Culture medium for metal chronic toxicity experiment and application thereof

Abstract

The present application belongs to the technical field of zooplankton culture, and particularly relates to a culture medium for metal chronic toxicity experiment and application thereof. The culture medium provided by the present application 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, is conducive to reducing the influence of the components of the culture medium on the bioavailability and toxicity evaluation and determination results of metals, improving the accuracy and reliability of the evaluation results, and thus more accurately determining the chronic toxicity or bioavailability of rare earth elements, and is conducive to accurately exploring the long-term ecological risk of rare earth elements, and provides a new reference approach for metal environmental standard formulation and scientific environmental decision-making.

Description

Technical Field

[0001] This invention belongs to the field of zooplankton culture technology. More specifically, it relates to a culture medium for testing the chronic toxicity of metals and its application. Background Technology

[0002] Daphnia magna, with its relatively short lifespan, high reproductive rate, predominantly parthenogenetic reproduction, ease of obtaining genotype-identical monoclonal individuals, and high sensitivity to environmental changes, has become a key model organism in ecotoxicology research, widely used in aquatic ecosystem and metal toxicity assessment. In particular, chronic toxicity studies of Daphnia magna are considered an important tool for investigating the long-term biological effects of metal exposure in water and its potential impact on the ecological environment, achieved by monitoring biological indicators such as survival rate, growth status, and reproductive capacity.

[0003] However, current mainstream large-scale Daphnia chronic toxicity test 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. This complexation 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 have chosen to use aerated filtered tap water as experimental water, but this approach has two major drawbacks: first, the uncertainty of the composition of the filtered water, and second, the differences in the composition of tap water in different regions. Both of these points threaten the repeatability and accuracy of the experiment.

[0005] To address the aforementioned issues, Chinese patent application CN111357692A proposes an indoor breeding and cultivation method for *Daphnia breviscapus*. This method cultivates wild-type female *Daphnia breviscapus* in a specific breeding medium, followed by culturing juveniles in a stabilizing medium to ensure individual stability. This stabilizing medium consists of NaHCO3 (240 mg / L), CaSO4·2H2O (150 mg / L), MgSO4 (150 mg / L), and KCl (10 mg / L), with the pH maintained between 6.3 and 7.8. However, this patent still relies on a traditional medium containing EDTA during the breeding stage, failing to completely eliminate the interference problems caused by EDTA.

[0006] Therefore, there is an urgent need for a new type of culture medium for large-scale chronic toxicity experiments of Daphnia zearalensis to solve the above problems. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of existing large-scale daphne chronic toxicity test culture media, which usually contain chelating agents such as EDTA, which interfere with the accuracy and reliability of rare earth element toxicity and bioavailability tests, and to provide a culture medium.

[0008] The purpose of this invention is to provide the 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 objective of this invention is achieved through the following technical solution:

[0011] The culture medium composition includes: metal ions, Se. 4+ HCO3 - SO4 2- Cl - And vitamins; HCO3 - SO4 2- Cl - and vitamins;

[0012] The metal ion is Na. + Ca 2+ Mg 2+ K + ;

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

[0014] Existing toxicity testing media typically contain chelating agents such as EDTA, which can interfere with the accuracy and reliability of rare earth element toxicity and bioavailability tests. The media provided by this invention, however, does not contain EDTA or other chelating agents, while still meeting the normal reproduction and growth requirements of model organisms. It is suitable for long-term culture of model organisms, reducing the influence of the media's own components on the assessment results of metal bioavailability and toxicity, thus improving the accuracy and reliability of the assessment results. This allows for a more accurate determination of the chronic toxicity or bioavailability of rare earth elements, facilitating a more precise exploration of 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 of the culture medium satisfies one or more of the following conditions:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0034] (9) The concentration of the vitamin is 65–80 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 B vitamins include vitamin B1 and / or vitamin B1. 12 .

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

[0039] Furthermore, the Na + It is 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 + It is 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 - It is selected from one or more of potassium chloride, magnesium chloride, calcium chloride, and sodium chloride.

[0047] In a preferred embodiment, the culture medium comprises: NaHCO3 170–210 mg·L⁻¹ -1 CaSO4 70~115mg·L -1 MgSO4 90~145mg·L -1 KCl 5~15mg·L -1 Se 4+ 1~5μg·L -1 Vitamin B1 60-90 μg / L -1 Vitamin B 12 0.6~2μg·L -1 Vitamin H 0.5~1μg·L -1 .

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

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

[0050] Furthermore, the pH of the culture medium is adjusted appropriately according to the organism being cultured and the experimental purpose. Specific pH values ​​may be 5.8, 6.0, 6.3, 6.5, 7.2, 7.6, 8.0, or any range formed by the above values, such as 5.8–6.3, 6.0–6.5, 7.2–8.0, etc., but are not limited to these.

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

[0052] Furthermore, the zooplankton is a cladoceran zooplankton.

[0053] Furthermore, the cladocerans described are daphnia.

[0054] Furthermore, the daphnia include Daphnia macrocarpa.

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

[0056] S1. Set rare earth elements as the target metal;

[0057] S2. Obtain zooplankton from freshwater;

[0058] S3. Using the culture medium, prepare target metal solutions of different concentrations, expose zooplankton to the obtained target metal solutions, and determine the mortality rate, body length, initial reproduction time and reproduction rate of zooplankton at different exposure times to assess the toxicity of the target metal to zooplankton.

[0059] Furthermore, the zooplankton is a cladoceran zooplankton.

[0060] Furthermore, the cladocerans include daphnia.

[0061] Furthermore, the daphnia include Daphnia macrocarpa.

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

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

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

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

[0066] The culture medium for metal chronic toxicity testing provided by this invention does not contain EDTA or other chelating agents, while also meeting the needs of normal reproduction and growth of model organisms. It is suitable for long-term culture of model organisms and helps to reduce the influence of the culture medium's own components on the assessment results of the bioavailability and toxicity of metals, thereby improving the accuracy and reliability of the assessment results. This allows for a more accurate determination of the chronic toxicity or bioavailability of rare earth elements, which is beneficial for the precise exploration of the long-term ecological risks of rare earth elements and provides a new reference for the formulation of metal environmental standards and scientific environmental decision-making. Attached Figure Description

[0067] Figure 1 A statistical chart showing the impact of 21 days of Nd and Y exposure on the mortality rate of Daphnia magna.

[0068] Figure 2 A statistical chart showing the impact of 21 days of Nd and Y exposure on the growth of Daphnia macrocarpa.

[0069] Figure 3 A statistical chart showing the impact of 21 days of Nd and Y exposure on the first reproduction time of Daphnia macrocarpa.

[0070] Figure 4 A statistical chart showing the impact of 21 days of Nd and Y exposure on the total reproductive number of Daphnia macrocarpa.

[0071] Figure 5 A statistical graph showing the effect of using EDTA-free M7 medium on the survival rate of Daphnia macrocarpa. Detailed Implementation

[0072] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

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

[0074] The large Daphnia used in this invention comes from the Institute of Aquatic Biology, Jinan University, and a pure strain was obtained through long-term stable multi-generation cultivation in the laboratory.

[0075] Example 1: Composition of the culture medium used in the chronic toxicity test of metals

[0076] The culture medium used in the chronic toxicity test of metals comprises:

[0077] In a first aspect, the present invention provides a culture medium for a chronic toxicity test of rare earth elements on *Daphnia macrocarpa*, comprising:

[0078] A solution containing sodium bicarbonate (NaHCO3), calcium sulfate (CaSO4), magnesium sulfate (MgSO4), potassium chloride (KCl), selenium (Se), and mixed vitamins, wherein...

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

[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) The mixed vitamin solution includes: Vitamin B1: 75 μg·L -1 Vitamin B 12 1 μg·L -1 Vitamin H: 0.75 μg·L -1 .

[0086] As an optional approach, 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 (provides Se): 44 mg·L -1 ;

[0092] (6) The mixed vitamin solution includes: mixed vitamin B1: 750 mg / L -1 Vitamin B 12 10

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

[0094] When preparing the culture medium, each stock solution is added to water at dilution ratios of 1:400, 1:10, 1:500, 1:1000, 1:10000, and 1:10000, respectively. After adjusting the pH value to pH=6.0, the solution is equilibrated at 100 rpm for 12 hours before use.

[0095] Example 2: Application of culture medium in metal chronic toxicity test

[0096] The application method of the culture medium for chronic toxicity testing of metals includes the following steps:

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

[0098] S2. Obtaining zooplankton from freshwater: *Daphnia macrocarpa* was selected and cultured in a constant-temperature incubator at 20°C with 10% light and a 12-hour light-to-dark cycle. If the culture medium used daily differs from that obtained in Example 1, the parent daphnia need to be acclimatized to the water quality and feeding conditions in the culture medium obtained in Example 1 before the experiment to avoid stress on the experimental daphnia from the new culture medium. Specifically, the commonly used culture medium and the culture medium obtained in Example 1 are mixed at a 9:1 volume ratio as a transitional culture medium. The ability of adult individuals to stably reproduce is considered an indicator of *Daphnia macrocarpa*'s adaptation to water quality. The mother and third-generation juveniles are separated, and the content of the culture medium is gradually increased at a volume ratio of 10-20%. The acclimatization process takes approximately two months. Before the experiment, the parent daphnia are acclimatized in the culture medium, and non-first-parity juveniles are collected for the experiment (daphnia age <24h).

[0099] S3-1. Using the culture medium obtained in Example 1, prepare exposure solutions of Y and Nd at different concentrations, with exposure concentrations set at 0.02, 0.05, 0.1, 0.2, 0.5, 0.8, and 1.0 mg·L⁻¹. -1 Set up one control group (without rare earth metals), and set up 10 parallel groups for each concentration. After adjusting the pH value to pH=6.0, equilibrate at 100rpm for 12h before use.

[0100] S3-2. Place a healthy, newly hatched large Daphnia daphnia in a beaker containing 30 mL of exposure solution and culture it individually. Feed it Chlamydomonas reinhardtii daily at a concentration of 5 × 10⁻⁶ for the first three days. 4 cell / mL, 10 on the third day 5 Cell / mL. Before feeding, Chlamydomonas reinhardtii in the exponential growth phase was centrifuged at 3500 rpm for 6 minutes, three times, and the supernatant was removed before resuspending in the culture medium. Cell density was then measured. The exposure solution was reconstituted and replaced every two days to maintain the stability of the exposure concentration.

[0101] S3-3. During the experiment, observe the appearance and survival status of the parent daphnia daily, promptly remove newly born daphnia and dead parent daphnia, and record the parent daphnia mortality rate, the number of daphnia born, and the time of first reproduction. After the experiment, measure the body length of all surviving parent daphnia.

[0102] The results are as follows Figures 1-2 As shown, chronic toxicity experiments were conducted using the culture medium obtained in Example 1. The control group maintained normal growth with no mortality, while *Daphnia macrocarpa* exposed to Nd and Y were affected to varying degrees, especially showing significant mortality in the later stages, exhibiting a concentration (dose)-response relationship. The effect of rare earth elements on *Daphnia macrocarpa* growth was characterized by promotion at low concentrations and inhibition at high concentrations. Compared to Nd, *Daphnia macrocarpa* showed stronger sensitivity to Y. Figure 3 , 4The results showed that the average number of surviving offspring produced by each surviving parent daphnia in the control group was greater than 60, meeting the quality control standards for daphnia reproduction experiments set by the Organization for Economic Cooperation and Development (OECD). This indicates that the culture medium obtained in Example 1 does not affect the normal growth and reproduction of daphnia and is suitable for long-term cultivation. Consistent with the effects of rare earth elements on the growth of daphnia, the effects of rare earth elements on the reproductive capacity of daphnia also showed a promoting effect at low concentrations and an inhibiting effect at high concentrations, and that Y was more toxic to daphnia than Nd. This demonstrates that the culture medium of this application can be used to cultivate model organisms for evaluating the toxicity or bioavailability of rare earth elements.

[0103] The above results indicate that the culture medium for the chronic toxicity test of metals provided by this invention does not contain EDTA or other chelating agents, while still meeting the needs of normal reproduction and growth of model organisms. It is suitable for long-term culture of model organisms and helps reduce the influence of the culture medium's own components on the assessment results of rare earth element toxicity, improving the accuracy and reliability of the assessment results, thereby more accurately determining the chronic toxicity of rare earth elements. Comparative Example 1: Application of the culture medium for the chronic toxicity test of metals.

[0104] Compared to Example 2, *Daphnia macrocarpa* was cultured using EDTA-free M7 medium. The formulation of the EDTA-free M7 medium is as follows: CaCl2·2H2O: 294 mg·L⁻¹ -1 MgSO4·7H2O: 123 mg·L -1 KCl: 5.8 mg·L -1 NaHCO3 64.8 mg·L -1 Na₂SiO₃·9H₂O: 5 mg·L⁻¹ -1 K2HPO4: 0.184 mg·L -1 KH2PO4: 0.143 mg·L -1 H3BO3: 0.715 mg·L -1 NaNO3: 0.274 mg·L -1 .

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

[0106] The results are as follows Figure 5 As shown, the survival of Daphnia magna was significantly affected when cultured in M7 medium without EDTA, with a survival rate of 0% after 17 days of exposure. This indicates that removing EDTA from the standard M7 medium is detrimental to the growth and survival of Daphnia magna.

[0107] In summary, this invention ensures accurate assessment of the bioavailability and toxicity of target metals by modifying the culture medium used for exposure. It simulates long-term exposure of rare earth elements in nature using aquatic model organisms (such as Daphnia magna), eliminates interference from the components of the culture medium itself, and improves the accuracy and reliability of experimental results. This allows for a more accurate determination of the chronic toxicity or bioavailability of rare earth elements, which is beneficial for the precise exploration of the long-term ecological risks of rare earth elements.

[0108] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A culture medium, characterized in that, For the cultivation of daphnia, the culture medium comprises the following components: NaHCO3 170~210 mg·L⁻¹. -1 CaSO4 70~115 mg·L -1 MgSO4 90~145 mg·L -1 KCl 5~15 mg·L -1 Se 4+ 1~5 μg·L -1 Vitamin B1 60~90 μg·L -1 Vitamin B 12 0.6~2 μg·L -1 Vitamin H 0.5~1 μg·L -1 The culture medium does not contain chelating agents; the pH of the culture medium is 5.8-8.

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

3. The use of the culture medium according to claim 1 or 2 in the culture of daphnia.

4. A method for predicting the toxicity of rare earth elements, characterized in that, The method includes the following steps: S1. Set rare earth elements as the target metal; S2. Obtain freshwater daphnia; S3. Using the culture medium described in claim 1 or 2, prepare target metal solutions of different concentrations, expose daphnia to the obtained target metal solutions, and determine the mortality rate, body length, initial reproductive time and reproductive rate of daphnia at different exposure times to assess the toxicity of the target metal to daphnia.

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