Data collection
Water quality parameters were measured six times during the experimental
period, on day 0, 27, 37, 44, 51 and 58. Conductivity, temperature and
dissolved oxygen concentration (DO) were measured using a HACH portable
multi-parameter meter (HQ40d, HACH, USA). Total nitrogen (TN) and total
phosphorus (TP) were determined using spectrophotometry after digestion
with K2S2O8 in an
autoclave (120 °C, 1.1 kg cm-2) for 30 minutes,
measuring at wavelengths 220 and 275 nm for TN, and 700 nm for TP
(UV-2800, Unico, China, GB 11894-89, GB 11893-89). At the end of the
experiment, water was sampled from each experimental container and
filtered onto GF/C filters to analyze chlorophyll a (Chl a) content as
an indicator of phytoplankton biomass. Periphyton Chl a content (µg Chl
a per cm2 area) was determined by brushing off a 9
cm2 (3 × 3 cm) area in the middle of one side wall
after draining the water, filtering the brushed water through Whatman
GF/C filters. Chl a content was determined spectrophotometrically
after ethanol extraction (HJ 897-2017). Snails in each container were
collected manually and wet weight was determined. Ten P. crispuswere randomly selected in each container to measure internode length.
All macrophytes were harvested with shoot and root separately cleaned
and blotted dry to measure wet weight, after which they were dried in an
oven at 60 °C for 48 hours. Dried plant samples were ground individually
in a 2 ml tube on a mill
(MiniBeadbeater-16, Biospec
Products, USA). Plant total C and N were determined on an elemental NC
analyzer (Flash EA 1112, CE
Instruments, Italy). P content was determined by incinerating and
digesting the organic P with
K2S2O8 (same to water TP
analysis), and then measuring the dissolved phosphate concentration on a
spectrophotometer (Cleverchem380,
DeChem-Tech., Germany).