Rapid Detection of Nitrate in Aquatic Products by K-model of Characteristic Absorbance Points

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长江中下游地区浅水湖泊生源要素的生物地球化学循环

长江中下游地区浅水湖泊生源要素的生物地球化学循环一、本文概述Overview of this article本文旨在深入探讨长江中下游地区浅水湖泊生源要素的生物地球化学循环。

长江中下游地区作为中国的重要经济和文化中心，其浅水湖泊生态系统对于区域生态环境和经济发展具有至关重要的影响。

本文将对这一区域内浅水湖泊中的生源要素（如碳、氮、磷等）的生物地球化学循环过程进行系统的阐述和分析。

This article aims to explore in depth the biogeochemical cycles of biogenic elements in shallow lakes in the middle and lower reaches of the Yangtze River. As an important economic and cultural center of China, the shallow lake ecosystem in the middle and lower reaches of the Yangtze River has a crucial impact on the regional ecological environment and economic development. This article will systematically elaborate and analyze the biogeochemical cycling process of biogenic elements (such as carbon, nitrogen, phosphorus, etc.) inshallow lakes in this region.我们将概述长江中下游地区浅水湖泊的基本特征，包括湖泊的水文条件、生态环境和生源要素的分布状况。

在此基础上，我们将深入探讨这些湖泊中生源要素的生物地球化学循环过程，包括生源要素的输入、转化、输出和积累等关键环节。

液相色谱串联质谱临床检测方法的开发与验证

液相色谱串联质谱临床检测方法的开发与验证·189·检验医学2019年3月第34卷第3期Laboratory Medicine，March 2019，V ol. 34，No. 3液相色谱串联质谱临床检测方法的开发与验证中国医师协会检验医师分会临床质谱检验医学专业委员会摘要：液相色谱串联质谱（LC-MS/MS）是近年来发展极为迅速的新技术。

该技术结合了液相色谱的高分离性能和质谱的高敏感性、高特异性等优势，被广泛应用于化工、生物、医药、食品、临床医学、环境等领域。

在临床检验和诊断领域，LC-MS/MS作为传统诊断技术的补充，可提供更为准确、可靠的依据，使许多疾病得到准确、快速的诊断。

文章结合目前已公布的LC-MS/MS方法相关验证指南、文献及实际操作经验，系统介绍了LC-MS/MS方法开发的关键流程，并以25-羟基维生素D3 [25（OH）D3]为实例介绍方法验证的关键要素，为LC-MS/MS技术临床应用方法的建立、验证和实施提供参考。

关键词：液相色谱串联质谱；方法开发；方法验证Consensus of method development and validation of liquid chromatography-tandem mass spectrometry in clinical laboratories Clinical Mass Spectrometry Committee，Chinese Medical Doctor Association of Laboratory Medicine.Abstract：Liquid chromatography-tandem mass spectrometry （LC-MS/MS）is an emerging technology which has developed rapidly in recent years. It has combined the separation properties of liquid chromatography and the high sensitivity and specificity of mass spectrometry，which is widely applied in various areas，such as chemistry，biology，pharmaceutical science，food，clinic and environmental science. Especially in the field of clinical laboratory and diagnostics，LC-MS/MS used as a complement to traditional diagnostic techniques can often provide more accurate and reliable testingresults in accurate and rapid diagnosis of diseases. In this review，some internationally published LC-MS/MS method validation guidelines，related literature and practical experience were summarized，and some key processes of LC-MS/MS development were introduced. Using 25-hydroxyvitamin D3 [25（OH）D3] as an example，the key elements of method validation were reviewed，in order to provide a reference for the establishment，veri?cation and implementation of LC-MS/MS.Key words：Liquid chromatography-tandem mass spectrometry；Method development；Method validation 文章编号：1673-8640（2019）03-0189-08 中图分类号：R446.1 文献标志码： A DOI：10.3969/j.issn.1673-8640.2019.03.001近年来，各种检验新理论和新技术不断涌现，极大地推动了临床检验学科的发展。

生态鱼缸观察日志

生态鱼缸观察日志英文回答：Ecological Fish Tank Observation Log.Day 1:Today, I set up my ecological fish tank. I carefully arranged the rocks and plants to create a natural habitat for the fish. I added water and turned on the filter and heater. The water temperature is set at 26 degrees Celsius, which is ideal for the fish I plan to keep. I also added some fish food to start the nitrogen cycle.中文回答：生态鱼缸观察日志。

第一天：今天，我设置了我的生态鱼缸。

我仔细地摆放了岩石和植物，以营造一个适合鱼类生活的自然栖息地。

我加入了水并打开了过滤器和加热器。

水温设置在摄氏26度，这对我计划饲养的鱼类来说是理想的。

我还加入了一些鱼食来启动氮循环。

Day 2:Today, I noticed that the water in the fish tank has become slightly cloudy. This is a normal occurrence during the initial stages of setting up a new tank. It is caused by the growth of beneficial bacteria that help break down the fish waste. I will continue to monitor the water parameters and make sure they are within the appropriate range.中文回答：第二天：今天，我注意到鱼缸中的水变得稍微浑浊了。

超分子溶剂萃取

第42 卷第 5 期2023 年5 月Vol.42 No.5559~567分析测试学报FENXI CESHI XUEBAO（Journal of Instrumental Analysis）超分子溶剂萃取/超高效液相色谱－串联质谱法测定血浆中他克莫司含量谢以清1，2，吕悦广2，孟宪双2，雷海民1*，马强2*（1．北京中医药大学中药学院，北京102488；2．中国检验检疫科学研究院，北京100176）摘要：该文建立了血浆中免疫抑制剂他克莫司（TAC）的超分子溶剂（SUPRAS）萃取/超高效液相色谱－串联质谱分析方法。

通过单因素实验结合响应面设计对超分子溶剂组成、用量及涡旋萃取时间等关键因素进行优化后，血浆样本以正戊醇、四氢呋喃和水形成的超分子溶剂进行高效萃取。

萃取液经Waters ACQUITY UPLC BEH C18（50 mm × 2.1 mm，1.7 μm）色谱柱分离后，在电喷雾质谱正离子模式下，以多反应监测（MRM）模式对他克莫司进行测定，内标法定量。

结果表明，他克莫司在0.5 ~ 30 ng/mL质量浓度范围内的线性关系良好，相关系数（r）为0.998 6；方法检出限和定量下限分别为0.1、0.5 ng/mL；在低、中、高3个加标水平下，平均回收率（n = 3）为91.9% ~ 99.9%，相对标准偏差（RSD）为1.7% ~ 5.7%。

所建立的方法快速、灵敏、稳定，适用于血浆中他克莫司的准确测定。

关键词：他克莫司；免疫抑制剂；超分子溶剂；血浆；超高效液相色谱－串联质谱中图分类号：O657.7；R917文献标识码：A 文章编号：1004-4957（2023）05-0559-09Determination of Tacrolimus in Plasma by Supramolecular Solvent Extraction/Ultra-high Performance Liquid Chromatography－Tandem Mass SpectrometryXIE Yi-qing1，2，LÜ Yue-guang2，MENG Xian-shuang2，LEI Hai-min1*，MA Qiang2*（1．School of Chinese Materia Medica，Beijing University of Chinese Medicine，Beijing 102488，China；2．Chinese Academy of Inspection and Quarantine，Beijing 100176，China）Abstract：An analytical method for the determination of tacrolimus（TAC） in blood plasma was estab⁃lished by supramolecular solvent（SUPRAS）extraction combined with ultra-high performance liquid chromatography－tandem mass spectrometry．After optimizing the key factors such as the composition and amount of SUPRAS，and vortex extraction time through single factor experiment and response sur⁃face design，blood plasma samples were extracted efficiently with SUPRAS formed by pentanol，tetra⁃hydrofuran and water．The extract was separated on a Waters ACQUITY UPLC BEH C18column （50 mm × 2.1 mm，1.7 μm），analyzed by electrospray ionization mass spectrometry in positive ion mode under multiple reaction monitoring（MRM） mode，and quantified by internal standard method．Experimental results demonstrated that there was a good linear relationship for TAC in the concentration range of 0.5－30 ng/mL，with a correlation coefficient（r） of 0.998 6．The limit of detection（LOD）and quantitation（LOQ） were 0.1 ng/mL and 0.5 ng/mL，respectively．The average recoveries（n = 3）at low，medium and high spiked concentration levels ranged from 91.9% to 99.9%，with relative stan⁃dard deviations（RSDs） of 1.7%－5.7%．The proposed method is rapid，sensitive and stable，and it was suitable for the accurate determination of TAC in blood plasma.Key words：tacrolimus；immunosuppresive agent；supramolecular solvent；plasma；ultra-high performance liquid chromatography－tandem mass spectrometry免疫抑制剂是用于抑制机体免疫力的药物，多用于抑制肝肾移植术后的免疫反应，以及治疗变态反应性和自身免疫性疾病，如类风湿关节炎、红斑狼疮等［1－3］。

快速消解分光光度法测定高氯废水中COD

快速消解分光光度法测定高氯废水中COD陈燕12毕军平12刘沛12秦迪岚12刘艳菊12龙雯琪12(1.湖南省生态环境监测中心，长沙410019#.国家环境保护重金属污染监测重点实验室"长沙410019)摘要：快速消解分光光度法利用COD快速测定仪，用较少取样量，缩短消解时间，操作步骤简单,20min可反馈结果，能大大提高工作效率&该方法检出限、精密度和准确度均能满足各种水质COD测定的要求&使用抗高氯试剂能准确测定含Cl浓度大于1000mg/L的高氯废水COD,表明快速法可替代重铬酸盐法和氯气校正法，大大提高高氯废水COD测定的工作效率，是一种简单、快速、准确、环保的检测方法，适合大力推广使用&关键词：快速消解分光光度法COD重铬酸盐法高氯废水DOI：10.3969/j.issn.1001—232x.2021.03.012Determination of COD in high chlorine wastewater by rapid digestion spectrophotometry.ChenYan1,2,Bi J unping1'2,Liu Pei1'2,Qin DHan1'2,Liu Yanju1'2,Long Wenqi1'2((1.Hu,nan Ecology and Environment Monitoring Center,Changsha410019,China;2.State Environmental Protection Key Laboratory of Monitoring for Heavy Metal Pollutants,Changsha410019,China)Abstract:Rapid digesting spectrophotometry uses COD rapid measuring instrument,which has the advantages of less sampling amount and simple operation procedures"and can submit results in20minutes,by which the working efficiency can be significantly improved.The detection limit,precision and accuracy of the rapid method can meet the requirements of COD determination in various water quality samples.By using anti-high chlorine reagent,the COD of high chlorine wastewater containing Cl-concen-tration more than1000mg/L can be accurately determined.It shows that the rapid method can replace the dichromate method and chlorine correction method,and remarkably improve the efficiency of COD determination of high chlorine wastewater.Key words:Rapid digestion spectrophotometry；COD；Dichromate method；High chlorine wastewater化学需氧量(Chemical Oxygen Demand,即COD)是指在强酸并加热条件下，用重铬酸钾作为氧化剂处理水样时消耗氧化剂的量&COD常作为衡量水中有机物质含量的指标，也是我国实施污染物排放总量控制的指标之一&氯离子能被重铬酸盐氧化，并能与反应催化剂作用生成沉淀，是影响.COD结果的主要因素&高氯废水是指氯离子浓度大于1000mg/L的废水&在对废水进行COD监测时常遇到水中氯离子含量过高的情况，如处理不当，则会使结果不真实甚至影响实验进展&在进行COD测定时，要避免氯离子含量过高带来的结果误差&《水质化学需氧量的测定重铬酸盐法》(HJ828—2017)(以下简称重铬酸盐法)和《水质高氯废水化学需氧量的测定氯气校正法》(HJ/T70—2001)是环境监测机构常用的COD检测方法，这两种方法虽然准确度高，稳定性好，但操作繁琐，耗时较长，至少4〜5h才能出结果，甚至更长，给工作带来很多不便+旧。

溶解氧对硝化反硝化的影响

溶解氧对硝化反硝化的影响英文回答：Dissolved oxygen (DO) plays a crucial role in the nitrification-denitrification processes. Nitrification, the oxidation of ammonia to nitrite and nitrate, is an aerobic process that requires the presence of DO. In contrast, denitrification, the reduction of nitrate to nitrogen gas, is an anaerobic process that occurs in the absence of DO.The interplay between DO and nitrification-denitrification has significant implications for wastewater treatment and environmental management. In wastewater treatment plants, maintaining optimal DO levels isessential for efficient removal of nitrogen. High DO concentrations promote nitrification, while low DO concentrations favor denitrification. By controlling DO levels, it is possible to optimize nitrogen removal and minimize the release of harmful nitrogen compounds into the environment.In natural ecosystems, DO fluctuations can influence the rates and pathways of nitrification-denitrification. In aquatic environments, DO levels can vary seasonally or diurnally, affecting the microbial communities responsible for these processes. In soils, DO concentrations can be influenced by factors such as soil moisture content and aeration, which in turn can impact the balance between nitrification and denitrification.DO also affects the production of nitrous oxide (N2O), a potent greenhouse gas. Nitrous oxide can be produced as an intermediate during both nitrification and denitrification. Under high DO conditions, nitrification dominates, resulting in reduced N2O production. Conversely, under low DO conditions, denitrification becomes more dominant, leading to increased N2O production.中文回答：溶解氧 (DO) 对硝化反硝化过程有着至关重要的影响。

地表水氨氮含量测定过程中的注意事项

5 000 mL 。

实验室根据采集水样的多少自行选择规格。

采集少量的水样通常选用2 500 mL 的规格。

下面就在湖泊某一指定深度采集三个平行水样为例来阐述有机玻璃采水器的使用注意事项。

采集水样根据要求是不能取湖面上方浮层，也不能将湖底的泥沙采集，所以一般是在湖深的1/2处进行取样。

当然取样前要用湖水将采水器和盛水器进行三次润洗，然后依次采集三个平行样。

有个问题很值得讨论：采水深度的确定。

根据有机玻璃采水器的工作原理即水是从采水器的下端进入的，所以采水深度确定时，要从采水器的底部算起。

在今年的工业分析与检验国赛试点赛上，多数参赛队对于采水深度的确定是错误的，他们没有搞清楚有机玻璃采水器的工作原理，直接从采水器上方开始算深度。

2 现场空白注意事项采集地表水检测氨氮含量时，通常空白是用无氨水来完成的。

要求把无氨水带到采集现场后，再进行空白取样，并且所加保护剂要和其他三个平行样所加的保护剂品种和量要尽可能一致。

采集空白样品要在现场完成，主要是为了使得空白和现场所处的温度，湿度完全一致，才能排除环境因素带来的误差。

3 保护剂的选择采集水样时，对于所加保护剂的要求是：经济并且对测定无干扰和无不良影响。

不同水样和不同的测定项目使用的保护剂要求是不一样的。

这里对于保护的使用级别是有严格要求的，为了避免系统误差，要求保护剂的纯度尽可能高，即保护剂0 引言地表水指的是陆地表面动态水和静态水的总称，主要包括液态水和固态水，主要有河流、湖泊、沼泽、冰川等[1]。

地表水是目前人类生活用水的主要来源之一，也是世界各国水资源的主要组成部分之一。

水溶液中的氨氮是游离的氮或离子氮，水中氨氮主要来源于天然水中的含氮物质的降解过程，还有目前另一个主要来源就是生活污水和工业废水。

氨氮在一定条件下可以转换成亚硝酸盐，生活中如果长期饮用含有氨氮的水，那么水中的亚硝酸盐会和人体内的蛋白质结合形成亚硝胺，是一种致癌物质，对人体的健康会造成很大的危害。

硝酸盐还原英语

硝酸盐还原英语Nitrate Reduction in EnglishNitrates are a class of chemical compounds that consist of a nitrogen atom bonded to three oxygen atoms. They are ubiquitous in the environment, playing a crucial role in various natural and anthropogenic processes. One of the most significant aspects of nitrates is their ability to undergo reduction, a process in which the nitrogen atom in the nitrate molecule is converted to a different form, often with important implications for environmental and industrial applications.The reduction of nitrates is a complex and multifaceted process that can occur through both biological and chemical pathways. In the natural environment, the reduction of nitrates is primarily driven by the activities of various microorganisms, such as bacteria and archaea, which utilize nitrates as an electron acceptor in their metabolic processes. This process, known as denitrification, is a crucial component of the global nitrogen cycle, as it helps to remove excess nitrates from aquatic and terrestrial ecosystems.The denitrification process involves a series of enzymatic reactions inwhich nitrates are sequentially reduced to nitrites, nitric oxide, nitrous oxide, and finally, molecular nitrogen. The enzymes responsible for these reactions are collectively known as nitrate reductases, and they are produced by a diverse array of microorganisms, including both heterotrophic and autotrophic species.The reduction of nitrates can also occur through chemical pathways, particularly in industrial and agricultural settings. In these contexts, the reduction of nitrates is often employed as a means of mitigating the environmental impact of nitrate-rich waste streams or as a method of producing valuable chemical products.One of the most prominent examples of the chemical reduction of nitrates is the production of nitric oxide (NO), a highly reactive gas that has a wide range of applications in both the industrial and medical fields. Nitric oxide is produced through the reduction of nitrates using various reducing agents, such as hydrogen gas or organic compounds. This process is particularly important in the production of fertilizers, as nitric oxide is a key precursor to the synthesis of ammonia, a critical component of many agricultural fertilizers.Another important application of nitrate reduction is the treatment of wastewater and the removal of excess nitrates from aquaticenvironments. In this context, the reduction of nitrates is often coupled with the use of biological denitrification processes, in which microorganisms are used to convert nitrates into harmless nitrogen gas. This approach is particularly valuable in addressing the problem of eutrophication, a phenomenon in which excessive nutrient inputs, including nitrates, can lead to the overgrowth of algae and the depletion of dissolved oxygen in aquatic ecosystems.The reduction of nitrates also plays a role in the production of certain pharmaceutical and industrial chemicals. For example, the reduction of nitrates can be used to synthesize various organic compounds, such as amines and nitriles, which are important building blocks for a wide range of chemical products.Despite the numerous benefits of nitrate reduction, there are also some potential drawbacks and challenges associated with this process. One of the most significant concerns is the potential for the formation of nitrite, a compound that can be toxic to both humans and the environment. Additionally, the reduction of nitrates can also lead to the production of other potentially harmful compounds, such as nitric oxide and nitrous oxide, which can contribute to air pollution and climate change.To address these challenges, researchers and industry professionals are continually working to develop more efficient andenvironmentally-friendly methods of nitrate reduction. This includes the development of new catalysts and enzymatic systems, as well as the optimization of existing processes to minimize the formation of unwanted byproducts.In conclusion, the reduction of nitrates is a complex and multifaceted process with a wide range of applications in both the natural and industrial realms. From the removal of excess nitrates in aquatic environments to the production of valuable chemical products, the reduction of nitrates plays a crucial role in shaping the world around us. As our understanding of this process continues to evolve, it is likely that we will see even more innovative and sustainable approaches to nitrate reduction in the years to come.。

QuEChERS-_气相色谱-_三重四极杆质谱法测定不同基质中氟虫腈及其3_种代谢物残留量

QuEChERS-气相色谱-三重四极杆质谱法测定不同基质中氟虫腈及其3种代谢物残留量陈浩（常德市食品检验所，湖南常德 415000）摘要：建立了气相色谱-串联三重四极杆质谱仪测定大米、茶叶、油麦菜和柑橘中氟虫腈及其3种代谢物残留量的方法。

使用改良后的QuEChERS 前处理技术进行样品的提取净化，结合GC-MS/MS 检测，内标法定量。

结果表明，在0.005～0.800 mg·L -1，4种不同基质中氟虫腈及其3种代谢物均呈现良好的线性关系，相关系数R 2＞0.996。

氟虫腈及其3种代谢物在4种不同基质中均存在不同程度的基质增强效应，在0.01 mg·kg -1、0.05 mg·kg -1、0.10 mg·kg -1 3个添加水平下，氟虫腈及其3种代谢物的平均回收率在88.3%～102.5%，相对标准偏差在2.6%～7.7%，方法定量限在0.5～1.0 μg·kg -1。

该方法操作简单、灵敏、准确，适用于大米、茶叶、蔬菜水果中氟虫腈及其代谢物的快速测定。

关键词：QuEChERS ；气相色谱-串联三重四极杆质谱法；氟虫腈；代谢物Determination of Fipronil and Its Three Metabolites in Different Matrices by QuEChERS Coupled with Gas Chromatography-Triple Quadrupole Mass SpectrometryCHEN Hao(Changde Institute for Food Control, Changde 415000, China)Abstract: A gas chromatography-tandem triple quadrupole mass spectrometry method has been established for the determination of ﬂuconazole and its three metabolites residues in rice, tea, rapeseed, and citrus. The improved QuEChERS pre-treatment technology was used for sample extraction and purification, combined with GC-MS/MS detection and internal standard quantification. The results showed that within the concentration range of 0.005～ 0.800 mg·L -1, there was a good linear relationship between fipronil and its three metabolites in four diﬀerent matrices, with a correlation coefficient of R 2＞0.996. Flufenitrile and its three metabolites exhibited varying degrees of matrix enhancement eﬀects in four diﬀerent matrices. At three levels of addition of 0.01 mg·kg -1, 0.05 mg·kg -1 and 0.10 mg·kg -1, the average recovery rates of ﬂufenitrile and its three metabolites ranged from 88.3% to 102.5%, with a relative standard deviation of 2.6% to 7.7%, and the quantitative limit of the method is 0.5～1.0 μg·kg -1. The method was found to be simple, sensitive, accurate and suitable for the rapid quantitative analysis of fipronil and its metabolites in in rice, tea, vegetables and fruits.Keywords: QuEChERS; gas chromatography-triple quadrupole mass spectrometry; fipronil; metabolite作者简介：陈浩（1987—），男，湖南常德人，硕士，工程师。

微生物英文文献及翻译—原文

Dynamic and distribution of ammonia-oxidizing bacteria communities during sludge granulation in an anaerobic e aerobic sequencing batch reactorZhang Bin a ,b ,Chen Zhe a ,b ,Qiu Zhigang a ,b ,Jin Min a ,b ,Chen Zhiqiang a ,b ,Chen Zhaoli a ,b ,Li Junwen a ,b ,Wang Xuan c ,*,Wang Jingfeng a ,b ,**aInstitute of Hygiene and Environmental Medicine,Academy of Military Medical Sciences,Tianjin 300050,PR China bTianjin Key Laboratory of Risk Assessment and Control for Environment and Food Safety,Tianjin 300050,PR China cTianjin Key Laboratory of Hollow Fiber Membrane Material and Membrane Process,Institute of Biological and Chemical Engineering,Tianjin Polytechnical University,Tianjin 300160,PR Chinaa r t i c l e i n f oArticle history:Received 30June 2011Received in revised form 10September 2011Accepted 10September 2011Available online xxx Keywords:Ammonia-oxidizing bacteria Granular sludgeCommunity development Granule sizeNitrifying bacteria distribution Phylogenetic diversitya b s t r a c tThe structure dynamic of ammonia-oxidizing bacteria (AOB)community and the distribution of AOB and nitrite-oxidizing bacteria (NOB)in granular sludge from an anaerobic e aerobic sequencing batch reactor (SBR)were investigated.A combination of process studies,molecular biotechniques and microscale techniques were employed to identify and characterize these organisms.The AOB community structure in granules was substantially different from that of the initial pattern of the inoculants sludge.Along with granules formation,the AOB diversity declined due to the selection pressure imposed by process conditions.Denaturing gradient gel electrophoresis (DGGE)and sequencing results demonstrated that most of Nitrosomonas in the inoculating sludge were remained because of their ability to rapidly adapt to the settling e washing out action.Furthermore,DGGE analysis revealed that larger granules beneﬁt more AOB species surviving in the reactor.In the SBR were various size granules coexisted,granule diameter affected the distribution range of AOB and NOB.Small and medium granules (d <0.6mm)cannot restrict oxygen mass transfer in all spaces of the rger granules (d >0.9mm)can result in smaller aerobic volume fraction and inhibition of NOB growth.All these observations provide support to future studies on the mechanisms responsible for the AOB in granules systems.ª2011Elsevier Ltd.All rights reserved.1.IntroductionAt sufﬁciently high levels,ammonia in aquatic environments can be toxic to aquatic life and can contribute to eutrophica-tion.Accordingly,biodegradation and elimination of ammonia in wastewater are the primary functions of thewastewater treatment process.Nitriﬁcation,the conversion of ammonia to nitrate via nitrite,is an important way to remove ammonia nitrogen.It is a two-step process catalyzed by ammonia-oxidizing and nitrite-oxidizing bacteria (AOB and NOB).Aerobic ammonia-oxidation is often the ﬁrst,rate-limiting step of nitriﬁcation;however,it is essential for the*Corresponding author .**Corresponding author.Institute of Hygiene and Environmental Medicine,Academy of Military Medical Sciences,Tianjin 300050,PR China.Tel.:+862284655498;fax:+862223328809.E-mail addresses:wangxuan0116@ (W.Xuan),jingfengwang@ (W.Jingfeng).Available online atjournal homepage:/locate/watresw a t e r r e s e a r c h x x x (2011)1e 100043-1354/$e see front matter ª2011Elsevier Ltd.All rights reserved.doi:10.1016/j.watres.2011.09.026removal of ammonia from the wastewater(Prosser and Nicol, 2008).Comparative analyses of16S rRNA sequences have revealed that most AOB in activated sludge are phylogeneti-cally closely related to the clade of b-Proteobacteria (Kowalchuk and Stephen,2001).However,a number of studies have suggested that there are physiological and ecological differences between different AOB genera and lineages,and that environmental factors such as process parameter,dis-solved oxygen,salinity,pH,and concentrations of free ammonia can impact certain species of AOB(Erguder et al., 2008;Kim et al.,2006;Koops and Pommerening-Ro¨ser,2001; Kowalchuk and Stephen,2001;Shi et al.,2010).Therefore, the physiological activity and abundance of AOB in waste-water processing is critical in the design and operation of waste treatment systems.For this reason,a better under-standing of the ecology and microbiology of AOB in waste-water treatment systems is necessary to enhance treatment performance.Recently,several developed techniques have served as valuable tools for the characterization of microbial diversity in biological wastewater treatment systems(Li et al., 2008;Yin and Xu,2009).Currently,the application of molec-ular biotechniques can provide clariﬁcation of the ammonia-oxidizing community in detail(Haseborg et al.,2010;Tawan et al.,2005;Vlaeminck et al.,2010).In recent years,the aerobic granular sludge process has become an attractive alternative to conventional processes for wastewater treatment mainly due to its cell immobilization strategy(de Bruin et al.,2004;Liu et al.,2009;Schwarzenbeck et al.,2005;Schwarzenbeck et al.,2004a,b;Xavier et al.,2007). Granules have a more tightly compact structure(Li et al.,2008; Liu and Tay,2008;Wang et al.,2004)and rapid settling velocity (Kong et al.,2009;Lemaire et al.,2008).Therefore,granular sludge systems have a higher mixed liquid suspended sludge (MLSS)concentration and longer solid retention times(SRT) than conventional activated sludge systems.Longer SRT can provide enough time for the growth of organisms that require a long generation time(e.g.,AOB).Some studies have indicated that nitrifying granules can be cultivated with ammonia-rich inorganic wastewater and the diameter of granules was small (Shi et al.,2010;Tsuneda et al.,2003).Other researchers reported that larger granules have been developed with the synthetic organic wastewater in sequencing batch reactors(SBRs)(Li et al., 2008;Liu and Tay,2008).The diverse populations of microor-ganisms that coexist in granules remove the chemical oxygen demand(COD),nitrogen and phosphate(de Kreuk et al.,2005). However,for larger granules with a particle diameter greater than0.6mm,an outer aerobic shell and an inner anaerobic zone coexist because of restricted oxygen diffusion to the granule core.These properties of granular sludge suggest that the inner environment of granules is unfavorable to AOB growth.Some research has shown that particle size and density induced the different distribution and dominance of AOB,NOB and anam-mox(Winkler et al.,2011b).Although a number of studies have been conducted to assess the ecology and microbiology of AOB in wastewater treatment systems,the information on the dynamics,distribution,and quantiﬁcation of AOB communities during sludge granulation is still limited up to now.To address these concerns,the main objective of the present work was to investigate the population dynamics of AOB communities during the development of seedingﬂocs into granules,and the distribution of AOB and NOB in different size granules from an anaerobic e aerobic SBR.A combination of process studies,molecular biotechniques and microscale techniques were employed to identify and char-acterize these organisms.Based on these approaches,we demonstrate the differences in both AOB community evolu-tion and composition of theﬂocs and granules co-existing in the SBR and further elucidate the relationship between distribution of nitrifying bacteria and granule size.It is ex-pected that the work would be useful to better understand the mechanisms responsible for the AOB in granules and apply them for optimal control and management strategies of granulation systems.2.Material and methods2.1.Reactor set-up and operationThe granules were cultivated in a lab-scale SBR with an effective volume of4L.The effective diameter and height of the reactor was10cm and51cm,respectively.The hydraulic retention time was set at8h.Activated sludge from a full-scale sewage treat-ment plant(Jizhuangzi Sewage Treatment Works,Tianjin, China)was used as the seed sludge for the reactor at an initial sludge concentration of3876mg LÀ1in MLSS.The reactor was operated on6-h cycles,consisting of2-min inﬂuent feeding,90-min anaerobic phase(mixing),240-min aeration phase and5-min efﬂuent discharge periods.The sludge settling time was reduced gradually from10to5min after80SBR cycles in20days, and only particles with a settling velocity higher than4.5m hÀ1 were retained in the reactor.The composition of the inﬂuent media were NaAc(450mg LÀ1),NH4Cl(100mg LÀ1),(NH4)2SO4 (10mg LÀ1),KH2PO4(20mg LÀ1),MgSO4$7H2O(50mg LÀ1),KCl (20mg LÀ1),CaCl2(20mg LÀ1),FeSO4$7H2O(1mg LÀ1),pH7.0e7.5, and0.1mL LÀ1trace element solution(Li et al.,2007).Analytical methods-The total organic carbon(TOC),NHþ4e N, NOÀ2e N,NOÀ3e N,total nitrogen(TN),total phosphate(TP) concentration,mixed liquid suspended solids(MLSS) concentration,and sludge volume index at10min(SVI10)were measured regularly according to the standard methods (APHA-AWWA-WEF,2005).Sludge size distribution was determined by the sieving method(Laguna et al.,1999).Screening was performed with four stainless steel sieves of5cm diameter having respective mesh openings of0.9,0.6,0.45,and0.2mm.A100mL volume of sludge from the reactor was sampled with a calibrated cylinder and then deposited on the0.9mm mesh sieve.The sample was subsequently washed with distilled water and particles less than0.9mm in diameter passed through this sieve to the sieves with smaller openings.The washing procedure was repeated several times to separate the gran-ules.The granules collected on the different screens were recovered by backwashing with distilled water.Each fraction was collected in a different beaker andﬁltered on quantitative ﬁlter paper to determine the total suspended solid(TSS).Once the amount of total suspended solid(TSS)retained on each sieve was acquired,it was reasonable to determine for each class of size(<0.2,[0.2e0.45],[0.45e0.6],[0.6e0.9],>0.9mm) the percentage of the total weight that they represent.w a t e r r e s e a r c h x x x(2011)1e10 22.2.DNA extraction and nested PCR e DGGEThe sludge from approximately8mg of MLSS was transferred into a1.5-mL Eppendorf tube and then centrifuged at14,000g for10min.The supernatant was removed,and the pellet was added to1mL of sodium phosphate buffer solution and aseptically mixed with a sterilized pestle in order to detach granules.Genomic DNA was extracted from the pellets using E.Z.N.A.äSoil DNA kit(D5625-01,Omega Bio-tek Inc.,USA).To amplify ammonia-oxidizer speciﬁc16S rRNA for dena-turing gradient gel electrophoresis(DGGE),a nested PCR approach was performed as described previously(Zhang et al., 2010).30m l of nested PCR amplicons(with5m l6Âloading buffer)were loaded and separated by DGGE on polyacrylamide gels(8%,37.5:1acrylamide e bisacrylamide)with a linear gradient of35%e55%denaturant(100%denaturant¼7M urea plus40%formamide).The gel was run for6.5h at140V in 1ÂTAE buffer(40mM Tris-acetate,20mM sodium acetate, 1mM Na2EDTA,pH7.4)maintained at60 C(DCodeäUniversal Mutation Detection System,Bio-Rad,Hercules,CA, USA).After electrophoresis,silver-staining and development of the gels were performed as described by Sanguinetti et al. (1994).These were followed by air-drying and scanning with a gel imaging analysis system(Image Quant350,GE Inc.,USA). The gel images were analyzed with the software Quantity One,version4.31(Bio-rad).Dice index(Cs)of pair wise community similarity was calculated to evaluate the similarity of the AOB community among DGGE lanes(LaPara et al.,2002).This index ranges from0%(no common band)to100%(identical band patterns) with the assistance of Quantity One.The Shannon diversity index(H)was used to measure the microbial diversity that takes into account the richness and proportion of each species in a population.H was calculatedusing the following equation:H¼ÀPn iNlogn iN,where n i/Nis the proportion of community made up by species i(bright-ness of the band i/total brightness of all bands in the lane).Dendrograms relating band pattern similarities were automatically calculated without band weighting(consider-ation of band density)by the unweighted pair group method with arithmetic mean(UPGMA)algorithms in the Quantity One software.Prominent DGGE bands were excised and dissolved in30m L Milli-Q water overnight,at4 C.DNA was recovered from the gel by freeze e thawing thrice.Cloning and sequencing of the target DNA fragments were conducted following the estab-lished method(Zhang et al.,2010).2.3.Distribution of nitrifying bacteriaThree classes of size([0.2e0.45],[0.45e0.6],>0.9mm)were chosen on day180for FISH analysis in order to investigate the spatial distribution characteristics of AOB and NOB in granules.2mg sludge samples wereﬁxed in4%para-formaldehyde solution for16e24h at4 C and then washed twice with sodium phosphate buffer;the samples were dehydrated in50%,80%and100%ethanol for10min each. Ethanol in the granules was then completely replaced by xylene by serial immersion in ethanol-xylene solutions of3:1, 1:1,and1:3by volume andﬁnally in100%xylene,for10min periods at room temperature.Subsequently,the granules were embedded in parafﬁn(m.p.56e58 C)by serial immer-sion in1:1xylene-parafﬁn for30min at60 C,followed by 100%parafﬁn.After solidiﬁcation in parafﬁn,8-m m-thick sections were prepared and placed on gelatin-coated micro-scopic slides.Parafﬁn was removed by immersing the slide in xylene and ethanol for30min each,followed by air-drying of the slides.The three oligonucleotide probes were used for hybridiza-tion(Downing and Nerenberg,2008):FITC-labeled Nso190, which targets the majority of AOB;TRITC-labeled NIT3,which targets Nitrobacter sp.;TRITC-labeled NSR1156,which targets Nitrospira sp.All probe sequences,their hybridization condi-tions,and washing conditions are given in Table1.Oligonu-cleotides were synthesized andﬂuorescently labeled with ﬂuorochomes by Takara,Inc.(Dalian,China).Hybridizations were performed at46 C for2h with a hybridization buffer(0.9M NaCl,formamide at the percentage shown in Table1,20mM Tris/HCl,pH8.0,0.01% SDS)containing each labeled probe(5ng m LÀ1).After hybrid-ization,unbound oligonucleotides were removed by a strin-gent washing step at48 C for15min in washing buffer containing the same components as the hybridization buffer except for the probes.For detection of all DNA,4,6-diamidino-2-phenylindole (DAPI)was diluted with methanol to aﬁnal concentration of1ng m LÀ1.Cover the slides with DAPI e methanol and incubate for15min at37 C.The slides were subsequently washed once with methanol,rinsed brieﬂy with ddH2O and immediately air-dried.Vectashield(Vector Laboratories)was used to prevent photo bleaching.The hybridization images were captured using a confocal laser scanning microscope (CLSM,Zeiss710).A total of10images were captured for each probe at each class of size.The representative images were selected andﬁnal image evaluation was done in Adobe PhotoShop.w a t e r r e s e a r c h x x x(2011)1e1033.Results3.1.SBR performance and granule characteristicsDuring the startup period,the reactor removed TOC and NH 4þ-N efﬁciently.98%of NH 4þ-N and 100%of TOC were removed from the inﬂuent by day 3and day 5respectively (Figs.S2,S3,Supporting information ).Removal of TN and TP were lower during this period (Figs.S3,S4,Supporting information ),though the removal of TP gradually improved to 100%removal by day 33(Fig.S4,Supporting information ).To determine the sludge volume index of granular sludge,a settling time of 10min was chosen instead of 30min,because granular sludge has a similar SVI after 60min and after 5min of settling (Schwarzenbeck et al.,2004b ).The SVI 10of the inoculating sludge was 108.2mL g À1.The changing patterns of MLSS and SVI 10in the continuous operation of the SBR are illustrated in Fig.1.The sludge settleability increased markedly during the set-up period.Fig.2reﬂects the slow andgradual process of sludge granulation,i.e.,from ﬂocculentsludge to granules.3.2.DGGE analysis:AOB communities structure changes during sludge granulationThe results of nested PCR were shown in Fig.S1.The well-resolved DGGE bands were obtained at the representative points throughout the GSBR operation and the patterns revealed that the structure of the AOB communities was dynamic during sludge granulation and stabilization (Fig.3).The community structure at the end of experiment was different from that of the initial pattern of the seed sludge.The AOB communities on day 1showed 40%similarity only to that at the end of the GSBR operation (Table S1,Supporting information ),indicating the considerable difference of AOB communities structures between inoculated sludge and granular sludge.Biodiversity based on the DGGE patterns was analyzed by calculating the Shannon diversity index H as204060801001201401254159738494104115125135147160172188Time (d)S V I 10 (m L .g -1)10002000300040005000600070008000900010000M L S S (m g .L -1)Fig.1e Change in biomass content and SVI 10during whole operation.SVI,sludge volume index;MLSS,mixed liquid suspendedsolids.Fig.2e Variation in granule size distribution in the sludge during operation.d,particle diameter;TSS,total suspended solids.w a t e r r e s e a r c h x x x (2011)1e 104shown in Fig.S5.In the phase of sludge inoculation (before day 38),H decreased remarkably (from 0.94to 0.75)due to the absence of some species in the reactor.Though several dominant species (bands2,7,10,11)in the inoculating sludge were preserved,many bands disappeared or weakened (bands 3,4,6,8,13,14,15).After day 45,the diversity index tended to be stable and showed small ﬂuctuation (from 0.72to 0.82).Banding pattern similarity was analyzed by applying UPGMA (Fig.4)algorithms.The UPGMA analysis showed three groups with intragroup similarity at approximately 67%e 78%and intergroup similarity at 44e 62%.Generally,the clustering followed the time course;and the algorithms showed a closer clustering of groups II and III.In the analysis,group I was associated with sludge inoculation and washout,group IIwithFig.3e DGGE proﬁle of the AOB communities in the SBR during the sludge granulation process (lane labels along the top show the sampling time (days)from startup of the bioreactor).The major bands were labeled with the numbers (bands 1e15).Fig.4e UPGMA analysis dendrograms of AOB community DGGE banding patterns,showing schematics of banding patterns.Roman numerals indicate major clusters.w a t e r r e s e a r c h x x x (2011)1e 105startup sludge granulation and decreasing SVI 10,and group III with a stable system and excellent biomass settleability.In Fig.3,the locations of the predominant bands were excised from the gel.DNA in these bands were reampliﬁed,cloned and sequenced.The comparative analysis of these partial 16S rRNA sequences (Table 2and Fig.S6)revealed the phylogenetic afﬁliation of 13sequences retrieved.The majority of the bacteria in seed sludge grouped with members of Nitrosomonas and Nitrosospira .Along with sludge granula-tion,most of Nitrosomonas (Bands 2,5,7,9,10,11)were remained or eventually became dominant in GSBR;however,all of Nitrosospira (Bands 6,13,15)were gradually eliminated from the reactor.3.3.Distribution of AOB and NOB in different sized granulesFISH was performed on the granule sections mainly to deter-mine the location of AOB and NOB within the different size classes of granules,and the images were not further analyzed for quantiﬁcation of cell counts.As shown in Fig.6,in small granules (0.2mm <d <0.45mm),AOB located mainly in the outer part of granular space,whereas NOB were detected only in the core of granules.In medium granules (0.45mm <d <0.6mm),AOB distributed evenly throughout the whole granular space,whereas NOB still existed in the inner part.In the larger granules (d >0.9mm),AOB and NOB were mostly located in the surface area of the granules,and moreover,NOB became rare.4.Discussion4.1.Relationship between granule formation and reactor performanceAfter day 32,the SVI 10stabilized at 20e 35mL g À1,which is very low compared to the values measured for activated sludge (100e 150mL g À1).However,the size distribution of the granules measured on day 32(Fig.2)indicated that only 22%of the biomass was made of granular sludge with diameter largerthan 0.2mm.These results suggest that sludge settleability increased prior to granule formation and was not affected by different particle sizes in the sludge during the GSBR operation.It was observed,however,that the diameter of the granules ﬂuctuated over longer durations.The large granules tended to destabilize due to endogenous respiration,and broke into smaller granules that could seed the formation of large granules again.Pochana and Keller reported that physically broken sludge ﬂocs contribute to lower denitriﬁcation rates,due to their reduced anoxic zone (Pochana and Keller,1999).Therefore,TN removal efﬁciency raises ﬂuctuantly throughout the experiment.Some previous research had demonstrated that bigger,more dense granules favored the enrichment of PAO (Winkler et al.,2011a ).Hence,after day 77,removal efﬁciency of TP was higher and relatively stable because the granules mass fraction was over 90%and more larger granules formed.4.2.Relationship between AOB communities dynamic and sludge granulationFor granule formation,a short settling time was set,and only particles with a settling velocity higher than 4.5m h À1were retained in the reactor.Moreover,as shown in Fig.1,the variation in SVI 10was greater before day 41(from 108.2mL g À1e 34.1mL g À1).During this phase,large amounts of biomass could not survive in the reactor.A clear shift in pop-ulations was evident,with 58%similarity between days 8and 18(Table S1).In the SBR system fed with acetate-based synthetic wastewater,heterotrophic bacteria can produce much larger amounts of extracellular polysaccharides than autotrophic bacteria (Tsuneda et al.,2003).Some researchers found that microorganisms in high shear environments adhered by extracellular polymeric substances (EPS)to resist the damage of suspended cells by environmental forces (Trinet et al.,1991).Additionally,it had been proved that the dominant heterotrophic species in the inoculating sludge were preserved throughout the process in our previous research (Zhang et al.,2011).It is well known that AOB are chemoau-totrophic and slow-growing;accordingly,numerous AOBw a t e r r e s e a r c h x x x (2011)1e 106populations that cannot become big and dense enough to settle fast were washed out from the system.As a result,the variation in AOB was remarkable in the period of sludge inoculation,and the diversity index of population decreased rapidly.After day 45,AOB communities’structure became stable due to the improvement of sludge settleability and the retention of more biomass.These results suggest that the short settling time (selection pressure)apparently stressed the biomass,leading to a violent dynamic of AOB communities.Further,these results suggest that certain populations may have been responsible for the operational success of the GSBR and were able to persist despite the large ﬂuctuations in pop-ulation similarity.This bacterial population instability,coupled with a generally acceptable bioreactor performance,is congruent with the results obtained from a membrane biore-actor (MBR)for graywater treatment (Stamper et al.,2003).Nitrosomonas e like and Nitrosospira e like populations are the dominant AOB populations in wastewater treatment systems (Kowalchuk and Stephen,2001).A few previous studies revealed that the predominant populations in AOB communities are different in various wastewater treatment processes (Tawan et al.,2005;Thomas et al.,2010).Some researchers found that the community was dominated by AOB from the genus Nitrosospira in MBRs (Zhang et al.,2010),whereas Nitrosomonas sp.is the predominant population in bioﬁlter sludge (Yin and Xu,2009).In the currentstudy,Fig.5e DGGE proﬁle of the AOB communities in different size of granules (lane labels along the top show the range of particle diameter (d,mm)).Values along the bottom indicate the Shannon diversity index (H ).Bands labeled with the numbers were consistent with the bands in Fig.3.w a t e r r e s e a r c h x x x (2011)1e 107sequence analysis revealed that selection pressure evidently effect on the survival of Nitrosospira in granular sludge.Almost all of Nitrosospira were washed out initially and had no chance to evolve with the environmental changes.However,some members of Nitrosomonas sp.have been shown to produce more amounts of EPS than Nitrosospira ,especially under limited ammonia conditions (Stehr et al.,1995);and this feature has also been observed for other members of the same lineage.Accordingly,these EPS are helpful to communicate cells with each other and granulate sludge (Adav et al.,2008).Therefore,most of Nitrosomonas could adapt to this challenge (to become big and dense enough to settle fast)and were retained in the reactor.At the end of reactor operation (day 180),granules with different particle size were sieved.The effects of variation in granules size on the composition of the AOBcommunitiesFig.6e Micrographs of FISH performed on three size classes of granule sections.DAPI stain micrographs (A,D,G);AOB appear as green ﬂuorescence (B,E,H),and NOB appear as red ﬂuorescence (C,F,I).Bar [100m m in (A)e (C)and (G)e (I).d,particle diameter.(For interpretation of the references to colour in this ﬁgure legend,the reader is referred to the web version of this article.)w a t e r r e s e a r c h x x x (2011)1e 108were investigated.As shown in Fig.5,AOB communities structures in different size of granules were varied.Although several predominant bands(bands2,5,11)were present in all samples,only bands3and6appeared in the granules with diameters larger than0.6mm.Additionally,bands7and10 were intense in the granules larger than0.45mm.According to Table2,it can be clearly indicated that Nitrosospira could be retained merely in the granules larger than0.6mm.Therefore, Nitrosospira was not present at a high level in Fig.3due to the lower proportion of larger granules(d>0.6mm)in TSS along with reactor operation.DGGE analysis also revealed that larger granules had a greater microbial diversity than smaller ones. This result also demonstrates that more organisms can survive in larger granules as a result of more space,which can provide the suitable environment for the growth of microbes(Fig.6).4.3.Effect of variance in particle size on the distribution of AOB and NOB in granulesAlthough an inﬂuence of granule size has been observed in experiments and simulations for simultaneous N-and P-removal(de Kreuk et al.,2007),the effect of granule size on the distribution of different biomass species need be revealed further with the assistance of visible experimental results, especially in the same granular sludge reactors.Related studies on the diversity of bacterial communities in granular sludge often focus on the distribution of important functional bacteria populations in single-size granules(Matsumoto et al., 2010).In the present study,different size granules were sieved,and the distribution patterns of AOB and NOB were explored.In the nitriﬁcation processes considered,AOB and NOB compete for space and oxygen in the granules(Volcke et al.,2010).Since ammonium oxidizers have a higheroxygen afﬁnity(K AOBO2<K NOBO2)and accumulate more rapidly inthe reactor than nitrite oxidizers(Volcke et al.,2010),NOB are located just below the layer of AOB,where still some oxygen is present and allows ready access to the nitrite produced.In smaller granules,the location boundaries of the both biomass species were distinct due to the limited existence space provided by granules for both microorganism’s growth.AOB exist outside of the granules where oxygen and ammonia are present.Medium granules can provide broader space for microbe multiplying;accordingly,AOB spread out in the whole granules.This result also conﬁrms that oxygen could penetrate deep into the granule’s core without restriction when particle diameter is less than0.6mm.Some mathematic model also supposed that NOBs are favored to grow in smaller granules because of the higher fractional aerobic volume (Volcke et al.,2010).As shown in the results of the batch experiments(Zhang et al.,2011),nitrite accumulation temporarily occurred,accompanied by the more large gran-ules(d>0.9mm)forming.This phenomenon can be attrib-uted to the increased ammonium surface load associated with larger granules and smaller aerobic volume fraction,resulting in outcompetes of NOB.It also suggests that the core areas of large granules(d>0.9mm)could provide anoxic environment for the growth of anaerobic denitriﬁcans(such as Tb.deni-triﬁcans or Tb.thioparus in Fig.S7,Supporting information).As shown in Fig.2and Fig.S3,the removal efﬁciency of total nitrogen increased with formation of larger granules.5.ConclusionsThe variation in AOB communities’structure was remarkable during sludge inoculation,and the diversity index of pop-ulation decreased rapidly.Most of Nitrosomonas in the inocu-lating sludge were retained because of their capability to rapidly adapt to the settling e washing out action.DGGE anal-ysis also revealed that larger granules had greater AOB diversity than that of smaller ones.Oxygen penetration was not restricted in the granules of less than0.6mm particle diameter.However,the larger granules(d>0.9mm)can result in the smaller aerobic volume fraction and inhibition of NOB growth.Henceforth,further studies on controlling and opti-mizing distribution of granule size could be beneﬁcial to the nitrogen removal and expansive application of granular sludge technology.AcknowledgmentsThis work was supported by grants from the National Natural Science Foundation of China(No.51108456,50908227)and the National High Technology Research and Development Program of China(No.2009AA06Z312).Appendix.Supplementary dataSupplementary data associated with this article can be found in online version at doi:10.1016/j.watres.2011.09.026.r e f e r e n c e sAdav,S.S.,Lee, D.J.,Show,K.Y.,2008.Aerobic granular sludge:recent advances.Biotechnology Advances26,411e423.APHA-AWWA-WEF,2005.Standard Methods for the Examination of Water and Wastewater,ﬁrst ed.American Public Health Association/American Water Works Association/WaterEnvironment Federation,Washington,DC.de Bruin,L.M.,de Kreuk,M.,van der Roest,H.F.,Uijterlinde,C., van Loosdrecht,M.C.M.,2004.Aerobic granular sludgetechnology:an alternative to activated sludge?Water Science and Technology49,1e7.de Kreuk,M.,Heijnen,J.J.,van Loosdrecht,M.C.M.,2005.Simultaneous COD,nitrogen,and phosphate removal byaerobic granular sludge.Biotechnology and Bioengineering90, 761e769.de Kreuk,M.,Picioreanu,C.,Hosseini,M.,Xavier,J.B.,van Loosdrecht,M.C.M.,2007.Kinetic model of a granular sludge SBR:inﬂuences on nutrient removal.Biotechnology andBioengineering97,801e815.Downing,L.S.,Nerenberg,R.,2008.Total nitrogen removal ina hybrid,membrane-aerated activated sludge process.WaterResearch42,3697e3708.Erguder,T.H.,Boon,N.,Vlaeminck,S.E.,Verstraete,W.,2008.Partial nitriﬁcation achieved by pulse sulﬁde doses ina sequential batch reactor.Environmental Science andTechnology42,8715e8720.w a t e r r e s e a r c h x x x(2011)1e109。

Filter media

Filter mediaAquatic life generates a variety of wastes which can rapidly reach toxic levels. Tap water can also introduce undesirable elements. These wastes and bi-products must be removed. Water purification cannot occur without some type of filter material. The proper use of effective and specific media for particular tasks is vital in aquarium filtration. We separate these types into mechanical, biological, adsorptive and chemical filter media.Mechanical filter media work much like a sieve trapping particulate waste as it passes through the material.EHFIMECHA coarse mechanical filter material which is to be used as bottom layer filtering. Its hollow ceramic design creates eddies which disburse the water into many paths. It traps large debris while creating an even flow of water for subsequent layers.EHFIFIXA medium mechanical filter material which is to be placed between EHFIMECH on the bottom and the subsequent biological or chemical filter layers. It traps debris which has passed through the previous layer and acts as a divider.For models 2211, 2213, 2215/22176, 2222, 2224, 2226, 2228 a special pad is recommended.Filter padSeparates different filter media layers.EHFISYNTHA completely neutral filter wool. The fine phenol free structureof this synthetic material traps tiny particles of dirt. It should beloosely placed as the last layer in your filter arrangement. Formodels 2222, 2224, 2226, 2228 we recommend a specialEHFISYNTH pad.Biological filtrationA build-up of toxic nitrogenous wastes is a natural result of all aquarium inhabitant's life processes. In nature, the body of water is large enough to dissipate or dilute these wastes. Some will even be converted into usable energy by living organisms. Within an aquarium however, without a powerful biological breakdown of these toxins, the fish literally poison themselves. This is the reason why biological filtration is the most desirable of all aquarium filtration.This type of filtration is the purification of the aquarium water by using living organisms, such as nitrifying bacteria. These desirable bacteria will attach themselves to all hard surfaces within the aquarium system. They use the toxins as a food, converting harmful toxins such as ammonia and nitrite into less harmful nitrate. Nitrate can then be removed by performing regular water changes.After prolonged use, mechanical and chemical filter media will act in a biological manner. They provide some surface area for bacterial colonization but this is actually very limited. In order to provide the greatest amount of surface area for bacteria colonies, filter media such as EHFISUBSTRAT or EHFILAV should be used.EHFISUBSTRATTo create biologically sound water as found in nature, you need EHFISUBSTRAT. Biological filtering is based on a natural decomposition of harmful substances using helpful bacteria. They convert ammonia and nitrite into relatively non-toxic nitrate. The efficiency of biological filtration is limited by the media that bacteria are growing on. With over 450 ml per litre (22, 000sq. ft. per lmp. gal. / 18, 3000 sq. ft. per U.S. gal.) EHFISUBSTRAT is a specially designed sintered glass.Bacteria are able to stick better to a surface which has a complex pore system. EHFISUBSTRAT has been specially developed to offer optimum sites for bacteria colonization. The effectiveness of these bacteria is linked to how much oxygen and toxins can flow by. With faster decomposition of toxins compared to other media. Highly effective, economically priced, it is the best biological media available to aquarium hobbyists.EHFILAVA naturally occuring porous volcanicrock which is ideal for biological filtrationwhere large debris is present such asponds and highly stocked aquariums.This natural product is pre-tested forimpurities before pachaging to ensurethat it is free of toxins.Adsorptive filter mediaAdsorptive filtration is a process in which dissolved substances are captured by solid bodies such as carbon. These dissolved substances can be harmful to aquatic life. They are generally of chemical origin such as chlorine in tap water, alkaline residues of aquarium medication and even some dissolved metals. However the toxic substances accumulate and the absorption capacity of the carbon is limited. With all types of carbon, the effectiveness is limited to a short period of time. They must then be removed from the filter, to avoid having the adsorbed substances washed back into the aquarium. We recommend that carbon be used for short -term only and for a specific purpose. After the inital aquarium setup or to remove medications or additives, turbidity, water discoloration and odor.EHFIAKTIVThis product offers the highest adsorption. It is alkaliactivated, acid-washed, pH neutral, heavy metal free andformed in a special compression mold to perform a rapid,highly effective adsorption of harmful substances andresidues of medication.EHFIKARBONA high quality filter carbon. It is recommended for short-termuse in freshwater aquariums.Chemical filter mediaEHFITORFA specially treated media, whichmakes the water more acidic andlowers the carbon hardness. Thepeat filter is recommedable for softwater fishes in order to achieve atropical aquatic environment. Justlike in the case of all other additivesthe changed water property has tobe regularly monitored.Aquatic life generates a variety of wastes which can rapidly reach toxic levels. Water purification cannot occur without some type of filter material. The proper use of effective and specific media for particular tasks is vital in aquarium filtration. Ideal water conditions very much depend on the correct use of filter media. We separate these types into mechanical, biological, adsorptive and chemical filter media.EHEIM filter pads are used to separate the different layers in standard filters.EHFISYNTH filter pad for mechanical fine cleaning, forthe EHEIM professionel filters series.EHEIM filter cartridges made of special foam materialsprovide a large surface area. They can be changed in afew simple steps and after their "running-in" time theynot only function mechanically but also biologically. Inaddition, there are also active carbon cartridges, whichare ideally suited for adsorption.。

eDNA技术在水生态学研究中的发展和应用

总751期第十七期2021年6月河南科技Henan Science and TechnologyeDNA技术在水生态学研究中的发展和应用王赛王团团黄丽波（广东天鉴检测技术服务股份有限公司，广东深圳518101）摘要：随着分子生物学的快速发展，eDNA（environmental DNA）技术已成为一种新的监测手段，在国际上被广泛应用。

eDNA是指生物体通过唾液、粪便、分泌物和皮肤脱落等释放到环境中的无细胞DNA，因此，eDNA 的采集和检测具有环境友好、易于取样和灵敏度高等优点，可以提供实时的物种分布和群落多样性信息。

本文从eDNA的发展历程、研究方法及其在水生态学中的应用（如监测入侵种和濒危物种，追踪物种生活史过程，推算种群的丰度和生物量，反映生态系统结构变化）等方面进行介绍，阐明eDNA技术在生态环境监测中的适用性和重要性，指出当前eDNA技术的优势与不足之处，并展望了eDNA在生态学和环境学领域的科研前景。

关键词：生态系统；生物多样性；入侵种；濒危物种；生活史过程中图分类号：Q178文献标识码：A文章编号：1003-5168（2021）17-0125-05 Development and Application of eDNA Technology inWater Ecology ResearchWANG Sai WANG Tuantuan HUANG Libo（Skyte Testing Services Guangdong Co.,Ltd.，Shenzhen Guangdong518101）Abstract:Due to the rapid development of molecular biology,eDNA(environmental DNA)technology has become a new monitoring method and is widely used in the world.eDNA refers to cell-free DNA released into the environment through saliva,feces,secretions and skin shedding,therefore,the collection and detection of eDNA has the advantag⁃es of environmental friendliness,easy sampling and high sensitivity,and can provide real-time information on spe⁃cies distribution and community diversity.In this paper,the development history,research methods and application of eDNA in water ecology(such as monitoring invasive species and endangered species,tracing the life history pro⁃cess of species,calculating the abundance and biomass of population,and reflecting the change of ecosystem struc⁃ture)are introduced,and the applicability and importance of eDNA technology in ecological environment monitoring are expounded,and the advantages and disadvantages of current eDNA technology are pointed out,and the research prospect of eDNA in ecology and environmental science is prospected.Keywords:ecosystem；biodiversity；invasive species；endangered species；life history process近50年来，随着人口数量的不断增加和城市化的快速扩张，野生动植物的生存空间日益受到威胁，越来越多的物种开始趋于濒危甚至灭绝，人和自然的和谐关系被打破。

海洋馆维生系统操作流程

海洋馆维生系统操作流程The operation of a marine aquarium's life support system is a critical aspect of maintaining a healthy and sustainable environment for the marine life within the facility. 海洋馆生命维持系统的操作是维持设施内海洋生物健康和可持续环境的关键方面。

This system encompasses a range of equipment and processes that work together to managethe water quality, temperature, and overall well-being of the aquatic inhabitants. 这个系统涵盖了一系列设备和流程，它们共同工作以管理水质、温度和水生生物的整体健康状况。

The operation flow of a marine aquarium's life support system involves several key steps that mustbe carefully managed to ensure the optimal conditions for the marine life. 海洋馆生命维持系统的操作流程涉及几个关键步骤，必须进行精心管理，以确保海洋生物的最佳生存条件。

One of the first steps in the operation flow of a marine aquarium'slife support system is the monitoring of water quality. 海洋馆生命维持系统操作流程中的第一步是监测水质。

同位素内标法-超高效液相色谱-串联质谱法快速测定水产品中地西泮残留

分析检测同位素内标法-超高效液相色谱-串联质谱法快速测定水产品中地西泮残留马承鸿1,2，陈晓嘉1,2，吴家玲1,2，何雯倩3，周芳梅1,2（1.广东省食品质量监督检验站，广东广州 510000；2.广东省食品工业研究所有限公司，广东广州 510000；3.江苏广海检验检测有限公司，江苏南通 226121）摘要：目的：建立同位素内标法-超高效液相色谱-串联质谱法快速测定水产品中地西泮残留量的方法。

方法：在样品中加入地西泮-D5，采用乙腈涡旋提取，QuEChERS法除脂净化后，取上清液过滤上机。

使用phenomenex Luna C18色谱柱分离，超高效液相色谱串联四极杆质谱模式，多反应离子监测方式检测，内标法定量。

结果：在0.1～10.0 μg·L-1线性关系良好，相关系数大于0.995，检出限为0.05 μg·kg-1，定量限为0.15 μg·kg-1，回收率（n=6）为95.8%～110.3%，相对偏差（n=6）为0.6%～8.5%。

结论：该方法具有简便快速、灵敏度高、结果准确的优点，适用于大批量水产品中地西泮残留量的准确快速测定。

关键词：超高效液相色谱-串联质谱法；地西泮；内标法；QuEChERS；水产品Rapid Determination of Diazepam Residues in Aquatic Products by UPLC-MS/MS with Isotope Internal Standard Method MA Chenghong1,2, CHEN Xiaojia1,2, WU Jialing1,2, HE Wenqian3, ZHOU Fangmei1,2(1.Guangdong Food Quality Supervision and Inspection Station, Guangzhou 510000, China;2.Guangdong Food Industry Institute Co., Ltd., Guangzhou 510000, China;3.Jiangsu Guanghai Inspection and Testing Co., Ltd., Nantong 226121, China)Abstract: Objective: A rapid method for the determination of diazepam residues in aquatic products by internal standard isotope-ultra-high performance liquid chromatography-tandem mass spectrometry was established. Method: By adding diazepam-D5 to the samples, followed by vortex extraction with acetonitrile and QuEChERS-based defatting, the supernatant was obtained and filtered. The analytes were separated on a Phenomenex Luna C18 column, and detected by UPLC coupled with quadrupole mass spectrometry in multiple reaction ion monitoring mode, employing an internal standard method for quantification. Result: The linear relationship was good in the range of 0.1 ～10.0 μg·L-1, the correlation coefficient was greater than 0.995. The limit of detection was 0.05 μg·kg-1, limit of quantification was 0.15 μg·kg-1, the recovery rate (n=6) was 95.8%～110.3%, and the relative deviation (n=6) was 0.6%～8.5%. Conclusion: The method is simple, rapid, sensitive and accurate, which is suitable for the accurate and rapid determination of diazepam residues in large quantities of aquatic products.Keywords: ultra-high performance liquid chromatography-tandem mass spectrometry; diazepam; isotope internal standard method; QuEChERS; aquatic products地西泮属于苯二氮卓类药物，为第二代催眠药。

敲除tp53_对斑马鱼高温耐受能力和运动能力的影响

生物技术进展 2023 年第 13 卷第 4 期 580 ~ 587Current Biotechnology ISSN 2095‑2341研究论文Articles敲除tp53对斑马鱼高温耐受能力和运动能力的影响杨倩婷，田晓英，张俊芳，韩兵社 *上海海洋大学水产与生命学院，水产种质资源发掘与利用教育部重点实验室，水产科学国家级实验教学示范中心，上海 201306摘要：研究了敲除tp53基因对斑马鱼高温耐受能力和运动能力的影响。

通过RT -qPCR 、Western blot 等技术发现高温下野生型的斑马鱼tp53表达上调，暗示tp53可能在高温下发挥作用，而敲除tp53基因的斑马鱼在高温下的存活时间降低。

进一步研究发现，tp53-/-斑马鱼在高温下ROS 水平升高（P <0.05），ATP 水平降低(P <0.001)，γH2A.X 蛋白水平显著升高。

通过苏木精-伊红（hematoxylin -eosin ，HE ）染色发现tp53-/-斑马鱼高温下肝脏组织充血更严重。

Danio Vision 斑马鱼行为轨迹分析显示34 ℃下tp53-/-斑马鱼的运动能力减弱。

研究结果表明，tp53在高温下对斑马鱼的高温耐受能力和运动能力起正向调节作用，为斑马鱼高温耐受机制和运动行为学研究提供了新思路。

关键词：斑马鱼；高温；tp53；运动能力；肝DOI ：10.19586/j.20952341.2023.0039 中图分类号：Q958.8 文献标志码：AEffect of tp53 Knockout on High Temperature Tolerance and Swimming Capacity in ZebrafishYANG Qianting ， TIAN Xiaoying ， ZHANG Junfang ， HAN Bingshe *National Demonstration Center for Experimental Fisheries Science Education ， Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources ， Ministry of Education ， College of Fisheries and Life Science ， Shanghai Ocean University ， Shanghai 201306， ChinaAbstract ：The aim of the study was to investigate the effect of tp53 knockout on high temperature tolerance and swimming capac⁃ity in zebrafish. Through RT -qPCR ， Western blot and other technologies ， it was found that the expression of tp53 in wild -type ze⁃brafish at high temperature was upregulated ， suggesting that tp53 may play a role at high temperature ， while zebrafish with the tp53 gene knockout had a reduced survival time at high temperature. Further studies found that tp53-/- zebrafish had increased ROS levels （P <0.05）， decreased ATP levels （P <0.001）， and significantly increased levels of γH2A.X protein at high tempera⁃tures. It was found by hematoxylin -eosin （HE ） staining that the liver tissue hyperemia of tp53-/- zebrafish was more severe at hightemperatures. Danio Vision behavior trajectory analysis showed that tp53-/- zebrafish exercise capacity was weakened at 34 ℃. The results showed that tp53 has a positive effect on the high temperature tolerance and swimming capacity of zebrafish ， which provided a new idea for the study of high temperature tolerance mechanism and exercise behavior in zebrafish.Key words ：zebrafish ； high temperature ； tp53； swimming capacity ； liver季节性温差会导致水体温度发生变化。

鱼缸里的生物课这本书读后感

鱼缸里的生物课这本书读后感英文回答：After reading the book "The Biology of the Aquarium," I was truly fascinated by the intricate ecosystem that exists within a fish tank. The author did a fantastic job of explaining the various interactions between different organisms, from the fish to the plants to the beneficial bacteria.One of the key takeaways for me was the importance of maintaining a balanced and healthy environment in the aquarium. Just like in nature, every organism plays a crucial role in the ecosystem, and any imbalance can have detrimental effects on the entire system. For example, if there are too many fish in the tank, the waste they produce can build up and create toxic conditions for the other inhabitants. This reminded me of the saying "everything in moderation" – a principle that definitely applies to aquarium keeping.I also learned a lot about the nitrogen cycle and how essential it is for the well-being of the fish. The book explained how beneficial bacteria break down ammonia from fish waste into less harmful substances like nitrite and nitrate. This process is vital for maintaining waterquality and ensuring the health of the aquatic life. It made me realize the importance of patience in aquarium keeping, as it takes time for the nitrogen cycle to establish itself properly.Overall, reading this book has deepened my appreciation for the complexity of life in a fish tank. It has inspired me to be more diligent in caring for my own aquarium and to strive for a harmonious balance among all the inhabitants.中文回答：读完《鱼缸里的生物课》这本书后，我对鱼缸内部存在的复杂生态系统感到非常着迷。

合理使用化肥的英语作文

合理使用化肥的英语作文Title: The Rational Use of Fertilizers。

In modern agriculture, the use of fertilizers plays a crucial role in enhancing crop productivity and meeting the ever-growing global food demand. However, the excessive and improper use of fertilizers can lead to adverse environmental and health effects. Therefore, it is imperative to emphasize the rational and responsible use of fertilizers to maximize their benefits while minimizing their negative impacts.First and foremost, it is essential to understand the significance of fertilizers in agriculture. Fertilizers provide essential nutrients such as nitrogen, phosphorus, and potassium, which are vital for plant growth and development. By replenishing soil nutrients, fertilizers help improve soil fertility, leading to increased crop yields and quality.However, indiscriminate use of fertilizers can resultin several detrimental effects. One of the most significant concerns is water pollution. Excessive application of nitrogen-based fertilizers can lead to nitrate leachinginto groundwater or runoff into surface water bodies. High levels of nitrates in water can cause eutrophication, leading to algal blooms and oxygen depletion, which in turn harm aquatic ecosystems.Furthermore, the overuse of fertilizers can disrupt the balance of nutrients in the soil, leading to nutrient imbalances and soil degradation. This can result in decreased soil fertility over time, requiring even higher fertilizer inputs to maintain crop productivity—a vicious cycle that can have long-term negative consequences for agricultural sustainability.To address these challenges, it is crucial to promote the rational use of fertilizers through various strategies. Firstly, farmers should adopt precision agriculture techniques to apply fertilizers more efficiently. Byutilizing technologies such as soil testing, remote sensing,and variable rate application systems, farmers can tailor fertilizer applications to specific soil and crop requirements, thereby minimizing wastage and reducing environmental impacts.Moreover, integrated nutrient management practices should be encouraged, combining the use of chemical fertilizers with organic sources such as compost, manure, and crop residues. This not only helps reduce the reliance on synthetic fertilizers but also improves soil health and biodiversity, promoting sustainable agriculture in the long run.Education and awareness programs are also essential to promote responsible fertilizer use among farmers. By providing training on proper fertilizer application techniques, environmental conservation, and the importance of soil health, farmers can make informed decisions to optimize fertilizer use while minimizing negative impacts.Government policies and regulations play a crucial role in promoting the rational use of fertilizers. Implementingmeasures such as subsidies for soil testing, promoting best management practices, and enforcing restrictions onfertilizer application rates can incentivize farmers to adopt sustainable practices and reduce environmental pollution.In conclusion, while fertilizers are indispensable for modern agriculture, their excessive and improper use can have detrimental consequences for the environment and human health. Therefore, it is imperative to promote the rational use of fertilizers through precision agriculture, integrated nutrient management, education, and policy interventions. By adopting these measures, we can ensure sustainable agricultural practices that balance the needfor increased food production with environmental stewardship and long-term soil health.。

双波长分光光度法同时测定溶液中的硝酸根和碘离子

双波长分光光度法同时测定溶液中的硝酸根和碘离子张慧芳;郭探;李权;叶秀深;吴志坚【摘要】食品和环境样品中往往同时含有硝酸根和碘离子,用紫外分光光度法直接测定硝酸根或碘离子时,二者相互干扰.为此建立了主、次波长分别为220.0、231.5 nm的等吸收点双波长紫外分光光度法测定溶液中的硝酸根和共存的碘离子.当溶液中硝酸根的浓度范围在0～0.12 mmol/L,碘离子的浓度在0～0.10 mmol/L时,主、次波长下的吸光度差值A220-231.5与溶液中硝酸根的浓度CNO3-呈良好线性关系,线性方程为A220-231.5=2.9958 CNO3-+0.0016(R2=0.99994)；其中A220( NO3-)=3.6099 CNO3-+0.0084(R2=0.99994),利用吸光度的加和性:A220 (I-)=A220-A220 (NO3-)=10.7394 CI-+0.0029(R2=0.99994),间接得到碘离子含量CI-.硝酸根和碘离子的平均相对标准偏差分别为0.6％、0.2％,回收率分别为99.5％～102％、99.9％～100％.方法简便快捷,可用于溶液中微量硝酸根和碘离子的同时测定.【期刊名称】《中国无机分析化学》【年(卷),期】2011(001)004【总页数】5页(P24-28)【关键词】硝酸根;碘离子;双波长分光光度法【作者】张慧芳;郭探;李权;叶秀深;吴志坚【作者单位】中国科学院青海盐湖研究所,西宁810008;中国科学院研究生院,北京100049;中国科学院青海盐湖研究所,西宁810008;中国科学院研究生院,北京100049;中国科学院青海盐湖研究所,西宁810008;中国科学院青海盐湖研究所,西宁810008;中国科学院青海盐湖研究所,西宁810008【正文语种】中文【中图分类】O657.32;TH744.12+21 引言工业废水、农田排水的不合理排放以及化肥使用量的不断增加，致使大量硝酸盐进入地下水［1－4］，也导致很多蔬菜中的硝酸盐含量超标［5］。

保护水生动物英语作文

Protecting Aquatic Animals is an essential duty for every individual as they play a crucial role in maintaining the balance of our ecosystem. Here are some key points to consider when writing an essay on this topic:1. Introduction to Aquatic Animals: Begin by introducing the variety of aquatic animals, such as fish, whales, dolphins, turtles, and various species of marine life that inhabit our oceans, rivers, and lakes.2. Importance of Aquatic Animals: Explain their significance in the food chain, their contribution to biodiversity, and the ecological services they provide, such as water purification and nutrient cycling.3. Threats to Aquatic Animals: Discuss the various threats that aquatic animals face, including overfishing, pollution, habitat destruction, climate change, and the introduction of invasive species.4. Consequences of Not Protecting Aquatic Animals: Highlight the potential consequences of not taking action to protect these species, such as the collapse of ecosystems, loss of biodiversity, and the disruption of the food chain.5. Legislation and Conservation Efforts: Mention the laws and regulations that have been put in place to protect aquatic animals, such as the Endangered Species Act, and the efforts of organizations dedicated to marine conservation.6. Individual Actions: Suggest ways in which individuals can contribute to the protection of aquatic animals, such as reducing plastic use, supporting sustainable seafood choices, and participating in beach cleanups.7. Technological Advancements: Discuss how technology can aid in the protection of aquatic animals, such as using drones for monitoring marine life and employing satellite technology to track and protect migratory species.8. Education and Awareness: Emphasize the importance of educating the public about the value of aquatic animals and the need for their protection to foster a sense of responsibility and stewardship.9. International Cooperation: Point out the need for international cooperation in marine conservation efforts, as many aquatic species migrate across international waters.10. Conclusion: Conclude by reiterating the importance of protecting aquatic animals andthe collective responsibility of humanity to ensure their survival for future generations. Remember to use clear and concise language, provide specific examples, and maintain a logical flow throughout your essay.。

碳氮氧稳定同位素技术在水生态环境中的应用

第 35 卷　第 1 期环　境　科　学　研　究Vol.35，No.1 2022 年 1 月Research of Environmental Sciences Jan.，2022碳氮氧稳定同位素技术在水生态环境中的应用杨　蓉1,2，李　垒2*1. 北京市南水北调水质监测中心, 北京　1000972. 北京市水科学技术研究院, 北京　100048摘要：稳定同位素技术是研究环境和生态系统中元素循环途径的重要方法. 稳定同位素的丰度变化反映了自然界和生物体内混合、分馏双重作用的结果，因此可作为指标计算混合物的来源贡献，或研究造成分馏的化学反应和生物代谢路径. 从20世纪中期确立稳定同位素的基础原理，70年间该技术在地球化学、环境科学、生态学、微生物学、食品科学等领域获得了大量有价值的成果. 其中，水体作为自然环境的重要组成和人类社会的重要资源，已有诸多研究涉及稳定同位素在水环境中污染物溯源、水生态系统元素迁移转化、水生生物营养来源和营养关系等方面的应用. 通过梳理常见的碳、氮、氧稳定同位素在水环境和水生态领域的研究进展，发现污染物和食物来源分析已不局限于定性识别，基于数学模型的混合物组分定量评估方法正得到越来越多的应用；同时，为开展水体脱氮强度和通量估算、水生生物营养级计算及食物网分析，精确测量18O、15N和13C的富集程度以及通过试验和调研获取运算所需的基础参数都是关键步骤. 虽然在实际应用中存在待完善之处，但稳定同位素技术的前沿研究仍昭示了其整体化、精细化的发展方向. 未来与计算科学的方法学进步相结合，将为水科学研究提供更有力的技术支撑.关键词：稳定同位素；分馏；多元混合模型；硝酸盐溯源；食性分析；营养级中图分类号：X52文章编号：1001-6929（2022）01-0191-11文献标志码：A DOI：10.13198/j.issn.1001-6929.2021.07.03Applications of Stable Carbon, Nitrogen, and Oxygen Isotope Techniques in Aquatic Environment and EcologyYANG Rong1,2，LI Lei2*1. Beijing Water Quality Monitoring Center for South-to-North Water Diversion, Beijing 100097, China2. Beijing Water Science and Technology Institute, Beijing 100048, ChinaAbstract：Stable isotope technique is an important method to study element cycle in the environment and ecosystems. The change in the abundance of stable isotopes reflects the results of mixing and fractionation in nature and organisms. Therefore, it can be used as an indicator to calculate the contribution of mixture sources, or study the chemical reactions and biological pathways that cause fractionation. Since the establishment of the basic principles of stable isotopes in the middle of the last century, this technology has obtained many valuable achievements in geochemistry, environmental science, ecology, microbiology, food science and other fields in the past 70 years. Among them, there have been many studies on the application of stable isotopes to evaluate the pollutant sources, element migration and transformation, food sources and nutritional relations in the aquatic environment that is an essential component of nature and a significant resource of human society. By summarizing the research progress of common carbon, nitrogen and oxygen stable isotopes in the field of water environment and ecology, it is found that the analysis of pollutants and food sources is not limited to qualitative identification. In fact, quantitative assessment methods of mixture components based on mathematical models have been increasingly used. Meanwhile, to evaluate the intensity and flux of denitrification, or perform trophic level calculation and food-web analysis, it is important to measure the enrichment degree of 18O, 15N and 13C accurately, and obtain appropriate basic parameters for model through experiments and survey. Although some aspects still need to be optimized in practical applications, the research frontier of stable isotope technology shows its integrated and refined development direction. Combined with methodological advances in computational science, stable isotopes will provide more powerful technical support for water science research.收稿日期：2021-04-24 修订日期：2021-06-19作者简介：杨蓉(1987-)，女，山西大同人，高级工程师，博士，主要从事水环境监测及评价研究，ygrg@.*责任作者，李垒(1980-)，男，辽宁朝阳人，教授级高级工程师，博士，硕导，主要从事水生态环境评估和污染生态修复研究，ll@基金项目：国家自然科学基金项目(No.41301540)Supported by National Natural Science Foundation of China (No.41301540)Keywords：stable isotope；fractionation；multivariate mixed model；nitrate source identification；diet analysis；trophic level稳定同位素指核结构稳定、不发生或不易发生放射性衰变以及半衰期极长的一系列核素. 1912年，英国物理学家John Thomson发现天然氖由20和22两种不同质量数的原子组成，首次证明了常态元素稳定同位素的存在. 该技术的发展始于20世纪中期的稳定同位素地球化学领域，自然界常见的氢、碳、氮、氧、硫等元素都有超过一个的稳定同位素，研究者测定各类天然物质中元素的同位素组成，分析变化规律并推测原因所在.近年来，稳定同位素技术在地球化学、环境科学、生态学、微生物学、食品科学等领域得到了广泛应用[1-3]. 应用重点并非探究同位素丰度的分布特征，而是将其作为观测手段研究过程、开展溯源. 物理、化学和生物过程都可造成稳定同位素的丰度差异，反之稳定同位素化学性质相同，其变化不影响过程，因此可作为示踪剂为回溯环境、生物等多种因素对元素的共同作用提供科学有效的依据.水是生物不可或缺的基础物质，水生态环境不仅是环境科学和生态学的研究对象，其污染状况、物种分布等信息也是水资源管理的重要支撑. 随着色谱质谱联用和傅里叶变换核磁共振技术的诞生，同位素的检测手段愈加丰富，研究成本大幅降低，已有大量研究涉及稳定同位素在水体污染物溯源、水生生物食物网研究等不同方向的应用. 聚焦研究中最常见的碳、氮、氧稳定同位素，介绍了分馏和混合的基础理论及模型. 水质分析方面，以氮素和硝酸盐为例探讨了氮氧稳定同位素以及以IsoSource和SIAR为代表的混合模型在污染物迁移转化和溯源中的应用；水生态方面，基于碳氧稳定同位素特征、浓缩系数和基线生物选择综述了该技术在营养级、生态位和食性分析中的应用.1 稳定同位素分馏和混合的基础理论1.1改变同位素丰度变化的驱动力−分馏作用1.1.1分馏作用的定义同位素中子数的差异使其质量和原子间结合力不同，也导致了地球化学及生物化学过程中扩散和反应速率的差异.最终表现为同位素分馏(isotopic fractionation)效应，即一种元素的同位素以不同比例在不同物相中分配. 通常而言，轻同位素优先参与化学反应，造成动力学非平衡分馏[4]. 而当反应已达到化学平衡，不同化合物对重同位素的竞争导致了热力学平衡分馏.分馏效应形成了具有不同同位素丰度的物质库. 2H、13C、15N和18O等重同位素在环境中的绝对丰度低，一般接近或低于1%. 为了放大细微变化，便于比较，一般用样品和国际公认标准物质同位素比值的千分差描述稳定同位素含量，记作δ. δ值越大(越正)表示重同位素越多，越小(越负)表示轻同位素越多[5].1.1.2自然分馏自然界的物理过程和化学反应均会造成同位素分馏. 以氮素为例，挥发、扩散、水解、固氮、硝化、反硝化等反应体系具有不同的同位素分馏特征. 氨化作用造成的分馏仅为±1‰左右. 但硝化作用会在反应物里富集15N，浓缩系数在−29‰~−12‰之间；氨挥发也会导致NH4+中15N含量上升，氮的轻同位素更易于以NH3的形式挥发[6-7]. 李荣富等[8]总结了15N在氮循环过程各环节中的分馏系数，并指出并不是所有环节的同位素分馏都得到了清晰阐述，如硝酸盐异化还原、厌氧氨氧化的分馏效应就暂未明确.1.1.3营养分馏生物的新陈代谢会导致同位素分馏. 呼吸和排泄优先排出营养元素的轻同位素，造成食物中的重同位素在捕食者体内富集. 研究[9-10]表明，13C富集系数通常为0~1‰，而15N的浓缩系数通常为3‰~4‰，并随着食物链进一步放大. 因此，与食物组成相近的δ13C 常被用于追溯水体中捕食者的食物组成和贡献，富集程度更高的δ15N则用于开展水生态系统的营养水平研究[11-14].1.2计算源贡献的理论及方法−多源混合模型分馏和混合是稳定同位素研究中的两个重要概念. 分馏作用导致物质库之间的同位素丰度差异，而多个物质库的相互混合进一步造成环境中稳定同位素的复杂分布. 混合模型用数学方法推断混合物中各物质库的贡献率，被广泛应用于污染物定量溯源和捕食者食性分析.作为最基础的混合模型，质量平衡模型(mass-balance model)可用于n种同位素和n+1个来源的溯源计算. 最常用的是2种同位素和3个来源的定量评估，可以避免单一同位素的区分度不够(如NO3−中的15N). 但也有研究[15]指出质量平衡模型未考虑不确定性的影响，在混合物具有多种来源时不能使用. 为解决多来源的问题，2003年，Phillips等[16]通过对质量平衡方程组的反复迭代提出了IsoSource模型. IsoSource模型适用于n种同位素和>n+1个源，获得192环　境　科　学　研　究第 35 卷的不是点估计，而是分布. 2008年，Parnell等开发的SIAR(stable isotope analysis in R)模型引入了不确定性和同位素分馏的考虑，其基于狄利克雷分布和贝叶斯算法，结合似然函数和逻辑先验分布计算贡献率.吴文欢等[17]对质量平衡模型、IsoSource模型和SIAR 模型的适用情境和使用技巧进行了详细论述.除质量平衡模型、IsoSource模型和SIAR模型外，IsoError、IsoEconc、IsotopeR、MixSIR等模型也在污染物溯源中有所应用，Hopkins[18]等和冯建祥等[19]均比较了它们的原理、特征和优缺点. 但在实际使用中为避免误用，还需仔细评估背景资料是否充分，科学问题、试验设计和模型选择是否合理[20].2 氮氧双同位素在水体氮素迁移转化及溯源中的应用2.1反硝化脱氮氮循环是生物地球化学循环中的重要环节，生态系统中氮的输入、转化、利用和消除都是被长期探讨的科学课题. 氮氧双同位素用于脱氮过程研究，提供了用稳定同位素探讨元素迁移转化的范例.厌氧条件下反硝化菌有偏好地利用轻同位素转化成N2和N2O，造成15N和18O富集在剩余的NO3−中，二者的同位素分馏系数比值约为2，即δ15N升高1‰，δ18O相应上升0.5‰[6]. Xue等[7]认为，实际情况下15N 和18O以1.3∶1～2.1∶1的比例富集都可以推论反硝化的发生. 水体脱氮过程中氮氧同位素分馏系数如表1所示. Panno等[26]、Houlton等[28]分别试算了森林和密西西比河中因反硝化损失的氮量，为研究氮素迁移转化提供了新方法.2.2硝酸盐溯源我国许多地方的地表水和地下水正在面临硝酸盐污染[29-30]. 六成分图、派珀图等是硝酸盐污染分析的经典方法[31]，但随着同位素方法的建立和推广，氮氧双同位素被广泛用于氮源定性识别. 1998年Kendall等[6]根据大量试验结果提出了利用氮氧双同位素示踪不同来源硝酸盐的经验方法，利用δ15N的差异区分来自肥料和降水中NH4+硝化、土壤氮素、有机肥与污水的硝酸盐，再通过δ18O的差异区分来自硝酸盐肥料和大气降水的硝酸盐. 该方法得到了后续研究的证实[7,32-33].氮氧同位素溯源研究通常需要结合水化学和其他同位素数据一起进行[34]. 例如，Min等[35]采用δ15N 联合NO3−浓度解释了生活污水和化肥对韩国洛东江流域地下水硝酸盐的贡献；Nyilitya等[36]利用NO3−、Cl−、硼(boron, B)浓度和δ11B探讨了肯尼亚基苏木城和Kano平原地下水硝酸盐来源及转化；Wang等[37]比较了Cl−浓度与NO3−浓度、NO3−/Cl−的相关性，以讨论云南程海氮素的来源和反硝化的发生；Wang等[38]在赣江流域分析了δ15N与NO3−浓度、NO3−/Cl−的关系. 此外，研究地点的背景信息(如土地使用情况)[31,38]、排放负荷和入水负荷[39]等也常被用作综合分析.在氮源定性识别的基础上，量化污染源贡献率的定量评估方法也逐渐受到青睐. 研究者将一系列数学模型用于地表水和地下水的硝酸盐来源探讨，得到氮源的相对贡献. 近年来，IsoSource和SIAR模型在我国各地得到了越来越多的应用(见表2).表 1 水体脱氮过程中氮氧同位素分馏系数对比Table 1 Fractionation factors of nitrogen and oxygen stableisotope in aquatic denitrification采样区域水体类型氮氧同位素分馏系数比值数据来源溪流岸边带地下水 1.5:1文献[21]河流岸边带地下水 1.3:1文献[22]城市含水层地下水 1.9:1文献[23]湖泊深水层地表水 1.8:1文献[24]脱氮菌培养系统模拟淡水/海水1:1(兼性厌氧反硝化菌)、1.6:1(光合异养菌)文献[25]森林地区土壤溶液 1.5:1文献[26]森林地区土壤溶液 1.7:1 ~ 3.6:1文献[27]表 2 稳定同位素在硝酸盐定量溯源中的应用Table 2 Application of stable isotopes in nitrate source identification研究区域水体类型模型名称主要结论数据来源江苏滆湖地表水质量平衡模型临近城市的河流工业废水对硝态氮贡献率为76%~82%；远离工业区的样点硝酸盐来源主要是农业面源和生活污水，二者之和在60%~80%之间文献[39]德国瓦尔诺河地表水质量平衡模型灌溉用水、地下水和大气降水对瓦尔诺河水中硝态氮的贡献率分别为86%、11%和3%文献[40]山西汾河地表水IsoSource模型丰水期临汾段和M5采样点污染源主要是粪便和生活污水，占比为40%~72%，其余采样点化肥贡献率高，为45%~62%；枯水期粪便和污水是所有采样点硝态氮的主要来源，占比为40%~73%文献[41]第 1 期杨　蓉等：碳氮氧稳定同位素技术在水生态环境中的应用1933 碳氮双同位素在水生生物营养谱构建和食性研究中的应用3.1营养级估算稳定同位素在营养生态学的应用始于20世纪90年代中期. 以营养分馏为基础，聚焦水生生物体内的15N含量可反映生态系统中不同物种的营养水平. Zanden等[59]发现传统食物分析和稳定同位素法得到的342种鱼类的营养级无显著性差异，证明了新方法的可靠性. 稳定同位素计算结果用连续数值标示生物在食物网中所处位置，与传统方法获得的正整数相比更能反映其在生态系统中的真实状况[60].捕食者和食物之间较确定的δ15N差值是稳定同位素用于营养级关系计算的基础，根据实际需要选用有针对性的浓缩系数和基线生物是开展研究的前提. Minagawa等[10]的试验表明15N浓缩系数的平均值±标准差为3.4‰±1.1‰，Post[61]和Mccutchan等[62]基于文献调研的结果分别为3.4‰±0.98‰和2.2‰±0.18‰，相差不大. 多种原因造成了浓缩系数的差异，如Mccutchan等[62]发现，针对不同的食物结构和捕食者部位，15N呈现出不同的富集模式；Zanden等[63]按照生物分类(鱼类和无脊椎动物)、栖息地(海水和淡水)、调查方式(实验室和野外)和食物类型(肉食和　续表 2研究区域水体类型模型名称主要结论数据来源内蒙古乌梁素海地表水IsoSource模型化肥和土壤氮相关的农业活动对春季湖水硝酸盐的贡献率为43.7%；生产生活污水是其他3个季节硝酸盐氮的主要来源，贡献率分别为51.3%、38.8%和40.2%文献[42]山东王河地下水库地下水IsoSource模型库区中硝酸盐主要来源于化肥和生活污水，二者的平均贡献率分别为61.44%和33%文献[43]广西寨底地下河地下水IsoSource模型硝酸盐来源以化肥、动物粪便和生活污水为主，且距离居民区较远时化肥贡献比例较高，距居民区较近时动物粪便和生活污水贡献比例更高文献[44]重庆中梁山槽谷区地下水IsoSource模型雨季土壤氮、粪便及污水、降水和化肥中的氨氮对硝酸盐的平均贡献率分别为26%、21%和45%，旱季分别为20%、47%和19%文献[45]重庆青木关地下河地下水IsoSource模型不同采样点的硝酸盐污染特征不同，临近居民区和养殖区的采样点主要污染来源分别为土壤有机氮(48.3%)和粪便污水(59.6%)文献[46]山东桓台地下水IsoSource模型和SIAR模型硝酸盐主要来源于化肥(45%~46.9%)和污水(40.7%~51.7%)，畜禽养殖也有一定贡献(2.6%~10.7%)文献[47]河北洋河地表水SIAR模型硝酸盐来源贡献表现为粪便生活污水(45.37%)>土壤氮(41.39%)>降水和化肥中的氨氮(13.24%)文献[48]陕西浐河灞河地表水SIAR模型硝酸盐来源贡献表现为污水及粪便>土壤有机氮>化肥>降雨，其中污水及粪便在浐河灞河的贡献率分别为30%和36%文献[49]安徽滁州地表水SIAR模型无论雨季和旱季，粪便和生活污水(28%~36%)及土壤氮(24%~27%)都是硝酸盐的主要来源文献[50]浙江长兴地表水SIAR模型12月硝酸盐来源以粪便和生活污水为主，占比为52%；5月以化肥为主，占比为37%文献[51]山东潘庄引黄灌区地下水SIAR模型粪便和生活污水的贡献率最高，为35.1%~80.5%，其次是化肥，占比为4.9%~31.7%文献[52]河北洋河地下水SIAR模型硝酸盐氮来源分别为土壤氮(44.36%)、粪便及污水(43.35%)、无机化肥及工业废水(9.24%)文献[53]贵州会仙湿地地下水SIAR模型动物粪便及生活污水、化肥、土壤氮是主要的硝态氮来源，贡献率差别不大，平均值分别为39.1%、32.2%和28.5%文献[54]比利时弗兰德斯地表水SIAR模型粪便和生活污水是主要的硝态氮来源，冬季贡献率为9%~85%，夏季贡献率为8%~59%文献[55]美国匹兹堡地表水SIAR模型94%的硝酸盐来自污水；暴雨情况下34%的硝酸盐来自大气降水文献[56]希腊阿索波斯盆地地表水和地下水SIAR模型离城市和工业区越远，城市和工业废水对硝酸盐贡献越大，反之化肥和粪便贡献越大文献[57]韩国京畿道地下水SIAR模型农业用地的硝酸盐主要来源于化肥(贡献率为35%~71%)，其次是有机肥料(包括堆肥，贡献率为39%~49%)文献[58]194环　境　科　学　研　究第 35 卷植食)比较了15N的浓缩系数，发现后二者有显著性差异(P<0.05)，但整体在3‰左右；万祎等[64]和蔡德陵等[65]分别通过渤海湾水生生物网调查和鳀鱼养殖试验得出了3.8‰和2.5‰的15N浓缩系数并得到应用[64,66]，但很多研究还是采用3.4‰[67-69].通常情况下浮游植物的数量变动剧烈，不适合反映水域时间和空间的平均信息，因此基线生物多选用研究水域中常年存在、食性单一的浮游动物或底栖动物[70]，如魏虎进[66]等、李红燕[71]均以水体浮游动物优势种中华哲水蚤、太平洋纺锤水蚤、针刺拟哲水蚤作为基线生物，以其δ15N值为基线值. 也有研究[61]认为，浮游动物易受外界干扰、季节波动明显，应选择个体较大、生活周期长的螺类和双壳类，如已有实际应用的福寿螺[68]、铜锈环棱螺[72]、珠蚌[73]、栉孔扇贝[69]、翡翠贻贝[70]等. 在国内，稳定同位素技术曾被用于三峡库区、太湖等淡水水域以及象山港、胶州湾、流沙湾等海域的连续营养谱构建，所调查的生物、使用的基线生物和浓缩系数等如表3所示.表 3 稳定同位素在国内水域营养谱构建中的应用Table 3 Application of stable isotopes in domestic trophic position calculation 采样区域水域类型受试生物基线生物浓缩系数/‰数据来源小江库湾淡水POM、固着藻类、软体动物、水生植物、鱼类不详 3.4文献[67]东太湖淡水苦草、浮游生物、底栖动物、鱼类铜锈环棱螺、河蚬 3.4文献[72]象山港海水浮游生物、SOM、POM、底栖生物、鱼类浮游动物 2.5文献[66]东营、烟台潮间带海水海草、底栖动物、鱼类青蛤、异白樱蛤 3.4文献[74]胶州湾海水浮游生物、底栖动物、鱼类中型浮游动物 3.4文献[75]大连近岸海水底栖动物、鱼类栉孔扇贝 3.4文献[69]枸杞岛海水浮游生物、SOM、POM、底栖生物、鱼类小型浮游桡足类 3.4文献[76]流沙湾海水浮游生物、底栖大型海藻、贝类、虾蟹、头足类、鱼类翡翠贻贝 2.5文献[70]中国澳门海水软体动物、虾蟹、鱼类、鸟类颗粒有机物 3.4文献[77]上海市崇明区海水底栖动物绿螂 2.9文献[78]3.2生态位研究利用δ13C-δ15N散点图量化营养结构和生态位的方法为食物网研究提供了新思路. 在用δ13C-δ15N二维坐标关联同位素含量和营养功能群的基础上[79]，提出δ15N差值(δ15N range, NR)，δ13C差值(δ13C range, CR)、总面积(total area, TA)、平均离心距离(mean distance to centroid, CD)、平均最邻近距离(mean nearest neighbor distance, NND)和平均最邻近距离标准差(standard deviation of nearest neighbor distance, SDNND)6个指标，分别表示营养层次、食源多样性、占据生态位或食物网中营养多样性的总量、营养多样性平均水平、群落的整体密度和营养生态位分布的均匀程度[80]. 该方法量化了食物网的营养结构多样性程度和冗余度，有利于评价生态系统中每个物种的功能及生态位变化.张文博等[81]和谢斌等[82]使用δ13C-δ15N散点图研究了海洋渔场中小型消费者的碳氮稳定同位素比值，分别分析了华南海陵湾、陵水湾两个水域和连云港海州湾不同季节的水生生物NR、CR、TA、CD、NND、SDNND的时空变化. 盖珊珊等[83]和俞雅文等[84]将该方法用于不同生物的生态位研究，分别探讨了两种鱼类和两种蟹类生态位的宽度和重叠程度. 其中，盖珊珊等[83]除NR、CR和TA外，还计算了标准椭圆面积(standard ellipse area, SEA)和营养生态位重叠面积(overlap area, OA). SEA被认为比TA更好，且更不易受样本数量干扰[85]. OA量化了生态位的重叠程度，能更好地评价不同物种利用食物资源的竞争强弱.3.3食物源分析胃含物分析是开展食性研究的经典方法，通过直接解剖动物胃肠道分析残留食物的组成，以此了解其食物来源. 实际应用中发现其具有一系列局限性，包括样本量小时偶然性强、不适于难以解剖的小型动物、只能反映较短时间内的摄食状况、食物残渣偏向难消化的食物类型等[86]. 与之相比，生物体内某些组织的稳定同位素具有较长的周转时间，可用来研究长生命周期内消费者对食物的代谢和吸收[87-88].与定性判断氮源类似，可通过对比捕食者和不同来源食物的同位素值分析食物组成. 例如，当几种潜在食源的δ13C值差异显著，消费者的δ13C落在颗粒有机物和固着藻类之间，可认为这二者是主要食物[67]. 张波等[89]根据生物的δ13C值由栖息水层加深逐渐增大，提出了崂山湾鳅虎鱼不同生命阶段摄食不同深度水生生物的变化规律；崔莹等[90]结合中华绒第 1 期杨　蓉等：碳氮氧稳定同位素技术在水生态环境中的应用195螯蟹各发育阶段特点和碳氮同位素比值分析其洄游期食物组成，强调了结合稳定同位素与生活习性开展分析的必要性. 定量分析方面，混合模型同样被用于食物贡献率计算，稳定同位素在水生生物食性研究中的应用如表4所示. 此时要注意通过胃含物、文献查阅等传统方法确定食源范围，避免取回的样品缺失重要的食物来源；另外，尤其针对15N等营养分馏较明显的同位素，需要选取合适的分馏系数[97].表 4 稳定同位素在水生生物食性研究中的应用Table 4 Application of stable isotopes in energy sources analysis同位素模型名称主要内容数据来源C、N质量平衡模型计算了5种捕食者2~3个食物来源的贡献，C和N的营养分馏系数分别取1.3‰和3.3‰文献[91]C、N质量平衡模型计算了陆源植物碎屑、丝状藻类和水生昆虫对两种淡水螯虾的贡献. C和N的营养分馏系数分别取0.8‰和3.4‰文献[92]C IsoSource模型分析了沉积有机物(SOM)、悬浮颗粒有机物(POM)和浮游植物3种食源对海州湾牧场28种消费者的贡献文献[82]C IsoSource模型分析了石莼、浒苔、海带、POM、SOM和浮游植物6种食源对象山港牧场34种消费者的贡献文献[66]C IsoSource模型分析了C3植物、C4植物和微型藻类3种食源对3种鱼类和1种虾类的贡献文献[93]C IsoSource模型分析了底层鱼类、中下层鱼类、中上层鱼类、虾类、蟹类和头足类共6类28种食源对江豚的食物贡献比例文献[94]C、N SIAR模型分析了8类19种潜在食源在精养和共作两种养殖模式下对克氏原螯虾的贡献.C和N采用甲壳类的分馏系数，分别为1.3‰和3.9‰文献[68]C、N SIAR模型分析了POM、浮游动物、鱼类和甲壳类4种食源对闽江口凤鲚的贡献.C和N的营养富集因子分别为0.4‰和3.4‰文献[95]C、N、S SIMMR模型26种食源被分成无脊椎动物4组和鱼类3组，考察了其对半干涸河口3种主要鱼类的食物贡献. C、N和S的营养分馏系数分别为1.0‰、3.3‰和0文献[96]4 其他特定化合物稳定同位素技术利用氮氧同位素进行硝酸盐溯源是针对化合物(或离子)中某元素开展的稳定同位素分析(compound-specific isotope analysis, CSIA)应用之一. 在环境科学领域，CSIA也被用于卤代烃、多环芳烃等有机污染物的溯源[98]. 而在微生物系统结构和生物化学过程解析方面，Ohkochi等[3]分离纯化了日本Kaiike湖中的光合色素并测定其碳氮稳定同位素组成，解释了与色素相关的自养微生物的生活深度、同化途径和生物功能. Isaji等[99]测定了意大利某盐场中营养盐、叶绿素的δ15N变化，以此探讨底栖微生物中氮素的转化途径及铵态氮在初级生产中的循环利用.上文所述碳氮同位素分析食源和营养级的研究都是将个体或组织(如肌肉)作为对象，检测同位素的整体丰度. 目前已知15N浓缩的主要原因是氨基酸代谢中的脱氨基反应有较明显的同位素分馏，导致排泄出更轻的氮同位素[10]. 因此有研究者分离作为生物标记物的氨基酸并测定其δ15N，从中获得与食物链相关的信息，其理论基础是：生物体内以谷氨酸为代表的一类氨基酸可显著富集15N(约8‰)，而苯丙氨酸等的15N含量随食物链富集程度较弱(约0.4‰)，二者的δ15N值携带了浓缩系数和基线的双重信息[100]. 另有研究[101]认为，利用谷氨酸、脯氨酸、天冬氨酸、赖氨酸等多种氨基酸的δ15N计算营养级可能比仅采用谷氨酸和苯丙氨酸更为准确. 基于氨基酸氮元素的CSIA方法已在食物网生态学中有所应用[102]；与此类似，脂肪酸碳元素的CSIA为海洋食物网的食源分析研究、尤其是海底热液系统中能量来源等方面提供了诸多信息[103].随着研究的进一步深入，还出现了针对化合物特定位置元素开展的稳定同位素分析(position-specific isotope analysis, PSIA)，以获得更详细的分子内同位素分布信息[104]. 例如，NO2中氮素的PSIA方法可被用于研究硝化和反硝化过程，且在一定程度上推算二者发生的比例[105]. PSIA可以解答CSIA提出的问题，加深人们对元素在环境中归趋与去向的理解.5 结论与展望稳定同位素技术为研究自然和生物过程中的元素迁移转化提供了新手段，其在水环境和水生态领域得到快速发展，在模型方法逐渐完善的同时积累了大量有价值的数据. 但在实际应用中，还存在以下需要深入解决的问题.a) 磷是水生态系统中的重要元素，与水域富营养化关系密切. 然而除31P外，磷的其他同位素均具有放196环　境　科　学　研　究第 35 卷。