CN116165183A - Biomarker for risk assessment of marine pollutants in aquatic ecosystem - Google Patents
Biomarker for risk assessment of marine pollutants in aquatic ecosystem Download PDFInfo
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Abstract
The invention discloses a biomarker for marine pollutant risk assessment of an aquatic ecosystem. Belongs to the technical field of biological monitoring environment. The invention examines the toxicity mechanism of two typical marine pollutants on the marine Synechococcus PCC7002 strain, screens out the biomarker for environmental pollutant risk assessment from the growth and photosynthesis parameters obtained in the toxicity test, performs obvious differential analysis by a variance analysis method, combines with IC on the basis of Pearson correlation coefficient analysis 50 Value screening out novel efficient toxicology biomarker delta F IP . The sample pretreatment method in the marine pollutant risk assessment method of the aquatic ecosystem is simple,the chlorophyll fluorescence analyzer can be used for in-situ detection of algae strains, the stability of the results is good, the repeatability is high, the risk of marine harmful substances can be accurately estimated and predicted, and early prediction and early warning of marine pollutant risk estimation are provided.
Description
Technical Field
The invention relates to the technical field of biological monitoring environments, in particular to a biomarker for risk assessment of marine pollutants of an aquatic ecosystem.
Background
The 21 st century is called the "century of the ocean", and protection of the marine environment is an important component in the development of sustainable development strategies. With the increasing demand for chemicals in industrial and agricultural production and the prosperity of marine transportation, the number of chemicals in global marine transportation increases by 2.15 million tons per year. Among them, most chemicals are considered as dangerous and toxic substances, and the risk of leakage during transportation thereof is also increasing. Once leaked into the marine environment, dangerous and toxic substances pose a serious threat to living beings, humans and the surrounding environment. The increasing frequency of marine transport activities of dangerous and toxic substances has raised concerns about their potential damage to the ecological environment. Therefore, the biological accumulation effect and the toxic effect of the marine pollutants are studied deeply, and the development of an effective strategy for rapidly monitoring the marine pollutants in the aquatic ecosystem has important significance.
Microalgae are the main component of the primary productivity of the marine ecosystem, and play an important role in maintaining the energy balance of the aquatic ecosystem. Thus, microalgae toxicity tests are believed to provide early warning signals for aquatic ecosystems. Synechococcus sp.PCC7002 is a single-cell marine microalgae with wide salt, has certain heat resistance and high temperature resistance, and is one of the most rapid growth strains in cyanobacteria. These superior characteristics make PCC7002 algal strains a potential test organism for risk assessment of marine pollutants in aquatic ecosystems.
To date, the development of effective and rapid tools for monitoring the toxic effects of marine pollutants is a major limiting factor. Biomarker methods have been successfully applied to environmental monitoring and assessment, and screening for suitable toxicological biomarkers is critical to assessing potential risk of toxicity of marine pollutants in aquatic ecosystems. Previous studies have explored a variety of biomarkers sensitive to marine pollutants, such as metallothionein, 7-ethoxymethyl red O-deethylase, and antioxidant enzyme activity, among others. In view of the key role of photosynthesis in maintaining energy balance in aquatic ecosystems, it is urgent to mine photosynthetic indicators as novel biomarkers for detecting ocean pollutants in aquatic ecosystems. Chlorophyll fluorescence technology plays a unique role in measuring the physiological state of plants and photosynthesis. Early studies showed that harmful substances can reduce the activity of photosystem II and prevent photosystem I from being photo-inhibited. Thus, the growth and photosynthesis parameters obtained from the toxicity test can provide a risk assessment for marine pollutants in the aquatic ecosystem, aid in environmental decisions, and facilitate the development of novel biomarkers for rapid detection of marine pollutants in the aquatic ecosystem.
In summary, providing a novel high-efficiency biomarker for marine pollutant risk assessment of aquatic ecosystems is a problem in the art that needs to be solved.
Disclosure of Invention
In view of this, the present invention provides a biomarker for marine pollutant risk assessment of an aquatic ecosystem.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a biomarker for marine pollutant risk assessment of an aquatic ecosystem, the biomarker being δf IP ；
δF IP The difference in fluorescence intensity between point I and point P in the OJIP fluorescence induction curve.
Further, the screening process includes the steps of:
(1) The method comprises the following steps of carrying out an acute toxicity test for Synechococcus PCC7002 strain for 110 hours by using marine pollutants, wherein the marine pollutants are benzyl alcohol and phenethyl alcohol, and the method comprises the following steps:
(11) Biological culture of algal strains: the culture medium of the algae strain is A + The culture medium is placed in an illumination incubator for culture.
(12) Exposure test of marine pollutants: preparing benzyl alcohol and phenethyl alcohol test liquid with certain concentration gradient, dividing the algae strain into 4 groups, wherein 1 group is a control group, and the other 3 groups are test groups, setting 3 parallel groups in each group, respectively placing into an illumination incubator for culturing, and sampling at 110 hours.
(13) Determination of algal strain biomass: the determination of algae strain biomass adopts turbidity colorimetric method. The test uses an ultraviolet-visible spectrophotometer to measure the absorbance (OD) 730 )。
(14) Determination of chlorophyll fluorescence parameters of algal cells: the chlorophyll fluorescence characteristics of algal cells were measured by a chlorophyll fluorescence meter (Aqua Pen-C AP-C100).
The beneficial effects of adopting above-mentioned scheme are: the two marine pollutants have obvious toxic effects on the growth and photosynthetic performance of the PCC7002 algae strain, and the toxic effects are concentration-dependent. Meanwhile, the stress of marine pollutants damages the structure of the algae strain oxygen release complex, inhibits the transfer of electrons in a photosynthetic electron transfer chain, and influences the absorption, transfer and transfer of light energy by a photosynthesis reaction center.
Based on the technical scheme, the invention also makes the following improvements:
the marine pollutant carries out 110-hour acute toxicity test on the Synechococcus PCC7002 strain, analyzes the change condition of biochemical indexes related to growth metabolism and photosynthetic metabolism, and obtains a series of biomarkers for marine pollutant risk assessment.
(2) A screen for novel high-efficiency biomarkers for marine pollutant risk assessment of an aquatic ecosystem, comprising the steps of:
(21) A range of biomarkers obtained from acute toxicity tests were screened for potential biomarkers for marine contaminant risk assessment by analyzing the dose-response relationship of biomarker response to marine contaminant concentration.
(22) Significant variance analysis is carried out by a variance analysis method, and based on Pearson correlation coefficient analysis, the combination of IC 50 And (5) screening out novel high-efficiency toxicology biomarkers.
By the method, a novel high-efficiency biomarker delta F for marine pollutant risk assessment of the aquatic ecosystem is screened out IP 。
Compared with the prior art, the invention has the beneficial effects that: according to the marine pollutant risk assessment method for the aquatic ecological system, the sample pretreatment method is simple, the chlorophyll fluorescence instrument can be used for carrying out in-situ detection on the algae strain, the result stability is good, the repeatability is high, the risk of marine harmful substances can be accurately assessed and predicted, and early prediction and early warning of marine pollutant risk assessment are provided.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the effect of two marine pollutants on the growth condition of a PCC7002 strain in example 1 of the present invention, wherein A, B, C and D represent the growth condition of a benzyl alcohol treated strain, a physical image of a benzyl alcohol treated strain and a physical image of a benzyl alcohol treated strain, respectively;
FIG. 2 is a graph showing the effect of two marine pollutants on the chlorophyll fluorescence induction curve of the PCC7002 strain in example 1 of the present invention, wherein graph A shows the benzyl alcohol treatment results and graph B shows the benzyl alcohol treatment results;
FIG. 3 shows the effect of two marine pollutants on the chlorophyll fluorescence parameters of a PCC7002 strain in example 1 of the present invention, wherein A is the values of the benzyl alcohol treated fluorescence parameters ETo/TRo, ETo/ABS, VJ, mo and Sm, B is the values of the benzyl alcohol treated fluorescence parameters ETo/TRo, ETo/ABS, VJ, mo and Sm, and C is the benzyl alcohol (left) or benzyl alcohol (right) treated fluorescence parameters W k The D graph shows the fluorescence parameter delta F after benzyl alcohol treatment JI And delta F IP The E graph shows the fluorescence parameter delta F after phenethyl alcohol treatment JI And delta F IP The F diagram is the values of fluorescence parameters Fv/Fm and PIabs after benzyl alcohol treatment, the G diagram is the values of fluorescence parameters Fv/Fm and PIabs after benzyl alcohol treatment, and the H diagram is the values of fluorescence parameters ABS/RC, TRo/RC, ETo/RC, DIo/RC and Fo/Fm after benzyl alcohol (left) or benzyl alcohol (right) treatment;
FIG. 4 is a graph showing the effect of two marine pollutants on the inhibition rate of different biomarkers in example 2 of the invention, wherein graph A shows the benzyl alcohol treatment result and graph B shows the benzyl alcohol treatment result;
FIG. 5 is a graph showing the analysis of the Pearson correlation coefficient between various growth and photosynthesis metrics and two marine contaminant concentrations in example 2 of the present invention;
FIG. 6 is a schematic representation of the toxic mechanisms of two marine pollutants on the growth metabolism and photosynthetic metabolism of PCC7002 algal strains.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The required medicament is a conventional experimental medicament and is purchased from a commercial channel; the test methods not mentioned are conventional test methods and will not be described in detail herein.
The toxicity potential test of the PCC7002 algae strain proves the concentration dependency effect of two marine pollutants on the growth and photosynthetic performance of the PCC7002 algae strain so as to examine the toxicity mechanism. The growth and photosynthesis parameters obtained from toxicity tests were analyzed for significant differences by analysis of variance (ANOVA) and based on Pearson correlation coefficient analysis, combined with the sensitivity of the different parameters to marine pollutants (IC 50 ) Screening out novel efficient biomarker delta F for marine pollutant risk assessment of aquatic ecosystem IP 。
Example 1 toxicity mechanism of two Marine pollutants on PCC7002 algal strains
(1) Biological culture of algal strains
The experimental test organism is sea Synechococcus PCC7002, and the culture medium of the strain A + The culture medium is placed in an illumination incubator for culture. Wherein the culture temperature is 35+ -1deg.C, and the illumination intensity is 80 μmol photons m -2 s -1 The light-dark period was 14h:10h and the rotational speed was 50rpm.
(2) Exposure test of toxic compounds
Taking a plurality ofCell culture flasks, to which a number of autoclaved A were added, respectively + The final concentrations of benzyl alcohol and phenethyl alcohol in each flask were 0.00, 0.50, 1.00 and 1.50g/L, respectively, and each group was repeated 3 times. Then PCC7002 algae strain in logarithmic growth phase is added, shake is carried out uniformly, and OD is initialized 730 The value was 0.350, and the cells were cultured in an illumination incubator and sampled at 110 h.
(3) Determination of algal Strain biomass
The determination of algae strain biomass adopts turbidity colorimetric method. The test uses an ultraviolet-visible spectrophotometer to measure the absorbance (OD) 730 ) The test result data are analyzed by adopting univariate analysis of variance (ANOVA) and Duncan multiple comparison test.
(4) Determination of chlorophyll fluorescence parameters of algal cells
The chlorophyll fluorescence characteristics of algal cells were measured by a chlorophyll fluorescence meter (Aqua Pen-C AP-C100). Before measurement, algae cells of different treatment groups are taken and utilized A + Absorbance OD of each treatment group with medium 730 Adjusted to the same level and placed in a dark environment for 30 minutes, and the measurement operation was performed in the dark environment.
The chlorophyll fluorescence parameter information used is shown in table 1.
TABLE 1 chlorophyll fluorescence parameter information
The effect of two marine pollutants on growth of PCC7002 algal strains is shown in figure 1. With the increasing concentration of the two marine pollutants tested, the growth of the algae strain is significantly inhibited. When the two marine pollutants are increased to 2g/L, the algal strains are not good or can not survive basically. Thus, the subsequent experiments examined only the toxic effects of three concentration gradients of 0.50, 1.00, 1.50 g/L.
The effect of two marine pollutants on the chlorophyll fluorescence induction profile of the PCC7002 algal strain is shown in figure 2. With increasing treatment concentrations of both marine pollutants, the chlorophyll fluorescence induction profile of PCC7002 algal strains appears to decrease in sequence.
The effect of two marine pollutants on chlorophyll fluorescence parameters of the PCC7002 algal strains is shown in figure 3. With the increasing treatment concentration of two marine pollutants, W k Fluorescence parameters such as DIo/RC, fo/Fm, sm and the like show positive correlation with marine pollutant treatment concentration, while OD 730 、Fv/Fm、PIabs、ETo/ABS、δF JI 、δF IP The isosulfuorescence parameters show a negative correlation with the marine contaminant treatment concentration, they are potential biomarkers for marine contaminant risk assessment.
In conclusion, through toxicity potential tests on the PCC7002 algae strain, two marine pollutants are found to have obvious toxicity effects on the growth and photosynthetic performance of the PCC7002 algae strain, and the toxicity effects are concentration-dependent. Meanwhile, the stress of marine pollutants damages the structure of the oxygen-releasing complex of the algae strain, inhibits the transfer of electrons in a photosynthetic electron transfer chain, and influences the absorption, transfer and transfer of light energy of a photosynthesis reaction center. Most important is the acquisition of a range of biomarkers for marine contaminant risk assessment.
Example 2 dose-response relationship of biomarker response to toxic compound concentration
A range of biomarkers obtained from toxicity assays were screened for potential biomarkers for marine contaminant risk assessment by analyzing the dose-response relationship of biomarker response to marine contaminant concentration. Significant variance analysis is carried out by a variance analysis method, and based on Pearson correlation coefficient analysis, the combination of IC 50 And (5) screening out novel high-efficiency toxicology biomarkers.
The results of the two marine contaminant and different biomarker significance analysis and the linear correlation analysis are shown in table 2.
Table 2 two marine pollutant and different biomarker significance and linear correlation analyses
The effect of two marine pollutants on inhibition rates of different biomarkers is shown in FIG. 4, the abscissa of FIG. 4 shows the treatment concentration of marine pollutants, the ordinate shows the inhibition rates of different biomarkers, and the colored boxes respectively represent the ICs of different biomarkers 50 Values. With increasing treatment concentrations of the two marine pollutants, significant differences in inhibition rates of the potential biomarkers obtained in example 1 occurred. IC binding to different biomarkers 50 Values we found δF IP Is of (2) 50 The minimum value indicates δF IP The index is more sensitive to both marine pollutants.
By the method, a novel high-efficiency biomarker delta F for marine pollutant risk assessment of the aquatic ecosystem is screened out IP 。
The method for preprocessing the samples is simple, can use the chlorophyll fluorescence instrument to perform in-situ detection on the algae plants, has good result stability and high repeatability, can accurately evaluate and predict the risk of marine harmful substances, and provides early prediction and early warning for marine pollutant risk evaluation.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (6)
1. A biomarker for marine pollutant risk assessment of an aquatic ecosystem, wherein the biomarker is δf IP ；
δF IP The difference in fluorescence intensity between point I and point P in the OJIP fluorescence induction curve.
2. A biomarker for marine pollutant risk assessment of an aquatic ecosystem according to claim 1, wherein the screening process comprises the steps of:
(1) The marine pollutant carries out 110-hour acute toxicity test on the marine Synechococcus PCC7002 strain, analyzes the change condition of biochemical indexes related to growth metabolism and photosynthetic metabolism, explores the toxicity mechanism of the marine Synechococcus PCC7002 strain, and obtains a series of biomarkers for marine pollutant risk assessment;
(2) Significant variance analysis is carried out by a variance analysis method, and based on Pearson correlation coefficient analysis, the combination of IC 50 And (5) screening out novel high-efficiency toxicology biomarkers.
3. The biomarker according to claim 1, wherein the test method of growth metabolism in step (1) is turbidity and the test index is absorbance value OD 730 。
4. The biomarker according to claim 1, wherein the test method of photosynthetic metabolism in step (1) is chlorophyll fluorescence, and the test index is a series of fluorescence parameters.
5. The biomarker of claim 1, wherein the panel of biomarkers screened in step (1) is OD 730 、Fv/Fm、PIabs、ETo/ABS、δF JI 、δF IP 。
6. The biomarker of claim 1, whereinWherein the novel high-efficiency biomarker screened in the step (2) is delta F IP 。
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| CN116818995A (en) * | 2023-04-18 | 2023-09-29 | 安徽启威生物科技有限公司 | Multi-class bactericide product stability test platform based on big data analysis |
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| CN116818995A (en) * | 2023-04-18 | 2023-09-29 | 安徽启威生物科技有限公司 | Multi-class bactericide product stability test platform based on big data analysis |
| CN116818995B (en) * | 2023-04-18 | 2024-04-16 | 安徽启威生物科技有限公司 | Multi-class bactericide product stability test platform based on big data analysis |
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