Mixed cellulose ester membrane filter

During the period between June and October, which was the high-occurrence season for waterborne cryptosporidiosis and giardiasis^{26 (link),27 (link),47 ,48} , water samples were collected from 4 sampling sties (as shown in Fig. 1) at ten-day intervals and a total of 52 samples were obtained. The sampling site 1# is close to the zoo in the south of the West lake and site 4# in the north, site 2# is located in the swimming area in the East lake and site 3# is near the cruise ship terminal. Each sample was collected using a 20-L plastic container. After collection, the samples were transported on ice to the laboratory immediately and parasitic pathogens in the samples were concentrated by a membrane filter dissolution method described previously^{49 (link)}. Briefly, water samples were filtered and then the mixed cellulose ester membrane filter (diameter, 142 mm; pore size, 1 μM; Advantec MFS, Inc. Japan) was dissolved in acetone solutions, followed by centrifugation at 1,050 × g for 10 min at 4 °C. The packed pellets were resuspended in a suitable volume (2–10 mL) of distilled water. Half of the resuspension from each sample was used for protozoan enumeration and the other half for DNA extraction.

Sewage samples were concentrated 80-fold via the membrane adsorption and elution
method as described previously14 (link)15 (link)16 (link). Briefly,
800 ml of the sewage was centrifuged at
3200 × g for 30 min
at 4 °C. MgCl₂ solution (2.5 M)
was added to the supernatant to a final concentration of 0.05 M, and
the pH was adjusted to 3.5 with hydrochloric acid (0.5 M). The
solution was filtered through a 0.45 μm mixed cellulose
ester membrane filter (ADVANTEC). Subsequently, the membrane was cut into small
pieces and absorbents on the membrane were eluted with addition of
10 ml 3% beef extract solution followed by ultrasonication for
3 min. After centrifuge at 3
000 × g for 30 min,
the supernatant was filtered through a 0.22 μm filter
and was ready for RNA extraction.

An adsorption-elution method was used to concentrate viruses from sewage samples as described previously [14 (link)]. Briefly, sewage sample (1 L) was centrifuged at 3000 × g for 30 min at 4 °C. MgCl₂ (2.5 mol l⁻¹) was added to the supernatant to a final concentration of 0.05 mol l⁻¹, and the pH was adjusted to 3.5 by hydrochloric acid (0.5 mol l⁻¹). After filtered through a mixed cellulose ester membrane filter (0.45 μm, ADVANTEC, Tokyo, Japan), the membranes were cut into pieces. The viruses were eluted from the membranes, using 10 ml of a 3 % beef extract solution (pH = 9), followed by ultrasonication for 5 min. The eluted solution was centrifuged at 3000 × g for 30 min, and the supernatant was filtered through a 0.2 μm filter (PALL, Ann Arbor, MI, USA) prior to RNA extraction.

Concentration of the dissolved oxygen was measured using the method of Winkler as described previously [94 ]. Organic matter, total P and total N were determined in unfiltered water. Si, PO₄³⁻, NO₃⁻, NO₂⁻ and NH₄⁺ were analysed in water samples filtered through mixed cellulose ester membrane filters (Advantec, Tokyo, Japan), with a pore diameter 0.45 μm. Concentration of the nutrients was determined using a spectrophotometer UNICO-2100 (Dayton, NJ, USA) according to [138 ]: PO₄³⁻ was identified by the Denigès–Atkins method in modification with tin chloride, NH₄⁺ was detected by the indophenol method [139 (link)] and silica with a method based on measuring the intensity of the yellow colour accompanying the formation of the silicomolybdic acid complex. NO₃⁻ and NO₂⁻ concentration was measured by high performance liquid chromatography (EcoNova, Novosibirsk, Russia) with UV detection on an inverse-phase column modified with octadecyltrimethylammonium bromide [94 ,95 (link)]. Total N and P were measured spectrophotometrically after oxidation with persulfate. Permanganate oxidation index [140 ] and bichromate oxidation demand were used to assess organic substances content [141 (link)]. Chlorophyll a concentration was determined as described previously [66 (link)].