dietary exposure to organotin compounds …organotin compounds (ots) ΟΤsare the most used...
TRANSCRIPT
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
AOAC Europe Workshop 4
OTs
Bioaccumulation
up to××××105
AOAC Europe Workshop 5
Toxic effects of OTs
IMPOSEX
Inhibitors of the female hormones synthesis
Shell thickening
Effects on the Ca2+ metabolism
AOAC Europe Workshop 6
1. Seafood
2. Products of daily use:
• materials in contact with food
• fabrics• plastics (water pipes)
Human exposure to OTs:
AOAC Europe Workshop 7
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?
?
AOAC Europe Workshop 8
•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
AOAC Europe Workshop 9
ΟΤ-SAFE CONSORTIUM
AOAC Europe Workshop 10
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
AOAC Europe Workshop 11
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
AOAC Europe Workshop 12
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
AOAC Europe Workshop 13
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)
AOAC Europe Workshop 14
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
168937366.1Fish (Quality
B), fresh
159541366.0Fish (Quality
A), fresh
Rate of consumers
(%)
ConsumersConsumption
(g/person/day)
Mean high (95%)
Total Population
Consumption
(g/person/day)
Mean high (95%)
Food name
AOAC Europe Workshop 15
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
AOAC Europe Workshop 16
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)
AOAC Europe Workshop 17
• 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)
AOAC Europe Workshop 18
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
AOAC Europe Workshop 19
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%
AOAC Europe Workshop 20
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
AOAC Europe Workshop 21
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
AOAC Europe Workshop 22
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)
Geo. meanMedianMeanRange
AOAC Europe Workshop 23
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)
Geo. meanMedianMeanRange
AOAC Europe Workshop 24
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
AOAC Europe Workshop 25
RISK ASSESSMENT
1st Scenario:
Mean Exposure of Total Population:
High Exposure of Total Population:
)]70([
)]/(_[)]/(_[][
kgbw
daygnConsumptioMeanggOTsGeoMeanDI
×=
µ
)]70([
)]/%(95__[)]/(_[][
kgbw
daygnConsumptioHighggOTsHighDI
×=
µ
AOAC Europe Workshop 26
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
AOAC Europe Workshop 27
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
AOAC Europe Workshop 28
DAILY DIETARY INTAKES
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
Daily intake/bw(µg/day/kg bw)
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
AOAC Europe Workshop 29
DAILY DIETARY INTAKES
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
Daily intake/bw(µg/day/kg bw)
Mean High (95%)
Concentration (ng/g)
Mean High
OTsConsumption (g/person/day)Mean High(95%)
Food name
199% of TDI
5% of the total
population:
600.000 people
AOAC Europe Workshop 30
DAILY DIETARY INTAKES
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
Daily intake/bw(µg/day/kg bw)
Mean High (95%)
Concentration(ng/g)
Mean High
OTsConsumption (g/person/day)Mean High(95%)
Food name
267% of TDI !
5% of the 41% of
The total population:
246.000 people
AOAC Europe Workshop 31
DAILY DIETARY INTAKES
High consumers (14%)
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
Daily intake/bw(µg/day/kg bw)
Mean High (95%)
Concentration(ng/g)
Mean High
OTsConsumption (g/person/day)Mean High(95%)
Food name
189% of TDI
5% of the 14% of
The total population:
84.000 people
AOAC Europe Workshop 32
DAILY DIETARY INTAKES
High consumers (13%)
0.007040.84ΣBTs3412CAN
0.007040.84ΣΟΤ3412CAN
-< LOD< LODΤPhT3412CAN
0.00920.000718.874.23MBT3412CAN
0.54250.00631116.9936.61DBT3412CAN
-< LOD< LODTBT3412CAN
Daily intake/bw(µg/day/kg bw)
Mean High (95%)
Concentration(ng/g)
Mean High
OTsConsumption (g/person/day)Mean High(95%)
Food name
217% of TDI
5% of the 13% of
The total population:
78.000 people
AOAC Europe Workshop 33
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
AOAC Europe Workshop 34
(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
AOAC Europe Workshop 35
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
AOAC Europe Workshop 36
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)
AOAC Europe Workshop 37
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
AOAC Europe Workshop 38
THANK YOU VERY MUCH!
?