Effects of Fungicide Iprodione and Nitrification Inhibitor

Effects of Fungicide Iprodione and Nitrification Inhibitor Effects of fungicide iprodione and nitrification inhibitor 3, 4-dimethylpyrazole phosphate on soil enzyme and bacterial properties Manyun Zhang a, b, à ¯Ã‚ Ã¢â‚¬ ºÃƒ ¯Ã¢â€š ¬Ã‚ ªÃƒ ¯Ã‚ Ã‚ , Weijin Wang a, c, Yaling Zhang a, Ying Teng b, à ¯Ã‚ Ã¢â‚¬ ºÃƒ ¯Ã¢â€š ¬Ã‚ ªÃƒ ¯Ã‚ Ã‚ , Zhihong Xu a, à ¯Ã‚ Ã¢â‚¬ ºÃƒ ¯Ã¢â€š ¬Ã‚ ªÃƒ ¯Ã‚ Ã‚  a Environmental Futures Research Institute, School of Natural Sciences, Griffith University, Brisbane, Queensland 4111, Australia b Key Laboratory of Soil Environment and Pollution Remediation, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008, China c Department of Science, Information Technology and Innovation, Dutton Park, Queensland 4102, Australia Abstract Agrochemical applications may have unintended detrimental effects on soil bacteria and soil health. However, limited studies have been conducted to evaluate the effects of repeated fungicide applications and interactive effects of different agrochemical applications on soil bacteria. In this study, an incubation experiment was established to evaluate the potential influences of fungicide iprodione and nitrification inhibitor 3, 4-dimethylpyrazole phosphate (DMPP) on soil enzyme and bacterial properties. Weekly iprodione applications decreased the activities of all enzymes tested, and single DMPP addition inhibited soil ÃŽ ²-glucosidase and urease activities. Compared with the blank control, bacterial 16S rRNA gene abundance significantly decreased following repeated iprodione applications, but increased after DMPP application. After 28 days of incubation, the treatment of iprodione and DMPP applications had higher bacterial 16S rRNA gene abundance than the counterpart with i prodione applications alone, and the similar trends were also observed in the Shannon index. Repeated iprodione applications significantlyincreased the relative abundance of Proteobacteria, but decreased the relative abundances of Chloroflexi and Acidobacteria. The combined applications of iprodione and DMPP increased the number of members of Gaiellaceae, Microbacteriaceae, Nocardioidaceae and Methylobacteriaceae. Simultaneously, bacterial community structure was significantly changed by repeated iprodione applications, alone or together with the DMPP. These results showed that repeated iprodione applications exerted negative effects on soil enzyme activities, bacterial biomass and community diversity. Moreover, relative to iprodione applications alone, extra DMPP application had the potential to alleviate the toxic effects of iprodione on soil bacterial biomass and community diversity. Keywords: agrochemicals; soil enzyme; 16S rRNA gene; bacterial community diversity and structure Introduction Fungicides play important roles in ensuring the crop quality and yield in modern agriculture (Maltby et al., 2009; Sabatier et al., 2014). The infections and phytopathies caused by fungi are the major problems and threats in agricultural production, which leads to the intensification in applications of fungicidal agrochemicals (OMaille, 2015). Previous research has shown that in some developing countries, fungicide application dosages were as high as 8 kg ha-1 y-1 (Liu et al., 2015). Iprodione, as a broad-spectrum fungicide, has been widely used in the intensive agriculture to control phytopathies of cash crops, and iprodione residuals have already been detected in water (Goewie et al., 1985; Sauret et al., 2006), soils (Leistra and Arrià «nne, 2004) and vegetable and fruit (Picà ³ et al., 2004; Juanà ¢Ã¢â€š ¬Ã‚ Garcà ­a et al., 2005; Angioni et al., 2012). According to a report of the Pesticide Data Program, U.S. Department of Agriculture (2014), the detected amount of iprodion e was the highest among all agrochemicals, and iprodione was the only fungicide detected in baby food. Besides the crop phytopathies caused by fungal pathogens, the lower utilization efficiency of nitrogen (N) fertilizer and nitrous oxide (N2O) emission are also worldwide problems in agricultural production (Clough et al., 2007; Menà ©ndez et al., 2012). As a result, nitrification inhibitors are sometimes applied to enhance the utilization efficiency of N fertilizer. The most widely used nitrification inhibitor in recent years is 3, 4-dimethylpyrazole phosphate (DMPP) (Menà ©ndez et al., 2012; Florio et al., 2014). The fungicide iprodione and nitrification inhibitor DMPP may be simultaneously applied into soils in some circumstances. Fungicides are designed to control fungal pathogens, but their lethal effects are not constrained to fungi only (Duah-Yentumi and Johnson, 1986; Muà ±oz-Leoz et al., 2011; Schnug et al., 2015). Once entering into agricultural soil, fungicides and their degradation metabolites may have detrimental effects on soil bacteria and, hence, the overall soil environment. There have been increasing research interests in the impacts of iprodione on environmental safety (Leistra and Matser, 2004; Verdenelli et al., 2012; Morales et al., 2013), because the iprodione is widely applied into agricultural soils at high dosages. Previous studies generally focused on the impacts of a single iprodione application, whereas few studies paid attention to the effects of repeated iprodione applications which occurs in intensively managed cropping systems. Furthermore, iprodione and other agrochemicals (such as DMPP) may be applied into agriculture soils simultaneously. To the best of our knowledge, few stud ies have been conducted to evaluate the interactive effects of different agrochemicals. Researches are, therefore, required to better understand the effects of combined iprodione and DMPP applications on soil bacterial properties. In this study, the iprodione and DMPP were applied into an agricultural soil. Soil enzyme activity, bacterial 16S rRNA gene abundance and bacterial community structure were determined. The main objectives were to (1) assess the effects of iprodione and DMPP on soil enzyme activities; (2) evaluate the impacts of agrochemicals on soil bacterial biomass via determining bacterial 16S rRNA gene abundance; (3) reveal the responses of soil bacteria at different taxa to the agrochemical applications; and (4) compare the potential impacts of iprodione and DMPP applications on soil bacterial community structure. This study will improve our current understanding of the ecological risks of iprodione and DMPP applications, alone or together, on soil nutrient cycling and bacterial properties. 2. Materials and methods 2.1. The chemicals and soil samples A commercial wettable powder formulation of iprodione (Bayer Crop Science, Hangzhou, China) and a chemical reagent DMPP (purity > 97.0%; CIVI-CHEM, Shanghai, China) were used for soil treatments. Soil samples were taken from a farmland (36.78†² N, 118.67†² E) located in Shandong Province, China. The surface soil (0-20 cm) was collected, air-dried at room temperature, mixed thoroughly and sieved (< 2 mm) prior to use. The selected physical and chemical properties of the soil were as follows: sand (50-2000 ÃŽ ¼m), 31.4  ± 1.4%; silt (2-50 ÃŽ ¼m), 36.9  ± 0.8%;   clay (< 2 ÃŽ ¼m), 31.7  ± 0.6%; soil pH (in water), 7.19  ± 0.05; organic carbon (C) content, 10.0  ± 0.1 g kg-1; total N content, 0.93  ± 0.01 g kg-1; Olsen-P, 28.8  ± 0.2 mg kg-1;   NH4OAc-K, 69.9  ± 1.5 mg kg-1; cationic exchange capacity,   16.9  ± 0.4 cmol kg-1. All treatments were added with urea at 200 mg N kg-1 dry soil before the iprodione or DMPP application so that enough substrate ( NH4+-N) was available for soil nitrification (DMPP is generally applied with the urea in agriculture). 2.2. Experimental design Four treatments were used in this study: Treatment 1 (CK), without any iprodione or DMPP applications; Treatment 2 (IPR), weekly iprodione applications at 1.5 mg kg-1 dry soil (the frequency followed the instructions); Treatment 3 (DAA), nitrification inhibitor DMPP application at 2 mg kg-1 dry soil (equivalent to 1% of applied urea-N) at commencement; and Treatment 4 (I+D), weekly iprodione and initial DMPP applications as described in treatments 2 and 3. Each treatment was prepared in triplicates. The chemicals were dissolved in double distilled H2O (ddH2O) and then applied into the test soil. Sixty glass bottles (4 treatments 5 sampling time 3 replications) were filled with the treated soil at 150 g dry weight per bottle. Soil moisture was adjusted to 60% of the water holding capacity and was maintained by the additions of ddH2O. The soil samples were then incubated at 28  °C in the dark, and after 0, 7, 14, 21 and 28 days of incubation, soil samples were collected after mixin g thoroughly in each bottle for analyses of soil enzyme and bacterial properties. 2.3. Soil enzyme activity and geometric mean of assayed enzymes activities Soil ÃŽ ²-glucosidase activity was determined using a soil enzyme assay kit (Catalogue No. HK000218, Toyongbio Company, Shanhai, China). The analytical method was based on that soil ÃŽ ²-glucosidase can hydrolyze p-nitrophenyl-ÃŽ ²-D-glucoside to generate p-nitrophenol. The determination method followed the manufacturers protocol, after treated with toluene, soil samples were incubated with the p-nitrophenyl-ÃŽ ²-d-glucoside and citrate-phosphate buffer (pH = 6.0) for 1 h at 37  °C. The reaction product (p-nitrophenol) concentration was determined with a spectrophotometer at 410 nm, and the results were expressed as ÃŽ ¼g p-nitrophenol g-1 dry soil d-1. Soil urease and phosphatase (acid phosphatase and alkaline phosphatase) activities were determined with the commercially available quantitative analytical kits (Jiancheng Bioengineering Institute, Nanjing, China). In the analytical kit of soil urease (Catalogue No. T017), urea was used as the substrate that can be hydrolyzed by soil urease to generate NH 4+-N. Prior to the urease determination, the test soils were treated with the toluene, and then they were incubation with the urea and citrate buffer (pH = 6.7) for 24 h at 37  °C. The concentration of NH4+-N generated from urea hydrolysis was determined via the indophenol blue method, and soil urease activity was expressed as ÃŽ ¼g NH 4+-N g-1 dry soil d-1. Soil phosphatase activity was determined with disodium phenyl phosphate as the enzyme reaction substrate. Soil phosphatase could hydrolyze the disodium phenyl phosphate at different pH conditions, and in the analytical kits of soil phosphatase and alkaline phosphatase (Catalogue No. T008 and T009), the buffers were acetate buffer (pH = 5) and borate buffer (pH = 9.4), respectively.   After 24 h of incubation at 37  °C, enzyme actions were terminated, and released phenol was determined at 660 nm. Soil phosphatase activities were expressed as ÃŽ ¼g phenol g-1 dry soil d-1. Soil arylsulphatase activity was determined according to the method of Floch et al. (2009) with minor modifications. P-nitrophenyl-sulfate was used as the reaction substrate, and the buffer was 0.5 M acetate (pH = 5.8). After 1 h of incubation, the reaction was halted by the additions of 0.5 M CaCl2 and 1 M NaOH, and the p-nitrophenol concentration was measured with a spectrophotometer at 410 nm. The soil arylsulphatase activity was expressed as ÃŽ ¼g p-nitrophenol g-1 dry soil d-1. The geometric mean of assayed enzymes activities (GMEA) was calculated to integrate data from variables that have different units and variation range. The calculations were made by following Hinojosa et al. (2004). where Glu, Ure, AcP, AlP and Ary were soil ÃŽ ²-glucosidase, urease, acid phosphatase, alkaline phosphatase and arylsulfatase activities, respectively. 2.4. Soil DNA extraction and real time quantitative PCR (qPCR) The genomic DNA of soil samples was extracted from approximately 0.5 g of soil with a Fast DNA SPIN Kit for Soil (MP Biomedicals, Cleveland, OH, USA). Soil bacterial biomass was revealed via determining bacterial 16S rRNA gene abundance, and qPCR was performed to assess bacterial 16S rRNA gene abundance with the universal primers 515F (5-GTGCCAGCMGCCGCGG-3) and 907R (5-CCGTCAATTCMTTTRAGTTT-3). The standard curve of qPCR was obtained by performing serial dilutions of the plasmid containing bacterial 16S rRNA gene. Each qPCR reaction was prepared in a 20.0 ÃŽ ¼L of solution consisting of 10.0 ÃŽ ¼L of SYBR ® Premix Ex Taqâ„ ¢ (TaKaRa Biotech, Dalian, China), 7.6 ÃŽ ¼L of sterile ddH2O, 2.0 ÃŽ ¼L of soil template DNA and 0.4 ÃŽ ¼L of universal primers. The thermocycling conditions were as follows: 95  °C for 3 min, followed by 45 cycles at 95  °C for 10 s, 56  °C for 30 s, 72  °C for 30 s, and then plate reading. The melting curve analyses were conducted by gradually heatin g the PCR mixtures from 65  °C to 95  °C with the determination of SYBR green signal. Negative control was run with sterile ddH2O as the template. After testing the diluted soil DNA suspension, there were no inhibitions detected for the qPCR, and the amplification efficiency of the target gene was 114.52%, with R2 > 0.995. 2.5. The Illumina MiSeq and sequenced data analysis At the end of incubation, soil bacterial community was also analyzed with the Illumina MiSeq platform. After amplifying the V4 region of bacterial 16S rRNA gene with the primers 515F/907R, the PCR products were purified and then subjected to the Illumina Miseq platform (Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China) to sequence nucleic acid bases of PCR products. The raw reads were de-multiplexed and filtered via the QIIME (version 1.17) with reference to the following standards: (1) The approximately 300 bp reads were truncated at the end side, receiving an average quality score of < 20 over a 10 bp sliding window, and shorter sequence reads (truncated length < 50 bp) were discarded; (2) raw reads with vague bases were discarded; and (3) only sequences with > 10 bp overlap were assembled with reference to the overlapped sequences, and the unassembled reads were unwanted. Operational taxonomic units (OTUs) were clustered within a 0.03 difference via the UPARSE (version 7.1 http://drive5.com/uparse/) for assessing community richness (Ace and Chao1 richness estimators) and community diversity (Shannon and Simpson indices). The richness estimators Ace and Chao1 were nonparametric and abundance-based estimators, which could predict the true value of taxa based on the proportion of rare taxa in a sample and were suitable for community richness ana lysis (Sogin et al., 2006; Gihring et al., 2012). The Shannon and Simpson indices were used for heterogeneity assessment, and the main difference between them was in the calculation method of taxa abundance. 2.6. Statistical analysis Two-way analysis of variance was conducted to detect significant differences among treatments, sampling times and their interactions, and Duncans multiple range test was used to compare statistical significances (P < 0.05) of the means among different treatments at each sampling time and among different incubation time in a specific treatment. Linear discriminant analysis effect size (LEfSe) method (http://huttenhower.sph.harvard.edu/galaxy/root) was employed to reveal biomarkers of soil bacteria among the treatments. The sequencing data were further processed to assess similarities and discrepancies of the whole bacterial community structure among different treatments using the principal coordinate analysis (PCoA) and the unweighted pair group method with arithmetic mean (UPGMA). 3. Results 3.1. Soil enzyme activities and GMEA Soil enzyme activities were significantly affected by the treatments, but the interactions between treatments and sampling time were not significant for the ÃŽ ²-glucosidase and alkaline phosphatase activities (Table S1). The ÃŽ ²-glucosidase, urease and arylsulfatase activities in the CK treatment increased significantly during the first 7 days of incubation (P < 0.05, Fig. 1). By the end of the incubation, the soil enzyme activity in the CK treatment rose from 1004  ± 51 to 1278  ± 133 ÃŽ ¼g p-nitrophenol g-1 dry soil d-1 for ÃŽ ²-glucosidase, from 60.9  ± 4.9 to 323.0  ± 19.6 ÃŽ ¼g NH4+-N g-1 dry soil d-1 for urease, from 698  ± 129 to 802    ± 76 ÃŽ ¼g phenol g-1 dry soil d-1 for acid phosphatase, from 2.21  ± 0.12 to 2.23  ± 0.13 ÃŽ ¼g phenol g-1 dry soil d-1 for alkaline phosphatase, and   from 20.9  ± 2.7 to 28.8  ± 2.3 ÃŽ ¼g p-nitrophenol g-1 dry soil h-1 for sulphatase. After 7 days of incubation, there were no significant differences in soil enzyme a ctivities between IPR and CK treatments, with the exception of acid phosphatase activity. However, soil enzyme activities in the IPR treatment decreased after repeated iprodione applications. DMPP application had no significant effects on phosphatase and arylsulphatase activities during the whole incubation period, but ÃŽ ²-glucosidase and urease activities were consistently inhibited by the DMPP application (89.8% and 76.5% % of the control after 28 days of incubation, respectively). It is interesting to note that, after 28 days of incubation, the activities of soil enzymes in the I+D treatment tended to be higher than those in the IPR treatment (Fig. 1). As shown in Table 1, the GMEA in the CK treatment increased significantly during the first week and remained relatively stable during the following three weeks of incubation. However, compared with the CK treatment, the GMEA was negatively affected by repeated iprodione applications, and the GMEA in the DAA treatment also tended to be lower than their counterparts from 7 days to the end of the incubation (88.9% of the control after 28 days of incubation). Consistent with the trend presented in Fig.1, the GMEA in the I+D treatment was significantly higher than its IPR counterpart after 28 days of incubation (P < 0.05). 3.2. Soil bacterial 16S rRNA gene abundances Both the treatments and sampling time could significantly affect soil bacterial 16S rRNA gene abundances (Table S1). The gene abundances in the CK treatment increased from 3.89-109 to 1.64-1010 copies g-1 during the 28 days of incubation (Fig. 2). A single iprodione application generated negligible effect on soil bacterial 16S rRNA gene abundance after 7 days, but repeated iprodione applications significantly (P < 0.05) decreased soil bacterial 16S rRNA gene abundance, relative to the CK treatment. At the end of the experiment, the bacterial 16S rRNA gene abundance in the IPR treatment was 6.55-109 copies g-1 dry soil, which was the lowest of the four treatments. The gene abundances in the DAA treatment tended to be higher than those in the CK treatment, especially from 14 days onwards. Relative to the iprodione applications alone, extra DMPP application at the commencement increased the gene abundances, after 28 days of incubation, bacterial 16S rRNA gene abundance in the I+D treatm ent was significantly (P < 0.05) higher than that from the IPR treatment. 3.3. Soil bacterial community diversity A total of 624067 raw sequences (> 200 bp) was obtained from the four treatments, and the average length of valid sequences was 396.71. The similarities and differences among OTUs of the four treatments were demonstrated in a four-set Venn diagram (Fig. 3). The unique OTUs were 12, 16, 13 and 22 for the CK, IPR, DAA and I+D treatments, respectively, and the four treatments shared 1653 OTUs. After 28 days of incubation, the Ace and Chao1 estimators, and Shannon index of the CK treatments were 1863  ± 37, 1853  ± 24 and 5.55  ± 0.15, respectively (Table 2). The IPR treatment had the lowest estimator Chao1 (1772  ± 17) and Shannon index (5.40  ± 0.05) among the four treatments, while the I+D had the highest estimator Chao1 and Shannon index. The Simpson index showed no significant differences among the four treatments. Relative to the CK treatment, DMPP application did not exert adverse effects on richness estimators and diversity indices, and DMPP applications had the potential to increase the Shannon indices. 3.4. Soil bacterial community structure The OTUs could be assigned into 11 predominant phyla which were in the following ranking orders: Proteobacteria, Actinobacteria, Gemmatimonadetes, Chloroflexi, Firmicutes, Acidobacteria, Bacteroidetes, Planctomycetes, Nitrospirae, Saccharibacteria and Verrucomicrobia, and the relative abundances of these phyla varied among the different treatments (Fig. 4A). The phylum Proteobacteria was the most abundant, comprising approximately 37.2%, 45.9%, 36.4% and 39.8% OTUs for the CK, IPR, DAA and I+D treatments, respectively. The phylum Actinobacteria was the second most abundant, and the proportions varied from 14.5% to 21.9%. There were no significant differences of relative abundances across all the predominant phyla between the CK and DAA treatments (Fig. 4B). However, compared with the CK treatment, repeated iprodione applications significantly (P < 0.05) increased the relative abundance of phylum Proteobacteria, but decreased the relative abundances of Chloroflexi and Acidobacteria. A t the genus level, the genera Micromonospora, Gemmatimonas, Haliangium and Bacillus accounted for large proportions in the twelve soil samples (Fig. 5). 3.5. Comparison of bacterial community structure LEfSe analysis demonstrated that there were significant associations among predominant bacterial taxa in the four treatments (Fig. 6). The predominant bacterial taxa were the Opitutaceae and Xanthomonadales families in the CK treatment, the Xanthomonadaceae, Erythrobacteraceae, Sphingomonadaceae Sphingomonadales and Rhodocyclaceae families in the IPR treatment, the Streptomycetales Order and the Gemmatimonaceae and Cytophagaceae families in the DAA treatment and the Gaiellaceae, Microbacteriaceae, Nocardioidaceae, and Methylobacteriaceae families in the I+D treatment. The results of LEfSe analysis further revealed that, consistent with the relative abundances shown in Fig. 4, the phylum Protebacteria increased following iprodione applications. A two-dimensional PCoA plot of bacterial community structure explained 68.2% of the total variance, with the PCoA1 having a greater power of separation (accounting for 57.7%). The two-dimensional PCoA demonstrated that the treatments without iprodione applications (CK and DAA treatments) resulted in a shift to the left along PCoA1 (Fig. 7A). There was no significant difference in the PCoA values (both PCoA1 and PCoA2) between the CK and DAA treatments, and this suggested that soil bacterial community structure was not significantly altered by the DMPP application. However, iprodione applications alone (IPR) or together with the DMPP (I+D), shifted the bacterial community to the right side along PCoA1. The IPR and I+D treatments were located in the opposite directions of the origin, with the I+D treatment having lower PCoA2 values. Consistent with the trends presented in the PCoA plot, four clusters could be grouped for these twelve soil samples (Fig. 7B): Cluster 1 contained the samp les of the DAA treatment, CK_1 and CK_2, but the CK_3 sample alone was classified into Cluster 2; Cluster 3 consisted of the samples of I+D treatment, and the samples of IPR treatment were all grouped in Cluster 4. 4. Discussion 4.1. Effects of iprodione applications on soil enzyme and bacteria As a soil xenobiotic, the fungicide iprodione displayed toxicity to soil bacteria on various aspects including the activity, biomass and community diversity (Duah-Yentumi and Johnson, 1986; Verdenelli et al., 2012). Previous researches have also revealed that the principal degradation metabolite 3, 5-dichloroanniline is more biologically toxic and stable than its parent compound iprodione (Athiel et al., 1995). Consequently, both the fungicide and its degradation metabolites have the potential to inhibit the non-target bacteria. On the other hand, the broad-spectrum fungicide could inhibit the flourish of fungi, which can also exhibit the indirect impacts on soil bacteria community (Muà ±oz-Leoz et al., 2011; Verdenelli et al., 2012). The ÃŽ ²-glucosidase, urease, phosphatase, and arylsulfatase are essential in the cycling of C, N, P and S in soil, respectively (Muà ±oz-Leoz et al., 2011). The activities of these enzymes tended to decline after repeated iprodione applications, highlighting the severe impacts of repeated iprodione applications and perhaps the accumulations of its metabolites on soil nutrient cycling. The fungicides could negatively affect soil enzyme activities as a result of: (1) directly reducing the biomass of soil microbes that produce enzymes, (2) competing for the active sites of enzymes with substrates, (3) decreasing the substrate bio-availability through the reaction with substrates, and (4) reacting with the enzyme-substrate complexes (Wang et al., 2009). In the IPR treatment, soil enzyme activities and bacterial 16S rRNA gene abundances decreased concurrently. We postulated that the declines of soil bacterial biomass caused by iprodione applications might have resulted in the decreases of soil enzyme activity. Soil bacterial 16S rRNA gene (both the abundance and community diversity) has been used as an important ecophysiological index for assessing soil contamination (Sipilà ¤ et al., 2008; Bell et al., 2014). In this study, soil bacterial 16S rRNA gene abundance was not significantly affected by the first iprodione application, but decreased with repeated iprodione applications (Fig. 2). The result was consistent with earlier finding of Duah-Yentumi and Johnson (1986) that the impacts of iprodione on soil microbial biomass differed between single and repeated applications. The reasons for these phenomena might be that accumulations of the fungicides and perhaps their degradation products following repeated application increased its eco-toxicity (Trabue et al., 2001; Zhang et al., 2016). Direct measurement of soil bacterial community could reveal shifts in the diversity due to fungicide applications, which might not be detectable by measuring overall bacterial activities and biomass (Lupwayi et al., 2009). Repeated iprodione applications resulted in consistent reductions in the values of bacterial alpha diversity (Table 2). The result was in agreement with the finding of Verdenelli et al. (2012) that iprodione application had significantly negative impacts on microbial community diversity in both agricultural and grassland soils. Moreover, repeated iprodione applications led to declines in the relative abundances of phyla Chloroflexi and Acidobacteria (Fig. 4). The Chloroflexi is associated with the second step of soil nitrification and plays key roles in soil N cycling (Sorokin et al., 2012). As decomposers in soil environment, Acidobacteria could degrade torganic matters derived from plants and soil animals, maintaining soil nutrient cycling and energy flow (Ward e t al., 2009). These changes in the relative abundances of functional bacteria indicated that iprodione applications might slow down soil organic matter turnover and soil nitrification.In this study, we found that repeated iprodione applications, alone or together with the DMPP, caused significant changes in the soil bacterial community structure. In contrast, Wang et al. (2004) showed that a single iprodione application at lower dosage was not detrimental to the soil bacterial community. Given that iprodione was often repeatedly applied at high dosages in intensive agricultural systems, shifts in bacterial community as observed in this study could lead to a series of alternations in soil microbial communities and soil C and nutrient cycling. Therefore, more attention should be paid to the long-term ecotoxic effects caused by repeated applications. 4.2. Effects of DMPP application on soil enzyme activities and bacteria Compared with the CK treatment, DMPP application resulted in significantly lower soil urease activity, (Fig. 1), which was largely responsible for the decline in GMEA in the DAA treatment. It is interesting to note that soil ÃŽ ²-glucosidase, as a proxy for soil organic matter mineralization capacity, was also negatively affected by the DMPP application. Maienza et al. (2014) revealed that DMPP application had adverse impacts on the growth of soil heterotrophic bacteria and fungi. All these results suggested that DMPP might have the potential to slow down soil organic matter decompositions, and this could get supports from previous research results that DMPP could reduce soil carbon dioxide (CO2) and methane (CH4) emissions (Weiske et al., 2001; Maris et al., 2015). The LEfSe analysis revealed that Gemmatimonadetes and Cytophagia families increased following DMPP application (Fig. 6). The Gemmatimonadetes containing photosynthesis genes could assimilate CO2 into organic material via phototrophic pathway and transform solar radiation into metabolic energy, which plays an important role in the increase of soil organic matter content (Zeng et al., 2014). Some strains of the Cytophagaceae family have the nifH gene and have the potential to increase soil N content by biological N fixation (Xu et al., 2014). Dong et al. (2013b) also reported that DMPP application could significantly increase soil nifH gene abundances. These results indicated that apart from inhibiting soil nitrification, DMPP application might have the potential to improve soil C and N contents via (1) decreasing CO2, CH4 andNOX emission;(2) slowing down soil organic matter decompositions; (3) promoting the flourish of some functional microorganisms; and (4) promoting activity of N-fixing bacteria. Consequently, although soil urease and ÃŽ ²-glucosidase were inhibited, the whole soil bacterial biomass increased following DMPP application (Fig. 2), and this is also one of the positive effects generate d by DMPP application. An increase in soil bacterial biomass is commonly found in parallel with increases in community diversity (Weinbauer et al., 2007; Torstensson et al., 2015). Based on the data presented in Table 2, we found that DMPP application promoted, rather than decreased, soil bacterial community diversity, which is consisted with the result of Dong et al. (2013a). Furthermore, both the PCoA and UPMGA indicated that a large proportion of soil bacterial community in the DAA treatment overlapped with that in the C

Ebara Balance Scorecard and Communication Plan Essay

Ebara Technology Incorporated Component Division is a subsidiary of Ebara Corporation. Ebara Technology is known as a Research and Development focus company, whose main subject matter is on research and development for its business operations. Ebara will always campaign for research and development to increase added value to the product and over and above research and development related cost reduction and manufacturing technologies, toward improving it competitiveness and profitability, (R&D – Ebara (n.d.)). Finance The financial perspective has the strategic objective in areas of the market shares, revenues and costs, profitability, and competitive position. On the financial viewpoint of the new F-REX 600 millimeter Chemical Mechanical Polishing System will increase the sales growth anywhere from 10% to 20%. This modification or enhancement will dictate the end results of decreasing in the cost, increase higher quality and speed, for a prompt response to customers’ needs. The operating cost will decrease anywhere from 15% to 25% for the reason that on common parts the volume will increase and with contracts already in place the forecast will go up and the price will go down. A small number of ideas turn out to be profitable for the reason of research and development, and pre-marketing costs of transforming a promising idea into a profitable product. Customer In the customer value perspective we want to look at how the customers see us in all these areas, customer retention or turnover, customer satisfaction and customer value. Our goal of retention rate is to recognize the turnover and to monitor the performance over time. â€Å"The main objective is not to be at zero, however to be at a position that is supporting our goals for  growth† (Customer Retention Strategy | Marketing MO. (n.d.)). Developing plans to increase customer loyalty and decrease turnover. Our company will set up meeting on a quarterly basis with customers with the agenda being directed at on-time delivery percent of sale from the new product, and feedback from the customers. On time delivery will be recorded and reviewed to communicate to our shareholders and employees. The company as a whole will be working with all current customers to maintain a goal 98% or better delivery performance. Feedback whether it is positive or negative from our customer is vital action for the growth of the company. Internal Business Process The internal business process is to construct a streamline process for the manufacturing process. Manufacturing objective is make sure they have create a useful and efficient process that will imposed check points at all operating locations. Paperwork will be turned in and the information is loaded into the database. The target is recorded and a weekly or monthly report is run. These reports are called Key Performance Indicator (KPI) or Key Success Indicator (KSI). â€Å"These reports are a set of quantifiable measures that the company or an industry uses to gauge or compare performance in team meeting of the strategic and operational goal† (Key Performance Indicators (KPI) Definition | Investopedia. (n.d.). These reports will be posted in all appropriate departments and meetings are set-up for discussion to talk about great performance or what we need to improve on. Learning and Growth The learning and growth of the company will depend widely on the knowledge and training of all employees. In order to achieve our vision production must be able to be ready for changes and the ability to improve. Our goal will be focus in two different areas; the first will be the production employees and the second will be document control. Our intention is for all managers and executives to keep and open communication with every employee in the company and all outsourcing suppliers of all pending and up and coming changes. Every employee and the outsourcing suppliers will go through a training program on how to build all areas of F-REX 600 millimeter Chemical Mechanical Polishing System.. Our aspiration would be to ensure  that each outside service and each and every in-house employee are trained and certified in their specified areas. Eventually we would want to see some cross-training in all the areas. On the Document Control side will also be following the same step listed above, but because changes will need to be made rapidly and some changes will need to be phased in. This department must be knowledgeable of all aspect of the Engineering Changes Notices and the Engineering Changes Orders. The goal for this department is to be conscious and mindful of Engineering Changes Orders and Engineering Changes Notices. The focus would be to make certain they have all signatures and it is approval through all levels. We will need to proceed by requiring training, cross training and certification of each of employees in this department. Conclusion The supposition of the balanced scorecard we covered all four areas: The financial side with building of the F-REX 600 millimeter Chemical Mechanical Polishing System we will be decreasing the cost, increase higher quality and speed, where the customer is getting their needs met. This proposal will bring profit because of the research and development, and pre-marketing cost changing ideas into a profitable product. Secondly on the customer perspective side, the goal is increase customers loyalty and always looking for ways to decrease the turnover rate. Customer satisfaction and customer value by encouraging face to face meeting to keep client/customers informed. The internal process is where we will find ways to improve the process. Key Performance Indicator will be set up in difference department so that each department knows how they are performing. And last is the learning and growth of the employees. We will focus on the production employees and Document Control . Training and c rossing training is a major area because of all the changes that will forthcoming. Document Control will be overwhelmed with countless changes and will need to be prime and ready for the surplus. References Customer Retention Strategy | Marketing MO. (n.d.). Retrieved from http://www.marketingmo.com/strategic-planning/customer-retention-strategy/ Key Performance Indicators (KPI) Definition | Investopedia. (n.d.). Retrieved October 16, 2014, from http://www.investopedia.com/terms/k/kpi.asp R&D – EBARA – Global Website. (n.d.). Retrieved September 26, 2014, from http://www.ebara.com/global/en/company/rd.html

Counseling Ethics Essay

Ethics are based on philosophical principles and these guidelines assist a practitioner in making the best possible decisions for the welfare of the clients and the practitioner himself. Ethics are normative or critical in nature and concern human conduct and moral decision. Morality describes decision making and judgement by an individual concerning an incident or human behaviour. This is greatly influenced by the values he or she has acquired or formed as a result of external influence or indoctrination. Value is an enduring belief that a specific that a specific end-state of conduct is desirable (McLeod, 1998). Terminal and instrumental values are two types of values where the former refer to the desired end-state of existence, for example wisdom and the later refer to the mode of conduct that leads to it, for example broad-mindedness .Values then influence and determine the decisions we choose to make in our daily lives. In providing an effective, therapeutic therapy, a practitioner helping a client encountering dilemma in decision- making may adopt the eight – step model approach to think through the ethical problems (Corey, Corey & Callanan, 2007). The steps of the model are described as follows: Step 1- Identify the problem or dilemma. In the first step the existence of the problem must be recognised. The nature of the problem has to be ascertained. Identify if it is an ethical, legal, moral, professional or clinical problem. The practitioner’s and the client’s insights regarding the problem must be examined. Consultation with the client can begin at this stage as problems are being identified. Looking at the problem from different perspectives is useful as most ethical dilemmas are complex. Step 2 – Identify the potential issues involved. From the collected information, irrelevant ones must be discarded. The critical issues must be noted and described. The welfare of those involved; their rights and responsibilities must be evaluated. Ethical principles relevant to the problem must be identified and examined with the client. In doing so the moral principles namely, autonomy, non-maleficence, beneficence, justice and fidelity must be considered and applied to the  situation. The safety and welfare of the client and practitioner cannot be compromised while potential issues are being sorted out. Step 3- Review the relevant ethics codes. The practitioner must seek guidance that can be found from the professional codes of ethics. They provide a basis for accountability, and through their enforcement, provide protection for clients from unethical practices (Corey, 1997). Practitioner must also examine the agreeability of his values with the relevant codes. Should they be in conflict, he must have a rationale to support his stance. It is essential to consider congruency of these values and ethics with those of the client’s. The practitioner must ensure clarity of the ethical codes and if they are applicable with the state laws of the region. Step 4- Know the applicable laws and regulations. The practitioner should be up to date with the specific and relevant laws that apply to the ethical issue. This is particularly critical in situations which deal with keeping or breaching of confidentiality, reporting of child or elder abuse, record keeping, assessment, diagnosis, issues pertaining to dangers to self or others and the grounds of malpractice. Step 5- Obtain consultation. Consulting with colleagues to obtain different perspectives on the problems is generally considered to be helpful. Seeking legal counsel for legal questions is prudent along with consulting a person with an expertise in an unfamiliar culture to serve a client from that culture. In addition the practitioner must understand current rules and regulations of the agency or organization that he or she is working for. It is wise for the nature of the consultation and suggestions provided to be documented. These records would illustrate the practitioner’s attempt to adhere to the community’s standard practice. Step 6- Consider possible and probable courses of action. At this point a list of a variety of courses of action may be identified  through brainstorming. The practitioner could discuss with the client as well as other professionals the available options. The possibilities could be identified for probable courses of actions and these should be documented. Step 7- Enumerate the consequences of various decisions. From the various possible courses of actions, implications of each course must be examined. The questions of who will be affected and to what extent will the client’s decision to pursue the actions affect them must be carefully examined. Again using the fundamental moral principles as a framework, the client must collaborate with the practitioner to ascertain the probable outcomes and consequences. If new ethical issues arise from the selected course of action, a re-evaluation of the action must be pursued. Step 8- Decide on what appears to be the best course of action. Careful consideration of all information received from different sources deliberately and with sensitivity to cross cultural issues is critical before making the best decision. Once making the decision, informing the supervisor, implementing and documenting the decision follows. Reflecting on the experience considering any follow up action could result in finding a solution for the client. While the procedural steps may help in resolving ethical matters, some implications may be noted. Firstly, the client enters a collaborative relationship with the practitioner. The implication is that the client with the practitioner’s help must draw out the details of the problem. This implicates that the client should refrain from coveting relevant information to enable an accurate analysis of the issue. This is to ascertain the true nature of the problem whether it is an ethical, legal, moral, professional, or clinical one. The different perspectives of the problem must be explored. What are the insights the client and practitioner have regarding the problem? (Corey et al., 2007). Failing which the consequence is an unnecessary delay in resolving the problem as there will be an inaccurate analysis of the situation. Secondly, in identifying the potential issues, all the persons involved in the problem must be identified. The implication of failing to identify any one individual who may be affected by the decision of the client would be unethical. The welfare, rights and responsibilities of those affected by the decision might create a different set of problems. The decision would then have to be reversed and a new course of action would have to be pursued. It is to therefore necessary to explore to what extent the course of the action will affect the client and the others (Corey et al., 2007). Next the values and ethics of the client and the practitioner must be evaluated and the degree of congruency noted. This implicates that the relevant ethical principles that are identified to the problem should not be in conflict with those of the client and the practitioner. If there are disagreements, then they must be supported with a rationale. If necessary, guidance must be sought from the relevant organization to clarify the professional codes to the particular problem. Otherwise consequently, the client’s decision may violate the ethical codes relevant to the issue. The client must be informed of the relevant and most recent laws or regulations that apply to the situation. He must look out for any law or regulations that have a bearing on the situation. The implication of his ignorance is that he may run into problems with the law. The practitioner too must abide by the rules, regulations and policies of the workplace. When in doubt practitioner must seek professional advice. The client must be informed of legal issues related to confidentiality, abuse of the vulnerable, record keeping and grounds for malpractice. If the practitioner discovers a criminal act by a client for example, sex with an under-aged girl he has the moral responsibility to report him. The practitioner has the ethical responsibility to discuss with the client on the implications of his actions before reporting the incident. The client must understand the implications of his actions that violate the law. The fundamental moral principles may be considered as framework for evaluating the consequences of the given course of action. The client must  decide the principles that apply to the situation specifically and prioritise them. By thinking through these ethical principles, professional can better evaluate their options in such complex situations. Prioritising the principles can help the client and practitioner to work through the steps of the decision-making model (Elizabeth, 2010). There are implications however when prioritizing one over another. The practitioner encourages the client to exercise autonomy i.e. making a free choice. In doing so, the client must have the concept of doing no harm or non-maleficence and acting in justice (Elizabeth, 2010). Conflict can arise when subscribing to justice which may result in the necessity of treating an individual differently. Though not easy to apply equal weightage to all the principles, it will help to explore an ethical dilemma and resolve it with the least damage to the welfare of those affected. The model may be useful when clients seek help in making decisions in their life regarding relationship issues. A client may be caught in a loveless marriage and be involved in an extra-marital affair and seek advice to take the next step in his life. The dilemma of whether to dissolve his marriage and move on with his life or stay in the marriage to fulfil his duties towards his wife and children is one that needs careful consideration. A divorce would mean breaking up of his family and causing a disruption in the relationship with his children. Staying in the marriage would mean the sacrifice of his love life. Analysing his situation using the model can shed light to clarify the implications of his actions and weigh the consequences. The model may help clients to make decisions at their place of work regarding conflicting work practices. An office worker may be tormented by the wrong practices of her fellow colleagues. She may be facing a dilemma as to whether to report her colleagues to the management or turn a blind eye to the situation. The model can help the client to analyse the situation and evaluate her moral values. She would then be able to make an ethical decision that would do least harm to those involved in the situation. In conclusion, the eight – step model can be a useful tool in helping a practitioner to guide clients to make sound decisions that do not have  conflict with their ethics and are aligned with the laws and regulations of the region. In doing so the practitioner must ensure that he or she is operating in the best interest of the clients.

Garden Of The Gods A State Park - 2133 Words

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