Assessing a low-dose copper treatment for dreissenid mussels: effects on zebra mussel (Dreissena polymorpha) population

Assessing a low-dose copper treatment for dreissenid mussels: effects on zebra mussel (Dreissena polymorpha) population | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Assessing a low-dose copper treatment for dreissenid mussels: effects on zebra mussel (Dreissena polymorpha) population Matthew T. Barbour, James A. Luoma, Angelique Dahlburg, Todd J. Severson, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6205885/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract We conducted and evaluated a low-dose copper treatment (applied as EarthTec QZ) to suppress zebra mussel ( Dreissena polymorpha Pallas 1771) veliger abundance and settlement in a 66.3 ha bay in Lake Minnetonka (Hennepin County, MN) over a 3-y period. We maintained a mean (standard deviation [SD]) concentration of 83.0 (10.3) µg/L as copper over the 10-d treatment period, much lower than the maximum allowable 1 mg/L as copper. Veliger density was reduced from 6.0 veligers/L before treatment to 0.3 veligers/L following the treatment period. Posttreatment zebra mussel settlement was 1900 times lower in the treated bay compared to an untreated bay days after the treatment despite similar pretreatment veliger densities. Veliger density and settlement remained suppressed nearly 2 y following the treatment. Sampling for adult zebra mussels within the treated bay returned variable results but survival of caged adult zebra mussels indicated ~ 30% treatment-related mortality. Copper in surface waters returned to near pretreatment concentrations 90 d after treatment. Our study demonstrates that low-dose applications of a copper molluscicide can effectively reduce zebra mussel veliger densities and settlement. aquatic invasive species population control chemical control tool application technique Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Zebra mussels ( Dreissena polymorpha Pallas 1771) are an invasive, freshwater mollusk that have caused significant economic and ecological impacts outside their native range (Strayer 2009). Prolific reproduction, planktonic larvae (veliger), and rapid growth rates have facilitated the rapid spread and establishment of zebra mussel populations in waterbodies across North America and Europe (Ackerman et al. 1994; Johnson and Carlton 1996; Aldridge et al. 2004). Zebra mussels are highly efficient filter feeders capable of altering food webs and energy pathways in many systems, resulting in limited food availability for native planktivores, including larval fish and zooplankton (MacIsaac 1996; Higgins and Vander Zanden 2010; McEachran et al. 2018; Hansen et al. 2020). Zebra mussel infestations have been associated with reductions in recreation, local economy, and property values due to biofouling of water infrastructure, boats, docks, and beaches (ISAC 2016). Resource managers have limited options for controlling established zebra mussel populations in open water. EarthTec QZ, an acid-stabilized liquid ionic copper formulation, is one of two copper products registered for open water applications (Earth Science Laboratories, Inc. 2021). Since 2014, EarthTec QZ has been used in six rapid-response treatments conducted in Minnesota to attempt the eradication of newly introduced populations in isolated areas within lakes (Lund et al. 2017; Barbour et al. 2018; MNDNR 2021). The rapid-response treatments targeted the maximum allowable concentration of EarthTec QZ based on labeled restrictions (up to 1.0 mg/L as total copper [background + applied]); however, successful prevention of population establishment has failed in most early-detection, rapid-response cases. Often mussels are later found outside the isolated treatment areas resulting in populations establishing in the treated waterbody despite an efficacious treatment within the treatment area. Such was the case for Christmas Lake and Lake Minnewashta in Minnesota (Lund et al. 2017; Dickhart and Edgcumbe 2018). An EarthTec QZ treatment in a quarry lake demonstrated that much lower copper concentrations can effectively reduce and possibly eradicate established zebra mussel populations. Billmeyer Quarry (Lancaster County, PA; 12 ha) was treated with three near-shore applications of EarthTec QZ targeting 0.2 mg/L as copper over 37 d and produced 100% mortality of caged zebra mussels after 40 d (Hammond and Ferris 2019). Zebra mussels were not detected the following 6 y with eDNA analysis, plankton tows for veligers, or surveys for adult mussels. Treatment of a 30-acre lake in Illinois in 2021 with a single dose of EarthTec QZ at 0.24 mg/L as copper resulted in 100% mortality of adult zebra mussels in cages around the waterbody (Hammond et al., 2022). Veligers are reportedly more sensitive than adults to copper by an order of magnitude (Kennedy et al. 2006). Claudi et al. (2013) reported that an 84-h exposure to 50 µg/L as copper prevented veliger settlement. Similarly, McCartney (2016) reported 17 h lethal concentrations to kill 50% (LC 50 ) and 99% (LC 99 ) of veligers with EarthTec QZ were 64 and 18 times lower, respectively, than the reported LC values for adult zebra mussels exposed to copper from a different product, Cutrine Ultra. Zebra mussels reportedly spawn when water temperatures exceed 12 °C (Ram et al. 1996). Gametes are released into the water column for external fertilization and the developing veligers are planktonic for 2 to 3 weeks before settling on and attaching to substrate (Ram et al. 1993, 1996). By targeting peak periods of veliger production (i.e., spawn events), much lower copper concentrations could be used to effectively reduce veliger density thereby reducing zebra mussel recruitment while also minimizing non-target impacts and copper accumulation in a waterbody (McCartney 2016). Our objectives were to evaluate the effectiveness of a low-dose treatment (~6% of maximum allowable treatment concentration) for reducing settlement of zebra mussels in an established lake population and to characterize veliger and settlement density over 3 y following the treatment. A companion paper reports results of the low-dose copper on nontarget species over 3 y after treatment. Methods Study Area Lake Minnetonka (Hennepin County, MN; 5879 ha), is within the metropolitan area of Minneapolis-St. Paul and has a highly developed shoreline. Zebra mussels were first detected in Lake Minnetonka in 2010 and have since become well established throughout the waterbody (MNDNR 2021). We selected 2 bays within the lake, one as a reference and one for treatment with EarthTec QZ. Robinson Bay (~37.2 ha), the reference bay, has a maximum depth of 19.1 m and substrate dominated by gravel and cobble (Figure 1). St. Albans Bay (66.3 ha), the treatment bay, had a maximum depth of 11.3 m and substrate composed of primarily silt and organic material. Additionally, St. Albans Bay is more hydrologically isolated from the main body of Lake Minnetonka, connected with a narrow outlet, whereas Robinson Bay is more open and has no obstructions to the main body of the lake. We selected five sampling sites within each bay at depths of ~3.5 m for collection of pre- and post-treatment samples and for deployment of test organisms. Application EarthTec QZ was applied from a flat-deck boat outfitted with a delivery system that monitored the application rate and total applied volume of product during treatments (Supplement A). We used a novel application technique which attempted to isolate the treatment to the epilimnion by exploiting thermal stratification with the idea of targeting planktonic veligers while decreasing the overall amount of copper required. The depth of the thermocline was determined the day before each application with a thermocline sensor that measured depth and temperature simultaneously (model: PS-2151; Pasco Scientific, Roseville, CA). Mean water temperature was used to determine water density at 0.1 m depth increments with the calculation from McCutcheon et al. (1993). Water density calculations were then used to determine relative thermal resistance (RTR) between the 0.1-m depth increments. The thermocline for each sampling point ( n = 3) was defined as the depth with the greatest RTR (Vallentyne 1957). Existing bathymetric data acquired from the Minnehaha Creek Watershed District (Minnetonka, MN) was used to estimate water volumes above 0.3048 m (1 ft) thermocline increments in ArcMap (version: 10.7; ESRI, Redlands, CA). The estimated water volume above the mean thermocline depth was then used to calculate the volume of EarthTec QZ required for application. EarthTec QZ was applied a total of five times over a 10-d period with applications made on alternate days (i.e., every other day). The target concentration for the first application was 100 µg/L as copper followed by a sustained target of 60 µg/L as copper for the remaining four applications. The initial concentration and subsequent “bump” applications were designed to maintain the target concentration due to copper uptake by the environment (e.g., sediment binding and vegetation uptake; Welsh and Denny 1980). The protocol and application complied with the product label and a permit issued by the Minnesota Department of Natural Resources (permit number 2019-0758). Treatment Assessment Copper Concentration Dissolved copper concentrations were determined in the field from composite samples of 0.45 µm filtered water taken from the five sample sites and analyzed with a spectrophotometer (model: DR3900, Hach Company, Loveland, CO) and porphyrin test kits (product: 2603300, Hach Company). Field measurements of copper were solely used to determine volumes of EarthTec QZ required for subsequent “bump” applications to maintain the targeted concentration. We used inductively coupled plasma-optical emission spectroscopy (ICP-OES; model: 5110; Agilent Technologies, Santa Clara, CA) to determine copper concentrations in water samples collected before, during, and after the treatment. Samples were collected with a drill-powered, peristaltic pump fitted with an inline filter holder and glass-fiber filter (Whatman 934-AH, 47 mm dia.). Filtered samples (15 mL) were acidified with 1153 µL of nitric acid (ACS grade, 70%; Sigma-Aldrich) and transported to the Upper Midwest Environmental Sciences Center (UMESC; La Crosse, WI) for analysis. Samples in St. Albans Bay were collected hourly for 12 h on application days and once per day on non-application days. Samples in Robinson Bay were collected once daily during the treatment. Sampling of copper in both bays during subsequent years was performed in coordination with other sampling efforts (i.e., n = 3 for Robinson and 7 for St. Albans between 2020 and 2022). Robinson Bay was not sampled in 2022. Mean concentrations were calculated by sample collection date. Veliger Abundance Veliger abundance was estimated from vertical plankton tows collected in both bays 3 to 4 d before the first application and again at 1 d and 14 d after the final application. Samples were collected in both bays during July and August in 2020 and 2021. In 2022, veligers were only sampled in St. Albans Bay at three time points during June, July, and August. We used a 30 cm diameter plankton net with 50 µm mesh sample cup (Aquatic Research Instruments, Hope, ID) and collected triplicate vertical tows near each sampling site. Tows extended from the thermocline to the surface. Samples were preserved in 70% ethanol and transferred to an independent lab (RMB Environmental Laboratories, Bloomington, MN) for enumeration (Supplement B). Veliger abundance was calculated from tow length, net opening area, and veliger counts. Juvenile Settlement Veliger settlement was assessed from plate samplers deployed at the sampling sites ( n = 30/bay; n = 6/site). Samplers were deployed in mid-May, about 2 months before treatment, to allow biofilm development and pretreatment settlement. Samplers were constructed of 4 PVC plates (15 × 15 cm) stacked vertically along a large eyebolt and spaced by 2.5 cm PVC pipe pieces (~2.5 cm o.d.). The samplers were fixed to a 1 m threaded rod extending vertically from a concrete pad (~0.6 × 0.6 m; Figure 2). Half of the settlement samplers ( n = 3/site) were retrieved from each sampling site approximately 30 d and 90 d after the final application (4 September and 30–31 October 2019, respectively). In 2020, 2021, and 2022, we followed the same timeline with deployment in mid-May and retrieval of 3 samplers per site in both late August or early September and October. Upon retrieval, each sampler was disassembled, both sides of plates were scraped with a razor blade and removed materials were preserved in 70% ethanol. We enumerated the number of zebra mussels under stereomicroscopy (10-40X). The top and bottom plates were omitted from sample enumeration because they were most likely to have been disturbed during retrieval and were accessible to predation. Samplers that overturned during deployment, encrusted with sponge growth, or accidentally spilled during handling were omitted from the analysis (n = 15). Resident Population and Adult Mortality We monitored the resident zebra mussel population density with two methods. We used a petite ponar to sample the benthic invertebrate community, including zebra mussels (native community described and analyzed in Dahlburg et al., IN REVIEW ). Ponar samples were collected in duplicate at each sampling location concurrent with plankton tow sampling. Secondly, a contracted diver (Minnesota Divers, Excelsior, MN) conducted dive surveys to estimate zebra mussel abundance before treatment and 30 d posttreatment. Three 10-m transect lines were placed in 2 to 6 m of water at 4 locations within each bay. Quadrats (0.25 m 2 ) were placed on alternating sides of the transect at regularly spaced intervals ( n = 12 quadrats/transect). All zebra mussels within the quadrats were collected, sorted into live and dead, frozen, and later enumerated. To monitor treatment efficacy, adult zebra mussels were caged and placed at each sampling location ( n = 5/bay). About 48 h before the first EarthTec QZ application adult zebra mussels were collected from substrate in Robinson Bay and removed by cutting the byssal threads with a scalpel. Mussels were held overnight in a submerged mesh bag suspended from a pier in St. Albans Bay. Suitability for testing was determined the next day by challenging the adductor muscle response to gentle pressure applied to opposing valves. Mean (SD) shell length of mussels used was 16.4 (2.4) mm ( n = 50) . Zebra mussels ( n = 50) were indiscriminately distributed into semi-rigid plastic mesh bags (15.2 × 10.2 ×25.4 cm; w × d × h), secured to the top of a native mussel cage, and submerged at each sampling site. Caged adult zebra mussel mortality was assessed 1 d after the treatment period. Mortality was defined as failure to respond to probing and failure to resist opening when pressure was applied to opposing valves. Water Chemistry We collected daily water samples from 1 m below the water surface at each sampling site with a Van Dorn sampler (model: 1120-G45; Wildco, Yulee, FL). Beginning in 2020, we sampled water chemistry at each sampling site at three times between June and August. We measured water quality parameters directly in the Van Dorn sampler including dissolved oxygen and pH with a portable water quality meter (model: HQ40d; Hach Company), temperature with a digital thermometer (model: Mk4; ThermoWorks Company, American Fork, UT), and specific conductance (μS/cm at 25 °C) with a 1-cm conductivity cell and meter (model: AP75; Fisher Scientific Company, Pittsburg, PA). Additional subsamples of water were collected from the Van Dorn sampler and used to determine total hardness and alkalinity with a spectrophotometer (model: DR3900, Hach Company) and test kits (kit numbers: TNT 869 and TNT 870, respectively; Hach Company). Data Analysis We conducted all statistical analysis and created data visualizations in R version 4.1.1, declaring statistical significance at α = 0.05 (Wickham 2016; R Core Team 2021). Mixed-effects analysis of variance models were constructed with the lme4 package to analyze differences within the bays at each sampling time and between sampling times within each bay (Bates et al. 2015; Lenth 2021). For each model, sampling time and bay were fixed effects and sampling location was a random effect nested within each bay (Montgomery 2017). The natural logarithm of veliger abundance per liter was the response variable and a positive constant (1) was added prior to computation to eliminate errors associated with calculating the logarithm of zero. The cube root of zebra mussel settlement was used as the response variable to stabilize the variation within sampling locations. Adult zebra mussel mortality was compared with logistic mixed-effects modeling ( lme4::glmer ) as an odds ratio. Resident zebra mussel populations enumerated in ponar samples and SCUBA surveys were normalized to mussels/m 3 and summarized. Results Application The 10-d treatment of St. Albans Bay started on 22 July 2019 and ended on 31 July 2019. EarthTec QZ was applied on alternate days for a total of 5 applications during the 10-d treatment. The mean (standard deviation) thermocline depth ranged from 5.73 (0.12) to 6.67 (0.47) m and the estimated water volume treated ranged from 2,188,197 to 2,369,924 m 3 . A total of 7286 L of EarthTec QZ was applied over the treatment period (Table 2). Table 2. Volumes of EarthTec QZ applied to St. Albans Bay (66.3 ha) of Lake Minnetonka and nominal doses of applications (Hennepin County, MN) in late July 2019. Date Volume applied (L) Nominal dose a (µg/L as Cu 2+ ) 22 July 3453 94 24 July 1179 32 26 July 1103 30 28 July 248 7 30 July 1303 39 Total 7286 199 b a Based on estimated epilimnion volume of 2.2 million m 3 . b Estimated dose if applied in one application. Treatment Assessment Copper Concentration Background copper concentrations were below the analytical limit of quantification (4.7 µg/L as copper) in both bays immediately before the first application. Daily mean (SD) copper concentration over the course of the treatment period in the treated bay was 83.0 (10.3) µg/L as copper (Figure 3). Copper steadily declined after the treatment from 82.7 (20.9) µg/L on 1 August to 2.92 (3.15) µg/L by 29 October 2019, with only one sample above the limit of quantification. We observed copper in the reference bay at one site on July 22 and 23 at 23.4 and 20.5 µg/L as copper, respectively. On 1 August 2019, elevated copper concentrations were measured at 4 sampling sites in the reference bay (range: 14.5 – 46.1 µg/L as copper). We observed higher than background concentrations of copper in both bays in 2021, two years after our applications. There were no unusual trends or anomalies observed in the monitored water chemistry parameters (Supplement C). Veliger Abundance Veliger abundance was similar between the reference and treated bays before the treatment ( F 56.7 = 1.31; p = 0.1943; Figure 4). Veliger abundance in St. Albans Bay (treated) was significantly reduced in comparison to the reference bay 1 d following the treatment ( F 56.7 = -14.64; p < 0.0001) and remained reduced ( p ≤ 0.0080) until August 2021 ( F 56.7 = -1.60; p = 0.1157). Mean (SD) veliger abundance in St. Albans Bay was reduced from 6.0 (2.8) veligers/L before treatment to 0.3 (0.5) and 0.1 (0.3) veligers/L 2 and 14 d following the last application, respectively. Mean veliger abundance remained ≤ 0.6 veligers/L in St. Albans Bay through 2021, more than 2 y following the treatment. Veliger abundance in St. Albans Bay was decreased compared to pretreatment conditions until July 2022 ( F 93.2 = 3.19; p = 0.0580). Veliger abundance remained similar during the August 2022 sampling with a mean (SD) 6.0 (2.5) veligers/L. Juvenile Settlement Mean (SD) settlement in the St. Albans Bay (treated) was 50.8 (51.4) mussels/m 2 compared to 107,838 (35,739) mussels/m 2 in the reference bay in October 2019 (Figure 5). Zebra mussel settlement was significantly decreased in the treated bay compared to the reference bay through 2021 ( p < 0.0001). Settlement gradually increased in St. Albans Bay each year following the treatment and was similar to Robinson Bay by October 2021 ( F 37.2 = -1.40; p = 0.1687). By 2022, settlement in St. Albans was similar to that in Robinson Bay reaching a mean (SD) of 89,133 (23,989) mussels/m 2 . Settlement within Robinson Bay was greater during the August or September (i.e., early) sampling than it was during the October (i.e., late) sampling each year ( p < 0.0001) and the same trend was noticeable in St. Albans Bay with exception of 2020. Resident Population and Adult Mortality Resident zebra mussel densities were highly variable throughout the study. Overall, the petite ponar method resulted in observations of greater densities, up to 8224 mussels/m 2 in Robinson Bay in July 2020, than did the SCUBA surveys which resulted in a maximum 980 mussels/m 2 observed in St. Albans Bay 5 d before the treatment in 2019. The SCUBA sampling method was more consistent in finding adult zebra mussels in St. Albans Bay with only one sampling effort resulting in no mussels found in July 2020. Both methods found adult zebra mussels in Robinson Bay at all sampling times whereas the ponar method did not return a single zebra mussel in St. Albans Bay until August 2021, 2 y after the treatment. Both sampling methods returned a high degree of variation. Stocked adult zebra mussels had a reduced odds of survival related to the treatment (odds ratio = 0.09, p < 0.0001). Mean (SD) adult survival in the treated bay was 68.0% (23.0) compared to 96.0% (2.4) in the reference bay. Discussion Our observations of decreased veliger abundance and settlement in St. Albans Bay, concurrent with spikes in veliger densities in Robinsons Bay, indicates a strong likelihood of a direct treatment related effect. We have no reason to think that the timing of zebra mussel reproduction would differ between the bays as they are connected waterbodies with similar temperature trends. As such, the changes in veliger abundance in Robinson Bay indicates a seasonal peak that is ideal for the timing of our application with objectives of targeting veliger abundance and settlement. Unexpectedly, we observed prolonged decreases in veliger abundance and settlement through 2020 and into 2021 as a result of the treatment. We anticipated the relatively unaffected adult population (68% survival) in the bay to continue reproducing at a rate similar to the surviving population (i.e., 68% veliger production relative to pretreatment density). However, we observed a greater decrease in veliger abundance and settlements for 2 years following the treatment. It is possible the differences in adult survival and subsequent veliger densities were due to delayed mortality in the adult population as a result of the treatment since our assessments of caged adults were conducted only 1 d after the treatment. Our surveys of the resident population may be too variable to conclude if delayed mortality occurred. Extended monitoring of adult mortality and investigation of copper effects on reproduction in zebra mussels could broaden the understanding of treatment efficacy and help inform decisions on the frequency of treatments. If less-than-lethal exposures to copper affect adult reproductive success it would be an added benefit, consequently extending treatment effects on veliger density and settlement beyond the treatment period. The 30% mortality we observed in stocked zebra mussels is comparable with reported toxicological endpoints from laboratory studies. Luoma et al. ( 2018 ) reported an LC 50 of 125 µg/L as copper after chronic, 14-d exposures at 22°C. By comparison, our treatment was a chronic exposure to around 83.0 µg/L as copper for approximately the same time period albeit warmer temperatures. Incorporating our results and lessons learned into management practice is dependent upon management objectives and strategies. Our treatment applied less copper than most previous zebra mussel control projects (URS Group Inc. 2009; Lund et al. 2017 ; Barbour et al. 2018 ; Hammond and Ferris 2019 ); however, our treatment was population suppression, whereas the goals of the projects cited were eradication. Our study evaluated a long-term management strategy rather than all-out population eradication. These alternative management strategies and objectives range from localized functional eradication to regional propagule suppression, both of which focus on reducing population over longer timeframes and would benefit from decreased effects on nontargets during treatments. Functional eradication is a management strategy to reduce or remove the ecosystem function and therefore the ecological impacts of a population which does not necessarily require the removal or complete eradication of the target species (Green and Grosholz 2020 ). This conceptual framework has yet to be examined in the context of zebra mussels but has shown effectiveness with other aquatic invasive species (Adams et al. 2021 ; Britton et al. 2023 ). At a larger scale, propagule suppression focuses on containment on a regional basis and would include treatments in targeted waterbodies to reduce propagule pressure on uninfested waters (Lampert and Liebhold 2022 ). Regional suppression and control strategies are more complex, requiring information on potential propagule sources, routes of transport, and optimization of efforts (Hauser and McCarthy 2009 ; Bossenbroek et al. 2020 ; Nishimoto et al. 2021 ). Our examination of this copper treatment provides more in-depth assessment of a zebra mussel control treatment earlier studies by monitoring zebra mussel populations for 3 y post-treatment and, when paired with the accompanying paper (Dahlburg et al, IN REVIEW ), nontarget communities. However, we did identify several shortcomings of our methodology. We identified potential sampling bias in our surveys of resident populations within St. Albans Bay which limited detection of treatment related effects to the caged zebra mussels. More frequent veliger sampling from June through August would also improve determination of peak veliger production in the years following treatment. Both of these nuances are easily addressed for any future work by investing in pretreatment surveys to identify the location of resident populations and by increasing sampling frequency. One interesting anomaly we noticed was the decrease in the density of mussels on settlement plates from the early to later collection time. This has implications for the timing of sampler retrieval as data are more accurate since it considers the higher mortality rates among juveniles. We did not quantify biomass on settlement plates in this study, but it is likely that the decrease in density of mussels on the plates was inversely correlated to increased biomass. Future treatment assessments and monitoring would benefit from collecting data in a manner to support Before-After Control-Impact analysis (Conner et al. 2016 ); unfortunately, limited resources prevented enough pretreatment sampling to meet those requirements. In conclusion, the 10-d treatment of St. Albans Bay in July 2019 with a mean (SD) dose of 83.0 (10.3) ug/L as Cu 2+ corresponded with a decline in zebra mussel veliger abundance and settlement lasting into 2021. Concurrent veliger densities from an untreated bay during the same timeframe indicate the treatment occurred during a spawning event and comparative data show decreased veliger abundance and settlement in the treated bay while untreated values remained fairly consistent year to year. Knowledge gaps remain about the sublethal impacts adult zebra mussel, effects on nontarget communities (e.g., phytoplankton), and habitat to better understand potential lasting effects of the treatments on reproduction, population dynamics, environmental fate of the applied copper, and mobility of copper in the food web. Further, the topics of functional eradication and suppression for containment may be worth investigating with similar treatment strategies. Declarations Disclosures This project was funded by the Minnesota Environmental and Natural Resource Trust Fund administered through the Legislative-Citizens Commission on Minnesota Resources, USGS invasive species appropriated funds, the University of Minnesota Aquatic Invasive Species Research Center, and Hennepin County. The project was completed under permits issued by the Minnesota Department of Natural Resources (application permit number 2019-0758 and prohibited invasive species permit number 436) and the Hennepin County Sherriff’s Office Water Patrol Unit (temporary structure permit). Disclaimer Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government Competing Interests David Hammond is employed by Earth Science Laboratories, Inc the producer of EarthTec QZ used in the study discussed in the paper. David contributed technical assistance and guidance on product use and did not have a role in data collection or analysis. Author Contribution MB prepared the original draft.All authors reviewed and edited versions of this manuscript.BB and MB performed the formal analysis.JL and DW secured funding and managed the project.MB, AD, MM, JW, TS, JL, and DW conducted the project.DH and BB provided technical assistance and guidance. Acknowledgement The authors thank Gabe Jabbour (Tonka Bay Marina, Tonka Bay, MN) for his invaluable assistance and hospitality in facilitating the application, storage of EarthTec QZ, and use of his marina. The authors also thank Justin Smerud and Todd Johnson (USGS-UMESC) for their assistance in the field, and Justin Schueller (USGS-UMESC) for conducting the ICP-OES analysis for copper samples. The authors acknowledge the support and outreach efforts of the Lake Minnetonka Conservation District, the cities of Deephaven and Greenwood, the Minnehaha Creek Watershed District, and the Lake Minnetonka Association. The project was completed under permits issued by the Minnesota Department of Natural Resources (application permit number 2019-0758 and prohibited invasive species permit number 436) and the Hennepin County Sherriff’s Office Water Patrol Unit (temporary structure permit). 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Environ Monit Assess 188:14. https://doi.org/10.1007/s10661-016-5526-6 Dahlburg AD, Barbour MT, Luoma JA, Severson TJ, Wise JK, Meulemans MJ, Hammond D, Phelps NBD, Waller DL (IN REVIEW) Assessing low-dose copper treatment for dreissenid mussels: effects on nontarget organisms. Environ Man Dickhart D, Edgcumbe A (2018) Rapid response to zebra mussel infestation 2017: Second treatment: Lake Minnewashta, Carver County, MN. MNDNR, Carver County, MN Earth Science Laboratories, Inc. (2021) EarthTec label. https://www3.epa.gov/pesticides/chem_search/ppls/064962-00001-20151020.pdf Green SJ, Grosholz ED (2020) Functional eradication as a framework for invasive species control. Front Ecol Environ. https://doi.org/10.1002/fee.2277 Hammond D, Bland J, Johnson S (2022) Apparent eradication of zebra mussels ( Dreissena polymorpha ) from an entire lake using low doses of acid-stabilized ionic copper. 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U.S. Department of the Interior, Washington, DC Johnson LE, Carlton JT (1996) Post-establishment spread in large-scale invasions: Dispersal mechanisms of the zebra mussel Dreissena Polymorpha . Ecology 77:1686–1690. https://doi.org/10.2307/2265774 Kennedy AJ, Millward RN, Steevens JA, et al (2006) Relative sensitivity of zebra mussel life stages to two copper sources. J Gt Lakes Res 32:596–606 Lampert A, Liebhold AM (2022) Optimizing the use of suppression zones for containment of invasive species. Ecol Appl 33:. https://doi.org/10.1002/eap.2797 Lenth RV (2021) emmeans : Estimated marginal means, aka least-squares means Lund K, Cattoor KB, Fieldseth E, et al (2017) Zebra mussel ( Dreissena polymorpha ) eradication efforts in Christmas Lake, Minnesota. Lake Reserv Manag 1–14. https://doi.org/10.1080/10402381.2017.1360417 Luoma J, Severson TJ, Barbour MT, Wise JK (2018) Effects of temperature and exposure duration on four potential rapid-response tools for zebra mussel ( Dreissena polymorpha ) eradication. Manag Biol Invasions 9:425–438. https://doi.org/10.3391/mbi.2018.9.4.06 MacIsaac HJ (1996) Potential abiotic and biotic impacts of zebra mussels on the inland waters of North America. Am Zool 36:287–299. https://doi.org/10.1093/icb/36.3.287 McCartney MA (2016) Field evaluation of toxicity of low-dose molluscicide treatments for zebra mussel veliger larvae - potential applications in lake management. Minnesota Aquatic Invasive Species Research Center, Saint Paul, MN McCutcheon SC, Martin JL, Barnwell TO (1993) Chapter 11: Water quality. In: Maidment DR (ed) Handbook of Hydrology, 1st edn. McGraw Hill, New York, NY, p 11.1-11.69 McEachran MC, Trapp RS, Zimmer KD, et al (2018) Stable isotopes indicate that zebra mussels ( Dreissena polymorpha ) increase dependence of lake food webs on littoral energy sources. Freshw Biol 64:183–196. https://doi.org/10.1111/fwb.13206 [MNDNR] Minnesota Department of Natural Resources (2021) Pilot projects to control zebra mussels. https://www.dnr.state.mn.us/invasives/aquaticanimals/zebramussel/pilot_project.html Montgomery DC (2017) Design and Analysis of Experiments, 9th edn. John Wiley and Sons, Hoboken, NJ Nishimoto M, Miyashita T, Yokomizo H, et al (2021) Spatial optimization of invasive species control informed by management practices. Ecol Appl 31:e02261. https://doi.org/10.1002/eap.2261 R Core Team (2021) R : A language and environment for statistical computing Ram JL, Crawford GW, Walker JU, et al (1993) Spawning in the zebra mussel (Dreissena polymorpha ): Activation by internal or external application of serotonin. J Exp Zool 265:587–598. https://doi.org/10.1002/jez.1402650515 Ram JL, Fong PP, Garton DW (1996) Physiological aspects of zebra mussel reproduction: Maturation, spawning, and fertilization. Am Zool 36:326–338. https://doi.org/10.1093/icb/36.3.326 Strayer DL (2009) Twenty years of zebra mussels: lessons from the mollusk that made headlines. Front Ecol Environ 7:135–141. https://doi.org/10.1890/080020 URS Group Inc. (2009) Zebra mussel eradication project. 55 CES/CEV, Offut Air Force Base, NE Vallentyne JR (1957) Principles of modern limnology. Am Sci 45:218–244 Wickham H (2016) ggplot2 : Elegant Graphics for Data Analysis. Springer-Verlag, New York, NY Additional Declarations Competing interest reported. David Hammond is employed by Earth Science Laboratories, Inc the producer of EarthTec QZ used in the study discussed in the paper. David contributed technical assistance and guidance on product use and did not have a role in data collection or analysis. Supplementary Files Targetsupplement.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 27 Jun, 2025 Reviews received at journal 27 Jun, 2025 Reviewers agreed at journal 24 Jun, 2025 Reviews received at journal 16 Apr, 2025 Reviewers agreed at journal 26 Mar, 2025 Reviewers agreed at journal 25 Mar, 2025 Reviewers invited by journal 25 Mar, 2025 Editor assigned by journal 24 Mar, 2025 Submission checks completed at journal 13 Mar, 2025 First submitted to journal 11 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6205885","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":438357891,"identity":"bf25de46-515e-41f6-8d5f-4ae35bbf0ecb","order_by":0,"name":"Matthew T. Barbour","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYDACCQYGZhDNT7oWyQaStRgcIFaH7uzmZ58LKg7nG99If/zyB0OtHEG9ZneOGc+eceaw5bYbOWbWPAzHjQlruZFgzMzbdtjA7EYOmzEDw7HEmQ0EtaR/BmsxnpH+zPAHcVpyILYYSCQYP+BhqEnsJ6AD6JczxcwzzqQbSJx5Y8bMY3DAmGAEmd1u38xcUGFtwN+e/vjjj4o6OTZCWpABmwSDwWFSNACj9AMDQx1pWkbBKBgFo2BEAACt8z/kL9CC6AAAAABJRU5ErkJggg==","orcid":"","institution":"U.S. Geological Survey","correspondingAuthor":true,"prefix":"","firstName":"Matthew","middleName":"T.","lastName":"Barbour","suffix":""},{"id":438357892,"identity":"e6b5be85-3869-49b0-8e63-61058a47b71a","order_by":1,"name":"James A. Luoma","email":"","orcid":"","institution":"U.S. Geological Survey","correspondingAuthor":false,"prefix":"","firstName":"James","middleName":"A.","lastName":"Luoma","suffix":""},{"id":438357893,"identity":"fe3fb298-6024-40f3-b282-3b2504b10855","order_by":2,"name":"Angelique Dahlburg","email":"","orcid":"","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Angelique","middleName":"","lastName":"Dahlburg","suffix":""},{"id":438357894,"identity":"7c282cf9-ae99-46c5-a5b8-ce00c29ef406","order_by":3,"name":"Todd J. Severson","email":"","orcid":"","institution":"U.S. Geological Survey","correspondingAuthor":false,"prefix":"","firstName":"Todd","middleName":"J.","lastName":"Severson","suffix":""},{"id":438357895,"identity":"72f9b94d-3b6d-4fab-acf9-a870aaa43010","order_by":4,"name":"Jeremy K. Wise","email":"","orcid":"","institution":"U.S. Geological Survey","correspondingAuthor":false,"prefix":"","firstName":"Jeremy","middleName":"K.","lastName":"Wise","suffix":""},{"id":438357896,"identity":"053ee8b5-7836-40d7-9c0d-dfa1bb69ccfd","order_by":5,"name":"Matthew J. Meulemans","email":"","orcid":"","institution":"U.S. Geological Survey","correspondingAuthor":false,"prefix":"","firstName":"Matthew","middleName":"J.","lastName":"Meulemans","suffix":""},{"id":438357897,"identity":"9f960feb-6895-4c56-b200-6d5dc9462961","order_by":6,"name":"Barbara Bennie","email":"","orcid":"","institution":"University of Wisconsin-La Crosse","correspondingAuthor":false,"prefix":"","firstName":"Barbara","middleName":"","lastName":"Bennie","suffix":""},{"id":438357899,"identity":"116b17b1-8061-4d59-a9cd-a7b1d38fd09a","order_by":7,"name":"David Hammond","email":"","orcid":"","institution":"Earth Science Laboratories, Inc","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Hammond","suffix":""},{"id":438357902,"identity":"87858acc-6149-4447-b6d4-ec62ef27aad5","order_by":8,"name":"Diane Waller","email":"","orcid":"","institution":"U.S. Geological Survey","correspondingAuthor":false,"prefix":"","firstName":"Diane","middleName":"","lastName":"Waller","suffix":""}],"badges":[],"createdAt":"2025-03-11 18:23:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6205885/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6205885/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80361982,"identity":"8ad89e62-c256-4307-b72c-89dc5baef2ad","added_by":"auto","created_at":"2025-04-11 04:12:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":270272,"visible":true,"origin":"","legend":"\u003cp\u003eStudy area for the assessment of low-dose copper treatment on zebra mussel populations. Black circles represent sampling site locations within each bay.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6205885/v1/20ca5958ea2f4473c6796a71.png"},{"id":80362269,"identity":"82d51da9-8255-40b7-ab2d-c0f598e5b171","added_by":"auto","created_at":"2025-04-11 04:20:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":416777,"visible":true,"origin":"","legend":"\u003cp\u003eZebra mussel settlement plates deployed at each sampling location in the spring and retrieved in late summer and early fall.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6205885/v1/3e421fbe8a18c73bcb223787.png"},{"id":80362275,"identity":"ee492572-931a-4600-bd25-e2af2f71a577","added_by":"auto","created_at":"2025-04-11 04:20:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":181796,"visible":true,"origin":"","legend":"\u003cp\u003eDaily mean copper concentration in St. Albans (treated; black circles) and Robinson (reference; red triangles) bays. St. Albans Bay was treated with EarthTec QZ for 10 d starting on July 22, 2019. The sustained target treatment concentration was 60 µg/L as copper and the realized mean (SD) treatment concentration was 83.0 (10.3) µg/L as copper for the 10-d treatment period. Error bars represent 1 SD. The left pane covers the treatment year with axis labels formatted as YYYY-MM and the right pane includes extended monitoring through 2022.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6205885/v1/12c3409b68805032e7cca270.png"},{"id":80362740,"identity":"51a88354-9224-4242-8957-a845dd2d5a8e","added_by":"auto","created_at":"2025-04-11 04:28:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":123687,"visible":true,"origin":"","legend":"\u003cp\u003eMean veliger density in St. Albans (black circles) and Robinson (red triangles) bays. St. Albans Bay was treated with low-dose copper applications in late July 2019. Robinson Bay was sampled as a reference. No sampling was conducted in Robinson Bay in 2022. Error bars represent 1 SD about the mean.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6205885/v1/d089dd41ad3608b61ae74baf.png"},{"id":80361986,"identity":"971b67ff-4a10-466b-8e02-d92e49e46520","added_by":"auto","created_at":"2025-04-11 04:12:51","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":135260,"visible":true,"origin":"","legend":"\u003cp\u003eMean zebra mussel settlement in St. Albans (black circles) and Robinson (red triangles) bays. St. Albans Bay was treated with low-dose copper applications in late July 2019. Robinson Bay was sampled as a reference. No sampling was conducted in Robinson Bay in 2022. Error bars represent 1 standard deviation about the mean. Zebra mussel settlement represented as thousands per m\u003csup\u003e2\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-6205885/v1/458b3c7e2e91066a222b98da.png"},{"id":80363610,"identity":"b26d9fb1-353c-4cac-a311-a0d543f893ce","added_by":"auto","created_at":"2025-04-11 04:44:51","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1750257,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6205885/v1/33f0ce27-6f8f-496e-ac46-14536efa87e5.pdf"},{"id":80361987,"identity":"077052c0-5332-4525-8e67-00bcd3ae2346","added_by":"auto","created_at":"2025-04-11 04:12:51","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":584921,"visible":true,"origin":"","legend":"","description":"","filename":"Targetsupplement.docx","url":"https://assets-eu.researchsquare.com/files/rs-6205885/v1/8c081b300e28b0ddc90d69cb.docx"}],"financialInterests":"Competing interest reported. David Hammond is employed by Earth Science Laboratories, Inc the producer of EarthTec QZ used in the study discussed in the paper. David contributed technical assistance and guidance on product use and did not have a role in data collection or analysis.","formattedTitle":"Assessing a low-dose copper treatment for dreissenid mussels: effects on zebra mussel (Dreissena polymorpha) population","fulltext":[{"header":"Introduction","content":"\u003cp\u003eZebra mussels (\u003cem\u003eDreissena polymorpha\u003c/em\u003e Pallas 1771) are an invasive, freshwater mollusk that have caused significant economic and ecological impacts outside their native range\u0026nbsp;(Strayer 2009). Prolific reproduction, planktonic larvae (veliger), and rapid growth rates have facilitated the rapid spread and establishment of zebra mussel populations in waterbodies across North America and Europe\u0026nbsp;(Ackerman et al. 1994; Johnson and Carlton 1996; Aldridge et al. 2004). Zebra mussels are highly efficient filter feeders capable of altering food webs and energy pathways in many systems, resulting in limited food availability for native planktivores, including larval fish and zooplankton\u0026nbsp;(MacIsaac 1996; Higgins and Vander Zanden 2010; McEachran et al. 2018; Hansen et al. 2020). Zebra mussel infestations have been associated with reductions in recreation, local economy, and property values due to biofouling of water infrastructure, boats, docks, and beaches\u0026nbsp;(ISAC 2016).\u003c/p\u003e\n\u003cp\u003eResource managers have limited options for controlling established zebra mussel populations in open water. EarthTec QZ, an acid-stabilized liquid ionic copper formulation, is one of two copper products registered for open water applications\u0026nbsp;(Earth Science Laboratories, Inc. 2021). Since 2014, EarthTec QZ has been used in six rapid-response treatments conducted in Minnesota to attempt the eradication of newly introduced populations in isolated areas within lakes\u0026nbsp;(Lund et al. 2017; Barbour et al. 2018; MNDNR 2021). The rapid-response treatments targeted the maximum allowable concentration of EarthTec QZ based on labeled restrictions (up to 1.0 mg/L as total copper [background + applied]); however, successful prevention of population establishment has failed in most early-detection, rapid-response cases. Often mussels are later found outside the isolated treatment areas resulting in populations establishing in the treated waterbody despite an efficacious treatment within the treatment area. Such was the case for Christmas Lake and Lake Minnewashta in Minnesota\u0026nbsp;(Lund et al. 2017; Dickhart and Edgcumbe 2018). An EarthTec QZ treatment in a quarry lake demonstrated that much lower copper concentrations can effectively reduce and possibly eradicate established zebra mussel populations. Billmeyer Quarry (Lancaster County, PA; 12 ha) was treated with three near-shore applications of EarthTec QZ targeting 0.2 mg/L as copper over 37 d and produced 100% mortality of caged zebra mussels after 40 d\u0026nbsp;(Hammond and Ferris 2019). Zebra mussels were not detected the following 6 y with eDNA analysis, plankton tows for veligers, or surveys for adult mussels. Treatment of a 30-acre lake in Illinois in 2021 with a single dose of EarthTec QZ at 0.24 mg/L as copper resulted in 100% mortality of adult zebra mussels in cages around the waterbody (Hammond et al., 2022).\u003c/p\u003e\n\u003cp\u003eVeligers are reportedly more sensitive than adults to copper by an order of magnitude\u0026nbsp;(Kennedy et al. 2006).\u0026nbsp;Claudi et al. (2013)\u0026nbsp;reported that an 84-h exposure to 50 µg/L as copper prevented veliger settlement. Similarly,\u0026nbsp;McCartney (2016)\u0026nbsp;reported 17 h lethal concentrations to kill 50% (LC\u003csub\u003e50\u003c/sub\u003e) and 99% (LC\u003csub\u003e99\u003c/sub\u003e) of veligers with EarthTec QZ were 64 and 18 times lower, respectively, than the reported LC values for adult zebra mussels exposed to copper from a different product, Cutrine Ultra.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eZebra mussels reportedly spawn when water temperatures exceed 12 °C (Ram et al. 1996). Gametes are released into the water column for external fertilization and the developing veligers are planktonic for 2 to 3 weeks before settling on and attaching to substrate (Ram et al. 1993, 1996). By targeting peak periods of veliger production (i.e., spawn events), much lower copper concentrations could be used to effectively reduce veliger density thereby reducing zebra mussel recruitment while also minimizing non-target impacts and copper accumulation in a waterbody (McCartney 2016). Our objectives were to evaluate the effectiveness of a low-dose treatment (~6% of maximum allowable treatment concentration) for reducing settlement of zebra mussels in an established lake population and to characterize veliger and settlement density over 3 y following the treatment. A companion paper reports results of the low-dose copper on nontarget species over 3 y after treatment.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch2\u003eStudy Area\u003c/h2\u003e\n\u003cp\u003eLake Minnetonka (Hennepin County, MN; 5879 ha), is within the metropolitan area of Minneapolis-St. Paul and has a highly developed shoreline. Zebra mussels were first detected in Lake Minnetonka in 2010 and have since become well established throughout the waterbody (MNDNR 2021). We selected 2 bays within the lake, one as a reference and one for treatment with EarthTec QZ. Robinson Bay (~37.2 ha), the reference bay, has a maximum depth of 19.1 m and substrate dominated by gravel and cobble (Figure 1). St. Albans Bay (66.3 ha), the treatment bay, had a maximum depth of 11.3 m and substrate composed of primarily silt and organic material. Additionally, St. Albans Bay is more hydrologically isolated from the main body of Lake Minnetonka, connected with a narrow outlet, whereas Robinson Bay is more open and has no obstructions to the main body of the lake. We selected five sampling sites within each bay at depths of ~3.5 m for collection of pre- and post-treatment samples and for deployment of test organisms.\u003c/p\u003e\n\u003ch2\u003eApplication\u003c/h2\u003e\n\u003cp\u003eEarthTec QZ was applied from a flat-deck boat outfitted with a delivery system that monitored the application rate and total applied volume of product during treatments (Supplement A). We used a novel application technique which attempted to isolate the treatment to the epilimnion by exploiting thermal stratification with the idea of targeting planktonic veligers while decreasing the overall amount of copper required. The depth of the thermocline was determined the day before each application with a thermocline sensor that measured depth and temperature simultaneously (model: PS-2151; Pasco Scientific, Roseville, CA). Mean water temperature was used to determine water density at 0.1 m depth increments with the calculation from\u0026nbsp;McCutcheon et al. (1993). Water density calculations were then used to determine relative thermal resistance (RTR) between the 0.1-m depth increments. The thermocline for each sampling point (\u003cem\u003en\u003c/em\u003e = 3) was defined as the depth with the greatest RTR\u0026nbsp;(Vallentyne 1957). Existing bathymetric data acquired from the Minnehaha Creek Watershed District (Minnetonka, MN) was used to estimate water volumes above 0.3048 m (1 ft) thermocline increments in ArcMap (version: 10.7; ESRI, Redlands, CA). The estimated water volume above the mean thermocline depth was then used to calculate the volume of EarthTec QZ required for application.\u003c/p\u003e\n\u003cp\u003eEarthTec QZ was applied a total of five times over a 10-d period with applications made on alternate days (i.e., every other day). The target concentration for the first application was 100 \u0026micro;g/L as copper followed by a sustained target of 60 \u0026micro;g/L as copper for the remaining four applications. The initial concentration and subsequent \u0026ldquo;bump\u0026rdquo; applications were designed to maintain the target concentration due to copper uptake by the environment (e.g., sediment binding and vegetation uptake; Welsh and Denny 1980). The protocol and application complied with the product label and a permit issued by the Minnesota Department of Natural Resources (permit number 2019-0758).\u003c/p\u003e\n\u003ch2\u003eTreatment Assessment\u003c/h2\u003e\n\u003ch3\u003eCopper Concentration\u003c/h3\u003e\n\u003cp\u003eDissolved copper concentrations were determined in the field from composite samples of\u0026nbsp;0.45 \u0026micro;m filtered water taken from the five sample sites and analyzed with a spectrophotometer (model: DR3900, Hach Company, Loveland, CO)\u0026nbsp;and\u0026nbsp;porphyrin test kits (product: 2603300, Hach Company). Field measurements of copper were solely used to determine volumes of EarthTec QZ required for subsequent \u0026ldquo;bump\u0026rdquo; applications to maintain the targeted concentration.\u003c/p\u003e\n\u003cp\u003eWe used inductively coupled plasma-optical emission spectroscopy (ICP-OES; model: 5110; Agilent Technologies, Santa Clara, CA) to determine copper concentrations in water samples collected before, during, and after the treatment. Samples were collected with a drill-powered, peristaltic pump fitted with an inline filter holder and glass-fiber filter (Whatman 934-AH, 47 mm dia.). Filtered samples (15 mL) were acidified with 1153 \u0026micro;L of nitric acid (ACS grade, 70%; Sigma-Aldrich) and transported to the Upper Midwest Environmental Sciences Center (UMESC; La Crosse, WI) for analysis. Samples in St. Albans Bay were collected hourly for 12 h on application days and once per day on non-application days. Samples in Robinson Bay were collected once daily during the treatment. Sampling of copper in both bays during subsequent years was performed in coordination with other sampling efforts (i.e., n = 3 for Robinson and 7 for St. Albans between 2020 and 2022). Robinson Bay was not sampled in 2022. Mean concentrations were calculated by sample collection date.\u003c/p\u003e\n\u003ch3\u003eVeliger Abundance\u003c/h3\u003e\n\u003cp\u003eVeliger abundance was estimated from vertical plankton tows collected in both bays 3 to 4 d before the first application and again at 1 d and 14 d after the final application. Samples were collected in both bays during July and August in 2020 and 2021. In 2022, veligers were only sampled in St. Albans Bay at three time points during June, July, and August. We used a 30 cm diameter plankton net with 50 \u0026micro;m mesh sample cup (Aquatic Research Instruments, Hope, ID) and collected triplicate vertical tows near each sampling site. Tows extended from the thermocline to the surface. Samples were preserved in 70% ethanol and transferred to an independent lab (RMB Environmental Laboratories, Bloomington, MN) for enumeration (Supplement B). Veliger abundance was calculated from tow length, net opening area, and veliger counts.\u003c/p\u003e\n\u003ch3\u003eJuvenile Settlement\u003c/h3\u003e\n\u003cp\u003eVeliger settlement was assessed from plate samplers deployed at the sampling sites (\u003cem\u003en\u003c/em\u003e = 30/bay; \u003cem\u003en\u003c/em\u003e = 6/site). Samplers were deployed in mid-May, about 2 months before treatment, to allow biofilm development and pretreatment settlement. Samplers were constructed of 4 PVC plates (15 \u0026times; 15 cm) stacked vertically along a large eyebolt and spaced by 2.5 cm PVC pipe pieces (~2.5 cm o.d.). The samplers were fixed to a 1 m threaded rod extending vertically from a concrete pad (~0.6 \u0026times; 0.6 m; Figure 2).\u003c/p\u003e\n\u003cp\u003eHalf of the settlement samplers (\u003cem\u003en\u003c/em\u003e = 3/site) were retrieved from each sampling site approximately 30 d and 90 d after the final application (4 September and 30\u0026ndash;31 October 2019, respectively).\u0026nbsp;In 2020, 2021, and 2022,\u0026nbsp;we followed the same timeline with deployment in mid-May and retrieval of 3 samplers per site in both late August or early September and October. Upon retrieval, each sampler was disassembled, both sides of plates were scraped with a razor blade and removed materials were preserved in 70% ethanol. We enumerated the number of zebra mussels under stereomicroscopy (10-40X). The top and bottom plates were omitted from sample enumeration because they were most likely to have been disturbed during retrieval and were accessible to predation. Samplers that overturned during deployment, encrusted with sponge growth, or accidentally spilled during handling were omitted from the analysis (n = 15).\u003c/p\u003e\n\u003ch3\u003eResident Population and Adult Mortality\u003c/h3\u003e\n\u003cp\u003eWe monitored the resident zebra mussel population density with two methods. We used a petite ponar to sample the benthic invertebrate community, including zebra mussels (native community described and analyzed in Dahlburg et al., \u003cem\u003eIN REVIEW\u003c/em\u003e). Ponar samples were collected in duplicate at each sampling location concurrent with plankton tow sampling. Secondly, a contracted diver (Minnesota Divers, Excelsior, MN)\u0026nbsp;conducted dive surveys\u0026nbsp;to estimate zebra mussel abundance before treatment and 30 d posttreatment. Three 10-m transect lines were placed in 2 to 6 m of water at 4 locations within each bay. Quadrats (0.25 m\u003csup\u003e2\u003c/sup\u003e) were placed on alternating sides of the transect at regularly spaced intervals (\u003cem\u003en\u0026nbsp;\u003c/em\u003e= 12 quadrats/transect). All zebra mussels within the quadrats were collected, sorted into live and dead, frozen, and later enumerated.\u003c/p\u003e\n\u003cp\u003eTo monitor treatment efficacy, adult zebra mussels were caged and placed at each sampling location (\u003cem\u003en\u003c/em\u003e = 5/bay). About 48 h before the first EarthTec QZ application adult zebra mussels were collected from substrate in Robinson Bay and removed by cutting the byssal threads with a scalpel. Mussels were held overnight in a submerged mesh bag suspended from a pier in St. Albans Bay. Suitability for testing was determined the next day by challenging the adductor muscle response to gentle pressure applied to opposing valves. Mean (SD) shell length of mussels used was 16.4 (2.4) mm (\u003cem\u003en\u003c/em\u003e = 50) . Zebra mussels (\u003cem\u003en\u003c/em\u003e = 50) were indiscriminately distributed into semi-rigid plastic mesh bags (15.2 \u0026times; 10.2 \u0026times;25.4 cm; w \u0026times; d \u0026times; h), secured to the top of a native mussel cage, and submerged at each sampling site. Caged adult zebra mussel mortality was assessed 1 d after the treatment period. Mortality was defined as failure to respond to probing and failure to resist opening when pressure was applied to opposing valves.\u003c/p\u003e\n\u003ch2\u003eWater Chemistry\u003c/h2\u003e\n\u003cp\u003eWe collected daily water samples from 1 m below the water surface at each sampling site with a Van Dorn sampler (model: 1120-G45; Wildco, Yulee, FL). Beginning in 2020, we sampled water chemistry at each sampling site at three times between June and August. We measured water quality parameters directly in the Van Dorn sampler including dissolved oxygen and pH with a portable water quality meter (model: HQ40d; Hach Company), temperature with a digital thermometer (model: Mk4; ThermoWorks Company, American Fork, UT), and specific conductance (\u0026mu;S/cm at 25 \u0026deg;C) with a 1-cm conductivity cell and meter (model: AP75; Fisher Scientific Company, Pittsburg, PA). Additional subsamples of water were collected from the Van Dorn sampler and used to determine total hardness and alkalinity with a spectrophotometer (model: DR3900, Hach Company) and test kits (kit numbers: TNT 869 and TNT 870, respectively; Hach Company).\u003c/p\u003e\n\u003ch2\u003eData Analysis\u003c/h2\u003e\n\u003cp\u003eWe conducted all statistical analysis and created data visualizations in \u003cem\u003eR\u003c/em\u003e version 4.1.1, declaring statistical significance at \u0026alpha; = 0.05 (Wickham 2016; R Core Team 2021). Mixed-effects analysis of variance models were constructed with the \u003cem\u003elme4\u003c/em\u003e package to analyze differences within the bays at each sampling time and between sampling times within each bay (Bates et al. 2015; Lenth 2021). For each model, sampling time and bay were fixed effects and sampling location was a random effect nested within each bay (Montgomery 2017). The natural logarithm of veliger abundance per liter was the response variable and a positive constant (1) was added prior to computation to eliminate errors associated with calculating the logarithm of zero. The cube root of zebra mussel settlement was used as the response variable to stabilize the variation within sampling locations. Adult zebra mussel mortality was compared with logistic mixed-effects modeling (\u003cem\u003elme4::glmer\u003c/em\u003e) as an odds ratio. Resident zebra mussel populations enumerated in ponar samples and SCUBA surveys were normalized to mussels/m\u003csup\u003e3\u003c/sup\u003e and summarized.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eApplication\u003c/h2\u003e\n\u003cp\u003eThe 10-d treatment of St. Albans Bay started on 22 July 2019 and ended on 31 July 2019. EarthTec QZ was applied on alternate days for a total of 5 applications during the 10-d treatment. The mean (standard deviation) thermocline depth ranged from 5.73 (0.12) to 6.67 (0.47) m and the estimated water volume treated ranged from 2,188,197 to 2,369,924 m\u003csup\u003e3\u003c/sup\u003e. A total of 7286 L of EarthTec QZ was applied over the treatment period (Table 2).\u003c/p\u003e\n\u003cp\u003eTable 2. Volumes of EarthTec QZ applied to St. Albans Bay (66.3 ha) of Lake Minnetonka and nominal doses of applications (Hennepin County, MN) in late July 2019.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eDate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eVolume applied (L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNominal dose\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003cp\u003e(\u0026micro;g/L as Cu\u003csup\u003e2+\u003c/sup\u003e)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e22 July\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e3453\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e94\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e24 July\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1179\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e26 July\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1103\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e28 July\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e248\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e30 July\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e1303\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e39\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cem\u003eTotal\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cem\u003e7286\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cem\u003e199\u003csup\u003eb\u003c/sup\u003e\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003csup\u003ea\u003c/sup\u003eBased on estimated epilimnion volume of 2.2 million m\u003csup\u003e3\u003c/sup\u003e.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"3\" valign=\"bottom\"\u003e\n \u003cp\u003e\u003csup\u003eb\u003c/sup\u003eEstimated dose if applied in one application.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003ch2\u003eTreatment Assessment\u003c/h2\u003e\n\u003ch3\u003eCopper Concentration\u003c/h3\u003e\n\u003cp\u003eBackground copper concentrations were below the analytical limit of quantification (4.7 \u0026micro;g/L as copper) in both bays immediately before the first application. Daily mean (SD) copper concentration over the course of the treatment period in the treated bay was 83.0 (10.3) \u0026micro;g/L as copper (Figure 3). Copper steadily declined after the treatment from 82.7 (20.9) \u0026micro;g/L on 1 August to 2.92 (3.15) \u0026micro;g/L by 29 October 2019, with only one sample above the limit of quantification. We observed copper in the reference bay at one site on July 22 and 23 at 23.4 and 20.5 \u0026micro;g/L as copper, respectively. On 1 August 2019, elevated copper concentrations were measured at 4 sampling sites in the reference bay (range: 14.5 \u0026ndash; 46.1 \u0026micro;g/L as copper). We observed higher than background concentrations of copper in both bays in 2021, two years after our applications. There were no unusual trends or anomalies observed in the monitored water chemistry parameters (Supplement C).\u003c/p\u003e\n\u003ch3\u003eVeliger Abundance\u003c/h3\u003e\n\u003cp\u003eVeliger abundance was similar between the reference and treated bays before the treatment (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e56.7\u0026nbsp;\u003c/sub\u003e= 1.31; \u003cem\u003ep\u003c/em\u003e = 0.1943; Figure 4). Veliger abundance in St. Albans Bay (treated) was significantly reduced in comparison to the reference bay 1 d following the treatment (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e56.7\u0026nbsp;\u003c/sub\u003e= -14.64; \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001) and remained reduced (\u003cem\u003ep\u003c/em\u003e \u0026le; 0.0080) until August 2021 (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e56.7\u0026nbsp;\u003c/sub\u003e= -1.60; \u003cem\u003ep\u003c/em\u003e = 0.1157). Mean (SD) veliger abundance in St. Albans Bay was reduced from 6.0 (2.8) veligers/L before treatment to 0.3 (0.5) and 0.1 (0.3) veligers/L 2 and 14 d following the last application, respectively. Mean veliger abundance remained \u0026le; 0.6 veligers/L in St. Albans Bay through 2021, more than 2 y following the treatment. Veliger abundance in St. Albans Bay was decreased compared to pretreatment conditions until July 2022 (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e93.2\u0026nbsp;\u003c/sub\u003e= 3.19; \u003cem\u003ep\u003c/em\u003e = 0.0580). Veliger abundance remained similar during the August 2022 sampling with a mean (SD) 6.0 (2.5) veligers/L.\u003c/p\u003e\n\u003ch3\u003eJuvenile Settlement\u003c/h3\u003e\n\u003cp\u003eMean (SD) settlement in the St. Albans Bay (treated) was 50.8 (51.4) mussels/m\u003csup\u003e2\u003c/sup\u003e compared to 107,838 (35,739) mussels/m\u003csup\u003e2\u003c/sup\u003e in the reference bay in October 2019 (Figure 5). Zebra mussel settlement was significantly decreased in the treated bay compared to the reference bay through 2021 (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001). Settlement gradually increased in St. Albans Bay each year following the treatment and was similar to Robinson Bay by October 2021 (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e37.2\u0026nbsp;\u003c/sub\u003e= -1.40; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.1687). By 2022, settlement in St. Albans was similar to that in Robinson Bay reaching a mean (SD) of 89,133 (23,989) mussels/m\u003csup\u003e2\u003c/sup\u003e. Settlement within Robinson Bay was greater during the August or September (i.e., early) sampling than it was during the October (i.e., late) sampling each year (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001) and the same trend was noticeable in St. Albans Bay with exception of 2020.\u003c/p\u003e\n\u003ch3\u003eResident Population and Adult Mortality\u003c/h3\u003e\n\u003cp\u003eResident zebra mussel densities were highly variable throughout the study. Overall, the petite ponar method resulted in observations of greater densities, up to 8224 mussels/m\u003csup\u003e2\u003c/sup\u003e in Robinson Bay in July 2020, than did the SCUBA surveys which resulted in a maximum 980 mussels/m\u003csup\u003e2\u003c/sup\u003e observed in St. Albans Bay 5 d before the treatment in 2019. The SCUBA sampling method was more consistent in finding adult zebra mussels in St. Albans Bay with only one sampling effort resulting in no mussels found in July 2020. Both methods found adult zebra mussels in Robinson Bay at all sampling times whereas the ponar method did not return a single zebra mussel in St. Albans Bay until August 2021, 2 y after the treatment. Both sampling methods returned a high degree of variation. Stocked adult zebra mussels had a reduced odds of survival related to the treatment (odds ratio = 0.09, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001). Mean (SD) adult survival in the treated bay was 68.0% (23.0) compared to 96.0% (2.4) in the reference bay.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur observations of decreased veliger abundance and settlement in St. Albans Bay, concurrent with spikes in veliger densities in Robinsons Bay, indicates a strong likelihood of a direct treatment related effect. We have no reason to think that the timing of zebra mussel reproduction would differ between the bays as they are connected waterbodies with similar temperature trends. As such, the changes in veliger abundance in Robinson Bay indicates a seasonal peak that is ideal for the timing of our application with objectives of targeting veliger abundance and settlement.\u003c/p\u003e \u003cp\u003eUnexpectedly, we observed prolonged decreases in veliger abundance and settlement through 2020 and into 2021 as a result of the treatment. We anticipated the relatively unaffected adult population (68% survival) in the bay to continue reproducing at a rate similar to the surviving population (i.e., 68% veliger production relative to pretreatment density). However, we observed a greater decrease in veliger abundance and settlements for 2 years following the treatment. It is possible the differences in adult survival and subsequent veliger densities were due to delayed mortality in the adult population as a result of the treatment since our assessments of caged adults were conducted only 1 d after the treatment. Our surveys of the resident population may be too variable to conclude if delayed mortality occurred. Extended monitoring of adult mortality and investigation of copper effects on reproduction in zebra mussels could broaden the understanding of treatment efficacy and help inform decisions on the frequency of treatments. If less-than-lethal exposures to copper affect adult reproductive success it would be an added benefit, consequently extending treatment effects on veliger density and settlement beyond the treatment period. The 30% mortality we observed in stocked zebra mussels is comparable with reported toxicological endpoints from laboratory studies. Luoma et al. (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) reported an LC\u003csub\u003e50\u003c/sub\u003e of 125 \u0026micro;g/L as copper after chronic, 14-d exposures at 22\u0026deg;C. By comparison, our treatment was a chronic exposure to around 83.0 \u0026micro;g/L as copper for approximately the same time period albeit warmer temperatures.\u003c/p\u003e \u003cp\u003eIncorporating our results and lessons learned into management practice is dependent upon management objectives and strategies. Our treatment applied less copper than most previous zebra mussel control projects (URS Group Inc. 2009; Lund et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Barbour et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Hammond and Ferris \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e); however, our treatment was population suppression, whereas the goals of the projects cited were eradication. Our study evaluated a long-term management strategy rather than all-out population eradication. These alternative management strategies and objectives range from localized functional eradication to regional propagule suppression, both of which focus on reducing population over longer timeframes and would benefit from decreased effects on nontargets during treatments. Functional eradication is a management strategy to reduce or remove the ecosystem function and therefore the ecological impacts of a population which does not necessarily require the removal or complete eradication of the target species (Green and Grosholz \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This conceptual framework has yet to be examined in the context of zebra mussels but has shown effectiveness with other aquatic invasive species (Adams et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Britton et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). At a larger scale, propagule suppression focuses on containment on a regional basis and would include treatments in targeted waterbodies to reduce propagule pressure on uninfested waters (Lampert and Liebhold \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Regional suppression and control strategies are more complex, requiring information on potential propagule sources, routes of transport, and optimization of efforts (Hauser and McCarthy \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Bossenbroek et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Nishimoto et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur examination of this copper treatment provides more in-depth assessment of a zebra mussel control treatment earlier studies by monitoring zebra mussel populations for 3 y post-treatment and, when paired with the accompanying paper (Dahlburg et al,\u003cem\u003eIN REVIEW\u003c/em\u003e), nontarget communities. However, we did identify several shortcomings of our methodology. We identified potential sampling bias in our surveys of resident populations within St. Albans Bay which limited detection of treatment related effects to the caged zebra mussels. More frequent veliger sampling from June through August would also improve determination of peak veliger production in the years following treatment. Both of these nuances are easily addressed for any future work by investing in pretreatment surveys to identify the location of resident populations and by increasing sampling frequency. One interesting anomaly we noticed was the decrease in the density of mussels on settlement plates from the early to later collection time. This has implications for the timing of sampler retrieval as data are more accurate since it considers the higher mortality rates among juveniles. We did not quantify biomass on settlement plates in this study, but it is likely that the decrease in density of mussels on the plates was inversely correlated to increased biomass. Future treatment assessments and monitoring would benefit from collecting data in a manner to support Before-After Control-Impact analysis (Conner et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e); unfortunately, limited resources prevented enough pretreatment sampling to meet those requirements.\u003c/p\u003e \u003cp\u003eIn conclusion, the 10-d treatment of St. Albans Bay in July 2019 with a mean (SD) dose of 83.0 (10.3) ug/L as Cu\u003csup\u003e2+\u003c/sup\u003e corresponded with a decline in zebra mussel veliger abundance and settlement lasting into 2021. Concurrent veliger densities from an untreated bay during the same timeframe indicate the treatment occurred during a spawning event and comparative data show decreased veliger abundance and settlement in the treated bay while untreated values remained fairly consistent year to year. Knowledge gaps remain about the sublethal impacts adult zebra mussel, effects on nontarget communities (e.g., phytoplankton), and habitat to better understand potential lasting effects of the treatments on reproduction, population dynamics, environmental fate of the applied copper, and mobility of copper in the food web. Further, the topics of functional eradication and suppression for containment may be worth investigating with similar treatment strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDisclosures\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was funded by the Minnesota Environmental and Natural Resource Trust Fund administered through the Legislative-Citizens Commission on Minnesota Resources, USGS invasive species appropriated funds, the University of Minnesota Aquatic Invasive Species Research Center, and Hennepin County. The project was completed under permits issued by the Minnesota Department of Natural Resources (application permit number 2019-0758 and prohibited invasive species permit number 436) and the Hennepin County Sherriff\u0026rsquo;s Office Water Patrol Unit (temporary structure permit).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclaimer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAny use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003cp\u003eDavid Hammond is employed by Earth Science Laboratories, Inc the producer of EarthTec QZ used in the study discussed in the paper. David contributed technical assistance and guidance on product use and did not have a role in data collection or analysis.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMB prepared the original draft.All authors reviewed and edited versions of this manuscript.BB and MB performed the formal analysis.JL and DW secured funding and managed the project.MB, AD, MM, JW, TS, JL, and DW conducted the project.DH and BB provided technical assistance and guidance.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors thank Gabe Jabbour (Tonka Bay Marina, Tonka Bay, MN) for his invaluable assistance and hospitality in facilitating the application, storage of EarthTec QZ, and use of his marina. The authors also thank Justin Smerud and Todd Johnson (USGS-UMESC) for their assistance in the field, and Justin Schueller (USGS-UMESC) for conducting the ICP-OES analysis for copper samples. The authors acknowledge the support and outreach efforts of the Lake Minnetonka Conservation District, the cities of Deephaven and Greenwood, the Minnehaha Creek Watershed District, and the Lake Minnetonka Association. The project was completed under permits issued by the Minnesota Department of Natural Resources (application permit number 2019-0758 and prohibited invasive species permit number 436) and the Hennepin County Sherriff\u0026rsquo;s Office Water Patrol Unit (temporary structure permit).\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData analyzed and described in this work are accessible through the USGS ScienceBase repository at https://doi.org/10.5066/P14JBUQU.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAckerman JD, Sim B, Nichols SJ, Claudi R (1994) A review of the early life history of zebra mussels (\u003cem\u003eDreissena polymorpha\u003c/em\u003e): comparisons with marine bivalves. Can J Zool 72:1169\u0026ndash;1179. https://doi.org/10.1139/z94-157\u003c/li\u003e\n\u003cli\u003eAdams JV, Birceanu O, Chadderton WL, et al (2021) Trade-offs between suppression and eradication of sea lampreys from the Great Lakes. J Gt Lakes Res 47:S782\u0026ndash;S795. https://doi.org/10.1016/j.jglr.2021.04.005\u003c/li\u003e\n\u003cli\u003eAldridge DC, Elliott P, Moggridge GD (2004) The recent and rapid spread of the zebra mussel (\u003cem\u003eDreissena polymorpha\u003c/em\u003e) in Great Britain. Biol Conserv 119:253\u0026ndash;261. https://doi.org/10.1016/j.biocon.2003.11.008\u003c/li\u003e\n\u003cli\u003eBarbour MT, Wise JK, Luoma JA (2018) A bioassay assessment of a zebra mussel (\u003cem\u003eDreissena polymorpha\u003c/em\u003e) eradication treatment. U.S. Geological Survey, Reston, VA\u003c/li\u003e\n\u003cli\u003eBates D, Maechler M, Bolker B, Walker S (2015) Fitting linear mixed-effects models using \u003cem\u003elme4\u003c/em\u003e. J Stat Softw 67:1\u0026ndash;48. https://doi.org/10.18637/jss.v067.i01\u003c/li\u003e\n\u003cli\u003eBossenbroek JM, Kraft CE, Nekolai JC (2020) Prediction of long-distance dispersal using gravity models: Zebra mussel invasion of inland lakes. 12\u003c/li\u003e\n\u003cli\u003eBritton JR, Lynch AJ, Bardal H, et al (2023) Preventing and controlling nonnative species invasions to bend the curve of global freshwater biodiversity loss. Environ Rev 31:310\u0026ndash;326. https://doi.org/10.1139/er-2022-0103\u003c/li\u003e\n\u003cli\u003eClaudi R, Prescott TH, Coffey H (2013) Efficacy of EarthTec algicide for control of quagga and zebra mussels\u003c/li\u003e\n\u003cli\u003eConner MM, Saunders WC, Bouwes N, Jordan C (2016) Evaluating impacts using a BACI design, ratios, and a Bayesian approach with a focus on restoration. Environ Monit Assess 188:14. https://doi.org/10.1007/s10661-016-5526-6\u003c/li\u003e\n\u003cli\u003eDahlburg AD, Barbour MT, Luoma JA, Severson TJ, Wise JK, Meulemans MJ, Hammond D, Phelps NBD, Waller DL (IN REVIEW) Assessing low-dose copper treatment for dreissenid mussels: effects on nontarget organisms. Environ Man\u003c/li\u003e\n\u003cli\u003eDickhart D, Edgcumbe A (2018) Rapid response to zebra mussel infestation 2017: Second treatment: Lake Minnewashta, Carver County, MN. MNDNR, Carver County, MN\u003c/li\u003e\n\u003cli\u003eEarth Science Laboratories, Inc. (2021) EarthTec label. https://www3.epa.gov/pesticides/chem_search/ppls/064962-00001-20151020.pdf\u003c/li\u003e\n\u003cli\u003eGreen SJ, Grosholz ED (2020) Functional eradication as a framework for invasive species control. Front Ecol Environ. https://doi.org/10.1002/fee.2277\u003c/li\u003e\n\u003cli\u003eHammond D, Bland J, Johnson S (2022) Apparent eradication of zebra mussels (\u003cem\u003eDreissena polymorpha\u003c/em\u003e) from an entire lake using low doses of acid-stabilized ionic copper. Proceedings of International Conference on Aquatic Invasive Species (ICAIS), Oostende, Belgium, April 18-22, 2022.\u003c/li\u003e\n\u003cli\u003eHammond D, Ferris G (2019) Low doses of EarthTec QZ ionic copper used in effort to eradicate quagga mussels from an entire Pennsylvania lake. Manag Biol Invasions 10:500\u0026ndash;516. https://doi.org/10.3391/mbi.2019.10.3.07\u003c/li\u003e\n\u003cli\u003eHansen GJA, Ahrenstorff TD, Bethke BJ, et al (2020) Walleye growth declines following zebra mussel and \u003cem\u003eBythotrephes\u003c/em\u003e invasion. Biol Invasions 22:1481\u0026ndash;1495. https://doi.org/10.1007/s10530-020-02198-5\u003c/li\u003e\n\u003cli\u003eHauser CE, McCarthy MA (2009) Streamlining \u0026lsquo;search and destroy\u0026rsquo;: cost-effective surveillance for invasive species management. Ecol Lett 12:683\u0026ndash;692. https://doi.org/10.1111/j.1461-0248.2009.01323.x\u003c/li\u003e\n\u003cli\u003eHiggins SN, Vander Zanden MJ (2010) What a difference a species makes: a meta-analysis of dreissenid mussel impacts on freshwater ecosystems. Ecol Monogr 80:179\u0026ndash;196\u003c/li\u003e\n\u003cli\u003e[ISAC] Invasive Species Advisory Committee (2016) Invasive species impacts on infrastructure. U.S. Department of the Interior, Washington, DC\u003c/li\u003e\n\u003cli\u003eJohnson LE, Carlton JT (1996) Post-establishment spread in large-scale invasions: Dispersal mechanisms of the zebra mussel \u003cem\u003eDreissena Polymorpha\u003c/em\u003e. Ecology 77:1686\u0026ndash;1690. https://doi.org/10.2307/2265774\u003c/li\u003e\n\u003cli\u003eKennedy AJ, Millward RN, Steevens JA, et al (2006) Relative sensitivity of zebra mussel life stages to two copper sources. J Gt Lakes Res 32:596\u0026ndash;606\u003c/li\u003e\n\u003cli\u003eLampert A, Liebhold AM (2022) Optimizing the use of suppression zones for containment of invasive species. Ecol Appl 33:. https://doi.org/10.1002/eap.2797\u003c/li\u003e\n\u003cli\u003eLenth RV (2021) \u003cem\u003eemmeans\u003c/em\u003e: Estimated marginal means, aka least-squares means\u003c/li\u003e\n\u003cli\u003eLund K, Cattoor KB, Fieldseth E, et al (2017) Zebra mussel (\u003cem\u003eDreissena polymorpha\u003c/em\u003e) eradication efforts in Christmas Lake, Minnesota. Lake Reserv Manag 1\u0026ndash;14. https://doi.org/10.1080/10402381.2017.1360417\u003c/li\u003e\n\u003cli\u003eLuoma J, Severson TJ, Barbour MT, Wise JK (2018) Effects of temperature and exposure duration on four potential rapid-response tools for zebra mussel (\u003cem\u003eDreissena polymorpha\u003c/em\u003e) eradication. Manag Biol Invasions 9:425\u0026ndash;438. https://doi.org/10.3391/mbi.2018.9.4.06\u003c/li\u003e\n\u003cli\u003eMacIsaac HJ (1996) Potential abiotic and biotic impacts of zebra mussels on the inland waters of North America. Am Zool 36:287\u0026ndash;299. https://doi.org/10.1093/icb/36.3.287\u003c/li\u003e\n\u003cli\u003eMcCartney MA (2016) Field evaluation of toxicity of low-dose molluscicide treatments for zebra mussel veliger larvae - potential applications in lake management. Minnesota Aquatic Invasive Species Research Center, Saint Paul, MN\u003c/li\u003e\n\u003cli\u003eMcCutcheon SC, Martin JL, Barnwell TO (1993) Chapter 11: Water quality. In: Maidment DR (ed) Handbook of Hydrology, 1st edn. McGraw Hill, New York, NY, p 11.1-11.69\u003c/li\u003e\n\u003cli\u003eMcEachran MC, Trapp RS, Zimmer KD, et al (2018) Stable isotopes indicate that zebra mussels (\u003cem\u003eDreissena polymorpha\u003c/em\u003e) increase dependence of lake food webs on littoral energy sources. Freshw Biol 64:183\u0026ndash;196. https://doi.org/10.1111/fwb.13206\u003c/li\u003e\n\u003cli\u003e[MNDNR] Minnesota Department of Natural Resources (2021) Pilot projects to control zebra mussels. https://www.dnr.state.mn.us/invasives/aquaticanimals/zebramussel/pilot_project.html\u003c/li\u003e\n\u003cli\u003eMontgomery DC (2017) Design and Analysis of Experiments, 9th edn. John Wiley and Sons, Hoboken, NJ\u003c/li\u003e\n\u003cli\u003eNishimoto M, Miyashita T, Yokomizo H, et al (2021) Spatial optimization of invasive species control informed by management practices. Ecol Appl 31:e02261. https://doi.org/10.1002/eap.2261\u003c/li\u003e\n\u003cli\u003eR Core Team (2021) \u003cem\u003eR\u003c/em\u003e: A language and environment for statistical computing\u003c/li\u003e\n\u003cli\u003eRam JL, Crawford GW, Walker JU, et al (1993) Spawning in the zebra mussel \u003cem\u003e(Dreissena polymorpha\u003c/em\u003e): Activation by internal or external application of serotonin. J Exp Zool 265:587\u0026ndash;598. https://doi.org/10.1002/jez.1402650515\u003c/li\u003e\n\u003cli\u003eRam JL, Fong PP, Garton DW (1996) Physiological aspects of zebra mussel reproduction: Maturation, spawning, and fertilization. Am Zool 36:326\u0026ndash;338. https://doi.org/10.1093/icb/36.3.326\u003c/li\u003e\n\u003cli\u003eStrayer DL (2009) Twenty years of zebra mussels: lessons from the mollusk that made headlines. Front Ecol Environ 7:135\u0026ndash;141. https://doi.org/10.1890/080020\u003c/li\u003e\n\u003cli\u003eURS Group Inc. (2009) Zebra mussel eradication project. 55 CES/CEV, Offut Air Force Base, NE\u003c/li\u003e\n\u003cli\u003eVallentyne JR (1957) Principles of modern limnology. Am Sci 45:218\u0026ndash;244\u003c/li\u003e\n\u003cli\u003eWickham H (2016) \u003cem\u003eggplot2\u003c/em\u003e: Elegant Graphics for Data Analysis. Springer-Verlag, New York, NY\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-management","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"emvm","sideBox":"Learn more about [Environmental Management](http://link.springer.com/journal/267)","snPcode":"267","submissionUrl":"https://submission.nature.com/new-submission/267/3","title":"Environmental Management","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"aquatic invasive species, population control, chemical control tool, application technique","lastPublishedDoi":"10.21203/rs.3.rs-6205885/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6205885/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe conducted and evaluated a low-dose copper treatment (applied as EarthTec QZ) to suppress zebra mussel (\u003cem\u003eDreissena polymorpha\u003c/em\u003e Pallas 1771) veliger abundance and settlement in a 66.3 ha bay in Lake Minnetonka (Hennepin County, MN) over a 3-y period. We maintained a mean (standard deviation [SD]) concentration of 83.0 (10.3) \u0026micro;g/L as copper over the 10-d treatment period, much lower than the maximum allowable 1 mg/L as copper. Veliger density was reduced from 6.0 veligers/L before treatment to 0.3 veligers/L following the treatment period. Posttreatment zebra mussel settlement was 1900 times lower in the treated bay compared to an untreated bay days after the treatment despite similar pretreatment veliger densities. Veliger density and settlement remained suppressed nearly 2 y following the treatment. Sampling for adult zebra mussels within the treated bay returned variable results but survival of caged adult zebra mussels indicated\u0026thinsp;~\u0026thinsp;30% treatment-related mortality. Copper in surface waters returned to near pretreatment concentrations 90 d after treatment. Our study demonstrates that low-dose applications of a copper molluscicide can effectively reduce zebra mussel veliger densities and settlement.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"Assessing a low-dose copper treatment for dreissenid mussels: effects on zebra mussel (Dreissena polymorpha) population","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-11 04:12:46","doi":"10.21203/rs.3.rs-6205885/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-27T09:21:02+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-27T09:01:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"245555657249329182491988554339997678795","date":"2025-06-24T07:40:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-16T22:29:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"62279829889376691516122987639241053219","date":"2025-03-26T22:35:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"66699846340297559564190393310995756555","date":"2025-03-25T11:44:34+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-25T08:59:05+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-24T23:38:05+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-13T10:32:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Management","date":"2025-03-11T18:21:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-management","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"emvm","sideBox":"Learn more about [Environmental Management](http://link.springer.com/journal/267)","snPcode":"267","submissionUrl":"https://submission.nature.com/new-submission/267/3","title":"Environmental Management","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"616bda9c-76e5-45e0-b75d-7e935652486c","owner":[],"postedDate":"April 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-07-12T22:38:10+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-11 04:12:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6205885","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6205885","identity":"rs-6205885","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}