Introduction

Petromyzon marinus (Linnaeus, 1758; sea lamprey) is a parasitic fish native to the Atlantic Ocean that entered the Laurentian Great Lakes in the early to mid-1900s, leading to significant declines in native fish populations (Smith and Tibbles, 1980; Docker and others, 2021). In response to the economic and ecological threat posed by sea lamprey, the Great Lakes Fishery Commission (GLFC) was established in 1955 by a treaty between the United States and Canada. The GLFC was tasked with developing and implementing a program to control sea lamprey populations (Siefkes and others, 2013). Among its earliest initiatives, the GLFC funded research that led to the discovery of 3-(trifluoromethyl)-4-nitrophenol (TFM), a pesticide selective for sea lamprey, commonly referred to as a lampricide (Applegate and others, 1958). A few years later, Howell and others (1964, 1980) demonstrated that incorporating 0.5 to 2 percent niclosamide by mass significantly reduced the amount of costly TFM used per treatment while maintaining selectivity for the target species. Since then, applications of TFM or TFM combined with niclosamide have been used to target larval sea lamprey in tributaries throughout the Great Lakes. These treatments have become the cornerstone of the GLFC’s Sea Lamprey Control Program (SLCP; Sullivan and others, 2021).

Over the past 35 years, the SLCP has employed two niclosamide formulations in combination with TFM. Bayluscide 70-percent wettable powder, registered as a lampricide in 1990, required specialized delivery techniques that limited its use to streams with easy access and sufficient turbulence to ensure proper mixing and distribution. In addition to these logistical constraints, handling the powder was labor-intensive and posed an inhalation hazard to application personnel. To overcome these challenges, a bayluscide 20-percent emulsifiable concentrate (EC) was registered in 2003 (Luoma and others, 2024; U.S. Environmental Protection Agency [EPA], undated). While EC addressed the difficulties associated with the powdered formulation, it introduced new concerns due to the inclusion of N-methyl-2-pyrrolidone, a solvent identified by the EPA as a developmental and reproductive toxin (EPA, 2022). The presence of N-methyl-2-pyrrolidone raised worker safety concerns and contributed to application equipment degradation and clogging, necessitating frequent cleaning or replacement. To address these issues, scientists at the U.S. Geological Survey (USGS) Upper Midwest Environmental Sciences Center partnered with Battelle UK (Hampshire, United Kingdom) to develop a water-based alternative: a bayluscide 20-percent suspension concentrate (SC). This formulation retains the active ingredient used in both previous products (niclosamide ethanolamine salt, also called bayluscide) and incorporates inert components approved by both the EPA and the Health Canada Pest Management Regulatory Agency (PMRA). Furthermore, a comparative study found no difference in toxicity between the SC and EC formulations towards larval sea lamprey, while SC exhibited slightly reduced toxicity to nontarget species. These findings suggest improved environmental safety and favorable selectivity with the newer formulation (Luoma and others, 2024).

This report summarizes a field performance evaluation of SC conducted by the USGS and the U.S. Fish and Wildlife Service (FWS) on the Indian River, a tributary to the Manistique River in Schoolcraft County, Michigan, in October 2024. The primary objectives of the evaluation were to quantify lampricide concentration profiles resulting from the application of SC alone and in combination with TFM, and to assess the formulation’s compatibility with existing SLCP application procedures and equipment.

Methods

Niclosamide and TFM Concentration Verification

Grab water samples were collected from four evenly spaced positions across the width of the stream at a designated sample collection site located 0.8 km downstream from the lampricide application point. The stream width at this location was approximately 35 meters (m). Therefore, samples were taken at distances of approximately 4.4 m and 8.8 m from each stream bank, as shown in figure 1.

Triplicate water samples were collected in 20-milliliter (mL) glass scintillation vials immediately prior to the start of the lampricide application, at 30-minute intervals throughout the treatment, and again 30 minutes after the application ceased at hour 8. This final sampling point accounted for the time required for the lampricides to travel from the application site to the downstream collection site.

Approximately 500-mL grab samples of lampricide-free water were also collected directly above the application point at hours 0, 4, and 8. This water was used to prepare six control samples at these three time points. In addition, six field-fortified samples containing 15 µg/L niclosamide were prepared at hours 0 and 4, and six more containing 15 µg/L niclosamide and 1.5 mg/L TFM were prepared at hours 5 and 8. These samples were prepared at hour 5 rather than hour 4 to be consistent with the time it took for the lampricides to travel from the application point to the sample collection site.

Following collection, samples were solid phase extracted (SPE) for cleanup and preservation using 1 cubic centimeter Oasis HLB cartridges containing 30 milligrams (mg) of sorbent (Waters Corporation, Milford, Massachusetts). The SPE cartridges were conditioned with 2 mL of high-performance liquid chromatography (HPLC)-grade methanol under a gentle vacuum of 16–27 kilopascals (kPa), followed by a drying period of 1 minute under 50–57 kPa. Subsequently, 2 mL of sample or blank water was gently drawn through the cartridges. The samples were then gently eluted from the cartridges and directly into HPLC vials using 2 mL of eluent solution (0.1 percent ammonium hydroxide in HPLC-grade methanol). Vials were stored in a portable refrigerator at 4 degrees Celsius (°C) in the dark until they were transported to the Upper Midwest Environmental Sciences Center (La Crosse, Wisconsin) for analysis. Within 3 days of collection, samples were quantified on an Agilent 1260 HPLC (Agilent Technologies, Inc., Santa Clara, California) equipped with a Kinetex, 2.6 micrometer, EVO–C18, 50×2.1-millimeter liquid chromatography column (Phenomenex, Inc., Torrance, California) coupled to an Agilent 6460 triple quadrupole tandem mass spectrometer detector in electrospray ionization mode. The column compartment was set to 50 °C. The injection volume for all samples and standards was 1–4 microliters. Mobile phase solution A was prepared by combining 200 mL of liquid chromatography–mass spectrometry (LC–MS)-grade methanol, 800 mL of 18.2 megaohms water, and 385 mg of reagent-grade ammonium acetate. Mobile phase solution B was prepared by combining 1,000 mL of LC–MS-grade methanol and 385 mg of reagent-grade ammonium acetate. Using a flow rate of 0.60 milliliters per minute (mL/min), gradient chromatography was applied starting with an A:B ratio of 80:20. Upon sample injection, a 1.50-minute gradient was applied to reach a final A:B ratio of 20:80 that was held for 0.75 minutes. A 0.05-minute gradient was applied to return to the original A:B ratio of 80:20, where it remained for the duration of the 3.00-minute run. Detector settings are provided in table 2.

Table 2.    

Instrument settings for Agilent 6460 triple quadruple tandem mass spectrometer used to quantify niclosamide and 3-(trifluoromethyl)-4-nitrophenol concentrations for a field performance evaluation of a bayluscide 20-percent suspension concentrate.

[°C, degrees Celsius; L/min, liters per minute; PSI, pounds per square inch; m/z, mass divided by ion charge]

Table 2.    Instrument settings for Agilent 6460 triple quadruple tandem mass spectrometer used to quantify niclosamide and 3-(trifluoromethyl)-4-nitrophenol concentrations for a field performance evaluation of a bayluscide 20-percent suspension concentrate.

Instrument setting	Niclosamide	TFM
Gas temperature (°C)	350	350
Gas flow (L/min)	9	9
Nebulizer (PSI)	25	25
Sheath gas temperature (°C)	400	400
Sheath gas flow (L/min)	11	11
Precursor ion (m/z)	325.1	206.0
Product ion (m/z)	288.9	176.0
Product ion (m/z)	170.9	160.0

Results and Discussion

Stream discharge and water chemistry conditions in the Indian River during the field evaluation are summarized in table 3. Minimal variation was observed for these parameters between the lampricide application point and the sample collection site. As a result, no adjustments to the target concentrations of niclosamide or TFM were required due to changes in water chemistry. However, SC delivery was temporarily halted 30 minutes into the evaluation following the observation of a potential nontarget fish mortality event near the application hose. Lampricide delivery resumed at hour 1.5 after a deer carcass located upstream from the hose was removed. The carcass had created an eddy that caused localized pooling of SC, resulting in the mortality of several Ambloplites rupestris (Rafinesque, 1817; rock bass). Upon resuming treatment, the target niclosamide concentration was reduced to 55 percent of the sea lamprey MLC, or 6.18 μg/L, as a precautionary measure to prevent further nontarget impacts. At hour 3.5, the concentration was increased to 70 percent of the MLC (7.87 μg/L) to initiate co-application with TFM-HP Sea Lamprey Larvicide. Both niclosamide and TFM concentrations were maintained at 70 percent MLC for the remainder of the field trial. No additional fish mortality was observed.

Mean test chemical recovery (±1 standard deviation) for field-fortified control samples was 114 (±3) percent for niclosamide and 106 (±3) percent for TFM. These recovery values were used to adjust the reported lampricide concentrations for water samples obtained at the sample collection site.

During the initial phase of the application, when SC was applied alone, the target concentration of 55 percent of the MLC (6.18 μg/L) was reached and slightly exceeded by hour 2.5, 1 hour after SC delivery resumed following the resolution of the unintended fish mortality event. Between hours 2.5 and 4, the mean niclosamide concentration (±1 standard deviation) was 6.7 (±0.3) μg/L. Although this value was approximately 8 percent above the 55 percent MLC target, concentrations were trending downwards and approaching the target prior to the co-application of the TFM-HP Sea Lamprey Larvicide at hour 4.

SC diluted readily with stream water using standard lampricide application protocols and equipment. This facilitated an even distribution of niclosamide across the stream width, as evidenced by the high degree of precision (less than 2 percent relative standard error) associated with each mean niclosamide concentration data point between hours 2.5 and 4 (fig. 2A).

Similarly, SC combined readily with TFM-HP Sea Lamprey Larvicide, enabling the target concentration of 70 percent of the MLC to be reached within the first hour of the second 4-hour phase of the field evaluation. Target concentrations of both niclosamide and TFM were maintained at steady-state levels with high precision (fig. 2). Between hours 5.5 and 8, the mean (±1 standard deviation) concentrations of niclosamide and TFM were 8.2 (±0.2) μg/L and 0.75 (±0.03) mg/L, respectively (fig. 2). These values were within 5 percent of the target concentrations for both lampricides and resulted in an actual TFM-to-niclosamide application ratio of 100:1.09 by mass, closely aligning with the intended ratio of 100:1 by mass. In addition, lampricide distribution across the stream width was consistent during this period, as indicated by relative standard error values of less than 1.5 percent for both niclosamide and TFM. The field evaluation was concluded at hour 8, as USGS and FWS personnel determined that sufficient data had been collected to demonstrate that SC can be used to successfully execute a lampricide treatment within the standard 12-hour application timeframe.

Overall, the field evaluation demonstrated that SC is compatible with existing SLCP application protocols and equipment. Personnel reported that SC was easily diluted with stream water and mixed effectively with TFM-HP Sea Lamprey Larvicide. These observations align with the lampricide concentration profiles presented in figure 2, which show that target concentrations of niclosamide and TFM were achieved rapidly and with high precision, supporting the successful execution of the field application.

Summary

Petromyzon marinus (Linnaeus, 1758; sea lamprey) is a parasitic, invasive fish of the Laurentian Great Lakes. Since the late 1950s, the Great Lakes Fishery Commission has administered an integrated Sea Lamprey Control Program (SLCP) that relies extensively on the lampricidal chemicals 3-(trifluoromethyl)-4-nitrophenol (TFM) and niclosamide. The currently registered niclosamide formulation, a bayluscide 20-percent emulsifiable concentrate, contains the solvent N-methyl-2-pyrrolidone, which has been associated with worker safety concerns and has contributed to equipment degradation and clogging during application. To address these issues, the U.S. Geological Survey (USGS), in collaboration with Battell UK, developed a water-based alternative: a bayluscide 20-percent suspension concentrate (SC). However, before pursuing regulatory approval for SC as a lampricide, a field performance evaluation was conducted on October 9, 2024, on the Indian River, a tributary to the Manistique River in Schoolcraft County, Michigan. The objective was to assess the formulation’s compatibility with SLCP application procedures and equipment, and to evaluate its ability to deliver precise lampricide concentrations in a timely manner.

During the trial, SC was applied both independently and in combination with TFM. When applied alone, the formulation diluted readily with stream water, achieving the target niclosamide concentration within 1 hour of application. Moreover, concentrations remained within 8 percent of target values, with less than 5 percent variation across the width of the stream, demonstrating consistent and uniform distribution. When co-applied with TFM at a target mass ratio of 100:1 (TFM:niclosamide), SC mixed effectively with TFM, and target concentrations for both lampricides were achieved within 1 hour. Concentrations remained within 5 percent of target values and were evenly distributed across the stream transect, with less than 1.5-percent variation. These results support continued efforts by the USGS to pursue the registration and adoption of SC as a new tool for controlling invasive sea lamprey in the Great Lakes.