Evaluating the Safety and Efficacy of Pseudomonas fluorescens Strain CL145A to Control Dreissenid Mussels
There is an immediate need for effective and safe tools to prevent the spread of planktonic larval dreissenids (quagga Dreissena rostriformis bugensis and zebra mussels D. polymorpha) and to rehabilitate and protect native unionid habitats by controlling existing dreissenid mussel populations in and around the Great Lakes. More than half of the 78 native unionid species in Minnesota, Wisconsin, Iowa, Missouri, Illinois, Indiana, and Ohio are listed as endangered, threatened, or of special concern . Although the US Fish and Wildlife Service (USFWS) considers zebra mussels to be an immediate threat to freshwater unionids in the Upper Mississippi River System, there is no effective tool to control established zebra mussel infestations in native unionid mussel beds. Not only do quagga and zebra mussels harm native mussels, they also disrupt the entire aquatic food change with potentially devastating effects to multiple fish species, including sport fish.
Ecologists at the New York State Museum Field Research Laboratory (NYSM) discovered a strain of the common soil bacterium Pseudomonas fluorescens (Pf-CL145A) originally isolated from river sediments in North America that had selective activity against zebra mussels. Early in this process, NYSM scientists discovered that killed cells of Pf-CL145A had similarly activity as live Pf-CL145A cells against dreissenid mussels, thus all future studies and development will be completed using killed cells of Pf-CL145A. When ingested by filter-feeding zebra and quagga mussels, a cellular component of Pf-CL145A targets the digestive gland of dreissenid mussels , ultimately resulting in their death. As part of the development of the Pf-CL145A killed-cell product at NYSM, a series of non-target laboratory and mesocosm trials were conducted to assess its specificity to zebra and quagga mussels. These acute toxicity trials included a variety of aquatic organisms, including a freshwater ciliate Colpidium colpoda, zooplankton Daphnia magna, freshwater amphipod Hyalella azteca, bluegill Lepomis macrochirus, fathead minnow Pimephales promelas, brown trout Salmo trutta, blue mussels Mytilus edulis and adult unionids Eastern elliptio Elliptio complanata, Eastern lampmussel Lampsilis radiata, creek heelsplitter Lasmigona compressa, giant floater Pyganodon grandis, creeper Strophitus undulates, Eastern floater Pyganodon cataracta. Significant mortality in these non-target organisms did not occur even when exposed to levels of the Pf-CL145A killed-cell product that was lethal to zebra mussels (i.e., exposed animal mortality did not differ from that of untreated controls).
The Pf-CL145A killed-cell product is currently being developed as the commercial product Zequanox™ by Marrone Bio Innovations (MBI; Davis, CA). It was registered by the US Environmental Protection Agency (EPA) on July 29, 2011 to control dreissenid mussels in defined discharges. Studies, including those to assess potential human health risks, were completed by MBI and reviewed by EPA as part of the registration process. Living cells of Pf-CL145A (technically the active ingredient) applied to rats through the skin (dermal), in feed (oral), and by inhalation caused no adverse effect and were non-pathogenic to rats. One measure of toxicity, the oral LD50 (the dose expected to be lethal to 50% of the exposed population) had a value of >5000 mg/kg, placing Pf-CL145A in the lowest risk category for pesticide products. The EPA concluded from the available information that Zequanox™ to be “tolerance exempt ”, one of the highest safety levels assigned by EPA to pesticide products.
This project, funded through a competitive grant award from the EPA’s Great Lakes Restoration Initiative and through appropriated funds from the U.S. Fish and Wildlife Service (USFWS) and the U.S. Geological Survey (USGS), is a collaborative effort between the NYSM, the USGS Upper Midwest Environmental Sciences Center (UMESC) and the USFWS Genoa National Fish Hatchery (GNFH). The project is independent of MBI in all aspects other than providing Zequanox™ for experimental use and does not receive funding from any non-profit organizations or state natural resource agencies. Though Pf-CL145A has proven effective in experiments designed to evaluate its use to control dreissenid mussels in pipes, little research has been conducted to evaluate its potential for controlling existing dreissenid mussel populations in open waters or to prevent the distribution of dreissenid mussel veligers in transported water (i.e., the water in a fish hatchery distribution truck). Comprehensive research is required to determine the potential of Pf-CL145A as a dreissenid management tool for natural aquatic habitats. Determining this potential may ultimately allow this emerging technology to be used to support ecosystem restoration and rehabilitation efforts in and around the Great Lakes.
The three objectives of this project are to:
- Evaluate the acute toxicity of the Pf-CL145A commercial product formulations on freshwater fish and unionids from the Great Lakes and Mississippi River.
- Prevent the spread of dreissenids by developing and demonstrating treatment protocols to use the Pf-CL145A-based product to kill 100% of the planktonic larvae (veligers) in contained systems such as fish transport water and within fish hatcheries.
- Develop application methods for using Pf-CL145A to reduce dreissenid populations within native unionid restoration structures and within native unionid beds. Assess implementation of potential integrated pest management strategies that incorporate the Pf-CL145A-based product as an aid in native unionid restoration.
Objective 1: Evaluate the acute toxicity of the Pf-CL145A commercial product formulation on freshwater fish and unionids from the Great Lakes and Mississippi River.
This project will specifically assess the sensitivity of multiple species of native fish (Table 1) and unionid mussels (Table 2) to the formulated Pf-CL145A product to evaluate its safety, significantly expanding the diversity of non-target organism sensitivity information available. Not only will these trials expand the sensitivity information already available for Pf-CL145A by testing more species of fish and unionids, but also by obtaining the first evaluation of the sensitivity of early life stages of native fish (<1 year-old) and unionids (i.e., glochidia and juvenile [<48-h post glochidia stage] mussels). This critical information will allow unionid conservationists to make informed choices on whether, when, and how to apply this tool to reduce potential impacts on unionids or their fish hosts. The toxicity trials will be conducted at UMESC with mussels (and mussel propagation expertise) provided by GNFH. The acute toxicity of the commercial product formulation of Pf-CL145A to fish and unionids will be assessed following standard testing guidelines for acute toxicity testing wherever possible and will conform to the strict data recording procedures required by Good Laboratory Practice regulations. The information developed in these trials will be essential for potential users of this tool to make informed management decisions as part of Integrated Pest Management (IPM) programs to limit dreissenid mussel populations and support unionid conservation and restoration efforts.
Objective 2: Prevent the spread of dreissenids by developing and demonstrating treatment protocols using the Pf-CL145A-based product to kill 100% of the planktonic larvae (veligers) in contained systems such as fish transport water or within fish hatcheries.
Once established in a water body, dreissenid mussels are virtually impossible to eradicate without affecting the entire ecosystem. Therefore, preventing the spread of zebra and quagga mussels is the most effective and safest means of control. The larval stages of dreissenids, including the feeding veliger stage, are microscopic and swim suspended in the water column for up to three weeks before settling on surfaces on lake or river bottoms. Water containing these larval planktonic dreissenids can inadvertently be transferred to new water bodies by recreational users (i.e., in live wells and bait buckets). Although many fish hatchery managers treat transfer water with salt solutions to kill dreissenid larvae, having another tool to eliminate these larvae from the water without harming the organisms being transferred (e.g. fish or unionids) would be beneficial. Preliminary experiments indicate that dreissenid veligers are even more susceptible to the Pf-CL145A-based product than are attached stages (i.e., juvenile and adult mussels). The NYSM will develop and demonstrate (at bench scales) easy-to-implement veliger treatment protocols. Those protocols will then be “field” tested in collaboration with UMESC and GNFH by evaluating the treatments in commonly used fish transfer units.
Objective 3: Develop application methods for using Pf-CL145A to reduce dreissenid populations within unionid restoration structures and within unionid beds. Assess implementation of potential integrated pest management strategies that incorporate the Pf-CL145A-based product as an aid in native unionid restoration.
Zebra and quagga mussels are an imminent threat to unionid survival in and around the Great Lakes. Some natural resource managers consider existing dreissenid mussel populations to be the greatest threat to unionid populations, many of which are listed as threatened or endangered. Recent evidence indicates that dreissenid mussels are not only a threat to native mussels but they are disrupting the entire Great Lakes ecosystem – from the microscopic organisms at the bottom of the food web all the way up to the most desirable game fish such as coho, lake trout and whitefish. Since there are currently no safe and effective ways to manage dreissenids in unionid beds, restoration efforts have focused on removing attached dreissenids from unionid shells, relocation, and propagation. Propagation cages holding thousands of juvenile unionids, can become encrusted with dreissenids and reduce water circulation (and food for unionids) within the structure, and dreissenid mussels can attach to the juvenile unionids themselves. Lake Pepin (Pool 4 of the Mississippi River) provides an excellent example of the impact of zebra mussels on unionid mussel propagation – unionid propagation in Lake Pepin was extremely successful prior to zebra mussel infestation but was discontinued due to the intense fouling of cages and native juvenile mussels after zebra mussel populations exploded in Lake Pepin.
In close coordination with the Minnesota Department of Natural Resources (DNR), the Wisconsin DNR, the U.S. Army Corps of Engineers and others, pilot field trials will be conducted to evaluate the efficacy of the Pf-CL145A product to control zebra mussels attached to native unionids or mussel propagation cage materials. The pilot studies will be completed in the UMESC mobile assay trailer, a self-contained mobile laboratory. Water from a local water body will be pumped through test vessels enclosed in the mobile laboratory where zebra mussels, attached to mussel propagation cage material and to native unionids, will be exposed under controlled conditions to the Pf-CL145A product. Test water containing the Pf-CL145A product will be disposed of according to procedures established by EPA or the state regulatory agency. Five test sites will be selected from Minnesota and Wisconsin lakes and rivers that have established zebra mussel populations in consultation with the respective state DNR. Mussel propagation cage material will be artificially “seeded” with zebra mussels in the fall of 2011. Pilot field trials will be completed in summer 2012 after non-target studies have been completed. Data from the non-target studies will be summarized and provided to the respective state DNR prior to conducting pilot field efficacy studies. If the pilot trials are successful, studies in 2013 will focus on in-water applications to unionid mussel beds or propagation cages.
A joint study between the USGS UMESC, New York State Museum Field Research Laboratory and the US Fish and Wildlife Service Genoa National Fish Hatchery.
For more information about this project, please contact:
USGS Upper Midwest Environmental Sciences Center
2630 Fanta Reed Road
La Crosse, Wisconsin 54603
USGS Upper Midwest Environmental Sciences Center
2630 Fanta Reed Road
La Crosse, Wisconsin 54603