2.3.3.
Experiment III – determination of exudate activity
Our
study also investigated whether allelopathically active com-
pounds
were naturally exuded from C. australis and P. crispus into
the
surrounding medium, in this case lake water. Fresh macrophytes
were carefully washed free of debris and attached algae with
tap
water then submerged in lake water (20 g FW 2 L
) and incubated
for 48 h in eight different small glass aquaria (four replicates
for
each macrophyte). Four controls were also prepared by incubating
the same volume of the lake water without macrophytes. The
incubation
water was subsequently filtered over glass filters (GF/C
Whatman)
and isopore polycarbonate membrane filters (0.22 m
pore
size, Millipore) to remove suspended particles and possible
bacterial
contaminants. The filtrates (100 mL) were added to four
replicate
250 mL Erlenmeyer flasks (for each macrophyte and for
the
control) containing 50 mL BG-11 medium inoculated by A. variabilis
at an initial cell density of 7 × 10
6
cells mL
−1
. Nutrient levels
at
the site of field collection (Symes, P., pers. comm. March 2012), as
well
as nutrients in the BG-11 medium, were high enough to support
the growth of microalgae throughout the experiment, hence
additional
nutrients were not required. The flasks were kept in the
CT
room for 10 d. The algal cell densities were counted every second
day
using a haemocytometer to determine numbers of cells mL
−1