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1
Incorporation of bioavailability
Patrick Van Sprang – ARCHE
OECD Workshop on Metals Specificities in Environmental Hazard Assessment, Paris, 7-8 september 2011
2• Metals are found in different forms in the environment • These are referred to as metal “species”• Changing in the environment is called “ (chemical) speciation” or
“transformation”• Kinetics and chemical speciation under environmentally relevant
conditions crucial for PNEC derivation & read-across• Important point: Not all metal species are toxic
Introduction
3
– with inorganic ligands (OH-, CO32-, HCO3
-,..)
– with dissolved NOM2 (measured as DOC3: humic and fulvic acids) each of these processes may reduce metal bioavailability/toxicity
Dissolved Metal ComplexesDissolved Metal Complexes
Metals exist in the environment…
– Free-ion forms tend to bind to biological ligands, e.g.physiologically active sites at the gill these species mainly causes metal toxicity
Dissolved Free Metal Dissolved Free Metal
– adsorbed to suspended solids (POC1 or mineral surfaces) each of these processes may reduce metal bioavailability/toxicity
Particulate MetalParticulate Metal
1POC: Particulate Organic Carbon2NOM: Natural Organic Matter3DOC: Dissolved Organic Carbon
4For terrestrial and sediment systems, the concentration of a metal that is determined after destruction of the mineral matrix. For aqueous systems: the total amount of metal present, including the fraction sorbed to particles and to dissolved organic matter and the fraction in the mineral matrix; = particulate (sorbed + precipitated) + dissolved (inorganic complexes + organic complexes + free ionic forms)
Total Metal ConcentrationTotal Metal Concentration
Metal toxicity can be expressed as…
Dissolved Metal Concentration*Dissolved Metal Concentration*most often, the dissolved fraction in ecotoxicity tests refers to the fraction that passes through a filter of 0.45 µm. = inorganic complexes + organic complexes + free ionic forms
* It should be noted, however, that this definition may not necessarily refer to the metals in solution. In the range of 0.01- 0.45 µm colloid inert particles containing metal ions that remain suspended, may still exist;
5
- the degree to which a metal species is available to interact with the biotic ligand (e.g. physiologically active sites at the gill) to exert its effect.
= free ionic forms (mainly)
Bioavailable Metal ConcentrationBioavailable Metal Concentration
- Biotic Ligand* Model (BLM): assumes that both the free metal ion activity and the interaction of the available cationic forms with the organism reflect the toxicity.
- Free Ion Activity Model (FIAM): assumes that the free metal ion activity reflects the chemical reactivity and toxicity of the metal
Metal toxicity can be expressed as…
* A "biotic ligand" is a biochemical receptor that is metal-binding and is treated similarly to other ligands in the exposure water, except that it is on the organism. An example of a biotic ligand is a fish gill.
6
Why incorporate bioavailability in CSR of metals?
• NOEC/EC10 in laboratory test media which often maximizes bioavailability (e.g. low DOC in water; low OC in soil) may not reflect ‘real environment’ (rivers may have different DOC, pH) !
• Database often contains NOEC/EC10 obtained in test media with widely varying chemistry (= very different bioavailability) which toxicity values to select (species mean ?, lowest NOEC/EC10 ?) ?
• Generic/uncorrected SSD does not represent ‘intrinsic sensitivity’ alone but rather a mix of ‘intrinsic sensitivity’ + bioavailability effects toxicity values should therefore be normalized towards similar physico-chemical conditions !
7
Why inorporate bioavailability in CSR of metals?
Incorporationbioavailability
Concentration (µg/l)
Cum
ula
tive D
istr
ibuti
on
Funct
ion (
%)
Generic PNEC
Concentration (µg/l)
Cum
ula
tive D
istr
ibuti
on
Funct
ion (
%)
Normalized PNEC
• Bioavailability models ‘remove’ the variability in sensitivity due to differences in physico-chemistry
8
Does bioavailability matter in EU waters ?
0
100
200
300
400
01 02 03 05 06 07 07 08 09 10 11
48h-
EC
50 in
µg
Cu/
L648
Acute effects (LC50 in µg/l) of copper to Daphnia magna,tested in 11 different EU surface waters
Factor 30 difference in acute Cu-toxicity across EU surface waters !!
De Schamphelaere et al., 2002
Sampling location
9
Soil pH (CaCl2) Nitrific. Glucose Maize Barley Tomato Eisenia Folsomia
Houthalen 3.6 87 24 24 55 32Zegveld 4.1 196 226 226 966 619Montpellier 4.1 38 59 71 27 78 113Rhydtalog 4.2 68 603 91 48 179 510Jyndevad 4.5 26 14 31 87Kövlinge II 5.1 39 37 109 27 282 22Aluminusa 5.6 62 191 185 47 309 103Borris 5.6 89 16 46 110 54 31 183Woburn 6.1 104 555 297 416 136 303 884Ter Munck 6.7 97 97 54 103 103 169 298Souli ** 7.0 253 425 169 283 283 378 559Marknesse 7.6 66 148 84 233 233 299 583Brécy 7.5 156 457 801 504 504 609 941Cordoba 2 7.6 196 712 712 396 72 514 875Cordoba 1 7.6 61 190 68 105 105 195 79Guadalajara 7.7 33 54 411 192 192 312 542
Does bioavailability matters in EU soils ?Chronic effects of nickel (NOEC/EC10 in mg/kg) to soil
organisms/processes tested in 16 different EU surface soils
Factor between 10-45 difference in chronic Ni-toxicity across EU soils !!
10TOTAL METAL LEVELS (MONITORING DATA)
KD, SS
Ca, pH, DOC,…(speciation model)
SEM, AVS
Toxicity-based models (Biotic Ligand Model, Regression Models,…)
PHYSICO-CHEMICAL SPECIATION MODELLING
SEDIMENT
TotalMe-concentration
WATER
TotalMe-concentration
Porewater or free ion model
Free ionicMe-fraction
BIOAVAILABILITY ASSESSMENT MODELLING
Bioavailable Metal Fraction
Biogeochemical Region X1 Biogeochemical Region X2 Biogeochemical Region Xn
DissolvedMe-fraction
SEM – AVSMe-fraction
SOIL
TotalMe-concentration
TOTAL METAL LEVELS (MONITORING DATA)
KD, SS
Ca, pH, DOC,…(speciation model)
SEM, AVS
Toxicity-based models (Biotic Ligand Model, Regression Models,…)
PHYSICO-CHEMICAL SPECIATION MODELLING
SEDIMENT
TotalMe-concentration
SEDIMENT
TotalMe-concentration
WATER
TotalMe-concentration
WATER
TotalMe-concentration
Porewater or free ion model
Free ionicMe-fraction
BIOAVAILABILITY ASSESSMENT MODELLING
Bioavailable Metal Fraction
Biogeochemical Region X1 Biogeochemical Region X2 Biogeochemical Region XnBiogeochemical Region X1 Biogeochemical Region X2 Biogeochemical Region Xn
DissolvedMe-fraction
SEM – AVSMe-fraction
SOIL
TotalMe-concentration
SOIL
TotalMe-concentration
Approaches for bioavailability ?
11
1. Transformation from total to soluble fraction - approach
12
2. Use of speciation models - approach
13
3. Toxicity related bioavailability models: approach
The BLM requires a description of water chemical parameters that can influence metal toxicity: - pH- DOC (a convenient measure of NOM)- Major ions: Calcium, Magnesium- Others: e.g. Sodium (Cu)
14
MeOH+
MeCO3
Me-DOC
pH
[Me] on ‘biotic ligand’
Toxic effect
Water Organism
H+
pH
Me2+
Ca2+
Na+
Mg2+
‘biotic ligand’ e.g. gill, cell surface
Speciation (WHAM)Intrinsic sensitivity
Competition (log K’s)
Log KCaBL
Log KMgBL
Log KNaBL
Log KHBL
Log KHBL
15
De Schamphelaere & Janssen, 2002
R2 = 0.9672
R2 = 0.9559
0
1020
30
40
5060
70
0.0 0.5 1.0 1.5 2.0
Cation activity (mM)
48h
EC50
(Ni
2+) (
µM) Acute
R2 = 0.9918
R2 = 0.8086
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0.0 0.5 1.0 1.5
Cation activity (mM)21
d EC
50 (N
i2+
) (µM
) Chronic
Ca Mg
BLM: development (1)3. Toxicity related bioavailability models
16 De Schamphelaere et al., 2002
Sampling of waters
Chemical analyses (pH, DOC, Ca, Na,…)
Adding ≠ concentrationsDetermine toxicity
Test
BLM
Factor 2
BLM: validation (1)3. Toxicity related bioavailability models
17
Factor 10 to 30 variability in toxicity… reduced to factor 2 in > 90% of the cases
10
100
1000
10000
10 100 1000 10000
observed EC50 (µg/L)
pre
dic
ted
EC
50 (
µg
/L)
Daphnia - acute - Cu
Daphnia - chronic -Cu
Daphnia -acute -Zn
Daphnia - chronic -Zn
Daphnia - acute - Ni
Field cladocerans -acute - CuRainbow trout -chronic - Zn
..for invertebrates and fish
BLM: validation (2)3. Toxicity related bioavailability models
18
10
100
1000
10000
10 100 1000 10000
observed EC50 (µg Cu/ L)
pred
icte
d EC50 (
µg C
u/L)
P. subcapitataChlorella sp.C. reinhardtiiP. subcapitata (NOEC, fi eld)
..for algae
Factor 10 to 30 variability in toxicity… reduced to factor 2 in > 90% of the cases
BLM: validation (3)3. Toxicity related bioavailability models
19
BLM: similar response across metals ? (1)
InvertebratesAlgae Fish
Invertebrates, fish & algae
NO
EC
(µ
g/l Z
n)
NO
EC
(µ
g/l Z
n)
algae
pHDOC
invertebrates fish
pH
NO
EC
(µ
g/l Z
n)Invertebrates, fish
& algae
NO
EC
(µ
g/l Z
n)
Hardness
Ca > Mg
Invertebrates, fish & algae
NO
EC
(µ
g/l N
i)
NO
EC
(µ
g/l N
i)
Invertebrates, fish, algae
pHDOC
Mg > Ca
Invertebrates, fish & algae
NO
EC
(µ
g/l N
i)Hardness
- Zn
- Ni
3. Toxicity related bioavailability models
20
Invertebrates, fish & algae
NO
EC
(µ
g/l C
u)
NO
EC
(µ
g/l C
u)
algae
pHDOC
invertebrates fish
pH
NO
EC
(µ
g/l C
u)
Hardness does not
significantly affect chronic
toxicity
- Cu
- Toxicity response = f(organism; phys-chem parameter)- Toxicity response = pH: similar for algae; different for
invertebrates & fish= DOC: similar for all
organisms= H: ± similar for all
organism (> Ca for Zn; > Mg for Ni; less significant for Cu)
BLM: similar response across metals ? (2) 3. Toxicity related bioavailability models
21
BLM: applicability domain
• BLMs developed & validated within 90th % of phys.-chem from EU waters and should therefore only be applied within such boundaries !!
• Specific conditions outside boundaries need special attention (e.g. model extrapolation, additional specific testing….
3. Toxicity related bioavailability models
22• BLM developed for limited number of species:
– P. subcapitata (green alga)– D. magna/C. dubia (cladoceran, invetebrate),– O. mykiss/P. promelas (fish)
• Ecotoxicity database contains NOEC/EC10 for other species/taxonomic groups (e.g. molluscs, rotifers, insects)
• Given that individual development for all existing aquatic species is impossible, can a BLM developed for one species be used for another species?...
• Extrapolation assumes similar mechanism of actions (e.g. similar stability constants between the cations (Ca, Mg, H) and the biotic ligands, similar site of action)
BLM: extrapolation across other species ? (1)3. Toxicity related bioavailability models
23
23
Read across?
Read across?
Read across?
BLM model fish (rainbow trout)
BLM model algae (Raphidocelis)
BLM model water flea (Daphnids)
BLM: extrapolation across other species ? (2)3. Toxicity related bioavailability models
24
How ?: perform ‘spot checking’ of the BLMs for species for which no validation had been undertaken.
BLM: extrapolation across other species ? (3)
- Insect: Chironomus tentans- Rotifer: Brachionus calyciflorus- Molluscs: Lymnaea stagnalis- Higher plant: Lemna minor
BLM predictions were within a factor ± 3
3. Toxicity related bioavailability models
25
BLM: Implementation in risk assessment (1)
Yes
Yes
No
No
3. Toxicity related bioavailability models
26
Eco-region HC5
0
10
20
30
40
50
60
70
80
90
100
1 10 100 1000 10000
NOEC (µg/l)
perc
entil
e
Scenario ditchThe Netherlands
Scenario lake Monate Italy
Scenario river Rhine The Netherlands
Scenario river Otter United Kingdom
Scenario river Teme United Kingdom
Scenario acid lake Sweden
Scenario river Ebro Spain
10,9/11,8 µg/l
57,3 µg/l25,4 µg/l
17,5 µg/l
14,7 µg/l9,9 µg/l
DOC = 2,5 3,2 3,7 2,8 2,5 8,0 12,0 mg/lpH = 7,7 8,1 8,2 7,8 6,7 7,6 6,9
BioFwater,X NOECrefNOECx
2. Calculate the bioavailability factors (BioF) for the BLM species
PECbioavailable=PEC * BioFwater,X
1. Normalise the NOEC for the BLM species towards site specific conditions (NOECx) and towards EU reference water chemistry conditions (NOECref)
3. Select the highest BioF for the BLM species
4. - Calculate the bioavailable PEC concentration
- Use D. magna/C. dubia BLM to normalise all other invertebrates (e.g. molluscs, rotifers,..)- Use O. mykiss/P. promelas to normalize all fish/amphibians - Use R. subcapitata to normalize all other algae
BLM: Implementation in risk assessment (2)
3. Toxicity related bioavailability models
- Or calculate the bioavailable PNEC concentration
PNECbioavailable=PNECgeneric /BioFwater,X
27
- Coastal/open sea waters are characterised by… - high pH (typically between 7.8–8.3), high salinity (35‰), high ionic strength. - DOC levels may vary considerably between marine waterbodies- Freshwater and marine organisms face very different iono- and osmo-regulatory
issues related to living in either a very dilute or concentrated salt environment. freshwater BLMs can NOT directly be used for marine environments
- Me-DOC binding freshwater different then marine waters = Speciation modelling to be modified with the ionic strength
DOC normalization if applicable = bioavailability correction = not species-specific
27
4. Bioavailability models in marine waters
28
4. Bioavailability models in marine waters
Model accuracy - Bioavailability prediction within a factor of 2
Toxicity - DOC regressions for 6 marine species