DIETARY EXPOSURE TO ORGANOTIN COMPOUNDS …ORGANOTIN COMPOUNDS (OTs) ΟΤsare the most used...

DIETARY EXPOSURE TO ORGANOTIN COMPOUNDS IN GREECE FROM SEAFOOD

CONSUMPTION

Nikolaos S. Thomaidis

University of AthensDepartment of Chemistry

Laboratory of Analytical Chemistry

ORGANOTIN COMPOUNDS (OTs)ΟΤs are the most used organometallic compounds

AOAC Europe Workshop 3

Fate of OTs in the marine environment

R4Sn

R3Sn+(X=OH-)

R2Sn2+

RSn3+

R= Me, Bu, Ph,Oct


OTs

Bioaccumulation

up to××××105


Toxic effects of OTs

IMPOSEX

Inhibitors of the female hormones synthesis

Shell thickening

Effects on the Ca2+ metabolism


1. Seafood

2. Products of daily use:

• materials in contact with food

• fabrics• plastics (water pipes)

Human exposure to OTs:


OTs IN FOOD

Little is known about human exposure

OTs: Endocrine Disrupting Compounds

WHO: TDI for TBT 0.25 µg/day/kg bw,

TPhT 0.5 µg/day/kg bw

EFSA: TDI for ΣOTs (BTs+TPhT+DOT)

0.25 µg/day/kg bw

�What is the dietary exposure to ΟΤs?

�Do EU has to take legislative measures?

?


•SCOOP Task 3.2.13: Assessment of the dietary exposure to organotins compound of the population of the EU Member States

2001 – 2002

•OT-SAFE: Sources, consumer exposure and risks of organotin contamination in seafood

2002 – 2004

EU initiatives


ΟΤ-SAFE CONSORTIUM


ΟΤ-SAFE ObjectivesThe overall goal of that project was to assess if there was a reason for concern regarding human exposure to TBT through seafood from the European market. There were three major objectives:

To compile a uniform EU-wide database on TBT levels in seafood from major fishing grounds and shellfish farms

To determine the fate of TBT in seafood during preparation (e.g., cooking, baking, frying, etc.)

To perform a risk assessment from dietary exposure to OTs


PURPOSE OF THIS STUDY

�The determination of OTs in seafood that are

consumed by Greek population

�The estimation of the dietary exposure of the

Geek population to these compounds from the

consumption of contaminated seafood


PLANNING

There was no data for OTs in Greece before 2001

� Development and validation of a method for the determination of ΟΤs in seafood

� Seafood analysis (High consumption and high [ΟΤs])

Sampling of seafood that are widely consumed

in Greece:

Food Balance Sheets: Data from national seafood

production and imports – exports patterns (1999)

Household budget surveys (HBS): DAFNE data base


Food Consumption Data

Household budget survey (1998-99):

• average and consumers only availability

(g/person/day) of selected food items

• 14-day food accounts diary

• 6258 households

• all-year round to allow for seasonal variability

in food consumption

• Geographical Level: National (urban+rural)


Average availability (g/person/day) of selected

food items among the Greek population

148939365.5Fish (Quality

A, B and C),

frozen

4195417118Fish (Quality

C), fresh


B), fresh


A), fresh

Rate of consumers

(%)

ConsumersConsumption

(g/person/day)

Mean high (95%)

Total Population

Consumption

(g/person/day)

Mean high (95%)

Food name


Average availability (g/person/day) of selected

food items among the Greek population

133412101.5Canned fish,

fish roe,

caviar, fish

pies

9.87131243.4Frozen

seafood

(incl. snails)

6.49537182.6Fresh

seafood

(incl. snails)

Rate of

consumers

(%)

ConsumersConsumption

(g/person/day)

Mean high (95%)

Total Population

Consumption

(g/person/day)

Mean high (95%)

Food name


SAMPLING (1)

Period: 8/2001 – 6/2006TOTAL : 98 samples

• fish A : n=6 – fresh fish Qual. A (3 sea bass, 3 sea

bream)

• fish B : n=2 – fresh fish Qual. B (2 hakes)

• fish C : n=13 – fresh fish Qual. C (8 anchovies, 5

sardines)

• fish D : n=13 – frozen fish (5 swordfish, 1 shark, 4

cod, 2 topes, 1 perch)


• ML A : n=37 – fresh molluscs (8 stripped venus, 6 bearded horse mussels, 19 Mediterraneanmussels , 3 scallops, 1 hard clam)

• ML B : n=19 – frozen molluscs (5 octopus, 6squids, 1 cuttlefish, 3 mussels, 4 shrimps)

• CAN : n=8 – canned and processed seafood (4 tuna, 2 processed anchovies, 1 smoked mackerel, 1 sardine)

SAMPLING (2)


Acidic extraction with NaBEt4

PentylationPentylation with with GrignardGrignard

reagentreagent0.1g of sample

Leaching of OTs in sonication bath (15 min) with 10 ml of 0.05% tropolone/MeOH+ internal standard (TPrT)

+ 1 ml of HCl

Centrifugation

Add 0.7 ml of ΝΗ3 + adjustment of pH at 5 (10 ml of Ac-buffer)

+ 1 g of ΝaCl

Derivatization with 1 ml of NaBet4 0.15% and extraction (10 min) in hexane (2 ml)

Centrifugation

Determination by GC-FPD46 samples analysed

Determination by GC-MSD28 samples analysed

Sample (20mg of fat – max. 5g)

Homogenised for 15 s after the addition of 5 ml of H2O and adjustment of pH at 1.5-2.2 (with

solutions of HCl and NaOH)

Add 0.5 g NaCl + 12 ml tropolonesolution (0.02 % in n-pentane) +

homogenised for 30 s

Add 12 ml tropolone solution + ultrasonic bath for 5 min + shaken for 5 min at 250 cpm + add of internal

standard + adjustment of volume to 1 ml

Ultrasonic bath for 5 min + shaken for 5 min at 250 cpm + dried with Na2SO4.

Determination by GC-MSD24 samples analysed

Derivatization with 1.5 ml of Grignard reagent + cooling in an ice bath + add of 2 ml of hexane + adjustment of volume to 1 ml

Purification over a chromatographic column filled with 5 g alumina and 1 cm layer of NaSO4, previously conditioned with 5 ml of n-hexane/diethylether

(80/20 v/v) + elution with 15 ml of n-hexane/diethylether (80/20 v/v) + drying under N2flow + adjustment of end volume to 1 ml with

hexane


QUALITY ASSURANCE – VALIDATION – CRM 477 ANALYSIS

1.04±0.100.921.50±0.14-TPhT

1.36±0.150.971.31±0.211.50±0.28MBT

1.30±0.141.341.68±0.351.54±0.12DBT

1.77±0.132.162.29±0.302.20±0.19TBT

Concentrations found with GC-MSD analysis after pentylationwith Grignardreagent (mg kg-1) (n=5)

Concentrations found with GC-MSD analysis after acidic extraction with NaBEt4 (mg kg-1)

Concentrations found with GC-FPD analysis after acidic extraction with NaBEt4 (mg kg-1) (n=6)

Certified concentration(mg kg-1)

DETERMINATION OF ΟΤs

RSDR : 10-21%


DETERMINATION OF ΟΤs

3

4

5

6

7

8

9

10

11

0 2 4 6 8 10 12 14 16

t (min)

mV

MBT

DBT

MPhT

TBT

DPhT

TPhT

50 µg L-1

HP-1 (25m × 0.32 mm)

LIMITS OF DETECTIONLIMITS OF DETECTION

11310TPhT

176MBT

1136DBT

3154TBT

GC-IT-MSD analysis after pentylation with Grignardreagent (ng g-1)

GC-MSD analysis after acidic extraction with NaBEt4 (ng g-1)

GC-FPD analysis after acidic extraction with NaBEt4 (ng g-1)

C < LOD →→→→ LOD/2


CONCENTRATIONS FOUND

ΤΒΤ (ng/g)

---<15.0CAN (n=8)

3.592.005.44<1.00 – 29.0ML B (n=19)

17.117.029.8<4.00 – 109.3ML A (n=37)

5.817.509.40<4.00 – 54.2Fish D (n=13)

27.438.074.1<4.00 – 491.0Fish C (n=13)

27.327.627.629.3 – 31.2Fish B (n=2)

6.907.507.92<4.00 – 12.5Fish A (n=6)

Geo. meanMedianMeanRange


DΒΤ (ng/g)

36.649.4173.1<10 – 1117CAN (n=8)

8.516.5049.0<1.00 – 229.9ML B (n=19)

18.815.043.15.00 – 366.1ML A (n=37)

21.66.5067.6<10 – 371.2Fish D (n=13)

4.205.005.45<1.00 – 20.0Fish C (n=13)

---<13Fsh B (n=2)

---<13Fish A (n=6)



MΒΤ (ng/g)

4.233.505.33<6 – 18.9CAN (n=8)

3.093.507.16<1.00 – 30.9ML B (n=19)

7.847.8717.81.10 – 151.0ML A (n=37)

5.133.506.54<7 – 21.5Fish D (n=13)

2.741.507.08<1.00 – 49.0Fish C (n=13)

---<7Fish B (n=2)

2.742.754.72<1.00 – 17.3Fish A (n=6)



TPhT (ng/g)

---<13CAN (n=8)

4.305.0013.9<1.00 – 90.4ML B (n=19)

8.305.0018.21.80 – 201.3ML A (n=37)

6.006.506.04<13Fish D (n=13)

7.696.0010.54.40 – 49.0Fish C (n=13)

---<13Fish B (n=2)

---<13Fish A (n=6)

Geo.meanMedianMeanRange


RISK ASSESSMENT

1st Scenario:

Mean Exposure of Total Population:

High Exposure of Total Population:

)]70([

)]/(_[)]/(_[][

kgbw

daygnConsumptioMeanggOTsGeoMeanDI

×=

µ

)]70([

)]/%(95__[)]/(_[][

kgbw

daygnConsumptioHighggOTsHighDI

×=

µ


RISK ASSESSMENT

2nd Scenario (worst case scenario):

Mean Exposure of the Consumers:

High Exposure of the Consumers :

)]70([

)]/(_[)]/(_[][

kgbw

daygnConsumptioMeanggOTsGeoMeanDI

×=

µ

)]70([

)]/%(95__[)]/(_[][

kgbw

daygnConsumptioHighggOTsHighDI

×=

µ


DAILY DIETARY INTAKES

Total Population

0.05180.00003201.38.30TPhT182.6ML A

0.001643.77ΣBTs182.6ML A

0.03880.0003151.07.84MBT182.6ML A

Daily intake/bw(µg/day/kg bw)

Mean High (95%)

Concentration(ng/g)

Mean High

OTsConsumption (g/person/day)

Mean High(95%)

Food name

0.001952.08ΣOTs182.6ML A

0.09410.0007366.118.84DBT182.6ML A

0.02810.0006109.317.10TBT182.6ML A



High consumers (6.4%)

0.023143.77ΣBTs9537ML A

0.027652.08ΣΟΤ9537ML A

0.27320.0044201.38.30ΤPhT9537ML A

0.20500.0041151.07.84MBT9537ML A

0.49690.010366.118.84DBT9537ML A

0.14840.0090109.317.10TBT9537ML A


Mean High (95%)

Concentration(ng/g)

Mean High

OTsConsumption (g/person/day)Mean High(95%)

Food name

199% of TDI

5% of the 6.4% of

The total population:

38.400 people



Total Population

0.008834.31ΣBTs7118Fish C

0.010842.00ΣΟΤ7118Fish C

0.04970.002049.007.69ΤPhT7118Fish C

0.04970.000749.052.74MBT7118Fish C

0.02030.001120.004.20DBT7118Fish C

0.49800.0070491.027.36TBT7118Fish C


Mean High (95%)

Concentration (ng/g)

Mean High


Food name

199% of TDI

5% of the total

population:

600.000 people



High consumers (41%)

0.020134.31ΣBTs9541Fish C

0.024642.00ΣΟΤ9541Fish C

0.06650.004549.007.69ΤPhT9541Fish C

0.06660.001649.052.74MBT9541Fish C

0.02710.002520.004.20DBT9541Fish C

0.66640.0160491.027.36TBT9541Fish C


Mean High (95%)

Concentration(ng/g)

Mean High


Food name

267% of TDI !

5% of the 41% of


246.000 people




0.018132.51ΣBTs8939Fish D

0.021538.51ΣΟΤ8939Fish D

< LOD< LODΤPhT8939Fish D

0.02730.002921.435.13MBT8939Fish D

0.47190.0120371.2021.57DBT8939Fish D

0.06890.003254.205.81TBT8939Fish D


Mean High (95%)

Concentration(ng/g)

Mean High


Food name

189% of TDI

5% of the 14% of


84.000 people




0.007040.84ΣBTs3412CAN

0.007040.84ΣΟΤ3412CAN

-< LOD< LODΤPhT3412CAN

0.00920.000718.874.23MBT3412CAN

0.54250.00631116.9936.61DBT3412CAN

-< LOD< LODTBT3412CAN


Mean High (95%)

Concentration(ng/g)

Mean High


Food name

217% of TDI

5% of the 13% of


78.000 people


MAXIMUM DAILY DIETARY INTAKES

OF HIGH CONSUMERS

(µg /kg bw/day)

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

Fis h A

(n=6)

Fis h B

(n=2)

Fis h C

(n=13)

Fis h D

(n=13)

ML A

(n=37)

ML B

(n=19)

CAN

(n=8)

TBT

DBT

MBT

TP hT

TDI


(µg /kg bw/day)

0,0000

0,2000

0,4000

0,6000

0,8000

1,0000

1,2000

Fis

h A

(n=

6)

Fis

h B

(n=

2)

Fis

h C

(n=

13

)

Fis

h D

(n=

13

)

ML

A

(n=

37

)

ML

B

(n=

19

)

CA

N

(n=

8)

TBT

DBT

MBT

TP hT

ΣBTs

ΣOTs

TDI

MAXIMUM DAILY DIETARY INTAKES

OF HIGH CONSUMERS


CONCLUSIONS (1)

At least one OT was found in 83 of 98 samples analysed

Total populationDietary intakes were lower than TDIMaximum dietary intakes were lower than TDI except for TBT from the group Fish C (sardines – anchovies)

High consumersDietary intakes were lower than TDI for the mean high consumerMaximum dietary intakes were lower than TDI except for TBT for the group Fish C, for DBT for groups Fish D, ML A, CAN and for TPhT for group ML A


For a person to be at risk of exceeding the TDI as a result of seafood consumption, the person has to be a high consumer of seafood and has to consume seafood with a higher-than-average concentration of OTs

There is a need for extensive monitoring and more accurate food consumption data

CONCLUSIONS (2)


Acknowledgements

EU (OT-SAFE – QLK1-CT-2001-01437)

Postgraduate Students, Univ. Athens, GR:

Stavroula Chandrinou

Costas Pappas

IVM, Free Univ. Amsterdam, NL

Jan-Willem Wegener

Dept. of the Environment, Univ. the Aegean, GR

Athanasios Stasinakis


THANK YOU VERY MUCH!

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DIETARY EXPOSURE TO ORGANOTIN COMPOUNDS …ORGANOTIN COMPOUNDS (OTs) ΟΤsare the most used...

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