Investigations into various sample preparation techniques for the production of
shellfish laboratory reference materials for Paralytic Shellfish Poisoning toxins
Andrew Turner, Robert Hatfield, Andy Powell and Wendy Higman
Centre for Environment, Fisheries and Aquaculture Science (Cefas), Barrack Road, Weymouth, Dorset, DT4 8UB, UK.
Biological testing
Alexandrium Mass Cultures
and Oyster Feeding
•
•
•
Strains: CCMP 1598, CCMP 1846, CCMP 1719.
12 Tubular Bag, 360L reactor; 14L:10D cycle; 17º C.
Algae added gradually to shellfish held at 17º C over 5 days.
Homogeneity of treated and non-treated mussel and native oyster
LRMs as determined by RSDs (n=12, duplicate analysis).
Toxins
GTX 1,4
NEO
C 1,2
dcSTX
GTX 2,3
GTX 5
STX
Total
Mussels Wet
frozen
5%
5%
2%
2%
1%
4%
2%
2%
Mussels
AS
9%
8%
3%
5%
3%
5%
3%
5%
N. Oyster
Wet frozen
- - 8%
- 7%
8%
4%
5%
N. Oyster
AS
- - 5%
- 8%
6%
8%
6%
Sample preparation
Wet frozen aliquots: Mussel and native oyster materials
homogenised and aliquoted (6g) prior to testing.
Gamma irradiation: 6g aliquots (native oyster and mussel) LRMs
submitted to irradiation at 3, 6, 13, 18.1 kGy (Isotron, UK).
Artificial stabilisers (AS): Wet homogenate (native oyster and
mussels) treated with Ethoxyquin, Ampicillin, Oxytetracycline and
Erythromycin (0.02% w/w).
High pressure processing (HPP): Homogenate, split into 6 subsamples, each divided further into triplicates. Each set of triplicate
samples subjected to HPP under variable conditions (AFBI,
Northern Ireland).
Microbial testing of mussel and native oyster LRMs 1 week and 6
months after sample treatment (incubation between 1 and 7 days
at 25oC, units CFU/g; LOD = 5 CFU/g).
Initial testing during week 1
Incubation
3 kGy
6 kGy
12 kGy
18 kGy
Control
Mussels
1 day 7 days
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
20 >30000
Native oysters
1 day
7 days
<LOD
>3000
<LOD
<LOD
<LOD
>30
<LOD
480
195 >300000
Re-test after 6
months
Native
Mussels
oysters
3 days
3 days
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
>700 >300000
HPP
Gamma
LRMs tested using a total viable count (TVC) method. Duplicate
100μl aliquots of serial log10 dilutions of LRM sample spread
onto the surface of plate count agar (Oxoid CM0325) plates
and incubated at 25±1°C for 1 day and 7 days prior to visual
examination. Testing was repeated on LRMs stored for 6 months.
PSP toxin concentra�ons and PSP toxicity normalised to
control
Experiments have been conducted at Cefas to contaminate a
variety of shellfish species through feeding experiments with toxic
Alexandrium in order to produce a range of suitable reference
materials for the validation and implementation of quantitative
analytical PSP methods. Post-production work concentrated on
determining the homogeneity and stability of oyster and mussel
materials, initially focussing on the potential use as laboratory
reference materials for application to the AOAC 2005.06 HPLC-FLD
method. A number of different techniques were investigated to
potentially prolong the lifetime of the materials, including the use
of different storage conditions, artificial matrix stabilisers and the
use of gamma irradiation.
HPLC-FLD chromatograms of Pacific oyster LRMs
following peroxide oxidation of C18 extracts of
travel control and HPP treated samples
HPLC-FLD chromatograms of native oyster samples
following peroxide oxidation of C18 extracts of a)
control oyster b) 18 kGy irradiated oyster
1.40
PSP toxin concentrations and total STX eq. measured in native
oyster samples after gamma irradiation at 4 dose levels (± 1 sd).
1.20
1.00
C1,2
0.80
1.40
GTX2,3
0.60
STX
0.40
Total STX
eq.
0.20
0.00
Control
3 kGy
6 kGy
12 kGy
Effects of HPP processing conditions on PSP toxin concentrations
1.20
Toxin concentra�ons (normalised against control)
Introduction
18 kGy
1.00
GTX 1,4
0.80
NEO
C 1,2
0.60
GTX 2,3
STX
Total
0.40
0.20
0.00
200MPa, 5°C
400MPa, 5°C
600MPa, 5°C
200MPa, 35°C
400MPa, 35°C
600MPa, 35°C
Controls
HPP Processing Condi�ons
HPP processing conditions.
Set temperature
5°C
5°C
5°C
35°C
35°C
35°C
Sample analysis
PSP toxin testing
•
•
•
12 randomly selected aliquots analysed in duplicate for
homogeneity testing
LRMs stored at -80oC, -20oC and +4oC for 9 time points
over a 12-month period in order to assess the stability of
the materials. Classical stability assessment performed to
determine potential issues during the assessment period
LRMs extracted and analysed using AOAC 2005.06 method.
PSP toxin concentrations quantified in terms of µg saxitoxin
dihydrochloride equivalents per 100g flesh (µg STX eq./100g).
Stability of PSP toxins in mussels over 12 months
following a variety of sample processing techniques
1.40
Stability of PSP toxins in native oysters over 12 months
following a variety of sample processing techniques
STX
1.20
GTX23
2.00
1.60
1.00
1.40
0.80
0.60
1.00
-20°C
0.40
+4°C
0.20
AS +4°C
0.00
AS -20°C
0
100
200
300
1.80
400
NEO
1.60
1.40
Gamma 3kGy
Gamma 6kGy
Gamma 12 kGy
1.20
Gamma 18kGy
1.00
•
1.20
-80°C reference
•
•
0.80
0.60
0.40
•
0.20
0.00
0
50
100
150
200
250
300
350
400
C1,2
1.60
1.40
•
•
1.20
•
1.00
0.80
0.80
Conclusions
•
1.80
Response relave to -80C reference
Set Time
5 min
5 min
5 min
5 min
5 min
5 min
Response rela�ve to -80C reference
Set Pressure
200 MPa
400 MPa
600 MPa
200 MPa
400 MPa
600 MPa
Gamma, AS and Frozen
Sample
HPP1
HPP2
HPP3
HPP4
HPP5
HPP6
Gamma irradiation at 4 dose levels effectively eliminates
biological activity
No effects on PSP toxin concentrations following gamma
irradiation, HPP or addition of artificial stabilisers
Homogenous LRMs created following the above treatment
Most of the toxins stable following the majority of treatment
protocols
Some evidence for instability of some toxins in some LRMs,
seemingly improved in irradiated samples.
Stability assessment incorporates relatively high levels of
method repeatability
Further work continuing to assess short and long term stability
following reverse isochronous design
Additional work ongoing investigating effects of freeze drying
on oyster LRMs
0.60
0.60
0.40
0.40
0.20
0.20
0.00
0.00
0
100
200
Time (days)
300
400
0
50
100
150
200
Time (days)
250
300
350
400
Acknowledgements
Cefas Seedcorn project funding, InterAct partnership funded by the Department
of Business Innovation and Skills. Martin Dawson, Isotron, UK, Mark Linton and
Margaret Patterson, AFBI, Northern Ireland. Lewis Coates for technical help.
Contact: [email protected]