CN110988323A - Method for evaluating toxicity of total sediments by utilizing daphnia magna - Google Patents
Method for evaluating toxicity of total sediments by utilizing daphnia magna Download PDFInfo
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- 239000013049 sediment Substances 0.000 title claims abstract description 191
- 241001494246 Daphnia magna Species 0.000 title claims abstract description 102
- 231100000419 toxicity Toxicity 0.000 title claims abstract description 56
- 230000001988 toxicity Effects 0.000 title claims abstract description 56
- 238000000034 method Methods 0.000 title claims abstract description 37
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 78
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 37
- 239000000243 solution Substances 0.000 claims abstract description 27
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 26
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 26
- 239000000661 sodium alginate Substances 0.000 claims abstract description 26
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 26
- 238000011065 in-situ storage Methods 0.000 claims abstract description 25
- 238000010790 dilution Methods 0.000 claims abstract description 24
- 239000012895 dilution Substances 0.000 claims abstract description 24
- 239000001110 calcium chloride Substances 0.000 claims abstract description 22
- 229910001628 calcium chloride Inorganic materials 0.000 claims abstract description 22
- 238000002156 mixing Methods 0.000 claims abstract description 16
- 238000004132 cross linking Methods 0.000 claims abstract description 15
- 239000011259 mixed solution Substances 0.000 claims abstract description 12
- 238000005406 washing Methods 0.000 claims abstract description 12
- 238000007873 sieving Methods 0.000 claims abstract description 8
- 238000004108 freeze drying Methods 0.000 claims abstract description 7
- 238000012258 culturing Methods 0.000 claims abstract description 4
- 230000004071 biological effect Effects 0.000 claims abstract description 3
- 238000007865 diluting Methods 0.000 claims abstract description 3
- 238000000227 grinding Methods 0.000 claims abstract description 3
- 231100000518 lethal Toxicity 0.000 claims abstract description 3
- 230000001665 lethal effect Effects 0.000 claims abstract description 3
- 238000003756 stirring Methods 0.000 claims abstract description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 33
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 22
- 239000008367 deionised water Substances 0.000 claims description 17
- 229910021641 deionized water Inorganic materials 0.000 claims description 17
- 239000002904 solvent Substances 0.000 claims description 14
- 239000006228 supernatant Substances 0.000 claims description 14
- 239000002957 persistent organic pollutant Substances 0.000 claims description 10
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 9
- 238000005119 centrifugation Methods 0.000 claims description 9
- 229910001385 heavy metal Inorganic materials 0.000 claims description 9
- 229910052760 oxygen Inorganic materials 0.000 claims description 9
- 239000001301 oxygen Substances 0.000 claims description 9
- 238000002137 ultrasound extraction Methods 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 8
- 238000004140 cleaning Methods 0.000 claims description 7
- 238000001035 drying Methods 0.000 claims description 7
- 239000003517 fume Substances 0.000 claims description 7
- 238000005273 aeration Methods 0.000 claims description 6
- 239000011575 calcium Substances 0.000 claims description 6
- 239000003960 organic solvent Substances 0.000 claims description 6
- 231100000331 toxic Toxicity 0.000 claims description 5
- 230000010355 oscillation Effects 0.000 claims description 4
- 239000002689 soil Substances 0.000 claims description 4
- 238000005286 illumination Methods 0.000 claims description 3
- 231100000252 nontoxic Toxicity 0.000 claims description 3
- 230000003000 nontoxic effect Effects 0.000 claims description 3
- 230000002588 toxic effect Effects 0.000 claims description 3
- VTYYLEPIZMXCLO-UHFFFAOYSA-L calcium carbonate Substances [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 2
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 2
- 230000003203 everyday effect Effects 0.000 claims description 2
- 230000008774 maternal effect Effects 0.000 claims description 2
- 239000003973 paint Substances 0.000 claims description 2
- 230000008186 parthenogenesis Effects 0.000 claims description 2
- 231100000572 poisoning Toxicity 0.000 claims description 2
- 230000000607 poisoning effect Effects 0.000 claims description 2
- 239000000523 sample Substances 0.000 description 38
- 231100000820 toxicity test Toxicity 0.000 description 13
- 238000002474 experimental method Methods 0.000 description 6
- 230000002401 inhibitory effect Effects 0.000 description 6
- 239000002245 particle Substances 0.000 description 6
- 231100000673 dose–response relationship Toxicity 0.000 description 5
- 239000003344 environmental pollutant Substances 0.000 description 5
- 231100000719 pollutant Toxicity 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 239000011159 matrix material Substances 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 231100000403 acute toxicity Toxicity 0.000 description 3
- 230000007059 acute toxicity Effects 0.000 description 3
- 235000010410 calcium alginate Nutrition 0.000 description 3
- 239000000648 calcium alginate Substances 0.000 description 3
- 229960002681 calcium alginate Drugs 0.000 description 3
- OKHHGHGGPDJQHR-YMOPUZKJSA-L calcium;(2s,3s,4s,5s,6r)-6-[(2r,3s,4r,5s,6r)-2-carboxy-6-[(2r,3s,4r,5s,6r)-2-carboxylato-4,5,6-trihydroxyoxan-3-yl]oxy-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylate Chemical compound [Ca+2].O[C@@H]1[C@H](O)[C@H](O)O[C@@H](C([O-])=O)[C@H]1O[C@H]1[C@@H](O)[C@@H](O)[C@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@H](O2)C([O-])=O)O)[C@H](C(O)=O)O1 OKHHGHGGPDJQHR-YMOPUZKJSA-L 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 238000010998 test method Methods 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- 241000256137 Chironomus tentans Species 0.000 description 2
- 241000238578 Daphnia Species 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 231100000215 acute (single dose) toxicity testing Toxicity 0.000 description 2
- 238000011047 acute toxicity test Methods 0.000 description 2
- 239000012496 blank sample Substances 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 231100000614 poison Toxicity 0.000 description 2
- 125000005575 polycyclic aromatic hydrocarbon group Chemical group 0.000 description 2
- KMUONIBRACKNSN-UHFFFAOYSA-N potassium dichromate Chemical compound [K+].[K+].[O-][Cr](=O)(=O)O[Cr]([O-])(=O)=O KMUONIBRACKNSN-UHFFFAOYSA-N 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000003440 toxic substance Substances 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 240000009108 Chlorella vulgaris Species 0.000 description 1
- 235000007089 Chlorella vulgaris Nutrition 0.000 description 1
- 241001162206 Hyalella azteca Species 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 231100000132 chronic toxicity testing Toxicity 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 231100000584 environmental toxicity Toxicity 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229920005547 polycyclic aromatic hydrocarbon Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 231100000816 toxic dose Toxicity 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5082—Supracellular entities, e.g. tissue, organisms
- G01N33/5085—Supracellular entities, e.g. tissue, organisms of invertebrates
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Abstract
The invention provides a method for evaluating the toxicity of total sediments by utilizing daphnia magna, which comprises the following steps: 1) freeze-drying the sediment, grinding and sieving to obtain a sediment sample; 2) detoxifying the sediment sample to obtain an in-situ blank sediment sample, and diluting the corresponding sediment sample by using the in-situ blank sediment sample to obtain a plurality of groups of mixed samples with equal mass but different sediment sample concentrations; 3) respectively stirring and uniformly mixing the mixed samples with a sodium alginate solution to prepare a plurality of groups of standby mixed solutions; 4) adding multiple groups of standby mixed liquor into multiple groups of calcium chloride solutions at a constant speed respectively, crosslinking to form an immobilized total sediment, washing, transferring into standard dilution water containing daphnia magna, continuously culturing and periodically recording the death number of the daphnia magna; 5) and calculating the lethal middle concentration LC50 of the sediment according to the biological effect of the daphnia magna and the diluted proportion of the sediment, and judging the toxicity grade of the sediment. The method for determining the toxicity of the total sediments by utilizing the daphnia magna is simple and rapid and is sensitive to determination.
Description
Technical Field
The invention relates to a total sediment toxicity evaluation method by utilizing daphnia magna, belonging to the field of ecological safety evaluation.
Background
After the pollutants are discharged from the river channel, the pollutants are finally deposited in the sediments, and under proper conditions, the pollutants are released into the overlying water again, so that the sediments become a secondary pollution source in the water environment. Therefore, the Whole Sediment Toxicity test (WST) is of great importance in the ecological safety evaluation of river channels. The WST test generally uses a 10-day acute toxicity test of telepoda (Hyalella Azteca) or a 4-6 week chronic toxicity test of Chironomus tentans (Chironomus tentans) to determine sediment ecotoxicity, requiring a great deal of time and labor. Daphnia magna (Daphnia magna) is one of the most common model organisms in aquatic toxicity tests, and has the advantages of high growth speed, short breeding cycle and high sensitivity to toxic substances. However, the experiment of using daphnia magna directly for total sediment toxicity can hardly be realized, mainly because the suspended sediment particles can cause problems of breathing, feeding and movement for daphnia magna. Therefore, in order to enable a rapid diagnosis of sediment toxicity using daphnia magna, the elutriation water of the sediment is used as a medium to react the sediment toxicity. The preparation of the elutriation water comprises the steps of violently shaking a mixed liquid of water and sediment with a fixed ratio, and centrifuging (filtering) to obtain a supernatant, wherein the steps simulate the process of releasing toxic substances in the sediment into the supernatant water to the greatest extent possible, so that the elutriation water becomes a reliable and most widely-used sediment toxicity test medium. However, since the hydrophobic contaminants bound to the sediment particles (or carbon particles) are removed during the centrifugation/filtration process, a decrease in the toxic concentration in the elutriation water is caused, underestimating the actual risk of contaminating the sediment.
Disclosure of Invention
The technical problem is as follows: the invention aims to provide a method for evaluating the toxicity of total sediments of daphnia magna, which utilizes sodium alginate gel with mild preparation process, excellent mass transfer performance and no biotoxicity to fix sediment particles to obtain an immobilized total sediments, and the immobilized total sediments are applied to daphnia magna toxicity experiments, so that the daphnia magna is prevented from being damaged by sediment entering solution, pollutants in the sediments are efficiently released, and the accurate determination of the toxicity effect of the sediments is realized.
The technical scheme is as follows: the invention provides a method for evaluating the toxicity of total sediments by utilizing daphnia magna, which comprises the following steps:
1) freeze-drying the sediment, grinding and sieving to obtain a sediment sample;
2) detoxifying the sediment sample to obtain an in-situ blank sediment sample, and diluting the corresponding sediment sample by using the in-situ blank sediment sample to obtain a plurality of groups of mixed samples with equal mass but different sediment sample concentrations;
3) respectively stirring and uniformly mixing a plurality of groups of mixed samples with equal mass but different sediment sample concentrations with a sodium alginate solution to prepare a plurality of groups of standby mixed solutions;
4) adding multiple groups of standby mixed liquor into a calcium chloride solution at a constant speed respectively, crosslinking to form an immobilized total sediment, washing the immobilized sediment, transferring the washed immobilized sediment into standard dilution water containing daphnia magna, continuously culturing, and periodically recording the death number of daphnia magna;
5) and calculating the lethal middle concentration LC50 of the sediment according to the biological effect of the daphnia magna and the diluted proportion of the sediment, and judging the toxicity grade of the sediment.
Wherein:
the sediment in the step 1) is the sediment or soil of a river channel.
Detoxifying the sediment sample in the step 2), acquiring an in-situ blank sediment sample, wherein the sediment is collected from the same place, and the specific steps of detoxifying the sediment sample are as follows: adding the sediment sample into a calcium chloride solution, oscillating and centrifuging to remove supernatant, cleaning with deionized water (preferably 3 times) to remove soluble heavy metals and residual calcium chloride, performing ultrasonic extraction with acetone and dichloromethane (preferably 3 times), centrifuging, cleaning with deionized water (preferably 3 times) to remove organic pollutants and residual organic solvent, and drying with a fume hood to obtain an in-situ blank sediment sample; the mass-volume ratio of the sediment sample to the calcium chloride solution is 5-10 g:50mL, and the mass-volume ratio of the calcium chloride to the solvent water in the calcium chloride solution is 1-2 g:100 mL; the mass volume ratio of the sediment sample to acetone is 5-10 g:30mL, and the mass volume ratio of the sediment sample to dichloromethane is 5-10 g:30 mL; the step of removing the supernatant after oscillation and centrifugation is to oscillate for 4-8 hours under the condition that the oscillation speed is 150-300 rpm, and remove the supernatant after centrifugation under the condition of 7000-12000 rpm; and then, ultrasonically extracting by using acetone and dichloromethane respectively, centrifuging, and cleaning by using deionized water, wherein the ultrasonic extraction time is 10-30 min, and the centrifugation speed is 7000-12000 rpm.
The multiple groups of mixed samples with equal mass but different sediment sample concentrations mean that the total dry weight of sediment samples and in-situ blank sediment samples in the mixed samples is 0.5-1.5 g, and the mass ratios of the sediment samples in the mixed samples are 0%, 25%, 50%, 75% and 100% respectively;
the mass-volume ratio of sodium alginate to solvent water in the sodium alginate solution in the step 3) is 2-5 g:100mL, and a plurality of groups of mixed samples with equal mass but different sediment sample concentrations in the step 3) are respectively stirred and mixed with the sodium alginate solution uniformly, wherein the mass-volume ratio of the mixed samples to the sodium alginate solution is 1g: 5-10 mL.
Adding the standby mixed solution into a calcium chloride solution at a constant speed in the step 4), and in the process of forming the immobilized total sediment through crosslinking, the adding speed at the constant speed is 0.5-5 drops/second, the mass-to-volume ratio of calcium chloride to solvent water in the calcium chloride solution is 2-5 g:100mL, the crosslinking temperature in the process of forming the immobilized total sediment through crosslinking is 25 +/-2 ℃, the crosslinking time is 20-35 min, and the mixture is rinsed with water for 1-3 times;
the daphnia magna in the step 4) is a non-first-fetus healthy young daphnia magna which is derived from the same maternal line and bred for more than 3 generations in parthenogenesis and has the birth time less than 24 hours; and 4) during the continuous culture process, the daphnia magna is not fed.
The pH value of the standard dilution water in the step 4) is 7.8 +/-0.2 and is CaCO3The hardness is measured to be 250 +/-25 Mg/L, and the ratio of Ca to Mg is 3.8-4.2: 1; dissolved oxygen is more than or equal to 3.0mg/L, and aeration is carried out for more than 24 hours before use.
The continuous culture and the regular recording of the death number of the daphnia magna in the step 4) mean that the daphnia magna is continuously cultured for 24-96 hours, the death number of the daphnia magna is recorded every 24-48 hours, the water temperature is controlled to be 18-22 ℃ in the culture process, the illumination is carried out for 8-16 hours every day, and the dissolved oxygen is not lower than 3.0 mg/L.
Step 5), the judgment of the toxicity grade of the sediment is judged according to the following formula:
toxicity unit TU 1/LC50 formula 1
When the toxicity unit TU is less than 0.4, the grade is I, and the paint is nontoxic; when the toxicity unit is more than or equal to 0.4 and TU is less than 1, the grade is II, and the toxicity is slight; when the toxicity unit is more than or equal to 1 and TU is less than 10, the grade is III, poisoning; when the toxicity unit is more than or equal to 10 TU and less than 100, the grade is IV and the toxicity is high; when the toxic unit TU is more than or equal to 100 and grade V, the toxicity is severe.
Has the advantages that: compared with the prior art, the invention has the following advantages:
1. according to the invention, the sodium alginate gel is used for fixing sediment particles to obtain the immobilized total sediment, and the immobilized total sediment has good holding capacity and can prevent the sediment particles from falling off for the second time and entering the solution;
2. the preparation process of the immobilized total sediment is mild, the calcium alginate matrix has no biotoxicity and does not influence the growth of daphnia magna, and the pore diameter of the surface of the calcium alginate matrix is 5-200 nm, so that pollutants in the sediment can be efficiently released;
3. compared with the daphnia magna experimental method for washing water, the method for determining the toxicity of the total sediment by using the daphnia magna is more accurate and sensitive, and is quicker and simpler than the traditional total sediment toxicity test method.
Detailed Description
The invention is further described with reference to specific examples, but without thereby limiting the scope of protection of the invention.
Example 1
A method for evaluating the toxicity of total sediments by utilizing daphnia magna comprises the following steps:
1. freeze-drying the sediment of the river channel by using a freeze dryer, and sieving to obtain a sediment sample;
2. weighing 5g of sediment sample, adding 50mL of calcium chloride (1%, m/v) solution, oscillating at 150rpm for 4h, centrifuging at 7000rpm to remove supernatant, washing three times by using deionized water to remove soluble heavy metals and residual calcium chloride, then respectively adding 30mL of acetone and 30mL of dichloromethane, performing ultrasonic extraction for three times for 10min, centrifuging at 7000rpm, washing three times by using deionized water to remove organic pollutants and residual organic solvents, and drying by using a fume hood to obtain in-situ blank sediment;
3. mixing the sediment samples and the in-situ blank sediment to obtain a group of mixed samples with total dry weight of 0.5g and different sediment sample proportions, wherein the mass ratios of the sediment samples in each group are respectively 100%, 75%, 50%, 25% and 0%;
4. respectively mixing the diluted mixed sample with a sodium alginate solution (the mass volume ratio of the sodium alginate to the solvent water is 2g:100mL) according to the mass volume ratio of 1g:5mL, and uniformly mixing to prepare a standby mixed solution;
5. extruding the standby mixed solution into a calcium chloride solution (the mass volume ratio of calcium chloride to solvent water is 2g:100mL) by a 10mL injector at a constant speed of 5 drops/second respectively, forming a fixed deposit with the crosslinking duration of 25min at the temperature of 25 ℃, taking out the fixed deposit, and rinsing with water for 1-3 times;
6. the washed fixed sediment was mixed with 50mL of standard dilution water (pH 7.8. + -. 0.2, as CaCO)3The hardness is measured to be 250 +/-25 Mg/L, the Ca/Mg ratio is 4:1, the aeration is carried out for more than 24 hours, the dissolved oxygen is more than or equal to 3.0Mg/L), the mixture is transferred into different small beakers of 100mL, and each cup is added with 10 daphnia magna (the same parent offspring of the same age is cultured for three generations, and then the daphnia magna is born for 6-24 hours);
7. moving the small beaker into a biochemical incubator, setting the temperature at 20 +/-2 ℃, illuminating for 16h, observing and recording the death number of daphnia magna in 24h and 48h, and judging that the standard for inhibiting the daphnia magna is a light shaking beaker, and the daphnia magna is considered to be dead after not swimming within 15 s;
8. a dilution concentration-mortality curve (dose-response curve) is drawn by using origin (Ver.2018), and a logarithmic fit (four-parameter logistic fitting) or a linear fit (linear fitting) is selected according to the curve to calculate the sediment dilution ratio causing 50% death of daphnia magna and a 95% confidence limit SPSS (Ver.20).
Example 2
A method for evaluating the toxicity of total sediments by utilizing daphnia magna comprises the following steps:
1. taking soil, freeze-drying by using a freeze dryer, and sieving to obtain a sediment sample;
2. a10 g sample of the sediment was weighed, added with 50mL of calcium chloride (2%, m/v) solution, shaken at 300rpm for 8h, centrifuged at 12000rpm to remove the supernatant, and washed three times with deionized water to remove the soluble heavy metals and the remaining calcium chloride. Then respectively adding 30mL of acetone and 30mL of dichloromethane, performing ultrasonic extraction for three times within 30min, centrifuging at 12000rpm, then using deionized water, cleaning for three times to remove organic pollutants and residual organic solvent, and drying in a fume hood to obtain an in-situ blank deposit;
3. mixing the sediment sample and the in-situ blank sediment sample to obtain a group of mixed samples with total dry weight of 1.5g and different sediment sample proportions, wherein the sediment sample proportions in each group are respectively 100%, 75%, 50%, 25% and 0%;
4. mixing the mixed sample with a sodium alginate solution (the mass volume ratio of the sodium alginate to the solvent water is 5g:100mL) according to the mass volume ratio of 1g:7.5mL, and uniformly mixing to prepare a standby mixed solution;
5. respectively extruding the standby mixed solution into a calcium chloride solution (the mass volume ratio of calcium chloride to solvent water is 5g:100mL) by using a 10mL syringe at 0.5 drop/second, forming a fixed deposit by crosslinking for 35min at the temperature of 25 ℃, taking out the fixed deposit, and rinsing for 1-3 times by using water;
6. the washed fixed sediment was mixed with 50mL of standard dilution water (pH 7.8. + -. 0.2, as CaCO)3The hardness is measured to be 250 +/-25 Mg/L, the Ca/Mg ratio is 4:1, the aeration is carried out for more than 24 hours, the dissolved oxygen is more than or equal to 3.0Mg/L), the mixture is transferred to a small 100mL beaker, and 10 daphnia magna (the same parent offspring in the same age is cultured for three generations and then the daphnia magna which is young for 6-24 hours is born) are added into each beaker;
7. and (3) moving the small beaker into a biochemical incubator, setting the temperature at 20 +/-2 ℃, illuminating for 16h, and observing and recording the death number of the daphnia magna at 24h and 48 h. Judging that the standard for inhibiting the daphnia magna is a light shaking beaker, and judging that the daphnia magna does not move within 15s and dies;
8. a dilution concentration-mortality curve (dose-response curve) is drawn by using origin (Ver.2018), and a logarithmic fit (four-parameter logistic fitting) or a linear fit (linear fitting) is selected according to the curve to calculate the sediment dilution ratio causing 50% death of daphnia magna and a 95% confidence limit SPSS (Ver.20).
Example 3
A method for evaluating the toxicity of total sediments by utilizing daphnia magna comprises the following steps:
1. freeze-drying the sediment of the river channel by using a freeze dryer, and sieving to obtain a sediment sample;
2. weighing 5g of sediment sample, adding 50mL of calcium chloride (1%, m/v) solution, oscillating at 150rpm for 4h, centrifuging at 7000rpm to remove supernatant, washing three times by using deionized water to remove soluble heavy metals and residual calcium chloride, then respectively adding 30mL of acetone and 30mL of dichloromethane, performing ultrasonic extraction for three times for 10min, centrifuging at 7000rpm, washing three times by using deionized water to remove organic pollutants and residual organic solvents, and drying by using a fume hood to obtain in-situ blank sediment;
3. mixing the sediment samples and the corresponding in-situ blank sediment samples to obtain a group of mixed samples with total dry weight of 1g and different sediment sample proportions, wherein the proportion of the sediment samples in each group is respectively 100%, 75%, 50%, 25% and 0%;
4. mixing the diluted mixed sample with a sodium alginate solution (the mass volume ratio of the sodium alginate to the solvent water is 2g:100mL) according to the mass volume ratio of 1g:10mL, and uniformly mixing to prepare a standby mixed solution;
5. respectively squeezing the mixed solution into calcium chloride solution (the mass volume ratio of calcium chloride to solvent water is 2g:100mL) at 5 drops/s with a 10mL syringe, crosslinking at 25 deg.C for 25min to form fixed deposit, taking out the fixed deposit, rinsing with water for 1-3 times
6. The fixed deposits after the moistening are respectively mixed with50mL of Standard dilution Water (pH 7.8. + -. 0.2, as CaCO)3The hardness is measured to be 250 +/-25 Mg/L, the Ca/Mg ratio is 4:1, the aeration is carried out for more than 24 hours, the dissolved oxygen is more than or equal to 3.0Mg/L), the mixture is transferred to a small 100mL beaker, and 10 daphnia magna (the same parent offspring in the same age is cultured for three generations and then the daphnia magna which is young for 6-24 hours is born) are added into each beaker;
7. and (3) moving the small beaker into a biochemical incubator, setting the temperature at 20 +/-2 ℃, illuminating for 16h, and observing and recording the death number of the daphnia magna at 24h and 48 h. Judging that the standard for inhibiting the daphnia magna is a light shaking beaker, and judging that the daphnia magna does not move within 15s and dies;
8. a dilution concentration-mortality curve (dose-response curve) is drawn by using origin (Ver.2018), and a logarithmic fit (four-parameter logistic fitting) or a linear fit (linear fitting) is selected according to the curve to calculate the sediment dilution ratio causing 50% death of daphnia magna and a 95% confidence limit SPSS (Ver.20).
Example 4
A method for evaluating the toxicity of total sediments by utilizing daphnia magna comprises the following steps:
1. freeze-drying the sediment of the river channel by using a freeze dryer, and sieving to obtain a sediment sample;
2. a10 g sample of the sediment was weighed, added with 50mL of calcium chloride (2%, m/v) solution, shaken at 300rpm for 8h, centrifuged at 12000rpm to remove the supernatant, and washed three times with deionized water to remove the soluble heavy metals and the remaining calcium chloride. Then respectively adding 30mL of acetone and 30mL of dichloromethane, performing ultrasonic extraction for three times within 30min, centrifuging at 12000rpm, then using deionized water, cleaning for three times to remove organic pollutants and residual organic solvent, and drying in a fume hood to obtain an in-situ blank deposit;
3. mixing the sediment samples and the corresponding in-situ blank sediment samples to obtain a group of mixed samples with total dry weight of 1g and different sediment sample proportions, wherein the proportion of the sediment samples in each group is respectively 100%, 75%, 50%, 25% and 0%;
4. mixing the diluted mixed sample with a sodium alginate solution (the mass volume ratio of the sodium alginate to the solvent water is 5g:100mL) according to the mass volume ratio of 1g:10mL, and uniformly mixing to prepare a standby mixed solution;
5. respectively extruding the standby mixed solution into a calcium chloride solution (the mass volume ratio of calcium chloride to solvent water is 5g:100mL) by using a 10mL syringe at 0.5 drop/second, forming a fixed deposit by crosslinking for 35min at the temperature of 25 ℃, taking out the fixed deposit, and rinsing for 1-3 times by using water;
6. the washed fixed sediment was mixed with 50mL of standard dilution water (pH 7.8. + -. 0.2, as CaCO)3The hardness is measured to be 250 +/-25 Mg/L, the Ca/Mg ratio is 4:1, the aeration is carried out for more than 24 hours, the dissolved oxygen is more than or equal to 3.0Mg/L), the mixture is transferred to a small 100mL beaker, and 10 daphnia magna (the same parent offspring in the same age is cultured for three generations and then the daphnia magna which is young for 6-24 hours is born) are added into each beaker;
7. and (3) moving the small beaker into a biochemical incubator, setting the temperature at 20 +/-2 ℃, illuminating for 16h, and observing and recording the death number of the daphnia magna at 24h and 48 h. Judging that the standard for inhibiting the daphnia magna is a light shaking beaker, and judging that the daphnia magna does not move within 15s and dies;
8. a dilution concentration-mortality curve (dose-response curve) is drawn by using origin (Ver.2018), and a logarithmic fit (four-parameter logistic fitting) or a linear fit (linear fitting) is selected according to the curve to calculate the sediment dilution ratio causing 50% death of daphnia magna and a 95% confidence limit SPSS (Ver.20).
Example 5 comparison of the washing water toxicity test of daphnia magna and the toxicity test of daphnia magna total sediment of the present invention
1. Cultivation of daphnia magna
Daphnia magna (Daphnia magna) is from institute of aquatic organisms of Chinese academy of sciences, cultured in tap water aerated for more than 48h, and fed with fresh Chlorella vulgaris. And (3) taking offspring of the same parents at the same age, culturing for three generations, and then using young daphnia for 6-24 h after birth as daphnia magna for experiments. Experiments were performed using an illuminated incubator to maintain the same conditions: 20 +/-2 ℃, pH 7.0-8.0, 16h illumination and 8h darkness. The sensitivity was tested using potassium dichromate prior to the experiment.
2. Acquisition of sediment before and after river channel dredging and in-situ blank sample preparation
In 2017, in 10 months, river surface sediments are collected at four positions in a polluted river (Wu-Cheng-Zhou city, China). The deposits were named deposits a1, B1, C1 and D1. In 8 months in 2018, 4 sediments are collected at the same position in the later period of river dredging, and are named as sediments A2, B2, C2 and D2 respectively. All deposits used in the toxicity test were freeze-dried and sieved through a 100 mesh sieve.
3. Experiment of acute toxicity of large daphnia elutriation water
Preparation of Standard dilution Water: adding 11.76g of CaCl2·2H2Dissolving O in water to constant volume of 1L, and adding 4.93g MgSO4·7H2Dissolving O in water to constant volume of 1L, and adding 2.59g NaHCO3Dissolving in water to constant volume of 1L, dissolving KCl 0.25g in water to dilute to 1L, mixing the above solutions 25mL, adjusting pH to 7.8 + -0.2 with sodium hydroxide solution or hydrochloric acid solution, aerating for 24 hr before use, and dissolving oxygen no less than 3.0 mg/L.
50g of the freeze-dried sediment was mixed with 500mL of deionized water (1:10), and then the mixture was shaken (150rpm) in the absence of light at room temperature for 10 hours, and centrifuged (5000g) to take the supernatant. The elutriation water at each point position is provided with 5 concentration gradients, and the elutriation water is respectively diluted to 0 percent (all standard dilution water), 25 percent, 50 percent, 75 percent and 100 percent (all standard dilution water) by standard dilution water. 50mL of diluted elutriation water was added to a 100mL glass beaker, 10 daphnia magna were added to each beaker, three in parallel. And moving the beaker into a biochemical incubator, setting the temperature at 20 +/-2 ℃, illuminating for 16h, and observing and recording the death number of the daphnia magna at 24h and 48 h. And judging that the standard for inhibiting the daphnia magna is a light shaking beaker, and judging that the daphnia magna does not move within 15s and dies.
4. Acute toxicity test of daphnia magna total sediment
Deposit in situ blank control: weighing 10g of sediment sample (dry weight, after freezing and sieving), adding 50mL of calcium chloride (1% m/v), shaking at 200rpm for 4h, centrifuging to remove supernatant, washing with deionized water, centrifuging, and extracting for three times to remove soluble heavy metal and CaCL2Then respectively adding 30mL of acetone and 30mL of dichloromethane, ultrasonically extracting for three times for 15min each time, centrifuging at 10000rpm for 10min each time, removing organic pollutants, washing with deionized water for three times, and ventilating in a fume hoodAnd (5) drying.
Each sediment sample was diluted with its corresponding in situ blank sample, where 0%, 25%, 50%, 75% and 100% percent of the final dry weight of 1g sediment was prepared (0% is all in situ blank), 7.5mL of 3% (m/v) sodium alginate solution and 1.0g of diluted soil and sodium alginate solution were stirred well, and the mixed material was injected into 50mL of 4% (m/v) CaCl using a 10mL syringe2Solution, hardened for 35min, rinsed three times, the fixed sediment and 50mL of standard dilution water were transferred to 100mL small beakers, each with 10 daphnia magna, matrix control (CK1, with only 7.5mL of hardened sodium alginate): only 7.5ml of hardened sodium alginate and 10 daphnia magna are added into the beaker, and the three are parallel.
And (3) moving the beaker into a biochemical incubator, setting the temperature at 20 +/-2 ℃, illuminating for 16h and dark for 8h, and observing and recording the death number of the daphnia magna at 24h and 48 h. And judging that the standard for inhibiting the daphnia magna is a light shaking beaker, and judging that the daphnia magna does not move within 15s and dies.
5. Test results
TABLE 1 physicochemical Properties of the sediment before and after dredging
PAHs:16U.S.Environmental Protection Agency(U.S.EPA)polycyclicaromatic hydrocarbons
The CK1 group (only 7.5mL of hardened sodium alginate is added) does not die of daphnia magna, and the hardened calcium alginate matrix is proved to have no biotoxicity to daphnia magna. Aiming at the toxicity test of the same polluted sediment, when the proportion of the original sediment is less than 100%, the fatality rate detected by using the full sediment toxicity test method of daphnia magna is greater than that of the elutriation water, probably because the concentrations of polycyclic aromatic hydrocarbon and other organic pollutants in the sediment are higher, most of the substances have hydrophobicity, limited organic matters are released into water in the 10-hour elutriation process, and the part of the organic matters are further lost in the centrifugation process, so that the toxicity of the elutriation water is reduced.
6. Data processing
A dilution concentration-mortality curve (dose-response curve) is drawn by using origin (Ver.2018), and a logarithmic fit (four-parameter logistic fitting) or a linear fit (linear fitting) is selected according to the curve to calculate the sediment dilution ratio causing 50% death of daphnia magna and a 95% confidence limit SPSS (Ver.20).
TABLE 2 toxicity grading standards
TABLE 3 acute toxicity and grade of toxicity of 48h daphnia magna for A, B, C, D sediments before and after dredging measured using the fixed sediments method
Table 4 acute toxicity and toxicity rating of daphnia magna 48h for A, B, C, D sediments before and after dredging measured using the elutriation method
The heavy metal concentration of dredged sediment is obviously reduced (table 1), and the corresponding toxicity measured by using the elutriation water is non-toxic. Whereas the total deposit method shows that the deposit is still slightly toxic. Generally speaking, the elutriation water can truly reflect the toxicity of heavy metals and hydrophilic organic pollutants in the sediment, but the toxicity of the elutriation water to hydrophobic organic pollutants is lack of effective expression. The daphnia magna total sediment toxicity test method comprehensively reflects sediment toxicity through longer release time and better release capacity.
7. Conclusion
The total sediment toxicity test of the daphnia magna uses a smaller amount of sediment, a simpler mode is adopted, a more sensitive toxicity result than that of washing water is obtained, and meanwhile, the adopted in-situ blank sediment can reflect the toxicity effect on the daphnia magna when the sediment is not polluted, so that the toxicity result is more accurate and has environmental reality, and the method for the total sediment toxicity test of the daphnia magna is proved to have practical application value.
Claims (10)
1. A method for evaluating the toxicity of total sediments of daphnia magna is characterized by comprising the following steps:
1) freeze-drying the sediment, grinding and sieving to obtain a sediment sample;
2) detoxifying the sediment sample to obtain an in-situ blank sediment sample, and diluting the corresponding sediment sample by using the in-situ blank sediment sample to obtain a plurality of groups of mixed samples with equal mass but different sediment sample concentrations;
3) respectively stirring and uniformly mixing a plurality of groups of mixed samples with equal mass but different sediment sample concentrations with a sodium alginate solution to prepare a plurality of groups of standby mixed solutions;
4) adding multiple groups of standby mixed liquor into multiple groups of calcium chloride solutions at constant speed respectively, crosslinking to form an immobilized total sediment, washing the immobilized sediment, transferring the washed immobilized sediment into standard dilution water containing daphnia magna, continuously culturing, and periodically recording the death number of daphnia magna;
5) and calculating the lethal middle concentration LC50 of the sediment according to the biological effect of the daphnia magna and the diluted proportion of the sediment, and judging the toxicity grade of the sediment.
2. The method for evaluating the toxicity of total sediments by using daphnia magna according to claim 1, characterized in that: the sediment in the step 1) is the sediment or soil of a river channel.
3. The method for evaluating the toxicity of total sediments by using daphnia magna according to claim 1, characterized in that: detoxifying the sediment sample in the step 2), acquiring an in-situ blank sediment sample, wherein the sediment is collected from the same place, and the specific steps of detoxifying the sediment sample are as follows: adding the sediment sample into a calcium chloride solution, oscillating and centrifuging to remove supernatant, cleaning with deionized water to remove soluble heavy metals and residual calcium chloride, performing ultrasonic extraction with acetone and dichloromethane respectively, centrifuging, cleaning with deionized water to remove organic pollutants and residual organic solvents, and drying with a fume hood to obtain an in-situ blank sediment sample.
4. The method for evaluating the toxicity of the total sediments by using daphnia magna according to claim 3, characterized in that: the mass-volume ratio of the sediment sample to the calcium chloride solution is 5-10 g:50mL, wherein the mass-volume ratio of the calcium chloride to the solvent water in the calcium chloride solution is 1-2 g:100 mL; the mass volume ratio of the sediment sample to acetone is 5-10 g:30mL, and the mass volume ratio of the sediment sample to dichloromethane is 5-10 g:30 mL; the step of removing the supernatant after oscillation and centrifugation is to oscillate for 4-8 hours under the condition that the oscillation speed is 150-300 rpm, and remove the supernatant after centrifugation under the condition of 7000-12000 rpm; the method is characterized in that acetone and dichloromethane are respectively used for ultrasonic extraction, and the mixture is cleaned by deionized water after centrifugation, wherein the ultrasonic extraction time is 10-30 min, and the centrifugation speed is 7000-12000 rpm.
5. The method for evaluating the toxicity of total sediments by using daphnia magna according to claim 1, characterized in that: the multiple groups of mixed samples with equal mass but different concentrations of the sediment samples in the step 3) mean that the total dry weight of the sediment samples and the in-situ blank sediment samples in the mixed samples is 0.5-1.5 g, and the mass ratios of the sediment samples in the mixed samples are 0%, 25%, 50%, 75% and 100% respectively; the mass-volume ratio of sodium alginate to solvent water in the sodium alginate solution in the step 3) is 2-5 g:100mL, and a plurality of groups of mixed samples with equal mass but different sediment sample concentrations in the step 3) are respectively stirred and mixed with the sodium alginate solution uniformly, wherein the mass-volume ratio of the mixed samples to the sodium alginate solution is 1g: 5-10 mL.
6. The method for evaluating the toxicity of total sediments by using daphnia magna according to claim 1, characterized in that: adding multiple groups of standby mixed liquor into a calcium chloride solution at a constant speed in the step 4), and in the process of forming the immobilized total sediment through crosslinking, the adding speed at the constant speed is 0.5-5 drops/second, the mass-to-volume ratio of calcium chloride to solvent water in the calcium chloride solution is 2-5 g:100mL, the crosslinking temperature in the process of forming the immobilized total sediment through crosslinking is 25 +/-2 ℃, the crosslinking time is 20-35 min, and the mixture is rinsed with water for 1-3 times.
7. The method for evaluating the toxicity of total sediments by using daphnia magna according to claim 1, characterized in that: the daphnia magna in the step 4) is a non-first-fetus healthy young daphnia magna which is derived from the same maternal line and bred for more than 3 generations in parthenogenesis and has the birth time less than 24 hours; and 4) during the continuous culture process, the daphnia magna is not fed.
8. The method for evaluating the toxicity of total sediments by using daphnia magna according to claim 1, characterized in that: the pH value of the standard dilution water in the step 4) is 7.8 +/-0.2 and is CaCO3The hardness is measured to be 250 +/-25 Mg/L, and the ratio of Ca to Mg is 3.8-4.2: 1; dissolved oxygen is more than or equal to 3.0mg/L, and aeration is carried out for more than 24 hours before use.
9. The method for evaluating the toxicity of total sediments by using daphnia magna according to claim 1, characterized in that: the method is characterized in that: the continuous culture and the regular recording of the death number of the daphnia magna in the step 4) mean that the daphnia magna is continuously cultured for 24-96 hours, the death number of the daphnia magna is recorded every 24-48 hours, the water temperature is controlled to be 18-22 ℃ in the culture process, the illumination is carried out for 8-16 hours every day, and the dissolved oxygen is not lower than 3.0 mg/L.
10. The method for evaluating the toxicity of total sediments by using daphnia magna according to claim 1, characterized in that: step 5), the judgment of the toxicity grade of the sediment is judged according to the following formula:
toxicity unit TU 1/LC50 formula 1
When the toxicity unit TU is less than 0.4, the grade is I, and the paint is nontoxic; when the toxicity unit is more than or equal to 0.4 and TU is less than 1, the grade is II, and the toxicity is slight; when the toxicity unit is more than or equal to 1 and TU is less than 10, the grade is III, poisoning; when the toxicity unit is more than or equal to 10 TU and less than 100, the grade is IV and the toxicity is high; when the toxic unit TU is more than or equal to 100 and grade V, the toxicity is severe.
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