Fluorescence analysis of copper(II) and zinc(II) binding behaviour of fulvic acids from pig slurry and amended soils D. HERNA ´ NDEZ a , C. P LAZA a , N. S ENESI b & A. P OLO a a Centro de Ciencias Medioambientales, Consejo Superior de Investigaciones Cientı ´ficas, Serrano 115 dpdo., 28006 Madrid, Spain, and b Dipartimento di Biologia e Chimica Agroforestale ed Ambientale, University of Bari, Via Amendola 165/A, 70126 Bari, Italy Summary The evaluation of the mechanisms and extent of interaction of Cu(II) and Zn(II) with fulvic acids (FAs) is of importance for understanding the mobility and bioavailability of these metals in pig slurry (PS)- amended soils. In this work, a fluorescence titration method and a single site model were used to charac- terize the interaction of Cu(II) and Zn(II) with FAs isolated from PS, from soils amended with 90 and 150 m 3 ha –1 year –1 of PS for 7 years and from the corresponding unamended soil. The binding capaci- ties of FAs and the strengths of metal ion-FA complexes were larger for Cu(II) than for Zn(II). With respect to the unamended control soil FA, PS-FA was characterized by smaller binding capacities and stability constants. The binding capacities and affinities of PS-amended soil FA were intermediate between those of unamended soil FA and PS-FA, and tended to decrease with increasing amounts of PS applied to soil, thus suggesting a partial incorporation of FA fractions of PS into native soil FA. Introduction Animal manures, such as pig slurry (PS), have traditionally been applied to soil as a means of increasing soil organic matter and plant nutrient contents (Senesi et al., 2006). However, this practice has recently raised serious environmental concerns due to the presence in PS of Cu(II) and Zn(II) ions used abun- dantly as pig feed additives, which may reach excessive amounts in PS-amended soils, endangering soil and water quality (Giusquiani et al., 1998; Nicholson et al., 1999; De la Torre et al., 2000; Taboada-Castro et al., 2002). Metal ion mobility and bioavailability in soil is extensively controlled by soil organic matter (Lindsay, 1979; Sposito, 1994; Sparks, 2002), and especially humic substances, of which humic acids (HAs) and fulvic acids (FAs) represent two major fractions. These materials are the most important soil organic ligands in terms of metal binding capacity due to their large content of oxygenated functional groups, including various car- bonyl, carboxyl, phenolic, alcoholic and enolic hydroxyl groups (Stevenson, 1994; Tipping, 2002). For these reasons, the knowl- edge of the effects of PS application on the composition, struc- ture and metal ion binding properties of native soil HA and FA is a key requirement for understanding and predicting the trans- port, retention and bioavailability of Cu(II) and Zn(II) ions in PS-amended soils. Recently, Herna´ndez et al. (2006) investigated the effect of seven annual consecutive additions of PS at two rates (90 and 150 m 3 ha –1 year –1 ) on the compositional, structural and func- tional properties of soil HA. Further, fluorescence spectros- copy, a well-established, reliable and invaluable means for examining interactions of HS with metal ions (e.g. Senesi, 1992; Fukushima et al., 1997; Elkins & Nelson, 2001, 2002; Wu et al., 2004), was used to evaluate and compare the Cu(II) and Zn(II) binding behaviour of the PS-HA fraction, native soil HA, and PS-amended soil HAs. The FA fraction differs from the HA fraction mainly in terms of its smaller molecular weight, carbon content and aromaticity, larger content of oxygenated functional groups and greater sol- ubility and mobility in soil (Stevenson, 1994; Senesi & Loffredo, 1999). Because of these different properties, FAs can form metal complexes that are more soluble, bioavailable and mobile than those formed by HAs. Generally, HAs tend to immobilize and accumulate metal ions in soil solid phases, whereas FAs can act as metal ion carriers in soil solution (Stevenson, 1994; Tipping, 2002). Since PS application to soil is expected to modify not only the HA fraction, but also the FA fraction, the objectives of this work were to determine and compare: (a) the Cu(II) and Zn(II) Correspondence: C. Plaza. E-mail: c.plaza@ccma.csic.es. Received 27 April 2007; revised version accepted 8 May 2007 900 # 2007 The Authors Journal compilation # 2007 British Society of Soil Science European Journal of Soil Science, August 2007, 58, 900–908 doi: 10.1111/j.1365-2389.2007.00932.x