weather stations across New Zealand has led to identification of the stable isotope ..... the origin and migration routes of Monarch butterflies (Hobson et al., 1999; ...
A stable isotope rainfall map for the protection of NZ’s biological and environmental resources
Prepared For: CDRP Isotopes in Precipitation Project Steering Group MAF Biosecurity and Department of Conservation January 2011
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Contents Contact Details: ...................................................................................................................................................2 Executive Summary .............................................................................................................................................3 Introduction .........................................................................................................................................................4 Background ..........................................................................................................................................................5 The Present Study................................................................................................................................................7 Sampling Methodology .......................................................................................................................................7 Sample Preparation .............................................................................................................................................8 Oxygen in Water ..................................................................................................................................................8 Deuterium in water .............................................................................................................................................8 Methods ..............................................................................................................................................................9 Results ...............................................................................................................................................................10 The New Zealand Meteoric Water Line .............................................................................................................12 Deuterium Excess ..............................................................................................................................................14 Example Maps ...................................................................................................................................................15 Conclusions ........................................................................................................................................................18 Appendix 1 Application in Burn Pine Log Beetle Case ......................................................................................19 Appendix 2: Understanding Stable Isotopes .....................................................................................................28 Appendix 3 Previous Work (adapted from Biosecurity NZ project B0108) ......................................................30
Contact Details: This report was prepared by : Associate Professor Russell Frew and Dr Robert Van Hale Department of Chemistry University of Otago Dr Tony Moore Department of Surveying University of Otago
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Executive Summary NZ investigators currently have no tool to provide a definitive answer to the question: “Where did this specimen come from?”
MAF, through Biosecurity NZ (BNZ) and the NZ Food Safety Authority, and the Department of Conservation have identified the need for a tool that will rapidly and reliably identify the geographic origin of flora and fauna. This document reports on the work to this end as funded by the CDRP from a bid jointly proposed by MAF and DoC with support from the Combined Law Agency Group (CLAG). This research provides the underlying dataset from which tools may be developed to allow point of origin claims to be verified for a wide range of biological materials. The basis for such tools is a stable isotope map of NZ rainfall which will be used to establish and verify origin of specimens of interest to at least unique island (New Zealand) levels of accuracy.1 There currently exist large-scale maps at low to moderate resolution of isotopes in precipitation for the whole world, based on a global database administered by the IAEA (IAEA, 2001; Bowen and Revenaugh, 2003).
There is very little data for the Pacific region in the IAEA database. This project builds on the limited isotope map established by BNZ and extends the map to encompass all of NZ. This ensures that the map is not only useful to BNZ and DoC, but will provide a valuable resource to a range of NZ departments and agencies. The project is aligned with international programmes of research utilising stable isotopes and is supported by the USDA and CSIRO and aligns strongly with other NZ research being developed with EU agencies.
Rainwater collection sites were selected to sample the range of environments found in New Zealand. The selection of sample sites was constrained by the location of conventional meteorological weather stations so that this data could be collected with every water sample. Maps of precipitation isotopic composition have been produced using established methodology and geographic correlates of isotopic variability. These products imply that H and O isotopes have potential use in applications to constrain the source of materials within New Zealand. In particular, the maps suggest several regions which differ greatly in their precipitation isotopic composition and from which many biological materials should be isotopically distinct. The maps suggest that Coastal regions on the North Island are largely unique in their precipitation isotopic composition, as are the higher-elevation regions of the South Island. Inland North Island locations, in contrast, have precipitation isotopic compositions similar to those at many coastal South Island sites. Within each Island, strong coast to interior gradients in the 2H and 18O values of precipitation should be conducive to constraining the source of materials. 1
For an overview of stable isotopes see Appendix 2
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Introduction Information about the point of origin of natural materials, organisms, or of products manufactured from natural materials is extremely useful for a range of investigations:
Establishing the point of origin of invasive species is crucial for the effective management of biosecurity risks. In particular, being able to determine whether an individual insect trapped in the course of biosecurity monitoring is part of an established colony or a recent arrival enables the appropriate deployment of resources in response. The measurement of the stable isotope composition of biologic material such as insect keratin offers a technique to determine the point of origin of the insect2.
Forensic examination of agricultural produce, either in export from, or import to New Zealand, when the point of origin has been called into question. Light stable isotope analysis is proving useful in tracing the origins of primary produce such as beef, honey, tea and wine (see the special issue of Food Chemistry, 118, 2010, for recent examples)
As part of the Cross Departmental Research Project (CDRP), analysis of rainwater collected from weather stations across New Zealand has led to identification of the stable isotope composition of precipitation throughout the country. Stable isotope data from January 2007 to December 2009 have been collected for approximately 50 sites across New Zealand. Further data from a reduced number of sites was collected up to June 2010. This data has been processed using a Geographic Information System (GIS), with the aim of producing maps displaying the isotopic variability of rainfall across New Zealand. The data was composed of delta values for the stable isotope ratios of hydrogen and oxygen acquired from each station’s monthly rainfall. The work in this report aims to contribute to the CDRP’s aims of establishing a tool for determining the point of origin of biological materials, with the mapping procedure supplying predictions of isotopic composition in areas where rainfall is not currently sampled or analysed. This document reports on the achievements of the project in terms of the dataset produced. Some preliminary interpretation is presented as is an application where this dataset has been used to help ascertain the origin of a pest insect in a biosecurity case. Work on the interpretation and modelling of the data is underway and will be the subject of future reports and data products.
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Background Project Vision: Government enforcement agencies will have access to a robust tool that will improve management and response to biosecurity threats and incursions; increase capabilities in apprehending environmental criminals; provide a strong basis from which to provide answers regarding origin of NZ produce and enhance collaborative operational and research programmes that require point of origin information.
Aim: To provide a high-resolution map of the stable isotopic composition (18O and 2H) of rainfall throughout New Zealand. Reason: The stable isotopic composition of refractory components of an organism can be used to ascertain where that organism originated. This has been proven in recent pilot studies to determine the origin of painted and light brown apple moths2. Provision of a rainfall database will generalise the technique so it may be readily applied to any species from any location in NZ, provided suitable sample material is obtainable. The lack of such a database is the single most critical issue limiting the full deployment of the isotope technology to Biosecurity point of origin applications. The purpose of this research was to develop a high resolution map of the stable isotope composition of precipitation throughout New Zealand. There currently exist large-scale maps at low to moderate resolution of isotopes in precipitation for the whole world, based on a global database administered by the IAEA (IAEA, 2001; Bowen and Revenaugh, 2003). This study has created New Zealand’s first high-resolution map of isotopes in precipitation. New Zealand has a complex topological and meteorological system, and is a narrow island stretched over a large latitude range. Therefore the relatively simple relationship between isotopic composition of precipitation and geographical location is not expected to hold as there will be many influences on rainfall composition.. The isotopic data for this map has been correlated with meteorological data, latitude, longitude and altitude to determine the key drivers of isotopic variation in New Zealand precipitation.
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Frew R.D. Effect of geographic origin on the isotopic composition of the Light Brown Apple Moth. Report to Biosecurity NZ, June 2006, 14 pp. Frew R.D., R. van Hale and T. Husheer. BRP/160/05 Effect of diet on the isotopic signature of the Light Brown Apple Moth. Report to Biosecurity NZ, August 2006, 24 pp. Frew R.D. Towards determining the point of origin of painted apple moths (Lepidoptera: Lymantriidae) using stable isotope ratio measurements. Invited address. Entomological Society of America annual meeting, San Diego, December 2007 5
The initial question in Biosecurity applications is ‘Where did this come from?’ i.e. ‘can we attempt with some degree of surety determine the geographic origin of given specimens?’ Point of origin and pathways and could then be better identified and appropriate action(s) taken. As an eradication or management programme progresses the question shifts to ‘Is it from the population being treated or is it a new incursion?’ This is a crucial question, particularly when the target population is in the advanced stages of collapse and aberrant specimens are trapped.
Presently the isotopic approach is able to provide evidence as to whether an individual came from a particular region provided appropriate comparison samples are available. Such an approach is limited to a yes/no answer and only for specimens where the reference samples have been measured i.e. it is species and location dependent.
The isotopic composition of the meteoric water is the primary geographical signal contained in the isotopic measurements. Almost all of the atmospheric precipitation is derived from evaporation from the surface ocean at temperate latitudes. The water vapour travels from low latitude toward the poles by atmospheric transport processes. The H and O isotopic composition of precipitation is primarily dependent on thermodynamic fractionation occurring at the point of condensation of the water vapour in a cloud. The water containing the heavier isotopes has lower vapour pressure and hence will condense out of the vapour phase relatively faster than the bulk water. The result is a tight relationship between isotopic composition and cloud temperature. In turn the main drivers of cloud temperature are altitude and latitude. Other effects such as elevation and the rain shadow of mountain ranges impart secondary effects on the actual composition of the precipitation arriving at the ground. In general precipitation becomes increasingly depleted in the heavier isotopes the further the vapour travels from the source region (equator). A similar process (termed Rayleigh distillation) occurs when the vapour moves over a continent. Weather patterns thus impart regional variability on rainfall composition that is reflected in the organisms that grow in that region. Organisms growing in a region will obtain their hydrogen isotopic signal either directly from the water or from their food source, which in turn obtains its isotopic signature from the water. Part of the oxygen isotope signal is obtained from the water, the other part of plant oxygen intake is from CO2 metabolism and this is essentially isotopically invariant.
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The Present Study NIWA have an extensive network of some 700 meteorological stations throughout the country. Ideally the stable isotopic composition of rainfall would be determined at each of these site with sampling resolution similar to the other meteorological parameters measured. However, this is logistically and financially prohibited and so the approach adopted for this study was to select a suitable number of these sites and collect monthly rainwater samples for 18O and 2H analysis. The key questions to be asked of this dataset are: 1. The extent of the spatial variability across the country, i.e. how discriminatory is the isotopic composition? Are there several disparate regions with similar compositions? 2. The temporal variability: How does the isotopic composition vary with season? How does this compare with the spatial variability? 3. Can the isotopic composition for a given time and region be modelled? The ultimate aim is to build a quantitative model that would be able to predict the composition of rainfall, given other meteorological data such as weather direction, season, temperature. Thus the composition of rainfall for the growing season of the food source for the species of interest could be predicted.
Sampling Methodology Monthly rainfall was collected in a 1.5 L PET bottle with a funnel hermetically attached to the bottle cap. The sampling bottle was held upright and protected from the environment by means of an upturned bucket pegged into the ground through which the sampling funnel protruded. The funnel size was calculated so that the bottle would not overfill in the event of a 1 in 10 year rainfall event for the particular sampling location. 40 mL of paraffin oil was placed in each collection bottle to prevent evaporation of the water.
Figure 1: The sampling system developed for this project. This one was deployed on Mt Cook hence required the additional cage to protect from the attention of kea
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Sample Preparation Monthly samples were couriered to the Project Manager who sub-sampled in a 15 mL glass bottles with aluminium caps and rubber seals. These subsamples were sent to the laboratory for analysis. A further subsample was taken and archived.
The isotopic composition of the water samples was determined using continuous flow isotope ratio mass spectrometry. Results were normalised to the VSMOW scale using three laboratory standards that had previously been calibrated with IAEA (International Atomic Energy Agency) standard waters: VSMOW, GISP and SLAP. The following methods were followed.
Oxygen in Water Oxygen in water was analyzed by equilibration with CO2 utilising a GasBench (Thermo Finnigan, Bremen, Germany) interfaced to a Delta Plus Advantage (Thermo Finnigan, Bremen, Germany). Three water standards of known value were run with each set of samples and the isotope ratios of the samples were normalized to the VSMOW scale. Oxygen isotope ratios are reported using delta notation. The delta value is the difference in the 18O/16O ratio between the sample and an international standard, expressed in parts per thousand (or per mil): Rsample
Rreference
1 1000
Where R is the 18O/16O ratio; VSMOW is Vienna Standard Mean Ocean Water A precision (1σ) of 0.1‰ was achieved on the control samples.
Deuterium in water Deuterium in water was analyzed by the reduction of small (0.3 L) aliquots of water to H2 gas over hot glassy carbon (Gong et al., 2007). The isotopic composition of the resulting gas was measured on the Delta V mass spectrometer (Thermo Finnigan, Bremen, Germany). Hydrogen isotope ratios are reported using delta notation. The delta value is the difference in the H/H ratio between the sample and an international standard, expressed in parts per 8
thousand (or per mil): Rsample
Rreference
1 1000
Where R is the ratio; VSMOW is Vienna Standard Mean Ocean Water Precision of 1‰ was routinely obtained for these samples. Three service providers were used in this project. Initially all samples were analysed at Isotrace NZ Ltd with a subset of randomly chosen sample also sent to the Rafter laboratory at Geological and Nuclear Sciences. Isotrace NZ Ltd ceased trading early 2008 and responsibility for the analyses passed to the Stable Isotope Laboratory in the Department of Chemistry, University of Otago. QA performance: Comparison between the Isotrace NZ Ltd and GNS results revealed good agreement for the hydrogen isotope values. However, a discrepancy was found in the oxygen isotope data. Further analysis of the data showed the Isotrace NZ Ltd data to be in error by between 0.7 and 1.4‰. It was initially thought this was a simple calibration error that could be resolved by reanalysis of a few samples from each batch. This proved not to be the case and so the Chemistry Department laboratory undertook to reanalyse all samples previously analysed by Isotrace NZ Ltd. The dataset presented here comprises the data confirmed to be in accord with the QA check performed by GNS.
Methods Monthly data (i.e. delta values) for each station were received and formatted in Microsoft Excel for use. Weather variables for each month from August 2007 to June 2010 were downloaded from the NIWA Climate Database (http://cliflo.niwa.co.nz). The variables available are: 1. MSLPress = Mean sea level pressure at 9am local day (hPa) 2. PET = 24-hour Penman potential evapotranspiration total from 9am local day (mm) 3. Rain = 24-hour rainfall total from 9am local day (mm) 4. RH = Relative humidity at 9am local day (%) 5. SoilM = 24-hour soil moisture index total from 9am local day (mm, positive = runoff, negative = soil moisture deficit) 6. TempEarth10cm = Earth temperature at 10cm depth at 9am local day (degC) 7. Radn = 24-hour global solar radiation total from midnight local day (MJ/m2) 8. Tmax = Maximum temperature over 24 hours from 9am local day (degC) 9. Tmin = Minimum temperature over 24 hours to 9am local day (degC) 9
10. VapPress = Vapour pressure at 9am local day (hPa) 11. WindSpeed = Average wind speed at 10m above ground level over 24 hours from midnight local day (m/s)
The vast amount of data makes it impractical to include all maps in this report and so maps for each variable and month are being prepared for deposit in an electronic repository. Such maps are displayed in addition to the interpolated surfaces in order to facilitate interpretation of the isotope data.
Results 58 rainfall collection stations were used in the course of this study. Not all of them were used at once, there was reallocation of sampling resources during the study based on the review of information as data was collected. Intensive sample collection was conducted in 2008 and 2009 and the number of stations was reduced to 12 in 2010. A total of 1398 samples were collected and successfully analysed for 18O. The 2H data for three of these samples was lost (i.e. a sufficiently precise result was not obtained) making a total of 1395 samples successfully analysed for this parameter. The stable isotope ratios of water ranged from -16.33‰ to -0.11‰ (median -5.96‰, average 6.23‰, stdev 2.6‰) for 18O and from -123.4‰ to 5.54‰ (median -35.15‰, average -38.29‰, stdev 21‰) for 2H. This range and variation is large: it is approximately 70 times greater than the precision of the measurements for δ2H and more than 180 times the precision of 18O analyses. These results confirm that 2H and 18O measurements offer significant power to constrain the origin of biological materials from within New Zealand provided accurate maps of these parameters are available for individual months. The dataset produced in the present study will enable statisticallyrobust models using the relationships between isotope composition and the readily available meteorological parameter to be derived. These models will be used to create maps of isotope composition for times where no or little direct isotope measurements are available. Some models are available (e.g. Gabe Bowens Isoscapes) but while these work well in continental settings they have proven to be too inaccurate for application to Islands such as New Zealand. Indeed the conventional use of relationships between latitude and altitude only explains half of the variation in the New Zealand precipitation isotope composition3.
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B0108 Isotopic Mapping of New Zealand Precipitation, Biosecurity New Zealnd, 2008
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Figure 2: Rainfall collection stations across New Zealand.
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No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
Location Kaitaia Dargaville Whangarei Kaikohe Bay of Islands/Kerikeri Te Paki Station North Shore West Auckland South Auckland Waiheke Island Auckland Central NIL Helensville Thames Whitianga Great Barrier Island Hamilton Raglan Mt Maunganui Rotorua Murupara New Plymouth Taupo Gisborne Port Napier Port Havelock North Palmerston North Wellington Airport Hutt Valley Picton Nelson City
No. 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58
Location Takaka District Westport Kaikoura Hanmer Springs/Culverden NIL Rangiora Hokitika Lincoln Lyttelton Port Christchurch airport Timaru Peel Forest Port Chalmers Dunedin City Dunedin Airport (Outram) Roxburgh Queenstown Orepuki, Bluff Stewart Island Milford Sound Mount Cook Arthurs Pass Renwick, Marlborough Chatham Islands (not shown) East Cape Whakapapa Stratford
The New Zealand Meteoric Water Line Figure 3 show the full 2H and 18O data set for all samples. The values are highly correlated and cluster close to the Global Meteoric Water Line (GMWL) which is an approximation of the global average relationship as derived from data collected by the IAEA from 219 sites around the globe over many decades. The equation for the GMWL is; 2H = 8.17 (0.07) 18O + 11.27 ( 0.65) ‰ VSMOW4 The relationship defined by the full New Zealand dataset has a slightly lower slope and indistinguishable intercept to the GMWL. This Local Meteoric Water line (LMWL) is defined by the equation: 2H = 7.92 (0.05) 18O + 11.07 ( 0.31) ‰ VSMOW
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y = 7.92x + 11.07 R² = 0.96
0
2Hvsmow (‰)
-20 -40 -60 -80 -100 -120 -140 -20
-15
-10
-5
0
18Ovsmow (‰)
Figure 3: The relationship between the oxygen and hydrogen isotopic compositions for the complete dataset. The solid black line is the least-squares linear regression line. 4
Rosanski et al., (1993) Isotopic patterns in modern global precipitation. In: Continental Isotope indicators of Climate, American Geophysical Union Monograph.
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The NZ LMWL is quite distinct from the Australian one which is defined by the equation: 2H = 7.1 18O + 8.2 ‰ VSMOW5
While the overall New Zealand LMWL is very close to the GMWL there is interannual variability in both the slope and intercept. The annual relationships are presented in Figure 3. 20
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y = 8.43x + 11.61 R² = 0.98
0
-20
2Hvsmow (‰)
y = 7.90x + 11.54 R² = 0.94
0 -20
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y = 7.76x + 10.73 R² = 0.92
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y = 7.86x + 9.97 R² = 0.97
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Figure 4: Local meteoric Water Lines presented by yearly average. The annual averages presented in Figure 4 indicate seasonality in the composition. Note that 2007 only has data for August to December and 2010 has data for January to June. The same data are replotted in Figure 5 but separated into North and South Island. The variation in the regression line for the North Island (slope 7.3 – 7.8, intercept 8.6 – 10.6) was much lower than that of the South Island (slope 7.6 – 8.3, intercept 8.5 – 12.3). The more positive values were found in the North Island while the more negative values were from the South Island. The larger variability in the South Island is consistent with larger variability in climatic factors such as temperature experienced in the South.
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Liu et aal (2010) Stable isotopic compositions in Australian precipitation, JOURNAL OF GEOPHYSICAL RESEARCH, VOL. 115, D23307, doi:10.1029/2010JD014403
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0
20
20
South Island y = 8.31x + 9.80 R² = 0.98
0
2Hvsmow (‰)
-20
North Island y = 7.82x + 9.80 R² = 0.95
-40 -60
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North Island y = 7.31x + 8.72 R² = 0.91
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y = 7.53x + 8.60 R² = 0.97
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-20
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-20
South Island y = 8.10x + 12.28 R² = 0.94
0
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y = 7.6x + 8.52 R² = 0.91
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North Island y = 7.46x + 10.55 R² = 0.93
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Figure 5: Local meteoric Water Lines presented by yearly average with North and South Islands distinguished.
Deuterium Excess An important isotopic measure of the origin and history of a water sample is the deuterium excess (Dex) parameter, which is defined as: Dex = δ2H - 8 x δ18O (e.g., Gat, 1996) and is essentially a measure of how greatly the coupled isotope values for a water sample deviate from the GMWL. The global average value for Dex in precipitation is approximately +10‰. Two classes of factors influence the Dex values of individual precipitation samples or events. Precipitation in continental interiors exhibit significantly lower values and are typically associated with the partial evaporation of raindrops as they fall from the cloud base to the ground. Higher values of Dex usually 14
indicate that some amount of the precipitating water was sourced from re-evaporation from the land surface (i.e. ‘recycling’ from open water bodies, glaciers, or soils). For coastal and island sites, Dex values may be more closely related to ambient humidity levels in the atmosphere over adjacent oceanic regions. The average Dex value for New Zealand precipitation calculated across all monthly samples is +12‰, which is slightly greater than the global average. Dex values for the New Zealand samples ranged widely, from -7‰ to +29‰, indicating a wide range of climatological histories for precipitation events sampled by the network. Although the values of Dex for South Island samples were slightly more variable than those for the North Island sites (standard deviation = 5‰ vs. 4‰), the average South Island Dex value (+11‰) was more similar to the global mean than was the average North Island value (+14‰). This is consistent with the lower relative humidity of air over the Pacific Ocean from ~20° to 40° S from which westerly and northwesterly weather systems approach the North Island (Peixoto and Oort, 1996).
Example Maps This project has generated a vast amount of data from which a large number of maps can be produced. An electronic supplement to this report is being prepared that will allow users to choose the variable of interest and month, quarter or annual average. Figures 6 and 7 present some examples of these maps to illustrate the variability found in isotope composition. The maps are for January and June 2009. The surface values are represented by a rainbow colour scale and the coverage of the surface is across the majority of the country. The interpolation is limited by the location of the sampling stations; therefore, the coloured surface does not stretch beyond the outermost locations (extrapolation is not advised in these situations).
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Figure 6: Example spatial maps for isotope composition, temperature and rainfall for February 2009 16
Figure 7: Example spatial maps for isotope composition, temperature and rainfall for July 2009
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Conclusions
This project has successfully produced a dataset that is a valuable timeline of stable isotope data for New Zealand.
Nearly 1400 samples have been collected and successfully analysed for 2H and 18O.
The data are of high quality and match international standards for precision and accuracy (1‰ fo 2H ; 0.1‰ for 18O).
A high depgree of variation is found between regions in New Zealand confirming that 2H and 18O measurements offer significant power to constrain the origin of biological materials from within New Zealand.
In its present form the dataset can be used to determine variation between regions of interest. An example of such an application where this dataset has already been applied is appended to this report (Appendix I).
Work is underway to produce two key products from this datset; 1) detailed maps of isotopic and other meterological parameters, 2) statistical models for interpolation and prediction of isotope compsitoin for regions and/or times where direct measurements are unavailable.
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Appendix 1 Application in Burn Pine Log Beetle Case
Intercepted Burn Pine Longhorn beetle point of origin assessment using hydrogen stable isotope analyses.
Peter W. Holder and Russell Frew
The infestation of burnt pine longhorn beetle that caused the container ship Tatiana Schulte to be refused permission to enter Australia in early 2010, has been shown to be most likely to have originated in or around Auckland.
1. Background The cargo ship Tatiana Schulte, coming from new Zealand in early February 2010, was refused permission to enter Australia by the Australian Quarantine and Inspection Service (AQIS) following findings of many burnt pine longhorn beetle specimens on the ship. The burnt pine longhorn is a timber pest that occurs in New Zealand but not Australia and is a biosecurity risk to Australia's timber industries and native forests. (Maritime Today E-News Wednesday, February 10, 2010). The cost of returning the vessel to New Zealand has been substantial and the interception may impede container traffic from the Port of Auckland, at least in the short term6. To determine where in New Zealand the beetles boarded the vessel and thus manage this pest-risk pathway, MAF BNZ has contracted OritainTM to conduct hydrogen isotope analyses of the samples, provide an interpretation of the data and an independent technical opinion of the possible point of origin of the infestation. The burnt pine longhorn (Arhopalus ferus (Mulsant) [Cerambycidae]) (BPHL) attacks injured, fire damaged, or cut coniferous lumber. The larvae feed primarily in the cambium of the host timber, and development takes one to two years. Adults live for several weeks, and can occur in large numbers; they are active, including flying, from dusk to dawn, and are attracted to light. Adults shelter in crevices during the day (Hosking and Bain 1977). The infestation on the Tatiana Schulte consisted of only adult beetles, and the cargo was exclusively closed containers and no timber was included in the manifest. Therefore the infestation is considered likely not to be directly associated with the cargo, but to have flown on to vessel at either 6
In 2004 AQIS turned away another cargo vessel arriving from New Zealand following a similar detection of this beetle. 19
the point of loading, or during passage as the ship passed close to the coast of Northland. The first potential point of origin is where the ship was loaded - overnight at the Auckland container terminal, taking advantage of the anti-BPLH control practices at the port facility. The ship left Auckland at dawn, and travelled up the North-Auckland and Northland coasts, to pass close to the tip of Northland and the Three Kings Islands at dusk. Hence the Northland/ Three Kings Island regions are considered as the second potential point of origin of the infestation.
Figure 1: Map of upper North Island showing sampling locations.
2. Scientific Rationale The environmental variation of stable isotopes is a tool to geographically distinguish populations of animals (e.g. Cormie et al., 1994). Although the application of stable isotometry in entomological science is limited, previous work has demonstrated the potential of isotope signatures to determine the origin and migration routes of Monarch butterflies (Hobson et al., 1999; Dockx et al., 2004), and more recently the point of origin of biosecurity insect risk pests (Holder et al. 2010). The principle of the technique is that the stable isotope composition of an animal is determined by its diet which in turn contains the isotopic imprint of the environment in which it grew. For example the hydrogen isotopic composition of rain varies geographically in a predictable fashion; any organism growing in a region will take on the hydrogen isotopic composition of that region. If that signal is preserved in body tissue then measurements can be made to determine where the organism originated. In the case of insects the wings are composed of protein and chitin matrices that are metabolically inert following metamorphosis and adult emergence (Gratton and Forbes 2006). Thus wings provide a useful material for tracking where insects have originated.
The locations of interest are relatively close to each other (Te Kao, 34.63°S 172.92°E; Auckland, 37.23°S 175.04°E) and so differences in rainfall composition were not expected to be large. 20
Inspection of the CDRP Isotopes in Precipitation database showed the average difference in 2H for the annual rainfall between Te Kao and Auckland was -12‰, with Auckland exhibiting the lighter values expected for the higher latitude.
Figure 2: Annual average 2H values for 2009 from the CDRP Isotopes in Precipitation data Hydrogen stable isotopes have been used as a proxy for latitude in this study. Recent research has shown that greater geo-location discrimination of insect specimens is available for future studies by combining stable isotope signatures with heavy element radiogenic isotope and trace element concentration profiles, as these reflect the geology of the point of origin (Holder et al. 2010).
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3. Scientific Process 3.1 Design The data sets of Hobson and Wassenaar (1999), as well as those of Holder et al. (2010) have shown that δ2H in insects is highly variable within each place and so δ2H needs to be considered on a population level. Reference populations were collected from various sites within the regions of the two potential points of infestation. A minimum of 10 individual beetles from each of the reference populations were analysed and compared to 10 beetles from the Tatiana Schulte.
3.2 Material Type The adult beetles were dissected the whole RHS flight wing removed. This part of the insect body is considered least likely to undergo turn-over during the adult life stage (Gratton and Forbes 2006) and thus preserve the natal origin signal.
3.3 Analytical Methods The stable isotopic composition of samples was determined by isotope ratio mass spectrometry of the hydrogen gas prepared by combustion or high temperature pyrolysis. Results are reported in the standard delta (δ) notation: (
)
[
]
Where R is the ratio of the heavier isotope to the lighter one of the pair. Data are calibrated to the international references VSMOW for hydrogen. Hydrogen isotopes were determined on 0.15mg samples in silver cups, pyrolysed at 1450°C over glassy carbon. The isotopic composition of the resulting H2 gas was measured on a Thermo Finnigan Delta V mass spectrometer. The exchangeable hydrogen component of the samples is excluded from the δ2H values expressed below using the simultaneous differential equilibration method recently published by Sauer et al. (2009). I.e., the δ2H values used in this report are for non-exchangeable hydrogen and calibrated to external standards.
3.4 Statistical Analysis A one way ANOVA over the δ2H data of all three populations was conducted using the GenStat 12 package.
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4. Results 4.1 Caveat The data needs to be treated with caution. The mass spectrometer run gave precision for repeated samples that was outside of the lab norm of +/- 2‰. We have assumed that the analytical error will affect all samples equally – and so the statistical analysis is expected to be valid. Never-the-less, we will endeavour to repeat the analysis and verify the results as soon as practicable.
4.2 Data and statistical analysis The data are summarised in the tables and graph below, and given in full in the Appendix. Fischer’s Least Significant Differences values are given (α = 5%)
Table 1: Comparison of means Reference populations Auckland
Te Kao
Unknown/ Tatiana Schulte
mean
-93
-78
-99
replicates
12
11
10
1 std dev
4
10
8
s.e.
1
3
3
(-96)
(4)
Note that the mean value for the unknown is driven to much lighter values by one very light result. This has been included for completeness but is most likely an outlier. If it were excluded the mean values for the Auckland and Unknown samples are very similar (result with outlier excluded in brackets in table 1). The difference between the Te Kao and Auckland beetle 2H values (~ -15‰) is similar to and reflective of the differences found in the annual precipitation composition (-12‰).
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Graph 1: Three populations boxplot
-40
2H
-60
-80
-100
-120 Te Kao
Auckland
unknown
-140
There is significant within site variation in the Te Kao (Northland) and Tatiana Schulte beetle δ2H values, with the range in values covering approximately 30‰ at each site. This is consistent with other geo-referenced entomological δ2H data sets (Holder et al. 2010)7. Despite the overlapping δ2H ranges, the populations can be distinguished.
Table 2: Least significant differences (at 5%) Auckland
*
Te Kao
6.760
*
unknown
6.934
7.076
*
Auckland
Te Kao
unknown/ Tatiana Schulte
4.3 Interpretation The Northland and Auckland BPLH population are significantly different. Similarly, the Northland and Tatiana Schulte populations are (highly) significantly different. In contrast, the Auckland and Tatiana Schulte populations are different, but not significantly different (see note above about possible outlier in Unknown dataset).
7
2
This BPLH data set provides a valuable insight into the expression of δ H in insects. As BPHL develop over 1 - 2 years we had assumed that the within site variation would be smaller than other insects studied - that all have 2 -3 weeks feeding 2 periods; we had thought the latter insects reflected short term precipitation variation. The BPHL δ H data suggests that 2 the within site δ H variation is more likely due to individual specimen physiological differences.
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We therefore conclude that the δ2H data strongly indicates that the Tatiana Schulte BPHL infestation was not from Northland. Further, the δ2H data suggests that the Tatiana Schulte beetles are from a similar latitude as the Auckland reference BPLH population.
5. Conclusions δ2H has been used to contrast and compare three populations of BPLH, including those that were collected from the cargo ship Tatiana Schulte. The Tatiana Schulte population were similar to the Auckland reference population; yet was distinguished from the Northland reference population. The results suggests that the Tatiana Schulte BPHL specimens are from a similar latitude as the Auckland reference BPLH population. This is the first time this technology has been applied to this species of insect, and the first application of mass spectrometry to the identification of a biosecurity risk pathway.
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6. References Cormie AB, Schwarcz H, et al. 1994. Relation between hydrogen isotopic ratios of bone collagen and rain. Geochimica et Cosmochimica Acta 58(1): 377-391. Dockx C, Brower LP, et al. 2004. Do North American monarch butterflies travel to Cuba? - Stable isotope and chemical tracer techniques. Ecological Applications 14(4): 1106-1114. Gratton C, Forbes AE. 2006. Changes in δ13C stable isotopes in multiple tissues of insect predators fed isotopically distinct prey. Oecologia 147: 615–624 Hobson KA, Wassenaar LI, et al. 1999. Stable isotopes (delta D and delta C-13) are geographic indicators of natal origins of monarch butterflies in eastern North America. Oecologia 120(3): 397-404. Holder PW, Armstrong K, et al. 2010. Trace elements, radiogenic and stable isotopes as geo-location markers for biosecurity: Can the technology determine the origin of pests? Forensic Isotope Ratio Mass Spectrometry, Washington DC. April 12-14, 2010. http://bioprotection.org.nz/student-project/project-description/stepping-fightagainst-exotic-pest-insects
Hosking GP, Bain J. 1977. Arhopalus ferus (Coleoptera: Cerambycidae): its biology in New Zealand. New Zealand Journal of Forestry Science 7: 3-15. Sauer PE, Schimmelmann A, Sessions AL, Topalov K. 2009. Simplified batch equilibration for D/H determination of non-exchangeable hydrogen in solid organic material. Rapid Commun. Mass Spectrom. 23: 949–956
Disclamer Questions regarding the contents of this scientific report should be directed to: Associate Professor Russell Frew Department of Chemistry University of Otago P.O. Box 56 Dunedin The opinions provided in the Report have been prepared for the Client and its specified purposes. Accordingly, any person other than the Client, uses the information in this report entirely at its own risk. The Report has been provided in good faith and on the basis that every endeavour has been made to be accurate and not misleading and to exercise reasonable care, skill and judgment in providing such opinions.
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Appendix: BPHL δ2H data Place Te Kao
Riverhead
Latitude Longitude -34.63
-36.76
172.92
174.59
Unknown
2Hvsmow (‰)
Mean
Median
Std Dev
-76.7 -91.97 -82.38 -66.93 -78.82 -77.56 -86.52 -89.15 -60.95 -72.2
-78.3
-78.2
9.8
-94.32 -83.94 -98.96 -88.64 -86.54 -96.18 -94.26 -90.32 -95.48 -92.79 -97.13
-92.6
-94.3
4.7
-96.94 -97.45 -94.83 -102.2 -105.11 -93.31 -91.65 -119.83 -92.71 -93.56
-98.8
-95.9
8.6
-96.4
-94.8
4.6
with outlier removed
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Appendix 2: Understanding Stable Isotopes Stable isotopes are naturally occurring stable forms of elements with differing nuclear masses which determined by the number of protons and neutrons in a given element’s nucleus. As an example, carbon-12 (12C) is composed of 6 neutrons and 6 protons (12C comprises ~98.90% of all natural carbon); 13C is composed of 7 neutrons and 6 protons (and comprises ~1.10% of all C). The simplest isotope pair is hydrogen-1 and hydrogen-2 (deuterium), which is represented below.
Other elements with useful stable isotopic pairs are Nitrogen, Oxygen, Sulphur, and Chlorine (respectively H, N, O, S, & Cl). The additional mass of the heavier isotope causes a very subtle perturbation of a chemical system such that isotopes behave differently in biogeochemical processes. These effects lead to measurable partitioning of isotopes that provide information on the environment the sample material was produced in. Changes in the abundance of isotopes are very small. For example, natural carbon is around 1.11140% 13C, the changes we are interested in occur in the 6th decimal place. It therefore requires specialised mass spectrometers to make such measurements and results are determined and reported as ratios to international standards rather than dealing with cumbersome absolute abundances. Data are expressed using delta notation such that:
Rsample
Rreference
1 1000
Where R is the ratio of heavy to light isotope. Such data have units of permil (‰). The exact ratio of isotopes in any substance is dependent on the origin and history of that substance. This is because chemical processes impart a small but measurable alteration of the isotopic ratio of the product. For example, the ratio of hydrogen-2 isotopes (deuterium) in rainfall varies in a well-defined way with latitude, which is demonstrated below. 28
Thus, the deuterium ratio of natural products from one region will be different from those grown in another. The measurement of these ratios allows the region of origin to be identified. The recent development of hydrogen isotopes composition of feathers has provided a powerful tool in determining movement of migratory birds. The basis is that predictable continental-scale hydrological patterns in hydrogen isotopes in rainfall strongly correlate with hydrogen isotopes in locally grown feathers. This has also been utilised to determine the origin of monarch butterflies caught in Cuba. Metabolically inert tissues can be used to study seasonal or geographical movement because the isotope ratios reflect food-web connections at the time of tissue growth and remain unchanged despite animal movements (Hobson, 1999). Most metabolically inert tissues of interest are keratin based (e.g. baleen, claw, feather, hair, horn or nail) and, although some of these tissues continue to grow over a period of time, stable isotope ratios are locked into the keratin structure at the time of growth. Hydrogen isotopes are the exception; some non-carbon-bound hydrogen exchanges with hydrogen in ambient water vapour (Chamberlain et al., 1987) but this can be corrected for (Wassenaar and Hobson, 2000, 2003).
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Appendix 3 Previous Work (adapted from Biosecurity NZ project B0108) The scientific precedents which give impetus to mapping New Zealand’s isotopes in precipitation come from recent studies in human and animal migration, and from research on the point of origin of natural products. These research questions depend on prior knowledge of the isotopic character of precipitation across their study regions. To this end, the continuing work of collecting isotopes in precipitation data, is increasing our power to determine the point of origin of biological materials. Most species of butterflies and moths do not eat and this means that their biosynthetic makeup is complete before they chrysalize. Making use of this fact, hydrogen isotope ratios in butterfly wings have been used to determine the region of birth of individual specimens forming part of the annual monarch migration from North America to Mexico (Hobson, 1999). For this work, the analysis was made on the proteins of the wing membrane as this material was hypothesized to be most resistant to chemical alteration during the life of the insect. In a study to determine the origin of black-throated blue warblers wintering in the Caribbean, Chamberlain et al (1997) found the hydrogen isotope concentrations in black-throated blue warbler feathers were significantly correlated (r = 0.86, P = 0.001) with those in surface and rain waters at the localities where they were collected across north America. The hydrogen isotope data, combined with sulphur and strontium isotope data, provided a level of resolution allowing categorization of most breeding individuals in a species with a relatively restricted breeding range, such as the black-throated blue warbler, into one of three regional groups (southern, northern, midwestern/interior). The International Atomic Energy Agency (IAEA), in co-operation with the World Meteorological Organization (WMO), conducts an ongoing worldwide survey of oxygen and hydrogen isotope content in precipitation. The program was initiated in 1958 and became operational in 1961. The main initial objective was the systematic collection of basic data on isotope content of precipitation on a global scale to determine temporal and spatial variations of environmental isotopes in precipitation and, consequently, to provide basic isotope data for the use of environmental isotopes in hydrological investigations within the scope of water resources inventory, planning and development. (http://isohis.iaea.org) Several groups have used data from the IAEA network and literature sources to produce gridded estimates of the spatial distribution of isotope ratios of precipitation. Among these are maps produced by the IAEA in collaboration with University of Waterloo, Canada (http://isohis.iaea.org/userupdate/Waterloo/index.html), those produced by G. Bowen and collaborators (Bowen and Wilkinson, 2002; Bowen and Revenaugh, 2003; Bowen et al., 2005; www.waterisotopes.org), and 1 km resolution maps for the United States produced expressly for avian migration research (Meehan et al., 2004). 30
Included below are three significant journal publications regarding the stable isotope identification of flora and fauna. “Tracing origins and migration of wildlife using stable isotopes: a review” Hobson, K.A. (1999)
A current review (1999) regarding the application of stable isotope analysis to origin and migration of wildlife “Organisms moving between isotopically distinct food webs can carry with them information on the location of previous feeding.” “extremely useful in helping to track migration and movement of a wide range of animals from insects to birds and mammals.” Table 1 includes 25+ examples of where animal groups have been separated through stable isotope analysis
“Global application of stable hydrogen and oxygen isotopes to wildlife forensics” Bowen, G.J, Wassenaar, L.I and Hobson, K.A. (2005)
The authors use global precipitation to formulate an oxygen and hydrogen isotope map. This map is used to identify the source of bird feathers. “…the greatest potential for applying hydrogen and oxygen isotope forensics exists in mid- to high-latitude continental regions, where strong spatial isotope gradients exist.” Work undertaken is applied to wildlife forensics, but is equally exchangeable with criminal forensic studies. “…strong relationship between animal tissues and [hydrogen isotopes from precipitation] exists and has the power to statistically constrain the origin of unknown samples.”
“Using stable hydrogen and oxygen isotope measurements of feathers to infer geographical origins of migrating European birds” Hobson, K.A., Bowen, G.J., Wassenaar, L.I., Ferrand, K. and Lormee, H.
(2004)
The authors found a strong relationship between geographical region and stable isotopes of hydrogen across Europe. A clear relationship also exists between geographical region and oxygen isotopes Stable isotopic variations are independent of bird species.
The majority of point of origin stable isotope research has focused on criminal forensic applications. That is, matching substance A to substance B. The stable isotope matching of substances can be applied to any field of research where the target material is comprised of hydrogen, carbon, nitrogen, oxygen or sulphur. Many research papers have been published in this field, but three of interest are below:
31
“Geo-location of heroin and cocaine by stable isotope ratios” Ehleringer, J.R., Cooper, D.A., Lott, M.J. and Cook, C.S. (1999)
Stable isotope ratio of drugs were found to match the region of manufacture “…heroin exhibited more pronounced isotopic differences among regions, increasing its potential as a tool for geo-location and for sample-sample comparison.”
“Forensic isotope ratio mass spectrometry of packaging tapes” Carter, F.J., Grundy, P.L., Hill, J.C., Ronan, N.C., Titterton, E.L. and Sleeman, R. (2004)
Used carbon, oxygen and hydrogen isotope ratios to match samples of packaging tape Different components of tape (e.g. backing and adhesive) can be separated for individual isotope analysis “…the method has the ability to link samples of PSA tape originating from the same batch following a period of use. The technique has many potential forensic applications in linking material found at different times and locations, crime scenes, premises, etc.”
“Stable isotope analysis of safety matches using isotope ratio mass spectrometry – a forensic case study” Farmer, N.L., Meir-Augenstine, W. and Kalin, R.M. (2005)
Compared the carbon and hydrogen isotopes of a wooden match from a crime scene with matched found on a suspect “... from the *hydrogen isotope+ values obtained for the crime scene matches and seized matches of 144.5‰ and -65‰, respectively, it was concluded that the matches seized were distinctly different from those collected at the crime scene.”
References
Bowen, G.J, Wassenaar, L.I and Hobson, K.A. (2005) Global application of stable hydrogen and oxygen isotopes to wildlife forensics. Oecologia 143:337-348
Carter, F.J., Grundy, P.L., Hill, J.C., Ronan, N.C., Titterton, E.L. and Sleeman, R. (2004) Forensic isotope ratio mass spectrometry of packaging tapes. Analyst 129:1206-1210
Ehleringer, J.R., Cooper, D.A., Lott, M.J. and Cook, C.S. (1999) Geo-location of heroin and cocaine by stable isotope ratios. Forensic Science Internat. 106:27-35
Farmer, N.L., Meir-Augenstine, W. and Kalin, R.M. (2005) Stable isotope analysis of safety matches using isotope ratio mass spectrometry – a forensic case study. Rapid Communications in Mass Spectrometry 19:1-5
32
Hobson, K.A. (1999) Tracing origins and migration of wildlife using stable isotopes: a review. Oecologia 120:314-326
Hobson, K.A., Bowen, G.J., Wassanaar, L.I., Ferrand, K. and Lormee, H. (2004) Using stable hydrogen and oxygen isotope measurements of feathers to infer geographical origins of migrating European birds. Oecologia 141:477-488
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