# Land

- Based Evaluations of the

Severn Trent De Nora BalPure TM BP

- 1 000

Ballast Water Management System

Maritime(Environmental(Resource(Center



## June 2010

c/o Chesapeake Biological Laboratory

University of Maryland Center for Environmental Sc ience

PO Box 38, Solomons, Maryland 20688, USA

UMCES Technical Repor t Series: Ref. No. [UMCES]CBL 10

- 015MERC ER01

- 10

i 

## Notic e

The objective of this Maritime Environmental Resource Center ( MERC ) evaluation wa s to provide shipping lines, classificat ion societies, regulators

, and flag states with an independent

and credible assessment of treatment performance under realistic conditions. Therefore, the

ballast water treatment system was tested in accordance with the International Maritime

Organization (IMO) International Convention for the Control and Management of Ships’ Ballast

Water and Sediment (2004), Resolution MEPC.174(58) Guidelines for Approval of Ballast

Water Management Systems (G8) and Resolution MEPC.169(57) Procedure for Approval of

Balla st Water Management Systems T hat M ake U se of Active Substances (G9)

. The evaluation

was conducted under specific, predetermined, agreed

- upon protocols, criteria, and quality

assurance procedures to assess the treatment system's performance.

MERC does not l abel or list technologies as acceptable or unacceptable but will present

the results in a n objective way that can be used to determine regulatory compliance by

appropriate administrations, agencies o r certification societies. Subsequent data on the

techno logy's performance characteristics is presented to allow for compar ison with the IMO

Convention discharge standards, Regulation D

- 2

, Ballast Water Performance Standard

. MERC and the MERC Advisory Board do not provide certification for technologies, or

ce rtify that a technology will always operate as demonstrated . Additionally, no expressed or

implied guarantee is provided as to the performance of the technology

, or that a technology will

always operate at the levels verified. MERC does guarantee the lev els verified during the

evaluation under the conditions, circumstances , and operations encountered as fully independent

and credible.

This report has be en reviewed by the MERC Advisory Board and provided to Severn

Trent De Nora and MERC funding agencies p rior to public release. Mention of trade names or

commercial products does not constitute endorsement or recommendation by MERC.

Questions and comments should be directed to Dr. Mario Tamburri, tamburri@umces.edu.MERC ER01

- 10

ii 

### Table of Contents

Page No.

1. MER C Background and Objectives

................................ ................................ ............... 1

2

. Description of the Severn Trent De Nora Ballast Water Management System

............. 1

3

. Summary of IMO Standards

................................ ................................ .......................... 2

4. Summary of Test Protocol s

................................ ................................ ............................ 2

5

. Summary of Results

................................ ................................ ................................ ....... 4

6. Results Trial 1

................................ ................................ ................................ ................ 6

7. Results Trial 2

................................ ................................ ................................ ................ 8

8. Results Trial 3

................................ ................................ ................................ .............. 1 1

9. Results Trial 4

................................ ................................ ................................ .............. 1 4

10

. Results Trial 5

................................ ................................ ................................ .............. 1 7

11. Chemical Analysis of Discharge

................................ ................................ .................. 20

12

. Acknowledgements

................................ ................................ ................................ ...... 2 1

Appendix A. Vendor Interpretation

................................ ................................ ......................... 2 2

Note : Detailed Test Plan, Protocols and Standard Operating Procedures (SOPs) can be

downloaded from www. maritime

- enviro.org.MERC ER01

- 10

1 

### 1

. MERC Background and Objectives

The Maritime Environmental Resource Center (MERC) is a State of Maryland initiative

that provides test facilities, information, and decision tools to address key environmental issues

facing the in ternational maritime industry. The Center’s primary focus is to evaluate the

mechanical and biological efficacy, associated costs, and logistical aspects of ballast water

treatment systems and the economic impacts of ballast water regulations and managemen t

approaches. A full description of MERC ’s structure, products, and services can be found at

www.maritime

- enviro.org.

To address the need for effective, safe, and reliable ballast water treatment systems to

prevent the introduction of non

- native species, MERC has developed as a partnership between

the Maryland Port Administration (MPA), Chesapeake Biological Laboratory/ University of

Maryland Center for Environmental Science (CBL/UMCES), U.S. Maritime Administration

(MARAD), Smithsonian Environmental Resea rch Center (SERC), and University of Maryland

(UM D ) to provide independent performance testing and to help facilitate t he transition of new

treatment technologies to shipboard implementation and operations.

This report describe s the MERC evaluation of th e Severn Trent De Nora BalPureTM BP

1 000 Ballast Water Management System through objective and quality assured land

- based

testing ( dockside at a flow rate of 200m 3 

### /hr). The goal of this evaluation was to provide shipping

lines, regulators, classification s ocieties, and flag states with an independent and credible

assessment of treatment performance under realistic conditions. Therefore, the data and

information on performance characteristics cover s legitimate information to meet the

evaluation's objective, and performanc e is presented in a way to allow for comparison against the

International Maritime Organization (IMO ) International Convention for the Control and

Management of Ships' Bal last Water and Sediments (2004 ) , Regulation D

- 2 Ballast Water

Performa nce Standard

.

2

. Description of the Severn Trent De Nora (STDN) Ballast Water Management System

The STDN Ballast Water Treatment System (BWTS) applies established chlorination

technology to oxidize and disinfect aquatic invasive species. The BALPURE® BW TS is an

electrolytic process for the on

- site generation of dilute hypochlorite on demand without storage

during the ballasting operation and the neutralization of residual oxidants during the de

ballasting operation. Sodium hypochlorite is generated by m eans of electrolysis of seawater. In

this process, seawater flows through the electrolytic cells in a ratio of 1 part for every 100 parts

of ballast water. The system can operate effectively in brackish water to 15 PSU. Because the

amount of seawater req uired to effectively treat ballast water is small

, even fresh water ballast

can be treated by BALPURE® BWT with supplemental salt or stored seawater in designated

ballast tanks such as aft peak tanks. The current passing through the seawater causes the sa lt

(NaCl) and water (H 2 

### O) to form sodium hypochlorite (NaOCl) and hydrogen (H 2 

### ) as a secondary

by

- product. Hypochlorite solution and Hydrogen are produced and separated immediately upon

exit from the electrolytic cells. The weak hypochlorite solution (1 g /L) is injected back into the

ballast stream. Hydrogen that is separated from the hypochlorite is immediately diluted to less

than 1% hydrogen by forced air blowers and discharged to a safe location. The hypochlorite isMERC ER01

- 10

2 

### generated automatically on demand a nd is matched to the ballast flow rate and the oxidant

demand of the ballast water. Initial oxidant concentration is dosed at 1.5 times the TOC

concentration

, which remains effective for several days. T o minimize potential regrowth of

(micro) organisms a background level of chlorine is maintained (1 mg/L) until discharge. To

eliminate the release of chlorine

, sodium sul fite or bisulfite is added prior to discharge.

For this land

- based test, the water is filtered through a 40 micron BallastSafeTM BSFc

Automatic Electric Filter, Model BSFc

- V

- 1.6 prior to treatment. The sintered stainless steel

screen technology enables i t to remove zooplankton. BallastSafe’s filter features continuous

cleaning of large volumes of dirt during ballasting without interruption, and a reversible screw

system for smooth, reliable and rapid cleaning of the entire screen surface.

All test fac ility equipment and instrumentation was operated by MERC personnel. The

ballast water treatment system was operated by STDN.

3. Summary of IMO Standards

This evalua tion was designed to determine i f the BalPure ® BWMS c ould meet IMO D2

standards in accord ance with both the IMO Guidelines for Approval of Ballast Water

Management Systems (G8) and the Procedure for Approval of Ballast Water Management

Systems that make use of Active Substances (G9) . The IMO Convention performance standard

states that ships mu st discharge:

1) Less than 10 viable organisms per m 3



### , greater than or equal to 50 μ m in minimum

dimension;

2) Less than 10 viable organisms per ml, less than 50 μ m in minimum dimension and greater

than or equal to 10 μ m in minimum dimension and

3) Less th an the following concentrations of indicator microbes, as a human health standard:

1. Toxi genic Vibrio chol erae (serogroups O1 and O139), less than 1 colony forming unit

(cfu) per 100 ml

2. Escherichia coli

, less than 250 cfu per 100 ml ;

3. I ntestinal Ent erococci

, less than 100 cfu per 100 ml

.

4

. Summary of Test Protocols

The following is a brief summary of the testing approach and methods. For complete

details on protocols, data management, and quality control / quality assurance procedures for this

M ERC evaluation, please refer to the Test Plan for the Performance Evaluation of the Se vern

Trent De Nora BalPureTM BP

- 1 000 Ballast Water Management System (August 2009), available

for download at www.maritime

- enviro.org

. The protocols described below are b ased upon the IMO G8/G9 guidelines and the U.S.

Coast Guard supported ETV protocols under development. Any deviation from IMO G8 /G9

guidelines or draft ETV protocols were explained and justified in the Test Plan. MERC

evaluated the biological efficacy of the STDN ballast water treatment system onboard the U.S.

Maritime Administration ( MARAD ) Ro

- Ro vessel MV Cape Washington while docked in the

Port of Baltimore . The ballast system o n MV Cape Washington was modified to allow for water

at a flow rate of 4 00 m 3 

### /hr to be split equally at flow rates of 200 m 3 

### /hr. Just before this split,

challenge condition concentrations of total suspended solids (TSS) and particulate organic

carbon (POC) were augmented by injecting a concentrated slurry of Arizona test dust and humicMERC ER01

- 10

3 

### acid ( developed and validated by the Naval Research Laboratory, Key West, Florida ). The water

was then delivered simultaneously to either a “control” (untreated) ballast tank or a “treated”

(passing first through the STDN system) ballast tank. T hese two tanks on the MV Cape

Washington were used for the required holding time of five days and were essentially identical in

size and structure. Each tank was filled to approximately 250 m 3 

### for each test trial.

Temperature, salinity, dissolved oxygen, chlorophyll fluorescence, turbidity and pH were

measured every 15 minutes during the test trials by two identical multi

- parameter probes placed,

one each, into the control and test tanks. Initial inline samples of ballast water during the filling

of the co ntrol and test tanks were collected, filtered, and analyzed (using USEPA methods) for

the water quality parameters of particulate organic carbon (POC), dissolved organic carbon

(DOC) a nd total suspended solids (TSS) by the CBL/UMCES Nutrient A nalytical Ser vices

Laboratory (NASL).

A total of 10 identical 1.1 m 3 

### conical bottom mesocosms were also used for controlled

sampling during each trial. Using the mesocosms, five sequential, time

- integrated, continuous

samples were taken during: (A) initial filling of tanks, just prior to the split of control and treated

water ( T0 Control ), (B) initial filling of test tank, just downstream of the BALPURE system

during filling of test tank ( T0 Treated ), (C) during discharge of control water after a five

- day

holding time ( TF Control ), and (D) during discharge of treated water after a five

- day holding

time ( TF Treated ).

Immediately after filling, 1.0 m 3 

### of water in each mesocosm was filtered through a 35 μ m

plankton net to concentrate the zooplankton for qualitative and q uantitative analyses under a

dissecting microscope . T he proportion and total concentration of live versus dead organisms was

determined using standard movement and response

- to

- stimuli techniques within one hour of

collecting the individual samples. Zoopl ankton samples were also fixed and returned to the

laboratory for additional taxonomic evaluations

. Ten liters of well

- mixed, but unfiltered, water from each mesocosm were also collected

immediately after filling, to determine concentrations of organisms in the 10 to 50 micron size

class using four distinct methods: (A) One sub

- sample was fixed with standard Lugol’s solution

to determine total cell abundances under an inverted compound microscope using grid settlement

columns and phase contrast lighting

. (B) A second sub

- sample was stained using CMFDA (5

- chloromethylfluorescein diacetate) as a selective live /viable indicator

. Stained sub

- samples were

incubated and observed on a Sedgewick Rafter slide using a Leitz Laborlux S modified for

epifluorescence. (C) A third sub

- sample wa s filtered and frozen until analysis of total and active

chlorophyll

- a by the NASL

. (D) Finally

, a fourth sub

- sample was used to determine chlorophyll

a levels after allowed to regrow under favorable conditions. An increase in chlorophyll

, or

positive regrowth

, indicates that viable phytoplankton were in the samples, whereas chlorophyll

levels at or below detection limits of the laboratory analytical method suggests that there was no

viable phytoplankton.

Additional subsamples of unfiltered water were also collected from each mesocosm to

determine concentrations of total heterotrophic bacteria and three specific indicator pathogens, E.

coli , intestinal Enterococci , and toxigenic Vibrio cholerae . Total heterotrophic bacteria wer e

enumerated by spread plate method using NWRI agar. The presence and abundance of intestinal

Enterococci was determined using a commercially availabl e chromogenic substrate method.

C ulturable E. coli concentrations were determined using a standard USEPA method: membrane

filt ration on modified mTEC agar . Abundance s of total and toxigenic V. cholerae were

calculated by filtration and selection on TCBS agar and enumerated using a sp ecies

- specificMERC ER01

- 10

4 

### RNA colony blot and ctxA DNA colony blot hybridization . Via ble toxigenic V. cholerae was assayed with a commercial DFA kit specific for serogro up O1 using monoclonal antibodies

tagged w ith fluorescein isothiocyanate

. To evaluate the toxicity of treated water at the completion of each trial, samples from

each mesoc osm were collected and tested for acute and chronic toxicity, and for total residual

chlorine. Filter “backflush” (retenate/filtrate during initial treatment of water) was also tested for

total residual chlorine. The toxicity protocols and species used w ere consistent with the US EPA

methods for Whole Effluent Toxicity (WET). The algal species tested was Isochrysis galbana,

the fish species was the S heepshead minnow (Cyprinodon variegatus) while th e invertebrate

species was the M ysid shrimp (Americamysis b ahia). A chlorine concentration in samples was

analyzed immediately upon collection to avoid potential loss of oxidant with time

. 

### The Standard

Methods for the Examination of Water and Wastewater Low

- Level Amperometric Titration

method 4500

- Cl D and DPD Co lorimetric method 4500

- Cl G were used to measure Total

Residual Oxidants (TRO) and Total Residual Chlorine (TRC) 

### . A Fischer and Porter amperometric

titrator was also used for amperometric measurements.

5 . Summary of Results

Biological Performance

- The Severn Trent De Nora BALPURE Ballast Water

Management System dramatically reduced the numbers of live organisms in ballast water during

MERC land

- based testing in the Port of Baltimore. For all biological categories, the treatment

system consistently met IMO D2 discharge standards.

Discharge Toxicity

- Treated ballast water upon discharge was not chronically toxic to

either mysid shrimp or sheepshead minnows for any of the trials conducted. All five of these

samples, however, were chronically toxic to th e marine algal species Isochrysis galbana

. Additional testing of several dilutions of the treated ballast water was conducted on the last three

samples (STDN

- 03 through

- 05). EC 50 

### ’s, measured as a reduction in growth, were found at

22%, 42% and 53% for S TDN 03, STDN

- 04 and STDN

- 05 respectively.

Additional toxicity testing has revealed that under certain circumstances dechlorinated

estuarine water remains toxic to some types of algae. The continued toxicity of dechlorinated

water is species specific with several golden brown algal species ( Isochrysis galbana and

Pavlova lutheri ) exhibiting decreased growth after TRO is at or below detection (DPD method).

In contrast

, similarly treated water was non toxic to the diatom Phaeodactylum tricornutum

. Toxicity was found in samples that were chlorinated by either electrochlorination or sodium

hypochlorite addition; and aged for five days.

Toxicity in high initial TRO sa mples was found after dechlorination with one of two

sulfur

- based reducing compounds, sodium thiosulfate or sodium bisulfite. Neither of these

reducing compounds alone was found to be toxic at relevant concentrations with NOECs of 100

and 200 mg/l for sod ium thiosulfate and sodium bisulfite, respectively. Additional algal toxicity

tests are being conducted to investigate the cause, as well as the extent, of toxicity caused by

dechlorinated water. Results of this investigatio n are being submitted to a pee r

- reviewed journal

for publication.

Chemical Analysi s of Discharge

– Detailed chemical analyses of various consitients (e.g.,

chloroform, bromoform, trichloroacetic acid, etc.) of treated water upon discharge were

conducted by Analytical Laboratory Service s, Inc. ( www.analyticallab.com ). Summary of

reuslts can be found in Appendix B

.MERC ER01

- 10

5 

### Mechanical Performance

- The BALPURE system itself experienced no mechanical

failures during formal test trials.

QA/QC

- Finally , the MERC Quality Control and Quality Assurance pla n was completed

as described in the Test Plan. In summary, there were no adverse findings or problems requiring

corrective action in any of the audits. The testing, sampling, analyses and data handling for this

evaluation met or exceeded MERC test require ments.

* Additional datasets (e.g., in tank values for temperature, salinity, dissolved oxygen, etc., every

15 minutes during hold time) are available upon request.MERC ER01

- 10

6 

### 6. Results Trial 1 (STDN

- CAL): 15

- 20 October 2009

Physical Parameters

TSS mg/l D OC mg/l POC mg/l

T0 Initial Ave StDev Ave StDev Ave StDev

Conditions 20.6 4.1 3.96 0.31 1.997 0.271

Temp o C Salinity psu Dis. Oxygen mg/l Turbidity NTU

Ave StDev Ave StDev Ave StDev Ave StDev

T0 Control 16.97 0.02 13.26 0.01 7.11 0.06 8.16 0.86

T0 Treated 17.10 0.01 13.21 0.01 7.74 0.09 9.75 0.63

TF Control 16.24 0.02 13.57 0.00 7.09 0.17 1.21 0.09

TF Treated 16.27 0.01 13.36 0.00 8.31 0.01 4.20 0.14



### Live Organisms > 50 μ m

T0 Ave #/m 3 T 0 StDev #/m 3 T F Ave #/m 3 T F StDev #/m 3

Control 60,000 11,250 72,000 3,000

Treated ND N D 1.4 1.7

* Taxa listed in order of abundance.



### Live Organisms 10

- 50 μ m

Vital Stain Results

- It is important to note that the specific approach used underestimates

the abundances of live organisms in this size class because it has been found that not all live

organisms reliably take up the CMFDA stain.

T0 Ave #/ml T0 StDev #/ml TF Ave #/ml TF StDev #/ml

Control 258 50.6 4 2.4

Treatment 0 0 0 0 * TF Control * TF Treated

Spionid polychaete Rotifera

Copepod nauplii Spionid polychaete

Rotifera

Calanoida ( Acartia sp.)

Cirripedia nauplii

Turbellaria

Dominant species Type Other

Skeletonema costatum Diatom Chain

- forming

Thalassiosira sp. Diatom Chain

- forming

Oxyrrhis marina Dinof lagellateMERC ER01

- 10

7 

### Active Chlorophyll

- a

- Chlorophyll is used as ancillary data and as a general

presence/absence indicator of viable photosynthetic organism (MDL = 0.56 μ g/l).

T0 Ave μ g/l T 0 StDev μ g/l T F Ave μ g/l T F StDev μ g/l

Control 6.27 0.47 0.88 0.05

Treated 0.56 0.00 0.56 0.00

Regrowth Control 2.61 1.59

Regrowth Treated 0.56 0.00



### Live Microbes (cfu = colony forming units)

E. coli T0 Ave

cfu/100ml T0 StDev

cfu/100ml TF Ave

cfu/100ml TF StDev

cfu/100ml

Control 13.6 4.93 0.2 0. 45

Treated 0 0 0 0



### Enterococci T0 Ave

cfu/100ml T0 StDev

cfu/100ml TF Ave

cfu/100ml TF StDev

cfu/100ml

Control 11.28 3.05 12.08 5.16

Treated 0 0 0 0



### V. cholerae

– No detectable culturable toxigenic Vibrio cholerae were detected in any

samples durin g any of the trials.

Heterotrophic

Bacteria T0 Ave

cfu/100ml T0 StDev

cfu/100ml TF Ave

cfu/100ml TF StDev

cfu/100ml

Control 547 93.24 613 237.51

Treated 0.9 0.78 1.2 2.45



### Discharge Toxicity

Residual Chlorine Analysis

- Levels of residual c hlorine (or total residual oxidants

- TRO) were at or below the method detection limit (MDL) of 0.02 ppm in all TF t reated samples.

Acute Toxicity

- There were no statistically significant survival effects in any test for

either acutely tested species ( Mysid sh rimp or Sheepshead minnows).

Chronic Toxicity

- Results showed that ballast water discharge was not chronically toxic

to either mysids or sheepshead minnows. All samples, however, were chronically toxic to the

marine algal species Isochrysis galbana wit h a statistically significant inhibition in growth in

treated ballast discharge compared to controls (see details under Summary of Results on page 4).MERC ER01

- 10

8 

### 7. Results Trial 2 (STDN

- 01): 22

– 27 October 2009

Physical Parameters

TSS mg/l DOC mg/l POC mg/l

T0 Initial Ave StDev Ave StDev Ave StDev

Conditions 28.7 5.1 3.98 0.14 2.912 0.163

Temp o C Salinity psu Dis. Oxygen mg/l Turbidity NTU

Ave StDev Ave StDev Ave StDev Ave StDev

T0 Control 16.00 0.00 12.14 0.01 8.48 0.04 9.70 0.85

T0 Treated 16.02 0.00 12.17 0.01 8.59 0.05 8.92 0.64

TF Control 15.86 0.01 12.14 0.01 7.34 0.04 1.99 0.12

TF Treated 15.85 0.00 12.23 0.00 8.63 0.02 3.46 0.10



### Live Organisms > 50 μ m

T0 Ave #/m 3 T 0 StDev #/m 3 T F Ave #/m 3 T F StDev #/m 3

Control 70,000 5,625 30,000 1,500

Treated No data 0.2 0.4

* Taxa listed in order of abundance.



### Live Organisms 10

- 50 μ m

Vital Stain Results

- It is important to note that the specific approach used underestimates

the abundances of live organisms in this size class because it has been found that not all live

organisms reliably take up the CMFDA stain.

T0 Ave #/ml T0 StDev #/ml TF Ave #/ml TF StDev #/ml

Control 267 14.8 38 25.6

Treatment 0 0 0 0



### Lugol’s Total Counts

- This approach commonly overestimates the abundances of live

organisms in this size class because all individuals in fixed samples are counted unless obviously

dead (significant damage to organism). Lugol’s staining alone does not distinguish live from

dead cells. * TF Control * TF Treated

Copepoda nauplii Harpacticoida

Spionid polychaete

Calanoida ( Acartia sp.)

Cyclopoida ( Oithona sp.)

Harpacticoida

NematodaMERC ER01

- 10

9 T0 Ave #/ml T0 StDev #/ml TF Ave #/ml TF StDev #/ml

Control Diatom 980 90

1,133 60

Treated Diatom 75 8 104 15

Dinoflagellate 250 19 12

Note: 2

, 300/ml = Average counts for T

- Final

- Treated, for picoplankton <10 μ m.



### Active Chlorophyll

- a

- Chlorophyll is used as ancillary data and as a general

presence/absence indicator of viable photosynthetic organisms (MDL = 0.56 μ g).

T0 Ave μ g/l T0 StDev μ g/l *TF Ave μ g/l *TF StDev μ g/l

Control 4.20 0.56 1.39 0.2 7

Treated 0.56 0.00 0.56 0.00

Regrowth Control 38.23 22.23

Regrowth Treated 0.56 0.00



### Live Microbes (cfu = colony forming units)

E. coli T0 Ave

cfu/100ml T0 StDev

cfu/100ml TF Ave

cfu/100ml TF StDev

cfu/100ml

Control 30.8 16.29 6.2 3.6 3

Treated 0 0 0 0



### Enterococci T0 Ave

cfu/100ml T0 StDev

cfu/100ml TF Ave

cfu/100ml TF StDev

cfu/100ml

Control 31.65 6.38 15.54 1.31

Treated 0 0 0 0



### V. cholerae

– No detectable culturable toxigenic Vibrio cholerae were detected in any

samples during any of the trials.

Heterotrophic

Bacteria T0 Ave

cfu/100ml T0 StDev

cfu/100ml TF Ave

cfu/100ml TF StDev

cfu/100ml

Control 517 495.26 144.9 81.53

Treated 0 0 0 0 Dominant species Type Other

Skeletonema costatum Diatom Chain

- forming

Thalassiosira sp. Diatom Chain

- forming

Oxyrrhis marina Dinoflage llateMERC ER01

- 10

10 

### Discharge Toxicity

Residual Chlorine Analysis

- Levels of residual c hlorine (or tota l residual oxidants

- TRO) were at or below the method detection limit (MDL) of 0.02 ppm in all TF t reated samples.

Acute Toxicity

- There were no statistically significant survival effects in any test for

either acutely tested species ( Mysid shrimp or S heepshead minnows).

Chronic Toxicity

- Results showed that ballast water discharge was not chronically toxic

to either mysids or sheepshead minnows. All samples, however, were chronically toxic to the

marine algal species Isochrysis galbana with a stati stically significant inhibition in growth in

treated ballast discharge compared to controls (see details under Summary of Results on page 4).MERC ER01

- 10

11 

### 8

. Results Trial 3 (STDN

- 02): 29 October

– 3 November 2009

Physical Parameters

TSS mg/l DOC mg/l POC mg/l

T0 Initial Ave StDev Ave StDev Ave StDev

Conditions 37.0 3.3 3.84 0.24 4.242 0.505

Temp o C Salinity psu Dis. Oxygen mg/l Turbidity NTU

Ave StDev Ave StDev Ave StDev Ave StDev

T0 Control 15.79 0.02 10.68 0.01 7.69 0.30 6.07 0.91

T 0 Treated 15.83 0.01 10.64 0.01 8.66 0.07 7.22 0.39

TF Control 15.69 0.00 10.67 0.00 6.94 0.03 1.69 0.04

TF Treated 15.67 0.01 10.73 0.00 8.61 0.01 4.12 0.12



### Live Organisms > 50 μ m

T0 Ave #/m 3 T 0 StDev #/m 3 T F Ave #/m 3 T F StDev #/m 3

Control 79,50 0 3,750 49,500 4,500

Treated No data 0.4 0.9

* Taxa listed i n order of abundance.



### Live Organisms 10

- 50 μ m

Vital Stain Results

- It is important to note that the specific approach used underestimates

the abundances of live organisms in this size class because it has been found that not all live

organisms reliab ly take up the CMFDA stain.

T0 Ave #/ml T0 StDev #/ml TF Ave #/ml TF StDev #/ml

Control 165 75.2 23 9.2

Treatment 0 0 0 0



### Lugol’s Total Counts

- This approach commonly overestimates the abundances of live

organisms in this size class because all in dividuals in fixed samples are counted unless obviously

dead (significant damage to organism). Lugol’s staining alone does not distinguish live from

dead cells. * TF Control * TF Treated

Copepoda nauplii Harpacticoida

Calanoida ( Acartia sp

. ) Spionid polychaete

Spionid polychaete

Rotifera

Harpacticoida

Tintinnid

Cirripedia naupliiMERC ER01

- 10

12 T0 Ave #/ml T0 StDev #/ml TF Ave #/ml TF StDev #/ml

Control Diatom 523 119

Dinoflagell ate 455 125

Treated Diatom 276 55 3

Dinoflagellate 169 40 19

Note: 3,0 00/ml = Average counts for T

- Final

- Treated, for picoplankton <10 μ m.



### Active Chlorophyll

- a

- Chlorophyll is used as ancillary data and as a general

presence/absence indicator of viable photosynthetic organisms (MDL = 0.56 μ g).

T0 Ave μ g/l T0 StDev μ g/l TF Ave μ g/l TF StDev μ g/l

Control 2.31 0.11 0.56 0.00

Treated 0.56 0.00 0.56 0.00

Regrowth Control 0.56 0.00

Regrowth Treated 0.56 0.00



### Live Microbes (cfu = colony forming units)

E. coli T0 Ave

cfu/100ml T0 StDev

cfu/ 100ml TF Ave

cfu/100ml TF StDev

cfu/100ml

Control 19.4 3.16 14.6 3.51

Treated 0 0 0 0



### Enterococci T0 Ave

cfu/100ml T0 StDev

cfu/100ml TF Ave

cfu/100ml TF StDev

cfu/100ml

Control 14.84 3.11 16.08 7.15

Treated 0 0 0 0



### V. cholerae

– No detectable cu lturable toxigenic Vibrio cholerae were detected in any

samples during any of the trials.

Heterotrophic

Bacteria T0 Ave

cfu/100ml T0 StDev

cfu/100ml TF Ave

cfu/100ml TF StDev

cfu/100ml

Control 1

, 970 214.48 425 118.39

Treated 0.1 0.33 0 0 Dominant species Type Other

Skeletonema costatum Diatom Chain

- forming

Thalassiosira sp. Diatom Cha in

- forming

Oxyrrhis marina

Gyrodinium estuarale Dinoflagellate

DinoflagellateMERC ER01

- 10

13 

### Discharge Toxicity

Residual Chlorine Analysis

- Levels of residual c hlorine (or total residual oxidants

- TRO) were at or below the method detection limit (MDL) of 0.02 ppm in all TF t reated samples.

Acute Toxicity

- There were no statistically significant survi val effects in any test for

either acutely tested species ( Mysid shrimp or Sheepshead minnows).

Chronic Toxicity

- Results showed that ballast water discharge was not chronically toxic

to either mysids or sheepshead minnows. All samples, however, were c hronically toxic to the

marine algal species Isochrysis galbana with a statistically significant inhibition in growth in

treated ballast discharge compared to controls (see details under Summary of Results on page 4).MERC ER01

- 10

14 

### 9

. Results Trial 4 (STDN

- 03): 5

– 10 November 2009

Physical Parameters

TSS mg/l DOC mg/l POC mg/l

T0 Initial Ave StDev Ave StDev Ave StDev

Conditions 29.4 6.1 3.65 0.10 3.070 0.536

Temp o C Salinity psu Dis. Oxygen mg/l Turbidity NTU

Ave StDev Ave StDev Ave StDev Ave StDev

T0 Control 15.14 0.03 10.46 0.01 7.28 0.09 7.79 0.74

T0 Treated 15.13 0.01 10.44 0.01 8.13 0.13 8.18 0.74

TF Control 14.50 0.02 10.38 0.00 6.81 0.02 1.71 0.03

TF Treated 14.51 0.01 10.49 0.00 8.15 0.01 4.92 0.17



### Live Organisms > 50 μ m

T 0 Ave #/m 3 T 0 StDev #/m 3 T F Ave #/m 3 T F StDev #/m 3

Control 84,000 1,875 40,500 3,000

Treated No data 0.8 0.8

* Taxa listed in order of abundance.



### Live Organisms 10

- 50 μ m

Vital Stain Results

- It is important to note that the specific approach used underestimates

the abundances of live organisms in this size class because it has been found that n ot all live

organisms reliably take up the CMFDA stain.

T0 Ave #/ml T0 StDev #/ml TF Ave #/ml TF StDev #/ml

Control * 28 3.2

Treatment * 0 0

* No data available at T0



### Lugol’s Total Counts

- This approach commonly overestimates the abundances of l ive

organisms in this size class because all individuals in fixed samples are counted unless obviously

dead (significant damage to organism). Lugol’s staining alone does not distinguish live from

dead cells. * TF Control * TF Treated

Copepoda nauplii Rotifera

Rotifera

Spionid polychaete

Calanoida ( Acartia sp.)

Cirripedia nauplii

Harpactico idaMERC ER01

- 10

15 T0 Ave #/ml T0 StDev #/ml TF Ave #/ml TF StD ev #/ml

Control Diatom 1,223 128

Dinoflagellate 1,463 163

Treated Diatom 253 36 25

Dinoflagellate 408 33 34

Note: 1,200/ml = Average counts for T

- Final

- Treated, for picoplankton <10 μ m.

Dominant species Type Other

Gyrodinium estuaral e

Glenodinium danicum Dinoflagellate

Dinoflagellate

Skeletonema costatum Diatom Chain

- forming

Oxyrrhis marina

Apedinella sp.

Chaetoceros sp. Dinoflagellate

Chrysophyceae

Diatom



### Active Chlorophyll

- a

- Chlorophyll is used as ancillary data and as a gen eral

presence/absence indicator of viable photosynthetic organisms (MDL = 0.56 μ g).

T0 Ave μ g/l T0 StDev μ g/l TF Ave μ g/l TF StDev μ g/l

Control 6.51 2.24 1.19 0.05

Treated 0.56 0.00 0.56 0.00

Regrowth Control 4.05 4.87

Regrowth Treated 0. 56 0.00



### Live Microbes (cfu = colony forming units)

E. coli T0 Ave

cfu/100ml T0 StDev

cfu/100ml TF Ave

cfu/100ml TF StDev

cfu/100ml

Control 28.8 2.39 2.8 2.77

Treated 0 0 0 0



### Enterococci T0 Ave

cfu/100ml T0 StDev

cfu/100ml TF Ave

cfu/100ml TF StD ev

cfu/100ml

Control 8.7 5.05 4.94 1.35

Treated 0 0 0 0



### V. cholerae

– No detectable culturable toxigenic Vibrio cholerae were detected in any

samples during any of the trials.

Heterotrophic

Bacteria T0 Ave

cfu/100ml T0 StDev

cfu/100ml TF Ave

cfu/10 0ml TF StDev

cfu/100ml

Control 712 376.94 287.3 166.88

Treated 0.1 0.33 0 0MERC ER01

- 10

16 

### Discharge Toxicity

Residual Chlorine Analysis

- Levels of residual c hlorine (or total residual oxidants

- TRO) were at or below the method detection limit (MDL) of 0.02 ppm in all TF t reated samples.

Acute Toxicity

- There were no statistically significant survival effects in any test for

either acutely tested species ( Mysid shrimp or Sheepshead minnows).

Chronic Toxicity

- Results showed that ballast water discharge was not chronically toxic

to either mysids or sheepshead minnows. All samples, however, were chronically toxic to the

marine algal species Isochrysis galbana with a statistically significant inhibition in growth in

treated ballast discharge compared to contro ls (see details under Summary of Results on page 4).MERC ER01

- 10

17 

### 10

. Results Trial 5 (STDN

- 04): 12

– 17 November 2009

Physical Parameters

TSS mg/l DOC mg/l POC mg/l

T0 Initial Ave StDev Ave StDev Ave StDev

Conditions 38.3 5.6 4.04 0.17 7.198 0.369

Temp o C Salinity psu Dis. Oxygen mg/l Turbidity NTU

Ave StDev Ave StDev Ave StDev Ave StDev

T0 Control 12.90 0.02 9.49 0.04 10.23 0.10 8.96 0.96

T0 Treated 12.95 0.02 9.48 0.01 10.29 0.03 10.00 0.69

TF Control 14.51 0.00 9.44 0.00 4.20 0.07 1.14 0.12

TF Treated 14.52 0.00 9.47 0.00 9.88 0.03 4.40 0.13



### Live Organisms > 50 μ m

T0 Ave #/m 3 T 0 StDev #/m 3 T F Ave #/m 3 T F StDev #/m 3

Control 92,250 7,125 25,125 1,875

Treated No data 0 na

* Taxa listed in order of abundance.



### Live Organisms 10

- 50 μ m

Vital Stain Results

- It is important to note that the specific approa ch used underestimates

the abundances of live organisms in this size class because it has been found that not all live

organisms reliably take up the CMFDA stain.

T0 Ave #/ml T0 StDev #/ml TF Ave #/ml TF StDev #/ml

Control 6,315 523.1 2,139 158.8

Tre atment 6 1.5 0 0



### Lugol’s Total Counts

- This approach commonly overestimates the abundances of live

organisms in this size class because all individuals in fixed samples are counted unless obviously

dead (significant damage to organism). Lugol’s staining alone does not distinguish live from

dead cells. * TF Control * TF Treated

Copepoda nauplii None live observed

Spionid polychaete

Calanoida ( Acartia sp.)

Rotifera

Cirripedia Nauplii

Bivalvia veligerMERC ER01

- 10

18 T0 Ave #/ml T0 StDev #/ml TF Ave #/ml TF StDev #/ml

Control Diatom 1

, 886 84 398 87

Dinoflagellate 16

, 125 1

, 715 3

, 733 100

Treated Diatom 1

, 818 272 236 107

Dinoflagellate 15

, 437 1

, 819 812 316

Note: 19

, 000/ml = Average counts for T

- Final

- Treated, for picoplankton <10 μ m.

Dominant species Type Other

Gyrodinium estuarale Dinoflagellate

Prorocentrum minimum Dinoflagellate

Oxyhrris marina Dinoflagellate

Apedinella sp. Chrysophyceae

Chae toceros Diatom

Skeletonema costatum Diatom Chain

- forming

Gymnodinium sp.

Scenedesmus quadricauda

Asterionella glacialis Dinoflagellate

Chlorophyceae

Diatom



### Active Chlorophyll

- a

- Chlorophyll is used as ancillary data and as a general

presence/absenc e indicator of viable photosynthetic organisms (MDL = 0.56 μ g).

T0 Ave μ g/l T0 StDev μ g/l TF Ave μ g/l TF StDev μ g/l

Control 78.28 9.87 12.50 0.86

Treated 0.56 0.00 0.56 0.00

Regrowth Control 41.86 15.67

Regrowth Treated 0.56 0.00



### Live Microbes (cfu = colony forming units)

E. coli T0 Ave

cfu/100ml T0 StDev

cfu/100ml TF Ave

cfu/100ml TF StDev

cfu/100ml

Control 74.4 40.3 6.8 3.19

Treated 0 0 0 0



### Enterococci T0 Ave

cfu/100ml T0 StDev

cfu/100ml TF Ave

cfu/100ml TF StDev

cfu/100ml

Co ntrol 18.8 6.46 3.7 0.89

Treated 1.33 0.58 0 0MERC ER01

- 10

19 

### V. cholerae

– No detectable culturable toxigenic Vibrio cholerae were detected in any

samples during any of the trials.

Heterotrophic

Bacteri a T0 Ave

cfu/100ml T0 StDev

cfu/100ml TF Ave

cfu/100ml TF St Dev

cfu/100ml

Control 2

, 692 408.14 2136 467

Treated 0.1 0.33 0 0



### Discharge Toxicity

Residual Chlorine Analysis

- Levels of residual c hlorine (or total residual oxidants

- TRO) were at or below the method detection limit (MDL) of 0.02 ppm in all TF t reated samples.

Acute Toxicity

- There were no statistically significant survival effects in any test for

either acutely tested species ( Mysid shrimp or Sheepshead minnows).

Chronic Toxicity

- Results showed that ballast water discharge was not chronic ally toxic

to either mysids or sheepshead minnows. All samples, however, were chronically toxic to the

marine algal species Isochrysis galbana with a statistically significant inhibition in growth in

treated ballast discharge compared to controls (see det ails under Summary of Results on page 4).MERC ER01

- 10

20 

### 11

. Chemical Analysis of Discharge

Summary table for chemical analyses of treated water upon discharge.

______________________________________________________________________________MERC ER01

- 10

21 

### 12 . Acknowledgments

T he MERC Testing Team included J. Barnes, M. Carroll, J. Choi, D. Fisher, C. Grim, B.

Haley, A. Huq, R. Kanzleiter, T. Mullady, G. Ruiz, G. Smith, D. Sparks, M. Tamburri. E.

Taviani, M. Wilkinson, L. Yonkos, and G. Ziegler. Test protocols were developed in

consultation with the Great Ships Initiative (GSI), Bundesamt fuer Seeschifffahrt und

Hydrographie (BSH) and Royal Netherlands Institute for Sea Research ( NIOZ )

. We wish thank

the crew of the MV Cape Washington who provided critical input and support on all aspects of

these trials, and the Maryland Port Administration and U.S. Maritime Administration for funding

and supporting this ballast water treatment evaluation.

June 7, 2010

June 7, 2010

Date Approved By: Dr. Mario Tamburri

MERC Executive Director

Date Approved By: Ross Kanzleiter

MERC Program Coordinator and

Chief Engineer