Toxicity of a Traditional Molluscicide to Asian Clam Veligers

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1 Notes Toxicity of a Traditional Molluscicide to Asian Clam Veligers Megan Layhee,* Miho Yoshioka, Bahram Farokhkish, Jackson A. Gross, Adam J. Sepulveda M. Layhee, B. Farokhkish, A.J. Sepulveda U.S. Geological Survey, Northern Rocky Mountain Science Center, 2327 University Way Suite 2, Bozeman, Montana M. Yoshioka Contractor, 2327 University Way, Suite 2, Bozeman, Montana J.A. Gross U.S. Geological Survey, Northern Rocky Mountain Science Center, 2327 University Way Suite 2, Bozeman, Montana Present address: Smith-Root Inc NE Salmon Creek Ave. Vancouver, Washington Abstract Aquaculture and hatchery industries are in need of effective control methods to reduce the risk of spreading aquatic invasive species, such as the Asian clam Corbicula fluminea, through aquaculture and hatchery activities. The planktonic nature of Asian clam veligers enables this life stage to enter water-based infrastructure undetected, including hatchery trucks used to stock fish. Once in hatchery trucks, veligers can disperse overland and establish in previously uninvaded habitats. As a result, there is a need to develop techniques that result in veliger mortality but do not harm fish. In September 2012, we conducted laboratory trials to determine if a molluscicide (750 mg/l potassium chloride and 25 mg/l formalin) commonly used to kill zebra mussel (Dreissena polymorpha) veligers in hatchery trucks can also effectively kill Asian clam veligers. We exposed Asian clam veligers to this molluscicide for 1, 3, and 5 h in each of two water types: deionized water and filtered lake water. We found,20% mortality at the 1-h exposure period and 100% mortality at both the 3-h and 5-h exposure periods, regardless of water type. This laboratory study represents an important step toward reducing the spread of Asian clams by aquaculture facilities. Keywords: aquaculture; Asian clam; Corbicula fluminea; invasive species; molluscicide; veliger Received: April 13, 2013; Accepted: September 25, 2013; Published Online Early: December 2013; Published: June 2014 Citation: Layhee M, Yoshioka M, Farokhkish B, Gross JA, Sepulveda AJ Toxicity of a traditional molluscicide to Asian clam veligers. Journal of Fish and Wildlife Management 5(1): ; e x. doi: / JFWM-032 Copyright: All material appearing in the Journal of Fish and Wildlife Management is in the public domain and may be reproduced or copied without permission unless specifically noted with the copyright symbol ß. Citation of the source, as given above, is requested. The findings and conclusions in this article are those of the author(s) and do not necessarily represent the views of the U.S. Fish and Wildlife Service. * Corresponding author: mlayhee@usgs.gov Introduction Overland transport has facilitated the spread of aquatic invasive species between hydrographically isolated waters and resulted in irreversible changes to native species distribution and abundance (Johnson et al. 2001). For example, outbound transportation of hatchery fish by stocking trucks has facilitated the dispersal of invasive freshwater bivalves and gastropods (Edwards et al. 2002). The accidental transport of a single bivalve can result in the propagation of thousands of veligers (larvae) at fish stocking destinations. This concern underscores the need to develop realistic treatment options to prevent dispersal of invasive species, especially at early developmental stages that are difficult to visually detect, during the transportation of fish from hatcheries. No treatment options currently exist to prevent the dispersal of invasive Asian clams Corbicula fluminea by Journal of Fish and Wildlife Management June 2014 Volume 5 Issue 1 141

2 stocking trucks, even though this invasive species occurs in waters used by hatcheries. The Asian clam is indigenous to temperate and tropical regions of southern Asia, eastern and central Australia, and Africa (McMahon 2000). The first documented occurrence of the species in the United States was on the Pacific coast in the 1930s, most likely introduced by immigrants as a food source (Counts 1986). Since its introduction, Asian clams have spread south and east, posing both ecological and economic threats to invaded systems (Sousa et al. 2008). Asian clams have negatively impacted native bivalve abundance and diversity as a result of competition for food resources, by high reproductive capacity, and by degrading habitat through fouling of underwater substrate and bioturbation activity (Sousa et al. 2008). This species is also an economic nuisance to water-based infrastructure as a result of biofouling, a process in which they decrease the functional efficiency of raw-water intake facilities (McMahon 1983). The planktonic nature of Asian clam veligers enables this life stage to enter water-based infrastructure, including hatchery trucks used to stock fish, undetected (McMahon 1983). Unlike most Unionidae species, Asian clams do not require a fish host to incubate larvae (Kennedy and Huekelem 1985). Rather, offspring are retained within hermaphroditic adults until they reach the veliger stage, and once released into the water, the microscopic veligers can naturally disperse through the passive assistance of currents (Doherty et al. 1987). Once in hatchery water supplies, veligers can spread as a result of fish stocking practices. Interestingly, the nation s hatcheries, which frequently transport fish within and across state borders, can act as effective vectors of invasive aquatic species (Edwards et al. 2002). Currently, there are no protocols set by U.S. Fish and Wildlife Service hatcheries for the control of Asian clam veligers in fish stocking trucks. Since veligers are able to lodge themselves into gill rakers and orifices of fish, treatment methods must be effective at preventing veliger settlement while having no residual effect on the fish being transported. Several compounds have been shown to induce mortality in Asian clam veligers, including copper and asbestos (Doherty and Cherry 1988). However, these substances can be toxic to nontarget organisms and may not be suitable treatment agents for the removal of Asian clam veligers in fish aquaculture. Edwards et al. (2000, 2002) found that a mixture of 750 mg/l potassium chloride (KCl) and 25 mg/l formalin (37% formaldehyde) was effective at preventing the spread of invasive zebra mussel (Dreissena polymorpha) veligers in hatchery truck fish tanks, while having no adverse effect on transported fish. In the present study, we tested if this zebra mussel veliger control protocol can also be used as a molluscicide for Asian clams. The specific objectives of this research were 1) to determine the minimum exposure time needed to induce 100% mortality (lethal dosage) in Asian clam veligers using the zebra mussel protocol and 2) to assess if the lethal exposure time varies by water source. The latter objective is meant to assess the efficiency of this molluscide in different water sources (with varying water quality characteristics; e.g., conductivity [200 3,000 ms/cm]) used in hatchery stocking trucks. Results from this work will help hatchery and aquaculture facilities to prevent the spread of Asian clams to uninvaded waters. Methods Study site We conducted laboratory experiments from 14 to 15 September 2012 at Gavins Point National Fish Hatchery, located 5 km west of Yankton, South Dakota (Figure 1). Fish culture water flows to the hatchery through a single waterline from Lewis and Clark Lake, and from three wells (West Well, East Well, and North Well; C. Bockholt, U.S. Fish and Wildlife Service, personal communication). Adult spawning and veliger collection We collected accessible Asian clam adults from Lewis and Clark Lake. After transporting clams in lake water to the hatchery, we evenly divided clams into 15-mm meshlined spawning chambers in an aerated raceway. We used a chiller in a flow-through system to maintain a raceway temperature of 19 21uC (near optimal conditions for adult spawning of veligers [Doherty et al. 1987]). After documenting veliger release in spawning chambers, we microscopically examined them to assess health (marked by presence or absence of active ciliary beating [Harrison et al. 1984]) and abundance for experimental use. Once sufficient quantities of veligers were spawned (approximately 1,000 individual veligers), we consolidated the available veligers in aerated 2-L glass beakers at densities of approximately 250 veligers per beaker. By taking equal numbers of veliger individuals from each spawning chamber and thoroughly mixing them in the holding beakers, we ensured that the results were independent from maternal lineage. We siphoned 960 veligers out of the holding beakers and evenly distributed them in 50-mL exposure beakers at densities of 20 veligers/beaker; similar to procedures used by Edwards et al. (2000). Chemical treatment The chemicals and respective concentrations used in this study were identical to those that were previously confirmed to be effective at controlling zebra mussels (750 mg/l KCl + 25 mg/l formalin; Edwards et al. 2000, 2002). We held the chemical concentrations constant throughout the study, and manipulated exposure time and water type in which the chemical treatment was imposed. Stocking trucks from Gavins Point National Fish Hatchery transport fish to areas 1 8 h away from the hatchery. Therefore, we assessed veliger mortality after 1, 3, and 5 h of chemical exposure independently. We did not include an 8-h exposure time in the experiment because the effectiveness of the chemical treatment lasting longer than 5 h is not practical for relatively shorter durations of fish transit. To determine if water source is an important factor to account for when testing the chemical treatment, we used filtered water from Lewis and Clark Lake (conductivity = 824 ms/cm; ph = ) as one water type, and Journal of Fish and Wildlife Management June 2014 Volume 5 Issue 1 142

3 Figure 1. Gavins Point National Fish Hatchery is located 5 km west of Yankton, South Dakota, on the Missouri River and Lewis and Clark Lake. The hatchery provides fishery resources within the Missouri River system and midwestern states. (Modified from USFWS 2013.) deionized water (< 0 ms/cm conductivity; ph = 7.0) as the second water type. Hatchery trucks primarily use filtered lake water when stocking fish. We assessed the effects of exposure period and water type on veliger mortality with a factorial design (Figure 2), in which veligers were exposed to either a combination of 750 mg/l KCl + 25 mg/l formalin or a control (no molluscicide) for a period of 1, 3, or 5 h in either filtered lake water or deionized water. The 12 treatment combinations were replicated four times with 20 veligers placed in each replicate. At the end of the exposure period, we recorded percentage of mortality (100 6[number of dead veliger individuals per 20 veliger individuals]) as the response variable. Veliger mortality was defined as cessation of all movement and ciliary beating and lack of response to gentle prodding by a dissecting probe (Harrison et al. 1984). It was not possible for us to include a recovery period to detect latent mortality because it is difficult to transport veligers from test solutions to untreated lake water without harming them (Edwards et al. 2000). Statistical analyses To compare the percentage of mortality of Asian clam veligers across our treatment combinations, we used a 3- way ANOVA with chemical treatment, exposure time, and water type as factors. Because water type was not a significant factor, we reran the ANOVA using only chemical treatment and exposure time. We then used Tukey s honest significance difference for post-hoc tests after the ANOVA. We used a Bonferroni correction to adjust the significance level from a = 0.05 to a/15 = for the pairwise comparisons. We conducted all statistical analyses in R (R Core Team 2012). Results The chemical treatment and exposure period interacted to affect veliger mortality (F 2 = 1,656.09, P, ). Veligers exposed to the chemical treatment for both 3 and 5 h experienced 100% mortality, whereas those in the 1-h exposure group experienced,20% mortality (9 out of 80 died in deionized treatment water and 12 out Journal of Fish and Wildlife Management June 2014 Volume 5 Issue 1 143

4 Figure 2. Comparison of mean percentage of mortality ( ) of Asian clam veligers collected from Gavins Point National Fish Hatchery (14 15 September 2012) after 1, 3, and 5 h of exposure to the chemical treatment (750 mg/l potassium chloride and 25 mg/l formalin). For each water treatment (deionized and lake) and exposure duration (1, 3, and 5 h) there were a total of 80 veligers (four replicates of 20 veligers). of 80 died in lake treatment water; Figure 2). This difference in veliger mortality between 3- and 5-h exposure periods and 1-h exposure periods was statistically significant (Tukey s honest significance difference). Veligers exposed to the control treatment for 1-, 3-, and 5-h exposure periods had 0% mortality. Water type did not influence veliger mortality (F 2 = 0.77, P = 0.39). Discussion Our results demonstrate that the chemical treatment commonly used to kill zebra mussel veligers induced 100% mortality in Asian clam veligers exposed for 3 5 h, regardless of water source. One-hour exposure periods were not adequate, as they induced less than 20% mortality. Because we did not have any exposure times between 1 and 3 h, we cannot estimate the minimum exposure time needed for 100% mortality. Further research with exposure times between 1 and 3 h is needed to estimate the lethal dosage. The response of Asian clam veligers to the chemical treatment did not vary by water source. A major difference between our two water sources was specific conductivity; deionized and filtered lake water had conductivities of 0 and 824 ms/cm, respectively. All else being equal, veligers should be vulnerable to the chemical treatment within this conductivity range. We do not know how veligers will respond to the treatment in water conductivities exceeding 824 ms/cm, but related studies on other mussels suggest that our results should be applicable at higher conductivities. At Gavins Point National Fish Hatchery, zebra mussel veligers exposed to the same protocol experienced 100% mortality under water conductivities up to 3,000 ms/cm (C. Bockholt, U.S. Fish and Wildlife Service, personal communication). Mechanistically, when KCL at 750 mg/l is introduced into high-conductivity waters, freshwater mussels become intoxicated and exhibit shell gaping and have low to no ciliary activity in the gills (Edwards et al. 2000). These intoxicated behaviors can leave mussels defenseless against the osmotic stresses wrought by formalin and other chemicals. In addition to water conductivity, other factors may affect the lethality of chemical stressors to Asian clam veligers, including water temperature, veliger size and developmental stage, and flowing vs. stationary water supply (Doherty and Cherry 1988). To optimize robustness of this Asian clam protocol, we recommend testing the effects of these variables on veliger mortality. We carried out a small-scale laboratory experiment in 50-mL beakers, but the field application of the chemical treatment will occur in larger, more complex environments: large-volume (e.g., 750-L) tanks filled with multiple species of fish that are being transported by hatchery trucks. The assumption that our results can be scaled up to such environments that vary in water chemistry should be tested prior to field implementation. In addition, the effect of removing the treated truck water into streams or lakes and the effect it may have on native mussels and other aquatic species should be a part of any future studies. Nevertheless, the protocol that we tested caused 100% mortality when tested on zebra mussel veligers in both 5-mL test tubes and 750-L tanks Journal of Fish and Wildlife Management June 2014 Volume 5 Issue 1 144

5 (Edwards et al. 2000, 2002). Because the zebra mussel protocol was effective across spatial scales, it is now being nationally implemented for hatchery fish transport. Therefore, findings from this laboratory study may provide valuable information towards the advancement of Asian clam control in similar settings. Acknowledgments We thank the technical assistance of Brianna McDowell and Caitlyn Brendal for constructing the spawning chambers used to collect study units. We also thank Marc Jackson and Craig Bockholt of U.S. Fish and Wildlife Service for providing the laboratory space at Gavins Point National Fish Hatchery, as well as the anonymous reviewers and Subject Editor at the Journal of Fish and Wildlife Management. This project was funded by U.S. Fish and Wildlife Service and U.S. Geological Survey (USFWS Interagency Agreement number: 60181AN411). Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. References Counts CL III The zoogeography and history of the invasion of the United States by Corbicula fluminea (Bivalvia: Corbiculidae). American Malacological Bulletin Special Edition 2:7 39. Doherty FG, Cherry DS Tolerance of the Asiatic clam Corbicula spp. to lethal levels of toxic stressors: a review. Environmental Pollution 51: dx.doi.org/ / (88) Doherty FG, Cherry DS, Cairns J Jr Spawning periodicity of the Asiatic clam Corbicula fluminea in the New River, Virginia. American Midland Naturalist 117: doi: / Edwards WJ, Babcock-Jackson L, Culver DA Prevention of the spread of zebra mussels during fish hatchery and aquaculture activities. North American Journal of Aquaculture 62: doi: / (2000)062,0229:potsoz.2.3.co;2 Edwards WJ, Babcock-Jackson L, Culver DA Field testing of protocols to prevent the spread of zebra mussels Dreissena polymorpha during fish hatchery and aquaculture activities. North American Journal of Aquaculture 64: doi: / (2002)064, 0220:FTOPTP.2.0.CO;2 Harrison FL, Knezovich JP, Rice DW Jr The toxicity of copper to the adult and early life stages of the freshwater clam, Corbicula manilensis. Archives of Environmental Contamination and Toxicology 13: doi: dx.doi.org/ /bf Johnson LE, Ricciardi A, Carlton JT Overland dispersal of aquatic invasive species: a risk assessment of transient recreational boating. Ecological Applications 11: doi: / Kennedy VS, Huekelem LV Gametogenesis and larval production in a population of the introduced Asiatic clam, Corbicula sp. (Bivalvia: Corbiculidae), in Maryland. Biological Bulletin 168: biolbull.org/content/168/1/50.full.pdf+html. McMahon RF Ecology of an invasive pest bivalve; Corbicula. Pages in Hunter WD, editor. The Mollusca. Volume 6: ecology. Orlando, Florida: Academic Press. McMahon RF Invasive characteristics of the freshwater bivalve Corbicula fluminea. Pages in Claudi R, Leach J, editors. Nonindigenous freshwater organisms: vectors, biology and impacts. Boca Raton, Florida: Lewis Publishers. R Core Team R: a language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing. Sousa R, Antunes C, Guilhermino L Ecology of the invasive Asian clam Corbicula fluminea (Muller, 1774) in aquatic ecosystems: an overview. International Journal of Limnology 44: doi: org/ /limn: [USFWS] U.S. Fish and Wildlife Service Gavins Point Public Information. Available: gavinspoint/publicinfo.htm (January 2014). Journal of Fish and Wildlife Management June 2014 Volume 5 Issue 1 145