accumulation in HLP through the sequestration role of Cd to HSP and protect the metal-sensitive proteins in this fraction.The capacity of MT synthesis in the hepatopancreas and brain was not sufficient to reduce Cd accumulation in MSF.Therefore,the capacity of subcellular Cd handling exhibited an organ-specificity.3?When the detoxification capacity(MDF)was unsaturated,Cd could accumulated significantly in all putative metal-sensitive compartments(MSF),which did not support“the spillover model of metal toxicity”.4?The relative proportion of Cd that occurred as MT-Cd complexes in the kidney was much higher than that in the hepatopancreas,which could be the primary cause of the organ-specific effects of Cd on hepatopancreas and kidney mitochondrial function.5?Cd accumulation in hepatopancreas mitochondria in vivo had to reach a certain concentration threshold to cause an inhibitory effect on the state III respiration rate and that the intensity of such an inhibitory effect showed no increase with a further increase in mitochondrial Cd concentrations.This discovery suggested that the pattern of Cd toxic effects observed in isolated mitochondria during in vitro exposure could not represent the stress of Cd in natural environment.6?The rapid accumulation of Cd in HLP and L-M fractions of gill and kidney in a brief time led to serious impairment of ion regulation,which could be the primary cause of the death of fish exposed to Cd.