| With the rapid development of China’s intensive aquaculture, aquaculture and achieved good economic and social benefits, however, with the rapid development of the aquaculture industry, expanding the scale of fanning, pollution of water bodies, so also will be a variety of diseases to their disease problems have become more prominent, seriously hampered the healthy development of the industry. And people’s quality of aquatic products have become more sophisticated, not only the requirements of aquatic pollution-free green products, disinfectants prefer aquatic efficient, low toxicity pollution-free direction, a new type of disinfectant has been widely applied.Quaternary ammonium salt iodine disinfectant is composed of quaternary ammonium compounds and iodine complexation and become, the surfactant of quaternary ammonium salt can change the permeability of cell membrane was damaged, enzyme system, effects of pathogens The new supersedes the old., play and help through the effect of carrier. And with the iodine complex, mild, diluted iodine and surface active agent is still closely, ensure the stability of iodine and germicidal. The disinfectant is a compound disinfectant can kill most pathogens, including bacteria, Mycoplasma, etc., and not light, heat, temperature, organic matter, pH and the impact of water quality, long-term storage effect diminished, the disinfection of non-corrosive and irritating objects, has been widely used in livestock, aquaculture farmers Quaternary phosphonium salt is a new generation of surfactants developed in recent years, has good bactericidal properties, known as the traditional quaternary ammonium germicides replacement products, aquaculture disinfectant is also changing from quaternary ammonium salts of phosphorus to the season. Quaternary phosphonium salt iodine is iodized disinfectants quaternary phosphonium salt disinfectant products, with high efficiency, broad-spectrum, low toxicity, low-dose, good use of a wide range of pH and chemical stability, etc., more and more widely used special aquatic breeding. Scientific use of disinfectants in aquaculture Disease Control and Prevention, and the elimination of the source of infection, improve water quality has a positive meaning, but the study found that a variety of disinfectants for breeding object has some toxicity, detoxification of the liver is the largest vertebrate and nutritional metabolic organ, is the main object pharmacology, physiology, and toxicology studies, and has a large number of studies indicate that the toxicity of in vitro method for detecting liver cells do not have many of the advantages of live animals, such as cells uniformity is good, very similar genetically; drugs and direct role in the cell, fast response; methods can be combined with a variety of techniques detect drug effects; more economical than with fish, convenient, can be used as an alternative model of living donor liver tissue study pharmacology, toxicology, physiology and other aspects. In this study, using different liver cell separation methods, compared the effects of the number, viability and proliferation of different separation methods for in vitro liver cells, liver cells in vitro culture system established, then carp (Carassius auratus), eel (Monopterus albus), of sturgeon (Acipenser dabryanus) in vitro cultured hepatocytes as an in vitro model, were studied by MTT assay iodine and quaternary phosphonium quaternary ammonium salt iodine on three kinds offish liver cells cytotoxic effects of two disinfectants for farming the potential toxicity offish, the main research results are as follows:(1) The impact of the separation method eel liver cell activityUsing tissue explants adherent method and enzymatic digestion (trypsin, collagenase, trypsin-EDTA, trypsin-EDTA-collagenase) for the separation of eel (Monopterus albus) hepatocyte cell number, cell viability and cell proliferation impact. The results showed that enzymatic digestion than adherent tissue, where the number of trypsin-EDTA-collagenase digestion graded cells obtained up to (1.30±0.06)×107个/g, resulting trypsin digestion of cell viability up to (90.2±1.2)%, the number of trypsin-EDTA-collagenase digestion of the obtained times of up to living cells (1.10±0.05)×107个/g. Cell proliferation increased first and then decreased, the largest number of cells in48h,144h after cell number decreased significantly.(2) Establish three kinds of fish liver cells in vitro culture systemAfter trypsin digestion and MTT assay established three kinds of fish in vitro toxicity testing system, the results show that using0.25%trypsin digestion under25℃0.5-1h or digestive16-18h at4℃for adjusting the cell density of5×105个mL, was added at96-well plate and cultured for24h after toxicity testing disinfectants, wherein the reaction time of4hours MTT final concentration of1mg/mL.(3) Quaternary ammonium iodide toxicity study of three species of fish liver cellsUsing three kinds of fish liver cells in vitro toxicity testing model, the quaternary ammonium iodide for three kinds of fish liver cells24h toxicity using the MTT assay results show that with the increase of the concentration of quaternary ammonium iodide, three kinds of fish liver cell viability of cells decreased; quaternary ammonium iodide concentration in1.56×10-4ml/L, the carp liver cell viability was95.80±13%, there was a significant difference compared with the control group, cell viability and quarter Natural salt iodine concentration in a range of a few good linear relationship, the regression equation y=-8.4911n (x)+8.2236(R2=0.9931), its IC50=7.303×10-3ml/L; quaternary ammonium salt iodine concentration was1.56×10-4ml/L, the eel liver cell survival was97.10±2%, there was a significant difference compared with the control group, cell viability and quaternary natural logarithm of the concentration of iodine in certain range was a good linear relationship, the regression equation y=-10.561ln (x)+2.2904(R2=0.9919), its IC50=1.092×10-2ml/L; quaternary ammonium iodide concentration in4.70×10-4ml/L when reached sturgeon hepatocyte survival was89.28±2%, there was a significant difference compared with the control group, cell viability and quaternary natural logarithm of the concentration of iodine in a certain range showed a good linear relationship regression equation y=-10.421n (x)+3.5972(R2=0.9953), its IC50=1.162×10-2ml/L; according to their IC50shows, quaternary ammonium iodide for three kinds of fish toxicity of Carassius auratus> Monopterus albus> Dabry’s sturgeon.(4) Quaternary phosphonium salt iodine toxicity study of three species offish liver cellsUsing three kinds of fish liver cells in vitro toxicity testing model to study the quaternary phosphonium salt iodine for three kinds of fish liver cells24h toxicity by MTT results showed that with increasing iodine concentration of quaternary phosphonium salt, three kinds of fish liver cell viability of cells decreased; phosphate salt iodine concentration in the quarter was6.72×10-5ml/L, the carp liver cell survival was93.11±8%, there was a significant difference compared with the control group, cell viability and quarter Natural phosphate salt iodine concentration in a range of a few good linear relationship, the regression equation y=-9.00921n (x)-1.7095(R2=0.9914), its IC50=3.217×10-3ml/L; phosphors in the quarter salt iodine concentration was6.27×10-5ml/L, the eel liver cell survival rate was87.12±3%, there was a significant difference compared with the control group, cell viability and quaternary natural logarithm of the concentration of iodine in certain range was a good linear relationship, the regression equation y=-10.0971n (x)-6.372(R2=0.9842), its IC50=3.763×10-3ml/L; phosphate salt iodine concentration in the quarter was6.27×10-5ml/L when reached sturgeon hepatocyte survival was95.05±5%, there was a significant difference compared with the control group, the natural cell viability and quaternary phosphonium salt iodine concentration in a range of a few good linear relationship, which regression equation y=-8.8751n (x)+6.8141(R2=0.9968), its IC50=7.700×10-3ml/L; according to their IC50shows, quaternary ammonium iodide for three kinds of fish toxicity of Carassius auratus> Monopterus albus>Dabry’s sturgeon. |
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