DEVELOPMENT OF AN IN-VITRO PROCEDURE TO MEASURE THE AVAILABILITY OF SOIL-ASSOCIATED RADIONUCLIDES FOR UPTAKE AFTER INADVERTENT INGESTION BY HUMANS

S. Shaw and N. Green

National Radiological Protection Board, Chilton, Didcot, Oxon OX11 ORQ, UK

1 INTRODUCTION

The ingestion of soil by humans is relatively common and can be inadvertent or purposeful. Inadvertent ingestion of soil generally occurs through primitive living conditions or professions that have close and continual contact with soil. For example, dust may adhere to hands or plants and be subsequently transferred to the mouth and ingested. Inadvertent ingestion is of particular importance to children because they may be less meticulous about hygiene than adults. Purposeful ingestion of soil can occur as a result of pica (an eating disorder manifested by a craving to ingest any material not fit for food) or geophagy, a special case of pica where substances such as chalk, clay or earth are ingested.

Radiological assessments of the impact of routine and accidental releases of radionuclides into the environment generally take into account the dose from inadvertent ingestion of radionuclides associated with soil or sediment. However, a lack of relevant data has meant that radionuclides associated with soil or sediment are assumed to have the same availability for gut uptake as those incorporated into food. Studies investigating the availability of soil-associated radionuclides after ingestion have been mainly conducted on ruminant animals. The ruminant digestive tract is totally different to that of man, and so data from animal experiments may not be valid in the case of humans. The importance of inadvertent ingestion in radiological assessments can be illustrated using a recent study on the potential incursion of marine sediment inland in Northwest England. The results indicated that inadvertent ingestion of deposited material could account for a substantial fraction of the estimated dose, especially for children.

Studies to investigate the availability and the possible absorption of soil-associated radionuclides in humans have to be carried out in vitro. The digestive system in humans is difficult to simulate because of the complex processes involved. Many experiments designed to simulate human digestion have been carried out to determine the availability/absorption of trace elements and heavy metals in nutritional studies. These studies either used expensive complex equipment or were simple acid extractions carried out on a small scale.

This paper summarises the development of a relatively simple inexpensive in vitro digestion procedure (enzymolysis) that would be able to simulate in vivo conditions as closely as possible. The enzymolysis procedure was then used to determine the availability of specific radionuclides, for potential absorption across the gut, in a range of soil types. A full report is available at NRPB's web site.

2 MATERIALS

2.1 Soil Types and Radionuclides of Interest

Loam, peat and sand soils were chosen for this study to cover a broad spectrum of cultivated soils found in the UK. The soils were collected from NRPB's established lysimeter facility, set up in 1983 and containing elevated levels of 137Cs, 90Sr, 239Pu and 241Am. Collected soil was stored in a field moist condition at 4 °C until required; sub-samples of soil were air-dried before use.

2.2 Reagents

The porcine enzymes pepsin (catalogue number P-7000), pancreatin (catalogue number P-1750), α-amylase (catalogue number A-1376) and bile salts (catalogue number B-8756) were purchased from Sigma Chemicals. The enzymes are available from the manufacturer in different strengths and therefore the catalogue numbers have been given here for clarification. All other reagents used were Analytical Reagent grade or equivalent.

2.3 Equipment

The vessel used to carry out the digestion was a 2 litre magnetic culture vessel. The vessel had two access ports and contained a magnetic stirrer and baffles to aid mixing. A magnetic hotplate stirrer connected to an electronic contact thermometer was used to keep the solution well mixed and at a constant temperature of 37 °C (body temperature).

3 ANALYTICAL METHODS

3.1 Sample Preparation

The liquid and solid phases of a sample were separated by centrifugation immediately after completion of the enzymolysis procedure described in Section 4. The liquid phase was then decanted through a series of cellulose nitrate membrane filters (5.0, 0.65 and 0.45µm). The membrane filters were dissolved in acetone and evaporated to dryness for analysis with the solid phase.

3.1.1 Liquid Phase. The sample volume was reduced, the solution was acidified with 8 M nitric acid and poured into a suitable container for 137Cs determination, if required. After 137Cs measurement the solution was evaporated to dryness and ashed at 500°C. The ash was dissolved in the minimum volume of 8 M nitric acid. Any undissolved residue was separated from solution and treated with small amounts of a 3:1 hydrofluoric acid: nitric acid mixture, to ensure total dissolution, before re-dissolving in 8 M nitric acid and combining with the bulk sample.

3.1.2 Solid Phase. After separation from the liquid phase, the residue was evaporated to dryness and then dried at 105°C. The dried residue was lightly ground and packed into a suitable container for 137Cs determination, if required. After 137Cs determination the sample was then combined with the membrane filters and carefully ashed at 500°C. Dissolution of the ash was carried out as described in Section 3.1.1. If after the hydrofluoric acid: nitric treatment solids still remained, then a caustic fusion was carried out to obtain total dissolution. The caustic fusion procedure was a modification of the method originally described by Bock, the full method is given in NRPB W-17.

3.2 Sample Analysis

Caesium-137 was measured by gamma-ray spectrometry using hyperpure Ge detectors, housed in a purpose built facility and appropriately calibrated. The separation and purification of 90Sr was carried out using Sr resin (EIChroM Industries, Inc.) and based on a method developed by Shaw and Green. After a suitable period of ingrowth followed by separation, 90Sr was determined by beta counting of its 90Y daughter on a low background gas flow beta counter. The analytical methods used for 239Pu and 241Am were based on those described by Krey and Beck" and Ham' respectively. All of the analytical methods employed are in regular operational use at WB's laboratories and are carried out within formal agreement with the United Kingdom Accreditation Service (UKAS).

4 ENZYMOLYSIS PROCEDURE DEVELOPMENT

After evaluation of several published methods, two phase enzymolysis procedure was designed based on a method developed by McKay and Memmott. The first phase of the procedure simulated the conditions found in the stomach and the second phase those found in the small intestines. The full detailed enzymolysis procedure is given in NRPB W-17. Briefly, the stomach phase consisted of pepsin dissolved in a saline hydrochloric solution which was heated to 37°C and then air-dried soil added. The stomach phase was then incubated for 1 h. After 1 h, the simulated intestinal fluid (intestinal enzymes dissolved in a sodium bicarbonate solution) heated to 37°C was added to the stomach phase and the pH adjusted to 7.5 with sodium hydroxide. The intestinal phase was then incubated for 2 h. When the digestion was complete the solid and liquid phases were separated by centrifugation.

The experimental protocol was developed by carrying out various studies to ensure that parameters which could possibly affect the extraction of radionuclides from the soil were optimised. The incubation times were chosen to reflect retention times of food in the stomach and small intestines and were investigated to see if longer incubation times increased the amount of activity extracted from the soil. It was found that longer incubation times had little effect on the extraction of the soil-associated radionuclides. The extraction of the radionuclides in the different phases was also studied. These experiments showed that a two phase procedure was necessary to simulate in vivo conditions.

Whilst evaluating the enzymolysis procedure, problems with soil ageing were also discovered. It is common practice to collect a bulk sample of soil and store in a refrigerator when sub-samples are required for a series of experiments. The storage of soil was found to have an indirect effect on the amount of activity extracted from the soil, especially for 239Pu. Therefore, subsequent experiments used freshly sampled soil is used for experiments. The detailed enzymolysis procedure development is given in NRPB W-17.

5 AVAILABILITY OF SPECIFIC RADIONUCLIDES FOR UPTAKE

The enzymolysis procedure was used to determine the availability of 137Cs, 239Pu, 241Am and 90Sr in loam, peat and sand soils. Each experiment was carried out in quadruplicate to ensure reproducibility of the results. The results are given in Table 1.

The soil type did not have a marked effect on the amount of 137Cs and 241Am that was extracted, the range being between 1% and 3%. However, the amount of 239Pu and 90Sr extracted was dependent on soil type. For 239Pu, approximately 6% was extracted from loam and peat soils, whereas 11 % was extracted from sand soil. The effect of soil type was even greater for 90Sr, approximately 20% being extracted from peat soil compared to around 50% extracted from loam and sand soil. If the soil type is unknown it is recommended that the generalised availability factors 3%, 3%, 10% and 50% for 137Cs, 241Am, 239Pu and 90Sr respectively are used. There was little variation between the results of the quadruplicate samples indicating that the procedure gave reproducible data.