analytical methods and quality control in the speciation analysis of organotin compounds roberto...
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Analytical Methods and Quality Control in the Speciation Analysis of
Organotin Compounds
Roberto MorabitoENEA – UTS PROT
3rd Aegean Analytical Chemistry DaysPolihnitos, Lesvos, Greece
September 29 - October 3, 2002
ANALYTICAL METHODS FOR ENVIRONMENTAL ANALYSIS
levels for sediment and biological
Sensitive enough to determine contaminants downto environmental concentrations
- from ppt to ppb levels for water samples- from ppb to ppm samples
*
Able to discriminate among different chemical formsin case of speciation analysis *
Characterized by good precision and accuracy*
CRITICAL STEPS IN ENVIRONMENTALANALYSIS
• SAMPLING
• STORAGE
• TREATMENT
ExtractionDerivatizationClean up
• ANALYSIS
SAMPLING STRATEGIES
CHOICE OF SAMPLES
COLLECTION OF SAMPLES
NUMBER OF SAMPLES
SPATIA L VARIABILITY
TEMPORAL VARIABILITY
Filtered water - Particulate matter Undisturbed surface sediments
Biota
Sampling devices Sampling vessels
Representativeness of sampling point Representativeness of sampling area
Microlayer Interface water-sediment Vertical stratification Enclosed and flushed areas Dockyard activities
Tides - Seasons
Rn Sn X(4-n)Rn Sn X(4-n)Rn Sn X(4-n)Rn Sn X(4-n)
WHERE:
X = Cl-
F-
Br-
OH-
SH-
O2-
COOH-
R = ALKYL GROUPS
OR
ARYL GROUPS
M. Hoch, Appl. Geochem., 16, 719-743 (2001)
R4Sn R3SnX R2SnX2 RSnX3 SnX4R4Sn R3SnX R2SnX2 RSnX3 SnX4R4Sn R3SnX R2SnX2 RSnX3 SnX4R4Sn R3SnX R2SnX2 RSnX3 SnX4
a) Ultraviolet irradiation
b) Biological cleavage
c) Chemical cleavage
d) Thermal cleavage
e) Gamma irradiation
TBT Distribution in marine environment
AIR
MICROLAYER
(Organics, oils)
WATER Colloids
Chloride (TBTCl)
Hydroxide (TBTOH)
TBT Aquocomplexes (TBTOH2) Inorganicl
Carbonatic species suspended matter
Organic
suspended matter
SEDIMENTS
Samplingpoint
Water(surface)
ng l-1
Water(5m depth)
ng l-1
Musselsng g-1
dry weight
Bioconcentration factor Sedimentsng g-1
dry weight
Accumulation factor
TBT 15 22 2940 130,000—200,000 400 18,000—26,000B4 DBT 85 14 1290 15,000—90,000 60 650—4200
MBT — — 400 60
TBT 11 13 550 42,000—50,000A1 DBT 86 9 110 1300—12,500
MBT — — 140
TBT 19 12 350 18,400—29,000A5 DBT 123 16 220 1800—14,700
MBT — — 180
TBT 10 7 1010 100,000—150,000 580 58,000—83,000CE2 DBT 5 3 180 40,000—60,000 <50
MBT — — <50 <50
TBT 10 6 2270 230,000—380,000 750 75,000—125,000CW2 DBT 6 4 570 100,000—140,000 <50
B4 = mussel farm; A1 = dockyard activities; A5 = military harbour; CE2 =open Gulf outside the harbour (east side); CW2 = open Gulf outside theharbour (west side).
TABLE 4. Water, mussel and sediment concentrations (as Sn) in the La Spezia Gulf.Reprinted from Chiavarini S., Cremisini C. and Morabito R. (1991) UNEP/FA O/IAEA MAPTechnicalReport Series No. 59, pp. 179-187, UNEP, Athens 1991.
Effetto del TBT sui bivalvi
Ispessimento del guscio dovuto ad alterazione del
metabolismo del calcio
Imposexsovraimposizione di caratteri
sessuali maschili su gasteropodi di sesso femminile
TBT inibitore dello sviluppo di
ormoni femminili
C.M. Rees et al., Mar. Pollut. Bullet., 42, 873-878 (2001)
M. Ramon andM.J. Amor, Mar.Env. Res., 52,463-475 (2001)
A.C. Birchenough et al., Mar. Pollut. Bullet., 44, 534-543 (2002)
M. Ramon and M.J. Amor, Mar. Env. Res., 52, 463-475 (2001)
P. Michel et al., Mar. Pollut. Bullet.,42, 1128-1132 (2001)
Concentration of butyltin compounds in mussels from Sardinian coasts - First sampling (November 2001)
0
1000
2000
3000
4000
5000
6000
7000
8000
Cagliari B Cagliari Arbatax Calasetta Villasimius B Villasimius Olbia B Olbia Alghero Asinara Oristano
ng
/g d
ry w
eig
ht
as c
atio
n
TBTDBTMBT
R. Morabito et al., unpublished results
Concentration of butyltin compounds along the Sardinian coasts - Second sampling (April 2002)
0
500
1000
1500
2000
2500
3000
3500
4000
4500
Caglairi B Cagliari Arbatax Calasetta Villasimius Bianco SS Olbia Alghero Asinara Oristano
ng
/g d
ry w
eig
ht
as c
atio
n
TBTDBTMBT
R. Morabito et al., unpublished results
Concentration of butyltin compounds in mussels from Venice Lagoon - November 2001
0
1000
2000
3000
4000
5000
6000
1A2 2A1 5A1 7A1 9A1 10A1 12A1 13A2 14A1 15A1 16A1 17A1 18A2
ng
/g d
ry w
eig
ht
as
ca
tio
n
TBTDBTMBT
R. Morabito et al., unpublished results
Concentration of butyltin compounds in clams from Venice Lagoon - November 2001
0
500
1000
1500
2000
2500
2B1 3B1 6B1 7B1 8B1 9B1 10B1 11B1 12B1 13B1 14B1 15B1 16B2 17B1 18B1
ng
/g d
ry w
eig
ht
as
ca
tio
n
TBTDBTMBT
R. Morabito et al., unpublished results
TBT concentration levels in mussels and clams from Venice Lagoon - November 2001
0
1000
2000
3000
4000
5000
6000
2 7 9 10 12 13 14 15 16 17 18
ng
/g d
ry w
eig
ht
as
ca
tio
n
mussels clams
R. Morabito et al., unpublished results
DBT concentration levels in mussels and clams from Venice Lagoon - November 2001
0
500
1000
1500
2000
2500
3000
3500
2 7 9 10 12 13 14 15 16 17 18
ng
/g d
ry w
eig
ht
as
ca
tio
n
musselsclams
R. Morabito et al., unpublished results
MBT concentration levels in mussels and clams from Venice Lagoon - November 2001
0
200
400
600
800
1000
1200
2 7 9 10 12 13 14 15 16 17 18
ng
/g d
ry w
eig
ht
as c
atio
n
musselsclams
R. Morabito et al., unpublished results
B.S. Tselentis et al.,Mar. Pollut. Bullet.,38, 146-153 (1999)
B.S. Tselentis et al.,Mar. Pollut. Bullet.,38, 146-153 (1999)
B.S. Tselentis et al., Mar. Pollut. Bullet., 38, 146-153 (1999)
P. Michel et al., Mar. Pollut. Bullet.,42, 1128-1132 (2001)
B.S. Tselentis et al., Mar. Pollut. Bullet., 38, 146-153 (1999)
AIMS OF THE HIC-TBT PROJECT
• To evaluate incidence of imposex along the shipping lanes in North Sea, Atlantic Ocean and Mediterranean Sea
• To disseminate information on the TBT problem and to establish and to promote contacts with scientific community, policy makers, paint producers, NGOs, etc
a B
A
D E
C
Station Fem N Fem I Male N Total VDSI Species Imposex frequency
PAL-A 4 4 1 5 3.2 B. brandaris 100 PAL-B 5 5 2 7 3.8 B. brandaris 100 PAL-C 3 3 1 4 3.7 B. brandaris 100 TER-A 8 8 6 14 3.5 H. trunculus 100 TER-B 6 6 13 19 4.0 H. trunculus 100 TER-C 13 13 9 22 3.9 T. haemastoma 100 CAS-A 10 10 29 39 4.4 H. trunculus 100 CAS-B 4 4 2 6 3.7 H. trunculus 100 CAS-B 3 3 1 4 4.0 B. brandaris 100 CAS-C 3 3 1 4 3.0 H. trunculus 100 CAS-C 1 1 3 4 4.0 B. brandaris 100 USP-A 1 1 10 11 3.0 H. trunculus 100 USP-B 2 2 16 18 3.5 H. trunculus 100 USP-C 3 3 15 18 3.3 H. trunculus 100 USR 10 1 6 16 0.1 H. trunculus 10
Imposex data from the first sampling campaign (1999).Stations: PAL, Gulf of Palermo; TER, Gulf of Termini Imerese; CAS, Gulf of Castellammare; USP, Ustica Island outside harbour; USR, Ustica Island Sea Reserve; Fem N, number of females; Fem I, females with imposex; Male N, number of males; VDSI, Vas Deferens Sequence Index.
Station Fem N Fem I Male N Total VDSI Species Imposex frequency
PAL-A 11 11 5 16 4.0 H.trunculus 100 PAL-B 14 14 6 20 3.6 H.trunculus 100 PAL-C 12 12 5 17 3.8 H.trunculus 100 TER-A 4 4 13 17 1.8 H.trunculus 100 TER-B 9 9 12 21 1.8 H.trunculus 100 TER-C 16 13 7 23 1.8 H.trunculus 81 CAS-A 6 6 12 18 4.5 H.trunculus 100 CAS-B 4 4 13 17 3.5 H.trunculus 100 CAS-C 12 8 6 18 2.9 H.trunculus 67 USP-A 11 11 3 14 3.9 H.trunculus 100 USP-B 8 8 9 17 3.0 H.trunculus 100 USP-C 8 8 12 20 3.0 H.trunculus 100 USR-A 13 6 1 14 0.9 H.trunculus 46 USR-B 12 4 10 22 0.3 H.trunculus 33 USR-C 9 7 15 24 0.8 H.trunculus 78
Imposex data from the second sampling campaign (2000). Stations: PAL, Gulf of Palermo; TER, Gulf of Termini Imerese; CAS, Gulf of Castellammare; USP, Ustica Island outside harbour; USR, Ustica Island Sea Reserve; Fem N, number of females; Fem I, females with imposex; Male N, number of males; VDSI, Vas Deferens Sequence Index.
Correlation between TBT concentration and imposex stage in gastropods
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
4,5
5,0
0 10 20 30 40 50 60 70 80 90 100ng/g Sn d.w.
impo
sex
stag
e
TBT
S. Chiavarini et al., Chemosphere, in press
C.M. Barroso and M.H. Moreira, Mar. Pollut. Bullet., 44, 480-486 (2002)
C.M. Barroso and M.H. Moreira, Mar. Pollut. Bullet., 44, 480-486 (2002)
SAMPLING STRATEGIES
CHOICE OF SAMPLES
COLLECTION OF SAMPLES
NUMBER OF SAMPLES
SPATIA L VARIABILITY
TEMPORAL VARIABILITY
Filtered water - Particulate matter Undisturbed surface sediments
Biota
Sampling devices Sampling vessels
Representativeness of sampling point Representativeness of sampling area
Microlayer Interface water-sediment Vertical stratification Enclosed and flushed areas Dockyard activities
Tides - Seasons
Dep
t h
Typical Tributyltin concentration profile in the sediments
Resuspension
Reduced direct inputs
Degassing
SAMPLING STRATEGIES
CHOICE OF SAMPLES
COLLECTION OF SAMPLES
NUMBER OF SAMPLES
SPATIA L VARIABILITY
TEMPORAL VARIABILITY
Filtered water - Particulate matter Undisturbed surface sediments
Biota
Sampling devices Sampling vessels
Representativeness of sampling point Representativeness of sampling area
Microlayer Interface water-sediment Vertical stratification Enclosed and flushed areas Dockyard activities
Tides - Seasons
CRITICAL STEPS IN ENVIRONMENTALANALYSIS
•SAMPLING
•STORAGE
•TREATMENT
ExtractionDerivatizationClean up
•ANALYSIS
SELECTION OF METHODSFOR SPECIATION ANALYSIS
Stabilisation / Storage (examples)« Preserver the original speciation »
STABILISATION- Acidification (waters) -
- Lyophilisation (biota, sediments, etc.) -- Oven-drying (sediments) -
- Pasteurisation (sediments) -- Gamma-irradiation (biota, sediments,waters) -
STORAGE- Freezing recommended for butyl- and phenyl-tins (biota, sediments) -
- Storage at ambient temperature (n the dark) for MeHg -- Storage at 4°C in the dark for trimethyllead -
CRITICAL STEPS IN ENVIRONMENTALANALYSIS
•SAMPLING
•STORAGE
•TREATMENT
ExtractionDerivatizationClean up
•ANALYSIS
TREATMENT
CONTAMINATION
LOSSES
CHANGES IN SPECIATION(DECOMPOSITION – TRANSFORMATION OF ONE SPECIES
IN ANOTHER ONE BY DEGRADATION, OXIDATION OR REDUCTION PHENOMENA)
INCOMPLETE EXTRACTION
LOW YIELDS OF DERIVATIZATION
SELECTION OF METHODSFOR SPECIATION ANALYSIS
Extraction (examples)
ACIDSAcetic acid (butyltins)
Sulphuric acid, distillation (MeHg)Nitric acid/micro-wave (MeHg)
MIXTURE ACID/ORGANIC SOLVENTHydrochloric acid / toluene (MeHg, butyltins)
Hydrochloric acid / methanol (butyltins) Sulphuric acid / toluene (MeHg)
ORGANIC SOLVENTSToluene (MeHg)
Methanol / hexane (butyltins)Tropolone / pentane (butyltins)
OTHERSCO2 / methanol, supercritical fluid (MeHg)
CO2 / tropolone, supercritical fluid (butyltins) Protease / lipase (butyltins)
Tetramethylammonium hydroxide (butyltins)
COMPARISON OF 12 SELECTED EXTRACTION METHODS FOR THE
DETERMINATION OF ORGANOTINS IN BIOLOGICAL SAMPLES
•SAME SAMPLE (CRM 477)
•SAME OPERATOR
•SAME ANALYTICAL STEPS AFTER EXTRACTION
THE PERFORMANCE OF THE OVERALL METHOD CAN BE STRICTLY RELATED TO THE EFFICIENCY
OF THE EXTRACTION METHOD APPLIED
General conditions adopted in the considered extraction
methods
Methods Extracting solvent
Complexing agent
Acid Extraction technique
1 Methanol Tropolone HCl Ultrasonically shaken 2 Methanol Tropolone - Ultrasonically shaken 3 Methanol - - Ultrasonically shaken 4 Hexane Tropolone HCl Ultrasonically shaken 5 Toluene Tropolone - Ultrasonically shaken 6 Dichloromethane Tropolone HBr Mechanically shaken 7 Pentane Tropolone HBr Mechanically shaken 8 Hexane Tropolone - Refluxed 9 Dichloromethane Tropolone - Refluxed 10 Hexane - - Soxhlet extraction 11 Methanol - HCl Microwave oven 12 Methanol - HAcO Microwave oven
CONCLUSIONS
Parameters affecting extraction efficiency of butyl- and phenyl-tin compounds from biological samples are:
•Polarity of organic solvents•Presence of a complexing agent•Acidic conditions
The type of extraction technique doesn’t seem to affect the extraction efficiency
The best extraction efficiency for all organotin compounds considered was achieved by using a mixture of methanol, tropolone, HCl (<1M) under ultrasonic treatment
BCR 477 - TBT, DBT and MBT in mussel tissue
TBT DBT MBT1,96 1,32 1,932,23 1,56 1,411,92 1,31 1,572,04 1,40 1,640,17 0,14 0,262,20 1,54 1,500,19 0,12 0,27
AverageSD
Certified valueUncertainity
Concentration in mg/kg as cation
BCR 710 - TBT and DBT in oyster tissue(at the certification stage)
TBT DBT MBT113 86 105115 95 143127 109 96118 96 1158 11 25
133 82 n.c25 17
Concentration in g/kg as cation
AverageSD
Certified valueUncertainity
0,0
0,1
0,2
0,3
2 3 10 12 14 17a 17b 21 24 25 ASE Mean
Labs
TBT c
on
cen
trati
on
mg
/kg
Candidate BCR 710 - Oyster tissue
0,0
0,1
0,2
2 3 10 14 17a 17b 21 24 25 ASE Mean
Labs
DBT c
on
cen
trati
on
mg
/kg
Candidate BCR 710 - Oyster tissue
0,0
0,1
0,2
0,3
2 3 11 14 17a 17b 21 25 ASE Mean
Labs
MBT c
on
cen
tra
tio
n m
g/k
g
Candidate BCR 710 - Oyster tissue
SELECTION OF METHODSFOR SPECIATION ANALYSIS
Derivatisation (examples)« Transformation of a given compound
to enable a better separation »
HYDRIDE GENERATION (NaBH4)RnSn(4-n)+ -------- RnSnH(4-n) + H2
with n = 1,2,3 (R= methyl, ethyl or butyl)
ETHYLATION (NaBEt4)
GRIGNARD REACTIONS- Methylation - Ethylation
- Propylation- Butylation- Pentylation- hexylation
TBT DBT MBT DPhT TPhT0
20
40
60
80
100
120
%
Influence of the Grignardconcentration
(*) 0 minutes - (**) 40 minutes
2 M (*)
0.5 M (*)
0.5 M (**)
TBT DBT MBT DPhT TPhT0
20
40
60
80
100
120
%
Influence of mechanical agitation
shaking
not shaking
TBT DBT MBT DPhT TPhT0
20
40
60
80
100
120
%COMPARISON BETWEEN DIFFERENT
DERIVATIZATION TECHNIQUES
Grignard pentylation
Grignard ethylation
Tetraethylborate
SELECTION OF METHODSFOR SPECIATION ANALYSIS
Separation (examples)« necessary step, considering that the determination of
different chemical species, in general, cannot be performed with a sufficient selectivity (e.g. selective detection by AAS)
»GAS CHROMATOGRAPHY
- Packed column -- Capillary column -
HIGH PERFORMANCELIQUID CHROMATOGRAPHY (HPLC)
OTHERS- Cryoegenic trapping (cold trap) -
- Capillary electrophoresis -- Ion exchange microcolumns -
SELECTION OF METHODSFOR SPECIATION ANALYSIS
Detection (examples)
ELEMENT SPECIFIC- Atomic absorption spectrometry -
(electrothermal, flame, quartz furnace)
MULTI-ELEMENT- Inductively coupled plasma atomic emission spectrometry (ICP-AES) -
- Inductively coupled plasma mass spectrometry (ICP-MS) -
SPECIFIC OF ONE GIVEN SPECIES- Voltammetry -
NON SPECIFIC- Flame ionisation detection (FID) -
- Flame photometric detection (FPD) -- Electron capture detection (ECD) -
CALIBRATION- Stoichiometry, purity, primary and internal standards, calibration method (e.g. standard additions)
ANALYTICAL PROCEDURE FORMUSSELS AND SEDIMENT
SAMPLEca 500 mg dry weight add 50-500 ng
TPrT as internal std.
SONICATION15 ml CH 3OH/1 ml HCl conc./0.05%
tropolone - repeat twice
LIQUID/LIQUID PARTITIONINGadd 150 ml water - 30 ml CH 2Cl2
SOLVENT EXCHANGEto 1 ml isooctane by a gentle stream
of N2
GRIGNARD DERIVATIZATION1 ml PeMgBr 2M in diethyl ether
sediments/biotaSILICA GEL/FLORISIL CLEAN UP
1 g in a Pasteur pipette elution with2.5 ml of hexane:toluene (1:1)
GC ANALYSIS(FPD or MS)
ORGANOTIN ANALYSIS BY:
GC-FPD GC-MS
8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.000
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
12000
13000
14000
15000
16000
17000
18000
19000
20000
21000
22000
23000
24000
25000
26000
Time-->
Abundance
TIC: 2107C03A.D
RM 424 – Harbour sediment
Species Indicative value(g/kg)
Accepted sets ofresults
Techniques used
TBT 20 ± 5 8 GC-MS, GC-FPD, HPLC-ICPMS, GC-MIP-AES
DBT 53 ± 19 6 HG-GC-AAS, GC-MS,GC-FPD
MBT 257 ± 54 3 GC-MS, GC-FPD
BCR 462R – Coastal sediment
Species Certified valueg/kg)
Accepted sets ofresults
Techniques used
TBT 54 ± 15 5 HG-GC-AAS, GC-MS,GC-FPD, HPLC-IDMS
DBT 68 ± 12 7 HG-GC-AAS, GC-MS,GC-FPD, GC-MIPAES,
GC-ICPMS, HPLC-ICPMS
BCR 477 – Mussel tissue
Species Certified valueg/kg)
Accepted sets ofresults
Techniques used
TBT 2.20 ± 0.19 18 GC-QFAAS, HG-GC-AAS, GC-MS, GC-FPD,
GC-MIPAES,HPLC-ID-ICPMS,
HPLC-ICPMS,HPLC-ICPAES,
HPLC-Fluo
DBT 1.54 ± 0.12 15 GC-QFAAS, HG-GC-AAS, GC-MS, GC-FPD,
GC-MIPAES,HPLC-ICPMS
MBT 1.50 ± 0.28 8 GC-QFAAS, HG-GC-AAS, GC-MS, GC-FPD,
GC-MIPAES,HPLC-ICPAES,
BCR 646 – FRESHWATER SEDIMENT
Species Certified valueg/kg)
Accepted sets ofresults
Techniques used
TBT 480 ± 80 14 GC-MS, GC-FPD, GC-MIP-AES, GC-AED, GC-
QFAAS, HPLC-Fluo
DBT 770 ± 90 13 GC-MS, GC-FPD, GC-MIP-AES, GC-AED, GC-
QFAAS
MBT 610 ± 120 10 GC-MS, GC-FPD, GC-MIP-AES, GC-AED, GC-
QFAAS
TPhT 29 ± 11 7 GC-MS, GC-MIP-AES,GC-AED, HPLC-Fluo
DPhT 36 ± 8 8 GC-MS, GC-FPD, GC-MIP-AES, GC-AED
MPhT 69 ± 18 6 GC-MS, GC-FPD, GC-MIP-AES, GC-AED
CANDIDATE BCR 710 – OYSTER TISSUE*
Species Certified valueg/kg)
Accepted sets ofresults
Techniques used
TBT 135 ± 27 10 GC-MS, GC-FPD,GC-ICPMS,
HPLC-ID-ICPMS,GC-AED
DBT 82 ± 19 9 GC-MS, GC-FPD,GC-ICPMS, GC-AED
* The candidate BCR 710 has been proposed also for the certification of arsenobetaineand methylmercury.
CRMS AVAILABLE FOR ORGANOTIN SPECIATION ANALYSIS
MATERIAL PRODUCER SPECIES CERTIFIED VALUES
RM 424 –Harbour sediment
BCR TBT*DBT*MBT*
20 ± 5 g/kg)53 ± 19257 ± 54
BCR 462R –Coastal sediment
BCR TBTDBT
54 ± 15 g/kg)68 ± 12
BCR 477 –Mussel tissue
BCR TBTDBTMBT
2.20 ± 0.19 (mg/kg)1.54 ± 0.121.50 ± 0.28
BCR 646 –Freshwatersediment
BCR TBTDBTMBTTPhTDPhTMPhT
480 ± 80 g/kg)770 ± 90610 ± 12029 ± 1136 ± 869 ± 18
BCR 710** -Oyster tissue
BCR TBTDBT
135 ± 27 g/kg)82 ± 19
PACS-1 –Harbour sediment
NRCC TBTDBTMBT
1.27 ± 0.22***(mg/kg)1,16 ± 0,19***0,28 ± 0.17***
PACS-2 –Harbour sediment
NRCC TBTDBTMBT
0.98 ± 0.13*** (mg/kg)1.09 ± 0.15***0.45 ± 0.05***
NIES-11 –Fish tissue
NIES TBTTPhT*
1.3 ± 0.1*** (mg/kg)6.3***
* Indicative values** At the certification stage (The material has been proposed also for the certificationof arsenobetaine and methylmercury)*** As Sn