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been processed from each of the treated and untreated ballast tanks respectively. The sampling tanks are
designed to allow complete immersion of each net during the filtration process, thereby minimizing
trauma to filtered organisms. At the end of each trial (after five days), the control and treated ballast tanks
are drained and processed as described above, with the treated sample undergoing a second pass through
the UV irradiation unit (but not the filter) before sampling.
The proportion and total concentration of live versus dead organisms will be determined using standard
movement and response to stimuli techniques and this live/dead analysis will take place within two hours
of collecting the individual samples. Three m
3
is the volume collected for control water upon filling and
discharge from test tanks (high numbers of live organisms) and 7 m
3
is collected for treated water on
discharge after five days (presumably very few live organisms). Depending on concentrations,
quantification of zooplankton in initial samples (upon ballasting) and control samples may require
analysis of sub-samples and extrapolation to the entire 3 m
3
. Zooplankton samples will then also be
fixed with buffered, 10% formalin in 500ml Nalgene bottles and shipped to the Smithsonian
Environmental Research Center (SERC) for additional taxonomic evaluation. Total counts and general
taxonomic classification will be conducted under a dissecting microscope at 25X, except for some taxa,
which will be removed and identified using a compound microscope. Larval forms of invertebrates will
be identified to higher taxonomic levels such as order (e.g., Decapoda) suborder (e.g., Balanomorpha) or
class (e.g., Bivalvia). Adults will be identified to species in most cases.
B.4.2. Viable Organism 10 - 50 µm in size
A 75 L integrated sample will be collected as an unfiltered split sample in parallel with the >50µm
fraction. This sample will be the source water for all other analyses including the 10-50µm fraction. 2 l
from this integrated sample will be subject to detailed analysis and counting. Determination of
concentrations of viable organisms in this size class will be made using four distinct methods (described
below).
Analytical methods are described below and in SOPs. All live unfiltered samples will begin to be
examined within three hours of collection on the MERC Mobile Test Platform or nearby partner
laboratories, which combine state of the art facilities and depth of experience. Any preserved samples are
also transported to MERC partner laboratories for further analyses and taxonomic identification.
One 250 ml sub-sample will be stained using a combination of CMFDA (5-chloromethylfluorescein
diacetate) and FDA (fluorescein diacetate) as a selective live/viable indicator. Samples stained with
CMFDA+FDA, are incubated and observed on a Sedgewick Rafter slide using a Leitz Laborlux S
modified for epifluorescence. Cells are scored as live when showing strong fluorescence signature under
excitation (some cells also showed motility). This approach has been validated for use in the Chesapeake
Bay (Steinberg et al., 2011) and provides the data for comparison to the IMO D2 and USCG Phase 1
discharge standards.
As supporting information, three other sub-samples are analyzed. A second 250 ml is collected from the
initial 2 l and fixed with standard Lugol’s solution in a amber Nalgene bottles to estimate total cell
abundances (both live and dead) and for species identification under an inverted compound microscope
using grid settlement columns and phase contrast lighting. A third sub-sample is filtered (Whatman GF/F