Basic HTML Version
MERC Hyde Test Plan
12 April 2012
7
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 US EPA 1603
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).
Quantifying Physical Conditions:
Temperature, salinity, dissolved oxygen, chlorophyll fluorescence, turbidity (NTU) and
pH will be measured every 15 minutes during the test trials using two identical multi-parameter
probes (calibrated before each trial according to manufactures specification) deployed into both
the control and treated tanks. A third hand-held instrument will be used to measure temperature,
salinity, and dissolved oxygen of water (and other parameters as required) during uptake or
discharge.
Initial inline samples (three replicates, approximately 500 ml - 2 L each) of ballast water
during the filling of the control and treated tanks will be collected, filtered, and analyzed for the
water quality parameters of particulate organic carbon (POC), dissolved organic carbon (DOC),
and total suspended solids (TSS). Mineral Matter will be calculated. Similarly, inline samples
of challenge water will also be 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 details.
Treatment Toxicity:
Although the Hyde filter + UV systems does not employ an active substance, MERC will
conduct at least one set of toxicity tests during the first biological efficacy trial one trial. The
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, an invertebrate and a plant (algae) will be used in all ballast discharge tests.
Because the Chesapeake Bay is a mesohaline aquatic environment with salinities ranging from 5
to 25 psu, estuarine organisms will be used in these tests. The fish species used in the test will be
the sheepshead minnow (
Cyprinodon variegatus
), invertebrate will be a mysid (
Americamysis
bahia
; formerly
Mysidopsis bahia
) and the algal species will be
Isochrysis galbana
, 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).
Both acute and chronic data will be generated for each test. A dilution series, using
ambient water, will be run for each species. A total of 38 L samples will be collected at the time
of discharge from the treated tank. This includes enough water to do all of the test renewals.
Test water will be 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 will be conducted at the University of Maryland
Wye Research and Education Center toxicology laboratory and will be initiated within three to
four hours of the completion of a specific trial.
Toxicity endpoints will include survival in acute fish and invertebrate tests, survival and
growth in chronic fish and invertebrate tests, and population growth in chronic algal tests as