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MERC+GSI – DRAFT Test Plan
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using a one-tailed ANOVA followed by a Dunnet’s means comparison test (equal number of
replicates/treatment) or a T-Test with Bonferroni Adjustment (unequal replicates/treatment) to
determine differences from control data. If data do not pass the assumptions of normality or
homogeneity, a Steel’s Many-One Rank Test (equal replicates) or Wilcoxon Rank Sum Test with
Bonferroni Adjustment (unequal replicates) will be performed. A
p
value of 0.05 will be used
for all hypothesis tests; a
p
value of 0.01 will be used for testing assumptions of normality and
homogeneity of variance. Results from the chronic statistical analyses will provide NOECs,
LOECs, and EC25s for each ballast water treatment run.
Definition of Test Failure on the Grounds of Toxicity:
Permissible residual toxicity will follow the guidelines outlined by the EPA National
Pollutant Discharge Elimination System (NPDES) for issuance of a Vessel General Permit
(VGP) (full text is available at www.epa.gov/npdes/vessels; relevant sections on ballast
discharge toxicity are 5.8.1.2 and 15.2). Based on these criteria a
test trial will be considered a
failure on the grounds of residual toxicity upon discharge if acute lethality (as indicated by
determination of an LC50 of less than 100%) occurs in any test species. Determination of test
failure as a result of chronic toxicity will be based on EC25 analyses. An EC25 is a point
estimate of the toxicant concentration (expressed as percent effluent) that causes an observable
adverse effect in 25 percent of test organisms. Chronic test results will be calculated in TUc
(chronic toxicity units), where TUc = 100/EC25 (e.g., an EC25 of 100% (i.e., undiluted effluent)
would yield a TUc of 1.0). In order for a test trial to pass, chronic toxicity of discharged ballast
must not exceed 1.6 TUc for any species tested (equivalent to an EC25 of 62.5%). Calculation
of a TUc greater than 1.6 for any test species will constitute a test trial failure based on residual
toxicity within discharged ballast water.
Toxicity Quality Assurance:
Toxicity test acceptability (i.e., performance) criteria are presented in Tables 4 through 8.
The quality assurance procedures for the algae tests will follow those discussed in detail in
Section 13 of ASTM Designation E 1218-04 “
Standard Guide for Conducting Static Toxicity
Tests with Microalgae
” (ASTM, 2006). Any deviations from the quality assurance procedures
will be given in the final report.
GSI - The GSI’s toxicity 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 plant,
an invertebrate and a fish) that address the sensitive life-stage. The preference is to include both
a sub-lethal endpoint (growth or reproduction) and a survival endpoint.” The following work
plan will outline proposed methods to meet these IMO guidelines, using standard operating
procedures (Table 3) developed by GSI which are based on methods approved by the USEPA
(2002) and the American Society for Testing and Materials (ASTM, 2005).