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The Journal of Veterinary Science. Photon 114 (2013) 218-226 https://sites.google.com/site/photonfoundationorganization/home/the-journal-of-veterinary-science Original Research Article. ISJN: 1784-6372

The Journal of Veterinary Science Ph ton

The Bioremediation potential of Spirulina platensis and Lemna gibba L in Grass Carp, Ctenopharyngodon idella Exposed to Cadmium Toxicity Hussein A. Kaoud

a*, Sayed N. Abou Elgheit

b, Ahmed R. Eldahshan

a, Sherein Saeid

c

a

Department of Veterinary Hygiene and Environmental Pollution, Faculty of Veterinary Medicine, Cairo University, Egypt b National Institute of Oceanography and Fisheries, Egypt

c Department of Pathology, Faculty of Veterinary Medicine, Cairo University, Giza, Egypt

Article history: Received: 16 December, 2012 Accepted: 19 January, 2013 Available online: 01 June, 2013 Keywords: Bioremediation, Cadmium, Ctenopharyngodon idella, Histopathology, Lemna gibba L weed, Spirulina platensis Abbreviations: Cd: Cadmium, L gibba L: Lemna gibba L, S. plat: Spirulina platensis Corresponding Author: Kaoud H.A.* Perofessor Email: ka-oud@link.net Phone: 0201224207641 Elgheit S.N.A. Associate Perofessor Email: abouelghei@yahoo.com Phone: 0201228850343 Eldahshan A.R. Associate Perofessor Email: dr_a.rezk@yahoo.com Phone: 0201226109091 Saeid S. Associate Perofessor Email: Shereinpathology@gmail.com Phone: 0201000800189

Abstract The effect of cadmium (Cd) toxicity, its impact on histopathological changes, the median lethal concentration (LC50-96 h) and the bioremediation effect of Lemna gibba L and dried Spirulina platensis to grass carp, Ctenopharyngodon idella, were investigated through semi-static acute toxicity test developed with cadmium chloride (CdCl2). This study indicated that:1) Cd poisoning caused structural damage in the fish organs ,2) Lemna gibba L weed was effective in removing Cd from water and reducing its bioaccumulation in liver and muscular tissues of fish, 3) Addition of dried Spirulina platensis reduced significantly (P<0.05) the Cd level uptake as compared to fish exposed to Cd alone and 4) Addition of Lemna gibba L weed and dried Spirulina platensis were remediated the toxic effect of Cd and provided protection against the degenerative action of Cd in grass carp, Ctenopharyngodon idella. Spirulina platensis was effective in removing Hg from water and reducing Hg bioaccumulation in liver and muscular tissues of fish, as well as remediated the toxic effect of Hg and provided protection against the degenerative action of Hg in Nile tilapia, Oreochromis Niloticus. Citation: Kaoud H.A., Elgheit S.N.A., Eldahshan A.R., Saeid S., 2013. The Bioremediation potential of Spirulina platensis and Lemna gibba L in Grass Carp, Ctenopharyngodon idella Exposed to Cadmium Toxicity. The Journal of Veterinary Science. Photon 114, 218-226.

1. Introduction

Contamination of fresh waters with a wide range of pollutants has become a matter of concern over the last few decades (Vinodhini et al, 2008; Vutukuru, 2005; Dirilgen, 2001; Voegborlo et al., 1999; Canli et al., 1998). Natural aquatic systems may extensively be contaminated with heavy metals released from domestic, industrial and other man-made activities (Velez and Montoro, 1998; Conacher et al., 1993).

Heavy metal contamination may have devastating effects on the ecological balance of the recipient environment and a diversity of aquatic organisms (Vinodhini et al, 2008; Farombi, et al., 2007; Vosyliene and Jankaite, 2006; Ashraj, 2005) and become toxic when they are not metabolized by the body and accumulate in the soft tissues. Cadmium (Cd) is a well known heavy metal toxicant with a specific gravity 8.65 times greater than water (Lide, 1992). The target

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organs for Cd toxicity have been identified as liver, placenta, kidneys, lungs, brain and bones (Roberts, 1999). Cadmium, as other toxic metals, may set up conditions that lead to inflammation in arteries and tissues, causing more Ca

2+ to be drawn to

the area as a buffer, contributing to hardening of the artery walls with progressive blockage of the arteries and result in osteoporosis. Bioenhancement of Cd transfer along a food chain was studied by Seebaugh et al., (2005) and fish are reported to be used as biological indicators to assess water pollution (Rashed, 2001). In aquatic systems, as fish occupy the upper trophic level, there are greater chances of transferring Cd to higher organisms particularly to man. It is listed by the US Environmental Protection Agency (1984) as one of 126 priority pollutants and is carcinogenic to a number of tissues (Waalkes, 2000). River Nile, the absolute highest value of Cd for most form of life was 22.14 µgL

-1 recorded in

the surface water layer during summer as compare it's the absolute lowest value 8.8 µgL

-

1 was measured in the bottom layer during

spring 2009 (Toufeek, 2009). Phytofiltration using aquatic plants has promising potential for the ex situ and in situ clean-up of polluted waters. Recent advancements have also proven successful via the addition of matched microbe strains to the medium to enhance the resident microbe population's ability to break down contaminants (Adami et al., 2002). Removal of certain heavy metals from waste water by Lemna gibba L. has been reported by Kwan & Smith (1991); Buckley (1994); Miranda & IIangovan (1996) ;Wafaa (2007) and Kaoud et al,.(2011). Blue-green alga Spirulina platensis (S. platensis) is a promising object for studies of different metals absorption and accumulation processes to perform the function of biomediator. Cell wall of S. platensis consists of polysaccharides, proteins and lipids having lots of negative carboxyl and phosphate groups, which are the dominant binding sites of toxic and heavy metals cations (Vonshak, 1997). In the present study, short -term bioassays were designed to evaluate the influence of Lemna gibba L- plant and Spirulina platensis -microalgae on the reduction of cadmium in

water and its bioaccumulation in tissues of grass carp, Ctenopharyngodon idella exposed to cadmium toxicity. 2. Experimental Contamination of fresh waters with a wide range of pollutants has become a matter of concern over the last few decades. Phytofiltration using aquatic plants has promising potential for the ex situ and in situ clean-up of polluted waters. 2.1 Fish culture and management Healthy Grass Carp (Ctenopharyngodon idella) of 6-8 grams were collected during the late August and early September, 2010 from the ponds of fish farm, Abo-Hammad, and Sharkia, Egypt. They were collected locally and confined to large plastic aquaria bearing tap water for up to 7 days in the laboratory for acclimation. 2.2 Cadmium chloride Technical grade cadmium chloride (99% purity) was obtained from El-Nasr Chemical Company (Cairo, Egypt) and prepared in aquatic solution to provide the required concentrations of cadmium. Control test without cadmium was performed. 2.3 Determination of Lc50 2.3.1 Acute Toxicity Assays Laboratory bioassays were conducted to determine the 24 hrs, 48 hrs, 72 hrs and 96 hrs LC50 values for C. idella exposed to CdCl2. The experimental design and calculations for the acute toxicity were based on well-known procedures given by Finney (1978) and Sparks (2000). The tests were carried out in 20 Liters rectangular fiberglass aquaria filled with well-aerated tape water. Dissolved oxygen in each tank was maintained at close to saturation by aeration. The temperature in each aquarium was maintained at 25.5-27° C. The photoperiod was 12 hrs light length/day. The fish were visibly free of any deformities, lesions, or disease and were acclimated in tap water for 1 week prior to the experiment. Fresh water was adjusted to the desired parameters as follows: temperature 26.30±2.25° C, pH 7-7.8, dissolved oxygen 5-8 mg L

-1, salinity 2ppt

and hardness 100-1

50 mg L-1

CaCO3 with a photoperiod (10L: 14D cycle). The nominal concentrations of CdCl2 tested were 0 (control), 0.50, 1, 5, 10, 15 and 20 mg Cd L

-1. Gross fish mortality of each

concentration was recorded every 1 h for the first 12 hrs and every 2 hrs thereafter for 96

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hrs while the dead fish were removed every 3–8 hrs. Test solutions were replaced by fresh ones of the same respective concentrations every 24 h until 96 h of testing, according to the renewal method recommended in APHA (1998). The Grass Carp (C. idella) was not fed throughout the test. The control and each test concentration were tested in triplicate. The data obtained were statistically analyzed using the Trimmed Spearman Karber method (Hamilton et al., 1977) for estimating the median lethal concentration (LC50), and 1/4 of the LC50-96 h (5 mg Cd L

-1) was taken as the

safe Cd concentration (Sprague, 1971). 2.4 Tested Plant and microalgae 2.4.1 Lemna gibba L Duckweed species used was L. gibba L which was taken from Ganabiet-Tersa drain, Giza, Egypt. The duckweed was acclimatized to the laboratory conditions for one week before starting the experiments. 2.4.2. Microalgae Spirulina platensis, preserved in a laboratory at Faculty of Agricultural Life Sciences, Cairo University of Egypt, was used in this study. It was grown at 25 ± 2° C in Zarrouk liquid medium (Parada et al., 1998), for 8-10 days under white fluorescent light (90 mmol photon m-2s-1) with 14 h illumination. At the exponential growth phase, culture was filtered through filters 47 mm (diameter) (Whatman GF/C). The filter was put in a glass Petri dish in the oven under 35° C. for 3 days (Boussiba and Richmond, 1976). 2.5. Cd reduction Fishes were distributed randomly in 50 Liters rectangular fiber glass aquaria filled with water at a rate of 25 fish / aquarium. Dissolved oxygen in each tank was maintained at close to saturation by aeration. The temperature in each aquarium was maintained by means of thermostats. These aquaria were divided into five groups with three replicates each per group. The first group was free from Cd and plants and maintained as a control. The second groups were exposed to 5 mg of CdCl2 only (Equivalent to 1/4 96 h LC50). The third, fourth and fifth group were exposed to 5 mg CdCl2 L

-1 plus L. gibba L plant (1 g L

-1), dried

S. platensis (5 mg L-1

) and L. gibba L plus dried S. platensis in the third, fourth and fifth group, respectively. 2.6 Cd residue Cadmium residues were measured in water, liver and muscles according to method of

Eaton & Stinson (1983). The water samples were preserved by the addition of one ml of concentrated nitric acid per liter until the time of analysis. The water samples were filtered through 0.45µl membrane filter. The required volume (100 ml) of the filtrate was collected to measure cadmium levels in water samples by using Air/Acetylene Flame Atomic Absorption Spectrophotometer (UNICAM 696 AA Spectrometer). The analysis of tissue sample was represented by 0.5 gram of tissues dissected from the liver and muscles, then placed in a clean screw-capped tube and digested according to the method described by Finerty et al. (1990). The obtained solutions were then analyzed by using Air/ Acetylene Flame Atomic Absorption Spectrophotometer (UNICAM 696 AA Spectrometer). Fish were fed frequently on a diet containing 30% crude protein at a rate of 2% and 1% biomass of live body weight twice daily for 15 days. Siphoning three quarters aquariums was done every day for waste removal and replacing it by an equal volume of water containing the same concentration of Cd and plants. Dead fish were removed and recorded daily. 2.7. Histopathological examination Tissue specimens from fresh C. idella were taken (liver and muscles) and fixed in 15 % buffered neutral formalin. They were processed to obtain five micron thick paraffin sections then stained with Hematoxylin and Eosin (Bancroft et al., 1996) and examined under light microscope. 2.8. Statistical analysis The obtained data were subjected to analysis of variance according to Snedecor & Cochran (1982). Differences between means were done at the 5% probability level, using Duncan’s new multiple range test (Duncan, 1955).

3. Results and Discussion 3.1 Acute Toxicity Assays and LC50 Value The values of LC50 determined for grass Carp (C. idella) in the present study, according to the different exposure times, are shown in Fig. 1 and 2. The 96 h LC50 value is 20 mg CdCl2 L

-1.

The LC50 determined in the present study was lower to those reported by Gui-yan et al., (2007) for grass carp which was 24.05 mg·L

-1.

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Figure 1a: and Figure 1b: The 96 h value of LC50

(the simple graphic method) determined for grass

carp (Ctenopharyngodon idella) in the present

study, according to the different exposure times, is

20 mg CdCl2 L-1

Figure 1a:

Figure 1b:

The LC50-96h when compared with the results from other studies developed on cadmium toxicity to fish such as Yilmaz et al., (2004) for guppy (Poecilia 4a 4g44) and Kaoud et al., (2011) for tilapia (Oriochromis niloticus) Table 3. The higher values obtained by Gui-yan et al. (2007) for grass carp may be attributed to some differences in standard techniques that were adopted in their experiments. The acute toxicity of waterborne cadmium on aquatic organisms is highly variable even among phylogenetically closely related species and depends on metal speciation, being the free ion (WHO, 1992). 3.2 Histopathological alterations Histopathological biomarkers have been largely used in fish to identify and evaluate toxic effects of exposure to pollutants (Adam1, 2002; Rabitto et al., 2005; Oliveira Ribeiro et al., 2006). Recent studies have demonstrated cause-to-effect links between exposure to pollutants and the development of hepatic lesions. Histopathology of different C. idella tissues revealed that there were several histopathological changes in different organs as shown in the Fig. 3a, b, c, d, e and f. Gills. C. idella exposed to CdCl2 showed several pathological changes such as hyperplasia of gill rays, proliferation of lamellar epithelium, subepithelial edema and vacuolation of

cytoplasm of lining epithelium, focal loss of lamellar epithelium (Fig. 3a,b) and congestion of blood spaces. This was in agreement with previous report that cell proliferation, lamellar cell hyperplasia, lamellar fusion and epithelial lifting were observed in gill filaments of Oreochromis niloticus when exposed to lethal and sublethal concentrations of CdCl2 (Kaoud et al., 2011; Wong and Wong 2000). Ferrari et al., (2010) studied the effects of acute exposure for 96 h to a sublethal concentration of cadmium (0.30 mg L−1) on the surface gill morphology of the teleosteans Cyprinus carpio, Australoheros facetum and Astyanax, the main cadmium-induced gill damages in the three species were: structural alterations, edema and fusion of adjacent secondary lamellae; there was a remarkable disruption of the gill epithelium. Epithelial hyperplasia and subepithelial edema were observed in gills of Cyprinus carpio L. (Common carp) due to effect of sublethal concentration of 5 mg L

-1 of combined (Cd +

Pb + Cr + Ni) metal solution containing 1.25 mg L

-1 of each metal ion (1/10

th of LC50 / 48h)

(Vinodhini and Narayanan,2009). Gill is an important site for the entry of heavy metals that provokes lesions and gill damage such as hypertrophy, hyperplasia of epithelium cells and destruction of the lamellas structure (Bols et al., 2001; Lock and Overbeeke, 1981; Vinodhini et al, 2008). Gills are also reported to act as storehouse of cadmium in experimental studies (Allen, 1995; Tao et al., 2000; Fafioye et al., 2004; Ramesh and Nagaranjan, 2007; Kumar and Singh, 2010). Liver tissues of C. idella exposed to CdCl2 were showed congestion of sinusoids pyknotic nuclei and focal areas of necrosis (Fig. 3 c). Liver of C. carpio L. exposed to 5 mg L

-1 of

combined (Cd + Pb + Cr + Ni) metal solution showed congestion of few sinusoids and on some places, signs of early fibrosis (Vinodhini and Narayanan ,2009). Also, O. niloticus exposed to sublethal concentrations of CdCl2, showed histopathological changes in the liver viz., vacuolation of hepatocytes and nuclear pyknosis (Kaoud et al, 2011; Wong and Wong 2000). Cd accumulates in liver of fishes in high concentrations (De-Smet and Blust, 2001; Rangsayatorn et al., 2004). It also induces various pathological changes in liver tissues including engorgement of blood vessels, congestion, vacuolar degeneration of

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hepatocytes, necrosis of pancreatic cells and fatty changes in the peripancreatic hepatocytes (Rani and Ramamurthi, 1989; Dangre et al., 2010). Stentiford et al. (2003) and Stehr et al. (2004) observed that toxicopathic liver lesions in fish species are effective biological markers of chemical exposures. The presence of necrosis is in fact one of the most visible damages in tissues affected by a pollutant (Rabitto et al., 2005). According to Manahan (1991) the occurrence of necrosis is also a consequence of enzymatic inhibition, damages in the cellular membrane integrity, and disturbances in the synthesis of proteins and carbohydrate metabolism. Kidney showed hydropic swelling of the renal tubules, sometimes with pyknotic nuclei and many necrotic areas as well as swollen proximal epithelial cells with necrotic nuclei (Fig. 3 d, e). Cd is heavy metal and poses high toxicity at very low level of exposure and has acute and chronic effects on aquatic animal health and environment (Kumar and Singh, 2010). C. carpio L. exposed to 5 mg L

-1 of combined

(Cd + Pb + Cr + Ni) metal solution, kidney lesions showed macrophages with lipofuscin granules accumulated in the affected cells (Vinodhini and Narayanan ,2009). In fact, kidney is the principle target organ of cadmium toxicity and chronic cadmium exposure in almost all animal species is characterized by varying degree of renal damage (Wong and Wong 2000; Roméo et al., 2000; Shukla and Gautam, 2004; Kumar et al., 2006; Kumar et al., 2009; Vesey, 2010; Kumar and Singh, 2010; Kaoud et al, 2011; Wong and Wong 2000). Muscular tissues of C.idella exposed to CdCl2 showed several pathological changes (Fig. 3f). The changes observed were swelling in muscle bundles with aggregations of inflammatory cells (leucocytic infiltration) between them with focal areas of necrosis. Our results were similar to those reported by Vinodhini and Narayanan (2009) where,C. carpio L. exposed to 5 mg L

-1 of combined (Cd

+ Pb + Cr + Ni) metal solution, showed disappearance of striations, necrosis and hyalinised muscles tissue.

Figure 3a,b: C. idella exposed to CdCl2 showed several pathological changes such as hyperplasia of gill rays,

proliferation of lamellar epithelium, subepithelial edema and vacuolation of cytoplasm of lining epithelium, focal

loss of lamellar epithelium (H&E,X:400). Figure 3c: Liver tissues of C. idella exposed to CdCl2 were showed

congestion of sinusoids pyknotic nuclei and focal areas of necrosis (H&E, X: 200). Figure 3d: Kidney showed

hydropic swelling of the renal tubules, sometimes with pyknotic nuclei and many necrotic areas as well as

swollen proximal epithelial cells with necrotic nuclei (H&E, X: 200). Figure 3e: Muscular tissues showed normal

structure (H&E, X: 200). Figure 3f: The changes observed were swelling in muscle bundles with aggregations of

inflammatory cells (leucocytic infiltration) between them with focal areas of necrosis (H&E, X: 200)

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Table 1: Cadmium residue in water (mg Cd L-1

), liver and muscles (µg Cd g-1

dry weigh) of Grass Carp

(Ctenopharyngodon idella) exposed to Cd with and without Lemna gibba L plant or Spirulina platensis

MusMucles Liver Water Group

15 15 15 Days

0.029±0.007a 0.048±0.013

a 0.030±0.008

a Control (metal free water)

2.563±0.17b 10. 950±0.19

b 4.480±0.166

b Cadmium alone (5 mg L

-1)

2.32±0.091a 8.92±0.085

b 3.9 12 ±0.576

b Cadmium (5 mg L

-1)+ L. Gibba L

2.70±0.067b 9.90±0.018

c 2. 680 ±0.208

c Cadmium (5 mg L

-1)+ S. platensis

1.10±0.079a 4.18 ±0.024

d 1.999 ±0.135

d Cadmium (5 mg L

-1)+ L. Gibba L+ S. Platensis

The same letter in the same column is not significantly different at P<0.05.

Table 2: Acute toxicity of cadmium in various fish species

Reference Species LC50-96h (Cd mg L-1

)

Present study Ctenopharyngodon idella 20.00

Gui-yan et al. (2007) Ctenopharyngodon idella 24.05

Yilmaz et al.( 2004) Poecilia reticulate 31.7 0 Kaoud et al.(2011) Oriochromis niloticus 40

3.3 Cd Bioaccumulation Highest bioaccumulation of Cd was observed in liver followed by muscles, Table 1 and Fig.4. Addition of L. gibba L and dried S. platensis to Cd polluted media was reduced significantly (P<0.05). Cd level in aquarium’s water as compared to that of Cd alone, Table 1 where, Cd concentration in water alone (without plants) was 4.480±0.166 mg L

-1 but, it was

declined significantly (P<0.05) to 3 .912 ±0.576 ±0.576, 2.680 ±0.208 and 1.999 ±0.135 mg L

-1 with the addition of L. gibba L,

S. platensis, L. gibba L + S. platensis respectively. The highest amount of Cd residue was found in the liver after 15 days of exposure. Table 1 and Fig.4, also, showed that the uptake of Cd by liver was 10.0950±0.19 µg g-1. While it was declined significantly to 8.92±0.085; 9.90±0.018; 4.18 ±0.024 µg g

-1 in fish group exposed to Cd with

L. gibba L; S. platensis; L. gibba L + S. platensis, respectively. Similar trends were observed in fish muscles. Characteristic gross picture such as: excessive mucus layer on the gills, also showing pale, anemic, congestion and hemorrhages as well as necrotic picture in all internal organs, were confirmed by histopathological examination. Gross lesions percentages were recorded (Fig.4). Fish exposed to 1\4 of LC50 Cd (5 mg L-1) for 15 days, showed high percentage of pathological lesions in their gills, liver, kidneys and muscles; 45, 65, 22, 10 and 15% respectively. While, those exposed to Cd with L. gibba L or to Cd with dried S. platensis showed low percentage of pathological lesions in their gills, liver, intestine, muscles and kidneys; 10, 12, 2, 0 , 2 and 10, 5, 5, 0 , 3 ; respectively.

On the other hand fish exposed to Cd plus L. gibba L + S. platensis showed some pathological lesions only in their gills, liver and intestine (1, 1, and 1% respectively). The present study could be indicate that L. gibba L weed and dried S. platensis were effective in removing Cd from water with the reduction of Cd bioaccumulation in grass carp fish. These results suggest that L. gibba L weed and dried S. platensis could chelate Cd ions producing a stable complex, thus reducing the chance for metal uptake by tissues. These results are in agreement with Santschi (1988) who reported that any agent that can remove Cd from water helps to reduce the bioaccumulation of this metal in fish. These results are in agreement with those recorded by Jayaram & Prasad (2009) who observed the biosorption potential of Prosopis juliflora seed powder (PJSP) for lead (II) from aqueous solution at pH 6.0. Figure 4: Prevalence of histopathological pathological alterations.

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Microbial biomass-related technologies have also been tested for heavy metal removal from polluted water bodies (Volesky and Holan, 1995; Chang et al., 1997; Eccles, 1999). Cell wall components of microorganisms like polysaccharides, proteins and lipids offer many functional groups which can bind metal ions (Ari et al., 1999). Furthermore, it has been found that microalgae to be very effective biosorbents, as they possess a large surface area and high binding affinity (Roy et al., 1993). Blue-green alga S. platensis is a promising object for studies of different metals absorption and accumulation processes. Components found in the cell wall of Spirulina, such as peptydoglycan, teichuronic acid, teichoic acid, polysaccharides and proteins (Schiewer and Wong, 2000) which display mainly carboxylic, hydroxyl and phosphate groups and are the dominant binding sites of toxic and heavy metals cations (Aksu, 2002; Markai et al., 2003; Vonshak, 1997) may give algal wall binding properties. Conclusion This study showed that Lemna gibba L- weed and blue-green alga S. platensis might be helpful to reduce Cd from water and to eliminate metal from the fish body and in turn ameliorate the toxic effect of cadmium in water medium. References Adami G.M., Barbieri P., Fabiani M., Piselli S., Predonzani S., Reisenhofer E., 2002. Levels of cadmium and zinc in hepatopancreas of reared Mytilus galloprovincialis from the Gulf of Trieste (Italy). Chemosphere, 48 (7), 671 - 677. Aksu Z., 2002. Determination of the equilibrium, kinetic and thermodynamic parame-ters of the batch biosorption of nickel (II) ions onto Chlorella vulgaris. Process Biochem. 38, 89-99. Allen P., 1994. Changes in the haematological profile of the cichlid Oreochromis aureaus (Steindachner) during acute inorganic mercury intoxication. Comp. Biochemical. Physiology. 108C(1), 117-121. American Public Health Association (APHA) .,1998. Standard methods for the examination of water and wastewater. 20th ed. American Public Health Association, Washington, DC. Ari A.B., Mel M., Hasan M.A., Karim M.I.A., 1999. The kinetics and mechanism of lead (II) biosorption by powderized Rhizopus oligosporus. World J. Microbiol. Biotechnol. 15, 291-298.

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