CN102539398A - On-site real-time determination method for chlorophyll a of phytoplankton - Google Patents
On-site real-time determination method for chlorophyll a of phytoplankton Download PDFInfo
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- CN102539398A CN102539398A CN2011104075747A CN201110407574A CN102539398A CN 102539398 A CN102539398 A CN 102539398A CN 2011104075747 A CN2011104075747 A CN 2011104075747A CN 201110407574 A CN201110407574 A CN 201110407574A CN 102539398 A CN102539398 A CN 102539398A
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Abstract
The invention discloses an on-site real-time determination method for the chlorophyll a of phytoplankton. The on-site real-time determination method comprises the following steps of: firstly respectively correcting an in-vivo luminoscope and a GPS (Global Positioning System) navigator, and keeping the in-vivo luminoscope and the navigator to be in complete time synchronization; opening the in-vivo luminoscope and the navigator, and determining the time synchronization of the in-vivo luminoscope and the navigator again; then synchronously storing the space location data of the GPS navigator in a computer, simultaneously opening a water pump to continuously convey a water sample under test to the interior of the in-vivo luminoscope; determining the sampling frequency of the computer and the sampling frequency of the in-vivo luminoscope so that the computer and the in-vivo luminoscope synchronously take samples; and finally, leading a surveying ship to operate along a pre-designed route, and obtaining a space distribution map of the chlorophyll a of the phytoplankton in water to be tested in the same day according to a chlorophyll a concentration database obtained by the in-vivo luminoscope through sampling and a space location database obtained by the computer through sampling after finishing the whole sampling process.
Description
Technical field
The present invention relates to the real-time detection technique of planktonic algae chlorophyll a on the water surface, more particularly relate to a kind of on-site real-time assay method of planktonic algae chlorophyll a.
Background technology
Planktonic algae body inner chlorophyll a is one of most important index of expressing the algae bio amount, also is one of important indicator of estimating water body in lake eutrophication state.The AAS that at present domestic many employing national environmental protection portions " water and effluent monitoring analytical approach (the 4th edition) " and Ministry of Water Resources's standard " chlorophyllous mensuration (AAS) " (SL88-1994) are recommended is as the assay method of planktonic algae chlorophyll a; This method is through gather lake discrete point water sample at the scene; Take back the laboratory; A certain amount of sample is used filtering with microporous membrane, collect the vegetalitas planktonic organism, 90% acetone soln of usefulness extracts.After the extract centrifuging, the absorbance of mensuration 750,663,645 and 630nm is calculated chlorophyllous concentration, and this method sample collecting preservation and test procedure are following:
Sample collecting and preservation step:
1) sample should be captured in glass or the tygon bottle by phytoplankton quantitative sampling method.River, lake, reservoir are got 500ml, and 300ml is got in the pond.
2) after the sampling, sample should be placed on the frescade, keeps out of the direct sun, and preferably immediately water sample is carried out analyzing and processing.Place water sample like need, then answer lucifuge stored refrigerated (2～5 ℃), and every premium on currency appearance need add 1% magnesium carbonate suspending liquid, cause the pigment dissolving to prevent acidifying
Test procedure is following:
1) concentrates: in a certain amount of sample, add 0.2ml magnesium carbonate suspending liquid, after fully stirring, with the miillpore filter suction strainer of diameter 60mm.Behind the no moisture, also to continue to aspirate a few minutes in the filtrator.If time-delay is extracted, can be placed on freezing lucifuge storage in the exsiccator to the filter membrane that is loaded with concentrating sample.
2) extract: the filter membrane that will be loaded with concentrating sample is put into mortar, adds 3ml acetone soln (4.2) to the degree that filter paper soaks, and grinds filter membrane; Add 90% acetone soln more slightly; Grind filter membrane fully, the filter membrane and the acetone soln that will grind with 90% acetone soln then wash in the band plug centrifuge tube of band scale, make the cumulative volume of extract in the centrifuge tube be no more than 6ml; Cover pipe close, place 4 ℃ dark place to soak 24h.
3) centrifugal: as centrifuge tube to be put into hydro-extractor, with 4000r/min speed centrifuging 20min.Supernatant is moved in the 10ml tool plug graduated tube of demarcating, add a small amount of 90% acetone soln in the centrifuge tube of former extract, suspended sediment and centrifugal merges supernatant once more.This operation repetition 2～3 times till deposition does not contain pigment, is settled to 10ml with supernatant at last.
4) measure: getting supernatant in the colorimetric pool of 10mm or 50mm, is contrast solution with 90% acetone soln, reads the absorbance of wavelength 750,663,645 and 630nm, selects the optical path length or the dilutability of colorimetric pool, makes its optical density OD
633Greater than 0.2, less than 1.0.
From the absorbance of each wavelength, deduct the absorbance of wavelength 750nm,, be calculated as follows chlorophyllous dense as corrected absorbance D:
In the formula:
The concentration of Ca---chlorophyll a, mg/L;
The constant volume of V1---extract, mL;
V2---filter the volume of water sample, L;
The optical path length of L---colorimetric pool, mm;
D---corrected extract absorbance.
Adopt AAS; After gathering water sample at the lake discrete point and taking back the laboratory, through concentrating, steps such as extraction, centrifugal and mensuration, the data that obtain to sample; Below but this method exists shortcoming: 1) discrete sampling, the space randomness is too big; 2) sampling is big with the experimental work amount; 3) cost of determination is high, needs a large amount of chemical reagent, for example acetone, hydrochloric acid, magnesium carbonate; 4) water sample is preserved and is required height, and algae is not easy to preserve as live body; 5) the mensuration process is loaded down with trivial details, needs pre-service, mills; 6) analysis time long, generally need could measure the result afterwards in 24 hours with organic extractant solution; 7) because need to use volatile extraction medicament such as acetone, health there is certain influence.
In a word, existing AAS field sampling sample size is limited, and leaching process is loaded down with trivial details during experiment, and extraction time is longer, and sensitivity is lower.
Summary of the invention
To the above-mentioned defective that exists in the prior art; The on-site real-time assay method that the purpose of this invention is to provide a kind of planktonic algae chlorophyll a; Can be in for example highly dense continuous sampling in several hours of short time, and obtain mass data, when the distribution situation of in a few days findding out water floating algae to be measured.
For achieving the above object, the present invention adopts following technical scheme:
A kind of on-site real-time assay method of planktonic algae chlorophyll a, the concrete steps of this assay method are:
A. calibrate living body fluorescent appearance and GPS navigation appearance respectively, and check living body fluorescent appearance, GPS navigation appearance and system for computer time, the time of guaranteeing is synchronous fully;
B. will calibrate good living body fluorescent appearance, GPS navigation appearance and computing machine and move on the surveying vessel of the water surface to be measured, and carry out the equipment location, install and connect;
C. open living body fluorescent appearance and GPS navigation appearance, confirm the time synchronized of living body fluorescent appearance and GPS navigation appearance once more; Space orientation data sync with the GPS navigation appearance deposits in the computing machine then, opens water pump simultaneously water sample to be measured is delivered in the living body fluorescent appearance continuously;
D. confirm the SF of computing machine and the SF of living body fluorescent appearance, make both synchronized samplings;
E. surveying vessel is along pre-designed circuit operation; After accomplishing whole sampling process; According to the chlorophyll-a concentration database of living body fluorescent appearance sampling acquisition and the space orientation database of computer sampling acquisition, obtain planktonic algae chlorophyll a spatial distribution map in the water surface to be measured.
As further improvement project, said assay method also comprises chlorophyll-a concentration verification of data step F, and the concrete steps of this step F are:
F1. how much be divided into high, medium and low three types of waters according to the quantity of planktonic algae in unit area in the water surface to be measured;
F2. in above-mentioned three types of waters, each waters grab sample 6-10 bottle water sample, the when and where of each water sampling and the sampling time and the place of living body fluorescent appearance are synchronous;
F3. adopt AAS to the water determination planktonic algae chlorophyll-a concentration in the step F 2;
F4. adopt the linear regression statistic law that the planktonic algae chlorophyll-a concentration data of the identical time point gathered separately by living body fluorescent method and AAS have respectively been carried out after the comparative analysis, obtain both regression fit lines and both related coefficients;
If F5. the numerical value of both related coefficients is more than or equal to 0.8, the planktonic algae chlorophyll-a concentration database of then confirming to be obtained by the sampling of living body fluorescent appearance is as the chlorophyll-a concentration data in the planktonic algae chlorophyll a spatial distribution map in the water surface to be measured; If the numerical value of both related coefficients less than 0.8, then need be recalibrated the living body fluorescent instrument, confirm the applicability of living body fluorescent appearance in this water body.
Water sample to be measured among the said step C is taken from following 0.5 meter of the water surface to be measured.
Water sample to be measured among the said step C is taken from the horizontal direction from surveying vessel 1-2 rice.
The travelling speed of surveying vessel is consistent with the speed of drawing water of water pump in the said step e.
Compared with prior art; Adopt the on-site real-time assay method of a kind of planktonic algae chlorophyll a of the present invention, at first, calibrate living body fluorescent appearance and GPS navigation appearance respectively; And check living body fluorescent appearance, GPS navigation appearance and system for computer time, the time of guaranteeing is synchronous fully; Then, living body fluorescent appearance, GPS navigation appearance and computing machine that calibration is good move on the surveying vessel of the water surface to be measured, carry out the equipment location, install and connect; Then, open living body fluorescent appearance and GPS navigation appearance, confirm the time synchronized of living body fluorescent appearance and GPS navigation appearance once more; Space orientation data sync with the GPS navigation appearance deposits in the computing machine then, opens water pump simultaneously water sample to be measured is delivered in the living body fluorescent appearance continuously; Confirm the SF of computing machine and the SF of living body fluorescent appearance, make both synchronized samplings; At last; Surveying vessel is along pre-designed circuit operation; After accomplishing whole sampling process,, obtain planktonic algae chlorophyll a spatial distribution map in the water surface to be measured according to the chlorophyll-a concentration database of living body fluorescent appearance sampling acquisition and the space orientation database of computer sampling acquisition.Because planktonic algae body inner chlorophyll molecule can be launched the characteristic red fluorescence after by UV-irradiation, its fluorescence intensity is directly proportional with chlorophyll concentration, can adopt the living body fluorescent method to carry out the quantitative test of chlorophyll a in view of the above.Adopt the living body fluorescent appearance have highly sensitive, selectivity good and advantage such as easy and simple to handle.Combine advanced GIS space orientation technique again; So just can accomplish layering, measure the concentration of planktonic algae chlorophyll a in the water to be measured in real time, apace, for example realize that in the utmost point short time spatial distribution characteristic of algae chlorophyll a in the water surface scope to be measured measures in several hours.
The living body fluorescent method is compared with traditional AAS possesses following advantage: 1) living body measurement, and noiseless to sample; 2) real-time high-efficiency; 3) high sensitivity; 4) easy and simple to handle; 5) highly dense sampling in the short time obtains mass data.6) can adopt traditional assay method as replicate determination, guarantee the reliability of the chlorophyll-a concentration value of on-the-spot METHOD FOR CONTINUOUS DETERMINATION.
Description of drawings
Fig. 1 is the schematic flow sheet of the on-site real-time assay method of a kind of planktonic algae chlorophyll a of the present invention;
Fig. 2 is the schematic flow sheet of embodiments of the invention 1;
Fig. 3 is the structural representation of embodiment of the present invention embodiment 1 device therefor;
Fig. 4 is the planktonic algae chlorophyll a space distribution synoptic diagram of the water surface to be measured of embodiments of the invention 1;
Fig. 5 is the employing living body fluorescent method of embodiments of the invention 1 and the planktonic algae chlorophyll-a concentration linear regression analysis synoptic diagram that AAS obtains.
Embodiment
Further specify technical scheme of the present invention below in conjunction with accompanying drawing and embodiment.
See also the assay method 10 of a kind of planktonic algae chlorophyll a shown in Figure 1, the concrete steps of this assay method are:
11. calibrate living body fluorescent appearance and GPS navigation appearance respectively, and check living body fluorescent appearance, GPS navigation appearance and system for computer time, the time of guaranteeing is synchronous fully;
Move on the surveying vessel of the water surface to be measured 12. will calibrate good living body fluorescent appearance, GPS navigation appearance and computing machine, carry out the equipment location, install and connect;
13. open living body fluorescent appearance and GPS navigation appearance synchronously, confirm the time synchronized of living body fluorescent appearance and GPS navigation appearance once more; Space orientation data sync with the GPS navigation appearance deposits in the computing machine then, opens water pump simultaneously water sample to be measured is delivered in the living body fluorescent appearance continuously;
14. confirm the SF of computing machine and the SF of living body fluorescent appearance, make both synchronized samplings;
15. surveying vessel is along pre-designed circuit operation; After accomplishing whole sampling process; According to the chlorophyll-a concentration database of living body fluorescent appearance sampling acquisition and the space orientation database of computer sampling acquisition, obtain planktonic algae chlorophyll a spatial distribution map in the water surface to be measured.
Reliability for the chlorophyll-a concentration value of further guaranteeing on-the-spot METHOD FOR CONTINUOUS DETERMINATION can also adopt traditional assay method as replicate determination, and assay method of the present invention also comprises chlorophyll-a concentration verification of data step 16, and the concrete steps of this step 16 are:
161. how much be divided into high, medium and low three types of waters according to the quantity of planktonic algae in unit area in the water surface to be measured;
162. in above-mentioned three types of waters, each waters grab sample 6-10 bottle water sample, the when and where of each water sampling and the sampling time and the place of living body fluorescent appearance are synchronous;
163. adopt AAS to the water determination planktonic algae chlorophyll-a concentration in the step 162;
164. adopt the linear regression statistic law that the planktonic algae chlorophyll-a concentration data in the identical time place of being gathered separately by living body fluorescent method and AAS have respectively been carried out after the comparative analysis, obtain both regression fit lines and both related coefficients;
If 165. the numerical value of both related coefficients (related coefficient is generally more than or equal to 0.8) in the reasonable scope, the planktonic algae chlorophyll-a concentration database of then confirming to be obtained by the sampling of living body fluorescent appearance is as the chlorophyll-a concentration data in the planktonic algae chlorophyll a spatial distribution map in the water surface to be measured; If the numerical value of both related coefficients is (related coefficient is generally less than 0.8) in unreasonable scope, then need recalibrate the living body fluorescent instrument, confirm the applicability of living body fluorescent appearance in this water body.
Water sample to be measured in the said step 13 is taken from following 0.5 meter of the water surface to be measured.
Water sample to be measured in the said step 13 is taken from the horizontal direction from surveying vessel 1-2 rice.
The travelling speed of surveying vessel is consistent with the speed of drawing water of water pump in the said step 15.
See also Fig. 2, Fig. 3, Fig. 4 and embodiment 1 shown in Figure 5 again, the 10-AU-005-CE living body fluorescent appearance of the on-the-spot U.S. Turner Designs of use of Dianshan Lake brand adopts boating type to measure lake water algae chlorophyll a in Shanghai City, and the mensuration process is following:
1), comprises 10-AU living body fluorescent appearance 21 and GPS navigation appearance 22 at testing laboratory's internal calibration instrument.
2) check the system time of 10-AU living body fluorescent appearance 21, GPS navigation appearance 22 and computing machine 23, guarantee accurately and realize consistent.
3) all calibrations are good instrument and equipment is removed the in-site measurement ship 24 to the lake, installs, connects, arranges, by 27 pairs of the said equipment power supplies of accumulator.
4) open instrument simultaneously, confirm that system time is consistent, GPS navigation appearance 22 begins to get into the The real time measure state, and space orientation data (longitude and latitude) deposit in the computing machine 23 synchronously, and computing machine 23 is preserved frequency and can be set, and general 1-10 preserves a latitude and longitude value in second.
5) open water pump 25 serial samplings; Begin to get into the real-time continuous mensuration state of chlorophyll a; The chlorophyll-a concentration data deposit in the internal memory of 10-AU living body fluorescent appearance 21; 10-AU living body fluorescent appearance 21 SFs can be set, and general 1-10 preserves a concentration value in second, need to prove; 10-AU living body fluorescent appearance 21 SFs and computing machine 23 shelf space locator datas are that the frequency of longitude and latitude keeps guaranteeing the sampled data of 10-AU living body fluorescent appearance 21 and the consistance of sampling position synchronously.Simultaneously, surveying vessel 24 beginnings are moved along pre-designed circuit, and ship's speed should not be too fast, guarantees steadily.It should be noted that assay method of the present invention because site work aboard ship requires ship's speed preferably steady relatively, therefore do not suit to carry out at boisterous adverse weather.One end of plastic tube 26 connects the water inlet of 10-AU living body fluorescent appearance 21, and the other end of plastic tube 26 is preferably in the water surface 30 following 0.5 meter, and in the horizontal direction suitably away from 1 to 2 meter in surveying vessel, the interference of wave that causes when avoiding shipping agency to advance water inlet appearance.
6) in surveying vessel 24 moving process; Use a hydrophore water sample surplus the random acquisition 20 on the line; Preferably respectively in the high, medium and low waters grab sample of algae concentration; Preparation is taken back testing laboratory and is used traditional spectrophotometry chlorophyll-a concentration, is used for further checking the chlorophyll-a concentration value that 10-AU living body fluorescent appearance is measured.Water sample is preserved according to relevant national standard, must write down sample time and sampling spot (longitude and latitude) during each water sampling, and should the sampling time point should be identical with the time point that adopts 10-AU living body fluorescent appearance 21 to sample.Surveying vessel 24 starts in the process; Accomplish as far as possible steadily; Make the travelling speed of surveying vessel 24 close, guarantee that the water sample that 10-AU living body fluorescent appearance 25 is measured does not repeat as far as possible on a spatial point with the speed of drawing water of water pump 25 (be connected 10-AU living body fluorescent appearance, continuous water sampling flows).
7) generally in several hours, can accomplish full lake chlorophyll a flow assay, immediately all appts and water sample taken back testing laboratory after the completion.
8) on surveying vessel, the chlorophyll-a concentration database of 10-AU living body fluorescent appearance 21 internal memorys is imported in the computing machine 23; Realize time synchronized with on-the-spot spatial orientation information (longitude and latitude) database of preserving in the computing machine 23; Certainly; This step also can be got back to the laboratory and accomplished, and produces the chlorophyll a spatial distribution map (see figure 4) of full lake short time.
9) for the further reliability of checking 10-AU living body fluorescent appearance 21 samplings, in testing laboratory, can also use the chlorophyll-a concentration of traditional spectrophotometry water sample, the chlorophyll-a concentration in the corresponding place of preserving with 10-AU living body fluorescent appearance is checked (see figure 5); Wherein horizontal ordinate is for adopting the chlorophyll-a concentration of AAS, and unit is a mg/litre, and ordinate is for adopting the chlorophyll-a concentration of living body fluorescent method; Unit is a mg/litre; What each point of Discrete Distribution was represented is the same place or the moment, respectively by AAS (four wave bands: 750nm, 663nm; 645nm; 630nm, acetone extract) and the chlorophyll-a concentration value that obtained of living body fluorescent method sampling, adopt linear regression statistic law and based on the correlation analysis method of the SPSS v14.0 of statistical software; 28 chlorophyll-a concentration data to living body fluorescent method and traditional AAS are measured have separately been carried out comparative analysis; Table 1 is seen in correlation analysis result demonstration, and the Pearson correlation coefficient of two groups of data is 0.963, is remarkable positive correlation (p=0.000＜0.01).
There is following linear equation relation in the chlorophyll-a concentration data that two kinds of methods of linear regression statistical result showed are measured:
Y=0.657x+6.264, i.e. straight line among Fig. 5,
Wherein y is the chlorophyll-a concentration that the living body fluorescent method is measured, and x is the chlorophyll-a concentration of traditional spectrophotometry, and coefficient of determination R2 reaches 0.926.
Table 1
| The tradition AAS | The living body fluorescent method | ||
| The tradition AAS | Pearson correlation coefficient | 1.000 | 0.963 ** |
| Conspicuousness (two-tailed test) p | / | 0.000 | |
| Sample number N | 28 | 28 | |
| The living body fluorescent method | Pearson correlation coefficient | 0.963 ** | 1.000 |
| Conspicuousness (two-tailed test) p | 0.000 | / | |
| Sample number N | 28 | 28 |
*: the significance test value of related coefficient is less than 0.01
Those of ordinary skill in the art will be appreciated that; Above embodiment is used for explaining the object of the invention; And be not with opposing qualification of the present invention; As long as in essential scope of the present invention, all will drop in the scope of claim of the present invention variation, the modification of the above embodiment.
Claims (5)
1. the on-site real-time assay method of a planktonic algae chlorophyll a is characterized in that:
The concrete steps of this assay method are:
A. calibrate living body fluorescent appearance and GPS navigation appearance respectively, and check living body fluorescent appearance, GPS navigation appearance and system for computer time, the time of guaranteeing is synchronous fully;
B. will calibrate good living body fluorescent appearance, GPS navigation appearance and computing machine and move on the surveying vessel of the water surface to be measured, and carry out the equipment location, install and connect;
C. open living body fluorescent appearance and GPS navigation appearance, confirm the time synchronized of living body fluorescent appearance and GPS navigation appearance once more; Space orientation data sync with the GPS navigation appearance deposits in the computing machine then, opens water pump simultaneously water sample to be measured is delivered in the living body fluorescent appearance continuously;
D. confirm the SF of computing machine and the SF of living body fluorescent appearance, make both synchronized samplings;
E. surveying vessel is along pre-designed circuit operation; After accomplishing whole sampling process; According to the chlorophyll-a concentration database of living body fluorescent appearance sampling acquisition and the space orientation database of computer sampling acquisition, obtain planktonic algae chlorophyll a spatial distribution map in the water surface to be measured.
2. on-site real-time assay method according to claim 1 is characterized in that:
Said assay method also comprises chlorophyll-a concentration verification of data step F, and the concrete steps of this step F are:
F1. how much be divided into high, medium and low three types of waters according to the quantity of planktonic algae in unit area in the water surface to be measured;
F2. in above-mentioned three types of waters, each waters grab sample 6-10 bottle water sample, the when and where of each water sampling and the sampling time and the place of living body fluorescent appearance are synchronous;
F3. adopt AAS to the water determination planktonic algae chlorophyll-a concentration in the step F 2;
F4. adopt the linear regression statistic law that the planktonic algae chlorophyll-a concentration data of the identical time point gathered separately by living body fluorescent method and AAS have respectively been carried out after the comparative analysis, obtain both regression fit lines and both related coefficients;
If F5. the numerical value of both related coefficients is more than or equal to 0.8, the planktonic algae chlorophyll-a concentration database of then confirming to be obtained by the sampling of living body fluorescent appearance is as the chlorophyll-a concentration data in the planktonic algae chlorophyll a spatial distribution map in the water surface to be measured; If the numerical value of both related coefficients less than 0.8, then need be recalibrated the living body fluorescent instrument.
3. on-site real-time assay method according to claim 1 and 2 is characterized in that:
Water sample to be measured among the said step C is taken from following 0.5 meter of the water surface to be measured.
4. on-site real-time assay method according to claim 1 and 2 is characterized in that:
Water sample to be measured among the said step C is taken from the horizontal direction from surveying vessel 1-2 rice.
5. on-site real-time assay method according to claim 1 and 2 is characterized in that:
The travelling speed of surveying vessel is consistent with the speed of drawing water of water pump in the said step e.
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| CN103076297A (en) * | 2012-12-27 | 2013-05-01 | 河北科技大学 | Method for quickly and real-timely measuring water chlorophyll through replacing chlorophyll standard substance with microcystis aeruginosa |
| CN107132326A (en) * | 2017-05-04 | 2017-09-05 | 天津大学 | A kind of device of in-site detecting halomereid species and biomass |
| CN107228843A (en) * | 2017-05-08 | 2017-10-03 | 绍兴市水环境科学研究院有限公司 | A kind of calibration method applied to fluorescence method chlorophyll a on-line computing model |
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Application publication date: 20120704 |