University of Leeds for Red Tractor Assurance. Stable Isotopes Project Report. Prepared by: Professor Simon Bottrell

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1 University of Leeds for Red Tractor Assurance Stable Isotopes Project Report Prepared by: Professor Simon Bottrell

2 Stable Isotopes and Food Provenance Assurance A critical appraisal of the application of stable isotope methodologies to the provenance assurance of UK pork Prof. Simon Bottrell Chair in Environmental Isotope Geochemistry School of Earth and Environment University of Leeds Biography: Simon Bottrell studied Geology and Mineralogy at Oxford University before completing a PhD in the geochemistry of formation of gold deposits at the University of East Anglia and British Geological Survey. This involved the use of stable isotopes and gas geochemistry, and in the mid-1980s he established a new stable isotope laboratory at the University of Leeds. His research applies stable isotope studies in geochemistry, environmental science, ecology, sub-surface microbiology and contaminant biodegradation.

3 EXECUTIVE SUMMARY Stable isotope variation in meat samples is driven by two main causes: i) variation in the isotopic composition of the animal s diet (carbon, nitrogen and sulfur isotopes); ii) variation in the isotopic composition of water supplied to the animal (hydrogen isotopes, which follow a climatically-controlled global pattern and provides an effective discrimination between UK and much of mainland Europe). The two-teir test currently applied by BPEX is a highly effective way of exploiting the potential of the stable isotope approach. The second stage of the test is particularly important in the case of dietsourced components, due to a minority of producers who use different feed sources. Confidence in the approach could be further enhanced by testing to confirm the fidelity of the tissue-water hydrogen isotope signal when unprocessed meat (such as pork chops) is compared to primary abattoir meat samples. 1.1 Stable isotopes of light elements 1. SCIENTIFIC BACKGROUND AND RATIONALE The existence of multiple isotopes of a single element (i.e. atoms with different mass but the same atomic number and therefore the same chemical properties) was first recognized in the 1920s. Technological developments in the 1940s meant the by the early 1950s it was feasible to routinely measure variations in the abundance of isotopes with high precision. The light elements H, C, N and S are all key components of biomolecules and early investigations showed that their stable isotope ratios showed variations in the environment that were typically 100 or more times greater than the precision with which they could be analyzed. These early studies also identified the key processes that fractionate (i.e. discriminate between) these isotopes and lead to the range of isotopic ratios observed. More recent technological developments now permit the rapid automated measurement of isotope ratios (decreasing the unit cost) albeit at a small loss of analytical precision. 1.2 Reporting of stable isotopic data the delta ( ) scale In the case of the elements H, C, N and S the commonly analyzed isotopic ratio is that of a less abundant heavy isotope and a light isotope that is the dominant form of that element e.g. 13 C/ 12 C. For carbon, this ratio is always close to 0.011, so rather than report the measured ratio (where the variation would be in the fourth decimal) data are instead reported using the delta scale where: δ 13 C = (R sample R standard )/R standard (x 1,000 in = per mil or parts per thousand). The resulting delta (=δ) scale converts the natural range of variability into more manageable units of 0.1 to ~100. NB: that the value of the chosen standard will always be 0 on this scale and the sample data will range from positive values (for materials containing a greater abundance of the heavy isotope than the standard) to negative values (for materials containing a lower abundance of the heavy isotope than the standard). Standards used are internationally agreed. Of the isotope ratios analyzed in meat products δ 13 C, δ 15 N and δ 34 S all have analytical uncertainties around 0.3 by

4 online analysis. δd has a larger analytical uncertainty of around 2 to 3 but varies over a correspondingly wider range. When referring to isotopic compositions the terms heavy (= greater δ value or richer in the heavier isotope) and light (= lesser, or more negative, δ value or more depleted in the heavy isotope) are often used for convenience. 1.3 Trophic enrichment in heavy isotopes In general, consumers show a slight preference to assimilate the heavy isotopes in their diet and preferentially excrete or exhale the light isotopes. This leads to a small trophic enrichment in stable isotope ratios between consumer and diet that increases progressively upward through a food web. 1.4 Carbon isotopes ( 13 C/ 12 C or δ 13 C) Bulk carbon isotopic compositions of organisms closely reflect diet, with only a small (~1 ) enrichment in 13 C between food source and consumer. Within a complex organism, different types of biomolecule (e.g. fats and lipids) may show consistently different δ 13 C values (i.e there is a moreor-less constant offset in δ 13 C of a specific type of molecule from the average for the organism). There is a significant range of carbon isotopic variation in diet due to the different isotopic affects associated with the enzyme systems that mediate the C3 and C4 photosynthetic pathways. In northwest Europe indigenous plants and crops are C3 plants that have lighter carbon isotopic compositions (δ 13 C typically around -27 ). C4 plants have heavier carbon isotopic compositions (around -14 ) that (in north-west Europe) may reflect a component of imported feed crops (e.g. maize, sugar cane pulp). Amongst C3 and C4 plants there is a range of isotopic composition within each group that results from inter-specific differences and environmental effects (e.g. stressed plants tend to have heavier isotopic compositions compared to unstressed). 1.5 Nitrogen isotopes ( 15 N/ 14 N or δ 15 N) Bulk nitrogen isotopes in consumers also reflect the composition of diet, though there is a relatively large trophic shift (typically 2 to 3 ) and nitrogen isotopes are thus often used in studies of trophic relationships. Viewed at a worldwide perspective, nitrogen isotopes in terrestrial plants broadly reflect climatic zones, with higher δ 15 N values associated with hotter and dryer climates. However, this large-scale pattern masks considerable local-scale variability due to differences in soil hydrology and differences in soil microbial ecology. These factors affect the cycling of nitrogen within soils and thus the isotopic composition of nitrogen species available to plants. In agricultural areas these features can be modified by the input of large amounts of nitrogen in artificial fertilizers. Marine sources of nitrogen (e.g. fish-meal feedstocks) tend to be enriched in the heavy 15 N isotope relative to terrestrial sources. 1.6 Sulfur isotopes ( 34 S/ 32 S or δ 34 S) Sulfur isotopes in consumers again principally reflect the isotopic composition of diet. Terrestrial habitats have a wide range of environmental sulfur compositions (see below) but marine ecosystems utilize sulfur from sea-water sulfate that has a very narrow range of heavy sulfur isotopic compositions (close to +20 ). Thus heavy sulfur isotopic compositions in consumers are often used

5 as a marker for a large marine component in diet. However, this marker is non-unique as environmental sulfur in some terrestrial habitats could have similar compositions (see below). Sulfur in terrestrial plants closely reflects that available to plants from soil/soil-solution. This sulfur pool is derived from a mixture of sources. Natural sources are principally sulfur released by rock weathering and natural atmospheric inputs (rainfall and dry deposition). In general most rock weathering releases sulfur with lighter isotopic compositions than sea-water sulfate (typically in the range -10 to +10 and in continental interior areas natural atmospheric sulfur inputs are derived from dust weathering and volcanic gasses and have a similar range of isotopic compositions. These conditions would lead to a strong contrast between sulfur isotopic compositions from marine and terrestrial sources, however: i) in some areas the bedrock geology contains marine evaporite minerals (formed from the salts of ancient oceans) these weather to release what is essentially marine sulfur (with heavy sulfur isotopic compositions) into the terrestrial environment; ii) in the coastal areas of continents there is a large natural input of isotopically heavy marine sulfate to soils as wind-borne sea-spray aerosol. Thus some terrestrial environments can be characterized by sulfur with isotopic compositions similar to marine sources. This complex natural situation is overprinted by anthropogenic inputs to soils, principally diffuse atmospheric pollutants ( acid rain ), localized atmospheric pollution and agricultural sulfur inputs in fertilizers and slurries. In many areas the anthropogenic inputs overwhelm the natural and dominate the ecosystem sulfur and its isotopic composition. It should be noted that decreasing atmospheric sulfur emissions in the UK and elsewhere, in response to environmental legislation, means that the balance of inputs and therefore isotopic compositions at any location are likely changing at the present time. 1.7 Industrial diet Used in the context of wild animal studies the assumption that animal isotope compositions reflect a diet sourced from their local environment is usually valid. This may not be true for farmed animals where feed is bought in that may be from non-local sources. Thus animal diets may not reflect local environment but rather the region(s) that sourced their foodstuffs. This can have ramifications in several possible ways: i) there is a tendency for industrial diet to homogenize isotopic compositions across large cohorts that are fed similar diet; ii) such cohorts can be subject to change in isotopic composition if the source material for feedstock is changed; iii) animals may receive elements of their diet with isotopic compositions that are exotic, e.g. marine S and N isotopic compositions associated with use of fish-meal as a protein source. 1.8 Hydrogen isotopes ( 2 H/ 1 H or D/H or δd)

6 Hydrogen isotopes in water in animal tissues are generally thought to reflect the isotopic composition of water in the animal s environment. This generally follows the well-established pattern of variation in rainwater isotopic composition imposed by global-scale patterns of evaporation and precipitation in the hydrological cycle. Hydrogen isotopic compositions correlate well with average annual temperature with progressive depletion of the heavy (Deuterium, 2 H) isotope (giving more negative δd values) in cooler regions further from the Equator and at higher altitudes. Thus δd compositions will tend to be characteristic of a given area. However, this composition is not unique to a given location, there are essentially continuous bands or zones of similar rainfall isotopic composition stretching across the continents. Nonetheless, tissue water from animals raised under different climatic conditions should be characterized by different hydrogen isotopic compositions (with the caveat that farmed animals may be given drinking water that has been transported considerable distances, usually from higher altitude, cooler areas). Hydrogen isotopes in the proteinaceous component of animal tissue are more difficult to interpret. At the base of a food chain all protein is synthesized and the hydrogen used reflects the local environmental water (albeit with considerable offset in isotopic composition). Higher in a food chain animals may either synthesize new protein or assimilate it from diet and there is evidence of a considerable trophic enrichment for hydrogen isotopes in assimilated protein. So tissue protein hydrogen isotope compositions likely represent a mixed signal. Nonetheless, a cohort of animals raised on the same feed in the same conditions should all have similar tissue protein hydrogen isotope compositions that might be useful as an empirical diagnostic fingerprint. Hydrogen isotope compositions can also be measured in lipid components (this was not part of the report on the pork chops). This analysis is thought to more closely relate to the water hydrogen isotope composition than does the protein (albeit with some offset ). This is potentially important as lipid hydrogen is accumulated and stored over longer timescales than tissue water and inconsistency between lipid and tissue water hydrogen might give indication of long distance movement of animals at some point before slaughter. 1.9 Implications for isotopic compositions in pork Stable isotope rations of C, H, N and S exhibit variations that can relate to different sources of materials. Of the isotope ratios analyzed, only the hydrogen isotopes in tissue water do not relate to diet but relate to location of production (via a climatic control on rainwater compositions). The other isotopic compositions measured vary with feed source. However, the signals are non-unique; other geographical areas with similar climate will have similar rainfall isotopic compositions and feedstocks could also have similar compositions at different locations. However, an atypical isotopic composition could warn of a cause for concern about product origin. In the mainland UK the commonest pig diet is locally grown wheat and barley and extracted rape meal (and some imported soya). This will predominantly give a C3-photosynthesis carbon isotopic composition (light C isotopes) and N reflecting UK-sourced fertilizers. This should give rise to a relatively narrow range of isotopic compositions that can usefully characterize a majority of UK mainland sourced pork. This is reinforced by a relatively narrow range of hydrogen isotopic compositions in rainfall over most of the mainland UK that will be reflected in tissue water hydrogen isotope compositions.

7 However, some UK mainland pigs are raised on a diet of food industry waste/by-product. Isotopic compositions of such a diet will likely be very different from UK grown feeds and possibly highly variable. However, these animals should have similar UK tissue water hydrogen isotopic compositions. 2. THE BPEX APPROACH TO USE OF STABLE ISOTOPES The approach used by BPEX consists of a two-stage protocol. The first stage is to compare isotopic compositions of test samples against a reference database of British pork. The assumption is that this database captures the range of variability in the genuine product. If the test samples fall within this range then there is no evidence that the samples were sourced outside of the database area. It should be noted that does not constitute proof of origin animals fed on a similar diet in an area with similar climate could easily have an identical range of isotopic compositions. However, a sample with isotopic compositions outside of the database range would (correctly) give rise to suspicion of an exotic source. In this case, the protocol moves to a second stage. At the second stage the protocol requires comparison of the suspect meat isotopic compositions with the isotopic compositions of samples from the source farm(s) indicated by the paper audit trail for the meat product. This step is an essential refinement of the process, since it protects against the possibility that the isotopic compositions of the genuine source farm lie outside the range captured by the reference database. If the sample isotopic compositions differ from those of the identified source(s) then there is genuine cause for concern over the origin of the product sampled. 3. CALCULATION OF PROBABILITY OF TEST SAMPLE ORIGINATING FROM A GIVEN SOURCE REGION The approach used is to fit a probability distribution function (PDF) to the database of analyzed samples from a region by Principal Component Analysis (PCA). The chance that a given sample composition could have originated from the region described by that database is then determined by whether the test sample sits: i) inside the range described by the PDF ( high probability of origin in the region ); ii) outside the range described by the PDF (very low probability of origin in the region); iii) at the margin of the range described by the PDF (intermediate probability of origin in the region). In fact the description of high probability of origin in the region is somewhat misleading. The test can never uniquely establish a region of origin, only ever indicate that a sample is inconsistent with origin in the tested region. The result could be more properly described as no evidence for origin outside the region. In general the approach used is appropriate for the type of data under consideration. The PCA discrimination is likely to be driven primarily by two major variables: feed components (mainly C, N, S isotopes) and water source (tissue water H isotopes). The latter is likely a key discriminant

8 between animals raised in the British Isles versus animals raised in most of mainland Europe and thus an important part of the test when applied at the first Teir. Roger Young indicated that they consider the details of this approach to be part of their commercial intellectual property IP and not available for further discussion. 4. CRITIQUE AND COMMENTS At a first level the protocol used by BPEX is well-matched to the information that stable isotope analysis provides. The test against a generalized reference database can only ever be indicative. The second stage, that ensures direct comparison against identified source farm(s), gives a more robust determination of origin. Dangers 4.1That the reference database ranges become so wide that a substantial proportion of overseas product lies within them e.g. inclusion of maize-fed animals would substantially widen the δ 13 C range. Inclusion of large numbers of samples of food product-fed animals could widen the ranges of genuine isotopic compositions in the database. This would render the test progressively less able to identify genuine cases of concern at the first stage. Care needs to be taken to manage the database to allow effective discrimination at the first stage; actual verification can then be tested robustly at the second stage. 4.2 Volatility of tissue water and possible corruption of original isotopic composition - since tissue water hydrogen is the only isotope ratio that is not subject to variation with feed source, it is a particularly useful discriminant. However, water is volatile and loss of a proportion of water by evaporation leads to a change in the isotopic ratio in the remaining water that would later be analyzed; the residual water would be isotopically heavier (= less negative δd values). In many cases the original suspect sample has been obtained off the shelf whereas database and comparison samples have been obtained at the abattoir. There is thus a risk that processing and packaging of the meat (particularly vacuum packing) might evaporate water thereby modifying the tissue water isotopic composition and invalidating the comparison. NB-1: this is a possible source of systematic error that could be evaluated and eliminated by some simple tests. NB-2: the shelf vs abbatoir comparison is what was done in the case of the pork chops in the present case and the difference in the tissue water δd is in the direction that could be caused by evaporation. This IS NOT proof that this has happened, but it would increase confidence in that particular case and in future application of the methodology if this possibility were evaluated. Richard Young indicated that the current protocol treat pork chop off the shelf as an unprocessed meat product and currently make no correction to analyzed tissue water isotopic composition (as they would for processed products such as ham or bacon) and thus make a direct comparison. However, vacuum packing is used at intermediate stages in the dressing of such products and it would be wise to test whether this produces any affect (rather than assuming it does not, which appears to be the case at present). The effect is likely to be small, but safest to quantify this.

9 5. RECOMMENDATIONS Testing of processing effects (as described in 4.2 above) should be undertaken to understand the effect (if any) of meat handling on tissue water hydrogen isotopes. Management of primary database. This is key to ensuring a rigorous test at Teir 1. It may be that, in the first instance, outliers that arise due to different feeding practises in a minority of farms should be discarded in the Teir 1 test as this will give a greater chance of failing the test at this stage. These data would be incorporated at Teir 2 only if the paper trail of origin indicated possible provenance from an outlier producer. Active management and scrutiny of this database is required in order to ensure that it performs to requirement. Include as many indicated likely source farms as possible in Tier 2 comparison. The more of the source farms that there are in the primary database, the more rapidly the Tier 2 test can be applied using the archived data, rather than needing to resample and reanalyze in the first instance. This also gives greater specificity at Teir 2 as variability in diet will be directly tested (and thus a wider range of isotopic discriminants can be reliably used).