The MODELKEY project: Connections between chemical … · MODELKEY (511237-GOCE) is a research...

MODELKEY (511237-GOCE) is a research project funded by

http://www.modelkey.org

The MODELKEY project: Connections between chemical and ecological status in the Llobregat

River Basin

Girona, 3rd December 2010

Isabel Muñoz Julio C. López-DovalRoberta Carafa (URS)

Helena GuaschMarta Ricart Anna RomaníSergi Sabater

Damià BarcelóRikke BrixAntoni GinebredaMiren López de Alda



1. General data and previous knowledge of the Llobregat basin.

2. The Llobregat study in the framework of Modelkey

3. Design of a work plan: multiple approach. Strategies at two levels: community/ecosystem level, population/individual level.

Field approach: extensive and intensive field monitoring, multivariate analysis.

Laboratory approach: biofilm and invertebrate bioassays

4. Conclusions



General data

The main channel is 170 km long.

The drainage basin has an area of 5100km2

Two main tributaries: Cardener, Anoia.

Geology: mainly calcareous materials. Salt deposits (chloride, sodium, potassium) at Cardener tributary

More than 5 million people live at the Llobregat basin

Industrial, agricultural land uses



General data

Mean monthly discharge

JanuaryFebruary

March April May June JulyAugust

SeptemberOctober

NovemberDecember

m3

s-1

0

10

20

30

40

Hydrological regime: discharge fluctuations reflect Mediterranean climate. Very low summer discharge and storms in spring and autumn may cause important floods.



Previous evidences of the presence of emergent contaminants and estrogenic activity

EDCs and estrogenic activity(vitellogenin synthesis in feral carps and RYA) in the Llobregat river (2000)

Chemical analysis:- alkylphenol ethoxylates- alkylphenols- carboxylate derivatives- estrogens- progestogens

Environ. Sci. Technol. 34, 5076-5083, 2000.

Environ. Toxicol. Chem. 20(6), 1152-1158, 2001.

Environ. Toxicol. Chem. 21(10), 2146-2156, 2002.

Analyst 127, 1299-1304, 2002.

Int. J. Environ. Anal. Chem. 81(4) 295-313, 2001

General data



More: Carafa et al. Emerging tools for risk assessment

of key toxicants

multi substance Potentially Affected Fraction (msPAF)



Models for Assessing and Forecasting theImpact of Environmental Key Pollutants onMarine and Freshwater Ecosystems andBiodiversity

Three case studies: Scheldt, Elbe, Llobregat

Modelkey project



Chemical status

External concentration

Ecological status

Community Structure and function

Chemical analysis

Field

Sediment Water

Community parameters

EC

OLO

GY

CH

EM

ISTR

Y

Internal response

Individual, population,community

effect LaboratoryBiota

In-vivo biomarkers

In-vivo bioassaysTO

XIC

OLO

GY

Exposure - effect chain

+

Tools Matrix



Analytical parameters:• Physico-chemical parameters (pH, O2,

TSS, nutrients, OM, ionic composition,etc.)• Pesticides• Pharmaceuticals

Characterization of the geographical and temporal distribution of these pollution sources.

Relationships with the biological community structure and function.

Multivariate Analysis

Work plan: Multivariate exploratory data analysis

Biological parameters:• Biomass, density, species composition

•Chl, in-vivo fluorescence measurements,

extracellular polysaccharide content) and

heterotrophic measurements (extracellular

enzyme activities)

BIOFILM: ALGAE, BACTERIAINVERTEBRATES



A1A2

A3

LL2

LL3

LL4

LL1A1

A2

A3

LL2

LL3

LL4

LL1

Spring 2005

Autumn 2005

Spring 2006

Autumn 2006

EXTENSIVE SAMPLING

Sites distributed in a wide extension of the basin

Different hydrological periods

Field approach



Water (ng/L)

Triazines

Organophosphates

Phenylureas

AcidicChloroacetanilideThiocarbamate Sediment ng/g

Chloroacetanilides

Phenylureas

Acidic

Anilide

OrganophosphatesTriazines

Field approachPesticide results

Proportional composition of the different pesticide families was not equivalent in water and sediment



Concentration increase downstream.

Higher concentrations with low discharge.

June 2006

A1 A2 A3 LL1 LL2 LL3 LL40

10

20

30

40

50

60

June 2005

0

10

20

30

40

50

60

70analgesics and antinflam m atorieslip id regulatorsCarbam azepineantih istam inicsantib ioticsB blockers

October 2006

0

5

10

15

20

25

30

35

Pha

rmac

eutic

al c

once

ntra

tion

(μg/

L)

Field approachPharmaceutical results




Redundancy analysis (RDA): Variance explained 14.6 %

J. Hydrology (DOI: 10.1016/j.jhydrol.2009.08.014)



Biofilm metricPhysical and

chemical variablesPesticides

Chl-a 8.43 91.57

EPS 46.55 53.45

Ymax 22.25 77.75

Yeff 39.06 60.94

F1/F3 44.97 55.03

Glucosidase 91.15 8.85

Peptidase 97.43 2.57

Phosphatase 88.14 11.86

Phenylureas

Organophosphates

Biofilm metrics

Multivariate statistical analysis: Variance partitioning technique

J. Hydrology (2010) 363: 52-61


Fraction of explained covariance: 50%Temperature

SO4




Trac, 28 (5): 592-602 (2009)

Relationships between conventional diversity indices with the waterborne concentration of the herbicide Diuron.r = Pearson correlation.

Significant decrease of diatom diversity with Diuron in the river.



-0.4 1.0-0

.60.

8

TubificidaeEnchytraeidae Naididae

Other Ephemeroptera

Baetis

Caenis

Chironomus spp

Cricotopus sp

Cryptochironomus

Nanocladius

Orthocladiinae

Polypedilum Rheocricotopus

Stictochironomus

Tanytarsus

Tanypodinae-0.4 1.2

-0.6

1.0

Ibuprofen

Indomethacine

Propranolol

Temperature

A1A

A2A

A3A

LL1A

LL2A

LL3A LL4A

A1B

A2B

A3B

LL1B

LL2B

LL3B

LL4B

A1C

A2CA3C

LL1C

LL2C

LL3C

LL4C

Redundancy analysis (RDA). Variance explained 71%


Env.Tox.Chem., 28 (12): 2706-2714 (2009)



100050 j

j

ECPNEC =

Hazard quotient where cij is the concentration of compound j in sample i and PNECj the ‘Predicted Non Effect Concentration’for compound j, as described above:


Env. Int. 36:153-162 (2010)

y = -0,5183x + 1,7784R2 = 0,4946

0,000

0,500

1,000

1,500

2,000

2,500

-1,500 -1,000 -0,500 0,000 0,500 1,000 1,500log(HQ)

Shan

non

dens

ity (b

its)

y = -0,4701x + 1,6986R2 = 0,8625

0,000

0,500

1,000

1,500

2,000

2,500

-2,000 -1,500 -1,000 -0,500 0,000 0,500 1,000 1,500 2,000

log(HQ)

Shan

non

dens

ity (b

its)

y = -0,8119x + 1,9626R2 = 0,841

0,000

0,500

1,000

1,500

2,000

2,500

3,000

-0,800 -0,600 -0,400 -0,200 0,000 0,200 0,400 0,600 0,800 1,000 1,200 1,400

log(HQ)

Sha

nnon

den

sity

(bits

)

Fish toxicity vs. Shannon diversity

Daphnia toxicity vs. Shannon diversity

Algae toxicity vs. Shannon diversity

Correlations between log transformed total hazard indexes log(HQ) and invertebrate diversity index.

j

ijij PNEC

chq =



PESTICIDES PHARMACEUTICALS

FIELD Algae(Biofilm)

Effects on biofilmmetrics and diatomcommunity structure (spp composition, diversity).

No relationship

Invertebrates No relationship Higher invertebratedensity and biomass. Lower diversity.

Field approach



Biofilm experiments– Long term toxicity test Diuron – Short term toxicity test Diuron – Short term toxicity test estrone (not seen an effect (20mg/L)– Short term toxicity test b-blockers (effects at high concentrations

9mg/L)

End points and biomarkers:

diatom composition, chlorophyll a, photosynthetic activity, extracellular polymeric substances, bacterial mortality, enzymatic activities, antioxidant enzymes (catalases, peroxidases).

Laboratory approach



Laboratory approach

Invertebrate experiments– In vivo toxicity test with Diuron (Physella acuta)– In vivo toxicity test with Indomethacine and Propranolol (Chironomus

riparius) (10 days)– In vivo toxicity test with Indomethacine and Propranolol (Physella acuta)

End points and biomarkers:

Survival, growth, number of clutches, eggs development, lipid, protein and C N content, oxygen respiration rates, histological effects.



(a)

Diuron concentration (μg L-1)

0,0001 0,001 0,01 0,1 1 10

Bio

volu

me

(μm

3 cm

- 2)

5e+8

1e+9

2e+9

2e+9

3e+9

3e+9

4e+9

4e+9(b)


0,0001 0,001 0,01 0,1 1 10

Bio

volu

me

(μm

3 cm

- 2)

5e+8

1e+9

2e+9

2e+9

3e+9

3e+9

4e+9

4e+9

EC50 (day 8) = 0.087 μg/L EC50 (day 29) = 0.054 μg/L

Community level EC50 (biovolume of diatoms) = 0.05 µg/L

Laboratory approachPesticide results-Biofilm

Chemosphere,17: 1392-1401 (2009)



(a)


0,0001 0,001 0,01 0,1 1 10Pho

tosy

nthe

tic e

ffici

ency

at P

SII

(% o

f con

trol)

40

60

80

100

120

(b)


0,0001 0,001 0,01 0,1 1 10

Chl

-a (%

of c

ontro

l)

60

80

100

120

140

160

180

200

(c)


0,0001 0,001 0,01 0,1 1 10

Pep

tidas

e ac

tivity

(% o

f con

trol)

0

200

400

900

1200

(d)


0,0001 0,001 0,01 0,1 1 10

Live

bac

teria

(% c

ontro

l)

0

50

100

150

200

250

300

Day 1Day 8Day 22Day 29

-Photosynthetic efficiency decreased- Chl content increased-Composition of the diatom community changed

- Number of live bacteria decreased by 50%- Change Bacteria capacity to process organic matter

Laboratory approachPesticide results-Biofilm



0

2

4

6

8

10

12

initial final

mg

DW

per

ind

initial pop. C 5 50 500 1000

biomass

0

20

40

60

80

100

120

C 5 50 500 1000

num

ber o

f egg

s/eg

g sa

c

10

15

20

25

30

35

40

C 5 50 500 1000

num

ber o

f egg

sac

s

fertility

Laboratory approachPesticide results-Invertebrates

No significant effects of Diuron on growth and reproduction

Physella acuta


μg/L


http://www.modelkey.organimal exposed to

1000ug/L (tegument)non exposed animal

(tegument)

Laboratory approachPesticide results-Inv.

Hypertrophy of secretory cells



Laboratory approachPharmaceutical results-Biofilm

0

50

100

150

200

0 0,9 90 900 9000

Propranolol μg L-1

Bio

mar

ker r

espo

nse

in p

erce

ntag

e o

cont

rol

Peptidase

PhotosyntheticYield

PhotosyntheticYield max

Catalase

life/deadbacteria ratio

Bonnineau et al. Aquat. Tox, 100:346 (2010)

Effects only at high concentrations, but lower EC50 described



Laboratory approachPharmaceutical results-Inv.

Control, 0.4, 2, 20, 120, 400 μg/g sediment

pH=4.6 and 8.6

Two experiments: indomethacine and propranolol

Endpoints: mortality, biomass.

Chironomus riparius (insect larvae).



0

0,02

0,04

0,06

0,08

0,1

0,12

0,14

mg

DW

Biomass C. riparius Propranololtreatment

control

0,4 ug/g

2 ug/g

20 ug/g

120 ug/g

400 ug/g

10% 20%


0

0,05

0,1

0,15

0,2

0,25

mg

DW

Biomass C. riparius Indomethacinetreatment

control

0,4 ug/g

2 ug/g

20 ug/g

120 ug/g

400 ug/g

0

1,515

90

0

0,05

0,1

0,15

0,2

0,25

0,3

0,35

0,4

0,45mg DW

pH= 4.6

pH= 8.6

*

*



Indomethacine exposure1 week duration

Treatments:CONTROL, 0.5, 5, 50, 500 μg/L

Physella acuta (Gasteropoda, snail)




No significant differences in biomass.

No effects on reproduction were observed.


INDMT

0

2

4

6

8

10m

g D

W0 ug/L 0,5 ug/L5 ug/L 50 ug/L500 ug/L

no significant differences

INDMT

0

2

4

6

8

10m

g D


INDMT

0

2

4

6

8

10m

g D


no significant differences



PESTICIDES PHARMACEUTICALS

LABORATORY Algae(Biofilm)

Direct and indirect effects of Diuron

No effects

Invertebrates Histological sub-lethal effects

Sub-lethal effects. Increase of Chironomus sp. biomass with the anti-inflammatory indomethacine.

Laboratory approach

Laboratory assays confirm the relationships, found in the field,between pesticides and pharmaceuticals and biological community.



Simultaneous chemical and biological sampling allowed to use multivariate statistical techniques and to find preliminary and potential causal links.

The approach done in the Llobregat basin confirmed different causal relationships in the different biological communities (biofilm, invertebrates) under a multiple chemical stress.

Combination of field and laboratory approaches could be a desirable way to go on in future risk management decisions, mainly with the emerging substances.

Procedures that identify causality allow corrective action measures for habitat recovery and control.



Thank you for your attention

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The MODELKEY project: Connections between chemical … · MODELKEY (511237-GOCE) is a research...

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