Toxic Effects And Response Mechanism Of Tegillarca Granosa Under Warm Drainage Stress

In order to find out the effects of nuclear power plant warm water pollution on stress of aquatic animals,the influence of temperature and residual chlorine coupling on the acute toxicity and long-term stress effects on T.granosa were studied by simulating the residual chlorine and temperature rise effects of nuclear power plant warm water in laboratory.This study can give a reference for toxicity studies of marine organisms exposed to water temperature and residual chlorine combined pollution,and provide scientific basis for ecological risk assessment.The results are as follows:1.Toxic effects of water temperature and residual chlorine on embryonic development,planktonic larvae,juveniles and adults of T.granosa（1）the effects of water temperature and residual chlorine on embryonic development of fertilized eggs of T.granosa.The cleavage rate at 35℃（77.6%）was significantly lower than that at 29℃（92.2%）,and the embryos did not develop to the D-shaped larvae stage and stayed at the tambourine larval stage.With the increase of residual chlorine concentration,the cleavage rate and the D-shaped larvae hatching rate of T.granosa showed a decreasing trend and the malformation rate showed an increasing trend.Temperature significantly promoted the inhibitory effect of free residual chlorine on the hatching of the D-shaped larvae of T.granosa;temperature significantly promoted the inhibitory effect of combined residual chlorine on the cleavage rate,the D-shaped larvae hatching rate and the D-shaped larvae malformation rate of T.granosa.（2）The effects of water temperature and residual chlorine on the planktonic larvae of T.granosa.The growth rate（6.56μm/d）and the attachment rate（62.7%）of T.granosa planktonic larvae were significantly decreased at 35℃.With the increase of residual chlorine concentration,the growth rate and the attachment rate of T.granosa plankton larvae showed a decreasing trend.Temperature significantly affected the inhibitory effect of free residual chlorine on the attachment rate of T.granosa planktonic larvae;temperature significantly affected the inhibitory effect of combined chlorine on the growth rate and the attachment rate of T.granosa planktonic larvae.（3）The effects of water temperature and residual chlorine on T.granosa juveniles,T.granosa of one year old and T.granosa of two years old.The half lethal concentration of T.granosa:juveniles<T.granosa of one year old<T.granosa of two years old,the older the T.granosa,the more tolerant to residual chlorine.half lethal concentration at 35℃>30℃>25℃,T.granosa juveniles,T.granosa of one year old and T.granosa of two years old showed sensitivity to lower doses of residual chlorine at higher temperatures.2.Analysis of physiological metabolism and molecular response mechanism of T.granosa to water temperature and residual chlorine.A 2×6 two-factor experiment was designed according to the preliminary experimental results and the actual situation,and the medium-long term stress experiment was carried out for 20 days.（1）The effects of water temperature and residual chlorine on the physiological metabolism of T.granosa.the oxygen consumption rate of T.granosa in 6.66 mg/L（high）free residual chlorine group and 47.0 mg/L（high）combined residual chlorine group were increased significantly at 19℃on 10^th day,which were2.10 and1.65 times higher than that in the control group,respectively.With the extension of stress time,it dropped to the level of control group on 20^th day.Water temperature and residual chlorine had a significant interaction on the oxygen consumption rate of T.granosa.（2）The effects of water temperature and residual chlorine on the number and function of T.granosa blood cell.（1）water temperature can significantly affect the ROS level.The higher the temperature,the higher the ROS level.（2）The ROS level and DNA damage of T.granosa in 0.33 mg/L group were increased significantly on 20^th day,which were 2.2,2.9times and 1.8,2.2 times higher than those in the control group at 19 and 29℃,respectively;the ROS level in the 6.66 mg/L group increased significantly on 20^th day,it was 6.2 and 3.6times higher than that in the control group at 19℃and 29℃,respectively;The DNA damage increased;The number of blood cell,the cell activity and the hemocyte phagocytosis ratedecreased.（3）The DNA damage of T.granosa in 2.35 mg/L group were increased significantly on 20^th day,which were 2.4 and 5.5 times higher than those in the control group at 19 and 29℃,respectively;The ROS level and DNA damage of T.granosa in 47.0 mg/L group increased to the maximum value on 20^th day,which were 2.1,2.0 times and 6.0,8.1times higher than those in the control group at 19 and 29℃,respectively;The number of blood cell,the cell activity and the hemocyte phagocytosis rate decreased.（4）Two-factor analysis of variance showed that water temperature and residual chlorine had significant interaction on the parameters of the ROS level,the DNA damage and the hemocyte phagocytosis rate of T.granosa.（3）The effects of water temperature and residual chlorine on the enzymatic activities of T.granosa visceral mass.The SOD activity and ATP activity of T.granosa in 0.33 mg/L group were decreased significantly.The SOD activity of T.granosa in 6.66 mg/L group decreased to the minimum value of 36.4±8.4 U/（mg·prot）^-1 at 29℃on 20^th day.The ATP activity of T.granosa in 2.35 mg/L and 47.0 mg/L group were decreased significantly.water temperature and residual chlorine had significant interaction on the parameters of the ATP activity of T.granosa.（4）The effects of water temperature and residual chlorine on gene expression in T.granosa.Free residual chlorine showed an induction effect on SOD gene expression in T.granosa;combined residual chlorine showed an induction effect on SOD gene expression at low temperature,and showed first inhibited and then induced at high temperature.water temperature and residual chlorine had significant interaction on the parameters of the SOD gene expression of T.granosa.（5）The effect of water temperature and residual chlorine on the microstructure of T.granosa hepatopancreas tissue.Microscopic observation showed yellow substance deposition in hepatopancreas tissue,and the higher the concentration,the more yellow substance.（6）The effect of water temperature and residual chlorine on the intestinal flora of T.granosa.The mean number of OTUs of CRC group was 43.3%less than that in CK group.（P<0.05）.The visceral mass flora of the three different treatment groups had significant differences（P<0.05）in richness（the Ace index,the Chao index）and no significant differences in diversity（the Shannon index,the Simpson index）.After free residual chlorine and combined residual chlorine stress,the covariate network connectivity of T.granosa gut flora was reduced and the interactions between taxa were weakened.