Environmentally Safe Biological Eradication of Annual Narcotic Crops in Central Asia

(BIO-ERA)

Responsible scientist

Dr. Kanat Sarsenbaev, Institute of Botany and Phytointroduction (IBPK), National Academy of Sciences, Republic of Kazakhstan, Timiriazeva Street 42, Almaty, 480091, Kazakhstan (Phone ++7 3272 695095, E-Mail: almaty@itte.kz)

Participants

Dr. Quirico Migheli Istituto di Patologia vegetale, Università degli Studi di Sassari (UNISS), Via E. de Nicola 1, I-07100 Sassari, Italy (Phone ++39 079 229295, Fax ++39 079 2229316, E-Mail: migheli@agraria.unito.it) Dipartimento di Valorizzazione e Protezione delle Risorse Agroforestali (Di.Va.P.R.A.) - Patologia vegetale, Università degli Studi di Torino (UNITO), Via Leonardo da Vinci 44, I-10095 Grugliasco (Torino), Italy (Phone ++39 011 6708540, Fax ++39 011 6708541, E-Mail: migheli@agraria.unito.it)

Dr. Maria Chiara Zonno, Dr. Antonio Vurro, Istituto Tossine e Micotossine da Parassiti Vegetali - Consiglio Nazionale delle Ricerche (ITMPV), Viale Einaudi 51, I-70125 Bari, Italy (Phone: ++39 080 5481570, Fax: ++39 080 5486063, E-mail: itmpmz13@area.area.ba.cnr.it)

Dr. Khurshet Kharimovich Karimov, Institute of Physiology and Biophysics (IPBT), Academy of Sciences of Republic of Tajikistan, Presidium Academy of Sciences, Rudaki Street 33, Dushanbe, 734025, Republic of Tajikistan (Phone: ++7 377 216991, Fax: ++7 377 234917, E-mail: plant@td.silk.or)

Dr. Dzhumamurad Kurbanov, National Institute of Deserts, Flora and Fauna (NIDFF), Ministry of Nature Use and Environmental Protection of Turkmenistan, 15 Bitarap Turkmenistan Str., Ashgabad, Turkmenistan 744000 (Fax: ++993 12 353716, ++993 12 357364, E-mail: babaev@desert.ashgabad.su)

Dr. Echut Sabirovna Salieva, Botanical Institute and Botanical Garden (BIU), Uzbek Academy of Sciences, F. Chodjaeva 32 Street, Tashkent, 700143, Republic of Uzbekistan (Phone: ++7371 1627085, Fax: ++7371 1627938, E-mail post@botany.org.uz).

Dr. Iftikhar Ahmad, Crop Diseases Research Institute (CDRI), National Agricultural Research Centre, Pakistan Agricultural Research Council, P.O. Box 1031, Islamabad, Pakistan (Phone: ++92 51 241128, Res: ++92 51 252097/260514, E-Mail: ifti@cdri-isb.sdnpk.undp.org)

Summary

The present project is aimed at developing environmentally friendly methods to control the production of opium poppy (Papaver somniferum) and cannabis (Cannabis sativa) crops in the main cropping regions of Central Asia. The proposed system involves the isolation, characterisation, mass-production and application of fungal and bacterial microorganisms which are specifically pathogenic to the target plants to control these crops in an environmentally safe way. Such methodology would lead to a sustainable and long-term biological control of annual illicit narcotic crops, thus avoiding the large-scale application of unsafe herbicides, currently adopted by the governmental eradication programs.

Background

Opium poppy production

According to the International Narcotics Control Strategy Report, 1997, opium poppy cultivation in Southeast Asia's Golden Triangle region, the world's opium-rich area, reached 184,950 hectares in 1997. Burma (Myanmar) accounts for over 60 percent of world's opium poppy cultivation and opium gum production. Estimated production in Burma was 2,365 metric tons, enough to produce 236 metric tons of heroin. Production in Laos was 2l0 metric tons, or about nine percent of the Southeast Asian total. In Vietnam, the US government identified substantial new growth of the country's opium poppy cultivation. By the end of 1997, there were 6,150 hectares under cultivation potentially yielding 45 metric tons of opium gum.

In Southwest Asia, Afghanistan remains the world's second largest opium producer, and poppy cultivation reached 39,150 hectares in 1997. As the main source of the heroin consumed in Europe, Afghanistan is in a key position to affect heroin supplies throughout the continent. Opium poppy is currently Afghanistan 's leading cash crop and the Taliban faction control an estimated 95 percent of the areas in which it is grown. Pakistan's opium poppy cultivation in 1997 reached 4,100 hectares, despite broadened crop cultivation bans and assistance from the US and other donors. Estimated potential opium yield in Pakistan is 85 metric tons. India's illicit cultivation was 2,050 hectares potentially producing 30 metric tons of opium in 1997. No firm data exist about poppy cultivation or opium production in Iran. In 1992, approximately 3,500 hectares were cultivated, with a potential yield of 35 metric tons to 70 metric tons.

The illicit drug crop situation in Russia and the Central Asian countries formerly part of the Soviet Union is not well known. While some of these countries may be able to produce significant opium poppy harvests, data are not sufficient to identify and measure all suspected cultivation areas. The Central Asian countries of Kazakhstan, Kyrgyzstan, Tajikistan and Uzbekistan, formerly important poppy growing regions for the Soviet Union are well placed to be conduits for much of this drug traffic. Kazakhstan provides a bridge for Southeast Asian heroin to move to Europe and Russia from Asia. Uzbekistan produces opium crop, mostly in the Samarkand region near the border with Tajikistan. Cultivation of opium in Turkmenistan does occur particularly in remote mountain and desert areas. Although no statistics on the extent of such cultivation are available, authorities report that most opium is cultivated along the Iranian border in the Ahal Velayat (region), which includes Ashgabad, and in the eastern parts of Lebap and Mary Velayats.

In Egypt, opium poppy is cultivated in the Southern Sinai on a seasonal basis. The Anti-Narcotics General Administration (ANGA) has developed a full time Sinai Eradication Unit and is beginning to establish a database to track cultivation areas. ANGA also retains sources within the local Bedouin community in the Sinai to assist in uncovering growing locations. Egypt does not have a crop substitution program or a herbicide eradication strategy, although ANGA is pushing to implement the use of herbicides in controlling cultivation.

Colombia is the Western Hemisphere's largest grower of opium poppies, though it represents less than two percent world production. For 1997, Colombian opium poppy cultivation (mainly in the regions of Tolima, Huila, Cauca and Cesar) was essentially steady at 6,600 hectares and capable of yielding an estimated 66 metric tons of opium gum, or 6.3 tons of heroin, assuming no losses. Mexico is Latin America's second largest cultivator of opium poppies. After Mexican government eradication operations destroyed 8,000 hectares of poppy, there were 4,000 hectares available for exploitation by the drug syndicates, with an estimated potential yield of 46 metric tons of opium gum, or 4.6 metric tons of heroin. Venezuela's border with Colombia has made it a potential poppy growing country. Over the past four years, the eradication program has destroyed over 3,000 hectares of opium poppy in the Sierra de Perija region along the Colombian border.

Cannabis production

Cannabis cultivation is very important in Mexico, with 4,800 hectares with a potential yield of 2,500 metric tons in 1997. Mexican law enforcement agencies eradicated 10,500 hectares of cannabis in 1997. In Colombia's traditional cannabis growing zones, where intensive eradication in previous years had virtually destroyed the crop, there is a resurgence of cultivation in recent years, to an estimated 5,000 hectares. Jamaica's cannabis crop was 317 hectares in 1997, with a potential yield of 214 metric tons.

The Chu Valley of Kazakhstan remains the top cannabis growing area in Central Asia, capable of producing 500 metric tons of marijuana per year. There are reports, however, that due to increased police surveillance in the Chu Valley, marijuana plantations are being developed in the Dzhambyl Region of southern Kazakhstan. Cannabis grows wild in much of Kyrgyzstan. Kyrgyz officials estimated in 1994 that such wild growth of cannabis totalled approximately 60,000 hectares. Cannabis is cultivated illegally in Azerbaijan. Authorities discovered and destroyed 339 tons of cannabis and poppy under cultivation, mostly in southern Azerbaijan. Cannabis is also present in Turkmenistan.

In North Africa, cannabis is cultivated mainly in the Northern or Rif region of Morocco, although some is also grown in the Souss Valley of the south. Unofficial sources estimate that between 80,000-85,000 hectares are devoted to cannabis production, and claim that this number has increased by a factor of ten in the last decade. The average hectare of cannabis produces 2 to 8 metric tons of raw plant. The government of Morocco has stated that it is committed to the total eradication of cannabis production. Given the economic dependence of the Northern part of the country on cannabis (cannabis crops are estimated to yield two billion US dollars in revenues annually) eradication is only feasible if accompanied by a highly subsidised crop substitution program. Consequently, the GOM has not yet made a serious attempt at eradication. Cannabis is also grown in Egypt, mainly in the Northern Sinai.

Current eradication programmes and environmental concern

A five-stage grower-to-user chain links the drug producer with the consumer. At one end is the farmer growing opium poppies or cannabis; at the other is the heroin user. In between lie the processing (drug refining), transit (shipping), and wholesale distribution links. Counter-drug programs target the first three links of the chain: cultivation, processing, and transit. For drugs that are not completely synthetic, the best chance stands if the first stage is eliminated. When crops are destroyed or left unharvested, no drugs can enter the system. Governmental eradication programs mainly utilise non-selective, systemic herbicides, such as glyphosate or tebuthiuron. Chemical eradication is by far the most cost-effective means, but large scale eradication may not be politically, socially and environmentally feasible in many countries.

Concern about the medium- and long-term effect of continuous herbicide distribution in the cropping areas is increasing among the public opinion, the resident population and the environmental associations. In particular, the justified fear exists that these broad-spectrum compounds may dramatically alter the biodiversity of the ecosystem and affect the quality of ground water. Moreover, long distance transport through wind or rivers would spread these effects to large, non-target areas. Consequently, by maintaining the importance of an effective eradication campaign, we believe that there is a strong need for developing more sustainable, environmentally friendly methods for limiting the extension of illicit narcotic crop cultivation around the world.

Biological control

To date, the most biologically effective alternatives to chemical weed control agents that have been extensively evaluated are the plant pathogens, more specifically, plant pathogenic fungi. The use of plant pathogenic microorganisms as in the biological control of weeds is being constantly implemented in modern agricultural systems, and a number of commercial products have now met market’s requirements.

Ideally, biological control consists in the application of specific pathogens on the target plant. The possibility to use phytopathogenic fungi for the biological control of undesirable plants can be realised in two ways: the inundative and the inoculative method. The first consists in the application of massive doses of inoculum to the weed population to create a fast and high level of epidemic. The second method consists of the introduction of pathogens in a small area and its progressive diffusion in the field. Under appropriate climatic conditions, a pathogen might be rendered completely destructive to its host by applying a massive dose of inoculum at a particularly susceptible stage of weed growth. The application of an inundative dose of inoculum and its proper timing would shorten the lag period for inoculum build-up and pathogen distribution, essential for natural epidemics. The onset of an epidemic would destroy or strongly limit the development of the weed. To be successful in this approach, it must be possible to produce abundant and durable inoculum in artificial culture at a low price, the pathogen must be genetically stable and specific to the target plant, and it must be possible to infect and kill the weed in environments of reasonably wide latitude. Besides obvious ecological advantages (extreme target specificity, environmental safety and less drastic changes in the ecosystem), inundative biocontrol techniques generally have a number of cost advantages over conventional herbicides in terms of reduced registration requirements and cheap production (for example, by submerged culture fermentation).

Recently, fungal pathogens have been developed registered and marketed as mycoherbicides. They can be used in combination with other pesticides and have mainly been developed to control weeds in crops. De Vine, the first registered mycoherbicide in the USA, was registered in 1981 for use as a post-emergence direct spray and is marketed as a liquid formulation of chlamydospores of Phytophthora palmivora to control strangler vine (Morrenia odorata) in Florida citrus groves. Collego, a dry powdered formulation of Colletotrichum gloeosporioides f.sp. aeschynomene for the control of Northern jointwetch (Aeschynomene virginica) in rice and soybean was registered in 1982. Numerous other pathogens have been investigated as potential biocontrol agents for many annual and perennial plants and are now available in the market such as Puccinia caniculata against Cyperus aesculentus (Dr BioSedge), Chondrostereum purpureum against Prunus serotina in forestry (Stumpout) and bacteria as Xanthomonas campestris pv. poae against Poa annua.

The use of toxic metabolites produced by phytopathogenic fungi seems to be another promising approach to plant biocontrol. A wide number of fungal pathogens such as Drechslera spp. Alternaria spp. Ascochyta, Fusarium spp are able to produce toxic metabolites. The phytotoxins belong to different classes of compounds such as sesquiterpenoids, diketopiperazines, peptides etc. and can vary both in molecular size and structure. These compounds can be completely non-specific with respect to the host (i.e. triticone) or can have a high specificity similar to that of the producer micro-organism (i.e. maculosin). Many micro-organisms produce phytotoxic compounds with the potential for direct use as herbicides or as models for new structural classes of herbicides. For instance, the members of the genus Fusarium produce a wide array of phytotoxic compounds possessing a broad range of biological activities.

The biological control approach has been proposed to control narcotic plants. A self-sustainable, long-term programme for the control of narcotic plants would benefit from the introduction of natural antagonists.

A successful example is that of coca (Erythroxilum coca): a specific fungal pathogen, Fusarium oxysporum f. sp. erythroxyli, is currently being evaluated as a biocontrol agent by the US Department of Agriculture and proved effective in greenhouse and field tests. This pathogen causes vascular wilt symptoms and death in coca plants as soon as 7 weeks after soil infestation. Because Fusarium wilt becomes more severe with continuous cropping, and the pathogen often persists in infested fields for years after the crop is removed, a single application of biocontrol agent could be expected to have detrimental effects on coca production over the medium- and long-term.

Little work has been done so far in the search for new biocontrol agents on opium poppy and cannabis, despite the major impact of these crops on the international drug market. Pleospora papaveracea, Dendryphion penicillatum and Fusarium spp. are being studied to control P. somniferum, while F. oxysporum f. sp. cannabis is regarded as a promising mycoherbicide on C. sativa. A combination of crop-specific vascular and necrogenic foliar pathogens is considered to be the most reliable mean of biological control of illicit narcotic plants.

Proposal

The proposed project involves the following tasks (on a 5 years basis):

1. Isolation of new potential biocontrol agents on P. somniferum and C. sativa from different major growing areas in Central Asia (years 1-3)

2. Testing the new isolates under controlled laboratory conditions (years 1-3)

3. Selection of most effective pathogens, study of their epidemiology and mode of action (years 2-4)

4. Testing the efficacy of most promising isolates in experimental fields (years 3-4)

5. Risk assessment evaluation of the biocontrol agents (years 3-4)

6. Development of mass production and formulation systems (years 4-5)

7. Development of storage and delivery systems adapted to local conditions (years 4-5)

Work programme

The first phase would include a cooperation among researchers of the Institute of Plant Pathology, University of Sassari (UNISS), the Plant Pathology Section, Di.Va.P.R.A., University of Torino (UNITO), the Istituto Tossine e Micotossine da Parassiti Vegetali (ITMPV) of Bari, the Technical Service Branch of UNDCP (Supply Reduction Section, Research and Scientific Section, Field Offices), the local scientific partners and the forces involved in the governmental programs of eradication. During this phase the candidate biocontrol agents will be isolated from their respective hosts, and a first characterization will be carried out (taxonomic position, morphology, biological properties, pathogenicity under contained conditions, mode of action, symptoms, production of phytotoxic compounds, growth in vitro).

In a second phase, the biological control efficacy of selected microorganisms will be tested more thoroughly under contained conditions in Italy (UNITO, UNISS and ITMPV facilities) and in the growing areas (UNDCP Field Offices facilities, local scientific partner institutions). Glassshouse and small scale field experiments will be conducted in order to evaluate the behaviour of promising biocontrol agents by modifying environmental parameters such as temperature, humidity, host plant density, soil texture, etc.). The isolation and purification of host-selective phytotoxic compounds will be the major target of the ITMPV during this phase. Risk assessment studies will be carried out at UNITO and UNISS in order to discard any effective pathogen having harmful side-effects (broad host range, genetic instability, pathogenicity or allergenicity on non target organisms, etc.).

In the third phase, methods for mass production and formulation of most effective biocontrol agents will be developed by UNISS, UNITO and ITMPV, in consultation with Dr. Michael P. Greaves, Institute of Arable Crops Research (IACR), Long Ashton Research Station, University of Long Ashton, Bristol, BS18 9AF UK (Phone ++44 1275 549362, Fax ++44 1275 394007). Storage and delivery systems will be adapted to local conditions. The collaboration of UNDCP officers will be necessary to find the most effective delivery system for new biocontrol agents.

Target biocontrol agents. Ideally, the biological control agent should combine the following characteristics: high host selectivity; rapid growth in vitro; rapid spread through airborne propagules; ability to survive on and to be transmitted through seeds or propagative material; ability to produce resting spores and to survive saprophytically in the absence of the host plant; ability to survive in dry formulation; absence of toxicity, allergenicity, pathogenicity to non-target organisms. Within the proposed project, we will also look for pathogens debilitating rather than killing narcotic plants, in order to render the crop no more economically feasible. Attention will be devoted to soilborne pathogens having a saprophytic stage and producing resting spores, sclerotia, chlamydospores able to keep the inoculum active over long periods, even in the absence of the host plant. In other words, the goal would be to create a condition of long-term soil suppressiveness towards the narcotic crop.

Isolation and characterization. Field surveys will be conducted in some unexplored areas in Uzbekistan, Kazakhstan, Turkmenistan, Tajikistan and Pakistan where it may be possible to find ecological niches containing new pathogens infecting narcotic plants. Particular attention will be devoted to those pathogens causing intense damage on newly produced vegetative organs (necrotic spots, yellowing, distortion, bud necrosis, etc.) or lethal wilting, by maintaining complete specificity towards the target plant. Samples from diseased plants will be collected and the pathogens isolated on-site, whenever possible, to avoid any damage to biological material. Isolates will be collected from leaves, stems and seeds of diseased plants. Portions of plant tissue (from leaves, stems, roots and vessels) or seeds will be placed on Petri dishes with water agar or in moist chamber to isolate the pathogens. If present, the fungus will grow into the agar and will be then transferred on selective solidified media. The next step will be the macro and microscopic observation of fungal morphology for proper identification of the potential pathogens.

Pathogenicity assays. Fungal isolates will be first screened for pathogenicity under laboratory conditions and, at a second stage, under contained (glasshouse, growth chamber) conditions. Pathogenicity test will be carried out on different host/plant systems. Several types of colony forming units (mycelium fragments, conidia, chlamydospores, sclerotia) will be placed or sprayed on whole plants and on plant organs (leaves, stems). In case of soilborne pathogens, the inoculum will be mixed to soil at transplant or sowing. The virulence of fungal pathogens will be tested in a controlled environment under conditions that will permit moderate development of symptoms. The defined conditions include spore density, plant stage, duration of the period of high moisture after inoculation, and temperature. Plants will be examined after 2 days for spore germination on leaves and roots (using fluorescein staining and light microscopy), and after 7 and 14 days for symptom development. Further observation will be followed on the development of any disease symptom. The success of mycoherbicide applications may be increased by determining and treating the most susceptible developmental stage of the weed. The information gained can then be used to determine the most appropriate timing of mycoherbicide treatments in the field.

Growth requirements. Fungi, after identification, will be assayed for their thermal and nutritive requirement as well as for the best condition for inoculation (spores concentration, temperature) and for production of inoculum (on liquid culture and on solid medium). The spore density required for optimal disease development is an important criterion for efficacy assessment of novel isolates of a potential mycoherbicide. A low level of inoculum may result in localisation of infection and in the survival of plants, while an excessive inoculum, besides being economically unfeasible, may result in poor disease development due to autoinhibition of spores.

Risk assessment. Risk assessment studies will be carried out on most promising biocontrol agents to evaluate the effect of large-scale biomass application on local ecosystems. Among the main factors to be considered for an adequate assessment of adverse effects we will refer to: establishment and survival of released biocontrol agents, dispersal of released biocontrol agents, genetic stability, effects of the introduced microorganism on the resident microflora and fauna, (e.g. pathogenicity, virulence, allergenicity and toxicity towards humans, animals and plants), availability and applicability of effective containment systems. The host specificity of potential biocontrol agents will be tested in vivo under greenhouse conditions on different biotypes of the narcotic plants and on different crops. Crops and spontaneous plants will be selected considering botanical similarity and their diffusion in the particular region. Plant species that shall be tested will include species 1) related to the target plant, 2) not previously exposed to the biocontrol agent, 3) having similar secondary compounds and/or morphological similarities with the target plant, 4) attacked by related organisms and 5) recorded as hosts of the candidate agent. Pre-release survival studies will be conducted in contained environments, such as soil microcosms, growth chambers or glasshouses, where natural conditions can be simulated in a limited space. To achieve this goal, unambiguous detection of the target organism is necessary. This will be accomplished through the use of selectable markers (e.g., antibiotic or fungicide resistance, nutritional complementation, induced mutations which confer particular colony morphology) or via molecular techniques (PCR/hybridization, restriction fragment length polymorphisms, REP/ERIC/BOX-PCR fingerprinting, electrophoretic karyotyping, nucleic acid hybridization techniques). The dispersal of biocontrol agents has important biosafety implications and will be studied in this phase. Active motility of biocontrol agents should not play a great role for translocation over space. Such transport is mainly passive and can be brought about by biological (soil animals, developing plant roots) or, more efficiently, by physical (wind and water) factors. These vectors will be taken in particular consideration in the risk assessment studies. Genetic stability of biocontrol fungi is a key factor in their safety. Any evidence for genetic instability in contained, pre-release experiments should prompt a re-evaluation of the antagonist and its release, as a change in the genetic structure may eventually lead to a modification of the host range, thus representing a serious environmental hazard. The meiotic and mitotic stability of antagonistic fungi shall be determined through comparative Southern analysis of specific sequences by using DNA extracted from cultures derived from cells obtained before and after a release into the environment. In considering the biosafety of biocontrol agents that are able to differentiate into a sexual stage in nature, the presence of related phytopathogenic species in the introduction area, as well as the existence of common hosts that might support hybridization shall be carefully evaluated. The uncertainty should be more acceptable for microorganisms that lack a sexual reproductive system, as the frequency of genetic exchanges would be reduced. Released microorganisms should not cause any qualitative or quantitative alterations in microbial community structure. Displacement of indigenous microbial groups can be important if introduced organisms possess high fitness. Given the rapid decrease of introduced microbial populations in the environment, the probability of adverse effects may be small, but this potential has to be carefully assessed. To estimate the effects on microbial communities, the population dynamics of specific functional groups of fungi or bacteria will be evaluated, e.g. those linked to the processing of nitrogen, sulphur and phosphorous and mycorrhizae. The antagonist should also not affect humans and animals. In vitro growth at 35-38°C, pathogenicity in immunocompromized patients and allergenicity should be absent. Different in vitro toxicological tests will be used to evaluate the presence of toxic or mutagenic metabolites in culture filtrates: testing the inhibition of root development in tomato germlings, radial growth of the fungus Geotrichum candidum, toxicity towards larvae of the crustacean Artemia salina, or genotoxicity on germ cells of locust. Physical containment in small scale pre-release trials will be accomplished through physical barriers (growth chambers, enclosures, sealed windows, air locks, filters), in order to prevent the escape and dispersal of the organism. In addition, organisms will be neutralized at the end of the experiment by burning and biocide application, alone or in combination with tillage.

Phytotoxin production and characterization. Among the signals involved in the plant-pathogen interactions, an important role is played by the toxic metabolites produced by the pathogen. These metabolites can be an important factor of virulence, and can have a different behaviour with respect to the host, varying from strictly host-specific to completely non-specific compounds. Some of these compounds are produced by undesirable plants pathogens, and could be used as natural herbicides, in addition or in alternative to the use of the pathogens. The higher the toxin production, the higher the disease degree caused by the pathogen. The selected candidates for the production of toxic metabolites will be grown on different media and environmental conditions for the choice of the best conditions for the production of phytotoxins (solid and liquid cultures, shaken or static conditions, different temperatures). After the optimization of growth conditions of pathogens for toxin production, different biological assays to quantitatively evaluate the phytotoxicity of culture filtrates will be developed or adapted, using leaves, seedlings, cuttings, seeds, cells, protoplasts or calli, both of host plants and of test plants. The isolation of the phytotoxic metabolites from fungi culture filtrates is dependent on their chemical nature. For lipophylic substances we shall use extractions with appropriate organic solvent and purification by column chromatography or TLC. For high molecular weight hydrophilic substances (proteins, glycoproteins, polysaccharides) physical methods will be used such as precipitation with organic solvent and/or dialysis, gel filtration and/or ion exchange, affinity chromatography and HPLC. Similar methods may be used to purify polar or charged compounds. The purification step is followed by testing the phytotoxic activity of the fractions and pure toxins on host and non-host plants. The structure determination will be carried out using the spectroscopic organic methods and chemical methods. To define the chemical nature of the macromolecules chemical degradative methods associated to spectroscopic ones will be used. The determination of toxins structure in the solution will be performed using NMR methods. A knowledge of the toxin structure will permit to assay their phytotoxicity on host and non-host plants in vitro and in vivo and the toxicological activity also in relation to their role in the pathogenic process. Purification, identification and chemical characterization of pure phytotoxins will be carried out in consultation with Prof. Antonio Evidente, Dipartimento di Scienze Chimico-Agrarie, Università di Napoli "Federico II" (UNINA), Via Università 100, 80055 Portici, Italy.

Pre-release field experiments. After the selected pathogens will be found to be potentially effective and safe biocontrol agents, a detailed proposal will be prepared by the responsible scientist and submitted to the UNDCP. Pre-release field experiments shall be performed in Uzbekistan (at the UNDCP facilities), Kazakhstan (IBPK facilities) and Pakistan (CDRI facilities). Once the approval for release will be obtained, liberation of the pathogens will occur at preselected, relatively undisturbed sites where the target plant has been grown. Since climate exerts a profound effect on disease development, location of release sites and timing of releases to coincide with suitable environmental conditions will be extremely important. The release phase of the program will require cooperation of UNDCP officers, local scientific partners and landowners. Infestations of the target plant will need not be located and the release site must be maintained for some time with minimal disturbance. Qualitative and quantitative records of the status of the target crop before and after introduction of the biocontrol agents will be carried out by counting the progress of disease based on visual observations, life table analysis, computer simulation models and photographic records. Cyclic isolation of the pathogens will be also performed in order to monitor the stability of introduced populations.

Production, formulation and delivery of biocontrol agents. Methodology has been developed to produce mycoherbicides using solid state fermentation, submerged culture fermentation, or combination of both at different stages of the growth cycle of the fungus. The method to be used has to be adapted to the particular fungal organism and cannot be predicted at this stage. Accordingly, the type of formulation will depend on the antagonistic microorganisms that will be isolated, tested and selected during the first part of the project. Formulation in Pesta granules would serve as one of the options, based on the suitability of this technique for storage and delivery of pathogens on other narcotic crops. Particular emphasis will be devoted to the possibility of delivering soilborne biocontrol agents on viable coated seeds of well adapted, non-susceptible Leguminosae: by germinating and establishing in soil the seed would serve as a living carrier of the microbial inoculant, which may find its ecological niche in the rhizosphere. Oil formulations will be used in this case, in order to reduce the quantity of inoculum needed to coat the carrier seeds. Invert emulsions will be tested for foliar pathogens to retard evaporation and trap water in the spray mixture, thereby decreasing the amount of dew required for spore germination and infection to occur. Finally, where new host-specific phytotoxic compounds will be identified, the possibility to combine the fungal pathogen and reduced amounts of purified toxins or synthetic analogues will be evaluated

The following tasks will be realised:

1. Isolation of new biocontrol agents (UNISS + UNITO + local scientific partners + UNDPC Field Offices)

2. Microbial (pathosystem) characterisation (UNISS + UNITO + ITMPV + local scientific partners)

3. Formulation technology (UNISS + UNITO + ITMPV)

4. Effectiveness in contained environment (UNISS + UNITO + ITMPV + local scientific partners)

5. Effectiveness in field (local scientific partners, UNDCP Field Offices)

7. Risk assessment (UNISS + UNITO + ITMPV)

8. Release (UNDCP Field Offices)

Proposed countries for isolation of new candidate biocontrol agents

Cannabis: Kazakhstan, Turkmenistan

Opium poppy: Uzbekistan, Tajikistan, Pakistan

Wherever possible (Uzbekistan and Pakistan), all the on-site activities will be carried out in collaboration with UNDCP Field Offices personnel. Moreover, the proposed project would possibly represent a starting point for the establishment of new UNDCP Field Offices in Central Asia.

FINANCIAL REQUIREMENTS

Item	Amount	Corresponding to :	Institution
Travel/subsistence	**	Sample collection, isolation of pathogens, field experiments, coordination	UNISS, UNITO, IBPK, IPBT, NIDFF, BIU, CDRI
Equipment	**	Small laboratory equipment (spore traps, microfuges, microscopes)**	UNISS, IBPK, IPBT, NIDFF, BIU, CDRI
Consumables	**	Substrates and growing media, plasticware, fuel, plant material, fertilisers, pesticides, containers, molecular biology reagents, publications	UNISS, UNITO, ITMPV, IBPK, IPBT, NIDFF, BIU, CDRI
Other costs	**	Glasshouse experiments in Italy and in Kazakhstan, pre-release field experiments in Kazakhstan, Uzbekistan and Pakistan	UNISS, UNITO, ITMPV, IBPK, CDRI, UNDCP Field offices
External services	**	Activities at the UNDCP Field Office facilities, at the IACR, UK and at the UNINA, Italy	UNDCP Field Offices, IACR, UNINA
Overheads	**	Administration, telephone, fax, mail,	UNISS, UNITO, ITMPV, IBPK, IPBT, NIDFF, BIU, CDRI
Total			**

** To be defined at a later stage.

WORK PROGRAMME

Research Project

Title: REHABILITATION OF ENVIRONMENT AND POPULATION HEALTH IN SEMIPALATINSK NUCLEAR TEST SITE (on example of Kainar village)

OBJECTIVES

The project is aimed at growing "vitamin" gardens and purification contaminated soils by planting trees, shrubs and grasses with high capacity to accumulate radioactivity in the villages and towns in vicinity of Semipalatinsk nuclear test polygon. We will use results obtained from Kainar village as a benchmark as it is a typical village in Semipalatinsk polygon area. We suggest to proceed by planting more then 40 species and cultivars of fruit trees, shrubs and grass as well as metal-accumulating plants in the contaminated places of Kainar and Karaolen villages, Atomic lake, Degelen and an Experimental field. We suggest the following objectives of the research:

· testing suitability of 40 cultivars and species of trees, shrubs and grass for organization vitamin gardens and purification of contaminated soil from radioactivity in Kainar and Karaolen villages;

· probation of the new method of planting trees and shrubs in semiarid zone of Semipalatinsk polygon with help of phytomeliorants;

· development of a phytoremediation method for purification of contaminated soils in Semipalatynsk polygon;

· preparing a recommendation for greening Semipalatinsk villages, organizing vitamin gardens that will have a high content of antioxidants in fruit; improvement of the ecological situation and population health.

BACKGROUND

Most of the consequences from nuclear testing in Semipalatinsk polygon are known now. There are many long-living radionuclear contaminated places. There are extensive deposits of radioactive plutonium underground. In addition millions of inhabitance suffer from poor health. For rehabilitation of local people it is important to improve the ecological situation. There are different methods and approaches to tackle this problem. According to the 53rd UN Resolution our program is taken in as a part of a massive rehabilitation program for this area. Our main tasks are as follows.

1. to use plants for production of very cheap antioxidants such as fruit and vegetables, antioxidants are known for helping to decrease a level of diseases in the area;

2. to develop a new phytoremedation method, that will allow to purify soils using plants that have a high capacity to accumulate toxic heavy metals and radioactivity. That is a new direction of investigation for this region.

The flora in Semipalatinsk region is not rich with plants. They include Artemisia scoparia, Artemisia dracunculus, Artemisia sieversiana, Artemisia austriaca, Artemisia frigida, Artemisia gracilescens, Kochia scoparia, Lepidium latifolium, Heteropappus altaicus, Urtica dioica, Cannabis ruderale, Psathyrostachis juncea, Bromopsis innermis, Barteroa incana, Setaria viridis, Ceratocarpus arrenarius, Solanum dulcamara,Tlaspi arvense, Fumaria vaillantii, Polygonum aviculare, Stipa capillata, Stipa sareptana, Festuca valesiaca, Spiraea hypericifolia, Caragana pumila, Cleistogenes squarrosa,Helectotrichon desertorum, Gallium ruthenicum, Ephedra dystachia, Potentila acaulis, Ancantia igniaria, Dianthus rigidus, Phragmites australis, Calamogrostis epigeios, Atriplex cana. There are few populations of Juniperus sabina, Berberis sibirica, Betula pendula, Populus tremula, Delphinium dyctiocarpum, Agrostis vinealis, Ziziphora linopodioides, Achillea nobilis, Fragaria viridis. In the areas with high level of radioactivity the number of species does not exceed 10. The local population does not receive enough vitamins. That is related to short summer periods and absence of wild or cultured fruit gardens.

The system of botanical gardens of Kazakhstan (Institute Botany and Phytointroduction of the National Academy of Sciences) has an experience in greening the places with poor soil and sevire climate conditions, particularly those associated with desert areas. There is a range of plants that are well-adapted too hard conditions. For a number of years the scientists from botanical gardens have worked with antropogenic contaminated areas. They developed some effective methods for planting and growing metal-accumulating plants. That gives a possibility to purify the soil from heavy metals. The method we propose is cheaper and more effective than the traditional industrial methods.

SCIENTIFIC AND TECHNICAL DESCRIPTION

RESEARCH PROGRAMME

Plant Material

The work will be performed with rose plants such as Rosa canina and Rosa rugosa(4 cultivars), quince plants such as Chaenomeles japonica and honeysucle, Lonicera coerulea, Lonicera turtsaninovii, slum (2 cultivars), apple (6 cultivars), mountain ash as Sorbus sibirica, Viburnum opulus, barberry - Berberis sphaerocarpa, Berberis iliensis, nawtorn - Crataegus songarica, Crataegus oxyacanta, pine - Pinus silvestris, birch - Betula pendula, golden currant -Ribes aureum, soya - 1 local cultivar, vetch - 1 local cultivar, alfalfa - 1 local cultivar, Stipa capillata, Stipa sareptana, sunflower - Heliantus angustifolia, sorrel - Rumex acetosa, Salix caprea, Salix viminalis, Syringa vulgaris, Hyppophae rhamnoides (cultivars - Chuiskaia, Prevoschodnaia), poplar - Populus hybrida (2 cultivars).The seedlings and seeds will be taken from the Kazakh botanical gardens situated on the places with similar climate and soil conditions.

CLIMATE AND SOME FEARCHES OF SOIL

A preliminary evaluation of terrain in the territory of Semipalatinsk nuclear test site and in Kaynar village in particular in has been already carried out. That is done to establish Kaynar village as a benchmark facility for the model experiments.

The soil is white-chestnut, low power, not salted with sub-clay mechanical composition.

The climate is characterized by dryness and it is very continental. Summers are hot and dry, springs are short, dry and cool. Winters are severe with a steady snow integument, and strong

winds. Thus, it would be plausible to us a territory in droughty, semi- desert zone, in particular one near Chingistau mountain that offers several types of landscapes, conical-shaped hill and flatness. On a contour the terrain reminds a semi-desert part of Goby (Mollgolia) with alternation of arrays of small mountains, conical-shaped hill, divided by valleys. The terrain bounds with Karaganda area and they are on intersection 49-30 northern latitudes and 77-45 western longitudes.

Precipitation

An annual amount of falling out is minimum 349 mm. The greatest amount of 43 mm falls out in June. In July air heats reach 40⁰ C, therefore considerable proportion of precipitation is lost via evaporation. The greatest height of a snow integument in winter period is 8 cm.

Plants (vegetation)

The vegetation of deserted and semi-deserted zones consists predominantly of kxerophytes. In the terrain the dominant association is artemisia - cereals .

Population

In the territory there are 2 villages: Kainar and Karaolen with population of 515 and 38 court yards respectively. The common population is 2635 persons including 1330 children under 17 years.

Facilities (economy).

The main economic activity is sheep breeding.

Territory

The territory of arable lands by vegetation is 500 ha of grain fields, 7 ha of potato fields, 2 ha of vegetables, the remaining of 35310 ha is pasture (1300 ha of them are wetlands).

Task 1: Greening

Applicability of 40 cultivars and species of trees, shrubs and grasses for organization of vitamin gardens and purification of radioactivity contaminated soils in Kainar and Karaolen villages will be evaluated. The seedlings and seeds characterized by high productivity and resistance to different stresses will be taken from Kazakhstan botanical gardens. The growth, development and productivity of the plants will be studied.

Task 2: Plantation in the desert regions

The land in Semipalatinsk region is poorly suited for plantation of the garden trees and shrubs. There is not enough water, wind constantly blows and there are high summer and winter temperatures (in winter the temperature can fall to -40 C). The soil here is poor in microelements and rich in heavy clay. In these conditions a special treatment of the soil is required. We are going to add some microelements, lime, gyps, biogel (the latter preserves water during the vegetation season) and planting seedlings adapted to local conditions. The seedlings will be growing in special plastic bags or in the stony soils in special trenches with specially prepared soil .

Task 3: Purification of the land from radioactivity/

We shall apply a phytoremedation method for purification of the contaminated soils in Semipalatynsk polygon. To proceed we will plant more then 40 shrubs, trees and grass in the places with different levels of radioactivity ranging from 50 tol 3000 microrentgen per hour. During the vegetation period and in autumn we shall measure the level of accumulated radioactivity. On the basis of this data we shall chose the species with the highest reading of metal and radioactivity accumulation. The plants on the experimental sites will be planted according to our methods. The most attention will be given to accumulation of radioactivity by the fruit trees. We will prepare recommendations on plantation, nursing and discharge of plants that accumulate some quantity of radioactivity.

Task 4: Preparation of recommendations

Special recommendation will be prepared on greening of Semipalatinsk villages, organization of vitamin gardens with high content of antioxidants in fruits as well as improvement of ecological situation and population health. The recommendations will be applicable to different places of Semipalatinsk region. Two big vitamin gardens, one in Kainar and the other in Karaolen, will be established in 3 years

Task 5: Genetical consequences of nuclear tests at polygon plants.

In the course of the research different biochemical parameters of plants will be studied. We will conduct an analysis of the compound complex of enzymes, proteins and DNA of different species that grow in conditions with different radioactivity levels. We are expecting to get information about genetic changes in experimental plants and the directions of the revealed changes. It could be possible to find genes that are responsible for resistance to a high level of radioactivity.

Deliverables, application and dissemination of findings

The project can be accomplished in three years as the teams have already performed the necessarily preliminary work, including research of methods and technique for the above mentioned objectives. All participants are well experienced in this field of research. The results obtained by all contractors will be compared regularly in direct communications and at annual meetings. These discussions will be help in researching characteristics of plant adaptation to local conditions and phytoremediation in various radioactivity levels.

The project involves an initial meeting between the partners for coordination of the working program as well as annual meetings for presentation of findings. Annual intermediate and final reports will be send by the co-ordinators to the headquarters of International Hilfsfonds e.V.

The results will be published in scientific journals and reported at conferences. Common publications will indicate names of the corresponding authors and a reference to International Hilfsfonds e.V. financial support will be made.

Research Team

Team leader: Prof.Kanat Sarsenbaev, Institute Botany and Phytointroduction of the National Academy of Sciences, Almaty, Republic of Kazakhstan, chief laboratory of plant physiology, specialist in greening places with high levels of radioactivity and high heavy metals content in soil.

He is responsible for a preparing application and co-ordination of the project, contacts with the headquarters of International Hilfsfonds e.V. and participation in workshops are aimed at programme ,discussion and intermediate and final report presentation.

Other team members:

1. Prof. A. Seisebaev radiobiology specialist

2. Dr. O. K. Abdurachmanov radiobiology specialist

3. vacancy medicine specialist

4. PhD student molecular biology specialist

5. PhD student U.U.Esnazarov ecology specialist

6. Dr. A. V. Kokorev agriculture specialist

Technical staff: one molecular biologist laborant, two biochemic laborants, 10 workers and guards.

Team Tasks:

· plantation of 40 species and cultivars in Kainar village, Karaolen village, Degelen, Experimental field, Atomic lake

· development of new plantation and phytoremediation methods

· evaluation of the effect of low constant doses of radiation on local and experimental plants employing biochemical methods such as PCR, SDS-electrophoresis, isozymes analysis

· study the radioactivity accumulating activity of examined plants.

Workshop discussions are expected to contribute in exchanging ideas and preparing intermediate and final reports

Participation in common publications

MANAGEMENT

At the initial partners meeting, partners will discuss steps of the program and coordination of work in great details.

The seedlings and seeds will be delivered from botanical gardens of Institute of Botany and Phytointroduction to Kainar, Karaolen, Atomic lake, Experimental field, Degelen and Kurchatov. According to specially designed schemes the seedlings and seeds will be planted by the workers and participants of the project and be closely watched and analyzed for three years.

Each team member will perform experiments and analysis, prepare review of the results for meetings and write intermediate and final reports. Co-ordinators will be responsible for submitting the annual reports to International Hilfsfond e.V. The common publications will be prepared by separate teams members and discussed at the meetings. Two annual meetings are planned for discussion of results and preparation of intermediate reports and the third meeting is planned for preparation of the final report.

Summary

Rehabilitation of the contaminated areas and poor health population of Semipalatinsk polygon area is a very important task. The problem is aggravated by the fact that radioactive pollution has effect of passing genetic damages to the next generations. Now the present time of grandchildren whose grandfathers and grandmothers were irradiated by the radioactivity. According to the medical data there is a high percentage of Semipalatinsk polygon population suffering various diseases. To decrease the process it is very important to purify the soil from radioactive substances and provide people with highly active natural antioxidants such as fruit. The purpose of our investigation is to try to solve these problems.

The proposed rehabilitation method is very simple: growing of the special plants with a high metal accumulative capability and the antioxidants productivity. These plants will supply population by the vitamins and purify the soil. The variety of species presently used in greening and eat by the inhabitants is very poor. We have to select species and cultivars with high resistance to local hard climate conditions, high productivity and heavy metal binding activity. Before us nobody has worked on this acute problem of Semipalatinsk polygon. In case of positive decision we could use this method in another world regions, where there is problem with antropogenically contaminated soil, water, air and shortage of vitamins.

For effective plantation of chosen species we will used the following.

1. a special gel with high absorbent capacity

2. microelements

3. specially treated soil

4. watering system

All the biochemical parameters, the content of radioactivity in different plant organs will be scrutinized using sensitive modern methods. Besides the practical result we shall also work on some theoretical issues. During the study of the plants that are exposed to constantly low radiation doses, we expect to find out genetic changes. For us it is very interesting to know the direction of these changes, i.e. most sensitive sites metabolism of DNA and proteins. Action of low chronic doses of radiation is very common situation in the majority of big sites. The burned oil, petrol, coal (which are used as resources of energy) have a radioactivity. Our method of purification could also be put into service there.

The advantage of this project is based on a new approach to the rehabilitation problem by combining the phytoremediation method and a special technology of growing the fruit trees highly resistant wild species in order to improve the soil and health of people in Semipalatinsk polygon. The objective of this project is not only rehabilitation, but also to find the ways of changing a genotype that has been exposed to constant doses of radioactivity. The obtained results can be used in different regions where there are high levels of radioactivity.

. My budget will consist of:

year

1

2

3

1.Plant material,

transport 10

2.labor cost 5 5 5

3.equipment 15

4.traveling 10 5 5

in Kazakhstan

5.Traveling 2 2 2 between countries

6.Overhead 15% 9675

Total : 75000$

SCIENTIFIC PUBLICATIONS OF PROPOSAL PARTICIPANTS

Sarsenbaev K.N.,Imanbaeva A., Gourret J.P., Misset M.T.Biological effects of different oxidative stresses on plant species in Central Kazakhstan. 2-nd winter research conferences."Cellular adaptation to oxidative stress oxidative DNA damage. Abiotic models of antioxidant enzymes",Les Deux Aples,France,22-27,!, 1995

Sarsenbaev K.N.,Zaka R.,J.P.Gourret, Misset M.T. Effects biologiques d"une contamination radioactive chronique sur des population de Stipa sareptana (Poaceae,Arundinoideae) dans la region du polygone de Semipalatinsk, Kazakhstan, Proccedings of Rennes university,1995,N1,p.106-108

Sarsenbaev K.N., Imanbaeva A., Fursova N., Esnazarov U., Sarsembaeva M., Mambetkulova K. Plantation in SEVIL climatic conditions and antropogenic contaminated regions of Kazakhstan. In the materials of Second International Syposium "Afforestation in arid and desert areas applying the Kallidendron method,p.1-15, Brussels, Belgium,1999

Sarsenbaev K.N., Mambetkulova K.K., Sarsembaeva M.V., Esnazarov U.U., Resistance and adaptation of plants in SEVIL climatic conditions of Kazakhstan on the level of enzymes. Proceedings of National Academy of Sciences of Kazakhstan,N5,p.48-56,1999

Sarsenbaev K.N., Esnazarov U.U., Starikova LM., Sarsembaeva M.V., Seisebaev A.T.,Misset M.T., Gourret J.P. Influence of low chronical doses of radiation on compound complex of enzymes and proteins of Stipa cappilata from Semipalatinsk polygon. Abstracts of reports International conference on Non-Proliferation problems, Almaty-Kurchatov,p.82,1997

Sarsenbaev K.N., Esnazarov U.U., Starikova L.M., Jakupova G., Sarsembaeva M.V.,Fursova N., Seisebaev A.T.,Misset M.T.,Gourret J.P. Influence of low chronical doses of radiation on compound complex of enzymes and proteins of Stipa cappilata from Semipalatinsk polygon. In book: Radiation safety and social,ecological problems of Kazakhstan, p.76-83, Science Press,1998, Almaty,Kazakhstan

Sarsenbaev K.N., Esnazarov U.U., Starikova L.M., Sarsenbaeva M.V., Fursova N.,Jakupova G., Seisebaev A.T.,Misset M.T.,Gourret J.P. Low chronical doses of gamma irradiation - influence on different fractions of proteins of Stipa cappilata seeds. In bok: Radiation safety and social,ecological problems of Kazakhstan, p.83-87,Science Press,1998,Almaty,Kazakhstan.

Sarsenbaev K.N., Baitulin I.O., Dashkevich A.P., Ksembaev A.R., Rachimbaev I.R. Recomendations on greening the places near metallurgy plants of Kazakhstan regions of Altai,Almaty,1982,p.1-26

Sarsenbaev K.N., Mesentseva N.I., Baitulin I.O.,Sitnikova A.S. Recomendation on assortment gas resistannt herbs for industrial contaminated places of Central Kazakhstan,Karaganda,p.1-22,1986

Sarsenbaev K.N., Polymbetova F.A. Role of enzymes in resistance of plants. Pulish House Science, Al;maty,1986,181 p.

Sarsenbaev K.N.,Chenal C., M.T.Misset et all. Scientific report "Ecobiological consequences of radioactive pollution to nuclear tests performed between 1949 and 1989 in Semipalatinsk region(Kazakhstan). Rennes,France,1999, INTAS grant, Ref. N Intas-93-1421

Sarsenbaev K.N., Sitnikova A.S. Recomendations on greening of industrial metallurgy enterprises of Karaganda region. Karaganda,p.24,1982

Abdurachmanov O.K.,Filipova N.F.,Krukova L.M. Influence of ionizating radiation on some physiologycal parametres of Glyzyrriza glabra. J.Radiobiology,v.4,N5, 1974, p.787-789

Abdurachmanov O.K. Action physical and biological active substances on growth and development of Glyzyrriza . Monography,Almaty,1998,p.135

WORK PROGRAMME

RESEARCH PROJECT

TITLE: GENETIC EFFECTS IN NATURAL PLANT AND ANIMAL POPULATIONS EXPOSED TO CONTAMINATION AT ARAL SEA REGION (KAZAKHSTAN)

Objective: to study cytogenetic and molecular-genetic effects of chronic contamination in different indicator species of plants, insects, amphibians and small rodents and assess and forecast genetic effects of contamination impact on natural associations.

The Project objective will be achieved by implementing the following tasks:

· Describing the present status of the biocenosis, determining plant and animal biodiversity and selecting indicator species for genetic studies.

· Assessing the ecological situation in the natural habitat of selected organisms and determining the natural and artificial radionuclides (²³⁸U,¹³⁷Cs, ⁹⁰Sr, ²³⁹Pu, etc.), heavy metals (Zn, Cu, Cd, Fe, Pb, etc.) , Na⁺, K+, CI^-1 ,CO₃^-2 , SO₄^-2, pesticides, heptyle concentrations in the different compartments of biological chains “soil-plant-animal”.

· Studying cytogenetic, genetic and molecular-genetic effects of different local ecological stresses on different model species of plants and animals.

· Identifying and analyzing possible adaptive responses of individuals and populations to chronic exposure to the stresses, determining the mechanisms and extent of adaptation in different plant and animal individuals and populations according to their resistance, reproduction type, ploidity and other biological properties.

· Studying the relationship between various genetic alterations in exposed populations and possible ecological shifts as revealing such shifts is one of the basic issues of ecological regulation of the environment contamination by toxic substances.

· Developing basic principles for organization and operation of long-term comprehensive genetic monitoring at the Aral Sea Region (ASR)

Background & Justification

The analysis of genetic processes in natural populations and ecosystems is an essential element to evaluate the environmental impact and ecological consequences of toxin action in contaminated areas. A review of the literature shows that the assessment of genetic effects of environment contamination on man, fauna and flora is a very complicated problem (1, 2, 6, 12, 13, 15, 18, ). Many aspects have to be considered among which: molecular mechanisms of genetic damages and reparation on cellular and organism levels, manifestation of mutational alterations, accumulation of induced mutations and their elimination by populations of different species. Consequently, the assessment and prediction of the genetic effects of different contaninators in natural ecosystems is only possible if the required information about the impact of their acting at molecular, cellular, organism, population and biocenose levels is available.

So far, the ecological situation over the entire ASR territory is far from being completely depicted. The main causes of the Aral Sea economic crisis are the regulated flow of Syr-Daria and Amu-Darya rivers coincnding with the shallowness of the seventies; that sharp reduction of the Aral Sea water surface; the subsequent change of regional climate towards aridization and rise in continentality; the rise in irrigation and drinking water mineralization; the pollution of ecosystems by municipal waters and the residues (12, 13, 14) of agrochemicals (aggressive components – NH₄⁺, NO₂^- ,NO₃^- ,Zn, Cu, Pb, Cd , different pesticides, particularly HCCH – 0.01 – 0.34 mg/l; DDT – 0.04 – 0.70 mg/l; thiodan,Acrex, TCM-3, Baleiton, Methaphos); sedimentation of salts carried away from the dessicated bottom of the Aral Sea; the negative influence of the Baikonur cosmodrome on the ecosystems of the ASR, including pollution by very toxic component of rocket fuel – heptyle and dioxins (POP’s) from the fragments of launcher rocket fall on the ground, particular "Proton". We have to add the contamination undeground water by the residues of uranium (8) and lead industries (uranium extraction by the ground leaching metods is using in Karamurun, Irkol, Harasan of Syrdaryanskaya uranium province ) and acceleration of natural mutation process by the high concentrations of salts ( the average salinity of Aral Sea water was 32.89% in 1991 and 33.77% in 1992 ) too (we combined all this substances and influences under the words as - toxins or contaminators, or pollutants). Some ASR areas were exposed to severe contamination able to cause serious genetic effects. In particular, in ASR process of blood creation and immunity of the population is significantly damaged. The frequency of chromosome aberrations in the lymphatic corpuscles of perepheral blood (chromosome abberations on 1000 methaphases in the Aralsk and Jhalagash regions of the Kyzylorda are 1.1±0.18 against 0.4±0.2 in Almaty). Analysis of chlorinated persistent substances and heavy metals showed high level of pesticides, Pb, Cd in the blood lipids of children fron Aral Sea. They have considerable changes of genetical structure of cells (it was discovered according to stigma degree, violation of cell structure, nonhistone proteins, polymorphism of soluble proteins). However, the issue of genetic effects of chronic action of local contaminators for natural populations plants and animals inhabiting the ASR is in fact still open (1. 11, 18, 19).

At the same time, the world scientific community has nothing similar to the Aral Sea Region. This is world level ecological catastrophe The territory provides a natural ecological laboratory and opportunities to monitor biological and ecological processes. Systematic observations of biological impact on biological objects would allow to determine the effects of chronic toxic substances exposure at different organization levels of life – from molecules to populations. Long term monitoring is a determinative factor for revealing the regularities of nature self-cleaning and self-recovery, for studying mechanisms of living organism adaptation and determining resistance of species and the ecosystem as a whole.

As a result of such an integrated study , the present ecological status of biocenosis will be assessed, mechanisms and results of mutation processes in chronically exposed populations will be determined in relation with concentration of toxins, which presents in the environment and other particular features of the habitat and reproduction ways of organisms.

The results to be obtained within the framework of this Project are expected to lead to a wide range of direct and indirect applications:

· they will primarily serve to establish the basis for the organization of a global genetic monitoring of the ASR;

· they will help at developing relatively simple and easy-to-apply methods for biological monitoring of toxic substances impact that could be applied elsewhere and extended to other types of environmental pollution;

· they will also contribute to increase the present knowledge in the field of chronic low concentrations different pollutants impact on ecosystems and their components;

· they will update the current knowledge about mutation effects and dynamics, which principles can be extrapolated to mutations of human populations;

· they will provide scientific evidence to help at justifying exposure concentration limits under normal and emergency conditions and dose criteria for the rehabilitation of contaminated sites;

Moreover, the information gathered during the implementation of the Project will be of great importance for other desert regions of Kazakhstan. The monitoring data concerning the ASR will be made available to the international scientific community, namely through a to‑be‑created web page,

Scientific/Technical Description

Research programme

Studying the effects of pollution in natural populations and communities is an important issue as it is related to actual exposure of natural populations inhabiting ASR. Study of genetic effects of environment contamination is one among the issues of highest priority which RK Institutes are involved in. Contamination of biogeocenosis by complex of toxic products is considered as a new abiotic factor of organism habitat which affects natural communities. Thus, studying the organism response to complex action of different toxins due to long-term living in contaminated biogeocenosis can reveal new [Ж Н1] mechanisms and effects which would be impossible to detect under laboratory conditions and which would reveal new aspects of genetic risk of plant and animal exposure. Based on results obtained by plant and animal studies, one can indirectly assess the risk of similar genetic effects in man. Studying the response of organisms living in natural habitat to the impact of chronic exposure to toxins is of particular interest for defining criteria for the protection of the environment against different types of contaminators.. The issue of exposure to low doses of radiation, heavy metals, pesticides, heptyle is now gaining a particular importance in connection with a non-threshold linear concept in the field of biology. Meanwhile, issues of low dose impact on populations are of primary importance for analysis of contamination test consequences and for justifying exposure dose limits admitted for populations under normal and emergency conditions and defining rehabilitation criteria.

Despite several measurement campaigns and research programs, the ecological situation on the ASR is far from being completely depicted. So far, comprehensive contamination maps are not yet available, specially regarding the distribution of radionuclids, heavy metals, pesticides, salinity. Neither has a thorough scientific assessment of the genetic consequences for natural populations yet been performed by now. Today, almost forty years after the begining “cotton revolution” in USSR , the territory of the ASR may be used as a huge natural bio‑ecological laboratory. There, areas of different contaminates levels and spectrum provide an opportunity to study the environmental and geological behavior and transport of toxins. The stress impact on living organisms and ecosystems structure and functioning can be assessed. Quantifying the genetic effects in plant and animal populations exposed to chronic treatment by toxins doses in general, and on the ASR in particular, is quite important.

A number of the Project results could be of a particular interest for improving the knowledge of some mechanisms relevant of the polution protection of man. In fact, no threshold can be invoked regarding stochastic effects caused by low doses, particularly heptyle. The observation of natural populations of plants and animals chronically exposed to local toxins for almost 40 years will certainly provide valuable arguments to support the choice of a dose‑effect relationship in the low dose range (7).

Long-term toxical exposure of living organisms causes genetic alterations, which may lead to mutations, almost always deleterious. However, in chronically exposed population, adaptation mechanisms could be expressed with time. A prerequisite of organism adaptation to elevated stress background is the genetic heterogeneity of species and the selection of the most resistant individuals. Expression of adaptation mechanisms will be sought after in plants and animals chronically exposed for almost half‑a‑century.

Response of ecosystems and natural populations of plants and animals (not only single individuals) is a poorly documented topic in ecological research. Experimental treatment by toxins of natural biotopes also allowed such studies, but the number of ecosystems considered still remains very limited. Observations conducted on the ASR will provide valuable information of desert ecosystem response to long-term treatment by toxins and allow to identify monitoring indicators at different levels, from molecular-genetic to populational ones.

The use of plant as test systems in ecological-genetic studies allows obtaining an integrated assessment of mutagenic potential of chemical pollution, which is the most important criterion of ecological condition of the environment. Moreover, study of mutagenic effects of chronic polution on plants is important itself as plants are the most important part of the biosphere determining the well-being of the biosphere itself as a whole and that one of man as a part of the biosphere.

Among the insects, Chironomini are chosen to be studied as they are an essential component of the water and terrestrial biocenoses, provide self-cleaning of water bodies and support food chains of fish and birds. Chironomini's larvae are under constant impact of different pollution types and, therefore, at risk, as they live in bottom sediment absorbing different toxins. Chironomini's larvae posses polytene chromosomes that allow performing the cytogenetic analysis and assessing the effect of toxins at the chromosomal level. Study of natural mutation process in Chironomini and its alteration due to water body contamination can serve as an indicator of genetic destruction in water organisms.

Small rodents are widely found in the region of the Project interest. Therefore, analysis of genetic processes in small rodents is one of the most important elements of genetic monitoring. The proposed research project will contribute to assess the ecological risk indifferent areas and to set up rehabilitation priorities (9, 10, 13, 16, 17).

The present project is the first phase of a long-term study of pollution genetic effects on living organisms and the first stage of the comprehensive genetic monitoring of the ASR territory.

Expected Results and their Application

The present status of ATR vegetation and animal species diversity will be assessed; vascular plants will be listed and an inventory [Ж Н2] of ASR vertebrate species will be established.

- For the first time an [Ж Н3] assessment of ecological situation in places of natural populations habitat at the ASR will be made taking into account pollutants content and distribution in the environment, and dose burden of biological objects; [Ж Н4] patterns of toxic substances accumulation in plants and animals will be studied; particular features of animal habitat in contaminated biogeocenosis will be determined. For the first time a scientifically-ground assessment of genetic effects in natural populations of the ASR will be given based on experimental data obtained for a series of biological indicators using different criteria (direct, indirect, extrapolation and population); dose-dependence of genetic effects in various animal species will be studied under different exposure conditions .

- Chironomini communities (Diptera, Chironomidae) inhabiting ASR open water bodies will be identified using morphometric analysis and karyological identification; cytophotocards will be made for some unique Chironomini species and spectrum and frequency of inverse disc sequences will be determined as well as their genotypical combinations in each chromosome arm dominant in Aral populations as compared to other regions will be identified; particular features of cytogenetic structure, frequency of chromosome abnormalities will be identified as well as alterations in DNA nucleotide sequences and compound complex of certain enzyme systems and proteins depending upon the level of the water body contamination will be determined.

- Mutagens-induced abnormalities in somatic and generative cells of small rodents (Allactaga major Kerr., Allactaga saltator Ewersm., Citellus erythrogenus Brandt, Apademus agrarius Pall.) and reptiles (Еremias argata Pall., Lacerta agilis Linn.) continuously inhabiting contaminated ASR areas will be determined using up-to-date methods of cytogenetics and biochemical genetics.

.

- Possible ways of population adaptation to chronic ionizing radiation will be analyzed, a manifestation extent of adaptation changes in different plants and animals depending on their radiosensitivity, reproduction type, ploidy and other biological properties will be determined.

- Certain conclusions upon remote genetic consequences of polution for natural populations of organisms will be drawn.

- The major principles of organization and operation of genetic monitoring on the territory of the ASR will be elaborated; recommendations on use of contaminated biogeocenosis for scientific purposes will be developed.

The results to be obtained within the framework of this Project are expected to lead to a wide range of direct and indirect applications:

· they will primarily serve to establish the basis for the organization of a global genetic monitoring of the ASR;

· they will help at developing relatively simple and easy-to-apply methods for biological monitoring of polution impact that could be applied elsewhere and extended to other types of environmental stresses;

· they will also contribute to increase the present knowledge in the field of chronic low dose impact on ecosystems and their components;

· they will update the current knowledge about mutation effects and dynamics, which principles can be extrapolated to mutations of exposed human populations;

International exchange of Project results will assist in understanding the effects of potential contamination at other desert sites.

REFERENCES

1. Sultangazin U. Applied problems of sustainable development of the Aral Sea Region. Proceedings of International ,scientific conference, September 9 – 11, 1997, Almaty, “Tethis”, p. 5 – 7

2. Pachikin K., Yakunin G. Soil resources of the Kazakhstan part of the Aral Sea region. Proceedings of International ,scientific conference, September 9 – 11, 1997, Almaty, “Tethis”, p.11 – 19

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13. Dementyev V., Sokolov S., Askarov A.,Makarov D. Spatial-seasonal variations in the composition of the natural waters in a zone of waters in a zone of water change in the Syr-Darya delta. Proceedings of International ,scientific conference, September 9 – 11, 1997, Almaty, “Tethis”, p.65 – 68

14. Amirgaliev A. The evaluation of the present salty regime and the level of pesticide pollution of the Aral Sea. . Proceedings of International ,scientific conference, September 9 – 11, 1997, Almaty, “Tethis”, p.89 – 93

15. Dimeyeva L. Floristic and phytocenotic diversity of coastal ecosystems of the Aral Sera: present-day state and tendencies of change. Proceedings of International ,scientific conference, September 9 – 11, 1997, Almaty, “Tethis”, p.107 – 111

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SUMMARY

The consequences of drying Aral Sea is terrible. This is desertification of huge region, land salination, pollination due to the run-off pesticides, fertilisers, heavy metals, hyptyle, dioxins, radioactivity. Under influence of different and strong enough stresses the genetical changes of animals and plants is expectated process. It was in Semipalatinsk polygon, Chernobyl, Altay , near metallic plants and etc. The high level of diseases of man in Aral Sea Region is a first symptom of genetical damages. The teams of scientists who worked during long time in different nuclear polygons decided to approve existance of genetical changes animals and plants in Aral Sea Region. It was chosen the most sensitive and effective methods of analysis morpho-biochemical and genetical features of typical plants and animals, which were effective in radiobiological investigations..

The species will be studied on intact, organs, cell and molecular level. The high qualification and experience of teams members will help to find out the genetical transformation plants and animals under acting the local contaminators. The decision of this problem is very important, because explain high level diseases in this region and the causes of changing the flora and fauna of this Region.The Project implementation will allow to evaluate the potencial dangering of desertification and contamination area by toxic industrial compaunds. Project activities will support fundamental and applied research in the field of ecolgical genetics of natural populations and in the field of environment protection. The evaluation level of genetical transformation of animals and plants gives possibility to develop the system of social help to population on the basis degree of genetical risk to people who lives in the different parts of ASR.

GENETIC EFFECTS IN NATURAL PLANT AND ANIMAL POPULATIONS EXPOSED TO RADIOACTIVE CONTAMINATION AT SEMIPALATINSK TEST SITE (KAZAKHSTAN)

(Project Title)

Project Objective: to study cytogenetic and molecular-genetic effects of chronic ionizing radiation in different indicator species of plants, insects, amphibians and small rodents and assess and forecast genetic effects of radiation impact on natural associations.

The Project objective will be achieved by implementing the following tasks:

· Describing the present status of the biocenosis, determining plant and animal biodiversity and selecting indicator species for genetic studies.

· Assessing the radiation situation in the natural habitat of selected organisms and determining the natural and artificial radionuclides (⁴⁰K, ²³²Th, ²³⁸U,¹³⁷Cs, ⁹⁰Sr, ²³⁹Pu, etc.) and heavy metals (Zn, Cu, Mn, Fe, Pb, Ni, etc.) concentrations in the different compartments of biological chains “soil-plant-animal”.

· Studying cytogenetic, genetic and molecular-genetic effects of chronic ionizing radiation on different model species of plants and animals.

· Identifying and analyzing possible adaptive responses of individuals and populations to chronic exposure to ionizing radiation, determining the mechanisms and extent of adaptation in different plant and animal individuals and populations according to their radiosensitivity, reproduction type, ploidity and other biological properties.

· Studying the relationship between various genetic alterations in exposed populations and possible ecological shifts as revealing such shifts is one of the basic issues of ecological regulation of the environment contamination by radioactive products.

· Developing basic principles for organization and operation of long-term comprehensive genetic monitoring at the STS territory.

The analysis of genetic processes in natural populations and ecosystems is an essential element to evaluate the environmental impact and ecological consequences of ionizing radiation in contaminated areas. A review of the literature shows that the assessment of genetic effects of environment radioactive contamination on man, fauna and flora is a very complicated problem. Many aspects have to be considered among which: molecular mechanisms of genetic damages, radiation mutagenesis and reparation on cellular and organism levels, manifestation of mutational alterations, accumulation of induced mutations and their elimination by populations of different species. Consequently, the assessment and prediction of the genetic effects of ionizing radiations in natural ecosystems is only possible if the required information about the impact of radiation at molecular, cellular, organism, population and biocenose levels is available.

So far, the radioecological situation over the entire STS territory is far from being completely depicted. Some STS areas were exposed to severe radioactive contamination and high radiation doses able to cause serious genetic effects. However, the issue of genetic effects of chronic ionizing radiation for natural populations inhabiting the STS is in fact still open.

At the same time, the world scientific community has nothing similar to the Semipalatinsk Test Site. This territory provides a natural radiobiological laboratory and opportunities to monitor radiobiological and radioecological processes. Systematic observations of radionuclide transport and biological impact on biological objects would allow to determine the effects of chronic radiation exposure at different organization levels of life– from molecules to populations. Long term monitoring is a determinative factor for revealing the regularities of nature self-cleaning and self-recovery, for studying mechanisms of living organism adaptation and determining radioresistance of species and the ecosystem as a whole.

As a result of such an integrated study , the present ecological status of biocenosis will be assessed, mechanisms and results of mutation processes in chronically exposed populations will be determined in relation with exposure rate, radionuclide species present in the environment and other particular features of the habitat and reproduction ways of organisms.

The results to be obtained within the framework of this Project are expected to lead to a wide range of direct and indirect applications:

· they will primarily serve to establish the basis for the organization of a global genetic monitoring of the test site ;

· they will help at developing relatively simple and easy-to-apply methods for biological monitoring of radiation impact that could be applied elsewhere and extended to other types of environmental pollution;

· they will also contribute to increase the present knowledge in the field of chronic low dose impact on ecosystems and their components;

· they will update the current knowledge about mutation effects and dynamics, which principles can be extrapolated to mutations of exposed human populations;

· they will provide scientific evidence to help at justifying exposure dose limits under normal and emergency conditions and dose criteria for the rehabilitation of radiocontaminated sites;

· they will also provide sounded arguments for making decisions concerning the uncontrolled migration of organisms within and outside radioactively contaminated areas.

Moreover, the information gathered during the implementation of the Project will be of great importance for other regions of Kazakhstan where 40 so called "peaceful underground nuclear tests for industrial purposes" were conducted: "Galit", "Lira", "Batolit", "Region" and "Meridian".

The monitoring data concerning the STS will be made available to the international scientific community, namely through a to‑be‑created web page,

Foreign collaborators will be welcome to take part in the Project implementation. Their role includes not only exchange of scientific data but also joint studies using equipment and software provided. The cooperation would be certainly more effective if the level of collaborators’ interest would allow them to find their own financial sources for implementation of joint activities.

	Project Proposal	#
Section 3. DETAILED PROJECT INFORMATION
1. Introduction and Overview

Studying the effects of ionizing radiation in natural populations and communities is an important issue as it is related to actual exposure of natural populations inhabiting nuclear test sites. Similar situations and problems are also encountered within the entire nuclear fuel cycle and fissile material mining, processing, use and storage. Therefore, study of genetic effects of environment radioactive contamination is one among the issues of highest priority which RK NNC is involved in. Contamination of biogeocenosis by radioactive products is considered as a new abiotic factor of organism habitat which affects natural communities. Thus, studying the organism response to ionizing radiation due to long-term living in contaminated biogeocenosis can reveal new [Ж Н5] mechanisms and effects which would be impossible to detect under laboratory conditions and which would reveal new aspects of genetic risk of plant and animal exposure. Based on results obtained by plant and animal studies, one can indirectly assess the risk of similar genetic effects in man. Studying the response of organisms living in natural habitat to the impact of chronic exposure to ionizing radiation is of particular interest for defining criteria for the protection of the environment against ionizing radiation. Such standards together with radiation-hygienic regulations are very important for regulation of atomic energy use for peaceful purposes. The issue of exposure to low doses of ionizing radiation is now gaining a particular importance in connection with a non-threshold linear concept in the field of radiobiology. Meanwhile, issues of low dose impact on populations are of primary importance for analysis of nuclear test consequences and for justifying exposure dose limits admitted for populations under normal and emergency conditions and defining rehabilitation criteria.

Despite several measurement campaigns and research programs, the radioecological situation on the STS is far from being completely depicted. So far, comprehensive contamination maps are not yet available, specially regarding the distribution of ⁹⁰Sr and Pu isotopes. Neither has a thorough scientific assessment of the genetic consequences for natural populations yet been performed by now.

Today, fifty years after the first bomb test, the territory of the test site may be used as a huge natural radio‑bio‑ecological laboratory. There, areas of different radioactive levels and spectrum provide an opportunity to study the environmental and geological behavior and transport of radionuclides. The radiological impact of remote acute exposure, combined to a protracted, chronic exposure to «low‑to‑moderate» radiation dose rates, on living organisms and ecosystems structure and functioning can be assessed.

Quantifying the genetic effects in plant and animal populations exposed to chronic low radiation doses in general, and on the STS in particular, is quite important.

A number of the Project results could be of a particular interest for improving the knowledge of some mechanisms relevant of the radiation protection of man. In fact, no threshold can be invoked regarding stochastic effects caused by low irradiation doses. The establishment of a dose‑response relationship for man in the range of low doses is, however, impaired by the lack of data in these dose ranges (ICRP-60). Extrapolations from the responses observed for acute, high doses (Hiroshima and Nagasaki), unevenly distributed, high doses given to patients, Ra workers and populations exposed to elevated natural background (mainly high‑LET alpha contribution) are not fully convincing. The observation of natural populations of plants and animals chronically exposed to ionizing radiation (gamma contribution, ⁹⁰Sr and Pu) for almost 50 years will certainly provide valuable arguments to support the choice of a dose‑effect relationship in the low dose range.

Long-term radiological exposure of living organisms causes genetic alterations, which may lead to mutations, almost always deleterious. However, in chronically exposed population, adaptation mechanisms could be expressed with time. A prerequisite of organism adaptation to elevated radiation background is the genetic heterogeneity of species and the selection of the most radioresistant individuals. Expression of adaptation mechanisms will be sought after in plants and animals chronically exposed for half‑a‑century.

Response of ecosystems and natural populations of plants and animals (not only single individuals) is a poorly documented topic in radioecological research. Experimental irradiation of natural biotopes was performed at Brookhaven (USA) and Cadarache (Fr). Kyshtym and Chernobyl accidents also allowed such studies, but the number of ecosystems considered still remains very limited so far and only Kyshtym may be considered as an area submitted to long‑term chronic exposure. Observations conducted on the STS will provide valuable information of steppe ecosystem response to long-term irradiation and allow to identify monitoring indicators at different levels, from molecular-genetic to populational ones.

The use of plant as test systems in ecological-genetic studies allows obtaining an integrated assessment of mutagenic potential of both radioactive and chemical pollution, which is the most important criterion of ecological condition of the environment. Moreover, study of mutagenic effects of chronic radiation on plants is important itself as plants are the most important part of the biosphere determining the well-being of the biosphere itself as a whole and that one of man as a part of the biosphere.

Among the insects, Chironomini are chosen to be studied as they are an essential component of the water and terrestrial biocenoses, provide self-cleaning of water bodies and support food chains of fish and birds. Chironomini's larvae are under constant impact of different pollution types and, therefore, at risk, as they live in bottom sediment absorbing radionuclides and heavy metals. Chironomini's larvae posses polytene chromosomes that allow performing the cytogenetic analysis and assessing the radiation effects at the chromosomal level. Study of natural mutation process in Chironomini and its alteration due to water body radioactive contamination can serve as an indicator of genetic destruction in water organisms.

Small rodents are widely found in the region of the Project interest. Therefore, analysis of genetic processes in small rodents is one of the most important elements of genetic monitoring.

And finally, it should be said that local populations (hunters, shepherds, etc.) have access to the STS. In order to allow a safe exploitation of the former nuclear test site, rehabilitation is needed. The Kazak Government has adopted a rehabilitation project for the STS. This project is supported by the UN's Resolution adopted by the 52^nd UN General Assembly on December 16^th, 1997, concerning the "International Co-operation and Co-ordination of Population Rehabilitation and Environment Remediation and Economic Development of Semipalatinsk Region". The proposed research project will contribute to assess the radiological risk indifferent areas and to set up rehabilitation priorities.

The present project is the first phase of a long-term study of nuclear tests genetic effects on living organisms and the first stage of the comprehensive genetic monitoring of the STS territory.

Project Objective: to study cytogenetic and molecular-genetic effects of chronic ionizing radiation in different indicator species of plants, insects, amphibians and small rodents and assess and forecast genetic effects of radiation impact on natural associations.

Project Tasks. Basic Project tasks include:

· Describing the present status of the biocenosis, determining plant and animal biodiversity and selecting indicator species for genetic studies.

· Assessing the radiation situation in the natural habitat of selected organisms and determining the natural and artificial radionuclides (⁴⁰K, ²³²Th, ²³⁸U,¹³⁷Cs, ⁹⁰Sr, ²³⁹Pu, etc.) and heavy metals (Zn, Cu, Mn, Fe, Pb, Ni, etc.) concentrations in the different compartments of biological chains “soil-plant-animal”.

· Studying cytogenetic, genetic and molecular-genetic effects of chronic ionizing radiation on different model species of plants and animals.

· Identifying and analyzing possible adaptive responses of individuals and populations to chronic exposure to ionizing radiation, determining the mechanisms and extent of adaptation in different plant and animal individuals and populations according to their radiosensitivity, reproduction type, ploidity and other biological properties.

· Studying the relationship between various genetic alterations in exposed populations and possible ecological shifts as revealing such shifts is one of the basic issues of ecological regulation of the environment contamination by radioactive products.

· Developing basic principles for organization and operation of long-term comprehensive genetic monitoring at the STS territory.

Project Location. The Project proposed will be implemented by the RK NNC Institute of Radiation Safety and Ecology (2 Krasnoarmeiskaya St. 490021 Kurchatov), RK MSE Institute Botany and Phytointroduction

( 36D, Timiriazeva St. 480090 Almaty).

2. Expected Results and their Application

2.1 The present status of STS vegetation and animal species diversity will be assessed; vascular plants will be listed and an inventory [Ж Н6] of STS vertebrate species will be established.

2.2 For the first time an [Ж Н7] assessment of radioecological situation in places of natural populations habitat at the STS will be made taking into account radiation exposure doses, radionuclides content and distribution in the environment, and dose burden of biological objects; [Ж Н8] patterns of radionuclides accumulation in plants and animals will be studied; particular features of animal habitat in radioactively contaminated biogeocenosis will be determined. For the first time a scientifically-ground assessment of nuclear test genetic effects in natural populations of the STS will be given based on experimental data obtained for a series of biological indicators using different criteria (direct, indirect, extrapolation and population); dose-dependence of genetic effects in various animal species will be studied under different exposure conditions particularly under low dose exposure;

2.2.1 Chironomini communities (Diptera, Chironomidae) inhabiting STS open water bodies will be identified using morphometric analysis and karyological identification; cytophotocards will be made for some unique Chironomini species and spectrum and frequency of inverse disc sequences will be determined as well as their genotypical combinations in each chromosome arm dominant in Semipalatinsk populations as compared to other regions will be identified; particular features of cytogenetic structure, frequency of chromosome abnormalities will be identified as well as alterations in DNA nucleotide sequences and compound complex of certain enzyme systems and proteins depending upon the level of the water body contamination will be determined.

2.2.2 Radiation-induced abnormalities in somatic and generative cells of small rodents (Allactaga major Kerr., Allactaga saltator Ewersm., Citellus erythrogenus Brandt, Apademus agrarius Pall.) and reptiles (Еremias argata Pall., Lacerta agilis Linn.) continuously inhabiting radioactively contaminated STS areas will be determined using up-to-date methods of cytogenetics and biochemical genetics.

2.2.3 Dependence of mutations found in some STS plant species (Stipa sareptana Besk., Stipa cappilata, Hordeum Bogdan Wilensky, Agropyron eristatum В., Pinus silvestris I, Iris halopila Poll, etc.) on chronic exposure dose rate and radionuclide forms will be determined.

2.2.4 Possible ways of population adaptation to chronic ionizing radiation will be analyzed, a manifestation extent of adaptation changes in different plants and animals depending on their radiosensitivity, reproduction type, ploidy and other biological properties will be determined.

2.2.5 Certain conclusions upon remote genetic consequences of nuclear testing for natural populations of organisms will be drawn.

2.3 The major principles of organization and operation of genetic monitoring on the territory of the test site will be elaborated; recommendations on use of contaminated biogeocenosis for scientific purposes will be developed.

The results to be obtained within the framework of this Project are expected to lead to a wide range of direct and indirect applications:

· they will primarily serve to establish the basis for the organization of a global genetic monitoring of the test site ;

· they will help at developing relatively simple and easy-to-apply methods for biological monitoring of radiation impact that could be applied elsewhere and extended to other types of environmental pollution;

· they will also contribute to increase the present knowledge in the field of chronic low dose impact on ecosystems and their components;

· they will update the current knowledge about mutation effects and dynamics, which principles can be extrapolated to mutations of exposed human populations;

· they will provide scientific evidence to help at justifying exposure dose limits under normal and emergency conditions and dose criteria for the rehabilitation of radiocontaminated sites;

· they will also provide sounded arguments for making decisions concerning the uncontrolled migration of organisms within and outside radioactively contaminated areas.

International exchange of Project results will assist in understanding the effects of potential radioactive contamination at other nuclear test sites.

3. Meeting ISTC Goals and Objectives

The Project implementation will allow former weapon specialists to switch over to peaceful activity. Project results will be highly important for conversion of scientific and technical potential of the former Semipalatinsk Test Site. Besides, Project activities will support fundamental and applied research in the field of radiation genetics of natural populations in areas of nuclear testing and in the field of environment protection.

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