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MERC+GSI – DRAFT Test Plan
10
Quantifying Viable Indicator Pathogens:
A 1 l sample of water for each mesocosm/tub (a 5 l integrated sample for MERC and a 3 l
integrated sample for GSI) is collected to determine concentrations of total heterotrophic bacteria
and three specific indicator pathogens,
E. coli
, intestinal
Enterococci
, and toxigenic
Vibrio
cholerae
(Table 2 page 27). Total heterotrophic bacteria are enumerated by spread plate method
using NWRI agar 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). Abundance of total and toxigenic
V.
cholerae
are calculated by filtration and selection on TCBS agar and enumerated using species-
specific RNA colony blot (500
μ
l to 1 ml) and
ctxA
DNA colony blot (1-10 ml). Viable
toxigenic
V. cholerae
is 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).
Data Analysis:
Although multiple mesocosms, samples, and measures from each tank will be taken, to
avoid pseudo-replication, the unit of replication for statistical analyses is each trial (n = 5 or 6).
We assume that all measures for a single trial provide one estimate of treatment efficacy. Thus,
treatment efficacy for any biological parameter is estimated as changes found before and after
trial (percent reduction), and as the difference in concentration between treated water and IMO
standards. This approach controls for variation due to temporal changes in environmental
conditions.
6. Protocols for Evaluations of SiCURE System Discharge Toxicity
MERC - The MERC Testing Team members at the University of Maryland Wye
Research and Education Center (WREC) will evaluate the aquatic toxicity of the ballast water
discharge. The testing is designed to meet Section 5.2 of the Procedure for Approval of Ballast
Water Management Systems That Make Use of Active Substances (G9) as resolved by the
Marine Environmental Protection Committee of the International Maritime Organization (IMO,
2008). Section 5.2 states that, “The advantage of conducting toxicity testing on the ballast water
discharge is that it integrates and addresses the potential for interactions of the Active Substances
and Preparations with the possible by-products.” This section requires that, “these toxicity tests
should include chronic test methods with multiple test species (a fish, an invertebrate and a plant)
that address the sensitive life-stage. The preference is to include both a sub-lethal endpoint
(growth) and a survival endpoint.” The MERC approach to meet these IMO guidelines use test
methods and species employed by the EPA for Whole Effluent Toxicity (WET) testing of
effluents. These methods are approved by the EPA (2002) and the American Society for Testing
and Materials (ASTM, 2006). Personnel at WREC are vary familiar with these test species and
methods and conducted WET testing from 1986 to 2003 for the Maryland Department of the
Environment in support of its NPDES WET bioassay-monitoring program.