ACTAUNIVERSITATIS

UPSALIENSISUPPSALA

2014

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 1156

Organic phosphorus speciation inenvironmental samples

Method development and applications

JULIA V. PARASKOVA

ISSN 1651-6214ISBN 978-91-554-8981-6urn:nbn:se:uu:diva-228734

Dissertation presented at Uppsala University to be publicly examined in B41, BMC,Husargatan 3, Uppsala, Monday, 8 September 2014 at 09:15 for the degree of Doctor ofPhilosophy. The examination will be conducted in English. Faculty examiner: ProfessorWilliam T. Cooper (Florida State University).

AbstractParaskova, J. V. 2014. Organic phosphorus speciation in environmental samples. Methoddevelopment and applications. Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 1156. 57 pp. Uppsala: Acta UniversitatisUpsaliensis. ISBN 978-91-554-8981-6.

This thesis investigates the development of new methodology for the identification andquantification of organic phosphorus compounds in environmental samples.

Phosphorus is a vital element for primary production and one of the factors contributing toeutrophication. Eutrophication of aquatic systems leads to algal blooms, changes in ecologicalbalance and deteriorating water quality. Difficulties in studying organic phosphorus stem fromthe fact that organic phosphorus is present in the environment in a variety of forms andeach form may have different degradation and turnover time, having very different effects oneutrophication.

New methods for the quantification of phosphorus derived from three groups of organicphosphorus compounds were developed. For the determination of phosphorus derived fromDNA and phospholipids selective extraction was combined with digestion and colorimetricdetermination of the extracted phosphate. For quantification of inositol phosphates highperformance liquid chromatography was coupled with tandem mass spectrometry usingelectrospray ionization.

The methods were applied to studying the distribution of these compounds in a smallcatchment and in the case of DNA-P and phospholipid-P, the degradation of the fractions inlake sediments. The studies showed that phosphorus bound to DNA, phospholipids and inositolphosphates constitute a sizeable part of the total phosphorus in different environmental samples.The phospholipid-P fraction was the smallest one, accounting for, on average, only a few percentof the total phosphorus in the sample. Inositol phosphates were most prevalent in the soils, withinositol hexakisphosphate accounting for over 10% of the total phosphorus content. The highestcontent of DNA-P was found in sediments and it was shown that DNA-P degrades more rapidlythan phospholipid-P and therefore plays a more critical role in internal loading.

Keywords: Organic phosphorus, DNA, phospholipids, inositol phosphates, sediment, soil,extraction, liquid chromatography (LC), mass spectrometry (MS)

Julia V. Paraskova, Department of Chemistry - BMC, Box 576, Uppsala University, SE-75123Uppsala, Sweden.

© Julia V. Paraskova 2014

ISSN 1651-6214ISBN 978-91-554-8981-6urn:nbn:se:uu:diva-228734 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-228734)

Someone once told me that time was apredator that stalked us all our lives, but Irather believe that time is a companion whogoes with us on the journey and reminds us tocherish every moment because they'll nevercome again. What we leave behind is not as important as how we've lived.

Jean-Luc Picard (1994) Star Trek: Generations

“We may be able to substitute nuclear power for coal, and plastics for wood, and yeast for meat, and friendliness for isolation – but for phosphorus there is neither substitute nor replacement.”

Isaac Asimov

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Preface

The environmental problems we face today are numerous. Often discoveries which help improve our quality of life also lead to problems, and phosphorus is no exception. The implementation of phosphorus fertilizers has com-pletely changed our agricultural practices, where the increased food produc-tion makes it possible to feed the still increasing world population. On the downside, an unrestrictive or improper use of fertilizers, manures, domestic waste or phosphorus-containing detergents have all contributed to an uncon-trolled release of phosphorus back into the environment, causing eutrophica-tion.

Most, if not all environmental issues are often very complex. The difficulty in mitigating eutrophication lies partly in the fact that phosphorus is present nearly everywhere, in both organic and inorganic forms. It undergoes changes in form and solubility, becomes more or less bioavailable, gets transported and accumulated, though not necessarily where it is needed. Or-ganic phosphorus has taken a backseat to inorganic phosphorus in research, but in the last decades it has become apparent that it must be studied due to the prominent role it in the phosphorus cycle.

Analytical chemistry is a branch of chemistry that enables the study of many types of samples through innovation of new methods and instrumentation. Although it is possible to define it as the science of developing methodology for the determination and structure of substances, it needs to be seen as a science, and not just a way to perform analytical measurements. It is true that other fields of chemistry concerned with the fundamental description of sub-stances are relying on determination methods. It does not, however, make the field of analytical chemistry, less scientific.

The foundation of developing new methodology needs to be based on solid understanding of the problem at hand. However, a new method also needs to be experimentally validated and also applied to a real-life system, i.e. it should be of practical use. The overall goal of an analytical method is to be robust, sensitive and reproducible. Current organic phosphorus research limitations are associated with incomplete extractions, poor recoveries, un-certainty of the origin of the extracted analytes as well as overall low con-centrations.

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In this work, analytical chemistry is used not only as an investigation tool, but also as a field in which the pursuit of new knowledge is focused on de-veloping and optimizing new methods, which in turn can be used for the study of organic phosphorus in the environment.

This thesis is written as part of the requirements for obtaining a Ph.D. degree in analytical chemistry at Uppsala University, Uppsala, Sweden. It is com-prised of different studies, each of which deals with a special aspect of or-ganic phosphorus found in samples relevant for environmental research. These include the development of new methods for the determination of different forms of organic phosphorus and methods application on studying their propagation in the real world with the hope of increasing our under-standing of the behavior of organic phosphorus in aquatic and terrestrial systems. It is meant to also give a brief overview of the field of organic phosphorus research and it is the hope of the author that the results presented here will make, at the very least, a small contribution to help solving some of our environmental problems.

Planet Earth possesses the correct chemical combination and is situated at “just the right” distance from its sun that makes life possible. Phosphorus is an essential part of that combination. Named for its luminescence properties - giver of light - it would not be wrong to paraphrase the name as giver of life. If not for phosphorus we would not be here. There is no better reason to want to study it.

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List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Paraskova, J.V., Rydin, E., Sjöberg, P. J. R. (2013) Extraction

and quantification of phosphorus derived from DNA and lipids in environmental samples. Talanta, 115: 336–341

II Paraskova, J.V., Sjöberg, P. J. R., Rydin, E., (2014) Turnover of DNA-P and phospholipid-P in lake sediments. Biogeochemis-try, 119(1-3): 361-370

III Paraskova J.V., Jørgensen C., Reitzel K., Pettersson J., Rydin E., Sjöberg, P. J. R., (2014) Speciation of inositol phosphates in lake sediments by ion-exchange chromatography coupled with mass spectrometry, ICP-AES and 31P NMR spectroscopy. Manuscript

IV Paraskova, J.V., Sjöberg, P. J. R., Rydin, E., (2014) Organic phosphorus speciation in a small catchment: sinks and sources. Manuscript

Reprints of Papers I and II are made with kind permission from the respec-tive publishers, Elsevier & Springer Science and Business Media.

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The author’s contribution to the papers: Paper I: Planned the study together with the co-authors. Performed all of the experiments in the study and wrote the paper. Paper II: Planned the study and wrote the paper together with the co-authors. Performed all of the experiments in the study. Paper III: Planned the study together with the co-authors. Performed most of the HPLC-MS and HPLC-ICP-AES experiments. Had the main responsi-bility for coordinating the project and writing the paper. Paper IV: Planned the study together with the co-authors. Collected sam-ples, performed most of the experiments. Wrote the paper.

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Contents

Preface ............................................................................................................ 5 

Abbreviations ................................................................................................ 10 

Aims .............................................................................................................. 11 

1 Introduction ................................................................................................ 12 1.1  Phosphorus in the environment ....................................................... 12 1.2  Phosphorus in terrestrial ecosystems .............................................. 14 1.3  Phosphorus in aquatic ecosystems .................................................. 15 1.4  Organic phosphorus ........................................................................ 16 

1.4.1  Inositol phosphates ................................................................ 17 1.4.2  DNA ....................................................................................... 18 1.4.3  Phospholipids ......................................................................... 19 1.4.4  Challenges in organic phosphorus quantification .................. 19 

2  Methods ............................................................................................... 21 2.1  Sampling ......................................................................................... 21 2.2  Extraction ........................................................................................ 22 2.3  Sequential extraction ....................................................................... 22 2.4  Nuclear magnetic resonance spectroscopy ...................................... 23 2.5  Spectrophotometry .......................................................................... 25 2.6  Inductively coupled plasma ............................................................ 26 2.7  Ion-exchange chromatography ........................................................ 27 2.8  Mass spectrometry .......................................................................... 28 2.9  Hyphenation .................................................................................... 30 

2.9.1  HPLC hyphenated with ICP .................................................. 30 2.9.2  HPLC hyphenated with MS ................................................... 31 

3  Applications ......................................................................................... 35 

4  Acknowledgments ............................................................................... 41 

5  Svensk sammanfattning ....................................................................... 44 

6  Краткое содержание .......................................................................... 48 

7  References ........................................................................................... 50 

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Abbreviations

AMP adenosine monophosphate ATP adenosine triphosphate DNA deoxyribonucleic acid HPLC high performance liquid chromatography ICP inductively coupled plasma IP inositol phosphate m/z mass to charge MRM multiple reactions monitoring MRP molybdate reactive phosphorus MS mass spectrometry MS/MS tandem mass spectrometry NMR nuclear magnetic resonance PL phospholipid Q-Trap quadrupole ion trap TP total phosphorus UV ultraviolet

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Aims

The general aim of this thesis was to develop new strategies for the speci-ation of organic phosphorus in samples relevant for environmental research and test these strategies on real life systems.

Paper I describes a new method for the selective extraction and quantitative determination of phosphorus originating from orthophosphate diesters. In Paper II this method was applied to the comparative study of two lake sediments, exploring the degradability of phosphorus derived from DNA (DNA-P) and phospholipids (PL-P) with sediment depth. Paper III focused on designing a new way of identifying the predominant group of organic phosphorus, inositol phosphates (IPs), with the help of high performance liquid chromatography (HPLC) coupled with mass spectrometry (MS). This new approach was compared to the method most commonly used in phos-phorus research, namely 31P nuclear magnetic resonance spectroscopy (31P NMR). The goal of Paper IV was to determine the composition of the above mentioned organic phosphorus fractions in a variety of environmental sam-ples and applying the methods in a comprehensive study in attempts to re-veal which phosphorus forms contribute to eutrophication.

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1 Introduction

Phosphorus was discovered in the second part of the 17th Century by Hennig Brandt, a German alchemist in the search for the Philosopher’s Stone. [1] Since then it has been defining human history. Early applications of elemen-tal phosphorus were unproductive, since its only apparent usefulness was its ability to glow in the dark, a novelty which was used purely for entertain-ment. Remarkably, doctors used it as medicine, although it never cured any-one of anything. [2] Some of its noteworthy applications were in the produc-tion of matches, while in history’s low points it became the ‘Devil’s ele-ment’, widely used in various military applications, including chemical war-fare. [3] For millennia humans have used animal manures, human excreta and bones to improve soil fertility, yet the role of phosphorus in agricultural production was unknown. [4] Everything changed in the middle of the 18th century, when phosphorus was identified as an element essential for plant nutrition by Jusus von Liebig who showed that the inorganic salts of phos-phorus and nitrogen had a fertilizing effect on plant growth. [3] By then phosphate rock deposits were discovered and although their value as a min-eral sources of phosphorus was not recognized until later, the mining of phosphate rock started in 1867 in the USA and in 1873 in North Africa. [5] The commercial mining and production of phosphate rock really took off in the middle of the 19th century, [5] when it was recognized as an abundant and cheap source of phosphorus. In the 20th century phosphorus chemistry has become important for other reasons, becoming a success story in making insecticides and detergents. [2] However, it is undeniable that the vast eco-nomical significance of phosphorus as a natural resource is due to its impor-tance to modern agricultural productivity.

1.1 Phosphorus in the environment

The most abundant and stable form of phosphorus found in the environment is orthophosphate. [6] Literature dealing with phosphorus in the fertilizer industry often interchanges the terms phosphate and phosphorus. [5] Phos-phate refers to orthophosphate and different polyphosphates such as

, and .These forms can be found in phosphorus ores and

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are used in the making of inorganic fertilizers. The extent of the global phosphorus reserves has been a subject of concern, because the mineral re-serves cannot be replenished. The immediacy of the problem is, however, difficult to assess, because the depletion of those reserves may be hard to evaluate. [5] Even though phosphate rock can be found all around the world, 85% of the reserves are controlled by just five countries – South Africa, USA, Jordan, Morocco and China. [3] It is reasonable to assume that a grow-ing world population will lead to an increase in phosphorus consumption. Therefore the depletion of the existing reserves is expected to proceed and at a higher rate than presently. Furthermore, the reserves cannot be expanded or added to, which means that they will eventually come to an end.

The most remarkable thing about phosphorus is that it is a prerequisite for life as we know it, as all living things contain this element. Phosphate esters are the building blocks of the genetic material DNA and the reservoirs of biochemical energy, adenosine triphosphate (ATP) are phosphates. [7] The structural material of teeth and bones is made of calcium phosphate. In addi-tion, phosphorus plays numerous and crucial roles in biochemistry. [1]

A growing interest in understanding the dynamics of phosphorus in the envi-ronment is driven by two opposing issues. On one hand the availability of inorganic phosphorus as a natural resource is limited, and it has been pre-dicted that global known, low-cost reserves may already be depleted by the end of this century. [8, 9] On the other hand organic forms of phosphorus are abundant in the environment since the element is present in all living organ-isms, which means that there is a potential for its successful management and recycling. It has been therefore argued that research should focus on using accumulated soil organic phosphorus reserves and that there is an im-mediate need to develop new efficient ways for the utilization of manures, composts and wastes. [10, 11] The ecological significance of phosphorus have been recognized and studied for a long time. It has been linked to marine productivity already in the 1940s [12] and decades later, recognized as one of the key factors causing eutrophication. [13, 14] Eutrophication means the increase of nutrients, spe-cifically phosphorus and nitrogen, in aquatic systems, which, among other things, leads to algal blooms in the summer and water quality problems. When algae die, the cells they are made of decompose and accumulate at the bottom of water bodies. Other organisms that use organic matter as food, process the algae while consuming oxygen making the layer close to the bottom anoxic. That in turn makes it hard, if not impossible, for other organ-isms, such as fish, to live and function. Although eutrophication is a natural process, it is supposed to proceed at a much slower rate. Due to anthropo-genic activity, which releases an abnormal amount of nutrients into the eco-

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system, the process is much faster and accumulation rates of dead organic matter are larger than normal. Understanding how eutrophication works im-plies mapping the flow pathways of nutrients from their origin to the places they get deposited and studying their degradation and turnover rates.

The sources of phosphorus can be divided into point and nonpoint sources. [15] Point sources may include runoff from various human activities, such as waste disposal sites, animal feedlots, mines and industrial sites. Nonpoint sources may include a more diffuse runoff from various agricultural prac-tices and activities on land that generate contaminants on a larger scale, such as wetland construction and conversion. They may be triggered by heavy precipitation and in many instances are seasonally-dependent. Discharges from point sources are considered to be continuous and vary little over time. Consequently, they are possible to regulate. Nonpoint sources are both hard to define and mitigate, but it has been recognized that soil and water conser-vation practices can minimize phosphorus losses. [16, 17]

Phosphorus transfer to the aquatic environment may occur in both dissolved and particulate forms. Although it has been previously believed that the main source of phosphorus in the environment was in inorganic form, there is emerging evidence that organic forms of phosphorus can be as bioavailable. For example some studies have shown that organic phosphorus can be re-leased and converted into bioavailable form after enzymatic hydrolysis, [18] under increased salinity conditions [19] or under periods of phosphorus stress. [20] Primary production in both the terrestrial and in the aquatic envi-ronments incorporates phosphorus into organic matter. During plant and algal decay dissolved organic phosphorus is transported downstream, where some part is mineralized and becomes bioavailable.

Mitigating eutrophication is a difficult task, because at present we do not fully understanding the processes that govern the cycling and deposition of phosphorus between terrestrial and aquatic systems. We know that phospho-rus is a limiting nutrient, which means that when phosphorus is scarce, ac-tivities that lead to eutrophication slow down to a halt. However the major challenge is in figuring out which forms of phosphorus are bioavailable.

1.2 Phosphorus in terrestrial ecosystems What makes the cycling of phosphorus in the environment unique is the fact that phosphorus does not normally appear in gaseous phase. Phosphate in the environment does not have a significant atmospheric component, but only a biochemical and a geochemical one, [1] which is illustrated in Figure 1. The biochemical component is the incorporation of inorganic phosphorus into the

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living biomass and its transformations therein. The geochemical component is the weathering of igneous or sedimentary rocks, and the transport of phos-phate by water flow to larger water bodies, where it can be precipitated or once again incorporated into the food chain. [1]

Figure 1. A conceptual model of the cycling of phosphorus in the environment.

An important role in the function and regulation of natural and managed terrestrial ecosystems is played by soil. It provides physical support for the vegetation and serves as a reserve and regulator of nutrients and water. The greater part of phosphorus in the environment is thus found in the soil. [21]

1.3 Phosphorus in aquatic ecosystems Phosphorus transport and accumulation in aquatic systems occurs in both soluble and particular form. While the dissolved phosphorus is readily used up, particulate phosphorus can remain in an aquatic system for a long time. Phosphorus is accumulated in lake sediments and river beds, but is also cy-cled between the water and the sediment. The release of phosphorus from the sediment back into the water column, a process called internal loading, is perhaps one of the major factors contributing to eutrophication. [22-24] Nu-trient input affects every part of an aquatic system, including water quality, oxygen level and biodiversity (Figure 2). Since the deposition of organic matter within water bodies contributes to the accumulation of sediments,

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organic phosphorus is often quantitatively the most important form of phos-phorus in the sediment.[25] The degradable organic phosphorus forms repre-sent the fraction of the phosphorus that is being recycled. Depending on the physical, chemical and biological processes which take place in the sediment and the water column, internal loading is dependent on the type of organic phosphorus being recycled.

Figure 2. Illustration of the nutrient impact on an aquatic system.

1.4 Organic phosphorus

Organic phosphorus compounds are characterized by the presence of both carbon and phosphorus in the same molecule and are classified into ortho-phosphate esters, phosphonates and anhydrides. [26] Orthophosphate esters, the esters of phosphoric acid, are stable in the pH range of most soils, but hydrolyze readily at extremes of pH or in the presence of phosphatase en-zymes. Phosphoric acid is a moderately strong acid, and its esters have an ionizable proton with a pK of about 2. [26] That is why orthophosphate es-ters are almost completely ionized in the pH range of most soils. Phosphorus bonding is always covalent, and in nature phosphorus stays at its highest oxidation state, phosphorus(V). [27]

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The most abundant phosphorus compounds found in soil are shown in Figure 3. These are orthophosphate monoesters, principally inositol phosphates (IPs) and orthophosphate diesters, a group that includes nucleic acids and phospholipids (PLs). [26, 28-30]

Figure 3. Graphic representation of the relative abundance of organic phosphorus compounds found in soil, after [26].

1.4.1 Inositol phosphates The predominant group of orthophosphate monoesters, IPs, represents a group of phosphoric esters of hexahydroxy cyclohexane (inositol) [28, 31] containing from one to six phosphate groups. They are commonly denoted as IPx, where x represents the number of phosphates. IPs can appear in the form of nine stereoisomers, where the myo-inositol (InsPn) is the most common one, with a variety of phosphorylated derivatives and conformational iso-mers. [32] The salt of myo-inositol-1,2,3,4,5,6-hexakis dihydrogen phos-phate (InsP6), also called phytate, consists of a myo-inositol esterified with symmetrically distributed phosphate groups (Figure 4). [33]

Figure 4. Structure of myo-inositol-1, 2, 3, 4, 5, 6-hexakis dihydrogen phosphate.

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The distinguishing feature of phytate and its salts is its notable stability. In the environment it has the ability to form complexes with the divalent metal ions calcium(II), iron(II), magnesium(II) and zinc(II), hydroxides and or-ganic matter and thus accumulate in the soil. [34] Inositol phosphates can form insoluble salts in soils with low pH and thus become unavailable for plants. Alternatively IPs can precipitate with calcium in soils with high pH. [35] It is suggested that IPs are very stable in aquatic systems. They can be immobilized in sediments through binding to mineral particles or in the wa-ter through the formation of complexes with the above mentioned metal ions, preventing them from being metabolized by microorganisms. [36, 37]

1.4.2 DNA The macromolecule DNA, the building block of genetic material, can be found in virtually all living organisms on our planet. It consists of sugar molecules, deoxyriboses, which are connected to each other through phos-phodiester bridges, [38, 39] schematically illustrated in Figure 5. Some of the distinguishing and remarkable features of DNA are its length, mass and asymmetry. Even the smallest DNA molecules are long. Compared to the longest proteins, the smallest DNA is at least six times larger.

Figure 5. Structure of part of a DNA chain.

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In most soils the relative abundance of DNA is only a few percent of the total phosphorus, [40] but in some soils it can account for more than half of the extractable organic phosphorus. [41] In sediments the amounts of DNA may be substantial [42], and it has been shown that it degrades and contrib-utes to phosphorus cycling. [43]

1.4.3 Phospholipids Lipids are hydrophobic molecules which have many different biological functions. Because they are water-insoluble they are very different from other biomolecules, such as amino acids and proteins. Phospholipids (PLs), a class of lipids that contain phosphate, are the main component of cell mem-branes. It has been shown that the PLs found in soils are mainly principal phosphoglycerides, [30] in which the phosphorus group is esterified to the hydroxyl group of one of the following alcohols: serine, ethanolamine, cho-line, glycerol and inositol. [38] An example of a PL can be seen in Figure 6.

Figure 6. Structure of phosphatidyl choline, an example of a phospholipid.

All living matter contains PLs and they can therefore be found in soils in smaller amounts, although it is possible that their quantity has been underes-timated because they do not easily extract into the same medium as other organic phosphorus compounds. Lipids have been used as a measure of mi-crobial biomass in soils and sediments [44-46] and vice versa, microbially active soils have been shown to accumulate PLs. [47]

1.4.4 Challenges in organic phosphorus quantification The main difficulty associated with the determination of organic phosphorus in environmental samples is due to the fact that there are currently no direct measurements methods in existence. Organic phosphorus is most often esti-mated indirectly, using the difference between total phosphorus and inor-ganic phosphorus measurements. [48] Alternatively, an organic phosphorus pool is defined after a sequential extraction of differently bound inorganic phosphorus compounds is performed. [49]

Difficulties stem also from the fact that both inorganic and organic forms of phosphorus can exist in nature in different states, i.e. in solution or as solid

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particles, associated with soils and sediments or incorporated into plant ma-terial. Additionally, phosphorus can have a variety of chemical forms, i.e. dissolved, such as orthophosphates, condensed inorganic and condensed organic phosphorus, particulate forms, such as mineral and organic or mixed forms in complexes, hydroxides and clays. You can find phosphorus in DNA, proteins and cell membranes of living organisms, but also in synthetic organic phosphorus compounds such as insecticides, herbicides or plant growth regulators. [26] Furthermore, the chemical characteristics of those compounds can be quite different. Some of these forms are water-soluble, while others are fat-soluble. Some are very stable, enabling their storage in the soils. Other forms are labile, which means they can be transformed by microorganisms. Finally the more complex forms of phosphorus can degrade to the more simple forms. It is therefore of great importance to have accurate methods which can separate and differentiate the different forms of phospho-rus from one another, so that their behavior can be studied.

It is important to be able to identify the biologically active phosphorus frac-tion, but this is difficult because both organic and inorganic forms of phos-phorus may be bioavailable and organic phosphorus can easily transform and integrate into the microbial biomass. [50]

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2 Methods

The prerequisite for meaningful results in any research projects is not only a robust method and meticulous laboratory work, but also proper planning. In fact, many issues need to be addressed prior to both method development and application. The analytical procedure includes many different steps in-cluding, but not limited to, sample collection and preparation, analysis, data processing and interpretation of the results. It is equally important to know what should be analyzed as well as in what manner the analysis should be performed. The actual sampling procedure and sample preparation are two vital parts of the analytical process, because they can give rise to the largest variability in the results. Apart from the simplest analysis, such as for exam-ple temperature or conductivity measurements, environmental analysis re-quires extensive sample preparation. An ideal analytical technique would require no sample preparation and would allow measurements in situ. That is often not possible. That is why the goal of any sample preparation technique is to make it as simple as possible, with as few steps as possible to prevent loss or degradation of the analyte while simultaneously removing interfer-ences.

Important for all measurements are the selectivity and detection limits of the chosen method. They can be achieved in different ways in the different steps of the analytical procedure.

2.1 Sampling

The greatest source of variability within the analytical procedure is the sam-pling procedure. During sampling it is essential to consider the sampling location, the number and type of samples to be collected and the actual sam-pling procedure. The samples need to be representative of the area studied and homogeneous enough to enable proper handling. One of the questions environmental scientists need to address is whether the sample studied, which can weigh less than a gram, can answer questions about a sampling area of many square kilometers. It is also important to identify possible sources of contamination, decide on how the sample should be transported and stored if it is not analyzed immediately. Most of the work in this thesis

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deals with method development to which the choice of sampling procedures were not crucial. However, different types of samples, such as soil, sediment and compost, were used in Paper I to ensure that the method could easily be applied to a variety of matrices. In contrast, the method developed in Paper III focused on one type of sample, namely lake sediment, and that presented challenges in the application of the method in Paper IV.

2.2 Extraction An important part of sample preparation is the extraction procedure, which is the isolation of a compound of interest from a matrix. The purpose of the extraction is to separate the analyte from a complex sample, to clean it up from interfering compounds and to pre-concentrate analytes to a concentra-tion which can be detected by the selected measurement method. [51] There are techniques which are able to analyze different phosphorus compounds in solids, such as solid state NMR [52], however the technique’s resolution is rather low, which is why extraction prior to analysis with other methods is preferred.

Many analytical procedures cannot be performed without an extraction step. All the projects described in this thesis have used different extraction tech-niques. In Paper I different strategies have been assessed for the successful extraction of phosphorus bound to DNA and PLs. The main goal of the ex-traction of DNA was to optimize the cell lysis procedure in order to increase the yield. Liquid-liquid extraction with immiscible solvents was used for the extraction of PLs. The extraction efficiency was evaluated on samples spiked with standards. In Paper III the extraction procedure was adopted from sample preparation methods currently used for 31P NMR, but it was modified in Paper IV to better suit the determination technique.

2.3 Sequential extraction The first methods used for the quantification of phosphorus in environmental samples implemented sequential extraction of different forms of phosphorus, based on their chemical bonding. As such, the different forms were opera-tionally defined, i.e. they included various chemical species which shared common characteristics. In all fractionation methods the samples were sub-jected to extractants of increasing strength, both basic and acidic. Examples of extractants used in the first fractionation schemes are ammonium chloride (NH4Cl), ammonium fluoride (NH4F), sodium hydroxide (NaOH) and sulfu-ric acid (H2SO4). The fractions could be separated, based on phosphorus solubility, into pools of labile phosphorus, aluminum, iron or calcium bound

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phosphorus, and depending on the method, refractory phosphorus, recalci-trant phosphorus or residual phosphorus. The first comprehensive method was developed by Chang and Jackson in 1957, [53] which since then has been extensively used and modified by others. [54, 55] In addition to meas-uring various inorganic phosphorus pools, it allowed to indirectly determine organic phosphorus by calculating the difference between inorganic phos-phorus before and after digestion of the NaOH extracts. However, a major shortcoming of the method was that it could not assess plant available or-ganic phosphorus, which incidentally was also the labile phosphorus frac-tion.

A new fractionation scheme, specifically designed organic phosphorus, was proposed by Bowman and Cole in 1978. [56] The novelty in the method was distinguishing between labile, moderately labile, resistant and acid insoluble phosphorus. A few years later Hedley et al. put together the original method of Chang and Jackson with the organic phosphorus method of Bowman and Cole to identify ten different phosphorus fractions, which included inor-ganic, organic and microbial phosphorus. [57] It added new extractants, such as sodium bicarbonate (NaHCO3), hydrochloric acid (HCl) and hydrogen peroxide (H2O2) to the scheme in order to improve the extraction efficiency of organic phosphorus. The milder extractants produced fractions which were hypothesized to be biologically relevant to plants. Sequential fractionation methods have been in use for over 50 years and are still in use today, because they are very well-described and are easy to per-form with basic laboratory equipment. They provide a simple way to moni-tor different systems and make comparisons between systems possible. However, they are not specific for any particular group of organic phospho-rus, which in turn means that their role in the phosphorus cycle may be at best ambiguous.

2.4 Nuclear magnetic resonance spectroscopy One technique which has advanced our understanding of organic phosphorus in a variety of samples is 31P NMR. [58] It was first applied to study phos-phorus in grassland soils by Newman and Tate in 1980. [59] The method distinguishes groups of phosphorus based on specific resonance frequencies relative to an applied magnetic field and can thus differentiate between or-thophosphate, polyphosphate, pyrophosphate, orthophosphate monoesters, orthophosphate diesters, and phosphonates. [60] In practice, compounds are identified by comparison of their chemical shift relative to the external stan-dard, phosphoric acid (H3PO4), where the chemical shift of the standard is set to 0 ppm. [61] The main advantages of this technique is the ability to detect and measure signals of multiple phosphorus compounds simultane-

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ously (Figure 7) and distinguish between compounds with similar structural information. The technique is most commonly used together with NaOH extraction, which as a bonus, requires no additional sample clean-up prior to analysis, except centrifugation. The drawback of this particular extraction procedure lies in the usage of a harsh solvent with very high pH which may change the chemical structure of some compounds and hydrolyze the most labile ones. [58]

Figure 7. A solution 31P-nuclear magnetic resonance (31P NMR) spectrum of A. Sample from under grass and B. Grassland plant litter in an oak savannah, Northern California, showing the diversity of phosphorus species in environmental samples. (Unpublished results, Barbara Cade-Menun).

Other extraction procedures which are milder may not guarantee quantitative recovery. The determination technique, 31P NMR, has high detection limits and poor sensitivity, which prompts the need further sample preparation through concentration of the samples. Sample concentration can be achieved with freeze-drying, rotary evaporation or by drying under a stream of nitro-gen, but it has been shown that there is a risk of some sample loss and deg-radation. [32] An additional disadvantage of the NMR technique is that some of the chemical shifts, for example in the orthophosphate diester region, can overlap or be poorly resolved. Finally, the technique is rather time consum-ing.

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2.5 Spectrophotometry Spectrophotometry is an analytical technique which determines chemical concentrations with the help of light. [51] The principle of the measurement is based on the reflection or transmission properties of the material of inter-est which can be expressed as a function of wavelength. The basic setup of a spectrophotometric experiment is presented in Figure 8. It shows that light passes through a monochromator so that one wavelength can be selected. It then strikes a sample, which adsorbs some of the light. As a result, the ir-radiance of the original beam produced by the light source is reduced. The absorbance, measured by a spectrophotometer, is a logarithmic measure of the amount of light which is absorbed by the sample and is directly propor-tional to concentration.

Figure 8. A principal diagram of a spectrophotometer.

Traditionally phosphorus was determined spectrophotometrically by the molybdenum blue method of Murphy and Riley. [62] The method was first adopted for water matrices, but it has been used extensively on soil extracts. It is often called the molybdate reactive phosphorus (MRP). In principle, the free phosphate present in a sample reacts with molybdate ions under acidic conditions to produce molybdophosphoric acid. The acid is subsequently reduced, either with tin chloride or ascorbic acid, to form a molybdenum blue complex, which can be detected colorimetrically. [63] Color intensity is proportionate to the amount of phosphate which has formed the blue com-plex, ideally all the free phosphate available. However, the formation of the complex can be rather slow and obviously, incomplete color development leads to the underestimation of inorganic phosphorus. The addition of anti-mony(III) as a catalyst can speed up the color formation in the sample. [63] The primary advantages of the MRP method is that it is cheap, easily per-formed and has good sensitivity and low detection limits. One of the disad-vantages of the MRP method is that it is subject to interference from com-plex matrices, such as soil. The blue molybdate complex can be formed with elements, other than phosphorus, for example arsenic, chromium or silicon. [63, 64] Additionally some ions, such as fluoride, have been shown to slow down the color development. [64] When the method is used to quantify phosphorus in soil extracts, especially samples from mineral soil, there is a

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risk of overestimating the results by creating false positives from the matrix. [63]

Additionally, it is important to acknowledge that the MRP procedure was designed to determine inorganic orthophosphate-phosphorus, but it has been shown to possibly include loosely bound inorganic and organic phosphorus. [65] To address this problem and avoid overestimating the results, selective extraction can be implemented as a procedure preceding MRP measure-ments. This theory was tested in Paper I on the orthophosphate diester frac-tion, focusing on the selective extraction of DNA and PLs. In the newly de-veloped method, the extraction procedure was designed to selectively target DNA, followed by the removal of small molecules through ultrafiltration and the subsequent persulfate digestion which transformed the extracted DNA-P fraction into easily measurable phosphate. A second procedure focused on the selective extraction of PLs, followed by dry ashing and hydrochloric acid digestion, which transformed the PL-P into inorganic phosphorus. The iso-lated fractions each contained phosphorus from only one source. Although the actual determination method measured orthophosphate, the selectivity was achieved by sample preparation.

2.6 Inductively coupled plasma

Inductively coupled plasma (ICP) is an atomic spectroscopy technique in which a substance is broken down into atoms at very high temperatures. [51] To achieve temperatures of 6000-10000 K an induction coil is used to ionize argon gas with the help of radio-frequency. The sample is pumped into a nebulizer which transforms the liquid into an aerosol which is then trans-ported into the spray chamber, where the larger droplets are discarded to waste and a mist of finer droplets is transported further. In the plasma the solvent is evaporated and the sample is atomized and ionized. Figure 9 shows the basic design of an ICP instrument.

ICP is most commonly coupled with atomic emission spectroscopy (AES) or mass spectrometry (MS). The main advantage of ICP-AES and ICP-MS is their ability to perform multi-elemental analysis. Additionally, the linear range is broad and the detection limits are usually low, in the ppb range for AES and in the ppt range for MS, for virtually all elements except halogens and the elements found in the atmosphere, hydrogen, oxygen, nitrogen and carbon. The disadvantages of ICP-AES and ICP-MS are that the analysis method cannot differentiate between whole molecules or elements in differ-ent oxidation states. Also, some elements have spectral overlaps.

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ICP-AES is widely used today for routine inorganic phosphorus analysis, such as total phosphorus determination. In Paper III, ICP-AES was used to characterize an in-house IP standard.

Figure 9. A principle diagram of inductively coupled plasma.

2.7 Ion-exchange chromatography Chromatography is an analytical technique in which a number of constitu-ents within a sample are separated from one another due to differences in their distribution ratios between two phases. In liquid chromatography the analytes are dissolved into a liquid, the mobile phase, which is passed through a column filled with solid material, the stationary phase. In ion-exchange chromatography the stationary phase can be composed of, for ex-ample, silica particles with charged functional groups at the surface (ref) or cross-linked polymers containing ionized or ionizable groups. [66] Analytes introduced into the column are retarded within the column because of their stronger or weaker interaction with the functional groups.

Ion-exchange chromatography is the most suitable separation technique for IPs. [67, 68] The ion-exchange mechanism relies on the interaction between the phosphate ions (the anions) with the functional groups of the column (the cations). The strength of the anion-cation interaction is dependent on the number of phosphate groups in the IP. In a solution with different IPs pre-sent, IP6 with its six phosphate groups would have the strongest interaction

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Sample inlet

Ion source Mass analyzer Detector

Computer

and therefore the longest retention within the column and IP1 with only one phosphate group, would have the weakest interaction and the shortest reten-tion within the column.

In Paper III ion-exchange chromatography was coupled with MS for the purpose of the identification and speciation of IPs in sediment samples. In Paper IV the method was tested on a variety of environmental samples.

2.8 Mass spectrometry Mass spectrometry is a powerful analytical technique which can be used for the detection and determination of analytes in different types of samples. It has the capacity to determine the mass of an analyte and to reveal structural information by producing and detecting fragments of molecules. [69-71] Thus, the main advantage of MS is embedded in the specificity of the identi-fication. A mass analyzer can serve as a universal detector since it can be used to study any element or compound which can be ionized. Additional advantages of MS are the small sample size required for the analysis and good sensitivity. The drawback of the technique is encountered when com-plex samples are analyzed, in which the information obtained can be from many compounds and not necessarily from the compound of interest.

A mass spectrometer has several major components, schematically presented in Figure 10: a sample inlet, which allows the sample to be introduced into the system, an ion source which transforms the sample into gas-phase ions, a mass analyzer, which separates the ions according to their mass to charge (m/z) ratios and a detector, which generates signals that record the different ions. Additionally a vacuum system provides a collision-free path for the ions and a computer collects and stores the data.

Figure 10. A conceptual illustration of the major parts of a mass spectrometer.

Vacuum

Ions Ions

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Mass analyzers operate with the help of electric, magnetic or a combination of electric and magnetic fields. The different type of mass analyzers include Time-of-Flight (TOF), Quadrupole Ion Trap (Q-Trap), Magnetic-Sector, Transmission Quadrupole, Fourier transform ion cyclotron resonance (FT-ICR) and Orbitrap. Tandem mass spectrometry, often abbreviated MS/MS, uses two stages of mass spectrometry, each with an m/z-selective process. It allows to the separation to be conducted in series, either in time or in space. The most common configuration of such a system would be two quadrupole mass analyzers with a collision cell placed in between. The unique combina-tion of MS/MS allows for one stage to choose an ion and the second stage to explore the relationship of this ion to another ion, either one from which it was generated or one that it fragments into. [70] The instrument used in this work was a hybrid triple quadrupole/linear ion trap 3200 QTRAP®, sche-matically illustrated in Figure 11.

Figure 11. Illustration of the triple quadrupole configuration of the 3200 Q TRAP.

The main module, the quadrupole, as the name suggests, is comprised of four electrical poles, in between which an electric field is created. In a triple quadrupole the middle section is used as a collision cell instead of a mass analyzer. The collision cell is filled with a collision gas, with which the in-coming ions collide and thereby undergo decomposition or fragmentation. The amount of fragmentation can be controlled by varying the collision en-ergy, which is influenced by the electric field and the speed with which the ions are introduced into the cell. [69]

One of the common ways of using MS/MS is through multiple reaction monitoring (MRM), which is a way of monitoring the transition of a given precursor ion to a particular product ion. The fragmentation of intact mole-cules, which happens reproducibly and consequently, can be used as a way of identifying and quantifying compounds that are identical or very close to each other in mass. In previous work, Cooper et al. showed that all IPs tend to produce a common fragment ion in the form of . [67] In Paper III the tendency of IPs to fragment was used as a way of identifying them after separation with HPLC, however to further support correct identification, two MRM transitions for each IP were monitored, based on a neutral loss of

(m/z 80) or (m/z 98).

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2.9 Hyphenation

In analytical chemistry the coupling of different techniques is called hy-phenation. The term was first introduced as a way of referring to the on-line combination of a separation technique and a spectroscopic detection tech-nique. [72] However today, it can be applied to combinations of several chromatographic techniques, for example one for preparative purposes and another for separation purposes, a combination of an extraction procedure and a chromatographic separation or a chromatographic separation coupled with several detection techniques. The main advantage of hyphenation is that it provides the means to solve complex analytical problems. Additionally, combined techniques are often easier to automate, thus they save time.

2.9.1 HPLC hyphenated with ICP The successful combination of HPLC and ICP presents several advantages. ICP is a highly sensitive technique, although in some cases not selective enough. It would provide a signal for any phosphorus-containing molecule, but would not be able to distinguish organic and inorganic phosphorus. HPLC with the most common UV-detector can identify some phosphorus compounds, but not all. HPLC can, however, separate phosphorus-containing molecules based on size, charge or some other property. A suc-cessful separation would in effect differentiate one sample into parts which are separated from one another in time. Using a continuous signal from the ICP those parts can be measured and compared to each other. This concept was tested in Paper III where the hyphenation helped in the quantification of the components in an in-house standard of InsPn. The standard was prepared from the potassium salt of InsP6 and contained a mixture of the InsPn isomers with unknown concentrations. With the help fo HPLC the components were separated in time, while ICP-AES provided the means of measuring the signal coming from each component. Figure 12 shows a chromatogram of an InsPn standard, obtained by HPLC-ICP-AES.

On its own, this chromatogram provides limited information about the components in the sample. Although several peaks can be observed, the signal is rather noisy and the smaller components may not be distinguishable from the noise. Additionally, overlapping peaks appear as one peak and cannot be used for quantification purposes. Some quantative information can nevertheless be obtained. It can be assumed that the ICP provides uniform response for the different phosphorus forms, because in essence it measures the same thing, i.e. total phosphorus concentration for each component. The size of the different peaks and their relative proportion compared to the total area can be determined through integration. However, only a combination of

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different techniques can provide the quantitative information required for the complete characterization of the standard (see end of section 2.9.2).

Figure 12. Phosphorus signal of an in-house inositol phosphate standard, detected with HPLC-ICP-AES.

2.9.2 HPLC hyphenated with MS In order to overcome the limitations presented by the separation and detec-tion techniques on their own, they can be combined. The advantages of hy-phenating HPLC with MS include convenient sample handling, including automation, faster analysis time and reduced possibility of sample loss. Ad-ditionally, structural information pertaining to the different analytes can help distinguish different components.

At the same time, the hyphenating presents challenges, which are due to the inherent difference of the two techniques, where HPLC is liquid based and MS is not. The analytes which are commonly separated with HPLC are likely to be involatile, which makes them hard to transform into gas phase. The ways to overcome this challenge is to use volatile additives and organic modifiers in the mobile phase. Moreover, the eluent from the chroma-tographic column comes also in a liquid form and needs to be brought into gas phase so that it can be introduced into the mass spectrometer.

Electrospray ionization (ESI) enables the combination of HPLC and MS by providing an interface where a liquid sample can be transformed into gas phase. To produce ESI, a strong electric field is applied to a liquid passing through a capillary, dispersing it into small charged droplets, which trans-form into solvated gas phase ions. [73, 74] The sample is introduced into the MS with a help of a capillary, often made of stainless steel, which can be connected to the outlet of the HPLC system. An electric field is generated

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between the tip of the capillary and a counter electrode, thus ionizing the liquid. The ESI interface has an atmospheric-pressure region in which the desolvation of the liquid stream is assisted by a drying gas. The charged droplets shrink while the solvent evaporates and drift towards the counter electrode. Consequently, solvated gas-phase ions are produced (Figure 13).

Ideally the interface should not reduce the chromatographic performance of the system, so considerations need to be made identifying the optimal condi-tions for the hyphenation. The system can be operated in negative or positive mode, the potential can be decreased or increased, the desolvation can be assisted by heating and the flow of gas can be regulated. Paper III partially deals with the optimization of the parameters for the successful separation and detection of IPs in sediment extracts using HPLC-ESI-MS/MS.

Figure 13. Formation of solvated gas phase ions in the electrospray ionization (ESI) interface, presented here in negative mode.

Although HPLC-ESI-MS/MS provides good selectivity, when unknown compounds are studied quantification may not be possible. This is due to the fact that the relative response in the ESI is compound dependent, and cannot always be predicted. Using combination of different detection techniques different type of information can be obtained, and a combination of tech-niques may help both identify the component and quantify them. The in-house standard used in Paper III could not be completely characterized by only using HPLC-ICP-AES. However, when combined with HPLC-ESI-MS/MS that became possible. The different components and their elution order were identified with HPLC-ESI-MS/MS (Figure 14). The relative size of the different components in the standard was calculated by integrating the peaks obtained with HPLC-ICP-AES. That information was used to calcu-

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late the response factors for the different InsPn forms in the ESI-MS/MS. Subsequently calibration curves for each InsPn could be constructed.

Figure 14. Chromatogram of an in-house inositol phosphate standard, detected with HPLC-ESI-MS/MS.

It is important to note that the in-house standard prepared did not contain large amounts of InsP1 and InsP2, and it was not possible to obtain a high enough signal with ICP-AES in order to assess their relative contribution in the standard. However, their response factors could nevertheless be calcu-lated based on the response factors which were obtained for the higher order InsPn (Table 1). The response factor was inversely proportional to the num-ber of phosphate groups in the respective InsPn (Figure 15).

Table 1. ESI-MS/MS response fac-tors for inositol phosphates ranging from 0.3 to 46 µM InsPn. The re-sponse factors for InsP1 and InsP2 are interpolated.

Figure 15. Response factors for inositol phosphates in the in-house standards. The grey markers are the interpolated values for InsP1 and InsP2.

Analyte Response factor

InsP1 152.0

InsP2 125.5

InsP3 102.4

InsP4 68.6

InsP5 43.6

InsP6 22.4

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This example clearly demonstrates the advantage of combining different hyphenated techniques to solve complex analytical problems.

It has been acknowledged that the combination of HPLC and MS offers a promising new way of routinely analyzing different forms of organic phos-phorus in environmental samples. [28, 75, 76] Compared to e.g. 31P NMR, MS is much more sensitive, and when used in MS/MS mode offers out-standing selectivity. In Paper IV the method developed in Paper III was applied to the study of different InsPn forms in a small catchment, where a range of environmentally relevant samples, such as soil, sediment and ma-nure, was examined.

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3 Applications

Analytical chemistry plays an important role in the search for new identifica-tion and determination methods. Testing the developed methods in real life systems can provide valuable insights into the distribution of organic phos-phorus in the environment. The methods presented in the thesis demonstrate that in order to develop a complete picture of the distribution of phosphorus in the environment there is an urgent need for new methods.

Organic phosphorus accounts for 30 – 65 % of TP in most soils, although there are soils with very low and very high organic phosphorus content. [21, 77] Organic phosphorus is present in the soil in many different forms, for example in plants, animals and microbes and is taken up and returned to the soil in plant residues and animal manure.

Mineralization is the process of decomposition of organic matter, during which organic phosphorus is oxidized and becomes available to plants in phosphate form. The opposite process is called immobilization, during which inorganic phosphorus is taken up by plant roots and converted to organic compounds by microorganisms. The mineralization of organic phosphorus is influenced by its chemical properties.

The bioavailability of phosphorus to aquatic organisms is directly linked to the mineralization of organic phosphorus to inorganic phosphorus, [78] where bacteria has been linked to the desorption and adsorption mechanisms of phosphorus in the sediments. [79] Microorganisms can release phospho-rus from their cells, acting as a source of phosphorus, or immobilize phos-phorus, acting as a phosphorus sink. [80] The immobilized phosphorus is incorporated into different biological components within the organism. These components are the source of the available organic phosphorus when the organisms die. The mineralization process is dependent on the chemical properties of the organic material, which means that the time it takes for a compound to degrade and the end result may differ as some compounds may degrade completely and some may not. Theoretically this gives the basis for hypothesizing why some compounds may contribute more to eutrophication. The dynamics of organic phosphorus compounds in soils and sediments are still poorly understood because there is little knowledge in regards to their

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sorption to mineral particles. In soil, IPs adsorb to minerals and form rela-tively stable complexes which shield them from microbial degradation. [26] The adsorption capacity of DNA and PLs is lower than that of IPs, which contributes to their rapid degradation by phosphatases enzymes. [26] Yet it has been shown that DNA can be adsorbed to mineral surfaces and thus be-come shielded from enzymatic degradation. At the same time, soil properties can influence the adsorption as it has been shown that DNA adsorption de-creases with increasing pH. [81] Many types of sediments have neutral or slightly basic pH, which entails decreased adsorption of DNA due to repul-sive electrostatic interaction. The adsorption capacity of orthophosphate monoesters is dependent on the number of phosphate groups associated with the molecule, meaning that an IP with fewer phosphate groups would have lower sorption capacity than an IP with many phosphate groups. [26]

There are many different biological, chemical and physical factors that affect the turnover of different phosphorus compounds in the environment [21] and it is likely that under different conditions the mineralization and mobilization of phosphorus will be influenced in different ways. [82] It has been sug-gested that PLs in sediments may become bioavailable because of the pres-ence of phospholipase [20] and DNA is degraded by microbial DNase. [83, 84] It is reasonable to believe that the same group of compounds may de-grade faster in a suitable environment, and be a part of a temporary pool of phosphorus available to plants and microorganisms, while under different, less favorable conditions accumulate, creating a refractory pool of phospho-rus. A good way of exploring degradation rates is to study lake sediments, since they accumulate continuously. Sediment cores represent a repository of information on accumulated matter, both inorganic and organic. Exploring how different compounds behave with depth can give us answers about deg-radation rates. We know, for example, that organic matter settles at the bot-tom of the lake and is decomposed. A fraction of it is degraded very fast, on a time scale of days to weeks. However, a part of it may decompose so slowly that it becomes refractory on a time scale of hundred years. [79] DNA and PLs are essentially a part of the organic material, so it is reason-able to assume that some of the non-decomposed organic material in the sediment is also a source of DNA-P and PL-P and under the same premises, some of it will be temporary and some of it will be refractory. Additionally, it has been postulated that the mechanisms involved in the transformations of organic phosphorus to inorganic phosphorus and vice versa are different for oligotrophic and eutrophic lakes. In eutrophic lakes where there is excess of phosphate, phosphorus may be stored in bacteria until released under the right conditions or during hydrolysis, leading to higher proportion of tempo-rary phosphorus. In oligotrophic lakes a larger portion of phosphorus can be incorporated into not yet degraded organic material and refractory com-pounds. Bacteria, therefore, needs to compete for phosphorus and cannot

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store any excess. That in turn results in higher proportion of refractory phos-phorus. [85] Paper II, which was intended as an application of the method developed in Paper I deals with the degradation of DNA-P and PL-P in lake sediments.

The main conclusions of Paper II were that the two orthophosphate diester fractions are recycled in the sediment and that they represent a source of bioavailable organic phosphorus in aquatic systems. The study showed that the degradation of DNA-P and PL-P appears to be very different. As can be seen in figure 16 the distribution of both organic phosphorus fractions vary with depth not only depending on the compound, but also depending on the lake. In the mesotrophic Lake Erken the amounts of the two organic phos-phorus compounds are much higher than in the oligotrophic Lake Ånnsjön. Whereas the degradation of DNA-P and PL-P in Lake Ånnsjön is concen-trated in the top 5 cm of the sediment and then levels off to a steady value, in Lake Erken the degradation continues throughout the whole sediment core studied. Interestingly, the degradation of DNA appears to be faster even though both DNA and phospholipids are both diesters and the phosphate has the same type of chemical bonding. Despite the DNA’s complexity phos-phate derived from DNA appears to mineralize faster than phosphate derived from phospholipids.

Figure 16. Distribution of phospholipid-P (open markers) and DNA-P (solid mark-ers) in Lake Erken (left) and Lake Ånnsjön (right).

The development of a new method for phosphorus derived from DNA and PLs revealed a trend in the distribution pattern of these two diester phospho-rus compounds, which could not be achieved before with other methods, specifically with 31P NMR, the most common organic phosphorus determi-nation method used today. It supports the idea that that even though a com-mon and exhaustive extraction procedure for all organic phosphorus com-pounds is desirable, at present it is not possible. Additionally it shows that organic phosphorus is quantitatively a very important fraction of bioavail-

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able phosphorus in aquatic systems, and specifically DNA-P needs to be considered when internal loading is addressed.

There is contradictory evidence to the bioavailability of IPs in both aquatic and terrestrial systems. [86] On one hand IPs have been shown to accumu-late in sediments [87] and soils [88] suggesting that they are refractory. The immobilization of IPs in sediments through binding to mineral particles or in the water through the formation of complexes prevents them from being metabolized by microorganisms. [36, 37] On the other hand rapid degrada-tion of IPs under certain conditions suggests that they may become bioavail-able. A large difference has been observed between marine and estuarine sediments, where measured concentrations of IPs could have a tenfold dif-ference [86]. This could be due to the different transformation processes of IPs in different environments. The synthesis of phosphatases by different microorganisms is one pathway for IP degradation. Additionally, changes in solubility or anaerobic processes may have the effect on the degradability of IPs. For example, complete hydrolysis of InsP6 was achieved under anaero-bic conditions in a laboratory [89] and may be a likely explanation for the increased bioavailability of IPs in some aquatic environments.

The method developed in Paper III provided a new and faster way to exam-ine the distribution of InsPn in sediment extracts and was applied in Paper IV to study a range of different samples in a small catchment. The greatest challenge in the direct application of the method was dealing with different types of sample matrices. As suggested in other studies, we expected to con-firm that the IPs accumulated in soils were mainly InsP5 and InsP6. [90] Sur-prisingly, unexpectedly high signals for InsP1 were obtained in most of the samples, and in the bay sediment those were the dominant signals. Since the in-house standards used for the quantification of InsPn had low amounts of InsP1 and the response factor could only be calculated theoretically, analyti-cal quantification of InsP1 could not be performed. However, this finding may be very important and needs to be investigated further. It may be espe-cially significant for the samples collected from the bay sediment, where the total IP-P concentration may be very high, but cannot be attributed to InsP6. The quantification of InsP6 in all the other samples was on the other hand possible and was used as a marker for IP-P within the catchment.

The main goal of Paper IV, besides testing the IP-P method on a variety of samples, was to become the pilot study in the mapping of the speciation of organic phosphorus in a small catchment. The methods for the determination of DNA-P, PL-P, InsP6-P were combined for the first time in order to pro-vide insight into the distribution of these organic phosphorus fractions. Dif-ferent types of environmentally relevant samples have been included in the study, such as soil from agricultural fields, forest and pasture, manure and

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sediment from a ditch and a small forest creek (Table 2, Figure 17). Addi-tionally a sediment profile was collected from a semi-enclosed bay into which the catchment area empties. The bay is a part of the Baltic Sea and represents an area which is highly eutrophied.

Table 2. Properties of the samples included in the catchment study.

Sample site Sample type

Sample site characteristic

1 Soil Agricultural field 2 Soil Limed agricultural field 3 Soil Pasture field 4 Soil Forest 5 Sediment Ditch 6 Sediment Forest creek 7 Manure Horse manure 8 Sediment Bay sediment

Figure 17. Positioning of the sampling sites within the studied catchment.

The results showed that DNA-P, PL-P and InsP6-P represent a significant part of the total phosphorus content in all the samples. The PL-P was the smallest organic phosphorus fraction, accounting, on average, for 2% of the total phosphorus in the soils and 5% of the total phosphorus in the sedi-

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ments. A notable exception was the top layers of the bay sediment where the PL-P was close to 10% of the total phosphorus content. On average DNA-P accounted for 5% of the total phosphorus content in the soils and for just over 8% of the total phosphorus content in the sediments. However, the top sediments combined, averaged a striking 19% of the total phosphorus, indi-cating a major sink. This was not unexpected since DNA is found in both living and dead organic matter, and the water-sediment interface most likely provides extremely favorable conditions for bacterial growth. As mentioned previously the total IP-P content in the different samples could not yet be assessed, however, the InsP6-P was determined in all, but the bay sediment samples. The largest proportion of InsP6-P was found in the soils, although it was twice as high in the two soils not used for agricultural purposes. The ditch which was used to drain the agricultural fields had an almost identical InsP6-P content as the soil of those fields, while the forest creek had the same InsP6-P content as the adjacent forest soil. This interesting result may serve as an indication that the accumulation of InsP6-P in aquatic systems is closely related to the land use of the adjacent terrestrial systems. The major conclusion of Paper IV is that the three organic phosphorus compounds studied constitute a substantial part of the total phosphorus found in the dif-ferent parts of the catchment and therefore need to be addressed separately when developing mitigation strategies for dealing with eutrophication.

In summary, this thesis has demonstrated the need for the development of new methodology for studying the distribution of organic phosphorus in the environment. Organic phosphorus compounds can be found in both aquatic and terrestrial systems, their distribution and degradation is not uniform and therefore their impact on eutrophication may differ. It is the hope of the au-thor that the methods described here will ultimately contribute to the ongo-ing work striving to alleviate our eutrophication problems.

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4 Acknowledgments

Working on this PhD has been an unforgettable experience that I will cherish for the rest of my life. It would not have been possible if not for my supervi-sors Per Sjöberg and Emil Rydin. Thank you, above all, for giving me the opportunity to embark on this amazing journey, but also for your curiosity, questions and enthusiasm. I have learned so much and most importantly, grown both as a person and as a scientist.

Jean Pettersson, you are a terrific person to work with! I wish I had you as my supervisor as well. Thank you for all the help with running the ICP, but most importantly for being an open encyclopedia of everything worth know-ing and for gladly sharing it with me. Rolf Danielsson, thank you for your patience and positive attitude towards my limited statistical capabilities, for your help with experimental design and for making sense of my random errors. My Danish colleagues and co-authors Charlotte Jørgensen and Kasper Reitzel, it’s been a pleasure working with you. I sincerely hope to continue our collaborations in the future. Besides, we’ll always have Pa-nama! Bo Ek, thank you for helping me in the lab, I am really glad the pro-ject will live on with you!

This endeavor would have never succeeded if not for all the doctors who’ve preceded me, exemplifying the fact that “PhDs are nice” and that I “should get one” (in alphabetical order): dr. H. Cederlund, dr. J. Dernfalk, dr. L. Edström, dr. A. Friberg, dr. C. Fuller, dr. D. Fuchs, dr. A. Hermansson, dr. T. Lauer, dr. D. Lundberg, dr. A. Płaczek, dr. J. van Schaik, dr. N. Torapava and dr. K. Önneby. Thank you for leading by example! I’ve tried not to make the same mistakes you have.

I am eternally grateful to the founding members of the Journal Club for brilliant scientific discussions that have spurred publications.

My phosphorus friends Annika Svanbäck, Helena Andersson, Pia Kynkäänniemi at SLU, and Karin Johannesson at Linköping University, I have no idea how this thesis work would have turned out without you! Thank you for all the amazing discussions, for supplying me with soil sam-ples and for taking me to your field studies. It’s always been great fun!

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My roomies at the office who made the uncountable hours spent in front of the computer bearable: Anne-Li for always making me feel special, for be-ing my best lab partner and life coach ever and for your magic hands, Sra-vani, for being so cheerful and for sharing your marvelous recipes with me, Jörg and Marcus (Mark II), for your undying love of sarcasm. You’ve cer-tainly made life at the office entertaining! My other roomies: Ping, Micke and Torgny, thanks for letting me hang out in your office when mine felt lonely! Ping, you have been a wonderful friend. I’m so happy you thought gardening was a high class hobby and joined me in the field. Micke, thank you for always coming to my rescue in the mass lab. Torgny, you are possi-bly the funniest and friendliest colleague I’ve ever had. The department is lost without you, and I really missed you when you took a year off. Thanks for getting me addicted to running!

My favorite bachelor student Mieke, thank you for your remarkable attitude and dedication, for (almost) not breaking anything, for making me smile and for providing insights on life, family, Belgian beer and Mexican cooking. You’ve been such a good sport!

Fredrika, thank you for coming to all my presentations, your desire to un-derstand what I do has taught me to be more pedagogical. In the office, your support is invaluable because you are great at what you do!

The sport mafia: Torgny, Eva, Per and Micke with guest appearances of Malin, thank you for making and keeping me happy and healthy by engag-ing, competing and participating in a multitude of physical activities such as lifting heavy things, ping pong, biking, running, spraybar hanging and sushi eating. Completing Blodomloppet with the amazing blue team was one of the highlights of 2012! Eva, I hope you take over as a personal trainer and moti-vator after me .

The department of Analytical chemistry is an amazing work place thanks to all my past and present colleagues. Each and every one of you has contrib-uted to this being a fantastic experience for me. Jonas, your words of assur-ance and sound advice have always meant a lot! I should have listened to your more! Marit, thank you for your genuine interest in education! You are a strong leader who says the things that need to be said even if they do not want to be heard, you are an inspiration! Konstantin and Anna, thank you for practicing languages with me. Our multilingual talks keep my mind alert. Kumari, your contagious smile and kind words make it fun being in the lab! Erik, thanks for always lending a helping hand and for being great company on very early mornings. Lena, our philosophical discussions about science and life in general meant the world to me, thank you for being a great friend!

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The PhD and junior research gang, I hope you all know that this place would be lost without you!

The Green group that, regrettably for me, moved to Lund. Thank you Irene, for helping me with everything when I just started and Arwa, for teaching me how to write fantastic lab notes. Jiaying, my long-lost science twin, you rocked my world! I never thought I could have so much in com-mon with anyone else.

The long-term visitors, Björn and Sofie, who shared our lab and fikaroom, thank you for brightening up my day. It was always great fun talking to you, Björn, even if it was about bikes. You are still unmatched in your role as quizmaster! Sofie, your passion for reading is contagious. I cannot describe how much it meant to me to that you supplied me with the greatest stress-relief - books!

My Uppsala family, especially my best friends Johanna and Alexandra, my life would be so boring without you! You’ve always listened, comforted and encouraged my many endeavors. Thank you for putting up with me and let-ting me be myself. Daniel, your enthusiasm towards research and attitude to life has been incredibly inspiring! If it wasn’t for you I doubt that I would have studied chemistry at all. Harald, thank you for your great company, all the non-chemistry science talks and for getting me hooked on the 50mm. Katarina, thank you for being an inspiration in everything artistic and imaginative and when it comes to the most important things in life ♥!

Candy, I’ve looked up to you ever since we’ve met and can honestly say that I dared to take on the scientific world because of you!

Claudia, my kindred soul, you have the amazing ability to make everything wonderful. You attitude towards life is remarkable and knowing you makes me a better person.

My family, especially my sister Ellie, thank you for encouraging my need for life-long learning. You were the one who always believed in me. I would not be here otherwise! Tomas, my soul mate, thank you for the countless hours you spent discussing with me everything and nothing. I owe my sanity and peace of mind to you.

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5 Svensk sammanfattning

Målet med denna avhandling är att utveckla nya metoder för att identifiera och mäta de huvudsakliga formerna av organiskt bundet fosfor i olika miljö-prover.

Den okontrollerade läckagen av fosfor är en av de främsta orsakerna till övergödning och därmed ett av våra största miljöproblem. Övergödning på-verkar miljön på många olika sätt, men syns framför allt i akvatiska system där den ger upphov till algblomning, förändrad ekobalans och försämrad vattenkvalitet. Övergödning leder till syrebrist i vattnet och bottensedimen-tet, vilket gör att vissa djurarter dör. Att sjöarna växer igen är delvis en na-turlig process som dessvärre har påskyndats av människans påverkan. Ge-nom t.ex. jordbruk släpps stora mängder fosfor ut i miljön. För att minska övergödningen krävs det kunskap om vilka fosforföreningar som finns i oli-ka mark- och sedimenttyper, vilka av dessa som är tillräckligt stabila för att inte påverka miljön på ett negativt sätt och vilka som bryts ner och därmed görs biotillgängliga och bidrar till övergödningen. Tidigare trodde man att det var oorganiska fosforföreningar, huvudsakligen mineralfosfater, som orsakade övergödning, men numera anses det att även organiskt bundet fos-for kan utgöra en ansenlig del av den totala fosforomsättningen, samt att organisk fosfor kan vara minst lika biotillgänglig som den oorganiska.

En svårighet i samband med bestämningen av halten organisk fosfor i oli-ka prover beror på det faktum att det för närvarande inte finns några direkta mätmetoder. Mängden organisk fosfor uppskattas därför oftast indirekt, som skillnaden mellan den totala och den oorganiska fosforhalten. Utöver denna grova uppskattning är det även en utmaning att identifiera vilka fosforföre-ningar som är relevanta. Det vi vet idag om organisk fosfor är att det finns många olika typer av organiska fosforföreningar och att de finns i det när-maste överallt. De dominerande grupperna är olika organiska molekyler, t.ex. DNA och RNA som utgör levande organismers genetiska material, fosfolipider som finns i cellväggar, samt inositolfosfater, som spelar en av-görande roll i olika cellulära funktioner. Hur mycket fosfor som finns bun-den i de olika molekylerna beror på antalet fosfatgrupper som finns i respek-tive molekyl.

Mikroorganismer har utvecklat olika mekanismer för att tillgodogöra sig fosfatet som finns både i löst form i vatten, i fast form i jord eller sediment, men även den som finns bunden i organiska molekyler. När mikroorganis-mer bryter ner organiskt material utvinner de energi för sin biomasseproduk-

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tion och när en molekyl brutits ner blir den biotillgänglig, d.v.s. andra orga-nismer kan tillgodogöra sig den. Under nedbrytnings-processen sönderdelas det organiska materialet till allt mindre moleklyer och frigör näringsämnen, däribland fosfatet. Beroende på molekylernas kemiska sammansättning kan både nedbrytningshastigheten och slutresultatet variera då vissa enkla mole-kyler bryts ner snabbt och fullständigt, medan stora och komplicerade mole-kyler endast bryts ner delvis under en mycket längre nedbrytningstid. Detta gör att olika fosforinnehållande molekyler kan ha olika betydelse för över-gödningen, vilket ökar behovet av att studera dem var och en för sig.

Traditionellt har fosfor studerats genom stegvisa extraktioner som har de-lat upp den totala halten fosfor från ett prov i olika grupper. Med hjälp av ett antal sura och basiska extraktionslösningar delade man upp ett prov i grup-per med olika egenskaper. Extraktionslösningarna har ökande styrka och därför kan man förenkla den komplicerade sammansättningen genom att definiera lättlösliga och svårlösliga fosforföreningar. När man undersöker biotillgänglighet anses det nämligen ofta att ju mer lättlöslig en förening är desto mer biotillgänglig är den. Nackdelen med sådana sekventiella extrak-tioner är dock att man inte kan särskilja organiska från oorganiska fosforfö-reningar.

En alternativ teknik som har använts mycket de senaste två decennierna är s.k. kärnmagnetisk resonans, NMR. Denna teknik är möjlig tack vare fosfor-atomens egenskaper. Det är en icke-destruktiv teknik, d.v.s. man kan mäta på samma prov många gånger utan att förbruka provet och särskilja mellan minst sju olika klasser av organiska fosforföreningar samtidigt. Tekniken är dock inte känslig nog för att identifiera varje förening var för sig, i synnerhet om halterna är väldigt låga.

I början på 1960-talet utvecklade Murphy och Riley en bestämningsmetod för oorganisk fosfor. Metoden skapades för att mäta fosforhalten i vatten, men har använts i stor utsträckning även på extrakt från jordar, komposter, sediment och andra miljöprover. Ofta kallas den för molybdenblåttmetoden eftersom mättekniken går ut på att mäta ett blått komplex som bildas då den fria fosfaten i ett prov reagerar med molybdatjoner. Dessa kan detekteras med ett optiskt mätinstrument, en s.k. spektrofotometer, som med hjälp av ljusabsorbansskillnader identifierar olika ämnens halt i lösningar. Fördelarna med molybdenblåttmetoden är att den är billig, lätt att utföra och har god känslighet och låga detektionsgränser. I de flesta lösningar kan man omedel-bart se om man har fosfat, eftersom lösningen får en blå färg, och ju blåare provet är, detso mer fosfat finns det i det. Nackdelen med metoden är att den mäter i princip bara fria fosfater, men eftersom de intressanta organiska fos-forföreningarna innehåller fosfat, kan man faktiskt mäta dem med molyb-denblåttmetoden om man först lossar fosfatgrupperna från molekylerna.

I arbete I av denna avhandling skapades en ny metod för mätning av fos-for som härstammar från DNA och fosfolipider, betecknat DNA-P respekti-ve fosfolipid-P. Fokus har lagts på att utveckla provberedningsmetoder för

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att selektivt extrahera DNA och fosfolipider ur jord-, sediment- och kompostprover. Den viktigaste delen av metodutvecklingen var att försäkra sig om att andra fosforinnehållande föreningar hade renats bort innan analy-sen utfördes. Då de isolerade fraktionerna innehöll fosfor från en enda källa, antingen DNA eller fosfolipider, kunde man efter att hydrolysera provet mäta fosforhalten med den vanliga molybdenblåttmetoden. Hydrolys medför att bindingarna mellan moleklyerna bryts ner vilket gör att fosfaten inte läng-re är bunden i moleklyen.

Målet med analytisk kemi är att uveckla metoder som kan hjälpa oss lösa verkliga problem, d.v.s. en ny metod måste vara praktiskt användbar. För att demonstrera att DNA-P och foslfolipid-P metoden utvecklad i arbete I fun-gerade användes den i arbete II för att undersöka sediment i två svenska sjöar. Målet var att genom kartläggning av förändringen av de två organiska fosforföreningarnas halt med djupet kunna avgöra om DNA-P eller fosfoli-pid-P kunde omsättas. De två sjöarna som undersöktes var Ånnsjön, en när-ingsfattig sjö i norra Jämtland, och Erken, en näringsrik sjö i Uppland. Sjö-arna valdes till detta projekt eftersom de befinner sig i ett jämviktstillstånd vad gäller den externa tillförseln av näringsämnen och därför avspeglar se-dimentet vad som händer med fosforn med tiden. Projektet visade att det fanns tydliga trender i omsättningen av DNA-P och fosfolipid-P med avse-ende på djupet. I de övre sedimentlagren fanns det mycket mer fosfor bun-den till DNA och fosfolipider än i de nedre sedimentlagren, vilket betyder att dessa två föreningar bryts ner med tiden och avges till vattnet. Dessutom kunde det visas att DNA-P omsätts snabbare än fosfolipid-P. Detta var spe-ciellt viktigt för den näringsrika sjön Erken, där den snabba omsättningen av DNA-P kunde anses ge ett avsevärt bidrag till övergödning.

I arbete III skapades en ny metod för att identifiera och mäta inositolfos-fater i ett sedimentprov. Som i de föregående arbeten extraherades först se-dimentet för att få ut inositolerna i lösning. Dock är inositoler mycket stabila molekyler och det anses vara mycket svårt att hydrolysera dem. Därför kun-de inte en liknande metod som i arbete I användas. En annan utmaning med arbetet var att separera och detektera inositoler, eftersom det finns många olika varianter. En inositolfosfat är en ringformad molekyl som kan innehål-la från en till sex fosfatgrupper. Dessutom kan formen på ringen variera och ge upphov till flera olika varianter med samma antal fosfatgrupper. Ytterli-gare en svårighet med inositolfosfater är att de är svåra att detektera spektro-fotometriskt. Vid mätning med masspektrometri kan man skilja de olika varianterna av inositolfosfater genom att mäta förhållandet mellan deras massa och laddning, men för att kunna skilja dem åt i ett prov måste man först separera dem från varandra. Därför användes s. k. högupplösande väts-kekromatografi, HPLC, för att först separera de olika inositolerna från var-andra. Med hjälp av den nya metoden kunde inositoler som innehåller olika antal fosfatgrupper skiljas åt, men metoden var inte känslig nog för att skilja de olika ringformerna åt. Alla dessa former kunde dock påvisas i sediment-

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provet, och man kunde beräkna halten av den inositol som har sex fosfat-grupper, inositolhexakisfosfat (InsP6). Den anses dessutom vara den mest förekommande inositolfosfaten i miljöprover.

I arbete IV kombinerades de nya metoderna för DNA-P, fosfolipid-P och InsP6-P för att studera vilka organiska fosforformer som förekom i olika mark- och sedimenttyper i ett mindre avrinningsområde. Prover togs från jordbruks-, skog- och betesmark, hästgödsel, samt sediment från ett dike och en bäck. Eftersom avrinningsområdet mynnar ut i en vik i Östersjön studera-des även en sedimentprofil från viken. Resultaten visade att DNA-P, fosfoli-pid-P och InsP6-P utgörde en betydande del av det totala fosforinnehållet i alla prover. Nästan en tredjedel av all fosfor var bunden i de tre formerna i skogsbäcken och i det översta sedimentlagret i viken, vilket tyder på att or-ganisk fosfor transporteras effektivt med vatten och ackumuleras i sedimen-ten. Fosfolipid-P var den minsta fraktionen i alla prover, vilket säger oss att trots att fosfolipider finns överallt är deras bidrag till övergödningen troligen liten. DNA-P utgjorde den största andelen organisk fosfor i alla typer av sediment. Detta kanske inte är oväntat eftersom DNA finns i både levande och dött organiskt material. Den största andelen InsP6-P hittades däremot i jordarna. Detta tyder på att inositolfosfater är stabila föreningar som inte lossnar så lätt från jordpartiklarna. Samtidigt kunde man inte mäta InsP6-P i sedimentet från viken, trots att inositolfosfater borde finnas även där. För-klaringen till detta kan vara att det är bräckt vatten i viken och att det därför var svårt att tillämpa mätmetoden på sedimentproverna som hade högre salt-halt. En annan möjlig förklaring är att andra inositolfosfatformer var mer förekommande i just sediment. Av alla jordar fanns det mest organisk fosfor i hästhagen, men trots att det fanns mest fosfor i gödslet var det bara en tion-del som bestod av fosfor från de tre organiska föreningarna.

Sammanfattningsvis har denna avhandling visat att det behövs nya meto-der för att kunna studera förekomsten av organisk fosfor i miljön. Fosfor bunden i DNA, fosfolipider och inositolfosfater förflyttas, i vissa fall lätt, till akvatiska system, där det antingen kan ansamlas eller brytas ner och därmed bidra till oönskad övergödning. De metoder som tagits fram här, visar att det är möjligt att kvantifiera betydligt fler fosforinnehållande föreningar än man tidigare trodde var möjligt, vilket är ett steg i rätt riktning för att komma tillrätta med ett av vår tids största miljöproblem.

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6 Краткое содержание

Целью данной диссертации является разработка новых методов определения и измерения органически связанного фосфора в природных образцах.

Бесконтрольное распространение фосфора является одной из основных причин эвтрофикации, то есть насыщения водоемов веществами, которые способствуют росту биомассы. Эвтрофикация представляет серьезную экологическую угрозу, поскольку ее влияение на водоёмы вызывает цветение воды, дефицит кислорода, замор рыбы и ухудшение качества воды. Частичное зарастание озер – естественный процесс, который, к сожалению, ускоряется в результате деятельности человека. В частности, ведение сельского хозяйства приводит к попаданию больших количеств фосфора в окружающую среду. Для уменьшения последствий эвтрофикации необходимо знать, какие соединения фосфора присутствуют в разных почвах и донных отложениях, какие из них являются стабильными и не оказывают негативного влияния на окружающую среду, а какие легко разлагаются и способствуют эвтрофикации. Ранее считалось, что неорганические соединения фосфора, в основном минеральные фосфаты, являются основной причиной эвтрофикации. Однако в настоящее время доказано, что и органический фосфор может быть биодоступным.

Сложности изучения органически связанного фосфора обусловлены тем, что существует множество его разновидностей. В природе органический фосфор встречается в первую очередь в молекулах ДНК и РНК, которые служат основой генетического материала всех живых организмов, а также в фосфолипидах, составляющих клеточную оболочку, и инозитолфосфатах, запускающих множество биохимических реакций в клетке. Различные типы органического фосфора разлагаются по-разному и с разными скоростями, что в свою очередь обусловливает их различный вклад в эвтрофикацию.

В данной диссертации были разработаны два новых метода для количественного определения органического фосфора, входящего в состав (1) ДНК и фосфолипидов и (2) инозитолфосфатов. Первый метод был использован для изучения распределения органического фосфора, содержащегося в фосфолипидах и ДНК, в донных отложениях двух озер. Озеро Оншён в северной Швеции является олиготрофным, то есть содержит мало питательных элементов; озеро

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Эркен в центральной Швеции, напротив, эвтрофное, то есть богато питательными веществами. Разработанный метод (1) позволил установить, что количество органического фосфора, содержащегося в фосфолипидах и ДНК, уменьшается с глубиной донного осадка, то есть со временем происходит разложение. Важно отметить, что это справедливо для обоих озер.Также было установлено, что разложение ДНК происходит быстрее, чем разложение фосфолипидов, то есть фосфор из ДНК вносит больший вклад в эвтрофикацию.

Наряду с методом (1), метод (2) был использован в данной работе для картирования всех трех вышеописанных типов органического фосфора на територии водосбора небольших размеров. Образцы были отобраны из почвенного слоя леса, а также поля и пастбища, донных отложений дренажного рва и, наконец, залива Балтийского моря, являющегося стоком данного водосбора. Проведенное картирование выявило, что количество фосфора, связанного в фосфолипиды ограничено, максимальное количество фосфора, связанного в ДНК, находится во всех донных отложениях, тогда как фосфор, связанный в инозитолфосфаты, наиболее распространен в почвах. Стоит отметить, что вышеописанные три типа органического фосфора составляли до 30% от общего содержания всего фосфора.

Результаты данной работы демонстрируют необходимость тщательно учитывать органический фосфор при исследовании эвтрофикации. Разработанные методы определения различных типов органического фосфора могут быть полезными в дальнейшем изучении загрязнений водоемов и преодолении последствий эвтрофикации.

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