Sources of lead in blood from Bangalore (India) female adults using stable lead isotopes -- a pilot study

Brian L.Gulson*#, T. Venkatesh+, Jacqueline M. Palmer#, Herman Suil D’Souza+, Michael J. Korsch#

* Graduate School of the Environment, Macquarie University Sydney 2109 Australia; # CSIRO Division of Exploration and Mining, POB 136, North Ryde 1670, NSW Australia; +Department of Biochemistry and Biophysics, St. John’s Medical College, Bangalore, India

Abstract

We have analyzed blood samples from 20 pregnant Indian subjects from the city of Bangalore to assess the suitability of Indian subjects in a pregnancy study and assess the feasibility of using the lead isotopic method for determining sources and pathways of lead in Indian subjects. The isotopic compositions of the Indian subjects are quite similar to those in long-term Australian subjects and preclude their use in confirming that lead is mobilized from maternal bone during pregnancy and lactation. Blood lead concentrations range from 4.3 to 20.1 mg/dl with a geometric mean of 9.0 mg/dl. There is good agreement between the isotope dilution measurements for blood lead concentration and those obtained by the rapid and simpler ESA Lead Analyser 3010B method. The ²⁰⁶Pb/²⁰⁴Pb ratios range from 17.12 to 17.75 and showed major differences to the lead isotopic ratios that have been measured in Indian lead-zinc mineral deposits. If the lead used in Indian gasoline is from the same sources as used in Australian gasoline, then the similarity in isotopic ratios between blood of the Indian subjects and lead in gasoline indicates that gasoline is a major source of lead in the blood of these Bangalore women.

Introduction

Lead is one of the most researched metallic toxins and is well-recognised as a neurotoxin. Identification of the sources and pathways of lead into the environment in humans is critical for the most cost-effective implementation of remedial actions.

At a recent conference held in Bangalore India, the potential sources of lead in blood were identified as follows (Tandon, 1999):

1. Vehicle emissions using leaded gasoline

2. Lead battery-recycling plants

3. Lead smelting as in silver refining for jewellery and articles

4. Lead based paints and pigments

5. Printing presses

6. Ceramic pottery glazes

7. Lead contained in cosmetics and folk medicines

With respect to lead paint, van Alphen (1999) showed that of 24 selected Indian paints, 13 had concentrations exceeding 1% Pb by weight.

As part of an investigation to assess the suitability of Indian subjects migrating to Australia for our NIEHS-supported project, Biokinetics of Lead in Human Pregnancy, we analyzed 20 blood samples for high precision stable lead isotopes. An additional aim was to assess the possibility of using lead isotopes for identification of potential sources of lead in blood. A further aim was to provide quality control for the Indian blood lead analyses using the yardstick of isotope dilution.

Methods

Subjects. Pregnant females ranging in age from 20 to 25 years that attended St. John’s Medical College for routine checkups including blood sampling. Consent was given by subjects for an additional 5 mls of blood to be collected.

Sampling. Venous blood samples were collected in duplicate using plain vaccutainer tubes with EDTA anticoagulant supplied by Becton-Dickinson. One sample was measured using a 3010B ESA Lead Analyzer in Bangalore and the other was for isotope dilution measurements in Sydney, Australia.

Analytical methods used in Bangalore. From one tube, 100 ml of blood was immediately transferred to the meta-exchange reagents provided by the ESA Inc USA. Calibration of the 3010B analyser was done on a daily basis using calibration standards supplied by the company. High, medium and low controls supplied by Controx (USA) were used to check the efficiency of the methodology.

Analytical methods for isotope dilution. All sample preparation was performed in purpose-built low contamination laboratories ('clean rooms') incorporating features such as filtered air intake and laminar flow hoods. A ²⁰²Pb 'spike' solution of known isotopic composition and lead concentration was added to the blood aliquot to obtain the concentration of lead and isotopic composition of the unknown sample in the one analysis (the isotope dilution method). Lead was separated from interfering elements, such as Fe and Zn, by anion-exchange chromatography in a hydrobromic acid medium.

Fractions of the purified lead samples were loaded onto a rhenium filament using the silica gel technique and analyzed for lead isotope composition on a thermal ionisation mass spectrometer. The precisions we allocate our data are ±0.2% (2 s) on the ²⁰⁶Pb/²⁰⁴Pb and ±0.1% on the ²⁰⁸Pb/²⁰⁶Pb and ²⁰⁷Pb/²⁰⁶Pb ratios. Data are normalized to the accepted values of the international standard NBS (NIST) SRM 981, by applying a correction factor of +0.08% a.m.u. to allow comparisons between laboratories. A measurement of the environmental lead acquired by the sample throughout the entire preparation analysis procedure was obtained in the form of a lead 'blank' measurement. The amount of contamination detected in blanks was generally around 200pg for blood. This processing blank is in addition to that contributed by EDTA, described below.

Data analysis. A paired t-test was used with SPSS version 8.0 to assess if there was any significant difference between the duplicate isotope dilution analyses and isotopic ratios.

Results

Database. No systematic studies of sources and pathways of lead in the environment using stable lead isotopes have been carried out in India. In this pilot study, we have used published lead isotope data from Deb et al. (1989) for several lead-zinc mines in northwest India and unpublished data from the CSIRO database. As the same company apparently supplies tetra-alkyl lead for India and Australia, we have used data obtained for over ten years for gasoline and air in Sydney and Brisbane in our interpretation.

In Figure 1, the lead isotope results for blood from the female adults are compared with the 95% confidence ellipses for lead isotope data from mines from northwestern India. The data for the Indian mines are highly unusual compared with most other lead-zinc mines of similar geological type in the world. The isotopic data for most lead-zinc mines of so called "massive sulphide" variety lie on, or very close to, a Pb evolution curve which is depicted as a heavy dashed line in Figure 1. The Indian data lie on a linear trend extending to high ²⁰⁷Pb/²⁰⁴Pb ratios as a result of complex geological processes involved in the formation of these deposits. The data for one group of deposits lie close to the data for the blood but these deposits are 1/20 of the size of the other deposits whose data are depicted in Figure 1. It is assumed that the use of lead from these deposits is also very small, especially in Bangalore.

EDTA. As a check on the level of lead in the EDTA, several tubes were leached overnight with Millipore water and spiked with a ²⁰⁸Pb spike. The amounts of lead ranged from 0.8 to 1.6 nanograms but this is insignificant compared with the amounts of lead in blood of the subjects.

Blood results. The lead concentrations in blood range from 4.3 to 20.1mg/dl (arithmetic mean 9.9, geometric mean 9.0). There is good agreement between the blood lead concentrations measured by isotope dilution and ESA. A paired t-test shows that there is a statistically significant difference (p 0.008) with the results for the ESA being about 6% higher than for those obtained by isotope dilution.

The blood results show a spread in the isotopic ratios from 17.12 to 17.75 (arithmetic and geometric means 17.41) which conforms to a linear trend. The blood lead isotopes lie at one end of the ellipse that represents the isotopic ratios measured in gasoline in Australia over the past ten years.

Discussion

Sources of lead in blood. There are no lead isotope data available for the various sources of lead in India, such as for example, the lead used in jewellery making and battery recycling. If however, such lead came from the mines whose data are illustrated in Figure 1, and this was the exposure pathways for the lead in blood of these subjects, there should be a shift in the isotopic ratios towards the high ²⁰⁷Pb/²⁰⁴Pb ratios as shown in Figure 1. This is not the case as there is minimal shift of the data towards a higher ²⁰⁷Pb/²⁰⁴Pb ratio. The similarity of blood lead isotope data and that for gasoline used in Australia is interpreted to mean that a majority of lead in the blood of the Bangalore subjects is derived from gasoline. The spread in isotopic ratios probably signifies differential exposure of the subject to various lead sources, which could not only include those listed earlier, but may also include diet. This interpretation assumes that lead from the smaller Ambaji-Deri deposits is not used extensively in Bangalore.

Future research. Although based on very small numbers, these conclusions can be confirmed by undertaking an investigation into the other sources of lead in India, and a comparison made with subjects who live in, for example, a rural village where use of lead in gasoline is minimal.

Suitability of Indian subjects for the pregnancy study. There is only a small isotopic difference between Indian subjects and those in long-term Australian subjects so that is not possible to use Indian subjects for our pregnancy study.

Acknowledgments: We thank: Dr Abraham George of The George Foundation for logistical support in obtaining the blood samples and BLG thanks him also for support in attending the Bangalore meeting; Admiral Dawson for providing a summary location map and production figures for the Indian mines.

References

Deb M, Thorpe RI, Cumming GL, Wagner PA. Age, source and stratigraphic implications of Lead isotope data for conformable, sediment-hosted, base metal deposits in the Proterozoic Aravalli-Delhi orogenic belt, Northwestern India.

Tandon SK. 1999. Some identified sources of lead in India. Lead Poisoning Prevention & Treatment: implementing a national program in developing countries. Ed Abraham M. George, 221-224. The George Foundation Bangalore India.

Van Alphen M. 1999. Lead in paints and water in India. Lead Poisoning Prevention & Treatment: implementing a national program in developing countries. Ed Abraham M. George, 265-272. The George Foundation, Bangalore India.

Caption for figure

Figure 1. Conventional ²⁰⁷Pb/²⁰⁴Pb versus ²⁰⁶Pb/²⁰⁴Pb isotope ratio plot, comparing the blood lead results with data for mines from Deb et al and unpublished CSIRO data. The solid dashed line is the lead evolution curve on which the data for most major lead-zinc mineral deposits from around the world lie. The data for the mines are enclosed by 95% confidence ellipses. The analytical precision of the isotopic ratio measurements are shown in the upper left-hand corner of the plot.