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MERC ER02-10
3
allow for water at a flow rate of 400m
3
/hr to be split equally at flow rates of 200 m
3
/hr. Just
before this split,
challenge condition concentrations of total suspended solids (TSS) and
particulate carbon (POC)
were augmented by injecting a
concentrated slurry of Arizona test dust
and humic acid (developed and validated by the Naval Research Laboratory, Key West, Florida).
The water was then delivered simultaneously to either a “control” (untreated) ballast tank or a
“treated” (passing first through the SiCURE
TM
system) ballast tank. These two tanks were used
for the required holding time of five days and were essentially identical in size and structure.
Each tank was filled to approximately 250 m
3
for each test trial.
Physical Parameters -
Temperature, salinity, dissolved oxygen, chlorophyll fluorescence,
turbidity and pH were measured every 15 minutes during the test trials by two identical multi-
parameter probes placed, one each, into the control and test tanks. Initial inline samples of ballast
water during the filling of the control and test tanks were collected, filtered, and analyzed (using
USEPA methods) for the water quality parameters of particulate organic carbon (POC),
dissolved organic carbon (DOC) and total suspended solids (TSS) by the CBL/UMCES Nutrient
Analytical Services Laboratory (NASL).
Sampling -
A total of 10 identical 1.1 m
3
conical bottom mesocosms were also used for
controlled sampling during each trial. Using the mesocosms, five sequential samples were taken
during: (A) initial filling of tanks, just prior to the split of control and treated water (T0 Control),
(B) initial filling of test tank, just downstream of the SiCURE
TM
system during filling of test tank
(T0 Treated), (C) during discharge of control water after a five-day holding time (TF Control),
and (D) during discharge of treated water after a five-day holding time (TF Treated).
Live Organisms > 50
µ
m
-
Immediately after filling, 1.0 m
3
of water in each mesocosm
was filtered through a 35 µm plankton net to concentrate the zooplankton for qualitative and
quantitative analyses under a dissecting microscope. The proportion and total concentration of
live versus dead organisms was determined using standard movement and response-to-stimuli
techniques within one hour of collecting the individual samples. Zooplankton samples were also
fixed and returned to the laboratory for additional taxonomic evaluations.
Live Organisms 10 - 50
µ
m
-
Fifteen to twenty liters of well-mixed, but unfiltered water
from each mesocosm were also collected immediately after filling, to determine concentrations
of organisms in the 10 to 50 micron size class using three distinct methods: (A) A sub-sample
was stained using a combination of CMFDA (5-chloromethylfluorescein diacetate) and FDA
(fluorescein diacetate) as selective live/viable indicators. Stained sub-samples were incubated
and observed on a Sedgewick Rafter slide using a Leitz Laborlux S modified for epifluorescence.
(B) A second sub-sample was fixed with standard Lugol's solution for use in taxonomic
identifications and to serve as a backup sample.
(C) A third sub-sample was filtered and frozen
until analysis of total and active chlorophyll-a by the NASL. (D) Finally, a forth sub-sample was
used to determine chlorophyll-a levels after allowed to regrow under favorable conditions. An
increase in chlorophyll, or positive regrowth, indicates that viable phytoplankton were in the
samples, whereas chlorophyll levels at or below detection limits of the laboratory analytical
method suggests that there was no viable phytoplankton.
Live Microbes -
Additional subsamples of unfiltered water were also collected from each
mesocosm to determine concentrations of total heterotrophic bacteria and three specific indicator
pathogens,
E. coli
, intestinal
Enterococci
, and toxigenic
Vibrio
cholerae
. Total heterotrophic
bacteria were enumerated by spread plate method using NWRI agar. The presence and
abundance of intestinal
Enterococci
was determined using a commercially available
chromogenic substrate method. Culturable
E. coli
concentrations were determined using a