Diatom and geochemical indicators of acidification in a tropical forest stream, singapore 5

Chapter Five METHODOLOGY 5.1 Overview 76 5.2 Field Methods 76 5.3 Laboratory Methods 5.3.1 Stratigraphical Description 5.3.2 Organic Carbon Content 5.3.3 Diatom Analysis 5.3.4 Geochemical Analysis – Total Sulphur 5.3.5 Geochemical Analysis – Lead, Zinc, Potassium, Sodium, Iron and Manganese 77 77 78 80 81 83 75 5.1 Overview Chapter five contains a description of the methodology employed with this study and has been subdivided into field methods and laboratory methods. Field methods cover the processes involved in the collection of a sedimentary core from the study site. Laboratory methods detail how a stratigraphical description of the core is provided along with the steps involved in determining the organic carbon content of the core. It then discusses the technique used to separate diatoms from the sedimentary matrix and mount them onto slides. Lastly the chapter covers the different sediment digestions involved in obtaining sulphur and other trace metals concentrations within the core, briefly explaining how the concentration levels were determined on an Inductively Coupled Plasma – Optical Emissions Spectrometer (ICP-OES). 5.2 Field Methods Sedimentary sequences can vary in thickness and complexity with depth at the same site, making sedimentary history hard to trace when viewing an individual core (Lowe and Walker, 1997). The time period of interest in this study – the past century – is often contained within the top 50cm of sediment or less (Smol, 2008). These recent sediments are challenging to collect as they are often unconsolidated and have a high water content, potentially exceeding 95% water by weight (Smol, 2008). Thus, to ensure sample representativeness, a rod-driven piston interface corer with a chamber length of 40cm and a diameter of approximately 6.5cm was used to extract 3 cores from the study area – Core A, Core B and Core C (Plate 4-3). Core A was 14cm in length and extracted from the side of the stream, Core B was 15cm in length, extracted from the middle of the stream and Core C was 17cm long and located just in front of the brick dam. A piston corer was chosen as it functions well in the majority of lake sediments and does not cause the 76 displacement of sediments. It is also best suited to retain the uppermost portion of the sedimentary profile as it operates by creating a seal that prevents the sediments from washing out of the tube (Aaby and Digerfeldt, 1986; Glew et al, 2001). Upon collection, the core barrels were sealed and transported back to the NUS Geography Laboratory for sub-sectioning and subsequent analysis. 5.3 Laboratory Methods Once a core has been brought to the surface, it can either be preserved in its entirety or sub-sampled in the field (Glew et al, 2001). As more accuracy can be obtained through the use of a core extruding apparatus, which would have been unwieldy to transport to Jungle Falls Valley, it was decided that core barrels should instead be transported back to and sub-sectioned in the laboratory. A decision also had to be made regarding the temporal resolution of analysis. The selected resolution will affect the sub-sampling interval, with a higher temporal resolution requiring a smaller interval (Smol, 2008). Much of this decision is also based on the diameter of the core barrel itself. This is because enough sediment is needed in each sub-sample for multiple analytical techniques including diatom analysis, organic carbon content measurements and geochemical analysis. It was decided that a sampling interval of 1cm was suitable for this study. Cores were extruded immediately upon arrival at the Geography Laboratory and sealed in plastic Ziploc® bags. Samples were then stored in a refrigerator prior to analysis. 5.3.1 Stratigraphical Description While there did not appear to be different layers within the sediment as the depositional environment had not altered significantly over the duration of the core, it is still important to provide visual descriptions of the cores as a foundation for any subsequent analysis that occurs. This would also allow potential comparison between sites and the “establishment of a general picture of 77 sedimentary deposits which could lead to a better understanding of them” (Kershaw, 1997: 67). A modified Troels-Smith system, based on Kershaw (1997) was used to provide this detailed lithostratigraphic description. This system was selected as it is widely utilised in paleolimnological research, requires no background knowledge of any specific natural science, is quick, simple and can be applied in most geographical and depositional environments (Kershaw, 1997). It is a lithostratigraphic system based solely on description and comprises three parts – physical factors, humicity and deposit elements (Aaby and Berglund, 1986). The physical factors are subdivided into the degree of darkness, the degree of stratification, the degree of elasticity and the degree of dryness of the sediment (Kershaw, 1997). Humicity is “the degree of disintegration of the organic substance, regardless of the way this disintegration has taken place, and of what substances resulted from it” (von Post and Granlund, cited in Aaby and Berglund, 1986: 233). Lastly, the deposit elements are the “nature and proportion of the elements composing the deposit” (Kershaw, 1997: 63). For the majority of these features, a five-point scale which ranges from 0 to 4 is used. 0 represents the complete lack of the feature and 4 is the maximum value for it (Kershaw, 1997). See appendix A for a more detailed description of the Troels-Smith scheme. 5.3.2 Organic Carbon Content There are numerous methods available to determine organic carbon content (for review, see Hesse, 1971) and percentage loss on ignition (%LOI) is used here as it is the most practical and straightforward one (Gale and Hoare, 1991). While there should not be significant variations in organic carbon content within the sediment cores from BTNR, it is worth measuring as organic carbon content may affect interpretations of geochemical analysis results (Urban, 1994) 78 and any variation present would also provide a practicable and straightforward method to enable multiple cores to be correlated (Flower et al, 1988). Firstly, the porcelain crucibles to be used for %LOI analysis were weighed (M1). Approximately 5g of each sample was then placed in these porcelain crucibles before being left in an oven at 105oC to dry for 24hrs. The crucibles were then moved into a desiccator and the contents were cooled to room temperature before another weighing (M2). Care was taken to minimise the time the crucible contents were exposed to air in order to minimise any increase in mass as the sample equilibrates with laboratory humidity. A furnace was preheated to 500oC before the crucibles were placed in it for 24hrs. The crucibles were then moved into the desiccator again to cool to room temperature. While Gale and Hoare (1991) recommends placing the samples in a furnace at 430oC for 24hrs, a temperature of 500oC was used to ensure complete ignition of plant organic matter based on a study by Oh (2000). As the study by Oh (2000) was conducted in Singapore, on similar sediments, it was felt that the higher temperature for %LOI testing was appropriate. Finally, the crucibles were weighed a third time (M3). Percentage loss on ignition (%LOI) is then calculated using the following formula: %LOI = 100[(M2 – M1) – (M3 – M1)] / (M2 – M1) Prior to testing sediments for organic carbon content, new and soiled crucibles were fired in the furnace at 550oC for a minimum of 5hrs to ensure that none of the loss in mass during ignition is due to the loss of contaminants or because of changes in the physical nature of the crucible. 79 5.3.3 Diatom Analysis Battarbee et al’s (2001) procedure for preparing and mounting diatoms from lake sediments has been employed in this study with slight modifications. To separate the diatoms from the sedimentary matrix, approximately 2 grams of wet sediment was placed in a beaker and a small amount of 19% H2O2 added. Fresh samples were used as oven drying can result in diatom breakage (Battarbee et al, 2001). When foaming, if any, had subsided, 75-100ml of 19% H2O2 was added and the beaker was heated on a hotplate until all the organic matter had reacted. If samples experienced a vigorous reaction, and levels of H2O2 got low, the beakers were topped up with more H2O2. This procedure took around 5-6hrs. If sediments needed to be washed down the side of the beakers due to strong and foaming reactions, deionised water was used. Subsequently, the samples were diluted three times (beakers were filled with deionised water and the sediments within were allowed to settle overnight before this water was removed using a water aspirator) prior to mounting on slides. Following dilution, 400µl of each diatom suspension was dropped onto a clean coverslip by pipette and left overnight for the diatoms to settle and water to evaporate. Once dry, a drop of Naphrax was placed on a glass slide and the coverslip inverted onto it with the dried diatoms over the drop. The slide was heated intermittently on a hotplate at around 100oC for 20mins to remove the toluene in the Naphrax and then left to cool. Once the toluene in Naphrax is removed, it has a refractive index of 1.73, ideal for diatoms analysis (Battarbee et al, 2001). Prepared slides were checked to ensure that the coverslip did not move when pushed with a finger. Two slides were prepared for each sample to provide replicates if necessary. Diatoms were identified using an Olympus BX40 system microscope under x400 magnification. Unfortunately, there are no diatom identification keys 80 for the region, and diatoms from studies within Asia are identified using keys from Europe and North America (such as Van Iperen et al, 1993; Horton et al, 2007 and Liu et al, 2011). As these keys were unavailable for this study, diatoms were identified using previous studies on Singapore diatoms by Wah (1988) and Oon (2010), along with a study of diatoms in low-alkalinity lakes in North America (Camburn and Charles, 2000), and various online sources including the Royal Botanic Garden Edinburgh (RBGE, 2010), the Academy of Natural Science in Philadelphia (ANSP, 2011), the University of Colorado Boulder (2011) and Newcastle University (2011a). Diatom counting was then carried out along continuous traverses. While diatoms are usually counted until a predetermined number is reached (usually between 300-600; Battarbee et al, 2011), in this study, the entire slide was counted as concentrations within were low. 5.3.4 Geochemical Analysis – Total Sulphur The use of a CHNS analyser is currently the preferred technique for measuring the sulphate content of sediments. However, without access to such equipment, it was decided that total sulphur would be determined gravimetrically. In gravimetric analysis, the aim is to convert the sulphur in the sediment to barium sulphate and weighing the amount of BaSO4 in the sample. This involves the addition of barium chloride solution to the acid extract of the sediment. The precipitate of BaSO4 is then collected, dried and weighed before sulphate content is calculated from the mass of the material used in the analysis and the mass of barium sulphate precipitated (BSI, 1990). Gravimetric analysis is one of the oldest analytical techniques and is the classical approach to the determination of sulphate (Gale and Hoare, 1991; FNU, 2009). Unfortunately, based on preliminary tests, the sulphate content of the sediments from Bukit Timah nature reserve were too low to measure gravimetrically as the quantity of sediment 81 required to yield results was above what was available after other analysis was conducted. It was therefore decided that total sulphate content would be measured using an ICP-OES. While this technique is not widely employed in paleolimnological investigations of acidification, Ryu et al (2006) used it with success in their study of the sulphur biochemistry of sediments from Owens Dry Lake in California. The effectiveness of this technique was examined by Sah and Miller (1992), though they were applying it to biological tissues. Sah and Miller (1992) found that digesting samples using 70% HNO3 and 30% H2O2 gave complete recovery of sulphur. Bukit Timah sediment samples were first oven dried at 40oC before being crushed and sieved with a 2.00mm mesh to remove large clasts and macroorganic matter such as roots. 0.5g of each sample was then digested in a mixture of 7ml HNO3 (68%) and 1ml H2O2 (30%) by microwave heating. While Sah and Miller (1992) used 4ml of HNO3 and 4ml of H2O2 in their digestion, they stated that using more than 2ml of H2O2 increases the risk of explosive venting. Furthermore, it is potentially dangerous to mix HNO3 and H2O2. Thus, based on application notes provided by the manufacturer of the microwave digestion system used – Milestone – of which usage of H2O2 did not exceed 1ml and was mixed with 7ml of HNO3, it was determined that a mixture of 7ml HNO3 and 1ml of H2O2 would be ideal. Prior to microwave digestion, the samples were left in a fumehood to react for 30mins to reduce the danger of explosive venting of the microwave vessels. Microwave temperature increased to 180oC in 10mins, and maintained at 180oC for 15mins. A reagent blank was run with each digestion to ensure no contamination occurred during the digestion process. Following digestion, as the sample contained particulates that may affect chemical analysis, 82 samples were centrifuged at 3000rpm for 15mins. Samples were then diluted 50 times prior to measurement by ICP-OES. A PerkinElmer® OptimaTM 8300 ICP-OES was used to measure sulphur concentrations. This machine has a duel view – axial and radial. The axial view measures the samples face-on and is thus ten times more accurate than the radial view which is from the side. Radial viewing is used only when sample concentrations are high and thus, axial view was employed in this study. Using argon, nitrogen and compressed air, the machine takes approximately 80mins to warm up before the plasma can be ignited. Following ignition, the system was run with deionised water for 30mins in order to allow the plasma to stabilise. The machine was then calibrated using a blank of deionised water followed by sulphur standard solutions of 5ppm, 10ppm, 25ppm, 50ppm and 100ppm. Deionised water was used for the calibration blank as the sulphur standard was in a water matrix. Finally, reagent blanks were measured before the digested core samples were run. The ICP-OES was programmed to replicate each reading three times before averaging the results. These replicates can be compared to ensure accuracy in readings. Random samples were also run twice to provide another verification of values recorded. Following each reading, the machine was flushed with deionised water for a minimum of 10secs to clear the system. 5.3.5 Geochemical Analysis – Lead, Zinc, Potassium, Sodium, Iron and Manganese The concentration of the above elements were measured by ICP-OES as well. The procedure used for the acid digestion of these sediment samples is based on the United States Environmental Protection Agency’s (EPA) Method 3501A – microwave assisted acid digestion of sediments, sludges, soils, and oils 83 (EPA 3501A, 2007). An alternative method was required for sulphur digestion as this method is not applicable to the measurement of sulphur concentrations. It should be noted that this method does not accomplish total decomposition of the sample and that the extracted analysed concentrations may not reflect the total content in the sample. This is because hydrofluoric acid, which is capable of dissolving silicates (EPA 3052, 1996), is not used. According to EPA Method 3052, “samples with lower concentrations of silicon dioxide ([...]... levels emitted at 5ppm, 10ppm, 25ppm, 50 ppm and 100ppm for these six other elements, and applying this calibration to the sulphur data UDA dataset, the concentrations of these elements in the sulphur sample digestions can be determined 85 It is preferable that all elements to be measured are in the initial calibration standards, rather than having to calibrate the machine a second time to apply to the... unique wavelength, the system also captures the full spectrum of wavelength data within the sample As a result, elements not measured in the initial instrument run can be calculated subsequently through a reprocessing of the UDA dataset With regard to sulphur analysis, the machine was programmed to measure sulphur at a wavelength of 181.975nm By recording the amount of light a sample emits at this wavelength,... rest of the protocol for the measurement of lead, zinc, sodium, potassium, iron and manganese concentrations follows that of sulphur above The PerkinElmer® system employed in this study also has the added option of “Universal Data Acquisition (UDA)” This means that while a sample is being analysed for the calibrated elements specifically input into the system, each element being recognised from a unique... of changing environmental and equipment conditions, the second calibration was carried out immediately after the last sulphur sample, C19, was run Through UDA and reprocessing, the sulphur concentrations in the multi-element sample digestions were also determined This data reprocessing served two purposed Firstly, should the trace metal concentrations and profiles in the original and reprocessed dataset... reprocessed data This is because environmental and equipment conditions could vary between runs which then affects the calibration of the equipment and consequently the results This does not mean that elemental concentrations will always be different in different runs, as the equipment calibration will negate environmental and equipment variables However, if a machine is calibrated on a different day to sample... the wavelengths of Pb (wavelength 220. 353 nm), Zn (wavelength 206.200nm), K (wavelength 766.490nm), Na (wavelength 58 9 .59 2nm), Fe (wavelength 238.204nm) and Mn (wavelength 257 .610nm) These wavelengths are based on both the recommendations of PerkinElmer® and the United States EPA Method 6010C – Inductively Coupled Plasma – Atomic Emission Spectrometry (EPA 6010C, 2000) After calibrating the machine to... sample measurements, the calibration may not be effective Unfortunately, the multi-element standard solution did not contain sulphur as one of the elements Furthermore, the multi-element standard solution was in 1mol/l HNO3 and the sulphur element standard solution was in water, and therefore, should not be mixed together Thus, the ICP-OES had to be calibrated in two different sets However, to address... knowing how much light is emitted at 5ppm, 10ppm, so on so forth, the machine is then able to determine the concentration of sulphur in a sample When UDA is activated, not only is the light level emitted at the sulphur wavelength measured, data for all other wavelengths is also measured Thus, upon conclusion of the sulphur instrument run, the data was reprocessed to record the light level emitted at... concentrations and profiles in the original and reprocessed dataset be similar, this would provide a verification of the results obtained Secondly, it would also enable an evaluation of the ability of the more unorthodox sulphur digestion method – the mixture of 1ml of H2O2 and 7ml of HNO3 – to provide a total recovery of trace metals in the sediments 86 ... in each sub-sample for multiple analytical techniques including diatom analysis, organic carbon content measurements and geochemical analysis It was decided that a sampling interval of 1cm was... using previous studies on Singapore diatoms by Wah (1988) and Oon (2010), along with a study of diatoms in low-alkalinity lakes in North America (Camburn and Charles, 2000), and various online... sedimentary matrix, approximately grams of wet sediment was placed in a beaker and a small amount of 19% H2O2 added Fresh samples were used as oven drying can result in diatom breakage (Battarbee