Comparative Biochemistry and Physiology Part C 125 (2000) 157–164
Histopathological changes in gills of the estuarine crab
Chasmagnathus granulata (Crustacea-Decapoda) following
acute exposure to ammonia
Mauro de Freitas Rebeloa,*, Enrique M. Rodriguezb, Euclydes A. Santosa,
Martı ´n Ansaldoc
aDept. Cie ˆncias Fisiologicas, Lab. Zoofisiologia, Fundac ¸a ˜o Uni?ersidade Federal do Rio Grande, CP 474, Rio Grande, RS,
bDepartment of Biological Science, Animal Physiology Laboratory, Uni?ersity of Buenos Aires, Pab. II. Ciudad Uni?ersitaria,
1428 Buenos Aires, Argentina
cArgentine Antartic Institute, Cerrito 1248, 1010 Buenos Aires, Argentina
Received 26 November 1998; received in revised form 16 September 1999; accepted 27 September 1999
Histopathological effects of ammonia on the gills of the estuarine crab Chasmagnathus granulata (Dana, 1851) were
evaluated after acute exposure to ammonia concentrations around LC50value (17.85 Mm). Disruption of pilaster cells
and a subsequent collapse of gill lamellae were the main effects observed. Ephitelial necrosis and hyperplasia were also
detected. Significant (P?0.05) increases in pCO2and lactate, and significant decreases of pO2were detected in the
haemolymph of ammonia-exposed crabs. These changes suggest that the observed histopathological damage affected gas
exchange, possibly leading to death. © 2000 Elsevier Science Inc. All rights reserved.
Keywords: Ammonia; Crab; Toxicity; Histopathology
crustaceans use gills for respiratory, ionoregula-
tory and excretory functions. The estuarine crab
Chasmagnathus granulata lives in salt marshes of
the Patos Lagoon (32oS, 52oW), Brazil, where
environmental ammonia concentrations can be
raised by industrial pollution, domestic sewage
and drastic changes of environmental conditions,
such as water temperature and salinity. In some
areas of the lagoon, ammonia concentrations in
the pore water, where crabs make their burrows,
can reach values as high as 1.2 mM (total ammo-
nia) (Baumgarten et al., 1990). Previous work on
osmoregulatory capacity of this crab suggests that
osmotic balance is not seriously affected by am-
monia. Despite the high tolerance to sublethal
concentrations, the crab exhibits a small resis-
tance time to ammonia concentrations close to the
corresponding 96 h LC50value (Rebelo et al.,
1998). Thus, we postulated that rapid and direct
damage of the gills, and not the relatively slow
breakdown of a physiological mechanism, would
be the responsible for the death of the animals.
*Corresponding author. Present address: Lab. Radioiso ´to-
pos, Cicade Universita ´ria, IBCCF-UFRJ CCS, 21940–900 Rio
de Janeiro, RJ, Brazil. Tel./fax: +55–21–5615339.
E-mail address: firstname.lastname@example.org (M. de Freitas Rebelo)
0742-8413/00/$ - see front matter © 2000 Elsevier Science Inc. All rights reserved.
M. de Freitas Rebelo et al. / Comparati?e Biochemistry and Physiology, Part C 125 (2000) 157–164158
Decapod crustaceans present two major kinds of
gills, anterior and posterior. Anterior gills have
thin ephitelial cells (1–5 ?m thick, protruding into
the haemolymph space), suitable for gas ex-change.
In contrast, most ephitelial cells of the posterior
gills are thick (10–20 ?m), called ionocytes, due to
their ionoregulatory functions. The ionocytes pos-
sess a well developed system of apical leaflets,
alternating with extracelular subcuticle spaces.
Above and between the folds, there is a great
number of mitochondria. Deep basolateral infold-
ings are located at the basolateral membrane,
together with elongate mitochondria for active
ionic transport (Taylor et al., 1992).
The main objective of this work was to deter-
mine histopathological alterations caused by am-
monia to the gills of C. granulata, and whether this
might be related to haemolymph osmotic imbal-
ances and gas exchange.
2. Materials and methods
Adult males of C. granulata were collected from
the salt marshes surrounding the Patos Lagoon
(Southern Brazil) and kept for acclimation in the
laboratory for 30 days under controlled conditions
(temperature: 20°C; salinity: 20‰; pH 7.0; pho-
toperiod: 14L:10D; feed ground beef every other
day ad libitum).
Animals were exposed to ammonia (as ammo-
nium sulfate) for 96 h at the following concentra-
tions: 0 (control), 16.5 and 27.5 mM. Unless
otherwise noted, all ammonia concentrations refer
to total ammonia (ionized plus unionized forms).
Ammonia free seawater was obtained at the uni-
versity’s aquaculture station and used to prepare
test solutions. Small flasks filled with 4.0 l of test
solution, containing 10 crabs each, were used for
exposing the animals to ammonia. Test solutions
were renewed every 24 h.
At the end of the exposure period, the 3rd and
8th pairs of gills were quickly dissected and fixed
in alcoholic Bouin solution for 24 h; they were then
transferred to 70% ethanol until further processing.
Gills were dehydrated in a progressive alcohol
series and embedded in Paraplast. Four to 5 m?
sections were prepared with a Zeiss HM 350
microtome and stained with hematoxylin and
pathologies were performed in a Reichert–Polivar
2 microscope. A rough quantification of the histo-
pathological effects was made by counting the
number of affected lamellae by each specific
pathology, in relation to the total number of
lamellae in each gill. Only the main pathologies
(necrosis, hyperplasia and pilaster cells disruption)
were computed. One lamella was considered af-
fected (and thus counted) despite the effect seemed
more or less severe. Statistical calculations were
made by means of Kruskal–Wallis ANOVA
(Sokal et al., 1979).
and quantification of gill
2.2. Physiological determinations
Determinations of lactate in the haemolymph
were made by means of an enzymatic (lactate-oxi-
dase) kit (Sigma no. 735). Haemolymph concentra-
tions of K+, Na+and Ca2+were determined by
flame photometry (DIGIMED NK-2004), and
chlorine concentration by titration (JENWAY
model PCLM3). Haemolymph samples were taken
previously to gill dissection from the blood sinus at
the base of the 3rd and/or 4th pair of pereiopods.
For determination of pH, pO2 and pCO2,
haemolymph samples were analyzed by means of
a BMS3 Mk2 blood microsystem (Radiometer),
thermostatted at 20°C.
A one way ANOVA was used to test significant
effects among the experimental groups (concentra-
tions and control), followed by multiple compari-
sons of means by the Tukey procedure (Sokal et
As shown in Fig. 1A, normal lamellae of control
gills, from both 8th and 3rd gill pairs exhibit a very
thin cuticle, a single layer of epithelial cells and the
characteristic pilaster cells. Posterior gills (8th
pair), show typical ionocytes. In contrast, exposed
gills exhibit collapsed lamellae, due to disruption
of pilaster cells, as shown in Fig. 1B. A detail of
this pathology can be seen in Fig. 2.
Epithelial necrosis and hyperplasia were also
observed (Fig. 3). These pathologies together with
the absence of pilaster cells, led to collapse of the
M. de Freitas Rebelo et al. / Comparati?e Biochemistry and Physiology, Part C 125 (2000) 157–164159
entire lamellae (Figs. 4 and 5). Unfortunately, not
always the collapsed lamellae exhibited a different
thick from healthy lamellae, preventing the use of
this variable as a good parameter of effect. The
reduced intraepithelial space caused by disappear-
ance of pilaster cells was more than fully compen-
sated by the enlargement of necrosed epithelial
tissue and cuticle.
Pathologies are quantified in Table 1. For both
respiratory (3rd) and osmoregulatory (8th) gills,
the percentage of lamellae exhibiting each one of
the consideredpathologieswas significantly
higher (P?0.05) in exposed crabs than in con-
trols, with the exception of the hyperplastic lamel-
lae of the 3rd gill from crabs exposed to 16.5 mM
of ammonia. Both, 3rd and 8th gills, were affected
by ammonia to the same extent.
3.2. Physiological experiments
No significant differences were found in pH
among treatments, while a significant increase of
pCO2and lactate, together with a significant de-
crease of pO2, were detected at the highest ammo-
Fig. 1. (A) Lamellae from the 8th gill pair of C. granulata. (A) in control animals showing normal structures like ionocytes (IC) and
pilaster cells (PC). (B) in animals exposed to 16.5 mM of total ammonia showing collapsing of lamellae after necrosis and disruption
of pilaster cells.
M. de Freitas Rebelo et al. / Comparati?e Biochemistry and Physiology, Part C 125 (2000) 157–164160
Fig. 2. Lamellae from the 8th gill pair of C. granulata exposed to 16.5 mM of total ammonia. Picture exhibits the disruption of pilaster
cells (indicated by arrows).
Fig. 3. Lamellae from the 3rd gills pair of C. granulata exposed to 16.5 mM total ammonia. Affected lamellae show hyperplasia (HYP),
necrosis (NCR) and detached cuticle (DTC).
nia concentration with respect to control (Table
Compared to controls, sodium and calcium lev-
els significantly increased at the highest ammonia
concentrations, while chloride level significantly
increased but only at 16.5 mM. No significant
differences were detected in potassium concentra-
tion. Haemolymph ion concentrations are pre-
sented in Table 3.
There are several reports in the literature on the
histopathological effects of pollutants in the gills
of crustaceans and fish (Papathanassiou, 1985;
Evans et al., 1988; Richmonds et al., 1989; Bati-
cados et al., 1991; Bigi et al., 1996; Randi et al.,
1996; Medesani et al., 2000). Nevertheless, just a
few of them are related to ammonia concentra-
M. de Freitas Rebelo et al. / Comparati?e Biochemistry and Physiology, Part C 125 (2000) 157–164161
tions (Paul et al., 1997; Winkaler et al., 1998).
Cardoso et al. (1996) detected changes in cells
and branchial tissue of larvae and juveniles of
Lophiosilurus alexandri, exposed to concentra-
tions of unionized ammonia close to the LC50
values. Paul et al. (1997), reported for the
ephitelial linings of the operculum of a catfish
massive and quick damage due to necrosis and
sloughing of the outer surface epithelial cells
with degeneration and disappearance of club
cells, after acute exposure to ammonium sulfate.
Winkaler et al. (1998), also found gill lamellae
fusion to be an effect of ammonia in fish ex-
posed to 0.41 mM of gaseous ammonia. They
also reported severe aneurysm as a specific effect
of such exposure.
Fig. 4. Lamellae from the 8th gill pair of C. granulata exposed to 16.5 mM of total ammonia. Only the nucleus of pilaster cells remains,
close to the necrotic epithelia of the lamellae.
Fig. 5. Lamellae from the 8th gill pair of C. granulata exposed to 27.5 mM total ammonia. The necrotic lamellae finally collapse after
total disruption of pilaster cells.
M. de Freitas Rebelo et al. / Comparati?e Biochemistry and Physiology, Part C 125 (2000) 157–164162
Percentage of affected lamellae (?SD) in the gills of C. granulata exposed to 16.5 and 27.5 mM of total ammoniaa
Total ammonia (mM) Necrosed lamellaeHyperplastic lamellae Cell disrupted lamellae
8th Gill pair
3rd Gill pair
aNumber of counted gills in brackets.
Mean and standard error (number of crabs) of haemolymphatic pH, pCO2and pO2of C. granulata, following acute exposure to
aPCO2and pO2as mmHg, lactate as mM. The same letter, for each variable, indicates absence of significant differences (P?0.05).
Mean and standard error (number of crabs) of haemolymphatic sodium (Na+), potassium (K+), calcium (Ca2+) and chloride (Cl−)
concentrations in C. granulata, following acute exposure to ammoniaa
aLevels of ions as meq/l. The same letter, for each variable, indicates absence of significant differences (P?0.05).
In C. granulata, we detected extensive struc-
tural alterations, involving cellular hyperplasia,
necrosis and disruption of pilaster cells after acute
exposure to ammonia. The histopathological ef-
fect of ammonia on gill epithelium showed to be
gill independent, since we found similar effects in
lamellae from both 3rd and 8th gills. This led to
considerable thickening of the gill epithelium and
reduction of haemolymph spaces resulting in re-
striction of respiratory gas exchange as shown by
hypoxia and hypercapnia.
A significant increase of haemolymph pCO2
and lactate, together with a decrease of pO2, was
verified.Thesame correlation, betweengill
pathologies (such as necrosis and hyperplasia) and
physiological imbalances in pO2, pCO2and lac-
tate concentration were also observed after acute
exposure of C. granulata to parathion (Medesani
et al., 2000).
Although a marked haemolymph acidosis was
seen after parathion exposure (Medesani et al.,
2000), no changes in pH were observed after
ammonia exposure. It is known that ammonia
diffuses through cells as NH3, acting as a base
and, therefore, an increase in pH was expected.
An opposing effect would be expected as a func-
tion of high pCO2. Thus, a compensatory effect of
increased ammoniaconcentrationin the
M. de Freitas Rebelo et al. / Comparati?e Biochemistry and Physiology, Part C 125 (2000) 157–164163
haemolymph (raising pH), and high pCO2(de-
creasing pH) may have occurred. Low pCO2
and pH have been reported in two shrimp spe-
cies exposed to ammonia, and the formation of
urea from ammonia, and CO2suggested (Chen
and Cheng, 1993a,b).
High ammonia concentrations in the medium
can raise cellular pH in C. granulata, and inhibit
the activity of Na+/K+-ATPase by as much as
80% (Bianchini et al., 1999). Together with the
substitution of K+by NH4
ion by Na+/K+-ATPase, which can take place
under exposure to high ammonia concentrations
(Towle et al., 1987), this could lead to a de-
crease in intracellular potassium. Since K+is
one of the main ions involved in the regulation
of cell volume, this process could be related to
Concerning ion balance involving the poste-
rior gills, no decrease in any ion level was de-
tected in the haemolymph of exposed crabs as
expected if the ion regulatory mechanisms were
inhibited. This is in accordance to the high tol-
erance to ammonia shown previously by C.
granulata (Rebelo et al., 1998) and is probably
related to its great osmoregulatory capacity.
Despite the high values of LC50and ammonia
concentrations to which the crabs have been ex-
posed in thiswork,
haemolymph is always lower than in the envi-
ronment, suggesting an efficient mechanism to
eliminate excess ammonia (Rebelo et al., 1998).
This mechanism does not seem to be affected by
sublethal ammonia concentrations, since no sig-
nificant effects on the haemolymph ionic con-
centrations have been found in previous studies
(Rebelo et al., 1998). Our results suggests that
the lethal effect of ammonia is a result of dam-
age to gas exchange mechanisms as consequence
of the gill pathologies observed.
+and the transported
We wish to thank Carina Lo ´pez for helping
with the preparation of histological material,
Paula Rodrı ´guez Moreno with the toxicity ex-
periments and Dr Luiz Nery for manuscript re-
view. This work was partially supported by a
grant from the University of Buenos Aires
(UBACYT 94-97 program). EAS is a productiv-
ity fellow of the Brazilian CNPq (Process no.
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