小秦岭某金矿区农田土壤重金属污染评价

小秦岭金矿区是中国第二大产金区,金矿开采、选冶活动会将重金属释放到环境中,对土壤环境造成污染。本研究系统采集了133件农田土壤样及邻区非矿业活动区的土壤对照样,结果表明,研究区农田土壤中Hg、Pb、Cd污染明显。以邻区表层土壤重金属含量作为对比评价矿区土壤污染的对比值,除Cr外,Hg、Pb、Cd、Cu、Zn、As累积超标率分别为96.90%、100%、60.10%、12.03%、78.90%、69.17%。Hg、Pb、Cu、Zn、Cd超过国家土壤环境质量二级标准的样本超标率分别为43.16%、12.8%、7.52%、3.01%、2.25%。土壤重金属综合污染指数表明,36.08%的土壤样本受到了轻度以上污染。Hg是农田土壤中最主要的重金属污染物,超标倍数均值为4.71,受Hg污染的农田面积为136.3km2,占到研究区面积的51.76%。土壤污染极其严重,矿区环境污染治理迫在眉睫。     

芜湖市不同功能区土壤重金属污染状况与环境质量评价

通过对芜湖市工业区、商业区、休闲区和居住区土壤重金属污染现状的调查与分析,分别以本地区自然土壤重金属元素自然含量的平均值和国家土壤环境质量标准作为评价标准,计算出各功能区土壤单因子污染指数,并采用主成分分析法确定权重,对各功能区的土壤环境质量进行综合评价。结果表明:芜湖市各功能区土壤Cd、Hg、Cu、Pb、Cr、Zn含量均超过本地区自然土壤重金属元素自然含量的平均值。其中工业区土壤中Cd、Hg和Cu,商业区土壤中Cd和Hg,休闲区和居住区土壤中Cd均超过国家土壤环境质量标准。Cu、Hg、Pb、Cr、Cd污染均表现为工业区最大,商业区次之,Zn在各功能区含量差异不大。土壤中重金属含量上层高于下层,且在商业区和居住区差异明显。土壤环境质量居民区尚清洁,休闲区和商业区中度污染,工业区重度污染。     

长期污灌农田土壤重金属污染及潜在环境风险评价

以西安市某典型污灌区农田土壤为研究对象,分析长期污水灌溉对表层土壤重金属含量及富集状况的影响,采用内梅罗指数法和Hakanson潜在生态危害指数法对其污染现状及潜在环境风险进行评价。结果表明：长期污灌已导致农田土壤Cd、Cr、Cu、Hg、Ni、Pb和Zn7种重金属相对自然背景有不同程度累积,其富集比例依次为100%、82.69%、100%、100%、80.77%、98.08%和100%,仅有土壤As平均含量低于其背景水平;以国家土壤环境质量标准二级限量值作为污染评价阈值,其中Cd和Hg污染表现突出,按其污染指数平均值排序为Cd〉Hg〉Ni〉Cu〉Zn〉As〉Cr〉Pb;土壤重金属综合潜在环境风险为＂强＂等级,Hg、Cd的环境影响占据主导;随污灌年限增长,离灌渠越近,农田土壤重金属的污染水平和环境风险越高。鉴于该区土壤重金属已呈现较强生态危害性,应及时采取必要防治措施,调整土地利用结构,确保农田环境及农产品安全生产。     

北京市农田土壤重金属年际差异分析与评价

通过对2005年至2009年北京市农田土壤重金属含量的持续监测,分析了该地区农田土壤中As、Hg、Cu、Cd、Cr和Pb等重金属的统计特征和年度变化趋势,并结合国家土壤环境质量标准,对农田环境质量进行了评价。结果表明,2005年至2009年间,As、Hg、Cu、Cd、Cr和Pb等重金属中,Hg和Cr的含量有逐年上升的趋势,Hg的平均含量逐年为0.053、0.066、0.070、0.072 mg.kg-1和0.079 mg.kg-1,Cr平均含量为50.0、52.4、55.3、58.6 mg.kg-1和62.1 mg.kg-1。其他重金属含量上下波动,无上升或下降的变化趋势。结合重金属含量统计和单因子评价结果,重金属的污染程度依次为Cd〉Hg〉Cu〉Pb〉Cr〉As。北京农田整体处于清洁状态,但存在着个别的Hg和Cd污染。     

安徽省某县农田土壤重金属污染及潜在生态风险评价

以安徽省沿江某县农田土壤为研究对象,通过野外调查采集土壤样品199个,分析了土壤中主要重金属元素含量,采用地积累指数法和Hakanson潜在生态风险指数法,并与GIS相结合评价了研究区农田土壤重金属潜在生态风险。结果表明:Cd、Pb、Cr、As、Hg等5种重金属元素的平均值分别为0.302、61.17、87.13、12.89 mg·kg~(-1)以及0.139 mg·kg~(-1),相较于《土壤环境质量标准》的二级标准只有Cd的平均值略超标;相较于当地土壤元素背景值则全部元素都超出范围;研究区土壤各种重金属元素出现污染的点位占比分别为:Cd 85.43%、Hg 76.38%、Pb 59.30%、As 26.63%以及Cr 19.60%。污染程度由强到弱依次为CdHgPbAsCr;研究区各种重金属潜在生态风险指数由高到低分别为:HgCdAsPbCr,其中Hg和Cd占主导地位;研究区综合潜在生态风险指数介于83.83~6 987.87之间,平均值为250.76,有13.57%的点位处于强或以上风险,应该引起相关部门的重视。     

河北板蓝根产地土壤-植物中镉铅汞砷含量特征及其污染评价

采用野外调查和室内分析相结合的方法,研究了河北省安国市和蔚县板蓝根产地土壤-植物中Cd、Pb、Hg、As含量特征及其在菘蓝中的累积特性,并对板蓝根产地土壤和草药中Cd、Pb、Hg、As污染状况进行了评价,为该地区安全、合理地发展中草药生产提供数据支撑和科学依据。结果表明,安国市和蔚县板蓝根产地土壤中重金属Cd、Pb、Hg、As含量差异不大,土壤重金属含量的变异系数在11.70%~97.65%。以《土壤环境质量标准》(GB 15618—1995)一级标准值进行评价,综合污染指数评价结果显示45%板蓝根种植区土壤Cd、Pb、Hg、As污染等级为警戒限,其他处于清洁水平;而以《土壤环境质量标准》二级标准值进行评价,种植区单项污染指数及综合污染指数结果均0.7,土壤环境清洁。此外,菘蓝地上部(大青叶)Cd、Pb、Hg、As平均含量分别为0.22 mg·kg~(-1)、0.89 mg·kg~(-1)、0.04 mg·kg~(-1)、0.25 mg·kg~(-1),对重金属的富集能力表现为CdHgPbAs;菘蓝地下部(板蓝根)Cd、Pb、Hg、As含量均值分别为0.14 mg·kg~(-1)、0.57 mg·kg~(-1)、0.04 mg·kg~(-1)、0.26 mg·kg~(-1),对重金属的富集能力表现为CdHgAsPb。所有菘蓝样品中Pb、Hg、As含量均未超出《药用植物及制剂进口绿色行业标准》(WM2—2001),大青叶9.09%样品中Cd超标,且Cd平均污染指数0.7,属警戒限污染等级。因此,在中药材GAP(良好的农业规范)产地环境质量评价时,除板蓝根产地土壤完全符合土壤环境质量二级标准外,也不应忽视板蓝根和大青叶吸收和累积重金属的自身特性。     

郑州农区土壤重金属污染与蔬菜质量相关性探析

以单因子污染指数和综合污染指数为评价方法,对郑州市郊区3种不同灌溉方式农田土壤进行重金属(Hg、Cr、Cd、Pb、As、Cu、Zn)污染监测和蔬菜质量调查。结果发现,郑州市郊区的土壤均未超过GB15618—1995《土壤环境质量标准》二级标准限值;部分蔬菜已受到重金属的污染,含量超过国家食品卫生蔬菜类标准;土壤中的Zn、Cu、Cd、Cr污染与多数蔬菜的污染呈显著的正相关,而Pb、Hg污染在多数蔬菜中均表现为与土壤污染无相关性。     

东平湖湖区农田土壤重金属污染评价

为考察东平湖湖区农田土壤的重金属污染状况,为农业活动和东平湖的水质安全提供支持,2014年在湖区农田中采集15个土壤样品,测定其中Pb、Cr、Cd、As和Hg 5种重金属的浓度,采用综合污染指数、地累积指数和潜在生态风险指数评价土壤的重金属污染状况,并用surfer软件对农田土壤的生态风险进行分区。结果表明:农田土壤中5种重金属的浓度基本都满足土壤环境质量标准的I~II类,主要富集物质为Hg,富集系数为2.50;综合污染指数为0.41,污染级别为1级;地累积指数为-1.23~2.63,污染级别为1~4级;潜在生态风险总指数为168,表明土壤具有中度的生态危害;分区结果显示湖区东北部的生态危害相对较重,需要进行进一步的污染源调查处置。     

滩涂围垦区土壤重金属调查及生态风险评价——以盐城市弶港镇为例

以苏北盐城弶港镇滩涂围垦土壤为研究对象,进行了重金属Cr、Pb、As、Cd、Hg的测定及其风险评价,为我国沿海滩涂种植业的发展提供依据,并为其他国家和地区相关研究提供参考。结果表明,该区域土壤中5种重金属含量均未超过《国家土壤环境质量标准》（GB15618—1995）一级标准限制,农田土壤Cr、As、Cd含量较高,郊区菜地土壤Pb、Hg含量较高;重金属Cr、Pb、As、Cd含量在不同土地利用类型间存在显著性差异;Hg含量差异不显著。采用地积累指数法进行评估可知,土壤没有受到单项重金属污染。依据内梅罗综合指数法评估可知,潮间带沉积物、工业区土壤、滩涂围垦熟化的土壤没有受到重金属污染;郊区菜地与农田土壤的污染等级已达到警戒线,污染水平属于＂尚清洁＂。Hakanson潜在生态风险评价结果表明,区域内土壤重金属处于无风险等级,尚不具有潜在生态风险。     

微生物对土壤Cd Pb和Zn生物有效性的影响研究

采用土壤盆栽模拟试验方法,研究了接种不同微生物对重金属富集植物——印度芥菜修复土壤中Cd、Pb、Zn的作用效果。结果表明,接入菌株JA27、JC55、JC40不仅显著促进植物的生长,提高印度芥菜的生物量,降低了土壤pH,并且对土壤Cd、Pb、Zn产生活化作用,使土壤Cd、Pb、Zn有效态含量显著增加,增强印度芥菜对土壤Cd、Pb、Zn吸收量,显著提高了富集植物的修复效果。以上3个处理使印度芥菜地上部Cd、Pb、Zn吸收量分别提高了117%～137%、37%～62%、9%～15.1%。接种JB37对土壤Cd、Pb、Zn产生钝化作用,并且抑制印度芥菜对土壤Cd、Pb、Zn的吸收。JB37处理印度芥菜地上部Pb、Zn吸收量分别降低了72.5%、27%,对Cd吸收量无显著影响。     

低氮和干旱胁迫对富士和秦冠生长及氮素利用的影响

【目的】以富士（Fuji）、 秦冠（Qinguan）嫁接在平邑甜茶（Malus hupehensis Rehd.）上的当年生盆栽苗为试验材料,采用砂培方法,研究了缺氮胁迫和干旱对富士和秦冠生长情况、 光合参数、 植株各部位氮磷钾含量及氮素利用效率的影响,分析比较了低氮干旱条件下富士和秦冠生长及氮素利用的差异,以期为果树生产高效肥水利用提供理论指导。【方法】试验共设四个处理: 正常氮正常水（ZZ）、 低氮正常水（DZ）、 正常氮干旱（ZG）、 低氮干旱（DG）。氮素和水分均设置两个水平,分别为正常氮（6 mmol/L NO-3-N）、 低氮（0.3 mmol/LNO-3-N）、 正常供水（保持盆中砂子相对含水量为饱和含水量的80%~85%）、 干旱处理（保持盆中砂子相对含水量为饱和含水量的60%~65%）。【结果】富士和秦冠的生物量（茎和叶）、 株高茎粗等生长指标以及光合速率、 气孔导度、 蒸腾速率均为正常氮正常水（ZZ）＞低氮正常水（DZ）＞正常氮干旱（ZG）＞低氮干旱（DG）,并且相对应处理下秦冠的以上指标均高于富士;正常供水下,缺氮处理使富士、 秦冠的根冠比比正常氮处理均有所增加,富士提高了2.05%,秦冠提高了22.40%。富士和秦冠的氮、 磷、 钾含量均表现出正常氮正常水（ZZ）＞低氮正常水（DZ）＞正常氮干旱（ZG）＞低氮干旱（DG）; 氮、 钾元素含量在植株各部位的分布顺序依次是叶＞根＞茎,磷元素则是根＞叶＞茎;光合氮素利用效率（PNUE）和氮素利用效率表现为秦冠处理之间差异极显著,富士处理之间差异不显著;秦冠的PNUE和NUE明显高于富士,在低氮正常水（DZ）处理下,秦冠氮肥利用率比富士高42.07%,在低氮干旱（DG）处理下高64.14%;低氮胁迫下富士和秦冠的NUE显著提高,并且秦冠提高的幅度高于富士。【结论】施用氮肥能够显著提高富士与秦冠的干物质量,同等水肥条件下,秦冠生长优于富士;水分亏缺会减少叶片对氮的吸收,干旱条件下适度增施氮肥,可提高果树的抗旱能力;低氮干旱胁迫下秦冠的生长指标、 光合指标及氮素利用效率指标均优于富士,表现出较强的抗低氮干旱胁迫的能力。     

Pasture Vegetation Elemental Analysis by Laser-Induced Breakdown Spectroscopy

Laser-induced breakdown spectroscopy (LIBS) is a new technique for the analysis of plant material. This study investigates the application of LIBS to pasture-based plant samples. The LIBS measurements were obtained from pelletized pasture samples (100 samples) that had been also analyzed by inductively coupled plasma–optical emission spectroscopy (ICP-OES) following microwave digestion for calibration and comparison purposes. Comparisons for elements sodium (Na), potassium (K), magnesium (Mg), calcium (Ca), manganese (Mn), iron (Fe), copper (Cu), zinc (Zn), boron (B), phosphorus (P), and sulfur (S) showed that LIBS could be used for almost all the standard profile total elements with concentrations down to low mg/kg levels (observed error of Na: 0.024 percent, K: 0.18 percent, Mg: 0.016 percent, Ca: 0.073 percent, P: 0.017 percent, Mn: 31 mg/kg, Fe: 150 mg/kg, Zn: 6.6 mg/kg, and B: 1.1 mg/kg). Elemental analysis at less than mg/kg levels was not possible using LIBS. The elements S and Cu were particularly difficult to analyze with reliability using LIBS at the concentration levels found in the plant samples. Replacing microwave digestion and subsequent ICP analysis with a direct analysis of dried plant samples using LIBS has the potential to improve the productivity and reduce the cost of testing.     

Faculty positions:Center for Agricultural Resources Research,Chinese Academy of Sciences

正The Center for Agricultural Resources Research(CARR),the Institute of Genetics and Developmental Biology(IGDB),Chinese Academy of Sciences,invites applicants for several research group leader positions.CARR is one of the research organizations in Chinese Academy of Sciences(CAS).We seek nominations and applications from individuals who have expertise and a record of accomplishment in research areas related to ecology,agro-hydrology,     

Macroinvertebrates in North American tallgrass prairie soils: effects of fire, mowing, and fertilization on density and biomass

The responses of tallgrass prairie plant communities and ecosystem processes to fire and grazing are well characterized. However, responses of invertebrate consumer groups, and particularly soil-dwelling organisms, to these disturbances are not well known. At Konza Prairie Biological Station, we sampled soil macroinvertebrates in 1994 and 1999 as part of a long-term experiment designed to examine the effects and interactions of annual fire, mowing, and fertilization (N and P) on prairie soil communities and processes. For nearly all taxa, in both years, responses were characterized by significant treatment interactions, but some general patterns were evident. Introduced European earthworms (Aporrectodea spp. and Octolasion spp.) were most abundant in plots where fire was excluded, and the proportion of the total earthworm community consisting of introduced earthworms was greater in unburned, unmowed, and fertilized plots. Nymphs of two Cicada genera were collected (Cicadetta spp. and Tibicen spp.). Cicadetta nymphs were more abundant in burned plots, but mowing reduced their abundance. Tibicen nymphs were collected almost exclusively from unburned plots. Treatment effects on herbivorous beetle larvae (Scarabaeidae, Elateridae, and Curculionidae) were variable, but nutrient additions (N or P) usually resulted in greater densities, whereas mowing usually resulted in lower densities. Our results suggest that departures from historical disturbance regimes (i.e. frequent fire and grazing) may render soils more susceptible to increased numbers of European earthworms, and that interactions between fire, aboveground biomass removal, and vegetation responses affect the structure and composition of invertebrate communities in tallgrass prairie soils.     

Variation in quality parameters between and within 14 Nordic tree fruit and berry species

Abstract

A 3-year study was carried out to investigate quality parameters in 14 tree fruit and berry species grown in southern Norway. The species were blueberry, apple, aronia, sour cherry, sweet cherry, red raspberry, strawberry, blackcurrant, gooseberry, red currant and elderberry, harvested along with wild bilberry, cloudberry and lingonberry. Significant differences between species were identified for all quality parameters. The coefficient of variation between species was lowest for pH (12.5%), dry matter (18.9%) and soluble solids (25.3%), followed by titratable acids (59.3%), total phenolics (83.8%), antioxidant capacity FRAP (85.7%) and antiradical power by the DPPH-assay (97.8%), total monomeric anthocyanins (132%) and ascorbic acid (137%). Average coefficient of variation within species were lower and ranged from 4 (pH) to 62% (ascorbic acid). Only the FRAP values were significantly affected by harvesting year with lower levels in 2004 than in 2005 and 2006. There were significant interactions between species and harvesting year for dry matter, soluble solids, pH, ascorbic acid and FRAP. The results indicate generic ranges in composition within species independent upon growing location and climate, and the composition of the tree fruits and berries is not likely to deviate from these ranges. It is concluded that desirable composition of tree fruits and berries and their products should primarily be achieved by selection among species rather than searching fors broadened variation within individual species.     

Potassium Fixation and Release Characteristics in Rhizosphere and Nonrhizosphere Soils for a Rapeseed–Rice Cropping Sequence

Potassium (K) fixation and release in soil are important factors in the long-term sustainability of a cropping system. Changes in K concentration and characteristics of K fixation and release in rhizosphere and nonrhizosphere soils in the rapeseed (Brassica napus L.)–rice (Oryza sativa L.) rotation were investigated using a rhizobox system. The concentrations of different forms of K in both rhizosphere and nonrhizosphere soils decreased with plants compared to without plants, regardless of K fertilizer application. Potassium uptake by crops mainly came from the rhizosphere soil. In the treatment without K fertilizer (–K), the main form of K supplied by the soil to the crops was 1.0 mol L^?1 nitric acid (HNO₃) nonextractable K, followed by nonexchangeable K, and then exchangeable K. In the treatment with K fertilizer (+K), the main K forms supplied by the soil to the crops were exchangeable K and nonexchangeable K. The amount and rate of K fixation after one cycle of the rapeseed–rice rotation was greater in rhizosphere soil than in nonrhizosphere soil. The amount and rate of K fixation of soil in the +K treatment were significantly less than in the –K treatment. The cumulative amounts of K released with 1.0 mol L^?1 ammonium acetate (NH₄OAc) and 1.0 mol L^?1 HNO₃ extraction increased with the increasing numbers of extractions, but the K-releasing power of soil by successive extraction decreased gradually and finally became almost constant. The release of K was less in rhizosphere soil than in nonrhizosphere soil. The release of K in the +K treatment was similar to that in the –K treatment in rhizosphere soil, but the K release in nonrhizosphere soil was greater with the +K than the –K treatment. Overall, the information obtained in this study will be helpful in formulating more precise K fertilizer recommendations for certain soils.     

Electron donor and pH relationships for biologically enhanced dissolution of chlorinated solvent DNAPL in groundwater

Biologically enhanced dissolution offers a method to speed removal of chlorinated solvent dense non-aqueous-phase liquid (DNAPL) sources such as tetrachloroethene (PCE) and trichoroethene (TCE) from aquifers. Bioremediation is accomplished by adding an electron donor to the source zone where fermentation to intermediates leading to acetic acid and hydrogen results. The hydrogen and possibly acetic acid are used by dehalogenating bacteria to convert PCE and TCE to ethene and hydrochloric acid. Reductive dehalogenation is thus an acid forming process, and sufficient alkalinity must be present to maintain a near neutral pH. The bicarbonate alkalinity required to maintain pH above 6.5 is a function of the electron donor: 800 mg/L of bicarbonate alkalinity is sufficient to achieve about 1.2 mM TCE dechlorination with glucose, 1.7 mM with lactate, and a much higher 3.3 mM with formate. Laboratory studies indicate that in mixed culture, formate can be used as an electron donor for complete conversion to ethene, contrary to pure cultures studies indicating it cannot. Various strategies can be used to add electron donor to an aquifer for DNAPL dehalogenation while minimizing pH problems and excessive electron donor usage, including use of injection-extraction wells, dual recirculation wells, and nested injection-extraction wells.     

Biodiversity and ecogeography of wild Lactuca spp. in some European countries

Staff members of the Department of Botany of Palacký University in Olomouc and Gene Bank Department – Workplace Olomouc, Research Institute of Crop Production in Prague, Czech Republic, conducted an expedition in seven European countries (Austria, Czech Republic, France, Germany, Italy, the Netherlands, Switzerland) in August/September 1999 to collect wild Lactuca spp. germplasm and study its geographic distribution, ecology and biodiversity. During the mission, more than 600 locations were visited resulting in the collection of 602 seed samples (accessions) of wild Lactuca species and 13 seed samples of related genera (Chondrilla and Mycelis). Lactuca serriola f. serriola, L. serriola f. integrifolia, L. saligna and L. viminea subsp. chondrilliflora were prevalent in southern Europe (Italy, France), however, only L. serriola was common in central and western Europe (Austria, Czech Republic, Germany, Netherlands, Switzerland). The greatest diversity of Lactuca species was found in France, where also the most seed samples (165) were collected. The most characteristic habitats with a high density of Lactuca spp. populations were observed along roads and highways, grassy ditches, ruderal communities, and dust-heaps. Natural infections by powdery mildew (Erysiphe cichoracearum) and downy mildew (Bremia lactucae) on some wild Lactuca spp. were observed. Recent observations concerning the geographic distribution, population structure, habitats, and natural occurrence of diseases of Lactuca spp. are discussed. This assemblage of genetic resources of Lactuca spp. can serve as the basis of future studies of species diversification, spatial population structure, plant microevolution, domestication processes, and genetic variability of host-parasite interactions.     

Responses of native legume desert trees used for reforestation in the Sonoran Desert to plant growth-promoting microorganisms in screen house

Three slow-growing legume trees used for desert reforestation and urban gardening in the Sonoran Desert of Northwestern Mexico and the Southwestern USA were evaluated whether their growth can be promoted by inoculation with plant growth-promoting bacteria (Azospirillum brasilense and Bacillus pumilus), unidentified arbuscular mycorrhizal (AM) fungi (mainly Glomus sp.), and supplementation with common compost under regular screenhouse cultivation common to these trees in nurseries. Mesquite amargo (Prosopis articulata) and yellow palo verde (Parkinsonia microphylla) had different positive responses to several of the parameters tested while blue palo verde (Parkinsonia florida) did not respond. Survival of all tree species was over 80% and survival of mesquite was almost 100% after 10 months of cultivation. Inoculation with growth-promoting microorganisms induced significant effects on the leaf gas exchange of these trees, measured as transpiration and diffusive resistance, when these trees were cultivated without water restrictions.     

Increase in auxiliary photoprotective photosynthetic pigments in wheat seedlings induced by Azospirillum brasilense

Inoculation of wheat seedlings with the plant growth-promoting bacterium Azospirillum brasilense Cd was immobilized in alginate microbeads and, without applying any stress, significantly increased the quantity of several photosynthetic pigments, such as chlorophyll a, chlorophyll b, and the auxiliary photoprotective pigments violaxanthin, zeaxanthin, antheroxanthin, lutein, neoxanthin, and β-carotene. This resulted in greener plants with no apparent visible stress. After monitoring the quantity of photosynthetic pigments for 4 weeks, we observed that inoculated plants had higher quantities of pigments in shoot and stem. The greatest difference in the quantity of all pigments between inoculated and noninoculated plants occurred in the first week of growth. Regardless of treatment, the quantity of pigments in stems was three to four times less than the quantity of these pigments in shoots. Application of Azospirillum, either as liquid inoculant or as alginate microbeads, did not alter the positive effect of the bacteria on pigment production or the positive response of the plants towards A. brasilense Cd inoculation.