Evaluation method for safe use of disinfectant for aquatic products

Abstract

The invention belongs to the technical field of disinfectant use evaluation, and particularly relates to an evaluation method for safe use of an aquatic disinfectant. The harmful bacteria (aeromonas hydrophila, vibrio harveyi, aeromonas veronii and nocardia) and beneficial bacteria are experimented, the harmful bacteria are used for exploring the concentration of the disinfectant, and the beneficial bacteria assist in verifying the reasonability of the concentration of the disinfectant, so that a proper disinfectant concentration range is obtained. The killing effect and the killing concentration of common pathogenic bacteria in aquaculture are explored through a common aquatic product disinfectant, meanwhile, the production concentration in the actual ecological environment is screened, and the appropriate and recommended use concentration of the aquatic product disinfectant is obtained. The method plays a scientific reference role in guiding reasonable use of the disinfectant and avoiding propagation of multiple drug-resistant bacteria.

Description

Technical Field

[0001] The present invention belongs to the technical field of the evaluation of the use of disinfectants, and particularly relates to a method for evaluating the safe use of disinfectants for aquatic products. Background Art

[0002] In the past decade, there have been many studies on the types and disinfection effects of aquatic disinfectants at home and abroad. At present, in aquaculture, the use of drugs is imitative, blind and arbitrary. Aquaculture farmers lack a clear understanding of the dosage, usage and withdrawal period of drugs, resulting in over-dose abuse or misuse of drugs, which not only fails to effectively treat diseases, but may also cause secondary infections or deteriorate the aquaculture environment. Some aquaculture farmers tend to increase the dosage to pursue quick results, but ignore the toxicity of drugs to aquatic animals and the negative impacts on the water environment, such as the increase of ammonia nitrogen and the death of algae. The main mechanism of action of these disinfectants is to destroy the cell membrane or cell wall of pathogenic microorganisms, interfere with their metabolism, and make them lose their activity, so as to achieve the purpose of disinfection. However, while these disinfectants kill pathogenic microorganisms, they may also have certain impacts on the water quality environment and fish. For a single disinfectant, the reasonable application of the disinfectant is crucial for aquaculture. A large number of studies have shown that when the disinfectant is applied unreasonably, it will enhance the drug resistance of aquatic organisms, and the bacteria in the aquaculture environment have strong adaptability and can continuously evolve through a series of mechanisms with the change of the environment and the application of disinfectants. And this evolution makes bacteria develop drug resistance to disinfectants. Generally speaking, if a certain kind of bacteria can survive or grow in a disinfectant with a concentration exceeding the common concentration range, then it has tolerance or resistance to the disinfectant. Bacterial resistance to disinfectants will greatly reduce the killing efficiency of disinfectants and seriously affect the killing of pathogens in the environment. Therefore, like antibacterial drug resistance, bacterial tolerance to disinfectants has become one of the greatest threats to public health in the 21st century. And too low a concentration of the disinfectant will reduce the disinfection effect of the disinfectant. Therefore, the importance of studying the optimal disinfection concentration of disinfectants is highlighted.

[0003] In response to this problem, many scholars have done a lot of research on the effects and concentrations of disinfectants. It is found that 19 common aquatic disinfectants have antibacterial effects on Flavobacterium columnare from fish, among which benzalkonium bromide has the best antibacterial effect on Flavobacterium columnare, and glutaraldehyde has a poor effect. In addition, there are also studies showing that the combination of glutaraldehyde and benzalkonium bromide is stronger than the other two bactericides in terms of spore killing activity and segment recognition destruction. The bactericidal effect is enhanced after the combination of glutaraldehyde and benzalkonium bromide.

[0004] In addition, the disinfection effect of disinfectants is not only related to the concentration but also to the content of organic matter in the water body. Research has found that within the same time period, as the concentration of organic matter increases, the sterilization rate of the new king disinfectant decreases. It is also pointed out that the main reasons for the impact of organic matter on the disinfection effect of disinfectants are that organic matter combines with disinfectants to form insoluble compounds, or organic matter adsorbs disinfectants and reacts chemically with them, causing the disinfectants to lose their disinfection function, or organic matter acts on microorganisms and provides mechanical protection to microorganisms, thus reducing the disinfection effect of disinfectants on microorganisms.

[0005] Regarding the toxicity of disinfectants, the research has clarified that the LC50 values of glutaraldehyde for koi at 24 h, 48 h, 72 h, and 96 h are 0.671, 0.656, 0.635, and 0.621 mg / L respectively, the safe concentration (SC) is 0.062 mg / L, and the effective action time of the drug is about 48 h. From this, it is inferred that glutaraldehyde is a highly toxic substance to koi. And histological observations have found that the gill tissues of the dead fish are severely damaged. In the case of benzalkonium bromide, the research has found that the LC50 values of benzalkonium bromide for juvenile Onychostoma sima at 24 h, 48 h, 72 h, and 96 h are 83.18, 81.28, 77.63, and 63.10 mg / L respectively, and the SC is 6.31 mg / L, indicating that benzalkonium bromide is a moderately toxic drug to juvenile Onychostoma sima. In order to detect the effects of the two disinfectants, glutaraldehyde and benzalkonium bromide, on the prevention and treatment of diseases in Rhodeus sinensis, the semi-static bioassay method was used to determine the acute toxicity effects of glutaraldehyde and benzalkonium bromide on Rhodeus sinensis. It has been proven that glutaraldehyde is a low-toxic drug to Rhodeus sinensis and benzalkonium bromide is a moderately toxic drug, and their common concentrations are both lower than the safe concentration, making them suitable as drugs for the prevention and treatment of diseases in Rhodeus sinensis. A study on rainbow trout has shown that at a dose of 0.05 - 0.30 mg / L of PAA, growth, survival rate, and feed conversion rate are not affected. The above experiments illustrate that disinfectants have certain toxicity, but using them at appropriate concentrations will not cause harm.

[0006] The common disease pathogens in the process of aquaculture mainly include viruses, bacteria, and parasites. Among them, bacterial diseases mainly include gill rot caused by Flexibacter columnaris and Aeromonas hydrophila infection, and ulcer disease caused by Aeromonas hydrophila infection. And research has also shown that Nocardiosis caused by Nocardia seriolae is also one of the common diseases that infect Micropterus salmoides. Currently, since most aquaculture animal health enterprises recommend the use of disinfectants based on enterprise standards, the recommended use concentrations vary among enterprises in production, which has to a certain extent promoted the abuse of disinfectants in production. Unreasonable disinfectant concentrations can cause negative impacts such as environmental damage, pathogen drug resistance, and damage to the health of farmed fish.

[0007] After a large amount of data query, it is found that the existing concentration standards for the same disinfectant are not uniform, and in the experiments to determine the reasonable dosage and concentration of disinfectants, the concentration of test bacteria is usually 10 5 -10 8 CFU / mL, which does not match the actual number of colonies in the actual ecological environment (outdoor pond water), resulting in uncertainty in the disinfection effect. Summary of the Invention

[0008] In view of the above problems, the purpose of the present invention is to provide a method for evaluating the safe use of disinfectants for aquatic products.

[0009] The technical content of the present invention is as follows:

[0010] The present invention provides a method for evaluating the safe use of disinfectants for aquatic products, including the following steps:

[0011] Determine the disinfection target, select the disinfectant, set the concentration gradient, pre-experiment design, disinfection effect evaluation, disinfectant toxicity evaluation, determine the safe dose, on-site application and detection, optimization and adjustment;

[0012] The disinfection target includes common pathogenic bacteria in aquaculture, including Aeromonas hydrophila, Vibrio harveyi, Aeromonas veronii, and Nocardia;

[0013] It also includes beneficial bacteria, Bacillus;

[0014] The disinfectants include glutaraldehyde, benzalkonium bromide, and glutaraldehyde-benzalkonium bromide;

[0015] The effect evaluation includes the following two methods:

[0016] A. Determine the inhibitory effect of the disinfectant on bacteria in aquaculture water according to MIC and MBC;

[0017] 1) Determine the minimum bactericidal concentration MIC and minimum bacteriostatic concentration MBC of the selected disinfectant against the test bacteria;

[0018] 2) Configure the concentration of the used disinfectant according to the MIC and MBC values;

[0019] 3) Adjust the number of test bacteria to the order of magnitude of the bacteria in the aquaculture water and then add it to the water sample;

[0020] 4) Subsequently, add disinfectants with different concentrations, measure OD600, and calculate the growth inhibitor;

[0021] Growth inhibition rate = (positive control well - experimental well) / (positive control well - negative control well) × 100%.

[0022] B. Use PCR technology to detect the residual amount of microbial DNA after treatment with the disinfectant;

[0023] Amplify the target DNA fragment of the microorganism by specific primers to detect the residual amount of microorganism DNA after disinfection treatment;

[0024] If the disinfectant effectively kills the microorganism, its DNA cannot be amplified due to cell rupture or degradation. By comparing the DNA concentration or amplification signal before and after treatment, the bactericidal effect can be quantified.

[0025] The toxicity evaluation of the disinfectant is as follows: using luminescent bacteria as the indicator organism, evaluating the toxicity of the disinfectant based on the principle that toxic substances inhibit the luminescence intensity of bacteria. The evaluation formula is as follows:

[0026] Inhibition rate of bacterial luminescence intensity (%) = [(luminescence value of the blank group - luminescence value of the sample group) / luminescence value of the blank group] × 100%. When the inhibition rate > 20%, it is determined that the disinfectant has biological toxicity;

[0027] Luminescent bacteria such as Vibrio fischeri contain a luciferase system, which releases blue-green light (wavelength 490 nm) through oxidation-reduction reactions (involving NADH and FMN). The luminescence intensity is positively correlated with the bacterial metabolic activity. Toxic substances (such as excessive disinfectant) damage the bacterial cell membrane or inhibit enzyme activity, resulting in a decrease in luminescence intensity. The inhibition rate and the toxic concentration show a dose-effect relationship.

[0028] The determination of the safe dose is evaluated by combining the indoor disinfection effect and the disinfection effect of the on-site aquaculture substrate.

[0029] The beneficial effects of the present invention are as follows:

[0030] A method for evaluating the safe use of an aquaculture disinfectant according to the present invention conducts experiments on harmful bacteria (Aeromonas hydrophila, Vibrio harveyi, Aeromonas veronii, Nocardia) and beneficial bacteria. The harmful bacteria are used to explore the disinfectant concentration, and the beneficial bacteria are used to assist in verifying the rationality of the disinfectant concentration, aiming to obtain an appropriate disinfectant concentration range. And it is experimentally confirmed according to the present invention that the change in the number of the four bacteria is not linearly related to the disinfectant concentration. The present invention explores the killing effect and killing concentration of common aquaculture disinfectants on common pathogenic bacteria in aquaculture, and at the same time screens the production concentration in the actual ecological environment to obtain the appropriate and recommended use concentration of the aquaculture disinfectant. The present invention has scientific reference significance for guiding the rational use of disinfectants and avoiding the spread of multi-drug resistant bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is the experimental bacterium growth inhibition effect diagram of the aquaculture disinfectant on different bacterial liquid concentrations;

[0032] Figure 2 It is the experimental bacterium temperature influence growth inhibition effect diagram of the aquaculture disinfectant on different experimental bacteria.

[0033] In the figure, Reagent 1: glutaraldehyde reagent; Reagent 2: benzalkonium bromide reagent; Reagent 3: glutaraldehyde-benzalkonium bromide mixed reagent. Detailed implementation mode

[0034] The present invention will be further described in detail below through specific implementation cases and accompanying drawings. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the protection scope of the present invention. After reading the present invention, various equivalent modifications made by those skilled in the art fall within the scope defined by the appended claims of this application.

[0035] Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents on the conventional market.

[0036] Example 1

[0037] A method for evaluating the safe use of a disinfectant for aquatic products includes the following steps:

[0038] Determine the disinfection target, select the disinfectant, set the concentration gradient, pre-experiment design, disinfection effect evaluation, disinfectant toxicity evaluation, determine the safe dose, on-site application and detection, optimization and adjustment;

[0039] The disinfection targets include common pathogenic bacteria in aquaculture, including Aeromonas hydrophila, Vibrio harveyi, Aeromonas veronii, and Nocardia;

[0040] It also includes beneficial bacteria, Bacillus;

[0041] The disinfectants include glutaraldehyde, benzalkonium bromide, and glutaraldehyde-benzalkonium bromide;

[0042] The effect evaluation includes the following two methods, such as Example 2 and Example 3.

[0043] Example 2

[0044] Determine the minimum bactericidal concentration MIC and growth inhibition rate MBC of the disinfectant against aquatic pathogenic bacteria

[0045] 1) Dilute glutaraldehyde, benzalkonium bromide, and glutaraldehyde-benzalkonium bromide into working solutions of 16384 mg / L with pure water respectively, and then filter them through a 0.22 μm filter;

[0046] 2) Prepare the bacterial suspension

[0047] Take the colonies in the logarithmic phase and prepare them into a concentration of 1×10 3CFU / mL bacterial solution was inoculated into 20 mL of liquid medium at an inoculation amount of 1%. Add 2 - 3 mL of PBS to a glass sample bottle, scrape bacteria with an inoculation loop and add them to the sample bottle, shake well, and compare the turbidity with a turbidimeter tube against the background of a turbidimeter card. After the turbidities are close, the bacterial solution with the corresponding concentration is obtained;

[0048] 3) The disinfectant was diluted by the two-fold dilution method and serially diluted on a 96-well plate

[0049] First, add 50 μL of liquid medium to each well. Add 50 μL of the working solution to the first column, then pipette 50 μL from the first column and add it to the second column, and so on until the remaining 3 columns are used as controls. At this time, the content of the disinfectant in each column is 16384, 8192, 4096, 2048, 1024, 512, 256, 128, 64, 32... mg / L;

[0050] Then, inoculate 50 μL of the bacterial solution. At this time, the content of the disinfectant in each column is 4096, 2048, 1024, 512, 256, 128, 64, 32, 16, 8, 4, 2, 1 mg / L. The positive control group is only added with the bacterial solution and the culture medium, without the disinfectant. The negative control group is only added with the disinfectant and the culture medium, without the bacterial solution. The blank control is only added with the culture medium. Place them in an incubator and culture for 24 hours. The experiment is repeated 3 times.

[0051] Determine the MIC

[0052] When the positive control shows bacterial growth (the culture medium becomes turbid), the negative control shows no bacterial growth (the culture medium is clear), the lowest concentration of the disinfectant without bacterial growth in the experimental group is the MIC of the disinfectant against the bacterium;

[0053] Determine the MBC

[0054] Select an appropriate amount of the mixed solution (30 μL) without bacterial growth in the above experiment, spread it on the culture medium, and culture for 24 hours. The concentration without colony growth is the MBC of the disinfectant against the bacterium.

[0055] The test results are shown in Table 1 and Table 2:

[0056] Table 1 Results of minimum inhibitory concentration MIC

[0057]

[0058]

[0059] Table 2 Results of minimum bactericidal concentration MBC of growth inhibition rate

[0060]

[0061] As can be seen from Table 1, benzalkonium bromide reagent can exhibit good antibacterial effects at relatively low concentrations even with a relatively low concentration of test bacteria, and the antibacterial effect is significant. The antibacterial effect of glutaraldehyde reagent is relatively poor against test bacteria compared with the three disinfectants;

[0062] As can be seen from Table 2, compared with the other two reagents, benzalkonium bromide reagent can achieve ideal bactericidal effects at a relatively low concentration, and the bactericidal effect is significant.

[0063] Example 3

[0064] Using PCR technology to detect the residual amount of microbial DNA after treatment with disinfectants

[0065] Amplify the target DNA fragment of the microorganism (such as 16S rRNA gene, fungal ITS region or spore-specific gene) through specific primers to detect the residual amount of microbial DNA after treatment with disinfectants. If the disinfectant effectively kills the microorganism, its DNA will not be amplified due to cell rupture or degradation. By comparing the DNA concentration or amplification signal before and after treatment, the bactericidal effect can be quantified.

[0066] 1) Sample treatment

[0067] Disinfectant treatment group: Mix the bacterial suspension (106 CFU / mL) with the three disinfectants for 10 min. Add a neutralizer to terminate the reaction (0.5% sodium thiosulfate), and centrifuge (12,000 rpm, 5 minutes) to collect the bacterial cells.

[0068] Control group:

[0069] Positive control: Bacterial suspension not treated with disinfectant.

[0070] Negative control: Sterile water.

[0071] 2) DNA extraction

[0072] Use a DNA extraction kit to extract DNA from the samples of the treatment group and the control group.

[0073] Measure the DNA concentration (Nanodrop) and adjust it to the same volume (50 μL).

[0074] 3) PCR amplification.

[0075] 4) Result detection

[0076] Take 5 μL of the PCR product for 1.5% agarose gel electrophoresis (120 V, 30 minutes).

[0077] Observe the bands with a gel imaging system and analyze the brightness of the target fragment (such as quantifying using ImageJ software).

[0078] 5) Data analysis

[0079] In the positive control group, clear bands should appear, and in the negative control group, no bands should be present.

[0080] Compare the band brightness of the disinfectant treatment group with that of the positive control to determine the DNA residue amount.

[0081] Example 4

[0082] The toxicity assessment of the disinfectant is as follows: Using luminescent bacteria as the indicator organism, the toxicity of the disinfectant is evaluated based on the principle that toxic substances inhibit the luminescence intensity of bacteria. The operation is as follows:

[0083] 1) Strain resuscitation and activation

[0084] Take colonies in the logarithmic phase and prepare a bacterial solution with a concentration of 1×108 CFU / mL. Inoculate it into a 20 mL liquid medium at an inoculation amount of 1%. Place it in a constant temperature shaker (20 °C) for activation for 30 minutes and set aside.

[0085] 2) Setting of disinfectant concentration gradient

[0086] Dilute the three disinfectants with 3% NaCl solution into a series of concentrations, and dilute them according to multiples of the stock solution, 1 / 2, 1 / 4, 1 / 8, and 1 / 16.

[0087] 3) Exposure reaction and luminescence detection

[0088] Experimental group: Take 100 μL of disinfectant solutions with different concentrations and add them to a 96-well plate. Set 3 replicates for each concentration. Add 100 μL of the activated bacterial solution (final volume 200 μL), mix well and start timing immediately. React for 15 minutes under light-shielded conditions at 20 °C.

[0089] Control group: Negative control: 100 μL of 3% NaCl solution + 100 μL of bacterial solution.

[0090] Positive control: 100 μL of 3% ZnSO4 solution + 100 μL of bacterial solution.

[0091] 4) Determination of luminescence intensity

[0092] Use a bioluminescence detector to measure the luminescence intensity (relative luminescence unit, RLU) of each well, and the integration time for each well is 1 - 5 seconds.

[0093] 5) Data processing

[0094] Calculate the inhibition rate: Inhibition of bacterial luminescence intensity (%) = [(luminescence value of the blank group - luminescence value of the sample group) / luminescence value of the blank group] × 100%. When the inhibition rate > 20%, it is determined that the disinfectant has biological toxicity.

[0095] Example 5

[0096] Determination of basic experimental data before and after using disinfectants in actual aquaculture ponds

[0097] 1) Determination of colony counts before and after catching fish and after using disinfectants

[0098] The aquaculture base has two ponds (B7 and B8) for California bass, each covering 8 mu (approximately 1.5 acres) and 2.5 meters (approximately 1.5 acres) of water. Pond B7 holds approximately 50,000 fish, while Pond B8 holds approximately 34,000. Water samples were collected from both ponds before and after catching the fish, and 30 minutes after disinfectant application.

[0099] The viable bacteria were counted using the plate colony counting method.

[0100] a. Take three 15 mL centrifuge tubes and add 9 mL of PBS to each tube.

[0101] b. Take 1 mL of water sample and add it to the first tube and mix well;

[0102] c. Take 1mL from the previous tube and transfer it to the next tube and mix well. -1 , 10 -2 , 10 -3 ;

[0103] d. Take 30 μL of the diluted water sample and spread it on a plate. Make two plates for each dilution; incubate at 30℃ for 24 hours and then count.

[0104] The results before and after catching fish and disinfecting the aquaculture pond with disinfectants are shown in Table 3:

[0105] Table 3 Colony counts before and after disinfection of aquaculture ponds

[0106]

[0107] It can be seen from the table that the total number of colonies before catching fish ranges from (2.7 to 2.9)×10 2 CFU / mL, after catching fish (3.4~3.6)×10 2 CFU / mL. The total colony count in the aquaculture pond after disinfection ranges from (3 to 5) × 10 1 CFU / mL, the disinfectant can eliminate up to 90% of bacteria in aquaculture ponds.

[0108] 2) Determination of water quality indicators before and after catching fish and after using disinfectants

[0109] a. Measure temperature, pH and dissolved oxygen on site during sampling;

[0110] b. After the water samples are sent back to the laboratory, the COD, total nitrogen, total phosphorus and nitrite indicators will be measured.

[0111] The experimental results of water quality indicators before and after disinfection of aquaculture ponds are shown in Table 4:

[0112] Colony Count before and after Disinfection of Aquaculture Ponds in Table 4

[0113]

[0114] As can be seen from Table 4, after the use of the disinfectant, the disinfectant played a role in purifying the water quality to a certain extent, played an important role in water treatment, and could effectively kill germs and viruses in the water, ensuring a reduction in the number of various pathogens in the water source. Among them, the total nitrogen, total phosphorus, and chemical oxygen demand all decreased significantly after disinfection, and the ammonia nitrogen index also decreased slightly, indicating that the disinfectant played a certain role in purifying the water quality.

[0115] Example 6

[0116] Determination of the Disinfection Experiment of the Disinfectant on Experimental Bacteria in the Actual Aquaculture Pond Water

[0117] 1) Prepare the concentration of the disinfectant used in the experiment in advance according to the MIC and MBC experimental result data in Example 2;

[0118] 2) Prepare the experimental bacteria into a bacterial solution and add it to the water sample to make the bacterial solution concentration in the water body 10 3 CFU / mL;

[0119] 3) Then add disinfectants with different concentrations. After adding the samples, place them in an incubator at 30 °C overnight and measure OD600. Calculate the growth inhibition rate according to the following formula:

[0120] Growth inhibition rate = (positive control well - experimental well) / (positive control well - negative control well) * 100%;

[0121] As Figure 1 shown, for the actual disinfection effect experiment results of the disinfectant on experimental bacteria (in turn Aeromonas hydrophila, Aeromonas veronii, Vibrio harveyi, Nocardia, and Bacillus), different ideal usage concentrations of different disinfectants when different bacteria break out: when Aeromonas hydrophila breaks out in the water body, the glutaraldehyde reagent has the best usage effect when the usage concentration is 4 mg / L; the benzalkonium bromide reagent has the best disinfection effect when the usage concentration is 4 mg / L during the outbreak of Aeromonas hydrophila, Aeromonas veronii, and Bacillus in the water body; the glutaraldehyde-benzalkonium bromide reagent has the best disinfection effect when the usage concentration is 4 mg / L during the outbreak of Aeromonas hydrophila, Aeromonas veronii, and Vibrio harveyi in the water body. Generally speaking, relatively speaking, the benzalkonium bromide reagent and the glutaraldehyde-benzalkonium bromide reagent can cope with more experimental bacteria outbreak situations at lower usage concentrations.

[0122] According to the MIC data of the disinfectant on experimental bacteria at the above different bacterial solution concentrations, convert the recommended usage concentration (mg / L) of the disinfectant obtained in the laboratory into the recommended usage concentration (g / 666.7 m3 ) to obtain the recommended actual production concentrations of the three disinfectants in this experiment under normal use. As can be seen from Table 5:

[0123] Table 5 Recommended usage concentrations of disinfectants in actual production (g / 666.7m 3 )

[0124]

[0125] Example 7

[0126] Experimental determination of the temperature influencing factors of disinfectants

[0127] 1) Prepare in advance according to the ideal disinfectant concentration obtained in Example 6. Take the three disinfectants into centrifuge tubes respectively, and set the temperature conditions to 15±1°C, 25±1°C, and 35±1°C respectively. After treatment according to the above conditions, reach the required test temperature;

[0128] 2) Shake the five experimental bacteria to the logarithmic growth phase, and dilute and adjust the bacterial liquid concentration to 1×10 3 CFU / mL according to the number of bacteria in the actual aquaculture pond in Example 5;

[0129] 3) Use the 96-well plate method (the same method as in Example 3) to measure the growth inhibition rate, and set 3 replicate groups.

[0130] As Figure 2 shown, the experimental results of the temperature influencing factors of the actual disinfection effects of the three aquaculture disinfectants on five different experimental bacteria (in order, Aeromonas hydrophila, Aeromonas veronii, Vibrio harveyi, Nocardia, and Bacillus). It can be seen that the disinfection effects exerted by the disinfectants at different temperatures will vary. Based on this, how to adjust the concentration in different temperature ranges to achieve the best disinfection effect. The experimental results are shown in Tables 6, 7, 8, 9, 10, and 11. Combining the above temperature influencing factor data, the scientific guiding concentrations in nearly different seasons are obtained.

[0131] Table 6 Growth inhibition rate of Aeromonas hydrophila at different temperatures (%)

[0132]

[0133] Table 7 Growth inhibition rate of Aeromonas veronii at different temperatures (%)

[0134]

[0135]

[0136] Table 8 Growth inhibition rate of Vibrio harveyi at different temperatures (%)

[0137]

[0138] Table 9 Growth inhibition rate of Nocardia at different temperatures (%)

[0139]

[0140]

[0141] Table 10 Growth inhibition rate of Bacillus at different temperatures (%)

[0142]

[0143] Table 11 Recommended usage concentrations of three disinfectants in different seasons (mg / L)

[0144]

[0145]

[0146] As can be seen from Table 6, in the experimental results of the growth inhibition effects of the three disinfectants on Aeromonas hydrophila at different dose concentrations, the glutaraldehyde reagent and the benzalkonium bromide reagent showed the best antibacterial effects in the experimental group with a concentration of 2 mg / L; the glutaraldehyde-benzalkonium bromide reagent showed the best antibacterial ability at a concentration of 3 mg / L. As can be seen from Table 7, in the experimental results of the growth inhibition effects of the three disinfectants on Aeromonas veronii, the ideal experimental concentrations of the glutaraldehyde reagent and the benzalkonium bromide reagent for antibacterial effects on the experimental bacteria were 4 mg / L; the ideal usage concentration of the glutaraldehyde-benzalkonium bromide reagent for this experimental bacteria was 2 mg / L. As can be seen from Table 8, Table 9, and Table 10, the experimental results of the growth inhibition effects of the three disinfectants on Vibrio harveyi, Nocardia, and Bacillus showed that the ideal experimental concentrations of the glutaraldehyde reagent and the glutaraldehyde-benzalkonium bromide reagent for the best growth inhibition effects on Vibrio harveyi and Nocardia were both 4 mg / L; the concentration at which the benzalkonium bromide reagent showed the most ideal growth inhibition effect was 2 mg / L.

[0147] As shown in Table 11, the ideal experimental usage concentrations corresponding to the five experimental bacteria treated with the above disinfectants at different temperatures are the specific recommended usage concentrations of the disinfectants in different aquaculture production seasons.

Claims

1. A method for evaluating the safe use of a disinfectant for aquatic products, characterized in that, It includes the following steps: Determine the disinfection target, select the disinfectant, set the concentration gradient, design the preliminary experiment, evaluate the disinfection effect, evaluate the toxicity of the disinfectant, determine the safe dose, apply and detect on-site, and optimize and adjust.

2. The evaluation method for the safe use of the aquatic disinfectant according to claim 1, wherein, The disinfection target includes common pathogenic bacteria in aquaculture, including Aeromonas hydrophila, Vibrio harveyi, Aeromonas veronii, and Nocardia.

3. The evaluation method for the safe use of the disinfectant for aquatic products according to claim 1, characterized in that, It also includes beneficial bacteria, Bacillus.

4. The evaluation method for safe use of the aquatic disinfectant according to claim 1, wherein The disinfectants include glutaraldehyde, benzalkonium bromide, and glutaraldehyde-benzalkonium bromide.

5. The evaluation method for the safe use of the aquatic disinfectant according to claim 1, characterized in that, The effect evaluation includes: A. Determine the inhibitory effect of the disinfectant on bacteria in aquaculture water according to MIC and MBC; 1) Determine the minimum bactericidal concentration MIC and minimum bactericidal concentration MBC of the selected disinfectant against the test bacteria; 2) Configure the concentration of the disinfectant used according to the MIC and MBC values; 3) Adjust the number of test bacteria to the order of magnitude of the bacteria in the aquaculture water and then add it to the water sample; 4) Subsequently, add disinfectants with different concentrations, measure OD600, and calculate the growth inhibitor; Growth inhibition rate = (positive control well - experimental well) / (positive control well - negative control well) × 100%.

6. The evaluation method for safe use of aquatic disinfectants according to claim 1 or 5, characterized in that, The effect evaluation also includes: B. Use PCR technology to detect the residual amount of microbial DNA after disinfectant treatment; Amplify the target DNA fragment of the microorganism through specific primers to detect the residual amount of microbial DNA after disinfectant treatment; If the disinfectant effectively kills the microorganism, its DNA will not be amplified due to cell rupture or degradation. By comparing the DNA concentration or amplification signal before and after treatment, the bactericidal effect can be quantified.

7. The evaluation method for the safe use of the aquatic disinfectant according to claim 1, characterized in that, The evaluation of the toxicity of the disinfectant is to use luminescent bacteria as the indicator organism and evaluate the toxicity of the disinfectant based on the principle that toxic substances inhibit the luminescence intensity of bacteria. The evaluation formula is as follows: Bacterial luminescence intensity inhibition rate (%) = [(luminescence value of the blank group - luminescence value of the sample group) / luminescence value of the blank group] × 100%. When the inhibition rate > 20%, it is determined that the disinfectant has biological toxicity.

8. The evaluation method for safe use of the aquatic disinfectant according to claim 1, wherein The determination of the safe dose is evaluated by combining the indoor disinfection effect and the on-site aquaculture substrate disinfection effect.

Images