Studies On The Ecotoxicity Of Roxarsone Residue

The organoarsenic roxarsone, originally used to aid in the control of coccidious as a additive added to poultry feed, is now used for growth stimulation, improved feed conversion, better feathering, increased egg production, and pigmentation. Roxarsone and arsanilic acid are two organoarsenic compounds permitted as animal feed additives in China, the former is used more than the latter. Roxarsone is excreted mainly as its parent drug in animal manure. During the 42-days growth period, each broiler excretes about 150 mg of roxasone, resulting in a total As concentration of 10-50 mg/kg in poultry litter. Poultry or livestock litter, which is a mixture of animal manure and bedding material, has widespread used as a fertilizer in the world. The roxarsone run into environment with the use of manure fertilizers, which usually be not treated by detoxicity technical process in China, may affect the quality of soil and surface or underground water.Heat shock proteins (HSPs) are severial families of proteins which organisms synthesize by inducing of stressors. HSPs is highly conserved, and play a important role in the survival of stressed cells and stabilization of internal environment. HSPs are usually identified for classes, namely the HSP90 family, the HSP70 family, the HSP60 family and the small molecular HSPs family. In HSPs, the HSP70 family is the most conversed, the most important family and more response to stress than the other family of HSPs. Studies show HSP70 can be used as a biomarker to several stresses, and is more used in the monitoring of environment pollution.The experimental doses of roxarsone are designed based on the level of roxarsone residue in animal letter. The ecotoxicity of roxarsone residue on soil were experimented by follow aspects. Firstly, the effects of different concentration of roxarsone residue on respiration, the NO₂-N concentration and the NH3-N content of experimental soil were studied. At the same time, the activity of phosphorlase, catalase, proteinase and urease of soil treated by roxarsone were also tested. Secondly, the vegetables of pakchoi cabbage and tomato were plated in the polluted soil treated by different level roxarsone. The growth parameters were measured and the changes of As concentration in these plants were monitored. The concentration curve of roxarsone in tested soil were graphed . Thirdly, the adsorption and mobility of roxarsone in soil were analyzed with various leaching solutions and with or no organic matter. Finally, the tissue specific induction of zebrafish HSP70 response to roxarsone was made for the use of HSP70 biomarker in the environment pollution .In the experiments of roxarsone pollute to soil, six groups were divided, the normal control, 20, 50, 80, 150 and 250 mg/kg roxarsone respectively. The effects of roxarsone residues on soil biochemical aspects (soil respiration, soil ammonification and soil nitrification) and the activity of phosphorlase, urease, catalase and proteinase of soil were measured. The results show that the effects of roxarsone residues were variable on these processes. The soil respiration was some enhanced during 4～6d after treating by roxarsone, but there was no significant difference(P>0.05). Comparing with the control group, the inhibitory effects on soil nitrification were more obvious with the roxarsone dose increased. The 80 mg/kg and 150mg/kg groups had significantly inhibitory effects over the whole experiment periods(P<0.05). The soil ammonification was weakened in 20 mg/kg and 50mg/kg roxarsone groups after treated 2d(P<0.05), and was strongly weakened at 80mg/kg and 150mg/kg doses(P<0.01). The activities of four enzymes were not decreased obviously, but were enhanced in some time. These results suggest that roxarsone residues affect the function of soil microbes, implying that the nitrogen cycle of soil treated may be inhibited.Pot experiments, in which a chicken manure with different concentrations of roxarsone were utilized in a garden soil, were conducted to study the effect of roxarsone pollution soil on growth of pakchoi cabbage and tomota as well as their roxarsone uptake. Results show weight, total length and leaf length of pakchoi were decreased at the more roxarsone dose in soil, but the length of root may be no affected visibly. Below the dose of 80mg/kg, roxarsone appear obviously enhancing effect to the weight and total length of tomota while it seems inhibitory phenomina at the higher level of 80mg/kg. The root length was reduced clearly at 150, 250mg/kg roxarsone in soil. The maximum uptake (total arsenic) in pakchoi and tomota come forth the time of 50 days planted. The elimination rate in tomota seem more rapidly than in pakchoi, the total arsenic content in tomota was below 3mg/kg up the day of 90th plated while the content in pakchoi was more than 12mg/kg till the plant had planted in soils treated by 150 and 250mg/kg roxarsone for 100 days. It is seemd that the accumlation of pakchoi to roxarsone may be higher than tomota to roxarsone.The high performance liquid chromatography method was developed for determination of roxarsone in soil. The chromatographic separation was performed on a C18 column (150×3.9mm, 4μm) using 0.05mol/L KH₂PO₄ : CH₃OH : 10%CH₃ COOH (80 :10 :10) as mobile phase at a rate of 0.8ml/min, temperature of 30℃, injection sample size of 20μl and UV detection at 264nm. The results demonstrated that a linear calibration curve of y=17.726x was prepared for roxarsone, the linear range was 0.10²0.0μg/mL with linear relative coefficient R2=0.9991. The average recoveries of different addition levels of 0.2, 2.0, 10.0mg/kg were 81.60±3.68%, 84.12±3.25% and 92.82±2.38% respectively with lower than 7% of RSD. The LOD of 0.04mg/kg and LOQ of 0.2mg/kg were observed. At 60th day planted, the roxarsone content in polluted soil was more than 6mg/kg (6.42～22.86mg/kg). It shows that the rate of roxarsone elimination may be slow, the half life of roxarsone in soil is about 22.6⁴4.9 days if the elimination is simulated on linear remove model.Using batch experiments, the sorption characteristics of roxarsone to Ap and Bt soils in our campus garden were investigated. Results demonstrated that roxarsone sorbed more to Bt soils than to Ap soil, but only showed moderate to low sorption onto experimental soils. The adsorption isotherms of roxarsone in soils fitted the Fetter linear equation well, and the adsorption values of roxarsone, calculated by Fetter equation, ranged from 0.1941 to 0.5979. Further calculation and analys were about KOC , the mean partial free energy and the free energy of adsorption of roxarsone on soil performed higher translocation in soils. Furthermore, it was found that the Kd values increased with decreasing of organic matter (OM ) , cation exchange capacity (CEC)content and the pH values of soil solution. It indicated that OM , CEC contents and the pH of soil solutions were the dominating factors influencing the roxarsone retention in all of the systems.For the mobility of roxarsone in soils, soil columns were leached with H2O, 0.01mol/L CaCl₂, 0.01mol/L EDTA-Na₂, and with or no poultry litter extract (PLE) solutions. The leaching effect of water to Ap and Bt2 soils was more than other two solution, so was the 0.01mol/L EDTA-Na₂ to Bt1 soil. The leaching rates of roxarsone with above three leaching reagents to soil added PLE were more than those without PLE, implying that the PLE decreased sorption of roxarsone in soils. Those three leaching solutions may leach out 72.3%～100.4% roxarsone from soils. Yet, there was not clear accumulation of roxarsone in soils. It was found that the transfer of roxarsone in soil showed obvious water solubility, therefore roxarsone may move into surface water or underground water.In the induction experiment of HSP70 in zebrafish exposed to roxarsone, the immunohistochemical analysis (SABC method) showed HSP70 positive express in gill, gonad and liver of zebrafish. The positive signals of HSP70 were extensively localized on the gonad and liver, particularly on the basilar part, branchialarch, epithelial cells of branchial plate in gill, stroma cell of gonad and in the cytoplasm of red cell. Furtherly, RT-PCR and real-time RT-PCR amplifing the hsp70, hsc70 and hsf1 mRNA were curried out becouse HSC70 and HSF1 were also closely related to stress. The quantitative results displaied the peak of hsp70, hsc70 and hsf1 mRNA express appeared at 12h exposed to roxarsone in gill, gonad and liver tissues. Comparing the mRNA express of same gene in normal control, change folds were 52.89, 34.83 and 59.31 fold of hsp70, hsc70 and hsf1 respectively in gill tissue, 26.43, 13.70 and 17.53 in gonad, 4.59, 3.82 and 3.32 in liver. Based on the relative abundance of hsp70, hsc70 and hsf1 mRNA at peak time, the express level of three genes was the highest in gill, moderate in gonad and the lowest in liver. From the time sequence, the induction express of these genes in gill was start-up firstly, in liver secondly and in gonad lastly. On all accounts, the HSP70 and reletive heat shock proteins appear a trend as abiomarkers use for further study in environment pollution of organoarsenic compounds.