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MERC ER01-12
4
analyzed for UV transmittance using a UV254 P200 RealTech field spectrophotometer, with
subsamples provided to Hyde for their own analysis of UV transmittance. See SOPs for
additional details.
Live Organisms >50µm in size:
The sampling system consists of two sets of paired canisters, each designed to
accommodate a 35 µm (50 µm diagonally) mesh plankton net used to collect the >50 µm size
fraction. One pair handles water from the treated ballast tank and the other pair handles water
from the control tank. The paired sampling canister/net arrangement allows for the residual from
the cod-end of one net from each pair to be processed for examination while filtration continues
via the other net, thereby avoiding clogging. In this way unimpaired filtration back and forth
between each pair of nets continues until a total of 3 to 7 m
3
has been processed from each of the
treated and control ballast tanks respectively. The sampling canisters 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 three day hold times), the control and treated
ballast tanks were drained and processed as described above, with treated water first passing
through the Hyde system for additional UV irradiation (but not filtration) prior to sampling.
The proportion and total concentration of live versus dead organisms > 50
µ
m were
determined using standard movement and response to stimuli techniques and this live/dead
analysis took place within two hours of collecting the individual samples. Three m
3
was the
volume collected for control water upon filling and discharge from test tanks (high numbers of
live organisms) and 7 m
3
was collected for treated water on discharge after three-day hold times
(presumably very few live organisms). Depending on concentrations, quantification of
organisms > 50
µ
m in initial samples (upon ballasting) and control samples may have required
analysis of sub-samples and extrapolation to the entire 3 m
3
. The > 50
µ
m samples was then also
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 was conducted under a dissecting microscope at 25X, except for
some taxa, which were removed and identified using a compound microscope. Larval forms of
invertebrates were identified to higher taxonomic levels such as order (e.g., Decapoda) suborder
(e.g., Balanomorpha) or class (e.g., Bivalvia). Adults were identified to species in most cases.
Live Organism 10 - 50 µm in size:
A 100 L integrated sample for both treated and control were collected as an unfiltered
split sample in parallel with the > 50 µm fraction. This sample was the source water for all other
analyses including the 10-50 µm fraction. Two liters from this well-mixed, integrated sample
was subject to three distinct analyses and counts (described briefly below and in detail in SOPs).
All of these live unfiltered samples were processed or examined within three hours of
collection on the MERC Mobile Test Platform or nearby partner laboratories. All preserved
samples were also transported to MERC partner laboratories within three hours, for further
analyses and taxonomic identification.
One 250 ml sub-sample was stained using a combination of CMFDA (5-
chloromethylfluorescein diacetate) and FDA (fluorescein diacetate) as a selective live/viable
indicator. Samples stained with CMFDA+FDA, were incubated and observed on a Sedgewick
Rafter slide using a Olympus IX-51 inverted phase/fluorescent microscope. Cells were scored as
live when showing strong fluorescence signature under excitation (some cells also show