CN114674765B - Test method for toxicity effect of oxygen-containing polycyclic aromatic hydrocarbon substances on aquatic organisms - Google Patents
Test method for toxicity effect of oxygen-containing polycyclic aromatic hydrocarbon substances on aquatic organisms Download PDFInfo
- Publication number
- CN114674765B CN114674765B CN202210187246.9A CN202210187246A CN114674765B CN 114674765 B CN114674765 B CN 114674765B CN 202210187246 A CN202210187246 A CN 202210187246A CN 114674765 B CN114674765 B CN 114674765B
- Authority
- CN
- China
- Prior art keywords
- oxygen
- polycyclic aromatic
- daphnia magna
- concentration
- zebrafish
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 title claims abstract description 68
- 229910052760 oxygen Inorganic materials 0.000 title claims abstract description 68
- 239000001301 oxygen Substances 0.000 title claims abstract description 68
- 125000005575 polycyclic aromatic hydrocarbon group Chemical group 0.000 title claims abstract description 62
- 231100000331 toxic Toxicity 0.000 title claims abstract description 45
- 238000010998 test method Methods 0.000 title claims abstract description 42
- 239000000126 substance Substances 0.000 title abstract 2
- 241001494246 Daphnia magna Species 0.000 claims abstract description 72
- 241000252212 Danio rerio Species 0.000 claims abstract description 48
- 230000002588 toxic effect Effects 0.000 claims abstract description 44
- 238000000034 method Methods 0.000 claims abstract description 11
- 210000004027 cell Anatomy 0.000 claims description 73
- 230000000694 effects Effects 0.000 claims description 45
- CHADEQDQBURGHL-UHFFFAOYSA-N (6'-acetyloxy-3-oxospiro[2-benzofuran-1,9'-xanthene]-3'-yl) acetate Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(OC(C)=O)C=C1OC1=CC(OC(=O)C)=CC=C21 CHADEQDQBURGHL-UHFFFAOYSA-N 0.000 claims description 24
- 210000002257 embryonic structure Anatomy 0.000 claims description 24
- 241000196321 Tetraselmis Species 0.000 claims description 22
- 241000195493 Cryptophyta Species 0.000 claims description 21
- 238000002835 absorbance Methods 0.000 claims description 21
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 18
- 239000001963 growth medium Substances 0.000 claims description 18
- 241001621356 Lampriformes Species 0.000 claims description 17
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 claims description 16
- 238000002474 experimental method Methods 0.000 claims description 16
- 238000009395 breeding Methods 0.000 claims description 15
- 230000001488 breeding effect Effects 0.000 claims description 15
- 210000000170 cell membrane Anatomy 0.000 claims description 15
- 239000006228 supernatant Substances 0.000 claims description 15
- 230000035699 permeability Effects 0.000 claims description 13
- 241000238578 Daphnia Species 0.000 claims description 11
- 229930002875 chlorophyll Natural products 0.000 claims description 10
- 235000019804 chlorophyll Nutrition 0.000 claims description 10
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 10
- 230000012010 growth Effects 0.000 claims description 10
- 230000002354 daily effect Effects 0.000 claims description 9
- 239000000203 mixture Substances 0.000 claims description 9
- 206010030113 Oedema Diseases 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
- 241000251468 Actinopterygii Species 0.000 claims description 6
- 108090000371 Esterases Proteins 0.000 claims description 6
- 238000004820 blood count Methods 0.000 claims description 6
- 230000010261 cell growth Effects 0.000 claims description 6
- 229930002868 chlorophyll a Natural products 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 230000012447 hatching Effects 0.000 claims description 6
- 210000003128 head Anatomy 0.000 claims description 6
- 239000012535 impurity Substances 0.000 claims description 6
- 230000000366 juvenile effect Effects 0.000 claims description 6
- 210000001161 mammalian embryo Anatomy 0.000 claims description 6
- 238000005192 partition Methods 0.000 claims description 6
- 230000000243 photosynthetic effect Effects 0.000 claims description 6
- 239000000049 pigment Substances 0.000 claims description 6
- 230000001850 reproductive effect Effects 0.000 claims description 6
- 208000000875 Spinal Curvatures Diseases 0.000 claims description 4
- 230000004720 fertilization Effects 0.000 claims description 4
- 210000001325 yolk sac Anatomy 0.000 claims description 4
- 108010051152 Carboxylesterase Proteins 0.000 claims description 3
- 102000013392 Carboxylesterase Human genes 0.000 claims description 3
- 241000195628 Chlorophyta Species 0.000 claims description 3
- 241000223785 Paramecium Species 0.000 claims description 3
- 208000012641 Pigmentation disease Diseases 0.000 claims description 3
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 claims description 3
- 241001464837 Viridiplantae Species 0.000 claims description 3
- 230000003698 anagen phase Effects 0.000 claims description 3
- 230000021164 cell adhesion Effects 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
- 238000003776 cleavage reaction Methods 0.000 claims description 3
- 208000027744 congestion Diseases 0.000 claims description 3
- 238000011161 development Methods 0.000 claims description 3
- 230000018109 developmental process Effects 0.000 claims description 3
- 230000003203 everyday effect Effects 0.000 claims description 3
- 230000005284 excitation Effects 0.000 claims description 3
- 230000009036 growth inhibition Effects 0.000 claims description 3
- 230000007062 hydrolysis Effects 0.000 claims description 3
- 238000006460 hydrolysis reaction Methods 0.000 claims description 3
- 230000001418 larval effect Effects 0.000 claims description 3
- 238000012417 linear regression Methods 0.000 claims description 3
- 238000005259 measurement Methods 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 230000003287 optical effect Effects 0.000 claims description 3
- 230000001776 parthenogenetic effect Effects 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 230000029553 photosynthesis Effects 0.000 claims description 3
- 238000010672 photosynthesis Methods 0.000 claims description 3
- 230000019612 pigmentation Effects 0.000 claims description 3
- 230000035935 pregnancy Effects 0.000 claims description 3
- 230000007017 scission Effects 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 239000010902 straw Substances 0.000 claims description 3
- 239000000725 suspension Substances 0.000 claims description 3
- 108700024526 zebrafish sox32 Proteins 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims 1
- 231100000419 toxicity Toxicity 0.000 abstract description 7
- 230000001988 toxicity Effects 0.000 abstract description 7
- 238000001514 detection method Methods 0.000 abstract description 6
- 238000005516 engineering process Methods 0.000 abstract description 6
- 241000251539 Vertebrata <Metazoa> Species 0.000 abstract description 4
- 230000036541 health Effects 0.000 abstract description 4
- 230000001665 lethal effect Effects 0.000 abstract description 4
- 230000035945 sensitivity Effects 0.000 abstract description 4
- 241000124008 Mammalia Species 0.000 abstract description 3
- 241001062954 Clinopodium Species 0.000 abstract 1
- 230000035764 nutrition Effects 0.000 abstract 1
- 235000016709 nutrition Nutrition 0.000 abstract 1
- 238000012360 testing method Methods 0.000 description 13
- HUKPVYBUJRAUAG-UHFFFAOYSA-N 7-benzo[a]phenalenone Chemical compound C1=CC(C(=O)C=2C3=CC=CC=2)=C2C3=CC=CC2=C1 HUKPVYBUJRAUAG-UHFFFAOYSA-N 0.000 description 8
- 230000001228 trophic effect Effects 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 238000001061 Dunnett's test Methods 0.000 description 2
- 238000012313 Kruskal-Wallis test Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000000090 biomarker Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000004062 sedimentation Methods 0.000 description 2
- 230000002110 toxicologic effect Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 1
- 206010012559 Developmental delay Diseases 0.000 description 1
- 101001121408 Homo sapiens L-amino-acid oxidase Proteins 0.000 description 1
- 102100026388 L-amino-acid oxidase Human genes 0.000 description 1
- 206010074268 Reproductive toxicity Diseases 0.000 description 1
- 101100012902 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) FIG2 gene Proteins 0.000 description 1
- 206010042602 Supraventricular extrasystoles Diseases 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000002485 combustion reaction Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002957 persistent organic pollutant Substances 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000007696 reproductive toxicity Effects 0.000 description 1
- 231100000372 reproductive toxicity Toxicity 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 231100000048 toxicity data Toxicity 0.000 description 1
- 231100000759 toxicological effect Toxicity 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Molecular Biology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biomedical Technology (AREA)
- Engineering & Computer Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
技术领域Technical Field
本发明属于水生生物检测技术领域，特别是涉及一种含氧多环芳烃类物质对水生生物毒性效应的试验方法。The invention belongs to the technical field of aquatic organism detection, and particularly relates to a test method for the toxic effects of oxygen-containing polycyclic aromatic hydrocarbons on aquatic organisms.
背景技术Background Art
含氧多环芳烃，简称OPAHs，为主要来源于化石染料或生物质的不完全燃烧以及环境中母体多环芳烃的二次转化，近年来，国内外对OPAHs在环境中的污染水平和风险日趋关注。水体通常是持久性有机污染物的沉积地，而OPAHs相较于母体多环芳烃更高的极性和更低的蒸气压使OPAHs具有更高的流动性和沉降速度，这意味着OPAHs相较于母体多环芳烃可能更易进入水环境中，并对水环境稳态和水生生物造成持续的不利影响，因此水环境中OPAHs的污染应受到足够重视。但总体来说，目前对OPAHs的研究主要集中在OPAHs的健康毒性方向，对于PACs对水生生态系统的潜在危害知之甚少。水生生物的毒理学效应是评价污染物对生态系统暴露风险及体现生态环境质量的重要指标之一，但目前各研究中OPAHs对水生生物暴露毒性的测试方法、测试生物以及指标并不一致，毒性数据难以进行比较，并且仅以一种营养级生物为受试对象进行毒理学实验，可能会使我们低估OPAHs的毒性效应；仅以致死率为毒性指标会使我们忽视OPAHs的亚致死毒性效应。因此，研发提供一种科学的研究OPAHs对水生生物毒性效应的试验方法是本领域技术人员亟需解决的问题。Oxygenated polycyclic aromatic hydrocarbons, referred to as OPAHs, are mainly derived from the incomplete combustion of fossil dyes or biomass and the secondary transformation of parent PAHs in the environment. In recent years, both at home and abroad, there has been increasing concern about the pollution level and risks of OPAHs in the environment. Water bodies are usually the sedimentation sites of persistent organic pollutants, and the higher polarity and lower vapor pressure of OPAHs compared to parent PAHs give OPAHs higher fluidity and sedimentation rates, which means that OPAHs may be more likely to enter the water environment than parent PAHs, and cause continuous adverse effects on the homeostasis of the water environment and aquatic organisms. Therefore, the pollution of OPAHs in the water environment should be given sufficient attention. But in general, current research on OPAHs mainly focuses on the health toxicity of OPAHs, and little is known about the potential hazards of PACs to aquatic ecosystems. The toxicological effect of aquatic organisms is one of the important indicators for evaluating the exposure risk of pollutants to ecosystems and reflecting the quality of the ecological environment. However, the test methods, test organisms, and indicators of OPAHs exposure toxicity to aquatic organisms in various studies are not consistent, and toxicity data are difficult to compare. In addition, toxicological experiments using only one trophic level organism as the subject may lead us to underestimate the toxic effects of OPAHs; using only the lethality as a toxicity indicator may lead us to ignore the sublethal toxic effects of OPAHs. Therefore, developing a scientific test method for studying the toxic effects of OPAHs on aquatic organisms is an urgent problem that technicians in this field need to solve.
发明内容Summary of the invention
本发明提供了一种含氧多环芳烃类物质对水生生物毒性效应的试验方法，解决了以上问题。The present invention provides a test method for the toxic effects of oxygen-containing polycyclic aromatic hydrocarbons on aquatic organisms, which solves the above problems.
为解决上述技术问题，本发明是通过以下技术方案实现的：To solve the above technical problems, the present invention is achieved through the following technical solutions:
本发明的一种含氧多环芳烃类物质对水生生物毒性效应的试验方法，包括含氧多环芳烃对斜生四链藻的毒性效应的试验方法、含氧多环芳烃对大型溞的毒性效应的试验方法以及含氧多环芳烃对斑马鱼的毒性效应的试验方法；The present invention provides a test method for the toxic effects of oxygen-containing polycyclic aromatic hydrocarbons on aquatic organisms, including a test method for the toxic effects of oxygen-containing polycyclic aromatic hydrocarbons on Tetraselmis obliquus, a test method for the toxic effects of oxygen-containing polycyclic aromatic hydrocarbons on Daphnia magna, and a test method for the toxic effects of oxygen-containing polycyclic aromatic hydrocarbons on zebrafish;
其中，含氧多环芳烃对斜生四链藻的毒性效应的试验方法包括如下步骤：The test method for the toxic effects of oxygen-containing polycyclic aromatic hydrocarbons on Tetraselmis obliquus includes the following steps:
S1、前处理：在无菌条件下接种适量处于对数增长期的斜生四链藻藻液于锥形瓶中使得锥形瓶中最终藻液体积为50mL，并且初始藻细胞数约为(2–5)×103cells/mL，加入OPAHs，用透气膜封口，打乱顺序置于光照培养箱(14h光照：10h黑暗、23±2℃)中培养96h，每日摇动5–6次并更换位置以减少光照不均匀和细胞贴壁对细胞生长产生的影响；S1. Pretreatment: Under sterile conditions, inoculate an appropriate amount of Tetraselmis obliquus algae solution in the logarithmic growth phase into a conical flask so that the final algae solution volume in the conical flask is 50 mL, and the initial algae cell number is about (2-5)×10 3 cells/mL, add OPAHs, seal with a breathable membrane, and place in a light incubator (14 h light: 10 h dark, 23±2℃) in a random order for 96 h. Shake 5-6 times a day and change the position to reduce the effects of uneven light and cell adhesion on cell growth;
S2、含氧多环芳烃对斜生四链藻生长速率的影响：培养96h后，于650nm波长下，测定藻悬浮液的吸光度，通过细胞浓度-吸光度关系公式可计算出细胞浓度，与对照组进行比较，可计算出各浓度组藻细胞生长抑制率；S2. Effects of oxygen-containing polycyclic aromatic hydrocarbons on the growth rate of Tetraselmis obliquus: After 96 hours of cultivation, the absorbance of the algae suspension was measured at a wavelength of 650nm. The cell concentration was calculated by the cell concentration-absorbance relationship formula. By comparing with the control group, the growth inhibition rate of algae cells in each concentration group was calculated.
S3、含氧多环芳烃对斜生四链藻细胞膜通透性的影响：利用二乙酸荧光素FDA其本身无荧光，但可穿透细胞膜并被细胞中的非特异性酯酶分解产生荧光素的特性测定细胞膜通透性，荧光素是亲水性的，不容易迁移穿过健康的细胞膜，因此，所产生荧光素的荧光强度既反映了酯酶活性又反映了细胞膜的完整性；培养96h后，从光照培养箱中取出藻液，离心藻细胞并使其重新在培养液中悬浮以获得4×105cells/mL的最终细胞浓度，避光适应室温20℃；以丙酮为溶剂制备FDA溶液现配现用，取10mL藻液于小管中并添加10μL的FDA溶液，最终FDA的浓度为3×10-6M，混匀使FDA和藻细胞能充分接触，用荧光分光光度计监测前10min内荧光强度的变化情况；仪器参数如下：时间扫描，600s；激发波长Ex＝485nm；发射波长Em＝530nm；仪器每隔1s记录一次荧光强度，通过对荧光强度与时间进行线性回归拟合(R2≥0.950)，其斜率为FDA被酯酶分解为荧光素的平均速率，以此数据来代表藻细胞的通透性；在没有细胞的对照组中，荧光强度始终为0，未监测到FDA水解；所有浓度组均有三个平行，在4h的测量期内，藻类处于黑暗中，细胞生长量和细胞通透性变化量忽略不计；S3. Effect of oxygen-containing polycyclic aromatic hydrocarbons on cell membrane permeability of Tetraselmis obliquus: The cell membrane permeability was determined by using the characteristics of fluorescein diacetate (FDA), which has no fluorescence itself but can penetrate the cell membrane and be decomposed by nonspecific esterases in the cell to produce fluorescein. Fluorescent material is hydrophilic and does not easily migrate through healthy cell membranes. Therefore, the fluorescence intensity of the produced fluorescein reflects both the esterase activity and the integrity of the cell membrane. After 96 hours of culture, the algal solution was taken out from the light incubator, the algal cells were centrifuged and resuspended in the culture medium to obtain a final cell concentration of 4×10 5 cells/mL, and the cells were protected from light and adapted to room temperature of 20°C. The FDA solution was prepared using acetone as the solvent and used immediately. 10 mL of the algal solution was taken into a small tube and 10 μL of the FDA solution was added. The final FDA concentration was 3×10 -6 M. The mixture was mixed to allow FDA and algal cells to fully contact each other. The changes in fluorescence intensity within the first 10 minutes were monitored using a fluorescence spectrophotometer. The instrument parameters were as follows: time scan, 600 s; excitation wavelength Ex = 485 nm; emission wavelength Em =530nm; the instrument records the fluorescence intensity every 1s, and the linear regression fitting (R 2 ≥0.950) between the fluorescence intensity and time is performed, and the slope is the average rate at which FDA is decomposed into fluorescein by esterase, and this data is used to represent the permeability of algal cells; in the control group without cells, the fluorescence intensity is always 0, and no FDA hydrolysis is detected; all concentration groups have three parallels, and during the 4h measurement period, the algae are in the dark, and the cell growth and cell permeability changes are negligible;
S4、含氧多环芳烃对斜生四链藻光合色素的影响：光合色素密切参与捕获、转递光能等过程，是斜生四链藻进行光合作用的基础，叶绿素a和总叶绿素含量能够一定程度上反映出生物进行光合作用的能力；培养96h后，收集10mL斜生四链藻藻液于离心管中，在4000rpm下离心10min，移除上清液收集藻细胞，加入5mL90％(V/V)丙酮溶液，用封口膜封口并用锡纸遮光，充分混匀后在4℃的黑暗条件下提取24h，8000rpm离心10min清除颗粒获得上清液；以90％的丙酮溶液为参比，测量上清液在663nm、645nm和750nm处的吸光度，上清液在750nm处吸光度需小于0.05，以此来排除溶液内杂物对吸光度测定的干扰；利用以下公式计算叶绿素a和总叶绿素的含量：S4. Effects of oxygen-containing polycyclic aromatic hydrocarbons on photosynthetic pigments of Tetraselmus obliquus: Photosynthetic pigments are closely involved in the capture and transfer of light energy, and are the basis for photosynthesis of Tetraselmus obliquus. Chlorophyll a and total chlorophyll content can reflect the ability of organisms to photosynthesize to a certain extent. After 96 hours of cultivation, 10 mL of Tetraselmus obliquus algae solution was collected in a centrifuge tube and centrifuged at 4000 rpm for 10 minutes. The supernatant was removed to collect the algal cells, and 5 mL of 90% (V/V) acetone solution was added. , seal with sealing film and shield with tin foil, mix thoroughly and extract at 4℃ in the dark for 24h, centrifuge at 8000rpm for 10min to remove particles and obtain supernatant; use 90% acetone solution as reference, measure the absorbance of supernatant at 663nm, 645nm and 750nm, the absorbance of supernatant at 750nm should be less than 0.05, so as to eliminate the interference of impurities in the solution on absorbance determination; calculate the content of chlorophyll a and total chlorophyll using the following formula:
Ca＝(12.7A663nm-2.69A645nm)/ρC a =(12.7A 663nm -2.69A 645nm )/ρ
C＝(8.02A663nm+20.2A645nm)/ρC=(8.02A 663nm +20.2A 645nm )/ρ
式中，Ca、C——叶绿素a浓度和总叶绿素浓度，μg/cells；Where, Ca , C——chlorophyll a concentration and total chlorophyll concentration, μg/cells;
A663nm、A645nm——上清液在663nm和645nm处的吸光度；A 663nm , A 645nm ——absorbance of supernatant at 663nm and 645nm;
ρ——藻细胞浓度，cells/mL。ρ——algae cell concentration, cells/mL.
进一步地，所述含氧多环芳烃对大型溞的毒性效应的试验方法包括如下步骤：Furthermore, the test method for the toxic effects of oxygen-containing polycyclic aromatic hydrocarbons on Daphnia magna comprises the following steps:
P1、挑选出同一批健康的、非初次生殖且具体为15–20日龄的母溞，单独放置于烧杯中，实验前24h停止喂食，在适宜条件下母溞通过孤雌繁殖产下幼溞，收集6–24h产下的新生幼溞；每个浓度组设10个平行，每个平行中有1只幼溞及50mL培养液，每天投喂一次(1–5)×105cells/mL浓度的新鲜绿藻，每隔一天更新一次溶液，培育21d；P1. Select the same batch of healthy, non-primiparous, 15-20-day-old female Daphnia gracilis and place them separately in a beaker. Stop feeding 24 hours before the experiment. Under suitable conditions, the female Daphnia gracilis gives birth to juvenile Daphnia through parthenogenetic reproduction. Collect the newly born juvenile Daphnia gracilis 6-24 hours later. Set up 10 parallels for each concentration group. Each parallel contains 1 juvenile Daphnia gracilis and 50 mL of culture solution. Feed them with fresh green algae at a concentration of (1-5) × 10 5 cells/mL once a day. Renew the solution every other day and cultivate for 21 days.
P2、含氧多环芳烃对大型溞繁殖指标的影响：在21d的实验周期内，每天监测亲本大型溞怀胎、产胎的时间，记录新生幼溞的数量并将其移除，从而可以得到初次产胎时间、初次产溞数、21d产胎次数及产溞总数等繁殖指标数据，此外，利用上述指标的数据，根据公式利用逐步逼近法可计算出种群内禀增长率的精确值；P2. Effects of oxygen-containing polycyclic aromatic hydrocarbons on the reproductive indicators of Daphnia magna: During the 21-day experimental period, the time of pregnancy and birth of the parent Daphnia magna was monitored every day, and the number of newborn larvae was recorded and removed, so as to obtain the reproductive indicator data such as the time of first birth, the number of first births, the number of births in 21 days and the total number of births. In addition, using the data of the above indicators, the exact value of the population intrinsic growth rate can be calculated according to the formula using the stepwise approximation method;
式中，r——内禀增长率；In the formula, r is the intrinsic growth rate;
x——日龄，d；x——age in days, d;
n——21日龄；n——21 days old;
lx——x龄大型溞的存活率；l x ——survival rate of Daphnia magna at age x;
mx——x龄大型溞的繁殖率；m x ——reproduction rate of Daphnia magna at age x;
P3、含氧多环芳烃对大型溞发育指标的影响：在21d的实验周期内，每日监测大型溞是否蜕壳，统计21d内大型溞的蜕壳次数；将大型溞转移到血球计数板上并通过移除培养液使大型溞暂时固定，在光学显微镜下进行拍摄，使用Image J图像分析软件测量大型溞的体长，大型溞体长被定义为从头顶处即眼睛上方区域到尾棘处的长度；P3. Effects of oxygen-containing polycyclic aromatic hydrocarbons on the developmental indicators of Daphnia magna: During the 21-day experimental period, the molting of Daphnia magna was monitored daily, and the number of molting of Daphnia magna within 21 days was counted; Daphnia magna was transferred to a blood cell counting plate and temporarily fixed by removing the culture medium, photographed under an optical microscope, and the body length of Daphnia magna was measured using Image J image analysis software. The body length of Daphnia magna was defined as the length from the top of the head, i.e., the area above the eyes, to the caudal spines;
P4、含氧多环芳烃对大型溞摄食能力的影响：21d后，将大型溞转移到含有已知浓度的新鲜藻细胞的培养液中，并设置无大型溞的对照组，12h后移去大型溞，用紫外分光光度计测定培养液吸光度以确定剩余藻细胞浓度，摄食能力定义为平均每只大型溞在1h内的滤食率，根据下式计算；P4. Effects of oxygen-containing PAHs on the feeding capacity of Daphnia magna: After 21 days, Daphnia magna were transferred to a culture medium containing fresh algal cells of known concentration, and a control group without Daphnia magna was set up. Daphnia magna were removed after 12 hours, and the absorbance of the culture medium was measured with an ultraviolet spectrophotometer to determine the concentration of remaining algal cells. The feeding capacity was defined as the average filtration rate of each Daphnia magna within 1 hour, calculated according to the following formula;
式中，F——平均每只大型溞的滤食率，mL/h；Where, F is the average filtration rate of each Daphnia magna, mL/h;
V——培养液体积，mL；V——culture medium volume, mL;
C0、Ct——实验结束后，无大型溞的对照组的藻细胞浓度及各实验组的藻细胞浓度，cells/mL；C 0 , C t —— the algal cell concentration of the control group without Daphnia magna and the algal cell concentration of each experimental group after the experiment, cells/mL;
N——大型溞个数；N——number of Daphnia magna;
t——实验持续时间，h。t——Experiment duration, h.
进一步地，所述含氧多环芳烃对斑马鱼的毒性效应的试验方法包括如下步骤：Furthermore, the test method for the toxic effects of oxygen-containing polycyclic aromatic hydrocarbons on zebrafish comprises the following steps:
Q1、斑马鱼产卵受精及胚胎收集：在繁殖前夜，将雄鱼和雌鱼以2：1的比例放置在繁殖盒中并用隔板隔开，黑暗条件下放置；繁殖当日清晨，抽出隔板，通过增加光照强度、适当提高室内温度、在繁殖盒内放置假绿色植物等方法刺激产卵，繁殖盒分为内外两缸，内缸底部有孔隙，使得所产胚胎可自然下落至外缸底部，避免被亲鱼吞食；产卵后将内缸取出，用吸管吸出胚胎并用相同温度的水清洗胚胎两次，在显微镜下观察胚胎，剔除未受精胚胎和杂质，选择正常发育的卵裂期胚胎进行后续实验；Q1. Zebrafish spawning, fertilization and embryo collection: On the eve of breeding, male and female fish were placed in a breeding box at a ratio of 2:1 and separated by partitions in dark conditions; in the early morning of the breeding day, the partitions were removed, and spawning was stimulated by increasing the light intensity, appropriately raising the indoor temperature, and placing fake green plants in the breeding box. The breeding box was divided into two inner and outer tanks. There were holes at the bottom of the inner tank so that the embryos could naturally fall to the bottom of the outer tank to avoid being swallowed by the parent fish; after spawning, the inner tank was taken out, the embryos were sucked out with a straw and washed twice with water of the same temperature, the embryos were observed under a microscope, unfertilized embryos and impurities were removed, and normally developed cleavage-stage embryos were selected for subsequent experiments;
Q2、空白组和浓度组均包含3个平行，每个平行中有40个胚胎，将正常发育处于卵裂期(≤2hpf)的胚胎转移到预先加入了溶液的无菌24孔板中，每孔中有一个胚胎和2mL溶液，溶液由预先曝气24h的培养液配成；从5dpf开始，将孵化的幼鱼转移至6孔板中，每个孔包含1条幼鱼和10mL溶液，此时可开始喂食，每天用草履虫和市售极细饲料混合投喂一次；实验周期为15d，在此期间，每日更换溶液以维持暴露浓度的稳定和充足的溶解氧；Q2, the blank group and the concentration group all contained 3 parallels, each with 40 embryos. The embryos in the cleavage stage (≤2hpf) of normal development were transferred to a sterile 24-well plate pre-added with a solution, with one embryo and 2mL of solution in each well. The solution was prepared from a culture medium that had been aerated for 24 hours. Starting from 5dpf, the hatched fry were transferred to a 6-well plate, with one fry and 10mL of solution in each well. Feeding could be started at this time, with a mixture of paramecium and commercially available ultra-fine feed once a day. The experimental period was 15 days, during which the solution was replaced daily to maintain a stable exposure concentration and sufficient dissolved oxygen.
Q3、含氧多环芳烃对斑马鱼孵化能力的影响：72hpf时，在显微镜下，对每个个体进行观察，若斑马鱼仍未孵化且存活，则认为出现孵化延迟现象；Q3. Effects of oxygenated PAHs on the hatching ability of zebrafish: At 72 hpf, each individual was observed under a microscope, and if the zebrafish had not hatched and survived, it was considered that hatching delay occurred;
Q4、含氧多环芳烃对斑马鱼心率的影响：斑马鱼在胚胎阶段及早期幼鱼阶段身体透明，在显微镜下可以清晰地观察到斑马鱼身体内部心脏跳动，因此在48及72hpf时，利用显微镜测量斑马鱼在30s内的心跳次数；Q4. Effects of oxygen-containing PAHs on zebrafish heart rate: Zebrafish are transparent in the embryonic and early larval stages, and the heartbeat inside the zebrafish can be clearly observed under a microscope. Therefore, at 48 and 72 hpf, the number of zebrafish heartbeats within 30 seconds was measured using a microscope.
Q5、含氧多环芳烃对斑马鱼体长的影响：15d实验结束时，每个浓度随机抽取15条幼鱼，使用100mg/L MS222对斑马鱼幼鱼进行麻醉，将幼虫放在血球计数板上，调整其姿势使其身体呈一条直线，放于显微镜下进行拍照，使用Image J软件计算幼鱼的体长；Q5. Effect of oxygenated PAHs on zebrafish body length: At the end of the 15-day experiment, 15 larvae were randomly selected from each concentration and anesthetized with 100 mg/L MS222. The larvae were placed on a blood cell counting plate and their posture was adjusted so that their bodies were in a straight line. They were placed under a microscope and photographed, and the body length of the larvae was calculated using Image J software.
Q6、含氧多环芳烃对斑马鱼形态的影响：每日在显微镜下观察斑马鱼胚胎/幼鱼，若斑马鱼出现心包水肿、卵黄囊水肿、充血、色素沉着、脊柱弯曲、头尾畸形等情况，则认为斑马鱼发生畸形。Q6. Effects of oxygenated PAHs on zebrafish morphology: Zebrafish embryos/larvae were observed under a microscope daily. If the zebrafish showed pericardial edema, yolk sac edema, congestion, pigmentation, spinal curvature, head and tail deformities, the zebrafish were considered to be deformed.
本发明相对于现有技术包括有以下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
本发明选用斜生四链藻为代表的浮游植物、大型溞为代表的无脊椎浮游动物、斑马鱼为代表的脊椎动物，三种不同营养级生物为受试生物，有别与采用哺乳动物等检测的人体健康风险技术以及采用单一水生生物检测的生态风险技术，且本技术方案以多种不同的亚致死毒性效应取代致死效应作为判定含氧多环芳烃毒性的指标，具有敏感性更高、科学适用性好等显著优势。The present invention selects phytoplankton represented by Tetraselmis obliquus, invertebrate zooplankton represented by Daphnia magna, and vertebrates represented by zebrafish, three organisms of different trophic levels as the test organisms. This is different from the human health risk technology that uses mammals and the like for detection and the ecological risk technology that uses a single aquatic organism for detection. In addition, the present technical solution uses a variety of different sublethal toxic effects instead of lethal effects as indicators for determining the toxicity of oxygen-containing polycyclic aromatic hydrocarbons, and has significant advantages such as higher sensitivity and good scientific applicability.
当然，实施本发明的任一产品并不一定需要同时达到以上所述的所有优点。Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
为了更清楚地说明本发明实施例的技术方案，下面将对实施例描述所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.
图1为本发明含氧多环芳烃对斜生四链藻的毒性效应的试验方法中含氧多环芳烃中的苯并蒽酮对斜生四链藻细胞膜通透性的影响的柱状图；FIG1 is a bar graph showing the effect of benzanthrone in oxygen-containing polycyclic aromatic hydrocarbons on the cell membrane permeability of Tetraselmis obliquus in the test method for the toxic effect of oxygen-containing polycyclic aromatic hydrocarbons on Tetraselmis obliquus of the present invention;
图2为本发明含氧多环芳烃对斜生四链藻的毒性效应的试验方法中苯并蒽酮对斜生四链藻细胞叶绿素含量的影响的柱状图；FIG2 is a bar graph showing the effect of benzanthrone on the chlorophyll content of Tetraselmis obliquus cells in the test method for the toxic effects of oxygen-containing polycyclic aromatic hydrocarbons on Tetraselmis obliquus of the present invention;
图3为本发明含氧多环芳烃对大型溞的毒性效应的试验方法中苯并蒽酮对大型溞的繁殖毒性影响的柱状图；3 is a bar graph showing the effect of benzanthrone on the reproductive toxicity of Daphnia magna in the test method for the toxic effect of oxygen-containing polycyclic aromatic hydrocarbons on Daphnia magna of the present invention;
图4为本发明含氧多环芳烃对大型溞的毒性效应的试验方法中苯并蒽酮对大型溞内禀增长率的影响的柱状图；4 is a bar graph showing the effect of benzanthrone on the intrinsic growth rate of Daphnia magna in the test method for the toxic effect of oxygen-containing polycyclic aromatic hydrocarbons on Daphnia magna of the present invention;
图5本发明含氧多环芳烃对大型溞的毒性效应的试验方法中苯并蒽酮对大型溞蜕壳次数和体长的影响FIG5 Effects of benzanthrone on the number of molting times and body length of Daphnia magna in the test method for the toxic effects of oxygen-containing polycyclic aromatic hydrocarbons on Daphnia magna in the present invention
图6为本发明含氧多环芳烃对大型溞的毒性效应的试验方法中苯并蒽酮对大型溞摄食能力的影响的柱状图；6 is a bar graph showing the effect of benzanthrone on the feeding ability of Daphnia magna in the test method for the toxic effect of oxygen-containing polycyclic aromatic hydrocarbons on Daphnia magna of the present invention;
图7为本发明含氧多环芳烃对斑马鱼的毒性效应的试验方法中苯并蒽酮对斑马鱼心率的影响的柱状图；FIG7 is a bar graph showing the effect of benzanthrone on the heart rate of zebrafish in the test method for the toxic effect of oxygen-containing polycyclic aromatic hydrocarbons on zebrafish of the present invention;
图8为本发明含氧多环芳烃对斑马鱼的毒性效应的试验方法中苯并蒽酮对斑马鱼形态的影响的柱状图。8 is a bar graph showing the effect of benzanthrone on zebrafish morphology in the test method for the toxic effects of oxygen-containing polycyclic aromatic hydrocarbons on zebrafish of the present invention.
具体实施方式DETAILED DESCRIPTION
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其它实施例，都属于本发明保护的范围。The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
本发明的一种含氧多环芳烃类物质对水生生物毒性效应的试验方法，包括含氧多环芳烃对斜生四链藻的毒性效应的试验方法、含氧多环芳烃对大型溞的毒性效应的试验方法以及含氧多环芳烃对斑马鱼的毒性效应的试验方法；本发明试验的个数据均表示为平均值±标准差。利用OriginPro 8软件对生物各项指标和含氧多环芳烃即OPAHs浓度作柱状图分析，并对各浓度组和对照组指标进行显著性分析(GraphPad Prism 9)，使用Shapiro-Wilk方法检验数据的正态性并使用Bartlett方法检验方差的同质性，根据检验结果，选择合适的检验方法(如Dunnett检验、Kruskal-Wallis检验)来检验是否有显著性差异，p≤0.05则认为数据之间具有显著性差异。含氧多环芳烃本具体实施例是以并蒽酮(BEZO)为例；The present invention discloses a test method for the toxic effects of oxygen-containing polycyclic aromatic hydrocarbons on aquatic organisms, including a test method for the toxic effects of oxygen-containing polycyclic aromatic hydrocarbons on Tetraselmis obliquus, a test method for the toxic effects of oxygen-containing polycyclic aromatic hydrocarbons on Daphnia magna, and a test method for the toxic effects of oxygen-containing polycyclic aromatic hydrocarbons on zebrafish; the individual data of the test of the present invention are all expressed as mean ± standard deviation. OriginPro 8 software is used to make a bar graph analysis of various biological indicators and oxygen-containing polycyclic aromatic hydrocarbons, i.e., OPAHs concentrations, and a significance analysis is performed on the indicators of each concentration group and the control group (GraphPad Prism 9). The Shapiro-Wilk method is used to test the normality of the data, and the Bartlett method is used to test the homogeneity of the variance. According to the test results, a suitable test method (such as Dunnett test, Kruskal-Wallis test) is selected to test whether there is a significant difference. If p≤0.05, it is considered that there is a significant difference between the data. The specific embodiment of oxygen-containing polycyclic aromatic hydrocarbons takes benzathrone (BEZO) as an example;
其中，如图1-2所示，含氧多环芳烃对斜生四链藻的毒性效应的试验方法包括如下步骤：As shown in Figure 1-2, the test method for the toxic effects of oxygen-containing polycyclic aromatic hydrocarbons on Tetraselmis obliquus includes the following steps:
S1、前处理：在无菌条件下接种适量处于对数增长期的斜生四链藻藻液于锥形瓶中使得锥形瓶中最终藻液体积为50mL，并且初始藻细胞数约为(2–5)×103cells/mL，加入OPAHs，用透气膜封口，打乱顺序置于光照培养箱(14h光照：10h黑暗、23±2℃)中培养96h，每日摇动5–6次并更换位置以减少光照不均匀和细胞贴壁对细胞生长产生的影响；S1. Pretreatment: Under sterile conditions, inoculate an appropriate amount of Tetraselmis obliquus algae solution in the logarithmic growth phase into a conical flask so that the final algae solution volume in the conical flask is 50 mL, and the initial algae cell number is about (2-5)×10 3 cells/mL, add OPAHs, seal with a breathable membrane, and place in a light incubator (14 h light: 10 h dark, 23±2℃) in a random order for 96 h. Shake 5-6 times a day and change the position to reduce the effects of uneven light and cell adhesion on cell growth;
S2、含氧多环芳烃对斜生四链藻生长速率的影响：培养96h后，于650nm波长下，测定藻悬浮液的吸光度，通过细胞浓度-吸光度关系公式可计算出细胞浓度，与对照组进行比较，可计算出各浓度组藻细胞生长抑制率；S2. Effects of oxygen-containing polycyclic aromatic hydrocarbons on the growth rate of Tetraselmis obliquus: After 96 hours of cultivation, the absorbance of the algae suspension was measured at a wavelength of 650nm. The cell concentration was calculated by the cell concentration-absorbance relationship formula. By comparing with the control group, the growth inhibition rate of algae cells in each concentration group was calculated.
S3、含氧多环芳烃对斜生四链藻细胞膜通透性的影响：利用二乙酸荧光素FDA其本身无荧光，但可穿透细胞膜并被细胞中的非特异性酯酶分解产生荧光素的特性测定细胞膜通透性，荧光素是亲水性的，不容易迁移穿过健康的细胞膜，因此，所产生荧光素的荧光强度既反映了酯酶活性又反映了细胞膜的完整性；培养96h后，从光照培养箱中取出藻液，离心藻细胞并使其重新在培养液中悬浮以获得4×105cells/mL的最终细胞浓度，避光适应室温20℃；以丙酮为溶剂制备FDA溶液现配现用，取10mL藻液于小管中并添加10μL的FDA溶液，最终FDA的浓度为3×10-6M，混匀使FDA和藻细胞能充分接触，用荧光分光光度计监测前10min内荧光强度的变化情况；仪器参数如下：时间扫描，600s；激发波长Ex＝485nm；发射波长Em＝530nm；仪器每隔1s记录一次荧光强度，通过对荧光强度与时间进行线性回归拟合(R2≥0.950)，其斜率为FDA被酯酶分解为荧光素的平均速率，以此数据来代表藻细胞的通透性；在没有细胞的对照组中，荧光强度始终为0，未监测到FDA水解；所有浓度组均有三个平行，在4h的测量期内，藻类处于黑暗中，细胞生长量和细胞通透性变化量忽略不计；S3. Effect of oxygen-containing polycyclic aromatic hydrocarbons on cell membrane permeability of Tetraselmis obliquus: The cell membrane permeability was determined by using the characteristics of fluorescein diacetate (FDA), which has no fluorescence itself but can penetrate the cell membrane and be decomposed by nonspecific esterases in the cell to produce fluorescein. Fluorescent material is hydrophilic and does not easily migrate through healthy cell membranes. Therefore, the fluorescence intensity of the produced fluorescein reflects both the esterase activity and the integrity of the cell membrane. After 96 hours of culture, the algal solution was taken out from the light incubator, the algal cells were centrifuged and resuspended in the culture medium to obtain a final cell concentration of 4×10 5 cells/mL, and the cells were protected from light and adapted to room temperature of 20°C. The FDA solution was prepared using acetone as the solvent and used immediately. 10 mL of the algal solution was taken into a small tube and 10 μL of the FDA solution was added. The final FDA concentration was 3×10 -6 M. The mixture was mixed to allow FDA and algal cells to fully contact each other. The changes in fluorescence intensity within the first 10 minutes were monitored using a fluorescence spectrophotometer. The instrument parameters were as follows: time scan, 600 s; excitation wavelength Ex = 485 nm; emission wavelength Em =530nm; the instrument records the fluorescence intensity every 1s, and the linear regression fitting (R 2 ≥0.950) between the fluorescence intensity and time is performed, and the slope is the average rate at which FDA is decomposed into fluorescein by esterase, and this data is used to represent the permeability of algal cells; in the control group without cells, the fluorescence intensity is always 0, and no FDA hydrolysis is detected; all concentration groups have three parallels, and during the 4h measurement period, the algae are in the dark, and the cell growth and cell permeability changes are negligible;
S4、含氧多环芳烃对斜生四链藻光合色素的影响：光合色素密切参与捕获、转递光能等过程，是斜生四链藻进行光合作用的基础，叶绿素a和总叶绿素含量能够一定程度上反映出生物进行光合作用的能力；培养96h后，收集10mL斜生四链藻藻液于离心管中，在4000rpm下离心10min，移除上清液收集藻细胞，加入5mL90％(V/V)丙酮溶液，用封口膜封口并用锡纸遮光，充分混匀后在4℃的黑暗条件下提取24h，8000rpm离心10min清除颗粒获得上清液；以90％的丙酮溶液为参比，测量上清液在663nm、645nm和750nm处的吸光度，上清液在750nm处吸光度需小于0.05，以此来排除溶液内杂物对吸光度测定的干扰；利用以下公式计算叶绿素a和总叶绿素的含量：S4. Effects of oxygen-containing polycyclic aromatic hydrocarbons on photosynthetic pigments of Tetraselmus obliquus: Photosynthetic pigments are closely involved in the capture and transfer of light energy, and are the basis for photosynthesis of Tetraselmus obliquus. Chlorophyll a and total chlorophyll content can reflect the ability of organisms to photosynthesize to a certain extent. After 96 hours of cultivation, 10 mL of Tetraselmus obliquus algae solution was collected in a centrifuge tube and centrifuged at 4000 rpm for 10 minutes. The supernatant was removed to collect the algal cells, and 5 mL of 90% (V/V) acetone solution was added. , seal with sealing film and shield with tin foil, mix thoroughly and extract at 4℃ in the dark for 24h, centrifuge at 8000rpm for 10min to remove particles and obtain supernatant; use 90% acetone solution as reference, measure the absorbance of supernatant at 663nm, 645nm and 750nm, the absorbance of supernatant at 750nm should be less than 0.05, so as to eliminate the interference of impurities in the solution on absorbance determination; calculate the content of chlorophyll a and total chlorophyll using the following formula:
Ca＝(12.7A663nm-2.69A645nm)/ρC a =(12.7A 663nm -2.69A 645nm )/ρ
C＝(8.02A663nm+20.2A645nm)/ρC=(8.02A 663nm +20.2A 645nm )/ρ
式中，Ca、C——叶绿素a浓度和总叶绿素浓度，μg/cells；Where, Ca , C——chlorophyll a concentration and total chlorophyll concentration, μg/cells;
A663nm、A645nm——上清液在663nm和645nm处的吸光度；A 663nm , A 645nm ——absorbance of supernatant at 663nm and 645nm;
ρ——藻细胞浓度，cells/mL。ρ——algae cell concentration, cells/mL.
如图3-6所示，其中，含氧多环芳烃对大型涵的毒性效应的试验方法包括如下步骤：As shown in Figure 3-6, the test method for the toxic effects of oxygen-containing polycyclic aromatic hydrocarbons on large culverts includes the following steps:
P1、挑选出同一批健康的、非初次生殖且具体为15-20日龄的母涵，单独放置于烧杯中，实验前24h停止喂食，在适宜条件下母涵通过孤雌繁殖产下幼涵，收集6-24h产下的新生幼涵；每个浓度组设10个平行，每个平行中有1只幼溞及50mL培养液，每天投喂一次(1–5)×105cells/mL浓度的新鲜绿藻，每隔一天更新一次溶液，培育21d；P1. Select healthy, non-primiparous, 15-20-day-old mother culms from the same batch and place them separately in a beaker. Stop feeding 24 hours before the experiment. Under suitable conditions, the mother culms give birth to young culms through parthenogenetic reproduction. Collect the newborn young culms born 6-24 hours later. Set up 10 parallels for each concentration group. Each parallel contains 1 young Daphnia gracilis and 50 mL of culture solution. Feed them with fresh green algae at a concentration of (1-5) × 10 5 cells/mL once a day. Renew the solution every other day and cultivate for 21 days.
P2、含氧多环芳烃对大型溞繁殖指标的影响：在21d的实验周期内，每天监测亲本大型溞怀胎、产胎的时间，记录新生幼溞的数量并将其移除，从而可以得到初次产胎时间、初次产溞数、21d产胎次数及产溞总数等繁殖指标数据，此外，利用上述指标的数据，根据公式利用逐步逼近法可计算出种群内禀增长率的精确值；P2. Effects of oxygen-containing polycyclic aromatic hydrocarbons on the reproductive indicators of Daphnia magna: During the 21-day experimental period, the time of pregnancy and birth of the parent Daphnia magna was monitored every day, and the number of newborn larvae was recorded and removed, so as to obtain the reproductive indicator data such as the time of first birth, the number of first births, the number of births in 21 days and the total number of births. In addition, using the data of the above indicators, the exact value of the population intrinsic growth rate can be calculated according to the formula using the stepwise approximation method;
式中，r——内禀增长率；In the formula, r is the intrinsic growth rate;
x——日龄，d；x——age in days, d;
n——21日龄；n——21 days old;
lx——x龄大型溞的存活率；l x ——survival rate of Daphnia magna at age x;
mx——x龄大型溞的繁殖率；m x ——reproduction rate of Daphnia magna at age x;
P3、含氧多环芳烃对大型溞发育指标的影响：在21d的实验周期内，每日监测大型溞是否蜕壳，统计21d内大型溞的蜕壳次数；将大型溞转移到血球计数板上并通过移除培养液使大型溞暂时固定，在光学显微镜下进行拍摄，使用Image J图像分析软件测量大型溞的体长，大型溞体长被定义为从头顶处即眼睛上方区域到尾棘处的长度；P3. Effects of oxygen-containing polycyclic aromatic hydrocarbons on the developmental indicators of Daphnia magna: During the 21-day experimental period, the molting of Daphnia magna was monitored daily, and the number of molting of Daphnia magna within 21 days was counted; Daphnia magna was transferred to a blood cell counting plate and temporarily fixed by removing the culture medium, photographed under an optical microscope, and the body length of Daphnia magna was measured using Image J image analysis software. The body length of Daphnia magna was defined as the length from the top of the head, i.e., the area above the eyes, to the caudal spines;
P4、含氧多环芳烃对大型溞摄食能力的影响：21d后，将大型溞转移到含有已知浓度的新鲜藻细胞的培养液中，并设置无大型溞的对照组，12h后移去大型溞，用紫外分光光度计测定培养液吸光度以确定剩余藻细胞浓度，摄食能力定义为平均每只大型溞在1h内的滤食率，根据下式计算；P4. Effects of oxygen-containing PAHs on the feeding capacity of Daphnia magna: After 21 days, Daphnia magna were transferred to a culture medium containing fresh algal cells of known concentration, and a control group without Daphnia magna was set up. Daphnia magna were removed after 12 hours, and the absorbance of the culture medium was measured with an ultraviolet spectrophotometer to determine the concentration of remaining algal cells. The feeding capacity was defined as the average filtration rate of each Daphnia magna within 1 hour, calculated according to the following formula;
式中，F——平均每只大型溞的滤食率，mL/h；Where, F is the average filtration rate of each Daphnia magna, mL/h;
V——培养液体积，mL；V——culture medium volume, mL;
C0、Ct——实验结束后，无大型溞的对照组的藻细胞浓度及各实验组的藻细胞浓度，cells/mL；C 0 , C t —— the algal cell concentration of the control group without Daphnia magna and the algal cell concentration of each experimental group after the experiment, cells/mL;
N——大型溞个数；N——number of Daphnia magna;
t——实验持续时间，h。t——Experiment duration, h.
如图7-8所示，其中，含氧多环芳烃对斑马鱼的毒性效应的试验方法包括如下步骤：As shown in Figures 7-8, the test method for the toxic effects of oxygenated polycyclic aromatic hydrocarbons on zebrafish includes the following steps:
Q1、斑马鱼产卵受精及胚胎收集：在繁殖前夜，将雄鱼和雌鱼以2：1的比例放置在繁殖盒中并用隔板隔开，黑暗条件下放置；繁殖当日清晨，抽出隔板，通过增加光照强度、适当提高室内温度、在繁殖盒内放置假绿色植物等方法刺激产卵，繁殖盒分为内外两缸，内缸底部有孔隙，使得所产胚胎可自然下落至外缸底部，避免被亲鱼吞食；产卵后将内缸取出，用吸管吸出胚胎并用相同温度的水清洗胚胎两次，在显微镜下观察胚胎，剔除未受精胚胎和杂质，选择正常发育的卵裂期胚胎进行后续实验；Q1. Zebrafish spawning, fertilization and embryo collection: On the eve of breeding, male and female fish were placed in a breeding box at a ratio of 2:1 and separated by partitions in dark conditions; in the early morning of the breeding day, the partitions were removed, and spawning was stimulated by increasing the light intensity, appropriately raising the indoor temperature, and placing fake green plants in the breeding box. The breeding box was divided into two inner and outer tanks. There were holes at the bottom of the inner tank so that the embryos could naturally fall to the bottom of the outer tank to avoid being swallowed by the parent fish; after spawning, the inner tank was taken out, the embryos were sucked out with a straw and washed twice with water of the same temperature, the embryos were observed under a microscope, unfertilized embryos and impurities were removed, and normally developed cleavage-stage embryos were selected for subsequent experiments;
Q2、空白组和浓度组均包含3个平行，每个平行中有40个胚胎，将正常发育处于卵裂期(≤2hpf)的胚胎转移到预先加入了溶液的无菌24孔板中，每孔中有一个胚胎和2mL溶液，溶液由预先曝气24h的培养液配成；从5dpf开始，将孵化的幼鱼转移至6孔板中，每个孔包含1条幼鱼和10mL溶液，此时可开始喂食，每天用草履虫和市售极细饲料混合投喂一次；实验周期为15d，在此期间，每日更换溶液以维持暴露浓度的稳定和充足的溶解氧；Q2, the blank group and the concentration group all contained 3 parallels, each with 40 embryos. The embryos in the cleavage stage (≤2hpf) of normal development were transferred to a sterile 24-well plate pre-added with a solution, with one embryo and 2mL of solution in each well. The solution was prepared from a culture medium that had been aerated for 24 hours. Starting from 5dpf, the hatched fry were transferred to a 6-well plate, with one fry and 10mL of solution in each well. Feeding could be started at this time, with a mixture of paramecium and commercially available ultra-fine feed once a day. The experimental period was 15 days, during which the solution was replaced daily to maintain a stable exposure concentration and sufficient dissolved oxygen.
Q3、含氧多环芳烃对斑马鱼孵化能力的影响：72hpf时，在显微镜下，对每个个体进行观察，若斑马鱼仍未孵化且存活，则认为出现孵化延迟现象；Q3. Effects of oxygenated PAHs on the hatching ability of zebrafish: At 72 hpf, each individual was observed under a microscope, and if the zebrafish had not hatched and survived, it was considered that hatching delay occurred;
Q4、含氧多环芳烃对斑马鱼心率的影响：斑马鱼在胚胎阶段及早期幼鱼阶段身体透明，在显微镜下可以清晰地观察到斑马鱼身体内部心脏跳动，因此在48及72hpf时，利用显微镜测量斑马鱼在30s内的心跳次数；Q4. Effects of oxygen-containing PAHs on zebrafish heart rate: Zebrafish are transparent in the embryonic and early larval stages, and the heartbeat inside the zebrafish can be clearly observed under a microscope. Therefore, at 48 and 72 hpf, the number of zebrafish heartbeats within 30 seconds was measured using a microscope.
Q5、含氧多环芳烃对斑马鱼体长的影响：15d实验结束时，每个浓度随机抽取15条幼鱼，使用100mg/L MS222对斑马鱼幼鱼进行麻醉，将幼虫放在血球计数板上，调整其姿势使其身体呈一条直线，放于显微镜下进行拍照，使用Image J软件计算幼鱼的体长；Q5. Effect of oxygenated PAHs on zebrafish body length: At the end of the 15-day experiment, 15 larvae were randomly selected from each concentration and anesthetized with 100 mg/L MS222. The larvae were placed on a blood cell counting plate and their posture was adjusted so that their bodies were in a straight line. They were placed under a microscope and photographed, and the body length of the larvae was calculated using Image J software.
Q6、含氧多环芳烃对斑马鱼形态的影响：每日在显微镜下观察斑马鱼胚胎/幼鱼，若斑马鱼出现心包水肿、卵黄囊水肿、充血、色素沉着、脊柱弯曲、头尾畸形等情况，则认为斑马鱼发生畸形。Q6. Effects of oxygenated PAHs on zebrafish morphology: Zebrafish embryos/larvae were observed under a microscope daily. If the zebrafish showed pericardial edema, yolk sac edema, congestion, pigmentation, spinal curvature, head and tail deformities, the zebrafish were considered to be deformed.
其中，图8中，箭头指示明显畸形，包括心包/卵黄囊水肿(*)、脊柱弯曲/尾部畸形/发育迟缓(黑色箭头)、充血( )；Among them, in Figure 8 , arrows indicate obvious deformities, including pericardial/yolk sac edema (*), spinal curvature/tail deformity/developmental delay (black arrow), and congestion ( );
所有数据均表示为平均值±标准差。利用OriginPro 8对生物各项指标和含氧多环芳烃浓度作柱状图分析，并对各浓度组和对照组指标进行显著性分析(GraphPad Prism9)，使用Shapiro-Wilk方法检验数据的正态性并使用Bartlett方法检验方差的同质性，根据检验结果，选择合适的检验方法(如Dunnett检验、Kruskal-Wallis检验)来检验是否有显著性差异，p≤0.05则认为数据之间具有显著性差异。All data are expressed as mean ± standard deviation. OriginPro 8 was used to make a bar graph analysis of the biological indicators and oxygen-containing polycyclic aromatic hydrocarbons concentrations, and the indicators of each concentration group and the control group were analyzed for significance (GraphPad Prism9). The Shapiro-Wilk method was used to test the normality of the data and the Bartlett method was used to test the homogeneity of variance. According to the test results, appropriate test methods (such as Dunnett test, Kruskal-Wallis test) were selected to test whether there were significant differences. p≤0.05 was considered to be significantly different between the data.
本发明选用斜生四链藻(浮游植物)、大型溞(无脊椎浮游动物)、斑马鱼(脊椎动物)三种不同营养级且敏感性高的水生生物作为受试对象。The present invention selects three aquatic organisms of different trophic levels and high sensitivity, namely, Tetraselmis obliquus (phytoplankton), Daphnia magna (invertebrate zooplankton), and zebrafish (vertebrate) as test subjects.
(2)本发明以多种不同的亚致死毒性效应取代致死效应作为判定含氧多环芳烃即OPAHs毒性的指标。(2) The present invention uses a variety of different sublethal toxic effects instead of lethal effects as indicators for determining the toxicity of oxygen-containing polycyclic aromatic hydrocarbons, namely OPAHs.
有益效果：Beneficial effects:
本发明选用斜生四链藻为代表的浮游植物、大型溞为代表的无脊椎浮游动物、斑马鱼为代表的脊椎动物，三种不同营养级生物为受试生物，有别与采用哺乳动物等检测的人体健康风险技术以及采用单一水生生物检测的生态风险技术，且本技术方案以多种不同的亚致死毒性效应取代致死效应作为判定含氧多环芳烃毒性的指标，具有敏感性更高、科学适用性好等显著优势。The present invention selects phytoplankton represented by Tetraselmis obliquus, invertebrate zooplankton represented by Daphnia magna, and vertebrates represented by zebrafish, three organisms of different trophic levels as the test organisms. This is different from the human health risk technology that uses mammals and the like for detection and the ecological risk technology that uses a single aquatic organism for detection. In addition, the present technical solution uses a variety of different sublethal toxic effects instead of lethal effects as indicators for determining the toxicity of oxygen-containing polycyclic aromatic hydrocarbons, and has significant advantages such as higher sensitivity and good scientific applicability.
以上公开的本发明优选实施例只是用于帮助阐述本发明。优选实施例并没有详尽叙述所有的细节，也不限制该发明仅为所述的具体实施方式。显然，根据本说明书的内容，可作很多的修改和变化。本说明书选取并具体描述这些实施例，是为了更好地解释本发明的原理和实际应用，从而使所属技术领域技术人员能很好地理解和利用本发明。本发明仅受权利要求书及其全部范围和等效物的限制。The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.
Claims (3)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210187246.9A CN114674765B (en) | 2022-02-28 | 2022-02-28 | Test method for toxicity effect of oxygen-containing polycyclic aromatic hydrocarbon substances on aquatic organisms |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210187246.9A CN114674765B (en) | 2022-02-28 | 2022-02-28 | Test method for toxicity effect of oxygen-containing polycyclic aromatic hydrocarbon substances on aquatic organisms |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114674765A CN114674765A (en) | 2022-06-28 |
| CN114674765B true CN114674765B (en) | 2024-10-15 |
Family
ID=82071953
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210187246.9A Active CN114674765B (en) | 2022-02-28 | 2022-02-28 | Test method for toxicity effect of oxygen-containing polycyclic aromatic hydrocarbon substances on aquatic organisms |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114674765B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103728284A (en) * | 2013-09-11 | 2014-04-16 | 中国科学院安徽光学精密机械研究所 | Water comprehensive toxicity rapid detection method based on algae chlorophyll fluorescene |
| CN110724724A (en) * | 2019-10-08 | 2020-01-24 | 南京信息工程大学 | A method for calculating half-effect concentration of ecotoxicity effect of environmental pollutants |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2377013B (en) * | 2001-06-06 | 2003-06-11 | Univ Surrey | Toxicity measurement |
-
2022
- 2022-02-28 CN CN202210187246.9A patent/CN114674765B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103728284A (en) * | 2013-09-11 | 2014-04-16 | 中国科学院安徽光学精密机械研究所 | Water comprehensive toxicity rapid detection method based on algae chlorophyll fluorescene |
| CN110724724A (en) * | 2019-10-08 | 2020-01-24 | 南京信息工程大学 | A method for calculating half-effect concentration of ecotoxicity effect of environmental pollutants |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114674765A (en) | 2022-06-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Gavaia et al. | Osteological development and abnormalities of the vertebral column and caudal skeleton in larval and juvenile stages of hatchery-reared Senegal sole (Solea senegalensis) | |
| Gavaia et al. | Comparing skeletal development of wild and hatchery‐reared Senegalese sole (Solea senegalensis, Kaup 1858): evaluation in larval and postlarval stages | |
| Mdegela et al. | Effect of pond management on prevalence of intestinal parasites in Nile Tilapia (Oreochromis niloticus) under small scale fish farming systems in Morogoro, Tanzania | |
| Small et al. | The roles of oxygen and ammonia in the symbiotic relationship between the spotted salamander Ambystoma maculatum and the green alga Oophila amblystomatis during embryonic development | |
| Shearer et al. | VIII. On the experimental hybridization of echinoids | |
| Englund et al. | Valve movement of Anodonta anatina and Unio tumidus (Bivalvia, Unionidae) in a eutrophic lake | |
| CN109601487B (en) | Drosophila melanogaster tumor model and application thereof | |
| Gumm et al. | Asexuals looking for sex: conflict between species and mate-quality recognition in sailfin mollies (Poecilia latipinna) | |
| CN114674765B (en) | Test method for toxicity effect of oxygen-containing polycyclic aromatic hydrocarbon substances on aquatic organisms | |
| CN115290615B (en) | High-flux method for detecting microplastic content in marine organism | |
| CN118303347A (en) | Method for testing toxicity of polyhalocarbazole as environmental pollutant by using zebra fish | |
| Sharp et al. | Effects of the duration of exposure to mercuric chloride on the embryogenesis of the estuarine teleost, Fundulus heteroclitus | |
| Herdman et al. | Oysters and Disease: An Account of Certain Observations Upon the Normal and Pathological Histology and Bacteriology of the Oyster and Other Shellfish | |
| Murakami et al. | Environmental DNA emission by two carangid fishes in single and mixed-species tanks | |
| CN115624512A (en) | Extraction method and application of centella asiatica with efficacy of removing red | |
| Zhu et al. | Paternal bisphenol A exposure alters craniofacial cartilage development in rare minnow (Gobiocypris rarus) descendants | |
| Dufour et al. | Prenatal reduction of E14. 5 embryonically fate‐mapped pyramidal neurons in a mouse model of autism | |
| CN106069940A (en) | A kind of cave coral indoor sexual reproduction method | |
| CN111713435A (en) | A developmental neurotoxicity assessment method for wastewater | |
| CN116064827A (en) | Kit and screening method for screening method of inflammation-relieving efficacy of daily-use chemicals for infants and children | |
| Luker et al. | Laboratory exercises in Zoology | |
| Paine et al. | Studies in bacteriosis. III. A bacterial leaf-spot disease of Protea cynaroides, exhibiting a host reaction of possibly bacteriolytic nature | |
| Harcotă et al. | Culture Techniques for Acartia clausi from the Romanian Black Sea coastal waters | |
| CN112540168B (en) | Detection method for combined toxicity of perfluorooctanoic acid and atrazine | |
| Swain et al. | Ontogeny of skeletal ossification in Labeo calbasu (Hamilton, 1822) using differential fluorescence staining technique |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |