Interaction between earthworms and NPs

Earthworm-soil system was studied to assess whether NPs provided at chronic dose through the diet could exert metabolic stress or toxicity. For this purpose earthworms were exposed to the NPs by placing contaminated horse manure on the top of the soil to simulate sewage sludge disposal.

2.8.1.

Experimental design

L. rubellus was exposed to Ag NPs, Ag+, Co NPs and Co2+ through its diet for five weeks. Ten adult earthworms were kept in terrarium with 500g of soil mix (see section 2.2 for the soil characteristics) at 65% of WHC and fed once a week with ground horse manure (0.5g dry weight of manure per worm per week). Horse manure, from a non-medicated horse, was spiked 24 hours prior to feeding, with a water solution of NPs and ions (as nitrate) to reach the 65% of WHC. The concentration of both nanoparticle and ion solutions was 10mg of pollutant kg−1 dry horse manure for all substances. The experiment was carried out in quadruple. Every week the earthworms were counted and weighed to assess growth and survival. After five weeks of exposure to contaminated food, earthworms of three boxes of each treatment were transferred to Petri dishes for two days in order to empty their gut and then prepared for further analysis (Fig. 2.3). The earthworms of the fourth box were moved to another soil, having the same characteristics of the previous one, and fed for another month with unpolluted food (Fig. 2.3). After such period, earthworms were transferred into Petri dishes for two days in order to empty their gut, and then prepared for further analysis (Fig. 2.2).

28 2.8.1.1. Chemical and biochemical soil analysis

At the end of the experiment soil samples were prepared for chemical and biological analysis as previously described in section 2.6.1.1 and 2.6.1.2, respectively. Differently from the abovementioned method, in this experiment soil respiration was determined by measuring the CO2

evolving from soil incubated under standard conditions. Briefly, 10g of soil at 50% of WHC was placed in 125mL glass bottles at 25°C, and the cumulative CO2 accumulated in the headspace after

3-day incubation was determined by a gas chromatograph (Trace GC, Thermo Electron, USA) equipped with a thermal conductivity detector (TCD).

In addition, PLFAs were determined on soils and earthworms faeces according to the modified Bligh and Dyer method (White et al. 1979). Briefly, at the end of the first time of exposure, 5g of soil and about 0.5g of earthworm faeces were extracted with a single-phase mixture of chloroform/methanol/citrate buffer on a horizontal shaker (250 rpm) for 3 hours at room temperature. After centrifugation (3000g, 5 minutes) the supernatant was transferred to another glassware tube and the soil vortexed and re-extracted for another 3 hours with an additional volume of extractant. The combined supernatant was split into two phases by adding citrate buffer and chloroform and left overnight to obtain separation. The CHCl3 layer was then transferred to a new

tube and dried by using a rotavapor. Phospholipids were separated from neutral lipids and glycolipids by using silicic acid columns. Neutral lipids and glycolipids were eluted with chloroform and acetone separately. Phospholipids were obtained from methanol elution and dried by using the rotavapor. A mild alkaline methanolysis was used to convert phospholipids into Fatty Acid Methyl Esters (FAMEs) (Guckert et al. 1985). FAMEs were recovered with a n- hexane/chloroform (4:1, v/v) mixture, reduced to dryness by rotavapor and re-dissolved in 200 µL of n-hexane. FAMEs were detected on a gas chromatograph (Focus-GC, Thermo Scientific, USA) equipped with a flame ionization detector and a fused-silica capillary column Mega-10 (50m x 0.32mm I.D.; film thickness 0.25µm). The GC temperature progression was: initial isotherm at 115°C for 5 minutes, increase at a rate of 1.5°C per minute from 115 to 230°C, and final isotherm at 230°C for 2 minutes. Both injection port and detector were set up at 250°C, respectively and Helium at 1mL min-1 in a constant flow mode was used as carrier. The injected volume was 1L in a splitless mode. Nonadecanoic acid methyl ester (19:0; cat no. N-5377) was used as an internal standard for quantification of FAMEs. The identification of the peaks was based on comparison of retention times to known standards (Supelco Bacterial Acid Methyl Esters mix cat no. 47080-U and Supelco 37 Component FAME mix cat no. 47885-U). The relative abundance of detected FAMEs

29 was expressed as mol %. The fatty acid nomenclature used was that described by Frostegård et al. (1993, 1996).

2.8.1.2. Earthworm tissue analysis

At the end of the first and second step of the experiment, some specimens were dried at 105°C, after 48 hours of depuration, and the metal concentration in the tissues was estimated by ICP-OES after digestion with HNO3 and H2O2 (4:1 v/v) in the microwave oven with the program above

described for the vegetable tissues (see section 2.7.1.1)

Earthworms exposed for 5 weeks to the NPs were analysed for tissue fatty acid content by the following standard procedure described by Kennedy (1994). About 150mg of earthworm sub- samples were weighed in 10mL glass test tubes, 1mL of 4 N NaOH in 50% methanol was added and then the mixture was heated for 30 minutes at 100°C in a water bath. After cooling at room temperature, 2mL of 6 N HCl in methanol was added for methylation of dissolved fatty acids in a water bath at 80°C (10 minutes). Then 1mL hexane/methyl-tert-butyl ether (1:1, v/v) was added and lipids extracted by shaking for 10 minutes. The organic phase was transferred to a new test tube and the extraction was repeated. The combined organic phase was washed once with 0.25 N NaOH, and subsequently transferred to 2mL vials for analysis on the gas chromatograph as above described. The degree of unsaturation D was calculated according to (Kates 1986):

D = ∑ (% mono-unsaturated + 2 * % di-unsaturated + 3 * % tri-unsaturated + …)/100.

In addition, earthworms were analysed to evaluate the any changes in morphology or apoptotic frequency in their tissues. After the second step, for each treatments and box (Fig. 2.2), five purified earthworms were stored at 4°C in a test tube containing 4% paraformaldehyde in 0.1M phosphate buffered solution (pH 7.4). The earthworms were dehydrated in ascending concentration alcohols, and paraffin Paraplast (Bio-Optica, Italy) embedded. Groups of four serial sections (5µm thick) were either stained for histological observations by Alcian Blue (Serfőző & Elekes 2010) and Hematoxylin-eosin (Bio-Optica, Italy) (Gambardella et al. 2010). Apoptosis was assessed on the evidence of morphological characteristics, such as chromatin condensation with 4', 6-DiAmidino-2- PhenylIndole (DAPI) staining that is a fluorescent stain that binds strongly to A-T rich regions in DNA, as well as by a fluorescein-conjugated TUNEL test (terminal deoxy nucleotidyl transferase- mediated nick end labelling, Roche, Germany) (Ferrando et al. 2005). For fluorescence observations, nuclear DAPI counterstaining (1:1000, Molecular Probes, The Netherlands) was carried out. Negative control was performed by incubating sections with the Label Solution,

30 containing the nucleotide mixture without the transferase enzyme. Sections were examined under a Leica optical microscope (Leica, Germany) and visualized with a Leica software program using TIFF image formats.