**8. Future perspectives**

fumarate, an intermediate of the trycarboxylic acid cycle. Research conducted with the same earthworm (*E. fetida*) and other families of organic compounds revealed a different metabolic response (Brown et al., 2010; Lankadurai et al., 2012), confirming the capability of metabolo‐ mics to discriminate the metabolic pathways involved in the response to a particular toxic compound. Moreover, the results strongly suggest that sets of biomarkers might be soon

The relevance of these and future studies on the development of risk assessment strategies is aggravated by the inherent risk of soil contamination for human health. Soil contaminants may be responsible for health effects costing millions of euros. Health problems range from cancer (arsenic, asbestos, dioxins), to neurological damage and lower IQ (lead, arsenic), kidney disease (lead, mercury, cadmium), and skeletal and bone diseases (lead, fluoride, cadmium). Overall, few studies have been conducted on the toxicity of complex chemical mixtures in soils. The effects of the soil and organisms within it upon organic pollutants are unknown. The data currently available correspond mostly to short-term studies and high level exposure of these chemicals, which is less relevant to the potential low-level, long term health impacts on living

sufficiently reliable as for their implantation in in toxicity standardized test.

**7. Metabolites related to soil contamination with heavy metals**

of metal detoxification and tolerance (Hall, 2002; Hall and Williams, 2003).

anhydrase from marine diatoms has been reported (Lane and Morel, 2000).

The uptake of excess metal ions is toxic to most organisms, and the biochemical impact of metal ions on the cells varies with the chemistry of the element as their chemical nature. In plants, phytotoxicity of heavy metals in most parts can be attributed to symplastic accumulation of heavy metals, such as the cytosol and chloroplast stroma. Metal-induced changes in develop‐ ment are the result of either a direct and immediate impairment of metabolism or signaling processes that initiate adaptive or toxicity responses that need to be considered as active processes of the organism. Transport processes have been recognized as a central mechanism

Some metals, for example, Zn and Cu, are essential for normal plant growth and development as they serve as structural and functional components of specific proteins. Other metals, for example, Cd and Pb, have no known function in plants although a Cd requirement for carbonic

Upon exposure to metals, organisms often synthesize a set of diverse metabolites that accu‐ mulate to concentrations in the millimolar range, particularly specific amino acids, such as proline and histidine, peptides such as glutathione and phytochelatins (PC), and the amines spermine, spermidine, putrescine, nicotianamine, and mugineic acids that can be detected as response to these metals exposure. The advance of toxicogenomics in relation to organic contaminants is significantly ahead of the equivalent research in metal contaminated soil (Table 1). Nevertheless, research conducted up to date has yielded a number of biomarkers representative of the biological response of soil microorganisms to metals toxicity. Thus, soil contamination with Pb has been related with an enhancement of lipid metabolism (Sanchez-Hernandez, 2006) and more directly with reduction of tyrosine levels (Wu et al., 2013).

organisms near to contaminated soil.

472 Environmental Risk Assessment of Soil Contamination

The main objective of metabolomics implementation in soil risk assessment is to meet the continuously increasing demand of safety data from human and ecological risk assessments. Accordingly, regulatory programs worldwide are currently incorporating tests with endpoints that involve the effects of chemicals and the impact in specific metabolic pathways (Ankley et al., 2006). Toxicological end-points can be general biological responses such as survival or weight loss (Baylay et al., 2012), but specific biomarkers provide the accuracy that was classically elusive for test with living organisms

Several issues immediately arise from the summary here presented, such as the need to perform field toxicological test, with natural soils rather than use artificial soils, as was the case with some of the studies listed in Table 1. Ecotoxigenomics can also benefit from the incorpo‐ ration of further analytical techniques. Techniques based on mass spectrometry are certainly required to understand the mechanisms involved in the alteration of metabolic pathways as response to toxicants. However, for screenings which merely require the detection of differ‐ ences between metabolic phenotypes, optical methods such as FT-IR would be suitable, particularly if extremely high sample throughput is required (Bundy et al., 2009). Although no data was available in the existing literature, Figure 2 illustrates the change in the fingerprint of organic compounds in a soil amended with different sources of carbon collected 10 after the application. While some of the groups of compounds might be merely related to the sources of carbon added, the variations in the signal associated to polysaccharides (600-1000 cm-1) can be associated to changes in the metabolic fingerprint of the soil system and therefore linked to microbiological activity in soil. Overall, the introduction of these results seeks to encourage further characterization of families of compounds in intact soil (or functional pools such as aggregates) in relation with soil processes, an approach that can find immediate application in the assessment of biological responses to toxic compounds in soil.

Figure 2. Absorption spectra obtained by Fourier transform infrared spectroscopy (FTIR) for an agricultural soil (S), soil amended with fresh residues (a): dry leaf litter (SL), peanut shell (SP), maize residue (SM), and soil amended with biochar (b) derived from those feedstocks (BL, BP or BM). Spectra presented (after 10 d incubation) are the average of 5 spectra obtained for different samples of each treatment. Hernandez-Soriano et al., unpublished data. **Figure 2.** Absorption spectra obtained by Fourier transform infrared spectroscopy (FTIR) for an agricultural soil (S), soil amended with fresh residues (a): dry leaf litter (SL), peanut shell (SP), maize residue (SM), and soil amended with bio‐ char (b) derived from those feedstocks (BL, BP or BM). Spectra presented (after 10 d incubation) are the average of 5 spectra obtained for different samples of each treatment. Hernandez-Soriano et al., unpublished data.

The variability of biological responses has been one of the main obstacles for their implementation in standardized risk assessment. However, the examination of changes in biological processes by accurate analytical techniques and powerful statistical tools has launched a new era in our understanding of the soil processes. The The variability of biological responses has been one of the main obstacles for their implemen‐ tation in standardized risk assessment. However, the examination of changes in biological

possibility of identifying the most sensitive metabolites for a certain toxicant and develop a tailored standardized

MCH-S thanks The University of Queensland for a postdoctoral research fellowship. JCJ-L thanks the European

The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the

Amiard-Triquet, C., Amiard, J.C., Rainbow, P.S., 2012. Ecological Biomarkers: Indicators of Ecotoxicological

Ankley, G.T., Daston, G.P., Degitz, S.J., Denslow, N.D., Hoke, R.A., Kennedy, S.W., Miracle, A.L., Perkins, E.J., Snape, J., Tillitt, D.E., Tyler, C.R., Versteeg, D., 2006. Toxicogenomics in Regulatory Ecotoxicology.

Baylay, A.J., Spurgeon, D.J., Svendsen, C., Griffin, J.L., Swain, S.C., Sturzenbaum, S.R., Jones, O.A.H., 2012. A metabolomics based test of independent action and concentration addition using the earthworm Lumbricus

Boer, M., Ellers, J., Gestel, C.M., Dunnen, J., Straalen, N., Roelofs, D., 2013. Transcriptional responses indicate attenuated oxidative stress in the springtail Folsomia candida exposed to mixtures of cadmium and

research program Marie Curie (FP7-PEOPLE-2011-IOF) for his PIOF-GA-2011-301550 grant.

test is the ultimate goal pursued.

**Acknowledgments** 

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processes by accurate analytical techniques and powerful statistical tools has launched a new era in our understanding of the soil processes. The possibility of identifying the most sensitive metabolites for a certain toxicant and develop a tailored standardized test is the ultimate goal pursued.
