## 2 Materials and Methods
### Sampling Methodology  
Indigenous organisms and sediments were collected in the Borromean Gulf, in front of the town of Carmine Inferiore (that was outside the area directly affected by industrial Hg polluting sources), and at one site in Lake Mergozzo (Figure 1). Sediment samples were collected using a 5-L or a 40-L Van Veen grab depending on specific sampling purposes: recovery of indigenous organisms for Hg bioaccumulation, collection of sediments for standardized bioaccumulation tests in the laboratory and recovery of indigenous organisms for taxonomical identification (Figure 2). The whole study was coordinated by the Piedmontese Environmental Protection Agency (ARPA Piedmont, Omega, Italy) and a synopsis of the actual activities carried out at the sampling sites is provided in Table 1.  
Figure 1: Lake Maggiore watershed (Italy/Switzerland): white box = position of the chlor-alkaly plant; black dots = sampling sites for indigenous chironomids, oligochaetes and bottom sediments; white stars = location of sediment cores collected during previous studies [21; 22]. See text for details and Table 1 for site codes. Indigenous chironomids and oligochaetes for Hg analysis were recovered only in Spring 2010 by sieving sediment samples on a 250 \(\upmu\)m nylon mesh net directly in the field and then kept in lake water with a thin layer of sediments. Tubicolous chironomids and oligochaetes were sorted in the laboratory, purged in filtered (<1 \(\upmu\)m) Lake Maggiore water for 6 h, blot dried and frozen pending lyophilisation and analysis (Figure 2). Aliquots of the same sediments were sieved through 2 mm and 50 \(\upmu\)m for determination of total Hg. The use of a 40-L grab sampler was necessary to recover from sediments a sufficient amount of organisms for one Hg analysis of pooled indigenous organisms. The choice of tubicolous chironomids chironomini and oligochaetes tubificidae is related to their feeding habits that make these organisms suitable proxies for studying different habitat conditions. Most tubificids stay with their caudal end protruding out of the top surface sediments (head-down) and serve as a proxy for the strict within-sediment habitat conditions. By contrast, the tube-building chironomids chironomini feed on freshly deposited or on suspended matter [17; 23; 24] without leaving their tubes and keeping their head exposed to the water (head-up). They serve as a proxy for water-sediment interface habitat conditions. Sediment samples for Hg bioaccumulation tests in the laboratory were collected in spring 2010, sieved at 2 mm, frozen to kill indigenous organisms and then thawed for preparation of batch exposures in the laboratory (Figure 2 and Figure S1). Aliquots of homogenized, sieved sediments were also freeze-dried and analysed for total Hg (Figure 2). As will be discussed later in the text, differences in the mesh-size of sieves were without effect on the total Hg content measured in sediment aliquots. Organisms for taxonomic identification were recovered in spring and autumn 2010 following the Italian national standardized method [25] according to the Water Framework Directive [26]. The autumn sampling was performed to verify that benthic organisms with a terrestrial adult stage (which include chironomids) were not partially or completely absent from sediments collected in spring due to the emergence of their images. The methodology provided for taking samples along transects at different depths representing the littoral, sublittoral and deep zones of a lake. In the present study, littoral stations (less than 25 m deep) were excluded because tubicolous chironomids and oligochaetes could be more easily found at higher depths. Three transects were, therefore, considered (one in front of Pallanza municipality, one in front of Baveno, and one in front of Carmine Inferiore), each one including a sub-littoral (SL) and a profundal (P) station (Table 1 and Figure 1).