STEP 2006 Application Form

Section 4.0: Proof of Ballast Water Treatment Performance

1

4.0 PROOF OF BALLAST WATER TREATMENT PERFORMANCE

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 ballast 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 taxonomic 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 BWT 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 technologies, 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 physical 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 precede 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, the 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 meaningful 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, however, 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 technology 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 other 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 arranged 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, pilot-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 background 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 provides 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 Treatment Performance

2

components. Any reference 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. 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 components 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 identifiers 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 reference 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 

entering 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), and 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 Treatment Performance

3

technologies should also be worked out at this stage. For example, bench scale tests would be 

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 subsections 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 experimental 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 (circle 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 the 

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 concentration 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 ”Location 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 “copepods”. 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 its 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 these references as alphabetically as 

“B-a”, “B-b”, “B-c”, etc., indicating a series of Bench 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 information 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 days

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 

facilities 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

Bacillariophyceae

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

Heterotrophic flagellate

<100%

<100%

<100%

Phytoplankton 94% Total bacteria

Vibrio cholerae

100%

100%

Artemia 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, 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 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 references 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 studies 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 replicates 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 effectiveness 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 estuarine 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 ClO2. The time course of action of ClO2 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 ClO2 (1, 5, 10 and 20 ppm) were measured 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 microbiology, 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 during 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 ClO2 could be 

detected in any of the treatments. It was determined that, under the bench-scale conditions 

used, an initial concentration of 5-ppm ClO2 provided sufficiently effective eradication of coastal 

planktonic communities to proceed further, with no significant increased effectiveness at 10 ppm 

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 ClO2 eradicated 

nearly 98% of all zooplankters after 24 hours, with survival occurring only and minimally in shellbearing 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 minimize 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 presented in Table 4-1.

Partial results from reference B-a are presented in table 4-1 for the inactivation effectiveness on 

plankton of ClO2 at an initial concentration of 5 mg.L-1 in seawater, seven days after treatment. 

All functional groups suffered from the presence of ClO2 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 days. 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 ClO2 long 

enough to survive the initial challenge and then thrive on the organic matter generated by the 

death of all organisms. Nevertheless the overall effect of ClO2 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 initial 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 original 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 plate 

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 treatment 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 even 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 detection 

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 

ClO2 treatment was only 61% effective 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 the 

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 ClO2 treatment in seawater 

favored decapsulation and hatching and, by the time the embryos emerged, there was no or 

insufficient ClO2 left to inactivate them. This experiment with Artemia cysts proved too artificial 

to be useful.

4.2 Literature Summary of Laboratory 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 fullsize unit operations, for example, dockside or large tank size. These tests are sometimes 

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, there was little benefit to pilot studies. Ecochlor proceeded directly to installing a fullscale 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 Pilot scale testing citations.

4.3 Literature 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, aquaria). 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 analytical 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 testing 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 as those used in Tables 4-1 and 4-2, except 

that Table 4-3 specifies that the Applicant identify references alphabetically as “F-a”, “Fb”, “F-c”, etc., indicating a series of Full scale testing citations.
STEP 2006 Application Form

Section 4.0: Proof of Ballast Water Treatment Performance

Table 4-3. Summary, Prior Experiments, Literature, and Studies, Full- Scale. Summary of literature, reports and web site information on the removal/inactivation efficiency of the components or combined 

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

Gastropoda

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 experiments 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 design, methods, analysis and data 

are presented. Unless absolutely necessary, do not list secondary sources, defined as reports where summaries from primary references 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 studies 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 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 applicability 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 ClO2; the control tank was filled at the same time, but without any 

addition of ClO2. The experiment was designed to follow the effect of ClO2 on temperate marine 

bacterial and planktonic coastal communities (from the Newark Bay, NJ) by documenting 

survival and potential for subsequent 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 reported 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 EcochlorTM 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 ClO2 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 oC and 

350 μM dissolved organic carbon, ClO2 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 ClO2 reduced bacteria abundance 

to non-detectable levels within the first 24 hours, whereas bacteria 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 autotrophic biomass resided 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 Thallassiosira sp.) in two of six cultures inoculated from the treatment 

water 96 hours after the application of ClO2. However visual observations by microscopy of a 

plankton tow did not reveal any live organisms in the treatment tank at T=96 h. The 

zooplankton 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 dominant 

copepod species. In contrast, there was 100% mortality in the ClO2-treated tank. Based on a 
STEP 2006 Application Form

Section 4.0: Proof of Ballast Water Treatment Performance

10

comparison between the treatment and control tanks, the ClO2 treatment was effective at killing 

zooplankton from 9 major taxonomic groups.

4.4 Summary Experimental 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 experiments 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 benchscale test literature and technical reports should have been referenced in Table 4.1 and 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 Sub-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 effectiveness 

(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 “Component”, 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 be 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: Proof 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 conducted. 

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 parallel 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”, refers 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, 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 bench test studies were conducted

 Bench Test #1: GSO, University 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: Location 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 End point

Ballast Water Treatment System

Zooplankton Taxa in 5 mg.L-1 ClO2 (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 3350 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

z4 PELECYPODA “ #1 32195 34840 T = 24 h 7 days 98.6% 93.5% 1 2

z5 GASTROPODA “ #1 8220 6700 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

z4 PELECYPODA, 

GASTROPODA and 

POLYCHAETA

“ #2 5312 7250 - 7 days - 100% 1 2

Phytoplankton in 5 mg.L-1 ClO2 (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: Proof of Ballast Water Treatment Performance

13

p2 

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

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

p3 DINOPHYCEAE microscopy #1 qualitative 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 qualitative in 50 mL - 7 days - 100% 1 2

Bacteria in 5 mg.L-1 ClO2 (100.mL-1 for CFU and g C.L-1.day-1 for bacterial production)

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 

106

5.94 x 

106 - 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: Proof 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 Duration 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 ClO2

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 ClO2 (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 qualitative in 50 mL T = 48 h 7 days 100% 100% 1 2

pr2 heterotrophic 

EUGLENOPHYCEAE microscopy #1 qualitative in 50 mL T = 48 h 7 days 100% 100% 1 2

pr3 heterotrophic 

nanoflagellates microscopy #1 qualitative 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 qualitative in 50 mL T = 48 h 7 days present n.o. 1 2

Pr5 CILIOPHORA

heterotrophic 

DINOPHYCEAE and 

heterotrophic 

nanoflagellates

microscopy #2 qualitative in 50 mL - 7 days - present 1 2

Viruses in 5 mg L-1 ClO2 (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 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)
STEP 2006 Application Form

Section 4.0: Proof of Ballast Water Treatment Performance

15

 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 Liter, etc)

 5 The number of hours or days that a sample is held after treatment 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 individual 

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 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 4-4a, 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 ClO2 treatments. 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 

were 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 ClO2 addition), 1, 2, 4, 12, 48 hours and after 5 days. 

Table 4.4a presents results obtained for the 5 mg.L-1 ClO2 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 insufficient. 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 ClO2 was prepared by dissolving a ClO2

source material (V-charge 1L-3000 pack, Vulcan Technologies) in deionized water. The ClO2 

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

ClO2) prior to addition to the treatment containers. For the time course, samples (150 mL) were 

collected in polyethylene bottles and immediately titrated for ClO2 concentration to the end-point 

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

Zooplankton: The concentration of each live and dead taxa in each 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: Proof 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 ClO2 treatment as demonstrated by microscopy and MPN.

Chlorophyll biomass was measured in duplicate filtered samples (from 50 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 ClO2 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 detected 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: 3H-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 ClO2, filter sterilized (0.2 μm), 

infected with ca. 60,000 cfu.mL-1 and then dosed with ClO2. 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

ClO2.

Virus: For the estimation of virus survival rate after exposure to ClO2, 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 ClO2.

As mentioned 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 described in Bench Test #1, consisted of replicate ClO2 treatments of 0, 1, 5, 10, and 

20 mg.L-1 with sampling limited to initial (day 0, prior to ClO2 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 metanaupliar and naupliar stages. Taxonomic identification of 

phytoplankton and protozoans and measurements of chlorophyll biomass were made initially 
STEP 2006 Application Form

Section 4.0: Proof of Ballast Water Treatment Performance

18

and after 7 days for all treatments. Given that the ClO2 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 for 

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 ClO2. 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 nevertheless reported. Some protozoans were still present 7 

days after treatment: an Euplotes-like ciliate, heterotrophic nanoflagellates and a nonphotosynthetic 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: Proof 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 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 4.1.

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 narrative 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 ClO2 at inactivating summer planktonic communities. The 1 mg 

L-1 ClO2 was judged to achieve insufficient eradication.

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

as a function of ClO2 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 ClO2 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 ClO2 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, during 

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 ClO2

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: Proof 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 ClO2 

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 results 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 ClO2 concentrations during Bench 

Test #2. All treatments were effective in 

eradicating the bacteria, as 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 technology 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 fullscale system on Atlantic 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

ClO2 concentration (mg.L-1)

CFU.mL-1

Initial

After 7 days
STEP 2006 Application Form

Section 4.0: Proof 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, if 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.

Explanations 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 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 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 description 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 this 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 inactivation 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: Proof 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 team 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 evaluating 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.

Explanations for terms used in Tables are 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 psu40o43’N 74o9’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 # 

organisms 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 stages 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

z2Pseudodiaptomus 

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

Z4 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: Proof 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

GASTROPODA “ #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), μg.L-1 for chlorophyll biomass) (n.o.: not observed or detected at that sampling time) 

<10 μm

p1 unidentified 

microflagellates

: 

Motility #1 0 0.012 0

4 days

n. o. 100% 1 1 F-a

10 to 50 μm

BACILLARIOPHYCEAE

:

p2 Cylindrotheca 

closterium

Color 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

p4 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 Unidentified 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) 

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 days 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 

106

1.38 x 

106 0 4 days 33% -66%
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Section 4.0: Proof of Ballast Water Treatment Performance

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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 Liter, 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: Proof of Ballast Water Treatment Performance

26

4.6 Summary Experimental Data for Full-Scale Ballast Water Treatment System 

(continued)

• Provide a brief narrative description 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 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 4-6a, 4-6b, 

etc.

The experiment listed in Table 4-6a was designed to determine the effectiveness 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 ClO2 generator that produces a concentrated solution of 1500 mg.L-1 of 

ClO2 that is injected via venturi eductors and mixed directly into 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 ClO2 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 (40o43’N 

74o9’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 fractionated 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 To, S, pH, DO C, N, DOC, chl a, ATP bacteria phytoplankton zooplankton

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

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

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

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

96 To, S, pH, DO C, N, DOC, chl a, ATP bacteria phytoplankton zooplankton
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Section 4.0: Proof of Ballast Water Treatment Performance

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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 PhytoplanktonPotential for growth

0 3H-leucine uptake F/8-Si, K/8

24 3H-leucine uptake F/8-Si, K/8

96 3H-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 oC, salinity = 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 ballast 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 and 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 

subsequently analyzed with a carbon and nitrogen analyzer (Carbo Erla NA1500). 

Water for chlorophyll a (triplicates) was first size fractionated (total, <50 μm and <10 μm) 

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-filtered 

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 3H-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 ColilertTM

and EnterolertTM, respectively (MPN-based quantification). Bacterial protein production 

(duplicates) was quantified using the 3H-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 assessment 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 results; 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) were inoculated in f/8 (designed to promote the growth of 
STEP 2006 Application Form

Section 4.0: Proof 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 oC) and under 

275 μmol photons.m-2.s-1. The biomass 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 tank 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 major 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 were 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 Zooplankton 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 ClO2 concentration 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 oC 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 ClO2 reduced bacteria abundance to nondetectable levels within the first 24 hours, whereas bacteria still proliferated 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 the 

treatment tank following exposure to ClO2 (duplicate samples).
STEP 2006 Application Form

Section 4.0: Proof of Ballast Water Treatment Performance

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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)

μg L-1

<10

10 to 50

>50 μm

μm

μm

ClO2 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)

μg L-1

<10

10 to 50

>50

μm

μm

μm

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 ClO2.

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 statistically 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 control 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 data 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 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 4.3.

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 this box blank and continue.

(2) Provide a brief narrative description.
STEP 2006 Application Form

Section 4.0: Proof of Ballast Water Treatment Performance

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4.7 Program Summary for BWT Experiments at All Scales

• Provide a summary of the results of 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 techniques, 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 treatment 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 fullscale experiments. Results show that the chlorophyll biomass exposed to 5 mg.

L-1 ClO2

decreased much more rapidly than and to a much 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. The removal rate for the zooplankton community was 

100% at the full scale demonstration.

The assessment of the efficacy of the 5-mg.L-1 ClO2 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 ClO2

comes from results of the full scale experiment confirming previously obtained bench-scale 
STEP 2006 Application Form

Section 4.0: Proof of Ballast Water Treatment Performance

31

data, where the zooplankton community (>50 μm) 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 to 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 chemicals 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 consistant 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, 

Washington.

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 100 C and begins to approach 00 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: Proof 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 ballasting

Residual 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 ballasting

Residual ClO2 in ppm

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

November, water temp 120 C November, water temp 100 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 ballasting

Residual 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 ballasting

Residual ClO2 in ppm

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

 November, water temp 120 C December, water temp 50 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 ballasting

Residual 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 ballasting

Residual ClO2 in ppm

 

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

 December, water temp 250 C December, water temp 260 C
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Section 4.0: Proof of Ballast Water Treatment Performance

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Ecochlor has contracted Nautilus Environmental to conduct 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 expected 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 incorporated into the Atlantic Compass and Moku Pahu. Completion of this 

work is anticipated to be completed in the 3rd or 4th 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 byproduct. 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 will 

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 shipboard 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:

Water Source Test Location Cl02 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 Cl02 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 Lab 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 Island.
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Section 4.0: Proof of Ballast Water Treatment Performance

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Water Source Test Location Cl02 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 testing 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 ballasting 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 LC50 was found to be in excess of 100% of the 

treated sample.

In the Bivalve Larval Development Test, the EC50 was found to be in excess of 100% of 

the treated sample.

In the Mysid Shrimp Acute Toxicity Test, the most sensitive organism, the LC50 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) Coordinator 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 report 

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. 

Candidates for the target discharge concentration include in order of preference:

1. A target discharge concentration will be set at two times the LC50 as long as the LC50 

is no more than three times the LC25. If it does not seem likely from the data that two 

times the LC50 would produce a complete effect, then the target discharge concentration 

will be set at three times the LC50 as long as the LC50 is no more than two times the LC25. 

If the bivalve test is the most sensitive, the EC50 and EC25 for combined survival and 

development will be used as described for the LC50 and LC25.

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 the most sensitive species (Mysid Shrimp), 2X the LC50 equals 

102.4% of the treated sample. Also, the LC50 is 2.5 times (less than 3 times) the LC25.
STEP 2006 Application Form

Section 4.0: Proof of Ballast Water Treatment Performance

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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 very 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 2650 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 on 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 EC50 25 ug/l Static

Daphnia Intoxication Immobile 48hr EC50 290 ug/l F-thru

Daphnia Intoxication Immobile 48hr EC50 14.6 ug/l Static

Daphnia Intoxication Immobile 48hr EC50 1400 ug/l Static

The great variability of these tests suggests that there are other factors effecting 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 other 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: Proof of Ballast Water Treatment Performance

36

- Confirmation of the effect of chlorite generated from the Ecochlor 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 previously 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 chlorite 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 rapidly 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.