BENCH-SCALE TESTS OF THE NEWMAN ZONE OS BALLAST WATER DEOXYGENATION TREATMENT

This technical report presents findings from bench-scale tests evaluating the performance of the Newman Zone OS Ballast Water Deoxygenation Treatment, hereafter Newman Zone OS, developed by RNAS Remediation Products of Brooklyn Center, Minnesota, USA. Bench-scale evaluation began in November 2022 and ended in December 2022 at the Lake Superior Research Institute (LSRI) of the University of Wisconsin-Superior (UWS) in Superior, Wisconsin, USA. The treatment consisted of Newman Zone OS, a concentrated solution which uses a Vitamin C-based chemistry with added food grade buffers and catalysts, to quickly produce anaerobic water. Following a retention period, the anaerobic water is then rapidly oxygenated through the addition of an oxidizing agent (i.e., hydrogen peroxide), potentially destroying any remaining microorganisms. The technology would treat a ship’s ballast water as it is taken onboard the vessel and reoxygenate the water prior to being discharged to a receiving body of water in a Great Lakes port. Water-only, aquatic degradation experiments were conducted in lab water and amended lab water to assess whether or not organic carbon, suspended solids and UV-transmittance had an effect on the Newman Zone OS treatment. Due to elevated residual concentrations of hydrogen peroxide following reoxygenation of treated water, dose effectiveness and chronic residual toxicity testing were not conducted

BENCH-SCALE TECHNICAL REPORT

This technical report presents findings from bench-scale tests evaluating the ability of electron beam treatment to kill aquatic algae and zooplankton, specifically examining delayed mortality within 48 hours following treatment. This evaluation was the first attempt to assess the effect of electron beam treatment on hatch rates of Daphnia magna ephippia. Researchers from the Lake Superior Research Institute (LSRI) of the University of Wisconsin-Superior (UWS) in Superior, Wisconsin, USA traveled to the Fermi National Accelerator Laboratory, hereafter Fermilab, to conduct the bench-scale evaluation in May 2022. The test apparatus is a stationary copper electron beam accelerator that supplies a radiation dose expected to cause mortality in standard test organisms. Testing conducted in this evaluation was developed based on findings from earlier tests at Fermilab that demonstrated electron beam treatment was effective at causing complete mortality in E. coli at the lowest dose employed (1 kGy) and was effective at causing immediate mortality in D. magna and Eucyclops spp. at doses between 5 and 10 kGy (Polkinghorne et al., 2022). S. capricornutum was more resistant to immediate effects of electron beam treatment in initial tests (Polkinghorne et al., 2022). All experimental exposures were conducted at Fermilab. The irradiation process was conducted by Fermilab staff in the Illinois Accelerator Research Center’s (IARC’s) Accelerator Applications Development and Demonstration (A2D2) machine. Doses ranged from 1 to 50 kGy. Dose effectiveness testing was completed in lab water only, based on initial tests demonstrating minimal differences in treatment effect between high and low challenge water (i.e., low vs. high organic carbon content, suspended solids, and UV-transmittance). In samples examined 48 hours post-treatment, S. capricornutum had >99% mortality and Eucyclops spp. had 90% mortality at the 2 kGy dose. D. magna had 100% mortality at the lowest dose. No D. magna emerged from ephippia treated at any dose with the electron beam

RA2 P4 TECHNICAL REPORT

Understanding the impact of ballast water treatment and neutralization methods on receiving waters is critical to minimizing harmful effects of ballast discharge to the environment, specifically the freshwater of Lake Superior and surrounding Laurentian Great Lakes. While studies have been conducted to evaluate the effect of active substances and disinfection byproducts (DBP) produced by different Ballast Water Management Systems (BWMS) on marine waters, very little has been done to determine what impact these treatment systems could have on a highly utilized Great Lakes port like the Duluth-Superior Harbor, with its seasonally fluctuating organic carbon content and low percent transmittance. Throughout this project, the focus was on a BWMS that utilizes UV radiation combined with filtration as the primary treatment. A series of samples were collected at the Montreal Pier Ballast Treatment System Testing Facility, Superior, WI, during the evaluation of Great Lakes-compatible treatment systems. DBP concentrations and whole effluent toxicity tests were conducted to determine the toxicity of treated discharge water to living aquatic organisms representing three levels of the food web (plants, invertebrates, and vertebrates)

TESTS EXPOLORING THE EFFECTIVENESS OF ELECTRON BEAM TREATMENT FOR BALLAST WATER MANAGEMENT

This technical report presents findings from bench-scale tests evaluating the ability of electron beam treatment to inactivate aquatic organisms. This evaluation was the first attempt to assess the electron beam treatment as a potential treatment method for ballast water in the Laurentian Great Lakes. Researchers from the Lake Superior Research Institute (LSRI) of the University of Wisconsin-Superior (UWS) in Superior, Wisconsin, USA traveled to the Fermi National Accelerator Laboratory, hereafter Fermilab, to conduct the bench-scale evaluation in January 2022. The test apparatus is a stationary copper particle accelerator that supplies a radiation dose expected to cause mortality in standard test organisms. It has been previously demonstrated that electron beam treatment can cause direct damage by creating either single- or double-strand breaks in the DNA molecule beyond cell repair (Shehata, M.M.K. et al., 2011) or recovery (Lei, J. et al., 2020). All experimental exposures were conducted at Fermilab. The irradiation process was conducted by Fermilab staff in the Illinois Accelerator Research Center’s (IARC’s) Accelerator Applications Development and Demonstration (A2D2) machine. Doses ranged from 1 to 50 kGy. Dose effectiveness testing was completed in two water types using bacteria, algae, and zooplankton—Escherichia coli, Selenastrum capricornutum, Eucyclops spp., and Daphnia magna, respectively. The treatment caused complete mortality for E. coli in both water types at even the lowest dose employed. Eucyclops spp. and Daphnia magna also experienced complete mortality in both water types between 5 and 10 kGy. Selenastrum capricornutum was the most resistant to treatment effect in both water types, but did experience mortality at the highest doses. Algae examined a week after exposure for delayed mortality experienced high mortality rates at all doses

TEST EXPLORING THE EFFECTIVENESS OF THE ULTRASONICATOR INVASIVE SPECIES WATER TREATMNET FOR BALLAST WATER MANAGEMNET

This technical report presents findings from bench-scale evaluation of the ultrasonicator invasive species water treatment developed by UPIR LLC to inactivate aquatic organisms. This evaluation was the first attempt to assess this technology as a potential flow through or recirculating ballast water treatment method for use on ships within the Laurentian Great Lakes. The evaluation began in January 2022 and ended in February 2022. All analyses occurred at the Lake Superior Research Institute at the University of Wisconsin-Superior in Superior, Wisconsin, USA. According to the developer, the UPIR ultrasonicator invasive species water treatment system produces a unique cavitation and agitation environment within the treated water to destroy unwanted organisms. Biological effectiveness testing was completed with the bacteria, Escherichia coli and Enterococcus faecium, and algae, Selenastrum capricornutum, in dechlorinated laboratory water. The system was found to be ineffective in the treatment of bacteria and algae when the organisms were exposed to a single pass through the system at a flow rate of 6 gpm. However, when exposed to multiple passes through the system, the delayed mortality of bacteria increased, and effectiveness with algae remained low. Water quality parameters were minimally impacted by the ultrasonicator invasive species water treatment

EXPLORING OPTIONS FOR REAL-TIME WATER QUALITY MONITORING WITHIN GREAT LAKES PORTS

This technical report presents findings from the exploration of available environmental monitoring sensors, the piloting experience of long-term field deployment of the Proteus multiparameter sonde developed by Proteus Instruments, and the development of an accessible platform for viewing real-time data in support of uptake avoidance best management practices as defined by the U.S. Environmental Protection Agency’s 2013 Vessel General Permit. Project staff conducted a literature review and investigation of commercially available sensors and selected an instrument that consists of a multiparameter sonde—able to measure turbidity, chlorophyll-a, phycocyanin, and total coliform bacteria—, telemetry system, and solar panel. Long-term deployment of the Proteus began in April 2022 and ended in October 2022 at the Montreal Pier Testing Facility located on the Duluth-Superior Harbor in Superior, Wisconsin, USA. Data collected from the multiparameter sonde was transmitted and stored on Outpost.com and real-time data was made publicly available using Web AppBuilder for ArcGIS. This piloting experience has shown promise to provide real-time data in support of uptake avoidance practices

Evaluating a Most Probable Number Method for Assessing the Viability of Great Lakes Protists

To support type approval testing of ballast water management systems we evaluated freshwater viability assessments for protists from the Duluth-Superior harbor of western Lake Superior using the most probable number (MPN) method. Tests were performed using varying temperatures and growth media and were compared to standard microscopic methods for determining live organism densities. Tests were also performed focusing on growth series derived from harbor water, and during an actual land-based test of a treatment system being evaluated for efficacy. We determined that growth of protists during MPN experiments was especially favored under higher temperatures and a growth medium comprising a 50 % solution of Bold Modified Basal Media. This medium also supported the growth of the greatest number of protist taxa. Based on microscopic analysis of live protists use of a treatment system during land-based testing reduced protist densities from 554 – 3000 cells/mL in the untreated water to 12 – 52 cells/mL after treatment. Corresponding assessments using the MPN method estimated respective densities of 1651 – 6060 cells/mL and 0 – 2.8 cells/mL, indicating that MPN likely overestimated viable cells in ambient harbor samples while it underestimated cell densities in treated samples. As asserted in the MPN protocols we confirmed that MPN-estimated protist densities were similar to densities in the protist size class that includes only cells strictly 10 – 50 µm in minimum dimension; protist densities including cells <10 µm were much higher than MPN estimates. However, based on all evaluations of freshly acquired samples containing a wide range of starting densities there was no correlation between MPN- and microscopy-determined densities, regardless of size class. Based on all testing, certain protist taxa were poorly favored during MPN grow-out periods (e.g., the chrysophyte Mallomonas), while others (e.g., free-living centric diatoms) tended to thrive, though there was substantial variability in taxonomic selectivity among tests. These findings contribute important freshwater data to the field of efficacy testing of ballast water treatment systems

Ballast Eye

This technical report presents findings from freshwater verification tests evaluating the performance of the Satake Ballast Eye Viable Organism Analyzer VOA1000K compliance monitoring device, hereafter Ballast Eye. Ballast Eye was developed by Satake Corporation of Hiroshima, Japan. The compliance monitoring device evaluation began in August 2020 and ended in December 2020 at the Lake Superior Research Institute (LSRI) of the University of Wisconsin-Superior (UWS) in Superior, Wisconsin, USA. Ballast Eye estimates the number of viable organisms and associated risk based on IMO D-2 ballast water discharge standards in the ≥10 and <50 µm (nominally protists) and ≥50 µm (nominally zooplankton) regulated size classes by measuring the fluorescence pulse number from fluorescein diacetate (FDA) stained organisms within a water sample. The verification testing was composed of three phases. Phase I testing was completed in two water types with laboratory-cultured organisms in the two regulated size classes, utilizing the single-celled protist Haematococcus pluvialis and colonial protist Scenedesmus quadricauda, and the zooplankton Daphnia magna and Eucyclops spp. Phase II was completed using naturally occurring Great Lakes organisms in the Duluth-Superior Harbor of western Lake Superior in the two regulated size classes. Phase III testing was completed using Duluth-Superior harbor water and ambient organisms before and after treatment with a ballast water treatment technology (BWT) during three land-based trials. Data from all phases were analyzed for precision, accuracy, and reliability. Quantification/detection limits were calculated for Phase I data. Phase I testing showed Ballast Eye was able to accurately estimate the number of zooplankton in high and low transparency water, while protist concentrations were not accurately determined. Phase II testing showed Ballast Eye was unable to accurately estimate the number or risk of ambient zooplankton or protists in Duluth-Superior harbor water. Phase III testing showed that Ballast Eye was able to accurately classify risk of ambient zooplankton or protists within uptake and treated discharge samples collected during land-based ballast water treatment technology testing at the Montreal Pier Facility located on the Duluth-Superior harbor.LSRI-GWRC would like to thank Satake Corporation (Hiroshima, Japan) and MOL Techno-Trade Ltd. (Tokyo, Japan) for their application to our laboratory-based testing program and for providing Ballast Eye and the expendable supplies for analysis. Hiroki Ishizuki, Yoshinori Tazoe, and Shinya Fushida provided operational training support prior to the start of testing and were instrumental in helping to troubleshoot technical/operational issues that occurred during testing. This work was supported by a grant from the United States Department of Transportation Maritime Administration’s Maritime Environmental and Technical Assistance Program

FASTBALLAST

This technical report presents findings from bench-scale verification tests evaluating the performance of the FastBallast compliance monitoring device in freshwater. FastBallast was developed by Chelsea Technologies Ltd. of Surrey, UK. The evaluation of the FastBallast compliance monitoring device began in August 2020 and ended in December 2020 at the Lake Superior Research Institute (LSRI) of the University of Wisconsin-Superior (UWS) in Superior, Wisconsin, USA. The FastBallast device employs Single Turnover Active Fluorometry (STAF) to rapidly quantify living organisms in ballast water samples in the ≥10 µm and <50 µm (nominally protists) regulated size class, providing an indication of compliance or exceedance of the International Maritime Organization (IMO) International Convention for the Control and Management of Ships’ Ballast Water and Sediments Regulation D-2 Ballast Water Performance Standard (2004). Verification testing composed of three phases in which results using the FastBallast device were compared to results using microscopic methods. Phase I testing was completed in two water types with laboratory-cultured organisms in the protist regulated size class, utilizing the single-celled protist Haematococcus pluvialis and colonial protist Scenedesmus quadricauda. Phase II testing was completed using naturally occurring Great Lakes organisms in the Duluth-Superior Harbor of Lake Superior. Phase III testing was completed using Duluth-Superior harbor water an ambient organism before and after treatment with a ballast water treatment (BWT) technology during three land-based trials. Data from all phases were analyzed for precision, accuracy, and reliability. Quantification/detection limits were calculated using data from Phase I testing. Phase I testing showed that FastBallast was effective at quantifying single-celled protists but was less accurate at counting colonial protists. Increased turbidity and carbon content slightly impacted FastBallast results, however, both water types displayed strong correlations to microscopic counts. FastBallast results were lower than microscopic counts in all trials of Phase I. Phase II testing showed strong correlations between the FastBallast results and microscopic results of protists collected from the Duluth-Superior Harbor, however the counts reported by FastBallast were 4 to 10 times greater than the microscopic counts. Phase III testing showed FastBallast accurately measured uptake and treated discharge water from samples collected during a land-based BWT technology evaluation. FastBallast counts were more similar to the density of protist entities ≥10 µm in any dimension than they were to live density of individual protist cells comprising entities ≥10 µm in minimum dimension. The device was found to have minor operational issues but was found reliable for measuring organisms within the protist size class.LSRI-GWRC would like to thank Chelsea Technologies Ltd. (Surrey, UK) for their application to our laboratory-based testing program and for providing the FastBallast device and the expendable supplies for analysis. Mary Burkitt-Gray and Kevin Oxborough at Chelsea Technologies Ltd. provided operational training support prior to the start of testing and were also instrumental in helping to troubleshoot technical/operational issues that occurred during testing. This work was supported by a grant from the United States Department of Transportation Maritime Administration’s Maritime Environmental and Technical Assistance Program

BallastWISE

This technical report presents findings from freshwater verification tests evaluating the performance of the MicroWISE BallastWISE compliance monitoring device, hereafter BallastWISE. BallastWISE was developed by MicroWISE, located in Ebeltoft, Denmark. The compliance monitoring device evaluation began in August 2020 and ended in December 2020, at the Lake Superior Research Institute (LSRI) of the University of Wisconsin-Superior (UWS) in Superior, Wisconsin, USA. BallastWISE utilizes separate chambers to enumerate organisms in each of two regulated size classes, ≥10 and <50 µm (nominally protists) and ≥50 µm (nominally zooplankton). Cameras and optical chambers capture video and track motility through software analysis for the zooplankton size class. Fluorescence microscopy evaluates chlorophyll containing organisms in addition to motility tracking in the protist size class. The verification testing was composed of three phases. Phase I testing was completed in two water types with laboratory-cultured organisms in the two regulated size classes, utilizing the single-celled protist Haematococcus pluvialis and colonial protist Scenedesmus quadricauda, and the zooplankton Eucyclops spp. and Daphnia magna. Phase II testing was completed using naturally occurring Great Lakes organisms in the Duluth-Superior Harbor of Lake Superior in the two regulated size classes. Phase III testing was completed using Duluth-Superior Harbor water and ambient organisms before and after treatment with a ballast water treatment technology (BWT) during three land-based trials. Data from all phases were analyzed for precision, accuracy, and reliability. Quantification/detection limits were also calculated from Phase I data . Phase I testing showed BallastWISE was effective at quantifying single-celled protists to within about 20% of the microscopic counts, but undercounted colonial protists. Colonial protist entity counts were close to microscopic entity counts suggesting that individuals within the colonies were not resolved. High total suspended solids (TSS) and (DOC) may slightly reduce BallastWISE sensitivity to protists. BallastWISE overcounted zooplankton in both species tested in both high and low TSS/DOC by between 150% and 420%. Phase II testing from the Duluth-Superior Harbor showed BallastWISE counts of natural assemblages of protists strictly in the ≥10 and <50 μm size class to be slightly below microscopic counts by about 35% and with high precision. Zooplankton were overestimated by BallastWISE by roughly 40% and with considerably more variation compared to microscopic counts. Phase III testing showed low BallastWISE accuracy and precision in untreated protist and zooplankton samples. This may have been caused by organism densities higher than the device’s effective upper limit of detection in the zooplankton samples, but further investigation would be needed to determine the cause of low accuracy and precision in protist analysis. BallastWISE accurately measured treated protist samples as 0 cells/mL in agreement with strict microscopic counts, but overcounted treated zooplankton samples in 2 out of 3 tests, possibly due to the method of treatment. A number of operational issues made enumeration of zooplankton unreliable, but improvements (e.g., software updates, guidance on device operation) from the developer over the period of this assessment have already improved performance. BallastWISE shows promise as a useful device for detecting and measuring protists and zooplankton in the Great Lakes as additional improvements are made.LSRI-GWRC would like to thank MicroWISE (Ebeltoft, Denmark) for their application to our laboratory-based testing program and for providing the BallastWISE system and the expendable supplies for analysis. Pia Haecky, MicroWISE CEO, and Nicholas Blackburn, MicroWISE Software Development, provided operational training support prior to the start of testing and were also instrumental in helping to troubleshoot technical/operational issues that occurred during testing. This work was supported by the United States Maritime Administration (United States Department of Transportation; Washington, D.C)