Arsenic BCR Three Step Sequential Extraction and Microbial Community's Response Research in Soil Polluted by Steel Smelting with Depth

Five soil samples in different depths of a site in the close vicinity of a Wuhan iron and steel factory (Core 1) were collected. One uncontaminated soil profile (Core 2) was taken for reference purpose. The soil arsenic content and speciation vertical distribution patterns were analyzed by inductively coupled plasma mass spectrometry, and BCR sequential extraction protocol. The results showed that the arsenic content in Core 1 was remarkably higher than Core 2 in same depth, which indicated that microbial communities were very sensitively to indicate arsenic distribution and toxicity. Additionally, principal component analyses also revealed that the arsenic exchangeable fraction play an important role to indicate arsenic ecotoxicity in mining soil and sediment.  Environmental concern on arsenic (As) contamination has increased in the past few decades due to the increased awareness of as risk to plants, animal and human health. In most previous researches, focuses are mainly on investigating the total arsenic content in soil and sediment. This method is useful for evaluating the arsenic burden, however arsenic mobility depends strongly on the specific chemical species or bonding involved. consquently sequential extraction procedures which is a good compromise method that gives information on the environmental contamination risk[1,2] Today, the Community Bureau of Reference (BCR) sequential extraction procedures have been widely applied to soils and aquatic sediments to characterize their chemical phase associations [3 4] Despite experimental uncertainty with all presently available methods for solid phase metal fractionation [5, 6] they are probably the most useful tool for predicting longterm adverse effects from contaminated solid material [7] Most analytical and bioanalytical chemical techniques can be explored to assess the results of sequential extraction and then to confirm the distribution patterns of metals in soil profiles  These researches can help us understand the geochemical processes governing arsenic mobilization and potential ecological significance. Additionally, the feedback of soil microcosm on arsenic ecotoxicity is a very useful indicator to assess the hazard and toxicity of arsenic to our environment. The main purpose of this research was to investigate the arsenic vertical speciation distribution in steel smelting soil profiles using a BCR three step sequential extraction procedure and also to examine a rapid response of microbial community by colony forming unit protocol, respectively. It is important to study how arsenic speciation changes with soil depth and their influence to microbial response. In the end, by compared contaminated and uncontaminated soil profile, these analyses would be helpful to develop our cognition in arsenic dynamic and potential ecotoxicity from contaminated sludge and soil.  Two soil profiles were collected in Wuhan in Wuhan iron and steel factory, they were immediately put into plastic bags to prevent oxidation in the material at 277 K. All samples from one depth were 0020 4th Annual International Conference on Material Science and Engineering (ICMSE 2016) (c) 2016. The authors Published by Atlantis Press mixed. And then, they were immediately put into plastic bags to prevent oxidation in the material at 277 K. Afterwards, these samples were immediately transported back to the laboratory. In the laboratory, after air dried and homogenized by sieving to less than 2 mm to remove roots and large objects in sterile condition, soil samples were stored at 277 K. Each soil sample was divided into two portions for biological and physicochemical studies, respectively. The portion for biological properties determination was air dried at 301 K, and the portion for physicochemical properties determination was oven dried at 373 K. Arsenic Sequential Extraction Procedure First step (Exchangeable and weak acid soluble fraction) 1 g soil sample was extracted with 40 mL of acetic acid (0.11 M, pH 2.82) solution by shaking in a mechanical, end over end shaker at 30 10 rpm for 16 h at room temperature. The extract was separated from the solid residue by centrifugation at 3000 rpm for 20 min. The supernatant was collected into a beaker. The residue was treated similarly by adding doubly deionised water (10 mL x 2) as described above, but shaking only for 15 min. The supernatant was added to the beaker while taking special care to avoid any introduction of the solid residues. Second step (Reducible fraction) 40 mL of 0.5 M hydroxylammonium chloride solution (adjusted to pH 1.5 with 2 M nitric acid) was added to the residue from the first step, and the mixture was shaken 30 10 rpm for 16 h at room temperature. The acidification of this reagent was completed by adding a 2.5 (v/v) 2 M HNO3 solutions. The extract was separated and the residue was washed as in the first step. Third step (Oxidizable fraction) 10 mL of 8.8 M hydrogen peroxide solution was carefully added into the residue from the second step. The mixture was digested for one hour at room temperature and another hour at 358 K, and the volume was reduced to less than 2 mL. A second aliquot of 10 mL of H2O2 was added, the mixture was digested for 1 h at 358 K, and the volume was reduced to about 1 mL. The residue was extracted with 50 mL of ammonium acetate (1 M adjusted to pH 2.0 with conc. nitric acid) were added into the residue. The mixture was shaken on a magnetic stirrer at the speed of 30 rpm for 16 h at room temperature. Arsenic determination of extracts 2 mL of HNO3 (65 was added into the final extracts acquired from each step and the solutions were evaporated to almost dryness. Finally, 20 mL of aqua regia (3:1 conc. HCl/conc. HNO3) were added to the extracted and followed by microwave assisted digestion. The arsenic content of the digested extracts were determined by the quadrupole ICP MS system. Control samples were also processed and they were treated in exactly the same way as the test samples. In each experiment, four replicate test samples and two control samples were processed simultaneously. Blank tubes (containing reagent but no soils) were also taken through each complete BCR procedure.  The number of living microorganisms of mixed population was estimated by viable count on serial spread plates. Briefly, 0.1 mL of each serial dilution of sample suspension was spread over an agar (2 plate with beef extract peptone medium (0.05 beef extract, 0.05 peptone, 0.03 NaCl, w/v) containing 0.05 fungicidine for culturing bacteria, Martin's medium (1 glucose, 0.5 peptone, 0.1 K2HPO4, 0.05 MgSO4*7H2O, and 0.003 w/v Rose Bengal) containing 0.003 of streptomycin for culturing fungus (Aon M A et al. 2001) and Gause's No. 1 synthetic medium (0.1 KNO3, 2 soluble starch, 0.05 K2HPO4, 0.05 MgSO4*7H2O, 0.05 NaCl, 0.001 w/v FeSO4) for culturing actinomycetes(Zheng S X et al. 2007 Six continuous 10 fold dilutions of each sample were prepared starting with 9.0 mL sterilized phosphate buffered saline and 1.0 g of soil sample. The soil samples were taken in a sterile flask and all planting process were aseptically performed. After inoculation, plates (duplicates) were incubated either aerobically (total bacteria, fungi, and actinomycetes) or under microaerophilic conditions at 301 K. Microaerophilic incubation was carried out in an anaerobic glovebox (Coy Laboratories Products, Toepffer GmbH, Goppingen, Germany) Latter procedures were triplicate. All plates were incubated at 301 K in the 0021 4th Annual International Conference on Material Science and Engineering (ICMSE 2016) (c) 2016. The authors Published by Atlantis Press dark until colonies appeared (3 days for bacteria and 5 days for fungi and actinomycetes) The CFUs varied from 10 to 100 for fungi and from 30 to 300 for prokaryotes. Characterization All data were done in triplicate and mean values were taken for statistical analysis. The results obtained from various methods were analyzed by one factor ANOVA and followed by the t test using the Excel data analyses tool (Microsoft, Redmond, WA, USA) The statistical significance level was set at P 0.05. Principal component analyses (PCA) and correlation analyses (CA) were performed using the software version 12.0 SPSS for Windows (Chicago, IL, USA) and Origin 7.5 (OriginLab, MA, USA) respectively. A correlation was deemed to be significant when r 0.5. PCA is the simplest of the true eigenvector based multivariate analyses and can been considered as revealing an internal structure of the data in a way that can well explain the variance in the data.  In this study, arsenic fractionation pattern and vertical speciation distribution were analyzed by BCR three step sequential extraction procedure and depicted in Table 1. Table 1 Arsenic extraction yields of BCR three step sequential extraction Sampling depth (cm) Core 1 Exchangeable Reducible Oxidisable Residual 0 20 17 21 30 32 20 40 14 30 20 36 40 60 10 29 22 39 60 80 8 28 22 42 80 100 6 26 24 44 Sampling depth (cm) Core 2 Exchangeable Reducible Oxidisable Residual 0 20 20 25 25 30 20 40 18 26 21 35 40 60 17 27 19 37 60 80 15 24 20 41 80 100 10 25 22 43 The soil as content was divided into four different fractions after the BCR three step sequential extraction procedure. Szakova (1999) reported that 4 arsenic was extractable in the first step of the procedure, 9 and 12 arsenic were extracted in the second and third steps, respectively, and 75 arsenic remained in the residual fraction. Similarly, all four different fractions of arsenic in the soil samples were analyzed in this research and summarized in Table 1. It was found that the arsenic content in the exchangeable fraction was highest at the soil surface layer and it was then gradually decreased wi