9ο Πανελλήνιο Συμπόσιο Ωκεανογραφίας & Αλιείας 2009 - Πρακτικά, Τόμος ΙΙ
CELLULAR RESPONSES OF THE FARMED SEA BASS
Dicentrarchus labrax TO COPPER OXIDE ANTIFOULANTS
Cotou E., Henry M., Rigos G., Alexis M.
Institute of Aquaculture, Hellenic Centre for Marine Research, ecotou@ath.hcmr.gr, morgane@ath.hcmr.gr,
grigos@ath.hcmr.gr, malexi@ath.hcmr.gr
Abstract
The main objective of this study was to assess if the levels of the copper released from the antifouling painted nets used nowadays in the fish farming may exert oxidative stress on the farmed sea bass Dicentrarchus labrax. We estimated the cellular
responses of D. labrax after exposure of seven days to antifouling painted net by multiple biomarkers including lipid peroxidation (measured as TBARS), glutathione S tranferase activity (GST), respiratory burst (measured as chemiliminenscent
response) and acetylcholinesterase activity (AChE). The concentration levels of released copper from the antifouling painted
net were detected by atomic absorption spectroscopy (AAS). The measured copper concentrations in the water samples
released from the antifouling painted net were not adequate to produced significant oxidative stress since all the biomarkers used shown no significant responses. In order to understand the lack of significant responses at sublethal concentrations
further studies must be performed on the oxidative defense mechanisms and the accumulation processes.
Keywords: TBARS, GST, AChE, chemiluminenscene, oxidative stress.
1. Introduction
The sea bass D. labrax is one of the most important Mediterranean cultured fish species.
Antifoulants are paints used to avoid the biofouling process on the structures and cage nets in fish
farming industry. As they are not directly used on food-producing fish, they do not fall under the
maximum residue limit (MRL) system. However, where they are used, fish are exposed to antifoulants
for months. Antifoulants used in the past were based on heavy metals such as tin and chrome which
were found to have high toxic effects on organisms (Michel & Averty, 1999). Nowadays the 99%
of these paints are formulated with biocides including copper and they are sold under many trade
names. Copper is their main active ingredient which is used as cuprous oxide (Cu2O). Copper as
an essential trace metal for living organisms can become toxic when elevated concentrations are
introduced into the environment (Marr et al., 1996; Karan et al., 1998). Thus, it is listed under the
EU Dangerous Substances legislation, and its release into natural environment may be controlled
under discharge limits. As a trace metal it plays an important role in cellular metabolism acting as
co-factor in a number of important enzymes. Moreover, at the cellular level, copper can interfere
with several metabolic pathways and thereby can induce different cellular responses.
Copper has been well described as a promoter of oxidative stress by catalyzing the formation of
highly reactive oxygen species (ROS), such as HO- radical through the Haber-Weiss reaction (Matés,
2000) and generating peroxidation of membranes lipids (Chan et al., 1982) and DNA alternations
(Ozawa et al., 1993). Lipid peroxidation (LP) is a very important consequence of oxidative stress.
Oxidative stress is an imbalance between the production of ROS and the cells’ ability to reduce
ROS, detoxify reactive intermediates, and/or repair damage that may occur in cellular molecules.
Detoxification processes occur by a superfamily of soluble enzymes, the glutathione S-transferase
(GST) (van der Oost et al., 2003), GST also functions in cellular transport (van der Oost et al.,
2003) and defense against oxidative damage and peroxidative products of DNA and lipids (George,
1994). Moreover, there is evidence that copper affects phagocyte function in a complex manner,
stimulating or inhibiting production of ROS depending on copper concentration (Muhvich et al.,
1995). Phagocytosis is the most common reaction of cellular defense, and is generally recognized
as a central and important way to eliminate microorganisms or foreign particles (Bachere et al.,
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1995). When phagocytes are activated they produce ROS in a process called ‘respiratory burst’.
Acetylcholinesterase (AChE) is a crucial enzyme in the nervous system of both vertebrates and
invertebrates, where it is responsible for the degradation of the neurotransmitter acetylcholine in the
synaptic cleft. AChE activity is mainly inhibited by xenobiotics like organophosphate and carbamate
pesticides (Varo et al., 2002) but recent evidences also suggest AChE activity inhibition by metals
including copper (Frasco et al., 2005). The aim of the present study was to assess if the levels of
the copper released from the antifouling painted nets can exert oxidative stress on the farmed fish
D. labrax.
2. Materials and Methods
2.1. Chemicals
Painted nets with the antifoulant Flexgard™ were purchased from IchthioalieftikI S.A.
All other chemicals were obtained from Sigma Chemical Co.
2.2. Experimental design
Sea bass D. labrax (average weight of 160 g) were purchased from a fish farm (Selonta) and
transferred to the facilities of Nutrition and Fish Pathology Laboratory at the Hellenic Centre for
Marine Research, in Athens where they were acclimated for 2 weeks prior to initiation of the study.
During the experiment three cylindroconical fiberglass tanks of 250 l were receiving seawater in
an open circuit with a salinity of 38%o at a flow rate of 70 l/h. Oxygen content was kept close to
saturation levels, photoperiod followed a 12 h dark – 12 h light cycle and water temperature was at
ambient levels (22-25 oC). At day zero, twenty four fishes were placed in two of the tanks (12 fishes
per tank), while a piece of net (1m2, with mesh size 10mm) painted with the antifoulant Flexgard
was placed in the third tank without fishes. For the first 96 hours (4 days) samples of seawater were
collected from the third tank in order to measure the amount of Cu released in the seawater from the
painted net. After the 96 hours the painted net was placed in one of the tanks with the fishes, while
in the other an unpainted net of similar size was placed and was used as control. For the next six
days fishes were exposed to the nets (painted and unpainted) without any changes of the physical
parameters (temperature, oxygen, photoperiod, flow rate). Fishes were not fed during the exposure
period. Samples of seawater were collected on days fifth and eleventh for Cu measurements. At the
end of the experiment all fishes were placed in ice-cold water.
2.3. Samples and analyses
As soon as fishes were calm, blood samples were collected for the respiratory burst assay which
was monitored by measuring the chemiluminescence emission (CL). Subsequently, the whole livers
were rapidly dissected from each fish, frozen in liquid nitrogen and kept at -80 oC till the glutathione
S transferase (GST) and the lipid peroxidation products quantified as thiobarbituric acid reactive
substances (TBARS) were estimated. Also, samples of muscles were sliced and stored at -20 oC
for analysis of acetylcholinesterase (AChE). Chemiluminenscence (CL) was performed according
to Marnila et al. (1995) in the presence of zymosan and luminol (5-amino-2.3-dihydro-1,4phthalazinedione) to amplify the CL response. The samples of livers and muscles were homogenized
in a glass-teflon Potter-Elvehjem homogenizer. Their extracts were centrifuged at 10.000 g for 20
min and their supernatants were collected for the enzymatic determinations. For TBARS detection
samples of liver were homogenized in an ultra turrax homogenizer. Homogenization buffer for GST
was containing 0.1 M phosphate buffer, pH 7.4, 0.15 M KCl, 1mM DTT, 1mM EDTA, 0.1 mM PMSF
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9ο Πανελλήνιο Συμπόσιο Ωκεανογραφίας & Αλιείας 2009 - Πρακτικά, Τόμος ΙΙ
and 20% glyserin. Analysis was performed according to Habig et al. (1974) adapted for microtiter
plate as described by Frasco & Guilhermino (2002). Homogenization buffer for AChE was Tris/
HCl (0.1 M, pH 7). Analysis of AChE was performed according to method of Ellman et al. (1961)
as modified by Galgani and Bocquene (1991) adapted for microplate assay. For TBARS detection,
0.5 g of liver tissue of each fish was homogenized and measured according to method of Hu et al.
(1989). Protein determination was made according to Bradford (1976). Copper concentration levels
in the seawater samples were detected by atomic absorption spectroscopy (AAS) using a Varian
spectrophotometer.
2.4. Statistical analysis
Independent samples t tests were used to check differences among the means of treatments. All
statistical analyses were carried out using SPSS 11.0 for Windows software. A significant level of
0.05 was used. Results are presented as % mean.
3. Results
Copper concentration levels in the seawater samples are shown in Figure 1. There was an abrupt
increase of copper levels up to 315 ppb the first 24 hours. Then, till 96 hours, the concentration levels
were reduced to 200 ppb, while afterwards copper levels were maintained at constant concentration
(185 ppb) till the end of the experiment.
Copper (ug/l)
400
96
200
0
0
40
80
120
160
200
240
280
Time (hours)
Fig. 1: Copper concentration levels released from copper oxide antifouling painted net during a period of eleven days.
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100,6
100
100,2
GST activity
(%)
AChE activity
(%)
80
60
40
20
0
99,4
99,0
Control
Antifoulant
Control
100
Antifoulant
105
90
TBARS (%)
% Chemiluminescence
99,8
80
70
100
95
Control
Control
Antifoulant
1
Antifoulant
Fig. 2: Multiple biomarkers responses of D. labrax exposed to copper oxide antifouling painted net for seven days.
As indicated in Figure 2, the 5,68% induction of TBARS observed in the liver of the fishes
exposed to antifouling painted net was not significantly different to TBARS of fishes exposed to the
unpainted net (control). Likewise, the respiratory burst activity (chemiluminescence) reduction of
14% was also not significant. Besides, an induction of 0.23 % GST activity and a reduction of 13%
of AChE activity did not show any significant difference between the two groups as well.
4. Discussion
During the whole experimental study excessive concentration levels of copper were released
from the antifouling painted net with the highest concentrations observed during a period of three
days. This justifies the decision for the recommended practice by the suppliers to fish farmers to
maintain the antifouling paint nets in the sea (without fishes) for 72 hours before they use them in
their fish farms. Yet, copper concentrations released were prolonged till the end of the experimental
study (11 days) at levels much higher above the levels of 1 ug/l recommended by the EU Directive
for the implementation of water quality criteria.
However, the measured copper concentration levels in the water samples indicated no ability
to produce significant oxidative stress since the biomarkers used to evaluate the cellular responses
between the exposed fishes to painted and unpainted nets showed no significant responses. Other
authors have also reported lack of significant cellular responses. Varo et al (2007) indicated absence
of significant responses for GST and AChE activities when they exposed Sparus aurata fingerlings
to sublethal concentrations (250-500-1500 ppb) of copper sulphate for 24 hours. They assumed,
however, that the absence of significance was due to relatively short time of exposure. However,
this assumption could not be applied to our results as D. labrax was exposed to copper levels for
relatively longer period (7 days). Dautremepuits et al. (2004) found no effect in GST activity in the
head kidney and liver of carps exposed to 250 ppb of copper regardless the length of exposure (4 or 8
days). Another reason may be attributed to accumulation of copper in the liver and muscles. Sanchez
et al. (2005) working with three-spined stickleback, showed that copper induced oxidative stress in
fish liver before significant metal accumulation could be detected and suggested the existence of
homeostatic regulation mechanism. Peyghan et al. (2003) when exposed the common carp Cyprinus
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9ο Πανελλήνιο Συμπόσιο Ωκεανογραφίας & Αλιείας 2009 - Πρακτικά, Τόμος ΙΙ
carpio to copper in short and long- term experiments, showed accumulation in liver and muscle after
only one day of exposure to 1 mg/l. Accumulation in liver and muscle may follow osmoregulatory
imbalance (Hansen et al., 2006). Copper have been shown to have an in vivo inhibitory toxic effect
on the production of reactive oxygen substances (luminal-dependent chemiluminescence) from
the anterior kidney of D. labrax (Bennani et al., 1996). AChE inhibition has been correlated with
peroxidation in a number of studies (Yang & Dettbarn, 1996; Yang et al., 1996; Uner et al., 2006).
Moreover, Song et al. (2006) have described AChE deactivation in carp brain as a result of oxidative
damage caused by hexachlorobenzene exposure. Under these circumstances, it seems to substantiate
the hypothesis of a rather incipient degree of oxidative stress and that the lack of significant responses
may be related to a balance between the production of reactive oxygen species (ROS) and the cells’
ability to reduce ROS and detoxify reactive intermediates. Further studies on the oxidative defense
mechanisms and accumulation processes should be undertaken to understand the lack of significant
cellular responses at sublethal concentrations of metals.
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