The Silver Bullet Water Treatment System: A Solution for Shrimp Aquaculture

The Silver Bullet Water Treatment System: A Solution for Shrimp Aquaculture Written by: Sonja Ingmanson February 13, 2014 1

Table of Contents: I. Executive Summary...3 II. III. Background: Vibrio parahaemolyticus, EMS/AHPND...3 Description of the Silver Bullet Water Treatment System...4 IV. University of Arizona, Aquaculture Pathology Lab Study: Silver Bullet effect on V. parahaemolyticus and shrimp survival:...6 V. CIAD Sinaloa, Mexico Study: on Silver Bullet: V. parahaemolyticus and shrimp survival.....7 VI. CIAD Hermosillo, Mexico Study: The Effect of Silver Bullet on V. parahaemolyticus...9 VII. VIII. IX. Other advantages of the SBWTS for the shrimp aquaculture industry. 9 Summary and Conclusion...10 References 11 2

I. Executive Summary The Silver Bullet Water Treatment System (SBWTS) has proved repeatedly that it is a promising tool for the shrimp aquaculture industry in its ongoing battle against pathogenic bacteria like Vibrio parahaemolyticus (V. parahaemolyticus). Multiple studies have been conducted by unaffiliated, non-biased certified laboratories in which the SBWTS was found to successfully reduce the amount of V. parahaemolyticus, maintain those bacterial counts at low levels and promote shrimp survival. The SBWTS generates its disinfectant from ambient air onsite, so does not involve the risks and dangers associated with storing and handling toxic, harmful chemicals. The SBWTS has proven to be safe to shrimp and is designed in a way that is easy, user friendly and affordable. Employing a SBWTS for shrimp aquaculture is a valid approach for protecting jobs, profits and shrimp from pathogenic contaminants that are otherwise devastating the shrimp aquaculture industry worldwide. II. Vibrio parahaemolyticus and EMS/APHND Vibrio parahaemolyticus, which has virulent and benign strains, causes Acute Hepatopancreatic Necrosis Disease (AHPND) or Early Mortality Syndrome (EMS) in shrimp (Lightner et al. Webinar 2013). AHPND damages the digestive system of shrimp and causes mortality, often within thirty days of stocking. V. parahaemolyticus tolerates a range of salinities, ph and temperatures, readily attaches to marine plankton and may be spread by ocean currents (Chamberlain 2013). At extremely dense populations, the colonies coordinate the release of a communication chemical (a potent toxin) through a process known as quorum sensing (Hardman et al., Lightner et al. Webinar 2013). Studies suggest that this quorum sensing toxin may be released when the V. parahaemolyticus populations reach 10 6 cfu/ml (Silver Bullet Studies: UA and Hermosillo CIAD, Lightner et al. Webinar 2013). This disease has caused significant loss for the shrimp aquaculture industry worldwide. This catastrophic loss is seen in shrimp production, jobs and profit. According to shrimp aquaculture experts in their December 2013 presentation for the Global Aquaculture Alliance meeting in Vietnam, Dr. Lightner and Dr. Chamberlain stated the following: In 2013, the global production of shrimp declined by about 15% from 2011 levels, but considering that the industry was expected to grow by about five percent a year, production was actually 23% below market expectations. In rough numbers, global production of farmed shrimp is about four million metric tons a year. If it were down 25%, that s a million metric tons. A million metric tons is a billion kilograms. Assuming a value of five dollars a kilogram, that s a five billion dollar loss to the industry at the farm gate. 3

III. How the SBWTS Works The SBWTS uses a very strong and effective disinfection method called an Advanced Oxidation Process (AOP). This type of water treatment technology is widely recognized for its powerful oxidizing capacity as a disinfectant, including by the United States Environmental Protection Agency (EPA) (EPA Guidance Manual). AOPs are generally accepted as being very efficient at eliminating many contaminants such as bacteria, viruses, protozoan cysts, persistent organic compounds (i.e. many pesticides, herbicides, industrial and synthetic compounds, pharmaceuticals, etc.), and general organic/inorganic matter (EPA Guidance Manual, Rashed et al.). AOPs are defined as technology that uses a combination of ozone (O3), hydrogen peroxide (H2O2), and/or UV lights to generate highly reactive hydroxyl (OH-) and oxygen free radicals (O-) to oxidize various contaminants in water (EPA Guidance Manual, Glaze et al. 1987, Kleiser et al., Kommineni et al., Munter). The SBWTS uses this combination of methods to create OH- and O- radicals for disinfection; the appearance and components of which are shown in Figure 1 below. Figure 1: The appearance and components of the Silver Bullet Unit. The hydroxyl and oxygen radicals have higher oxidation powers than chlorine and ozone (See Figure 2), so have a faster reaction time in which they aggressively attack virtually all organic contaminants (EPA Guidance Manual, Glaze et al. 1987, Munter). 4

Oxidation Potential (V) Relative Oxidizing Strength of Oxidizers 3.5 3 2.5 2 1.5 1 0.5 0 Oxidizers Fluorine Atomic Oxygen Hydroxyl Radical Ozone Hydrogen Peroxide Perhydroxyl Radicals Permanganate Chlorine Dioxide Hypochlorous Acid Chlorine Bromine Figure 2: This figure shows the extremely high oxidizing strength of hydroxyl and oxygen radicals over more conventional disinfectants like ozone and chlorine (EPA Handbook. AOPs, Kommineni et al.). The hydroxyl and oxygen radicals react very aggressively with contaminants by a chain reaction exchange of electrons (Munter). This reaction quickly breaks down contaminants (i.e. organic matter) into carbon dioxide and water (complete mineralization), while leaving a residual amount of H2O2; beneficial as a low level biocide (Rashed et al. 2005, Kommineni et al.). Awards: 2012: Best Venture award from the U.S. Department of Energy s National Renewable Energy Laboratory (NREL) for its cost effective and environmentally responsible water treatment solution. 2013: Silver Bullet Corporation was named the High Impact Cleantech Company of the Year by the Colorado Cleantech Industries Association. Certifications: UL Listed NSF 61 certified Can supply LEED credits for existing buildings and new construction Can be used in an ISO 14001 environmental management plan Manufactured in an ISO 9001 certified facility ASHRAE member EPA registered biocide device 5

IV. University of Arizona, Aquaculture Pathology Lab Study: Silver Bullet effect on V. parahaemolyticus and shrimp survival In August 2013, Dr. Donald Lightner and his team at the University of Arizona Aquaculture Pathology Lab conducted a preliminary study to determine if the SBWTS would be safe for shrimp and have an effect on eliminating V. parahaemolyticus. The results proved that the SBWTS was indeed successful at reducing the levels of V. parahaemolyticus after only very small doses without causing harm to the shrimp (see figure 3). With only 60 seconds of total exposure to the Silver Bullet (SB) treatment in each vessel, the total kill rate was as follows; Vessel A= 13,900 Colony Forming Units/ml (cfu/ml) (from an initial dose of 10 4 cfu/ml), Vessel B= 57,000 cfu/ml (from 10 5 cfu/ml), and Vessel C= 620,000 cfu/ml (from 10 6 cfu/ml). The graph below shows the decrease in V. parahaemolyticus in just 60 seconds in vessel A: Figure 3: After only 15 seconds of a dose of the SBWT gas, a decrease in the number of V. parahaemolyticus colonies was observed, with a much greater additional decline after 45 more seconds of treatment. 90% of the shrimp survived after 6 days in Vessels A (initial exposure= 10 4 cfu/ml) and B (initial exposure= 10 5 cfu/ml); a much higher survival rate than with no treatment (see Table 1 below). However 100% of the shrimp died in Vessel C after 4 days (initial exposure= 10 6 cfu/ml); still a pronounced delayed effect compared to shrimp exposed to that quantity of bacteria with no SB treatment (see Table 1). Evidence suggests that when populations of V. parahaemolyticus become so numerous to reach 10 6 cfu/ml, they begin to secrete a toxic chemical used for communication among the colonies, called quorum sensing (Hardman et al., Lightner et al., Webinar 2013). It is thought that despite the disinfection approach, shrimp experience extremely high rates of mortality following exposure to this virulent toxin. 6

Table 1: This table shows the higher survival rates of shrimp exposed to high levels of the pathogenic bacteria, V. parahaemolyticus, following treatment with SBWTS. Vessel A had 90% survival, whereas shrimp with no treatment have a 30% survival. Vessel B had 90% survival, where shrimp with no treatment usually have 0% survival. Vessel C had 0% survival, where untreated shrimp usually experience the same, however treatment delayed the onset of disease and death by 4 days. Note that the SB dose was extremely low (only 60 seconds) for this study. To summarize this study, Dr. Lightner s team at the Aquaculture Pathology Laboratory at the University of Arizona found that even with extremely small doses of SBWTS, it is effective at decreasing levels of pathogenic V. parahaemolyticus bacteria, while enhancing shrimp survival. The proceeding studies with two CIAD laboratories in Mexico demonstrate that the SBWTS can keep V. parahaemolyticus to levels below the virulent 10 6, while keeping shrimp alive significantly longer than without treatment. V. CIAD Sinaloa, Mexico Study: on Silver Bullet: V. parahaemolyticus and shrimp survival An additional study was conducted in December 2013 by Dr. Bruno Gomez and his team at the Federal Research Center on Nutrition and Development (CIAD) in Sinaloa, Mexico. In this study, it was found that the SBWTS was successful at reducing and controlling Vibrio parahaemolyticus growth over a 47 day testing period, while keeping the shrimp alive (93% survival) (see Figure 4 and 5). 7

Population of vibrios in water: Figure 4: The population of V. parahaemolyticus in water with shrimp remained low and controlled in the tank treated continuously with SB (blue line), compared to much more sporadic variation in the tank with shrimp and no water treatment (red line). Population of vibrios in sediment: Figure 5: The population of V. parahaemolyticus in the sediment of the SB treated tank with shrimp remained low and controlled (blue line), compared to much more sporadic variation in the sediment from the untreated tank with shrimp (red line). 8

VI. CIAD Hermosillo, Mexico Study: The Effect of Silver Bullet on V. parahaemolyticus In February 2014, Dr. Silvia Gomez at the CIAD laboratory in Hermosillo, Mexico ran a test that verified the kill rates of the SBWTS on virulent quantities of V. parahaemolyticus. Results showed that the SB dropped the V. parahaemolyticus populations from 10 5 to zero in less than 30 minutes and from 10 6 to zero in 3.5 hours (see Figure 6). Figure 6: In 30 minutes, SB caused the V. parahaemolyticus population to drop from 10 5 to zero. In 3.5 hours, SB caused the VP population to drop from 10 6 to zero. VII. Other advantages of SBWTS for the shrimp aquaculture industry a. Increased aeration and oxygen The SBWTS diffuses oxygenated gases into the aquaculture water. The need for aeration and oxygenation in shrimp habitats is widely used and necessary for shrimp health and survival (Leon 2013). b. Can potentially replace Disodium Ethylene Diamine Tetraacetate (EDTA) One of the first industry applications of the SBWTS was in cooling towers for buildings because the technology not only kills pathogens, but it keeps calcium ions dissolved in water. For cooling towers, keeping calcium ions dissolved in water is essential in helping to prevent scaling and corrosion on metal components. This mimics the purpose for use of EDTA to keep calcium dissolved in water to 9

promote the uptake of calcium ions for shrimp shell hardening and growth (Hatcheries Mortalities 2013, Licop, Taslihan). It is postulated that shrimp with harder, healthier shells may stand a better chance at warding off pathogenic bacterial biofilms from forming (Hatcheries Mortalities 2013, Licop, Taslihan). c. Can be used in conjunction with bioflocs and/or probiotics The SB application has been designed to apply low quantities of disinfecting gas into aquaculture water. This approach is to ensure the shrimp are not harmed and to pose minimal impact to the beneficial constituents of the aquaculture ecosystem (Probiotics and Shrimp Farming 2005). It has been proven that the SBWTS can preserve low levels of beneficial bacteria, while helping to control the growth of and minimize pathogenic bacteria. d. Benefits over other treatments like chlorine, UV, ozone Oxidation with AOPs has been found to be an important alternative to chlorination, because oxidation with OH- and O- does not result in toxic chlorinated organic compounds or other toxic Disinfection By-Products (DBPs) (Chen et al. 1997, EPA Guidance Manual, Rashed et al.). Since the SBWTS uses only ambient air for disinfection, there is no risk or danger associated from storing or handling harmful or toxic chemicals like ozone or chlorine. AOPs are also known for their ability to eliminate odor causing compounds like geosmin and MIB, which could affect shrimp flavor (EPA Guidance Manual). e. Affordability The AOP water treatment method is highly recognized as an extremely effective disinfectant, however cost efficiency and usability have been its main obstacles in the past (EPA Guidance Manual, EPA Handbook, Kommineni et al.). Few industries requiring water treatment could afford the pricey applications of an AOP system and it was difficult to come by skilled enough operators to oversee the technology. The SBWTS s patented engineering is unique in that it has finally made an AOP application that is easy to use and affordable. VIII. Summary and Conclusion In summary, the SBWTS has been proven to be a substantial and promising response to contamination in the international shrimp aquaculture industry. V. parahaemolyticus is affecting farms around the world, causing loss of shrimp, jobs and profit. The SBWTS has repeatedly and successfully reduced and controlled V. parahaemolyticus growth while enhancing shrimp survival rates. The SBWTS is an exceptional response for the shrimp farming industry to reduce contamination, preserve shrimp survival, protect jobs and promote a profitable farming season. 10

IX. References 1. Chamberlain, George. The Global Aquaculture Advocate (The Global Magazine for Farmed Seafood). Editor, Darryl Jory. GOAL 2013 Review: Challenge Health Management/Program Focuses on Perfect Killer EMS. Volume 16, Issue 6, Page 14, November/December 2013. 2. Chen, J., et al., PhD. Thesis, Advanced Oxidation Technologies: Photocatalytic Treatment of Wastewater, Universitair docent bij het subdepartment milieutechnologie, Holland, 1997. 3. EPA Guidance Manual. Alternative Disinfectants and Oxidants. Chapter 7: Peroxone (Ozone/Hydrogen Peroxide). April 1999. Pg 1-21. 4. EPA Handbook. Advanced Photochemical Oxidation Processes. December 1998. 5. Glaze, W. H., Kang, J.W. & Chapin, D.H. The Chemistry of Water Treatment Processes Involving Ozone, Hydrogen Peroxide and UV- Radiation. Ozone: Sce. Eng., 1987, 9, 335-352. 6. Hatcheries Mortalities. New Caledonia. A Discussion. November 27- December 13, 2013. Bob Rosenberry, Shrimp News International, December 16, 2013. 7. Murias, A. FIS United States. Shrimp Disease Has Catastrophic Impact in Sonora. January 8, 2014. 8. Hardman, A. M., Stewart, G. S. A. B., Williams, P. Quorum sensing and the cell-cell communication dependent regulation of gene expression in pathogenic and nonpathogenic bacteria. Antonie van Leeuwenhoek 74: 199-210, 1998. 9. Kleiser, G., Frimmel, F.H. Removal of precursors for disinfection by-products (DBPs)- differences between ozone- and OH- radical-induced oxidation. The Science of the Total Environment 256 (2000) 1-9. 10. Kommineni, S., Zoeckler, J., Stocking, A., Liang, S., Flores, A., Kavanaugh, M., Rodriguez, R., Browne, T., Roberts, R., Brown, A., Stocking. Advanced Oxidation Processes. Chapter 3.0. Literature Review. 11. Leon, Armando, interviewed by Bob Rosenberry, Shrimp News International. World Aquaculture Society Meeting, Nashville, Tennessee, USA (February 21-25, 2013). February 21, 2013. 12. Licop, S. R. Sodium-EDTA effects on survival and metamorphosis ofpenaeus monodon larvae. El Sevier. Aquaculture. Volume 74, Issues 3-4, 15 November 1988, pages 239-247. 13. Lightner, D., Chamberlain, G. Early Mortality Syndrome: Managing the Perfect Killer. A webinar organized by the Global Aquaculture Alliance. Bob Rosenberry, Shrimp News International. Ho Chi Minh City, Vietnam. December 10-13, 2013. 14. Munter, Rein. Advanced oxidation processes- current status and prospects. Proc. Estonian Acad. Sci. Chem., 2001. 50, 2, 59-80. 15. Probiotics and Shrimp Farming (Online Forum Discussion). Shrimp News International. October 1, 2005. Summarized by Bob Rosenberry. 16. Rashed, I. G., Hanna, M. A., El-Gamal, H.F., Al-Sarawy, A. A., Wali, F. K. M. Overview on Chemical Oxidation Technology in Wastewater Treatment. Ninth International Water Technology Conference IWTC9 2005, Sharm El-Sheikh, Egypt. 17. Taslihan, A., Sunaryanto. Proceedings. Shrimp Culture Industry Workshop. Jepara 11

(Indonesia). Brackishwater Aquaculture Development Centre, Jepara, Central Java. Pest and disease management in p. Monodon culture. FAO Corporate Document Repository. 25-28 Sep 1989. 12