RA2 P3 TECHNICAL REPORT

United States Coast Guard and International Maritime Organization rules developed to minimize the spread of aquatic nuisance species (ANS) in the ballast of commercial ships regulate indicator microbes, protists and zooplankton, determining protist survival using staining techniques and zooplankton survival based on organism motility. The density of eggs and resting stages in ballast discharge are not regulated when assessing the effectiveness of ballast water treatment systems although they can be present in meaningful densities, potentially increasing the likelihood of ANS spread. To date, viability of freshwater zooplankton resting stages has been determined by grow out methods which are time consuming and use light, temperature and media regimens that may not be appropriate for all species. Stains have been used to assess viability of freshwater protists in the regulated size class, as well as salt water zooplankton resting stages. This paper examines the effectiveness of two stains, aniline blue and TO-PRO-1 Iodide, at assessing viability of Daphnia magna ephippia and Brachionus calyciflorus cysts

Kria Ionizer Test Report

This technical report presents findings from bench-scale tests evaluating the performance of the Kria Ionizer Superoxide Generator Model DG-100, hereafter Kria Ionizer, developed by EcoUSA, LLC of Denver, Colorado, United States of America. This evaluation was the first to assess the Kria Ionizer as a potential, in-tank, recirculating ballast water treatment method for the Laurentian Great Lakes. The evaluation began in May 2019 and ended in September 2019 at the Lake Superior Research Institute (LSRI) of the University of Wisconsin-Superior (UWS) in Superior, Wisconsin, USA. The treatment technology uses atmospheric oxygen to create superoxide (O2-). According to the developer, the injection of this superoxide into water not only creates a supersaturated oxygenated environment but can also promote cavitation and microbubble reactions upon discharge. The system can be used for oxygenation of fish farms and for remediation of water bodies containing harmful cyanobacteria, wastewater bacteria, and organic contaminants. Previous testing has been completed in the lab and field setting for these previously mentioned applications, but the testing described herein is the first testing evaluating Kria Ionizer’s potential application for ballast water treatment. The ability of the Kria Ionizer to increase dissolved oxygen and oxidation-reduction potential in a 1000-L treatment tank was tested at two water temperatures (~10°C and ~25°C) using both dechlorinated laboratory water and amended dechlorinated laboratory water. Water-only experiments were conducted and showed that dissolved oxygen increased with time in both dechlorinated laboratory water and amended dechlorinated laboratory water at both temperatures. Dissolved oxygen in ~10°C water had higher concentrations than in ~25°C water. ORP measurements conducted during testing with both water temperatures and types did not show a high degree of correlation in ORP values with increased treatment time. During treatment it was also determined that ozone and hydrogen peroxide were not produced as by-products. Biological effectiveness testing was completed with the algae, Selenastrum capricornutum and bacteria, Escherichia coli and Enterococcus faecium, in dechlorinated laboratory water. The system was found to be ineffective in the treatment of algae and bacteria when operated for approximately 30 minutes, and ineffective in treatment of algae when operated for 6 hours. However, when operated for 24 hours results suggest that the Kria Ionizer was effective against bacteria.LSRI-GWRC would like to thank EcoUSA, LLC (Denver, Colorado, USA) for their application to our laboratory-based testing program and for providing the Kria Ionizer Model DG-100 ballast water treatment (BWT) testing unit. Mike Mangham of EcoUSA, LLC provided installation and operational training and support prior to the start of and during testing. The results and conclusions contained in this report reflect the scientific data and opinions of the LSRI-GWRC research team. The technology developer did not review or provide comments on this report. This work was supported by the United States Maritime Administration (United States Department of Transportation, Washington, D. C.) and the United States Environmental Protection Agency’s Great Lakes Restoration Initiative

Tests of the Amglo Kemlite Bench-scale Ballast Water Management System

This technical report presents findings from bench-scale tests evaluating the performance of the Amglo Kemlite Bench-Scale Ballast Water Management System (BWMS), hereafter Amglo Kemlite BWMS, developed by Amglo Kemlite Laboratories Inc. of Bensonville, Illinois. The Amglo Kemlite BWMS applies a patented light separation technology to treat ballast water with pulsed poly-chromatic light. The light separation technology employs high-energy, pulsed xenon flash lamps. The Amglo Kemlite BWMS is a bench-scale prototype with a single production-sized lamp and power supply installed in an appropriately sized unit for laboratory evaluation

Tests of the LED Light Activated Titanium Dioxide Bench-Scale Ballast Water Treatment Process

This technical report presents findings from bench-scale tests evaluating the performance of the LED Light Activated Titanium Dioxide Technology, hereafter LED TiO2, developed by YJB LED Professional Services of Crosslake, Minnesota, USA. Researchers conducted the bench-scale evaluation beginning in July 2019 and ending in September 2019 at the Lake Superior Research Institute (LSRI) of the University of Wisconsin-Superior (UWS) in Superior, Wisconsin, USA. The LED TiO2 treatment process applies light emitting diodes (LED) to activate a photocatalytic coating that creates a bacteriostatic, fungistatic, and algastatic environment. Biological effectiveness testing was completed with the algae, Selenastrum capricornutum and pathogen indicator organisms, Escherichia coli and Enterococcus faecium in lab water. The system was found to be effective at treating microbes in highly-transparent/low-suspended solids water, but was less effective at treating algae

NBOT 2.5 HP Report

This technical report presents the bench-scale evaluation of the Nano Bubble Ozone Technology 2.5-horsepower unit (NBOT 2.5-HP) developed by NanoClear Group Inc. of Rockville, Maryland. This evaluation was the first to assess NBOT 2.5-HP as a potential in-tank, recirculating ballast water treatment method for the Laurentian Great Lakes. The evaluation began in September 2019 and ended in March 2020. All analyses occurred at the Lake Superior Research Institute (LSRI) at the University of Wisconsin-Superior (UWS) in Superior, Wisconsin, USA. The NBOT 2.5-HP uses cavitation to create ultrafine microbubbles (nanobubbles) containing ozone (O3) generated by the system. According to the developer, the resulting ozone and hydroxyl radical biproducts destroy all chemicals containing activated functional groups (aldehydes, ketones, amines, nitrates, etc.), RNA, DNA, peptides, steroids, as well as activated organic compounds (herbicides and pesticides), and microbial toxins. The ability of NBOT 2.5-HP to increase dissolved ozone in a 1,000-L treatment tank was tested at two water temperatures (~15°C and ~25°C) using both dechlorinated laboratory water (LW) and the more challenging amended dechlorinated laboratory water (LW-TMH). In LW, NBOT 2.5-HP increased ozone (<15 minutes) upon treatment and reached equilibrium after approximately 2 hours of treatment under both temperature conditions. In LW-TMH, no increase in ozone was observed initially upon treatment. Instead, ozone increased after approximately 2 hours and reached equilibrium after 5 to 7 hours under both temperature conditions. Degradation rates of dissolved ozone in LW and LW-TMH were examined at two water temperatures (~15°C and ~25°C). In general, ozone degradation rates were lower at 15°C than at 25°C while degradation occurred more rapidly in LW-TMH than in LW. Biological effectiveness tests examined the ability of NBOT 2.5-HP to induce mortality in biological organisms over time in both LW and LW-TMH. Three classes of organisms were tested: bacteria (Escherichia coli and Enterococcus faecium), green algae (Selenastrum capricornutum), and zooplankton (D. magna neonates, D. magna ephippia, and Eucyclops spp.). In LW, the algae and bacteria experienced 100% mortality, or no live organisms (a count of <1 MPN/100 mL) after 30 minutes of treatment. In LW, D. magna neonates and Eucyclops spp. experienced 100% mortality after 30 – 60 minutes of treatment. In LW-TMH, the algae and E. coli experienced 100% mortality, or no live organisms (a count of <1 MPN/100 mL) after 240 minutes of treatment. In LW-TMH, only one sample replicate had an E. faecium count of 3 MPN/100 mL at 240 minutes and no live organisms were detected after 390 minutes of treatment. In LW-TMH, D. magna neonates and Eucyclops spp. experienced 100% mortality after 240 – 390 minutes of treatment. In both water types, the D. magna ephippia had a hatch rate of 22.5 - 36% following treatment. These results demonstrate that NBOT 2.5-HP is effective at inducing mortality in a wide range of organisms within size classes regulated in ballast water discharge in two different water qualities. Chronic Residual Toxicity (CRT) testing examined the potential for water treated with NBOT 2.5-HP to cause toxicity to organisms in receiving water upon discharge. This testing was conducted using LW treated with the NBOT 2.5-HP system. Three classes of organisms were tested: green algae (Selenastrum capricornutum), zooplankton (Ceriodaphnia dubia), and vertebrate (Pimephales promelas). No statistically significant effects on growth, survival or reproduction were seen.LSRI-GWRC would like to thank the American Marine University Research Institute Inc. for their application to our laboratory-based testing program and for providing the Nano Bubble Ozone Technology 2.5 horsepower (NBOT 2.5-HP) ballast water treatment (BWT) testing unit. Brian Domrese of NanoPure Tech provided installation and operational training and technical support prior to the start of and during testing. Dr. Peter Moeller of the National Oceanic and Atmospheric Administration’s (NOAA) National Ocean Service (NOS) in Charleston, South Carolina provided technical advice and assistance during implementation of bench scale testing. This work was supported by the United States Maritime Administration (United States Department of Transportation; Washington, D. C.) and the United States Environmental Protection Agency’s Great Lakes Restoration Initiative

NBOT 3 HP Report

This technical report presents the bench-scale evaluation of the Nano Bubble Ozone Technology 3-horsepower unit (NBOT) developed by NABAS Group Inc. of Rockville, Maryland. This evaluation was the first to assess NBOT as a potential, in-tank, recirculating ballast water treatment method for the Laurentian Great Lakes. The evaluation began in March 2019 and ended in June 2019. All analyses occurred at the Lake Superior Research Institute (LSRI) at the University of Wisconsin-Superior (UWS) in Superior, Wisconsin, USA. The treatment technology uses cavitation to create ultrafine microbubbles (nanobubbles) containing ozone (O3) generated by the system. According to the developer, the resulting ozone and hydroxyl radical biproducts destroy all chemicals containing activated functional groups (aldehydes, ketones, amines, nitrates, etc.), RNA, DNA, peptides, steroids, as well as activated organic compounds (herbicides and pesticides), and microbial toxins. The ability of NBOT to increase dissolved ozone and oxidation-reduction potential in a 1000-L treatment tank was tested at two water temperatures (~10°C and ~25°C) using both dechlorinated laboratory water and amended dechlorinated laboratory water. Ozone levels observed to be generated by NBOT were lower than anticipated based on observations by Dr. Peter Moeller of the National Oceanic and Atmospheric Administration’s (NOAA) National Ocean Service (NOS) who was utilizing a newer model of NBOT. Biological dose effectiveness testing was not completed, per the developer’s request, due to below expected levels of ozone.Great Lakes Restoration Initiative; Maritime Administratio

B-QUA

This technical report presents findings from bench-scale verification tests evaluating the performance of the B-QUA Quick Ballast Water Monitoring Kit, hereafter B-QUA, in freshwater. B-QUA was developed by LuminUltra Technologies Ltd. of New Brunswick, Canada. Researchers began conducting the bench-scale evaluation in October 2019 and ending in February 2020 at the Lake Superior Research Institute (LSRI) of the University of Wisconsin-Superior (UWS) in Superior, Wisconsin, USA. The monitoring kit utilizes adenosine triphosphate (ATP) and size fractionation to quantify living organisms in marine, brackish, and freshwater. The measurement of ATP is one of the indicative analyses to test for gross compliance with the D-2 ballast water management standard under the International Maritime Organization’s (IMO) Ballast Water Management (BWM) Convention, which applies to countries outside of the United States, including Canada (IMO, 2015). Two phases of testing were done. Phase I testing was completed in two water types using cultured organisms in the three regulated size classes, utilizing the pathogen indicator organisms Escherichia coli and Enterococcus faecium, the algae Haematococcus pluvialis and the zooplankton Ceriodaphnia dubia and Daphnia magna. Phase II testing was completed using naturally occurring Great Lakes organisms in the Duluth-Superior Harbor of Lake Superior in two of the regulated size classes. Phase I testing showed high correlation of B-QUA’s luminometer output (i.e., cATP values) with microscopic counts for the algae Haematococcus pluvialis (≥10 μm to 50 μm size class) in both water types. However, the B-QUA system was unable to detect E. coli or E. faecium (<10 μm size class) at levels above the D2 regulatory value in either water type in Phase I. Phase II correlation between B-QUA cATP values and microscopic counts was good for natural assemblages of phytoplankton and zooplankton in Duluth-Superior harbor water.This work was supported by the United States Maritime Administration (United States Department of Transportation; Washington, D.C) and the United States Environmental Protection Agency’s Great Lakes Restoration Initiative

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