CN105628781A - Isotopic tracing method for fate characteristics of organic substances in aquaculture pond - Google Patents
Isotopic tracing method for fate characteristics of organic substances in aquaculture pond Download PDFInfo
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Abstract
According to an isotopic tracing method for fate characteristics of organic substances in an aquaculture pond, stable isotopes of biological samples and no-biological samples are measured according to the fractionation effect of the stable isotopes for the purpose of analyzing flow characteristics of nutrient substances in an ecological system. The method comprises the steps of 1 selection and pretreatment of the samples for measurement, 2 dry grinding and sample packing, 3 arrangement of a mass spectrometer, 4 addition to and measurement of the packed samples in the step 2, and 5 output of a result and processing of data. According to the carbon and nitrogen international master standard correction result, the actual value of the carbon and nitrogen isotopes in the samples is output. According to the method, experience is summarized from all kinds of stable isotope measurement methods, and the method which can easily, conveniently and accurately measure the stable isotopes in the aquaculture pond samples in a standard mode is extracted out.
Description
Technical field
The inventive method technology belongs to ecosphere, is specifically related to a kind of method measuring cultivating pool ecosystem stable isotope, analyzes pond material cycle and flow of energy situation accordingly.
Background technology
The trophic structure of food web, material cycle and flow of energy are the integral parts of ecosystem research, and along with the development of science and technology, deepening continuously of research contents and improving constantly that result of study requires, traditional method can not meet requirement. Since the sixties in 20th century, the applied research of stable isotope makes a breakthrough. In recent years, stable isotope technology was applied in field of ecology research as a kind of important research means. Organism takes food the different food in source, cause in body isotropic substance composition difference through accumulation, stable isotope analysis based on this at wildlife food resource, Habit at selection, migrate, the structure of physiologic status assessment, nutritive substance distribution in vivo, aquatic food webs and the ecosystem obtains in studying with flow of energy etc. and applies more and more widely.
Compared with traditional animal feeding habits analytical procedure, this technology has obvious advantage. Not only can carrying out looking back investigation and analysis to the material obtained, this is that other feeding habits analytical procedures do not have again. Simultaneously by the foundation of isotopic mass equilibrium equation, it is possible to food source analysis is carried out qualitative, quantitative accurately and calculates. According to special model, system can be analyzed.
At present, there is a large amount of examples applied about stable isotope technology in field of ecology. Such as, utilize carbon and nitrogen stable isotope technical study aquatic food webs structure and trophic level relation, hydrocoles food transition, the Migration distribution etc. of hydrobiont, even to this day, its function almost penetrates into every field, industry, becomes the strong instrument promoting scientific progress, the national economic development. But, fully, accurately utilize this technology, premised on reasonable, reliable take off data. At present before isotopic analysis, very big difference is there is in the step such as standard operation about the process of sample, operation of entering sample, instrument in each research, so far also not formed and the preservation of standard accordingly of different biological species and pretreatment process and unified analytical model, selecting for the investigator of this technology of application has certain difficulty during method. The result that different treatment method and mixture model obtain is also variant, this makes Stable isotope ratio mutually comparing between each research there is very big difficulty, also being not easy to combine in each research different biological isotopic ratios goes to build complete food web model, so, the accuracy of data is very important by good stable isotope measurement technology.
Summary of the invention
The present invention provides a kind of method measuring cultivating pool ecosystem stable isotope, it is possible to simplicity, stdn, reliably measure isotropic substance.
An aspect of the present invention, provide a kind of cultivating pool organic substance and return the characteristic isotope tracer technique that becomes, fractionation effect according to stable isotope, measures the flow performance that stable isotope that is biological and abiotic sample analyzes ecosystem Middle nutrition material, comprises the steps:
(1) for the selection of sample that measures and pre-treatment
A, make sample for the Selection of fishes muscle of back tissue measured;
B, for the band shell animal that measures as the part removing shell during prey animal as sample, for the band shell animal that measures as the whole individuality comprising shell during predator all as sample;
C, select whole individuality as sample for the zooplankton measured;
D, select whole individuality as sample for the zoobenthos measured;
Particulate organic matter (POM) sample in E, water body obtains after filtering waters to be measured water body by 0.7 ��m, aperture glass fiber filter, and described glass fiber filter is in advance through 450-550 DEG C of calcination 4 hours;
F, remove surface impurity for the terrestrial plant that measures and waterplant after whole individuality as sample;
G, sediment sample and the uniform region of the fish movement of the sample containing inorganic carbonate on 1 meter more than, offshore limit gather and obtain, and drip with hydrochloric acid and add process after removing impurity, until not having bubble to produce, or adopt concentrated hydrochloric acid suffocating treatment;
(2) drying and grinding and bag sample
A, drying and grinding: after the sample of acquisition is completely dry, grind to form uniform powder;
B, again drying: the sample powder of acquisition is dried 2-4 hour before the assay again;
C, bag sample: powdered samples is accurately taken in tinfoil paper cup by the mass range analytical balance of test request, choose a kind of sample as internal standard substance, every 9 sample room bags interior standard specimen, the sample wrapped is placed in corresponding sample panel (stability for during timing optical viewer test sample), and record;
(3) mass spectrometric setting
A, open mass spectrograph, vacuum pump and computer, control vacuum pump by computer and regulate mass spectrometric vacuum tightness: regulate vacuum tightness (HVac) to e-009mBar;
B, open gas cylinder valve and check the gaseous tension of gas cylinder;
C, open elemental analyser and hunt leak;
D, by computer regulated furnace temperature and flow velocity: 900 DEG C, left stove, 680 DEG C, right stove, chromatographic column 50 DEG C, flow velocity 140ml/min;
E, before temperature, square frame is beaten hook, removes hook �� Send �� OK before SetInstrumenttostand;
F, confirmation intensification and flow velocity are normal, after having heated up, first open vacuum valve, then open ion source, stablize test sample after 10min until ion source;
(4) sample pipetting volume will wrapped in step (2), measures;
(5) output of result and the process of data
According to carbon nitrogen international standards correction result, export sample carbon nitrogen isotope actual value.
Above-described method, wherein, determining sample type according to food web composition, concentrated hydrochloric acid suffocating treatment described in step (1) is stifling 4 hours of the hydrochloric acid adopting concentration to be 12mol/L, drips with hydrochloric acid and add the hydrochloric acid that process adopts 1ml/L after described removal impurity.
Above-described method, wherein, determines sample type according to food web composition, and the drying and grinding step in step (2) comprises vacuum-drying, lyophilize or 40-70 DEG C of oven drying at low temperature.
Above-described method, wherein, determines sample type according to food web composition, takes fish tissue and zoobenthos about 1.5mg in the bag sample step in step (2), plant 2-3mg, POM are 15mg, plant plankton 15mg, settling 15-20mg.
Above-described method, wherein, determining sample type according to food web composition, the gaseous tension of the gas cylinder described in step (3) is adjusted to nitrogen 0.3Mpa, carbonic acid gas 0.3Mpa, air 0.2Mpa, helium 0.3Mpa, oxygen 0.3Mpa respectively.
Above-described method, wherein, determine sample type according to food web composition, step (3) also comprises the verification step of isotope mass spectrometer preci-sion and accuracy, specifically comprises:
The determination of A, precision: the isotope mass spectrometer having held machine successfully, first verifies its precision, by choosing a kind of sample at random as internal standard substance, repeats 10��12 times and measures carbon and nitrogen stable isotope �� X value, calculate �� respectively13C and ��15The standard deviation of N, verifies mass spectrometric precision;
The survey of the determination of B, accuracy: choice criteria TTB-1 relatively, by the degree of agreement accuracy of judgement degree of the value that records and standard value.
Above-described method, wherein, determines sample type according to food web composition, by adding, sample device adds sample to the sample step that adds in step (4) from sample holes port, add 2min after sample, evacuation of air, select to start test sample after data output paths accurately by computer.
Above-described method, wherein, determines sample type according to food web composition, and the international standards described in step (5) is USGS24 and USG26.
The present invention sums up experience from various stable isotope determination method, refine out a kind of method that can accurately measure cultivating pool sample stable isotope, do not only illustrate the detailed process of test sample, also indicate the problem measuring in cultivating pool sample carbon and nitrogen stable isotope process and should be noted that simultaneously, to the sample choice easily causing isotropic substance �� value to change (as fish body muscle is selected, the collection etc. selecting and adhering to algae of zoobenthos) and pre-treatment (zoobenthos is foster clearly, settling acidifying etc.) etc. step carried out scientific and reasonable standardization, sample choice is carried out according to the feature of flowing of isotropic substance in pond ecosystem and the stability of sample, ensure the reliability of measuring result.
The technology of the present invention process is easy, easily handles execution, and has strict standardization and science.
The technology of the present invention has related to a series of processes measuring the sample pre-treatments of sample stable isotope and measuring to mass spectrograph, and systematicness is high, and method is comprehensive.
Accompanying drawing explanation
Fig. 1 is the mensuration C of offer in the embodiment of the present invention, the mensuration schema of N stable isotope.
Fig. 2 is the detecting instrument preci-sion and accuracy provided in the embodiment of the present invention and small lobsters C, N isotopic ratio figure repeating 9 mensuration. The mean value of carbon stable isotope and standard deviation are respectively-26.609 �� and 0.088 ��; The mean value of nitrogen stable isotope and standard deviation are respectively 13.855 �� and 0.113 ��.
Fig. 3 is the cultivating pool isotopic analysis model data figure made according to the SIAR software package for isotopic analysis in the embodiment of the present invention, and Group1-5 refers to grass carp, flathead, crucian carp, black carp, silver carp respectively; Other seven kinds is source.
Embodiment
The advantage of the solution of the present invention and its all respects below in conjunction with drawings and Examples, the specific embodiment of the present invention is described in more details, can be understood better. But, embodiment described below and example are only the objects illustrated, instead of limitation of the present invention.
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
The step of the mensuration stable isotope of the present invention is as follows:
(1) selection of sample and pre-treatment
Select 3 four large Chinese carp polycultured ponds in region, Xinzhou, Wuhan, acquire the settling in pond, POM, attachment algae, fish dorsal muscle, the feed thrown something and fed, forage, zoobenthos.
Sedimental collection: sit canoe and gather three sediment samples with the gloomy sludge mining device of Peter near the cornerwise mid point in pond and from the both sides of pond bank about 2m, load in the sealed bag marked, put into insulation can to take back 60 DEG C, laboratory baking oven and dry to constant weight, the dilute hydrochloric acid of the sediment sample 1mol/L after mortar is pulverized soaks acidifying, successive soaking twice or thrice, until not having bubble to produce.
The collection of the particulate organic matter (POM) in water body: GF/C glass fiber filter is the organic substance of calcination 4h to remove on filter membrane in the retort furnace of 450-550 DEG C, gather cultivating pool water sample, large-scale zooplankton and algae is removed in advance with the plankton net of 113 ��m, then by the GF/C membrane filtration of water sample Vacuum filtration device after calcination, until filtered water stream slowly time, take out filter membrane and also dry at 60 DEG C.
The collection of attachment algae: get the attachment algae being attached to the surfaces such as edge, pond pasture and water, stone, bamboo pole with toothbrush brush, wash in distilled water, filtering water sample with the WhatmanGF/C glass fiber filter of 450-550 DEG C of calcination 4h under the low pressure of vacuum filtration machine, filter membrane is dried at 60 DEG C.
The collection of fish muscle sample: the adult fish gathering various nursing from pond, often kind of 3-5 bar, loads in the insulation can putting ice cube in advance well, takes back laboratory and gets fish dorsal muscle one fritter on dissection platform, remove skin, puts into 50ml centrifuge tube 60 DEG C and dry to constant weight.
The collection of input: feed and forage directly gather around bait machine and pond, loads sealed bag and takes back laboratory, dries to constant weight for 60 DEG C.
The collection of zoobenthos: gather top layer, pond bed mud with the gloomy sludge mining device of Peter near the cornerwise mid point in pond and from the both sides of pond bank about 2m, on-the-spot by 60 object screen cloth washing and filtering, washing rear residue loads in sealed bag, add a certain amount of water-band and go back to laboratory, from bed mud, pick out zoobenthos and identify, classify according to the big class such as shellfish, spiral shell class, oligochaetes and midge class, the zoobenthos of same species is mixed together, it is positioned in distilled water and spends the night, then dry to constant weight for 60 DEG C. When spiral shell class is as food, shell is removed, it is necessary to explanation, because band shell is biological as the not easily prey animal absorption of shell during prey animal, removes shell anti-tampering, derive from food as shell during predator, therefore stay when stable isotope is analyzed.
Containing significant quantities of fat in shellfish and zooplankton body, be necessary that fat processes during isotope assay, this example does not collect them, and the sample lipid content gathered is all little, therefore without the need to removing fat.
(2) drying and grinding and bag sample
A, drying and grinding: all samples to be all dried to dry-matter, concrete grammar has vacuum-drying, lyophilize and 40-70 DEG C of oven drying at low temperature, wherein vacuum lyophilization and oven drying at low temperature effect are all good, then sample is ground to form uniform fine powder, not only representative but also can react fully and carry out.
B, dry again: powdered sample also may can absorb the moisture in air depositing in process, so sample dries 2-4 hour (60 DEG C) again before formal test sample.
B, bag sample: accurately take in tin cup by the mass range analytical balance of test request by powdered samples, fish tissue and zoobenthos about 1.5mg, plant 2-3mg, POM are about 15mg, plant plankton about 15mg, settling 15-20mg. The tinfoil paper cup of suitable size is selected to wrap, it is noted that gas of leaving a blank of trying not inside the sample wrapped, for ensureing accuracy, choose a kind of sample as internal standard substance, empirically, every 9 sample room bags interior standard specimen, the sample wrapped to be placed in corresponding sample panel, and makes a record.
(3) checking of isotope mass spectrometer preci-sion and accuracy
Open mass spectrograph, vacuum pump and computer, open the software Aquisiton in computer, regulate vacuum tightness HVac to e-009mBar, FVac to e-003mBar;
Open gas cylinder, first open total valve, then open dividing potential drop valve (Mpa);
Note: total valve thoroughly to be opened, check that whether gaseous tension is enough simultaneously.
| Nitrogen | Carbonic acid gas | Air (general nitrogen) | Helium | Oxygen |
| 0.3 | 0.3 | 0.2 | 0.3 | 0.3 |
Open elemental analyser, with suds leak detection, dry.
Heat up with EA software, E.A.#2 �� EditElementalAnalyzerparameters �� furnace temperature: 900 DEG C, left stove, 680 DEG C, right stove, chromatographic column 50 DEG C, flow velocity 140ml/min.
Before temperature, square frame is beaten hook, removes hook �� Send �� OK before SetInstrumenttostand.
Whether click ViewElementalAnalyzerStatus observation intensification and flow velocity situation be normal.
After intensification completes, first opening vacuum valve, then open ion source, treating that 10min stablized by ion source namely can test sample.
Add sample, test sample: add sample and enter to add sample device, add from sample holes port;
Then in software Aquisiton, find sample table, write each parameter; Choosing data, preserve, start (starting after waiting 2min after adding sample, evacuation of air), choose the folder to be preserved, amendment filename, the parameter shown in select File name, other do not change, hit OK;
Note: choose data to be chosen full line at Far Left constantly, at most must not more than 32 row (corresponding autopipette 32 holes).
The determination of mass spectrograph precision: the isotope mass spectrometer having held machine successfully, first verifies its precision, and method chooses a kind of sample at random as internal standard substance, repeats about 10 times and measures carbon and nitrogen stable isotope �� X value, calculates �� respectively13C and ��15The standard deviation of N, verifies mass spectrometric precision, and the more little precision of standard deviation is more high. The �� of document report13The precision of C can reach �� 0.2 ��, ��15The precision of N can reach �� 0.5 ��.
Repeat small lobsters C, N isotope value measured for 9 times:
The relative standard deviation of two isotope measure results, all much smaller than 5%, shows that the precision of Instrument measuring nitrogen and carbon isotope ratios method is very high. Isotopic ratio is such as Fig. 1.
The determination of mass spectrograph accuracy: in experimentation, the application selects the survey to domestic proposed standard TTB-1 relatively. The standard value that TTB-1 provides: ��13C is 0.58 ��, ��15O is-8.49 ��, and the actual result recorded of the application is: ��13C is 0.57 ��, ��15O is-8.61 ��, and carbon isotope coincide very well with the standard value provided. Thus can determining, the application uses CarloErbaEA-1110 elemental analyser and DELTR-plus isotopic ratio mass spectrum (IRMS) instrument combined instrument (ThermoFinnigan company) Measurement sensibility carbon isotope, has very high accuracy.
(4) sample and isotope assay is entered
Owing to the mensuration of isotropic substance is relatively stricter to process control, so the process packaging of sample wants specification, prevent contaminated. During setting-out, first open the lid of elemental analyser sample injection disc, sample one to one with tweezers careful put into hole, discharge the sample checking in a lower opening and whether leaking setting-out product and repetition, bonnet upper cover out of question, process action is brisk.
After setting-out completes, sample quality, corresponding hole number, numbering are input in the form of computer operation software, check again before preservation and whether make mistakes, then click " beginning ", after the position that file is preserved and filename set, start test sample.
(5) this method is utilized to record four large Chinese carp polycultured ponds sample C, N isotropic substance composition (see Fig. 3)
The isotropic substance composition of Xinzhou District, table 1 Wuhan polycultured ponds sample
As can be seen from the table, the pond fish carbon isotope value measured according to this method is relatively, nitrogen isotope value differs greatly, this is that isotopic fractionation causes, the nutrient ecological position thus calculated and their feeding habits also meet (0��4 grade of criteria for classifying), illustrate that this method has science, reliability; The biological nutrition level calculated by data also with major part document in mark basically identical.
Last it is noted that above-mentioned example is only for example of the present invention is clearly described, and not to the restriction of the mode of enforcement. For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description. Here without the need to also cannot all enforcement modes be given exhaustive. And the apparent change thus amplified out or variation are still among protection scope of the present invention.
Claims (8)
1. cultivating pool organic substance returns the characteristic isotope tracing method that becomes, it is characterised in that, according to the fractionation effect of stable isotope, measure the flow performance that stable isotope that is biological and abiotic sample analyzes ecosystem Middle nutrition material, comprise the steps:
(1) for the selection of sample that measures and pre-treatment
A, make sample for the Selection of fishes muscle of back tissue measured;
B, for the band shell animal that measures as the part removing shell during prey animal as sample, for the band shell animal that measures as the whole individuality comprising shell during predator all as sample;
C, select whole individuality as sample for the zooplankton measured;
D, select whole individuality as sample for the zoobenthos measured;
Particulate organic matter sample in E, water body obtains after filtering waters to be measured water sample by 0.7 ��m, aperture glass fiber filter, and described glass fiber filter is in advance through 450-550 DEG C of calcination 4 hours;
F, remove surface impurity for the terrestrial plant that measures and waterplant after whole individuality as sample;
G, sediment sample and the uniform region of the fish movement of the sample containing inorganic carbonate on 1 meter more than, offshore limit gather and obtain, and drip with hydrochloric acid soln and add process after removing impurity, until not having bubble to produce, or adopt concentrated hydrochloric acid suffocating treatment;
(2) drying and grinding and bag sample
A, drying and grinding: after the sample of acquisition is completely dry, grind to form uniform powder;
B, again drying: the sample powder of acquisition is dried 2-4 hour before the assay again;
C, bag sample: powdered samples accurately taken in tinfoil paper cup by the mass range analytical balance of test request, choose a kind of sample as internal standard substance, every 9 sample room bags interior standard specimen, and the sample wrapped is placed in corresponding sample panel, and record;
(3) mass spectrometric setting
A, open mass spectrograph, vacuum pump and computer, control vacuum pump by computer and regulate mass spectrometric vacuum tightness: regulate vacuum tightness to e-009mBar;
B, open gas cylinder valve and check the gaseous tension of gas cylinder;
C, open elemental analyser and hunt leak;
D, by computer regulated furnace temperature and flow velocity: 900 DEG C, left stove, 680 DEG C, right stove, chromatographic column 50 DEG C, flow velocity 140ml/min;
E, before temperature, square frame is beaten hook, removes hook �� Send �� OK before SetInstrumenttostand;
F, confirmation intensification and flow velocity are normal, after having heated up, first open vacuum valve, then open ion source, test sample after ion source is stable;
(4) sample pipetting volume will wrapped in step (2), measures;
(5) output of result and the process of data; According to carbon nitrogen international standards correction result, export sample carbon nitrogen isotope actual value.
2. the method for claim 1, it is characterized in that, sample type is determined according to food web composition, concentrated hydrochloric acid suffocating treatment described in step (1) is stifling 4 hours of the hydrochloric acid adopting concentration to be 12mol/L, drips with hydrochloric acid and add the hydrochloric acid that process adopts 1mol/L after described removal impurity.
3. the method for claim 1, it is characterised in that, determine sample type according to food web composition, the drying and grinding step in step (2) comprises vacuum-drying, lyophilize or 40-70 DEG C of oven drying at low temperature.
4. the method for claim 1, it is characterised in that, determine sample type according to food web composition, taking fish tissue and zoobenthos about 1.5mg in bag sample step in step (2), plant 2-3mg, POM are 15mg, plant plankton 15mg, settling 15-20mg.
5. the method for claim 1, it is characterized in that, determining sample type according to food web composition, the gaseous tension of the gas cylinder described in step (3) is adjusted to nitrogen 0.3Mpa, carbonic acid gas 0.3Mpa, air 0.2Mpa, helium 0.3Mpa, oxygen 0.3Mpa respectively.
6. the method for claim 1, it is characterised in that, determine sample type according to food web composition, step (3) also comprises the verification step of isotope mass spectrometer preci-sion and accuracy, specifically comprises:
The determination of A, precision: the isotope mass spectrometer having held machine successfully, first verifies its precision, by choosing a kind of sample at random as internal standard substance, repeats 10��12 times and measures carbon and nitrogen stable isotope �� X value, calculate �� respectively13C and ��15The standard deviation of N, verifies mass spectrometric precision;
The survey school of the determination of B, accuracy: choice criteria TTB-1, by the degree of agreement accuracy of judgement degree of the value that records and standard value.
7. the method for claim 1, it is characterized in that, sample type is determined according to food web composition, by adding, sample device adds sample to the sample step that adds in step (4) from sample holes port, add 2min after sample, evacuation of air, selects to start test sample after data output paths accurately by computer.
8. the method for claim 1, it is characterised in that, determine sample type according to food web composition, the international standards described in step (5) is USGS24 and USG26.
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| CN107941649A (en) * | 2017-11-15 | 2018-04-20 | 青海民族大学 | The detection method of the inorganic nitrogen uptake rate of common microalgae in aquaculture pond |
| CN111060634A (en) * | 2019-12-31 | 2020-04-24 | 东华理工大学 | Method for measuring ratio of soluble organic carbon to soluble total nitrogen isotope in water |
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