INTRODUCTION

The field of nanotechnology is a rapidly growing industry that has the potential to have an enormous impact on the economy,society,and environment,owing to the large number of nanotechnology products being produced.Indeed,the demand for nano-based products has increased over the last few years,leading to growing concerns about their impact on the environment(Nowack and Bucheli,2007).Nanoparticles can be engineered to have specific sizes,shapes,and properties,which consequently determine their optical,electrical,magnetic,and chemical characteristics;nanoparticles have a greater surface area per unit mass than larger particles,which generally makes them more reactive(Dinesh et al.,2012).

The ecotoxicological properties and risks of nanoparticles have yet to be characterized fully.However,many nanoparticles have been reported to possess antibacterial properties and to directly affect microbial communities in soil(Vance et al.,2015).Nanoparticles can affect soil microorganisms through i)direct effects(i.e.,toxicity),ii)changes in toxin or nutrient availability,iii)interactions with nontoxic organic compounds,or iv)interactions with toxic organic compounds(Aruguete and Hochella,2010).Even though the toxicity mechanisms of most engineered nanoparticles remain unclear,putative underlying mechanisms include the disruption of membranes or membrane potential,protein oxidation,genotoxicity,the interruption of energy transduction,reactive oxygen species(ROS)formation,and the release of toxic constituents(Mishra and Kumar,2009).

6.实验室设备比较多，要注意节能，计算机设备可以设置电源保护，电脑几分钟不用自动切换到休眠状态，同时实验教师做到实验室课程结束及时关闭电源，并养成报废、淘汰设备拆件做维修备件再利用的习惯。

There is increasing evidence that nanoparticles released from industries can harm both aquatic and terrestrial habitats(Schlich and Hund-Rinke,2015).Silver ions,for example,have been reported to harm bacteria and inhibit bacterial respiration.They can interact with sulfur(S)-and phosphorus(P)-containing enzymes in the bacterial cell wall and may disturb their activity(Morones et al.,2005).Silver(Ag)ions may also enter bacterial cells as calcium(Ca)ions and may bind to S-and P-containing molecules,such as DNA.In addition,Ag ions may inhibit DNA replication(Feng et al.,2000).Therefore,it is not surprising that Ag ions also inhibit the denitrification activity of bacteria(Throb¨ack et al.,2007).Furthermore,silver nanoparticles(AgNPs)have been reported to affect earthworm reproduction,as well as enzyme activity of soils.The physiochemical characteristics of soil,such as pH,ionic composition,texture,organic matter content,and temperature,can affect the chemistry,mobility,bioavailability,and toxicity of nanoparticles(Schlich and Hund-Rinke,2015).

Soil enzymes play important roles in regulating the cycles of carbon(C),nitrogen(N),S,P,and other nutrients by mediating biochemical reactions involved in the decomposition of organic matter.Soil enzyme activities are often related to the organic matter content,physical properties,microbial activity,and microbial biomass of soil,thereby serving as an early indicator of changes in soil health(Dick et al.,1996;Bowles et al.,2014).

Two sizes(10 and 50 nm)of polyvinylpyrrolidone(PVP)-functionalized AgNPs(0.02 mg mL−1in water)were obtained from Sigma Aldrich Co.(USA)and characterized using ultraviolet-visible(UV-vis)spectroscopy.The 10-nm AgNPs exhibited maximum absorption at 390 nm(molecular weight∶107.87;density∶0.996 g mL−1at 25 °C),whereas the 50-nm AgNPs exhibited maximum absorption at 425 nm(molecular weight∶107.87;density∶1.001 g mL−1at 25 °C).

Significant interactions between treatment and incubation time were observed for all enzymes in the sandy loam(P＜0.0001).The acid phosphatase activity with AgNP treatment was consistently lower than that of the control for all incubation periods.There was no significant difference in the acid phosphatase activity between 1600 and 3200 µg Ag kg−1dry soil,after one hour and one month of incubation;however,the AgNP concentration had a significant and negative effect after one week(Table II).In addition,the 50-nm AgNPs failed to affect the acid phosphatase activity after one hour of incubation,but did in fluence the enzyme activity after one week and one month.The AgNP size had no significant effect on the enzyme activities.

MATERIALS AND METHODS

Table III summarizes the enzyme activities of the silt loam in response to AgNP treatment.The 50-nm AgNPs at the higher concentration significantly reduced the soil acid phosphatase activity compared to the control after one hour,but the activity showed a slight increase,though not statistically significant,with the 10-nm AgNPs.The AgNP treatment increased the acid phosphatase activity after both the one-week and one-month incubation periods,regardless of AgNP size or concentration(Table III).The AgNP treatment also significantly affected the β-glucosidase activity,regardless of AgNP size,AgNP concentration,or incubation time(Table III).

The soils were treated with either 0(control),1600,or 3200 µg Ag kg−1dry soil by applying 0,240,or 480µL of AgNP solutions(aqueous dispersion).Then,the treated soil samples were homogenized and incubated for one month.This was performed for each AgNP size,and the enzyme activities of each soil were measured after one hour,one week,and one month of incubation.The activities of three of the enzymes(acid phosphatase,β-glucosidase,and arylsulfatase)were measured,as described by Tabatabai(1994).Standardized protocols that included incubation at 37°C for a fixed time period at a specific buffer pH range,as well as the inclusion of necessary cofactors,were followed for each of the enzyme assays(Eivazi and Tabatabai,1977,1988).Meanwhile,β-glucosaminidase activity was determined using a method developed by Parham and Deng(2000).Briefly,2 mL 0.1 mol L−1acetate buffer(pH 5.5)and 0.5 mL 10 mmol L−1p-nitrophenyl N-acetyl-β-D-glucosaminidase(pNNAG)solution were added to 0.5 g soil.The mixtures were incubated at 37°C for 1 h,and the reaction was stopped using 0.5 mL 0.5 mol L−1calcium chloride(CaCl2)and 2 mL 0.1 mol L−1tris(hydroxymethyl)aminomethane(THAM)buffer(pH 12).The resulting solutions were filtered and measured for color intensity at 420 nm.The activities were measured in triplicate,along with a control.The absorbance of the products formed in each enzyme assay was measured with a Thermo Genesys spectrophotometer(Thermo Scientific,USA),and all substrates used in the assays were obtained from Sigma Aldrich Co.

The present study investigated the effects of silver nanoparticles on soil enzymes that are known to play a critical role in the mineralization of C,N,P,and S in soil(Tabatabai,1994)and are sensitive to changes in soil quality.Four enzymes were chosen in this study∶acid phosphatase,β-glucosidase,β-glucosaminidase,and arylsulfatase.The activity of acid phosphatase was studied because it catalyzes the hydrolysis of various organic and inorganic phosphomonoesters and is,therefore,important in soil P mineralization and plant nutrition(Eivazi and Tabatabai,1977).The activity of β-glucosidase,the most predominant glycosidase in soil,was studied because it is involved in the last limiting step of cellulose degradation(Eivazi and Tabatabai,1988),and that of βglucosaminidase,a key enzyme in the hydrolysis of N-acetyl-β-D-glucosamine residues from chitooligosaccharides(Parham and Deng,2000),was studied because of its role in converting chitin into amino acids,which is considered important in soil C and N cycling.The activity of arylsulfatase was studied because it is generally used to investigate the mineralization of organic S in soil.

1）经过去边框处理之后，计算去边框之后的车牌尺寸，宽高分别为w、h，设定一个标志位flag并初始化为 False。定义两个列表，Lborder[]、Rborder[]，分别用于存储各字符的左右边界。定义两个阈值threshlod1=5、threshlod2=15（分别对应于字符开始结束和字符宽度的阈值）。进入步骤2）。

Enzyme activity data were statistically analyzed using two-way analysis of variance(ANOVA).Tukey’s multiple comparison test was used to compare differences in the means.Statistical differences were tested at P≤0.05.Data were analyzed using SAS 9.4(SAS Institute,2013).

RESULTS

Both soil type and incubation time significantly affected enzyme activity(Table I).Therefore,the results of each soil type and incubation time are separated.

本文结合试验对红粘土剖面的常量元素和微量元素作出分析和对比研究，对红粘土剖面粘土矿物特征作出分析，并从中探讨了红粘土的形成机理及成土过程中的影响因素所在。

Sandy loam

The overall objective of the present study was to determine the effects of different sizes and concentrations of AgNPs on the activities of soil enzymes over one-month incubation.

Overall,the activity of β-glucosidase was signi ficantly reduced during incubation periods of one hour,one week,and one month with the exception of lowerconcentration(1600 µg Ag kg−1dry soil)and 50-nm AgNPs compared with control(Table II).The AgNPs significantly decreased the β-glucosaminidase activity as compared with the control after one hour for both sizes,whereas the 50-nm AgNPs showed increased,though not statistically significant,activity at the higher concentration after one week and one month of incubation.There was no difference in the β-glucosaminidase activity between the 10-and 50-nm AgNPs(Table II).The arylsulfatase activity was significantly reduced by AgNP treatment,regardless of incubation time or AgNP size,and there was no difference in the arylsulfatase activity between the 10-and 50-nm AgNPs(Table II).

Silt loam

Topsoil samples(0–15 cm)of two soils were collected from a field(38°31′34.3′′N 92°08′27.9′′W)at the George Washington Carver Farm,Lincoln University of Missouri,USA.The first soil was a Wrengart-Gatewood silt loam( fine-silty,mixed,active,mesic Fragic Oxyaquic Hapludalf),with a pH of 5.8,3.4%organic matter,cation exchange capacity of 11.7 cmolc kg−1,and 20%clay,whereas the second soil was an Elk sandy loam(Ultic Hapludalt),with a pH of 6.0,2.1%organic matter,cation exchange capacity of 15.7 cmolckg−1,7%clay,and 76%sand.The soil samples were thoroughly mixed,air dried,passed through a 2-mm sieve,and stored in sealed plastic bags for enzyme determination.

后方法教学的“特定性”、“实践性”和“可行性”是“在一个共生的环境中相互交织、相互促动、整体增效”[4]，构成了后方法教学概念基础，本文旨在依据后方法理论的三大参数，探讨后方法理论对会展英语教学的启示。

2014年，郝哲便开始在马合农场开展羊肚菌栽培技术研究。他远赴四川、云南学习取经，前往全国多地林区采集野生羊肚菌种质资源，全程悉心呵护、跟踪监测培育，换来的却是“未出菇”的结果。“科研工作，可能千百次失败才能换来一次成功，失败是成功之母嘛！”郝哲说。此后的实验中，他们选择了多处羊肚菌生长点，在半个多月的时间里废寝忘食，强化对羊肚菌野生环境的调查，不分昼夜监测土壤、空气的湿度、温度及营养成分，并在设施内模拟试验，终于在2016年获得成功，成就了首个在北方风沙区实现羊肚菌人工栽培的成功案例。

The AgNP treatment had inconsistent effects on the β-glucosaminidase activity after one hour of incubation,but significantly increased the activity after one week and one month(Table III).In contrast,the arylsulfatase activity was significantly affected by Ag-NP treatment after one hour and one week,with the 50-nm AgNPs increasing the enzyme activity and the 10-nm AgNPs reducing the enzyme activity,and the arylsulfatase activity was significantly reduced by Ag-NP treatment after one month,regardless of AgNP size or concentration(Table III).

TABLE I Results of analysis of variance for the effect of silver nanoparticles(AgNPs)on the activity of four enzymes in two soils collected from a field at the George Washington Carver Farm,Lincoln University of Missouri,USA

Variable Degree of freedom F value P value Soil type 368495.65 ＜0.0001 Incubation time 2 10260.74 ＜0.0001 Soil type×incubation time 2 6778.25 ＜0.0001 AgNP concentration 2 446.60 ＜0.0001 Soil type×AgNP concentration 2 285.46 ＜0.0001 Incubation time×AgNP concentration 4 902.76 ＜0.0001 Soil type×incubation time×AgNP concentration 4 606.76 ＜0.0001 AgNP size 1 304.90 ＜0.0001 Soil type×AgNP size 1 187.70 ＜0.0001 Incubation time×AgNP size 2 762.68 ＜0.0001 Soil type×incubation time×AgNP size 2 529.40 ＜0.0001 AgNP concentration×AgNP size 2 180.43 ＜0.0001 Soil type×AgNP concentration×AgNP size 2 127.09 ＜0.0001 Incubation time×AgNP concentration×AgNP size 4 216.95 ＜0.0001 Soil type×incubation time×AgNP concentration×AgNP size 4 140.62 ＜0.0001 1

TABLE II Activities of selected enzymes of the sandy loam treated with silver nanoparticles(AgNPs)of different concentrations and sizes after one hour,one week,and one month of incubation

a)Different letters within each column indicate significant differences(P ＜ 0.05).

AgNPs One-hour incubation Concentration Size Acid phosphatase β-glucosaminidase β-glucosidase Arylsulfataseµg Ag kg−1dry soil nm mg p-nitrophenol released kg−1soil h−1 0(control) 40.6aa) 2.2a 9.7a 8.2a 1600 10 35.9b 1.5bc 8.6b 6.1b 50 36.5ab 1.9ab 9.2a 5.8c 3200 10 34.6bc 1.7b 8.1b 5.8c 50 38.1a 1.7b 8.2b 5.2c AgNPs One-week incubation Concentration Size Acid phosphatase β-glucosaminidase β-glucosidase Arylsulfataseµg Ag kg−1dry soil nm mg p-nitrophenol released kg−1soil h−1 0(control) 40.3a 2.1a 9.2a 8.5a 1600 10 35.5a 1.9ab 8.1b 5.2b 50 26.8b 1.8b 8.2b 5.7b 3200 10 28.9b 1.9ab 8.9b 4.7c 50 20.1b 2.9a 7.8c 5.5b AgNPs One-month Incubation Concentration Size Acid phosphatase β-glucosaminidase β-glucosidase Arylsulfataseµg Ag kg−1dry soil nm mg p-nitrophenol released kg−1soil h−1 0(control) 39.9a 2.2a 9.6a 8.3a 1600 10 27.2bc 1.8b 7.6ab 3.7c 50 24.4bc 1.9ab 6.7c 4.2bc 3200 10 22.6c 1.9ab 8.9ab 2.9c 50 24.6bc 2.4a 6.8c 3.8c

DISCUSSION

The physiochemical characteristics of soil,such as pH,ionic composition,and texture,can in fluence the mobility,bioavailability,and toxicity of pollutants that are similar to nanoparticles(Hund-Rinke et al.,2012),and the transport and bioavailability of AgNPs can be affected by soil type,pH,ion content,and organic matter content.Nanoscale metals,such as AgNPs,are toxic to soil ecosystems,especially at low concentrations,and have been reported to significantly inhibit soil enzyme activity(Peyrot et al.,2014).Similarly,Khan et al.(2007)reported that heavy metals(Cd and Pb)inhibited soil enzyme activities and that the extent of inhibition was affected by both heavy metal concentration and incubation period.They also found that soil bacterial community structure,as determined using polymerase chain reaction and denaturing gradient gel electrophoresis,was affected by heavy metal contamination and that high levels of Cd contamination caused the greatest change.

Previous studies have also shown that AgNPs can significantly inhibit soil dehydrogenase activity.Cao et al.(2016),for example,tested the effects of Ag-NPs(0.024,0.24,4.80,9.60 µg g−1dry soil)on the wetland plant Typha orientalis Presl and found that AgNPs inhibited the activities of all exoenzymes tested.Dinesh et al.(2012)reported that AgNPs exerted negative effects on soil denitrifying bacteria.

The adsorption of nanoparticles onto soil organic matter can drastically reduce nanoparticle mobility and,thereby,reduce their bioavailability to soil microorganisms.However,Schlich and Hund-Rinke(2015)reported that there was no relationship between organic carbon content and AgNP toxicity in five types of soils.The organic carbon in the soils analyzed by Schlich and Hund-Rinke(2015)varied from 0.93%to 3.85%,whereas the sandy loam soil tested in the present study had 2.1%organic matter.Therefore,it is possible that the low content of organic matter of the soils used in the present study allowed the AgNPs to remain bioavailable to the microbial community of the soil and,therefore,to reduce the soil enzyme activity.

The effects of nanoparticles on enzyme activity can also be affected by soil type and texture.For example,Schlich and Hund-Rinke(2015)reported that there was a clear relationship between AgNP toxicity and soil particle size,that AgNP toxicity increased with increasing sand content,and that AgNP toxicity decreased with increasing clay content.Therefore,the high sand content of the sandy loam soil(76%sand)used in the present study may explain the negative effects of AgNPs on the activities of all four enzymes examined.Meanwhile,in the silty loam,which contained 20%clay and 3.4%organic matter,acid phosphatase activity was significantly reduced after the one-hour incubation with 50-nm AgNPs,but increased after the one-hour incubation with 10-nm AgNPs,and after one week and one month,the activity was increased,regardless of AgNP size or concentration.There was no significant difference in the effects of the 10-and 50-nm AgNPs on the acid phosphatase activity.Meanwhile,the β-glucosidase activity was reduced after the one-hour incubation with 10-nm AgNPs,regardless of AgNP concentration.These findings support those of Schlich and Hund-Rinke(2015)in that there is a clear relationship between AgNP toxicity and soil particle size.Indeed,H¨ansch and Emmerling(2010)also reported that AgNP treatment reduces microbial biomass,increases basal respiration,and slightly reduces leucine aminopeptidase activity in soil.They attributed these effects to the toxicity of AgNPs to soil microorganisms and to reduced substrate use efficiency.

本系统通过独特的视角提醒人们关注日常居室环境，避免室内污染对身体健康在不被感知的情况下造成的损害。采用方便、简单的设计实现室内环境数据的采集和呈现，并通过共享到云服务器为环境大数据分析提供依据。同时也考虑后期维护上比较容易，成本低、效率高。

In a study by Asadishad et al.(2017),the activities of five extracellular enzymes that are important in nutrient cycling were measured in soils treated with citrate-coated gold nanoparticles(nAu)and polyvinylpyrrolidone(PVP)-coated nAu(PVP-nAu)of three different sizes(5,50,and 100 nm)and different concentrations.They found that at low nanoparticle concentration(0.1 mg nAu kg−1dry soil),decreasing the size of PVP-nAu resulted in an increased stimulation of soil enzyme activity.However,in the present study,there were no significant differences in the effects of the two AgNP sizes(10 and 50 nm).Furthermore,the effects of the two AgNP concentrations(1600 and 3200 µg Ag kg−1dry soil)were sometimes positive,and sometimes negative,and there was no clear or consistent trend.Therefore,further studies are needed to determine the mechanisms by which AgNPs reduce soil enzyme activity.In order to elucidate the factors involved and to determine the extent to which AgNPs can affect soil ecosystems,these studies should involve testing different soil types and investigating the effects of AgNPs on soil microbial communities and plant growth.

TABLE III Activities of select enzymes of the silt loam treated with silver nanoparticles(AgNPs)of different concentrations and sizes after one hour,one week,and one month of incubation

a)Different letters within each column indicate significant differences(P ＜ 0.05).

AgNPs One-hour incubation Concentration Size Acid phosphatase β-glucosaminidase β-glucosidase Arylsulfataseµg Ag kg−1dry soil nm mg p-nitrophenol released kg−1soil h−1 0(control) 332aa) 57a 150a 125a 1600 10 362a 46b 131b 84b 50 305ab 51ab 145a 121a 3200 10 360a 42b 112c 92ab 50 186b 55a 133b 110ab AgNPs One-week incubation Concentration Size Acid phosphatase β-glucosaminidase β-glucosidase Arylsulfataseµg Ag kg−1dry soil nm mg p-nitrophenol released kg−1soil h−1 0(control) 333b 55b 151a 124a 1600 10 343ab 61ab 36cd 101ab 50 477a 76a 43c 122a 3200 10 442a 59b 47bc 44c 50 396ab 72a 44c 119a AgNPs One-month incubation Concentration Size Acid phosphatase β-glucosaminidase β-glucosidase Arylsulfataseµg Ag kg−1dry soil nm mg p-nitrophenol released kg−1soil h−1 0(control) 336b 58b 152a 122a 1600 10 511a 74ab 76ab 37c 50 502a 82a 67ab 42bc 3200 10 501a 72ab 70ab 29c 50 465ab 65b 68ab 38c

ACKNOWLEDGEMENTS

This study was funded by the National Institute of Food and Agriculture,United States Department of Agriculture(No.1007450).

REFERENCES

Aruguete D M,Hochella M F.2010.Bacteria-nanoparticle interactions and their environmental implications.Environ Chem.7:3–9.

Asadishad B,Chahal S,Cianciarelli V,Zhou K,Tufenkji N.2017.Effect of gold nanoparticles on extracellular nutrientcycling enzyme activity and bacterial community in soil slurries:role of nanoparticle size and surface coating.Environ Sci Nano.4:907–918.

Bowles T M,Acosta-Mart´ınez V,Calder´on F,Jackson L E.2014.Soil enzyme activities,microbial communities,and carbon and nitrogen availability in organic agroecosystems across an intensively-managed agricultural landscape.Soil Biol Biochem.68:252–262.

Cao C,Huang J,Cai W S,Yan C N,Jiang Y D,Peng C.2016.Effect of silver nanoparticles on the rhizosphere exoenzyme of T.orientalis Presl.In Int’l Association of Structural Engineering&Mechanics(IASEM),Korea Advanced Institute of Science&Technology(KAIST)(ed.)Proceedings of 2016 World Congress on Advances in Civil,Environmental,and Materials Research.IASEM,KAIST,Jeju Island.pp.1–12.

Dick R P,Breakwell D P,Turco R F.1996.Soil enzyme activities and biodiversity measurements as integrative microbiological indicators.In Doran J W,Jones A J(eds.)Methods for Assessing Soil Quality.Soil Science Society of America,Madison.pp.247–271.

Dinesh R,Anandaraj M,Srinivasan V,Hamza S.2012.Engineered nanoparticles in the soil and their potential implications to microbial activity.Geoderma.173-174:19–27.

Eivazi F,Tabatabai M A.1977.Phosphatases in soils.Soil Biol Biochem.9:167–172.

Eivazi F,Tabatabai M A.1988.Glucosidases and galactosidases in soils.Soil Biol Biochem.20:601–606.

Feng Q L,Wu J,Chen G Q,Cui F Z,Kim T N,Kim J O.2000.A mechanistic study of the antibacterial effect of silver ions on Escherichia coli and Staphylococcus aureus.J Biomed Mater Res.15:662–668.

H¨ansch M,Emmerling C.2010.Effects of silver nanoparticles on the microbiota and enzyme activity in soil.J Plant Nutr Soil Sci.173:554–558.

Hund-Rinke K,Schlich K,Klawonn T.2012.In fluence of application techniques on the ecotoxicological effects of nanomaterials in soil.Environ Sci Eur.24:30.

Khan S,Cao Q,Hesham A E,Xia Y,He J Z.2007.Soil enzymatic activities and microbial community structure with different application rates of Cd and Pb.J Environ Sci.19:834–840.

Mishra V K,Kumar A.2009.Impact of metal nanoparticles on the plant growth promoting rhizobacteria.Digest J Nanomater Biostruct.4:587–592.

Morones J R,Elechiguerra J L,Camacho A,Holt K,Kouri J B,Ram´ırez J T,Yacaman M J.2005.The bactericidal effect of silver nanoparticles.Nanotechnology.16:2346–2353.

Nowack B,Bucheli T D.2007.Occurrence,behavior and effects of nanoparticles in the environment.Environ Pollut.150:5–22.

Parham J A,Deng S P.2000.Detection,quantification and characterization of β glucosaminidase activity in soil.Soil Biol Biochem.32:1183–1190.

Peyrot C,Wilkinson K J,Desrosiers M,Sauv´e S.2014.Effects of silver nanoparticles on soil enzyme activities with and without added organic matter.Environ Toxicol Chem.33:115–125.

SAS Institute.2013.SAS/STAT User’s Guide.Version 9.4.SAS Institute,Cary.

Schlich K,Hund-Rinke K.2015.In fluence of soil properties on the effect of silver nanomaterials on microbial activity in five soils.Environ Pollut.196:321–330.

Tabatabai M A.1994.Soil enzymes.In Weaver R W,Angle S,Bottomley P,Bezdiecek D,Smith S,Tabatabai A,Wollum A(eds.)Methods of Soil Analysis.Part 2.Microbiological and Biochemical Properties.Soil Science Society America,Madison.pp.775–833.

Throb¨ack I N,Johansson M,Rosenquist M,Pell M,Hansson M,Hallin S.2007.Silver(Ag+)reduces denitrification and induces enrichment of novel nirK genotypes in soil.FEMS Microbiol Lett.270:189–194.

Vance M E,Kuiken T,Vejerano E P,McGinnis S P,Hochella M F Jr,Rejeski D,Hull M S.2015.Nanotechnology in the real world:redeveloping the nanomaterial consumer products inventory.J Nanotechnol.6:1769–1780.