# Soil Hg Contamination Impact on Earthworms'
## Materials and Methods  
### Experimental Set-up  
Two experiments were performed in 5-L polypropylene pots (19 cm in diameter) with perforated lids, where soil and earthworms were incubated. For both experiments, soils were collected (in October 2015 for experiment 1 and October 2016 for experiment 2) in a field near the town of Baron in Canton Valais, Switzerland (46\({}^{\circ}\)18\({}^{\prime}\)10.6\({}^{\prime}\) N, 7\({}^{\circ}\)48\({}^{\prime}\)34.2\({}^{\prime}\) E), where long-term severe Hg pollution had been reported [24; 26; 27; 28]. The pollution originated from sediments of a canal that received a large amount of Hg (estimated at 50 to 250 tons) from ca. 1930 until 1976, released from industrial waste located upstream along the canal. Subsequently, the sediments from the canal were used to flatten and improve the nearby fluvial soils for pasture on several occasions between ca. 1930 and 1990. Experiment 1 tested the effect of a Hg contaminated soil layer on the microbial communities of earthworm, cast and soil samples. In experiment 1, a total of 12 pots were used. Six pots were filled with filled with 15 cm of a mix of 70% garden soil and 30% quartz sand. The other 6 pots were filled with 10 cm of the mixed soil, followed by a 5-cm layer of Hg-contaminated soil collected at the polluted site. Total Hg in the contaminated soil averaged 36.1 \(\pm\) 4.8 \(\upmu\)g Hg g\({}^{-1}\) and methyl-mercury (MeHg) averaged 4.6 \(\pm\) 0.5 \(\upmu\)g g\({}^{-1}\) (Table 1). Fine-cut iceberg lettuce (28 g) was placed on the soil surface of each pot every four days. Earthworms (_Lombricus terrestris_; origin Canada) were purchased in a local fishing shop as it was logistically impossible to collect local ones at the time of the experiment. Earthworms were kept in the uncontaminated mixed soil for a week for acclimation, and their gut systems were emptied by keeping the earthworms in boxes with only wet tissue for 2 days before the start of the experiment. Four worms were subsequently placed in each experimental pot and kept there in the dark at 20 \({}^{\circ}\)C for 10 days. The total mass of the four earthworms averaged 17.2 g per pot. At the end of the experiment, the earthworms were washed, their gut emptied again for 2 days following the same protocol before they were shock-frozen with liquid nitrogen. The frozen earthworm tissue was subsequently ground into powder to obtain homogenized samples. Earthworm casts were collected from the soil surface of each pot at the end of the experiment, weighed, homogenized and stored at \(-\)20 \({}^{\circ}\)C until further analysis. Experiment 2 tested the effect of Hg contaminated soil on the gut microbiome of different parts along the earthworm digestive track. In experiment 2, a total of 16 pots were filled completely with soil from the vicinity of the Hg-polluted site in Raron. Half of the pots (8) were filled with highly Hg-contaminated soil (36.1 \(\upmu\)g Hg g\({}^{-1}\)) collected very close to the contaminated canal, where soil Hg levels were previously found to be the highest [24]. The remaining eight pots were filled with uncontaminated soil (0.25 \(\upmu\)g Hg g\({}^{-1}\)) collected about 100 m away from the canal. Earthworms (_Lombricus terrestris_) were collected from the vicinity of the WSL research institute in Birmensdorf, Canton Zurich, Switzerland (47\({}^{\circ}\)21\({}^{\prime}\)43.9\({}^{\prime}\) N, 8\({}^{\circ}\)27\({}^{\prime}\)21.4\({}^{\prime}\) E), and their gut systems were emptied for two days. Four earthworms were then added to each pot, with a total biomass per pot of 8.9 g at the