CN104120179A - Detection method for concentration of petroleum hydrocarbons in ocean - Google Patents
Detection method for concentration of petroleum hydrocarbons in ocean Download PDFInfo
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- CN104120179A CN104120179A CN201410318328.8A CN201410318328A CN104120179A CN 104120179 A CN104120179 A CN 104120179A CN 201410318328 A CN201410318328 A CN 201410318328A CN 104120179 A CN104120179 A CN 104120179A
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- petroleum hydrocarbon
- concentration
- detection method
- phascolosoma
- ocean
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
Abstract
The invention discloses a detection method for the concentration of petroleum hydrocarbons in ocean by utilizing phascolosoma esculenta. The method includes a plurality of steps: phascolosoma esculenta feeding, crude oil water-soluble solution preparation, petroleum hydrocarbon concentration gradient culture solution preparation, petroleum hydrocarbon exposure and sampling, single cell gel electrophoresis analysis and data analysis. The detection method is wide in application scope, short in experiment flow, good in safety, and high in sensitivity and accuracy, can perform visibility study on damage conditions of DNA of phascolosoma esculenta coelomic fluid cells in a single cell level, and converts and calculates to obtain the concentration of the petroleum hydrocarbons in the ocean.
Description
Technical field
The present invention relates to ocean environment toxic substance concentration studies field, relate in particular to a kind of biological detecting method to ocean Petroleum Hydrocarbon concentration.
Background technology
The complex mixts that petroleum hydrocarbon is comprised of hydrocarbon polymer, mainly by hydrocarbon composition, especially light aromatic hydrocarbon material and derivative thereof.Petroleum hydrocarbon has toxicity to marine organisms, is mainly to make biological nutrition and transporting confusion reigned, during high density, even can cause biological mortality, to Marine ecosystems, can cause great destruction.Particularly high Polycyclic aromatic hydrocarbons toxicity is the strongest, some component has carinogenicity, can make fish produce oily stink, oil is bonded on the fish gill or on fish-egg, can make it death by suffocation very soon, or hatching is affected, main harm is, wherein contain carcinogenic hydrocarbon, after the enrichment of fish shellfish, can be detrimental to health by food chain.Offshore oil exploitation in recent years and shipping accident occur again and again, the fast-developing and industry of seagoing vessel transport trade, a large amount of discharges of sanitary sewage in addition, and ocean environment Petroleum Hydrocarbon pollutes day by day serious, has even threatened halobiontic existence.
Phascolosoma is commonly called as Hai Ding, extra large agate locust, sandworm, mud garlic, native bamboo shoot etc., is under the jurisdiction of sipunculan door, leather bag Sipunculida, leather bag siphon-worm order, leather bag Sipunculidae, leather bag Sipunculus
, be the endemic species of China, be distributed widely in the provinces and regions such as Zhejiang, Fujian, Guangdong, Guangxi and Hainan.Phascolosoma is the kind in marine fishery resources with Important Economic meaning; in recent years because Coastal environments are polluted aggravation and people excessively adopt and catch; cause resource slump of disastrous proportions, therefore, the conservation of resources of Phascolosoma has started to be subject to people's attention.
At present, the method for research ocean Petroleum Hydrocarbon concentration has a lot, mainly contains ultraviolet spectrophotometry, weighting method, infrared spectrophotometry, spectrophotofluorimetry and Gas chromatography etc.Ultraviolet spectrophotometry is because sensitivity is low, invalid to stable hydrocarbon, cyclic hydrocarbon, is relatively suitable for the mensuration of enriched sample Petroleum Hydrocarbon concentration.Weighting method research long flow path, sensitivity is low, is only suitable for measuring the water sample of oleaginousness more than 10mg/L, and extract and solvent evaporation process in easily vapor away, the precision of method is different and difference is very large with operational condition and operator's skill level.Infrared spectrophotometry comprises NDIR (Non-Dispersive Infrared) absorption spectrophotometry and two parts of infrared spectroscopy, wherein the former advantage is that apparatus structure is simple, measurement has good circulation ratio, shortcoming is straight-chain paraffin and the naphthenic hydrocarbon that can only study in petroleum hydrocarbon, can not study benzene homologues, the latter can measure the total concn of petroleum hydrocarbon exactly, but when measuring the concentration of fish shellfish Petroleum Hydrocarbon, if in fish shellfish animal grease have residual, as easy as rolling off a log being interfered.The sensitivity of spectrophotofluorimetry research petroleum hydrocarbon concentration is very high, but it only can measure the concentration of benzene homologues in petroleum hydrocarbon, cannot measure the concentration of straight-chain paraffin and straight-chain paraffin in petroleum hydrocarbon, and the fluorescent emission intensity of different substances is widely different, measurement result is subject to oil product composition in sample to affect very large.Vapor-phase chromatography is by after the separation of petroleum hydrocarbon chromatographic column, studies respectively different petroleum hydrocarbon components, highly sensitive, but because petroleum hydrocarbon composition is very complicated, is not suitable for the mensuration of petroleum hydrocarbon total concn.
Summary of the invention
In order to solve the problems of the technologies described above, the invention provides a kind of detection method to ocean Petroleum Hydrocarbon concentration, this detection method is applied widely, and experiment flow is short, and security is good, and sensitivity and accuracy are high.
The technical solution adopted in the present invention is:
A detection method for ocean Petroleum Hydrocarbon concentration, is characterized in that: comprise the following steps:
1) by healthy Phascolosoma respectively from be added with the petroleum hydrocarbon of different concentration known and the nutrient solution of testing sample is cultivated certain hour altogether;
Detect the Coelomocytes DNA damage situation of Phascolosoma under different training conditions, according to the concentration of known petroleum hydrocarbon, determine the numerical relation between DNA damage situation and petroleum hydrocarbon concentration; 2) according to definite numerical relation, calculate the concentration of sample Petroleum Hydrocarbon to be checked.
Preferably, described numerical relation is determined by the mode of drawing standard curve.
Preferably, the configuration step of described nutrient solution is: by crude oil and after filtration the natural sea-water after sterilizing be mixed in proportion, follow after ultrasonic dispersion standing, draw lower floor's water as mother liquor, by the freezing preservation of mother liquor, the petroleum hydrocarbon solution that adds isopyknic several gradient concentrations of a certain amount of seawater preparation with petroleum hydrocarbon mother liquor, the same a collection of ooze that adds subsequently equivalent mixes.
Preferably, the ratio of the natural sea-water after described crude oil and after filtration sterilizing is 1:10-1:8, and described ultrasonic jitter time is 1h-1.5h, and time of repose is 4-5h, and freezing temp is 3 ℃-4 ℃, and described gradient concentration number is 6-8.
Preferably, described cultural method is: by the Phascolosoma of body weight 2 ~ 3g, in the plastic tank that Mare Humorum mud is housed, support and within one week, treat that it is in stable condition, get healthy Phascolosoma random packet, put into respectively prepared petroleum hydrocarbon nutrient solution for every group 20 and cultivate 7d ~ 10d, duration of test water temperature stability is 20 ℃ ~ 25 ℃, and at the 0th, 1,2,4,7 day that tests, obtains the Coelomocytes of Phascolosoma.
Preferably, the detection method of described DNA damage situation is single cell gel electrophoresis analysis, and it comprises and adopts the film-making of three layers of paving glue method, lysis, untwists and electrophoresis, neutralization, dyeing, 6 steps of microscopy.
Preferably, the damage status analysis of described mouthful of leather bag siphon-worm Coelomocytes cell DNA adopts the analysis of SPSS software statistics.
The invention has the beneficial effects as follows: the present invention is by using single cell gel electrophoresis investigative technique, the DNA damage situation to Phascolosoma in unicellular level that realized is carried out visuality research, and then draw the concentration of ocean Petroleum Hydrocarbon, relative prior art (as gradient sedimentation technology, the technology of untwisting and elution technique), has that experiment process is short, security good, an advantage such as accuracy and sensitivity height.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described further:
Fig. 1 is the schema of detection method of the present invention.
Fig. 2 is the petroleum hydrocarbon contaminated Phascolosoma Coelomocytes comet figure that is not subject to of the present invention;
Fig. 3 is the comet figure of Phascolosoma Coelomocytes after 4d in the petroleum hydrocarbon nutrient solution of the 0.90mg/L of being exposed to of the present invention.
Embodiment
It should be noted that, in the situation that not conflicting, embodiment and the feature in embodiment in the application can combine mutually.
As shown in Figure 1, a kind of detection method of Phascolosoma to ocean Petroleum Hydrocarbon concentration of utilizing of the present embodiment, comprise the nursing of Phascolosoma, the preparation of crude oil water-soluble solution, the preparation of petroleum hydrocarbon concentration gradient nutrient solution, six large steps such as the exposure of petroleum hydrocarbon, single cell gel electrophoresis analysis and data analyses.Wherein single cell gel electrophoresis research also comprise film-making, lysis, untwist, a plurality of steps such as electrophoresis, neutralization, dyeing, microscopy.
The nursing of A, Phascolosoma:
Preferably the Phascolosoma of body weight 2 ~ 3g, supports in the plastic tank of Mare Humorum mud is housed, and support and within one week, treat that it is in stable condition, and it is standby to select energetic healthy individual experiment.General activity is normal, to the sensitive Phascolosoma of irritant reaction, be energetic individuality.
The preparation of B, crude oil water-soluble solution:
Crude oil is pressed to V(crude oil): V(natural sea-water after filtering)=1:10 mixes, and ultrasonic dispersion 1h, draws lower floor's water as mother liquor after standing 4h, and mother liquor is placed in Brown Glass Brown glass bottles and jars only and saves backup in 4 ℃.
When natural sea-water filters with the filter membrane of 0.45 μ m, to remove the objectionable impuritiess such as settling in seawater.The concentration of mother liquor Petroleum Hydrocarbon is measured with reference to the method (ultraviolet-visible spectrophotometry) of Marine monitoring standard (GB 17378.4-2007).
The preparation of C, petroleum hydrocarbon concentration gradient nutrient solution:
Get the beaker of 6 1000ml, prepare respectively 6 parts of petroleum hydrocarbon solution that concentration is different, by a certain amount of petroleum hydrocarbon mother liquor and certain volume sea water mixing, the cumulative volume that makes the mixed solution in beaker is 500ml, and the ultimate density of petroleum hydrocarbon is respectively 0,0.05,0.30,0.50,0.70 and 0.90mg/L.Subsequently same a collection of water ratio 40% ooze of equivalent is added respectively in above mixed solution, mix.
Arranging according to being national Seawater Quality Standards of this step Petroleum Hydrocarbon concentration gradient, one, two class water Petroleum Hydrocarbon concentration≤0.05 mgL
-1, three class water Petroleum Hydrocarbon concentration≤0.30mgL
-1, four class water Petroleum Hydrocarbon concentration≤0.50 mgL
-1.
The exposure of D, petroleum hydrocarbon:
Get 120 of healthy Phascolosomas, be divided at random 6 groups, put into respectively the seawater nutrient solution containing different concns petroleum hydrocarbon that step C prepares for 20 every group and cultivate 7d, duration of test water temperature stability is 25 ℃.And sampling for the 0th, 1,2,4,7 day in experiment.3 Phascolosomas are chosen in each sampling at random, take the Coelomocytes of Phascolosoma with 1ml disposable syringe.In disposable syringe, extract in advance equivalent antithrombotics, sample is placed in to the 1.5ml centrifuge tube of precooling, through Trypan Blue, examine under a microscope cell survival rate (cell survival rate > 90%), using the not PBS damping fluid adjusting cell density of calcium-magnesium-containing is 1 * 10
6mL
-1, can be used for single cell gel electrophoresis research experiment.
E, single cell gel electrophoresis research:
1. adopt the film-making of three layers of paving glue method: it is the 1st layer of glue that normal fusing point agarose (NMA) solution that the massfraction of take is 0.8% is laid on slide glass.Get Phascolosoma Coelomocytes suspension and massfraction and be 0.6% low melting-point agarose gel proportionally 1:7～1:3 mix (approximately 1000～3000 of the number of cells on each film), drip in the 1st layer above glue, with cover glass, flatten rapidly, be placed in 4 ℃ and solidify, this is the 2nd layer of glue.After the 2nd layer of gelling is solid, get the low melting-point agarose solution that 75 μ L massfractions are 0.6% and be layered on the 2nd layer above glue, covered, puts and on ice bag, transfers in 4 ℃ of refrigerators immediately, makes it to solidify.
2. lysis: after gel is dry, throw off cover glass, slide glass level immerses and fills in the rectangular box of cell pyrolysis liquid.Consisting of of cell pyrolysis liquid: 2.5molL
-1naCl, 0.1 molL
-1na
2eDTA, 0.01 molL
-1tutofusin tris (Tris), the sarcosyl that massfraction is 1%, pH=10, add volume fraction and are 1% Triton X-100 (TritonX-100) and 10% dimethyl sulfoxide (DMSO) (DMSO) before use.In 4 ℃ of lucifuge cracking 2h, after cracking finishes, the PBS damping fluid rinsing of slide glass use precooling 3 times.
Under lysis effect, cytolemma, nuclear membrane and other microbial film are destroyed, and intracellular RNA, protein and other composition are leaked in gel, are diffused into subsequently in cell pyrolysis liquid, but core DNA still keeps being wound around Huan district to be attached on remaining nuclear skeleton, and stays original position.
3. untwist and electrophoresis: slide glass is placed in Horizontal electrophoresis tank side by side, add electrophoretic buffer (1 mmolL of the new preparation of precooling
-1na
2eDTA, 0.3molL
-1naOH, pH > 13), after 4 ℃ of lucifuge unwindase 13 0 min, switch on power, electrophoresis 30min under 25V, 30mA.
4. neutralize, dyeing, microscopy: after electrophoresis finishes, take out slide glass, put into Tris-HC1 damping fluid (pH=7.5) and 15min.Every glue drips the 30 μ gmL of 50 μ L
-1ethidium bromide (EB) aqueous solution, covered, dyeing 20min.(green range) observations taking pictures under the fluorescent microscope of 510～560 nm wavelength, magnification is 10 * 20 times.
Shown in Fig. 2 and Fig. 3, be respectively and be not subject to the comet figure of petroleum hydrocarbon contaminated Phascolosoma Coelomocytes and be exposed in the petroleum hydrocarbon nutrient solution of 0.90mg/L the comet figure of Phascolosoma Coelomocytes after 4d.Phascolosoma Coelomocytes in Fig. 2 presents fluorescence head circular, rule, without conditions of streaking, illustrates that cell ruptures; In Fig. 3 there is being similar to the hangover of comet in cell, and this shows that cell DNA received damage.
F, SPSS software data are analyzed.With Casp software analysis comet image, 13 DNA damage criterions such as long, tail long, head DNA content and per-cent, afterbody DNA content and per-cent, head area, tail area, tail square, Olive tail square to the end, wherein, tail square is the product of tail length and DNA per-cent, is an overall target.In the present embodiment, inquired into the variation of tail length and 2 indexs of tail square.Every Phascolosoma Coelomocytes is made 1 glue, 30 cells of every glue stochastic analysis, and the tail that counts the petroleum hydrocarbon contaminated lower cell comet of different concns is grown and back range.The average tail length of comet and the back range in table 1 and table 2, listed after different time, different concns petroleum hydrocarbon are processed change.Can find out that in control group, average comet tail length and the back range variation range of cell is little, the Coelomocytes of the Phascolosoma of processing through the petroleum hydrocarbon nutrient solution of different concns is along with the prolongation in treatment time, comettail mean length and back range increase gradually, and the DNA damage of cell is tending towards serious; In addition, for the cell through the same treatment time, along with the increase of petroleum hydrocarbon concentration, comettail is longer.Comprehensive above known, the DNA damage toxic action of ocean Petroleum Hydrocarbon to Phascolosoma, along with the increase of petroleum hydrocarbon concentration and the prolongation of action time, poisons more serious.Thus, can indirectly draw by analyzing the degree of impairment of the Coelomocytes DNA of Phascolosoma the concentration of ocean Petroleum Hydrocarbon.
?
the impact that table 1 petroleum hydrocarbon is long on Phascolosoma Coelomocytes comet tail
the impact of table 2 petroleum hydrocarbon on Phascolosoma Coelomocytes comet back range
More than that better enforcement of the present invention is illustrated, but the invention is not limited to described embodiment, those of ordinary skill in the art also can make all equivalent variations or replacement under the prerequisite without prejudice to spirit of the present invention, and the distortion that these are equal to or replacement are all included in the application's claim limited range.
Claims (7)
1. a detection method for ocean Petroleum Hydrocarbon concentration, is characterized in that: comprise the following steps:
1) by healthy Phascolosoma respectively from be added with the petroleum hydrocarbon of different concentration known and the nutrient solution of testing sample is cultivated certain hour altogether;
Detect the Coelomocytes DNA damage situation of Phascolosoma under different training conditions, according to the concentration of known petroleum hydrocarbon, determine the numerical relation between DNA damage situation and petroleum hydrocarbon concentration; 2) according to definite numerical relation, calculate the concentration of sample Petroleum Hydrocarbon to be checked.
2. detection method according to claim 1, is characterized in that: described numerical relation is determined by the mode of drawing standard curve.
3. detection method according to claim 1, it is characterized in that: the configuration step of described nutrient solution is: by crude oil and after filtration the natural sea-water after sterilizing be mixed in proportion, follow after ultrasonic dispersion standing, draw lower floor's water as mother liquor, by the freezing preservation of mother liquor, the petroleum hydrocarbon solution that adds isopyknic several gradient concentrations of a certain amount of seawater preparation with petroleum hydrocarbon mother liquor, the same a collection of ooze that adds subsequently equivalent mixes.
4. detection method according to claim 3, it is characterized in that: the ratio of the natural sea-water after described crude oil and after filtration sterilizing is 1:10-1:8, described ultrasonic jitter time is 1h-1.5h, time of repose is 4-5h, freezing temp is 3 ℃-4 ℃, and described gradient concentration number is 6-8.
5. detection method according to claim 1, it is characterized in that: described cultural method is: by the Phascolosoma of body weight 2 ~ 3g, in the plastic tank that Mare Humorum mud is housed, support and within one week, treat that it is in stable condition, get healthy Phascolosoma random packet, put into respectively prepared petroleum hydrocarbon nutrient solution for every group 20 and cultivate 7d ~ 10d, duration of test water temperature stability is 20 ℃ ~ 25 ℃, and at the 0th, 1,2,4,7 day that tests, obtains the Coelomocytes of Phascolosoma.
6. detection method according to claim 1, it is characterized in that: the detection method of described DNA damage situation is single cell gel electrophoresis analysis, it comprises and adopts the film-making of three layers of paving glue method, lysis, untwists and electrophoresis, neutralization, dyeing, 6 steps of microscopy.
7. detection method according to claim 1, is characterized in that: the damage status analysis of described mouthful of leather bag siphon-worm Coelomocytes cell DNA adopts the analysis of SPSS software statistics.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104715674A (en) * | 2015-03-19 | 2015-06-17 | 青岛海洋地质研究所 | Seabed hydrocarbon leakage simulation experiment device and experiment method thereof |
| CN106814050A (en) * | 2016-12-13 | 2017-06-09 | 大连海洋大学 | The method of oil hydrocarbon content in ultrasonic fluoroscopic examination Polychaeta body |
-
2014
- 2014-07-04 CN CN201410318328.8A patent/CN104120179B/en active Active
Non-Patent Citations (2)
| Title |
|---|
| G.ROESIJADI ET AL: "Uptake of Hydrocarbons from Marine Sediments Contaminated with Prudhoe bay Crude Oil:Influence of Feeding Type of Test Species and Availability of Polycyclic Aromatic Hydrocarbons", 《JOURNAL OF THE FISHERIES RESEARCH BOARD OF CANNADA》, 31 December 1978 (1978-12-31), pages 610 * |
| 高业田: "重金属Cd和Hg对可口革囊星虫的毒理学效应", 《硕士论文全文数据库 农业科技辑》, no. 03, 15 March 2013 (2013-03-15), pages 38 - 47 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104715674A (en) * | 2015-03-19 | 2015-06-17 | 青岛海洋地质研究所 | Seabed hydrocarbon leakage simulation experiment device and experiment method thereof |
| CN104715674B (en) * | 2015-03-19 | 2017-04-12 | 青岛海洋地质研究所 | Seabed hydrocarbon leakage simulation experiment device and experiment method thereof |
| CN106814050A (en) * | 2016-12-13 | 2017-06-09 | 大连海洋大学 | The method of oil hydrocarbon content in ultrasonic fluoroscopic examination Polychaeta body |
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