| Methylmercury(Me Hg)is a common persistent organic pollutant in the ocean posing potential threats to biological processes of marine organisms as well as human food safety,which has attracted widespread attention worldwide.In this study,flounder(Paralichthys olivaceus)at early life stage(ELS)was used for Me Hg toxicity tests.Acute toxicity tests for flounder embryos and larvae(0-24 ?g/L,48 h for embryos;0-30 ?g/L,96 h for larvae)were performed to calculate the lethal concentration of 50%(LC50)of Me Hg for the embryos and larvae,respectively.Subacute toxicity test(0-15 ?g/L;embryos-yolk-sac larvae,128 h)was performed to analyze the toxic effects of Me Hg on the development,survival and growth of embryonic-larval flounder.The effects of Me Hg on the antioxidant defenses,the level of lipid peroxidation(LPO),immune responses and growth of flounder larvae were analyzed through a chronic toxicity test(0-10.0 ?g/L;embryos-settling larvae,35 d).A chronic toxicity test for flounder juveniles(0-20.0 ?g/L,30 d)was also performed to analyze the antioxidant defenses and immune responses in different tissues,characteristics of Me Hg accumulation and the growth of juveniles under Me Hg exposure.These results can help us to better understand the toxic effects of Me Hg on the growth,development and survival as well as the main physiological functions of flounder at ELS,and to investigate the mechanisms underlying the toxicity of Me Hg on fish.Additionally,this study can provide scientific basis for the study of compensatory mechanism of fish resources under marine pollution,and for the construction of detection system of marine pollution.The results of acute toxicity tests showed that the 24 h-and 48 h-LC50 values of Me Hg for the embryos were 17.4 and 15.3 ?g/L,respectively.The 24 h-,48 h-,72 hand 96 h-LC50 values of Me Hg for the larvae were 22.3,20.7,19.0 and 16.3 ?g/L,respectively.These results demonstrated that flounder embryos were more sensitive to Me Hg toxicity than were the larvae.The results of subacute toxicity test for embryonic-larval flounder showed that Me Hg exposures at ?13 ?g/L increased accumulative mortality of embryos and larvae as well as larval morphological deformities;reduced final total length of larvae;and inhibited yolk absorption rate of larvae.These four endpoints were sensitive to Me Hg and their responses were dose-dependent,which could be used as effective bioindicators for assessing the toxic effects of Me Hg to the ELS of flounder.However,the accumulative hatchability of flounder embryos did not significantly differ between Me Hg concentrations and the control,but the hatching rate of the embryos at 15 ?g/L was significantly increased relative to the control.These results suggested that Me Hg exposure in the subacute test at concentrations <LC50 values of embryos and larvae did not reach the threshold at which it starts to affect the hatchability of flounder embryos.However,Me Hg exposure at 15 ?g/L could prolong the hatching time and adversely affect the hatching process of embryos.After chronic test for flounder larvae,Me Hg accumulation in the larvae was positively correlated with Me Hg concentration in a dose-dependent manner.Moreover,Me Hg exposure at ?1.0 ?g/L concentrations reduced larval growth.Compared to the controls,the superoxide dismutase(SOD)activity did not significantly differ among Me Hg concentrations,while the activities of catalase(CAT)and glutathione reductase(GR)were significantly increased at 10.0 ?g/L concentration,and the glutathione peroxidase(GPx)activity as well as lipid peroxidation(LPO)level were significantly increased at ?1.0 ?g/L concentrations.The corresponding antioxidant-related genes were upregulated under Me Hg exposure(cat and gpx at 10.0 ?g/L;gr at ?1.0 ?g/L),while the transcription of sod was not significantly affected by Me Hg exposure.Furthermore,lysozyme(LZM)content was significantly increased,but immunoglobulin M(Ig M)content was significantly decreased at 10.0 ?g/L concentration relative to the controls.As Me Hg concentration increased,the immune-related genes were significantly upregulated(hsp70 at 0.1 and 10.0 ?g/L;lzm and il-1? at ?1.0 ?g/L;tnf-? and il-6 at 10.0 ?g/L),while igm was significantly downregulated at ?0.1 ?g/L concentrations.Overall,Me Hg exposure induced oxidative stress at ?1.0 ?g/L concentrations and caused immunotoxicity at 10.0 ?g/L concentration,respectively.Moreover,the transcription of selected genes correlated with the corresponding antioxidant-or immune-related biochemical markers to defense against oxidative stress and immunotoxicity induced by Me Hg exposure.These markers were sensitive to Me Hg toxicity and could be used as effective indicators to assess the toxicity of Me Hg to the ELS of fish.After chronic test for flounder juveniles,Me Hg accumulation in vivo showed a significant concentration-dependent and tissue specificity tendency.In particular,Me Hg accumulation in different tissues increased significantly as Me Hg concentration increased as followed in the order of liver>gills>spleen>muscle.The level of LPO in the liver was higher than those in the gills and muscle,and the activities or contents of antioxidants(e.g.,SOD,CAT,Glutathione S-transferase(GST)and Glutathione(GSH))in the liver were more sensitive to Me Hg exposure than those in the gills and muscle.Specifically,the activities or contents of these four antioxidants in the liver were significantly induced at low Me Hg concentrations,whereas inhibited at high exposure.Furthermore,CAT activity in the gills and muscle showed no significant difference among Me Hg concentrations and the controls.However,the activities or contents of the other three antioxidants in the gills also showed a trend the same to the liver.Meanwhile,the activities or contents of SOD,GST and GSH in the muscle were all significantly induced at each Me Hg concentration compared to those in the controls.Additionally,the activities or contents of acid phosphptase(ACP),alkaline phosphatase(AKP),LZM and Ig M responded differently to Me Hg exposure at different concentrations.Generally,ACP,AKP and LZM in the gills,liver and spleen can better reflect the concentration-dependent and tissue specificity tendency of immune responses to Me Hg exposure.Finally,the growth of juvenile was significantly inhibited at 20.0 ?g/L.Therefore,the activities or contents of antioxidants(SOD,CAT,GST and GSH)as well as the activities or contents of ACP,AKP and LZM in different tissues of flounder juvenile can all be used as effective bioindicators for the toxicity study of Me Hg on marine fish. |
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