CN111122774A - Method for evaluating ecological risk safety threshold of toxic microalgae - Google Patents
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Abstract
The invention belongs to the field of ocean monitoring, and discloses a method for evaluating a safety threshold of ecological risks of toxic microalgae, which comprises the steps of culturing toxic microalgae of different sea areas and obtaining toxicity data, determining toxicity values of toxic microalgae such as LC50 or LC30 and the like according to toxicity research results, evaluating the ecological risks of the toxic microalgae by adopting an ecological model evaluation method of a species sensitivity distribution curve method, determining an acceptable effect level end point (HC5) according to the toxicity equivalent of the toxic microalgae and the toxicity values of experimental organisms, evaluating the water ecological species sensitivity risks of the toxic microalgae with different densities, providing a model which is reliably ensured by 95% of species of a system, and quantitatively stating the regional ecological risks of the toxic microalgae. The method fills the blank at home and abroad, the general method for evaluating the ecological risk of the toxic microalgae is not influenced by the biological variability of the algae, different water areas can be evaluated by modeling from the initial separation and cultivation, the evaluation is accurate, the method is suitable according to local conditions, the universality is strong, the operation is simple, and the application range is wide.
Description
Technical Field
The invention belongs to the field of marine environment monitoring, and particularly relates to red tide microalgae ecological risk evaluation.
Background
Toxic microalgae are widely distributed in China as reticuloendothelial algae, Alexandrium mimutum, Rimadia protothecoides, and the like.
The reticuloendotheliota (protoceratitis reticulata) is a tiny marine planktonic dinoflagellate, which can explosively reproduce to form red tide under proper ecological environment conditions. Red tide microalgae (Gymnosphaera reticulata) can secrete and generate Yessotoxins (YTXs), and the polycyclic polyether marine microalgae toxins can be accumulated in filter feeding organisms such as shellfish and the like. Toxicological results suggest that YTXs may cause damage to the heart, nervous system, immune system, etc. The European Union stipulates that the limiting standard for YTXs in shellfish and the like is 3.75 mg/kg. The original cauliflower is widely distributed in China and is a main source of patinopecten yessoensis toxin in North yellow sea of China. Researches show that the cauliflower has toxic or inhibiting effect on other organisms such as zooplankton, microalgae and the like, and in a water body with red tide, cells of the cauliflower can influence the biological composition structure of the water body, so that the cauliflower has ecological risk.
Alexandrium minutum (Alexandrium minutum) is a tiny marine planktonic dinoflagellate, and can be explosively propagated to form red tide under proper ecological environment conditions. The red tide microalgae Alexandrium mimutum and other Alexandrium species can secrete Paralytic Shellfish Poisoning (PSP) which can accumulate nitrogen-containing virulent marine microalgae toxins in filter organisms such as shellfish. The results of toxicological studies show that PSP can block cell nano-ion channels, so that the face, muscles and the like of a user are paralyzed, and the severe user can die. The European Union stipulates that the PSP restriction criterion in shellfish and the like is 0.8 mg/kg. The species including Alexandrium mimutum is widely distributed in China sea area, can poison or inhibit other organisms such as coexisting zooplankton, and has ecological risk because cells of Alexandrium mimutum and other Dunaliella mimutum can influence the biological composition structure of water body in water body with red tide.
Prorocentrum lima (Prorocentrum lima) is a tiny benthic dinoflagellate of the ocean, and can be explosively propagated to form a red tide under appropriate ecological environment conditions. The prorocentrum lima can secrete Diarrheic Shellfish Poisoning (DSP), mainly contains Okadaic Acid (OA) and fin algae toxin (DTXs), and can accumulate cyclic ether marine microalgae toxin in filter-feeding organisms such as shellfish. Toxicological results indicate that OA and DTXs can cause digestive system poisoning, diarrhea, abdominal pain and the like of eaters, and diarrheic shellfish poison can also increase the occurrence of digestive tract tumors. The European Union stipulates that the DSP limit criterion in Bobei et al is 0.2 mg/kg. The prorocentrum limanii is widely distributed in China, can poison or inhibit other organisms such as coexisting zooplankton and the like, and has ecological risks because cells of the prorocentrum limanii can influence the biological composition structure of a water body in a water body with red tide.
Ecological Risk Assessment (ERA) is a process for predicting the possibility of toxic and harmful effects of environmental pollutants on an ecosystem, and is a new research hotspot following the development of human health risk assessment. The ecological risk evaluation is an important means for quantifying the ecological hazards of toxic and harmful substances, and finally, a safe concentration threshold value or a risk value is obtained, so that a reference basis is provided for making an environmental management decision and a standard or a benchmark related to the environmental management decision.
The application of species sensitivity distribution in ecotoxicology is mainly ecological risk research on environmental pollutants such as heavy metals, pesticides, polycyclic aromatic hydrocarbons and the like. As in the literature: research on the water quality standard of aquatic organisms of inorganic mercury in China by applying a species sensitivity distribution method, the cynanchum paniculatum and the like, the science of environment, 2012,32(2): 440-449; evaluation of ecological risks of triclocarban on the freshwater environment of China by applying species sensitivity distribution, Wangzhen and the like, ecological and rural environmental reports, 2017,33(10): 921-927. There are 2 risk studies of blue-green algae toxins in the toxic algae sector, respectively: evaluation of ecological risks of microcystins and nitrogen-polluted water based on species sensitivity distribution, a standard of gem and the like, an application ecology bulletin, 2014,25(4) 1171-1180; species sensitivity distribution evaluation of algal toxin ecological risks in water environment, Zhu Xiao Yi, etc., ecological toxicology bulletin 2016, 11(3): 131-; the health risk evaluation of the microalgae toxins in the shellfish in China is carried out in the literature 'distribution hazard and risk evaluation of offshore toxic microalgae and toxins thereof in China' [ marine environmental science 2016, 35(5): 787-; the ecological risk evaluation of the marine toxic microalgae in China has no research report, and the main reason for analysis is that species sensitivity distribution depends on the toxicity perfection degree of documents and databases, the requirements on the stability and universality of toxic substances are high, the biological variability of the algae is high, the universality is low, the integrity and the universality of related data in the documents and the databases at home and abroad are also low, and the species sensitivity distribution cannot be adopted for evaluation.
Disclosure of Invention
The invention aims to solve the problem of blank evaluation of the ecological risk of the marine toxic microalgae at present, provides a method for evaluating the ecological risk safety threshold of the toxic microalgae, does not depend on the perfection degree of toxicity data in the existing database, can deal with the instability and variability of the toxic microalgae, and has the advantages of simple operation and accurate evaluation.
The technical scheme adopted by the invention for realizing the purpose is as follows: a method for evaluating the ecological risk safety threshold of toxic microalgae is characterized in that target toxic microalgae in different sea areas are cultured and toxicity data are obtained, the ecological risk of the toxic microalgae is evaluated by adopting an ecological model evaluation method of a species sensitivity distribution curve (SSD) according to toxicity values of toxic microalgae LC50 or LC30 and the like determined by toxicity research results, an acceptable effect level end point (HC5) is determined according to the toxicity equivalent of the toxic microalgae and the toxicity values of experimental organisms, the ineffective stress concentration for protecting 95% of species is determined, and the regional ecological risk of the toxic microalgae is quantitatively explained.
Further, the evaluation is performed as follows:
s1 separation and culture of algae: separating target toxic microalgae from a pre-evaluation sea water body or obtaining toxic microalgae from other sources, and realizing pure monoclonal culture in a laboratory;
acquisition of S2 toxicity data: carrying out marine organism toxicity test by using pure toxic microalgae to obtain toxicity values LC50, EC50, LC30 or EC30 and the like;
construction of the S3 model: using SSD evaluation software (SSD-Generator), obtaining a toxic algae toxicity value based on the acquired toxicity data, collecting the toxicity value reported in the literature, screening the toxicity data of each species according to the principle of risk evaluation technical schema, calculating to obtain sensitivity fitting parameters of all the species, evaluating the fitting degree, drawing a sensitivity distribution curve of the toxic algae to all the species, obtaining an HC5 value which protects 95% of the species from being influenced as a safety threshold value, and evaluating the ecological risk of the toxic algae and the algal toxins in the regional water body.
Further, the method for evaluating the ecological risk safety threshold of the toxic microalgae further comprises the following steps:
s4 model quality evaluation, wherein specific species are removed by collecting literature and toxicity data obtained by S2, the number of the evaluated species is not less than 5, and the evaluated species belong to 3 or more nutrition levels as much as possible; screening acute toxicity data which are in accordance with the ecological environment characteristics of China and are about patinopecten yessoensis toxin, okadaic acid toxin and paralytic shellfish poison, and taking the arithmetic mean value of a plurality of toxicity data of the same species; based on the obtained acute toxicity data of the 3 kinds of algal toxins, a concentration value (or a logarithmic value) of the toxicity data is plotted against quantiles arranged by the concentration, SSD-Generator software is used for calculating all species sensitivity fitting parameters of the 3 kinds of algal toxins, and the fitting degree is 1 at most; drawing a sensitivity distribution curve of each algae toxin (converted by the toxin production amount of the single algae cell) to all species; HC5, which protected 95% of the species, was calculated as a safety threshold to assess the ecological risk of each dunaliella.
Further, acquisition of the S2 toxicity data: wherein the marine organism is selected from one or more of phytoplankton, zooplankton and fish.
The invention provides a method for determining a toxic microalgae ecological risk safety threshold in the field of ocean monitoring, fills the blank at home and abroad, provides a general toxic microalgae ecological risk safety evaluation method, is not influenced by algae biological variability, can be evaluated by modeling from the initial separation and cultivation in different water areas, is accurate in evaluation and can be made according to local conditions, and has the characteristics of strong universality, simple operation and wide application range.
Drawings
FIG. 1 is a graph of the sensitivity profiles of the toxins from all species of Japanese scallops of example 1.
FIG. 2 is a graph showing the sensitivity distribution of all species to paralytic shellfish poisoning in example 2 of the present invention.
FIG. 3 is a graph showing the sensitivity profiles of all species of example 3 of the present invention to okadaic acid.
Detailed Description
The present invention will be described in further detail with reference to specific examples, but the present invention is not limited to the specific examples.
Example 1
Ecological risk safety threshold assessment of the angular cauliflower:
the specific evaluation steps are as follows:
s1 separation and culture of algae: picking single microalgae cell from burst and accretion natural seawater, collecting reticular original angle algae from Dalian Hainan sea area, and picking single-cell algae strain with capillary for purification and culture. Pure culture was carried out using 96-well plates and raw seawater (0.45 μm membrane filtration), then gradually replaced with 24-well plates and finally transferred to erlenmeyer flasks and cultured with desilication f/2 broth. The other conditions are that the temperature is 15 ℃, the illumination intensity is 4000lux, and the light-dark ratio is 12h:12 h. An appropriate amount of culture medium was added every other week, and the growth was observed daily with an inverted microscope (Olympus IX71, Japan) during the culture.
Acquisition of S2 toxicity data: the Chaetoceros reticuloensis, the golden algae, the chlorella, the large Platymonas subcordata and the Thalassiosira pseudonana are cultured in a marine germplasm bank laboratory. Performing sterile inoculation, and culturing the inoculated cauliflower in a light culture box at 15 ℃ under 4000lx and in a light-dark period of 12h:12 h. Culturing Chlorella, Platymonas subcordiformis, Chrysophyta japonica, and Thalassiosira pseudonana in a light culture box at 20 deg.C and 4000lx with a period of 12h:12 h.
Continuously aerating frozen brine shrimp eggs at 25ppt and 25 ℃ for about 24 hours to collect brine shrimp larvae. Feeding chlorella to obtain young brine shrimp, continuously aerating, and culturing for about 20 days to obtain metamorphosed adult brine shrimp.
The medaka in seawater is incubated and cultured in a national marine environment monitoring center laboratory, the salinity is 30ppt, the medaka is cultured in artificial seawater at the temperature of 20 ℃, newly incubated brine shrimps are fed, and the illumination period is 12h:12 h. Selecting active healthy medaka juvenile fish within three days after the fish eggs are hatched as a test organism.
Counting of Dunaliella cells: shaking up the cultured algae solution, diluting 1mL of algae solution by 10 times to 10mL, adding Luge reagent for cell fixation, and shaking up. 100 mu L of the shaken algae solution is put into a 10X 10 grid counting plate and placed in a microscope to count algae cells by adopting a snake counting method. Each sample was counted three times and finally averaged. Every two days, the number was counted.
Golden algae (Isochrysis galbana), Chlorella (Chlorella vulgaris), Platymonas subcordata (Platymonas heoglolandica), and Thalassia pseudonana (Thalassiosira pseudodonana) were counted using a hemocytometer.
Firstly, counting the cells of algae in algae liquid, and extracting the algae toxin according to a corresponding method.
Toxicity test of juvenile brine shrimp (medaka): adding reticular original caulerpa of different volumes into a twelve-hole plate, adding seawater to the total volume of 4mL, respectively adding 10 brine shrimps into each hole, and covering a cover plate. Three parallel experiments (including seawater blank) were performed for each gradient. The number of shrimp deaths per well plate observed under the stereoscope was recorded after 24h, 48h (or 72h, 96h), respectively, based on the immobility of the larvae tentacles. The semi-lethal concentration of the brine shrimp was calculated by log-probability unit of concentration (LC 50).
The toxicity test of adult brine shrimp is as above, and the total volume is 50mL by using a 50mL beaker.
Growth inhibition experiments with non-toxic algae: respectively counting the cauliflower, the large platymonas platensis, the pseudo-short Alternaria, the golden algae and the chlorella, and extracting the algal toxin and determining the toxin of the cauliflower. Placing different amounts of original cauliflower into sterilized 75mL conical flask, wherein the concentration of nontoxic experimental algae is 5 × 104cell/mL was used as background value, and sterilized artificial seawater was added to a total volume of 50 mL. Three parallel experiments are respectively carried out on each Dunaliella viridis gradient, and co-culture is carried out. A blank control group is arranged additionally, and cultured in a light incubator with 20 ℃, 4000lx and 12h period, the incubator is shaken once in the morning and at the evening every day, and the nontoxic algae are counted after 4 days. The half-inhibitory concentration (EC50) of the cauliflower toxic algae to the growth of four non-toxic algae is calculated.
Construction of the S3 model: the patinopecten yessoensis toxin (converted according to single algae cytotoxin amount and toxic algae density measured by toxin) obtained by the steps comprises 6 species and 7 groups of toxicity data; the SSD-Generator software is input to calculate that all species sensitivity fitting parameters of the angular cauliflower (based on the patinopecten yessoensis toxin) are 0.9747, which shows that the model applied by the software has better fitting degree to the toxicity data, and the sensitivity distribution curve of the algal toxin to all species is drawn, as shown in figure 1. The safety threshold for protection of 95% of species is 81/mL reticular gonioan density.
The method for evaluating the ecological risk safety threshold of the toxic microalgae is used for evaluating the ecological risk of the reticular original horn algae in the sea area of the great-even long sea, the abundance of the reticular original horn algae cells in the sea area water body is up to 31730 cells/L in 4 months in 2014, and the safety threshold obtained by the model is used for confirming that the annual reticular original horn algae does not threaten the ecological risk of the sea area and is lower than the safety threshold. The Heteropappus californicus in the island area 2011-2017 is phytoplankton dominant species in winter and spring, the abundance value range is 0-8978/L, the species are all in the safe range, and ecological harm is not caused to most species.
S4 model quality evaluation, wherein specific species are removed by collecting literature and toxicity data obtained by S2, the number of the evaluated species is not less than 5, and the evaluated species belong to 3 or more nutrition levels as much as possible; screening 7 groups of acute toxicity data of 6 species of patinopecten yessoensis toxin which are in line with the ecological environment characteristics of China, and taking the arithmetic mean value of a plurality of toxicity data of the same species; based on the obtained acute toxicity data, plotting the concentration value (or the logarithm value) of the toxicity data to the quantile arranged by the concentration, and calculating all species sensitivity fitting parameters of the patinopecten yessoensis toxin by using SSD-Generator software, wherein the fitting parameters are 0.9747; drawing a sensitivity distribution curve of the patinopecten yessoensis toxin (converted by the toxin yield of the Dunaliella unicellular) to all species; HC5 for protecting 95% of species is calculated as a safety threshold value to evaluate the ecological risk of each Dunaliella, which shows that the fitting degree is good and the evaluation model quality is good.
Example 2
Ecological risk safety threshold assessment of Alexandrium mimutum:
the specific evaluation steps are as follows:
s1 separation and culture of algae: alexandrium mimutum used in this study was purchased as a pure species, and was always cultured in this laboratory under a condition of 20 ℃ and 4000lx after sterilized seawater inoculation, and the Alexandrium mimutum was subjected to scale-up culture in a light incubator with a period of 12h:12 h.
Acquisition of S2 toxicity data: the experimental organisms (brine medaka, golden algae, chlorella, Pandalus marmoratus and Thalassiosira pseudonana) were obtained as above. Algal cell counts were as above. And (4) extracting and analyzing paralytic microalgae toxin contained in the Alexandrium mimutum according to standard specifications. Toxicity testing methods are as above. And calculating to obtain a toxicity value LC50 or EC 50.
Construction of the S3 model: the paralytic shellfish toxin (converted according to the single algae cytotoxin amount and the toxin density measured by the toxin) obtained by the steps comprises 5 species, 5 groups of toxicity data, and 10 species with 12 toxicity values reported in the literature are merged; the SSD-Generator software is input to calculate that all species sensitivity fitting parameters of Alexandrium mimutum (calculated according to paralytic shellfish poisoning) are 0.9381, which shows that the model applied by the software has better fitting degree to the toxicity data, and a sensitivity distribution curve of the algae toxins to all species is drawn, as shown in FIG. 2. The safety threshold for protection of 95% of species is approximately 1/mL density of micro-alexandrium alga.
The method for evaluating the ecological risk safety threshold of the toxic microalgae is used for evaluating the ecological risk of the Alexandrium in the sea area of the great-even long sea, the Alexandrium in the water body of the sea area is distributed throughout the year, and 2011-2017 data of the swertia islands show that the Alexandrium in the water body of the sea area is highest in abundance, which is as high as 971/L on average in autumn and winter (9-11 months) every year, and is 1350/L in 2015 11 months. The safety threshold value obtained by the model is used for confirming that the Alexandrium (including Alexandrium mimutum) in the sea area forms threat to the ecological risk of the sea area, and the safety threshold value is higher than the safety threshold value in some months, so that the survival of some sensitive species of organisms can be threatened or toxic hazard can be caused.
And (3) evaluating the quality of the S4 model, wherein all species sensitivity fitting parameters obtained by simulating the toxicity value are 0.9381, which shows that the fitting degree is good and the evaluation model quality is good.
Example 3
Ecological risk safety threshold assessment of prorocentrum lima:
the specific evaluation steps are as follows:
s1 separation and culture of algae: the raw dinoflagellate lima used in the research is purchased pure seeds, the raw dinoflagellate lima is always cultured in a seed preservation way in a laboratory, and after the raw dinoflagellate lima is inoculated by sterilized seawater, the raw dinoflagellate lima is cultured in an illumination incubator with the conditions of 20 ℃, 4000lx and the period of 12h:12h in an expansion way.
Acquisition of S2 toxicity data: the experimental organisms (brine medaka, golden algae, chlorella, Pandalus marmoratus and Thalassiosira pseudonana) were obtained as above. Algal cell counts were as above. Extracting and analyzing diarrheic shellfish toxin contained in the Prorocentrum limanii according to standard specifications. Toxicity testing methods are as above. And calculating to obtain a toxicity value LC50 or EC 50.
Construction of the S3 model: the diarrheic shellfish toxin (converted according to the single algae cytotoxin amount and the toxic algae density measured by the toxin) obtained by the steps comprises 6 species and 8 groups of toxicity data; the SSD-Generator software is input to calculate that the sensitivity fitting parameter of all species of the Prorocentrum lima (calculated according to the diarrheic shellfish toxin) is 0.9539, which shows that the model applied by the software has better fitting degree to the toxicity data, and a sensitivity distribution curve of the algae toxin to all species is drawn, as shown in FIG. 3. The safety threshold for protection of 95% of species is approximately 1/mL of lima prorocentrum density.
The method for evaluating the ecological risk safety threshold of the toxic microalgae is used for evaluating the ecological risk of the lyme-prorocentrum in the large-coptis-ocean sea area, and monitoring data shows that the lyme-prorocentrum exists in the sea area water body in 2016 and 2017, the density is very low, the highest abundance of the lyme-prorocentrum in the sea area water body in 2016 and 8 is 5 cells/L, and the safety threshold obtained by the model is used for confirming that the lyme-prorocentrum in the sea area does not threaten the ecological risk of the sea area. The red tide of the Prorocentrum limani is developed in the east China sea of Zhanjiang in 2010 3 months, and the density of the Prorocentrum limani in seawater is up to 5.0005 multiplied by 106The number per liter seriously threatens the survival of most species in the water body and has great ecological risk.
And (3) evaluating the quality of the S4 model, wherein all species sensitivity fitting parameters obtained by simulating the toxicity value are 0.9539, which shows that the fitting degree is good and the evaluation model quality is good.
Claims (3)
1. A method for evaluating the ecological risk safety threshold of toxic microalgae is characterized in that target toxic microalgae in different sea areas are cultured and toxicity data are obtained, the ecological risk of the toxic microalgae is evaluated by adopting an ecological model evaluation method of a species sensitivity distribution curve method according to toxicity values of the toxic microalgae such as LC50 or LC30 and the like determined according to toxicity research results, an acceptable effect level end point is determined according to the toxicity equivalent of the toxic microalgae and the toxicity values of experimental organisms, the reliable invalid stress concentration of 95% of species in a system is ensured, and the regional ecological risk of the toxic microalgae is quantitatively explained.
2. The method of claim 1, wherein the evaluation comprises the following steps:
s1 separation and culture of algae: separating target toxic microalgae from a pre-evaluation sea water body or obtaining toxic microalgae from other sources, and realizing pure monoclonal culture in a laboratory;
acquisition of S2 toxicity data: carrying out marine organism toxicity test by using pure toxic microalgae to obtain toxicity values LC50, EC50, LC30 or EC 30;
construction of the S3 model: using SSD evaluation software (SSD-Generator), obtaining a toxic algae toxicity value based on the acquired toxicity data, collecting the toxicity value reported in the literature, screening the toxicity data of each species according to the principle of risk evaluation technical schema, calculating to obtain sensitivity fitting parameters of all the species, evaluating the fitting degree, drawing a sensitivity distribution curve of the toxic algae to all the species, obtaining an HC5 value which protects 95% of the species from being influenced as a safety threshold value, and evaluating the ecological risk of the toxic algae and the algal toxins in the regional water body;
further, the method for evaluating the ecological risk safety threshold of the toxic microalgae further comprises the following steps:
the S4 model quality evaluation, through gathering the literature and toxicity data obtained from S2, rejecting the special species, evaluating the number of the species not less than 5, and belonging to 3 or more nutrition levels as much as possible, screening the acute toxicity data values of patinopecten yessoensis toxin, okadaic acid toxin and paralytic shellfish poison which accord with the ecological environment characteristics of China, and taking the arithmetic mean value of a plurality of toxicity data of the same species; calculating all species sensitivity fitting parameters of the 3 algal toxins by using SSD-Generator software based on the obtained acute toxicity data of the 3 algal toxins, and drawing a sensitivity distribution curve of each algal toxin (converted by the toxin yield of a single cell of the toxophyceae) to all species; HC5, which protected 95% of the species, was calculated as a safety threshold to assess the ecological risk of each dunaliella.
3. The method for assessing the ecological risk safety threshold of toxic microalgae according to claim 2, wherein the obtaining of the toxicity data of S2 is: wherein the marine organism is selected from one or more of phytoplankton, zooplankton and fish.
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