Basic HTML Version
MERC Hyde Test Plan
12 April 2012
6
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 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 require analysis
of sub-samples and extrapolation to the entire 3 m
3
. The
> 50
µ
m
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.
Viable Organism 10 - 50 µm in size:
A 75 L integrated sample will be collected as an unfiltered split sample in parallel with
the > 50 µm fraction. This sample will be the source water for all other analyses including the
10-50 µm fraction. Two liters from this well-mixed, integrated sample will be subject to three
distinct analyses and counts (described briefly below and in detail in SOPs).
All of these live unfiltered samples will be processed or examined within three hours of
collection on the MERC Mobile Test Platform or nearby partner laboratories. All preserved
samples are also transported to MERC partner laboratories within three hours, 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 Olympus IX-51 inverted phase/fluorescent microscope . Cells are scored as
live when showing strong fluorescence signature under excitation (some cells also show
motility). This approach has been validated for use in the Chesapeake Bay (Steinberg et al.,
2011) and provides the data for comparison to discharge standards.
As supporting information, two other sub-samples are analyzed. A second 250 ml is
collected from the integrated 75 L sample and fixed with standard Lugol’s solution in amber
Nalgene bottles to estimate total cell abundances (but not live versus 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 0.7 µm pore, 25 mm diameter
membrane) and frozen (-20
o
C) until analysis of total active chlorophyll-a by the CBL/UMCES
Nutrient Analytical Services Laboratory using US EPA Methods 445.0 for
extractive/fluorometric techniques.
Viable Bacteria and Indicator Pathogens:
An unfiltered 1 L sample of water sub-sampled from an integrated 75 L sample will be
analyzed to determine concentrations of total heterotrophic bacteria and three specific indicator
pathogens,
E. coli
, intestinal
Enterococci
, and toxigenic
Vibrio
cholera
(described briefly below
and in detail in SOPs).
Total heterotrophic bacteria will be enumerated by spread plate method using MA or
R2A agar according to
Standard Methods for the Examination of Water and Wastewater
(21
st
edition, 2005). The presence and abundance of
E. coli
and intestinal
Enterococci
is determined
using a commercially available chromogenic substrate method (IDEXX Laboratories, Inc.; Noble