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MERC ER01-12
5
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 were analyzed. A second 250 ml was
collected from the integrated 100 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 was 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 100 L sample was
analyzed to determine concentrations of total heterotrophic bacteria and three specific indicator
pathogens,
E. coli
, intestinal
Enterococci
, and toxigenic
Vibrio
cholerae
(described briefly below
and in detail in SOPs).
Total heterotrophic bacteria was enumerated by spread plate method using MA 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
was determined using a
commercially available chromogenic substrate method (IDEXX Laboratories, Inc.; Noble et al.
2003) and 100 ml water sample aliquots. Additionally, concentrations of culturable
E. coli
and
intestinal
Enterococci
were determined using a standard method (US EPA 1603 for
E. coli
, US
EPA 1106 for
Enterococci
) namely membrane filtration on mTEC agar (
E. coli
) (10 ml and 100
ml) and mEA agar (
Enterococcus
) (10 ml and 100 ml). Finally, the presence of viable cells of
V.
cholerae
O1 was preliminary assayed with a commercial DFA kit specific for serogroup O1
(New Horizons Diagnostics) using monoclonal antibodies tagged with fluorescein isothiocyanate
(FITC) (Hasan et al. 1994). Samples showing the presence of O1 positive strains were tested for
total and toxigenic
V. cholerae
by filtration, selection on TCBS agar, and enumeration using
species-specific RNA colony blot (1 ml) and
ctxA
DNA colony blot (1ml).
Treatment Toxicity:
Although the Hyde BWMS does not employ an active substance, one set of toxicity tests
during a biological efficacy trial was completed. MERC toxicity testing is designed to meet
IMO G9 requirements and uses test methods and species employed by the EPA for Whole
Effluent Toxicity (WET) testing of effluents (EPA 2002 and ASTM 2006). A fish (sheepshead
minnow
Cyprinodon variegatus
), an invertebrate (mysid
Americamysis bahia
), and an algae
(dinoflagellate
Isochrysis galbana
) were used for both acute and chronic toxicity tests, all listed
as estuarine test species in EPA’s Short-term Methods for Estimating the Chronic Toxicity of
Effluents and Receiving Waters to Marine and Estuarine Organisms (EPA, 2002).
A 38 L sample was collected at the time of discharge from the treated tank, which
included enough water to do all of the test renewals. Test water was stored in large HDPE
containers and held at 4°C in the dark to retain as much of the initial toxicity as possible. All of
the tests were conducted at the University of Maryland Wye Research and Education Center
toxicology laboratory and were initiated within three to four hours of sample collection.
Toxicity endpoints included survival in acute fish and invertebrate tests, survival and
growth in chronic fish and invertebrate tests, and population growth in chronic algal tests as