Accumulation of Heavy Metals in Freshwater Organisms: Assessment of Toxic Interactions

Turk J Chem 25 (2001) , 173 – 179. c TÜBİTAK Accumulation of Heavy Metals in Freshwater Organisms: Assessment of Toxic Interactions∗ Neylan DİRİLGEN Boğaziçi University, Department of Chemistry, Bebek P.K.2, İstanbul-TURKEY Received 10.10.2000 Heavy metals are continuously released into the terrestrial environment by natural sources and human activities. The uptake and accumulation of heavy metals by plants promotes a mechanistic understanding of the biological significance of particular metal concentrations and distributions in biota. The toxicity of chromium, zinc, copper and cobalt ions and their binary mixtures are studied at varying test levels using duckweed as the test organism. The accumulation of metal ions are determined by atomic absorption spectroscopy. The type of toxic interactions in binary mixtures is assessed as ‘synergistic’, ‘antagonistic’ and ‘additive’ by a statistical approach. Key Words: metal accumulation, heavy metal interactive effects, duckweed Introduction Diverse industrial wastes have aggravated the problem of water pollution. This problem becomes complex because of the non-degradability of inorganic pollutants like heavy metals1 . Metals have received particular attention among other non-degradable toxic chemicals because of their adverse effects on aquatic life forms2,3 . To control water pollution, the immediate problems have to be solved by adopting alternative technologies to chemical-specific tools which suit low capital availability and minimum manpower. There has been considerable interest in using aquatic plants for removal of various pollutants, including heavy metals, from water bodies because of their fast growth rate and simple growth requirements, which are favorably compared to those of fish4,5 . Moreover, aquatic plants are particularly important in heavy metal pollution studies, since the analysis of these plants can give an indication of the state of water environment to which they have been exposed6 . The common duckweed, Lemna minor, is potentially useful as an indicator of pollution because of its ability to integrate and rapidly monitor the pollutants’ variations in the water. Moreover, they tolerate unstable environmental conditions and exhibit high sensitivity to heavy metal toxicity7 . The majority of published data concerning the heavy metal removal potential by aquatic plants is focused on single metal ∗ This paper has beed presented at MBCAC III (3rd Mediterranean Basin Conference on Analytical Chemistry) 4-9 June, 2000 Antalya-Turkey 173 Accumulation of Heavy Metals in Freshwater Organisms:..., N. DİRİLGEN effects. However, aquatic organisms in natural systems are exposed to mixtures of metals, which may substantially add to, multiply or suppress the effects of single components. The purpose of the present study is to assess the interactive metal accumulation effects in binary combinations as ‘additive’, ‘antagonistic’, or ‘synergistic’. The aim of this study was to evaluate metal enrichment ability and toxicity to assess the feasibility of using duckweed as an indicator of metal contamination in aquatic ecosystems. In this study, the method that is followed is composed of two major parts: Part 1 involved testing of binary combinations of Zn+Co, Zn+Cu, Zn+Cr, Cu+Cr, Cu+Co and Cr+Co at various concentration levels, and Part 2 involved assessing toxic interactions by statistical testing of the difference between metal accumulation in binary mixtures and in single components. Experimental Reagents and Supplies Duckweed plants were subcultured from original stocks, maintained in our laboratory since 1988. Continuous illumination of stock and test cultures was made with fluorescent tubes of Philips TLD 36W/54 having an intensity of 40 µEm−2 s−1 at plant level. Several different test protocols are available for duckweed, as reported by Huebert et al.8 , ASTM9 and Cowgill and Milazzo10 ; the method used in this study was based on static conditions. Duckweed plants were subcultured from an original stock in full-strength Jacob culture medium11 . Sixty-four milligrams (wet weight) of bright green and healthy duckweeds were measured out and rinsed carefully with distilled water. Duckweeds were then placed in 200-mL of metal test solution contained high-quality glass jar and covered with aluminum foil to exclude side lighting and with a watch glass to prevent evaporation. The test solutions were adjusted to pH 6.0-6.5 with 0.1 M KOH or HCl. Temperature was kept at 25-27◦C. On the seventh day of frond incubation, plants were washed three times with distilled water and weighed. The experimental set for each binary mixture consisted of control samples and five replicates of the test sample. Consequently, plants were dried at 80◦C and digested in 3 mL of concentrated HNO3 . Metal accumulation in the plant body was measured by a flame atomic absorption spectrometer (FAAS), Varian SpectrAA Model 250 Plus. An air-acetylene flame was used. Working conditions for the metal ions, i.e., wavelength, concentration ranges and typical sensitivity values, are as follows, respectively: Cu (324.7 nm), 2-8 mg/L, 0.04 mg/L Zn (213.9 nm), 0.4-1.6 mg/L, 0.009 mg/L Co (240.7 nm), 3-12 mg/L, 0.066 mg/L Cr (357.9 nm), 2-8 mg/L, 0.055 mg/L Samples were diluted to a suitable final volume with double-distilled water prior to FAAS measurements. Cr determinations were made with a flameless atomic absorption spectrometer equipped with a carbon rod atomizer (CRA), Varian Techtron Model 1200. Interference in the air-acetylene flame from Cu, Mg and Ca has been reported and the extent of interference is strongly dependent on the flame stoichiometry. Also, Co and Fe have been found to cause depression of the Cr signal12. In the CRA technique, samples were diluted accordingly to suit the working conditions of 0.01-0.1 mg/L. Standards were prepared from 10 mg/L Cr and diluted with 20% NaCl to prepare 0.025, 0.05 and 0.1 mg/L concentration levels. Binary test levels were selected with respect to the previously reported data of the EC50 (mg/L) of 174 Accumulation of Heavy Metals in Freshwater Organisms:..., N. DİRİLGEN each metal component13 , i.e., the effective toxicant concentration to induce 50% growth inhibition. Binary metal combinations were prepared basically in two main groups. Group I consisted of sets with varying equimolar concentrations of the metal ions within the range of EC50 of each metal component. Group II consisted of sets with combinations where the concentration of one metal ion was kept constant while the other one was varied. Stock solutions of 1000 mg/L of CoCl2 and K2 Cr2 O7 were prepared from reagent grades, and the dichromate solution was acidified with sulfuric acid to maintain a relatively stable Cr (VI) species. Stock solutions of 1000 mg/L of zinc and copper were prepared from high purity metals by dissolving them in 1:1 (v:v) HCl (12 M) and 1:1 (v:v) HNO3 (16 M). Metal accumulation in the plant body was re (...truncated)