Pseudo-total concentrations of elements
were assayed after HNO3:H2O2 digestion in an autoclave (Wenzel
et al., 2001). Extracts were filtered (num. 42 filter paper, Whatman)
and diluted with milli-Q water. The extractable trace element content
of the soils was obtained by shaking 2 g of soil with 20 mL of 0.1 M
(NH4)2SO4 for 4 h, then the suspension was filtered and the filtrate
analyzed (Vázquez et al., 2008). Extraction was performed in triplicate
at the beginning and the end of the toxicity test.
Element concentration in samples of leachates and soils (total and
extractable) was analyzed by atomic absorption spectrometry (Perkin
Elmer Analyst 800 for Cd, Cu and Zn) or atomic fluorescence (P S Analytical
10.055 Millennium Excalibur System). Three analytical replicates
were measured per sample.
2.6. Statistical analysis
The data were analyzed statistically using STATGRAPHICS software
(Version 5.0). Statistically significant differences for chemical and
toxicological data were established by analysis of variance (ANOVA)
with Fisher's least significant difference procedure (LSD, P b 0.05). Logprobit
methods were used to calculate L(E)C50. Linear regression
analysis was performed for chemical soil analysis and toxicity data.