Journal of Radioanalytieal Chemistry, VoL 15 (1973) 71-- 78

N E U T R O N A C T I V A T I O N ANALYSIS A N D T O X I C O L O G Y

H. SMITH

The Department of Forensic Medicine, The University of Glasgow, Glasgow, G12 800 (Scotland)

Introduction

Neutron activation analysis is now a well established method for the detection and estimation of the elements. It is a particularly effective method when ap- plied to biological material because the matrix does not interfere by becoming active or by transformation to other species of interest.

The method has been applied to forensic toxicology where the interest is in the detection of the elements in human tissue. These elements may be present as one or more of the following categories.

(1) Elements necessary for life and the levels controlled by life processes; (2) Normal environmental contaminants present by chance and reflecting mainly

diet and the general atmosphere; (3) Abnormal environmental contaminants present by chance and reflecting heavy

local contamination~ (4) Deliberate exposure for medical reasons; (5) Deliberate exposure for homicidal reasons; (6) Direct external contamination from the environment without absorption and

management by biological processes.

The following paper outlines the application of thermal neutron activation anal- ysis to these categories and discusses problems and possible findings.

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H. SMITH: NEUTRON ACTIVATION AND TOXICOLOGY

S e n s i t i v i t y

When using neutron ac t iva t ion analysis there is considerable control over sensi t iv- i ty depending on the following factors.

(1) Choice of isotope. Many e lements produce more than one rad ioac t ive species,

(2) Choice of ac t iva t ion t ime. The increase in ac t iv i ty is l inear up to one half l i fe and thereafter ha l f as much again for each ha l f life. Only in excep t iona l cases is i t worth i r radiat ing beyond one ha l f l i fe ,

(3) Choice of decay t ime. Radioact ivi ty fal l by ha l f for each half l i fe period.

This property is used to al low species with ha l f l ives shorter than the sought for m a t e r i a l to decay to lower values and is of assistance in the separations,

(4) Choice of ac t iva t ion conditions. The intensity of the act ivat ing species may .be chosen so that greater or lesser numbers of nuclear reactions take p lace resulting in a greater or lesser rad ioac t iv i ty in the sought for e lement ,

(5) Choice of de tec t ion conditions. There are various de tec tor types ava i l ab le with different sensit ivit ies. Some of the devices allow several e lements to be measured at the same t ime by forming a gamma-spec t rum. This is usually less sensi t ive than measurement of one isotope at a t ime.

With these points in mind i t is possible to list (Table 1) the sensit ivit ies commonly ava i lab le when a reasonable select ion of conditions is made . The value quoted may be extended by a factor of 100 with faci l i t ies now ava i l ab le but this requires extra effort and the use of more powerful thermal neutron sources than usual.

Table 1 Trace e l emen t sensit ivity

Sensit ivi ty range, /~ g Element

O. 000009-0. 000001

O. 00009 -0. 00001

O. 0009 -0. 0001

O. 009 -0. 001

-0. 01

-0. I -I

O. 09

0.9 9

Dy, Eu In, Lu, Mn A1, As, Cu, Ho, Ir. La, Pr, Re, Sm, V

Au, Br, Co, Er, Ga, Ge, Hg, K, Na, Nb, Pd, Rh, Rb, Sb, Se, St, Ta, Tb, U, W, Y, Yb Ag, Ba, Cd, Cs, Ce, CI, Gd, Hf, Mo, Nd, Ni, Os, Ru, Se, Si, Te, Tin, Ti, Zn Bi, Cr, Mg, P, Pt, Sn, TI, Zr Ca, Fe, Pb, S

Note: Thevalues quoted here are ior irradiation in a thermal neutron flux of 1012 n " cm -2 �9 �9 s~c "1 for 1 week or to saturation, whichever is the shorter.

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H. SMITH: NEUTRON ACTIVATION AND TOXICOLOGY

Methods

�9 Two methods are used in the f ield of forensic ac t iva t ion analysis. The first requires lestruction of the sample followed by a chemica l separat ion which results in a r ad io -

:heroical ly pure species being isolated. This technique gives very high sensitivity and :he cer ta inty of ident i ty found by examina t ion of the properties of the radiat ion i. e.

mergy and ha l f - l i f e . The second method depends on the direct invest igat ion of the mdamaged mate r i a l af ter i r radiat ion by the technique of gamma-spec t rome t ry . This ~llows m u l t i - e l e m e n t analyses to be made but results in a somewhat lower sensit ivi ty.

In genera l the first method is used for tox ico logica l purposes and the second meth -

xl for t race e leroent distribution studies such as those used for ident i f ica t ion purposes. 3oth methods are described extensively in the l i tera ture and a good basic text book t s t h a t o f B o w e n and G i b b o n s . 1

Applications

The appl icat ions are those of a powerful ana ly t i ca l technique. Those described here are typ ica l of the wide scope ava i l ab le and have been deal t with under the head - ings, described in the introduction, which separates the pr incipal sources of human exposure.

E l e m e n t s n e c e s s a r y f o r l i f e

The major part of the matr ix e lements of b io logica l mater ia ls do not become rad io- ac t ive and this means that the minor or t race constituents are more easily analysed. In the human organism a number of t race elements are present because they are es- sent ia l for l i fe processes. As a result of this the organism controls these e lements and

maintains them at a constant l eve l . In other words analyses of tissue for such e lements wi l l result in a pred ic tab le leve l being found. For example if dry heart is analysed for copper a l eve l of 16.5 /~g/g (standard deviat ion + 3.7) wil l be found. This indi- cates that there is a narrow distribution range. Many other examples are given in a paper on essential and non-essent ia l t race e lements by L i e b s c h e r and S m i t h . 2 Two important considerations arise from this si tuation. The first is that most of the population have s imi lar essential t race e l emen t levels. This means that the use of these e lements for the ident i f ica t ion of human tissue is l imi ted . The second is that

there is a reasonably s table base leve l against which to interpret any ana ly t i ca l f ind- ings. If values of 3 or 4 t imes the a r i thmet ic mean are found then there is no doubt that overexposure has taken p lace unless, of course, there is some disease condi t ion

present in which the b io logica l control has fa i led . An example of such a condit ion involving copper (Wilson' s disease) is described by F e 11 et a l . 3

Act iva t ion analysis is a useful technique in investigations of this type because la rge

numbers of samples can be handled. The analyst is also certain of the ident i ty of the e lement being measured.

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H. SMITH: NEUTBON ACTIVATION AND TOXICOLOGY

N o r m a l e n v i r o n m e n t a l c o n t a m i n a n t s

It may be assumed that the environment contains traces of a l l the elements and as a result b io logica l systems supported by the environment also conta im traces of al l the e lements . Those e lements necessary for l i fe processes have a l ready been

dealt with. The remainder are present as contaminants . This, in effect , means that any b io logica l system wil l reflect its loca l environment . For example it is possible to restrict the human environment to such an extent that the concentrat ion of non- essential t r ace elements in tissue samples soon become almost iden t ica l . 4 Possibly

this might be the case in some comple te ly isolated communi t ies but in general the food taken is from so many different sources and change of environment is so rapid that a wide concentrat ion distribution is found,

Important considerations of this sect ion are the facts that some non-essent ia l t race e lements can substitute for some essential t race e lements and occas ional ly there may

be large loca l accumulat ions . Examples of these are the concentrat ions of several e lements such as ant imony in the lymph nodes of the lungs, 5 and the large concen- trations of mercury often found in lddney. 6

A number of surveys have been made to establish base leve l concentrations for these acc iden ta l ly included elements in tissue. In the work published by L i e b s c h e r and S m i t h 2 i t is shown that the distribution pattern is wide and skew-shaped fol low-

ing a log normal pat tern, However, i f the geometr ic mean and standard deviations are found then a reasonable working base may be established.

As in the previous sect ion ac t iva t ion analysis is useful here because of the ab i l - i ty to handle la rge numbers of samples and the cer ta inty of identi ty of the c lement being measured.

A b n o r m a l e n v i r o n m e n t a l c o n t a m i n a n t s

The human subject may be exposed to excessive amounts of the elements as a result of e i ther industrial operations or de l ibera te non med ica l self exposure. The e lement causing most concern at the moment is mercury. The exposure may be high causing large scale poisoning reactions or i t may be low with results which are difficuR to evalua te . High exposure is soon followed by recognit ion of symptoms and tracing of the source but low leve l exposure may be present for extended periods without recognit ion. A smal l number of the population appear to be sensit ive to mercury 7 and this has occas ional ly led to the discovery that a long term low leve l exposure has been going on. Recently foUowing one or two cases of mercury poison- ing in dentists L e n i h a n et a l . 8 invest igated mercury in the dental surgery and found that dentists are at risk, A s imi lar invest igat ion involving self adminis t ra t ion of mercury has also been invest igated recent ly . Occas ional ly symptoms of mercury poisoning were found in coloured subjects presenting themselves in an African hos- p i t a l The common factor in these poisonings was a cosmet ic "blanching" agent which

74 I . Rc~llocmaL Chem. 15 (1973)

H. SMITH: NEUTRON ACTIVATION AND TOXICOLOGY

contained mercury. A survey of people using this material showed that significant absorption was taking place and that a number of subjects were suffering mild symp- toms of poisoning.

Both examples of contamination described required the use of activation analysis because large numbers of samples were being analysed and because available sam- pies were of small size (a few mg). Only very small specimens of tissue may be taken from living subjects without undue discomfort.

D e l i b e r a t e e x p o s u r e f o r m e d i c a l r e a s o n s

A number of t race elements axe administered therapeutically to adjust the intake of a particular element or for the direct action some of them have on various organs or because some active substance is more readily available or stable in combination with a particular element . For example, iron is given to counter iron deficiencies, arsenic is occasionally used as a therapeutic agent and insulin is often administered as a zinc compound. The ' t ox i c ' element most widely used as a therapeutic agent is antimony. This is used on a massive scale for treatment of bilharziasis. Activation analysis has been used by M o 1 o k h i a and S m i t h 9,10 to investigate the antimony levels found in various tissues during treatment and to foUow the excretion pattern. One of the investigations involved the measurement of antimony distribution within the parasites. 11 This required the analysis of ppm levels in samples weighing in the region of a few /~g. This order of sensitivity is only available using activation anal- ysis.

The properties of activation analysis used in these investigations are certainty of identity, very high sensitivity involving the use of carrier techniques and freedom from interference after irradiation.

D e l i b e r a t e exposure for homicidal reasons

Poisoning is not a very popular method for destroying human life and certainly poisoning by me, am of the simple salts of the elements is rare. The strongest proof of criminal administration of poison is established by detection and absolute identifi- cation of the mater ia l in food, vomited or excreted matter or tissue. It is necessary to show that the materials found are not present by chance and are in concentrations outside the limi" of normal contamination. The required information can be obtained by simple activation analysis techniques. However. poisoning is not an easy process because fatal doses are ouly very approximately known and conditions of administra- tion and personal reaction are very variable. The result is that one or two attempts are often made before the fatal dose is administered. These previous attempts can be discovered by analysis of hair or better a single hair from the vic t im' s head. Hair forms an approximate record of trace element exposure, as reflected by the blood

J'. RadloanaL C.hcm. 1J (1973) 75

H. SMITH: NEUTRON ACTIVATION AND TOXICOLOGY

stream, against t ime . Major exposures such as a t tempted murder by means of arsenic 12

can be picked out easily and an approximate exposure t ime ca lcu la ted .

This type of work can benefi t from ac t iva t ion analysis because the ident i ty of the measured mate r ia l can be f i rmly established. The sensit ivity of ac t iva t ion analysis

is necessary for single hair analysis.

E x t e r n a l c o n t a m i n a t i o n

External contaminat ion can arise e i ther from contaminat ion of the subject or contaminat ion of ma te r i a l between sampling and analysis. The la t te r is e l imina ted by careful working. If a subject is in a contamina ted environment then i t is probable

that there wil l be some degree of external contaminat ion . It is necessary to be ab le to dec ide whether the levels found in mater ia ls such as hair , nai l and skin are caused by external contamina t ion or by internal contaminat ion , i . e . absorption and distribu- t ion by the blood s t ream. The problem can be reduced to some extent by a careful choice of samples . For example , a group of people showed mercury levels of 10.7 ppm in head hair and 66.7 ppm in finger nai l . 8 When pubic hair was analysed a leve l of 3 .9 ppm was found. Toe nai l gave a l eve l of 3 .5 ppm. This shows that mater ia ls protected from external contaminat ion by clothes g ive a much truer picture of levels due to al~orlxion and deposition. The leve ls in a control group from the same envi = ronment but not handling mercury were head hair 3 .5 ppm, pubic hair 1 .6 ppm, f inger nail 3 .5 ppm. toe nai l 1 .6 ppm. To take this a step further the type of shoe

worn controls the exposure of toe nai l . The above survey included the following results.

The average toe nail mercury levels of subjects wearing sandals or open- toed shoes was 9 .9 ppm. For subjects wearing closed shoes it was 3 .8 ppm and for subjects where both styles were worn the average was 5 .2 ppm.

Act iva t ion analysis was useful in this type of invest igat ion because large numbers of smal l samples from l iving subjects are ava i l ab le and only a sensit ive method can be used. For sect ional hair analysis where one to f ive mm of a single hair are ana- lysed at a t ime only act ivat ion analysis has the necessary sensit ivi ty.

Discussion

In the interpretat ion of any result each of the six sources must be comidered singly and in groups. A par t icular e lement under comidera t ion may be invest igated in the

following manner. Is the e l emen t necessary for l i fe or not? This is not a s imple question as the role, i f

any, of many e lements is not known. If the e l emen t can be p laced in one or other of the

categories the next fact required is an average value and the distribution about this

value found in the general populat ion. More often than not a true set of values is difficult to obtain.

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H. SMITH: NEUTRON ACTIVATION AND TOXIODIL)GY

Is the element present due to accidental contamination from an enriched envi- ronment~ This question requires a knowledge of the living and working environment and if possible comparison studies with materials subjected to the same conditions. It is essential to distinguish between this type of contamination and the situation where deliberate (even if this is not realised) self administration is taking place. A detailed look at the subjects' use of cosmetics, medicines and food is required.

If a high value for the sought for element is found in any material (e.g. hair) which

is subject to external contamination, an attempt should be made to distinguish be- tween this contamination and metabolised deposits.

Is the element present due to deliberate exposure for homicidal reasons~ This can only be assumed if all other sources have been definitely eliminated. Two other pieces of information may be of value. These are the finding of distribution patterns in the hair suggesting earlier exposure incidents or the typical repeating cycle of illness at home followed by recovery in hospital.

Is there any possibility of contamination during or after sampling~ It is necessary to be sure that the biological material is not contaminated by contact with the envi- ronment including knives and storage containers before activation.

When satisfactory ausT#ers are obtained for these points, making due allowance for the technique itself, then and only then is the investigation complexe.

Conclusion

Neutron activation analysis is a good analytical tool for application to toxicology. The advantages are as follows:

(1) Identification of the spieces being measured. This removes the possibility of the presence of other "interfering" materials.

(2) High sensitivity. This allows many analyses to be made on tissue from living subjects and makes possible accurate analyses of minute samples from single hairs or of low weight organisms.

(3) Ease of working. After the materials are irradiated contamination ceases to be a problem and significant weights of the sought for materials can be added so that there is no need for the use of microchemical techniques. This also allows the analysis to be performed rapidly - typically about 50 analyses per day.

References

1. H. I, M. B owen, D. Gibbons , Radioactivation Analysis, Oxford University Press, London, 1963.

2. K . L i e b s c h e r , H . S m i t h , Arch, Environm. Health, 17 (1968) 881. 3. G . S . F e l l , R . A . H o w i e , H . S m i t h , I. Clin. Path., 21(1968) 8.

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H. SMITH: NEUTRON ACTIVATION AND TOXIODLOGY

4. H. S m i t h , Proc. 1st Intern. Conf. on Forsenic Activation Analysis, San Diego, 1966. 5. M . M . M o l o k h i a , H . S m i t h , Arch. Environm. Health,15(1967) 745. 6. R . A . H o w i e 0 H. S m i t h . J. Forensic Sci. Soc.. 7(1967)90. 7. K . O . P r y k h o l m , Acta Odnnt. Seand.. 15 Suppl. (1957) 22. 8. J . M . A . L e n i h a n . H . S m i t h , W . H a r w e y , Proc. IAEA Syrup. onNuclear

Activation Techniques in the Life Sciences, Ljubljana, 1972. 9. M . M . M o l o k h i a , H . S m i t h , Bull. Wld. Hcalfi~Org., 40 (1969) 123.

10. M . M . M o l o k h i a , H. S m i t h , J. Trop. Med. Hyg.. 72 (1969)222. 11. M . M . M o l o k h i a , H . S m i t h , Ann. Trop. Med. Parasitol., 62 (1968)158. 12. H. S m i t h , J. Forensic Sci. Soc., 4(1964) 192.

78 ,1". Radloanal. Chem. 15 (1973)