International Journal of Environment Science and Technology Center for Environment and Energy Research and Studies (CEERS) ISSN: 1735-1472 EISSN: 1735-2630 Vol. 2, Num. 1, 2005-2006, pp. 35-40

International Journal of Enviornmental Science and Technology, Vol. 2, No. 1, Spring, 2005, pp. 35-40

Toxic effect of Pb, Cd, Ni and Zn on Azolla filiculoides in the International Anzali Wetland

^1*M. Khosravi, ¹M. Taghi Ganji and ²R. Rakhshaee

¹Department of Applied Chemistry, Islamic Azad University, North Tehran Branch, Tehran, Iran
²Department of Applied Chemistry, Islamic Azad University, Rasht Branch, Rasht, Iran
*Corresponding Author, E-mail: drm_khosravi@ yahoo.com

Code Number: st05005

Abstract

The limitation of plant growth in the polluted mediums can be used as a factor to determine of plant tolerance and the toxic effect of these mediums. In this work, the effect of Pb²⁺, Cd²⁺, Ni²⁺ and Zn²⁺ (individually) on Azolla filiculoides growth in the aqueous solution and using this method to water post treatment were studied. During 15 days the biomass the fresh Azolla with initial mass of 20 g was grown on the nutrient solution containing these metal ions, each in a concentration 4 mg/l. The presence of these ions, caused about 25%, 42%, 31% and 17% inhibition of biomass growth, respectively, in comparison to Azolla control weight which had not heavy metals. The water salinity of 1, 2 and 4 g. NaCl/l decreased the removal of these heavy metals about 4-7%, 20-24% and 40-55%, respectively. The addition of total dissolved solids (TDS) from 50 to 300 ppm. (as CaCO₃) into the samples of containing heavy metals increased Azolla growth, but decreased the control Azolla growth.

Keywords: Azolla filiculoides, bioaccumulation, living biomass, heavy metals

Introduction

Heavy metals are among the most dangerous substances in the environment, because of their high level of durability and harmfulness to live organisms. Biosorption is the accumulation of heavy metals using microorganisms (such as bacteria and fungi) and photosynthetic life (such as algae, aquatic and emergent plants). Biosorption using living aquatic plants (phytoremediation) is a relatively new technology to solve the problem of heavy metal pollution. In the process of phytoremediation pollutants are collected by plant roots and either decomposed to less harmful forms (for example CO₂ and H₂O) or accumulated in the plant tissues. Thus, phytoremediation is environment friendly, inexpensive and can be carried out in polluted places (remediation in situ) plus the products of decomposition do not require further utilization (Roy, et al., 1992, Sternberg and Dorn, 2002).

There are two general mechanisms associated with the separation of dissolved metals from water using aquatic plant biomass. The first is a fast metabolism (within minutes) independent surface reaction that has been modeled as a diffusion process and ends when the soluble metal ions bind or sorb to the outer cell wall of the biomass. The second is a slow metabolism (within hours or days) dependent cellular uptake that has been modeled as a mass transfer process from the outer cell wall to the cell or cell wall interior (Cho, et al.,1994 and Axtell, et al., 2003).

The advantage of using living cells over non-living biomass to remove heavy metals is that living cells work as well as dead when the metal concentration is low, and the living cells can generate new biomass through growth allowing the second removal mechanisms to occur. The major disadvantage is the toxic effect the metals can have on the organism; therefore, the using non-living biomass is preferred to remove the high concentration of heavy metals (Wang and Wood, 1984).

Azolla is a small aquatic fern. In fact, it is a symbiotic pair of Azolla filiculoides and a heterocystous blue-green alga Anabaena azollae. It has been used as a fertilizer in botanical gardens because of nitrogen-fixing capability (Peters and Meeks, 1989). Azolla has been used for several decades as green manure in rice fields. On the other hand, it has negative effects on the aquatic ecology due to its capable of colonizing rapidly to form dense mats over water surfaces.

Controlling its reproduction has been deemed necessary in some Azolla-abundant areas like South Africa (Ashton and Walmsley, 1976) and the north part of Iran.

In this regard, the development of an Azolla-based biosorbent for wastewater treatment, especially in developing countries, may benefit environmental problems, by removing heavy metals from water using this weed (Zhao, et al., 1999).

The non-living Azolla filiculoides has been shown to be able to effectively adsorb Cr (III), Cr (VI), zinc (II) and nickel (II) from solutions and electroplating effluent (Zhao, et al., 1997, 1998 and 1999) and gold (III) from aqueous solution (Antunes, et al., 2001). We had shown that the removal of heavy metals could be increased by activation of the non-living Azolla filiculoides using H₂O₂/MgCl₂ (Taghi Ganji, et al., 2005).

The kinds of living biomass also have been shown to be able to effectively remove heavy metals. This process decreases the growth ability of biomass that it depends on the toxic quantity of each heavy metal ion. For instance, Azolla caroliniana can remove Hg (II), Cr (III) and Cr (VI) from municipal waste water (Bennicelli, et al., 2004). Microspora and Lemna minor also to be able to remove Pb²⁺ and Ni²⁺ from aqueous solution (Axtell, et al. 2003).

In this study, the toxic effect of Pb²⁺, Cd²⁺, Ni²⁺and Zn²⁺ on the living Azolla filiculoides by determining of the biomass growth and the presence effect of water’s NaCl and total dissolved solids (TDS) in this process were studied.

Materials and Methods

The experiment was performed in a number of flasks as batch biosorption experiments. 4 ml IRRI solution as a commercial nutrient without nitrates was added to each jar (because Azolla used nitrogen provided by the cyanobacteria Anabaena azollae) (Ladha, et al., 1992).

IRRI medium contained K₂SO₄ (174 µg/ml), CaCl₂ (147 µg/ml), MgSO₄ (169 µg/ml), H₃O₄ (144 µg/ml), Fe chelate (3 µg/ml), NaH₂PO₄ (138 µg/ml), CuSO₄ (0.16 µg/ml), MnCl₂ (3.6 µg/ml), ZnSO₄ (0.4 µg/ml), NaMoO₄ (0.8 µg/ml), H₃BO₃ (5.6 µg/ml), CoCl₂ (0.1 µg/ml) and glucose (500 µg/ml).

The Pb²⁺, Cd²⁺, Ni²⁺ and Zn²⁺ (metals under experiments) stock solutions were prepared by dissolving their corresponding the salts of Pb(NO₃)₂, CdCl₂.2.5H₂O, NiCl₂ and ZnSO₄ (analytical grade from Merck) in deionised water. TDS also was provided by dissolving the salts of Na₂SO₄.10H₂O, CaCl₂.2H₂O, MgCl₂.6H₂Oand NaHCO₃ (Merck) in deionised water. The heavy metal solutions (volume 3 l) were introduced with known concentrations (C_o) 4 mg/l into the flasks (each solution contained one metal ion) except one of the flask that was used as a control. Viz. the control solution, containing only nutrient medium and biomass.Fresh Azolla filiculoides (as living biomass) was collected from the surface of the Anzali International Wetland in the north part of Iran. The amounts of Azolla (20 g.) were washed with deionised water for 1 min and were then added to each flask.

During 15 days as the experiment period the following parameters were maintained: pH 7.0 ± 0.2, water temperature (25 ± 2 °C), photoperiod 16/8 (8 h. by day and night under light of fluorescent lamp), agitation rate 50rpmfor 6hin each day.At an interval of 48 h, the biomass obtained was collected, weighed (fresh mass), and at the end of 15 day of cultivation it was dried at 70 °C until no further weight loss within 8 days (as dry mass). Dried matter of Azolla filiculoides was digested with 0.2 M. HNO₃ for determination of metal content in biomass. The analysis of heavy metal content in the solution and biomass were performed by a Shimadzu Model AA680 Flame Atomic Absorption Spectrophotometer (Japan).

Results

Azolla growth

Heavy metals as the growth inhibitors

As shown in Figure 1, fresh Azolla mass was increased during experiment period. The initial Azolla mass and metals concentration were 20 g. and 4 mg/l (for each heavy metal, individually), respectively.

The control Azolla grows with the higher rate so that after 15 days, its fresh weight was 56.3 g., while at same time, the Azolla mass in the containing samples containing Pb²⁺, Cd²⁺, Ni²⁺ and Zn²⁺ were 42.3 g., 32.7 g., 38.6 g. and 46.8 g., respectively.

In other words, at the end of experiment period (after 15 days), the presence of Pb²⁺, Cd²⁺, Ni²⁺ and Zn²⁺ caused a distinct limitation of Azolla filiculoides growth relative to a control sample about 25%, 42%, 31% and 17% less growth, respectively. It is considerable that Zinc and nickel are both essential trace elements, required in only small amounts to perform various coenzyme and regulatory functions unlike lead and cadmium (Salt and Prince, 2002). According to Figure 1 is appeared that the toxic effect of heavy metals on Azolla filiculoides growth is the following arrangement: Cd²⁺ > Ni²⁺> Pb²⁺ >Zn²⁺.

Effect of water’s TDS

Figure 2 shows the effect of TDS addition into samples on the biomass growth. This TDS consisted of Ca²⁺, Mg²⁺, Na⁺, HCO₃^-, Cl^- and SO₄^2- ions. The relation of each ion value to other ions value was selected about same at the different quantities of TDS. The comparisons were performed with due attention to the Azolla mass in the control and samples of without TDS after 15 days (section 1.1).

As can be seen, the control Azolla growth was decreased by increasing TDS, so that using TDS of 50 up to 300 ppm (as CaCO₃) decreased Azolla growth in quantities of 2.3 g. (4.0%) to 12.7 g. (22.5%), respectively. It may be due to the much more presence of cations and anions that have inhibitor effect in the nutrient uptake, especially, carbohydrates (Ladha, et al., 1992). On the other hand, the increasing of TDS increased the Azolla samples growth which containing heavy metal ions. In other words, using TDS of 50 up to 300 ppm (as CaCO₃) increased Azolla growth in quantities of 2.0-8.9 g. (4.7-21.0%), 2.7-7.4 g. (8.2-22.6%), 3.7-8.9 g. (9.5-23.0%) and 3.0-10.5 g. (6.4-22.4%) in Pb²⁺, Cd²⁺, Ni²⁺and Zn²⁺ solutions, respectively.

Heavy metals uptake by Azolla

Water post treatment

As can be seen from Figure 3, the removal of Pb²⁺, Cd²⁺, Ni²⁺ and Zn²⁺ (with initial concentration of 4 mg/l) after 15 days reached to about 61%, 57%, 68% and 74%, respectively. This experiment shows that the living Azolla filiculoides can be used to purification of polluted water by these heavy metals and at the mentioned conditions.

Figure 4 shows the effect of water salinity (NaCl) on the removal of heavy metal (C_o of 4 g/l) by living Azolla after 15 days. As can be seen, the addition of 1, 2 and 4 g NaCl /l (Merck) decreases the removal of heavy metal ions. These removal percentages are as follows, respectively: Pb²⁺ about 57%, 41% and 22%; Cd²⁺ about 51%, 34% and 19%; Ni²⁺ about 62%, 46% and 26%; Zn²⁺ about 69%, 52% and 19%.

Accumulation in Azolla mass

Figure 5 shows the contents of metals under examination in Azolla filiculoides (dry mass). To do this, the initial concentrations (C_o) were selected 0.1, 0.5 and 1 mg/l. and the biosorption time was 15 days. Heavy metal contents in biomass were determined by digestion of Azolla dried mass with 0.2M HNO₃. Pb²⁺ content was on the level 86, 320 and 586 mg/kg (dry mass) for these initial concentrations, respectively. The other metal ions were accumulated in the following amounts: 75, 165 and 371 mg. Cd²⁺/kg (dry mass); 93, 450 and 1010 mg. Ni²⁺/kg (dry mass); 110, 841 and 1260 mg.

Zn²⁺/kg (dry mass) for the same initial concentrations of metal ions, respectively. It had been shown that plant cadmium uptake was metabolically mediated, and appears to be competitive with zinc uptake (Grant, et al., 1998).

Discussion and Conclusion

The results obtained suggest that the living Azolla filiculoides has the capacity to accumulate large quantities of the heavy metals such as Pb²⁺, Cd²⁺, Ni²⁺ and Zn²⁺. This study showed that Azolla filiculoides has growth ability in the solutions containing these metal ions with the initial concentrations of 4 mg/l. within 15 days, although its growth was limited relative to Azolla control. The heavy metals concentration in solution can be modeled using the following material balance describing the time dependency of concentration (as a slow metabolism) with a first order differential equation as (Rahmani and Sternbetg, 1999):

where Cs is the metal ion concentration (mg/l.), m_x the wet (fresh) mass of plant (g), r_s the consumption rate of metal ion (mg metal ion /g biomass /h), and V the volume of water in experiment (l), that

r_s = ε _s (-r_x)

wherer_x is the disappearance rate of biomass (g biomass /g biomass /h), and ε _s the amount of metal ion per unit biomass (mg metal ion / g biomass).

using TDS up to 300 ppm. (as CaCO₃) increased Azolla growth up to 21.0%, 22.6%, 23.0% and 22.4% in Pb²⁺, Cd²⁺, Ni²⁺and Zn²⁺ solution, respectively. It can be due to the less diffusion of heavy metal ions into the biomass cells because of the presence and diffusion of ions with less harm (TDS) for the biomass growth. On the other hand, the addition of NaCl decreases the removal of heavy metal ions that can be due to the higher mobility and diffusion of Na⁺ and Cl^– relative to heavy metal ions (Rai and Rai, 2003).

Moreover, it was determined that the more growth of biomass was nearly led to more removal. The toxic effect of the individual metal ions was the following arrangement: Cd²⁺ > Ni²⁺ >Pb²⁺> Zn²⁺, respectively.

Acknowledgements

The authors are grateful to the Gilan Province Research Center of bony Fishes, Dr. M. Arvand and Mrs R. Mazaheri for their aids.

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