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EXTERNAL AND INTERNAL CONTAMINATION

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1 EXTERNAL AND INTERNAL CONTAMINATION

2 Radioactive sources Radioactive sources are present in the sealed (normally non spreadable) and unsealed form (spreadable): Gammagraphy essentially uses as sources iridium-192 and sometimes cobalt-60 Neutrongraphy uses sources of neutrons like californium-252 or an americium/beryllium couple. Betagraphy uses beta sources like carbon-14. Chemical and biological radiation-treatment uses gamma radiation sources cobalt-60 or caesium-137. Radioactive sources are present in the sealed (normally non spreadable) and unsealed form (spreadable). Industrial applications of sealed sources comprise essentially radiography, radiometric measuring instruments and radiation-treatment. Research laboratories use the majority of available radioactive sources which are associated with unusual radio-elements by function of their specific activity. Non-destructive control sources are used to produce radiographs of materials. Gammagraphy essentially uses as sources iridium 192 and sometimes cobalt 60.The activity of the most usual sources is significant, the order of 3,000 gigabecquerels (GBq). Gammagraphs are widely spread pieces of equipment (there are more than 600 in circulation in France). The source in a grammagraph is a small steel cylinder measuring about 2cm long with a diameter of 5mm. These small dimensions lead to underestimation of the potential dangers that they represent when handled by persons who have not had prior warning of the danger. They can be the source of serious irradiation accidents. Neutrongraphy uses sources of neutrons like californium 252 or an americium/beryllium couple. Neutrongraphy is used to radiograph hydrogen containing materials. Betagraphy, using beta sources like carbon 14, is cited to be remembered. Radiation meters use a radioactive source/detector pair. By measuring the absorption of ionizing radiation by a material, you can measure the value of a parameter. Meters to measure thickness and density exist, the sources of which have an average activity of the order of 30GBq. Humidity meters, for research into very light elements like water or hydrocarbons in geological prospecting use portable neutron sources of the same type as those used in neutrongraphy. Chemical and biological radiation-treatment uses gamma radiation. Chemistry under ionizing radiation employs very strong sources (cobalt 60 or cesium 137), in excess of 37,000 GBq for the treatment of some plastics or for the radio-sterilization of medical products, foodstuffs or cosmetics. These sources must conform to special legislation for their safe use. Sealed sources can also be found in other uses, for example in old lightning conductors, electrostatic charge elimination, or yet still, in some smoke detectors. These sources always have very low activity. Non-sealed sources are not frequently found in industrial sectors. These are essentially used as tracers in hydrological studies, inspection of wear of machine parts, searching for leaks and the manufacture of radio-luminescent objects. In the medical sphere, sealed radioactive sources are used in radiotherapy to carry out treatment in situ (Brach therapy) or remotely (external radiotherapy). In Europe the sources for brachytherapy are iridium or caesium, both being gamma emitters. These sources have activities below 37GBq. For external radiotherapy cobalt 60 has replaced caesium 137. These are physically very small sources (a few centimeters) having major activities above 37,000GBq (several thousand curies).

3 Radioactive sources in medicine
In medicine there are three uses for non-sealed radioactive nuclides: Biological analyses: radio-markers have been replaced progressively by non-radioactive markers. Medical imaging: nuclear medicine department use radio-pharmaceuticals for diagnostics, which are ingested by the patient to obtain an image of the tissue or organ while it is functioning. Therapy: radio-pharmaceuticals can constitute the treatment itself, for example iodine 131 for the treatment of thyroid cancer. In medicine there are three uses for non-sealed radio-elements. Biological analyses: radio-markers have been replaced progressively by non-radioactive markers. The radio-elements used are very varied. The quantities generally employed are small to limit the risks. Medical imaging: nuclear medicine department use radio-pharmaceuticals for diagnostics, which are ingested by the patient to obtain an image of the tissue or organ while it is functioning. Therapy: radio-pharmaceuticals can constitute the treatment itself, for example iodine 131 for the treatment of thyroid cancer. The quantities of radio-elements in the non-sealed form employed by the nuclear medicine service can be significant: to treat thyroid cancer you use about 3.7 GBq per patient.

4 Contamination risk The probability of external and internal radioactive contamination has increased with the use of radionuclides in research projects, medical applications, industrial processes and nuclear power plants. Of about 400 recorded radiation accidents, there were only a few with significant radioactive contamination of persons. Chernobyl in 1986 and Goiania in 1987 are the most important among them. Both these accidents resulted in a few dozen victims who required internal and external decontamination. Management strategies require knowledge of the physical and chemical characteristics of the radionuclides, their metabolism in humans, and the methods of accelerating their elimination from the body. Treatment should be a team effort that includes physicians, health physicists, analytical chemists, toxicologists, and dosimetry specialists.

5 Contamination sources
reactor accidents involving damage to the core leading to combined inhalation-peroral entry into the body of a mixture of fission products (primarily of volatile caesium and iodine isotopes) accidental violation of the regulations or procedures governing work with radioactive substances, especially in form of powder or solution  accidents involving leaking or damaged sealed sources (i.e. 192Ir, 137Cs, 60Co, 226Ra)  errors in dosage of radionuclides used for diagnostic and therapeutic purposes accidents involving the fabrication and reprocessing of nuclear fuels and the transportation and disposal of radioactive waste External and internal can arise from a number of circumstances, such as: reactor accidents involving damage to the core leading to combined inhalation-peroral entry into the body of a mixture of fission products (primarily of volatile caesium and iodine isotopes). Contamination of workers and members of the public with radionuclides may occur due to: accidental violation of the regulations or procedures governing work with radioactive substances, especially in form of powder or solution  accidents involving leaking or damaged sealed sources (i.e. 192Ir, 137Cs, 60Co, 226Ra)  errors in dosage of radionuclides used for diagnostic and therapeutic purposes  accidents involving the fabrication and reprocessing of nuclear fuels and the transportation and disposal of radioactive waste

6 Chernobyl accidents There were two reactor accidents which are known to have caused measurable contamination of the public with radionuclides: (1) the Windscale reactor accident in the UK in October and (2) the Chernobyl nuclear power plant accident in the Soviet Union in April 1986.

7 Goiania accident Area of contamination – 4 000 000 m2
The Goiânia radiological accident resulted in the highest level of 137Cs internal contamination ever recorded (2-3 GBq). There were 249 contaminated persons, all members of the general public, 129 with internal contamination. In one case, doses from external and internal exposure were similar and the internal dose played a significant role in the victims' death. In 1985, a radiotherapy clinic, built at Goiânia in Brazil, moved to a new location. Without referring to the authorities it left behind a piece of radiotherapy equipment loaded with a caesium 137 source. On 13 September 1987 two people entered the disused clinic and removed the cask containing the source. Once they had returned home, they tried for two to three hours to dismantle it, but had to abandon their task because they were overcome by nausea and vomiting and, for one of them, diarrhoea. This syndrome lasted 4 to 5 days. On 15 September one of the two men saw a doctor for oedema of the hand, vertigo and a digestive syndrome. The doctor attributed the symptoms to an allergic reaction related to food poisoning. On 18 September the two men tried again to dismantle the cask and, in so doing, broke the window of the source (the window having a thickness of one millimetre). They took a powder out of the casket. After they succeeded in separating the door, which was a protection enclosure, they decided to sell the fragments to a scrap merchant who lived in the neighbourhood. In the evening the scrap merchant noticed a bluish light escaping from the source enclosure. He decided then to take the object back home, thinking it was precious. During the course of the following three days the whole neighbourhood came to see and touch the “magic” object. In the meantime, part of the source in the form of particles, with the size of grains of rice and which turned to powder when handled, were distributed to his relatives and friends. Several friends covered their skin with this sparkling substance which resembled that used in carnivals. On 21 September the wife of the scrap merchant presented with a gastro-intestinal syndrome labelled “food poisoning”. Her mother came to care for her and returned home with the powder. Over several days the number of people exposed and contaminated continued to grow. Meanwhile, one of the two subjects who had dismantled the source was hospitalised for severe cutaneous lesions in a department specialising in tropical sicknesses. On 28 September, that is to say a fortnight after the theft of the source the scrap merchant’s wife, who was convinced that the shiny powder was responsible for her and her friends’ sickness, went to the “vigilancia sanitaria” with the remains of the source. She explained to a doctor that her family was in the process of dying because of what she had in her bag and gave it to him. She was hospitalised in the tropical sicknesses department where she found that a certain number of her relatives and friends were there already. Examination of the objects discovered very significant radioactivity: the dose rate measured with a detector mounted on a telescopic arm showed 0.4 Gy/hr at 1 metre distance. Calculations enabled assessment of the quantity of caesium still present in the source to be less than 10% of the initial activity, which meant that some 45 TBq (1012 Bq), ie. more than 1,000 curies ( ten or so grams of caesium) had contaminated the people and the environment. Confronted with this veritable ecological and health catastrophe the authorities sealed and evacuated the most contaminated zones on 29 September basing their actions on the personal stories of the people directly implicated and on summary detection. The people were grouped together in a tent at the neighbouring Olympic stadium to carry out radiological and medical triage and to avoid spread of the contamination. The press began to take an interest in the event. On 30 September the population of the town was worried and gathered at the entrance to the Olympic stadium to be examined. In total 112,800 people were examined. This accident, which was sparsely covered by the media at the time, has had major consequences: 249 contaminated people (120 contaminated only on their clothing, 129 externally and internally contaminated), 20 people who had been hospitalised because of their clinical condition, 4 deaths (of which the scrap merchant’s wife was one), 85 contaminated houses, of which 7 had to be demolished, 45 public places (pavements, squares, shops, bars) were decontaminated. A temporary site for the storage of waste had to be established to dispose of the 3,500 m3 of radioactive waste products removed at the time of the restoration. Collective anxiety was expressed about management of the crisis and there was significant disorganisation of the economy. Today the Goiânia region is still suspect in the Brazilian collective unconscious. Area of contamination – m2 249 contaminated (137Cs) persons, 129 with internal contamination, 4 deaths

8 Contamination sources in nuclear accidents
In the immediate vicinity of a nuclear accident (the near field), exposure could begin immediately if the released plume is at a low level. Main route of exposure is inhalation. Further away from the site of the accident (the far field), the main route of exposure would be ingestion of contaminated food and drink, particularly milk. Exposure by these routes could last longer, cover a larger area, and affect a larger population than exposure in the near field.

9 External radionuclide contamination
External contamination: radioactive materials in form of dust, solid particles, aerosols or liquid, become attached to skin or clothes External contamination occurs when a radioactive material, in the form of dust, solid particles, aerosols or liquid, becomes physically attached to skin or clothes. Considerable contamination of clothing can occur during an accident or during the immediate cleanup. Wet clothing impregnated with radioactive substances brings the contaminant into more intimate skin contact, thereby increasing the skin dose, as was experienced in the Chernobyl accident. Beta emitting radionuclides are the most hazardous, and can cause serious burns of the skin and underlying tissue.

10 External contamination
The risks linked to external exposure of the skin differ according to the type of radiation: alpha emitting radio-elements do not a priori present any risk by external contamination, beta emitting radio-elements do present a special risk because they entail exposure which is almost exclusively of the skin, gamma emitting radio-elements pose the same problems by external contamination as in external exposure, external contamination by a neutron emitting radio-element is impossible. External contamination reveals a secondary potential risk of internal contamination by inhalation, ingestion or breaking and penetration of the skin. External contamination Every deposition of radio-elements on the skin or its secondary features (hair, nails, etc), following fall-out or direct contact with radio-elements from a non sealed source, constitutes external contamination or external skin exposure. Consecutive irradiation is continuous when external decontamination has not been carried out, even when the person is no longer exposed to the source of contamination. The risks linked to external exposure of the skin differ according to the type of radiation: alpha emitting radio-elements do not a priori present any risk by external contamination, beta emitting radio-elements do present a special risk because they entail exposure which is almost exclusively of the skin, gamma emitting radio-elements pose the same problems by external contamination as in external exposure, external contamination by a neutron emitting radio-element is impossible. External contamination reveals a secondary potential risk of internal contamination by inhalation, ingestion or breaking and penetration of the skin.

11 People involved in the Chernobyl accident in 1986
The Chernobyl accident, 26 April 1986, involved the very serious irradiation and contamination of a number of people working in the power station and those who immediately intervened, it provides a good example of external contamination. More than 200 patients were hospitalized in the hours following the catastrophe. Before this massive flood of victims, the initial efforts to manage the situation were limited to treatment of the symptoms a full assessment of lesions, treatment of traumatic lesions and summary external decontamination. The latter fact was revealed to be particularly damaging as all the patients had external contamination by fission products, such as Cs-137, Sr-90, or I-131, all of them being beta and some gamma emitters. Beta emitters on the skin caused severe radiological burns with complex development. It is estimated that 5 of the 28 premature deaths following the accident were attributable in part to radiological burns. These 5 deaths, and perhaps others, could doubtless have been avoided if good external decontamination of all the exposed subjects had been carried out in the shortest time. People involved in the Chernobyl accident The Chernobyl accident, 26 April 1986, involved the very serious irradiation and contamination of a number of people working in the power station and those who immediately intervened, it provides a good example of external contamination. More than 200 patients were hospitalized in the hours following the catastrophe. Before this massive flood of victims, the initial efforts to manage the situation were limited to treatment of the symptoms (anti-emetics, sedatives and potassium iodide), a full assessment of lesions, treatment of traumatic lesions and summary external decontamination. The latter fact was revealed to be particularly damaging as all the patients had external contamination by fission products, such as caesium 137, strontium 90, or iodine 131, all of them being beta and some gamma emitters. Beta emitters on the skin caused severe radiological burns with complex development. It is estimated that 5 of the 28 premature deaths following the accident were attributable in part to radiological burns. These 5 deaths, and perhaps others, could doubtless have been avoided if good external decontamination of all the exposed subjects had been carried out in the shortest time.

12 External contamination triage
Triage following known or suspected radiation accidents includes both medical and radiological considerations Medical triage should be based upon local procedures for medical management of persons involved in accidents with radioactive materials and on considerations dependent on the severity of injuries. Treatment in life threatening conditions has priority over considerations for exposure to or contamination with radioactive materials. Following medical stabilization of the patient’s condition, careful radiological assessment can be directed to determining the presence of both external and internal contamination. Triage following known or suspected radiation accidents includes both medical and radiological considerations. Medical triage should be based upon local procedures for medical management of persons involved in accidents with radioactive materials and on considerations dependent on the severity of injuries. Treatment in life threatening conditions has priority over considerations for exposure to or contamination with radioactive materials. Following medical stabilization of the patient’s condition, careful radiological assessment can be directed to determining the presence of both external and internal contamination.

13 External contamination measurement
Proper monitoring of patient can detect and measure alpha, beta or gamma emitters; radiation type depends on isotope in contaminant There are a number of survey instruments available for beta-gamma detection and measurement. The great majority of instrument probes allow for discrimination between beta and gamma radiation and levels should be recorded both separately and as a total count. It is important to remember that some low energy beta emitters exist and special probes are required for their detection. Thus, the assistance of the experienced health or medical physicist is important. Alpha particles travel only a few centimetres in air and up to 40 micrometers in tissue and as such cannot penetrate the cornified epithelium. The low penetrating power of the alpha particles, therefore, dictates that alpha surveys must be accomplished with thin window probes, minimal absorbing material between the detector and source, and a limited physical distance between the probe and the surface monitored. Alpha monitor

14 Radiological survey Initial external contamination survey (of skin, eyes, lips) should be made with instruments adequate for the particular situation As soon as possible after an accident has occurred an initial external contamination survey (of skin, eyes, lips) should be made with instruments adequate for the particular situation. If there is any doubt regarding the radioactive materials involved, both a beta-gamma and alpha monitor should be used. The results of the radiological survey should be recorded on an anatomical chart and made a part of the patient's medical record. The probe can be held about one inch from the surface monitored and may be covered with a plastic or rubber glove to prevent contamination of the probe and subsequent false readings. If the probe cover becomes contaminated (evidenced by false increase in background) it should be changed. The results of all surveys should be recorded on anatomical charts (including clear personal data, date and time). The results of the radiological survey should be recorded on an anatomical chart and made a part of the patient's medical record

15 Quick `frisk’ 112 000 persons monitored in Goiania at olympic stadium
A slow, thorough survey of the entire body should permit radiological triage yielding two groups. One group consists of individuals with no detectable external contamination, and the other group with radioactive contamination of skin, hair or wounds. Survey should include techniques and instrumentation for detecting alpha, beta, and gamma radiations or any combination thereof. If large numbers of persons require radiological survey, such as occurred in the Goiânia (Brazil) 137Cs accident, a quick `frisk' may be initially used instead of a total body survey. The 'frisk' should include the hands, feet (and shoes), face and hairy top of the head. Quick frisks must always be followed by a more complete body survey. Individuals found to be externally contaminated should have their clothing removed, shower, dry, and then be resurveyed. Once decontaminated to the extent possible, clean clothing should be made available. Any wounds must be considered contaminated unless proven otherwise. Some of them may result in incorporation of radioactive material in the body. persons monitored in Goiania at olympic stadium

16 Decontamınatıon Skin decontamination should be undertaken to decrease the risk of skin beta burns, to lower the risk of internal contamination of the patient, and to reduce the potential of contaminating caretakers and the environment. After the patient's clothing is removed, showering or washing the patient with detergent and lukewarm water is 95 per cent effective. Soap and detergents emulsify and dissolve contamination. Gentle brushing or the use of an abrasive soap or abrasive granules may be indicated. They physically dislodge some contamination held by skin protein, or remove a portion of the horny layer of the skin. Addition of a chelating agent helps by binding the contaminant in a complex as it is freed from the skin. Keep in mind that the stratum corneum of the epithelium is replaced every days. Thus, contamination that is not removed and is not absorbed by the body will be sloughed within one-two weeks.

17 Decontamination techniques
Many cases of skin decontamination will be performed by non-medical personnel at or near the accident scene. When initial cleansing methods are not effective, the patient should be referred to a physician. The physician's decision on decontamination procedures should be based on an understanding of the physical and biological principles involved. Success in achieving the objectives stated above requires thoughtful appraisal of the level of residual contamination, rate of decontamination, and condition of the skin - factors that change as the cleansing procedures proceed.  The area of skin contaminated must be clearly determined using appropriate monitoring techniques. After monitoring, this should be covered to prevent spread of contamination. Contaminated skin on the head or near any body orifice is decontaminated followed by other areas. In the latter case, areas of highest contamination have first priority. Swipes or swabs of contaminated areas should be taken for analysis to determine amounts and identity of radionuclides, and decontamination effectiveness. Strict contamination control procedures should be used in handling these skin swipes/swabs.

18 Decontamination procedures
Start with gentle stream of warm water Use mechanical action of flushing and/or friction of cloth, sponge or soft brush For showering, begin with the head and proceed to the feet Keep materials out of eyes, nose, mouth and wounds Use waterproof draping to limit spread Cover uncontaminated area with plastic sheet and tape edges Begin with the least aggressive techniques and the mildest agents and progress to more aggressive ones. Whatever the procedure, take care to limit mechanical and chemical or thermal irritation of the skin. Aggressive rubbing or shaving should be avoided since they tend to cause abrasion and erythema and can lead to internal contamination. Complete decontamination is not always possible because some radioactive material can remain fixed on the skin surface. Decontamination should be only as thorough as practical. The decontamination procedure stops when the radioactivity level cannot be reduced any more.

19 Decontamination techniques and control
Use single inward movements or circular motion Then rinse area with tepid water and gently dry using the same motions After drying, premonitory skin to determine effectiveness of decontamination Decontamination can be accomplished by moving from the outer edge of a contaminated area toward the centre. Single inward movements can be used or a circular motion with increasingly smaller circles. The process may be repeated 2 or more times using a clean, soft surgical brush, 4 x 4 gauze pads, sanitary napkins, etc., each time. Afterwards, the area should be rinsed with tepid water and gently dried using the same motions working inward. After drying, the skin should be remonitored to determine the effectiveness of decontamination. If two or three washing/drying procedures are used (especially if aggressive techniques are used) and if the contamination level is not decreased, stop the procedure and seek expert consultation. Likewise, if hypaeremia or irritation appears, stop the procedure and seek expert consultation. It should be recognized that the stronger agents are normally used after a certain amount of skin irritation has already occurred. It is a common mistake to underestimate the potential for skin irritation until too late. Particular care should be taken on the more sensitive and thin skin areas, and also areas of skin grafts or skin donor sites.

20 Decontamination procedures: body orifices
Consideration: Orifices need special attention because absorption of radioactive material more rapid than through skin Procedures: Oral cavity: brush teeth with toothpaste, frequently rinse mouth with 3% citric acid Pharyngeal region: gargle with 3% H2O2 Swallowed radioactive materials: gastric lavage Nose: rinse with tap water or physiological saline Mouth Nostrils

21 Decontamination procedures: body orifices
Eyes: rinse by directing stream of water or physiological saline from inner to outer canthus while avoiding contamination of nasolacrimal gland Ears: - rinse externally with water - rinse auditory canal using ear syringe Eyes Ears

22 Useful therapeutic agents for skin decontamination
Common soap or detergent solution for skin and hair; low acidity (pH ~5) recommended Chelating agents: solution of EDTA 10% for skin or hair contamination with transuranium, rare earth and transition metals DTPA 1% in aqueous acid solution (pH ~4) for washing skin after contamination with transuranics, lanthanides or metals (cobalt, iron, zinc, manganese) Soaps and detergents can emulsify and dissolve contamination and are frequently all that are needed for decontamination of skin. Abrasive soap or abrasive granules (i.e. cornmeal) dislodge some contamination physically held by skin protein or remove a portion of the horny layer. In any case, tepid water (never hot) should be used for decontamination. Chelating agents, sodium hypochlorite, and titanium dioxide (an abrasive) have been successfully used in previous contamination accidents. The abrasive agents must not be used on the face. While additional chemical techniques are seldom necessary, they can be used but only with care and expert advice.

23 Useful therapeutic agents for skin decontamination
Potassium permanganate 5% aqueous solution should be used carefully not recommended for face, natural orifices and genital regions use when conventional washing ineffective follow with application of reducing agent, then rinse with water Hydroxylamine or sodium hyposulfite 5% freshly prepared aqueous solutions reducing agents - apply after KMn04 or Lugol, then wash with water Potassium permanganate, an oxidizing agent, and subsequent application of sodium acid sulphite to neutralize the permanganate, will remove a portion of the horny layer. In cases where contamination is localized in thick, horny areas more aggressive methods can be used but again with expert advice. Fine sandpaper can be used to remove localized contamination. Dermabrasion should be used with great care and only after expert consultation. Sticky tapes have limited usefulness since they tend to remove the horny layer rapidly and can increase percutaneous absorption

24 Useful therapeutic agents for skin decontamination
Antiphlogistic topical ointment: To be applied for fixed contamination, especially useful for contamination of fingers Isotonic saline solution for eyes Isotonic 1.4% bicarbonate solution for removing uranium from body Lugol solutions for iodine contamination Acetic acid solution (pH 4 to 5) or simply vinegar for decontamination of 32P Antiphlogistic topical ointment: To be applied for fixed contamination; must be kept hours under an occlusive dressing; creates an osmotic gradient which pumps the radionuclide from the skin towards the outside; especially useful for finger contamination. Isotonic saline solution for eyes. Isotonic 1.4% bicarbonate solution for removing uranium contamination. Lugol solutions (50 mg iodine and 100 mg potassium iodide per millilitre) for decontamination of radioactive iodine compounds from the skin. It has to be followed by application of sodium hyposulphite and then rinsed with water. Acetic acid solution (pH 4 to 5) or simply vinegar for contamination by 32P; wash and then rinse with water.

25 Internal contamination
Occurs when people ingest, inhale or are injured by radioactive material. The most frequent points of entry are by inhalation and wounds. Metabolism of non-radioactive analogue determines radionuclide’s metabolic pathway. Radionuclides obey the same principles of toxicity as do non-radioactive toxins. Basically, toxins are absorbed into the body and then distributed throughout it or, for some chemicals, concentrated in critical organs. A toxicant may undergo metabolic changes in the liver or in target organs and become more active or less active metabolites. Ultimately, the toxicant is eliminated through the body's excretory mechanisms. Internalization consists of intake, distribution, and metabolism.

26 Paints for luminous watch faces
In the 1920s and 1930s the clock making industry used radium 226 and 228 in radio-luminescent paint for watches. At this time, the risk from alpha emitting radio-elements was almost unknown. The workers who painted the luminous faces had the bad habit of tapering their brushes with their lips. Every time they did this they ingested several becquerels of radium. The fact that radium and calcium are chemical homologues, resulted in rare bone cancers appearing starting from the 20s, in the form of carcinoma of the sinus of the face. An epidemiological enquiry demonstrated the link between exposure to radium and the risk of bone cancer in 2,403 workers, whose ingestion of quantities of radium could be evaluated. 64 were suffering from osteosarcoma whereas 2 cases of this type of cancer would have been expected statistically. Paints for luminous watch faces This unfortunate experience illustrates well the risk of internal contamination . In the 1920s and 1930s the clock making industry used radium 226 and 228 in radio-luminescent paint for watches. At this time, the risk from alpha emitting radio-elements was almost unknown. The workers who painted the luminous faces had the bad habit of tapering their brushes with their lips. Every time they did this they ingested several becquerels of radium. The fact that radium and calcium are chemical homologues, resulted in rare bone cancers appearing starting from the 20s, in the form of carcinoma of the sinus of the face. An epidemiological enquiry demonstrated the link between exposure to radium and the risk of bone cancer in 2,403 workers, whose ingestion of quantities of radium could be evaluated. 64 were suffering from osteosarcoma whereas 2 cases of this type of cancer would have been expected statistically.

27 Extent of hazard Factors determining extent of contamination hazard:
Amount of radionuclides Energy and type of radiation Biological and radiological half-life Critical organ Chemical and physical properties of radionuclide The extent of a contamination hazard is determined by a number of factors: Amount of radionuclide Energy and type of radiation Duration of external/internal exposure Effect on critical organs: - Some internalized chemicals, including the radionuclide sodium-25, are evenly distributed throughout the human body. - Others have an affinity for critical organs, or target organs, where they concentrate. For example, the critical organ for lead is bone. Although lead is initially distributed in soft tissue organs, including the kidneys, liver, and red blood cells, it is redistributed to bone where it forms long lasting compounds and is slowly eliminated. - The critical organ for the iiodine isotopes is the thyroid gland and, for the uranium isotope, the critical organs are kidneys, liver and bone. Chemical makeup of the radionuclide: Heavy metals like uranium are toxicc for the kidneys and liver. Different radioactive isotopes and chemical compounds of uranium have different toxicity.

28 Intake routes In order of decreasing frequency, contaminants enter the body by four principle routes: Inhalation: Particularly likely with explosion or fire Particle characteristics important (size, chemical composition, solubility in body fluids) Ingestion: Critical for general public after accidental environmental release Wound contamination Absorption The usual routes for internal contamination are through inhalation, ingestion, and wounds but it may also occur by percutaneous absorption. Mechanical, chemical, or thermal injury to the skin during decontamination may also lead to internal contamination.

29 Inhalation Fate of inhaled particles dependent on physicochemical characteristics Soluble particles (3H, 32P, 137Cs) absorbed directly into circulatory system Insoluble particles (Co, U, Ru, Pu, Am) are cleared by lymphatic system or by mucociliary apparatus above alveolar level. Most secretions reaching pharynx swallowed, enter gastrointestinal system The inhalation pathway is the primary intake route for radioactive contamination. It is particularly likely when an explosion or fire occurs. Gaseous material or particulate matter may be inhaled and subsequently be absorbed or deposited throughout the respiratory tract. After deposition, absorption will depend on the chemical solubility of the contaminant. Soluble particles will be absorbed either into the blood stream directly or pass through the lymphatic system with ultimate movement into the circulatory system. Examples of such radionuclides are 3H, 32P, 137Cs, radioiodines and some chemical forms of 90Sr. Insoluble particles are transferred from the site of deposition by phagocytosis (lymphatic circulation) and cell migration.The mucociliary apparatus will clear insoluble particles that are deposited within the respiratory tract above the alveolar level. Secretions containing the particles that reach the pharynx, are subsequently swallowed and enter the gastrointestinal system. Insoluble particles are usually metallic oxides or metals. Examples are the oxides of Co, U, Ru, Pu Am.

30 Deposition and clearance from respiratory tract
Contaminant's particle size determines deposition in respiratory tract Particles <5 microns in diameter may reach alveolar area Particles >10 microns too large to pass into alveoli, deposited in upper airways Insoluble particles, until cleared from the respiratory tract, will continue to irradiate surrounding tissue. Large particles deposited in the upper bronchi will be cleared from the respiratory tract within one hour. Clearance of insoluble particles from the alveolar spaces may take up to 1500 days. In the alveoli, fibrosis and scarring are more likely to occur due to the localized inflammatory response to foreign bodies.

31 Pulmonary effects Irradiated lung tissue Pulmonary fibrosis
Radiation doses in the Gy also produce potentially life threatening pulmonary effects of respiratory insufficiency and pneumonitis, which will be seen days after exposure. Pneumonitis is likely caused by a complex of factors, including breakdown of vascular permeability, fluid imbalance, free radical tissue interactions, infectious agent, biological and chemical toxin damage, and inhalation injury from heat, smoke, and fumes. Irradiated lung tissue Pulmonary fibrosis

32 Ingestion All swallowed radioactive material enters digestive tract
primarily from contaminated food and water secondarily from respiratory tract Absorption from the gastrointestinal tract depends on chemical make-up and solubility of contaminant Ingestion of radionuclides is uncommon in industrial facilities or laboratories. However, it could be of importance for the general public. A striking example is the accidental ingestion of radiocaesium from a dismantled radiation therapy source by persons in Goiânia, Brazil. For large scale radiation accidents involving nuclear power reactors, the possibilities of ingesting milk or vegetables contaminated with radioiodine and meat, milk and vegetables contaminated with radiocaesium are of concern. All swallowed radioactive material, whether primarily from contaminated food and water, or secondarily from the respiratory tract, will enter the digestive tract, and will be handled like any other element. Absorption from the gastrointestinal tract depends on the chemical make-up of the contaminant and its solubility.

33 Parameters of ingestion
Gastrointestinal absorption < 10 % for most elements Elements of high absorption: radium (20%) strontium (30%) tritium (100%) iodine (100%) caesium (100%) Radioiodine and caesium are rapidly absorbed; plutonium, radium and strontium are not. The GI tract is considered the target organ for ingested insoluble radionuclides. The large intestine receives the greatest radiation exposure due to its slower transit time. The clearance time for the gastrointestinal tract is approximately 24 hours for individuals who maintain a high fibre diet. Individuals on a low fibre diet have a slower transit time, which may extend up to 5 days. Even in these individuals, insoluble alpha particle emitters will not cause significant injury because the exposure time within the critical organ is relatively short.

34 Any wound considered contaminated until proven otherwise
Wound contamination Any wound considered contaminated until proven otherwise Any wound must be considered contaminated until proven otherwise. All wounds must therefore be meticulously debrided and evaluated for the presence of radioactive contaminants when they occur in a radiological environment. Solubility, pH, tissue reactivity and particle size are factors that determine the speed of absorption from a wound. The higher the solubility, the faster the absorption rate. Insoluble particles of smaller size may be cleared by phagocytosis and enter the lymphatic system. If the contaminate is highly acidic, local tissue coagulation will occur decreasing the dispersion rate. For example, human wounds that contain depleted uranium may develop cystic lesions that decrease the uranium absorption rate. This has been demonstrated in Gulf War veterans wounded by depleted uranium shrapnel. Studies in these veterans and in animal models have demonstrated that uranium from these wounds is slowly distributed to the liver and kidneys. Open fracture demonstrates wound contamination with depleted uranium shrapnel

35 Percutaneous absorption
Generally, radionuclides do not cross intact skin, so uptake by this route does not occur Most important exceptions are: tritium, iodine, caesium Skin wounds, including acid burns, abrasive scrabbing, create portal for particulate contamination to subcutaneous tissue, bypassing epithelial barrier Skin absorption occurs primarily through passive diffusion. The physical barrier of the epithelium is resistant to particulate matter. Very few radionuclides can penetrate the skin to any appreciable degree. Tritiated water is the primary exception to this since, like any other water, it can pass readily through the skin. All substances have a skin permeability rate dependent on their relative solubility in both lipids and water. Care must be taken during decontamination, as skin that has been mechanically damaged by repeated abrasive scrubbing will allow for greater absorption. Skin that has been exposed to certain chemicals, for example, dimethyl sulphoxide (DMSO), will also be more permeable. Skin wounds, including acid burns, create a portal for any particulate contamination to come into direct contact with the subcutaneous tissue, bypassing the epithelial barrier.

36 Distribution and deposition
Iodine Once a radionuclide is absorbed, it crosses capillary membranes through passive and active diffusion mechanisms and is then distributed throughout the body. Radionuclides follow the same pharmacokinetics as do stable (non-radioactive) chemicals. For example, Sodium-25 (25Na) is evenly distributed throughout the body after resorption while iodine-131 (131I) has a strong affinity for the thyroid gland, the critical organ for iodine deposition. Uranium's primary sites for deposition are the liver and bones. The rate of distribution to each organ is related to the metabolism of the organ and the affinity of the radionuclide for naturally occurring chemicals within the organ. The liver, kidney, adipose tissue and bone have higher capacities for binding chemicals due to their high protein and lipid make-up. Uranium

37 Metabolism A radionuclide will be metabolized according to its chemical properties and will be excreted either in its original state, or as a metabolite. The biological half-life of a radionuclide is as important as its radiological half-life in determining the significance of the exposure. Most ingested heavy metal nuclides will pass through the gastrointestinal tract unchanged. Excretion is determined by the relative solubility of the compound and its delivery to the appropriate organ. Most elimination of absorbed nuclides will occur through the urinary tract. Other primary routes of excretion are through the liver and lungs. Minor routes of elimination include sweat, saliva or milk. In general, compounds that are water soluble are excreted through the urine, while lipid soluble compounds are secreted via the bile into the intestine. There is a lot of variability in elimination, depending on individual factors, including bowel motility, metabolism and diet. Diagram of intake, metabolism and excretion of radionuclides

38 Internal contamination measurement: direct methods
A number of methods are used to detect contamination and to estimate its extent. The methodology for assessing intake will depend on the systemic target organs or tissue, radionuclide emissions and excretion functions. For instance: the assessment of 3H, 32P, 90Sr which are pure beta emitters is possible from urine analysis. 131I (in the thyroid) or 137Cs (whole body) can be assessed in-vivo by measuring their gamma-ray emission. Direct contamination measurement Direct measurement methods include total body and partial body counters that also detect skin and wound contamination. The advantage of direct measurement is that it does not require the use of excretory rates for estimation of contamination. A major disadvantage is that measurements are influenced by external contamination and background radiation levels. Total and partial body counters measure beta and gamma radiation powerful enough to reach the body surface. Partial body counters are used for chest and thyroid measurements. Chest counters detect respiratory tract levels of contaminants such as plutonium and uranium. Alpha contamination measurement usually relies on detection of the gamma/beta radioactivity of daughter products or other contaminants. Whole body counters Thyroid uptake system

39 Indirect contamination measurement
Indirect measurement of contamination includes nasal swipes to determine respiratory intake of radioactive aerosols, and also urine and faeces sampling to establish internal contamination Alpha and beta emitters, the most hazardous internal contaminants, detected through bioassay sampling Accurate bioassays require carefully executed sampling over time and knowledge of type and time of contamination Skin swipes and nasal swipes are used to estimate the extent and type of contamination . Nasal swipes are taken bilaterally, using moistened, cotton-tipped applicators to swab the nostrils. The swabs are then placed individually in test tubes or envelopes, which are labelled with the subject's name and the sample collection time and date. The swipes are sent to a facility with a laboratory counter where contamination can be measured. Detection of radioactive material in both nostrils indicates respiratory inhalation. Unilateral contamination usually indicates surface contamination only. Bioassay sampling of urine and faeces provide indirect measurement of intake of radioactive materials to the body. Radioactivity and concentration of the nuclide in urine and faeces depends on individual metabolic and clearance rates. Extrapolation curves to estimate internal contamination are based on average human metabolic and clearance rates.

40 Bioassay sampling Bioassay sampling and excretion data are the principal methods of determining the presence of alpha and pure beta emitters, which are the most hazardous internal contaminants. Initial samples to be used to establish baseline levels of urine and faecal radioactivity should be obtained from a patient as soon as the medical condition allows. Measures should be taken to avoid accidental contamination of samples. For example, contaminated clothing from the victim should be removed and initial skin decontamination steps should be accomplished before sampling, and gloves should be worn by all personnel handling capture containers. Bioassay accuracy depends on baseline levels, multiple post-exposure samples, and knowledge of the precise time of contamination and of the type of contaminant. The activity in the urine represents the absorbed portion of the radionuclides. The activity in the faeces represents: ingested and unabsorbed material the unabsorbed portion of radionuclides physically cleared from the nasopharynx or the tracheobronchial system radionuclides cleared from the body via bile and gastrointestinal secretion.

41 Managment of internal contamination: First Action
Life threatening conditions have priority over considerations of radioactive exposure or contamination. Attention to vital functions and control of haemorrhage take priority Contamination levels almost never serious hazard to personnel for time required to perform lifesaving measures and decontamination When significant levels of radioactive materials are incorporated, pathological consequences may result and make emergency treatment particularly important. However, this must never take priority over treatment of life threatening conditions and of acute injuries. Following medical stabilization, careful radiological assessment can be performed to determine the presence of both external and internal contamination. It is important to note: Contaminated patients do not represent a direct hazard to health care providers. Lifesaving procedures should never be delayed regardless of the level of contamination.

42 Treatment of internal contamination
Treatment procedures: the sooner started – the more effective In practice, initial treatment decisions based on accident history rather than careful dose estimates Early recognition of internal contamination provides the greatest opportunity to remove the contaminant and reduces the potential for harm. Committed dose is the time integral of the dose rate, which decreases as radioactivity diminishes. If the therapeutic action is performed promptly, the benefit in reducing the dose may be significant. In practice, the critical initial treatment decisions have to be based on the accident history, rather than dose estimates. Additional treatment should then be based on more detailed estimates of the maximum credible accident, information from air and surface sampling, and early reports of in-vivo measurements and bioassay. The basic premise for treatment of persons with internal contamination is to reduce the absorbed radiation dose and hence the risk of possible future biological effects. The means available must be used to reduce absorption and internal deposition; and enhance excretion of absorbed contaminants. Both are most effective when begun at the earliest possible time after exposure.

43 Basic principles of treatment
Reduce absorption and internal deposition Enhance excretion of absorbed contaminants Respiratory contamination Little can be done to reduce lung absorption and deposition. Expectorants and mucolytics have not proven effective in increasing the transit rate of insoluble particles to the posterior pharynx. The deposition of insoluble particles may cause fibrosis and pulmonary scarring with resultant pulmonary restrictive disease. Lung lavage has been used in several patients. This is accomplished under general anaesthesia, with a double-cuffed, double-lumen endotracheal tube. One cuff is inflated within the trachea and the other cuff is inflated in one main stem bronchus to isolate the two lungs. One lung is filled with irrigating fluid and allowed to drain by gravity drainage. This is repeated two to three times for each lung. The greatest risk in this procedure is the general anaesthesia, not the fluid-filled lung. Lung lavage is used infrequently and the risk/benefit should be considered carefully. Gastrointestinal contamination The gastrointestinal system is more accessible and amenable for decontamination. Stomach lavage, if done early after ingestion of the contaminant, may be effective. Irrigating solution and gastric contents should be retained and sent for dosimetry analysis. Emetics can be used in conscious patients soon after ingestion. Syrup of Ipecac, followed by large amount of drinking water may induce vomiting. Apomorphine subcutaneously may also be used as an emetic. Magnesium sulphate forms insoluble sulphates with some radioactive materials, reducing absorption. Adsorbents like activated charcoal are used with some success to reduce chromium absorption. Aluminium containing antacids bind with strontium, decreasing the uptake of strontium. Alginates, made from brown sea algae (kelp) and used commonly as a bulking agent in ice cream products, decrease the uptake of strontium as well. The antacid Gaviscon contains 200 milligrams of alginate per tablet. Ten grams of alginate decreases the absorption of strontium by a factor of 10.

44 Current methods of treatment of internal contamination
- Saturation of target organ, e.g. potassium iodide for iodine isotopes - Complex formation at site of entry or in body fluids followed by rapid excretion, e.g. DTPA for Pu isotopes - Acceleration of metabolic cycle of radionuclide by isotope dilution, e.g. water for 3H - Precipitation of radionuclide in intestinal lumen followed by faecal excretion e.g. barium sulphate administration for 90Sr - Ion exchange in gastrointestinal tract, e.g. prussian blue for 137Cs The physician may encounter any number of radioactive contaminants following accidental release. Historically, however, only a relatively small number of radionuclides have commonly been encountered in potentially serious accidents. These include 3H, 60Co, 90Sr, 137Cs, 131I, 226Ra, 235U, 238U, 238Pu, 239Pu, and 241Am. Treatment for internal contamination requires a knowledge of the potential risk involved. Treatment is closely linked with metabolic information. The urgency and importance of the treatment depends on the efficiency of the therapeutic method and the amount of the contaminant. Current methods to remove radioactive contaminants involve:  - saturation of the target organ e.g. potassium iodide for iodine isotopes  - complex formation at the site of entry or in body fluids followed by rapid excretion, e.g. DTPA for Pu isotopes  - acceleration of the metabolic cycle of the radionuclide by isotope dilution, e.g. water for 3H  - precipitation of the radionuclide in the intestinal lumen followed by faecal excretion e.g. barium sulphate administration for 90Sr - ion exchange in the gastrointestinal tract, e.g. Prussian blue for 137Cs.  For practical reasons, it is convenient to consider soluble and non-soluble materials separately. Indeed the metabolic behaviour and therapeutic actions are mainly governed by this duality.

45 Diluting agents: water for tritium - 3H
Single exposures are treated by forced fluid intake: Enhanced fluid intake e.g. water, tea, beer, milk has dual value of diluting tritium and increasing excretion (accelerated metabolism) Biological half-life of tritium days Forcing fluids to tolerance (3-4 L/day) reduces biological half-life to 1/3-1/2 of normal value Diluting agents Tritium follows the pathway of water in the body, penetrates skin, lungs, and GIT, either as tritiated water (HTO) or in the gaseous form. Tritium contamination can be treated by forced fluid intake. Enhanced fluid intake e.g. water, tea, beer, milk has the dual value of diluting the tritium and increasing excretion (accelerated metabolism). This treatment reduces the biological half-life of tritium by a factor of two.

46 Ion exchange: prussian blue for 137Cs
137Cs - physical half-life Tp=30 years; biological half-life in adults average Tb=110 days, in children 1/3 of this Prussian blue effective means to reduce body's uptake of caesium, thallium and rubidium from the gastrointestinal tract Dosage of prussian blue: one gram orally 3 x daily for 3 weeks reduces Tb to about 1/3 normal value Prussian blue Caesium-137 is easily absorbed by the body through ingestion, inhalation, and skin penetration. Its physical half-life is 30 years, and its biological half-life is 110 days. It emits beta and penetrating gamma radiation, and it is distributed uniformly throughout the body. Prussian blue is ferric ferrocyanide. This chemical is not absorbed by the gastrointestinal tract (GIT) and works through two modes of action. It decreases the absorption of many radionuclides from the GIT and removes some radionuclides from the capillary bed surrounding the intestine and prevents their reabsorption. Prussian blue is most effective when given early after ingestion and serially, for 2-3 weeks, thereafter. In Goiania (Brazil, 1987) following the release and human incorporation of caesium-137 from a medical radiation source, prussian blue was used to treat contaminated human victims. Prussian blue decreases the biological half-life of caesium-137 to 30 per cent.

47 Chelation agents: DTPA for heavy metals and transuranic elements
Ca-DTPA is 10 times more effective than Zn-DTPA for initial chelation of transuranics. Must be given as soon as possible after accident After 24 hours, Ca-DTPA and Zn-DTPA equally effective Repeated dosing of Ca-DTPA can deplete body of zinc and manganese Chelation agents Chelators are mobilizing agents; they enhance the elimination of metals from critical organs. Chelators are organic compounds (ligands) that exchange less firmly bonded ions for metal ions. The stable complex, chelator and metal, is then excreted by the kidney. Hence, chelators are effective means for decorporation of radioactive heavy metals. Diethylenetriaminepentaacetic acid (DTPA), is generally more effective in removing many of the heavy metal, multivalent radionuclides. The chelates formed with many heavy metals are water soluble and excreted via the kidneys. The DTPA metal complexes are more stable and less likely to release the radionuclide before excretion. After intravenous administration, the agent is excreted rapidly with about 50% appearing in the first hour. Treating promptly with the highest recommended dose of the chelate produces the greatest enhancement of the radionuclide excretion. The effectiveness of treatment at later times following a radionuclide uptake is directly related to the solubility of the metal in vivo and its presence in the extracellular spaces. Actinides (plutonium, americium, curium, and californium), all have long biological half-lives. Inhalation is approximately 75% of industrial exposures. If the compound is soluble (nitrate, citrate, fluoride), it is ultimately translocated from the lungs to terminal disposition sites (bone and liver). Ca-DTPA and Zn-DTPA chelation therapy is the treatment of choice.

48 Dosage of Ca-DTPA and Zn-DTPA
1 g iv. or inhalation in a nebulizer Initially: 1 g Ca-DTPA, repeat 1 g Zn-DTPA daily up to five days if bioassay results indicate need for additional chelation Pregnancy: First dose Zn-DTPA instead of Ca-DTPA As with most chelators, it is more effective the earlier it is given. Both salts can be given intravenously or as a nasal inhalant. Dose recommendations are, for adults, 1 gram in 100 to 250 mL of normal saline infused intravenously slowly, over 3 to 4 minutes, and repeated on 5 successive days per week. Given as aerosol, 1 gram in a 4 mL vial is placed in a nebulizer, and the entire volume is inhaled over 3 to 4 minutes, repeated daily. With repeated dosing, Ca-DTPA can deplete the body of zinc and, to a lesser extent, manganese. Zinc-DTPA replacement therapy is recommended when repeated dosing is done due to loss of the body's zinc stores. Contraindications for Ca-DTPA: pregnancy, infancy, nephrosis and bone marrow depression. Teratogenicity and fetal death have occurred in mice. No serious toxicity in humans has been reported when used in recommended doses. When given repeatedly with short intervals for recovery, nausea, vomiting, diarrhoea, chills, fever, pruritus, and muscle cramps have been noted.

49 Additional chelating agents
Dimercaprol (BAL) forms stable chelates, and may therefore be used for the treatment of internal contamination with mercury, lead, arsenic, gold, bismuth, chromium and nickel Deferoxamine (DFOA) effective for chelation of 59Fe Penicillamine (PCA) chelates with copper, iron, mercury, lead, gold. Superior to BAL and Ca-EDTA for removal of copper (Wilson’s disease) Chelation therapy has been used for lead, mercury, arsenic, and other heavy metals. Chelators are most effective prior to the binding of a toxicant in the critical organ but can be used with variable results after target organ acquisition. Before, during and after chelation therapy, pertinent measurements of radioactivity in the body should be made to determine the efficacy of treatment. By the fifth day, evaluate the bioassay data of urine and faeces samples and the total-body and chest count measurements. Continuation of therapy is determined by assessing chelation yield with remaining body burden. .

50 Treatment of uranium contamination
In any route of internal contamination, treatment consists of slow intravenous transfusion of 250 mL of isotonic 1.4 % sodium bicarbonate Local treatment: for skin contamination, wash with isotonic 1.4% solution of sodium bicarbonate Inhalation is the usual route of occupational exposure. Average biological half-life is 15 days. 85% of retained U resides in bones. In acidic urine, uranyl ion makes complex with renal tubuli surface proteins and causes acute tubular necrosis (ATN). Chemical toxicity for kidneys is the basis for occupational exposure limits. Alkalinization of urine reduces the chance of ATN.

51 Summary of lecture Attend to life-threatening injuries first
Earlier skin decontamination decreases degree of beta burns, lowers risk of internal contamination, reduces chance of further contamination Goal of internal contamination treatment: decrease uptake into circulatory system, decrease deposition in critical organs, increase excretory rate contaminant Health physicists and medical specialists should advise on risks and benefits of decorporation Quiz 1. What is the primary mode of intake of a radiocontaminant? a) dermal absorption b) wound absorption c) inhalation d) via GIT 2. Prussian blue is effective in reducing the body burden from: a) plutonium b) uranium c) caesium d) plutonium and uranium 3. Body burden from intaken radionuclides can the most completely be estimated by: a) whole body counting (WBC) and contamination survey b) WBC and measurments of radionuclides in urine, faeces and thyroid gland c) WBC, metabolite and thermoluminescence measurements d) WBC, metabolite measurement in urine and fecal metabolite measurement 4. Which of the following reflects an incorrect substance/target organ relationship? uranium/bone b) iodine/thyroid c) lead/bone d) caesium/gut 5. Select the false statement. a) Potassium iodide (KI) increases the rate of thyroid hormone production b) KI should be given as soon as possible following exposure to I-131 c) Although rare, side effects, including rhinitis, conjuctivitis, and skin rashes, may occur due to the use of KI d) Effectiveness of KI is time dependent following exposure to I-131

52 kindly given by doctor Elena Buglova, were used
Lecture is ended THANKS FOR ATTENTION In lecture materials of the International Atomic Energy Agency (IAEA), kindly given by doctor Elena Buglova, were used


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