CN103616489A - Method for testing typical antibiotics wastewater toxicity by using zebra fishes - Google Patents
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Abstract
The invention belongs to the field of environmental toxicology, and relates to a method for testing typical antibiotics wastewater toxicity by using zebra fishes. According to the method, zebra fishes are used as to-be-tested organisms, and are exposed in typical antibiotics wastewater, the short-term or long-term toxic effect of the typical antibiotics wastewater to the zebra fishes is quantitatively tested through analyzing the acute toxicity and subacute toxicity of the typical antibiotics wastewater to the zebra fishes, the acute toxicity is described by using the 96h medium lethal concentration (96h LC50) (typical antibiotics wastewater percent by volume percent), and the subacute toxicity is described by fish body biochemical indexes such as superoxide dismutase (SOD) activity, peroxidase (POD) activity and malondialdehyde (MDA) content. By adopting the method, the toxicity characteristics and toxicity levels of the typical antibiotics wastewater can be analyzed, tested and quantitatively described, and the problem that conventional physicochemical indexes can not reflect biotoxicity of the typical antibiotics wastewater can be solved, and results obtained by analyzing, testing and describing can be used as biotoxicity monitoring and evaluating indexes of the typical antibiotics wastewater.
Description
Technical field
The invention belongs to ecotoxicology field, relate to a kind of method of utilizing zebra fish to test typical antibiotic waste water toxicity.
Background technology
Antibiotic waste water be a class colourity high, containing the many high concentrated organic wastewaters of difficult degradation and bio-toxicity material.National environmental protection in July, 2010 cloth promulgated by the ministries or commissions of the Central Government < < pharmaceuticals industry pollution discharge standard > >, wherein stipulated to take emission limit, monitoring and monitoring requirement that microbiotic etc. is main fermentation class pharmaceuticals industry enterprise water pollutant.The discharge index that relates to toxicity only has acute toxicity index (HgCl
2toxic equivalent), method of testing is photobacteria method, this index only can reflect that whether antibiotic waste water is toxic to photogen, but photogen is unicellular lower eukaryote, its result can not represent the impact of antibiotic waste water on higher organism, needs to find the bio-toxicity that new method and index are evaluated antibiotic waste water for this reason.
Summary of the invention
The present invention, for solving the problem of present antibiotic waste water bio-toxicity quantitative and qualitative analysis assessment, provides a kind of method that adopts zebra fish to test typical antibiotic waste water toxicity.The method adopts acute toxicity, subacute toxicity test, and two kinds of indexs of quantitative and qualitative analysis are described the bio-toxicity of typical antibiotic waste water, make up the deficiency of antibiotic waste water bio-toxicity monitoring index.
Thinking of the present invention is that employing short-term (96 hours) exposure chamber, measures and obtain the acute toxicity of typical antibiotic waste water to zebra fish, with 96h LC
50(the percent by volume % of typical antibiotic waste water) describes, and adopts long-term (20 days) exposure chamber, and subacute toxicity represents with one group of Antioxidant Indexes of fish body.
Judge whether the virose criterion of tool is typical antibiotic waste water to zebra fish: determination of acute toxicity obtains 96h LC
50(the percent by volume % of typical antibiotic waste water); Subacute toxicity is whether typical antibiotic waste water is remarkable on the impact of zebra fish fish body Antioxidant Indexes.If exposure group and blank group significant difference: think that typical antibiotic waste water has subacute bio-toxicity.Exposure group and blank group difference are not remarkable: think that typical antibiotic waste water is without subacute bio-toxicity.According to above-mentioned criterion, assess typical antibiotic waste water to the pathotype of zebra fish and toxic level, thereby can determine pathotype (belonging to acute toxicity or subacute toxicity) and the toxic level (level of significance of toxicity) of typical antibiotic waste water.
The present invention is achieved by the following technical solutions:
A kind of method of utilizing zebra fish to test typical antibiotic waste water toxicity, the method adopts zebra fish In vivo assay Cells, utilize respectively short-term (96 hours) and long-term (20 days) exposure chamber to describe typical antibiotic waste water to the acute toxicity of zebra fish and subacute toxicity, judge whether the virose criterion of tool is typical antibiotic waste water to zebra fish: acute toxicity is with 96h LC50(typical case antibiotic waste water percent by volume %) describe; Subacute toxicity is described with one group of Antioxidant Indexes;
Utilize short-term (96 hours) exposure chamber, measure and obtain the acute toxicity of typical antibiotic waste water to zebra fish, determination of acute toxicity obtains 96h LC
50(typical antibiotic waste water percent by volume %); With 96h LC
50acute toxicity is described;
Utilize long-term (20 days) exposure chamber, subacute toxicity represents with one group of Antioxidant Indexes of fish body, then utilizes significant t-test, and the difference between check exposure group and blank group, as t < t
0.05 (n), during P > 0.05, think there was no significant difference; Work as t
0.05 (n)≤ t < t
0.01 (n), i.e. 0.01 < P≤0.05 o'clock, thinks significant difference; As t > t
0.01 (n), P≤0.01 o'clock, thinks that difference is extremely remarkable;
This method of testing is, if exposure group and blank group significant difference (0.01<P<0.05 or P<0.01), think that typical antibiotic waste water has subacute bio-toxicity, if exposure group and blank group difference are not remarkable, think that typical antibiotic waste water is without subacute bio-toxicity.
The described zebra fish that utilizes is tested the method for typical antibiotic waste water toxicity, and it comprises following concrete steps:
(1) setup test is biological, select zebra fish (Zebra Fish) for biological subject, male and female individuality half and half, zebra fish is after 5% saline solution sterilization, with the dechlorination tap water of aeration 48h, in laboratory, raise and train 7 days, in preliminary experiment the previous day and duration of test, refuse feeding, avoid bait to exert an influence to test result;
(2) setup test condition, testing liquid pH value is 7.0～8.5; Total hardness is that 240～250mg/L(is with CaCO
3meter), test temperature is 23～25 ℃; Dissolved oxygen DO is higher than 5mg/L; Adopt natural lighting; Test period is 96h(preliminary experiment and acute toxicity testing duration) or the 20d(sub-acute toxicity test duration); Test method is hydrostatic formula (every 24h changes a test solution), at least measures water temperature, hardness, pH value, the dissolved oxygen content of 1 testing liquid every day;
(3) carry out preliminary experiment, in the glass aquarium of 50cm * 20cm * 25cm, hold 20L testing liquid, be divided into 5～6 exposure groups, exposure group concentration represents with the percent by volume containing typical antibiotic waste water in testing liquid.Three kinds of typical antibiotic waste water exposure groups arrange respectively as follows: penicillin wastewater exposure concentrations group is 5%, 10%, 15%, 20%, 25% and 30%; Terramycin wastewater exposure concentrations group is 0.1%, 0.2%, 0.3%, 0.4%, 0.5% and 0.6%; Streptomysin waste water exposure concentrations group is 1%, 3.2%, 10%, 32%, 100%; Do not establish parallel group, each exposure group is put 5 tail zebra fishs, tests continuously 96h, records the dead fish number in each container every day at least twice and takes out in time dead fish; Calculate preliminary experiment result: minimum 24h absolute lethal concentration (the percent by volume % of typical antibiotic waste water) and the highest 96h are without dead concentration (the percent by volume % of typical antibiotic waste water).
Penicillin wastewater preliminary experiment result: minimum 24h absolute lethal concentration and the highest 96h are respectively the percent by volume % of 20%(penicillin wastewater without dead concentration) and the percent by volume % of 10%(penicillin wastewater).
Terramycin wastewater preliminary experiment result: minimum 24h absolute lethal concentration and the highest 96h are respectively the percent by volume % of 0.1%(terramycin wastewater without dead concentration) and the percent by volume % of 0.5%(terramycin wastewater).
Streptomysin waste water preliminary experiment result: minimum 24h absolute lethal concentration and the highest 96h are respectively the percent by volume % of 100%(streptomysin waste water without dead concentration) and the percent by volume % of 10%(streptomysin waste water).
(4) acute toxic test method is according to the test result of described step (3), between 24h absolute lethal concentration (the percent by volume % of typical antibiotic waste water) and the highest 96h are without dead concentration (the percent by volume % of typical antibiotic waste water) by etc. logarithmic interval 5 exposure groups are set, exposure group concentration represents with the percent by volume containing typical antibiotic waste water in testing liquid.Three kinds of typical antibiotic waste water exposure groups arrange respectively as follows: penicillin wastewater exposure concentrations group is 10%, 12.57%, 15.81%, 19.88%, 24.99%; Terramycin wastewater exposure concentrations group is 0.1%, 0.15%, 0.22%, 0.33%, 0.5%; Streptomysin waste water exposure concentrations group is 17.78%, 23.71%, 31.62%, 42.17%, 56.23%.Each exposure group is established 2 parallel group, and blank group is set, amount to 11 groups, each exposure group is put into 8 tail zebra fishs at random, test period is 96h, test findings represents with LC50 (the percent by volume % of typical antibiotic waste water), every 24h, changes 1 testing liquid, adopts bandit's formula method and two kinds of methods of probit method to calculate 96h LC
50(the percent by volume % of typical antibiotic waste water) and 95% confidence limit thereof.
Acute toxic test result is: penicillin wastewater 96h LC
50for 13.46%(bandit formula method) and 13.66%(probit method); Terramycin wastewater 96h LC
50for 0.22%(bandit formula method) and 0.19%(probit method); Streptomysin waste water 96h LC
50for 25.56%(bandit formula method) and 29.51%(probit method).
(5) subacute toxicity method of testing arranges subacute exposure group with reference to the test result of step (4), and exposure group concentration is at 1/6 96h LC
50with 1/2 96h LC
50between, 5 exposure groups are set, penicillin wastewater, terramycin and streptomysin exposure concentrations group are 1/256 96h LC
50, 1/64 96hLC
50, 1/16 96h LC
50, 1/4 96h LC
50, 1/2 96h LC
50.Exposure group concentration represents with the percent by volume containing typical antibiotic waste water in testing liquid.Each exposure group arranges 2 parallel group, and 1 blank group amounts to 16 groups.Open-assembly time is 20 days, chooses healthy zebra fish, male and female individuality half and half, and each organizes 15 tail zebra fishs, changes water every day.5 exposure groups were got biological sample respectively at the 4th, 8,12,16 and 20 days and are analyzed, each each exposure group is got respectively 2 tail zebra fishs, mensuration POD is active, SOD is active and MDA content, test result is that exposure group is compared with blank group, POD is active, SOD is active and MDA content there were significant differences;
Described testing liquid is the mixed solution of typical antibiotic waste water and dechlorination tap water.
Test result is that the POD of contrast exposure group and blank group is active, SOD is active and MDA content, utilizes significant t-test, and the difference between check exposure group and blank group, as t < t
0.05 (n), during P > 0.05, think there was no significant difference; Work as t
0.05 (n)≤ t < t
0.01 (n), i.e. 0.01 < P≤0.05 o'clock, thinks significant difference; As t > t
0.01 (n), P≤0.01 o'clock, thinks that difference is extremely remarkable.
Method of testing of the present invention is, if exposure group and blank group significant difference (0.01<P<0.05 or P<0.01), think that typical antibiotic waste water has subacute bio-toxicity, if exposure group and blank group difference are not remarkable, think that typical antibiotic waste water is without subacute bio-toxicity.
The remarkable result that the present invention compared with prior art has is:
The inventive method can be carried out analytical test and quantitative description to the toxic characteristic of typical antibiotic waste water and toxic level, solve conventional physical and chemical index, as the typical antibiotic waste water bio-toxicity that chemical oxygen demand (COD) (COD), biochemical oxygen demand (BOD), pH etc. can not reflect, the index that can be used as typical antibiotic waste water bio-toxicity monitoring simultaneously and evaluate.
Embodiment
The present invention is a kind of method of utilizing zebra fish to test typical antibiotic waste water toxicity, and it comprises the following steps:
(1) setup test biology selects zebra fish (Zebra Fish) for biological subject, male and female individuality half and half.Zebra fish, after 5% saline solution sterilization, is raised and train in laboratory 7 days with the dechlorination tap water of aeration 48h, in preliminary experiment the previous day and duration of test, refuses feeding, avoids bait to exert an influence to test result.
(2) setup test condition, testing liquid pH value is 7.0～8.5; Total hardness is that 240～250mg/L(is with CaCO
3meter), test temperature is 23～25 ℃; Dissolved oxygen DO is higher than 5mg/L; Adopt natural lighting; Test period is 96h(preliminary experiment and acute toxicity testing duration) or the 20d(sub-acute toxicity test duration); Test method is hydrostatic formula (every 24h changes a test solution), at least measures water temperature, hardness, pH value, the dissolved oxygen content of 1 testing liquid every day.
The following stated testing liquid is the mixed solution of typical antibiotic waste water and dechlorination tap water.
(3) preliminary experiment method, in the glass aquarium of 50cm * 20cm * 25cm, holds 20L testing liquid, is divided into 5～6 exposure groups, and in exposure group, the concentration of typical antibiotic waste water is described by its percent by volume.Three kinds of typical antibiotic waste water exposure groups arrange respectively as follows: penicillin wastewater exposure concentrations group is 5%, 10%, 15%, 20%, 25% and 30%; Terramycin wastewater exposure concentrations group is 0.1%, 0.2%, 0.3%, 0.4%, 0.5% and 0.6%; Streptomysin waste water exposure concentrations group is 1%, 3.2%, 10%, 32%, 100%; Do not establish parallel group, each exposure group is put 5 tail zebra fishs, tests continuously 96h, records the dead fish number in each container every day at least twice and takes out in time dead fish.Calculate preliminary experiment result: minimum 24h absolute lethal concentration (the percent by volume % of typical antibiotic waste water) and the highest 96h are without dead concentration (the percent by volume % of typical antibiotic waste water).
Preliminary experiment result: the 24h absolute lethal concentration that penicillin wastewater is minimum and the highest 96h are respectively 20% and 10% without dead concentration.The 24h absolute lethal concentration that terramycin wastewater is minimum and the highest 96h are respectively 0.1% and 0.5% without dead concentration.The 24h absolute lethal concentration that streptomysin waste water is minimum and the highest 96h are respectively 100% and 10% without dead concentration.
(4) acute toxic test method is according to the test result of described step (3), between 24h absolute lethal concentration and the highest 96h are without dead concentration by etc. logarithmic interval 5 exposure groups are set.In exposure group, the concentration of typical antibiotic waste water is described by its percent by volume.Three kinds of typical antibiotic waste water exposure groups arrange respectively as follows: penicillin wastewater exposure concentrations group is 10%, 12.57%, 15.81%, 19.88%, 24.99% and blank group; Terramycin wastewater exposure concentrations group is 0.1%, 0.15%, 0.22%, 0.33%, 0.5% and blank group; Streptomysin waste water exposure concentrations group is 17.78%, 23.71%, 31.62%, 42.17%, 56.23% and blank group.Each exposure group is established 2 parallel group, and blank group is set, and amounts to 11 groups, and each exposure group is put into 8 tail zebra fishs at random, and the test period is 96h, and test findings represents with LC50, every 24h, changes 1 testing liquid, calculates 96h LC
50(the percent by volume % of typical antibiotic waste water) and 95% confidence limit thereof.
Acute toxic test result is: penicillin wastewater 96h LC
50for 13.46%(bandit formula method) and 13.66%(probit method); Terramycin wastewater 96h LC
50for 0.22%(bandit formula method) and 0.19%(probit method); Streptomysin waste water 96h LC
50for 25.56%(bandit formula method) and 29.51%(probit method).
(5) subacute toxicity method of testing, with reference to the test result of step (4), arranges 5 exposure groups, and exposure group concentration is at 1/6 96h LC
50with 1/2 96h LC
50between, penicillin wastewater, terramycin and streptomysin exposure concentrations group are 1/256 96h LC
50, 1/64 96h LC
50, 1/16 96h LC
50, 1/4 96h LC
50, 1/2 96h LC
50.In exposure group, the concentration of typical antibiotic waste water is described by its percent by volume.Each exposure group arranges 2 parallel group, and 1 blank group amounts to 16 groups.Open-assembly time is 20 days, chooses healthy zebra fish, male and female individuality half and half, and each organizes 15 tail zebra fishs, changes water every day.5 exposure groups were got biological sample respectively at the 4th, 8,12,16 and 20 days and are analyzed, and each exposure group is got respectively 2 tail zebra fishs at every turn, and mensuration POD is active, SOD is active and MDA content.
Test result is that the POD of contrast exposure group and blank group is active, SOD is active and MDA content, utilizes significant t-test, and the difference between check exposure group and blank group, as t < t
0.05 (n), during P > 0.05, think there was no significant difference; Work as t
0.05 (n)≤ t < t
0.01 (n), i.e. 0.01 < P≤0.05 o'clock, thinks significant difference; As t > t
0.01 (n), P≤0.01 o'clock, thinks that difference is extremely remarkable.
This method is thought, if exposure group and blank group significant difference (0.01<P<0.05 or P<0.01), think that typical antibiotic waste water has subacute toxicity to zebra fish, if exposure group and blank group difference are not remarkable, think typical antibiotic waste water to zebra fish without subacute toxicity.
Example 1
Take penicillin wastewater below as example, the embodiment of the toxotest method that the present invention proposes is described.
Select children zebra fish in age (Zebra Fish) for test organisms, zebra fish is provided by Hebei Medical University's Experimental Animal Center, and body is long is (34.05 ± 0.43) mm, and body weight is (0.24 ± 0.03) g.Experimental fish enters laboratory after 5% saline solution sterilization, with the dechlorination tap water of 48h aeration, raises and train 7 days in laboratory, in experiment the previous day and duration of test, refuses feeding, avoids bait to exert an influence to experiment.Test condition meets the demands.
(1) preliminary experiment method, in the glass aquarium of 50cm * 20cm * 25cm, holds 20L testing liquid.Testing liquid is the mixed liquor of penicillin wastewater and dechlorination tap water.
Trial test arranges 6 exposure groups, exposure group represents with the percent by volume of contained penicillin wastewater in testing liquid, exposure group is 5%, 10%, 15%, 20%, 25%, 30%, do not establish parallel, each exposure group is put 5 tail zebra fishs, continuous experiment 96h, calculates minimum 24h absolute lethal concentration (penicillin wastewater percent by volume %) and the highest 96h without dead concentration (penicillin wastewater percent by volume %).
Preliminary experiment result: penicillin wastewater continues to carry out 96h to the preliminary experiment of zebra fish, obtaining minimum 24h absolute lethal concentration is 20%; The highest 96h is without dead concentration 10%.
(2) acute toxicity test method is according to trial test result, between 24h absolute lethal concentration and the highest 96h are without dead concentration by etc. logarithmic interval 5 exposure groups are set, exposure group represents with the percent by volume of contained penicillin wastewater in testing liquid, penicillin wastewater is respectively 10%, 12.57%, 15.81%, 19.88%, 24.99% to the acute toxicity exposure group setting of zebra fish, each exposure group is established 2 parallel group, and 1 blank group is set, each exposure group is put into 8 tail zebra fishs, male and female individuality half and half at random.Test period is 96h, and test findings is with LC50 LC
50(penicillin wastewater percent by volume %) represents.For preventing bait impact, testing not feeding of the previous day and duration of test.
The acute toxicity tests: when zebra fish is exposed in testing liquid, according to the mortality ratio of zebra fish, adopt respectively karber's method and probit method to calculate the LC of this testing liquid to zebra fish
50(penicillin wastewater percent by volume %) (in Table 1 and table 2).
Table 1 karber's method gained penicillin wastewater is to zebra fish acute toxicity value
From table, utilize the penicillin wastewater of karber's method calculating to zebra fish 96h LC
50(penicillin wastewater percent by volume %) is respectively 13.46%.Along with the increase of open-assembly time, the LC of penicillin wastewater to zebra fish
50value reduces.Infer thus, along with the increase of open-assembly time, penicillin wastewater increases the toxicity of zebra fish.
Table 2 probit method gained penicillin wastewater is to zebra fish acute toxicity value
Note: y is zebra fish mortality ratio probit, and x is concentration group concentration logarithm.
From table, utilize the penicillin wastewater of probit method calculating to zebra fish 96h LC
50(penicillin wastewater percent by volume %) is respectively 13.66%.In testing liquid, penicillin wastewater concentration, open-assembly time and mortality ratio have good correlativity, and exposure concentrations is larger, and toxicity is larger; Open-assembly time is longer, and toxicity is larger.
The table 3 industrial waste water toxic grade criteria for classifying
Note: toxic unit characterizes with Tua, Tua=1/LC
50, regulation is when Tua<0.3, and waste discharge is to receiving the hydrobiont of water body without acute Toxicity Influence.
With reference to the American industry wastewater toxicity classification criteria for classifying (in Table 3), utilize industrial waste water 96h LC
50concentration range and acute toxicity unit (Tua) carry out divided rank, can find out that penicillin wastewater belongs to poisoning.
According to penicillin wastewater to zebra fish 96h LC
50(penicillin wastewater percent by volume %), carries out the sub-acute toxicity test of penicillin wastewater to zebra fish, and test period is 20 days, choose the zebra fish of healthy individual, male and female individuality half and half, is divided into 6 groups (comprising 5 exposure groups and 1 blank group) by it, at random specifically in Table 4.Each exposure group arranges 2 parallel group, and every group of 15 tail zebra fishs, change water every day, respectively at 4d, and 8d, 12d, 16d, 20d, measures that its musculature SOD is active, POD is active, MDA content.
The subacute toxicity test exposure group of table 4 penicillin wastewater to zebra fish
Testing liquid different time on the impact of zebra fish musculature POD, SOD activity and MDA content as shown in table 5～table 7.
The impact of table 5 testing liquid on zebra fish musculature POD activity
Note: * represents that there were significant differences; * indicates utmost point significant difference.
The impact of table 6 penicillin wastewater on zebra fish musculature SOD activity
Note: * represents that there were significant differences; * indicates utmost point significant difference.
The impact of table 7 penicillin wastewater on zebra fish musculature MDA content
Note: * represents that there were significant differences; * indicates utmost point significant difference.
Utilize significant t-test, the difference between check exposure group and blank group, as t < t0.05 (n), during P > 0.05, can think there was no significant difference; As t0.05 (n)≤t < t0.01 (n), during 0.01 < P < 0.05, can think significant difference; As t > t0.01 (n), during P < 0.01, can think that difference is extremely remarkable.
From table, zebra fish is exposed to testing liquid 20 days, in contrast exposure group and blank group zebra fish musculature, POD is active, SOD is active and MDA content, find all to occur significant difference (0.01 < P < 0.05) and utmost point significant difference (P < 0.01), according to the above results, show, penicillin wastewater has subacute toxicity to zebra fish.
Claims (2)
1. utilize zebra fish to test a method for typical antibiotic waste water toxicity, it is characterized in that,
The method adopts zebra fish In vivo assay Cells, utilize respectively short-term 96 hours and the long-term i.e. exposure chamber of 20 days to describe typical antibiotic waste water to the acute toxicity of zebra fish and subacute toxicity, judge whether the virose criterion of tool is typical antibiotic waste water to zebra fish: acute toxicity is with 96h LC
50describe; Subacute toxicity is described with one group of Antioxidant Indexes;
Utilize short-term exposure chamber, measure and obtain the acute toxicity of typical antibiotic waste water to zebra fish, determination of acute toxicity obtains 96h LC
50; With 96h LC
50acute toxicity is described;
Utilize long term exposure mode, subacute toxicity represents with one group of Antioxidant Indexes of fish body, then utilizes significant t-test, and the difference between check exposure group and blank group, as t < t
0.05 (n), during P > 0.05, think there was no significant difference; Work as t
0.05 (n)≤ t < t
0.01 (n), i.e. 0.01 < P≤0.05 o'clock, thinks significant difference; As t > t
0.01 (n), P≤0.01 o'clock, thinks that difference is extremely remarkable;
This method of testing is, if exposure group and blank group significant difference, while being 0.01<P<0.05 or P<0.01, think that typical antibiotic waste water has subacute bio-toxicity, if exposure group and blank group difference are not remarkable, think that typical antibiotic waste water is without subacute bio-toxicity.
2. the method for utilizing zebra fish to test typical antibiotic waste water toxicity as claimed in claim 1, is characterized in that, it comprises following concrete steps:
(1) setup test is biological, and selecting zebra fish is biological subject, male and female individuality half and half, zebra fish is after 5% saline solution sterilization, with the dechlorination tap water of aeration 48h, in laboratory, raise and train 7 days, in preliminary experiment the previous day and duration of test, refuse feeding, avoid bait to exert an influence to test result;
(2) setup test condition, testing liquid pH value is 7.0～8.5; Total hardness is with CaCO
3count 240～250mg/L, test temperature is 23～25 ℃; Dissolved oxygen DO is higher than 5mg/L; Adopt natural lighting; Test period is 96h or 20d; Test method is hydrostatic formula, and every 24h changes a test solution, at least measures water temperature, hardness, pH value, the dissolved oxygen content of 1 testing liquid every day;
(3) carry out preliminary experiment, in the glass aquarium of 50cm * 20cm * 25cm, hold 20L testing liquid, be divided into 5～6 exposure groups, exposure group concentration represents with the percent by volume containing typical antibiotic waste water in testing liquid.Three kinds of typical antibiotic waste water exposure groups arrange respectively as follows: penicillin wastewater exposure concentrations group is 5%, 10%, 15%, 20%, 25% and 30%; Terramycin wastewater exposure concentrations group is 0.1%, 0.2%, 0.3%, 0.4%, 0.5% and 0.6%; Streptomysin waste water exposure concentrations group is 1%, 3.2%, 10%, 32%, 100%; Do not establish parallel group, each exposure group is put 5 tail zebra fishs, tests continuously 96h, records the dead fish number in each container every day at least twice and takes out in time dead fish; Calculate preliminary experiment result: minimum 24h absolute lethal concentration and the highest 96h are without dead concentration;
Penicillin wastewater preliminary experiment result: minimum 24h absolute lethal concentration and the highest 96h are respectively 20% and 10% without dead concentration;
Terramycin wastewater preliminary experiment result: minimum 24h absolute lethal concentration and the highest 96h are respectively 0.1% and 0.5% without dead concentration;
Streptomysin waste water preliminary experiment result: minimum 24h absolute lethal concentration and the highest 96h are respectively 100% and 10% without dead concentration;
(4) acute toxic test method is according to the test result of described step (3), between 24h absolute lethal concentration and the highest 96h are without dead concentration by etc. logarithmic interval 5 exposure groups are set, exposure group concentration represents with the percent by volume containing typical antibiotic waste water in testing liquid.Three kinds of typical antibiotic waste water exposure groups arrange respectively as follows: penicillin wastewater exposure concentrations group is 10%, 12.57%, 15.81%, 19.88%, 24.99%; Terramycin wastewater exposure concentrations group is 0.1%, 0.15%, 0.22%, 0.33%, 0.5%; Streptomysin waste water exposure concentrations group is 17.78%, 23.71%, 31.62%, 42.17%, 56.23%; Each exposure group is established 2 parallel group, and blank group is set, amount to 11 groups, each exposure group is put into 8 tail zebra fishs at random, test period is 96h, test findings represents with LC50, every 24h, changes 1 testing liquid, adopts bandit's formula method and two kinds of methods of probit method to calculate 96h LC
50and 95% confidence limit;
Acute toxic test result is: penicillin wastewater 96h LC
50for with Kou Shifa, calculate 13.46% and with probit method, calculate 13.66%; Terramycin wastewater 96h LC
50for with Kou Shifa, calculate 0.22% and with probit method, calculate 0.19%; Streptomysin waste water 96h LC
50for with Kou Shifa, calculate 25.56% and with probit method, calculate 29.51%;
(5) subacute toxicity method of testing arranges subacute exposure group with reference to the test result of step (4), and exposure group concentration is at 1/6 96h LC
50with 1/2 96h LC
50between, 5 exposure groups are set, penicillin wastewater, terramycin and streptomysin exposure concentrations group are 1/256 96h LC
50, 1/64 96hLC
50, 1/16 96h LC
50, 1/4 96h LC
50, 1/2 96h LC
50, exposure group concentration is to represent containing the percent by volume of typical antibiotic waste water in testing liquid, and each exposure group arranges 2 parallel group, and 1 blank group amounts to 16 groups.Open-assembly time is 20 days, chooses healthy zebra fish, male and female individuality half and half, and each organizes 15 tail zebra fishs, changes water every day.5 exposure groups were got biological sample respectively at the 4th, 8,12,16 and 20 days and are analyzed, each each exposure group is got respectively 2 tail zebra fishs, mensuration POD is active, SOD is active and MDA content, test result is that exposure group is compared with blank group, POD is active, SOD is active and MDA content there were significant differences;
Described testing liquid is the mixed solution of typical antibiotic waste water and dechlorination tap water.
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| CN104833784A (en) * | 2015-05-19 | 2015-08-12 | 山东建筑大学 | Method for determining biotoxicity of water quality based on zebrafish movement velocity change |
| CN104849425A (en) * | 2015-05-19 | 2015-08-19 | 山东建筑大学 | Method for determining water quality biotoxicity |
| CN108106891A (en) * | 2017-11-15 | 2018-06-01 | 河海大学 | A kind of preparation method of colloid original solution for acting on pollutant bio-toxicity and its application |
| CN108120812A (en) * | 2017-12-14 | 2018-06-05 | 常州大学 | In a kind of dyeing waste water in groups biological toxicity tests and differentiate appraisal procedure |
| CN108196022A (en) * | 2018-01-29 | 2018-06-22 | 江南大学 | A kind of method for measuring disinfection by-products joint-biotoxicity in waste water after disinfection |
| CN108254522A (en) * | 2018-02-05 | 2018-07-06 | 环境保护部华南环境科学研究所 | A kind of method using Tang's fish monitoring water quality toxicity |
| CN108410948A (en) * | 2018-03-02 | 2018-08-17 | 南京大学 | A kind of toxicity tests method of evaluation advanced oxidation processes ecological security |
| CN110208483A (en) * | 2019-07-11 | 2019-09-06 | 上海海洋大学 | A kind of ocean shell-fish Ecology Toxicity test Methods |
| CN110275005A (en) * | 2019-07-11 | 2019-09-24 | 上海海洋大学 | A kind of marine fishes Ecology Toxicity test Methods |
| CN113035356A (en) * | 2021-03-03 | 2021-06-25 | 首都医科大学 | Evaluation method for damage of cardiovascular and nervous systems by atmospheric particulates |
| CN113884413A (en) * | 2021-08-25 | 2022-01-04 | 中国环境科学研究院 | Method for testing toxicity effect of gasoline vehicle exhaust particles on aquatic organisms |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103301479A (en) * | 2013-05-07 | 2013-09-18 | 南京工业大学 | Method for evaluating acute toxicity of drug by using model animal zebra fish fry |
| CN103392660A (en) * | 2013-07-24 | 2013-11-20 | 广东省微生物研究所 | Exposure device for fish embryos and larva fish for long-term chronic toxicity tests |
-
2013
- 2013-11-28 CN CN201310625401.1A patent/CN103616489A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103301479A (en) * | 2013-05-07 | 2013-09-18 | 南京工业大学 | Method for evaluating acute toxicity of drug by using model animal zebra fish fry |
| CN103392660A (en) * | 2013-07-24 | 2013-11-20 | 广东省微生物研究所 | Exposure device for fish embryos and larva fish for long-term chronic toxicity tests |
Non-Patent Citations (3)
| Title |
|---|
| 杨雷: "鱼类测试抗生素废水和菌渣生物毒性的试验研究", 《中国优秀硕士学位论文全文数据库》 * |
| 赵风云 等: "工业废水生物毒性的研究进展", 《工业水处理》 * |
| 高小辉 等: "水质的生物毒性检测方法", 《净水技术》 * |
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| CN104142384A (en) * | 2014-08-01 | 2014-11-12 | 山东省科学院生物研究所 | Method for screening active compounds capable of protecting or improving renal functions |
| CN104374883A (en) * | 2014-11-25 | 2015-02-25 | 安徽省环境科学研究院 | Application of zebra fish in detection of reproduction toxicity of BBP (Butyl Benzyl Phthalate) and method for rapidly detecting reproduction toxicity of BBP |
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| CN108410948B (en) * | 2018-03-02 | 2022-03-18 | 南京大学 | Biotoxicity experimental method for evaluating ecological safety of advanced oxidation process |
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| CN110208483A (en) * | 2019-07-11 | 2019-09-06 | 上海海洋大学 | A kind of ocean shell-fish Ecology Toxicity test Methods |
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| CN113035356A (en) * | 2021-03-03 | 2021-06-25 | 首都医科大学 | Evaluation method for damage of cardiovascular and nervous systems by atmospheric particulates |
| CN113035356B (en) * | 2021-03-03 | 2023-12-08 | 首都医科大学 | Method for evaluating damage of atmospheric particulates to cardiovascular and nervous system |
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