Toxicity Effects of Piscicide CFT Legumine™ (5% Rotenone) on
Lithobates sp. of New Mexico
Report to
New Mexico Department of Game and Fish
Share with Wildlife (#13-516-0000-00043)
from
C.A. Caldwell
U.S. Geological Survey, New Mexico Cooperative Fish and Wildlife Research Unit
Box 30003, MSC 4901, Las Cruces, New Mexico 88003
Guillermo Alvarez
New Mexico State University, Department of Fish, Wildlife and Conservation Ecology
Box 30003, MSC 4901, Las Cruces, New Mexico 88003
Kenneth G. Boykin
New Mexico State University, Center for Applied Spatial Ecology
Box 30003, MSC 4901, Las Cruces, New Mexico 88003
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 nontarget 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 (3740) 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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Toxicity Effects of CFT Legumine on Lithobates sp.
February 2014
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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DRAFT
Toxicity Effects of CFT Legumine on Lithobates sp.
February 2014
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
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DRAFT
Toxicity Effects of CFT Legumine on Lithobates sp.
February 2014
acclimatized 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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Toxicity Effects of CFT Legumine on Lithobates sp.
February 2014
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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Toxicity Effects of CFT Legumine on Lithobates sp.
February 2014
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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Toxicity Effects of CFT Legumine on Lithobates sp.
February 2014
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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Toxicity Effects of CFT Legumine on Lithobates sp.
Stage 21
Stage 22
Stage 23
Stage 24
Stage 25
Stage 28
Stage 29
Stage 30
Stage 34
Stage 35
February 2014
Early group: pre-limb
Stage 26
Stage 27
Mid-group: limb bud formation
Stage 31
Stage 32
Stage 33
Stage 36
Late group: toe differentiation of hind limb
Stage 37
Stage 38
Stage 39
Stage 40
Froglet: includes all development prior to front limbs
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 nonrenewal concentrations of CFT Legumine (5% rotenone).
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Toxicity Effects of CFT Legumine on Lithobates sp.
February 2014
Table 1. Toxicity of commercial rotenone products to amphibians.
Organism
Concentration
Threshold
Compound
Reference
Southern 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 h
field exposure
(100%
mortality)
CFT Legumine
5% Rotenone
Billman et al.
(2011)
Columbia spotted frog
(R. luteiventris)
adults
1.0 mg/L
24 h
field exposure
(no observed
mortality)
CFT Legumine
5% Rotenone
Billman et al.
(2011)
Boreal toad
(Anaxyrus boreas)
tadpoles
1.0 mg/L
CFT Legumine
5% Rotenone
Billman et al.
(2011)
Leopard frog
(R. pipiens)
Unknown stage of juveniles
Leopard frog
(R. pipiens)
Unknown stage of juveniles
Leopard frog
(R. pipiens)
Unknown stage of tadpoles
Leopard frog
(R. pipiens)
Unknown stage of tadpoles
Leopard frog
(R. pipiens)
Unknown stage of tadpoles
Tiger salamander
(Ambystoma tigrinum)
Unknown gilled stage
Tiger salamander
(A. tigrinum)
Unknown stage of tadpoles
7.3 mg/L
24 h
field exposure
(100%
mortality)
24 h LC₅₀
Dri-Noxfish
7.9 mg/L
24 h LC₅₀
Dri-Noxfish
4.6 mg/L
96 h LC₅₀
Dri-Noxfish
3.2 mg/L
96 h LC₅₀
Dri-Noxfish
0.1 mg/L
8-24 h
(100%
mortality)
8-24 h
(toxic but not
fatal)
8-24 h
(100%
mortality)
5% Rotenone
Farringer (1972)
(from Bradbury
1986)
Farringer (1972)
(from Bradbury
1986)
Farringer (1972)
(from Bradbury
1986)
Farringer (1972)
(from Bradbury
1986)
Hamilton (1941)
(from Bradbury
1986)
Hamilton (1941)
(from Bradbury
1986)
Hamilton (1941)
(from Bradbury
1986)
0.02 mg/L
0.1 mg/L
11
5% Rotenone
5% Rotenone
DRAFT
Toxicity Effects of CFT Legumine on Lithobates sp.
February 2014
Table 2. Clutch identification of Lithobates chiricahuensis obtained from captive refugia on
the Ladder Ranch, Sierra County, New Mexico.
Clutch ID
Date obtained
Estimated
Number of
Eggs in Clutch
900
Hatch
Date
Estimated
Hatch Rate
10 May
LC-02-13
(2 Jun)
900
LC-03-13
(14 Aug)
LC-04-13
(25 Sep)
LC-01-13
(3 May)
85%
Estimated
Number of
Tadpoles
~750
Number of
Tadpoles Used
in Tests
700
6 Jun
75%
~650
600
700
20 Aug
80%
~500
445
800
29 Sep
85%
~680
*
*Heating malfunction in the NMSU greenhouse resulted in the majority of the tadpoles
perishing.
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DRAFT
Toxicity Effects of CFT Legumine on Lithobates sp.
February 2014
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.
Clutch ID
LC-01-13
(3 May)
GS 21-25
12-15 Jun
Indoor
-
GS 26-30
23-26 Jun
GS 31-36
12-15 Jul
GS 37-40
24-27 Jul
GS 37-40
-
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)
10-13 Sep
-
17-20
Oct
8-9 Nov
**8-11 Nov
25-26 Nov
20-23
Aug
-
28-31
Aug
10-13 Sep
-
24-25 Sep
LP-01-13
(15 Jul)
* Represents the results of one toxicity test conducted in laboratory conditions (Indoor).
** One 48 h LC50 was performed but deemed unacceptable.
13
DRAFT
Toxicity Effects of CFT Legumine on Lithobates sp.
February 2014
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%.
Species
Gosner Stage
CFT Legumine (5%)
(mg/L)
Nominal Rotenone
(ug/L)
L. chiricahuensis
GS 21-25
Early: Pre-limb bud formation
L. chiricahuensis
GS 26-30
Mid: Limb bud formed
L. chiricahuensis
GS 31-36
Late: Toe differentiation hind limb complete
0.42 mg/L
(± 0.125, n=3)
21.0 µg/L
1.03 mg/L
(± 0.073, n=3)
51.5 µg/L
1.30 mg/L
(± 0.037, n=2)
65.0 µg/L
L. chiricahuensis
GS 37-40
Early froglet: Prior to front limb development
3.24 mg/L
(± 0.045, n=2)
162.0 µg/L
L. chiricahuensis** (Sub-lethal Exposure)
GS 37-40
Early froglet: Prior to front limb development
3.40 mg/L
(3.13-3.70)
170.0 µg/L
L. pipiens
GS 21-25
Early: Pre-limb bud formation
L. pipiens
GS 26-30
Mid: Limb bud formed
L. pipiens
GS 31-36
Late: Toe differentiation hind limb complete
1.30 mg/L
(1.21-1.41)
65.0 µg/L
1.26 mg/L
(1.16-1.36)
63.0 µg/L
3.06 mg/L
(2.86-3.27)
153.0 µg/L
L. pipiens (Sub-lethal Exposure)
GS 37-40
Early 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.
14
DRAFT
Toxicity Effects of CFT Legumine on Lithobates sp.
February 2014
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-urostylelength (SUL, mm), and average weights (g) observed among individuals upon completion of
metamorphosis.
Control
SUL
Weight
(mm)
(g)
3 mg/L
SUL
Weight
(mm)
(g)
Days
Days
4 mg/L
SUL
Weight
(mm)
(g)
ID
Days
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)
15
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Toxicity Effects of CFT Legumine on Lithobates sp.
February 2014
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).
16
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Toxicity Effects of CFT Legumine on Lithobates sp.
February 2014
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