NON-INVASIVE SAMPLING

TECHNICAL FIELD

[ 0001 ] This invention relates to a method for non-invasive sampling of plant transpiration products for detecting the presence of one or more chemical analytes. The method further relates to gradient mapping of the presence, absence or concentration of one or more analytes within a specified location, as well as to a system and method for doing so.

BACKGROUND

[ 0002 ] The following discussion of the background art is intended to facilitate an understanding of the present invention only. The discussion is not an acknowledgement or admission that any of the material referred to is or was part of the common general knowledge as at the priority date of the application.

[ 0003 ] Environmental assessments aim to measure the presence or absence of target analytes, chemicals, elements, markers, or compounds in an environment. Assessments known to the Applicant usually take the form of destructive, invasive sampling, such as digging up tracts of land, taking soil or sub-soil samples by drilling (which can be time consuming, require specialised labour, and drilling machines which may be difficult if not impossible to transport to a site), or disturbing landscapes to obtain samples for laboratory testing.

[ 0004 ] Most biological toxicology sampling methods require removing partial or full plant material from within plants including leaf, root and trunk cores, or the euthanising of animals for muscle, skeletal or organ tissue.

[ 0005 ] In certain instances, this shortcoming means that such tests are not only environmentally invasive and destructive, but they compound the very problem they were employed to monitor in the first instance, i.e. damage to the environment.

[ 0006 ] Biogeochemistry has not been routinely adopted for sampling, partly because biotic mechanisms of analyte migration from soil and sub-soil (including groundwater) to plant extremities and exudates are poorly understood. [ 0007 ] The following invention seeks to propose possible solutions, at least in part, in amelioration of the above shortcomings in the field of the invention.

SUMMARY OF THE INVENTION

[ 0008 ] Broadly, the Applicant has shown that it is possible to assess the presence, absence, and/or concentration of analytes in the soil or sub-soil structures by non-invasively and non-destructively screening for the presence, absence, and/or concentration of the analytes in plant transpiration products or exudates.

[ 0009 ] According to one aspect of the invention, there is provided a method of assessing the presence or absence of one or more analytes in an environment or site to be assessed, the method including the steps of: obtaining one or more transpiration samples from one or more plants within the environment or site to be subjecting the one or more transpiration samples to an analytical method suitable for detecting the presence or absence of the one or more analytes to be assessed.

[ 0010 ] The invention thus includes, in one aspect thereof, a method of assessing the presence or absence of one or more analytes in an environment or site to be assessed, the method including the steps of: obtaining one or more transpiration samples from one or more plants within the environment or site to be assessed; and detecting the presence or absence of the one or more

analytes to be assessed.

[ 0011 ] In this way, non-destructive and non-invasive testing, assessment, or sampling can be conducted either as a snapshot or for an extended period, or both during monitoring operations. This allows for future samples to be taken from the same plants so as to track presence, contamination, remediation, or cleanup operations in the environment or site being assessed. [ 0012 ] As such, the method provides a method of determining the presence, absence, or concentration of analytes within a plant by sampling transpiration products from said plant.

[ 0013 ] In one example, the method may include at least partly enclosing at least a part of a plant with an enclosure capable of retaining a transpiration sample. Typically, such an enclosure may be a plastic bag, although it is within the scope of the invention that other types of enclosures may also be used such as bottles, vessels, sleeves, pouches, sachets, or the like.

[ 0014 ] The part of the plant that is enclosed (or partly enclosed) may be a part that is associated with transpiration processes, such as one or more leaves, stems, fruit and/or flowers, or a part of the plant with stomata present for the release of transpiration.

[ 0015 ] The enclosures may be left on the plants for 1 to 72 hours, typically 3 to 48 hours, with the most typical assessment time being 6 hours . This may be varied according to the type of analyte being sampled as well as the types of plant forming part of the assessment.

[ 0016 ] Typically, the method may include the step of partly enclosing at least part of a plant at a specific time of a transpiration cycle of said plant. This may includes an extended time period for monitoring purposes. Generally, this will occur within the first 4 to 8 hours, sometimes 6 hours, after sunrise.

[ 0017 ] When assessing an environment or site, the enclosing of an array of plants may be substantially synchronous, i.e. occur at approximately the same time. In other words, when plants in a site or environment are to be assessed, they may all be enclosed at approximately the same time.

[ 0018 ] The enclosures or parts thereof may undergo analysis to determine if there is any influence by the devices on transpiration results of the target chemical . This also includes the step of washing the enclosures with, or exposing them to, an inert substance to determine any extraneous influence caused by the enclosure itself. The inert substance may be distilled water or any other standardised and inert substance known to persons skilled in the field of analytical chemistry, i.e. a blank rinsate .

[ 0019 ] A blank sample may also be collected. The blank sample may include the inert substance mentioned above. The inert substance may be placed within the collection device, then passively rolled over a similar part of the plant for a short period of time to replicate the transpiration movement across the plant material, to consider the presence of atmospheric and historic accumulations of the target chemical on the plant surface. This blank can also be used to determine historic and atmospheric accumulations as a point of interest .

[ 0020 ] The analyte may be an inorganic analyte. Furthermore, the analyte may be non-auric.

[ 0021 ] The enclosure may have an internal coating that aids in the capture and/or retention of transpiration products. As such, the enclosure may have an inert interior surface, such as a plastics material, or it may have one or more interior surfaces coated, or at least partially coated, with a material capable of interacting and capturing a particular analyte, depending on the analyte to be sampled .

[ 0022 ] In one embodiment, the interior may be coated with an inert material that readily relinquishes associated transpiration products upon certain pre-determined (or desired) conditions such as heating, cooling, or washing. One such coating may include one or more carbonaceous material, such as activated carbon or activated charcoal and derivatives thereof. The enclosure may be transparent or opaque. Specifically, the enclosure may be made of a material, or coated with a material internally and/or externally, which is UV resistant or near-UV resistant, so as to preserve photosensitive analytes.

[ 0023 ] The enclosure further may have an identifier associated therewith representative of any suitable automatic identification and data capture (AIDC) technology, such as an ID tag, barcode, or RFID tag, which allows for correct mapping and testing of samples in a desired environment. Accordingly, an enclosure may include a transmitter to enable rapid identification and retrieval thereof, especially in dense vegetative conditions. The transmitter may be configured to include geo-spatial positioning functionality. The transmitter may in turn be used to compile a map of an area to be sampled for tracking and audit purposes, which may, following testing of each sample, be used to build up a gradient map of one or more analytes, markers (or compounds) that are being monitored, sampled, or screened .

[ 0024 ] As such, the method may include the further step of configuring a processor via a suitable mapping software programme to create a concentration gradient map.

[ 0025 ] The method may be used to detect the presence, absence, or concentration of elements and uncomplicated compounds such as reduced or oxidised elements, and more complex compounds.

[ 0026 ] As such, the method provides an assessment, particularly a quantitative assessment of the concentration of analytes in plants and/or the soil or sub-soil features within which the plants are growing .

[ 0027 ] Assessment of the presence, absence, and concentration of gradients may be done using laboratory-based analytical techniques, or by in situ analysis, depending on the analyte to be sampled. As such, the techniques may be based on analytical chemistry techniques including, but not limited to:

• Titrimetry based on the quantity of reagent needed to react with the analyte;

• Electroanalytical methods, including potentiornetry and

voltaminetry;

• Spectroscopy, based on the differential interaction of the

analyte along with electromagnetic radiation;

• Chromatography

• Gravimetric analysis

• Microscopy; and

• Radioanalytical chemistry [0028] According to a further aspect of the invention there is provided a method of compiling a gradient map illustrating the presence, absence, concentration, or point source detection of an analyte in an environment, site, or area to be assessed, the method including the steps of: conducting an assessment in accordance with the method of the invention as described herein;

using data obtained from the assessment to extrapolate a gradient of the presence, absence, or concentration of analyte; and

mapping the gradient to a representation of the environment or area assessed.

[0029] As such, the extrapolated gradient may be correlated with geo-spatial positioning data to represent analyte concentrations and presence or absence across the assessed environment or area.

[0030] The invention includes, in another aspect thereof, an enclosure for capturing plant transpiration products as described hereinbefore. The enclosure may include, on at least part of an interior surface thereof, a coating that aids in the capture and/or retention of plant transpiration products . The enclosure may contain further features, as described hereinbefore.

BRIEF DESCRIPTION OF THE DRAWING

[0031] Further features of the present invention are more fully described in the following description of several non-limiting embodiments thereof. This description is included solely for the purposes of exemplifying the present invention. It should not be understood as a restriction on the broad summary, disclosure or description of the invention as set out above. The description will be made with reference to the accompanying drawing in which:

Figure 1 is a representation of an analyte gradient map generated using the method in accordance with one aspect of the invention.

DESCRIPTION OF EMBODIMENTS [ 0032 ] The following modes, given by way of example only, are described in order to provide a more precise understanding of the subject matter of a preferred embodiment or embodiments.

[ 0033 ] In the figures, incorporated to illustrate features of an example embodiment, like reference numerals are used to identify like parts throughout the figures.

[ 0034 ] Transpiration is the evaporation of water from plants. It occurs chiefly at the leaves while their stomata are open for the passage of C0 ₂ and 0 ₂ during photosynthesis. More particularly, transpiration is the process of water movement through a plant and its evaporation from aerial parts, such as leaves, stems, fruits and flowers .

[ 0035 ] As used herein, the term "plants" includes members of the kingdom Vegetabilia (i.e. Metaphyta or Plantae) , including, but not limited to moss, algae, fungi, also including other multicellular eukaryotes that undergo transpiration.

[ 0036 ] In accordance with one aspect of the invention, transpiration from plants is collected and analysed to detect target chemicals, commonly termed "analytes". Results are then reported and mapped to create concentration gradient maps and hotspots of the target chemicals/analytes .

[ 0037 ] As used herein, the term "assessing" may refer to monitoring, exploring, detecting, screening, bioassaying, sampling by non-invasive and/or non-destructive techniques, phytoscreening, phytoforensics , as well as techniques used for the assessment of ecotoxicology, bioaccumulation, rehabilitation, amelioration, phytoremediation, or bioremediation .

[ 0038 ] When electing an environment, sample site, project site, or area to be assessed, the following factors are considered:

(i) site selection: based on client information, landscape function (i.e. groundwater assessment, surface water assessment, soil type, geology, climate, topography and current, historic, and future land use) ;

(ii) timing of assessment: based on client requirements, seasonality, daily fluctuations, and presence or absence of vegetation

(iii) method of assessment: based on client requirements, type of enclosure to be used, number of plants to be assessed, type of plant to be assessed, number of samples to be collected per plant, part of plant to be assessed (flowers, leaves, stems, etc.), time that enclosures are left on plants

[ 0039 ] Individual plants are selected to detect target chemicals or analytes. Transpiration is collected from the plants using enclosures in the form of container, vessels or plastic bags, which are bound to the plant (s) to be assessed by hook and look strips, cable ties, rope, thread or any such material for fastening. The enclosures (i.e. vessels, containers, or bags) are typically left on the plants for 1 to 72 hours, sometimes longer for monitoring purposes, with typical assessment times being 6 hours.

[ 0040 ] At the expiry of the desired time, a corner of the container is removed and the transpiration product is drained off. Surprisingly, the Applicant has found that a wide array of analytes can be assessed in this way, without damaging plants or taking parts of plants (or whole plants) to be assessed.

[ 0041 ] The method can be applied to detect the presence, absence, or concentration of elemental chemicals and compounds. So far trials include lead, zinc, copper, arsenic, mercury, cyanide, calcium, potassium, sodium, boron, chloride, nickel, phosphorus, sulphur, molybdenum, tungsten, rubidium, strontium, vanadium, thorium, iron, uranium, nitrogen, lithium, ammonia, ammonium, sulphate, phosphate, selenium, silicon, titanium, aluminium, barium, magnesium, manganese and volatile compounds such as hydrocarbons and associated compounds and persistent compounds such as perfluorinated compounds. As such, the method provides a quantitative assessment of the concentration of analytes in plants and/or the soil or sub-soil within which the plants are growing. [ 0042 ] The assessment may have differing and multiple target analytes, depending on the type(s) of analyte(s) required to be assessed and the historic land use of the site.

[ 0043 ] The enclosures (i.e. collection devices in the form of vessels, bottles, bags, or the like) each may have a unique identifier that can be logged in a database. When the collection devices are drained into sample containers, each such container has a matching or corresponding identifier.

[ 0044 ] In this way, a gradient map can be built up of the presence, absence, or concentration of analytes once the samples have been analysed and mapped to specific areas using either a plot map of where the plant samples were located, or by having a transmitter included in the enclosure which can transmit coordinates and be tracked via GRPS or GIS, or by field operatives deploying the devices with a hand held GPS instrument.

[ 0045 ] As such, each bag or enclosure has an identifier associated therewith representative of any suitable automatic identification and data capture (AIDC) technology, such as an ID tag, barcode, or RFID tag, which allows for correct mapping and testing of samples in a desired environment.

[ 0046 ] Accordingly, an enclosure includes a transmitter to enable rapid identification and retrieval thereof, especially in dense vegetative conditions. The transmitter is be configured to include geo-spatial positioning functionality and is used to compile a map of the sampled area for tracking and audit purposes. This output or data can then, following testing of each sample, be used to build up a gradient map of one or more analytes (or markers or compounds) that are being monitored, sampled, or screened for.

[ 0047 ] The method in accordance with one aspect of the invention therefore extends to the configuring of a processor via a suitable mapping software programme to create a concentration gradient map, as is represented graphically in Figure 1.

[ 0048 ] Sample analysis typically is done by a laboratory, although the use of field kits, drones, hand held applications or any other mobile analysis units is specifically included in the scope of this invention, including automating sample collecting and analysis. Analysis is typically accomplished using spectrophotometric techniques, although the type of analysis used would be dictated by the analytes being sampled or assessed for. Analysis techniques in one embodiment of the invention include inductively coupled mass spectrometry (ICP-MS), but in other embodiments may also include any one or more techniques selected from the group comprising:

• Titrimetry based on the quantity of reagent needed to react with the analyte;

• Electroanalytical methods, including potentiometry and

vo11ammetry;

• Spectroscopy, based on the differential interaction of the

analyte along with electromagnetic radiation;

• Chromatography

• Gravimetric analysis

• Microscopy; and

• Radioanalytical chemistry

EXAMPLE 1

[ 0049 ] Study conducted at an abandoned mine site to trial a method of the invention to detect lead concentration in plant transpiration products (hereinafter referred to as "exudate") . The soil found at the site is a deep red sandy loam with a gravel lag at the surface. This site is recognized as having elevated background levels of lead as a result of historic mining activities and therefore seen as a suitable site for trials of heavy metal detection and correlation studies between exudate analyte concentration and soil concentration.

[ 0050 ] The site inspection was used to design the sampling regime which was based on proximity to the abandoned mine site and presence of appropriate vegetation to conduct the trial. Eucalyptus horistes mallees were found to be in a relatively similar distance from the disturbed land of interest. With the exception of the tree Acacia rostellifera at Site 1 and Eucalyptus dolichocera used for Site 6, the remaining sites used the mallee Eucalyptus horistes for the trial.

Trees with suitably abundant leaves and healthy condition were chosen at relatively even spacing along the site's circumference. [ 0051 ] Due to the presence of a small hill to the east-north-east of the site and time efficiency, it was decided to choose and prepare the sample sites directly after the site inspection. This would also ensure that the samples had been influenced by the full morning light for maximum sunlight exposure for exudate collection and transport to the laboratory for analysis.

[ 0052 ] Exudate sample collection devices were left overnight to be exposed in the sunlight from first light to noon (12pm WST) . Exudate was drained carefully into non-acidified plastic bottles by creating a slight incision in the collection bag with Teflon coated scissors. Samples were placed immediately into a container filled with ice bricks and taken to the refrigerators overnight before being sent the next morning to an independent laboratory for testing.

[ 0053 ] Total lead (Pb) concentrations were analysed by the independent laboratory to determine the method of the invention' s sensitivities to the presence of lead in plant exudate. As the mine is not in operation and the tailings heap is encased by earth, it was determined that field filtration was not required. The independent laboratory employed the WL272 (Issue 9.0) method for the analysis of water samples. Organic material was digested with 1% v/v nitric acid and solubilisation of metals with both nitric and hydrochloric acid to form water-soluble compounds. Ionisation, atomisation and determination of elements were conducted using Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) .

[ 0054 ] Results from the independent laboratory were presented spatially using Google Maps. These maps were overlayed with topographic contours within a GDA94 datum (see Figure 1 for a graphical representation) .

[ 0055 ] Total lead concentrations at the site ranged from 0.02mg/L at Site 1 (XEN_X_01) to 1.3mg/L at Site 8 (XEN_X_08) . The reading from Site 8 is 65 times higher than the reading at Site 1. The total mean lead concentration for the 9 sites were 0.266mg/L, however with the outliers of Sites 1 and 8 removed, the mean reading for the remaining 7 sites is 0.1534mg/L. Table 1 : Total Lead (Pb) Concentration in mg/L for the site using the technique of the invention

[ 0056 ] Site 1 is located within an open relic mine pit on the opposing side of the tailings heap. The site location would not receive direct surface water runoff from the heap with the obstruction of the mine pit. This site also used a different genus and species of tree, Acacia rostellifera, which presents a possible difference in phytoscreening and attenuation capacity, which could also account for the lowest reading of (0.02mg/L) .

[ 0057 ] Site 2 is within a grove of Eucalyptus horistes that would receive moderate runoff from the tailings heap. The reading of 0.16mg/L is in close alignment with the project's average and median total lead concentration. The foliage found on this specimen was not in abundance and the tree population in the area appeared to be in average health with some signs of stress.

[ 0058 ] Site 3 was the second closest tree to the tailings heap and is clearly within a position of receiving high surface water runoff in a direct line from the tailings heap. The reading of 0.38mg/L is also the second highest reading of total lead found. The tree specimen did not appear to be in healthy condition.

[ 0059 ] Sites 4 and 5 are located on a slight rise and beyond an elongated depression that would divert surface water runoff away from the tree specimens, possibly accounting for the lower readings. These sites along with Site 3 are in the lowest lying portion of the landscape within the project area. The tree specimens at these sites were mature with extensive foliage and branch coverage.

[ 0060 ] Site 6 is in a position to receive moderate runoff within a grove of Eucalyptus dolichocera , similar to Site 2 that had a similar reading. The specimen was particularly healthy with abundant foliage.

[ 0061 ] Site 7 is within range to receive surface water runoff from the tailings heap and was a less mature plant with signs of stress. The site was also under the influence of a hardened gravel track. These surface dynamics may account for the site having the third highest reading of 0.29mg/L.

[ 0062 ] Site 8 is located closest to the lead tailings heap and is in a slight depression that could accumulate and pool surface water runoff from the heap. The tree specimen was in moderate health condition with abundant foliage, though crown foliage was showing signs of stress, with the highest reading of 1.3mg/L, being over three times the level of the next highest reading from Site 3 (0.38mg/L) . This suggests the sample site topography and proximity to the tailings heap result in significantly higher total lead concentrations.

[ 0063 ] Site 9 is located at the bottom of a steep slope preceding a rise from the tailings heap, which would therefor receive little direct runoff from the heap. The tree specimen was also in average health with branchlets and leaves in relatively poor condition. The site had the third lowest reading of total lead of 0.33mg/L.

[ 0064 ] Sites 1, 4, 5 and 9 had obstructions to surface water runoff and therefore exhibited lower readings of total lead concentrations. Sites in closer proximity to the tailings heap also showed relatively higher levels of total lead concentrations. Site 8 was in the closest vicinity to the heap and was possibly influenced by accumulating surface water runoff. The results suggest that the tailings heap is directly impacting on total lead levels in vegetation exudate which is in close proximity to the heap and/or receiving direct surface water runoff from the heap. With the location of the project site being within a natural drainage line, additional sampling to detect the presence of total lead in vegetation downstream would further support this theory. [ 0065 ] The results from this trial validate the method of the invention for detecting lead concentrations in vegetation.

EXAMPLE 2

[ 0066 ] This trial was conducted to trial detection sensitivities of inorganic and trace elements in plant exudate (transpiration products) of an estate's vineyards and olive plantations, to validate the possible use of the method in the agricultural sector.

[ 0067 ] Calcium, potassium and boron were the elements identified as the target analytes for this project, these elements being important to fertilizer application and product yield.

[ 0068 ] Grapevines and olive trees were selected based on appropriate site location, healthy vigour of trees and exposure to sunlight. Varietals of grapevines {Vitis vinifera) included Chardonnay and Semillon, whilst the olive tree varietals (Oiea europea) used for sampling were Leccino at Olive Site 1 and the remaining olive sites were the Correggiola varietal .

[ 0069 ] Exudate collection devices were prepared and placed from

08:00 and collected at 14:00 on a relatively warm day (approximately 37 degrees centigrade) . The early collection of samples was imperative due to conservative water use of olive trees from stomata apertures on hot days. Exudate samples were drained carefully into non-acidified plastic bottles by creating a slight incision in the collection bag with Teflon-coated scissors. Samples were placed immediately into a container filled with ice bricks and taken by field staff to refrigerators, prior to being sent for independent chemical analysis.

[ 0070 ] Dissolved (filterable) calcium, potassium and boron was analysed by an independent laboratory to determine the method' s sensitivities to the presence of inorganic and trace elements in plant exudate. As the site had implemented some foliage fertilizing practices, it was decided that the samples would be filtered at the independent laboratory. The independent laboratory employed the WL272

(Issue 9.0) method for analysis of water samples. Dissolved samples were filtered through a 0.45 micron filter. Determination of elements was conducted using Inductively Coupled Plasma Mass Spectroscopy (ICP- MS) .

[0071] Results from the independent laboratory were sent directly to an independent GIS expert to append the findings to a map of the site. These maps were overlayed with topographic contours within a GDA94 datum.

[0072] Calcium concentrations from grapevines at the site ranged from 3.6 mg/L at Site 3 (XUL_G3) to 5.4 mg/L at Site 5 (XUL_G5), with a mean concentration of 4.02 mg/L (sd: 0.7855) and a median of 3.7 mg/L .

[0073] Potassium concentrations from grapevines ranged from 6.5 mg/L at Site 1 (XUL_G1) to 33 mg/L at Site 5 (XUL_G5) . The reading from Site 5 is an obvious outlier and is influencing the data. Mean concentration for Potassium from the grapevines sampled was 13.56 mg/L with a large standard deviation of 11.0631 mg/L with a median of 8.8 mg/L. Boron concentrations from grapevines ranged from 0.047 mg/L at Site 4 (XUL_G4) to 0.078 mg/L at Site 1 (XUL_G1), with a mean concentration of 0.0618 mg/L (sd: 0.0148) and a median of 0.06 mg/L.

[0074] Calcium concentrations for the olive trees sampled ranged from 7.9 mg/L at Site 5 (XUL_OL5) to 14 mg/L at Sites 1 and 3 (XUL_0L1 and XUL_OL3) . Mean concentration for Calcium from the olive trees sampled was 11.58 mg/L (sd = 2.5459) with a median of 11 mg/L. Potassium concentrations from olive trees ranged from 13 mg/L at Site 4 (XUL_OL4) to 30 mg/L at Site 3 (XUL_OL3), with a mean concentration of 18 mg/L (sd: 6.892) and a median of 16 mg/L. Boron concentrations from the olive trees sampled ranged from 0.059 mg/L at Site 5

(XUL_OL5) to 0.11 mg/L at Site 4 (XUL_OL4), with a mean concentration of 0.0852 mg/L (sd: 0.0235) and a median of 0.077 mg/L.

[0075] Potassium standard deviations appear to be high due to the presence of 2 large outlying readings within a small data set. Means and medians amongst the data are similar with the exception of Potassium in the grapevines sampled.

Table 2 : Dissolved and Filtered Calcium, Potassium and Boron concentration in mg/L for the test site Site Name Calcium (mg/L) Potassium (mg/L) Boron (mg/L)

XUL Gl (C) 3.6 12 0.078

XUL G2 (S) 3.9 8.8 0.048

XUL G3 (C) 3.5 6.5 0.060

XUL G4 (S) 3.7 7.5 0.047

XUL G5 (S) 5.4 33 0.076

XUL OL1 (L) 14 16 0.077

XUL OL2 (C) 11 14 0.070

XUL OL3 (C) 14 30 0.11

XUL OL4 (C) 11 13 0.11

XUL OL5 (C) 7.9 17 0.059

[ 0076 ] There does not appear to be any correlation between element concentrations and site locations, or any abiotic influence including topography. The olive site 5 (XUL_OL5) accounted for the lowest reading for both calcium and boron by a considerable margin. As this was the only site that was situated within a plantation and not at the periphery, the inferences that can be made from this observation is that there is a possible shading or competitive affect from the nearby olive trees . The shading affect could impact the collection of exudate from the sample tree's leaves. The competitive affect with other olive trees in intensive populations could impact the tree' s capacity to assimilate and access elements and nutrients at the root zone.

[ 0077 ] The inverse observation was made from the grapevine site 5

(XUL_G5), which accounted for the highest reading for both calcium and potassium, again by a considerable margin. The point of difference in this site location is that it is situated at the edge of a grapevine row alongside a pasture and within the vicinity of a natural tributary. This positioning could account for differing soil profiles, water access and nutrient regimes.

[ 0078 ] Olive tree sites 3 and 4 have similar site variables being in the vicinity of the natural drainage line. Site 3 (XUL_OL3) had the equal highest recordings of all three elements. Site 4 (XUL_OL4) had the highest reading of Boron and the lowest reading of Potassium. There is no obvious difference in readings between the olive tree varietals . [0079] The findings of this trial demonstrate a high expression of inorganic and trace elements in the samples collected. This validates the method of exudate analysis in detecting these target elements. Implications for the site rests in the dynamics within the olive plantations themselves, that is, differences in expressed elements due to shading and competition, which could impact on mineral and water uptake and plant vigour, which ultimately influences exudate dynamics.

[0080] The impacts of adjacent land-use, including natural ecosystem dynamics, also possibly impact the expression of elements in plant exudate/transpiration products.

EXAMPLE 3

[0081] Acacia and Eucalyptus trees were selected based on appropriate site location, healthy vigour of trees and exposure to sunlight .

[0082] Exudate collection devices were prepared and placed from

06:00 and collected at 14:00 on a relatively warm day. Exudate samples were drained carefully into non-acidified plastic bottles by creating a slight incision in the collection bag with Teflon-coated scissors. Samples were placed immediately into a container filled with ice bricks prior to being sent for independent chemical analysis.

[0083] Dissolved (filterable) trace elements including copper, mercury, and zinc were analysed by an independent laboratory to determine the method' s sensitivities to the presence of trace elements in plant exudate. As the site had implemented some foliage fertilizing practices, it was decided that the samples would be filtered at the independent laboratory. The independent laboratory employed the WL272

(Issue 9.0) method for analysis of water samples. Dissolved samples were filtered through a 0.45 micron filter. Determination of elements was done using Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) .

Table 3: Filtered Copper, Mercury, and Zinc concentration in mg/L for the test site

[0084] All targeted analytes exhibited a decreasing trend in concentration from a possible contamination source (XPt_X_Tl_01 to XPt_X_Tl_03 ) , thus exhibiting a concentration gradient from a possible point source.

Example 4

[0085] After field evaluation, the transpiration enclosures (also referred to herein as transpiration devices) were deployed the same afternoon, to be left overnight. The transpiration samples were collected approximately 24 hours later. All devices collected more than the volume required for laboratory analysis, with volumes held in the devices being between 100 and 300 millilitres. Blanks were collected at a "middle sub-site" for each site.

[0086] Filtration of the samples was not required in the field.

Samples were frozen as soon as sampling was completed to counteract the dynamic nature of ammonia, which can be readily oxidized/reduced by chemical and biological processes. An independent laboratory employed the WL272 (Issue 9.0) method for the analysis of total metals in the water samples. Methods WL119 and WL293 were employed for the analysis of chloride and sulphate and for the analysis of ammonia, method WL239 was employed. To establish any possible influence of the transpiration collection bags on the samples collected in the field, a sample bag was given to the laboratories to test for the presence and concentration of the chemicals targeted during the trials.

[0087] Total chloride concentrations at Site 1 ranged from below the detectable limit of 10 mg/L at sub-site TL5SBS04 to 23 mg/L at sub-site TR N 03. The reading from the blank was higher than any other readings from Site 1 (24 mg/L), giving rise to the theory that chemical accumulations had been dislodged in the process. Total sulphate was only detected at the two sub-sites OBI and TR_N_03 with concentrations of 6 mg/L and 8 mg/L respectively. Sub-Site Total Total Bore Name Total Total Name Chloride Sulphate Chloride Sulphate

(CI) (S04) (CI) (S04) (mg/L) (mg/L) (mg/L) (mg/L)

OBI 21 6 OBI 3300 1500

MR45 11 <5 MR45 / MR46 2400 / 280 / 120

1300

BLANK 1 24 <5

TR N 03 23 8 PB08 145.7 57.3

TL5SBS04 <10 <5 TL5 170 17

[ 0088 ] Total ammonia concentrations at Site 2 ranged from 0.35 mg/L at sub-site 1 OB6 to 9.2 mg/L at sub-site MR35. Again the reading from the blank was high at 3.5 mg/L. Total sulphate was only detected at the sub-site MR35 of 200 mg/L, which is comparable to the groundwater concentrations of sulphate found near this location at MR36 of 264.9 mg/L, which was thus a highly correlative result. The blank sample also found sulphate at relatively high levels of 33 mg/L.

[ 0089 ] At Site 3, no arsenic was found in the plant transpiration.

Total molybdenum concentrations ranged from 0.006 mg/L at MR65S to 0.073 mg/L at MR43. The blank sample was again highest at 0.11 mg/L. Again, some of these levels are similar in concentration to the groundwater levels. Total nickel was detected from the two sub-sites MR43 and MR138 with concentrations of 0.033 mg/L and 0.053 mg/L respectively . Sub-Site Total Total Total Bore Total Total Total Name Arseni Molybdenum Nicke Name Arseni Molybdenum Nicke c (As) (Mo) 1 c (As) (Mo) (mg/L) 1

(mg/L) (mg/L) (Ni) (mg/L) (Ni)

(mg/L (mg/L ) )

MR103 <0.005 <0.005 <0.00 MR22 0.036 0.082 <0.02

5 5

MR43 <0.005 0.073 0.033 MR43 0.024 <0.025 <0.02

5

MR65S <0.005 0.006 <0.00 MR65S 0.004 <0.025 -

5

BLANK 3 <0.005 0.11

0.016

MR138 <0.005 0.019 0.053 MR31 / 0.068 0.066 / <0.02

MR33 / <0.025 5 /

0.004 <0.02

5

[0090] Total ammonia concentrations at Site 4 were 3.3 mg/L and

2.6 mg/L with 9.9 mg/L expressed in the blank sample. No arsenic was detected at the site. The control sites results for most chemicals analysed were below detectable limits . Ammonia was detected at sub- site 2 and 3 at 0.14 mg/L and 0.013 mg/L respectively. Chloride was found at sub-site 2 at a concentration of 17 mg/L. The presence of ammonia could be explained by the differing soil dynamics and presence of pedestrian tracks. A depression at sub-site 2 could also accumulate other materials used in the construction of the pedestrian path with chloride accumulations from the main road and traffic influencing the chloride present at this sub-site.

Total

Sub-Site Total Total Bore Name Ammonia Total Name Ammonia Arsenic (NH3-N) Arsenic

(NH3-N) (As) (mg/L) (As) (mg/L) (mg/L) (mg/L)

CSWetlandl 3.3 <0.005 MR48 (N/A)

BLANK 4 9.9 <0.005 MR49 (N/A)

CSWetland2 2.6 <0.005 MR39 0.38 0.006

Sub-Site Name Total Total Total Total Total Total

Ammonia Chlorid Sulfat Arseni Molybdenum Nickel (NH3-N) e (CI) e c (As) (Mo) (Ni) (mg/L) (mg/L) (S04) (mg/L) (mg/L) (mg/L)

(mg/L)

CSBRPOl <0.010 <10 <5 <0.005 <0.005 <0.005

CSBRP02 0.14 17 <5 <0.005 <0.005 <0.005

CSBRP03 0.013 <10 <5 <0.005 <0.005 <0.005

[0091] Regression analyses of these results have demonstrated a correlation of plant transpiration chemicals with recent groundwater data supplied. Control sites were generally demonstrating no detectable presence of target chemicals with the exception of anomalous readings of chloride and ammonia at relatively similar levels found at a correlative site.

Example 5

Antimony geological exploration

Transpiration samples were taken from an Acacia species in Western Australia. The location was based on the surroundings of a possible antimony deposit and undertaken and supervised by senior geologists employing existing biogeological techniques such as leaf and soil sampling to assist in establishing putative correlations of all samples. Sites TYA009, TYA010 and TYA011 were control sites for the operation .

Copper Total mg/L 0.004 0.011 0.008 0.007

Gold Total mg/L < 0.001 < 0.001 < 0.001 < 0.001

Lead Total mg/L < 0.001 < 0.001 < 0.001 < 0.001

Magnesium Total mg/L 4.4 13 14 10

< < <

Mercury Total mg/L 0.0001

0.0001 0.0001 0.0001

Nickel Total mg/L < 0.001 0.001 < 0.001 < 0.001

Phosphorus Total mg/L 0.7 1.2 1 0.57

Potassium Total mg/L 14 36 25 12

Silver Total mg/L < 0.001 < 0.001 < 0.001 < 0.001

Sodium Total mg/L 11 17 19 9.8

Strontium Total mg/L 0.12 0.42 0.63 0.26

Sulphur Total mg/L 12 41 38 28

Thorium Total mg/L < 0.001 < 0.001 < 0.001 < 0.001

Tungsten Total mg/L < 0.001 < 0.001 < 0.001 < 0.001

Uranium Total mg/L < 0.001 < 0.001 < 0.001 < 0.001

Zinc Total mg/L 0.01 0.047 0.03 0.01

Sample Reference Units TYA005 TYA006 TYA007 TYA008

Arsenic Total mg/L < 0.001 < 0.001 < 0.001 < 0.001

Calcium Total mg/L 59 12 19 35

Copper Total mg/L 0.004 0.006 0.002 0.003

Gold Total mg/L < 0.001 < 0.001 < 0.001 < 0.001

Lead Total mg/L < 0.001 < 0.001 < 0.001 < 0.001

Magnesium Total mg/L 17 5.5 4.8 12

< < < <

Mercury Total mg/L

0.0001 0.0001 0.0001 0.0001

Nickel Total mg/L < 0.001 < 0.001 < 0.001 < 0.001

Phosphorus Total mg/L 1.8 0.41 0.46 0.58 Potassium Total mg/L 35 11 17 12

Silver Total mg/L < 0.001 < 0.001 < 0.001 < 0.001

Sodium Total mg/L 39 4.2 5 18

Strontium Total mg/L 0.49 0.09 0.14 0.36

Sulphur Total mg/L 47 15 18 27

Thorium Total mg/L < 0.001 < 0.001 < 0.001 < 0.001

Tungsten Total mg/L < 0.001 < 0.001 < 0.001 < 0.001

Uranium Total mg/L < 0.001 < 0.001 < 0.001 < 0.001

Zinc Total mg/L 0.014 0.019 0.014 0.032

Tungsten Total mg/L < 0.001 < 0.001 < 0.001

Uranium Total mg/L < 0.001 < 0.001 < 0.001

Zinc Total mg/L 0.018 0.031 0.012

[ 0092 ] The Applicant has found, surprisingly, that not only metal or metalliferous compounds can be detected in transpiration product, but also other inorganic compounds and trace elements. Importantly, the Applicant has found that it is possible to detect the presence, absence, or concentration of analytes in plants based on the transpiration product or exudate, with the corresponding presence, absence, or concentration of analytes in the soil or sub-soil in which the plant is growing. This extends, in certain circumstances, to analytes in ground water, marsh areas, and wetlands, depending on the geography and types of plants being assessed.

[ 0093 ] Usefully, plant exudates, including transpiration products, can be transported domestically without inspection by airplane, so testing and transport of samples can be accomplished easily and quickly, even from remote areas. The sampling also circumvents quarantine delays and is effectively a water-based filtered sub-soil expression, making laboratory analysis efficient.

[ 0094 ] A further aspect of the invention provides a method of compiling a gradient map illustrating the presence or absence of a marker to be assessed, as shown in Figure 1. The method, in one embodiment, includes the steps of sampling exudates (transpiration products), determining the geo-spatial position of the plants that were sampled, and using the data obtained from the assessment to extrapolate a gradient of the analyte to be assessed, and mapping the analyte gradient to a representation of the area assessed. In this way, the Applicant has shown that it is possible to create a map of target analyte concentration based on the analytes found in transpiration products. The extrapolated gradient is thus correlated with geo-spatial positioning data to represent analyte concentrations and presence or absence and possible chemical point source across and within the assessed area. [ 0095 ] As a result, the Applicant believes it advantageous that the method of the invention allows safer, easier, more productive, sampling and assessment, with greater rapidity than many existing techniques of which the Applicant is aware and with less, if any, damage to the ecosystem.

[ 0096 ] Optional embodiments of the present invention may also be said to broadly consist in the parts, elements, systems, methods, and features referred to or indicated herein, individually or collectively, in any or all combinations of two or more of the parts, elements or features, and wherein specific integers are mentioned herein which have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

[ 0097 ] It is to be appreciated that reference to "one example" or

"an example" of the invention is not made in an exclusive sense. Accordingly, one example may exemplify certain aspects of the invention, whilst other aspects are exemplified in a different example. These examples are intended to assist the skilled person in performing the invention and are not intended to limit the overall scope of the invention in any way unless the context clearly indicates otherwise .

[ 0098 ] It is to be understood that the terminology employed above is for the purpose of description and should not be regarded as limiting. The described embodiment is intended to be illustrative of the invention, without limiting the scope thereof. The invention is capable of being practised with various modifications and additions as will readily occur to those skilled in the art.

[ 0099 ] Various substantially and specifically practical and useful exemplary embodiments of the claimed subject matter are described herein, textually and/or graphically, including the best mode, if any, known to the inventors for carrying out the claimed subject matter. Variations (e.g. modifications and/or enhancements) of one or more embodiments described herein might become apparent to those of ordinary skill in the art upon reading this application. [ 00100 ] The inventor (s) expects skilled artisans to employ such variations as appropriate, and the inventor (s) intends for the claimed subject matter to be practiced other than as specifically described herein. Accordingly, as permitted by law, the claimed subject matter includes and covers all equivalents of the claimed subject matter and all improvements to the claimed subject matter. Moreover, every combination of the above described elements, activities, and all possible variations thereof are encompassed by the claimed subject matter unless otherwise clearly indicated herein, clearly and specifically disclaimed, or otherwise clearly contradicted by context.

[ 00101 ] The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate one or more embodiments and does not pose a limitation on the scope of any claimed subject matter unless otherwise stated. No language in the specification should be construed as indicating any non-claimed subject matter as essential to the practice of the claimed subject matter.

[ 00102 ] The use of words that indicate orientation or direction of travel is not to be considered limiting. Thus, words such as "front", "back", "rear", "side", "up", down", "upper", "lower", "top", "bottom", "forwards", "backwards", "towards", "distal", "proximal", "in", "out" and synonyms, antonyms and derivatives thereof have been selected for convenience only, unless the context indicates otherwise. The inventor (s) envisage that various exemplary embodiments of the claimed subject matter can be supplied in any particular orientation and the claimed subject matter is intended to include such orientations .

[ 00103 ] The use of the terms "a", "an", "said", "the", and/or similar referents in the context of describing various embodiments (especially in the context of the claimed subject matter) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. [ 00104 ] Moreover, when any number or range is described herein, unless clearly stated otherwise, that number or range is approximate. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value and each separate sub-range defined by such separate values is incorporated into the specification as if it were individually recited herein. For example, if a range of 1 to 10 is described, that range includes all values there between, such as for example, 1.1, 2.5, 3.335, 5, 6.179, 8.9999, etc., and includes all sub-ranges there between, such as for example, 1 to 3.65, 2.8 to 8.14, 1.93 to 9, etc.

[ 00105 ] Accordingly, every portion (e.g., title, field, background, summary, description, abstract, drawing figure, etc.) of this

application, other than the claims themselves, is to be regarded as illustrative in nature, and not as restrictive; and the scope of subject matter protected by any patent that issues based on this application is defined only by the claims of that patent.