IMPACT OF THE FIRE ANT PESTICIDE HYDRAMETHYLNON (AMDRO®) ON
LOGGERHEAD SEA TURTLE REPRODUCTIVE SUCCESS AND HATCHLING
QUALITY
by
Heather Smith
A Thesis Submitted to the Faculty of
The Charles E. Schmidt College of Science
in Partial Fulfillment of the Requirements for the Degree of
Master of Science
Florida Atlantic University
Boca Raton, Florida
May 2019
Copyright 2019 by Heather Smith
ii IMPACT OF THE FIRE ANT PESTICIDE HYDRAMETHYLNON (AMDRO®) ON
LOGGERHEAD SEA TURTLE REPRODUCTIVE SUCCESS AND HATCHLING
QUALITY
by
Heather Smith
This thesis was prepared under the direction of the candidate's thesis advisor, Dr. Michael Salmon, Department of Biological Sciences, and has been approved by all members of the supervisory committee. It was submitted to the faculty of the Charles E. Schmidt College of Science and was accepted in partial fulfillment of the requirements for the degree of Master of Science. SUPERVISe}? COMMITTEE: 1£u.L-« £'&-~ MichaelS lmon, Ph.D. The»r.'!t. 'lM"ft..l
rrault, Ph.D. C -T s Advisor ~ u.Jq.....-.~------~anette Wyneken, PllD. S~t:.~ Sarah Milton, Ph.D.
Rodney Murphey, Ph. Chair, Department of81olo · Ataifii.Ph.D.~- Dean, Charles E. Schmidt College of Science ~&LS~~'" ~ted 25~~/fJ Khaled Sobhan, Ph.D. Date Interim Dean, Graduate College
iii ACKNOWLEDGEMENTS
Thank you to my thesis advisor, Dr. Michael Salmon, who was ready with advice and
encouragement whenever it was needed. I would also like to thank my co-adviser, Dr. Justin
Perrault, for giving me the opportunity to work on this project and for his guidance throughout its duration. Thank you to my thesis committee: Dr. Jeanette Wyneken and Dr.
Sarah Milton, for their thoughtful comments and critiques.
Thank you to the staff at Loggerhead Marinelife Center, especially Christina
Coppenrath, Sarah Hirsch, Adrienne McCracken, Jennifer Reilly, and Heather Seaman, who pulled many late nights and early mornings to complete this project.
Thank you to my lab mate, Shelby Hoover, for her hard work, dedication, organization, and good nature. I don’t know how everything would have worked without her.
In addition to these individuals, I would also like to thank Dr. Thomas Kodadek’s lab at Scripps Research Institute for allowing us to use their facilities for our sample analyses.
Scott Simanski, especially, went above and beyond all expectations.
Finally, thank you to the Sea Turtle Conservancy. This project was funded in part by a grant awarded from the Sea Turtle Grants Program (#18-002R). The Sea Turtle Grants
Program is funded from proceeds from the sale of the Florida Sea Turtle License Plate.
Without this grant, this project could not have been completed.
iv ABSTRACT
Author: Heather Smith
Title: Impact of the fire ant pesticide hydramethylnon (AMDRO®) on loggerhead sea turtle reproductive success and hatchling quality
Institution: Florida Atlantic University
Thesis Advisor: Dr. Michael Salmon
Degree: Master of Science
Year: 2019
Invasive fire ants are aggressive predators of ground nesting birds and reptiles
and are spreading rapidly throughout tropical and temperate climates. Fire ants have been
known to prey on a variety of reptile species, including threatened loggerhead sea turtles.
The granular fire ant bait AMDRO® is being used on sea turtle nesting beaches to protect
nests and hatchlings from these predators, however no studies have been conducted to thoroughly assess its effect on any reptile species. In this field study, I examined the impact of AMDRO® on hatching and emergence success, body condition, and orientation
behavior in loggerhead sea turtles (Caretta caretta) in Juno Beach, Florida, USA.
Pesticide granules were placed in a one-foot diameter circle directly above nest chambers
during the final 5-10 days of incubation, which is representative of typical field
applications of this pesticide on nesting beaches. Two controls were used in this study:
cornmeal granules in soybean oil served as the vehicle control, and a second group of
untreated control nests were left to incubate naturally, undisturbed. After a natural
v emergence, hatchlings were collected to calculate a body condition index (BCI). For a
subset of the nests, 20 hatchlings were collected to perform orientation assays to assess
the hatchlings’ ability to orient correctly toward the ocean, a visually mediated process
that could be altered by visual impairments resulting from ADMRO® exposure. Three days following a mass emergence event, nests were excavated to collect hatching and emergence success data. Sand samples were collected to determine if the toxicant persisted in the environment or penetrated the egg chamber. Analyses indicated that the toxicant had no effect on hatchling morphology, hatching success, or emergence success.
It also had no effect on the ability of hatchlings to orient toward the ocean. However, the pesticide granules attracted more predators than were seen at control nests. Thus, while
AMDRO® might not directly impact reproductive success or hatchling behavior, it had
the unanticipated effect of possibly increasing nest vulnerability to predators.
vi IMPACT OF THE FIRE ANT PESTICIDE HYDRAMETHYLNON (AMDRO®) ON
LOGGERHEAD SEA TURTLE REPRODUCTIVE SUCCESS AND HATCHLING
QUALITY
LIST OF FIGURES ...... ix
LIST OF TABLES ...... x
INTRODUCTION ...... 1
Background ...... 2
Marine Turtle Toxicology ...... 7
MATERIALS AND METHODS ...... 9
Nest Selection and Treatment Categories ...... 9
Quantifying Hatching and Emergence Success ...... 11
Measuring Hatchling Body Condition ...... 11
Testing Sea-Finding Orientation ...... 12
Analyzing Sand Samples ...... 13
RESULTS ...... 15
Hatching and emergence success ...... 15
Hatchling body condition ...... 16
Sea-finding behavior ...... 16
Sand Samples ...... 17
DISCUSSION ...... 18
vii Nest success ...... 18
Pesticide permeability and hatchling body condition ...... 20
Orientation behavior ...... 21
Sand samples ...... 22
CONCLUSIONS...... 24
REFERENCES ...... 34
viii
LIST OF FIGURES
Figure 1. Map of the study site ...... 25
Figure 2. Hatching success across treatment groups ...... 26
Figure 3. Emergence success across treatment groups ...... 27
Figure 4. Body condition index across treatment groups ...... 28
Figure 5. Angle of orientation across treatment groups ...... 29
ix LIST OF TABLES
Table 1. Mean hatching and emergence success by treatment group ...... 30
Table 2. Total predator observations by treatment type ...... 31
Table 3. Mean body condition index by treatment group ...... 32
Table 4. Mean angle of orientation by treatment group ...... 33
x INTRODUCTION
Sea turtles face a multitude of anthropogenic threats that have led to dramatic
reductions in population sizes from historic levels (Allison et al. 2009). These threats
include light pollution (McFarlane 1963), climate change (Laloe et al. 2016), by-catch
from the seafood industry, and dredging activity (Robins 1995). In addition to these
challenges, sea turtles face predation pressure from invasive species as well as natural
predators (Fowler 1979). The impact of predators, both native and introduced, on sea
turtle nests is a continuing concern for biologists and one that warrants considerable
attention, given that many sea turtle populations are continuing to decline (Allison 2009).
The predaceous red imported fire ant (Solenopsis invicta; hereafter, RIFA) is an
invasive species that is now considered a significant threat at some sea turtle nesting
beaches, especially low-density nesting areas where nest predators can significantly
impact population dynamics. The Florida Fish and Wildlife Conservation Commission
(FWC) currently recommends fire ant removal by either pouring ~300 L of near-boiling
water onto mounds or by physically removing mounds with a shovel and bucket (FWC
2016). These methods are not only impractical at many nesting sites, but also potentially
risky to wildlife management personnel and the sea turtle nests they are trying to protect.
An alternative to physical removal of the nest is use of the pesticide hydramethylnon (known commonly by the brand name AMDRO®) to eradicate fire ant
mounds within 1.5 m to sea turtle nests. Data show that a reduction in RIFA populations
1 through the use of hydramethylnon leads to an increase in herpetofaunal abundance
(Allen et al. 2017). However, hydramethylnon is also known to be mildly to highly toxic to some vertebrate species (Lim 2004) although the effect of this pesticide on sea turtles generally, and hatchlings in particular, remains unknown.
This study investigated the effect of hydramethylnon on the nest survival and hatchling behavior of loggerhead sea turtles to determine if the application of this pesticide on sea turtle nests is a safe alternative to the physical removal of fire ant mounds. The project was undertaken to help wildlife management personnel make informed decisions with regards to use of this pesticide and ant removal on vulnerable sea turtle nesting beaches. While the incidence of ant predation on sea turtle nests on densely-populated South Florida beaches is low, the information gleaned from this study could have a broad impact and potentially inform conservation efforts worldwide, including those areas where fire ant predation is a serious threat to local sea turtle populations.
Background
Red imported fire ants are predators that have infested the majority of sea turtle nesting beaches on the East Coast of the United States and are known to prey on the eggs, developing embryos, and recently hatched young of a variety of reptilian species, including freshwater turtles (Allen et al. 2001; Buhlmann et al. 2001), gopher tortoises
(Dziadzio et al. 2016), racerunner lizards (Mount et al. 1981), rough green snakes
(Conners 1998), and caimans (Marcó et al. 2013). Fire ants are often present between the dune and wrack lines, where sea turtles are primarily nest (Allen et al. 2001) (Fig. 2).
Ants are likely attracted to the surface disturbance and cloacal mucous content of fresh
2 nests, and will excavate tunnels to monitor them for the emergence of hatchlings (Allen et al. 2001; Buhlmann & Coffmann 2001). Since at least 1979, anecdotal and experimental evidence have shown that fire ants readily prey on sea turtle nests. Ants have been observed feeding on recently pipped eggs and newly emerged hatchlings of loggerhead sea turtles (Parris et. al 2002) (Fig. 3).
The presence of fire ants can significantly impact hatchling emergence success and survival. Moulis (1997 in Allen et al. 2001) found that loggerhead sea turtles experience a 15% decrease in emergence success when nests are infested with fire ants at the time of emergence. Using the freshwater turtle Pseudemys nelsoni as a surrogate species, Allen et al. (2001) reported an average 33% reduction in hatchling survival from nests exposed to RIFAs compared to controls. Mortality due to fire ants may be also be significantly underestimated; 50% of P. nelsoni hatchlings that were ultimately killed by
S. invicta had successfully emerged from their nests and reached the water before perishing from ant stings suffered while exiting the nest (Allen et al. 2001). Likewise,
Krahe et al. (2003) found that hatchling loggerhead sea turtles which emerged successfully from their nests suffered increased mortality from even single ant stings.
Hatchling sea turtles delay their emergence to avoid predators and high daytime temperatures, which can be lethal (Mrosovsky 1968). Hatchlings congregate just below the surface of the sand until sand temperatures cool, and then emerge synchronously at night (Godfrey & Mrosovsky 1997). This behavior alleviates their risk to some predators and to harmful daytime temperatures but may put them at a higher risk of predation or lethal injury by fire ants, necessitating the physical removal of ant mounds or the use of pesticides such as hydramethylnon at nesting beaches with high RIFA densities.
3 Hydramethylnon (C25H24F6N4) is a pesticide in the trifluoromethyl
aminohydrazone class of insecticides. It was first registered with the United States
Environmental Protection Agency in 1980 and is used in granule or gel form as an
insecticide against ants, cockroaches, termites, crickets, and silverfishes (National
Pesticide Information Center 2002). The most widely recognized form of
hydramethylnon is the product AMDRO®, which is commonly used for the control of fire
ants on residential lawns. The key characteristic of hydramethylnon is delayed toxicity,
which allows time for worker ants to bring the bait back to the mound and share it with
the queen, effectively killing the entire colony. Hydramethylnon exhibits its toxic effects
on insects by acting as an inhibitor of the electron transport chain in mitochondria,
blocking cellular respiration. The onset of effects takes place approximately 24 h after
consumption, with lethargy being the initial symptom and death following within a week.
Initial reports suggested that hydramethylnon was selectively toxic only to insects with
chewing mouthparts, but subsequent research has shown that toxicity is not limited to
arthropods (Hollingshaus 1987). The molecular structure of hydramethylnon includes guanidine compounds that are known to be inhibitors of oxidative phosphorylation in a wide range of vertebrates, including humans (Hollingshaus 1987; Pressman 1963), suggesting that there could be a mechanism for a toxic effect on sea turtles as well.
Laboratory results indicate that hydramethylnon exhibits biphasic degradation
through abiotic photolysis with a half-life in soil of 4 days for the initial phase and 30 days for the second phase; the half-life in water is <1 h. Experimental data have loosely mirrored laboratory results, with half-lives ranging from 3 to 55 days in field conditions depending on location and climate (EPA 1998). Because sunlight is the primary catalyst
4 for degradation, sunnier climates may reduce the environmental persistence of this compound. When exposed to direct sunlight, hydramethylnon in AMDRO® has a half-life of just 12 h (Vander Meer et al. 1982). Hydramethylnon exhibits a high soil binding affinity, resulting in low dispersal through soil (Lim 2004), but the inorganic nature and high permeability of the beach sand characteristic of sea turtle nesting sites may allow for increased leaching, potentially allowing the pesticide to reach the egg chamber despite its low solubility in water (Huddleston 1996).
Ingested orally, hydramethylnon is mildly toxic to birds and mammals and is known to inhibit cellular respiration in in-vitro hamster ovary cells. It also causes chronic reproductive anomalies in mice and rabbits including 10% reductions in mouse fetal mass and 25% reductions in rabbit fetal mass compared to controls (Lim 2004; Hollingshaus
1987). Similar effects in sea turtles could be detrimental to survival, considering that smaller turtles are more susceptible to predation by gape-limited predators, and a sea turtle’s odds of survival rise dramatically with size (Salmon et al. 2015). Oral hydramethylnon has been shown to have significant negative effects on rats, including lethargy, carcinogenesis, weight loss, hair loss, and increased mortality (Lim 2004). In
Holstein calves, oral hydramethylnon caused leukopenia (a reduction in white blood cells) after just two weeks and led to significantly decreased rectal temperatures, indicating an effect on thermoregulation (Evans 1984).
Hydramethylnon is considered highly toxic to freshwater fish (rainbow trout, channel catfish, and bluegill) and has been shown to bioaccumulate in the bluegill sunfish
Lepomis macrochirus (Lim 2004), although its low solubility and rapid degradation in water should mitigate potential impacts to aquatic species. Elevated doses of dermal
5 hydramethylnon resulted in reduced food intake, weight loss, reduced platelet counts, reduced heart weight, and increased liver weight in rabbits (EPA 1998). Hydramethylnon is also known to affect the eyes of rabbits, causing eye irritation, corneal redness, and discharge (Tomlin 2003). Alterations in visual perception could be detrimental to the survival of hatchling sea turtles exposed to hydramethylnon, since hatchlings primarily use visual cues to orient from the nest to the surf zone in the initial phase of their offshore migration (Salmon et al. 1992). Because hatchling sea turtles remain just below the surface of the sand for extended periods, waiting for sand temperatures to decrease to initiate their emergence, they may experience dermal and ocular exposure to ant baits placed on surface sand.
These findings suggest the potential for a myriad of negative impacts of hydramethylnon on developing and newly hatched sea turtles, although there are currently no data on the effects of hydramethylnon on the behavior or physiology of any reptile species. Biomarkers of pollutant exposure range from early signals (at the molecular, cellular, and physiological levels) to late effects (at the population, community, and ecosystem levels). It is important to detect the effects of environmental pollutants before they reach the population level, as by then it is often too late to reverse any negative effects (Kaviraj et al. 2014). Marine turtles are considered bioindicators of pollution levels in the ocean environment (Andreani et al. 2008). Early studies on the effects of contaminants to which marine turtles are exposed are important in order to mitigate potential negative outcomes for these already imperiled species.
6 Marine Turtle Toxicology
Environmental pollutants were identified in 2010 as a global priority for sea turtle
research (Hamann et al. 2010). Understanding of the toxicological impacts of
contaminants in sea turtles is in its infancy, with most of the existing work having been
done within the last ten years (Finlayson et al. 2016). There are currently no data on the
uptake of hydramethylnon by sea turtles, but there is a growing body of information
suggesting that pesticides can and do impact the nest survival and hatchling behavior of
turtle species.
Environmental contaminants can also be absorbed by embryos during incubation.
Reptile eggshells range on a continuum from hard-shelled to parchment-shelled. The parchment-shelled eggs laid by sea turtles are leathery in appearance and contain more channels than hard-shelled eggs (Packard and Packard 1980 in Rimkus 1996). Kusuda et al. (2013) classified the eggs of testudines into six distinct categories based on results from electron microscopy analyses and X-ray diffractometry on the structure of the shells, with Type I being the most porous and Type 6 being the most waterproof.
Loggerhead eggs were categorized Type II, classified by the numerous channels that fully penetrate the calcareous layer of the eggshell, making the embryo potentially susceptible to environmental contaminants. Thinner, more porous eggs are also more permeable to water, requiring humid conditions in order to maintain their proper volume and promote hatchling development (Packard et al. 1982). This trait could confer a considerable disadvantage to sea turtle eggs exposed to pesticides, as many environmental contaminants are water soluble. Hydramethylnon is only slightly soluble in water, but
7 applications of the pesticide are generally close enough to egg chambers that small
amounts of the pesticide could conceivably enter the nest, especially after a heavy rain.
Experimental data have suggested that parchment-shelled eggs are more
permeable to environmental toxicants, including pesticides, than hard-shelled eggs (Wu et al. 2016). The freshwater painted turtle (Trachemys scripta) lays parchment-shelled eggs which are similar to sea turtle eggs. Those eggs are negatively affected by applications of the pesticide deltamethrin during stage 17 of development, with hatchlings exposed to the pesticide experiencing reduced swimming speeds (Wu et al.
2016). Snapping turtle (Chelydra serpentina) eggs incubated in soil mixed with the
pesticide tefluthrin, fertilizer ammonia, and soil fumigant metam sodium resulted in
increased hatchling mortality and morphological abnormalities above certain threshold
levels. The application of other pesticides and herbicides at field levels had no effect,
suggesting differential uptake for various environmental contaminants (de Solla et al.
2011; de Solla et al. 2013). Exposure to high levels of the herbicide glyphosate causes
decreased hatching success, body mass, and righting response times in the red-eared
slider turtle (Trachemys elegans), although standard field levels of this chemical are
relatively safe for embryos (Sparling et al. 2006).
Together, this body of research suggests that there could exist a mechanism for
the embryonic absorption of hydramethylnon in loggerhead sea turtles. However,
experimental data are needed for confirmation. Here, I ask three questions: (1) Do sea
turtle nests (eggs, hatchlings, or both) exposed to AMDRO® experience reduced hatching
and emergence success? (2) Does exposure to AMDRO® adversely alter the body
condition of hatchlings? (3) Does exposure to AMDRO® impair sea-finding orientation?
8 MATERIALS AND METHODS
Nest Selection and Treatment Categories
I conducted this study along a 9.6 km stretch of beach adjacent to Loggerhead
Marinelife Center (LMC) in Juno Beach, Florida USA from June – August of 2018 (Fig.
4). This site has an extraordinarily high nesting density of loggerhead sea turtles.
125 loggerhead nests were selected for this study from 18 – 29 June, 2018. Study
nests were marked in the early morning after the night they were deposited using standard sea turtle survey practices (FWC 2016). The exact location of the egg chamber was validated by digging into the sand until the top of the egg chamber was located. A wooden stake served as the clutch marker and was placed 30 cm due west of the center of the chamber. The study site was divided into six zones, all <1.7 km in length. Each morning, nests within each zone were randomly assigned to different treatment groups to minimize changes in environmental conditions across nest sites.
Cornmeal soaked in soybean oil is the carrier for hydramethylnon in AMDRO®
(with the soybean oil being the primary fire ant attractant); therefore, these two
substances were chosen as the vehicle control. Treatments included a natural control
group that was left to incubate undisturbed, two cornmeal control groups, and two
AMDRO® experimental treatment groups, with 25 nests in each group. Cornmeal control
1 and AMDRO® treatment 1 were treated three times (on days 45, 47, and 49 of
incubation). Cornmeal control 2 and AMDRO® treatment 2 were treated six times (on
days 39, 41, 43, 45, 47, and 49 of incubation). The vehicle control and AMDRO® were
9 kept separate to prevent contamination. To treat each cornmeal control nest, 25 g of
cornmeal was combined with 5 ml of soybean oil and spread inside a 30.5 cm diameter
circle directly above the nest egg chamber between 1800 – 2200 h. AMDRO® nests were treated likewise with 25 g of AMDRO®. The AMDRO® or cornmeal granules were then
covered with a very thin layer of sand to prevent wind from carrying the substance away.
This study design was intended to replicate standard practices for the application
of AMDRO® to sea turtle nests. This pesticide is typically placed on the nest in the later
stages of development when it provides more effective protection against ant predation.
Based on data collected by LMC surveys, loggerhead turtles on Juno Beach typically
incubate for approximately 51 days. During the course of the treatment regimen,
however, nests began to emerge unexpectedly early (some after only 44 days incubation).
To ensure that nests were not missing treatments entirely, the treatment schedule was
amended on August 5, 2018 by shortening the entire treatment schedule by two days for
the remainder of nests.
Nests were monitored for possible emergences beginning at day 45 of incubation.
Wooden restraining cages (53 cm long x 53 cm wide x 8.5 cm high) covered with black
plastic mesh were placed over the egg chambers of pre-emergent nests. The beach was then patrolled using all-terrain vehicles (ATVs) from 1100 until 0600, in an effort to monitor for hatchling emergence. All nests, regardless of the day of incubation, were also monitored by the LMC morning survey team between 0600 and 1100 for evidence of an emergence so that the exact date of emergence could be determined.
10 Quantifying Hatching and Emergence Success
Nests were observed by morning surveyors every morning between 0600 and
1100 for the duration of incubation. Any events that may have affected nest production
(e.g., depredations or sand accruement) were noted. Nests were excavated and
inventoried 3 days after the mass emergence event. Hatched eggs were counted as the
number of >50% eggshell fragments remaining in the nest. Unhatched eggs less shelled albumen globs (without yolks) were also counted, as well as the number of live and dead pipped eggs (eggshell torn with hatchling inside) and live and dead hatchlings. Hatching success was evaluated by dividing the number of hatched eggs by the total clutch size; emergence success was evaluated by dividing the number of emerged hatchlings by the total clutch size (Miller 1999). Data were analyzed using a one-way ANOVA to check for differences in means between treatments using R version 3.5.1 (R Core Team 2018).
Nests were also monitored for signs of predators. Due to the treatment schedule, some ADMRO® and cornmeal treatment nests were observed more frequently than the
natural control nests. However, all nests were observed every day from incubation day 45
onward, so analysis of predator data was confined to observances beginning at day 45.
The number of nests with predator observances (tracks, burrows, or predators
themselves) from day 45 to emergence was compared using chi-square tests.
Measuring Hatchling Body Condition
Up to ten hatchlings were collected from each nest to evaluate an average body
condition index (BCI) for each nest and to check for morphological abnormalities.
Hatchlings were allowed to emerge naturally and were only collected once they had fully
emerged from the nest and started to crawl towards the ocean. Hatchlings were placed
11 into small plastic coolers lined with a thin layer of moist sand and taken to LMC to obtain
morphometric data. Hatchlings were weighed using a digital scale. Straight carapace
length (SCL), straight carapace width, and body depth were measured using digital
calipers. BCI was determined using the formula adapted from Bjorndal et. al. (2000):
mass (g) BCI= x 10,000 SCL3 (cm) � � Due to the non-independence of hatchlings from the same nest receiving the same
treatment, a linear mixed effects model (LMM) and likelihood ratio test were used to
determine if there was a significant effect of treatment type (AMDRO®, cornmeal, or
natural control) or number of treatments on hatchling BCI. Nest treatment was included
as a fixed variable with nest ID included as a random variable. Data were analyzed using
the lme4 package in R version 3.5.1 (Bates 2015).
Testing Sea-Finding Orientation
Up to 20 hatchlings from each nest were transported in small coolers to a remote
area of beach, selected because it was shielded by vegetation from artificial lighting, to
conduct arena assays (Rientsma et al. 2014). A circle 4 m in diameter (the “arena”) was
drawn in the sand between the surf zone and dune. The arena sand was leveled with a
broom and any debris was removed. Hatchlings that were actively crawling inside their
cooler were placed in small groups of 6 or fewer turtles in a shallow (1-3 cm) pit centered
in the arena; they almost immediately crawled out of the pit and moved toward the arena
boundary. As they moved the turtles left flipper tracks in the sand that were used to
determine where they exited the arena. Their orientation angle was determined using an
electronic compass that measured where the turtle crossed the arena boundary, relative to
12 the center of the arena. After each turtle had been tested once, they were taken to another
dark beach location and released.
Rayleigh tests were used to determine if each nest as a whole was significantly
oriented towards a particular angle, and Watson-Williams tests were used to determine if
there were significant differences in the mean angle of orientation between treatment
groups. Data were analyzed using package circular in R version 3.5.1 (Agostinelli &
Lund 2017).
Analyzing Sand Samples
Two false nests were constructed and treated with AMDRO® so that sand samples could be taken without disturbing the eggs, embryos or hatchlings in real nests, and without hatchlings disturbing the surface treatments. These nests were marked with 4 stakes that designated a 1 x 1 m2 area on the beach that was then dosed with AMDRO®.
One nest was dosed with 25g of AMDRO® placed in a 30.5 cm circle, drawn in the "nest"
center, on days 45, 47, and 49 after staking. The second nest was similarly dosed with 25 g of AMDRO® at days 39, 41, 43, 45, 47, and 49 after staking. On day 51, sand samples
were taken at the surface and at increasing depths at 2.5 cm increments down to a depth
of 47 cm, the average nest depth for loggerhead turtles. Latex gloves were changed after
every sample to prevent cross contamination from upper layers of sand.
Samples were placed into Ziplock® bags, stored at -80°C until analysis, and then
analyzed using liquid chromatography-mass spectrometry (LC-MS). A 12.6 mg sand sample was combined with 1 ml of acetonitrile (ACN) to extract hydramethylnon present in the sand. The sand-ACN solution was then sonicated for 15 minutes and filtered into
LC-MS vials. LC-MS was carried out on an Agilent 1100 system equipped with an
13 Agilent ZORBAX StableBond 80Å C18, 4.6 x 100 mm, 3.5 µm HPLC column
(#861953-902) LC/MSD SL. The mobile phase was comprised of buffer A (95/5
H2O/CH3CN containing 0.1% formic acid) and buffer B (95/5 CH3CN/H2O containing
0.1% formic acid).
All LC-MS samples were run on a 15-min gradient from 100% A to 100% B controlled by an autosampler. A wash was performed with high performance liquid chromatography (HPLC) grade water after every 5 samples. An AccuStandard® analytical reference standard for hydramethylnon was used for quality control.
14 RESULTS
Hatching and emergence success
Of the 125 nests that were originally marked, 14 were removed from the study as
these clutches could not be located after the initial verification. This was primarily due to
sand accruement after the remnants of tropical storm Beryl hit Juno Beach. Emergences
occurred earlier than expected (mean incubation = 48.43 days), leading to nests receiving
a continuum of treatments ranging from 1 to 6. Nests were defined as
AMDRO®/Cornmeal treatment 1 nests if they received ≤ 3 treatments or
AMDRO®/cornmeal treatment 2 nests if they received ≥ 4 treatments. Hatching success shown by all nests (n=111) ranged between 62 – 70 % with a mean of 65 ± 2 % (Fig. 2).
Emergence success ranged between 59-67 %, with a mean of 61 + 2 % (Fig. 3). There
were no significant differences among the treatment groups in either hatching success
(ANOVA: F = 0.319, p = 0.865) or emergence success (ANOVA: F = 0.336, p = 0.853)
between treatment groups (Table 1).
Nest predators were observed at both AMDRO®-treated nests and cornmeal-
treated nests more often than natural control nests (Table 2) (Fisher’s exact test, p =
0.0326). The most frequently observed predator was the Atlantic ghost crab, Ocypode
quadrata. Ghost crabs, tracks, burrows, or a combination of all three were observed at
26.7% of treatment nests (n = 23) compared to 8.0 % of natural control nests (n = 2), but
this finding was insignificant (Fisher’s exact test, p = 0.0581). Unhatched eggs were
found to be predated by ghost crabs during excavations, although it is difficult to attribute
15 predations to ghost crabs with certainty. There was no significant difference between the
number of ghost crab observations at ADMRO® treated nests (n = 14) compared to
cornmeal treated nests (n = 9) (Fisher’s exact test, p = 0.1437).
AMDRO® and cornmeal treatments also attracted more birds than were seen at the
natural control nests, however this finding was not significant (Fisher’s exact, p = 0.195).
Bird tracks were observed at 9.3% of treatment nests (n = 8) compared to 0% of control
nests. Birds are visual predators and were likely attracted to the sight of the cornmeal or
AMDRO® granules. Birds were more prevalent at AMDRO® treated nests (n = 7) than
cornmeal treated nests (n = 1) (Fisher’s exact test, p = 0.023).
Ants were observed at 5.8% of treatment nests (n = 5) compared to 1.5% of
natural control nests (n = 1), but the sample size was too small to show a significant
effect (Fisher’s exact test, p = 1.00). Mammal predation was very low, with only 1
cornmeal nest and one natural control nest depredations.
Hatchling body condition
Morphometric data were collected from a sample of 457 hatchlings from 55 nests.
Mean BCI across all nests was 2.34 ± 0.01 g/mm3. Mean BCI across all treatments ranged between 2.25 and 2.44 g/mm3 (Table 2). There was no significant effect of
treatment type or number of doses on the BCI of any of these groups (LMM, likelihood
ratio test: p = 0.1022). No dermal abrasions or ocular irritations were visible on any of the
hatchlings (Fig. 3)
Sea-finding behavior
Orientation data were collected from 28 nests. All nests were significantly
oriented towards a singular direction (Rayleigh test, p < 0.01). One AMDRO® treatment 2
16 nest partially disoriented in a non-random way: hatchlings were significantly oriented toward the southeast (mean orientation angle = 124.05°, Rayleigh test: p < 0.01). This was likely due to urban lighting from Juno Beach at this location (Zone 3) reflecting on a large cloud located to the southeast. A second, darker location (Zone 9) with very little urban lighting and a tall tree barrier on the dune was chosen for the remainder of the study to eliminate variation caused by the reflection of urban lights on clouds. All nests assayed at this location oriented significantly toward the east (n = 24, mean = 88.62°,
Rayleigh test: p < 0.01). Mean orientation angle across all treatments ranged between
88.58° to 94.03° (Table 3). There was no significant difference in the mean angle of
orientation between the treatment groups (Watson-Williams test: p = 0.4126). However,
AMDRO® treatment 2 nests had higher variation (SD = 0.42) than other treatment groups
(Table 3, Fig. 3).
Sand Samples
A compound with the molecular weight of hydramethylnon (494.5 g/mol) was
detected at both false nests, with no other substances detected near that molecular weight,
indicating that hydramethylnon persisted in the environment for at least 48 h after dosing.
In the AMDRO treatment 1 nest, hydramethylnon was detected at the surface and in all
samples down to 5 cm in depth. In the AMDRO treatment 2 nest, hydramethylnon was
detected in the surface sand and in all samples down to 10 cm in depth.
17 DISCUSSION
The results of this study indicate that use of AMDRO® on nesting beaches has no
directly negative impact on loggerhead sea turtle hatching success, emergence success,
body condition, or orientation behavior. However, possible indirect negative impacts are
discussed below.
Nest success
The mean hatching and emergence success observed in this study are consistent
with previous literature on hatchling production from south Florida nesting beaches
(Brost et al., 2015).
An increase in nest predators was an unexpected finding in this study. There was
no significant difference between the number of ghost crab observations at ADMRO®
treated nests (n = 14) compared to cornmeal treated nests (n = 9) (Fisher’s exact test, p =
0.1437). This finding is consistent with previous research showing that ghost crabs primarily locate food by smell (Wellins et al. 1989, Harris et al. 2019). The scent of the cornmeal carrier in AMDRO® could be a ghost crab attractant. While an increase in ghost
crab activity did not lead to a noticeable reduction in nest success in this study, ghost crab
predation could be a serious problem on other nesting beaches. On small oceanic islands,
ghost crabs are the primary nest predators of sea turtles. Marco et al. (2015) found that 33
eggs per nest, on average, were predated by ghost crabs on the island of Boa Vista, one of
the largest loggerhead rookeries in the world and the largest in the Cape Verde Islands.
They also determined that nests were most vulnerable to ghost crab predation after 40
18 days of incubation. This coincides with the currently recommended timeframe for application of fire ant baits to nesting beaches.
Birds were observed at more treatment nests than control nests. Birds are visual predators and were likely attracted to the sight of the cornmeal or AMDRO® granules.
Birds were more prevalent at AMDRO® treated nests than cornmeal treated nests. This
finding is expected, considering that sea birds are primarily visual predators. While
hatchling sea turtles primarily emerge at night, some diurnal bird species are known to
prey on sea turtle hatchlings that emerge in the early morning or whose nests are exposed
(Burger & Gochfield 2014).
A further consideration is that an increase in ghost crab and avian predators to our
nest sites did not translate to a reduction in hatching or emergence success because these
predators often kill hatchlings as they make their way from their nest to the ocean, after
they have successfully emerged from the nest. Erb (2016) estimated nest-to-surf mortality
in loggerhead hatchlings from several nesting beaches on the east coast of Florida. Ghost
crabs were found to be the predominant predators on a majority of nesting beaches
surveyed. Hatchlings killed between the nest and the water line do not affect
measurements of hatching or emergence success, and so estimates of hatching or
emergence success are possibly underestimating the negative impact of placing a
potential predator attractant on or near the nest.
As expected, AMDRO® also attracted fire ants, however fire ant predation rates are low on Juno Beach and it was therefore difficult to show a significant relationship between pesticide treatments and ant depredation rates. One nest was nearly completely predated by ants. This nest emerged early (46 days) after receiving 1 dose of AMDRO®
19 at day 45. Prior to treatment, there were no signs of fire ants at this nest. During nest
checks following treatment, ants were observed covering the surface of the nest and
digging tunnels downward. During excavation, 66 eggs were discovered pipped but dead,
and the emergence success of this nest was 3.41%. The ants disappeared between the
time of emergence and excavation date, presumably killed by the AMDRO®.
No studies have examined the efficacy of AMDRO® at increasing the
reproductive success of sea turtles. In this study, no positive effect of AMDRO® on the
emergence success of loggerhead sea turtles was shown. Fire ants are not commonly
abundant at Juno Beach, so no conclusions can be drawn about the pesticide's efficacy
based on this study. However, anecdotal evidence indicates that caution should be used
when administering this pesticide at sea turtle nesting beaches. Because hydramethylnon
exhibits delayed toxicity, with lethal effects taking up to a week to occur, placing the
pesticide too late during incubation could have the unintended consequence of drawing
fire ants to the nests and only killing them after the turtles have emerged. The sight or
smell of the cornmeal/soybean oil carrier could also be an attractant to other unexpected
predators (e.g. crabs, birds), which could translate into a reduction in hatchling
productivity at nesting beaches with higher predator densities than Juno Beach (Clam
Pass Park South, Florida; “B” Key, Collier County, Florida) (Allen et al. 2001).
Pesticide permeability and hatchling body condition
There was no apparent effect of AMDRO® on the hatchling body condition of
loggerhead sea turtles. This study focused on the potential effect of AMDRO® when the
pesticide was placed on the nest during the later stages of embryonic development. The study design mimicked standard practices for application of the pesticide, which is
20 thought to be most effective at the end of incubation, ensuring there are no ant predators
at the time of hatching (when embryos break the egg shell [pipping] and become most
vulnerable to ants). Potential impacts of exposure during early development have not
previously been explored. Additionally, at many nesting beaches, nests are not
exhaustively marked and monitored. As such, AMDRO® could be unwittingly applied on
or near a nest that is in the early stages of development. However, its low water solubility
does not facilitate permeation through beach sand despite the low organic content of
sand.
During the course of the pesticide dosing schedule in this study, Juno Beach received 19.08 cm of rainfall. Monthly averages for Juno Beach are 15.80 cm in July and
15.49 cm in August (National Oceanic and Atmospheric Administration; Climate-
Data.org) so the study site received a higher than normal amount of rainfall throughout the duration of the study. Even if the pesticide could negatively impact early embryonic development or physiology, the results of this study indicate that the pesticide is highly unlikely to penetrate the sand deeply enough to reach the eggs, even under conditions of higher-than-normal rainfall.
Orientation behavior
The orientation behavior observed in this study is consistent with reports from previous studies dealing with the orientation of loggerhead sea turtles on the southeast coast of Florida (Salmon et al., 1995; Rientsma et al. 2014). Even hatchlings from the
AMDRO® treatment 2 nest that partially disoriented towards the southeast at the original assay location were behaving as expected: they were moving towards a light source. This indicates that the hatchlings used in this study were behaving normally and are likely
21 representative of normally-orienting loggerhead turtles. All nests that were assayed under
the darkest of conditions, with no moon and very little light in the sky, they were able to
orient correctly towards the water. If exposure to AMDRO® had produced visual
impairment, an ability orient so accurately under these low light conditions would be
unlikely. I conclude that AMDRO® had no behaviorally significant effect on the visually-
mediated process of sea finding in loggerhead sea turtle hatchlings. The higher variation
in the AMDRO® treatment 2 group (Fig. 4) is due to the single disorientation event from
Zone 3. Given these data, it is highly unlikely that AMDRO® has a significant effect on
the visually-mediated process of sea finding in loggerhead sea turtle hatchlings.
Sand samples
Based on previous literature on the environmental half-life of hydramethylnon,
our finding that hydramethylnon was present in surface sand was unsurprising. Under
sunny southeast Florida conditions, the expected half-life of hydramethylnon should be approximately 12 h (Vander Meer et al. 1982). However, the second stage of the biphasic degradation exhibited by hydramethylnon can take 30 days to complete. It is also possible that by covering the pesticide granules with a thin layer of sand, the compound was protected from degradation by photolysis, leading to an increased environmental persistence that is atypical for sunny conditions but still well within previous laboratory and field estimates (EPA 1998). Multiple treatments in succession, with each application covered by a thin layer of sand, may have also contributed to the unexpected depths at which hydramethylnon was detected. Those observations might have led to an overestimate of the potential for hydramethylnon to leach downward through the sand.
Even taken at face-value, however, the results of this study indicate that hydramethylnon
22 is unlikely to reach the egg chamber of loggerhead sea turtles and for that reason, is unlikely to pose a threat to embryonic development.
23
CONCLUSIONS
The findings of this study indicate that while the use of AMDRO® on nesting
beaches likely does not represent a direct threat to the development, reproductive success,
or behavior of loggerhead sea turtles, caution should still be exercised when applying this
pesticide to nesting beaches due to the potential for attracting predators to the nest site.
On some rookery beaches, such as Juno Beach, the incidence of predators is low enough that this attractant might not result in a noticeable reduction in hatchling productivity.
However, not all nesting beaches have the same predator profile, and the use of this pesticide should be weighed heavily against the type and abundance of predators on the individual nesting beach in question. I hope that this study will help to inform wildlife management personnel, with the result that they select the best option with regard to the use of this pesticide at sites where sea turtles place their nests.
24
Figure 1. Map of the study site, Juno Beach, FL USA. Adapted from Perrault et al. (2011).
25
Figure 2. Hatching success for each of the five groups: AMDRO® treatment 1 (≤ 3 doses), AMDRO® treatment 2 (≥ 4 doses), Cornmeal treatment 1 (≤ 3 doses), Cornmeal treatment 2 (≥ 4 doses), and the natural control (0 doses). Vertical lines represent the range of values, boxes represent the interquartile range (IQR), and the horizontal line represents the sample median. Dots represent outliers that are >1.5 IQR above or below the first or third quartile.
26
Figure 3. Emergence success for each of the five groups: AMDRO® treatment 1 (≤ 3 doses), AMDRO® treatment 2 (≥ 4 doses), Cornmeal treatment 1 (≤ 3 doses), Cornmeal treatment 2 (≥ 4 doses), and the natural control (0 doses). Vertical lines represent the range of values, boxes represent the interquartile range (IQR), and the horizontal line represents the sample median. Dots represent outliers that are >1.5 IQR above or below the first or third quartile.
27
Figure 4. Hatchling body condition Index (BCI) for each of the five groups: AMDRO® treatment 1 (≤ 3 doses), AMDRO® treatment 2 (≥ 4 doses), Cornmeal treatment 1 (≤ 3 doses), Cornmeal treatment 2 (≥ 4 doses), and the natural control (0 doses). Vertical lines represent the range, boxes represent the IQR (interquartile range), and the horizontal line represents the sample median. Dots represent outliers that are >1.5 IQR above or below the first or third quartile.
28
Figure 5. Orientation angles for the AMDRO® treatment groups (above) and control groups (below). Normal orientation in south Florida should approximate 90°. All crawls were significantly oriented towards the east.
29
Table 1. Mean hatching success and emergence success by treatment group. N = number of nests. + = All values are mean ± se.
Hatching Emergence Treatment N + Success (%)+ Success (%)
AMDRO® 1 20 63.1 ± 7.3 59.1 ± 7.0
AMDRO® 2 20 70.0 ± 4.3 66.9 ± 4.4
Cornmeal 1 24 64.3 ± 4.8 60.0 ± 4.7
Cornmeal 2 22 62.0 ± 5.7 59.9 ± 5.8
Natural Control 25 66.9 ± 5.1 63.7 ± 5.4
All nests 111 65.23 ± 2.42 61.90 ± 2.44
30
Table 2. Numbers of predator observations by treatment type. N = total number of nests in each group. Total observations from each treatment type do not sum to Total in last column due to repeat nests.
Treatment N Ghost crab Bird Ant Mammal Total
AMDRO 40 15 7 1 0 20
Cornmeal 46 9 1 4 1 14
Control 25 2 0 1 1 4
31
Table 3. Mean Body Condition Index (BCI) by treatment group. N = number of nests. + = All values are mean + standard error
+ Treatment N Mean BCI
AMDRO® 1 10 2.38 ± 0.02
AMDRO® 2 11 2.33 ± 0.02
Cornmeal 1 12 2.33 ± 0.02
Cornmeal 2 10 2.44 ± 0.02
Natural Control 12 2.25 ± 0.02
All nests 55 2.34 ± 0.01
32
Table 4. Mean orientation angle of each treatment group with standard deviation. + = All values are mean + standard error
N N Standard Treatment +Mean (°) (nests) (hatchlings) deviation
AMDRO 1 7 134 88.58 ± 1.44 0.29
AMDRO 2 6 117 90.61 ± 1.98 0.42
Cornmeal 1 5 94 93.03 ± 2.05 0.31
Cornmeal 2 5 99 91.55 ± 1.95 0.30 Natural 5 86 92.79 ± 1.66 0.27 control
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