Content uploaded by Joycelyn Cagatin Jumawan
Author content
All content in this area was uploaded by Joycelyn Cagatin Jumawan on Mar 14, 2023
Content may be subject to copyright.
Journal of Hazardous Materials Advances 10 (2023) 100275
Contents lists available at ScienceDirect
Journal of Hazardous Materials Advances
journal homepage: www.elsevier.com/locate/hazadv
Butachlor at environmentally relevant concentrations induces partial
feminization in male Luzon wart frog Fejervarya vittigera Wiegmann, 1834
Generose G. Salvani, Joycelyn C Jumawan
∗
Department of Biology, College of Mathematics and Natural Sciences, Caraga State University, Ampayon, Butuan City, 8600 Philippines
Keywords:
Herbicide
Amphibians
testicular oocyte formation (TOF)
Exposure to herbicides and pesticides is linked to adverse eects on amphibian populations. Butachlor
(C
17
H
26
ClNO
2
) is one of the common herbicides used in rice elds in the Philippines and Southeast Asia. The
Luzon wart frog Fejervarya vittigera is abundant at high densities in rice elds in the Philippines. Adult F. vittigera
are exposed to agriculturally relevant concentrations of butachlor for seven days under laboratory conditions to
assess the eect of exposure on the histology of the testis and ovaries. Six groups: dechlorinated water (control),
0.4 mg/L, 0.8 mg/L, 1.6 mg/L, 3.2 mg/L, and 4.8 mg/L butachlor were used as test treatments, and morphol-
ogy of the oocytes and seminiferous tubules and spermatogenic cells in adult F. vittigera . Partial feminization, as
seen by the appearance of testicular (perinucleolar) oocytes, was observed in the testis of frogs exposed to real-
istic concentrations of butachlor. Overall results suggest that butachlor may interfere with normal reproductive
conditions and may cause irregularities in the male reproductive tissues and the hormonal milieu.
Introduction
Chemical contamination from herbicide and pesticide applications
have been linked as a contributing factor to the decline of amphibian
populations ( Geng et al., 2005 ; Davidson, 2004 ). Evidence suggests that
many herbicides can act as endocrine disruptors in wildlife, including
amphibians ( Hayes et al., 2006a , 2010a ). Non-target aquatic organisms
such as frogs thriving in paddy elds are often exposed and thus be-
come susceptible to agrochemicals, such as herbicide residues, because
of their highly permeable skin, the development of eggs and larvae in
the water which ultimately decreases recruitment of amphibian pop-
ulations ( Hayes et al., 2002 , 2006b ). Aquatic habitats contaminated
with endocrine-disrupting chemicals may signicantly aect amphib-
ian populations since these organisms typically pass through critical
hormone-regulated developmental stages in the aquatic environment
( Hayes et al., 2006b ).
Butachlor (N-(butoxymethyl) − 2 chloro-N-2, 6 diethyl acetanilide) is
one of the widely used pre-emergence herbicides on rice elds as it is
highly eective and low toxic ( Lin et al., 2021 ). The half-life of butachlor
in non-sterile soil is 2.67–29.79 days, diering in dierent soil environ-
ments. However, the residual butachlor may negatively harm non-target
organisms in the environment due to its extensive use ( Kaur and Goyal
2020 ; Jolodar et al., 2021 ). Studies indicated that commercially rele-
vant concentrations of butachlor have adverse eects on distinct devel-
∗ Corresponding author.
E-mail address: jcjumawan@carsu.edu.ph (J.C. Jumawan) .
opmental stages. In the Philippines, the invasive and abundant wild cane
toad Rhinella marina , exposure to butachlor caused stunted growth and
thyroid endocrine disruption ( Shuman-Goodier et al., 2021 ). In Taiwan,
environmentally realistic concentrations (0.05- 3.2 mg/L) of butachlor
had adverse eects on the survival, development, and time to metamor-
phosis in the alpine cricket frog Fejervarya limnocharis tadpole ( Liu et al.,
2011 )
The native Luzon wart frog Fejervarya vittigera provides important
services in rice elds in the Philippines. The species is widely distributed
and is considered "Least Concern" ( IUCN Red List 2018 ) and is harvested
for human consumption and provides an additional source of income
for farmers in most rice elds in the country ( Shuman-Goodier, 2019 ).
Exposure of F. vittigera tadpoles at Gosner stages 25–27 for 14 days
to butachlor at 0.2 mg/L did not signicantly aect its development
and survival ( Shuman-Goodier et al., 2017 ), but no studies were pur-
sued to bridge the gap on the potential eect of this herbicide on go-
nads of adults. This study aims to determine the eect of butachlor at
agriculturally relevant concentrations on the histology of the testis and
ovaries of adult F. vittigera under laboratory conditions. These are es-
sential information updates on the eect of herbicides on the gonads
of F. vittigera and other amphibians thriving, particularly in agricul-
tural areas. Here, we report that butachlor, at environmentally rele-
vant concentrations, induces partial feminization in the testis of adult
F. vittigera .
https://doi.org/10.1016/j.hazadv.2023.100275
Received 30 December 2022; Received in revised form 5 March 2023; Accepted 6 March 2023
2772-4166/© 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
( http://creativecommons.org/licenses/by-nc-nd/4.0/ )
G.G. Salvani and J.C. Jumawan Journal of Hazardous Materials Advances 10 (2023) 100275
Materials and methods
Animal collection and acclimatization
Collection and the number of F. vittigera individuals were sanctioned
by the Department of Environment and Natural Resources (DENR)
Caraga Region through issuing a Gratuitous Permit (R13–2018–47).
Sexually mature F. vittigera (Mean body length: 62.26 mm, range 52–
73 mm) were collected from a remote rice paddy eld in Brgy Santo
Nino, Butuan City, Philippines, during the beginning of the rainy sea-
son. The rice eld was owned by a single family who practiced tradi-
tional farming without pesticides or herbicides. Frogs were caught us-
ing a hand net after spotting with articial light. Specimens were trans-
ported to the laboratory in plastic boxes with water to provide adequate
moisture. Frogs were acclimatized in the laboratory for seven days at
room temperature was maintained at 27 °C under a 12/12 h light/dark
cycle ( Kuwagata et al., 2008 ).
Butachor exposure
A commercial formulation of butachlor (60% active ingredient, N-
butoxy-methyl-2 chloro-2 ′ 6 ′ -diethylactinilide, Monsanto Co. USA) was
utilized. Six treatment groups: Control (Distilled, dechlorinated water)
and Butachlor at 0.4 mg/L, 0.8 mg/L, 1.6 mg/L, 3.2 mg/L, and 4.8 mg/L
were utilized. The 3.2 mg/l is in reference to Liu et al. (2011) ’s maxi-
mum concentration applied to Gosner stage 26 of Fejervarya limnocharis
under laboratory conditions. The recommended application rate of bu-
tachlor in rice paddy water was 4.8 mg/L ( Geng et al., 2005 ). All concen-
trations were prepared in 200 mL for each LLDPE (Linhui) food-grade
plastic container (14 ″ L ×10 ″ W ×12 ″ H) in triplicates, each housing
two similarly sized adult F. vittigera of the same sex (Total: Males:36; Fe-
males 36). Water with butachlor concentrations was changed every day
for seven days. Containers were covered with a screen for proper venti-
lation. Survival rates and external morphology of amphibians were ob-
served daily. Body weights were obtained on the rst and seventh days
of exposure. Frogs were fed with commercial garden worm Eudrilus sp.
(1 frog: 3 worms) every afternoon for seven days. Room temperature
was maintained at 27 °C under a 12/12 h light/dark cycle, same as dur-
ing the acclimatization.
Histological processing and examination
At the end of the 7th day, specimens were sacriced through double
pithing at the spinal cord to elicit the loss of the righting reex and lack
of response to toe pinch ( Othman et al., 2016 ). Frogs were incised from
the pubis to the sternum, with the thoracic and coelomic cavity opened
to enable direct viewing of the gonads (ovary and testis), which were
immediately isolated and preserved immediately in Bouin’s uid and
transferred into 70% ethanol after 48 h ( Smits et al., 2014 ). Four tissue
sections were prepared for each replicate gonad of frogs exposed to the
dierent concentrations. Gonads were sagitally sectioned every 5 µm us-
ing a rotary microtome and stained with Haematoxylin and Eosin (H&E).
Examining ovary characteristics involved viewing oogenic cells in the
ovary and comparing diameters (µm) of representative mature, sec-
ondary, and primary growth oocytes ( n = 60 per replicate slide) across
treatment groups. In ovary sections, oocytes were categorized based on
the size of the cells and the presence of an enlarged germinal vesicle
(oocyte nucleus) in frogs ( Curi et al., 2021 ). Comparison of diameters
(µm) of representative mature, secondary, and primary growth. Repre-
sentative spermatogenic cells in individual seminiferous lobules and a
comparison of coverage of oogenic and spermatogenic cells in testicular
sections in cases of testicular feminization were examined ( Hayes et al.,
2003 , 2010a ; Du Preez et al., 2009 ). Cross-sections of lobules were also
noted for the abundance of compact spermatozoa bundles or the ab-
Fig. 1. Mean (X-Y) diameter (µm) of mature, secondary, and primary growth oocytes in F. vittigera unexposed and exposed to butachlor. ( n = 6 females per treatment
group; 1440 follicles assessed per treatment group). Superscripts with dierent letters indicate signicant dierence at ≤ 0.05. Values are means ± SE.
2
G.G. Salvani and J.C. Jumawan Journal of Hazardous Materials Advances 10 (2023) 100275
sence thereof. All tissue sections were photographed under a Leica MZ6
stereomicroscope with a DV Leica EC4 and the Leica Application Suite
version 3.2.0.
Statistical analysis
ANOVA was used to compare the mean (X-Y) diameter (µm) of pri-
mary, secondary, and tertiary (mature) growth oocytes between treat-
ments. Post-hoc comparisons were done using the Tukey method. The
signicance level was set at 0.05. All data were analyzed with SPSS 11.0
software programs (SPSS, Chicago, IL, USA).
Results and discussion
A 25% mortality was observed in F. vittigera , each exposed to 0.8 –
4.8 mg/L butachlor as early as on the rst to the third day of exposure.
No mortality was observed in the control group and frogs exposed to
0.4 mg/L butachlor. Overt signs of morbidity in the skin in the form of
lesions and swelling were observed in 90% of frogs exposed to butachlor.
Gonad histology
Typically, the ovary of F. vittigera is paired and multi-lobed, located
near the kidney. No notable deformities were observed in the egg fol-
licles across treatments; hence, an additional comparison of the mean
diameter of oocytes was performed ( Fig. 1 ). The average diameter of
primary growth oocytes in females exposed to 4.8 mg/L butachlor was
signicantly bigger ( P = 0.001) compared to the control group. In con-
trast, the average diameter of secondary oocytes exposed to butachlor
was signicantly bigger compared to the control ( P < 0.0001). The mean
diameter of mature oocytes of F. vittigera exposed to 1.6 mg/L, 3.2 mg/L,
and 4.8 mg/L butachlor was signicantly larger ( P < 0.0001).
Fig. 2. Testicular tissue of F. vittigera unexposed (A-B) and exposed to butachlor (C-F). A . Compact seminiferous lobule with asynchronous spermatogenesis; control;
B . Inset of A showing a lobule lumen containing spermatozoa (SZ) with spermatids (ST) in the periphery. C-D .Testicular section dominated by testicular perinucleolar
(PN) oocytes scanty seminiferous lobules (white circles) from frogs exposed to butachlor beginning 3.2 mg/L (C) until 4.8 mg/l butachlor (D). E . A Testicular PN
oocyte beside a seminiferous lobule containing spermatids (ST). F . A seminiferous lobule lumen with mature spermatozoa (ST) in a PN-dominated testicular section;
4.8 mg/l butachlor. ST– spermatids, SZ– spermatozoa, PN-Perinucleolar oocytes; Scale bar = A , - D = 50 µm; E-F 20 µm. H.
3
G.G. Salvani and J.C. Jumawan Journal of Hazardous Materials Advances 10 (2023) 100275
Testis of F. vittigera unexposed to butachlor showed compact seminif-
erous lobules. Various spermatogenic cells were seen undergoing asyn-
chronous spermatogenesis ( Fig. 2 A,B). Fig. 2 C,D show testicular sections
dominated by testicular perinucleolar (PN) oocytes scanty seminifer-
ous lobules (white circles) from frogs exposed to butachlor beginning
3.2 mg/L (C) until 4.8 mg/l butachlor. Observation of feminization was
noted starting at 3.2 mg/L ( Fig. 2 E, F) to 4.8 mg/L butachlor, where a
more widespread number of perinucleolar oocytes versus spermatogenic
cells were seen.
In most amphibians, sex is genetically determined by homomorphic
sex chromosomes ( Bachtrog et al., 2014 ). Nonetheless, sexual dieren-
tiation in amphibians is highly sensitive and plastic to sex steroid hor-
mones. For example, the sexually dimorphic expression of steroidogenic-
related genes, such as the cytochrome 17 𝛼-hydroxylase/17,20 lyase
(cyp17a1), responsible for the conversion of progestogens to andro-
gens and the cytochrome P450 19 (cyp19a1), that converts androgens
to estrogens, and ( Navarro-Martín et al., 2012 ). Various environmen-
tal factors (pH, temperature, and specic pollutants) can inuence sex
determination in both tadpole and adult amphibian life history stages
( Ruiz-García et al., 2021 ; Nakamura, 2009 ) given that the permeable
amphibian skin can take up and absorb pollutants, especially with water-
mediated exposure ( Trudeau et al., 2020 ). Treatment of males with es-
trogens during early embryonic development was shown to cause sex
reversal or the formation of an ovotestis ( Navara, 2018 ). Rhinella marina
tadpoles exposed to butachlor exhibited increased expression of tr 𝛽, but
not another TH-responsive gene, Krüppel-like factor 9 (klf9) ( Shuman-
Goodier et al., 2017 ). In Xenopus laevis , butachlor increased T3 and T4
levels and up regulated tr 𝛽expression while promoting metamorphosis
( Li et al., 2016 ).
Most studies reporting gonadal abnormalities and hermaphroditism
in male amphibians highlight the pesticide Atrazine as an en-
docrine disrupting agent ( Hayes et al., 2002 , 2011 ) causing fem-
inization and demasculinization in X. laevis ( Hayes et al., 2006b ,
2011 ), Rana pipiens ( Hayes et al., 2002 ) and Hoplobatrachus rugulo-
sus ( Trachantong et al., 2013 ). Male X. laevis that are partially femi-
nized represent hermaphroditism rather than the females being mas-
culinized because of the given immutability of the female X. laevis ovar-
ian dierentiation ( Hayes et al., 2002 ). Adult R. pipiens collected from
atrazine-applied rice elds also had testicular oocytes ( Hayes et al.,
2003 ). Atrazine exposure in the laboratory resulted in testicular oogen-
esis and even induced growth (vitellogenesis) of the oocytes in slower-
developing males but had no eect in females ( Hayes et al., 2002 ,
2010b ).
Butachlor is among the most common herbicides used in rice elds.
Mounting evidence indicates it is an endocrine disruptor that aects
the development of wildlife at environmentally relevant concentra-
tions ( Abigail et al., 2015 ; Li et al., 2016 ; Shuman-Goodier et al.,
2017 ). Butachlor altered the genes in mRNA expression involving the
hypothalamic-pituitary-thyroid (HPT) axis, which increases the thy-
roid hormone level of X. laevis tadpoles causing developmental toxi-
city and thyroid endocrine disruption in a time and dose-dependent
manner ( Li et al., 2016 ). Butachlor at 0.87 mg/L concentration used
by Liu et al. (2011) for F. limnocharis tadpoles in a 96 h LC
50
study
was lower than the recommended dosage of 4.8 mg/L of application to
paddy elds, and yet, this low dosage has already documented genotox-
icity. Cane toads ( Rhinella marina ) exposed to commercial formulation
of butachlor, Machete EC weighed less than controls and had smaller
thyroid glands, thyrocyte cells, and more individual follicles ( Shuman-
Goodier et al., 2021 ). Incidences of testicular oocyte follicles (intersex)
in the testes of adult Limnodynastes fletcheri caught in rice bays were
reported ( Spolyarich et al., 2011 ).
There is scarce literature on the cause-and-eect evidence of bu-
tachlor on the testicular feminization (presence of oocytes in seminif-
erous lobules) of adult amphibians. The rice paddy frog of the genus Fe-
jervarya often forms stable populations in agricultural areas ( Liu et al.,
2011 ; Shuman-Goodier et al., 2017 ). The formation of perinucleolar
oocytes in the testis of adult F. vittegera exposed to environmentally rele-
vant concentrations of butachlor highlights the potential of F. vittigera as
a sentinel species for studies on environmental health eects attributed
to agrochemical exposure.
Conclusions
Partial feminization in the testis of F. vittigera after seven days of ex-
posure to realistic concentrations in eld applications–3.2 and 4.8 mg/L
butachlor has important implications as to the safety of this herbicide
to amphibians thriving in rice elds. This Philippine-endemic species
thrive in major islands of the country. Gonadal abnormalities such as
testicular oocyte formation can covertly undermine the native F. vit-
tigera populations by inhibiting the capacity of adults to reproduce. As
the current study is short-term, the capacity of fertilization success from
feminized males and unaected females and the impacts of the exposure
on mating behaviors have yet to be explored in future studies.
Fund information
The research is supported by personal funds for thesis and has no
existing external support.
Author’s contributions
GS performed experimentations, JCJ provided the facility, and ap-
proved the study design. Both authors contributed to the writing and
approved the nal version of the manuscript.
Funding
The research has no external funds.
Declaration of Competing Interests
The authors declare that they have no competing personal or nan-
cial interests to inuence this research output.
Data availability
Data will be made available on request.
Acknowledgments
We thank Dr. Rafe Brown for verifying our specimen and the Philip-
pine Kidney Dialysis Foundation Histopathology Division for the histo-
logical preparations of our gonad samples.
References
Abigail, M.E.A., Samuel, S.M., Ramalingam, C., 2015. Addressing the environmental im-
pacts of butachlor and the available remediation strategies: a systematic review. Int.
J. Environ. Sci. Technol. 12, 4025–4036. doi: 10.1007/s13762- 015- 0866- 2
.
Bachtrog, D., Mank, J.E., Peichel, C.L., Kirkpatrick, M., Otto, S.P., Ashman, T.L.,
Hahn, M.W., Kitano, J., Mayrose, I., Ming, R., Perrin, N., Ross, L., Valenzuela, N.,
Vamosi, J.C., 2014. Tree of sex consortium. Sex determination: why so many ways of
doing it? PLoS Biol. 12 (7), e1001899. doi: 10.1371/journal.pbio.1001899
.
Curi, L.M., Peltzer, P.M., Attademo, M.A., Lajmanovich, R.C., 2021. Alterations in gonads
and liver tissue in two neotropical anuran species commonly occurring in rice elds
crops. Water Air Soil Pollut. 232 (5). doi: 10.1007/s11270- 021- 05164- 6
.
Davidson, C., 2004. Declining downwind: amphibian population declines in California and
historical pesticide use. Ecol. Appl. 14, 1892–1902. doi: 10.1890/03-5224
.
Du Preez, L.H., Kunene, N., Hanner, R., Giesy, J.P., Solomon, K.R., Hosmer, A., Van Der
Kraak, G.J., 2009. Population-specic incidence of testicular ovarian follicles in Xeno-
pus laevis from South Africa: a potential issue in endocrine testing. Aqua Toxicol. 95
(1), 10–16. doi: 10.1016/j.aquatox.2009.07.018
.
Geng, B.R., Yao, D., Xue, Q.Q., 2005. Acute toxicity of the pesticide dichlorvos and the
herbicide butachlor to tadpoles of four anuran species. Bull. Environ. Contam. Toxicol.
75, 343–349. doi: 10.1007/s00128- 005- 0759- z
.
Hayes, T.B., Haston, K., Tsui, M., Hoang, A., Haeele, C., Vonk, A., 2002. Feminization of
male frogs in the wild. Nature 419, 895–896. doi: 10.1038/419895a
.
4
G.G. Salvani and J.C. Jumawan Journal of Hazardous Materials Advances 10 (2023) 100275
Hayes, T., Haston, K., Tsui, M., Hoang, A., Haeele, C., Vonk, A., 2003. Atrazine-induced
hermaphroditism at 0.1 ppb in American leopard frogs ( Rana pipiens ): laboratory
and eld evidence. Environ. Health Perspect. 111 (4), 568–575. doi: 10.1289/ehp.
5932
.
Hayes, T.B., Case, P., Chui, S., Chung, D., Haeele, C., Haston, K., Tsui, M., 2006a. Pesti-
cide mixtures, endocrine disruption, and amphibian declines: are we underestimating
the impact? Environ. Health Perspect. 114 (Suppl 1), 40–50. doi: 10.1289/ehp.8051
.
Hayes, T.B., Stuart, A.A., Mendoza, M., Collins, A., Noriega, N., Vonk, A., Johnston, G.,
Liu, R., Kpodzo, D., 2006b. Characterization of atrazine-induced gonadal malforma-
tions in african clawed frogs ( Xenopus laevis ) and exogenous estrogen (17 𝛽-Estradiol):
support for the demasculinization/feminization hypothesis. Environ. Health Perspect.
114, 134–141. doi: 10.1289/ehp.8067
.
Hayes, T.B., Khoury, V., Narayan, A., Nazir, M., Park, A., Brown, T., Gallipeau, S.,
2010a. Atrazine induces complete feminization and chemical castration in male
African clawed frogs ( Xenopus laevis ). Proc. Natl Acad. Sci. 107 (10), 4612–4617.
doi: 10.1073/pnas.0909519107
.
Hayes, T.B., Falso, P., Gallipeau, S., Stice, M., 2010b. The cause of global amphibian de-
clines: a developmental endocrinologist’s perspective. J. Experiment. Biol. 213 (6),
921–933. doi: 10.1242/jeb.040865
, 2010 .
Hayes, T.B., Anderson, L.L., Beasley, V.R., De Solla, S.R., Iguchi, T., Ingraham, H., Will-
ingham, E., 2011. Demasculinization and feminization of male gonads by atrazine:
consistent eects across vertebrate classes. J. Steroid Biochem. Mol. Biol. 127 (1–2),
64–73. doi: 10.1016/j.jsbmb.2011.03.015
.
IUCN SSC Amphibian Specialist Group, 2018. Fejervarya Vittigera . The IUCN Red List of
Threatened Species 2018 doi: 10.2305/IUCN.UK.2018- 2.RLTS.T58294A114915259.
en
.
Jolodar, N.R., Karimi, S., Bouteh, E., Balist, J., Prosser, R., 2021. Human health and eco-
logical risk assessment of pesticides from rice production in the Babol Roud River
in Northern Iran. Sci. Total Environ. 772, 144729. doi: 10.1016/j.scitotenv.2020.
144729
.
Kaur, R., Goyal, D., 2020. Biodegradation of butachlor by Bacillus altitudinis and identi-
cation of metabolites. Curr. Microbiol. 77, 2602–2612
.
Kuwagata, T., Hamasaki, T., Watanabe, T., 2008. Modeling water temperature in rice
paddy for agro-environmental research. Agric. For. Meteorol. 148, 1754–1766. doi: 10.
1016/j.agrformet.2008.06.011
.
Li, S., Li, M., Wang, Q., Gui, W., Zhu, G., 2016. Exposure to butachlor causes thyroid
endocrine disruption and the promotion of metamorphosis in Xenopus laevis . Chemo-
sphere 152, 158–165. doi: 10.1016/j.chemosphere.2016.02.098
.
Lin, Z., Pang, S., Zhou, Z., Wu, X., Bhatt, P., Chen, S., 2021. Current insights into
the microbial degradation for butachlor: strains, metabolic pathways, and molecu-
lar mechanisms. Appl. Microbiol. Biotechnol. 105 (2021), 4369–4381. doi: 10.1007/
s00253- 021- 11346- 3
.
Liu, W.Y., Wang, C.Y., Wang, T.S., Fellers, G.M., Lai, B.C., Kam, Y.C., 2011. Impacts
of the herbicide butachlor on the larvae of a paddy eld breeding frog ( Fejer-
varya limnocharis ) in subtropical Taiwan. ecotoxicol. 20, 377–384. doi: 10.1007/
s10646- 010- 0589- 6
.
Nakamura, M., 2009. Sex determination in amphibians. In: Seminars in Cell & Develop-
mental Biology. Academic Press, pp. 271–282 Vol. 20, No. 3
.
Navara, K.J., 2018. Mechanisms of environmental sex determination in sh, amphibians,
and reptiles. Choosing Sexes. Fascinating Life Sciences. Springer, Cham doi: 10.1007/
978- 3- 319- 71271- 0 _ 10
.
Navarro-Martín, L., Velasco-Santamaría, Y.M., Duarte-Guterman, P., Robertson, C., Lanc-
tôt, C., Pauli, B., Trudeau, V.L., 2012. Sexing frogs by real-time PCR: using aro-
matase (cyp19) as an early ovarian dierentiation marker. Sex Dev 6 (6), 303–315.
doi: 10.1159/000343783 .
Othman, M.S., Khonsue, W., Kitana, J., Thirakhupt, K., Robson, M.G., Kitana, N., 2016.
Morphometric and gravimetric indices of two populations of rice frog ( Fejervarya lim-
nocharis ) naturally exposed to dierent environmental cadmium levels. J. Sains Kesi-
hatan Malaysia 14 (2), 57–64. doi: 10.17576/jskm- 2016- 1402- 07
.
Ruiz-García, A., Roco, Á.S., Bullejos, M., 2021. Sex dierentiation in amphibians: eect
of temperature and its inuence on sex reversal. Sex. Dev. 15 (1–3), 157–167. doi: 10.
1159/000515220
.
Shuman-Goodier, M.E., Singleton, G.R., Propper, C.R., 2017. Competition, and pesticide
exposure aect development of invasive ( Rhinella marina ) and native ( Fejervarya vit-
tigera ) rice paddy amphibian larvae. ecotoxicol. 26 (10), 1293–1304. doi: 10.1007/
s10646- 017- 1854- 8
.
Shuman-Goodier, M., 2019. Illuminating Amphibian Nightlife in Rice Agro-Ecosytems
(Doctoral dissertation, Northern Arizona University).
Shuman-Goodier, M.E., Singleton, G.R., Forsman, A.M., Hines, S., Christodoulides, N.,
Daniels, K.D., Propper, C.R., 2021. Developmental assays using invasive cane toads,
Rhinella marina , reveal safety concerns of a common formulation of the rice herbicide,
butachlor. Environ. Pollut. 272, 115955. doi: 10.1016/j.envpol.2020.115955
.
Smits, A.P., Skelly, D.K., Bolden, S.R., 2014. Amphibian intersex in suburban landscapes.
Ecosphere 5 (1), 11. doi: 10.1890/ES13- 00353.1
.
Spolyarich, N., Ross, V., Hyne, R.V., Wilson, S.P., Palmer, S.G., Byrne, M., 2011. Mor-
phological abnormalities in frogs from a rice-growing region in NSW, Australia,
with investigations into pesticide exposure. Environ. Monit. Assess. 173, 397–407.
doi: 10.1007/s10661- 010- 1395- 6 .
Trachantong, W., Promya, J., Saenphet, S., Saenphet, ..K., 2013. Eects of Atrazine Herbi-
cide on Metamorphosis and Gonadal Development of Hoplobatrachus rugulosus . Maejo
Int. J. Sci. Technol. 7, 60–71
.
Trudeau, V.L., Thomson, P., Zhang, W.S., Reynaud, S., Navarro-Martin, L., Langlois, V.S.,
2020. Agrochemicals disrupt multiple endocrine axes in amphibians. Mol. Cell. En-
docrinol. 513, 110861. doi: 10.1016/j.mce.2020.110861
.
5
