5
To estimate total cell abundances (live and dead) and for species identification, 3 more
replicate samples were collected from each time-integrated cylinder in 500 ml, opaque Nalgene
bottles and fixed with 5 ml of Lugol’s solution plus 1 mL of formaldehyde. They were examined
under an inverted compound microscope using grid settlement columns and phase contrast
lighting.
A third sub-sample from each cylinder was immediately filtered (Whatman GF/F 0.7 µm
pore, 25 mm diameter membrane), then frozen (-20
o
C) until analysis of total and 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:
Three replicate unfiltered samples of water were immediately collected into 1 l bottles
from both the treated and the control 100 l time-integrated cylinders 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 the spread plate method using MA agar.
(
Standard Methods for the Examination of Water and Wastewater
(21
st
edition, 2005), Method
9215
C,
Heterotrophic Plate Count
,
Spread Plate Method
,
and
EPA NSCEP document:
EPA/600/R-10/146,
Generic Protocol for the Verification of Ballast Water Treatment
Technologies,
NSF International, September 2010, Version 5.1.)
The presence and abundance of
E. coli
and intestinal
Enterococci
was determined using
two chromogenic substrate methods, IDEXX Colilert-18 and Enterolert (IDEXX Laboratories,
Inc.; Noble et al. 2003). Each analyses start with a 100-ml water sample aliquot.
Finally, the presence of viable cells of
V. cholerae
O1 and O139 was preliminary assayed
with a commercial DFA kit specific for serogroups O1 and O139 (New Horizons Diagnostics)
using monoclonal antibodies tagged with fluorescein isothiocyanate (FITC). Samples showing
the presence of O1 and/or O139 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 BIO-SEA 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 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 total of 38 l samples were 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 glass
carboys 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 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