Lead and strontium isotopes as palaeodietary indicators in the Western Cape of South Africa

FUNDING: National Research Foundation (South Africa) (grant no. 84407) We analysed the isotopic compositions of bioavailable strontium (Sr) and lead (Pb) in 47 samples of animals and plants derived from the various geological substrates of southwestern South Africa, to explore the utility of these isotope systems as dietary tracers. Measurements were made using high-resolution multi-collector inductively-coupled-plasma mass spectrometry (MC-ICP-MS). 87Sr/86Sr could efficiently discriminate between geologically recent sediments of marine origin in near-coastal environments and older geologies further inland. However, 87Sr/86Sr was not able to distinguish between the Cape Granite Suite and the Cape System (Table Mountain sandstones), whereas Pb isotopes could, demonstrating the utility of this hitherto underused isotope system. Bioavailable 87Sr/86Sr in near-coastal terrestrial environments is influenced by marine input, whereas Pb isotopic ratios are not, because of low concentrations of Pb in seawater. There is considerable potential to use Pb isotopes as a dietary and palaeodietary tracer in near-coastal systems in fields as diverse as archaeology, palaeontology, wildlife ecology and forensics.


Introduction
We examined strontium ( 87 Sr/ 86 Sr) and lead ( 206 Pb/ 204 Pb, 207 Pb/ 204 Pb and 208 Pb/ 204 Pb) isotopes as biogeochemical tracers for studying diet and landscape usage in the (semi-)arid, coastal regions of southwestern South Africa, with application in both contemporary and ancient (archaeological and palaeontological) contexts. Consumer body tissues record the isotopic composition of food and water ingested in life. 1 Where these isotopes vary across the landscape, they provide a natural tracer of diet and migration. We measured Sr and Pb concentrations and isotopic compositions in animals and isotope compositions in plants collected from the major geological substrates of southwestern South Africa (shales, sandstones, granites and recent marine-derived sands), ranging in age from pre-Cambrian to Quaternary (Figure 1). We were thus able to characterise isotope ratios of bioavailable Sr and Pb for each substrate. Our work expands on previous studies of 87 Sr/ 86 Sr isotopes as a (palaeo)dietary indicator in this region [2][3][4][5] ; however, this study is the first to investigate Pb isotopes for this purpose. In addition, we aimed to determine the utility of Pb isotope measurements on archival samples that were collected during the time when leaded petrol was in use in South Africa. This is important because there is a large body of materials in museum and other collections that can be drawn from in future studies.

Sr isotopes in the geosphere and biosphere
Sr 2+ substitutes for Ca 2+ in minerals including plagioclase feldspar, calcite, dolomite, aragonite, gypsum and, most importantly regarding archaeological materials, apatite in bones and teeth. 87 Sr/ 86 Sr in biological materials is increasingly widely used to track animal migrations 1 , in forensics 6 , and in archaeology and palaeontology 7 . 87 Sr is radiogenic ( 87 Rb → 87 Sr, t 1/2 = 88x10 10 years), whereas 86 Sr is not. 8,9 87 Sr/ 86 Sr therefore increases gradually through time and is highest in geologically ancient rocks, and those with high Rb contents relative to Sr. 9,10 Sr is released from rocks through chemical weathering and moves (without fractionation of 87 Sr/ 86 Sr) from the source rock into the soils and groundwater. 9,11,12 Different components of rocks with different 87 Sr/ 86 Sr may weather at different rates, so bioavailable 87 Sr/ 86 Sr may differ from the average underlying bedrock. 13 Measurement of local animals and plants is the best way to characterise bioavailable 87 Sr/ 86 Sr 13,14 because Sr passes through the food chain from plants to animals and humans without significant fractionation of its isotopes 7,11,15 . 87 Sr/ 86 Sr in soil and water may be altered by admixing of non-local Sr from rivers flowing through different geologies, precipitation and wind-blown dust. 16 Sr is homogeneously distributed in the ocean, with a residence time of 2x10 7 years and a concentration of 7.62 ppm. 12 An important limitation of the Sr isotope system worldwide is the tendency of coastal terrestrial areas to have 87 Sr/ 86 Sr values reflecting the composition of present-day seawater at 0.709241±0.000032. 17 This is due to the presence of geologically recent marine-derived calcareous sediments with high fractions of shell 3,4 , and Sr contributed by sea spray and mists 16 .

Pb isotopes in the geosphere and biosphere
Pb has four stable, naturally occurring isotopes, of which 206 Pb ( 238 U → 206 Pb, t 1/2 = 4.47x10 9 years) 9 , 207 Pb ( 235 U → 207 Pb, t 1/2 = 0.70x10 9 years) 9 , and 208 Pb ( 232 Th → 208 Pb, t 1/2 = 14.01x10 9 years) 9 are all radiogenic. 204 Pb is not radiogenic and is therefore a good reference isotope. 206 Pb/ 204 Pb, 207 Pb/ 204 Pb and 208 Pb/ 204 Pb can increase over geological timescales and are highest in geologically ancient rocks, and those with high elemental U and Th content relative to Pb. 18 204 Pb may suffer isobaric interference from 204 Hg which, if not corrected for, can pose a problem in inductively-coupled-plasma mass spectrometry (ICP-MS). That being the case, Pb isotopic ratios over 206 Pb are often used.
Like Sr, bioavailable Pb moves through the food chain without significant fractionation of its isotopes 19,20 from the source bedrock via chemical weathering to soils and groundwater, and is then taken up by plants through their roots 21 . Industrial discharges and atmospheric transport and deposition of airborne Pb increase the Pb levels in soils, surface waters and the food chain. 22 Pb in modern rainwater seems to be mainly from airborne particles derived from industrial sources, most of which appears to be taken up by surface soils. 23 The introduction of alkyl-lead as an antiknock agent in petrol resulted in raised atmospheric Pb levels worldwide. 24 In South Africa, leaded petrol reached its peak between the 1980s and 1990s. 25 Since 1996, unleaded petrol has been available to motorists and its use gradually increased until 2006, when all leaded petrol was phased out and only lead-free petrol was available in South Africa.
In the oceans, Pb is not homogeneously distributed and has a much shorter residence time (80-100 years) 26 than Sr (2x10 7 years) 12 , resulting in Pb fluctuating with time as well as space. When comparing the Pb concentration and isotopic ratios of current surface waters in the South Atlantic Ocean to those measured in the 1990s, a decrease in the Pb concentration can be observed from 29 pmol/kg in 1990 27

Pb and Sr as palaeodietary tracers in southwestern South Africa
A number of archaeological and palaeontological studies have analysed Pb isotopes in bones and teeth for examining past mobility and geographical origins of archaeological specimens. [30][31][32][33] Progressively more studies are comparing Pb with Sr isotopes, and concluding that whilst Pb and Sr isotopic systems alone can provide valuable information, a combination of the two techniques is a very powerful tool. 30,32,33 Extensive research has been done on the bioavailable and wholerock 87 Sr/ 86 Sr in southwestern South Africa ( Sr/ 86 Sr. They reported values for shales (0.7178-0.7179) and sandstones (0.7154-0.7175) based on a limited number of samples from carefully chosen sites some distance from the coast, where the soils derived from the underlying geological formations. As a result, this study showed a clear separation between the values for shales and sandstones, and those for near-coastal marine sands (0.7094-0.7117). 2 Copeland et al. 4 and Lehmann et al. 5 also assessed bioavailable 87 Sr/ 86 Sr by analysing plants from the south coast and animal bone and teeth samples from the west coast of southern Africa. They employed much wider-ranging sampling strategies and included samples from shales, granites and sandstones near the coast, with significant marine Sr input. This is reflected in the very broad 87 Sr/ 86 Sr ranges, with marine Sr input contributing to the lower extremes: 0. Limited research has been done on whole-rock or bioavailable Pb isotopic ratios in southwestern South Africa. Soderberg

Sample collection
The details of the samples analysed are given in Supplementary table 1. Those collected specifically for this project comprise a variety of bones and teeth from animals that had recently died natural deaths, as well as some plants. As the goal of this study was to characterise bioavailable Pb and Sr isotopic ratios, diversity in the plant and animal species is irrelevant. It is, however, important to avoid cultivated areas where artificial fertilisers may have been used. Most samples were collected in the last few years, and therefore date from the post-2006 era of unleaded petrol. The sample set includes a few samples from the 1980s, when leaded petrol was still in use in South Africa, but these samples are from remote areas where there is likely to have been little influence from motor vehicle emissions. Small mammals from De Hoop Nature Reserve were trapped and euthanised in 2010 for a previous study. 3 The set of samples was derived from all of the major geological substrates of the Western Cape. Figure 1 is a geological map showing the sample collection locations.

Sample preparation
Bones and teeth were lightly sanded to remove superficial contamination. Pieces weighing approximately 50 mg were placed in vials filled with MilliQwater in an ultrasonic bath for about 10 min, then left to dry on watch glasses in an oven at 40 °C overnight, after which they were ready for chemical processing. As most teeth were from small animals, they were processed as 'whole-tooth' samples. In only two cases (both antelope teeth) were dentine and enamel separated and processed individually.
Plant samples were placed in quartz crucibles (uncovered) in a muffle furnace at an initial temperature of 300 °C and the temperature was increased by 100 °C every hour until a temperature of 650 °C was reached; thereafter the samples were left overnight. Possible Pb loss through volatilisation was minimised by increasing the temperature of the furnace gradually and keeping it well below the boiling point of Pb (1749 °C). The resulting ashed samples were ground to a fine powder using a mortar and pestle. Approximately 50 mg of each ash was weighed out (masses were recorded) and placed in a 7-mL Teflon vial.
The combined Sr-Pb elemental separation method used in this study is based on that of Pin et al. 36 , with minor modifications (see supplementary material for laboratory protocol). Sr and Pb, present in only trace amounts, were concentrated and matrix elements were removed by passing the samples through Savillex Teflon columns filled with Sr.Spec resin (Eichrom), using 0.05 M HNO 3 . Samples were processed in batches of eight, along with a total procedural blank and a reference material (NM95 in-house carbonate standard for the bone and tooth samples, and ALR33G in-house basalt standard for the plantderived mineral ash samples).

Measuring Sr and Pb concentrations and isotope ratios
Elemental concentrations of Sr and Pb were determined on a Thermo X-series II quadrupole ICP-MS, to assess the quantity of sample required for isotopic analysis. Because there is no published Sr or Pb concentration data for NM95, the in-house standard solutions were run as unknowns to assess accuracy. Calibration curves were obtained using artificial multielement standards, from which standard solutions were made.
Isotopic ratios of Sr and Pb were determined on a NuPlasma HR multicollector (MC)-ICP-MS from Nu Instruments. Samples were introduced into the MC-ICP-MS as solutions, using the Nu Instruments DSN-100 desolvating nebuliser. Solution analysis typically requires at least 50 ng of the element of interest, achieved through Sr-Pb elemental separation chemistry as described above.
The separated Sr fraction for each sample, dissolved in 2 mL 0.2% HNO 3 , was diluted to 200 ppb Sr for isotope analysis. Analyses were referenced to bracketing analyses of NIST SRM987, using an 87 Sr/ 86 Sr reference value of 0.710255. All Sr isotope data were corrected for

Sr and Pb concentrations
Sr concentrations of samples analysed here were in the range of 111-1862 ppm, while Pb concentrations were in the range of 0.012-2.30 ppm. As shown in Figure 2, all bone samples had Sr concentrations below 900 ppm and Pb concentrations below 1 ppm, while the wholetooth samples had Sr concentrations up to about 1900 ppm with Pb concentrations below 0.8 ppm. This finding is as expected, given that whole-tooth samples consist largely of enamel, with a much higher mineral content than bone. The 10 samples with [Sr]>1000 ppm were rock hyrax (dassie) whole-tooth samples from the Cape Supergroup, Karoo sediments and Namaqua-Natal metamorphic province, as well as the vlei rat tooth from Bredasdorp sediments. Of the entire sample set, only seven samples had Pb concentrations above 0.5 ppm. In the two cases in which the dentine and enamel of the tooth were separated, Pb concentrations were higher in dentine than in enamel, as seen in previous studies. 30 In addition, the Pb concentrations were higher in the dentine compared with the individual's bone.  Samples from coastal marine sands ('Cenozoic deposits' in Figure 1) had 87 Sr/ 86 Sr close to the marine value of 0.7092 17 , reflecting the marineshell-rich coastal sands and the influence of sea spray. It is clear from Figure 4 that the 87 Sr/ 86 Sr values of the samples from the Lead and strontium isotopes as a dietary tracer Page 5 of 8 87 Sr/ 86 Sr of both their and our samples from Table Mountain sandstones against distance from the coast shows increasing 87 Sr/ 86 Sr as one moves further inland (Figure 4), i.e. falling off of marine-derived Sr. The effect of marine-derived Sr appears to extend as far as 40 km inland. Similar results have been reported by other researchers 38,39 ; the magnitude of the effect depends on atmospheric circulation and is greater in soils with low Sr concentrations. Setting aside samples from older substrates close to the coast (e.g. Cape granites at Vredenburg Peninsula), the older substrates (Cape granites,  Figure 5 shows the ranges of bioavailable 87 Sr/ 86 Sr found in this study compared with those found in previous studies. We report much narrower Sr isotopic ranges for each substrate than Copeland et al. 4 and Lehman et al. 5 , although the same analytical methods were applied using the same analytical facility. In this study, the ranges of the Cape and Karoo geologies are distinct, whereas Copeland et al. 4 found them to overlap. This difference may in part be a sample population effect, as sample populations in this study (n=8 for the Karoo, 15 for Cape Supergroup) were smaller than those of Copeland et al. 4 (50 and 35 respectively).

Sr isotopic ratios
The bioavailable 87 Sr/ 86 Sr ranges for the older geological substrates from northwestern South Africa were as follows: 0.734004-0.755445 for the Namaqua-Natal metamorphic province and 0.726132 for the sample from the Richtersveld. High values are consistent with the underlying older Mesoproterozoic rocks, comprising highly deformed ultrametamorphic rocks, gneisses and migmatites. 40

Pb isotopic ratios
The new bioavailable 208 Pb/ 204 Pb, 207 Pb/ 204 Pb and 206 Pb/ 204 Pb ranges for the geological substrates from the Western Cape (from youngest to oldest geological age) are given in Table 2. The Cape granites at Rooiheuwel farm have narrower bioavailable Pb isotopic ranges compared with the Karoo and Cape samples and show a slight offset from the rest of the samples. This offset is only seen in Figure 6a ( 207 Pb/ 204 Pb vs 206 Pb/ 204 P) and not in Figure 6b ( 208 Pb/ 204 Pb vs 206 Pb/ 204 Pb). This result is not unexpected, as the Cape granites are known to have high concentrations of U and Th relative to Pb. 41,42 The half-life of 235 U → 207 Pb (0.70x10 9 years) is much shorter than that of 238 U → 206 Pb (4.47x10 9 years) and 232 Th → 208 Pb (14.01x10 9 years), therefore the initial production of 207 Pb is much more rapid than 206 Pb and 208 Pb. 43 This results in the initial rapid increase in the 207 Pb/ 204 Pb ratio of a geological system, as observed here for the Cape granites. For the Namaqua-Natal metamorphic province, the two samples from the granites on Dabidas farm plot between the Cape granites and the rest of the samples, while the two samples from the ultrametamorphic rocks of the Namaqua National Park had very different Pb isotopic ratios from the rest of the samples ( Figure 6).  Figure 8 shows that the red circled points (samples from the 1980s leaded petrol era) cover the same range as the non-circled points, so there appears to be no contribution from leaded petrol. These 1980s samples were collected from national parks in the Western Cape, or in coastal areas where emissions from motor vehicles are much lower than in urban areas.

Conclusions
This study has added to our database of measurements of bioavailable 87 Sr/ 86 Sr from the Western Cape Province of South Africa. 87 Sr/ 86 Sr can efficiently discriminate between coastal-marine environments and older geological substrates lying further inland. Organisms living on older geological substrates close to the coast have lowered 87 Sr/ 86 Sr as a result of marine Sr input. This decreases with increasing distance from the coast; the effects may be seen up to 40 km inland. 4 87 Sr/ 86 Sr measurements alone cannot distinguish between the Cape Granite Suite and the Cape Northern Cape Provinces, compared with the marine Pb isotopic signal, as measured in South Atlantic surface water sampled in 2010. 28 Errors are included within the sizes of the points as plotted. Refer to Figure 3 for geological substrates.
Supergroup (Table Mountain sandstones), whereas Pb isotopes can, as shown in this study. Pb isotopic ratios of terrestrial plants and animals living close to the coast are distinct from seawater values. There does not appear to be significant alteration from marine-derived Pb in sea spray or similar sources. Pb is much less abundant in seawater than Sr 12,28 , which could explain why the marine contribution to bioavailable Sr in the terrestrial environment is much greater than the contribution to bioavailable Pb. Ultimately, Pb isotope data can give valuable information on palaeolandscape usage, and can be used as an additional isotope system to extend interpretations based solely on Sr isotopes.
Samples collected from relatively remote localities in the 1980s had Pb isotope ratios similar to those of more recent samples from the same geologies, and distinct from leaded petrol. They do not appear to be compromised by contamination from leaded petrol. It should therefore be possible to use historical samples, e.g. from museum collections, in studies of this kind.
In conclusion, we have demonstrated the value of using a combination of both 87 Sr/ 86 Sr and Pb isotope systems in coastal terrestrial environments to trace mobility or migration and landscape usage. This use has applications in archaeology, palaeontology, studies of animal migration, wildlife forensics and more.