CN105651748A - Method for quantitatively analyzing enrichment and distribution of micro plastics in aquatic organisms - Google Patents
Method for quantitatively analyzing enrichment and distribution of micro plastics in aquatic organisms Download PDFInfo
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- CN105651748A CN105651748A CN201610030237.3A CN201610030237A CN105651748A CN 105651748 A CN105651748 A CN 105651748A CN 201610030237 A CN201610030237 A CN 201610030237A CN 105651748 A CN105651748 A CN 105651748A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N2021/6417—Spectrofluorimetric devices
Abstract
The invention discloses a method for quantitatively analyzing enrichment and distribution of micro plastics in aquatic organisms and belongs to the field of environment pollutant detection. The method is a method for quantitatively analyzing micro plastics in aquatic organisms based on the fluorescence tracer technology. The method includes the steps that tested fluorescent micro plastics and mode aquatic organisms are selected for poisoning experiments; target tissue samples are acquired; tissue is decomposed through nitric acid, and the volume of obtained decomposition liquid is made constant; fluorescent micro plastics turbid solutions in different concentration gradients are prepared, and a standard curve is obtained through fluorescence values measured by a fluorescence spectrometer; the fluorescent value of a sample solution is measured, and the content of micro plastics in the corresponding tissue samples is obtained based on the standard curve. By means of the technical scheme, micro plastics ingested in aquatic organisms can be accurately and quantitatively measured, and the purpose of detecting enrichment and distribution of micro plastics in aquatic organisms is achieved.
Description
Technical field
The invention belongs to environmental contaminants detection field, the method that the micro-plastics of a kind of quantitative analysis are enriched with distribution in aquatile body specifically, is a kind of micro-plastic grain quantitative detecting method based on fluorescent tracer technique.
Background technology
Plastics are used widely because of its high stability, persistency and corrosion resistance, and substantial amounts of plastics use and discharge and bring day by day serious pollution to environment, especially small size plastic garbage. Micro-plastics (Microplastics, MPs) refer to the particle diameter plastic grain less than 5 ��m, fragment, fiber etc., derive from industry raw plastic material, personal care articles or by big particle diameter plastic degradation. Invertebrates (shell-fish, bivalve software class, hirsutism link class), vertebrates (seabird, ocean fishes) even mammal (whale) can take in MPs, and MPs can be delivered to high trophic level by food chain from low nutrition level, thus ecological environment is had a negative impact.
Be widely present and the potential hazard of MPs cause mondial research boom. Current research emphasis is in that to illustrate MPs enrichment in aquatile body and distribution. Existing detection method, based on qualitative, carry out observing counting mainly through microscope, and then obtains MPs substantially situation in vivo. Show methodical being disadvantageous in that: (1) processing procedure exists subjectivity. When microscope observes counting, distinguish MPs and other similar substances (such as gravel) exist subjectivity and are difficult to distinguishing, be easily generated deviation. (2) experimental result can only be qualitative, it is impossible to carries out accurate quantitative analysis.
Fluorescent tracer technique is due to high sensitivity and selectivity, often it is used to the position of object observing thing and carries out quantitative measurement, and this technology is applied to detection by quantitative MPs enrichment in aquatile body and distribution, it is possible to it is greatly improved the precision of detection, and there is no relevant report.
Summary of the invention
Can not the difficult problem of accurate analysis MPs enrichment in aquatile body and distribution for existing method, the present invention provides method the method that the micro-plastics of a kind of quantitative analysis are enriched with distribution in aquatile body to realize mainly by fluorescent tracer technique, detection sensitivity is high, reproducible.
The technical solution used in the present invention is as follows:
The micro-plastics of a kind of quantitative analysis are enriched with the method for distribution in aquatile body, it is characterised in that said method comprising the steps of:
(1) exposure experiment: select fluorescently-labeled micro-plastic grain as tested material, preparation contamination substance solution system, using deionized water as blank, choose aquatile as biological subject, carry out exposure experiment;
(2) sample collecting: after contamination end cycle, dissect biological subject, gather destination organization organ, weigh after lyophilization;
(3) tissue is cleared up: is placed in concentrated nitric acid by the lyophilizing tissue in step (2) and clears up, and has cleared up rear constant volume, prepares testing sample solution;
(4) standard curve: the micro-plastic solution of fluorescence of preparation variable concentrations gradient, fluorescence spectrophotometer measures the fluorescence intensity of each solution after arranging particular excitation wavelength and launching wavelength, draw out standard curve with fluorescence intensity and corresponding micro-plastics concentration;
(5) sample determination: exciting and launching under wavelength in step (4), measures the fluorescence intensity of testing sample solution, calculates respective concentration according to standard curve, and after concentration is multiplied by constant volume, volume obtains micro-plastic content;
(6) content conversion: micro-plastic content that step (5) obtains contrasts with corresponding tissue dry weight, conversion obtains the content of micro-plastics in unit mass tissue, and the result of wherein contamination group sample to deduct the background value of blank group, obtains final result.
In above scheme, in step (1), the cycle of exposure experiment is one week.
Further, in step (2), gathering after destination organization organ, by the tissue lyophilization that collects 72 hours, the weight obtained of weighing was dry weight.
Preferably, the destination organization organ gathered in above step (2) is any one in hydrobiological liver, intestinal, the cheek or combination, is more preferably the combination of Different Organs.
In the methods of the invention, the condition clearing up tissue in step (3) is to be added in 1mL concentrated nitric acid by lyophilizing tissue, clears up 2h at 70 DEG C.
Preferably, the excitation wavelength of fluorescence spectrophotometer and fluoroscopic examination and transmitting wavelength respectively 418nm and 518nm.
Beneficial effect: the present invention is based on fluorescent tracer technique, it is provided that the micro-plastics of a kind of quantitative analysis are enriched with the method for distribution in aquatile body. This inventive technique scheme has the advantage that equipment is simple, easy and simple to handle, and treatment conditions are gentle, and method is highly sensitive, reproducible. The fluorescent tracer technique used is highly sensitive, can effectively fluorescence MPs be carried out quantitatively, reaches to measure purpose. The method compensate for the deficiency that MPs is enriched with distribution detection method at present in aquatile body, improve prior art subjectivity in MPs dosing process and deviation, realize effective detection by quantitative, being conducive to the enrichment in various aquatile bodies of the accurate analysis micro-plastics and the regularity of distribution, the bio-toxicity for disclosing micro-plastics provides basic data.
Accompanying drawing explanation
Fig. 1 is the flow chart of technical solution of the present invention.
The canonical plotting of 5 ��m of-MPs-PS of Fig. 2 fluorescence.
The canonical plotting of 20 ��m of-MPs-PS of Fig. 3 fluorescence.
Fig. 4 fluorescence MPs-PS in the Brachydanio rerio gill containing spirogram.
Fig. 5 fluorescence MPs-PS in Brachydanio rerio liver containing spirogram.
Fig. 6 fluorescence MPs-PS in Brachydanio rerio intestinal containing spirogram.
Detailed description of the invention
The invention discloses the method that the micro-plastics of a kind of quantitative analysis are enriched with distribution in aquatile body, the step of the method is as follows:
(1) exposure experiment: selecting tested substance is fluorescently-labeled micro-plastic grain, preparation contamination substance solution system, using deionized water as blank, choose biological subject, carry out exposure experiment;
(2) sample collecting: after contamination end cycle, dissect biological subject, gather destination organization organ (such as the cheek, liver, intestinal etc.), claims dry weight after lyophilization 72h;
(3) tissue is cleared up: be placed in 1mL concentrated nitric acid by the lyophilizing tissue in step (2), 70 DEG C clear up 2h after be settled to 5mL with ultra-pure water, obtain testing sample solution;
(4) standard curve: the micro-plastic solution of fluorescence of preparation variable concentrations gradient, fluorescence spectrophotometer measures the fluorescence intensity of each solution after arranging particular excitation wavelength and launching wavelength, draw out standard curve with fluorescence intensity and corresponding micro-plastics concentration;
(5) sample determination: exciting and launching under wavelength in step (4), measures the fluorescence intensity of testing sample solution, calculates respective concentration according to standard curve, and after concentration is multiplied by constant volume, volume obtains micro-plastic content.
(6) content conversion: micro-plastic content that step (5) obtains contrasts with corresponding tissue dry weight, conversion obtains the content of micro-plastics in unit mass tissue; The result of wherein contamination group sample to deduct the background value of blank group, obtains final result.
In such scheme preferably, described step (1) uses fluorescently-labeled micro-plastic grain.
, described step (3) is cleared up the condition of tissue: lyophilizing tissue adds 1mL concentrated nitric acid, clears up 2h for 70 DEG C in such scheme preferably.
In such scheme preferably, the drafting of described step (4) standard curve: the micro-plastic solution of fluorescence of preparation variable concentrations gradient, with fluorescence intensity and corresponding micro-plastics concentration drawing standard curve, for the micro-plastic content in detection by quantitative tissue.
Below in conjunction with detailed description of the invention, the invention will be further described.
Embodiment
The micro-plastics of quantitative analysis are enriched with the experiment of distribution in aquatile body and comprise the steps.
(1) exposure experiment: with fluorescently-labeled micro-plastic polystyrene granule (MPs-PS) as tested material, animal subject be ripe healthy Brachydanio rerio (Daniorerio, 5 monthly ages, 0.29 �� 0.02g), be randomly divided into contamination group and matched group, often group comprise 3 parallel, each 5 fishes of parallel use, the contamination cycle is 7 days. Matched group uses the aeration tap water of ultraviolet disinfection, contamination group uses 5 ��m and MPs-PS(5 ��m of-MPs-PS of 20 ��m of fluorescence, 20 ��m of-MPs-PS, excitation wavelength 418nm respectively, launch wavelength 518nm), by the ultraviolet disinfection aeration tap water preparation 20mg/LMPs-PS system consistent with matched group. Other experiment conditions are as follows: temperature: 24 �� 1 DEG C, pH:7.2 �� 0.5, dissolved oxygen: 6.6 �� 0.3mg/L, electrical conductivity: 0.256 �� 0.005mS/cm, hardness: 185 �� 9mg/LCaCO3��
(2) sample collecting: after contaminating 7 days, dissects contamination group and matched group Brachydanio rerio, gathers destination organization organ (gill, liver, intestinal), each parallel in the tissue of 5 fishes mix as a sample, lyophilization 72h claims dry weight, and makes a record. As a preferred scheme, it is possible to the different organ and tissue of every fish is mixed as a sample.
(3) tissue is cleared up: by a sample lyophilizing be placed in 1mL concentrated nitric acid in a organized way, 70 DEG C clear up 2h after be settled to 5mL with ultra-pure water, obtain testing sample solution.
(4) standard curve: the micro-plastic solution of fluorescence of preparation variable concentrations gradient, adopts fluorescence spectrophotometer (HITACHI company, Japan), arranges excitation wavelength and transmitting wavelength is 418nm and 518nm, measure the fluorescence intensity of each solution, drawing standard curve.
(5) sample determination: exciting and launching under wavelength at 418nm and 518nm, measures the fluorescence intensity of testing sample solution, calculates respective concentration according to standard curve, and after concentration is multiplied by constant volume, volume obtains micro-amount of plastic.
(6) content conversion: the micro-amount of plastic obtained with corresponding tissue dry weight is made ratio, convert obtain the content of micro-plastics in unit mass tissue; The result of wherein contamination group sample to deduct blank group background value, obtains final result.
Below the result in above-described embodiment is analyzed.
The standard curve of fluorescence MPs-PS arranges standard curve that Concentraton gradient is 0mg/L, 1mg/L, 3mg/L, 5mg/L, 8mg/L, 10mg/L, two kind of particle diameter MPs-PS as shown in Figures 2 and 3.
(1) MPs-PS quantitative analysis in the Brachydanio rerio gill
According to above-mentioned quantitative detecting method, obtain the content of MPs-PS in the Brachydanio rerio gill, as shown in Figure 4.
(2) MPs-PS quantitative analysis in Brachydanio rerio liver
According to above-mentioned quantitative detecting method, obtain the content of MPs-PS in Brachydanio rerio liver, as shown in Figure 5.
(3) MPs-PS quantitative analysis in Brachydanio rerio intestinal
According to above-mentioned quantitative detecting method, obtain the content of MPs-PS in Brachydanio rerio intestinal, as shown in Figure 6.
Below schematically technical solution of the present invention and embodiment thereof being described, this description does not have restricted, and the embodiment related to also is one of embodiments of the present invention, and actual biological subject and tissue are not limited thereto. So, if those skilled in the art is enlightened by it, when without departing from technical solution of the present invention objective, without creatively designing the embodiment similar to this technical scheme, the protection domain of technical solution of the present invention all should be belonged to.
Above in conjunction with the drawings and specific embodiments, embodiments of the present invention are described in detail, but the invention is not restricted to above-mentioned embodiment, in the ken that art those of ordinary skill possesses, it is also possible to make a variety of changes under the premise without departing from present inventive concept.
Claims (7)
1. the method that the micro-plastics of quantitative analysis are enriched with distribution in aquatile body, it is characterised in that said method comprising the steps of:
(1) exposure experiment: select fluorescently-labeled micro-plastic grain as tested material, preparation contamination substance solution system, using deionized water as blank, choose aquatile as biological subject, carry out exposure experiment;
(2) sample collecting: after contamination end cycle, dissect biological subject, gather destination organization organ, weigh after lyophilization;
(3) tissue is cleared up: is placed in concentrated nitric acid by the lyophilizing tissue in step (2) and clears up, and has cleared up rear constant volume, prepares testing sample solution;
(4) standard curve: the micro-plastic solution of fluorescence of preparation variable concentrations gradient, fluorescence spectrophotometer measures the fluorescence intensity of each solution after arranging particular excitation wavelength and launching wavelength, draw out standard curve with fluorescence intensity and corresponding micro-plastics concentration;
(5) sample determination: exciting and launching under wavelength in step (4), measures the fluorescence intensity of testing sample solution, calculates respective concentration according to standard curve, and after concentration is multiplied by constant volume, volume obtains micro-plastic content;
(6) content conversion: micro-plastic content that step (5) obtains contrasts with corresponding tissue dry weight, conversion obtains the content of micro-plastics in unit mass tissue, and the result of wherein contamination group sample to deduct the background value of blank group, obtains final result.
2. the method that the micro-plastics of quantitative analysis as claimed in claim 1 are enriched with distribution in aquatile body, it is characterised in that in step (1), the cycle of exposure experiment is a week.
3. the method that the micro-plastics of quantitative analysis as claimed in claim 1 are enriched with distribution in aquatile body, it is characterised in that in step (2), after gathering destination organization organ, by the tissue lyophilization that collects 72 hours, the weight obtained of weighing was dry weight.
4. the method that the micro-plastics of quantitative analysis as claimed in claim 3 are enriched with distribution in aquatile body, it is characterised in that the destination organization organ gathered is any one in hydrobiological liver, intestinal, the cheek or combination.
5. the method that the micro-plastics of quantitative analysis as claimed in claim 4 are enriched with distribution in aquatile body, it is characterised in that the destination organization organ gathered is the combination of hydrobiological liver, intestinal, the cheek.
6. the method that the micro-plastics of quantitative analysis as claimed in claim 1 are enriched with distribution in aquatile body, it is characterised in that the condition clearing up tissue in step (3) is to be added in 1mL concentrated nitric acid by lyophilizing tissue, clears up 2h at 70 DEG C.
7. the method that the micro-plastics of quantitative analysis as claimed in claim 1 are enriched with distribution in aquatile body, it is characterised in that the excitation wavelength of fluoroscopic examination and transmitting wavelength respectively 418nm and 518nm.
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| CN106645049A (en) * | 2016-09-30 | 2017-05-10 | 大连海洋大学 | Method for detecting plastic content of marine organism |
| CN107328622A (en) * | 2017-07-06 | 2017-11-07 | 南京大学 | A kind of method for preparing the micro- plastics of bar-shaped fluorescence labeling |
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| CN107328622A (en) * | 2017-07-06 | 2017-11-07 | 南京大学 | A kind of method for preparing the micro- plastics of bar-shaped fluorescence labeling |
| CN107449655A (en) * | 2017-08-15 | 2017-12-08 | 浙江工业大学 | The pre-treating method of micro- plastics in a kind of identification marine product |
| CN107957490A (en) * | 2017-11-17 | 2018-04-24 | 中国水产科学研究院黄海水产研究所 | The experimental provision and application method of the micro- plastics ecological niche on-spot study of seawater |
| CN107957490B (en) * | 2017-11-17 | 2020-04-03 | 中国水产科学研究院黄海水产研究所 | Experimental device for seawater micro-plastic ecological niche in-situ research and using method |
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