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0.7 µm pore, 2.5 cm diameter membrane) and frozen (-80
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 (see Appendix D). Finally, a fourth sub-sample is used to provide a
qualitative measure of phytoplankton growth potential by determining chlorophyll levels after samples are
allowed to regrow under favorable conditions. Algae specific vitamins, minerals, and nutrients (Guillard
1975, F/2 formulation) are added to three 250 ml sub-samples from both control and treated water
collected upon discharge after the five-day hold time and are placed in a standard algal culture light-dark
regimen for five to six days, prior to extractive chlorophyll-a analysis (described above).
B.4.3. 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
cholerae
.
Total heterotrophic bacteria will be enumerated by spread plate method using
marine agar (MA) or R2A
agar for freshwater
according to
Standards 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 et al. 2003) and
10 ml and 100 ml water sample aliquots. Additionally, concentrations of culturable
E. coli
and intestinal
Enterococci
are determined using a standard USEPA method, namely, membrane filtration on mTEC agar
(
E. coli
) (1 ml, 10 ml and 100 ml) and mEA agar (
Enterococcus
) (10 ml and 100 ml). Finally, the
abundance of total and toxigenic
V. cholerae
will be determined by filtration and selection on TCBS agar
and enumerated using species-specific RNA colony blot (500 µl to 1 ml) and
ctxA
DNA colony blot (1-10
ml). Viable toxigenic cells of
V. cholerae
are 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).
B.4.4. Quantifying Physical Conditions
Temperature, salinity, dissolved oxygen, chlorophyll fluorescence, turbidity and pH will be measured
every 15 minutes during the test trials by two identical multi-parameter probes (calibrated before each
trial according to manufactures specification) placed, one each, into the control and test tanks. A third
hand-held instrument will be used to measure temperature, salinity, and dissolved oxygen of water in each
uptake or discharge as they occur.
Initial inline samples (three replicates, 500 ml - 2 l each) of ballast water during the filling of the control
and test tanks will also be collected, filtered, and analyzed for the water quality parameters of particulate
organic carbon (POC), dissolved organic carbon (DOC), and total suspended solids (TSS). See SOPs for
details.