Organic Phosphorus Compounds in Aquatic Sediments172647/FULLTEXT01.pdfaquatic sediments [4-7] but...

ACTA

UNIVERSITATIS

UPSALIENSIS

UPPSALA

2008

Digital Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 560

Organic Phosphorus Compoundsin Aquatic Sediments

Towards Molecular Identification with MassSpectrometry

HEIDI DE BRABANDERE

ISSN 1651-6214ISBN 978-91-554-7306-8urn:nbn:se:uu:diva-9319

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Just keep swimming… Dory

List of Papers

The thesis is based on the following papers, which are referred to in the text by their Roman numerals: I. Sediment phosphorus extractants for 31P-NMR analysis; a quantitative evaluation. Joakim Ahlgren, Heidi De Brabandere, Kasper Reitzel, Emil Rydin, Adolf Gogoll & Monica Waldebäck. Journal of Environmental Quality. 2007. 36: 892-898. II. Sediment extraction and clean-up for organic phosphorus analysis by electrospray ionisation tandem mass spectrometry. Heidi De Brabandere, Per J.R. Sjöberg, Rolf Danielsson, Joakim Ahlgren, Emil Rydin & Monica Waldebäck. Talanta. 2007. 74: 1175-1183. III. Screening for Organic Phosphorus Compounds in Aquatic Sediments by Liquid Chromatography Coupled to ICP-AES and ESI-MS/MS. Heidi De Brabandere, Niklas Forsgard, Lena Israelsson, Jean Petterson, Emil Rydin, Monica Waldebäck & Per J. R. Sjöberg. Analytical Chemistry. 2008. 80: 6689-6697. IV. Degradation rates of organic phosphorus in lake sediment. Kasper Reitzel, Joakim Ahlgren, Heidi De Brabandere, Monica Waldebäck, Adolf Gogoll, Lars Tranvik & Emil Rydin. Biogeochemistry. 2007. 82: 15-28. V. Organic phosphorus composition in Baltic Sea sediment: origin and degradability on a molecular level. Heidi De Brabandere, Kasper Reitzel, Monica Waldebäck, Per J.R. Sjöberg & Emil Rydin. Manuscript. Reprints made with kind permission of ASA-CSSA-SSSA, Elsevier, American Chemical Society and Springer Science and Business Media.

Author’s contribution Paper I: Participated in the planning of the study, performing the experiments and writing the article. Paper II: Planned the study together with the co-authors and had the main responsibility for writing the article. Performed all the experiments in the study. Paper III: Planned the study together with the co-authors and had the main responsibility for writing the article. Performed all the experiments in the study together with Niklas Forsgard. Paper IV: Participated in performing the experiments and writing the article. Paper V: Planned the study together with the co-authors and had the main responsibility for writing the article. Performed all the experiments in the study.

Contents

1. Role of analytical chemistry in the search for environmentally important phosphorus......................................................................................9

2. Eutrophication...........................................................................................12

3. Phosphorus................................................................................................15 3.1. Phosphorus in the environment .........................................................15

3.1.1. Phosphorus in biota....................................................................16 3.1.2. Phosphorus in products..............................................................16

3.2. Internal loading of phosphorus in aquatic systems............................16 3.3. Phosphorus compounds.....................................................................17

4. Analysis ....................................................................................................21 4.1. Sample collection ..............................................................................22 4.2. Extraction ..........................................................................................23 4.3. Sample preparation............................................................................26 4.4. Separation..........................................................................................28 4.5. Detection: a closer look.....................................................................31

4.5.1. Total phosphorus .......................................................................32 4.5.2. ICP-AES ....................................................................................32 4.5.3. 31P-NMR ....................................................................................34 4.5.4. Mass spectrometry .....................................................................37

4.5.4.1. Electrospray ionisation.......................................................38 4.5.4.2. Mass analyser.....................................................................39

5. Data analysis and result interpretation ......................................................45

6. Conclusions and future aspects .................................................................46

7. Acknowledgements...................................................................................47

8. Summary in Swedish ................................................................................49

9. References.................................................................................................52

Abbreviations

ACN AcetonitrileAMP Adenosine MonophosphateAq. AqueousATP Adenosine TriphosphateBD Bicarbonate-buffered DithioniteCID Collision-Induced DissociationDiester-P Orthophosphate diestersDNA Deoxyribonucleic AcidDNA-P DNA-associated PhosphorusEDTA Ethylenediaminetetraacetic AcidEPI Enhanced Product Ion scanESI Electrospray IonisationFT-ICR Fourier Transform Ion Cyclotron ResonanceIC Ion ChromatographyICP-AES Inductively Coupled Plasma Atomic Emission Spectroscopy IDA Information Dependent AcquisitionIP-LC Ion Pair Liquid ChromatographyMonoester-P Orthophosphate monoestersMRP Molybdenum Reactive PhosphorusMS Mass SpectrometryMS/MS Tandem Mass Spectrometrym/z Mass to charge ratioNaOH Sodium hydroxidePCA Principal Component AnalysisPGC Porous Graphitic CarbonP-lipid PhospholipidODS, C18 OctadecylsilanePLE Pressurised Liquid Extraction31P-NMR 31Phosphorus Nuclear Magnetic ResonancePoly-P PolyphosphatePyro-P PyrophosphateQ-TRAP® Quadrupole Trap RNA Ribonucleic acidrpm Rounds per minuteSEC Size Exclusion Chromatographyw/v Weight to VolumeZIC®-pHILIC Zwitterionic® Hydrophilic Interaction Liquid Chromatography

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1. Role of analytical chemistry in the search for environmentally important phosphorus

Eutrophication can cause severe algae blooms and other environmental problems affecting water quality. The importance of these problems has been recognised by the international community e.g. the Helsinki Commission (HELCOM) for the protection of the Baltic Sea or ILEC (International Lake Environment Committee) gathering information on the water quality of lakes all around the world. A general definition of eutrophication is the overenrichment of nutrients, mainly phosphorus (P) and nitrogen (N), in both terrestrial and aquatic ecosystems. However, the focus in this thesis is on eutrophication in aquatic systems. Typical problems arising from a state of aquatic eutrophication are cyanobacteria blooms and oxygen depletion in the water body.

In a number of aquatic systems (mainly in lacustrine and brackish waters) P is the limiting nutrient, so an increase in P concentration results in an increase in primary production (e.g. cyanobacteria) in the water [1]. Moreover, P compounds present in the water column can, after settling to the sediment, be buried permanently or be released back into the water column after mineralisation. The latter process, known as internal loading makes eutrophication a difficult process to reverse, since reducing external P inputs is not sufficient. Organic P compounds are known to form a substantial part of the P forms to be mineralised and released [2, 3]. Phosphorus compounds contributing to internal loading can originate from different sources on land (farmland, forests, etc.) or from primary production in the lake and can therefore be expected to have different composition and thus different degradability. It is therefore necessary to monitor these compounds accurately. Much attention has been paid to inorganic P compounds in aquatic sediments [4-7] but little to organic P compounds, even though it is well known that they play an important role in the overall P cycle [2]. The aim of this thesis was therefore to characterise sediment organic P compounds. However, their analysis is complicated by the complexity of the sediment matrix and the lack of extraction methods specific for organic P compounds, rendering it necessary to combine several analysis methods of different selectivity (analysis of specific compounds, compound groups or elements). Traditionally, the organic P pool is quantified as one single pool of compounds in a sequential extraction scheme where the main focus is on

10

inorganic P compounds [8]. In recent decades, studies focusing on organic P compounds have e.g. used 31P nuclear magnetic resonance spectroscopy (31P-NMR) to identify and quantify the P binding groups [9, 10]. It is likely that the individual P compounds contributing to one kind of P binding group have different degradability and make different contributions to internal loading. Complementary and more selective data on the structure of these organic P compounds on a molecular level can therefore give a more thorough understanding of the P turnover in the environment. The focus of this thesis was therefore on the development of an analysis method enabling mass spectrometric detection resulting in mass to charge (m/z) information on both the P compound and its fragments.

However, when proceeding from sediment to actual structural identification, it is important to consider the analytical chemistry chain as shown in Figure 1.

Figure 1. The analytical chemistry chain and examples for each step related to this thesis.

One of the major questions in analytical chemistry is whether the analysis results actually reflect the composition of the original sample or not. Each of the steps presented in Figure 1 can give rise to uncertainty but are usually necessary for analysis. The structure of this thesis is primarily based on the chain depicted, with the focus on the sample, i.e. the sediment and organic P compounds present in the sediment (Sections 2 and 3). To enable analysis of

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these organic P compounds, sediment has to be brought from the bottom of a lake or sea to the water surface with minimum alteration and therefore a section of this thesis is devoted to sampling techniques (Section 4.1). Most commonly, organic P compounds are extracted from sediments to enable analysis with solution-based detection methods. The wide variety of extraction solvents available have their own pros and cons, as regards the different detection techniques (Section 4.2). Specific sample preparation techniques are needed to obtain e.g. sufficiently high analyte concentration or to remove particles prior to detection (Section 4.3). For easier analysis of organic P compounds, it is beneficial to separate the P compounds from each other and from other matrix compounds in space and time by various separation techniques (Section 4.4). There are a number of different detection techniques that are commonly used in this area and certain of these specific detection techniques and methods were selected for use in this thesis (Section 4.5). After analysis, data have been produced that depending on the amount, will have to be analysed in order to interpret the results and draw conclusions (Section 5).

The methods used and the results obtained in Papers I-V are summarised in this thesis. The main focus in Paper I was improvement of the extraction of organic P compounds from the sediment as evaluated with 31P-NMR. With the present non-specific extractants typically used to extract a whole range of different organic P compounds, many other compounds are co-extracted. This places high demands on sample preparation, separation and the selectivity and sensitivity of the detection methods. In Paper II, a sample preparation method was developed to enable direct infusion of the sediment extract in the mass spectrometer. Moreover, a thorough statistical study was carried out to identify where P losses occurred. Paper III presents a chromatographic separation method and its potential for identification of sediment organic P compounds in combination with mass spectrometry (MS). In an attempt to understand the organic P cycle in the environment, both an established analysis method (Paper IV) and the new method (Paper V) were applied to phytoplankton, settling particles and sediment from Lake Erken in Upland, Sweden (Paper IV) and to several lakes (Upland, Sweden) and Landsort in the Baltic Sea Proper (Paper V).

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2. Eutrophication

As organic P in aquatic sediments is closely related to the maintenance of a state of eutrophication [3, 5, 11-13], this section is solely devoted to this subject.

The term ‘eutrophy’ originates from the greek word ‘eutrophia’ which means ‘nourish’ and is now used to refer to a system rich in nutrients. High nutrient levels usually lead to high primary production in aquatic systems, although nutrient levels are not the only factor regulating this. Eutrophication means the alteration of the production of an aquatic system along a line in the direction from low to high values, i.e. from an oligotrophic state (low primary production) towards an eutrophic state [14], due to nutrient enrichment exceeding the normal uptake capacity of that aquatic system. The word is mostly used when anthropogenic overenrichment occurs with unwanted ecosystem consequences as described in Figure 2 [15]. An increase in nutrients, mainly N and/or P, can lead to higher primary production e.g. algae and can thus result in less transparent water. Due to lack of light, benthic vegetation may die, leading to an increase in organic matter on the bottom of the aquatic system. Certain types of algae that dwell below the surface also decrease due to lack of light, resulting in loss of biodiversity. Breakdown of organic matter by microorganisms requires primarily oxygen. A rapid decrease in oxygen levels can result in fish death, leading to a direct increase in organic matter. A decrease in water transparency can favour certain fish species, again resulting in loss of biodiversity. Moreover, the fragile food web can be altered in a way that promotes phytoplankton (algal) growth, again leading to a further increase in organic matter. Anoxic conditions and loose sediments (due to a decrease in benthic vegetation) promote the recycling of sediment P into the water column, resulting once more in an increase in nutrients.

The degree of each pathway in this cycle is very individual for each specific aquatic system. It depends largely on the concentration of nutrients and on factors influencing this, such as the inflow of oxygenated water or water with lower nutrient concentrations. There are two main P fluxes, external loads of P (coming from outside the water body) and internal loads of P (recycled from the sediment into the water column). Both fluxes have to

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be reduced simultaneously in order to decrease the total P concentration in the water. This aspect is discussed in more detail in Section 3.

Figure 2. A schematic overview on the circle of eutrophication.

Another drawback related to eutrophication is toxin production by cyanobacteria, posing health risks for animals and humans. Besides environmental and health risks, there is also a great financial downside to eutrophication-induced problems. The tourist industry suffers from dirty and/or polluted recreational waters due to e.g. restricted or no swimming and fishing possibilities. Globally, drinking water resources are endangered and require additional water treatment or in the worst case, alternative water supplies (Turner [16] and references therein).

Lake Erken, the study site in Papers I-IV, is a moderately eutrophied lake in Sweden. Nutrient loads have been constant since measurements started in the 1930s due to a small and constant number of inhabitants in the catchment area, 10% of which is low intensity farmland. The lake stratifies during the summer period, resulting in oxygen depletion during this period.

The Baltic Sea, where samples were taken for Paper V, contains seven of the ten largest areas of dead sea bottom [17, 18] and its eutrophication-related problems have been ongoing for several decades [19]. As far back as 1992, all the countries around the Baltic Sea united in the Helsinki Commission (HELCOM) to protect the Baltic Sea environment. Preventing the expansion of dead sea bottom areas is now high on the agenda. In 2005, a panel of foreign experts met in Sweden to assess the main causes of the eutrophication problems in the Baltic sea and on the west coast of Sweden [19] and suggested a range of measures to improve the situation. Phosphorus concentrations proved to be of vital importance in many of the affected

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areas. Despite the external inputs of P having been substantially decreased, problems remain due to internal recycling of P buried in the sediment. The Helsinki Commission has recognised the seriousness of the situation and have drawn up a HELCOM Baltic Sea Action Plan to transform the Baltic Sea from its eutrophic state to its original oligotrophic state [20] and to solve other environmental problems (e.g. hazardous compounds/pollution). The plan mainly includes measures to decrease P and N input from all countries surrounding the Baltic Sea.

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3. Phosphorus

3.1. Phosphorus in the environment Phosphorus originally enters the biosphere by mining or erosion of the mineral apatite in bedrock. Since the volatility and solubility of these compounds are low, the main P fluxes occur via dust particles into the atmosphere and suspended solid particles in water streams [21]. Phosphates are used in many different products such as fertilisers and detergents, and P from these kinds of products contributes to the anthropogenic P input in water bodies. Figure 3 shows the main fluxes of P compounds in the environment.

Figure 3. P sources in the environment. Adapted from Ahlgren [22]. Graphics by Andreas Dahlin.

Generally, two types of P sources can be recognised, diffuse (or non-point) sources and point sources, where diffuse sources represent the main P dis-charges in developed countries [23]. Examples of diffuse sources include

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leaching of P compounds from agricultural land due to heavy rainfall after application of fertiliser and P emissions from houses in sparsely populated areas not connected to the municipal wastewater treatment system. In addi-tion to these anthropogenic diffuse P sources, there are also natural diffuse P sources such as erosion and leaching from minerals and forests (Figure 3). Point source discharges come mainly from industries and municipal waste-water outlets, but in Sweden P discharges have decreased substantially in the last couple of decades due to advances in wastewater treatment [19]. Phos-phorus compounds entering a water body may be buried in the sediment and/or recycled back into the water column via internal loading. They may also be assimilated by biota in the water column and released again to the water or transported to the sea via rivers and streams.

3.1.1. Phosphorus in biota Phosphorus plays an important role in the biological metabolism of plants and animals. P is essential for catalysis of metabolic processes through enzymes and in the energy supply to cells through ATP (adenosine triphosphate). Phosphorus is also found in the cell membrane in the form of P-lipids. Furthermore, phosphates form part of DNA where they can constitute up to 10% of the weight [24] and are also found in other nucleic acids such as RNA (ribonucleic acid) and mRNA (messenger ribonucleic acid). Phosphates make up the structure of bones and teeth for animals and play a vital role for cell growth and reproduction in bacteria and plankton.

3.1.2. Phosphorus in products The majority of commercial phosphates are used in fertilisers, in the form of ammonium and calcium phosphate and in laundry and dishwasher detergents, in the form of sodium phosphate. In Sweden, the annual consumption of sodium tripolyphosphate is about 4000 tonnes for laundry detergents and about 2000 tonnes for dishwasher detergents [25]. Worldwide, about 148000 tonnes of P in all forms, mainly as phosphates, were produced in 2005, about 80% of which were used in the fertiliser industry [26]. Phosphates are also used in all kinds of synthetic products such as plastics, dyes (as binding agent), flame retardant products (as additive) and lubricants. Phosphates are even present in food products such as egg, milk powder, soft cheese and cola drinks.

3.2. Internal loading of phosphorus in aquatic systems Phosphorus compounds that have entered the sediment, can be buried permanently or can be degraded and released into the water column (internal

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loading). This process of internal loading is important for the trophic status of the water body since P is considered the limiting nutrient in many aquatic systems [1, 19, 27].

The P compounds are released to the water column through various chemical and physical pathways. Release of P has been related to the presence of metals such as iron (Fe) [4] and aluminium (Al) [5]. Redox potential has long been considered the most important pathway of P release from sediments to water [4, 28]. This is mainly true where P compounds are bound to Fe(III) via oxygen (Fe-O-P) and where anoxic conditions can reduce Fe(III) to Fe(II), with release of the P compounds as a consequence [4, 29]. However, it has been shown more recently that Fe(II) can immobilise P compounds to some extent [30]. Furthermore, oxic conditions do not necessarily lead to complete retention of P by the sediment [31, 32]. Since bacteria are dependent on electron acceptors (see below) for utilisation of organic matter, including organic P compounds, redox potential also plays an important role here as the bacteria reduce the electron acceptors [33]. Microorganisms adapted to aerobic conditions use oxygen (O2) as their electron acceptor, while anaerobic microorganisms will use nitrate ions (NO3

-), sulphate ions (SO42-), carbon dioxide (CO2) or manganese (Mn(IV))

and iron (Fe(III)) compounds as electron acceptors. This is especially interesting when extra inputs of NO3

- (mainly from agricultural land) or SO4

2- (through burning of fossil fuels) enter the aquatic system. Input of NO3

- can reduce the release of P since microorganisms can use NO3- as an

electron acceptor prior to Fe(III). Sulphate ions, on the other hand, can increase P release due to formation of dihydrogen sulphide (H2S), possibly reducing Fe(III), followed by precipitation as iron sulphide (FeS) [32, 34]. Another important chemical factor is pH, which influences the adsorption of P compounds on clay minerals [35].

After P compounds have been mobilised by chemical factors, they are transported into the water column by physical and biological factors such as diffusion and bioturbation by benthic organisms. In shallow waters, wind-induced resuspension can also be an important factor [36].

3.3. Phosphorus compounds Research on sediment P has been proceeding over a long period of time, initially with the focus mainly on inorganic P forms. However, in the 1980s, the focus moved to organic P forms and, with the introduction of the 31P-NMR technique, different P compound groups in sediment extracts could be identified (Table 1).

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Table 1. General structure and examples of P compound groups identified in sediments using 31P-NMR. Modified from Ahlgren [22].

* R represents organic side chains, not necessarily identical. The main groups are orthophosphate (Ortho-P), pyrophosphate (Pyro-P), polyphosphate (Poly-P), orthophosphate monoesters, (Monoester-P), orthophosphate diesters (Diester-P) and phosphonates.

Ortho-P is the most common form of P in sediments and it is this ionic form that is utilised by primary producers. Many organic P compounds are eventually degraded to Ortho-P in the aquatic environment. Pyro-P is a single inorganic molecule consisting of two phosphate groups, while Poly-P

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can be a variety of compounds, both organic and inorganic. Of the organic P compound groups, Monoester-P seems to be the most abundant in lacustrine and brackish waters. Monoester-P compromises a large group of compounds including sugar phosphates, P-lipids, mononucleotides and inositol phosphates with different degrees of phosphorylation. In general, Monoester-P is one of the more recalcitrant (i.e. stable) P compound groups in lacustrine sediments (e.g. [13, 37], Paper IV). Diester-P generally contains three different fractions, DNA-P, Teichoic P and P-lipids of which DNA-P usually forms the largest fraction [3] and is known to substantially contribute to the P cycling in deep sea sediment [24]. Poly-P and Pyro-P are considered the most labile compounds in lake sediments ([9, 13, 38], Paper IV). Phosphonates differ from the other P compounds due to their direct C-P bond and are thought to be more stable compounds [39]. Phosphonates are usually not found in aquatic environments with anaerobic conditions [22], and were not detected in the studies presented in this thesis.

The different organic P compound groups mentioned above are also found in humic acids (Paper IV), which are complex mixtures of organic compounds. Humic acids are mainly formed during degradation of vascular plant tissue [14]. They tend to be stable and therefore accumulate in soils and sediment. It is important to know whether humic acids can form a major sink of P or whether the associated P compounds are as readily available for release to the water column as non-associated P compounds. This question was therefore addressed in Paper IV. The different P compound groups detected in humic acids showed a slower decrease with sediment depth than the non-associated P compounds. However, since they did not reach a constant level of concentration they cannot be considered a major sink of P, but merely more stable and more slowly degrading than non-associated P compounds.

More recently, the structural identification of individual sediment organic P compounds has gained importance but examples are still scarce. Suzumura & Kamatani [40] analysed inositol hexaphosphate using 1H-NMR and gas chromatography (GC). Their findings suggest that inositole hexaphosphate is susceptible to biological degradation and is therefore not a significant component of the stable organic P pool in marine sediments. Their results also indicate that inositole phosphate is probably of allochthonous origin in the marine environment studied [41]. Inositol phosphate has also been studied by enzymatic hydrolysis followed by spectrophotometric detection, and has been shown to be a stable compound in fresh and brackish water [37]. The P-lipid phosphatidylcholine, isolated from sediment pore waters and extracts, has been analysed with chemiluminescence via an enzymatic reaction [42]. In addition, several sediment P-lipids have been identified by a flow-blending extraction combined with liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS) [43]. Phospholipids with different fatty acid composition could in this way be traced as coming

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from microorganisms and plant waxes. In Paper V, the mass to charge ratio of 114 P compounds was detected with LC-ESI-MS/MS. Several compounds were identified as being nucleotides (see Section 4.5.4.), probably degradation products of RNA. By following their respective mass to charge throughout the sediment depth, some were found to be more stable than others and thus less prone to contribute to internal P loading, than others.

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4. Analysis

As can be seen from the analytical chemistry chain in Figure 1, the previous sections mainly provide an introduction to the sample (first step in Figure 1) and the new knowledge, contributed by Papers I-V in these areas. However, the actual analysis represents a process, from sediment to results about P concentration in the sediment, and on to the identity of P compound groups or individual P compounds. Section 4 discusses every step in this process from sample collection to detection, with the main focus on extraction, sam-ple preparation and separation in combination with mass spectrometric de-tection. However, the more conventional analysis methods are also dis-cussed. An overview of the different analysis pathways used for analysis of P compounds in sediment is given in Figure 4.

Figure 4. Overview of different analysis pathways for P compounds in sediments, leading to complementary information.

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4.1. Sample collection In any research on real-life samples, the material of interest often has to be sampled and transported to the laboratory. The sampling process is generally considered the greatest source of variation in the analytical chain. Caution should thus be exercised when sampling sediment (Papers I-V), phytoplankton (IV) or settling material (Paper IV), so that the actual sampling process and subsequent transportation alter the chemical and physical properties of the sample as little as possible. In cases where the results of sample analysis are used to draw chemical or physical conclusions about e.g. a lake, one should also pay attention to when and where samples are taken, so that they are representative of the lake in question. It is thus important to carefully define the parameters to which analysis results relate.

In Papers I-V sediment samples were always taken on accumulation bottoms, the deepest point in a water body where there is a correlation between the depth of the sediment and the sediment age. This was necessary since the aim in Papers IV and V was to study the turnover rate of P compound groups and P compounds, respectively. In an accumulation bottom, compounds or compound groups found both at the surface and in the deeper sediment can be considered more stable than compounds or compound groups only found in the surface sediment.

Undisturbed sediment cores can be sampled with gravity core samplers. The many different models of gravity core samplers have a common basic set-up. They consist of one or more plastic, transparent tubes, with weights attached to carry the tubes down to the sediment. During the passage of the sampler through the water, the water must be able to pass freely through the tube in order to prevent the creation of pressure waves that can disturb the sediment. Gravity ensures that the tube enters the sediment, and when the sampler is being pulled up a locking mechanism retains the sediment in the tube. The models used in the studies reported were a Willner sampler (Paper I–V) and a Gemini twin core sampler (Paper V) (Figure 5).

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Figure 5. Two different types of gravity core samplers. (A) Gemini twin core sampler and (B) Willner sampler. Adapted from Ahlgren [22].

Phytoplankton (Paper IV) were sampled with 40 μm plankton nets in the upper 1 m of the lake. Settling material (Paper IV) was collected during one month with sediment traps at two different depths in the water column. In Papers I-IV the sample area was Lake Erken, a well-examined lake east of Uppsala, Sweden (see Section 2). The samples used in Paper V were taken in Landsort in the Baltic Sea Proper, and in four different lakes in Upland (Sweden), of which two lakes had a forest catchment and the other two lakes had an agricultural catchment.

Dating of the sediments to enable calculation of turnover rates as in Paper IV (Lake Erken) and V (Baltic Sea) was done by measurement of 137Caesium (Cs) activity. Caesium dating is based on the distribution of radioactive Cs in some parts of Sweden and the Baltic Sea after the Chernobyl disaster in 1986. The layer which gives the highest radioactive Cs concentration corresponds to 1986, and from that the sediment accumulation rate can be calculated and thus also the date of the other layers.

4.2. Extraction Since it is hard to analyse P compounds in sediment or soils as such, an extraction of the compounds into a liquid phase facilitates their analysis. There is a technique available for analysing compounds present in solids, solid state 31P-NMR [44], but since its resolution is rather low, extraction is preferred, despite the risk of it changing the compounds of interest.

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Conventional analysis of sediment P compounds is based on an extraction procedure such as that proposed by Psenner & Pucsko [8]. In this procedure, different extraction solvents such as water, bicarbonate-buffered dithionite (BD), hydrochloric acid (HCl) and sodium hydroxide (NaOH, alkaline) are applied in sequence onto the sediment to extract the following different operationally defined P groups in order of appearance: porewater-P and loosely sorbed P; P forms where release is sensitive to low redox potential (e.g. P adsorbed to Fe and Mn); P bound to Al, and organic P. The extracts are then measured according to the molybdenum reactive P (MRP) method [45], see Section 4.5.1. Pettersson et al. [46] provides an overview of the many different extraction procedures. They emphasise that with the fractionation procedures, only operationally defined P groups are obtained, i.e. defined according to the extractant used in each step, and that errors occur when linking these operationally defined P groups to certain chemical P forms. The use of the many different procedures also complicates the comparison of results from different studies. Common to all these methods is that organic P compounds are detected as one single pool as a part of the NaOH extraction step. More specifically, the organic P pool is calculated as the difference in MRP of the NaOH extract before and after a digestion step according to Menzel & Corwin [47].

Since the 1980s, analysis of sediment organic P with 31P-NMR has become increasingly common. Alkaline extraction solvents such as NaOH [13, 48] or ethylenediaminetetraacetic acid (EDTA) in combination with NaOH ([3, 38]) are commonly used for the extraction of sediment organic P for subsequent 31P-NMR analysis and have their basis in the more conventional fractionation procedure as discussed above. The mechanism of extraction with NaOH or other alkaline solvents is based on replacing polyvalent bridging ions (e.g. Fe3+) between mineral particles and organic P compounds with monovalent ions such as sodium (Na+) which are less effective in functioning as bridging ions [49]. This theory is based on the assumption that most organic P compounds are bound to mineral particles via e.g. iron oxides (Fe-O-P) [50, 51]. In an attempt to improve the extraction yields of organic P compound groups and their NMR spectra, a great variety of extractants, sometimes in combination with pre-extractants, has emerged. Common pre-extractants are EDTA and BD, which are thought to respectively chelate or reduce metal ions onto which the organic P compounds are bound.

The variety of pre-extractants and extractants complicates the comparison of results from different studies. Therefore a comparative study of some common alkaline extractants, in combination with pre-extractants, was performed (Paper I). The highest total P yield was obtained with EDTA as pre-extractant (30 min.) and NaOH as main extractant (16 h), whereas using BD as pre-extractant (1 h) in combination with NaOH as main extractant resulted in the highest yield for Poly-P. In Paper IV, BD was used as a pre-

25

extractant mainly to remove porewater P and P bound to reducible metals (i.e. Fe and Mn). In Paper II only the main extraction step with NaOH (0.1 mol L-1) was performed to avoid additional ion suppression in ESI (Section 4.5.4). In Paper III, ion suppression by the use of EDTA was avoided by an online separation step prior to ionisation. For the study in Paper V, again only NaOH was used to be in accordance with previous extractions since these were performed before the findings in Paper I.

However, the use of alkaline solvents such as NaOH with a pH of 13 for a period of 16 hours can induce hydrolysis of labile compounds such as RNA [52, 53], and this should be considered when interpreting results. Paper I showed that mainly Poly-P is hydrolysed when NaOH is used as the main extractant without pre-extractant. The use of a pre-extractant (BD or EDTA) reduces this phenomenon but the highest Poly-P yield is obtained with BD as pre-extractant. Both EDTA and BD most likely inactivate metal ions (e.g. divalent calcium ions, Ca2+) which are thought to catalyse degradation processes [54]. These problems have also been discussed for organic P compounds in soil [49]. Some P-lipids and certain phosphonates found in soil are less stable depending on the extraction time and solvent strength compared with Monoester-P, organic Poly-P and the phosphonate 2-aminoethyl phosphonic acid which are quite stable under alkaline conditions. Paper III identified a series of nucleotides that could originate from alkaline hydrolysis of RNA [52, 53].

Another important factor to consider is the final concentration of the compounds of interest in the extract. For example, the relatively low sensitivity of 31P-NMR analysis requires a high P concentration in the NaOH extract. One way is to use a high solid to solvent (weight to volume, w/v) ratio during the extraction of the sediment. However, this can reduce the extraction efficiency relative to P extractions from fractionation procedures where lower ratios are used. Therefore a compromise has to be made between high concentration and high extraction efficiency. Ahlgren [22] compared 31P-NMR spectra from sediment extracts with a solid to solvent ratio of 1:3 and 1:25 (w/v). The 1:3 ratio resulted in the best resolved 31P-NMR spectrum and has been used in several studies (e.g. [9, 10, 55], Paper I-V). Moreover, studies using high sediment to solvent ratios are usually performed on dry samples, while studies using low ratios are usually performed on wet samples. The sediment particle to solvent ratio might thus be similar. In addition, drying of sediment samples is not advisable, since it can cause changes in organic P composition [44]. Another possibility to improve the sediment P extraction yield is to perform several extraction steps (e.g. three) with smaller volumes of solvent. This requires a thorough study of the total extraction time needed, as the conventional extraction time for alkaline solvents such as NaOH is 16 hours or overnight. Several extraction steps with the same length of time would thus be highly

26

impractical and would further increase problems with hydrolysis of the organic P compounds.

As P compounds can associate with humic acids, which are extracted from the sediment with NaOH, it is of interest to separate these humic acids from the NaOH extract. Since little is known about P compounds associated with these stable humic acids, an attempt was made to isolate and analyse them in Paper IV. In brief, the NaOH sediment extract was acidified with sulphuric acid (H2SO4) [56]. The precipitate was then removed by centrifugation and redissolved in 0.1 mol L-1 NaOH in preparation for total P measurement and 31P-NMR analysis. As humic acids are complex mixtures of organic compounds, there is no clear definition of origin or degradability. A possible explanation for the precipitation of these humic acids in an acid environment is that the NaOH solution extracts charged organic compounds which remain in suspension due to intermolecular repulsions. In the acid environment, the negatively charged organic compounds are neutralised and Van der Waals forces attract the compounds, forming colloids and resulting in precipitation due to size [14].

Since alkaline extractants can give rise to artifacts in the sediment organic P compounds, there is a need to find alternative extraction methods that are faster and that allow the use of milder solvents. Pressurised liquid extraction (PLE) is an interesting alternative where both higher pressure and higher temperatures can be applied. Preliminary results [57] show that it is possible to extract one-third of the organic P compounds with only 10 minutes of extraction time at 75 °C and 50 bar with NaOH as extraction solvent, compared with the 16 hours required in the conventional extraction method with NaOH. The organic P amount is calculated as the difference in MRP of the NaOH extract before and after a digestion step according to Menzel & Corwin [47]. Longer extraction times and/or higher temperatures than those mentioned above should be avoided due to the degradation risk. Preliminary tests with acetonitrile (ACN) and isopropanole resulted in very low extraction yields after a 10-minute extraction but could still be of interest for the extraction of specific P compounds, e.g. more hydrophobic compounds such as P-lipids. These promising results warrant further research on sediment organic P compound extraction with PLE.

4.3. Sample preparation Sample preparation is necessary to simplify the sample matrix prior to detection and in this way increase the sensitivity and selectivity of the analysis method. At the same time, the number of steps in sample preparation should be minimised because each additional preparative step involves losses of the compounds of interest. Considering the non-selective (for P compounds) sediment extraction with NaOH, it is obvious that a large

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amount of matrix compounds are co-extracted. Reducing the matrix compounds and/or increasing the P compound concentration are thus key points for further analysis.

Sample preparation of NaOH sediment extracts depends on the subsequent detection method used. Section 4.5 discusses the different detection techniques used in Papers I-V. The use of inductively coupled plasma atomic emission spectroscopy (ICP-AES) requires no special sample preparation of the NaOH sediment extract other than removal of particles by centrifugation to prevent clogging of the nebuliser.

For 31P-NMR analysis, the extract needs to be concentrated. However, as discussed previously, high solid to solvent ratio as a sole measure for obtaining the desired concentration is not sufficient for 31P-NMR analysis. An additional concentration step is needed and the most common of these are rotary evaporation [38, 48, 58] and freeze-drying [59]. Reitzel et al. [55] compared freeze-drying and rotary evaporation as concentration steps. Some P groups decreased significantly during freeze-drying and the total P losses were higher than with rotary evaporation. Therefore the NaOH extract was concentrated 25-fold (Papers I and II) or 10-fold (Paper III-V) with rotary evaporation and subsequently centrifuged to remove particles that cause signal broadening in the NMR spectrum, decreasing the resolution of the spectrum.

Prior to ESI-MS/MS, more thorough sample preparation is necessary. If the NaOH sediment extract is to be injected directly, as in Paper II, the amount of Na ions and particles should be reduced. The former is necessary to prevent ion suppression and the latter to prevent clogging of the ESI orifice, both of which result in low signals. It is also necessary to concentrate the extract to obtain sufficiently high signals for the compounds of interest.

Two techniques for removing Na ions were considered: ion exchange and barium (Ba) precipitation, the latter according to Llewelyn et al. [60] and Cooper et al. [61]. Details of the ion exchange procedure can be found in Paper II under Experimental. The Ba precipitation procedure was initially used for extraction of pentaphosphate and hexaphosphate from alkali extracts from soils [62, 63]. Llewelyn et al. [60] used an adjusted method for the extraction of organic P compounds from natural waters and simultaneously preconcentrating the sample compared with the original sample volume. This procedure was tested for the removal of Na ions and successfully reduced the amount of Na ions. However 33% (n = 4) of the P compounds were lost, as opposed to less than 1% (n = 4) losses with the ion exchange method (Table 1 in Paper II). As the ion exchange method was less time-consuming and resulted in lower P losses, this method was preferred in Paper II. However, the Ba precipitation procedure warrants further investigation as it can provide a solution to analysis problems caused by the many matrix compounds in an alkaline sediment extract.

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In Papers II, III and V, the concentration step was performed by rotary evaporation for the above-mentioned reasons, followed by centrifugation at 10000 rpm for particle removal to prevent clogging in the tubing connecting the syringe with the ESI interface. The concentration step resulted in a viscous liquid which was hard to spray when injected directly into the ESI interface as in Paper II. Normal filter paper with a pore size of 45 μm was considered, as were mass cut-off filters. The compounds retrieved in the filtrate after mass cut-off filtration did not exceed a certain mass, narrowing the mass window for MS analysis and simplifying the mass spectra. Mass cut-off filters were therefore preferred over normal filter papers in Paper II. The effect of this sample preparation procedure is further discussed in Section 4.5.3 in relation to NMR, as the procedure was evaluated by calculating the yield of the different P compound groups measured with 31P-NMR after each sample preparation step.

When a chromatographic separation is performed prior to ESI-MS/MS (Papers III and V) sample preparation can be simplified, as the chromatographic column acts as a filter for interfering matrix compounds. A simple concentration by rotary evaporation followed by a 0.45 μm syringe filtration prior to column injection is sufficient. The problem with Na ions can be avoided by switching the outlet from the chromatographic column away from the inlet to the ESI interface when the Na ions are eluting.

4.4. Separation Liquid chromatography (LC) separates compounds in time depending on their properties, e.g. size, charge and structural binding groups. This is of special interest since the overall aim of the studies presented in this thesis was to identify specific organic P compounds. Separation of these compounds in time further increases the sensitivity and selectivity in MS and enables the individual compounds to be detected one by one.

A comprehensive overview of different chromatographic separation techniques applied in the natural organic P area can be found in McKelvie [2]. Size exclusion chromatography (SEC) was one of the first serious attempts to separate organic P compounds. It was e.g. used to demonstrate the presence and importance of inositol phosphates in preconcentrated lake water [64], and for revealing the presence of inositol hexaphosphate in wetland soils [65]. Ion exchange chromatography (IC) has been used for separation of P compounds in soil solutions [66] and for separation of inositol phosphates in sediments [40]. Partition chromatography such as normal phase chromatography has mainly been used for the separation of P-lipids in sediments [43].

However, to the best of my knowledge, no work has been done on the chromatographic separation of organic P compounds in alkaline sediment

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extracts. Therefore different chromatographic separation methods were tested for their potential use in the separation of sediment organic P compounds. The compatibility of mobile phases with subsequent detection methods such as ICP-AES and ESI-MS/MS has to be borne in mind throughout.

Pre-tests with SEC and IC resulted only in one unresolved peak. Many of the P organic compounds are polar and thus difficult to retain with reversed phase LC such as C18. However, the possibility of using C18 with ion pair (IP) reagents such as tributylamine, N,N-dimethylhexylamine and hexylamine was explored. These IP-LC tests resulted in several resolved chromatographic peaks against ESI-MS/MS (Section 4.5.4), but none of the chromatograms had the same amount of resolved peaks as with the porous graphitic carbon (PGC) used in Paper III. This is probably due to interference of the ion pair reagents in the ESI (Section 4.5.4). Moreover, with IP-LC it was necessary to lower the pH of the highly alkaline sediment extract (pH = 13) to 5.5. Few stationary phases can withstand a high pH but decreasing the pH of a small volume of sediment extract is tedious work and is hard to perform with analytical accuracy. Careful control of the pH is important to avoid loss of analytes due to precipitation, which can occur below pH 4. It was therefore of interest to use stationary phases that can withstand extreme pH, such as zwitterionic® hydrophilic interaction LC (ZIC®-pHILIC), and PGC. The ZIC®-pHILIC material was also interesting due to its ability to separate polar and hydrophilic compounds, which most of the sediment P compounds are (not P-lipids). Compared with normal phase liquid chromatography, lower amounts of organic modifier can be used, avoiding problems with dissolving the analyte. A limitation with ZIC®-pHILIC regarding analysis of NaOH sediment extracts is that it provides poor retention unless the extract is diluted with organic modifier, which counteracts efforts to preconcentrate the analytes. Separation of organic P compounds in alkaline sediment extracts with ZIC®-pHILIC coupled to ESI-MS/MS did not compete with PGC in amount of resolved peaks and had low repeatability. Pre-tests with PGC coupled to ICP (Section 4.5.3) resulted in several chromatographic peaks and repeatable results. PGC also allows the use of relatively high contents of organic modifiers and still retains even very polar analytes. To my knowledge, it has not previously been used in the determination of natural organic phosphorus compounds in the aquatic environment. PGC has been used for separation of standard phosphonic acids in one study [67].

The PGC material [68] is composed of flat sheets of carbon atoms bound in a hexagonal arrangement (Figure 6) and numerous publications describe its properties [69-71]. Its special retention behaviour for polar analytes has been ascribed to the polar retention effect on graphite [70, 71] and it provides an alternative and attractive complement to IC [72].

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Figure 6. Crystal structure of PGC, showing the three-dimensional graphite arrangement. Graphics by Andreas Dahlin.

The retention of non-polar and geometric isomers depends on several factors. The strength of interaction between a hydrophobic analyte molecule and the PGC surface largely depends on the structural planarity of the analyte molecule. The higher the planarity of the molecule, the more interaction with the flat PGC surface due to charge transfer and dispersion forces [73]. PGC is particularly selective with respect to geometrical isomers and closely related substances [74-76]. This was observed in Paper III where structural isomers of some nucleotides were separated in time, e.g. 5’-AMP and 3’-AMP. In general, highly structured and rigid molecules are less retained on PGC than flexible molecules of the same molecular weight. Researchers have also found that non-polar analytes are strongly retained on PGC [75]. Compared with the conventional octadecylsilane (ODS) materials, more organic modifier is required to elute an analyte from a PGC column as shown in Paper III, where a gradient gradually increasing from mobile phase A containing 1% ACN to mobile phase B containing 80% ACN, was needed to elute the bulk of compounds. Some compounds do not seem to elute at all and it is thus likely that these are non-polar compounds (Paper III, Section 4.5.2).

Due to the high degree of freedom in the choice of mobile phase conditions, the coupling to MS detection is greatly simplified. However, PGC is an electrical conducting stationary phase and it is not recommended to connect the PGC column directly to a high voltage ESI interface. This would induce a current through the PGC column and most likely result in redox reactions affecting both the PGC particles and the analytes [77]. It is therefore important to insert a ground point between the conducting PGC stationary phase and the ESI interface, i.e. ground the union as seen in Figure

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7, which illustrates the recommended set-up for a PGC column coupled to a ESI-MS interface as used in Papers III and V.

Figure 7. Recommended set-up for a PGC column coupled to ESI-MS.

4.5. Detection: a closer look The conventional method for detection of Ortho-P is the molybdenum reactive phosphorus (MRP) method with spectrophotometric detection [45]. This is a good method when working with the extraction procedure of e.g. Psenner and Pucsko [8], but can give an overestimation of the Ortho-P concentration due to hydrolysis of organic P compounds present in the extract under the acidic conditions of the MRP method [78, 79]. Moreover, no information can be obtained on specific P compounds and therefore more selective and sensitive methods are of interest.

In the beginning of the 1980s, the more specific detection method of 31P-NMR started to be used in the area of environmental research and was of particular interest for characterisation of organic P compound groups. Its potential is shown in Table 1 (Section 2) and it was the main detection method used in Papers I and IV. A detailed description of the technique and its use in the area of sediment organic P can be found in Ahlgren [22].

As for measuring the total concentration of elements, e.g. P in aqueous solution, it is equally correct and faster to use ICP-AES instead of the MRP method. This feature was used in Papers I to IV to calculate the extraction yields by measuring total P concentration in the sediment extracts and comparing this with the total P amount in the sediment. In Paper I elements such as Al, Mn and Fe were also measured since the presence of these metals can affect the P extraction yield. In Paper III the separation of the organic P compounds extracted from sediments was optimised by coupling the LC column to ICP-AES. Phosphorus compounds eluting from the column could thereby be detected and quantified with ICP-AES and the separation efficiency could be evaluated.

The above-mentioned methods are not sufficient to provide more information about the identity of individual sediment organic P compounds

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in order to follow their cycle in the aquatic environment. Although all are extremely valuable, the MRP method, ICP-AES and 31P-NMR can mainly quantify Ortho-P, elements and structural binding groups respectively.

With MS, on the other hand, and more specifically ESI-MS/MS, it is possible to obtain the molecular weight of the individual P compounds and their fragments and in this way get a hint about their respective elemental composition. This technique was therefore used in Papers II, III and V to identify specific P compounds from alkaline sediment extracts.

The determination of total P content in the sediment and total P concentration in the sediment extract (aq.) is discussed below, as is the use of ICP-AES, 31P-NMR and MS in the field of organic P compounds in aquatic sediments.

4.5.1. Total phosphorus When discussing total P it is crucial to distinguish between the total P content in the sediment and the total P concentration in the sediment extract (aq.). Without breakdown of the P compounds, it is not possible to extract all of the P compounds in the sediment. The total P content in the sediment is therefore determined by acid hydrolysis at 340 ºC which converts all bound phosphate to free phosphate. The concentration of free phosphate can either be measured by the MRP method or by ICP-AES. The MRP method, used in Papers III and IV, is based on the reaction of free phosphate with ammonium molybdate, resulting in a yellow complex. When ascorbic acid is added to the complex it turns blue, and since its intensity follows the Lambert-Beer law within a certain concentration range, measurement with spectrophotometry is possible. The blue complex is measured at a wavelength of 882 nm. This method was originally developed by Murphy & Riley [45] and a variation of this method can be found in Koroleff [80]. In Papers I-III and V, the total P concentration in the sediment was determined with ICP-AES after acid hydrolysis. Both the MRP method and ICP-AES can be used to determine the total P concentration in aqueous solution e.g. a sediment extract.

4.5.2. ICP-AES Besides converting components of samples to atoms or simple elementary

ions, AES atomisers also excite a fraction of these species to higher electronic states. Because of rapid relaxation of the excited species, UV-VIS line spectra are produced. These are useful for qualitative and quantitative elemental analysis. The ICP source is the most commonly applied plasma source. Detailed information on the working principle of the ICP-AES technique can be found in Skoog et al. [81].

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ICP-AES was used to measure total P concentrations in extracts studied in Papers I-IV. In Paper III, a LC method for the separation of sediment organic P compounds was developed by coupling the LC column with ICP-AES.

The LC-ICP-AES method (Paper III) with PGC as stationary phase resulted in eight chromatographic peaks containing P compounds (Figure 8). Since ICP-AES is element-selective and not P compound-selective, it is not possible to resolve chromatographically overlapping peaks. Therefore, the chromatographic peaks observed in Figure 8 most likely do not represent individual P compounds but rather several.

To preconcentrate the compounds of interest on the beginning of the column, a sample loop with large volume (250 μL) was combined with a linear gradient of the mobile phase containing 1% ACN for 3 minutes (Table 1 in Paper III) and a flow rate of 200 μL min-1. The on-column concentration step in combination with the preconcentration step (10-fold) prior to injection improved the P signal intensity. Although ICP-AES is capable of detecting low P concentrations, the sensitivity for P can vary with the ACN content of the mobile phase due to increased background emission at similar wavelengths to P.

Figure 8. Chromatogram of P compounds extracted from Lake Erken sediment (0-1 cm) with EDTA�NaOH detected with LC-ICP-AES and PGC as stationary phase (Paper III).

From the chromatogram in Figure 8, it was possible to quantify the separated peaks as exemplified with surface sediment from Lake Erken. The

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sediment total P content of the surface layer (0-1 cm) was 1.7 mg P g-1 dry weight according to the Murphy & Riley method [45]. Peak 0 represents 23% and peaks 1 to 8 together represent 1.4% of the surface sediment total P content (Figure 8). Since peak 0 contains compounds that are not retained by the PGC material, these are most likely small compounds and/or highly charged compounds and therefore have little interaction to the hexagonal carbon sheets [69-71]. The NaOH extracted 37% of the surface sediment total P content, and according to Paper I, 35% of this is present as Ortho-P. It is thus likely that a large amount of this Ortho-P is present in peak 0. This was confirmed by a 31P-NMR experiment on a fraction containing peak 0 which indicated that Ortho-P and Monoester-P are present (results not shown). It is therefore of great interest to continue the development of a separation step for these P compounds, but this was not addressed in Paper III. It is also worth noting that 13% of the surface sediment total P content is not detected after elution from the column. One possible explanation is that these P compounds have a very strong interaction with the carbon sheets of the PGC column and thus are likely to be large molecules with long carbon chains and little charge. Another, or a rather complementary, explanation is that some of these P compounds become separated from each other and elute, but have a concentration under the detection limit of the ICP-AES instrument.

4.5.3. 31P-NMR Since the 1980s, 31P-NMR has been used for the measurement of P compounds in natural samples. With this technique it is possible to detect different kinds of P groups as seen in Table 1 (Section 2). A typical 31P-NMR spectrum of a sediment extract is shown in Figure 9.

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Figure 9. Typical 31P-NMR spectrum of an extract from Lake Erken surface sediment, with NaOH as main extractant and BD as pre-extractant (Paper I).

The general mechanism of nuclear magnetic resonance is described in e.g. Canet [82] or Veeman [83]. A description of 31P-NMR in the area of organic P in the environment is provided in Cade-Menun [44, 84] and references therein. The basic principle of NMR is shown in Figure 10. A NMR tube containing the sample is placed into a strong magnet. A radio frequency pulse is applied to the sample resulting in data which after Fourier transformation results in a typical NMR spectrum.

Figure 10. Basic principle of a NMR experiment. The sample (a) is inserted into the magnet (b). The experiment (c, application of radio frequency pulse) gives data (d, in the form of free induction decay) which are translated into a spectrum (e) through Fourier transformation. Adapted from Ahlgren [22].

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As mentioned earlier (Section 4.2), 31P-NMR was used for quantifying extracted P compound groups from sediment with different extraction solvents (Paper I). Using 31P-NMR, it was clearly shown that Diester-P, most likely representing DNA-P, disappeared after mass cut-off filtration (Paper II). The 31P-NMR spectra of the NaOH extract after ion exchange and of the extract after mass cut-off filtration are shown in Figure 11. Note that the signal to noise ratio of the different peaks is about the same in both spectra except for the peak of DNA-P and Teichoic-P, which disappeared completely in the 31P-NMR spectrum for the extract after concentration and filtration. Some of the P compound groups decrease after sample preparation in comparison with the Ortho-P peak. This indicates that these P compound groups are partially broken down, resulting in the release of Ortho-P.

Figure 11. Influence of sample preparation prior to ESI-MS/MS analysis on the P compound groups from a NaOH sediment extract, measured with 31P-NMR (Paper II).

31P-NMR also proved to be a valuable tool for determining degradation rates of organic P compound groups in lake sediments (Paper IV). In parallel, P compound groups found in humic acids and assumed to be more stable, were also analysed with 31P-NMR.

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4.5.4. Mass spectrometry Current techniques such as 31P-NMR only provide information about the degradability and release of P compound groups. It is likely that the individual P compounds contributing to one group will have different degradability and make different contributions to internal loading. Identification of specific organic P compounds would therefore promote understanding of P turnover in the environment. With MS it is possible to obtain fundamental properties of molecules, e.g. their molecular weight, and with special types of MS instruments it is also possible to determine the molecular weight of their structural fragments. MS includes a wide variety of techniques for ionisation and mass analysis of sample analytes. Apart from variations in ionisation techniques and mass analysers, all mass spectrometers have the following general set-up in common (Figure 12).

Figure 12. Flow-chart on the basic components of a mass spectrometer.

The inlet is usually some kind of separation step, such as liquid chromatography as in Paper III and V or gas chromatography. However it is also possible to perform direct infusion by which the sample is directly transferred into the ion source without a prior separation step as in Paper II. Via the ion source, ions are introduced into the mass analyser (Figure 12). From there, the ion signals can be enhanced via an ion transducer, which results in a mass spectrum (data output) after conversion of the signal in the signal processor. A comprehensive overview of the wide variety of ion sources is given in de Hoffmann [85]. A suitable ion source can be chosen, depending on the application. More specifically, the development of ‘soft’ ionisation techniques such as ESI has made it possible to look at high-molecular weight and low-volatility molecules [85, 86]. Moreover, ESI induces minimum fragmentation, which facilitates the identification of entire compounds. In related studies, ESI as ion source has been combined with a number of different mass analysers. For instance, ESI-MS (quadrupole mass analyser) has been applied to identify dissolved organic matter (DOM) [87, 88]. In an attempt to characterise organic phosphorus in natural waters, ESI-Fourier transform ion cyclotron resonance-MS (ESI-FTICR-MS) was used [60, 61]. With ESI coupled to a time-of-flight mass analyser (ESI-TOF-MS), inositol hexaphosphate has been detected in wetland soils [65]. Furthermore, as mentioned previously, P-lipids in sediments have been determined with ESI-MS/MS (triple quadrupole mass analyser) [43]. With tandem mass spectrometry (MS/MS) it is possible to detect parent ions of selected

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fragments after collision-induced dissociation (CID). This technique has been applied in the determination of phosphopeptides [89, 90].

In Paper II, we opted for an ESI-triple quadrupole instrument since this provided the best available compromise between tandem MS and the tough matrix of the sediment extracts. The quadrupole ion trap (Q-TRAP®) system used in Paper III gave enhanced identification possibilities during one single LC run. This set-up was also used in Paper V to follow the turnover of the detected P compounds with Baltic Sea Proper sediment depth and to study their potential source. The ionisation technique and the scan modes used for the respective mass analysers are discussed in detail below.

4.5.4.1. Electrospray ionisation As P compounds are non-volatile and the risk of degradation during the ionisation process has to be minimised except under controlled circumstances, ESI seemed to be the perfect solution since it is a desorption method that can deal with non-volatile or thermally unstable compounds [85]. Desorption methods are methods that directly transform the solid or liquid samples into gaseous ions, without any intermediate steps. As a result, spectra are greatly simplified and in positive ion mode often show only ions corresponding to the protonated molecule, while in negative ion mode (Papers II, III and V) they show ions corresponding to deprotonated molecules. However, a drawback with ESI in general is the need for relatively clean samples, free from e.g. salts that can result in ion suppression during the ionisation process. Therefore, a procedure for removal of Na ions was included in the sample preparation method in Paper II.

ESI takes place under atmospheric pressure and sometimes at elevated temperatures (about 50 ºC). As P compounds are more easily deprotonated than protonated, ionisation in this work was always performed in negative ion mode (Papers II, III and V) and therefore the general principle of ESI is explained here for ionisation of compounds into negatively charged ions (Figure 13). Of course, most of the steps in the ESI working principle are valid in both negative and positive ion mode. ESI consists of a stainless steel needle containing a silica capillary in which a solution of the sample is pumped through at a rate of 1 to 10 μL min-1. The actual spray is created by applying a strong electric field in the order of 106 V m-1 to the liquid through the capillary. It is obtained by a potential difference of 3 to 6 kV between the capillary and the counter-electrode, in this case the interface plate. The electric field in its turn creates a charge accumulation in the liquid at the capillary tip. When the surface tension is overcome, the liquid tip breaks into highly charged droplets. As the droplets get smaller, because of evaporation of the solvent, their charge density becomes greater and desorption of ions takes place (Figure 13). A gas introduced coaxially with the capillary limits dispersion of the droplets in space and simultaneously facilitates droplet

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formation and the use of higher liquid flow rates. On entering the MS, the droplets pass through a heated curtain gas to remove the last solvent molecules.

Figure 13. Electrospray ionisation. Graphics by Andreas Dahlin, adjusted by Niklas Forsgard.

4.5.4.2. Mass analyser The output of an ion source is a stream of positive or negative ions that are accelerated into the mass analyser. The function of the mass analyser is to separate ions with different mass-to-charge ratios. Ideally, the analyser should be able to distinguish between ions with minute mass differences. Passage of a sufficient number of ions (ion transmission) is necessary in order to yield measurable ion currents.

Quadrupole mass analyser A quadrupole instrument consists of four parallel rods (often cylindrical) that serve as electrodes (Figure 14).

Figure 14. Quadrupole with cylindrical rods. Graphics by Andreas Dahlin.

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One pair of opposite rods is connected to the positive side of a variable direct current (DC) source and the other pair to the negative side. Variable radio-frequency alternating current (AC) potentials are then applied to each pair of rods, 180 degrees out of phase. Ions are accelerated into the space between the rods with a potential of 1 to 10 V. Simultaneously, the alternating and direct current potentials on the rods are increased with a constant ratio, so ions that do not have a certain m/z value strike the rods and are converted to neutral molecules. Only ions in a limited range of m/z values reach the transducer. Quadrupole instruments can normally resolve ions that differ in mass by one unit.

Tandem mass spectrometry Tandem mass spectrometry is a useful tool for the identification of ion structures and for analysis of complex samples, providing both high selectivity and sensitivity. Tandem mass spectrometry in quadrupole instruments requires two mass resolving quadrupoles and a collision cell in between, usually a quadrupole. The basic principle is shown in Figure 15. Several scanning modes are possible, such as precursor ion scan (also named ‘parent ion scan’), product ion scan (also named ‘daughter ion scan’), neutral loss and selected reacting monitoring. In Paper II, precursor ion scan was used for tracing phosphate-containing compounds [89, 90].

Figure 15. Basic set-up of a triple quadrupole instrument: Q1 – first mass analyser; q2 – collision cell; Q3 – second mass analyser; D – detector.

While ions in Q1 are scanned over a whole mass range, Q3 is set to transmit a certain product ion. Q1 was set to scanning between mass to charge (m/z) 75 and 800; q2 was set to fragmenting ions coming from Q1 and Q3 was set to transmit ions coming from q2 with m/z 79 = [PO3]- or m/z 97 = [H2PO4]- (dihydrogenphosphate ion). The fragmentation step in q2 is also called collision-induced dissociation (CID) and is performed by letting the ions collide with an inert gas, e.g. N2 (g).

The effect of parent ion scanning with MS/MS on a sediment NaOH extract as opposed to normal scanning with MS is shown in Figure 16. Note the increased selectivity, i.e. reduced number of m/z signals after parent ion scanning (right pane), as opposed to normal scanning between m/z 75 and

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800 (left pane). In the left pane it is not possible to know which ion signal represents phosphate-containing compounds.

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Figure 16. Left: normal scan in negative mode on a sediment NaOH extract. Right: parent ion scan of 97 m/z ([H2PO4]-) on a sediment NaOH extract in negative ion mode.

As the aim in Paper III was to determine m/z on both the parent ions of the phosphate ion and their respective fragment ions simultaneously, a Q-TRAP® system was used instead of a triple quadrupole instrument. Moreover, a liquid chromatographic (LC) system was coupled online to the system prior to ionisation. A Q-TRAP® system has the same basic set-up as the triple quadrupole system (Figure 15). The only difference is that Q3 can function as a linear ion trap enabling trapping and fragmentation of product ions coming from q2. The acquisition method used to exploit this feature is called information-dependent acquisition (IDA). The selection of phosphate-containing compounds is carried out in the same way as in a standard triple quadrupole instrument; Q1 is set to scanning, q2 to fragmentation and Q3 to transmission of m/z 97 (i.e. [H2PO4]-). In a short time span, however, the different quadrupoles can switch scan mode, where Q1 is set to m/z 346 as in Figure 15, q2 to fragmentation and Q3 to first trap the fragments and then scan. This is called an enhanced product ion (EPI) scan.

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The method is exemplified in Figure 17. Pane A shows the total ion chromatogram (TIC; all ions containing the dihydrogenphosphate fragment ion with m/z 97). During the run, the software picks out signals above a certain intensity and fragments the compounds (in q2), followed by trapping and scanning of the fragments in Q3. The mass spectrum detected at a retention time (tR) of 26.3 minutes is shown in pane B, while pane C shows the EPI of the phosphate-containing molecule eluting at this time.

Figure 17. LC-ESI-MS/MS data from separation of an EDTA�NaOH surface (0-1 cm) sediment extract (Lake Erken). Pane A shows the total ion chromatogram on the precursors of m/z 97 ([H2PO4]-). Pane B is a spectrum taken at 26.3 minutes showing the precursors of m/z 97 detected at that very time. Pane C is an enhanced product ion scan on m/z 346 (pane B) (Paper III).

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The detailed structural information obtained with this method enabled several organic P compounds in Lake Erken sediment extract to be identified (Paper III). Some compounds were verified as being nucleotides (Figure 18). This method was applied to sediment extracts in Paper V.

Figure 18. Nucleotides with verified compound identity present in Lake Erken sediment extract.

With suitable Q-TRAP® software, particular product ions such as m/z 211 representing the sugar phosphate in a nucleotide can be extracted, resulting in an extracted ion chromatogram (XIC) (Figure 6 in Paper III). A promising application of this method was also performed in Paper III. To gain an insight into P compound turnover, sediment extracts from two depths were run with the method presented. The TIC on the precursors of m/z 97 (i.e. [H2PO4]-) of a 0-1 cm sediment extract and a 29-30 cm sediment extract are shown in Figure 19.

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Figure 19. A and B are the total ion chromatograms from the precursors of m/z 97 ([H2PO4]-) as monitored with LC-ESI-MS/MS on a EDTA�NaOH Lake Erken sediment extract at depth 0-1 cm and 29-30 cm respectively. C and D are the XIC (extracted ion chromatogram) on m/z 346 from A and B respectively. Note the difference in intensity between C and D (Paper III).

From the TIC it is not easy to see any clear difference between the samples. However, on extracting the signal for AMP (m/z 346; panes C and D in Figure 19) a significant difference (approx. 10-fold decrease); in signal strength was observed for the deep sediment, indicating substantial degrada-tion over time in the sediment. A decrease with depth was also noted in the upper 10 cm of the Baltic Proper sediment for 5’-AMP, 5’-UMP and a com-pound with m/z 353 (Paper V).

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5. Data analysis and result interpretation

Analytical chemistry is not only the development of actual analysis methods but also to a great extent the evaluation of the data obtained. Without correct interpretation of data, all analysis methods are useless. With large data sets in particular, it can be hard to ‘extract’ the actual results at first glance and more advanced data analysis is required. Details of the statistical analyses performed can be found in Papers I-V.

In Papers I, II and V, principal component analysis (PCA) was used to identify the parameters responsible for the variation in the data. This made it easy to pick out the extraction solvent resulting in the highest yield and, more importantly, to verify the P compound groups on which this depended (Paper I). In Paper II a variety of statistical analyses were carried out in addition to PCA to describe the variation in the results and to measure the repeatability and reproducibility of the analysis method. The main goal was to map the steps in the analysis method that caused variation and to determine the extent of this variation.

In Paper V, PCA on all the P compounds detected at the different sampling sites showed the extent of relationship or similarity between these sites. The two extracts from the lake sediment with agricultural catchment had a similar composition of detected P compounds but differed from all other sampling sites. The two extracts from the lake sediment with forest catchment did not have a similar composition of detected P compounds and also differed from the other sampling sites. The three extracts from three different sediment depths in the Baltic Sea Proper showed a trend-wise difference. Non-parametric statistical analysis (based on ranks, Figure 4 in Paper V) revealed the ion signals (representing certain P compounds) that were mainly responsible for the differences or similarities. For example, 12 compounds were found to substantially increase their share in detected P compounds with Baltic Sea Proper sediment depth.

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6. Conclusions and future aspects

More detailed understanding of the biogeochemical P cycle is of major importance in order to decrease the negative consequences of eutrophication in a sustainable and long-term manner. Although P bound into organic compounds is considered to be of major importance in many aquatic systems, research in this area has experienced difficulties due to lack of appropriate analytical techniques.

The use of 31P-NMR has increased knowledge of the partial structure and binding sites of organic P compounds and of their origin and turnover. However, individual organic P compounds within one group detected with 31P-NMR can have different degradability. The LC-ESI-MS/MS developed in this thesis proved to be a powerful tool for detection of this individual organic P compounds and has for the first time been used to study the turnover of specific compounds within a sediment profile. This is a great step forward compared to more conventional analysis methods and paves the way for a more thorough understanding of the turnover of specific organic P compounds and of the P cycle in the aquatic environment in general.

More specifically, the identification of a broader range of sediment organic P compounds and their quantification is required. This can be obtained by more selective extraction methods resulting in less matrix compounds and especially causing less degradation. Another aspect to this is the development of complementary separation techniques taking care of non-retained compounds on PGC.

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7. Acknowledgements

Great thanks to the department of Analytical Chemistry and to the dept. of Limnology for enabling this Ph.D. financially. When I started almost five years ago, I could never have imagined meeting so many great and inspiring people. What follows is a mere glimpse of all the people that I’m grateful to.

My (co-)supervisors:

Monica Waldebäck, for all help with writing this thesis, for research discussions, for giving me so much of your time, for your inspiration and enthusiasm about chemistry, for sharing all your wisdom of life… Emil Rydin, for all research discussions before and during the writing of this thesis, for your time and enthusiasm, for fun and interesting sampling trips and for seeing the potential in every little bit of result or mass spectrum. Per ‘J.R.’ Sjöberg, for all invaluable help with mass spectrometry, for help with the writing of my papers, for all input during the writing of this thesis, for research discussions and for appreciating Belgian beers. Rolf Danielsson for doing all the statistical analysis and taking time to try and make me understand. My co-authors:

Joakim Ahlgren, for convincing me to come back to Sweden, for all help with research and all discussions, for reading through my thesis, for all fantastic trips in Sweden with your wife Ulla, for talking me into some great conference trips,… Kasper Reitzel, thanks for all email discussions during the writing of this thesis, for joining our sedimentomics group, for a great time in Uppsala, Ødense and Berlin. Niklas Forsgard, for help with pictures and for support whenever I managed to destroy the ICP. Jean Pettersson for all ICP-time, didn’t mean to ruin it…Adolf Gogoll, for all NMR time. ‘My’ exam-students:

Lena Israelsson and Katrien Eeckhout, for doing a great job with interesting results despite all kinds of diffuclties!

Barbro N, for all administrative help and for always being nice no matter how

stupid my questions were or how many times you already had answered them before. Yngve, for all the help with labsupplies and nice conversations about life, sorry to have bothered you so many times during your lunch or fika break, I hope you’ll forgive me ;-). Bosse, for all computer help.

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My roommates: Sara B, Jocke, Jörg, Erik P and Sofia, I really enjoyed sharing the office with you. Remember, take care of the plants! The Green Team (present and former members) for all advice on my thesis and for reading through my papers, for fun and interesting meetings, for research discussions and for being nice and inspiring people. To all colleagues for a nice working place, for fun Friday Spraybars and afternoon fikas, for really cool and fun department trips (special thanks to Jean for organising them), research discussions and discussions about life, sports, the Belgian scandals and much more. My former colleagues, for pictures (Andreas), for encouragement, for a fun research environment, for innebandy, dart tournaments, Friday pubs…

And now some people not related to work! My friends in Uppsala: ‘Döbelnsgänget’, for great friendship, filmevenings (thank you Martin!), numerous parties and for helping me over the shock of seeing my 11 square meter room for the first time. Alvaro and Dominic, for all nice lunches outside of BMC and for all the great parties. Alvaro, for all Capoeira training, nice Spanish food (even though it requires patience) and because I can just come and visit you without warning! Kristina, Anna and Franziska, because you’re great people, with great ideas, loads of energy, made me look at the world with different (critical) eyes and have encouraged and believed in me during my Ph.D. studies. Anna, for numerous telephone calls and all moral support. My Belgian friends, An&Ties, Kenneth&Ilse, Arne&Febe, Dimitri, Nathalie and Sarah for trying to meet me whenever I’m in Belgium. I have fond memories of your visits! My family: Aan mijn moeder, bedankt voor alle telefoonuren om mij op de hoogte te houden van wat er allemaal in België gebeurt. Ma&Pa, Lieve&Patrick, Céleste, Camille, Babette, Nancy&Dimitri, Mila en Nore; Hilde&Tom, Victor, Léon en Achiel; bedankt om mijn thuiskomst steeds weer even gezellig en aangenaam te maken! Dan, for making it even more fun being in Sweden and for loving me the way I am. Your family, for all relaxing weekends at your home away from all must-dos.

Thanks! Heidi De Brabandere 1st of October 2008, Uppsala

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8. Summary in Swedish

Varför studera just P (fosfor) föreningar, som är organiska, och varför leta efter de på bottnen av sjöar och hav? Det finns flera skäl till detta som är tätt involverade i varandra. Till att börja med är P, tillsammans med kväve (N), ett viktigt näringsämne för primärproduktionen (växtplanktonproduktionen) i akvatiska miljöer. P är ofta ett tillväxtbegränsande ämne, det vill säga, finns det mer P så ökar primärproduktionen. För att få bättre förståelse för vad som reglerar tillgången på P behöver P cykeln i akvatiska miljöer kartläggas. P koncentrationen i t.ex. en sjö bestäms både av externbelastning, d.v.s. in-flöde utifrån (t.ex. lantbruksavrinning, rester efter kommunal vattenrening, etc.) och det som kallas internbelastning, d.v.s. P som efter att ha tillförts i vattnet har lagrats i sedimentet och under vissa förhållanden återigen kan frigöras till vattnet. Därför är kunskapen om P också viktig vad gäller över-gödningsproblematiken. Övergödning av vatten betyder tillförsel av en för stor mängd av näringsämnen (främst N och P) vilket ofta leder till extremt stor primärproduktion, vilket yttrar sig som t.ex. algblomning. Detta kan leda till hälsofaror p.g.a. toxiska ämnen som vissa alger producerar, minskning i biodiversitet samt syrebrist och ökning av syrefria bottnar. Internbelastning innebär dessutom att en minskning av tillförsel av P till det övergödda sy-stemet ofta inte räcker för att vända övergödningsspiralen.

Även om P i vatten och dess omsättning är ett väl studerat ämne, så har den största delen av forskningen fokuserats på oorganiska P föreningar, löst fosfat, samt dess bindningar till järn, aluminium och kalcium. Däremot har det inte gjorts så mycket forskning vad gäller karakterisering av organiska P föreningar trots att de har visat sig vara en stor och viktig del i den total P omsättningen. Det som har gjorts vad gäller organiska P föreningar i sedi-ment har gjorts med 31P nukleär magnetisk resonans (31P-NMR). Denna tek-nik mäter hur P är bunden i en molekyl och på så sätt kan man skilja på föl-jande P grupper; orthofosfat monoestrar, orthofosfat diestrar, orthofosfat och pyro- och polyfosfat. På så sätt har omsättningen av de olika P grupperna kunnat följas i miljön t.ex. från land till vatten, eller från sedimentet till vatt-net. Detta kan i längden ge en ökad förståelse om P cykeln. Däremot ger 31P-NMR inga uppgifter om strukturen av en enskild P molekyl. Detta är nöd-vändigt eftersom de olika P föreningar som utgör en P grupp antagligen har olika grader av nedbrytbarhet och alltså kommer att bidra olika mycket till internbelastningen. Vilka P föreningar är det som ger mest upphov till in-ternbelastning och vilka borde man därför fokusera på när mängderna blir

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för höga och ger upphov till övergödningsproblem? Dessutom är det också viktigt att veta vilka deras källor är; åkermark, skog eller andra källor på land.

Ett bra val för att studera enskilda molekyler är masspektrometri, en tek-nik där varje enskild molekyl kan detekteras genom att mäta massa över laddning av joniserade molekyler. Den masspektrometerteknik som använ-des i Papper II, III och V kräver att molekylerna är i lösning, alltså behöver de organiska P föreningarna extraheras från sedimentet. Att använda tradi-tionella extraktionsmetoder, vilka oftast är alkaliska extraktionsamedel (lös-ningar), är inte helt oproblemtiskt. Alkaliska extraktionsmedel, oftast base-rad på natriumhydroxid (NaOH), kan hydrolysera vissa organiska P före-ningar. Detta skulle kunna undvikas genom att använda sig av analysmetoder som inte kräver att föreningarna befinner sig i lösning utan att de kan vara kvar i den fasta fasen, sediment. Tyvärr ger de befintliga metoderna såsom fast-fas-NMR inte tillräcklig hög upplösning så det som återstår är att försö-ka optimera befintliga extraktionsmetoder eller hitta på nya. I Papper I har olika befintliga alkaliska extraktionsmedel utvärderats utifrån total P utbyte och enskilda P grupper mätt med 31P-NMR. Förextraktion med EDTA följd av extraktion med NaOH gav högst total P utbyte medan för-extraktion med BD optimerade utbytet av en viss organisk P grupp (poly-P).

Ytterligare en aspekt att ta hänsyn till, är att det är svår att injicera ett smutsigt sediment extrakt, med många matriskomponenter och relativ få organiska P föreningar som är av interesse, direkt in i en masspektrometer. Dessutom ska molekylerna joniseras före en mass spektrometrisk detektion. En bra joniseringsmetod som anses vara ’mjuk’ för molekylerna, d.v.s. inte slår sönder de i mindre delar, är t.ex. elektrospray jonisering (ESI). Jonise-ringsprocessen i ESI fungerar mindre bra om det t.ex. finns många Na-joner som hindrar P molekylerna att komma fram till mass spektrometer delen. Papper II är därför helt ägnat åt utvecklingen av en provupparbetningsme-tod som tar bort Na-joner, höjer organiska P föreningars koncentration och avlägsnar några av de största matrisföreningarna genom använding av filter. Studien beskriver hur stora olika felkällor är och var de uppstår. Under arbe-tet med denna artikel blev det snart tydligt att separation av de olika organis-ka P föreningarna, från varandra och från olika matriskomponenter, med hjälp av vätske kromatografi (LC), skulle öka selektivitet och känslighet. Då skulle även behovet av provupparbetning före analys minska, dessutom ökar möjligheten till identitetsbestämning av organiska P föreningar, eftersom de eluerar separerat från varandra. Utvecklingen av denna metod beskrivs i Papper III, som vätskekromatografisk kolonn användes en PGC (poröst grafit kol) kolonn som tål det höga pH:t i sedimentextraktet och som dessut-om kan separera isomeriska föreningar. Med hjälp av standarder identifiera-des främst olika nukleotider som kan tänkas vara nedbrytningsprodukter som bildas vid alkalisk hydrolys av en RNA sträng.

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I Papper IV följs omsättningen av P föreningar med vissa bindnings-grupper (P grupper, mätt med 31P-NMR) från vattenytan till 40 cm djupt i sedimentet där djupet motsvarar en viss ålder (desto djupare, desto äldre). På så sätt fick vi fram att olika P grupper bröts ner olika snabbt och vi fick en uppfattning om vilka P grupper som på så sätt frilägger P och därgenom ökar den interna belastningen. Medan i Papper V följs omsättningen av enskilda organiska P molekyler genom detektion med LC-ESI-MS/MS metoden som beskrivs i Papper III. I detta projekt studerades fyra sjöar med två olika avrinningsområden (två skogssjöar och två lantbrukssjöar) och tre olika se-dimentdjup i Egentliga Östersjön (ytsediment skiktet, 0-1 cm, 4-5 cm djup och 9-10 cm djup). Vi fann olika mönster på de extraherade och detekterade organiska P föreningar beroende på avrinningsområde och sedimentålder i Egentliga Östersjön. Olikheter i omsättningshastighet kan förklara dessa mönster. Vi lyckades detektera enskilda massor över laddning på molekyler som förekom såväl i skogssjösediment som djupare ner i Östersjösediment, båda är miljöer där man väntas se mer redan nedbrutna molekyler. Kunskap om nedbrytbarheten av olika organiska P föreningar från olika källor utgör grunden för att förstå vilka källor som läcker organiska P föreningar och för att i förlängningen skapa en effektiv hantering av övergödningen.

Sammanfattningsvis har jag studerat omsättningen av organisk P med 31P-NMR och optimerat extraktions-, provupparbetnings- och separationsmeto-der som möjliggör masspektrometrisk detektion på molekylärnivå med en nyutvecklad LC-ESI-MS/MS metod. Med denna metod har jag funnit ett antal organiska fosforföreningar i Östersjösedimenten, några har visat sig vara stabila, och andra inte d.v.s. de har brutits ner. Detta är ett stort steg framåt jämförd med tidigare analysmetoder och öppnar vägen till ökad kun-skap om enskilda P molekylers omsättning i miljön, vilket i förlängningen ger ökad förståelse för hela P cykeln i akvatisk miljö.

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