Research Proposal

Studying the geochemistry of Bald Cypress tree rings provides the opportunity to study anthropogenic impacts on local water resources. Considering the rapid increase in urbanization and implementation of infrastructure in the greater Austin area, understanding these impacts will be invaluable in managing our natural stream waters.

This study intends to analyze a tree core from a Bald Cypress tree located along Waller Creek, running through central Austin. The core will be analyzed by both LA-ICP-MS and solution-based ICP-MS in order to determine any analytical differences. Of particular importance to our methodology will be the sample preparation. For the study, analytically prepared standards of beech wood and valerian root will be used.  For solution-based analysis, the core be split into 5 year sections to allow for specific time periods to be analyzed. For proper analysis the cellulose/lignin matrix of the core will need to be digested and minimized.  It is expected that the study will indicate whether such analysis has the ability to determine changes in trace element concentrations within the tree growth rings, and whether this may be utilized to record anthropogenic changes in natural stream water. The methodology and results should also demonstrate whether one method is more suitable for larger studies of growth rings, by considering the spectral results and the rigor of the sample preparation.

Specific Research Objectives

By examining concentrations of target analytes associated with anthropogenic inputs, we hope to reconstruct a timeline of increasing urbanization and development in the local area. From Beal et al (2020), we observe differences in trace element concentrations between urban and rural stream waters, as well as markedly different values for municipal waters. Therefore, in this study we will focus our analysis on the concentrations of Al, Ca, Fe, Sr, and Ba, given that metal uptake can occur from a tree’s water sources.  Additionally, we will analyze Pb, which can be an important indicator of anthropogenic pollution, such as from car batteries and previously burned, but phased out around 1973, leaded car fuels, respectively (US EPA 1973). However due to the ease of analysis by quadrupole ICP-MS, the whole amu mass range will be taken for each sample. 

The methodology for this project will involve running the same tree core for both LA-ICP-MS analysis and solution-based analysis. For LA, a methodology will be developed in order to systematically run ablation along each set of growth rings and collect concentrations for a running time series. This method is very valuable for tree core analysis in that it will allow for rapid analysis of cores, with little sample preparation required.  For solution-based ICP-MS, we will need to be able to dissolve sections of the core to again produce a time-series of concentrations. By running both types of analysis, we hope to determine if laser ablation is an effective and efficient method, when compared to the more established solution mode methods. In other words, refining the laser ablation method will allow for more efficient and fast determinations of trace element concentrations in tree rings, with what we hope is a similar accuracy to that of solution mode, but without the lengthy sample prep.  This will enable easier analyses of elemental concentration proxies in nature and therefore enable an easier understanding of human pollution and management of local water resources.

Justification of Significance

As urbanization increases, the potential for human wastes and outputs, such as municipal water, to infiltrate natural systems inevitably increases (Moiseenko et al. 2019). This can be traced by quantifying the levels of trace elements and heavy metals in natural systems that might not otherwise be seen. Although Moiseenko et al. (2019) note that trace element/heavy metal pollution has recently been decreasing in the developed countries of Europe and North America, largely due to stricter environmental regulations, past production of these trace elements can become taken up by biological processes and incorporated into biochemical cycles, such as those present in trees and plants.  When accepting water or nutrients from the soil, plants may pick up these anthropogenically inputted elements and “trap” them into organic materials, such as the wood in trees, which will vary over time depending on levels of urbanization, with more developed time frames showing higher concentrations of trace metals  (Salmani-Ghabeshi et al. 2021). 

We anticipate the concentrations of the analytes of interest  to be related to the concentrations in the water and in the soil. Future studies of the soil would be required and need to be compared with the concentrations found in the tree wood. In the future it also would be of value to consider the contributions that the pollution in the air might play in the concentrations of heavy metal trace elements in the trees (Zaanouni et al. 2018).  The changes in concentrations, if any, will guide our research as to how effective trees are at recording local environmental changes. Yu et al. (2009) have demonstrated an effective method for analyzing tree samples for wet trace elemental analysis, but with Chinese mahogany trees and using ICP-AES. Creating a similar effective method for LA-ICP-MS analysis for Bald Cypress trees will be a valuable tool for quick and accurate sample preparation in geochronological studies. For anticipated accuracy of the results, we expect to see similar results to that of Perone et al. (2018), given the similarities between the instrumentation employed. We predict these values to be in the range of anywhere from 1-1000 ppm  or around 3 orders of magnitude for the various analytes being considered in our study.

Review of Relevant Work

Despite being somewhat of a niche topic, some significant and applicable, to our purposes, research has been performed on measuring the concentration of trace elements and/or metals in tree ring/core samples using ICP-MS and similar analysis methods. Some of the relevant research was mentioned above and a few more studies have been summarized below. 

Rich et al. (2012) showed that it is possible to determine isotopic ratios and elemental concentrations of Cedar of Lebanon tree rings using multicollector – ICP-MS. For geochronological studies MC-ICP-MS is one of the most common instruments used for isotopic geochronogocial studies where very high precision is required to distinguish between isotopes. However, for isotopic applications purification, as performed by Rich et al. (2012) with column extraction techniques, some form of purification is required, which is time consuming and only allows for analysis of usually one element, like Sr in this study. In their study Rich et. al. found some issues with digestion of the wood cores, requiring multiple repeat digestions following the 4 step nitric acid and hydrogen peroxide procedure. Their study highlights some of the issues that can come about in the sample preparation procedure and some of the benefits of using quadrupole ICP-MS over other detector types. 

Various solution mode ICP-MS studies on tree cores have been conducted, most using a similar sample digestion procedure to Rich et al. (2012) above. Another somewhat less common method involves ashing the wood before primary digestion. This method is especially useful in that it removes the number of occasions in which the sample has to be contaminated, in that it requires less external digestive solutions. A rather unique use of this technique was made by Jeon et al. (2020), which employed the use of a direct mercury analyzer (DMA) to analyze Hg in tree rings, which although is a different instrument entirely, highlights the benefits of ashing.  The study below makes use of the more general ashing technique and applies it to ICP-MS. 

Watmough et al. (1995) performed a solution mode analysis of sycamore tree rings using ICP-MS. In their case the purpose was evaluating the fluctuations in metal concentrations in the tree as a result of pollution from a nearby factory and whether the short term pollution releases from the factory would be reflected in the dendrochronology. They note several difficulties in the study including certain affinities in certain tree rings for certain metals, the lateral movement of metals between the rings, and the loss of short term changes in concentration due to the need to group rings together to gather enough material to obtain a sufficiently high resolution in analysis. For standards this study used a ground sample of reference wood (hop-hornbean). For sample prep, they first ashed the samples and then dissolved them using 70% nitric acid.  They analyzed the cores for Mg, K, Ca, Mn, P, Fe, Cu, Ni, Zn, Sr, Cd and Pb concentrations. Recovery for the elements was found to be quite good 80% and 120%, except for phosphorus, which was only 60%. The results from the ring analyses seemed to have few issues (, except with the aforementioned probable contamination from other parts of the tree, such as the bark, making some of the core unusable in the study. For most of the analytes in the tree rings however, the concentrations were within detectable limits and showed that solution mode ICP-MS was an effective method and that tree rings could be effective tools for performing dendrochronology studies. 

Many other studies have proven that analyzing tree rings chronologies by solution mode ICP-MS is effective and are doable with few issues. However, given the relative ease of sample prep for laser ablation ICP-MS, many other studies have attempted using laser ablation to perform similar wood concentration chronology studies.  Laser ablation brings with it different issues than solution mode. One of the issues is how to obtain a standard with a comparable matrix and in some cases whether this is necessary for accurate analysis, as is investigated in many of the following studies. 

Garbe-Schönberg et al. (1997) used laser ablation ICP-MS to analyze tree-ring profiles in pine and birch trees. To prep the tree samples sanding was performed to smooth out the surface therefore improving the ablation of the samples. The material used for sanding is a possible source of contamination for any study involving wood, given that sanding is often a required step. In the case of Garbe-Schönberg et al., they performed measurements on the abrasive paper material to determine if it contained any of the measured analytes.  For calibration the study used Nist 610 and 612 analytical grade glass beads. In this study the analytes of interest were Cu, Pb, Al, Mg, Ba, Co, Ni, As, Sb, Se, and Sn. Due to low concentrations of the desired analytes, the laser ablation step had to be performed in triplicate for each ring. Despite ablating in triplicate Co, Ni, As, Sb, Se, and Sn were not found to have concentrations within the detection limits of the instrument. Ba and Mg were not found to vary among the tree rings. The study concludes by declaring that laser ablation was an effective method, but that due to various factors surrounding the way in which individual trees bring in elements, comparisons between trees in an area for pollution analysis may not be very reliable for modelling pollution in an area.  They do note however that this can greatly depend on the specific environment under consideration and the levels of pollution in the area, which for their study area in an heavy industry area of Russia was very high. 

Looking at a more recent study, Perone et al. (2017) used laser ablation to measure trace element concentrations of Cr. Cu, Hg, Mb, Ni, Pb, Tl, U, V, W, and Zn in downy oak trees in north central Italy. In their study the same NIST 612 glass standard was used as Garbe-Schönberg et al. (1997). However, they determined that this would not be an effective reference standard for the wood, given its crystalline/uniform matrix, so they only considered relative concentrations to the 13C internal standard. During the laser ablation procedure they used a spot ablation procedure with a dimension of 257 μm, however they took into consideration that this parameter would vary based on the specific laser in use and the type of wood. The specific analytes on consideration were Co, Cu, Cr, Pb, Hg, Mo, Ni, W, Tl, U, V, and Zn. The results of the experiment showed that for some of the analytes (Co, Mo, Ni, and Pb) showed increasing concentrations, which were detectable using laser ablation ICP-MS, over the time period. Spot LA-ICP-MS was able to effectively describe the historical variations in the wood cores. 

As with any ICP-MS analysis, depending on one’s analytes of interest, various interferences are present in the generated spectra and have to be accounted for.  Look at the table below for the possible interferences. The previous studies investigated don’t go into much detail on what interferences were actually found to be present in the analysis. 

Analyte (Considering only major isotopes)	Possible Interference
Al	14N2, 10B17O, 9Be17O, 12C15O, 13C14N, 13C2H, 12C13CH2, 10B16O, 10B17O
Ca	40Ar, 38ArH2, 24Mg16O, 12C14N2, 29Si11B, 30Si10B
Fe	40Ar16O, 38Ar18O, 40Ar15NH, 38Ar17OH, 40Ca16O, 19F218O, 39K17O, 40K16O, 23Na33S, 37Cl18OH, 28Si2, 46Ca10B, 42Ca14N
Sr	40Ar48Ca, 70Zn18O, 7Li81Br, 176Lu2+, 175Lu2+, 176Yb2+, 87RuH
Ba	57Fe81Br
Pb	192Pt16O, 191Ir17O, 190Pt18O

Looking at the possible interferences above, we anticipate many of the larger m/z interferences will be minimized by the collision reaction cell (CRC) employed by the Agilent ICP-MS instrument used in our study or by optimizing the laser and solution mode parameters during tuning. However, it will become evident during analysis and setup if these interferences are present in our data. Matrix effects are also important to consider, and are partially what makes analysis by LA-ICP-MS so difficult, due to the complexity and inconsistency of the cellulose/lignin matrix. 

Materials and Methods

In order to collect results that span a suitable time period and have the ability to show temporal changes in our analytes, we will examine one tree core collected from Waller Creek. The core, as shown below, has been dated through tree-ring counting. 

 

Firstly, the core will be split into two halves, using a sharp and clean blade. One half of the core will be analyzed using LA-ICP-MS. For the LA-ICP-MS half, the sample will be sanded down using smooth grit sandpaper to help improve uniformity of ablated surfaces. For analysis, two standards will be used, Beech Wood (IPE:240) and Valerian Root (IPE:143), both which have been purchased from WEPAL/QUASIMEME and pressed into a powder by myStandards. One standard will be used for calibration, and the other for quality control. The standards were chosen to reflect both the matrix of a tree core (for example, high lignin) and suitable concentrations of our analytes for analysis using ICP-MS.

Concentrations of analytes in standards: 

Analytes	Concentration in Beech Wood (mg/Kg ± StdDev or MAD)	Concentration in Valerian Root (mg/Kg ± StdDev or MAD)
Al	4.60 ± 1.495 **	652 ± 61.7
Ca	1250 ± 117	2230 ± 189
Fe	7.20 ± 3.183 *	2960 ± 441
Sr	6.91 ± 0.391 *	14.3 ± 0.85 *
Ba	35 ± 2.11	17.4 ± 2.14 *
Pb	0.058 ± 0.02416 **	2.71 ± 0.391

Legend: Consensus (none); Indicative (*); Informative (**)

 

Standards were professionally ground and pressed into pellets to achieve maximum homogeneity. Laser ablation will be carried out in specific intervals to ablate each set of rings multiple times. This should provide an average set of measurements for each analyte for each time period. Overall, the running time for laser ablation, for both the standard and sample, should be short. 

For solution-based ICP-MS, the core will be split using a sharp, clean blade and split into 5 year increments. Each increment will be ground and weighed, then dissolved to digest the cellulose content of the sample. We will follow a modified procedure outlined in Rich et al (2012) to digest our samples without the use of a microwave:

For solution-based ICP-MS, we will follow a modified procedure outlined in Rich et al (2012) to digest our samples without the use of a microwave:

1. 0.1–0.2 g ground Bald Cypress wood will be accurately weighed and transferred into a Teflon beaker. 
2. 4 ml of 14 M HNO3 and 2 ml of H2O2 (analytical grade) will be added and the beakers placed on a hotplate at 90 C for 12 h. 
3. The samples will be evaporated to dryness at 100 C and then taken up in 1.5 ml of 14 M HNO3 and 4.5 ml of 12.6 M HCl, then heated on a hotplate for 24 h at 110 C. 

Considering our tree core, this will produce 9 increments of 5 years spanning the time period between 2020 and 1975.

 

 

 

Possible Outcomes

While the results of the experiment are not known at this point, our main concern is the comparison between the concentrations, sensitivities, and detection limits we are able to achieve/detect for both the laser ablation and solution modes of ICP-MS analysis. Our hope is that they appear somewhat similar and indicate that LA-ICP-MS can be effectively and quickly used to determine concentrations of trace elements in tree samples, without some of the more tedious steps required for solution methods (cutting, grinding, acid digestion, etc.)  However, if the methods achieve different results, we will try to quantify and explain the differences. 

 

Timeframe and Budget

The proposed study will run an adequate number of samples, including the use of calibration standards, to assess any changes in the proposed trace elements/heavy metals. Since only one tree core will be analyzed, the majority of sample preparation will be focused on preparing the dissolved samples and the preparation of the cellulose calibration standard. Samples will be prepared over a one month period to be ready by November 11, 2021. 

Analysis costs:

Type	Cost	#	Total cost
LA analysis	$73/hr	4	$292
Solution analysis	$17/sample	12	$204

Total cost = $496