Effect of interactions between nickel and other heavy metals on the soil microbiological properties J. Wyszkowska, E. Boros, J. Kucharski University of Warmia and Mazury in Olsztyn, Olsztyn, Poland ABSTRACT A pot greenhouse experiment was performed to determine the effect of contamination with nickel interacting with other heavy metals on the microbiological properties of soil. The study was conducted on samples of soils classified under natural conditions as typical Eutric Cambisol developed from heavy loamy sand and typical Eutric Cambisol developed from light silty loam. Soil material was contaminated with nickel in the amount of 50 and 200 mg Ni2+/kg. The treatments with 200 mg Ni2+/kg were additionally contaminated with other heavy metals (Zn2+, Cu2+, Pb2+, Cd2+, Cr6+), in the amount of 50 mg/kg soil. The following treatments, in which the soil was contaminated with hea- vy metals applied alone or in combinations, were compared in the study: Ni, Zn, Cu, Pb, Cd, Cr, NiZn, NiCu, NiPb, NiCd, NiCr, NiZnCu, NiZnPb, NiZnCd, NiZnCr, NiZnCuPb, NiZnCuCd, NiZnCuCr, NiZnCuPbCd, NiZnCuPbCr, NiZnCuPbCdCr. The experiment was carried out in four replications. A microbiological analysis was performed on days 28 and 56. The tested crop was oat. It was found that the impact of particular heavy metals on microbiolo- gical properties of soils depended on their type, interactions between nickel and zinc, copper, lead, cadmium and chromium (VI), date of analysis and soil species. Soil contamination with heavy metals reduced the population size of Azotobacter spp. The counts of other microbial groups, i.e. copiotrophic bacteria, spore-forming copiotrophic bacteria, oligotrophic bacteria, spore-forming oligotrophic bacteria, ammonifying bacteria, nitrogen immobilizing bacteria, cellulose-decomposing bacteria, Arthrobacter spp., Pseudomonas spp., actinomyces and fungi, showed va- ried susceptibility to heavy metals. Keywords: soil microbes; heavy metals; soil Heavy metals are permanently bound to the Fertilization of sewage sludge as well as composts sorption complex, which may result in their ac- from different wastes, and also wide utilization of cumulation in soil. Heavy metals exert a signifi- this metal in paper, food and chemical industry are cant effect on soil microbes and soil processes, the causes of local contamination of environment thus disturbing the biological equilibrium of soil, with this element. Nickel is used for production of followed by soil degradation (Huang and Shindo laboratory apparatus, medical instruments, steel 2000). Babich and Stotzky (1997) demonstrated melting, artificial materials, electrodes, and bat- that heavy metals are highly toxic to soil microbes. teries, and to cover metal objects (Kabata-Pendias The impact of heavy metals on microorganisms and Pendias 2001). The wide use of nickel in dif- and on enzymatic activity depends, among oth- ferent branches of industry exposes environment ers, on soil pH, content of organic and mineral to its uncontrolled emission into atmosphere, colloids, as well as on the type of heavy metals water and soil. Its influence on microbiological and their chemical properties (Kucharski and properties of the soil is less recognized than that Wyszkowska 2004). of other heavy metals. Nickel is one of the most toxic heavy metals. Its Literature (Pandey and Sharma 2002) provides geochemical properties are similar to cobalt and abundant information on the influence of single iron. Geochemical properties of nickel result in heavy metals on soil metabolism as well as on similar distribution and influence of this element the growth and development of different plant in environment. Nickel in soils appears most often species. However, data on combined effects of on the second and the third degree of oxidation. several heavy metals on the microbiological and 544 PLANT SOIL ENVIRON., 53, 2007 (12): 544–552 biochemical properties of soil as well as on plants the soil uncontaminated with heavy metals was are scarce (Giridhara and Siddaramappa 2002); still studied as well. in a natural environment heavy metal pollution is Prior to the establishment of the experiment, in most cases caused by some heavy metals. soil samples were weighed on an individual basis, Hence, the aim of the present study was to de- fertilizer components were added and the soil was termine the impact of nickel interacting with other contaminated with heavy metals. The samples heavy metals (zinc, copper, lead, cadmium, chro- were mixed thoroughly and put into pots. Oat mium) on the microbiological activity of soil. In (cv. Bajka) was sown when a moisture content level this investigation all heavy metals were applied of 60% capillary water capacity of soil was reached. alone in the dose of 50 mg/kg, whereas nickel as After emergence oat plants were thinned to 12 per a less recognized element was used in two doses: pot. The plants were collected at the panicle dif- 50 and 200 mg/kg. 200 mg Ni2+ was a background ferentiation stage (day 56 of the experiment) and of soil contamination for application of other their mass was determined. Simultaneously, soil heavy metals. samples were taken for a microbiological analysis. A microbiological analysis of soil was also per- formed on day 28, and the obtained results are MATERIAL AND METHODS presented as means of two measurements. For microbiological analyses 10 g of soil samples was The experiment was performed in a greenhouse. taken. All analyses were done in 6 replications Plastic pots were filled with 3 kg of typical Eutric for each sample. Cambisol developed from heavy loamy sand and 3 kg The analysis included the determination of the of typical Eutric Cambisol developed from light silty counts of oligotrophic bacteria, copiotrophic bacte- loam. The detailed characteristics of soils are given ria, spore-forming oligotrophic bacteria and spore- in Table 1. The experiment was carried out in four forming copiotrophic bacteria – on the Hattori’s replications. All treatments were regularly fertilized medium (Hattori and Hattori 1980); ammonifying with macro- and micronutrients, as follows (pure bacteria, nitrogen immobilizing bacteria and cel- component in mg/kg soil): N – 100 [CO(NH2)2], lulose-decomposing bacteria – on the Zaborowska’s P – 44 [KH2PO4], K – 83 [KH2PO4 + KCl], medium (Zaborowska et al. 2006); Azotobacter Mg – 20 [MgSO4.7 H2O], Cu – 5 [CuSO4.5 H2O], spp. – by the method of Fenglerowa (Fenglerowa Zn – 5 [ZnCl 2], Mn – 5 [MnCl 2.4 H 2O], 1965); Arthrobacter spp. – on the Mulder and Mo – 5 [Na2MoO4.2 H2O], B – 0.33 [H3BO4]. Antheumisse’s medium (Mulder and Antheumisse Different heavy metals contamination was the main 1963); Pseudomonas spp. – on the Mulder and 2+ variable experimental factor: Ni (NiCl2.6 H2O) – 50 Antheumisse’s medium containing nystatin (Mulder 2+ or 200 mg/kg soil and 50 mg/kg soil of Zn (ZnCl2), and Antheumisse 1963); actinomyces – on the 2+ 2+ Cu (CuCl 2), Pb [Pb(CH 3COO).3 H 2O], Kuster and William’s medium containing nystatin 2+ 6+ Cd (CdCl2.2.5 H2O), Cr (K2Cr2O7). The treat- and actidion (Parkinson et al. 1971); and fungi – on ments contaminated with 200 mg Ni2+/kg were the Martin’s medium (Martin 1950). additionally contaminated with the following heavy The results were verified statistically by the metals: Zn, Cu, Pb, Cd, Cr, ZnCu, ZnPb, ZnCd, Duncan’s multiple range test and a two-factorial ZnCr, ZnCuPb, ZnCuCd, ZnCuCr, ZnCuPbCd, analysis of variance, using the Statistica software ZnCuPbCr, ZnCuPbCdCr. In this investigation (StatSoft, Inc. 2003). Table 1. Some physicochemical properties of soils used in the experiment Granulometric composition (mm) Hh S Corg Soil species 1–0.1 0.1–0.02 < 0.02 pHKCl mmol(+)/kg soil (g/kg) % hls 66 17 17 6.90 11.25 89.30 7.50 lsl 42 32 26 7.00 8.77 159.00 11.15 hls – heavy loamy sand; lsl – light silty loam; Hh – hydrolytic acidity; S – sum of exchangeable basic cations; Corg – organic carbon content PLANT SOIL ENVIRON., 53, 2007 (12): 544–552 545 RESULTS AND DISCUSSION and depended on the type of heavy metal, soil species and date of analysis, as confirmed by the The elements used in the experiments and the coefficients of correlation between the doses of interactions between nickel and these elements nickel and other heavy metals (Zn2+, Cu2+, Pb2+, changed the population size of all tested microbial Cd2+, Cr6+), mean oat yield and the counts of soil groups (Tables 2–4). This impact was different microbes (Table 5). Table 2. Effect of soil contamination with nickel and other heavy metals on the counts of oligotrophic and copiotrophic bacteria (cfu/kg dm) 8 7 8 8 Olig × 10 Oligp × 10 Cop × 10 Copp × 10 Metals* soil species hls lsl hls lsl hls lsl hls lsl 0 83 ± 3 79 ± 1 32 ± 1 42 ± 1 49 ± 3 68 ± 10 49 ± 3 53 ± 4 Ni50 99 ± 10 74 ± 6 24 ± 3 36 ± 3 55 ± 5 68 ± 12 24 ± 4 75 ± 7 Ni200 91 ± 7 100 ± 4 29 ± 3 136 ± 18 195 ± 11 81 ± 8 64 ± 3 56 ± 2 Zn 49 ± 7 61 ± 2 19 ± 1 44 ± 2 188 ± 14 79 ± 10 72 ± 12 80 ± 5 Cu 66 ± 8 131 ± 2 12 ± 2 132 ± 8 138 ± 20 91 ± 4 68 ± 7 90 ± 6 Pb 65 ± 11 105 ± 5 16 ± 3 120 ± 14 206 ± 11 92 ± 2 50 ± 5 50 ± 7 Cd 35 ± 4 57 ± 3 16 ± 2 112 ± 9 204 ± 21 65 ± 6 47 ± 4 76 ± 12 Cr 90 ± 6 90 ± 5 40 ± 2 86 ± 9 303 ± 37 70 ± 1 42 ± 5 51 ± 3 NiZn 64 ± 13 59 ± 1 29 ± 4 93 ± 5 121 ± 4 71 ± 5 36 ± 4 59 ± 2 NiCu 89 ± 5 154 ± 4 26 ± 4 122 ± 9 130 ± 12 57 ± 5 51 ± 5 87 ± 13 NiPb 70 ± 8 114 ± 7 13 ± 2 118 ± 11 171 ± 11 89 ± 25 45 ± 1 91 ± 10 NiCd 123 ± 11 136 ± 1 18 ± 4 99 ± 5 166 ± 25 81 ± 2 59 ± 6 81 ± 9 NiCr 199 ± 14 189 ± 6 33 ± 3 61 ± 6 175 ± 13 108 ± 8 46 ± 4 76 ± 10 NiZnCu 79 ± 12 142 ± 8 31 ± 4 57 ±