Aquatic Toxicology 74 (2005) 160–171 The toxic effect of phthalate esters on immune responses of giant freshwater prawn (Macrobrachium rosenbergii) via oral treatment Wen-Liang Chen, Hung-Hung Sung ∗ Department of Microbiology, Soochow University, Taipei 111, Taiwan, ROC Received 23 November 2004; received in revised form 28 March 2005; accepted May 2005 Abstract A previous in vitro study has indicated that four phthalate esters (PAEs) could damage hemocytes and decreases the cellular immunity of prawns [Sung, H.H., Kao, W.Y., Su, Y.J., 2003 Effects and toxicity of phthalate esters to hemocytes of giant freshwater prawn, Macrobranchium rosenbergii Aquat Toxicol 64, 25–37] The aim of this study was to investigate the in vivo effect of four PAEs, diethyl phthalate (DEP), dihexyl phthalate (DHP), dipropyl phthalate (DPrP) and diphenyl phthalate (DPP) on the defense system of the giant freshwater prawn, M rosenbergii PAE dissolved in corn oil was continuously fed to prawns for days and five immune parameters (total hemocyte count, THC; ratio of granulocytes to hyalinocytes, G/H; intrahemocytic total phenoloxidase activity, POT ; intracellular superoxide anion (O2 − ) production; transglutaminase (TGase) activity) were separately detected on days 1, and In addition, mortality was determined on days and after challenging the prawns with Lactobacillus garvieae In comparison with untreated prawns, the results showed that DHP demonstrated the lowest toxicity in that it only influenced the PO activity and O2 − production before days after treatment and caused 6.6% mortality on day DEP decreased G/H, POT and TGase activity on day and reduced THC, G/H and POT and caused 16.6% mortality on day 4; however, on day 8, it increased O2 − production and caused no mortality In the DPrP-treated group, a reduction of all the immune reactions apart from TGase activity and 22.2% mortality were detected on day As for the effect of DPP, results showed that it decreased all the immune parameters apart from THC on days and 4, but caused no mortality on day 4; but on day 8, an increase of O2 − production and 17.7% mortality were detected These results indicated that the immune reactions of prawns were variable due to the different toxic effects of PAEs In addition, it was found that, on day after treatment, the three PAEs, DHP, DPrP and DPP increased O2 − production and did not influence the other four reactions, but mortality was detected in these groups These results suggest that other physiological responses may also be affected to increase the susceptibility of prawns to pathogens © 2005 Elsevier B.V All rights reserved Keywords: Phthalate esters; Endocrine disruptor (ED); Chemical pollutant; Prawn; Immunity; Toxicity Introduction ∗ Corresponding author Tel.: +886 28819471x6860; fax: +886 28831193 E-mail address: hhsung@scu.edu.tw (H.-H Sung) 0166-445X/$ – see front matter © 2005 Elsevier B.V All rights reserved doi:10.1016/j.aquatox.2005.05.008 In many countries of the world (e.g Taiwan), prawn cultivation has an economic importance in the W.-L Chen, H.-H Sung / Aquatic Toxicology 74 (2005) 160–171 aquaculture industry In Taiwan, production of the giant freshwater prawn, Macrobrachium rosenbergii, reached a peak (16,196 t) in 1991, but decreased in 1992 and 1993 and was only 7223 t in 1999 (Primavera, 1997; Rosenberry, 1998) The decline in production of farmed prawns resulted from outbreaks caused by yeast infection in the cool season and bacterial infection in the hot season (Hsu, 1993; Cheng and Chen, 1998) However, poor pond management or deterioration of water quality can also induce disease outbreaks and thus, decrease production (Liao, 1989; Chamberlain, 1997) Previous studies have demonstrated that the appearance of harmful factors, such as toxins and changes in dissolved oxygen, salinity or temperature, created an environment which increased the susceptibility of prawns to disease (Lightner, 1988, 1996; Lightner and Redman, 1998; Brock, 1992; Brock and Lightner, 1990; Le Moullac et al., 1998; Cheng et al., 2002) Other studies have reported that both the heavy metals released from sediments and the contamination of shrimp farms due to pesticides and pollutants from agriculture or industrial activities may decrease the resistance of the shrimp to disease (Primavera, 1993; Flaherty and Karnjanakesorn, 1995) Phthalate esters (PAEs) are widely used industrial chemicals which serve as important additives to impart flexibility to polyvinyl chloride (PVC) resins and become widely diffused in the environment (Jobling et al., 1995) via the manufacturing process PAE concentrations have been reported in the range of 0.1–300 g/l for surface marine waters (Mayer et al., 1972; Giam et al., 1978; Gledhill et al., 1980; Fatoki and Vermon, 1990) and freshwater sites (Gledhill et al., 1980) and from 0.1 ng/g to 100 g/g for river sediments (Thuren, 1986; Tan, 1995) In Taiwan, PAEs have been found to be widely distributed in river water and sediment and soil (Liu et al., 2000; Yuan et al., 2002) and four PAEs, diethyl phthalate (DEP), dibutyl phthalate (DBP), benzyl butyl phthalate (BBP) and di-(2-ethylhexyl) phthalate (DEHP), have also been found to accumulate in fish (Chang et al., 2004) The United States Environmental Protection Agency (USEPA) and its counterparts in several other countries have classified the most commonly occurring PAEs as priority pollutants and endocrine disrupting compounds (ECPI, 1996) Numerous experiments have shown that bioaccumulation of PAEs occurs in the aquatic and terrestrial food chain (reviewed from 161 Staples et al., 1997a) DEHP has been shown to be absorbed, metabolized and largely accumulated in the tissue of a penaied shrimp via oral administration and this process had a linear relationship with the dose according to the dose range studied (Hobson et al., 1984) Many studies have demonstrated the acute toxicity and chronic toxicity of phthalate esters to microorganisms, algae, aquatic invertebrates and fish (reviewed by Staples et al., 1997b) In crustaceans, hemocytes play a crucial role in non-specific cellular immunity against pathogens and parasites, which function as the primary immune responses including phagocytosis, encapsulation, nodule formation and cytotoxic mediation (Anderson, 1992) The circulating phagocytic hemocytes have received considerable attention as the primary cellmediated immunity mechanism Sung and Song (1996) have indicated that phagocytosis is performed by hyalinocytes in prawns According to previous studies (Săoderhăall, 1982; Ratcliffe et al., 1985; Smith and Săoderhăall, 1991), several proteins associated with the hemocyte prophenoloxidase-activating system (PAS), which is released from induced semigranular and granular cells, play an important role in non-self recognition and host defense for elimination of foreign particles in the body cavity of crayfish and other crustaceans (Săoderhăall et al., 1994) Furthermore, a coagulation system is essential in invertebrates to prevent excess blood loss from a wound and to obstruct microorganisms, which would otherwise invade the wound In the wound area, the clottable protein oligomerizes to prevent hemolymph loss through breaks in the exoskeleton and dissemination of bacteria throughout the body In crayfish hemocytes, both semigranular and granular cells, as well as the muscle tissues contain TGase (Hisanori et al., 1997) Therefore, the phagocytic activity, the activation of PAS and TGase may be used as the defense indicators in crustaceans, including cultured prawns (Rodr´ıguez and Moullac, 2000) Before the study of Sung et al (2003), which indicated that PAEs could damage hemocytes and reduce the cellular immunity of prawns by means of in vitro exposure experiments, no one knew the effects of PAEs and their derivates on the defense reactions of crustaceans such as cultured prawns Therefore, in this study, we further investigated the effects of four PAEs, diethyl phthalate (DEP), dihexyl phthalate (DHP), dipropyl phthalate (DPrP) and diphenyl 162 W.-L Chen, H.-H Sung / Aquatic Toxicology 74 (2005) 160–171 phthalate (DPP), which are highly toxic to the hemocytes of giant freshwater prawns (M rosenbergii) (Sung et al., 2003) on the defense functions of prawns given these PAEs orally for days Five immune parameters, comprising total hemocyte count (THC), ratio of granulocytes to hyalinocytes (G/H), intrahemocytic total phenoloxidase activity (POT ), intracellular superoxide anion (O2 − ) production and transglutaminase (TGase) activity, were used to evaluate the effect of PAEs on prawns; this was further defined by recording the resultant mortality when the prawns were challenged with a pathogen purchased from Life Technologies Inc (GIBCO BRL 21200-076) The four PAEs were separately dissolved in corn oil to a concentration of 10,000 ppm as a stock solution, which was stored at room temperature prior to the experiments 2.2 Animals 2.1 Chemicals and preparation 2.2.1 Acclimation M rosenbergii, weighing 15–20 g/prawn and purchased on separate days from local prawn farms, were acclimated in 360 l glass aquaria containing fresh pond water at 28 ◦ C for at least days prior to the experiments Prawns were fed with synthetic feed pellets twice a day, an amount equivalent to 5% of their body weight The stocking densities were maintained at 20 prawns per aquarium Four phthalate esters were used in this study as shown in Fig These were diethyl phthalate (Chem Service Co., O-525), dihexyl phthalate (Chem Service Co., Pt-21), diphenyl phthalate (Aldrich Chem Co., 10588-0) and dipropyl phthalate (Chem Service Co., F2158) The hemocyte-culture medium, M-199, was 2.2.2 Oral treatment with phthalate esters For each type of PAE, prawns were divided into three experimental groups and one control group The three experimental groups were continuously fed with the PAE for 1, and days, respectively Each prawn was fed with 100 l of the PAE (1 g/l) once a day Materials and methods Fig The chemical structures and properties of the four phthalate esters used in this experiment MW, molecular weight; (–) no detection Data of MW and aqueous solubility are cited from the review of Staples et al (1997b) and the study of Cousins and Mackay (2000), respectively W.-L Chen, H.-H Sung / Aquatic Toxicology 74 (2005) 160–171 using a syringe with a soft silicon tube The control group was fed with an equal volume of corn oil 2.3 Preparation of hemolymph and hemocyte samples To evaluate the immune reactions of PAE-treated prawns, both hemolymph and hemocyte samples were prepared A hemolymph sample (0.5 ml) was drawn from the first abdominal segment of each prawn with a 25 G hypodermic needle containing 0.5 ml of anticoagulant (10 mM Tris–HCl, 100 mM trisodium citrate, 10 mM EDTA, 82 mM glucose, 20 mM NaCl, pH 7.56) with an osmolarity of 420 ± 20 mOsm/kg Hemocyte suspensions were prepared according to a procedure described by Song and Hsieh (1994) Briefly, the hemolymph sample was centrifuged at 300 × g for 10 at ◦ C and the resultant hemocyte pellet was suspended in ml of calcodylate (CAC) buffer (pH 7.0) or M-199 medium with an osmolarity of 420 ± 20 mOsm/kg Hemocyte concentrations were adjusted according to different experiments Only, cell suspensions with a viability of 85% or more, tested by trypan blue exclusion (0.05% in 0.01 M PBS), were used to determine the immune reactions in this study The hemolymph sample was used to determine the total hemocyte count, the differential hemocyte count (DHC) and transglutaminase activity The hemocyte sample in CAC buffer or M-199 medium was used to examine the intrahemocytic total phenoloxidase activity and the production of superoxide (O2 − ), respectively 2.4 Determination of defense responses 2.4.1 Total hemocyte count and differential hemocyte count The total number of hemocytes in a mixture of 10 l of hemolymph and trypan blue (Sigma, T-6164; 0.05% trypan blue in 0.01 M PBS, pH 7.56, osmolarity 420 ± 20 mOsm/kg) was counted with a hemocytometer (Hausser Scientific, Bright-Line) under a light microscope (NIKON, ECLIPSE, E800) at a magnification of 100× As for determining the differential hemocyte count, 10 l of hemolymph was fixed with an equal volume of 20% neutralized formaldehyde for at room temperature After adding 20 l of 0.5% Evans’ Blue (Sigma, E-2129), 20 l of mixture was smeared 163 on a cover glass (18 mm × 18 mm) The numbers of both hyalinocytes (HC) and granulocytes (GC; composing semigranular and granular cells) were counted for a total of 50–100 cells with a light microscope at a magnification of 400× The ratio of GC to HC was calculated by the formula: G/H = number of GC/number of HC The values given in this study were the means of average relative THC or G/H ± the standard deviation (S.D.) of the mean of three replicates from more than 18 individuals All of the values of average relative THC or G/H from both experimental (PAE-treated) group and control (corn oil-treated) group were compared to the untreated group The value of relative THC or G/H was calculated using the formula: THC or G/H of PAE-treated prawn/THC or G/H of untreated prawn 2.4.2 Intrahemocytic total phenoloxidase activity Before assay of the phenoloxidase activity, hemocyte lysate supernatant (HLS) of prawn was prepared according to procedures described by Sung et al (1996) Briefly, the hemocyte suspension in 0.01 M CAC buffer was homogenized using a sonicator (Vibra cell, AC-600) equipped with a microtip and centrifuged at 43,000 × g for 30 at ◦ C and the HLS was then collected The resultant HLS was used as an enzyme source and its protein concentration was determined by the Protein assay Kit II (Bio-Rad, USA) Intrahemocytic total phenoloxidase activity, which resulted from all the intrahemocytic prophenoloxidase (proPO) being completely catalyzed to form PO when HLS was treated with trypsin, was assayed as described by Sung et al (2004) After a mixture of 25 l of HLS and an equal volume of trypsin solution (1 mg/ml of 0.01 M CAC buffer; Sigma, T-4665) was incubated at 30 ◦ C for 15 min, 200 l of freshly prepared substrate solution, 0.01 M of l-3,4-dihydroxyphenylalanine (lDOPA, Sigma, D-9628) in CAC buffer, was added and reacted for The optical absorbance at 490 nm was measured One unit of enzyme activity was defined as an increase in absorbance of 0.001/(min mg) of protein (Săoderhăall and Unestam, 1979) The values given in this study were the means of the average relative POT activity (RA) ± the standard deviation of the mean of three replicates from more than 18 individuals The value of RA was calculated using the formula: POT of HLS from PAE-treated prawn/POT of HLS from untreated prawn 164 W.-L Chen, H.-H Sung / Aquatic Toxicology 74 (2005) 160–171 2.4.3 NBT assay Since the production of superoxide anions (O2 − ) and H2 O2 contribute to the initiation of a proinflammatory event, in this study, O2 − production assayed by NBT reduction was used as a defense parameter This assay was conducted as described by Song and Hsieh (1994) Reactions occurred in flat-bottomed 96-well microtiter plates, with each well coated with 100 l of poly-l-lysine solution (0.2%, Sigma P-1274), at room temperature for 30 Hemocyte suspension (100 l) was added to each well (106 cells/well) and cytocentrifuged (Kubota, KN-70) at 300 × g for 10 at ◦ C After removing the supernatant, 100 l of zymosan A (from Saccharomyces cerevisiae; Sigma Z-4250) suspension (107 particles/well) was added and the mixture was incubated at 28 ◦ C for 30 After washing with M-199, the hemocytes were stained with 200 l nitroblue tetrazolium solution (NBT, 0.15% in M-199) for 30 at 28 ◦ C The staining reaction was terminated by removing the NBT solution and then adding 200 l of absolute methanol (Merck) After three washings with 70% methanol, the hemocytes were air-dried and coated with a solution of 120 l KOH (2 M) and 140 l dimethyl sulfoxide (DMSO) to dissolved cytoplasmic formazan, and then, the mixture was measured at 630 nm with a microplate reader (Molecular Device, Emax) In order to determine the reproducibility of the results, hemocytes collected from more than 18 prawns were individually assayed The ratio of OD630 from the treated hemocytes to the OD630 of the untreated hemocytes was used as an index for comparing the effects of different PAEs on both O2 − generation and reductase activity, since either O2 − or cellular reductase can reduce NBT to the monoformazan (Tarpey and Fridovich, 2001) The TGase activity assay was performed according to procedures described by Seiving et al (1991) First, 200 l of the casein solution (1 mg/ml of sodium carbonate buffer at pH 9.8) was added to each well of a flat-bottomed 96-well microtiter plate Before washing with washing buffer (50 mM Tris–HCl, 0.15 M NaCl and 0.1% Tween-80) containing mg/ml of dithiothreitol (DTT; Amersham Pharmacia Biotech), they were incubated at room temperature overnight Thereafter, a hemolymph sample (100 l) was added to one caseincoated well and then serially diluted two-fold with 50 mM Tris–HCl (osmolarity 420 ± 20 mOsm/kg) After supplementation with 100 l of reagent Ca2+ (6 ml of 5.8 U/ml of thrombin, ml of 50× diluted biolinated casein, ml CaCl2 ), the mixture was incubated at 37 ◦ C for 20 Following washing twice with washing buffer, 100 l of streptavidin-labeled alkaline phosphatase (Sigma, S2890) solution was added and then incubated at 28 ◦ C for 45 After washing to remove the unbound biotin, 100 l of pnitrophenylphosphate solution (1 mg/ml) was added and incubated at 37 ◦ C for 30 Color development was measured at a wavelength of 405 nm Instead of the hemolymph sample, a guinea pig liver TGase (Sigma, T5398) solution was added as a standard enzyme to calculate the standard curve of OD405 versus enzyme activity (unit/mg) The protein concentration of hemolymph samples was determined by the Protein Assay Kit II (Bio-Rad, USA) The values given in this study were the means of average relative TGase activity ± the standard deviation of the mean of three replicates from more than 18 individuals The value of relative TGase activity was calculated using the formula: TGase activity of PAE-treated prawn/TGase activity of untreated prawn 2.4.4 Transglutaminase activity The biotin-labeled casein used as a substrate in the transglutaminase activity assay was prepared according to procedures described by Song et al (2003) Briefly, a mixture of 200 l of biotinamidocaproyl hydrazide (Sigma, B-3770) solution (100 mg/ml of DMSO) and 10 ml of casein (Sigma, C-5890) solution was stirred at room temperature for 12 h and then dialyzed in 50 mM Tris–HCl buffer (pH 7.4) in a 14 K dialysis tube at ◦ C overnight After dialysis, the biotin-labeled casein solution was diluted 20× with 50 mM Tris–HCl buffer (pH 7.4) and stocked at ◦ C 2.5 Susceptibility of prawns to pathogen 2.5.1 Preparation of bacterial suspension The pathogen Lactococcus garvieae used in the study was isolated from diseased M rosenbergii with whitish musculature syndrome (Cheng and Chen, 1998; Chen et al., 2001) and was a kind gift from Dr Winton Cheng (Department of Aquaculture, National Pingtung University of Science and Technology) A stock kept in 50% glycerol at −20 ◦ C was thawed at 37 ◦ C for min; ml of the stock was inoculated to a 250 ml flask containing 50 ml of brain heart infusion W.-L Chen, H.-H Sung / Aquatic Toxicology 74 (2005) 160–171 broth (BHIB, Difco) and incubated overnight at 28 ◦ C, 150 rpm Subsequently, ml of bacterial solution was subcultured into 50 ml of BHIB and the mixture was incubated at 28 ◦ C until bacterial growth reached the late-log phase Following centrifugation at 3000 × g and ◦ C for 15 min, the pellet was washed once and suspended in sterile 0.01 M phosphate-buffered solution (0.01 M PBS, osmolarity 420 ± 20 mOsm/kg, pH 7.56) The concentration of bacterial cells was adjusted to × 108 cells/ml via a cell counting method (Petroff Hausser Counting Chamber, Hausser Scientific Co., USA) using a light microscope at a magnification of 1000× 2.5.2 Challenge experiment In the challenge experiment for each PAE, each batch of prawns was divided into four groups with 12 or 15 prawns in each group; two groups were continuously fed with PAE for days and the other two groups were fed for days On day after treatment, the two 4-day-treated groups were divided into one experimental group, which was injected with 100 l of the bacterial suspension at the dose of 105 cells/g of prawn (1/10 LD50 ) and one corresponding control group, which was injected with an equal volume of sterile PBS The injection dose was able to cause infection of healthy untreated prawns, but not induce death (Sung and Sun, 2002) The same challenge experiment was performed on day after treatment After injection, prawns were held in aquaria at 28 ◦ C with aeration The number of dead prawns was recorded twice daily, until no prawns died for days Mortality percentages were calculated using the formula: (total number of dead prawns number of non-specific death)/(total number of prawns number of non-specific death) × 100%, where the non-specific death represents the number of prawns that died within the first 12 h after injection 2.6 Detection of anorexia To evaluate whether the effect of PAEs on prawn immunity is caused indirectly by an influence on prawn appetite, in both PAE-treated and untreated groups, the amount of synthetic feed pellets taken by prawns was recorded After feeding, the quantity of remaining synthetic feed pellets was calculated at h intervals for 12 h 165 2.7 Statistics Outbreed prawns were used as samples in this study and the physiological status of each sample used was significantly different All results from the experiments, including the five immune parameters, total hemocyte count, ratio of granulocytes to hyalinocytes, intrahemocytic total phenoloxidase activity, intracellular superoxide (O2 − ) production and transglutaminase activity, as well as the mortality of prawns challenged with the pathogen, also showed great variations among individuals Therefore, the data from the means of the average relative value of three replicates from at least 15 individuals were statistically analyzed with regard to the effects of PAEs on immune reactions and the susceptibility of prawns by using ANOVA and Duncan’s multiple range tests with a specified significance level of p < 0.05 Results To determine whether prawn appetite is affected by corn oil or PAE treatment, the amount of synthetic feed pellets taken by prawns was recorded during 12 h In this observation period, corn oil- and PAEtreated prawns took fewer pellets in the first h and recovery of normal appetite was observed during the second h Furthermore, we still observed but did not record the data during the whole experimental period Appetite did not differ between the treated and untreated groups Therefore, in this study, the effects of PAEs on immune responses are apparently independent of prawn appetite Following the feeding of prawns with PAEs, five immune parameters were detected on days 1, and The total hemocyte counts were not different from corresponding control groups on days and after treatment with the four PAEs; however, a significant reduction in the percentage of THC was detected in the DEP- and DPrP-treated groups (p < 0.05) on day 4, the figures being 77.7 and 72.7%, respectively (Table 1) As for the ratio of granulocytes to hyalinocytes, it was found that the percentage of G/H was decreased by 20.7 and 25.3% on days and 4, respectively, after DEP treatment, by 23.5% on day in the DPP-treated group and by 34.5% on day in the DPrP group (p < 0.05) (Table 2) 166 W.-L Chen, H.-H Sung / Aquatic Toxicology 74 (2005) 160–171 Table Changes in total hemocyte count (THC) of prawns given phthalate esters (PAEs) orally Days of treatment Relative THC (×100%) Controla (n = 18) (n = 26) (n = 18) 100 ± DEP 22.5N DHP 109 ± 10.5N 22.6N 18.5b 100 ± 100 ± 28.3N 77.7 ± 100.3 ± 27.0N DPrP 113 ± 24.7N DPP 92.8 ± 19.1 18.0N 98.6 ± 102.9 ± 18.9N N 121.7 ± 24.2N a 104.0 ± 35.7N 92.1 ± 30.6N 72.7 ± 19.3 91.2 ± 19.4 N DEP, diethyl phthalate; DHP, dihexyl phthalate; DPrP, dipropyl phthalate; DPP, diphenyl phthalate; n, the number of prawns used in this experiment for each treated group; N, no difference from the corresponding control a Prawns in control group were fed corn oil without PAE b The data from the means of relative value of three replicates from more than 18 prawns decrease were statistically analyzed using ANOVA and Duncan’s multiple range tests with a specified significance level of p < 0.05; the error bars represent standard deviation (S.D.) As shown in Fig 2, the total phenoloxidase activity was reduced by 16.2 and 29.7% on days and 4, respectively, after DEP treatment (p < 0.05), but was no different from the corresponding control group on day In the other three PAE-treated groups, a significant reduction of POT was detected on day (p < 0.05), but neither on days nor As for the expression of superoxide (O2 − ) production and reductase activity (superoxide/reductase) analyzed by NBT assay, the results showed that it was not affected on days and after DEP treatment, but was significantly enhanced on day (p < 0.05) (Fig 3) In the other three PAEtreated groups, the expression of superoxide/reductase was decreased on both days and 4, but increased on day after PAE treatment (Fig 3) Transglutaminase activity was significantly reduced on day after DEP and DPP treatment, by 33.5 and 42%, respectively; however, there were no differences from the corresponding control groups on days and (p < 0.05) (Fig 4) No change in TGase activity was found in Table Changes in ratio of granulocytes to hyalinocytes (G/H) of prawns given phthalate esters (PAEs) orally Days of treatment Relative G/H (×100%) Controla (n = 18) (n = 26) (n = 18) 100 ± 26.6N 25.9N 100 ± 100 ± 21.7N DEP DHP 16.3b 79.3 ± 74.7 ± 21.2b 154.4 ± 108.8N DPrP 18.3N 86.0 ± 86.2 ± 18.7N 144 ± 124.2N DPP 25.0N 90.5 ± 65.5 ± 25.2b 134.3 ± 76.3N 77.5 ± 19.1b 87.4 ± 34.0N 141.5 ± 121.5N DEP, diethyl phthalate; DHP, dihexyl phthalate; DPrP, dipropyl phthalate; DPP, diphenyl phthalate; n, the number of prawns used in this experiment for each treated group; N, no difference from the corresponding control a Prawns in control group were fed corn oil without PAE b The data from the means of relative value of three replicates from more than 18 prawns decrease were statistically analyzed using ANOVA and Duncan’s multiple range tests with a specified significance level of p < 0.05; the error bars represent standard deviation (S.D.) Table Susceptibility of PAE-treated Macrobrachium rosenbergii to Lactococcus garvieae Days of treatment (n = 12) (n = 15) Mortalitya (%) Controlb DEP DHP DPrP DPP 0±0 0±0 16.6 ± 0±0 0±0 6.6 ± 22.2 ± 5.8 20 ± 0±0 17.7 ± 3.26 DEP, diethyl phthalate; DHP, dihexyl phthalate; DPrP, dipropyl phthalate; DPP, diphenyl phthalate; n, the total number of prawns used in the challenge experiment for each PAE-treated group a Percentage of mortality was calculated by the following formula: death rate (%) = (total number of deaths − number of non-specific deaths)/(total number of prawns − number of non-specific deaths) The data were the means of mortality ± the standard deviation (S.D.) of three replicates from more than 12 prawns b PAE-treated prawns were injected with sterile PBS without bacterial cells W.-L Chen, H.-H Sung / Aquatic Toxicology 74 (2005) 160–171 Fig Changes in intrahemocytic total phenoloxidase activity (POT ) of prawns on days 1, and after PAE treatment DEP, diethyl phthalate; DHP, dihexyl phthalate; DPrP, dipropyl phthalate; DPP, diphenyl phthalate The data from the means of average relative POT ± the standard deviation (S.D.) of three replicates from more than 18 prawns were statistically analyzed using ANOVA and Duncan’s multiple range tests with a specified significance level of p < 0.05; the error bars represent standard deviation D, significant decrease compared to activity of the corresponding control; N, no difference from activity of corresponding control either the DHP- or the DPrP-treated group With further statistical analysis of Pearson correlation, the POT , superoxide/reductase and TGase activity in this study were shown to have a significant positive correlation with THC, percentage of granulocyte and G/H (p < 0.01) Finally, to evaluate the effects of PAEs on the susceptibility of prawns to pathogens, prawns were challenged with L garvieae (×105 cells/g of prawn) via injection on days and after PAE treatment and the subsequent mortality was determined As shown in Table 3, on day after treatment, the mortality of DEPand DPrP-treated prawns was 16.6 and 22.2%, respectively, and all of the DHP- and DPP-treated prawns survived However, on day 8, the DHP- and DPP-treated groups had mortalities of 6.6 and 17.7%, respectively In addition, the DPrP-treated group continued to have a mortality of 20% In this experiment, the mortality of all control groups was 0% (Table 3) 167 Fig Changes in intrahemocytic superoxide production in prawns on days 1, and after PAE treatment assessed by nitroblue tetrazolium (NBT) assay DEP, diethyl phthalate; DHP, dihexyl phthalate; DPrP, dipropyl phthalate; DPP, diphenyl phthalate The data from the means of average relative OD630 of three replicates from more than 18 individuals were statistically analyzed using ANOVA and Duncan’s multiple range tests with a specified significance level of p < 0.05; the error bars represent standard deviation (S.D.) D, significant decrease; E, enhancement, compared to activity of the corresponding control; N, no difference from activity of the corresponding control Discussion Increasing evidence has indicated that many substances, which are degraded from chemical pollutants but not biologically decomposed in sewage treatment works, are often not acutely toxic to exposed aquatic animals when they are emitted into water, but lead to a chronic intoxication resulting in tissue alterations, including the formation of neoplasias There is a developing awareness that in both fish and mollusks, diseases in populations are linked to environmental changes or coastal marine pollution There is considerable evidence to support links between environmental changes (including contaminants), non-infectious diseases and a depression of the immune system (Durnier and Siwicki, 1993; Pipe and Coles, 1995) 168 W.-L Chen, H.-H Sung / Aquatic Toxicology 74 (2005) 160–171 Fig Changes in hemolymph transglutaminase (TGase) activity of prawns on days 1, and after PAE treatment DEP, diethyl phthalate; DHP, dihexyl phthalate; DPrP, dipropyl phthalate; DPP, diphenyl phthalate The data from the means of average relative TGase activity ± the standard deviation (S.D.) of three replicates from more than 18 prawns were statistically analyzed using ANOVA and Duncan’s multiple range tests with a specified significance level of p < 0.05; the error bars represent standard deviation D, significant decrease compared to activity of the corresponding control; N, no difference from activity of the corresponding control In crustaceans, environmental stress from pollutants seems to be an important factor in determining the reduction of immunocompetence and is signalled by the appearance or increased prevalence of disease (Victor et al., 1990; Smith and Johnston, 1992) Effects include infection pressure from facultative microbial pathogens and reduced resistance to infection (Sindermann, 1979) Exposure of the common shrimp, Crangon crangon, to contaminated sediments, which contained numerous compounds including polychlorinated biphenyls (PCBs), polynuclear aromatic hydrocarbons (PAHs) or heavy metals, has shown that the exposed shrimp displayed an elevation in recoverable hemolymph volume and a reduction in total hemocyte count; biochemical assays also indicated reduced hemocyte phenoloxidase (PO) activity (Smith et al., 1995) In addition, the freshwater prawn, Macrobrachium idea, exposed to g/l of mercuric chloride for 30 days, exhibited hyperplastic gill lamellae engorged with hemocytes; the hemocytes were released into the interlamellar spaces through necrotic regions and then covered the entire gill lamellae (Victor et al., 1990) These findings indicate that chronic exposure to contaminated sediment has a marked effect on host defense in marine crustaceans Phthalate esters, considered as endocrine disrupting chemicals (EDCs), are found in various environmental and biological samples (Mayer et al., 1972; Giam et al., 1978; Gledhill et al., 1980; Thuren, 1986; Fatoki and Vermon, 1990; Tan, 1995; Yin and Su, 1996; Staples et al., 1997a) Previous studies have demonstrated that acute toxicity and chronic toxicity of phthalate esters were limited in lower PAEs with alkyl chain lengths