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Histological effects of a combination of heavy

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Article in Aquatic Toxicology October 2008
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Aquatic Toxicology 89 (2008) 152157

Contents lists available at ScienceDirect

Aquatic Toxicology
journal homepage: www.elsevier.com/locate/aquatox

Histological effects of a combination of heavy metals on Pacic white shrimp

Litopenaeus vannamei juveniles
Martin G. Fras-Espericueta a , Selene Abad-Rosales b , Aide C. Nevrez-Velzquez a , Isidro Osuna-Lpez a ,
Federico Pez-Osuna c , Rodolfo Lozano-Olvera b , Domenico Voltolina d,
a

Laboratorio de Estudios Ambientales, Facultad de Ciencias del Mar, Universidad Autnoma de Sinaloa, Paseo Claussen s/n, Mazatln, Sinaloa, Mexico
Centro de Investigacin en Alimentacin y Desarrollo, Unidad Mazatln en Acuicultura y Manejo Ambiental, Sbalo Cerritos s/n, Mazatln, Sinaloa, Mexico
Universidad Nacional Autnoma de Mxico, Instituto de Ciencias del Mar y Limnologa, Unidad Acadmica Mazatln, Mazatln, Sinaloa, Mexico
d
Centro de Investigaciones Biolgicas del Noroeste, Laboratorio de Estudios Ambientales UAS-CIBNOR, P.O. Box 1132, 82000 Mazatln, Sinaloa, Mexico
b
c

a r t i c l e

i n f o

Article history:
Received 25 February 2008
Received in revised form 23 May 2008
Accepted 19 June 2008
Keywords:
Histological effects
Metals
Gills
Epipodites
Hepatopancreas

a b s t r a c t
Exposure to different levels of a mixture in equitoxic concentrations of Cd, Cu, Fe, Hg, Mn, Pb and Zn, equivalent to between 5 and 0.5% the individual respective 96-h median lethal concentrations (0.050.005 AF:
application factors) caused dose- and time-dependent damages to the hepatopancreas, gills, epipodites
and midgut tissues of Litopenaeus vannamei juveniles. After between 1 and 34 weeks, there was between
50 and 100% loss of the hepatopancreas R cells and of the tubules regular structure. Gill alterations were
observed between 2 and 3 weeks with 0.050.025 and 0.005 AF, respectively. Epipodites showed timeand dose-dependent increasing degrees of melanization, and hemocytic enteritis was observed with 0.025
and 0.01 AF. Exposure to 0.05 and 0.025 AF caused also 50 and 12% mortalities after 2 and 3 weeks respectively, showing that even at these low levels the mixture of these metals may have a mid-term lethal
effect. For this reason, the assessment of risk and of safe levels of toxic substances added to any natural environment through human or natural sources, should not neglect the effects on biological systems
caused by the interaction of minute amounts of toxicants, which would be harmless unless present in
combination.
2008 Elsevier B.V. All rights reserved.

1. Introduction
In spite of the national policy directed to safeguard the environment and to protect public health, there is increasing evidence
that the levels of heavy metals are increasing throughout northern Mexico (Garca-Rico et al., 2004). In particular, the coastal
water bodies of the Gulf of California (NW coast of Mexico)
receive industrial and municipal efuents, as well as those of
the >2.30 106 ha dedicated to intensive agriculture with high
use of agrochemicals (INEGI, 1994), which are the main factor of
deterioration of the coastal lagoons of NW Mexico (Carvalho et
al., 1996). These support important traditional sheries (between
17 and 20% of the total catches landed along the Pacic coast:
Espino-Barr and Cruz-Romero, 2006) as well as more than 90%
of the national shrimp farming developments which, according to 2003 ofcial gures (SAGARPA, 2004), occupied more
than 60 103 ha of ponds, concentrated in the coastal states of

Corresponding author. Tel.: +52 669 981 0377; fax: +52 669 981 4643.
E-mail addresses: voltolin04@cibnor.mx, voltolina@mzt.megared.net.mx
(D. Voltolina).
0166-445X/$ see front matter 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquatox.2008.06.010

Sinaloa (41,557 ha = 69.3% of the total pond surface) and Sonora

(13,941 ha = 23.2%).
Materials introduced into the environment come from anthropogenic and natural sources and in most situations they are
complex mixtures, which may cause environmental damage
(Barata et al., 2006). The presence of metals in the marine/estuarine
aquatic environment is of ecotoxicological concern, because they
may alter some physiological processes of the organisms, such as
osmoregulation, respiration and growth.
The concentrations of chemicals that do not affect the structure and function of natural ecological systems (safe level) are
generally calculated from the known median lethal concentration
(usually 96-h LC50) of single compounds, multiplied by empiirical application factors (AF, usually 0.1 and 0.01 for non-persistent
and persistent toxicants, respectively: Environment Canada, 1999;
Castillo, 2004), and the resulting low concentrations are supposed
to be harmless when only the chosen toxicant is present in the
environment.
However, mixtures of metals at concentrations well below their
individual sublethal values may affect the growth and reproduction
of aquatic organisms (Enserink et al., 1991; Verslycke et al., 2003)
and in particular there is little information on the effects of metal

M.G. Fras-Espericueta et al. / Aquatic Toxicology 89 (2008) 152157

mixtures for the white shrimp Litopenaeus vannamei, which is the

most important species for Mexican aquaculture.
The aim of this work was to investigate if theoretically safe levels of a mixture of seven heavy metals might cause histological
and structural damages to the gills, epipodites, hepatopancreas and
midgut of white shrimp L. vannamei juveniles.

2. Materials and methods

L. vannamei juveniles (56.5 cm) were obtained from a commercial shrimp farm and acclimated during 3 days to the experimental
conditions in a common container, after which 20 shrimp obtained
at random from the common holding tank were placed in each of
ve 120 l plastic containers and maintained for three additional
days under the same conditions.
During this stage and for the rest of the experiment the organisms were kept with a 12:12 h light:dark photoperiod and fed
pelleted shrimp food twice daily. Excess food and faeces were
removed during the 100% daily water exchanges with 1-m ltered
seawater treated with activated charcoal during the acclimation
period, and with the respective test solutions thereafter.
The metals chosen for the experiment were Cd, Cu, Fe, Mn,
Pb and Zn, because their presence in the edible tissues of shrimp
and sh caught in commercial shery along the Northwest coastal
zone of Mexico has been previously reported by Pez-Osuna and
Fras-Espericueta (2001) and Fras-Espericueta et al. (2005). Their
background concentrations, determined with an atomic absorption
spectrophotometer (Varian, Spectra AA), were 0.32, 2.5, 0.7, 1.05,
1.06 and 5.5 g/l, for Cd, Cu, Fe, Mn, Pb and Zn, respectively.
Stock solutions of Cd, Cu, Fe, Hg, Mn, Pb and Zn in concentrations
equivalent to the respective 96-h LC50 values (Fras-Espericueta
et al., 2001, 2003) were prepared with the water-soluble salts
CdCl2 , CuCl2 , FeCl3 6H2 O, HgCl2 , MnCl2 4H2 O, PbCl2 and ZnCl dissolved in Milli-Q deionized water. The experimental mixtures were
obtained adding to the water of the shrimp containers the appropriate volume of each stock solution, in order to obtain equitoxic
concentrations equivalent to progressive dilutions, from 5 to 0.5% of
each individual 96-h LC50 value (0.050.005 AF) (Table 1). Throughout the experiment, the mean values of salinity, DO, pH and total
ammonia were 35.4 0.2 ppt, 6.5 0.3 mg/l, 8.1 0.1 and 5 1 g/l,
respectively.
The experiment lasted 28 days; every week, four organisms of
each AF were injected in the hepatopancreas and in the anterior and
posterior abdominal regions with Davidsons solution and left in the
same solution for 24. Longitudinal body sections were dehydrated
in 70, 80, 96 and 100% ethanol, cleared with xylene, embedded

153

Table 1
Median lethal (96-h LC50) and nominal concentration of each metal for each application factors (AF)
Metal

96-h LC50

Cd
Cu
Fe
Hg
Mn
Pb
Zn

2,490
37,300
44,200
1,230
130,000
134,000
2,080

AF
0.005

0.01

0.025

0.05

12.45
186.5
221
6.15
650
670
10.4

24.9
373
442
12.3
1300
1340
20.8

62.25
932.5
1105
30.75
3250
3350
52

124.5
1865
2210
61.5
6500
6700
104

All values are in g/l.

in parafn and 5-m thin sections were obtained with a conventional microtome. Each sample was re-hydrated, stained with
hematoxylin-eosin (Odendaal and Reinecke, 2007) and observed
under a microscope.
Depending on the size of the tissues, the number of elds
observed in the slides prepared with the samples obtained from
each organisms varied from 60, 31, 30 and 6 for hepatopancreas,
gills, midgut and epipodites, respectively (240, 124, 120 and 24
elds observed every week for each treatment), in each case covering the whole longitudinal section of each tissue.
Following the scale suggested by Zodrow et al. (2004), the degree
of histological damage observed each week in each treatment was
scored according to the percentage of the total elds with histological damage found out of the total observed in the four samples
of each treatment (severe, moderate, mild and none = 100%; >75%,
<25% and 0% of the elds with histological damage, compared to
the control sections).
3. Results
Survival was 100% in the controls and with 0.01 and 0.005 AF,
the respective percentages of survival were 50 and 88% with 0.025
and 0.05 AF after the second and third week, but the respective
mortalities were 100% between weeks 2 and 3 (0.025 AF) and 3 and
4 (0.05 AF).
The results of the histological observations are summarized in
Table 2, which shows that, even with the lowest AF, moderate alterations were observed in gills and hepatopancreas between weeks 2
and 3. The hepatopancreas of the organisms exposed to the highest
AF showed acute damage by the end of the rst week and there was
an evident time, as well as dose related trend to increasing degrees
of alteration in most if not in all tissues.

Table 2
Histological damages observed in the hepatopancreas (HP), gills (GL), epipodites (EP) and midgut (MG) of Litopenaeus vannamei after exposure to the metal mixture with
different application factors (AF)
Tissue

Pathology

AF
0.005

HP

Reduction of R cells
Inltration of hemocytes
Sloughing of epithelial cells
Reduction of B cells

0.01

0.025

0.05

W1

W2

W3

W4

W1

W2

W3

W4

W1

W2

W3

W4

W1

W2

W3

W4

++

++
+
+

++
+
+
+

++
+
+
++

+++
+
+
++

+
+
+
+

++
++
+
++

+++
++
+
++

NS
NS
NS
NS

+++
++
+
++

+++
++
++
++

NS
NS
NS
NS

NS
NS
NS
NS

GL

Abnormal dilation of the lacunae

++

++

+++

++

++

NS

+++

NS

NS

EP

Inltration of hemocytes
Focal melanization

++

++

+++

++
++

++

+
++

NS
NS

NS
NS

NS
NS

MG

Hemocytic enteritis

+++

+++

NS

NS

NS

() no histopathology; (+) histopathology in <25% of elds; (++) histopathology in >75% of elds; (+++) histopathology in all elds. NS: sample not available due to 100%
mortality.

154

M.G. Fras-Espericueta et al. / Aquatic Toxicology 89 (2008) 152157

Fig. 1. (a) Hepatopancreas tissue of control shrimp after 4 weeks. Scale bar: 20 m. (b) Reduction of stored lipid vacuoles and modication of regular structure (*) in
hepatopancreas R cells after 1 week exposure to 0.05 AF. Scale bar: 20 m. (c) Absence of vacuoles of B and R cells and sloughing of epithelial cells (arrow), causing
modication of the normal structure of the hepatopancreas after 3 weeks exposure to 0.025 AF. Scale bar: 20 m. (d) Necrotic cells (arrow), sloughing of epithelial cells and
vacuole reduction of B cells in the hepatopancreas of shrimp exposed during 4 weeks to 0.01 AF. Scale bar: 20 m.

After 1 week, the hepatopancreas of all shrimp exposed to

0.05 AF showed a total reduction of R cells, inltration of hemocytes
and sloughing of epithelial cells causing loss of regular structure. In
addition, reduction in the secretory vacuoles was observed in the
B cells (Fig. 1a and b).
With 0.025 AF, 75% of the organisms showed mild reduction of
R cells and of secretory vacuoles in B cells, as well as inltration
of hemocytes after the rst week. The degree of damage increased
gradually, and atrophy of R cells and loss of the tubules regular
structure were observed after the second and third week (Table 2,
Fig. 1c).
After the rst week, the hepatopancreas of shrimp exposed to
0.01 AF showed inltration of hemocytes. By the second there was
reduction of B and R cells, loss of regular structure and sloughing of
epithelial cells and by the third all organisms showed separation
between myoepithelium and epithelium. After the fourth week,
75% of the shrimp showed moderate and severe losses of B and
R cells, 25% had inltration of hemocytes and presence of necrotic
cells, as well as atrophy and sloughing of epithelial cells (Fig. 1d).
After 3 weeks in 0.005 AF, 25% of the hepatopancreas samples
showed moderate reduction of R cells, as well as mild tubular atrophy and sloughing of epithelial cells; by the fourth week, 25% of the
shrimp showed low inltration of hemocytes.
By week 2, the gills of all organisms exposed to 0.05 and 0.025 AF
showed moderate to severe alterations of their regular structure;
with 0.01 AF there was also an abnormal dilation of the lacunae of
gill lamellae (Fig. 2b). Between the second and third week, the same
effect was observed with 0.005 AF.
Starting on the rst week, the epipodites of all shrimp in
0.05 AF showed inltration of hemocytes, and focal melanization
was observed in those kept at 0.025 AF. By the third week, 50% of
the organisms showed moderate focal and multifocal melanization.

Fig. 2. (a) Gill lamellae of control shrimp after 4 weeks. Scale bar: 20 m. (b) Abnormal dilation of the lacunae of the secondary laments of gill lamellae and loss
of regular structure of the columnar epithelium (arrow) of shrimp after 1 week
exposure to 0.01 AF. Scale bar: 20 m.

M.G. Fras-Espericueta et al. / Aquatic Toxicology 89 (2008) 152157

Fig. 3. (a) Epipodite of control shrimp after 3 weeks. Scale bar: 20 m. (b) Hemocytic inammation and melanized layers of hemocytes in the cuticular epithelium
epipodite (arrow) after 4 weeks exposure to 0.01 AF. Scale bar: 20 m.

No alterations were observed after the rst week in 0.01 AF, but after
the second 50% showed moderate multifocal melanization (Fig. 3b),
which became severe by the end of experiment (Table 2).
With 0.005 AF, no effects were noted in the epipodites during
the rst and second weeks, but moderate local melanization was
observed by the end of the third week. No additional histological
damage was observed after the fourth week.
In the midgut, severe hemocytic enteritis (multiple layers of
hemocytes in the mucose surface) was observed in all shrimp
exposed for 3 weeks to 0.025 AF. After the same time of exposure, this effect was observed with lower severity with 0.01 AF
(Fig. 4b), but it became severe the following week. This pathology affected also the anterior and posterior midgut caecum, with
necrotic mucosal epithelia replaced by multiple layers of hemocytes, some of them melanized between the folds of the caecum.
4. Discussion
There is evidence that survival of L. vannamei postlarvae may be
as high as 80100% after 96 h exposure to single metal solutions
in concentrations close or lower than 50% the acute toxic level, and
between two and three orders of magnitude higher than those used
in the present experiment (survival = 100% with 12 mg/l Cu, 30 mg/l
Fe, 0.5 mg/l Hg, and 1 mg/l Zn, 20% mortality with 1 mg/l Cd and 10%
with 60 mg/l Pb: Fras-Espericueta et al., 2001, 2003).
Therefore, the mortalities caused by the mixture of metals in
individual concentrations equivalent to 5 and 2.5% the acute toxic
level conrm their synergistic effect, which may affect the survival
of aquatic organisms even at these low levels, as was shown with
the nematode Caenorhabditis elegans by Chu and Chow (2002) and
with the crustacean Neomysis integer by Verslycke et al. (2003).

155

Fig. 4. (a) Control organism after 4 weeks: lumen of midgut. Scale bar: 20 m. (b)
Shrimp exposed during 3 weeks to 0.025 AF: hemocytic inltration of the mucose
epithelium in the lumen of midgut (*). Arrow: remnants of pyknotic hemocytes close
to narrow lumen with circulating hemocytes. Scale bar: 20 m.

Exposure to heavy metals may cause cytological, cytochemical and ultrastructural alterations to the hepatopancreas of
crustaceans, which has been identied as a target organ in ecotoxicological research because of its important role in detoxication
and excretion of xenobiotic compounds.
In particular, Manisseri and Menon (1995), Correia et al. (2002),
Yang et al. (2007) and Li et al. (2007) described Cu-induced histological damages to the hepatopancreas of the shrimp Metapenaeus
dobsoni, the amphipod Gammarus locusta, the crab Eriocheir sinensis and the prawn Macrobrachium rosenbergii, respectively; while
Zn induced effects on the histological structure of the isopod Porcellio laevis (Odendaal and Reinecke, 2007).In addition, Khler et al.
(1996) pointed out that Cd, Pb and Zn altered the hepatopancreas
structure in the isopod Porcellio scaber, and Cd has been related
to an unbalanced energy budget as well as to impaired nitrogen
metabolism and excretion of the mysid Leptomysis lingvura (Gaudy
et al., 1991). This might affect several vital functions, because the
hepatopancreas plays a primary role in the metabolism and energy
budget, which could be one of the factors affecting our experimental organism.
Gills are the rst target of waterborne pollutants due to their
constant contact with the external environment, and several
authors have described gill lesions consequent to metal exposure in
different aquatic organisms, such as sh (van Heerden et al., 2004)
and molluscs (Rodrguez de la Rua et al., 2005).
In decapods, histological alterations of gill tissues similar to
those of this study have been reported for higher concentrations
of single metals (Cd, Cu, Pb and Zn) in Penaeus (Melicertus) duorarum (Couch, 1977), Marsupenaeus japonicus (Soegianto et al.,
1999a,b), Caridina sp. (Ghate and Mulherkar, 1979), Procambarus
clarkii (Torreblanca et al., 1989), Macrobrachium hendersodyanum

156

M.G. Fras-Espericueta et al. / Aquatic Toxicology 89 (2008) 152157

(Patil and Kaliwal, 1989), Carcinus maenas (Nonnotte et al., 1993),

E. sinensis (Yang et al., 2007) and M. rosenbergii (Li et al., 2007).
These pathologic alterations in structure might impair gill functions, resulting in hypoxia (Li et al., 2007), which could explain the
deaths observed in L. vannamei exposed to 0.05 and 0.025 AF.
The epipodites of L. vannamei were more susceptible to the mixture of metals than gills. This coincides with the results by Soegianto
et al. (1999a), who observed loss of regular structure and necrosis
in the epipodites of M. japonicus after exposure to 0.24 mg/l Cd,
which seemed to be in direct relation to the external metal concentration and to the time of exposure, while the epipodites of shrimp
exposed to 0.011 mg/l Cu were completely altered (Soegianto et
al., 1999a,b). In our study, the epipodites of L. vannamei showed
melanized areas, which could have caused disruption of physiological processes, because epipodites have an important function in
osmoregulation.
Hemocytic enteritis or other histological damages to the gastrointestinal tract may be caused by some algae related to red tides
and some forms of Gram-negative bacteria (Lightner, 1996) or by
pollutants (TCDD), as shown by Spitsbergen et al. (1988) with the
rainbow trout (Salmo gairdneri). Zodrow et al. (2004) pointed out
that midgut alterations in different organisms could be observed
after long-term exposure to pollutants. The presence of Hg in the
midgut of the shrimp Crangon crangon have been reported by
Andersen and Baatrup (1988), and Andres et al. (2002) have shown
that uxes of waterborne Hg from the tissue to the internal medium
were slightly higher in the intestine than in the gill. In this study,
hemocytic enteritis was observed only after long-term exposure to
0.01 and 0.025 AF, showing that time course studies should be used
to elucidate the responses to the presence of waterborne metals in
L. vannamei.
Because of the greater allocation of energy to protect cells and
tissues from the accumulation and toxic effect of pollutants, the
presence of metals in the environment may result in increased susceptibility to any stress agent, including diseases (Liao et al., 2006).
In Mexico this might cause serious economic problems especially
for shrimp aquaculture, because most shrimp farms and hatcheries
are located along the NW Mexican coast, which receives considerable quantities of metals from several human as well as natural
sources (Fras-Espericueta et al., 2008).
In agreement with Chapman et al. (1996) and Crane and
Newman (2000), our results indicate that risk assessments and/or
safe levels of toxic substances in the aquatic environment should
not neglect their interactions, since the combination of minute
amounts of each toxicant may generate a far more severe impact
than that expected from their individual effect and that therefore
regulations must be based on reliable studies indicating the concentrations with no effect on reproduction, growth or on any other
physiological process.
Acknowledgments
Work supported by CECYT 2003 Sinaloa and PROFAPI-UAS. H.
Bojrquez-Leyva gave technical assistance.
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