### STEP 2006 Application Form

Section 4.0: Proof of Ballast Water Treatm ent Performance



### 1 4.0 P ROOF OF 

### B ALLAST 

### W ATER 

### T REATMENT 

### P ERFORMANCE



### The acceptance of a BWT into STEP is based upon the ability of the Applicant to make and support claims of treatment effectiveness relative to the removal/inactivation of the diversity of taxa present in bal last water. This requires the presentation of test data quantifying the effectiveness of either physical removal or mortality/inactivation of organisms produced by the proposed BWT. Preventing biological invasions thorough ballast water discharge focuses on the number of viable organisms discharged per cubic meter of de

- ballasted water. Therefore, biological testing, background data and proposed testing under STEP (see Section 5), must address the number of viable organisms across a diversity of taxonomi c groups. It is understood that data may not be available for all taxonomic groups at the time of the application; however, taxa specific information is needed for full evaluation of a BWT system. In addition, demonstrating the ability for the proposed B WT system to meet the NVIC treatment criterion is an essential component of entry into STEP.

The best approach is prior experimentation at smaller scales leading to the full

- scale test. The first step is laboratory

- scale work on the core treatment technol ogies, resulting in optimized dose/response curves, minimum required treatment times, and confirmation of the appropriateness of the selected assays and other metrics. Next, pilot

- scale testing (e.g., on a barge or at a shoreside site) should address phys ical scale

- up questions such as sufficient mixing, assessment of settling issues, operations and maintenance needs, system controls, and finalization of analytical procedures and sampling protocols for full

- scale test programs.

Those steps typically prec ede full

- scale installation and experimentation, which should confirm prior system engineering, operations testing, and biological effectiveness experiments. If either laboratory

- or pilot

- scale steps have not been executed prior to full scale testing, th e Applicant should offer a reasoned, scientific rationale for proceeding to full

- scale, that is, that the treatment has a high probability of performing effectively, that the test protocols are in

- place, and the larger scale experiment(s) will provide mean ingful data.

There may be latitude in the matter of small

- scale tests for proven water treatment technology, with relevant laboratory results derived from others and the employment of standard experimental procedures at full scale. Such latitude, howeve r, is not appropriate when either the treatment system or the proposed experimental methods for the onboard tests are novel or unproven. In such cases, the Applicant must satisfy a higher burden of proof and must demonstrate the feasibility of new technol ogy or experimental methods at small scale. In addition, the Applicant must document that the analytical and test protocols are fully operational within the proposed Test Team.

Section 4 requests literature, experimental data, and data available from othe r sources (technical reports, unpublished manuscripts) in order to gather, in a consistent manner, the background information necessary to perform an evaluation of the status of the BWT system being proposed for inclusion into STEP. The sub

- sections are a rranged to organize data on the technology (components and full system) from all sources, regardless of the water treatment application, and to partition these data sources by the three scales of testing typical of technology development: bench

- scale, pilo t

- scale, full

- scale (ship

- board and shore

- side). Within

each sub

- section there are summary tables to be filled out by the Applicant. These tables are to reduce the amount of text required, to help reduce redundancy and to allow rapid evaluation of

the ba ckground data supporting the Applicant’s BWT system. In the case of well

- developed technologies or components, it is important that the Applicant include only key literature needed to support claims made.

In Sub

- sections 4.1, 4.2, and 4.3, the Applicant p rovides demonstrative data and information from testing or use of equipment relevant to either the full BWT system or any of its

### STEP 2006 Application Form

Section 4.0: Proof of Ballast Water Treatm ent Performance



### 2 components. Any reference or data source may be included, whether the tests were on ballast

water or from other water treatmen t industries, or whether tests were conducted by the Applicant’s team or others. Each sub

- section should include sufficient literature and data sources relevant to components or systems that provide background to the Applicant’s BWT
system:

- Sub

- section 4.1, bench

- scale testing

- Sub

- section 4.2, pilot

- scale testing

- Sub

- section 4.3, full

- scale testing (shipboard and shore

- side)

In sub

- sections 4.4, 4.5, and 4.6, the Applicant provides experimental data exclusively from testing of the specific BWT compon ents or the full BWT system. It is expected that most such data will be from testing conducted by the Applicant’s team. Each sub

- section should include all available literature and data sources, organized test scales as for sub

- sections 4.1 through 4.3:

- Sub

- section 4.4, bench

- scale testing

- Sub

- section 4.5, pilot

- scale testing

- Sub

- section 4.6, full

- scale testing (shipboard and shore

- side)

The Applicant should bear in mind the following instructions for completion of Section 4:

1. Sub

- Sections 4

- 1, 4

- 2, 4

- 3 allow inclusion of all information regardless of water treatment application as long as they are relevant to the technology proposed for STEP; Sub

- Sections

4

- 4, 4

- 5, 4

- 6 allow inclusion of ONLY information from testing on the STEP specific BWTS or

its specific components.

2. The application should include any relevant dose/response information, particularly by taxa. It is not expected that all levels of testing in both sets of tables will always be available, only that sufficient information be available to ensure that the performance goals of the proposed BWT System will be met under STEP.

3. Only pre

- STEP experimentation for which data are available is relevant, not future experiments or experiments currently underway for which data are not available.

4. Only primary references should be included, that is, those that are the original source of the data rather than those which only reference the primary work or include later reproductions of figures and tables,.

5. The application should include all reference iden tifiers as requested in the tables for each

sub

- section.

6. Appendix B should include hard copies and if available .pdf files of all references, annotated with the reference identifiers that the Applicant uses in Tables 4

- 1 through 4

- 6.

4.1 Literature Summary of Laboratory Experiments; Bench Scale

The sub

- section summarizes data from existing bench

- scale literature and other available data sources (technical reports, unpublished manuscripts) relevant to either the full BWT system or any of its components. Any ref erence or data source may be included, whether the tests were on ballast water or from other water treatment industries or whether tests were conducted by the Applicant’s team or others. The summary focuses primarily on Table 4

- 1, with the Applicant

enter ing text in response to specific questions in the Text Boxes provided.

Bench

- scale experiments are meant to establish the abilities of individual treatment components’ core technologies to accomplish the desired operations (proof of concept experiments), a nd to determine the key operational variables (design experiments). The analytical methods, assays, and other metrics employed to measure performance of the core

### STEP 2006 Application Form

Section 4.0: Proof of Ballast Water Treatm ent Performance



### 3 technologies should also be worked out at this stage. For example, bench scale tests would b e used to determine if centrifugal force will sufficiently remove microscopic organisms, the UV dose/inactivation curve for target species, or the chlorine demand and required residual for

source waters.

At the bench

- scale, equipment is generally small in scale, and the experiments are often batch in nature. Data from larger prototype or full

- scale equipment testing should be placed in sub

sections 4

- 2 and 4

- 3, respectively.

If an Applicant did not conduct small or bench scale testing, it is important that a clear rationale is presented offering the assurance that the treatment system has a reasonable chance of performing effectively, and that the larger scale experiment(s) will provide meaningful data.

Well

- proven treatment technology and/or standard expe rimental procedures are clearly desirable aspects in such cases, as would be relevant laboratory results from elsewhere, if they

exist.

TEXT BOX : Has small scale testing been conducted on the treatment system or its components by your team or by others (c ircle 1)? Yes No

**•** If the answer is “No”, proceed to sub

- section 4.2.

**•** If “Yes”, then, complete Table 4

- 1. Note the following explanations of terms used in

Table 4

- 1:

o “BWT unit tested” (or component(s) tested) column, requests specification of th e single component or process or multi

- component system or process tested, that relates to the BWT components or system in this application.

o “BWT Unit Specifications” refers to the type of process tested. The request is for

information on dose or concentr ation or filter mesh size, etc.

o “Testing source water” refers to the type of water used in the tests (harbor, coastal, estuarine, lake, ocean). If possible the actual source

o ”Locati on of Tests” refers to the test facility or laboratory where the testing was conducted. For example, Naval Research Laboratory Key West. water should be noted, as well as any known specifics on water chemistry.

o The heading “Taxa” appears under each of the major taxonomic functional headings (i.e., zooplankton, phytoplankton). The concept is to pair the specific taxonomic level or group tested with the level of removal/inactivation (column to right). For example, a reference may have tested a component of the proposed BWT on zooplankton, only identified to the level of “copepod s”. The copepods would be entered paired with the appropriate removal/inactivation. However, a reference may have conducted work on a species level on 10 phytoplankton species. In this case the 10 species would be individually listed each paired with it s reported removal/inactivation

rate.

o “Literature Reference” refers to primary references, that is, those sources that include the original data, not reports or papers that merely reproduce earlier work. The table specifies that the Applicant identify the se references as alphabetically as

“B

- a”, “B

- b”, “B

- c”, etc., indicating a series of B ench scale testing citations.

### STEP 2006 Application Form

Section 4.0: Proof of Ballast Water Treatment Performance



### Table 4

- 1. Summary, Prior Laboratory Experiments, Literature and Studies, Bench

- Scale . Summary of literature, reports and web site infor mation on the removal/inactivation efficiency of the components or combined systems. Add additional rows as needed.

BWT Unit Tested (1) BWT Unit Specifications (2) Testing

Source Water (3) Location

of Test Lab (4) Literat

ure Refere nce (5) Test

Grou p (6 ) Zooplankton

in 5 mg L - 1 Protozoa

in 5 mg L - 1 Phytoplankton

in 5 mg L - 1 Bacteria

in 5 mg L - 1 Viruses

in 5 mg L - 1 Cysts/Resting Stages

in 5 mg L - 1 Other

in 5 mg L - 1

Taxa (7) at t = 7 days EFF % (8)* Taxa (7) at t = 7 days EFF % (8)* Taxa

at t = 7 d ays EFF % (8)* Taxa at t = 7 days EFF % (8)* Taxa

at t = 7

days EFF % (8)* Taxa at t = 7 days EFF % (8)* Taxa at t = 7 days EFF % (8)*

Chlorine

dioxide 0, 1, 5 and 10 mg L - 1

, dose

- response and timecourse Enriched Narragansett Bay (30.5 psu) MERL

facilit ies at GSO, University

of Rhode

Island B

- a TT Copepoda Ctenophora Pelecypoda Gastropoda Polychaeta

Other 100%

100%

93.5%

83%

100%

84% Ciliophora Heterotrophic dinoflagellate Heterotrophic flagellate <100%

100%

<100% All phytoplankton Bacillariophy ceae Dinophyceae Cyanophyceae >89%**

>78%

100%

100% Total bacteria

Vibrio cholerae 0% ***

100% Coliphage 100%

Chlorine

dioxide 0, 1, 5, 10 and 20 mg L - 1

, dose

- response

at end

- point Enriched Narragansett Bay (30.5 psu) MERL

facilities at GSO, URI B

- a TT Pelecypoda Gastropoda Polychaeta 100%

100%

100% Ciliophora Heterotrophic dinoflagellate

Heterotro phic flagellate <100%

<100%

<100% Phytoplankton 94% Total bacteria

Vibrio cholerae 100%

100% Arte mia 61%



### Literature and references cited:

B

- a Oviatt, C. A., P. E. Hargraves, R. P. Kelly, M. Kirs, L. Maranda, B. S. Moran, D. M. Outram, D. C. Smith, B. K. Sullivan and K. J. Whitman. Toxicity of chlorine dioxide to ballast water flora and fauna in bench scale assays. Final Report to Ecochlor, 56 pp.

B

- b
Note: Adjust the Column / Row dimensions to fit information provided.

(1) Indicate component tested (e.g., UV treatment apparatus, f iltration apparatus, etc.).

(2) Indicate the specifications of the BWT Unit/Component dose, filter size, etc.
(3) Source where water used in testing was obtained (e.g., Bay water, harbor water, etc.)., include salinity if available.

(4) Location of the test facility where experiments were conducted.

(5) Use letter (B

- a to B

- z) identifiers in this column and list references at bottom (e.g., Primary Scientific literature, reports, web sites, etc.). Primary sources should be given, defined as those where the experimental design, methods, analysis and data are presented. Unless absolutely necessary, do not list secondary sources, defined as reports where summaries from primary r eferences are presented.

(6) Enter "TT" if experiments conducted by v endor Test Team; Enter "O" for other if experiments were conducted by an external test team. Relates to who conducted the st udy. (7) The major taxa tested. In some studies may be general categories such as "copepods," "polychaetes," etc., in other stu dies more specific taxa or even individual species may have been tested, such as Artemia or Fecal coliform bacteria, , etc. (8) The range of removal or inactivation rates (efficiency of system either as % removal/inactivation). If multiple tests or rep licate s were conducted give the range of removals, such as 80%

- 95% or 2

- 3 logs. * Time = seven days after exposure **Limit of detection for % effectiveness on day 7 as measured by MPN was >89% or 10 organisms . (100mL - 1 ) *** Contamination during sampling prior to day 7 was demonstrated (see experiment 2)

### STEP 2006 Application Form

Section 4.0: Proof of Ballast Water Treatment Performance

**•** Provide a brief narrative description (one or two paragraphs each) of bench

- scale testing

references included in Table 4

- 1. The purpose is to indicate the applicability of the data to gauging the effectivenes s of the proposed BWT system.

TEXT BOX: Annotated bibliography

Reference B

- a describes results of two sets of experiments conducted to document the effectiveness of three initial concentrations of chlorine dioxide (1, 5 and 10 ppm) at eradicating

summer e stuarine planktonic communities in duplicate enclosures mimicking conditions in a ballast tank (temperature and darkness). Controls were held under similar conditions, but without any addition of ClO 2



### . The time course of action of ClO 2 

### was monitored for each concentration at 0, 1, 2, 3, 6, 12, 24, 48, 120,168 and 240 hours for most measurements. The second experiment is a repeat of the previous one but without the time course, where the effect

of four concentrations of ClO 2 

### (1, 5, 10 and 20 ppm) were mea sured at one end

- point, seven days after treatment. This second experiment was specifically designed to minimize contamination during sampling. The authors are research scientists and faculties with recognized expertise in seawater chemistry, marine micr obiology, phytoplankton and zooplankton biology.

TEXT BOX: Brief narrative describing the present status of dose/response relative to the proposed BWT process. Only data from bench

- scale should be discussed.

Chlorine dioxide decreased exponentially durin g the course of the first experiment, approximating first

- order kinetics with a half

- life of 5.8 hours for the 5

- and 10

- ppm initial

treatment and 1.1 hour for the 1

- ppm treatment. After a seven

- day period, no ClO 2 

### could be detected in any of the treatmen ts. It was determined that, under the bench

- scale conditions used, an initial concentration of 5

- ppm ClO 2 

### provided sufficiently effective eradication of coastal planktonic communities to proceed further, with no significant increased effectiveness at 10 p pm as measured by many parameters. The 1

- ppm treatment was not sufficient to inactivate a large proportion of the plankton. It was shown that an initial concentration of 5

- ppm ClO 2 

### eradicated nearly 98% of all zooplankters after 24 hours, with survival o ccurring only and minimally in shell

bearing animals. A second more stringent experiment, where ctenophores were removed to minimize grazing effects, led to a measured 100% effectiveness after 7 days. In most cases of measurements made for phytoplankton abundance and viability, the experiments revealed either 100% effectiveness or levels close to or below detection limits of the assays. Bacteria and viruses were totally inactivated after 24 hours and even after 7 days when precautions were

taken to minim ize contamination.

TEXT BOX: Brief summary (synthesis) of the key findings of all biological testing of removal/inactivation from bench scale testing of the BWT process (system, components, process). This synthesis should be linked to the references pres ented in Table 4

- 1.

Partial results from reference B

- a are presented in table 4

- 1 for the inactivation effectiveness on plankton of ClO 2 

### at an initial concentration of 5 mg

. 

### L - 1 

### in seawater, seven days after treatment. All functional groups suffered from t he presence of ClO 2 

### and most were eradicated, which was displayed as a 100% effectiveness on viruses, bacteria (including Vibrio cholerae ), most phytoplankton and zooplankton. Heterotrophic flagellates were not totally eliminated, especially

after seven d ays. It is suspected that, being closely associated with detritus and organic matter (‘marine snow’), enough of them would be somewhat protected from the effect of ClO 2 

### long enough to survive the initial challenge and then thrive on the organic matter gen erated by the death of all organisms. Nevertheless the overall effect of ClO 2 

### was particularly striking when

### STEP 2006 Application Form Section 5.0: STEP Study Plan



### 6 residual live populations were compared to initial populations. Seven days after treatment, the zooplankton community was reduced to 2% of its in itial value (experiment #1), with bivalve, polychaete and gastropod larvae completely eradicated (experiment #2), while concentrations of control populations were 87% of their initial values. The chlorophyll

- a concentration was reduced to <1% of its origi nal value, while control populations still had 5% of their initial values. Bacterial growth as observed on a variety of media was always 0% of initial values when care was taken to avoid contamination between samplings, while controls showed complete plat e coverage. Similarly, viruses (PFU mL
- 1 

### ) were 0% of initial values, compared to a 72% reduction

in the control.

It was noticed that more than one variable per functional group needed to be measured to give a complete picture of the effectiveness of the t reatment over time. This was especially true for phytoplankton, as measurement of chlorophyll only gave an indication of total algal biomass, microscopy provided analysis by taxa and some indication of viability but with limited quantitative assessment ev en after concentrating samples by centrifugation. Analysis by Most Probable Number (MPN) was quantitatively helpful on a taxonomic basis, although it required an extensive number of multiwell plates and had statistical limitations, thus our limit of detec tion of 10 organisms

. 

### (100 mL - 1 

### ).

The difficulty in readily obtaining cysts and resting stages for experimental purposes lead us to use the commercially available cysts of Artemia salina as proxy for this functional group. The

ClO 2 

### treatment was only 61% e ffective but it served to demonstrate that Artemia cysts are poor alternatives to dinoflagellate cysts and other resting stages because of their particular life cycle. The cysts survive in a dry state and many float when added to water or accumulate on th e container walls, unless aeration is provided. They hatch within a day or so upon rehydration. Furthermore, decapsulation (removal of shell from embryos) is achieved in hatchery by using bleach to improve the hatching process. Thus we believe that the ClO 2 

### treatment in seawater favored decapsulation and hatching and, by the time the embryos emerged, there was no or

insufficient ClO 2 

### left to inactivate them. This experiment with Artemia cysts proved too artificial

to be useful.

4.2 Literature Summary of La boratory Experiments; Pilot Scale

Pilot

- scale experiments are generally conducted on prototype or medium scale technology units or components and are usually performed with higher volumes and frequently under conditions of flow (rather than the batch tests typical of bench

- scale tests). These experiments are generally conducted to resolve unforeseen issues encountered in the scale

- up of processes, and the conversion of the technology from batch to semi

- batch and continuous operations. System controls and materials of construction should be worked out at this stage. Although not necessarily full

- scale, the equipment may be large enough to simulate the performance of full

size unit operations, for example, dockside or large tank size. These tests are somet imes

associated with mesocosms.

While analytical methods should be consistent with bench

- scale tests (assuming they were properly investigated and adapted during bench

- scale testing), field sampling techniques (collection, compositing, storage and shipping ) should be worked out at this stage.

TEXT BOX : Has pilot

- scale testing been conducted on the general technology system or its components by your team or by others (circle 1)? Yes No See note below.

### STEP 2006 Application Form Section 5.0: STEP Study Plan



### 7 Note:

Lab scale experiments and full

- scale shipboard tests have been conducted. Because of the success of the bench scale data, the proven effectiveness of chlorine dioxide in numerous land based water disinfection applications, and the ability of the same system to treat up to 2500 MT per hour, t here was little benefit to pilot studies. Ecochlor proceeded directly to installing a full

scale system on Atlantic Compass . Therefore, Table 4

- 2 has been omitted.

**•** If the answer is “No”, proceed to sub

- section 4.3.

**•** If “Yes”, then, complete Table 4

- 2.

The terms used in Table 4

- 2 are the same as those used in Table 4

- 1, except that Table

4

- 2 specifies that the Applicant identify references alphabetically as “P

- a”, “P

- b”, “P

- c”, etc., indicating a series of P ilot scale testing citations.

4.3 Literatur e Summary of Full

- scale Experiments

In this section, the Applicant presents information on the full

- scale testing of treatment systems or components, in ballast water treatment or other water treatment applications (such as wastewater, water supply, aquar ia). Tests referenced may include both shipboard and shoreside sites. The concept is to provide data and references that support the biological effectiveness of the technology in tests at the scale of the shipboard application proposed for

STEP.

While ana lytical methods should be consistent with bench

- scale and pilot

- scale tests (assuming they were properly investigated and adapted during these phases), the approaches to be used under STEP should be documented in this stage.

TEXT BOX : Has full

- scale testi ng been conducted on the general technology system or its components by your team or by others (circle 1)? Yes No

**•** If the answer is “No”, proceed to sub

- section 4.4.

**•** If “Yes”, then, complete Table 4

- 3.

The terms used in Table 4

- 3 are the same a s those used in Tables 4

- 1 and 4

- 2, except

that Table 4

- 3 specifies that the Applicant identify references alphabetically as “F

- a”, “F

b”, “F

- c”, etc., indicating a series of F ull scale testing citations.

### STEP 2006 Application Form

Section 4.0: Proof of Ballast Water Treatment Performance



### Table 4

- 3. Summary, Prior Experiments, Literatur e, and Studies, Full

- Scale . Summary of literature, reports and web site information on the removal/inactivation efficiency of the components or combin ed systems. Add additional rows as needed.

BWT Unit Tested (1) Test Program Literat

ure Refere nce (5) Test

Grou p (6) Zooplankton Protozoa Phytoplankton Bacteria Viruses Cysts/Resting Stages Other

Component
or system BWT Unit Spec (2) Testing

Source Water (3) Location

of Test Lab (4) Taxa (7) EFF % (8)* Taxa (7) EFF % (8)* Taxa EFF % (8)* Taxa EFF % (8) * Taxa EFF

% (8)* Taxa EFF

% (8)* Taxa EFF % (8)*

Shipboard full

- scale studies

Chlorine

dioxide (EcochlorTM BWT system) 5 mg L - 1 initial Newark, NJ Harbor, 20 psu Atlantic Compass F

- a TT Copepoda Mysidacea Amphipoda Decapoda Cirripedia Pelecypoda

G astropoda Polychaeta

(larvae)

Turbellaria 100%

100%

100%

100%

100%

100%

100%

100%

100% Tintinnids Heterotrophic dinoflagellate 100%

100% Diatoms Dinoflagellates Microflagellates 100%

100%

100% Total coliforms

E. coli Heterotrophs

Vibrios 71%

100%

0%

100%

Other types of full

- scale studies

Literature and references cited:

F

- a 

### Maranda, L., R. G. Campbell, D. C. Smith and C. A. Oviatt. Summer Field Test of the Ecopod aboard the M/V Atlantic Compass. Final Report to Ecochlor, Inc., 22 pp

F

- b
Note: Adjust the Column / Row dimensions to fit information provided.

(1) Indicate component tested (e.g., UV treatment apparatus, filtration apparatus, etc.)

. (2) Indicate the specifications of the BWT Unit/Component dose, filter size, etc.
(3) Source where water used in testing was obtained (e.g., Bay water, harbor water, etc.)., include salinity if available.
(4) Location of the test facility where e xperiments were conducted.

(5) Use letter (F

- a to F

- z) identifiers in this column and list references at bottom (e.g., Primary Scientific literature, reports, web sites, etc.). Primary sources should be given, defined as those where the experimental de sign, methods, analysis and data are presented. Unless absolutely necessary, do not list secondary sources, defined as reports where summaries from primary r eferences are presented.

(6) Enter "TT" if experiments conducted by vendor Test Team; Enter "O" for other if experiments were conducted by an external test team. Relates to who conducted the study.
(7) The major taxa tested. In some studies may be general categories such as "copepods," "polychaetes," etc., in other stud ies more specific taxa or even individual species may have been tested, such as Artemia or Fecal coliform bacteria, , etc.
(8) The range of removal or inactivation rates (efficiency of system either as % removal/inactivation). If multiple tests o r replicates were conducted give the range of removals, such as 80%

- 95% or 2

- 3 logs. * Time = five days after exposure

### STEP 2006 Application Form

Section 4.0: Proof of Ballast Water Treatment Performance

**•** Provide a brief narrative description (one or two paragraphs each) of full

- scale testing

references included in Table 4

- 3. The purpose is to indicate the applicabil ity of the data to gauging the effectiveness of the shipboard BWT system.

TEXT BOX: Annotated bibliography

.

Reference F

- a describes results of experiments conducted in summer 2005 aboard an operating commercial containership/Ro/Ro vessel, Atlantic Compass (Atlantic Container Line), to verify the effectiveness of the Ecochlor BWT system at eradicating estuarine planktonic communities under actual shipping conditions. The treatment tank received an initial concentration of 5 mg

. 

### L - 1 

### ClO 2 

### ; the control tank was filled at the same time, but without any

addition of ClO 2



### . The experiment was designed to follow the effect of ClO 2 

### on temperate marine bacterial and planktonic coastal communities (from the Newark Bay, NJ) by documenting survival and potential for subse quent growth of mid

- Atlantic species as invasive organisms over

a four

- day period. The comparison continued while the ship sailed onto Baltimore, MD and back to Newark, NJ. The authors are some of the same scientists involved in bench

- scale experiments r eported in B

- a above.

TEXT BOX: Brief narrative describing the present status of dose/response relative to the proposed BWT process. Only data from full

- scale should be discussed. If no relevant studies exist, enter “NONE”.

NONE

TEXT BOX: Brief summary (synthesis) of the key findings of all biological testing of

removal/inactivation from full

- scale testing of the BWT process (system, components, process). This synthesis should be linked to the references presented in Table 4

- 3.

Testing the Ecoc hlor TM BWT system aboard the Atlantic Compass allowed for the evaluation of sampling constraints, logistics and experimental design in addition to providing data on the effectiveness of the biocide against natural communities under real shipping conditions. It was demonstrated with water from Newark Bay that an initial ClO 2 

### concentration of 5 mg

. 

### L - 1 

### was as effective at eliminating bacterial and planktonic populations in all size fractions, as was found in prior laboratory phases of the technology development. With water temperature at 24 o 

### C and 350 μM dissolved organic carbon, ClO 2 

### residuals lasted for ca. 6 hours, for an initial oxidant demand (C x T) of 171 mg

. 

### min

. 

### L - 1 

### . The application of 5 mg

. 

### L - 1 

### ClO 2 

### reduced bacteria abundance

to non

- detectable levels within the first 24 hours, whereas b acteria still proliferated in the control tank. After two days, some bacterial growth was observed. Phytoplankton biomass was virtually eliminated in all size fractions, by comparison to the observed biomass in the control tank. The bulk of the autotrop hic biomass resid ed in the <10

- μm size fraction with concentrations, as measured by chlorophyll

- a, unexpectedly low. This size fraction rebounded slightly toward the end of the experiment, as demonstrated by the moderate growth of a small centric diatom (possibly a Thalla ssiosira sp.) in two of six cultures inoculated from the treatment water 96 hours after the application of ClO 2 

### . However visual observations by microscopy of a plankton tow did not reveal any live organisms in the treatment tank at T=96 h. The zooplankto n community (>50 μm) at the study site was comprised of a summer, estuarine community that is typical of this region. Zooplankton in the control tank were in very good condition and many species appeared to be actively reproducing, including all 3 dominan t copepod species. In contrast, there was 100% mortality in the ClO 2 

### - treated tank. Based on a

### STEP 2006 Application Form

Section 4.0: Proo f of Ballast Water Treatment Performance



### 10 comparison between the treatment and control tanks, the ClO 2 

### treatment was effective at killing zooplankton from 9 major taxonomic groups.

4.4 Summary Experime ntal Data for Bench

- Scale Ballast Water Treatment System

In this section, the Applicant provides additional detail on the removal or inactivation of organisms in bench

- scale tests, for the specific BWT system proposed for STEP or its components, in experim ents specific to ballast water treatment. All studies referenced in this

section should also be listed in Table 4.1.

Sub

- section 4.4 presents bench

- scale data (as defined in sub

- section 4.1) specific to the testing of the treatment system (Table 4.4a), or its individual components (Table 4.4b, 4.4c, etc.), proposed for STEP.

The Applicant should note that, at this point in this STEP Application, all of the relevant bench

scale test literature and technical reports should have been referenced in Table 4.1 a nd been placed in Appendix B.

TEXT BOX : Has bench

- scale testing been conducted on your STEP specific technology system or its components by your team or by others (circle 1)? Yes No

**•** If the answer, in the Text Box above is “No”, proceed to S ub

- section 4.5.

**•** If “Yes” then complete Table 4

- 4a, for bench

- scale BWT system test data, and, if pertinent, the series 4

- 4b, 4

- 4c, etc. for tests evaluating individual treatment components. Testing at bench

- scale may include estimates of biological effect iveness (removal/inactivation) under either batch or continuous flows.

Note the following explanations of terms used in the Tables in Sub

- section 4

- 4:

o “ BWT system ”, refers to a multi

- component treatment unit, results for which belong in Table 4.4a.

o “ Comp onent ”, refers to a single treatment process either alone or as experimentally isolated as part of multi

- component test. For example, UV is a component within a filtration/UV treatment system. Tests including the

combination of filtration and /UV would b e system tests belonging in Table 4.4a. Testing that samples the UV unit alone or takes samples just pre

- and post

- UV are “component tests”, belonging in Table 4

- 4.b or 4

- 4.c.

o “ Test Taxa ”, refers to the organisms tested. The test taxa are partitioned by functional groups (zooplankton, phytoplankton, bacteria, etc). Under each functional heading, the Applicant is to list the taxonomic identifier used in the study referenced. For example, under zooplankton, z1 may be “copepods” in one study or “ Calanus ” (genus) or Calanus finmarchicus (species) in another, or even “all zooplankton larger than a given size” in another. Under bacteria, the response may be “heterotrophic bacteria” (trophic designation) or Vibrio cholera (species related to IMO standards) or some other taxon of interest.

o “ Viability Assay Method ”, refers to the method used to determine if an organism is dead or inactivated or unable to reproduce. For example, zooplankton may be assayed using movement or response to physical stimuli; bacteria may be assayed by MPN or plate count culture methods.

### STEP 2006 Application Form

Section 4.0: Proo f of Ballast Water Treatment Performance



### 11 o “ Test Platform ”, refers to the number of the “Bench Test Study#_” filled in by the Applicant in the row below the Table Description, which indicated the laboratory where the bench testing was conducte d.

o “ Treated ”, refers to test water that has passed or will pass through the Treatment System (Table 4

- 4a) or individual treatment component (Table 4

- 4b,c).

o “Control”, refers to water that is not treated by the technology, but which has been handled in p arallel with the treated water, thus includes all experimental

effects exclusive of treatment.

o “ Holding ”, refers to whether the viability samples were assayed without holding time or were held, for example to simulate holding in a ballast tank.

o “Refs”, ref ers to primary references and uses the same identifiers as in Table 4.1 and Appendix B.

### STEP 2006 Application Form

Section 4.0: Proof of Ballast Water Treatment Performance

Table 4

- 4a. Summary of Existing Bench

- Scale Test Data for the STEP BWT System. List primary references which present the experimental design, methods and data, avo id listing multiple references that cite the same data set. As necessary, rows should be added under major taxa heading to provide a full list of the taxonomic breakdown.

Indicate where the bench test studies were conducted

Bench Test #1: GSO, Univer sity of Rhode Island Location of Testing (source water): Narragansett Bay, RI (with added plankton)

Bench Test #2: GSO, University of Rhode Island Location of Testing (source water): Narragansett Bay, RI (with added plankton)

Bench Test #3: L ocation of Testing (source water):

Test Taxa 1 Viability Assay

Method 2 Test Platform 3 Initial # organisms in Test

4 Test Duration 5 % Removal/

Inactivation

over control # Experime

nts # Reps /Expt. Refs

Treated Control T = 24 h End point T = 24 h E nd point



### Ballast Water Treatment System

Zooplankton Taxa in 5 mg

. 

### L - 1 

### ClO 2 (m - 3 )

All zooplanakton Motility, neutral red staining,
response to physical stimuli #1 69000 67000 T = 24 h 7 days 97.4% 94.4% 1 2

COPEPODA z1 Calanoid nauplii “ #1 4110 33 50 T = 24 h 7 days 93.6% 100% 1 2

z2 Non

- calanoid “ #1 7535 5360 T = 24 h 7 days 100% 100% 1 2

z3 CTENOPHORA “ #1 8220 8040 T = 24 h 7 days 100% 100% 1 2

z 4 PELECYPODA “ #1 32195 34840 T = 24 h 7 days 98.6% 93.5% 1 2

z5 GASTROPODA “ #1 8220 670 0 T = 24 h 7 days 97.3% 83% 1 2

z6 POLYCHAETA “ #1 6165 6030 T = 24 h 7 days 96.5% 100% 1 2

z7 Other “ #1 2055 2680 T = 24 h 7 days 100% 84% 1 2

z 4 PELECYPODA,

GASTROPODA and

POLYCHAETA “ #2 5312 7250

- 7 days

- 100% 1 2



### Phytoplankton in 5 mg

. 

### L - 1 

### C lO 2 (100

. mL - 1 for MPN, presence/absence in 50 mL for microscopy, μg

. L - 1 for chlorophyll, n.o.: not observed)

p1 All phytoplankton MPN #1 875 T = 24 h 7 days >99%* >89%* 1 2

### STEP 2006 Application Form

Section 4.0: Proo f of Ballast Water Treatment Performance



### 13 p2

BACILLARIOPHYCEAE MPN #1 808 T = 24 h 7 days >99%* >78% 1 2

p3 DINOPH YCEAE MPN #1 78 T = 24 h 7 days >89%* >81%* 1 2

p3 DINOPHYCEAE microscopy #1 qualita

tive in 50 mL T = 48 h 7 days 100% 100% 1 2

p4 CYANOPHYCEAE microscopy #1 “ “ T = 48 h 7 days n. o. 100% 1 2
p5 Chlorophyll biomass Chlorophyll #1 12.87 10.5 T = 24 h 7 days 99% 76% 1 2
p5 Chlorophyll biomass Chlorophyll #2 21.54 20.7

- 7 days

- 94% 1 2
p6

BACILLARIOPHYCEAE microscopy #2 qualita

tive in 50 mL

- 7 days

- 100% 1 2



### Bacteria in 

### 5 mg

. 

### L - 1 

### ClO 2 (100

. mL - 1 for CFU and g C

. L - 1. day - 1 for bacterial prod uction)

b1 Total bacteria % area covered
on peptone agar and R2A agar in

seawater #1 100 100 T=24 h 7 days 100% 0%** 1 2

b2 Vibrio cholerae

nonO

- 1; ATCC#55332 % area covered

on beef extract/peptone

agar plates #1 100 100 T=24 h 7 days 100% 100% 1 2

b3 Bacterial growth rate 3 H

- leucine incorporation #1 5.3 x 10 - 6 4.9 x 10 - 6 T=24 h 7 days 100%

- 208%** 1 2

b1 Total bacteria % area covered
on peptone

agar #2 80 95

- 7 days

- 100% 1 2

b2 Vibrio cholerae CFU on beef extract/peptone

agar plates #2 6.03 x 10 6 5.94 x

10 6 - 7 days

- 100% 1 2

b3 Bacterial growth rate 3 H

- leucine incorporation #2 6.9 x 10 - 5 8.1 x 10 - 5

- 7 days

- 98.2% 1 2

### STEP 2006 Application Form

Section 4.0: Proo f of Ballast Water Treatment Performance



### 14 Table 4

- 4a (continued)

Test Taxa 1 Viability Assay

Method 2 Test Platform 3 Initial # organisms in Test

4 Test Durat ion 5 % Removal/

Inactivation # Experime

nts # Reps /Expt. Refs

Treated Control T = 24 h End point T = 24

h End point



### Cysts and Resting Stages in 5 mg L - 1 

### ClO 2

cr1 Artemia salina Mobility of nauplii and of

metanauplii #2 0.1 g 0.1 g

- 7 days

- 61% 1 2



### Protozoa in 5 mg L - 1 

### ClO 2 (100

. mL - 1 for MPN, presence/absence in 50 mL for microscopy, n.o. not observed, b.d. below detection)

pr1 heterotrophic

DINOPHYCEAE microscopy #1 qualita

tive in 50 mL T = 48 h 7 days 100% 100% 1 2

pr2 hete rotrophic

EUGLENOPHYCEAE microscopy #1 qualita

tive in 50 mL T = 48 h 7 days 100% 100% 1 2

pr3 heterotrophic nanoflagellates microscopy #1 qualita

tive in 50 mL T = 48 h 7 days present present 1 2

pr3 heterotrophic nanoflagellates MPN #1 15 T = 24 h 7 days 100% present 1 2

pr4 CILIOPHORA MPN #1 0 T = 24 h 7 days b.d. present 1 2

pr4 CILIOPHORA microscopy #1 qualita

tive in 50 mL T = 48 h 7 days present n.o. 1 2

Pr5 CILIOPHORA heterotrophic

DINOPHYCEAE and heterotrophic nanoflagellates mic roscopy #2 qualita

tive in 50 mL

- 7 days

- present 1 2



### Viruses in 5 mg L - 1 

### ClO 2 (PFU mL - 1 )

v1 RNA bacteriophage

MS2 Top

- agar overlay method with competent

E. coli #1 7500 7450 T = 24 h 7 days 100% 100% 1 2

1 Under each major taxonomic groupi ng add rows to show the taxa tested, (copepods, diatoms, etc, or specific species). 2 Give the assay used to determine viability relative to each test taxa (e.g., motility for zooplankton, heterotrophic viabl e plate counts for bacteria, etc)

### STEP 2006 Application Form

Section 4.0: Proo f of Ballast Water Treatment Performance



### 15

3 En ter the number of the bench test study indicated above.
4 The number of individuals at the start of the viability test for each taxa (# bacteria per mL, # copepods per Liter, etc)
5 The number of hours or days that a sample is held after treatmen t before assay to determine treatment effectiveness *Limit of detection for % effectiveness as measured by MPN was >98.7% on day 1 and >89% on day 7 **Bacterial contamination suspected during sampling and it was later demonstrated to be the case

6 If the BWTS is comprised of more than 1 component (e.g. filtration+UV or filtration+biocide), and removal effectiveness on indiv idual components has been determined, then fill in the component description and then fill in the appropriate data in Tables 4

- 4b, etc.

### STEP 2006 Application Form

Section 4.0: Proof of Ballast Water Treatment Performance

4.4 Summary Experimental Data for Bench

- Scale Ballast Water Treatment System (continued)

**•** Provide a brief narrative description of the experiments listed in Tables 4

- 4a, 4

- 4b, etc, the equipment tested, experimental design, and assays for determining

removal/inactivation rates and treatment effectiveness. Also indicate the conditions and

source waters under which treatment was conducted. The data from the individual components should be put in the context of the overall system’s inactivation rates a nd the Applicant should directly link the data in the tables to the narrative. The goal is to focus on the experimental data specific to the BWTS for STEP, its level of uncertainty and the validity of the assay methods.

TEXT BOX: Brief narrative descript ion of the experiments listed in Tables 4

- 4a, 4

- 4b, etc.

The experiment for Bench Test #1 (July 2002) was performed in the dark in a temperature controlled environmental chamber (20.5°C) and consisted of duplicate 0 (control), 1, 5, and 10

mg . 

### L - 1 

### ClO 2 

### tre atments. Polyethylene containers (75.7 L) were filled with unfiltered seawater (30.5 ppt) collected from Narragansett Bay (41° 29.53’N; 71° 25.12’W) to 40 L, then continuously mixed at 30 rpm. Equal volumes of concentrated phytoplankton and zooplankton

w ere added to increase the plankton biomass to bloom conditions. The communities were continuously mixed at 30 rpm for the duration of the experiment. Most samples were collected at t = 0 (prior to ClO 2 

### addition), 1, 2, 4, 12, 48 hours and after 5 days.

Table 4.4a presents results obtained for the 5 mg

. 

### L - 1 

### ClO 2 

### treatment, given that this concentration was just as efficient in removing the planktonic community as were higher concentrations and that the 1 mg

. 

### L - 1 

### concentration was deemed generally insuffi cient. All data

in the column ‘% removal/inactivation over control’ of Table 4

- 4a are reported as percentages

over the control value at the indicated time of sampling.

Chlorine Dioxide: A stock solution of ca. 3000 mg

. 

### L - 1 

### ClO 2 

### was prepared by dissolvin g a ClO 2 

### source material (V

- charge 1L

- 3000 pack, Vulcan Technologies) in deionized water. The ClO 2 

### concentration was verified by amperometric titration with phenylarsine oxide (Method SM 4500

- ClO 2 

### ) prior to addition to the treatment containers. For the t ime course, samples (150 mL) were collected in polyethylene bottles and immediately titrated for ClO 2 

### concentration to the end

- point as indicated by zero amps. Precision was typically ±5%.

Zooplankton: The concentration of each live and dead taxa in ea ch treatment and control container was determined by microscopy using 1% neutral red stain and/or motion as a response to prodding. There were no significant differences between concentrations of animals in initial treatments and initial controls (ANOVA, p=0.988).

Phytoplankton: Assays were conducted for Most Probable Number (MPN

. 

### 100 mL - 1 

### ), chlorophyll, and taxonomic identification by microscopy. The MPN assay was conducted in 24

- well polystyrene non

- tissue culture treated plates with a four

- dilution series (1:1, 1:10, 1:100, 1:1000) and five replicates. MPN observations were made with a dissecting microscope under dark field (63X) for the microbiota of the experimental containers. Given that the limit of detection was 10 organisms per 100 mL, under the chosen conditions, a response of 0

– 0

– 0

– 0 (i.e. no growth at any dilution) meant that 100% removal could never be statistically demonstrated, thus the reported values of >99% or >89%, the variation between the two being

### STEP 2006 Application Form

Section 4.0: Proo f of Ballast Water Treatment Performance



### 17 explained by the different number of organisms in the control. Even with five replicates at four dilutions for each set of treatments, the error bars associated with each evaluation were often times very large, an inherent limitation of this assay. It is noteworthy to report that dinoflagellates, organisms often responsible for the formation of harmful algal blooms, were efficiently eradicated by the 5 mg

. 

### L - 1 

### ClO 2 

### treatment as demonstrated by microscopy and MPN. Chlorophyll biomass was measured in duplicate filtered samples (from 5 0 to 250 mL) by the standard method of Yentsch and Menzel (1963) with filters soaked in 90% buffered acetone. Chlorophyll concentration decreased with time in the controls as well as in the treatments, although the rates were significantly different. The levels were below 0.5 μg

. 

### L - 1 

### within 24 hours

in all ClO 2 

### treatments, whereas control containers did not reach that level until 7 days. The 76% removal for chlorophyll reported for day 7 reflects the very low concentrations encountered

under treatment as well as control conditions. Treated phytoplankton exhibited less than 1% of initial chlorophyll concentration (measurements were close to the detection limit of the assay); the equivalent value under the control conditions was 5%. No recovery was detect ed within the period of observation.

For identification of taxa, the microbiota was concentrated (50 to 2) by centrifugation, identified and evaluated for living/dead condition using phase

- contrast and epifluorescence microscopy. The data were reported as presence/absence.

Bacteria: 3 

### H

- leucine incorporation (Smith and Azam 1992) was used to determine bacterial growth rates and the number of colony forming units (cfu) grown on peptone agar and R2A agar was used for total bacterial abundance. Although the populations were totally inactivated after 24 hours, there was significant subsequent growth (this was subsequently demonstrated to be

due to cross

- contamination. However, the samples used for testing viruses and Vibrio cholerae

were isolated from the containers in duplicate sterile polystyrene tubes to reduce the possibility

of contamination. For the survival estimate of V. cholerae (non

- O1; ATCC #55332), aliquots (100 mL) of water were taken from each tank prior to dosing with ClO 2 

### , filter sterilize d (0.2 μm), infected with ca. 60,000 cfu

. 

### mL - 1 

### and then dosed with ClO 2 

### . Aliquots (100 μL) from each treatment were plated at intervals on beef extract/peptone agar to determine V. cholerae

survival. Total eradication was demonstrated as no colonies were observed at the 5 mg

. 

### L - 1



### ClO 2



### .

Virus: For the estimation of virus survival rate after exposure to ClO 2 

### , the RNA bacteriophage MS2 was added to the same bottles containing the V. cholerea samples at an initial concentration of ca. 7500 pfu

. 

### mL - 1 

### . At each time point, sub

- samples were added to competent host cells ( Escherichia coli ) and virus survival was determined by quantifying the resultant plaque forming units (pfu) using the top

- agar overlay method. The removal was total at the 5

mg . 

### L - 1 

### ClO 2



### .

As ment ioned earlier, the use of more than one variable per functional group provided a reliable

assessment of the effectiveness of the treatment over time.

The second experiment (Bench Test #2, August 2002), performed under conditions similar to

the ones descri bed in Bench Test #1, consisted of replicate ClO 2 

### treatments of 0, 1, 5, 10, and 20 mg

. 

### L - 1 

### with sampling limited to initial (day 0, prior to ClO 2 

### addition) and final (day 7) times to avoid possible cross contamination. A separate experiment was conducted to test hatching rate

of Artemia salina ; dried cysts (0.1 g) were added to duplicate 1

- L Erlenmeyer flasks filled with seawater just after dosing each of the five treatments. Aliquots of Artemia culture were counted after 7 days for live / dead metanaupli ar and naupliar stages. Taxonomic identification of phytoplankton and protozoans and measurements of chlorophyll biomass were made initially

### STEP 2006 Application Form

Section 4.0: Proo f of Ballast Water Treatment Performance



### 18 and after 7 days for all treatments. Given that the ClO 2 

### was effective in killing most zooplankton

taxa in Bench Test #1, Bench Test #2 focused on bivalves, gastropods, and polychaetes. The ctenophores and other large predators were removed prior to enrichment of the plankton community to each container, in order to minimize the effect of grazing. All samples used f or testing abundance and viability of bacteria were initially isolated from the containers in duplicate sterile polystyrene tubes to reduce the possibility of contamination from outside sources. The assays were the same as those used in Bench test #1.

Results from Bench Test #2 for 5 mg

. 

### L - 1 

### treatment were particularly convincing in demonstrating

the effectiveness of ClO 2 

### . The zooplankton community was totally eradicated. The chlorophyll biomass in the treatment was barely above detection levels of the assay, while a removal of 100% was documented by microscopy. The bacterial community was totally inactivated. As explained in Text box following Table 4.1, the use of Artemia cysts proved too artificial to be useful; a removal rate of 61% is nevertheles s reported. Some protozoans were still present 7 days after treatment: an Euplotes

- like ciliate, heterotrophic nanoflagellates and a non

photosynthetic Gymnodinium sp. They likely survived the initial challenge, then were able to thrive by feeding on the accumulated detritus and dead material.

### STEP 2006 Application Form

Section 4.0: Proo f of Ballast Water Treatment Performance



### 19 **•** Indicate in the text box whether dose

- response data at bench scale are available for the BWT system or components and provide a brief narrative description of relationship of

dose to treatmen t effectiveness by taxonomic group for the BWT System proposed for STEP. Dose/response graphs should be included if available or applicable. The purpose is to bring forward in more detail these relationships for the specific STEP BWT system as opposed to the general data in Sub

- section 4.1.

TEXT BOX: (1) Are there bench

- scale dose/response data specific to your BWTS for STEP (circle): Yes No

If Yes is circled proceed to (2), if No, leave the rest of this box blank and continue.

(2) Provide brief narrat ive description.

Dose response graphs (Figures 1, 2, 3 and 4) are included for zooplankton, phytoplankton and bacteria as examples of the data obtained in bench tests #1 and #2 demonstrating the effectiveness of 5 and 10 mg L - 1 

### ClO 2 

### at inactivating summer planktonic communities. The 1 mg

L - 1 

### ClO 2 

### was judged to achieve insufficient eradication.

Figure 1. (Left) Live zooplankton abundance (L - 1 

### )

as a function of ClO 2 

### concentration and time after exposure (day 0, dark blue; day 1, white; and day 7, dark red) in Bench Test #1.

Figure 2. (Right) Chlorophyll biomass (μg

. 

### L - 1 

### ) as

a function of ClO 2 

### concentration and time after exposure (day 0, dark blue; day 1, white; and day 7, dark red) in Bench Test #1.

Figure 3. (Left) Example of enumeration (L - 1 

### ) for the taxonomic breakdown of the zooplankton community

as a function of ClO 2 

### concentration and time (day 0, dark blue; day 1, white; day 7, dark red). Panel A represents the concentration of live bivalve (Pelecypoda) and panel B, live ctenophores, duri ng Bench Test #1. Similar results were obtained during Bench Test #2 with bivalves, polychaetes and gastropods totally eliminated at 5, 10 and 20 mg L - 1



### . First Experiment ClO 2

0 2 4 6 8 10 12 14

Control 1 ppm 5 ppm 10 ppm 0

1 d

7 d First Experiment Zooplankton

0 10 20 30 40 50 60 70 80

Control 1 ppm 5 ppm 10 ppm 0 d

1 d

7 d

0 10 20 30 40

Control 1 ppm 5 ppm 10 ppm

0 4 8 12

Control 1 ppm 5 ppm 10 ppm 

### A

B

### STEP 2006 Application Form

Section 4.0: Proo f of Ballast Water Treatment Performance



### 20 Figure 4. (Right) Bacterial abundance (% areal plate coverage) at day 0 (dark blue) and day 7 ( red) after treatment with a range of ClO 2 

### concentrations. The samples were collected from the treated containers at time 0 and kept isolated in test tubes. Samples collected directly from the treatment containers at day 7 (turquoise) gave a negative resu lts only at 10 and 20 mg L - 1 

### . (Bench Test #2)

Figure 5. (Left) Abundance of Vibrio cholerae (CFU mL
- 1 

### ) in seawater

- based cultures exposed to a range of ClO 2 

### concentrations during Bench

Test #2. All treatments were effective in eradicating the bacteria, a s only the controls allowed subsequent growth, thus demonstrating the very high effectiveness of the treatment.

4.5 Summary Experimental Data for Pilot

- Scale Ballast Water Treatment System

In this section, the Applicant provides more detailed information on removal/inactivation of organisms from pilot

- scale BWTS test data from the specific BWT system proposed for STEP or its components under testing specific to ballast water treatment. All studies referenced in this

section should also be listed in Table 4.2.

Sub

- section 4.5 presents pilot

- scale data (as defined in sub

- section 4.2) specific to the testing of the treatment system (Table 4.5a), or its individual components (Table 4.5b, 4.5c, etc.), proposed for STEP. The Applicant should note that, at this point in this STEP Application, all of the relevant pilot

- scale test literature and technical reports should have been referenced in Table 4.2 and been placed in Appendix B.

TEXT BOX : Has pilot

- scale testing been conducted on your STEP specific technolog y system or its components by your team or by others (circle 1)? Yes No See note below.

Note:

Lab scale experiments and full

- scale shipboard tests have been conducted. Because of the success of the bench scale data, the proven effectiveness of chlorine dioxide in numerous land based water disinfection applications, and the ability of the same system to treat up to 2500 MT per hour, there was little benefit to pilot studies. Ecochlor proceeded directly to installing a full

scale system on Atl antic Compass as illustrated in the next section. Therefore, Tables 4.5 a, b

and c have been omitted. Bench test #2 - **Vibrio cholerae**

0 10000 20000 30000 40000

0 0 1 1 5 5 10 10 20 20

ClO 2 concentration (mg
. L -1 )CFU.mL-1 Initial

After 7 days

### STEP 2006 Application Form

Section 4.0: Proo f of Ballast Water Treatment Performance



### 21 **•** If the answer, in the Text Box above is “No”, proceed to Sub

- section 4.6

**•** If “Yes” then complete Table 4

- 5a, for pilot

- scale BWT system test data, and, i f pertinent,

the series 4

- 5b, 4

- 5c, etc. for tests evaluating individual treatment components. Testing at pilot

- scale may include estimates of biological effectiveness (removal/inactivation)

under either batch or continuous flows.

E xplanations for terms used in Tables are found in Section 4

- 4

4.5 Summary Experimental Data for Pilot

- Scale Ballast Water Treatment System (continued)

**•** Provide a brief narrative description of the experiments listed in Tables 4

- 5a, 4

- 5b, etc, the equipment tested, experimental design, and assays for determining

removal/inactivation rates and treatment effectiveness. Also indicate the conditions and

source waters under which treatment was conducted. The data from the individual components should be put in the context of the ov erall system’s inactivation rates and the Applicant should directly link the data in the tables to the narrative. The goal is to focus on the experimental data specific to the BWTS for STEP, its level of uncertainty and the validity of the assay methods.

TEXT BOX: Brief narrative description of the experiments listed in Tables 4

- 5a, 4

- 5b, etc.

N/A

**•** Indicate in the text box whether dose

- response data at pilot scale are available for the BWT system or components and provide a brief narrative d escription of relationship of dose to treatment effectiveness by taxonomic group for the BWT System proposed for STEP. Dose/response graphs should be included if available or applicable. The purpose is to bring forward in more detail these relationships for the specific STEP BWT system as opposed to the general data in Sub

- section 4.2.

TEXT BOX:

(1) Is there pilot

- scale dose/response data specific to your BWTS for STEP (circle): Yes No

If Yes is circled proceed to (2), if No, leave the rest of t his box blank and continue.

(2) Provide a brief narrative description.

N/A

4.6 Summary Experimental Data for Full

- Scale Ballast Water Treatment System

In this section, the Applicant provides additional detail on the removal or inactivati on of organisms in full

- scale tests, for the specific BWT system proposed for STEP or its components, in experiments specific to ballast water treatment. All studies referenced in this section should

also be listed in Table 4.3.

Sub

- section 4.6 presents full

- scale data (as defined in sub

- section 4.1) specific to the testing of the treatment system (Table 4.6a), or its individual components (Table 4.6b, 4.6c, etc.), proposed for STEP.

### STEP 2006 Application Form

Section 4.0: Proo f of Ballast Water Treatment Performance



### 22 The Applicant should note that, at this point in this STEP Application, all of the relevant full

- scale test literature and technical reports should have been referenced in Table 4.3 and been placed in Appendix B.

TEXT BOX : Has full

- scale testing been conducted on your STEP specific technology system or its components by your t eam or by others (circle 1)? Yes No

**•** If the answer, in the Text Box above is “No”, proceed to Sub

- section 4.6

**•** If “Yes” then complete Table 4

- 6a, for full

- scale BWT system test data, and, if pertinent,

the series 4

- 6b, 4

- 6c, etc., for tests evaluat ing individual treatment components. Testing

at full

- scale should include estimates of biological effectiveness (removal/inactivation) under continuous flow conditions at normal shipboard ballast system flow rates.

E xplanations for terms used in Tables a re found in Section 4.4.

### STEP 2006 Application Form

Section 4.0: Proof of Ballast Water Treatment Performance

Table 4

- 6a. Summary of Existing Full

- Scale Test Data for the STEP BWT System. List primary references which present the experimental design, methods and data, avoid listing multiple references that cite the same data set. As necessary, rows should be added under major taxa heading to provide a full list of the taxonomic breakdown.

Indicate where the full

- scale test studies were conducted

Vessel or Shoreside Facility #1: Atlantic Compass Location of Testing (source water): Newark Harbor, 20 psu

40 o 43’N 74 o 9’W

Vessel or Shoreside Facility #2: Location of Testing (source water):

Vessel or Shoreside Facility #3: Location of Testing (source water):

Test Taxa 1 Viability Assay

Method 2 Test Platform 3 Initial #

organ isms in Test

4 Test Duration 5 % Removal/

Inactivation # Experime

nts # Reps /Expt. Refs

Treated Control Initial End point T = 24

h End point



### Ballast Water Treatment System
Zooplankton Taxa (m - 3 ) (copepods identified to naupliar and copepodite stag es and male and female for copepodite VI) (n.o.: not observed at that sampling time)

COPEPODA

z1 Acartia tonsa Motility, neutral red staining,
response to physical stimuli #1 0 5407

0 4 days 100% 100% 1 1 F

- a

z2 Pseudodiaptomus

coronatus “ #1 0 1000 0 4 days 100% 100% 1 1 F

- a

z3 Oithona similis “ #1 0 242 0 4 days 100% 100% 1 1 F

- a

Z 4 Centropages

hamatus “ #1 0 22 0 4 days 100% 100% 1 1 F

- a

Z5 Eurythemora

herdmani “ #1 0 11 0 4 days 100% 100% 1 1 F

- a

Z6 Unidentified calanoid “ #1 0 0 0 4 days 100% 100% 1 1 F

- a

MYSIDACEA “ #1 0 264 0 4 days 100% 100% 1 1 F

- a

AMPHIPODA “ #1 0 11 0 4 days 100% 100% 1 1 F

- a

DECAPODA “ #1 0 0 0 4 days 100% 100% 1 1 F

- a

### STEP 2006 Application Form

Section 4.0: Proo f of Ballast Water Treatment Performance



### 24 CIRRIPEDIA “ #1 0 22 0 4 days 100% 100% 1 1 F

- a

PELECYPODA “ #1 0 88 0 4 days 100% 100% 1 1 F

- a

G ASTROPODA “ #1 0 22 0 4 days 100% 100% 1 1 F

- a

POLYCHAETA (larvae) “ #1 0 0 0 4 days 100% 100% 1 1 F

- a

TURBELLARIA “ #1 0 33 0 4 days 100% 100% 1 1 F

- a


### Phytoplankton (mL - 1 ), **μ** g . L - 1 for chlorophyll biomass) (n.o.: not observed or detected at that sampling time) 4 days

<10 **μ** m p1 unidentified microflagellates : Motility #1 0 0.012 0 4 days

n. o. 100% 1 1 F

- a

10 to 50 **μ** m BACILLARIOPHYCEAE :

p2 Cylindrotheca

closterium Co lor and shape of chloroplats #1 0 0.006 0 4 days 100% 100% 1 1 F

- a

p3 Thalassiosira sp. “ #1 0 0.024 0 4 days 100% 100% 1 1 F

- a

p 4 Melosira sp. “ #1 0 0.012 0 4 days 100% 100% 1 1 F

- a

p5 Actinoptychus “ #1 0 0.006 0 4 days n. o. 100% 1 1 F

- a

p6 Unid entified pennate

diatoms Gliding

movement, shape and color of chloroplasts #1 0 0.006 0 4 days

100% 100% 1 1 F

- a

DINOPHYCEAE: p7 Scrippsiella sp. Motility, shape

and color of chloroplasts,

Evan’s blue

staining #1

0 0.006 0 4 days

100% 100% 1 1 F

- a

p8 Total chlorophyll

biomass fluorimetry #1 0 1 0 4 days 96.4% 67% 1 1 F

- a



### Bacteria (100 mL - 1

, dpm

. mL - 1. h - 1 for growth rate) 4 days

b1 Total coliform IDEXX Colilert #1 0 1398 0 4 days 100% 71.3% 1 2 F

- a

b2 E. coli IDEXX Colilert #1 0 56.5 0 4 da ys 100% 100% 1 2 F

- a

b3 Heterotrophs Zobell marine agar (plates) #1 2,000 >500,000 0 4 days >99.6% 0% 1 2 F

- a

b4 Vibrios TCBS agar (plates) #1 0 11,500 0 4 days 100% 100% 1 2 F

- a

b5 Bacterial growth rate 3 H

- leucine incorporation #1 0.34 x

10 6 1.38 x

10 6 0 4 days 33%

- 66%

### STEP 2006 Application Form

Section 4.0: Proo f of Ballast Water Treatment Performance



### 25 Table 4

- 6a (continued)

Test Taxa 1 Viability Assay

Method 2 Test Platform 3 Initial # organisms in Test

4 Test Duration 5 % Removal/

Inactivation # Experime

nts # Reps /Expt. Refs

Treated Control Initial End point T = 24

h End point



### Protozoa (mL - 1 ) (not observed or detected at sampling time)

pr1 Tintinnids (e.g. Helicostomella) Motility,

Evan’s blue staining #1 0 0.006 0 5 days 100% n. o. 1 1 F

- a

pr2 Heterotrophic dinoflagellates Motility,

Evan’s blue staining #1 0 0.006 0 5 days 100% n. o. 1 1 F

- a

1 Under each major taxonomic grouping add rows to show the taxa tested, (copepods, diatoms, etc, or specific species). 2 Give the assay used to determine viability relative to each test taxa (e.g., motility for zooplankton, heterotrophic viable plate counts for bacteria, etc)
3 Enter the number of the bench test study indicated above.
4 The number of individuals at the start of the viability test for each taxa (# bacteria per mL, # copepods per Lite r, etc)
5 The number of hours or days that a sample is held after treatment before assay to determine treatment effectiveness

### STEP 2006 Application Form

Section 4.0: Proo f of Ballast Water Treatment Performance



### 26 4.6 Summary Experimental Data for Full

- Scale Ballast Water Treatment System (continued)

**•** Provide a brief narrative descripti on of the experiments listed in Tables 4

- 6a, 4

- 6b, etc, the equipment tested, experimental design, and assays for determining

removal/inactivation rates and treatment effectiveness. Also indicate the

conditions and source waters under which treatment was conducted. The data from the individual components should be put in the context of the overall system’s inactivation rates and the Applicant should directly link the data in the tables to the narrative. The goal is to focus on the experimental data speci fic to the BWTS for STEP, its level of uncertainty and the validity of the assay

methods.

TEXT BOX: Brief narrative description of the experiments listed in Tables 4

- 6a, 4

- 6b,

etc.

The experiment listed in Table 4

- 6a was designed to determine the effectiv eness of the Ecochlor water treatment system at eliminating potentially invasive species that could be introduced via the discharge of ballast water. This preliminary testing allowed evaluation of sampling constraints, logistics and experimental design in addition to providing data on the effectiveness of the biocide against natural communities under real shipping

conditions. This self

- contained system generates and delivers chlorine dioxide to treat water coming into ballast tanks and had previously been installed aboard an operating commercial containership and RO/RO, Atlantic Compass

. The Ecochlor unit is composed of a ClO 2 

### generator that produces a concentrated solution of 1500 mg

. 

### L - 1 

### of

ClO 2 

### that is injected via venturi eductors and mixed directly int o incoming seawater being piped to ballast tanks at a calibrated rate to obtain a concentration of 5 mg

. 

### L - 1



### . The experiment was conducted in July 2005 and involved the treatment of one ballast tank, while a second tank was used as a control (no replicates were available in this first phase). The experiment was designed to follow the effect of ClO 2 

### on temperate marine bacterial and planktonic coastal communities by documenting survival and potential for subsequent growth of mid

- Atlantic species as invasive organisms. Bacterial and planktonic populations from the Newark Channel, just off Newark Bay, NJ (40 o 

### 43’N

74 o 

### 9’W) were exposed to an initial concentration of 5 mg

. 

### L - 1 

### of chlorine dioxide inside the treatment tank of the docked Atlantic Compass, while the control tank received untreated

water. The comparison between treatment and control continued for a four

- day period while the ship sailed onto Baltimore, MD and back to Newark. Time intervals and parameters measured are listed in text

- box tables 1 and 2

. Table 1. Schedule of sampling aboard the RO/RO Atlantic Compass in July 2005, following treatment of ballast water with five parts per million of chlorine dioxide. Time 0 is defined as the time of completion of the filling process. Tows were size fra ctionated to

obtain <10

- μm, 10

- to

- 50

- μm and >50

- μm fractions of the plankton communities.

Time

(h) (YSI probe) whole water whole

water Tows Tows

0 T o 

### , S, pH, DO C, N, DOC, chl a , ATP bacteria phytoplankton zooplankton

12 T o 

### , S, pH, DO C, N, DOC, chl a

, ATP bacteria phytoplankton zooplankton

24 T o 

### , S, pH, DO C, N, DOC, chl a , ATP bacteria phytoplankton zooplankton

48 T o 

### , S, pH, DO C, N, DOC, chl a , ATP bacteria zooplankton

72 T o 

### , S, pH, DO C, N, DOC, chl a , ATP bacteria zooplankton

96 T o 

### , S, pH , DO C, N, DOC, chl a , ATP bacteria phytoplankton zooplankton

### STEP 2006 Application Form

Section 4.0: Proo f of Ballast Water Treatment Performance



### 27 Table 2. Schedule of whole

- water sampling for activity parameters requiring the use of isotopes and incubations performed at the University of Rhode Island.

Time (h) Bacteria PhytoplanktonP otential for growth

0 3 

### H

- leucine uptake F/8

- Si, K/8

24 3 

### H

- leucine uptake F/8

- Si, K/8

96 3 

### H

- leucine uptake F/8

- Si, K/8

At the dock, whole water samples were first collected at the depth of water intake into the ballast tanks (temperature = 24.4 o 

### C, sal inity = 19.1 psu, pH 7.3, dissolved oxygen = 4.71 mg

. 

### L - 1 

### , chlorophyll biomass = 0.86 μg . 

### L - 1 

### ). Physical parameters were obtained with a YSI 600 multiprobe sensor. Vertical phytoplankton and zooplankton tows sampled the water column for identification of organisms larger than 10 and 50 μm, respectively (zooplankton = 1099 m - 3 

### ). For the ba llast tanks, whole water was collected with a

Kemmerer bottle from mid

- depth, a statistical analysis having previously determined no significant difference in the values of physical parameters at three depths. The plankton was sampled by lowering a 10

- μm a nd a 50

- μm plankton nets five times up and down the water column of each tank to cover an estimated volume of 168 L, diaphragm pumps having been found unsatisfactory.

Water was filtered in duplicates for particulate carbon and nitrogen (POC/PON) and subseq uently analyzed with a carbon and nitrogen analyzer (Carbo Erla NA1500). Water for chlorophyll a (triplicates) was first size fractionated (total, <50 **μ** m and <10 **μ** m) before filtration and freezing until analysis by fluorimetry. Duplicate samples for ATP

were size

- fractionated, filtered, extracted immediately in 5% trichloroacetic acid (TCA) and frozen for later analysis using the luminometer method for the Promega’s ENLITEN ®


### ATP assay. Triplicate samples for dissolved organic carbon (DOC) were pre

- filte red through 0.22 μm and preserved with sodium azide. Measurements of DOC followed the method of high

- temperature catalytic oxidation (TOC

- V CHS Shimadzu).

Bacteria

- The response of the bacterial community was assessed in two ways: i) Control and Treatment water plated in duplicates on seawater

- based growth media and incubated, and ii) bacterial protein production based on 3 

### H

- leucine uptake. The total number of colony forming units (CFU) was used to determine the efficacy of chlorine dioxide in killing bacteria that are capable of growing on the media. The evaluation of E.

coli and enterococci was accomplished using Idexx enzyme

- based test kits Colilert TM



### and Enterolert TM 

### , respectively (MPN

- based quantification). Bacterial protein production (duplicates) was quantified using the 3 

### H

- leucine assay (Smith and Azam 1992).

Heterotrophic protists and phytoplankton

- Heterotrophic protists and phytoplankton were observed for viability by microscopy of water mounts of the communities. Viability was determined by qualitative ass essment of cell color (mostly chloroplasts), shrinking or deterioration of the cytoplasm within cell walls and inability to swim for swimming cells. The use of the live/dead dye Evan’s blue (1% final concentration) was abandoned as it yielded mixed result s; it worked as expected with dinoflagellates, but less well with certain species of diatoms. Quantitative assessment of population abundance was determined on preserved whole

- water samples (T=0) or on preserved samples (1% formaldehyde:acetic acid final concentration) quantitatively collected by plankton tows and enumerated with Sedgwick

- Rafter chamber. Potential for growth for phytoplankton followed a protocol similar to the one used by Sutherland et al. (2001), where triplicate water samples (2 mL) wer e inoculated in f/8 (designed to promote the growth of

### STEP 2006 Application Form

Section 4.0: Proo f of Ballast Water Treatment Performance



### 28 organisms other than diatoms) and k/8 (designed to promote the growth of diatoms) growth media (20 mL) and incubated at the prevailing temperature (25 o 

### C) and under 275 μmol photons

. 

### m - 2. 

### s - 1 

### . The bioma ss of the resulting mixed cultures was evaluated after 5 days by measuring chlorophyll content and composition by microscopy.

Zooplankton

– A total of 5 hauls (13

- cm diameter, 50

- μm mesh plankton net) for the

control tank and 10 hauls for the treatment tan k were taken at each sampling period, which sampled a total of 91 and 182 liters, respectively. The entire sample from each tank was immediately examined live under a dissecting microscope to determine the proportion of living/non

- living organisms in the m ajor taxonomic groups. As with phytoplankton, the vital stain neutral red was found mostly unreliable. Thus the organisms’ appearance and condition (coloration and evidence of physical damage), swimming behavior, and response to mechanical manipulation w ere recorded and used collectively to assess their viability. After the initial examination, the whole sample was preserved in 4% buffered formaldehyde solution for a more complete taxonomic analysis upon return following standard protocols (ICES Zooplankt on Methodology Manual). The whole sample was enumerated and the most abundant forms were identified to the highest taxonomic level possible (species and stage) and the rarer forms to more basic taxonomic groups.

It was demonstrated that an initial ClO 2 

### co ncentration of 5 mg

. 

### L - 1 

### was as effective at eliminating bacterial and planktonic populations as was found in prior laboratory phases of the technology development. With water temperature at 24 o 

### C and 350 μM dissolved organic carbon, chlorine dioxide residuals lasted for ca. 6 hours, for a CxT value of 171

mg . 

### min

. 

### L - 1 

### . The application of 5 mg

. 

### L - 1 

### ClO 2 

### reduced bacteria abundance to non

detectable levels within the first 24 hours, whereas bacteria still proli ferated in the control tank (Text Box Figures 1 A and B). After two days, some bacterial growth was observed. Phytoplankton biomass was virtually eliminated in all size fractions, by comparison to the observed biomass in the control tank (Figure 2 A and B). In contrast to the control tank, there was immediate and near 100% mortality of zooplankton (>50 μm) in the treatment tank (Table 3).

A B

Total coliform colonies

0 200 400 600 800 1000 1200 1400

0 12 24 48 72 96

Time (h)CFU 100 mL-1 Control

Treatment Treatment Vibrios

0 2,000 4,000 6,000 8,000 10,000 12,000 14,000 16,000

0 12 24 48 72 96

Time (h)cfu 100 mL-1 Control

Treatment Treament



### Figure 1. Timecourse of coliform bacteria (A) and Vibrio colonies (B) in the control and in t he treatment tank following exposure to ClO 2 

### (duplicate samples).

### STEP 2006 Application Form

Section 4.0: Proo f of Ballast Water Treatment Performance



### 29 A B

Control tank - chl **a** concentrations

-0.1 0.1 0.3 0.5 0.7 0.9

0 12 24 48 72 96

Time (h)**μ**g L-1 <10

10 to 50

>50 **μ** m

**μ** m

**μ** m ClO 2 Treatment tank - Chl **a** concentrations

-0.1 0.1 0.3 0.5 0.7 0.9

0 12 24 48 72 96

Time (h)**μ**g L-1 <10

10 to 50

>50 **μ** m

**μ** m **μ** m



### Figure 2. Timecourse of chlorophyll

- a concentration in three size fractions in the control (A) and in the treatment tank (B) following an initial dosage of 5 mg L - 1 

### ClO 2



### .

Table 3. Total zooplankton abundance (number m - 3 

### ) in the control and treatment tanks over the course of the study.

Time = 0 12 h 24 h 48 h 72 h 96 h

Control 7121 5703 6615 2945 2846 1901

Treatment 0 0 0 5 0 0

Although some minor but statistica lly significant recovery occurred in coliform bacteria four days after treatment and to a very minor extent in the autotrophic plankton, the system was deemed very effective at controlling potentially invasive species by comparison to the non

- treated contr ol tank. It is possible that biofilms in the tanks provided refuge for the organisms that survived. Laboratory experiments conducted in

more controlled conditions (no biofilms) showed no such regrowth.

**•** Indicate in the text box whether dose

- response da ta at full scale are available for the BWT system or components and provide a brief narrative description of relationship of dose to treatment effectiveness by taxonomic group for the BWT System proposed for STEP. Dose/response graphs should be included i f available or applicable. The purpose is to bring forward in more detail these relationships for the specific STEP BWT system as opposed to the general data

in Sub

- section 4.3.

TEXT BOX:

(1) Is there pilot

- scale dose/response data specific to your BWT S for STEP (circle):

Yes No

If Yes is circled proceed to (2), if No, leave the rest of this box blank and continue.

(2) Provide a brief narrative description.

### STEP 2006 Application Form

Section 4.0: Proo f of Ballast Water Treatment Performance



### 30 4.7 Program Summary for BWT Experiments at All Scales

**•** Provide a summary of the results o f the experimental program at various scales and interpretation establishing the efficacy of the treatment approach (in terms of actual concentrations achieved and the percent difference between controls and treatments), the accuracy of the analytical tech niques, appropriateness of the measured variables in predicting the system performance, and the efficacy of achieving similar results with a full

- scale shipboard system.

**•** Provide evidence that the proposed BWT system has satisfied the NVIC ballast

water tr eatment criterion or will meet the NVIC in the primary testing under STEP, that is, that the system has accomplished 98% removal effectiveness for organisms >50 μm in preliminary experiments, and will likely perform similarly during shipboard trials under STEP.

TEXT BOX:

(A) Summary of experimental results and interpretation:

Overall , the treatment was quite effective in the first 24 hours at eliminating living biomass in all size fractions which remained inactivated, except for some components of the bacterial and protozoan populations which showed some recovery, either at the bench scale or in the ballast tank. However, when the treatment was applied under laboratory conditions with sterile techniques to minimize contamination (which is certainly not possible for ballast tanks), no recovery was found, thus suggesting that the eventual recovery was likely the result of biofilms in the tanks providing refuge for the organisms that survived. Laboratory experiments conducted in more controlled conditions (no biofilms) showed no such regrowth (section 4.4 text

- box figure 4). Another significant relevant and compelling finding of these experiments comes from the total eradication of vibrios and viruses.

The phytoplankton component (or autotrophic biomass of <50 μm) decreased with time under control as well as under treatment conditions during the course of both bench and full

scale experiments. Results show that the chlorophyll biomass exposed to 5 mg

. 

### L - 1 

### ClO 2 

### decreased much more rapidly than and to a muc h greater extent than the biomass under control conditions. Under treatment conditions, chlorophyll concentrations generally

reached near

- detection levels for the assay and observations by microscopy did not detect any viable photosynthetic organisms. Th e removal rate for the zooplankton community was

100% at the full scale demonstration.

The assessment of the efficacy of the 5

- mg . 

### L - 1 

### ClO 2 

### treatment on planktonic populations was established by measuring a variety of parameters of biomass and activity of the planktonic community to cover sizes from viruses to macrozooplankton. The overall performance of the treatment is thus not the result of the evaluation of a single or just a few parameters, but it comes from measurements using standard methodology in aquatic microbial and planktonic ecology .

B) Summary of evidence that BWT system can satisfy the NVIC treatment criterion:

The most convincing evidence of the efficacy of treating ballast water with 5 mg

. 

### L - 1 

### ClO 2 

### comes from results of the full scale exp eriment confirming previously obtained bench

- scale

### STEP 2006 Application Form

Section 4.0: Proo f of Ballast Water Treatment Performance



### 31 data, where the zooplankton community (>50 **μ** m) was totally and immediately eliminated by the treatment, with no resurgence after four days (reported above in section 4.6, text

- box table 3), by comparison t o a control tank where various crustaceans, mollusks and worms

were still alive. This 100% removal rate is thus well above expectations required by STEP policy and guidance in NVIC 01

- 04 for a treatment performance of 98% removal for organisms larger than 50 micrometers. From an initial zooplankton concentration of 7,000 organisms

. 

### m - 3 

### , the 100% removal was based on assessing the abundance and viability of all organisms collected in 90 (control) or 180 L (treatment).

**•** Provide data and interpretation from the experimental program on chemical residuals in the treated water, both for disinfection chemicals and their byproducts. Discussion should include a description of the chemicals (and doses used) and byproducts, the time

- course of decay/loss of said che micals and byproducts, their projected concentration in ballast water upon discharge, and the conditions under which the results were derived (salinity, temperature, etc.), if

available.

Include specifics of results at all scales tested, and references to residuals in section 3 (Table 3

- 2b) and Section 6.2.

TEXT BOX: Discussion of chemical and byproduct residuals.

Dosage and residual :

The addition / injection of chlorine dioxide is done during the loading of the ballast water. A target dosage of 5.0 ppm is added to the incoming ballast water into the main ballast line. Within 30 minutes, or the quickest opportunity to capture a sample out of a ballast tank, the chlorine dioxide concentration is typically between 1.0 ppm and 2.0 ppm. This

has been consi stant in the testing through actual ballasting operations in Newark, New Jersey, Baltimore, Maryland and Portsmouth, Virginia, San Francisco, California as well as in laboratory testing of waters from Halifax, Nova Scotia, Houston, Texas and Seattle,

Washi ngton.

Residual Decay:

The decay of the remaining chlorine dioxide residual is temperature dependent. Through the actual shipboard testing of the chlorine dioxide decay, we have found that as the ballast water temperature drops below 10 0 

### C and begins to ap proach 0 0 

### C, the slope of the decay is modified and a small residual lasts longer. The following graphs show the decay of chlorine dioxide in November of 2004 in Newark, Baltimore, Portsmout and Djibouti, Africa.

### STEP 2006 Application Form

Section 4.0: Proo f of Ballast Water Treatment Performance



### 32 ClO2 Decay Rate
Baltimore, November 2004 Atlantic Compass, ballast tank 6DBP

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

0 2 4 6 8 10 12 14 16 18 20 hours since ballastingResidual ClO2 in ppm ClO2 Decay Rate
Maher Terminal, November, 2004 Atlantic Compass, ballast tank 6DBP

0 0.1 0.2 0.3 0.4 0.5 0.6

0 5 10 15 20 25 30 hours since ballastingResidual ClO2 in ppm

Decay in Baltimore, Initial dosage of 5. 0 ppm Decay in Newark, Initial dosage of 5.0 ppm

November, water temp 12 0 C November, water temp 10 0 C

ClO2 Decay Rate
Portsmouth, November 2004 Atlantic Compass, ballast tank 6DBP

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

0 1 2 3 4 5 6 7 8 9 10 11 12 hours since ballastingResidual ClO2 in ppm ClO2 Decay Rate
Baltimore, December 2004 Atlantic Compass, ballast tank 6DBP

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1

0 5 10 15 20 25 30 35 40 45
hours since ballastingResidual ClO2 in ppm

Decay in Portsmouth, Initial dosage of 5.0 ppm Decay in Baltimore, Initial dosage 5.0 ppm

November, water temp 12 0 C December, water temp 5 0 C

ClO2 Decay Rate Djibouti Africa, December 2005 M/V Moku Pahu, #1 wing tank, starboard

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4

0 5 10 15 20 25

hours since ballastingResidual ClO2 in ppm ClO2 Decay Rate Djibouti Africa, December 2005 M/V Moku Pahu, #3 wing tank, starboard

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4

0 5 10 15 20 25

hours since ballastingResidual ClO2 in ppm

Decay in Djibouti, Initial Dosage of 5.0 ppm Decay in Djibouti, Initial Dosage of 5.0 ppm

December, water temp 25 0 C December, water temp 26 0 C

### STEP 2006 Application Form

Section 4.0: Proo f of Ballast Water Treatment Performance



### 33 Ecochlor has contracted Nautilus Environmental to c onduct a detailed decay study to determine the expected relationship between temperature and decay. From this work

and our on

- going field work, a model will be incorporated into the Ecochlor PLC to allow us to predict (and adjust if necessary) the expecte d point where the chlorine dioxide

residual will be non

- detect.

The modifications to the PLC logic as well as incorporating a temperature sensor to measure the incoming ballast water temperature will be incorporated in all future designs and will be incorp orated into the Atlantic Compass and Moku Pahu. Completion of this work is anticipated to be completed in the 3 rd 

### or 4 th 

### quarter of 2006.

By product issues

– Chlorine :

The Eka process for generating chlorine dioxide does not produce any chlorine

By product issues

– Chlorite : as a by

p roduct. This was one of the main reasons for using this technology for ballast water treatment. Other methods that use chlorine or sodium hypochlorite to generate the chlorine dioxide will be less than 100% efficient and have some chlorine present in the treatment solution. The presence of this chlorine would contribute to the formation of some chlorinated compounds that are undesirable.

Chlorine dioxide use will form some chlorite. As the chlorine dioxide decays, there wil l be between 50% and 80% of the initial dosage remaining as chlorite. This chlorite level will also decay over time as it is reacted upon by various compound (organics, metals) in

the water.

Ecochlor’s testing has found in laboratory and through shipboa rd testing of chlorite in ballast water there is between 25% and 50% of the initial dosage.

Nova Chem Laboratories Inc, Oxford, Ohio using Ion Chromatography (USEPA 300.1) found the chlorite levels in the following samples:

Wat er Source Test Location Cl0 2 

### Dosage Chlorite Ion (mg/l) Discharge Toxicology Testing

San Francisco AMEC Envir Labs 5.0 2.31

San Francisco AMEC Envir Labs 5.0 2.09

This is the concentration that was found to be present during the discharge toxicology testing conducted by AMEC Environmental per the protocols set by Washington State Department of Ecology.

Water Source Test Location Cl0 2 

### Dosage Chlorite Ion (mg/l) Bench Scale Testing at the University of Rhode Island

Narrag. Bay URI Lab 1.0 ppm < 0.15

Narrag. Bay URI La b 5.0 ppm 0.43

Narrag. Bay URI Lab 10 ppm 0.72

It is speculated that the lower chlorite levels were due to the high level of organisms in the test containers that were being studied in the effectiveness testing conducted by the University of Rhode I sland.

### STEP 2006 Application Form

Section 4.0: Proo f of Ballast Water Treatment Performance



### 34 Water Source Test Location Cl0 2 

### Dosage Chlorite Ion (mg/l) Shipboard Testing

– M/V Atlantic Compass

Newark DB6

– 1 5.0 ppm 2.48

Newark DB6

– 2 5.0 ppm 2.45

Newark DB6

– 3 5.0 ppm 2.35

These samples were taken during the URI field testi ng aboard the Atlantic Compass in July of 2005.

Chlorite and seawater

The testing conducted by Nautilus Environmental (formerly AMEC Environmental) shows the impact (or lack there of) of a chlorite residual on marine organisms. The tests

simulated a balla sting of San Francisco Bay water (more specifically water from the San Pablo Bay

- Carquinez Strait off the Benicia Dock) where the target dosage of 5.0 ppm of chlorine dioxide was added. The toxicology tests were run immediately after the chlorine

dioxide residual was found to be less than detection limit.

In the Topsmelt Acute Toxicity test, the LC 50 

### was found to be in excess of 100% of the treated sample.

In the Bivalve Larval Development Test, the EC 50 

### was found to be in excess of 100% of

the treated sam ple.

In the Mysid Shrimp Acute Toxicity Test, the most sensitive organism, the LC 50 

### was found to be 51.2% of the treated sample.

The criteria set by the Department of Ecology in Washington State regarding the approvable limits was authorized by WAC 220

- 77

- 095 Interim ballast water discharge standard approval process. In part:

The results of the proposed ballast water biocide definitive toxicity testing must be submitted to the Department of Ecology Water Quality Program’s Whole Effluent Toxicity (WET) Coord inator for calculation of the toxic threshold and determination of the target discharge concentration. The results of range

- finding tests must also be included if they were used to determine the most sensitive species prior to definitive testing. The rep ort must also propose an onboard method for accurately measuring either biocide concentration or a meaningful surrogate in order to verify that the biocide is near the target discharge concentration just prior to beginning ballast water discharge.

Candida tes for the target discharge concentration include in order of preference:

1. A target discharge concentration will be set at two times the LC 50 

### as long as the LC 50 

### is no more than three times the LC 25 

### . If it does not seem likely from the data that two

ti

mes the LC 50 

### would produce a complete effect, then the target discharge concentration

will be set at three times the LC 50 

### as long as the LC 50 

### is no more than two times the LC 25



### . If the bivalve test is the most sensitive, the EC 50 

### and EC 25 

### for combined sur vival and development will be used as described for the LC 50 

### and LC 25



### . 2. A target discharge concentration will be set at three times the LOEC if a concentration at or between two to four times the LOEC caused complete mortality.

Using criteria #1 and th e most sensitive species (Mysid Shrimp), 2X the LC50 equals 102.4% of the treated sample. Also, the LC 50 

### is 2.5 times (less than 3 times) the LC 25



### .

### STEP 2006 Application Form

Section 4.0: Proo f of Ballast Water Treatment Performance



### 35 Based on this work, Ecochlor will propose that the decomposition products of an initial target dosage of 5.0 ppm of chlorine dioxide will be an acceptable discharge

concentration.

Chlorite and fresh water

Ecochlor does not have a vessel that currently takes on fresh water ballast and discharges that fresh water treated ballast in a fresh water port. This is the combination

that would be of most concern from the chlorite residuals.

The most sensitive species to chlorite in fresh water appears to be Daphnia magna. Daphnia magna are a fresh water species and not found in marine environments. Daphnia are also ver y sensitive to salt (sodium chloride). The USEPA 1988 Ambient water quality criteria for chloride ( Office of Water, Washington, DC. EPA

- 440

- 5

- 88

- 001) reports the Species Mean Acute Value

– the geometric mean of available LC50 datapoints

– for Daphnia is 26 50 mg/l chloride. Typical seawater has chloride levels in excess of 30,000 mg/l.

In fact, the discharge of mid

- ocean exchanged ballast water would be very toxic to daphnia in a fresh water port.

A recent review of the effects of chlorite concentrations o n Daphnia magna show a significant variance in the data. The PAN Pesticides Database (in Appendix) shows the following:

Name Effect Measurement Study Endpoint Toxic Exp

Time Dose Type



### Daphnia Intoxication Immobile 48hr EC 50 

### 25 ug/l Static

Daphnia Intoxication Immobile 48hr EC 50 

### 290 ug/l F

- thru

Daphnia Intoxication Immobile 48hr EC 50 

### 14.6 ug/l Static

Daphnia Intoxication Immobile 48hr EC 50 

### 1400 ug/l Static

The great variability of these tests suggests that there are other factors effect ing the endpoints and toxic dose. Some of these factors are as follows:

- What were the experimental pH values? The water pH effects the characteristics of the chlorite (lower pH’s

– below 4.0

– will increase the toxic effect of chlorite).

- Where there o ther impurities in the water? Sodium chloride is very toxic to Daphnia

magna.

- Is “immobile” the best measurable endpoint? Some subjective aspects are possible when determining “immobile” vs a more definitive “mortality” endpoint. Also, an organism that was classified as “immobile” may recover after the 48 hour period when the chlorite concentration is depleted.

- How was the chlorite generated? Possibly, the laboratory technique is adding variables into the experimental design.

Path Forward:

Based on the concerns over Daphnia, Ecochlor will investigate several items before seeking treat fresh water ballast and discharge into fresh water ports. These items

include but will not be limited to:

### STEP 2006 Application Form

Section 4.0: Proo f of Ballast Water Treatment Performance



### 36 - Confirmation of the effect of chlorite generated from the E cochlor treatment system on Daphnia magna. A suggestion to take actual treated ballast water to conduct the tests would provide the most reliable data

- Investigate the pH values, chloride content and other impurities that may have been present in the pr eviously generated data.

- Conduct other toxicological tests of species of interest to Federal / State and Local Agencies in Fresh water ports and harbors.

- Modify (if necessary) the Ecochlor treatment methodology in Fresh Water Ports to incorporate a ch lorite reduction step. This may include the use of a second treatment chemical (acidified sodium sulfite, ascorbic acid or other) to reduce / eliminate the chlorite before discharging of the ballast water.

Chlorite degradation :

An important factor in the persistence of chlorite in the environment deals with the impact of sunlight. Within the past few years, researchers [Wang 1998] determined that chlorite solutions degraded rapidly (half

- life ~ hours) under laboratory fluorescent lighting

and even more ra pidly in direct sunlight (half

- life ~ minutes). This was verified in a field trial at the Los Angeles Country Reservoir [Stolarik 1999] in which chlorine dioxide was applied to control algal blooms. Chlorine dioxide was applied at night to give adequate reaction time with the algae present. Within four hours after sunrise, levels of both chlorine dioxide and its primary degradation byproduct, chlorite ion, were below

detection limits.