CN104535632B - Method for determining IC50 value in water body employing combination of mixed bacteria and electrochemical technology - Google Patents
Method for determining IC50 value in water body employing combination of mixed bacteria and electrochemical technology Download PDFInfo
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- CN104535632B CN104535632B CN201510043733.8A CN201510043733A CN104535632B CN 104535632 B CN104535632 B CN 104535632B CN 201510043733 A CN201510043733 A CN 201510043733A CN 104535632 B CN104535632 B CN 104535632B
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Abstract
The invention discloses a method for determining an IC50 value in a water body employing combination of mixed bacteria and an electrochemical technology, relates to a method for determining toxicity of water body, and aims at solving the problems that an existing method for detecting toxicity of the water body is long in elapsed time, high in cost and narrow in application range. The method comprises the following steps: determining a standard current output value; 2 determining a current output value after toxic substances are added; and 3 calculating the IC50 value, evaluating the toxicity degree in the water body through the IC50 value, so as to finish determination of the IC50 value in the water body by combination of the mixed bacteria and the electrochemical technology. The method is simple to operate, short in time, and low in energy consumption; moral controversy caused by biological detection of the toxicity of the water body is avoided; online real-time detection can be achieved; the method has potential application value in the fields of wastewater treatment, living water and edible liquid quality; and the method for determining the IC50 value in the water body employing combination of the mixed bacteria and the electrochemical technology can be obtained.
Description
Technical field
The present invention relates to a kind of method measuring water body toxicity.
Background technology
With scientific and technical progress and economic developing rapidly, the life production that water environment pollution gives people brings huge
Threat.Traditional detection method of toxicity life-time service organism is directly observed as subject, necessarily brings the cycle long, expense
The problems such as height and moral controversy.Electrochemical Detection is the method for detection water body toxicity ideal at present, and Chinese scholars are adopted
With single culture, assessment is made to water pollutant toxicity, up to the present, be used mixed bacteria as subject research also
Seldom, the industrial wastewater and in real life and sanitary sewage be all complicated component, the water body example of difficult degradation containing heavy metal.
Content of the invention
The invention aims to the method solving existing detection water body toxicity exists, and time-consuming, costly and range of application
Narrow problem, and ic in a kind of mixing thalline combined with electrochemical technical measurement water body is provided50The method of value.
Ic in a kind of mixing thalline combined with electrochemical technical measurement water body50The method of value, is specifically realized by the following steps
:
First, the mensure of normalized current output valve: by 2ml activated sludge, 0.8ml 160mmol/l～200mmol/l
k3fe(cn)6It is added in centrifuge tube with 0.2ml～1ml distilled water, then add ph value to be 7.4 in centrifuge tube
The pbs buffer solution of 0.2mol/l, to centrifuge tube, the cumulative volume of mixture is 4ml；Again temperature be 30 DEG C～40 DEG C and
Mixing speed is stirring reaction 30min～60min under conditions of 200r/min～300r/min, then is centrifuged, and obtains
Supernatant liquid after centrifugation；Test the current output value of the supernatant liquid after centrifugation using three-electrode system, obtain
Standard limit value i0；
2nd, the current output value after toxicant is added to measure:
(1) 2ml activated sludge, 0.8ml 160mmol/l～200mmol/l are added in, being centrifuge tube 1. to numbering
K3fe(cn)6, 0.2ml～1ml water sample to be measured and toxicant；The pbs buffering adding the 0.2mol/l for 7.4 for the ph value is molten
Liquid, makes the cumulative volume that numbering is mixture in centrifuge tube 1. be 4ml；
Numbering described in step 2 (1) is that the concentration of the mixture toxic material in centrifuge tube 1. is
0.05mmol/l；
(2) 2ml activated sludge, 0.8ml 160mmol/l～200mmol/l are added in, being centrifuge tube 2. to numbering
K3fe(cn)6, 0.2ml～1ml water sample to be measured and toxicant；The pbs buffering adding the 0.2mol/l for 7.4 for the ph value is molten
Liquid, makes the cumulative volume that numbering is mixture in centrifuge tube 2. be 4ml；
Numbering described in step 2 (2) is the concentration of the mixture toxic material in centrifuge tube 2. is 0.10
mmol/l；
(3) 2ml activated sludge, 0.8ml 160mmol/l～200mmol/l are added in, being centrifuge tube 3. to numbering
K3fe(cn)6, 0.2ml～1ml water sample to be measured and toxicant；The pbs buffering adding the 0.2mol/l for 7.4 for the ph value is molten
Liquid, makes the cumulative volume that numbering is mixture in centrifuge tube 3. be 4ml；
Numbering described in step 2 (3) is that the concentration of the mixture toxic material in centrifuge tube 3. is
0.15mmol/l；
(4) 2ml activated sludge, 0.8ml 160mmol/l～200mmol/l are added in, being centrifuge tube 4. to numbering
K3fe(cn)6, 0.2ml～1ml water sample to be measured and toxicant；The pbs buffering adding the 0.2mol/l for 7.4 for the ph value is molten
Liquid, makes the cumulative volume that numbering is mixture in centrifuge tube 4. be 4ml；
Numbering described in step 2 (4) is that the concentration of the mixture toxic material in centrifuge tube 4. is
0.20mmol/l；
(5) 2ml activated sludge, 0.8ml 160mmol/l～200mmol/l are added in, being centrifuge tube 5. to numbering
K3fe(cn)6, 0.2ml～1ml water sample to be measured and toxicant；The pbs buffering adding the 0.2mol/l for 7.4 for the ph value is molten
Liquid, makes the cumulative volume that numbering is mixture in centrifuge tube 5. be 4ml；
Numbering described in step 2 (5) is that the concentration of the mixture toxic material in centrifuge tube 5. is
0.25mmol/l；
(6), respectively by numbering be 1. the mixture in centrifuge tube 5. temperature be 30 DEG C～40 DEG C and stirring speed
Spend for stirring reaction 30min under conditions of 200r/min～300r/min～60min, then be centrifuged, obtaining numbering is
1. arrive the supernatant liquid after centrifugation 5.；The use of three-electrode system test No. is the upper strata after the centrifugation 1. arrived 5.
The current output value of liquid, obtains carrying current value i after adding toxicant1、i2、i3、i4And i5；
3rd, calculate ic50Value:
With ic50Value is as the evaluation index evaluating water body toxicity；
1., calculating numbering first with following computing formula is the suppression 1. arriving centrifuge tube toxic material 5. to water sample
Rate ai% processed:
Ai%=(i0-ii)/i0× 100%；
In above-mentioned formula:
i0For standard limit value i obtaining in step one0, unit is a；
iiFor carrying current value i adding after toxicant obtaining in step 21、i2、i3、i4And i5, unit is a；
Calculating numbering according to above-mentioned formula is the suppression ratio difference 1. arriving centrifuge tube toxic material 5. to water sample
For a1, a2, a3, a4 and a5；
②、ic50Calculating:
Respectively with number be 1. concentration 0.05mmol/l of the mixture toxic material in centrifuge tube 5.,
0.10mmol/l, 0.15mmol/l, 0.20mmol/l and 0.25mmol/l are as abscissa or with log (0.05) mmol/l, log
(0.10) mmol/l, log (0.15) mmol/l, log (0.20) mmol/l and log (0.25) mmol/l is as abscissa, to compile
Number for 1. arriving centrifuge tube toxic material 5. suppression ratio a1, a2, a3, a4 and a5 to water sample as vertical coordinate, obtain a little
A, point b, point c, point d and point e；Point a, point b, point c, point d and point e are fitted, obtain matched curve, vertical seat in matched curve
Mark suppression ratio is that the concentration value of corresponding abscissa toxicant when 50% is ic50Value；Complete a kind of mixing thalline knot
Close electrochemical techniques and measure ic in water body50The method of value.
The principle and advantage of the present invention:
First, the present invention combines the natural mixing thalline activated sludge of water body, using electrochemical method, with k3fe(cn)6As
Human intermediary body, with ic50Value, as the index of water body toxicity assessment, proposes a kind of quick and comprehensive water-body poison can be described comprehensively
The method of property；The toxicity size of toxicant is with ic50It is worth for weighing, the ic in water body50Value is that in water body, total amount of bacteria is down to one
The concentration of half toxicant used；By comparing the ic in water body50Value, we can determine whether water body toxic level；Logical
Cross the defects such as the method for the present invention can overcome traditional toxicity assay to take, expensive, range of application is narrow it is adaptable to exist for a long time
The comprehensive water-body toxicity monitoring of line, and realize sensitive, the quick detection of live toxicity, the real information of sample toxicity is provided；This
The activated sludge that invention uses is mixed bacteria, has more met the detection demand to water body in real life；
2nd, the present invention is simple to operate, save time low consumption, it is to avoid the moral controversy being caused due to biological detection water body toxicity,
And it is capable of on-line real time monitoring；In wastewater treatment, the detection field of domestic water and edible liquid quality has potential
Using value；
3rd, as electron acceptor, reduzate is easily surveyed, process is simple, result to adopt the potassium ferricyanide to replace oxygen in solution
Accurately；
4th, the present invention detects that water body toxic method cost price is cheap and is easy to get, and has competitive advantage；
5th, the present invention adopts this natural mixed bacteria of activated sludge as subject, overcomes single culture to certain poison
Property material blunt；
6th, the present invention adopts electrochemistry chronoamperometry determination limit electric current, and save time low consumption；
7th, the species of the measurable toxicant of the present invention extensively, has a extensive future；
Eight present invention can be implemented in the detection of the comprehensive water-body toxicity of line；
9th, the method passing through the easy on-line testing water body toxicity of the present invention, as potential with electrochemical method assessment
Water body toxicity, is applied in the inspection of daily life water quality.
The present invention can obtain ic in a kind of mixing thalline combined with electrochemical technical measurement water body50The method of value.
Brief description
Fig. 1 is the current output value curve chart that test one step 2 (6) obtains；In figure 1 is to compile in test one step 2 (6)
The electric current output curve diagram of the supernatant liquid after the centrifugation in centrifuge tube number 1., 2 is numbering in test one step 2 (6)
The electric current output curve diagram of the supernatant liquid after the centrifugation in centrifuge tube 2., 3 is to number 3. in test one step 2 (6)
Centrifuge tube in centrifugation after supernatant liquid electric current output curve diagram, 4 is that in test one step 2 (6) numbering is 4.
The electric current output curve diagram of the supernatant liquid after centrifugation in centrifuge tube, 5 be in test one step 2 (6) numbering 5. from
The electric current output curve diagram of the supernatant liquid after centrifugation in heart pipe；
Fig. 2 is the matched curve figure that test one step 3 obtains.
Specific embodiment
Specific embodiment one: present embodiment is ic in a kind of mixing thalline combined with electrochemical technical measurement water body50Value
Method, be specifically realized by the following steps:
First, the mensure of normalized current output valve: by 2ml activated sludge, 0.8ml 160mmol/l～200mmol/l
k3fe(cn)6It is added in centrifuge tube with 0.2ml～1ml distilled water, then add ph value to be 7.4 in centrifuge tube
The pbs buffer solution of 0.2mol/l, to centrifuge tube, the cumulative volume of mixture is 4ml；Again temperature be 30 DEG C～40 DEG C and
Mixing speed is stirring reaction 30min～60min under conditions of 200r/min～300r/min, then is centrifuged, and obtains
Supernatant liquid after centrifugation；Test the current output value of the supernatant liquid after centrifugation using three-electrode system, obtain
Standard limit value i0；
2nd, the current output value after toxicant is added to measure:
(1) 2ml activated sludge, 0.8ml 160mmol/l～200mmol/l are added in, being centrifuge tube 1. to numbering
K3fe(cn)6, 0.2ml～1ml water sample to be measured and toxicant；The pbs buffering adding the 0.2mol/l for 7.4 for the ph value is molten
Liquid, makes the cumulative volume that numbering is mixture in centrifuge tube 1. be 4ml；
Numbering described in step 2 (1) is that the concentration of the mixture toxic material in centrifuge tube 1. is
0.05mmol/l；
(2) 2ml activated sludge, 0.8ml 160mmol/l～200mmol/l are added in, being centrifuge tube 2. to numbering
K3fe(cn)6, 0.2ml～1ml water sample to be measured and toxicant；The pbs buffering adding the 0.2mol/l for 7.4 for the ph value is molten
Liquid, makes the cumulative volume that numbering is mixture in centrifuge tube 2. be 4ml；
Numbering described in step 2 (2) is that the concentration of the mixture toxic material in centrifuge tube 2. is
0.10mmol/l；
(3) 2ml activated sludge, 0.8ml 160mmol/l～200mmol/l are added in, being centrifuge tube 3. to numbering
K3fe(cn)6, 0.2ml～1ml water sample to be measured and toxicant；The pbs buffering adding the 0.2mol/l for 7.4 for the ph value is molten
Liquid, makes the cumulative volume that numbering is mixture in centrifuge tube 3. be 4ml；
Numbering described in step 2 (3) is that the concentration of the mixture toxic material in centrifuge tube 3. is
0.15mmol/l；
(4) 2ml activated sludge, 0.8ml 160mmol/l～200mmol/l are added in, being centrifuge tube 4. to numbering
K3fe(cn)6, 0.2ml～1ml water sample to be measured and toxicant；The pbs buffering adding the 0.2mol/l for 7.4 for the ph value is molten
Liquid, makes the cumulative volume that numbering is mixture in centrifuge tube 4. be 4ml；
Numbering described in step 2 (4) is that the concentration of the mixture toxic material in centrifuge tube 4. is
0.20mmol/l；
(5) 2ml activated sludge, 0.8ml 160mmol/l～200mmol/l are added in, being centrifuge tube 5. to numbering
K3fe(cn)6, 0.2ml～1ml water sample to be measured and toxicant；The pbs buffering adding the 0.2mol/l for 7.4 for the ph value is molten
Liquid, makes the cumulative volume that numbering is mixture in centrifuge tube 5. be 4ml；
Numbering described in step 2 (5) is that the concentration of the mixture toxic material in centrifuge tube 5. is
0.25mmol/l；
(6), respectively by numbering be 1. the mixture in centrifuge tube 5. temperature be 30 DEG C～40 DEG C and stirring speed
Spend for stirring reaction 30min under conditions of 200r/min～300r/min～60min, then be centrifuged, obtaining numbering is
1. arrive the supernatant liquid after centrifugation 5.；The use of three-electrode system test No. is the upper strata after the centrifugation 1. arrived 5.
The current output value of liquid, obtains carrying current value i after adding toxicant1、i2、i3、i4And i5；
3rd, calculate ic50Value:
With ic50Value is as the evaluation index evaluating water body toxicity；
1., calculating numbering first with following computing formula is the suppression 1. arriving centrifuge tube toxic material 5. to water sample
Rate ai% processed:
Ai%=(i0-ii)/i0× 100%；
In above-mentioned formula:
i0For standard limit value i obtaining in step one0, unit is a；
iiFor carrying current value i adding after toxicant obtaining in step 21、i2、i3、i4And i5, unit is a；
Calculating numbering according to above-mentioned formula is the suppression ratio difference 1. arriving centrifuge tube toxic material 5. to water sample
For a1, a2, a3, a4 and a5；
②、ic50Calculating:
Respectively with number be 1. concentration 0.05mmol/l of the mixture toxic material in centrifuge tube 5.,
0.10mmol/l, 0.15mmol/l, 0.20mmol/l and 0.25mmol/l as abscissa or with log (0.05) mmol/l,
Log (0.10) mmol/l, log (0.15) mmol/l, log (0.20) mmol/l and log (0.25) mmol/l as abscissa, with
Numbering is 1. to arrive centrifuge tube toxic material 5. suppression ratio a1, a2, a3, a4 and a5 to water sample as vertical coordinate, obtains
Point a, point b, point c, point d and point e；Point a, point b, point c, point d and point e are fitted, obtain matched curve, vertical in matched curve
Coordinate suppression ratio is that the concentration value of corresponding abscissa toxicant when 50% is ic50Value；Complete a kind of mixing thalline
Ic in combined with electrochemical technical measurement water body50The method of value.
The principle and advantage of present embodiment:
First, present embodiment combines the natural mixing thalline activated sludge of water body, using electrochemical method, with k3fe(cn)6
As human intermediary body, with ic50Value, as the index of water body toxicity assessment, proposes a kind of quick and can illustrate that water body is comprehensive comprehensively
The method closing toxicity；The toxicity size of toxicant is with ic50It is worth for weighing, the ic in water body50Value is total amount of bacteria fall in water body
The concentration of the toxicant used by half；By comparing the ic in water body50Value, we can determine whether water body toxic water
Flat；The defects such as traditional toxicity assay is time-consuming, expensive, range of application is narrow can be overcome by the method for present embodiment, fit
Monitor for comprehensive water-body toxicity online for a long time, and realize sensitive, the quick detection of live toxicity, the true of sample toxicity is provided
Real information；The activated sludge that present embodiment uses is mixed bacteria, has more met the detection demand to water body in real life；
2nd, present embodiment is simple to operate, save time low consumption, it is to avoid because the morals that biological detection water body toxicity causes are striven
View, and it is capable of on-line real time monitoring；In wastewater treatment, the detection field of domestic water and edible liquid quality has latent
Using value；
3rd, as electron acceptor, reduzate is easily surveyed, process is simple, result to adopt the potassium ferricyanide to replace oxygen in solution
Accurately；
5th, present embodiment detects that water body toxic method cost price is cheap and is easy to get, and has competitive advantage；
5th, present embodiment adopts this natural mixed bacteria of activated sludge as subject, overcomes single culture to certain
Plant the blunt of toxicant；
6th, present embodiment adopts electrochemistry chronoamperometry determination limit electric current, and save time low consumption；
7th, the species of the measurable toxicant of present embodiment extensively, has a extensive future；
8th, present embodiment can achieve the detection of online comprehensive water-body toxicity；
9th, the method passing through the easy on-line testing water body toxicity of present embodiment, as potential with electrochemical method
Assessment water body toxicity, is applied in the inspection of daily life water quality.
Present embodiment can obtain ic in a kind of mixing thalline combined with electrochemical technical measurement water body50The method of value.
Specific embodiment two: present embodiment with specific embodiment one difference is: the activity described in step one
The od value of mud is 1.375.Other steps are identical with specific embodiment one.
Specific embodiment three: present embodiment with one of specific embodiment one or two difference is: step 2 (1),
Used in step 2 (2), step 2 (3), step 2 (4) and step 2 (5), the od value of activated sludge is 1.375.Other
Step is identical with specific embodiment one or two.
Specific embodiment four: present embodiment with one of specific embodiment one to three difference is: institute in step one
The condition determination of the use three-electrode system stated is pt electrode for working electrode, and reference electrode is ag/agcl electrode, to electrode is
The three-electrode system of au electrode, setting voltage is the limit under the conditions of 0.45v, sweep time 100s and scanning speed 100mv/s
Electric current.Other steps are identical with specific embodiment one to three.
Specific embodiment five: present embodiment with one of specific embodiment one to four difference is: in step 2 (6)
The condition determination of described use three-electrode system is pt electrode for working electrode, and reference electrode is ag/agcl electrode, to electrode
For the three-electrode system of au electrode, setting voltage is 0.45v, the pole under the conditions of sweep time 100s and scanning speed 100mv/s
Threshold currents.Other steps are identical with specific embodiment one to four.
Specific embodiment six: present embodiment with one of specific embodiment one to five difference is: will in step one
2ml activated sludge, the k of 0.8ml 180mmol/l～200mmol/l3fe(cn)6It is added to centrifugation with 0.2ml～1ml distilled water
In test tube, then in centrifuge tube add the 0.2mol/l for 7.4 for the ph value pbs buffer solution, the mixture to centrifuge tube
Cumulative volume be 4ml；Stir under conditions of temperature is 35 DEG C～37 DEG C and mixing speed is 240r/min～270r/min again
Reaction 30min～40min, then be centrifuged, obtain the supernatant liquid after centrifugation；Using three-electrode system test from
The current output value of the supernatant liquid after heart separation, obtains standard limit value i0.Other steps and specific embodiment one to five
Identical.
Specific embodiment seven: present embodiment with one of specific embodiment one to six difference is: in step 2 (6)
Respectively by numbering be 1. the mixture in centrifuge tube 5. temperature be 35 DEG C～37 DEG C and mixing speed be 240r/min
Stirring reaction 30min～40min under conditions of～270r/min, then be centrifuged, obtain numbering the centrifugation being 1. arrive 5.
Supernatant liquid after separating；The electric current the use of three-electrode system test No. being the supernatant liquid after the centrifugation 1. arrived 5. is defeated
Go out value, obtain carrying current value i after adding toxicant1、i2、i3、i4And i5.Other steps and specific embodiment one to six
Identical.
Specific embodiment eight: present embodiment with one of specific embodiment one to seven difference is: institute in step 2
The numbering stated is the mixture that 1. toxicant in the mixture in centrifuge tube 5. is cd and pb, and 1. number be
To in the mixture of centrifuge tube 5., the concentration of cd and the concentration of pb are equal.Other steps and specific embodiment one to seven phase
With.
Specific embodiment nine: present embodiment with one of specific embodiment one to eight difference is: step 3 2. in
Matched curve correlation coefficient r2≥0.95.Other steps are identical with specific embodiment one to eight.
Specific embodiment ten: present embodiment with one of specific embodiment one to nine difference is: step 3 2. in
The coordinate of described point a is (0.05mmol/l, a1) or (log0.05 (mmol/l), a1)；The coordinate of described point b is
(0.10mmol/l, a2) or (log0.10 (mmol/l), a2)；The coordinate of described point c be (0.15mmol/l, a3) or
(log0.15(mmol/l)、a3)；The coordinate of described point d is (0.20mmol/l, a4) or (log0.20 (mmol/l), a4)；
The coordinate of described point e is (0.25mmol/l, a5) or (log0.25 (mmol/l), a5).Other steps and specific embodiment
One to nine is identical.
Using tests below checking beneficial effects of the present invention:
A kind of test one: ic in mixing thalline combined with electrochemical technical measurement water body50The method of value, specifically presses following step
Suddenly complete:
First, the mensure of normalized current output valve: by 2ml activated sludge, 0.8ml 160mmol/l～200mmol/l
k3fe(cn)6It is added in centrifuge tube with 0.2ml～1ml distilled water, then add ph value to be 7.4 in centrifuge tube
The pbs buffer solution of 0.2mol/l, to centrifuge tube, the cumulative volume of mixture is 4ml；Fast with stirring for 37 DEG C in temperature again
Spend for stirring reaction 30min under conditions of 240r/min, then be centrifuged, obtain the supernatant liquid after centrifugation；Make
Test the current output value of the supernatant liquid after centrifugation with three-electrode system, obtain standard limit value i0；
2nd, the current output value after toxicant is added to measure:
(1) 2ml activated sludge, 0.8ml 160mmol/l～200mmol/l are added in, being centrifuge tube 1. to numbering
K3fe(cn)6, 0.6ml water sample to be measured and toxicant；Add the pbs buffer solution of the 0.2mol/l for 7.4 for the ph value, make
Numbering is the cumulative volume of mixture in centrifuge tube 1. is 4ml；
Numbering described in step 2 (1) is that the concentration of the mixture toxic material in centrifuge tube 1. is
0.05mmol/l；
(2) 2ml activated sludge, 0.8ml 160mmol/l～200mmol/l are added in, being centrifuge tube 2. to numbering
K3fe(cn)6, 0.6ml water sample to be measured and toxicant；Add the pbs buffer solution of the 0.2mol/l for 7.4 for the ph value, make
Numbering is the cumulative volume of mixture in centrifuge tube 2. is 4ml；
Numbering described in step 2 (2) is that the concentration of the mixture toxic material in centrifuge tube 2. is
0.10mmol/l；
(3) 2ml activated sludge, 0.8ml 160mmol/l～200mmol/l are added in, being centrifuge tube 3. to numbering
K3fe(cn)6, 0.6ml water sample to be measured and toxicant；Add the pbs buffer solution of the 0.2mol/l for 7.4 for the ph value, make
Numbering is the cumulative volume of mixture in centrifuge tube 3. is 4ml；
Numbering described in step 2 (3) is that the concentration of the mixture toxic material in centrifuge tube 3. is
0.15mmol/l；
(4) 2ml activated sludge, 0.8ml 160mmol/l～200mmol/l are added in, being centrifuge tube 4. to numbering
K3fe(cn)6, 0.6ml water sample to be measured and toxicant；Add the pbs buffer solution of the 0.2mol/l for 7.4 for the ph value, make
Numbering is the cumulative volume of mixture in centrifuge tube 4. is 4ml；
Numbering described in step 2 (4) is that the concentration of the mixture toxic material in centrifuge tube 4. is
0.20mmol/l；
(5) 2ml activated sludge, 0.8ml 160mmol/l～200mmol/l are added in, being centrifuge tube 5. to numbering
K3fe(cn)6, 0.6ml water sample to be measured and toxicant；Add the pbs buffer solution of the 0.2mol/l for 7.4 for the ph value, make
Numbering is the cumulative volume of mixture in centrifuge tube 5. is 4ml；
Numbering described in step 2 (5) is that the concentration of the mixture toxic material in centrifuge tube 5. is
0.25mmol/l；
(6) respectively numbering is, that 1. the mixture in centrifuge tube 5. is that 37 DEG C and mixing speed are in temperature
Stirring reaction 30min under conditions of 240r/min, then be centrifuged, it is 1. upper to after centrifugation 5. for obtaining numbering
Layer liquid；The current output value the use of three-electrode system test No. being the supernatant liquid after the centrifugation 1. arrived 5., obtains
Add carrying current value i after toxicant1、i2、i3、i4And i5；
3rd, calculate ic50Value:
With ic50Value is as the evaluation index evaluating water body toxicity；
1., calculating numbering first with following computing formula is the suppression 1. arriving centrifuge tube toxic material 5. to water sample
Rate ai% processed:
Ai%=(i0-ii)/i0× 100%；
In above-mentioned formula:
i0For standard limit value i obtaining in step one0, unit is a；
iiFor carrying current value i adding after toxicant obtaining in step 21、i2、i3、i4And i5, unit is a；
Calculating numbering according to above-mentioned formula is the suppression ratio difference 1. arriving centrifuge tube toxic material 5. to water sample
For a1, a2, a3, a4 and a5；
②、ic50Calculating:
Respectively with number be 1. concentration 0.05mmol/l of the mixture toxic material in centrifuge tube 5.,
0.10mmol/l, 0.15mmol/l, 0.20mmol/l and 0.25mmol/l, as abscissa, are tried with numbering the centrifugation being 1. arrive 5.
Pipe toxic material obtains point a, point b, point c, point d and point e to suppression ratio a1, a2, a3, a4 and a5 of water sample as vertical coordinate；
Point a, point b, point c, point d and point e are fitted, obtain matched curve, in matched curve, vertical coordinate suppression ratio is 50% when institute
The concentration value of corresponding abscissa toxicant is ic50Value；Complete a kind of mixing thalline combined with electrochemical technical measurement water
Ic in body50The method of value；
Step 3 2. described in point a coordinate be (0.05mmol/l, a1)；The coordinate of described point b is
(0.10mmol/l、a2)；The coordinate of described point c is (0.15mmol/l, a3)；The coordinate of described point d is (0.20mmol/
l、a4)；The coordinate of described point e is (0.25mmol/l, a5).
Fig. 1 is the current output value curve chart that test one step 2 (6) obtains；In figure 1 is to compile in test one step 2 (6)
The electric current output curve diagram of the supernatant liquid after the centrifugation in centrifuge tube number 1., 2 is numbering in test one step 2 (6)
The electric current output curve diagram of the supernatant liquid after the centrifugation in centrifuge tube 2., 3 is to number 3. in test one step 2 (6)
Centrifuge tube in centrifugation after supernatant liquid electric current output curve diagram, 4 is that in test one step 2 (6) numbering is 4.
The electric current output curve diagram of the supernatant liquid after centrifugation in centrifuge tube, 5 be in test one step 2 (6) numbering 5. from
The electric current output curve diagram of the supernatant liquid after centrifugation in heart pipe；The concentration of toxicant is bigger as can be seen from Figure 1, to bacterium
Body Repiration restraint is stronger, and in respiratory, the amount of electrons of transfer is fewer, thus the amount meeting of the potassium ferrocyanide being reduced
Reduce, carrying current will accordingly reduce.
Fig. 2 is the matched curve figure that test one step 3 obtains；As can be seen from Figure 2, the ic that 2. test one step 3 obtains50Value
For 0.0876mmol/l, ic50Value is less, illustrates that the toxicity in water body is bigger, and therefore in test one, the water sample to be measured of test has
Toxicity.
Claims (10)
1. ic in a kind of mixing thalline combined with electrochemical technical measurement water body50The method of value is it is characterised in that a kind of mix thalline
Ic in combined with electrochemical technical measurement water body50The method of value is specifically realized by the following steps:
First, the mensure of normalized current output valve: by the k of 2ml activated sludge, 0.8ml 160mmol/l～200mmol/l3fe
(cn)6It is added in centrifuge tube with 0.2ml～1ml distilled water, then add the 0.2mol/l for 7.4 for the ph value in centrifuge tube
Pbs buffer solution, to centrifuge tube the cumulative volume of mixture be 4ml；Again temperature be 30 DEG C～40 DEG C and mixing speed
For stirring reaction 30min under conditions of 200r/min～300r/min～60min, then it is centrifuged, obtain centrifugation
Supernatant liquid afterwards；Test the current output value of the supernatant liquid after centrifugation using three-electrode system, obtain standard limit
Value i0；
2nd, the current output value after toxicant is added to measure:
(1) add the k of 2ml activated sludge, 0.8ml 160mmol/l～200mmol/l in, being centrifuge tube 1. to numbering3fe
(cn)6, 0.2ml～1ml water sample to be measured and toxicant；Add the pbs buffer solution of the 0.2mol/l for 7.4 for the ph value, make
Numbering is the cumulative volume of mixture in centrifuge tube 1. is 4ml；
Numbering described in step 2 (1) is the concentration of the mixture toxic material in centrifuge tube 1. is 0.05mmol/
l；
(2) add the k of 2ml activated sludge, 0.8ml 160mmol/l～200mmol/l in, being centrifuge tube 2. to numbering3fe
(cn)6, 0.2ml～1ml water sample to be measured and toxicant；Add the pbs buffer solution of the 0.2mol/l for 7.4 for the ph value, make
Numbering is the cumulative volume of mixture in centrifuge tube 2. is 4ml；
Numbering described in step 2 (2) is the concentration of the mixture toxic material in centrifuge tube 2. is 0.10mmol/
l；
(3) add the k of 2ml activated sludge, 0.8ml 160mmol/l～200mmol/l in, being centrifuge tube 3. to numbering3fe
(cn)6, 0.2ml～1ml water sample to be measured and toxicant；Add the pbs buffer solution of the 0.2mol/l for 7.4 for the ph value, make
Numbering is the cumulative volume of mixture in centrifuge tube 3. is 4ml；
Numbering described in step 2 (3) is the concentration of the mixture toxic material in centrifuge tube 3. is 0.15mmol/
l；
(4) add the k of 2ml activated sludge, 0.8ml 160mmol/l～200mmol/l in, being centrifuge tube 4. to numbering3fe
(cn)6, 0.2ml～1ml water sample to be measured and toxicant；Add the pbs buffer solution of the 0.2mol/l for 7.4 for the ph value, make
Numbering is the cumulative volume of mixture in centrifuge tube 4. is 4ml；
Numbering described in step 2 (4) is the concentration of the mixture toxic material in centrifuge tube 4. is 0.20mmol/
l；
(5) add the k of 2ml activated sludge, 0.8ml 160mmol/l～200mmol/l in, being centrifuge tube 5. to numbering3fe
(cn)6, 0.2ml～1ml water sample to be measured and toxicant；Add the pbs buffer solution of the 0.2mol/l for 7.4 for the ph value, make
Numbering is the cumulative volume of mixture in centrifuge tube 5. is 4ml；
Numbering described in step 2 (5) is the concentration of the mixture toxic material in centrifuge tube 5. is 0.25mmol/
l；
(6) respectively numbering is, that 1. the mixture in centrifuge tube 5. is that 30 DEG C～40 DEG C and mixing speed are in temperature
Stirring reaction 30min～60min under conditions of 200r/min～300r/min, then be centrifuged, obtaining numbering is 1. to arrive
5. the supernatant liquid after centrifuge tube centrifugation；It is the centrifuge tube centrifugation 1. arrived 5. using three-electrode system test No.
Supernatant liquid current output value after separating, obtains carrying current value i after adding toxicant1、i2、i3、i4And i5；
3rd, calculate ic50Value:
With ic50Value is as the evaluation index evaluating water body toxicity；
1., calculating numbering first with following computing formula is the suppression ratio 1. arriving centrifuge tube toxic material 5. to water sample
Ai%:
Ai%=(i0-ii)/i0× 100%；
In above-mentioned formula:
i0For standard limit value i obtaining in step one0, unit is a；
iiFor carrying current value i adding after toxicant obtaining in step 21、i2、i3、i4And i5, unit is a；
According to above-mentioned formula calculate numbering be 1. arrive centrifuge tube toxic material 5. to the suppression ratio respectively a1 of water sample,
A2, a3, a4 and a5；
②、ic50Calculating:
Respectively with number be 1. concentration 0.05mmol/l of the mixture toxic material in centrifuge tube 5.,
0.10mmol/l, 0.15mmol/l, 0.20mmol/l and 0.25mmol/l are as abscissa or with log (0.05) mmol/l, log
(0.10) mmol/l, log (0.15) mmol/l, log (0.20) mmol/l and log (0.25) mmol/l is as abscissa, to compile
Number for 1. arriving centrifuge tube toxic material 5. suppression ratio a1, a2, a3, a4 and a5 to water sample as vertical coordinate, obtain a little
A, point b, point c, point d and point e；Point a, point b, point c, point d and point e are fitted, obtain matched curve, vertical seat in matched curve
Mark suppression ratio is that the concentration value of corresponding abscissa toxicant when 50% is ic50Value；Complete a kind of mixing thalline knot
Close electrochemical techniques and measure ic in water body50The method of value.
2. ic in a kind of mixing thalline combined with electrochemical technical measurement water body according to claim 150The method of value, it is special
Levy and be that the od value of the activated sludge described in step one is 1.375.
3. ic in a kind of mixing thalline combined with electrochemical technical measurement water body according to claim 150The method of value, it is special
Levy the od being activated sludge used in step 2 (1), step 2 (2), step 2 (3), step 2 (4) and step 2 (5)
Value is 1.375.
4. ic in a kind of mixing thalline combined with electrochemical technical measurement water body according to claim 150The method of value, it is special
Levying the condition determination being the use three-electrode system described in step one for working electrode is pt electrode, and reference electrode is ag/
Agcl electrode, to electrode for au electrode three-electrode system, voltage is 0.45v, and sweep time is 100s, and scanning speed is
100mv/s.
5. ic in a kind of mixing thalline combined with electrochemical technical measurement water body according to claim 150The method of value, it is special
Levying the condition determination being the use three-electrode system described in step 2 (6) for working electrode is pt electrode, and reference electrode is
Ag/agcl electrode, to electrode for au electrode three-electrode system, voltage is 0.45v, and sweep time is 100s, and scanning speed is
100mv/s.
6. ic in a kind of mixing thalline combined with electrochemical technical measurement water body according to claim 150The method of value, it is special
Levy and be the k of 2ml activated sludge, 0.8ml 180mmol/l～200mmol/l in step one3fe(cn)6With 0.2ml～1ml
Distilled water is added in centrifuge tube, then adds the pbs buffer solution of the 0.2mol/l for 7.4 for the ph value in centrifuge tube, extremely
In centrifuge tube, the cumulative volume of mixture is 4ml；Being 35 DEG C～37 DEG C in temperature again is 240r/min～270r/ with mixing speed
Stirring reaction 30min～40min under conditions of min, then be centrifuged, obtain the supernatant liquid after centrifugation；Use
Three-electrode system tests the current output value of the supernatant liquid after centrifugation, obtains standard limit value i0.
7. ic in a kind of mixing thalline combined with electrochemical technical measurement water body according to claim 150The method of value, it is special
Levy and be in step 2 (6) to be that 1. the mixture in centrifuge tube 5. is 35 DEG C～37 DEG C and stir in temperature by numbering respectively
Mixing speed is stirring reaction 30min～40min under conditions of 240r/min～270r/min, then is centrifuged, and is compiled
Number for 1. arriving the supernatant liquid after centrifuge tube centrifugation 5.；The use of three-electrode system test test No. is 1. to arrive 5.
Supernatant liquid current output value after centrifuge tube centrifugation, obtains carrying current value i after adding toxicant1、i2、i3、
i4And i5.
8. ic in a kind of mixing thalline combined with electrochemical technical measurement water body according to claim 150The method of value, it is special
Levy and be that the numbering described in step 2 is that 1. the toxicant in the mixture in centrifuge tube 5. is cd's and pb
Mixture, and to number be that 1. in the mixture of centrifuge tube 5., the concentration of cd and the concentration of pb are equal.
9. ic in a kind of mixing thalline combined with electrochemical technical measurement water body according to claim 150The method of value, it is special
Levy the correlation coefficient r of the matched curve in being step 3 2.2≥0.95.
10. ic in a kind of mixing thalline combined with electrochemical technical measurement water body according to claim 150The method of value, its
The coordinate of the point a described in being characterised by step 3 2. is (0.05mmol/l, a1) or (log0.05 (mmol/l), a1)；Described
Point b coordinate be (0.10mmol/l, a2) or (log0.10 (mmol/l), a2)；The coordinate of described point c is
(0.15mmol/l, a3) or (log0.15 (mmol/l), a3)；The coordinate of described point d be (0.20mmol/l, a4) or
(log0.20(mmol/l)、a4)；The coordinate of described point e is (0.25mmol/l, a5) or (log0.25 (mmol/l), a5).
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