Effects of an organophosphorus insecticide, trichlorfon, on hematological parameters of the giant freshwater prawn, Macrobrachium rosenbergii (de Man)

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1 Aquaculture 243 (2005) Effects of an organophosphorus insecticide, trichlorfon, on hematological parameters of the giant freshwater prawn, Macrobrachium rosenbergii (de Man) Shinn-Pyng Yeh a, Tzeng-Gan Sung b, Chin-Chyuan Chang b, Winton Cheng a, *, Ching-Ming Kuo c a Department of Aquaculture, National Pingtung University of Science and Technology, Pingtung 912, Taiwan, ROC b Institute of Tropical Agriculture, National Pingtung University of Science and Technology, Pingtung 912, Taiwan, ROC c Marine Research Station, Institute of Zoology, Academia Sinica, Ilan 262, Taiwan, ROC Received 19 February 2004; received in revised form 25 October 2004; accepted 26 October 2004 Abstract Hemolymph osmolality, ion concentration, acid base balance, and immune parameters in Macrobrachium rosenbergii (15 20 g) were measured after 8 days of exposure to mg l 1 trichlorfon. A significant depression in hemolymph osmolality and Cl 1 contents were observed with exposure to 0.3 mg l 1 trichlorfon. Similarly, hemolymph ph, HCO 3, and TCO 2 decreased significantly when prawns were exposed to concentration of greater than 0.2, 0.1, and 0.1 mg l 1 trichlorfon, respectively. A notable increase in the hemolymph pco 2 was observed with 0.3 mg l 1 trichlorfon. However, no significant differences in hemolymph oxyhemocyanin or protein levels were observed among prawns with mg l 1 trichlorfon. These findings may have resulted from histological changes in the respiratory epithelium and/or the inhibitory action of the nervous system of respiration. Phenoloxidase activity in the hemocytes of prawns decreased significantly with exposure to greater than 0.2 mg l 1 trichlorfon. This indicates that trichlorfon reduces the immune ability of M. rosenbergii. D 2004 Elsevier B.V. All rights reserved. Keywords: Macrobrachium rosenbergii; Trichlorfon; Hemolymph; Osmolality; Ions; Oxyhemocyanin; Protein; Acid base balance; Immunity 1. Introduction * Corresponding author. Tel.: x 6224; fax: address: winton@mail.npust.edu.tw (W. Cheng). Macrobrachium rosenbergii is a large prawn of the family Palaemonidae indigenous to tropical freshwater and brackish water habitats of the Indo-Pacific (George, 1969). This species inhabits a wide range of salinity levels during its life cycle and is found in /$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi: /j.aquaculture

2 384 S.-P. Yeh et al. / Aquaculture 243 (2005) waters ranging from 0x to 18x salinity. During the reproductive season, adults migrate from freshwater habitats to estuarine regions where the eggs hatch and planktonic larval development occurs (Nelson et al., 1977). However, it is commercially important in the world primarily as an inland species (New, 1995). Potentially damaging compounds are used in shrimp culture, such as disinfectants, therapeutics, feed additives, algicidals, pesticides, and fertilizers. These chemicals may cause biological damage to all life stages during shrimp production. Biochemical responses in penaeids to contaminants were reviewed by Bainy (2000) and Lignot et al. (2000). Several studies previously demonstrated disruption of the normal osmotic and ionic balance after exposure to pollutants (Caldwell, 1974; Inman and Lockwood, 1977; Neufeld and Pritchard, 1979), and the osmoregulatory capacity has been proposed as a potential indicator of the physiological condition and a stress indicator among crustaceans (Boitel and Truchot, 1989; Charmantier et al., 1989; Young-Lai et al., 1991; Lin et al., 1993; Bambang et al., 1995a,b). Boitel and Truchot (1989) indicated that copper at sublethal and lethal concentrations affects the hemolymph acid base balance and ion concentrations in the shore crab Carcinus maenas kept in undiluted seawater. Hemolymph hemocyanin, proteins, and the acid base balance of crustaceans were altered by exchanges in ambient salinity, temperature, dissolved oxygen (Truchot, 1983; Ferraris et al., 1986), ammonia-n (Chen and Cheng, 1993a), nitrite-n (Chen and Cheng, 1995), and saponin (Chen and Chen, 1996). Several quantitative, rapid, and easy procedures are used to evaluate the expression of the immune response of shrimp. For example, the hemogram and two cellular mechanisms, the radical oxygen intermediates (ROIs) generated during postphagocytic events, and phenoloxidase (PO) activity have been considered potential markers. Environmental contaminants have been reported to cause variations in total haemocyte counts (THCs), differential hemocyte counts (DHCs) (Truscott and White, 1990; Victor et al., 1990; Smith and Johnston, 1992; Le Moullac and Haffner, 2000), phenoloxidase activity (Truscott and White, 1990; Smith and Johnston, 1992, Smith et al., 1995), and superoxide anions (Le Moullac et al., 1998) in crustaceans. In M. rosenbergii, Cheng and Chen (2000) indicated that both THCs and phenoloxidase activity were altered by ph, temperature, and salinity. Phenoloxidase activity and superoxide anions were also affected under ammonia-n (Cheng and Chen, 2002) and copper sulfate (Cheng and Wang, 2001) stress. It is not uncommon to observe intensive use of insecticides in agricultural areas adjacent to prawn farming zones. Metabolic changes observed in seawater and freshwater crustaceans exposed to organophosphorus (OP) insecticides create widespread disturbances in general physiological processes, such as enzymatic activities (Bhagyalakshmi et al., 1984; Omkar and Shukla, 1985; Repetto et al., 1988), oxidative metabolism (Bhagyalakshmi et al., 1984), oxygen consumption (Pawar and Katdare, 1984; Cebrian et al., 1992), and osmoregulation (Lignot et al., 1997). Trichlorfon [O,O-dimethyl (1-hydroxy 2,2,2-trichloroethyl) phosphonate] is one of the most commonly used compounds, which is an OP pesticide with high solubility (14%) and moderate toxicity. OP insecticides, used massively and repeatedly because of their rapid degradation in the environment, are potential threats to nontarget species, such as fish, crabs, and shrimp (Tronczynski, 1990). In this study, we attempted to determine the effects of different concentrations of trichlorfon over long exposure periods on the hemolymph and immune parameters of M. rosenbergii. 2. Materials and methods 2.1. Experimental design M. rosenbergii (15 20 g in the intermolt stage) were obtained from a commercial farm in Pingtung, southern Taiwan, and acclimated in the laboratory for 2 weeks before experimentation. Two prawns were kept in each 60-l glass aquarium containing 40 l of test solution at different concentrations of trichlorfon (0, 0.1, 0.2, and 0.3 mg l 1 ). Test solutions were renewed daily, and the experiment lasted for 8 days. Each test solution was conducted in quadruplicate. Prawns were fed twice daily with a formulated prawn diet (Shinta Feed Company, Pingtung, Taiwan). During the experiment, water temperature was maintained at 28F1 8C, ph at , total hardness at

3 S.-P. Yeh et al. / Aquaculture 243 (2005) mg l 1, osmolality at 2 mosm kg 1,Na + at 0.5 mmol l 1,Ca 2+ at 0.09 mmol l 1, and Mg 2+ at 0.34 mmol l 1. However, the concentrations of K + and Cl were too low to be determined Effects of trichlorfon on the hemolymph parameters of M. rosenbergii Hemolymph samples were taken by inserting a syringe (25 G1W) into the ventral sinus of the prawns. Hemolymph protein was determined using the Bio-Rad Protein Assay Kit no (Bio- Rad Laboratories, Richmond, CA, USA) using bovine albumin (molecular weight, 66,000) as a standard (Bradford, 1976). To measure oxyhemocyanin, 100 Al of hemolymph was immediately diluted with 900 Al distilled water, and the absorbance was measured at 335 nm (characteristic of oxyhemocyanin) using a Hitachi (Tokyo, Japan) U-2000 spectrophotometer. The concentration of oxyhemocyanin was calculated based on the methods of Nickerson and Van Holde (1971) and Hagerman (1983). The ratio of oxyhemocyanin to protein was calculated by dividing the concentration of oxyhemocyanin (mmol l 1 )by that of protein (mmol l 1 ), which was converted from mg ml 1 to mmol l 1 by dividing by 66 (Chen and Cheng, 1993b). Hemolymph osmolality and medium osmolality were measured by injecting a 20-Al sample into a micro-osmometer (Model 3MO plus, Advanced Instruments, Norwood, MA, USA). To determine Na +,K +, and Cl, 100 Al of a hemolymph sample was immediately injected into an Ion Selective Electrode Analyzer (Medica EasyLyte PLUS, USA). For determination of hemolymph Ca 2+ and Mg 2+, hemolymph was diluted with distilled water to onetenth and one-half, respectively, and measured using a Blood Biochemical Analyzer (Humalyzer 2000, Human, Germany). Hemolymph ph, PCO 2, and PO 2 levels were determined immediately by injecting hemolymph (40 Al) into a ph/blood-gas analyzer (Stat Profile phox Analyzer, NoVa Biomedical, USA) with the thermostat set to 37 8C and corrected automatically to a value at 25 8C (NOVA, 1998). Hemolymph HCO 3 and TCO 2 were calculated based on the formulae from the data for ph and PCO 2 (NOVA, 1998) Effect of trichlorfon on the immune parameters of M. rosenbergii Hemolymph (100 Al) was withdrawn from the ventral sinus of each prawn into a 1-ml syringe (25 G1W) containing 0.9 ml of an anticoagulant solution (0.114 M trisodium citrate, and 0.1 M sodium chloride; ph 7.45, osmolality 490 mosm kg 1 ). A drop of hemolymph was placed on a hemocytometer, and the total hemocyte count (THC) was measured using a microscope. Phenoloxidase activity was measured spectrophotometrically by recording the formation of dopachrome produced from l-dihydroxyphenylalanine (l-dopa; Herández-López et al., 1996). The diluted hemolymph was centrifuged at 300g at 4 8C for 10 min, the supernatant fluid discarded, and the pellet was rinsed, resuspended gently in cacodylate-citrate buffer (0.01 M sodium cacodylate, 0.45 M sodium chloride, 0.10 M trisodium citrate; ph 7.0), and then centrifuged again. The pellet was then resuspended in 200 Al cacodylate buffer (0.01 M sodium cacodylate, 0.45 M sodium chloride, 0.01 M calcium chloride, and 0.26 M magnesium chloride; ph 7.0). One hundred microliters of the cell suspension was incubated with 50 Al of trypsin (1 mg ml 1 ), which served as an elicitor, for 10 min at C. Fifty microliters of l-dopa (3 mg ml 1 ) was added, followed by 800 Al of cacodylate buffer added 5 min later. The optical density at 490 nm was measured using a Hitachi U-2000 spectrophotometer (Tokyo, Japan). The control solution, which consisted of 100 Al of cell suspension, 50 Al cacodylate buffer (to replace the trypsin), and 50 Al of l-dopa, was used for the background phenoloxidase activity in all test solutions. The background phenoloxidase activity optical density values were in the range of 0.02 to The phenoloxidase activity optical density values of prawns for all test conditions were expressed as dopachrome formation in 50 Al of hemolymph Statistical analysis Effects of trichlorfon on protein, oxyhemocyanin, the ratio of oxyhemocyanin/protein, osmolality, Cl, Na +,K +,Ca 2+,Mg 2+, total hemocytes, and phenoloxidase activity were analyzed statistically by Duncan s

4 386 S.-P. Yeh et al. / Aquaculture 243 (2005) multiple range test using version 8.2 Statistical Analysis System (SAS; SAS, 2001). Prior to the Duncan s multiple range tests, all data were applied to a normal distribution test. 3. Results 3.1. Effect of trichlorfon on hemolymph parameters of M. rosenbergii Osmotic and electrolyte concentrations Hemolymph osmolality of prawns following 8 days of exposure to 0.3 mg l 1 trichlorfon had significantly decreased by 5.1% compared to prawns kept in the control solution (0 mg l 1 ; Fig. 1A). Hemolymph chloride of prawns exposed to 0.3 mg l 1 was significantly lower than that of prawns exposed to 0.2, 0.1, and 0 mg l 1 after 8 days. Hemolymph chloride had significantly decreased by 8.2% as compared to that of prawns held in the control solution (Fig. 1B). However, hemolymph sodium, potassium, calcium, and magnesium concentrations did not significantly differ among prawns exposed to 0 to 0.3 mg l 1 and were in the range of , , , and mmol l 1, respectively (Fig. 1C F) Oxyhemocyanin and protein levels No significant difference was found in the oxyhemocyanin or protein levels or the ratio of oxyhemocyanin to protein of prawns following 8 days of Fig. 1. Hemolymph osmolality (mosm kg 1 ; A), Cl (mmol l 1 ; B), Na + (mmol l 1 ; C), K + (mmol l 1 ; D), Mg 2+ (mmol l 1 ; E), and Ca 2+ (mmol l 1 ;F)ofM. rosenbergii after 8 days of exposure to different concentrations of trichlorfon. Each bar represents the mean value from eight samples with the standard error. Bars with different letters significantly differ ( pb0.05).

5 S.-P. Yeh et al. / Aquaculture 243 (2005) Fig. 2. Hemolymph protein levels (mg ml 1 ; A), oxyhemocyanin (mmol l 1 ; B), the ratio of oxyhemocyanin to protein (%; C), and po 2 (mm Hg; D) of M. rosenbergii after 8 days of exposure to different concentrations of trichlorfon. Statistical descriptions are the same as in Fig. 1. Fig. 3. Hemolymph ph (A), pco 2 (mm Hg; B), TCO 2 (mmol l 1 ; C), and HCO 3 (mmol l 1 ;D)ofM. rosenbergii after 8 days of exposure to different concentrations of trichlorfon. Statistical descriptions are the same as in Fig. 1.

6 388 S.-P. Yeh et al. / Aquaculture 243 (2005) exposure to of 0, 0.1, 0.2, and 0.3 mg l 1 trichlorfon, which were in the range of mmol l 1, mg ml 1, and %, respectively (Fig. 2A C). Although, no significant difference in hemolymph PO 2 was noted among the prawns exposed to 0 to 0.3 mg l 1 trichlorfon, it slightly decreased for prawns exposed to 0.3 mg l 1 trichlorfon (Fig. 2D) Acid base balance The hemolymph ph of prawns had significantly decreased following 8 days of exposure to 0.2 and 0.3 mg l 1 trichlorfon as compared to prawns held in 0.1 mg l 1 trichlorfon and the control group. Hemolymph HCO 3 and TCO 2 had significantly decreased in prawns following 8 days of exposure to 0.1 to 0.3 mg l 1 trichlorfon. Hemolymph HCO 3 had decreased by 5.6%, 5.6%, and 7.6%, and hemolymph TCO 2 had decreased by 6.6%, 5.6%, and 7.7% for prawns following 8 days of exposure to 0.1, 0.2, and 0.3 mg l 1 trichlorfon, respectively, compared to prawns kept in the control solution. On the contrary, hemolymph PCO 2 of prawns following exposure to Fig. 4. Total hemocyte count in the hemolymph (A) and phenoloxidase activity (B) in hemocytes of M. rosenbergii after 8 days of exposure to different concentrations of trichlorfon. Statistical descriptions are the same as in Fig mg l 1 trichlorfon had significantly increased by 38.3% compared to prawns held in the control solution (Fig. 3A D) Effect of trichlorfon on the immune response of M. rosenbergii No significant difference in the THC of prawns was observed among treatments. The means of THC varied from (99.0F2.21)10 5 to (104.90F3.37)10 5 cells ml 1 (Fig. 4A). Phenoloxidase activity of prawns exposed to 0.2 and 0.3 mg l 1 was significantly lower than those exposed to 0.1 mg l 1 and the control group. However, the phenoloxidase activity of prawns following 8 days of exposure to 0.2 and 0.3 mg l 1 trichlorfon had significantly decreased by 25.0% and 26.1%, respectively, compared to prawns kept in the control solution (Fig. 4B). 4. Discussion Hematological parameters, such as hemolymph protein, hemocyanin, osmolality, ion compositions, total hemocyte counts, differential hemocyte counts, phenoloxidase (PO) activity, and superoxide anion (O 2 ) vary with molt cycle in crustaceans (Mercaldo- Allen, 1991; Chen and Cheng, 1993b; Chen and Chia, 1997; Cheng et al., 2001; Cheng et al., 2003). Hemolymph protein and oxyhemocyanin contents of M. rosenbergii were highest in the premolt stage (D 0 / D 1 ) and lowest in the postmolt period (stage A), while hemolymph osmolalities, as well as Cl,Na +, and K + levels, were all lower during the postmolt and elevated during intermolt stages (Cheng et al., 2001). In addition, M. rosenbergii displayed the lowest THC and PO activity in the D3 and A stages, respectively, while both were highest in the C stage (Cheng and Chen, 2001; Cheng et al., 2003). All prawns used in this study were individually examined to determine molt stages (Peebles, 1977), and only those at intermolt (stage C) were used to minimize intrinsic variations. In crustaceans, gills are important organs of respiration as well as of osmoregulation (Mantel and Farmer, 1983; Pequeux, 1995). Previous studies suggested that epipodites are involved in the osmoregulation of penaeid shrimp (Bouaricha et al., 1991;

7 S.-P. Yeh et al. / Aquaculture 243 (2005) Lignot et al., 1997). Keeping Marsupenaeus japonicus juveniles in seawater (1100 mosm kg 1 >37 ppt) and diluted seawater (550 mosm kg 1 >19 ppt), Lignot et al. (1997) reported that fernitrothion decreased the osmoregulatory capacity (OC=difference between the hemolymph osmotic pressure and the osmotic pressure of the medium) at both lethal and sublethal concentrations, and the effect was dosedependent. They also indicated that histopathological changes in the epipodites and in gill lamellae (hemocytic congestion, gill lamellae necrosis, and the accumulation of particles surrounding the gill lamellae) were observed with lethal concentrations of fenitrothion. In general, organophosporous compounds produce specific inhibition of acetylcholinesterase (AchE), which in some cases is accompanied by inhibition of NET (neuro target esterase). Modifications in NTE activity (esterase activity at ph 8) are responsible for the apparition of the syndrome of delayed neurotoxicity induced by some organophosphorous compounds (Johnson, 1977; Repetto et al., 1988). Repetto et al. (1988) reported that NTE activity was inhibited by progression of time of exposure to 0.1 Ag ml 1 trichlorfon, which showed an effect typical of some organophosphates. Lignot et al. (1998) also reported that AchE activity decreased in Litopenaeus stylirostris treated with fenitrothion. In this study, hemolymph osmotic pressure of prawns had significantly decreased following exposure to concentration of 0.3 mg l 1 trichlorfon as compared to the controls after 8 days. It is possible that the decrease resulted from either histological changes or direct inhibition of the osmoregulation mechanisms, but further clarification is required. In addition, the rate of decrease of the hemolymph chloride concentration was close to that of osmotic pressure. Thus, it is suggested that the decrease in hemolymph osmolality mainly results from a decrease in the hemolymph chloride concentration. In crustaceans, exposure to pollutants not only disrupts the ionic balance but also induces extracellular acid base changes. A large disturbance of the hemolymph acid base balance was found in shore crabs C. maenas exposed to copper. At the sublethal level of 0.5 mg l 1 copper, Boitel and Truchot (1989) indicated that the blood ph of C. maenas slowly but significantly decreased from 7.80 to a minimum of 7.45 by 12 days, and then recovered to values not significantly different from the control by 20 days. Blood PCO 2 transiently decreased during the first 4 days, resumed control values at 8 days, and remained at this level for the remainder of the exposure period. Bicarbonate concentration rapidly decreased from to 4.79 meq l 1 at 4 days, and then slowly recovered and reached a value not significantly different from the controls at 20 days. However, the lactate concentration remained very low. Thus, they suggested that the acidosis was nonlactic and was partly compensated for by transitory hypocapnia, which may be linked to increased gill ventilation. At a toxic level of copper, respiratory gas exchanges are additionally affected, most probably because of anatomical gill damage (Boitel and Truchot, 1989). Lignot et al. (1997) indicated that after 96 h of exposure to fenitrothion at a concentration higher than 0.5 Ag l 1, particles had accumulated between the branchial lamellae of Penaeus japonicus. The presence of particles surrounding the gill lamellae may be a consequence of a lack of ventilation in the branchial cavity due to the inhibitory action of the pesticide on the nervous system. In this study, hemolymph ph, HCO 3, and TCO 2 of prawns decreased significantly upon exposure to trichlorfon at greater than 0.2, and 0.1 and 0.1 mg l 1, after 8 days. Hemolymph pco 2, on the contrary, had significantly increased in prawns following 8 days of exposure to 0.3 mg l 1 trichlorfon. However, no significant difference in hemolymph po 2 was found among prawns exposed to 0 to 0.3 mg l 1 trichlorfon, but it had slightly decreased in prawns at a concentration of 0.3 mg l 1. These changes may be related to decreased ventilation and impeded respiratory gas exchange, leading to respiratory disturbances, resulting from inhibition of respiratory mechanisms and from damage to respiratory organism epithelial cells. Decreases in ph and HCO 3 of the hemolymph induced an increase in the PCO 2 level benefiting excretion of CO 2 in hemolymph resulting in a decrease in TCO 2. Unfortunately, the lactate level of the hemolymph was not determined. Circulating hemocytes are also known to be affected by extrinsic factors like temperature, ph, salinity, dissolved oxygen, and ammonia in several species of decapod crustaceans (Truscott and White,

8 390 S.-P. Yeh et al. / Aquaculture 243 (2005) ; Le Moullac et al., 1998; Le Moullac and Haffner, 2000, Cheng and Chen, 2001). M. rosenbergii reared at temperatures of and C had significantly higher THC values than those reared at and C, and prawns reared at a ph of had significantly higher THC values than those reared at phs of and (Cheng and Chen, 2000). There were no significant differences in THC among prawns at the beginning and after 168 h of exposure to ammonia-n in the range of mg l 1 (Cheng and Chen, 2002) or to nitrite-n in the range of mg l 1 (Cheng et al., 2002). There were no significant differences in THC and DHC among prawns at the beginning, or following 96 h of exposure to copper sulfate in the range of mg l 1 (Cheng and Wang, 2001). This study also indicated that no significant difference in THC was observed among the prawns after an 8-day exposure to trichlorfon concentrations in the range of mg l 1. Smith and Johnston (1992) reported that common shrimp C. crangon following exposure to PCB 15 (polychlorinated biphenyl 15) produced significant decreases in THC and phenoloxidase activity. Both the phenoloxidase activity and THC of M. rosenbergii were significantly higher at ph and C and were significantly lower at ph and , C, and in freshwater (Cheng and Chen, 2000). Phenoloxidase activity was significantly lower for M. rosenbergii exposed to 0.55, 1.68, and 3.18 mg l 1 ammonia-n (Cheng and Chen, 2002), to 0.1, 0.2, 0.3, and 0.4 mg l 1 copper sulfate (Cheng and Wang, 2001), and to 0.2 and 0.3 mg l 1 trichlorfon after 8 days in this study. Le Moullac and Haffner (2000) indicated that the defense functions belonging to phenoloxidase activity and peroxinectin are reduced at the level of gene expression in L. stylirostris when exposed to ammonia. It is expected that a similar reaction may occur in trichlorfon-exposed M. rosenbergii. In conclusion, the observation that trichlorfon in water reduces the hemolymph osmolality, Cl, ph, HCO 3, and TCO 2, and increases PCO 2 of M. rosenbergii suggests that trichlorfon disturbs osmoregulation and the acid base balance of prawns. In addition, a decrease in the hemocyte phenoloxidse activity and respiratory burst suggests that the immune resistance of prawns might be depressed by trichlorfon. Acknowledgements The paper was supported by the National Science Council (NSC B ), Republic of China. Special thanks go to the respected referees and to Ms. Ya-Chuan Wong for her proofreading. References Bainy, A.C.D., Biochemical responses in penaeids caused by contaminants. Aquaculture 191, Bambang, Y., Charmantier, G., Thuet, P., Trilles, J.P., Effect of cadmium on survival and osmoregulation of various developmental stages of the shrimp Penaeus japonicus (Crustacea: Decapoda). Mar. Biol. 123, Bambang, Y., Charmantier-Daures, M., Trilles, J.P., Charmantier, G., Effect of copper on survival and osmoregulation of various developmental stages of the shrimp Penaeus japonicus Bate (Crustacea: Decapoda). Aquat. Toxicol. 33, Bhagyalakshmi, A., Sreennivasula Reddy, P., Ramamurthi, R., In vivo sub-acute physiological stress induced by sumithion on some aspects of oxidative metabolism in the freshwater crab. Water Air Soil Pollut. 23, Boitel, F., Truchot, J.P., Effects of sublethal and lethal copper levels on hemolymph acid base balance and ion concentrations in the shore crab Carcinus maenas kept in undiluted seawater. Mar. Biol. 103, Bouaricha, N., Charmantier, G., Charmantier-Daures, M., Thuet, P., Trilles, J.P., Ontogenèse de l osmorègulation chez la crevette Penaeus japonicus. Cah. Biol. Mar. 32, Bradford, M.M., A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal. Biochem. 72, Caldwell, R.S., Osmotic and ionic regulation in decapod Crustacea exposed to methoxychlor. In: Vernberg, F.J., Vernberg, W.B. (Eds.), Pollution and Physiology of Marine Organisms. Academic Press, New York, pp Cebrian, C., Andreu-Moliner, E.S., Fernandez-Casalderrey, A., Ferrando, M.D., Acute toxicity and oxygen consumption in the gills of Procambarus clarkii in relation to chlorpyrifos exposure. Bull. Environ. Contam. Toxicol. 49, Charmantier, G., Bouaricha, N., Charmantier-Daures, M., Thuet, P., Trilles, J.P., Salinity tolerance and osmoregulation capacity as indicators of the physiological state of penaeid shrimps. Spec. Publ.-Eur. Aquac. Soc. 10, Chen, J.C., Chen, K.W., Hemolymph oxyhemocyanin, protein levels, acid base balance, and ammonia and urea excretions of Penaeus japonicus exposed to saponin at different salinity levels. Aquat. Toxicol. 36, Chen, J.C., Cheng, S.Y., Hemolymph osmolality, acid base balance and shift of ammonotelic to ureotelic excretory pattern of Penaeus japonicus exposed to ambient ammonia. Comp. Biochem. Physiol. 106C,

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