analytical methods and quality control in the speciation analysis of organotin compounds roberto...

Analytical Methods and Quality Control in the Speciation Analysis of

Organotin Compounds

Roberto MorabitoENEA – UTS PROT

[email protected]

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


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


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


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


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


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


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)

SAMPLING STRATEGIES

CHOICE OF SAMPLES


NUMBER OF SAMPLES




Biota




Tides - Seasons

Dep

t h

Typical Tributyltin concentration profile in the sediments

Resuspension

Reduced direct inputs

Degassing

SAMPLING STRATEGIES

CHOICE OF SAMPLES


NUMBER OF SAMPLES




Biota




Tides - Seasons


•SAMPLING

•STORAGE

•TREATMENT


•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 -


•SAMPLING

•STORAGE

•TREATMENT


•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


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


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



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 (**)


20

40

60

80

100

120

%

Influence of mechanical agitation

shaking

not shaking


20

40

60

80

100

120

%COMPARISON BETWEEN DIFFERENT

DERIVATIZATION TECHNIQUES

Grignard pentylation

Grignard ethylation

Tetraethylborate


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 -


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)


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



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



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*



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

analytical methods and quality control in the speciation analysis of organotin compounds roberto...

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