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MERC+GSI – DRAFT Test Plan
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Test samples will be collected at the time of discharge from the MERC facility. Samples
will be collected by the MERC staff for analysis of both the efficacy of treatment at eliminating
organisms from the ballast water and to investigate residual toxicity at discharge as described
above. For the suite of toxicity tests, a volume of 38 L (10 gallons) must be collected. 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.
Portions of this sample will be used each day to serve as the renewal water for the bioassay.
Sub-samples of each will also be sent to a certified chemistry laboratory (TBD) for analysis of
disinfection byproducts. MERC Testing Team will collect/deliver all samples for chemical
analysis and manage analytical results but costs of chemical analysis will be covered by
Siemens.
Summaries of the proposed test methods are given in Tables 4 through 8 (page 28). All
of the tests will be conducted at the WREC toxicology laboratory. Since chlorine degrades
rapidly, all toxicity tests will be initiated within two hours of the completion of a specific trial.
Pilot studies have demonstrated that there is no measurable difference in chloronated water held
for five days and then either tested within 30 minutes of collection or after a two hour holding
and transport time. Standard EPA (2002) and ASTM (2006) methods that have used in at
WREC since 1987 to conduct Whole Effluent Toxicity tests and single compound toxicity tests,
will be employed. The survival and growth end-points from these tests are those required by the
G9 document in Section 5.2.4 (IMO, 2008). The algae test represents a true population growth
test.
In addition to the ballast water discharge efficacy testing, sampling and toxicity testing of
the water in the test tank will also be conducted on a minimum of one of the six test trials.
Sampling will be done at the time of tank filling/treatment, and at one day and five days. The
analytical and bioassay methods described above will be used to analyze these different time
point samples.
Statistical Analyses:
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
required in Section 5.2.4 of the G9 (IMO, 2008). Tests are designed with a dilution series to
allow calculation of daily LC50 (concentration yielding 50% lethality) values from acute and
chronic mortality data. In addition, chronic tests will include sufficient treatment replication to
allow calculation of NOEC (no observable effect concentration), LOEC (lowest observable
effect concentration) and EC25 (percent concentration yielding a 25% effect) values for all
toxicity endpoints as required in Section 5.2.5 of the G9 (IMO, 2008). Statistical analyses will
be performed using ToxCalc statistical software (TSS, 2006) according to methods from USEPA
(2002) and ASTM (2006) guidance documents. Briefly, LC50s at daily intervals will be
calculated from survival data using the Probit Method if an adequate dose response is achieved.
If an adequate dose response is not achieved (e.g., only one partial mortality between the
concentration causing 100% mortality and that causing 0% mortality), the Trimmed Spearman-
Karber Method will be used. Chronic data will be tested using a Probit Method (EC25) and by
analysis of variance (ANOVA) with means testing (NOEC/LOEC). Prior to ANOVA testing
chronic data will be tested for normality using the Shapiro-Wilk’s Test and for homogeneity of
variance using the Bartlett’s Test. Survival data will be arcsine square root transformed prior to
analysis. If normally distributed and homogeneous survival and growth data will be analyzed