PR-8-93 Chalk River, Ontario AEGL-4213

PR-8-93

Chalk River, Ontario

AEGL-4213 ATOMIC ENERGY OF CANADA LIMITED Chalk River Nuclear Laboratories

PROGRESS REPORT

January 1 to March 31, 1972

Biology and Health Physics Division

PR-B-93

Chalk River, Ontario

June 1972

AECL-4213 PROGRESS REPORT

January 1 to March 31, 1972

Biology and Health Physics Division

Director - Dr. A.M. Marko

Scientific Administrative Officer*

Secretary - Mrs. B.L. Phillips

Staff reporting to Divisional Office . . - Mr. M.I. Gibson Mr. L.G. Hunter

CONTENTS

1. Summary - Dr. A.M. Marko 1 - 5

Z. Biology Branch - Dr. A.M. Marko 7-23

3. Population Research Branch - Dr. H.B. Newcombe 25-28

4. Environmental Research Branch - Dr. C.A. Mawson 29-41

5. Health Physics Branch - Mr. G. Cowper 43-63

* Administrative assistance provided by F.P. Blackstein of Physics Division BIOLOGY AND HEALTH PHYSICS DIVISION SUMMARY

A.M. MARKO

Biology

Considerable progress has been achieved in all the current projects in the Biology Branch. At the molecular level, a method for establishing the order of certain genes in RNA molecules has been made possible by the purification of an enzyme which degrades RNA from one end only. Studies with E_^ coli have been concerned with measurement of the breakdown of DNA membrane complex following irradiation under different conditions. Two chemical compounds, which are known to interfere -with the function of the cell membrane, also increase the number of breaks in the irradiated DNA. In the bacterium M_. radiodurans, the detailed chemical reactions of two enzymes to digest damaged DNA has been -worked out; these enzymes are believed to be involved in repair

processes. Another enzyme, DNA polymerase from M. radiodurans,

which repairs the defect in the damaged part of the DNA molecule has been

further characterized by finding what chemical agents can inhibit its

activity.

In the yeast. S_. pombe, the inhibition of repair by caffeine hat.

been used in the study of recovery from uv- and y-radiation. The

results show that repair requires a complete growth medium and is

dependent on temperature. The effects of chemical mutagens on yeast -2-

have proved that replicating instabilities are common, and yeast cells

can mutate to stable phenotypes or replicate as a mutant,which continues

to be genetically unstable. In bacteriophage, gene mapping has indicated

the region on the DNA responsible for "superinfection exclusion", a pro-

cess which occurs when bacteria are reinfected with phage, and results

in the exclusion of the second phage among the progeny. Mutants induced

in phage by chemicals, by uv-> and by ionizing radiation, have been

screened for their ability to exclude superinfecting phage.

In the wasp Dahlbominus, the irradiation of carmine eye colour

mutants has shown that reversion to the wild phenotype is possible, This

is a good indication that the eye colour mutants do not arise as a result

of chromosome rearrangements or deletions but as true point mutations.

Leukemia in rats has b^en chemically induced, and the charac-

teristics of the disease have been shown to be comparable to that found

in irradiated animals.

Population Research

Analyses of data from the experiments with irradiated trout

sperm, carried out at Jasper, have now revealed a mixture of "harmful"

and "beneficial" effects of the lower doses used (i.e. 25 and 50 rad).

Although such exposures produce congenital malformations, they also

lower the likelihood that eggs will fail to develop embryos, and the like-

lihood of death among the embryos that are formed. Higher dose to the -3-

sperm (of 200 and 400 rad) have unconditionally harmful effects with respect to all three end points.

Work is continuing on the production of indexes of British

Columbia births, in a multiplicity of sequences, designed to facilitate integration of diagnostic information on congenital and hereditary disease from handicap, hospital, and death records, into individual and family histories of disease.

As part of a pilot study of cancer following multiple fluoroscopy, designed initially at CRNL and now being carried out by the National

Cancer Institute, a phantom has been constructed and sent to NCI for the

purpose of measuring the dose-depth curve for two old fluoroscope machines that are still in intermittent use at Toronto Hospital Weston.

Dosimeters are being provided by the Health Physics Branch at CRNL.

Environmental Research

Discharges of radioactive wastes into the environment via the

stack and effluent streams entering the Ottawa River have remained

within the CRNL Working Limits, which are far below the recognized

Safety Limits. Discharges to the river continue to be a small fraction of

the fission products carried by the river, originating from weapons

fallout,

Analytical work on environmental samples collected during the -4-

Spring and Summer of 1971 continued throughout the quarter and will

shortly be completed.

Winter work on aquatic biology beneath ice cover was carried

out at Maskinonge and Perch Lakes and preparations were made for the

installation of summer meteorological equipment at Ferch Lake.

Water and mineral balance studies in the Perch Lake Basin

continued, and the number of elements studied in the leaching survey

has been increased with the acquisition of new equipment and elaboration

of improved techniques.

Contract work by the Trois Rivieres Campus of the Universite

du Quebec on the effects of discharge of heated effluent by the Gentilly

nuclear power station proceeded successfully. Good liaison between the

Branch and University staff is producing a pleasing unity of interests

and excellent personal collaboration.

Health Physics

Certain monitoring procedures for contamination control require

large area radiation detectors and this requirement has hitherto been met

by the use of thin plastic scintillators and photo-multipliers which are

both expensive and bulky. An attractive alternative detector now available

from a commercial source is a 23 mg/cm^ window GM counter having a

sensitive area of 250 cm2. The application of this counter to hand, foot

and clothing monitoring is now being examined. -5-

Manufacture of the automatic thermoluminescence dosimeter

(TLD) reader is proceeding in industry and large-scale field trials of dosimeters are continuing with 150 workers being monitored. TLD's have also been used to determine diagnostic doses received by patients in the CRNL hospital.

The development of techniques for monitoring of tritium in air and water continues. A more rugged detector assembly capable of with- standing accidental over-pressures has been developed for the plastic scintillator plate water-monitor, and at the same time the feasibility of continuous monitoring using a liquid scintillator at very low flow rates

(for reasons of economy) is being examined. With a consumption of only

100 litres of scintillator per year, it appears possible to monitor tritium- in-water levels as low as 100 nCi/litre.

The modified tritium-in-air ion chambers, which compensate both for gaseous fission product and external gamma radiations, are in satisfactory operation at Douglas Point.

An area monitor capable of operation in pulsed high energy radiation fields from accelerators as well as fields from radioactive sources is being developed using a plastic scintillator and photomultiplier whose mean current will be processed to give digital outputs of both do.se rates and integral doses. -7-

BIOLOGY

A. M. Marko, Acting Branch Head

CONTENTS

2. 1 Staff (1 January to 31 March 197 2)

BIOCHEMISTRY AND MOLECULAR BIOLOGY

2. 2 Comparison of RNA Molecules in Animal Cells by Oligo- nucleotide Analysis

2. 3 Study of Rapidly Sedimenting, Heterogeneous RNA in Animal and Yeast Cells

2. 4 Search for Genes Corresponding to Poly A Sequences Found in Messenger and Heterogeneous Nuclear RNA

2. 5 Radiation Biochemistry of E. coli

a) DNA membrane association

b) Radiation sensitizing agents

2. 6 Protection of Rat Thymus Cells

2. 7 Induction of Leukaemia in Rats

2. 8 Nucleases of M. radiodurans

a) 5 -nucleases b) Exonucleases 2. 9 Repair of Radiation Damage in Bacteria and Yeast

a) The DNA polymerase from Micrococcus rad_io_- durans b) Recovery of S. pombe from radiation damage -8-

GENETICS, DEVELOPMENT AND POPULATION STUDIES

2. 10 Genetic Control of Radiation Sensitivity in Schizosaccharo-

myces pombe

2. 11 Replicating Instabilities In Schizosaccharomyces pombe

2. 12 Liquid Holding Recovery and Spontaneous Cell Death in the Normal and Radiation-sensitive Strains of Schizosaccharomyces pombe a) Modification of radiation-induced mutational damage b) Differentiation between dead and inactivated cells

2. 13 The Effect of Dark Repair Inhibitors On the Growth of

Irradiated S. pombe Cells

2. 14 Super infect ion Exclusion by Bacteriophage T4

2- 15 Polyacrylamide Gel Electrophoresis of Bacteriophage T4

2. 16 Radiation Genetics in Insects - Dahlbominus 2. 17 Radiation Injury - Rhodnius

2. 18 Radiation Studies on Biting Flies

2. 19 PUBLICATIONS -9-

2.1 STAFF

Acting Branch Head - A.M. Marko

BIOCHEMISTRY AND MOLECULAR BIOLOGY

RNA Molecules

H.C. Birnboim Assistants B.V. Coakley (Miss) M.D.B. Cecil (Mrs.)

Radiation Biochemistry

D.K. Myers Assistant L.D. Johnson K. G. Chetty Repair of DNA. in Bacteria

R.E.J. Mitchel Assistant K.M. Baird

Enzymatic Repair of Radiation Damage

N.E. Gentner Assistant M.M. Werner (Mrs.)

GENETICS, DEVELOPMENT AND POPULATION STUDIES

Radiation Repair in Insects

W.F. Baldwin Assistants G.D. Chant P. A. Knight A.G. Knight

Microbial Genetics

A. Nasim Assistants C.E. Grant (Miss) B.P. Smith

M.M. Shahin

Bacteriophage T4 Genetics

J. D. Childs Assistant L, A. Chant (Mrs. )

Secretarial - M. E. Swallow (Mrs. )

Biological attendants - 2 -10-

2. 1 continued

Attached Staff

M. M. Shahin, an NRC Postdoctoral fellow, is working with A Nasim.

K. G. Chetty, a trainee under the Colombo Plan, is working with D. K. Myers.

2. 2 COMPARISON OF RNA MOLECULES OF ANIMAL CELLS BY OLIGONUC LEO TIDE ANALYSIS - H. C. Birnboim and B. V. Coakley

Experiments in progress are designed to establish the order of ribosomal "genes" for 18S and 28S RNA. The principle of the method for establishing the order of the genes is illustrated by reference to the model of 45S RNA.

5 , i ABC | 1 ^1 1 1 3' X 18S X 28S X Model of 45S RNA (approximately 12 x 103 nucieotides)

45S RNA is a large molecule made within the nucleolus of cells. It is a "precursor" molecule, that is, it is present in the cell for only a short time following which it is extensively modified. In the course of the modification, the 18S and 28S regions are con- served whereas the regions marked X are superfluous and are destroyed. Previously, we have isolated fragments (oligonucleotides) A-F from 45S RNA as well as from mature 18S and 28S RNA. The relative positions of 18S and 28S RNA within the 45S RNA molecule can be established by partially digesting the 45S RNA molecule with a highly specific exonuclease (an enzyme which attacks only from one end). For example, in the model shown, if an exonuclease which attacks from the 3 end is used and one half the molecule is digested, then oligonucleotides D, E, and F would be missing, and A, B, and C w^ald be present in the remain- ing half of the molecule. This would establish that 28S rRNA (which contains D, E, and F) is nearer the 3* end and that 18S rRNA (which contains A, B, and C) is nearer the 5r end of the molecule. At the present time we are purifying an enzyme which will have the high degree of specificity required for this experi- ment. If such an enzyme were available, it could be useful in the analysis of other genes and gene products. -11-

STUDY OF RAPIDLY SEDIMENTING, HETEROGENEOUS RNA IN ANIMAL AND YEAST CELLS - H. C. Birnboim and M. D. B. Cecil

Heterogeneous nuclear RNA (HnRNA) is a class of metabolic ally active RNA molecules found in the nucleus of most eukaryotic organ- isms. This class of RNA is particularly intriguing; it is widely found in nature but no specific function has been assigned to it. In- direct evidence has led to speculation that it is involved in very fundamental cellular processes, viz. gene expression and regulation. Part of the evidence has been the recent demonstration that these molecules contain polyadenylate sequences, as do functional messenger RNA molecules. We have previously shown that a class of molecules resembling HnRNA of animal cells can be isolated from yeast cells. However, the size of these molecules •was sensitive to temperature and salt conditions used to extract them. This has led us to examine more critically HnRNA from animal cells. For most of these studies, we employed dimethyl sulfoxide (DMSO), an organic solvent which has been used to denature (break hydrogen-bonds but not covalent bonds) DNA and RNA molecules. It has been found that the apparent size of HnRNA depends upon the specific conditions of DMSO treatment. Our results explain conflicting reports from other laboratories. We have been able to show that HnRNA molecules can reproducibly be dis- sociated with DMSO under appropriate conditions: molecules which sediment at 45-50S are shifted only slightly in position, whereas 30S molecules are converted to a broad distribution from 20-80S. The unresolved question at the present time is whether 1) those molecules which dissociate were originally intact but were broken during their isolation, 2) were originally small but were aggregated during their isolation, or 3) we are dealing with a new pathway in the processing of HnRNA

SEARCH FOR GENES CORRESPONDING TO POLY A SEQUENCES FOUND IN MESSENGER AJMD HETEROGENEOUS NUCLEAR RNA H. C. Bimboim and R. E. J. Mitchel

Nothing further to report. (For latest information on this project, see PR-B-92, AECL #4145.)

RADIATION BIOCHEMISTRY OF E. COLI - D. K. Myers, K. G. Chetty and L. D. Johnson

a) DNA-membrane association

Previous experiments suggested that the radiation-induced degradation of DNA in E. coli cells is dependent upon association of the DNA -12-

2. 5 continued

with the cell membrane. We are currently developing a method for measuring the amount of DNA-membrane complex under different conditions. The :ell wall is weakened by partial enzymic hydrolysis and the cells are subsequently lyssd in a weak detergent, Brij 58. When the lysate is centrifuged briefly - through a column of 20% sucrose overlaying a column of 60% sucrose, free DNA remains near the top of the centrifuge tube while DNA-membrane complex accumulates near the top of the 60% sucrose column. The relative amounts of DNA in each position are measured by radioactive label incorporated in the DNA

b) Radiation-sensitizing agents

In order to determine whether the effect of radiation-sensitizing agents was mediated via damage to the cell membrane, we have examined the effects of two other compounds on radiation-induced lethality and on radiation-induced breaks in the DNA of E. coli cells. The two compounds tested, sodium deoxycholate and ethylenediamine tetraacetate, were specifically chosen not as radiation sensitizers but as agents which should produce disfunction of the cell membrane. Apparent increases in the number of breaks in the DNA were in fact observed when these compounds were present during irradiation of the cells. However, the increase in radiation lethality was not always proportional to the apparent increase in DNA damage when different compounds were compared. This lack of correlation may be due to the fact that these compounds alter membrane function reversibly whereas other radiation sensitizers produce irreversible effects on cell metabolism.

2. 6 PROTECTION OF RAT THYMUS CELLS - D. K. Myers and K. G. Chetty

Rat thymus cells are susceptible to interphase death within several hours after exposure to low doses of ionizing radiation. In vitro experiments have shown that cell death can be delayed by a variety of chemicals: adenine, nicotinamide, caffeine and alloxan are all effective to varying degrees. In the presence of these compounds, adenosine triphosphate levels are maintained and the degradation of adenosine derivatives to hypoxanthine in the irradiated cells is prevented. We are currently studying the effect of these compounds on different enzymes in the thymus cells in order to determine the enzymic basis for this protective action. The enzymes selected for initial study are adenosine deaminase, adenosine monophosphate deaminase and cyclic-adenosine monophosphate phosphodiesterase. An effort will be -13-

2. 6 continued

made to correlate the inhibitory effect of various compounds on these enzymes with their protective action on irradiated thymus cells.

2. 7 INDUCTION OF LEUKAEMIA IN RATS - D. K. Myers

Currently we are inducing leukaemia by repeated intravenous injections of dimethylbenzanthracene, following a procedure described in the literature. The types of leukaemia and other tumors produced by this chemical are being compared with those which are being studied in our irradiated animals.

2. 8 NUCLEASES OF M. RADIQDURANS - R. E. J. Mitchel and K. M. Baird

a) 5 -Nucleotidase

A 5'-nucleotidase has been shown to be bound to the cell membrane of M. radiodurans. The enzyme can be released by detergent treatment and after several purification steps, including chromatography on Sephadex G-200 and DEAE cellulose, the enzyme preparation contains only two proteins. These two proteins are resistant to further separation. The enzyme has a marked tendency towards aggregation and requires the presence of detergent during all purification procedures.

Magnesium and calcium ions stimulate the hydrolysis of 5 - nucleotides but a tightly bound metal ion, as yet unidentified, may be necessary for activity. Incubation with EDTA causes a slow irreversible loss of activity and any exposure to a cation exchanger also inactivates the enzyme.

The ability of this enzyme to deal with various structurally modified substrates is now being investigated. The enzyme has an absolute specificity for nucleoside-5 -phosphomonoesters. Neither 21- nor 31-phosphates serve as substrates, nor do 51-pyrophosphates. Purine nucleotides are attacked more rapidly than pyrimidine nucleotides with a preference for adenylic acid. Modifications to the sugar portion of the nucleotide also influence cleavage. The rate of hydrolysis of 5*-nucleotides is slowed by the presence of a 2 -hydroxyl. If the sugar is further modified by the formation of a borate adduct with the 21-, 31-vicinal hydroxyls of a 5'-ribonucleotide, the rate of hydrolysis becomes very slow and may reflect the equilibrium of the borate adduct. -14- 2. 8 continued

Additional information pertaining to the enzymes1 requirements in the base portion of the nucleotide has been obtained by the chemica] alteration of deoxy-5*-guanylic acid. This nucleotide has been modified by reaction with glyoxal, which attacks the nitrogens at the N- 1 position and the 2-amino position, forming an additional 5 membered ring on the guanine. This new structure almost completely inhibited the action of the nucleotidase; long digestion times and high levels of enzyme were required to detect any hydrolysis. This modified deoxyribonucleotide was further altered by the formation of a borate complex with the vicinal hydroxyls of the new 5 membered ring. This further modification completely prevented phosphate hydrolysis by the 5'-nucleotidase. A comparison was made with the well-known 5*-nucleotidase from snake venom. The glyoxal-borate modification of deoxy 5'-guanylic acid also completely prevented hydrolysis by this venom enzyme.

b) Exonuclease

The membrane bound exonuclease of M. radiodurans has been further investigated for its ability to degrade damaged DNA, This enzyme has a high degree of preference for single stranded DNA, digesting it completely to 5'-nucleotides. If single stranded DNA is irradiated with uv light, adjacent thymine residues are dimerized. These dimers do not stop hydrolysis by the exonuclease. The enzyme is able to proceed through these damaged regions and continue digesting the DNA

To determine if the exonuclease could digest DNA with other types of damage, single stranded DNA was modified at guanine residues by reaction with glyoxal, as described above. This gly-oxylated DNA was further damaged by formation of borate adducts with the glyoxal- guanine bases. The exonuclease, when added to this modified DNA, was able to digest it, releasing the corresponding modified deoxy 5-guanylic acid.

In comparison, snake venom exonuclease which is known to be unable to digest uv-irradiated DNA, was also unable to degrade past the glyoxal-borate blocked guanylic acid residues of the modified DNA.

2. 9 REPAIR OF RADIATION DAMAGE IN BACTERIA AND YEAST - N. E. Centner and M. M. Werner

a) The DNA polymerase from Micrococcus radiodurans

The DNA polymerase from the highly radiation-resistant bacterium Micrococcus radiodurans is being characterized as part of a program -15-

9 continued

concerned with assessment of its role in the repair of radiation- induced DNA damage in this organism.

The use of EDTA to inactivate nucleases has yielded considerably higher apparent activities in crude extracts. The DNA polymerase activity is quite labile, which has hindered attempts at extensive purification- The enzyme from DEAE-Cellulose chromatography appears to be free of DNase activity, and has been employed for enzymological studies.

p_-Chloromercuribenzoate does not inhibit the reaction rate, which suggests that there is no essential sulfhydryl group required for activity; in this respect it is similar to the DNA polymei-ase I (Kornberg enzyme) from E. coli.

No inhibition is observed with any of the four deoxyribonucleoside-5'-monophosphates (TMP, dAMP, dGMP, dCMP), at concentrations as high as 2. 5 mM, or with 5f-AMP.

The M. radiodurans DNA polymerase has a high affinity for the deoxyribonucleoside-5 -triphosphates. With the other three dXTP's

present at 33(JLM, the following Km's were obtained: for TTP,^6uM; for dATP, ^3|oM; for dGTP,^ 1,JM; and for dCTP,~ l-2uM.

The ribonucleoside-5'-triphosphates cannot substitute for the deoxyribonucleoside-5 -triphosphates in the reaction catalysed by the DNA polymerase. The reaction catalysed by this enzyme is, however, inhibited by ATP (50% inhibition at">l. 3mM) and by UTP (50% inhibition at-3mM), but not by GTP or CTP. The mechanism of this inhibition does not appear to involve complexation with the cofactor Mg++, which is essential for DNA polymerase activity. Experiments are being conducted to ascertain the kinetic nature of the inhibition by ATP and UTP. The physiological significance of such inhibition is under question.

b) Recovery of Schizosaccharomyces pombe from radiation damage

Caffeine has been shown to inhibit repair in various systems. An enhanced sensitivity to uv inactivation when caffeine v/as present in the post-irradiation plating medium has been used as evidence for dark repair.

Plating wild type S. pombe on caffeine-containing plates after uv-irradiation is known to result in lower survival than if plated on -16-

2. 9 continued

the same medium without caffeine. Caffeine has also been shown to inhibit recovery from v-radiation.

Knowledge of the conditions necessary for recovery of S. pombe from radiation-induced damage is necessary to define optimal conditions under which to study the involvement of various repair enzymes in this process. We have developed a technique by which to examine the effect of various parameters on the repair process by looking at the recovery of S. pombe from susceptibility to caffeine inhibition of repair.

An example will serve to illustrate the basic experimental plan: S. pombe , after 1200 ergs/mm2 uv, yields«10% survival if plated~on YPG and/vO. 3% survival if plated on YPG-0. 1% caffeine. If irradiated cells are incubated in liquid YPG medium and plated at various times thereafter on YPG-0. 1% caffeine (which serves to stop repair at that time), one can observe the time course of recovery between these two levels of survival. Such a dose induces a division delay of about 11 hours; we are concerned with the recovery events before this first post-irradiation division. The overcoming of the inhibition by caffeine represents the recovery or repair process. It should be noted that this technique, ie, the overcoming of the inhibitor block, should be adaptable to any organism and any inhibitor of repair in order to allow examination of the requirements for recovery. The results for S. pombe follow. After --ither uv- or v-irradiation, recovery was 80-90% accomplished within 3 hours of incubation at 30°C in rich medium (YPG). This repair required a complete growth medium. S. pombe minimal medium gave a time course of recovery somewhat more rapid than that of the rich medium. A carbon source (2% glucose) by itself gave only a slight degree of recovery. No recovery whatsoever was observable with non-nutritive solutions such as water.

The effect of temperature on the recovery process supplied evidence for the enzymatic nature of the repair. The optimal temperature of incubation was 25°-30°C. At lower temperatures (15°C and 7. 5°C) the recovery process proceeded more slowly, and maximal recovery was not attained; at higher temperatures (36°C), the initial rate of recovery was higher, but then decreased considerably after 2 hours incubation.

This system is currently being exploited to ask a question of extreme importance with regard to repair in S. pombe ; is new protein synthesis, ie, synthesis of repair enzymes, necessary for the recovery process, or does the cell utilize its pre-existing complement of enzymes (that are present during normal growth) to accomplish repair. -17-

2. 9 continued

If, for example, inhibition of protein synthesis in the post-irradiation medium decreased the time course of recovery significantly, the answer would be that new protein synthesis was necessary for repair, and by adding the inhibitor of protein synthesis at various times after irradiated cells were put in the rich medium, one could determine the period of time required for synthesis of new repair enzymes.

Two commonly used inhibitors of protein synthesis, puromycin and chloramphenicol, at concentrations as high as 200 (xg/ml, were ineffective with S. pombe. Cycloheximide, however, at 25 (xg/ml, inhibited growth completely. This concentration of cycloheximide' had only a slight effect on the recovery of uv-irradiated cells from caffeine inhibition of repair, which would prompt a conclusion that new protein synthesis was not essential for repair, in spite of the requirement for a complete growth medium. When, however, the time course of 14C- alanine incorporation into protein was examined, it was found that 25 |j.g/ml cycloheximide did not shut off protein synthesis until about 60 minutes; this would be an entirely adequate time in which to accomplish synthesis of new repair enzymes. Therefore, higher concentrations of cycloheximide, such that protein synthesis is shut off immediately (and yet low enough so that plating efficiency is not affected) will have to be employed. Alternately, the cells could be pre-incubated in 25 jig/ml cycloheximide in H2O before irradiation and subsequent dilution into the rich medium (also containing cycloheximide) necessary for recovery. The feasibility of both these approaches is under examination, and once the experimental design necessary for complete shut-off of protein synthesis is defined, it should be possible to determine whether or not new protein synthesis is required for recovery of S. pombe from radiation damage.

A similar study will be made with Micrococcus radiodurans; such a definition of the time course of repair, and of the requirements for the repair process, would serve to define the conditions under which to examine possible alterations in the level of certain repair enzymes concomitant with recovery.

2. 10 GENETIC CONTROL OF RADIATION SENSITIVITY IN SCHIZOSACCHAROMYCES POMBE - A, Nasim and B. P. Smith

Dark Repair Inhibitors in Yeast

The highly radiation sensitive single oene mutants as well as double mutants showing super-sensitivity to uv are being used in •18-

2. 10 continued

current experiments. Many agents are known to inhibit dark repair in bacteria, thus causing marked reduction in colony forming ability when these are present in the post-irradiation plating medium. One possible way of assessing the dark repair capacity of the radiosensi- tive mutants of yeast would be to study their response to inhibitors of dark repair. In the super sensitive mutants if the enhanced sensitivity results from an absence of dark repair, then the survival of such mutants should not be markedly influenced by the presence of an inhibitor. In preliminary experiments a number of such inhibitors like acuf la vine, acudine orange, hydroxyurea, quinacrine and chloroquine have been tested for their suitability in such studies. A search for an inhibitor which may be specific for either uv or X-ray damage in yeast is also being carried out. By using this method it should be possible to test the large number of single gene sensitive mutants and distinguish a smaller class among such mutants where the radiation sensitivity has resulted from a specific block in steps involved in dark repair of radiation damage.

2. 11 REPLICATING INSTABILITIES IN SCHIZOSACCHAROMYCES POMBE - A. Nasim and C. Grant

The experiments concerning site specificity of such instabilities (for details see PR-B-92, AECL #4154) have now been concluded. The data show that ethylmethanesulfonate induced instabilities in the mutational system involving forward mutations are confined to the same site within a given line. Such evidence has been obtained from both complementation and intragenic recombination experiments. A search for mutational systems other than those concerned with the biosynthesis of adenine has been undertaken to study instabilities in other parts of the genomes. The use of mating type locus in S. pombe has given positive results. The homothallic condition hgg gives a blue coloration with iodine on sporulating medium. When a parental strain h+ mating type is treated with a mutagen, plated on sporulation medium and exposed to iodine vapours the homothallic mutants give complete and mosaic mutants characterised by blue colour. Such primary mosaics have been shown to yield more mosaics on replating than would be expected of instabilities. Similar tests are now in progress for another visual system of scoring mutants using the Galactose fer- mentation ability as the genetic marker. Data so far imply that replicating instabilities are of wide occurrence and are associated with many different kinds of mutational events. A mutation is not always a change from one stable phenotype to another stable phenotype. Very often mutagenic treatments produce an unstable state which can mutate -19-

2. 11 continued

to any one of the two stable phenotypes or even replicate as an instability and produce the unstable state during subsequent cell divisions.

2. 12 LIQUID HOLDING RECOVERY AND SPONTANEOUS CELL DEATH IN THE NORMAL AND RADIATION-SENSI TIVE STRAINS OF SCHIZOSACCHAROMYCES POMBE - M. M. Shah in

The first part of this study has now been completed, and for progress in this project, see Quarterly Report PR-B-92, AECL #4154.

Previous results suggest that in this organism any significant recovery from radiation-induced damage may only be initiated in a rich medium and occurs mainly during the first post-irradiation round of replication. The negative liquid holding effect observed could thus be attributed to a kind of radiation-induced damage, not amenable to repair when cells are held in a non-nutritive solution.

Experiments in progress at present are designed to obtain more data on the underlying mechanism of liquid holding recovery as these relate to the known mechanisms of dark repair in microorgan- isms.

a) Modification of radiation-induced mutational damage

Both lethal and mutational damage induced by radiation exposure are known to be repaired by cellular repair processes. The inactivation and cell death represent a nonspecific biological end-point, whereas the damage expressed as mutations results from specific alterations in the genetic material. By using a variety of yeast strains including a few mutator strains which are characterised by having high rates of spontaneous mutability, the effect of holding cells in different nutritive and non-nutritive media is under study. This approach should also reveal whether any component of the damage leading to marked negative liquid holding effect (earlier observed in our experiments in the non- nutritive solution) is genetic in nature. Both uv and v-rays are being used in such mutation studies.

b) Differentiation between dead and inactivated cells

Microscopic studies using vital dye methylene blue in a cell suspension are now in progress to determine the stage at which the -20-

2. 1 2 continued

irradiated cells are inactivated or die after exposure to radiation and during the course of liquid holding. The stage at which cell death occurs should show whether irradiated cells are dying immediately after treatment or if the colony-forming ability is decreasing due to later damage. This damage may be caused by pl±yciological factors affecting metabolic unbalance in the non-nutritive medium. The technique is being followed to study the effect of sucrose and NaCl which in the earlier experiments were found to reduce the colony-forming ability of unirradiated cells.

2. 13 THE EFFECT OF DARK REPAIR INHIBITORS ON THE GROWTH OF IRRADIATED S. POMBE CELLS - M. M. Shahin

The effect of many agents which act as dark repair inhibitors has not been investigated in yeast. This study is concerned with the examination of the influence of dark repair inhibitors such as caffeine, chloroquine and acriflavine on the growth of irradiated S. pombe cells in liquid media of different compositions.

2. 14 SUPERINFECTION EXCLUSION BY BACTERIOPHAGE T4 - J. D. Childs and L. A, Chant

If Escherichia coli is infected with bacteriophage T4 and then superinfected with T4 a few minutes later, the superinfecting phage is excluded from the progeny of the subsequent burst. This process is known as superinfection exclusion. A mutant of bacteriophage T4 (exc 29) has been isolated which is deficient in its ability to exclude superinfecting phage. Wild type T4 can exclude at least 90% of superinfecting T4 phage, whereas this mutant is able to exclude only 30% of superinfecting phage. Previously it has been shown that the region of T4 responsible for superinfection exclusion by incomplete genomes of T4 is located between or close to genes 42 and 43. To determine whether the site of mutation in exc 29 is in this region, exc 29 was crossed to a strain of T4 with amber mutations in genes 56, 41, 42, 43, 44 and 46, which are located in that order on the genetic map. Recombinants were isolated, which had resulted from single crossovers in any one of the 5 intervals between genes 56 and 46, and these were tested for their ability to exclude. Those which had inherited genes 42 and 43 from exc 29 failed to exclude, whereas those which inherited this region from the other parent were able to exclude superinfecting phage. This confirmed that the gene responsible for exclusion is located between genes 42 and 43. -21- 2. 15 POLYACRYLAMIDE GEL ELECTROPHORESIS OF BACTERIOPHAGE T4 - J. D. Childs and H. C. Birnboim

Polyacrylamide gel electrophoresis has been used to separate a variety of macromolecules, cell components and small spherical viruses (maximum diameter 225 A). We have attempted to apply this method to bacteriophage T4, a. large virus with a complex structure (approximate dimension 650 A x 2000 A), and to see whether a small variant of T4 (approximately 650 A x 1700 A) can be separated from the normal size T4. It has been found that T4 particles will move electrophoretically through gels if the pore size is sufficiently large and clumping of phage particles prevented. This was accomplished by appropriate choice of acrylamide concentration in preparing gels and a buffer. When acrylamide concentration was less than 1. 8%, gellation did not occur; above 2. 1%, phage particles did not enter the gel. Magnesium ions (0. 6 - 3 mM) were necessary to prevent clumping. The phage migrated through the gel as a band visible by light scattering or by ultraviolet adsorption. Thus far, separation of normal and small T4 phage particles has not been detected, which is surprising in view of the known ability of gel electrophoresis to separate molecules on the basis of size. The inability to separate these virus particles which differ in length by 15% is not understood at the present time. Further experiments will be done to measure the viability of the virus on elution from the gel and its rate of migration in different voltage gradients.

2. 16 RADIATION GENETICS IN INSECTS - DAHLBOMINUS - W. F. Baldwin, G. D. Chant, A. G. Knight and P. A. Knight

Tests with Dahlbominus to determine if eye colour mutants arise as true point mutations are continuing, and the irradiation of females from a homogeneous line of carmine eyes show that reversion (the production of back mutations - to wild type) does occur with this colour. In 47, 358 males from carmine females exposed to 500R, five wild type and one black eye mutation were recovered. In addition to the usual four bright eye colours, individuals showing new eye colour phenotypes were found, consisting of 9 green, 26 brown and 3 grey eye males. Analysis of the data demonstrated that eye colour frequency in irradiated carmine eye females was identical to irradiated wild type (frequencies of 199/243 x 10"5 - Pi. 0. 05). Body malformations believed to be the result of radiation-induced chromosome rearrangements, did not appear, however, to occur at the same rate; the results showed a statistical difference between the two groups (310/289 x 10"5- P<0. 05). It may be that the mutagenic effects of radiation due to chromosome damage may be critical in populations derived from mutant lines. These phenomena will be investigated in several additional mutant strains. -22-

2. 16 continued

The maintenance breeding of selected lines of 25 different mutations was completed during the period. This operation must be done on a routine basis at least two or three times a year, since Dahlbominus females will remain viable as pupae under cold storage conditions only for 3 to 4 month periods.

2. 17 RADIATION INJURY - RHODNIUS - W. F. Baldwin, G. D. Chant and A, G. Knight

See PR-B-92, AECL. #4154, section 2. 16 for progress on this phase of the work.

2. 18 RADIATION STUDIES ON BITING FLIES - W. F. Baldwin

Dr. W. F. Baldwin returned recently from Venezuela where he had been invited by the Ministerio de Sanidad y Asistencia Social, at Maracay, as a consultant on several different public health problems involving insect disease vectors. The visit involved meetings with public health officials at Maracay and Caracas and several consultations with parasitologists at both Carabobo University in Valencia and the Agricultural College at Maracay. Field trips with Dr. J. C. Gomez- Nunez included visits to the western section of the country south of the Andes where malaria is endemic, and to rain forest regions on the north coast near Puerto de Cabello in areas where leishmaniasis is spread by sand flies. At present, methods of attracting and trapping both malarial mosquitoes and sand flies are unsatisfactory, and traps developed at CRNL employing CO2 and light as attractants are now being tested at field stations in these locations. If the traps are satisfactory, dispersal studies using insects tagged with P32 will be possible. Further plans for marking vectors of Chagas disease with radioactive isotopes (platinum iridium) were discussed, and new work on this project will be initiated in the near future.

2. 19 PUBLICATIONS

W. F. Baldwin Linear dose effect response for mutations in mature oocytes of the wasp Dahlbominus. Radiation Res. 49, 190-196 (1972). -23-

2. 19 PUBLICATIONS, continued

N. E. Gentner Evidence that Gly-tRNAG \n is physiologically important donor in formation of Glycyl-lipopolysaccharide in E. coli. Biophysical Soc. Abstracts, 16th Annual Meeting, p. 108a (1972) - Abstract.

D. K. Myers Protein and RNA synthesis in radiation-sensitive strains of yeast. Mutation Res. _14, 112-114(1972).

J. D. Childs (H. R, Warner, D. P. Snustad, J. F. Koerner and J. D. Childs) Identification and genetic characterization of mutants of bacteriophage T4 defective in the ability to induce exonuclease A. J. of Virology 9... 399-407,(1972). -25-

POPULATION RESEARCH

Howard B. Newcombe

CONTENTS

3. 1 Staff (1 January to 31 March 1972)

3.2 Aims of the Branch

HEREDITARY CHANGES IN VERTEBRATES

3.3 Low-dose effects in fish embryos from irradiated sperm

3.4 Chromosome cytology of irradiated fish

METHODS FOR DERIVING HUMAN DATA ON DISEASES LIKELY TO BE INCREASED BY IRRADIATION -- DEVELOPMENT OF THE INFORMATION SOURCES

3. 5 Organization of birth and marriage records

3.6 Ascertainment of congenital malformations through hospital records

PROPOSALS FOR STUDIES OF HUMAN POPULATIONS

3.7 Cancer following multiple fluoroscopy

OTHER ACTIVITIES

3. 8 Publications -26"

3.1 STAFF

Branch Head - H.B. Newcombe

Hereditary changes in vertebrates

Assistant J.F. McGregor

Methods for deriving human data on diseases likely to be increased by irradiation

Assistant M.E. Smith (Miss)*

Secretarial - B.L. Phillips (Mrs.)

On six months unpaid leave from 10 September 1971 to continue studies at Queen's University.

3.2 AIMS OF THE BRANCH

The Branch attempts to obtain biological data with which to assess the consequences of exposing large numbers of people to low levels of radiation. Information has been derived from irradiated and unirradiated populations of rats and fish, and essentially new methods have been developed at CRNL for extracting data for man from existing records of procreation and health.

HEREDITARY CHANGES IN VERTEBRATES

3, 3 LOW-DOSE EFFECTS IN FISH EMBRYOS FROM IRRADIATED SPERM - H.B. Newcombe and J.F. McGregor

Significant reductions in embryo mortality (p = 0.001) have been observed in rainbow trout following exposures of sperm to doses of 25 and 50 rad of gamma radiation. This "beneficial" effect of radiation is more apparent in the early period of embryo -27-

3. 3 continued

development than during the later stages. Higher doses of 200 and 400 rad caused an increase in embryo death. The evidence argues against the idea that the dose-effect curve for early mortality in the first generation after irradiation is necessarily linear, or even consistent in sense, over a range of low doses.

Extreme distortions of trout embryos so that they resemble mixed tumours have been observed occasionally following irradiation of the sperm, and in the control series. The data are currently being analysed.

3.4 CHROMOSOME CYTOLOGY OF IRRADIATED FISH - J.F. McGregor andH.B. Newcombe

This work has been interrupted for a number of months because of failure of the equipment for incubating the cell cultures. It is expected to begin again in April.

METHODS FOR DERIVING HUMAN DATA ON DISEASES LIKELY TO BE INCREASED BY IRRADIATION - DEVELOPMENT OF THE INFORMATION SOURCES

3.5 ORGANIZATION OF BIRTH AND MARRIAGE RECORDS - H.B. Newcombe and M.E. Smith (with N. R. Burnham, Mathematics and Computation Branch)

Work on the sorting of birth index summary records into multiple sequences is continuing. The sequencing by year and registration number has been completed but will need to be re- done because of two out-of-sequence records. The "double soundex" sequence (father's surname followed by mother's maiden surname, in phonetically coded form) has been completed but the file will need to be divided between two tapes for ease of handling. The sequence by child's surname ("single soundex" sequence) has been completed, and the sorting by birth date has been partially completed. The final versions of these birth index files will be used to integrate diagnostic information from handicap, hospital, and death records, into individual and family histories. -28-

3.6 ASCERTAINMENT OF CONGENITAL MALFORMATIONS THROUGH HOSPITAL RECORDS - M.E. Smith and H.B. Newcombe

Work on this undertaking will be resumed when Miss Smith returns in May.

PROPOSALS FOR STUDIES OF HUMAN POPULATIONS

3.7 CANCER FOLLOWING MULTIPLE FLUOROSCOPY

Assistance has been given to the National Cancer Institute of Canada in the form of a chipboard phantom with which to measure the dose rates, at different depths, from two old fluoroscopy machines at Toronto Hospital Weston. Lithium fluoride dosimeters for this work have been supplied by the Health Physics Branch.

OTHER ACTIVITIES

3.8 PUBLICATIONS

McGregor, J.F. and Newcombe, H.B. Dose-response relationships for yields of major eye malformations following low doses of radiation to trout sperm. Radiation Research 49, 155-169 (1972); AECL #4037. -29-

ENVIRONMENTAL RESEARCH BRANCH

C.A. Mawson

4.1 Staff 4.2 Off-Site Monitoring 4.3 Liquid Disposal Area 4.4 Liquid Effluent Monitoring 4.5 Tritium Survey 4.6 Survey Summary 4.7 Fate of Radionuclides in Aquatic Ecosystems 4.7.1 Metabolism of Radiostrontium by Fish 4.7.2 Stable Cobalt Analysis 4.7.3 Effects of Chlorinated Hydrocarbons on Ca/Sr Metabolism of Fish 4.7.4 Accumulation of Radionuclides by Vascular Aquatic Plants 4.8 Perch Lake Basin Studies 4.8.1 60Co in Perch Lake 4.8.2 90Sr in Perch Lake 4.9 St. Lawrence River Studies 4.10 Perch Lake Evaporation Study 4.11 Atmospheric Dispersion studies 4.12 Environmental Pollution Studies 4.13 Neutron Activation Analysis 4.14 Permanent Storage Of High-Level Wastes 4.15 Effects Of Nutrient Enrichment On Primary Production 4.16 Environmental Radioactivity studies 4.17 Evaporation Studies 4.18 Staff Publications

For Figures 1, 2 and 3 see end of section. -30-

4.1 STAFF

Branch Head - C.A. Mawson

Biological and Physical Studies I.L. Ophel Assistants: CD. Fraser J.M. Judd S-R. Gentner (Mrs.) J.W. McMahon Assistant: A.E. Docherty

Meteorological Studies P.J. Barry Assistants: P.C. Jay R.E. Légère D.P. Wildsmith

Chemical Studies W.E. Grummitt Assistants: G.G. Gunter G. Lahaie L.A. Mask H.M. McLaughlin (Mrs.) J.L. Young

Soil Studies and Radiochemical Analysis W.F. Merritt Assistant: B.A. Risto

Isotope Studies R.W. Durham1

R.W. Durham attached to Canada Centre for Inland Waters, Burlington. Terminated March 31, 1972. -31-

Evaporation Studies 2 F.A. PrantPrantll (Miss) Assistant: E. Robertson (Mrs.)

Secretarial 3 D.J. TerMarsch (Mrs.)

CERN European Organization for Nuclear Research, joined the staff as an Assistant Research Officer May 12, 1971 for a period of two years.

Mrs. D.J. TerMarsch joined the Environmental Research Branch on March 1, 1972. -32-

4.2 OFF-SITE MONITORING - W.E. Grummitt

Precipitation samples collected from the area around the Nuclear Power Demonstration (NPD) Station, Rolphton, and from Deep River were analyzed for 90Sr and 137Cs. Monthly composite samples of water from the Ottawa River, collected at Rolphton, Deep River and Pembroke, were also analyzed for 137Cs and other gamma emitting nuclides, tritium and 90sr. The results are shown in Figure 1.

The main source of activity in these samples continues to be weapons fallout.

4.3 LIQUID DISPOSAL AREA - W.E. Grummitt

Weekly samples and flow readings were taken at weirs on the surface streams carrying contaminated seepage water from the Liquid Disposal Area into Perch and Maskinonge Lakes'•*•' . Samples were also taken from the Perch Lake outlet stream. Radiochemical analyses of monthly composite samples were performed. 60 3H are still the main contaminants.

4.4 LIQUID EFFLUENT MONITORING - W.E. Grummitt

At CRNL, four liquid effluent streams discharge radioactivity to the Ottawa River. Three of these, the Process and imhoff sewers plus the "Foundation" storm sewer, drain the active inner area, perch cre draining Perch Lake and the disposal areas, also flows into the river. Each of these is sampled regularly and is analysed for individual nuclides. The total amount of radioactivity discharged to the Ottawa River per day is shown in Figure 2.

At no time did the average concentrations of radioactivity in the effluents exceed 1% of the ICRP 40-hour occupational MPC for water.

'see map. Figure 3 -33-

4.5 TRITIUM SURVEY - W.E. Grununitt and G. Lahaie

Average concentrations of tritium in three streams within the exclusion area are as follows:

Estimated H-Ci/1 Ci/year

Bldg. 610 drainage 30 300 Duke Stream 1.7 ~100 Main Stream 0.4

The Bldg. 610 stream flows directly into the river. Duke Stream discharges through Maskinonge Lake; Main Stream discharges through Perch Lake.

4.6 SURVEY SUMMARY

No significant contamination of the environment resulting from operations at CRNL has been detected in these surveys.

4.7 FATE OF RADIONUCLIDES IN AQUATIC ECOSYSTEMS - I.L. Ophel, J.M. Judd and S-R. Gentner

4.7.1 Metabolism of Radiostrontium by Fish - No work carried out on this project during the quarter.

4.7.2 Stable Cobalt Analysis - An atomic absorption spectro- photometer has been purchased. After a preliminary screening two makes of instrument were selected for prolonged testing. One of these was selected for pur- chase and has now been installed. Methods of preparing, ashing, complexing and extracting large-volume (20 litre) water samples for cobalt analysis are being investigated.

4.7.3 Effects of Chlorinated Hydrocarbons on ca/Sr Metabolism of Fish - Work has continued on testing and modification of equipment for maintaining continuous, sub- lethal concentrations of chlorinated hydrocarbons in experimental tanks. -34-

4.7.4 Accumulation of Radionuclides by Vascular Aquatic Plants - The relative importance of roots and leaves as pathv?ays for ion absorption are being studied with 5 3 a radioactive cation (32po4)3~ and anion ( ^Fe) ^. Three types of rooted aquatic plants are being used, namely: floating-leaved, submerged bottom-rooted and submerged trailing-rooted.

4.8 PERCH LAKE BASIN STUDIES - I.L. Ophel and CD. Fraser

4.8.1 Co in Perch Lake - Aquatic, semi-aquatic and terrestrial mammals were trapped at the lake-shore during the winter. Body burdens of 60Co and concentrations in food (stomach contents) were measured. an 4.8.2 ?uSr in Perch Lake - The animals listed above (and stomach contents) are being analysed for stable Sr and Ca by flame emission spectrophotometry.

4.9 ST. LAWRENCE RIVER STUDIES - I.L. Ophel

Under a research contract with AECL a group from the Universite du Quebec at Trois-Rivieres is studying the effects of heated effluent from the Gentilly nuclear power station on the chemical, physical and biological conditions of the St. Lawrence River. Up to the end of December, 1971, they have results for approximately 18 months of measurements - ten months of these data were collected before AECL support began. Most of these results have now been tabulated and analysed statistically using the Universite du Quebec computer facilities. Their analysis shows no significant differences in chemical, physical and biological conditions between two upstream and five downstream sampling stations either during the pre-operational phase or the low-power phase of reactor operations.

4.10 PERCH LAKE EVAPORATION SPUDY - P.J. Barry

The period was split on quality control and analys- ing of data collected during the summer field programme. -35-

4.11 ATMOSPHERIC DISPERSION STUDIES - P.J. Barry

Routine measurements were continued throughout the year.

4.12 ENVIRONMENTAL POLLUTION STUDIES - P.J. Barry

Theoretical work continued. These studies revealed areas where further experimental information is required to complete calculations of the radiation dose implications to people as a result of the release of radioactive wastes in a variety of environmental situations.

4.13 NEUTRON ACTIVATION ANALYSIS - W.F. Merritt

A new ADC has been installed in the 4096-channel analyzer and it is finally operating satisfactorily. The detector resolution has been improved to 2.4 kw at 1.33 MeV and it is now feasible to operate at 0.5 keV/channel. Five more elements (Sin, Nd, Yb, Lu, Hf) can now be estimated in water samples.

Irradiation tests on the polyethylene capsules used as sample holders indicate that the present irradiation time of 4 hours (equivalent to 3 x 1018 neutrons) is the maximum that can be expected, since they becoma brittle or shatter in the rabbit on longer exposure. If longer irradiation times,are required, quartz capsules would have to be used and the higher backgrounds due to impurities in the quartz could tend to minimize any gains in sensitivity.

4.14 PERMANENT STORAGE OF HIGH-LEVEL WASTES'- W.F. Merritt

The 1971 results of the glass block test disposal indicate that the daily leaching rate i,.s now about half of what it was when last reported-in 1966. A report is being prepared. i -36-

4.15 EFFECTS OF NUTRIENT ENRICHMENT ON PRIMARY PRODUCTION - J.W. McMahon

Laboratory studies have been carried out to determine the effects of addition of nutrients to natural lake water. Results from short-term experiments (4 hours) gave no indication of increases in carbon fixation. Bioassay studies are now being carried out over longer periods and at different temperatures, preliminary findings indicate that increased watsr temperature and the presence of some added nutrients may adversely influence 14c primary fixation.

Data collected in the summer of 1971 on phyto- plankton species succession and abundance in the polyethylene columns in Maskinonge Lake and in the lake itself have been analysed. This information is relevant to the study of primary production in the oligotrophic waters of Maskinonge Lake.

Techniques have been worked out for determining total organic carbon content of lake water. water samples were freeze-dried, encapsulated and their total carbon determined with a Leco carbon analyser. This phase of the study is in co-operation with the General Chemistry Branch at CRNL.

4.16 ENVIRONMENTAL RADIOACTIVITY STUDIES - R.W. Durham

Work continued on the development of a method for the analysis of lake sediments for fallout radionuclides. High chemical yields can now be obtained for Ru, Zr, Ce, Sr and Cs when these elements are added as carriers. Experiments with added y-ray emitting radioisotopes of these elements helped to determine the cause of poor separation of some elements .

A trial of the method on a surface sample (0-2 cm) of Lake Ontario sediment showed no detectable levels 144 of 1O6RU Or 95Zr, barely detectable ce, but readily -37-

measurable 137Cs and 90Sr. A core from Lake Erie Western basin has been sectioned at 1 cm intervals down to 20 cm and analyses are being done on each section. A preliminary survey of the top seven dried sections with a Ge(Li) detector and pulse height analyzer showed iJ7Cs and 144Ce to be present, along with daughter products of radium and thorium.

The Ge(Li) detector was standardized for two sample geometries using standard solutions obtained from Amersham/Searle. A curve of efficiency versus energy was determined using the method of Verplanke (Nucl. Insts. and Methods, 9£ (1971) 557) to correct for coin- cident summing of Y~rays« The accuracy of the stand- ardization is being checked by analyzing a standard lake water sample spiked with low levels (pCi/t) of several radionuclides, distributed by Argonne National Laboratory.

4.17 EVAPORATION STUDIES - F.A. Prantl and E. Robertson

A new approach to determination of evaporation from lakes is based upon tracer isotope measurement.

Evaporation and condensation experiments were carried out with deuterated and tritiated water. For general use in the field a tritium tracer method is being developed. For this purpose the fractionation factors between the phases of tritiated water were measured.

Under equilibrium conditions of 0°C the factors are 0.84 between vapour and solid phase, 0.97 between liquid and solid, and 0.86 between vapour and liquid.

During evaporation from open water surfaces into the atmosphere or moisture condensation in an open system the fractionation factors measured were larger than those under equilibrium conditions. This effect has been studied systematically as a function of various environmental parameters. One of the most important is atmospheric humidity. -38-

Models and formulae have been developed for calculation of the fractionation factor under environmental conditions. They were tested using field data collected over three years. Agreement within the accuracy of measurement was found between predicted and measured data.

In combination with our previously developed formulae this allows us to calculate the net loss of tritium from open water basins into the atmosphere and the isotope concentration of water vapour formea in the Perch Lake Basin.

4.18 STAFF PUBLICATIONS

BARRY, P.J. "Atlas of Energy Budgets of Plant Leaves" - Book Review - (D.M. Gates and LaVerne E. Papian). Submitted to Boundary Layer Meteor- ology in January, 1972. -39-

137 ROLPHTON Cs ROLPH TON 90Sr 0.8 1,2 pCi/'l

0.4 0.6

i i i i i i i i JFMAMJJASOND J F H A H J J A S 0 N D

DEEP RIVER 137Cs 0.8 1.2 pCi/l

0.4 0.6

i i i j j i i i i i i i i i l—i JFMAMJJASQHD JFMAMJJASONO

90 PEMBROKE 137Cs )KE Sr 0.8 1.2 pCi/l

0.4 0.6

i ii i • • • i • JFMAMJJASONO J F M A M J J A S 0 H 0 FIGURE 1 OTTAWA RIVER WATER -40-

28 15i- 9O NOMINAL /3 S 200 - 12 -

9 m Ci/d m Ci/d 100 6

i i i i i i i I i i .i II II II JFMAMJ JASOND JFMAMJJASOND

27 15 a 137 Cs 12 12

9 m Ci/d m Ci/d

I I I I I I I I I I I JFMAMJ JASOND JFMAMJJASOND

10:- 10 106 Ru 239pu B m Ci/d mg/d 6

2 O: M A M J J A S 0 N D J F M A M J J A S 0 NO Fl JURE 2 RADIONUCLIDES DISCHARGED TO OTTAWA RIVER Outer Area of AECL Chalk River Nuclear Laboratories -43-

HEALTH PHYSICS BRANCH by G. Cowper

5.1 Staff 5.2 Spark Counting Technique for Damage Track Dosimeter Measurements 5.3 Fast Spectra Resulting from Moderation of Fission Neutrons 5.4 Counting Rh X-Rays from Thick Foils 5.5 Rh Gamma Rays from Inelastic Neutron Scattering 5.6 Thermoluminescence Dosimetry (TLD) 5.7 Semiconductor Detectors 5.8 Geiger Counters 5.9 Monitoring for Tritium in Air 5.10 Monitoring for Tritium in Water 5.11 Portable Fluorimeter 5.12 Health Instruments begun in the First Quarter of 1972 5.13 Health Instruments in Progress in the First Quarter of 1972 5.14 Health Instruments completed in the First Quarter of 1972 5.15 Miscellaneous 5.16 Detection of Iodine in Air 5.17 Microscopic Flying-Spot Scanner 5.18 Specimen Preparation for Heavy Atom Imaging by Phase Contrast Electron Microscopy 5.19 Routine Dose Monitoring 5.20 Publications -44-

5.1 STAFF

G. Cowper - Branch Head Secretarial N. Rod (Mrs.) Draftsmen B.A. MacDonald (1) J.H. Sneddon (1) Assistant L.S. Larkin Film R.M. Rondeau (Miss) Monitoring J.M. Vincent (Mrs.)

W.G. Cross P.J. Bunge H. Ing Assistants . D.J. Robertson

A.R. Jones A.H. Ohno Assistants W.F. Richter

R.V. Osborne A.S. Coveart H.M. Johnson (2) Assistants N.W. Tepley

R.M. Holford J.G. Plato Assistant

(1) On loan from Drawing Office. (2) National Research Council Post-Doctoral Fellow.. -45-

5.2 SPARK COUNTING TECHNIQUE FOR DAMAGE TRACK DOSIMETER MEASUREMENTS - W.G. Cross

Study of the mechanism of spark discharges through damage track holes in thin insulator foils (PR-B-92, AECL-4154) has continued. Sparks were found to be not independent of one anoth2r but correlated in both position and time. Since this may result in more than one spark occurring in a. hole, the voltage is not reapplied until a definite time (1 to 100 ms) after each spark, in order to reduce the effects of residual ionization. Under some circumstances, sparks are quenched by evaporation of the Al electrode rather than by discharge of the capacitance.

Apparatus has been constructed for making spark replicas in gases other than air and at pressures from 0.001 to 5 torr. The foil can be put under the desired atmosphere within a few seconds. Carrying out the initial discharge (for opening up the holes) at high pressure gave more reproducible results than doing this at atmospheric pressure. Minimum sparking voltage and minimum hole diameter in the replica were obtained in He at about 4 torr. This permits counting up to 5000 holes/cm2 with 10% losses caused by over- lap of holes.

5.3 FAST SPECTRA RESULTING FROM MODERATION OF FISSION NEUTRONS - H. Ing and W.G. Cross Knowledge of the neutron spectrum is important for the determination of neutron dose. In a criticality accident, the original fission spectrum would be modified by scattering in the surrounding material. In previous work (1) we have examined spectra from typical criticality configurations and have shown that for many such arrangements simple models predict the spectrum with adequate accuracy for dose determinations .

(1) W.G. Cross and H. Ing, Abstracts of the Sixteenth Annual Meeting of the Health Physics Society, Health Physics 21, 45 (1971). -46-

Here we study spectra from similar configurations using the Monte Carlo approach with the . purposes of finding the limitations of the results using simple theory and of providing additional results with which they can be compared. So far the calculations have been limited to changes in the fission spectrum caused by moderation in H2O or D2O. The fission source was located either at the center of, or uniformly distributed in, spherical moderators of varying radii and neutrons that escape were analyzed over the energy range 1 eV to 10 MeV.

With a point source at the center, a thickness of water 10 cm or larger gives good agreement with the "equilibrium" shape predicted by simple theory. Below 10 cm, the intermediate energy neutrons are building up to the equilibrium spectral shape with increasing H2O thickness. For D2O, good agreement with simple theory is obtained for a radius of 20 cm. Below this value there are fewer keV neutrons than expected while at radii over 20 cm there are more. When the source is uniformly distributed, the escape spectrum for a fixed radius is harder than that for the source at the center. Although an equilibrium shape is also reached for about 10 cm of water, it is not approximated as closely by the simple theory. Calculations for D20 moderators with a dispersed fission source are in progress.

5.4 COUNTING Rh X-RAYS FROM THICK FOILS - H. Ing and W.G. Cross Dose determinations using the 103Rh(n,n')103mRh reaction require counting 20 keV X-rays from foils in which self-absorption is the order of 50%, in approximately 2TT geometry. Detection efficiencies for a circular detector and coaxial disc sources of various diameters and at various distances have been calculated for thicknesses in which self- absorption is important. These calculations have been checked by measuring (a) the relative efficiency at distances between 0.15 and 90 mm from the 38-mm diameter Nal detector and (b) the attenuation, in 2TT geometry, of Ru X-rays by various thicknesses of Rh. The results agree with the calculations to ± 2%. -47-

5.5 Rh GAMMA RAYS FROM INELASTIC NEUTRON SCATTERING - H Ing

No comprehensive calculation of the 103Rh(n,n') 103mRh excitation function can be found in the literature although the cross section has been accurately measured (D . A preliminary curve was computed by E. Vogt in 1962 (2) using the energy levels below 600 keV. He obtained an energy variation in rough agreement with experimental data below 600 keV, but pointed out that the curve was unreliable at higher energies because the required energy levels were not known.

We have studied the higher levels of 103Rh by inelastic scattering of 2.8 MeV neutrons from the d(D,n)3 He reaction, so that such calculations may be extended. Rh foils (18.8 g) were placed at the end of a colliiuator consisting of a 1.25 cm diameter hole through a 30 cm thick polyethylene slab. Coincidences were measured between neutrons scattered inelastically at 90° to the incident collimated beam and the resulting gamma radiation, also at 90°. The gamma detector was a 40 cc Ge(Li) crystal while neutrons were detected with a stilbene crystal operated with n-y pulse shape discrimination. The background run was made by substituting a carbon scatterer, of appropriate mass, for the Rh sample.

Preliminary analysis of data yields the gamma rays shown in table 1. Several of the gamma rays have been observed in recent work in radioisotope decay, inelastic proton scattering, Coulomb excitation or combinations of these.

(1) J.P. Butler and D.C. Santry, Atomic Energy of Canada Limited report No. AECL-3043 (1968).

(2) E. Vogt, private communication to A. Fowler (1962). -48-

Table 1

Gamma rays observed Other experiments where gamma rays have (keV) been identified Beta decay (P,P*) Coulomb excitation 295 X X X 357 X X 490 X 498 X 563 X 611 X 695 710 803 X 843 X 899

5.6 THERMOLUMINESCENCE DOSIMETRY (TLD) - A.R. Jones A random selection of 150 workers is now monitored with TLD rather than photographic film dosimeters in order to obtain operating experience under typical conditions, prior to full-scale use of TLD. A practical method of radiation exposure control is by withdrawing a worker when his quartz film electrometer indicates three quarters of the administrative control limit of 600 mR for a 2-week monitoring period. When using film dosimeters, this procedure occasionally leads to "overexposures", i.e. film dosimeter readings in excess of 600 mR. In the test with TLD's, involving 750 individual exposures, no such overexposures have occurred. This experience bears out the findings of tests of the relative response of film, TLD and quartz film electrometers. An, important advantage of the TLD system was shown in one instance when a quartz film electrometer was found to indicate an off-scale reading. The worker's TLD was recovered and processed within 15 minutes. -49-

Contamination of the TLD's with luminescent particles sensitive to UV light has been found to give spurious results. Black tape is now being placed over the hole at the back of the photobadge to prevent UV exposure and it will also serve to inhibit accumulation of particulate contamination. A rectangular hole will be cut in the photobadge to expose the identity code on the TLD and will enable the user to check that he has been given the correct dosimeter. Measurements have been made, using TLD's, of diagnostic exposures of patients at the Plant Hospital. Using four 0.035" thick LiF TLD's, a standard deviation of about 10% is attainable with doses at the 10 mrad level. Construction of the automatic TLD readers has begun at the Canadian Admiral Corporation Limited with delivery promised in August. Samples of four thousand thick (0.035") and six thousand thin (0.010") TLD's have been checked for uniformity of sensitivity. The thick dosimeters were within the ± 10% specification but some of the thin ones were outside their 15% specification. The sensitivity was well correlated with thickness and weight and the supplier has agreed to take them back and submit them, and the balance of the order, to 100% testing of weight as well as sample testing of sensitivity.

5.7 SEMICONDUCTOR DETECTORS - A.R. Jones RCA Research Laboratories have supplied six sample reach-through diodes which were about twice as sensitive as and more uniform than earlier Samples. They were found to operate at a low voltage, 300 volts, which lies within the plateau for 6 keV radiation. Pulse height analysis of spectra of 6 keV X-rays and 14C beta radiation shows that the detectors may be used for detection but not spectroscopy of these radiations. A further doubling of sensitivity would make the detectors more widely applicable in dosimetry. -50-

Ten diffused pn junction detectors provided by RCA for incorporation into beta meters (AEP-5210) were found to be noisy. Others of the same type and made at about the same time were also noisy. These have been replaced by RCA with satisfactory detectors, but they were unable to find the cause. This type of detector has otherwise behaved reliably over long periods.

5.8 GEIGER COUNTERS - A.R. Jones A study has begun on the use of large area (250 cm2) thin window (23 mg/cm2) counters for hand, foot and laundry monitoring. Compared with photomultiplier tubes, they are easier to shield because of their thin flat shape, and the attendant electronics is simpler. The latter is important because it can reduce construction and maintenance costs. Their counting life is limited because they are quenched with organic vapour but this disadvantage may be overcome by arranging to bias them only while in use. Furthermore, the duty cycle of hand and foot monitors is very low.

A very low sensitivity, short dead time, Geiger counter has been developed by ITT-Standard which can be used in fields of up to 300 R/h with counting loss errors of less than 20%. This performance is about three times better than that of the Philips 18529 counter now used for high range instruments. Although the counter cathode is small the gas reservoir is qu^te large, which should give it a longer life in high fields.

5.9 MOdlTORIUG FOR TRITIUM IN AIR - R.V. Osborne and A.S. Coveart (a) Portable Monitor AEP-5215 Field testing of two instruments continues. (b) Area Monitor AEP-10101 with compensation against noble gases A pair of AEP-10101 chamber assemblies, modified as described in PR-B-92, AECL-4154, are now installed at Douglas Point Generating Station and are operating satisfactorily. (c) Area Monitor AEP-10107 Design of this monitor, outlined in PR-B-92,

-51-

AECL-4154, has continued. Performance of the temperature-stabilised electrometer has so far proved disappointing. Noise at the output in the range 0.01 to 1 Hz is at least 2 fA peak-to-peak with long-term drift (over several hours) an order of magnitude greater. This is too large for use with the proposed 2.7 litre ionization chambers. 5.10 MONITORING FOR TRITIUM IN WATER - R.v. Osborne (a) AEP-5252

One monitor has been commissioned at Pickering Generating Station and the monitor at NPD has been rebuilt to the current design. The main problem remains that of water filtration; weekly filter changes are operational- ly unacceptable. As an alternative method for producing clean samples, continuous distillation followed by condensation is being investigated. Fracture of two of the plastic detector cases has occurred; one, as a result of an over- pressure excursion above the specified limit of 100 kN/m2 (approximately 15 psig), the other because of a poor plastic joint. An alternative aluminum case with gasketed plastic windows has been designed; two are being built.

(b) Liquid Scintillation Methods A method for continuous flow monitoring of water streams using liquid scintillator at less than 0.2 cm3/min (i.e. less than 100 litre/y) : has: been conceived and is being studied in the laboratory. Running costs at this usage rate should be acceptable. Anticipated detection sensitivity1 is 100 nCi/litre in sampled water with1 ia response time of 5 minutes.

5.11 PORTABLE FLUORIMETER - A.S. Coveart ;..••. Ai simple: fluorimeter has been constructed to bes used; by the Environmental Research branch for Jfehei;d^tectioh of fluorescent dyes in flowing M streams;. , Theuxinit is battery-operated and reads fiuor^scence-dir^ctlyj over 4 decades on three :: ,' •,:.,::.v>'.iLa:n%i^Ci ^ahgea^^."iTheXelectrohics consists of a .photdraultiplier detecting the fluorescence -52- followed by a d.c. amplifier and meter. Natural fluorescence can be backed-off to give a reading from essentially a zero baseline. Total weight of the complete instrument is less than 25 lbs. 5.12 HEALTH INSTRUMENTS BEGUN IN THE FIRST QUARTER OF 1972 - A.R. Jones (a) Personal Contamination Monitor A monitor using large area Geiger counters (see section 5.8) is being developed. It comprises a central unit to which one or more hand or shoe units can be attached and contains the high voltage supplies, an audible alarm and a clothing contamination probe. Each hand and foot counted will give a visual alarm and also sounds an audible alarm in the central unit. The contamination probe indicates activity by a flashing light with the count rate shown digitally in the central unit.

The hand unit contains two counters for one hand and a background counter, all surrounded by a one-inch lead shield. The shoe unit will monitor each shoe independent- ly with separate visual indicators and will contain side shielding and background subtraction. The initial specifications follow: - Hand and shoe sections - 0.1 nCi/cm^ of beta contamination detectable in backgrounds up to 2.5 mR/h. Window thickness 23 mg/cm2. Average decision time: 0.4 s. - Clothing contamination probe has a 50 cm^ window, 12 mg/cra2 thick. Count rate scale . is 0-1000 c/s. The circuits and detectors have been tested and the lead shields for the hand monitor built. (b) Hide^ Range Area Monitor _The increased use of accelerators at AECL requires installed gamma dose rate meters with differentviCharaicstefistics than are found in existing instruments^, ^They must? be capable of giving an estimate of djoses to deep-seated organs sfrom high energy ^otpnsa^ olAeryenergetic; particles) Jarid to fthe sk^tn frpm short-range particles and photons. The monitors must be able to work in pulsed radiation fields, -53-

including the special case of single pulses. The design of a monitor to meet this need has begun. The initial specifications require a dose rate indication ->f 1 mrad/h-1000 rad/h in steady or pulsed fields (doses of 3 mrad/pulse or less) . Dose indication from 1 mrad-1000 mrad will also be given. Variable alarm levels for both dose and dose rate are needed. The monitor will comprise three parts. The head unit will contain a detector, a plastic scintillator and six-stage photomultiplier, and an analog-to-digital converter providing one pulse/urad. The scintillator will need a cap whose dimensions will vary according to the penetrating ability of the radiation being monitored. These pulses are fed to either (or both) an analog or a digital unit which may be remote. The analog unit will have two 3-decade ranges (mrad/h or rad/h) and be capable of driving a recorder and activating alarms. The digital unit will display doses from 1-1000 mrad and dose rates from 1 mrad/h to 1000 rad/h with numerical displays. This unit will have alarms on both dose and dose rate and digital output for printing or recording.

(c) Digital Pocket Alarm Following tests on a low sensitivity Geiger counter (see section 5.8), the design of a pocket alarming dosemeter/dose rate meter has begun. It employs a low power consumption high voltage supply and COSMOS digital circuitry, both of which, in low radiation fields, consume negligible power. Because the memory is digital, the dosimeter can be used at very low dose rates (as well as high ones greater than 200 R/h) so that they can be used continuously over long periods (up to 3 months) without resetting. It will also be able to read doses less than that required to sound the alarm. 5.13 HEALTH INSTRUMENTS IN PROGRESS IN THE FIRST QUARTER OF 1972 - A.R. Jones (a) Pocket Warning Dosimeter AEP-5248 The engineering prototype has been built and is under test in the laboratory. -54-

(b) Wide Range Beta Meter AEP-5257 Because of problems in the manufacture of diffused pn junctions (reported in section 5.7) and the promising results obtained with a low sensitivity Geiger counter (see section 5.8) it has been decided to study the possibility of using Geiger counters in place of the semiconductor detector. To cover the same range of beta and gamma ray dose rates, three GM counters are needed, but the costs of the two methods of detection including attendant electronics, are about the same. Energy dependence filters are being designed for the two gamma ray Geiger counters and measurements made on their performance. A perforated filter has proved impracticable for the insensitive counter because of the small dimensions of the cathode (2 x 1.5 mm). Instead, a simple lead filter has been found to work well. For measuring beta dose rates a small end window counter is employed. It is fitted with a perforated 0.5 mm tungsten shield which may permit the measurement of contact and distant beta dose rates. The circuitry for use with Geiger counters is being tested. The dose and dose rate alarm section is similar to that being developed for the digital pocket alarm.

(c) Finger Tip Beta Meter AEP-5265 Mechanical design is in progress. Construction of the engineering prototype has begun. (d) Digital Count Rate Meter AEP-5271 A laboratory prototype has been built. Mech- anical design has begun. ' (e) Foot Cbntarnxnaijon Monitors AEP-2159A and B The> design has been approved for prototype construction. An engineering prototype is being built by the Canadian Admiral Corporation Limited. -55-

5.14 HEALTH INSTRUMENTS COMPLETED IN THE FIRST QUARTER OF 1972 - A.R. Jones

(a) Multichannel Alpha-in-Air Monitor AEP-5266 This has been designed to provide the plutonium production facility with fifteen independent channels of plutonium-in-air monitoring. These have separate alarms which will be connected to a central alarm panel. They are grouped in fives with an additional unit which shows which of the five alarmed first. The range covered is 10 pCi to 10 nCi of alpha activity collected on a filter paper. With a sampling rate of 1 cubic foot/min this allows for the detection of 1 (MPC)a of any alpha activity in less than 8 hours. Except under conditions of high radon daughter concentrations coupled with high humidity, adequate discrimination against radon daughters is obtainable.

(b) Hand Monitors AEP-2158A and B The two versions (with and without background cancellation units) are complete, allowing their production at the Canadian Admiral Corporation Limited for TRRP to proceed. Measurements have been made on the sensitivity of these monitors to 60Co (the beta ray end point energy is 0.3 MeV) . The monitors are set up for 30% efficiency for a 90Sr/90Y source (beta-ray end point energies, 0.5 and 2.2 MeV) and the efficiency for 60co with the existing photomultiplier is 5%. Using an alternative type, which now can be used in place of the existing one, this efficiency can be raised to 11% without encountering noise problems. This was obtained with a sample supplied by the manufacturer and may not be typical.

5.15 MISCELLANEOUS - A.R. Jones (a) Monitoring for 239Pu on Fuel Pencils Engineering design has been supplied with design information for monitoring 500 mm fuel pencils in 15 minutes. The monitor would detect activity at the level of lO1"5 yCi/cm^ and give some information about its location. -56-

Remote Monitoring with the Portable Area Monitor AEP-5233A " ~~~~ The monitor has an internal detector and was not designed for remote operation, except with a remote meter. For use with Slowpoke one monitor has been adapted by placing the detector on the end of a five-foot cable. This is a simple modification and appears to be satisfactory at. least for short cable runs. 5.16 DETECTION OF IODINE IN AIR - R.M. Holford The prototype of the iodine-in-air monitor AEP-5217 has remained in the NRX reactor building for the entire period. It has been operational for much of this time but interpretation of its readings is made difficult by several- sources of interference.

Firstly there is a fairly strong flux of energetic gamma rays from the reactor itself. These can easily penetrate the shielding of the radiation detector in the monitor, but since they are fairly constant in intensity, they can be corrected for by using background subtraction.

Another source of background radiation is short- lived fission products in the air being sampled by the monitor. True gases, such as argon, are not retained by the filter mechanism, but particulate matter is retained and is a significant problem at present. At least one of the isotopes involved, J-PATC, emits gamma rays at almost the same, energy as 131l (360 keV) and therefore the only solution to this problem is the use of a glass-fibre particulate filter in the inlet to the monitor. Unfor- tunately such a filter also traps a fraction of the radioiddine, found experimentally to be about 15%.

Even a particulate filter cannot cure the third problem, that of the other shorter-lived isotopes of; iodine. The significant isotopes appear £o be 1321^ 133i, 134If 135L 82Br andJ$ci5have also been observed, but may be produced ;byj activation rather than by nuclear fission. These isotopes are also hazardous - the; (MPC)a:for i33jvis only 3.3 times that for 131i - so tEataJprJ*he-:detector to give a positive reading for these isotopes is not undesirable. However, if background subtraction is being used to compensate for an external gamma -57-

field, these other radioiodines may actually give negative readings on the monitor and mask the presence of J-JJ-I.

The present system of background subtraction uses part of the gamma spectrum above 800 keV energy. While this is satisfactory for external gamma fields, it is difficult to choose part of the spectrum without including one of the prominent lines of one of the shorter-lived radioiodines. An investigation of the response of the detector to mono-energetic gamma sources of different energy is being undertaken so that its response to more complicated spectra can be predicted and a more satisfactory method of background compensation devised.

5.17 MICROSCOPIC FLYING-SPOT SCANNER - R.M. Holford Several test systems have been devised to permit examination of the parts of the instrument, in order to find the causes of the various drifts, noise signals and non-linearities which are at present limiting its performance. Firstly, by using timing pulses from the instrument and a precision voltage source, controlled by MOSFET switches, a simulator for the video signal has been constructed. This is fed into the logarithmic amplifier in place of the video signal from the photomultiplier and can therefore be used to check the operation of the logarithmic amplifier, the integrator and the digital output. These tests were 'largely satisfactory, although the useful dynamic range of the logarithmic amplifier was found to &e less than expected. Within, this range, the output was found to depend only on £he ratio of the simulated ' signal' and 'background', voltages, and nbt'oh their absolute value.

If the output of the video simulator is used to drive a light-emitting diode, placed near the pKbtpmultiplier, this component and the video amplifier can be included in the test. ; Unfortunately the light output of the diode is apparently not strictly proportional to the input current and this restricts the usefulness of this test system. A final test will be to operate the system as1 a conventional flying-spot scanner with a fairly lai^ge ph^ptographic spot and good light collection. 1 x This test Should reveal the performance; of the -58-

system freed from the limitation imposed by statistical noise. Preliminary checks on this have already shown that noise due to phosphor non-uniformity is also present and will require correction. 5.18 SPECIMEN PREPARATION FOR HEAVY ATOM IMAGING BY PHASE CONTRAST ELECTRON MICROSCOPY - H.M. Johnson (a) Introduction The experimental approach to single atom imaging in the electron microscope was outlined in the previous report PR-B-92, AECL-4154. Mellitic acid (Cl9HgO12) has been used as the organic matrix which can be stained by heavy metal ions such as thorium and uranyl (U02++), attaching to the carboxyl groups (C00~) in a symmetric array. Heavy metal complexes (uranium and thorium) of mellitic acid have been supported on thin carbon films, themselves supported on thick holey carbon films. Since both of these films are important in the microscopy at this level and since their production differs from existing procedures, a description of their preparation is provided here.

(b) Production of a Holey Net Films were formed from a solution of 0.5% (w/v) Formvar (polyvinyl formal) and 0.2% (v/v) heptane in chloroform. A new glass microscope slide was vigorously wiped with lens tissue and placed vertically in 20 m£ of the solution in a 100 m?, cylinder. The slide was permitted to wet (1 min) , was field to drain just above the, solution (1 min), then removed and air-dried-.,'"for five minutes. vAfter s^drin|g the slide'edge witii a razor blade, tne plas^itsLc film was '"£ Ibated of f By obliguely; lowering; this slide into distilled water. Standard 400 mesh microscope grids were placed on the film and then covered with filter paper. The assemblage was removed, after sinking, and air-dried. Each side of the plastic film was coated with a 200 A thick layer of carbon, evaporated from spectroscopic grade graphite (10~4 torr, 15 cm source distance).

(c) Production of Thin Carbon Films Thin carbon overlay films (less than 50 A in thickness) have supported the heavy atom specimens* While other substrates have been tried (e.g. beryllium, -59- bare Pormvar), shattering and tearing of the films was observed and a return has been made to the use of carbon.

These films were produced by the indirect evaporation of spectroscopic grade graphite on freshly cleaved mica. A glass plate (3-1/4" x 4-1/4") was interposed between the evaporation source (resistance- heated graphite rod) and the mica. The best films were formed when no deliberate attempt was made to provide reflections of the evaporated carbon. Evap- orations were carried out in an Edwards, oil diffusion pumped evaporator at 3 x 10~5 torr.

Estimates of film thicknesses were obtained from the measurement of the optical density of a film fragment. This measurement was based on the calib- ration provided by Moretz et_ aJL. (1968) . At high resolution, these thin films have a granularity due to randomly arranged graphite crystallites. It is against such granularity that all electron microscopic images must be interpreted. A support film of reduced granularity is of utmost importance in the field and work is in progress to achieve this. An anomaly in the electron scattering of very thin films, first reported by Johnson and Parsons (1969) has been verified by Williams and Glaeser (1972) in the course of the latters1 exploration of ultra thin carbon films. This indicates that future attempts to model the carbon support film in calculations of image contrast must respect a non-linearity in scattering amplitude for film thicknesses less than fifty angstroms.; Williams and Glaeser (1972) also undertook gas ehromato- graphic,analysis of carbon films prepared in a 'normal evaporator1 and found hydrogen and nitrogen levels below detection, suggesting that the fear of hydrocarbon contamination of thin films formed in 'normal evaporators' is unfounded. It should also be pointed out that the density of evaporated carbon films as determined by Williams and Glaeser (1972) is abnormally large (3.1 g/cnr*) , disagreeing with the value of 1.98 ± 0.03 g/cm3 obtained by Johnson and Parsons (1969) and being inconsistent with the density of graphite (2.25 /3 -60-

(d) Microscopy Electron microscopy has continued using this multilayered specimen. The distribution of tri- uranyl benzene hexacarboxylic acid has been increased to 1013 molecules/cm2. New computer calculations based onethe phase contrast imaging of uranium atoms, 10 A apart as in the mellitic acid complex, have guided the microscopy.

References Johnson, H.M. and Parsons, D.F. (1969), J. Micro- scopy 9_0_, 199. Moretz, R.C., Johnson, H.M. and Parsons, D.F. (1968), J. Appl. Phys. 39_, 5421. Williams, R.C. and Glaeser, R.M. (1972), Science 175, 1000. 5.19 ROUTINE DOSE MONITORING (a) Film Monitoring - R.M. Rondeau In the fourteen-week period ending March 3 for CRNL workers and February 25 for WNRE and Power Projects workers, and in the calendar year to these dates, the following distribution of radiation exposures occurred.

(i) External gamma radiation - CRNL Exposure Fourteen Weeks Calendar Year Range (Roeritgehs) Nbv of Total No. of Total lion i to red Exposure Monitored Exposure Persons Man-R Persons Man-R No exposure 1954 0 1994 0 0.02 - 0.49 470 96.04 500 99.19 0.50 - 0.99 147 104.46 108 72.23 1.00 - 1.49 37 43.16 16 17.76 1.50 - 1.99 9 15.14 1 1.64 2.00 - 2.59 2 4.50 0 0 -61-

External gamma radiation - WNRE

Exposure Fourteen Weeks Calendar Year Range (Roentgens) No. <^f Total No. of Total Monitored Exposure Monitored Exposure Persons Man-R Persons Man-R No exposure 676 0 694 0 0.02 - 0.49 131 21.39 128 20.87 0.50 - 0.99 37 24.78 23 14.54 1.00 - 1.49 1 1.07 0 0

External gamma radiation - Power Projects Exposure Fourteen Weeks Calendar Year Range L (Roentgens) No. of Total No. of Total Monitored Exposure Monitored Exposure Persons Man-R Persons Man-R ! j... i i 185 0 194 0 ' No exposure • 34 5.48 28 2.96 0.02 - 0.49 8 5.96 5 3.44 0.50 - 0.99 1 1.13 1 1.13 1.00 - 1.49 ;

(ii) External beta radiation - CRNL

Exposure Fourteen weeks Calendar year Range Rads No. of Total No. of Total Monitored Exposure Monitored Exposure Persons man-rads Persons man-rads

No dose 2525 0 2543 0 0.02 - 0.49 60 12.78 52 9.69 0.50 - 0.99 19 12.77 16 10.65 1.00 1*49 . 8 9.75 3 3.07 1.,50 1.99 2 3.17 1 1.73 2,,00 2.49 ! 2 4.02 2 4.02 2.50 - 2v99 i 0 0 0 0 3.00 - 3.49 1 3.34 1 3.34 3.50 - 3.99 I 2 7.85 1 3.88 -62-

External beta radiation - WNRE

Exposure Fourteen Weeks Calendar Year Range Rads No. of Total No. of Total Monitored Exposure Monitored Exposure Persons man-rads Persons man-rads No dose 832 0 836 0 0.02 - 0.49 10 2.52 8 2.21 0.50 - 0.99 1 0.71 .0 0 1.00 - 1.49 1 1.45 1 1 .4^) 1.50 - 1.99 1 1.77 0 0 _J . _

The average exposures are therefore:

In fourteen weeks 101 mR y per monitored CRNL worker 56 mR y per monitored WNRE worker 55 mR Y Per monitored PP worker 396 mR Y per exposed CRNL worker 280 mR Y per exposed WNRE worker 292 mR Y per exposed PP worker 571 mrad 3 per exposed CRNL worker 496 mrad 3 per exposed WNRE worker In the calendar year 73 mR Y per monitored CRNL worker 42 mR Y per monitored WNRE worker 33 mR Y per monitored PP worker 305 mR Y per exposed CRNL worker 235 mR Y per exposed WNRE worker 221 mR Y per exposed PP worker 479 mrad 3 per exposed CRNL worker 407 mrad 3 per exposed WNRE worker. In the fourteen-week period ending March 3, 1972, the 600 jnR control limit for two-weekly exposure to gamma radiation was exceeded on 11 occasions and the 1600 mrad control limit for two-weekly exposure to beta radiation was exceeded on 4 occasions by CRNL workers.

In the fourteen-week period ending February 25, 1972, the 600 mR control limit for two-weekly exposure to gamma radiation was exceeded on 4 occasions by Power Projects workers. -63-

(b) Hand Monitoring with Thermoluminescent Dosimeters - L.S. Larkin~~ ~

In the fourteen-week period ending March 11, 1972. and in the calendar year to this date, the following dose distributions occurred.

Dose Range Number of persons Number of persons Rad exposed in 14 weeks exposed in calendar year 0.02 _ 0.49 34 34 0.50 — 1.99 28 28 2.00 - 4.00 8 8 4.00 — 5.00 4 4 5.00 - 6.00 1 1 over 6 .00* 3 3

* 9.85, 13.55 and 6.35.

5.20 PUBLICATIONS A.R. Jones, A Small Alpha Monitor using a Surface Barrier Detector, Health Physics 22, 99-102 (1972); also Atomic Energy of Canada Limited report No. AECL-4000. A.R. Jones, A Personal Dosimeter System Based on Lithium Fluoride TLD's; Proc. Third International Conference on Luminescence Dosimetry, RisjzJ, Denmark, Vol. 2. Also Atomic Energy of Canada Limited report No. AECL-4115. Additional copies of this document may be obtained from Scientific Document Distribution Office Atomic Energy of Canada Limited Chalk River, Ontario, Canada

Price - $2.00 per copy

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