1
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Efficacy of two approaches for disinfecting surfaces and water
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infested with quagga mussel veligers
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Christine M. Moffitt1*, Amber Barenberg2, Kelly A. Stockton2, and Barnaby J. Watten3
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1
US Geological Survey, Idaho Cooperative Fish and Wildlife Research Unit, Department of
Fish and Wildlife Sciences, University of Idaho, Moscow, Idaho, USA,
cmoffitt@uidaho.edu; 208-885-7047 (phone); 208-885-9080 (fax)
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2
Department of Fish and Wildlife Sciences, University of Idaho, Moscow, Idaho, USA;
Bare6282@vandals.uidaho.edu; stoc4872@vandals.uidaho.edu; 208-885-7139 (phone)
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3
US Geological Survey, S.O. Conte Anadromous Fish Research Center, Turners Falls,
Massachusetts, 01376-0796, bwatten@usgs.gov; 413-863-3800
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* Corresponding author, Christine M. Moffitt
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Chapter in “Biology and Management of Invasive Quagga and Zebra Mussels in the Western
United States”, edited by Wai Hing Wong and Shawn Gerstenberger
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This draft manuscript is distributed solely for purposes of scientific peer review. Its content is
deliberative and predecisional, so it must not be disclosed or released by reviewers. Because the
manuscript has not yet been approved for publication by the U.S. Geological Survey (USGS), it
does not represent any official USGS finding or policy."
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Abstract
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Disinfection tools and protocols are needed to meet an array of applications to reduce the
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probability of transferring invasive mollusk species as hitchhikers. Applications include
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rapid disinfection of recreational equipment and boats, to disinfecting water and equipment
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used in fire suppression, to tools safe for hatchery and aquaculture operations. In replicated
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laboratory trials, we tested the lethality of concentrations of three chemical solutions on
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quagga mussel veligers (Dreissena rostriformis bugensis). Aqueous solutions of pH 12 were
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created with NaOH or Ca(OH)2 and tested at 15 and 22°C and three aqueous concentrations
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of Virkon® Aquatic were tested at 22°C. We observed mortality of veligers was faster in
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warmer temperatures and in solutions of Ca(OH)2. We observed complete mortality in
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solutions prepared with Ca(OH)2 within 10 min of exposure, and within 30 min of exposure
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in solutions prepared with NaOH. We found solutions of 5 g/L of Virkon® Aquatic, used as
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a disinfectant in aquaculture operations, killed all veligers within a 10 min exposure. We
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conclude that all chemicals show promise as disinfectants, and use of Ca(OH)2 or NaOH to
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elevate the pH of disinfecting solutions may provide an economical and environmentally
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acceptable way to disinfect large surfaces or tanks.
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Introduction
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Global transportation of commodities and human movements pose challenges to natural
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resource managers, as the risks of transporting alien organisms are compounded with each
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vehicle and vector. In addition, many altered ecosystems are more vulnerable to invasion.
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Among the highly successful invader alien organism are the mollusks, as they are armored
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with shells that protect them from desiccation, and have great capacity to be considered as
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engineers of systems in which they establish. Many vectors and vehicles including, fish, birds
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and mammals, aquarium traders, ship ballast water, recreationalists, agency personnel, and
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other water users can distribute invasive mollusks to new locations (Bowler 1991; Alanso
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and Castro-Diez 2008; Bruce and Moffitt 2010).
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Quagga mussels (Dreissena rostriformis bugensis Andrusov, 1897), native to Eastern
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Europe, were accidently introduced into the Laurentian Great Lakes in North America in the
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1980s in ballast water (Vanderploeg 2002; Strayer 2009). The major pathway of quagga
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mussel introduction from the Great Lakes to other locations in North America has been
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assumed to be recreational boaters (Johnson et al. 2001, 2006; Leung et al. 2006; Mueting
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and Gerstenberger 2011). It is hypothesized that quagga mussels were introduced into Lake
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Mead sometime before 2007 in bilge water (with bait or live wells) by a boat from the Great
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Lakes region (Wong and Gerstenberger 2011).
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Activities such as fire suppression and fish stocking are of concern in Western North
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America as they have high risks of transfer of invasive mollusks (Bruce et al. 2009; Culver et
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al. 2000; Edwards et al. 2000; Britton and Dingman 2011). Fish hatcheries in Colorado,
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Arizona, and Nevada are now infested with quagga mussels, and such facilities are
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constrained to reduce the risk of transporting veligers with movement of fish, or equipment
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between locations. Western states use helibucket, pumps and other operations for fire
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suppression that use water from nearby open water sources. The US Forest Service, Bureau
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of Land Management and interagency fire management teams are concerned that raw water
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samples from infested areas could introduce invasive mollusks into new areas (Britton and
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Dingman 2011).
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Once established, quagga mussels can affect ecosystems and damage infrastructure of
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cooling pipes, water intake pipes, head gates, and hatchery facilities (Pimentel et al. 2000,
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2005; Peyer et al. 2009; Wong and Gerstenberger 2011). There is a need for tools that can be
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used to address both small scale to large scale disinfection needs. Since these invasive
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mollusk species continue to have a high probability of successful transport to new locations,
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effective and safe methods of disinfection for various conditions and circumstances are
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needed. Innovative tools are needed and economic constraints for purchase of chemicals and
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infrastructure used in the disinfection process can be limiting factors (Karatayev et al. 2007)
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Often highly effective disinfectants are expensive, and/or they have substantial
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environmental consequences if used in large quantities. Chemicals that can be used in large
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scale applications must be less expensive, and have attributes that would allow for safe
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neutralization and disposal.
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Different life history stages have different survival times, but it has been recently reported
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that even quagga veligers can remain alive for several days to weeks in moist environments
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depending on temperature (Choi et al. 2013). Studies conducted by Britton and Dingman
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(2011) found that a 10 minute exposure of 3% concentration of Sparquat 256® sanitation
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was sufficient to kill 100% of tested veligers held 60 min post treatment. These and similar
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quaternary ammonia reagents have been reported to be effective in killing mussel veligers
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and other invasive species in and around fish hatchery environments (Waller et al. 1996;
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Mitchell and Cole 2008; Oplinger and Wagner 2009). However, widespread use of
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quaternary ammonia compounds has caused concerns as they can affect aquatic and soil
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systems (Garcia et al 2001; Li and Brownawell 2010; Sarkara et al. 2010), and their
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persistence has increased concerns regarding their genotoxic effects (Ferk et al. 2007). Other
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compounds have been considered, and Edwards et al. (2000) used KCL and formalin to
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reduce risks of invasive zebra mussels. Antifouling compounds are also of great interest to
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protect infrastructure in aquaculture and industry but environmental consequences must be
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considered (Guardiola et al. 2012). Chlorine and other oxidizing reagents are effective tools
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for disinfection (Brady et al. 1996) but the residual compounds have environmental
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consequences for water supplies and aquatic organisms (Watson et al. 2012). Recent studies
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reported the efficacy of a proprietary copper chemical EarthTec® to kill and prevent
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settlement of veligers (Watters et al. 2013), but dosages tested were above those considered
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safe for most aquatic organisms.
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Ship ballast is well recognized as a source of invasive organisms throughout the globe (Briski
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et al. 2012), and recent studies by Starliper and Watten (2013) show promise with the
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hydroxide alkalinity as a tool to reduce microbial populations. Starliper and Watten (2013)
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report progress exploring with other collaborators (Cangelosi et al. 2013) the use of
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hydroxide alkalinity (i.e. with chemical addition of sodium hydroxide) as a ballast
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decontaminant to meet or exceed the new ballast water standards in decontaminating
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organisms, along with other important criteria associated with its use including cost
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effectiveness, mixing characteristics, safety and ease of use for crew members and
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neutralization. The use of NaOH has been successfully used as a dairy disinfection tool for
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milking equipment to avoid use of chlorine (Gleeson et al. 2013). Hydrated lime [Ca(OH)2]
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has been reported to be effective for disinfecting wastewater (Grabow et al. 1978; Scanar et
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al. 2001) and is used widely in endodonics to prevent infection (Orstavik et al.1991; Siqueira
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and Lopes 1999; Athanassiadis et al. 2007).
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Virkon® Aquatic (reformulated from Virkon® S in 2007) is one of very few US
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Environmental Protection Agency-registered disinfectants labeled for use in aquaculture
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facilities (Mainous et al. 2010; DuPont 2011) for use on bacterial, fungal and viral pathogens.
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Active ingredients of Virkon® Aquatic are potassium monopersulphate and sodium chloride,
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21.9% (Mitchell and Cole 2008). The mode of action is by oxidizing proteins and other
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components of cell protoplasm, resulting in inhibition of enzyme systems and loss of cell-
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wall integrity (Curry et al. 2005). The recommended concentration for disinfecting most
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surfaces for a majority of the organisms is a 10 g/L concentration with an exposure time of at
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least 5 min (Western Chemical 2008).
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The objectives of our study were to compare the mortality response of quagga veligers in
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disinfectant strength solutions of aqueous pH 12 made with Ca(OH)2 or NaOH, and in
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solutions of an approved hatchery disinfectant, Virkon® Aquatic. In addition we explore the
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relative costs, environmental risks and benefits, and suitability for each disinfectant for use
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on gear, boats, tanks and other applications.
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Methods
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Study Location and Source of Organisms
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All tests were conducted at Willow Beach National Fish Hatchery, Willow Beach, Arizona.
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Tests were conducted from 13 through 22 October 2009 (Virkon trials), and 17 through 25
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November 2012 (elevated pH). Veligers for tests were collected with a 30 µm mesh plankton
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tow net hung from the grating system of a raceway head box for 30 – 60 min. The contents of
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the cod end were then filtered through a 500 µm nylon mesh screen into a 500 mL Nalgene
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sample bottle. The contents of the sample bottle were allowed to settle at room temperature
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(22°C) for 1-3 h. Condition of veligers was assessed for living, healthy veligers; plankton
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tow samples with greater than 90% living veligers were retained for testing. The veligers for
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tests of elevated pH were concentrated additionally by collecting settled filtrate on ~100 μm
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mesh nylon fabric and gently placing this into a 1.8 L plastic container and rinsing with
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squirt bottles of aerated well water.
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Test Chemicals
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We weighed ~ 0.6 g/L NaOH (Fisher Chemical, Pittsburgh, PA, Lot 060432) or Ca(OH)2 (J.
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T. Baker Chemical, Phillipsburg, NJ, Lot G43364), dissolved into aerated well water to reach
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a final solution pH of 12. The test solutions were filtered to remove particulates, and placed
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into Nalgene carboys to acclimate to test temperature overnight. The pH of each solution
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was verified with a Hach sensION platinum portable pH meter, or YSI 556 multiprobe.
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Virkon® Aquatic (lot # 2258523) (Western Chemical, Ferndale, WA) was measured (0.01 g)
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and mixed in volumetric flasks with well water at 18°C. Solutions were placed at test
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temperatures for at least an hour to acclimate and activate. The test concentrations of 2.5, 5,
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and 20 g/L were verified with Virkon® Aquatic test strips (Western Chemical, Ferndale,
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WA).
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Experimental Design
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Elevated pH – A 2 mL sample from the bottom of the settling container with veligers was
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removed with a disposable plastic pipette and was placed into a replicate 120 mL beaker. We
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then added 100 mL of test solution rapidly and recorded the starting time. Tests were
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conducted at 15°C and 22°C. A flow through raceway trough was used to maintain
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temperature for tests at 15°C. Tests at 22°C were conducted on the laboratory bench top.
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Exposures were terminated at intervals of 2, 5, 10, 20, and 30 min. At the end of each time
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interval the contents of each container were poured through a stainless steel mesh spoon lined
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with ~ 100 μm nylon mesh. After veligers were collected on the mesh fabric, we used well
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water acclimated to the test temperature to rinse veligers of any residual test solution. After
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the rinse process the nylon mesh was removed from the spoon and reversed over the top of
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one of a labeled glass Petri dish and rinsed additionally to remove all veligers. Veligers were
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maintained in the glass Petri dish and allowed to recover for 24 hours at test temperature
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before final assessment of mortality. As a control, we held and handled replicated containers
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of veligers not exposed to test compounds. To determine surviving veligers, the glass petri
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dishes were placed under a dissecting microscope and the number of live and dead veligers
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was counted. Veligers were considered alive if cilia were moving and moving or spinning
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parts could be seen inside the shell. Veligers were considered dead if shells were open or
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empty, internal parts had been crystallized, or no reaction to a disturbance was seen.
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Virkon trials – We used a serological pipette to transfer 1 mL of settled sample water
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containing veligers from the bottom of the sample bottle into a 150 mL glass beaker. We
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then added 9 mL of stock Virkon® Aquatic concentration to achieve a final concentration of
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2.5, 5, or 20 g/L. Veligers were assessed at 5, 10, 15, or 20 minutes of exposure by removing
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3 mL of test solution from the bottom of a test beaker and pipetted into a 30 mm petri dish.
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A dissecting microscope was used to locate and enumerate veligers from each test solution.
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Veligers were examined for mortality, and then all veligers were placed in aerated well water
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for 24 h recovery. Final assessment of survival was recorded after 24 h with the aid of a
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Sedgewick rafter microscope slide and compound microscope. No movement of cilia, visible
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organs, and darkening or crystallization of the veliger constituted mortality. Tests were
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conducted at room temperature (22°C).
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Data Analysis
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In our tests of elevated pH, we compared the frequency of live and dead veligers by test
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chemical and test temperature in exact chi square tests of independence and in log linear
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categorical models by duration of exposure. For the test intervals where only one chemical
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was tested we report simple exact frequency probabilities to compare the outcome of live and
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dead by test temperature. In tests of Virkon Aquatic, we compared the frequency of live and
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dead veligers in test concontrations of 2.5 and 5 g/L for exposure times of 5 and 10 minutes.
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All statistical analyses were conducted in SAS version 9.2 (SAS Institute, Cary, NC).
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Results
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We found rapid mortality of all veligers tested at elevated pH (Figure 1). We found no effect
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of test temperature after 2 minutes exposure in Ca(OH)2 (χ2= 1.7, DF= 1; P > 0.18; Table 1).
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In exposures lasting 5, 10 and 20 minutes tested with both chemicals, we found significant
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differences in the likelihood rations of live and dead between Ca(OH)2 and NaOH (Table 2).
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We found mortality was significantly faster in the warmer water temperatures at 5 minute
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exposures (P < 0.001; Tables 1 and 2). After 10 minutes the differences between temperature
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were not as pronounced (P = 0.069), but we detected a significant test chemical by
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temperature interaction. The likelihood ratios of survival after 20 minutes of exposure to
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both chemicals were highly significant between temperature and test chemical. In veligers
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exposed to pH 12 created with Ca(OH)2, we recorded complete mortality at both water
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temperatures, but 3% of veligers held at 15°C in NaOH survived (Tables 1 and 2).
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Throughout our trials we observed little to no mortality in control tests of veligers that were
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handled similarly, but not exposed to elevated pH.
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Veligers exposed to Virkon® Aquatic at 2.5 g/L died between 10 to 15 minutes (Table 3),
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but concentrations of 5 g/L were more effective, killing veligers more rapidly. The frequency
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of live and dead veligers compared between concentrations of 2.5 and 5 g/L at 5 minutes
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were significantly different (χ2= 30.49; P < 0.001). The frequency of live and dead veligers
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between the two concentrations after a 10 minute exposure were not significantly different
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(χ2 = 2.52; P > 0.285). Our test system had no effect on veliger survival, as all controls were
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alive during the testing and recovery time.
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Discussion
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Both solutions of pH 12 made with NaOH and Ca(OH)2 were effective, killing veligers,
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although the cooler water temperature took longer time to 100% mortality. Similar success
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with laboratory trials of disinfection of a suite of microorganisms was conducted by Starliper
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and Watten (2013). They compared colony forming units as an endpoint of efficacy for a
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suite of microorganisms tested at pH 10 – 12 with NaOH, and achieved 100% killing of all
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Gram-negative and Gram-positive cultures. In recently reported shipboard trials conducted
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by the Great Ships Initiative the test treatment of pH 12 with NaOH, large reductions in the
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density of live organisms ≥ 50 μm were achieved in the treatment discharge samples over
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those observed in control discharge samples (Cangelosi et al. 2013).
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Claudi et al. (2012) evaluated the effect of a range of pH on mussel settlement and mortality
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and determined that dreissenid mussels have a relatively narrow range of pH tolerance, with
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the optimum range being 7.5 to 9.3. Other factors affect the settlement of mussels (Chen
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2011). The rapid mortality in our tests with veligers is likely also related to the size of the
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organisms tested. Mitchell and Cole (2008) found solutions of pH 12–13 made with
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hydrated lime and sodium hydroxide were not effective as reagents to kill adult sized faucet
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snails after a 1 hour exposure.
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Our tests with Virkon Aquatic were also very effective. Quagga mussel veligers were killed
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by concentrations of 5 g/L after a short exposure of 5 to 10 minutes. Our results of the
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quagga mussel veliger toxicity support O’ Connor et al. (2008) report, which showed that 5
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mg/L of Virkon® Aquatic caused 100% abnormal embryo development in Sydney rock
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oysters Saccostrea glomerata.
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Stockton and Moffitt (2013) found a 15–20-min bath application of 20 g/L Virkon® Aquatic
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was a reliable tool to disinfect boot surfaces infested with New Zealand mudsnails
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Potamopyrgus antipodarum and other aquatic invertebrates. Mitchell et al. (2007) reported
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100% mortality in red-rim melania Melanoides tuberculata at concentrations greater than 1
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g/L for 24 h (Mitchell et al. 2007). However, faucet snails Bithynia tentaculata exposed to
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concentrations of 2 g/L Virkon® for 1 -24 hr showed little mortality (Mitchell and Cole
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2008).
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Our tests with Virkon® Aquatic were conducted at one temperature and additional tests at a
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range of water temperatures are needed. The active ingredient in Virkon® Aquatic is
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potassium permonosulfate and sodium chloride (Western Chemical 2008; Mitchell and Cole
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2008). Potassium and sodium gradients control the neuron response in humans (Starr and
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Taggert 1998). A neuron response was first identified in sea slug or sea hare Aplysia
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californica mollusks (Russell and Brown 1972). Fisher et al. (1991) reported D. polymorpha
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has a low tolerance for elevated potassium concentrations and this can destroy the integrity of
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the mussel gill epithelium, leading to asphyxiation.
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Trials conducted in our laboratory indicated Virkon® Aquatic was safe for use in and around
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vertebrates in aquaculture environments (Stockton 2011). Further testing of the efficacy of
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Virkon® Aquatic on other aquatic invasive species is recommended to enable broad-
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spectrum use. The use of Virkon as a disinfectant was comparable to the results obtained
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using Sparquat 256®, but Virkon is easily deactivated by organics into simple salts that pose
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little environmental risk (Stockton and Moffitt 2013).
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Determining the appropriate tool for disinfection requires understanding the costs, risks and
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environmental consequences of each of these treatments. At the present time, high-pressure
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hot water spray has been recommended as an effective means for boat decontamination of
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attached juvenile and adult mussels (Comeau et al. 2011; Zook and Phillips 2012). The
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addition of an elevated pH treatment could improve the effectiveness of this treatment, as the
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chemical would be effective in areas that were not adequately covered with elevated
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temperatures. The use of elevated pH could also be effective in treating water for fire
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suppression activities. The solutions were easily prepared and used approximately 0.6 g/L
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dissolved in well water to achieve the pH target. Natural resource interest groups and
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regulatory agencies have made it clear that safe and non-chemical alternatives for controlling
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mussel fouling are preferred (Britton and Dingman 2011).
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Acknowledgements
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We thank Mark Olson, Thomas Frew, Giovanni Cappellii, and Andrew Flaten at Willow
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Beach National Fish Hatchery, Arizona, for their support and assistance with sampling and
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accommodations for these trials. This research was supported with funding from the Fish and
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Wildlife Service, Paul Heimowitz, Project officer; and the US Geological Survey, Work
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Order 153 in collaboration with the National Park Service. We are grateful to X and X
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reviewers for assistance in evaluating earlier drafts of this manuscript. The use of trade
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names or products does not constitute endorsement by the U.S. Government
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20
Table 1. Summary of number veligers dead or alive and percent survival (in parentheses) in tests
of Ca(OH)2 and NaOH by time interval, temperature and test compound, replicates combined.
Veligers exposed to NaOH were not scored at 2 minutes, and Ca(OH)2 were not scored at 30
minutes.
Chemical
15 °C
Dead
22°C
Live
Dead
Live
2 min
Ca(OH)2
21
185
116
56
(11)
(89)
(67)
(32)
5 min
Ca (OH)2
NaOH
182
25
150
5
(88)
(12)
(97)
(3)
45
39
24
31
(54)
(46)
(43)
(56)
0
10 min
Ca(OH)2
NaOH
137
0
84
(100)
(0)
(100)
70
12
97
(85)
(15)
(100)
0
21
Table 1, continued.
Chemical
15 °C
Dead
22°C
Live
Dead
Live
110
0
20 min
Ca(OH)2
60
0
(100)
NaOH
(100)
68
2
72
(97)
(3)
(100)
0
30 min
NaOH
53
(100)
0
78
(100)
0
22
Table 2. Summary of maximum likelihood analysis of variance log linear comparisons of
independence for frequency of live and dead veligers, tested by interval for differences attributed
to chemical tested, and test temperature.
Source
DF
Chi-Square
Pr > ChiSq
5 minutes exposure
Temp
Chemical
Chemical*Temp
1
1
1
12.28
90.79
0.44
0.0005
<0.0001
0.5094
Likelihood ratio
4
306.44
<0.0001
Ten minute exposure
Temp
Chemical
Chemical*Temp
1
1
1
3.32
90.79
0.44
0.0685
<0.0001
<0.0001
Likelihood ratio
1
45.4
<0.0001
Twenty minute exposure
Temp
Chemical
Chemical*Temp
1
1
1
32.67
17.19
0.25
<0.0001
<0.0001
0.6199
Likelihood ratio
1
78.88
<0.001
23
Table 3. Summary of live and dead quagga mussel veliger in trials of three concentrations of
Virkon® Aquatic, for durations of 5 to 20 minutes exposure. Trials were conducted at room
temperature, ~ 22°C.
Concentration
(g/L)
2.5
Exposure
(min)
Number
dead
Number
alive
Percent
mortality
5
3
7
30
10
31
1
96.9
15
20
0
100
20
20
0
100
5
110
7
94
10
80
0
100
20
5
30
0
100
0(control)
5
0
60
0
10
0
20
0
15
0
20
0
20
0
30
0
5.0
24
15°C
100
80
60
Ca(OH)2
40
NaOH
Percent Mortality
20
0
0
5
10
15
20
25
30
35
25
30
35
22°C
100
80
60
40
20
0
0
5
10
15
20
Exposure Time (min)
Figure 1. Summary of percent mortality of veligers versus time of exposure in replicated trials of
Ca(OH)2 and NaOH at pH 12 at two temperatures.