RPCV (2012) 107 (581-582) 75-84
R E V I S TA P O R T U G U E S A
DE
CIÊNCIAS VETERINÁRIAS
Evolution of fatty acid profile and Condition Index in mollusc bivalves
submitted to different depuration periods
Avaliação do perfil de ácidos gordos e do Índice de Condição em moluscos
bivalves submetidos a diferentes períodos de depuração
Francisco Ruano1, Paula Ramos1, Mário Quaresma2*,
Narcisa Maria Bandarra1, Isabel Pereira da Fonseca2
1
Instituto do Mar e da Atmosfera, IP (IPMA)/Instituto Nacional dos Recursos Biológicos (IPIMAR/INRB)
Av. de Brasília, 1449-006 Lisboa, Portugal
2
Centro de Investigação Interdisciplinar em Sanidade Animal (CIISA),
Faculdade de Medicina Veterinária, Av. Universidade Técnica, Pólo Universitário Alto da Ajuda, 1300-477 Lisboa, Portugal
Summary: In the outbreaks of foodborne diseases, bivalves are
an important source of infection for man. Bivalve depuration
warrants a significant improvement in microbiological quality
and economical value, but bivalves are submitted to important
physiological and traumatic stress, which cause significant losses
and changes in nutritional composition of the final product.
This issue has been discussed among the shellfish industry, as a
handicap of this method. Whole content of cupped oyster, blue
mussel, common edible cockle, and carpet shell clam were used
to evaluate the effect of long depuration periods on their
Condition Index (CI) and lipid composition. Data showed that
bivalve depuration was inexorably associated with loss of a
considerable percentage of the CI and lipid reserves, which was
associated with the decline in the nutritional quality. Loss in the
CI was directly related with losses in total fatty acids, but no
correlation was observed between n-3 PUFA losses and losses
in other parameters. The 72 hour depuration period induces a
considerable loss of total fatty acid contents (11-25%).
Considering the n-3 PUFA losses, mussel was the specie with
the lowest loss (10.7% of total n-3 PUFA), all other species in
study had superior losses of n-3 PUFA, all above 20% of the
initial content. The loss of n-3 PUFA, as EPA and DHA, can be
considered the major negative consequence of depuration, since
it contributes to the loss of prime quality fatty acids important
to human health.
Keywords: bivalves, depuration, lipid profile, Condition Index
Resumo: Os bivalves, enquanto organismos filtradores, desempenham papel importante no decurso de surtos de doenças
transmitidas através de alimentos. A depuração dos bivalves
garante uma melhoria da qualidade microbiológica e do valor
económico embora cause stress fisiológico e traumático
indutores de perdas significativas e alterações na composição
nutricional do produto final. Esta questão tem sido discutida na
indústria do marisco como uma desvantagem do método. Este
trabalho procurou avaliar o efeito de longos períodos de depuração sobre o índice de condição corporal e a composição
lipídica de ostra, mexilhão, berbigão e amêijoa. Os resultados
obtidos mostraram que a depuração dos bivalves foi inexoravelmente associada à perda de uma percentagem considerável do
índice corporal e das reservas lipídicas, o que se traduz numa
*Correspondência: [email protected]
diminuição da qualidade nutricional. A redução no índice de
condição relacionou-se directamente com a diminuição dos ácidos gordos totais, embora não tenha sido observada correlação
entre as perdas de n-3 PUFA e de outros parâmetros. Um período de depuração de 72 horas induziu uma perda considerável
(11-25%) dos ácidos gordos totais. Considerando as reduções
de n-3 PUFA, o mexilhão foi a espécie com menor perda (10,7%
do total de n-3 PUFA) enquanto as outras espécies em estudo
apresentaram reduções superiores e acima de 20% do conteúdo
inicial. As reduções de n-3 PUFA, assim como de EPA e de
DHA, podem ser consideradas como as consequências mais
negativas da depuração, uma vez que contribuem para a
diminuição de ácidos gordos benéficos para a saúde humana.
Palavras-chave: bivalves,depuração, perfil lipídico, Índice de
Condição
Introduction
Bivalves can concentrate in different organs (gills,
digestive gland) and in haemolymph, several harmful
elements from the water column, such as xenobiotics,
biotoxins, microbial and parasitic forms including
oocysts and other parasitic forms, being for this reason
a potential source of contamination and infection for
man (Ramos et al., 2005; Pereira da Fonseca et al.,
2006). Among infectious agents commonly found in
bivalves, the enteric parasite Cryptosporidum parvum,
is of particular importance to human patients with
immunodeficiency or depressed defensive mechanisms (Stark et al., 2009).
In EU, the Regulation (EC) 854/2004 has been
adopted by all member states. Accordingly to such
Regulation, mollusc bivalves production areas can be
classified into three different classes A, B and C. This
classification focuses on a major parameter, the faecal
coliform bacteria contami-nation, as an index of global
microbial contamination.
75
Ruano F et al.
Most of Portuguese bivalves production is gathered
from open sea beds along the coast, located in Class A
areas. On the other hand, the most important component of aquaculture production comes from parks in
interior waters (estuaries and coastal lagoons), most of
them classified as Class B in the last official classification, which compels these aquaculture bivalve
production to a depuration process, in order to reduce
bacteriological contamination levels to less than 230
coliforms per 100 g of flesh. The depuration procedure assures a significant improvement in bivalve
microbiological quality, which otherwise should be
sent to the industrial transformation.
The depuration process is performed in sterile sea
water, during periods of time from 24 to 72 h, being
considered an efficient method to clean up bivalves,
even in the cases of contamination by Cryptosporidium oocysts (Ramos et al., 2005). However, physiological and traumatic stress can be induced during
depuration, causing weakness and deaths on bivalves,
especially when exposed for long periods (Ruano et
al., 1998). Important changes on both nutritional
composition and organoleptic characteristics of the
final product have been argued among the shellfish
industry, as a handicap of this procedure, but there is
no available scientific data confirming such claims.
The lack of information concerning the consequences
of depuration procedure on bivalve quality has compelled us to identify and quantify the effect of long
periods of depuration on live bivalves. In order to
assess the depuration outcome, we have evaluated the
effect of different depuration periods on the CI and
fatty acid composition of four different bivalve
species, namely the Portuguese cupped oyster (Crassostrea angulate; Lamarck, 1819), the blue mussel
(Mytilus edulis; Linnaeus, 1758), the edible cockle
(Cerastoderma edule; Linnaeus, 1758), and the carpet
shell clam (Venerupis pullastra; Montagu, 1803).
Material and methods
Sampling
In this study we selected four bivalve species collected from different origins. The Portuguese cupped
oyster, Crassostrea angulate (Lamarck, 1819), from
natural beds of Sado estuary; blue mussel, Mytilus
edulis (Linnaeus, 1758), from rafts in Albufeira
coastal lagoon; common edible cockle, Cerastoderma
edule (Linnaeus, 1758), from natural beds in Sado
estuary and carpet shell clam, Venerupis pullastra
(Montagu, 1803) from natural beds in Tagus estuary.
Bivalves were collected during the low tide before
each depuration procedure, roughly cleaned and
washed from mud and sand with sea water and transported to the lab in net bags, inside an isothermal
cooling box. These species were selected due to their
76
RPCV (2012) 107 (581-582) 75-84
different feeding strategies and filtering capacities,
they also colonize distinct niches in the aquatic
ecosystem. All the samples were collected during
the same week at May of 2007, except oysters which
were collected at the end of November 2007 in areas
classified as B.
Depuration procedure
Each species was submitted to an equal depuration
procedure, running from 0 to 72 hours period, using a
licensed industrial compact depuration circuit
DEPURMAR®, with a capacity to depurate 180 kg of
bivalves every 48 h. The equipment, assembled in
close circuit, includes a 1.5 m3 fibber glass water tank,
an electric pump, a mechanic filter, a sterilisation
chamber with two lower pressure tubes of UV lamps
(25W – 254nm) and a water cooler system. The sea
water flows through bivalves by a superficial oxygenated shower and by a bottom current with a flow
rate of 1 m3/h, removing the foam from the surface and
faeces and pseudo faeces from the bottom. Thereafter,
the water flows through a foam filter to remove the
suspended matter from the water column, then passes
through the cooler to maintain the water temperature
previously programmed and then through a UV chamber to be sterilised and finally returns into the tank.
Before each assay, salinity and water temperature of
the depuration circuit was adjusted with the bivalve
origin conditions. All the bivalves were separated
from clusters (mussels and oysters), brushed, washed
using sterilised sea water (sterilisation of sea water in
the particular conditions of the experiment, means the
reduction of bacteriological contamination to less than
100 MPN/100 mL) and then distributed per several
plastic trays and stored inside the depuration tank into
two layers in densities on a maximum of 10 kg/m3.
During the adaptation period (first hour) a visual
check-out was performed, to verify the opening movement of their valves, the exteriorisation of siphons,
tentacles or tactile cilia of the mantle, depending on
the species, in order to assure that bivalves reinitiated
their filtration process.
Bivalves of each species were randomly divided into
six replicates for the experiment. Two replicates were
used to determine the initial and final Condition Index
(CI) at 0 h and 72 h respectively and four more replicates were taken to the assessment of Lipid Profile (0 h,
24 h, 48 h and 72 hours), as summarised in Table 1.
Total lipids quantification
Total lipids were determined according to the
Association of Official Analytical Chemists methods
(AOAC, 2005). Briefly, total lipids were determined
with the Soxhlet extraction method using ethyl ether
(99.7%, Merck). The results were expressed in g per
100 g of wet weight.
Ruano F et al.
RPCV (2012) 107 (581-582) 75-84
Table 1 - Sample size used for determination of the Condition Index (CI), lipid analyses and total mortality during the depuration
essays
Depuration time (hours)
Species (Total Sample)
0
72
0
24
Lipid analyses
CI
Oyster (150)
Clam
(124)
Mussel (150)
Cockle (150)
25
26
25
25
25
18
24
23
25
18
24
24
Chromatography analysis
The whole content of bivalves used for lipid analysis was removed from the shell, stirred and frozen
at -26 ºC in Petri dishes, covered with Parafilm® pellicle and then lyophilized.
The analysis of fatty acids was based on the experimental procedure described by Lepage and Roy
(1986) and modified by Zvi et al. (1988). The fatty
acid methyl esters (FAME) were analysed in a CP
3800 Varian gas chromatograph, equipped with an
auto-sampler and fitted with a flame ionisation detector (FID). The separation was carried out with helium
as carrier gas in a DBWax Polyethylene glycol column
(30 m 0.25 mm id) programmed to start at 180 ºC for
5 min, heating at 4 ºC/min for 10 min and hold up at
220 ºC for 25 min, with a detector at 250 ºC. A split
injector (100:1) at 250 ºC was used. Fatty acid methyl
esters were identified by comparison of their retention
time with those of chromatographic Sigma standards.
Quantification was done using the area of the internal
standard 21:0. The total unsaturation index (UI) was
generated by summing the individual fatty acid unsaturation indices, which were calculated by multiplying
the number of double bonds of each fatty acid by its
percentage and dividing by 100 (Huynh and Kitts,
2009).
Condition Index quantification
The CI was assessed by calcula-ting the value of
meat dry weight, dividing it for the volume of the shell
cavity (Lawrence and Scott, 1982). The dry weight
was determined by weighting after draining the meat
IC 0h
IC 72h
Loss (%)
-45
350
300
IC
250
-30
200
loss
%
150
-15
100
50
0
0
Oyster
Clam
Mussel
Cockle
Figure 1 - Evolution of the CI of four bivalve species, between
0 h and 72 h depuration period and percentual variation
48
72
Mortality
number of specimens
25
25
18
18
23
22
23
22
25
18
22
21
0 (0%)
8 (6.4%)
10 (6.7%)
12 (8.0%)
in filter paper for 15 minutes at room temperature and
then drying it in an incubator at 100 ºC, during 12 h,
until reaching a constant weight. The volume of shell
cavity was calculated by the difference between the
volume of entire live bivalves and the volume of
empty shells (± 1 ml). The CI was calculated using the
expression:
CI=
Dw(g)x100
Sc(mL)
Dw - dry weight of meat, Sc - Shell cavity volume.
Results and discussion
Condition Index
The CI values along the 72 hours of the depuration
shows a considerable decrease in all species studied
(Figure 1), as consequence of starvation imposed by
the depuration. However, the decrease in the CI associated with weight loss was variable among species in
study, suggesting that some species should react to the
depuration process in a more sensible or resistant
manner, depending on their energy reserves and probably on their capacity to reduce the metabolic rate, as
physiologic response to starvation (Bayne, 1973).
The study results suggest that mussels and cockles
were the most sensible species, showing a sharp
decrease in their CI during the 72 h period, passing
from 114.5 to 84.3 and from 310 to 220.7, losing
26.3% and 28.8% of their CI, respectively for mussels
and cockles. On the other hand, oysters and clams
were apparently more resistant to depuration, which is
confirmed by a moderate decrease in their CI values
(110.9 to 90.1 and from 223.3 to 191.8 for oysters and
clams, respectively) that was responsible for the loss
of 18.8% and 14.2% of their CI, respectively. Such
result was consistent with the mortality rates, which
were higher in for mussels and cockles.
Bivalve energetic reserves are dependent of environment conditions and the quantity and quality of
seston, which is highly variable throughout the year.
Carbohydrates, particularly glycogen, are considered
to be the main energy source in adult marine bivalves,
and are important for gamete formation and maintenance of adult condition during periods of nutritive
77
Ruano F et al.
stress (De Zwaan and Zandee, 1984). Therefore, it was
expectable that bivalves with higher energetic reserves
(glycogen, triacylglycerols and protein) should be
more tolerant to depuration than bivalves with lower
energetic reserves.
Beyond previous explanations, differences in the
energetic reserves between species should be dependent
of different ecologic and biological characteristics
being important factors conditioning differences in the
accumulation of energy reserves and their response to
starvation, such as: 1) differences in the quality and
quantity of seston are important determinants of food
resources of bivalves and these factors are highly
variable in shallow marine environments conditioning
seasonal differences and differences between different
habitats (Biandolino et al., 2008); 2) differences in the
feed behaviour and the specific characteristics of food
intake, namely filtration capacity and clearance rates
between species (Vilela, 1950; Heral et al., 1980;
Henry, 1987); 3) diversified food web between buried
bentonic species, as clams cockles and even oysters
possess a diversified food web; 4) the reserve cycles in
bivalves indicate a complex interaction between food
and temperature and between growth and spawning
cycle (Fernández-Reiriz et al., 2004); 5) differences
between oysters and the other species could also be
associated with differences in the biological cycle at
the time of bivalve gathering.
Initial fatty acid composition
The fatty acid profile of blue mussel, carpet shell
clam, edible cockle and the Portuguese cupped oyster
obtained immediately after their collection (depicted
on Table 2), revealed a similar fatty acid profile in all
bivalve species. PUFA (46.9-49.0%) was the dominant
group followed by SFA (28.2-31.1%) and MUFA
(17.7-19.5%). Prime fatty acids, of the saturated and
monounsaturated groups were palmitic acid (15-19%
of total fatty acids) and palmitoleic (2.2-7.3% of total
fatty acids) as principal elements of the SFA and
MUFA fatty acid groups. While eicosapentaenoic
(EPA) and docosahexaenoic (DHA) acids (13-16%
and 10-14% of total fatty acids, for EPA and DHA,
respectively) were prime representatives of the
n-3PUFA group, the araquidonic acid (ARA; 20:4n-6)
was the major representative of the n-6 PUFA family
(2-3.68% of total fatty acids). The comparison of
bivalve species fatty acid profile shows that clam was
the bivalve specie with the highest percentage of
PUFA (49%). Such superiority in PUFA was consequence of the superior percentage in the long chain
polyunsaturated fatty acids (LCPUFA) of the n-3
series, as EPA, DHA and docosapentaenoic acid (DPA).
The superior prevalence of such n-3 PUFA has been
previously reported in other studies (Chu et al., 1990;
Napolitano et al., 1997; Reuss and Poulsen, 2002;
Dalsgaard et al., 2003). The n-3/n-6 ratio presented the
78
RPCV (2012) 107 (581-582) 75-84
lowest level in cockle (3.45) and the highest in oyster
(10.0).
Fatty acid composition of neutral lipids reflects the
diet and can be used as a trophic biomarker in
bivalves, while the fatty acid composition of the polar
lipids is strongly regulated and reflects the membrane
requirements (Copeman and Parrish, 2003). As these
structural lipids are the minor fraction, the fatty acid
profile of total lipids, presented in this work, provides
simultaneously an overall nutritional evaluation and
Table 2 - Fatty acid composition and partial sums (expressed as
g/100 g total fatty acids) of blue mussel, carpet shell clam,
edible cockle and the Portuguese cupped oyster immediately
after collection
Fatty acids
14:0
i-14:0
15:0
i-15:0
16:0
i-16:0
ai-16:0
17:0
18:0
19:0
20:0
∑SFA
15:1
16:1n-9
16:1n-7
16:1n-5
17:1
18:1 n-9
18:1 n-7
18:1 n-5
20:1 n-9
20:1 n-7
22:1 n-11
22:1 n-9
∑MUFA
16:2 n-4
16:3 n-4
16:3 n-3
18:2 n-6
18:3 n-6
18:3 n-3
18:3 n-4
18:3 n-3
18:4 n-3
20:2 n-6
20:4 n-6
20:4 n-3
20:5 n-3
22:2 n-6
22:4 n-6
22:5 n-6
22:5 n-3
22:6 n-3
∑PUFA
(n-3)/(n-6) ratio
n.d. – not detected
Clam
2.22
n.d.
0.40
n.d.
15.64
0.84
0.59
0.59
6.75
1.20
n.d.
28.23
0.34
0.30
5.26
0.14
n.d.
2.07
4.80
n.d.
1.11
n.d.
0.78
2.91
17.71
0.73
0.49
6.93
0.47
1.11
0.29
n.d.
n.d.
n.d.
1.02
2.84
0.38
16.17
n.d.
1.42
0.58
2.47
14.12
49.02
5.22
Cockle
1.70
n.d.
0.52
n.d.
15.40
0.83
0.93
0.55
8.06
1.32
1.78
31.09
1.00
1.52
3.27
0.87
n.d.
0.84
2.82
n.d.
2.56
0.53
2.77
2.57
18.75
1.54
0.64
7.66
0.87
n.d.
1.00
1.12
n.d.
n.d.
1.12
3.68
0.58
13.13
n.d.
3.25
1.48
1.78
9.96
47.81
3.45
Mussel
2.67
n.d.
0.55
n.d.
15.95
1.04
0.78
0.27
4.48
n.d.
0.93
26.67
n.d.
0.41
7.27
n.d.
0.68
1.01
2.70
0.08
2.21
1.24
n.d.
3.90
19.50
0.71
0.16
8.94
1.28
0.26
0.84
1.29
n.d.
n.d.
0.22
3.12
n.d.
15.65
1.14
n.d.
n.d.
1.64
11.88
47.13
6.49
Oyster
2.26
0.77
n.d.
0.39
19.37
0.40
n.d.
2.01
5.11
0.30
0.46
31.07
n.d.
0.26
2.23
n.d.
n.d.
2.67
5.42
n.d.
1.60
3.89
n.d.
3.00
19.07
0.47
0.75
4.47
1.52
0.31
2.67
0.31
2.67
3.91
n.d.
2.04
0.78
15.78
n.d.
n.d.
n.d.
1.02
10.19
46.89
10
Ruano F et al.
offers information regarding trophic biomarkers
present in food web. In this logic the saturated fatty
acids 14:0, 16:0 and 18:0 are of little value as biochemical markers since they are synthesized by most
organisms and occur in all algae groups in variable
levels of concentrations. Among these three SFA, 16:0
is the most constant over the life cycle of seafood and
is considered a key metabolite in the synthesis of fatty
acids (Nunes et al., 2003). On the other hand, the
presence of several other fatty acids, combinations
of them or ratios have been used as biomarkers for
particular sources of organic matter, helping researchers to estimate the relative contribution of diatoms,
bacteria, dinoflagellates, or terrestrial organic matter to
food web of marine species such as bivalves (Budge
et al., 2001; Alfaro et al., 2006; Volkman, 2006;
Biandolino et al., 2008). Several studies have been
using fatty acids as trophic biomarkers. Useful fatty
acid biomarkers have previously been defined as those
fatty acids that are synthesized at low trophic levels
but remain relatively unchanged when transferred
throughout the food chain (Reuss and Poulsen, 2002).
The predominance of n-3 PUFA, of 20 and 22
carbons, in the fatty acid profiles of all bivalve species
in study suggest that marine phytoplankton is their
major food source. Phytoplankton are dominated by
the polyunsaturated fatty acids of the n-3 family with
20 and 22 carbons, reaching in some conditions 50%
of total lipids (Chu et al., 1990; Napolitano et al.,
1997; Dalsgaard et al., 2003). EPA and DHA are,
therefore, biomarkers of phytoplankton. EPA is the
predominant fatty acid found in diatoms while DHA is
predominantly produced by dinoflagellates (Parrish et
al., 2000). All the bivalves in study showed a predominance of EPA over DHA, such result suggests that
diatoms or other phytoplanktonic organisms rich in
n-3 PUFA, and EPA in particular, as haptophytes, were
important elements in the food web of all bivalve
species in study.
Several fatty acids, namely the 15:0, 17:0 and all
branched fatty acids are produced primarily by aerobic
and anaerobic bacteria (Parkes and Taylor, 1983;
Caudales and Wells, 1992; Harvey and Macko, 1997),
therefore the sum of these fatty acids has been used as
bacterial biomarkers. The results of our study revealed
that the sum of odd and branched fatty acids were
below 4% of total fatty acids, a common result for fish
and marine invertebrates (Copeman and Parrish,
2003). Among species in study, oyster was the one
with the highest percentage (3.6% of all fatty acids),
while clam was the species with the lowest percentage
of odd and branched fatty acids (2.4% of total fatty
acids). Terrestrial plant contribution to bivalve food
web could be evaluated by the presence of linoleic and
linolenic acids and the very long chain (>24 carbons)
fatty acids (Harvey, 1994; Santos et al., 1994;
Colombo et al., 1997), because they are found in high
amounts in most terrestrial plants (Napolitano et al.,
RPCV (2012) 107 (581-582) 75-84
1997; Budge and Parrish, 1998). The biomarkers for
terrestrial plant (18:2n-6 and 18:3n-3) represented just
a minor percentage of bivalves fatty acid profile,
ranging from 2.1% of total fatty acids in the mussel to
0.8% of all fatty acids in clam.
In what concerns n-3/n-6 ratio, the calculated values
in cockle were similar to that previously referred for
the same species (Bandarra et al., 2004). Oyster had
the highest n-3/n-6 ratio, giving this species a relevant
importance in the nutritional point of view.
Depuration effect on bivalve nutritional quality
The analysis of fatty acid profile before, during and
after depuration revealed important and original data
concerning the response of bivalves to starvation
imposed by the depuration procedure. Loss in total
fatty acid contents was associated with a variable
decline in the major fatty acid groups (Figure 2).
The fatty acid composition assessment was considered the appropriate approach to evaluate bivalve
response to depuration-induced starvation and estimate the consequent nutritional loss. Bivalve species
in study showed different lipid mobilization pattern
during the depuration period (Figure 2). Starvation
imposed by the 72 h depuration period and the elimination of gut content were responsible for a massive
fatty acid decrease in a percentage that was variable
between species (10.8%, 19.8%, 24.6% and 24.7% of
total fat contents, for mussel, cockle, clam and oyster
respectively).
However, the quantification of fatty acid recruitment throughout the depuration period revealed three
distinct mobilization patterns, since oyster has registered a total fatty acid mobilization of 4.24 mg/g
of fresh tissue, which was almost twice the amount
registered by clam (2.49 mg/g of fresh tissue) and four
times the amount recorded in mussel and cockle (0.83
and 0.98 respectively). Such differences in the amount
of fatty acid mobilization during the 72 h period, do
not appear to be dependent of total lipid reserves
but on the daily energy maintenance requirements
necessary for each species.
The 72 h depuration period (Figure 2) was responsible
for considerable losses in all major fatty acid groups,
50%
45%
40%
35%
30%
25%
20%
15%
10%
5%
0%
B
Clam
Cockle
Mussel
Oyster
FAME
SFA
MUFA
PUFA
Figure 2 - Total fatty acid loss (TL) during the 72 hour depuration period (presented as % of total contents lost in the overall
depuration period) for total FAME and major fatty acid groups
(SFA, MUFA and PUFA)
79
Ruano F et al.
revealing important species-dependent differences.
Mussel was the bivalve species with the smallest
losses in all major fatty acid groups (9.0% of SFA;
12.1% of MUFA and 11.2% of PUFA). On the other
hand, oyster was the species registering the highest
percentual loss of PUFA (26.7% loss of total PUFA),
while clam suffered the highest loss in both SFA and
MUFA (27.4% and 29.0% of total SFA and MUFA
respectively). Cockle has shown an intermediate
position concerning the percentage of fatty acid losses
during depuration (21.3% of SFA; 14.1% of MUFA
and 21.2% of PUFA).
In general, the biochemical composition of bivalves
is dependent of three major factors: 1) different
biologic characteristics between species in study,
conditioning differences in the feed behaviour and the
specific characteristics of food intake, namely filtration capacity and clearance rates between species
(Vilela, 1950; Heral et al., 1980; Henry, 1987); 2) the
quality and quantity of seston and 3) state of sexual
maturity (Fernández-Reiriz et al., 2007). Therefore,
biological differences between bivalve species and
differences in the habitats from where bivalve species
in study were collected should be responsible for
differences in bivalve composition, namely glycogen
and lipid reserves. Such differences represent a possible explanation for differences observed in the group
of post-spawning species (clam, cockle and mussel)
concerning fatty acid mobilization throughout depuration. On the other hand, differences in the sexual maturity between oyster and other bivalve species should
also be part of possible explanation for differences
found between species.
Chronological assessment of the depuration effect
in bivalve lipid composition
In order to better understand bivalve response to
long depuration periods, the fatty acid composition of
each bivalve species was evaluated at 24, 48 and 72
hours of depuration. The depuration procedure outcome on bivalve lipid fraction was examined in a
chronological approach showing that each bivalve
species has a unique reaction pattern to starvation
(Figure 3). Considering total fatty acids as the prime
parameter to evaluate lipid mobilization, it was possible to observe three distinctive patterns: i) mussel was
the most resistant species to starvation imposed by
global depuration (with the loss of just 10.7% of total
fatty acids), ii) cockle and clam suffered massive lipid
recruitment during the first 24 hours of the depuration
procedure (with the loss of 16.1% and 12.8% of total
fatty acids, respectively), the fatty acid recruitment
persisted until the end of the depuration period but in
a lower rate than observed in the first 24 h period; iii)
oyster revealed the highest tolerance to starvation and
subsequent lipid mobilization during the first 48 hours
(with the loss of 2.1% of total fatty acids). However,
80
RPCV (2012) 107 (581-582) 75-84
the low fatty acid recruitment of the first 48 hours
vanished in the time period comprised between 48 and
72 hours of depuration, which was characterized by a
massive fatty acid mobilization and the loss of 22.6%
of total fatty acids.
Taking into account that this study encloses an
extreme depuration time (72 hours), which is above
the depuration period normally used in this industry, it
was considered essential to evaluate the depuration
effect throughout the depuration procedure. The
species in study revealed a distinctive fatty acid mobilization throughout the first 24 hours period of the
depuration procedure (Figure 4). Considering the first
24 hours period, it was observable that starvation
imposed by depuration was responsible for variable
fatty acid mobilization among species in study,
reflected on total fatty acid loss (2.4% in oyster, 5.1%
in mussel, 12.8% in clam and 16.1% in cockle).
Oyster was the species with minor lipid mobilization
in all fatty acid groups (consuming 1.4% of total
PUFA, 3.7% of total MUFA and 3.0% of total SFA
reserves). Mussel was the species with the second
lowest lipid mobilization, on that period, in all fatty
acid groups (loosing 4.9% of total PUFA, 4.9% of
total MUFA and 5.6% of total SFA reserve). On the
other hand, clam and cockle displayed a lipid mobilization pattern considerable above previously
Clam
Cockle
Mussel
Oyster
100%
95%
90%
85%
80%
75%
0
24
48
72
Hours
Figure 3 - Chronological mobilization of total fatty acids
during depuration
Clam
Cockle
Mussel
Oyster
25%
20%
15%
10%
5%
0%
FAME
SFA
MUFA
PUFA
(n-3)PUFA (n-6)PUFA
Figure 4 - Percentual mobilization of total FAME and fatty acid
groups in the first 24 hours of depuration
Ruano F et al.
described for oyster and mussel. Clam lipid recruitment was primarily concentrated on SFA and MUFA,
(consuming 17.6% of total SFA and 22.8% of total
MUFA and just 5.1% of total PUFA), while cockle had
a lipid recruitment that was transversal to all fatty acid
groups (18.8% of total SFA, 13.5% of total MUFA and
15.3% of total PUFA). The results of this study
revealed that oyster was the species with highest lipid
recruitment in the 72 hours depuration period and the
lowest lipid recruitment throughout the first 24 and 48
hours periods. Such contrasting result was a consequence of intensive lipid mobilization in the last 24
hours period (from 48-72 hours), suggesting that the
energy requirements for the first 48 hours were
accomplish by glycogen mobilization, resulting in the
exhaustion of glycogen reserves. Furthermore, lipid
mobilization was the following step of energy recruitment, but results also suggest that oyster had few lipid
reserves at time of collection. Such suggestion is sustained by the analysis of fatty acid recruitment: 1)
PUFA were the prime fatty acid group used for energy
conversion; 2) 89% of the n-3 PUFA total loss took
place in the last 24 hours period, 3) DHA, a fatty acid
associated with structural-type functions (Bergé and
Barnathan, 2005), was the n-3 PUFA submitted to the
highest percentage of mobilization.
On the other hand, the lipid mobilization of cockle
was almost limited to the first 24 hours period, but the
percentages of total lipid and n-3 PUFA lost in this
period were the highest among the species in analysis.
Such results suggest a low content of glycogen
reserves at time of collection and the almost exhaustion of lipid reserves. Mussel and clam species reveal
an intermediary pattern of lipid mobilization, characterized by a continuous lipid mobilization throughout
depuration.
Effect of depuration on n-3 PUFA contents and
nutritional quality
Considering the inevitability of nutritional loss
associated with the depuration procedure and subsequent lipid decrease, it was considered useful to
understand the relationship between depuration period
and loss of n-3 PUFA, which are prime fatty acids to
human nutrition. The results depicted on Figure 5
shows that the nutritional loss associated with n-3
PUFA elimination during depuration was variable and
dependent on the specie.
The overall amount of n-3 PUFA loss during the
global depuration procedure allowed us to establish
four different patterns concerning the temporal loss of
n-3 PUFA (Figure 5): (1) oyster revealed minimum
mobilization rate of n-3 PUFA during the first 48
hours of depuration (3.2% in the first 24 hours period
and 7.6% in the second 24 hours period); (2) clam
showed a moderate mobilization rate of n-3 PUFA
RPCV (2012) 107 (581-582) 75-84
during the first 48 hours of depuration, revealing a
similar recruitment rate in the first and second 24
hours periods, 20.8% and 19.4% respectively; (3)
mussel revealed an intense mobilization rate of n-3
PUFA in the first and third 24 hours periods, with
minimum recruitment in the period comprised
between the 24 and 48 hours of depuration (47.2%,
1.0% and 51.9% in the first, second and third period
of 24 hours); (4) cockle showed maximum mobilization rate at the beginning of the depuration period
(first 24 hours), minimum recruitment in the second
24 hours period and the remaining in the third 24
hours period (77.4% of all n-3 PUFA were mobilized
in the first 24 hours period, 1.8% in the second 24
hours period and the remaining 20.8% in the last 24
hours period of depuration).
The percentage of loss of major n-3 PUFA during
depuration, depicted in Figure 6, shows that α-linolenic acid was the n-3 PUFA with superior mobilization
in all species in study, with the exception of oyster.
Among the n-3 LCPUFA, there was no common
pattern of recruitment, since EPA was preferentially
mobilized by clam and cockle, while DHA and DPA
suffered a superior mobilization in mussel and oyster,
respectively.
Clam
Cockle
24
48
72
Mussel
Oyster
0%
20%
40%
60%
80%
100%
Figure 5 - Chronological loss of (n-3)PUFA throughout depuration periods (24h, 48h and 72h), estimated as percentage of
overall loss during complete depuration procedure (72 hours)
100%
90%
80%
70%
18:3n-3
20:5n-3
22:5n-3
22:6n-3
60%
50%
40%
30%
20%
10%
0%
Clam
Cockle
Mussel
Oyster
Figure 6 - Percentual loss of major (n-3) PUFA (presented as %
of total contents lost in the overall depuration period)
81
Ruano F et al.
RPCV (2012) 107 (581-582) 75-84
The partial sums of major fatty acid groups and
families and their nutritional ratios before and after
depuration are depicted on Table 3. The results show
that bivalves submitted to a longer period of starvation
(72 hours) appeal to a massive lipid recruitment, loosing
considerable percentage of total fatty acids, variable
with species (10,8% in mussel to 24,7% in oyster).
The amount of total lipid loss was consequence of
fatty acid recruitment during starvation and elimination of gut content, and was transversal to all major
fatty acid groups (Table 3). Considering the loss of
PUFA and n-3 PUFA in particular, data showed that
oyster was the leading species in losses, losing 1.95 g
of PUFA /100g of fresh flesh, which were predominantly from the n-3 family and long chain (1.95 g/100
g of flesh and 1.3 g/100 g of flesh, for total n-3 PUFA
and n-3 LCPUFA). Taking into account that the
Portuguese cupped oyster, used in this study had an
average edible portion of 10.39 g, it is possible to
estimate that a single oyster, lost an average 202 mg of
n-3 PUFA and 135 mg of EPA+DHA.
The depuration procedure was associated with slight
variation in both the (n-3)/(n-6) and the unsaturation
index, which either increased or decreased depending
on the species. EPA and DHA are of particular importance to support optimal brain/visual performance,
cardiovascular care, and other health conditions for
young and old alike, but they are almost absent in terrestrial food products (Uauy and Valenzuela, 2000;
Huynh and Kitts, 2009). Despite being imperative for
human health and nutrition, the n-3 PUFAs are not
effectively produced in humans by their precursor
α-linolenic acid (Burdge et al., 2002; Burdge and
Wootton, 2002) and must consequently be consumed
from foods which are naturally rich in n-3 fatty acids,
especially marine animals that are the primary source
of EPA and DHA (McLean and Bulling, 2005).
The health beneficial effects of long-chain n-3
PUFA are well documented and, overall, increased
intake reduces risk of cardiovascular disease (CHD)
(Kris-Etherton et al., 2002; Ian Givens and Gibbs,
2008) and contributes to the alleviation of inflammatory conditions such as rheumatoid and osteoarthritis
(Gibson and Gibson, 1998) and asthma (Emelyanov et
al., 2002). Recent evidence also points to a role in
reducing age-related decline in cognitive function (van
Gelder et al., 2007).
The American Heart Association (AHA) has recommended that people without coronary heart disease
(CHD) should eat a variety of fish, preferably oily fish
(salmon, tuna, mackerel, herring and trout), at least
twice a week. On the other hand, the AHA advises
people with a previously documented CHD to
consume 1 g of EPA+DHA per day, preferably from
oily fish, while the ingestion of 2-4 g of EPA+DHA
per day are recommended for patients with elevated
plasma levels of triglycerides.
It is important to emphasize the contribution of
bivalve species in study as a source of n-3 PUFA.
Despite their low fat content, bivalve represents an
important resource of n-3 PUFA, as EPA and DHA.
The comparison of n-3 PUFA contents of Atlantic and
Mediterranean fish with bivalve species in studied
revealed two important characteristics: 1) the total
content of n-3 PUFA found in bivalves is in between
the n-3 PUFA contents found in oil fish (as anchovy,
mackerel, tuna and rainbow trout; ranging from
0.4-1.6 g/100 g of muscle tissue) and white fish (as
bass, sea pike, swordfish, plaice and red sea bream;
0.2-0.4 g/100 g of muscle tissue); 2) the bivalve
species in study had a DHA/EPA ratio below 1
(ranging from 0.7-0.9), revealing that EPA and DHA
are present in similar concentrations, while fish
possess a considerable higher rate of DHA/EPA,
thanks to a superior content of DHA over EPA
(Soriguer et al., 1997; Passi et al., 2002). Therefore,
bivalves in study represent an important source of
n-3 PUFA with similar content of major n-3 PUFA
(EPA and DHA).
The loss of n-3 PUFA, as EPA and DHA, can be
Table 3 - Total lipids (g/100g fresh flesh), partial sums of fatty acids (mg/g fresh flesh) and nutritional ratios (n-3/n-6, P/S and UI)
all values presented as mean values before – after depuration (% variation)
SFA
MUFA
PUFA
n-3 PUFA
EPA+DHA
n-6 PUFA
Total FA1
Total lipids
ratios
P/S2
(n-3)/(n-6)
UI3
1
Before
3,18
2,13
4,80
3,97
2,98
0,75
10,12
1.4
Clam
After
2,31
1,52
3,81
3,16
2,37
0,57
7,63
1.1
%
27,4%
28,6%
20,7%
20,4%
20,4%
24,3%
24,6%
21.4%
Before
1,58
0,95
2,43
1,82
1,23
0,53
4,96
0.7
Cockle
After
1,24
0,82
1,91
1,44
0,96
0,42
3,97
0.6
%
21,1%
13,0%
21,6%
21,2%
21,4%
20,0%
19,8%
14.3%
Before
2,20
1,60
3,86
3,30
2,32
0,50
7,65
1.1
Mussel
After
1,99
1,41
3,42
2,95
2,07
0,44
6,82
1,0
%
9,4%
11,7%
11,3%
10,7%
10,7%
12,5%
10,8%
9.1%
Before
5,52
3,51
8,15
7,13
4,77
0,71
17,16
2.4
Oyster
After
4,20
2,76
5,98
5,18
3,47
0,54
12,92
1.9
%
23,9%
21,4%
26,7%
27,3%
27,2%
23,5%
24,7%
20.8%
1,51
5,30
2,55
1,65
5,57
2,44
-9,2%
-5,2%
4,3%
1,54
3,47
2,37
1,54
3,42
2,31
0,6%
1,4%
2,5%
1,75
6,63
2,41
1,72
6,76
2,43
2,0%
-2,1%
-0,8%
1,48
10,03
2,20
1,42
9,54
2,09
3,7%
4,9%
5,0%
Total FA –Total fatty acids; 2PUFA/SFA; 3UI – Unsaturation Index.
82
Ruano F et al.
considered the major negative consequence of depuration, since it contributes to the loss of prime quality
fatty acids, with important health benefits.
Considering the study results and subsequent
analysis of data, it is possible to conclude that depuration effects on bivalve lipid fraction seems to be a
matter of interest taking into account the importance
of seafood fatty acid composition to human diet and
differences among bivalve species.
The intercrossing of results from the Index of
Condition and lipid variation throughout the depuration shows some apparent controversy between data,
since bivalve species with less variation in their IC,
clam and oyster, were those with superior mobilization
of their lipid reserves. Considering that lipid mobilization should be associated with body emaciation
and the subsequent decrease in the IC, such result
suggest that the decrease in IC is associated with the
mobilization of other energetic reserves as glycogen
or even protein.
Data shows that the bivalve depuration procedure is
inexorably associated with the loss of a considerable
amount of the initial lipid content. Such loss in the
lipid fraction is linked to a decline in their nutritional
quality. Differences in between species in analysis
suggest that the loss of half the n-3 PUFA after a 48
hours depuration period is inevitable in species as
mussel and clam, but in other species as cockle and
oyster, even the shorter depuration period of 24 hours
is associated with a considerable loss.
To avoid the negative effect of the depuration, especially in the most sensitive species, we believe that
actual depuration procedure could be improved by the
correct adjustment of the depuration time to the level
of contamination of bivalves and also by proceed with
a transposition step to more clean areas, including to
class "A" areas for bivalves with high levels of contamination before putting them into the depuration/
expedition circuit.
Bibliography
Alfaro AC, Thomas F, Sergent L, Duxbury M (2006).
Identification of trophic interactions within an estuarine
food web (northern New Zealand) using fatty acid bio
markers and stable isotopes. Estuarine, Coastal and Shelf
Science, 70, 271-286.
AOAC (2005). Official Methods of Analysis of AOAC
International, 18th Edition. Latimer G, Horwitz W, Eds.;
AOAC: Gaithersburg. AOAC International, 3000 p.
Bandarra NM, Calhau MA, Oliveira L, Ramos M, Dias MG,
Bartolo H, Faria MR, Fonseca MC, Gonçalves J, Batista I,
Nunes ML (2004). Composição e valor nutricional dos
produtos da pesca mais consumidos em Portugal.
Publicações avulsas do IPIMAR, Vol. 11. IPIMAR,
Lisboa, 103.
Bayne B (1973). Aspects of the metabolism of Mytilus
edulis during starvation. Netherl. J Sea Res, 7, 399-410.
Bergé J-P and Barnathan G (2005). Fatty Acids from Lipids
RPCV (2012) 107 (581-582) 75-84
of Marine Organisms: Molecular Biodiversity, Roles as
Biomarkers, Biologically Active Compounds, and
Economical Aspects. Adv Biochem Engin/Biotechnol, 96,
49-125
Biandolino F, Prato E, Caroppo C (2008). Preliminary
investigation on the phytoplankton contribution to the
mussel diet on the basis of fatty acids analysis. JMBA, 88,
1009-1017.
Budge SM and Parrish CC (1998). Lipid biogeochemistry of
plankton, settling matter and sediments in Trinity Bay,
Newfoundland. II. Fatty acids. Org Geochem, 29, 15471559.
Budge SM, Parrish CC, McKenzie CH (2001). Fatty acid
composition of phytoplankton, settling particulate matter
and sediments at a sheltered bivalve aquaculture site. Mar
Chem, 76, 285-303.
Burdge GC, Jones AE, Wootton SA (2002). Eicosapentaenoic and docosapentaenoic acids are the principal
products of alpha-linolenic acid metabolism in young
men. British J Nutr, 88, 355-363.
Burdge GC and Wootton SA (2002). Conversion of
a-linolenic acid to eicosapentaenoic, docosapentaenoic
and docosahexaenoic acids in young women. British J
Nutr, 88, 411-420.
Caudales R and Wells JM (1992). Differentiation of free-living anabaena and nostoc cyanobacteria on the basis of
fatty-acid composition. Int J System Bacterial, 42, 246251.
Chu FL, Webb KL, Chen J (1990). Seasonal changes of
lipids and fatty acids in oyster tissues (Crassostrea
virginica) and estuarine particulate matter. Comp
Biochem Physiol Part A: Physiology, 95, 385-391.
Colombo JC, Silverberg N, Gearing JN (1997). Lipid
biogeochemistry in the Laurentian Trough. 2. Changes in
composition of fatty acids, sterols and aliphatic hydrocarbons during early diagenesis. Organic Geochem, 26, 257274.
Copeman LA and Parrish CC (2003). Marine lipids in a cold
coastal ecosystem: Gilbert Bay, Labrador Marine Biol,
143, 1213-1227.
Dalsgaard J, St John M, Kattner G, Muller-Navarra D,
Hagen W (2003). Fatty acid trophic markers in the pelagic
marine environment. Adv Marine Biol, Vol 46. Academic
Press Ltd, London, 225-340.
Darriba S, Juan F, Guerra A (2005). Energy storage and
utilization in relation to the reproductive cycle in the razor
clam Ensis arcuatus (Jeffreys, 1865). Ices J Marine Sci,
62, 886-896.
De Zwaan A and Zandee DI (1984). Body distribution and
seasonal changes in glycogen content of the common sea
mussel Mytilus edulis. Comp Biochem Physiol A, 43,
53-58.
Emelyanov A, Fedoseev G, Krasnoschekova O, Abulimity A,
Trendeleva T, Barnes PJ (2002). Treatment of asthma with
lipid extract of New Zealand green-lipped mussel: a
randomised clinical trial. Eur Respiratory J, 20, 596-600.
Fernández-Reiriz MJ, Pérez-Camacho A, Delgado M,
Labarta U (2007). Dynamics of biochemical components,
lipid classes and energy values on gonadal development
of R. philippinarum associated with the temperature and
ingestion rate. Comp. Biochem. Physiol. - Part A:
Molecular and Integrative Physiology, 147, 1053-1059.
Fernández-Reiriz MJ, Pérez Camacho A, Pérez Corbacho E,
83
Ruano F et al.
Labarta U (2004). Processos fisiológicos y disponibilidad
de alimento del mejillón en las rias gallegas. In: m. In
Bateeiros, mejillón, (Ed.), Una perspectiva bioeconómica.
CIEF - Fundación Caixa Galicia, 49-77.
Gibson SLM and Gibson RG (1998). The treatment of
arthritis with a lipid extract of Perna canaliculus: a
randomized trial. Complem Therapies Med, 6, 122-126.
Harvey HR (1994). Fatty acids and sterols as source markers
of organic matter in sediments of the North Carolina
continental slope. Deep Sea Res. Part II: Topical Studies
in Oceanography, 41, 783-796.
Harvey HR and Macko SA (1997). Kinetics of phytoplankton
decay during simulated sedimentation: changes in lipids
under oxic and anoxic conditions. Org Geochem, 27, 129140.
Henry M (1987). Glande digestive de la palourde Ruditapes
decussatus (L.). Recherches ultrastructurales, cytochimiques, écophysiologiques et écotoxicologiques. Vie Mar, 1,
1-407.
Heral M, Razet D, Maestrini S, Garnier J (1980).
Composition de la matière organique particulaire dans les
eaux du bassin de Marennes-Oléron : apport énergétique
pour la nutrition de l'huître. In: CIEM International
Council for the Exploration of the Sea, Copenhagen,
Denmark, 14.
Huynh MD and Kitts DD (2009). Evaluating nutritional
quality of pacific fish species from fatty acid signatures.
Food Chem, 114, 912-918.
Ian-Givens D and Gibbs RA (2008). Current intakes of EPA
and DHA in European populations and the potential of
animal-derived foods to increase them. Proc Nutr Soc, 67,
273-280.
Kris-Etherton PM and Harris WS (2002). Appel for the
Nutrition. Fish Consumption, Fish Oil, Omega-3 Fatty
Acids, and Cardiovascular Disease. Circulation, 106,
2747-2757.
Lane JM (1986). Allometric and biochemical studies on
starved and unstarved clams, Rangia cuneata (Sowerby,
1831). J Exp Marine Biol Ecol, 95, 131-143.
Lawrence DR and Scott GI (1982). The determination and
use of condition index of oysters. Estuaries, 5, 23-27.
Lepage G and Roy CC (1986). Direct transesterification of
all classes of lipids in a one-step reaction. J Lipid Res, 27,
114-120.
McLean CH and Bulling KR (2005). Differences in lipid
profile of New Zealand marine species over four seasons.
J Food Lipids, 12, 313-326.
Napolitano GE, Pollero RJ, Gayoso AM, MacDonald BA,
Thompson RJ (1997). Fatty acids as trophic markers of
phytoplankton blooms in the Bahia Blanca estuary
(Buenos Aires, Argentina) and in Trinity Bay (Newfoundland, Canada). Biochem System Ecol, 25, 739-755.
Nunes ML, Bandarra NM, Batista I (2003). Fish products:
Contribution for a healthy food. Electron. J Environ
Agricult Food Chem, 2, 453-457.
Official controls on products of animal origin intended for
human consumption (2004). Official Journal of the
European Union L 139 of 30 April. Regulation (EC) No
854/2004 of the European Parliament and of the Council
of 29 April 2004. http://eur-lex.europa.eu/LexUriServ
84
RPCV (2012) 107 (581-582) 75-84
Parkes RJ and Taylor J (1983). The relationship between
fatty-acid distributions and bacterial respiratory types in
contemporary marine-sediments. Estuarine Coastal Shelf
Sci, 16, 173-180.
Parrish CC, Abrajano TA, Budge SM, Helleur RJ, Hudson
ED, Pulchan K, Ramos C (2000). Lipid and phenolic biomarkers in marine ecosystems: Analysis and applications.
Marine Chem, 5, 193-223.
Passi S, Cataudella S, Di Marco P, De Simone F, Rastrelli L
(2002). Fatty Acid Composition and Antioxidant Levels in
Muscle Tissue of Different Mediterranean Marine Species
of Fish and Shellfish. J Agricult Food Chem, 50, 73147322.
Pereira da Fonseca I, Ramos P, Ruano F, Duarte A, Costa JC,
Almeida AC, Falcão ML. Fazendeiro MI (2006). Efficacy
of Commercial Cleansing Procedures in Eliminating
Cryptosporidium parvum Oocysts from Bivalves. J Euk
Microbiol, 53, S49-S51.
Ramos P, Pereira da Fonseca IM, Ruano F, Duarte A, Correia
da Costa JM, Almeida AC, Falcão ML, Fazendeiro MI
(2005). Avaliação da eficácia da depuração comercial em
várias espécies de bivalves contaminados experimentalmente com oocistos de Cryptosporidium parvum. Acta
Parasitol Portuguesa, 12, 51-52.
Reuss N and Poulsen L (2002). Evaluation of fatty acids as
biomarkers for a natural plankton community. A field
study of a spring bloom and a post-bloom period off West
Greenland. Marine Biol, 141, 423-434.
Rodríguez-Moscoso E and Arnaiz R (1998). Gametogenesis
and energy storage in a population of the grooved carpetshell clam, Tapes decussatus (Linné, 1787), in northwest
Spain. Aquaculture, 162, 125-139.
Ruano F, Brito BGP, Nunes MC, Carneiro A (1998). Ensaios
de certificação de um tanque experimental de depuração
de bivalves. Relat Cient Téc Inst Invest Pescas Mar, 31, 20.
Santos V, Billett DS, Rice AL, Wolff GA (1994). Organic
matter in deep-sea sediments from the Porcupine Abyssal
Plain in the north-east Atlantic Ocean. I--Lipids. Deep Sea
Res Part I: Oceanographic Research Papers, 41, 787-819.
Soriguer F, Serna S, Valverde E, Hernando J, MartinReyes
A, Soriguer M, Pareja A, Tinahones F and Esteva I (1997).
Lipid, protein, and calorie content of different Atlantic
and Mediterranean fish, shellfish, and molluscs commonly
eaten in the south of Spain. Eur J Epidemiol, 13, 451-463.
Stark D, Barratt JLN, van Hal S, Marriott D, Harkness J and
Ellis JT (2009). Clinical Significance of Enteric Protozoa
in the Immunosuppressed Human Population. Clin
Microbiol Rev, 22, 634-650.
Uauy R and Valenzuela A (2000). Marine oils: the health
benefits of n-3 fatty acids. Nutrition, 16, 680-684.
Van Gelder BM, Tijhuis M, Kalmijn S and Kromhout D
(2007). Fish consumption, n-3 fatty acids, and subsequent
5-y cognitive decline in elderly men: the Zutphen Elderly
Study. Am J Clin Nutr, 85, 1142-1147.
Vilela H (1950). Vida bentónica de Tapes decussatus (L.).
Arquivos do Museu Bocage, 21, 1-120.
Volkman J (2006). Lipid Markers for Marine Organic
Matter. Hdb Env Chem Vol. 2, Part N (2006), 27-70.
Zvi C, Avigad V and Amos R (1988). Effect of environmental conditions on fatty acid composition of the red alga
Porphyridium cruentum: correlation to growth rate. J
Phycology, 24, 328-332.