CN101131384A - Method for detecting subaqueous acute biological toxicity using photobacteria - Google Patents
Method for detecting subaqueous acute biological toxicity using photobacteria Download PDFInfo
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Abstract
A method to detect the acute biology toxicity by the photobacterium is to prepare the new photobacterium culture firstly, then to adjust the pH of tested water: the pH is 4.5 when the Cu density is higher than other metal and organic matter; the pH is 5.4 when the water sample does not contain Cu and other metal density is higher than the organic matter; the pH is 7.0 when the organic matter in water is higher than metal density. The ZnSO4 is as reference toxicity and matching a series of standard solution which are set in tube which is added in the photobacterium culture and detect the relative luminance. The luminance change of test water is expressed by the relative luminance. The computing formula is: the sample relative luminance = sample tube luminance/reference tube luminance. The invention uses the photobacterium not the toxicity to detect the acute biology toxicity of water.
Description
Technical field
The present invention relates to a kind of detection method of biological technical field, specifically is a kind of method of utilizing photobacteria to detect acute biological toxicity in the water.
Background technology
Photobacteria is a kind of special marine bacteria, and it can be survived in the water of cleaning and be luminous, and its luminous power can weaken when water quality is subjected to polluting.Utilize the difference of photobacteria luminous intensity in different polluted water bodies can show the situation of water quality biological acute toxicity.Adopt the photobacteria method that water quality is monitored and have short, highly sensitive characteristics of time, and can also remedy the deficiency of conventional physics and chemistry monitoring, therefore widely adopted.Present traditional photobacteria monitoring method exist as a result reappearance not high, use extremely toxic substance mercuric chloride higher as toxicity object of reference toxicity, cause problem such as environmental pollution easily.Therefore be necessary existing detection method is further improved, reduce object of reference toxicity, optimize detection method.
Through the prior art literature search is found, GB/T 15441-1995 " the mensuration photobacteria method of State Standard of the People's Republic of China's acute toxicity of water quality " regulation, be remarkable negative correlation (P≤0.05) based on photobacteria relative rediance and water sample toxic component total concentration, thereby can decide the relative rediance of water sample by bioluminescence degree instrumentation, represent its acute toxicity level with this.The acute toxicity of water quality level selects for use suitable reference toxicant mercuric chloride concentration (is unit with mg/L) to characterize, with the relative HgCl of specimen
2Concentration is divided toxic grade.Mercuric chloride is extremely toxic substance, and buying needs loaded down with trivial details link, all has certain risk in the use and after using.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of method of utilizing photobacteria to detect acute biological toxicity in the water is provided, make it can replace extremely toxic substance mercuric chloride, and can effectively improve the testing result precision as the toxicity object of reference.The present invention adopts the relatively low ZnSO of toxicity
4Replace mercuric chloride HgCl
2As the toxicity reference, be determined by experiment ZnSO
4With HgCl
2The toxicity ratio, when water sample toxicity is divided, change accordingly, avoided the use of extremely toxic substance, and used photobacteria (commercially available prod) and reach the same purpose that the water quality acute biological toxicity is measured.
The present invention realizes by following technical method.The present invention specifically may further comprise the steps:
The first step, the preparation of the fresh bacteria suspension of photobacteria
Detecting used photobacteria is photobacterium phosphoreum T3 microspecies (Photobacteriumphosphoreum T3 spp. is the commercially available prod, and Nanjing soil is buied in the Chinese Academy of Sciences).Before detecting to photobacteria screen, the inclined-plane is cultivated and shake bottle bacterium liquid and cultivate and obtain initial luminosity and be not less than 1.5 * 10
6The fresh bacteria suspension of RLU/S, standby.
Second step, the processing of specimen and dilution
The biological acute toxicity that the present invention monitored is not considered the influence of pH, therefore needs to get rid of the pH factor.To test water sample and CK (contrast) (sodium chloride 3g/100mL) pH is adjusted into: the Cu amount that contains in the water sample is 4.5 when being higher than other metals and organic content, not containing Cu in the water sample is 5.4 when other tenor is higher than organic content simultaneously, is 7.0 when organic content is higher than tenor in the water sample;
The test water sample adopts distilled water diluting without exception.Before the constant volume, add NaCl by constituting NaCl 3g/100mL concentration.
In the 3rd step, the fresh bacteria suspension of photobacteria adds
Under 20～25 ℃ of conditions, draw the fresh bacteria suspension of photobacteria for preparing by the first step, the fresh bacteria suspension of stable photobacteria is added each testing tube one by one, cover bottle cap, put upside down for several times, leave standstill the back and measure luminosity with hand with micro syringe.
The 4th step, the calculating of specimen acute toxicity
Reference toxicant adopts ZnSO
4, place the 5mLCK pipe respectively behind the preparation reference toxicant series standard solution, add the fresh bacteria suspension of photobacteria of first step preparation in every pipe, fully carry out relative rediance behind the mixing and measure.Each is tested the luminous intensity variations of water sample and represents that with relative rediance computing formula is: the luminosity (RLU/S) of the luminosity of sample hose relative rediance=sample hose (RLU/S)/CK (contrast) pipe.
Relative rediance during according to different reference toxicant concentration, with the relative rediance is the longitudinal axis, reference toxicant concentration is the transverse axis mapping line linearity match of going forward side by side, and by the dependent equation between reference toxicant concentration and the relative rediance, can calculate relative rediance and reduce by 50% o'clock institute's test sample product with ZnSO
4The EC that concentration is represented
50Value.By with HgCl
2For the parallel experiment of reference poisonous substance can obtain HgCl
2The toxicity of solution is with concentration ZnSO
412.458 times of solution, converting with this can get specimen with HgCl
2The acute toxicity of expression.
The present invention adopts the relatively low ZnSO of toxicity
4Replace extremely toxic substance mercuric chloride as the toxicity reference, be determined by experiment ZnSO
4With HgCl
2The toxicity ratio, when water sample toxicity is divided, change accordingly, avoided the use of extremely toxic substance, and used photobacteria and reach the same purpose that the water quality acute biological toxicity is measured.Select ZnSO simultaneously
4Reference replaces HgCl as toxicity
2Not only reduce the detection cost, weakened toxicity, enlarged monitoring range, and had stability preferably.With HgCl
2During for the poisonous substance reference, the measurement sensitivity of photobacteria is 0.025mg/L; And with ZnSO
4During for the poisonous substance reference, can obviously improve the measurement sensitivity of photobacteria, less than 0.01mg/L.
Embodiment
Below embodiments of the invention are elaborated: present embodiment is being to implement under the prerequisite with the technical solution of the present invention, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment adopts the ZnCl of three kinds of reference poisonous substance: 0.00mg/L～2.00mg/L
2Series standard solution; 0.00mg/L the ZnSO of～2.25mg/L
4The HgCl of series standard solution and 0.5～6mg/L
2Series standard solution compares experiment.Whole enforcement implementation procedure is as follows:
1. prepare to implement material by following requirement, wherein reference toxicant adopts ZnCl
2, ZnSO
4And HgCl
2Three kinds (these three kinds of chemical substances all have commercially available, but HgCl
2Belong to extremely toxic substance, need public security department to examine and special conveying);
The test bacterial classification: photobacterium phosphoreum T3 microspecies (Photobacterium phosphoreum T3spp.), Chinese Academy of Sciences's Nanjing soil the commercially available prod that can buy.
Dilution: 3%NaCl, 2%NaCl.
Single tube luminous detection instrument Tube Luminometer Sirius V3.1; Constant temperature oscillator, incubator, high-temperature sterilization device; 10 μ L or 20 μ L micropipettors, 1mL syringe, volumetric flask, triangular flask etc.
2. implementation method
2.1 the preparation of the fresh bacteria suspension of photobacteria
(1) screening bacterial classification: get the institute's photobacteria bacterial classification of purchasing coating and cultivate 48h, the various bacterium colony bacteriums of picking carry out microscopy, pick out the bacterium colony of required photobacteria.
(2) slant strains is cultivated: get photobacteria before the mensuration and pick out first generation inclined-plane on fresh inclined-plane, cultivate the second generation inclined-plane of transferring immediately behind the 24h for 20 ± 0.5 ℃, cultivate 24h for 20 ± 0.5 ℃, pick out 20 ± 0.5 ℃ on third generation inclined-plane again and cultivate behind the 12h standby.Each inoculum concentration is no more than an oese.
(3) shaking bottle bacterium liquid cultivates: get the nearly ring of third generation slant strains, be inoculated in the 250mL triangular flask that the 50mL nutrient solution is housed, 20 ± 0.5 ℃, it is standby that 184rpm/min cultivates 12～14h down.
(4) nutrient solution is diluted to every milliliter of 108～109 cells, initial luminosity is not less than 1.5 * 10
6RLU/S, standby.
2.2 the processing of sample and dilution
The biological acute toxicity of being monitored is not considered the influence of pH, therefore needs to get rid of the pH factor.To test water sample and CK (contrast) (sodium chloride 3g/100mL) pH is adjusted into: the Cu amount that contains in the water sample is 4.5 when being higher than other metals and organic content, not containing Cu in the water sample is 5.4 when other tenor is higher than organic content simultaneously, is 7.0 when organic content is higher than tenor in the water sample; The correction of disturbing for coloured sample determination is with reference to the detailed rules and regulations of GB/T 15441-1995.
Sample adopts distilled water diluting.Before the constant volume, add NaCl by the concentration that constitutes NaCl 3g/100ml.
2.3 the fresh bacteria suspension of photobacteria adds
Under 20～25 ℃ of conditions, draw the fresh bacteria suspension of 10 μ L photobacterias with micro syringe, the fresh bacteria suspension of stable photobacteria put in order by sample add each testing tube (tool standard grinding port plug one by one, for the frit of making color comparison tube is made, make by professional glass apparatus factory), cover bottle cap, put upside down for several times with hand, leave standstill the back and adopt single tube luminous detection instrument to measure luminosity.
2.4. the calculating of specimen acute toxicity
Reference toxicant adopts ZnCl
2, ZnS0
4And HgCl
2Three kinds, place the 5mLCK pipe respectively behind the preparation reference toxicant series standard solution, add the fresh bacteria suspension of photobacteria of preparation in the step 2.1 in every pipe, fully carry out relative rediance behind the mixing and measure.The luminous intensity variations of each standard model represents that with relative rediance computing formula is: the luminosity (RLU/S) of the luminosity of sample hose relative rediance=sample hose (RLU/S)/CK (contrast) pipe.
Relative rediance during according to different reference toxicant concentration, with the relative rediance is the longitudinal axis, reference toxicant concentration is the transverse axis mapping line linearity match of going forward side by side, by the dependent equation between reference toxicant concentration and the relative rediance, can calculate the concentration that relative rediance reduces by 50% o'clock institute's test sample product is EC
50Value.
3. each is carried out 10 groups of parallel laboratory tests with reference to the pairing standard model of poisonous substance, calculate their EC respectively
50Value.The gained result is carried out statistical analysis, and testing result is as shown in table 1.
The replication result of table 1 toxicity reference
| Classification/group number | Minimum value | Maximal value | Mean value | Standard deviation | The coefficient of variation/% |
| ZnCl 2EC 50 ZnSO 4EC 50 HgCl 2EC 50 | 0.968 1.032 0.086 | 1.202 1.21 0.103 | 1.1048 1.1499 0.0923 | 0.065 0.054 0.005 | 5.88 4.71 5.37 |
In conjunction with the coefficient of variation analysis of 3 embodiment, ZnSO
4Minimum is 4.71%, and data are more neat; Secondly be HgCl
2, be 5.37%; That maximum is ZnCl
2, be 5.88%.Through the comparison of 10 groups of data, HgCl
2The toxicity of solution is with concentration ZnSO
412.458 times of solution.Be not difficult to find out, select ZnSO
4Reference replaces HgCl as toxicity
2Not only reduce implementation cost, weakened toxicity, enlarged monitoring range, and had stability preferably.
Claims (7)
1. a method of utilizing photobacteria to detect acute biological toxicity in the water is characterized in that, may further comprise the steps:
The first step, the preparation of the fresh bacteria suspension of photobacteria: detecting used photobacteria is photobacterium phosphoreum T3 microspecies, before detecting to photobacteria screen, the inclined-plane is cultivated and shake bottle bacterium liquid and cultivate and obtain initial luminosity and be equal to, or greater than 1.5 * 10
6The fresh bacteria suspension of RLU/S, standby;
Second step, the processing of specimen and dilution: will test water sample and contrast pH being adjusted into: the Cu amount that contains in the water sample is 4.5 when being higher than other metals and organic content, not containing Cu in the water sample is 5.4 when other tenor is higher than organic content simultaneously, is 7.0 when organic content is higher than tenor in the water sample;
In the 3rd step, the fresh bacteria suspension of photobacteria adds: get the fresh bacteria suspension of photobacteria of first step preparation, the fresh bacteria suspension of stable photobacteria is added each testing tube one by one, cover bottle cap, put upside down for several times with hand, leave standstill the back and measure luminosity;
The 4th step, the calculating of specimen acute toxicity: adopt ZnSO
4As reference toxicant, place pipe respectively behind the preparation reference toxicant series standard solution, the fresh bacteria suspension of photobacteria that adds first step preparation in every pipe, fully carrying out relative rediance behind the mixing measures, each is tested the luminous intensity variations of water sample and represents that with relative rediance computing formula is: the luminosity of the luminosity/control tube of sample hose relative rediance=sample hose.
2. the method for utilizing photobacteria to detect acute biological toxicity in the water according to claim 1 is characterized in that, in the first step, and the preparation of the fresh bacteria suspension of described photobacteria, concrete steps are:
(1) screening bacterial classification: get the coating of photobacteria bacterial classification and cultivate, the various bacterium colony bacteriums of picking carry out microscopy, pick out the bacterium colony of required photobacteria;
(2) slant strains is cultivated: get photobacteria before the mensuration and pick out first generation inclined-plane on fresh inclined-plane, cultivate the second generation inclined-plane of transferring immediately behind the 24h for 20 ± 0.5 ℃, cultivate 24h for 20 ± 0.5 ℃, pick out 20 ± 0.5 ℃ on third generation inclined-plane again and cultivate behind the 12h standby;
(3) shaking bottle bacterium liquid cultivates: get the nearly ring of third generation slant strains, be inoculated in the 250mL triangular flask that the 50mL nutrient solution is housed, 20 ± 0.5 ℃, it is standby that 184rpm/min cultivates 12～14h down;
(4) nutrient solution is diluted to every milliliter of 108～109 cells, initial luminosity is equal to, or greater than 1.5 * 10
6RLU/S, standby.
3. the method for utilizing photobacteria to detect acute biological toxicity in the water according to claim 2 is characterized in that, in the step (1), and described cultivation, its time is 48h.
4. the method for utilizing photobacteria to detect acute biological toxicity in the water according to claim 2 is characterized in that, in the step (2), each inoculum concentration is equal to or less than an oese.
5. the method for utilizing photobacteria to detect acute biological toxicity in the water according to claim 1 is characterized in that, in second step, the processing of described specimen and dilution, be specially: the test water sample adopts distilled water diluting, before the constant volume, adds NaCl by constituting NaCl 3g/100mL concentration.
6. the method for utilizing photobacteria to detect acute biological toxicity in the water according to claim 1, it is characterized in that, in the 3rd step, the described fresh bacteria suspension of photobacteria of getting first step preparation is meant: draw the fresh bacteria suspension of photobacteria for preparing by the first step with micro syringe under 20～25 ℃ of conditions.
7. the method for utilizing photobacteria to detect acute biological toxicity in the water according to claim 1, it is characterized in that, in the 4th step, the calculating of described specimen acute toxicity, be specially: the relative rediance during according to different reference toxicant concentration is the longitudinal axis with the relative rediance, and reference toxicant concentration is the transverse axis mapping line linearity match of going forward side by side, by the dependent equation between reference toxicant concentration and the relative rediance, calculate relative rediance and reduce by 50% o'clock institute's test sample product with ZnSO
4The EC that concentration is represented
50Value is by with HgCl
2For the parallel experiment of reference poisonous substance can obtain HgCl
2The toxicity of solution is with concentration ZnSO
412.458 times of solution, with this convert specimen with HgCl
2The acute toxicity of expression.
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