EULIMNOS: EULIMNOS: Successful International Successful International

Cooperation in WBCCooperation in WBC

LotharLothar ErdingerErdingerUniversity of HeidelbergUniversity of Heidelberg

Prof. Lefter Kashta, Shkodra

Common topic of Interest

The LakeThe Lake

ConsortiumConsortium

University ofGraz

University of HeidelbergGermany

University of ShkodraAlbania

University of Montenegro

CG

HRK/Stability Pact

CETIPodgorica

CG

University ofLjubljanaSlovenia

Örebro University Sweden

University ofSalzburgAustria

University ofPristina

CG

University ofSouthhampton

UK

Some HistorySome History2001 2001 11stst Conference, ShkodraConference, Shkodra

Initiation of Scientific CooperationInitiation of Scientific CooperationJan. 2002Jan. 2002 22ndnd Conference, HeidelbergConference, HeidelbergMar. 2002Mar. 2002 Workshop, PodgoricaWorkshop, PodgoricaSept. 2002Sept. 2002 33rdrd Conference, PogradecConference, PogradecSept. 2003Sept. 2003 Participation in SETAC Conference, HeidelbergParticipation in SETAC Conference, HeidelbergFeb. 2004Feb. 2004 44thth Conference, ShkodraConference, Shkodra20042004--20082008 Common Participations in SETAC ConferencesCommon Participations in SETAC Conferences20072007 Successfull application within Successfull application within 20092009 Opening of Ecological Educational Center and Opening of Ecological Educational Center and

Biological Station, VirpazarBiological Station, VirpazarOngoingOngoing Exchange of Scientists and Students for shortExchange of Scientists and Students for short--

term integrated projectsterm integrated projects

Our WebsiteOur Website

Basic AimsBasic Aims

Basis forWater Management

Protection ofthe Lake

Implementation ofLaboratories

Common Scientific Projects

ImprovementOf Skills

CooperationCooperation

Tools: Integrated AssessmentTools: Integrated Assessment

Assessment ofFauna

Assessment ofFlora

Biotests of Water and Sediments

Analysis ofBioavailableCompounds

MicrobiologicalData for

Water Quality

ChemicalData for

Water Quality

Physico-chemical Parameters

PROJECTSPROJECTSUmbrella ProjectUmbrella Project

Monitoring of several sampling sites on both sides of the lake Monitoring of several sampling sites on both sides of the lake for basic chenical and microbiological Parameters.for basic chenical and microbiological Parameters.

Biological monitoring of Fauna and FloraBiological monitoring of Fauna and FloraTaxonomic studies on biodiversity of algae, waterplants, fish, Taxonomic studies on biodiversity of algae, waterplants, fish, and other species.and other species.

Triade ProjectTriade ProjectIntegrative assessment of water and sediment quality at Integrative assessment of water and sediment quality at selected sites on the lake.selected sites on the lake.

SPMD ProjectSPMD ProjectDevelopment and application of innovative sampling Development and application of innovative sampling methods for bioavailable compounds.methods for bioavailable compounds.

Lake monitoringLake monitoring

Industry

Houshold

Aerial

Oxygen Watercolour

pH

Turbidity

Alkalinity

Algae growth

Microbial growth

Nutrients

Temperature, Irradiation

Umbrella ProjectUmbrella Project

BenefitBenefitDescription of actual lake situation by scientific data.Description of actual lake situation by scientific data.Starting point and basis for furthergoing investigations.Starting point and basis for furthergoing investigations.

DevelopmentDevelopmentPossibly: Installation of permanent monitoring.Possibly: Installation of permanent monitoring.

Outcome so farOutcome so farBasic information on some routine parameters Basic information on some routine parameters –– basic basic evaluation of ecological conditions.evaluation of ecological conditions.

Future activitiesFuture activitiesIntegration into a wider monitoring concept which must be Integration into a wider monitoring concept which must be supported by national authorities.supported by national authorities.

PROJECTSPROJECTSUmbrella ProjectUmbrella Project

Monitoring of several sampling sites on both sides of the lake Monitoring of several sampling sites on both sides of the lake for basic chenical and microbiological Parameters.for basic chenical and microbiological Parameters.

Biological monitoring of Fauna and FloraBiological monitoring of Fauna and FloraTaxonomic studies on biodiversity of algae, waterplants, fish, Taxonomic studies on biodiversity of algae, waterplants, fish, and other species.and other species.

Triade ProjectTriade ProjectIntegrative assessment of water and sediment quality at Integrative assessment of water and sediment quality at selected sites on the lake.selected sites on the lake.

SPMD ProjectSPMD ProjectDevelopment and application of innovative sampling Development and application of innovative sampling methods for bioavailable compounds.methods for bioavailable compounds.

Biological monitoring of Flora and FaunaBiological monitoring of Flora and Fauna

BenefitBenefitScientific interest in both universities, long tradition, strongScientific interest in both universities, long tradition, strongbasis.basis.

DevelopmentDevelopmentStrengthening of cooperation between scientists on both Strengthening of cooperation between scientists on both sides of the lake.sides of the lake.

Outcome so farOutcome so farOngoing broadening of scientifically interesting specific Ongoing broadening of scientifically interesting specific knowledge.knowledge.

Future activitiesFuture activitiesFurther integration in ecotoxicological research activities.Further integration in ecotoxicological research activities.

PROJECTSPROJECTSUmbrella ProjectUmbrella Project

Monitoring of several sampling sites on both sides of the lake Monitoring of several sampling sites on both sides of the lake for basic chenical and microbiological Parameters.for basic chenical and microbiological Parameters.

Biological monitoring of Fauna and FloraBiological monitoring of Fauna and FloraTaxonomic studies on biodiversity of algae, waterplants, fish, Taxonomic studies on biodiversity of algae, waterplants, fish, and other species.and other species.

Triade ProjectTriade ProjectIntegrative assessment of water and sediment quality at Integrative assessment of water and sediment quality at selected sites on the lake.selected sites on the lake.

SPMD ProjectSPMD ProjectDevelopment and application of innovative sampling Development and application of innovative sampling methods for bioavailable compounds.methods for bioavailable compounds.

Triade ProjectTriade Project

BenefitBenefitApplication of modern ecotoxicological tools on a new Application of modern ecotoxicological tools on a new environment.environment.

DevelopmentDevelopmentInstallation of basic tools for ecotoxicological assessment in Installation of basic tools for ecotoxicological assessment in both university laboratories.both university laboratories.

Outcome so farOutcome so farProof of toxic compounds in sediment extracts by biotest Proof of toxic compounds in sediment extracts by biotest methods.methods.

Future activitiesFuture activitiesDevelopment of further skills, development of individual Development of further skills, development of individual research activities.research activities.

SEESEE--ERA NETERA NET

PROJECTSPROJECTSUmbrella ProjectUmbrella Project

Monitoring of several sampling sites on both sides of the lake Monitoring of several sampling sites on both sides of the lake for basic chenical and microbiological Parameters.for basic chenical and microbiological Parameters.

Biological monitoring of Fauna and FloraBiological monitoring of Fauna and FloraTaxonomic studies on biodiversity of algae, waterplants, fish, Taxonomic studies on biodiversity of algae, waterplants, fish, and other species.and other species.

Triade ProjectTriade ProjectIntegrative assessment of water and sediment quality at Integrative assessment of water and sediment quality at selected sites on the lake.selected sites on the lake.

SPMD ProjectSPMD ProjectDevelopment and application of innovative sampling Development and application of innovative sampling methods for bioavailable compounds.methods for bioavailable compounds.

SPMD ProjectSPMD Project

BenefitBenefitDevelopment and application of modern, scientifically Development and application of modern, scientifically challenging sampling technique .challenging sampling technique .

DevelopmentDevelopmentInstallation of basic tools for SPMD application in both Installation of basic tools for SPMD application in both university laboratories.university laboratories.

Outcome so farOutcome so farProof of toxic compounds in water phase, relevance not Proof of toxic compounds in water phase, relevance not known so far.known so far.

Future activitiesFuture activitiesDevelopment of further skills, development of individual Development of further skills, development of individual research activities.research activities.

EstablishmentEstablishment

•• Of a sustainable research and education Of a sustainable research and education facility localted directly on the lakeside...facility localted directly on the lakeside...

Ecological Station VirpazarEcological Station Virpazarfounded by A. and S. Perovicfounded by A. and S. Perovic

SummarySummary

With the help of HRK a cooperation network between With the help of HRK a cooperation network between Universities from Shkodra, Podgorica, Heidelberg, and Universities from Shkodra, Podgorica, Heidelberg, and Graz has been established.Graz has been established.A routine monitoring program has been initiated.A routine monitoring program has been initiated.New and modern tools of ecotoxicological risk New and modern tools of ecotoxicological risk assessment have been implemented and used to assess assessment have been implemented and used to assess Lake Shkodra.Lake Shkodra.Biotests show some interesting results mainly for Biotests show some interesting results mainly for sediments.sediments.Reasons for some highly toxic effects are yet unclear.Reasons for some highly toxic effects are yet unclear.

AcknowledgementAcknowledgement

We gratefully appriciate the help of We gratefully appriciate the help of HRK and Stability Pact for HRK and Stability Pact for

$upport$upport

Some DetailsSome Details

M10

M7

M1

M2M3

M4

M5

M6M8M9

A1A2

A3

A5

A4

Routine Routine Monitoring Monitoring --MicrobiologyMicrobiology

Sampling Sites - Albania

A1 A2 A3 A4 A5

Col

ony

Cou

nt

0

200

400

600

800

1000

1200

MPNTotal coliformsPseudomonas

Sampling Sites - Montenegro

M1 M2 M3 M4 M5 M6 M7 M8 M9 M10

Col

ony

Cou

nt

0

1000

2000

3000

4000

MPNTotal Coliforms

Some Chemical Monitoring Some Chemical Monitoring Data for M4Data for M4

(Radus)(Radus)

Month

Tota

l Col

iform

Cou

nt

0

500

1000

1500

2000

2500

3000

3500

4000

Oxy

gen

[mg/

l]

5

6

7

8

9

10

11

pH

7,6

7,7

7,8

7,9

8,0

8,1

8,2

Alk

alin

ity [m

mol

/l]

2,4

2,6

2,8

3,0

3,2

3,4

3,6

3,8

4,0

4,2

4,4

M10 M10 -- KarucKaruc

Month

May June July August September October

TC

0

1000

2000

3000

4000

NTU

0,6

0,8

1,0

1,2

1,4

1,6

1,8

2,0 Month

May June July August September October

TC

0

1000

2000

3000

4000

mg/

l

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1,0

Month

May June July August September October

TC

0

1000

2000

3000

4000

pH

7,5

7,6

7,7

7,8

7,9

8,0

8,1

Alkalinity

Turbidity

COD

pH

EcotoxicologyEcotoxicology

The SedimentThe Sediment

Fate of Toxic CompoundsFate of Toxic Compounds

Toxic compounds

Outtake

Mineralisation

Metabolisation

Adsorption

Bioaccumulation

Significance of SedimentsSignificance of Sediments

Toxic Compounds

Sediments = Memory of the Lake

Food Chain

Sediment Triad

Bioassays

Fieldstudies

Chemicalanalysis

Bioassay-directedfractionation (BdF)

Extract

Bioassays

Fractionation

Active fractions

Hexa

n

Hexa

n - D

CM 2

:1

EtO

H

Ace

ton

DCM

Hexa

n - D

CM 1

:2

Hexa

n - D

CM 1

:1

DCM

- A

ceto

n 1:

1

0

200

400

600

800

1000

NR50

in m

g /m

L

Ch. analysis

IdentificationAbundance

0

10000

20000

30000

40000

0 20 40 60

time

Bioassays

Fieldstudies

Chemicalanalyses

Integrative

assessment

What are Bioassays good for?What are Bioassays good for?

Standardized Bioassays show the presence Standardized Bioassays show the presence of compounds with a certain toxic effect.of compounds with a certain toxic effect.Bioassays are sum parameters for the Bioassays are sum parameters for the measurement of toxic chemicals.measurement of toxic chemicals.Results of Bioassays are the starting point Results of Bioassays are the starting point for the identification of toxic compounds.for the identification of toxic compounds.

Biotesting of Sediment SamplesBiotesting of Sediment SamplesAmes Ames –– Test (several tester strains)Test (several tester strains)Comet Comet –– Assay (several eucaryontic cell lines)Assay (several eucaryontic cell lines)CytotoxicityCytotoxicity

Microtox assay using bacteriaMicrotox assay using bacteriaNeutral Red Assay using eucaryontic cellsNeutral Red Assay using eucaryontic cells

DHA Contact AssayDHA Contact AssayDehydrogenase ActivityDehydrogenase Activity

EROD AssayEROD AssayEthoxyresorufinEthoxyresorufin--OO--deethylasedeethylase(Indicator for Dioxin(Indicator for Dioxin--like compounds, AHH inducing like compounds, AHH inducing potency)potency)

YES AssayYES AssayHormonHormon--like activity using yeast cells with human receptorslike activity using yeast cells with human receptors

More Sampling......More Sampling......

Sites for

Sediment Sampling

Sediments – Memory of a lake

EROD Induction in Liver Cell Culture as a Biomarker of Dioxin-like Activity

Liver Cell Culture

Addition of medium and sample

Exposure: 48 hHomogenisation

EROD and protein

EC25TCDD determination

Reader 10-1 100 101 102 1030

10

20

30

40

50

60

70

Concentration extracted sample(mg/ml medium)

% o

f max

imum

ER

OD

indu

ctio

n

DioxinDioxin--like Organic Compounds like Organic Compounds (Dioxins, (Dioxins, DibenzofuransDibenzofurans, PCBs), PCBs)

Cl

Cl

Cl

Cl

O

Cl

Cl

Cl

Cl

O

O

Cl

ClCl

Cl

Polychlorinated Biphenyls TCDD

DibenzofuranPolycyclic aromatic hydrocarbon (PAH)

EROD Activity of Sediment ExtractEROD Activity of Sediment Extract(Radus (Radus -- Montenegro)Montenegro)

0.1 1 10 100

1

2

3

4

5

6

7

8

EC25: 2 mg/ml

T1 48h

2,3,7,8 TCDD

control

mg/ml

ERO

D a

ctiv

ity

Sediment contact assay

Arthrobacterglobiformis

Aquatox HD

Resazurin595 nm

Resorufin620 nm

Dehydrogenases

Controlls 4-NP Extract

w/o w w/o w w/o w

DHA contact testC

0,1250,2

50,5

12PC

T1T2T3

0

25

50

75

100

Inhi

bitio

n [%

]

Sediment [g]

Inhibition of microbial dehydrogenasesynthesis vs. concentration of sediment solid phase (Arthrobacterglobiformis)

YES YES ––Assay: SedimentsAssay: Sediments

T3

0

0,5

1

1,5

2

2,5

3

0,001 0,01 0,1 1 10

Extract Equivalent (g)

adju

sted

abs

orba

nce

at 5

40 n

m

T3upper 99% confidence intervallow er 99% confidence intervalE2 response

0

0,5

1

1,5

2

2,5

3

0,001 0,01 0,1 1 10

Extract Equivalent (g)

adju

sted

abs

orba

nce

at 5

40 n

m

T1upper 99% confidence intervallower 99% confidence intervalE2 response

0

0,5

1

1,5

2

2,5

3

0,001 0,01 0,1 1 10

Extract Equivalent (g)

adju

sted

abs

orba

nce

at 5

40 n

m

T2upper 99% confidence intervallower 99% confidence intervalE2 response

SPMDSPMD--ApproachApproach

Sampling of Bioavailable Organic Sampling of Bioavailable Organic CompoundsCompoundsAnalysis of Individual componentsAnalysis of Individual componentsBiotestingBiotesting

EROD EROD -- AssayAssayYES YES -- AssayAssay

Sampling Sites for SPMDSampling Sites for SPMD

AdriaticSea

Albania

MontenegroPodgorica

LakeSkadar/Shkodra

Shkodra

Bar

Virpazar

Golubovci

Koplik

R. Moraca

R.Buna

4

N

10 Km

ShirokaZogaj

Plavnica

Dajlon

5

6

12

3

0

2000

4000

6000

8000

10000

12000

Site 1 Site 2 Site 3 Site 4 Site 5 Site 6

Sum

PP

-PA

Hs

(ng/

SP

MD

)

Results of EROD Assay from SPMD Results of EROD Assay from SPMD DialysatesDialysates

0.01 0.1 1 10 1000

2

4

6

8

10

12

14

EC25: 36 %

Site 1

TCDD

DMSO control

SPMD dialysate concentration

ERO

D a

ctiv

ity

0.01 0.1 1 10 1000

2

4

6

8

10

12

14

EC25: 100 %

Site 2

TCDD

DMSO control

SPMD dialysate concentration

ERO

D a

ctiv

ity

0.01 0.1 1 10 1000

2

4

6

8

10

12

14

EC25: 22 %

Site 3

TCDD

DMSO control

SPMD dialysate concentration

ERO

D a

ctiv

ity

0.01 0.1 1 10 1000

2

4

6

8

10

12

14

EC25: n. d.

Site 4

TCDD

DMSO control

SPMD dialysate concentration

ERO

D a

ctiv

ity

0.01 0.1 1 10 1000

2

4

6

8

10

12

14

EC25: 42 %

Site 5

TCDD

DMSO control

SPMD dialysate concentration

ERO

D a

ctiv

ity

0.01 0.1 1 10 1000

2

4

6

8

10

12

14

EC25: 32 %

Site 6

TCDD

DMSO control

SPMD dialysate concentrationER

OD

act

ivity

TEQTEQ‘‘s for SPMD Sampless for SPMD Samples

0.0

0.1

1.0

10.0

100.0

1000.0

10000.0

1 2 3 4 5 6Site

TEQ

s (p

g/m

l)

Bio-TEQs PAH-TEQs

R2 = 0.74

0200400600800

10001200140016001800

3000 5000 7000 9000 11000

Sum PP-PAHs (ng/SPMD)TC

DD

-EQ

(pg/

ml)

YES YES -- ActivityActivity

0

0.5

1

1.5

2

2.5

3

0.01 0.1 1 10 100SPMD dialysate concentration

abs

orba

nce

at 5

40 n

m

Site 1E2 maximum response

0

0.5

1

1.5

2

2.5

3

0.01 0.1 1 10 100SPMD dialysate concentration

abs

orba

nce

at 5

40 n

m Site 2E2 maximum response

0

0.5

1

1.5

2

2.5

3

0.01 0.1 1 10 100SPMD dialysate concentration

abs

orba

nce

at 5

40 n

m

Site 3E2 maximum

response

0

0.5

1

1.5

2

2.5

3

0.01 0.1 1 10 100SPMD dialysate concentration

abs

orba

nce

at 5

40 n

m Site 4

E2 maximum response

0

0.5

1

1.5

2

2.5

3

0.01 0.1 1 10 100SPMD dialysate concentration

abs

orba

nce

at 5

40 n

m

Site 5E2 maximum response

0

0.5

1

1.5

2

2.5

3

0.01 0.1 1 10 100

abs

orba

nce

at 5

40 n

m

SPMD dialysate concentration

Site 6E2 maximum response

SummarySummary

With the help of HRK a cooperation network between With the help of HRK a cooperation network between Universities from Shkodra, Podgorica, Heidelberg, and Universities from Shkodra, Podgorica, Heidelberg, and Graz has been established.Graz has been established.A routine monitoring program has been initiated.A routine monitoring program has been initiated.New and modern tools of ecotoxicological risk New and modern tools of ecotoxicological risk assessment have been implemented and used to assess assessment have been implemented and used to assess Lake Shkodra.Lake Shkodra.Biotests show some interesting results mainly for Biotests show some interesting results mainly for sediments.sediments.Reasons for some highly toxic effects are yet unclear.Reasons for some highly toxic effects are yet unclear.

AcknowledgementAcknowledgement

We gratefully appriciate the help of We gratefully appriciate the help of HRK and Stability Pact for HRK and Stability Pact for

$upport$upport

Microbiological Monitoring Microbiological Monitoring ––Shkodra UniversityShkodra University