Bactericidal Effect and Clinical Application of 20% Sodium Dichloroisocyanurate Powder

Chengxin LYU Youjiang DIAO Shijin GUO

Abstract ［Objectives］This study was conducted to investigate the formulation stability of 20% sodium dichloroisocyanurate powder and its killing effect on common pathogenic bacteria in the animal breeding environment and to provide a scientific basis for veterinary clinical application.

［Methods］ The stability of 20% sodium dichloroisocyanurate powder was tested through an accelerated test and a long-term test. In the accelerated test, 20% sodium dichloroisocyanurate powder and sodium hypochlorite were stored in 37 ℃ constant temperature incubators for 90 d, and the available chlorine contents were compared. In the long-term test, 20% sodium dichloroisocyanurate powder was sealed and stored at room temperature, and its effective chlorine content and pH were measured regularly. In order to test its bactericidal effect in clinical use, 20% sodium dichloroisocyanurate powder, lime milk and 5% dilute glutaraldehyde solution were sprayed in groups to disinfect empty sheds in a pig farm, and the total colony count, Escherichia coli colony count, Staphylococcus aureus colony count and fungal colony count were used as testing indexes to comprehensively evaluate the disinfection effects on microorganisms in the air of the empty sheds and the surface of objects inside the sheds after disinfection.

［Results］ The 20% sodium dichloroisocyanurate powder had an available chlorine loss rate of 7.92% in the accelerated test, and the loss rates of available chlorine content and pH of 20% sodium dichloroisocyanurate powder in the long-term test (240 d) were 1.52% and 1.87% at room temperature, respectively. In the test to detect the sterilization effects on the pig farm, the total colony count, E. coli colony count, S. aureus colony count and fungal colony count of samples treated with 20% sodium dichloroisocyanurate powder were lower than those of other two groups (P<0.05), and the total colony count of samples disinfected with 20% sodium dichloroisocyanurate powder after cleaning the dirt was 20 times lower than that of samples disinfected directly without cleaning. In conclusion, 20% sodium dichloroisocyanurate powder was stable and effective in disinfection, and the presence of organic matter had little effect on the disinfection effect.

［Conclusions］ This study will provide important reference for the application of 20% sodium dichloroisocyanurate powder in veterinary clinic.

Key words Sodium dichloroisocyanurate; Stability; Disinfection; Clinic application

Received: June 16, 2022  Accepted: August 18, 2022

Chengxin LYU (2000-), female, P. R. China, major: veterinary medicine.

*Corresponding author. E-mail: gsj7877@126.com.

Environmental disinfection is an important measure for the biosecurity control of animal husbandry. It can prevent the spread of diseases by eliminating pathogenic microorganisms in the environment［1］. As an efficient disinfectant, sodium dichloroisocyanurate (C3N3O3Cl2Na) has characteristics of wide antimicrobial spectrum, fast disinfection speed, stable properties, small pH change, good water solubility, high active chlorine content, convenient transportation, good safety, high efficiency and low cost［2］. It is often used for disinfection of the environment and drinking water, and serves as a drinking water disinfectant recognized by the International Health Organization［3］. The disinfection mechanism is that sodium dichloroisocyanurate powder dissolves in water to form hypochlorous acid (HClO) and cyanuric acid (C3H3N3O3)［4］, of which hypochlorous acid molecules enter the cells of microorganisms and achieve a bactericidal effect through the chlorination reaction or oxidation reaction, which denatures bacterial proteins, changes membrane permeability, disturbs the physiology and biochemistry of the enzyme system, and affects the synthesis process of DNA［5］. Studies have shown that sodium dichloroisocyanurate has a strong killing effect on Newcastle disease virus, infectious bursal disease virus, avian influenza virus, foot and mouth disease virus, PRRSV, African swine fever virus, Escherichia coli, Salmonella and Staphylococcus. In this study, with the 20% sodium dichloroisocyanurate powder preparation produced by Shandong Lvdu Ante Animal Veterinary Drug Industry as the research object, the stability of the product and its killing effect on common pathogens in animal breeding environment were discussed, providing a scientific basis for veterinary clinical application.

Materials and Methods

Experimental materials

The experimental materials used: 20% sodium dichloroisocyanurate powder (Shandong Lvdu Ante Animal Veterinary Drug Industry 20210927); sodium hypochlorite (Shandong Lvdu Ante Animal Veterinary Drug Industry 20220120); quicklime (Shijiazhuang Huilu Building Materials Co., Ltd.), 5% dilute glutaraldehyde solution (Lvdu Ante Animal Veterinary Drug Industry 20210904); 301 type electric heating constant temperature incubator (Longkou Xianke Instrument Co., Ltd.); PHS-3EPH meter (Leici); SJ-CJ type ultra-clean workbench (Suzhou Zuoyou Purification Technology Co., Ltd.); high-pressure steam sterilizer (Ailaibao Technology Co., Ltd.); 301 type electric heating constant temperature incubator (Longkou Xianke Instrument Co., Ltd.); electronic balance (Shanghai Sartorius Instrument Co., Ltd.); nutrient agar medium (Beijing Land Bridge Technology Co., Ltd.); MacConkey agar (Beijing Land Bridge Technology Co., Ltd.); Baird-Parker medium (Beijing Zhonghai Biotech Co., Ltd.); agar powder (Beijing Solarbio Life Sciences Co., Ltd.).

Stability test

The stability test was carried out with available chlorine and pH value indexes［6-7］.

Accelerated test

The 20% sodium dichloroisocyanurate powder and sodium hypochlorite were respectively placed in a 37℃ constant temperature incubator for 90 d. The solution for effective chlorine content detection was prepared freshly for use. If the loss rate of available chlorine is less than or equal to 10%, the shelf life of the product can be set as 2 years; and if the loss rate of available chlorine is greater than or equal to 10%, the shelf life of the product is one year［8］.

③ Loss of available chlorine: Loss of available chlorine (mg/L)=Available chlorine before the test-Available chlorine after the test;

④ Loss rate of available chlorine: Loss rate of available chlorine (%)=［Loss of available chlorine /Available chlorine before the test］×100%.

Stability test

A room temperature sampling method was adopted for detection. The 20% sodium dichloroisocyanurate powder was stored at room temperature in a sealed state. Its available chlorine content and pH were checked every 30 d.

Determination method of available chlorine content

The available chlorine was detected by the indirect iodometric method of sodium thiosulfate recommended by the Chinese Veterinary Pharmacopoeia Committee［9］. An appropriate amount of sample was precisely weighed, and added in a 250 ml iodine bottle. Then, 100 ml of water was added to dissolve the sample, and 3 g of potassium iodide was added. The obtained solution was shaken to dissolve the potassium iodide, and then added with 20 ml of sulfuric acid solution (1→5). The solution was sealed, shaken and stood in a dark place for 5 min. The stopper and the inner wall of the bottle were washed with 5 ml of water, and titration was performed with 0.1 mol/L sodium thiosulfate to the near end point, that is, when the color turned yellow. At this time, 2 ml of starch indicator was added, after which the solution turned blue, and the titration was continued until the blue disappeared. The titration results were corrected with a blank test.

Content equivalent to the labeled amount(%)=V×T×CrW×Cs×1 000×100

In the formula, V is the volume of the titrant consumed by the test product (ml); T is the titer of the titrant according to the substance to be tested (g/ml); Cr is the actual concentration of titrant; W is the sample mass (mg); and Cs is the standard concentration of titrant.

Note: Each 1 ml of sodium thiosulfate titration solution (0.1 mol/L) is equivalent to 3.545 mg of Cl.

Disinfection effect test

Experimental treatment

Pig house situation: The test was carried out in a medium-sized pig farm in Binzhou City, Shandong Province. There were eight pig houses in total, with a single building stocking 500 pigs in a north-south direction. Four empty houses were randomly selected, one in each group, for disinfection treatment. The treatment methods for different groups are shown in Table 1.

During sampling, the doors and windows were closed for 15-30 min, and the number of indoor personnel, temperature and humidity was recorded. Five collection points were selected from each building, and five samples were collected in parallel at each collection point［10］.

Blank control: The pig house was comprehensively sprayed and disinfected from top to bottom directly using 20% sodium dichloroisocyanurate powder 1∶1 000 without physical cleaning.

Air microbial detection method: According to the natural sedimentation method reported by Wu et al.［8］, when a nutrient agar plate is exposed to the air, microorganisms will naturally settle on the nutrient agar plate through the action of gravity, and the number of colonies is obtained by counting the number of colonies after growing into colonies through laboratory culture［2］. The counting method was according to Part 3 of the National Standard Examination Methods for Public Places: Airborne microorganisms (GB/T 18204. 3-2013)［11］. Three points were evenly selected along the long axis of each pig house, and two points were selected at the same distance from the center of the pig house on a diagonal line, and a total of five sample points were collected［12］. A medium plate was placed at each sampling point 50-60 cm from the ground, opened and exposed to the air for 30 min to collect airborne microorganisms (total colony count, Escherichia coli, Staphylococcus aureus, and fungi). Ground, wall, railing detection method: After a cotton swab was soaked with neutralizer, an area of 25 cm2 (length and width of 5 cm) was selected to wipe once by constantly changing the swab surface, and then, the cotton end of the cotton swab was put into a sampling liquid.

Detection indexes

The collected samples were cultured (Table 2), and the medium used was the test medium.   The four detection indexes (total colony count, E. coli, S. aureus, and fungi) were counted simultaneously to judge the removal of microorganisms in each empty house by different disinfection modes. The counting method of the total number of colonies refers to Determination of the Total Colony Count (GB 4789.36-2016); the counting method of E. coli adopted the plate counting method, referring to Escherichia coli Counting (GB 4789.38-2012); the calculation method of S. aureus referred to the Determination Technology of Staphylococcus aureus (GB 4789.10-2010); and the calculation method of fungi was according to Counting of Viable Bacteria on Agar Plate (GB4789.39-2008).

Bacterial assay method

From a 10 ml of stock solution, 1 ml was taken from in a clean bench and diluted by 10-1.

① First, 9 ml of sterile saline was added to test tubes 1 to 11, and 1 ml of the sample to be tested was added in test tube 1, and the liquid was mixed by blowing and beating. From test tube 1, 1 ml of was pipette and added to test tube 2, and the liquid was mixed by blowing and beating. The same operation was repeated until test tube 11, from which 1 ml was drawn and discarded.  Test tube 12 was set as a blank control which was added with 1 ml of sterile normal saline.

② From each of four dilutions with appropriate dilution ratios and blank control, 1 ml was added to sterile medium. Then, 20 ml of medium was poured at a temperature of (45.5±1) ℃ onto the medium, and mixed well with it. The plates were laid flatly until the medium was solidified, and then inverted.

③ The medium was placed in a constant temperature incubator and cultivated.

④ Colonies were counted under an oil microscope. The total colony count without spreading growth between 30-300 CFU was taken.

Total colony count (CFU/cm2)=The number of colonies on the plate×Dilution factor÷25 (cm2, sampling area)［13］.

The results are shown in Table 5.

Conclusions and Discussion

Stability test

Available chlorine content is one of the quality standards for judging chlorine-containing disinfectants［14］. It can be seen from Table 3 that the loss rate of available chlorine of 20% sodium dichloroisocyanurate powder at 37 ℃ for 90 d was 7.92%, indicating that the loss rate of the active ingredient content of sterilization was less than 10%, so the shelf life could be set as two years. The loss rate of available chlorine of sodium hypochlorite at 37 ℃ for 90 d was 16.02%, so the shelf life of sodium hypochlorite was less than two years［15］. By comparison, it could be seen that 20% sodium dichloroisocyanurate powder had a longer shelf life and more stable properties. The results in Table 4 showed that after 20% sodium dichloroisocyanurate powder was stored at room temperature for 240 d, the available chlorine content and pH value were slightly reduced, and the loss rates were 1.52% and 1.87%, respectively. The data showed that the variation range of available chlorine content of the product is small, and the product properties are stable.

Discussion on the experiment for detecting the bactericidal effect on the pig farm

① It could be intuitively understood by counting the total number of bacteria in Table 5 that there were also a lot of bacteria in the environment after removing the dirt and dust. In the cleaning work of farms, if we simply clean up the garbage and dirt, it is far from enough. In order to achieve the environmental standard of low bacterial concentration in farms, suitable disinfectants must be used. The three disinfectants selected in this study are often used in the disinfection of breeding environments such as the ground, fences, and walls of sheds［16］. It can be seen from Table 5 that the total colony accounts with the use of disinfectants decreased significantly, and the value of total colony account decreased by 10 000 to 100 000 times［17］. From the counting results, the total number of bacteria in the samples disinfected with 20% sodium dichloroisocyanurate powder in group III was relatively small, and the overall number of bacteria decreased by 100 000 times, and was dozens of times lower than the number of bacterial colonies in groups II and IV; the E. coli colony count in the samples disinfected with 20% sodium dichloroisocyanurate powder was 100 000 times lower than that without disinfectants, which is basically consistent with the report of Zhang et al.［18］, and the value was 10 times lower than that of group II, and similar to the value of group IV; and the S. aureus colony count before unsterilization and the fungal colony count themselves were very low. The S. aureus colony count after disinfection with 20% sodium dichloroisocyanurate powder was 12 times lower than that without disinfectant, 2 times lower than that of group II, and similar to that of group IV; and the fungal colony count disinfected with 20% sodium dichloroisocyanurate powder decreased by 1 times compared with the data without disinfectant, and the decrease times was similar to the data of groups II and IV.

② The effects of physical cleaning on the sterilization effect of 20% sodium dichloroisocyanurate powder: The total colony account obtained from the blank control was 5.61×106, and the total colony account obtained from sterilization after physical cleaning was 2.84×105. The difference was nearly 20 times, which is basically consistent with the study results of Song et al.［19］.

The above results showed that 20% sodium dichloroisocyanurate powder was very stable during the shelf life, and its disinfection effect was better than those of lime milk and 5% dilute glutaraldehyde. The presence of organic matter had little effect on the disinfection effect of 20% sodium dichloroisocyanurate powder.

In summary, 20% sodium dichloroisocyanurate powder had stable properties and good sterilization and disinfection effect, and the existence of organic matter had little influence on the disinfection effect. This study provides a useful reference for the further promotion and application of 20% sodium dichloroisocyanurate powder in veterinary clinics.

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