Vol.2, No.7, 748-759 (2010) Natural Science
http://dx.doi.org/10.4236/ns.2010.27094
Environmental stress upon hepatopancreatic cells
of freshwater prawns (Decapoda: Caridea)
from the floodplain of Paraná River
Pablo Collins
Instituto Nacional de Limnología (CONICET-UNL) Ciudad Universitaria, Pje El Pozo s/n, 3000 Santa Fe Santa Fe Argentina TE
0054-0342-4511645 int 113, Fac. de Bioq. y Cs. Biol. UNL, Fac. de Tecnología y Ciencias, UADER;
Received 10 March 2010; revised 11 April 2010; accepted 17 April 2010.
ABSTRACT
In order to evaluate the influence of stressed
environments on hepatopancreatic cells of fre-
shwater prawns, Macrobachium borellii Nobili,
1896 and Palaemonetes argentinus Nobili 1901,
(Crustacea, Decapoda, Palaemonidae) were
collected at three different aquatic environ-
ments with different relationship to urban de-
velopment in Argentina. Furthermore the effects
of several cypermethryn concentration on hepa-
topancreatic cell of M. borellii and P. argentinus
were evaluated in a laboratory assays. The “N°
1” lake (Santa Fe Argentina) which is more af-
fected by the anthropogenic pressure and “Don
Felipe” lake which is still not strongly by human
activities were the studied sites from the flood-
plain of Paraná river. While Alejandra lake was
the intermedia effects sites. Different damaged
ultrastructures were found in F- and R-cells of
prawns in the stressed lake. The predominant
features were: disrupted the microvillous border,
swelled mitochondria, reduction of endoplasmic
reticulum, dyctiosomes, glycoproteins, desna-
turalization of vacuole membrane and prema-
ture autolysis. Moreover the F-cell number was
higher in the environment near to city than in
the others sites. Similar effects were observed
in the cypermethryn assays. The observations
clearly indicate that the ultrastructure of midgut
gland in the both palaemonids varies depending
on the site from which animals are collected and
the biocid presence. So, in this case it can be
stated that the hepatopancreas histology of fre-
shwater prawns is a good tool to monitor the
impact of a stressed environment upon fresh-
water prawns.
Keywords: Palaemonidae; Midgut Gland; Shrimp;
Hepatopancreas; Environmental Stress
1. INTRODUCTION
Freshwater prawns are an important and abundant group
in the floodplain of the Paraná River, mainly the species
Macrobrachium borellii Nobili, 1896 and Palaemonetes
argentinus Nobili, 1901. Both prawns have as habit the
lotic and lentic environment from La Pampa region, Ar-
gentina [1]. This area is characterized by intensive farm-
ing, and urban-industries development. Thus the aquatic
environment gathers several elements which affect the
quality of the rivers and lakes, mainly at the rain events
[2]. The nutrient increase, toxins presence and others
xenobiotic input, such as biocid and heavy metals, pro-
voke changes in the normal conditions that according to
inflow regime (sewage of various degree of treatment,
thermal regime, weather conditions and land use) affects
the quality conditions of the receptors [3,4].
In the last decades there have been many studies about
the influences of exogenous factors upon crustaceans,
being the majority of these researches made as labora-
tory tests. Such works revealed important basic data, and
it can be refered to in case of evaluating the environ-
mental influences upon hepatopancreas. There exists a
close correlation with different kind of stresses and the
ultrastructure of hepatopancreas [5-17]. Some products
and/or household waste, such as industrial discharges,
pesticides or sewage are transformed and degraded
through various reactions with other biotic and abiotic
components, when they get into the river. These ele-
ments may alter the hepatopancreatic cells and it could
be used as biological markers so as to assess the toxic
properties of the environmental contaminants [18-20].
The aim of the present work is to compare the ultra-
structure of the hepatopancreatic cells of M. borellii and

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P. argentinus freshwater prawns at three natural enviro-
nments with different quality water, in relationship with
the biocid use, human activities or land use.
2. MATERIALS AND METHODS
2.1. Sampling Prawns
Juveniles, males and females of M. borellii and P. argen-
tinus (Crustacea, Decapoda, Palaemonidae) were sam-
pled in the lakes “Don Felipe” (31° 39' S, 60° 41' W),
“Alejandra” (31° 45' S, 60°31' W) and “N° 1” (31° 40' S,
60°30' W) (Santa Fe, Argentina) during a time of low
and high water phases of the flooding cycle (winter and
summer, respectively) (Figure 1), and they samples
were immediately taken to laboratory.
2.2. Sites of Study
The three lakes are placed in the Paraná River floodplain,
and they have different relations with human activities
and land use. In this river, the water level declined from
summer to winter, with a new flooding pulse in spring.
They have an average annual discharge of 18,000 m³/s
and a peak of 60,000 m³/s [21]. The flooding water of
the lentic bodies in the alluvial valley dilutes the biotic
densities of the communities, and during the isolation or
low water phase these concentrations recover rapidly.
The animal densities show important oscillations am-
ong the lakes depending on its volume, distance to the
river, amplitude-longitude of flooding pulse and time of
water residence [22-24].
“Don Felipe” is an oxbow lake close to the Colastiné
River which is a secondary branch directly connected to
the main channel (Figure 1). This fact changes its vol-
ume depending on the input of water flow from the Co-
lastiné River. In the isolated phase, its maximum and
mean depth is 2 m and 0.66 m respectively [25]. The
main industrialized cities occur about 400 km away from
studied site. Most of the wasted material (e.g. from food
industries) are rapidly dissolved when it reaches the
flood valley.
“Alejandra lake” is an overflow lake with a direct
connection to the Coronda River with a total length of
900 m, 1.8 m depth and it has an area of 75,000 m2 ap-
proximately (Figure 1). The land use near this lake is
low with a scarce impact on the environment. It is a
weekend residential area but it is near to Santa Fe and
Santo Tome cities.
“N° 1” is an abandoned meander scroll lake (Figure 1)
with a 0.55 m depth average during the isolated phase
(Collins, unpublished) located close to the Salado river
(a river with high dissolved contents salt), and its vol-
ume changes depending on the input of water flow from
the Salado and Santa Fe rivers. During the high water
period, the water Salado River is retained by the Paraná
system. The Salado River passes through a series of in-
dustries and farmlands in which biocides and fertilizers
are applied upon them. Moreover industrial discharges
and waste-water emitted from the cities (Santa Fe and
Santo Tomé) are poured into the last sections of the river.
2.3. Measurement of Water Parameters
The target variables to analyze in each sampling event
were taken in the top layer and three meters from the
coast line. These were water temperature; conductivity;
pH; transparency; nitrate; ammonia; orthophosphate;
dissolved oxygen; demand biological of oxygen 5
(DBO5); glyphosate; lead and copper. The samples
were collected in polyethylene bottles and preserved at
2-4ºC to posterior analysis in some cases. Temperature,
conductivity, pH and dissolved oxygen were taken in
situ with digital sensors (Hanna HI9143, HI991003,
HI9033). Transparency was measured with Secchi disc.
All the analytical methods, conformed to EPA methods
[26] or Standard Methods [27], were determined by a
spetrophotometer Metrolab330 (nitrate; ammonia; or-
thophosphate), a spetrophotometer Perkin Elmer (limit
4 ug/L) (heavy metals) and with a high precision liquid
cromatography (limit 0.2 ug/L) (biocid). The parame-
ters were sampled weekly in a month during summer
and winter.
2.4. Hepatopancreatic Cells Evaluation with
TEM
Dissections of the midgut glands were done for three
juveniles, males and females of both prawns (M. borellii
and P. argentinus) in intermolt stage according to Drach
and Tchernigovtzeff [28] from the three lakes. Adults
without gonadal maturation evidence were used. Disse-
ctions were conducted under a binocular microscope and
placed in 2% cold glutaraldehyde solution at pH 7.4 for
2 hr at 1-4°C. The tissues were then washed in several
changes of Sörensen buffer followed by postfixation in
2% osmium tetroxide solution for 2 hr at 1-4°C. After
dehydration in graded ethilic alcohol the material was
embedded in (ERL) araldite resin. Sections with gold
interference colours were obtained using a Reichert-Jung
Ultramicrotome with a glass and a diamond knife and
then mounted on coated copper slot grids. The hepato-
pancreas sections in the grids were then double stained
in uranyl acetate (saturated solution in 70% methanol, 4
h) followed by lead citrate (5 min) and viewed in a Sie-
mens Electron Microscope 101. The cell types were
identified and quantified in each observed section (5 for
each specimen).

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Figure 1. Study sites in the floodplain of Paraná Medio River (Santa Fe, Argentina). A) Main rivers and cities
indicating the three sampling environments (1: Don Felipe, 2: N° 1, 3: Alejandra). B) Main rivers in the de la
Plata Systems. C) Seasonal variation of water level in the Paraná Rriver (Santa Fe port limnigraph) (arrows
indicated the sampling time).

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2.5. Laboratory Assays
In order to evaluate on the hepatopancreatic cell of the
prawns the effects of cypermethrin, an extensively bio-
cid used in the farms and urban region, was done a labo-
ratory assays. The biocid was used in its commercial
form (Sherpa®: cypermethrin). Prawns (M. borellii and
P. argentinus) were kept in plastic containers of 0.9 m2
and 40 l with a density of 55 ind/m. The nominal sub-
lethal concentrations (cypermethrin (CY): 0.01; 0.001
and 0.0001 µg CY/l) [29] were applied only once. The
protocol was: first a continuous flow of water without
biocid of 1 ml/s during 15 days in all containers. After
that, the solutions with biocid were applied in the same
flow rate (1 ml/s) during one day. Later on the flow re-
turned to water without cypermethrin. The control group
was treated the similar forms in order to flow rate and
experience time. The prawns were daily fed with fish
muscle “ad libitum” and the mortality was registered in
all containers. The experience lasted 45 days. Each
seven day, the prawn hepatopancreas in intermolt phase
were dissected, and they were processed by a routine
histological method (dehydrated in alcohol series and
embedded in paraffin wax). They were cut into thin sec-
tions of 6 μm thickness by a rotative microtome. The
hystological cuts of these organs were stained with
haematoxylin and eosin to observe of the biocide effects
in the light microscope (5 cut for each prawns). The
hepatopancreatic cells were identificated and quantifi-
cated.
2.6. Data Analysis
Water quality parameters of each environment were
compared with ANOVA and Tukey post-test. In the same
way, differences in hepatopancreatic cell number were
evaluated with ANOVA. In the assays, survival and he-
patopancreatic cell data from control and experimental
groups were analysed with ANOVA in conjunction with
Tukey post-test too. All data were previously evaluated
to normality and homoscedasticity [30].
3. RESULTS
3.1. Environment Quality
In all the lakes, significant differences (ANOVA p < 0.05)
were registered for some parameters and sampling time
(Table 1), being the conductivity in “N° 1” lake (near
the Salado River) the highest unlike. Furthermore, these
differences were high for both sampling periods, with a
decrease values in summer during flooding period (Ta-
ble 1). The pH was not similar among all the sites, but
differences between floods and drought phases occur
only in the environment near to the Coronda River. Dis-
solved oxygen did not differ in the three lakes, being the
values lower during winter. Nutrients, such as ortho-
phosphorous, were different between Don Felipe and the
other lakes, corresponding high values in summer (Table
1). Nitrate values got the highest level in the Coronda
River (Alejandra lake). The unlike data of winter and
summer were not relevant (p > 0.05). Ammonia concen-
tration oscillated from 0.006 (Don Felipe summer) to
0.069 (N° 1 winter), showing difference between sam-
pling time. The unlike values among sites was not sig-
nificantly (Table 1). DBO5 was bigger in the environ-
ment near the Coronda River, receiving its water from
the Salado and Santa Fe Rivers (Figure 1). In summer,
all lakes have a distinct DBO5 but without statistically
significance, except in Don Felipe lake. In low water
phase (Table 1), glyphosate, residues of glyphosate, and
lead were registered in the N° 1 lake near the Santa Fe
and Santo Tomé cities.
Table 1. Mean and standard deviations of environmental parameters measured in the three lakes (Don Felipe, N° 1 and Alejandra
lakes) and two sampling times.
Don Felipe lake N° 1 lake Alejandra lake
winter summer winter summer winter summer
Temperature °C 14.8 ± 1.63 b 23.7 ± 1.11 b 13.6 ± 1.56 b 23.8 ± 0.97 b 14.7 ± 1.12 b 23.9 ± 1.12 b
Conductivity μS cm-1 150 ± 30.3a b 104 ±19.3 a b 5580 ± 1337.9 a b994 ± 29.8 a b 261 ± 55.2 a b 128 ± 17.3 a b
Transparency cm 27.5 ± 2.38 b 16.7 ± 3.79 b 29.5 ± 3.00 b 16.7 ± 3.79 b 31.8 ± 7.89 b 13.0 ± 5.29 b
pH 6.9 ± 0.29 a 6.7 ± 0.05 a 7.7 ± 0.20 a 7.5 ± 0.14 a 7.3 ± 0.12 a b 6.8 ± 0.08 a b
Dissolved oxygen ml l-1 8.8 ± 1.20 b 6.4 ± 1.09 b 9.2 ± 0.60 b 5.9 ± 2.69 b 9.6 ± 1.23 b 7.9 ± 0.29 b
DBO5 ml l-1 3.1 ± 1.68 ab 1.8 ± 0.69 ab 3.3 ± 2.25 a 3.7 ± 0.52 a 4.5 ± 4.96 a 4.2 ± 0.87 a
nitrate mg l-1 1.5 ± 0.28 1.8 ± 0.25 1.5 ± 0.61 1.5 ± 0.31 2.3 ± 1.20 2.8 ± 1.31
ammonia mg l-1 0.026 ± 0.018 b 0.009 ± 0.006 b0.037 ± 0.028 b 0.013 ± 0.015 b0.023 ± 0.017 b 0.012 ± 0.002 b
orthophosphate mg l-1 1.6 ± 1.06 a 1.1 ± 0.62 a 0.7 ± 0.27 a b 2.1 ± 0.20 a b 0.6 ± 0.64 a b 2.6 ± 0.33 a b
glyphosate* μg l-1 < 0.2 < 0.2 3.2 < 0.2 < 0.2 < 0.2
Pb μg l-1 < 4 < 4 49 < 4 < 4 < 4
Cu μg l-1 < 5 < 5 55 7.2 < 5 < 5
Significant differences according to ANOVA and Tukey post test (p < 0.05) a) among environment; b) between sampling times; *glyphosate and their
metabolite (AMPA).

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3.2. Hepatopancreatic Cells
There have been alterations in the midgut glands of ju-
veniles, males and females of both prawns species from
“N° 1” lake of Los Sapos island near Santo Tomé and
Santa Fe cities and Alejandra lake. The variability of the
damage degree was important. This depended on the
prawn origen, and the seasons (low and high water level
and temperature). The major alterations for both prawn
species were observed in the F- and R-cells, and the per-
centage of the cell type was differed from those prawns
in Don Felipe lake (mainly F-cell) (Table 2). Some pra-
wns from Alejandra lake show similar alterations than
those from N° 1 lake. The difference was observed in the
affected cell number, and/or in the intensity of the altera-
tion. Both prawn species showed similar hepatopan-
creatic cell frequency in all lakes and seasons (Table 2).
In apical zone of the alterated F-cells showed a dam-
aged microvilli border. The endoplasmic reticulum
(Figure 2(f)) and the mitochondria were more abundant
and swelled, and the cristae of the last was damaged and
increased in number compared with the mitochondria of
those prawns from Don Felipe lake. Endoplasmic re-
ticulum was either broken up into vesicles and/or exhib-
its dilated profiles (Figures 2(a), (c), (f), (h) and (i)).
The glycoproteins granules and others organelles were
increased too, e.g. in the basal region. Amorphous cor-
puscles with high electron density were found in the
cytoplasm (Figures 2(a), (b), (f) and (g)). In the most
affected F-cells (mainly in N° 1 lake) organelles lysis,
including cell nucleus were observed without breaking
the wall cell (Figures 2(b), (d) and (g)). The normal
F-cells (M. borellii and P. argentinus) belonging to “Don
Felipe” lake showed abundant rER without dilated pro-
files in the apical zone. The organelles have a homoge-
neous distribution, e.g. the cytoplasm contains glyco-
protein (or free ribosomes), peroxisomas, vesicles and
few and small mitochondria. In the basal region, it is
more frequently found the presence of Golgy systems
with distended cisterns, and surrounded by small and
dense vesicles.
The R-cells of prawns from N° 1 and Alejandra lakes
were affected in similar ways. The main alterations were:
in the apical zone of R-cells, the microvilli border was
somewhat shortened and sometimes it lacked the micro-
villar core filaments (Figures 3(a), (b) and (e)). The
endoplasmic reticulum was disrupted and the number of
residual bodies increased. The mitochondria were swell-
ed and sometimes its walls became deteriorated (Figures
3(b), (e), (f) and (g)). The lipid vacuole had an abrading
multilayered membrane (Figures 3(b) and (e)). Material
with high electron density was observed near this vacu-
ole in the apical zone (Figure 3(a)). R-cells with pre-
mature autolizing appearance were frequent (Figures
3(d), (f) and (g)). In the basal region, there were few
glycoprotein granules, and only the mitochondria was
increased and swelled, and its cristae was damaged
(Figures 3(c), (d) and (g)). The normal R-cells of
prawns from Don Felipe Lake have in apical zone a
large number of mitochondria, vesicles and glycoprotein,
which are prolonged in concentrated below the apical
plasma membrane. The undamaged microvillous border
was regular in all the extensions. In the basal region endo-
plasmic reticulum (rER and sER), glycoprotein granules
and many mitochondria were observed. The B-cells did
not show alterations that differ from the normal of hepa-
topancreatic cell cycles (Figures 4(a) and (b)) except af-
fections in some B-cells, main in N° 1 lake, where auto-
lising of organelles were registered (Figures 4(c) and (d)).
Other changes, such as basophyles vacuole groups and
differents residuals bodies, were observed in apical zone
of the F-cells in both species. Moreover, evident autolis-
ing of the organelles (Figures 5(a), (b) and (e)) and in-
definable elements associated with residuals bodies in
R-cells with abundant and distended cisterns of endo-
plasmic reticulum were frequent in the studied species
from N° 1 lake (Figures 5(c) and (d)).
3.3. Low and high water Level and
Temperature
The alterations were detected for both seasons and in the
same studied sites. The highest number of damaged cell
was registered in winter with low water level and tem-
Table 2. Hepatopancreatic cell frequency of two prawns and three lakes (Don Felipe, N° 1 and Alejandra lakes) during two sampling
times (winter and summer) (540 total observed sections).
Don Felipe lake N° 1 lake Alejandra lake
M. borellii winter summer winter summer winter summer
F-cell 29 ± 1.6 a 32 ± 1.1 a 49 ± 6.6 ab 43 ± 5.7 ab 38 ± 2.2 a 39 ± 6.1 a
R-cell 35 ± 3.3
a 39 ± 4.3 a 25 ± 5.9 a 23 ± 6.8 a 32 ± 5.2 a 37 ± 4.6 a
B-cell 36 ± 2.8 ab 29 ± 1.9 ab 26 ± 3.4 ab 34 ± 7.9 ab 30 ± 8.9 ab 24 ± 2.0 ab
P. argentinus
F-cell 32 ± 6.3 ab 25 ± 3.1 ab 54 ± 5.6 ab 46 ± 4.7 ab 36 ± 3.2 a 33 ± 1.2 a
R-cell 37 ± 5.3 a 43 ± 4.3 a 23 ± 3.9 ab 31 ± 4.8 ab 32 ± 5.5 a 37 ± 4.6 a
B-cell 31 ± 2.3 a 32 ± 3.7 a 23 ± 3.0 a 23 ± 6.3 a 32 ± 4.7 a 30 ± 1.2 a
Significant differences according to ANOVA and Tukey post test (p < 0.05). a among environment; b between sampling times.

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Figure 2. F-cell of Macrobrachium borellii and Palaemonetes argentinus from Alejandra (a and f re-
spectively) and N°1 lakes (b and g resepctively). Apical cell zone of M. borellii in N° 1 lake with lysis
appearence of mitchondria, endoplasmic reticulum and glycoprotein granules (d). Basal zone of F-cell in
M. borellii (c) sampled at Alejandra and P. argentinus cell (h and i) from both lakes with vesicles, golgi
bodies and glycoprotein granules, endoplasmic reticulum forms cistern and vacuolizing dictyiosomes.
Abbreviations: F-cell (F), rough endoplasmic reticulum (rer), golgi bodies (g), high electron density
corpuscles (ac), lumen cell (l), microvillous border (mb), mitochondria (m), vesicles (v), ribosomes (r),
peroxisomes (p), nucleus (n), wall cell (w). Bars: 0.1 μm approximately.
Figure 3. R-cell of Macrobrachium borellii and Palaemonetes argentinus affected in different levels from
Alejandra and N° 1 lakes. Apical zone with microvillous border deteriorated, endoplasmic reticulum and
shortened and mitochondria swelled, small vesicles, high electron density corpuscles and lipid vacuole with
a multilayered membrane disrupted (a, b, e, f). Basal zone with endoplasmic reticulum vesiculated (rER
and sER), glycoprotein granules and mitochondria swelled (in some R-cells were found a disrupted wall
mitochondria) or with lysis appearance (c, d). Abbreviations: R-cell (R), rough endoplasmic reticulum (rer),
smooth endoplasmic reticulum (ser), high electron density corpuscles (ac), lipid vacuole (li), microvillous
border (mb), mitochondria (m), ribosomes (r), nucleus (n), wall cell (w). Bars: 0.1 μm approximately.

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Figure 4. B-cell of Macrobrachium borellii and Palaemonetes argentinus from Alejandra and N° 1
lakes. Two moment of cell cycle without stress evidence (a, b) but in some cell was observed organ-
elles autolysis in cytoplasm (c, d). Abbreviations: B-cell (B), rough endoplasmic reticulum (rer),
smooth endoplasmic reticulum (ser), autosomes (au), lumen cell (l), microvillous border (mb), mito-
chondria (m), vesicles (v), lisosomes (ly), wall cell (w). Bars: 0.1 μm approximately.
Figure 5. Others hepatopancreatic cell evidence of stress in Macrobrachium borellii and Palae-
monetes argentinus from Alejandra and N° 1 lakes. Apical zone of F-cells with undefinible resid-
ual bodies and organelles autolysis. (a, b, e) In R-cells with differents vesicles without lipid con-
tent and swelled endoplasmc reticulum (c, d, e). Abbreviations: F-cell (F), R-cell (R), rough endo-
plasmic reticulum (rer), residual bodies (rb), microvillous border (mb), mitochondria (m), vesicles
(v), wall cell (w). Bars: 0.1 μm approximately.

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perature (Table 2). The percentage of affected prawns
was not different (ANOVA p > 0.005) between the species
M. borellii and P. argentinus. The “N° 1” lake had the
highest percentage of alterated cells in prawns with 75%,
32% in Alejandra lake and only 9% in Don Felipe lake
approximately. In this last one, the alterations coincided
with the observations indicated as starvation effects (lipid
dropplets with multilayered wall in R-cells). Moreover,
the cell type frequency varied with the prawn origen (Don
Felipe, N° 1 and Alejandra lakes), being the frequency of
F-cells higher in prawns from N° 1 lake during samples of
winter than other origin (Table 2).
In summer the number of affected cells disminished in
all the lakes (45 % in “N° 1” lake, 26% in Alejandra lake
and only 2% in Don Felipe lake approximately), and the
cell type frequency was similar (Table 2).
3.4. Laboratory Assays
In both prawns, damaged hepatopancreatic cells by the
pyretroid have had a relationship with the tested concen-
trations, being the cell frequency in each tested concen-
trations differents with the control at the end of the ex-
perience (ANOVA F(0.05, 2, 22) = 11.31; p < 0.0021).
The F-cells were more abundant than all the others
(Figure 6). The difference among C2, C3 and control, in
the F-cell percentage, occurred during the second week
whereas in the C1 it was in the third week (Figure 6). In
the experience, the F-cell presence oscillated with
changes significant among treated groups (Tukey’s p >
0.05). The B-cell percentage in all cypermethrin concen-
trations were low, 12% (C1) and 19% (C3).
Prawn survivals in treated groups varied significantly
during the 35 days of assays (ANOVA F(0.05, 2, 22) =
350.64; p < 0.0001, Tukey`s p < 0.05), and in the control
group there had low mortality (Figure 6)
4. DISCUSSION
The hepatopancreas of both freshwater prawns (M.
borellii and P. argentinus), which consists of four cell
types, undergoes ultrastructural changes depending on
the quality of the environment. This fact was observed in
the F-, R- and B-cells in different intensities. In these
cells, microvilli border, endoplasmic reticulum and mi-
tochondria were the most affected ultrastructure together
with and the increase of residual bodies number. More-
over premature autolysis development was observed.
On the one hand, three measured parameters have dif-
fered, being the conductivity the most variable. Trans-
parency reflected a great density of algae in the “N° 1”
lake before flooding period (November-April), which
could be due to a nutrient rise. Also, this coincides with
high DBO5 values. In the studied environments, differ-
ent algae species can produce phytotoxins, exposing the
prawns to this toxic stress. This situation could probably
activate the antioxidant defences in the hepatopancreas
leading to multiple oxidative processes and an effective
detoxification [31]. The cytoskeleton could be destroyed
by an increase in protein phosphorylation level, occur-
ring cell necrosis [32]. The immune system responds
with a melanization process which could act as a possi-
ble ROS scavenger [33]. Besides, the B-cell showed an
intracell digestion what carried out, possibly, with poste-
rior organelles degeneration in all cells, such as it was
suggested in exposed crabs to microcystins [34]. More-
over the large vacuole of B-cells contains digestive en-
zimes, e.g. cathepsin L (MeCatL) [35]. It was recognized
that in transicional cells F/B occurs P-glycoprotein
(P-gp), suggesting a function in specialized cells for ac-
cumulation and elimination of toxic compounds [36].
On the other, many substances may be toxic taking
into account their nature and amount, which are being
released by industries and cities to fresh waters and/or
the washing of farmlands during rains [37]. In these
waste waters, the heavy metals could be important quan-
titatively, and these are toxic elements for decapods [38,
39]. Moreover, these xenobiotic elements were present
in sites near to the cities, in other studies, several heavy
Table 3. Heavy metals measurements in sediment of several sites of Salado river close to the more affected sampling
area (from a work [40]).
Loc. Cu Zn Pb Cd Cr Fe Mn
1 19.54 21.29 19.47 n.d 55.18 107.1 793.05
2 0.78 0.05 1.9 n.d. 1.23 190.18 17.09
17 1.03 0.21 1.03 nd 0.64 nd 1.77
16 21.06 25.29 13.96 nd 9.71 560.92 182.26
values in µg/g; Localities: (1) Salado river in Motorway bridge (2 km up the river of “N° 1” lake approximately), (2) Salado river in Santo
Tomé-Santa Fe bridge (200 m down the river of “N° 1” lake approximately), (17) Salado river in Recreo city (10 km up the river of “N° 1”
lake, approximately), (16) Las Prusianas river in Grutly (50 km up the river of “N° 1” lake, approximately).

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Figure 6. F-cell frequency and survival percentage of
freshwater prawns (Macrobrachium borellii and Pa-
laemonetes argentinus) in the 35 days laboratory as-
says with several cypermethryn concentrations. Solid
line: control groups; dash and point line: 0.01 µg
CY/l groups; point line: 0.001 µg CY/l groups; da-
shed line: 0.0001 µg CY/l groups).
metals were registered at different points of the Salado
River sediments (Table 3), being a possible cause of
potential stress effects. Another parameter that might
provoke alterations in the prawns hepatopancreas is
temperature but this do not suggest any alteration, due to
the fact that lipid consumption for thermal stress occurs
at 29°C in M. borellii [41], and the samplings were taken
during spring (September-December) with lower tem-
peratures.
Some of the observed changes in the hepatopancreatic
cells were correlated with the starvation process and
xenobiotics effects which were described for other de-
capods [5,6,8,9,18,42,43]. The affected prawns from N° 1
lake and the assay had similar evidence of stress in the
hepatopancreas. The pyretroid is an biocid that it is used
in non-urban and urban environments, and it could po-
tentially get into the aquatic systems by different ways
(e.g. perfution, rain drenage). This could induce to ROS
production and to the formation of oxidative tissues in
the hepatopancreas of prawns, while their cells have
different biochemicals mechanisms to protect them-
selves from oxidative damage among which the glu-
tathione plays an important role [44]. Different intrinsic
factors such as specific capacity to stress tolerance, size,
nutritional condition and extrinsic factors (e.g. tempera-
ture, conductivity, ph) are important to evaluate the po-
pulational effects in a natural environment. The high
percentage of F-cells of exposed prawns to cypermethrin
could indicate that some detoxification mechanisms is
activated, including a more activity of the endoplasmic
reticulum and ribosomes in this cell type than in others.
The low number of B-cell could be interpreted as an
increase of the exoxitation of the B-cells to hepatopan-
creas lumen. Moreover another efluents, e.g. oil product
(PAH), provoked a size disminish in the hepatopancreas
cells [43]. Furthermore, cell number varies significantly
during the molt cycle (mainly, R-cell and F-cell) linked
with a change in the enzimatic activity (ATPasas and Na
/K ATPasas) [45], considering only the prawns in inter-
molt phase (c) by even.
The variation in the ultrastructure could have affected
the absorption mechanisms and protein synthesis as ob-
served by Papathanassiou and King [42]. However we
could say that these prawns can tolerate some degree of
environmental stress due to the fact that populations of
these species were found in “Laguna N° 1” lake with
similar sizes [46] to those taken from environment with
low human interference (Don Felipe lake) [25]. Thus we
can infer that there must be some detoxification mecha-
nisms such as those described by several authors [18,38,
47-51], where the F-cells could have a main rol. In these
works it was also included the capture of transuranic
elements, the deactivation of insecticides by rER or re-
moval of metals by granule formulation and its excretion
as detoxification pathways. These routes, together with
that of the complete renewal of the epithelium occurring
after several mitotic pulses [52] and/or some other me-
chanisms which involve a increase mitochondrial activ-
ity, could have allowed the presence of M. borellii and P.
argentinus in this environment.
However, the detoxification is only feasible when the
intensities of stressors do not exceed a particular thresh-

P. Collins / Natural Science 2 (2010) 748-759
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757
old value, otherwise it produces cellular damage [13]
and then the point-of-no return could be reached, such as
it was observed in starved individuals of Penaeus mono-
don [9], and in this study according to the evidence in
the cypermethrin assay.
5. CONCLUSIONS
The damage cell is irreversible to live of this prawn
when the values achieved determined concentrations or
stress level. The F-cell was increased with the intensities
of the stressors such as mechanisms of detoxification.
This involves a growth in numbers and size of rER, ri-
bosomes, and mitochondria. Therefore, the reserves in
the vacuole of R-cell are used to contribute to consume
energy in the detoxification pathway. This justified the
starvation conditions that is observed in some prawns.
The midgut glands of M. borellii and P. argentinus
reacts to water quality variations, and the structural
changes in cells and tissues represent the most sensitive
level. It is showed the synergistic impact of an element,
its metabolites and other exogenous and endogenous
factors showing the hepatopancreas as a good tool in the
determination of a stress areas of the natural environ-
ment.
6. ACKNOWLEDGEMENTS
The study was supported by a grant CONICET PIP 6275 (2006-2007).
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