1. « Mise au point d’une systématique de caractérisation des interactions Matière Organique Naturelle Dissoute (MOND) – Contaminants métalliques »
Thèse de Doctorat soutenue par:
M. Yoann LOUIS
En vue d’obtenir le titre de Docteur de l’Université du Sud Toulon-Var
Directeurs de Thèse :
Dr. S. MOUNIER Université du Sud Toulon Var – PROTEE
(PROcessus de Transferts et d'Echanges dans l'Environnement)
Dr. D. OMANOVIĆ Institut Ruđer Bošković – LPCT
(Laboratory for Physical Chemistry of Traces)
Université du Sud Toulon Var novembre 2008
Subvention N° 03/ /T
Matière Organique NAturelle en miLIeu SAlé

2. Analytical protocol improvements Concentrated Marine DNOM study
SUMMARY
Introduction
Analytical protocol improvements
Concentrated Marine DNOM study
Natural Estuarine ecosystem study
Conclusions & perspectives
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

3. Introduction
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

4. Trace metals in the environment
ATMOSPHERE
Metals
Metals
AQUATIC ENVIRONMENT
(Coastal and estuarine system)
SOILS
Metals
Metals
WATER
SEDIMENTS
Anthropogenic origin
Natural origin
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

5. Trace metals in the environment
Toxic metals:
not needed
Pb, Hg, Cd, …
Toxicity ≠ Total concentration
When metal became toxic ?
 depend on its speciation
Concentration increase
“Oligoelements”:
necessary for metabolism
Cu, Fe, F, Mg, Mn, Zn, …
Metals
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

6. Trace metals speciation in the water column
Filtration (0.45µm)
“Not” or less
bioavailable
Particulate > 0.45 µm
Dissolved < 0.45 µm
Could be bioavailable
Micro-organisms (bacteria, virus,…)
Dissolved metal speciation
Inorganic Ligands
Cl-, NO3-, SO42- … M M
bioavailable M
OH-
Water column
M n+
Organic and Inorganic Particules
Organic Ligands
EDTA, DNOM … M
“Not” or less bioavailable
Generally toxic for biota
Metal trap: Less toxic
[MTOTAL] ≠ [MTOXIC]
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

7. Anthropogenic activities Plants, animals, µorganisms decomposition
DNOM Origin?
Heterogeneous origins  heterogeneous and complex structure
Anthropogenic activities
Humification&
Polymerization
 DNOM modifications
Plants, animals, µorganisms decomposition
River input
Photosynthesis
Bacterial activity
degradation
Phytoplankton activity
Representation of a simplified NOM
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

8. DNOM speciation “Analytical speciation” “Mechanistical speciation”
“structural” determination
separation and analysis
Dialysis, UF GFAAS
CFFFF, HPSEC ICP-MS
HPLC, GC CV-AFS
C18, Chelex Voltammetry
… ...
Specific components determination
Less usable for metal behavior prediction
Interactions characterization
ISE
Voltammetry
Fluorescence Quenching …
Results usable in speciation codes for prediction
(for example MOCO from IFREMER)
No Functional characterization
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

9. + + + DNOM speciation H+ M M L L H+ Comp Comp M
DNOM-Metal interaction study
Used to describe the DNOM reactivity
kM1
KMthermo M +
kM-1 M L
DNOM L
DNOM +
KHthermo H+ +
KCompthermo
Comp
Continuous model: NICA-Donnan
Discrete model: WHAM
Comp
For 1 DNOM: All K and [LiT] determined = “Chimiotype”
 For Metal-DNOM interaction study: Need a technique to measure only M or
Assuming a kinetic of 1st order
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

10. Analytical tool used to measure trace metal: DPASV
3 electrodes system:
Counter electrode (Pt)
Reference electrode (Ag/AgCl/KClsat)
Working electrode (Hg)
Stirrer
Purging (N2)
Metal addition M
DNOM L
Metal-Ligand Complex M M
Direct measurement of
free & inoganic copper fraction =
electrolabile fraction
(= bioavailable fraction)
Escan
Edep
Edep
After tdep = 5 min
Escan
Voltamogram
Reduction Step
Oxydation step e-
I=f([M]) M M M M
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

11. Discrete fitting of experimental data with PROSECE program
Metal logarithmic scale titration (Garnier et al., 2004, Env. Technol. 25, )
Discrete fitting of experimental data with PROSECE program
(Speciation calculus + optimization)
pCuT
pCuLab
Determination of Kequilibrium, [LT]
Determination of k1, k-1, [LT]
 New characterization of the DNOM: reactivity
[Metal added]: From nM to µM
Data at equilibrium  Kequilibrium, [LT]
Kinetic  k1, k-1, [LT]
For each point:
2h of equilibrium
Measurements every 6 min.
Metal complexed by DNOM
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

12. Pseudopolarography measurements (Nicolau et al., 2008, ACA 618, 35-42)
Measured if:
UV, pH=2,
Edep << Edep for CC
Labile fraction = Free + inorganic fraction : bioavailable
Dissociable organic fraction: Probably not bioavailable
Not measured fraction = electroinactive in the Edep range used
Voltammograms
Pseudopolarogram
Edep for CC
measurements
Deposition potential (V)
Scanning potential (V)
Intensity (nA)
Direct ML complex reduction
Labile fraction
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

13. Goals of the study Analytical protocol determination
adapted to low [DOC] and [Metal]
Real complex natural ecosystem study
Improvements:
Technical
Analytical
Mathematical
“Model DNOM” definition
Based on the concentrated sample from GDR MONALISA
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

14. Analytical Protocol determination
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

15. Technical and mathematical improvements
Limit adsorption (Teflon use)
Precise metal additions (automatic pumps 500µl)
Avoid pollution with additions (tubing separation)
Avoid evaporation (N2 wet purging)
Mathematical baseline and peak definition
Multi-PROSECE (more optimization loop & confidence interval calculus)
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

16. Analytical improvement: Surface Active Substances (SAS) interferences (Louis et al., 2008, ACA 606, 37-44) e-
Edep e-
Escan
I  [Cu]meas  [LT]
Distorded shape
-0.45V with SAS
Escan
-0.45V without SAS
SAS
SAS M M
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

17. Analytical process to get rid of SAS interferences during the stripping step
Additional experiment A.C Voltametry (Phase angle: 90° measure of capacitive current)
Max. Ads.
At pzc
Classical used tdep
Edep = -0.6V
Eacc= V + 3sec at -1.6V Eacc= -1.6 V
Eacc= V
Classical Edep used for Cu - +
Edep = -0.6V
+ 3s at -1.6V
Edep = -1.6V
ΔI ↑ = Itcap- It0cap = SAS quantity ↑
Full circles Edep=60 s
Dotted circles Edep=60 s + 1s at -1.6V
Triangles Edep =60s (After UV)
High influence of SAS at tdep = 300 s and Edep ≈ V
Only 1% of the total deposition time (297s)
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

18. Influence of these SAS on the apparent [LT]
Without 3sec
[LT]= 335 nM
logK=6.17
With 3sec
[LT]= 160 nM
logK=6.47
Ruzić linearization
[LT] change from 335 nM to 160 nM
 Artificially « Hidden Metal » by SAS  bad speciation determination  toxicity
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

19. Analytical protocol for DNOM study
Filtration
at 0.45µm
DOC
Total Metal
UV at pH2
Salinity or majors ions by Ionic Chromatography
Log addition window determination :
Add1= 10% Mini
Final conc.:
1mgC/L  1µM
10 mgC/L  10µM
Pseudo  Edep
(Optional)
H+ , Ca2+ competition
(After Chelex)
Log additions at Edep,
Kinetic experiment
PROSECE
Raw sample
Potentiometry
(Chelex)
(1)
(2)
(4)
(3)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
For concentrated samples
“Chimiotype”
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

20. Study of a natural seawater sample (MONALISA project) (Article submitted to Marine Environmental Research)
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

21. Sampling site: Balaguier Bay
Military port
> 100 nM
~ 15 nM
~ 5 nM
Site interest:
Coastal Semi-Closed Area under anthropogenic influences
Goal: Give standard DNOM usable in metal speciation/transport program
1000L seawater sampling (online filtration and nanofiltration and reverse osmosis concentration by GDR MONALISA, ISM-LPTC: E. Parlanti, PhD of Arnaud Huguet).
Concentrated from 1000 L to ~10 L, [DOC]final= 30.4 mg/L
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

22. 60% 40% 70% 30% Potentiometry on Concentrated DNOM 265.2 181.2 446.4
(Garnier et al., 2004, Water Research, 38, )
LH1
LH2
LH3
LH4
Carboxylic like
Phenolic like
Total acidic Sites
LHiT (meq/molC)
210 ± 10.8
54 ± 2.4
80.4 ± 1.2
100.8 ± 1.2
265.2
181.2
446.4
pKa
3.6 ± 0.1
4.8 ± 0.1
8.6 ± 0.1
12.0 ± 0.4
Carboxylic-like
Phenolic-like
60%
40%
/2.7
70%
30%
165.3
Lu and Allen (2002) : Suwanee River
(also concentrated by RO)
(Letizia and Gnudi, 1999)
PROSECE Fitting for 4 types of acidic sites (DOC=1.2mmolC.L-1).
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

23. Exploratory experiment: Pseudopolarography coupled with logarithmic addition
Edep = -0.5V
2nd site saturation
1st site saturation
Estimation of a [1st site]: 90% x 2.5µM = 2.25 µM (= 1.87 meq/molC)
Estimation of a [2nd site]: 50% x 25µM - [1st site]: = µM (= 8.54 meq/molC)
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

24. Log addition and Cu2+ competition with H+ and Ca2+
≈ 2µM of Cu bound to specific sites
Strong affinity toward proton  phenolic-like sites
Strong affinity of copper for the studied DNOM
% Cu lab
Model Marine DNOM complexing parameters = DNOM “Chimiotype”
(Comparable to standards OM used in NICA-Donnan /WHAM models, obtained for soil/river extracted OM)
% Cu lab
[LM1] = 0.03 meq/molC
Ca additions pH
LH1
LH2
LH3
LH4
LHiT (meq/molC)
210 ± 10.8
54 ± 2.4
80.4 ± 1.2
100.8 ± 1.2
pKa
3.6 ± 0.1
4.8 ± 0.1
8.6 ± 0.1
12.0 ± 0.4
[Cu]T = 12.5µM, pH = 8.2  [Cu]T = 4µM.
Edep = -0.5V, pH = 8.2, DOC = 1.2mmolC.L-1.
Strong complexing site specific to copper
Hight calcium competition effect
Comparison of 2 different Edep (-0.5V and -1.5V).
Total metal binding site density LMT 12
Total acidic sites density
446
LM1
LM2
LMiT (meq/molC)
1.72
±0.13
10.25
±2.7
logKCuL
9.9
±0.1
6.9
logKCaL
2.5
±0.4
5.5
±0.6
pKa
8.6
8.2
±0.3
Closed to values estimated with pseudo coupled with log add.
~3% of
(= Buffle, 1988)
Phenolic-like sites
Complexing parameters determined after simultaneous fitting by PROSECE of the 3 experiments
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

25. MINEQL simulation of natural DNOM according to determined model marine DNOM
Experimental points
DNOM simulated by Mineql adjusting only [DOC]
Difference between modeled DNOM and experimental points
<<5%
seawater sample treated with Chelex (DOC = 0.09 mmolC.L-1); pH = 8.2, Salinity 37.
Correct simulation validating the characterization protocol
DNOM reactivity is not strongly modified by concentration step
Model DNOM determined usable
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

26. Simulation of copper speciation for the studied marine environment
Condition: Majors ions for salinity of 38, DOC = 0.09 mmolC.L-1, Cutot = 14.8nM
80% of total copper complexed as organic forms
>90% found in several paper: Influence of SAS ?
specific behavior of the studied DNOM and high copper content
Natural marine water conditions
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

27. Estuarine DNOM Study (Article submitted to Marine Chemistry)
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

28. Sampling site: Krka, Croatia (2007&2008)
Sampling in the water column:  gradient of salinity
Brackish
FSI layer
Seawater
Low tide on Adriatic sea  stratified estuary
Pristine watershed
Potential anthropogenic inputs in estuary
On site measurements in nearby laboratory
Challenge is to give data on speciation and kinetic in this natural area
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

29. Simplified protocol used
Filtration
at 0.45µm
DOC
Total Metal
UV at pH2
Salinity or majors ions by Ionic Chromatography
Log addition window determination :
Add1= 10% Mini
Final conc.:
1mgC/L  1µM
10 mgC/L  10µM
Pseudo  Edep
(Optional)
H+ , Ca2+ competition
(After Chelex)
Log additions at Edep,
Kinetic experiment
PROSECE
Raw sample
Potentiometry
(Chelex)
(1)
(2)
(4)
(3)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
No concentration step and no use of Chelex
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

30. Salinity, DOC and Copper profiles
(Elbaz-Poulichet F. et al., 1991)
1.78 in may 1988
Same curve shape for 2007  & 2008 
Oligotrophic freshwater  Very few carbon content, DOC est. < DOC sea  low anthrop. inputs
Non conservative behavior: Bigger amount of metal & DOC in the FSI  “special layer”
Additional source of DOC in the FSI: can be due to an exacerbated biological activity (Svensen et al, 2006)
Increase of copper in the FSI: particulate/dissolved metal exchange due to salinity increase
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

31. Comparison of the kinetic and at Equilibrium approach
Data obtained for only 1 sample: Example from Salinity 11, April 2007
Log K at Equilibrium
Log K kinetic
Average of Log K kinetic
Average of LT kinetic
LT kinetic
LT at Equilibrium
Fitting at Equilibrium
Data at Equilibrium
Average of Log k1 kinetic
Log k1 kinetic
Good agreement between the constants obtained at equilibrium and with the kinetic approach
Fitted Kinetic data
Kinetic data
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

32. Comparison of the kinetic and at Equilibrium approach
Good agreement between constants determined at equilibrium or kinetic
At equ: Higher M/L ratio  better [L] determination
Kinet.: more points after each addition less equilibrium dependent, kinetic parameters determined k1
Apparent overestimation of Kinetically determined logK (or underestimation of the at equ. approach) due to:
kinetic first point estimation of Culab at t0
Is the solution at equilibrium with the at equ. Approach
Both approaches are complementary
k-1
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

33. Complexing parameters results
& In MINEQL:
83%
Expected variation with salinity
Organic Cu
90 to 99%
Observed variation
strong (,) and weak (,) ligands, 2007 (,) and 2008 (,),
In dotted line in inset: toxicity limit of 10 pM (Sunda et al., 1987).
Difference  Autochthonous DNOM production in the estuary
Higher affinity for ligands from seawater origin
In the FSI: Higher inorganic and free copper content (up to 20pM) in spite of [ligands] increase
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

34. Simulation of DNOM reactivity under a Cu contamination
2008
2006
t50%
t99%
Omanović et al., 2006
time
Used for prediction
≈ 2h30
≈ 4h30
Higher [Metal] in summer due to traffic of touristic boats
Calculated free copper concentration potentially toxic for µorganisms at the surface in summer
Lower reactivity of the FSI DNOM
Compared to hydrodynamics variations tequ. are quite long  system probably not at equilibrium in the estuary
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

35. Comparison of the measured DNOM vs. the model DNOM
Determined with the simplified protocol
simulated from model marine DNOM
Higher free [Cu] with the use of model DNOM > toxicity limit
Bigger difference for marine sample  ≠ DNOM behavior between Toulon & Šibenik
Model DNOM not sufficient, even if DNOM is from same origin  This ≠ show the necessity to study samples representatives of the studied system
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

36. Conclusion
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

37. Conclusions and perspectives
NEW use of “3sec” for DNOM analysis remove SAS interferences
Determined protocol  NEW direct analysis of coastal natural samples at low [DOC] and [M]
 complexing parameters determination + NEW Kinetic parameters (reactivity prediction)
model DNOM usable in environmental contaminant speciation/transport programs
Standard DNOM hardly usable to model DNOM behavior of a complex environment
Use of the determined protocol for specific ecosystem understanding
Main improvement needed: Voltammograms automatic fitting
Deeper analysis of pseudopolarograms,
On site measurements (DGT) and comparison of data
Actually protocol applied on a depth profile from Dycomed (Dyfamed site) …
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

38. Application of the method to an “oceanic” depth profile
5 nM
First results shows:
At natural [Cu]: Cufree under toxicity limit
until simulated total [Cu] up to 5nM
Surface DNOM is less complexant
Still analyzing samples (Dycomed 15) and need to treat all kinetics data…
Need to make a connection with on site measurments (Chlorophyll, COT, fluorescence … )
Introduction – Analytical protocol – Marine DNOM study – Estuarine DNOM Study - Conclusion

39. Merci à tous de votre attention !
Balaguier Bay (Toulon, France)
Martinska (Šibenik, Croatia)
Merci à tous de votre attention !
Special thanks to my Directors:
Dr. Mounier S.
Dr. Omanović D.
To the Jury’s members:
Prof. Marmier N. Prof. Riso R.
D.R. Elbaz-Poulichet F. D.R. Cossa D. Dr. Garnier C.
And members from PROTEE (USTV) and LPCT (RBI) labs
Salle de délibération réservée pour le Jury de 15 à 17 H en Y’211.

41. Representation of baseline determination problem

42. Sensitivity
Intensitypeak or Areapeak = Sensitivity x [Metal]labile

43. A.C. Voltammetry at “90°”
Capacitive current
Faradic current
π/2 =
90°

44. Kinetic at (to) low and (to) high metal content
To much noise at low [M]/[L]
Not enough curvature at high [M]/[L]

45. Thermodynamic and Conditional stability constants

46. General principle of PROSECE
(« PRogramme d’Optimisation et de SpEciation Chimique en Environnement ») Garnier et al., Analytica Chimica Acta, 2004, 2005
Composed of two modules:
Algorithm of chemical speciation
. equilibrium resolution by a Newton-Raphson algorithm
. minimisation of the mass-balance deviation
Algorithm of optimisation
use of a modified simplex
 parameters values (complexation, acidic, fluorescence …)
adjustment to simulate the experimental titrations

47. Comparison to experimental results (pH and/or pM)
Principle of (multi-)PROSECE
(« PRogramme d’Optimisation et de SpEciation Chimique en Environnement ») Garnier et al., Analytica Chimica Acta, 2004, 2005
Quasi-particles (Sposito, 1983) parameters. Initial values
Speciation calculation
chemical speciation algorithm NO
modified Simplex evolution of ALL quasi-particles parameters
Comparison to experimental results (pH and/or pM)
optimisation algorithm
iteration
Bias test
YES
Optimised values
Modified with multi-PROSECE
SD calculus
Determination of the parameters which better depict the analysed DNOM properties

48. PARAFAC results C1: Humic like (peak A) C3: Marine Humic like (peak M)
C2: Protein like (Tryptophane)