A Pilot Study Testing a Natural and a Synthetic Molluscicide for Controlling Invasive Apple Snails (Pomacea maculata) Heather M. Olivier, Jill A. Jenkins, Mark Berhow & Jacoby Carter
Bulletin of Environmental Contamination and Toxicology ISSN 0007-4861 Bull Environ Contam Toxicol DOI 10.1007/s00128-015-1709-z
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Author's personal copy Bull Environ Contam Toxicol DOI 10.1007/s00128-015-1709-z
A Pilot Study Testing a Natural and a Synthetic Molluscicide for Controlling Invasive Apple Snails (Pomacea maculata) Heather M. Olivier1 • Jill A. Jenkins1 • Mark Berhow2 • Jacoby Carter1
Received: 2 March 2015 / Accepted: 25 November 2015 Ó Springer Science+Business Media New York (outside the USA) 2015
Abstract Pomacea maculata (formerly P. insularum), an apple snail native to South America, was discovered in Louisiana in 2008. These snails strip vegetation, reproduce at tremendous rates, and have reduced rice production and caused ecosystem changes in Asia. In this pilot study snails were exposed to two molluscicides, a tea (Camellia sinensis) seed derivative (TSD) or niclosamide monohydrate (PestanalÒ, 20 ,5-dichloro-40 -nitrosalicylanilide, CAS #73360-56-2). Mortality was recorded after exposure to high or low concentrations (0.03 and 0.015 g/L for TSD, 1.3 and 0.13 mg/L for niclosamide). The TSD induced 100 % mortality at both concentrations. Niclosamide caused 100 % and 17 % mortality at high and low concentrations respectively. These molluscicides were also tested on potential biocontrol agents, the red swamp crayfish (Procambarus clarkii) and redear sunfish (Lepomis microlophus). No crayfish mortalities occurred at either concentration for either chemical, but sunfish experienced 100 % mortality with TSD (0.03 g/L), and 21 % mortality with niclosamide (0.13 mg/L). Keywords Tea seed
Invasive species Niclosamide Pomacea
The spread of invasive species threatens the survival of wetland communities (Zedler and Kercher 2005).
& Heather M. Olivier
[email protected] 1
U.S. Geological Survey, Wetland and Aquatic Research Center, 700 Cajundome Boulevard, Lafayette, LA 70506, USA
2
U.S. Department of Agriculture, Agricultural Research Service, 1815 N. University Street, Peoria, IL 61604, USA
Economic losses caused by invasive species are high, whether generated through direct effects on agriculture or indirect effects via reductions in biodiversity or ecosystem services (Pimentel et al. 2005). In 1979, Pomacea apple snails from South America were introduced first to Taiwan, then to other Asian countries as an inexpensive protein source (Sin 2006). However this market was unsuccessful, culturing snails was abandoned, and snails escaped from holding pins into rice farms and natural wetlands causing millions of dollars in damage (Sin 2001). The most important components of wetland structure are the aquatic plants (Burlakova et al. 2008), and invasive apple snails have greatly reduced submerged aquatic vegetation in the wetlands they have invaded (Carlsson and Lacoursie`re 2005; Carlsson et al. 2006). By the early 1980s three non-native apple snail species were sold or traded as pets in the US. These species subsequently escaped and established populations in eleven states as of 2006 (Howells et al. 2006) (see Fig. 1 for P. maculata range, US Geological Survey Nonindigenous Aquatic Species Database ). In the 1990s, concerns arose because apple snails appeared in the rice-growing areas of Texas (Rawlings et al. 2007). In Florida, their populations began to overtake those of the native apple snail, raising concerns about their effect on the threatened Snail Kite (Rostrhamus sociabilis) (Cattau et al. 2010). Invasive Pomacea species also serve as intermediate hosts to parasites infective to humans (Hollingsworth and Cowie 2006) and wildlife (Kim et al. 2002). For example, a recent study found that a fraction of the apple snails in canals surrounding the New Orleans area tested positive for rat lungworm (Teem et al. 2013). As apple snails continue to spread, methods will be needed to control their populations. In this pilot study, we tested two molluscicides, one natural and one synthetic, for
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Author's personal copy Bull Environ Contam Toxicol Fig. 1 Species collection map showing the range of invasive Pomacea maculata in the US, generated from the US Geological Survey Nonindigenous Aquatic Species website (2014)
Fig. 2 a Mature Pomacea maculata withdrawn in its shell. b A female P. maculata laying eggs on a fiberglass holding tank
their effectiveness on P. maculata (Fig. 2) and on potential biological control species. First a plant-based product was tested, saponin-containing powdered tea (Camellia sinensis) seed. Saponins can cause hemolysis in exposed animals, and tea seed powder has been successfully used in Malaysia to treat golden apple snail (P. canaliculata) infestations in rice fields (Sin 2002). Tea seed powder is a byproduct of tea oil production that is relatively inexpensive and easy to obtain. Then a commercially available synthetic molluscicide, niclosamide monohydrate, (PestanalÒ, 20 ,5-dichloro-40 -nitrosalicylanilide, CAS #7336056-2, also known as Bayer 2353), was tested. It is an anti-
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helminthic used in human and veterinary medicine (Andrews et al. 1987), is recommended by the World Health Organization for the control of snails that cause schistosomiasis transmission (WHO s1993), is a more selective biocide than the currently approved copper sulfate (Oliveira-Filho et al. 2004), is non-persistent in the aquatic environment, and has a half-life of 1.1–3.9 days (Andrews et al. 1987; Calumpang et al. 1995). Niclosamide has been approved by the US Fish and Wildlife Service to decrease trematode infection in catfish ponds by eliminating the intermediate host, ram’s horn snails (Heliosoma sp.)(Terhune et al. 2003). The niclosamide formulation
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BayluscideÒ has been used in conjunction with other pesticides to eliminate sea lampreys (Petromyzon marinus) in the Great Lakes, though it is currently under review for this use (Dawson 2003). In our pilot study, adult snails and egg masses were exposed to a saponin-containing tea seed derivative (TSD) and niclosamide, and mortality was recorded. Biological control, the use of natural enemies to combat pest species (Strong and Pemberton 2000), has been successfully applied in Kenya by using red swamp crayfish (Procambarus clarkii) against schistosome-transmitting snails (Hofkin et al. 1991). Two non-target species that could serve as biocontrol agents were also exposed, red swamp crayfish and redear sunfish (Lepomis microlophus). Both are native to Louisiana and are cultured species. Niclosamide has been previously found to be harmful to some species of invertebrates, fish, and amphibians (Andrews et al. 1987), but not to crayfish (Sanders 1977), and TSD can be similarly harmful to fish and amphibians (Sin 2002). Our primary question was which pesticide was more effective at killing the snails without harming non-target species.
Methods and Materials Snails used in this study were collected near Schriever, Louisiana (29°420 2600 N, 90°540 1300 W). The species used in this study was genetically sequenced and found to be P. maculata. In the past these were often confused with the phenotypically similar P. canaliculata (Hayes et al. 2012). Snails were maintained indoors in a round *500 L fiberglass tank with a 12 h light cycle, at *24°C. The tank was aerated; water was recirculated, and drained and refilled as needed to maintain water quality. Herbaceous material dredged from a local pond was provided ad libitum with supplementary lettuce and other vegetables. Control measures to prevent escape included a tank covering of wire mesh and drains distant from outfalls. The TSD is commercially available outside of the US and has the trade name ‘Harvestwinner’. It was labelled as containing *25.03 % saponin (crude fiber – 25.65 %, crude protein – 8.24 %, fat residue – 1.79 %, ash – 2.81 %, moisture – 7.53 %, starch – 5.87 %, sugar – 12.03 %, and impurities – 11.02 %). Chemical compositions of natural, seed-derived products vary. This particular product was produced similarly to others that have been used successfully to control apple snails (Sin 2002). Akinpelu et al. (2012) found that saponin inhibited acetylcholinesterase activity in snails. This TSD was extracted in 50:50 dimethylsulfoxide and methanol and subjected to highperformance liquid chromatography (HPLC) using a gradient of acetonitrile/water and monitored at 210 nm. Chromatograms showed a mixture of compounds, with
several peaks in the saponin range indicating various sugar substitutions with a total concentration of 15 % by weight. To prepare exposure solution the TSD was dissolved at 0.015 g/L (low concentration) and 0.030 g/L (high concentration; label application rate) in degassed tap water. Study subjects were adults with an operculum width of at least 22 mm. Twelve snails and six adult crayfish, each in individual polypropylene containers, were exposed to 2 L of treatment solution at low or high concentration, or to control treatment (degassed tap water). The 54 total containers were aerated, randomly arranged, and maintained at 24°C on a 12 h light cycle with no water renewal. Water quality parameters (including dissolved oxygen, temperature, conductivity, salinity, and pH) were measured with a YSI model 85 water quality meter (YSI Inc., Yellow Springs, OH) at 0 and 24 h after exposure. Water quality measures did not vary greatly among treatments. Visual assessments on animal condition were also made at 0, 24, 48, and 72 h, at which point the experiment was terminated. Based on whether the snail was out of its shell, withdrawing upon prodding with a blunt probe, or unresponsive to prodding, snails were recorded as live, likely live, or dead, respectively. If closed, the aperture was pried open with a probe, and the individual was prodded; likely live individuals slowly closed the aperture, dead ones did not. Crayfish that reacted to prodding were considered live. Analytical standard niclosamide monohydrate, PestanalÒ, (99.9 % pure, Sigma-Aldrich, St. Louis, MO), was dissolved in 250 lL of ethanol and diluted in 2 L degassed tap water for exposures at 0.13 mg/L (low) and 1.3 mg/L (high; Pomacea LC50 as per Oliveira-Filho and Paumgartten 2000). Control exposures contained 250 lL of ethanol. Snails and crayfish were exposed similarly as above, and water quality and visual assessments were recorded. The mode of action of niclosamide on snails is believed to be a reduction in oxygen uptake and carbohydrate metabolism (Andrews et al. 1987). Redear sunfish were similarly exposed to the high concentration of TSD (9 treated, 6 control), and 3 crayfish were also exposed. Because the high concentration of niclosamide was above the LC50 for zebrafish (Danio rerio; BayluscideÒ product info.) sunfish were only exposed to the low concentration (14 treated, 8 control). Using a hand-held sprayer, snail egg masses were saturated with the high concentration of each chemical treatment (n = 11 TSD, n = 18 niclosamide). The egg masses were monitored along with two control groups of masses which were not sprayed (removed from the tank [dry control, n = 13], or still in the holding tank [tank control, n = 9]). The endpoint was the percent of eggs that hatched per mass. To determine the effect of spraying an unpaired one-tailed t test with unequal variances was
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applied to the data. All statistics were performed using Statistical Analysis SystemÒ version 8 (SAS, Cary, NC 1999). Actual concentration assessments weren’t performed during exposure, but in a separate experiment compound recovery attempts at 0, 24, 48, and 72 h were performed with high-performance liquid chromatography at 330 nm for niclosamide and 254 nm for a standard tea extract (Sigma; cat. no. T5550). Chemical standards were prepared in methanol at 1 and 5 lg/lL respectively. Niclosamide was diluted to 1 lg/mL in 30 mL water and the tea extract to 20 lg/mL in 30 mL water, then frozen and -80°C, then freeze-dried to concentrate. Samples were resuspended in 2 mL methanol from which 25 lL were injected to the column. Starting at 0 h and throughout, 66.3 % of niclosamide was recovered. Starting at 0 h, the major peak noted from the standard curve for tea extract in methanol was not apparent, but approximately 60 chemical entities were separated over the 50 min for each time period. Thus, under these conditions, the concentration of the single compound niclosamide was consistently bioavailable at 66 %, but the tea extract changed chemical form after water exposure, thus disallowing precise identification and quantification of each of the chemical components due to its complex nature.
Results and Discussion Both concentrations of TSD (0.015 and 0.030 g/L) induced 100 % mortality in adult apple snails by 72 h (Fig. 3). Although mortality was apparent at each concentration by 24-48 h, the final determination of death was made at 72 h. Niclosamide acted faster than TSD, where 80 % of snails died within 24 h, and 100 % were dead by 48 h in the high concentration (1.3 mg/L) (Fig. 3). However in the low concentration of niclosamide (0.13 mg/L), 16.6 % of snails were dead by 72 h. All apple snails and 94 % of crayfish survived in the control treatments. Crayfish survived exposure to both concentrations of both TSD and niclosamide up to 72 h (Fig. 3). Of nine sunfish exposed to the high dose of TSD, 100 % were dead within 3 hours, and all control treatments survived. From the niclosamide exposure, 21 % of treated fish died at 72 h, and all controls survived (Fig. 3). No statistical analysis was necessary for these results. Neither TSD nor niclosamide significantly affected hatching success of egg masses (p = 0.467 and 0.427 respectively). Other physical, biological, and chemical control options for slowing the spread of apple snails could be explored. Physical control, via collection of adults and crushing of egg masses, can potentially cause the least damage to natural environments, but can be prohibitively labor
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intensive. In Hawaii and Southeast Asia, ducks are used in agricultural fields to help eliminate small adult snails (Sin 2001). Effective biological control organisms are readily available, can focus specifically on the target species, and do not present their own invasion risk. Although the red swamp crayfish is not a snail specialist, it is a native species, and is already an aquaculture commodity often cocultured with rice in Louisiana. Likewise, the native redear sunfish feed on small snails and apple snail hatchlings (Carothers and Allison 1968). In laboratory experiments (data not shown), we found that both the crayfish and sunfish consumed small apple snails (smaller than fish gape size), though fully grown adults would be too large for either species. Because closely related P. canaliculata can aestivate for months (Little 1968) to survive dry conditions, this adaptation is important to consider when determining the timing of possible P. maculata control actions in agricultural settings after drainage, as chemical spraying will not affect individuals that have already burrowed into soil. One such strategy that may be employed in rice fields might include handpicking of adults, treatment with niclosamide or a TSD, followed by the introduction of biocontrol agents such as crayfish or sunfish following the period of chemical breakdown. The label directions for BayluscideÒ do not currently recommend human consumption of exposed organisms for at least 12 months after treatment, though more testing is needed. Calumpang (1994) found varying concentrations of niclosamide in the flesh of different fish species collected from treated aquaculture ponds, but did not find niclosamide residue in prawns or in rice (Calumpang et al. 1995) at harvest time when taken from treated fields. These control organisms could not be harvested for human consumption, but may be used in conjunction with chemical control so that treatment concentrations need not be as high. The TSD was equally effective at killing apple snails at both concentrations, but niclosamide was more effective at the higher concentration. When each molluscicide was tested on crayfish, no mortality was observed with either. Niclosamide is known to be harmful to some fish species (Calumpang et al. 1995), and we found niclosamide caused 21.4 % mortality in sunfish. Also, TSD caused 100 % sunfish mortality within only 3 h of exposure at the lowest concentration. These data show that both of these molluscicides would likely cause negative environmental effects on nontarget fish species and of course on any native mollusks, a group which is particularly at risk in North America (Ricciardi and Rasmussen 1999), if applied to natural wetlands. Before choosing to use either chemical in a natural setting it would be important to balance the benefit of apple snail reduction with the possible loss of native fish and invertebrate species. A chemical and/or biological treatment, paired with a mechanical treatment,
Author's personal copy Bull Environ Contam Toxicol Fig. 3 Percent mortality of a apple snails (Pomacea maculata), b redear sunfish (Lepomis microlophus), and c red swamp crayfish (Procambarus clarkii) at 72 h post-exposure to molluscicides at two concentrations or control. NT indicates concentration not tested for one of the molluscicides
such as crushing egg masses, may be effective at slowing the spread of apple snails through agricultural areas, thereby limiting their environmental effects. Our results
reflect laboratory conditions, not field conditions, and additional studies (including longer exposure durations with additional species and more concentrations) would be
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needed prior to the application of these chemicals to eliminate invasive apple snails. Acknowledgments We thank Eric Theall and Sergio Merino of IAP World Services for technical support, Matt Cannister of USGS for map production, and Tim Ruth of the LA Department of Wildlife and Fisheries (LDWF) for snail collection. Compliance with Ethical Standards This project was supported in part by the USGS Invasive Species Program. Any use of trade, product, or firm names is for descriptive purposes only and does not imply endorsement by the US Government. Conflict of interest
The authors report no conflicts of interest.
Human and Animal Rights This study followed animal protocols in accordance with taxon-specific guidelines (Use of Fishes in Research Committee 2014).
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