Regulation Potential Of Water-soluble Substances Derived From Dendranthema Indicum On Phytoplankton Structure In Aquaculture Waters

With the increasing demand for aquatic products,the aquaculture industry has developed rapidly.Due to intensive culture and unscientific management,the imbalance of phytoplankton structure and harmful cyanobacteria blooms in aquaculture waters caused by eutrophication are increasingly prominent.The current countermeasures have their own merits and demerits.Compared with the traditional chemical algal control products,plentiful plant-derived active substances are more easily degraded,becoming one potential ecological technology to prevent and control harmful cyanobacterial blooms.Water extracts of a terrestrial medicinal plant wild chrysanthemum（Dendranthema indicum）flowers（WCF）showed strong inhibitory effects on Microcystis aeruginosa in our previous screening experiments.In this paper,the responses of different phytoplankton species,basic characteristics of active substances and their aquatic biosafety were further investigated to evaluate the regulation potential of water-soluble substances from D.indicum on phytoplankton structure.The main experimental results are as follows:（1）The effects of water extracts from D.indicum on two strains of M.aeruginosa and threestrains of green algae were analyzed by setting different concentration levels and different extraction conditions to evaluate the regulation feasibility of water extracts from D.indicum on phytoplankton community structure.The results showed obvious concentration-dependent inhibitory effects on M.aeruginosa by water extracts from D.indicum.However,the growth of Chlorella vulgaris was promoted by low concentration(C≤2.0 g·L^-1)and inhibited by high concentration(4.0 g·L^-1≤C≤8.0 g·L^-1)of the water extracts.When the extract concentration was 1.0 g·L^-1,it had the highest promotion effects on C.vulgaris growth.When the extraction time was 8h,the inhibitory effects of water extracts from D.indicum showed highest inhibition on the growth of M.aeruginosa.When the concentration of D.indicum was 1.0g·L^-1,the growth and photosynthesis of two strains of M.aeruginosa were significantly inhibited,while that of three strains of green algae was significantly promoted.The experiment with the contactless coculture with D.indicum and the experiment with the direct addition of water extracts both showed same results.（2）The basic characteristics of the water-soluble active substances in D.indicum were investigated from three aspects:thermal stability,molecular weight and polarity.The mechanism of water-soluble substances from D.indicum on M.aeruginosa was also studied.There was no significant difference in responses of M.aeruginosa to water-soluble substances extracted from D.indicum in boiling water and normal temperature water,which indicates that the active substances of D.indicum have a good thermal stability.Inhibitory effects of water-soluble substances from D.indicum that penetrate through dialysis bags with different molar weight cut-off on M.aeruginosa were compared,and there was no significant difference among three kinds of dialysis bags.It indicates that the molecular weight of active substances in D.indicum was less than 3500 Da.Solid phase extraction with single-step elution and gradient elution of water-soluble substances from D.indicum was conducted,and all the elution fractions were subjected to inhibition tests on M.aeruginosa.The methanol fraction showed the strongest inhibitory effects on M.aeruginosa,indicating that polarity of the active substances of D.indicum were relatively strong.It was also observed that water-soluble substances of D.indicum reduced the content of chlorophyll a,destroyed the structure of PSII reaction center,decreased the original light energy conversion efficiency and affected photosynthetic electron transport efficiency.It indicates that the water-soluble substances in D.indicum inhibit the photosynthesis of M.aeruginosa.（3）The Daphnia magna and Cyprinus carpio were selected as the test organisms to assessaquatic biosafety of water-soluble substances in D.indicum preliminarily.Acute toxicity of water extract from D.indicum was studied for juvenile and mature D.magna and juvenile C.carpio.The results showed that the 24 h LC₅₀ of water extract from D.indicum was 3.415 g·L^-1 and 4.781 g·L^-1 on juvenile and mature D.magna,respectively.It was 1.518 g·L^-1 for juvenile C.carpio.It indicates that the toxicity of water extract from D.indicum is the least to mature D.magna.These preliminary results showed juveniles of aquatic organisms were more sensitive to water-soluble active substances of D.indicum.Hence,it is essential to consider dosage and timing when using water-soluble active substances of D.indicum to control harmful cyanobacterial blooms and regulate phytoplankton structure.For example,to avoid incubation period and the larval stage of aquatic organisms.Long-term exposure experiments need to further examine the effects of D.indicum on the growth and reproduction of non-target aquatic organisms.