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Immune response in the tilapia, Oreochromis mossambicus on exposure to tannery effluent
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Ecotoxicology and Environmental Safety 68 (2007) 372–378 www.elsevier.com/locate/ecoenv
Immune response in the tilapia, Oreochromis mossambicus on exposure to tannery effluent$ M. Prabakarana, C. Binuramesha, Dieter Steinhagenb, R. Dinakaran Michaela, a
Centre for Fish Immunology, Postgraduate and Research, Department of Zoology, Lady Doak College, Madurai-625 002, Tamil Nadu, India b Fish Disease Research unit, Centre for Infection Medicine, School of Veterinary Medicine, Bu¨nteweg 17, 30559 Hannover, Germany Received 26 February 2006; received in revised form 2 November 2006; accepted 18 November 2006 Available online 29 January 2007
Abstract The objective of the study was to investigate the effect of chronic exposure to sublethal concentrations of tannery effluent (TE) on the specific immune response and nonspecific immunity in tilapia, Oreochromis mossambicus. The effluent from the tannery was collected directly from a chrome-tanning factory situated in Dindigul district, Tamil Nadu, India. Apart from chromium (88.2 ppm), the effluent contained appreciable amount of calcium carbonate and sodium sulphate. Groups of fish (45–50 g) were exposed to 0.0053, 0.053 or 0.53% [0.1%, 1% or 10% LC50] of TE for 28 days. The specific immune response of fish was assessed by antibody response to heat-killed Aeromonas hydrophila by ELISA and bacterial agglutination assay. Nonspecific immune mechanisms were assessed in terms of serum lysozyme activity, production of intracellular reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) by peripheral blood leucocytes (PBL). The results indicate that chronic exposure of fish to 0.53% of TE, significantly suppressed antibody response, nonspecific serum lysozyme activity, and ROS and RNI production. Exposure to 0.053% (1% LC50) of TE also caused a similar suppressive effect though at a lesser degree. In conclusion, the study shows, that exposure to sublethal concentrations of TE, can lead to adverse effects on selected immune reactions in tilapia. Further, these findings may be important in terms of monitoring fish health and risk assessment during periods of fluctuating levels of pollutants in the natural and farm environments. r 2006 Published by Elsevier Inc. Keywords: Tannery effluent; Lysozyme; ELISA; Antibody response; RNI; ROS; Tilapia
1. Introduction Heavy load of various industrial pollutants in the freshwater aquatic systems pose a serious threat to the health of wild and cultured fish by affecting their immune system. The leather-tanning industry is one of the major polluters of freshwater bodies of many developing countries including India (Tare et al., 2003). The Indian leather industries are significant in terms export and employment opportunities for people of economically weaker sections (Tare et al., 2003; Naidu, 2000). There are more than 1900 chrome leather tanning facilities in India most of which are $ The experimental animals were maintained in accordance with internationally accepted principles for laboratory animal use and care, as per national and institutional guidelines. Corresponding author. Fax: +91 452 2521333. E-mail address: [email protected] (R. Dinakaran Michael).
0147-6513/$ - see front matter r 2006 Published by Elsevier Inc. doi:10.1016/j.ecoenv.2006.11.016
located near major river banks (Khwaja et al., 2001). The characteristics of tannery effluent (TE) include high concentrations of chromium, sulphides (113.9736 mg L1), total suspended solids (846.1 mg L1) and sodium sulphate (776.8 mg L1) (Tare et al., 2003). It also creates high biological and chemical oxygen demand (BOD and COD). Among the above said constituents, the heavy metal, chromium has been found in higher proportions (Walsh and O’Halloran, 1998). Many tanning units in India are situated along the banks of rivers where the concentration of hexavalent chromium has been frequently exceeded the permissible limits for the discharge wastewater from a tannery unit into the receiving stream (Khwaja et al., 2001). Chromium has been found to range from 3.0 to 11.2 mg L1 against the permissible limit of 0.5 mg L1 (Tare et al., 2003; Koteswari and Ramanibai 2003; Dubey et al., 2001; Jawahar et al., 1998). Chromium has been shown to affect humoral and cell-mediated immunity by
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decreasing levels of antibody (Arunkumar et al., 2000) and splenic plaque-forming cell number (Prabakaran et al., 2006), by reducing proliferation of splenic lymphocytes and increase the susceptibility to bacterial infection in fish (Khangarot et al., 1999; Prabakaran et al., 2006). Earlier, Sudhan and Michael (1995) reported that sublethal concentrations of TE suppressed the primary and secondary antibody responses to bovine serum albumin (BSA) in a dose-dependent manner, in Oreochromis mossambicus. Reports on the effect of TE on the nonspecific immunity in fish are very sparse. Fish and their immune system may also represent an important scientific tool in the monitoring of environmental quality, particularly immunotoxic environmental pollution. Hence, the present study was aimed at investigating the direct effect of sublethal concentrations of TE on the specific immune response in terms of antibody level and nonspecific immune mechanisms in terms of serum lysozyme activity, intracellular reactive oxygen species (ROS) production and reactive nitrogen intermediates (RNI) production in O. mossambicus. 2. Materials and methods
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Groups of fish were exposed to 0.0053%, 0.053% and 0.53% of TE for 28 days by static renewal bioassay method. The test solutions were replaced once in every 2 days and the water quality parameters were monitored regularly. The data showed a pH range of 5.370.3 and dissolved oxygen 8.670.3 mg L1 in tanks containing 0.53% of TE, for 0.053% of TE the pH was 6.470.2 and dissolved oxygen 8.170.5 mg L1, for 0.0053% of TE the pH was 6.970.5 and dissolved oxygen 7.370.7 mg L1. After this period, immunological assays were performed keeping the fish in TE. The specific immune response was assessed by antibody response against heat-killed Aeromonas hydrophila using bacterial agglutination assay and ELISA. To study the kinectics of primary antibody response, the fish were sampled till day 56 (till day 28 post-immunization). Nonspecific immune mechanisms were assessed by serum lysozyme activity, ROS production by NBT reduction and RNI in terms of nitric oxide (NO) production by peripheral blood leucocytes (PBL).
2.3. Antigen preparation and administration Heat-killed A. hydrophila bacterin was prepared following the method of Karunasagar et al. (1997). Briefly, overnight cultures of A. hydrophila (1 108 cells mL1) in tryptone soy broth (Himedia, India) were exposed to 60 1C in a water bath for 1 h and spun down at 800 g for 15 min. The pellet was washed twice with phosphate buffered saline (PBS). The pellet was resuspended to contain 1 108 cells in 0.2 mL of PBS. Fish were administered with 0.2 mL of the heat-killed bacterin through intraperitoneal route.
2.1. Fish 2.4. Serum and antiserum collection O. mossambicus (outbred, wild strain) grown extensively in ponds was procured from a local farmer, was acclimated to the ambient, uncontrolled laboratory water temperature of 2872 1C under natural photoperiod for 2 weeks in 165 L fibre-reinforced plastic tanks. All tanks were fitted with a closed water recirculation system using external biofilter (Eheim-2213, Germany) to maintain the optimal water quality parameters, i.e. pH 7.370.2; dissolved oxygen 5.270.1 mg L1. Only male fish weighing 45–50 g were used in the present study since all-male monosex tilapia culture is practiced all over the world (Dan and Little, 2000). All the fish were fed ad libitum once a day with a balanced fish diet (Prabakaran et al., 2006) prepared in the laboratory. The ingredients of fish diet includes dried fish—42 g, groundnut oil cake—20 g, tapioca flour—15 g, wheat flour—15 g, blood meal—5 g, mineral mix—2 g, vitamins—1 g. Except for vitamins and mineral mix, all other ingredients were dried, powdered, mixed well and sterilized for 30 min. To this mixture vitamins, mineral mix and sterile water (300 mL kg1) was added. The mixture was then pressed through fine dye to obtain filamentous pellets. The pellets dried and stored till use.
2.2. Tannery effluent exposure A total of 250 L sample of chrome tanning effluent was collected 1 week prior to the experiment from the direct outlets of chrome tanning factory situated in Dindigul, Tamilnadu, India, during a period of normal operation. The sample was transported in full sealed polyethylene containers to our laboratory where it was mixed thoroughly in a large fibre reinforced plastic tank, filtered to remove the debris and then stored at 4 1C in full sealed polythene barrels. Apart from chromium (88.2 ppm measured by atomic absorption spectrophotometer) the effluent contained appreciable amounts of calcium carbonate (91.4 mg L1 CaCO3) and sodium sulphate (67.4 ppm). In order to select the range of sublethal levels of TE for exposure studies, the 96 h LC50 was determined by a static bioassay method (Doudoroff et al., 1951) following probit analysis (Finney, 1964). The 96 h LC50 of TE for O. mossambicus was found to be 5.3% (4.67 mg L1 of chromium) of TE. After assessing the lethal concentration, ranges of sublethal levels of 0.0053%, 0.053% and 0.53% (0.1%, 1% and 10% of LC50 of TE, respectively) of TE were selected for the study.
For both serum and antiserum collection, 200 mL of blood was drawn from common cardinal vein (Michael et al., 1994) and whole bleeding procedure was completed within 1 min. The blood was collected in serological tubes. The clot was stored at 20 1C and was spun down at 400 g for 10 min. The separated serum was stored in sterile eppendorf tubes at 20 1C until further use. For bacterial agglutination assay the sera were de-complemented by incubating at 47 1C for 30 min in a water bath (Sakai, 1981).
2.5. Leucocyte preparation For PBL separation, the blood was collected by cardinal vein puncture (Michael et al., 1994) into syringes filled with blood collecting medium (RPMI-1640 supplemented with 50,000 IU L1 sodium heparin, 1,00,000 IU L1 penicillin, 100 mg L1 streptomycin). Peripheral blood leucocytes were separated from blood by density gradient using Lymphoprep (Nycomed, Oslo, Norway) followed by centrifugation (800 g, 20 min) as described by Miller and McKinney (1994). Cell suspensions were washed thrice with wash medium (RPMI-1640 supplemented with 10,000 IU L1 sodium heparin, 1,00,000 IU L1 penicillin and 100 mg L1 streptomycin) for 10 min at 700 g and resuspended in culture medium (RPMI-1640 supplemented with 3% [v/v] of pooled, heat inactivated (30 min, 47 1C) tilapia serum, 1,00,000 IU L1 penicillin, 100 mg L1 streptomycin and 4 mM L-glutamine (all chemicals: Biochrom AG, Germany). Numbers of viable cells were enumerated and adjusted to 40 106 mL1 using cell culture medium.
2.6. Antibody titration by bacterial agglutination assay For detecting the effect of sublethal concentrations of TE on the antibody response by bacterial agglutination assay, eight fish per group (n ¼ 8/group) were maintained. After 28 days of TE exposure, the fish were administered intraperitoneally with heat-killed A. hydrophila bacterin containing 1 108 cells in 0.2 mL of PBS per fish (Karunasagar et al., 1997). An unimmunized control group was also maintained under similar conditions as the experimental group. All the fish were repetitively bled at
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regular intervals of 7 days post-immunization till day 28. Antibody titres against heat-killed A. hydrophila was performed in 96-well ‘‘V’’ bottom microtitre plate by a method published by Sakai et al. (1993) with slight modification. Briefly, 25 mL of serum was added to the first well and twofold serial dilutions were made with PBS. A volume of 25 mL of heat-killed A. hydrophila suspension (1 109 cells mL1) prestained with crystal violet was added to each well. The microtitre plate was incubated at 37 1C overnight. The highest dilution of serum sample that showed detectable macroscopic agglutination was recorded and expressed as log2 antibody titre of the serum.
2.7. Enzyme linked immunosorbant assay (ELISA) Serum antibodies to A. hydrophila were measured using an indirect ELISA method described by Delamare et al. (2002) with minor modifications (Binuramesh et al., 2005). Microtitre plates were coated with 100 mL well1 of whole bacterial cells. The bacteria were earlier harvested from overnight liquid culture, washed thrice with 0.15 mol L1 NaCl solution, and adjusted to 5 107 cells mL1 in 0.05 M carbonate bicarbonate buffer (pH 9.6). The plates were incubated overnight at 4 1C, and then washed thrice with PBS containing 0.05% (v/v) Tween 20 (PBST). Non-specific binding sites were blocked with 100 mL well1 of 1% (w/v) BSA in PBS. The plates were incubated at 28 1C for l h and washed thrice with PBS-T. One hundred microlitres of test serum samples (diluted to l: 30 in PBS-T) were added to triplicate wells and incubated for l h at 28 1C. After incubation, the plates were washed thrice with PBS-T and 100 mL of rabbit anti-tilapia polyclonal antibody (prepared and purified in the laboratory) diluted 1:100 using PBS, was added to each well. The plates were allowed to stand for 1 h at 37 1C followed by washing thrice with PBS-T. Then, 100 mL of goat anti-rabbit IgG antibody, conjugated with horseradish peroxidase (Sigma, USA) diluted 1:2000 in PBS, was added to each well and incubated for 1 h at 37 1C. The plates were again washed thrice with PBS-T. The colouring reaction was developed by adding 100 mL of 3,30 ,5,50 - tetramethyl benzidine (Sigma, USA) mixed with 0.1% [v/v] of hydrogen peroxide. The reaction was stopped with 25 mL of 1 M sulphuric acid and read at 450 nm using ELISA plate reader. The mean absorbance value for triplicate wells was used to express serum antibody level.
2.8. Serum lysozyme assay For lysozyme assay, eight fish per group (n ¼ 8/group) were maintained. All the fish were repetitively bled at regular intervals of 4 days till day 40 starting after 28 days of effluent exposure. Serum lysozyme assay was determined by using turbidimetric assay developed by Parry et al. (1965) with the microplate adaptation method of Hutchinson and Manning (1996). A suspension of 0.3 mg mL1 Micrococcus lysodeikticus in 0.05 M sodium phosphate buffer (pH 6.2) was used as substrate. Ten microlitres of sera were added to 250 mL of the bacterial suspension and the reduction in absorbance at 490 nm was determined after 0.5 and 4.5 min of incubation at 28 1C in a microplate reader (Model–680 Biorad, USA). One unit of lysozyme activity was defined as a reduction in absorbance of 0.001 min1 (Ellis, 1990).
2.9. Production of reactive oxygen species To study the effect of chronic exposure to TE on the ROS production by phagocytes, five fish per group (n ¼ 5/group) were maintained. The fish were exposed to different concentrations of TE for 28 days. After this period, ROS production was measured by using PBL at regular intervals of 4 days until day 40. The intracellular respiratory burst activity was determined by the nitroblue tetrazolium (NBT) reduction assay of Secombes (1990) with minor modifications. PBL (1 106 per well) were incubated with 25 mL of nitroblue tetrazolium (NBT, 1 g L1) in 175 mL culture medium for 2 h at 28 1C. The supernatants were removed and the cells were fixed with 100% [v/v] methanol for 5 min. Each well was washed
twice with 125 mL of 70% [v/v] methanol. The fixed cells were allowed to air dry. The reduced NBT (in the form of formazan) was dissolved using 125 mL of 2 N potassium hydroxide (KOH) and 150 mL dimethyl sulphoxide (DMSO) per well. Optical density was recorded spectrophotometrically at 650 nm.
2.10. Reactive nitrogen intermediates production assay To study the effect of chronic exposure to TE on the RNI in terms of NO production, five fish per group (n ¼ 5/group) were maintained. The fish were exposed to different concentrations of TE for 28 days. After this period, NO production by PBL was estimated at regular intervals of 4 days until Day 40. Preparation of leucocytes was performed as described earlier. The release of NO by PBL in the medium was measured using Griess reaction. The Griess reagent indicates the presence of nitrite as a surrogate of NO (Green et al., 1982). Peripheral blood leukocytes were cultured for 96 h at 28 1C and 50 mL culture supernatant was collected and transferred to a separate microtitre plate. To each well containing the culture supernatant, 50 mL of Griess reagent (1% sulphanilamide, 0.1% Nnaphthyl-ethylenediamine, 2.5% (v/v) phosphoric acid) was added. After incubation for 10 min, the optical density was recorded spectrophotometrically at 570 nm in a microplate reader. Molar concentrations of NO2 were calculated from a standard curve generated from a graded series of NaNO 2 concentrations in culture medium.
2.11. Statistical analysis The data were expressed as arithmetic mean7standard error (SE). Student’s t-test was performed to determine the level of significance in the difference between means of two groups. One way ANOVA was also used to test for group differences, and Tukey HSD post hoc test was used to determine which groups were significantly different from the rest. In all analyses, P-values below 0.05 were considered significant.
3. Results 3.1. Effect of tannery effluent on the specific immune response 3.1.1. Bacterial agglutination Fig. 1, with antibody response expressed as log2 titre shows that 0.53% and 0.053% of TE, dose-dependently suppressed (Po0.01, 0.02) antibody titre to heat-killed A. hydrophila on day 7 and 14 post-immunization. On day 21 and 28, fish exposed to 0.53% of TE showed a significant (Po0.01, 0.02) suppression of antibody titre. However, exposure to the lowest concentration (0.0053% TE) did not have any significant effect on the antibody titre. 3.1.2. ELISA The antibody response against A. hydrophila measured by ELISA showed that 0.53% of TE was suppressed (Po0.02) on day 7 after immunization. A significant (Po0.01, 0.02) reduction in antibody response compared to the control was found in the groups exposed to 0.53% and 0.053% of TE on day 14 and 21 post-immunization (Fig. 2). On day 28, fish exposed to 0.53% of TE showed significant (Po0.02) suppression of antibody response. Here also, exposure to the lowest concentration (0.0053% TE) did not have any significant effect on the antibody titre.
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Fig. 1. Effect of chronic exposure to tannery effluent (TE) on the antibody response to heat-killed Aeromonas hydrophila assayed by bacterial agglutination test in Oreochromis mossambicus. Each point represents the arithmetic mean (n ¼ 8/group)7SE. (**Po0.02; ***Po0.01).
3.2. Effect of tannery effluent on nonspecific immunity 3.2.1. Serum lysozyme activity The results of serum lysozyme activity (Fig. 3) suggest significant reduction of lysozyme activity on day 32, 36 and 40 in the group chronically exposed to 0.053% and 0.53% of TE (Po0.01, 0.05). Exposure to the lowest concentration (0.0053% of TE) did not have any significant effect on the serum lysozyme activity on any day of testing. 3.2.2. Reactive oxygen species production assay The results of ROS production suggest that significant reduction of ROS production was observed on day 32, 36 and 40 (Po0.02) in the group exposed to 0.53% of TE. Exposure to 0.053% of TE significantly (Po0.05) reduced the ROS production on all the days tested except for day 36. This was due to a slight reduction of ROS production in the control on day 36. Exposure to the lowest concentration tested did not have any significant effect on the ROS production on any day of testing (Fig. 4). 3.2.3. Effect of tannery effluent on the RNI production in O. mossambicus The RNI production was measured in terms of NO production by PBL. It is evident that significant reduction (Po0.02) of NO production in the group exposed to
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Fig. 2. Effect of chronic exposure to tannery effluent (TE) on the antibody response to heat-killed Aeromonas hydrophila assayed by ELISA in Oreochromis mossambicus. Each point represents the arithmetic mean (n ¼ 8/group)7SE. (**Po0.02; ***Po0.01).
0.53% of TE was observed on all the days (Fig. 5) whereas exposure to 0.053% of TE significantly (Po0.05) reduced the NO production on day 32 and 40. Exposure to lowest concentration did not have any significant effect on the NO production on any day of testing. 4. Discussion The results of this study clearly show that chronic exposure to sublethal concentrations of TE suppressed specific and nonspecific immunity in O. mossambicus. The results of experiments on antibody response indicate that chronic exposure to 0.53% (10% LC50) of TE significantly suppressed the antibody titres against heat-killed A. hydrophila and exposure to 0.053% (1% LC50) of TE had a similar suppressive effect on the peak day (day 14) though at a lesser degree. Similar findings have been reported earlier (Sudhan and Michael, 1995) on the suppression of primary and secondary immune responses against BSA in O. mossambicus exposed to 2.5% to 10% LC50 of TE for 21 days. The suppressive effect might be due to the immunosuppressive effect of chromium present in the effluent (Sudhan and Michael, 1995). Chromium has been shown to suppress the humoral immune response against BSA in O. mossambicus (Arunkumar et al., 2000)
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Fig. 3. Effect of chronic exposure to tannery effluent (TE) on the serum lysozyme activity in Oreochromis mossambicus. The fish were sampled 2 days prior to TE exposure and at regular intervals of 4 days after 28 days of TE exposure till day 40. Each point represents the arithmetic mean (n ¼ 8/group)7SE. (*Po0.05; ***Po0.01).
and sheep red blood cells (SRBC) in fresh water Catfish, Saccobranchus fossilis (Khangarot et al., 1999). Recently, Prabakaran et al. (2006) reported that chromium at concentrations of 0.5 and 5 mg L1 decreased antibody production against A. hydrophila bacterin and the number of antibody producing cells. Chromium can interact with lymphocyte cell surface proteins thereby altering responses of these cells to various stimuli (Snyder and Valle, 1991). Chromium has also been shown to induce DNA strand breakage (Borges and Wetterhan, 1989). Further, chromium can also react with cell surface receptors for mitogen, block lymphocyte proliferation and inhibit immunoglobulin production (Koller, 1980). Serum lysozyme activity was used as a parameter to assess the influence of chronic exposure to TE on the humoral component of the nonspecific defense mechanism of O. mossambicus. Sanchez-Dardon et al. (1999) have reported that heavy metal pollution affects lysozyme levels causing alterations in immunoregulatory functions in fish. The present study reveals chronic exposure to both 0.035% (1% LC50) and 0.53% (10% LC50) of TE has decreased serum lysozyme activity. Earlier literature reveals the fact that decreased level of lysozyme is common in fish exposed to metals or pollutants. Fletcher (1986) observed that decreased plasma lysozyme activity in plaice (Pleuronectes platessa) after exposure to mercury.
Fig. 4. Effect of chronic exposure to tannery effluent (TE) on the production of reactive oxygen species by peripheral blood leucocytes in Oreochromis mossambicus. The fish were sampled 2 days prior to TE exposure and at regular intervals of 4 days after 28 days of TE exposure till day 40. Each point represents the arithmetic mean (n ¼ 5/group)7SE. (*Po0.05; **Po0.02).
Production of ROS in phagocytes is a potent cytotoxic mechanism against bacteria and protozoan pathogens. Exposure of fish to environmental contaminants has been shown to modulate fish phagocytic ROS production (Zelikoff, 1994; Zelikoff et al., 1996; Roszell and Anderson, 1996). When exposure to a toxicant causes a decrement in ROS production, it may reduce an animal’s ability to effectively kill ingested pathogens thereby increasing the host’s susceptibility to infectious agents (Elasser et al., 1986; Zelikoff, 1994). Present study reveals that chronic exposure to 0.053% and 0.53% of TE decreased intracellular ROS production. Bols et al. (2001) have reviewed similar suppressive effect of metals or pollutants on respiratory burst activity in fish. Copper inhibited respiratory burst activity in rainbow trout leucocytes (Elasser et al., 1986). Cadmium inhibited PMA-stimulated respiratory burst activity in trout peritoneal macrophages exposed to a concentration of 2 ng mL1 for 30 days (Zelikoff et al., 1995). Fish phagocytes are able to produce NO in response to intracellular infection, by the action of inducible nitric oxide synthase (iNOS). Campos-Perez et al. (2000) reported that secretion of NO by activated macrophages of rainbow trout in response to bacterial infection. Results from the present study on the effect of TE on RNI in terms
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The results of this study reemphasize the importance of integration of immunological assays into environmental monitoring with reference to industrial effluents. It also demonstrates the sensitivity of immune mechanisms in relation to environmental contamination, indicating their possible use as immunological indicators of chromium and other heavy metal pollution in and around heavy metal based industries. Among the nonspecific parameters tested for immunomodulation, serum lysozyme activity assay seems to be the more sensitive, inexpensive and less timeconsuming than other assays used in this study. Acknowledgment The study was supported by the grants from Volkswagen foundation, Hannover, Germany and Council of Scientific Industrial Research (CSIR), India. References
Fig. 5. Effect of chronic exposure to tannery effluent (TE) on the reactive nitrogen intermediates in terms of NO secretion by peripheral blood leucocytes in Oreochromis mossambicus. The fish were sampled 2 days prior to TE exposure and at regular intervals of 4 days after 28 days of TE exposure till day 40. Each point represents the arithmetic mean (n ¼ 5/ group)7SE. (*Po0.05; **Po0.02; ***Po0.0).
of NO production reveal that chronic exposure to 0.053% and 0.53% of TE suppressed the intracellular RNI production. Steinhagen et al. (2004) reported a similar suppression of NO secretion on in vitro exposure to 20 mmol L1 Cr (VI) for 96 h in head kidney leucocytes of carp (Cyprinus carpio). TE contains sulphides, ammonical nitrogen, neutral salts and mainly excess chromium along with oil and grease. Recently, Prabakaran et al. (2006) reported that chromium at concentrations of 0.5 and 5 mg L1 had decreased nonspecific immunity in terms of serum lysozyme activity and RNI. The discharge of TE into ambient water resources may affect the physical and chemical characteristics of water there by posing a major threat to many aquatic organisms. An implication of this finding on the potential effects of TE underlines the necessity and need for treating chrome-tanning effluent before discharge.
5. Conclusion In general, the study clearly indicates that chronic exposure to sublethal concentrations i.e. 0.053% and 0.53% (1% and 10% LC50) of TE had suppressed the specific and nonspecific immune mechanisms in the O. mossambicus.
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Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S., 1982. Analysis of nitrate, nitrite and (15N) nitrate in biological fluids. Anal. Biochem. 126, 131–138. Hutchinson, T.H., Manning, M.J., 1996. Seasonal trends in serum lysozyme activity and total protein concentration in dap (Limanda limanda L.) sampled from Lyme Bay, UK Fish. Shellfish. Immunol. 6, 473–482. Jawahar, A.J.K., Ponselvan, J.K.S., Chinnadurai, M., Annadurai, G., 1998. Pollution Inventory for Tanneries in Ranipet and Vaniyambadi in Tamil Nadu. Indian J. Environ. Prot. 18, 418. Karunasagar, I., Ali, A., Otta, S.K., Karunasagar, I., 1997. Immunisation with bacterial antigens: Infections with motile Aeromonads. Dev. Biol. Stand. 90, 135–141. Khangarot, B.S., Rathore, R.S., Tripathy, D.M., 1999. Effects of chromium on humoral and cell-mediated immune responses and host resistance to disease in a freshwater Catfish, Saccobranchus fossilis (Bloch). Ecotoxicol. Environ. Saf. 43, 11–20. Khwaja, A.R., Rashmi, S., Tandon, S.N., 2001. Monitoring of Ganga water and sediments vis-a`-vis tannery pollution at Kanpur (India): a case study. Environ. Monit. Assess. 68, 19–35. Koller, L.D., 1980. Immunotoxicology of heavy metals. Int. J. Immunopharmacol. 2, 269–279. Koteswari, Y.N., Ramanibai, R., 2003. The effect of tannery effluent on the colonization rate of plankters: a microcosm study. Turk. J. Biol. 27, 163–170. Michael, R.D., Srinivas, S.D., Sailendri, K., Muthukkaruppan, V.R., 1994. A rapid method for repetitive bleeding in fish. Indian J. Exp. Biol. 32, 838–839. Miller, N.W., Mc Kinney, E.C., 1994. In vitro culture of fish leukocytes. In: Hocachka, P.W., Mommsen, T.P. (Eds.), Biochemistry and Molecular Biology of Fishes 3. Elsevier, Amsterdam, pp. 341–353. Naidu, A.S., 2000. Indian Leather Industry in 21st Century: Challenges and Opportunities. Yojana 3, 13. Parry, R.M., Chandan, R.C., Shahani, K.M., 1965. A rapid and sensitive assay of muramidase. Proc. Soc. Exp. Biol. 119, 384–386. Prabakaran, M.P., Binuramesh, C., Steinhagen, D., Michael, R.D., 2006. Immune response and disease resistance of Oreochromis mossambicus to Aeromonas hydrophila after exposure to hexavalent chromium. (in press). Roszell, L.E., Anderson, R.S., 1996. Effect of in vivo pentachlorophenol exposure on Fundulus heteroclitus phagocytes: modulation of bactericidal activity. Dis. Aquat. Org. 26, 205–211. Sakai, D.K., 1981. Heat inactivation of complements and immune hemolysis reactions in rainbow trout, masu salmon, coho salmon, goldfish and tilapia. Bull. Jap. Soc. Sci. Fish 47, 565–571.
Sakai, M., Atsuta, S., Kobayashi, M., Kawakami, H., Yoshida, T., 1993. The cell surface hydrophobicity and hemagglutinating properties of the bacterial fish pathogen, Pasteurella piscicida. Bull. Eur. Ass. Fish Pathol. 13, 168–170. Sanchez-Dardon, J., Voccia, I., Hontela, A., Chilmonczyk, S., Dunier, M., Boermans, H., Blakely, B., Fournier, M., 1999. Immunomodulation by heavy metals tested individually or in mixtures in rainbow trout (Oncorhynchus mykiss) exposed in vivo. Environ. Toxicol. Chem. 18, 1492–1497. Secombes, C.J., 1990. Isolation of salmonid macrophages and analysis for their activity. In: Stolen, J.S., Fletcher, T.C., Anderson, D.P., Roberson, B.S., van Muiswinkel, W.B. (Eds.), Techniques in Fish Immunology. SOS Publications, New Jersey, pp. 137–152. Sudhan, T., Michael, R.D., 1995. Modulation of humoral immune response by tannery effluent in Oreochromis mossambicus (Peters). Indian J. Exp. Biol. 33, 793–795. Snyder, C.A., Valle, C.D., 1991. Lymphocyte proliferation assays as potential biomarkers for toxicant exposures. J. Toxicol. Environ. Health 34, 127–139. Steinhagen, D., Helmus, T., Maurer, S., Michael, R.D., Leibold, W., Scharsack, J.P., Skouras, A., Schuberth, H.J., 2004. Effect of hexavalent carcinogenic chromium on carp Cyprinus carpio immune cells. Dis. Aquat. Org. 62, 155–161. Tare, V., Gupta, S., Bose, P., 2003. Case studies on biological treatment of tannery effluents in India. J. Air Waste Manage. Assoc. 53, 976–982. Walsh, A.R., O’Halloran, J., 1998. Accumulation of chromium by a population of mussels (Mytilus edulis (L.)) exposed to leather tannery effluent. Environ. Toxicol. Chem. 17, 1429–1438. Zelikoff, J.T., 1994. Immunological alterations as indicators of environmental metal exposure. In: Stolen, J.S., Fletcher, T.C. (Eds.), Modulators of fish immune response. SOS Publications, New Jersey, pp. 101–110. Zelikoff, J.T., Bowser, D., Squibb, K.S., Frenkel, K., 1995. Immunotoxicity of low level cadmium exposure in fish: an alternative animal model for immunotoxicological studies. J. Toxicol. Environ. Health 45, 235–248. Zelikoff, J.T., Wang, W., Islam, N., Flescher, E., Twerdok, L.E., 1996. Heavy metal-induced changes in antioxidant enzymes and oxyradical production by fish phagocytes: Application as biomarkers for predicting the immunotoxic effects of metal-polluted aquatic environments. In: Stolen, J.S., Fletcher, T.C., Bayne, C.J., Secombes, C.J., Zelikoff, J.T., Twerdok, L.E., Anderson, D.P. (Eds.), Modulators of fish immune response. SOS Publications, Fair Haven, New Jersey, pp. 425–442.
Ecotoxicology and Environmental Safety 68 (2007) 372–378 www.elsevier.com/locate/ecoenv
Immune response in the tilapia, Oreochromis mossambicus on exposure to tannery effluent$ M. Prabakarana, C. Binuramesha, Dieter Steinhagenb, R. Dinakaran Michaela, a
Centre for Fish Immunology, Postgraduate and Research, Department of Zoology, Lady Doak College, Madurai-625 002, Tamil Nadu, India b Fish Disease Research unit, Centre for Infection Medicine, School of Veterinary Medicine, Bu¨nteweg 17, 30559 Hannover, Germany Received 26 February 2006; received in revised form 2 November 2006; accepted 18 November 2006 Available online 29 January 2007
Abstract The objective of the study was to investigate the effect of chronic exposure to sublethal concentrations of tannery effluent (TE) on the specific immune response and nonspecific immunity in tilapia, Oreochromis mossambicus. The effluent from the tannery was collected directly from a chrome-tanning factory situated in Dindigul district, Tamil Nadu, India. Apart from chromium (88.2 ppm), the effluent contained appreciable amount of calcium carbonate and sodium sulphate. Groups of fish (45–50 g) were exposed to 0.0053, 0.053 or 0.53% [0.1%, 1% or 10% LC50] of TE for 28 days. The specific immune response of fish was assessed by antibody response to heat-killed Aeromonas hydrophila by ELISA and bacterial agglutination assay. Nonspecific immune mechanisms were assessed in terms of serum lysozyme activity, production of intracellular reactive oxygen species (ROS) and reactive nitrogen intermediates (RNI) by peripheral blood leucocytes (PBL). The results indicate that chronic exposure of fish to 0.53% of TE, significantly suppressed antibody response, nonspecific serum lysozyme activity, and ROS and RNI production. Exposure to 0.053% (1% LC50) of TE also caused a similar suppressive effect though at a lesser degree. In conclusion, the study shows, that exposure to sublethal concentrations of TE, can lead to adverse effects on selected immune reactions in tilapia. Further, these findings may be important in terms of monitoring fish health and risk assessment during periods of fluctuating levels of pollutants in the natural and farm environments. r 2006 Published by Elsevier Inc. Keywords: Tannery effluent; Lysozyme; ELISA; Antibody response; RNI; ROS; Tilapia
1. Introduction Heavy load of various industrial pollutants in the freshwater aquatic systems pose a serious threat to the health of wild and cultured fish by affecting their immune system. The leather-tanning industry is one of the major polluters of freshwater bodies of many developing countries including India (Tare et al., 2003). The Indian leather industries are significant in terms export and employment opportunities for people of economically weaker sections (Tare et al., 2003; Naidu, 2000). There are more than 1900 chrome leather tanning facilities in India most of which are $ The experimental animals were maintained in accordance with internationally accepted principles for laboratory animal use and care, as per national and institutional guidelines. Corresponding author. Fax: +91 452 2521333. E-mail address: [email protected] (R. Dinakaran Michael).
0147-6513/$ - see front matter r 2006 Published by Elsevier Inc. doi:10.1016/j.ecoenv.2006.11.016
located near major river banks (Khwaja et al., 2001). The characteristics of tannery effluent (TE) include high concentrations of chromium, sulphides (113.9736 mg L1), total suspended solids (846.1 mg L1) and sodium sulphate (776.8 mg L1) (Tare et al., 2003). It also creates high biological and chemical oxygen demand (BOD and COD). Among the above said constituents, the heavy metal, chromium has been found in higher proportions (Walsh and O’Halloran, 1998). Many tanning units in India are situated along the banks of rivers where the concentration of hexavalent chromium has been frequently exceeded the permissible limits for the discharge wastewater from a tannery unit into the receiving stream (Khwaja et al., 2001). Chromium has been found to range from 3.0 to 11.2 mg L1 against the permissible limit of 0.5 mg L1 (Tare et al., 2003; Koteswari and Ramanibai 2003; Dubey et al., 2001; Jawahar et al., 1998). Chromium has been shown to affect humoral and cell-mediated immunity by
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decreasing levels of antibody (Arunkumar et al., 2000) and splenic plaque-forming cell number (Prabakaran et al., 2006), by reducing proliferation of splenic lymphocytes and increase the susceptibility to bacterial infection in fish (Khangarot et al., 1999; Prabakaran et al., 2006). Earlier, Sudhan and Michael (1995) reported that sublethal concentrations of TE suppressed the primary and secondary antibody responses to bovine serum albumin (BSA) in a dose-dependent manner, in Oreochromis mossambicus. Reports on the effect of TE on the nonspecific immunity in fish are very sparse. Fish and their immune system may also represent an important scientific tool in the monitoring of environmental quality, particularly immunotoxic environmental pollution. Hence, the present study was aimed at investigating the direct effect of sublethal concentrations of TE on the specific immune response in terms of antibody level and nonspecific immune mechanisms in terms of serum lysozyme activity, intracellular reactive oxygen species (ROS) production and reactive nitrogen intermediates (RNI) production in O. mossambicus. 2. Materials and methods
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Groups of fish were exposed to 0.0053%, 0.053% and 0.53% of TE for 28 days by static renewal bioassay method. The test solutions were replaced once in every 2 days and the water quality parameters were monitored regularly. The data showed a pH range of 5.370.3 and dissolved oxygen 8.670.3 mg L1 in tanks containing 0.53% of TE, for 0.053% of TE the pH was 6.470.2 and dissolved oxygen 8.170.5 mg L1, for 0.0053% of TE the pH was 6.970.5 and dissolved oxygen 7.370.7 mg L1. After this period, immunological assays were performed keeping the fish in TE. The specific immune response was assessed by antibody response against heat-killed Aeromonas hydrophila using bacterial agglutination assay and ELISA. To study the kinectics of primary antibody response, the fish were sampled till day 56 (till day 28 post-immunization). Nonspecific immune mechanisms were assessed by serum lysozyme activity, ROS production by NBT reduction and RNI in terms of nitric oxide (NO) production by peripheral blood leucocytes (PBL).
2.3. Antigen preparation and administration Heat-killed A. hydrophila bacterin was prepared following the method of Karunasagar et al. (1997). Briefly, overnight cultures of A. hydrophila (1 108 cells mL1) in tryptone soy broth (Himedia, India) were exposed to 60 1C in a water bath for 1 h and spun down at 800 g for 15 min. The pellet was washed twice with phosphate buffered saline (PBS). The pellet was resuspended to contain 1 108 cells in 0.2 mL of PBS. Fish were administered with 0.2 mL of the heat-killed bacterin through intraperitoneal route.
2.1. Fish 2.4. Serum and antiserum collection O. mossambicus (outbred, wild strain) grown extensively in ponds was procured from a local farmer, was acclimated to the ambient, uncontrolled laboratory water temperature of 2872 1C under natural photoperiod for 2 weeks in 165 L fibre-reinforced plastic tanks. All tanks were fitted with a closed water recirculation system using external biofilter (Eheim-2213, Germany) to maintain the optimal water quality parameters, i.e. pH 7.370.2; dissolved oxygen 5.270.1 mg L1. Only male fish weighing 45–50 g were used in the present study since all-male monosex tilapia culture is practiced all over the world (Dan and Little, 2000). All the fish were fed ad libitum once a day with a balanced fish diet (Prabakaran et al., 2006) prepared in the laboratory. The ingredients of fish diet includes dried fish—42 g, groundnut oil cake—20 g, tapioca flour—15 g, wheat flour—15 g, blood meal—5 g, mineral mix—2 g, vitamins—1 g. Except for vitamins and mineral mix, all other ingredients were dried, powdered, mixed well and sterilized for 30 min. To this mixture vitamins, mineral mix and sterile water (300 mL kg1) was added. The mixture was then pressed through fine dye to obtain filamentous pellets. The pellets dried and stored till use.
2.2. Tannery effluent exposure A total of 250 L sample of chrome tanning effluent was collected 1 week prior to the experiment from the direct outlets of chrome tanning factory situated in Dindigul, Tamilnadu, India, during a period of normal operation. The sample was transported in full sealed polyethylene containers to our laboratory where it was mixed thoroughly in a large fibre reinforced plastic tank, filtered to remove the debris and then stored at 4 1C in full sealed polythene barrels. Apart from chromium (88.2 ppm measured by atomic absorption spectrophotometer) the effluent contained appreciable amounts of calcium carbonate (91.4 mg L1 CaCO3) and sodium sulphate (67.4 ppm). In order to select the range of sublethal levels of TE for exposure studies, the 96 h LC50 was determined by a static bioassay method (Doudoroff et al., 1951) following probit analysis (Finney, 1964). The 96 h LC50 of TE for O. mossambicus was found to be 5.3% (4.67 mg L1 of chromium) of TE. After assessing the lethal concentration, ranges of sublethal levels of 0.0053%, 0.053% and 0.53% (0.1%, 1% and 10% of LC50 of TE, respectively) of TE were selected for the study.
For both serum and antiserum collection, 200 mL of blood was drawn from common cardinal vein (Michael et al., 1994) and whole bleeding procedure was completed within 1 min. The blood was collected in serological tubes. The clot was stored at 20 1C and was spun down at 400 g for 10 min. The separated serum was stored in sterile eppendorf tubes at 20 1C until further use. For bacterial agglutination assay the sera were de-complemented by incubating at 47 1C for 30 min in a water bath (Sakai, 1981).
2.5. Leucocyte preparation For PBL separation, the blood was collected by cardinal vein puncture (Michael et al., 1994) into syringes filled with blood collecting medium (RPMI-1640 supplemented with 50,000 IU L1 sodium heparin, 1,00,000 IU L1 penicillin, 100 mg L1 streptomycin). Peripheral blood leucocytes were separated from blood by density gradient using Lymphoprep (Nycomed, Oslo, Norway) followed by centrifugation (800 g, 20 min) as described by Miller and McKinney (1994). Cell suspensions were washed thrice with wash medium (RPMI-1640 supplemented with 10,000 IU L1 sodium heparin, 1,00,000 IU L1 penicillin and 100 mg L1 streptomycin) for 10 min at 700 g and resuspended in culture medium (RPMI-1640 supplemented with 3% [v/v] of pooled, heat inactivated (30 min, 47 1C) tilapia serum, 1,00,000 IU L1 penicillin, 100 mg L1 streptomycin and 4 mM L-glutamine (all chemicals: Biochrom AG, Germany). Numbers of viable cells were enumerated and adjusted to 40 106 mL1 using cell culture medium.
2.6. Antibody titration by bacterial agglutination assay For detecting the effect of sublethal concentrations of TE on the antibody response by bacterial agglutination assay, eight fish per group (n ¼ 8/group) were maintained. After 28 days of TE exposure, the fish were administered intraperitoneally with heat-killed A. hydrophila bacterin containing 1 108 cells in 0.2 mL of PBS per fish (Karunasagar et al., 1997). An unimmunized control group was also maintained under similar conditions as the experimental group. All the fish were repetitively bled at
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regular intervals of 7 days post-immunization till day 28. Antibody titres against heat-killed A. hydrophila was performed in 96-well ‘‘V’’ bottom microtitre plate by a method published by Sakai et al. (1993) with slight modification. Briefly, 25 mL of serum was added to the first well and twofold serial dilutions were made with PBS. A volume of 25 mL of heat-killed A. hydrophila suspension (1 109 cells mL1) prestained with crystal violet was added to each well. The microtitre plate was incubated at 37 1C overnight. The highest dilution of serum sample that showed detectable macroscopic agglutination was recorded and expressed as log2 antibody titre of the serum.
2.7. Enzyme linked immunosorbant assay (ELISA) Serum antibodies to A. hydrophila were measured using an indirect ELISA method described by Delamare et al. (2002) with minor modifications (Binuramesh et al., 2005). Microtitre plates were coated with 100 mL well1 of whole bacterial cells. The bacteria were earlier harvested from overnight liquid culture, washed thrice with 0.15 mol L1 NaCl solution, and adjusted to 5 107 cells mL1 in 0.05 M carbonate bicarbonate buffer (pH 9.6). The plates were incubated overnight at 4 1C, and then washed thrice with PBS containing 0.05% (v/v) Tween 20 (PBST). Non-specific binding sites were blocked with 100 mL well1 of 1% (w/v) BSA in PBS. The plates were incubated at 28 1C for l h and washed thrice with PBS-T. One hundred microlitres of test serum samples (diluted to l: 30 in PBS-T) were added to triplicate wells and incubated for l h at 28 1C. After incubation, the plates were washed thrice with PBS-T and 100 mL of rabbit anti-tilapia polyclonal antibody (prepared and purified in the laboratory) diluted 1:100 using PBS, was added to each well. The plates were allowed to stand for 1 h at 37 1C followed by washing thrice with PBS-T. Then, 100 mL of goat anti-rabbit IgG antibody, conjugated with horseradish peroxidase (Sigma, USA) diluted 1:2000 in PBS, was added to each well and incubated for 1 h at 37 1C. The plates were again washed thrice with PBS-T. The colouring reaction was developed by adding 100 mL of 3,30 ,5,50 - tetramethyl benzidine (Sigma, USA) mixed with 0.1% [v/v] of hydrogen peroxide. The reaction was stopped with 25 mL of 1 M sulphuric acid and read at 450 nm using ELISA plate reader. The mean absorbance value for triplicate wells was used to express serum antibody level.
2.8. Serum lysozyme assay For lysozyme assay, eight fish per group (n ¼ 8/group) were maintained. All the fish were repetitively bled at regular intervals of 4 days till day 40 starting after 28 days of effluent exposure. Serum lysozyme assay was determined by using turbidimetric assay developed by Parry et al. (1965) with the microplate adaptation method of Hutchinson and Manning (1996). A suspension of 0.3 mg mL1 Micrococcus lysodeikticus in 0.05 M sodium phosphate buffer (pH 6.2) was used as substrate. Ten microlitres of sera were added to 250 mL of the bacterial suspension and the reduction in absorbance at 490 nm was determined after 0.5 and 4.5 min of incubation at 28 1C in a microplate reader (Model–680 Biorad, USA). One unit of lysozyme activity was defined as a reduction in absorbance of 0.001 min1 (Ellis, 1990).
2.9. Production of reactive oxygen species To study the effect of chronic exposure to TE on the ROS production by phagocytes, five fish per group (n ¼ 5/group) were maintained. The fish were exposed to different concentrations of TE for 28 days. After this period, ROS production was measured by using PBL at regular intervals of 4 days until day 40. The intracellular respiratory burst activity was determined by the nitroblue tetrazolium (NBT) reduction assay of Secombes (1990) with minor modifications. PBL (1 106 per well) were incubated with 25 mL of nitroblue tetrazolium (NBT, 1 g L1) in 175 mL culture medium for 2 h at 28 1C. The supernatants were removed and the cells were fixed with 100% [v/v] methanol for 5 min. Each well was washed
twice with 125 mL of 70% [v/v] methanol. The fixed cells were allowed to air dry. The reduced NBT (in the form of formazan) was dissolved using 125 mL of 2 N potassium hydroxide (KOH) and 150 mL dimethyl sulphoxide (DMSO) per well. Optical density was recorded spectrophotometrically at 650 nm.
2.10. Reactive nitrogen intermediates production assay To study the effect of chronic exposure to TE on the RNI in terms of NO production, five fish per group (n ¼ 5/group) were maintained. The fish were exposed to different concentrations of TE for 28 days. After this period, NO production by PBL was estimated at regular intervals of 4 days until Day 40. Preparation of leucocytes was performed as described earlier. The release of NO by PBL in the medium was measured using Griess reaction. The Griess reagent indicates the presence of nitrite as a surrogate of NO (Green et al., 1982). Peripheral blood leukocytes were cultured for 96 h at 28 1C and 50 mL culture supernatant was collected and transferred to a separate microtitre plate. To each well containing the culture supernatant, 50 mL of Griess reagent (1% sulphanilamide, 0.1% Nnaphthyl-ethylenediamine, 2.5% (v/v) phosphoric acid) was added. After incubation for 10 min, the optical density was recorded spectrophotometrically at 570 nm in a microplate reader. Molar concentrations of NO2 were calculated from a standard curve generated from a graded series of NaNO 2 concentrations in culture medium.
2.11. Statistical analysis The data were expressed as arithmetic mean7standard error (SE). Student’s t-test was performed to determine the level of significance in the difference between means of two groups. One way ANOVA was also used to test for group differences, and Tukey HSD post hoc test was used to determine which groups were significantly different from the rest. In all analyses, P-values below 0.05 were considered significant.
3. Results 3.1. Effect of tannery effluent on the specific immune response 3.1.1. Bacterial agglutination Fig. 1, with antibody response expressed as log2 titre shows that 0.53% and 0.053% of TE, dose-dependently suppressed (Po0.01, 0.02) antibody titre to heat-killed A. hydrophila on day 7 and 14 post-immunization. On day 21 and 28, fish exposed to 0.53% of TE showed a significant (Po0.01, 0.02) suppression of antibody titre. However, exposure to the lowest concentration (0.0053% TE) did not have any significant effect on the antibody titre. 3.1.2. ELISA The antibody response against A. hydrophila measured by ELISA showed that 0.53% of TE was suppressed (Po0.02) on day 7 after immunization. A significant (Po0.01, 0.02) reduction in antibody response compared to the control was found in the groups exposed to 0.53% and 0.053% of TE on day 14 and 21 post-immunization (Fig. 2). On day 28, fish exposed to 0.53% of TE showed significant (Po0.02) suppression of antibody response. Here also, exposure to the lowest concentration (0.0053% TE) did not have any significant effect on the antibody titre.
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Fig. 1. Effect of chronic exposure to tannery effluent (TE) on the antibody response to heat-killed Aeromonas hydrophila assayed by bacterial agglutination test in Oreochromis mossambicus. Each point represents the arithmetic mean (n ¼ 8/group)7SE. (**Po0.02; ***Po0.01).
3.2. Effect of tannery effluent on nonspecific immunity 3.2.1. Serum lysozyme activity The results of serum lysozyme activity (Fig. 3) suggest significant reduction of lysozyme activity on day 32, 36 and 40 in the group chronically exposed to 0.053% and 0.53% of TE (Po0.01, 0.05). Exposure to the lowest concentration (0.0053% of TE) did not have any significant effect on the serum lysozyme activity on any day of testing. 3.2.2. Reactive oxygen species production assay The results of ROS production suggest that significant reduction of ROS production was observed on day 32, 36 and 40 (Po0.02) in the group exposed to 0.53% of TE. Exposure to 0.053% of TE significantly (Po0.05) reduced the ROS production on all the days tested except for day 36. This was due to a slight reduction of ROS production in the control on day 36. Exposure to the lowest concentration tested did not have any significant effect on the ROS production on any day of testing (Fig. 4). 3.2.3. Effect of tannery effluent on the RNI production in O. mossambicus The RNI production was measured in terms of NO production by PBL. It is evident that significant reduction (Po0.02) of NO production in the group exposed to
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Fig. 2. Effect of chronic exposure to tannery effluent (TE) on the antibody response to heat-killed Aeromonas hydrophila assayed by ELISA in Oreochromis mossambicus. Each point represents the arithmetic mean (n ¼ 8/group)7SE. (**Po0.02; ***Po0.01).
0.53% of TE was observed on all the days (Fig. 5) whereas exposure to 0.053% of TE significantly (Po0.05) reduced the NO production on day 32 and 40. Exposure to lowest concentration did not have any significant effect on the NO production on any day of testing. 4. Discussion The results of this study clearly show that chronic exposure to sublethal concentrations of TE suppressed specific and nonspecific immunity in O. mossambicus. The results of experiments on antibody response indicate that chronic exposure to 0.53% (10% LC50) of TE significantly suppressed the antibody titres against heat-killed A. hydrophila and exposure to 0.053% (1% LC50) of TE had a similar suppressive effect on the peak day (day 14) though at a lesser degree. Similar findings have been reported earlier (Sudhan and Michael, 1995) on the suppression of primary and secondary immune responses against BSA in O. mossambicus exposed to 2.5% to 10% LC50 of TE for 21 days. The suppressive effect might be due to the immunosuppressive effect of chromium present in the effluent (Sudhan and Michael, 1995). Chromium has been shown to suppress the humoral immune response against BSA in O. mossambicus (Arunkumar et al., 2000)
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Fig. 3. Effect of chronic exposure to tannery effluent (TE) on the serum lysozyme activity in Oreochromis mossambicus. The fish were sampled 2 days prior to TE exposure and at regular intervals of 4 days after 28 days of TE exposure till day 40. Each point represents the arithmetic mean (n ¼ 8/group)7SE. (*Po0.05; ***Po0.01).
and sheep red blood cells (SRBC) in fresh water Catfish, Saccobranchus fossilis (Khangarot et al., 1999). Recently, Prabakaran et al. (2006) reported that chromium at concentrations of 0.5 and 5 mg L1 decreased antibody production against A. hydrophila bacterin and the number of antibody producing cells. Chromium can interact with lymphocyte cell surface proteins thereby altering responses of these cells to various stimuli (Snyder and Valle, 1991). Chromium has also been shown to induce DNA strand breakage (Borges and Wetterhan, 1989). Further, chromium can also react with cell surface receptors for mitogen, block lymphocyte proliferation and inhibit immunoglobulin production (Koller, 1980). Serum lysozyme activity was used as a parameter to assess the influence of chronic exposure to TE on the humoral component of the nonspecific defense mechanism of O. mossambicus. Sanchez-Dardon et al. (1999) have reported that heavy metal pollution affects lysozyme levels causing alterations in immunoregulatory functions in fish. The present study reveals chronic exposure to both 0.035% (1% LC50) and 0.53% (10% LC50) of TE has decreased serum lysozyme activity. Earlier literature reveals the fact that decreased level of lysozyme is common in fish exposed to metals or pollutants. Fletcher (1986) observed that decreased plasma lysozyme activity in plaice (Pleuronectes platessa) after exposure to mercury.
Fig. 4. Effect of chronic exposure to tannery effluent (TE) on the production of reactive oxygen species by peripheral blood leucocytes in Oreochromis mossambicus. The fish were sampled 2 days prior to TE exposure and at regular intervals of 4 days after 28 days of TE exposure till day 40. Each point represents the arithmetic mean (n ¼ 5/group)7SE. (*Po0.05; **Po0.02).
Production of ROS in phagocytes is a potent cytotoxic mechanism against bacteria and protozoan pathogens. Exposure of fish to environmental contaminants has been shown to modulate fish phagocytic ROS production (Zelikoff, 1994; Zelikoff et al., 1996; Roszell and Anderson, 1996). When exposure to a toxicant causes a decrement in ROS production, it may reduce an animal’s ability to effectively kill ingested pathogens thereby increasing the host’s susceptibility to infectious agents (Elasser et al., 1986; Zelikoff, 1994). Present study reveals that chronic exposure to 0.053% and 0.53% of TE decreased intracellular ROS production. Bols et al. (2001) have reviewed similar suppressive effect of metals or pollutants on respiratory burst activity in fish. Copper inhibited respiratory burst activity in rainbow trout leucocytes (Elasser et al., 1986). Cadmium inhibited PMA-stimulated respiratory burst activity in trout peritoneal macrophages exposed to a concentration of 2 ng mL1 for 30 days (Zelikoff et al., 1995). Fish phagocytes are able to produce NO in response to intracellular infection, by the action of inducible nitric oxide synthase (iNOS). Campos-Perez et al. (2000) reported that secretion of NO by activated macrophages of rainbow trout in response to bacterial infection. Results from the present study on the effect of TE on RNI in terms
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The results of this study reemphasize the importance of integration of immunological assays into environmental monitoring with reference to industrial effluents. It also demonstrates the sensitivity of immune mechanisms in relation to environmental contamination, indicating their possible use as immunological indicators of chromium and other heavy metal pollution in and around heavy metal based industries. Among the nonspecific parameters tested for immunomodulation, serum lysozyme activity assay seems to be the more sensitive, inexpensive and less timeconsuming than other assays used in this study. Acknowledgment The study was supported by the grants from Volkswagen foundation, Hannover, Germany and Council of Scientific Industrial Research (CSIR), India. References
Fig. 5. Effect of chronic exposure to tannery effluent (TE) on the reactive nitrogen intermediates in terms of NO secretion by peripheral blood leucocytes in Oreochromis mossambicus. The fish were sampled 2 days prior to TE exposure and at regular intervals of 4 days after 28 days of TE exposure till day 40. Each point represents the arithmetic mean (n ¼ 5/ group)7SE. (*Po0.05; **Po0.02; ***Po0.0).
of NO production reveal that chronic exposure to 0.053% and 0.53% of TE suppressed the intracellular RNI production. Steinhagen et al. (2004) reported a similar suppression of NO secretion on in vitro exposure to 20 mmol L1 Cr (VI) for 96 h in head kidney leucocytes of carp (Cyprinus carpio). TE contains sulphides, ammonical nitrogen, neutral salts and mainly excess chromium along with oil and grease. Recently, Prabakaran et al. (2006) reported that chromium at concentrations of 0.5 and 5 mg L1 had decreased nonspecific immunity in terms of serum lysozyme activity and RNI. The discharge of TE into ambient water resources may affect the physical and chemical characteristics of water there by posing a major threat to many aquatic organisms. An implication of this finding on the potential effects of TE underlines the necessity and need for treating chrome-tanning effluent before discharge.
5. Conclusion In general, the study clearly indicates that chronic exposure to sublethal concentrations i.e. 0.053% and 0.53% (1% and 10% LC50) of TE had suppressed the specific and nonspecific immune mechanisms in the O. mossambicus.
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