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MERC ER01-10
3
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 STDN system) ballast tank. These two tanks on the
MV Cape
Washington
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.
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).
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, time-integrated, continuous
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 BALPURE 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).
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.
Ten 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 four distinct methods: (A) One sub-sample was fixed with standard Lugol’s solution
to determine total cell abundances under an inverted compound microscope using grid settlement
columns and phase contrast lighting. (B) A second sub-sample was stained using CMFDA (5-
chloromethylfluorescein diacetate) as a selective live/viable indicator. Stained
sub-samples were
incubated and observed on a Sedgewick Rafter slide using a Leitz Laborlux S modified for
epifluorescence.
(C) A third sub-sample was filtered and frozen until analysis of total and active
chlorophyll-a by the NASL. (D) Finally, a fourth 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.
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 standard USEPA method: membrane
filtration on modified mTEC agar. Abundances of total and toxigenic
V. cholerae
were
calculated by filtration and selection on TCBS agar and enumerated using a species-specific