Introduction

The commercial and sport fisheries in the Laurentian Great Lakes collapsed after the invasion and establishment of the nonnative parasitic Petromyzon marinus (sea lamprey) in the early to mid-1900s (Smith and Tibbles, 1980; Siefkes, 2017; Wilkie and others, 2019). In response, the Great Lakes Fishery Commission (GLFC) was formed in the mid-1950s and charged with developing and implementing a program to eradicate or control sea lamprey in the Great Lakes (Siefkes, 2017; Wilkie and others, 2019; Sullivan and others, 2021). Soon after, an intensive chemical screening program resulted in the discovery of the lamprey-selective pesticide known as TFM (4-nitro-3-[trifluoromethyl]phenol, also known as 3-trifluoromethyl-4-nitrophenol; Applegate and others, 1958). TFM treatments of larval lamprey nursery streams began in 1958 and a robust, binational chemical control program was active in all the Great Lakes by the mid-1980s (Sullivan and others, 2021). Howell and others (1964) determined that coapplying as much as 2 percent niclosamide (2′,5-dichloro-4′-nitrosalicylanilide) with TFM substantially reduced the amount of TFM required to kill larval sea lamprey while maintaining selectivity in a broad range of water chemistries. The pesticide known as Bayluscide (2′,5-dichloro-4′-nitrosalicylanilide ethanolamine salt), a more water-soluble salt formulation of niclosamide, was integrated into the program to reduce the amount of TFM required during stream treatments and a bottom-release granular formulation was developed for use in population assessment surveys and for treating lentic areas (Boogaard and others, 2003; Wilkie and others, 2019; Sullivan and others, 2021). A Bayluscide 70-percent wettable powder formulation was developed and used as a TFM additive during stream treatments and was followed by a Bayluscide 20-percent emulsifiable concentrate (EC) formulation. The U.S. Fish and Wildlife Service first registered the Bayluscide 20-percent EC formulation in the United States and Canada in 2003 (U.S. Environmental Protection Agency registration number 6704-92, Health Canada registration number 27407). The 20-percent EC formulation improved worker safety by eliminating the inhalation hazard associated with the 70-percent wettable powder formulation. However, degradation of application tubing, clogging of application equipment, difficult application equipment cleaning, and worker safety concerns related to the solvent in the formulation, N-methyl-2-pyrrolidone, prompted efforts to develop a suitable replacement.

Scientists from the U.S. Geological Survey Upper Midwest Environmental Sciences Center (UMESC; La Crosse, Wisconsin) and Battelle UK, Ltd. (Havant, Hampshire, United Kingdom) collaborated to develop a Bayluscide 20-percent suspension concentrate (SC) formulation. To aid product registration and transition, the Bayluscide SC formulation was intentionally developed using approved pesticide inert ingredients and the same concentration of active ingredient as the Bayluscide EC formulation.

The purpose of this report is to communicate comparative toxicity data for the Bayluscide 20-percent SC formulation and the Bayluscide 20-percent EC formulation to support regulatory decisions. The specific objective of the study was to determine and compare the lethal concentrations (LC) of the 20-percent SC and 20-percent EC formulations required to kill 25, 50, and 99.9 percent of larval sea lamprey, Oncorhynchus mykiss (rainbow trout), Perca flavescens (yellow perch), and Lepomis macrochirus (bluegill) using stream treatment mimicking 12-hour continuous-flow exposures.

Methods

Bayluscide 20-percent EC and Bayluscide 20-percent SC produced by the Coating Place, Inc. (Verona, Wisconsin) and Battelle UK, Ltd. (Havant, Hampshire, United Kingdom), respectively, were used as the test articles. Reagent grade niclosamide was acquired from MilliporeSigma (Burlington, Massachusetts) and used as the reference article for the high-performance liquid chromatography analysis of test article concentrations in water samples.

Test Animals

Test animal collection, handling, and testing were completed under procedures approved by the UMESC Animal Care and Use and Biosecurity Committees. A total of 600 test animals of each species were used in the tests. Nontarget test fish were reared at and obtained from the UMESC fish culture facility. Larval sea lamprey were collected in June 2023 from the Chocolay River (Marquette County, Michigan) using backpack electrofishers (AbP–2, ETS Electrofishing Systems, LLC, Madison, Wis.) and the methods described in U.S. Fish and Wildlife Service “Larval Assessment Sampling Protocol Using the AbP–2 Backpack Electrofisher In Great Lakes Streams” (Larval Assessment Task Force, 2019). After collection, larval lamprey were transported to UMESC in temperature-adjusted well water within a sealed, insulated, and oxygenated 370-liter (L) fish transportation tank. The larval lamprey were maintained within a biosecure facility in a modified recirculating Living Stream system (model LS-510, Frigid Units, Inc., Toledo, Ohio) that contained 500 L of well water, a floating bead biofilter, and approximately 8 centimeters (cm) of sand to allow for larval lamprey burrowing (fig. 1). Larval lamprey were offered a daily ration consisting of a mixture of Microvert Invertebrate Food (Kent Marine, Franklin, Wis.), instant dry yeast (Fleischmann’s, Oakbrook Terrace, Illinois), chlorella powder (Nutricost Organic, Vineyard, Utah), and spirulina powder (Nutricost Organic, Vineyard, Utah).

Toxicity Tests

Three independent continuous-flow toxicity tests were completed with each Bayluscide formulation and fish species using the methods described by the American Society for Testing and Materials (ASTM International, 2014) and the “Standard Operating Procedures for Application of Lampricides in the Great Lakes Fishery Commission Integrated Management of Sea Lamprey (Petromyzon marinus) Control Program” (Barber and Van Kempen, 2024). Exposures were 12 hours in duration, and the test animals remained in the test chambers with untreated flow-through well water for an additional 12 hours to assess latent mortality. Tests were completed using continuous-flow diluter systems constructed according to Bills and Johnson (1992; fig. 2). Well water was used in all tests, and untreated water chemistry properties had a mean pH, dissolved oxygen (DO), temperature, alkalinity, and hardness of 7.95 standard units, 9.35 milligrams per liter (mg/L), 13.4 degrees Celsius (°C), 141 mg/L as calcium carbonate calcium carbonate, and 174 mg/L as calcium carbonate, respectively. Each diluter system consisted of nine serially diluted test chambers and an untreated control chamber. The test systems mimicked stream treatments because they steadily increased exposure concentrations over 2–3 hours until they plateaued at peak levels that were then maintained for about 9 hours (fig. 3).

The individual glass test chambers (25 cm wide x 49 cm long x 30.5 cm high) served as the experimental units. Each test chamber contained 10 test animals and 20 L of test water. Test chambers were supplied with a continuous flow of test water at a rate of 500 milliliters per minute, which resulted in about one test chamber volume exchange every 40 minutes. Stock solutions of the Bayluscide test articles were delivered to the diluter mixing boxes using digital peristaltic chemical delivery pumps (Masterflex L/S model 07551–20, Cole-Parmer Instrument Company, Barrington, Ill.). The Bayluscide test articles were mixed and serially diluted with well water flowing from the head box and then delivered to the test chambers (fig. 2). Serial dilutions resulted in each successive Bayluscide concentration being approximately 80 percent of the previous concentration.

Results

Test animal were of the same origin and their sizes were similar between formulations (table 1). Water quality properties were also similar among tests and remained well within suitable parameters (ASTM International, 2014). Mean DO ranged from 9.1 to 9.5 mg/L with corresponding oxygen saturations greater than 85-percent, mean pH values ranged from 7.93 to 8.09, and mean water temperatures ranged from 12.4 to 14.0 °C (table 2).

In all test replicates, control survival was 100 percent, at least one test chamber had partial mortality, and at least one chamber had complete mortality. Two-parameter log-logistic dose-response curves for species and formulation (fig. 4) were used to estimate the LC25, LC50, and LC99.9 for each species (fig. 5). No differences between formulations were detected in the sea lamprey LC values or the LC99.9 values of the same nontarget species. Although differences exist among the LC25 and LC50 values for nontarget species, the differences were minimal and the suspension concentrate formulation was less toxic to nontarget species.

Discussion

The presence of a small quantity of an antimicrobial in the Bayluscide SC formulation warranted a comparative toxicity evaluation against the currently registered Bayluscide EC formulation to provide evidence that could be used to determine the suitability of the Bayluscide SC formulation for inclusion in the GLFC binational sea lamprey control program.

There were nonsignificant differences in the toxicity between the formulations in the target species, sea lamprey. Differences in the LC25 and LC50 values of the pesticide formulations within nontarget species were minimal and no differences were detected in the LC99.9 values of the same species (fig. 5). In contrast to sea lamprey, the SC formulation was less toxic to nontarget species than the EC formulation in all but one case (bluegill LC99.9; fig. 5). The toxicity of both formulations is comparable to the toxicity of niclosamide reported by Marking and Hogan (1967) and Dawson and others (1977).

Although only speculative, the observed differences in Bayluscide formulation toxicity could be related to inert ingredients. The EC formulation contains a surfactant and an organic solvent, which may increase niclosamide solubility and uptake, resulting in higher toxicity. Although minimal, the differences in formulation toxicity may provide increased protection of nontarget species (figs. 4 and 5). Additional investigation may determine if the coapplication of SC formulation with TFM will increase protection to nontarget species. Lake sturgeon (Acipenser fulvescens) is a culturally important species and they are also known to be sensitive to lampricides (Boogaard and others, 2003). Determining if the coapplication of the SC formulation with TFM would increase protection to lake sturgeon could benefit the GLFC binational sea lamprey control program by reducing treatment-related effects to this important species.

The coapplication of Bayluscide with TFM during larval sea lamprey stream treatments can reduce the amount of TFM required by as much as 50 percent while maintaining a selective toxicity profile (Associate Committee on Scientific Criteria for Environmental Quality, 1985; Dawson, 2003). The reduced amount of TFM required for an effective treatment because of the addition of Bayluscide can result in substantial savings. Our tests demonstrate the SC formulation has a similar toxicity profile to the EC formulation. The similar toxicity profile, water soluble characteristics, and more environmentally friendly inert ingredients indicate the SC formulation could be a suitable pesticide for incorporation into the GLFC binational sea lamprey control program. The SC formulation has potential to improve worker safety and reduce application problems and expenses associated with equipment deterioration and cleanup.

Conclusion

This report describes the results of comparative toxicity tests of Bayluscide formulations to inform regulatory decisions for a new Bayluscide 20-percent suspension concentrate (SC) formulation. Continuous-flow and stream treatment mimicking laboratory exposures were completed. Then, comparisons were made between the resulting concentrations lethal to 25, 50 and 99.9 percent of the test animals calculated for each formulation and species. The results demonstrate that the toxicity of the new Bayluscide 20-percent SC formulation does not differ from the toxicity of the Bayluscide 20-percent emulsifiable concentrate formulation to larval sea lamprey and it is slightly less toxic to nontarget species. The results support the continued evaluation and potential registration of the new Bayluscide 20-percent SC formulation for use in the binational sea lamprey control program.