CN105713952A - Biotoxicity assessment method of oilfield chemicals - Google Patents
Biotoxicity assessment method of oilfield chemicals Download PDFInfo
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Abstract
The invention discloses a biotoxicity assessment method of oilfield chemicals. The assessment method comprises the following contents: (1) photobacterium recovery: preparing a bacterium suspension from photobacterium by virtue of a sodium chloride solution, and recovering the photobacterium in an ice water bath; (2) synchronized culture: conducting activating culture on the recovered bacterium suspension which is inoculated in an activating culture medium; in the later period of the activating culture, adding a growth inhibitor to a bacterium culture solution, and conducting synchronized culture; and after the synchronized culture, collecting bacterium cells; and (3) toxicity assessment: preparing a bacterium suspension, which has a certain luminescence amount, from the bacterium cells by virtue of the sodium chloride solution, and adding a luminescence promoter for toxicity assessment analysis. According to the method disclosed by the invention, the tested photobacterium, which is relatively high in luminescence stability, can be obtained through such modes as strain recovery, synchronized culture, steady-state screening and the like; and by adding the luminescence promoter in the process of toxicity detection, individual luminescence differences are weakened, and the stability and repeatability of a toxicity assessment result are guaranteed.
Description
Technical field
The invention belongs to environmental technology field, relate to a kind of bio-toxicity evaluation methodology to Oilfield Chemicals.
Background technology
Day by day strong along with the increasingly stringent of world wide environment preservation law regulation and Public Environmental Awareness, the environmental conservation evaluation problem that Oilfield Chemicals (mainly includes drilling fluid additive and system, completion fluid additive and system, oilfield sewage treatment agent etc.) receives common concern and attention.Abroad, as the U.S., Britain, Canada, Norway, Holland etc. have all formulated the environment protection index about Oilfield Chemicals (mainly drilling fluid and garbage thereof).In China, the bio-toxicity problem of Oilfield Chemicals is increasingly subject to pay attention to, carry out rejected well drilling liquid to halobiontic toxotest, and tentatively established the experimental arrangement of oil base drilling fluid toxicity assessment, but the research about Oilfield Chemicals toxicity test has been started late.The research of Oilfield Chemicals toxicity test starts from the nineties initial stage, up to now, there is no unified Oilfield Chemicals toxicity test standard and grade scale.
Nineteen ninety-five, State Bureau of Environmental Protection promulgated " water quality acute biological toxicity Luminous bacteria " i.e. GB GB/T15441-1955, and began at whole nation implementation March 1 nineteen ninety-five.It is the standard method measuring water quality acute biological toxicity.Environmental monitoring master station of CNPC is based on " water quality acute biological toxicity Luminous bacteria ", with reference to EPA oppossum shrimp biological assays, establish the method for testing and the bio-toxicity stage division that utilize Luminous bacteria to measure drilling fluid and additive bio-toxicity, and draft as benchmark and establish China National Petroleum Corporation (CNPC) company standard Q/SY111 2007, i.e. " Oilfield Chemicals, drilling fluid bio-toxicity classification and detection method Luminous bacteria ".
Luminous bacteria is based on a kind of evaluation methodology that the inhibition of the luminosity of photobacteria is designed by toxicant, has easy and simple to handle, and the detection cycle is short, it is easy to the features such as execute-in-place, widely used by multiple industries.CN201110090246.9 discloses the assay method of a kind of Photobacterium Phosphoreum Toxicity, and the method comprises the following steps: 1) actication of culture: dissolved by the lyophilized powder of photobacterium phosphoreum, available after being inoculated on inclined-plane to go down to posterity 3 times；2) cultivation of strain: the strain inoculating loop gone down to posterity is inoculated into fluid medium, 20 DEG C, 180rpm cultivates 12h；3) balance of bacterium solution: pipette appropriate bacterium solution in 20mL3%NaCl solution, controls light value measurement range between 50-500 ten thousand, stirs 40min under 20 DEG C of conditions；4) application of sample and luminous intensity detection: the bacterium solution continuous sampling device having stirred 40min is added the 3%NaCl blank of 200 μ L to 800 μ L and the 3%NaCl contaminated liquid of 800 μ L Concentraton gradient, on swirl mixing device, 2min is mixed immediately after application of sample, under 20 DEG C of conditions, after standing 15min, sample cell is inserted mensuration luminous intensity in fluorescence detector；5) double cross mistake counter point calculates the sample suppression ratio to luminous intensity.CN201010537039.9 discloses the method for the long-term preservation of a kind of photobacteria strain, rapid fluid resuscitation.By developing special antibacterial freezing media and recovery solution, it is achieved the long-term of photobacteria strain preserves and rapid fluid resuscitation, had both saved testing cost, and had also substantially reduced the time of toxotest, the immediate toxicities test for chemical pollutant provides possibility.
The bio-toxicity grade of current Oilfield Chemicals judges that being mainly based upon the chemical agent luminous quantity to photobacteria suppresses degree.There is autonomous metabolism and trained reflex mechanism due to photobacteria, luminous quantity is as the unique Testing index of bio-toxicity, it is very big by thalli growth metabolism and environmental effect, and the difficulty of accurate quantification is very big, and therefore the stability of photobacteria detection method, repeatability are not high.
Summary of the invention
For the deficiencies in the prior art, the invention provides a kind of Oilfield Chemicals bio-toxicity evaluation methodology.The method obtains the confession examination luminescent bacteria bacteria suspension with higher stability of photoluminescence by modes such as strain recovery, synchronizing culture, stable state screenings；In toxicity detection process, add luminous promoting agent, thus weakening individual emission difference, it is ensured that the stability of toxicity assessment result and repeatability.
Oilfield Chemicals bio-toxicity evaluation methodology of the present invention, including following content:
(1) luminescent bacteria recovery: luminescent bacteria sodium chloride solution is configured to bacteria suspension, recovers in ice-water bath；
(2) synchronizing culture: by recovery bacterial suspension inoculation in activation medium, carries out activation culture；In the activation culture later stage, bacterial culture fluid adds growth inhibitor, carries out synchronized culture；After synchronized cultivation, collect somatic cells；
(3) toxicity assessment: with sodium chloride solution, somatic cells is configured to the bacteria suspension with certain luminous quantity, and adds luminous promoting agent, for toxicity assessment analysis.
In the inventive method, the luminescent bacteria adopted is a class strain with special luminous metabolic pathway, including vibrio, Photobacterium, different brevibacterium, and wherein preferred photobacterium phosphoreum.
In the inventive method, the luminescent bacteria adopted can be from commercially available luminescent bacteria lyophilized powder, it is also possible to is inclined-plane or the strain of liquid preservation.
In the inventive method, thalline resuscitation process uses mass concentration to be 1.5%～3.0% sodium chloride solution, recovery time 2min～10min.After recovery, bacteria suspension luminous quantity reaches more than 600mV and namely meets test requirements document.
In the inventive method, described activation culture based formulas is tryptone 0.1wt%～0.5wt%, yeast extract 0.2wt%～0.5wt%, glycerol 0.1wt%～0.3wt%, potassium dihydrogen phosphate 0.05wt%～0.2wt%, disodium hydrogen phosphate 0.05wt%～0.2wt%, sodium chloride 0.2wt%～0.5wt%, pH6.5.The activation culture time is 12h～18h.
In the inventive method, by adding the mode of growth limitation composition and growth inhibitor in activation medium, carry out growth synchronization and regulate.Selected growth inhibitor is that nucleic acid synthesis suppresses or cell division inhibitor, as being one or more in 5-fluorodeoxyuridine, hydroxyurea, cytosine arabinoside, methotrexate, adenine, guanine, thymus pyrimidine, Colchicine, Demecolcine etc..Different with specific reference to the growth inhibitor selected, it is determined that its addition, it is possible to achieve synchronizing culture.
In the inventive method, after luminescent bacteria cultivates 5h～16h in adding growth inhibitor bacterial culture fluid, it is centrifuged processing, it is thus achieved that bacterial sediment.Centrifugal rotational speed is 500rpm～1500rpm, centrifugation time 10min～20min.
In the inventive method, the bacterial sediment of collection carried out 2～4 times clean with the sodium chloride solution that mass concentration is 2%～3%, and be configured to luminescent bacteria bacteria suspension, evaluate for drilling fluid bio-toxicity.
In the inventive method, adding mass concentration in luminescent bacteria bacteria suspension is 0.1%～0.3% luminous promoting agent.Luminous promoting agent is the aldehydes of more than hexose or eight carbon, it is preferable that glucose, octanal.
Compared with prior art, Oilfield Chemicals bio-toxicity evaluation methodology of the present invention processes step by effective thalline synchronous growth, obtain the conforming somatic cells of growth, eliminate the impact on thalline luminous quantity of the thalli growth difference, simultaneously according to thalline principle of luminosity, detection bacteria suspension with the addition of luminous promoting agent, improve luminous intensity and stability of photoluminescence, be more beneficial for improving stability and the repeatability of toxicity assessment result:
(1) existing toxicity assessment scheme is to change the index as toxicity detection using the luminous quantity of luminescent bacteria, owing to luminescent bacteria is the organism with voluntary physiological metabolic regulation, it is thus in the thalline of different growing stages, its luminous quantity is in constantly change, the luminous total amount undulatory property of its performance is very big, as Testing index, the stability of its result, accuracy can be subject to significant problem.The present invention is in the way of thalline synchronized culture, it is thus achieved that the confession examination thalline of synchronous growth, thus improve the stability of testing result.
(2) in existing drilling fluid system, rich organic principle of supporting is less, and after adding luminescent bacteria, its luminous quantity is in progressively downward trend, thus the judgement that impact is to its toxicity.Therefore, this programme with the addition of luminous promoting agent in detection bacteria suspension so that it is luminous quantity maintains a constant level, is conducive to the accuracy that its toxicity judges.
Detailed description of the invention
Illustrate below in conjunction with the embodiment detailed process to the inventive method and effect, but be not limited to following example.In the present invention, wt% is mass fraction.
Luminescent bacteria used in the present embodiment is commercially available photobacterium phosphoreum lyophilized powder.Drilling liquid toxicity evaluation test process is according to China National Petroleum Corporation (CNPC) company standard Q/SY111 2007, and namely " Oilfield Chemicals, drilling fluid bio-toxicity classification and detection method Luminous bacteria " carries out.
Embodiment 1
(1) take commercially available photobacterium phosphoreum T3 microspecies (PhotobacteriumphotoreumT3spp.) lyophilized powder 0.5g, be dissolved in the sodium chloride solution of 1mL mass concentration 2.5wt%.Recovery 5min in ice-water bath；
(2) by recovery bacterial suspension inoculation in 50mL activation medium, in 20 DEG C, activation culture 18h；
(3), after activation 18h, in culture medium, 5 μ g Demecolcines, synchronized culture 7h are added；
(4) after synchronized culture 7h, 1000rpm is centrifuged 10min, abandons supernatant, takes bacterial sediment, cleans twice with 3% sodium chloride solution, and recentrifuge obtains bacterial sediment；
(5) thalline is dissolved in 10mL and contains 3wt% sodium chloride, the buffer solution of 0.1wt% glucose, be configured to bacteria suspension；
(6) take in 10 μ L bacteria suspensions addition 2mL3wt% sodium chloride solutions and carry out luminous quantity mensuration.
Experimental result is as shown in the table.
Embodiment 2
(1) take commercially available photobacterium phosphoreum T3 microspecies (PhotobacteriumphotoreumT3spp.) lyophilized powder 0.5g, be dissolved in the sodium chloride solution of 1mL mass concentration 2.5wt%.Recovery 5min in ice-water bath；
(2) by recovery bacterial suspension inoculation in 50mL activation medium, in 20 DEG C, activation culture 18h；
(3), after activation 12h, in culture medium, 25mg thymus pyrimidine, synchronized culture 16h are added；
(4) after synchronized culture 16h, 500rpm is centrifuged 20min, abandons supernatant, takes bacterial sediment, cleans three times with 3wt% sodium chloride solution, and recentrifuge obtains bacterial sediment；
(5) thalline is dissolved in 10mL and contains the buffer solution of 3wt% sodium chloride, 0.2wt% octanal, be configured to bacteria suspension；
(6) take in 10 μ L bacteria suspensions addition 2mL3wt% sodium chloride and carry out luminous quantity mensuration.
Experimental result is as shown in the table.
Embodiment 3
(1) take commercially available photobacterium phosphoreum T3 microspecies (PhotobacteriumphotoreumT3spp.) lyophilized powder 0.5g, be dissolved in the sodium chloride solution of 1mL mass concentration 2.5wt%.Recovery 5min in ice-water bath；
(2) by recovery bacterial suspension inoculation in 50mL activation medium, in 20 DEG C, activation culture 18h；
(3), after activation 16h, in culture medium, 25mg adenine, synchronized culture 16h are added；
(4) after synchronized culture 16h, 1200rpm is centrifuged 10min, abandons supernatant, takes bacterial sediment, cleans three times with 3wt% sodium chloride solution, and recentrifuge obtains bacterial sediment；
(5) thalline is dissolved in 10mL and contains the buffer solution of 3wt% sodium chloride, 0.2wt% glucose, be configured to bacteria suspension；
(6) take in 10 μ L bacteria suspensions addition 2mL3wt% sodium chloride and carry out luminous quantity mensuration.
Experimental result is as shown in the table.
Embodiment 4
With embodiment 1 acquisition luminescent bacteria bacteria suspension, for the bio-toxicity evaluation of 5% calcium chloride drilling liquor system.Toxicity assessment test procedure is according to China National Petroleum Corporation (CNPC) company standard Q/SY111 2007, and namely " Oilfield Chemicals, drilling fluid bio-toxicity classification and detection method Luminous bacteria " carries out.Experimental result is as shown in the table.
Relative luminosity, wherein ESampleRefer to the luminous quantity of laboratory sample, EPKRefer to the luminous quantity of control sample.
Comparative example 1
With (1) in embodiment 1～(4) experimental implementation, it is thus achieved that bacterial sediment, and thalline is dissolved in 10mL containing in 3wt% sodium chloride solution, do not use luminous promoting agent, detect for luminous quantity, experimental result is as shown in the table:
Compared with embodiment 1, thalline luminous quantity is relatively low, it was demonstrated that luminous promoting agent may advantageously facilitate the recovery of thalline secondary, thus improving luminous quantity.
Comparative example 2
With (1), (2), (4), (5) experimental implementation in embodiment 1, obtain bacterial sediment, do not use growth inhibitor Demecolcine, it is thus achieved that thalline be dissolved in the 10mL buffer solution containing 3wt% sodium chloride, 0.1wt% glucose, detecting for luminous quantity, experimental result is as shown in the table:
Compared with embodiment 1, the undulatory property that the existence of thalline luminous quantity is unordered, it was demonstrated that synchronization process is conducive to improving thalline stability of photoluminescence.
Comparative example 3
(1) take commercially available photobacterium phosphoreum T3 microspecies (PhotobacteriumphotoreumT3spp.) lyophilized powder 0.5g, carry out recovering, activating according to CN201110090246.9 publicity pattern；
(2) take in 10 μ L bacteria suspensions addition 2mL3% sodium chloride and carry out luminous quantity mensuration.
Experimental result is as shown in the table:
Compared with embodiment 1, the thalline of this patent report is recovered, the method for activation, and its thalline luminous quantity exists bigger undulatory property.
Comparative example 4
According to China National Petroleum Corporation (CNPC) company standard Q/SY111 2007, namely total Test process in " Oilfield Chemicals, drilling fluid bio-toxicity classification and detection method Luminous bacteria ", carries out bio-toxicity evaluation to 5% calcium chloride drilling liquor system.Result is as follows:
Compared with embodiment 4, the change of relative luminosity does not have regularity, therefore can not clearly judge its toxicity range.
Claims (12)
1. the bio-toxicity evaluation methodology of an Oilfield Chemicals, it is characterised in that include following content:
(1) luminescent bacteria recovery: luminescent bacteria sodium chloride solution is configured to bacteria suspension, recovers in ice-water bath；
(2) synchronizing culture: by recovery bacterial suspension inoculation in activation medium, carries out activation culture；In the activation culture later stage, bacterial culture fluid adds growth inhibitor, carries out synchronized culture；After synchronized cultivation, collect somatic cells；
(3) toxicity assessment: with sodium chloride solution, somatic cells is configured to the bacteria suspension with certain luminous quantity, and adds luminous promoting agent, for toxicity assessment analysis.
2. in accordance with the method for claim 1, it is characterised in that: the luminescent bacteria described in step (1) is vibrio, Photobacterium or different brevibacterium.
3. in accordance with the method for claim 2, it is characterised in that: the luminescent bacteria adopted is photobacterium phosphoreum.
4. the method described in claim 1,2 or 3, it is characterised in that: the luminescent bacteria adopted comes from the strain of commercially available luminescent bacteria lyophilized powder or inclined-plane or liquid preservation.
5. in accordance with the method for claim 1, it is characterised in that: in step (1), thalline resuscitation process uses mass concentration to be 1.5%～3.0% sodium chloride solution, recovery time 2min～10min.
6. in accordance with the method for claim 1, it is characterized in that: the activation culture based formulas described in step (2) is tryptone 0.1wt%～0.5wt%, yeast extract 0.2wt%～0.5wt%, glycerol 0.1wt%～0.3wt%, potassium dihydrogen phosphate 0.05wt%～0.2wt%, disodium hydrogen phosphate 0.05wt%～0.2wt%, sodium chloride 0.2wt%～0.5wt%, pH6.5；The activation culture time is 12h～18h.
7. in accordance with the method for claim 1, it is characterised in that: the growth inhibitor described in step (2) is nucleic acid synthetic inhibitor or cell division inhibitor.
8. in accordance with the method for claim 7, it is characterised in that: the growth inhibitor described in step (2) is one or more in 5-fluorodeoxyuridine, hydroxyurea, cytosine arabinoside, methotrexate, adenine, guanine, thymus pyrimidine, Colchicine, Demecolcine etc..
9. in accordance with the method for claim 1, it is characterised in that: after in step (2), luminescent bacteria cultivates 5h～16h in adding growth inhibitor bacterial culture fluid, being centrifuged processing, centrifugal rotational speed is 500rpm～1500rpm, centrifugation time 10min～20min.
10. in accordance with the method for claim 1, it is characterised in that: the somatic cells of collection is carried out 2～4 times and cleans by the sodium chloride solution being 2%～3% with mass concentration in step (2).
11. in accordance with the method for claim 1, it is characterised in that: adding mass concentration in step (3) bacteria suspension is 0.1%～0.3% luminous promoting agent, and luminous promoting agent is the aldehydes of more than hexose or eight carbon.
12. in accordance with the method for claim 11, it is characterised in that: described luminous promoting agent is glucose or octanal.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108051428A (en) * | 2018-01-26 | 2018-05-18 | 杭州绿洁水务科技股份有限公司 | A kind of water-quality test method, apparatus, online Toxicity Monitoring instrument |
| CN112198150A (en) * | 2020-07-07 | 2021-01-08 | 山东省临沂生态环境监测中心 | Method for monitoring polluted water body by bioluminescence method |
| CN117611039A (en) * | 2023-12-13 | 2024-02-27 | 上海多宁生物科技股份有限公司 | Dry powder cell culture medium transportation stability assessment method and transportation device |
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| CN103487555A (en) * | 2013-09-27 | 2014-01-01 | 桂林理工大学 | Method for analyzing long-term microplate toxicity of environmental pollutants on photobacterium phosphoreum |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102465097A (en) * | 2010-11-10 | 2012-05-23 | 中国科学院生态环境研究中心 | Method for long-term preservation and rapid recovery of luminous bacterium strain |
| CN103487555A (en) * | 2013-09-27 | 2014-01-01 | 桂林理工大学 | Method for analyzing long-term microplate toxicity of environmental pollutants on photobacterium phosphoreum |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108051428A (en) * | 2018-01-26 | 2018-05-18 | 杭州绿洁水务科技股份有限公司 | A kind of water-quality test method, apparatus, online Toxicity Monitoring instrument |
| CN112198150A (en) * | 2020-07-07 | 2021-01-08 | 山东省临沂生态环境监测中心 | Method for monitoring polluted water body by bioluminescence method |
| CN112198150B (en) * | 2020-07-07 | 2022-10-25 | 山东省临沂生态环境监测中心 | Method for monitoring polluted water body by bioluminescence method |
| CN117611039A (en) * | 2023-12-13 | 2024-02-27 | 上海多宁生物科技股份有限公司 | Dry powder cell culture medium transportation stability assessment method and transportation device |
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