Phosphorus-31 NMR of Sediment Phosphoüpids

by

Emma EIizabeth Watts

Department of Chemistry

submined in partiai fulfilment of the requirements for the degree of

Master of Science

FacuIty of Graduate Studies The University of Western Ontario

London, Ontario August 1999

Q Emma E. Watts 1999

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Abstract

Phosphorus-3 1 nuclear magnetic resonance spectroscopy (NMR) has been used to

examine sedunent phospholipids which ate believed to be indicative of the sediment

microbial community. Two isolated watersheds in southwestern Ontario were investigated

for phospholipid abundance and richness. The resuits obtained show seasonal variations

consistent with the existing fiterature on soil microbes. Both abundance and richness aIso

show well-de£ined spatial variation. The two sites show remarkably different phospholipid

patterns. Generaily, atypical behaviour in any sample set may be attributed to human

disturbance but some differences appear to be due to physical characteristics such as

turbidity. In general, the results show that NMR is a promising technique to monitor changes

in soif microbid communities.

The signincant problems we face cannot be solved at the same ievel o f thinkuig we were at when we created them.

Albert Einstein

1 would like to thank Dr. Ron Martin for his guidance and support throughout the

course of this thesis. A special th& to Dr. Phü Dean and Dr. Sammy Sammynaiken for

their instruction and advice with the NMR T would iike also Like to thank Dr, Kee

Dewdney, Dr. Anwar Maun, Dr. Rob Schincariol and A n k a Williamson for their

coliaborative research and insights into the two study areas. This work would not have been

possible without the guidance of Mr. Ross Davidson, ML Mark Biesinger and Ms. Mary

Jane Walzak on the various instruments at Surface Science Western. FinaiLy, a special

thanks to Mark, Gina and Dave whose support and fnendship made the whole process so

much easier.

Table of Contents

. . Certi£icateofExamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . u

... Absixact ...-.....,.....-...........--.-............-..------..-.......iir

Acknowledgements ...................-...--.-œœœ..-.---...~...-........~v

....................................................... Tableofcontents vi

ListofFigures . . . . . . . . . . . . . . . . . . . . . . . . . . . . - . . . . . . . - . . . . . . . . . . . - . . . . . . . . xi

-.- ListofTables ......-..........-...-.--..........-....-.---.-.-.--...--xrrr

ListofAbbreviations ................................-.-................ xiv

1,Introduction . . . . . . . . . . . . . . . . . . . - . . . . . . . . .~. . , . . . . . . . -- . . . -- . . . . . . - . . . - l

1.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - . . . . . . . . . . . . . t . . . . . . 1

1.2 The History of Nuclear Magnetic Resonance Spectroscopy of Soils ...... - 4

............................. 1.3 Phosphorus in Freshwater Ecosystems - 5

1.4 TheStudyAreas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1.4.1 The OId Ausable River Channel .......................... - 7

1.4.2 The Kirk Cousins Management Area ....................... -9

.......................................... 1.5 Research Objectives - 1 1

................................................... 1.6 References 13

2. Instnimentation . . œ . . . . . . . . . . . . . - . . . . . . . . . . - . . . . . . . . - . . . . - . . . . . . œ - . . - . l 6

................. 2.1 Nuclear Magnetic Resonance (NMR) Spectroscopy -16

........... 2.2 Inductively Coupled Plasma - Mass Spectrometry (ICP-MS) 19

.......................... 2.3 X-ray Photoelectron Spectroscopy W S ) -21

...................................... 2.4 Ion Chromatography (IC) -23

................................. 2.5 Electrospray Mass Spectrometry -23

....................................... 2.6 X-ray Diffraction (XRD) -24

2.7 Static Secon dary Ion Mass Spectrometry (SIMS) .................... -25

................................................... 2.8 References 25

............................................... 3 . Phospholipid Analysis -27

.................................. 3.1 Site Seleciion and Description -27

.......................... 3.1.1 The Old Ausabie River Channel 27

...................... 3.1.2 The Kirk Cousins Management Area -28

3.2 Experimental ................................................. 28

.................................... 3.2.1 Sediment Collection 28

................................. 3.2.2 Phospholipid Extraction 30

............................ 32.3 "P NMR ~eagent Preparation 31

................. 3 .2.4 Sample Preparation for "P NMR Analysis -31

................................ 3 .2.5 "P NMR Spectroscopy - 3 2

............................ 3.2.6 Phospholipid Quantincation - 3 2

.............................. 3.2.7 Phospholipid Identification 33

...................................... 3.2.8 X-ray Dfiaction 36

............................................ 3.2.9 Matenals 37

........................................ 3.3. Resuits and Discussion 37

3.3.1 The Old Ausable River Channel .......................... 37

...................... 3.3.2 The Kirk Cousins Management Area -41

vii

.................................... 3 -4 Phospholipid Quantification -46

3 -5 Phospholipid Identification ..................................... -48

3.6 Conchsions .................................................. 49

.................................................. 3.7 References - 5 0

................................ 4 . Water Analysis for Dissolved Phosphonis -52

4.1 Introduction .................................................. 52

................................................. 4.2 Expehental 52

.................................... 4.2.1 Sample Collection -52

4.22 Sample Preparation for ICP-MS Analysis ................... 53

........................................ 4.3 Results and Discussion -53

4.3.1 The Old Ausable River Channel ......................... -53

4.3.2 The Kirk Cousins Management Area ...................... - 5 5

4.4 Conclusions ................................................. - 5 8

................................................... 4.5 References 58

...................................... 5 . Ancillary Identification Techniques 60

.................................................. 5-1 Introduction 60

5.2 Experimental ................................................. 60

............................ 5.2.1 Thin Layer Chromatography 60

......................... 5.2.2 Electrospray Mass Spectrometry - 6 1

........................ 5.2.3 X-ray Photoelectron Spectroscopy -61

................... 5.2.4 Static Secondary Ion Mass Spectrometry - 6 2

............................................ 5.2.5 Materials 62

5.3 Results and Discussion ........................................ -62

5.3.1 Thui Layer Chromatography ............................ - 6 2

5.3.2 Electrospray Mass Spectrometry ......................... -63

5.3.3 X-ray Photoelectron Spectroscopy ......................... 65

5.3.4 Static Secondary Ion Mass Spectrometry ................... - 6 5

5.4 Conclusions ................................................. -67

5.5 References .................................................. -67

6 . Phosphoiipid . Calcite Interactions ....................................... 68

6.2 Experime rital ................................................. 68

6.2.1 Methodoiogy ........................................ -68

6.2.2 Ion Chromatography .................................. -69

6.2.3 Materiais ........................................... -70

6.3 Results and Discussion ........................................ -70

6.3.1 Standards and Controls ................................ - 7 0

.............. 6.3.2 Potassium Acid Phosphate, Proof of Principle - 7 2

6.3.3 Phosphatidic Acid ..................................... 74

6.3 -4 Diethyl Phosphate ..................................... 77

6.4 Conclusions .................................................. 77

................................................... 6.5 References 78

7 . Conclusions and Future Work .......................................... - 7 9

7.1 Conclusions .................................................. 79

.................................................. 72FutureWork 81

.......................... Appendix 1: Individual Phospholipid Concentrations - 8 2

AppendixII:NMRSpectra ............................................... 85

....................................................... CUmculumVita 108

List of Finures

Figure Description page

1.1 Stnicture of Phosphatidic Acid ...................................... - 3

1.2 Major Classes of Phospholipids ............... ,, ..................... - 4

.................... ........... 1.3 Map of the Old Ausable River Channel ,, 8

1.4 Map of the Kirk Cousins Management Area ............................ 10

2.1 Schematic of an KP-MS .......................................... -20

2.2 The photoelectric effect, iliustrated for the emission of a phosphorus 2pelectron ....................................................... 22

...................... 2.3 Schematic of an X-ray photoelectron spectrometer -22

3.1 NMR spectra of phosphofipid standards; run 9 and nin 10 with the . .................................. addition of phosphatidylinositoL .. 35

3 -2 NMR spectra of Mudhoie South. s p ~ g 98. Mudhoie South. sumrner 98. and Burley Bridge North, summer 98 .............................. -40

3 -3 NMR spectra of a sample fiom Canoe Docks North, autuma and same sample foilowing 100 days outside in OARC water (Balcony

......................................................... sample) 42

3.4 XRD spectrum of suspended particdate matter fkom Pond Mu in summer98 ....................................................... 45

3.5 NMR spectni of Canoe Docks North samples; amalgamated sample. s p ~ g 98. single sampie. spring 98. spring 99 .......................... -47

4.1 Typical relationship between dissolved phosphorus and phospholipid .......................................... abundance in the OARC - 5 4

4.2 Relationship between dissolved phosphorus and phospholipid abundance at each site within the KCMA ............................. -57

5.1 Electrospray mass spectrum of unknown phospholipid foliowing TLC ....................................................... separation 64

6.1 XPS nwey scans showing the increase in adsorbed phosphate as the concentration of KH2P04 in solution increases _ - . . , . - - - - . , . , . . . ,. .. . .. . -73

6.2 Raman spectra of a cfean calcite crystal and a crystai exposed to a solution of KH,PO,. Adsorbed phosphate is ùidicated on the exposed sample by the peak at 927.7 cm-' . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . -75

6.3 High resolution XPS spectra of the phosphorus (2p) peak for the standard sample of phosphatidic acid (PA) and for a calcite d a c e exposed to 10 ppm solution of PA for 24 hours. The fitted peak is the overd contribution fiom both the P(2pd and the P(2p3,3 peaks . . . . . . . . -76

Table Description page

3.1 Seasonal and spatial patterns of total phospholipid abundance and ............................................. nchness in the OARC 38

3 -2 Seasonal and spatial patterns of total phospholipid abundance and ............................................ richness in the KCMA .44

4.2 Seasonal and spatial changes in total dissolved phosphorus in the ......................................................... KCMA 56

5.1 Signifïcant mass hgments and their assignments fiom the positive ....................................... secondary ion mass spectrum -66

6.1 Surface composition of standard samples in atomic percent as determioed by X-ray photoelectron spectroscopy (WS). Phosphorus (2p) binding energies for each sample are presented in the far right

........ column. Binding energies are referenced to carbon (1s) at 285.0 eV 71

List of Abbreviations

amu BBN BBS CDN CDS EDTA ESCA ES1 FAA FID GPC IC ICP-MS KCMA LPA LPC LPG MHN MHS MU NMR OARC 3'P PA PC PE PG PI rf Rc RMM RMS SIMS SM TLC TP u XPS XRD

atomic mass unit Burley Bridge North Burley Bridge South Canoe Docks North Canoe Docks South ethylenediaminetetraacetic acid electron spectroscopy for chernical anaiysis electrospray ionization fatty acid analysis fiee induction decay greatest phospholipid abundance ion chromatography inductively coupfed plasma - mass spectrometry KUk Cousins Management Area lysophosphatidic acid Lysophosphatidylcholine iysophosphatidylglycerol Mudhole North Mudhole South Pond Mu nuclear magnetic resonance spectroscopy Old Ausable River Channel phosphonis-3 1 phosphatidic acid phosphatidylcholine phosphatidy Iethanolamine phosphatidylglycerol phosphatidylinositol radio fiequency retardation factor Regina Mundi Middle Regina Mundi South secondary ion mass spectrometry sphingomy elin thin layer chromatography Turtle Pond unknowll X-ray photoelectron spectroscopy X-ray diffraction

xiv

1- Introduction

1.1 Background

Sediment microorganisms play an important role in the decomposition of organic

material,' the degradation of po~utants'" and the recychg ofnutrients? An evahation of

the biomass and composition ofthe microbial community is therefore essentid in assessing

soil quality and pollution impacts on an ecosystem The direct quantification of microbiai

biomass in sediment is constrained by the difnculty of isolatïng the microbes? Moreover,

the time involved in isolathg and identifjing Merent bacteria, h g i , algae and protozoa

makes it unreaiistic to process the nurnber of samples required to adequately define a redistic

ecosystem. However, the cellular membranes of microorganisms are largely composed of

phospholipids which are, in principle, characteristic of each celi type? A profile of sediment

phospholipids might therefore be used to fingerprint the microbial community. Since

phospholipids have a short haKlife in sedunent they can provide good estimates of viable

biomass at any given time as there should be no caq-over nom year to year.' In this way,

phospholipids may be usefiil short term indicators of ecosystem health.

There is sigdicant iïterature on pollution induced changes in the microbial

co~nmuniSh)~~~ and on a varîety of analytical methods used for its evaluation?None of

the traditional methods is completely satisfactory.

Culture methods, for instance, have many limitations. In fact, it is estimated that ody

a small percentage of soiI microorganisms can be cultured in the laboratory and these

analyses have o&n underestimated microbial populations in soil.' Culture studies have

2

however aided in the interpretation of results obtained by other methods. Culturing samples

has helped to elucidate the phosphoiipid and the phospholipid fatty acid composition of

specific microorganisms under a variety of external ~tresses.~ This work needs to be taken

out of the laboratory and tested under naturd condit io~s.~~

The popular fatty acid analysis (FAA) has proven to be one of the most useftl

methods available to evaiuate a microbid ~omrnuni ty .~ '~ This analysis examines microbial

Lipids indirectiy through their fatty acid compositioa FAA is a sensitive technique consisting

of severai separations of the iipid extracts as weii as the derived fatty acid methyl esters.

Zelies and Bai4 give a detaiied account of the solid phase extractions, chemical

derivatizations and of the h a 1 measurements by gas chromatography - mass spectrometry

required for F M

Unfortunately, FAA tends to examine the total iipid content of a sample rather than

solely its phospholipid content (aithough it is sometimes referred to as the phospholipid fatty

acid analysis). Phospholipids are generally regarded as providing a better characterization

of the current microbiai community. The phospholipids are degraded rapidly followiog cell

death compared to other cell iipids.' As a result, the fatty acids present may represent an

integration of materiai fiom the current living microbes and those previously deposited in the

sample. In addition, the many chernical steps involved in the FAA procedure rnight destroy

the integrity of the onginal soi1 sample. A method capable of faster sample turnover with

fewer chemical manipulations is necessary.

'lP nuclear magnetic resonance (NMR) spectroscopy is a powedùl tool capable of

analysing phospholipids extracted nom sediment directly. "P is the ody stable isotope of

phosphorus and has a nuclear spin of 95, making it sensitive to NMR Each phospholipid

contains a single phosphoms atom in its polar head group that can be detected. Structures

of typical phospholipids are shown in Figure 1.1 and 1 -2. NMR spectroscopy investigates

the nature of the phosphorus atom by monitoring the interaction of its nuclear magnetic

moment with radio fkequency electromagnetic radiation in a strong magnetic field In

p"ciple, each phospholipid species may be identifiai and quantiited. "P NMR wiII be

discussed in detail in chapter 2.

Figure 1.1 : Structure of Phosphatidic Acid

Phosphatidic acid is the simplest form ofa phospholipid. The length of the fatty acid

side chains and the degree of unsaturation Vary, t in Figure 1.1. Additional classes of

phospholipids are characterized by the substituent es teaed to the phosphate group of the

acid, $ in Figure 1.1. Some examples are shown in Figure 1 -2.

Phosphatidy lserine

Figure 1.2: Major Classes of Phospholipids. The classes listed above are obtained by the addition of the accompanyhg groups to phosphatidic acid.

1.2 The History of Nuclear Magnetic Resonance Spectroscopy of Soi1

Nuclear magnetic resonance spectroscopy was discovered over 50 years ago.lL

Although it was weil established for organic chernical analysis by 1965, only recent

technologicd advancements have permitted its routine application in soil studies.ll Early

work using NMR to investigate soil organic matter was carried out in 1963 when Barton and

S~hnitzer'~ pubiished their analysis of methylated humic acids ushg proton NMR Since

then, superconducàng magnets with high fields and the computing power of Fourier

transform techniques have extended the application of NMR to less sensitive nuclei such as

5

lL These developments aiso permitted the analysis of cornplex, heterogeneous soi1

smplesl 1, 13'14 and dihite phospholipid sediment extracts.' Early phospholipid experiments

using NMR did not provide sutncient resolution for the accurate quantification of

individual phospholipids. This poor resolution is caused by the presence of paramagnetic

cations CO O rdinated to the phosphodiester fùnctional groups of the p hospholipids. l5 These

ions may broaden or quench the resonance si@ by inducing "P NMR relaxationL6

Improvements in the sediment extraction process have Wtuaily eliminated this problem by

using phosphate and ethylenediaminetetraacetic (EDTA) acid as chelating agents to remove

cations.* There are as yet few Literature references that may be used to assess the quantitative

reliability ofNMEL when phosphate aud EDTA are used in sample preparation. This research

seeks to add to the limited information available on the use of 31P NMR in the identification

and quantification of sediment phospholipids.

1.3 Phosphorus in Freshwater Ecosystems

Phosphorus is an essential nutrient for plants and is ofien the limiting nutrient in

freshwater systems.17 Its importance in the process of eutrophication has been emphasized

repeatedly.lbLg Anthropogenic inputs represent the greatest contribution of phosphorus to

natural waters." Sewage, agricultura1 runoff and detergents are ali major contributors.

Naturaliy, in the absence of direct human impact, atmospheric deposits and mineral

weathering represent its principal s0urces.l

Phosphoms exists in fieshwater bodies in both the organic and inorganic particdate

and dissolved forms. It ia primarily the dissolved inorganic phosphorus that is viewed as

6

bi~available.'~ The retention of available phosphorus is a important part of the phosphoms

cycle, con t rohg both water quality and soil fertiiity. Biotic factors contrr'buting to its

retention include uptake by vegetation, plankton, periphyton and micr~or~anisms.'~ Abiotic

processes such as sedimentation, chemicai precipitation and chernical adsorption also aid in

the attenuation of phosphorus in aqyatic systems." An explanation of the phosphoms cycle

wilI not be provided here. The interesteci reader might note that VymazalZL and Kramef7

give a comprehensive review of the processes involved. Reddy et ai-" also give a detailed

account of phosphorus retention in streams and wetlands-

This research will focus on phosphorus sequestration by the microbiai community

and its relation to phosphorus in the water column In systerns with high organic inputs such

as waste effluent loadings and detrital plant matter deposition, microorganisms play a major

role in phosphoms retention?O Newbold et aLn demonstrated that microbial uptake could

be responsible for up to 91% of the total phosphorus retention in a woodland Stream. This

excessive up take of phosphate by bacteria and its subsequent release under unfavourable

conditions have been weil documented? The knowledge has now been put to use as a

cheap and natural alternative for phosphorus removal f?om water in sewage treatment

plants.526

1.4 The Study Areas

Study areas were selected to minimize extraneous variables such as multiple pollution

sources. Two very dEerent isolated watersheds were investigated. The Old Ausable River

Channel has been shown to be a phosphorus poor system with precipitation and groundwater

7

seepage as its ody water sources.* The Kidr Cousins Management Area consists ofover 20

kettle ponds in wetland, forest and meadow ecosystems." Other than atmospheric

deposition, the oniy possible sources of phosphoms in this system are local- This area has

some sampling sites presumed to be pristine while others are impacted by human efnuent

inputs. A single, direct source of human effluent fiom Regina Mun& College at the Kirk

Cousins site is the major Merence between this site and the Old Ausable River ChannelI

1.4.1 The Old Ausable River Channel

The Old Ausable River Channel (OARC) was chosen as it is a relatively simple,

isolated system that is vulnerable to eutrophication. It is only 14 km in length, with a flow

rate averaging less than 1 cm/s and a maximum depth of 2.5 metres." A map of the Old

Ausable can be found in Figure 1 -3.

The Old Ausable River Channel begins in Grand Bend, Ontario, and flows southwest,

parallel to Lake Huron In 1892, the construction of a canal completely separated the old

channel fiom the Ausable River." Today, the Old Ausable is fed exclusively by precipitation

and groundwater seepage? Its water level is maintained by a series of culverts and a dam

The channel travels through the rare oak savanna ecosystem of the Pinery Provincial Park

and continues southwest to the park boundary, just south of Port Franks. It then rejoins the

Ausable River, flowing West into Lake Huron_

An interdisciplinary study of the Old Ausable was initiated due to concems

surroundhg the abundant plant life in the river during the summer. The Pinery boasts over

a million visitors each year and the deterioration of the Old Ausable threatens the Park's

Figure 1.3: Map of the Old Ausable River Channel." Produced under Licence h m Her Majesty the Queen in Rîgk of Canada. with permission of Natural Resources Canada.

9

annual incorne. The underwater vegetation is often so dense that most aquatic actMties are

unappealuig. Many private houses also Iine the baaks of the Old Ausable between Grand

Bend and the noahem boundary ofthe Park Overflow fiom septic tanks, nuiofffkom lawns

and driveways and the increased human activity within the Park may be affecting water

q~ality.~O

Favourable growing conditions are provided by the slow ruovin& shdow waters

which receive ample sunlight and remain warm aii summer. D7Ulisse and ~ a u n ~ ' have

shown that some areas of the channel are stagnant and in faet, show signs of a lentic

ecosystem. Large sections ofthe river have low turbidlty, aüowiag sunlight to reach the river

bottom and causïng waterternperatures to nse. Therefore, even modest nutrient inputs could

cause accelerated vegetative growth. The Old Ausable is akeady showing more advanced

signs of natural eutrophication. The taste and odour problems associated with anoxie water

are apparent.

1.4.2 The Kirk Cousins Management Area

The Kirk Cousins Management Area (KCMA) is located in London, Ontario, just

south of Highway 401, east of Wellington road. The KCMA, established in May 1982,

consists of 25 ha of si@cant wetland and 16 ha of meadow. It represents an important

headwater source for the Kettie Creek watershed. There are over 20 kettle ponds in the area,

established by successive glacier melts 13,000 - 11,000 years ago? They occupy the height

of land between Lake Erie and Lake Huron and are therefore isolated fiom al1 but local

pollution sources. A map of this region c m be found in Figure 1-4.

I l

In 1963, Regina Mundi Coiiege was built on the edge of the management area as a

junior semhuy- In 1965, it was expanded to admit residentiai, non-seminary students and

by 1973, student enrollment totalied 152- Raw sewage fiom the coiiege once entered a pond

in the management area but it now passes through a treatment plant designed for phosphorus

removd and ultraviolet disinfection prior to discharge into the adjacent pond. Aithough a

treatment plant had been in place since the schooI was bd t , it was poorly maintaineci and

out of s e ~ c e for many years. An upgraded treatment p l a t was approved in August 1995.

The Ministry of the Environment now requires routine operational testïng of the fàcility and

enforces etnuent discharge requirements. In 1983, Regina Mundi was converted fkom a

residentid school to a day school. Its current staff and student population is 1426.~' Since

precipitation is the sole source of water for this area, the impact of the wastewater on the

pond's microbid cornxnety can be examined.

1.5 Research Objectives

The phospholipid distribution in sediments can, in principle, serve as an indicator of

the composition and health of the microbial biomass. Since NMR may be the most powemil

technique currently available to probe phosphoiipids, the fist objective of this study is (1)

to evaluate the utility of "P NMR in the qualitative and quantitative assessrnent of sediment

phosphoiipids in two weli defhed, distinct and environmentally important systems. This

evaluation will be achieved in part by (2) detennining whether or not seasonal and spatial

changes c m be observed at either of the rtudy areas usiag NMR. Since seasonai changes in

the microbial biomass are well docu~nented?~~ any fdure of NMR spectra to show

12

systematic changes would suggest that the techaiwe is seriously flawed. Ifseasonal changes

are detected by NMR, spatial changes c m be interpreted as changes in the microbial

comunity. Both richness and abundance of phospholipids will be considered. These terms

are given the foliowing narrow definitions for the purpose of this study. Abundance is

d e h e d as the total amount ofphospholipid present while richness represents the number of

different phosphofipids present in the sample-

The choice of study sites in the KCMA will d o w (3) a cornparison of the

phospholipid abundance and richaess in ponds r e c e h g human effluent with that in ponds

not exposed to a comparable pollution source.

(4) A single expennient is also camed out to explore the rate of naturai degradation

of phospholipids to determine which phospholipids, if any, niMve the winter.

Since the positive identification of a phospholipid revealed by NMR c m only be

obtained by standard addition, (5) attempts will be made to identay individual phospholipids

both by standard addition and by other techniques uicluding electrospray mass spectrometry.

(6) The relationship between the abundance of sediment phospholipids and the

concentration of dissolved phosphorus in the water co lum will be examined. ICP-MS is

combined with NMR in this portion of the research.

Finally, X-ray photoelectron spectroscopy facilities are available and (7) will be used

to explore phospholipid adsorption onto calcium carbonate, one of the most abundant

minerals in each study area.

1.6 References

1. O. A. Chadwick, L. A Derry, P. M, Vtousek, B. J- Huebert, and L. O. Hedin. Naf~re,

397,49 2 (19W).

2- T. Pemanen, H- Fritze, P. Vanhaia, O. Kïikkiiii, S. Newonen, and E. AppL E m

Microbiol., 64,2173 (1998)-

3. E. B u M. Diaz-Ravlna, k Frosteghd and C. Campbell, AppL Env. Microbid, 64,

238 (1998).

4. L. Zeiles and Q. Baï, Sait BioL Biochem., 25,495 (1993)-

5- D. C. White, Microbes in their Natural Environments, J. H- Slater, R Whittenbury and

J. W. T. Wmpenny, Eds., Cambridge University Press, Cambridge, 37-66, 1983.

6- D. Langworthy, R Stapieton, G. Sayler, and R Findlay, Appt- E m Microbiol., 64,3 422

(1998).

7. S. M. PfXher, A V. Palumbo, T. Gibson, D. B. Ringelberg, and J. F. McCarthy, Appl.

Biochern. Biotech., 63, 775 (1997).

8. L. Bardygula-Nonn, J. L. Kaster and T. Glonek, Lipirls, 30, 1047 (1 995).

9. N. Rajendran, O. Matsuda, Y. Urushigawa, and U. Simidu, Appl. Em. Microbid, 60,

248 (1994)-

10. N. Rajendran, O. Matsuda, N. hamura, and Y. Urushigawa, Appl. Env. Microbiol., 58,

562 (1992).

I 1. C . M. Preston, Soil Science, 161, 144 (1996).

12. D. H. R Barton and M. Schnitzer, Nahrre, 198, 217 (1963).

13. 2. R Hinedi, A C . Chang, and R W. K- Lee, J Environ. Q d , 18,323 (1989).

14

14. M. Nègre, M. Gennarï, C. Crechio, and P. RuggÏero, Soli Sci-, 159, 199 (1 995).

15. T. Glonek, "P NMR Spectral Properties in Compound Characterization and Structural

Anaiysis, L- D. Quin and J. G. Verkade, Eds., VCH Publishers, New York, 283-294,

1994.

16. T. O. Henderson, A- W. Kruski, L. G. Davis, T. Glonek, and A M. Scanu,

Bfucherni.sfry, 14, I9 15 (1975).

17. J. R Kramer, S- E. Herbes, and H- E- Men, Nutnents in Natural Waters, H- E. Ailen

and J. R Kramer, Eds., John Wdey& Sons Inc., New York, 51-100, 1972.

18. A F. Bartsch, Role of the Federal Govemment in Controhg Nutrïents in Naturai

Waters presented at the American Chernical Society Symposium on Nutrients in Nitturat

Waters, Los Angeles, Mach 28-Apd 2, 1972.

19. J. R Valentyne, Cm- Res. Dev., 49,36 (1 970)

20. K. R Reddy, R H. Kadlec, E. Flaig, and P. M. Gale, Critical Reviews in Environmental

Science anci Technologv, 29, 83 (1999).

21, J, Vymazal, Algae and EIement Cycling: in Wetlaads, Lewis Publishers, London, 257-

271, 1995,

22. D. J. Newbold, J. W- Elwood, R V. O'Nedi, and A L. Sheldon, Ecology, 64, 1249

(1983).

23. M. H. Deinema, M. Van Loosdrecht, and A Scholten, Water Sci. Technol., 17, 119

(1985).

24. Y. Corneau, K. J. Hall, R E. W- Hanrock, and W. K- Oldham, Water, 20, 15 11 (1986).

25. Fuhs, G. W. and M. Chen, Microb. E c d , 2, 119 (1975)-

26. G. V. R Marais, R E. Loewenhal, and B. M- Simon, Wder Sci- T e c h i - , 15, 15

(1983).

27. J. N. Steinbach, Hvdmeolow of the Old Ausable River Channel (OARCI Watershed,

Grand Bend Ontario, MSc- Thesis, University of Western Ontario, London, Ontario,

1999,

28. Public records fiom the Ministry of Environment and Energy-

29. M. A- Maun, RA Schiacariol and R R Martin, Biolow. Hvdrogeolow and Chemistry

of the Old Ausable River Channel Watershed, Unpublished Progress Report for the

Acadernic Development Fund, The University of Western Ontario, London, Ontario,

1998.

30, A. D'Ulisse and M- A Maun, The Ecolow of The Old Ausable River Channel Flowinq

Throueh the Grand Bend and Pinerv Dune Svstem, Unpublished Report, Department

of Plant Sciences, University of Western Ontario, London, Ontario, 1993.

3 1. Mapping SeMces Branch of Geomatics Canada, Natural Resources Canada, 40 Pf4 O

6" edition and 40 P/5 Q 4h edition, 1985.

32. D. A. Bossio, K. M- Scow, N. Gunapala, and K J. Graham, Microb. Ecol., 36,1(1998).

33. M. Wood, Soi1 Biolow, Blackie and Sons Ltd-, New York, 80-82, 1989.

2. Instrumentation

2.1 Nuclear Magnetic Resonance (NMR) Spectroscopy

Nuclei with non-integer nuclear spins assume quantized orientations in strong

magnetic fields. Transitions between these orientations can be induced with a radio

fiequency electrornagnetic field. The fiequencies at which absorption of radio fiequency

energy occurs can be used to ident* individual atoms and their chernicd state. These

properties are exploited in NMR. An excellent review of the basic principles of NMR

spectroscopy is given by R S. Macornber' and by R K. Harris?

NMR takes advantage of the magnetic properties of certain nuclei. Nuclei with

unpaired (or odd numbers of) protons or neutrons wili have a spin associated with them.

Nuclei with a net spin, such as hydrogen-1 (nuclear s p i . O = %), deuterium (I = l), carbon-

13 (I = %), and phosphorus-3 1 (I = %), have a magnetic moment. When nuclei (I + 0) are

placed in a strong magnetic field (Bo), they wiii adopt 21 + 1 energy States, For nuclei with

I = %, tWO states wi l l occur, parailel (I = %) and antipardel (I = - 54) to the magnetic field.

Since the antiparaliel arrangement is energeticdy less favourable, there wiIl be slightly more

atoms aligned paralle1 to the field than anti-parallel, according to the Boltzmann distributioe

The energy difference (AE) between the two states (EL for the +% state and E, for the -%

state) is equal to:

AE=&- E,=)iyB,

where h is Planck's constant divided by 2x, Bo is the magnetic field, and y is the

gyrornagnetic ratio. This ratio, a collection of nuclear properties, descnbes how much spin

17

state energies of a given nucleus vary 6 t h changes in an extemai magnetic field. The

gyromagnetic ratio is different for a l l magnetic nuclei. Therefore, by v q i n g the field

strength @,), one may huie the NMR experiment to specifk nuclei and M. The sïze of y

plays an important role in the sensitivity of a nucleus, with a larger y (such as that for 'H)

giving a larger AE than a smaiier y (such as that for 3'P).

Before nuclei in these Herent spin states cm absorb electromagnetic radiation, they

must be osciüating in a periodic motion. The nuclei, although statisticaily h e d up with Bo,

are actudy arranged at some angle fiom it. Each nucieus precesses around the axis of Bo

at a characteristic fiequency calied the Larmor fiequency (a).

o = yB,

If a second magnetic field (BI) is generated perpendicular to Bo and this field is oscillating

at the Larmor frequency, the magnetic moments of the nuclei wili Line up and precess with

BI about Bo. When this occurs, the precession of the net magnetic moment can be detected

by Faraday induction within the receiver coil. During irradiation by B,, energy will be

absorbed by the nuclei, prornoting E, to E, by a spin f ip. When the rate of promotion of

nuclear spins fiom the Iower to the higher energy state equals that of r e m to the lower

energy state, as by stimulated emission, no M e r absorption of energy occurs and the

system is said to be saturated. BI is pulsed to prevent saturation, ailowing the nuclei to retum

to their normal Boltzmann distribution in between pulses. The time needed to retum to this

state is known as the spin-Iattice or longitudinal relaxation tirne (T,). Another relaxation

process also occurs; spin-spin relaxation. This is the individual nuciear magnetic moments

rehirning to a random arrangement around the axis of B, The time required to do this is

18

cailed the spin-spin or trausvene relaxation tirne (Ta.

The magnitude of the external magnetic fie'd that the nucleus experiences is aEected

by shielding ftom the surrounding electron cloud and also by electron density in other parts

of the molecule. Changes in electron shielding give rise to nuclei with different Larmor

precession fiequemies which in turn give rise to the chemicai shift In the earlier years of

NMEt, these different nuclei were detected by sweeping B,. With the advent of more

powemil cornputers, al1 fiequencies are analyzed si.multaneously using a brief but powerful

pulse of monochromatic radio fiequency (rf) electromagnetic radiation (Fourier transform

m). A complex rf signal (interference pattern) that incorporates the precession and

relaxation of al1 nuclei is received. This is the fiee induction decay (FID) signal. The

Fourier transform is then employed to convert this signal ftom the time domain to the

fiequency domain, which gives the normal NMR spectm.'

The effective magnetic field that a nucleus experiences can also be affected by the

rnagnetic spin state of neighbourbg atoms. A neighbouring atom with 1 = % will slightly

increase the magnetic field felt by the nucleus and vice versa for an atom with 1 = - % . In

a sarnple, roughly equivalent amounts of each spin state will be found. This will give rise

to two equal peaks for this nucleus. More neighbours (with 1 + O) will give rise to more

combinations of the effective magnetic field experienced by the nucleus, resulting in

additional resonance signals. This is known as coupling. In this work we wish to avoid

coupling of our target nuclei C'P) vvîth surrounding protons. A decoupler is used to this end.

Another magnetic field is added that targets the range of proton resonance fkequencies. This

field causes proton spin flips to occur very rapidly so that the neighbouring ''P experiences

19

only an average of the two magnetic fields, yielding only one signai for each chemically

different phosphorus nucleus-

2.2 Inductive& Coupled Plasma - Mus Spectrometay (ICP-MS)

hductively coupled plasma - mas spectrometry is weU suited for the analysis of

elements at trace levers. This is particuiarly useful in this study since concentration of

phosphorus in water samples c m be very low. The detection I imits for phosphorus for the

ICP-MS instrument used are currently in the 50 ppt range?

The liquid sample is pumped into a nebulizer where it is transformed into an aerosol.

Spray droplets smailer than 10 microns in diametter (to promote high efficiency desolvation,

volatilization, and atomhtion) are then passed on through to the plasma torch. Larger

droplets condense ont0 the walls of the water cooled spray chamber. The inductively

coupied plasma torch then heats the aerosol to approximately 5700 to 6300 K. The ICP torch

consists of three concentric quartz tubes that are surrounded by an induction or load coil. An

inert gas phsrna is used so that Little reaction between analyte and the gas occurs. The quartz

tubes shape and constrain the plasma as well as direct the aerosol through its centre. The

load coil supplies radio fiequency (rf) power which is coupled to the plasma inductively and

maintains the plasma The plasma is initially generated with the introduction of energetic

electrons by the bnef application of a Tesla coil. Within the torch, the aerosol loses solvent,

bonds are broken and the excitation and ionization of the elements occur. Ions are then

extracted fiom the plasma torch, focussed, and accelerated toward the entrance of the mass

spectrometer. Within this interface region, differential pumping reduces the pressure of the

Figure 2.1 : Schematic of an ICP-MS6

1. ICP ion source; 2. interface, including sampler and skimmer cones; 3. transfer and focussing optics; 4. acceleration and beam focussing; 5. entrance slit; 6. electromagnet; 7. electrïc sensor; 8. exit slit; 9. conversion dynode; 10. electron multiplier.

Reprinted with the permission of The Amencan Chernical Society.

21

system. Ions enter a double focussing, reverse geometry magnetic sector m a s spectrometer

for mass separation and detection (Figure 2.1)-

2.3 X-ray Photoelectron Spectroscopy (XPS)

X-ray photoelectron spectroscopy (XPS) , d s o known as electron spectroscopy for

chemical analysis (ESCA), provides chemical information about the d a c e of a sample.

Probing depths range fiom about three to five nanometres and ail elements with an atomic

number greater than three (lithium) cm be detected,

In WS, a monochromatic beam of X-rays is directed at the d a c e of a sample. An

X-ray striking the sarnple can penetrate to a depth of a seveml micrometers. This incident

photon can Uiteract with an electron, causing it to be emitted (Figure 2.2). An electron

ejected fkom depths greater than a few nanometres is usually attenuated by the bulk of the

sample. However, an electron emitted nom a depth of less than a few nanometres can exit

the surface without signincant loss of kinetic energy. The kinetic energy of this

photoelectron c m then be measured using a concentric hemispherical analyser (Figure 2.3)

and the original binding energy of that electron c m be obtained using the following equation:

E,=hu - Eb- @,

where Eb is the binding energy of the electron, hu is the energy of the incident photon, E, is

the kinetic energy of the exiting electron and & is the spectrorneter work fünction.

The resulting electron bindiog energies can be related to specifk orbitals ofelements,

allowing for elemental identification of species on the surface of a sample. The binding

energies are ais0 indicative of the chemical state of the elements present Changes in the

Phosphorus

O vacuum

d 3p - binding 14 3s ---v O

energy - - - - * -photoelectron (eV) 130 2p - -

photoelectron process

Figure 2.2: The photoelectric effect, illustrated for the emission of a phosphorus 2p electron.

. . - (Monochromator) - - - e-Xf

\ -- - - - \: -t------==;;i(?smple -L&------- Gr: ' \ \ \

Detector

I Cornputer

Figure 2.3: Schematic of an X-ray photoelectron spectrometer.

23

electronic configuration of the valence electrom due to bonding will affect the binding

energy of the core electrom. These changes can be detected and allow for chernical state

information to be obtained?

2.4 Ion Chromatography (IC)

Ion chrornatography involves the separation of ions through their distribution between

two phases, a mobile phase (the eluent) and a stationary phase (the column). As the anaiyte

ions in the eluent proceed through the column they replace eluent ions on the stationary

phase. The strength of the ion-stationary phase interaction wiil determine the length of stay

of the ion on the column. As the analyte ion is replaced by an eluent ion, the analyte ion

rnoves M e r d o m the length of the coiumn. This continuai process results in the

separation of the various analyte ions?

At the end of the column, the ions are usually detected by a conductivity detector.

Quantification is achieved by cornparison to a calibration curve made fiom the analysis of

standard solutions of the ions of interest,

2.5 Electrospray Mass Spectrometry

Electrospray ionkation ('SI) is a relatively new method of ionization that permits

the formation of charged ions of high molecular weight molecules such as peptides or

proteins. Multiple charges on these molecules bring them into the analysis range of standard

mass spectrometes. Wiîh ESI, a solution of the anaLyte molecules are moved tbrough a

needle that carries a high voltage (t5.5 kV). At the end of the needle the solution is

24

nebulued into smaü charged droplets that are expelled into a s m d vacuum chamber.

Solvent ions evaporate into the vacuum leaving the analyte ions behind. The analyte ions are

then directed, using a series of lenses, into a quadruple mass spectrometer for analysis?

2.6 X-ray Dinraction @RD)

X-ray difEaction is a powerful tool used to characterize crystalline substances such

as geologic materiais, metals and cerami~s.'~ X-ray powder *action is usefid for geologic

materials in that it c m distinguish between different forms of chemically sunilar compouods

@olymorphs). in XRD, an X-ray beam is used to interogate the sample. As the beam

passes through the crystal lattice of the sample, the X-rays will be diaacted. DBkction is

defined as "a divergence of light fiom its initial line of tra~el".~~ When the X-rays are

scattered by the atoms in the crystal, most of the X-rays will interfere destructively.

However, some will interfere constructively giving bright areas of X-rays in a specinc

pattern, a d-action pattern. This pattern can be captured either by film or electronic

means. Using Bragg's Law, information about the type of various crystal structures in a

sample can be deduced." Bragg's law is as follows:

d = 2d sin0

where n is an integer, A. is wavelength of the incoming X-ray, d is the interplaner distance

in the crystal and 8 is the angle of incidence (and diffraction).

2.7 Static Secondary Ion Mass Spectrometry (SIMS)

Static SIMS uses a focussed ion beam to sputter the rippermost monolayers of a soiid

surface. Sputtered elemental and molecuiar ions are then focussed into a suitable mass

spectrometer to be analysed. Fragmentation patterns can then be used to determine the

species present Detection limits for most species are in the Low ppm range.13

2.8 References

1. R. S. Macornber, A Com~lete Introduction to Modem NMR Soectrosco~~, Wiley, New

York, 1-103, 1988,

2. R. K. h r r i s , Nuclear Mametic Resonance S~ec t rosco~v~ Longman Scientific &

Technical, Essex, 1986.

3. D. A. S koog and J. J. Leary, Princi~les of Instrumental Analysis, Saunders, Fort Worth,

3 17, 1992.

4. 1. B. Lamert, H- F. Shurvell, D. Lightner and R. G. Cooks, Introduction to Organic -

Suectroscoti~, Macmillan, New York, 9-99, 198%

5. W. A. Strobel and W. R. Heineman, Chemicai Instrumentation: A Svstematic A~~roach ,

John Wiley & Sons, New York, 673-722, 1989.

6. L. Moens and N. Jakubowski, Ana[ytical Chemistry News & Features, 25 lA-Z6A,

Apnl 1, 1998.

7. D. Briggs, and M. P. Seah, Eds., Practicai Surface Analvsis: Vol. 1, Wiey, New York,

1-83, 1983-

8. H. A. Strobel and W. R Heineman, Chernical Instnunentation: A Systematic A~~roach,

John Wiley & Sons, New York, 863-959,1989.

9. R C . Cole, Ed., Electromrav Ionization Mass S-pectrometry: Fundarnentals,

hstrumentation & Ab~lications, Wiley, 4-60, 1997-

10. ASM Handbook Conmittee, Metds Handbook. Vol. 1 O. Materials Characterizaiion,

Amencan Society for Metais, Metals Park, 325-343, 1 986.

I 1. R. A. Sherway, Ph~sics for Scientists and Eneineers, Saunders, Philadelphia, 870,1986.

12. B. D. Cullity, Elements of X-ray Difiction, Addison-Wesley, Reading, 1 - M , I 978.

13. D. Briggs, A. Brown, and J. C. Vickerman, Handbook of Static Second- Ion Mass

Spectrometrv (SIMS), John Wiley & Sons, Chichester, 3-1 1, 1989.

3.1 Site Selection and Description

3.1.1 The Old Ausable River Channel

Three sampling sites were chosen based on their accessibility and location dong the

Iength of the river- These sites are indicated in Figure 1.3 - Samples were coliected on both

the north and south side of each site.

The source of the river is locally cailed the Mudhole. This name is retained to

ident* this site throughout this thesis. It was chosen due to its residential location in Grand

Bend where pollution inputs fiom septic systems are suspected to be hi&. A road traverses

the river at this point, Iirnitîng its fiow. The waters are shalIow however, reaching a

maximum depth of only 0.5 metre during the spring. As a result, the Mudhole North, located

on the no& side of the road, experiences dramatic changes throughout the year. The

riverbed fieezes during the winter and in Iate summer may dry out completely. The Mudhole

South is much deeper (maximum depth, 2.5 metres) and is heavily lïttered with detntal plant

material.

The second sampiing site, the Canoe Docks, is located at the river's only dam. The

water level is highest at this site and an accumulation of organic material behind the dam is

evident. Anthropogenic activity is Likely also greatest at this site within the Pinery Provincial

Park since it provides river access for canoeing, fishing and swimming. The Canoe Docks

South also has facilitated access in the fonn of a small dock,

A third sampling site was chosen close to the mouth of the river at the Burley Bridge.

28

Water levels at this site remain fiirly constant throughout the year and a culvert prevents an

interruption to the river's flow.

3.1.2 The Kirk Cousins Management Area

Three kettle ponds were chosen for study in this region; Regina Mundi Pond, Pond

Mu and Tude Pond- Figure 1-4 indicates the location of these ponds in the area.

Regina Mundi Pond, the largest of the three, is located directly behind Regina Mundi

Coilege. Aithough it now receives treated sewage fiom the school, waste once entered the

pond without treatment, One sampling site was chosen near the waste effluent output and

another about 300 metres fiom this site, at the southern most tip of the pond.

Pond Mu is the smaliest of the three ponds. It is surrounded by meadow and was

chosen due to its apparent isolation fiom Regina Mundi Pond.

Turtle Pond is located in a forested area of the KCMA and also appears isoIated fiom

Regina Mundi Pond.

3.2 Experimental

3.2.1 Sediment Collection

Sediment corers were designed in consultation with Dr. M. Powell fiom the

Department of Earth Sciences, They were made fiom polyvinylchlox-ide tubing, two metres

in length and 5.7 centimetres in diameter. Each corer was bevelled on the inside at one end

by Irold Schmidt in the machine shop of the Physics and Astronomy Department to create a

thin, sharp edge.

29

Sediment cores were coilected fkom the OARC in the second month of every season

fiom October 1997 until April1999. The Kirk Cousins ponds were sampled from October

1998 un61 July 1999. The corer was driven into the sediment and the bevel ensured that the

sample remained intact when the corer was subsequendy removed Ideally, these samples

were collected fiom a water depth of 0.5 me= The top 5 cm of sediment was retained for

analysis. There was some selection however. Grave1 portions of the nverbed &en darnaged

the corers and prevented m e r penetration of the nverbed and on occasion evaporation

reduced water levels to such an extent that the water depth critenon could not be met- As a

result, in the second year of sampling, a shovel was used for sample collection.

Generally, several samples fiom any given site were amalgamated into a single

sample both to ensure that sufncient phospholipid could be extracted for subsequent analysis

and to produce a sample that was representative of a selected area. Once each season

additionai samples were coilected and analysed for cornparison. Al1 sediment specimens

were seaied in plastic and fiozen in-situ with dry ice to ensure the preservation of the material

before analysis,

Phospholipids are regarded as short term biomarkers with a comparatively short h a -

life in the environment. Since the sampling protocol used in this work required sampling

over a twelve month period, a simple experiment was designed to establish which, if any,

phospholipids swvived the winter andor what degradation products could be identified in

the sample. Ln the fd of 1998, an amalgamated Canoe Docks North sample was divided in

haK One portion was analysed immediately while the other was left outside in a bucket of

O A K water for 100 days before analysis, the "balcony sampleyy.

30

3.2.2 P hosp holipid Extraction

The phospholipid extraction procedure used in thîs study is based on the method that

was modined for sediment analysis by Bardyguia-Nonn et ai- The original procedure was

developed for biological tissue analysis by Foich et al.' It provides a quantitative separation

of a phospholipid mixture that may be used to ident* sediment types and to monitor

pollution.

Sediment specimens were airdriedand passed through a 500 jun soii sieve to remove

any detrital plant material and macrouivertebrate fauna. 40 g samples of drïed sediment were

then added to 600 ml of a chloroform / methanol(2:l) solution and mixed vigorously for 24

hours using a Burreil Wrist Action Shaker. The mixture was filtered in two steps through

VWR Scientific qualitative filters and Gelman nylon membrane nIters (pore size, 0.45 pm).

This two step process ensured the removal of suspended particdates which decreased the

signal to noise in the nuclear magnetic resonance spectrum. 120 ml of scrubbing reagent (0.2

M K-EDTA and 0.2 M KH2P0,) were added to the extract and the mixture was shaken for

five days. The scrubbing reagent is required to remove any ofthe paramagnetic ions that may

broaden or quench the NMR resonance signals. M e r scrubbing, the mixture was placed into

a separatory funne1 for 24 hours to completely separate the organic and aqueous phases. The

organic phase was then collected and evaporated to dryness at 37°C. The sample was

redissolved in 3 0 ml of the chloroform / methanol mixture and then placed hto a separatory

b e l . 6 ml of 0.2 M K-EDTA were added and the mixture was shaken and left to separate.

Any residual K-EDTA in the chloroform phase was compietely removed in this second

scmbbing step. The organic phase was recovered a second time and nui through a Varian

31

Bond Elut silica column to concentrate the phospholipid. The column was prepared and the

phosphoiipids were eluted according to the procedure outhed by Zeiles and ai? The

sample was once again evaporated to dryness in preparation for 3'P EIEYIR analysis. A yellow

to dark brown extract is produced.

33.3 "P NMR Reagent Preparation

The Cs-EDTA was prepared by titrating a suspension of the acid with cesium

hydroxide until the last crystal of EDTA dissolved. A final pH of 6.0 was determined using

a Corning 220 pH meter. The solution was then evaporated to dryness. The Cs-EDTA was

redissolved in distilled, deionized water to produce a 1 .O M solution. This reagent is reported

to be stable indefinitely at C?

3.2.4 Sample Preparation for "P NMR Analysis

The phospholipid sample was prepared in a 10 mm NMR tube in deuterated

chloroform at a concentration of 25 mg/ml. 0.5 ml of rnethanol and 0.5 ml of aqueous

Cs-EDTA were then added. Tetraphenylphosphonium bromide was used as an interna1

standard for chemicd ski? reference and quantification. The mixture was shaken thoroughly

and the aqueous layer of methanol and Cs-EDTA was left to separate to the surface of the

sarnple. The aqueous layer need not be removed as it does not affect the experiment and

serves to minimize the evaporation of the organic solvents durhg analy sis. Samples may be

stored refrigerated at 5 OC for up to four months and still produce identical NMR spectra.

32

3.2.5 3'P NMR Spectroscopy

The NMR instrument used in this study was a Varian XL-300 system operating at

12 1 -42 MHz for "P with a magnetic field of 7.2 Tesla The spectra were recorded at ambient

temperature and at a spin rate of 15 Hz. A proton broadband decoupler was applied to

prevent any mutliplets due to 'H - 3'P interactions. Each resonance signai represents one

phosphoiipid species-

The spectrometer scan conditions used were: single pulse sequence; pulse width, 8-6

ps (74 O); spectral window, S000 Hz; fiee-inductiondecay size, 12,800 channels; acquisition

tirne, 1.28 s; number of acquisitions, 40,000 - 70,000; line broadening, 1.000. The total

andysis thne per sample is 12 - 24 hours.

3.2.6 Phospholipid Quantification

The material extracted fkom the sediment contains phospholipids, otherceli iipids and

organic material which is soluble in the chioroform/methanol mixture. Accordingiy, a simple

weighing of the extract does not provide a good mesure of the mass of phospholipid in the

sediment.

Unfortunately, an accurate quantification of total phospholipid cannot be obtained by

integrating the entire spectral region for phospholipids. Some spectra have broad baselines

which are not iniproved by M e r scmbbing. The baseline is reported to be characteristic

of polymenzed and crosslinked phospholipids.' An integration incorporating this baseiine

will elevate concentrations. It c m however be corrected with modem NMR instruments.

Individual phospholipid concentrations were determined through the spectral

33

integration of each resonance signal foliowed by cornparison to a suitable reference under

f U y relaxed NMR conditions. Totai abundance was determined by adding the individuai

phospholipid concentrations in each sample. In this thesis, concentration is reported in moles

of phosphorus per gram of dry sediment Appendix 1 presents the individuai phospholipid

concentrations at each site thmughout the year. Total moles or mass of a particuiar

phosphofipid cannot be reported without c o ~ t i o n of phospholipid identity- In addition,

the fatty acid side chaîns do not affect the chemical shift of phosphorusL and as such each

resonance signal represents a particuiar phospholipid with a variety of side chains of dieerent

molecular weight. An estimation of average molecular weight would be necessary to provide

the approximate mass of each phospholipid present.

Some phospholipids were not quantified due to the integration ditFculties

encountered fiom extensive spectral overlap- In these cases, their concentrations are included

in the value reported for the main peak. Appendk II provides the complete spectral library

for the sites in this study. The spectra should not be compared visudy for relative

phospholipid concentrations as peak widths Vary and vertical scales ciiffer. These spectra

shouid only be used for a cornparison of chexnical shifts and sample richness.

3.2.7 Phospholipid Identification

The experimental and instrumental parameters used in this study are not identical to

those reported in the literahire and consequentiy the chemical s h f i reported here do not

correspond to fiterature values. Phosphoiipid identification was therefore attempted by

standard addition.

34

In the chIorofodmethanol reagent, chemicai shift does not depend on the length of

the fatty acid side chahs of the phospholipids ifthey are longer than six carbon atoms or on

the presence of double bonds.' Shifts c m Vary however, dependhg on a number of other

factors- NMR field strength and analysis temperatures have an effect on chemical shift.

Phosphatidic acid aiso shows a chemical shift dependence with changes in pH between pH

5 and 13.' The increase in solution acidity hcreases the shielding of the phosphorus

nucleus ,6

Some lipids exhibit a linear shift with the log of total phospholipid concentration

where chemical shifk decreases with increasing concentration. Lipids that are negatively

charged show this dependence as well as ethanolamine plasmaIogen. Zwitterionic and

neutrai lipids do not demonstrate thk dependence?

Small differences in sample environment evoke substantial NMR shift changes.

Figure 3.1 shows the effect of adding phosphatidylinositol to a mixture of pure phosphoiipids

in chloroform- The chemicai shifts of these standards are not comparable to the sample

spectnim due to the complex matrix composition of sediment which may alter shifts

substantially ,

The presence of cations in solution can aiso cause shift variations. Ammonium and

the allcali metals cause only rninor shift changes but the effects of sodium on phosphatidic

acid can be c~nsiderable?*~' Shifts Vary widely with the alkaline earth cations and can cause

broadening, especially with magnesium.7*8 Transition metais and trivalent cations have k e n

shown to cause extensive broadenuig or q~enching"'~ by paramagnetic relaxati~n.'~ In the

case of soils and sediment, iron is &en the principle cation involved. This can complicate

LPA 6 = 1.977 pprn LPG 6 = 1.905 pprn PG S = 1.348 pprn PA 6 = 1224 ppm PE 6 = 0.898 pprn S M 6 =0.838 pprn LPC 6 = 0.627 pprn PC 6 = 0.03 1 pprn

LPA 6 = 2.052 pprn LPG 6 = 1.860 pprn PG 6 = 1.330 pprn PA 8 = 1.252 pprn PE S =0.876 pprn S M 6 = 0.830 pprn LPC S = 0.616 pprn PI 6 = 0.325 pprn PC 6 = 0.030 pprn

-- --

Figure 3.1 : NMR spectra of phosphoiipid standards; (top) nin 9 and (bottom) run 1 O with the addition of phosphatidylinositol.

36

quantification as well as identification due to peak overlap and the difficulties in the accurate

determination of integration Limits. Fortunately, very s m d changes in solvent composition

c m cause substantid chemicai s W . Studies altering the ratios of chlorofom to methanol

to water have found that adequate separation of broad, overlapping resooance signals can be

achieved, ' Changes in the countercation of the EDTA salt in the aqueous layer of the NMR

reagent alters the chernical shifts of anionic phospholipids. However, phosphoiipids

containing a covalentiy bonded positively charged group do not show a countercation

dependent shift.' The Cs-EDTA salt is used exclusively in the NMR reagent in this study.

Many of these chemical shift problems may be prevented. Field strength, temperature

and solution pH can al i be controiled. Moreover, scrubbing with EDTA and phosphate

should minimize the cations in solution. The greatest concern in this work is the variability

of phospholipid concentration and matrix composition in environmental samples since the

phospholipid NMR signals are dependent on these uncontrolled variables,

3.2.8 X-ray Diffkaction

Suspended particdate material fiom the Kirk Cousins ponds was finely ground for

XRD analysis. Analyses were performed by Ms. K. Law fiom the Department of Earth

Sciences. A Rigaku X-ray Diffractometer equipped with a copper X-ray tube source was

used for the analyses presented in this work. The copper cathode was held at 20 kV and the

tube current was 40 mA-

37

3.2.9 Materiais

Reagent grade extraction solvents were purchased fkom Caledon Laboratories Inc.

99 % pure phospholipid standards were provided by Sigma Chemical Company with the

exception of phosphatidylinositol which was suppiïed by Fluka Biochimika. Deuterated

chloroforrn fiom Cambridge Isotope Laboratories, tetraphenylphosphonium bromide and

cesium hydroxide fiom Aldrich Chemical Company Inc. and ethyf enediaminetetraacetic acid

fkom BDH Inc. were also purchased for these experiments. AU reagents were used as

received fiom the suppliers.

3.3 Results and Discussion

3.3.1 The OId Ausable River Channel

An examination of Table 3.1 reveals a seasonal phospholipid pattern in both total

rïchness and total abundance- A minimum is observed for both indicators in winter which

caries into spring, with a maximum during the summer and autumn. Microbial activity,

measured using soi1 respiration rates, has been shown to slow considerably duriog the winter

months in temperate climates while the comrnunîty thrives under warm conditions." That

is not to Say that some types of microbes cannot survive cold temperatures. Some bacteria

thive in arctic environments while others are able to flourish in hot, arid ~limates.'~ Studies

suggest that it is the temperature fluctuations that have a more pronounced effect on the

microbial cornmunity than constant extceme condition^.'^ In temperate climates, few

microbes survive the cold of winter but wann nimmer temperatures provide optimal growing

conditions. Plant litter in the fâll supplies additional nutrients to the microbes for ce11

1: TOTAL

Spring Autumn 1 Winter 1

Table 3.1: Seasonal and spatial patterns of total phospholipid abundance and richness in the OARC, Richness indicates the number of different phospholipids preçent in the sample. Abundance indicates the total amount of phospholipid in the sample. Abundance is measured in moles P/g of dried sediment. GPC, greatest phospholipid contribution, represents the individual phospholipid contributing the greatest amount to the total abundance in %, a refer to list of abbreviations berror of 30 %

39

maintenance, growth and repli~ation.'~ An increase in the amount of organic material is

generaily indicative of an increase in microbial biorna~s.'~ Variations in the organic content

of the d a c e sediment at each site may also suggest spatial variation in microorganisms

within the OARC. Figure 3.2 ilIustrates the seasonal and spatial changes in richness

observed in the Old Ausable River Channel-

Aimost every sampling site in the OARC exhi'bits the same richness and abundance

pattern as shown in Table 3.1. The Mudhole is an anomaiy. This site is most impacted by

humau activity and experiences dmmatic changes in water content throughout the year. It

often dries completely by the end of the summer and the sediment fieezes during the winter.

Drying and re-wettuig have been s h o w to cause more pronounced changes in the rnicrobial

community than fieezuig and thawing. Drying kiiis some of the microbial population while

re-wetting causes rapid growth." The Mudhole North exhibits drylwet cycles. When the

system dries out completely, as it did in September 1998, there is a marked drop in

phospholipid abundance and richness. Re-wetting under favourable growth conditions wodd

not have occurred until the temperatures rose in the spring. It might be noted that the

Mudhole sites are also compromised by drainage tiles and locd road work.

The Canoe Docks sequence exhibits the highest richness and abundance in this

system. This may be attributed to the interrupted flow at the river's dam. Sedimentation

above the dam, at the Canoe Docks North site, creates an environment rich in organic

material which resuits in greater abundance and richness in the microbial biomass. Increased

oxygenation and material dispersion around the dam spillway favours a vigorous microbial

commmity at the Canoe Docks South site.

Abundance: 2-4 nmol P/g dry sediment Richness: 1

--

Abundance: 12.8 nmol P/g dry sediment Richness: 9

Abundance: 8.9 nmol P/g dry sediment Richness: 3 E

I

Figue 3 -2: NMR spectra of (top) Mudhole South, spring 98, (Iniddle) Mudhole South, sumnier 98, and (bottom) Burley Bridge North, sumoier 98.

41

GPC in Table 3.1 indicates that it is possible for two samples to have the same

richness while one sample is dominated by a single phospholipid. It is tempting to subdivide

rkhness into two categones but it is important to note that GPC may sirnply be a samphg

artifact. Nevertheless, phospholipid richness may be a good measure of the diversity of the

microbial community.

A detaïied quantification of all the phospholïpids appearing in the NMR spectra is

provided in Appenduc 1. Phospholipids that have not been identined have been assigned a

letter for later reference. Those for which a positive identification has ken obtained are

labelled with an abbreviation of their chemical name. A List of the names correspondhg to

these abbreviations is found on page xiv.

E and K are the most abundant and persistent phosphoiipids. Figure 3.3 compares the

autumn Canoe Docks North sample analysed in October to the same Canoe Docks North

sample which was ieft outside in OARC water for 100 days. K persists weH into the winter

while the ten other phospholipids present appear to have decomposed.

3.3.2 The Kirk Cousins Management Area

The Kirk Cousins Management Area is a more complex system than the Old Ausable.

The ponds thernselves are not equivalent as they dEer in d a c e area, depth, and shade

cover, as well as in flora and fauna. DBerences in system turbidity and evaporation M e r

complicate the phospholipid results fiom each pond. In addition, Regina M u d i Pond

receives waste effluent, one of the reasons we chose this site.

The overall pattern shows the greatest phospholipid abundance in spring and winter

Richness: I I Abundance: 28.8 nmol P/g dry sediment E

I

Abundance: Richness: 1

2.4 mol P/g dry sediment

Figure 3 -3 : NMR spectra of a sample fkom (top) Came Docks North, autumn and (bottom) same sample following 100 days outside in OARC water (Balcony sample).

43

and the greatest richness in summer, autumn and winter (TabIe 3 2 ) . This behaviour con-

with that observed in the OARC. We attn'bute this ciifference partly to the hirbidity caused

by wind and wave action observed in each of the ponds. Smali bottom dweliïng catfïsh

called madtoms (Notuncs *nus) may also contribute to the resuspension of the fine cIay

sedirnents. Dr. K. Dewdney 6rom the Department of Zoology has identified a population of

madtoms which may be unique to Pond Mu.

Figure 3 -4 is an X-ray dif5action pattern of the suspended particuiate material in Pond

Mu in the Sumner. Silica, calcite, feldspars and clays are some of the major mineral

components. This particuiate matter provides abundant surface area to scavenge microbes.

Many species of bacteria, caiied penphytes, characteristicdy grow while attached to solid

surfaces'" and clays are known to provide adsorption sites for microbes.l5 This results in a

decrease in the observed sediment phospholipid. In addition, turbidity reduces light intensity

which affects the photosynthetic zone. As this study deah with the littoral-benthic zone, a

region dong the shore where light ofien penetrates to the pond bottom, light reduction may

result in a decline in mauy sediment microorganisms. The effect of light on the microbiota

in estuarine sediment was an increase in biomass in many microbial classes, with the greatest

effect on the population of diatoms, a type of algae.16 In winter, when the ponds fieeze, wave

action ceases and the suspended particdates settle out, frnally contributing to the mass of

sediment that is collected and analysed.

The second perturbation to this system is effluent fiom Regina Mundi College. This

has two effects on the neighbouring pond. It adds bactena and phosphorus and maintains the

water level even when the other ponds are aected by drought. Abundance is always higher

Spring

Table 3.2: Seasonal and spatial patterns of total phospholipid abundance and richness in the KCMA. Richness indicates the number of different phospholipids present in the sample. Abundance indicates the total amount of phospholipid in the sample. Abundance is measured in moles P/g of dried sediment. GPC, greatest phospholipid contribution, represents the individual phospholipid contributing the greatest amount to the total abundance in %. a refer to list of abbreviations benor of 30 % 'net detected

D - dolomite G - gypsum M - muscovite 1 - illite

- -

Q-9- F - feldspars C - calcite Q

C

Figure 3.4: XRD spectnun of suspended particdate matter fiom Pond Mu in summer 98.

46

at one of the sampIiag sites withui this pond, no doubt because of the input.

Pond Mu and Turtie Pond are the most difficult to explain. It would appearthat Pond

Mu was most afEected by drought, It is the smallest, possibly the warmest pond and the most

susceptible to evaporation. Pond Mu is also very shdow, making it especiaüy vulwrable

to hirbidity. Turtle Pond is the le& turbid and appears to contain the most detrita1 0rgan.k

matter- It is aIso the most shaded in summer which might explain the low richness found at

thïs site. Sampling artifacts may confise our interpretation.

Appendix 1 shows the quantification of the individual phosphoiipids fond in each

sample from the KCMA. Once again, phospholipids K and E are the most abundant and

persistent. Ln fact, unlike the OARC, no other phospholipids show a strong presence in these

ponds.

3.4 Phospholipid Quantification

Concentration variability in samples fiom the same site in the same season is to be

expected in environmental studies. An estimation of sampling error was obtained through

the anaiysis of multiple Canoe Docks samples. The analysis of five samples fkom the Canoe

Docks North during the spring gave a sampling error of 10 %. This error might only be

reduced by additional sampling. Figure 3.5 iiiustrates the sample consistency seen in richness

and abundance for the Canoe Docks site. One M e r sample was collected in the subsequent

spring of 1999 for comparison. Although abundance has increased, the richness and relative

phospholipid concentrations are surprisingly consistent.

Determinate or systematic error was assessed at 20 %. The greatest contribution to

- . - -

Abundance: 11.8 m o l P/g dry sedhent Richness: 5

Abundance: 13.8 nmol P/g dry sediment Richness: 6

Abundance: 21 -8 nmoI P/g dry sediment Richness: 7

Figure 3.5: NMR spectra of Canoe Docks North samples; (top) amalgamated sample, spring 98, (middle) single sarnple, spring 98, (bottom) spring 99.

48

this was in weighuig the s m d mass of intemal standard. Integration e m r is inciuded in this

value and is based on five integrations of a siugle sample. Integration problerns inciude the

poor baselines encountered in severai spectra and peak overlap due to broad resonance

signals. Once again, s m d changes in solvent composition c m elicit substantial shift changes

in some phospholipids. This c m be used to achieve better separation of resonance signais

for accurate quantification"

The quantincation procedure was verifïed in the sediment matrix. 1 mg of 99 % pure

phosphatidylcholine was added to the Mudhole North winter sample due to its low nchness.

Normal extraction and quantification procedures were used yieIding a 93 % recovery.

3.5 Phospholipid Identification

It is now possible to distinguish between many of the major groups of microbes by

their signature lipids." Phosphatidylglycerol is found predominantly in bacteria whiie

phosphosphingolipids are rare in bacteria but have k e n found among the anaerobic

fermenters. Plasmalogens are signature lipids for anaerobic bacteria and are rarely found in

higher plants, fish or moliuscs. Methane fonning bacteria predominantly contain

phosphatidy linositol and the di and tetra-phytanyl gl y cerol ether phospholipids. '' The identification of signature iïpids, used in the Lipid fatty acid analysis, has aiiowed

researchers to leam a great deal about the effects of pollutants on the microbial community.

For example, soi1 acidity has been shown to have a greater effect on bacterid biomass than

on fbngi and some reports have even shown an increase in fungi.I8 PAH contaminated

sediments were discovered to be nch in aerobic gram-negative bacteria and in the

49

heterotrophic microeukaryotes which feed on thm. Ambient sedlments were emiched in

gram-positive bacteria and anaerobic gram-negative bacteri;t19

Before any conclusions may be drawn about the health of the OARC or the KCMA,

phospholipid identification must be complete. So far, phosphatidylethanolamine,

phosphatidylcholine and lysophosphatidylcholine have been identified by standard addition.

Further attempts at identification are outlined in chapter 5.

3.6 Conclusions

The Old Ausable River Channel shows strong variation in microbial activity witb a

maximum in the summer/autumntumn This agreement with the existing literatm on temperate

zone microbes shows that NMR phospholipid andysis provides a means of monitoring the

microbid communïty.

The Kirk Cousins Management Area shows anomalous seasonal behaviour. We

believe the major cause is the high turbidity of the water either because of wind and wave

action a d o r bioturbation by bottom dweliïng fish.

The Regina Mundi Middle and South sites show the greatest abundance in the Kirk

Cousins system. We attribute this primarily to effluent input but note that the flow fiom

Regina Mundi College maintains the water level in this pond so it is not subject to drought.

Pond Mu is the most subject to drought while Turtle Pond is more shaded. These

factors may explai. our observations in these ponds.

Phosphatidylethanolamine, phosphatidylcholine and lysophosphatidylcholine have

been positively identified. E and K, the most persistent and widespread phosphoiipids, have

yet to be identified- This may uidicate a persistent rnicrobid source.

FinaIly, the baicony sample c o n f b s that the phospholipids have a rapid turnover in

sediments, Future work will focus on monitoring short-term changes in phospholipid

profiles.

3.7 References

I

L. Bardygula-Nom, J. L. Kaster and T. Glonek, LipiriS, 30, 1047 (1995).

J. Folch, M. Lees, and G. H. SIoane Stanley, J Biol- Chern-, 226,497 (2957).

L. Zeiles and Q- Bai, Soil BioL Biochem-, 25,495 (1993).

P. Meneses and T. Glonek, J. Lipid Res., 29,679 (1988).

T. Glonek, Phos~horus-3 1 NMR Spectral Properties in Cowound Characterization and

Structural Analvsis, L- D. Quin and J. G. Verkade, Eds., VCH Publishers, Inc., New

York, 283-294,1994-

M. M. Crutchfield, C. H. Dungan, J. H. Letcher, V. Mark, and J. R Van Wazer, 3 ' ~

Nuclear Mametic Resonance: Topics in Phos~honis Chemistrv. Volume 5, John Wiley

and Sons, Inc., New York, 1967.

T. E. Merchant and T. Glonek, Lipiak, 27,551 (1992).

K. Panchaluigam, S. Sachedina, J. W. Pettegrew, and T. Glonek, Int J. Biochem., 23,

1453,1991.

A. J. R. Cosello, T. Glonek, and J. R Van Wazer, Inorg. Chern., 15,972 (1976).

T. O. Henderson, A. W. Druski, L, G. Davis, T. Glonek, and A. M. Scanu, Biochemisby,

14. 1915 (1975).

51

1 1. M. Branca, N- Cdeddu, M- Fnrianu, and M. V. Serra, Anal- Biochem., 232,1(1995).

12- M. Wood, So2 Biolom, Blackie and Sons Ltd,, New York, 80-82, 1989.

13. N. S. Subba Rao, Soil Microoreanisms and Plant Growth, 3" Edition, Science

Publishers, Inc,, New Delhi, 21-50, 1995.

14. M. J. PeIczar, Jr., R. D. Reid, E. C. S. Chan, Microbiotoa: 4h Edition, McGraw-Hill,

Inc., New York, 724-746, 1977.

15. S. J- MaIcohn and S. O, Stanley, Sediment Mi~robiolow~ D. B. Nedwell and C. M.

Brown, Eds., Academic Press, London, 2, 1982.

16. R. J. Bobbie, J. S. Nickels, G. A. Smith, S. D. Fazio, R H. Findiay, W. M. Davis, and

D. C. White, Appl. Env- Microbiol., 42, 150 (198 1).

17. D. C . White, Microbes in their Natutal Environments, J. H. Slater, R. Whittenbury and

5. W. T. Wimpenny, Eds., Cambridge University Press, Cambridge, 37-66, 1983.

18. T. Pennanen, H. Fritze, P. Vanhala, O. Kukkilii, S. Neuvonen, and E. BiZth, Appl. Em-

Microbiol., 64,2 173 (1 998).

19. D. E. Langworthy, R. D. Stapleon, G. S. Sayler, and R. H. Findlay, Appl. E m

Microbiol., 64,3422 (1998).

4. Water Anaivsis for Dissoived Phos~horus

4.1 Introduction

Dissolved phosphorus is defined as that portion of phosphorus in the water column

that wili pass through a 0.45 pm filter.' Aithough the dissolved inorganic phosphorus is

generaily considered to be available to plants, both the inorganic and organic forms may be

assimilated by the microbial commUnity.l Microorganisms play a major role in the

sequestration of dissolved phosphorus in systems receiving high inputs of organic matter.

Systems such as the OARC receive significant leaf litter nom the shoreline littoral zone

whiie the KCMA has organic and phosphorus loading problems associated with waste

effluent Under these conditions, microorganisms are capable of incorporating phosphorus

into theïr cellular components to form polyphosphate, allowing them to survive alternathg

oxidizing and reducing environments.' Under conditions that disrupt the microbial

community, a rapid release of phosphorus has been observed?

Inductively coupled plasma mass spectrometry has been employed to determine the

total dissolved phosphorus at each site in the two study areas. These data have been used to

explore the relationship between dissolved phosphorus and microbial community abundance.

4.2 Experimental

4.2.1 Sample Collection

125 ml water samples were collected at each sarnpling site in the Old Ausable River

Channel in the second month of every season fiom April1998 mtil January 1999. The Kirk

53

Cousins Management Area was sampled nom July 1998 untii April 1999. Samples were

coliected fkom a water depth of 0.25 metre, halfway between the surface of the water and the

sedimedwater interface. The samples were coiiected in the same sampihg locations as the

sediment collection. Clean nalgene bottles were rinsed several times in the river prior to

being fïiied and capped. Gloves were wom to prevent phosphorus contamination.

43.2 Sam ple Preparation for ICP-MS Analysis

Water samples were fltered through Gelman nylon nIter membranes (pore size, 0.45

pm) to remove suspended particdates. After filtration the samples were analyzed by C.

Bradley on a Finnigan MAT ELEMENT ICP-MS at the London Health Sciences analytical

facility at University Hospital.

4.3 Results and Discussion

4.3.1 The Old Ausable River Channel

Disso lved phosphorus is negatively correlated with phospholipid abundance. When

phospholipid abundance is high, aqueous dissolved phosphorus is relatively low. Figure 4.1

illustrates the typical relationship observed at each of the sampling sites? The dissolved

phosphorus data can be found in Table 4.1. In spring, as the system begins to regenerate,

phosphorus is slowly removed fkom the water column and is assimilated by the microbial and

plant communities. Dissolved phosphorus is at a minimum during the autumn when

' There is a displacement in the dissolved phosphorus maxima af the Mucihole site. Due to this anomalous behaviour, attributed to drying, fieezhg and human activity, the behaviour of the other sites is considered to be typical of the undisturbed Old Ausable system,

54

the microbiai community is most active. Findy, during the winter rnonths, plants and

microorganisms die, releasing phosphorus back into the water column. Dissolved

phosphorus reaches a maximum during the spring which resttvts the cycle.

Total Dissolved Phosphorusb @pb) I ( Fall -

DRY 24 4.3 2.8 6.8 23

Table 4.1 : Seasonal and spatial changes in total dissolved phosphorus in the OARC.

"refer to list abbreviations benor of 15 %

- -

Dissolved Pl Phospholipid Relationship Buriey Bridge North

Season - R - e - Dissolved Phosphorus Phospholipid

Figure 4.1 : Typical relationship between dissolved phosphorus and phospholipid abundance in the OARC.

55

4.3.2 The Kirk Cousins Management Area

The pattern of dissolved phosphorus in the KCMA contrasts that observed in the

OARC. Values soar during the autumn as seen in Table 4.2. This atypical behaviour is

partially due to the high turbidity in the ponds during the summer and autumn. The

suspended particdate materiai provides adsorption sites for both periphytes and

orthophosphates. ' Investigations into the exchange ofphosphorus across the sedimentlwater

interface have shown that wave action and wind-induced turbidity can cause a substantiai

release of phosphorus into the water column.' Both the iiberation of adsorbed phosphate as

well as the mWng of sediment pore water with the water column contribute to the large

increase in total dissolved phosphorus-' Moreover, the suspension ofsediment particles with

signifïcant phosphorus buiding capacity may aid in its subsequent re-sedimentation, causing

a substantial decrease during the whter months when wave action has ceased. The hirbidity

induced death of the microbiai community, resulting fkom the reduction in light intensity,)

may aIso cause a large release of phosphorus. Figure 4.2 austrates the relationship between

phospholipid abundance and dissolved phosphorus at each of the sampling sites in the

KCMA. Almost every site shows a negative correlation with maximum dissolved

phosphorus and minimum phosphoiipid in the autumn.

However, the Regina Mundi South site shows a positive correlation. Both total

dissolved phosphorus and phospholipid abundance reach a maximum in the autumn. There

may be a reduction in turbidity at this site because extensive reed beds damp out wave action,

resulting in a local increase in sediment phospholipid (as discussed in section 3.3.2). The

trend in dissolved phosphonis foliows that of the entire pond due to diffision. Further

sampling wïii be required to adequately explain this anomaiy.

It should aiso be noted (see Table 4.2) that Pond Mu contains the highest levels of

dissolved phosphorus in the autumn, up to eight times higher than the other two ponds. The

death and decay of the madtom catnsh that inhabit this pond may be a contributhg factor.

Their deaîh has been linked to a toxin produced by a blue-green aigae, Aenrginosa sp., that

was identified Î n water samples fiom the pond by Dr. K. Dewdney . Regina Mundi and Tuale

Ponds do not have a madtom population.

The high temperatures and low precipitation rates in the summer of 1998 resulted in

a substantiai decrease in the volume of Pond Mu thus increasing the concentration of

dissolved phosphorus species. Evaporaîion did not have a marked effect on either of the

other ponds. Regina Mundi's water Levels are artifïcidy maintained by the effluent fiom

Regina Mundi Coiiege and Turtle Pond is &cientiy sheltered by surrounding trees to

reduce water Ioss by evaporation.

RMM RMS MU TP

Total Disaolved Phosphomsb (ppb)

Table 4.2: Seasonal and spatial changes in total dissolved phosphorus in the KCMA.

"refer to List of abbreviations benor of 15 %

Dissolved P/ Phospholipid Relationship Regina-Mundi Middle A

T i

- Season - z

+ Dissolved Phosphorus . Phospholipid

DissoIved P/ Phospholipid Relationship Pond Mu

Season - DissoIvcd Phosphonrs . PhosphoIipid

Dissolved P/ Phosphoiipid Relationship Regina-Mundi South w A

-- a

Season h - Dissoived Phosphorus Phospholipid

Dissolveci P/ PhosphoIipid Relaüonship Turtle Pond

- Dissolved Phosphonis Phospholipid

Figure 4.2: The relationship between total dissolved phosphonis and phospholipid abundance at each site within the KCMA.

4.4 Conclusions

There is good negative correlation between phospholipid abundance and aqueous

phosphorus in the Old Ausable system. This is consistent with seasonal cycles in sediment

microbiai activity.

The Old Ausable exhibits minimum aqueous dissoIved phosphorus and maxUnum

phospholipid abundance in the autuInn. This is consistent wïth maximum plant and

microbiai growth prior to the omet of winter.

The Mudhole represents an anomalous site both because of direct human impacts

such as road building as weli as fieezing and drying of the shaüow water and sediment at this

site.

n i e KCMA sites also show a negative correlation between aqueous dissolved

phosphorus and sediment phospholipid abundance- However, the dissolved phosphorus

maximum occurs in the autumn. We attribute this singular behaviour to turbidity caused by

wave action with a strong interaction between dissolved phosphorus and suspended mineral

matter. The Regina Mundi South site alone shows a positive correlation. We suggest this

effect is due to a reduction in turbidity at this site fiom wave damping by reed beds.

4.5 References

1. K. R. Reddy, R H. Kadlec, E. Flaig, and P. M. Gale, Critical Reviews in Environmental

Science and Technology, 29,83 ( 1 999).

2. B. Bostrom, J. M. Andersen, S. Fleischer, and M. Jansson, HydrobioZoga, 170,229

(1988).

59

3- R. J, Bobbie, J. S. Nickels, G. A- Smith, S. D. Fazio, R H. Finàiay, W. M. Davis, and

D. C. White, Appl. Env. Microbiol, 42, 150 (1981).

5. Ancillarv Identification Techniaues

5.1 Introduction

Since NMR is a poor technique for identifLing phospholipids unless standards are

readily available, other techniques have been employed for identification. Attention has

been focussed on phospholipids E and K because of their abundance and persistence. An

autumn Mudhole North sample showed the simplest spectrum, containing only one

concentrated phospholipid corresponding to E. Significant effort was expended on its

purification and identification.

5.2 Esperimental

5.2.1 Thin Layer Chromatography

Following the phosphoiipid extraction procedure, the phospholipid mixture was

dissolved in a smali volume of chlorofonn to produce a concentrated solution of 50 mg/ml.

Silica gel 60F, TLC plates were suppiied by EM Science in 20 X 20 cm sheets. The plates

were eut into strips 3 cm wide and 20 cm long. The concentrated solution was spotted onto

the TLC stnps 2 cm fiom the bottom of the plate using glas capillary tubes. The

phospholipids were eluted in a developing chamber containing a solvent mixture of 30 mi

chloroform, 34 ml ethanol, 8 mi distilled water and 35 mi triethylamine.' The TLC plate was

withdrawn after 2-3 hours, when the solvent fiont was 1 cm fkom the top of the plate. The

plate was lefi to air dry for 24 hours and was then cut in half dong its length. One half of

the plate was developed with Dittmer and Lester phosphorus spray.' Compounds containhg

61

a phosphate ester stained blue with a v i d detection Iimit reported at 5 nmol phospholipid?

The plate was allowed to dry for at least one hour until the blue colour was M y developed.

A phospholipid standard was also run to test the procedure. The two halves of the TLC plate

were then compared. The upper and lower llmits of the location of the blue stain was marked

on the unsprayed half of the plate, The phospholipid sample dong with the silica were

scraped fiom the unsprayed plate and placed ioto a test tube. Chloroform was added to

dissolve the phospholipid. Findy, the mixture was filtered through a Gelman nylon

membrane (pore size, 0-45 jm) to remove the silica The filtrate was then ready for M e r

andysis.

5.2.2 Electrospray Mass Spectrometry

The phospholipid separated by 1ZC was prepared in a 0.5 mM NaOH solution of

chioroform, methanol and water (45:45: 10). These reagents were added only just pnor to

analysis to prevent the hydrolysis of the phospholipid. The spectrometer used was a TOBY

prototype instrument made by Sciex, Concord, Ontario, a single quadrupole electrospray

mass spectrometer-

5.2.3 X-ray Photoelectron Spectroscopy

An aliuninum stub was metallographically polished with a 3 p m diamond grit paste

and sonicated in methanol. One &op of the filtered phospholipid sample nom TLC was

dried onto the polished aliiminum stub. The stub was mounted onto a . XPS sample holder

for analysis. The SSL-100 ESCA Spectrometer at Surface Science Western equipped with

62

an electron flood gun for charge neutralization and an Ai Ka X-ray source was used.

5.2.4 Static Secondary Ion Mass Spectrometry

The same sample that was initidy analysed by XPS was transferred to a custom buiit

static SIMS for analysis- This instrument is based on the VG ZAB-2F reverse geometry

magnetic sector mass spectrometer and uses an fei gal.Lum ion source. The primary ion beam

was run at 25 keV with a current of 0- 1 UA (static conditions)- A 0.5 mm x 0.5 mm area of

the surface was anaiysed. The mass resolution was set low (apertures at widest setting) to

obtain the largest signal possible. Positive secondary ions fkom mass 0-600 amu were

analy sed-

5.2.5 Materials

The solvents used for thin layer chromatography and electrospray mass spectrometry

were supplied by Caledon Laboratories Ltd. 99 % pure phospholipid standards were

purchased fiom Sigma Chemical Company and used as received-

5.3 Results and Discussion

5.3.1 Thin Layer Chromatography

The initial extracted phospholipid was light brown in colour. Foliowing separation

by TL,C, the phospholipid / chIoroform solution was colourless. The pigmented components

in the original extract can be seen near the solvent fiont on the TLC plate. A mixture of

phospholipids may require additional separations as the deeply pigmented samples may

63

o v e m the top haif of the plate. A retardation factor (Rd of 0.35 was determîned for

phospholipid E in the autumo Mudhole North sample. No other blue stained matenal was

obsewed.

5.3.2 Electrospray Mass S pectrometry

Earlier work using eiectrospray mass spectrornetry for an weparated sample gave

a spectrum containing a broad background around the central peak. It was felt that an

improved spectnun couid be obraiwd ifa purified sample was used. The autumn Mudhole

North sample that had k e n separated by TLC was analysed (Figure 5.1). One sharp peak

at 502.5 amu was found and the broad background had been removed Two very minor

peaks were detected at 474.5 and 518.5 m u . These two peaks may be representative of

changes in the fatty acid side c h a h of the phospholipid. The peak at 5 18.5 amuis indicative

of the addition of an oxygen atom to the molecule. The peak at 474.5 amu ciiffers fiom the

main peak by a mass of 28 amu which is indicative of a CO m e n t .

It should be noted that the mass of a sodium atom has been subtracted iÎom the mass

values on the observed spectrum to give the values mentioned above. The addition of

sodium hydroxide to the analysed solution gives nse to an added sodium in the molecule.

Previous work by Dr. Fred Possmayer using this procedure has shown that only one sodium

atom is associated with each phospholipid.'

Figure 5.1 : Electrospray mass spectmm of unknown phospholipid following TLC separaîïon.

65

5.3.3 X-ray Photoelectron Spectroscopy

XPS survey analysis of the dned TLC phospholipid extract was undertaken to explore

the possibüity that heteroatoms, such as nitrogen or sulphur were present. None were

detected. This hnits the possible phospholipids to those without heteroatoms. Simiificant

amounts of silicon were detected- This is attribut& to silica fkom the TLC plate.

5.3.4 Static Secondary Ion Mass Spectrometry

Electrospray mass spectrornetry gives information on only the parent mass peak,

accordingly static secondary ion mass spectrometry (SIMS) was employed to obtain a mass

fragment pattern- If a suitable mass hgrnent pattern codd be acquired, the structure of

various side chains, fùnctional groups and perhaps the entire molecule might be elucidated-

Although most peaks in the spectnim c m be related to organic fiagments, strong

peaks at 23 and 28 amu reveal the presence of sodium and possibly siiicon respectively.

Mass 28 could also be CO but silicon is more liicely based on the previous XPS results. The

majority of the intense fhgments are below 60 a m . A list of some of the more prominent

peaks and possible assignments are given in Table 5.1. Most peaks are indicative of

hydrocarbon fiagments, however some mass fragments are indicative of the incorporation

of oxygen into the organic portion of the molecule (mas 45 and 59). Higher molecdar

weight fiagments, in particular a cluster of fiagments at 1 89,19 1, and 193 (unassigned), are

indicative of the larger fatty acid side chah of the molecule. However, assignment of these

peaks are difEcult without other significant hgments.

1 14 (CH,

Mass Assignment

117 (Unknown

191 1 Unknown

193 1 Unknown

Table 5.1 Significant mass fragments and their assignrnents fiom the positive secondary ion mass spectnim,

67

5.4 Conctusions

No positive identification of phospholipids was made other than those for which

NMR standards were available. Electrospray MS suggests that phospholipid E has a mass

o f 502.5 amu.

5.5 References

1. J. C. Touchstone, 5. C. Chen, and K. M. Beaver, Lipidr, 15,61 (1980).

2. 1. C . Dittmer and R. L. Lester, J. Lipid Res., 5, 126 (1964).

3. L. Konermam, Department of Chemistry, personal communication.

6. Phos~boü~id - Calcite Interactions

6.1 Introduction

An X-ray photoelectron spectroscopy @PS) study of the interaction of a calcite

surface with various phospholipids was undertaken to investigate the possible adsorption of

phospholipids on mineral d a c e s . Such adsorption would change the nature of the

sedirnent/water interface and might help explain the mechanism of phosphorus

muieralization. XPS was employed because ofits surface sensitivity and its ability to detect

changes the chemical state of elements. h addition, W S has been used previously to study

phosphate adsorption onto goethite.'" Calcite (CaCOJ was used in this study because it is

a major mineral in both the Old Ausable River Channel and in the various ponds Ui the Kirk

Cousins Management Area-

6.2 ExperimentaI

6.2.1 Methodology

A crystal or powder sample of calcite was placed in a 50 ml solution of varying

concentration for varying tune periods. Potassium acid phosphate, phosphatidk acid, and

diethyl phosphate solutions ranging fiom 0.01 ppm to 10 ppm were used as possible

adsorbent. Exposure times of solid CaCO, to solutions containing the adsorbing species

ranged fiom 10 minutes to 4 weeks. The solution was saturated with CaCO, to minimize the

dissolution of the adsorbing calcite crystals and adjusted to a pH of 8 to simulate conditions

at the two study areas. The sample was shaken using a Burrell Wrist Action Shaker to ensure

69

uniform distribution of the various solutes over the entire d a c e of the calcite sample. The

crystai was then removed fiom the solution. It was washed bnefly (5 seconds) with distilled

deionized water, dried, placed on a vacuum compatible, conductive carbon based adhesive

for analysis by X-ray photoelectron spectroscopy W S ) . Control caicite samples were

prepared in the sarne rnanner as above without the addition of the adsorbing species. A

nickel grid was placed approximately 1 mm over the sample. The nickel grid in conjunction

with an electron flood guo is used for sample charge compensation! For survey analyses,

a pass energy of 150 eV and a spot size of 600 Pm was used. For high resolution spectra, a

pass energy of 50 eV and a spot sùe of 600 Pm was used. Standard samples of various

phospholipids and powdered calcite samples were mounted for XPS analysis by pressing the

solids samples into indium foil. The SSL-100 ESCA Spectrometer at Surface Science

Western equipped with an eiectron flood gun for charge neutralization and an Al Ka X-ray

source was used.

6.2.2 Ion Chromatography

Following the removai of the calcite crystals, the tiltrate was analyzed by ion

chromatography to examine the remaining ionic species. A Dionex DX-100 Ion

Chromatograph equipped with a Dionex IonPac AS 14 4 mm anion exchange column was

used for this work. A suppressor membrane was used to neutralize buffer s a k in the eluent

solution. The use of a suppressor wili Iower background conductivity, thereby increasing

analyte sensitivity.

6.2.3 Materials

Nahiral calcite samples were supplied by Dr. M. Powell fkom the Department of

Earth Sciences. Synthetic calcite and pure phospholipid samples were suppiied by Sigma

Chernical Company in 99 % purity and used as received Potassium acid phosphate was

purchased fiom Caledon Laboratories Ltd,

6.3 Resuïts and Discussion

6.3.1 Standards and Controls

Standard samples of phospholipids were anaiyzed by XPS (see Table 6.1). It was

hoped that heteroatoms (atoms other than carbon, oxygen and phosphorus), such as nitrogen

in L-a-Phosphatidylethanolamine, couid aid in the identification of the adsorbed

phospholipid species. Phosphorus (2p) binding energies are simdar for all standard samples

ranging from 133.8 to 134.5 eV. This range is too narrow to ailow for identification of

phospholipids from P(2p) binding energies. This was expected but was an important

exercise nonetheless. Changes in these values should be observed if any reaction on the

surface or decomposition of the phosphate group takes place. These baseline values will

help to determine if chernical changes have occurred.

Initial investigations were also carried out to determine if a suitable sample of calcite

could be found. Most minerals are far fiom king pure cornpounds, thus, other species are

often found. This will be especialiy tme at grain boundaries where many species wiU

accumulate. Two samples of calcite were chosen; Iarger crystals (-3-5 mm diameter) o f

Iceland spar, considered to be the purest nahval calcite available,' and a powder of a 98%

71

pure synthetic calcite. XPS anaiysis of a fieshly fÏactured surface of Iceland spar gave a clean

calcite spectnim containhg o d y carbon, oxygen and calcium- However, spectra of

unfiactured surfaces of Iceland spar varied in theïr relative cleanIiness. SmaU arnounts of

sodium, silicon, nitrogen, and sulphur on the outer surface of the sampie were occasionally

detected. The use of extra elements, such as nitrogen and sulphur, for the identification of

some phospholipids may prove ciifficuit ifthey are present on the d a c e of the untreated

calcite. Analysis of the synthetic calcite powder revealed only carbon, oxygen and calcium.

Sample C O

L-a-Phosphatidic Acid (Sodium 81.8 12.0 Salt)

L-a-Lysophosphatidic Acid, Oleoy L 73 -2 16-1 (Sodium Salt)

Sphuigomyelin

L-a-Phosphatidylethanolamine

(Sodium Salt)

1 Palmitoyl (Sodium Salt) 1 1

Table 6.1 : Surface composition of standard samples in atomic percent as determined by X- ray photoeiectron spectroscopy (XE'S). Phosphorus (2p) binding energies for each sample are presented in the farright column. Binding energies are referenced to carbon (1s) at 285.0 eV-

6.3.2 Potassium Acid Phosphate, Proof of Principle

Phosphate minerai adsorption has k e n studied extensively and is weii known?'

Potassium acid phosphate (=?Po4) solutions were used to determiue ifadsorbed phosphate

couid be detected on the calcite surface by XPS. Phosphou was detected on both calcite

samples after exposure to 1 O-* M to 1 Od M solutions with exponire times ranging fiom 10

minutes to 5 days- As exposure times and concentrations increased so did the amount of

phosphonis detected on the sample d a c e s (see Figure 6.1). One important observation was

that significantly more phosphorus was detected on the surface of the Iceland spar than on

the powdered calcite sample. There are three possible explmations for this. The larger

surface area of the powdered sample ailows for more adsorption of the phosphate, effectively

Iowering the phosphate concentration of the solution at a faster rate. The phosphate becomes

thinly spread over the large d a c e area of the powder. The crystal sample adsorbs much

less total phosphate and as such, is exposed to a higher solution concentration for a longer

period of tirne. This allows a more complete coverage of phosphate on its surface.

Secondly, fieshly cleaved Iceland spar surfaces may be more reactive as the crystals tend to

cleave dong defect boundaries. Finally, this observation may also be due to the way each

sample is analysed by the XPS. Only the nirface of the crystal sample is analyzed with the

IceIand spar but with the powdered sarnple, some of the background indium foii surface may

be analyzed as well. The adventitious carbon component of ihe indium foi1 then adds to the

amount of carbon detected, effectively l o w e ~ g the amount of phosphorus- Use of the

powder was also more tedious because the powder had to be filtered foilowing adsorption.

In addition, very srnail amounts of powder were used and loss of sample during filtration and

Control CaCO,

300 275 250 225 200 175 150 125 1 O0

Binding Energy (eV)

Figure 6.1 : XPS survey scans showing the increase in adsorbed phosphate as the concentration of KH,PO, in solution increases.

handling can be a problem.

A Raman spectrum of a treated calcite surface confirmed the presence of adsorbed

orthophosphate (yp04). A strong broad peak centred at 930 cm-' was found on the reacted

surface that was not present on the unreacted surface of calcite (Figure 6.2)- A literature

value of 938 cm-' is given for orthophosphate?

6.3.3 Phosphatidic Acid

Phosphatidic acid (PA) consists of a phosphate head group and two fatty acid side

chains (see Figure 1.1). The presence of fatty acid side c h a h mean that steric factors wili

play a role in the molecule's reactivity on calcite surfaces. Beyond the detection of the

adsorbed phospholipid it was hoped that d a c e changes and perhaps adsorbed degradation

products might be detected. PA was first detected using a 10 ppm solution and 24 hours of

exposure. No phosphorus was detected at lower concentrations even for much longer periods

of exposure (up to 4 weeks). The weak phosphorus (2p) signal was at approximately 134.2

eV, essentiaily unchanged from the standard sarnple (Figure 6.3). Since the concentrations

used were well above those encountered in the natural system, this research avenue was

abandoned.

In retrospecf the adsorbed species could be phosphate and not phosphatidic acid.

High resolution scans of other elements (carbon, oxygen and calcium) did not provide any

further information. Ion chromatography of the solution could have confirmed the presence

of phosphate and therefore the hydrolysis of phosphatidic acid. In addition, M e r XPS

Wavenumbers (cm-')

Figure 6.2: Raman spectra of a clean calcite crystal (bottom) and a crystal exposed to a solution of -PO, (top). Adsorbed phosphate is indicated on the exposed sarnple by the peak at 929.7 cm-l.

Binding Energy (eV)

Figure 6.3 : High resolution XPS spectra of the phosphom (2p) peak for the standard sarnple of phosphatidic acid (PA) and for a calcite d a c e exposed to 10 ppm solution of PA for 24 hours. The fitted peak is the overaU contribution fiom both the P(2p,,-J and P ( 2 ~ 3 d peaks.

77

experiments might show slight differences in phosphorus (2p) binding energies,

distinguishing between adsorbed phosphate and phosphatidïc acid.

63.4 Diethyl Phosphate

It was then decided to attempt adsorption with the smaitest phosphate ester that could

be obtained, diethyl phosphate. It was speculated that adsorbed species could be detected on

the calcite surface fiom solutions at realistic concentrations. Initial experiments with low

concentrations and short exposure times failed to reveal any adsorbed phosphom species.

It was not until longer exposure times were used that adsorbed phosphom was detected (1

ppm, 4 week exposure). Phosphorus (2p) binding energies for the adsorbed species ranged

fiom 133.4 to 133 -7 eV. This is on the lower end of the values obtained for the standard

samples.

The long exposure times and the low bînding energy obtained for the phosphonis (2p)

peak suggested that the diethyl phosphate ester had hydrolyzed in the slightly basic solution.

Ion chromatography of the solution revealed an orthophosphate ion peak. It is therefore

likely that the adsorbed phosphorus species is orthophosphate and not diethyl phosphate.

Literature binding energy values for phosphate range nom 132.4 to 134.2 eV.'' This

corresponds to the observed binding energies.

6.4 Conclusions

XPS yields adsorption data consistent with the known behaviour of the phosphate1

calcite system. The attempts reported here to use W S to study the possible adsorption of

78

phosphoiipids on calcite are inconclusive. In the cases where phosphorus was detected, IC

analysis of the solutions used showed the presence of phosphate, a probable hydrolysis

product.

6.5 References

1- R. R. Martin, and R. S t C. Smart, Soil Sci Sc. Am. J,, 51,54 (1987).

2. R. EL Martin, R St C. Smart, and K. Tazaki, Soi2 Sci. Soc. Am. J., 52,1492 (1988).

3. R G. Jonasson, R. R Martin, M. E. Giuliacci, and K. Tazaki, J Chem. Soc., Fmu&y

Trans. 1,84,2311 (1988).

4. B. V. Crist, Handbooks of Monochromatic XPS S~ectra: Vol. 1. - The Elements and

Native Oxides, XPS International Inc., Kawasaki, lapan, xïii, 1997.

5. M. Powell, Department o f Earth Sciences, personal communication.

6. C. Colombo, V. Barr&, J. Torrent, Geochimica et Cosmochimica Acta, 58,126 1 (1 994).

7. M. A. Maiiti, R. A. Fassam, 1. R. Henderson, J. Chem Tech. Biotechnol-, 58,3 87 (1993).

8. E. Gonzalez-Pradas, M. Villafranca-Shchez and A. Gallego-Campo, J. Chem Tech.

Biotechnol., 54,29 1 ( 1 992)-

9. W. P. Griffith, Inf'rared and Raman S~ectrosco~v of Lunar and Terrestriai Mllierals, C.

Karr, Ed., Academic Press, New York, 3 17, 1975.

10. J. F. Moulder, V. F, Stickle, P. E. Sobol, and K. D. Bomber, Handbook of X-ray

Photoeiectron SDectrosco~v, Perkin-Elmer, Eden Prairiey Minnesota, 1992-

7. Conclusions and Future Work

7.1 Conclusions

NMR analysis of phospholipids in the OARC show seasonai changes consistent with

the existing üterature. This may be taken as validation of the NMR anaiysis of

sediment phospholipids as an indicator of the microbial comrnunïty.

The spatial distribution of phosphoiipids in the Old Ausable system can be readiiy

explained in terms of hown sediment characteristics. This may be taken as furthet

c o ~ a t i o n of the utility of3'P NMR as a monitor of secliment mlcrobial commmities.

The Mudhole North site, known to undergo anomalous fieezing and drying events as

well as being subjected to human impacts, shows a discontinuity in the established

phospholipid pattern.

The phospholipid richness also shows seasonal variation with maximumrichness in the

autumn,

Sediment phosphoiipids in the K M . . do not display a we1l defined seasonai cycle.

This behaviour is attributed to the extreme turbidity observed in this system as well as

phosphate input to Regina Mundi Pond fiom the coiIege.

XRD of the suspended matter in the Kirk Cousins ponds system confkms the presence

of mineral matter known to interact strongly with dissolved phosphorus species.

In the Kirk Cousins system, phosphoiipid richness is significantly Iower than in the

OARC. W e it is tempting to attribute this ciifference either to effluent fiom Regina

Mundi College and/or water turbidity, the ponds studied each have unique, individual

characteristics. This diversity argues against any simple interpretation of these results.

80

8. In the Old Ausable River Channel there is a negative correlation between sediment

phospholipid abundance and aqueous dissolved phosphorus. This is consistent with a

mechanism where microbiota sequester phosphorus during optimum growing

conditions. Phosphorus is subsequentiy released when the microbiota die in winter.

9. In the Kirk Cousins Management Area the same negative correlation is obsenred,

however, the maximum dissolved phosphorus concentration is dispIace& occurring in

autumn rather than wiuter/sprhg. This is almost certainly due to maximum turbidity

in autumn. Both bioturbation and ememe reductions in water levels in one of the ponds

may be contributing factors.

10. Regina Mundi South represents the only site with a positive correlation between

aqueous dissohed phosphorus and sediment phospholipid abundance. We suggest this

effect is due to a reduction in turbidity at this site fiom wave damping by reed beds.

1 1. Phoqhatidyiethanolamine, phospha t idy lcho l Lysophosphatidylcholine have been

identified by standard addition.

12. Phospholipids K and E are the most persistent and abundant.

13. The balcony sample confirms the suggestion that phospholipids are short-lived in the

environment.

14. XPS shidies of possible phospholipid adsorption suggest that there is no direct

interaction between p hospholipids and calcium carbonate. However, phosphatidk acid

appears to be adsorbed at concentrations in the order of 10 ppm.

81

7.2 Future Work

Nattuaiiy fhture work shouid include additionai sampling at the study sites. The

balcony sample suggests that a weii defiued experiment shouid be designed to monitor the

rate of phospholipid loss between the auhunn and spring.

Now that we are confident that individuai phosphoiïpids can be extracted by thin

Iayer chromatography, an attempt should bernade to i d e n e these species using ion trapping

and laser hgmentation foiiowed by mass spectrometry. Such an experiment is being

designed and wïii be undertaken with Dr- G, Wiiiett, University of South Waies, Australia-

&mendix 1

Individuai Phosphoiipid Concentrations

Seasonal and spatial changes in individual phospholipid concentrations for the Old Ausable River Channel, Phospholipid concentration is reported in nmol P/g dry sediment with an error of 30 %. OL - overlapping peak. Iis concentration is included in the value for the main peak (see Appendix II), arefer to list of abbreviations

a 1 ni* 1 swa / wlva d , ~ ntu swli w~lr d ~ , MI smlr I

A ~ ~ e n d k

NMR Spectra

G 6 = 1.471 pprn J 6 = 1.180 pprn K 6 = 1.081 pprn M 6 = 0,975 pprn PC 6 = 0.064 pprn

MudhoIe North, Spring 98

U 8 = 1.833 pprn E 6 = 1.643 pprn F 6 = 1.588 ppm H 6 = 1.390 pprn 1 8 = 1.277 pprn J 8 = 1.198 pprn K 6 = 1,109 pprn M 6 = 1.010 pprn PC 6 = 0.079 pprn

Mudhole North, Summer 98

Mudhole North, Autumn 98

E 6 = 1.699ppm PC 6 = 0.070 ppm

Mudhole North, Wmter 98

K S = 1,165 pprn

Mudhole South, Spring 98

E S = 1.688 pprn F 6 = 1.613 ppm H 6 = 1.426ppm 1 6 = 1.342 pprn J 6 = 1.232 pprn K 6 = 1.139ppm PE S = L.100ppm M 6 = 0.969 pprn PC 6 = 0.079 pprn

Mudhole South, S u m e r 98

D 6=1,722ppm E 6 = 1.588 ppm F 6 = 1.490 ppm K 6 = 1,075pprn PC 6 = 0,055 ppm

Mudhole South, Autumu 98

E 8 = 1.647 ppm K 8=1.121 ppm

- . - -

Mudhole South, Winter 98

E 6 = 1.522ppm J 6 = 1.191 pprn K 6 = 1.095 ppm PE 6 = 1.050 ppm M 6 = 0.904 pprn

Canoe Docks North, Spring 98

D 6 = 1.718 pprn E 6 = 1.624 pprn G 6=1.462ppm H 6 = 1.361 pprn J 6 = 1.180ppm K 6 = 1.081 pprn PE 8 = 0.988 pprn Q 6=0.127ppm PC 6 = 0.075 pprn

. -- --

Canoe Docks North, Sumer 98

A 6 = 2.870 ppm E B 6=1.915ppm D 6 = 1.677 ppm E 25 = 1.613 pprn G 8 = 1.431 pprn J 6 = 1.146 pprn K 6 = 1.015 pprn M 6 =O.gIS pprn LPC 8 = 0-613 pprn Q 6 =0.109ppm PC 6 = 0.051 pprn

Canoe Docks North, Autumn 98

E 8 = 1.696 ppm c

K 6 = 1.117ppm PC 6 = 0.059 ppm

-

Canoe Docks North, Winter 98

E 6 = 1.673 pprn K 6 = 1.167 pprn PE 6 = 1.140 pprn

Canoe Docks South, Sprïng 98

B 6 = 1.859 pprn E 6 = 1.554ppm F 6 = 1.450 pprn K 6 = 1.043 pprn Q 6 = 0.095 pprn PC 6 = 0.043 pprn

Canoe Docks South, Summer 98

93

B 6 = 1.895 pprn C 6 = 1.768 pprn D 6 = 1.661 ppm E 6 = 1,580 pprn G S=1,404ppm J 6 = 1.219 ppm K 6 = 1.033 pprn M 6 = 0.884 pprn Q 8=0.115ppm PC 6 =0.041 pprn

Came Docks South, Autumn 98

E S = 1.733 ppm F 6 = 1.673 pprn K 6 = 1.165 pprn PE 6 = 1.124 pprn N S = 0.93 1 pprn P S = 0.627 pprn Q 8 = 0.135 pprn PC 6 = 0.075 pprn

-

Canoe Docks South, Winter 98

E 6=1.628ppm J 6=1.226ppm K K 6 = 1,128 ppm PE 6 = 1.083 pprn LPC 8 = 0.703 ppm PC 6 = 0.066 ppm

I ~ ' ~ ~ l = - g L ~ " " r ~ ' ' ~ I B " ' I " " I ' ~ " I = ~ l B ~ ~ . . . ~ " ' . ( 4.0 3.5 3-0 2-5 2.0 i-5 1.0 0 -5 O .O -0.5 PRI

Burley Bridge North, Sprïng 98

E 6 = 1.633 ppm K 6 = 1.095 ppm PC S = 0.069 ppm

Burley Bridge North, Summer 98

O 8 = 1.689 pprn E 6 = 1.588 ppm E G 6 = 1.443 pprn H 6=1.335ppm J 6=1.149ppm K 6=l.O53 pprn M 6 = 0.905 pprn PC 6 = 0.049 pprn

~ " " I ' " ' i ~ . c = l ~ ' - . ~ . - . . r . . r - l - - m = I - . ' . J ' . . . , ' . . , 4.0 3.5 3.0 2-5 2. O 1.5 1.0 0.5 0.0 4 - 5 PP)I -1.0

Burley Bridge North, Autumn 98

E 6 = 1.752ppm K 8 = 1.213 pprn PE 6 = 1.195 pprn

Burley Bndge North, Winter 98

K S = 1.262 ppm

Budey Bridge South, Sprhg 98

B 6 =2.013 ppm E E 6 = 1-693 ppm K 6 = 1.168 ppm PC 6 = 0.085 ppm

--

Burley Bridge South, Summer 98

B 6 = 1.933 pprn E 6 = 1.653 ppm G 6 = 1.517 pprn H 6 = 1.367 pprn 1 6 = 1.231 pprn K 6 = 1.095 pprn

-

Burley Bridge South, Autumn 98

E 6 = 1.736 pprn K 8 = 1.165 pprn

Burley BrÏdge South, Winter 98

E 6 = 1.629 pprn J 6 = 1.232 pprn K 6=1.138ppm PE S = 1 .OS9 pprn M 6 = 0.998 ppm PC S = 0.079 ppm

Canoe Docks North, Single Sample, Sprhg 98

E 6 = 1.722 pprn H 6 = 1.426 ppm J 8 = 1.260 pprn K 6 = 1.188 pprn M 6 = 1.043 pprn Q 6 =O.l58 pprn PC 6 = 0.098 pprn

Canoe Docks North, Spring 99

Balcony Sample

K 6 = 1.080 ppm

Regina Mundi Pond Middle, Spring 99

D 6 = 1.717ppm E 6 = 1.609ppm G 6 = 1.477 ppm J 6 = 1.221 ppm K 6 = 1.085 ppm M 6 = 0.979 ppm

Regina Mundi Pond Middle, Summer 98

Regina Mundi Pond Middle, Autumn 98

E 6 = 1.617 ppm K 6 = 1.095 ppm

Regina Mundi Pond Middle, Witer 98

K S= 1.195 ppm L 6 = 1-180 ppm

Regina Mundi Pond South, Spring 99

E 6 = 1.624 ppm K 8 = 1.090 ppm

Regina Mundi Pond South, Summer 98

K 6 = 1.095 ppm

Regina Mundi Pond South, Autumn 98

E 6 = 1.629 ppm K 6 = 1.100ppm

Regina Mundi Pond South, Whter 98

E 6 = 1,792 ppm K 8 = 1.241 ppm

--

Pond Mu, Spring 99

E 8 = 1.617ppm K 6 = 1.060 ppm

Pond Mu, Srunmer 98

E 6=1-732ppm K 6 = 1.154 ppm PE 6 = 1.122 ppm

Pond Mu, Autumn 98

E 6 = 1.683 ppm F 6 = 1-635 pprn K 8 = 1.141 ppm

Pond Mu, Winter 98

K 6 = 1.079 ppm

Turtie Pond, Spring 99

Turtle Pond, Sumer 98

Turtle Pond, Autumn 98

K 6 = 1-138 ppm

TurtIe Pond, Winter 98