Research articles
 

By Dr. Rachid Rouabhi
Corresponding Author Dr. Rachid Rouabhi
Biology, Tebessa University - Algeria 12000
Submitting Author Dr. Rachid Rouabhi
TOXICOLOGY

Metalotoxicity, Ni, Biochemical parameters, Respiration, Cytotoxicity

Rouabhi R. Influence of Nickel on Growth, Movement Speed, Respiratory Metabolism, and Biochemical Parameters of Paramecium sp.. WebmedCentral TOXICOLOGY 2011;2(12):WMC002676
doi: 10.9754/journal.wmc.2011.002676

This is an open-access article distributed under the terms of the Creative Commons Attribution License(CC-BY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
No
Submitted on: 19 Dec 2011 09:15:22 AM GMT
Published on: 19 Dec 2011 03:33:13 PM GMT

Abstract


Living things require different concentrations of metals such as iron, Cu and Zn, whose consumption is crucial for the metabolism, large amounts of these metals induces a phenomenon called Metalo-toxicity.
This study is intended to investigate thechronic toxicity of nickel on specie of freshwater ciliatesParamecium sp. After exposing standard culture to a soluble compound of nickel (NiCl2.6H2O) at three selected concentrations 15, 30 and 60 ppm in strictly controlled conditions of temperature, light and darkness.
The results show an inhibitory effect of Ni on the growth and mobility of protozoa according the time.
The determination of biochemical parameters level (protein and carbohydrates) shows an increase in these parameters compared with controls contrarily to the lipids level where there is a decrease compared to the control.
It is noted that the concentration of 60 ppm of Ni increases the rate of these parameters unless the other two concentrations.
Effect of nickel "Ni" on the respiratory metabolism showed inhibition of O2 consumption in concentrations 15 and 60 ppm in contrast to the 30 ppm concentration where there is a stimulation of respiration.

Introduction


The history of heavy metals has not beenwell written. It seems closely related to civilization. Gold, silver, copper helped produce the first coins. Without heavy metals, there would be no drinking water in Rome by the lead pipes. Neither, paint because old paint withstood the time with incorporated metal pigments or stained glass windows in cathedrals, or mirrors, tin amalgam of tin and mercury. The man used the heavy metals and continued to use them. Sometimes, to excess often unconscious or worse, in good conscience. If heavy metals make civilization, they can also undo because heavy metals are also toxic power.Heavy metals are a class of toxics and their toxic effects are varied and depended on the chemical state. Poisoning by some heavy metals such as mercury and lead can lead to phenomena of autoimmunity (Masclet, 2005).
Heavy metals are toxic to most microorganisms at certain concentrations. In freshwater environments, heavy metals are discharged from final effluents of waste treatment plants, and are common pollutantsof sewage, particularly where there is input of industrial waste. High concentrations often can be traced to specific industrial sources (Lester, 1983).
When metals are discharged with effluent, they may result in severe contamination of downstream ecosystems. In an aquatic environment, metals occur both in the dissolved or soluble fraction and in particulate matter. The mechanisms by which heavy metals affect the microorganisms is not clear. It has been suggested that they may block enzyme systems or interfere with some essential cellular metabolites of bacteria and protozoa (Morgan and Lackey, 1958).Metals also act on the cell membrane or interfere with cytoplasmic or nuclear functions after entry into the cell (Larsen and Nilsson, 1983).
In aquatic systems, nickel is usually in the form of Ni2+. The form in which it is present in water dependson pH. Nickel compounds found in the aquatic environment are generally identified and reported as total nickel, although this range of compounds reaching the aquatic environment because of anthropogenic emissions includes both soluble salts as insoluble oxides or metallic nickel dust. In the state of current knowledge, there are no Ni compounds that are present exclusively in the aquatic environment.Nickel in the divalent state can form a wide range of compounds and is the only important oxidation state of nickel (other oxidation states such as nickel (+4) may be present in some complex oxides) (Pichard, 2005). Thus, nickel is very poorly absorbed by most living organisms, but is largely associated with particulate matter (Kabata-Pendias and Pendias, 1992 ATSDR, 1997).
Nickel and its compounds cause a variety of cancer in rodents and are listed as possible causativeagents for occupational or environmental cancer in man (Dunnick et al., 1995), but its toxicity toward aquatic organisms is not well known.
Ciliated protozoa represent a basic component of microorganisms of aquatic environments, where they play critical roles both quantitatively and qualitatively (Fenchel, 1987). These microorganisms mediate the flow of biological substances and energy from one trophic level to the next (Sherr et al., 1988).
Ciliated protozoa arevery numerous both in aquatic environments and in all types of biological treatment systems (Madoni et al., 1993, 1996; Amann et al., 1998); they play an important role in purification and overall regulation of the entire aquatic community. It has been demonstrated that ciliates improve the quality of effluents through their involvement in the regulation of the bacterial biomass, by the removal of most of the dispersed bacteria (Curds et al., 1968; Madoni et al., 1994; Madoni, 2002, 2003; Salvado´ et al., 1995).
In this context, the ciliate assay has become a valuable tool for detection of environmental disturbance and for assessment of the trophic state (Cairns and Pratt, 1989).
This study is an estimate of the growth and cellular division, some morphophysiological parameters and biochemical, of paramecium treated with three concentrations of nickel (15, 30 and 60ppm).

Materials and Methods


Cells culturing and treatments
Paramecium sp was used in the logarithmic phase of growth. The cells were grown at exponential phase in Proteose Peptone Yeast Medium (PPY), 2% proteose peptone and 5% yeast extract at pH 7.0-7.5, at 24±2 °C. The density of Paramecium sp culture was adjusted in fresh PPY in order to obtain at least 104 cells per ml. Before the experiments on respiration metabolism, the cells were washed with fresh culture medium and were resuspended at the concentration of 5x104 cells ml-1 in 200ml flask; we take 1 ml to test in oxygraph (each time we added the appropriate concentration of Nickel to the reactive chamber by microsyringe). The cells were not exposed to Ni were used as controls. For the evaluation of Ni effect on Paramecium population growth, we added the metal in culture medium before the addition of paramecium cells, the used cells are starved for 96 h to become encysted. After the regeneration in culture medium that contains metal, we investigate the effect on growth.
Chemical preparation
Nickel in soluble state (NiCl2.6H2O) is prepared in distilled water at three concentrations 15, 30 and 60 ppm.
Growth measurement
The growth of Paramecium sp.,population density is estimated by Foissner (1999) method, modified by Rouabhi et al. (2006b) (Optic density at ?=600nm), on aliquots of 2.5ml of cultures for each concentration with 3 repetitions.We used distilled water as white control, the cell number was determined by counting every cell present in 1 ml sample using a microscope and Petri box (Cutler et al., 2005a).
Swimming speed measurement
Using a micrometer graduated microscopewe can calculate the rate for which the cell can pass through a number of graduations (distance), and the chronometer allows us to note the time of movement.
Respiratory metabolism
Protistes respiration is estimated using Clark’s electrode (oxygraph), described by Djebar and Djebar, (2000), which go until nanomol of oxygen consumption. Paramecia at logarithmic phase (5x104 cells/ml) from homogenous culture were obtained and 1ml of culture is put inside reaction room of oxygraph, then we added the metal at appropriate concentrations 15, 30 or 60 ppm, each trial was repeated 3 times, the respiration kinetic is followed for 20 minutes (after that time, cells become no stables) (Djebar and Djebar, 2000). The results are registered directly as graphs on computer screen linked to the oxygraph.
Biochemical parameters
The determination of biochemical parameters (carbohydrate, protein and fat) is made during the 09th day, to evaluate the effect of nickel on the general metabolism of protozoa. The methods are: Method Duchateau and Floking, (1959) quantified for carbohydrates, the method of Bradford (1976) for proteins and method of Goldsworthy et al. (1972) for lipids.
Extraction of carbohydrates, fats and proteins according to Shibko et al. (1966):
Samples of the control series, and treated are placed in a tube containing 1 ml of trichloroacetic acid (TCA) 20%, after a first centrifugation (5000 rev/min for 10 min), supernatant is used for determination of total carbohydrates. To the first clot 1 ml of the ether / chloroform (1V/1V) is added and after a second centrifugation (5000 rev/min for l0 min), the supernatant II will be used for the assay of lipid and the clot 2 is dissolved in NaOH (0.1N) for the determination of proteins.
Statistical study
All the experiments were repeated three times or more, and the results were expressed as mean and standard deviation (SD) values. We use Minitab 16.1.1 software to make simple two-way ANOVA test with two criteria (treatment and time) and the test of Dunnett for comparison between the control and treated cells

Results and Discussion


Heavy metals are toxic to most microorganisms at certain concentrations. In freshwater environments, heavy metals are discharged from final effluents of waste treatment plants, and are common pollutantsof sewage, particularly where there is input of industrial waste.
The impact of Nickel on the population growth of Paramecium is shown in (Fig. 1). Indeed, Ni has an inhibitory effects on the population density growth of protozoa in concentration-dependent manner, the highest concentration inhibits strongly the growth of Paramecium (pSpirostomumteres(0.17 mg Ni per l, 24-h LC50) and Paramecium bursaria (0.36 mg Ni/l, 24-h LC50) showed the highest sensitivity to the nickel, while Euplotes patella(7.7 mg Ni/l, 24-h LC50)was the most tolerant species.
Microorganisms are highly sensitive to chemicals in aqueous environment. However, paramecia cells are relatively resistant to high concentration of acrylamide (Takahashi et al., 2005).Dorange et al. (1995) showed that there is no toxic effects appeared at doses less than 8 µg/ml. Some inhibitor effects are shown at 9 and 10 µg/ml of cadmium.
Impact of Ni on parameciummovement speed is presented in figure 02. A clear decrease in speed is appeared in dose-dependent manner this is possibly due to the direct impact of Ni on ciliary body of protozoa or direct impact on general metabolism which reflect on ATP production and general energy function(Libri, 2010; Litim and Djoudi, 2010).
The effect of Nickel on the respiratory metabolism of Parameciumwas investigated for 5 minutes (acute toxicity) using Hansatech electrode described by Djebar and Djebar (2000), the results are illustrated in (Fig. 3). It is to note that 15 and 60 ppm of Nickel decrease the O2 consumption according to the controls while, the medium concentration (30 ppm) enhance the cellular respiration. This result is explained if we base on the detoxification/metabolisation mechanisms by mono-oxygenases enzymes, where the cells consummate O2 to make the substrate more hydrophilic so eliminated by/with water. These enzymes are coupled with substrate in the cells treated with 15 and 60 ppm of Nickel, but saturated or blocked in cells treated with the other concentration (30 ppm) (Rouabhi et al., 2006b). The decrease of oxygen consumption in the highest concentration of Novaluron is also a signification of the reduced number of cells because we started from the same number of cells.
Obtained results from dosage of biochemical parameters show an increase of proteins and carbohydrates level, decrease of lipids level. Bonias (1999) showed a decrease of total proteins level in hepatocytes of rat treated with Cd and Ni and this is against our findings. Proteins level is increased in protozoa because of the phenomenon of resistance and the synthesis of resistance proteins (Rouabhi, 2007). Increase in total carbohydrates level is due probably to the direct impact of nickel on general metabolism and this is confirmed in our results on movement speed because of the leak of ATP (Libri, 2010). The decrease in lipids after treatment by nickel is in dose-dependence manner and it is due probably by general effect on metabolism, Lawton andDonalson(1991) showed a total decrease in lipids level of chicken liver after treatment with heavy metals, also the biotransformation of lipids to carbohydrates due to the absence of ATP is a very probable hypothesis.
In conclusion, our study showed that the highest concentrations of Ni caused a dose-dependent growth inhibition of Parameciumpopulation. The results suggest that the antiproliferative effect of Ni may be mediated by reducing the vital process of cells, also by the direct effect on mitochondria and lashes, so Ni has toxic effects on Paramecium by reduction of growth, perturbation of respiratory metabolism, and perturbation of biochemical parameters. All results showed that this metal has an ecotoxicological effects on this non-target organism and ecosystem. It is to note that the used concentrations are high compared with concentrations found to effect aquatic crustaceans and insects, this due to the response and the physiology of Paramecium cells which are comparable to human and high organisms.
These results fill the gap concerning the effect of heavy metal on non-target organisms (protozoa)..

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Reviews
5 reviews posted so far

Rouabhi Influence of Nickel
Posted by Dr. Marie Bourgeois on 19 Feb 2012 10:29:29 PM GMT

Ecotoxicology
Posted by Dr. Rasheed Rabhellah Mahmoud on 14 Jan 2012 09:42:00 AM GMT

Ecotoxicology
Posted by Prof. Prasunpriya Nayak on 31 Dec 2011 01:57:52 PM GMT

Nickel
Posted by Dr. Prateek Rastogi on 30 Dec 2011 08:48:33 AM GMT

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