Electronic Supplementary Material

Earthworm metabolomic responses after exposure to aged PCB contaminated soils

Melissa Whitfield Åslund†, Myrna J. Simpson†*, André J. Simpson†, Barbara A. Zeeb‡, andAllison Rutter§,

†Department of Physical and Environmental Sciences, University of Toronto, 1265 Military Trail Toronto, Ontario, Canada M1C 1A4

‡Department of Chemistry and Chemical Engineering, Royal Military College of Canada, Kingston, Ontario, Canada

§School of Environmental Studies, Queen’s University, Kingston, Ontario, Canada

*Corresponding author, Phone: +1 416-287-7234, Fax: +1 416 287-7279, Email:

S1.Analysis of soil PCB concentrations

PCBs present in soil samples were extracted and quantified using procedures adapted from EPA Methods 3540C and 8082A (EPA 1996; EPA 2007). Prior to analysis, approximately 10 g (wet weight) was sub-sampled from each soil for the determination of percent dry weight (EPA 1996). A separate subsample (10 g wet weight) was collected from each soil, accurately weighed, mixed with 10 g sodium sulfate (Na2SO4; min 99.5% purity, Fisher Scientific), and spiked with decachlorobiphenyl (DCBP, min 99.2% purity, Sigma Aldrich) as a surrogate standard. Soil samples were extracted in a soxhlet apparatus for 4 hours at 4-6 cycles per hour in 250 mL of dichloromethane(DCM, Pesticide grade, Fisher Scientific).Following extraction, sample extracts were concentrated by rotoevaporation, the solvent exchanged for hexane, and then extracts were applied to a Florisil column (1000 mg/ 6 mL, Fisher Scientific)for cleanup.Analysis of extracts for total Aroclorswas then performed using an Agilent 6890 Plus gas chromatograph equipped with a micro-63Ni electron capture detector (GC/μECD), a SPB™-1 fused silica capillary column (30 m, 0.25 mm ID x 0.25 μm film thickness) and HPChem station software.The carrier gas was helium, at a flow rate of 1.6 mL·min-1.Nitrogen was used as the makeup gas for the electron capture detector (ECD).Detection limits were 0.1 mg·kg-1. All values were reported as mg·kg-1 dry weight. Total soil PCB concentrations were corrected for extraction efficiency using the surrogate standard percent recovery and calculated on a dry weight basis by comparison to observed soil percent dry weight (EPA 1996).

For every nine samples extracted and subsequently processed, one analytical blank (Ottawa sand), one control sample (a blank sample spiked with a known amount of either Aroclor 1254 or 1260), and one analytical duplicate were also extracted and processed.None of the analytical blanks contained any PCBs at concentrations above detection limits (0.1 μg·g-1) and all control samples were between 70 to 130% of the expected value.Relative standard deviations between the samples and their analytical duplicate were below 30% for all results.

S2:Soil water holding capacity (WHC)

Water holding capacity (WHC) was determined after Parent (Parent and Caron 1993). Water was mixed into approximately 10 g of soil until a slurry was formed and allowed to settle for one hour.The slurry was added to a glass funnel lined with filter paper (Whatman, Grade 1) which was then covered with aluminum foil and allowed to drain into a beaker for 24 hours. The mass of the soil at maximum water holding capacity (gwhc) was recorded.Samples were then dried for 24 hours at 105°C and the mass of dry soil (gdry) also recorded. Soil WHC was determined gravimetrically by dividing the mass of water in the soil at WHC by the mass of oven-dry soil, i.e. WHC = (gwhc – gdry)/gdry.

S3:Soil pH

To measure soil pH, 50 mL of deionized water was added to 10 g of air-dried soil (1:5 soil to water ratio), shaken for 30 minutes on an orbital shaker and allowed to settle for one hour.The pH of the supernatant was then measured using an Accumet basic AB15 pH meter (Fisher Scientific).

S4:Permutation testing

The significance of each PLS model was estimated through response permutation testing (Alam et al. 2010; Eriksson et al. 2006). In this method, the X table (the quotient normalized NMR spectra) remained fixed, and the order of the Y vector was randomly permuted 400 times. Each time, a new PLS model was fitted and Q2Y calculated, providing a reference distribution of the Q2Y statistic. Confidence in the validity of the PLS model is increased if the PLS models built using the permuted dataset consistently give lower Q2Y values than the original model (Eriksson et al. 2006).

S5:Cross-validated predictions of y-variables and histograms of Q2Y values from 400-fold permutation tests

Tables S1- S4 present the cross-validated predictions and permuted Q2Y values for the PLS models presented in Tables 2a and 2b in the main text. For each PLS model, two figures are presented:

Cross-validated predictions:Average predictions of y-values () given spectra iby the PLS model derived during the leave one out cross-validation procedure with spectra iomitted for PLS models with optimized number of components (See Tables 2a, 2b).The solid line indicates a linear regression between the actual and predicted values. Error bars represent the standard error of the mean.In PLS models which include the unspiked artificial control treatment, the mean predicted values for this treatment are indicated with an unfilled marker (∆).

Histograms of permuted Q2Y values:Histogram of PLS regression Q2Y values for cross-validated PLS models with optimized number of components (See Tables 2a, 2b) Distribution was constructed using 400 permutations of the Y table. The Q2Y observed in the optimized model using the unpermuted Y table is also indicated.

References

Alam TM, Alam MK, Neerathilingam M, Volk DE, Sarkar S, Shakeel Ansari GA, Luxon BA (2010) 1H NMR metabonomic study of rat response to tri-phenyl phosphate and tri-butyl phosphate exposure. Metabolomics 6:386-394

Eriksson L, Johansson E, Kettaneh-Wold N, Trygg J, Wikström C, Wold S (2006) Multi- and Megavariate Data Analysis Part I Basic Principles and Applications. Umetrics, Umeå , Sweden

Parent LE, Caron J (1993) Physical Properties of Organic Soils. In: Carter MR (ed) Soil sampling and methods of analysis. Lewis Publishers, Boca Raton, Florida, pp 450-451

United States Environmental Protection Agency (EPA) (2007) Method 8082A Polychlorinated Biphenyls by Gas Chromatography. In: Test Methods for Evaluating Solid Waste, Physical/Chemical Methods. National Technical Information Service (NTIS), U.S. Department of Commerce, Springfield, VA

United States Environmental Protection Agency (EPA) (1996) Method 3540C Soxhlet Extraction. In: Test Methods for Evaluating Solid Waste, Physical/Chemical Methods. National Technical Information Service (NTIS), U.S. Department of Commerce, Springfield, VA

Table S1: Cross-validated predictions and permutation histograms for PLS models constructed using earthworm metabolic data collected after two days of exposure, with the artificial soil treatment included

Artificial soil treatment included? / Length of exposure / Y-variable / Cross-validated predictions / Histograms of permuted Q2Y values
Yes / 2 days / [PCB] / /
Yes / 2 days / pH / /
Yes / 2 days / Total soil C / /
Yes / 2 days / Inorganic C / /
Yes / 2 days / Organic C / /

Table S2: Cross-validated predictions and permutation histograms for PLS models constructed using earthworm metabolic data collected after 14 days of exposure, with the artificial soil treatment included

Artificial soil treatment included? / Length of exposure / Y-variable / Fig. 1 Cross-validated predictions / Fig. 2 Histograms of permuted Q2Y values
Yes / 14 days / [PCB] / /
Yes / 14 days / pH / /
Yes / 14 days / Total soil C / /
Yes / 14 days / Inorganic C / /
Yes / 14 days / Organic C / /

Table S3: Cross-validated predictions and permutation histograms for PLS models constructed using earthworm metabolic data collected after two days of exposure, with the artificial soil treatment excluded

Artificial soil treatment included? / Length of exposure / Y-variable / Fig. 1 Cross-validated predictions / Fig. 2 Histograms of permuted Q2Y values
No / 2 days / [PCB] / /
No / 2 days / pH / /
No / 2 days / Total soil C / /
No / 2 days / Inorganic C / /
No / 2 days / Organic C / /

Table S4: Cross-validated predictions and permutation histograms for PLS models constructed using earthworm metabolic data collected after 14 days of exposure, with the artificial soil treatment excluded

Artificial soil treatment included? / Length of exposure / Y-variable / Fig. 1 Cross-validated predictions / Fig. 2 Histograms of permuted Q2Y values
No / 14 / [PCB] / /
No / 14 / pH / /
No / 14 / Total soil C / /
No / 14 / Inorganic C / /
No / 14 / Organic C / /

Figure S1:

Fig. S1 Sample 500 MHz1H NMR spectrum of E. fetidatissue extract collected and processed using methods described in the primary text.

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