## Land

- Based Evaluations of the Maritime Solutions, Inc.

Ballast Water Treatment System

Maritime(Environmental(Resource(Center



## 20 November 2009

c/o Chesapeake Biological Laboratory

PO Box 38, Solomons, Maryland 20688, USA

U MCES Technical Repor t Series: Ref. No. [UMCES]CBL 09

- 138MERC ER0 2

- 09

i 

## Notic e

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

, and flag states with an independe nt

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 Manageme nt of Ships’ Ballast

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

Water Management Systems (G8)

. The evaluation was conducted under specific, predetermined,

agreed

- upon protocols, criteria, and quality assurance pro cedures to assess the treatment system's

performance.

MERC does not label 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

technology'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 MER C Advisory Board do not provide certification for technologies, or

certify 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 wil l

always operate at the levels verified. MERC does guarantee the levels 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 Adviso ry B oard and provided to MSI and

MERC funding agencies prior 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, tamburr i@umces.edu.MERC ER0 2

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ii 

### Table of Contents

Page No.

1. MERC Background and Objectives

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

2

. Description of the MSI Ballast Water Treatment System

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

3

. Summary of IMO Standards

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

4

. Summary of Test Protocol s

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

5

. Summary of Results

................................ ................................ ................................ ....... 3

6

. Results Trial 1

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

7

. Resul ts Trial 2

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

8

. Results Trial 3

................................ ................................ ................................ ................ 9

9

. Results Trial 4

................................ ................................ ................................ .............. 11

10

. Results Trial 5

................................ ................................ ................................ .............. 13

11

. Acknowledgements

................................ ................................ ................................ ...... 16MERC ER02

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### 1

. MERC Background and Objectives

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

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

facing the international 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 management

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 reliabl e 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 Scien ce (CBL/UMCES), U.S. Maritime Administration

(MARAD), Smithsonian Environmental Research 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 shipb oard implementation and operations.

This report describe s the MERC evaluation of the Maritime Solutions Inc. ( MSI) Ballast

Water Treatment System through objective and quality assured land

- based testing ( dockside at a

flow rate of 200 m 3 

### /hr). The goal o f this evaluation was to provide shipping lines, regulators,

classification societies, and flag states with an independent and credible assessment of treatment

performance under realistic conditions. Therefore, the data and information on performance

char acteristics 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 Shi ps' Bal last Water and

Sediments (2004 ) , Regulation D

- 2 Ballast Water Performance Standard

.

2

. Description of the MSI Ballast Water Management System

The MSI Ballast Water Treatment System (UV), patent pending, is designed to meet

IMO D2 discharge standa rds for low to moderate fl ow rate shipboard applications. The

treatment utilizes Ballast Safe Filtration Company's proprietary self

- cleaning filter design to

separate the components of the influent ballast water in its primary treatment stage. As a

prima ry treatment, the filter is intended to remove silt and s ediment, organic materials and

organisms > 25 μ m (nominal) in size from the influent ballast water . The remaining filtered

water stream is then treated by Hanovia UV In

- Line UV units in a secondary treatment stage to

address the remaining organisms < 25 μ m

. In addition to its two treatment stages, the MSI

System (UV) has been fully integrated and is controlled by a proprietary ABB Instrumentation

water quality monitoring and flow control system desi gned to assure and document effective

treatment by continuously monitoring a number of water quality parameters including total

suspended solids (TSS) and UV transmission rate, automatically adjusting flow rate to assure

proper treatment, and recording all required water quality and system operation parameters.

Because the treatment system was a prototype, it was operated at all times by members of the

MSI or ABB staff. All evaluation system equipment and instrumentation, excluding the

treatment system, w as operated by MERC personnel.MERC ER02

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2 

### 3. Summary of IMO Standards

This evalua tion was designed to determine if the MSI system can meet IMO D2 standards

in accordance with both the IMO Guidelines for Approval of Ballast Water Management Systems

(G8)

. The IMO C onvention performance standard states that ships must 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 than 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 co li

, less than 250 cfu per 100 ml ;

3. I ntestinal Enterococci

, 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 qualit y control / quality assurance procedures for this

MERC evaluation, please refer to the Test Plan s for the Performance Evaluation of the Maritime

solutions, Inc. Ballast Water Treatment System (April 2008 and March 2009 ), available for

download at www.marit ime

- enviro.org

. The protocols described below are based upon the IMO G8 guidelines and the U.S. Coast

Guard supported ETV protocols under development. Any deviation from IMO G8 or ETV were

explained and justified in the Test Plan. MERC evaluated the bio logical efficacy of the MSI

ballast water treatment system onboard the MARAD 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 00m 3 

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

### /hr and delivered

simultaneously to either a “control” (untreated) ballast tank or a “treated” (p assing first through

the MSI system) ballast tank. These two tanks 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 control and test tanks were collected, filtered, and analyzed for the water quality

parameters of particul ate organic carbon (POC) , dissolved organic carbon (DOC

– only in 2009 )

, and total suspended solids (TSS).

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 sequenti al 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 MSI system during filling of test tank ( T0

Treated ), (C) during discharg e 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 quantitative analyses under a

dissecting microscope . T he proportion and total concentration of live versus dead organisms wasMERC ER02

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3 

### determined using standard movement and response

- to

- stimuli techniques within one hour of

collecting the individual samples. Zooplankton samples were also fixed and returned to the

laboratory for additional taxonomic evaluations

. Ten liters of unfiltered water from each mesocosm were also collected immediately after

filling, to deter mine 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 colu mns 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 favora ble 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 phytop lankton.

Additional 1

- liter 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 cho lerae

. Total heterotrophic

bacteria were enumerated by spread plate method using NWRI agar according to Standards

Methods for the Examination of Water and Wastewater . The presence and abundance of E. coli

and intestinal Enterococci was determined using a commercially available chromogenic substrate

method (IDEXX Laboratories, Inc.; Noble et al. 2003) and 10 ml and 100 ml water sample

aliquots. Additionally, concentrations of culturable E. coli and intestinal Enterococci were determined using a standard US EPA method, namely, membrane filtration on mTEC agar ( E.

coli ) (1 ml, 10 ml and 100 ml) and mEA agar ( Enterococcus ) (10 ml and 100 ml). Abundance of

total and toxigenic V. cholerae were calculated by filtration and selection on TCBS agar and

enumerated us ing species

- specific RNA colony blot (500 μ l to 1 ml) and ctxA DNA colony blot

(1

- 10 ml). Viable toxigenic V. cholerae was assayed with a commercial DFA kit specific for

serogroup O1 (New Horizons Diagnostics) using monoclonal antibodies tagged with fluorescein

isothiocyanate (FITC) (Hasan et al. 1994).

5. Summary of Results *

The MSI Ballast Water Treatment System dramatically reduced the numbers of live

organisms in ballast water during MERC land

- based testing in the Port of Baltimore. For most

biological categories, the treatment system consi stently met IMO D2 discharge standards.

However, while large reductions in the abundances of organisms greater than 50 μ m were found,

the T

- Final average numbers of live zooplankton (> 50 μ m) were not consistently below 10 μ m

in treated water after the 5

- day holding time. T he MSI system also experienced minor

mechanical failures at different points during the testing process (broken in

- line sensor housing

and tripping of a circuit breaker) . However, the failures were easily resolved with small

modificati ons or repairs

.

* Complete datasets and further performance information is available upon request.MERC ER02

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4 

### 6. Results Trial 1 (MSI

- 05

- 08): 18

- 23 September 2008

Physical Parameters

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

T0 Initial Ave StDev Ave StDev Ave StDev

Conditi ons 5.0 0.5

- - 1.123 0.129

Note: Did not analyze for DOC in 2008

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

Ave StDev Ave StDev Ave StDev Ave StDev

T0 Control 24.5 0.0 11.7 0.0 3.9 .5

- -

T0 Treated 24.6 0.2 11.6 0.1 3.7 0.3

- -

TF Con trol 24.5 0.1 11.7 0.0 3.8 0.1

- -

TF Treated 24.7 0.1 11.6 0.1 3.3 0.1

- -

*Data not recorded in 2008



### 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 31

, 175 4

, 238 55

, 050 10

, 828

Treated 107 4.16 6 1.87

* Taxa listed in order of abundance.



### Live Organisms 10

- 50 μ m

V

ital S tain Re sults

Note: the CMFDA stain technique commonly underestimates the abundance of live

organisms in this size class because not all live organisms reliably take up this stain.

T0 Ave #/ ml T 0 StDev #/ ml T F Ave #/ ml T F StDev #/ ml

Control 530.7 122.5 11. 2 2.3

Treated 7.3 0.6 0.6 0.7 * TF Cont rol * TF Treated

Nauplii (copepod) Nematoda

Copepoda, Harpacticoida nauplii (copepod)

Copepoda, Cyclopoida Turbellaria

Copepoda, Calanoida

Nematoda

RotiferaMERC ER02

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5 

### 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).

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

Control Diatom 7

, 734 1

, 280 3

, 063 457

Dinoflagellate 578 223 134 49

Treated Diatom * 149 52

Dinoflagellate * 6 8

* Not yet ana lyzed

Note: 454/ml = Average counts for T

- Final

- Treated, for picoplank ton <10 μ m.

Dominant species Type

Leptocylindrus minimum Diatom

Gyrodinium estuariale Dino

Eutreptia veridis Euglenid Flagellate

Thalassiosira sp. Diatom

Amphora sp. Diatom

Gonyaulux sp. (danicum?) Dino

Gyrodinium spirale Dino

Amphidinium sphenoide s Dino

misc. Tinntinnids Ciliates



### 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

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

Control 23.70 5.39 3.45 2.19

Treated *

- 0.46 0.05

Regrowth Control

- - 11.37 5.33

Regrowth Treated

- - 0.04 0.01

*No initial treated data collected



### Live Indicator Pathogens

CFU = colony forming u nits



### E. coli

T0 Ave

cfu / 100ml T 0 StDev

cfu/100ml T F Av e

cfu/100ml T F StDev

cfu / 100ml

Control 19.4 12.7 0.6 0.89

Treated *

- 0 0

*No initial treated data collectedMERC ER02

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6 

### Enterococci

T0 Ave

cfu / 100ml T 0 StDev

cfu/100ml T F Ave

cfu/100ml T F StDev

cfu / 100ml

Control 5.66

- 13.58

-

Treated *

- 0

-

*No initial t reated data collected

. No StDev reported

.



### V. cholerae

No detectable culturable toxigenic Vibrio cholerae were detected in any samples during

any of the trials.

Total Heterotrophic Bacteria

T0 Ave

cfu / 100ml T 0 StDev

cfu/100ml T F Ave

cfu/100ml T F StDev

cfu / 100ml

Control 31,833 7,591 98,288 90,638

Treated *

- 6,936 6,586

*No initial treated data collected



### 7. Results Trial 2: (MSI

- 06

- 08) 2

- 7 Oct 2008

Physical Parameters

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

T0 Initial Ave StDev Ave StDev Ave StDev

Condi tions 7.4 0.4

- - 0.474 0.042

Note: Did not analyze for DOC in 2008

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

Ave StDev Ave StDev Ave StDev Ave StDev

T0 Control 22.8 0.2 14.9 0.3 1.4 1.0

- -

T0 Treated 22.6 0.5 14.4 0.1 0.9 0.1

- -

TF Control 22.1 0.1 15.7 0.1 3.8 0.2

- -

TF Treated 22.4 0.1 14.7 0.1 4.5 0.8

- -

*Data not recorded



### 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 61

, 100 8

, 600 30

, 625 6

, 027

Treated 1370 120 2170 523

Note: Increa se in numbers for T

- Final treated counts likely due to hatch

- outs of the egg bearing poicelistome

copepod.MERC ER02

- 09

7 * Taxa listed in order of abundance.



### Live Organisms 10

- 50 μ m

V

ital S tain Results

Note: the CMFDA stain technique commonly underestimates the abundance of live

organ isms in this size class because not all live organisms reliably take up this stain.

T0 Ave #/ ml T 0 StDev #/ ml T F Ave #/ ml T F StDev #/ ml

Control 54.7 6.5 2.6 0.3

Treated 1.5 0.1 0.5 0.3



### Total Counts

This approach commonly overestimates the abundan ces of live organisms in this size

class because all individuals in fixed samples are counted unless obviously dead (significant

damage to organism).

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

Control Diatom 849 176 520 161

Dinoflagellate 1 16 39 18 12

Treated Diatom * 83 16

Dinoflagellate * 4 4

*Not yet analyzed

Note: 365 #/ml = Average counts for T

- Final

- Treated, for picoplankton <10 μ m.

Dominant species Type

Leptocylindrus minimum Diatom

Gyrodinium estuariale Dino

Chaetoce ros socialis Diatom

Thalassiosira sp. Diatom

Amphora sp. Diatom

Navicula didyma Diatom

misc. Pennates Diatom

misc. Tinntinnids Ciliate * TF Control * TF Treated

nauplii (copepod) nauplii (copepod)

Polychaeta (larvae) Copepoda, Cyclopoida

Copepoda, Cyclopoida Copepoda, Poicelistomo ida

Copepoda, Poicelistomoida

Copepoda, Calanoida

Rotifera

BivalviaMERC ER02

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8 

### Active Chlorophyll

- a Chlorophyll is used as ancillary data and as a general presence/ absence indicator of

viab le photosynthetic organism. MDL = 0.56 μ g

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

Control 1.52 0.10 

### 0.35 0.04

Treated *

- 

### 0.22 0.02

Regrowth Control

- - 

### 4.39 0.90

Regrowth Treated

- - 

### 0.01 0.00

*No initial treated data collected



### Live Indicator Pathogens

CFU = colony forming units



### E. coli

T0 Ave

cfu/100ml T0 StDev

cfu/100ml TF Ave

cfu/100ml TF StDev

cfu/100ml

Control 10.6 13.56 1.4 1.52

Treated *

- 0 0

*No initial treated data collected



### Enterococci

T0 Ave

cfu/100ml T 0 StDev

cfu/100ml TF Ave

cfu/100ml TF StDev

cfu/100ml

Control 24.84

- 14.28

-

Treated *

- 0

-

*No initial treated data collected

. No StDev reported

.



### V. cholerae

No detectable culturable toxigenic Vibrio cholerae were detected in any samples during

a ny of the trials.

Total Heterotrophic Bacteria

T0 Ave

cfu / 100ml T 0 StDev

cfu/100ml T F Ave

cfu/100ml T F StDev

cfu / 100ml

Control 12,150 6,954 11,920 6,633

Treated *

- 7,860 8,308

*No initial treated data collectedMERC ER02

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9 

### 8

. Results Trial 3: (MSI 01

- 09) 1 6

- 21 April 2009

Physical Parameters

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

T0 Initial Ave StDev Ave StDev Ave StDev

Conditions 7.33 0.50 3.62 0.24 0.84 0.07

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

Ave StDev Ave StDev Ave StDev Ave StDev

T0 C ontrol 10.3 0.3 9.8 0.1 8.4 0.1 4.4 0.2

T0 Treated 10.4 0.1 9.8 0.0 8.4 0.5 4.2 0.1

TF Control 13.1 0.1 10.0 0.1 8.6 0.1 1.6 0.1

TF Treated 13.3 0.1 9.3 0.1 9.5 0.1 3.2 0.1



### 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 179

, 000 49

, 518 127

, 933 21

, 624

Treated 200 288 8 5

*Taxa listed in order of abundance.



### Live Organisms 10

- 50 μ m

V

ital S tain Results

Note: the CMFDA stain technique commonly underestimates the abundance of live

organisms in this size class because not all live organisms reliably take up this stain.

T0 Ave #/ ml T 0 StDev #/ ml T F Ave #/ ml T F StDev #/ ml

Control 72.7 38.5 22.3 3.7

Treated 10 4 0.4 0.1 * TF Control * TF Treated

Calanoida ( Eurytemora affinis ) Calanoida

Copepoda nauplii Harpacticoida

various eggs Copepod nauplii

Cirrepedia nauplii

Rotifera

Ploychaeata (Spionidae)

Harpac ticoidaMERC ER02

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10 

### 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).

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

Control Diatom 41 17 48 18

Dinoflagellate 21 12 16 4

Treated Diatom 22 15 3 2

Dinoflagellate 10 10 1 1

N ote: 2552 #/ml = Average counts for T

- Final

- Treated, for picoplankton <10 μ m.

Dominant species Type

Amphora sp. Diatom

Gyrodinium estuariale Dino

Thalassiosira sp. Diatom

Heterocapsa rotundatum Dino



### Active Chlorophyll

- a Chlorophyll is used as ancill ary data and as a general presence/ absence indicator of

viable photosynthetic organism. MDL = 0.56 μ g

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

Control 6.20 0.51 2.99 0.38

Treated 5.25 0.46 2.44 0.14

Regrowth Control

- - 60.00 27.3 1

Regrowth Treated

- - 0.07 0.09



### Live Indicator Pathogens

CFU = colony forming uni ts



### E. coli

T0 Ave

cfu / 100ml T 0 StDev

cfu/100ml T F Ave

cfu/100ml T F StDev

cfu / 100ml

Control 22.40 2.70 0.20 0.45

Treated 0.00 0.00 0.00 0.00



### Enterococci

T0 Ave

cf

u / 100ml T 0 StDev

cfu/100ml T F Ave

cfu/100ml T F StDev

cfu / 100ml

Control 21.14 8.43 10.12 4.93

Treated 0.2 0.45 0.00 0.00



### V. cholerae

No detectable culturable toxigenic Vibrio cholerae were detected in any samples during

any of the trials.MERC ER02

- 09

11 

### Total Heterot rophic Bacteria

T0 Ave

cfu / 100ml T 0 StDev

cfu/100ml T F Ave

cfu/100ml T F StDev

cfu / 100ml

Control 1

, 593.00 4

, 796.51 556.00 1

, 672.98

Treated 75.30 22.74 162.00 39.25



### 9. Trial 4 (MSI

- 02

- 09 ) 23

- 28 April 2009

Physical Parameters

TSS mg/l DOC mg/l PO C mg/l

T0 Initial Ave StDev Ave StDev Ave StDev

Conditions 30.13 3.69 3.57 0.34 1.10 0.04

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

Ave StDev Ave StDev Ave StDev Ave StDev

T0 Control 11.3 0.1 10.4 0.2 6.4 0.3 8.6 0.3

T0 Treated 11.5 0 .1 10.5 0.3 6.4 0.3 8.8 0.3

TF Control 13.8 0.1 10.5 0.0 7.4 0.1 1.3 0.4

TF Treated 13.8 0.1 10.3 0.1 8.4 0.1 5.7 0.2



### 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 117

, 000 32

, 786 47

, 167 3

, 329

Treated 158 53 * 5 16 103

*Increase in treatment numbers due to an increase in newly hatched cirrepedia nauplii.

* Taxa listed in order of abundance. * TF Control * TF Treated

Calanoida ( Eurytemora affinis ) Calanoida

Copepoda nauplii Cirrepedia nauplii

Cirrepedia nauplii Copepoda nauplii

Gastropod larvae Ro tifera

various eggsMERC ER02

- 09

12 

### Live Organisms 10

- 50 μ m

V

ital S tain Results

Note: the CMFDA stain technique commonly underestimates the abundance of live

organisms in this size class because not all live organ isms reliably take up this stain.

T0 Ave #/ ml T 0 StDev #/ ml T F Ave #/ ml T F StDev #/ ml

Control 1

, 259 88 68 20

Treated 91 26 12 5



### Total Counts

This approach commonly overestimates the abundances of live organisms in this size

class because all indi viduals in fixed samples are counted unless obviously dead (significant

damage to organism).

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

Control Diatom 764 176 211 80

Dinoflagellate 255 47 106 24

Treated Diatom 324 137 38 6

Dinofla gellate 128 46 18 2

Note: 2489 #/ml = Average counts for T

- Final

- Treated, for picoplankton <10 μ m.

Dominant species Type

Heterocapsa rotundatum Dino

Gyrodinium estuariale Dino

Prorocentrum minimum * Dino

Chaetoceros sp. Diatom

misc. Copepod nauplii

Microcystis sp.* (4

- 5 μ m ) Cyanobacteria

* Know harmful algal species.



### 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

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

Control 10.22 0.44 1.11 0.17

Treated 8.76 1.41 5.56 0.25

Regrowth Control

- - 16.39 5.58

Regrowth Treated

- - 0.12 0.03MERC ER02

- 09

13 

### Live Indicator Pathogens

CFU = colony forming units



### E. coli

T0 Ave

cfu / 100ml T 0 StDev

cfu/100 ml T F Ave

cfu/100ml T F StDev

cfu / 100ml

Control 19.40 26.57 1.40 0.89

Treated 0.00 0.00 0.00 0.00



### Enterococci

T0 Ave

cfu / 100ml T 0 StDev

cfu/100ml T F Ave

cfu/100ml T F StDev

cfu / 100ml

Control 61.44 11.48 13.72 1.94

Treated 0 0.00 0.00 0.00



### V. chol erae No detectable culturable toxigenic Vibrio cholerae were detected in any samples during

any of the trials.

Total Heterotrophic Bacteria

T0 Ave

cfu / 100ml T 0 StDev

cfu/100ml T F Ave

cfu/100ml T F StDev

cfu / 100ml

Control 533.00 1

, 624.82 271.25 947.93

Treated 92.80 18.14 116.88 24.22



### 10. Trial 5 (real trial MSI

- 03

- 09) 30 April

– 5 May 2009

Physical Parameters

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

T0 Initial Ave StDev Ave StDev Ave StDev

Conditions 9.27 5.66 3.95 0.10 1.22 0.17

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

Ave StDev Ave StDev Ave StDev Ave StDev

T0 Control 17.32 0.03 7.41 0.00 8.86 0.13 2.8 0.1

T0 Treated 17.51 0.00 7.34 0.00 9.13 0.01 2.8 0.1

TF Control 15.73 0.01 7.49 0.00 6.77 0.02 1.3 0.1

TF Treated 15.81 0.00 7.33 0. 00 7.33 0.09 1.6 0.1MERC ER02

- 09

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 113

, 500 9

, 674 51

, 500 4

, 515

Treated 38 7 * 27 11

* All live individuals found were Acartia

.

* Taxa listed in order of abundance.



### Live Organisms 10

- 50 μ m

V

ital S tain Results

Note: the CMFDA stain technique commonly underestimates the abundance of live

organisms in this size class because not all live organisms reliably take up this stain.

T0 Ave #/ ml T 0 StDev #/ ml T F Ave #/ ml T F StDev #/ m l

Control 1,944 174 280 35

Treated 149 27 11 3



### 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 orga nism).

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

Control Diatom 417 186 281 47

Dinoflagellate 799 173 202 40

Treated Diatom 134 45 21 0

Dinoflagellate 285 66 17 7

Note: 1547 #/ml = Average counts for T

- Final

- Treated, for picopl ankton <10 μ m. * TF Control * TF Treated

Calanoida ( Acartia tonsa ) Copepoda nauplii

Cirrepedia nauplii Cirrepedia nauplii

Copepoda nauplii Calanoida ( Acartia tonsa)

Polychaeta (Spionidae) Polychaeta (Spionidae)

Harpacticoida Harpacticoida

various eggsMERC ER02

- 09

15 * Know harmful algal species.



### 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

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

Control 23.10 1.84 2.21 0.19

Treated 18.80 1.39 7.23 0.10

Regrowth Control

- - 11.75 5.53

Regrowth Treated

- - 0.17 0.12



### Live Indicator Pathogens

CFU = colony forming units



### E. coli

T0 Ave

cfu / 100ml T 0 StDev

cfu/100ml T F Ave

cf

u/100ml T F StDev

cfu / 100ml

Control 2.20 1.10 1.00 1.71

Treated 0.00 0.00 0.00 0.00



### Enterococci

T0 Ave cfu

cfu / 100ml T 0 StDev

cfu/100ml T F Ave

cfu /100ml T F StDev

cfu / 100ml

Control 2.62 1.17 3.9 0.84

Treated 0 0.00 0.00 0.00



### V. cholerae

No dete ctable culturable toxigenic Vibrio cholerae were detected in any samples during

any of the trials.

Total Heterotrophic Bacteria

T0 Ave

cfu / 100ml T 0 StDev

cfu/100ml T F Ave

cfu/100ml T F StDev

cfu / 100ml

Control 208.75 716.73 192.20 33.08

Treated 116.88 24.22 145.30 44.39 Dominant species Type

Gyrodinium estuariale Dino

Prorocentrum minimum * Dino

Heterocapsa rotundatum Dino

Gymnodinium sp. Dino

Gyrodinium sp. Dino

Amphidinium sp. Dino

Amphora sp. Diatom

Microcystis sp.* (4

- 5 μ m) Cyanobacteria

misc. T inntinnids CiliatesMERC ER02

- 09

16 

### 11 . Acknowledgments

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.C. Wilkinson, L. Yonkos, and G. Z iegler. We wish thank the crew of the M/V Cape

Washington who provided critical input o n all aspects of these trials, and the Maryland Port

Administration and U.S. Maritime Administration for funding and supporting this ballast water

treatment evaluation.

November 20 , 2009

November 20 , 2009



### Date Approved By: Dr. Mario Tamburri

MERC Executive Director

Date Approved By: Ross Kanzleiter

MERC Progra m Coordinator and

Chief Engineer