The effect of size and age on depuration rates of diarrhetic

shellfish toxins (DST) in mussels (Mytilus edulis L.)

A. Duinker a,*, M. Bergslien a,1, Ø. Strand b, C.D. Olseng c,d, A. Svardal b,2

a National Institute of Nutrition and Seafood Research (NIFES), PO Box 2029 Nordnes, 5817 Bergen, Norwayb Institute of Marine Research, PO Box 1870 Nordnes, 5817 Bergen, Norway

c Norwegian Institute of Water Research (NIVA), Nordnesboder 5, 5005 Bergen, Norwayd Department of Biology, University of Bergen, Jahnebakken 5, 5020 Bergen, Norway

Received 13 March 2006; received in revised form 22 June 2006; accepted 3 October 2006

www.elsevier.com/locate/hal

Harmful Algae 6 (2007) 288–300

Abstract

Depuration or elimination of diarrhetic shellfish toxins (DST) was followed for 73 days in 1- and 2-year-old mussels. The age

groups also differed in size, providing a broad approach to studying the effect of the differences in physiology accompanying the

differences in size. Content of DST was analysed both on groups and individual mussels. Environmental variables were measured to

evaluate their effect on depuration.

We found no significant differences in elimination rate of DST between 1- and 2-year-old mussels under natural conditions. This

suggests that size and age do not affect the elimination rate of the DST. The present study is the first study on the effect of age and

size on the elimination rate of algal toxins in bivalves. The natural variations in food levels and temperature were not found to affect

the elimination rate of DST.

The digestive gland weights in the 1-year-old mussels increased four times while the DST content per individual decreased eight

times. This demonstrated that dilution of toxins due to tissue growth could have an important contribution to declines in toxin

concentrations. Changes in tissue mass are affected by environmental variables via growth or starvation, and when such changes

lead to concentration or dilution of toxins this does not reflect the accumulation or removal of toxins from the tissues. We hence

suggest that when evaluating the actual elimination capacity of the mussels, as in the present study, the total content of toxins per

individual should be used, rather than toxin concentrations.

The 1-year-old mussels had faster growth compared to the 2-year-old mussels in both total soft tissue and digestive glands. The

mechanism of DST elimination is still unknown. If this process involves metabolism of the toxins, one could expect the rates of

elimination to follow overall metabolic rates. However, the results from the present study suggest that large differences in growth

rates, which also include difference in feeding and metabolic rates, do not affect the elimination rate of DST. Our results support the

assumption that the depuration rates cannot be accelerated, even in artificial systems, as a cost-effective way to solve the problem

with toxic mussels for the industry.

# 2006 Elsevier B.V. All rights reserved.

Keywords: Diarrhetic shellfish toxins; DST; DSP; Depuration; Elimination; Environmental factors; Growth; Mussels; Mytilus edulis; Physiology

* Corresponding author. Tel.: +47 55 90 51 00; fax: +47 55 90 52 99.

E-mail address: duinker@nifes.no (A. Duinker).1 Present address: Trysil-Knuts gate 43, 4021 Stavanger, Norway.2 Present address: Section for Pharmacology, Arm. Hansens hus,

Haukelandsv. 28, 5021 Bergen, Norway.

1568-9883/$ – see front matter # 2006 Elsevier B.V. All rights reserved.

doi:10.1016/j.hal.2006.10.003

1. Introduction

Diarrhetic shellfish toxins (DST) are produced in

dinoflagellates of the genera Dinophysis and Prorocen-

trum (Yasumoto et al., 1978; Murata et al., 1982), and

A. Duinker et al. / Harmful Algae 6 (2007) 288–300 289

Table 1

Estimated age and mean shell size for the mussel groups at start of the

experiment (�S.D.)

Mussel group Estimated age (months) Shell length (cm)

1-Year-old 8 2.83 � 0.36

2-Year-old 20 4.66 � 0.44

Control 20 5.58 � 0.56

bivalves including mussels accumulate the toxins from

ingestion of the algae (Haamer et al., 1990; Sidari et al.,

1998; Morono et al., 2003). Excretion of the toxins can

be studied when the producers are no longer in the

water. Though not as well studied as for the Paralytic

Shellfish Toxins (PST) (Bricelj and Shumway, 1998),

excretion or depuration of DST has been studied for

several decades. However, the mechanism for the

depuration is not known, and there are discrepancies in

the literature with respect to the effect of various

environmental and physiological factors on the depura-

tion rates. Some studies suggest that the quantity or

quality of the food sources affect the depuration rates

(Marcaillou-Le Baut et al., 1993; Blanco et al., 1999),

while other studies in field and laboratory suggest that

neither food levels, salinity or temperature affect the

depuration rates (Svensson, 2003a; Svensson and

Forlin, 2004). A study of mussel groups with different

lipid levels also failed to affect the depuration rates and

concluded that processes insensitive to short-term

manipulations of the external environment regulate

the depuration of DST (Svensson and Forlin, 2004). The

only consistent differences in depuration rates have

been seen between different seasons (Svensson, 2003b),

with faster depuration rates in summer compared to

autumn.

In the present study, an alternative approach to

studying the effects of external factors on depuration

was chosen. Depuration of DST was followed in two

groups of mussels that differed in age and size. The size

of mussels affect their size-specific ingestion rates

(Thompson and Bayne, 1974; Navarro and Winter,

1982), routine oxygen consumption (Bayne and Newell,

1983), maintenance ration (Winter and Langton, 1976),

net growth efficiency (Navarro and Winter, 1982) and

scope for growth (Thompson and Bayne, 1974). The

two age and size groups hence provide a broad approach

to studying effects of internal physiological processes

on depuration. Content of DST was analysed both in

groups and individual mussels. The mussels were kept

in the sea. A control group of toxin-free mussels was

followed in parallel in order to monitor for additional

toxin uptake during the depuration period. Phytoplank-

ton composition and abundance, temperature and

salinity were monitored in order to also evaluate the

effects of natural variations of these environmental

variables on depuration.

2. Materials and methods

Blue mussels (Mytilus edulis) were collected on 26

February 2003 from a commercial longline mussel farm

situated outside Frønningen (Sognefjorden, Western

Norway). Based on data from a nearby farm, the

mussels were expected to retain diarrhetic shellfish

toxins (DST) from ingestion of Dinophysis spp. the

previous autumn (personal communication, P. Hov-

gaard, Sogn og Fjordane University College, Norway).

Two age classes of mussels were collected. All mussels

were collected from the same longline, but from

different ropes. Mussels from the 2001 settlement are

termed 2-year-old mussels. The collectors containing

these mussels had experienced second-set of spat from

the spawning in 2002. However, the majority of these

younger mussels had subsequently been removed from

the 2001 settlement by size-grading and resocking

during late autumn 2002, so that the majority of the

mussels from these ropes that were harvested for the

present study originated from the 2001 settlement. Size-

grading as described below further decreased the chance

of incidences of younger mussels. Mussels from the

2002 settlement were termed 1-year-old mussels. The

mussels that were collected were separated by a

declumper (mod. B1, Talleres Aguin, Spain) and

graded. The smallest and largest mussels in both age

groups were discarded in order to obtain the mid-range-

sized mussels (Table 1). The mussels were transported

in chilled condition to the laboratory facilities (Institute

of Marine Research, Bergen) within 48 h of collection

from the farm, and kept for 14 days in flow-trough tanks

supplied by seawater from 90 m depth (salinity 34.5%and temperature 9 � 0.5 8C). Mussels representing the

control group were collected from a longline farm in

Hardangerfjorden. The mussel stock had been approved

for commercial harvest and was regarded as toxin-free.

These mussels arrived at the laboratory on 7 March.

On 14 March, the mussels were placed into plastic

socks of the pergolari-type (Intermas, Spain), and

suspended from an experimental longline outside the

laboratory. Socks of 3 m lengths were deployed with

50 cm intervals from 1 to 4 m depth, varying �0.5 m

due to the tidal range. In order to equalize biomass

density in the two groups, they were socked in densities

corresponding to 1200 and 600 mussels per meter for

the 1- and 2-year-old mussels, respectively. The socks

A. Duinker et al. / Harmful Algae 6 (2007) 288–300290

were distributed into three replicates per group, and due

to the difference in density, one sock per replicate was

used for the 2-year-old mussels and two socks per

replicate were used for the 1-year-old mussels. The

control group consisted of one replicate with 1000

mussels distributed in two socks.

Mussels were sampled on day 0 (14 March), 3, 7, 12,

20, 26, 40, 54 and day 73 (26 May). Each replicated

sample consisted of 50 mussels from different depths

along the socks. Mussels completely covered in byssal

threads or mussels that were trapped inside the socks

were not sampled. For each replicate, the mussels were

counted and steamed, and total meat weights and shell

weights were used for calculations of average weights

per individual. The steaming was according to the

standard protocol for preparing mussels for food safety

analyses at that time. The soft tissue was stored at

�20 8C. The digestive glands were dissected from

frozen and partly thawed pieces of soft tissue, weighed

and later analysed for DST.

Individual samples were taken on day 5 (19 March)

and day 74 (27 May), consisting of 30 mussels from

each age group on each date. The mussels were

steamed, and individual weights of shell and soft tissue

were measured. Individual samples of soft tissue were

stored at �20 8C for subsequent DST analysis.

2.1. Algae, temperature and salinity

Integrated water samples (0–6 m) for quantitative

microscopy of phytoplankton and net hauls (0–10 m)

were taken at time intervals of 2–5 days in the trial period

from 5 March to 27 May. Water samples were preserved

with Lugol solution (2% final concentration) and net

hauls with formaldehyde (Throndsen, 1978). About

25 ml of the integrates water samples were sedimented

using Untermohl technique (Hasle, 1978) and counted in

an inverted light microscope (Leica DM IRB, Wetzlar

GmbH, Germany) at 125–500�magnification. Net hauls

were checked for toxin producing alga using a Leitz,

Aristoplan light microscope (Wetzlar, Germany).

Specific carbon biomasses were computed for the

dominating algal species based on equations given by

Strathmann (1967). Cell volumes in this equation, were

based on cell measurements of 40 cells taken from four

different samples or net hauls measured at 400–500�magnification in light microscope (Leica DM IRB,

Wetzlar GmbH, Germany or Leitz, Aristoplan, Wetzlar,

Germany) using geometrical equations given by Hansen

(1992) or Hillebrand et al. (1999).

Salinity, temperature and fluorescence were mea-

sured using a STD/CTD instrument (SD204, Saiv AS,

Norway) submersed from 0 to 20 m depth every 2–5

days. In addition, temperature was monitored at 2.5 m

depth every 60 min using a Tinytag1 temperature

logger (Intab Interface-Teknik AB, Sweden).

2.2. Analysis of diarrhetic shellfish toxins (DST)

The hepatopancreas from 50 mussels were homo-

genized for about 1 min (Buhler Homogenizer HO 4/A).

A portion of 1 g homogenate was extracted with 9 ml

methanol:water (8:2) for 10 min using a Stuart rotator

(Bibby Sterilin Ltd., Staffordshire, UK) at maximum

velocity. For analysis on individual mussels, the

hepatopancreas was homogenized directly in 80%

MeOH using a Virtis tempest IQ 500W homogenizer.

The samples were then centrifuged at 3000 rpm. About

500 ml of these supernatants were passed through a

0.22 ml Spin-X centrifuge filter (Corning Incorporated,

NY, USA) by centrifugation at 3000 rpm for 3 min, and

the filtrate was used for analysis of DTX-1 and OA on

LC/MS.

The amount of esterified forms of DSP toxins was

determined by adding 400 ml of a 1 M sodium

hydroxide solution to 2 ml of filtered supernatant.

The vial containing this mixture was placed in a Stuart

Rotator, which was run at maximal velocity for 1 h.

About 500 ml of 1 M HCl was then added to stop the

reaction. About 2 ml of hexane was added and the

sample extracted for 3 min using the Stuart Rotator. The

sample was centrifuged at 3000 rpm for 3 min for

separation of the two phases and the upper phase

(hexane) removed and transferred to waste. To the lower

phase 1 ml water and 2 ml dichloromethane were added

and the sample extracted for 3 min using the Stuart

Rotator. Following centrifugation for 3 min at

2800 rpm, the lower phase (dichloromethane) was

transferred to a new vial and the upper phase extracted

once more with 2 ml dichloromethane. The combined

dichloromethane phases were evaporated to dryness

using a Turbovap LV (temperature 35 8C), resolved in

2 ml 80% MeOH and filtered trough a 0.22 mm Spin-X-

centrifuge filter at 3000 rpm for 3 min. The samples

were now ready for LC–MS analysis.

The LC system was an Agilant (Palo Alto, CA)

HP1100 with binary pump, variable volume injector and

a thermostated auto sampler. HPLC separation was

conducted at 24 8C using an isocratic solvent mixture,

methanol:25 mM ammonium acetate (pH 5.80) (80:20).

A Hypersil BDS C-18 column (3 mm, 2.1 mm �100 mm); flow rate 0.1 ml/min was used. About 10 ml

of the sample was injected. The mass spectrometer

used was a Micromass Quattro LC Tandem MS/MS

A. Duinker et al. / Harmful Algae 6 (2007) 288–300 291

(Micromass UK Limited, Manchester, UK) equipped

with electro spray source. Desolvation temperature was

300 8C and desolvation gas flow rate 650 l/h. Selected ion

monitoring (SIR) for data acquisition and negative ion

detection was used (OA: m/z 803.5; DTX-1: m/z 817.5).

Analyst software (MassLynx NT) was used for HPLC

system control, data acquisition, and data processing.

Okadaic acid (OA) and 35-methylokadaic acid

(DTX-1) were purchased from Alexis Biochemicals,

Lausen, Switzerland. CRM-DSP-MUS, a certified

reference mussel/microalgal material (11.0 mg OA

and 0.96 mg DTX-1/g homogenate), was obtained from

National Research Council, Halifax, Canada. All other

chemicals used were analytical grade.

Half-life was calculated for each replicate according

to the formula; t1/2 = ln 2/l; t1/2 = half-life, where lrepresents the slope of the curve.

2.3. Statistics

t-Tests were used for comparison between DST

concentrations and half-lives between the two groups of

mussels. Simple correlations were used to compare

toxin burden and digestive gland weight on the

individual mussel sample. StatisticaTM 6.0 (Statsoft,

Inc., USA, 2004) was used for the analyses.

3. Results

3.1. Growth

The mean soft tissueweights of the 2-year-old mussels

decreased during the first week of the experiment, while

the digestive glands increased during this period (Fig. 1).

In the 1-year-old mussels, both these compartments

increased during this period. Both age groups showed

faster growth of the digestive glands in the beginning and

end of the study, compared to stagnant or decreasing

weights between 21 March and 21 April.

The 1-year-old mussels had faster growth compared to

the 2-year-old mussels in both total soft tissue and

digestive glands. The digestive glands increased 4.4

times in the 1-year-old mussels and 2.0 times in the 2-

year-old mussels, while total soft tissue increased 3.1

versus 1.3 times in the 1- and 2-year-old mussels,

respectively. The 1-year-old mussels grew 1.4 times

faster with respect to shell weight. Consequently, the size

differences between the age groups decreased throughout

the study, from 7.1 to 2.9 times higher soft tissue weights,

from 5.3 to 2.4 times higher digestive gland weights and

from 6.3 to 4.2 times higher shell weights in the 2 years

compared to the 1-year-old mussels.

3.2. Diarrhetic shellfish toxins

In the present study the sum of dinophysistoxin-1

(DTX-1) and okadaic acid (OA) is referred to as DST.

DTX-3 and esterified DTX-1 were not detected in the

mussel samples. At the start of the experiment, the 1-

year-old mussels had more than three times higher

concentrations of DST in total soft tissue (t test,

p < 0.001) with approximately 2400 mg DST kg�1

compared to the 2-year-old mussels that had

700 mg DST kg�1 (Fig. 2). The difference between

the age groups decreased throughout the experiment,

and at the end there was no significant difference

( p = 0.13). In the 1-year-old mussels, toxin concentra-

tion in total soft tissues decreased steadily throughout

the study. In the 2-year-old mussels, concentrations in

soft tissue were stagnant during the first week of the

study, coinciding with the decrease in soft tissue

weights in this group (Fig. 1). In the digestive glands, on

the other hand, toxin concentrations decreased steadily

throughout the study in both age groups (Fig. 2).

Concentrations of DST in the digestive glands were

significantly higher in the 1-year-old mussels at the start

of the experiment ( p < 0.01) with 14,000 mg DST kg�1

compared to about 5500 mg DST kg�1 in the 2-year-old

mussels, and there was no significant difference at the

end of the study ( p = 0.42). The total decrease in DST

concentration in the digestive gland from start to end

values was significantly and 2.6 times larger in the 1

year compared to the 2-year-old mussels ( p < 0.01).

Contrary to the toxin concentrations, the 2-year-old

mussels had higher total toxin content per mussels

(toxin burden) compared to the 1-year-old mussels

(Fig. 2). Toxin burden decreased eight times in both age

groups from start to end of the study.

There was no significant difference in half-life for

toxin concentration in the digestive glands between the

two age groups ( p = 0.055), with 35.2 days in the 1-

year-old mussels compared to 44.4 days in the 2-year-

old mussels. There was also no significant difference

with respect to half-lives for toxin burden ( p = 0.56),

with 49.7 days in the 1-year-old mussels and 52.5 days

in the 2-year-old mussels. Fig. 3 shows log transformed

concentration and toxin burden over time. The slopes of

the regression lines are the basis for the calculations of

half-lives.

No DST was detected in the control mussels during

the trial period.

In the individual samples, there were no significant

correlations between DST concentrations and digestive

gland weights within the groups ( p = 0.33–0.57),

except for the large mussels on day 74. In this group,

A. Duinker et al. / Harmful Algae 6 (2007) 288–300292

Fig. 1. Mean weight of total soft tissue and digestive gland for 1- and 2-year-old mussels. n = 3 from pooled samples of 50 mussels each. Vertical

bars indicate standard deviation.

there was a negative correlation between size and toxin

concentration ( p = 0.002) that was no longer seen when

removing outliers with toxin concentrations above

1200 mg/kg and digestive glands less than 0.3 g

( p = 0.40). Combining the two age groups gives the

same picture as between the groups in the time-series.

There is a significant negative correlation between size

and toxin concentration on day 5 ( p = 0.013) (Fig. 4),

which is still significant when removing outliers with

more than 20,000 mg/kg ( p = 0.036). The correlation is

not longer significant on day 74 ( p = 0.052). Individual

data on toxin burden for the two age groups combined

also show the same trend as the time-series, with

positive correlation between size and toxin burden on

day 5 ( p < 0.001) but not on day 74 ( p = 0.053). The

averages between the two groups were significantly

different on day 5 (t test, p = 0.01) but not on day 74

( p = 0.66).

The variation among the individuals within each

group at the two dates in the individual samples ranged

from 1.7 to 2.8 times in terms of 95% confidence

intervals, and from 10 to 34 times difference from

minimum to maximum values, calculated both from

concentrations and from toxin burden. The correspond-

ing coefficients of variation (CV) ranged between 70

and 120%. The two age groups overlap both in digestive

gland weights and in toxin concentrations.

3.3. Environment

Fluorescence measurements and estimates of carbon

biomasses show two distinct peaks in algal biomass, one

A. Duinker et al. / Harmful Algae 6 (2007) 288–300 293

Fig. 2. DST concentration in total soft tissue and digestive gland and total content of DST per mussel (toxin burden) for 1- and 2-year-old mussels.

n = 3 from pooled samples of 50 mussels each. Vertical bars indicate standard deviation.

A. Duinker et al. / Harmful Algae 6 (2007) 288–300294

Fig. 3. Log values for DST content over time expressed as concentration in the digestive glands and total content per mussel (toxin burden) for 1- and

2-year-old mussels. Common regression lines for the three replicates per age group are indicated.

in mid March to the beginning of April and a second in

May. Mainly two species were dominating during this

period. In March and April colonies of the prymnesio-

phyte Phaeocystis spp. were dominating (maximal

concentration of 11.4 million cells/L 21 March) (Fig. 5).

The diatom Skeletonema spp. was found throughout the

trial period and dominated during the biomass peak in

May (maximal concentration of 7.8 million cells/L 21

May).

Dinophysis spp. was found in trace concentrations

well below the regulatory level given by Norwegian

authorities during the trial period, except for one sample

taken 1 day before termination of the experiment (26

May) were Dinophysis acuminata concentration was

2200 cells/l, which is above the regulatory level of

900 cells/l.

Temperature at 3 m depth was about 5 8C in March

and the first week of April. A thermocline developed in

mid April and the temperature at 3 m depth increased to

around 8 8C for the rest of the study. A stable salinity

profile was seen throughout the trial period except for

the low salinity (approximately 17–23%) seen in the

last period of the experiment.

4. Discussion

In the present study, we found no significant

differences in elimination rate of DST between 1-

A. Duinker et al. / Harmful Algae 6 (2007) 288–300 295

Fig. 4. Individual samples on day 5 and 74 with DST concentrations in the digestive glands against digestive gland weights in 1- and 2-year-old

mussels.

Fig. 5. Estimates of food levels given as total carbon concentrations (mg/l) and the contribution of the dominating algae species in the water samples

throughout the study.

A. Duinker et al. / Harmful Algae 6 (2007) 288–300296

and 2-year-old mussels under natural conditions. This

suggests that size and age do not affect the elimination

rate of the DST. The present study is the first detailed

study on the effect of age and size on the depuration rate

of algal toxins in bivalves. Blanco et al. (1999) found an

inverse relationship between decreases in DST con-

centrations in mussels and body weight, and suggested

that smaller mussels had higher depuration capabilities

compared to larger mussels. However, the correlation

was not consistent, and a possible dilution effect was not

accounted for as discussed below.

The two groups of mussels that differed in age and

size showed large differences in specific soft tissue

growth. These differences in size and growth were

probably associated with differences in various phy-

siological rates between the two age groups. Such

differences in physiological rates with size were not

measured in the present study but can be found in

literature. In general, large differences in both ingestion

rates, oxygen consumption, maintenance ration and

scope for growth can be found for large spans in mussel

sizes, and typically the maximum differences are

between two- and four-fold (Thompson and Bayne,

1974; Winter and Langton, 1976; Navarro and Winter,

1982; Bayne and Newell, 1983). For comparison with

our two age groups, however, relevant sizes should be

considered. At the start of the study, the two groups had

approximately 100–300 mg and 700–900 mg dry-tissue

weight throughout the study in the 1- and 2-year-old

mussels, respectively, assuming a dry-weight percen-

tage of 30% for the steamed meat weights. Considering

the relative differences in various size-measures

between the two groups, it should be relevant to

consider data from literature on mussels of 250 and

1000 mg dry-tissue weights, a four times difference,

which also gives a comparable modelling of growth

according to the discussion below.

Size-specific ingestion rates generally decrease with

size. Navarro and Winter (1982) found 1.7–1.8 times

faster and Thompson and Bayne (1974) found 2.5 times

faster ingestion rates in 250 mg compared to 1000 mg

dry soft tissue mussels (recalculations of the data

presented by these authors). This relationship is stable

over the whole range in food concentrations tested in

these two studies. Also size-specific routine oxygen

consumption generally declines with size as reviewed

by Bayne and Newell (1983). Applying the formula

y = axb with a typical b of 0.75 (Bayne and Newell,

1983) gives 1.4 times higher specific oxygen consump-

tion in 250 compared to 1000 mg mussels, which is

consistent with the values from Navarro and Winter

(1982). This comparison should be roughly relevant to

the age classes in the present study, although the b-

values can vary within different age-classes (Camacho

et al., 2000). On the other hand, differences in oxygen

consumption are not always found with size, as

demonstrated by Sukhotin et al. (2002). Further,

maintenance ration generally decreases with size,

reflecting high metabolic rates for small mussels

(Winter and Langton, 1976), and applying the formula

fitted for data presented by these authors gives

maintenance rations of 1.72 versus 1.61% of dry

weight for 250 and 1000 mg mussels, respectively. With

respect to net growth efficiency, the proportion of

assimilated energy available for production, further

calculations based on the data from Navarro and Winter

(1982) reveals 1.2 times higher growth efficiency in

250 mg compared to 1000 mg mussels at the various

food levels investigated.

All these factors can be summarised in the scope for

growth. Like the underlying physiological processes,

size-specific scope for growth declines with size as

demonstrated by Thompson and Bayne (1974). From

their data, relative growth rates in calories per day per

total soft tissue calories can be calculated, revealing

maximum differences of 2.7 versus 0.27% per day for

250 and 1000 mg mussels, respectively, at their second

highest food level. Further calculations give 2.2 versus

1.1 times increase in soft tissue calories after for

instance 30 days. This corresponds to twice as fast

growth in the smaller mussels, which is similar to the

observations in the present study, and hence indicates

that the data and comparisons used here are adequate. In

summary, differences in size-specific growth between

size-classes of mussels comparable to the present study

are mainly based on considerably higher size-specific

ingestion rates, enhanced by smaller but probably also

markedly higher size-specific oxygen consumption

rates in the smaller compared to larger mussels. We

found no differences in toxin elimination between the

age and size groups in the present study. Our

observations hence support previous studies suggesting

that feeding rate has no effect on the toxin elimination

(see discussion below). We also suggest that physio-

logical rates associated with differences in oxygen

consumption do not affect toxin elimination, which may

involve a range of processes. It is not known whether the

toxins are metabolised or not during the elimination

period. If metabolic routes were involved, one could

expect the elimination rates to be influenced by overall

metabolic rates. Our results do not support this. Further

studies are needed to elucidate this.

The more than three times higher DST concentration

in the 1-year-old mussels at start of the study can also be

A. Duinker et al. / Harmful Algae 6 (2007) 288–300 297

explained by the higher ingestion rate in smaller

mussels as discussed above, with higher size-specific

ingestion of toxic algae resulting in higher toxin

concentrations. The difference in DST concentration is

larger than the around two times difference in ingestion

rate accompanying a two times difference in growth as

discussed above. However, the difference in size

decreased throughout the study, and the size difference

must have been larger at the time of toxin accumulation,

hence explaining the larger difference in toxin

concentration. Similarly, higher concentrations in

smaller mussels have been observed for DST by Aalvik

and Framstad (1981) and for paralytic shellfish toxins

(PST) by Morono et al. (2001).

The mussels experienced large variations in algal

concentrations throughout the study, with 5–10-fold

differences in total cell carbon concentrations. These

variations where large enough to affect growth of the

digestive glands in both age groups. The periods with

fast growth in the first and last part of the study

coincided with high concentrations of diatoms. The

period with stagnant or decreasing digestive gland

weights from late March to late May seemed to be

initiated by high concentrations of the prymnesiophyte

Phaeocystis followed by low algal concentrations. The

Phaeocystis bloom may have caused starvation of the

mussels since colonies of these algae as we observed in

the present study can block the filtration in mussels

(Pieters et al., 1980; Smaal and Twisk, 1997). The

temperature variations were smaller compared to the

annual variation, with only a 3 8C increase in late April.

Altogether, the environmental variations did not seem to

affect the elimination rates, and the toxin content

decreased steadily throughout the study.

The mechanism by which the mussels eliminate DSP

toxins is unknown, although a few models have been

suggested. Several authors have suggested that food

availability is the main factor affecting depuration rates

(Haamer et al., 1990; Sampayo et al., 1990; Marcaillou-

Le Baut et al., 1993), and Blanco et al. (1999) proposed

that increased feeding rate and hence defaecation rate

will increase the rate of toxin elimination. This model

seems inappropriate to account for the results in the

present study since we found no differences in DST

elimination despite large differences in growth rates and

hence ingestion rates as discussed above. Strohmeier

et al. (2005) found differences within a mussel farm

during a detoxification period, with lower DST

concentrations in mussels with higher condition index

in areas with higher food supply. It was suggested that

higher food supply could have increased depuration

rates, but it was emphasised that depuration rates were

not measured and that other explanations were possible.

Svensson (2003a,b) and Svensson and Forlin (2004)

found no effect of food levels and defaecation rates as

well as temperature and salinity on detoxification rates

of M. edulis in various experiments, and the toxin

content declined under all conditions. Detoxification of

DST occurs in the field at stable rates also throughout

the winter with low food levels and temperatures both in

Swedish (Svensson and Forlin, 2004) and Norwegian

(personal communication, T. Aune, Norwegian Veter-

inary College) waters, with no marked effect of the

spring phytoplankton bloom. As discussed above, the

present results suggest that overall metabolic rates

linked to oxygen consumption, associated with differ-

ences in growth rates, do not influence DST elimination.

The only consistent differences that have been reported

are seen with different seasons, with shorter half-lives in

August–September in both field and laboratory com-

pared to November studies in different years (Svensson,

2003b). Combined with our results, it seems that the rate

of detoxification is influenced by some factor that varies

with season but that is not affected by environmental

conditions or differences in ingestion rates, oxygen

consumption rates or growth rates that can be found

between different age groups. The most marked

changes associated with season are the seasonal

changes in maturation, with associated changes in

oxygen consumption that can be more than five-fold

(Devooys, 1976; Sukhotin, 1992; Hatcher et al., 1997),

but also associated with changes in energy storage and

expenditure (Thompson, 1984; Cartier et al., 2004). In

order to understand the mechanism involved, physio-

logical studies should be supplied with information

about the cellular and sub-cellular localisation of the

toxins (Svensson and Forlin, 1998, 2004).

The toxin concentrations in the individual samples

showed large variation, which could be due to variations

in food availability and ingestion of toxic algae within

the collectors from which the mussels were harvested

prior to the experiment. Variations in feeding behaviour

or kinetics in uptake or elimination of toxins among

individuals may also contribute to the variation, as

discussed by White et al. (1993). The variation in the

present study in terms of coefficients of variations was

higher but in the same order as the variation reported for

PST in various species of shellfish by White et al. (1993)

and references in therein. There was no clear correlation

between size and toxin concentration within the groups.

This was probably due to the combination of this large

variation and too small size-range within each group.

Logically, when combining the age groups on each date

the same picture is seen as in the time-series, with

A. Duinker et al. / Harmful Algae 6 (2007) 288–300298

significantly decreasing toxin concentration with size in

the beginning of the experiment but not in the end. The

variation in the data was also characterised by the

presence of outliers giving as high differences as 10–34

times in both concentrations and toxin burden, which is

consistent with up to 16 times differences in OA

concentration for Swedish mussels within the same

collectors (Edebo et al., 1988). This large variation

should be considered when deciding sample sizes for

both scientific and food safety purposes.

There were several precautions that had to be taken

in order to compare the capacity of toxin elimination in

the two groups of mussels. The first precaution was

using only the digestive gland for DST analysis, in order

to avoid differences in toxin concentrations in whole

soft tissue due to different spawning patterns. A

spawning was seen in the 2-year-old mussels during

the three first samplings as a decrease in soft tissue

weights while the digestive gland was increasing, which

was not seen in the 1-year-old mussels. Since DST is

almost exclusively located in the digestive gland

(Murata et al., 1982; Edebo et al., 1988), a spawning

will result in an increase in DST concentration in the

total soft tissue independent of the toxin elimination. No

strong effect was seen of the spawning, which was

probably only partial. Toxin concentrations in soft

tissue in the 2-year-old mussels were stagnant during

the period of increase in soft tissue weights. If no

spawning had taken place these concentrations would

probably have decreased, as seen in the digestive gland.

A significant difference in total decrease in DST

concentration was seen between the two mussel groups,

with more than two times larger decline in the 1-year-old

mussels. From this perspective, it seems that there are

obvious differences in detoxification rates between the

two mussel groups. However, the two-fold difference in

start values (see discussion below) and the exponential

nature of the toxin decline curve require further

precautions for this comparison. During an exponential

decline, the absolute declines are faster with higher

values, decreasing asymptotically towards lower values.

Logarithmic transformation of an exponential curve

gives a linear curve with the same elevation – which gives

the rate of decrease – for high and low values, and hence

eliminates the problem of different start values.

The faster decrease associated with the higher

concentrations in the 1-year-old mussels does not

necessarily reflect a higher capacity for toxin elimina-

tion in this group. Calculations of half lives, on the other

hand provides such a logarithmic transformation that is

independent of differences in start values. Another

solution to this problem is using percentage decrease in

relation to start values as presented by Svensson and

Forlin (2004).

Both mussel groups had the same pattern of digestive

gland growth throughout the study. However, while the

2-year-old mussels doubled the digestive gland weight

during the experiment, the 1-year-old mussels had a

digestive gland weight 4.4 times higher at the end of the

experiment than at the start. Similarly, the 1-year-old

mussels increased almost two times faster in total soft

tissue compared to the 2-year-old mussels. This affects

the depuration rate in terms of concentration in the way

that the toxins were more than two times more diluted in

the 1 year compared to the 2-year-old mussels, which

gives a false impression on the loss of toxins in this

group, regardless of the mussels’ potential ability to

actually eliminate the toxins. The four times increase in

digestive gland mass in the 1-year-old mussels

compared to the eight times decline in toxin burden

demonstrates that dilution due to growth can have an

important contribution to declines in toxin concentra-

tion if growth is fast. To illustrate such effects of toxin

dilution due to changes in tissue mass, one can imagine

comparing a group of starving mussels with decreasing

tissue mass, to a group of well-fed and growing mussels.

Toxin concentrations would increase in the starving

mussels and decrease in the growing mussels even if the

toxin content in the animals is constant, and the

different changes in toxin concentrations would not

reflect the actual elimination of toxins from the mussels.

Similar dilution of algal toxins has also been demon-

strated for PST in mussels and clams (Bricelj et al.,

1990; Bricelj and Cembella, 1995) and discussed by

Blanco et al. (2005). The third precaution in order to get

a proper interpretation was, therefore, to consider the

decline in the total amount of DST per mussel, termed

as toxin burden. This procedure eliminates the problem

with different extent of toxin dilution due to differences

in growth, although this was not demonstrated with

significant differences in half-lives for the decrease in

toxin concentrations in the present study. The 50 and 53

days half-lives in toxin burden give a more correct

measure of the elimination of toxins in the 1- and 2-

year-old mussels, respectively, than the 35 and 44 days

half-lives in toxin concentrations. When focusing on the

actual elimination of toxins in the animals, the use of

toxin burden will eliminate problems with changes in

toxin concentrations due to all kinds of changes in tissue

mass, including spawning, starvation and differences in

growth. Svensson (2003a) found that an increase in

toxin concentration in a group of starving mussels

disappeared when considering total content of DST.

Other publications on detoxification of bivalves focus

A. Duinker et al. / Harmful Algae 6 (2007) 288–300 299

on decline in concentrations, which, according to the

discussion above, include both the factor of toxin

elimination and the factor of dilution due to positive or

negative growth of soft tissue. Without information

about growth it is not possible to discriminate between

actual toxin elimination rates and dilution effects that

are caused by environmental factors that affect growth.

5. Conclusion

Neither size, age nor natural variations in food levels

and temperature were found to significantly affect the

elimination rate of diarrhetic shellfish toxins (DST).

The mechanism for depuration of DST is unknown.

The results from the present study support earlier

studies suggesting that depuration rates cannot be

stimulated by manipulations of environmental factors.

When the two age classes were compared, it was found

that large differences in growth rates, which also

include difference in feeding and metabolic rates, did

not affect the elimination rate of DST. This strengthens

further the assumption that the depuration rates cannot

be accelerated, even in artificial systems, as a cost-

effective way to solve the problem with toxic mussels

for the industry.

Acknowledgements

This manuscript is based on the master thesis of

Morten Bergslien at the University of Bergen and

NIFES. Thanks to Torunn Eide for analysis of DST and

patience during training in sample preparations. Thanks

to Peter and Kjartan Hovgaard for providing boat,

machinery and expertise for collection of the mussels

for the experiment. The project was founded by an

internal project at NIFES and analysis costs were

supported by the Norwegian Food Safety Author-

ities.[SES]

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