Mechanism Of The Inhibitory Action Of Allelochemical Dibutyl Phthalate On Algae Gymnodinium Breve

Frequent outbreaks of red tide has become a worldwide marine environmental problem, garnering more and more attention from the global society. In recent years, red tides in China sea area show characteristics of high frequency, long time lasting, large scale and a early fist time during a year, resulting in ecological disruption, aquaculture lost and even human health risk. Among different physical, chemical and biological technologies treating with red tide algae, allelopathy and allelochemical form macrophytes show a distinctive competitive advantage, for the easy getting and high yield of the rough materials of macrophytes, the environmental friendly feature from the species-specific sensitivity working style and the proper amount of usage from their low effective values. But the new allelopathy algaecide agent is not widely used, because of the uncertainty of the inhibitory mechanism of the allelochemical. Recent studies disclosing the potential mode, mainly focused on the influence on the algae photosynthetical system, the destruction on algae membrane, the disruption of the balance of the anti-oxidation system, the inactivation of the extracellular enzyme and the disturbance on the reprodution of the algae. The recent study paid more attention on the results of the allelopathy, like the changes of the enzyme activity and the status of oxidative stress. For the especial oxidative stress, it is widely mentioned as it could be related to many environmental factors, but whether the oxidative stress in algae was an original reason from the disruption on electron transfer chain, or it is a subsequential consequence of the inactivation of the anti-oxidation system, is not distinguished. And in fact this limits the practical application of the allelochemical in red tide algae control. The determination of the target cellular organ in algae will give a directive function in the practical application of the allelopathy algaecide agent.Dibutyl phthalate (DBP) was reported in our previous study to be an effective allelochemical in macrophyte Ulva pertusa(EC50,72h 1.1mg/L). However, mechanisms underlying the effect are still not revealed. In the present study, the algae ultrastructure, lipid peroxidation (malonaldehyde, MDA) and the reactive oxygen species (ROS) level, including·OH, H₂O₂ and O₂·^- , in G. breve cells under DBP exposure were detected. Further researches, including activities of SOD isoforms and effects of rotenone(an inhibitor of mitochondrial respiration), diuron(an inhibitor of photosynthesis) and dicumarol(an inhibitor of the plasma electron) on the DBP induced ROS production, were made to determine the ROS production site or the target site of the DBP.The results show an increase in malondialdehyde content, and for the 3mg/L DBP treated algae culture a peak value occurred at 72h, which is about 2.3 times than the control, suggesting that the algae was endured to oxidative stress. TEM pictures show that almost all normal cell organelles are indistinguishable. DBP triggered the synthesis of reactive oxygen species(ROS), and with the increase of DBP concentration·OH and H₂O₂ contents accumulated, as for the 3mg/L DBP treated algae cultures,·OH showed a peak of 33U/mL at 48h, which was about 2.4 times higher than the controlled, and H₂O₂ contents was about 250 nmol/(107cells) at 72h, which was about 5 times higher and also was the highest during the whole culture. Further researches show that the activity of CuZn-SOD(main cytosolic isoform) under DBP exposure is higher than the control, but for the Mn-SOD(mitochondrial isoform), its activity has a significant inhibition. Rotenone(an inhibitor of mitochondria electron transport chain complexⅠ) decrease the DBP induced ROS production, and dicumarol(an inhibitor of electron transport chain in the plasma membrane) stimulates the DBP induced ROS production. Taken all together, the results demonstrate DBP induced over production of ROS in G. breve causing damage on subcellular structure, and mitochondria seems to be the main target sites of DBP. The mechanism of the action of DBP on the algae G. breve growth is a worthy study of theoretically instructive senses considering its further application in algaecide.