Culture medium for metal chronic toxicity experiment and application thereof

Abstract

The invention belongs to the technical field of zooplankton culture, and particularly relates to a culture medium for a metal chronic toxicity experiment and application of the culture medium. The culture medium provided by the invention does not contain EDTA (Ethylene Diamine Tetraacetic Acid) or other chelating agents, can meet the requirements of normal reproduction and growth of model organisms, is suitable for long-term culture of the model organisms, is beneficial to reducing the influence of components of the culture medium on evaluation and determination results of the bioavailability and toxicity of metals, improves the accuracy and reliability of the evaluation results, and has a wide application prospect. Therefore, the chronic toxicity or bioavailability of the rare earth elements can be more accurately determined, the long-term ecological risk of the rare earth elements can be accurately explored, and a new reference approach is provided for metal environment standard formulation and scientific environment decision making.

Description

Technical Field

[0001] The invention belongs to the technical field of zooplankton culture, and more specifically, relates to a culture medium for metal chronic toxicity experiments and application thereof. Background Art

[0002] Daphnia magna has become a key model organism in ecotoxicology research due to its relatively short life cycle, high reproductive rate, parthenogenesis, easy acquisition of monoclonal individuals with consistent genotypes, and high sensitivity to environmental changes. It is widely used in aquatic ecosystems and metal toxicity assessment. In particular, the chronic toxicity test of Daphnia magna is regarded as an important tool to explore the biological effects of long-term exposure to metals in water and its potential impact on the ecological environment, which is achieved by monitoring biological indicators such as its survival rate, growth and reproductive capacity.

[0003] However, the current mainstream Daphnia magna chronic toxicity test culture media, such as Elendt M4 and Elendt M7, contain ethylenediaminetetraacetic acid (EDTA), which poses a challenge in toxicity studies involving rare earth elements. EDTA has the ability to form complexes with rare earth elements, which not only affects the bioavailability of rare earth elements, but may also interfere with their toxicity performance, thereby weakening the accuracy and reliability of experimental results.

[0004] In addition, some researchers choose to use aerated filtered tap water as experimental water, but this approach has two major flaws: one is the uncertainty of the filtered water composition, and the other is the difference in the composition of tap water in different regions. Both of these pose a threat to the repeatability and accuracy of the experiment.

[0005] In response to the above problems, Chinese patent application CN111357692A proposed an indoor breeding and cultivation method for Daphnia brevis, which cultivates wild female Daphnia brevis through a specific breeding culture medium, and then switches to a stable culture medium to continue culturing young Daphnia to ensure the stability of the individual state. The stable culture medium is composed of NaHCO3 (240 mg / L), CaSO4·2H2O (150 mg / L), MgSO4 (150 mg / L), and KCl (10 mg / L), and the pH value is maintained between 6.3 and 7.8. Despite this, the patent still relies on traditional culture media containing EDTA during the breeding stage, and fails to completely avoid the interference problem caused by EDTA.

[0006] Therefore, a new culture medium for chronic toxicity experiments of Daphnia magna is urgently needed to solve the above problems. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide a culture medium to overcome the defects and shortcomings of the existing Daphnia magna chronic toxicity test culture medium which usually contains chelating agent components such as EDTA, which interferes with the accuracy and reliability of the toxicity and bioavailability experiments of rare earth elements.

[0008] The object of the present invention is to provide application of the culture medium.

[0009] Another object of the present invention is to provide a method for predicting the toxicity of rare earth elements.

[0010] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0011] The composition formula of the culture medium includes: metal ions, Se 4+ 、HCO3 - 、SO4 2- , Cl - and vitamins; HCO3 - 、SO4 2- , Cl - and vitamins;

[0012] Wherein, the metal ion is Na + , Ca 2+ Mg 2+ , K + ;

[0013] The Na + The concentration is 40~70mg·L -1 ; the Ca 2+ The concentration is 18~40mg·L -1 ; The Mg 2+ The concentration is 18~35mg·L -1 ; The K + The concentration is 2~8mg·L -1 ; The Se 4+ The concentration is 1-5 μg·L -1 ; The HCO3 - The concentration is 110~160mg·L -1 ; The SO4 2- The concentration is 120~200mg·L -1 ; the Cl - The concentration is 2~8mg·L -1 ; The concentration of the vitamin is 50 to 100 μg·L -1 ; The culture medium does not contain a chelating agent; The pH of the culture medium is 5.8-8.

[0014] Existing toxicity test culture media usually contain chelating agents such as EDTA, which will interfere with the accuracy and reliability of toxicity and bioavailability experiments of rare earth elements. The culture medium provided by the present invention does not contain EDTA or other chelating agents, and can also meet the normal reproduction and growth requirements of model organisms. It is suitable for long-term cultivation of model organisms, which is conducive to reducing the impact of the culture medium's own components on the evaluation and determination results of metal bioavailability and toxicity, and improving the accuracy and reliability of the evaluation results, thereby more accurately determining the chronic toxicity or bioavailability of rare earth elements, which is conducive to more accurately exploring the long-term ecological risks of rare earth elements, and providing a new reference for the formulation of metal environmental standards and scientific environmental decision-making.

[0015] Preferably, the composition formula of the culture medium meets one or more of the following conditions:

[0016] (1) The Na + The concentration is 45~62mg·L -1 ; and / or

[0017] (2) Ca 2+ The concentration is 20~35mg·L -1 ; and / or

[0018] (3) Mg 2+ The concentration is 18~30mg·L -1 ; and / or

[0019] (4) K + The concentration is 2.6~6.5mg·L -1 ; and / or

[0020] (5) Se 4+ The concentration is 1-3 μg·L -1 ; and / or

[0021] (6) HCO3 - The concentration is 120~150mg·L -1 ; and / or

[0022] (7) SO4 2- The concentration is 140~180mg·L -1 ; and / or

[0023] (8) Cl - The concentration is 2.5~6mg·L -1 ; and / or

[0024] (9) The concentration of the vitamin is 60 to 85 μg / L -1 .

[0025] More preferably, the composition formula of the culture medium satisfies one or more of the following conditions:

[0026] (1) The Na + The concentration is 48~56mg·L -1 ; and / or

[0027] (2) Ca 2+ The concentration is 24-32 mg·L -1 ; and / or

[0028] (3) Mg 2+ The concentration is 22-28 mg·L -1 ; and / or

[0029] (4) K + The concentration is 3.5~5.2mg·L -1 ; and / or

[0030] (5) Se 4+ The concentration is 1.5~2.5μg·L -1 ; and / or

[0031] (6) HCO3 - The concentration is 130~145mg·L -1 ; and / or

[0032] (7) SO4 2- The concentration is 150~175mg·L -1 ; and / or

[0033] (8) Cl - The concentration is 2.8~4.5mg·L -1 ; and / or

[0034] (9) The concentration of the vitamin is 65 to 80 μg / L -1 .

[0035] Furthermore, the vitamins include B vitamins and / or vitamin H.

[0036] Preferably, the mass ratio of the B vitamins to vitamin H is (60-190):1, more preferably (70-140):1, and even more preferably (80-120):1.

[0037] Furthermore, the vitamin B group includes vitamin B1 and / or vitamin B 12 .

[0038] Preferably, the vitamin B1 and vitamin B 12The mass ratio is (30-150):1, more preferably (40-110):1, further more preferably (55-105):1, and most preferably (90-105):1.

[0039] Furthermore, the Na + Selected from one or more of sodium bicarbonate, sodium sulfate and sodium chloride.

[0040] Furthermore, the Ca 2+ Selected from calcium sulfate, calcium chloride or hydrates of the above calcium salts.

[0041] Furthermore, the Mg 2+ Selected from magnesium sulfate, magnesium chloride or hydrates of the above magnesium salts.

[0042] Furthermore, the K + Selected from one or more of potassium chloride, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate.

[0043] Furthermore, the Se 4+ Selected from sodium selenite and / or potassium selenite.

[0044] Furthermore, the HCO3 - Selected from sodium bicarbonate and / or potassium bicarbonate.

[0045] Furthermore, the SO4 2- Selected from magnesium sulfate, calcium sulfate, sodium sulfate or hydrates of the above sulfates.

[0046] Furthermore, the Cl - Selected from one or more of potassium chloride, magnesium chloride, calcium chloride and sodium chloride.

[0047] As a preferred embodiment, the composition formula of the culture medium includes: NaHCO3 170-210 mg·L -1 、CaSO4 70~115mg·L -1 、MgSO4 90~145mg·L -1 、KCl 5~15mg·L -1 、Se 4+ 1~5μg·L -1 , Vitamin B1 60~90μg·L -1 , Vitamin B 12 0.6~2μg·L -1 , Vitamin H 0.5~1μg·L -1 .

[0048] As a more preferred embodiment, the composition formula of the culture medium includes: NaHCO3 180-200 mg·L -1、CaSO4 80~110mg·L -1 、MgSO4 110~130mg·L -1 、KCl 6~12mg·L -1 、Se 4+ 1.5~2.5μg·L-1, vitamin B1 65~80μg·L -1 , Vitamin B 12 0.8~1.5μg·L -1 , Vitamin H 0.6~0.9μg·L -1 .

[0049] Furthermore, as a more preferred embodiment, the composition formula of the culture medium includes: NaHCO3 185-198 mg·L -1 、CaSO4 90~105mg·L -1 、MgSO4 115~125mg·L -1 、KCl 7~9mg·L -1 、Se 4+ 1.8~2.2μg·L-1, vitamin B1 70~80μg·L -1 , Vitamin B 12 0.8~1.2μg·L -1 , Vitamin H 0.7~0.8μg·L -1 .

[0050] Furthermore, the pH of the culture medium is appropriately adjusted according to the cultured organisms and the purpose of the experiment. The specific pH may be 5.8, 6.0, 6.3, 6.5, 7.2, 7.6, 8.0, etc., or an interval range formed by any of the above values, such as 5.8-6.3, 6.0-6.5, 7.2-8.0, etc., but is not limited thereto.

[0051] The invention also protects the use of the culture medium in culturing plankton.

[0052] Furthermore, the zooplankton is cladoceran zooplankton.

[0053] Furthermore, the cladoceran zooplankton is a Daphnia pulex.

[0054] Furthermore, the Daphnia includes Daphnia pulex.

[0055] The present invention also protects a method for predicting the toxicity of rare earth elements, the method comprising the following steps:

[0056] S1. Setting rare earth elements as target metals;

[0057] S2. Obtain zooplankton from fresh water;

[0058] S3. Use the culture medium to prepare target metal solutions of different concentrations, expose zooplankton to the obtained target metal solutions for culture, and measure the mortality, body length, first reproduction time and reproduction rate of zooplankton at different exposure times to evaluate the toxicity of the target metal to zooplankton.

[0059] Furthermore, the zooplankton is cladoceran zooplankton.

[0060] Furthermore, the cladoceran zooplankton includes daphnia.

[0061] Furthermore, the Daphnia includes Daphnia pulex.

[0062] Furthermore, the rare earth element includes one or more of neodymium, yttrium, lanthanum, cerium, and praseodymium.

[0063] Furthermore, the concentration of the target metal solution is 0.01 to 5 mg·L -1 .

[0064] Preferably, the concentration of the target metal solution is 0.02 to 1 mg·L -1 .

[0065] Compared with the prior art, the present invention has the following beneficial effects:

[0066] The culture medium for metal chronic toxicity experiments provided by the present invention does not contain EDTA or other chelating agents, and can also meet the normal reproduction and growth requirements of model organisms. It is suitable for long-term culture of model organisms, and is beneficial to reducing the influence of the culture medium's own components on the bioavailability and toxicity assessment results of metals, thereby improving the accuracy and reliability of the assessment results, thereby more accurately determining the chronic toxicity or bioavailability of rare earth elements, which is beneficial to accurately exploring the long-term ecological risks of rare earth elements, and providing a new reference approach for the formulation of metal environmental standards and scientific environmental decision-making. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 This is a statistical chart showing the effect of Nd and Y exposure for 21 days on the mortality of Daphnia magna.

[0068] Figure 2 This is a statistical chart showing the effects of Nd and Y exposure for 21 days on the growth of Daphnia magna.

[0069] Figure 3 This is a statistical chart showing the effect of 21 days of Nd and Y exposure on the first reproduction time of Daphnia magna.

[0070] Figure 4 This is a statistical chart showing the effect of Nd and Y exposure for 21 days on the total reproductive number of Daphnia magna.

[0071] Figure 5 This is a statistical graph showing the effect of using M7 medium without EDTA on the survival rate of Daphnia magna. DETAILED DESCRIPTION

[0072] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0073] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0074] The Daphnia magna used in the present invention comes from the Institute of Hydrobiology of Jinan University, and is a pure strain variety obtained through long-term stable multi-generation cultivation in the laboratory.

[0075] Example 1 Composition of the culture medium for metal chronic toxicity test

[0076] The composition of the medium for metal chronic toxicity experiment includes:

[0077] In a first aspect, the present invention provides a culture medium for a chronic toxicity experiment of rare earth elements to Daphnia magna, the components of which include:

[0078] Sodium bicarbonate (NaHCO3), calcium sulfate (CaSO4), magnesium sulfate (MgSO4), potassium chloride (KCl), selenium (Se), mixed vitamin solution, among which,

[0079] The specific components and concentrations of the culture medium are:

[0080] (1) NaHCO3: 192 mg·L -1 ;

[0081] (2) CaSO4: 95 mg·L -1 ;

[0082] (3) MgSO4: 120 mg·L -1 ;

[0083] (4) KCl: 8 mg·L -1 ;

[0084] (5) Se: 2 μg·L -1 ;

[0085] (6) Mixed vitamin solution includes: Vitamin B1: 75 μg·L -1 , Vitamin B 12 :1μg·L -1 , Vitamin H: 0.75 μg·L -1 .

[0086] As an optional solution, the following stock solutions can be prepared:

[0087] (1) NaHCO3: 76800 mg·L -1 ;

[0088] (2) CaSO4·2H2O: 1200 mg·L -1 ;

[0089] (3) MgSO4·7H2O: 122860 mg·L -1 ;

[0090] (4) KCl: 8000 mg·L -1 ;

[0091] (5) Na2SeO3 (providing Se): 44 mg·L -1 ;

[0092] (6) Mixed vitamin solution includes: Mixed vitamin B1: 750 mg·L -1 , Vitamin B 12 :10

[0093] mg·L -1 , Vitamin H: 7.5mg·L -1 .

[0094] When preparing the culture medium, each stock solution was added to water at a dilution ratio of 1:400, 1:10, 1:500, 1:1000, 1:10000, and 1:10000, respectively, and the pH value was adjusted to pH=6.0 and then balanced at 100 rpm for 12 hours for use.

[0095] Example 2 Application of culture medium for metal chronic toxicity experiment

[0096] The method for using the culture medium for metal chronic toxicity test comprises the following steps:

[0097] S1. setting rare earth elements neodymium (Nd) and yttrium (Y) as target metals;

[0098] S2. Obtain zooplankton in fresh water: select biological Daphnia gracilis, place it in a constant temperature incubator at 20°C, 10% light, and a light cycle of 12h light: 12h dark. If the culture medium used daily is different from the culture medium obtained in Example 1, the parent Daphnia gracilis needs to be acclimated to the water quality and feeding conditions in the culture medium obtained in Example 1 before the experiment begins, so as to avoid the new culture medium from causing stress to the experimental Daphnia gracilis. The specific process is: mix the daily culture medium and the culture medium obtained in Example 1 in a volume ratio of 9:1 as the culture medium for the transition period, and take the ability of adult individuals to stably reproduce offspring as a sign that Daphnia gracilis has adapted to the water quality. Separate the mother and the third generation of newborn Daphnia gracilis, and at the same time gradually increase the content of the culture medium by 10-20% volume ratio. The acclimation process takes about 2 months. Before the experiment, the parent Daphnia gracilis was acclimated in the culture medium, and non-first-born Daphnia gracilis (Daphnia age <24h) were collected for the experiment;

[0099] S3-1. The medium obtained in Example 1 was used to prepare exposure solutions of different concentrations of Y and Nd, with the exposure concentrations set to 0.02, 0.05, 0.1, 0.2, 0.5, 0.8 and 1.0 mg·L -1 , set up a control group (no rare earth metal added), set up 10 parallel groups for each concentration, adjust the pH value to pH = 6.0 and then balance at 100 rpm for 12 hours for use.

[0100] S3-2. A healthy newborn Daphnia magna was placed in a beaker containing 30 mL of exposure solution and cultured separately. Chlamydomonas reinhardtii was fed daily at a feeding concentration of 5×10 4 cell / mL, 10 after the third day 5 Before feeding, Chlamydomonas reinhardtii in the exponential growth phase was centrifuged at 3500 rpm for 6 minutes, centrifuged 3 times, and the supernatant was removed and resuspended in the culture medium to measure the cell density. The exposure solution was reconfigured and replaced every two days to maintain the stability of the exposure concentration.

[0101] S3-3. During the experiment, the appearance and survival of the parent daphnia were observed every day, the newborn larvae and dead parent daphnia were removed in time, and the mortality rate of parent daphnia, the number of larvae produced and the time of first reproduction were recorded. After the experiment, the body length of all surviving parent daphnia was measured.

[0102] The results are as follows Figures 1-2 As shown, the culture medium obtained in Example 1 was used to conduct chronic toxicity experiments. The control group maintained normal growth without death, while the Daphnia magna exposed to Nd and Y were affected to varying degrees, especially in the later stage, with a large number of deaths, and showed a concentration (dose)-effect relationship. The effect of rare earth elements on the growth of Daphnia magna was promoted at low concentrations and inhibited at high concentrations. Compared with Nd, Daphnia magna showed a stronger sensitivity to Y. Figure 3 , 4The results show that the average number of surviving young Daphnia produced by each surviving parent Daphnia in the control group is greater than 60, which meets the quality control standards of the Organization for Economic Cooperation and Development (OECD) for the reproduction test of Daphnia magna, indicating that the culture medium obtained in Example 1 does not affect the normal growth and reproduction of Daphnia magna and is suitable for long-term culture of Daphnia magna. Consistent with the effect of rare earth elements on the growth of Daphnia magna, the effect of rare earth elements on the reproductive capacity of Daphnia magna is also manifested as low concentration promotion and high concentration inhibition, and Y is more toxic to Daphnia magna than Nd. This shows that the culture medium of the present application can cultivate model organisms for evaluating the toxicity or biological effectiveness of rare earth elements.

[0103] The above results show that the culture medium for metal chronic toxicity experiment provided by the present invention does not contain EDTA or other chelating agents, and can also meet the normal reproduction and growth requirements of model organisms, is suitable for long-term culture of model organisms, and is conducive to reducing the influence of the culture medium's own components on the evaluation results of the toxicity of rare earth elements, improving the accuracy and reliability of the evaluation results, and thus more accurately determining the chronic toxicity of rare earth elements.

[0104] Compared with Example 2, the M7 medium without EDTA was used to culture Daphnia magna. The formula of the M7 medium without EDTA was as follows: CaCl2·2H2O: 294 mg·L -1 ;MgSO4·7H2O: 123mg·L -1 ; KCl: 5.8 mg·L -1 ;NaHCO364.8mg·L -1 ;Na2SiO3·9H2O:5mg·L -1 ; K2HPO4: 0.184mg·L -1 ; KH2PO4: 0.143mg·L -1 ; H3BO3: 0.715mg·L -1 ;NaNO3: 0.274mg·L -1 .

[0105] The other steps and parameters are the same as those in Example 2.

[0106] The results are as follows Figure 5 As shown in the figure, the survival of Daphnia magna was significantly affected when the M7 medium without EDTA was used to culture Daphnia magna, and the survival rate of Daphnia magna was 0% after 17 days of exposure. This shows that removing EDTA from the conventional M7 medium is not conducive to the growth and survival of Daphnia magna.

[0107] In summary, the present invention ensures accurate assessment of the bioavailability and toxicity of target metals by improving the culture medium used for exposure, simulates long-term exposure of aquatic model organisms (such as Daphnia magna) to rare earth elements in nature, eliminates interference from the components of the culture medium itself, and improves the accuracy and reliability of experimental results, thereby more accurately determining the chronic toxicity or bioavailability of rare earth elements, which is conducive to accurately exploring the long-term ecological risks of rare earth elements.

[0108] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A culture medium, characterized in that The composition formula of the culture medium includes: metal ions, Se 4+ 、HCO3 - 、SO4 2- , Cl - and vitamins; Wherein, the metal ion is Na + , Ca 2+ Mg 2+ , K + ; The Na + The concentration is 40~70mg·L -1 ; the Ca 2+ The concentration is 18~40mg·L -1 ; The Mg 2+ The concentration is 18~35mg·L -1 ; The K + The concentration is 2~8mg·L -1 ; The Se 4+ The concentration is 1-5 μg·L -1 ; The HCO3 - The concentration is 110~160mg·L -1 ; The SO4 2- The concentration is 120~200mg·L -1 ; the Cl - The concentration is 2~8mg·L -1 ; The concentration of the vitamin is 50 to 100 μg·L -1 ; The culture medium does not contain a chelating agent; The pH of the culture medium is 5.8-8.

2. The culture medium according to claim 1, characterized in that The composition formula of the culture medium meets one or more of the following conditions: (1) The Na + The concentration is 45~62mg·L -1 ; and / or (2) Ca 2+ The concentration is 20~35mg·L -1 ; and / or (3) Mg 2+ The concentration is 18-30 mg·L -1 ; and / or (4) K + The concentration is 2.6~6.5mg·L -1 ; and / or (5) Se 4+ The concentration is 1-3 μg·L -1 ; and / or (6) HCO3 - The concentration is 120~150mg·L -1 ; and / or (7) SO4 2- The concentration is 140~180mg·L -1 ; and / or (8) Cl - The concentration is 2.5~6mg·L -1 ; and / or (9) The concentration of the vitamin is 60 to 85 μg / L -1 .

3. The culture medium according to claim 1, characterized in that The vitamins include B vitamins and / or vitamin H.

4. The culture medium according to claim 3, characterized in that The mass ratio of the B vitamins to vitamin H is (60-190):

1.

5. The culture medium according to claim 1, characterized in that The vitamin B family includes vitamin B1 and / or vitamin B 12 .

6. The culture medium according to claim 5, characterized in that Vitamin B1 and vitamin B 12 The mass ratio is (30~150):

1.

7. The culture medium according to claim 1, characterized in that The composition formula of the culture medium includes: NaHCO3 170-210 mg·L -1 、CaSO4 70~115mg·L -1 、MgSO4 90~145mg·L -1 、KCl 5~15mg·L -1 、Se 4+ 1~5μg·L-1, vitamin B1 60~90μg·L -1 , Vitamin B 12 0.6~2μg·L -1 , Vitamin H 0.5~1μg·L -1 .

8. The culture medium according to claim 7, characterized in that The composition formula of the culture medium includes: NaHCO3 180-200 mg·L -1 、CaSO4 80~110mg·L -1 、MgSO4 110~130mg·L -1 、KCl 6~12mg·L -1 、Se 4+ 1.5~2.5μg·L-1, vitamin B1 65~80μg·L -1 , Vitamin B 12 0.8~1.5μg·L -1 , Vitamin H 0.6~0.9μg·L -1 .

9. Use of the culture medium according to any one of claims 1 to 8 in culturing plankton.

10. A method for predicting the toxicity of rare earth elements, characterized in that: The method comprises the following steps: S1. Setting rare earth elements as target metals; S2. Obtain zooplankton from fresh water; S3. Use the culture medium described in any one of claims 1 to 8 to prepare target metal solutions of different concentrations, expose zooplankton to the obtained target metal solutions for culture, and measure the mortality, body length, first reproduction time and reproduction rate of zooplankton at different exposure times to evaluate the toxicity of the target metal to zooplankton.

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