Assessing low-dose copper treatment for dreissenid mussels: effects on nontarget organisms

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Dahlberg, Matthew T. Barbour, James A. Luoma, Todd J. Severson, and 5 more This is a preprint; it has not been peer reviewed by a journal.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-6205980/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Zebra mussels ( Dreissena polymorpha ), an invasive dreissenid mussel, have been established and caused considerable effects in many North American aquatic ecosystems. In response, copper-based pesticides have been used to manage zebra mussel populations. We evaluated the effects of a low-dose copper-based molluscicide for zebra mussel suppression on nontarget species in Lake Minnetonka (Minnesota, USA). Our study evaluated nontarget effects before and after treatment. Chlorophyll- a concentration increased in both the treated and reference bays 1 and 14 d posttreatment. Zooplankton community composition changed in both bays over the course of this study; zooplankton abundance and diversity initially decreased in the treated bay but gradually recovered and was back to pretreatment and reference bay levels after one year. We observed no significant differences in benthic invertebrate abundance or diversity between the treated and reference bays, although abundance and diversity estimates were dynamic and uncertain. Among caged organisms, copper bioaccumulation was higher in both mussel species than in fish, and among fish, was highest in fathead minnow ( Pimephales promelas ). These findings contribute to our understanding of the potential effects of copper-based pesticides on aquatic ecosystems and provide insights for zebra mussel management. bioaccumulation community response nontarget effects aquatic invasive species control tool Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Zebra mussels ( Dreissena polymorpha ) were introduced to the Laurentian Great Lakes of North America in the mid-1980s and have since spread to and established in hundreds of waterbodies across North America (Mackie, 1991 ). Outside their native range, zebra mussels alter food webs, change water clarity, biofoul water treatment and power generation facilities, and more (MacIsaac, 1996 ; Connelly et al., 2007 ; Strayer, 2009 ; McEachran et al., 2018 ; Hansen et al., 2020 ; Fantle-Lepczyk et al., 2022 ). To address and mitigate these effects, managers are turning to an increasing number of control strategies (Fernald and Watson, 2014 ; Barbour et al. 2018 ; Lund et al. 2018 ; Hammond and Ferris, 2019 ; Dahlberg et al., 2023 ). One such method of control is the application of copper-based pesticides (Claudi et al. 2014 ; Luoma et al. 2018 ; Hammond and Ferris 2019 ; Hammond et al. 2022 ). Copper-based pesticides have been used for over a century in lake management, largely to control algae, although also as an herbicide and molluscicide (Hanson and Stefan, 1984 ; Kamrin 1997 ; Dahlberg et al. 2023 ). As early as 2004, copper-based pesticides were used to control zebra mussels in open water (Claudi et al., 2014 ; Luoma et al., 2018 ; Hammond and Ferris, 2019 ). Copper-based pesticides available today include copper sulfate (CuSO 4 ), Cutrine-Ultra (EPA registration number 67690-98; SePro Corp.), Natrix (67690-81; SePro Corp.), and EarthTec QZ (64962-1; Earth Science Laboratories, Inc.). Of those, only EarthTec QZ and Natrix are registered by the U.S. Environmental Protection Agency (USEPA) as molluscicides for dreissenid mussel control. In aquatic environments, copper affects gilled organisms by rapidly binding to gill membranes where it interferes with osmoregulatory processes by competing with essential monovalent cations, blocking Na + uptake at transport sites, or inhibiting Na + /K + ATPase (Grosell and Wood, 2002 ; Paquin et al., 2002 ). At high concentrations of copper, gilled organisms can become deficient in ions such as Na + and Cl − , causing a cascade of physiological responses including cardiovascular collapse and mortality (Kamunde and Wood, 2004 ). Copper exposure is also linked to increased tissue concentrations of reactive oxygen species (ROS), which may lead to DNA damage (Bopp et al., 2008 ). Copper can be toxic to fish (De Oliveira-Filho et al., 2004 ; Wagner et al., 2017 ), zooplankton (Havens, 1994 ; Williams et al., 2015 ), chironomids (Kosalwat and Knight, 1987 ; Warrin et al., 2009 ), snails (Brix et al., 2011 ; Carmosini et al., 2018 ), and other aquatic organisms (Zyadah and Abdel-Baky, 2000 ; Murray-Gulde et al., 2002 ; Wang et al., 2007 ). Although numerous studies describe the effects of copper on a wide range of freshwater organisms, few studies have evaluated how open water copper-based pesticide applications affect organisms in dynamic ecosystems (Dahlberg et al., 2023 ). We conducted a low-dose copper-based zebra mussel suppression treatment in 2019 in a 66.3 ha bay (St. Albans Bay) of Lake Minnetonka, Minnesota, and returned to monitor post-treatment conditions in 2020, 2021, and 2022 (Barbour et al., in review). Our objectives were to describe the short- and long-term effects of low-dose copper treatment on nontarget organisms and to compare copper uptake (concentration) in tissues of zebra mussels and native fish and mussels. Details of the application and results of the targeted zebra mussel population control and monitoring are reported in Barbour et al. (in review). Methods Study sites and treatment application Lake Minnetonka (Hennepin County, Minnesota, USA; 5879 ha) is located within the metropolitan area of Minneapolis-St. Paul and is a highly urbanized and popular recreational lake. Zebra mussels were first confirmed in the lake in 2010 and have since become well-established throughout the lake (MN DNR, 2024). In 2019, from July 20 through July 30, a largescale zebra mussel suppression treatment using EarthTec QZ was conducted in St. Albans Bay (66.3 ha) of Lake Minnetonka (refer to Barbour et al., in review, for detailed treatment information; Fig. 1 ). Briefly, EarthTec QZ was applied via a flat-deck boat at an initial target concentration of 100 µg Cu/L, with regular bump treatments to maintain a dose of 60 µg Cu/L for 10 d. St. Albans Bay is in the southeast corner of the lake and is connected to the main body of the lake through a narrow boating channel. Robinson Bay (~ 37.2 ha) of Lake Minnetonka was used as the untreated reference site (control bay). Robinson Bay is located on the east-central side of the lake and has no obstructions to the main body of the lake. Longer-term effects of the treatment were monitored during the summer field seasons (May-October) of 2020 (both bays), 2021 (both bays), and 2022 (St. Albans Bay only). Within each bay, we indiscriminately selected five sites in water ~ 3-m deep for deployment of test organisms. Phytoplankton (Chlorophyll-a) We measured chlorophyll- a and Secchi disk transparency as proxies for phytoplankton abundance. In year 1 (2019, our treatment year), we collected triplicate water samples at 2 d pretreatment, 1 d posttreatment, and 14 d posttreatment at each sampling site in each bay using a Van Dorn sampler (1 m depth, n = 5 sites per bay). Samples were transferred to amber glass bottles, stored on ice, and delivered to a contract laboratory (RMB Environmental Laboratories, Bloomington, Minnesota) within 24 h for chlorophyll- a analysis with a fluorometric method (Supplement 1). In years 2–4 (2020–2022), we collected triplicate water samples again (July and August of years 2 and 3, and June and August of year 4). Samples were collected and analyzed using the same methods by the U.S. Geological Survey (USGS) Upper Midwest Environmental Sciences Center (UMESC; La Crosse, Wisconsin). For statistical analysis, all chlorophyll- a concentrations below the detection limit (1.0 µg/L) were assigned one-half the detection limit (0.5 µg/L; Helsel, 1990 ). Secchi disk transparency was measured at each sampling site in triplicate, concurrent with chlorophyll sampling. Zooplankton monitoring The zooplankton community present in each bay at the time of sampling was determined from the same plankton tow samples collected for veliger enumeration; refer to Barbour et al., in review, for complete methods. Samples were collected using a 30-cm diameter plankton net with a 50-µm mesh sample cup (Aquatic Research Instruments, Hope, Idaho). In year 1 (2019), we collected triplicate tow samples at 1 d pretreatment, 2 d posttreatment, and 14 d posttreatment at each sampling site ( n = 3 tows at 5 sites per bay). In years 2–4, we collected triplicate tow samples in July and August (the reference bay was not sampled in year 4). Samples were preserved in 70% ethanol and transferred to a contract laboratory (RMB Environmental Laboratories) for enumeration. Zooplankton were enumerated and identified to the family-level under microscopy (RMB Environmental Laboratories; Supplement 1). Organism density (individuals per liter) was calculated from tow length and net opening area. From years 1–3, we conducted vertical plankton tows from the thermocline (mean depths 5.73–6.67 m) to the surface. In year 4, vertical tows were conducted from 3-m depth to the surface. Benthic invertebrate monitoring The benthic invertebrate community of each bay was sampled with a petite ponar (model: 1728-G30; Wildco, Yulee, Florida). In 2019, we collected duplicate samples at 1 d pretreatment, 2 d posttreatment, and 14 d posttreatment at each sampling site ( n = 5). In 2020, 2021, and 2022 we collected duplicate samples in July and August at the same five sites. To sort invertebrates from the sediment, all ponar grabs were elutriated for 10 min (Magdych 1981 ) then washed through a series of mesh screens (final mesh: 500 µm). Finally, samples were preserved in 70% ethanol. Benthic invertebrates were enumerated and identified to family level (RMB Environmental Laboratories). Caged fish and mussels We evaluated the effects of the copper treatment on four species of native fish that occur in the lake: yellow perch ( Perca flavescens ), fathead minnow ( Pimephales promelas ), bluegill ( Lepomis macrochirus ), and largemouth bass ( Micropterus salmoides ); and one species of native mussel, fatmucket ( Lampsilis siliquoidea ). Fish (young of year) and fatmuckets (juvenile, 1 y old) were reared at UMESC. We measured total length and mass of a subsample ( n = 20) of each fish species and measured the maximum shell length (anterior-posterior axis parallel to the hinge line) of a subsample of fatmuckets ( n = 20; Table 1 ). Fish and mussels were transported with appropriate permits from UMESC to the study area in oxygen-overlaid bags and acclimated onsite to lake temperature (~ 5°C increase) by floating the bags in coolers with lake water for > 1.5 h. For each fish species, individuals were indiscriminately distributed into holding cages (30.5 × 91.4 cm, dia × h; 0.48 cm 2 mesh netting; n = 5 sites per bay, n = 4 cages per site, n = 20 individuals per cage with one species per cage) and submerged at each sampling site ~ 18 h before the first application. The cages were suspended ~ 0.5 m below the water surface and secured to a floating PVC frame positioned around each sampling buoy. Twenty fatmuckets were indiscriminately placed into holding cages (15-L plastic buckets; n = 5 sites per bay) with openings on each side (15.2 × 20.3 cm) and in the lid (12.7 × 12.7 cm), covered by mesh (0.48 × 0.48 cm) to allow water exchange. Approximately 4 cm of sand was placed in each cage for mussels to position and burrow. The cages were placed on the lakebed at each sampling site ( n = 5 sites per bay). Table 1 Mean (standard deviation) mass and total length of fishes and shell length of bivalves caged in study areas during a low-dose copper treatment for zebra mussel veligers. Species Mass (g) Length (mm) n Bluegill 2.62 (0.57) 56.35 (4.23) 20 Fathead minnow 2.75 (0.86) 60.20 (6.14) 20 Largemouth bass 2.03 (0.39) 56.95 (3.61) 20 Yellow perch 4.77 (1.85) 81.75 (9.15) 20 Fatmucket - 21.45 (2.07) 20 Zebra mussel - 16.37 (2.37) 50 Adult zebra mussels were placed next to fatmucket cages for comparison, as described in Barbour et al. (in review). Briefly, adult zebra mussels were collected from encrusted rocks found in Robinson Bay 2 d pretreatment by severing byssal threads with a scalpel. Collected zebra mussels were held overnight in a submerged mesh bag suspended from a pier in St. Albans Bay. Suitability for testing was determined 1 d pretreatment by challenging the adductor muscle response to gentle pressure applied to opposing valves. We measured shell length on a subset of 50 zebra mussels (Table 1 ). Fifty zebra mussels were indiscriminately distributed into semi-rigid plastic mesh bags (15.2 × 10.2 × 25.4 cm; w × d × h; n = 5 sites per bay), secured to the top of a native mussel cage, and submerged at each sampling site. One cage per species ( n = 4 fish species, n = 1 fatmucket, n = 1 zebra mussel) was deployed at each sampling site ( n = 5 sites per bay) the day before the first EarthTec QZ application. Cages were retrieved 2 d after the last application. We calculated survival based on the number of stocked individuals, not the number recovered, because dead organisms may have decomposed before cage retrieval. Damaged cages with suspected escape of test organisms were omitted from the analyses ( n = 2 cages). Copper Bioaccumulation We indiscriminately sampled a subset of each species from the cages at each sampling site for tissue copper analysis. Whole, live fish were individually packaged in centrifuge tubes and frozen ( n = 5 fish/species/site, with 5 sites per bay). When fewer than five fish were alive in a cage, we preserved all remaining live animals for tissue copper analysis. Soft tissues were removed from fatmuckets and frozen in centrifuge tubes ( n = 5 mussels per site, 5 sites per bay). Fifteen live zebra mussels were indiscriminately collected from each sampling site, the soft tissue was removed from the shell, and pooled into three composite samples per sampling site ( n = 5 mussels per sample). Organisms and tissues were rinsed with clean well water to remove copper residues from exterior surfaces. Tissues were placed on ice, transported to UMESC and frozen at 20 ºC until analysis. Samples were then shipped to the Iowa State University Veterinary Diagnostics Laboratory (Ames, Iowa) for analysis with a digestion and inductively coupled plasma method (Supplement 1). Analysis We conducted all statistical analyses and created data visualizations in R version 4.1.1, utilizing the lme4 and vegan packages (Bates et al. 2015 ; R Core Team, 2019; Oksanen et al. 2020), and interpreted differences as significant when α < 0.05. We constructed mixed-effects analysis of variance models to assess the effect of copper treatment on chlorophyll- a concentration, benthic invertebrate abundance and diversity, and zooplankton abundance and diversity. We used logistic mixed-effects modeling as an odds ratio to assess the effect of the treatment on nontarget species mortality. We constructed mixed-effects analysis of variance models using transformed concentration values as the response variable for survival to determine the effect of the treatment on the tissue copper concentrations of our caged organisms. Transformations were performed to stabilize residual variance. Copper concentrations in the tissue of largemouth bass, yellow perch, bluegill, fatmucket, and zebra mussel were natural log transformed. Fathead minnow concentrations were inverse log transformed. We compared differences between sampling times within each bay and between the bays at each sampling time. In each model, sampling time and bay were treated as fixed effects, and sampling location was treated as a random effect nested within each bay (Montgomery, 2017 ). Zooplankton and benthic invertebrate community structures at family level were compared across all sampling times within each bay using permutational analysis of variance (PERMANOVA) for comparisons. Results Phytoplankton (Chlorophyll-a) The mean chlorophyll- a concentration was similar in the reference and treatment bays before treatment ( p = 0.22, Fig. 2 ). At 1 d posttreatment, the treatment bay had significantly higher chlorophyll- a concentrations than the reference bay ( p < 0.01) and at 14 d posttreatment the reference bay had significantly higher concentrations ( p < 0.01). In 2020, chlorophyll- a concentrations were significantly higher in the treated bay compared to the reference bay ( p 0.56). Secchi disk transparency was inversely related to chlorophyll- a concentration (Fig. 2 ). Zooplankton community Before treatment, zooplankton abundance was higher in the treated bay than the reference bay ( p 0.999) and 14 d ( p = 0.175) posttreatment samplings. In the treated bay, zooplankton abundance decreased between pretreatment and 1 d posttreatment ( p = 0.002). Zooplankton abundance was similar at 14 d posttreatment to 1 d posttreatment ( p = 0.982) but higher than pretreatment abundance ( p = 0.056). By August of 2020, one year posttreatment, zooplankton abundance was again higher in the treated bay than in the reference ( p < 0.001). Zooplankton abundance was similar between the two bays in July 2021 ( p = 0.129) and higher in the treated bay compared to the reference bay in August 2021 ( p = 0.004). In 2022, zooplankton abundance was similar in the treated bay in every comparison except August 2021 to July 2022 ( p values all > 0.355, except when August 2022 had higher abundance than July 2022 and p = 0.052). Zooplankton diversity was higher in the reference bay than the treated bay pretreatment ( p = 0.0002; Fig. 4 ). Following treatment, diversity was stable in the reference bay (all p > 0.999) and remained higher than in the treated bay ( p = 0.000). In the treated bay, diversity decreased between pretreatment and 1 d posttreatment ( p = 0.000). Diversity remained similarly low at 14 d posttreatment ( p > 0.999 when comparing 1 d to 14 d posttreatment). By August of 2020, however, diversity in the treated bay increased and was similar to that in the reference bay ( p = 0.54). Treatment bay diversity remained similar to the reference bay in 2021 (both p values > 0.82) and diversity decreased in 2022 from its 2021 levels (excluding July 2021 compared to August 2022, all p values < 0.05; July 2021 compared to August 2022 p = 0.18), with June 2022 levels similar to immediately post treatment ( p = 0.89). The shift in zooplankton abundance and diversity was reflected in changes in species community composition in both bays (Fig. 5 ). Species composition changed in both treated and reference bays immediately following treatment (early- and mid-August 2019). Among specific native zooplankton taxa, Daphniidae and Diaptomidae decreased most in the treated bay, although densities did not remain low in 2020 and 2021. Not all families were affected by treatment. For example, in 2019, Cyclopidae densities in the treated bay closely mirrored the densities in the reference bay with mean densities deviating by < 2 per L at sampling times. In the reference bay, Cyclopidae family densities changed the most throughout the observed period; densities ranged from 68.4 per L in July 2020 to 14.2 per L in July 2021. Other families, such as Leptodoridae and Temoridae, had consistent densities throughout the observation period. Overall, zooplankton communities within each bay differed at nearly every sampling date, highlighting the natural variability of populations independent of treatment (PERMANOVA; only four comparisons had p > 0.05, n = 46 for the treated bay and n = 22 for the reference bay). Within the reference bay, each July composition was different from every other July composition (PERMANOVA; p ≤ 0.002 for n = 3 measurements), each August was different from every other August (PERMANOVA; p = 0.001 for each of n = 3 mid-August measurements), and an early August assessment in 2019 was also different from a mid-August assessment that same year (PERMANOVA; p = 0.001). Similarly, within the treated bay, each July composition was different from every other July composition (PERMANOVA; p ≤ 0.002 for n = 6 measurements), each August was different from every other August (PERMANOVA; ≤ 0.002 for each of n = 6 mid-August measurements). An early August assessment in 2019, conducted immediately following treatment, was also different from a mid-August assessment that same year, conducted two weeks post-treatment (PERMANOVA; p = 0.001). Benthic invertebrate community Benthic invertebrate abundance in the reference bay was similar throughout the study period (all p > 0.10; Fig. 6 ) with the exception that August 2020 had higher abundance than pretreatment ( p = 0.06). Within the treated bay, benthic invertebrate abundance was similar at every observation (all p > 0.62). Benthic invertebrate diversity did not change in the reference bay (all p > 0.12; Fig. 7 ). Within the treated bay, benthic invertebrate diversity was only different in July 2021 (all other p > 0.97). July 2021 had significantly lower diversity than was observed pretreatment, immediately posttreatment, and during August 2021 (all p < 0.03). As benthic invertebrate abundance and diversity changed throughout the sampling periods, species community compositions shifted within each bay (Fig. 8 ). Community composition was most represented by 13 families, including Dreissenidae. Thirty of the taxonomic groups were represented by 0–2 organisms on most sampling dates. The majority of organisms represented 13 taxonomic groups. No difference was detected in community structures within bays between pretreatment and 1 d post treatment (PERMANOVA; p ≤ 0.015), nor between 1 d posttreatment and 14 d posttreatment ( p ≥ 0.051), although the pretreatment communities of both bays were significantly different than the communities 14 d post treatment (PERMANOVA; p ≤ 0.015). Both bays were characterized by organisms in the Chironomidae, Hyalellidae, and Asellidae families. Caged fish and mussels Fathead minnow and adult zebra mussels both had reduced survival odds in the treatment bay (odds ratio [OR] = 0.030, p = 0.0066 and OR = 0.09, p < 0.0001, respectively). Fathead minnow mean survival was 84.0% (26.1 standard deviation (SD)) in the reference bay but only 38.0% (24.1 SD) in the treated bay. Similarly, zebra mussel mean survival was 96.0% (2.4 SD) in the reference bay but only 68.0% (23.0 SD) in the treated bay. Survival and recovery of fish species other than bluegill was low (< 89%) in both bays, and two fish cages from Robinson Bay were omitted from analysis due to damage and likely escape of test animals (Table 2 ). Fatmucket mortality was only observed in the reference bay ( n = 4 mussels). Table 2 Mean (standard deviation) percent survival and percent recovery of caged test animals in Robinson (reference) and St. Albans (treated) Bays after the low dose copper treatment period; n = number of cages recovered. Species Robinson Bay St. Albans Bay Survival % recovery n Survival % recovery n Bluegill 100.0 (0) 100.0 (0) 4 91.0 (4.2) 91.0 (6.5) 5 Fathead minnow a 84.0 (26.1) 87.0 (26.4) 5 38.0 (24.1) 53.0 (37.5) 5 Largemouth bass 73.8 (45.9) 81.3 (34.2) 4 71.0 (35.4) 76.0 (29.9) 5 Yellow perch 46.0 (27.5) 85.0 (11.2) 5 35.0 (26.7) 87.0 (15.7) 5 Fatmucket 96.0 (4.2) 100.0 (0) 5 100.0 (0) 99.0 (2.2) 5 Zebra mussel a 96.0 (2.4) 99.6 (0.9) 5 68.0 (23.0) 100.0 (0) 5 a Statistical difference in survival between bays (α = 0.05). Tissue copper concentrations were greater for all species in the treated bay than the reference bay (Table 3 ). Mean tissue copper concentration was greatest in zebra mussels (40.85 µg/g), followed by native mussels (26.41 µg/g). Of the fish species, fathead minnow had the greatest mean tissue copper concentration. Tissue copper concentration in fathead minnow from the treated bay varied widely but averaged an order of magnitude greater than those in the reference bay (7.53 versus 0.75 µg/g, respectively). Table 3 Mean (standard deviation) tissue copper concentration (µg/g) measured in test organism tissues from Robinson (reference) and St. Albans (treated) Bays following treatment with low dose copper. Robinson Bay St. Albans Bay Species Tissue copper (µg/g) n Tissue copper (µg/g) n Bluegill a 0.51 (0.40) 21 0.93 (0.42) 25 Fathead minnow a 0.75 (0.10) 25 7.53 (12.29) 20 Largemouth bass a 0.52 (0.07) 16 0.97 (0.23) 22 Yellow perch a 0.50 (0.21) 22 2.70 (1.30) 20 Fatmucket a 1.22 (0.28) 25 26.41 (7.93) 25 Zebra mussel a 3.00 (0.86) 15 40.85 (10.17) 15 a Statistical difference between bays (α = 0.05). Discussion This study assessed the short- and long-term effects of a low-dose copper treatment on nontarget organisms. We compared the copper uptake (concentration) in the tissues of zebra mussels and native fish and mussel species and closely monitored the changes in chlorophyll- a and zooplankton and benthic invertebrate populations. We found the lake system to be highly dynamic with positive and negative shorter- and longer-term treatment-induced changes, as well as changes that were likely the result of natural processes. We observed a short-term increase in chlorophyll- a accompanied by a decrease in Secchi disk transparency in the treated bay immediately (1 d) posttreatment, indicative of an algae bloom. This was unexpected because copper is used as an algicide (Aryal 2018 ). This may have occurred because treatment coincided with an algal bloom that had already begun when treatment was initiated. Additionally, the elevated chlorophyll- a concentrations observed in 2020 in the treated bay may be related to the reduction in zebra mussels that year. However, our data do not capture the phytoplankton community structure needed to better understand or identify the drivers behind the chlorophyll- a increase. Both chlorophyll- a concentration and Secchi disk transparency returned to near pretreatment levels within two weeks posttreatment. Overall zooplankton abundance and diversity decreased following treatment (1 d and 14 d posttreatment), although taxa were affected differently. For example, while Daphniidae and Diaptomidae abundance decreased, Cyclopidae abundance was unchanged. This overall decline was expected, as previous studies reported zooplankton species — particularly Daphniidae — to be highly sensitive to copper (McIntosh and Kevern, 1974 ; Naddy et al., 2002 ). These declines and changes in species could have trophic-level effects on algae and fish (Dettmers et al., 2003 ; Zhao et al. 2008 ), warranting further research in this area. However, because this lake has been affected by zebra mussels for more than a decade (MN DNR 2024), zooplankton communities have likely already been altered from a pre-infestation state. Additionally, because zooplankton are sensitive to changes in nutrient concentrations and predator-prey interactions, their decline may not have been solely treatment-related (McIntosh and Kevern, 1974 ; Naddy et al., 2002 ). One year after treatment, zooplankton overall abundance had surpassed pretreatment abundance. These data, combined with our PERMANOVA analysis, indicate a full recovery of the community, consistent with past research documenting similar rebounds after copper exposures (McIntosh and Kevern, 1974 ). The variability observed in zooplankton community structure within both bays fits descriptions of naturally chaotic planktonic community structure (Benincà et al., 2008 ). Abundance may therefore be a more responsive metric than community structure when assessing how a treatment changes the overall zooplankton community over short time periods such as this single treatment, four-year study. Overall, the zooplankton community recovered faster than the zebra mussel population (refer to Barbour et al., in review), although zooplankton abundance and diversity estimates were dynamic. Given this, it may be important for managers to identify their goals and select treatment plans accordingly. For example, managers interested in increasing biodiversity and improving natural communities in a waterbody may expect an initial decline in zooplankton species and anticipate a recovery within a calendar year. Alternatively, managers seeking to maximize fish production may want to avoid copper treatment to not limit food availability, or time the treatment to not overlap with when larval fish are most reliant on zooplankton. Benthic invertebrate abundance and diversity had no observed effect in response to treatment. Community structure in the treatment bay also remained unchanged, except for one different community present in July of 2021. We were unable to compare the communities of the treatment and reference bays in a meaningful way because substrate type varied between the bays and benthic communities are known to change with substrate (Tolonen et al., 2001 ; White and Irvine, 2003 ). Although previous research indicates benthic invertebrates are susceptible to copper (Montz et al., 2011 ), changes in abundance and community structure may also be attributed to the boom-bust life histories of some species during the project period (Galatowitsch 2014 ). Considering life history dynamics would provide important insight when planning copper-based treatments to avoid critical life stages and help to understand how treatment effects differ between organisms that live in versus on the sediment as well as how timing may affect different species. For example, a benthic invertebrate species with a slower and seasonal reproductive cycle may have delayed recovery, especially if they are sensitive to copper, because these species cannot reproduce until the following year. One family with seasonal reproduction, Hyalellidae, has been shown to be highly sensitive to copper concentrations similar to those in our treatment. One species in that family, Hyalella azteca , has a reported 48-h lethal concentration 50 (LC50) of 87 µg/L in similar water chemistry as observed in our treated bay (Schubauer-Berigan et al., 1993 ). In our study, we observed a family-level decline among Hyalellidae immediately following treatment, which was likely due to the copper exposure. Species in Hyalellidae, such as Hyalella azteca , are a food source for fish species, including bluegill (Camacho and Thacker 2013 ), and reductions in that family may have repercussions for the larger trophic structure within a waterbody. Among species with varying reproduction and emergence times, however, attributing abundance changes to treatment is more difficult. For example, Chironomidae have varied reproduction timing. One species in that family, Chironomus riparius , has a reported 24-h LC50 for first instars of 2.09 mg/L as copper in soft water (8 mg/L as CaCO 3 ; Béchard et al., 2008 ). Although we observed a decline in this family following treatment, because these species’ emergence times are not seasonally restricted, the decline in larval and pupal chironomids possibly corresponded to a major adult emergence event rather than the treatment. Posttreatment monitoring in subsequent years found that community structures more closely resembled pretreatment conditions, but further research may determine whether any response is directly attributable to treatment. The effects of copper treatment on fish mortality were confounded by low survival in both bays, likely from environmental stressors (e.g., housing, high boat traffic, predator species observed immediately outside cages). Fathead minnow were the only native test species that experienced treatment-related mortality, although control survival was < 90%. Among the fish species, fathead minnow had the highest tissue concentrations of copper. Although we cannot say for certain that those tissue copper concentrations were related to low survival, waterborne copper exposure is known to cause a loss of branchial Na + and Cl − , which triggers a cascade of physiological responses culminating in cardiovascular collapse (Kamunde and Wood, 2004 ). Of note, the fish evaluated in this study were caged and subjected to the full dose of the treatment. In the natural environment, we would expect fish to move away from physiological stressors to areas of less stressful refuge (Svecevičius, 1999 ; Ezeonyejiaku et al., 2011 ). In contrast to fish, fatmucket mortality was zero in the treatment bay; the only fatmucket deaths occurred in the reference bay. Although our experiment does not show treatment-related mortality among fatmuckets, past research has reported newly transformed juvenile fatmucket LC50 values well below our treatment mean copper concentration (e.g., 96-h EC50 values of survival in reconstituted hardwater reported by Wang et al. 2009 were 31–44 µg Cu/L). Fatmuckets possibly could have experienced delayed mortality not found in our data, which could be an area of future research. Copper bioaccumulation was higher in both zebra mussels and fatmuckets than in fish species, in agreement with past research (Wang and Rainbow, 2008 ). Adult zebra mussels had the highest tissue copper concentration of the species in this study (Table 3 ). Unlike the fatmucket mussels, elevated tissue copper concentration in zebra mussels was accompanied by 32% mortality in the treated bay (Table 2 ). These results are consistent with a 10-d laboratory study using comparable copper concentrations that found zebra mussel tissues contained 40–60 µg Cu/g (Mersch et al., 1993 ; Le et al., 2021 ). The effects of copper differ among organ systems in freshwater fish, zebra mussels, and other species (Gundacker 1999 ; Malhotra et al., 2020 ; Le et al., 2021 ). Histological or molecular examination of organisms or organ systems could determine sublethal physiological effects of low-dose copper that may predispose animals to secondary or chronic stressors. Potential future directions Further research is warranted to investigate the optimal frequency and duration of treatments, considering factors such as the specific ecosystem characteristics and the life history traits of nontarget organisms. For example, implementing a treatment plan with no-treatment intervals longer than 1 year could allow zooplankton and benthic invertebrate communities to rebound. However, waiting three years or more may provide zebra mussels a chance to recolonize and fully recover from the treatment, indicating there may be an optimal timeframe for treatment that supports nontarget species recovery without allowing zebra mussels to rebound. By allowing nontarget species communities sufficient time to recover, managers could potentially develop more pragmatic management practices for suppressing zebra mussels with minimal long-term effects. Additionally, management decisions on invasive mussel control require managers to determine a goal for population suppression and align it with the chosen treatment strategy and its consequences for the conservation of native species and ecosystem health. For example, complete eradication of established zebra mussel populations may not be feasible with a low-dose copper treatment; however, if the goal is ecosystem restoration, or maintaining ecosystem function, a low-dose copper treatment could reduce zebra mussel density below a level of impact while also accounting for the health of native communities. Gaining a better understanding of the trade-offs between the benefits of zebra mussel control and the potential nontarget effects will be crucial in developing sustainable and ecologically sound management approaches. Conclusion The copper treatment in our study was aimed to reduce zebra mussel recruitment and settlement while minimizing treatment effects to nontarget species. Phytoplankton showed a short-term population increase following treatment, as measured through chlorophyll- a concentration. Zooplankton experienced an overall decline in abundance and diversity, although taxa responded differently, and estimates were dynamic and uncertain, and both abundance and diversity recovered within one year. Some benthic invertebrate families also decreased in abundance along with overall diversity and appeared to recover within two years. Fish species were difficult to assess due to low survival rates in our experimental cages, although we did observe that fish accumulated less copper than mussels. Overall, these findings indicate that although some limited non-target effects were caused by a low-dose copper treatment, it may be a viable strategy for future management controlling invasive mussels while minimizing collateral damage to the ecosystem. Declarations Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. Data Availability The data in support of this publication are available at https://doi.org/10.5066/P14JBUQU (Barbour et al., 2025). Acknowledgements We are grateful to Justin Schueller and Courtney Kirkeeng (USGS-UMESC) for their Inductively Coupled Plasma analysis of our copper samples, and Justin Smerud and Todd Johnson (USGS-UMESC) for their assistance with sample collections. We thank Gabriel Jabbour (Tonka Bay Marina, Tonka Bay, Minnesota) for his invaluable assistance and hospitality in facilitating the application and storing the EarthTec QZ and allowing us to use his marina. We thank Dr. Barbara Bennie (University of Wisconsin La Crosse) for her assistance with statistical analyses. We are grateful to the cities of Deephaven and Greenwood, Minn., as well as the Minnehaha Creek Watershed District and Lake Minnetonka Association for their support and outreach efforts. Funding Funding was provided by the Minnesota Environment and Natural Resources Trust Fund as recommended by the Minnesota Aquatic Invasive Species Research Center (MAISRC) and the Legislative-Citizen Commission on Minnesota Resources (LCCMR), and the State of Minnesota. Funding support for this project was also provided by the U.S. Geological Survey Ecological Missions Area Biological Threats and Invasive Species Research Program and by Hennepin County (Minn.). Funding for the MAISRC Zebra Mussel Research Fellowship that supported AD was provided by the Fletcher Family Foundation, Pelican Lakes Association of Crow Wing County, and Bay Lake Improvement Association. Competing interests AD, MB, JL, TS, JW, BB, NP, and DW declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. DH is an employee of Earth Science Laboratories, manufacturer of the molluscicide utilized in this study. This study explains the product’s effects on target and nontarget species. DH contributed to the design of the treatment protocol but not in collection or analysis of data. Author contributions Conceptualization: James A. Luoma, Diane L. Waller, and Nicholas B. D. Phelps Data curation: Matthew T. Barbour, Todd J. Severson, Jeremy K. Wise, and Matthew Meulemans Formal analysis: Matthew T. Barbour Funding acquisition: James A. Luoma, Diane L. Waller, and Nicholas B. D. Phelps Investigation: Angelique D. Dahlberg, Matthew T. Barbour, James A. Luoma, Todd J. Severson, Jeremy K. Wise, Matthew Meulemans, and Diane L. Waller Methodology: James A. Luoma, Diane L. Waller, David Hammond, Nicholas B. D. Phelps, and Matthew T. Barbour Project administration: James A. Luoma, Diane L. Waller, and Nicholas B. D. Phelps Resources: David Hammond Supervision: James A. Luoma, Diane L. Waller, and Nicholas B. D. Phelps Validation: Matthew T. Barbour Visualization: Matthew T. Barbour Writing – original draft: Matthew T. Barbour and Angelique D. Dahlburg Writing – review & editing: James A. Luoma, Todd J. Severson, Jeremy K. Wise, Matthew Meulemans, Nicholas B. D. Phelps, and Diane L. Waller Ethics approval 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). The use and treatment of test organisms in this study was approved by the Animal Care and Use Committee at UMESC (Study plan AEH-18-LowCu-01), and hatchery reared organisms were certified specific pathogen free by the U.S. Fish and Wildlife Service’s La Crosse Fish Health Center (La Crosse, Wisconsin). References Aryal, D., 2018. Evaluating the effectiveness of algaecide in a continuous flow through system. Thesis, University of Akron. 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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 Nontargetsupplement.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 14 May, 2025 Reviews received at journal 06 May, 2025 Reviews received at journal 15 Apr, 2025 Reviewers agreed at journal 27 Mar, 2025 Reviewers agreed at journal 26 Mar, 2025 Reviewers invited by journal 25 Mar, 2025 Editor assigned by journal 24 Mar, 2025 Submission checks completed at journal 12 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. 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Wise","email":"","orcid":"","institution":"U.S. Geological Survey","correspondingAuthor":false,"prefix":"","firstName":"Jeremy","middleName":"K.","lastName":"Wise","suffix":""},{"id":427774015,"identity":"c494e17f-1926-4e9f-9623-01b1c7b70f49","order_by":5,"name":"Matthew Meulemans","email":"","orcid":"","institution":"U.S. Geological Survey","correspondingAuthor":false,"prefix":"","firstName":"Matthew","middleName":"","lastName":"Meulemans","suffix":""},{"id":427774016,"identity":"1f98a34d-608e-4ef4-aefd-f4f62f7fdb8b","order_by":6,"name":"David Hammond","email":"","orcid":"","institution":"Earth Science Laboratories, Inc","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"","lastName":"Hammond","suffix":""},{"id":427774017,"identity":"113d4646-0d8e-4ab2-a61e-18de47e7691d","order_by":7,"name":"Nicholas B. D. Phelps","email":"","orcid":"","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Nicholas","middleName":"B. D.","lastName":"Phelps","suffix":""},{"id":427774018,"identity":"5139a798-50ac-4602-8ecb-aad5f34e69ff","order_by":8,"name":"Diane L. Waller","email":"","orcid":"","institution":"U.S. Geological Survey","correspondingAuthor":false,"prefix":"","firstName":"Diane","middleName":"L.","lastName":"Waller","suffix":""}],"badges":[],"createdAt":"2025-03-11 18:38:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6205980/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6205980/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78530923,"identity":"c0a00eb1-4240-4a5f-a56f-03b94b0d3410","added_by":"auto","created_at":"2025-03-14 14:05:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":125658,"visible":true,"origin":"","legend":"\u003cp\u003eStudy area indicating sampling sites (Barbour et al., in review).\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-6205980/v1/3f9741e73fa218b34f5a5a0d.png"},{"id":78530925,"identity":"15010278-f4d0-4a8f-81db-07497275df3d","added_by":"auto","created_at":"2025-03-14 14:05:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":134654,"visible":true,"origin":"","legend":"\u003cp\u003eMean chlorophyll-a concentration (μg/L; \u003cem\u003en\u003c/em\u003e = 3) and Secchi disc depth (m; \u003cem\u003en\u003c/em\u003e = 3) in a reference (red triangle) and copper-treated (black circle) bay in Lake Minnetonka, Minnesota. Bars represent one standard deviation. “Z” symbols denote that the Secchi disc hit lake bottom for all readings. Treatment occurred from 7/20/2019-7/30/2019; refer to Barbour et al. (in review) for complete details. Measurements were not taken in the reference bay in 2022.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-6205980/v1/36525d0080dbfa9defdf5e34.png"},{"id":78530707,"identity":"efe4879a-cfb0-48f4-b139-c8565da18162","added_by":"auto","created_at":"2025-03-14 13:57:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":154988,"visible":true,"origin":"","legend":"\u003cp\u003eZooplankton abundance (#/ L; \u003cem\u003en\u003c/em\u003e = 3 tows at 5 sites per bay in 2019-2022) in a reference (red triangle) and copper-treated (black circle) bay in Lake Minnetonka, Minnesota. Treatment occurred from 7/20/2019-7/30/2019; refer to Barbour et al. (in review) for complete details. Measurements were not taken in the reference bay in 2022.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6205980/v1/2fdbb417a49d652cd53d7f73.png"},{"id":78530710,"identity":"2d67ddfe-0b14-412c-ab88-54c3770b07b8","added_by":"auto","created_at":"2025-03-14 13:57:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":136821,"visible":true,"origin":"","legend":"\u003cp\u003eZooplankton diversity (Shannon diversity index; \u003cem\u003en\u003c/em\u003e = 3 tows at 5 sites per bay in 2019-2022) in a reference (red triangle) and copper-treated (black circle) in Lake Minnetonka, Minnesota. Treatment occurred from 7/20/2019-7/30/2019; refer to Barbour et al. (in review) for complete details. Measurements were not taken in the reference bay in 2022.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6205980/v1/d62ca746fb3f38670115d9b4.png"},{"id":78532289,"identity":"78b54299-819f-481b-a454-714a6bc3c34b","added_by":"auto","created_at":"2025-03-14 14:13:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":407784,"visible":true,"origin":"","legend":"\u003cp\u003eZooplankton mean abundance (#/L; \u003cem\u003en\u003c/em\u003e = 3 tows at 5 sites per bay in 2019-2022) by family in a reference a (red triangle) and copper-treated (black circle)bay in Lake Minnetonka, Minnesota. Bars represent one standard deviation. Treatment occurred from 7/20/2019-7/30/2019. Measurements were not taken in the reference bay in 2022. Zooplankton were identified to family, except Class Ostracoda.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6205980/v1/921e2512c1bbcfe793d77217.png"},{"id":78530711,"identity":"7ddd1e32-6597-4917-88ed-9b321f03b78f","added_by":"auto","created_at":"2025-03-14 13:57:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":154729,"visible":true,"origin":"","legend":"\u003cp\u003eBenthic invertebrate abundance (#/ m\u003csup\u003e2\u003c/sup\u003e; \u003cem\u003en\u003c/em\u003e = 2 petite ponar grabs at 5 sites per bay in 2019-2022) in a reference (red triangle) and copper-treated (black circle) bay in Lake Minnetonka, Minnesota. Bars represent one standard deviation. Treatment occurred from 7/20/2019-7/30/2019; refer to Barbour et al. (in review) for complete details. Measurements were not taken in the reference bay in 2022.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6205980/v1/73bdb34a0b1ae5d8d616ee77.png"},{"id":78530930,"identity":"d29d458d-d16b-4ba1-9101-4e762db6d14d","added_by":"auto","created_at":"2025-03-14 14:05:11","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":128919,"visible":true,"origin":"","legend":"\u003cp\u003eBenthic invertebrate diversity (Shannon diversity index; \u003cem\u003en\u003c/em\u003e = 2 petite ponar grabs at 5 sites per bay in 2019-2022) in a reference (red triangle) and copper-treated (black circle) in Lake Minnetonka, Minnesota. Bars represent one standard deviation. Treatment occurred from 7/20/2019-7/30/2019; refer to Barbour et al. (in review) for complete details. Measurements were not taken in the reference bay in 2022.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6205980/v1/9f887ebad0cb746cf4b9ef5a.png"},{"id":78532496,"identity":"7b2584ca-e0fa-4611-80d5-4d1d58b3215c","added_by":"auto","created_at":"2025-03-14 14:21:11","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":469986,"visible":true,"origin":"","legend":"\u003cp\u003eBenthic invertebrate mean family abundance (#/L; \u003cem\u003en\u003c/em\u003e = 2 petite ponar grabs at 5 sites per bay in 2019-2022) by family in a reference (red triangle) and copper-treated (black circle) bay in Lake Minnetonka, Minnesota. Bars represent one standard deviation. Treatment occurred from 7/20/2019-7/30/2019. Measurements were not taken in the reference bay in 2022.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-6205980/v1/30a69359bcfc06e7eba71dc6.png"},{"id":78533739,"identity":"c411d1a4-7b3a-49e3-b054-c6c04f89870f","added_by":"auto","created_at":"2025-03-14 14:29:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2463580,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6205980/v1/6f2c05a1-08e1-497d-b781-64fcec9a0754.pdf"},{"id":78530924,"identity":"b4cd5490-72fa-4b72-b99a-6628b3f96a07","added_by":"auto","created_at":"2025-03-14 14:05:10","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":21307,"visible":true,"origin":"","legend":"","description":"","filename":"Nontargetsupplement.docx","url":"https://assets-eu.researchsquare.com/files/rs-6205980/v1/a0c9267add420d8fe55a301e.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 low-dose copper treatment for dreissenid mussels: effects on nontarget organisms","fulltext":[{"header":"Introduction","content":"\u003cp\u003eZebra mussels (\u003cem\u003eDreissena polymorpha\u003c/em\u003e) were introduced to the Laurentian Great Lakes of North America in the mid-1980s and have since spread to and established in hundreds of waterbodies across North America (Mackie, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e1991\u003c/span\u003e). Outside their native range, zebra mussels alter food webs, change water clarity, biofoul water treatment and power generation facilities, and more (MacIsaac, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Connelly et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Strayer, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; McEachran et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Hansen et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Fantle-Lepczyk et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). To address and mitigate these effects, managers are turning to an increasing number of control strategies (Fernald and Watson, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Barbour et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Lund et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Hammond and Ferris, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Dahlberg et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). One such method of control is the application of copper-based pesticides (Claudi et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Luoma et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Hammond and Ferris \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Hammond et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCopper-based pesticides have been used for over a century in lake management, largely to control algae, although also as an herbicide and molluscicide (Hanson and Stefan, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1984\u003c/span\u003e; Kamrin \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Dahlberg et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). As early as 2004, copper-based pesticides were used to control zebra mussels in open water (Claudi et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Luoma et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Hammond and Ferris, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Copper-based pesticides available today include copper sulfate (CuSO\u003csub\u003e4\u003c/sub\u003e), Cutrine-Ultra (EPA registration number 67690-98; SePro Corp.), Natrix (67690-81; SePro Corp.), and EarthTec QZ (64962-1; Earth Science Laboratories, Inc.). Of those, only EarthTec QZ and Natrix are registered by the U.S. Environmental Protection Agency (USEPA) as molluscicides for dreissenid mussel control.\u003c/p\u003e \u003cp\u003eIn aquatic environments, copper affects gilled organisms by rapidly binding to gill membranes where it interferes with osmoregulatory processes by competing with essential monovalent cations, blocking Na\u003csup\u003e+\u003c/sup\u003e uptake at transport sites, or inhibiting Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e ATPase (Grosell and Wood, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Paquin et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). At high concentrations of copper, gilled organisms can become deficient in ions such as Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e, causing a cascade of physiological responses including cardiovascular collapse and mortality (Kamunde and Wood, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Copper exposure is also linked to increased tissue concentrations of reactive oxygen species (ROS), which may lead to DNA damage (Bopp et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Copper can be toxic to fish (De Oliveira-Filho et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Wagner et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), zooplankton (Havens, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Williams et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), chironomids (Kosalwat and Knight, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1987\u003c/span\u003e; Warrin et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), snails (Brix et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Carmosini et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), and other aquatic organisms (Zyadah and Abdel-Baky, \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Murray-Gulde et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Although numerous studies describe the effects of copper on a wide range of freshwater organisms, few studies have evaluated how open water copper-based pesticide applications affect organisms in dynamic ecosystems (Dahlberg et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe conducted a low-dose copper-based zebra mussel suppression treatment in 2019 in a 66.3 ha bay (St. Albans Bay) of Lake Minnetonka, Minnesota, and returned to monitor post-treatment conditions in 2020, 2021, and 2022 (Barbour et al., in review). Our objectives were to describe the short- and long-term effects of low-dose copper treatment on nontarget organisms and to compare copper uptake (concentration) in tissues of zebra mussels and native fish and mussels. Details of the application and results of the targeted zebra mussel population control and monitoring are reported in Barbour et al. (in review).\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy sites and treatment application\u003c/h2\u003e \u003cp\u003eLake Minnetonka (Hennepin County, Minnesota, USA; 5879 ha) is located within the metropolitan area of Minneapolis-St. Paul and is a highly urbanized and popular recreational lake. Zebra mussels were first confirmed in the lake in 2010 and have since become well-established throughout the lake (MN DNR, 2024). In 2019, from July 20 through July 30, a largescale zebra mussel suppression treatment using EarthTec QZ was conducted in St. Albans Bay (66.3 ha) of Lake Minnetonka (refer to Barbour et al., in review, for detailed treatment information; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Briefly, EarthTec QZ was applied via a flat-deck boat at an initial target concentration of 100 \u0026micro;g Cu/L, with regular bump treatments to maintain a dose of 60 \u0026micro;g Cu/L for 10 d. St. Albans Bay is in the southeast corner of the lake and is connected to the main body of the lake through a narrow boating channel. Robinson Bay (~\u0026thinsp;37.2 ha) of Lake Minnetonka was used as the untreated reference site (control bay). Robinson Bay is located on the east-central side of the lake and has no obstructions to the main body of the lake. Longer-term effects of the treatment were monitored during the summer field seasons (May-October) of 2020 (both bays), 2021 (both bays), and 2022 (St. Albans Bay only). Within each bay, we indiscriminately selected five sites in water\u0026thinsp;~\u0026thinsp;3-m deep for deployment of test organisms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePhytoplankton (Chlorophyll-a)\u003c/h3\u003e\n\u003cp\u003eWe measured chlorophyll-\u003cem\u003ea\u003c/em\u003e and Secchi disk transparency as proxies for phytoplankton abundance. In year 1 (2019, our treatment year), we collected triplicate water samples at 2 d pretreatment, 1 d posttreatment, and 14 d posttreatment at each sampling site in each bay using a Van Dorn sampler (1 m depth, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5 sites per bay). Samples were transferred to amber glass bottles, stored on ice, and delivered to a contract laboratory (RMB Environmental Laboratories, Bloomington, Minnesota) within 24 h for chlorophyll-\u003cem\u003ea\u003c/em\u003e analysis with a fluorometric method (Supplement 1). In years 2\u0026ndash;4 (2020\u0026ndash;2022), we collected triplicate water samples again (July and August of years 2 and 3, and June and August of year 4). Samples were collected and analyzed using the same methods by the U.S. Geological Survey (USGS) Upper Midwest Environmental Sciences Center (UMESC; La Crosse, Wisconsin). For statistical analysis, all chlorophyll-\u003cem\u003ea\u003c/em\u003e concentrations below the detection limit (1.0 \u0026micro;g/L) were assigned one-half the detection limit (0.5 \u0026micro;g/L; Helsel, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). Secchi disk transparency was measured at each sampling site in triplicate, concurrent with chlorophyll sampling.\u003c/p\u003e\n\u003ch3\u003eZooplankton monitoring\u003c/h3\u003e\n\u003cp\u003eThe zooplankton community present in each bay at the time of sampling was determined from the same plankton tow samples collected for veliger enumeration; refer to Barbour et al., in review, for complete methods. Samples were collected using a 30-cm diameter plankton net with a 50-\u0026micro;m mesh sample cup (Aquatic Research Instruments, Hope, Idaho). In year 1 (2019), we collected triplicate tow samples at 1 d pretreatment, 2 d posttreatment, and 14 d posttreatment at each sampling site (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3 tows at 5 sites per bay). In years 2\u0026ndash;4, we collected triplicate tow samples in July and August (the reference bay was not sampled in year 4). Samples were preserved in 70% ethanol and transferred to a contract laboratory (RMB Environmental Laboratories) for enumeration. Zooplankton were enumerated and identified to the family-level under microscopy (RMB Environmental Laboratories; Supplement 1). Organism density (individuals per liter) was calculated from tow length and net opening area. From years 1\u0026ndash;3, we conducted vertical plankton tows from the thermocline (mean depths 5.73\u0026ndash;6.67 m) to the surface. In year 4, vertical tows were conducted from 3-m depth to the surface.\u003c/p\u003e\n\u003ch3\u003eBenthic invertebrate monitoring\u003c/h3\u003e\n\u003cp\u003eThe benthic invertebrate community of each bay was sampled with a petite ponar (model: 1728-G30; Wildco, Yulee, Florida). In 2019, we collected duplicate samples at 1 d pretreatment, 2 d posttreatment, and 14 d posttreatment at each sampling site (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5). In 2020, 2021, and 2022 we collected duplicate samples in July and August at the same five sites. To sort invertebrates from the sediment, all ponar grabs were elutriated for 10 min (Magdych \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1981\u003c/span\u003e) then washed through a series of mesh screens (final mesh: 500 \u0026micro;m). Finally, samples were preserved in 70% ethanol. Benthic invertebrates were enumerated and identified to family level (RMB Environmental Laboratories).\u003c/p\u003e\n\u003ch3\u003eCaged fish and mussels\u003c/h3\u003e\n\u003cp\u003eWe evaluated the effects of the copper treatment on four species of native fish that occur in the lake: yellow perch (\u003cem\u003ePerca flavescens\u003c/em\u003e), fathead minnow (\u003cem\u003ePimephales promelas\u003c/em\u003e), bluegill (\u003cem\u003eLepomis macrochirus\u003c/em\u003e), and largemouth bass (\u003cem\u003eMicropterus salmoides\u003c/em\u003e); and one species of native mussel, fatmucket (\u003cem\u003eLampsilis siliquoidea\u003c/em\u003e). Fish (young of year) and fatmuckets (juvenile, 1 y old) were reared at UMESC. We measured total length and mass of a subsample (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;20) of each fish species and measured the maximum shell length (anterior-posterior axis parallel to the hinge line) of a subsample of fatmuckets (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;20; Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Fish and mussels were transported with appropriate permits from UMESC to the study area in oxygen-overlaid bags and acclimated onsite to lake temperature (~\u0026thinsp;5\u0026deg;C increase) by floating the bags in coolers with lake water for \u0026gt;\u0026thinsp;1.5 h. For each fish species, individuals were indiscriminately distributed into holding cages (30.5 \u0026times; 91.4 cm, dia \u0026times; h; 0.48 cm\u003csup\u003e2\u003c/sup\u003e mesh netting; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5 sites per bay, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 cages per site, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;20 individuals per cage with one species per cage) and submerged at each sampling site\u0026thinsp;~\u0026thinsp;18 h before the first application. The cages were suspended\u0026thinsp;~\u0026thinsp;0.5 m below the water surface and secured to a floating PVC frame positioned around each sampling buoy. Twenty fatmuckets were indiscriminately placed into holding cages (15-L plastic buckets; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5 sites per bay) with openings on each side (15.2 \u0026times; 20.3 cm) and in the lid (12.7 \u0026times; 12.7 cm), covered by mesh (0.48 \u0026times; 0.48 cm) to allow water exchange. Approximately 4 cm of sand was placed in each cage for mussels to position and burrow. The cages were placed on the lakebed at each sampling site (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5 sites per bay).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean (standard deviation) mass and total length of fishes and shell length of bivalves caged in study areas during a low-dose copper treatment for zebra mussel veligers.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMass (g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLength (mm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003en\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBluegill\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.62 (0.57)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e56.35 (4.23)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFathead minnow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.75 (0.86)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e60.20 (6.14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLargemouth bass\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.03 (0.39)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e56.95 (3.61)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYellow perch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4.77 (1.85)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e81.75 (9.15)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFatmucket\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e21.45 (2.07)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZebra mussel\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16.37 (2.37)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e50\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAdult zebra mussels were placed next to fatmucket cages for comparison, as described in Barbour et al. (in review). Briefly, adult zebra mussels were collected from encrusted rocks found in Robinson Bay 2 d pretreatment by severing byssal threads with a scalpel. Collected zebra mussels were held overnight in a submerged mesh bag suspended from a pier in St. Albans Bay. Suitability for testing was determined 1 d pretreatment by challenging the adductor muscle response to gentle pressure applied to opposing valves. We measured shell length on a subset of 50 zebra mussels (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Fifty zebra mussels were indiscriminately distributed into semi-rigid plastic mesh bags (15.2 \u0026times; 10.2 \u0026times; 25.4 cm; w \u0026times; d \u0026times; h; \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5 sites per bay), secured to the top of a native mussel cage, and submerged at each sampling site.\u003c/p\u003e \u003cp\u003eOne cage per species (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 fish species, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1 fatmucket, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1 zebra mussel) was deployed at each sampling site (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5 sites per bay) the day before the first EarthTec QZ application. Cages were retrieved 2 d after the last application. We calculated survival based on the number of stocked individuals, not the number recovered, because dead organisms may have decomposed before cage retrieval. Damaged cages with suspected escape of test organisms were omitted from the analyses (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2 cages).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eCopper Bioaccumulation\u003c/h2\u003e \u003cp\u003eWe indiscriminately sampled a subset of each species from the cages at each sampling site for tissue copper analysis. Whole, live fish were individually packaged in centrifuge tubes and frozen (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5 fish/species/site, with 5 sites per bay). When fewer than five fish were alive in a cage, we preserved all remaining live animals for tissue copper analysis. Soft tissues were removed from fatmuckets and frozen in centrifuge tubes (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5 mussels per site, 5 sites per bay). Fifteen live zebra mussels were indiscriminately collected from each sampling site, the soft tissue was removed from the shell, and pooled into three composite samples per sampling site (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5 mussels per sample). Organisms and tissues were rinsed with clean well water to remove copper residues from exterior surfaces. Tissues were placed on ice, transported to UMESC and frozen at 20 \u0026ordm;C until analysis. Samples were then shipped to the Iowa State University Veterinary Diagnostics Laboratory (Ames, Iowa) for analysis with a digestion and inductively coupled plasma method (Supplement 1).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnalysis\u003c/h3\u003e\n\u003cp\u003eWe conducted all statistical analyses and created data visualizations in R version 4.1.1, utilizing the \u003cem\u003elme4\u003c/em\u003e and \u003cem\u003evegan\u003c/em\u003e packages (Bates et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; R Core Team, 2019; Oksanen et al. 2020), and interpreted differences as significant when α\u0026thinsp;\u0026lt;\u0026thinsp;0.05. We constructed mixed-effects analysis of variance models to assess the effect of copper treatment on chlorophyll-\u003cem\u003ea\u003c/em\u003e concentration, benthic invertebrate abundance and diversity, and zooplankton abundance and diversity. We used logistic mixed-effects modeling as an odds ratio to assess the effect of the treatment on nontarget species mortality. We constructed mixed-effects analysis of variance models using transformed concentration values as the response variable for survival to determine the effect of the treatment on the tissue copper concentrations of our caged organisms. Transformations were performed to stabilize residual variance. Copper concentrations in the tissue of largemouth bass, yellow perch, bluegill, fatmucket, and zebra mussel were natural log transformed. Fathead minnow concentrations were inverse log transformed. We compared differences between sampling times within each bay and between the bays at each sampling time. In each model, sampling time and bay were treated as fixed effects, and sampling location was treated as a random effect nested within each bay (Montgomery, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Zooplankton and benthic invertebrate community structures at family level were compared across all sampling times within each bay using permutational analysis of variance (PERMANOVA) for comparisons.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePhytoplankton (Chlorophyll-a)\u003c/h2\u003e \u003cp\u003eThe mean chlorophyll-\u003cem\u003ea\u003c/em\u003e concentration was similar in the reference and treatment bays before treatment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.22, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). At 1 d posttreatment, the treatment bay had significantly higher chlorophyll-\u003cem\u003ea\u003c/em\u003e concentrations than the reference bay (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and at 14 d posttreatment the reference bay had significantly higher concentrations (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In 2020, chlorophyll-\u003cem\u003ea\u003c/em\u003e concentrations were significantly higher in the treated bay compared to the reference bay (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). In 2021, there was no significant difference between the two bays (\u003cem\u003ep\u003c/em\u003e values\u0026thinsp;\u0026gt;\u0026thinsp;0.56). Secchi disk transparency was inversely related to chlorophyll-\u003cem\u003ea\u003c/em\u003e concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eZooplankton community\u003c/h2\u003e \u003cp\u003eBefore treatment, zooplankton abundance was higher in the treated bay than the reference bay (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In the reference bay, zooplankton abundance remained similar between pretreatment and 1 d (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.999) and 14 d (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.175) posttreatment samplings. In the treated bay, zooplankton abundance decreased between pretreatment and 1 d posttreatment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.002). Zooplankton abundance was similar at 14 d posttreatment to 1 d posttreatment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.982) but higher than pretreatment abundance (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.056). By August of 2020, one year posttreatment, zooplankton abundance was again higher in the treated bay than in the reference (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Zooplankton abundance was similar between the two bays in July 2021 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.129) and higher in the treated bay compared to the reference bay in August 2021 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.004). In 2022, zooplankton abundance was similar in the treated bay in every comparison except August 2021 to July 2022 (\u003cem\u003ep\u003c/em\u003e values all \u0026gt;\u0026thinsp;0.355, except when August 2022 had higher abundance than July 2022 and \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.052).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eZooplankton diversity was higher in the reference bay than the treated bay pretreatment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0002; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Following treatment, diversity was stable in the reference bay (all \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.999) and remained higher than in the treated bay (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000). In the treated bay, diversity decreased between pretreatment and 1 d posttreatment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.000). Diversity remained similarly low at 14 d posttreatment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.999 when comparing 1 d to 14 d posttreatment). By August of 2020, however, diversity in the treated bay increased and was similar to that in the reference bay (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.54). Treatment bay diversity remained similar to the reference bay in 2021 (both \u003cem\u003ep\u003c/em\u003e values\u0026thinsp;\u0026gt;\u0026thinsp;0.82) and diversity decreased in 2022 from its 2021 levels (excluding July 2021 compared to August 2022, all \u003cem\u003ep\u003c/em\u003e values\u0026thinsp;\u0026lt;\u0026thinsp;0.05; July 2021 compared to August 2022 \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.18), with June 2022 levels similar to immediately post treatment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.89).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe shift in zooplankton abundance and diversity was reflected in changes in species community composition in both bays (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Species composition changed in both treated and reference bays immediately following treatment (early- and mid-August 2019). Among specific native zooplankton taxa, Daphniidae and Diaptomidae decreased most in the treated bay, although densities did not remain low in 2020 and 2021. Not all families were affected by treatment. For example, in 2019, Cyclopidae densities in the treated bay closely mirrored the densities in the reference bay with mean densities deviating by \u0026lt;\u0026thinsp;2 per L at sampling times. In the reference bay, Cyclopidae family densities changed the most throughout the observed period; densities ranged from 68.4 per L in July 2020 to 14.2 per L in July 2021. Other families, such as Leptodoridae and Temoridae, had consistent densities throughout the observation period.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOverall, zooplankton communities within each bay differed at nearly every sampling date, highlighting the natural variability of populations independent of treatment (PERMANOVA; only four comparisons had \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05, \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;46 for the treated bay and \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;22 for the reference bay). Within the reference bay, each July composition was different from every other July composition (PERMANOVA; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.002 for \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3 measurements), each August was different from every other August (PERMANOVA; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001 for each of \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3 mid-August measurements), and an early August assessment in 2019 was also different from a mid-August assessment that same year (PERMANOVA; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001). Similarly, within the treated bay, each July composition was different from every other July composition (PERMANOVA; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.002 for \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6 measurements), each August was different from every other August (PERMANOVA; \u0026le; 0.002 for each of \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6 mid-August measurements). An early August assessment in 2019, conducted immediately following treatment, was also different from a mid-August assessment that same year, conducted two weeks post-treatment (PERMANOVA; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBenthic invertebrate community\u003c/h2\u003e \u003cp\u003eBenthic invertebrate abundance in the reference bay was similar throughout the study period (all \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.10; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) with the exception that August 2020 had higher abundance than pretreatment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.06). Within the treated bay, benthic invertebrate abundance was similar at every observation (all \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.62). Benthic invertebrate diversity did not change in the reference bay (all \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.12; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Within the treated bay, benthic invertebrate diversity was only different in July 2021 (all other \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.97). July 2021 had significantly lower diversity than was observed pretreatment, immediately posttreatment, and during August 2021 (all \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.03).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs benthic invertebrate abundance and diversity changed throughout the sampling periods, species community compositions shifted within each bay (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Community composition was most represented by 13 families, including Dreissenidae. Thirty of the taxonomic groups were represented by 0\u0026ndash;2 organisms on most sampling dates. The majority of organisms represented 13 taxonomic groups. No difference was detected in community structures within bays between pretreatment and 1 d post treatment (PERMANOVA; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.015), nor between 1 d posttreatment and 14 d posttreatment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026ge;\u0026thinsp;0.051), although the pretreatment communities of both bays were significantly different than the communities 14 d post treatment (PERMANOVA; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.015). Both bays were characterized by organisms in the Chironomidae, Hyalellidae, and Asellidae families.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCaged fish and mussels\u003c/h2\u003e \u003cp\u003eFathead minnow and adult zebra mussels both had reduced survival odds in the treatment bay (odds ratio [OR]\u0026thinsp;=\u0026thinsp;0.030, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0066 and OR\u0026thinsp;=\u0026thinsp;0.09, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, respectively). Fathead minnow mean survival was 84.0% (26.1 standard deviation (SD)) in the reference bay but only 38.0% (24.1 SD) in the treated bay. Similarly, zebra mussel mean survival was 96.0% (2.4 SD) in the reference bay but only 68.0% (23.0 SD) in the treated bay. Survival and recovery of fish species other than bluegill was low (\u0026lt;\u0026thinsp;89%) in both bays, and two fish cages from Robinson Bay were omitted from analysis due to damage and likely escape of test animals (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Fatmucket mortality was only observed in the reference bay (\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 mussels).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean (standard deviation) percent survival and percent recovery of caged test animals in Robinson (reference) and St. Albans (treated) Bays after the low dose copper treatment period; n\u0026thinsp;=\u0026thinsp;number of cages recovered.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c4\" namest=\"c2\"\u003e \u003cp\u003eRobinson Bay\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003eSt. Albans Bay\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSurvival\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e% recovery\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003en\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSurvival\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e% recovery\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003en\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBluegill\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100.0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100.0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e91.0 (4.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e91.0 (6.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFathead minnow\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e84.0 (26.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e87.0 (26.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e38.0 (24.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e53.0 (37.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLargemouth bass\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e73.8 (45.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e81.3 (34.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e71.0 (35.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e76.0 (29.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYellow perch\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e46.0 (27.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e85.0 (11.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e35.0 (26.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e87.0 (15.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFatmucket\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e96.0 (4.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100.0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e100.0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e99.0 (2.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZebra mussel\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e96.0 (2.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e99.6 (0.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e68.0 (23.0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e100.0 (0)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"8\" nameend=\"c8\" namest=\"c1\"\u003e \u003cp\u003e\u003csup\u003ea\u003c/sup\u003e Statistical difference in survival between bays (α\u0026thinsp;=\u0026thinsp;0.05).\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTissue copper concentrations were greater for all species in the treated bay than the reference bay (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Mean tissue copper concentration was greatest in zebra mussels (40.85 \u0026micro;g/g), followed by native mussels (26.41 \u0026micro;g/g). Of the fish species, fathead minnow had the greatest mean tissue copper concentration. Tissue copper concentration in fathead minnow from the treated bay varied widely but averaged an order of magnitude greater than those in the reference bay (7.53 versus 0.75 \u0026micro;g/g, respectively).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMean (standard deviation) tissue copper concentration (\u0026micro;g/g) measured in test organism tissues from Robinson (reference) and St. Albans (treated) Bays following treatment with low dose copper.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eRobinson Bay\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eSt. Albans Bay\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTissue copper (\u0026micro;g/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003en\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTissue copper (\u0026micro;g/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003en\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBluegill\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.51 (0.40)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.93 (0.42)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFathead minnow\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.75 (0.10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.53 (12.29)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLargemouth bass\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.52 (0.07)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.97 (0.23)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYellow perch\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.50 (0.21)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.70 (1.30)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFatmucket\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.22 (0.28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e26.41 (7.93)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZebra mussel\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.00 (0.86)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e40.85 (10.17)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c6\" namest=\"c1\"\u003e \u003cp\u003e\u003csup\u003ea\u003c/sup\u003e Statistical difference between bays (α\u0026thinsp;=\u0026thinsp;0.05).\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study assessed the short- and long-term effects of a low-dose copper treatment on nontarget organisms. We compared the copper uptake (concentration) in the tissues of zebra mussels and native fish and mussel species and closely monitored the changes in chlorophyll-\u003cem\u003ea\u003c/em\u003e and zooplankton and benthic invertebrate populations. We found the lake system to be highly dynamic with positive and negative shorter- and longer-term treatment-induced changes, as well as changes that were likely the result of natural processes.\u003c/p\u003e \u003cp\u003eWe observed a short-term increase in chlorophyll-\u003cem\u003ea\u003c/em\u003e accompanied by a decrease in Secchi disk transparency in the treated bay immediately (1 d) posttreatment, indicative of an algae bloom. This was unexpected because copper is used as an algicide (Aryal \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This may have occurred because treatment coincided with an algal bloom that had already begun when treatment was initiated. Additionally, the elevated chlorophyll-\u003cem\u003ea\u003c/em\u003e concentrations observed in 2020 in the treated bay may be related to the reduction in zebra mussels that year. However, our data do not capture the phytoplankton community structure needed to better understand or identify the drivers behind the chlorophyll-\u003cem\u003ea\u003c/em\u003e increase. Both chlorophyll-\u003cem\u003ea\u003c/em\u003e concentration and Secchi disk transparency returned to near pretreatment levels within two weeks posttreatment.\u003c/p\u003e \u003cp\u003eOverall zooplankton abundance and diversity decreased following treatment (1 d and 14 d posttreatment), although taxa were affected differently. For example, while Daphniidae and Diaptomidae abundance decreased, Cyclopidae abundance was unchanged. This overall decline was expected, as previous studies reported zooplankton species \u0026mdash; particularly Daphniidae \u0026mdash; to be highly sensitive to copper (McIntosh and Kevern, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; Naddy et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). These declines and changes in species could have trophic-level effects on algae and fish (Dettmers et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), warranting further research in this area. However, because this lake has been affected by zebra mussels for more than a decade (MN DNR 2024), zooplankton communities have likely already been altered from a pre-infestation state. Additionally, because zooplankton are sensitive to changes in nutrient concentrations and predator-prey interactions, their decline may not have been solely treatment-related (McIntosh and Kevern, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1974\u003c/span\u003e; Naddy et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). One year after treatment, zooplankton overall abundance had surpassed pretreatment abundance. These data, combined with our PERMANOVA analysis, indicate a full recovery of the community, consistent with past research documenting similar rebounds after copper exposures (McIntosh and Kevern, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1974\u003c/span\u003e). The variability observed in zooplankton community structure within both bays fits descriptions of naturally chaotic planktonic community structure (Beninc\u0026agrave; et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Abundance may therefore be a more responsive metric than community structure when assessing how a treatment changes the overall zooplankton community over short time periods such as this single treatment, four-year study.\u003c/p\u003e \u003cp\u003eOverall, the zooplankton community recovered faster than the zebra mussel population (refer to Barbour et al., in review), although zooplankton abundance and diversity estimates were dynamic. Given this, it may be important for managers to identify their goals and select treatment plans accordingly. For example, managers interested in increasing biodiversity and improving natural communities in a waterbody may expect an initial decline in zooplankton species and anticipate a recovery within a calendar year. Alternatively, managers seeking to maximize fish production may want to avoid copper treatment to not limit food availability, or time the treatment to not overlap with when larval fish are most reliant on zooplankton.\u003c/p\u003e \u003cp\u003eBenthic invertebrate abundance and diversity had no observed effect in response to treatment. Community structure in the treatment bay also remained unchanged, except for one different community present in July of 2021. We were unable to compare the communities of the treatment and reference bays in a meaningful way because substrate type varied between the bays and benthic communities are known to change with substrate (Tolonen et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; White and Irvine, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Although previous research indicates benthic invertebrates are susceptible to copper (Montz et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), changes in abundance and community structure may also be attributed to the boom-bust life histories of some species during the project period (Galatowitsch \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Considering life history dynamics would provide important insight when planning copper-based treatments to avoid critical life stages and help to understand how treatment effects differ between organisms that live in versus on the sediment as well as how timing may affect different species.\u003c/p\u003e \u003cp\u003eFor example, a benthic invertebrate species with a slower and seasonal reproductive cycle may have delayed recovery, especially if they are sensitive to copper, because these species cannot reproduce until the following year. One family with seasonal reproduction, Hyalellidae, has been shown to be highly sensitive to copper concentrations similar to those in our treatment. One species in that family, \u003cem\u003eHyalella azteca\u003c/em\u003e, has a reported 48-h lethal concentration 50 (LC50) of 87 \u0026micro;g/L in similar water chemistry as observed in our treated bay (Schubauer-Berigan et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). In our study, we observed a family-level decline among Hyalellidae immediately following treatment, which was likely due to the copper exposure. Species in Hyalellidae, such as \u003cem\u003eHyalella azteca\u003c/em\u003e, are a food source for fish species, including bluegill (Camacho and Thacker \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), and reductions in that family may have repercussions for the larger trophic structure within a waterbody.\u003c/p\u003e \u003cp\u003eAmong species with varying reproduction and emergence times, however, attributing abundance changes to treatment is more difficult. For example, Chironomidae have varied reproduction timing. One species in that family, \u003cem\u003eChironomus riparius\u003c/em\u003e, has a reported 24-h LC50 for first instars of 2.09 mg/L as copper in soft water (8 mg/L as CaCO\u003csub\u003e3\u003c/sub\u003e; B\u0026eacute;chard et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Although we observed a decline in this family following treatment, because these species\u0026rsquo; emergence times are not seasonally restricted, the decline in larval and pupal chironomids possibly corresponded to a major adult emergence event rather than the treatment. Posttreatment monitoring in subsequent years found that community structures more closely resembled pretreatment conditions, but further research may determine whether any response is directly attributable to treatment.\u003c/p\u003e \u003cp\u003eThe effects of copper treatment on fish mortality were confounded by low survival in both bays, likely from environmental stressors (e.g., housing, high boat traffic, predator species observed immediately outside cages). Fathead minnow were the only native test species that experienced treatment-related mortality, although control survival was \u0026lt;\u0026thinsp;90%. Among the fish species, fathead minnow had the highest tissue concentrations of copper. Although we cannot say for certain that those tissue copper concentrations were related to low survival, waterborne copper exposure is known to cause a loss of branchial Na\u003csup\u003e+\u003c/sup\u003e and Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e, which triggers a cascade of physiological responses culminating in cardiovascular collapse (Kamunde and Wood, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Of note, the fish evaluated in this study were caged and subjected to the full dose of the treatment. In the natural environment, we would expect fish to move away from physiological stressors to areas of less stressful refuge (Svecevičius, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Ezeonyejiaku et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn contrast to fish, fatmucket mortality was zero in the treatment bay; the only fatmucket deaths occurred in the reference bay. Although our experiment does not show treatment-related mortality among fatmuckets, past research has reported newly transformed juvenile fatmucket LC50 values well below our treatment mean copper concentration (e.g., 96-h EC50 values of survival in reconstituted hardwater reported by Wang et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2009\u003c/span\u003e were 31\u0026ndash;44 \u0026micro;g Cu/L). Fatmuckets possibly could have experienced delayed mortality not found in our data, which could be an area of future research. Copper bioaccumulation was higher in both zebra mussels and fatmuckets than in fish species, in agreement with past research (Wang and Rainbow, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAdult zebra mussels had the highest tissue copper concentration of the species in this study (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Unlike the fatmucket mussels, elevated tissue copper concentration in zebra mussels was accompanied by 32% mortality in the treated bay (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These results are consistent with a 10-d laboratory study using comparable copper concentrations that found zebra mussel tissues contained 40\u0026ndash;60 \u0026micro;g Cu/g (Mersch et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Le et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe effects of copper differ among organ systems in freshwater fish, zebra mussels, and other species (Gundacker \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; Malhotra et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Le et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Histological or molecular examination of organisms or organ systems could determine sublethal physiological effects of low-dose copper that may predispose animals to secondary or chronic stressors.\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePotential future directions\u003c/h2\u003e \u003cp\u003eFurther research is warranted to investigate the optimal frequency and duration of treatments, considering factors such as the specific ecosystem characteristics and the life history traits of nontarget organisms. For example, implementing a treatment plan with no-treatment intervals longer than 1 year could allow zooplankton and benthic invertebrate communities to rebound. However, waiting three years or more may provide zebra mussels a chance to recolonize and fully recover from the treatment, indicating there may be an optimal timeframe for treatment that supports nontarget species recovery without allowing zebra mussels to rebound. By allowing nontarget species communities sufficient time to recover, managers could potentially develop more pragmatic management practices for suppressing zebra mussels with minimal long-term effects.\u003c/p\u003e \u003cp\u003eAdditionally, management decisions on invasive mussel control require managers to determine a goal for population suppression and align it with the chosen treatment strategy and its consequences for the conservation of native species and ecosystem health. For example, complete eradication of established zebra mussel populations may not be feasible with a low-dose copper treatment; however, if the goal is ecosystem restoration, or maintaining ecosystem function, a low-dose copper treatment could reduce zebra mussel density below a level of impact while also accounting for the health of native communities. Gaining a better understanding of the trade-offs between the benefits of zebra mussel control and the potential nontarget effects will be crucial in developing sustainable and ecologically sound management approaches.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe copper treatment in our study was aimed to reduce zebra mussel recruitment and settlement while minimizing treatment effects to nontarget species. Phytoplankton showed a short-term population increase following treatment, as measured through chlorophyll-\u003cem\u003ea\u003c/em\u003e concentration. Zooplankton experienced an overall decline in abundance and diversity, although taxa responded differently, and estimates were dynamic and uncertain, and both abundance and diversity recovered within one year. Some benthic invertebrate families also decreased in abundance along with overall diversity and appeared to recover within two years. Fish species were difficult to assess due to low survival rates in our experimental cages, although we did observe that fish accumulated less copper than mussels. Overall, these findings indicate that although some limited non-target effects were caused by a low-dose copper treatment, it may be a viable strategy for future management controlling invasive mussels while minimizing collateral damage to the ecosystem.\u003c/p\u003e"},{"header":"Declarations","content":"\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\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe data in support of this publication are available at https://doi.org/10.5066/P14JBUQU (Barbour et al., 2025).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Justin Schueller and Courtney Kirkeeng (USGS-UMESC) for their\u0026nbsp;Inductively Coupled Plasma analysis of our copper samples, and Justin Smerud and Todd Johnson (USGS-UMESC) for their assistance with sample collections. We thank Gabriel Jabbour (Tonka Bay Marina, Tonka Bay, Minnesota) for his invaluable assistance and hospitality in facilitating the application and storing the EarthTec QZ and allowing us to use his marina. We thank Dr. Barbara Bennie (University of Wisconsin La Crosse) for her assistance with statistical analyses. We are grateful to the cities of Deephaven and Greenwood, Minn., as well as the Minnehaha Creek Watershed District and Lake Minnetonka Association for their support and outreach efforts.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding was provided by the Minnesota Environment and Natural Resources Trust Fund as recommended by the Minnesota Aquatic Invasive Species Research Center (MAISRC) and the Legislative-Citizen Commission on Minnesota Resources (LCCMR), and the State of Minnesota. Funding support for this project was also provided by the U.S. Geological Survey Ecological Missions Area Biological Threats and Invasive Species Research Program and by Hennepin County (Minn.). Funding for the MAISRC Zebra Mussel Research Fellowship that supported AD was provided by the Fletcher Family Foundation, Pelican Lakes Association of Crow Wing County, and Bay Lake Improvement Association.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAD, MB, JL, TS, JW, BB, NP, and DW declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. DH is an employee of Earth Science Laboratories, manufacturer of the molluscicide utilized in this study. This study explains the product\u0026rsquo;s effects on target and nontarget species. DH contributed to the design of the treatment protocol but not in collection or analysis of data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: James A. Luoma, Diane L. Waller, and Nicholas B. D. Phelps\u003c/p\u003e\n\u003cp\u003eData curation: Matthew T. Barbour, Todd J. Severson, Jeremy K. Wise, and Matthew Meulemans\u003c/p\u003e\n\u003cp\u003eFormal analysis: Matthew T. Barbour\u003c/p\u003e\n\u003cp\u003eFunding acquisition: James A. Luoma, Diane L. Waller, and Nicholas B. D. Phelps\u003c/p\u003e\n\u003cp\u003eInvestigation: Angelique D. Dahlberg, Matthew T. Barbour, James A. Luoma, Todd J. Severson, Jeremy K. Wise, Matthew Meulemans, and Diane L. Waller\u003c/p\u003e\n\u003cp\u003eMethodology: James A. Luoma, Diane L. Waller, David Hammond, Nicholas B. D. Phelps, and Matthew T. Barbour\u003c/p\u003e\n\u003cp\u003eProject administration: James A. Luoma, Diane L. Waller, and Nicholas B. D. Phelps\u003c/p\u003e\n\u003cp\u003eResources: David Hammond\u003c/p\u003e\n\u003cp\u003eSupervision: James A. Luoma, Diane L. Waller, and Nicholas B. D. Phelps\u003c/p\u003e\n\u003cp\u003eValidation: Matthew T. Barbour\u003c/p\u003e\n\u003cp\u003eVisualization: Matthew T. Barbour\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; original draft: Matthew T. Barbour and Angelique D. Dahlburg\u003c/p\u003e\n\u003cp\u003eWriting \u0026ndash; review \u0026amp; editing: James A. Luoma, Todd J. Severson, Jeremy K. Wise, Matthew Meulemans, Nicholas B. D. Phelps, and Diane L. Waller\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 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). The use and treatment of test organisms in this study was approved by the Animal Care and Use Committee at UMESC (Study plan AEH-18-LowCu-01), and hatchery reared organisms were certified specific pathogen free by the U.S. Fish and Wildlife Service\u0026rsquo;s La Crosse Fish Health Center (La Crosse, Wisconsin).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAryal, D., 2018. Evaluating the effectiveness of algaecide in a continuous flow through system. Thesis, University of Akron.\u003c/li\u003e\n\u003cli\u003eBarbour, M.T., Luoma, J.A., Dahlberg, A., Severson, T.J., Wise, J.K., Meulemans, M.J., Bennie, B., Hammond, D., Waller, D., IN REVIEW. Assessing a low-dose copper treatment for dreissenid mussels: effects on zebra mussel (\u003cem\u003eDreissena polymorpha\u003c/em\u003e) population. Environ Man\u003c/li\u003e\n\u003cli\u003eBarbour, M.T., Luoma, J.A., Dahlberg, A., Severson, T.J., Wise, J.K., Meulemans, M.J., Waller, D.L., 2025. 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Science of The Total Environment 806, 151318. https://doi.org/10.1016/j.scitotenv.2021.151318\u003c/li\u003e\n\u003cli\u003eFernald, R.T., Watson, B.T., 2014. Eradication of zebra mussels (\u003cem\u003eDreissena polymorpha\u003c/em\u003e) from Millbrook Quarry, Virginia: Rapid response in the real world, in: Quagga and Zebra Mussels: Biology Impacts and Control. CRC Press LLC, Boca Raton, FL, pp. 195\u0026ndash;213.\u003c/li\u003e\n\u003cli\u003eGalatowitsch, M. L., 2014. Invertebrate life-history trade-offs and dispersal across a pond-permanence gradient. University of Canterbury.\u003c/li\u003e\n\u003cli\u003eGrosell, M., Wood, C.M., 2002. Copper uptake by rainbow trout gills. The Journal of Experimental Biology 205, 1179\u0026ndash;1188. https://doi.org/10.1242/jeb.205.8.1179\u003c/li\u003e\n\u003cli\u003eGundacker, C., 1999. Tissue-specific heavy metal (Cd, Pb, Cu, Zn) deposition in a natural population of the zebra mussel \u003cem\u003eDreissena polymorpha\u003c/em\u003e pallas. Chemosphere 38, 3339\u0026ndash;3356. https://doi.org/10.1016/S0045-6535(98)00567-0\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. Oral presentation at the International Conference on Aquatic Invasive Species, 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. Management of Biological Invasions 10, 500\u0026ndash;516. https://doi.org/10.3391/mbi.2019.10.3.07\u003c/li\u003e\n\u003cli\u003eHansen, G.J.A., Ahrenstorff, T.D., Bethke, B.J., Dumke, J.D., Hirsch, J., Kovalenko, K.E., LeDuc, J.F., Maki, R.P., Rantala, H.M., Wagner, T., 2020. Walleye growth declines following zebra mussel and \u003cem\u003eBythotrephes\u003c/em\u003e invasion. 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Accessed 3 January 2025.\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\u003eMontz, G.R., Hirsch, J., Rezanka, R., Staples, D.F., 2011. Impacts of copper on a lotic benthic invertebrate community: response and recovery. Journal of Freshwater Ecology 25, 575\u0026ndash;587. https://doi.org/10.1080/02705060.2010.9664407\u003c/li\u003e\n\u003cli\u003eMurray-Gulde, C.L., Heatley, J.E., Schwartzman, A.L., Rodgers, J.H., 2002. Algicidal effectiveness of Clearigate, Cutrine-Plus, and copper sulfate and margins of safety associated with their use. Archives of Environmental Contamination and Toxicology 43, 19\u0026ndash;27. https://doi.org/10.1007/s00244-002-1135-1\u003c/li\u003e\n\u003cli\u003eNaddy, R.B., Stubblefield, W.A., May, J.R., Tucker, S.A., Hockett, J.R., 2002. The effect of calcium and magnesium ratios on the toxicity of copper to five aquatic species in freshwater. 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Bulletin of Environmental Contamination and Toxicology 64, 740\u0026ndash;747. https://doi.org/10.1007/s001280000066\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":"bioaccumulation, community response, nontarget effects, aquatic invasive species, control tool","lastPublishedDoi":"10.21203/rs.3.rs-6205980/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6205980/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eZebra mussels (\u003cem\u003eDreissena polymorpha\u003c/em\u003e), an invasive dreissenid mussel, have been established and caused considerable effects in many North American aquatic ecosystems. In response, copper-based pesticides have been used to manage zebra mussel populations. We evaluated the effects of a low-dose copper-based molluscicide for zebra mussel suppression on nontarget species in Lake Minnetonka (Minnesota, USA). Our study evaluated nontarget effects before and after treatment. Chlorophyll-\u003cem\u003ea\u003c/em\u003e concentration increased in both the treated and reference bays 1 and 14 d posttreatment. Zooplankton community composition changed in both bays over the course of this study; zooplankton abundance and diversity initially decreased in the treated bay but gradually recovered and was back to pretreatment and reference bay levels after one year. We observed no significant differences in benthic invertebrate abundance or diversity between the treated and reference bays, although abundance and diversity estimates were dynamic and uncertain. Among caged organisms, copper bioaccumulation was higher in both mussel species than in fish, and among fish, was highest in fathead minnow (\u003cem\u003ePimephales promelas\u003c/em\u003e). These findings contribute to our understanding of the potential effects of copper-based pesticides on aquatic ecosystems and provide insights for zebra mussel management.\u003c/p\u003e","manuscriptTitle":"Assessing low-dose copper treatment for dreissenid mussels: effects on nontarget organisms","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-14 13:57:06","doi":"10.21203/rs.3.rs-6205980/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-05-14T13:58:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-06T08:57:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-15T09:45:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"42844789465612893995009990498113337369","date":"2025-03-27T09:54:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"273764854429257726046429909240390418368","date":"2025-03-26T05:42:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-25T08:56:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-24T23:37:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-12T10:58:59+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Management","date":"2025-03-11T18:32:52+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":"March 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-06-25T23:23:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-14 13:57:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6205980","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6205980","identity":"rs-6205980","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}