THE APPLICATION OF EXCITATION-EMISSION FLUORESCENCE SPECTROPHOTOMETRY TO THE MONITORING OF DISSOLVED ORGANIC MATTER IN UPLAND CATCHMENTS IN THE UNITED KINGDOM. THE APPLICATION OF EXCITATION-EMISSION FLUORESCENCE SPECTROPHOTOMETRY TO THE MONITORING OF DISSOLVED ORGANIC MATTER IN UPLAND CATCHMENTS IN THE UNITED KINGDOM. by Lucy Bolton A thesis submitted to the University of Newcastle upon Tyne in partial fulfilment of the requirements for the degree of Doctor of Philosophy in the School of Geography, Politics and Sociology School of Geography, Politics and Sociology University of Newcastle upon Tyne, U.K. NE1 7RU June 2003 Declaration I hereby certify that the work described in this thesis is my own, except where otherwise acknowledged, and has not been submitted previously for a degree at this or any other University Lucy Bolton Acknowledgements I would like to thank my supervisors, Dr Andy Baker and Prof. Malcolm Newson, for their expert guidance, encouragement and help with sampling and funding. I am also grateful to everyone who helped with fieldwork and water sampling, especially Watts Stelling and Howard Waugh at Coalburn and Chris Rix at Assynt and all the organisations involved in the work at Coalburn I would also like to thank all my friends and colleagues who made it worthwhile and especially Trev, without whom it wouldn’t have happened. The University of Newcastle helped to fund this project. 4 Abstract This study details the investigation into the use of spectrophotometric methods, principally excitation emission fluorescence spectrophotometry, in the monitoring of dissolved organic matter (DOM) in upland catchments. A protocol for the storage and analysis of DOM solutions was designed. To minimise deterioration immediate analysis was recommended. Long term storage, by freezing, resulted in significant and unpredictable alteration of the spectrophotometric properties. A post analytical correction was applied to overcome concentration related interferences. Solutions were analysed at natural pH, with consideration of the influence this property has on the spectrophotometric properties of DOM. Two study areas: the Coalburn Experimental Catchment (Northumberland) and the Loch Assynt area (Sutherland) were monitored. Spatial assessment of surface waters indicated that the distribution of DOM spectrophotometric properties was related to the influence of inorganic material in soils. This was observed as DOM in runoff from peat dominated areas, compared to non-peat, the former DOM having greater aromaticity or higher molecular weight. Distinct DOM spectrophotometric properties were observed in rainwater and throughfall and DOM from fresh and partially degraded spruce needles had a unique spectrophotometric signal. The two study areas exhibited limited variations in DOM properties, when compared to DOM from a wider range of sources. The mean estimated export DOC of from the Coalburn Experimental Catchment was 22.00 gm-2a-1 but the rate varied through the year. DOM spectrophotometric properties in both study areas varied seasonally exhibiting production and flushing periods with changes in catchment conditions. Discharge relationships indicated DOM sources in peat dominated area, however, these sources are only important when hydrologically active. A mild aqueous extraction method, to obtain dissolved organic mater from peat, was designed. This method obtained DOM, which reflected the distribution of spectrophotometric properties in related surface water. The method was applied to peat profiles from both study areas and the spectrophotometric properties of the DOM indicated relatively homogenous peat derived DOM. Peat DOM depth variations were observed and in some cases related to the presence of litter and inorganic layers. There was a broad spectrophotometric change with depth indicating increased aromaticity or molecular weight. 5 Chapter 1. Introduction and Literature Review This thesis presents the study of dissolved organic matter (DOM) using spectrophotometric techniques. The use of such methods, especially excitation emission matrix (EEM) fluorescence spectrophotometry, in the study of DOM has become widespread in the last 10 years. These studies have not concentrated in detail on DOM in upland areas at high resolution. The following chapters describe the spectrophotometric examination and characterisation of DOM composition, sources and processes from two such areas. The importance of DOM in upland catchments is two fold, firstly the negative impact it’s presence and composition has upon drinking water quality and secondly on habitats. With future predictions of climate change these aspects become more important as estimates of DOC in rivers indicate an increase in exports. It is therefore essential to be able establish accurate concentrations and compositions of DOM. There are many methods for this, however each has drawbacks, in addition to benefits. DOM is a complex aquatic component and thus requires extensive isolation and sample preparation prior to analysis. This study applies a method, fluorescence spectrophotometry, which does not require isolation and maintains the natural associations by analysis bulk samples. Also it is quick, easy and cheap method, when previously been applied to DOM studies (Baker, 2001). This is the first detailed use of these analytical techniques for the detailed examination of DOM in upland areas and also the first detailed examination of DOM in a forested peat catchment. The study aims both to utilise spectrophotometric properties to investigate DOM composition, but also to consider flow paths and DOM sources. The aims of this study are presented in each chapter with respect to the specific aspects of the research presented therein. The aims relate to the investigation into spatial and temporal variations in aquatic and peat DOM in locations in the UK: the Coalburn Experimental Catchment (Northumberland) and the Loch Assynt area (Sutherland). Variations in DOM were monitored using EEM spectrophotometry and UV-visible absorbance analytical methods and a further aim of the study is to assess 6 these methods. Utilising such methods provides the ability to analyse DOM in situ incorporating the multiple interactions with other aquatic components. The following chapter presents an overview of the literature regarding the current understanding and importance of DOM in the environment and the methods employed in the monitoring of it. A summary of the use of spectrophotometric methods in the analysis of DOM is also detailed as are the field areas monitored. 1.1 Dissolved organic matter Dissolved organic matter is ubiquitous in soil and aquatic ecosystems. In aquatic environments natural organic matter (NOM) ranges in concentration from 0.5 mgL-1 DOC in alpine streams to 100 mgL-1 in wetland draining streams (Spitzy and Leenheer, 1991; Frimmel, 1998). An operational classification is applied to NOM, between particulate organic matter (retained on 0.45µm filter) and dissolved organic matter (DOM) (Aiken et al., 1985; Spitzy and Leenheer, 1991). Organic carbon occurs bound into organic molecules and the terms DOM and DOC, are used interchangeably in the literature (Eatherall et al., 1998). In aquatic environments, NOM is composed of carbon compounds and related nitrogen or phosphorus compounds (Spitzy and Leenheer, 1991). In addition to naturally derived organic matter there are many classes of anthropogenically derived DOM in aquatic systems. These components are derived from specific sources such as agriculture or sewage, or can be present as pollutants such as pesticides, petroleum products and industrial effluents (Manahan, 1994). This work is concerned with NOM and DOM is used to denote dissolved NOM in both soil and aquatic environments. DOC is used to indicate the concentration of dissolved organic carbon. Riverine DOM is composed of a variety of substances, which vary in time and space. Approximately 25% of DOM is fully characterised; this comprises amino acids, nucleic acids, carbohydrates, hydrocarbons, fatty acids and phenolic compounds (Spitzy and Leenheer, 1991; Thomas, 1997) the rest being composed of humic substances (HS). Estimates of the amount of HS in aquatic DOM are in the region of 40-60% (Senesi, 1993) and 50-70% (Thurman, 1985). In soil systems HS are closely 7 associated with non-humic components and inorganic material involving multiple interactions and aggregations (MacCarthy, 2001). 1.1.1 Humic substances Humic substances (HS) are natural, complex, macromolecular substances that form from the breakdown of plant and animal debris and are ubiquitous in soil, sediments and water (Thurman, 1985; MacCarthy, 2001). HS are the main components of soil organic matter (~80%) but due to the complexities of formation processes and composition they are not completely described (Hayes and Clapp, 2001; MacCarthy, 2001). Aiken et al. (1985) defined HS as: “A general category of naturally occurring heterogeneous organic substances that can generally be characterised as being yellow to black in colour, of high molecular weight, and refractory”. This definition is still considered to be valid, however the refractory nature may only exist in protected environments, (Hayes, 1998) and the term “high molecular weight” is not always applicable (Hayes, 1997). MacCarthy (2001) has proposed a more recent definition relating to the basic principles of HS. This addresses questions about the nature of the composition and formation of HS: “Humic substances comprise an extraordinarily complex,