Basic HTML Version
MERC ER02-09
3
determined using standard movement and response-to-stimuli techniques within one hour of
collecting the individual samples. Zooplankton samples were also fixed and returned to the
laboratory for additional taxonomic evaluations.
Ten liters of unfiltered water from each mesocosm were also collected immediately after
filling, to determine concentrations of organisms in the 10 to 50 micron size class using four
distinct methods: (A) One sub-sample was fixed with standard Lugol’s solution to determine
total cell abundances under an inverted compound microscope using grid settlement columns and
phase contrast lighting. (B) A second sub-sample was stained using CMFDA (5-
chloromethylfluorescein diacetate) as a selective live/viable indicator. Stained
sub-samples were
incubated and observed on a Sedgewick Rafter slide using a Leitz Laborlux S modified for
epifluorescence.
(C) A third sub-sample was filtered and frozen until analysis of total and active
chlorophyll-a by the NASL. (D) Finally, a fourth sub-sample was used to determine chlorophyll-
a levels after allowed to regrow under favorable conditions. An increase in chlorophyll, or
positive regrowth, indicates that viable phytoplankton were in the samples, whereas chlorophyll
levels at or below detection limits of the laboratory analytical method suggests that there was no
viable phytoplankton.
Additional 1-liter subsamples of unfiltered water were also collected from each
mesocosm to determine concentrations of total heterotrophic bacteria and three specific indicator
pathogens,
E. coli
, intestinal
Enterococci
, and toxigenic
Vibrio
cholerae
. Total heterotrophic
bacteria were enumerated by spread plate method using NWRI agar according to
Standards
Methods for the Examination of Water and Wastewater
. 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 10 ml and 100 ml water sample
aliquots. Additionally, concentrations of culturable
E. coli
and intestinal
Enterococci
were
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
were 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
was 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).
5. Summary of Results*
The MSI Ballast Water Treatment System dramatically reduced the numbers of live
organisms in ballast water during MERC land-based testing in the Port of Baltimore. For most
biological categories, the treatment system consistently met IMO D2 discharge standards.
However, while large reductions in the abundances of organisms greater than 50
µ
m were found,
the T-Final average numbers of live zooplankton (> 50
µ
m) were not consistently below 10
µ
m
in treated water after the 5-day holding time. The MSI system also experienced minor
mechanical failures at different points during the testing process (broken in-line sensor housing
and tripping of a circuit breaker). However, the failures were easily resolved with small
modifications or repairs.
* Complete datasets and further performance information is available upon request.