脱氧雪腐镰刀菌烯醇、黄曲霉毒素B_1和玉米赤霉烯酮对秀丽隐杆线虫的联合毒性研究

脱氧雪腐镰刀菌烯醇(DON)、黄曲霉毒素B_1(AFB_1)和玉米赤霉烯酮(ZEN)是最为常见的粮食真菌毒素,易共存于谷物产品和动物饲料中,而目前对其联合毒性的研究较少,且研究结果并不完全一致。为探究DON、AFB_1和ZEN的联合毒性作用,本论文以秀丽隐杆线虫(C.elegans)为模型,分别评估了毒素混合物AFB_1+DON、AFB_1+ZEN、DON+ZEN和AFB_1+DON+ZEN对C.elegans的生长发育(体长)和生殖能力(产卵量)的毒性作用,并用Chou-Talalay模型来判定毒素混合物的相互作用类型。研究表明,AFB_1、DON和ZEN单独染毒C.elegans时,其毒作用强弱为AFB_1ZENDON。联合染毒时,AFB_1+DON对C.elegans产生协同作用,而DON+ZEN则产生拮抗作用;AFB_1+ZEN对体长(24 h)和产卵量的毒作用随着暴露浓度的增加,由弱拮抗变为协同作用,而在毒素暴露48 h后,对线虫的生长发育呈协同作用;AFB_1+DON+ZEN除在EC50-24 h和EC75-24 h时对体长产生明显的毒性增强作用外,其他普遍表现出拮抗作用。由此表明,DON、AFB_1和ZEN对C.elegans的联合毒性作用与剂量和时间相关。     

洱海沉积物不同分子量溶解性有机氮空间分布及光谱特征

利用超滤技术对洱海沉积物溶解性有机氮(DON)进行分子量分级,探究了DON各组分空间分布特征,并运用紫外-可见吸收光谱和三维荧光光谱对其结构进行了表征.结果表明,洱海沉积物总DON含量ω(DON)为23.46—61.40 mg·kg-1,平均值为37.19 mg·kg-1,空间分布总体呈现南部>北部>中部的趋势;分子量大于1K Da的大分子组分ω(>1KDa)占ω(DON)的比例为79.1%—93.0%,即洱海沉积物DON是以大分子为主.沉积物DON各组分的芳香性及腐殖化程度较高,芳香环取代基中活性官能团种类较多,且南部芳香性及腐殖化程度最高,北部芳香环取代基种类最多;大分子组分DON含有类富里酸荧光物质,其来源于陆源输入和微生物降解共同作用.由此可见,洱海沉积物DON主要由大分子物质组成,其腐殖化程度较高且含类富里酸荧光物质,而此类DON对藻类的生物有效性较低,这可能是洱海沉积物氮含量较高而上覆水氮浓度较低的原因之一,即沉积物DON分子组成及其结构特征可在一定程度上作为湖泊营养水平的衡量指标.     

淡水水体溶解有机氮对有毒藻种的生物有效性

溶解有机氮(Dissolved organic nitrogen,DON)是多数天然水体中溶解氮的主要组成部分。天然水体DON是许多微生命体包括有毒藻种的氮营养源,在供水安全以及水体富营养化等方面的生态环境效应不容忽视。文章系统地介绍了淡水水体DON含量与来源、生物有效性与估算方法,以及对有毒藻种生长的影响。DON的来源是影响水体中DON含量动态特征的关键因素。DON来源包括陆地径流,植物碎屑,土壤淋溶液,沉积物释放,大气沉降,藻类、大型植物、细菌与细胞死亡或自我分解,微型及大型浮游动物捕食和排泄、分泌物释放等。研究表明约有12%～72%的DON可迅速被生物所利用,具显著差异,究其原因可能是其来源组成、化学本质(分子质量与极性)、测试生物组成、是否有细菌作用等因素造成的。不同藻种具有不同氮源利用能力,DON对藻类生长具有直接或间接的作用,并可能影响藻类群落结构(有毒藻类成为优势种)。考虑到水环境保护与饮用水安全供水的重要性,未来研究应重视淡水水体DON生物有效性与其化学本质的揭示,尤其是对有毒藻种。     

淡水水体溶解有机氮对有毒藻种的生物有效性

溶解有机氮（Dissolved organic nitrogen,DON）是多数天然水体中溶解氮的主要组成部分。天然水体DON是许多微生命体包括有毒藻种的氮营养源,在供水安全以及水体富营养化等方面的生态环境效应不容忽视。文章系统地介绍了淡水水体DON含量与来源、生物有效性与估算方法,以及对有毒藻种生长的影响。DON的来源是影响水体中DON含量动态特征的关键因素。DON来源包括陆地径流,植物碎屑,土壤淋溶液,沉积物释放,大气沉降,藻类、大型植物、细菌与细胞死亡或自我分解,微型及大型浮游动物捕食和排泄、分泌物释放等。研究表明约有12％～72％的DON可迅速被生物所利用,具显著差异,究其原因可能是其来源组成、化学本质（分子质量与极性）、测试生物组成、是否有细菌作用等因素造成的。不同藻种具有不同氮源利用能力,DON对藻类生长具有直接或间接的作用,并可能影响藻类群落结构（有毒藻类成为优势种）。考虑到水环境保护与饮用水安全供水的重要性,未来研究应重视淡水水体DON生物有效性与其化学本质的揭示,尤其是对有毒藻种。     

光照对湖泊上覆水DON影响机制及环境学意义

利用三维荧光光谱和紫外-可见光谱技术,通过室内模拟实验研究了光照对洱海上覆水溶解性有机氮(DON)影响,经平行因子分析法(PARAFAC)和荧光区域积分法(FRI)解析了DON含量、结构组分变化特征,并探讨了其环境学意义.结果表明:(1)未加汞光照条件下,洱海上覆水DON含量随光照时间延长呈波动上升趋势,NH_4~+与DON含量呈显著负相关(R2=0.94,P0.05),即NH_4~+与DON之间存在相互转化,且光照可能促进了NH_4~+向DON的转化;(2)加HgCl_2后实验组与对照组SUVA254(1.78、1.85)、A253/A203(0.35、0.34)、E2/E3(5.85、5.77)及SR(1.03、1.14)均值差别不大,未加HgCl_2实验组较对照组SUVA254、A253/A203、E2/E3值有一定差别,表明光照主要是通过微生物作用,进而影响DON特征,表现为光照增强了DON芳香环取代基结构的复杂程度,并且使得羰基、羧基、羟基和酯基种类有所增多;(3)PARAFAC识别出类蛋白质物质(T峰)和类富里酸物质(A峰)两类组分,表明腐殖质类物质与蛋白质类物质之间可能存在相互转化,且微生物所发挥的作用较为明显.以上结果表明,光照可增强生物活性,进而影响对DON的转化和降解.     

水中天然含氮有机物的形成、迁移转化及分布

天然含氮有机物是水环境中的重要组成部分,其在天然水体中的形态及分布对环境质量有显著影响.本文围绕水中天然含氮有机物在氮循环中的地位、迁移转化以及其在国内主要水域中的分布情况,对天然含氮有机物的研究现状进行了梳理.我国不同水域中溶解性含氮有机物(DON)浓度相差较大;其中水体中DON浓度一般在1.0 mg·L^-1以下;沉积物中DON浓度通常为几十至几百mg·kg^-1.水体DON以分子量<1 kDa的有机物为主,主要成分是尿素、氨基酸等物质.沉积物DON以分子量<1 kDa和>30 kDa的有机物为主,其中前者主要由芳构化程度较高的氨基酸等小分子有机物构成,后者以腐殖质类为主.水体中部分胺类物质本身具有一定毒性,游离氨基酸等DON还是卤乙腈、卤代酰胺、卤代硝基甲烷、卤化氰和亚硝胺等含氮消毒副产物的重要前体物.由于水体中许多含氮有机物具有生物可利用性,有机氮可能是引起水体富营养化的重要原因之一.     

中国与欧洲禾谷镰刀菌DON毒素HPLC定量比较分析

选用4个来自中国、7个来自欧洲的代表性禾谷镰刀菌菌株,经脱氧雪腐镰刀菌烯醇(DON)毒素特异引物鉴定,确认其具有产生DON毒素的遗传物质基础后,接种于PDB培养基培养7d,从培养基上清中经硅胶柱分离、纯化DON毒素,经HPLC定量分析表明,纯化的禾谷镰刀菌DON毒素,与购自公司的DON毒素标准样品一样,其HPLC检测谱峰清晰明显,基线平稳,无干扰,保留时间为9.5min左右;供试菌株的DON毒素含量分布在0.023~1.934μg/mL之间,德国菌株F703产毒量最大,比位居第二的中国菌株5005(1.232μg/mL)高57%;其余的6个欧洲菌株中,除意大利菌株Lor9(0.128μg/mL)略低于另一个中国菌株7105(0.135μg/mL)外,均比3个中国菌株(4020、7105、8029)的产毒量大.欧洲镰刀菌产生DON毒素的能力远远大于中国菌株,这说明欧洲菌株长期在欧洲生态环境下已演变形成其特有的高毒素代谢类型,我国有必要严防欧洲禾谷镰刀菌入侵,加强对来自欧洲的禾谷类粮食及其产品的镰刀菌和毒素的检疫和监控;同时,本研究建立的毒素样品制备与HPLC检测体系可用于准确定量分析样品的DON毒素.图3表2参14     

热带滨海区不同土地利用背景下土壤溶解性有机氮的组成与粒径分布特征

溶解态有机氮(DON)是土壤中活跃的氮库,其生态环境行为与它的化学组成和粒径分布密切相关。为评估热带滨海区不同土地利用方式对不同粒径中土壤溶解性有机氮组成特征的影响,从水稻田、橡胶园、菜园和果园采集土壤样品,通过一系列微滤和超滤(0.7,0.45,0.2,0.1μm,100,10,1 kDa)对土壤溶解性有机氮分级,并使用连续流动分析仪、三维荧光光谱和红外光谱研究了滤液中溶解态有机氮、无机氮的含量及荧光组分和有机官能团特征。结果表明,4种土地利用背景下土壤DON值的范围为5.25-10.88 mg·kg^-1,其大小顺序为水稻>菜园>果树>橡胶,且DON与溶解性总氮(DTN)的比值范围为26.08%-67.11%,其中橡胶土最高,水稻土最低;不同粒径下4种土地利用类型土壤DON主要集中在<100 kDa的粒径中,其值范围为4.85-9.48 mg·kg^-1,占全量的85.89%-92.41%。三维荧光光谱(3D-EEMs)及平行因子分析表明,4种土地利用背景下土壤DON含有两种类腐殖质组分及一种类蛋白质组分,且以类腐殖质组分为主,占比54.00%-77.67%;类蛋白组分对土地利用变化敏感,且随着粒径的减小,类蛋白组分占比增加,在<1 kDa组分中比例最高。红外光谱结果表明,4种土地利用背景下土壤DON主要在6个位置有相似的吸收峰,包含3410 cm^-1、1636 cm^-1、1402 cm^-1、1138-1035 cm^-1、673 cm^-1、602 cm^-1,不同土地利用背景下各吸收峰的透光度不同,强度最大的吸收来自游离的胺类N-H伸缩振动;水稻、菜园土壤DON芳香物质含量较高,结构较复杂。了解DON的组成与粒径分布对土地利用的响应,对进一步研究其生态环境行为具有重要意义。     

胶州湾不同季节尿素分布特征和来源探讨

根据2012年7月(丰水期)、2012年11月(平水期)、2013年3月(枯水期)和2013年5月(枯水期)在胶州湾海域进行的4次调查资料,分析了胶州湾不同季节表层海水中尿素的分布特征和可能来源,并初步探讨了胶州湾尿素和溶解有机氮(DON)的生物可利用性.结果表明,胶州湾尿素含量(以N计)分布在0.16—26.22μmol·L-1范围内,平均为5.39±5.21μmol·L-1,尿素在DON中的占比在0.02—0.89范围内,平均为0.21±0.21.胶州湾不同季节尿素空间分布呈现明显的斑块状,高值区主要位于胶州湾北部和东北部.胶州湾尿素含量呈现明显的季节差异,枯水期尿素含量及其在DON中的占比均明显高于平水期和丰水期.胶州湾丰水期和平水期尿素含量和分布主要决定于陆源输入,且与氨氮具有相似的来源和转化过程.胶州湾不同季节尿素浓度及其在DON占比都较高,表明胶州湾DON具有较高的生物可利用性,对该海域浮游植物生长影响不容忽视.     

冻融对湿地土壤可溶性碳、氮和氮矿化的影响

通过室内模拟试验,研究了不同冻融循环过程(-5-5℃或-25-5 ℃)对沼泽湿地土壤可溶性有机碳(DOC)、可溶性有机氮(DON)以及土壤有机氮矿化过程的影响.结果表明,随着冻融次数的增加,土壤DOC和DON含量呈先增加后降低趋势,土壤DOC含量在冻融1次(-5-5 ℃或-25-5 ℃冻融循环处理)后达最大值,而土壤DON分别在冻融2次(-5-5 ℃冻融循环处理)和4次(-25-5 ℃冻融循环处理)后达最大值.这说明在短期内冻融交替对土壤DOC和DON含量的影响较明显.冻结温度和冻融次数显著影响土壤有机氮矿化过程,且-25-5 ℃冻融循环比-5-5 ℃冻融循环矿化累积量高.冻融循环促进了土壤有机氮的矿化,有利于土壤有效氮的累积,为春季植物生长提供足够的氮素,对维持湿地生态系统稳定具有重要意义.     

Application of a 15N tracer method to the study of dissolved organic nitrogen uptake during spring and summer in Chesapeake Bay

The dissolved organic nitrogen (DON) pool in marine waters contains a diverse mixture of compounds. It is therefore difficult to accurately estimate planktonic uptake of DON using the limited number of radiolabeled compounds commercially available. We describe a method to estimate DON uptake rates using ¹⁵N-labeled DON recently released from phytoplankton. To make ¹⁵N-labeled DON, we incubated surface water with ¹⁵NH ₄ ⁺ and then isolated the DON, including any recently released DO¹⁵N, with ion retardation resin. This DON was then added to a freshly collected water sample from the same environment to quantify the rate of DON uptake. The technique was applied to investigate rates of DON uptake relative to inorganic nitrogen in the mesohaline Chesapeake Bay during May 1990 and August 1991. The May experiment took place after the spring bloom, and rates of DON uptake [ranging from 0.31 to 0.53 g-atom (g-at) Nl^-1 h^-1] often exceeded rates of NH ₄ ⁺ and NO ₃ ^- uptake combined. The rates of DON uptake at this time were higher than estimated bacterial productivity and were not correlated with bacterial abundance or bacterial productivity. They were, however, correlated with rates of NO ₃ ^- uptake. In May, we estimate that only 7 to 32% of DON uptake was a result of urea utilization. In contrast, in August, when regenerated nutrients predominate in Chesapeake Bay, rates of DON uptake (ranging from 0.14 to 0.51 g-atom Nl^-1 h^-1) were an average of 50% of the observed rates of NH ₄ ⁺ uptake. Consistent with the May experiment, rates of DON uptake were not correlated with bacterial production. A sizable fraction of DON uptake, however, appeared to be due to urea utilization; rates of urea uptake, measured independently, were equivalent to an average of 74% of the measured rates of DON uptake. These findings suggest that, during both periods of study, at least a fraction of the measured DON uptake may have been due to utilization by phytoplankton.     

Examining the coupling of carbon and nitrogen cycles in Appalachian streams: the role of dissolved organic nitrogen

Although regional and global models of nitrogen (N) cycling typically focus on nitrate, dissolved organic nitrogen (DON) is the dominant form of nitrogen export from many watersheds and thus the dominant form of dissolved N in many streams. Our understanding of the processes controlling DON export from temperate forests is poor. In pristine systems, where biological N limitation is common, N contained in recalcitrant organic matter (OM) can dominate watershed N losses. This recalcitrant OM often has moderately constrained carbon:nitrogen (C:N) molar ratios (approximately 25-55) and therefore, greater DON losses should be observed in sites where there is greater total dissolved organic carbon (DOC) loss. In regions where anthropogenic N pollution is high, it has been suggested that increased inorganic N availability can reduce biological demand for organic N and therefore increase watershed DON losses. This would result in a positive correlation between inorganic and organic N concentrations across sites with varying N availability. In four repeated synoptic surveys of stream water chemistry from forested watersheds along an N loading gradient in the southern Appalachians, we found surprisingly little correlation between DON and DOC concentrations. Further, we found that DON concentrations were always significantly correlated with watershed N loading and stream water [NO3-] but that the direction of this relationship was negative in three of the four surveys. The C:N molar ratio of dissolved organic matter (DOM) in streams draining watersheds with high N deposition was very high relative to other freshwaters. This finding, together with results from bioavailability assays in which we directly manipulated C and N availabilities, suggests that heterotrophic demand for labile C can increase as a result of dissolved inorganic N (DIN) loading, and that heterotrophs can preferentially remove N-rich molecules from DOM. These results are inconsistent with the two prevailing hypotheses that dominate interpretations of watershed DON loss. Therefore, we propose a new hypothesis, the indirect carbon control hypothesis, which recognizes that heterotrophic demand for N-rich DOM can keep stream water DON concentrations low when N is not limiting and heterotrophic demand for labile C is high.     

Plant diversity, CO2, and N influence inorganic and organic N leaching in grasslands

In nitrogen (N)-limited systems, the potential to sequester carbon depends on the balance between N inputs and losses as well as on how efficiently N is used, yet little is known about responses of these processes to changes in plant species richness, atmospheric CO2 concentration ([CO2]), and N deposition. We examined how plant species richness (1 or 16 species), elevated [CO2] (ambient or 560 ppm), and inorganic N addition (0 or 4 g x m(-2) x yr(-1)) affected ecosystem N losses, specifically leaching of dissolved inorganic N (DIN) and organic N (DON) in a grassland field experiment in Minnesota, USA. We observed greater DIN leaching below 60 cm soil depth in the monoculture plots (on average 1.8 and 3.1 g N x m(-2) x yr(-1) for ambient N and N-fertilized plots respectively) than in the 16-species plots (0.2 g N x m(-2) x yr(-1) for both ambient N and N-fertilized plots), particularly when inorganic N was added. Most likely, loss of complementary resource use and reduced biological N demand in the monoculture plots caused the increase in DIN leaching relative to the high-diversity plots. Elevated [CO2] reduced DIN concentrations under conditions when DIN concentrations were high (i.e., in N-fertilized and monoculture plots). Contrary to the results for DIN, DON leaching was greater in the 16-species plots than in the monoculture plots (on average 0.4 g N x m(-2) x yr(-1) in 16-species plots and 0.2 g N x m(-2) x yr(-1) in monoculture plots). In fact, DON dominated N leaching in the 16-species plots (64% of total N leaching as DON), suggesting that, even with high biological demand for N, substantial amounts of N can be lost as DON. We found no significant main effects of elevated [CO2] on DIN or DON leaching; however, elevated [CO2] reduced the positive effect of inorganic N addition on DON leaching, especially during the second year of observation. Our results suggest that plant species richness, elevated [CO2], and N deposition alter DIN loss primarily through changes in biological N demand. DON losses can be as large as DIN loss but are more sensitive to organic matter production and turnover.     

Changes in the dissolved nitrogen pool across land cover gradients in Wisconsin streams

Increases in anthropogenic nitrogen fixation have resulted in wide-scale enrichment of aquatic ecosystems. Existing biogeochemical theory suggests that N enrichment is associated with increasing concentrations of nitrate; however, dissolved organic nitrogen (DON) is often a major component of the total dissolved nitrogen (TDN) pool in streams and rivers, and its concentration can be significantly elevated in human-influenced basins. We examined N concentrations during summer base flow conditions in 324 Wisconsin streams to determine whether DON was a significant component of TDN and how its relative contribution changed across a gradient of increasing human (agriculture and urban) land use for 84 of these sites. Total dissolved nitrogen varied from 0.09 to 20.74 mg/L, and although DON was significantly higher in human-dominated basins relative to forested and mixed-cover basins, its concentration increased relatively slowly in response to increasing human land cover. This limited response reflected a replacement of wetland-derived DON in low-N streams by anthropogenic sources in human-dominated sites, such that net changes in DON were small across the land use gradient. Nitrate-N increased exponentially in response to greater human land cover, and NH4-N and NO2-N were present at low levels. Nitrite-N exceeded NH4-N at 20% of sites and reached a maximum concentration of 0.10 mg/L. This examination suggests that basic mechanisms driving N losses from old-growth forests subject to N saturation also shape the summertime N pool in Wisconsin streams, in addition to other processes dictated by landscape context. The overwhelming role of human land use in determining the relative and absolute composition of the summertime N pool included (1) rapid increases in NO3-N, (2) limited changes in DON, and (3) the unexpected occurrence of NO2-N. High (>3 mg/L) TDN conditions dominated by NO3-N, regardless of landscape context or forms of N inputs, indicate a state of "N hypersaturation", which appears to be increasingly common in human-influenced streams and rivers. Many sites in agriculturally rich areas had NO2-N and NO3-N concentrations that, if sustained, are at chronically toxic levels for sensitive aquatic biota, suggesting that N enrichment now has local consequences for resident stream biota in addition to contributing to coastal eutrophication.     

Growth of marine planktonic diatoms on inorganic and organic nitrogen

A study was conducted to determine if coastal diatoms from eutrophic waters (Werribee, Port Phillip Bay, Australia) are able to grow better than diatoms from oligotrophic waters (Bass Strait, Australia) on organic nitrogen compounds as their principal nitrogen sources. Eight clones of marine planktonic diatoms, belonging to 5 species (Skeletonema costatum, Asterionella japonica, Nitzschia closterium, Coscinodiscus sp., and Fragilaria sp.), were incubated with inorganic (either nitrate or ammonia) or organic (either urea, uric acid, alanine, aspartic acid, glutamic acid, glycine, serine, threonine, or valine) nitrogen sources and growth response was measured under high and low light intensities. All clones grew well on the organic as well as thorganic nitrogen compounds under both light regimes. Intraspecific differences were not great, as no appreciable difference was noted between clones from oligotrophic and eutrophic waters. The two negatively-charged amino acids, aspartic and glutamic acids, were somewhat less effective in supporting growth of some clones than were the other amino acids. Virtually all of the dissolved organic nitrogen (DON) compounds tested were utilizable for algal growth. Further, all clones appeared able to utilize at least some natural DON (uncharacterized) for cell division; in 1980, DON represented 97% of total nitrogen in Bass Strait and 83% of total nitrogen in Werribee waters. The results are consistent with previous findings on algal utilization of certain DON compounds and indicate comparable abilities of cells from oligotrophic and eutrophic coastal waters to assimilate these nutrients.Publication No. 307 in the Ministry for Conservation, Victoria, Environmental Studies Series