level delivery date - baselineeurope.eu · edilus) are compared. the levels of toxins are compared...

BASELINE - www.baselineeurope.eu

Project funded under the Food, Agriculture and Fisheries, and Biotechnology theme (KBBE)

Collaborative project Large-scale integrating project

Project acronym BASELINE

Project title Selection and improving of fit-for-purpose sampling procedures for specific foods and risks

Grant Agreement number 222738

Date of latest version of Annex I 05/07/2010

Del No Deliverable name WP no Lead

participant Nature Dissemination

level Due delivery date

D 1.4 Assessment of an alternative method for surveillance of toxins in sea water

1 NVI R PU 42

Delivery Date: 19/04/2013 Project co-ordinator: Prof. Gerardo Manfreda

Alma Mater Studiorum – Università di Bologna

Tel: +39 051 209785 E-mail: [email protected] Project website address www.baselineeurope.eu

2



TABLE OF CONTENTS INTRODUCTION ........................................................................................................ 3

Objective of the deliverable ...................... ...................................................................... 3

Sub-task 1.4.1 New sampling method for surveillance of toxins in shellfish ...... 4

Objectives: ....................................... ................................................................................ 4

Deliverables: ..................................... ............................................................................... 4

Materials and methods ............................. ....................................................................... 4 Study areas: ..................................................................................................................................... 4

Sampling scheme: .................................. ......................................................................... 6 Passive sampling disks ................................................................................................................... 6 Extraction of toxins from disks ......................................................................................................... 7 Preparation of shellfish homogenates ............................................................................................. 7 LC-MS/MS analysis ......................................................................................................................... 8

Results ........................................... ................................................................................... 8 Algal counting: ............................................................................................................................... 14

Conclusion .............................................................................................................. 16

References .............................................................................................................. 17

3



INTRODUCTION

Objective of the deliverable Phycotoxins are periodically found in shellfish due to accumulation from toxin producing algae

(Toyofuku, 2006). “Blooming” periods of such algae appear to become more frequent and in an

increasing number of areas along the European coasts Analysis of biotoxins in the shellfish flesh is

required to determine the safety of the product for consumption. However, analysis of shellfish is

time consuming, technically demanding and expensive, so that it is not ideal as a tool for monitoring

the progress of toxigenic blooms. Phytoplankton monitoring has the ability to provide early warning

of toxin problems, but is subject to some serious limitations; it is relatively labour intensive, requires

specialised laboratory and expertise, provides only a snapshot of the algal population at the time of

sampling, and is limited to toxins which have definitively been linked to particular toxins.

Nevertheless, the combination of phytoplankton monitoring and shellfish analysis has historically

provided a reasonable degree of protection to shellfish consumers in Norway and elsewhere

(Hallegraeff, 1993; van Egmond et al., 1993; Batoreu et al., 2005)

Alternatives have been sought to improve marin biotoxin monitoring. Of these, passive sampling

methods have shown much promise as tools for measuring aqueous concentrations of a wide range

of priority pollutants including algal toxins. Some of the general features of different passive

sampling devices have previously been reviewed (Stuer-Lauridsen, 2005; Vrana et al., 2005). In

comparison to traditional water sampling, passive samplers offer the ability to integratively sample a

range of environmental contaminants over an exposure period, to mimic biological uptake while

potentially avoiding heterogeneity and clean-up problems implicit with biological matrices (Verhaar,

H. J. M. et al. 1995; Kot-Wasik et al., 2007). Recently, (MacKenzie et al., 2004) introduced the idea of

monitoring algal toxins by passively adsorbing them directly from seawater using solid-phase

adsorbents.

Chemical analysis of the algal toxins from the passive sampling devices is less time-consuming than

for shellfish. Sample preparation is rapid and simple, and few interfering components are present in

the sample extracts. The objectives of this study are to assess this technique which may reduce

monitoring costs and provide improved spatial and temporal sampling opportunities. In addition, this

approach could substantially reduce the need for a large proportion of routine shellfish monitoring

tests by reducing the number of expensive chemical analysis or reducing the number of mouse bio

assays by ethical reasons.

4



Sub-task 1.4.1 New sampling method for surveillance of toxins in shellfish Assessment of an alternative method for surveillance of toxins in sea water.

Objectives: A study was planned in August-October to study spatial and temporal distribution of phycotoxins in

blue mussels within a shellfish farm in Norway. The aim was to provide data for validation of spatial

and temporal variation in toxin distribution, algae concentration and mussel biomass, and to develop

sampling plans to query characteristics of a lot of blue mussels. The sampling plans were designed to

ensure defensible, statistically valid decision making regarding the acceptance or rejection of a lot.

Additionally, data from time integrated sampling of disks was compared with toxin profiles in blue

mussels to evaluate the use of passive samplers for predicting dangerous levels of toxins in mussels.

Moreover, the passive samplers were used for analyzes in 3 other shellfish farms of surrounding

waters aiming to get early warning in case of algal blooms. These are important aspects within

development of improved sampling strategies in BASELINE.

Deliverables: Data from spatial and time integrated sampling of disks with subsequent extraction and LC/MS

analysis for OA/DTXs are presented, although data are also available for AZAs, PTXs, YTXs, spirolides

and pinnatoxins . Profiles of accumulated toxins in the disks and toxin profiles in blue mussels (M.

edilus) are compared. The levels of toxins are compared with results from the Norwegian Food Safety

Authorities who monitor blue mussels every second week and algae every week in the same area.

Materials and methods Study areas:

Norgeskjell was first established in 1983 in Aafjord (fig 1), producing fresh, blue mussels. Today’s

production is up to 1000 tons fresh blue mussels yearly, and their market is both domestic and

abroad, mainly Europe. They grow mussels along coastlines in ropes suspended in water (fig 2). The

company is situated in Aafjord near Askerholmen, where the Norwegian food safety Authorities

monitor blue mussels every second week and algae every week.

The mussel farming company Snadder og Snaskum AS started production in 1980 and has long

experience in the industry, and a rich tradition in promoting fresh cultured mussels in Norway. They

harvest rope-grown mussels mainly in Aafjord and Verrasundet fjords.

These two companies represent half of the Norwegian blue mussel production.

5



Figure 1 Localization of the shellfish farms

Figure 2 Rope-grown mussels in Aafjord

6



Sampling scheme: At the production site A (fig 1), passive sampling discs were attached to a fixed point at 1 and 6m

depths and at different positions along the lines (fig 3), and immersed in the sea for one week. The

disks were then sampled, sealed in air-tight plastic bags, and shipped to the laboratory for analysis.

Simultaneously, mussels (2 kg) were harvested at the same sites.

In addition, discs and shellfish were sampled along the fjord in production sites B, C and D (fig 1) to

investigate the possible development and the distribution of an algal bloom over time to investigate

the potential of the discs as early warning tools for detection of toxic algal blooms.

Figure 3 A schematic drawing of the production site in Aafjord. The sampling sites for discs and blue mussels are numbered A1-1 to A1-9 and A6-1 to A6-9 at 1 and 6m depth respectively.

Passive sampling disks

Passive samplers were constructed from 100-mm nylon mesh (Sefar AG, Heiden, Switzerland) folded

in half, a 75 mm diameter plastic embroidery frame (Permin,Copenhagen, Denmark) and HP-20 resin

(DIAION HP-20, Mitsubishi Chemical Corporation, Tokyo, Japan). The resin (3.0 g) was placed

between the two layers of nylon mash, and clamped tightly in the embroidery frame so as to form a

thin layer of resin between the layers of mesh. A No. 2 fishing swivel (Mustad, Gjøvik, Norway) was

attached to the outer ring of the embroidery frame to provide a point of attachment during

deployment (Fig. 4). The resin was activated by soaking the packed disk in methanol for 15 min and

washing in deionized water, as described in the resin-manufacturer’s instructions. The activated

passive sampling disks were placed in an air-tight plastic bags and stored cold (but not below 0 C)

prior to and after deployment in the sea.

7



Figure 4 Fully assembled passive sampling disk (E), and its component parts: (A) 100 mm nylon mesh; (B) HP-20 resin; (C) inner and (D) oute r rings of a 75 mm diameter embroidery ring with (F) a No. 2 fishing swivel attached

Extraction of toxins from disks

The embroidery ring was opened, and the used resin was quantitatively transferred to a 25 mL Varian

Bond-elute reservoir fitted with a 20 mm nylon frit (Varian, Palo Alto, CA) and washed free of salts

with 30–50 mL deionized water. Excess water was drawn from the column by application of a

vacuum. MeOH was added to the column and the resin was stirred gently then left to stand for 15

min. The column was then eluted slowly (0.5–1 drop/s) and when finished, the process was repeated

with another 10 mL MeOH. Finally, an additional 3 mL MeOH was pushed through to flush the

column, and the combined eluate evaporated to dryness in vacuo. The residue was dissolved in 1.0

mL 80% MeOH, centrifuged, and the supernatant analyzed by LC-MS/MS analysis.

Preparation of shellfish homogenates

Mussels (1 kg) were homogenized in an Ultra turrax blender . A fraction of the homogenate (1.00 ±

0.05g) was transferred to a 15 mL falcon tube and Whirleymixed after adding 9 mL of MeOH until

homogeneity. The tube was then centrifuged for 10 min at 3000g in a Beckman centrifuge. The

supernatant was transferred to another falcon tube and the volume was adjusted to 10 mL with

MeOH. 200 µL of the filtered (spinnex filter –Galaxy minister, VWR) supernatant was added 50 µL of

water and transferred to HPLC vials for LC-MSMS analysis.

8



LC-MS/MS analysis

Liquid chromatography was performed on a Symmetry C18 column (3µm, 50 × 2.1 mm) (Waters,

Milford, MA, USA), using a Waters 2670 HPLC module. Separation was achieved using a linear

gradient starting with MeCN–water (35:65, both containing 2 mM ammonium formate and 0.01%

formic acid) rising to 100% MeCN over 10 min. Isocratic elution with 100% acetonitrile was

maintained for 5 min before the eluent was switched back to 35% acetonitrile, at 0.3 mL/min. The

HPLC system was coupled to a Quattro Ultima Pt triple quadrupole mass spectrometer operating

with an electrospray ionization (ESI) interface (Waters Micromass, Manchester, UK). Typical ESI

parameters were a spray voltage of 3.5 kV, desolvation temperature at 250 °C, source temperature

at 100 °C and cone gas and desolvation gas at 40 and 600 L/h of N2, respectively. The mass

spectrometer was operated in MS/MS mode with argon as collision cell gas at 1 × 10-3 Torr.

Ionization and MS/MS collision energy settings (typically 35–45 eV) were optimized while

continuously infusing (syringe pump) 20 ng/mL of the toxins, at a flow rate of 3 µL/min.

Quantification of the analytes were performed with multiple reaction monitoring (MRM) in either

positive ionization mode; AZA-1 842.5>672.5, AZA-2 856.5>672.5, AZA-3 828.5>658.5, PTX-2

876.5>823.5, PTX-12 874.5>821.5 or negative ionization mode; OA 803.5>255.1, DTX-1 817.5>255.1,

DTX-2 803.5>255.1. All toxins were quantified using external calibration curves of standard

specimens dissolved in 80% MeOH.

Data were analyzed using the JMP® Software for Statistical Visualization (SAS Institute Inc). Log

transformations of data were employed where necessary, in order to allow the use of parametric

statistical methods. Parametric tests by Bartlett’s test for homogeneity of variance were used. The

probability of significance was set at α = 0.05. A Student’s T comparison test was used, to compare

both depths.

Results Sampling were done by employees at Norgeskjell. The study started august 7

th, and lasted until

October 26th

. Sampling dates are presented in tables 1- 12. Concentrations of the algal toxins

analyzed were generally low, below quantification levels or not detected throughout the period.

Individual mussels from each position were stored at -20°C and planned to be analyzed in order to

determine the variation of toxin levels at each depth within the shellfish farm. Due to the very low

levels both in the shellfish and passive samplers, it was decided not to go any further with this

approach.

Upon digestion of Dinophysis spp. Blue mussels metabolize variable amounts of OA/DTXs into esters.

Basic hydrolysis is therefore routinely performed to determine total amounts of OA/DTXs regarding

the acceptance or rejection of a lot before commercial trading. We performed basic hydrolysis on a

few samples to determine the levels of esterified OA/DTXs, but found no esterified forms of these

toxins. The hydrolysis step was therefore omitted for the rest of the samples.

The concentrations of OA in passive samplers and blue mussels are presented in tables 1, 2, 3 and 4:

9



Table 1 Concentrations of Ocadaic acid in passive s amplers from Aafjord shellfish farm at different positions illustrated in fig. 3. ND; not detected. Values designed 0,00 are below the quantification limit. Discs OA

Position Conc. (ng/ml)

Date

07.08.2012 14.08.2012 21.08.2012 04.09.2012 11.09.2012 18.09.2012 25.09.2012 18.10.2012 26.10.2012

A1-1 0,57 0,28 0,51 0,42 0,80 0,76 ND ND ND

A2-1 0,56 0,36 0,44 0,28 0,89 ND 0,67 ND ND

A3-1 0,94 0,36 1,91 0,32 ND 0,70 0,36 4,04 ND

A4-1 1,27 0,65 1,15 0,54 ND 0,35 0,90 ND 2,71

A5-1 1,49 0,24 2,01 0,00 0,23 0,62 0,35 ND 0,37

A6-1 0,58 0,10 1,19 0,65 1,05 0,44 0,54 0,53 0,21

A7-1 1,13 0,34 1,79 0,29 1,06 1,11 0,44 0,28 0,26

A8-1 1,52 0,46 1,77 0,48 0,31 0,32 0,84 0,48 0,32

A9-1 1,02 0,63 2,08 0,70 0,10 0,81 ND ND ND

B-1 1,99 0,12 ND 0,13 0,70 0,80 0,24 ND 0,36

C-1 0,56 0,23 ND 0,13 ND 0,16 0,70 0,36 0,36

D-1 0,65 0,57 ND 0,22 0,71 0,28 1,32 ND 3,15

A1-6 1,30 0,15 0,16 0,84 ND ND 0,74 ND ND

A2-6 0,50 0,08 0,02 0,17 0,32 ND 0,70 0,65 3,48

A3-6 0,74 ND 2,09 0,28 0,03 0,15 0,13 0,55 0,27

A4-6 0,18 0,36 1,31 0,19 0,51 0,33 0,16 0,26 3,34

A5-6 0,66 1,02 2,04 0,25 0,02 0,32 0,13 0,69 0,10

A6-6 0,80 0,25 2,19 0,31 0,30 0,15 0,19 0,68 4,19

A7-6 0,94 0,60 1,36 0,25 0,19 0,44 0,15 0,22 0,27

A8-6 0,93 0,58 1,40 1,26 0,00 0,11 0,22 0,18 ND

A9-6 0,48 0,44 1,26 0,37 1,17 0,29 0,19 0,51 0,36

B-6 ND 1,05 ND 0,07 0,90 0,62 0,12 0,59 0,40

C-6 ND 0,20 ND 0,18 0,45 1,22 0,46 7,75 ND

D-6 ND 0,20 ND 0,00 0,02 0,25 ND 0,54 0,11 Table 2 Mean concentrations (ng/mL) of OA in passiv e samplers during the study period at 1 and 6m. Discs OA

Mean 07.08.2012 14.08.2012 21.08.2012 04.09.2012 11.09.2012 18.09.2012 25.09.2012 18.10.2012 26.10.2012

1m 1,01 0,38 1,43 0,41 0,63 0,64 0,58 1,33 0,77

6m 0,73 0,43 1,31 0,43 0,32 0,25 0,29 0,47 1,72

10



Table 3 Concentrations of Ocadaic acid in blue muss els from Aafjord shellfish farm at different positions illustrated in fig. 3. ND; not detected. Values designed 0,00 are below the quantification limit. Blue mussels

OA

Position Conc. (ug/kg )

Date

07.08.2012 14.08.2012 21.08.2012 04.09.2012 11.09.2012 18.09.2012 25.09.2012 18.10.2012 26.10.2012

A1-1 0,00 ND 0,00 0,00 0,00 0,00 ND 0,00 0,41

A2-1 0,00 ND 0,40 0,00 0,00 0,00 ND 0,00 0,59

A3-1 0,00 ND 0,00 0,00 0,00 0,00 ND 0,00 0,00

A4-1 0,00 ND 0,00 0,00 0,00 0,80 ND 0,00 0,00

A5-1 0,00 ND 0,00 0,00 0,00 0,00 ND 0,00 0,00

A6-1 0,00 ND 0,00 0,00 0,00 0,00 ND 0,00 0,44

A7-1 0,00 ND 0,00 0,00 0,00 0,00 ND 0,00 0,11

A8-1 0,00 ND 0,00 0,00 0,00 0,00 ND 0,00 0,00

A9-1 0,00 ND 0,00 0,00 0,00 0,00 ND 0,00 0,00

A1-6 0,41 ND 0,00 0,00 0,00 0,00 ND 0,00 0,00

A2-6 0,29 ND 0,00 0,00 0,00 0,00 ND 0,00 0,00

A3-6 0,00 ND 0,00 0,00 0,00 0,00 ND 0,00 0,00

A4-6 0,00 ND 0,00 0,00 1,50 0,00 ND 0,00 0,74

A5-6 0,00 ND 0,68 0,00 0,00 0,00 ND 0,00 0,00

A6-6 0,00 ND 0,00 0,00 0,89 0,00 ND 2,89 0,00

A7-6 0,00 ND 0,00 0,00 0,00 0,00 ND 0,00 0,00

A8-6 0,00 ND 0,00 0,64 0,00 0,00 ND 0,00 0,00

A9-6 0,00 ND 0,00 0,00 0,00 0,40 ND 0,00 0,00

B 0,59 ND 0,00 0,00 0,00 0,00 ND 0,00 0,00

C 0,00 ND 0,00 1,14 0,00 0,00 ND 0,00 0,00

D 0,00 ND 1,59 0,00 0,00 0,00 ND 0,00 0,00 Table 4 Mean concentrations (µg/mL) of OA in blue m ussels during the study period at 1 and 6m. Blue mussels OA

Mean 07.08.2012 14.08.2012 21.08.2012 04.09.2012 11.09.2012 18.09.2012 25.09.2012 18.10.2012 26.10.2012

1m 0,00 ND 0,04 0,00 0,00 0,09 ND 0,00 0,17

6m 0,08 ND 0,08 0,07 0,27 0,04 ND 0,32 0,08

The levels of DTX1 in passive samplers and blue mussels are presented in tables 5, 6, 7 and 8:

11



Table 5 Concentrations of DTX1 in passive samplers from Aafjord shellfish farm at different positions illustrated in fig. 3. ND; not detected. Values designed 0,00 are below the quantification limit. Discs DTX-1


Date

07.08.2012 14.08.2012 21.08.2012 04.09.2012 11.09.2012 18.09.2012 25.09.2012 18.10.2012 26/29.10.2012

A1-1 0,0000 0,0909 0,18391304 0,1230 0,0891 0,0000 0,0891 0,0000 0,0000

A2-1 0,1065 0,0326 0,1578 0,0348 1,2461 0,0000 0,0000 0,0000 0,0000

A3-1 0,1543 0,1443 2,0487 0,0865 0,0000 0,0000 0,0000 2,3578 0,0000

A4-1 0,0778 0,0000 1,3939 0,0787 0,0000 0,0600 0,1248 0,0000 1,8217

A5-1 0,0000 0,1309 2,5304 0,0243 0,0348 0,0509 0,0348 0,0000 0,0635

A6-1 0,0000 0,0461 1,8265 0,0122 0,0383 0,0509 0,0000 0,1191 0,0474

A7-1 0,0000 0,0000 1,9865 0,0478 0,0961 0,0000 0,0717 0,0630 0,1578

A8-1 0,2052 0,0000 1,7996 0,0557 0,0000 0,0000 0,0617 0,1213 0,0296

A9-1 0,0509 0,1030 1,9183 0,0604 0,0465 0,0000 0,0000 0,0000 0,0000

B-1 0,1300 0,0000 0,0000 0,0387 0,2035 0,0770 0,0178 0,0000 0,2122

C-1 0,0913 0,0943 0,0000 0,0000 0,0000 0,0635 0,1670 0,0935 0,0800

D-1 0,0935 0,0000 0,0000 0,0109 0,1204 0,0261 0,0000 0,0000 1,5413

A1-6 0,1078 0,0000 0,0909 0,1004 0,0000 0,0000 0,0000 0,0000 0,0000

A2-6 0,1152 0,0000 0,1552 0,0174 0,0704 0,0000 0,0500 0,2865 2,5130

A3-6 0,1291 0,0787 1,8883 0,0648 0,0191 0,0909 0,0400 0,1543 0,0261

A4-6 0,0274 0,0787 1,0165 0,0000 0,0535 0,1261 0,0596 0,1096 1,9117

A5-6 0,0965 0,0000 1,6217 0,0683 0,0143 0,0196 0,0200 0,1048 0,0313

A6-6 0,2865 0,0000 1,9361 0,0000 0,0448 0,1196 0,0861 0,0830 2,9335

A7-6 0,0000 0,0000 1,2004 0,2504 0,0278 0,0617 0,0617 0,0809 0,0000

A8-6 0,0000 0,0713 1,1783 1,2696 0,0196 0,0000 0,0383 0,1183 0,0000

A9-6 0,2474 0,0539 1,1826 0,0322 1,2843 0,1074 0,1191 0,0896 0,0517

B-6 0,0000 0,0235 0,0000 0,0352 0,0000 0,1270 0,0000 0,1952 0,1030

C-6 0,0000 0,0552 0,0000 0,0217 0,0852 0,2935 0,0943 4,8691 0,0000

D-6 0,0000 0,0617 0,0000 0,0000 0,0000 0,0374 0,0000 0,2448 0,1143 Table 6 Mean concentrations (ng/mL) of DTX1 in pass ive samplers during the study period at 1 and 6m. Discs DTX-1

Mean 07.08.2012 14.08.2012 21.08.2012 04.09.2012 11.09.2012 18.09.2012 25.09.2012 18.10.2012 26.10.2012

1m 0,07 0,06 1,54 0,06 0,17 0,02 0,04 0,30 0,24

6m 0,11 0,03 1,14 0,20 0,17 0,06 0,05 0,11 0,83

12



Table 7 Concentrations of DTX1 in blue mussels from Aafjord shellfish farm at different positions illustrated in fig. 3. ND; not detected. Values designed 0,00 are below the quantification limit. SHELLFISH

DTX-1

Position Conc. (ug/kg SM)

Date

07.08.2012 14.08.2012 21.08.2012 04.09.2012 11.09.2012 18.09.2012 25.09.2012 18.10.2012 26/29.10.2012

A1-1 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00

A2-1 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00

A3-1 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 1,51

A4-1 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,71

A5-1 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00

A6-1 0,00 0,00 0,00 0,00 0,00 0,00 0,00 1,43 0,00

A7-1 3,35 0,00 0,00 0,00 0,00 0,00 0,00 1,66 2,18

A8-1 0,00 0,00 0,00 0,00 2,33 0,00 0,00 0,00 0,00

A9-1 2,70 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00

A1-6 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00

A2-6 2,69 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00

A3-6 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,75

A4-6 1,94 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00

A5-6 3,43 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00

A6-6 0,75 0,00 0,00 0,00 1,18 0,00 0,00 0,00 0,00

A7-6 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00

A8-6 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00

A9-6 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00

B 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00

C 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00

D 2,66 0,00 0,00 0,00 2,49 0,00 0,00 0,00 0,00 Table 8 Mean concentrations (µg/mL) of DTX1 in blue mussels during the study period at 1 and 6m. Blue mussels DTX-1

Mean 07.08.2012 14.08.2012 21.08.2012 04.09.2012 11.09.2012 18.09.2012 25.09.2012 18.10.2012 26.10.2012

1m 0,67 0,00 0,00 0,00 0,26 0,00 0,00 0,34 0,49

6m 0,98 0,00 0,00 0,00 0,13 0,00 0,00 0,00 0,08

The levels of DTX2 in passive samplers and blue mussels are presented in tables 9, 10, 11 and 12:

13



Table 9 Concentrations of DTX2 in passive samplers from Aafjord shellfish farm at different positions illustrated in fig. 3. ND; not detected. Values designed 0,00 are below the quantification limit. Discs DTX-2


Date

07.08.2012 14.08.2012 21.08.2012 04.09.2012 11.09.2012 18.09.2012 25/27.09.2012 18.10.2012 26.10.2012

A1-1 0,0000 0,0165 0,01434783 0,0000 0,1035 0,0000 0,0000 0,0000 0,0000

A2-1 0,0283 0,0000 0,0000 0,0078 0,0830 0,0000 0,0000 0,0000 0,0000

A3-1 0,0000 0,0000 0,4678 0,0048 0,0000 0,0000 0,0178 0,0000 0,0000

A4-1 0,0000 0,0191 0,4843 0,0000 0,0000 0,0000 0,0000 0,0000 0,3674

A5-1 0,2043 0,0465 0,6565 0,0165 0,0052 0,0000 0,0343 0,0000 0,0387

A6-1 0,0487 0,0000 0,4739 0,0000 0,0000 0,1017 0,0000 0,0322 0,0217

A7-1 0,0000 0,0000 0,2152 0,0187 0,0000 0,0000 0,0270 0,0239 0,0091

A8-1 0,0000 0,0000 0,3752 0,0000 0,0330 0,0200 0,0000 0,0130 0,0187

A9-1 0,0000 0,0000 0,4317 0,0852 0,0000 0,0000 0,0000 0,0000 0,0000

B-1 0,3017 0,0483 0,0000 0,0009 0,0000 0,1965 0,0000 0,0000 0,0000

C-1 0,0352 0,0000 0,0000 0,0000 0,0000 0,0000 0,0000 0,0304 0,0000

D-1 0,0000 0,0000 0,0000 0,0000 0,0000 0,0000 0,0000 0,0000 0,4265

A1-6 0,0000 0,0000 0,1200 0,0000 0,0000 0,0000 0,0000 0,0000 0,0000

A2-6 0,0600 0,0600 0,0217 0,0000 0,0000 0,0000 0,0000 0,1078 0,4596

A3-6 0,0000 0,0000 0,5313 0,0700 0,0000 0,0000 0,0091 0,0387 0,0248

A4-6 0,0000 0,0000 0,3122 0,0000 0,0000 0,0874 0,0000 0,0000 0,4043

A5-6 0,0000 0,0000 0,5761 0,0157 0,0039 0,0000 0,0248 0,1378 0,0000

A6-6 0,0000 0,0000 0,4948 0,0091 0,0074 0,1065 0,0617 0,1452 0,5539

A7-6 0,0304 0,0000 0,2752 0,2391 0,0022 0,0000 0,0139 0,0126 0,0048

A8-6 0,0422 0,0304 0,2987 0,5474 0,0087 0,0000 0,0126 0,0200 0,0000

A9-6 0,0483 0,0422 0,2622 0,0087 0,2548 0,1965 0,0000 0,0000 0,0674

B-6 0,0000 0,0000 0,0000 0,0000 0,2013 0,0000 0,0000 0,0270 0,0000

C-6 0,0000 0,0000 0,0000 0,0017 0,0939 0,0000 0,0174 1,0504 0,0000

D-6 0,0000 0,0274 0,0000 0,0000 0,0152 0,0000 0,0000 0,0122 0,0000 Table 10 Mean concentrations (ng/mL) of DTX2 in pas sive samplers during the study period at 1 and 6m. Discs DTX-2

Mean 07.08.2012 14.08.2012 21.08.2012 04.09.2012 11.09.2012 18.09.2012 25.09.2012 18.10.2012 26.10.2012

1m 0,03 0,01 0,35 0,01 0,02 0,01 0,01 0,01 0,05

6m 0,02 0,01 0,32 0,10 0,03 0,04 0,01 0,05 0,17

14



Table 11 Concentrations of DTX2 in blue mussels fro m Aafjord shellfish farm at different positions illustrated in fig. 3. ND; not detected. Values designed 0,00 are below the quantification limit. Blue mussels

DTX-2

Position Conc. (ug/kg SM)

Date

07.08.2012 14.08.2012 21.08.2012 04.09.2012 11.09.2012 18.09.2012 25.09.2012 18.10.2012 26.10.2012

A1-1 0,00 1,75 0,00 0,00 0,00 0,00 0,00 0,00 0,00

A2-1 0,00 0,65 0,00 0,14 0,51 0,00 0,00 0,00 0,45

A3-1 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00

A4-1 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00

A5-1 0,00 0,00 0,00 0,15 0,00 0,00 0,00 0,00 0,00

A6-1 0,00 0,90 0,00 0,00 0,00 0,00 0,00 0,00 0,00

A7-1 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00

A8-1 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00

A9-1 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00

A1-6 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00

A2-6 0,00 0,00 0,00 0,00 0,00 0,65 0,00 0,00 0,00

A3-6 0,00 0,00 0,00 0,00 0,00 0,89 0,00 0,00 0,00

A4-6 0,00 1,38 0,00 0,00 0,00 0,00 0,00 0,00 0,00

A5-6 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00

A6-6 0,00 3,49 0,00 1,04 0,00 0,48 0,00 0,00 0,00

A7-6 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00

A8-6 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00

A9-6 0,00 1,00 0,00 0,00 0,00 0,00 0,00 0,00 0,39

B 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00 0,00

C 0,00 0,00 0,00 0,00 0,00 0,00 0,00 1,73 0,00

D 0,00 0,00 0,00 0,00 0,00 0,24 0,00 0,00 0,00 Table 12: Mean concentrations (µg/mL) of DTX2 in bl ue mussels during the study period at 1 and 6m. Blue mussels

Mean 07.08.2012 14.08.2012 21.08.2012 04.09.2012 11.09.2012 18.09.2012 25.09.2012 18.10.2012 26.10.2012

1m 0,00 0,37 0,00 0,03 0,06 0,00 0,00 0,00 0,05

6m 0,00 0,65 0,00 0,12 0,00 0,22 0,00 0,00 0,04

The highest mean level of toxins in blue mussels (DTX1) was 1 µg/kg. The maximum residual levels

for OA/DTXs are 160 µg/kg. No correlation between toxin levels in passive samplers and shellfish

were found. No significant differences were found between different depths neither in the passive

samplers or the shellfish. This is not unexpected since most of the toxin levels were below

quantification levels. In our earlier study in this project, a clear correlation between levels in discs

and blue mussels was demonstrated during algal blooms.

Algal counting:

The Norwegian Food Safety Authority (NFSA) runs a monitoring program of algae toxins in mussels

and dietetic advice to the public. The aim of the program is on weekly basis, to advice public on the

risk associated with consumption of wild mussels. The program concentrates primarily on

Alexandrium spp. and Dinophysis spp. which are the major producers of Paralytic Shellfish Poisoning

15



(PSP) and Diarrheic Shellfish Poisoning (DSP). From the 38 stations covering the Norwegian coast

from the Oslo fjord northwards to the Varrangerfjord near the Russian border, one of the stations,

Askerholmen (see table 13) is situated at Aafjord, near the shellfish farm. During the study period no

toxin producing algae were detected in the water samples. The chemical analysis report only present

concentrations above 20 µg/kg for OA/DTXs. All the samples were below this limit. This is in

agreement with our results.

Table 13 Overview from the Norwegian Food safety Au thority’s national monitoring program for shellfish 2012. Data are extracted for the stud y period and showing data for Askerholmen, nearby Aafjord shellfish farm.

Norwegian Food Safety AuthorityNational monitoring program for shellfish 2012

Watersample, Cells/L Toxins µg/kg Shellfish

location Date Week

Din. acumin

ata

Din. acut

a Din.

norvegica

Protoceratium reticula

tumAzadinium

spp.Pseudon.

spp.

Pseudon.

seriata.-gr.

Alex. spp.

Alex. tamare

nseAlex. Spp DSP YTX AZA PTX PSP ASP

Askerholmen 07.08.2012 32 nd nd nd nd nd nd nd nd nd nd <20 <20 <20 <20 <100 <500

14.08.2012 33 nd nd nd nd nd nd nd nd nd nd <20 <20 <20 <20 <100 <500








regulatory limits, algae Regulatory limits, toxinsWatersample (cells/L) DSP 160 µg / kgAlexandrium tamarense/minutum 200 PSP 400 µg STX/ kg Alexandrium spp. concideration ASP 20000 µg / kgPseudonitzschia spp. 1000 000* AZA 160 µg / kgProtoceratium reticulatum 1000 * PTX 160 µg / kgDinophysis acuta 200/100** YTX 1000 µg / kgDinophysis acuminata 1000Dinophysis norvegica 4000

* Basis for hygenic concideration**three weeks in a row

16



Conclusion It was not possible to assess the method for passive harvesting/sampling of toxins from sea water

due to the absence of toxigenic algal species during the study period. However, the same toxins were

detected in the discs and in shellfish. This study demonstrates that use of this technique could

substantially reduce the need for a large proportion of routine shellfish monitoring tests by reducing

the number of expensive chemical analysis or reducing the number of mouse bio assays by ethical

reasons. This is because within any monitoring programme the great majority of tests are negative

(as in this study) and a rapid screening method applied to a extracts from passive sampling devices

(possibly in combination with a conventional phytoplankton examination) would suffice to provide an

assurance that growing areas are uncontaminated. Moreover, this approach would lower the

monitoring costs considerable. In our study, sampling of shellfish from 6m was quite labor intensive

due to the weight of the mussel ropes. Whereas the discs easily were lifted by hand, mussels had to

be lifted by crane. Together with the sampling, the transport of shellfish to the laboratory was the

main cost in this study. In contrast, passive sampling devices can be sent by mail.

Toxins detected also included spirolides and pinnatoxins. The alga that produces pinnatoxins is still

unknown, which means that phytoplankton monitoring can only provide effective monitoring for

toxins when the identity of the toxigenic species is known, whereas this information is not necessary

when using passive sampling devices. Passive sampling is a useful tool to monitor waters if shellfish

are unavailable and for analyzes of surrounding waters to get early warning in case of algal blooms.

These are important aspects within development of improved sampling strategies in BASELINE

17



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level delivery date - baselineeurope.eu · edilus) are compared. the levels of toxins are compared...

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