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Histopathological changes in gills of the estuarine crab Chasmagnathus granulata (Crustacea-Decapoda) following acute exposure to ammonia
Comparative Biochemistry and Physiology Part C 125 (2000) 157 – 164
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Histopathological changes in gills of the estuarine crab Chasmagnathus granulata (Crustacea-Decapoda) following acute exposure to ammonia Mauro de Freitas Rebelo a,*, Enrique M. Rodriguez b, Euclydes A. Santos a, Martı´n Ansaldo c a
Dept. Cieˆncias Fisiologicas, Lab. Zoofisiologia, Fundac¸a˜o Uni6ersidade Federal do Rio Grande, CP 474, Rio Grande, RS, 91206 -900, Brazil b Department of Biological Science, Animal Physiology Laboratory, Uni6ersity of Buenos Aires, Pab. II. Ciudad Uni6ersitaria, 1428 Buenos Aires, Argentina c Argentine Antartic Institute, Cerrito 1248, 1010 Buenos Aires, Argentina Received 26 November 1998; received in revised form 16 September 1999; accepted 27 September 1999
Abstract Histopathological effects of ammonia on the gills of the estuarine crab Chasmagnathus granulata (Dana, 1851) were evaluated after acute exposure to ammonia concentrations around LC50 value (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 (PB 0.05) increases in pCO2 and lactate, and significant decreases of pO2 were 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
1. Introduction Both osmoregulating and osmoconforming crustaceans use gills for respiratory, ionoregulatory and excretory functions. The estuarine crab Chasmagnathus granulata lives in salt marshes of the Patos Lagoon (32o S, 52o W), Brazil, where environmental ammonia concentrations can be raised by industrial pollution, domestic sewage and drastic changes of environmental conditions, *Corresponding author. Present address: Lab. Radioiso´topos, Cicade Universita´ria, IBCCF-UFRJ CCS, 21940–900 Rio de Janeiro, RJ, Brazil. Tel./fax: + 55–21–5615339. E-mail address: [email protected] (M. de Freitas Rebelo)
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 ammonia) (Baumgarten et al., 1990). Previous work on osmoregulatory capacity of this crab suggests that osmotic balance is not seriously affected by ammonia. Despite the high tolerance to sublethal concentrations, the crab exhibits a small resistance time to ammonia concentrations close to the corresponding 96 h LC50 value (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.
0742-8413/00/$ - see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 7 4 2 - 8 4 1 3 ( 9 9 ) 0 0 0 9 3 - 6
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M. de Freitas Rebelo et al. / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 157–164
Decapod crustaceans present two major kinds of gills, anterior and posterior. Anterior gills have thin ephitelial cells (1 – 5 mm thick, protruding into the haemolymph space), suitable for gas ex-change. In contrast, most ephitelial cells of the posterior gills are thick (10–20 mm), called ionocytes, due to their ionoregulatory functions. The ionocytes possess 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 infoldings 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 determine histopathological alterations caused by ammonia to the gills of C. granulata, and whether this might be related to haemolymph osmotic imbalances 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; photoperiod: 14L:10D; feed ground beef every other day ad libitum). Animals were exposed to ammonia (as ammonium sulfate) for 96 h at the following concentrations: 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 university’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.
2.1. Histopathology 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 mm sections were prepared with a Zeiss HM 350 microtome and stained with hematoxylin and
eosin. Determination and quantification of gill pathologies were performed in a Reichert–Polivar 2 microscope. A rough quantification of the histopathological 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 affected (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).
2.2. Physiological determinations Determinations of lactate in the haemolymph were made by means of an enzymatic (lactate-oxidase) kit (Sigma no. 735). Haemolymph concentrations 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 (concentrations and control), followed by multiple comparisons of means by the Tukey procedure (Sokal et al., 1979).
3. Results
3.1. Histopathology 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. / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 157–164
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 disappearance of pilaster cells was more than fully compensated 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 considered pathologies was significantly
159
higher (PB 0.05) in exposed crabs than in controls, with the exception of the hyperplastic lamellae 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 pCO2 and lactate, together with a significant decrease 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.
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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 2). Compared to controls, sodium and calcium levels 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 concentration. Haemolymph ion concentrations are presented in Table 3.
4. Discussion 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; Baticados 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. / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 157–164
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 concentrations 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
161
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 exposed 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.
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Table 1 Percentage of affected lamellae ( 9SD) in the gills of C. granulata exposed to 16.5 and 27.5 mM of total ammoniaa Total ammonia (mM)
Necrosed lamellae
Hyperplastic lamellae
Cell disrupted lamellae
8th Gill pair Control (3) 16.5 (10) 27.5 (11)
1.9%9 2.2 84.3%9 32.6 100%9 0.0
1% 91.6 69.1%947.8 72.7% 946.7
0% 90.0 90.6% 9 24.5 97.8% 9 7.3
Control (3) 16.5 (6) 27.5 (6)
3rd Gill pair 1.1% 9 1.1 95.6%9 7.4 70.4%9 38.3
0.4% 90.6 68.5% 948.9 5.6%96.1
0% 90.0 94.1% 9 9.2 87.2% 9 6.5
a
Number of counted gills in brackets.
Table 2 Mean and standard error (number of crabs) of haemolymphatic pH, pCO2 and pO2 of C. granulata, following acute exposure to ammoniaa Treatment
pH
pCO2
pO2
Lactate
Control 16.5 mM 27.5 mM
7.65 9 0.03 (18)b 7.6890.09 (6)b 7.6390.05 (13)b
3.32 9 0.21 (18)b 4.139 0.77 (6)b,c 4.589 0.35 (14)c
13.1 9 1.3 (13)b 11.3 9 2.0 (6)b,c 8.15 9 0.8 (14)c
48.0 9 22.6 (7)a 28.6 9 6.72 (6)a 178.8 959.5 (3)b
a
PCO2 and pO2 as mmHg, lactate as mM. The same letter, for each variable, indicates absence of significant differences (P\0.05).
Table 3 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 Treatment
Na+
K+
Ca2+
Cl−
Control 16.5 mM 27.5 mM
567.69 48.8 (10)b 646.19 50.3 (8)b,c 761.09 109.3 (3)c
13.69 0.6 (9)b 14.89 0.8 (7)b 13.69 2.3 (3)b
8.1 90.2 (9)b 9.4 9 0.1 (6)b 11.3 9 0.4 (3)c
436.7 916.9 (10)b 548.0 916.4 (8)c 479.0 923.2 (4)b,c
a
Levels 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 structural alterations, involving cellular hyperplasia, necrosis and disruption of pilaster cells after acute exposure to ammonia. The histopathological effect 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 restriction 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. The same correlation, between gill
pathologies (such as necrosis and hyperplasia) and physiological imbalances in pO2, pCO2 and lactate 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 function of high pCO2. Thus, a compensatory effect of increased ammonia concentration in the
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haemolymph (raising pH), and high pCO2 (decreasing pH) may have occurred. Low pCO2 and pH have been reported in two shrimp species exposed to ammonia, and the formation of urea from ammonia, and CO2 suggested (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 NH+ 4 and the transported ion by Na+/K+-ATPase, which can take place under exposure to high ammonia concentrations (Towle et al., 1987), this could lead to a decrease in intracellular potassium. Since K+ is one of the main ions involved in the regulation of cell volume, this process could be related to cell disruption. Concerning ion balance involving the posterior gills, no decrease in any ion level was detected in the haemolymph of exposed crabs as expected if the ion regulatory mechanisms were inhibited. This is in accordance to the high tolerance 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 LC50 and ammonia concentrations to which the crabs have been exposed in this work, ammonia in the haemolymph is always lower than in the environment, 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 significant effects on the haemolymph ionic concentrations have been found in previous studies (Rebelo et al., 1998). Our results suggests that the lethal effect of ammonia is a result of damage to gas exchange mechanisms as consequence of the gill pathologies observed.
Acknowledgements We wish to thank Carina Lo´pez for helping with the preparation of histological material, Paula Rodrı´guez Moreno with the toxicity experiments and Dr Luiz Nery for manuscript review. This work was partially supported by a grant from the University of Buenos Aires (UBACYT 94-97 program). EAS is a productiv-
163
ity fellow of the Brazilian CNPq (Process no. 300.967/87-5).
References Baticados, M.C.l, Tendencia, E.A., 1991. Effects of gusathion A on the survival and shell quality of juvenile Penaeus monodon. Aquaculture 93, 9 – 19. Baumgarten, M.G.Z., Niencheski, L.F., 1990. O estua´rio da laguna dos Patos: variac¸o˜es de alguns paraˆmetros fı´sico-quı´micos da a´gua e metais associados ao material em suspensa˜o. Cieˆncia. e Cultura. 42, 390 – 396. Bianchini, A., de Castilho, P.C., 1999. Effects of sinc exposure on oxigen consuption and gill Na+, K+ -ATPase of the estuarine crab Chasmagnathus granulata dana, (Decapoda-Grapsidae). Bull. Environ. Contam. Toxicol. 62, 63 – 69. Bigi, R., 1996. Efectos del cadmio sobre la fisiologı´a branquial de Chasmagnahtus granulata (Decapoda, Brachyura). Graduate Thesis, University of Buenos Aires. Cardoso, E.L., Chiarini Garcia, H., Ferreira, R.M.A., Poli, C.R., 1996. Morphological changes in the gills of Lophiosilurus alexandri exposed to un-ionized ammonia. J. Fish Biol. 49, 778 – 787. Chen, J.C., Cheng, S.Y., 1993a. Haemolymph osmolality, acid – base balance and shift of ammonotelic to ureotelic excretory pattern of Penaeus japonicus exposed to ambient ammonia. Comp. Biochem. Physiol. 106C, 733 – 737. Chen, J.C., Cheng, S.Y., 1993b. Haemolymph pCO2, hemocyanin, protein levels and urea excretions of Penaeus monodon exposed to ambient ammonia. Aquat. Toxicol. 27, 281 – 292. Evans, R.E., Brown, S.C., Hara, T., 1988. The effects of aluminum and acid on the gill morphology in rainbow trout, Salmo gairdneri. Environm. Biol. Fishes 22, 299 – 311. Medesani, D.A., Rodrı´guez, E.M., Ansaldo, M., 2000. Imbalances caused by parathion in hemolymphatic parameters of the estuarine crab Chasmagnathus granulata (Decapoda, Brachyura). Indian J. Invertebr. Zool. Aquat. Biol. (in press). Papathanassiou, E., 1985. Effects of cadmium ions on the ultraestructure of the gill cells of the brown shrimp Crangon crangon. Crustaceana 48, 6 – 17. Paul, V.I., Banerjee, T.K., 1997. Histopathological changes induced by ambient ammonia (ammonium sulfate) on the opercular linings of the catfish Heteropneustes fossilis. Dis. Aquat. Org. 28, 151 – 161. Randi, A.S., Monserrat, J.M., Rodrı´guez, E.M., Romano, L.A., 1996. Histopathological effects of cadmium on the gills of the freshwater fish, Macropsobrycon uruguayanae Eigenmann (Pisces, Atherinidae). J. Fish. Dis. 19, 311 – 322.
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Taylor, H.H., Taylor, E.W., 1992. Gills and lungs: the exchange of gases and ions. In: Harrison, F.W., Humes, A.G. (Eds.), Microscopic Anatomy of Invertebrates. Wiley-Liss, New York, pp. 203 – 293. Towle, D.W., Holleland, T., 1987. Ammonium ion substitutes for K+ in ATP-dependent Na+ transport by basolateral membrane vesicles. Am. J. Physiol. 252, R479 – 489. Winkaler, E.U., Martinez, C.B.R., 1998. Efeitos da amoˆnia na morfologia branquial de Astyanax bimaculatus. 5th Brazilian Meeting on Ecotoxicology. Itajaı´. Abstracts.
Rebelo, M.F., Santos, E.A., Monserrat, J.M., 1998. Effects of ammonia on osmo-ionregulation and its accumulation on haemolymph of Chasmagnathus granulata (Crustacea, Decapoda) Dana. Comp. Biochem. Physiol. 122A, 429–435. Richmonds, C., Dutta, H.M., 1989. Histopathological changes induced by malathion in the gills of the bluegill Lepomis macrochirus. Bull. Environ. Contam. Toxicol. 43, 123–130. Sokal, R.R., Rohlf, F.J., 1979. Biometrı´a. Principios y me´todos estadı´sticos en la investigacio´n biolo´gica. Madrid, H. Blume ediciones.
.
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Histopathological changes in gills of the estuarine crab Chasmagnathus granulata (Crustacea-Decapoda) following acute exposure to ammonia Mauro de Freitas Rebelo a,*, Enrique M. Rodriguez b, Euclydes A. Santos a, Martı´n Ansaldo c a
Dept. Cieˆncias Fisiologicas, Lab. Zoofisiologia, Fundac¸a˜o Uni6ersidade Federal do Rio Grande, CP 474, Rio Grande, RS, 91206 -900, Brazil b Department of Biological Science, Animal Physiology Laboratory, Uni6ersity of Buenos Aires, Pab. II. Ciudad Uni6ersitaria, 1428 Buenos Aires, Argentina c Argentine Antartic Institute, Cerrito 1248, 1010 Buenos Aires, Argentina Received 26 November 1998; received in revised form 16 September 1999; accepted 27 September 1999
Abstract Histopathological effects of ammonia on the gills of the estuarine crab Chasmagnathus granulata (Dana, 1851) were evaluated after acute exposure to ammonia concentrations around LC50 value (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 (PB 0.05) increases in pCO2 and lactate, and significant decreases of pO2 were 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
1. Introduction Both osmoregulating and osmoconforming crustaceans use gills for respiratory, ionoregulatory and excretory functions. The estuarine crab Chasmagnathus granulata lives in salt marshes of the Patos Lagoon (32o S, 52o W), Brazil, where environmental ammonia concentrations can be raised by industrial pollution, domestic sewage and drastic changes of environmental conditions, *Corresponding author. Present address: Lab. Radioiso´topos, Cicade Universita´ria, IBCCF-UFRJ CCS, 21940–900 Rio de Janeiro, RJ, Brazil. Tel./fax: + 55–21–5615339. E-mail address: [email protected] (M. de Freitas Rebelo)
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 ammonia) (Baumgarten et al., 1990). Previous work on osmoregulatory capacity of this crab suggests that osmotic balance is not seriously affected by ammonia. Despite the high tolerance to sublethal concentrations, the crab exhibits a small resistance time to ammonia concentrations close to the corresponding 96 h LC50 value (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.
0742-8413/00/$ - see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 7 4 2 - 8 4 1 3 ( 9 9 ) 0 0 0 9 3 - 6
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M. de Freitas Rebelo et al. / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 157–164
Decapod crustaceans present two major kinds of gills, anterior and posterior. Anterior gills have thin ephitelial cells (1 – 5 mm thick, protruding into the haemolymph space), suitable for gas ex-change. In contrast, most ephitelial cells of the posterior gills are thick (10–20 mm), called ionocytes, due to their ionoregulatory functions. The ionocytes possess 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 infoldings 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 determine histopathological alterations caused by ammonia to the gills of C. granulata, and whether this might be related to haemolymph osmotic imbalances 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; photoperiod: 14L:10D; feed ground beef every other day ad libitum). Animals were exposed to ammonia (as ammonium sulfate) for 96 h at the following concentrations: 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 university’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.
2.1. Histopathology 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 mm sections were prepared with a Zeiss HM 350 microtome and stained with hematoxylin and
eosin. Determination and quantification of gill pathologies were performed in a Reichert–Polivar 2 microscope. A rough quantification of the histopathological 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 affected (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).
2.2. Physiological determinations Determinations of lactate in the haemolymph were made by means of an enzymatic (lactate-oxidase) kit (Sigma no. 735). Haemolymph concentrations 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 (concentrations and control), followed by multiple comparisons of means by the Tukey procedure (Sokal et al., 1979).
3. Results
3.1. Histopathology 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. / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 157–164
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 disappearance of pilaster cells was more than fully compensated 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 considered pathologies was significantly
159
higher (PB 0.05) in exposed crabs than in controls, with the exception of the hyperplastic lamellae 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 pCO2 and lactate, together with a significant decrease 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.
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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 2). Compared to controls, sodium and calcium levels 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 concentration. Haemolymph ion concentrations are presented in Table 3.
4. Discussion 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; Baticados 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. / Comparati6e Biochemistry and Physiology, Part C 125 (2000) 157–164
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 concentrations 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
161
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 exposed 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.
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Table 1 Percentage of affected lamellae ( 9SD) in the gills of C. granulata exposed to 16.5 and 27.5 mM of total ammoniaa Total ammonia (mM)
Necrosed lamellae
Hyperplastic lamellae
Cell disrupted lamellae
8th Gill pair Control (3) 16.5 (10) 27.5 (11)
1.9%9 2.2 84.3%9 32.6 100%9 0.0
1% 91.6 69.1%947.8 72.7% 946.7
0% 90.0 90.6% 9 24.5 97.8% 9 7.3
Control (3) 16.5 (6) 27.5 (6)
3rd Gill pair 1.1% 9 1.1 95.6%9 7.4 70.4%9 38.3
0.4% 90.6 68.5% 948.9 5.6%96.1
0% 90.0 94.1% 9 9.2 87.2% 9 6.5
a
Number of counted gills in brackets.
Table 2 Mean and standard error (number of crabs) of haemolymphatic pH, pCO2 and pO2 of C. granulata, following acute exposure to ammoniaa Treatment
pH
pCO2
pO2
Lactate
Control 16.5 mM 27.5 mM
7.65 9 0.03 (18)b 7.6890.09 (6)b 7.6390.05 (13)b
3.32 9 0.21 (18)b 4.139 0.77 (6)b,c 4.589 0.35 (14)c
13.1 9 1.3 (13)b 11.3 9 2.0 (6)b,c 8.15 9 0.8 (14)c
48.0 9 22.6 (7)a 28.6 9 6.72 (6)a 178.8 959.5 (3)b
a
PCO2 and pO2 as mmHg, lactate as mM. The same letter, for each variable, indicates absence of significant differences (P\0.05).
Table 3 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 Treatment
Na+
K+
Ca2+
Cl−
Control 16.5 mM 27.5 mM
567.69 48.8 (10)b 646.19 50.3 (8)b,c 761.09 109.3 (3)c
13.69 0.6 (9)b 14.89 0.8 (7)b 13.69 2.3 (3)b
8.1 90.2 (9)b 9.4 9 0.1 (6)b 11.3 9 0.4 (3)c
436.7 916.9 (10)b 548.0 916.4 (8)c 479.0 923.2 (4)b,c
a
Levels 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 structural alterations, involving cellular hyperplasia, necrosis and disruption of pilaster cells after acute exposure to ammonia. The histopathological effect 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 restriction 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. The same correlation, between gill
pathologies (such as necrosis and hyperplasia) and physiological imbalances in pO2, pCO2 and lactate 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 function of high pCO2. Thus, a compensatory effect of increased ammonia concentration in the
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haemolymph (raising pH), and high pCO2 (decreasing pH) may have occurred. Low pCO2 and pH have been reported in two shrimp species exposed to ammonia, and the formation of urea from ammonia, and CO2 suggested (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 NH+ 4 and the transported ion by Na+/K+-ATPase, which can take place under exposure to high ammonia concentrations (Towle et al., 1987), this could lead to a decrease in intracellular potassium. Since K+ is one of the main ions involved in the regulation of cell volume, this process could be related to cell disruption. Concerning ion balance involving the posterior gills, no decrease in any ion level was detected in the haemolymph of exposed crabs as expected if the ion regulatory mechanisms were inhibited. This is in accordance to the high tolerance 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 LC50 and ammonia concentrations to which the crabs have been exposed in this work, ammonia in the haemolymph is always lower than in the environment, 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 significant effects on the haemolymph ionic concentrations have been found in previous studies (Rebelo et al., 1998). Our results suggests that the lethal effect of ammonia is a result of damage to gas exchange mechanisms as consequence of the gill pathologies observed.
Acknowledgements We wish to thank Carina Lo´pez for helping with the preparation of histological material, Paula Rodrı´guez Moreno with the toxicity experiments and Dr Luiz Nery for manuscript review. This work was partially supported by a grant from the University of Buenos Aires (UBACYT 94-97 program). EAS is a productiv-
163
ity fellow of the Brazilian CNPq (Process no. 300.967/87-5).
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