1H NMR-based metabolomic observation of a two-phased toxic mode of action in Eisenia fetida after sub-lethal phenanthrene exposure

Brian P. Lankadurai; David M. Wolfe; André J. Simpson; Myrna J. Simpson

doi:10.1071/EN10094

RESEARCH ARTICLE (Open Access)

Next Contents Vol 8(2)

¹H NMR-based metabolomic observation of a two-phased toxic mode of action in Eisenia fetida after sub-lethal phenanthrene exposure

Brian P. Lankadurai ^A , David M. Wolfe ^A , André J. Simpson ^A and Myrna J. Simpson ^A ^B

+ Author Affiliations

- Author Affiliations

^A Department of Chemistry, University of Toronto, 1265 Military Trail, Toronto, ON, M1C 1A4, Canada.

^B Corresponding author. Email: myrna.simpson@utoronto.ca

Environmental Chemistry 8(2) 105-114 https://doi.org/10.1071/EN10094
Submitted: 24 August 2010 Accepted: 2 December 2010 Published: 2 May 2011

Journal Compilation © CSIRO Publishing 2011 Open Access CC BY-NC-ND

Environmental context. Phenanthrene is a persistent soil contaminant, whose toxic mode of action in earthworms has not been fully examined. We adopt a metabolomics approach, using ¹H nuclear magnetic resonance (NMR) spectroscopy, to measure the response of earthworms to sub-lethal phenanthrene exposure. The results indicate that NMR-based metabolomics may be used to monitor responses to sub-lethal levels of contaminants and to delineate their toxic mode of action.

Abstract. ¹H NMR-based metabolomics was used to examine the response of the earthworm Eisenia fetida to sub-lethal phenanthrene exposure. E. fetida were exposed via contact tests to six sub-lethal (below the measured LC₅₀ of 1.6 mg cm^–2) concentrations of phenanthrene (0.8–0.025 mg cm^–2) for 48 h. Multivariate statistical analysis of the ¹H NMR spectra of earthworm tissue extracts revealed a two-phased mode of action (MOA). At exposures below 1/16th of the LC₅₀, the MOA was characterised by a linear correlation between the metabolic response and exposure concentration. At exposures ≥1/16th of the LC₅₀, the metabolic response to phenanthrene appeared to plateau, indicating a distinct change in the MOA. Further data analysis suggested that alanine, lysine, arginine, isoleucine, maltose, ATP and betaine may be potential indicators for sub-lethal phenanthrene exposure. Metabolite variation was also found to be proportional to the exposure concentration suggesting that NMR-based earthworm metabolomics is capable of elucidating concentration-dependent relationships in addition to elucidating the MOA of sub-lethal contaminant-exposure.

Additional keywords: concentration-dependence, contact tests, LC₅₀, metabonomics.

References

[1] J. G. Bundy, E. M. Lenz, N. J. Bailey, C. L. Gavaghan, C. Svendsen, D. Spurgeon, P. K. Hankard, D. Osborn, J. A. Weeks, S. A. Trauger, Metabonomic assessment of toxicity of 4-fluoroaniline, 3,5-difluoroaniline and 2-fluoro-4-methylaniline to the earthworm Eisenia veneta (Rosa): identification of new endogenous biomarkers. Environ. Toxicol. Chem. 2002, 21, 1966..
| 12206438PubMed |

[2] M. J. Simpson, J. R. McKelvie, Environmental metabolomics: new insights into earthworm ecotoxicity and contaminant bioavailability in soil. Anal. Bioanal. Chem. 2009, 394, 137.
| Environmental metabolomics: new insights into earthworm ecotoxicity and contaminant bioavailability in soil.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhsFWisb0%3D&md5=b57bc5aa80a551588927c245e903630bCAS | 19194697PubMed |

[3] S. A. E. Brown, A. J. Simpson, M. J. Simpson, Evaluation of sample preparation methods for nuclear magnetic resonance metabolic profiling studies with Eisenia fetida. Environ. Toxicol. Chem. 2008, 27, 828.
| Evaluation of sample preparation methods for nuclear magnetic resonance metabolic profiling studies with Eisenia fetida.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXktFGntLg%3D&md5=f8467651471cd45df8ab5ab4160d6142CAS | 18333692PubMed |

[4] C. A. Edwards, P. J. Bohlen, The effects of toxic-chemicals on earthworms. Rev. Environ. Contam. Toxicol. 1992, 125, 23..

[5] L. C. Fitzpatrick, R. Sassani, B. J. Venables, A. J. Goven, Comparative toxicity of polychlorinated-biphenyls to earthworms Eisenia foetida and Lumbricus terrestris. Environ. Pollut. 1992, 77, 65.
| Comparative toxicity of polychlorinated-biphenyls to earthworms Eisenia foetida and Lumbricus terrestris.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK38XksVaitLo%3D&md5=d0b0ccd388e0635384a0a4855289f267CAS | 15091979PubMed |

[6] M. R. Viant, Recent developments in environmental metabolomics. Mol. Biosyst. 2008, 4, 980.
| Recent developments in environmental metabolomics.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXhtFeitrfK&md5=c3993a0cee1e75c6a46b549a3f163f1bCAS | 19082136PubMed |

[7] M. Oldiges, S. Lutz, S. Pflug, K. Schroer, N. Stein, C. Wiendahl, Metabolomics: current state and evolving methodologies and tools. Appl. Microbiol. Biotechnol. 2007, 76, 495.
| Metabolomics: current state and evolving methodologies and tools.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXpt1ahsbs%3D&md5=df34d7a8117ff350a8b9f0d8abd438ddCAS | 17665194PubMed |

[8] Y. Y. Mosleh, S. Paris-Palacios, M. Couderchet, G. Vernet, Acute and sublethal effects of two insecticides on earthworms (Lumbricus terrestris L.) under laboratory conditions. Environ. Toxicol. 2003, 18, 1.
| Acute and sublethal effects of two insecticides on earthworms (Lumbricus terrestris L.) under laboratory conditions.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXhtFKjsLc%3D&md5=67f840669273a3a24eb9a5b6c050c319CAS | 12539138PubMed |

[9] E. F. Neuhauser, C. A. Callahan, Growth and reproduction of the earthworm Eisenia fetida exposed to sublethal concentrations of organic-chemicals. Soil Biol. Biochem. 1990, 22, 175.
| Growth and reproduction of the earthworm Eisenia fetida exposed to sublethal concentrations of organic-chemicals.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK3cXhvFegtL0%3D&md5=c9b3c7229eac85a21c88ff7d2931fc7fCAS |

[10] D. R. Ekman, Q. Teng, D. L. Villeneuve, M. D. Kahl, K. M. Jensen, E. J. Durhan, G. T. Ankley, T. W. Collette, Investigating compensation and recovery of fathead minnow (Pimephales promelas) exposed to 17 alpha-ethynylestradiol with metabolite profiling. Environ. Sci. Technol. 2008, 42, 4188.
| Investigating compensation and recovery of fathead minnow (Pimephales promelas) exposed to 17 alpha-ethynylestradiol with metabolite profiling.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXlt1Sks7o%3D&md5=ad652146af4f861f9db0b30febc09dfdCAS | 18589986PubMed |

[11] B. M. Beckwith-Hall, J. K. Nicholson, A. W. Nicholls, P. J. D. Foxall, J. C. Lindon, S. C. Connor, M. Abdi, J. Connelly, E. Holmes, Nuclear magnetic resonance spectroscopic and principal components analysis investigations into biochemical effects of three model hepatotoxins. Chem. Res. Toxicol. 1998, 11, 260.
| Nuclear magnetic resonance spectroscopic and principal components analysis investigations into biochemical effects of three model hepatotoxins.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1cXisVSiu7Y%3D&md5=5a11c1904758ede1c091bd76386f1b17CAS | 9548796PubMed |

[12] S. A. E. Brown, A. J. Simpson, M. J. Simpson, 1H NMR metabolomics of earthworm responses to sub-lethal PAH exposure. Environ. Chem. 2009, 6, 432.
| ¹H NMR metabolomics of earthworm responses to sub-lethal PAH exposure.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhsFSkurnK&md5=ce1e72442ee2dcc90157930d537ca8a4CAS |

[13] J. G. Bundy, M. P. Davey, M. R. Viant, Environmental metabolomics: a critical review and future perspectives. Metabolomics 2009, 5, 3.
| Environmental metabolomics: a critical review and future perspectives.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXisFSls7k%3D&md5=f8528d88bc47af243aadb4abbb08cdbbCAS |

[14] J. G. Bundy, D. Osborn, J. M. Weeks, J. C. Lindon, J. K. Nicholson, An NMR-based metabonomic approach to the investigation of coelomic fluid biochemistry in earthworms under toxic stress. FEBS Lett. 2001, 500, 31.
| An NMR-based metabonomic approach to the investigation of coelomic fluid biochemistry in earthworms under toxic stress.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXkvVemu7w%3D&md5=d53370733042d10c6abdb9500697faebCAS | 11434921PubMed |

[15] J. R. McKelvie, J. Yuk, Y. P. Xu, A. J. Simpson, M. J. Simpson, 1H NMR and GC/MS metabolomics of earthworm responses to sub-lethal DDT and endosulfan exposure. Metabolomics 2009, 5, 84.
| ¹H NMR and GC/MS metabolomics of earthworm responses to sub-lethal DDT and endosulfan exposure.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXisFSls7g%3D&md5=1688328b4ca9fcb0b33fd8ef8867b01bCAS |

[16] O. A. H. Jones, D. J. Spurgeon, C. Svendsen, J. L. Griffin, A metabolomics based approach to assessing the toxicity of the polyaromatic hydrocarbon pyrene to the earthworm Lumbricus rubellus. Chemosphere 2008, 71, 601.
| A metabolomics based approach to assessing the toxicity of the polyaromatic hydrocarbon pyrene to the earthworm Lumbricus rubellus.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXisVWjsbY%3D&md5=7445330843c7108859a9b535664969dfCAS | 17928029PubMed |

[17] S. A. E. Brown, J. R. McKelvie, A. J. Simpson, M. J. Simpson, 1H NMR metabolomics of earthworm exposure to sub-lethal concentrations of phenanthrene in soil. Environ. Pollut. 2010, 158, 2117.
| ¹H NMR metabolomics of earthworm exposure to sub-lethal concentrations of phenanthrene in soil.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXlvVent7w%3D&md5=040d4c79d9b40051a63f9b35826e7b24CAS | 20338676PubMed |

[18] S. Lundstedt, P. Haglund, L. Oberg, Simultaneous extraction and fractionation of polycyclic aromatic hydrocarbons and their oxygenated derivatives in soil using selective pressurized liquid extraction. Anal. Chem. 2006, 78, 2993.
| Simultaneous extraction and fractionation of polycyclic aromatic hydrocarbons and their oxygenated derivatives in soil using selective pressurized liquid extraction.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XivVels7g%3D&md5=d649d73c1bff64f02607a6d45d347530CAS | 16642985PubMed |

[19] M. J. Smith, T. H. Flowers, H. J. Duncan, J. Alder, Effects of polycyclic aromatic hydrocarbons on germination and subsequent growth of grasses and legumes in freshly contaminated soil and soil with aged PAHs residues. Environ. Pollut. 2006, 141, 519.
| Effects of polycyclic aromatic hydrocarbons on germination and subsequent growth of grasses and legumes in freshly contaminated soil and soil with aged PAHs residues.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XjtFGkt74%3D&md5=8ebbe7373b6db1ff074696fac6984352CAS | 16246476PubMed |

[20] M. K. Chung, R. Hu, K. C. Cheung, M. H. Wong, Pollutants in Hong Kong soils: polycyclic aromatic hydrocarbons. Chemosphere 2007, 67, 464.
| Pollutants in Hong Kong soils: polycyclic aromatic hydrocarbons.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXnslKktg%3D%3D&md5=c7a557d7ebb4c074b53a470cffe29fa9CAS | 17109918PubMed |

[21] C. Svendsen, D. J. Spurgeon, P. K. Hankard, J. M. Weeks, A review of lysosomal membrane stability measured by neutral red retention: is it a workable earthworm biomarker? Ecotoxicol. Environ. Saf. 2004, 57, 20.
| A review of lysosomal membrane stability measured by neutral red retention: is it a workable earthworm biomarker?Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3sXpsVSiu7g%3D&md5=b670defa639f48521c4261921ecf4ebeCAS | 14659363PubMed |

[22] R. van der Oost, J. Beyer, N. P. E. Vermeulen, Fish bioaccumulation and biomarkers in environmental risk assessment: a review. Environ. Toxicol. Pharmacol. 2003, 13, 57.
| Fish bioaccumulation and biomarkers in environmental risk assessment: a review.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD38XptFeqsro%3D&md5=e8f487a8d9b9c22d87eb26c4e9f1dfd8CAS |

[23] Earthworm acute toxicity tests, in OECD Guideline 207 1984 (Organisation for Economic Co-operation and Development: Paris).

[24] M. L. Hannam, S. D. Bamber, T. S. Galloway, A. J. Moody, M. B. Jones, Effects of the model PAH phenanthrene on immune function and oxidative stress in the haemolymph of the temperate scallop Pecten maximus. Chemosphere 2010, 78, 779.
| Effects of the model PAH phenanthrene on immune function and oxidative stress in the haemolymph of the temperate scallop Pecten maximus.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BC3cXhtValtL0%3D&md5=3b014970e052beedfca553d5e77eb8cbCAS | 20074773PubMed |

[25] C. M. Hurdzan, N. T. Basta, P. G. Hatcher, O. H. Tuovinen, Phenanthrene release from natural organic matter surrogates under simulated human gastrointestinal conditions. Ecotoxicol. Environ. Saf. 2008, 69, 525.
| Phenanthrene release from natural organic matter surrogates under simulated human gastrointestinal conditions.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1cXivVOitbs%3D&md5=b0500da9fb316623219bfb1ab7fd6470CAS | 17433439PubMed |

[26] Trimmed Spearman–Karber (TSK) Program, Version 1.5 1991 (Ecological Monitoring Research Division, Environmental Monitoring Systems Laboratory, US Environmental Protection Agency: Cincinnati, OH).

[27] M. A. Hamilton, R. C. Russo, R. V. Thurston, Trimmed Spearman–Karber method for estimating median lethal concentrations in toxicity bioassays. Environ. Sci. Technol. 1977, 11, 714.
| Trimmed Spearman–Karber method for estimating median lethal concentrations in toxicity bioassays.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaE2sXksl2it7s%3D&md5=290149ce379b8a45c8a01abd3db87d9fCAS |

[28] C. A. Edwards, J. E. Bater, The use of earthworms in environmental-management, in The 4th International Symposium on Earthworm Ecology 1990, pp. 1683–1689 (Pergamon-Elsevier Science Ltd: Avignon, France).

[29] A. J. Simpson, S. A. Brown, N. M. R. Purge, Effective and easy solvent suppression. J. Magn. Reson. 2005, 175, 340.
| Effective and easy solvent suppression.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXls1ygsrY%3D&md5=67f12730c50ea4ae54303eca7e7a36cfCAS | 15964227PubMed |

[30] J. G. Bundy, D. J. Spurgeon, C. Svendsen, P. K. Hankard, J. M. Weeks, D. Osborn, J. C. Lindon, J. K. Nicholson, Environmental metabonomics: Applying combination biomarker analysis in earthworms at a metal contaminated site. Ecotoxicology 2004, 13, 797.
| Environmental metabonomics: Applying combination biomarker analysis in earthworms at a metal contaminated site.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2MXhslyrs7o%3D&md5=95873eae73546df65733e9f724c0319dCAS | 15736850PubMed |

[31] L. Eriksson, E. Johansson, N. Kettaneh-Wold, J. Trygg, C. Wikstrom, S. Wold, Multi- and Megavariate Data Analysis Part 1: Basic Principles and Applications 2006 (Umetrics AB: Umea, Sweden).

[32] D. I. Broadhurst, D. B. Kell, Statistical strategies for avoiding false discoveries in metabolomics and related experiments. Metabolomics 2006, 2, 171.
| Statistical strategies for avoiding false discoveries in metabolomics and related experiments.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhsVWktL4%3D&md5=4228914b3a43ed4f6839bdf27ee981d6CAS |

[33] S. Wold, M. Sjostrom, L. Eriksson, PLS-regression: a basic tool of chemometrics. Chemometr. Intell. Lab. 2001, 58, 109.
| PLS-regression: a basic tool of chemometrics.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXotF2mtLw%3D&md5=3c29bb47eff6ccd96a20f683964223b9CAS |

[34] S. Wold, J. Trygg, A. Berglund, H. Antti, Some recent developments in PLS model building. Chemometr. Intell. Lab. 2001, 58, 131.
| Some recent developments in PLS model building.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3MXotF2mtLo%3D&md5=abfe021b5246cd72dcd79278fd9fe9e8CAS |

[35] S. Wold, L. Eriksson, Statistical validation of QSAR results, in Chemometrics Methods in Molecular Design (Ed. H. van de Waterbeemd) 1995, pp. 309–318 (VCH: Weinheim, Germany).

[36] M. B. Brown, A. B. Forsythe, Robust Tests for Equality of Variances. J. Am. Stat. Assoc. 1974, 69, 364.
| Robust Tests for Equality of Variances.Crossref | GoogleScholarGoogle Scholar |

[37] A. F. B. Boroujerdi, M. I. Vizcaino, A. Meyers, E. C. Pollock, S. L. Huynh, T. B. Schock, P. J. Morris, D. W. Bearden, NMR-based microbial metabolomics and the temperature-dependent coral pathogen Vibrio coralliilyticus. Environ. Sci. Technol. 2009, 43, 7658.
| NMR-based microbial metabolomics and the temperature-dependent coral pathogen Vibrio coralliilyticus.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXhtFCqtb7J&md5=aa9d7b13c4af1b842e9a80df0c7a98e6CAS | 19921875PubMed |

[38] D. R. Ekman, Q. N. Teng, D. L. Villeneuve, M. D. Kahl, K. M. Jensen, E. J. Durhan, G. T. Ankley, T. W. Collette, Profiling lipid metabolites yields unique information on sex- and time-dependent responses of fathead minnows (Pimephales promelas) exposed to 17 alpha-ethynylestradiol. Metabolomics 2009, 5, 22.
| Profiling lipid metabolites yields unique information on sex- and time-dependent responses of fathead minnows (Pimephales promelas) exposed to 17 alpha-ethynylestradiol.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXisFSls7Y%3D&md5=c8efcf49af259f8012808abba5505f65CAS |

[39] J. G. Bundy, H. C. Keun, J. K. Sidhu, D. J. Spurgeon, C. Svendsen, P. Kille, A. J. Morgan, Metabolic profile biomarkers of metal contamination in a sentinel terrestrial species are applicable across multiple sites. Environ. Sci. Technol. 2007, 41, 4458.
| Metabolic profile biomarkers of metal contamination in a sentinel terrestrial species are applicable across multiple sites.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXltVKqs70%3D&md5=676a6197a402726b6f710004b7bef7f8CAS | 17626452PubMed |

[40] W. Tuffnail, G. A. Mills, P. Cary, R. Greenwood, An environmental H-1 NMR metabolomic study of the exposure of the marine mussel Mytilus edulis to atrazine, lindane, hypoxia and starvation. Metabolomics 2009, 5, 33.
| An environmental H-1 NMR metabolomic study of the exposure of the marine mussel Mytilus edulis to atrazine, lindane, hypoxia and starvation.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD1MXisFSls7w%3D&md5=a82832ab67dc19ff63d6cf47a06a2146CAS |

[41] N. S. El-Shenawy, R. Greenwood, I. M. Ab-Del-Nabi, Z. I. Nabil, Effect of atrazine and lindane on the scope for growth of marine mussels Mytilus edulis. Acta Zool. Sin. 2006, 52, 712..

[42] P. H. Yancey, M. E. Clark, S. C. Hand, R. D. Bowlus, G. N. Somero, Living with water-stress – evolution of osmolyte systems. Science 1982, 217, 1214.
| Living with water-stress – evolution of osmolyte systems.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL38XlsFyisbw%3D&md5=442ff2b660e5ba77fdc5051b8f40c7faCAS | 7112124PubMed |

[43] S. A. S. Craig, Betaine in human nutrition. Am. J. Clin. Nutr. 2004, 80, 539..
| 15321791PubMed |

[44] L. Eriksson, P. L. Andersson, E. Johansson, M. Tysklind, Megavariate analysis of environmental QSAR data. Part I – A basic framework founded on principal component analysis (PCA), partial least squares (PLS), and statistical molecular design (SMD). Mol. Divers. 2006, 10, 169.
| Megavariate analysis of environmental QSAR data. Part I – A basic framework founded on principal component analysis (PCA), partial least squares (PLS), and statistical molecular design (SMD).Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XntFykt7o%3D&md5=13de0d6f1ca1b0ad92dd479293cfa9e8CAS | 16770514PubMed |

[45] L. Eriksson, P. L. Andersson, E. Johansson, M. Tysklind, Megavariate analysis of environmental QSAR data. Part II – Investigating very complex problem formulations using hierarchical, non-linear and batch-wise extensions of PCA and PLS. Mol. Divers. 2006, 10, 187.
| Megavariate analysis of environmental QSAR data. Part II – Investigating very complex problem formulations using hierarchical, non-linear and batch-wise extensions of PCA and PLS.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XntFyrs7Y%3D&md5=8ff0ac9b445aa216af2fa5e68c1f8feeCAS | 16802062PubMed |

[46] M. Henningsson, E. Sundbom, B. A. Armelius, P. Erdberg, PLS model building: a multivariate approach to personality test data. Scand. J. Psychol. 2001, 42, 399.
| PLS model building: a multivariate approach to personality test data.Crossref | GoogleScholarGoogle Scholar | 1:STN:280:DC%2BD38%2FksVCksg%3D%3D&md5=8cf9a75aba98270fd214f4f001fc704eCAS | 11771809PubMed |

[47] S. Carraro, S. Rezzi, F. Reniero, K. Heberger, G. Giordano, S. Zanconato, C. Guillou, E. Baraldi, Metabolomics applied to exhaled breath condensate in childhood asthma. Am. J. Resp. Crit. Care 2007, 175, 986.
| Metabolomics applied to exhaled breath condensate in childhood asthma.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXmsFyqsLw%3D&md5=5241dd5d76d2702d4f4dead3810fc350CAS |

[48] P. Geladi, B. R. Kowalski, Partial least-squares regression – a tutorial. Anal. Chim. Acta 1986, 185, 1.
| Partial least-squares regression – a tutorial.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaL28XmtVahs7c%3D&md5=ecdde10a303a213efaba96f43297bd65CAS |

[49] M. R. Viant, J. G. Bundy, C. A. Pincetich, J. S. de Ropp, R. S. Tjeerdema, NMR-derived developmental metabolic trajectories: an approach for visualizing the toxic actions of trichloroethylene during embryogenesis. Metabolomics 2005, 1, 149.
| NMR-derived developmental metabolic trajectories: an approach for visualizing the toxic actions of trichloroethylene during embryogenesis.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28Xht1SmsrjE&md5=1a48125c3ed554b0fc8b1e243c54947dCAS |

[50] O. Beckonert, H. C. Keun, T. M. D. Ebbels, J. G. Bundy, E. Holmes, J. C. Lindon, J. K. Nicholson, Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts. Nat. Protoc. 2007, 2, 2692.
| Metabolic profiling, metabolomic and metabonomic procedures for NMR spectroscopy of urine, plasma, serum and tissue extracts.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2sXhtlSlsr3E&md5=204325afd210fb611b89aad5cd844184CAS | 18007604PubMed |

[51] G. Balcerowska, R. Siuda, H. Czarnik-Matusewicz, Orthogonal signal correction to PLS modelling in application to spectral data. Acta Agrophysica 2005, 6, 7..

[52] M. Saint-Denis, J. F. Narbonne, C. Arnaud, E. Thybaud, D. Ribera, Biochemical responses of the earthworm Eisenia fetida andrei exposed to contaminated artificial soil: effects of benzo(a)pyrene. Soil Biol. Biochem. 1999, 31, 1837.
| Biochemical responses of the earthworm Eisenia fetida andrei exposed to contaminated artificial soil: effects of benzo(a)pyrene.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DyaK1MXmslGjtLs%3D&md5=b3fde7c598787852b3aab0d06ca1471bCAS |

[53] W. W. Zhang, Y. F. Y. F. Song, P. P. Gong, T. H. T. H. Sun, Q. X. Zhou, M. M. Liu, Earthworm cytochrome P450 determination and application as a biomarker for diagnosing PAH exposure. J. Environ. Monit. 2006, 8, 963.
| Earthworm cytochrome P450 determination and application as a biomarker for diagnosing PAH exposure.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD28XovFKlt7Y%3D&md5=b573a277fda5fa52daf2a21687de8587CAS | 16951757PubMed |

[54] R. K. Achazi, C. Flenner, D. R. Livingstone, L. D. Peters, K. Schaub, E. Scheiwe, Cytochrome P450 and dependent activities in unexposed and PAH-exposed terrestrial annelids. Comp. Biochem. Phys. C 1998, 121, 339..

[55] P. J. Brown, S. M. Long, D. J. Spurgeon, C. Svendsen, P. K. Hankard, Toxicological and biochemical responses of the earthworm Lumbricus rubellus to pyrene, a non-carcinogenic polycyclic aromatic hydrocarbon. Chemosphere 2004, 57, 1675.
| Toxicological and biochemical responses of the earthworm Lumbricus rubellus to pyrene, a non-carcinogenic polycyclic aromatic hydrocarbon.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD2cXpt1arsrg%3D&md5=d5fa0315324825ee1766746b27f1161dCAS | 15519413PubMed |

[56] J. J. Whyte, R. E. Jung, C. J. Schmitt, D. E. Tillitt, Ethoxyresorufin-O-deethylase (EROD) activity in fish as a biomarker of chemical exposure. Crit. Rev. Toxicol. 2000, 30, 347.
| Ethoxyresorufin-O-deethylase (EROD) activity in fish as a biomarker of chemical exposure.Crossref | GoogleScholarGoogle Scholar | 1:CAS:528:DC%2BD3cXmsVWjtrg%3D&md5=bdb7e906e747234f311a27928449aefcCAS | 10955715PubMed |

[57] R. F. Lee, Annelid cytochrome P-450. Comp. Biochem. Phys. C 1998, 121, 173..

[58] E. Gasteiger, C. Hoogland, A. Gattiker, S. Duvaud, M. R. Wilkins, R. D. Appel, A. Bairoch, Protein identification and analysis tools on the ExPASy server, in The Proteomics Protocols Handbook (Ed. J. M. Walker) 2005, pp. 571–607 (Humana Press: Totowa, NJ).