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Toxicity Effects of Piscicide CFT Legumine™ (5% Rotenone) onLithobates sp. of New Mexico

Report to

New Mexico Department of Game and Fish Share with Wildlife (#13-516-0000-00043)

from

C.A. CaldwellU.S. Geological Survey, New Mexico Cooperative Fish and Wildlife Research Unit

Box 30003, MSC 4901, Las Cruces, New Mexico 88003

Guillermo AlvarezNew Mexico State University, Department of Fish, Wildlife and Conservation Ecology


Kenneth G. BoykinNew Mexico State University, Center for Applied Spatial Ecology


DRAFT Toxicity Effects of CFT Legumine on Lithobates sp. February 2014

EXECUTIVE SUMMARY

Conservation through restoration of imperiled fishes can result in indirect benefits to amphibian populations through removal of invasive predatory species of fish. However, very limited information is available with regards to the effects of piscicidal applications to the non-target amphibians. Of particular interest in New Mexico is the federally-listed Chiricahua leopard frog (Lithobates chiricahuensis), and the Plains leopard frog (L. blairi) listed in New Mexico as a Species of Greatest Conservation Need. These two species co-occur throughout a watershed slated for native fish restoration using a piscicide. Toxicity trials using CFT Legumine (5% rotenone) were conducted on four larval stages of L. chiricahuensis and a surrogate species for L. blairi, the northern leopard frog (L. pipiens). Larvae of L. chiricahuensis exhibited greater sensitivity than the surrogate (L. pipiens) across all Gosner stages and both species exhibited decreasing sensitivity through tadpole development. The 48 h LC50 of CFT Legumine and its active ingredient (5% rotenone) was lowest (most toxic) in L. chiricahuensis at Gosner stages 21-25 (0.42 mg/L or 21.0 µg/L rotenone). In contrast, the 48 h LC50 for L. pipiens at these earliest Gosner stages was 1.30 mg/L (65.0 µg/L rotenone). In the later Gosner stages (26-30 and 31-36) of L. chiricahuensis, toxicity effects of CFT Legumine decreased to 1.03 mg/L (51.5 µg/L) and 1.30 mg/L (65.0 µg/L rotenone), respectively. In contrast, L. pipiens was less susceptible to the piscicide at the later Gosner stages (1.26-3.06 mg/L). While both species exhibited the least sensitivity after hind limbs were complete, L. chiricahuensis continued to be more sensitive to CFT Legumine (3.40 mg/L or 170 µg/L rotenone) than L. pipiens (3.86 mg/L or 193.0 µg/L rotenone). Late Gosner stage tadpoles (37-40) of both species demonstrated a delay in metamorphosis up to 90 days (total development time) when exposed for 48 hours to sublethal concentrations of CFT Legumine. Additional research is needed to evaluate the sublethal effects of the piscicide on metamorphosis in amphibians.

BACKGROUND

Amphibians are considered indicators of environmental health. More than a dozen species have disappeared from their historic ranges (Stebbins 2003) and considerable evidence suggests the global amphibian decline is the combined result of habitat loss, climate change, and invasive species (Houlihan et al. 2001; Skelly et al. 2003). Evidence has recently linked pesticides to declines in amphibian populations (Allran and Karasov 2000; Billman et al. 2011, 2012; Relyea et al. 2004). Of importance to regulatory agencies is the use of a piscicide (rotenone) to eradicate invasive non-native fishes from ponds, streams, and rivers. While rotenone’s effects on fishes have been described (see Ling 2003; McClay 2000), the acute and sublethal effects on non-target organisms such as amphibians are less known. Rotenone is a mitochondrial respiratory inhibitor with a variety of pesticidal uses, but is currently registered as

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a piscicide (USEPA 2007). Fish eradication projects are intended to restore native fish species which can indirectly benefit amphibian populations through removal of invasive predatory species of fish. However, indirect effects of piscicidal applications of rotenone have been recently linked to absence or loss of larval life stages of amphibian populations (presumably because of gills in the larval form) (Billman et al. 2011). Since 2007, registered use of rotenone as a piscicide is available in three commercial formulations: Prenfish™ Toxicant Liquid E.C. (EPA Reg. No. 655-422), Prentox™ Rotenone Fish Toxicant Powder (EPA Reg. No. 655-691), and Prentox CFT Legumine™ Fish Toxicant (EPA Reg. No. 655-899) (EPA 2007). Of these three, CFT Legumine™ (5% rotenone) is used in New Mexico to eradicate non-native fishes and is the only rotenone formulation reported in amphibian research (Billman et al. 2011; 2012).

The literature is varied with regards to toxicity effects of rotenone in amphibians (Table 1). Of note, the genus Rana was changed to Lithobates (Frost et al. 2008). Using standard 96 h LC₅₀ tests (lethal concentration for which 50% of the population responded), Chandler and Marking (1982) reported an LC₅₀ of 0.5 mg/L in Rana sphenocephala tadpoles using Noxfish™ thus identifying the gill-breathing larval stage as the most sensitive vertebrate to rotenone. During an effort to eradicate exotic African clawed frogs (Xenopus laevis), McCoid and Bettoli (1996) tested the efficacy of Prenfish™ (5-6 mg/L) to remove the invasive frog and found that only tadpoles were eliminated while the adults continued to reproduce in the ponds. Using Prenfish™ (5% active ingredient), Little and Calfee (2008; unpublished data) obtained a 96 h LC₅₀ of 0.79 mg/L for the Chiricahua leopard frog (Lithobates chiricahuensis) tadpole (Gosner stage 25). The authors suggested that high mortality would likely be observed in breeding areas if the recommended concentration of the rotenone formulation to eradicate fish was applied (2-5 mg/L). Using a 5% rotenone formulation (CFT Legumine), Billman et al. (2011), demonstrated early Gosner stages (21-25) of Columbia spotted frog (R. luteiventris) were highly vulnerable to 1.0 mg/L treatment while late Gosner stages (40-45) exhibited low mortality. Tadpoles of the boreal toad Anaxyrus boreas experienced high mortality across all age groups at 1.0 mg/L of CFT Legumine (Billman et al. 2012).

In New Mexico, the plains leopard frog (L. blairi) historically overlapped with the Chiricahua leopard frog (L. chiricahuensis) throughout Socorro, Chaves, Lincoln, and Sierra counties of New Mexico. Lithobates blairi is considered as a Species of Greatest Conservation Need, and populations in the Canadian, Mimbres, Pecos, Rio Grande, and Tularosa watersheds are considered vulnerable (NMDGF 2006). Lithobates chiricahuensis is federally-listed as threatened (USFWS 2002) due to 82% reduction throughout its historic range. Within the Rio Grande drainage, the species occurs in Alamosa Creek in Socorro County, and Cuchillo and Seco creeks in Sierra County. While L. chiricahuensis is reliant on permanent aquatic habitats, L. blairi occupies water bodies that are shallow and often temporary (Degenhardt et al. 1996). Of particular interest is the presence of L. chiricahuensis throughout the Las Animas watershed and its co-occurrence with a population of cutthroat trout introgressed with Yellowstone cutthroat trout (Oncorhynchus clarkii bouvieri) and rainbow trout (Oncorhynchus mykiss; Douglas and Douglas 2006). A piscicide treatment is planned to remove the introgressed

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cutthroat trout and replace with a pure population of Rio Grande cutthroat trout (Oncorhynchus clarki virginalis). Thus, the presence of the federally-listed amphibian species throughout a drainage slated for native fish restoration is of concern to private, state and federal agencies charged with managing for the conservation of more than one of these species. Federal and state fish biologists requested that toxicity testing be conducted on Lithobates sp. because of the planned fish restoration projects throughout areas containing populations of these native amphibians. Of particular concern is timing and duration of rotenone application during breeding and the effects on tadpole development and survival.

The goal of the research was to provide acute and potential long term (sublethal) toxicity effects of a rotenone formulation to management agencies to minimize the collateral effects in amphibian populations throughout areas slated for fish removal. Our objectives were to characterize the acute and chronic toxicity effects of a commercial rotenone formulation (CFT Legumine™ 5% Rotenone) on early life stages of Lithobates sp. native to New Mexico. Using concentrations relevant to piscicidal applications, we conducted a series of short (48 h) acute toxicity studies throughout four larval stages to determine the lethal concentration of a commercial rotenone formulation at which 50% (LC50) of the test populations died. From the results of these acute toxicity tests, we conducted a series of sub-lethal toxicity tests to determine the concentrations of the rotenone formulation on metamorphosis. We report delayed time to metamorphosis and mass and length of the froglet at metamorphosis.

MATERIALS AND METHODS

Test Animals- At the time of this report, toxicity tests have not been conducted on L. blairi. Drought followed by wildfire wiped out several populations of L. blairi that we had begun to sample. Prior to the fire and subsequent ash flow, we captured two L. blari tadpoles that are currently housed in our ranarium at New Mexico State University. We plan to rear these individuals (male and female) as a captive breeding pair for future toxicity testing, or, until wild populations of L. blari can be located. To complete our contractual obligations with Share with Wildlife, we conducted a series of toxicity tests on the northern leopard frog (L. pipiens) as a surrogate ranid, native to New Mexico. We obtained eggs through a commercial distributor (eNASCO, Wisconsin). Prior to toxicity tests, we evaluated hatch rate, growth rate, and time to metamorphosis of L. pipiens and deemed the species comparable to L. chiricahuensis under the same environmental conditions.

Recent discovery of a population of L. chiricahuensis in the Seco drainage, Sierra County, led to an agreement between the U.S. Fish and Wildlife Service and the Turner Endangered Species Fund to support captive propagation and management activities of the local population (McCaffery and Phillips 2012). As part of the agreement, a captive-breeding ranarium and tadpole rearing facility was implemented on the Ladder Ranch to supplement local wild populations of L. chiricahuensis. Egg clutches that were not slated for augmenting the wild population were transported to New Mexico State University where they were acclimatized

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to greenhouse conditions. Clutch identification was assigned and number of eggs within each clutch was estimated by eye (Table 2). Estimated hatch rates for each clutch varied resulting in a range of total numbers of tadpoles available for each of the toxicity tests (Table 3). Husbandry and Maintenance- In anticipation of the arrival of each egg clutch, de-chlorinated water was aerated in small aquaria. The eggs were received in gallon zip-lock bags and placed in aquaria (bags with eggs). The eggs were aerated within the original bag to allow the eggs to acclimatize slowly to greenhouse temperatures. Within 2-3 hours, the egg mass was gently transferred into the aquarium. The water was partially replaced (~50%) every other day from the date of arrival through hatching until hatching was complete. The hatchling larvae (2-3 days post-hatch) were then transferred to an aquarium containing 160 L of aerated and de-chlorinated water. Dissolved oxygen (mg/L) and temperature (oC) (Hach Model HQ40D53; Hach Co.) were monitored daily. Ammonia and nitrite (Total N mg/L; Hach Co.) were monitored weekly.

Prior to the toxicity tests, both air and water temperatures were monitored within the greenhouse. While ambient air temperatures within the greenhouse were set at 22.2ᵒC during winter months and 18.3ᵒC during summer months, data loggers (Hobo UX100, Onset Computer Corporation) revealed air temperatures within the greenhouse varied from 19 to 35ᵒC and water temperatures varied from 15 to 30ᵒC. The exception occurred October 17 when the majority of clutch LC-04-13 died due to a malfunction of the greenhouse heating system. An electronic component and a venting flap within the greenhouse heating system malfunctioned and an estimated 600 tadpoles from the clutch perished. To prevent additional loss of our animals due to equipment malfunction, NMSU installed an automated calling system (SensaPhoneSystem™) on 6 January 2014 to alert the caretaker, the farm manager, and the researchers of temperatures outside acceptable limits.

Feeding schedules and food items depended upon the stage of tadpole development and were modified following the dietary and husbandry requirements provided by Wright and Whitaker (2001). Both species of tadpoles were fed the same diet items at the same ratio based on tank density, tadpole weight, length, and stage of development. Feed was offered prior to the depletion of the yolk sac. Each day, 50 ml of liquefied spirulina, dried shrimp, and trout chow was distributed throughout the aquaria containing the developing tadpoles. This combination of feed items was modified from diets of other tadpole species and reflects high protein content (i.e., trout chow), high palatability (i.e., dried shrimp), and high plant content (i.e., spirulina). Water within each holding aquarium containing tadpoles was changed (no more than 50%) every other day. Tadpole densities were maintained at approximately 10-15 tadpoles/L. Upon reaching Gosner stage 23, tadpoles were transferred to larger holding tanks (1-2 tadpoles/L). At this stage, feeding schedules and food items changed slightly with the addition of 150 ml of the liquefied diet supplemented with algae wafers (10 wafers per 150 L).

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Experimental Design- Pilot studies also revealed the range of treatment concentrations using CFT Legumine (5% Rotenone) for the range of Gosner stages (Gosner 1960; see Figure). The earliest test group was represented by Gosner stages of development from 21 to 25 (pre-limb bud formation). The second (mid-stage) test group was represented by Gosner stages of development from 26 to 30 (limb bud formed). The third (late-stage) group was represented by Gosner stages of development from 31 to 36 (toe differentiation and hind limb development completed). The last test group included tadpoles at the climax of metamorphosis and represented Gosner stages from 37 to 40 (pre-forelimb development). Upon reaching one of four Gosner stages the tadpoles were exposed to static (non-renewal) concentrations of the CFT Legumine formulation for 48 h (ASTM 2007).

Test Solution Preparation- CFT Legumine test solutions were prepared on the start date of each test from a stock solution of 1.019 g/L using reversed-osmosis water. Test solutions were prepared and stored in dark glass air-tight containers in the laboratory and transported to the greenhouse in an ice-chest. The desired volume was pipetted from the stock solution into 1 Liter bottles containing aerated dechlorinated water. The water used to prepare the treatment solutions was the same water used for the controls which was municipal in origin and required dechlorination as well as aeration prior to use.

48 h Acute Toxicity Tests- Pilot tests were initially carried out to 96 h to assess toxicity through time. We knew that environmental degradation of rotenone would be directly influenced by sunlight (Cabras et al. 2002; Draper 2002; Jones et al. 1933), thus rotenone would be expected to hydrolyze (reduced). We observed no detectable differences in LC50 values at 48, 72, and 96 h and selected 48 h as the duration of our toxicity exposures. Five tadpoles were placed in 250 ml glass containers containing 200 ml of the solution (1 tadpole/40 ml) and tested in replicates of five (n=5) for a total 25 tadpoles per treatment. For both species, tadpoles of Gosner stages 21-25 were exposed to control (no toxicant), 1.0, 0.5, 0.25, and 0.125 mg/L CFT Legumine. Similarly, tadpoles of Gosner stages 26-30 and 31-36 were exposed to control (no toxicant), 2.0, 1.5, 1.0, and 0.5 mg/L CFT Legumine. For Gosner stages 37-40, tadpoles were exposed to control (no toxicant), 5.0, 4.0, 3.0, and 2.0 mg/L CFT Legumine. Tadpoles were not fed for 24 hours prior to the tests or throughout the 48 h tests. Dissolved oxygen (mg/L) and temperature (oC) were monitored daily; ammonia (mg/L as total N) and pH were measured at the end of each test. Dead tadpoles were removed and preserved in 70% ethyl alcohol. At the end of the tests, all surviving tadpoles were euthanized in a lethal concentration of Finquel™ and archived at -27oC for analyses of rotenone concentrations. The 48 h LC50 and the 95% confidence interval were calculated using the Trimmed Spearman-Karber method (Hamilton et al. 1977).

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48 h Acute Toxicity Tests: Greenhouse versus Laboratory- Using the same procedures described above for carrying out and analyzing the results, two 48 h toxicity tests were completed on L. chiricahuensis under controlled laboratory conditions to determine if toxicity was detectably different between greenhouse (sunlight and diel temperature swings) and laboratory (photoperiod and constant temperature) conditions. Tadpoles were not fed for 24 hours prior to the tests or throughout the 48 h tests. Dissolved oxygen and temperature were monitored daily; ammonia and pH were measured at the end of each test. Throughout the tests, dead tadpoles were removed and preserved in 70% ethyl alcohol; at the end of the tests, all surviving tadpoles were euthanized in a lethal concentration of Finquel™ and archived at -27oC for analyses of rotenone concentrations.

Sublethal Assays- A range of concentrations was selected to characterize the sublethal effects of CFT Legumine (5% Rotenone) on metamorphosis of L. chiricahuensis and L. pipiens tadpoles. Late-age tadpoles (Gosner stages 37-40) were targeted due to the number of survivors at the end of the 48 h acute exposures. Individual tadpoles were placed in 250 ml glass containers with 100 ml of the solution and tested in replicates of 15 tadpoles per treatment. For both species, tadpoles of Gosner stages 37-40 were exposed to control (no toxicant), 5.0, 4.0, 3.0 mg/L CFT Legumine. Tadpoles were not fed for 24 hours prior to the test or throughout the 48 h tests. At the end of each test, one-half of all survivors (treatments and controls) were each placed in 15 L aquaria containing rotenone-free dechlorinated water and a floating patch of plastic grass to allow emerging froglets to perch above the water level. The remaining half of the survivors was euthanized using Finquel™, preserved in 70% ethyl alcohol and archived at -27oC for analyses of rotenone concentrations. Aquarium water was renewed every three days and tadpoles/froglets were fed according to their development. Tadpoles were monitored closely and development recorded daily. These included forelimb emergence, tail reabsorption, disease, loss of appetite, sluggishness, and mortality. Upon completion of metamorphosis (indicated by reabsorption of the tail), the froglet was measured (snout-urostyle length, mm), photographed, assessed for malformations, lesions, or evidence of disease. The individuals were euthanized using Finquel™ and preserved in 70% ethyl alcohol and archived at -27oC for analysis of rotenone concentrations. At the time of this report (February 2014), analysis of rotenone concentrations using high-performance-liquid-chromatography has not been conducted on preserved water or tissue samples.

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RESULTS

We report both the nominal concentrations of CFT Legumine formulation (mg/L) and the active ingredient (5% rotenone, µg/L part per billion) (see Table 4). Lithobates chiricahuensis exhibited greater sensitivity than L. pipiens across all Gosner stages tested and both species exhibited decreasing sensitivity to CFT Legumine through tadpole development. The 48 h LC50 of CFT Legumine was lowest (and thus most toxic) in L. chiricahuensis at Gosner stage 21-25 (0.42 mg/L or 21.0 µg/L rotenone). In contrast, the 48 h LC50 for L. pipiens at these earliest Gosner stages was 1.30 mg/L (65.0 µg/L rotenone). The toxicity effects of CFT Legumine in L. chiricahuensis decreased to 1.03 mg/L (51.5 µg/L) and 1.30 mg/L (65.0 µg/L rotenone) in the later Gosner stages (26-30 and 31-36). In contrast, L. pipiens exhibited much lower toxicity thresholds at these later Gosner stages (1.26 – 3.06 mg/L). While both species exhibited the lowest toxicity effects after hind limbs were complete within the late Gosner stages, L. chiricahuensis was more sensitive to CFT Legumine (3.40 mg/L or 170 µg/L rotenone) than L. pipiens (3.86 mg/L or 193.0 µg/L rotenone).

Two 48 h toxicity tests were conducted concurrently under controlled laboratory conditions to determine if rotenone toxicity differed between greenhouse (sunlight and diel temperature swings) and laboratory (photoperiod and constant temperature) conditions. We observed a lower (thus more toxic) 48 h LC50 of 0.22 mg/L in L. chiricahuensis exposed to CFT Legumine in the laboratory when compared to the 48 h LC50 of 0.42 mg/L in greenhouse conditions. One test is anecdotal and does not necessarily indicate differences in toxicity; however, laboratory lighting conditions would have ameliorated photolysis of rotenone thereby reducing toxicity effects. The cautionary tale here is a careful comparison of laboratory-derived versus field-derived rotenone effects. Analysis of archived samples would relate degradation rates of rotenone with loss in toxicity. Water and tissue samples were collected and will remain archived for actual concentrations of rotenone if funding becomes available.

All tadpoles within the control groups of the sublethal tests completed metamorphosis within 18 days (average) of the end of the toxicity tests (see Table 5). At the time of this report (25 February 2014), one-half of the surviving L. chiricahuensis tadpoles from 3.0 mg/L treatments have completed metamorphosis while none of the surviving tadpoles from the 4.0 mg/L treatments have completed metamorphosis. Two tadpoles from the 4.0 mg/L treatment have yet to produce forelimbs. At the time of this report, there appears to be no detectable differences in growth (length and weight) of the controls compared to growth of tadpoles and metamorphs in the CFT Legumine treatments. No visible malformations or lesions were observed with only one tadpole succumbing to disease a few days after exposure to CFT Legumine.

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DISCUSSION

Early life stages of amphibians represent the most sensitive period of development to environmental stressors and contaminants. For example, growth in amphibian larvae was drastically affected by food availability, temperature (Petranka 1984), larval density (Warner et al. 1991), predator and competition (Werner and Anholt 1996), and use of synthetic toxicants (Carey and Bryant 1995). We observed acute toxicity effects as low as 21 µg/L rotenone in early life stages of L. chiricahuensis (throughout limb bud formation). Given the target concentration of rotenone in fishes of 50 µg/L, the earliest stages of L. chiricahuensis would experience lethal effects if present during piscicidal applications. Billman et al. (2011) observed tadpoles of Columbia spotted frog (L. luteiventris) and boreal toad (A. b. boreas) were highly sensitive to piscicidal treatment concentrations of CFT Legumine while mortality in late stage tadpoles and metamorphs was low. Presumably, the effects of rotenone are pronounced in gill-breathing stages of amphibians because gills offer a direct line of exposure to sites of toxic action due to the large surface area and very short diffusion distance of gill lamellae.

Survival may also be affected if metamorphosis is delayed. Slower development rates have several long-term effects that are not just limited to reduced size at time of metamorphosis. Lower growth rate was reflected in a reduced ability to swim away from predators (Goater et al. 1993) and smaller size at maturity was manifested in low egg production (Semlitsch et al. 1988; Wilbur and Collins 1973). The sublethal exposure trials began late September 2013 for L. pipiens and late November 2013 for L. chiricahuensis. We observed a delay in metamorphosis of nearly 90 days in L. chiricahuensis exposed to 3.0 and 4.0 mg/L CFT Legumine and a delay of nearly 40 days in L. pipiens. Delayed tadpoles were no larger or smaller than the controls at the time of metamorphosis. In addition, neither disease nor behavioral differences were manifested in the treated tadpoles. The sublethal trials were conducted late fall, and while tadpoles within the control groups for both species completed metamorphosis within the expected timeframe, one should not rule out the effects of timing such as seasonal photoperiod may have had in the delay of metamorphosis in tadpoles exposed to CFT Legumine. Sublethal toxicity studies should be replicated in spring 2014 to characterize the contribution of CFT Legumine versus environmental factors in the delay of metamorphosis.

This research demonstrates differences in levels of sensitivity of two species of Lithobates to a piscicide and emphasizes the importance of expanding protective guidelines to include other native amphibians (i.e., L. blairi). Differences in geographic distributions may also represent different toxicity responses to rotenone among ranids emphasizing an urgent need for the description of lethal concentrations as well as sub-lethal effects to these non-target organisms during fish removal. Furthermore, our research emphasizes the importance of timing in the application of a piscicidal treatment in relation to non-target amphibians and stage of development. It is important to note we report nominal concentrations of both CFT Legumine and its active ingredient (5% rotenone). Laboratory analysis of rotenone is relatively inexpensive and should be considered in future work to better characterize actual concentrations to effectively relate toxicity effects.

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Stage 21 Stage 22 Stage 23 Stage 24 Stage 25

Stage 26 Stage 27 Stage 28 Stage 29 Stage 30

Stage 31 Stage 32 Stage 33 Stage 34 Stage 35 Stage 36

Stage 37 Stage 38 Stage 39 Stage 40

Figure. Stages in early development of anurans based on Gosner (1960). These four stages were selected in Lithobates chiricahuensis and L. pipiens for 48 h exposure to static non-renewal concentrations of CFT Legumine (5% rotenone).

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Early group: pre-limb

Mid-group: limb bud formation

Late group: toe differentiation of hind limb

Froglet: includes all development prior to front limbs


Table 1. Toxicity of commercial rotenone products to amphibians.

Organism Concentration Threshold Compound ReferenceSouthern leopard frog(Rana sphenocephala)unknown larval stage

0.5 mg/L 96 h LC₅₀ Noxfish Chandler and Marking (1982)

African clawed frog(Xenopus laevis)

Adults/unknown stages of tadpoles

5-6 mg/L 100% mortality (tadpoles)

Prenfish McCoid and Bettoli (1996)

Chiricahua leopard frog(R. chiricahuensis) Gosner stage 25

0.79 mg/L 96 h LC₅₀ Prenfish Little and Calfee (2008) unpublished

data

Chiricahua leopard frog(R. chiricahuensis)Gosner stage 28-31

1.2 mg/L 96 h LC₅₀ Prenfish Little and Calfee (2008) unpublished

data

Columbia spotted frog(R. luteiventris)

Gosner stage 40-45

1.0 mg/L 24 hfield exposure

(100% mortality)

CFT Legumine 5% Rotenone

Billman et al. (2011)

Columbia spotted frog(R. luteiventris)

adults

1.0 mg/L 24 hfield exposure(no observed

mortality)



Boreal toad(Anaxyrus boreas)

tadpoles

1.0 mg/L 24 hfield exposure

(100% mortality)



Leopard frog(R. pipiens)

Unknown stage of juveniles

7.3 mg/L 24 h LC₅₀ Dri-Noxfish Farringer (1972) (from Bradbury

1986)Leopard frog(R. pipiens)

Unknown stage of juveniles



Unknown stage of tadpoles







0.1 mg/L 8-24 h(100%

mortality)

5% Rotenone Hamilton (1941) (from Bradbury

1986)Tiger salamander

(Ambystoma tigrinum)Unknown gilled stage

0.02 mg/L 8-24 h(toxic but not

fatal)


1986)Tiger salamander

(A. tigrinum)Unknown stage of tadpoles

0.1 mg/L 8-24 h(100%

mortality)


1986)

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Table 2. Clutch identification of Lithobates chiricahuensis obtained from captive refugia on the Ladder Ranch, Sierra County, New Mexico.

Clutch IDDate obtained

Estimated Number of

Eggs in Clutch

Hatch Date

Estimated Hatch Rate

Estimated Number of Tadpoles

Number of Tadpoles Used

in TestsLC-01-13(3 May)

900 10 May 85% ~750 700

LC-02-13(2 Jun)

900 6 Jun 75% ~650 600

LC-03-13(14 Aug)

700 20 Aug 80% ~500 445

LC-04-13(25 Sep)

800 29 Sep 85% ~680 *

*Heating malfunction in the NMSU greenhouse resulted in the majority of the tadpoles perishing.

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Table 3. Clutch identification and dates of toxicity tests and Gosner stages (GS) for Lithobates chiricahuensis (LC) and L. pipiens (LP) subjected to acute (48 h) and sublethal (48 h) toxicity tests.

* Represents the results of one toxicity test conducted in laboratory conditions (Indoor). ** One 48 h LC50 was performed but deemed unacceptable.

Clutch ID GS 21-25 Indoor GS 26-30 GS 31-36 GS 37-40 GS 37-40

LC-01-13(3 May)

12-15 Jun - 23-26 Jun 12-15 Jul 24-27 Jul -

LC-02-13(31 May)

2-5 Jul *1-4 Jul 16-19 Jul 5-8 Aug 16-19 Aug -

LC-03-13(14 Aug)

LP-01-13(15 Jul)

10-13 Sep

20-23 Aug

-

-

17-20 Oct

28-31 Aug

8-9 Nov

10-13 Sep

**8-11 Nov

-

25-26 Nov

24-25 Sep

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Table 4. Average (± standard error, sample size; 95% confidence intervals are reported in parenthesis when the tests were not replicated) lethal concentrations of 50% of the test populations (LC50) at 48 h using CFT Legumine (Rotenone 5%) on Chiricahua leopard frog (Lithobates chiricahuensis) and northern leopard frog (L. pipiens) at targeted Gosner stages (GS) in greenhouse conditions. Nominal concentrations of rotenone were obtained from CFT Legumine formulation assuming the active ingredient (rotenone) is 5%.

SpeciesGosner Stage

CFT Legumine (5%) (mg/L)

Nominal Rotenone (ug/L)

L. chiricahuensisGS 21-25Early: Pre-limb bud formation

0.42 mg/L(± 0.125, n=3)

21.0 µg/L

L. chiricahuensisGS 26-30Mid: Limb bud formed

1.03 mg/L(± 0.073, n=3)

51.5 µg/L

L. chiricahuensisGS 31-36Late: Toe differentiation hind limb complete

1.30 mg/L(± 0.037, n=2)

65.0 µg/L

L. chiricahuensisGS 37-40Early froglet: Prior to front limb development

3.24 mg/L(± 0.045, n=2)

162.0 µg/L

L. chiricahuensis** (Sub-lethal Exposure)GS 37-40Early froglet: Prior to front limb development

3.40 mg/L(3.13-3.70)

170.0 µg/L

L. pipiensGS 21-25Early: Pre-limb bud formation

1.30 mg/L(1.21-1.41)

65.0 µg/L

L. pipiensGS 26-30Mid: Limb bud formed

1.26 mg/L(1.16-1.36)

63.0 µg/L

L. pipiensGS 31-36Late: Toe differentiation hind limb complete

3.06 mg/L(2.86-3.27)

153.0 µg/L

L. pipiens (Sub-lethal Exposure)GS 37-40Early froglet: Prior to front limb development

3.86 mg/L(3.44-4.33)

193.0 µg/L

** represents an ongoing test at the time of this report.

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Table 5. Clutch identification for Lithobates chiricahuensis (LC) and L. pipiens (LP) at Gosner stages 37-40 subjected to sublethal 48 h toxicity tests of 3.0 and 4.0 mg/L CFT Legumine. Average number of days to reach completion of metamorphosis (Days), average snout-urostyle-length (SUL, mm), and average weights (g) observed among individuals upon completion of metamorphosis.

Control 3 mg/L 4 mg/LID Days SUL

(mm)Weight

(g)Days SUL

(mm)Weight

(g)Days SUL

(mm)Weight

(g)

LC-03-13 18.2 22 (± 2.3) 1.6 (±0.4) *21.0 *23 (±2) *1.7 (±0.5) ** ** **

LP-01-13 23.5 27 (± 4) 2.0 (±0.6) 29.2 26 (±3.4) 2.4 (± 0.8) 36.5 25 (±4.1) 2.1 (±0.5)

* indicates less than half of the tadpoles have completed metamorphosis (February 2014)** indicates no tadpoles have completed metamorphosis (February 2014)

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ACKNOWLEDGEMENTS

The research was funded through the New Mexico Department of Game and Fish Share with Wildlife Grant (#13-516-0000-00043). Additional support was provided by the Turner Endangered Species Fund, U.S. Fish and Wildlife Service – Ecological Services Field Office (Michelle Christman), New Mexico State - University, Department of Fish, Wildlife and Conservation Ecology, and U.S. Geological Survey- New Mexico Cooperative Fish and Wildlife Research Unit. Our heartfelt thanks go to Hanne Small (Turner Endangered Species Fund) and Dr. Carter Kruse (Turner Enterprises, Inc.) for providing egg clutches of L. chiricahuensis. CFT Legumine™ is a restricted pesticide. As such, Caldwell received State licensure (#61914) to purchase and use the pesticide from New Mexico Department of Agriculture. Caldwell holds a New Mexico Research and Collection permit (NMDGF #3033) to collect L. pipiens, and a Federal T&E permit (#TE-046517) to use L. chiricahuensis in toxicity tests. We received approval from the New Mexico State University Institutional Animal Care and Use Committee for the research (#2012-025).

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