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

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.. Thus, to ensure

Chapter Five METHODOLOGY

5.3.1 Stratigraphical Description 77

5.3.2 Organic Carbon Content 78

5.3.4 Geochemical Analysis – Total Sulphur 81

5.3.5 Geochemical Analysis – Lead, Zinc, Potassium, Sodium, 83

Iron and Manganese

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

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

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)

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

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

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

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 macro-organic 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,

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

(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 (<10% to 0%) may require much less hydrofluoric acid (0.5ml to 0ml)” (1996: 6) As previous studies

have documented the poor preservation of diatoms in Singapore (Taylor et al,

2001; Oon, 2010), and a preliminary investigation of diatoms within Bukit Timah Nature Reserve revealed that diatom concentrations there are low, silicon dioxide concentrations were deemed to be low enough to not require the use of hydrofluoric acid in the digestion of sediments

Similar to the technique used for total sulphur analysis, samples were oven dried at 40oC then crushed and sieved through a 2.00mm mesh 0.5g of each sample was then digested in 10ml of concentrated (65%) HNO3 by microwave heating Microwave temperatures increased to 180oC in 10mins, and maintained at 180oC for 10mins While EPA method 3051A specified that temperatures should increase to 175 ± 5oC in approximately 5mins, rather than 10mins, this time was lengthened in order to ensure that the sediment reaction was gradual rather than vigorous Following digestion, samples were centrifuged

at 3000rpm for 15mins then diluted 50 times before being run through the PerkinElmer® Optima TM 8300 ICP-OES

Axial view was also employed in this analysis and the machine was calibrated, at concentrations of 5ppm, 10ppm, 25ppm, 50ppm and 100ppm, using

a multi-element standard solution in 1mol/l HNO3 As such, the calibration blank was also a 1mol/l HNO3 solution, prepared by diluting concentrated HNO3 (68%)