Ecomonitoring in Rozhen Bulgaria

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BULGARIAN. SWISS BIODIVERSITY CONSERVATION PROGRAMME ROZHEN, SREDNOGORIEPROJEKT

ECOMONITORINGIN ROZHEN AND SREDNOGORIE I BULGARIA

Ministry of Environment SDC- SWISSAGENCY FOR NationalNature Protection Seruice DEVELOPEMENTAND COOPERATION Ecomonitoringin Rozhenand Srednogorier Bulgaria

Editors:Dimitar Peev. Klaus Ammann and Ari Artinian

Sofia 1997 CONTENT

1.FOREWORD

Pierre Galland Swiss Co-ordinatorof the BSBCPProject

2. INTRODUCTION

Dimitar Peev,Klaus Ammann Noteson thespecifics of Rozhenand Srednogorie control stations Dimitar Peev Formerexperience of ecomonitoringprograms as a basisfor the structureof the project. 3

3. METHODS

3.1.Population level Sfefan Kozhucharov, Dimitar Peev Biomonitoringof the environment:concept, structural scheme and patternof the information 4 Dimitar Peev, Sonra Tzoneva, Natalia Valjovska The roleof karyology,stomata, pollen and statisticalanalysis in the biomonitoringsystem of Bulgaria I lvanka Janeva An approachand resultsof thehydrobiological monitoring in Bulgaria 13 Kathrin Peter Methodologicaldevelopment and validationof indicatorsfor 10 integratedenvironmental observation Dencho Denchev, Atanas Kaimaktchiev, Kolishka Tsekova The micro-organismsas indicatorsof the environmentalpollution 27 3.2.Organismal level Klaus Ammann Multivariatecorrelation of depositiondata with lichendata 30 Roumiana Metcheva Smallmammals like bioindicators in zoologicalmonitoring 32 A/asko Atanassov The use of morphophysiologicaland cytogeneticalmethods in zoologicalmonitoring 34 Violeta Fakirova The fungias accumulatorsof contaminants 36 Dobri lvanov The use of lichensin biomonitoring 40 3.3.Macromolecular level Nina Bakardjieva Problemsand possibilitieswhen using physiological and biochemicaldata for biologicalmonitoring 42 Georgi Angelov lsoenzymesas bioindicatorsof environmentalpollution on the populationlevel 45 Simeon Popov, K. Stefanov, V. Bankova, Nedyalka Handjieva, K. Seizova, l. Elenkov Changesin lipidand secondaryplant metabolism and pollution bioindication 49 3.4.Abiotic level Urs Krdhenbiihl Measuringpollutants in plants,a chronologicalperspective in analyticalenvironmental chemistrv 52 EmitiaNikolova, Ari Artinian Energydispersive X-ray analysis and its versatileapplicability to environmentalsamples 55 Milen lovtchev Radioactivecontamination of the biosphere 62 4. RESULTS

4.1.Plant biomonitors Dimitar Peev,Ari Artinian, Emilia Nikolova, Sonia Tzoneva, NataliaValjovska Heavymetals and toxicelements contamination and pilotresults from some biotestsin phytomonitorsfrom Rozhenand srednogorie controlstations 66 Rilka Taskova, Dimitar peev, NedyatkaHandjieva, Simeon Popov changes in the concentrationsof iridoidglucosides in veronica speciesfrom Rozhenand Srednogorie 74 Nina Bakardjieva, Evgeni Ananiev, N. Christova. Biochemicalscreening of the effectof industrialpollution with veronicafrom Rozhenand srednogoriecontrol stations 79 Georgi Angelov lndustrialpollution lmpact on the isoenzymestructure of Rgzhen and Srednogoriepopulation s of Dactytisglomerata L. 86 4.2.Fungi and lichensas biomonitors Dobri lvanov contentof someelements in lichenfrom Rozhen background station 89 ii Violeta Fakirova, Emilia Nikolova, Ari Artinian Accumulationof the heavymetals and reactionsof selectedfungi speciesin Rozhenand Srednogorie 92 4.3.Micro-organisms as biomonitors Atanas Kaimaktchiev, Dencho Denchev, Kolishka Tsekova Microbialbiodiversity of soilfrom Srednogorie and Rozhen biomonitoringstations 97 4.4.Zoomonitors lvanka Janeva, Luchezar Pehlivanov, Yanka Vidinova, Stefan Stoichev, Violeta Tyufekchieva, Krassimir Kumanski A comparativeecological characterisation of lotic benthal zoocoenosesfrom two streamsunder different anthropogenic influencein Rozhenand Srednogorie 101 Roumiana Metcheva, Ari Artinian, Emilia Nikolova, Michaela Beltcheva, Roumiana Laleva Estimationof the environmentalquality using monitor species small mammalsfrom Rozhen and Srednogorie l. Bioaccumulationof toxic elementsand their influenceon some hystologicaland haematologicalindices. 113 Michaela Beltcheva, Roumiana Metcheva Estimationof theenvironmental quality using monitor species small mammalsfrom Rozhen and Srednogorie ll. Populationcharacferisfics and food preferencestn the monitor spec/es. 12o Nasko Atanassov Morphophysiologicaland cytogenetical analyses of smallrodents biomonitorspecies from the regionsof Srednogorieand regional backgroundstation "Rozhen" (Bulgaria) 1124

5. CONCLUSIONSAND OUTLOOK

5.1.Status and reactionof the biomonitorsin Rozhen andSrednogorie control stations Bilijana Ammann Strategiesand methodologyof ecosonderesults implementation in Bulgaria eB Dimitar Peev, Klaus Ammann, Ari Artinian, Roumiana Metcheva, Stefan Popov, Nina Bakardjieva The statusand prognosisfor the development of theenvironment in Rozhenand Srednogoriemonitoring station - Finaldiscussion and Conclusions 130

Acknowledgement rc2 Appendixes 133

iii FOREWORD

BIOMONITORINGPROJEET

The BulgartanSwiss Pi-ogramme is verydiverse. l-he aim of the bromonitoringcompo- nentwas to set up a commonmethodology between scientists from differentfields in order to assessthe impactof the pollutionat variouslevels of the livingorganisms, and to test the methodsin thefields. This has been done very successfully, as it is shownin the present report.Moreover, the same methocjshave been appliedin one other projectof the programme,in orderto assessthe quality of theforage, milk and meatproduced in a large protectedterritory of StaraPlanina. The projecthas then demonstratedhow its outputcan be usedas a complementof otherfield studies ; we wishit can be usedagain in thefuture as a toolto assessthe qualityof the habitatsin differentparts of the country.

I would like to expressmy gratitudeto the team of scientistsco-ordinated by Prof. DimitarPeev for theirexcellent job. I alsowish their results to be widelydisseminated and theirmethodology used as a modelin othercountries as well.

May,1997

Dr PierreGalland Pro Natura- SwissLeague for NatureProtection BSBCPmanager NOTESON THESPECIFICS OF ROZHENAND SREDNOGORIE CONTROLSTATIONS

DimitarPeev . KlausAmmann

1. CONCEPT 1.1 . Objective The two controlstations have to indicatetwo distinct situations in the bioticcomplexes as follows: Rozhencontrol station - practicallydistant from the industrialpollutant sources, in the middleof Bulgai'ia, s.c.in the middleof Balkanpeninsula. Srednogoriecontrol station - in the areawith very high air pollutionfrom Srednogoriecooper smelting factory. 1.2. Control stations In our understandingand vision as a controlstation can be definedas an areaor zonewith homogeneous ecologicalconditions and containingthe biotopsfor all biomonilorsto be sampled.The overallimpact should be comparablein compositionand origin.

2. GEOGRAPHY 2.1.Rozhen Rozhencontrol zone is situatedin Perelik- Prespapart of theWestern Rhodops Mountains in the natural forestand meadowsat 1100to 2000m a.s.l. 2.2.Srednogorie Srednogoriecontrol station is situatedin the foothillsof CentralBalkan south slopes at 700 - 800 m a.s.l. nearlycooper smelting factory.

3. CLIMATE

Rozhen Srednoqorie ln temperatemounlain climatic zone In thetranscontinental climatic zone Yearmax. to: 26-300 of the air Yearmax. t" . 18.9- 21.4"of the air Meanrainfall per year: 700 - 900mm Meanrainfall per year. 600 - 650 mm Snowcover: 80 - 100days Snowcover: 70 - 90 days Dominatingdirection of the wind:east - wesr Dominatinqdirection of the wind:south - north

4. VEGETATION 4.1.Rozhen Homogenousnatural dominated by Piceaexcelsa and Abies a/ba growing on limestonebasal rocks and mountain meadows with a dominanceof Agrostls tenuis,Fesfuca nigrescens,Nardus stricta, Dactylis glomerata and Veronicachamaedrys. 4.2.Srednogorie Naturalforests vegetation in the areainvestigated is practicallydestroyed. A smallfragment from former mixeddeciduous forests still exists. Secondary shrubby and herbaceouscomponents form a mosaicof dis- persedspots. The Prnusnigra and Robiniapseudoacatia plantations cover some territory of th'earea investigated. The areacan be takenas a true"lichen desert"

5. TYPEOF THE POLLUTINGEMISSION

Rozhen Srednooorie Backgroundtype, long distance pollution. Very close to main industrialpollution source, The nearestBulgarian pollution source is in the mainlycompressed of SO2,NOz, Cu andPb. Plovdivarea and it is isolatedbv mountainbarriers

6. SAMPLINGSTRATEGY All methodsof variousmicroanalitical levels described in chapter3 hasbeen applied within the framework of thetwo stationschosen on mixedsamples. 3 sampleshas beentaken at randomwithin a definedtest area (control sites) and beforemeasurement thesesamples has been mixed and homoqenised. FORMEREXPERIENCE IN ECOMONITORINGPROGRAMMES AS A BASISFOR THE CREATION OF THE PROJECT

DimitarPeev Instituteof Botany,BAS. Sofia Bulgaria

To detect,measure and assessthe changesin the statusof triodiversity,appropriate methods employing specificindicators of the biodiversitycharacteristics should be understoodas ourversionof biomonitoring. Duringthe period1979 - 1989was initiatedthe developmentof a phytomonitoringsubsystem within the frameof GSME (GlobalSystem for Monitoringof the Environment)under the controlof.the Council for Eco- nomicHelp of the formersocialist countries - Bulgaria,Czech Republic, USSR, Romania, and Poland.In its realisationInstitutes of the BulgarianAcademy of Sciences,the Ministryof the Environmentand othersimilar institutionsin the countriesmentioned above were engaged. The reactionsof the species-bioindicatorswere considered to be a very importantelement of the whole monitoringsystem which lead to the creationof controlstations as follows: . Controlstation "Steneio" - CentralBalkan . Controlstation "Rozhen" - WesternRhodops . Conlrolstation "Ropotamo" - Strandza Mt. Subsequently,during different periods of time,observations were madein the followingcontrol slations aswell: . "Boatin"- CentralBalkan . "Pirdop"- WesternBalkan . "Shkorpilovtzi"- Northern Black Sea coast In morethan 20 controlsites the reactionsand statusof 20 phytomonitorswith provedsensitivity were observedand assessed.The spectreof the biologicalparameters under study was createdon the basisof the followingelements: - speciescomponent in the ecologicaltransactions; - coenobitestructure and specificity, - phenologicaldynamics; - morphologicalchanges in the habitus; - changesin the quantityand quality of the stomataapparatus; - changesin the formand vitality of the pollengrains. The resultsachieved were reported in specialdiaries and documents-bearers. Parallelwith the observationsand experiments on the statusof the phytomonitorsrepresentative samples of rain,lake and riverwater were collected; soil from the controlsites, fresh plant samples from the phyto- monitorsfor the detectionof the heavymelals quantity accumulated in them. Parallelwith the phytomonitoringwork observation on a largenumber of zoomonitors(small mammals, riverand lake plankton, bents, etc.)was organised in a similarwayand with the samebearers of information. This experimentprovided the possibilityfor an assessmenlof the degreeand characterof the possible pollutantson bioindicatorsdifferent in theirsensitivity and level of biologicalorganisation. After 1989 the biomonitoringsystem collapsed. Some partial observations during 1991 - 1992carried oul on phytomonitorsin StrandzaMt. and the BlackSea coastcan not be regardedas a realcontinuation of the programmemen- tionedabove. ln spiteof this break,it couldbe definitelystated that duringthe period1979 - 1989were createdthe methodologyand organisationalbasis (stations, control sites, populations-phytomonitors, parameters were chosen,documents, etc.) of a modernecological monitoring. One of the basicshorlcomings of the systemwhich have existed up to nowwas thatthe macromolecular and chromosomallevels of biologicalorganisation were neglected.Thus, many importantdata aboutthe plantphysiology, accumulation of specificsubstances and changesin the karyotypesremained out of our attention.This fact itself leads to the laterevealing of the damageswhich reduces the efficiencyof the ecomonitoring. This practicaland theoreticalshortcoming was avoidedwhen the verticaland horizontalstructure of the project"Ecomonitoring Rozhen - Srednogorie"was created. BIOMONITORINGOFTHE ENVIRONMENT: CONCEPT, STRUCTURAL SCHEMEAND PATTERN OF THE INFORMATION

StefanKozhucharov, Dimitar Peev Instituteof Botany,BAS, Sofia, Bulgaria

1. THE STATUS OF BIOTIC COMPONENTSAND THEIR RESPONSETO BACKGROUNDAND IMPACT-GENERATEDCHANGES OF THE ENVIRONMENT.

Duringthe lasttwo decadesa globaltendency has beenobserved towards heavy investment into the systemsof biomonitoringon differentorganisational levels. The maintenanceand controlof ecologicalbal- anceand biologicalresources can be effectedonly on the basisof objectivebiomonitoring data. Underthe conditionsof multi-factorregulation of plant,animal and humanpopulations there is no clear idea abouttheir immediate response to the influenceof the componentsof the abioticenvironment. This assumptionis validfirst and foremost in thosecases when the parametersof thesecomponents do not con- formwith the standardsof Nature. Whatis the thresholdof enduranceunder the conditionsof pressure,which are the compensatorypossi- bilitiesof organicsystems, how rapidlythe natural(or closeto natural)balance of the environmentcould be restored,what are the reproductivedynamics and environmentalresources limits under the conditionsof backgroundor impact-generatedpollution. The answerto thesequestions can be foundonly on the basisof dataobtained through the.use of the efficientbiomonitoring system, with the supportof a wide rangeof spe- cialistsfrom various branches of the recentscience: chemists, physics, biologists, managers, s.o. BulgarianBiomonitoring system can be a partof NationalAutomatic System of EnvironmentalMonitoring (NASEMsystem), it has beenworked out for reading,measuring and prognosisof the reactionsof biomonitors (indicatorspecies) at the changeof the environment,under the pressureof the naturaldynamic factors or anthropopressure. The roleof thissubsystem is determinedby the necessityto preservethe optimalenvironment, biological diversity,genetic fund and humanpopulation. Without this subsystemwould have been reducedto mere registrationof facts,devoid of a clearlydefined need for retraction.Consequently, the effectivefunctioning of this subsystemdepends on the adequateregistration of correlalionbetween biotic and abioticunits of NASEMsystem. When the abioticmonitors are correctlychosen and co-ordinatedwithin the subsystemof ecological(abiotic) monitoring they haveto be regardedas a backgroundmaterial which serves as a basis forworkingout a subsystemof bioticmonitoring (flora, fauna, microbiological diversity of soil,etc.). This cre- atesthe conditionsfor moreefficient application of chemoanalyticaland bioanalyticaltechniques. So far we are unableto definethe limitof loadingfor speciesand communities(known also as Limit of Admissible Concentrations)which results from the applicationof variousuncalibrated methods. lt is impossibleto make biologicalobservalions without taking into account the chemicalreactions of plantsand animals,the antago- nisticreactions of microelements,the degreeof toleranceto high accumulationof pollutants,the relation betweensoil and plants,basic rock, soil, plants and animals,possible radioactivity, etc. Othenrvise, the inter- dependencebetween cause and effect(norm and pathology)cannot be correctlyunderstood, whereas this understandinglays the basisfor environmentalmonitoring. Workingout sucha conceptwe shouldbear in mindone veryimportant circumstance related to the selectionof sitesfor monitoring.The mainshortcoming already existed control sites was the fact that in most casesthey were chosen on the basisof hydrometeorologicaland landscape parameters, while the biological complexesused as monitorswere regardedas somethingcomplementary. For this reasonthey couldnot providesufficiently representative information. Whiledeveloping the presentconcept we shallmake an attemptto choosethe sitestaking into account variousfactors. Our choicewill be basedon comprehensiveanalysis of abioticparameters which in its turn requiresthe adequateknowledge about NASEM system. In practicalterms this approachwill allowto find moreeffective solutions for the problemsof separatedstations, to work out an obseruationschedule and to selectthe foundationfor eachsite. It shouldbe emphasisedthat Biomonitoringsubsystem has to be workedout takinginto account lhe entire complexof factors.lts verticalstructure depends on the subordinationto the Ministryof Environment, whilethe scopeof the horizontalstructure is determinedby the numberof specialisedworking teams from differentresearch units. The role of the BulgarianAcademy of Sciencesas a well-developedfunctioning institutionwill be quiteimportant for the fulfilment of thistask. The proposedhot spotsare arrangedin modelorder and their final specification will depend on the applicationof Appendix2 of the Statuteof the Int.Committee for PriorityProjects to the Ministryof Environment. + 1.A. The statusand dynamics oi tne brolii.cornl;i.lirer'ri - sfierirlrcairon cifbirrrnonitors and measi:reinentof theirreactions. 1.A.1.The levelof ecosystems(biological ciivei-sity spatial struciure), 1.A.2 Thelevel of populations(space and trme) 1.A 2.1.The number of populationsand their s;;etial structure, 1.4 2 2. Reproduction.restoraiion, nrortirlity iate. 1.A.3.Macromorphological and orgarric level (dose - ettect) '1 .4.3.1.Visually assessable darnage or deviations,the appearance, 1.4.3.2.Statistically assessable deviatii;ns (size. irrimber. frequency); 1.A.4.Macromorphologicai level (the raie of microtlr-i;iptatiorrsor rnutations); 1.A.5.Cellular level (Cytological levet), 1.A.5.1.Genetic status - chromosomes,clrrornosome structures, caryoiypological variations, differentiaI sta ining. enzyrr les and genctic regu iation ; 1.4.5.2.Physiological reactivity: Morphophysiological characteristics, Activity of pigments andphotosynthesis, haemoglobin, hormones and other specific substances; 1.A.5.3.Chemical reactivity. for irrstance lipid membranes 1.8. Thestatus and dynamics of environnrentand biological response under various conditions: 1.8.'1. The impact of heavymetals as pollutantsof air,water and soil;tests and correlation matrices: 1.8.2.Radiation impact, 1.B.3.The impactof chemicalreagents used in agriculture, '1.8.4. Anthropopressurecaused by tourism; 1.8.5.Various nonidentified and imoossible to be identifiedneoative effects

2. THEBIOMONITORING AND CONTROL SITES

lmpactmonitoring sites Backgroundmonitoring "Hotspots" Pirdop, Plovdiv, Nationalparks, reserves Backgroundnetwork (1 990) Assenovgrad,Vratsa, Bouras, Pirin,Rila, Vitosha (total 11 sites) CentralStara Planina,Southern 'acc to App.ME Black-SeaCoast, the Rhodopes (total3 sites) 2A.impact, local backqround,reqional backqround,reqional 28. pastures,river, ecosystems; forest.meadow. lake. sea forest,marsh, sea, coastal (sand) DOqS 2C. unprotectedhighly polluted Nationalparks: Rila, Pirin, Vito- reservesin the CentralStara territories sha,Southern Black-Sea Coast,Strandla Planina.Strandia, the Rhodooes 2D. extinct,rare and interesting form-generatingsites, unique extinctspecies and communities species communities.endemic. relicts (ordinary,rare) 2E.intensive economic use of medicinalplants, macromycetes, medicinalplants, rnaciomycetes, natural mammals, snails lumberingsnails, mammals. lurnbering (mainlyconifers) (mainlyconifers and decidLrous specres) 2F.high risk due to highconcen- moderaterisk due to misman- moderaterisk: locatiori within the trationof chemica agementduring the skiing boundariesof NP Steneto, pollutants seasonand tourist industry in Ropotamo.higher risk of thesummer pollutionfor Rozhen_region __

3. CRITERIA,STANDARDS, PARAMETERS

3A. The criteriaare chosenwith regardto life-determiningprocesses, narnely: nraiirtenance of the nLjnr. ber of populations,reproduction, variations in the geneticand automaticpfocesses related to survivaiof monitoredpopulations - assessment standards are defined in the courseof researchand are basedcln lor,g- termexperience accumulated for a concreteor relatedgroup of species

3B. Parametersof monitoring:number, ethnology, microtopography, seasonal dynamics (;rlierri;li;gy') stomatastructure and frequency,pollen apparatus (sterility- fertility), number of cnrornosomes.caryotype structure,differential staining of chromosomes(bending method, isoenzyme specification according to groupsof isoenzymes,activity of liprdmembranes and the synthesisof secondarymetabolites. photosyn- theticactivity and pigmentation apparatus. haematological and other changes in livingorganisms 3C. Registrationmethods: 3.C.1. In situwork aimed at the measurementof thefollowing parameters: phenologicaldynamics, migration, number and structure of populations,regular collection of samples(the latter requires heavy-duty motor vehicles) material' 3.C.2.Laboratory processing: microscopes, centrifuges, photo equipment, stainlng auxiliaryequipment, spectrometers, etc' andgraphic 3.C.3.Computer equipment: a programfor statisticaldata processing, word-processing programsfor digitaldata processtng. (3 repetitionsfor 3.D.The frequencyof fieldsample collection: once in 60 daysfrom 3 controlsites eachmonitored species). 3.E.Characteristics of inPut data: to the Writtentext - standardtype-written pages, tables (formatted and preparedaccording requirementsof the Contractor). 3.F.Layout of a loadingdocument 3.F. 1 . PhYtomonitoring 3.F.2.Zoomonitoring 3.F.3.Other models: microbiological, chemical, etc'

4. CO-ORDINATIONOF BIOMONITORINGSUBSYSTEM:

4.1.To usethe controlsites of NASEMwhen possible' controlsites 4.Z.Thespecificcharacter of Biomonitoringsubsystem requires to establishindependent dependingon the presenceof monitoredspecies.

5. BIOMONITORINGAND FUNDAMENTALRESEARCH obtainedwith a view 5.1.Some asPects of the researchprogramare routine ones and the resultsare of mere registrationof facts. 5.2.The necessity for working out new approaches, methodology,comprehensive analysis of data re- relevantto interdisciplinaryfields of chemistry, biology and physicsgive grounds to expect fundamental sults.

6. THE PROGRAMFOR STEP-BY-STEP INTRODUCTION

6.1.First steP - organisation 6.1.1.Setting up researchteams and definition of specifictasks 6.1.2.Working out the sYstemsof: . controlSites . controlDlots . controlspecies . controlparameters . experimentaland monitoring regime 6.2.Second steP - in situwork 6.2.1.Microtopographic mapping and number 6.2.2.Regular observations 6.2.3.SamPle collection (see6'2') 6.3.Third step - taboratoryprocessing - this step is partlyexecuted during the fieldwork 6.3.1. CaryologicalanalYsis 6.3.2.Differential staining 6.3.3.lsoenzYme analYsis 6.3.4.Photosynthetic and physiological activity and specificity 6.3.5.Other analYsis 6.3.6.Pollen analysis (fertility-sterility test) 6.3.7.The analysisof lipidmembranes and other active substances. 6.4.Fourth steP: 6.4.1.Data processing and statistics 6.4.2.Completion of standarddata carriers 6.4.3.Reoort to the Contractor. 7. CONCLUSION

The developedprogram given above is a resultfrcm long personal experience and communicationswith variousspecialists related with nature protection and environmental problems.

REFERENCES

Ancev,A., L. Yurukova,M. Markova.1987. Some resultsfrom the ecologicalmonitoring in Biospherereserve "Steneto", Central Stara planina. FirstNational Conference of the Problemsof BiologicalMonitoring, BAS, Plovdiv, 38-43. Kozhucharov,S., A. Petrova,L. Yurukova.1987. Some peculiaritiesof the grassspecies as a basictest for the phytomonitoring.First National Con- ferenceof the Problemsof BiologicalMonitoring, BAS, Plovdiv, 50-55. Kozhucharov,S., L. Yurukova. 1987. The contentof heavymetals in the soils,plants and waters in stationsof the backgroundmonitoring in Bulgaria.Problems of the BackgroundBiological monitoring. Second School of Biologicalmoni- toring,1984, BAS, 65-70. Kozhucharov,S., A. Petrova,L. Yurukova1985 Some aspeclsof usingplants as indicatorsof environmentalpollution, India, Symp. Biomonitoring StateEnviron., 1985. 98 - 105. Mechinev,T., P. Vassilev.1983. On the problemof key and criticalparameters for monitoringof natureenvironment. Third National Conferenceof Botany,S., BAS,972-979. Nikolov,N., L. Yurukova,V. Nikolov.1987. The contentof heavymetals in the phytomonitorsof Poaceae- Backgroundstation "Rozhen" flffest- ern RhodopesMountain). First National Conference of the Problemsof BiologicalMonitoring, BAS, Plovdiv,26-31. Peev.D.. S. Gerassimov.1980. The biologicalmonitoring - a backgroundfor the controlof the conditionand prognosisof the ten- denciesin the biospherechanges. Partizdat, Sofia, '195-201. Petrova,A., N. Andreev,V. Nikolov,E. Bozhilova,L. Yurukova.1983. Structureand organisationof the observationfor the controland the protectionof the flora and vegetation.Control station "Rozhen". Report to Ministryof environment. Yurukova,L., N. Nikolov,S. Kozhucharov.'1993. lmpactbioindicators studies in Bulgariaforthe 1985- 1990period. J. AquaticEcosystem Health. THEROLE OF KARYOLOGY,STOMATA, POLLEN AND STATISTICAL ANALYSIS INTHE BIOMONITORING SYSTEM OF BULGARIA

Dimitar Peev, SoniaTzoneva, Natalia Valjovska Instituteof Botany,Bulgarian Academy of Sciences

1.INTRODUCTION Populationlevel isthe basicone forthe estimationof the statusof the environment.The resistanceof the naturalpopulations against the negativeimpact depends on the sustainabledevelopment of the population structure- age,sexual, spatial, genetical and s.o. Asfaras our practiceshows, the observationsof varietyof parameters(using given methods) will give a satisfactoryinformation about the type of the responseof the investigatedpopulations. In the alreadyapplied scheme the followingbiotic parameters have beenused: 1) listingof the speciesin the controlsites; 2) visualestimation of the habitusof the monilors;3) microtopog- raphicmapping of the controlsites; 4) phenology;5) statisticsof the variability;6) pollenfertility: 7) stomata analysis;8) karyotypeanalysis. The alreadymentioned methods have been applied in the monitoringresearch in the period1982 - 1987. The informationis storedin Ministryof Environmentdata bank.During 1994 in the French- Bulgarianbio- monitoringproject OM2 in RilaMountain the 12 controlsites have been investigated (Peev and all, 1995). Periodicallythe heavymetals content was determinated too. The processof the increasingaccumulation of the heavymetals can be takenas a signalfor the beginningof the injuries.The visible necrosis, chlorosis, deformations of the shapeand s.o.are alreadyadvanced stageof that kindof injuries.The intermediaryperiod, well knownin the literatureas "nonvisible" effects of the injuries(diminishing of plantdimensions, slow ontogenic development, decreasing of biomassaccumula- tion, changesof the anatomyand ultrastructures)are very importantfor the timelydiagnosis. These pa- rametersform a complexof veryspecific indicators of the eventualanthropopression The correct"diagnosis" can be improvedby strictobservation of the ecologicalfactors and geneticaltolerance of the phytomonitors to the pollution.So, the chosenset of paramelersand the analyticallook on the correlationin lhe resultscan be a correctbasis for biologicalcontrol. That is the generalaim of the biomonitoring. The structure,organisation and observationof the impactin the backgroundconditions was theoretically proved,unified and appliedfor 5 yearsin Bulgaria.On the table 1 You are presentedthe locationof the backgreundlong range control stations in Bulgariaand the listof selectedphytomonitor species.

2. KARYOLOGY 2.'1.lntroduction The experimentallyproved mutagenic effects of the anthropogenicpollutants give the opportunityto use the karyologycalanalysis for the purposesof the phytomonitoring.lt is absolutelyinformative in the caseof drrectimpact and in the sametime it is informativein the backgroundcondilions too. Theoretically, there is no limitsfor a minimalconcentration dose provoking mutagenic effect. The reactionin that casecan be determinedas more"qualitatively" than "quantitatively". The structuralchanges in the karyotypescan be provokedfrom the rapidand strongchange in the environmentalconditions where the investigateddeme is located. 2.2. Material and methods Phytomonitorplants from nativelocalities in controlsites were collectedand germinatedin pots under greenhouseconditions. Root tips are takenand fixedin Clarkeafter pre-treatment in oxyquinolin.Chromo- some countsare takenafter maceration in 1n HCIand 50% aceticacid at roomtemperature and stainedin haematoxylineat 600.The permanentsquash slides were prepared after freezing and imbeddingin Euparal. The chromosometype is givenafter Whithe, Robertis, Nowinski and Sacz.elaborated by Kuzmanovand Kozhucharov(1967). 2.3. Resultsand discussion (table2) The resultsof the karyologycalanalysis (Ancev et al.,1987) of the phytomonitorsfrom the Stenetocontrol stationconfirmed the data about Stellarianemorum and Vacciniumvitis-idaea. ln the Luzulaluzuloides karyotypeB chromosomeand in the karyotypeof Calamagrostisarundinacea 28 chromosomescan be obtained.ln the knownliterature B chromosomesfromthesesoecies are notreferred. Duringthe summerof 1994the karyologicalresearch on the Rilaphytomonitors (Peev et all,1995) gave the followingresults: . Nardusstricta - (2n =24) beforethe dam Beli lskar . Nardusstricta - (2n =28) upperpart of the dam Belilskar . Nardusstricta - (2n=26) near the lowerMaritchino lake . Nardusstricta - (2n =24)second terrace upper Maljovitza chalet x a Nardusstricta - (2n =24)eastern slopes near the dam Beli lskar a Festucapicturata - (2n =14) upperpart of the dam Beli lskar a Soldanellahungarica - (2n =40)before the dam Belilskar

The chromosomenumber and karyotypesfrom these natural populations in the time of the investigation leadto the conclusionthat the environmentalimpact is withinthe limitsof the normaldegree. No extraordi- narychanges at the chromosomelevel have been obtained.

3. STOMATA 3.1. lntroduction The anatomicinvestigations analysing the changesprovoked by the air pollutionhave nearly100 years history.Some changes in the photosyntheticapparatus as: number of the basalepidermal cells, frequency at the stomata,thickness of the leaves,thickness of the palisadeparenchyma, the palisadecoefficient of, the volumeof the intercellularspace, green pigments and contentcan be usedas an indicatorand quantitative criteriafor the estimationof the impact.In ourcase we usedthe frequencyof stomataas approximatechar- acter.The stomatographicdescription of the plantsmaterials originated from polluted areas shows significant xeromorphizationexpressed by diminishingof the sizeof stomataand accompanyingcells, deformation of the shape.The observationof sucha deformationhave to be usedas a signalfor a further,more detailed ultrastrucluralinvestigation 3.2. Materialand methods The materialsfor stomataanalysis have been taken from the middlepart of the adultleaves, near to the central(main) vein. Follows a maceration(according.thp methodology) and staining.The stomatafrequency havetobe calculatedonminimum 10 samples 1 mm'eachone (Uzunova, 1982). 3.3. Results and discussion(table 2 and table 3) The dataof Stenetocontrol station (table 2) doesn'tshow any significantchanges in the structureof the stomalalapparatus (Ancev et al., 1987).When in the Ropotamostation (table 3) some improvementof the environmentalconditioncan be seen(Peev, 1990). These results can be referreddirectly to the normalfunc- tionof the photosyntheticsystem. ln the conditionof veryhigh degree of pollutionby Coppermetallurgic works "Srednogorie" in the 1983a changesof the stomatalfrequency is verywell expressedaccording to the distancefrom the works(Peev, Medarova,1994). ln the conditionof the air pollutionin the townof Rousethe followingphytomonitors have beenchosen: Taxusbaccata L., Sambucus nigra L., Tiliatomentosa Moench., Dactylisglomerata L. measuredin 3 control points(Kossev, 1993). The injuringof the stomatalapparatus correlated with the level.ofthe toxicityof the air.The tendency of increasingthe numberand drminishing the stomatalsize is clearlyexpressed.

4. Pollenfertility 4.1. lntroduction In the investigationson the embryologicalaftermath provoked by the pollutionof the environmentseveral deviations,correlated with the levelof the contaminationcan be obtained.In the caseof low impactof pollution a compensalorysystems played significant role and the responsehas a characterof adaptation.ln the caseof a verystrong and rapidpollution the highdegree of pollendegradation and sterilitycan be observed (Krusheva,1984). The increasingof the percentof the sterilepollen shows the necessityof the instantlycytoembryological investigationfor the establishmentof the populationstatus. 4.2. Material and methods The mixedsamples from the phytomonrtorshave to be taken(according the methodology).The difference in the colourof the sterileand fertile pollen grains is used.For the pollenstaining 4oh acetocarmin is used.lf necessary,the stainingwith iodine solution (when the exineis verythick) can be implemented.The degreeof fertilityis calculatedas a meanvalue from 1000 pollen grains. 4.3. Results and discussion (table2 and table 5) The resultof the estimationof the pollenfertility in Stenetocontrol station (Ancev et al., 1987)shows a normalmicrosporogenesis and reproductivepollen capacity in 5 of the phytomonitors(72-97%). This percenl howeveris low in the samplesfrom Stellarianemorum. Probably the injuriesin the reproductivesystem is compensatedby an extensivevegetative reproduction, providing the normaldevelopment of the population A normalmicrosporogenesis and reproductivepollen capacity in all phytomonitorsin Ropotamocontrol sta- tionwas established. (Peev, 1990). In the areaunder very strong anthropogenic impact the totallysterile plant populations are observed.So the populationfrom Genistaovata, located near metallurgic factory "Kremikovtzi" is 100%sterile, and the populationnearthe road before Backovo - monastery(Rhodops) shows the sameresult. (Krusheva, 1984).

5. MORPHOMETRICALTEST AND STATISTICS 5.1. lntroduction Severalexperimental works proved that the diminishingof the sizeand biomassis very informativeindi- catorfor the negativermpact. That is why,the resultsfrom the morphometrictest are necessaryin the phy- tomonitoringpractice. The morphologicallyinformative metric characteristics of the phytomonitorsare processedby standard statisticalmethods. The degreeof the variabilityis more or lessdetermined by the impactof the environmentalconditions. The naturalselective pressure (Heywood, 1967) in combinationwith some anthropogenic impact(anthropopression) provoke the reactionof the geneticsystem, which reflects in the increasingof the rateof mutationsand /or morphometricvariability. Some very rapid changes have led to the disruptiveselec- trveeffects within the populationsand this process can be describedstatistically. 5.2. Material and methods The samplesof phytomonitorspecies are correctly collected accordrng to the rulesof randomsampling. The charactermatrix consists of minimum8 charactersincluding size of the vegetativeand reproducliveorgans. The followingstatistical methods were used: simpledescriptive statistics including coefficient of variation; two-groupcomparison by the HottellingT2-test; principal component analysis (PCA) as a powerfulexplora- torytool for clusteringmetric characteristics in the samplesand the lineardiscriminant analysis (LDA) in orderto revealthe distinctionbetween groups. The statisticalanalyses were carried out by the BMDPpackage (Dixon,1990). 5.3.Resu/ts and discussion Usingthe alreadydescribed statistical techniques the morphometricvariability of 3 monitorsSesleria co- mosa,Phleum alpinum and Alopecurusriloensis from 5 controlsites under background conditions in Rila Mountainwas established(Peev and all. 1995). Havingin mindthe resultswe acceptthe studiedpopulation fragments with very highmorphological ho- mogenettyas elementsfrom well adapted and balancedhigh mountain system. The low degreeof correla- tionsupport an "open"ecological plasticity. According to the results(from the firstobservation), no signalfor a very"aggressive" pressure on the phytomonitorsexists at thatmoment.

6. CONCLUSTON The leavingcreatures response to the negativeimpact is a very intricateprocess. We are only goingto collectsome elements of the naturalleaving puzzle.lf the elementsdoesn't coincide - somethingwrong has happened,that is the aimof biomonitoring.The nextstep is - an actionagainst the pollutants.

APPLICATION

Table1. Controlstations and phytomonitorsin Bulgaria Table1.1. Control stations

RhodopsMountain CentralBalkan SouthernBlack sea coasl RilaMountain Rozhencontrol station Stenetocontrol station Roootamocontrol station Moussalacontrol station 3 controlsites 3 controlsites 3 controlsites 12 controlsites ), (750-800-300m2; (150-210m2) (685-324-180m2; (6 m- eacnone) 800-1200m a.s.l. 1400-1450m a.s.l. (0-5m a.s.l.) 1700-2925ln a.s.l. 8 phvtomonitors 6 phytomonrtors 9 phvtomonitors 14 phvtomonitors

Table1.2. Selected phytomonitors

Controlstations Selectedphvtomonitors Rozhencontrol station 1. Festucanigrescens Lam.; 2. Anthoxantumodoratum L.; 3. Viciacassubica L.; 4. Pinussylvestris L.; 5. Juniperusoxycedrus L.; 6. Dactylisglomerata L.; 7. Trisetumflavescens (L.) P.W.; 8. Rumexacetosa L. '1. Stenetocontrol station Luzulaluzuloides (Lam.) Dandy; 2. Stellarianemorum L.; 3. Juniperus sibiricaBurgsd.; 4. Vacciniumvitis-idaea L.; 5. Chamaecytisusausiriacus (L.) Link;6. Calamagrostisarundinacea (L.) Roth. l0 Roootamocontrol station 1. Cionui"aei"ecta (L.) Griseb., 2. Peucedanumarenarium W.K.; 3 Pancratium maritimumL 4 Juncuseffusum L , 5 Phragmitiscommunis Trin.: 6. Lepido- trichumuechtriiziarrum (Bornm.) Vel , 7 Galanthusnivalis L. 8. Cyclamen coumMill.. 9. lrispumila L Moussalacontrol station 1 Sesleriacomosa Vel., 2 Festucariloensis (Hack ex Hay.)Markgr.-Dannb.; 3. Alopecurusriloensrs (f'lack.) Pawl.4. Nardus stricta L., 5. Agrostiscapillaris L.,6. Calamagrostisaiundinacea (L)Roth; 7. Carexcurvula All.: 8. Carex '11. nigra(L )Rchb 9. l.Lrzulasp; 10 Rhinanthusalpinus Baumg.; Hypericum perforatumL.', 12 Acerpsetidoplatanus L.; 13. Dicranumscoparium Hedw.; 14.Pogonatum urniqerum (Hedw.) P Beauv.

Table2 Stene^tocontroi station - chromosomenumbers. pollen feflility (%) and stomata frequency (mm')

Phvtomonitors zn Pollenfertivitv Stomatafreouencv Luzulaluzuloides (Lam.) Dandy 12.12+1 B 79-97 45- 117* Stellarianemorum L. 26 44-96 20-66" Juniperussibirica Burgsd. 96-97 88-287*' Vacciniumvitis-idaea L. aA 85-9'1 196-377- Chamaecytisusauslriacus (L.) Link 84-96 68-287" Calamagrostisarundinacea (L.) Roth. ZA+Ze I z-Yo 91-181**

* numberof stomataon the lowerepidermis *" numberof stomataon the upperepidermis

Table3. Ropotamocontrol station - stomatafrequency (mm')

Stomatafrequency (mm') Stomatafrequency (nrm') Phytomonitors Upperepidermis Lowerepidermis 1987 1986 1985 1984 1987 1986 1985 1984 Pancratiummaritimum 107-360 118-290 0-320 200-350136-246 148-286 160-320 145-355 Peucedanumarenarium 181-760 190-7850-890 558-956348-641345-862 396- 924 458-962 Cionuraerecta 174-370186-384 6-494 230-520 n??_Ro7 849-986 984-1356 000-1 485 Erynqiummaritimum 104-'186124-198 0-200 140-220 70 -156 69 -160 78- 178 I J- tOZ

Table4. Srednogoriecontrol station - stomata(lower epidermis) rnm'

Phytomonirors Controlsites 1 2 4 5 6 a n Teucriumchamaedrys ll'rE 429 425 298 270 251 296 Fagussylvatica 441 434 4Ut 368 276 '7i Agrostiscapillaris 81 81 76 56 7) Fraqariavesca JYJ 400 375

Table5 Ropotamocontrol station - pollenfertility

Phytomonitors Pollenfertilitv % 1987 | 1986 I 1985 1984 Pancratiummaritimum 8e.70 I e6.78 | 87.00 | 89.00 Peucedanumarenarium 8713 I aorz I 87oo I 89.80 Cionuraerecta no stainingbecause of the verythic exine Ervnqiummaritimurn 84.00 | ao.+oI ag.oo I 89.50 REFERENCES

Ancev,A., L. V. Nikolov.1987. Some resultsfrom the ecologicalmonitoring in Biospherereserve "Steneto", Central Stara planina.First National Conference of the Problemsof BiologicalMonitoring, BAS, Plovdiv, 38-43. Dixon,W. J. (ed.).1990. BMDP StatisticalSoftware manual Univ. of CaliforniaPress, Los Angeles. Heywood,V. 1967. PlantTaxonomy. E. Arnold,1 15 p. Kossev,K. 1993. Structuraland functional changes of the photosyntheticapparatus of someplants species under air pollution.PhD Thesis, Autoreport. 29 p. Krusheva,R.1984. Influenceof the pollutionof the extertnalenvironment over the embryologyof someflower plants. Listenrecent theoretical and appliedaspects in the plantecology, BAS, S., 583-591. Kuzmanov,B., S. Kozhucharov.1967. Caryotypesof fourBulgarian Compositae species. - Compt.Rend. Acad. Bulg. Sci., 5, 469-472. Peev.D.. M. Medarova.'1984. The impactof the pollutionwith heavy metals from copper work "Srednogorie" on somemicro and macromorphologicalstruclures in vascularplants. (unpublished). Peev,D.1990. Developmentand implementation of the methodsfor biologicalmonitoring in the backgroundcon- trolstations "Ropotamo", "Steneto" and "Rojen". Reports to the Ministryof Environment1982- 1990. Peev,D., A. Artinjan,E. Nicolova,S. Tzoneva,N. Valjovska.1995. Phytomonitoringin Rila Mountain - 1994.L Microtopography,heavy metals, statistics and karyology of the controlpopulations. High Mountain Observatory Moussala OM2, No 3, 150-161. Peev,D., S. lvanceva,G. Angelov,M. Kourteva,P. Derebanova.1995. Phytomonitoringin Rila Mountain - 1994.ll. Flavonoids,isoenzymes and pigmentsof the control populations.High Mountain Observatory Moussala OM2, No 3,162-170. Uzunova,K., E. Palamarev.1982. The foliarepidermis studies of FagaceaeDumort. from the BalkanPeninsula l. CastaneaMill. and FagusL. Fitologija,13-26.

t2 AN APPROACHAND RESUTTSOF THE HYDROBIOLOGICAL MONITORINGIN BULGARIA

lvankaJaneva Instituteof Zoology,BAS

The purposefulhydrobiological monitr:ring inriestigations in Bulgariahave begun since 1977.These investigationsare based on observationsconducted on river and stagnantob- jects at Regional backgroundstations of "Rozhen","Ropotamo" /"Steneto"/, "Boatin" and "Shkorpilovtsy"(Russev,8., S. Gerasimov,1985; CSerasimov, S.. B. Russev, 1988; Abaku- mov,8., B. Russev,1988, Uzunov, Y., S Kovachev,1984). A complex of'approacheshave been used for estimationof the backgroundinfluences. These approachesdescribe the compositionand the structureof the basic biocoenosesthat occupylhe relevantreservoirs. The estimationis based on many years of professionalexperience and numerousinvestiga- tions of the reservoirsthat are exposedto an impactpollution. Some approaches have been used simultaneouslyto evaluate the saprobiological statusof the studiedstations. The use of the biocoenosesapproach is based on the principleof climax status of the ecologicalsystems. lt is knownthat each alive organism(or set of organismpopulation, bio- coenosis)is under the continuousinfluence of many abioticand bioticfactors. The connec- tions betweenabiotic and bioticparts of the ecosystemare determinedand accomplishedby constantphysical, chemical and biologicalprocesses which finallylead to an ecologicalbal- ance. The climax slatus of the ecosystemin this case is defined by stable processesthat change regularlyand periodicallymostly under seasonalclimatic changes or due to natural biologicalchanges that are connectedwith reproductionand development. Practically,an ecosystemat climaxstatus was representativewith maximumnumber of speciesthat require living conditionsadequate to these existingat the moment (e.9. high varietyof species)and a maximumreproduction coefficient of the species.The high variety of speciesis usuallyconnected with high balanceand low dominalion. The anthropologicalinfluence breaks the climaxstatus, resulting in a seriesof succes- sive changesthat aim to restorethe balance.This influencemay have impulsivecharacter and then it is brief However,it can be constantand chronic.Due to the unimportantchronic influences,the changesin the biocoenosesoccur withinthe basic species compositionand boil down to metabolicand behaviouralchanges of the specimen.Most frequentlythey influence a number of particularspecies (especially dominant and stenobiontspecies) or whole taxonomicgroups which bringschanges in the structure.As a resultof constantlyincreasing influence,the changesaffect and reflectthe basiccomposition of species. The data that describesthe compositionand the structureof the zoobenthoscoenoses has been collectedthrough monitoring investigations to estimatethe status of the river ecosystems.This is determinedby the specificityof the studiedrivers (in most cases plankton doesn'tgrow permanently).Moreover, the benthoslike an inhabitantof the bottom,indicates most objectivethe real and permanentcondition of the river in contrastwith the plankton that indicatesthe momentarystale of the runningwater. The investigationsinclude data about cumulatingof differentcontaminants (usually heavy metals) in differenttissues and organsof fish, but the methodsfor such kind of studiesare questionof anotherpaper. A set of indiceswere taken from each samplingpoint giving the characteristicsand the structureof benthoszoocoenoses: total numberof speciesand species by groups, species composition,relative number (total number, by groups,by species),the indicesof total species variety accordingto Margaleff(1958), dominants afler Simpson (1949); equalisation after Pielou(1966); species similarity according to Sorensen(1848) of speciesof the orders Ephemeropleraand Plecopterarelated. Determined was also the correlalionbetween the number of specimensand number of speciesof the orders Ephemeropteraand Plecoptera (Ks and K6) relatedto the total number of speciesand specimensof all gatheredinverte- bratesfrom the respectivepoint (Russev & Janeva,1987). In the meantimedata is collected about the numberof dominantspecies. The compositionand the structureof phyto and zoo- plankton'ssocieties (Najdenov, V., D. Says, 1989)were investigatedin order to study and evaluatethe changesoccurring in the ecosystemsof the stagnantreservoirs. The data were collectedfor the followingparameters: composition of species,number and biomass (total and by groups),dominant composition according to Kodjova(1970 ), a ratio among specific groups (by number and by biomass);size-agestructure (stage of development,size of an organism),horizontal and verticaldistribution, seasonal and yearlydynamics. Specificattention is paid to the dynamicsof the numbersand the biomassof particular species-monitorsthat most frequentlyhave good displayingproperties (stenobiont organisms). A set of biologicalapproaches is descrrbedin literatureto determinethe saprobical state of water reservoirsbased on bioindicatingpossibilities of the hydrobionts.The idea fcr usingthe organismslike indicatorsto determinethe statusof water environmentgrew up in 1850. The classicalsystem of the indicators,established by the German researchworkers Kolkwitzand Marssonin 1902, was publishedfor the first time in 1908. This system intro- duces the term "saprobity"- abilityof organismsto grow under differentorganic polluiion of water and the term "saprobionts"- the organismsliving in waste water. Since then the system of indicatorshas been developedcontinuously by differentspecialists. The system includesalso bioindicatorsdetermined by the Bulgariansaprobiologists: prof. Dr. B.Russevfor the Ephemeropteraorder, Research associate Jordan Uzunov (for the Oligochaeta);R.A. Stanoy Kovachevfor the Simuliidaefamily, Diptera order; R A. lvanka Janeva for the Baetis genus (Ephemeropteraorder) and Hirudineaclass (Russev et al,, 1e76). From all known saprobiologicalmethods the most commonlyused in practiceare the following:the method of saprobiologicalvalences created by Zelinka and Marvan in 196'1 throughthe modificationsof the methodof Rothschein,1962; the method created by Pantle and Buck in 1955 and modifiedby Sladecekin 1973;the bioticindex createdby Woodiwiss in 1972. The last method in our opinionalthough rather practical,is quick and doesn't require high level of experience.lt is inappropriatefor the goals of phonic monitoringstudies becausesome of its featurescould bring ratherwrong results(Janeva, 1., B. Russev,1987; Kovachev,S., 1987).The methodof saprobicvalences expanded by Rothscheinwas applied in the monitoringinvestigations of river stations.The method of Pantle and Buck was used for standingreservoirs. The estimationin both methods is completedwith dogotal results that correspondto the acceptedin practiceclassifications for saprobity-Xenosaprobity, Oli- gosaprobity,fJ-mesosaprobity, u-rTrososzrprobity and Polysaprobity.The method of saprobic valences,in our opinionis the most precisebecause it estimatesin full detailthe indicating possibilitiesof particularspecies and it influencesthe better indicatorsand the same time involvesthe individualnumbers of each of them. As it was mentionedabove, the hydrobiologicalmonitoring investigations have been carriedout in several phonic stations"Rozhen", "Ropotamo", "Boatin" and "Steneto".They include3lakes (the SmolyanLakes),one dam (the Yasna polyanadam) and 9 rivers - Shi- rokolushka,Trigradska. Dvoynitsa, Mladezhka, Fakiyska, Cherni Osum, Veleka, Chairska and Cherni Vit. The investigationsal the Smolyan Lakes and "Yasna polyana"dam were conducted by Prof. Naydenovand the scientificworkers D. Sais and R. Kalchev; for the Veleka,Mladezhka, Fakiyska and Dvoynirsarivers - by Y. Uzunov and S. Kovachev,for the Shirokolushka,Trigradska, Chairska, Cherni Osum and Cherni Vir rivers - by Prof. Russev and l. Janeva. The specificstations were investigatedin differentmonths and years in the periodfrom 1978to'1 990. Frequencyof observationswithin each year varies- one mirrimurn and six maximum.Data for the investigatedparameters are recordedon four types of forms: form for recordingthe resultsof periodicalstudies; form of zoomonitor;signal form of species threatenedwith extinctionand saprobiologicalform. The more importantsummarized resultsfrom the elevenyears of investigationsof riverstations (1980-1990) are releasedby Janeva and Russev in'1993.According to them the averagevalues of Sp are withinthe oligosaprobic level. The lower values measured in particularcases are assigned to the changes of climatic conditions(water quantityand temperature).The values of structural parametersand the natureof their dynamicscorrespond to the above conclusion.The nurn- ber of species the total relalivenumber, the total and individualvariety of speciesand the equalizationare ratherhigh and the dominationis low. That is an indicationof optimumliv- ing conditionsof zoocoenosesThe values of the coefficientsKs and Krrare high because the Ephemeropteraand Plecopteraorders are mass developed.The characterof dynamics of the structuralindices proves that the biocoenosesare stablestructures. They can endure the negative episodic influencesof the naturalclimatic factors high or low waters, more sensitivechanges in the watertemperature, hence of oxygencontent eic. These resultsallow the followingconclusions to be made: Regionalbackground monitoring stations in Bulgariaand in parirculartheir river sections used for the conductingof hydrobiologicalmonitoring are surtableand rneetthe necessary requiremenls. l-l Employedmetnods and crite;-iaaiiow tire gatlieringof data, which successfullychar- acterizesthe real state of i^iverecosystems irid ecolDgicalconditions ior their existence, They also are highiysensitive to the siighiestchanges of conditionsand can successfully be used for the purpcsesof backgroundniiirritcring

REFERENCES

Anakumov V. B Russev,1986. Ailaui the in',oS{r!1nii,:n by strbject "Workingout and implant the meihcdsoi biomonitoringof ccmpiexoa;:kgrotlnd stations". Probl backgr.monit. cond. environm.,Voil.16 (ed F. Rovinski),Hydrorneteoizdat, Leningrad.50-55. Gerassimov,S., B. Russev.1987 Gici:aiiiend aiid attainmentin the investigationby zoo- logicaland hydrobiologicaimoniior-rng in Bulgaria.-l'' Nat. Conf probl biol monit Plovdir,,22-24 1987.89-92 Kovachev.S. 198'l. lwo biotic indexesfor riversr:ontrol. colroarison with standardmeth- ods: pro ancjcon- In. Contemporarytrends of the Bulgarianzoolr:gy, BAS,252-256 Kozhova,O 1970.ForminE of phytoplankir":nof the Bratskodanr - In: Formingenvironmental conditionanci life in Bratskodam Sci. 26-160. Naydenov,V., D. Sais. 1989. Methodologicalrecommenclation by biomonitoringf reshwater ecosystemsof the frameworkof problemsXll scientific-technical contributionin CMEA "Globalsystem monitoring environment", Moskow, 1-66 Russev, 8., S. Gerassimov.1985 Generaltheoretical principles and approachesfclr reali- zation biomonitoring.,Ecological cooperation- News bulletinby probl. lll, "Preser- vationof the ecosystemsand landscapes",4, Bratislava,'10-19. Uzunov,J., S. Kovachev.1984. Some principleof the structureand organizationof the hydrobiologicalmonitoring of rivers,Hydrobiology, 21. 34-41. .1993 Janeva, 1.,B. Russev. Hydrobiologrcalmonitoring on river sectionsof the "Rozhen", Rhodopes Mountains,and "Boatin"("Steneto"), Balkan Mountains,regional background monitoringstalions,- Lauterbornia, 14, Dinkelscherben,79-83 Kolkwitz,R., M. Marsson. 1902. Grundsdtzef ir die biologischeBeurteilung des Wassers nach seinerFlora und Fauna.-Mitt. Kgl Pruef.Anst.f.Wasserversorg.,l ,33-72. Kolkwitz,R., M. Marsson. 1908. Okologieder planzlichenSaprobien.- Ber. deutsch. bot Ges., 26, 505-519. Margaleff,R. 1958.Temporal succesion spatial heterogenty in Phytoplankton- In: Perspec- tives in Marine Biology,Buzzali Trav. ed. Universityof CaliforniaProc. Berkley, 323-347 Pantle,R., H. Buck. 1955. Die biologischeUberwachung der Gewdsserund die Darstellrrng der Erqebnisse.-Gas.-und Wasserfach, 96, p. 604. Pielou. E. 1966. Speciesciiversity and patterndiversity in the study of ecologicalsticces- sions.-J.Theor Biol. 10. 370-383 London. Rothschetn,J. 1962 Grafickeznanornenie vysledkiv biologickeho hodnotenia cistoty vc0 - Veda a vyskLtnpraxi VUVH 9.1-64, Bratislava. Russev,B.,S.Kovatschev, I Janeva, M. Karapetkova,J.Uzunov, R. Detscherra.1976 Ver- treter der bulgarischenFluss fauna als limnosaprobeBioindikatoren - Hydrobiology 4, 3-16 Shanncn,S., W. Weaver '1963 The mathematicaltheory of communica- tion - 117 S , LJrbanaUniv. lllinoisPress. Simpson,E. 1949.Measurement of diversity.-Nature 163, 688, London. Sorensen,K. 1948 A method of establishinggroups of equal amplitude irr plant scciety based on similarityof speciescontent.- K.Danske Videsk.Selsk., 5, 1-34 Sladecek,V. 1973 Sysiem of lvaterquaiity from biologicalpoint of view.- Ergebn. Limnol, 7, 1-218. Sladecek,V., E Fjerdingstaad.H. Hawxes. 1971. Manual on Analysisfor Waler PollLitiori Control,Vlll. BiologicalExamination, World HealthOrganization, 1-280. Woodiwiss,F. 1964. The biologicalsystem of streamclassification used by the Trent River Board.-Chemistry and Industry,11, 443-447. Zelinka,M., P. Marvan.'1961. Zur Prazisierungdei biologischenKlassifikation cier Reinneit flieBenderGewasser.- Arch l'lvdrobiol 57 389-407. METHODOTOGICALDEVELOPMENT AND VALIDATION OF INDICATORS FORINTEGRATED ENVIRONMENTAL OBSERVATION Conceptof an Ecosondefeasibility study in UmikerSchachen (Canton of Aargau,Switzerland)

Dr. Kathrin Peter PULS.Berne. Switzerland

SUMMARY The SwissCommittee for EnvironmentalObservation (Schweizerische Kammission f6r Umweltbeobach' tung - SKUB)was commissionedby the SwissAcademy of the NaturalSciences and by the FederalDe- partmentfor the Environment,Forests and Landscape(Bundesamt ftir Umwelt, Wald und Landschaft- BUWAL)to developthe conceptof rntegratedecosystem-related environmental observation (ecosonde) over a six-yeardevelopment phase. The proposedobservation system takes the form of a nationalnetwork con- sistingof regionalobservation areas. The observationprocess is to be implementedin all environmental sectorsand is intendedto extend,complement and integrate the existing,generally sectorial, forms of observation.As well as dealingwith all actorsrelevant to the environmentand their emissionactivities, the ecosondeindicator system also deals with the effects,damage and environmental situation which result from this behaviour.The surveyedquantities are linkedon the basisof suspectedrelated effects. The ecosonde also makesit possibleto documentchanges in thesefunctions. The environmentalobservation system is, thus,conceived as a multi-facetedinstrument suitable for useon a political,administrative and private(e.9. insurancecompanies) level. This system's key wordsinclude early detection, control, steering instruments, tailoredimplementation and "sensitising"environmental reporling. The ecosondeconcept is to be imple- mentedfor the firsttime as partof the UmikerSchachen methodological study. The aim of this pilotstudy is to demonstratethe feasibilityof the conceptthrough its applicationin a specificarea, to researchthe meth- odologyused and to applythe initialresults on a praclical(administrations) and academic level. Investigationon the statusof integratedecological environmental observation have shown that attempts are alsobeing made in othercountries to linkecosystem research, long{erm ecological studies and institutionalenvironmental observation. This proposedstudy sees itselfvery much as part'of this trend and is rootedin the traditionof multi-disciplinaryecosystem research. Thus, it aimsto helpin the understandingof the processesand effectsat work in complexman-ecosystem relationships. In keepingwith this multi- disciplinaryapproach, the observationactivities in thisstudy are to be extendedto social,political and eco- '1. nomicsystems. The projectis dividedinto ten modules: Land-use;2. Economy;3. Socio-culture;4. Ad- ministrationand policyobservalion; 5. Geology/hydrology,6. Air; 7. Landscape;8a Biology;8b Controlof the implementationof the AlluvialSite Decree; 9. Forest. The way in vrhichland is usedreflects various economic, social, and politicalactivities which can give rise to potentialenvironmental impacts which in turnaffect various natural and landscapeprocesses. The social- scientificmodule will observe,record and analyseindicators which describe specific co-operating actors, theiractivities (market and regulations),definitive socio-cultural dimensions and the resultingemissions behaviour.The scientificmodules describe the effectsand problemsarising from this behaviourby meansof specificindicators which will be methodologicallyevaluated in the courseof the research.During the synthesis phaseof the project,the indicatorswill be combinedlo forma consistent,practical set of indicatorswhich are applicableto the entireUmiker Schachen system. This set of indicatorsshould also be applicableto othersystems. The plannedduration of the UmikerSchachen methodological study is three years,during whichit willinvolve close collaboration and co-operation between research and praxis. Key words:development of methodology,environmental indicators, integrated ecosystem-related environmentalobservation, environmental processes, long-term observation, ecosonde, ecosystem research, feasibilitystudy.

CURRENTSTATUS OF RESEARCHIN THE FIELD

Background In the late 1980's,complex environmental problems such as forestdamage and lhe accumulationof pollutantsin the soil led to the recognitionof the urgentneed for an environmentalobservation system whtch goes beyondsectorial boundaries. A betterunderstanding of environmentalprocesses and appropriate measuresto overcomeenvrronmental problems can onlybe reachedby way of a processwhich takes into accountthe cyclesand chainreactions set in motionby variousmedia. Thus, integrated environmental ob- servationcalls for the ecosystemperspective. Sincethe cominginto forceof the FederalAct on the Environment(1985)', the SwissFederation and Cantonsare undera legalobligation lo providea widerange of servicesin the areaof environmentalprotection.However, experience has shownthat the only measureswhich can be successfullyimplemented are

' '1 EnvironmentalProtection Act of October7th 993,SR 81401 l6 thosewhich are widely accepted by the populationancj iocal aulhorities, and that only those solutions which are tailoredto localcircumstances will receivesui:port at ttlis level.This appliesfor the protectionof both waterand air qualityand for the increasinglyimportant areas of natureand landscapeconservation. Due to their generalbinding force, nationai measurement ptans terrd io be greetedwith opposition.The political solutionsought to this p;"oblemtakes the form of difterentiateclexecution which takes accountof regional political.economic and culturalcharacteristics This kind of approachnecessitates differentiated evaluation and controlof environmentalquality Thus, observatiorr systems are requiredwhich take intoaccount both specificlocal features; at the sametime however,there is a demandfor activityal a representativenation- wide level.Most of the nationalobservation networks irnpienrent environmental observation on a sectorial basis2.However, it is possiblefor differentenvironmerrtal sectors to be sirnultaneouslyaffecied by changes in the environmentinduced by measuresintroduced for onesector. Ecosystem observation provides a better meansof evaluatingthe effectswhich occur outside the controlledparamelers than sectorialobservalion does. The successfulintrodtrctiorr of remedialrneasures is dependenton an awarenessof the problemwithin society,and this in turn is linkedwith the perceptiorrof the environmentalsituation. For this reason,integratedenvironmental observation must take economic and social processes into account. The SwissCommission for EnvironmentalObservation (SKUB) was foundedin 1988 with the specific taskof developingsuch a permanent,spatial and integrated environmental observation system' which would also recordunexpected long-term changes in the environment.lt was also establishedto supportthe re- searchof the causesof environmentalproblems and the interpretationof the measuresnecessary to allevi- ate them. The proposalfor an observationsystem has been in existencesince 1993; it fulfills- at least in abstracttefms - nationaland regionalrequirements and was developed in the contextof comparableefforts in neighbouringcountries".

Ihe status of integrated ecological environmental observation in other countries The followingsection will present a smallselection of the projectscurrently under way in othercountriess. Likethe UmikerSchachen methodological study, these projects are basedon ecosystemicand integrative approaches.The increasingpolitical focus on envirgnmentalproblems and accompanyingsearch for new scientificsolutions (ecosystem research, human ecology, long-term studies etc.) in variouscountries since the 1970'shave led to that (specializedand mainlyshort-term) basic research on ecologicalprocesses be complementedby interdisciplinaryand long-termecosystem-related projects. Such conceptshave mainly beingdeveloped in academiccontexts'. In spring1994, the SKUB co-ordinationoffice did sorre researchto establishthe statusof integrated ecologicalenvironmental observation in othercountries (SKUB 1994)'. The studyrevealed that variousef- fods are-nowunderway in somecountries to linkecosystem research and ecologicallong-term studies with institutionalenvironmental observation The German"Concept for ecosystemicenvironmental observation - pilot project for biospherereserves" is a typicalexample of sucha development.In early1992, the GermanFederal Environment Agency in Ber- lin commissionedan interdisciplinaryworking group made up of representativesfrom the Berchtesgaden NationalPark administrationand the Departmentof LandscapeEcology at the TechnicalUniversity in Mu- nichto implementthe ecologicalenvironmenlal observ-alion system'which had beendeveloped on the re- quest of the ExpertCouncil for Environmentallssues". The resultsof ecosystemresearch carried out in Berchtesgadenfor someyears (MAB 6) wereused as a basisfor the developmentof this system.The re- sultingsystem consists of a manualwith mapping and observationdimensiohs which are to be routinelysur- veyedthroughout the nation-widebiosphere reserves al a laterdate. This ecosystemic environmental obser- vationcorresponds in many aspectsto the basicconcept for the UmikerSchachen methodological study startingwith the speciallink and continuingwith the inclusionof the conceptof land use throughwhich the socio-economicfactors are integratedwith the ecosystem.Co-operation with this projectcould take a num-

'Knoepfel 1992on environmentalobservatron and environmental reporting In Switzerland in H.Weidner, R. Zieschank, P. Knoepfel (eds) 3 SNGenvironmentai observation working group in December1987. 4Grolimund,P.andPeler,K., 1994.lntegrlefte'kosystembezogeneumweltbeobachtung,KonzeptfIrdieEinf!hrungeinesBeobach- furgssystems,vdf Ztlrich. Shortreport also available: short report of March3rd 1993by theSwrss Academy of NaturalSciences (published in Germanand in French) t A moreextensive account of the currentstatus of environmentalreporting can be foundin H.Weidner, R. Zieschank,P. Knoepfel (eds.)1992 6 Excellentexamples of this include. Hellaweil 1991 and Likens 1987. 7 Comprlationof someforeign environmental observation instruments relating to integratedecosystem-related environmental observation:SKUB 31 .5.94. A shortsummarv can be found in the annex of Grolimundand Peter 1994. 8 lntermediatereport on the R+D projecl"l

I nterdis ci pl in ary ecolog i cal research The interestin interdisciplinaryecological research projects mainly originates from ecosystem research in the'1970's,in particulartheGerman Solling project". Interdisciplinary ecological research was consolidated in the 1980'swith the developmentof the UnescoMAB programmeand, aboveall, the contributionof the MAB-6programmelu 1in Switzerland: NFP-55). This also resulted in the developmentof approachesinvolv- ing co-operationand collaborationbetween the naturalsciences and humanitiesand in the use of system- supporttechniques (geographical information systems) and the system modelinginstruments. The co-

'o Shortdescrrption of the projectfor the researchproposal. Entwicktung eines lndikatorensystems fiir den Zustandder lJmweltin der B.undesrepublikDeutschland mit Praxistestfilr ausgewdhltelndrkatoren und Bezugsraume,Slatisches Bundesamt Wiesbaden 1995 ' ' Concernof TomorrowCOT 1988and NattonalEnvironmental Outlook NEO 1990-2010.Rijksinstituut Volgsgezondheid en mi- lieuhyygiene(RIVM) National Institute of PublicHealth and EnvironmentalProtection '' Environmentalobservation and Assessment Program; Program Guide: EPA-USA, Office of Researchand Development,1993. 13 EnvironmentData Centre (EDC) 1993: Manual for Integrated Observation, Programme Phase 1993-1 996; Environmental Report 5, NationalBoard of Watersand Environment,Helsinki tn NationalSwedrsh Environmental Protection Board. Monitor 1985. Stockholm 1985. lsEnvironmental ResearchNetwork(ECN):Information,January'lgg2andCoreMeasurementProtocols, 'u Otaft6lTlg3 LarrueC, Knoepfel,P.. final reporl. Evaiuation de la Potitique'frangaisede surveillance et de connaissancede l'6tatde l'environne- rnent,L'oeil and idheap,Chavannes 1993 " Ellenberg,Mayer and Schauermann, 1986 '" Messerli'l 989. l8 ordinationprojects within the SwissNalrr;lial Flescarc;'1 f r111rl's envirorrnlerttal prograrr rrlre (Scl/u/etpLtrikt Pto- grammUmwelt - SPP-L0represent the continuationof thistrerrd Sigrritrcant progress was madein ternrsof methodsarrd experlise through these prolects''. Few critical accolades hacl been awarded in the operative field(project planning arrd steering)- thrrs previoris expefrenues. above all corrcerning individuals who were .fIre involvedin interdisciptrr)afypro1ccts. can strll tre put to gouduse. co-ordinatedco-opr:ration between the sciertcesand humanitiessirll orrly exrsts orr a terrtatrveprelirrrrrrary level fhc proposedrrtulti-disciplirrary UnrikerSchachen nrethodolclgical study uffefs an opporttrnrlytor the develr;prnentof instrurnentsfor operative ptojectplanning and supervisroriand irrpiarlicular the clevelo;rrrrentand irnplemerrtationo1operating techniquesforsyslem-orienlcd arrd spatially lirikerl cu-rl1-reralron between the sciences and hurnanities'.

THESTATUS OF PROPERRESEARCH

The conceptof integ rated ecosystern-related envi ronrnental observati orr Irt responseto the task set at its inceptiorr.SKUB has developeda conceptfor rntegratedecosystem- relatedenvironmental observation urider the directionof the rnainapplicant of this proposaland the co- operationof fourdoctoral stud.ents-' antj a rrrarragementtearn. Various publications are now available on the conceptand rts developrnent Integratedecosystem-related environrnental observation is basedon interactiveecosystemic processes whichexist between the environment,humarr beings and their activities. The obseryationfocuses on all rele- vantaspects of the ecosystemand alsoincludes the socio-economic'andpolltical dimensions. The observation is implementedin exemplaryareas (ecosystems). For practicalreasons, observation activities must be restrictedto suchexemplary areas. An ecosondeis definedas an observationprogramme applied in a concreteobservation area on the basis of a hypotheticallyproven measurement system (ensemble of measurementand observationinstru- ments).The ecosondeembodies a highlysensitive spatially-adapted measurement network for integrated environmentalobservation. As wellas dealingwith all actorsrelevant to the environmenland theiremission activities,the ecosondeindicator system also deals with the effects,damage and environmentalsituation which resultfrom this behaviour.The surveyedquantities are linkedon the basisof suspectedrelated effects Fourbasic subject-specific programrnes constitute lhe coreof the long-termobservation programme. To ensurethat individualareas receive adequate treatment, the four programmesinclLrde a minimumcata- logueof specialsurveying and analysismethods for the observation.and the relevantparameters, evaluation standardsand qualitycontrol tools. The basicprogrammes were developedin areaswhere existing, long-termobservation activities have proven clearly inadequate and/or where it is appropriateto airnat ap- proacheswhich look beyond the specificarea in question.The basicprogrammes cover the areasof landscapeobservation, biology, the monitoringof the flow of materialsand the observationof the activitiesof administrativebodies Existingenvironmental data and surveysspecific to the projectwill constitutethe datafrarnework for the ecosonde.The ecosondeis not intendedto replaceexisting measurement networks and surveysbut to conr- plementthem and availof them in the observationof ecosystems.The ecosonde'stypical data sources in- cludeaerial photographs, archive nraterial, legal and administrativedocurnents and archivematerial. Eco- systemresearch, which provides new insightinto functional links, methodological and standardsforthe routine operationof the ecosondes,goes hand in handwith integratedobservation. The ecosondeprovrdes ecosystemresearch with a well-equipped,interdisciplinary data framework for the generationand testirrgof hypotheses. The proposedobservation system will be shap;edas partof a futurenational measufernent systern whrch shouldalso fulfil cantonal and international requirements. The environmentalobservation system is, thus,conceived as a multi-facetedinstrument suitable for use on a political,administrative and private(e.9. insurance companies) level. This system'skey wordsirrclude earlydetection, control, steering instrurnents, tailored implementation and "sensitising"environmental re- pofling. The ecosondealready exists in conceptualfonn.The nextstep wrll entail testing it on a practicallevel; due to its complexity,this processrepresents a signifrcantscientific and academicchallenge. The Umiker Schachenmethodological study is intendedto substantiateor disproveits conceptualand nrethodological feasibility.The preparatorywork on thisproject wasstarted in February1994 at the instigationof the SANW

National Park ecosonde feasibility study

" Ellenberg1991 '" idem 2'The basicprogrammes landscape (Glauser 1992), biology (Baroni 1992), material flow (clenck 1994 and 1995)and administrative observationWildi 1995)were developed as parlof fourdoctoral theses " Lehmann,P. 1985;Zimmermann 1988, Elsasser H andKnoepfel P (eds.)1990, Peter K andGrolimund Pl 1992Peter K andGro- limundP1993; SKUBl994,GrolimundP andPeterK, l994,Knoepfel P.andPeterK 1994 l() A secondthematically specificfeasibility study commissioned by SKUBISANWis alsobeing carried out in the SwissNational Park in additionto the UmikerSchachen methodological study. The NationalPark study willbe completedin spring1995. As part of the NationalPark ecosonde feasibility study, the basicdata alreadyavailable is beingevaiu- atedin termsof itssuitability for integration(data survey, spatial link, system relationships) and the additional datanecessary forthe operationof an ecosondewill also be examined.The resultsof this studywill be inte- gratedinto the UmikerSchachen methodological study and thus provide information on the comparabilityof the ecosondeapproach in a rural-urbanand rural-natural landscape context. In April1995, the mainapplicant of the currentproposal will participatern the drawingup of anotherre- searchproposal for the developmentof permanentindicator systems which will be rntegratedinto the Envi- ronmentand ClimateProgramme, Human Dimensions of EnvironmentalChange. This projectwill be co- ordinatedby the EnvironmentalPolicy Research Institute at the Free UniversityBerlin. Research institutes fromthe Netherlands(University of Leiden,Center of EnvironmentalScience) and Denmark(Department of Environment,Technology and Social Studies, Roskilde University Center) will also be involvedin thisproject This projectwill providean internationallink with the indicatordevelopment of the UmikerSchachen study andensure its conformity with other observation systems.

DETAILEDRESEARCH PLAN

Aims of the Umiker Schachen methodological study The UmikerSchachen methodological study will involvethe studyand developmentof methodsfor the conceptof an integrated,ecosystem-related environmental observation on the basisof a specificexample. The aim of this pilotstudy is to establishthe feasibitityof the conceptand the use of the resultsin both researchand praxis.The ecosondewill be operatedin UmikerSchachenfollowing completion of thethree-year methodologicaland integrationtests. A set of indicatorsspecific to UmikerSchachen will be definedon the basisof datawhich is, for the mostpart, already available and implementedon a permanentbasis. Given that the UmikerSchachen ecosonde is intendedto functionas part of a nationalobsewation network at a laterstage, the indicatorsel shouldbe compatiblewith those in otherareas.

The project The projectis dividedinto ten thematicmodules. This meansthat clearlydefinable working units have beencreated which can be assignedto the individualresearch teams. This detailed project structure is intendedto counteractthe complexityof the environmentsystem and to establishclearly defined tasks and responsibilities.The processesand individualcomponents relevant to the UmikerSchachen systern can, however,only be describedif all of the modulesare implemented.Our applicationincludes the minimum numberof subjectareas to be appliedin the studyarea. The omissionof one modulewould result in impor- tantgaps in the overallrepresentation of the situationand would make it impossibleto adequatelyjudge the variouseffect mechanisms at work.The ninemodules can be dividedinto three groups: Group1: Module1 Land-use: The way in whichland is usedreflects various economic, social, and politicalactivities which can give rise to potentialenvironmental impacts which in turnaffect various natural and landscapeprocesses. Group 2: Module2 Economy;3 Socio-culture;4 Administration and policy observation: These modulesfocus on economic,social and politicalprocesses. They observe,record and analyse indicatorswhich describespecific co-operating actors, their activities(market and regulations),definitive socio-culturaldimensions and resultingemissions behaviour. The steeringfactors for policyimplementation can be found here.The socio-culturalmodule examines the feedback-linkbetween environmental quality and its perceptionby the actors. Group 3: Module5 Geology/hydrology;6 Air; 7 Landscape;8a Biology;8b Controlof the implementation of the AlluvialSite Decree; 9 Forest: The scientificmodules describe the effectsand problemsarising from this behaviour by meansof specific indicatorswhich will be methodologicallyevaluated in the courseof the research.They are concernedwith naturaland landscape processes. The indicatorsrepresent the various parameters of the specificprocesses. It is alsointended to establisha material-flowmodule based on the SKUBmaterial-flow basic programme (Glenck1995) at a laterpoint in the methodologicalstudy.

Relevance of the individual modules within the methodological study Group1: Module 1 - Land-use:Land-use is a productof economic,social and politicalactivity and reflectsthe valuationof existingresources. lt transformssocial values and servicesinto naturaland landscapeprocesses(production, resource management and evaluation).The periodicactual use of the land,accurately recordeddown to the lastplot, forms the basisfor the use-relatedanalyses of the othermodules and is to be

20 made availablein the form of a geographicalinformation system. The actualuse^categories will be defined on the basisof the requirementsof allofthe modulesand existing use categories'" (spatialstatistics).

Group2: Module2 - Economy:The economymodule establishes the linkbetween economic parameters and the observedenvironmental effects, in particularin the areasof emissionsand the flow of materialsand energy (interfaceto group1 and 3 modules).The observationand explanation of the ecologicallyrelevant behaviour of economicactors is central.The concreteobject of the study is to establishthe extentto which environment-relevantdecisions by certainactor groups (companies, households, state bodies)are determinedby generaleconomic development, environmental policy measures or other factors(interface to socio-cultural module).This actor-relatedapproach provides the basisfor both polluter-orientedsolutions and for the pre- steeringof environmentaleffects in the senseof a preventativeprinciple..The economy module serves the furtherdevelopment of the ecosondeconcept to an integratedecological-economical environmental observationsystem. Module 3 - Socio-culture:This modulerecords data on the way in which the social system reactsto changesin the environment.The mainfocus is on the integrativeevaluation by membersof the populationof changesin natureand landscapein the contextof the formationof politicalwill. The modulestudies the way in which the cognitivedimension, interest orientation and affectiveor emotionaldimension become part of the processof the formationof politicalopinions and how these componentsare weightedwithin this process. This modulewill provideinput in the areaof emotions.Data on the cognitivedimension will be inte- gratedinto the Group3 scientificsurveys and the interestsof modules2 Economyand 4 Administrationand policyobservation. Module4 - Administrationand policyobservation: This module aims to providea permanentsupply of monitoringmaterial on the real environmentaleffects of stateactivities and the testingof the corresponding hypotheseswhich can be formulatedon the basisof existinginformation. The inclusionof administrationand policyobservation as a field in this studyreinforces the relevanceof the ecosondeas an instrumentfor the steeringof environment-relevantprocesses. State policiesare the embodimentof this steeringdimension. The expansionof the frameworkof the methodologicalstudy to includethe policydimension in the areasof natureprotection (Alluvial Site Decree)and clean-airwill facilitatethe developmentof policyproduct moni- toringwhich is of particularinterest when it comesto the definitionof politicalpriorities.'

Group3: Module 5 - Geology/hydrology:This moduleis basedon a fundamentalapproach. Firstly, it provides basic data for the other modulesand, secondly,it demonstratesthe interactionbetween hydrosphere and geosphereby meansof an integralobservation approach based on the exampleof the alluvialsite in a specificarea. The timeaxis will also become a dimensionof the observationas a resultof the multi-phasecom- plex developmentof the studyarea. The nextstep will involvethe evaluationof the relationshipsand effects of processesin the geosphereand hydrospherein the contextof the otherenvironmental areas represented in the project.The modulewill providedetailed insight of continuingrelevance for futureand permanentre- sourcemanagement in the region. Module 6 - Air: This moduledescribes the pollutionof air by individualpollutants, the accumulationof these pollutantsin lichenand the biologicaleffects of the overallprocess of air pollutionon lichens.The data representsparameters for the emissions-pollutionmechanism and the effectof the pollutionon livingorgan- ismsand communities.Data surveys in thisarea have hitherto been aimed at the definitionof the pollution and its effects.In the contextof the methodologicalstudy, these surueys are to be processedfor integration withother areas and will, thus, provide a newperspective. The biologicallichen indication method (lAP) and the bio-accumulationof lichensare integralparts of the basicbiology programme. These are established methods- thus,it will not be necessaryto developmethods for this project. Module7 - Landscape:The basiclandscape programme is beingimplemented as partof the landscape module(Glauser 1992). The landscapeobservation programme documents the multi-layeredtransformation of the landscapeand putsit intocontext with building, maintenance and otherfunctions as triggeringfactors. Real functionalchanges which affectthe landscape,for examplein farming,have a directeffect on the state of the environmentand thus representfactors which give riseto change. Module 8a - Biology: The biologicalmodule is concernedwith the developmentof a standardised methodfor the observationof vegetationand its dynamics.In the contextof the basic biologyprogramme (Baroni1993), this task corresponds to thevegetative mapping of the biotope.Pedological indicators will also be evaluatedto providea fasterindication of changesin the environment.The moduleexplains and demonstratesthe linksbetween vegetation, soil and soilfauna. This biologypart-module will ensurethe scientifl- cally-foundeddevelopment of the observationmethod for the BUWAL"Alluvial Site MonitoringProgramme".

23 BundesamtfUr Statistik 1992 2l As a complementto module8b, the controlof the implementationof the AlluvialSite Decree,this module alsoinvolves the controlof biotopedevelopment. Module8b - Controlof the implementationof the AlluvialSite Decree:This part-moduleis a partof the BUWALAlluvial Site Monitoring Programme. lt implementsthe controlof administrativeprotection measures.The work carriedout in UmikerSchachen on the AlluvialSite Monitoring Programme serves methodologicaldevelopment in predominantlystabilised alluvial ecosystems. These are typicalcharacteristics of the conditionof alluvialsites in centralSwitzerland. Module 9 - Forest:The forestmodute is part of the WSL "long-termforest ecosystem research" project. The condition,structural and functional variety of the riversidewoodlands represent both a responseto local abiotic-bioticsystemic interaction and the outcomeof managementpractices. Sustained forest management is definedin boththe ecologicaland social contexts. The modulesurveys location parameters in the context of riskestimation of riversidewoodland development against a backdropof changingenvironmental circumstances.The datashould be linkedwith the permanentSwiss forest observation programme and the forest inventory. The annexcontains an overviewof the numerousinterfaces between lhe modules.The interfacesclarify the multi-disciplinaryand interdisciplinarynature of the entireproject. The overviewcontains a list of vari- ableswhich the modulesrequire in the developmentof the hypotheses.These variables are dividedinto categorieswithin the threegroups. The categorieshave been adapted to the compendiumof Swissenviron- mentalstatistics (Federal Statistics Office, BfS). The statusof the individualmodules with respectto theirimplementation in the integratedecosystem- relatedenvironmental observation system differs. In the case of module6 - Air, for example,suitable and establishedpermanent observation methods are already available which can be usedin the testarea without needingmajor adaptation. ln the caseof module7 - Landscape,SKUB has developeda basicprogramme withinthe frameworkof its conceptualwork which has already been tested in the Ldgerntest area (Glauser 1992).Modulesl-Landuse,6-AirandT-Landscapeandpart-moduleSb-thecontroloftheimplementa- tion of the AlluvialSite Decreeare technicalmodules. They are basedon a practicalconcept and do not requirefurther theoretical research. The othermodules, module 2 - Economy,3 - Socio-culture,4 - Admini- strationand policyobservation, 5 - Geology/hydrology,8a - Biology,9 - Foreststill require intensive meth- odologicalresearch. These lasl modules represenlthe main body of the academicresearch to be carriedout by the methodologicalstudy. Chapters2.2.3 of the individualproject proposals contain precise descriptions of the plannedstudies and experimentswhich will be necessaryto the completionof the individualmodules. The module-internalap- proachesare alsodescribed and detailed information is providedabout methods already available to module directorsand areasfor which methods need to be develooed.

Synthesis The integrationof the variousobservation areas represents a majoracademic challenge. In additionto quantitativeeffect processes, this also invqlves the establishmentof linksbetween "soft" qualitative informa- tionand "hard"ouantitative statements. Duringthe synthesisphase, the indicatorswill be combinedto forma consistentset of practicalindicators applicableto the enlireUmiker Schachen system. In additionto functionalfactors, this set will also take statusand steeringfactors into account. The indicatorsare definedon the basisof the individualmodules. Theseindicators provide guaranteed statements applicable within the modulesand aboutthe linksbetween the differentmodules. There are a numberof methodswhich would be suitablefor use in the synthesis phase.The firststep will involvethe definitionof the paramelersrelevant to UmikerSchachen. Information basedon expertiseand experienceis crucialto thisprocess. The methodswhich could be usedinclude the Delphimelhod2a, the systemanalysis method developed by Vestelsand othermethods involving networked thinking".System dynamic modelling (Stella), which offers the possibilityof a soft-modellingbased system analysisis anotheralternative. The qualitativedescription of the assumedlinks acts as a basisfor the quan- titativedescription of the systemor itsparts. This is implementedthrough the mathematicalformulation of the observedlinks. The mathematicalmodel building (hard modelling) could be basedon the classicalanalytical methodand system identification - possibly combined''. The secondstep wouldinvolve the testingof the indicatorset for errorboundaries, ability to be repro- duced,sensitivity and specificity to the UmikerSchachen system, applicability to othersystems and areaand temporalvalidity. In additionto multivariatestatistics, retrospective tests. mathematical model building methodsand scenarioanalyses could be usedfor thispurpose.

'o DaenzerW.F and F. Huber (eds.) 1994. 25 Described in Vester 1980 and 1990. 'u UlrichH. and ProbstG.J.B. 1991 and ProbstG.J.B and Gomez P. 1993. " A detailed representationof this process can be found in Buhler K in Annex B. Models and theoreticalbasis can be found in Gro- limundP. and Peter K 1994 11 The projectgroup brings together a broadspectfuin of environmentalexpertise and researchexperience in the individualfields. The projectinvolves highly reputable researchers from all overSwitzerland and per- sonswith long years of practicalexperience in administrativeErodies and environmental consultancies.

The arganisation of the project The methodologicalstudy is a compiexproleci whictr places tiigh demancts on the academicand social competenceof participants.lrrtegration in the environmentalfield primarily irrvolves the linkingof scientific resultswith social-scientific studies Partrcular attention will be paidto communicationproc;esses in thisinter- disciplinaryproject The developmenlstatus of the indicators,rnethods and aiinsin the indrvidualobserv'ation areas differs considerably.In the caseof air,for example,proven indicators already exist for the effectsand are readyfor integration.The statusof the biologicaland socio-cultulemodules contrasls sharply with lhis and the first stepwill involvethe developmentof suitablemethods for theseareas. This will necessitatemutual agree- menton scientificaims and oraxis-orientedadministrative tasks A furtherpotential source of tensionexists between the differentadministrative levels of the Canton,Fed- erationand international institutions. The selectedorganisational form of the prcijecttakes account of thesemulti-faceted requirements and the concomitantrisks. The organisationof thefuture project should be basedon the followingbodies:

- A sponsorshipbody whrchunites the institutionsinvolved on a financiallevel. The sponsorshipbody uniteslhe usersof the integratedobservation system to be developed.The sponsorsinclude the threeFed- eralagencies BUWAL, BRP BfS,the cantonof Aargau,LEKUB, the SwissNational Research Fund, and, possiblyalso, some private institutions.

- A steering committeewhich consistsof representativesof the sponsoringinstitutions, The steering committeehas the taskof establishingthe projectin itsscientific and administrative-political environment and in the sponsoringinstitutions. lt is responsiblefor strategicdecisions and will monitorthe progressof the project.

- The project group whichincludes the projectmanagement and the moduleswith their directors and re- searcners. - The project managementis againdivided into technical management and subject-baseddata integration office The technicalproject management is responsiblefor operativedecisions, the coordinationand managementof the progressof the project.The tasksof the projectrnanagement include the nationaland interna- tionalnetworking and the marketingof the project.The projectmanagement will be the proyectinterface lvith the SwissNational Research Fund. The academicdirector of the projectis itsprincipal applicant. The dataintegration office is responsiblefor the integrationof the datafrom the varioussubject areas lt will producethe GIS conceptand buildup the GIS adviseand supervise the moduleresearchers and carry out researchinto the methodologicalaspects of the dataintegration. The supervisorof the dataintegration officeis to haveaccess to the GIScenter of theAargau Construction Department.. - The individualproject group members are responsiblefor lhe implementationof the modules andthey arefree to selectthp module-internaldirectorship. They will co-ordinate and managethe internalprogress of the moduleand they are also responsiblefor the processingof inter-hypotheses-o.Dunng the synthesis phasethey will be availablefor subject-specificco-operation on the indicatorset. Their tasksinclude the establishmentof the academicroots of the modules(including national and internationalresearch projects) andthe involvementof therelevant national and cantonal instances. Regularreporting and exchangeof informationbetween the projectbodies (steering committee, project managementand modules) is plannedas a wayof optimisingthe projectprocess:

The Umiker Schachenobservation area The UmikerSchachen observation area lies in the centralregion of the cantonof Aargau.The studype- rimeterof the methodologicalstudy embraces the five communitiesof Brugg,Umiken, Schinznach Bad SchinznachDorf and Villnachern. Umiker Schachen, an alluviallandscape area along the banksof theAare river.is locatedat the centreof the studyarea. In additionto thisalluvial site of nationalimport, the test pe- rimeterof the methodologicalstudy includes, the agglomerationof Bruggand the four communitiesrnen- tionedabove. The testsfor eachmodule have been adapted to the specificsize of the observationarea. The test perimeterapplies for all the modules,however, some modulesgo beyondit (e.9.economy, hydrology/geology).The basicdataforthe ecosondeis providedby existingobservation prograrnmes and existing

28 A distinctionis madebetween Inter-hypotheses and internal hypotheses The former are characterised by the factthat data rs requrrecj forthe hypothesis test which is gathernot only wrthrn one module but through the cooperatton wrth other modules jr data". Clarificationwith the cantonalauthorrties has revealedthat it would be possiblelo transfercurrent sectorialenvironmental observation to UmikerSchachen lt wouldalso be convenientto combinevarious currentdevelopments on nationallevel with the methodologicalstudy. An "AlluvialSite ObservationPro- gramme"is currentlybeing developed by BUWAL The inclusionof the developmentof this observationpro- grammein the plannedmethodological study substantiates an impoftantassumption of the ecosonde:the inclusionof the sectorialobseryation network in an integratedprogramme. The advantagefor the obserua- tion programmelies in the potentialcombination with other measurement systems offered by the ecosonde andwith complementary processes through the observationof the effectmechanism UmikerSchachen is a rnajornatural resource, a factwhich is reflectedin the inclusionof the areain the nationalinventory of importantalluvial sites. The conditionof the areais, however,influenced by a widevariety of uses:the use of hydro-electricpower changes the waterand balanceof alluvialdeposits. the banks have,forthe mostpart, been reinforced, the forestsare usedon a commercialbasis and the localpopulation usesthe areaas a leisureamenity. Umiker Schachen provides a typicalexample of the currentsituation of a centralalluvial site and the pressuressuffered by sucha systemdue to the widevariety of usesto whichit rs subjected. Alluvialsites are highlycomplex ecosystems. One of theirdistinctive features is the simultaneityof6ev- eralecological gradients: alluvial sites combine different development phases - frompioneering societieis and numeroussubsequent societies to the balancingend phaseof the climaxsociety. These stages are charac- terisedby differentdegrees of complexityand levelsof organisation.Functions like food chains and the en- tirespectrum of energeticrelationships, varying time scales and area distribution of regulatoryprocesses can occurin the sameecosystem. lt is thiscompleteness of processesand characteristicsthat makethe alluvia. sitean idealobject forthe generalisation of functional observation

TIMETABLEFOR THE ENTIRE PROJECT

The proposedduration of the entireproject is threeyears lt is dividedinto three phases which are subdi- videdinto sub-phases. These subphases constitute complete autonomous pro'cesses with gradualtargets Theend or eachsub-phase is markedby a milestonewhich rvill enable intermediary decisions on thefurther developmentof the project.This is linkedwith the allocationof furlherfinances. The phasedstructure is aimedat achievinga simplesystem for the steeringand controlof the projectand the finances.The phases buildon eachother (0) Contactphase (prior to the beginningof the methodologicalstudy) - Preparatorywork for the combinationof researchand practicalwork; co-ordinationdiscussions with authoritiesand in particularthe canton of Aargau. (1) Preparatoryphase (time required: 12 months) 1 a) - Existingdata collated and processed for hypothesistesting - Theconcept is producedforthe geographical information system and data integration. 1 b) - Missingdata surveyed. - Databasecreated and collected data processed for the database. (2)Module phase (time required: 12 months) 2 a) - Hypotheses(internal hypotheses) tested within the modules. 2b) - Hypotheses(inter-hypotheses) tested between the modules. (3)Synthesis phase (time required: 12 months) 3 a) -Researchfindings combined to form indicatorsets (parameters,methodological, measurement system)which correspond to practicalrequirements. 3 b) - The Umiker Schachenmethodological study obseryationconcept wiil be convertedto the ecosonoe.

The imporiance of the Umiker Schachenmethodological study in researchand praxis

The plannedproject is intendedto provideinformation on the feasibilityof the ecosondeconcept. To this end,actual methodological requirements with a conceptualdimension (hypotheses) are to be implementedin the form of six modules.The simultaneouspractical test constitutesa preconditionof thesemethods and conceptsfor the institutionalisationof observation on nationallevel and for the operationof ecosystemob- servation.This observation system will be adaptedto the Swisssituation and capableof beingnetworked al internationallevel The mainpractical contribution of the prolectlies in the followingfeatures, particularly the waythat

- it enablescomprehensive evaluation and monitoring of the environment;

:!'A selecttonof avatlabledata and informationon UmikerSchachen rs provrdedin the annex - rtenabies early deiection of damageand provrJes new insigiit into the linksbetween effecis as a basis for the cornprehensiveevaluation requii'ed ior integiaieieilviionmental reporting; - in additionto controllingthe politicaiimpier-neniaticn of ;"neasrires, it also providesresults ancj bases for the developmentof generalpolicy progranrmes and for the evaiuationof effectivemeasures. - it can be usedas a managementinsirument for re,clicilalimpierrreniation tasks; - ii enaolesihe integrationof giobaiissL:es witn regioiiai ieqLrirements, - it offersa weil-equippedlong-term data iaooi-atory io, lne snon-lsmrgeiieration of hypothesesin the areaof environmentalresearcfr. -T"he rmpcrtanceof ihe methcdologicalstijdy iies iri ir) irLjiTl€rr"ri;sLli-idgiiig ftrriciions anc integrationserv- icesbetween: o i-esearchanC practrCe; o socialand naturai screnics 3 secloi'ialand poiiticai-aiirninisiiativeareas . diiferentobservaticn arid policy levels. The testingof hypotheseson the basisof tne airiitirio pio,rifiea meihoiclcgicaldefinition for the as- suineClinks within ecosystems wiiich aie reveaiedthi-ough ecosysiem ;"esearch is at the heartof the Umiker Schachenmethodological stuciy. The studyrepieseiits ar fur'rheratiempt to reccrdand describqthe complexityof systenrs.it representsan importantstep in the directionof an improi,eounderstanding of functional links.The attemptto recoi^dthe changeand deveioprnent cf functionsancl to makethem accessibleto iong- term observationis alsocentrai. This representsa cruciaiacidition which ecosystemic observation can offer to otherobservation approaches. The sustaining of systernsis cioselylinked with functionality. The projectalso contributes to: - the observationof alluvialsites: the developmentcf a biologicalprogramme is scientificallysupported by datafrom other environmental sectors and can be netrvorkedwith Administration and.policy observation. - documentationand archiving:existing data in all of the relevantenvironmental seclors in the Umiker Schachenregion are beingcombined electronically and v'rill be avaiiableon a GlS.This is highlysignificant, not leastfor the administration("spaiial{emporal memory"). - the overallviewof a region:missing data will bre collected and gaps filled; - coordination:the datasurveys will be comparedso thatmultiple surveys and datagraveyards can be avoided.

LITERATURE

ArbeitsgruppeUmweitbeobachtung der SNG: "l 987 UmweltbeobachtungSchweiz, Programm fiii" eine integrieileokologische Raumbeobachiung, Bern, Dezember Baroni,P.: 1992 Observationde l'environnement.Contribution de la Biologied un conceptinterdisciplinaire pour ia sur- veillanced longterme de l'6tatde l'environnement.Universit6 de Lausanne. Bundesamtftlr Statistik 1992 Arealstatistik197911985: Kategorienkatalog, Statistik der Schweiz,Fachbereich 2 Raum,Landschaft unci Umwelt.Bern.. DaenzerW. F. undF. Huber(Hg): 1994 Systemsengineering, Methodik und Praxis; 8. verb.Auflage, Verlag lndustrielle Organisation Ziirich Ellenberg,H.: 1991 Erfahrungenbei interdisziplindrerOkosystemforschung im Solling seit 1966.In: Nievergelt,8., Scheurer Th.:Forschung in Naturreservaten.Publ. der Schw.Akad. der Natunviss.Nr4, UniversitdtverlagFribourE EllenbergH., Mayer und Schauermann: 1986 Okosystemforschung:Ergebnisse des Solling-Projektes 1966-1986"- Stuttgart; Uimer Elsasser,H., Knoepfel, P., (Hrsg.): 1990 Umweltbeobachtung;Schriftenreihe Wirtschafisgeografie und Raumplanungdes Geografischeninsiitutes der UniversitAtZtrrich Vol. 8 Glauser,P.: 1993 Landschaftsbeobachtung.Wirtschaftsgeographie und Raumplanung Vol. 16, Geogr. Inst. Uni'v. Zudch Gienck,E , 1994 Contributionsdes m6nagesurbains dans ie rn6tabolismechimique des filieresde d6sapprovisionnei;ien'i des bienssolides, Dissertation EPFZ Glenck,E , 1995 Ooservationr6gionale int6gr6e ei A longterme du m6taboiismemat6riel et chimiquede i'anthrcposphei'e et de I'envirorrneiTrefitabioticue. CSOE Heilawell,John M.: 1991

2i Developmentof a rationalefor monitoring,in: Monitoringfor Conservationand Ecology,ed. F.B. Golcj- smith,Capman & Hall,London Grolimund,P. undPeter, K., 1994: Integriertedkosystembezogene Umweltbeobachtung, Konzept f[]r die Einfiihrungeines Beobachtung- ssystems,vdf Ztirich Knoepfel,P.: 1992 Umweltbeobachtungund Umweltberichlerstattungin der Schweiz;in: H. Weidner,R. Zieschank,P Knc- epfel(Hg.): Umwelt-lnformation, Berrchterstattung und Informationssyslemein zw6lf Ldndern, WZB. Ber- lin KnoepfelP. und PeterK.: 1994 The swissconcept of integratedecosystem-related observation:the ecosonde; Report of the UNEP-HEM workshop:Harmonization of EnvironmentalData. Lehmann,P.: Januar 1985 Auswirkungender anthropogenenLuftbelastung auf den Menschenund seine nattlrlicheund kulturelie Umwelt, Forschungsstand,Forschungslt.icken, Abdeckung der Bedurfnisse,Bern, Schweizerische Naturforschende Gesellschaft. Likens,G.E. (Ed.): 1987 Long-TermStudies in Ecology.Approaches and Alternatives. Springer Verlag New York. MesserliP.: 1989 Menschund Naturim alpinenLebensraum; Chancen und Risiken PeterK. undGrolimund P. : Vom Sichtenund Sammeln,Der Auftrag der Schweizerischen Kommission fi.ir Umweltbeobachtung GAIA 1 (1992)no. 4 S. 232- 233. PeterK. undGrolimund P.: 1993 Okosonden:integrierte regionalisierte Umweltbeobachtung in GAIA 2 no.45.239 - 244. ProbstG.J.B. und Gomez P. 1993 VernetztesDenken: Gabler . Wiesbaden2. Aufl. RUSAG: 1995 Kurzberichtund Protokolleder lnterviewszur Abkldrungder Bedtirfnissedes KantonsAargau bezi.iglich Okosonde"Machbarkeitsstudie Umiker Schachen" zuhanden der SchweizerischenAkademie der Natur- wissenschaften,Bern. SKUB(Schweizerische Kommission fi.ir Umweltbeobachtung): 1993 Arealstatistikder Schweiz:Landnutzungskategorien im Vergleich mit europdischenLandnutzungsstatisti- ken und Erhebungenzur Raum-und Landnutzung auf Bundesebene.Bericht im Auftragdes Bundesam- tes fi.irStatistik. Bilro B. Berz,Bern. UlrichH. undProbst G.J.B.: '1991 Anleitungzum ganzheitlichenDenken und Handeln, 3. Auflage,Paul Haupt Verlag Bern VesterF.: '1990 AusfahrtVerkehr, Wilhelm Heyne Verlag, S.21, Mtinchen. VesterF.: 1980 Neulanddes Denkens; Mi]nchen. WeidnerH., Zieschank R., Knoepfel P. (Hg.):1992 Umwelt-lnformation,Berichterstattung und Informationssysteme in zw6lf Ldndern, WZB, Berlin. Wildi- Ballabio,E.: 1995 Observationdes activit6s6latiques li6es d l'environnement,Programme de baseet listede parametres, CSOE. Zimmermann,W.: Februar 1988 Umweltbeobachtungund und Umweltforschungin der Schweiz,Schriftenreihe Umweltschutz Nr. 80 und 81, Band| (Dokumentation),und Band ll (Umfrage),Bern, Bundesamt fi.ir Umweltschutz.

26 THEMICROORGANISMS AS INDIEATORSOF THEENVIRONMENTAL POLLUTION DenchoDenchev, AtanasKaimaktchiev, Kolishka Tsekova Instituteof Microbiology- Bulgarian Acaijemy of Sciences

1. A SI-IORTREV|EW OF THEPROELEM Hea,rymetals ai"e the main soil, air and watei poliutiirrrsTheir increasingquantity in tne biosphere "cnangethe structui"eof microbialcenosls, destroys rhern arrct cieates artificial agroecosystems" [1]. Tfie prc'bieinof heavyrnetals influence on soil[iricro{r[gai'risrns, plarrts arrd anirnals is very importantfor nowadays.The reasortis that in the soilare concentratedprocesses of organicsubstanc:es mineralization. The pollutantschange soii conditions and themselr;es have physical. chemical and biochemicaltransforma- lions.These new cnernical substances are the seccndai-ypc,llutants and makeworse the soil properties,the kineticsof soil niirogenand humuscreation Therefoi"e ttie analysisof physicaland chemicalproperiies of the soilis mostimportafit for testing of its pollution. The toric activityof heavymetals on the micruorganisrirsis widely discussed in the papers.Bishoff [2] ihoughtthat the toxicityspectrum included the primarygroMh inhibitionto total eliminationof microbial population.The internrediatereactions could be uitrastructuralmorphological changes and the changesin the metabolicpathways and enzyme acrivities. The microorganismsreact in differentways on ihe heavymatal, depending on theirspecies. For all the microbialcells these substances in a verylow concentralionsare necessaryfor theirvitality. In a highcon- centrationeach of thesemetals became a toxicones for them.The cell reactionsare connectedwith the changesin theirquantities ant speciesbelonging [3, 4]. Mostof the heavymetals inhibit respiration [5], pro- vokebiostatic effect [6] or reactlike a mutagen[7,8]. In somecases some resistant forms appeared which is geneticallydetermined or is in a connectionwith to cellwall permeability when pH is low [9] Mostsensible are cyanobacters.actinomyces and algae, and the mostresistant are fungr and some thionobacters [10, 11]. It is consideredthat eucariotesare more resistantto the toxicinfluence of the heavymetals than the pro- karyotes Some of the heavymetals rnhibit the enzymeactivities in the soil:amylase, dehydrogenase, urease, in- vertase,catalase [12,13,14]. That is whysome authors suggest index analogical to LD56where the pollut- o/o ant concentrationdecreases the cellphysiological activities with 50 or 25 [15,16]. Whenthe heavymetals are in the environmentthey have an immediateaction not on the soilonly, and on the plantsas well.Some data from the papersshowed that the numberof yeastcells on the greenparts of the plantsis increased.Tnat is the reasonsome epiphytic yeast species are usedas indicators[17]. The specialresistance to heavymetals are manifested in pigmentedmicroorganisms [18] orthose which producedcarbonic acids [19], because the organicacids are chelatingagents and easilycreate stable complexeswith metal ions. This could be usedas a mechanismfor their detoxification 2. ANALYSISOF OUR PRELIMINARYEXPER]MENTS In our researchwith yeastand mouldswhich are citricacid producers in the mediawiih ihe nighercon- centrationof cooperions the pigmentationof yeastand their preserved organic acid ability were checked out. The same effectwas not showndown in mediawith cadmium.Nevertheless, the criticalconcentration of cooperions were much morehigher compared to thoseof cadmium.In the bolh groupsof microorganisms therewas a changein the cellshape and size. lt wasessential that the groMhof alltested microbial species producingcarbonic acids and otherbiologically active substances was totallyinhibited by higherconcentfa- 'l). tionsof Cu2+,Cd2+ and p52+ iTable Thereis a strainof Aspergiltusniger, producer of glucoarnylase, wherecadmium inhibited the enzymeactivity but not the cell groMh.Therefore it could be assumedthai thereis a correlationbetween the organicacid production ability and the resistanceto metalions. This fact givesthe possibilitythese microorganisms to be usedas indicatorsto heavymetal pollution Because of this we can use the isolatesfrom naturalsources which have these abilities to introducethern in the pc,lluted regionsand to checkout their survival. DifferentmicroorEanisms could uptake heavy metals in concenlrationsmuch higherthan their nonnal requests.Increased quantities of heavymetals in mediabrought to accumulationoi theseions in nticrobial cellsin concentrationhigher than in the media1221. ln ourstudies (Table 2) 50 - 8O%of Cd2+was accurnu- latedin the yeastbiomass. This processstrongly depended on the species,the strain,the quantityof the metalin the medraefc. At lowermetal concentrations the accumulationin the cellswas considerablyhigher. In conclusionit couldbe saidthat microorganisms have a flexiblemetabolism and ciearlyexpressed mor- phologicaland biochemicalreactions. That is the reasonwe can use them as indicatorsfor antropogenic pollution.Their use for bionronitoringis possiblebecause of the populationreaction to successiveenviron- mentalalterations. The observation,the registrationand the analysisof thesereactions are the maintask for directprotec- tionof environmentfrorn xenobiotics using selected microbial strains.

LI Table1. Totalelimination of microbialgrowth in mediawith differentheavy metals.

Microorganism Producerof: Inhibitingconcentration Cu2+ Cd2+ pb2+ (mg.l-1)

Asp.niger 13 citricacid 89.3 9.2 Asp. niger 73 gluconicacid 102.0 7.4 Asp.niger 77 glucoamylase 51.0 18.5 Asp.teneus itaconicacid 51.0 18.5 Rhizopuschlnensis unknown 25.5 5.5 Pen. claviformae antibiotic 51.0 36.9 Sacch.lipolytica 21 crtrrcacid 280.5 14.6 1023.4 Sacch. lipolytica 43 citricacid 357.0 9.8 584.8 Sacch. lipolytica 58 unKnown 63.6 24.4 Rhodot.glutinis unknown 102.0 73.2 621.4 Candida utilis proteins 25.5 2.4 621.4 Candida scoftri proteins 76.4 14.6 365.5 Candidaacapsuligena proteins 76.4 24.4 804.1 Candida boidinii proteins 76.5 48.8 731.0 Sacch. cereivsiae bakeryeast 157.5 14.6 1096.5

Table2. Contentsof Cd2+in yeastcells after 72 hours cultivation

Yeastspecies Strain Cd2+in Biomass Concentrationof No. the media cd2+ in the biomass (mg/l) (s/l) (ms/g) (v")

Cand. tropicalis 46 48.7 5.18 4.90 52.1 Cand. tropicalis 46 58.2 3.31 8.36 47 3 Sacclr. lipolytica 40 2.4 3.99 0.38 62.5 Sacch. lipolytica 40 9.8 1.87 2.46 47.2 Candida utilis '115 2.4 3.65 0.54 80.5 Candida utilis 115 9.8 1.92 3.00 59.1

3. PURPOSESOF THESTUDY The mainpurpose of our researchis to findout the possibilitiesto usethe microbialproperties for indicationof antropogenicpollutants - heavy metals. In thisway the microorganismsas an activestructural part of naturalecosystems can be includedin the primarytrophic part of the systemfor effectiveenvironmental monitoring. In the courseof the investigationthe seasonkinetics of soil,water and epiphyticmicroflore about their quantitiesand speciesvariability, metabolic activities (enzymes and respiration)should be checkedout. The estimationwould be givento the populationadaptability to inhabitcertain environment and on the otherhand the presenceof heavymetals and petroleumproducts pollutants. This will givethe opportunityto checkout the optimumand the mostdisadvantage phase of microbialsuccession. The microbialpossibilities as de- structorsof thesetypes of pollutantswill be evaluated. 4, DESCRIPTIONOF THE METHODS It is difficultto imaginethe ecologyof plansand animals without studying the populationsin the nature.lt is clearthat in this way we can foundout the mostrmportant properties which provide the developmentof the objectsin the environment. Long time in the microbialecology dominates another approach: microbial populations were studied mainlyin experimentalconditions on specialnutnent media and the dataobtained were usedto extrapolate the relationshipsin nature.The disadvantagesof thismethod is evidentbut the currentmethods for investigationsof microorganismsdid not givethe possibilitiesfor checkingout the certainpopulations in natural habitats. Duringthe lastfew yearsmore accurateand rapidmethods for checkingthe speciesdiversity, quaniity and functionsin naturewere successfullyapplied. This givesthe possibilityto selectspecies and strains whichcould be usedas indicatorsfor differentantropogenic pollutants.

28 Fromtheoretical pclint of viewfoi workingout of the p,fo1ectG'K strategresaie used baseciort lr4acl..i1rrr hypothesiswhich is developedby many rrowadays airthors [20,21) The nrainpoint of the ideafor ecologicalstrategy rs rr a connectrorrwrth tne princ;ip;alof trticrcoiganrsnt optimalconstruction.'fhat means the cell will preserve itself if it is possibleto choiceand realize from all the potentialand possible multrtLrdes the one which is n d ccirrn€clionwith the rtrinimal perrnissible: price In thisaspect the oblectfLirtctiorr F cc.,trlrl be displayeci.rs sriri'r of groMh erlei'gyconsriiTrptrort A mainte- nanceI andorotection C F=A+B+ C The parametersA B arrrlC giveto the ot-rlecta grotip,,rf indiciltir-ris whtch cuttlrj be experirnettiailyde' termined. lf the ecosysterrrcorrditrons are be exanrinedin two levels:"saturated" and "non-satuiated" rrilhese two systemsa drfferentselection of specieswrll be realizedwhrch will depend on physicaland chemical proper- tiesof the systemand on the brologicaland gerretical possibrlities of the strair.tsThere is a sialernentwhich meansthat the populationcould not rraxirnrzed simultaneously both strategies K and R hrecairseof selfpro' tectiorrprincipal It is wellknown that the rnicr oorgarrisrrrs belonging to R arespeoes rrrultiplying with a highrate and that ts the reasonto suwiveirr this shorl ter.rns when the rredrais adverselyfor groMh.On the otherhand the K strategiesare slowly growirrg and are actrve for a longtirne In thisway the questionis to isolateand sepa- ratethese both groups and to be checkedout tlieirspecific properlies the maxirrialgroMh rate the lag- phaseduralron, the periodof activemetabolism. the saturationand inhibitionconstarrts interspecies rela- tionshiparrd so on. The praclicalsoiLrtron of problerns wrll be doneby usingmicrobiological methods for isolatronan quantrta tivechecking of fluorescencemicroscopy and selection orr nutrient media. For speciesidentrficatron Ameri' can system"Biolog" automatically testing yeasts and bacteriacould be used The rnetabolicactivily (enzymes,respiration) will be madeby spectrophotometryor specially constructed oxygen electrclde Foranalyzing of differentchemical substances in soil(heavy metals, nutrient substances) LC andatorilc absorptionspectrophotometn/ will be used Morphologicallychanged cells will be legisteredby light and electron microscopy 5. CONCLUSION Workingout the projectin thisway wrllgive the possibilitythis project to be includedin a programfor protectiortand speciesdiversity lt wrllgive an informationfor ecosystemstatus and will createprerequisrtes andcriterra for biomonitoling of antropogenic polluted regions in danger

REFERENCES '1980 l AndryukEl Proc.lnst Microbiol.& Virol.,Acad Scl.Kas SSR.26 79-90. (ln Russian) 2. BishoffB. EcotoxicolEnvionrn Safety6 No 2 157-162.1982 3 BabichH. G Stotzky.Envirorrrn Res.36 No 1.111-137.1985 4. DuxburyT. Adv.Microbiol Ecol .8.185-255 1985 '1985 5 ZellesL, I Schennerl.F KofleJ EnvironmSci Helth20 No.5.457-488, 6. Skvortsova| , S. Li. I Voro.leykina.Heavy metals in the environment. 121-125. i., 1980(ln RLissian) 7. FlesselC. Metalsas mutagenslnorg Nutr.Aspecfs Cancer, Proc lConf. Int Assos BioinorgSr-r California,4-L, 107-128, 1978 8 KondoI T Schikawa.H. Nakahara.J Bacteriol ,117,1-7.1974. 9 StarkeyR J Gen.Microb..78.217-225. 1973. 10.Bulavko G.l Rep.Sibirian Div Acad ScrSSSR Ser Biochem.1.579-86 1982 (ln Rrrssran) .1 1.Kurek E. A Francr.J BollagArch Environtn. Contatn. Toxicol ,2l ,1,106-111.1991 12.Panikova E, A. Perlsovskaya.Chernistry inthe agricLrlture i 3,12,1982 (ln Russian) 13.Doelman P., L Haamstra.Sol/ Biol. Broclrcm,'11 No 5 481-4851979 14.Heflkorn-Obst U H FrankForum Microbiol..3 No.6 376-378.1980. 15.Babich H R Bauley.G.Stotzky. Arch Environtn. Contarn. Toxicol.,12. No. 4 421-4261983. 16.RogersJ, S Li.Bull EnvironmContatn Toxicol..34 No 6,858-8651985 17.Levin S.. l. BabevaSol/ Scierrce. 6 97-101.1985 (ln Russrarr). 18. Skvortsova| , M. Leonova.Proc. lll All UnionConf. on lnvestigatiortof pollttion rnigratirtnsr/Lrsla/)L'es //l soiland marginal environtnenls 141 -149. 1985 (ln Russran) 19 ShravankumarC,Sivarama Sastry K. MaruthMohan P. Biotechol.Techn 14.1099-1202.1992 20. Van GemerdenH. J KuinenStrategies for growthand evolutionof microorganisnrsin oligotiopfrrc habitats.Heterotroplt Acl Sea . Proc NATO Adv Res lnst Microbiol Metabol and Cycl Org Matter Sea,Cascars N -Y. L 25-54 1984 21.Kojevin P A MtcrobialpopLtlations irtthe nature Mosk StateUniv. 135-1531989 (ln Russian) 22 BoldryM B A.CDeari Microbios Letter, 1980 105,111 MULTIVARIATECORRELATION OF DEPOSITIONDATA WITH LICHENDATA

K.Atnmann,R. Herzig,L. Liebend1rfer,K.Peter and M. Urech Geobotany,University of Berne,Switzerland Altenbergrain21, CH-301 3 Bern,Switzerland, email kammann@sgi. unibe.ch

1. INTRODUCTION,INTERDISCIPLINARY CONCEPT

Since1980 a groupof scientistsof the Universitiesof Berne, Lausanne, land-use planners and polrticians have been workingon an interdisciplinarystudy concerned with meteorology,air pollutionand land-use planningin Biel-Bienne,Switzerland (Swiss National Research Programme No. 14). Stttdy objects . Topography,land-use, wind roses for six locationswhich are representativeof conditionsfound within thestudy area. . Emissioninventory for SO2.All sourceshave beenconverted to a 500m grid.Largest emission has beenestimated to be near45 tonsper year, proportion of domesticheating with one exception7Sok or more. . SO: concentration(half hour values), recorded in two typicalstations (city center and southernslopes of nearbyJura mounlain chain) . Emissioninventory for NOx.Highest values some 50 tons N02 per year.Proporlion of traffichighest alonghighways and in citycentre. . Meanannual values of NO2(prg/cm'), Gaussian plume model: Mirroring the mainhighways and traffic activity. . Nighttimeand earlyafternoon fields of potentialtemperature and streamlines of boundarylayer air flow,typical for a highpressure weather situation with weac synoptic winds. Cool air streamfrom the ,,Taubenlochgorge" with clean mountain air disturbing picture of urbanpollution heavily . Regionaldistribution of calibratedlichen Index of AtmosphericPollution in the Bielarea dividedinto fivezones

2. SAMPLINGCONCEPT OF LICHENDATA

Basedon a modifiedgrid conceptafier Wildi 1981,528 sites(actually trees) where chosen at random, evenlydistributed within 48 gridunits of 100hectares and 52 gridunits of 25 hectareswithin the areaof Biel. A newlydeveloped ,,frequency grid" of 10subunits arranged in twovertical rows of 5 was attachedto the tree trunc,the sizefitting always half the circleof thetrunc.

Data collectedon the sites: . Geographicaland ecological data of the site . Treelrunc dala, such as species,texture of bark,inclination of truncetc. . Lichendata such as frequencyin '1'l classes,coverage in 7 classes,vitality in 3 classes,thallus damage in 3 classes,average age of populationin 3 classes,spectrum of species.

3. IMMISSIONDATA

Depositiondata of SO:, dust,NO3, Cl, Pb,Zn, Cd, Cu have been measured,for detailssee previous oublications.

4. DEVELOPMENTOF CALIBRATEDIAP METHOD

The aim of our biologicalstudy was to developa methodwich would later allow to map in detailair pollution impactby meansof a lichenstatistics calibrated to lhe depositiondata of eightpollutants as mentioned in 3. An evaluationby multiplelinear regression of 20 differentIAP formulas derived from the followingover- allformula:

fl Q,xC,xF, lAPl = : ------i=1 V,x D,

n = numberof lichenoer site I - Sum overlichen species ,and the following parameters, calculated according to formula .-10 Q - Toxitolerance(= averagecompanion species number for grven species) C - Coverage F - Sumof frequency. counted with the ten,,fr equency grrd" units V - Vitalrty D - thallusdarrrage

Evaluationof 20 formulasto calculatevarious IAP rndrces was arrangedaccorriing to bestRi lmLrttiple linearregression) Frequencyalorre included prooved to bethe bestfittirrg IAP formula.

t=1 lAPlo = !

An evenhigher R-'value was obtained with a slightlyreduced set of species,where. all pollutiontoler-ant specieswhere excluded. As a concequence,one can predict wrth a probabilityof some97% the total impact of airpollution. Themultiple linear regressiorr was done according to thefollowing formula.

lAP,= lJ,r+lll x (SOr;+ 11.x (NO')+ llrx (Cl)+ lJ4x(Dtot) + 1r.x (Pb)+ lJcx(cu) + lJ;x(Zn) + lisx(Cd) +.E

Dtot- Dustoverall precipitatron E - Residuevariation not explained with regression model lJ - correctionfactor for eachpollutant, lJ; initial correction factor

Stepby stepelimination of airpollution parameters revealed a minimumnumber of 4 to 5 parametersto guaranteea goodmodel (calibration) qualrty and in additionit showedthat there is no clearcut redundancy of anyone parameter.

5. CALIBRATEDIAP ZONE MAP

IAP zoneshave beenconstructed out of over500 lichenanalyses on trees in Bieland many otherre- gions.lt can clearlybe seenthat the lowestlichen activity (lichen desert) is registeredin the centreof citres

6. CONCLUSION

Multivariatecalibration of lichendata and pollution data has been achieved for the firsttime in Biel,then this has been repeatedseveral limes withinSwitzerland In one case.Quantitativedata have been used withinthe measuringnetwork of lhe Swissgovernment. In allcases the R- revealedto be highenough (t 95) for the followinginterpretation: All data supporta quantitativerelationship between lichen data and overallair pollutionGovernment agenciesmay thus be interestedin thisscreening method to delimitproblem areas at considerablylow costs

Furtherreading and more citations in '1989 Arbvon. C. andBrunold C Lichenphysiology and air pollution. l. Physiological responses of in situParmelia sulcata among air pollutionzones within Biel, Switzerlano HerzigR., M.Urech, L.Liebendorfer, K.Ammann, M.Guecheva and W.Landolt 1989 Lichensas Biologicallndicators of Air Pollutionin Switzerland.Passive Biomonitoring as a Partof IntegratedMeasuring System for MonitoringAir Pollution. LiebenddrferL., R.Herzig, M.Urech, and K.Ammann '1988 Staub-Reinhaltunoder Luft48. 233-238

il SMALLMAMMALS LIKE BIOINDICATORS INZOOLOGICAL MONITORING

RoumianaMetcheva Instituteof zoology,BAS

Rodentsand smallinsectivorous belong to the rnostabundant vertebrates living in the forestecosystems, Forthis reason, they became increasingly popular as bioindicatorsin studies on the effectof the industrial pollutantson livingorganisms. Bradley et al.(1978, cited after Martin and Coughtry) specify conditions that the smallmammals satisfy if theyare to be usedas indicatorsof metalpollution of the environmentThe animalsmust be small,easy to catch,they should inhabit a smallarea, live on a vegetablediet be adapted to theirhabitat and their life-span should be short.Small rodents satisfy these conditions. Bradley et al.think that out of three small rodentspecies - wood mouse(Apodemus sylvaticus), bank vole (Clethrionotnys glareoltts)and fieldvole (Microtusagrestis).A lot of otherauthors complement this list:for differentsrrall rodents from USA - Peromyscusmaniculafus, Mus musculusand Microtusochrogasfer -Raymond and Forbes(1975), Getz et al.(1977),Kisseberth et al.(1984). ForCanada Cloutier et al.(1985) suggesls Micro- tus pensylvanicus.In Europeamong most frequently used are'.Clethrionomys glareolus. Apodemus sylvaticus and Microtusagresfis $/Villiamson and Evans1972, Sawicka-Kapusta and Kozlowski1984. Johnson et al.1978).ForBulgariathislistwasenhancedbyGerasimovetal.(1987),Metchevaetal.(1987) Someother specificspecies for a givenregion were add like:yellow-necked wood mouse(Apodemus flavicollis) . pine vole (Pitymys subterrarteus).snow vole (Chionornysnivalis) and Gentler'svole (Microtusguentheri), and insectivorous:white toothed shrew (Crocidura leucodon) and commonshrew (Sorex araneus). These specieshave different places in thefood chains. The microtinerodents are herbivorous,the murine- omnivorous and the littleinsectivorous-carnivorous. These monitorspecies were used to estimatethe environmental stateapplying chemical. morphophysiological andgenetic methods.

1.ANALYTICAL TECHNIOUES

Two analyticalmethods were usedfor determinethe levelof toxic elements:atomic absorption spec- trometryand X-rayfluorescence analyses. The morpho- physiologicalmethods were alsotwo: morpho- physiologicalcharacteristics of the peripheralblood and morphophysiological indices of internalorgans. The cytogeneticmethods involved cytogenetic characteristics and chromosomal aberrations. Analyticalmethods were usedlike a firststep, because the toxicitydue to chronicexposure to very low dosesof metallictoxicants is difficultto diagnoseespecrally when chemicalor outwardsymptoms are not wellpronounced. Under these conditions different internal organs and also the whole carcass were analyzed to identifyand asses the dosage of thetoxicant. One of the most useful analytical method is the conventional flameatomic absorption spectrophotometry for determinationof tracemetals. "Flameless" atomic absorption has considerableadvantages over convention methods in sensitivityand requiredsample size Thistech- niqueis capableof analyzingmicroliter samples. The samples preparation for AAS demands dry mouldering at hightemperature or aciddigestion in Teflonor quartzvessels. The secondway avoidslosses from the volatilemetals like lead, but there are another circumstances which must be kept.The acidsmust [re soectral pureand the wholemineralizatiorr should be verycareful done, because of additionalpollution or quantity losses.This disadvantages were avoided using another analytic technique - X-rayfluorescence. This tech- niquepermits raptd and simultaneous analysis of a largenumber of metalsover a widerange of concentrations.Forthis method the samples must be dried by low-temperature mouldering (Lucky VeLrngopal, 1977).

2. HAEMATOLOGICALANALYSIS

Besidesthe chemicalanalysis different morphophysiologrcal investigations also were done One of thern is the standardclinical method for determining the haemoglobincontent. haematocrit and the morphological characterisationof erythrocytes (Kostelecka-Myrcha, 1967). In orderto controlgenetic damages a micronuclei test and a presenceof basophilicgranulation in erythrocyteswere tested(Metcheva et al., 1987).To illustratethese methodsresults from differentRegional Background Stations (RBS - selectednonpolluted territories)- Rozhen. Stene and Stranja and the impactstation of Srednogoriewill be presented.The results showthat the amounlsof the tesledelements-lead, cadmium, zinc and copperwere rather similar for the speciesfrom genusApodernus and Microtus as well.Only regarding copper higher accumulation was ob- servedat RBS"Strandla" lt canbe explainedwith the increasedgeochemrcal background in the referenceto thatelement. The contentsof leadand cadmium in the bodiesof the monitoringspecies from the polluled regionof Srednogoriewere 4-5 times higher than of the RBS (Metcheva et al.,1993) The haematocritvalue, the numberof leukocytesfrom the investigatedmonitor species show that the valuesvaried in similarlimits. There were no significantdifferences between the resultsobtarned from the RBS'sand the pollutedregion of Srednogorie.Nevertheless there were establishedsome morphological changesin the bloodcells Basophilicgranulation and presence of micronucleiin the erythrocytesfrom pe- tl ripheralblood of Apodemusftavicollis from Srednogorie Region have been found. The testscarried out on 10 animals of yellow-neckedmice show that the frequencyof erythrocyteswith basophilicgranulation was among5.7-26% and the frequencyof micronucleiwas between 14.5-24.1o/o. I n 1993-1994started a complexmonitoring project on Rilamountain. Forfirsttimeforthe determination of heavymetals and the toxicelements was useda X-rayfluorescence analyses. The wholebody and the liver of the monitoringspecies from three altitudes Mala Tzerkva (1000-1200), Maliovitza (1800) and peakMous- sala (2925)were investigated.The mosttoxic metals lead and cadmiumwere presentin two biotops- Mala Tzerkvaand peak "Moussala".The highestlead concentrationwas found in the carcassof the bank voles from Mala Tzerkva.The lead concentrationsin the carcassand in the liversfrom these two placeswere similar.Cadmium accumulation was determinedonly in the animalsfrom MalaTzerkva. This levelof heavy metals(Pb and Cd) in the bodyand in the liverof bankvoles is comparableto the amountsfound in small rodentsfrom different(polluted and unpolluted)regions in CentralEurope, England and Bulgaria(Metcheva et al.,1 995). The resultsof haematologicalindices show that the numberof erythrocytesin the bank vole from "Malio- vitza"(1800m) were higherthanin the samespecies from MalaTzerkva (1200m). There is an evidencefor geneticdamages expressed by presenceof micronuclei(Howell-Jolly bodies) in allthe monitorspecies. lt is considerednormal to find 1-3 erythrocytesper 100 cellswith micronucleiin the films of these species.The bankvoles from MalaTzerkva have higher than usualpathogenic erythrocytes in the peripheralblood. The percentagjein the otherspecies: yellow-necked mouse and snow vole were not so high.In comparisonwith the monitorspecies of rodentsfrom othernonpolluted regions (RBS) the percentageof the animalswith pathogenicchanges of erythrocytesis lowerthan 50. In conclusionsthe resultsfrom the presentedinvestigations confirm wild rodentsas bioindicatorsalso in unconlaminatedareas which could be usedas controlsites. Furthermore for monitoringenvironmental quality it is necessaryto prolongthis kindof investigations.

REFERENCES

Cloutier N.R.,F.V.Clulow,T.P.Lim,N.K.Dave.1985.Metal(Cu,Ni,Fe,Co,Zn,Pb) andRa-226 levelsin meadowvoles Microtus pennsylvanicus living on nickeland uraniummine tailing in Ontario,Canada: environmentaland tissue levels. Environ. Pollut. Ser.B, 10: 19-46. Gerasimov,S.,R.Mecheva,P.Dimitrova.l9ST.Theroleofeco-toxicologicalexperimentfortheselec- tion of bioindicatoryanimal species and indicativerndices in biologicalmonitoring.(in Russian). Prob- lemsof BackgroundEcol. Monitoring. Bulg. Acad. Sci. 37-41. G e t z, L. L., L. Verner,M. Prater.1977. Lead concentration in smallmammals living near highways. Envi- ron.Pollut. 13, 1 51-157. Jonso n, M S. R. D. Roberts,M. Hutton,M. J. lnskip.1978. Distribution of lead, zincand cadmiumin smallmammals from polluted environments. Oikos. 30, 153-159. K is s e b e rt h , W.C.,J. P. Sunberg,R. W. Nyober,J. D. Reynolds,S. C. Kasten,V. R. Beasley.1984. Industrialleadcontamination of an lllinoiswildliferefuge and indigenoussmall mammals. JAVMA, 185, 1309-1 31 3 Kostelecka-Myrcha A. 1967.Variationofmorpho-physiological indicesof bloodinClethrionomys glareolus(Schreber,1780). Acta theriol., 12, No 13,191-222. L u k e y, T. D., B. Venugopal.1977. Metal toxicity in mammals.Plenum Press. New York and London.238 pp. M a rt in, M. H., P. J. Coughtry.1982. Biological monitoring of heavymetal pollution. Applied Sci. Publish- ers,London - NewYork, 475 pp. M e t c h e v a, R., P. Dimitrova,S. Gerasimov,A. Mikhova.1987. Experimental studies of leadeffects on a fieldmouse used as biologicalindicators.(in Russian). Problems of backgroundmonitoring. Leningrad. 5, 124-131. Metcheva R.,AtanasovN., GerasimovS. 1993.The useof small rodentsin biological monitoring. In: Rodentset spatiumlV. Abstracts, Mammalia, 57, 4: 638 p. M e t c h e v a, R., M. Topashka-Ancheva,N. Atanassov,A. Artinian,V. Minkova.1995. Bioaccumulation and distributionof toxicelements, haematological indexes and chromosomalchanges in the organisms of indicatorspecies of small rodentsfrom Rila mountain.Observatoire montagne de Moussala OM-2,No 3,203-212. Raymond R.8., R. BForces1975. Leadin hairof urban and rural small mammals. Bull, environ. Cont. Toxicol.13, 551-553 Sawicka-Ka pu st a K.,J. Kozlowski.1984. Flowof heavymetalsthrough selected homeotherm con- sumers.(ln: Forestecosystems in industrialregions. Eds. W. Grodzinski,J. Weiner,P. F. Maycock). Spring er-Verl ag, Berlin-Heidel berg-New-York-Toki o.1 39-1 42.

.) .l THEUSE OF MORPHOPHYSIOLOGICALAND CYTOGENETICAL METHODSIN ZOOLOGICAL MONITORING fuaskoAlarrassov Inslituteof zoology.BAS

1. Morphopysiologicalindices of someinternal organs

The influenceof the environmentalfactors on the livingorganisms cause complex changes in theirme- tabolismand the total physiological condition To inveslrgatethis changes it is necessaryto choosethe mosl sensitiveindices Such are the absoluteand the relativeindices (weights) of anirlal'sinternal organs. Schwarzet al.(1968)named them "morphophysiological indices". because they possessspecial features: reflectthe environmentalchanges and they are criterion for the physiologicalfunctions in the animalorgan- ism Forthis purpose it is necessaryto compareanimals from the samespecies. in equalages and sex.but in differentecological conditions - for example:different altitude, climate, food diet etc. Small mammals are parlicularsuitable for suchtype of investigationsThey have relatively short live span widespread, easy to catchand are usedlike a modelin the monitoringinvestigations (Bradly et al.,1978, cited after Martin and Coughtry,1982). All collectingrodents with livetraps were individuallyweighted and dividedinto two weightgroups ap- proximatelyrepresenting age andsex classes.The animalsfrom the agegroup "adults" (no lessthan 10) weredissected to removethe intestinaltract (including contents) and internalorgans: liver, kidneys, spleen and adrenalQlands. These organs were weighted to 0 001 g and theirmorphophysiological indices were determined(Schwarz et al.,1968) like a relationbetween the organweight and the animal'sbody weight in ''/u'.In our previousinvestigations the mostinformative morphophysiological indices are shownthe following ones:these of liver,spleen, kidneys and adrenal glands (Gerassimov et al ,'l994). As a resultof oLrrinvestigations in specific nonpolluted regions called Regional Background Stations (RBS)-Rozhen, Steneto and Stranjaand the impactstation of Srednogoriehave been gained information as a basisfor someprimary conclusions: The comparisonof the datafrom the RBSwith that from Srednogorie Regionshow the highestvalues of adrenalglands, kidney and liverindices only at yellow-neckedmouse Theseconclusions have served as a modelof complexstudies for environmentalmonitoring and as a basis forcomparison of regionswith various levels of industrialpollution.

2. Cytogeneticalanalysis

Allthe organismsin the nature,including small rodents are characterized with a definitenumber of chromosomesand chromosomalmorphology , forming theirs karyotypes. lt is relativestabile characteristic. per- mitsto deterrninethe speciestaxa on the investigatedindividuals and cytogenetic stalus. The environmental pollutioncausing by the humanactivity concern in differentways the livingorganisms. These pollution effects twotypes of changes- heritableand nonheritable. Recently, a bigattention has been paid on the establishmentof thegenetic changes in theliving organisms suffering under the environmentalfactors (Deknut et al , '1980; 1977,Gerber et al . Degraeve,1981: Holterman et al., 1984;Chorvatovichova, Reichertova, 1985, Gerasimovet al. 1985,Belcheva, Topashka-Ancheva, 1987; Preston et al..1987; Sawicka-Kapusta et al., 1987.Lison, Lowris 1990 etc.). Thegenetic investigations have been done using routine cytogenetic methods for chromosomeanalyses. To determinechromosomal aberrations in the marrowcells wereprovided from 5 adultmales plus 5 adult females.The slide preparationswith well-spreadcomplete metaphases were obtainedfrom colchicine- blockedbone marrow cells from the femuraccording to a routinerrethod. After the hypotoniclreatment and fixation,flame dried bone marrowslides were stainedwith Giemsasolution (Macgregor, Varly 1986) In eachexperimental group 50 metaphasesper animal were analysed. '1995) Our last investigationswere from differentaltitudes in NationalPark Rila (Metchevaet al , The gainedprimary information is a basisfor comparisons.In the cellsof examinedanimals chromatid and chro- mosomebreaks and individualfragments. exchanges and ringswere observed.An averageof 8 aberrations/100cells in the samplesof yellow-neckedwood mouse collectedin the regionof MalaTzerkva were establtshed,but rnthe samplesfrom Maliovitzathe percentageof aberrantmetaphases was 2 Therewere no statisticallysignificant differences between the samplesfrom the two regionsbecause of the low number of investigatedanimals. Chromosome breaks, fragments and exchanges in lhe samplesof bankvoles from MalaTzerkva were observedThe comparisonof the resultsof cytogeneticalinvestigations of A. flaficollis from Rilawith the datafrom our previousstudies ( Metchevaet al., 1993)yielded as follows:The average percentageof aberrantmetaphases in the samplesof Maliovitzawere similarto those obtainedfrom the samplesof the RegionalBackground Statron at Boatin(in NationalPark Central Balkan). The averageper- centageof aberrantmetaphases in the samplesof the yellow-neckedmouse from MalaTzerkva and in the samplesof the samespecies from the impactregion of Srednogoriewere similar. An aberrationfrequency t_l notexceeding 2 to 3 % is regardedas normal.The aberratron frequency found in thesamples of theyellow- neckedmouse and bankvole from the Maliovrtzaarea can also be interpretedas normaland as occurring spontaneouslyHowever, the dataof the sanrplesof thesespecies from MalaTzerkva were differentand appearto be non-sponlarreotrs All thoseanintals investigaled belorrging to the rnonrtorspecres yellow-necked mouse and bankvole. wereobserved to havea nearlythree trmes htgner iate of chrornosornalaberrations in MalaTzerkva than rn Maliovitza. The Lrseof thesetwo sensitive,rnformative and representativemethods for monitorinoof the environmentalquality is oneof the best for long term plan

REFERENCES B e lc h e v a R, Topashka-AnchevaM 1987 Cylogeneticeffect of leadacetate on borremarrow cells of BALB/cymice(in bulgarian). l-st Nat Corif Probl Biol Monitoring,22-24 X.1987. Sofia. 202-207 . C h o rv a t o v i c h o v a D ReicheflovaE'1985 Theeffect of long-termexposure of mouseDBA/2 to magnesitedust on the frequencyof chromosomalaberrations in bonemarrow (in Slovak)Biologra (Bratislava)40 267-269. Degraeve N 1981Carcinogenic,teralogenicandnrutageniceffectsof cadmiumMutationRes86 115- 135 D e k n u t G., ColleA, GerberG B. 1977 Chromosornalabnormalities in lymphocytes from monkey poi- sonedwith lead MutationRes 45:77-83 Gerasimov S MetchevaRDimitrovaP 1985 Theroleof ecologo-toxicologrcal experimentsforthe selectionof broindicatoryanimal species and indicatrve indices in biologicalmonitoring.(in Russian) Problemsof BackgroundEcological Monitoring BAS, pp 37-41. G e ra s im o v S. DimitrovaP. AtanassovN.. Mikhailova V 1994.Themethod of morphophysiological indicesand its use in ecologicalinvestigations (inbulgarian) Ecologia, 25,53-57 '1980 G e rbe rG.G.,LeonardA, JacquetP Toxicitymutagenicityandteratogenicityof lead.Muiation Res 76 115-141 HoltermanW.F VogtP Copirsl.1984 Cadmium/zincrelationshipinkidneycorlexandmeta- lothioneineobservations on horsekidneys. Environ Res 35: 466-48'l . L is o n D, LowrisR 1990.in vitrocytotoxic effects of cobalt-containingdust on mouseperitoneal and ratalveolar macrophages. Environ Res 52. 187-198. M a cg reg o rG. VarlyD. 1986Metodyraboty s hromosornami zivotnich.(in Russian) Mir, Moskva,272 'pp M a rt in M. H. P J Coughtry1982 Biologicalmonitoiing of heavymetal pollution. Applied Sci Publishers. London- NewYork 475pp M etc h e va R.,AtanasovN., GerasirnovS l993 Theuse of smallrodentsin biological monitoring. In. Roderrset spatiurnlV Abstracts.Mammalia, 57 4: 638p M et c h e v a, R.,M. Topashka-Ancheva N.Atanassov, A Arlinian,V Minkova. 1995 Bioaccurnulationand distribution of toxicelements. haematological indexes and chromosomal changesin the organismsof indicatorspecies of srnallrodents from Rilamountain Observatoire montagnede MoussalaOM-2. No 3. 203-212 P resto n R. Dean8., GallowayS Holden H, McFeeAF,SheldyM. 1987. Mammalianin vivocytogenetic assay analysis of chromosomeaberrations in bonemarrow cells. MutationRes '189: 157-165 Sawicka-Ka pustaK., GoreckiA. LangeR. l98T.Heavymetalsin rodentsfrom pollutedforestsin SouthernPoland. Ecol. Polska. 35(2) 345-354 S c hv arlz S.S, SmirnovV, DobrinskiiD 1968.The method of morphophysiologicalindrces irrihe ecol ogy of terrestrialveftebrates USSR. Sverdlovsk. 553 pp. THEMUSHROOMS AS ACCUMULATORS OF CONTAMINANTS

VioletaFakirova Instituteof Botany,BAS.

1. PRELIMINARIES The mushroomshave entered the humanlife in the remotepast, due to theirvaluable nourishing quali- ties. Recently,it has been established,that they are able to absorbvarious chemical compounds including heavymetals. This propertyallows to usethem as contaminationindicators. The studiesin this directionbegan in the last fifteenyears and duringthe past decade,the mushrooms becameobject of intensiveinvestigations, due to increasingglobal pollution of the atmosphereand soil by heavy metals.In regionalaspect the studyof mushroomsas accumulatorsof heavy metalsare in different stages.In a numberof Europeancountries these problems are understudy since the seventies:Germany (Seeger,1976, 1978; Seeger et al., 1978,Enke et al., 1977;Laub et al., 1977;Dietl, 1987; Fischer, Hein 1990),Finland (Laaksovirta, Lodenius, 1979; Lodenius, Herranen, 1981;Lodenius et al.,1981;Kuusi et al. 1981;Liukkonen-Lilja et al., 1983;Othonen, 1982), Switzerland (StUve, Beson 1976; Stijve,1977; lrlet, Rie- der,1985;Quinche,1979a, b;1980a, b, c;1981,1982a,b, c;1983a,b;1987a, b:1988; Quinche etal., 1976),Austria (Mutsch et al., 1979;Rtlcker, Peer, 1988), Sweden (Tyler, 1982a, b; Rilhlinget al., 1984), Czechoslovakia(Lep5ov5, Mejstoik, 1988; LepSov5, Krdl, 1988), Poland (furnau, 1990), Yugoslavia (Byrne et al., 1976),Norway (Allen, Steinnes, 1978), ltaly (Bargagli, Baldi 1984) etc. Regretfully,many European countriesstill do not paythe necessaryattention to theseinvestigations.

2. HEAVYMETAL ACCUMULATION IN MUSHROOMS

The specificityof mushroomorganism to exist in the soil as perennialfungi mycelium,exposed to the effectsof heavymetals available in the soil,as well as the abilityof myceliumto form largeoverground fruit- bodiesin which these metalsare concentrated,makes the mushroomsa very suitableobject for indication (Dietl,1987; R|cker, Peer, 1988). Moreover,the mushroomsexhibit a tendencyto accumulateheavy metals on a largerscale than the higherplants growing in the samesurrounding. For example, the mercurycontent in somemushrooms could reachmore than 1OO-foldthat of the plants(Lodenius, Herranen, 1981). Another importantproperty, proven also experimentally,is the mushroomability to accumulateheavy metalsin much largerquantities than the soil.For instance,in some casesthe mercurycontent in rnush- roomscould be 30 - 550-foldhigherthan in the soilon whichthey grow (Seeger,1977). The accumulationof heavymetals in mushroomsvaries in wide rangewith respectto the kind of con- taminationand its quantity(Lodenius et al., 1981).Various data on heavymetal accumulation have been reportedfor definitekind of mushroomsfrom specific regions. Quinche, 1979a, b; 1980a,b, c; 1981, 1982a, b, c; 1983b;1987b; 1988; Quinche et al.,1976), reported in a seriesof papersanalysis results on 8 heavy metals,established in the fruit-bodyof variouskinds of mushroomsgathered from variouslocalities in Swit- zedandand France.Similar data havebeen obtained by otherauthors (Creight, Schfoeder, 1987; Stijve, Besson,1976; Tyler, 1982a: Mutsch et al.,1979; Laaksovirta, Lodenius, 1979; Lodenius et al.,1981) etc.

3. SIGNIFICANCEOF THE PROBLEM

A largepart of the mushroomsunder study, belong to the categoryof ediblemushrooms, so that the data on them have both scientificand practicalimportance. In a seriesof papersemphasis is placedon the serious dangerof heavymetal contamination in regionsadjacent to water plants,factories for scrap processing, metallurgicalworks producing copper, lead, mercury and others,in areasaround highways, or in park areas in denselypopulated citieswith intensive industry (Quinche, 1980b, c; 1982a;1987b; Laaksovirta, Alakuijala, 1978;Laaksovirta, Lodenius, 1979; Enke et al.,1977; Fischer, Hein, 1990; Kuusi et al., 1981;Lodeniuset al.,1981;Liukkonen-Lilja et al., 1982). The variousecological-trophic groups of mushroomsexhibit different ability to concentrateheavy metals. The highestdegree of accumulationof mercury,copper and cadmiumpossess the soil littersaprotrophes (Lodeniuset al.,1981; Laaksovirta, Lodenius, 1979). The wood-decaying fungi have the lowestaccumulation ability(Seeger, 1976; Sanglimsuwan et al.,1993). Indisputably,therd is a determinismof mushroomspecies with respectto accumulationof specificpollut- ers (Ohtonen,1982; Allen, Steinnes, 1978; Sanglimsuwan et al., 1993).Some species of the genusAgari- cus, particularlythose belongingto sectionF/ayescens are ableto accumulatecadmium with very high concentrations(Laub et al.,1977, Seeger, 1978; Dietel, 1987; Lodenius, 1981; Quinche, 1980a, c). A similar dependenceof mercuryaccumulation has been establishedin Agaricuscampester and Bolefusedulis. The speciesof genusLycoperdon (Gasteromycefesl exhibit a definitepreference toward the lead,whereas Mac-

3(r '1 rclepiotarhacacies ano l",i Dracaraaie oi.itsta..,lri-iEiii.;!:t,iii!.riet(,,rs of c-op[)er (Mutsch et 31, 979; Byrneet al., 1976) Sincethese species are irrdelyspfead iir Fi:rop:;an,i lisi:din lai'geextent for corrsutnmation,a problem ai-isesconcerriing the risx whicfiin tire r.;iir.titir;rrsui iutal tr,jrlutr{,noi naturee;

4, STATE.OF.THE-ARTIN BULGARIA 'The firstsystemaiic studies on accumulatingability of mrshroomsin Bulgariawere iniiiatedat the Insti- tute of Botany,Bulgarian Academy of Sciences,two yearsago. Two contaminatedareas were chosenfor this purpose:(1.) Metallurgical works Kremikovtzi producing steel, situated in closevicinity to uraniummine Bukhovo;the lastcircumstance would make possible future studies of radionucleiin the mushrooms,growing in thisregion; (ll.) Copper producing wcrks Zlatitza. In the firstyear of investigatron,due to an enormousdrought, only 20 speciesof mushroomswere gath- ered for analysis.In the secondyear the conditionswere morefavorable and the numberof specieswas increasedconsiderably to morethan 40. The studyof mushroomsamples was performedusing the method describedin the nextsubsection.

5. MATERIALSAND METHODS

Samplesof mushroomsfi^om the mostavailable species were collectedon each locaiiiy.Sampling was carriedout usingplastic bags. The sampleshave been stored in a refrigeratorunlil furlher treatrrient. They wei"enot washed with distilied water or dissolvingagents, but sny attachedperiphytic material was carefully scrapedoff. The watercontent was determinedat 40'C to constantweight. About 2 g of the poldered antJ dried materialwas treatedwith 15 ml nitricacid overnight.The wet-ashedprocedure rvas contrnued with heatingon a waterbath, following addrlion of hydrogenperoxide (5 ml in portions)till full digestioriThe fil- tratewas diluted with double distilled water to 50 ml.Aftei'cleanrng the soilsui-face samples (0.10 cil) were groundto passa 1 mm stainlesssteel sieve, dried (at 90'C for 48 hours)and treated (about 2 g) wiih a 3;'1 mixtureof perchloricand nitricacid (10 ml). The mixturewas heatedon a sandbath nearly to 0ryness.This procedurewas repeatedwith 10 ml hydrochloricacid. The digestedsoii sample was finallyfiltered and the filtratemade up to 50 ml.The solutionswere stored in plasticflasks. Duplicates of eachniushroorrt airci soii samplewere preparedindependently. The elementalanalyses have beendone by Atomrcenrission spec" trometry(AES) with the Inductivelycoupled plasma (lCP) using VARIAN - Libertyinstrument arid refeience materials,Analytical precision was checkedalso by replicating;deviation between the duplicateswas Delow 5% in allcases. The concentralionsare expfessed as mg/kgdry weight.

6. CONCLUDINGREMARKS

The resuitsof analysiscbtained in oui stLrCiesyriil be presentedin a numberof papersnow in ftrlalstaEe of prep:aration,We'uvould like to incicatea parlof the nroreiriieresting results aboui sonre rnushirroiiis stiid' ied: 1. Miriimumand maximum concentration of heavy metals in mushrooms.from poiluted and hachgrcrrrid areas(Table l) 2. Bavistaplumliea ani Marasmiusoreado.s - ihe most widelyspread species in both ccntanrirratedar- eas.showing different data for six elements (Tabie ll). Concludingwe shouidernphasize that the dataexpected from the futurestudies in thisoirecticri lvill be a substantialcontribution to the developrnentof biologicalenvironment monitoririg in Btitgaria,as a consttti.i€ni pai-iuf the globalecoiagi.al moniioring tJndoubteoly, a part of theseresults woirld bc r:f spci-:ialinteresi for tiretoxicology praciice in tlrrsr;ourtti1. i-; REFERENCES

AllenR., E. Steinnes,1978. Concentrations of somepotentially toxic metals and othertrace elements in wild mushroomsfrom Norway Chemosphere,4: 371-378. BargagliR., F. Baldi,1984. Mercury and methylmercury in higherfungi and theirreiation with the substratain a cinnabarmining area Chemosphere,13(9): 1 059-1 071 ByrneA.,RavnikV,, L. Kosta,1976. Traceelementconcentrationsin higherfungi Sci TotalEnviron.,6:65-78. CreightJ., D. Schroeder,1977. Cadmium, lead and nickelcontent ol Lycoperdonpelatum Pers.in a roadsideenvironment. Environ. Pollut.13:265-268 DietlG., 1987.Wildpilze speichern Schwermetalle. Umwelt, 1-2.2+26. EnkeM., MatschinenH., M. Achtzehn,1977. Schwermetallanreiche-rungen in Pilzen. Die Nahrung,2l(4).331-334. 'Fischer '1990. C.,B. Hein, Bleiund Cadmium in PilzenausWestberlin. Z. Mycol.,56(1): 159-166. lrletB., K. Rieder,1985. Cadmium und Bleiin Pilzenaus der Alpinen stufe der SchweizerAlpen. Mycologia Helvetica, 1(6): 393-399. KuusiT., LaaksovirtaK., Liukkonen-LiljaH., LodeniusM., S. Piepponen,1981. Lead, Cadmium, and MercuryContens of Fungiin the HelsinkiArea and in UnpollutedControl Areas. L. Lebensm.Unters. -Forsch. 173:261-267. LaaksovirtaK., M. Lodenius1979. Mercury content of fungiin Helsinki.Ann. Bot. Fennici, 16.208-212. 'l LaaksovirtaK., P. Alakuijala,978. Lead, cadmium and zinc contents of fungion lhe parkof Helsjnki.Ann. Bot.Fenn., 15: 25+257. LaubE.,Waligorski F., R.Woller,'1977,UberdieCadmiumanreiche-rung in Champignons. L. Lebensm.Unters.- Forsch. 164: 269-271. Lep5ovdA., V. Mejstoik,1988. Accumulation of traceelements in fruitingbodies of macrofungion the KruSn6hory mountains Czecho- slovakia.The Scienceof the TotalEnvironment, 76. 117-128. Lep5ovdA., R. Kr6l,1988. Lead and cadmiumin fruitingbodies of macrofungiin the vicinttyof a leadsmelter. The Scienceof the Total Environment,76: 129-1 38, Liukkonen-LiljaH., KuusiH., LaaksovirtaK., LodeniusM., S Piepponen,1983. Thd Effectof Lead ProcessingWorks on the Lead, Cadmiumand MercuryContens of Fungi.L. Lebensm.Unters.- Forsch, 176: 120-123. LodeniusM., KuusiT., LaaksovirtaK., Liukkonen-LiljaH,, S. Piepponen,1981. Lead, cadmium and mercurycontents of fungiin Mikkeli, SE Finland.Ann. Bot. Fennici, 18: 'l 83-186. LodeniusM.,M. Herranen,1981. Influence of a chlor-alkaliplant of the mercurycontentsoffungi. Chemosphere, 10(3): 313-318. MutschF., HorakO., H. Kinzel,1979. Spurenelemente in hdherenPilzen.Z. Pflanzenphisiol. Bd. 94: 1-10. OhtonenR., 1982.Mineral concentrations in someedible fungi and their relation to fruit-bodysize and mineralstatus of substrate.Ann. Bot.Fennici l9(4) : 203-209. QuincheJ.-P., 19794. Teneurs en quelques6l6ments traces du Lycoperdonperlatum. Bulletin romand de myeologie,10, 13-1 4. QuincheJ.-P., 19798. L'Agaricusbitorquis. un champignonaccumulateur de mercure,de s6l6niumet de cuivre.Revue suisse Vitic. Arboric.Hortic. 1 1(4): 189-1 92. QuincheJ.-P., 1980A. L'Agaricus silvicola. un champignonaccumulateur de m6tauxlourds. Bull. Suisse de Mycologie,9: 138-140. QuincheJ.-P., 19808. Teneurs en huit6l6ments traces du Tricholomageorgii. Bull. romand Myco. 3: 20. QuincheJ.-P., 1980C. Teneurs en quelques6l6ments traces de l'Agaicuscampester. Bull. romand de mycologie,2: 20. QuincheJ.-P., 1981. Teneurs en huit6l6ments traces de Marasmiusoreades. Bull. romand de mycologie,5: 20. QuincheJ.-P.,1982A Teneursenhuit6l6mentstracesdeLeplsfanebulais.Cahierdetransition1982A'MycologiaHelvetica,29-32. QuincheJ.-P., 19828. Accumulationsde m6tauxlourds et de s6l6niumchez quelquesesp-ces de champignonssup6rieurs. Bull. A.R.P.E.A.,111,42-6. QuincheJ.-P., 1982C, L'absorption de huit6l6ments traces par Lactarius piperatus. Bull. romand de mycologie,'l: 19. QuincheJ.-P., 1983A. Les teneurs en s6l6nniumde 95 esp'cesde champignonssup6rieurs et de quelquesterres. Schweiz. Landw. Fo. Rechercheagronom. en Suisse, 22(31 4): 137 -1 43. QuincheJ.-P., 19838. Les teneurs en huit6l6ments traces de Boletusedulis. Mycologia Helvetica, 1(2): 89-95. QuincheJ.-P., 19874. Le cadmium,un 6l6mentpr6sent en tracesdans les sols,les planteset les champignons.Revue suisse Agric. 1q4.71-77. QuincheJ.-P., 19878. Les teneurs en huit6l6ments traces de Lepistanuda. Mycologia Helvetica, 2(2): 173-181 . QuincheJ.-P., 1988. Teneurs en huit6l6ments traces d'Amanita muscaria. Bull. romand de mycologie,1: QuincheJ.-P., Bolay A., V. Dvorak,1976. La pollutionpar le mercuredes v6g6tauxet des solsde la Suisseromande. Revue suisse Agric.,8(5): 130-142. RUckerT., T. Peer, 1988.Pilzsoziologische Untersuchungen am Stubnerkogel(Gasteiner Tal, Salzburg,Osteneieh) unter BerUck- Sichtigung der Schwermetallsituation.Nova Hedwigia,47 (1 -2): 1 -38. RUhlingA., BaathE., NordgrenA., B. Siiderstrom,1984. Fungi in Metal-Contaminated.Ambio, 13(1): 3+36. SanglimsuwanS., YoshidaN., MorinagaT., Y. Murooka,1993. Resistance to and Uptakeof HeavyMetals in Mushrooms.J. ol Fer- mentationand Bioengineering,75(2): 112-114. . SeegerR., 1976.Quecksilbergehalt der Pilze.L. Lebensm.Unters.- Forsch. 160: 303-312. SeegerR., 1977.Quecksilber in jungenund alten Pilzen und in Pilzsporen.Deutsche lebensmittel+undschau, 73(5): 160-162. SeegerR., 1978.Cadmium in PilzenL. Lebensm.Unters.- Forsch. 166: 23-34. SeegerR., MeyerE., S. Sch6nhut,1978. Blei in Pilzen.L. Lebensm.Unters.- Forsch. 162: 7-10. StijveT., 1977.Selenium Content of Mushrooms.L. Lebensm.Unters.- Forsch. 164: 201-203. Turnau K., 1990. Heavy metal uptake by Armillaria/ufea growingin a Pino-Quercetumforest treated with cadmiumdust. Nova Hed- wigia,50(1 -2): 201 -2'l 1, TyferG., 19824.Accumulation and exclusionof metalsin Collybiaperonata and Amanita rubescens. Trans. Br. mycol.Soc., 79(2'):239- 245. TylerG. 19828.Metal accumulation by wood-decayingfungi. Chemosphere, 'l1(1 1): 1 141-1 146.

38 TableI of metalsln mushroom lrom anclba areas Soeciesof oollutinoareas Soeciesof controlareas Elements mtn max mrn max concentrationmushrooms concentrationmushrooms concentration mushrooms concentrationmushrooms Fe 55 Sur//usoranulatus 4 948 Lenzifessp. 20 Su//usluteus 1 864 Sui//us/uteus Mn 2.1 Su//usoranulatus 938 Lenzifessp. 3.9 Sui//usluteus 110 Mvcenarosella Cu 1.4 Tricholomarutilans 1 344 Bovistaplumbea 3.6 Coriolus hirsufus 627 Clitocvbeodora Zn 11 Tricholomarutilans 305 Bovistaolumbea 27 Coriolushirsutus 238 Lvcoperdonperlatum Pb < 1.0 Ticfioloma rutilans 50 Eovista plumbea < 1.0 manv species 9.6 Lvcoperdonperlatum cd < 0.14 Tricholomarutilans 27 Coprinusatramentarius < 0.18 Sui//usqranulatus 18 Cvstodermacarcharias As < 0.6 many species 41 Aoaricus silvicola < 0.5 manv soecies 31 Clitocvbeoeotropa

Tablell of heavymetals in two mushroomspecies (mg/kg dry weighl Kremikovtzi Zlalilza Control Elements mushrooms substrata mushrooms substrata mushrooms mrn max min max mln max mrn max mrn max

Bovisf a p lumbea

Fe 176 2235 17000 78 000 149 608 11000 16000 136 222 Mn 14 359 622 3 351 't7 48 220 866 12 59 Cu 48 204 25 14 926 387 1 344 350 5 1s7 65 117 Zn 93 184 92 8 195 106 305 33 135 124 160 Pb 2.3 50 26 16106 1.8 18 36 318 < 1.6 3.9 cd < 0.3 20 0.7 30 1.1 3.5 0.7 4.4 0.49 4.1 As 1.3 24 < 0.42 844 < 0.56 10 8.8 61 0.65 2.5

Marasmius oreades Fe 76 438 28 000 39 000 73 418 {1 000 16000 47 62 Mn 42 150 622 3 351 21 46 220 537 24 31 Cu 12 141 96 14 926 273 1163 3s0 5 157 54 86 Zn 79 1't9 265 I 195 124 160 33 128 67 98 Pb 1.3 2.0 256 16106 1.8 8.6 36 318 < 1.0 .2.0 cd 0.3 1.0 8.2 30 0.29 4.5 0.7 4.4 < 0.2 3.6 As 1.1 7.7 128 140 3 7.3 10 61 1.0 3.3

\o THEUSE OF LIEHENSIN BIOMONITORING Dobrilvarrcv BotanicalGarden - Varna SofiaUnrversrty, Bulgaria

The steadilyincreasing pollution caused by iritrc,duceoby man iiiorganrcand organicsubslances cue ill the increasingindustrialization and urbanizationduring the lastcentury, requires permanent global i-iioniio;'- ing of the of potentialpollutants and observatioriof the biologicaleffects which ihey induceon tire L,cos;js- tems.The accumulationof the changesin the envirorrmentcan be detachedbythe changesobserve:iJ irrthe' ecosystemsand respectivelyin the livingorganisms For this purposepopulations of speciesare usedha'v- ing highestsensibility to increasedpollution and lowestown naturalvariability lt has long beei-rknown ihai epiphyticlichens are extremelysensitive to atmosphericpollution, especially to SO2,and that iney can lli,, usedas bioindicatorsof air pollution(James 1973, Seaward 1989). Lichens are eXfemelyserisitive syirbr- oticorganisms, consisting of fungusand algae, which react to evenslightly polluted air Thejoint lrfe of their componentsis due to a veryfine regulation realized by helpof differentlichen cornpounds Furrgus and algae exchangecertain substances within themselves and this pi'ocess is facilitatedby the specificartatorrrcal and morphologicalstructure of lichen By thisreason it is veryeasy to ciisturbthe balancein the licherithal- lus.So the pollutionusually damages lichens, and as a resultthey can die.The registrationof certainana- tomical,morphological and physiologicalchanges or the lackof lichensit all enablesrelevant conclusions on the immissionsituation of the areawhere the examinedlichens live orare expectedto live. Sincethe secondhalf of the lastcentury some papers appeared in whichthe aulhorsdiscussed the farrt thatthe numberof lichenspecies in the bigtowns and the industrialcentres was verylow in comparisonwitn the unsettledterritories and the fact that the sensibilityof singlespecies to pollutionwas quite different /Nylander,1866/. Later Sernander /19261 for the firsttime determinedfor Stockholmthe so cold lichen zones:" lichendesert" /zone with high air pollutionand without lichens/, struggle zone lzone with medium air pollutionand poorlichen vegetation/ and normalzone lzone with low air pollutionand lichenvegetation that normallyoccurs at thatgeographical region/. In thatway the mappingof the townareas /in zones/according to the air pollutionon the baseof lichensbegan. Two very importantevents took placein the 60s whichhelped the developmentof lichenbioindication. The firstwas the experimentalevidence of thetoxic action of SO2,the mostimportant pollutant of the air on manylichen species. And the secondwasthe calculationof the firstlichen indexes /Trass, 1968; DeSloover andLe Blanc,1968;Le Blanc and DeSloover; 1970, DeSloover 1964/. Since then many bioindication studies usinglichens have been basedon the use of Indexof AtmosphericPurity /lAP/, accordingto whichthe higherthe IAPthe betterthe air quality.The otherindex that hasbeen suggested is the Indexof Poleotoler- ance/lP/ usedmainly in the republicsof theformer USSR. This index is the sum of classesof poleotolerance multipliedby the coverageof eachspecies devided by the totalcoverage of all species.According to this formulathe higherlP the morepolluted air /Trass, 1985/. The originalIAP allowsa quantitativenumerical evaluation of air qualityby meansof the number,frequencyand toxitoleranceof epiphyticlichens. lt has beenproposed for the firsttime by DeSloover(1964) and has beenslightly modified by Le BlancandDeSloover/19701. The IAPisthe sum of toxitolerancemulti- pliedby frequency. Duringthe lasttwenty years many variations of the Indexhave been used, giving different weight to the variablesenclosed /Hoffrnan, 1974, Moore.1974, Crespo et all.,1981, McCune, 1988/. The groupof Swiss authors/Ammannet al i987,Herzig etal. 1987, Liebendorferet al. '1988, Peterand Ammann, 1990, Herzig, 1990,Urech et al. 1990,199'1/tested the predictabilityof 20 differentIAP formulas by mulliplelinear regression analysis,comparing IAP valueswith the directmeasurement of 8 biologicallyrelevant air pollulants /SO2, NOx, Pb, Cu, Cd, Zn,Cland dust/and foundthe best correlation/R2=0.98, p-0.05/ for the formula IAP=Fin whichthe Indexis the sumonly of frequenciesof eachspecies. Both the bioincjicationmethods and the technicalmeasuring complement each other. The technically measured immission data, together with the presentedmethods are a valuabletechnique for modernintegrated immission control. The modificationof IAP=Fdiffers from all the othermodifications and from the originalformula in thatit substantiallyexcludes all elementsof subjectivity/connected with such variables as toxitolerance,ecological index, index of associa- tionetc./. All the indexesused correlate very well with the dataof the technicalair poliutionmeasurements. On the basisof the strongcorrelation found between IAP andthe combinedeffect of severalair pollutantsiAP val- uescan be convertedin to airqualiiy values. And in thatway according to the numericalvalues calculaied oir the basisof lichensgrowing in certainarea one can make a conclusionabout the qualityof air. The lichensdo not specificallyreact lo singletoxic components in the air, but ratherindicaie the integra- trvetoxic effect of a combinationof differentbiologically relevant pollutants. By the aid of the PassiveBio- monitonngit is possibleto determinewhich single pollutant affects the lichencommunity and seamsto be responsiblefor the degradationin lichenvegetation. Multi-element analysis on a selectedlichen species can enablesquantitative and qualitative conclusions on typeand amount of singleactive pollutants.

40 Generallythe two methodscomplement each other. With tne aid of IAP- methodan area-widecadastre of biologicaleffects of air pollutionis provided.On the otherside Passive biomonitoring leads to findingson the immissionstress by singlepollutants, by meansof extensivemulti-element analysis on lichenspecies, In thatway an IntegratedBiological Measuring System for MonitoringAir Pollutionappears and it consists of CalibratedLichen Indication Method and Passive Biomonitoring. In Bulgarialichen bioindic.ation methods using calculation of anytrind of Indexeshave not beenapplied at allfor any region.All whathas been done is a studyof lichenvegetation in the partof the townof Sofiaand its surroundingsto directionof Vitoshamountain. Here the authorhas not usedany kindof Indexesor stations,but has madean attemptof areazonation on the basisof subjectiveobservations on the decreasing of the lichenvegetation from the Vitotshamounlain to directionof thetown centre /Philipova, 19621. The secondstudy is ourpilot study of the lichenvegelation in the townof Varnaand itssurrounding area. There25 stationsscattered through the townwere chosenaccording to ihe presenceof nearly10 suitable trees,having diameter not less that 0.5 m at the highof 1.5m. We consideredthat the normallichen vegetation characteristicforthisregion potentially could be developedon suchtrees. Laterwe started investigation in 10 morestations situated in the North- North-Eastdirection from the townwhere the normallichen vege- tationwas supposedto be found.Twenty eight epiphytic lichen species belonging to familiesPhysciaceae and Parmeliaceaewere found as a resultof this study.The mostwidespread species were Phaeophyscia orbicularis,Physcia stellaris, Xanthoria parietina and others. Nearly half of the determinedspecies l13l were foliousand the same numberwere crustaceus. The big numberof foliousspecies found probably is due to the influenceof marineair and to thefact that these species are quite toxitolerant lichens. At that stage of investigationsno recordingsof frequency,coverage vitality etc have been done and no Indexeshave been calculated. So we stillcan not speak about the certainlichen zones in the townof Varna. But,nevertheless, it is interestingto mentionthat a widearea including the towncentre and the areaaround the sea portwas foundto be withoutany lichenvegetation at all.The stationswith more richvegetation (from five to ten species)were situatedat the end of the townwith the exceptionof the stationin the old cemetery. Much more richlichen vegetation was observedin the stationsNorth - Northeastfrom the town to the direction of the BlackSea resorts("St Konstantine" and "Zlatnipiasatsi"). Besidesthe lackof investigationsusing the IAP- methodin Bulgarianon multi-elementanalysis of lichen specieshas beendone. But mostof the specieswhich havebeen examined in differentparts of Europedo growin Bulgaria.Such toxitolerant folious species as Parmeliacaperata, P. sulcata,Hypogymnia physodes, some speciesfrom the generaPhyscia s.1.,Xanthoria etc very oftencan be foundin moderatelypolluted areasnear the townsand are suitablefor the analysismade in parallelwith the mappingof the regionac- cordingto the IAP- method. For applyingan IntegratedBiological Measuring System for MonitoringAir Pollutionin Bulganawe have to startwide investigations at severalpolluted areas. Such areas we havein Bulgariaenough: Varna-Devnja, Burgas,Medet, Kardjali etc. All this hasto be donefirst of all in the areas,where technical immission data are available,in orderto calibratethe IAP- valuesobtained in Bulgaria.The secondthing which has to be done is a multi-elementanalysis of somemonitoring lichen species from the stationsin the same area.In that way we can obtainprecise and detailedinformatibn of biologicaleffect of total air pollutionand of single pollutantsas well. Since, changesin lichensare correlatedwith the frequencyof lung and upper respiratorytract illness. lichen biomonitoringwould be a powerfultool to administratorsand politiciansinvolved in environmenial planning.

REFERENCES CrespoA., BarrenoE., SanchoE:, L. and BruenoA.G., 1981. Establicimentode una recjde volaracionde purezaatmosferiea en ia provinciade La Coruna/Espana/ mediante bioindicadores liquenicos. Lazaroa. V 3, p.289 - 31| DeSlooverJ.,1964. Vegetauxepiphytes et pollutionde I'air.Rev.Quest. Scient., V. 25, p.531 - 561. DeSlooverJ., Le BlancF. 1968.Mapping of atmosphericpollution on the basisof lichensensitivity. In Proc.Symp. Reeerrt Advances in TropicalEcology. Varansi, p. 42 - 56. GilbertO.L.,l965.Lichensasindicatorsof airpollutionintheTyneValley.Ecologyandtheindustrial society,Blackwell,Oxford, p 35.47 '1973. JamesP.W., Air pollutionand lichens.The Athlone Press. London o. 1 - 5. JohnV., 1988.Epiphytic lichens, climate and pollution in lzmir.Plants and pollutantsin developedand developingcountries, 12 p. HoffmanG.R. 1974. The influenceof a paperpulp millon the ecologicaldistribution of epiphyticcriptogams in the vicinityof Lewisioi'r ldahoand Clarkston,Washington. Environ. Pollut. V. 7, p. 283- 301. Le BlancF., DeSlooverJ., 1970, Relationbetween industrialization and the distributionand growthof epiphyticlichens and mossesin Montreal.Can.J. Bot.V.48, No7, p.1485-1496. McCuneB.,lgSE.Lichencommunitiesalong03 and SO2 gradientinlndianopolis.Bryologist.V.9l, p.223-228. MooreC.C.,l9T4.Amodificatronofthe"lndexofAtmosphericPurity"methodforsubstratedifferences.Lichenologist,V.6p l56-157. NylanderW. 1866. Les lichens du Jardindu Luxemburg.Bull. Soc. Bot, France, V. 13. p. 364- 372. PhilipovaL., 1962. L'influencedes conditionsecologiques de la villede Sofiasur la developpementet la distributiondes lichens.Ann Univ.Sofia, V. LVl, p. 83 - 96. SeawardM.R.D.,l9S9.Lichensaspollutionmonitors: adaptingtomodernproblems.Plantandpollutantsindeveiopecianci developing countries,lzmir, p. 35 - 47. TrassH.,1985. Classesof lichenpoleotoleranceandecological monitoring.Problemsof ecol.monit.andecosystemmodeling. V. Vll. p. 122- 137. 1l PROBLEMSAND POSSIBILITIESWHEN USING PHYSIOLOGICAL AND BIOCHEMICALDATA FOR BIOLOGICAL MONITORING

NinaBakardlieva Instituteof PlantPhysiology, BAS

Manycharacteristics of the metabolicstatus of the biologicalsystems are a necessarycomponent of the systemof biologicalmonitoring. Growing is the contributionof modernphysiology and biochemistryin this respect,which necessitates an exactestimate of the approachto suchdata. Otherwise the errorsand delu- sionswill be muchmore. Such an estimaleis possibleonly by meansof a thoroughevaluation of the biological statusof the system-objectwhen checking the typical dynamics of the physiologicalindices. Owing to this dynamicsthey may be a betterand faster indicator of the changesoccurred in the environrnelrt.But in order to be alsoan exactindicator it is necessaryto findthe wayfortheir use and interpretation. The appearanceof initialprimitive forms of lifemust be linkedwith the integrationof morethan one es- sentialfunctions of lifeinto a system,wherein they act jointly. Among them the catalytic,reproductive, infor- mational,electron transport, etc. functions could be mentioned.lt is namelythis initialcoordination of functionsand processeswhich presupposes a controlof theirintensity that marksthe beginningof the specific metabolismfor the biologicalsystem and the typicalfor lifeexchange of matterand energy.This is validfor all the levelsof lifeon Eafth,including the biosphereas well.These primary functions undergo an evolution, duringwhich their effectiveness and specificity increase and newintegral functions emerge. Moreover, in the courseof evolutionof lifean optimalcorrelation of functionsis beingachieved, specific for each organiza- tionallevel. This functional equilibrium may be the basisfor manyconclusions in the practicalactivity of man, relatedto the productivityof biologicalsystems and its regulation,to the preservationof ecosystems,etc The majormeaning of thisequilibrium is connectedwith the conceptionfor the preservationof the biosphere - the mostessentialtask on a globalscaleforthe preservationand development of lifeon Earth. It is veryimportant to findthe factorsand components which are importantfor maintainingthe equilibrium in the biocenosis.lt is namelythat hadto be usedfor characterizingthe statusof the systemand for monitoring. In orderto accomplishthis goal first of all mustbe definedwhat does physiological norm mean. lt is well knownthat the metabolicprocesses in the celland the entireplant are in a definiteequilibrium which is ge- neticallyand evolutionarydetermined, expressed generally as homeostasis. The biologicalhomeostasis is realizedat differentorganizational levels - cell,organ, organism, species, biocenosis,biosphere. At everyorganizational level exists some dynamic equilibrium which, however, does not excludechanges in the sublevels.On the contrary,being typical for a complexhierarchical system, they helpthe systemto combatsomewhat the changesprovoked by the environmentand to retainits biological specificity.That is why indicatorsfor changesof the speciesand biocenosis should be searchedfor aboveall at organismand suborganismlevel, especially at subcellular.Therefore, we must orientateourselves be- tweentwo tendenciesthat are somewhatand probablyseemingly contradictory: on one sidethe changesin a seriesof physiologicaland biochemicalprocesses as a resullof signalsfrom the environmentand on the other- the effectivenessof the systemto preserveits stableequilibrium. Attention should be drawnto such indiceswhich like signal lamps show dangerous changes in the environment,although still without violation of the naturalequilibrium of the processesin the examinedsystem, for instancethe biocenosis.Such quickly reactingand indicativeindices could be selectedfrom measurements already made of the physiologicaland biochemicalprocesses in the plants.As a matterof fact it is by changein the velocityof some metabolic processesthat the plantcell and the plantitself can retain the homeostasis. Characteristicfor the functionalindices is thatthey are not constant.They vary withindefinite intervals dependingon the limitsfor lifeof the system.Consequently, it is necessaryfirst of allto knowthe parameters which markthe variationswithin the limitsof the normaland the respectiveaverage value acceptedas a norm.An increaseof thisindicator shows that the systemuses the maximumof its potentialand vice versa - a decreaseaccounts for a partialelimination of the respectivesystem, which however could be favorablefor the cellorthe organism. Furthermoreit is necessaryto considerthe fact that the parameterswithin which a given physiological indicatorchanges are speciesspecific and depend on the phytogeniticposition of the givenspecies. There- fore, a more detailedcharacteristic is necessaryof the plantspecies set to be investigatedfor the goals of biologicalmonitoring.

12 It becomesclear that one oi tiie nosl difiilrtrniorllei'it5 rs to firJdthe sorcalledcc;ntrol and evaluatethe meaningof the changesoi a givenindicator for ttretotal status of the ptant,biocenr:sis and tne ecological characteristtcsof tne regiori.Naiurally, tnis does l)c,tmtsarr ttiat the physiolugicalindices ought not to be used.On the contrary- emphasizedrs oniy the rrecessityot a purposeiulinvestigation for the needsof monitoring.In orderto frndthe approachio theirr|ost exaot use. AnotherqLlestron of exlraordinaiyirrrpoitaric.e in this r;gse is the specificityof the metaticlicresponse cf the piantsio the changesin the envirorii-neritaniJ dtrfing stress situations Thrs rii factmeans rfiat owing to drasticcharrges lrr different tactots ot the errviionmerrttirc physiologicalarrd tlre bir-rctienrrcalirrdicators might undergosimilar changes. Ctinsequentiy the dalataken into account will give as arrswerabove all aboutthe r'eactionof the plants,while tire facior v,rhir;h has piovokL'(l this reaction will have to be identifiedseparately. May be, however,for the aimsof rni;nitoririgthis coLrld iie an advantage Frornwhat was saioso far rt becomesclear that for the estimateof the stateof the ecosysiemand moni- tciringit is betternot io usethe indicesslrowing the contentof a giverrcompouncl (ior instancechlorophyll, organicacids, etc.,1 but predorninantlythi:se showrng the eltectivenessof a givenprocess. The followirtgindices could be meritiorredarnorrg thern: effectrveness of the enzymesystems iinked with biosyntheticprocesses or elinrinatinghamrful influences of factorsfrorn the environment;effectiveness of photosynthesis,effectiveness of proteinsynthesizing apparatus; effective waiei balance,elc. This approach makesit possiblefor a selectionof suiiableindicators, giving information about tfie physiologicalstatus of the plantinvestigated Emphasizing on this approachI wantto underlinethat it must not be copsideredas an absolute.lt mightbe connectedwith values for the contentof a givengroup of compounds- for examplevuith provenprotective effect or otherfunctional characteristics helpful for the protectionof the organrsm.Special attentiondeserye the naturalcompounds linked with the phenornenonailelopathy, which have to be investi- gatedboth from the standpointof the metabolismof the plantproducing them and the plantthey are exerting influenceon. The followingtest systems could be markedfor usein brologicalmonitoring:

1. Determiningthe activityof peroxidasd,superoxidedismutase and catalase. These are enzymesvrith a key participationin the decompositionof the toxic for the plantcell forms of actirratedoxygen - a proouctof the normalmetabolism. Characteristic, however, is thatthe quantityof the activeoxygen forms is higher underthe influenceof stressfactors. That is why the effectiveaction of tne enzymecomplex is very importantfor the suruivalof the plant.Therefore, a changein the activityof the threeenzymes gives a fast informationfor eventswhich have taken place in the biocerrosis.On the otherhand - the degreeof quicr reactionof the systemis an indexfor the metabolicstatus of the plantspecies. Testing is appropriatetc be realizedduring a provocativeinfluence on the system.

2. Analysesof the isonzymecomposition of someenzyrnes, for exampleperoxidase, supercxidedismuiase and catalase,some dehydrogenases,etc. Essentialiri the case is that every isoenzyrnespecirr:in is characterizedby twogroups of isoenzymes.We demonstratedin ourinvesiigations that oneof the groups - the so-calledconservative forms' areless changing and are similarin the plantsat differentphytogeni- tic position.The secondgroup - the so-calledadaptive isoqrzymes - are speciesspecific and reactrnost stronglyto changesin the environment.Part of themcan demonstrate activity only at givenconditions oi the environment.Namely this group is mostperspective fiom the standpointof biologrcalrnonrtonng. The firstgroup ensures the realizationof the catalyticfunction, the secondgroup adapts the functionto ine peculiaritiesof metabolism;and is responsiblefor the preseruationof homeoslasisas most capabieto balancethe intensityof the function.The test criterionmay be the appearanceor disappearanceoi arr isoenzymeof the secondgroup, as wellas the drasticchange of activityin thatgroup.

3. Characteristicsof effectivenessof the photc;syntheticapparatus. For this aim - the changesin ti'repa- rametersof gas exchangein the leavescould be traced,as wellas the trarrsformationof lighi e iiergy anri the mechanismsof carbondioxrde assrmilation. ln the endwe musthave a notionabout the proouciivity of the photosyntheticapparatus. This could be achievedby applyingmodern biochemicai anci L,icrphysical methods.A provocativeinfluence could also be appliedin the case,which will give an ideaaboui ihe capacityof the photosyntheticapparatus.

+J 4. Effectivenessof the biosynthesisof RNA and proteinsin the cell.lt couldbe measuredby the introduction of labeledprecursors (aminoacids) in the totalprotein and by characterizingthe stateof polisomes'profile for a giventissue or species.One of the usefulmethods is studyingthe biosynthesesof proteinusing a standardtest in vitro.By the productof biosynthesisin vitrowe can checkthe appearanceof stressindu- cable specificproteins. The biosynthesisof RNA will be characterizedin a system of isolatednuclei by the activityof endogenousnucleic RNA polimerases-l and ll.

A few wordsabout the interpretationofthe dataabout the contentof chemicalelements in the plants.The quantityand correlationof mineralelements, especially metal ions in plantsis speciesspecific, conditioned by the evolutionof metabolism.This which could be nameda normalso varies within definite limits. That is why the estimateof the determinedvalues for contentof metal ions oughtto be consideredboth with the pointedvariations within the normalvalues and withthe changesof a givenbiochemical indicator - in this case most suitableis the enzymeactivity and namelythat of the adaptiveand fast reactingenzymes (as for examplethe peroxidase).There are sufficientproofs that the directinteraction of the peroxidasewith a series of metal ions is an importantmechanism of regulationof a seriesof metabolicprocesses, linked with changesin growthas well.This can alwayshappen in the cell containingan increasedquantity of an element which presupposesthe existenceof free ions. In our researchwe demonstratedthat the interaction betweenperoxidase and calcium ions plays an essentialrole in the transportof signalsabout changes in the environmentthrough the so-calledmechanism of secondmessenger in the plantcell. A highercontent of someelements in the planttissues allows in vivothe formationof complexeswith low-molecular compounds, above all aminoacids,which possesstheir own specificphysiological activity. The newlyformed complexes includethemselves actively in the metabolicreactions (especially the catalyticones) havinga certaincata- lyticactivity and beingeffectors of many enzymesystems. All this givesme groundto assumethat the data aboutthe contentof chemicalelements, especially of metalions in the soiland in the livingorganisms ought to be interpretedas a complexity,connected with concretephysiological and biochemicaldata. The proposedmetabolic tests, predominantly at a biochemicaland molecularlevel, could be elaborated by a Bulgarianteam with the necessaryqualification. The problemsdiscussed emphasize the needof a new approachwhen usingthe physiologicaland biochemicaldata for biologicalmonitoring with a viewto be moreinformative. Quite large perspectives and pos- sibilitiesare simultaneously open for collaborationand research work.

44 ISOENZYMESAS BIOINDIEATORSOF ENVIRONMENTALPOLTUTION

GeorgiAngelov nsiituieof Botany,BAS, Bulgaria

I. INTRODUCTION This reporlis devotedtc tfie appiicaiionof isoerizynrerlai-kers frrr the purposesof impactand backgroundmonitoring. It is knownthat industrialpollution causes sei"ious changes of environmerrtand naturaiecosystems. Nu- merousstudies, mainly on lree species,demonstrated that in regionspolluted by industrialactivities populationsof foresttrees modify their genetrc structure in responseto the alteredenvironmental conditions (Sin- clair 1969).Applying enzyme gene markeis,many authors(Scholz and Bergmann1984; Geburec et al., 1987)demonstrated that air pollutionand rtsconversion products can alterthe geneticslructure of monitor speciesby processeswhich are assumed to haveselective effects. Firstwill be representedresults of siudieson the populaiionstrucrture of two grassspecies Dichantium is- chaemumL. and Chrysopogongryllus t, in areasunder strong anthropogenic pressure (air pollutionand highconcentralions of heavymetals in the soii)and in areasunder a relativelyweak anthropogenic pressure, The aim of the studywas to findout possiblechanges in the populaiionstructure of D. ischaemumand Ch. gryllusunder strong industrial pollution by meansof isoenzymemarkers.

II. MATERIALAND METHODS Forthe studies,two selsof localpopulations were chosen The firstset consistedof populationslocalized close(1-5 km) to a coppersmelter nearthe town of Srednogorie(set S), the second- of localpopulations undera weakanthropogenrc pressure serving as a conlrol(set K). Totally.l04 living plants of D. ischaemum and 107 plantsof Ch. gryllusfrom set S populationsand 72, 101 plantsfrom set K populationswere collected. For isoenzymeanalysis, leaves extracts were obtained and subjectedlo verticalpolyacrylamide slab-gei electrophoresisaccording to Davis(1964) and Reisfeldet al. (1962).The enzymesesterase (EST), pei'oxi- dase (PER), glutamate oxaloacetatelransaminase (GOT), glucose 6-phosphate (G6PDl-i).6- phosphogluconatedehydrogenase (6PGDH) and malatedehydrogenase (MDH) were examined.Each isoformwas givena numbersymbol reflecting its gel mitrationin mm. Becauseof the lackof geneticanalysis, no alleleor genotypefrequencies were determined. The frequencyof each isoformin the populationsets, was calculated. The significanceof the differencesin the isoformfrequencies between two setsof populationswas evalu- atedusing Wilcoxon signed-rank test (Hollander and Wolfe 1973) at 5% level. The soil concentrationsof the heavymetals Pb, Zn, Cu. Co, Cd and Ni were determinedby atomieab- sorptionspecirophotometry.

III.RESULTS Heavymetal accumulation.The soilconcentrations of heavymetals in the tlvo sets of populatibnsare '1. '10 presentedin table lt was evidentthat the heavymetal concentrations in set S were 1,5 - tirneshigher than the concentrationsin set K. The analysisof the heavymetai concentrations in D ischaernun(table 2) revealedtwofold to tenfoldenhancement of heavymetal accumulation in the plantscollected from the set S populations.The correspondingdata for Ch.gryllus showed similar tendencies of heavymeial accumulation in the set S populations.In general,the obtaineddata indicated that set S populationsof D. ischaemumand Ch. gryllusare undera muchstronger anthropogenic pressure that set K populationsof bothspecies. Populationstructure. The anodalisoforms of ESTwere electrophoretically resolved onty. The cornpari- son of the set S and set K populationsof D. ischaemumand Ch. gryllusdemonstrated no significantdiffer. encesbetween them. The anodaland cathodalisoforms of PERwere eleclrophoretically fractionated. The populatiorrstrircture in regardto cathodalPER is presentedin table3. Bothspecies possessed ten isoforms.Some of thern" 2i3, 27 inD. ischaemum,33in bothspecies were monomorphic in set S. The comparisonof lhe isoforrnfrequei-i- ciesresulted in signific'antdifferences between two setsof populationsat 5% level. Regardingthe anodalPER isoforms, no significantdifferences between sel and sei K poprrlatiorisof hoth specieswere observed. 6-phosphogluconatedehydrogenase. The populationstructure of Ch. gryllusis shownin the nextiai:ie The comparisonbetween two setsof populationsrevealed significant differences at 5% levelwniie rio srgriifi- cantdifferences were found with respect to D. ischaemum. Regardingglutamate oxaloacetate transaminase (table 5) it was shownthat set S and set K poprrlations of bolhspecies differed srgnificantly at 5% level.

ii Malatedehydrcge nase Thc popuiationstiucture of D. ischaemumis shownin the nexttable. A totalof eightisoforms were resol,,ed in thrsspecies, two of them(13, 35) beginmonomorphic in set S and set K, respectively.There 'v,,,,eie significant differences at 5% levelbetween set S and set K populations.The correspondingdata for Cii gryliusshowed no significantdifferences of isoformfrequencies between two setsof populations. RegardingGlucose - 6 phospnatedehydrogeitase no significantdifferences between set S and set K of bothspecies were fouiro

IV.DISCUSSION The analysisof the pcpulationstructure of D ischaemumshowed that significantdifference occur be- tweentwo setsof populaiionsat threeout of six isoenzymesinvestigated - cathodal PER, GOT and MDH. The correspondingdata for Ch. grylluswere similar, Three out of six isoenzymesnamely, cathodal PER. GOT and 6-PGDHdemcnstrated significant differences between two setsof populations.These results are in goodagreement vvith data of Scholzand Bergmann(1984), Geburek et al. (1987),Prus-Glowaski and Novak-Bzowy(1989) for GOT, Miler-Stark (1985) for GOT and MDH. Someof the isoenzymesexamined (GOT, PER, MDH) have been shown to be involvedin metabolicpro- cessesaffected by gaseouspollutants (parlicularly SO2) as statedby Malhotraand Hocking(1976), Jager andKlein (1980). SO2 is a dominantgaseous pollutant in the investigatedregion. In the lightof the above- citesinvestigations, it is reasonableto supposethat SO2 is amongthe environmentalfactors influencing the populationstructure of the speciesinvestigates On the otherhand, as it was shown,the regionof Srednogorieis highlycontaminated by heavymetals. Sincethe 1950it has beenknown that certainplant species (mainly grasses), growing in suchheavy meta pollutedsites, shcw a distincttolerance to the metalsoccurring in the soil(Martin and Cougthrey1982). lt is importantto mentionthat the majorityof articleson the metaltolerance suggest that lhe requiredselection processadding to formationcf tolerantindividuals may haveacted over relativelyshort period of historical time.Thus, for example,Hogan and Rauser(1979) proposed that copper,nickel and cobalttolerance of Agrostisgigantea had occurredcver 70 years.Coughtrey and Martin(1978)) demonstraled that Holcusla- natushas developedtolerance after 45 yearsof industrialactivities. Thus, lhere is considerableevidence to suggestthat tolerance lo variousheavy metals can be demonstratedin someplant species after a remarka- bly shortperiod of timefrom the startof a contaminatingevent. Taking into account that the coppersmelter near Srednogorieis approximately45 years-old,it is reasonableto supposethat heavymetal pollution is anotherenvironmentalfactor influencing the population structure of D. ischaemumand Ch. gryllus growing in pollutedsites close to the srnelier The investigationof the pcpulationstructure of D ischaemumand Ch.gryllus allowed us to assumethat the selectionpressLri"e by someindusti"ial pollutants (mainly SO2 and heavymetals) leads to an increasein ihe propcrticttof tolei-antinoividuals in the set S populationsof bothspecies. lt is logicalto expectthat the enhaticementof ihe percentageof the toierantindividuals has an effecton the populationstructure of both speciesanci nray explain the alteredpopulation structure of D. ischaemumand Ch. gryllusunder strong an- thi'opogenicpressure Finally tnese i"esLiltscjemonstrated that the populationstructure of two grass speciesDichantium is- chaernuinand Ciriysopcgongryilus v,ras influenced by industrialpollution as revealedby isoenzymemarkers.The gaseouspoiiutant 3O2 anclthe heavymetals Pb, Zn, Cu, Co, Cd, Ni whichprevail in the investi- gateclregion are supposedto be amongthe majorenvironmental factors altering the populationstructure of bothspecies These observation on the populationstructure of D. ischaemumand Ch. gryllusunder a strong antfiiiipogetticpressure can be usedas an additionalmethod for biologicalmonitoringof the environment. Secrtnde^antple iliustrates tne applicationof isoenzymetechnique in the backgroundmonitoring. BoatinNationai Resenre was an experrmentalsite in the systemof backgroundmonitoring. F. nigrescens rrSSull€ 0t the br')tnOilit0t'S The aim of tne studywas io analyzethe isoenzymestructure of Boatinpopulati'on and its temporal crtartgesirr cor;-iparisorr to tne a,/eiaEelsoenzyme structure of F. nigrescensfound for its Bulgarianpopula- tions. In a pt'eviousstudy set ct 216plants belonging to 6 populations(P 6)wasanalyzed to revealtheaverage isoenzymestruciuie of F. nigresceris.A set of 50 plantsof Boatinpopulation was studiedfor threeconsecu- tiveyears (B 2, B 2, B 3). The expertmei'rtaiprocedrrres was the sameas in the previousinvestigation. The isoformsof EST,PER. GOTano ACF'H were resolved. Pair.wise comparisons between sets B 1,82, B 3 and P 6 weremade and the oifferencesbetween ttrenr were evaltlated by Wilcoxonsigned rank test at 5% level. A totalof elevenisoforrns of anodalEST were resolved (table 7). Someisoforms 32, 35, 37, 40 and 44 weremonornorphically-fixed Regardrng isoforrns 14, 16, 18,20 and24,there were some frequency fluctua- lions but pair-wisecompariserns showed statistically insignificant differences between the isoenzymestruc- tureof Boatinpopulation and the averageisoenzyme structure of F. nigrescens. -l(i RegardingGOT, PER andACPH similar results were obtained-no significant differences between B 1, B 2,B3andP6werefound. Summarizingthe results,it wasevident isoenzyme structure of Boatinpopulation is stableas indicatedby the insignificantdifferences between B 1, B 2 and B 3. On the otherhand, the isoenzymestructure of Boatin populationdid notdiffer statistically from the averageisoenzyme structure of F. nigrescensfound for its Bul- garianpopulations. lt is worthmentioning that BoalinNational Reserve is comparativelyclose (15 km) to coppersmelter of Srednogorie.As it was shown,populations of Dichantiumischaemum and Chrysopogon grylluslocalized close to Srednogoriesmelter were influenced by industrialpollution. Despite its closenessto Srednogoriesmelter, Boatin population proved to be stableand its isoenzymestructure did not differfrom the averageisoenzyme structure of F. nigrescens.The resultsof this study showedthat the isoenzyme markerscan be usedeffectively as an additional-approachforthe controlof the environmentin the systemof backgroundmonitoring.

REFERENCES

CoughtreyP., H. Martin.1978. Tolerance of Holcuslanatus to lead,zinc and cadmium in factorialcombina- tion.New Phytol., 81,147-154. DavisD. 1964.Disc electrophoresis. 2. Method and application to humanserum proteins. - Ann.N. Y. Acod. Sci..121 .404-427. GeburekTh., F. Scholz,W. Knabe,A. Vornweg.1987. Genetic studies by isoenzymegene loci on tolerance andsensivity in an air pollutedPinus sylvestris field trial. Silvae Genetica, 36, 49-58. HoganG., W. Rauser.1979. Tolerance and toxicityof cobalt,copper, nickel and zinc in clonesof Agrostis gigantea.New Phytol., 83. 655-663. HollanderM., D. Wolfe.1973.Nonparametric statistical methods. John Willey. New York. JagerH., H. Klein.1981. Biochemical and physiological effects of SO2 on plants.Angew. Botanik, 54,337- 346. MalhotraS., G. Hocking.1976. Biochemical and cytologicaleffects of sulphurdioxide on plantmetabolism. NewPhytol. ,76, 227-237. MartinM., P. Coughtrey.'1982. Biological monitoring of heavymetal pollution land and air.Applied Science Publishers.London, New York. Muller-Stark,G. 1985.Genetic differences between "tolerant" and "sensitive"beeches (Fagus sylvatica L.) in environmentallystressed adult forest stand. Silvae Genetica, 34,242-251. Prus-GlowackiW., R. Nowak-Bzowy.1989. Demographic processes in Pinussylvestris populations from regionsunder strong and weak anthropogenous pressure. Silvae Genetica, 38, 55-62. ReisfeldR., U. Lewis,D. Williams.1962. Disc electrophoresis of basicproteins and peptideson polyacryla- midegels. Nalure, 195,281-283. ScholzF., F. Bergmann.1984. Selective pressure by air pollutionas studiedby isoenzyme-gene-systemsin Nonruayspruce exposed to sulphurdioxide. Silvae Genetica, 33,238-245.

Table1. Soilconcentrations (pg/g dryweight) of heavymetals in set S andset K

Set Metal Pb Zn Cu Co Cd Ni

a 34 58 194 t5 1.2 31 K 15 40 19 3.4 0.8 12

Table 2. Heavymetal congentrations (pg/g dry weight) in set S andset K of D. ischaemum

Set Metal Pb Zn Cu Co cd Ni

11.2 58 75 11 0.3 31 K 4.2 zo 5.3 1.8 0.0 7.8

la RegardingGOT, PERand ACPHsimilar results were obtained-no significant differences between B 1, B 2,B3andPOwerefound. Summarizingthe results,it wasevident isoenzyme structure of Boalinpopulation is stableas indicatedby the insignificantdifferences between B 1, B 2 and B 3. On the otherhand, the isoenzymestructure of Boatin populationdid notdiffer statistically from the averageisoenzyme structure of F. nigrescensfound for its Bul- garianpopulations. lt is worthmentioning that BoatinNational Reserve is comparativelyclose (15 km) to coppersmelter of Srednogorie.As it was shown,populations of Dichantiumischaemum and Chrysopogon grylluslocalized close to Srednogoriesmelterwere influenced by industrialpollution. Despite its closenessto Srednogoriesmelter, Boatin population proved to be stableand its isoenzymestructure did not differfrom the averageisoenzyme structure of F. nigrescens.The resultsof this studyshowed that the isoenzyme markerscan be usedeffectively as an additional-approachforthe controlof the environmentin the systemof backgroundmonitoring.

REFERENCES

CoughtreyP., H. Martin.1978. Tolerance of Holcuslanatus to lead,zinc and cadmium in factorialcombina- tion.New Phytol., 81,147-154. DavisD. 1964.Disc electrophoresis. 2. Method and application to humanserum proteins. - Ann.N. Y. Acod. Sci.,121 ,404-427. GeburekTh., F. Scholz,W. Knabe,A. Vornweg.1987. Genetic studies by isoenzymegene loci on tolerance andsensivity in an air pollutedPinus sylvestris field trial. Silvae Genetica, 36, 49-58. HoganG., W. Rauser.1979. Tolerance and toxicity of cobalt,copper, nickel and zinc in clonesof Agrostis gigantea.New Phytol., 83, 655-663. HollanderM., D. Wolfe.1973.Nonparametric statistical methods. John Willey. New York. JagerH., H. Klein.1981. Biochemical and physiological effects of SO2 on plants.Angew. Botanik, 54,337- 346. MalhotraS., G. Hocking.1976. Biochemical and cytologicaleffects of sulphurdioxide on plantmetabolism. NewPhytol., 76, 227 -237 . MartinM., P. Coughtrey.1982. Biological monitoring of heavymetal pollution land and air.Applied Science Publishers.London, New York. '1985. Muller-Stark,G. Geneticdifferencesbetween "tolerant" and "sensitive"beeches (Fagus sylvatica L.) in environmentallystressed adult forest stand. Silvae Genetica, 34,242-251. '1989. Prus-GlowackiW., R. Nowak-Bzowy. Demographicprocesses in Pinussylvestris populations from regionsunder strong and weak anthropogenous pressure. Silvae Genetica, 38, 55-62. ReisfeldR., U. Lewis,D. Williams.1962.Disc electrophoresis of basicproteins and peptideson polyacryla- midegels. Nature, 195,281-283. ScholzF., F. Bergmann.1984. Selective pressure by air pollutionas studiedby isoenzyme-gene-systemsin Norwayspruce exposed to sulphurdioxide. Silvae Genetica, 33,238-245.

Table1. Soilconcentrations (;rg/g dry weight) of heavymetals in set S andset K

Set Metal Pb Zn Cu Co Cd Ni

S 34 58 194 IJ 1.2 31 K 1q 40 19 3.4 0.8 12

Table2. Heavymelal concentrations (pg/g dry weight) in set S andset K of D. ischaemum

Set Metal Pb Zn Cu Co Cd Ni

Q 11.2 58 11 0.3 31 K 4.2 zo q? 1.8 0.0 7.8

+1 A oo able 3. lsoformfrequencies of cathodalPER in set S and set K lsoform Species Sel 10 12 15 19 21 23 27 33 35 D. ischaemum a 0.51 0.86 0.44 0.00 0.26 0.00 1.00 1.00 1.00 0.14 K 0.71 0.92 0.59 0.00 0.18 0.00 0.98 0.96 0.98 0.23 Ch. gryllus a 0.46 0.42 0.56 0.43 0.45 0.65 0.00 0.63 1.00 0.00 K 0.44 0.45 0.22 0.31 0.19 0.71 0.00 0.34 0.68 0.00

able 4. lsoformfrequencies of 6 PGDHin setS andset K of D. ischaemum lsoform Set 19 21 23 29 31 ?2 35 e 0.90 0.70 0.93 0.65 0.52 0.12 K 0.47 0.40 0.63 0.50 0.24 v.z3 0.12

Table5. lsoformfrequencies of GOT in setS andset K lsoform Soecies Set 6 10 12 14 to 18 20 31 JJ 35 39 41 45 D. ischaemum 0.00 0.15 0.00 0.69 0.00 0.49 0.00 0.00 0.10 0.97 0.76 0.54 0.11 K 0.00 0.03 0.03 v.oz 0.35 0.87 v.zv 0.00 0.00 1.00 0.69 0.98 0.12 Ch. gryllus b o.o2 0.13 0.26 1.00 U.J3 0.24 0.00 o.22 0.49 1.00 0.18 0.02 0.28 K 0.00 0.00 0.05 0.69 0.04 U.JO 0.14 0.06 0.12 0.9'l 0.26 0.15 0.04

able 6. lsoformfrequencies of MDHin setS andset K in D. ischaemum lsoform Sel 13 15 tt 18 27 JU 32 35 S 1.00 0.61 0.63 0.04 0.69 0.67 0.28 0.00 K 0.76 0.68 0.65 0.00 0.25 0.00 0.00 1.00

aDIeble 7/. lsoenz slructurA ofF nrorescens- a nodalesterase lsoform Population 14 16 18 20 zz 24 32 JC 3l 40 44 B1 0.04 0.30 0.48 non 1.00 0.10 1.00 1.00 1.00 1.00 1.00 B2 0.02 0.24 0.54 noA 1.00 0.08 1.00 1.00 1.00 1.00 1.00 B3 0.10 0.20 0.50 0.86 0.90 0.26 1.00 1.00 1.00 1.00 1.00 P6 0.08 0.26 0.58 1.00 1.00 0.12 1.00 1.00 1.00 1.00 1.00 CHANGESIN LIPIDAND SECONDARY PLANT METABOLISM ANDPOLLUTION BIOINDICATION S.Popov, K. Stefanov, V. Bankova, N. Handiieva, K. Seizova, l.Elenkav instituteof OrganicCnemistry with Centre of Phytochernistry,Bulgarian Acaciemy of Sciences G.Kirnenov,LPopova Dep.Plant Physiology, Faculty of BiologyUtttversiiy of Soiia A.Atanasov, D. Djilianov De MonfclrtUniversity, Int. institute Plant Science Research arrd Institute of GeneticEngineering, AgriculturalAcademy, Kostinbrod

In the lastyears it was snownthat the nretabolisrnof the livingorganisnrs strongly depends on the environmentalfactors,including pollution. The significant changes obtairied may be of adaptivevalue or maybe attributedlo stressinduced degradation processes. They dependon the typeof the polluiantand on itscon- centration.Because these changes are connected with the physiologicalchanges caused by the pollution, they can be usedas an imporlanibioindicator for it whichwill give not onlyinforrnation about the pollution (compositionand concentrationof pollutants)but alsofor the daniage,caused by it and for the adaptation rnechanismsof livingorganisms. For suchinvestigations could be useddifferent types of rnetabolites- proteins, lipids, secondary metabolites,etc. We concentratedour effortson lipidsand secondarymetabolites because inese two groupsof compoundsare investigatedin the Centreof Phytochemistry,Institute of OrganicChemistry. Bulgarian Academyof Sciences.They are of bigecological significance. Poiar lipids (glycolipids and phospholipids)are importantconstituents of the cellmembranes and the changesof theircomposition will changethe bilayer permeabilitybarrier between the externaland internalenvironment (1). Analogous is the roleof sterolsin cell membranes.Other secondary metabolites, as polyphenols,alkaloids, terpenoids, steroids, etc., have importantdefensive functions in plantsand the changesin theirmetabolism, activated or inhibitedfrom differentpollutants, are of bigimpofiance for the livingorganisms. Investigationsin thisfield started mainly in the lastdecade. Till nowthere is very lirniteddaia abotttthe changesin the plantmetabolism caused by the environmentalstress but it is evidentthat the changesob- tainedcan be connectedwith the typeand concentration of the pollutantsand for this reascnthese changes couldbe usedfor biomonitoringof the pollutton. Recentlywe investigatedthe changesin the lipidand sterol metabolism in plants.including algae. caused by leadions, water stress, salinity and one herbicide(glyphosate). Changes of the lipidmetabolism of one musselcaused by totalwater pollution, were alsoinvestigated. The changesof somedefensive secondary metabolitesin tobacco,treated with glyphosate, appeared to be significant. Mostof our experimentswere connected with the changesof lipidand sterolmetabolism in plants,incu- batedwith lead ions.lt is knownthat heavymetals, including lead, are not essentialfor plantgi-oMh, but affectplant physiology, mainly photosynthesis and water utilization. ln somecases very low leadconcentfa- tionsstimulate plant development, but in generalthey inhibit the groMhof plants,especially this of roots(2) We foundthat afteran incubalionof Zea mayswith leadacetate stomatal resislance increased, wnile the rateof photosynthesisand transpiration decreased (3) Sucheffects were shown earlier for otherplants We founda significantdecrease of the concentrationof monogalactosyldiacylglycerols (MGDG) in ihe rootsof Phaseo/usvulgaris (4), which contain more than 90% of the accumulatedlead lt is known(5) that, becauseof its hexagonal(2)-structure, a reduced level of MGDGrnay indicate a higheidegree of controlof ionic permeabilityin the cell membranesand thus may have adaptivevalue. Similar, but not identical changes,were obtained in the MGDGconcentrations o'(Z. mays(3). The obtaineddifferences can be dueto the differencesin the carbondioxide assimilation mechanisms in bothplants (C3 and C4 mechanisms).This explanationis in agreementalso with the obtaineddifferences in the concentrationsof totalphospholrpicls (PL)in bothplants after lead treatment - theyincrease in Z. maysand decrease in P. vulgarisleaves. It is knownthat individualPL havedrfferent effects on cellmembranes ('l). Fotthis reasonwe per{ormed investigationsnot onlyon totalPL, but alsoon individualphospholipids. In bothplants the changesap- pearedto be identical(unpublished results). After lead treatment the concentrationsof phosphatidylglycerols (PG)decreased and these of phosphatidylcholines(PC) increased. Phosphatidatelies at a branchpoint of the glycerolipidbiosynthesis, which provides three routes foi iur thermetabolism. One of themleads to acidicPL, including PG, while another produces precursors for ihe biosynthesisof the zwitterionicPL, suchas PC. Fromour resultsit is evidentthat lead iortsparlially iniiibii the first biogeneticpathway, which causeddecreased PG concentrations.PG and PC are btlayerlip;itis whichdecrease the permeabilityof cellmembranes. Probably the increasedpermeability of the cellrnem- branescaused by PG decreasewill decrease the adaptationof the plantstowards pollutron, rnakiitg tire cell morevulnerable. lt seemsthat in orderto compensateit the relativeconcentration of PC (he otherbilayer PL) increasedafter lead treatment.

.+t It is knownthat the treatmentof piantswith heat,yrnetai ions ieadsto decreasedoermeability of cell membranes.One possibleexplanation is thatheavy meicl:; take part in the creationof bondsbetween ceilu- losefibbers through protein bridges, which leads tc delrcasecpermeability of cellmembranes (6) Our resultsshow that therecan be anotherreason fcr" tire decreased permeability of ihe cellmembranes it is evidentthat aflerthe leadtreatrnent of the investigatedpiants the sum of the relativec.oncentrations of pG and PC increased,which will cause a decreaseof the cellmembrane permeabiiity and this can enhance the adaptationof plantsto increasedlead concentratir:ns. Besidesthe changesin the relativeconcentratrons of individual me rnbrane lipids we foLindii:;i the:-e are significantchanges in theirfatty acid (FA) composition caused by leadions. In general,the ielatiireconcr;n- trationsof saturatedfatty acids and these with shorler chains decreased, whi!e there is a big irir:i'easeof ihe mostunsaturated FA in rnvestigatedplants - linolenicacid (18:3). Evidently lead icns stinrulate ihe dehydrc- genationand elongation of FA. Analogousexperiments have been performed with tobacco plants, treaied with one of wiclei."liised herl-i- cides- glyphosate(N-phosphonomethyl-glycine). lt is very effective and acts as inhibitorcf pciy;pirr,,nolbic' synthesis,as well as thisof proteinsand nucleic acids. Nothing is knownfor eventualchanges in ihe lipio compositionafter a treatmentwith glyphosate. We foundthai rntwo tobaccocuitivates spraying with glyphc- satecaused significant changes in the membraneiipirl composition, There was an increaseof ihe roncerr trationsof saturatedFA and decreasein ihe concentrstionsof MGDG. Bothchanges cause a dcc;eas.ci the cell membranepermeability, which was obsenredearlier in plantstreated vrith lead Evidentiytnis ;s .l commonreaction of the plantsin harmfulenvironment - decreased permeability of the cell membranepre- ventsthe cellfrom pollutants.This was in agreementwith the obtainedby us increasein the stignrastero! concentrationand decreaseof this in sitostercl.These changes also leadto a decreaseof the cell membranepermeability (7). It is importantto developglyphosate tolerant piant forms. Their availabiliiy will make possible the appirca- tion of this total herbicidein a selectivemanner. A treatmentof tobaccocallus in the regenerationmedia affordedregenerates. Analogously to controlsMGDG concentration decreased, which means decreased cell membranepermeability, but the concentrationof saturatedFA alsodecreased and this increasesthe membranepermeability. The treatmentof regenerates+rith glyphosate causes an increaseof MGDG concenti^a- tionsand decreaseof theseof unsaturatedfatty acids. The firstwill causeincrease of the cell membrane permeability,while the secondwill decrease it. The influenceof the pollutionon invertebratesappeared to be similar.We investigatedthe lipidcomposition of the fresh-watermussel Pseudoanodonta complanafa collected from two locationsin Danuberiver, possessingdifferent concentrations of pollutants.In the sarnpiefrom the more pollutedregion there was a sharp increasein the phosphatidylethanolamine(PE) concentrations,while these of PC decreased.In- creasedconcentrations of PE willincrease the cellmembrane permeabiiity, just oppositcto PC effect.PE is a biosyntheticprecursoi' of PC and probablythe pollution(or someindividual compound) affects the PL me- iabolism,inhibiting its last stage - methylationof PE to PC.The increasedcell membrane permeability is not compatiblewith maintenanceof a permeabilitybarrier between external and internalcompartments and thesechanges can affectthe adaptationof the mussels.The increasecjpermeabiliiy couid be nnoreor less loweredby the observeddecreased unsaturation in almostall lipidgroups as the pollutionincreases. This may be the adaptivereaciion of the musselstowards pollution and clisruptionin the lipidmetabclism caused by it (8) We investigatedalso the petrol- inducedchanges in the lipidand sterol cornposition of threemicro algae (9).Contrary to the above-mentionedresults here we found smailchanges in the lipidcomposition, but there werechanges in sterolcomposition which couid stabilize cell membranes. Evidently, at leastone of the componentsof the cellmembrane, sterols or polarlipids, musi changein orderto stabilizemembrane functions at new environmentalconditions. In our experimentson some desiccation-tolerantplants (10), lipids and steroisshowed substantial changes at differentwater deficits The algae,Chondria tenuissima, inhabiting waters with different salinity, showed lipid differences, which alsocould be explainedwith the adaptationto cjifferentenvircnment ('11). Whilethere are relativelybig numberof publicationson lipidchanges caused by environmentaistress, includingpollution, there are very limiteci number of invesiigationson the changesof secondarymetabolites at same conditions.In the courseof the abovementioned investigations on the effeciof glyphosateon io- baccocultivars we investigatedthe changesin the concentrationsof the mostimportant secoildary metabolites(polyphenols and alkaloids)in the same plantsand regenerants.before and afterthe treatrnentwith glyphosate. After treatmentof the controlplants and their regenerantswith the herbiciderrre found substantiai changesin the concentrationsof the abovementioned secondary metabolites. As expected,the biosynthesi:; of the polyphenolswas stronglyinhibited - rouiinedisappeared and concentrationsof the main poiyphenoi chlorogenicacid, were reduced.Surprisingly there was a sharpincrease of the concentrationsof another secondarymetabolite - the alkaloidnicotine. Aikaioids, as weil as poiyphenols,have defensive functions in the plantsagainst bacteria, fungi, viruses, insects and other herbivcres and evidently the reducedconcentra- 50 tionof anyof them will be harrnfulfor the plant. Glyphosate inhibits the biosynthesis of aromaticcompounds, so the concentrationsof polyphenolsin the treatedplants will be reducedand this will reducetheir defense In orderto surviveplant must intensrfy the biosynthesisof otherdefensive compounds and the obtainedin- creaseof nicotineconcentrations in treatedwith glyphosate tobacco plants and regenerantscan compensate the reducedconcentrations of polyphenolicdefensive compounds and can be acceptedas an adaptivereac- tionof thepiant It nrustbe mentionedthat ihe changesin the lipid,polyphenol and alkaloid composition in oneof theto- baccocultivars, Zlalna Arda, are rnoresigrrificant than in the secondcultivar, Nevrokop A24, which is in ac- corcianr:eorith the biggerresistance of thesecond cultivartowards glyphosate (12). Experiments vlth the Danuberiver rnussel (8) showedunambiguously that the differencesbetween the lipidccmpcsition of musselscollecteo frorn polluted and relativelypure waters are significantand can be usedfor biomonitoringof the polltrtionModel experiments with plants,incubated with differentpollutants ;hov'redthat in all casesthe changesare substantialand can be usedfor biomonitoring.Differences in the tnetaboiicchanges obtained with differentpollutants afford the identificationof some individualpollutants. Tl-trsmethod is slowerand moreexpensive than routine methods for moniloringof the pollution,but it have the advantagethat it r:onnectsthe degreeand characterof the pollutionwith the importantphysiological frrnctionsof theliving organisms Alsc. these investigationsgive informationabout the adaptivereactions of the organismstowards the polluticnIn the nearfuture we shallnot be ableto controlpollution and probably it willbe of interestto finda \.,ayto lir,,ewith it andthe adaptivernechanisms of plantsand animals can helpus. Thereare many caseswhen identicalorganisms (plants or invertebrates),collected from differentloca- tions,show significant differences in theirchemical composition Some authors try to explainthem with the existenceof "chemoraces",but our investigationsshow that differences in the environmentcan be a reason for the metabolicdifferences obtained. This can be of importancefor the manufacturingof biologicallyim- portantproducts from natural sources. because their concentrations and compositionwill strongly depend on the environmentalfactors, including the pollution.

ACKNOWLEDGEMENT Theseinvestigations have been completed with the financialsupport of the NationalFoundation for Sci- entificResearch (Contract X-307).

REFERENCES G B Anselland S Spanner (1982) Phosphatrdylserrne,phosphatidylethanolamine and phosphattdylcholine,in "Phos- pholiptds"(eds J N Hawthornand G B Ansell),pp 34-36, ElsevierBiomed Press, Amsterdam K tVithuchelian,S Rant and K Palrwai(1988) Differential action of Cu and Cd on chlorophyllbiosynthesis and nrtrate reductaseactrvity rn Vigna sinensrs L.,lndtan J PlantPhysiol ,31, 169-173 K Stefanov,l Popova, E Kamburova,T Pancheva,G Kimenov,L Kulevaand S Popov(1993) Lipid and sterol changes n Zeantays causedby leadrcns, Phytochent ,33,47-51 K Stefanov,I Popova, B Nikolova-Darryanova,G Kimenov and S Popov(1992) Lipid and sterolchanges in Phaseolus vulgariscaused by leadions Pltytochent31 3745-3748 PQuiper(1984)in Structure,Functionand Metabolismof Plant Lipids(eds. PASiegenthalerandWEichenberger), 9, p 525,Elsevrer, Amsterdam 6 tr,lTepfer and J Taylor(1981) fhe tnteractionof drvalentcatrons with pecticsubstances and theirinfluence on acid inducedcell wall loosening, Can J Botany,59, 1522-1525 7 M G.Guye(1987), Chtlling and age relatedchanges in the freesterol composition of Phaseolusvulgaris L prrmary leaves.Plant Sci 53.209-213 3 K Stefanov,B Budevska,O lvanov,N Genov,St Andreev and S Popov(1993) Lipidand aminoacid changes rn the mussel Pseudoanodontacontplanala caused by water pollution, Cornp Brochem Physrol . 105C.39-42 3 G Petkov,S Furnadzie';aand S Popov(1992), Petrol-induced changes rn the lipidand sterolcomposition of three microalgae, Phytochem 31 1165-1166 10 K Stefanov.Yu Markovska,G Kimenovand S Popov(1992) Lipid and sterolchanges in leavesof Haberlearhodo- pensisand Ramondaserbrca at transitionfrom biosrsinto anabiosisand vice versa causedby waterstress, Phytochem. 31, 2309-2314 11 K Stefanov,K Seizova,l Elenkor,,L Kuleva,S Popovand St Dimitrova(1994) Lipidcomposition of the red alga Chondriatenuissima, inhabiting waters with different salinity. Botanica Marina,37, 445-447 12 V Bankova,N Handleva,D Dlilianov,ZVassrleva, R.Bachvarova, AAtanasov, K Stefanovand S Popov(1995), Polyphenoland alkaloidchanges in glyphosate-treatedtobacco regenerants selected for herbicidetolerance, Z.Natur-forsch50c 313-375

ll MEASURINGPOLLUTANTS IN PLANTS,A CHRONOLOGICALPERSPECTIVE IN ANALYTICALENVIRON MENTAL CHEMISTRY

Urs Kriihenbtihl Laboratoryfor Radioand Environmental Chemistry , University of Bern

The goal of the investigationI will talk about is to understandthe dynamicof the pollutionof the environ- mentsince the industrialisation.In Europe this will comprise a timespan of about150 years

GENERALASPECTS OF DATING The datingof archivescan be realisedby measuringartificial and man made radionuclides Possible nuclidesto datesamples of the lasttwo hundredyears include Be-7 produced by the cosmicradiation, Cs- 137 introducedinto the atmosphereby test seriesof atomicbombs, and by nuclidesof the naturaldecay seriessuch as Pb-210. The timespan accessible by the useof the individualradio nuclide is a functionof it'shalf-life decay time. This timespan comprises 1/10 to 10times the halfhalf-life of the nuclideunder consideration. So it is possibleto datewith Pb-210, Tr: = 22years,samples with an ageof 2to 200years. In additionto theselection of the propernuclide further conditions must be fulfilledfor properuse of this method.First a constantinput is needed,and second the concentrationof the nuclideof interestmust not be alteredby laterprocesses (e.9. weathering,biodegradation)

GENERALASPECTS OF ANALYSISOF SAMPLES FROM THE ENVIRONMENT For naturalsamples it is quiteoften difficultto obtain a representativealiquot of the materialtobe studied. It is notwise to produceresults by an analyticalprocedure with an excellentprecision and accuracywhen the investigatedmaterial is veryheterogeneous (e.9. analytical precision 2ok, variabtlity of the studiedmaterial t 70%). When we want to obtainthe informationregarding the pollutionby heavymetals of a forestwe will not succeedby the analysisof the needlesof an individualtree. This is not a functionof the employedanalytical methodbut of the factthat the materialwas not sufficient homooeneous or not representativeat all.

Pre-treatment Collectedsamples should be submittedimmediately to the selectedchemical procedure to avoidsecon- daryalteration (losses, contamination). lf this is notpossible then the bestpre-treatment has to be evaluated. For plantmaterial deep freezing, freeze drying or dryingare possiblepre-treatments. In caseof the investigationof the specificationthe samplesshould be conservedby freezedrying. The demandsupon the pre- treatmentsample material is alsoa functionof the elementsto be studied.When the concentrationof mer- curyshould be investigatedit is not possibleto drythe sampleswithout losses at a temperatureof 80'C. It is impodantto submitlarger amounts of rnaterialof a sampleto the investigationwhen the materialis so homogeneous(50 mg of a finepowder may be representativefor the sampleto be studied,but when the sarnematerial is coarsegrained may be 500mg is needed.) In mostcases solutions are submitted to the effectivestep of chemicalanalysis. The digestionby acidsis the methodused most for plantmaterial. Recently, the digestionin Teflonbombs in presenceof HNO_rand H:Ozby microwave heating is recommended.The sameresult can be obtainedin an opensystem, but the amountand the composition of thementioned solutions have to be adapted. In allcases the completeness of the digestion, losses and contamination have to be controlled.

Methods Amongimportant anthropogenic elements Pb, Cd, Zn, Hg areall accessibleby measurementswith elec- trothermalatomic absorption spectroscopy. For Hg oftenthe moresensitive hydride technique is mandatory. OnlyZn showsa concentrationrange accessible by the normalatomic absorption speclroscopy. The disadvantageof this techniqueis the factthat only one elementat the time can be measured(of coursethe resultingsolution from the digestionof the samplematerial can be submittedto a secondrun for a differentelement). A very convenientmulti-element technique represents the ICP-AESmethod (inductive coupled plasma atomicemission spectroscopy). The concentrationsfound in naturaluncontaminated plant materials are oftenaround the detectionlimits of thistechnique. These limits can be effectivelylowered (by abouta factor of ten)by the employmentof an ultrasonicnebulizer. Mercury is in mostcases not detectable by thismethod. Forthe investigationby neutronactivation analysis the radiolyticdecomposition of the materialhas to be observedIn manycases the sensitivityof the instrumentalneutron activation analysis is sufficient.When this is notthe casethe chemicalseparation of the elementsof interestwill lower the detectionlimit cruciallv The

52 labourinvolved in the chemicalseoaration will Imit the nilit-ri]elor r,iertrentsand sarnirieswhicn can be han- dledoer unit of time.

INVESTIGATEDARCHIVES The investigationof environmentalarcirives have i;i;ii'rcii iiigir .:igitifii:arce iir ihe iast years !ce ccres, lakesediments of coralscan be consideredas classrcreseivcriis uf the prast.lin"iate Crnbrotrophic peat bogs werefirst investigated in Canadaand Scarrdina,,iiaarrd wete lorinrl 1,) reljreserit good reseir,rcirsto reconsti- tutethe anthropogenicinput of heavymetals Later,the rnot,rlityof th.:deposited ccntaminates was investi- gatedas a functionof the prevailingconditions witlrin the peat, It was realisedthat withinin an ombrotrophicpeat L,og hcrlloirvs an(1 hurrrrrocks behave differently since theirgroMh factors are notidentical An importantlink is the relationbetween the measirreddeposited arnount ot poilutantsand the measured concentrationinthe neighbouringatmosphere. For this it is also of inrerestto includeother biornr.'nitors such as lichensand mossesto comparetheir fixatiorrof contarninanlsand to studytheir integration fac-.tor over trme How iongwill it take untillichens or mossesshowthe latest concentration level of the pollutinghe'avy rnetais iir tlie air? ln our investigationwe collectedone speciesof lichens:ilypogymnia physodes. In the case of mosses Hylocomiumsplendens and Hypnum cupressiforme \^ref e collected At eachsampling site the imissionsituation is monitoredby measuringthe ciepositionuiith the Bergerhofl method In additionthe dust is collectedwith total filters and the size distributioncf aerosoisare collected withlow volume cascade Berner lmoactors.

ANALYSISOF PEATBOGS .100 With a mechanicaland a handdrill peat cores of 50 to cm lengthand 5 to I cm diameterwereccl- lected.To avoidalteration of the samplesduring transpon to our laboratorythe sampleswere frozen in the fielddirectly afterthe collection. For dissecting the coresthey urere cut by an electi'icsavr in the frozenstate Freezedrying was the nexlstep, follow by grindingin a ballrnrll. Afterwardsthe activityof the 4% gammaline at 46 KeVof Pb-210was measuredon an aliquoiof aboul2 g. Forthe chemicalanalysis 300 - 400 mg weredigested in HNO:and H2O2in a Teflontube by rnicrov/ave excitation.The concentrationsof the elementsof interestin the resuliinosoluiions were deterrnined either bv ICP-AESor by graphitetube atomic absorption spectroscopy.

Results The measurementof tne gammaactivity of Pb-2'10gave at the sameiime the activityof es-'i:i7 in the samplewhich grew in the season1986 the activityof Cs-137,deposited from the Chei"rrobyiaccideiit, ivas found.This indicates that the few percentof soliddust in lhe samplecontains sufficient ciay rninelals io im- mobilisecompletely the Cs-137. Evaluatingthe resultsof the datingit wasrealised, that the growihrate for hummocksanc huilo'lrsciffei-s by abouta factorof two (inmost locations). From this finding tne questionsarises for iir:wiong sircri a structure is stable.At thistime it is not understoodif the elevationabove see level infiuences tfrrs beliavioiii' Thedeposition of anthropogenicpollutants has not the identicalsignature for the 3 investigaiedelenicnt: It is obviousthat Pb Zn and Cd havenot the samesource lt is quitef.rositive to note thatin the latestsam- ples(from the last10 years) the measured concentrations have declining tendericy. This is a goodindicaticn aboutthe effectivenessof the emissionreduction measures rn the yearssirrce iiie late 1960ties(stacK-gas cleaningin industriesand waste burning installations aswell as the introductiorr oi urileaoed gasolirre;, A distinctdifference in the measuredconcentrations is obserued for samplesof differentorigrn depv;iurr-tg of the regionaland local situation regarding pollution. At higherelevations in theAlps the levelof antirrupr;- geniccontamination is generallysmaller The variation in depositionof the threeelemenis at the investrgaterJ sitesis a factorof 2for Zn andPb anda factorof 3 in the caseof Cd

MOSSESAND LICHENS Mossesand lichensare suitablebiomonitors for pollutioncontrol bel.ause iirey oittai;", i'tissi ol riicn;ruIri ent supplydirectly from atmospheric deposition. Lichensbeing perennial and having no developedcuticie are weii si:iteci as ilitegraioisftir uepositru,r-,,1 elementsoriginating of naturaland man-rnade sources At siteswiih highantliropogenic ioao of p;i;ll,r(arrts the distributionof lichensis quitelimited. Therefore. it is rrrsorne piaces quite difficLrli to collecis.riti-icrit materialfor the chemicalarialysis On the other'sideit is evidentto oe caiefulio collectiircilefrai lvhic-h r> representativefor a givensite. lt is derrorrstratedthat not well chosen inoividual sarnplt:s iiia'l va(y -irlse to a factorof two (selectionof 10 samplesand average).The lrchensare collectedfrorn ciifiercrit ritrc5 and fruiri all sidesaround the selectedspruce by crrttirrgsrlall branches.The tfeesselected grew rn lrie open in an areaof about50 x 50 m. In the laboratorylhe rnaterialwas carefuliy sorted so any adir.riirgrrtatcrial (c g barkand pineneedles) was removedfr c.'ni the licirens i , The mosssamples were selected from an openarea of about50 x 50 m. Inthe laboratorythe threeupper segmentsof Hylocomiurnsplendens representing the lastthree growittg seasons were cut andselected. The materialfrom the fieldexcursion were stored in a deepfreezer. After the sortingthe materiaiwas freeze-driedand thenground in a ballmill. An aliquotof about250 mg was digestedfor the chemicalanalysis.The resultingsolution was measuredby ICP-AEStechnique.

Results For low pollutionsampling sites in the BerneseOberland lichens show 3 to 4 timeshigher Zn concentra- tionsthan moss samplesfrom the same region.The correspondingratios for Pb are even higher:they amountto a factor4 to 6. For Cd the correspondingconcentration ratios are between2 and 3. Mossand lichensamples from ruralsites show completely different accumulation behaviour compared to urbanpol- lutedareas. There, the ratioof concentrationsof the measuredelements in lichensover rnossesapproach the valueof 1. A similarfinding was reportedsome yearsago in a Swedtshpublication by Foikeson.At a load of 100 ptg/gfor Zn in mosssamples the lichento mossratio is aboulone. For lead a ratioof one is reachedat a concentrationof 30 1tg/gin mosssamples. For Cd the ratioreaches unity'of aboui 0.5 rlgigin mosssamples. Onlywith the aid of calibrationmeasurements of thistype and the necessarycalibration factors is it pcs- sible to deduce imissionsituations at a samplingsite from the measuredconcentrations in lichensor mosses.

EVALUATIONOF IMISSIONS Forthe evaluationof the imissionsituation 3 kindsof equipmentwere employed. The Bergerhoffcollector accumulates wet and dry depcsition.This is e very inexpensivepassive sampling device.The two othersampler, active devices, need the useof pumps,which makes it moredemanding for installationsin the field: A totalfilter collects dust and aerosolsiarger than 0.45 pLm diameter. The low pres- surecascade impactor is calibratedfor an airflux of 30 | per minute.The 8 collectorplates sample aerosols with mean aerometricdiameter of 0.1;tmto 6 pm (witha factorof 2 largerdiameter from one plateto the other).

Results The concentralionsof a givenelement collected in a passiveor activesampling device varies with the seasonsvery significantly. The resultsof the totaldust collector changes by morethan a factorof 5. These resultsare of courseinfluenced by the weatherconditions prevailing in the samplingperiod (a lot of rainor a dry period,changing main wind direction). The resultsfor the aerosolcollector gives for the 3 elementsZn, Cd, and Pb a differentsize distribution. This rnay indicate that the 3 elementshave not the identicalsource. The maximumloacj is foundin mostcases in the fractionof 1 to 2 pm. The measuredconcentration varies by morethan a factorof 3 for the two sitesBurgmoos and Jaun. The Bergerhoffsampler, the totaldust col- lectorand the Bernerimpactor indicate a similartrend. Generally, the measuredconcentrations in the active samplersshow the samedistribution pattern as the biomonitorsconsidering the differentintegration behaviour.

SUMMARY Resultsof chemicalanalysis of plantmaterial allow to gaininformation on the air qualiiyduring the growing time of the investigatedsystem. Lichens and mossesare ideallysuited to monitorlocal and regional variationsof the depositionof pollutants.The studyof peatbogs can be usedto elaborateinformation about the dynamicof the anthropogenicpollution since industrialisation. For the reconstructionof historicair qualitydata it is importantto establishcalibration factors for the transferfunction of any pollutionin to lhe studiedbiomonitor. For this it is importantto measurethe imission situationat the investigatedsite by activesamplers. It wasdemonstrated, that each biomonitor shows an individualfixation behaviourfor polluting compounds. In furtherstudies results may be expectedwhich will allgwto statein whichform (specification)the meas- ureddeposited heavy metals can be found.

5l EDXRFANALYSIS AND ITSVERSATILE APPLICABILITY TO ENVIRONMENTALSAMPLES Emilia.Nikolova, Ari Artinian lnstituteof nuclearresearch and nuclear energy, BAS,

I. INTRODUCTION T-heapplication of mocierninstruniental inetiiorjs to tfiedeiennination of traceelements in environmental sampieshas promotedthe betterundersianding of ihc roleof ihis elernents.The acquaintingwith the concentrationsof heavymetals and toxicelements gives us the opportunityfor investigationof the distribution and dynamicsof chernicalelements accumulation in floraand fauna and their passingover the trophic chainsand alsofor evaluationof antropogenicinfluenc;e of toxicelements and technogenic transportation on the ecosystems. The energydispersive X-ray fluorescence analysis is one of the wellknown analytical methods as a pow- erfulanalytical tool for the qualitativeand quantitativedetermination of almostall chemicalelements in a sample,first of all as a rapidand reliablemethod.

II.PRINCIPLES AND INSTRUMENTATION In the EDXRFmethod, the sample,lo be analysedis excitedwith electromagneticradiation of sufficient energy.This leadsto the ejectionof electronsfrom the innershells of the atomspresent. Each vacancy is immediatelyfilled up by an electronfrom an oulershell while the differencein energybetween the two levels is generallyreleased in the formof an X-ray.The energyof the emittedX-ray is characteristicfor the ionized element, and fromthe intensitiesof the differentcharacteristic X-rays in the spectrum,the concentrationof the elementsin the samplecan be deduced.X- ray fluorescencerequires the sourceof radiation,the sample,which is irradiatedand the detectionsystem. With these the spectrometeris completeand through com- putersupport all necessarycalculations to transferthe intensitiesinto concentrationsand to identifythe elementscan be carriedout fromthe measuredspectrum. The powerof thisanalytical instrument is dueto the energydispersive detector, the simultaneousdeter- minationof all elements,Information is obtainedrapidly and no element,which is presentcan be overlooked. For standardexcitation the lowerdetection limits are in the rangepg/g (ppm). This is sufficientfor manyap- plications,but for some environmentalproblems these detection levels are too high.Recent developments forsolvingthis problems are the applicationof totalreflection [1] and the introductionof synchrotronradiation sourcesfor excitation[2,3].

III.SAMPLE AND SAMPLEPREPARATION The bestprocedure for samplepreparation is to leavethe sampleas it is, but in manycases this is unre- alistic.Any processto transferthe sampleon a substrateor preparationneeds safe treatment so as to guar- anteethat the processedsample is representativeof the original.Homogeneity of the sampleand contami- nationfree samplepreparation are the basicrequiremenls in eachtechnique. In particular,environmental samples,which we investigatein our lab are soils,sediments, leaves, needles, grass, moss, lichen, fishes, mousses.In orderto minimizethe errorfrom sample preparation we use only a dryingand grindingproce- dure.An amountof about3009 from the sampleis driedin microwaveoven for 10 minutes.Then this sample 'l0g is ground to a meshside about 0.1 mm andhomogenized. An amountof fromthe plantsand biological samplesand 209from soils and sediments are pressedin a specialisedsample carrier. Particular difficulties occurs,when very small amount from the sampleare available, as in the caseof internaltissue of fishesand mousses.ln this case an approximationcurye intensity versus sample mass (fig.1)is establishedfor nor- malizationof obtainedcharacteristic X-ray intensities.

IV. INSTRUMENTATION Two spectrometricsystems are availablein our lab for X-rayanalyses. The firstone (fig.2)is equipped with Si(Li) detectorwith25 ;"rmBe windowand 180eV energyresolution at 5.9 keV Mn-K,.line.The data are acquiredwith a multichannelanalyzer, interfaced to a personalcomputer (AT486/33MHz), that applies specializedsoftware,for speclra processing. This system is combinedwith an excitinghead, based on an annularsource Am" with activity3.7 GBq and threetypes of secondarytargets in two excitationmode - transmissionand reflectionin orderto selectthe desiredexciting energy - namelythe characteristicline of Mo (17.441ke\i), Sn (25.19keV) and Dy (45.985ke\l) [4].For determinationof heavyelements, which are essentialfor biologythe systemuse the Dy secondarytarget, that allows detection of elementsdown to the Z=50"This construciionpermits the measuremenlof greatnumber of elementswith a high sensitivityby achievingthe desirableexperimental conditions of lowbackground level and at a highcount rate. The secondsystem is equippedspecially for lightelement analysis with Si(Li)detector with 12.5prm Be windowand 160eV energyresolution for 5.9keV Mn-K,.line. An excitinghead, especially designed for lowZ elementsdeiermination (n9.3) is equippedwith three radionuclide sources Fe"' . In the caseof thisconstruc- 55 tion, the distancessource-sample and sample-detectorare greatlyreduced and optimized,which allows elementswith atomic number Z=13 lo 25 to be analyzed Duringthe measurementsof zoologicalsamples, traces from very essential for biologyelements like Cu, Zn, Mn, Pb wereoccurred. To becomehigher sensitivity and lower detection limit forthese elements another excitingsource Pu"" is builtin the samehead (fig. 3). lt'senergy of 13.6'13keV is verysuitable for excitingof elementswithZ=22 to 35. In thisway the all rangeof Zfrom 13lo 82 is coveredand eachsample can be measuredwith this three kind of excitation.

V. SPECTRUMPROCESSING To obtainresults the measuredspectra must be evaluatedas to line energiesand line intensities,with only the net intensityused for quantification.The correclnesswith whichthe peak areasfor the analyzed elementsare determinedis of crucialimportance in orderto obtainaccurate and reliableresults for their amounts.Different methods for energydispersiveX-ray spectra processing have been investigated [5 6] and now in softwarepackage three different methods for peakarea determination are included- narnelytotal peakarea, linear and non-linear least square algorithms, resolution enhancement procedures.

VI . CAL]BRATION An internationalstandard from lake sediment IAEA SL-1 and artificialstandard with about100 ppm concentralionsof the elementsCl, K, Fe, Se, Y, Mo, Cd addedto lightmatrix are usedto obtainthe sensitivity curvesfor Am(Dy)and for Pu excitation(fi9.4,5). A Belgianstandard FCG-15 is appliedfor verificationof thesecurves. The obtainedresults are in goodagreement with the referenceconcentrations forthe standard. As an examplesfig.6 show spectra from the internationalstandard SL-1 and from a multielementalartificial standardrespectively Forthe excitinghead with Fe" sourcesa setfrom geological standards and artificialstandards with S, Cl, Al areused to obtainthe sensitivitycurve, valid incoherent scattered radiation is included. Oncethe correlationbetween intensity and concentrations is found, comparable sample types, measured undersame conditiorrs can be quantified.At tracelevels the XRFtechniques provides a directproportionality betweenanalyte line net intensityand its concentration[7]. This holdsfor homogenoussample of a given matrixand eitherof constantor infinitethickness, i.e. of constantmass. In thisway, when the net intensities of theanalytical elements are found the unknown elemental concentrations can be calculated. As an examplethe obtainedconcentrations is shownin table 1. Typicalspectra from soil, plantsand zoologicalsamples are shown on figures7-9.

VII. ACCURACY,ERRORS AND DETECTIONLIMITS The totalerror of the analysisconsists of manyerrors, which can be provocateby: measurementof the tniensity,reproducibility of the samplepreparation and conversionof intensityinto concentration. The total relativeerror of a 7 - 10%is estimatedduring the calibrationprocedure as a standarddeviation error of a previouslyprepared and analyzed standards with known elemenl concentrations. The detectionlimits (DL)(the minimum amount, that can be analyzed)are determinedby the sensitivity and by the backgroundin the X-rayspectrum. The useof threekinds of excitationgive us the oppoflunityto reachdetection limit levels, which are enoughfor environmentalcontrol and monitoringpurposes. With a newtechniqueof totalreflection XRF a drasticallydecrease of thisvalues is possible[8].

VIII.CONCLUSIONS EDXRFis capableof detectingtraces of almostall elementsof the periodicsystem. The methodenables fast, non-destructivedirect analyses without lengthy sample pre-treatment and is well suitedfor environ- mentalinvestigations because of its sensitivityand lowerdetection limits achieved and alsobecause of the rapidand simultaneousacquisition of informationon the elementspresent. The softwareavailable permits quantitativedetermination of traceelements within a shorltime once the calibrationfunction for the systemis established.The optimizationof excitationparameters leads to the versatileapplications in environment monitoring

REFERENCES 1. WobrauschekP., Aiginger H., "Total reflection X-ray fluorescence spectrometric determinationof elements in nanogramamounts", Anal. Chem. 47, No.6, 1975, p.852 2. lidaA., SakuraiK.. YoshinagaA., GoshiY., "SR excitedXRF analysis,using total reflectionof X-rays", Nucl.lnstrum.Methods,A246, 1986, p 736 3. PellaP.A., Dobbyn R.C., "Total reflection energy dispersive XRF spectrometryusing monochromatic SR. applicationto Se in bloodserum", Anal. Chem. 60, 1988, p.684 4. ArtinianA.A., Stoev K.N., Vutchkov M.K., "A modelfor secondary transmission target Thickness optimrza- tionfor the purposes of EDXRFA",Bulg.J. of Physics,V16, No.2, 1989, p.201 56 StoevK.N., Vutchkov M.K., Nikolova E.L "Metrologrcaltesr c.rf cliffererri iirctriurjs i;l e;iei'g1 :risij€;:ri\.id )i rayspectra processing", Bulg.J. of Physics.V17, No5 1990.p 419 StoevK.N., Vutchkov M K, NikolovaE.L, "Exarninationof different non lrneai airg.,;r"ithins fc;'X-i;t siteciin processingwith personal computers" Bulg.J of PhysicsV17 No5 1990 p 4ul Plesh R and Thiele8., "Fundamentalsof the SrernensuorrrpLrter prograrns fcir X-ia;, jpeltfosi,opy", SiemenbAnalytical Application, Note #196 8. WobrauschekP., StrerliC.. GeorglR. LadisicfrW. "Totalleilectittn X.ray fli,iore.icei-ri-c anair.;is aiiij ii: versatileapplicabilityto environmeirtal samples". Proceeding of on Interrratiurral s),,firf'ro.)iuiri on "Appir- cationof isotopesand radiationconservation of the environnrent",Karlsruhe g-t: fu{aiin1gg2

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TRANSITIONTARGET MODE

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58 I

SENSITIVITYCURVE AM(DY) EXCITATION

Rank 19 Eqn 2050 Y=a+bx+8*dix 12=o996910052 DF Ad140993820104 FrtstdEr=o 134124145 Fstat=537 715701 a=.3 1091 1 69 b=0 54828803 c='0 00906'10267d=-5 3270831

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Figure 4

Sensitivitycurve Pu excitation

Rank'16 Eqn 6001 y=6+[v+6x2+dx3+ex4 t2=0.991467479DF Adjr2=0 990426928 FrtStdErF0.20048293 Fstat=1220 08592 a=-24645579 b=12.57 1351 c=-2. 1520529 d=0.1 6790754 e=-0.0048423733

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Figure5

59 250C a,l I I Ft rM,f, 2U0i - 80i. ll Multistandard StandardSL-l Source f,ry' SourceAm(Dy) Am(D1) - | f#Hv rt \ Meas.time3000s Ileas.time3000s I - i r5oo 60c I I [.

400", $ : IUULJ ,l \ 2AA Zn \t,r s" ' i,;'f,, \, 2,, t l, t nr' -. i 1n-\ ,t\ i \';sr '/.;'\-, \ ,..*'l '-"/**--,.-*,-* ,J \.r/-{.^r rus". ,4-'.- -./ .*

0t 50 lci 150 200 25c 5.0 10.0 15.0 20.0 25.0 Encr*\1Ke\') Energ] (ke\')

Figure6

TABLENo.1 Elementsconcentration in some biologicalsample from Rila Mountain Monitoringprogramme OM2, June- September1994

ELEMENT Unit Number (code) of the sample 1(z2l 2 (Z 121 3 (z 16) 4 (Z6l 5 (z 2e) AI ("/") 2.1 610.30 ei (%) 0.5r0.04 0.3610.05 P (ppm) 9137t731 6175t494 4292!327 78681624 6028!482 Q (ppm) 20461!1637 6997r560 4665r373 17465t13488828r706 CI (ppm) 59001600 zo tolzov 2690t270 3886r395 35891400 K (%) 1.5i0.13 1.45!0.12 0.73r0.051.3310.10 1.3310.1 0 Ca ("/") 2.25!0.15 0.07r0.010.06r0.01 1.8410.15 0.'12r0.0'l Ti (ppm) (ppm) Mn (ppm) 54!4 194x14 53i4 286116 Fe (ppm) 6514 1285!26 594!22 564141 2813!43

L,U (ppm) 377!19 10451206 41!6 580i105 Zn (ppm) 82!7 318!17 273!18 114r9 334!23 AS (ppm) 17!2 tzll Se (ppm) 10t1 3512 10r1 9r0 20!2 Br (ppm) 5514 5614 110i10 35!2 91rB Rb (ppm) 20t2 67r5 6115 104rg Sr (ppm) 6315 42!3 Cd (ppm) Pb (ppm) 811 I J=Z

(r() 2500 ;l

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Figure7

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00 50 100 150 20c 25C 00 5A 100 i5 I:rrclqi (kr:\'i Iint'rg{kc1 1

Figure9 RADIOACTIVECONTAMINATION OF THE BIOSPHERE Milen lovchev Institutefor NuclearResearch and Nuclear Energy, BAS

I. INTRODUCTION

The lifeon the Earthbegan and is evolvingunder the conditionsof continuousimpact of a natural radiationbackground which, during the whole Earthhistory and almosttill nowadays,has beenchar- acterisedby a constant,slightly changing value. Due to that and also becauseof the fact that the naturalradiation background mainly consists of a penetratingradiation which exerts an impacton all organsand iissues,living organisms do not possessspecial sense organs which would respondto ionizingradiation in a certainmanner. There are no appropriateadjusting mechanisms as well throughwhich our bodycould get adjustedto considerablechanges in the radiationlevel. Livingorganisms are exposed to externalirradiation mainly of cosmic,electromagnetic and neutron radiationand to internalirradiation caused by radionuclidesincorporated during respiration and with waterand food. All typesof ionizingradiation regardless of theirorigin - naturalor artificial,cause the same primary physicalprocesses of ionizationand excitationof atomsand moleculesin the irradiatedmaterial. The followingchemical and biochemicaleffects can entail biological impact, changes and injuries.So the radiotoxicityof the bulkof the radionuclidesexceeds many times the chemicaltoxicity of the corre- spondingstable isotopes.

2. RADIOACTIVITY!N THE BIOSPHERE

2.1.Natural radiation sources Morethan 60 radionuclidesare knownto occurnaturally in the environment.These are classified intotwo groupsaccording to theirorigin: cosmogenic and terrigenous. 2.1.1.Cosmogenic radionuclides The Earthis continuouslyexposed to ionizingradiation of cosmicorigin. The primarycosmic radiation consistsof lightatomic nuclei and high-energyprotons originating from the outerspace. The in- teractionof thoseparticles with gaseous atoms in the atmosphereleads to the continualproduction of at least'14radionuclides in the earth'senvelope. The cosmogenicradionuclides include tritium (H-3) and carbon-14(C-14) as theirprincipal members. 2.1.2. Terrigenous radionuclides The terrigenousradionuclides include long-lived primordial nuclides co-existing with their stable elemenlcounterparts which enter into the earth'scrust from its inception.The principalmembers in the abovecategory are K-40and Rb-87. Anothergroup of the primordialterrigenous radionuclides includes three actinideparent nuclides {Tn-232,U-235, U-238) and theirdescendant products which consistof about35 radioisotopesof Pb, Bi,Po, Rn, Ra, Ac, Th, Pa andU.

2.2. Radiationcontamination in terms of civilizationfactors (some aspects) A. Naturalradionuclides Miningand reprocessingof radioactiveraw materials(local contamination): nuclides of radioactive families; Powergeneration (TPP on coal and lignite):Ra-226, Rn-222, Po-210, Pb-210; Miningand reprocessingof phosphateminerals (phosphate fertilizers, construction materials): U- 238 anddaughter products; Construction(construction materials): Rn-222, Rn-220 and daughterproducts. B. Artificialradionuclides Severalhundreds of artificialradionuclides are releasedintentionally or unintentionallyin the result of nuclearweapon tests, as well as in the resultof the operationof researchand nucleartechnical facilities(NPPs, accelerators, plants for fuel reprocessing,RW repositoriesetc.) and accidents.The artificialradionuclides which are found in the environmentare classified into four categories depending mainlyon theirmode of production: - lightnuclides (H-3, C-14) - fissionproducts (Cs-137, Ce-144, Ru-106, l-131, Sr-90 etc) - transuraniumnuclides (Am-241, Np-237, Pu-238, Pu-239, Pu-240 etc. To predict an effectivedose equivalentfor the world population21 isotopesaccording to their ra- diotoxicityand theirrelative contribution tothetotaldose are usuallyused. The mostimportant of them are:l-131, Cs-137, Sr-90, Pu-239, Zr-95, Ru-106, C-14, H-3.

|)1. 3. RADIONUCLIDESIN THE BIOSPHERE

3.1.Radioactivity in the atmosphere Naturaland artificialradioactivity in the atmosphereis causedby radioactivegases and aerosols. The globaland local fallout, depending on the conditionsof explosionsduring nuclear tests, has led to the contaminationof large areas.The radioactivitylevel is affectedby seasonsand geographical changes. In the vicinityof nuclearfacilities the artificialradioactivity of the atmosphereis mandatoryand regularlymonitored.

3.2.Radioactivity in the hydrosphere Water radioactivitydepends on its originand chemicalcomposition. The enrichmentof naturalra- dionuclidesis conditionedby emanationprocesses, by leachingand dissolutionof mineralsub- stances.Artificial radionuclides get intothe hydrospherethrough radioactive fallout and disposedra- dioactiveliquid waste.

3.3.Radioactivity of soils The naturalsoil radioactivityis mainlydue to K-40,uranium, radium,thorium, and Rb-87.The contentof certainradionuclides varies within a very wide range and dependson many factors(soil formingrocks, chemical properties, soil compositionand structure,vegetation cover etc.).The radionuclides'behaviour, their migration in soil,their sorption and desorption,their transfer to plantsare conditionedby thesefactors. When the biosphereis contaminatedwith artificialradioactive substances, the soil plbysthe role of a powerfulnatural absorber thus forminga permanentsource of radionuclides.

3.4. Radioactivityin plants Radionuclidesuptaken by all organsof the overgroundparts and root systemfrom the soil, soil formingrocks, from the water and the air are accumulatedby the plantand get througha food chain intoan animalor humanbody. There is a certainlink betweenthe levelof the accumulatedradioactivity and the plantlocation in the phytogeneticsystem. According to the elevationof the naturalradioactivity the followingdependence can be built: Superiorangiospermae

4. MEASUREMENTOF THE ENVIRONMENTALRADIOACTIVITY

4.1. Trace analysis of radionuclidesin environmentalsamples includesin general the foliowing steps(C - chemical,P - physical,M - mathematicalcharacter):

1. Samplingand samples storage c 2. Samplespreparation c 3. Ashing 4. Ashfusion 5. Dissolvingof residue 6. Concentration 7. Separation c 8. Preparationof a measuringagent c 9. Measurement:a) integral;b) spectral P 10. Dataprocessing M 11. Evaluationof results M

63 4.2.General analytical problems A. Determinationof radionuclidesin extremelylow concentrations,tn most casescarrier-free, in samplesof differenttype, consrstency, chemical cornpusition etc. Necessity large amounts of materlal to be treated.Problems with the representativityof the analysedsamples. B. Selectionof appropriaternethods of sampling,storage and preparationof sampldsfor niinimiz- ing as wellas coverageof alltypes of errors.Obtaining of radioactiveequilibrium between genetically linkedradionuclides. C. Detectionlimit depends both on concentrationand decay constant of a correspondingnuclide.

4.3.Analytical methods A. Non-destructivephysical methods - spectfometrydosimetry etc., are appliedif possiblewhen traceanalysis is made; - theyare labour and time saving (cf 4.1 - items2-8 are dropped); - errorsare well covered.maximum elimination of errorsources could be obtained. The most irnportantcorrdition: the analyticalsignal should be well establishedwithin acceptable timeof measuring. B. Radiochemicalmethods (RCM) should be appliedonly when physicalmethods canttot produce undercertain conditions a satisfactorysolution of the analyticalproblem, namely: - when identifyingand detecting alpha- and beta-emitters; - when gamma-activitiesare very low (lowerthan detectablelevel when measureddirectly withinreasonably selected time): - with particularlycomplex mixtures even of gamma-emitterswith high activities,whert the resolutionof the detectorsused turns to be instrffrcient. The mainRCM advantagesare as follows: - sinrplifieddetermination of the chemicalyield for the wholeanalytical technique by usingra- dioindicatorsThis allows for partialapplication of fusion,enrichment and separation operations; - disturbancesof non-radioactivepollutants during the analysisare practicallyexcluded The mainRCM drsadvantages are as follows: - requirespecial laboratories and qualified staff; - labour-,time-, and cost-consuming, and are difficult to be automated.

5. RADIOECOLOGYIN THEINSTITUTE FOR NUCLEAR RESEARCH AND NUCLEARENERGY

5.1.Previous experience A. Laboratoryof RadiationDosimetry (LRD) - Since1961 environmental radiation monitoring of the IRT-2000Research Reactor in Sofia(refer- encepoint: Vitosha Mountain near Sofia) a) Totalalpha- and total betaactivity,Pu-(239+240),Sr-90 etc. ExampleLLD for Pu-(239+240) is 0 0037nrBq/g ash for 1009ashed soil sample 0.0185mBq/g ash for 209 ashed plant sample; b) Samplessoil, plants (annual , perennial,fruit), water (sewerage, cooling, waste, precipitatron): c) radiochemicallaboratory for the tfeatment of environmentalsamples, d) measuringequipment. LAS 34 low-ievel actlvity system (for beta-counttng), ORTECalpha-spectrometer, Si surface barrier detectors. - Thermoluminescencedosirnetry (TLD): conventional method, high performancelevel (participa- iionin 8 intercomparisons) a) gamma-raydetectors TL detectorsCaSO4 Dy (LRDproduction) cornbinedtracUTL detectors LR 1'15 Kodak Pathe detectors: b) Applicatron.sorl-gas (Rn) and daughter products, Th-230). - UV measurement(measurement of integralabsorbed energy, spectral sensitivity UV-B): a) calibratedto erytheme(skin) sensitivity; b) feasibilityto be ca[bratedto chlorophyllsensitivity to measurethe actualenergy absorbed by the plant. - lnternatiolalcollaboration of LRD: IAEA, JINR-Dubna, KfK Karlsruhe, OM2 jointFrench and Bul- garianProject (Ecology of RilaMountain) former CSSR, Hungary etc. - Participationin the Bulgarian-FrenchProject "MonitorinE and Managementof HighMountain Eco- systems"(OM2). Radioecologicalstate of the RilaMountain Natural Park (300 samples taken at the (r-l altitudes 1200 - 2925m) has been investigated.Data of the total beta-activityand gamma- spectrometryof rocks,soils, sediments, plants (annual and perennial),fish, amphibiansand small mammals(whole bodies and selectedorgans) has beenobtained [1-7]. The resultswell describe the contentof beta-emittersin essentiallinks of the ecosystemunder condilions of elevatedradiation backgroundin some points,which is mainlydue to the increasedconcentration of naturalradionuclides(human activities), as wellas artificialradionuclides because of the transboundaryatmospheric transfer.Soil-gas Rn-222 concentration was measured in the sameregions.

B. Radiochemistryand RadioecologyDepartment (RCRED): - padicipationin a numberof ecologicalprojects (NPP Kozloduy,closing down of uraniummining, expertassessment of agriculturalsoils etc.). a) high performancelevel of gamma-spectrometry(participation in a numberof interlaboratory comparisons)- natural and artificial radionuclides, b) radiochemicaldetermination of alpha-and beta-emitters. - measuringequipment. gamma-spectrometers(Ge- and Ge(Li) detectors) alpha-spectrometer.

5.2.Problems encounted: Comparativelylimited laboratory capacity (working places, measuring equipment) and thus limited numberof analyses.This imposes a carefulselection of samplesfor RC analysiswith regard to their relevancyand in viewof preliminaryactivity measurement results Financialdifficulties in procurementof: radioactivetracers and standards. selectivereagents and chemicals

REFERENCES

1. M.lovtchev,l. Mishev, L.Bogoeva, D. Sariev,M.Apostolova "Premier resultats de I'etudede la con- centrationen 137-Cs,134-Cs et plutoniumdans des echantillons preleves au montRila", OM2- Serie,Vol.1 , p.73-77, Sofia 1 993. 2. M lovtchev,l.Mishev, L.Bogoeva, M.Apostolova, D Sariev " Premierresullats de I'etudede la radio- activitebeta totald'echantillons preleves au montRila", OM2-Serie, Vol.1 .p.57-62, Sofia 1993 3. I Mishev,M. lovtchev,M.Guelev "Etudes radioecologiques dans le mont du Rila",OM2-Serie, Yol.2,p.3-27, Sofia '1994. 4. M.lovtchev,L.Bogoeva, M.Apostolova, M.Kostova, D.Sariev "Total Beta Activity of Samplesfrom RilaMountain", OM2-Serie, Vol 3, p.8-13,Sofia 1995. 5. M.lo\4chev,M.Kostova, R.Rousev, D.Sariev, M.Apostolova, L.Bogoeva "lnvestigation of the Total BetaActivity in Samplesfrom the Belylskar Valley. Rila Mountain", OM2-Serie. Vol.3, p 14-19, Sofia1995. 6. M.lovtchev,R. Metcheva,N.Atanassov, M.Apostolova, L.Bogoeva, M.Zivkov, G.Raikova-Petrova, M.Karapetkova"Total Beta Activity of IndicatorVertebrate Species from RilaMountain", OM2- Serie,Vol.3 , p.20-24,Sofia 1995. 7. D.Peev,M.lovtchev, L.Bogoeva, M.Apostolova, D.Sariev, S.Tzoneva, N.Valjovska "Phytomonitoring in RilaMountain -1994. lll. Total Beta Activity in Phytomonitorsfrom Control Populations", OM2- Serie,Vol.3 , o.172-176.Sofia 1995.

(r5 HEAVYMETALS AND TOXICELEMENTS CONTAMINATION AND PILOTRESULTS FROMSOME BIOTESTS IN PHYTOMONITORSFROM ROZHEN AND SREDNOGORIE CONTROLSTATIONS D. Peev,A. Artinjan*,E. Nikolova*.S. Tzoneva,N. Valjovska Instituteof Botany,BAS Acad.G. BonchevStr. bl. 23,1113 Sofia *lnstitutefor NuclearResearch and Nuclear Energy, BAS T2,Tzarigradskoshousse, blvd., 1184 Sofia

1,INTRODUCTION

Accordingthe theoreticalbase, developed in our previouspapers (Kozhuharov, Peev) the respondof tlre pollutioncan be discoveredin the differenllevel of the organisationof the phytomonitors.That rs way our attentionwas stressedon the "visible"changes in macromorphologicalcomplex as well in so not "easyvisi- ble" stomata,pollen and karyologicalcharacteristics of the chromosomes.The correlationwith data from accumulationof sometoxic elements, izoenzimes and other analysis give the possibilityto pointout the momentof firstprotective reaction orthe momentof the beginningof the firstnegative effect in the system,indi- vidualor populationlevel. Practically that is the mainobject of the biologicalmonitoring and control. In 1995,a set of controlsites and phytomonitorsaround copper smelting factory in Pirdop(Srednogorie) and in backgroundregion of Rozhen- Rhodopshave been selected. In 1996the investigationshave been carriedout on the sameset (Table.1).

Table1. Controlsitesand phytomonitorsin Rozhenand Srednogorie controlstations

Controlstations Sites Selectedphvtomonitors in the conlrolstations Rozhen 1. Smallobservatory (NAO1) Veronicachamaedrys L. 2. Centralsite (NAO2) V. officinalisL. 3. Traphopost V. austriacaL. 4. Rozhenmeadows Dactylis glomerata L. Dicranumscoparium Hedw. Ceratodonpurpureus (Hedw.) Brid. Srednogorie 1.Bridge/ Sv.Spas Veronicachamaedrys L. 2.Capture/ Sv.Spas V. officinalisL. Meadows/ Sv.Soas Dactylis glomerata L. 4.Maikai dete(1996) Ceratodonpurpureus (Hedw.) Brid. Pellia so.

2. MATERIALAND METHODS

2.1.Elements contamination A randomsampling nearly the phytomonitorsin Ar soil honzon,packing in sterilepaper bags are collected.A randomsampling of phytomonitorsin vegetativestatus "blossoming" from lhe samefragment of the populationinvestigated are collected. Using the EDXRFanalysis (Nikolova et al.,1993) the concentrationof heavymetals and toxicelements in soiland plant samples are determined.

2.2.Macromorphology and morphometry The comparisonof the macromorphologicaldescription of the phytomonitorsfrom the controlpopulation with alreadyexisting one FloraRepublicae Popularis Bulgaricae (Peev, 1995; Georgiev, 1963) and mono- graphs(Peev, 1974; Kozhuharov, 1986) gives the possibilityto be estimatedthe morphologicalstatus and the placeof the controlpopulations among the otherparts of the Bulgarianhyper population of the given species.Using the statisticstestatendency of an disruptiveeffects can be mentioned.

2.2.'1.Sampling for macromorphologicaltest A 15 matureindividuals, randomly chosen in the phaseof floweringand 15 individualsof selectedphyto- monitors (Veronica chamaedrys,Veronica officinalis and Dactylis glomerata)in the phase of fructification wereselected and strictlyvisually estimated according the followingcharacteristics. 1. defoliation;2. chlorosis / necrosis;3. type of dentationof the leaves;4. shapeand colourof flower;5. shapeof the fruitsand seeds. 66 2.2 2. Sarnpitngfc,r rrrorphoinetrpal test The rrrorphologicallyinfoimative irretric characteristics (leaves, racemes) ot Dactylisglcmerata are studied Two samplesfrom Rozhenand two samplesfrom Srednogorieare correctlycollected accorciing the rulesof randomsampling in 1995 Thesamples of sarnesites are collected in 1996 The morphornetricalcharactetrslics aie deterrninedon the basrsof measuremeittsof 50 specrrlensran- domlychosen from eachsarnple. V1 herghtof ilre plarrt(t.rlr); V2 diarneterof the slernunder the firstnode (mm):V3 numberofnodes.V4 leirgtlr of thelongestleaf (crn) V5 widthofthelongestleaf(min),V6 length of the paniclefrom the firstbranch to tne rop(cnr), V7 ntir.nberof groupsof cornposingspikes; Vb lengthol theglum (rnrri), V9 widthof thc gluin(rnrtr) V10 lerrgthof tfre lernma (rnrl) The statisticalarralyses wer-e calrred out by BMDPpaokage (Dixon 1990)and by the statisticalpackage STATISTICA

2.3.Micromorphology tesrs 23l Pollensarnpling A ntixedsarlples from the lit-rwci.-, i'itne individualsrnerrlrorted rnpoint 2.2.1 . wastaKen in acetocarminto the ntomentof staining.100 grainsfrorr 5 differentobservations are numberedTlie feitrlityis grvenas a rrreanvalues of thisfive calculations

2.3.2.Chromosome analysis In the two controlstations Rozhen and Srednogoriea randomcytodemes selection was applied So in Rozhenstation - NAO1,NAO2, Rozhen meadows (RM) and in Srednogorie- Spasl (SPl),Spas2 (SP2) and Zlalilzameadows (ZM) was chosen.A livingplants was taken and cultivatedin potsin glasshouseThe root tipsare fixed in oxyquinolinand the slidesare prepared applying squash techniques The chromosometype is givenafterWhithe, Robertis, Nowrnski and Sacz, elaborated by Kuzmanovand Kozhucharov (1967). After a comprehensiveanalysis of a ten slidesfrom eachdeme a commonkaryotypes was described stressingon the symmetryof the chromosomesand eventualdestruction (aberration, deficiency, centromer index,B chromosomesetc.) of comparingwith already known karyotype stricture (Peev, 1974; Kozhuharov, 1986).

3. RESULTS

3.1.Concentration of heavymetals and toxic elements The resultsof heavymetals and toxic elements concentration in soils,and phytomonitors(vascular plants andbryophytes) are presented intables 1,2,3,4 and 5.

Table1 . Concentrationof heavymeta ls and toxicelements (1 995) in ppmin theA.'' soil horizcr rr

Rozhencontrol station Srednogoriecontrol station z SmallOb- Central Traphopost Rozhen Bridge/ Sv. Meadows/ Capture/ Sv servatory site meadows Soas Sv. Spas Spas Cr 208t26 3651,143 274!98 57r10 305163 113!21 Mn 1281 161 131 5182 1250x123 1454153 620x72 2078t85 647t72 Fe 311 571485 39743t497 30740!387 17041!1772 28218!103 42181 1538 23076!871 Ni 174!44 207!18 171x10 61!17 86124 I JUIS 64.f10 Cu 62r8 62r5 cu+5 34t4 242t7 75t9 818t1 0 Zn 80r5 8713 8315 bdt / 48t4 75t5 AS 2815 16!2 29.3 28x3 29r3 Br tu-lz 15!2 Ilrz 1712 Rb 10011 0 10711 0 10011 0 6016 16611 2 120x10 119t10 Qr 49+3 zzlz 11 1tl0 98_Lg 9618 9418 88t8 Mo 611

Ag t1l

Cd trl Hg 7!2 Pb I v1:z lo+z 37x4 4315 47t3

67 lTable2. Concentrationof heavymetals and toxic elements (1996) in ppmin the A.,,soil horizon Rozhencontrol station Srednoqoriecontrol station Elemenl m.Obser Central Traphopost Rozhen Bridge/ eadowss Capture/ Maikai vatory site meaoows Sv. Spas Sv.Soas Sv Spas dete '100t10 133t1 0 20315 223t28 37!4 4315 230!25 13118 Mn 790r80 777t1 0 874!20 JZJlJU 472t55 1822!167 513151 / UJIO Fe oh 3.45r01 4.5110.13.2610.05 2.37t0.13.1610.084.7310.05 2.5110.05 2.73t01 Ni 75r8 90110 8416 4615 70+8 Izz+c 36+4 It7z Cu lELA 4515 ?n+? 214!3 67t7 721!10 384!21 Zn 6917 36+O 85+2 51+4 4813 18t2 18x2 zvrz zJ+ z z trz 29+3 2813 zv+3 I J:.2 Br 28t3 tlTz Rb 119t10 IJJAIU 116t10 tzo+ tu 166tl 2 120rl 0 119t10 IJO: IU Qr I Itz 108t9 329115 9618 94t9 8818 107t10 Mo 11 Ag trl

11 41 Cd tr I trl trl Hq Pb I z1:z 28!3 tJtz 4315 47x5 4515

Table 3. Concentrationof heavymetals and toxic elements in ppm in Veronicaphytomonitors

Rozhencontrol station Srednoooriecontrol station z V. chamaedrvs V. officinalis V. austriaca V. chamaedrvs V. officinalis '1995 1995 1996 1995 1996 1996 1995 1996 1995 I YVO

69r'1 8 ZJJ:J 41+10 8!2 22t4 '15t3 Mn Ioofo 44t4 160t40 186120 35r3 186r'15 26013 181110 Fe 488157 560t53 1624!204 613151720!57 684!24 10941'15413t12 39001187 2875!10 Ni 15t5 4t1 I z+z o tl '13016 tu I zrJ J+l 10!2 811 15t2 117!6 42x2 30213

Zn 81:TJ + zaaz 15115 o taJ 52!5 ZJTJ 92!4 3t1Z 79110 64!2 4a 4 /.{5 9!2 oal | +-z I Z.lZ ILT I

Br 8t1 5+1 Jfl I trz 4 A L4 Rb 14x1 33r1 ztrz IUI I 8r1 I J+Z Qr 35-3 26!2 34r3 J41 Z tzl l I t\z JCtJ 57t4 6815 11 Mo 2t1 trl 5tl ztI Aq

^,4 cd z!l 5t1 zTl Hg 5t1 7!2

9t1 o:l 10+1 01 | 3:t I J+l 611

Table4 Concentrationof heavymetals and toxic elements in ppmin D. glomerata

Rozhencontrol station Srednoqoriecontrol station Elements RozhenObservatory Rozhenmeadows Zlalilzameadows 19e5 | rggo 1995 1996 1995 1996 Cr 27!5 Mn f, t+o 306145 125!15 10411 0 Fe 417!11 36101473 42t2 1244+122 1861.15 Ni Iz7z t-u 3t1 42!3 26!4 c-'l Zn 5314 18!2 9219 4013 130x12 JU iJ As 7x2 8!2 14 -z Br tarl 7x1 tza I 21

(r li TableaDle4 (contrnual(continuation

Rb 3r1 t1l z5'il 18t2 8tl Sr 4t1 511 611 5+1 Mo Ag Cd Hg 1At2 Pb 7x1 l ttBt'l 1 z tTz 10r2

Table5. Concentraiionof heavymetals and toxic elements in ppnrIn mosses

Rozhencontrol station Srednoooriecontrol station Eiemeni D. scoaariurn C. purpureus C. purpureus Pellia sp. 1995 1996 1995 1996 1995 1996 1995 1996

Cr Prl 173x27 30t3 515r77 363t16 50r20 17!2 Mn 311i10 790118 440169 706126 1570t1 43 247!17 1367!27 9011I Fe 3357181 3378148 7972t448 240t11 3364611553 7771t84 6897169 2230t107 o Ni zJ+z 58i1 3 65r8 1.0x2 7!1 Cu 20!2 44t4 4+1 484!5 31!4 182!2 230!4 Zn 7516 45+2 685145 44!3 49!2 6014 91t5 4613 /{5 5r1 10r1 12!1 30!2 18!2 20L2 16tl Br 6i1 9t1 19t2 9!2 54!4 Rb 35t3 17!2 3513 31t3 5113 37!4 41.4 38r3 Sr 3814 35r3 76r5 3013 5713 62.4 58i4 25!2 Mo 4r.1 5rl Ag 2t1 cd Hq Pb 3614 JU-i5 3814 JU+J 3814 10x2 29r3

3.2.Macromorphology The resultsof macromorphologytests of V. chamaedrysand D. glomerataare presentedon tables6 & 7.

Tabfe 6. Macromorphologicaltestof Veronicachamaedrys s. str.

Control Rozhen Srednogorie station Years 1995 1996 1995 1996 Control NAO NAO RM NAO NAO RM SP1 SP2 ZM SP1 SP2 ZM oooulation I 2 1 2 defoliation nl nl nl nl n nl ++ ++ + chlorosis + n n nl ++ ++ + necrosrs + n n nl +++ +++ + dentation n n nl +++ +++ + flowershape n nl ni n n nl +++ +++ nl flowercolour n nl nl n n nl +++ +++ nl capsuleshape +++ +++ +++ +++ +++ +++ seedsshape + +++ +++ +++ +++ +++ +++ remarks irreqularstems and position

Legend:C) - no (changes)difference from the standard;+ - under5% of the individualsare affected;++ - 5 - 40%of individualsare affected; +++ - 40.1-75% % of individualsare affected; 75 - 100%;ni - no information

()Y Tabfe 7. Macromorphologicaltestof Dactylisglomerata s str

Control Rozhen Srednogorie* station Years 1995 1996 1995 1996 Control NAO NAO RM NAO NAO RM SP1 SP2 ZM SP1 SP2 ZM oooulation 1 2 1 2 defoliation +++ +++ ++ +++ +++ ++ chlorosis + +++ +++ ++ +++ +++ ++ necrosrs + +++ +++ ++ +++ +++ ++ dentation +++ +++ ++ +++ f++ ++ flowershape +++ +++ ++ +++ +++ ++ flowercolour +++ +++ ++ +++ +++ ++ fruitshaoe nl nl nl nl nl nl nl nl nt nl nl nl seedsshape +++ +++ ++ +++ +++ ++ remarks

Legend:C) - no (changes)difference from the standard;+ - under5% of the individualsare affected;++ - 5 - 4oo/oof individualsare affected; +++ - 40.1-75o/o % of individualsare affected: 75 - lOoo/oini- no infcrmation *This monitorspecies is presentedin the eontrolsite with singleindividuals doesn't forming a populations structure

3.3.Morphometrical tests The resultsof tested morphometricalcharacteristics of D. glomeratain Rozhencontrol station are presentedon figure1, in Srednogoriecontrol station on figure2.

Fig. l. Box Plot ol D. glomeratir lr,ledian; Box: 2-s1.,.7.s9": Whisker: Non-0iltlier Min, Non-(Jutlier Max

tr VI A v2 o V.J

tr v-s A V6 o V8 o A vl0

SITES

Legend:see V1-V10 in 2.2,2. Sites1,3- 1995;2,4-1996

70 I-ig 2. Bor PlQt of l) giomerata Median; Bor: 2-s:.,.7.5';,); Whisker Ni)n-(.)riilterNlF. N,rn"'()tiilirr I\ldx

1_ I

I tr rl a V2 : o V-J o \/4 D \,-5 aV0 ov7 o vti ov9 A Vl0

SITES

Legend:see V1-Y10 in 2.2.2. Sites5,7 - 1995;6, 8 - 1996

3.4.Stomata size (in p),frequency (mm') and pollenfertility The resultsof stomatasize, frequencyand pollenfertility of V. chamaedrysand D. glomerataare presentedon tables 8, 9, 10and 11.

Tabfe 8. Stomatasize (in ;t), frequency(mm') and pollenfertility of Veronicachamaedrys s.sfr. in Rozhen monitoringstation

Controlled Year Upperepidermis Lowerepidermis Pollen population width lenoth frequencv width lenqth frequencv % fertilitv NAOl 1995 17-24 22-25 27 20-22 22-35 198 960/o 1996 16-20 24-25 28 17-20 18-19 210 9SYo NAO2 1995 18-21 22-30 26 18-20 23-25 200 96Yo 1996 to-zz 20-2s zo 13-17 24-26 208 98% Rozhen 1995 15-19 20-26 28 15-18 25-30 160 96% meadows 1996 13-20 23-25 30 18-20 24-27 158 98%

Tabfe 9. Stomatasize (in pr),frequency (mm2) and pollenfedility of Dactytisgtomerata s. sfr. in Rozhen monitorinostation

Controlled Year Upperepidermis Lowerepidermis Pollen population width lenqth freouencv width lenoth freouencv % fertilitv NAOl 1995 30-36 50-54 250 45-48 52-59 198 980k 1996 25-28 48-52 260 43-46 50-52 200 96% NAO2 1995 28-32 35-39 281 55-61 66-76 164 98% 1996 30-36 38-43 186 55-60 70-74 180 97o/o Rozhen 1995 22-26 37-40 194 50-56 61-68 204 99% meadows 1996 z t-zc 40-48 168 42-49 50-57 202 98o/o

7l Tabfe 10. Stomatasize (in p), frequency(mm2) and pcllenfertility of Veronicachamaedrys s.str. in Sredno mon station Controlled Year Upperepidermis Lowerepidermis Pollen oooulation width lenqth freouencv width lenoth freouencv % fertilitv Spasl 1995 14-17 18-21 38 15-18 16-24 224 68% 1996 12-18 17-19 42 12-15 14-20 240 844/o Spas2 1995 15-19 16-22 51 11-16 14-22 261 82.5% 1996 14-16 15-21 16 10-13 20,31 296 81o/a

Tabfe 11. Stomatasize (in pr),frequency (mm2) and pollenfertility of Dactytisglomerata s.sfr. in Srednogo rie station Controlled Year Upperepidermis Lowerepidermis Pollen population width lenqth frequency width lenoth frequencv % fertilitv Spasl 1995 28-33 40-48 278 31-35 41-49 211 44o/o 1996 27-34 42-49 294 30-36 45-54 209 50Ya Spas2 1995 25-32 39-46 264 29-34 54-65 234 520k 1996 26-31 36-52 258 32-37 56-63 zto 640k Meadows 1995 28-34 39-50 296 31-38 49-58 231 78To Zlatilza 1996 25-35 44-51 292 26-29 47-56 245 80% 3.5.Karyotypology Theresults are presented on table 12,13,14 and 15

able 12. KaryotvpeoI Veronicachamaedrys s. str.in Rozhenmonitorinq station Controlleddeme Year Tvoe of chromosomes 2n NAOl 1995 SM 16 LSM10 46 4x= 32 1996 SM 16 LSM10 46 4x= 32 NAO2 1995 SM 16 SM 10 46 4x= 32 1996 SM 16 SM,lO A6 4x= 32 Rozhenmeadows 1995 SM 16 SHSM10 46 4x= 32 1996 SM 16 SHSM10 A6 4x= 32

able13. Ka otypeot Dactylts s. str.in Roznenmonitorlno station Controlleddeme Year Tvoeof chromosomes 2n NAOl 1995 LM 16 LSM8 A4 4x= 28 1996 LM 16 LSM8 44 4x=28 NAO2 1995 LM 16 LSM6+2 A4 4x= 28 1996 LM 16 LSM6+2 A4 4x= 28 Rozhenmeadows 1995 LM 16 LSM8 A4 4x=28 1996 LM 16 LSM8 A4 4x= 28

Table14. Ka ot veronrcacnamaedrys s. str.In srednogonemonttonng statton Controlleddeme Year Tvoe of chromosomes 2n Spasl 1995 SM 14 LSM12 46 4x= 32 1996 SM 14 LSM12 46 4x= 32 Spas2 1995 SM 14 SM 12 A6 (x= 32 1996 SM 14 SM 12 A6 4x= 32

Table 15. Karyotypeof Dact ratas. str.In Srednoriemonitonnq statlon Controleddeme Year Tvoe of chromosomes 2n Spasl 1995 SHM16 SHSM6 46 4x= 28 1996 SHM16 SHSM6 46 4x= 28 Spas2 1995 SHM16 SHSM8 42 4x= 26 1996 SHM16 SHSM8 A2 4x= 26 Mead6ws 1995 SHM16 SHSM4 A 8 4x= 28 Zlatilza 1996 SHM16 SHSM4 A 8 4x= 28

72 4. DISCUSSIONOF THERESULTS The resultreceived demonstrate the capacityof the bicmoniioringappraach. in sn:inerespects the';i are pilotone and onlysome existing tendency can be d;'ownihan r"eai ciiagno>ts

4,1.From the contaminationof heavymetals and toxic elenrent: The pictureof the contaminationof a largenurnber of eieri:enls(Cr, Fe , t'.li,Zn. As, Bi, RD,Mo, Ag. Cd, Hg)measured rn Rozhen and Srednogorie is similar.The onlysignificant difference can be seenin the levels of Cu and Mn whichvalues in Srednogorieare much higher. According this constatation lhe negativeimpact on the phytomonitoi'sis caused by ihe quantiiyof Cu andMri.

4.2.From the macrornorphologicaland morphometricaltests In Rozhenstation they are very near to the standardsdescribecj - normal {oliation, no necrosis,chlorosis, the flowerand capsuleshape are in the limitsof the nornlalrrariatrori. The specimensfrom Srednogoriestation are deeplyrnjurerJ - 10 4C% are with chlorosis,necrosis, irregular flowersand spikesor spikelets,seeds and s.o. Tlre variation shows a significanicoefficients out of the standards. The populationsin controlsites SP1 and SP2 are practicallyciesiroyed and the singleindividuals only exist. The morphometricalpicture of D. glomeratano significantdifferences between Rozhen and Srednogorie controlstation.

4.3.From the micromorphologytest 4.3.1. Stomalafrequency and pollen fertility The picturein Rozhenstation is similarto characteristicsfrom otherlocalities under background condition.The highpercent of fertilepollen show a normaldevelopmentof the populationinvestigated. The significantdecreasing of the size and increasingof the stomatafrequency proved a syndromeof xeromorphizationtypical for the plantsindividuals under air and soil pollution. The pollenfertility is about20% lowerlhan Rozhen.Evidently the systemrelated with photosynthesisfunction and normalpollination is al- readyinjured in Srednogorie. 4,3.2.Karyotypology The comparisonof the karyotypesobserved in Rozhenconfirms the alreadyknown one (Peev, 1974; Kozhuharov,1986). While the symmetryof the karyotypesobserved in Srednogorieis absolutelydifferent The most impressivedifference is in the biggernumber of acrocentricchromosomes. There is (not statistically confirmed)data aboutthe significantshortened of the totallength of the chromosomes.

5. CONCLUSTON

Althoughthe pilotcharacterof the studya followingoutputs can be mentioned: . The chosenmonilors react similarly. . The macromorphologyand micromorphologyparameters shows a similarpicture related with the differenttype and degree of the pollutionpressure in Rozhenand Srednogorie. . The controlpopulations in Rozhenare in goodcondition - withnormal morphological status and re- productivecapacity. o The populationstructure in Srednogoriestation is destroyed.That situation can be predictedhaving in mind the significantlyaffected systems in the separateindividuals. The gene pool of the species mentioneddoesn't exist and the survivinghas no chance. . The vegetationcover under pollution around Srednogorie smelting factory is significantlyendangered by extinctionto 1100m a.s.l.

REFERENCE

Dixon,W. J. (ed.).1990. BMDP StatisticalSoftware manual Univ. of CaliforniaPress, Los Angeles. Georgiev,T. 1963.Dactylis L. Floraof the People'sRepublicof Bulgaria. Vol. 1, S., Publ.House of the Bulg. Acad.Sci, 366-369. Kuzmanov,8., S. Kozhucharov.1967. Caryotypes of four BulgarianComposite species. - Compt.Rend Acad,Bulg. Sci., 5, 469-472. Nicolova,E., A. Artinian,J. Karamanova.1993. Determination par la methoderoentgenofluorescente cje tracesd'elements dans des echantillonspreleves dans la regiondu pic Moussala.In: Observatoirede Montagnede Moussala- OM2, S., 1,96-102. Peev,D. 1995.Veronica L. Floraof Republicof Bulgaria.Vol. 10, S., Publ.House of the Bulg.Acad. Sci, 142-188.

l-) CHANGESIN THE CONCENTRATIONSOF IRIDOIDGLUCOSIDES IN VERONICA SPECIESFROM POLLUTED AND NON-POLLUTEDREGIONS

Rilka Taskova',Dimitar Peev*, Nedyatka Handjieva*" and Simeon Popov** * Instituteof Botany,Bulgarian Academy of Sciences, *"lnstituteof OrganicChemistry with Centre of Phytochemistry,Bulgarian Academy of Sciences

I. INTRODUCTION

The iridoidsare a largegroup of naturalmonoterpene compounds containing a cyclopentanopyranring system.Several names have been applied to theseplant secondary metabolites and todaythe term"iridoid" is generallyaccepted, which refers to theirrelationship with the simplestcompound of this class,iridodial [1- 31.The firstiridoid was isolatedin the nineteenthcentury, but the basiciridoid skeleton was establishedonly in 1958[1-3]. In the generalsense, the iridoidsare dividedinto iridoids and secoiridoids[1-3]. These compoundsare distributed in the dicotyledons,especrally in the sympetalousplants and in someinsects [1-3]. The iridoidglycosides possess some valuablebiological activities as feedingattractants, stimulants. feeding deterrentsto birds and some insects,etc. Some pharmacologicalactivities as hepatoprotective, chologogic,anti-stress, antileukaemic, laxative, sedative, etc. were shown, which make them perspectrvefor practicalapplications. Less is knownabout their functions in plants.The antibacterialand fungicidal activities of theiraglycones, as well as the toxicityof the iridoidglycosides towards some insectsindicate their defen- siverole in the plants. Changesin the primarymetabolites, mainly in proteinsand lipids,caused by environmentalfactors, includingpollution [4,5], are discussed by manyauthors. Such changes are connectedwith the cellmembrane compositionand its functionsand usuallyhave an adaptivevalue dependingon the extentof the environmentalchange. They could be usedas bioindicatorsof the pollution.These investigations are relativelyslow and more expensivethan the convenientmethods. On the otherside, they givevaluable information on the influenceof the pollutionon the physiologicalchangesin the organisms,inhabiting the pollutedareas. Littleis knownabout the influenceof the environmenlalchanges on the concentrationsof the secondary metabolitesin the livingorganisms. Recently we establishedsome changesin the concentrationsof poly- phenolsand alkaloidsin two tobaccocultivars, treated with glyphosate[6]. Disappearanceof routineand lowerconientrations of chlorogenicacids were registeredafter treatment with the herbicide,connected with suppressionof the biosynthesisof the aromaticcompounds. The decreasein the concentrationof one de- fensivecompound (chlorogenic acid) accompanied by the increaseof anotherdefensive compound (nico- tine)could proceed to keepup the plantresistance. It is expectedthe biosynthesisof othersecondary metabolites to be influencedby changesin the environment.The iridoidshave a complexbiosynthetic pathway and some biosyntheticstages could be stimu- lated or inhibitedby the environmentalfactors. The subjectof this work are preliminarystudies on eventual quafitative and quantitativedifferences in lhe concentrationsof the iridoidglycosides in four Veronicaspecies grownin non-pollutedand pollutedregions.

II. MATERIALSAND METHODS

Plantmaterial. Plants from erghtnatural populations of four Veronicaspecies from non-pollutedand pollutedregions were collectedduring flowering (Table 1). Voucherspecimens were identifiedby Dr. D. Peev anddeposited in the herbariumof the Instituteof Botany(SOM) Sofia.

lsolation.Dry plants(200 g each sample)were extractedwith methanol.The iridoidswere isolatedand purifiedaccording to the convenientprocedures. Compounds 1-10 were identifiedOy 1H anC 13C NMR spectraand comparrsonwith authentic samples.

TLC analysis.Dried ground plants (400 mg) of the eightsamples were twice extractedwith methanolfor 24 h. Afterconcentration of the combinedextracts, water (4 ml) was addedand a threefoldextraction with dichloroethane(x 2 ml) was carriedout. The waterlayers were concentrated and dissqlvedin2 ml methanol:water(1:1). These solutionswere used forTLC. Alufolio silica gel 60F254precoated plates (Merck) were usedfor the analysis.Aliquots (5.0 prl) of the samplesolutions together with the standardsolutions (5.0 pl) wereapplied to the silicagel plates.The plateswere developed with chloroform:methanol:water (60:15:4 and 60'.22'.4,v/v/v) and afterevaporation of the solventstheplateswere placed in a chamberoversulphuryl chlo- ridevapours for 60 minand then heated at 120ofor 30 min.

'74 III.RESULTS AND DISCUSSION

As mentionedeight samplesfrom four Veronicaspecies were collectedfrom the non-pollutedregion Rozhenin the Rhodopesand the pollutedwith heavymetal contamination Srednogorie region in Sredna gora.The dataconcerning the origrnof the collectedsamples are summarised in Table1. The iridoidglycosides were isolaled as totaliridoid mixtures. After separation and purificationten individual components1-10were isolated in purestate and identifiedby spectralmethods (1H anO13C rufUR; and comparisonswith authenticsamples. The iridoidswere analysedby thin layerchronratography (TLC) on silicagel with two mobilephases (Table 2) in the presenceof standad iridoids.The lesspolar phase was suitablefor determinationof compounds1-7, while the polarone for compounds8, 9 and 10.

coocH3

HOCH2 HO OGlu H3 OGlu HOCH2

R 4

1: benzoyl 2. vanilloyl 3: isovanilloyl 5: p-hydroxybenzoyl 6: caffeoyl 7'. 3,4-dihydroxybenzoyl 9: OH I 0: 3"'-O-glucosyl-4"-hydroxycinnamoyl

The totaliridoids showed a tendencyfor higherconcentrations in the samplesfrom the non-pollutedregion (Rozhen).Habitat factors are not completelyexcluded. The individualiridoidt found in higherconcen- tralionsdiffered in the differentspecies. Common for all investigatedplants were ihe increasedconcentra- tionsof iridoidsfrom the last stepsof the iridoidbiosynthesis in Veronica(Fig.1) as catalpol(9) and its esters amphicoside(2), verminoside(6) and catalposide(5). ln V. arvensisand V. persicafrom the non-polluted regionlow increase of the aucubin(8) concentrations was registered. The obtaineddata are very scarceto explainlhe natureof the observedchanges. lt is possiblethe pollution in the Srednogorieto inhibitthe oxidises,taking part in the oxidationof the C-7 - C-8 doublebond to formthe epoxidering in catalpoland its esters.The reductionof the concentrationsof the totaliridoids in lhe same regionis an indicationthe terpenicbiosynthesis to be suppressedby the pollutionat an earlierstage. These preliminaryresults need confirmationby futureinvestigations on a considerablenumber of samples fromthe samegenus, including representatives of other genera or evenfamilies, in orderto be ableto make conclusionsabout the mechanismof the effectof the pollutionon the iridoidbiosynthesis and aboutthe possibilityto usethe iridoidsas indicatorsfor the pollution.Model experiments with differentconcentrations of heavymetals and with otherpollutants must be performed. Somefunctions of the plants,responsible for their adaptation towards pollution, could depend on the concentrationsof some secondarymetabolrtes. To checkthe eventualeffect of iridoidson the activityof plant peroxidazes,thirty iridoids were isolatedfrom differentplant sources (Table 3) and afterpurification tested in the laboratoryof Prof.N. Bakardjieva(lnstitute of PlantPhysiology, Bulgarian Academy of Sciences).The preliminaryresults showed that some of the iridoidglucosides stimulate the peroxidazeactivity. It couldbe concludedthat to usethe iridord-containingplants as bioindicatorsof the pollutionfurther ex- perimentsare needed,including experiments on the roleof iridoidsin plants.

75 ./

HO X"A, >-r\ eol I eol I y-Y" )-\'-o cinnamoyl-OcHZ oCtu benzoyFOcH2 6Ctu 2

p€H-benzoylO

6 I I cafieoy14"{l rcopyran osyfO protcatechoyO X/\ €ol I )-\-" oHcHz 6ctu .,0 7 I

Figure 1. Biogenesisof the iridoidglucosides in Veronica

'7',| Table 3. lridoidglucosides tested for activationof peroxidazes.

No Compound Plantsource Amount, mo 1 aucubin Plantago ?.9 2 catalpol Plantago 2.9 3 monomellitoside Plantago 1.7 4 mellitoside Plantago 3.1 5 kickxioside Kickxia 2.0 6 antirrinoside Linaria 2.4 7 linarioside Linaria 2.5 8 5-O-menthvlfolovlkickxioside Kckxia 1.4 I antirride Kckxia 2.1 10 mussaenosidicacid Kickxia 1.7 11 swertiamarine Gentiana 2.0 12 oentiopicrine Gentiana 2.0 13 sweroside Centaurium 2.0 14 V3 Galium 2.2 15 asoeruloside Galium 16 daohilloside Galium 17 asperulosidicacid Galium 2.1 18 desacetvlasperuloside Galium 2.0 19 desacetylasperulosidicacid Galium 3.7 20 monotrooeine Galium 2.3 21 scandoside Galium 2.0 22 oenioosidicacid Galium 2.2 23 secooalioside Galium 24 desacetylasperulosidicacid Galium 2.3 methvlester 25 mussaenoside Veronica 1.6 26 6-O-vanillovlcatalool Veronica 2.5 27 verproside Veronica 2.9 28 valtrate/isovaltrate Centranthus 3.6 29 didrovaltrate Valeriana 2.2 30 veronicoside Veronica 2.0

REFERENCES

1. Inouye,H.: (1991) lridoids In: Methods in PlantBiochemistry 7, p. 99-143.Academic Press Limited. 2. Jensen,R. S.: (1991)Plant iridoids, their biosynthesis and distributionin angiosperms- In: Harborne,J. B., Thomas-Barberan,F. A. (Eds.)Proc. Phytochem. Soc. Eur. 31, Ecol.Chem. & Biochem.of Plant Terpenoids,p. 133-158.Clarendon Press, Oxford. 3. Inouye,H and Uesato,S.: (1986)Biosynthesis of iridoidsand secoiridoids.- In: Herz,W. ef a/. Progr. Chem.Org. Nat. Prod. 50, p.169-236.SpringerVerlag, Wien. 4. Quiper,P.J.C.: (1984) Lipid metabolism of higherplants as a factorin environmentaladaptation. - In: Siegenthaler,P. A., Eichenberger,W.(Eds.): Structure, Functions and Metabolismof Plant Lipids.p. 525-530.Elsevier Science Publishers, Amsterdam . 5. Stefanov,K., Budevska,B., lvanov,O., Genov,N., Andreev, St., Popov, S.: (1993)Lipid and aminoacid changes in the mussel Pseudoanodontacomplanata caused by water pollution.Comp. Biochem, Physiol.105C, 39-42. 6. Bankova,V., Handjieva,N., Djilianov,D., Vassileva,2., Bachvarova,R., Atanasov,A., Stefanov,K., Popov,S.: (1995)Polyphenol and alkaloidchanges in glyphosate-treatedtobacco regenerants se- lectedfor herbicidetolerance. Z. fur Naturforsch.50c, 313-315.

78 BIOCHEMICALSCREENING OF THE EFFEGT OF INDUSTRIAL COPPER POLLUTIONUSING LEAVES OF VERONICASPECIES AS BIOMONITORS

N. T. Bakardjieva,E. D. Ananiev,N. Christova,K. Christov lnstituteof PlantPhysiology, Bulgarian Academy of Sciences

When a given plantis usedas a biomonitorit is presurnedthat it willgive some kindof informationabout its environment(Martin and Coughtrey 1982, Markert 1991, Witting 1993). The question arisesto find such suit- abie indices,which will providea fast and exactinformation. Morphological and cytogeneticcharacteristics have been used in this respect,as well as the concentrationsand accumulationof some pollutantsin the differentplant organsand tissues(Littleand Martin 1974,Leith and Markert1994). The studiesin the last aspectsincluded higher plants (Mager, 1983, Lamersdorf, 1988 and others),fungi (Mejstrik,Lepsova, 1993) mossesand ferns(Puckett, 1988, Richardson, 1988, Brown, 1991, Thoeni, L., J. Hertz,1987, Bruning and Kreeb,1993 and many others).But in all casesplants are not a passiveaccumulator of metalsor other pollutants.They reactto differentextreme situations by specificmetabolic mechanisms, which can be usedas indicesin biomonitoring. The accumulationof high,non-toxic or toxiccontents of different+netalsin plantcells provokes a varietyof interactionsbetween different metal ions and othercell constituents,for exampleproteins, other ligand part- nersbeing different small molecules as aminoacids,organic acids etc. (Bakardjieva, 1981 , 1986,Bakardjieva and Christova,1991, Streit and Stumm,1993 and others).As a resultconsiderable changes in metabolic reactionsmay occur.The newcomplex compounds formed may possiblypossess different and new biochemical functions,the functionof some enzymesmay be modifiedand lhe velocityof severalenzyme reactions may be changed.So the correctfunctioning of some biochemicalsystems and processesmay be destroyed. Some of them, which are very fast reactingto increasedcontent of metal ions in the cell may be used as biochemicalindices in the biomonitoring. Plant peroxidaseis one of the enzymes,which reactvery quicklyto changesin the environmentand to differentstress situations. Changes in peroxidaseactivity are indicativeand are studiedand discussedas a resultof metabolicdisturbances caused by pathogens,by temperaturestress and UV-lighteffects, high concentrationsof heavymetals, etc. (Bakardjieva,1986, Gaspar et all. 1988,1992, Campa, 1991, Kerby and Sommerville, 1992,van Asscheand Clijsters,1990, Castillo, 1992). The useof peroxidasefor this purposeis basedon the factthat the enzymeis includedin the protectiveenzyme complex of the plantcell, eliminating the toxicoxygen radicals produced under unfavorable external factors (Elstner, 1988, Bakardjieva et all. 1994, 1996).In this enzymecomplex peroxidases are functioningtogether with catalaseand superoxidedismutase (Elstner,1987). The threeenzymes are partnersin the formationof the metabolicresponse of the plantcell to changesin the environment,which are resultof toxicagents and differentkinds of pollutants.That is why it is logicalto assumethat this enzymecomplex as a wholeis very suitablefor biomonitoring.A role of catalase duringenvironmentalstress is reviewed byWillekens, Inze, van Montaguand van Camp(1995). Catalase is involvedin the responseof the plantcell to intensivelight, pathogens, chilling, high temperature etc., the individualisoforms being affecteddifferently. The peculiaritiesof SOD handlingwhen affectedby different stressfactors is reviewedby Bowler,van Montaguand Inze(1992). The changesin the enzymeactivity may be interpretedas a resultof bothactivation and additionalsynthe- sis of enzymeprotein. In our investigationswe characterizedthe activityand heterogeneityof peroxidaseand superoxidedismutase and alsochecked the rateof proteinbiosynthesis, the synthesisof RNA andthe endog- enousnucler RNA polymeraseactivity. These data allow to findsome answer for the physiologicalstatus of the plant at the pollutedregion and for the interpretationof enzymeassays. The observationthat the effectof oxidantsand an oxidativestress show a correlationwith the ratesof proteinturnover is stressedand discussed by Pacificiand Davies(1990). So we try to estimatethe importanceand the valueof thesebiochemical indices or systemsfor biological monitoring,respectively to havean earlyand fast information about heavy metal pollution. ln our caseVeronica specieswere used as biomonitors,leaf samples being taken from Rozhenregion and from Srednogorieregion The secondis characterizedby an industrialpollution and manytimes increased quantity of copperin the soil and in the plantswhich was found by ourcolleagues in thisinvestigation.

Materialsand Methods Preparation of enzyme extract Leavesfrom Veronicachamaedrys, V. officinalls and V.austriaca were sampledfrom their naturalhabitat at the two regionsin Bulgaria- from Rozenregion in the mountainRhodopes and Srednogorie region, the second beingcharacterized as a pollutedone, during June - August.All operationsfor preparingan enzymeextract were performedat 4oC.Forthe enzymesassay the leaveswere homogenized in 0.1 M Tris-HClbuffer, pH 7.8, containing0.1 mM EDTAor in 0.05Mphosphate buffercontaining 0.1 mM EDTA,pH 7.0.After 30 minthe 79 homogenateswere filtered through 4 layersof gauzeand centrifuged at 15000g for 30 minutes.The obtained supernatantwas dialyzedfor 24 h againsttwice deluted extraction buffer, and centrifugedagain for 20 min at 15000g. The supernatantwas usedfor characterizationof enzyme activity and isoenzymecomposition. The proteincontent of the enzymee)Cract was determined by the methodof Lowry(1951). Enzyme assays The activityof peroxidasewas measuredby ihe quantityof the productof guaiacoloxidation formed for 5 min at 420 nm. Catalaseactivity was characterized by the degreeof H-ra,exhaustion, measured at 260 nm spectrophotometrically.The SOD activitywas assayedby measuringits abilityto inhibitthe photochemica reductionof nitroblue tetrazolium (NBT) according to Beauchampand Fridovich(1971). The reactionwas startedby switchingon the light.After 10 minthe absorbanceof 560nm wasmeasured.- Separation of isoforms For separationof the isoformsof POD and SOD discelectrophoresis on 7.5o/o PAAG was performedusing Tris-glycineseparating buffer, pH 8.3,applying 5 mA pertube.The isoperoxidaseswerevisualized by incuba- tionthe gelsin guiacol-benzidinesolution for 5 min andaddition of HrO,to a finalconcentration 0.05%. The SOD patternswererevealed according to Beauchampand Fridovich (1971). In thetwo casesa densitometer scanningwas usedto registerthe isoforms. Am ino acid incorporation Discsfrom leaves of Veronicaofficinalis L. and Veronicachamaedrys L. (B mm diameter)were exposedto 185kBq.mll 14C-labelledaminoacid mixture (Amersham, UK). After 4 h al28"C the sampleswere thorouhly washedand groundin a mortarwith 80o/oacetone, centrifuged and the pelletswere extractedconsequently with100% acetone, petroleum ether and 5% TCA. TCA- insoluble protein was dissolved in 10%NaOH and its radioactivitywas measuredin toluene-basedscintilation counter Beckman. Estimation of RNA content and determination of RNA synthesis by in vivo labelling experimmenfs and me as urin g of radio activity RNAwas estimated according to the procedure of Wollgiehnand Parthier (1964). Approximately 39 samples werehomogenized with 80% ethanol in a mortar.After extraction of pigmentsand acid-soluble materials RNA was digestedwith 0.5N KOH at 37"Cfor t h and measuredspectrofotometrically bfter neutralizationwith HCl04. Leaf discs from V officinalisand U chamaedryswere labelledwith [3H]uridine(O.7.107 Bq.ml-1, 870 GBq.mmole-1)andfor 4 h at 28'C, washedin ice-cold50 mM sodiumacetate buffer, pH 5.66and ground in a mortarwith the samebuffer. The uptakeand incorporation of radioactivityinthe leafdiscs were determined as describedby Brayand Dasgupta(1976). Nucleiisolation and RNApolymerase assay Nucleiwere isolated from Veronicaleaves according to Ananievand Karagyozov(1984) and the endog- enousRNA polymerase activity was assayed acording to procedurrepublished by Ananiev et al.(1987). Briefly, nucleiwereincubated in a reactionmixture (60 pl) containing:40 mM Tris-HCl,pH 7.9;5 mM MgClr;50 mM (NHo),SOo;10 mM B-mercaptoethanol;0.4 mM eachof ATP,cTP andCTP; 7 pM [3H]uridine-5ttriphosphate (UTP)(185 GBq.mmole-1); nuclei (10 pg of DNA)and 20% glycerol.After 10 min of incubationat 25"Cthe radioactivemixture was pipettedon WhatmanNo 1 paperdisks presoaked in 0.1 M EDTA,precipitated with cold10% TCA andafler water elimination counted in toluene-basedscintilation mixture.

Resultsand Discussion The comparisonof the activitiesof POD, SOD and catalasein three Veronicaspecies (Table 1) show considerabledifferences. A very high catalaseactivity is typicalfor V. austriacaand V. officinalis.In these speciesPOD and SOD activitiesare lowerin comparisonwith that measuredin the V chamaedrys.The more balancedratio betweenthe three enzymesactivities in V. chamaed4ysshow that it is a suitableobject for biomonitoringstudies. So the responseof the defence enzyme complex to environmentalstress situations and alsoin the casewhen it is causedby increasedmetal ions content in plantswill be demonstratedbetter. The activitiesof peroxidase,catalase and superoxidedismutase in the leaves of V. chamaedrysfrom Rozenand Srednogorie regionswere mea-sured. Average data fortwo years are represented in fig.1and fig 2 A considerableincrease in activityof PODand catalase is typicalfor plantsfrom the Srednogorie,compared with that from Rozhen.SOD in plantsfrom the two areasdoes not showwell expresseddifferences. So a higheractivity of POD and catalaseis characteristicforthe pollutedregions as Srednogorie.lt may be supposedthat they can be usedas biochemicalindices. to definethe high metal ions concentrationin some reglons. After electrophoreticseparation of the enzymeextracts from V chamaedrysleaves 9-11 isoperoxidases wererevealed, which may be markedin twogroups - thefirst of themincludes 5 peroxidaseisoenzymes with lowelectrophoretic mobility and the secondgroup consists of 5-6peroxidase bands with middlemobility. For the isoenzymespectrum of V.chamaedrys, sampled at Rozhenarea is characteristicthat the secondgroup of peroxidasesshow higher or relativelyequal activity in comparisonwith the first group. Typical for V.chamaedrys samplesfrom Srednogorie is that the activityof the secondgroup of the isoenzymesis considerablylower and 80 thaltte actr.,rty3f mosrrsoperoxidases from the first group is clearlydominatrng ,,,l!'n"y 1: ::':t:* 113'13::y.tl,:I3!"-391[?f,i.]L" ??,li."l:11:.,"f ,11:.1,Y".,9'.?:P'=.?.f.lll: i;',T?j"::,1T:^i: !ras: i i's: r'.e harr|fLIl factorS. ::l'-.-'. :t)atnaeclrysischaracterizedby4isolornrs Thecharr.Jesrntherractrvtlyobservedrrlsamplillg an(l 1rsciiffrcrrlltotlsethemasindrcesinthe ,--.--,=:-:--l-l_Sr"O',ogorieregiotrsarenotsotyprcal--.. --a' 1 :'=:-''..i I frg3 showstne RNAcontent in theleaves of Veronrcachamaedrys L grownin the :-'- -'.'..:'-_:,-sof RozhenandSrednogorie.ltisclearly:eer,tnattheRNAcontentintheleavesof the - t .-': =,'-i " Srednogoriewasdecreased by about 309'. rn ccrrparison wrth that in the regiott of Roshett. S '.= ;'.1 a::ummulatronintfre cells of theleaves is a reslt of boththe processes of RNAsynthesis and Q\: r:l'.ralr.. v/efurlher studied the processof RNAsynthesrs lself by meansof the systemof in vitro R^.1Sr^thes s ,rrrsolaled nucler. The resultsconc.ernlng tht iolalerrdogenous RNA polyrneraseactivtty of .-.: ? s: a:er from leavesof Veronicaare presenteclrrr fig 4 As',an be seen.the tolalRNA polymerrase aCr i; '- l.e samLrlestrorn Srednogoriewas sigrriticantly decreased to 45%of thatin the samplescollected fr.rr lie reJ,3nof RozhenCompared with the values foi RNrrcontent, RNA polymerase activity was more se.?r?ly affected because of the fact that the polymerase system reflects more sensitively the extent of RNA synthes,srn the cell Thus.the inhrbitionof RlrlAaccumulatron inthe leaves of theplants from Srednogorie is duemarnly on the inhibitionof RNAsyrrthesis during the processof transcription.We couldnot exclude also anda possibleeffect of RNAdegradation on the RNAcontent by activationof the cellproteases, but that was rrota subjectof investigationin thiswork. In additionwe checkedthe rateof 3H-uridineincorporatron into total RNA of the cellsas a measureof RNA synthesisin vivo.The results(not shown here) revealed a veryclose correlation between the incorporalionof the labelledprecLrrsor of RNAsythesis and the valuesof RNAcontent and the activityof RNApolymerases in the samplesfrom the bothregions. Sincetotal RNA polymerase activity represents mainly (up to 85%)the activityof RNApolymerase I catalizing the synthesisof rRNAs,which are used for production of newribosomes, it wasreasonable to proposea close correlationof the effectof air and soil pollutionbetween RNA and proteinsynthesis. Fig.5 shows that the incorporationof 14C-aminoacid mixture into the soluble protein as a measurefor proteinsynthesis. As canbe expected,the incorporationof labelledperecursors into total cell proteinwas inhibitedin the plantmaterial obtainedin Srednogorie.The inhibitionof proteinsynthesis was approximatelyin the sameextent as thatof RNA synthesis.Therefore, it can be concludedthat it existsa close similarrtyin the effect of air and soil pollutionon boththe processesof RNAsynthesis and protein synthesis in the plantcells suffering the negative effectof pollution.The highersensitivity of thesecrucial biochemical processes to pollutionof heavymetals couldbe usedas a meansof biomonitoringof industrialareas poisoned by suchkind of pollutants. The plantsfrom Srednogorie regicn used in ourstudies are affected by a long{ermexposure to increased concentrationsof copperin the environment. lt is a resultof anthropogenicindustrial pollution. lt wasfound that in the tissuesof theseplants the coppercontent is considerablyhigher. This gives a goodbasis for evaluation of the possibilitiesto usebiochemical parameters for monitoring of man-mademetal stress in comparisonwith the datafor plantsfrom purearea as Rozhenis. According to Verkleijand Schat(1990) some physiological parametersare closely related to heavymetal stress syndromes. Our studies showed that biochemical induced characterizingthe metabolicfast response to environmentalstress and the physiologicalstatus of plantsmay be usefulin the bioindicationof heavymetal pollution. On the basis of ourresults it mayme assumedthat more informativeare the data about isoperoxidases complemenl and the biosynthesis of RNAand protein. They can be successfullyapplied in biomonitoringassays.

References AnanievE. D., KarakyozovL. K., "Comparativeeffect of sarkosyl,heparinand aurintricarboxilic acid on RNA polymeraseactivity in isolatedbarley nuclei.", Physiol. Ve1.,22,555-563, 1984. AnanievE. D., KaragyozovL. K., KaranovE. N.,"Effect of cytokininson ribosomalRNA gene expression in excisedcotylrdons of Cucurbitapepo L.", Planta, 170,510-378, 1987.. Bakardjieva,N. 1986.Metal ions control on activity,polyfunctionality and UV photosensitivityof plant peroxidase.In: Molecular and Physiological Aspects of PlantPeroxidase (eds. Greppin, H., Penel,C. and Gaspar,Th.) University of Geneva,143-154. N. Bakardjieva,N. Christova- Photoregulationof catalytic ascorbate oxidation and its dependence on metal ionsand some peptides in earlyand laterevolution - Plant Physiology, Sofia, 1991, #3, 29-34. Bakardjieva,N. T.,Christova, and N.,Christov, K. 1994.Effect of calciumand zinc ions on the thermosensitivityof peroxidase,superoxide dismutase and catalase in Scenedesmusacutus cells. Comp. rend.Acad. Bulg. Sci. v. 47,N 12,83-86. Bakardjieva,N.. N. Christova,K. Christov,1996. Effect of calciumand zinc ions on the sensitivityof peroxi- dasefrom mosses(Mnium sp.) and ferns (Polypodium vulgare) to hightemperature. Can. J.Bot.,74, 81 thatthe activityof mostisoperoxidases from the firstgroup is clearlydominating. It may be assurnedthat namelythe ratiobetween the activitiesof the two groupsof the isoperoxidasesis more indicativeabout the situationin the environnrent.The loweractivity of the secondgroup shows the presenceof someharmful factors. SODfrom V.chamaedtys is characterized by 4 isoformsThe changes in their activity observed in sampling leavesat Rozhenand Srednogorieregions are notso typicalancl it is difficultto usethem as indicesin the biomonitoringresearch. The data presentedin fig.3shows tne RNA contentin the leavesof Veronicachamaedrys L" grownin the bothstudied regions of Rozhenand Srednogorie.lt is clearlyseen that the RNA contentin the leavesof the plantscollected in Srednogoriewas decreased by about30% in comparisonwith that in the regionof Roshen. SinceRNA accummulationin the cellsof the leavesis a resultof boththe processesof RNA synthesisand RNA degradation,we furtherstudied the processof RNAsynthesis itself by meansof the systemof in vitro RNA synthesisin isolatednuclei. The resultsconcerning the totalendogenous RNA polymeraseactivity ot nucleiisolated from leavesof Veronicaare presentedin fig.4.As can be seen,the total RNA polymerrase activityin the samplesfrom Srednogorie was signif icantly decreased to 45%of thatin the samplescollected from the regionof Rozhen Comparedwith the valuesfor RNA content,RNA polymeraseactivity was more severelyaffected because of the facttirat the polymerasesystem reflects more sensitively the extentof RNA synthesisin the ceil.Thus, the inhibitionof RNAaccumulatlon in the leavesof the plantsfrom Srednogorie is due mainlyon the inhibitionof RNAsynthesis during the processof transcription.We couldnot excludealso anda possibleeffect of RNAdegradation on the RNAconlent by activationof the cellproteases, but that was not a subjectof investigationin thiswork. In additionwe checkedthe rateof 3H-uridineincorporation into total RNA of the cellsas a measureof RNA synthesisin vivo.The results(not shown here) revealed a veryclose correlation between the incorporalionof the labelledprecursor of RNAsythesis and the values of RNAcontent and the activityof RNA polymerasesin the samplesfrom the bothregions. Sincetotal RNA polymerase activity represents mainly (up to 85%)the activityof RNApolymerase I catalizing the synthesisof rRNAs,which are used for production of newribosomes, it wasreasonable to proposea close correlationof the effectof air and soil pollutionbetween RNA and proteinsynthesis. Fig.5 shows that the incorporationof 1aC-aminoacid mixture into the soluble protein as a measurefor proteinsynthesis. As canbe expected,the incorporationof labelledperecursors into total cell proteinwas inhibitedin the plantmaterial obtainedin Srednogorie.The inhibitionof proteinsynthesis was approximatelyin the sameextent as thatof RNA synthesis.Therefore, it can be concludedthat it existsa closesimilarity in the effectof air and soil pollutionon boththe processesof RNAsynthesis and protein synthesis in the plantcells suffering the negative effectof pollution.The highersensitivity of thesecrucial biochemical processes to pollutionof heavymetals couldbe usedas a meansof biomonitoringof industrialareas poisoned by suchkind of pollutants. The plantsfrom Srednogorie region used in ourstudies are affected by a long{ermexposure to increased concentrationsof copperin lhe environment.lt is a resultof anthropogenicindustrial pollution. ltwas found that in the tissuesof theseplants the coppercontent is considerablyhigher. This gives a goodbasis for evaluation of the possibilitiesto usebiochemical parameters for monitoring of man-mademetal stress in comparisonwith the datafor plantsfrom purearea as Rozhenis. According to Verkleijand Schat(1990) some physiological parametersare closely related lo heavymetal stress syndromes. Our studies showed that biochemical induced characterizingthe metabolicfast response to environmentalstress and the physiologicalstatus of plantsmay be usefulin the bioindicationof heavymetal pollution. On the basis of ourresults it mayme assumedthat more informativeare the data about isoperoxidases complement and the biosynthesis of RNAand protein. They can be successfullyapplied in biomonitoringassays.

References AnanievE. D., KarakyozovL. K., "Comparativeeffect of sarkosyl,heparinand aurintricarboxilic acid on RNA polymeraseactivity in isolatedbarley nuclei.", Physiol. Yeg., 22,555-563, 1984. AnanievE. D., KaragyozovL. K., KaranovE. N.,"Effect of cytokininson ribosomalRNA gene expression in excisedcotylrdons of Cucurbitapepo L.", Planta, 170,570-37 8, 1987.. Bakardjieva,N. 1986.Metal ions control on activity,polyfunctionality and UV photosensitivityof plant peroxidase.In: Molecular and Physiological Aspects of PlantPeroxidase (eds. Greppin, H., Penel,C. and Gaspar,Th.) University of Geneva,143-154. N. Bakardjieva,N. Christova- Photoregulationof catalytic ascorbate oxidation and its dependence on metal ionsand some peptides in earlyand laterevolution - Plant Physiology, Sofia, 1991, #3, 29-34. Bakardjieva,N. T.,Christova, and N.,Christov, K. 1994.Effect of calciumand zinc ions on the thermosensitivityof peroxidase,superoxide dismutase and catalase in Scenedesmusacutus cells. Comp. rend.Acad. Bulg. Sci. v. 47,N 12,83-86. Bakardjieva,N.. N. Christova,K. Christov,1996. Effect of calciumand zinc ions on the sensitivityof peroxi- dasefrom mosses(Mnium sp.) and ferns (Polypodium vulgare) to hightemperature. Can. J. Bot.,74, 81 1665-1 670. BeauchampC. H. and l. Fridovich,1971. Superoxrde dismutase: improved assay and an assayapplicable to acrylamidegels. Analyt. Bioch., 44,276-87. Bowler,C., M. van Montagu,D. Inze,1992. Superoxide dismutase and stress tolerance. ,Ann. Rev. Plant Physiol.v. 43:83-116. BrayC. M., DasguptaJ., "Ribonucleicacid synthesis and loss of viabilityin peaseeds"., Planta, 132,103- 108,1976. Brown,D. H.,1991. Symbiosis, v. 11,207-223. Bruning,F. and K. H. Kreeb,1993. Mosses as biomonitorsof heavymetalcontamination within urban areas, In:Plants as biomonitors(Ed. B. Markert),VCH - Weineim,N. Y.,Basel-Cambrigde, 395-401. Campa.A. 1991.Biologicalrole of plantperoxidases: known and potentialfunction. In: Peroxidasesin Chemistryand Biology(eds. Everse, J., Everse,K. E.,and Grisham, M. B ) vol. ll, CRC Press,25-50. Castillo,F.J. 1992 Peroxidases and stress. ln: PlantPeroxidases 1980-1990- Molecular, Biochemicaland PhysiologicalAspects (eds. Penel, C., Gaspar, Th., and Greppin, H.) University of Geneva,187-203. Elstner,E. 1987.Metabolism of activatedoxygenspecies. In: The BiochemistryofPlants, Acad. Press, v. 11,253-315. Elstner,E. F.,Wagner, G. A., andSchutz, W. 1988.Activated oxygen in greenplants in relationto stress situation.In: Current topics in PlantBiochemistry and Physiology, vol. 7, 159-187. Gaspar,Th., Kevers,C., Penel,C., Crevecoeur, M., andGreppin H. 1988.Biochemical characterization of normaland habituatedsugarbeet calli. Relationship with anatomy. Habituation and Organogenesis. PostdamerForsch 578, 21-30. Gaspar,Th., Hagege,D., Penel,C., Foidart,J. M.,and Greppin, H. 1992.Peroxidases and Plantcancerin the absenceof pathogens.In: Plant Peroxidases 1980-1990. (eds. Penel, C., Gaspar,Th., and Greppin, H.) Universityof Geneva,125-137. Kerby,K., and Summerville,S. C. 1992.Purification of an infection-relatedextracellular peroxidase from barley.Plant Physiol. 100, 397-402. Lamersdorf,N., 1988.Verteilung und akkumulation von spurenstoffen in waldokosystemen.Ber. Forsch - ZentrumWaldokosysteme, 4-36, Univ. Gotingen. 205 pp. Leith,H. and Markert,8.,1990. Elementconcentration cadasters in ecosyslems,VCH, Weinheim,448 pp. Little,P. and Martin,M, H., 1974.Biological monitoring of heavyrnetal pollution. Env. Pollut.,6, 1-19. LowryO., Rosebrough,A., Farr,A., Randall,R. 1951.Protein measurementwith Folin phenol reagent. J. Boil.Chem. 193, 680-685. Markert,B., 1991. Inorganicchemical investigations in the forestbiosphere reserve near Kalinin, USSR. Vegetatic,95, 127-135. Martin,M. H.,Coughtrey, P. J., 1982.Biological monitoring of heavymetal pollution. Appl. Sci. Publ., London,475 pp. Mayer,R., 1983.Interaction of forestcanopies with atmospheric constituents: aluminium and heavymetals. In:Effect of accumulationof air pollutionin forestecosystems. D. ReidelPubl. Comp., Dordrecht,47-55. Mejstrik,V. and Lepsova, A., 1993.Applicability of fungitothe monitoringof environmentalpollution by heavymetals. In: Plants as biomonitors(Ed. B. Markert),VCH - Weineim,N. Y.,Basel-Cambrigde, 365- 379. Pacifici,R. and K. Davies,1990. Protein degradation as an indexof oxidativestress. Meth. Enzymol., 189, 485-502. Puckett,K. J., 1988.In: Lichees, bryophytesand airquality. Eds.: Nach H. Wirth,Cramer, vo|.30,231-267. Richardson,D. H. S., 1988.BotanicalJ. of LinneanSoc., v.96,31-43. Thoeni.L., and Hertz,J. 1992.Applicability of the mossHypuum cupressiforme for biomonitoringof heavy metals.An investigationof siterequirements. Metal compounds in Environmenland Life,4, 137-151. VanAsshe, F., and Clijsters,H. 1990.Effects of metalson enzymeactivity in plants.Plant Cell and Environ- ment,13, 195-206. Verkleiij,J. A. C., H. Schat,1990. In: Heavy metaltolerance in plants:Evolutionary aspects (A. J. Schaw, eds.),Boca Raton: CRC Press,179-193. Willekens,H., D. Inze,M. van Montaguand W. van Camp,1995. Catalases in plants.Molecular Breeding, 1,207-228. Wittig,R., 1993.General aspects of biomonitoringheavy metals in plants.In: Plants as biomonitors(Ed. B. Markert),VCH - Weineim,N. Y.,Basel-Cambrigde, 3-29. WollgiehnR., ParthierB.,"Ein Beitrag zurquantitativen Bestimmung von Ribonucleinsaureund Protein in Blattern".,Flora, 154, 325-348. 1964.

82 I I rllrl *^-r- \ I I , '|.flr:- I l\lil \ '\ l. tl ti Ii/\ It \t\ tl \il \il . i'\i\l 'rl I \i.-' \11 \ll l',' '/\ \llr , i \ \' \lI I'i'i/ \. llI /'Jl /\J, I I t. l i., l,L il tl 5 illl I I lltl I lrl ill ilIt I ill i\ lll 11 i \ /-:- I ilr l\/ --',, \lI{I \ I \I It'\/ \tv\ \ I l, ll,i \ \l't ', l\r \/ ! \ v \f i/ t \, r I \ t1 I\ i i tl \ ,i ''\iti I 'lll I /llr il ' -/ \li /il : --- / \ll I l-t \ t

Fig.1.lsoenzyme complement of peroxidasein the leavesof Veronica Fig.2. lsoformsof superoxidein the leaves of Veronicachamaedrys chamaedrysfrom Rozhen region (up) and from Srednogorie (down). Peroxi- from Rozhenregion (up) and from Srednogorie(down). SOD was ex- dasewas extracted using Tris-HCl buffer containing 0.1 mM EDTA(left) or tractedusing Tris-HCl buffer containing 0.1 mM EDTA(left) or phos- phosphatebuffer containing 0.1 mM EDTA(right) phatebuffer containing 0.1 mM EDTA(right) Table1. Activityof peroxidase,superoxide dismutase and catalasein differentVeronicaspecies at Rozhen area- 1996

Calalase Peroxidase Supcroxide dis- Leaves front: % of hydrogen EU/rngprotein nlutase peroxideexhaustion EU/mg protein

Veronicachamaedrys -r.4 -17.-s

Veronicaofficinalis 0.5 77.8 21.6

Veronicaaustriaca la 87 ?1

Table 2. Activityof peroxidase,superoxide dismutase and catalasein Veronicachamaedris from Rozhen andSrednogorie regions.

Catalase Peroxidase Superoxidedismutase % oi hydrogenperoxide EU/mg protein EU/rng protein exhaustion Sample

Sredno- Sredno- Morr R&oiarr hzban Sredno- gorie gone gorie

Veronica 3.4 5.9 34.9 55.8 37.5 36.l chamaedrys leaves 100 t76.4 100 r59.8 t00 96.5

' 0l

() ?

0 C) s n')

Rozhen Srednogorie Fig. 3. Effectof air and soil pollutionon the contentof RNA in the leavesexcised from Veronicachamaedrys L. plants,grown in the regionof industrialarea of the townof srednogorie. 84 2.0

'7\ li

c0-{

il 1.0

! fli

Rodren Srednogorie

Fig.4. Effectof air and soil pollution.onthe synthesisof totalsoluble protein in the leavesexcised from Veronicachamaedrys L. plants,grown in the regionof Srednogorie.The proteinsynthesis was measuredby the incorporationof 14C-aminoacid mixtureinto thetotalcellsoluble protein (fordetails see Materials and methods).

2.0 ,- :

c.) o t5

-? 1.0

c.)

ti flut

Rozhen Srednosorie

Fig.5. Effectof air and soilpollution on the endogenousnuclear RNA polymeraseactivity. Nuclei were isolated from leavesexcised from V chamaedrysL. and V officinalisL. plants,grown in the regionsof Srednpgorie. 85 INDUSTRIALPOLLUTION IMPACT ON THE ISOENZYME STRUCTURE OF ROZHEN AND SREDNOGORIEPOPULATIONS OF DACTYLISGLOMERATA L.

GeorgiAngelov Instituteof Botany,Bulgarian Academy of Sciences

I.INTRODUCTION

Industrialpollution causes serious changes of environmentand naturalecosystems. Numerous studies clearlydemonstrated that the geneticstructure of plantpopulations in regionspolluted by industrialactivities is changedin responseto the alteredenvironmental conditions (Sinclair 1969: Meinartowicz 1983: Bergmann andScholz 1987: Geburek et al. 1987). RozhenObservatory (Rhodopes Mt.) is an experimentalsite in Bulgariansystem of backgroundmonitoring. Srednogorie(Sredna gora Mt.) is a site in Bulgariansystem of impactmonitoring. Dactylis glomerata L. is one of phytomonitorsin bothexperimental sites. The purposeof the presentstudy was to analysethe isoenzymestructure of Rozhenand Srednogorie populationsin comparisonto the averageisoenzyme structure of D. glomeratawhich is foundfor its Bulgar- ian populationsin orderto evaluatethe impactof pollutionon environmentin bothmonitoring stations.

II. MATERIALAND METHODS

For the studies,lhree sets of localpopulations were chosen.First set (R) was locatedin RozhenCbser- vatoryP5 plants).Second set (S) consistedof populationslocalised comparatively close (5-6 km.)to copper smelter of Srednogorie(67 plants).Third set (D) consistedof local populationsfrom different phyto- geographicalregions of Bulgaria.Totally 187 plantfrom set D were studiedin order to revealthe average isoenzymestructure which is characteristicfor Bulgarianpopulations of D. glomerata. Anodalisoforms of esterase(EST), and acid phosphatase(ACPH) were fractionatedfollowing procedure of Davis(1964). Cathodal isoformsof EST and PER were electrophoreticallyresolved according to Reisfeld et al. (1962).Details of experimentalprocedures were describedelsewhere (Angelov, 1986; 1992).Each isoformwas given a numericalsymbolwhich reflects its gel migrationin mm (Shumakerrand Babble,1980). Th'efrequency of eachisoform was calculatedas a percentageof plantspossessing it in'a givenpopulation. Pair-wisecomparisons between sets R, S and D were made andthe differencesbetween population sets were evaluatedby Wicoxonsigned-rank test (Hollanderand Wolfe, 1973).

III.RESULTS AND DISCUSSION

Totallyfifteen isoformsof anodalEST were found in D. glomerata(table 1). lsoform25 and 37 were monomorphicallyfixed(frequency of 1,00)in all sets studiedThere were some differencesbetween isoenzyme structureof populationsets analysed. lsoform 16 lackedin set S and isoform42was not foundin both S and R sets. Despiteof this, there were not statisticallysignificant differences between the isoenzyme structureof D, S and R setsof D. glomerata. Analysisof cathodalEST showedthe same results(table 2) - no significantdifferences between population sets examined. Sevenisoforms of cathodalPER wereelectrophoretically resolved (table 3). lsoforms32, 34 and 37 were invariantinallpopulationsets.Thefrequehcyofisoforms20,23,26and2Sfluctuatedbetweenthesets but statisticaltest appliedshowed no significantdifferences between D, S and R sets of D. glomerata. The isoenzymestructure of D. glomerata(ACPH) is presentedin table 4. In total eleven isoformsof ACPH were found.lsoform 37 was monomorphicallyfixed in all populationsets. There were some frequency fluctuations but pair wise comparisonsshowed insignificantdifferences between isoenzyme structure of threeset examined. The resultsof the presentstudy showed that isoenzymestructure of Rozhenpopulation (set R) did not differfrom the averageisoenzyme structure (set D) whichwas foundfor Bulgarianpopulations of D. glomerata These resultscorrespond to data for phytomonitorspecies Festuca nigrescens from Boatin National Reserve(Angelov, in press). Srednogoriepopulation (set S) provedto be unchangedin respectto the averagepopulation structure of D. glomerataas revealedby isoenzymemarkers analysed. In a previousstudy (Angelov1993) it was shownthat isoenzymestructure of phytomonitorsDichantium ischaemumand Chrysopogongryllus collected near by coppersmelter of Srednogoriewas differentfrom the averageisoenzyme structure of bcth specieswhich is characteristicfor their Bulgarianpopulations. lt was supposedthat Srednogoriepopulations of both phytomonilorswere stronglyinfluenced by industrialpollu- 86 tion. On the contrary,results of the presentstudy implied that Srednogoriepopulalions of D. glomeratawere not influencedby industt'ialpollution. In our opinion,relatively small samplesize and numberof enzymes usedon the one hand and comparativelylong distance between collection sites and sourceof industrialpol- lutionon the other hand can explainedat leastpartly for these results.Hypothetically it is possiblethat D. glomeratais more tolerantto industrialpollution than D. ischaemumand Ch. gryllusbut this statemenlneeds additionaltests to be provedor rejected. In conclusion,the resultsof the presentstudy showedthat genelic struclureof Rozhen populationof phytomonitorspecies D. glomeratais unchanged.This is an indicationthat the environmentin the regionof Rozhenmonitor station is not influencedby industrialpollution as judgedby isoenzymemarkers. The stateof environmentin Srednogorieregion is differentas revealedby isoenzymestudy of D. ischaemumand Ch. gryllus(Angelov 1993 and other relevant studies (Petrova et al.,unpubl. res.)..

REFERENCES

Angelov,G. 1986.Biosystematic study of naturalpopulations of Bulgarianspecies of genus FestucaL. The- sis,Sofia, (in Bulgarian). Angelov,G. 1992.Biosystematicstudy of speciesfrom genus Festuca L. lV. Electrophoreticanalysis of en- zymesperoxidase, acid phosphatase and catalase.- Fitologija,43. 58-68 Angelov,G. 1993.lmpact of industrialpollution on the populationstructure of Dichantiumischaemum and Chrysopogongryllus. -Agrochimica, 37, 68-76. Angelov,G. lsoenzymestructure of Festucanigrescens (Gramineae) from BoalinNational Reserve, Bul- garia.- Bocconea,in press. Bergmann,F., F. Scholz.1987.The impactof air pollutionon the geneticstructure of Nonvayspruce. - Sil- vae Genet.,36, 80-88. Davis,D. 1964.Disc electrophoresis. 2. Methodand applicationto humanserum proteins. - Ann. N. y. Acad Sci. 121, 404-421. Geburek,T., F. Scholz,W. Knabe,A. Vornweg.1987. Geneticstudies by isoenzymegene locion tolerance andsensivity in an airpolluted Pinus sylvestris field trial. - SilvaeGenet., 36, 49-57. Hollander,M., D. Wolfe.1973. Nonparametric statistical methods, John Willey, New York. Mejnartowicz,L. 1983.Changes in geneticstructure of Scotspine (Pinussylvestris) populations affected by industrialemission of fluorideand sulphurdioxide. - Genet.po|.,24, 424-432. Reisfeld,R., U. Lewis,D. Williams.1962. Disc electrophoresis of basicproteins and peptideson polyacryla- midegels. - Nature,195,281-283. Sinclair,W. 1969.Polluted air: potential new selective force in forest.- J. Forest.,67, 305-311. Shumaker,K., B. Babble.1980. Patterns of allozymesimilarity in ecologicallycentral and marginalpopula- tionsof Hordeumjubatum in Utah.- Evolution,94, 110-i17.

87 6 oo

Table 1. lsoenzymestructure of Dactylisglomerata - anodalesferase

Population lsoformsfrequencv Set 16 18 21 23 25 27 30 33 5I 40 41 42 44 47 51 D 0.05 0.08 0.56 0.30 1.00 0.47 0.53 0.89 1.00 0.30 0.47 0.05 0.86 0.39 0.30 S 0.00 0.00 0.29 0.38 1.00 0.57 0.46 n47 1.00 0.17 0.41 0.00 0.73 0.50 0.27 R 0.02 0.10 0.44 0.42 1.00 0.52 0.58 0.74 1.00 0.27 nqn 0.00 0.59 0.44 u.J /

Tabfe 2. lsoenzymestructure of Dactylisglomerata - cathodalesferase

Population lsoformsfrequencv Set 22 28 32 35 U 0.95 no? 0.05 0.10 0.82 0.87 0.02 0.27 R 0.79 0.81 0.12 0.18

Tabfe 3. lsoenzymestructure of Dactylisglomerata - cathodalperoxidase

Population lsoforms Set 20 23 26 28 32 34 37 D 0.07 0.23 0.25 0.14 1.00 1.00 1.00 S 0.00 0.15 0.37 0.28 1.00 1.00 1.00 R 0.13 0.27 0.40 0.20 1.00 1.00 1.00

Tabfe 4. lsoenzymestructure of Dactylisglomerata - acid phosphatase

Population lsoforms Set 10 12 14 16 18 24 27 29 33 a-7 40 D 0.15 0.19 0.27 0.17 0.21 0.32 0.40 0.65 0.38 1.00 0.48 e 0.27 0.05 0.39 0.29 0.13 0.47 0.57 0.32 0.43 1.00 0.51 R 0.10 0.23 0.11 0.22 0.32 0.37 4.25 0.48 0.51 1.00 0.44 CONTENTOF SOMEELEMENTS IN LIEHENFROM ROZHEN BACKGROUNDSTATION Dobri lvartov, VarnaBotanical oarderr

I. INTRODt.'CTION L,ichensare dependent almo:.;t erclusively on the atrriospirerefor irutlients,absorbing and retaining caiionsfrom very dilute solutiorrs like rain-water lt is well knc.rwnthat they have the abilityto accumu- late many airbornesubslances to concentrationsabove those in the environment.Sonie authors '1973; (James. Andersenet al., 1978;Pilegaard, 1979; Herzig et al , 1989a,1989b) showed that the concentrationsof trace eiementsfound inside lichen thali are directlycorrelated with environmental levelsof tneseelernents. II. STUDYAREA, MATERIALSAND METF{ODS In 1995a mixedsampies were taken from three iocations 0 nearthe Rozherrbackground station. in 1996samples from one locatioirwere taken and carefullyseparated to five one-speciessamples. All the sampleswere collected from spruce tree at the height1,5 - 2 m and analysedfor 29 in 1995 and24 in 1996elemenls by the eneigydispersive X-ray fluorescence analysis. III.RESULTS The concentrationsof metalsin the specimenscollected in 1995were representedin table 1 and ihosecollected in 1996in table2. The resultsof someelements were compared with the dataof other authorsin tables3 and4. Table1. Samplesfrom the regionof: A - NAORozhen, B - Mainobseruatory, e - nearthe bridge; a - valueof concentration.b - standarddeviation error. Element Unit A B a b a b a b AI %o 0.75 0.24 0.8 0.2 0.86 0.05 bl Yo 1.95 0.42 0.31 0.07 0.61 0.13 P (ppm) 906 105 (ppm) 1381 364 2130 20 2345 406 cl (ppm) 2524 528 716 88 3443 180 K (ppm) 5920 538 5171 96 5941 472 "Ca (ppm) 3108 640 1780 162 2507 286 Ti (ppm) 408 88 81 4 166 10 V (ppm) Cr (ppm) 58 2 Mn (ppm) 211 20 43 4 Fe (ppm) 5252 106 1874 336 2577 141 Ni (ppm) 25 5 3 1 3 1 Cu (ppm) 20 z 14 J Zn (ppm) 71 7 82 6 72 6 Ga (ppm) 5 1 As (ppm) 10 2 Se (ppm) I Br (ppm) I 1 4 I 12 2 4 Rb (ppm) 13 1 4 1 8 I Sr (ppm) 8 4 8 n 4 1 (ppm) 1 1 Zr (ppm) 19 2 4 1 6 1 Nb (ppm) Mo (ppm) Ag (ppm) 2 1 a cd (ppm) 5 I 1 1 Hq (ppm) I 2 Pb (ppm) 35 3 22 3 26 t

89 Table2. Samplesfrom: A - Bryoriaspp., B - Usneaspp., C - Hypogymniatubulosa, D - Everniadivari cata,E - Pseudeverniafurfuracea; a - valueof concentration,b - standarddeviationerror. Element Unit A B c D E

a D a b d b a b a b AI (ppm) 2146 13 712 100 3730 500 7013 800 4556 226 Ca (ppm) 337 63 815 100 3366 104 2164 416 2170 190 Ti (ppm) 111 12 V (ppm) Cr (ppm) 135 15 124 15 232 31 500 54 140 10 Mn (ppm) 46 7 48 2 107 20 115 18 63 b Fe (ppm) 774 32 946 82 3301 237 2212 56 1940 122 Ni (ppm) I 4 17 2 48 2 15 2 Cu (ppm) 2 4 3 1 I Zn (ppm) 45 4 34 I 64 5 57 5 70 7 Ga (ppm) 7 1 As (ppm) 5 1 ? 1 10 z 22 ? 10 Se (ppm) Br (ppm) 10 1 6 1 10 1 11 1 Rb (ppm) 5 I Sr (ppm) 2 1 2 1 3 1 (ppm) Zr (opm) 2 1 4 1 Nb (ppm) 7 1 Mo (ppm) 4 1 Aq (ppm) cd (ppm) 1 4 Hq (ppm) Pb (ppm) 10 z 19 z 12 2 21 z

Table3. Datareported: A - Loppiet al.,1993 from the surroundsof townof Pistoia;B - Bargagliet al., 1987,from background area; C - Bargaglietal., 1987 nearthe highway; D -Olmezet al.,1085 nearCoal-Fired Power Plant; Our dataof mixedsamples from: E - NAORozhen; F -Main observatory;near the bridge.

Element A FI c D E F G cd 0,53 1,20 3 1 Cr 2.11 6.80 6,90 3,80 58 Cu 12,05 6,90 11.10 20 14 Ho 0,09 0,34 0,32 I Ni 2.41 25 3 3 Pb 14.23 10,60 82,40 35 22 26 Zn 74,12 34,50 58,50 64.00 7'l 82 72

Table 4. Datareported by Hezig et al., 1988from the regionof Biel:A - criticalair pollution;B - high pollution;C-mediumpollution; D-lowpollution; E-verylowairpollution; F-background; Our data:G - Hypogympiatubulosa; Significant correlation between lAP18 and element contentsof H. physodesin Biel.

Element A B D E F \J H Ca 8470 14198 21279 30900 29395 25501 3366 +0,87 cd 1,10 1,07 0.76 0.96 0,59 0.22 Cr 7.20 12.56 4.90 4,67 2,68 1,75 232 Cu 31,05 23,02 16,96 13,05 9,67 .4,29 3 -0.78 Fe 1916 1804 1157 975 590 480 3301 -0,91 K 4538 4474 4455 4232 3216 3095 3730 Pb 180 94 60 51 31 28 19 -0,85 Zn 168 152 114 97 85 44 64 -0,73 90 lv.DlscussloN

The mixed samplesfrom B and C correlatevery well wrththe data of Loppi et al.,'1994with the exceptionof Hg contentin B. The valuesin A are high abovethe reporiedeven for the high polluted areas,with the exceptionof Zn lf we comparesome of our datafor H. tubulosawith thoseof Herzziget al., 1989for H. physodes, a very similarsystematically and ecologicallyspecies we noticea interestingconelation of concentrations of Zn. Pb, K and Cu with the data obiainedin the zonewith very low air pollutionand background.The concentrationsof Cr and Fe are highlyabove those reportedfrom the criticalpolluted zone.Our datafor concenlrationof Ca are muchlower than those of allthe zones. Hezic et al., 1989aroved the existenceof correlationbetween concentrations of Fe, Ca, Pb, Cu, Zn and the correspondingvalues of Indexof AtmosphericFurity (lAP18). More the correlationof the finstthree elementsare good and highlysiEnificant. Our data of concentrationsof theseelements showed the valuescorresponding to the valuescor- relatingwith the IAP characterisingthe zonewith very low air pollution, withthe exceptionof Ca - very low concentrationand Fe - very highconcentration. lt is interestingto nrentionthat the concentrations of Fe in the otherspecies especially Bryoria and Usneaare of quitelower values.

V. CONCLUSIONS

This firstinvestigation of concentrationsof some elementsin Bulgarianlichens gives us data from Rozhenmonitoring station that can be comparedwith future investigations. Some of our data shQwed thatthe areais withlow air pollution.

REFERENCES

Andersen,A., Hovmand,M.F. & Johnsen,l.: 1978.Atmospheric Heavy Metal Deposition in the Co- penhagenArea. Environ. Pollut. 17, 133-151 . James,P.W.: 1973. The Effectof Air PollutantsOtherthan Hydrogen Fluoride and SulphurDioxide on lichens.Air Pollutionand Lichens,The Athlone Press, London, pp 143-175. Pilegaard,K.: 1979.Heavy Metals in BuikPrecipitation and transplanted Hypogymnia physodesand Dicranoweisiacirrata in the Vicinityof DanishSteelworks. WaterAir Soil Pollut. 11,77- 91. Herzig,R., Liebendorfer,L., Urech,M., Amman,K., Guecheva,M& Landolt,W.: 1989a.Passive Bio- monitoringwith Lichensas a Part of an IntegratedBiological Measuring System for MonitoringAir Pollutionin Switzerland.Intern. J. Anal.Chem. 35, 43-57. Hezig, R., Urech,M., Liebendorfer,L., Amman,K., Guecheva,M& Landolt,W.: 1989b.Lichens as BiologicalIndicators of Air Pollutionin Switzerland:Passive Biomonitoring as a Part of an Inte- gratedBiological Measuring System for MonitoringAir Pollution;in: Lieth,H. and Markert,B. (Eds.), ElementConcentration Cadaster in Ecosystems,VCH, Weinheim/NewYorUBasel/Cambridge. Pp. 317-332. Bergagli,R., losco,F.P. & D'Amato,M.L.: 1987. Zonation of TraceMetals Accumulation in Three Spe- cies of EpiphyticLichens Belonging to the GenusParmelia. Cryptogamie, Bryol. Lichenol. 8, 331- 337. Olmez,1., Cetin Gulovali, L. & GordonG.E.: 1985. Trace ElementConcentration in LichenNear a Coal-FiredPower Plant. Atm. Environ.19, 1663-1869. Loppi,S.,Chiti, F., Corsini,A. & Bernardi,L.: 1994.Lichen Biomonitoring of TraceMetals inthe Pistoia Area (CentralNorthern ltaly). Environmental Monitoring and Assessment,29, 17-27.

9l MACROAND MICROELEMENTCONTENT IN MUSHROOMSFROM TI.IE REGIONS OF ZLATITSAAND ROZHENAS BIOMONITORSFOR POLLUTION

Violeta Fakirova *, En'tiliaNikolova"*, Ari Arlinian ** *lnstituteof Botany,Bulgarian Academy of Sciences "*lnstitutefor NuclearResearch and Nuclear Energy, Bulgarian Acadenty of Sciences

I.INTRODUCTION Regionalenvironmental pollution is an old problem,caused by humanactivities. At presentthe world- wide increaseof trace elementsis of generalinterest, especially when two regionsmust be cornpared- a pollutedone - wherea largefactory produces many wastes to a so calledRegional Background Statioi'r. Somespecies of higherfungi are well known for theirability to accumulatespecific elemenis. The possibilityof observingthe heavymetal element status of environment,using high fungi as biomonitorsgives informationon the qualityof this environmentand for the pollutionlevel. Fungi posses several good biomoni" toringfeatures: . Vastgeographical distributron of manyspecies and occurrence in natural,industrial, urban arid rural areas; . Abilityto accumulaleelements in higherthen the averageconcentrations for the speciesin unpollutedregion; . Simplemorphological structure and osmotrophicalmanner of nutrition,predetermine maxirrr;ri speedof metabolicprocesses; . Fine structureof the hyphi(diameter 2-4 pm),which providestheir penetrationin the soil rnici"c" pores,in contrastto the plants,where the finest fibber has a diameterabout 10ptm . Two years investigationof elementalconcentrations in 47 samplesfrom 29 high fungi specieswas carried.The sampleswere collectedfrom two differentregions: impacted area surrounding large coppei-factory,nearZlatitsa and a backgroundreference area nearthe NationalObservatory Station Rozhen in Middle Rodops.Most of the specieswere representedonly with one sample;only from 5 specieswe have more samplesavailable. Soil samples, collected under some of specieswere also analysed in orderto findcorre- lationbetween concentrations of someelements.

II. INSTRUMENTATION Energydispersive X-ray fluorescencemethod was used for determinationof 29 elementsin the fungi samples.The materialwas dried,ground and homogenised.Three spectrometric systems were appliedin orderto cover all rangeof elements,equipped with three different types of excitingsources - Fe"', Pu"o and Am2ot.The excitingionditionswere optimised in ordertoreach hignersensitivity and lowerdetectionlimits. The spectrometersare equippedwith Si(Li) detectorswith about180 eV energyresolution at 5.9 keV Mn K, line. The data were acquiredwith a multichannelanalyser, interfaced to personalcomputer. A specialised softwarewas appliedfordata processing.Standard samples from Orchardleaves N8S1571, Bowen's kale and Hay powderIAEA V-10 were usedto createsensitivity curves for each excitingsource in orderto calibratethe spectrometers. Other standardsamples were used for verificationof calibrationcuryes and the obtainedvalues were foundto be in a good agreementwith the referencevalues.

III.RESULTS AND DISCUSSION The concentrationsof the elementsin dependencgof theirlocality is shownin Fig.1-3. lt is easyto find out the very highconcentrations of someelements like Cu, Ag and Se near the copperfactory. Largerange of concentrations,especially for some certainelements were found for the fungi,collected in one andthe samelccality. The highestconcentrations were found in the followingfungi species: from Rozhen region: Pluteusatromarginafus (Konr.) Kuhn - 200 ppm Mn; Hygrophorusprafensls (Pers.ex Fr.) Fr. - 620 ppm Zrr Armillariamellea (Fr.) Kummer- 13 ppm Cd from Zlatitsa region: Bovistaplumbea Pers.:Pers. - 6260 ppm Fe, 1959ppm Cu,77 ppm Ag, 42 ppm Pb; Calvatiautriformis (Bull.ex.Pers.) Jaap - 41 ppmAs, 149 ppm Se. The resultsillustrate that some of the fungi specieshave affinityfor accumulationnot only for one ele" ment. For examplethe Bovisfaplumbea Pers.:Pers has affinity for the elementsFe, Cu, Ag and Pb ani Calvatiautriformis (Bull.ex.Pers.) Jaap - for As and Se.

92 A parallelanalyse was madefor threecouples of fungr.collected from the two regions(Table 1 ) The resultsshow, that Bovistaplumbea Pers Persfrom Zlatilsa region has accumulated6 times more Fe, 25 timesmore Cu and 4 tirnesmore Ag as comparedto the conc.entrationol the theseelements in the same speciesfrom Rozhenregion. Analogous relations were tound also for Marasrniusoreades (Bolt Fr)Fr frorrt Zlatitsaregion, where Crr and Ag concentrationswere 5 timeslarger as conrparedto the sarnespecies from Rozhenregion. lt is our opinionthat these two speciesare very suitableas biontonitorsin the passivebio- monitoringsystem for severalreasons - geneticaffinity for accumulationof some certarnelements spread distrrbutionin the ecosystemsand not highrequirements, regarding the ecologicalconditions, which is due to theirmorphological structure The bothspecies are typicalmeadow species ancj in trophicalsense they are relatedto the humussaprotrophs The analysisof soil samplesfrom thesetwo regionsconcerning these furtgi species, showing highei affinityfor accumulationsof someelements, confirm the conclusions,ihat a geneticdependence really exist Evenin the case.when these elements are in verysrnall concentrations in the soilsample or are underde- tectionlimits of the method,they are presentin highconcentrations in the fungisample Typicalexarrrple for suchsituation is Armillariamellea (Fr.) Kummer for accumulationof Cd in Rozhenregion where Cd werenot detectedin the soilsample, The abilityof Armillariamellea (Fr ) Kummerfor accurnulationof higharrrourrts of Cd is experimentallyproved by Turnau[2] and our resulls confirmed their conclusions. Very highvalues of Ag contents(77 ppm)were found in Bovistaplumbea Pers Pers and also of Se - (149ppm) in Calvatiautrifonnis (Bull.ex.Pers.) Jaap in comparisonthe contentsof theseeiements irrthe scil samplesfrom the pollutedZlatitsa region. The Ag and Se concentrationin all speciesare shownin fig 1 and fig.2where it is obviousthat these high concentrations were found only in Zlatitsaregron and onlyfor these two species.lt is very interesting, that in two otherspecies Paxillus involutus (Batsch )Fr and Prso/ltuhus arhiztts(Pers.)Rausch., located neX to Calvatiautrifonnis (Bull .ex. Pers ) Jaap- no tracesof Se werefound Thisfact once again proves the geneticdependence of Calvatiautriformis (Bull.ex.Pers.) Jaap to Se

IV.CONCLUSIONS Significantdifference in the concentrationsfor Cu, As, Se and Ag for the two regionsis observed.The conlentsof theseelements in Zlatitsaregion are eltremely high, which proves, that the Copperfactory really pollutethe region.As a conclusion,based on the presentedresults, we consider,that we have sufficient evidenceto includethe followingfungi in the categoryof the biomonitors: Armillaria mellea (Fr.) Kummer Bovista plumbea Pers.: Pers Calvatiautriformis (Bull. ex. Pers.)Jaap Marasmius oreades(Bolt.: Fr.) Fr Our resultsare in mainlenanceof the moreand moresupported in the latestyears opinion,ihat "theaf- finityfor accumulationis speciesspecific" [5].

V. RESUME An energydispersive X-ray fluorescent method is usedfor determinationof 29 elementsin 47 highfungi and5 soilsamples The distribution of 10 elementsis investigated. Mn, Fe ,Cu,Zn, As, Se,Ag, Cd andPb. The samplesare collectedin two differentregions: -impactedarea surrounding the largecopper factory, near Zlaiitsa; - backgroundreference afea near the NationalObservatory Station Rozhen in MiddleRodops. Big differencein the concentrationsfor Cu, As, Se and Ag for the two regionsis found.The contentsof theseelements in Zlatitsaregion are extremely high, which proves, that the Copperfactory really pollute the region.Genetic dependence for accumulationof certainelements for fourfungi species also is found.Armil- lariamellea (Fr.) Kummerfor Se, Bovistaplumbea Pers.: Pers and Marasmiusoreades (Bolt.:Fr)Fr for Ag andCalvatia utriformis (Bull.ex.Pers.) Jaap for Cd.The highaccumulation ability also for the otherelements giveus confidence, thatthese fourfungispecies are very suitable as biomonitorsin passivebiomonrtoring.

LITERATURE 1. FischerC.,Blei und Cadmium in PilzenausWestberlin,Z. Mycol., B.56 (1,1,1990,p.159-166 2. TurnauK., Heavymetal uptake by Armillarialutea growing in a Pino-Quercetumforest treated with cadmiumdust, Nova Hedwgia,50(1-2), 1990, p201-211 3. LepsovaA., KralR., Leadand Cadmiumin fruitingbodies of macrofungiin the vicinityof a leadsrreltei'. Sci.Total Environ., 76(2-3), 1988, 129-1 38 4. WilckeC., HubnerD., GrasserK., MeyerR., PietschH., Artspecifischeschwermetalaufnahme von Wild- pilzenund Zuchtchampignons, Trace Elem. Symp., Leipzig, 1989, p1545-1552. 5 DietlG.,Wildpilze speichern Schwermetalle, Umwelt, N1-2, 1987,p.24-26

9-+ ELEMENT SAMPLE CODE z Unit GS5 GS6 GS7 GR5 G22 GS9 G21 GR3 Value +l- Value +l- Value +l- Value +l- Value +l- Value +i- Value +i- Value +l- K (ppm) 21409 1803 26167 1028 20861 942 22990 1115 42685 1244 21220 1020 33195 588 21184 1153 (ppm) 1555 205 1280 206 2284 44 I +2. d5 1100 15 831 124 861 an 428 28

il (ppm) Jf,

1 a Mn (ppm) o IJ L o z

A4 a Fe (ppm) 327 27 580 26 1341 105 129 15 481 20 z Ni (ppm) a '161 Cu (ppm) 319 11 452 11 12 86 .z 48 z 25 x 1

Zn (ppm) 124 12 vo 5 88 105 80 z 195 6 2A I 106 z Ga (ppm) I 1 a a .) As (ppm) 22 ? 22 z 21 z 8 1 1 Se (ppm) 58 o 50 5 48 -7 Br (ppm) q 1 5 1 1 8 a ,1 1.1 Rb (ppmi 4 8 I 12. I 21 218 i:l 15 Sr (ppm) 4 1 a I 1 1 1 Y (ppm) Zr (ppm) z- 1 8 1 1 a 1 1 1 Nb (ppm) e,1 3 Mcr (ppm) 1 Ag (ppm) 75 5 27 ol 4 o 1 14 1 7 1 ,l ,'l (ppm) z L 1 1 2 til

q I Hg (ppm) 11 I I 1q a Pb (ppm) 16 1 2A 3 L Weight(g) 31 6.3tt 26.2 z.o 2.68 50.6 978 22.4i Sampletype Marasmius Marasmius Marasmius Marasmius Marasmius Lactarius Lactarius Lactanus Oreades Oreades oreades oreades oreacles resimus ptperatus deltciosus Region Zlatitsa Zlatitsa Zlatitsa Rozhen Rozhen Zlatitsa Rozhen Rozhen nearstack nearrail-station St.Spas St.Spas '12 SamplingData 6 05.96 18.10.96 9.96 4.1 0.96 25.9.95 20.9.96 25.9.95 4.10.96 ELEMENT SAMPLECODE z Unit GS2 GSlO Gl0 G2 Gl6 G28 Value +l- Value +l- Value +l- Value +l- Value +l- Value +l- K (ppm) s23 153 21075 897 34568 419 14460 1857 41281 2843 35764 337 Ca (ppm) 1778 204 1481 159 1598 11 1778 117 1057 68 1119 aq Ti (ppm) 248 123 13 JU z V (ppm) Cr (ppm) .l 16 a,t q Mn (ppm) 161 1 UI 4 Fe (ppm) 789 33 1538 2.'7 6260 J5C 237 46 975 90 174 16 Ni (ppm) q Cu (ppm) 1959 40 960 7 1577 50 102 43 ? JZ z Zn (ppm) 32 a 26 J 100 15 159 10 102 4 114 AS (ppm) 23 Se (ppm) 57 6 43 5 3t 4 z Br (ppm) q 1 Rb (ppm) 27 z 37 1 371 25 15 z Sr (ppm) q 4 1 8 1 1 Zr (ppm) 7 1 16 1 1 Mo (ppm) 3 1 Ag (ppm) 11 1 15 1 77 1 'l I 10 1 1 4 cd (ppm) 4 1 I 1 4 1 Hg (ppm) Pb (ppm) 42 4 o 1 /Veight(s) 9.94 23 13.87 8.24 1.11 2.8 Sampletype Bovista Bovista Bovista Bovista Bovista Bovista plumbea plumbea plumbea plumbea plumbea plumbea Region Zlatitsa Zlatitsa Zlatitsa Rozhen Rozhen Rozhen meadowSt. Spas rail-station faucetSt. Spas mainobservatory hut Rozhen Sampling Data 23.7.96 20.9.96 15.7.95 13.7.95 26.9.95 13.7.95 MICROBIALBIODIVERSITY OF SOILFROM SREDNOGORIE AND ROZHENBIOMONITORING STATIONS

AtanasKaimaktchiev. Denciic Denchev.K. Tsekova instituteof Mici"otriology,Bulgaliari Acaderny cf Scierrces.

i. iNTRODUCTIOt'l

Thetoxic acti"rity of heavymetals on themicrcorganisms rsv/adeiy discrrssed by manyauthors 11,2,3, 4, 5.6, 71.Nov; is well-kriownthat the tcxicityspectluni included ihe priniarygrowth inhibition to totalelimination of microbialpopulaiion [1] The intermediatereactior]s could be ullrastructui-almorphologrcal changes, the changesin themetabolic pathways and the enzyme activities t2,3 4,5.6, 71. in thisstudy lvas made attenipt to followthe totalmicrobial quantity and the micrcbialdistribution by the maingi'cups: bacteria. actinonryces, yeasts and moulds Sonreof lhe mostoften remarked and isolatedmi- croorganismswere determined as speciesand their LD5g for scmehea,ry nretal ions rnrere detected.

II. MATERIALSAND lllETHODS

Soilsamplesweretakenby usualmicrobiological procedures in Julyof 1995anci 'i996. froin Srednogorie and Rczhenbiomonitoring stations. The numberof sampleswere indicatedan Tabies1 and 2. Slandard mediaof Sartorius[8] for microbiologicaltesting were used: Standard TTC, Endo,Glucose-Tryptone, Wort, Sabouraud.Some modifiedby us selectiveculture media were also used.Safiorius filtration systems were appiied.Usual microscopic techniques were used. For determinationof heavymetal ions atomic absorption spectrophotometryof suitable diluted solutions was carried out.

III.RESULTS AND DISCUSSION

The data of the microbiologicalbiodiversity were presented on Tables1 and 2. lt shouldbe mentioned there not essentialdifferences in the quantityand the varietyof the micro-organismscomparing the data fromthe bothyears. The presenceof the fourmain groups micro-organisms - bacleria, actinomyces, yeasts and mouldscould be observed.In quantitativeterms it couldbe pointedout that the data for the separate groupsmicro-organisms were comparable. As a differencein ihe datafrom bothyears could be noticedthe appearanceof nitrificationand denitrification bacteria in the Srednogoriestation samples from 1996.Consid- erablythe numberof colibacteria in all samplesfrom Srednogorie (1996) was increased.Definiiely it could be asserledthat the quantityof the actinomyces(typical soil microorganisms)in all Srednogoriesamples weresignificantly less in comparisonwith Rozhen samples. The micro-organismsindicated on Table3 (determinedas species)successfully could be usedas microbial indicatorsfor the heavymetal pollLrtion of the soilof bothsample stations - Rozhenand Srednogorie. These strainswere typicalfor all samples.Morphologically they clearlydisiinguished by other micro- organismsin the samples.Their LD5g for cadmium,copper and leadwere relatively high fl-able 3) andthat is whythese strains could be usedfor boisorptionof the ionsof thesemetals. The most often remarkedstrains o'f Rhodotorulaglutinis could be describedmorphologically as follow: The streakculture is coralred to salmoncoloredor slightlyorange, the surfaceof coloniesis smooth,often with fine transversestriations, well developedpseudohyfae or true hyfaewith largedark pigmentedtelois- pores. The coloniesof Saccharomycescerevisae are butyrous,cream to slightlybrownish, slightly raise and smoolhwith lightstriations, often sectored. Pseudohyfae were formed, well branched.Microscopically asci containingone to fourellipsoidal ascospores could be watched. The strainsbelonging to the othersspecies, indicated on Table 3 were also typicaland couldbe de- scribedsuccessfully. The datafor the contentsof someheavy metals (well known as stronglytoxic for all groupsmicroorgan- isms)in Srednogorieand Rozhensamples (1995) were presentedon Table4. lt was evidentthat the concentrationsof Cu, Pb, and Sr in the Srednogoriesamples were higher comparing these ones from Rozhen biomonitoringstations. According to manyauthors [9, 10, 11] the ionscf Mn,Fe areless toxic for the groMh of manymicro-organisms. lt was acceptedthat the ionsof Ag and Hg arethe mosttoxic for almosiall microorganisms.These metals were missing in allsamples of bothbiomonitoring stations. Depending on the toxic actionon the micro-organismsit is possiblelo determineso called"toxic line" of some heavymetals. Of coursethere are some variabilityin theselines which is in directconnection with the speciesbelonging of '10 microbialstrains.Generally these lines look like as follow. Cu, Cd, Pb,Co, Sr, Cr etc.[9, 11 12].Proba- ()l blythe increasedcontents of Cu, Pb,and Sr wasthe reasonfor the decreasedquantity of actinomyces,am- monification,nitrification and denitrificationbacteria in Srednogoriesamples. lt is well knownthat these groupsof micro-organismshave very important role in the naturalcircle of substancesand especiallyin the soil.Evidently the toxicityof the heavymetals is dependingon theircontenls and chemicalform in the soil. Of coursemany factors could influence the heavyrnetal forms in the soil.Some of themare pH, ionstrength, the contentsof organicsubstances etc. By thesereasons investigating the soilmicrobial biodiversity is very importantto foreseemany and different factors.

REFERENCES

1. BishoffB., Ecotoxical. Environm. Safety, 6, No 2, 157-162,1982 2. BabichH., G. Stotzky,Environm. Res., 36, No 1,111-137, 1985 3. DuxburyT., Adv. Microbiol. Ecol., 8, '185-255,1985 4.ZellesL., l. Schennert,F. Korte,J. EnvironmSci.,20, No 5,457-488,1985 5. KurekE., A. Franci,J. Bolag,Arch. Environm. Contam. Toxicol.,21, No 1, '106-111,1991 6. DoelmanP., L. Haamstra, Soil Biol. Biochem., 11, No 5,481-485, 1979 7. Hertkorn-ObstU., H. Frank, Forum Microbiol.,3, No6,376-378, 1980 8. Sartorius,Publication No FM-4017-e95025 9. WilsonS., A. Dean,Microbios. Lett., '12,37-42,1977 10.SakaguchiT., T. Hirokoshi,A. Nakagima.Agr. Chem. Soc. Jap.,5'1,497-505,1977 11.Lester N., R. Parry,H. Dadd,Water Res., 13, '1055-1063, 1979 12.Yang H., L. Erlich,In: Intern. biodegr. symp.3rd, Kingston, Proc. p. 196,1976

9tt Table1. Quantityand microbial biodiversity in some soilsamples, BSBCP, July 1995.

Sample Totalbacterial Yeasts Moulds Actinomycetes Ammoni- Nitrificants Denitrificants Colibacteria number number ficants Rozhen: No1 7.0106 7.5fi4 2.5 104 4.5105 1 7 105 95101 2.0rc2 No2 1.3 105 4.5104 7.0fi4 8.5104 3.5103 4.5101 9.5101 .1.0 No3 8.5106 1.0rc4 2.0rc4 105 3.6103 4.0101 9.6101 Srednoqone. No1 6.9104 1.2 103 1.0 105 ln 102 1.1102 1.0 102 No2 8.5104 1.0 104 1.5 104 2.0103 o.u 101 4 No3 4.0104 4.0 104 2.0rc4 1.2102 30 101 6.0101 6.5103

Table2. Quantityand microbial biodiversity in some soilsamples, BSBCP, July 1996.

Sample Totalbacterial Yeasts Moulds Actinomycetes Ammoni- Nitrificants Denitrificants Colibacteria number number ficants Rozhen: No1 7.0105 7.5 104 4.5103 7.0105 2.5103 o_u101 2.5 102 No2 4.0106 8.0104 7.5rc4 1.1 105 2.0 103 13102 2.4 fi2 No3 4.5106 1.7105 2.0 104 8.0105 1.1104 13102 1.1103 No4 1.1106 3.0103 4.0rc4 3.0105 2.0 104 6.0101 Srednoqorie: No1 3.2rcs 4.5103 8.5103 1.4 103 1.2rc6 1.2102 1.6 103 1.0104 No2 2.3105 5.5103 2.0103 3.5rc2 6.0106 1.2102 2.5 104 3.0103 No3 3.0104 1.8 104 3 3 104 1.2102 2.5 102 1.0 103 No4 3.0105 2.0103 s.0103 2.4rc2 2.5 104 3.0102 1.7 103 Table3. LD56of somemould and yeast strains isolated by soilsamples, Srednogorie, July, 1995 and 1996

Micro-organism LD5g,mg litre-l ^, )+ cd2+ cu2+ PO'', Aspergillusniger 01 4.4 49.4 2370 Asperqillusniqer 02 5.1 48 2730 Asperqillusterreus 01 6.5 zo. I ztJv Rhizopusdelemar 1.6 11 '1590 Sacch aro mvcopsis Ii polvtic a 2.5 245.8 845 Rhodotorulaalutinis 12.2 526 597 Sacch aro mvces cerevislae 5.O 122 Candida utilis 19 7.9 581

Table4.Thecontentsofheavymetals(ppm)insamplesfromRozhenandSrednogoriebiomonitoringstations,BSBCP,Julyl995.

Element Cr I Mn Fe Cu Zn As \,u Pb Sr Rozhen: No1 247t30 1254t125 48765 40r.4 82r.6 10r3 1r1 44x5 129r.13 No2 207t21 1281t130 52860 62+6 80r5 10i3 42t5 71t7 '1315r135 No3 366r38 62430 6216 8716 32t5 3t2 53r5 Srednoqorie: No'l 57ro 619162 45628 242r24 48r.4 42t5 2t2 17r.4 120x12 No2 305131 2078!214 61532 75t7 75t5 52t6 12r3 11grj2 No3 112!12 647r65 42568 811110 82r.10 19r4 106i1 1 A COMPARATIVE ECOLOGICAL CHARACTERISATION OF LOTIC BENTHAL ZOOCOENOSESFROM TWO STREAMS UNDER DIFFERENTANTHROPOGENIC INFLUENCE

lvanka Janeva' , LuchezarPehlivanov* , Yanka Vidinova* Sfefan Stoichev' , Violeta Tyufekchieva* . Instituteof Zoology,Bulg. Acad. Sci.

KrassimirKumanski"" "* NationalMuseum of NaturalHistory, Bulg. Acad. Sci.

I. INTRODUCTION

Investigationshave been taken in the autumnof 1995 and 1996 at followingstations: Shirokolushkariver, the RhodopesMts., hydrometricstation 477 (ca. 10 km upstream from its outfall into Vucha river, districtof Devin),and three ones of Topolnitsariver, Transbalcanvalley - above Doushantsivillage, above Chavdarvillage and below Poi- brenevillage. Shirokolushkais righttributary of Vuchariver, which, subsequently, is a right confluent of Maritsariver. lts lengthis 29,2 km, with a drainagearea of 217,5 sq km'. The station is at a heightof 750 m a.s.l.This mountainriver flows through the regionof the "Rozhen" backgroundregional station and representsone of the establishedsites for hydrobiological backgroundmonitoring. The river has been investigatedin this connectionevery year in 1980-1990period, at a stationabove Shirokaluka v. This stationis locatedca. 10 km upstream from the one object of our explorationnow. The resulls were published by Janeva& Russev(1 993). Topolnitsariver is a left tributaryof Maritsa,with a length of 154,8 km and drainage area of 1789 sq km. The mid- sectionof the river passesclosely to the industrialregion of Pirdop-Zlatitsalowns, thusbeingsubject of correspondingpollution. The altitudeh ere is between650-400 m a.s.l.Topolnitsa is, furtheron, determineda zone of the objectsof impact hydrobiologicalmonitoring and has subsequentlybeen investigatedin the period 1984-1990.However, there are no data concerningthe resultsof the investigationsof its benthiczoocoenoses and the saprobiologicalstate. The only informationin this aspect could be founded in Metchevaet. a/. (1987),where data about heavy metals conlentsin five fish speciesinhabiting the Topolnitsariver are published. Present investigationscome to representa continuationof those carried out in the course of previousperiods and representan objectivepremise for tracing the eventual changesin the ecologicalcircumstances. Thus, the main goal of the paper now presentis to appeal an actualisedcharacteristics of the compositionand structureof benthalzoo- coenosesin the investigatedstations, as a sequenceof ecologicalconditions existing' now, includinghydrochemical parameters of the stream and saprobiologicalstate of the bottom.Additional information on the impact of anthropogeneinfluence becomes available from the analysisof heavymetals'content in macrozoobenthosand fishes. The methodologyof investigationsis similarto that that has alreadybeen used in monitoring investigationsof other Bulgarianrivers (Janeva, in print).lt is based on coenology and correspondswholly to the ecosystems'analysisof the qualityof water environment; thus, there appearsthe total complexof factorswhich determinethe anthropogenechar- acter of changes.At the same time, the stabilityof zoocoenosesagainst these impact factors could be evaluatedand controllingmeasures proposed. The contents of heavy metals and toxic elementsin zoobenthosand fishes were analysedby Dr. E. Nikolova and Dr. A. Artinian(lnstitute of NuclearResearch and NuclearEnergy by the method of XRF analysis,(Nikolova & Artinian,1993).Hydrochemical analysis of 1995 water samples of Topolnilsariver have beentaken in the Chemicallaboratory at the Instituteof Zoology, BAS, by the specialist Mr. lvan Bolev.

Here and furtheron hydrologicaldata are from the "Hydrologicalrefere nce-book of riversrn Republicof Bulgaria"(Ed. Head Departmentof Hydrol.& Meteorol.,S., Vols. t- tv, 1957-1984)

t0I II. RESULTSOF THE INVESTIGATION

RegionalBackground Station "Rozhen" Shirokolushkariver (hydrometricstation 477, above outfallinto Vucha r.)

11.1.Biodiversity

The stationis a typicalbig mountaintorrent, well correspondingto the hyporhitralzone of continentalfreshwaler streams. In course of the two investigationsof this stationa total of 58 taxa of species level have been establishedamong the benthon,they belong to one of the 13 basic groups (Table. 1). Most numerousare the representatrvesof the insect order Tricltoptera(20) and Ephemeroptera(12). They dominatealso in the total quantityof specimens.Among Ephemeropteradominant in both years are Baetisrhodani, Ecdyonurus gr. venosus,8ae- tis alpinus. DifferentTricltoptera are permanentlydominating in the station:among the almostorn- nivorous larvae of fam. Hydropsychidaesuch are Hydropsyche incognita (so far mentioned as H. pellucidulain prevailingpart of publicationsfrom differentsites of Bulgaria, beforeseparation of those close pair of speciesdone by Pitsh,1993)), as well as Hydro- psychesp cf. bulbifera(group of guttata)and H sp.gr instabilis(although the very high probabilitythese are bulbifera,resp. instabilisindeed, the lack of adults in the samples does not give a 100% warranteefor that ). Free living larvae of Rhyacophilanubila and Rh. obliterataare the most common representativesof predatorsof Rhyacophilidaehere Case-bearingTrichoptera, also abundanthere, are representedby both the sericostoma- tid Oecismusmonedula, and severallimnephilidis, the lathers hardly determinableafter immaturestages so far, but most probablybelonging to some Stenophylacini-genera,e.g Halesus, Micropterna).In the materials mentioned as "Limnephilidaegen. sp." most probablydominating are larvae of Drusus spp. (emergingmainly between May-August) and the autumntribe of Chaetopterygini,Chaetopteryx stankovici being the dominant,but surely not the only species.All these case-bearingcaddisflies represent the detritopha- gous and phytophagous Trichopteracomponent there.

11.2.Saprobiological state

On the basis of the saprobiologicalcharacterisation estimated by saprobiologicalindex by Rothscheinand saprobicindex by Pantle& Buck,the investigatedstation is qualified as oligosaprobeduring two yearsof investigation(Table.2). Besides B. rhodani, C. macrura, E. ignita (Ephemeroptera);H. incognita, H. bullbifera and Rh. nubila (Trichopteral,which are ecologicallybroad talentedindicators, inhabiting p- mesosaprobeor even (r- mesosaprobestreams, mosl of the rest of the benthic coe- nose comprisesspecies typical for the xeno-or oligosaprobezone.

11.3.Content of heavy metals and toxic elementsin zoobenthon

In 1995 the contentof iron (Fe), manganese(Mg), zinc (Zn), copper (Cu) and arsenic (As) is relativelyhigh (Table.3), while duringOctober 1996 all quantitiesare considerably lower. A presence of the toxic lead (Pb), cadmium (Cd) and selenium (Se) is not found.Only relativehigh quantityof strontium(Sr) is established.

11.4.Generalised ecological characterisation

Out of analysisof the speciescomposition, as well of the values of the structurepa- rametersand the saprobiologicalassessment of the station,it can be concludedthat the zoocoenosesare stableand ecologicallycomplete. lt is statedalso after a comparisonof the basic structuralparameters in differentperiods of investigations(Table.4).

lll. "sREDNoGoRlE" 102 Topolnitsa river (above Doushantsi v.; above Chavdar v.; below Poibrene v.)

lll. 1. Biological diversity

A total of 66 taxa of specieslevel belongingto 17 benthicgroups at the three stations are establishedduring the studies(Table.5). Two benthic groups are most numerous- order Ephemteroptera( 15) and Tricltoptera (15), followedby Plecoptera(7) and Chironomtdaefam. (Diptera)- 4. ln accordancewith relativequantity Oligochaeta is a donrinant,too. The Topolnitsariver at ihe first station has a hyporhithral-epipotamalappearance. Thus, togetherwith representatives,typical for the potamal(e g. H. incognita,H. bulbifera, H. modesta - Tricltoptera, E danica, E. ignita - Ephemeroptera) preference of rhithral(or obligatethere) are representedwith rnostspecies, although in lesserquantity Rhyacopltilacf. fasciata,H. tabacaruiand, above all, severallimnephilids (Potamophylax sp., Stenopltylacinigen. spp , Halesus sp. etc.) contributeto the transitive pattern of outlookof the station. The two last stationsshould easily be referredto the epipotamal,where lhe Hydropsy- che-complex and representativesof Baeticlae,Oligochaeta and Diptera are collected there.

lll. 2. Saprobiological state

Duringthe autumn 1995 Topolnitsariver above Doushantsiv. is oligosaprobic,and in 1996 - [)- mesosaprobic.The othertwo stationsduring these periodsare l']-mesosaprobic (Table.2). The values of the most hydrochemicalparameters of flowingwater characterisehigher cleanness- within the oligosaprobity(Table 6) In this respectvery high values of the water dissolvedoxygen make a great impression.The values of nitrrtesand phosphates at second and third stations(above Chavdarv. and below Poibrenev. respeclively)are pointingto a [1-mesosaprobity too. The waler of the both stationsis with higherhardness and contentof nitrites(polisaprobic values).

lll.3. Content of heavy metals and toxic elementsin benthon

Most quantitiesof Fe, Cr, Mn, Zn, Cu, Pb, Sr, Cd, Se and As in macrozoobenthonare established.Among the strongtoxic elementsupon living organismsAs, Pb, Cd, Sr, Se are with highestquantity. (Table 3)

lll. 4. Content of heavy metals and toxic elements in fish tissues

The shub (Leuciscuscephalus) and goudgeon(Gobio gobio) are used like indicating fish species.They are caughtat pointsabove Doushantsiv. (young-of-the-yearand older specimens)and below Poibrenev. (young-of-the-yearspecimens). The whole young-of- the-yearfish and separateorgans of the older (respectivelybigger) ones are examined. The attentionis orientedmainly towards five elements:Cu, As, Sr, Cd and Pb. No Cd content has been establishedin the tissues of young-of-the-yearfish. At the same time, in the specimensfrom this group,caught below Poibrene v., the concentration of As and especiallyof Cu is much higher, than in fish, caught at the point above Doushantsiv. (Table.7). However,it should be noted the considerableexcess of the content of Cu in the tissues of examinedfish in both points comparedwith the values, establishedin 1985/86(Metcheva et al.,1987), this excessat the point below Poibrenev. being very expressive.In our view, these resultsare suggestinga prolongedpollution, mainlyby Cu-salts,produced by the cooperworks nearly. A reversetrend in the distributionof the concentratronof Pb was registeredin the tissues of examinedfish, caught in both points(Table.7), which is more difficultto be explained.The comparisonwith the data, obtainedin 1985/86,shows, that the values of this index in young-of-the-yearfish from the conditionallyclear zone of the river remain almost unchanged,while the concentrationof Pb in the gills of the older specimensis considerablyhigher. lt appearsthat the resultsnow obtainedshould be connectedwith the influenceof backgroundpollution by Pb, accumulatedin the bottom sediments.The

l():l higherconlent of this elementin the gills of older fish is probablyone resultfrom its accumulationin the osseoustissue. A comparativelyhigh contentof Sr in the tissuesof fish is, evidently,connected with the presenceof Pb as well. lt is known that this elementis been accumulatedmainly in osseoustissue, what explainsa clearlyexpressed differentiation of its distributionin the bodies of fish from older age groups (Table 7) A comparisonof the Sr-contentin the tissuesof fish and in ones of zoobenthosfrom Doushantsiv. regionis a good exampleof its accumulatjngtrough the food chain.

lV. Compendious ecological characterisation of investigated stations

|V.1. Topolnitsa river, above Doushantsi v.

The ecologicalsituation there seem to be close to naturalone accordingto the zoocoenoses'composition,structural parameters, dominants and saprobiologicalassessment. The zoocoenosesare stable and ecologicallysufficient. By previousinvestigation (during 1983-1987period) - it was provedthat this stationhas an analogousecological characterisation(unpublished data of Kovachev& Uzunov).The value of the coefficientof speciessimilarity in 1995 and 1996 is too high - 45,71.There are minimumor completely absentquantities of heavy metalsin zoobenthon,only with an exceptionfor Sr.

|V.2. Topolnitsa river, above Chavdar v.

This station is situated below the outfalls of Medetska and Pirdopska rivers (main contaminators,which transfera pollutedwater from variousproductions), and above the outfallof Mirkovskariver (a clear riverwith rich faunisticcomponent). During the two periods of investigation,16 taxa of specieslevel belongingto 10 benthicgroups are established.In 1995 there are 10 taxa of specieslevel from 7 groups.Representatives of the Mollusca type and of the orders Plecoptera,Amphipoda, lsopoda, Odonata, have not been established. The order Dipterais representedwith a large numberof taxa, which are single at the rest of presentedgroups. According to their relativequantity, the representativesof the cfassof Oligochaetaare dominants,followed by Baetisrhodani (Ephemeroptera). In 1996 at this stationa total of 1'1taxa of specieslevel belongingto 7 groups are established. There is only one caddisfly-species(Trichoptera) and representativesof Ephemeroptera and Plecoptera are missing. Regarding to the total numbers, only the Oligochaeta- speciesare dominants.At first sight,these facts are not correspondingto [.]-mesosaprobity, which is supposedto be an "ecologicaloptimum". According to Kovachev'& Uzu- nov's unpublisheddata, no zoobenthonhas been establishedat this place, probablybe- cause of the strongtoxic effectof the runningwater presentedthere. This suggestionis proved by resultsof heavy metalscontent'analyses of water and some fishes (Metcheva et al., 1987),(Karapetkova, unpublished data). The resultsof our investigationsdemon- strate a low contentof heavy metalsin flowingwater and at lhe same time - an unusually high contentof these elementsin zoobenthonand fishes(Table 5, 7). In this connection the highercleanness (oligosaproby) of the runningwater could be explained. Therefore,our observationsduring the 1995-1996period document processes of zoocoenoses reslorationas a positiveresponse to the diminishingnegative anthropogene impact. Usually these processesare rather dynamic and inconstantwhich provokes a dynamicsin speciescomposition too. Probablyit is a reasonfor the low value of the coefficientof speciessimilarity - 20,00.

1V.3. Topolnitsa river, (below Poibrene v.

Duringthe two periodsof investigations28 taxa of speciesbelonging lo 12 basic zoo- benthic groups are established.Trichoptera, Ephemeroptera and Diptera orders have a large numbersof taxons.In 1995there are 17 taxonsfrom 8 groups.Ephemeroptera- and Trichoptera-species are dominants.Ephemeroptera order dominatesas regards to the total numbers(400 specimens).In 1996 the representativesof Oligochaetaare most numerous,but their numberis considerablylesser than in the previousstation (accordingly i0-t 160 and 2000 specimens;.In contraslto O/igochaerarn 1996- study lhe EphemeroStlera order is representedwith singlespecinrens r;f Baellsfuscafus The saprobiologicalstate of the staticrnis withirrtfre franresof [i-mesosaprobyduring the two-yearsperiod of investigationwhich is a considerablybetier state, comparedto the correspondinq1984-1990'period Acccirciirrqto unpublrsheddatil, Iopolttitsariver at that time, and sarnesecIion was polysaprobicto,r-ri-resosaprobiu whicir had been proved by species compositiori,the structureof zclococr.ros€sas well as by the heavy rnetals' quantities,accurnulated in the zoobenthonand fishes As in lhe previousstation, processes of stabilisationof ecolooicalstate are avarlatrlehere too

V. CONCLUSION

I he investigatedstalion in "Rozhen"-region is a typical oligosaprobicriver section. srluated in a zone ur)der background influenc:eThe cornpositionof the river fauna, slructureof benthic zoocoenoses,saprohriological assessnrent durirr g 1995-1996period as well as the dynamics of observed parametersirr comparisonwith past periods of studies( 1980-1990) confirm this conclusion The section of Topolnitsanver above Doushantsiv whrchis not under anlhropogene impactcould be similarlycharacterised. Topolnitsariver above Chavdarv. and belowPoibrene v. is ll-mesosaprobic.The fauna compositron,zoocoenoses struclure, as well as the hydrochemicalwater analysis from these stationsdefine the ecosystemsin a processof restorationafter a continuousnega- tive anthropogeneeffect. However, that cannot be acceptedas a !astingtrend since the two-year investigationssuggest an instabilityof the process.In spite of that the heavy metals'and toxic elements'(especiallyCu) contentin zoobenthonand fish in this area provethat the industrialimpact is stillconsiderable

VI. ACKNOWLEDGEMENTS

We thank to Dr. Nikolovaand Dr. Artinian(lNRNE, BAS) for leavingthe resultsof XRF analysis,to Dr. St Kovachevfor determrnationof Simuliidae(Diptera) material, as well as l. Botev for hydrochemicalanalysis

VII. REFERENCES

Metcheva.R, M. Karapetkova,S. Geiasimov.1987. An accuinulationof heavymetais in bioindicaicry speciesof vertebrateanimals from the Srednogorieregion.- l'' f.lai.Conf. Probl. Biol. Monrtor- tng.22-24 Ocl., Plovdiv, 174-177 (in Bulg.) Janeva.l, B. Russev 1993 Hydrobrologicalmonitoring on riversections on the "Rozhen"and "Boatin" .14, ("Steneto")regional background monitoring stations - Lauterbornia, Dinkelscherben,79-84. Janeva.| (lnprint). An approachand results of thehydrobiological monitoring in Bulgaria. Nikolova.E, A Artinian.Y. Karamanova1993 Determinationpar la methoderoentgenfluorescente de lracesd'6l6ments dans des echantillouspreleves dans la regiondu picde Moussala- Obser- vatoirede Montagnede MoussalaOM2.'1 . Pitsch,T. 1993 Zur Kenntnisder Hydropsyche pellucidula - Gruppe in Mitteleuropa(Trichoptera. Hydro- psychrdae).-Braueria, 2O, Lunz, 27 -32.

105 Table1 Zoobenthonfrom Shirokolushka river (HS477. above outfall into Vucha)

Group/ September Octomber Taxon 1995 1996 1 2

Turbellaria Dugesiagonocephala D. lugubris-polychroa

Nematoda Monchisterapaludicola

Oligochaeta indet.

Mollusca Gastropoda Ancylusfluviatilis Lammellibranchia Pisidiumsp. Sphaeriumsp.

Amphipoda Gammaridae Gammarusbalcanicus

lsopoda Asellusaquaticusus

Ephemeroptera Baetisalpinus B.fuscatus + B.melanonyx + B.muticus ? B.rhoda ni + B.scambus T B.vernus. Caenismacrura Ecdyonurussp. Epeorussylvicola Ephemeradanica Ephemerellaignita

Plecoptera Capniasp. Dinocrascephalotes + lsoperlaburesi 7 Leuctrasp. + Perlamarginata +

Coleoptera Elmissp. Stenelmissp. g.sp.imago

I06 Table1 (conlinuation 1 2

Trichoptera f ChaetopterYxstankovici HydropsychesP'.gr.guttata i H.sp".gr.guttata .t H sp.cf.bulbifera + H. sP.cf.incognrta +' + H. incogntta + + H.sP.gr.instabilis + Limnephilidae,gsP f LyPereducta + Oecisrnusmonedula + PolycentroPussP.cf + + flavoma-culatus RhyacoPhilanubila + + Rh. obliterata + Rh.sp.cf.tristis + Rh.sp.gr.vulgaris, ? cf. oblterata Sericostomatidaeg.sP. i + S.flavicorne ? SilosP. + + StenoPhYlacinig.sP. ? S. ,cf.MicroPternasP. + Diptera Blephariceridaeg.sP. Simuliidae Cnethabertrandi C. verna OdagmiarheoPhila Simuliumreptans ? Chironomidae ChironomusriParius i DicrotendiPesnervosus + Eukiefferiellagracei + PolypedilumPedestre + Ceratopogonidae Bezziasp. Athericidae Atherixsp.

Table2. Saprobiologicalassessment of the investigatedstations

"Rozhen" "Srednogorie" lndexof Shirokolushka Topolnitsadver Years nver Above Above Above Below saprobity Shirokaluka Doushantsi Chavdar Poibrene villaoe villaoe villaqe village 1995 7250-o 5713-lr 4977-1t

(Zelinka- 1 qqA 70,82-i 6A A1- r{ 46,42-| 44,98-lJ Marvan) 1995 1,'18-i 1,54-lr 1,62-lJ 1,88- ll DPU (Pantle-Buck) 1996 1.22-o 1,52-lr 1,78- ll 2,02-ll

I(17 Z

Table3. Contentsof heavymetals and toxic elernents (water and benthon ppm) of the investrgated stations

"Rozhen" "Srednogorie" Shirokolushkariver Topolnitsariver

Z AboveSh luka AboveDoushantsi AboveChavdar BelowPoibrene

1995 1996 1995 1996 1995 1996 1995 1996 berrthon benthon WAIEI benthon benthon WAICT benthon benthon WAICT benthon benthon t'- r 125!67 41!6 trace 6!2 trace 6t2 trace 108r35 233!32

Mn 1431i55 361r16 002 494!41 1250!120 004 1225!88 51 60i302 0,05 186016.1 67581650

Fe 21740t27 4 55541180 0.11 2810!205 51231460 0,15 4895115 861 01362 2511 4t1 192 1805011 707 6 Cu 77!5 42!2 trace 3814 7016 0.05 652r56 1137 !37 0.05 1058132 1627!134

Zn 573r10 202!5 0.01 405135 590t57 0,02 1280165 32316 002 618!25 547!57

I zaz 6r1 4x1 4!1 24!4 10011 0 49r5 1812

31r3 9r1 I zaz 40!2 19t2 9r1 31i3 10r1

4al 10!2 8r.2

Pb trace trace 20!2 0,2 23t2

Cd trace 3r1 trace 14!2 5i1 trace 11t1

Ag JTI Table.4.Structure Parameters and Saprobiological Index from Shirokolushka riverforthe 1980-1990 and 1995-1996periods

S H d e c K5 Kp Sp Station Date Totalnumber Individual Totalvariety Equalization Domination (S"pn"r- '(N"pn"t. of species varietyof of species Sp1""/S) Np1""/N) soecies

06.1980 13 2,8 6.4 0,77 0,24 46,15 42JA 81,83

04.1983 21 4,0 9,6 0.91 0,07 57.14 64.52 73,60

09.1984 25 3,8 10,2 0.82 0.10 56,00 69,23 69.39

10.1 985 24 3,0 10,0 0.66 0,24 33,33 70.77 72,23 Shirokolushka riverabove 09.1984 33 3,2 11,4 0,63 0,20 3S,89 47,30 69.13 Sh.luka village 08.1987 35 3,4 12,2 0.67 0,15 25.71 37.29 58.16

10.1987 32 3,8 13.2 0,77 0,11 34,38 54,88 71,00

10.1988 44 3,5 14,2 0.64 0,16 36,36 71,96 80,85

07.1989 38 4.0 15,0 0,76 0,1'! 28,95 67,93 64,86

05.1990 36 4,0 12,2 0,78 0,10 38,89 39,04 60.14 wP 477 (aboveoutfall 09.1995 37 2,2 9,76 0.44 0,33 27.78 90,47 72,50 intoBucha river) 10.1996 36 3,4 12.7 0.67 0,15 32.43 72,78 70,82 Table5. Zoobenthonfrom Tooolnitsa river orie") AboveDoushantsi vill. AboveChavdar vill. BelowPoibrene vill.

lx.1995 tx.1995

Nematoda Dorylaimusstagnalis i Monochustruncatus ?

Oligochaeta,indet.

Hirudinea Erpobdellaoctoculata Haemopissanguisuga

Mollusca Gastropoda Ancylusfluviatilis + Radixperegra ?

Lamellibranchia Pisidiumsp.

Amphipoda Gammaruskomareki Gammarussp.

lsopoda Asellusaquaticus

Fnhemprnntpre Baetisalpinus + B.buceratus I ? B.fuscatus + ? B.lutheri t B.rhodani i + B.scambus ? + B.vernus ? Caenismacrura Caenissp. + Ecdyonurussp. i t Epeorussylvicola ? + Ephemeradanica + + Ephemerellaignita ? + Habroleptoides + confusa Paraleptophlebia sub-marginata

Plecoptera Amphinemurasp. Capniasp. T lsoperlasp. i Leuctrasp. + Nemourasp. + Perlasp. + Perlodesintrrcata

Odonata indet.

ll0 able5 (corrlrnuatlon a A 1 J 6 7

Coleoptera ElmissP. Stenelmissp + Dytiscidae.g.sP + Heteroptera- indet.

Megaloptera Sialissp.

Trichoptera Hydropsyche bulbifera H sp.cf.bulbifera H sp.cf.instabilis H. modesta T H sp.cf.modesta H.spcf.incognita + H.sp.(grguttata) ? H spp + + H.in cog n ita i H.tabacarui - Potamophylaxsp. + Rhyacophilasp. i (gr.vulgaris) Rh.sp.cf.fasciata + Stenophylacini,g.sp. + St.,cf.Halesus sp

Diptera Blephariceridae Gen.sp i Tipulidae Tipulasp. + + Limoniidae + Gen.sp f Hexatomasp. Dixidae Gen.sp. + Simuliidae Gen sp. i Eusimuliumlatizonum i Chironomidae Chironomusriparius + + + Cryptochironomus defectus i + T Tanytarsusgregarius Tveteniacalvescens t + Ceratopogonidae BezziasP. + Tabanidae Tabanussp Empididae Gen.sp. Heterodromiasp. Fam.,oen.sp.

lll Table 6 . Hydrochemicalparameters of the watersamples from Topolnitsa river

Parameters AboveDoushantsi AboveChavdar BelowPoibrene villaoe villaoe villaoe

4 I z 4

02 - mQ/l 10,6 7,9 8,7

02' o/o 102,7 83,8 90,7

KMnOa 2,32 2,72 2,32 Oxidabilitv-mqO;/l Xf'lK - mg/l 9,2 13,6 11,3

HCO3- mg/l 45,7 to,z 149,4

Alcality- mgeqv/l 0,75 1,25 2,45

Ca - mg/l 8,02 90,18 100,2

Mg - mg/l 3,65 9,73 Iz,z

Hardness-dH" 1,96 14,86 16,8

NH4- mg/l 0,04 v,t z 0,06

NO2- mg/l 0,0'1 0,49 0,19

NO3-mg/l 0,29 5,98 7,06

Table 7. Contentof heavymetals and toxic elements (ppm) in fishtissues from Topolnitsariver

AboveDoushantsi villaoe BelowPoibrene villaoe Element young-of- gills bones muscles young-of-

the-year the-year

Cu t!. 1 13rl 253t11

As 10t2 12r2 8i1 15t2

Sr 92t5 14311 1 147*11 2x1 86i5

Pb 13t2 24!2

cd 9t2

t12 ESTIMATIONOF THE ENVIRONMENTALQUALITY USING MONITOR SPECIESSMALL MAMMALS FROM TWO REGIONS WITH DIFFERENT ANTROPOGENICINFLUENCES IN BULGARIA A. Bioaccumulationof Toxic Elementsand their Influenceon some Hystologicaland HaematologicalIndices.

RoumianaMetcheva-, Ari Artirilan"',Emilia Nikctlova", Michaela Bellcheva', Routtiaita Laleva* *lnstituteof Zoology,BAS, **lnstitutefor Nuclear Research and Nuclear Energy. BAS

I. INTRODUCTION

Regionalenvironmental contamination is a currentproblem for BulgariaThe purposeof the presenlstudy is to assesthe environmentalstate of two regionswith differentanthropogenic influence - the industrially pollutedSfednogorie and the lesseffected by pollutiorrarea of Rozhen- Rodops,using small mammals as monitorspecies. In additronmethods and criteria were worked out to be appliedin the biomonitoringinvesti- .1987, gations(Metcheva et al. Gerasimovet al. 1987,Metcheva, 1995). To demonstratethe effectof the industriallypollutants different type of analysishave been done: . Bioaccumulationand distribution of heavymetals and toxic elements . Hystologicalinvestigations ofsome organs and tissues . Haematologicalindices

II.MATERIAL AND METHODS

The monitorspecies .rn.,, r.r*als - commonvole (Microtusarvatis) and wood mouse (Apodemus sylvaticus)were caught using baited live and snap traps at a 1 km in the directionof the marginallyprevailing windfrom the copper- producecombine in Pirdop(Srednogorie) From the backgroundregion of Rozhen- Rodopsusing the same methodwere caughtalso commonvole, bank vole (ClethrionunysglareolLLs,), pine vole (Pitymyssubterraneas), yellow necked wood mouse (Apodemusflavicollis), and the commonshrew (Sorexaraneus). All the collectedadult animals were dissectedto removethe intestinaltract with contents,the internal organsand tissues:livers, kidneys, spleens, bones, blood and muscles.The sampleswere oven-driedat 60"C for approximatelythree days to obtainreference weight and analysedby X-rayfluorescence spec- trometerXRFI (Nikolovaet al.1993). For histologicalanalysis necroscopic material from some internal organs (liver, kidneys, spleen and adrenalglands) were fixed in 10o/oformalin. The preparedfrozen paraffin sections were painted with hematoxilin- eozin(Romeis, 1968). Bloodsamples were taken from the variousmonitor species from the sublingualvein in vivo The percentageof variousforms of cellswas determinedusing Gimsa stains. Hematocrit and ihe morphological characteristicsof erythrocyteswere determined by a standardclinicalmethods (Kostelecka - Myrcha 1967)

III.RESULTS AND DISCUSSION

Bioaccumulationand distrrbution of heavymetals and toxic elements in differentorgans and tissues in the monilorspecies of smallmammals The resultsfrom the industriallypolluted region of Srednogorieand the backgroundstation of Rozhenfor '1995 and'1996are presented on tables 1,2,3,4,5,6 and7. Comparisonof the dataof the toxicelements concentrations show that some differences among the bio- accumulationin the organismof the differentmonitor species from the two regionsexist ln the bodiesof the animalsfrom Srednogorieone of the mosttoxic element Cd was not foundin neitherof the internalorgans. The concentrationsof thiselement in the differentspecies from Rozhen varies from 1 - 3 ppm in the bodies of volesup to 15 ppm in the micebodies The highestvalues were found in the voleskidneys - from6 to 9 ppmand in the micernuscles - from 4 upto 16 ppm.Aspecial attention must be paidto the livers(17 ppm ) andkidneys (50 ppm) bioaccumulationof the common shrew (Sorex araneus). that occupies highertrophic level. The heavymetals concentrations of Hg and Pb were presentedin no dangerouslevels Hg was regis- teredfor the firsttime in the liverof woodmice ( 2 ppm) fromSrednogorie (Table 2). In the bodiesand in the internalorgans of Microtusanvalis from the sameregion lead was not foundbut in wood miceorganism the concentrationvaries from 3 ppmin the musclesup to 17 ppmin the kidneys.[able 2) The cornparisonwith the samemonitor species from the non-pollutedregion of Rozhenshows absence of leadin the organismof

lli commonvoles. (Table 5). This element varied from 2 pprnin the oodesto 5 pprnrn tiie iiversof woodrrice The concentrationswere significantly lower than that found in ihe satnespecres from Srednogorte (Table 6) Thebioaccumulation of lead in the other monitor species was very unevenly distributed O'able 3 Table4. Table7 ) The comparisonwith data obtained for Srednogorie 10 yearsago (Metcheva et al 1987)shov,t tirai rri the bodiesof the monitorspecies Microtus arvalis and Apodetnus sylvaticus the concentratiottsof Co Pb Cu -[he and Co were very closeto the valuesobtained in the presentstudy comparisonfor the sarnernonrtor speciesand elernents,again for the body but from regionof Rozhenshows littlediffererices in data (Metchevaet al. 1993).The presentinvestigation shows significant higher concerrti-atrcn for Cldand Pb in the bodyof thewood mice.

Table1 Concenlrationof heavymetals and toxic elements In mg/kgdry bodywerghi in the commonvole (Microtus arvalis) from Srednogorie region

Table2. Concentrationof heavymetals and toxic elements in mglkgdry bodyweight in thewood mouse (Apodemus sylvaticus)from Srednogorie region

ELEMENT BODY LIVER KIDNEY SPLEEN BONES MUSCLES BLOOD (-r

Mn tal '1 Fe 243t19 JJf,+JC 249x19 1032t134 11 1t10 136r1 1426!113 Ni zr:I Iz+J Cu 10!2 I u7:z I v-rz 36r8 20+4 '10619 Zn 109t4 76!4 91tl3 107!11 67r5 15r3 As 10!2 Se 1t1 1r1 Qr 14!2 3t1 311 Mo 7xl 5i'l Ag C.1

Hg zrl

Pb 5t1 ILl I t-rz 3!2 3x2

lll Table3 Concentrationof freavymetals and toxic elernerrts irr mg/kg dry bodyweighl n the prrtevole (Pttyrnyssubtenanetrs) frorn backgrourid regir-lrr ol Ruzfren

ELEMENT BODY LIVER KIDNEY SPLEEN BONES MUSCLES BLOOD Cr lJ1 | zrl 9t3 19f3

Mn tz-L I UIJ Jtrlf, Fe 417r33 11 09:76 OJIJ f J3 1435188 234\1 5 250t15 Ni Ar1 17t5 'l5t4 LU 911 8:.2 43i8 5x2 Ztt 124r7 tz3+3 I tf,to 98r14 123!15 59 16 11!2 o+l ctl Se )r tJtz trl 78r5 611 Mc 7t2 8t1 16x2 5t2 Ag C1 |

14 Cd 1 f 1 t-l 7t1 4+1 |ts Pb 16t2 3t2

Table4 Concenlrationof heavymetals and toxic elements in mg/kgciry body weight in the bank vole(Clethrionomys glareolus) from backgroundregion of Rozhen

ELEMENT BODY LIVER KIDNEY SPLEEN BONES MUSCLES BLOOD l"r 18r5 36r5 24t10 41 ^ Mn I tl:z 75110 Fe 268118 '13311715681101 1227!256 217t22 1891 t1 56 Ni 1+1 15r3 UU 15!2 Iz1 z lOl:Z 10!2 32t5 46x2 Zn 9614 tzo!5 11316 19011 1 120!45 8316 21x4 As tal Se ar 14!2 YlZ 2t1 48t5 Mo 4x2 Ao (.l I1l 8t1 1613 Hq Pb trl 5r1

Table5. Concentrationof heavymetals and toxic elements in mg/kgdry bodyweight in the commonvole (Microts arvalis)from background region of Rozhen

ELEMENT BODY LIVER KIDNEY SPLEEN BONES MUSCLES BLOOD Cr 2615 Mn 23t5 Fe 331t32 830t11 0 590r35 1521x152 296186 152!15 11 80145 Ni 5!Z 6!2 Cu R+? 18t5 25!4 4x2 Zn 101r9 137t3 120!4 521.5 102x10 50r5 19t2 n5 Se Sr 6+2 o-f z 4!2 Mo 3x2 Ag 9r3 iA 1+1 t1l 6r1 1t1 12+3 Hg ph

ll5 Table6. Concentrationof heavymetals and toxic elements in mg/kgdry bodyweight in the yellow-neckedwood mouse(Apodemus flavicollis)from background region of Rozhen

ELEMENT BODY LIVER KIDNEY SPLEEN Cr 9t2 Mn M 4015 Fe 330121 6516t308 758t19 1264t108 Ni 5+Z 6x2 UU 9x2 8!2 12x2 21t3 Zn 9812 119!4 120x5 148132

/-\5 Se 15!2 32t3 Mo 3+Z 5t1 5t1 Ao Cd 15!2 tStz 9t1 Hq Pb z+z 5t1

Table7. Concentrationof heavymetals and toxic elements in mg/kgdry bodyweight in the commonshrew (Sorex araneus) from background region of Rozhen

ELEMENT BODY LIVER KIDNEY SPLEEN Cr Mn Fe 415146 759161 349185 301t49 Ni LU 17!3 7!2 Zn 126!7 128x7 3013 34t3 As 5t1 Se \r otl Mo 1t1 5t1 13t2 As cd tal 17!3 50t7 8!2 Hq Pb | 5iz

IV. HYSTOLOGICALANALYSIS OF MONITORSPECIES

In all the investigatedinternal organs of the monitorspecies from Srednogorieregion morphological changeswere not found.Only in the liversof two woodmice (Apodemus sylvaticus) in the lobuleperiphery focallymphoid agglomerations were found. Similar pathologic changes in the liversis possiblyto be considered in presenceof heavymetals or toxicelements in the environment.Hollerer and Coduro(1977) and Johnsonand Roberts(1978) mentioned that in the smallrodents inhabiting industrially polluted area it is possibleto findsome poorlyexpressed distrophycal and necrobioticalchanges. In the monitorspecies from Srednogoriesuch changes was notfound The hystologicalanalysis of the internalorgans tissues of the animalsfrom Rozhenshow a normalmor- phologicalstate.

V. HAEMATOLOGICALINVESTIGATIONS

The resultsfrom the haematologicalanalysis provided in the autumnperiod of '1995and 1996are presentedon tab.8, 9, and10. Allthe investigated red blood parameters in the monitorspecies small rodents - miceand voles show that the haematologicalindexes - Hb,Er, Lc andHt werein the physiologicalstandard. The analysisof the whiteblood picture for the threeindividuals of the commonvole from Srednogorieand ll(r Rozhenshow abserrceof eosinophylic,basophilic and plasmocytecells. Neveflheless the numberof lym- phocytesin the volesfrom Srednogorie was higher than the numberin the cornmonvoles from Rozhen. The cclmparisonbetween the woodmice from the two regionsshow presence of singlebasophilic and Stab cells. Onlyin one pinevole individual from Rozhen the lymphocytecells richer in cytoplasmwere found. Some lymphocytesinclude 4 to 6 nucleoliin the nucleuswith prevailing of ripelymphocyte forms. The red bloodpiclure was assessed using of three inclices- hypochromy, anisocytosis and erythrocyteswith pathologicchanges 68 % of allthe investigatedvole species from Rozhen in 1995show nornrocytosis and normochromy. ln 32% from all investigatedanimals mild hypochromyand erythrocyteswith polychromatophiliccyto- plasmwere found. Pathologrc inclusions in erythrocyteswere not obserued. In 4 of Microtusspecies lhere were found 4 to 8 erythrocyteswith pathologicchanges - polychromato- phyliccytoplasm. In one vole it was found3 erythroblasts/1000 erythrocytes. lt meansdisturbance in the riperiessof the nucleus 50% of the investigatedcommon voles from Srednogorieshow normocytosisand normochromy.The otlter50% show normocytosisand mild hypochromywithout pathologic inclusions in erythrocytes.Harder normocytosisand hypochromyin 90%were observed in the woodmice. In the same percentagebasophilic granulatederythrocytes in micewere found. All the investigatedindividuals show slow hemoglobinoforming (Erwith polychromatophilic cytoplasm)

Table8 Red bloodcells morphology in differentmonitor species from the backgroundstation of Rozhenand the imoactstation of Srednoooriefor 1995and 1996vear.

ER WITH SPECIES YEAR N ANISOCYTOSIS HYPOCHROMY BASOPHILIC GRANULATION (o/oo)

ROZHEN

Pitymys 1995 normocytosis normochromy n subterraneus' Microtus 1995 o normocytosrs mild 0 arvalis hvpochromv Clethrionomys 199s normocytosis normochromy 0 olareolus Clethrionomys 1996 1 normo expressed 0 olareolus microcvtosis hvoochromv Apodemus 1996 1 normocytosrs normochromy flavicollis x two of the animalsare with mild normocytosis and mild hypochromy

SREDNOGORIE

Microtus 1995 8 mildnormo and normoand mild 5.8 arvalis* microcvtosis hvpochromv Apodemus 1995 mild normo and normoand mild 7.5 svlvaticus microcvtosis hvoochromv Apodemus 1996 z normocytosis normochromy 0 svlvaticus * ln all lhe Apodemusspecies erythrocytes with pathologicchanges polychromatophilic cvtoplasmwas observed.

I l'7 Table 9. White bloodpicture of the differentmonitor species from the backgroundstation of Rozhenand the impactstation of Srednogoriefor 1995year.

LYMPH. SG MO BA STO SPECIES YEAR N fi10e) fi10s) (x 1oe) fi 1o'g) (x 1o'g)

ROZHEN

Pitymys 1995 7 0.75t0.12 0.14r0.09 0.11t0.06 0 0 sbuterraneus* Microtus 1995 6 0.5510.120.4910.14 0.04r0.05 0 0 arvalis Clethrionomys 1995 3 0.7010.14 0 0.3010. 1 4 0 n olareolus Clethrionomys 1996 1 0.70 0.20 0.10 0 n olareolus Apodemus 1996 1 0.85 0.05 0.05 0.05 0 flavicollis

SREDNOGORIE

Microtus 1995 I 0.7810.13 0.18r0.120.04*0.04 0 0 arvalis* Apodemus 1995 5 0.7710. 1 0 0.0810.05 0.15+0.08 0 0 sv/vaticus Apodemus 1996 z 0.79 0.07 0.13 0 0.01 svlvaticus

Table 10. Bloodparameters in differentmonitor species from the backgroundstation of Rozhenand the impactstation of Srednogoriefor 1996year.

NUMBERSPECIES HB [G/L] HT ER [.1012/L] LEUC[.10e L]

ROZHEN

1 Clethrionomys 131.56 0.39 4.10 3.40 glareolus 1 Apodemus 154.33 0.46 4.90 4.00 flavicollis

SREDNOGORIE

2 Apodemus 135.35 0.39 4.10 3.30 sylvaticus

I 18 VI. CONCLUSIONS

The resultsobtained for the heavymetals and toxicelements bioaccumulation in the bodiesand someinternal organs and tissues of the investigatedmonitor species small mammals: voles, mice and shrewsfrom the two regionswith differentanthropogenic influence - Srednogorieand Rozhenshow rathersimilar amounts of the testedelements. The bioaccumulationof Cd in all the monitorspecies fromthe unpollutedregion of Rozhenhas probably a geochemicalorigin. In the analysedmonitor speciesfrom the industriallypolluted region of Srednogoriethis very toxic elemeni was absentat all.In the bodiesand in the internalorgans and tissuesof woodmice from Srednogoriehigher than in micefrom Rozhenquantity of Pb was established.Higher was alsothe concentrationof As - the main pollutant for Srednogorieregion. The hystologicalanalysis of the monitorspecies from the two investigatedregions show normal morphologicalstate. Only in the liversof the wood micefrom Srednogorie in the lobuleperiphery focal lymphoidagglomerations were found. In all the testedanimals the haematologicalindices were without pathologic changes, except for the wood micefrom Srednogorie.In 90%of thesemice basophilicgranulated erythrocytes were found. This is an evidencefor leadintoxrcation (Metcheva et al. 1987).

VII.REFERENCES

Gerasimov,S., R. Metcheva,P. Dimitrova.1987. The roleof eco-toxicologicalexperiments forthe selectionof bioindicatoryanimal species and indicativeindices in biologicalmonitoring. ln: Prob- lemsof BackgroundEcol. Monitoring. BAS. 37-41. Hollerer,G., E, Coduro1977. Zur Schwermetalkontaminatonvon einheimischen Wild. Z. Lebensmittel Untersuch.Forsch. 1 63(4), 260-263. Johnson,M.S., R.D. Robefts,1978. Distribution of lead,zinc and cadmiumin smallmammals from pollutedenvironments. Oikos 30(1), 153-159. Kostelecka- Myrcha,A. 1967.Vanation of morphophysiologicalindices of blood of Clethrionomys glareolus(Schreber 1780). Actiheriol. 12 (13),191-122. Metcheva,R., P. Dimitrova,S. Gerasimov,A. Mikhova.1987. Experimentalstudies of lead effects on a fieldmouse as biologicalindicators. Problems of BackgroundMonitoring. Leningrad.5,124-131. Metcheva,R., M. Karapetkova,S. Gerasimov.1987. Heavy metals contamination in bioindicatorspe- cies of vertebrateanimals from Srednogorieregion. First National Conference on the Problems of BiologicalMonitoring. 174-177. (in Bulg.) Metcheva,R., N. Atanasov,S. Gerasimov.1993. The use of smallrodents in biologicalmonitoring. Mammalia,57 (4),638. Metcheva,R. 1995.Small mammals like bioindicators in zoological monitoring. (in print) Nikolova,E. , A. Artinian,J. Karamanova.1993. Determination par la methoderoentgenfluorescente de tracesd'elements dans des echantillous preleves dans la regiondu picde Moussala.Obser- vatoirede Montagnede MoussalaOM2. 1, 96-102. Romies,B. 1968.Mikroskopishe Technik. MUnchen - \Men,R. OlenbourgVerlag.

l19 ESTIMATIONOF THE ENVIRONMENTALQUALITY USING MONITOR SPECIES SMALL MAMMALSFROM TWO REGIONSWITH DIFFERENT ANTROPOGENIC INFLUENCESIN BULGARIA

B. PopulationCharacteristics and Food Preferencesin the MonitorSpecies.

MichaelaBeltcheva, Roumiana Metcheva instituteof Zoology,BAS

I. INTRODUCTION

The anthropogenicinfluenced environments are often with damaged nature. There is a possrbility for somechanges on the populationparameters: number, density, fertility, sex and age structureetc. in monilorspecies small mammals. Detailed dietary anaiysis are importantfor furtherinvestigations on the traceand toxicelements through the trophicchains in the smallmammals populations. For this purposetwo regionsin Bulgariawith different anthropogenic influences were investigated- the industriallypolluted Srednogorie and considerably clean region of Rozhen- Rodops.

II. MATERIALAND METHODS

The monitorspecies small mammals - commonvole (Microtusarvalis) ano wood mouse (Apo- demussylvaticus) were caught using baited live and snap traps at a 1 km in the directionof the marginallyprevailing wind from the copper- producecombine in Pirdop(Srednogorie). From the backgroundregion of Rozhen- Rodopsusing the same methodwere caughtalso commonvole, bank vole (Clethrionomysglareolus), pine vole (Pitymyssubterraneusl, yellow neckedwood mouse (Apo- demus flavicollis),and the commonshrew (Sorex araneus). The populationsize of the investigated specieswas eslimatedaccording Zippin's method (Grodzinski et al. 1966). The foodspectra were determined by stomachcontent analyses using two differentmethods. The greenvegetable remains were estimatedby Holishova(1965) and the seedsby Bomford(1987). Afler24 hourspreliminary lightening in glycerinefrom the contentsof eachstomach 5 glycerine- gelatinemicroscopic slides were made.

III.RESULTS AND DISCUSSION

II l. 1. P opu Iatio n c h a racterrstics

The resultspresenting the structureand size of all caughtsmall mammalspecies from the two investigatedregions are shown on tableI . Apodemuss/vaticus and Microtusarvalis were caughtfrom Srednogoriein 1995, but next year the commonvole was absentfrom the totalcatch. The populationdensity of the two specieswas approximateiysimilar in 1995,and the younganimals were dominant. They composed about 85% of the miceand 66% of the volepopulation. The investigatedsex structureshow that malevoles were predominantin micepopulation (about 70% of thewhole population). The populationnumber of mice in 1996was considerablyless then in 1995.The lowernumber of caughtanimais does not givenot a possibilitylo calculatethe agestructure. The sex structure changed to the prevalenceof females. Considerablylarger number of indicatorspecies were caught in the surroundingsof Rozhen.Mr- crotus arvaliswas absentfrom the total catch in 1996.A tendencyfor decreasingthe population numberol Apodemusflavicollisand Pitymyssubterraneus in 1996 is shownon tab. 1. The population densityof Clethrionomysglareolus and Sorexaraneus were withoutchanges. The sex structure of the populationsof alltheinvestigating species forthe two years remains always the same.

ll?0 Table.1 Biodiversityand distribution of monitorspeeies small rodents from the impactstation of Srednogorieand the background station of Rozhen.

REGION MONITOR DENSITY AGESTRUETURE SEXSTRUCTURE SPECIES tND./100 SAD. AD. MALEFEMALE M:F TRAPS 1995 Srednogorie Apodemus 3.3 1.t 2 10 3 3.3:1.0 sylvaticus Microtus 3.9 B 4 6 ct 1.0:1.0 arvalis Rozhen Apodemus 4.0 14 6 6 1.0:1,0 flavicollis Microtus 5.1 18 5 4 1.3:1.0 arvalis Clethrionomys 2.8 10 3 6 1.0:2.0 glareolus Pitymys 5.1 18 I 10 1.3:1,0 sbterraneus Sorex 0.3 1 1 araneus 1996 Srednogorie Apodemus 1.0 2 3 1.0:1,5 sy/vaficus Rozhen Apodemus 1.3 2 2 1.0:1.0 flavicollis Clethrionomys 2.8 4 5 1.0:1.3 glareolus Pitymys 1.8 4 3 1.3:1.0 sbterraneus Sorex 0.3 1 araneus

llf. 2. Stomach contents

Stomachcontents for all the speciesfrom the two regionswere investigatedexcept for the commonshrew (fig. 1).

Srednogorie Resultsfor the commonvole and for the wood mouseshow that 86% of the total food were presented by green parts of Centaureatriumferata, Galium sp. and Fragaria vesca. In all stomachs of Apodemushitin parts from Arthropods and seedsof corn(Zea mais)were found. Rozhen Stomach contentsfrom all speciesfound with there exceptionof the common vole show that in the diet of mice seedsfrom speciesol Poaceaeand Asteraceaewere predominant- 72o/o.20o/o of stomach contents belongsto green parts of Fistula sp., Taraxacumofficinale and Alchemfl/asp. In some stomachsanimal food was found. In two vole speciesdominant food were green vegetable parts from Alchemillaand Traxacumtor Clethrionomysglareolus and Festucaand animal hitin for Pitymys.These analyses of stomachcontents were necessaryfor furtherheavy metal investigations in the diet.

t2l Figure1. Dietof the monitorspecies ( in %) fromthe two investigatedregions

Dietof the Commonvole from the regionof Srednogorie 14lo

Diet of the Wood mous from the region of Srednogorie

12% arA--=. ,,-5

Diet of the Common vole from the region of Rozen 240/o ,^. \) \,/ r -A\G Es*""p",r.-lHgreen parts -- | ElseedsLrseeos I

- 760/o

Diet of the Pine vole from the region of Rozen --\Z160/o / Er,'-'-"."",'* | , -: ,Eseeos \ // : llanimat food \-/ /:t :

t22 Diet of theWood mous from the reEion of Rozen

8o/o

[eo';;fi;',"I lEseeos I Llqr,g-rgg,gl

IV. CONCLUSIONS

The investigationson the dynamicparameters of the smallmarnrnals populations showed larger number of monitorspecies in the regionof Rozhen.The lowerdensity of all popuiationsin the secondyear is a result of the naturalcyclic decreasing of smallmammals populations number in 1996. The slomachcontent analysis showed more variable number of plantspecies in the diet of the animals caughtfrom the surroundingsof Rozhen.The investigationson the food preferencesare necessaryfor further heavymetal research through the foodchains.

V. REFERENCES

Bomford,M. 1987.Food Quality, Diet and Reproductionof HouseMice on lrrigatedCereal Farms. Ph. D. Thesis.Austral. Nat. Univ. Grodzinski,W., Z. Pucek,L. Ryszkowski.1966. Estimation of rodentnumbers by meansof probatingand intensiveremoval. Acta Theriol. 12 (13),191- 122. Holishova,V. 1965.Food of the overcountedpopulation of the bankvole Clethrionomysglareolus in a low- landforest. Zool. Listy 14 (1),15-28. Hollerer.G.. E. Coduro1977. Zur Schwermetalkontaminatonvon einheimischenWild. Z. LebensmittelUn- tersuch.Forsch. 163(4), 264-263. Romies,B. 1968.Mikroskopishe Technik. MUnchen - Wen, R. OlenbourgVerlag.

t23 MORPHOPHYSIOLOGICALAND CYTOGENETICALANALYSES OF SMALL RODENTS BIOMONITORSPECIES FROM THE REGIONSOF PIRDOPAND REG|ONAL BACKGROUNDSTATION "ROZHEN"

NaskoAfarrassov

Instituteof Zoology,BAS

I. INTRODUCTION Since1977 Bulgaria has begunto selectand work out appropriateRegional Background Stations (RBS) and methodsand criteriafor evaluationof environmentstate as well.After a complexstudy of some pro- spectiveregions and on the groundsof biologicaland ecological criteria the mostappropriate places for RBS have been selected.One of them is the RBS "Rozhen"in the Rhodops.The requirementfor maximalre- motenessfrom sources of noxiousnessemissions above all heavymetals and toxic elements has been kept. Pirdopis locatedin the hillsof CentralBalkan. The regionwas includedin the listof the districtsin Bul- gariawith the high levelof anthropogenicpressure as the cooper-zincfactory is locatednot far from the town. The effectof polymetalpowder depositions and gas emissions on the livingorganisms have been studied intensively.However the investigationshave been concentrated mainly on the bioaccumulationand the dis- turbancesin the functionof theirorgans,systems and karyotype (genome) are notstill understood. The aim of thiswork is to presentthe resultsof morphophysiologicaland cytogeneticalanalyses of small rodents'biomonitor species from the regionsof Pirdopand Regional BackgroundStation "Rozhen"(Bul- garia). II. MATERIALSAND METHODS Samplesof differentmonitor species from the RBS "Rozhen"and the regionof Pirdopwere coilecteci oncea yearin October1995 and 1996.The smallrodents were caught using bailed live traps at a 1-2km in the directionof the marginallyprevailing wind from the cooper-zincextracting factory near Pirdopand near the Astronomyobservatory at Rozhen.Yellow-necked (Apodemus flavicollis) and European(Apodemus sylvaticus)wood mice,common (Microtus arvalis), pine (Microtussubterraneus) and bank (Clethrionomys glareolus)voles were collected.Animals from the differentregions were individuallyweighted and divided intotwo weightgroups approximately representing age and sex classes. All collectinganimals from the agegroup "adults" were dissected to removethe intestinaltract (including contents),internal organs - liver,kidneys, spleen and adrenalglands. These organs wei"e weighted (o 0.001 g andtheir morphophysiological indices were determined (Schwarz et al.,1968). The geneticinvestigations have been done using routine cytogenetic methods for chromosomeanalysis. To determinechromosomal aberrations in the marrowcells adult males and femaleswere carriedout. The slidepreparations with well-spread complete metaphases were obtained from colchicine-blockedbone marrow cellsfrom the femuraccording to a routinemethod. The hypotonictreatment was carriedout in 0.075M KCIfor 20 min at 37"C.Fixation was carriedout withmethanol: glacial acetic acid (3:1).Flame dried bone marrowslides were stained with 5% Giemsasolution. In eachexperimental group 50 metaphasesper animal wereanalysed (Macgregor, Varly, 1986). The statisticalassesses were carriedout by standardstatistical methods(Parker, 1 973). III.RESULTS AND DISCUSSION An insufficientnumber of individualsas a resultof low populationdensity of smallrodents in October 1996was collected.That is the reasonthe morphophysiologicaland cytogeneticalanalyses only on the animalscollected in October1995 hasbeen done. lll.1. Morphophysiological analysis The trappedanimals have been treated in the laboratoryusing the methodof morphophysiologicalindi- cators(Schwartz et al., 1968).The commonvoles have beentrapped both in the RBS "Rozhen"and the surroundingsof Pirdoptown that's why the presenteddata on Table1 arefor this species only. On thisTable the meanvalues of the weightsof liver,spleen, kidneys, adrenal glands and their indices are comparedby t- test.The resultsare as following: Significantdifference between the meanvalues of aboveparameters for maleand female individuals has not beenestablished so the datafrom each region have been analysed in commonsample. The meanvalues of the animals'weightsfrom the two regionsdo notdiffer significantly, so we couldsug- gestthat the meanvalues of the weightsof the liver,spleen, kidneys, adrenal glands and theirindices show reliabledifferences ( t=2.3-3.9; p>0.05-0.01 ). The specificrole of malesand females in breedingprocess, in the populationdevelopment and surviving, as wellas environmentalconditions determine the populationmorphophysiological characteristics Schwartz, t21 '1984; 1977;Olenev, 1981; Bolshakov et al., Liro,1985; etc.). The enlargedinternal organs of the volesfrom the Pirdopregion and theirindices probably are resultedby intrapopulationalfaclors and eventualenviron- mentalimpact on the commonvole populationin this region.Investigations on housemouse (Mus mace- donicus)from the industrialpolluted surroundings of lead-zincfactory near Plovdiv (Asenovgrad town) show similarresults (Topashka-Ancheva et al , 1994).The obtainedresults from the Pirdopregion and RBS" Roz- hen"are a basefor futureirrvestigations on populationadaptations of smallmammals to anthropologically influencedenvironment, tll. 2. Cytogenetical analysis Cytogeneticalanalysis' results of the investigatedaninrals from the Pirdopregion and RBS"Rozhen" are presentedon Table2. Ihe comparisonof the datashows an increasedpercentage of aberrantcells ( t=3.2; p>0.05)in the animalsfrom the Pirdopregion. The percentageof the polyploidcells also is increasedbut the differenceis not significant.The analysisof the type of chromosomalaberrations shows that the chromosomal breaks,fragments and exchangesC/C type is predominant.All thesedata are similarto thoseestab- lishedby Deknutet al. (1977),Leonard&Lauwerys (1980), Gerber (1980), Degraeve (.1981), Dhur (1990), Topashka-Anchevaet al. ('1995)etc., which investigated the heavymetal influence causing chromosomal aberratronsin the cellsof laboratoryand wild rodents. Our resullsand the literaturedata suggestthat the cytotoxicand genotoxiceffecls of the polymetaldust dependmainly on the chemicalcharacteristics of the heavymetal content. Lisson&Lauwerys (1990) clearly demonstratedthat lhe polymetaldust particlesfrom a heavymetal producingfactory, (such as the Pirdop's plan) aremore toxic to peritonealand alveolar macrophages than the actionof eachcomponent alone. Apartthe commonvoles five wood mice (Apodemus sylvaticus) have been trapped within the Pirdopre- gionand havebeen analysed. The receivedmean values of percentagesof aberrantand polyploidcells also are high- 5.7x2.6and 3.0t1.4 (Table 2). Seven pine voles (Microtus subterraneus) trapped in RBS "Rozhen" have beenanalysed and the percentageof aberrantand polyploidcells is closeto thoseof commonvoles fromthe sameregion (Table 2). Neverthelessthe insufficientnumber of animalscollected in October1996 in this periodtwo individualsof wood mice(Apodemus sylvaticus) from the regionof Pirdopand one individualof wood mouse,one individual of pinevole (Microtussubterraneus) and one individualof bankvole (Clethrionomysglareolus) from the RBS "Rozhen"were trapped.The resultsof cytogeneticalanalysis of these individualsare presentedon Table3. Notwithstandingthe insufficientnumber of individualsfor statisticalanalysis, the establishedten- dencyof increasingthe chromosomalaberrations in animalsfrom the regionof Pirdopwas determined. IV. CONCLUSIONS All theseresults, being previous, supposed some environmentalpollution in Pirdopregion, but its level and influenceon the investigatedrodent populations will be an objectof future,more eltensive studies. V. REFERENCES Bolshakov,V.P., P. Kovalchuk,A. Yastrebov.1984. Energeticheskiiobmen polevok lego izmeneniav extremalnichusloviah. ANUSSR. Sverdlovsk. 235 p. (ln Russian). Degreave,N..1981. Mut.Res., 86, 115-135 Deknut,G.,G.Colle, G.Gerber. 1977. Mut. Res., 45, 77-83. Dhur,H., A.Roy, A. Sharma,G. Talukder.1990. Mut.. Res., 241,305-312. Gerber,G.G., A. Leonard,P. Jaquet.1980. Mut.. Res.,76, 115-141. Leonard,A., R. Lauwerys.'1980. Mut.. Res., 76, 227-239. Liro,A. 1985,Acta theriol., 30,24,359-377. Lisson,D., R. Lauwerys.1990. Environ.Res., 52, 187-198. Olenev,G.B. 1981, Jurn.ob.biol., 62, 4, 506-511.(ln Russian). Schwadz,S.S.,V.Smirnov, L, N. Dobrinskii.1968. Metod morfofiziologicheskich indicatirov. In:Trudi InstitutaEkologii rastenii ljivotnich. UF AN USSR.58, 387p.(in Russian). Schwartz,S.S.1977. In: Voprositeriologii. Moskow, Nauka,279-289. (ln Russian). Topashka-Ancheva,M., R. Metcheva,N. Atanassov.1994. In: Ekologia'94, Bourgas,3,21-26 (in Bulgarian). Topashka-Ancheva,M., F. Avramova,V. Simeonova,R. Metcheva,N. Atanassov,A. Angelova, M. Stambolova.1995. Ann. De I'Univ.De Sofia.St.Kl.Ohridski",88,4,69-80.

i25 Table 1. Meanvalues ( x to ) of absoluteand relativeweights of someinternal organs in Microtusarvalis species from the investigatedregions

Investigated Weightof Weightof Relative Weightof Relative Weightof Relative Weightof Relative region animals liver weightof spleen weightof kidneys weightof adrenal weightof ad. olx, (q) (q) liver ( ) (o) spleen(ol. ) (q) kidneys(7oo) qlands(o ) qlands(9bn ) RB Station "Rozhen"(n=8) 18.813.8 0.7410.33 28.812.0 0.0410.02 1.34t0.42 0.2210.03 10.610.9 0.006+0.001 0.33t0.09 The region of Pirdoo(n=6) 19.2t4.8 0.74!0.23 34.314.5 0.06J0.02 2.910.9 0.2610.05 12.4!1.8 0.01210.004 0.6510.20

Table2. Numberand frequency of chromosomeaberrations found in threespecies of smallmammals from Regional Background Station "Rozhen" andthe regionof Pirdopin October1995.

Species Analysed Type of aberrations % of cells % of polypl. Biotops N mitosis Chromatid Chromosome Fragments Exchanqes Rings withabera- cells breaks breaks c/t vt tionsx t o xto 1 2 3 4 q o I I 10 11 12 ,| 1F 50 2 1 a 4 Microtus arvalis 2M 50 1 z 4 1 I 2n=46,NF=72 3F 50 z I z 2 a The regionof 4M 50 1 L 1 8 L Pirdop 5M 50 2 i 1 0 a 6F 50 i 1 A o 5.7!2.6 4.3X2.1 1F 50 z 4 2F 50 i 2 2 Microtus arvalis 3M 50 1 1 2n=46.NF=72 4M 50 RegionalBack- 5M 50 4 groundstation 6M 50 "Rozhen" 7F 50 4 8M 50 , 4 + 14+.tn 2.5t 2.1

Legend'.M - male;F- female

l2(l Table2 contrnuation

I z 4 5 7 B I 1A 12

4 q Apodemus sylvaticus 1F 50 1 z z I 12 2n=48,Nfa=46 2M 50 2 I b 4 4 1 I I The regionof Pirdop 3M 50 2 I I I I 12 2. ') 4M 50 1 1 a o 2 4 5M 50 z I 1 1 12 10 10.016.4 5.0!2.4

1F 50 I z 4 Microtussubterraneus 2F 50 1 2 6

2n=52,NFa=56 3M 50 I 2 L RegionalBackground 4F 50 1 1 8 2 station"Rozhen" 5M 50 1 1 4 2 .l a 6M 50 I 2 7F 50 1 1 4 4 4.0!2.1 2.3! 1.2

Table3. Numberand frequency of chromosomeaberralions found in smallrodent species from Regional Background Station "Rozhen and region of Pirdooin Odober1996

Species Analysed Tvoe of aberrations % of cells % of plyploid Biotops mitosis Chromatid Chromosome Fragments Exchanges Rings with cells breaks breaks clc c/t t/t aberrations Apodemus sylvaticus q q 2n=48.Nfa=46 100 4 z ? Pirdoo Apodemus flavicollis 1 2n=48 Nfa=46 50 I L RBS "Rozhen" Pitymyssubtenaneus 2n=52 Nfa=60 50 z 4 6 RBS"Rozhen" Clethrionomysglareolus

2n=56 Nfa=56 50 1 I 4 2 RBS "Rozhen"

{l.J STRATEGIESAND METHODOLOGYOF ECOSONDERESULTS IMPLEMENTATIONIN BULGARIA

BiljanaPapazov Ammann, Tillierstr.46, CH-3005Bern, Switzerland, Tet.+ Fax+41 31 35170 33

The ,,newapproach" provides a paradigmfor the treatmentof socio-ecologicalsystems. For this purpose, the methodrecognising the wickednessof socio-ecologicalproblems be givenequal weight alongside the type of decisionmaking. lts conceptualframework has five kindsof questionscorresponding to a fivefold knowledge.This newframework helps the individualtodevelop self-respect and, though a betterapprecia- tronof hisown strengthsand limitations,to recognisefalse theories. Thereare lwo basicways of understandinglife: a) as obseruers:describing life without leading it; and: b) as participants:organising life and looking for ways in whichto influencethe lifesituation 'to 'to A consequenceof this is that know'and be ableto' appearto be contradictoryto eachother. This 'technical leadsto misunderstandingstypical of the society'. It is necessaryto bridgethis gap andto findchannels for communicationbetween the two humantypes 'to 'to mentionedabove: the observerand the participant.In thisway, the contradictionbetween know'and be ableto' can be reduced. The two humantypes - the observerand the participant- are expressionsof two differentworld views. We haveto makethe worldview clearbefore talking about, for example,strategies and methodologyof implementationof ecosonderesults in Bulgaria. Whenpreparing a choiceof alternativestomake a decision,the followingquestions must be answered: a) Whatis the problem? c) Whatare the alternatives? d) Howdo we comparethem? e) Howcan we reacha solution? This indicatesthat five kindsof knowledgemust be producedand combined,namely: factual, denotative, explanatory,instrumental and conceptual knowledge. The answersto thesequestions will produce an adequateprocess of decisionmaking and will reduce the needfor rational'prayer-books'. The industrialsociety is basedon the conceptof productivity.lt is thereforenecessary to answerthe followingquestions: a) Productivityfor increasingthe productionof what? b) Productionwith what purpose? c) Increaseof productivityat whatecological and human cost? The answersto thesequestions will make it clearwhich kind of developmentwe have chosen,which is the resultof the worldview that has been adopted. It is necessaryto think of a worldbased on pluralismof valuesand openness.Such a world can be achievedif answersare found to the followingquestions: What is the problem?What do we want?What are the alternatives?How do we comparethem? And howcan we reacha solution?as presentedabove. However,these answers must be regardedas provisionalconstructs which will helpus in reachinga state of adequatedecision making. Provisional constructs will help us to think accuratelyand to act efficiently withoutbecoming absolute'truths'. The scientific{echnicalway of thinking should not destroy openness. The consequenceof what I havesaid for the purposeof this paperis a callfor findingways of under- standingthrough comprehension as a strategyby treatmentof environmentalquestions.

FURTHERREADING:

Rittel,Horst: Planen,Entwer-fen, Design - AusgewiihlteSchriften zu Theorieund Methodik(Reuter W. Hrsg.).Kohl- hammer,Stuttgart 1992.

128 Developmentof the complex approachof theecological investigationin the syntheticalone

A COMPLEXINVESTIGATION nnIN TIIE DIFFERENTECOLOCICAL DISCIPLINES

ENVIRONMENTALASSESSMENT REGARDING THEACCUMULATION OFTHE POLLUTION AND PROGNOSIS

RISK ASSESSMENTON THEPOPULATION AND COMMUNITY LEVEL

- I ASSESSMENTOFI I rur PUBLTc I DEVELOPMENT DECISIONSAND OFTHE ACTIVITIESOF THERESPONSIBLE I ,ellf,frI LEGISLATION ORGANZATIONS POLITIC]AL SEQUELTO THE LECISLATION

't29 THE STATUSAND PROGNOSESFOR DEVELOPMENT OF ENVIRONMENT IN ROZHENAND SREDNOGOR]EMONITORRING CONTROL STATIONS FINALDISCUSSION AND CONCLUSIONS DimitarPeev, KlausAmmann, Ari,Minian, RoumianaMetcheva, StefanPopov, Nina Bakarjieva

1. ABOUTTHE DIFFERENCESBETWEEN THE 2 CONTROLSITES"

The two controlsites are chosenpurposefully in two significantlydifferent regions - Rozhen- with natural backgroundconditions and Srednogorie- highdegree of pollutionwith some heavymetals. The studiescarried out showthat the two stationsdo not differsignificantly in the numberof studiedele- ments. As it could be expected,the differencesare in the quanlities- Cu, Zn, As and much less Pb, and are emissionsfrom the Copperplant. The radioactivebackground of the two controlsites is withinthe limitsfor the respectivealtitudes.

2. BIOMONITORSENSITIVITY

2.1.Phytomonitors and fungi

2.1.1.Physiological and biochemicallevel.and effects. 2.1.1.1.Protein synthesis In Srednogorie30 % inhibitionof the transcriptionand translationof.proteins, hnn and some enzymes wasfound. 2.1.1.2.lsozymes quantitative qualitative ' No significantthe and differencesin the isozymespectre were foundin the control populationsof the two sites. 2.1.1.3.lridoids In Srednogoriesignificant inhibition of the iridoidsynthesis is foundrefening mainlyto the catalpoland its estersdue to the blockingof the oxidises. The speciesspecificity is preserved. 2.1.2.Micm morphological level and effects. 2.1.2.1.Chromosomes Statisticallyno significantfluctuations were found.The karyotypologyof the populationsin the two sites was similar. 2.1.2.2.Pollen High pollensterility in the zone at Srednogorie. 2.1.3.Macro morphological level and statistics. Visualinjuries on differentplant organs and partswere established in Srednogorie.This did not refleclon the levelof statisticaivariability. 2.1.4.Spatial structure of the populations 2.1.4.1.In Srednogorie- disperse to singleindividuals of higherplants, with normalgene flow. In Rozhen- densepopulations with activegene flow. 2.1.4.2.Lichens In Srednogorie- becauseof the highaerosoldensity of the air no lichenswerefound (lichendesert). In Rozhen- highquantity of lichensfrom 8 maingroups which corresponds to the atmosphericpurity (ln- dex AtmosphericPurity). Because of the shortinterval the studiesare onlytentative. 2.1.4.3.Fungi No possibilityfor parallelanalysis of the accuniuiationin pairspecies. A clearspecific selectivity emerges forthe accumulationof: Pb (8ovr'sfaplumbea), Cu (Bovistaplumbea), As (Calvariautriformis).

2.2.Zoomonitors

2.2.1.Hydrophauna and saprobe waters lmpoverishmentof the speciescontent in the main groups Ephemeroptera,Trichoptera and Diptera (about20 o/ofor Srednogorie). The waters in the Rozhenregion are oligosaprobous,and these in Srednogorie- oligomezosaprobous. There is a tendencyto improvementof the watersin Srednogorie. 2.2.2.Small mammals 2.2.2.1.Blood state

130 Statisticallyno signiflcantfluctuations in the stateof red and whiteblood elements was found in the zoomonitors.The studyof the samplesfrom genusApodemus pathogenic changes in the erythrocytecytoplasm was found. 2.2.2.2.Chromosomes Statisticallyinsignificant changes in the % of deletionsand aberrations,and autopolyploiddeformations werefound in Srednogorie.

2.3.Soil microorganisms

In Srednogorieinsignificant reduction to completeabsence of amonificators,cjenitrificators and nitrificators was found.Because of the higherquantity of Pb, Cu, Zn, As the soilcircum-rotation was damagedstrongly. It is provedthat the existingdifferences in the reactionsof the biomonitorsare limitedby the high ouantities of Cu,Zn, As accumulatedin the soil,water and biomonitors. The environmentalassessment in Rozhenshows lhat the processesunder observation are elementsof the naturaldynamics of the ecosystemsin this region. The breakingof the soil equilibriumin Srednogorieand the high accumulationof Cu, Zn and As in the biomonitorsare the mainnegative factorthat leads to deslructionof the ecologiealbalance in this zone.

t3l ACKNOWLEDGEMENT

The authorsexpress personal thanks to Mr. P. Gallandfor the successful collaborationand managementof this project.They reallyappreciate the valuable suggestionsand brilliantideas of Mr. K. Ammannduring the Arcutino'95workshop andthe projectrealization period. The scientificgroups thanks to the staff,leaded by Mrs,Konstantinova, for the kindnesstechnical and financial support. AUTHOR'SINDEX

Ari Artinian Luchezar Pehlivanav AtanasAtanassov Michaela Beltcheva Atanas Kaimaktchiev Milen lovtchev Bilijana Ammann N. Christova. D. Djilianov Alasko Atanassov Dencho Denchev NataliaValjovska Dimitar Peev Nedyalka Handjieva Dobri lvanov Nina Bakardjieva Emilia Nikolova, Pierre Galland Evgeni Ananiev Rilka Taskova G. Kimenov Roumiana Laleva Georgi Angelov Roumiana Metcheva l. Elenkov SimeonPopov l. Popova Sonia Tzoneva lvanka Janeva Stefan Kozhucharov K. Seizova StefanPopov K. Stefanov Sfefan Stoichev Kathrin Peter Urs Krdhenbiihl Klaus Ammann V. Bankova, Kolishka Tsekova Violeta Fakirova Krassimir Kumanski Violeta Tyufekchieva Yanka Vidinova

ORGANIZATIONAND INSTITUTIONS

BotanicalGarden - Varna,Sofia University, Bulgaria De MonfortUniversity, lnt. Institute Plant Science Research and Instituteof Genetic Engineering,Agricultural Academy, Kostinbrod Dep.Plant Physiology, Faculty of Biology,University of Sofia,Bulgaria Instituteof Botany,BAS, Sofia, Bulgaria Instituteof Microbiology,BAS, Sofia,Bulgaria Instituteof nuclearresearch and nuclear energy, BAS, Sofia, Bulgaria Instituteof OrganicChemistry with Centre of Phytochemistry,BAS, Sofia,Bulgaria Instituteof OrganicChemistry with Centre of Phytochemistry,BAS, Sofia, Bulgaria Instituteof PlantPhysiology, BAS, Sofia, Bulgaria lnstituteof Zoology,BAS, Sofia,Bulgaria Laboratoryfor Radioand EnvironmentalChemistry , University of Bern,Switzerland NationalMuseum of NaturalHistory, BAS, Sofia,Bulgaria PULS.Berne. Switzerland

122 HIERACHICAL SUBORDINATIONOF THE BSPCP

PRECIPITATION VEGETATION

T}IOT)IVERSITY FRESHWATERS SECOM)ARY HI(iI{ER I'LANTS FJL(X)D LI(;HT SI'EC]TRA METAT]0LITS PICTIITTE MICROTOP0GMPHY I'OLLEN SToMATA

MARINE FIEAVY ECoSYS'|EMS METALS TOTAL RAI)IOACTIVII'Y STATISTICS

TI}IOI)IVERSI'TY

PFIYSIOLOCiY T}IOI)IVI]RSITY FtIOI)IVERSII'Y

I'RO]TTN S\'}ITHESIS Inforrmtion databank

iiptg,s{[gell.:ll:

SpEC|:F|,G,,ANEYISES..IIIPRETRF$M'ENTS,,

LEGEND / ,. lry0rnrrggrotq)!;) T.BOTANY 2. SMALLMAJI,TALS 3.IIYDROBIOLOGY 1. MICROBIOLOGY s. ORGANIC CHEMISTRY 6 SPECTROSCOPY& ILACTIVTIY FTINCTIONAL SCIIEME 7.& 8. (MIERRE EARCH GROI]N' OF THE BSBCP

ACC'ORDING BSBCP WORKSHOPDEASIONS BWGARIA, ARCUNNO, 17 - 22. JWE 1995/ The Bulgarian Swiss Biodiversity Conservation Programme (BSBCP)is financedby SDC- the SwissAgency for Developmentand Co- operation and is realisedin partnershipwith the Bulgarianministry of environment,Bulgarian NGO-s, research institutes, Pro Natura- the Swiss League for Natural Protection, BurdLife Switzerland - the Swiss Association for the protection of birds (SVS) and World Conservation Union(IUCN). The main objective of the programme is to contribute to the conservationof Bulgariannature by helpingto implementthe "National biodiversity strategy" and the "National action plan for conservation of the most importantwetlands" by capacitybuilding and concrete action. The programmeis activein the followingfields: . Conservation,administration and policy. Training of managers for protectedareas; . Environmentaleducation; programmes for public relationsand collaboration;establishment of InformationCentres . Expanding and strengthening the Protected areas network; working out managementplans of existing Protectedareas and proposalsfor new Protectedareas; . Encouragingeco-agriculture and promotinginteraction between agricultureand natureprotection; . Assistance in the sustainabledevelopment of regions of the projects; developingeco-tourism policy; . Eco-monitoring

The Programme includes projects on the territories of Central Balkan, Eastern Rhodope, Dobrudja, Coastal Wetlands and works on biomonitoring and conseryationof the plant genetic resourcesin different regionsof Bulgaria.

This book contains an description of the experienceand the work alreadydone in the field of the biodiversityand eco-monitoringof polluted areas by some scientific and researchgroups from BulgarianAcademy of Sciences' Institutesand scientistsfrom Switzerland.lt includes also all reviews on investigation,carried out accordingthe BSBCPin Rozhenand Srednogorie The studies present the long-term activities - field and laboratory - connected with many aspects of environmentalobservation and estimation of pollution impact on bio- and zoo sphere. Two different places are chosen and a comparison of heavy and toxic metals concentration influence in biomonitors is made. Some proposals for government future investments and activities for environmental protection are given.

ENIGMAPRESS LTD. Sofia,Bulgaria