MORFOLOGIA DO ENCÉFALO E NEUROTOXICOLOGIA EMBRIONÁRIA DO 2,4-D EM Tropidurus Torquatus

UNIVERSIDADE FEDERAL DE GOIÁS – REGIONAL JATAÍ UNIDADE ACADÊMICA ESPECIAL DE CIÊNCIAS AGRÁRIAS PROGRAMA DE PÓS-GRADUAÇÃO EM BIOCIÊNCIA ANIMAL

LETÍCIA MENEZES FREITAS

MORFOLOGIA DO ENCÉFALO E NEUROTOXICOLOGIA EMBRIONÁRIA DO 2,4-D EM Tropidurus torquatus

(SQUAMATA: TROPIDURIDAE)

JATAÍ – GO 2020

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LETÍCIA MENEZES FREITAS

MORFOLOGIA DO ENCÉFALO E NEUROTOXICOLOGIA EMBRIONÁRIA DO 2,4-D EM Tropidurus torquatus (SQUAMATA: TROPIDURIDAE)

Dra. Mônica Rodrigues Ferreira Machado

Dra. Ana Paula da Silva Peréz

Dr. Suleyman Kaplan

Dissertação apresentada ao Programa de Pós-Graduação em Biociência Animal da Universidade Federal de Goiás, Regional Jataí, como requisito para obtenção do grau de Mestre em Biociência Animal.

JATAÍ – GO 2020

Dedico este trabalho à minha família e meus amigos. Sem vocês eu não estaria passando por mais esta etapa na minha vida. Também dedico à Fabiano Lima, que contribui imensamente com a realização deste sonho.

AGRADECIMENTOS

Agradeço imensamente à Fabiano Lima, pela orientação e grande ajuda, mesmo em momentos atribulados, e amizade sincera. Não seria a pessoa que sou hoje sem seu apoio, pois em cada momento juntos veio um novo aprendizado. Almejo ser uma profissional, e um ser humano, tão bom quanto você. Muitíssimo obrigada às professoras Mônica Machado e Ana Paula Perez, pela ajuda e apoio na elaboração deste trabalho, ao professor Benner Geraldo Alves, sem o qual estaria perdida na parte estatística e ao professor Suleyman Kaplan, que teve paciência para me ajudar a entender a técnica estereológica mesmo estando distante. A minha família sempre me apoiou nos estudos, entendendo que a dedicação exclusiva a faculdade, saindo cedo e muitas vezes voltando à noite para casa, é em busca de um futuro melhor. Meus pais sempre buscaram meios para que eu pudesse estudar sem passar nenhuma necessidade, vocês são minha inspiração. Minha avó Neuzita, embora sempre bravejando, foi parte essencial dessa jornada, me motivando e sentindo orgulho de minhas conquistas, que afinal são nossas. Obrigada aos meus amigos, principalmente Fabiano Neves, que mesmo distante sempre encontra palavras de motivação em momentos difíceis. Nos conhecemos de maneira não convencional, mas nossa conexão foi imediata e estamos sempre disponíveis ao outro em momentos de alegrias e tristezas. Agradeço a ajuda das técnicas Lília Cristina de Souza Barbosa, Sueisla Lopes Rezende Silva, Tracy Martina Marques Martins e Juliana Flávia Ferreira e Silva Paranaíba. Em todas as etapas vocês estiveram presentes e dispostas a compartilhar conhecimento para que eu tivesse sucesso nesta jornada. Agradeço a Fundação de Apoio à Pesquisa do Estado de Goiás – FAPEG pelo apoio financeiro durante este processo, pois foi essencial para que me dedicasse integralmente ao mestrado e pudesse continuar trilhando este caminho. Obrigada à UFG pela oportunidade de aprendizado e aprimoramento profissional durante todos os anos em que estive presente em suas salas e corredores. E também agradeço ao apoio e disponibilidade dos Laboratórios de Anatomia Humana e Comparada, de Morfofisiologia e de Pesquisas Médicas. Obrigada a todos vocês. O papel de cada um foi essencial para me tornar pessoa que sou hoje e vocês são uma inspiração quem almejo ser.

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“You want weapons? We’re in a library! Books! The best weapons in the world!” The Doctor

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RESUMO

Agrotóxicos são utilizados desde a antiguidade, porém, apenas no século passado teve início a produção sintética desses produtos, sendo o Brasil um dos maiores consumidores estas substâncias. O 2,4-D é um herbicida seletivo para ervas daninhas de folhas largas, amplamente utilizado em vários tipos de cultura. Em condições normais em animais, o 2,4-D é eliminado na urina, mas se a taxa de depuração renal for ultrapassada surgem efeitos adversos, inclusive neurotóxicos. Poucos estudos avaliam o efeito do 2,4-D sobre o desenvolvimento embrionário, sendo estes necessários pois organismos jovens são mais sensíveis que adultos. Com isso em mente visamos avaliar o efeito do 2,4-D DMA® 806 BR no desenvolvimento embrionário do encéfalo de Tropidurus torquatus. Também objetivamos descrever o encéfalo de Tropidurus torquatus como base para o estudo toxicológico. Esta espécie é abundante e por habitar lavouras pode contaminar-se com agrotóxicos naturalmente, fazendo-a um bom modelo animal. Animais adultos foram capturados e eutanaziados para a descrição macro- e microscópica do encéfalo. Fêmeas grávidas foram coletadas e mantidas em terrários até a oviposição. Os ovos foram distribuídos aleatoriamente em dois grupos, controle e 2,4-D. O substrato do grupo 2,4-D foi contaminado logo após a oviposição com uma dose correspondente à 1,5L p.c./ha, e no controle o substrato foi regado com a mesma quantidade de água destilada. De ambos os grupos foram coletados embriões nos dias 15, 30 e 60 após a oviposição. Os encéfalos dos embriões foram submetidos a inclusão em parafina para coloração com H.E. e foi estimado o volume do teto óptico através da técnica de estereologia, comparando-se os dois grupos. Foi observado um decréscimo do volume no grupo 2,4-D do dia 15, mas não foram encontradas diferenças do dia 30, sugerindo que o 2,4-D tem um leve efeito neurotóxico na dose recomendada, mas que pode ser reversível.

Palavras-chave: agrotóxico, embrião, herbicida, lagarto, réptil, sistema nervoso

ABSTRACT

Pesticides have been used since antiquity, nevertheless synthetic products emerged only last century and currently Brazil is the biggest consumer of these substances. 2,4- D is a selective herbicide for broad-leaved weeds, used in several types of crops. Usually 2,4-D is eliminated in urine in animals, but when the renal clearance rate is exceeded, adverse effects, including neurotoxic ones, appear. Few studies evaluate the effect of 2,4-D on embryonic development, which is necessary because young organisms are more sensitive than adults. With this in mind we aim to evaluate the effect of 2,4-D DMA® 806 BR on the embryonic development of the brain of Tropidurus torquatus. We also aimed to describe the brain of Tropidurus torquatus as the basis for the toxicological study. This species is abundant and by inhabiting crops it can naturally contaminate itself with pesticides, making it a good animal model. Adult animals were captured and euthanized for the macro- and microscopic description of the brain. Pregnant females were collected and kept in terrariums until oviposition. Eggs were randomly distributed into two groups, control and 2,4-D. The substrate of the 2,4-D group was contaminated shortly after oviposition with a dose corresponding to 1.5L/ha, and the control’s substrate was watered with the same quantity of water. Embryos were collected on days 15, 30 and 60 after oviposition. The embryos' brains were submitted to paraffin inclusion for H.E. staining and the volume of the optical tectum was estimated using stereology to compare both groups. A decrease in volume was observed in the 2,4-D group on day 15, but no differences were found on day 30, suggesting that 2,4-D has a mild neurotoxic effect at the recommended dose, which may be reversible.

Keywords: pesticide, embryo, herbicide, lizard, reptile, nervous system

LISTA DE ABREVIATURAS Página 5-HIAA – 5-hydroxyindolacetic acid ...... 08 5-HT – serotonin ...... 03 ac – anterior commissure ...... 51 AChE – acetylcholinesterase...... 18 ACP – alpha-cypermethrin ...... 04 aob – acessory olfactory bulb ...... 52 CAT – catalase ...... 18 ce – cerebellum ...... 51 CE – coefficient of error ...... 64 ch – cerebellar hemisphere ...... 52 chp – choroid plexus ...... 57 cl – cell layer ...... 57 CNS – central nervous system ...... 36 Con – control ...... 63 cp – cerebellar penducle ...... 52 CV – coefficient of variance ...... 64 DA – dopamine ...... 03 dc – dorsal cortex ...... 52 di – diencephalon ...... 51 DIN – dinotefuran ...... 06 dmc – dorsal medial cortex...... 53 dms – dorsal median sulcus ...... 58 DOPAC – dihydroxyphenylacetic acid ...... 08 dsa – dorsal sac ...... 57 dvr – dorsal ventricular ridge ...... 52 em – ependyma ...... 56 ep – epithalamus ...... 51 EPA – United States Environmental Protection Agency ...... 04 epl – external plexiform layer...... 56 EPSPS – enolpyruvylshikimate-3-phosphate synthase ...... 16 fl – flocculus ...... 52 fs – floccular sulcus ...... 58 xi

GLA – glufosinate-ammonium ...... 18 gll – glomerular layer ...... 57 grl – glanular layer ...... 57 GS – glutamine synthetase ...... 18 GST - glutathione-S-transferase ...... 19 ha – habenula ...... 55 hac – habebular commissure ...... 58 hc – hippocampal commissure ...... 51 HE - hematoxylin-eosin ...... 38 HTP – hypothalamus-pituitary-thyroid axis ...... 05 HVA – homovanillic acid ...... 08 hy – hypothalamus ...... 51 I – olfactory nerve ...... 52 II – optic nerve ...... 51 iii – third ventricle ...... 55 IMI – imidacloprid ...... 06 in – infundibulum ...... 52 ipl – internal plexiform layer ...... 56 ISO – International Organization for Standardization ...... 04 iv – fourth ventricle ...... 51 lc – lateral cortex ...... 53 LCT – lambda-cyhalothrin ...... 05 LDH – lactate dehydrogenase ...... 04 L-GLA – L-glufosinate-ammonium ...... 18 lp – lateral part of cerebellar hemisphere ...... 52 lv – lateral ventricle ...... 53 ma – mesencephalic aqueduct ...... 51 mb – midbrain ...... 51 mc – medial cortex ...... 51 mcl – mitral cell layer ...... 56 MDA – malondialdehyde ...... 18 MFB – medial forebrain bundle ...... 06 ml – molecular layer ...... 59 mo – medulla oblongata ...... 51 xii

mob – main olfactory bulb ...... 52 mp – median part of cerebelar hemisphere ...... 52 nAChRs – nicotinic acetylcholine receptors...... 06 ob – olfactory bulb ...... 51 oc – optic chiasm ...... 51 olv – olfactory ventricle ...... 56 onl – olfactory nerve layer ...... 56 op – olfactory penducle ...... 51 ot – optic tectum ...... 53 otr – optic tractum ...... 53 out – olfactory tubercle ...... 53 ov – optic ventricle ...... 55 pa – pallidum ...... 53 par – paraphysis ...... 58 pc – posterior commissure ...... 51 pcl – purkinje cell layer ...... 59 pe – parietal eye ...... 58 pm – pallial membrane ...... 53 po – pineal organ ...... 55 rf – reticular formation ...... 53 sac – stratum album centrale ...... 58 sc – spinal cord ...... 51 sep – septum ...... 51 sfgs – stratum fibrosum and griseum superficiale ...... 58 sfp – stratum fibrosum periventriculare ...... 58 sgc – statum griseum centrale ...... 58 sgp – stratum griseum periventriculare ...... 58 so – stratum opticum ...... 58 soc – supraoptical commissure ...... 51 SOD – superoxide dismutase ...... 18 sp – subpallium ...... 51 st – striatum ...... 53 ta – thalamus ...... 51 tcm – tectal commissure ...... 51 xiii

tg – tegmentum ...... 51 TMX – thiamethoxam ...... 06 ts – torus semicircularis ...... 53 V – trigeminal nerve ...... 53

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LISTA DE FIGURAS Página MACRO- AND MICROSCOPIC ANATOMY OF THE BRAIN OF Tropidurus torquatus Figure 1: A – Topography and macroscopic anatomy of the brain of Tropidurus torquatus...... 51 Figure 2: Macroscopic anatomy of the brain of Tropidurus torquatus...... 52 Figure 3: Transversal sections of the brain of Tropidurus torquatus...... 53 Figure 4: Sagittal sections of the brain of Tropidurus torquatus...... 54 Figure 5: Frontal sections of the brain of Tropidurus torquatus...... 55 Figure 6: Olfactory bulb of Tropidurus torquatus...... 56 Figure 7: Cortices of Tropidurus torquatus...... 57 Figure 8: Epithalamus of Tropidurus torquatus and associated structures...... 57 Figure 9: Optic tectum of Tropidurus torquatus...... 58 Figure 10: Cerebellum of Tropidurus torquatus...... 58

TOXICOLOGIA DE 2,4 NO DESENVOLVIMENTO DO ENCÉFALO DE Tropidurus torquatus Fig 1 Software Stepanizer ...... 64 Fig 2 Embryos of Tropidurus torquatus...... 66 Fig 3 Volume comparison of the optic tectum of Tropidurus torquatus from embryos exposed to water (control) and 2,4-D...... 67 Fig 4 Volume comparison of the optic tectum of Tropidurus torquatus from embryos exposed to water (control) and 2,4-D within the identified stages...... 68 Fig 5 Correlation analysis between the estimated brain volume and the developmental stages of the embryos of Tropidurus torquatus ...... 68

SUMÁRIO

Página LISTA DE ABREVIATURAS ...... x

LISTA DE FIGURAS ...... xiv

1. INTRODUÇÃO GERAL ...... 1

ANIMAL MODELS IN THE NEUROTOXICOLOGY OF 2,4-D ...... 3

Summary ...... 3 Introduction...... 4 Literature review ...... 4 Conclusion...... 10 References ...... 11 TOXICITY OF PESTICIDES IN LIZARDS ...... 14 Summary ...... 14 Introduction...... 14 Literature review ...... 16 Herbicides ...... 16 Insecticides...... 22 Fungicides ...... 29 Conclusion...... 29 References ...... 31 MACRO- AND MICROSCOPIC ANATOMY OF THE BRAIN OF Tropidurus torquatus ...... 35 Summary ...... 35 Introduction...... 36 Material and methods ...... 38 Macroscopic analysis ...... 38 Histologic analysis ...... 38 Results and discussion ...... 39 Olfactory bulbs ...... 39 xvi

Telencephalon ...... 41 Diencephalon ...... 42 Mesencephalon ...... 43 Hindbrain ...... 44 Acknowledgements ...... 46 References ...... 46 Figure legends ...... 51 DEVELOPMENTAL NEUROTOXICOLOGY OF 2,4-D IN Tropidurus torquatus ... 60 Abstract ...... 60 Acknowledgments ...... 60 Introduction...... 60 Materials and methods ...... 62 Collection and maintenance of lizards ...... 62 Egg collection, maintenance and herbicide exposure ...... 63 Histology...... 63 Stereology ...... 64 Statistical analysis ...... 64 Results ...... 65 Discussion ...... 68 References ...... 69 REFERÊNCIAS BIBLIOGRÁFICAS ...... 73 ANEXO 1 – Extrato da permissão de coleta do SISBIO ...... 75 ANEXO 2 – Aprovação no Comitê de Ética ...... 83

1 1. INTRODUÇÃO GERAL 2 3 Agrotóxicos diversos têm sido utilizados historicamente com fontes, 4 estruturas e meios de ação variados para eliminar organismos indesejados. 5 Estes produtos causam uma forte pressão seletiva em espécies alvo, porém sua 6 ação não é limitada, podendo afetar todo o ecossistema e estar relacionado com 7 perda de biodiversidade (ARONZON et al., 2014). Seu uso é particularmente alto 8 em regiões de clima tropical, pois este clima propicia o surgimento e propagação 9 de espécies que afetam a qualidade das lavouras. O Brasil possui esse perfil, 10 sendo um grande produtor e exportador agrícola, consumindo atualmente 20% 11 do total de agrotóxicos comercializados mundialmente (BOMBARDI, 2017), 12 valores estes que estão em constante crescimento. 13 Dentre os herbicidas mais utilizados no Brasil está o 2,4-D, tendo sido 14 comercializadas 57.389 toneladas em 2017 (IBAMA, 2018). Este herbicida é 15 seletivo, usado em pré e pós-emergência de várias culturas como cana-de- 16 açúcar, milho e soja, possuindo efeito sobre ervas daninhas de folhas largas 17 (ERTUG et al., 2014). Possui classificação toxicológica como extremamente 18 tóxico (Classe I) pela ANVISA ([2018]) e altamente solúvel na água e no solo, 19 sendo um risco para o meio ambiente e para o ser humano (HUY et al., 2017). 20 Em animais, nenhum órgão específico é alvo do 2,4-D e sua toxicidade 21 é dose dependente, com efeitos adversos sendo observados apenas se a taxa 22 de depuração renal for ultrapassada. Diversos efeitos do 2,4-D são relatados, 23 como neurotóxicos e teratogênicos, tanto em modelos animais quanto em casos 24 de intoxicação em humanos (SHARMA; RAJESH, 2018). Porém, ainda não 25 existem dados suficientes para definir sua teratogenicidade (LOOMIS et al., 26 2015). Embora seja recomendado o uso de indivíduos nos primeiros estágios de 27 desenvolvimento em investigações toxicológicas, pois são mais sensíveis a 28 agentes externos (USEPA, 2016), poucos são os estudos sobre os efeitos deste 29 herbicida sobre o desenvolvimento embrionário. 30 Animais não alvo podem sofrer com a exposição a agrotóxicos, como 31 Tropidurus torquatus (Wied, 1820). Popularmente conhecido por calango, a 32 espécie possui hábito terrestre e sua locomoção, permanência e oviposição em 33 solo contaminado pode contaminar seus espécimes e ovos. Também podem ser

1 expostos à poluentes por ingestão de alimentos com presença de resíduos 2 químicos. 3 Estudos variados utilizam T. torquatus como organismo modelo, devido 4 ao seu conjunto de características, por exemplo sobre dieta (SIQUEIRA et al., 5 2013), reprodução (ORTIZ et al., 2014) e temperatura (KIEFER et al., 2005) e 6 seu desenvolvimento embrionário normal pós-oviposição foi descrito por Py- 7 Daniel et al. (2017), com o total de 42 estágios e duração de aproximadamente 8 75 dias. 9 A espécie é abundante, distribuindo-se do Brasil à Argentina, e possui 10 ciclo reprodutivo sazonal, coincidindo com o período chuvoso (RODRIGUES, 11 1987; ORTIZ et al., 2014; PY-DANIEL et al., 2017). Essas características 12 facilitam um estudo com uma amostra significativa de embriões a cada ciclo 13 reprodutivo e que não afete sua população. 14 Embora o desenvolvimento embrionário de T. torquatus tenha sido 15 descrito, não existem informações detalhadas sobre seu encéfalo adulto, 16 adicionando-se a ausência de estudos sobre o efeito de defensivos agrícolas 17 sobre esta espécie, que habita regiões de grande produtividade agrícola, até 18 mesmo pela disponibilidade de insetos nessas regiões, sendo fonte abundante 19 de alimento para a espécie. Portanto, são susceptíveis ao contato por agentes 20 químicos desta natureza. 21 Objetiva-se analisar o efeito do herbicida 2,4-D na formulação comercial 22 DMA® 806 BR no desenvolvimento do encéfalo de T. torquatus, exposto a uma 23 dose única no começo do desenvolvimento, assim como descrever a anatomia 24 macro- e microscópica do encéfalo da espécie T. torquatus. Também procura- 25 se saber se a recomendação da quantidade de herbicida sugerida na bula do 26 produto é adequada para que não ocorram efeitos nocivos no meio ambiente e 27 aos organismos, especialmente durante o período de desenvolvimento. 28 Resultados obtidos com este modelo pela exposição a formulação 29 comercial de 2,4-D DMA® 806 BR, poderão ser extrapolados a outros animais, 30 incluindo humanos. 31 32 33 3

1 Animal Models in the Neurotoxicology of 2,4-D

2 Summary

3 2,4-D is a selective pre- and post-emergence herbicide used for several crops. It is

4 hazardous for the environment and a risk for humans, therefore several studies attempt to

5 evaluate its effects and consequences of its use. The nervous system is supposedly a target

6 for this herbicide and this comprehensive review gathers the information about animal

7 models that have been used for the study of the neurotoxicity of 2,4-D. The studies used

8 several different methods to evaluate the neurotoxicity of this herbicide, most of which

9 used rodents, mainly rats, two used fish and one used chicken eggs. The main behavioral

10 effect observed concerned alterations in locomotor patterns and reduced motor activity.

11 Biochemical analysis showed decreased levels of serotonin (5-HT) and increased levels

12 of its metabolites and dopamine (DA) and its metabolites have increased or decreased

13 levels depending on the brain area analyzed. Hypomyelination is also a possible effect of

14 2,4-D, when the exposure occurs during the proliferation and development of the

15 oligodendrocytes. The worst neuropathologic effects were observed in fish. Since most

16 studies focused in the neurotoxicity of 2,4-D in rodents, the effect it may have on other

17 species and groups of animals, especially with different physiology, is unclear and it

18 should be researched.

20 Keywords

21 2,4-D, herbicide, brain, behavior.

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1 Introduction

2 2,4-D is the International Organization for Standardization (ISO) name for 2,4-

3 Dichlorophenoxyacetic acid. 2,4-D is a selective herbicide, first synthesized in 1941, that

4 acts as an auxin, a plant growth hormone. It is absorbed by roots and leaves, causing

5 excessive growth which leads to death, being more effective in broad-leaved weeds.1-3 It

6 has pre- and post-emergence uses for crops such as rice, coffee, sugarcane, corn and

7 soybean. 4,5

8 It is classified in Brazil as extremely toxic (Class I) and highly soluble in water and soil,

9 this is a hazardous product for the environment (Class III) and a risk for humans.3,5 The

10 EPA (United States Environmental Protection Agency) considers the 2,4-D to have

11 moderate toxicity to birds and mammals and to be slightly toxic to fish. It is a light-yellow

12 powder used in various commercial formulations, of which the most used its the

13 dimethylamine salt.4-6

14 2,4-D toxicity has been tested in several animal models and many human intoxications

15 have been reported. The nervous system is supposed to be one of the target organs of

16 chlorophenoxy herbicides, such as 2,4-D, and effects related to loss of motor functions,

17 lethargy and behavioral alterations have been described.7-9 The objective of this literature

18 review is to analyze data in respect of the neurotoxicity of 2,4-D in animal models.

20 Literature review

21 The research was carried out using three scientific databases: Periódicos CAPES,

22 PubMed and SciELO. Several searches were made for titles and abstracts containing

23 several permutations of keywords which were: 2,4-D; 2,4-Dichlorophenoxyacetic;

24 Neurotoxic; Neurotoxicity; and Nervous System. The inclusion factors were studies 5

1 evaluating the toxicity of 2,4-D and the use of animal models. The exclusion factors were

2 scientific papers that did not use live animals (using cell cultures, for example) and studies

3 that did not address the effect of 2,4-D on the nervous system.

4 We found 41 articles (excluding redundant titles) and 12 of them fit the criteria stated

5 above.1,2,7-16 Of the 12 selected articles, 9 studied the effect of 2,4-D on rodents of

6 different strains (CD-1 mice7, Wistar8,10-13, Fischer 3441,14 and Sprague-Dawley rats15),

7 one of them also used New Zealand white rabbits7. Two studies evaluated the effect of

8 this herbicide in fish, Zebrafish (Danio rerio)16 and Guppy (Poecilia reticulata)9 and one

9 study used chicken eggs2.

10 Oral administration at doses ranging from 60-300 mg/kg in rats Wistar resulted in reduced

11 locomotion and rearing frequencies, as well as reduced motor activity and increased

12 immobility. The effects increased the higher the dose used.10 When administered via

13 gavage in corn oil to Fischer 344 rats, at doses of 15-250 mg/kg, the highest dose (250

14 mg/kg) resulted in incoordination and alterations in gait, mainly on the first day and limb

15 stiffness as well as reduced motor activity, only in the first day.7

16 For 1 year 2,4-D was available ad libitum in the diets (weekly for 13 weeks and then

17 every 4 weeks) of Fischer 344 rats at doses of 5-150 mg/kg day. The effects were observed

18 mostly in females of the highest dose group. The grip strength of the forelimbs increased

19 and there were no differences in motor activity. The retinas of females from the larger

20 dose group had bilateral retinal degeneration at the end of the study, characterized by the

21 loss of photoreceptor cell layers. There were no pathological changes in the nervous

22 system.7

23 The maternal behavior of Wistar nulliparous rats under 2,4-D oral exposure during the

24 gestational period was evaluated. The doses were approximately 15, 25 and 50

25 mg/kg/day. The appearance of the dams, and the number of pups and their survival were 6

1 not affected. However, some maternal behaviors were disrupted, the total was higher in

2 higher doses, such as decreased licking and nursing puppies, totally annulling the licking

3 of the anogenital region of the pups at all doses; latency and length of retrieval of pups;

4 dams leaving nest and increased time spent out of it.11

5 Fischer CDF 344 rats underwent dermal exposure for 2 h/day 5 days/week, of

6 approximately 150 mg (corresponding to approximately 34.6 mg/kg, according to the

7 pretreatment weight average – 230,4 ± 3.3 g) during 2 weeks and 111 mg (corresponding

8 to approximately 19.7 mg/kg, according to the pretreatment weight average – 177,4 ± 2

9 g) for 3 weeks and there were no neuropathologic consequences and the grip strength was

10 not affected.14

11 Unilateral intracerebral administration was tested in 3 regions of the brain of Wistar rats.

12 The result of 100 μg 2,4-D in the striatum was decreased exploratory and motor activities,

13 postural deviation and moderate ipsilateral circling response. The same amount (100 μg)

14 was injected in the accumbens, also resulting in decreased exploratory and motor

15 activities, circling response was not observed, but the total turn numbers were reduced;

16 In the medial forebrain bundle (MFB), 50 μg was injected and alterations in locomotion

17 activity were not observed, but ipsilateral circling response was.8

18 Behavioral changes were observed in guppies after exposure to 2,4-D. At a dose of 15

19 mg/L 24h less general activity and grouping at the aquarium corners were observed after

20 24h. At a dose of 30 mg/L, after 12 hours of treatment, there was shortness of breath,

21 sudden rotations and jumping. The effects were more severe at the highest dose of 45

22 mg/L, almost 6 h after dosing, it was observed loss of equilibrium, sudden movements,

23 grouping around the aeration area and color loss.15

24 The accumulation and regional distribution of 2,4-D was quantified by labeled [14C] 2,4-

25 D in New Zealand white rabbits, Sprague-Dawley rats and CD-1 mice. It was more 7

1 marked when there was a pre-exposure to the herbicide, with greater effect in increasing

2 doses. A pre-exposure dose of 250 mg/kg injected into the saphenous vein of rats, and

3 application of 8 μCi/kg of 2,4-D, increased [14C] presence in the brain and cerebrospinal

4 fluid, as well as in the liver, testes, lungs, heart and muscles, but activity decreased in the

5 kidneys and plasma, myotomy and lethargy were also observed in the animals.15 For both

6 mice and rabbits (intraperitoneally injected with pretreatment of 0, 40, 80 or 160 mg/kg,

7 the last dose only in rabbits, and treatment of 50 μCi/kg of [14C] 2,4-D), the areas with

8 greatest accumulation of 2,4-D were the brainstem, cerebellum and frontal cortex, and

9 the smaller were the hypothalamus and caudate nucleus. In pregnant mice females the

10 concentration found in the fetal brain was higher than in the maternal, but smaller when

11 compared with other tissues and blood plasma.7

12 It has been found that 2,4-D can be detected in the brain and serum of exposed animals

13 even at a small dose of 10 mg/kg. Through samples of the cerebrospinal fluid and choroid

14 plexus of New Zealand white rabbits, the source of the accumulation of 2,4-D in the brain

15 was checked. There were two hypotheses, increased entry in the brain or failure in its

16 elimination. The blood–brain barrier permeability did not change in CD-1 mice,

17 indicating another mechanism, which was better explained in the rabbits by the

18 competitive inhibition of 2,4-D elimination via active transport in the anionic system of

19 the choroid plexus.10,7 This mechanism is likely the same in fish and other species.

20 However, in early development the susceptibility of 2,4-D infiltration in the brain of

21 embryos is possibly higher until the blood-brain barrier is fully developed. Eutherian

22 animals are protected by the mother during this period, but oviparous animals may be

23 more vulnerable.

24 In neurochemical studies, 2,4-D at an oral dose of 200 mg/kg in Wistar rats, did not affect

25 the levels of dopamine (DA) and homovanillic acid (HVA) in the striatum, however 8

1 serotonin (5-HT) levels decreased and 5-hydroxyindolacetic acid (5-HIAA), a serotonin

2 metabolite, increased after 4 hours exposure. In the brainstem only 5-HIAA levels

3 increased. This indicates an increase in the functional activity of serotonin after the

4 application of 2,4-D.10 Maternal behavior alterations in Wistar rats orally exposed to 15,

5 25 and 50 mg/kg/day are associated with decreased 5-HT and increased DA levels in the

6 arcuate nucleus as well as decreased serum prolactin levels.11

7 With the injection of 100 μg of 2,4-D in the striatum of Wistar rats, similar effects were

8 observed concerning 5-HT, though there was a slight increase in HVA in the injected

9 hemisphere. There were no changes in monoamine levels. In rats injected with 100 μg of

10 2,4-D in the accumbens, HVA and 5-HT increased in the injected hemisphere whereas in

11 the other hemisphere injected with the vehicle, the levels decreased. When injected into

12 the MFB (50 μg), levels of DA and its metabolites, HVA and dihydroxyphenylacetic acid

13 (DOPAC) in the striatum decreased, 5-HT and 5-HIAA also decreased, but only after 7

14 days. Injection in the MFB caused more effects on the striatum than intrastriatal

15 application.8

16 It was evaluated whether neonatal exposure of 2,4-D throughout lactation alter the

17 myelination of rats Wistar. In rats, oligodendrocytes develop after birth and the deposition

18 of myelin occurs rapidly over two weeks. 2,4-D were administered in a dose of 100

19 mg/kg/day, in three different periods, 9-25, 9-15 and 15-25 postnatal days. A decrease in

20 total lipids in the brain was observed in the groups that were exposed up to the 25th day,

21 and their composition changed, the cholesterol esters levels increased 263% and

22 phospholipids (38.6% measuring phosphate) and free fatty acids (62.2%) levels

23 decreased, which may alter the fluidity and stability of cell membranes.12

24 There was hypomyelination in the above rats as a result of reduced galactolipids.12 Wistar

25 rats treated with subcutaneous injections of 2,4-D (doses 70 and 100 mg/kg every 48 h in 9

1 the dorsal region of the neck tested in several groups starting treatment at 7 or 12 postnatal

2 days and ending on days 17 or 25), also showed a significant decrease in ganglioside and

3 DNA levels in groups treated with higher doses for longer times.13

4 The hypomyelination was further explored by De Moro et al.9 using 2,4-D butyl ester in

5 fertilized chicken eggs. The contamination was topical with 3.1 mg/egg. No changes in

6 development and brain appearance were observed. The deposition of galactolipids have

7 decreased 30-40% due to changes in the level of cerebrosides (42-55%) and sulfatides

8 (32-37%). The cholesterol content, the total number of proteins and activity of the enzyme

9 CNPase, which is one of the important markers for the synthesis or demyelination of

10 myelin, was significantly lesser than control over development. The DNA content

11 decreased at first, but increased significantly from day 14, resulting in a decreased

12 protein/DNA ratio.

13 When the treatment is applied on chicken eggs from the 15th day, there is no change in

14 the myelin content.9 All this suggests that the vulnerable period is the proliferation and

15 development of the oligodendrocytes, which is a possible target for the action of 2,4-D,

16 either by reducing their capacity for lipid synthesis or by destroying formed myelin.9,12,13

17 Zebrafish is a widely used animal model and Ton et al.16 aimed to establish it as a model

18 for neurotoxicity, testing some compounds in embryos of the species, including 2,4-D.

19 Embryos exposed 6 hours post fertilization (hpf), up to 48 or 96 hpf at doses of 50-75 μM

20 (23.87-35.81 mg/L) presented decreased motility, slow heart rate (at 50 μM) and edema

21 of the heart (at 75 μM). These were also present at a dose of 200 μM (95.51 mg/L), as

22 well as short body and hemorrhage. The neurotoxic effects were increased apoptosis,

23 disrupted motor neuron growth, and reduction of axon projections to the optic tectum.

24 2,4-D is slightly teratogenic in Zebrafish, indicating that its neurotoxicity is specific

25 because it is not toxic for other animals.16 10

1 There were behavioral and neurotoxic effects on the spinal cord of guppies at three doses

2 (15, 30 and 45 mg/L), exposed for 72 and 96 hours. Survival rates after 96 h were 30, 60

3 and 90% respectively, and 100% for control. The effects observed were loss of neurons,

4 edema, degeneration of Nissl corpuscles, pycnotic nuclei, gliosis, intercellular spaces and

5 vacuolization. At the lower doses (15 and 30 mg/L), these effects were mainly mild or

6 absent at 72 h, whereas in 96 h most became moderate. At the highest dose (45 mg/L),

7 the effects were mild and moderate and at 72 h and most became severe at for 96 h, except

8 vacuolation (moderate).2

9 The route of exposure may affect the severity of the effects, given that dermal exposure

10 did not result any problems for the nervous system, even though there was a prolonged

11 time exposure (weeks), compared with a single oral exposure, which resulted in

12 alterations, mainly behavioral. The dose and animal used are also variables to be

13 considered. The experimental doses varied from 5-300 mg/kg for oral, dermal and

14 injected administration in rodents, while the exposure was topical in chicken eggs. For

15 experiments in fish, the dilution of the herbicide was made in water (varying from 15-95

16 mg/L). Intracerebral administration used very low doses (50-100 μg) and considering the

17 different exposure and doses, it is hard to make a correlation of its effects with other types

18 of administration.

20 Conclusion

21 The effect of 2,4-D is dose-dependent, the effects increasing with dose. The main

22 neurotoxic effects observed for 2,4-D were reduced motor activity, alterations in

23 locomotor patterns and disrupted behaviors; hypomyelination; changes in lipids, protein

24 and DNA levels; changes in serotonin (5-HT), dopamine (DA) and associated

25 compounds; and histopathological changes. 11

1 Labeled 2,4-D was used to show its accumulation and distribution, being more present in

2 the brainstem, cerebellum and frontal cortex. Its entrance and accumulation in the brain

3 was better explained by anionic system transport and no change in the blood-brain barrier

4 was detected.

5 Biochemical studies indicated that levels of 5-HT usually decreased while its metabolites

6 levels increased, and that levels of DA and its metabolites increased or decreased in

7 different brain regions.

8 Hypomyelination was present when the organisms were exposed during myelination.

9 This was evidenced by reduction in the levels of lipids due to 2,4-D, some of which are

10 important in myelination. Protein and DNA levels may possibly be reduced as an effect

11 of the herbicide.

12 Each species may respond differently to exposure to the herbicide, causing different

13 effects. Most neuropathologic effects were observed in fish, which appears to be more

14 sensible to 2,4-D. Among the studied animal models, the majority were rodents, being

15 necessary studies in other species and groups of animals, since they have different

16 metabolism and physiology.

18 References

19 1. Mattsson JL, Charles JM, Yano BL, et al. Single-dose and chronic dietary neurotoxicity

20 screening studies on 2, 4-dichlorophenoxyacetic acid in rats. Fundam Appl Toxicol 1997;

21 40: 111-9.

22 2. Uyanıkgil Y, Yalçınkaya M, Ateş U, et al. Effects of 2, 4-dichlorophenoxyacetic acid

23 formulation on medulla spinalis of Poecilia reticulata: A histopathological study.

24 Chemosphere 2009; 76: 1386-91. 12

1 3. Song Y. Insight into the mode of action of 2, 4‐dichlorophenoxyacetic acid (2, 4‐D) as

2 an herbicide. J Integr Plant Biol 2014; 56: 106-13.

3 4. Ertug ND, Akbulut C, Abar M, et al. The Histopathological Effects of 2, 4-

4 Dichlorophenoxyacetic Acid on Intestine Tissue of Zebrafish (Danio rerio). Elixir

5 Pollution 2014; 74: 27021-27024.

6 5. Brasil. Agência Nacional De Vigilância Sanitária. Monografia D27 - 2,4-D.

7 http://portal.anvisa.gov.br/registros-e-autorizacoes/agrotoxicos (2018, accessed 16

8 September 2018).

9 6. United States of America. United States Environmental Protection Agency. 2,4-D.

10 https://www.epa.gov/ingredients-used-pesticide-products/24-d (2019, accessed 26

11 March 2018)

12 7. Kim CS, Keizer RF and Pritchard JB. 2, 4-Dichlorophenoxyacetic acid intoxication

13 increases its accumulation within the brain. Brain Res 1988; 440: 216-26.

14 8. Bortolozzi A, De Duffard AM, Dajas F, et al. Intracerebral administration of 2, 4-

15 diclorophenoxyacetic acid induces behavioral and neurochemical alterations in the rat

16 brain. Neurotoxicology. 2001; 22: 221-32.

17 9. De Moro GM, Duffard R and De Duffard AM. Neurotoxicity of 2, 4-

18 dichlorophenoxyacetic butyl ester in chick embryos. Neurochem Res 1993; 18: 353-9.

19 10. Oliveira GH, Palermo‐Neto J. Effects of 2, 4‐dichlorophenoxyacetic acid (2, 4‐D) on

20 open‐field behaviour and neurochemical parameters of rats. Pharmacol Toxicol 1993; 73:

21 79-85.

22 11. Stürtz N, Deis RP, Jahn GA, et al. Effect of 2, 4-dichlorophenoxyacetic acid on rat

23 maternal behavior. Toxicology 2008; 247: 73-9. 13

1 12. Duffard R, Garcia G, Rosso S, et al. Central nervous system myelin deficit in rats

2 exposed to 2, 4-dichlorophenoxyacetic acid throughout lactation. Neurotoxicol Teratol

3 1996; 18: 691-6.

4 13. Rosso SB, Di Paolo OA, de Duffard AM, et al. Effects of 2, 4-dichlorophenoxyacetic

5 acid on central nervous system of developmental rats. Associated changes in ganglioside

6 pattern. Brain Res 1997; 769: 163-7.

7 14. Mattsson JL, Johnson KA and Albee RR. Lack of neuropathologic consequences of

8 repeated dermal exposure to 2, 4-dichlorophenoxyacetic acid in rats. Toxicol Sci 1986; 6:

9 175-81.

10 15. Elo HA and Ylitalo P. Distribution of 2-methyl-4-chlorophenoxyacetic acid and 2, 4-

11 dichlorophenoxyacetic acid in male rats: evidence for the involvement of the central

12 nervous system in their toxicity. Toxicol Appl Pharmacol 1979; 51: 439-46.

13 16. Ton C, Lin Y and Willett C. Zebrafish as a model for developmental neurotoxicity

14 testing. Birth Defects Res A Clin Mol Teratol 2006; 76: 553-67.

25 14

1 Toxicity of Pesticides in Lizards

2 Summary

3 Many threats exist to reptile populations, environmental pollutants being one of them.

4 Lizards and other reptiles are usually not taken into consideration in environmental risk

5 assessments, with the use of surrogate species for their estimates. Unfortunately, not all

6 pesticides have the same effects in the reptile species and on these surrogates, birds and

7 mammals, some being more toxic in lizards. This difference brings the need to evaluate

8 their toxicity in lizards to safeguard its protection. Studies in the last decades involving

9 contaminants’ toxicity in lizard species have increased, thus we proposed to gather these

10 information in this comprehensive review. Through searches in data bases about toxicity

11 of pesticides in lizards, sixteen scientific papers were found. Most studies investigated

12 locomotor performance, histopathology, oxidative stress, neurotoxicology and genetic

13 damage from diverse pesticides with different modes of action. Progress has been made

14 to acquire data on lizard ecotoxicology and more research is needed to cover more

15 variables, such as studies in the embryologic stage and different pesticides.

17 Keywords

18 Reptile, histopathology, oxidative stress, neurotoxicity, locomotor performance.

20 Introduction

21 Reptile populations are declining and their main threats are environmental

22 pollutants, global climate changes, habitat loss and degradation, invasive species and

23 disease/parasitism. Despite its significance, very little is understood from pollutant effects

24 in reptiles and the group has been highly overlooked in ecotoxicological investigations. 15

1 A paucity of data regarding exposure and effects of environmental contaminants on

2 reptiles present a challenge for assessing ecological risks to these species.1-3

3 Reptiles are usually not considered in environmental risk assessments under the

4 assumption that birds and mammals estimates would be good and safe surrogates for

5 them. Nevertheless, some pesticides are more toxic to the lizards than birds and mammals.

6 Due to reptile unique physiological and biological features, predicting the effects of

7 environmental contaminants on reptiles with toxicity parameters established to other

8 vertebrates may likely to be ineffective. More toxicological data is needed to determine

9 which pesticides provide a reasonable surrogate for reptiles.3,4-6

10 Lizards must be included in any realistic study on environmental toxicants, as

11 they comprise a large percentage of reptiles and are an important component in many

12 terrestrial and aquatic ecosystems, from which one in five lizard species is threatened

13 with extinction.4,7 Lizards are easily exposed to pesticides in several ways, including

14 ingestion of contaminated food, dermal exposure, inhalation, maternal transfer to

15 eggs/young and absorption by eggs of contaminants from surrounding environments.

16 Thus, many species are vulnerable to the adverse effects of pollutants, even though they

17 are not the target species, and these threats to their diversity need more attention from the

18 scientific community.8

19 Fortunately, in the last decades there has been an increase in ecotoxicological

20 studies involving lizards. We aimed to assess data concerning the toxicology of diverse

21 pesticides on lizard species, gathering here the main effects reported.

22 To this purpose, subjects and titles/abstracts containing the keywords “pesticide”

23 and “reptile” or “lizard” were searched. Two scientific databases were used, PubMed and

24 Periódicos CAPES. Inclusion factors were studies evaluating the toxicity of pesticides in

25 lizard species. Exclusion factors were scientific papers that did not evaluate pesticides 16

1 effects; studies without lizards as the animal model; and studies that did not involve a

2 laboratory-controlled experiment.

4 Literature Review

5 We found 48 articles (excluding redundant titles) and 16 of them fit the inclusion

6 criteria, half of which were published in the last five years. Most works (16 - 81.25%)

7 studied the effects of insecticides, from which one also studied a fungicide and three

8 (18,75%) evaluated the effects of herbicides. Oral gavage was the preferred method of

9 administration of the pesticides (9 - 56,25%), followed by direct or indirect dermal

10 exposition (4 - 25%), the remaining studies (18,75%) each had different methods. One

11 study injected it and another administered through the diet. Only one work used embryos

12 to test the pesticide and it was applied directly in the eggs. Information about the species,

13 route and period of exposure, doses and parameters evaluated are exposed in table 1.

15 Herbicides

16 Herbicides are synthetic or natural compounds used to eliminate unwanted

17 plants. Non-selective herbicides kill all plants on contact, while selective herbicides

18 control specific weeds. Current herbicides have several modes of action, including

19 inhibition of specific enzymes that cause the stop of a metabolic via, leading to the

20 organisms’ death.9,10 Glyphosate is a widely used herbicide, and it inhibits the activity of

21 the enzyme enolpyruvylshikimate-3-phosphate synthase (EPSPS) from the shikimic acid

22 pathway, which converts simple carbohydrate into aromatic amino acids.10,11 Glufosinate

23 ammonium inhibits glutamine synthetase, an enzyme responsible for the production of

24 glutamine, leading to the buildup of ammonia and impairment of photorespiration and

25 photosynthesis.12,13 17

1 Additives in commercial formulations of pesticides may be more toxic than the

2 active ingredient.14,15 Aronzon et al.14 suggest that the toxicity of commercial products is

3 relevant to risk assessments regarding environmental protection and human health,

4 because only the data of active ingredients are considered for regulatory purposes and the

5 effect on non-target species may be undervalued.

6 Two glyphosate formulations, Agpro Glyphosate 360 and Yates Roundup

7 Weedkiller were tested in adult Oligosoma polychroma (New Zealand common skink)

8 for changes in mass and temperature. The animals were sprayed with a 4 cm layer of loose

9 straw above the lizard to simulate vegetation cover. Neither glyphosate formulation

10 impacted the mass significantly. Specimens could choose a cold or hot spot in the

11 terrarium and those treated with Yates Roundup Weedkiller selected significantly higher

12 temperatures across three weeks after exposure, which was not observed in the Agpro

13 Glyphosate 360 group. This pesticide difference could be due to the different adjuvants

14 used in the formulations.15

15 Genotoxic effects of the herbicide glyphosate Roundup® has been evaluated in

16 neonates of Salvator merianae (Tegu lizard) after a single embryonic application at the

17 beginning of incubation using doses of 50, 100, 200, 400, 800 and 1600 μg/egg. The size

18 and growth of lizards at birth and after six months were not influenced by the herbicide.

19 Through the micronucleus test, nuclear abnormalities assay and comet assay, which are

20 biomarkers for genotoxic effects induced in erythrocytes, it was not observed any

21 teratogenic effects. The comet assay indicates early DNA damage which can later be

22 removed by DNA repair systems, while the other tests indicate harmful events. Only the

23 comet assay presented statistically significant differences between the groups, the effect

24 being worse in the 800 and 1600 μg/egg groups than in the 200 and 400 μg/egg groups.16 18

1 Glufosinate-ammonium (GLA) has a chiral center and a pair of chiral isomers.

2 Its herbicidal activity was ascribed to the L-Glufosinate-ammonium (L-GLA).13 Zhang et

3 al.13 hypothesized both isomers would have different effects due of their different

4 biological activity and bioaccumulation. Therefore, both enantiomers were tested in male

5 and female specimens of Eremias argus (Mongolian racerunners). A dose of 20 mg/kg

6 soil weight was mixed in the soil in which the experimental animals were kept for 60

7 days. GLA concentration in the brain was significantly higher, supporting the authors’

8 hypothesis that the isomers have different effects due to their difference in

9 bioaccumulation.

10 Neurotoxic effects were a possibility for GLA and L-GLA, since glutamate is also

11 a neurotransmitter and timidity and anxiety were observed in the lizards. Total number of

12 turning back, mean sprint velocity, body weight, brain weight and brain index were

13 reduced in all groups, especially males. Whereas no changes in length were observed. A

14 score to different neurotoxicity-related enzymes [Glutamine synthetase (GS),

15 Acetylcholinesterase (AChE), Na+/K+-ATPase], antioxidant system [Catalase (CAT),

16 Superoxide dismutase (SOD)], and Malondialdehyde (MDA) levels indicated that GLA

17 mainly acted on GS, AchE and CAT, while L-GLA pointed at AchE, Na+/K+-ATPase,

18 SOD and MDA levels. This study showed that both GLA and L-GLA could be neurotoxic

19 and cause oxidative stress, although the isomers have different toxic effects, possibly due

20 to different accumulation of the isomers in the lizard brain. It also reinforced the idea that

21 males may be more sensitive and vulnerable to exogenous contaminants.13 19

Table 1: Data from the articles analyzed in the review containing. AchE – Acetylcholinesterase; CAT – Catalase; GH – growth hormone; GHRH – growth hormone releasing hormone; GS – Glutamine synthetase; GST – glutathione-S-transferase; LDH – Lactate dehydrogenase; MDA – Malondialdehyde; SOD - Superoxide dismutase; SST. Reference Type of Pesticide Species Number Route of Period of Doses Parameters evaluated pesticide (n) of exposure exposure lizards

Carpenter et al., Herbicide Glyphosate Oligosoma 58 Sprayed in Once 144 mg/L Mass (once a week for 8 2016 polychrome subject water weeks); (covered with a thermoregulatory 4 cm layer of behavior (across 21 loose straw) days) Schaumburg et Herbicide Glyphosate Salvator 96 eggs Topical on egg Once 50, 100, Micronucleus test; al., 2016 merianae 200, 400, nuclear abnormalities 800, 1600 assay; comet assay (after μg/egg 6 and 12 months of life) Zhang et al., Herbicide Glufosinate- Eremias 90 Contaminated 60 days 20 mg/kg Locomotor perfomance 2019 ammonium; argus soil soil weight (after 14 days); brain L-glufosinate- accumulation of the ammonium herbicide; biochemical analysis (GS, AchE, CAT, SOD enzymes and MDA) Holem et al., Insecticide Malathion Sceloporus 50 Oral gavage Once 0.2, 2, 20, Locomotor performance 2006 occidentalis 200 mg/kg (24 h before dosing, 4, body 24, 120 and 312 h post- weight dose) Khan, 2005 Insecticide Cypermethrin; Calotes No data Injected Once 1µl Cholinesterase activity Malathion versicolor (after 24 h) 20

Holem et al., Insecticide Malathion Sceloporus 52 Oral gavage 81 days – 2.0, 20, Terrestrial and arboreal 2008 occidentalis every 27 100 mg/kg locomotor performance days body (24 h before dosing, 24, weight 288 and 576 h post- dose); growth and food consumption (before each feeding) DuRant et al., Insecticide Carbaryl Sceloporus 121 Oral gavage Once 2.5, 25, Terrestrial and arboreal 2007 occidentalis 250 mg/kg locomotor performance (24 h before dosing, 4, 24, and 96 h post-dose) Cakici and Insecticide Carbaryl Ophisops 64 Oral gavage Once 2.5, 25, Histopathology of the Akat, 2012 elegans 250 mg/kg digestive system (after 96 h) Cakici and Insecticide Carbaryl Ophisops No data Oral gavage Once 2.5, 25, Histopathology of testis Akat, 2012 elegans 250 mg/kg (after 96 h) Alexander et al., Insecticide Deltamethrin Meroles 24; 28 Sprayed on Once 17,5, 25 g Poisoning symptoms 2002 suborbitalis; subjects; (every 15 min after Pedioplanis sprayed on soil treatment for a day, then namaquensis every 4 h for 2 days) Talent, 2005 Insecticide Pyrethrin Anolis 140 Subjects were Once 300 mg/L Temperature (15, 20, 25, carolinensis dipped in the 30, 35, and 38ºC) solution influence on sensibility to pesticide (for 48 h) Chen et al., Insecticide Beta- Eremias 12 Oral gavage Once 20 mg/kg Biochemical analyses 2019 cypermethrin argus body (SOD, CAT, LDH, weight AChE enzymes and MDA) (after 96 h) 21

Chen et al., Insecticide Alpha- Eremias 144 Dietary 8 weeks 2, 20 Bioaccumulation of 2019 cypermethrin argus (contaminate mg/kg wet insecticide; biochemical mealworms) weight of analyses (SOD, CAT, mealworms GST enzymes and MDA); hormone levels; histopathology; reproductive output Chang et al., Insecticide Lambda- Eremias 81 Oral gavage 21 days – 10 mg/kg Bioaccumulation; 2018 cyhalothrin argus once a body thyroid gland lesions; week weight thyroid hormone levels; hypothalamus-pituitary- thyroid axis related gene expression Wang et al., Insecticide Dinotefuran; Eremias 48 Oral gavage 35 days – 20 mg/kg Distribution, hormone 2019 Thiamethoxam; argus twice a body concentrations (GH, Imidacloprid week weight GHRH and SST); histopathology Chang et al., Insecticide Flufenoxuron Eremias 18 Oral gavage 21 days – 50 mg/kg Thyroid hormone levels; 2017 argus once a thyroid gland lesions; week expression of hypothalamus-pituitary- thyroid axis related genes Chen et al., Fungicide Myclobutanil Eremias 12 Oral gavage Once 20 mg/kg Biochemical analyses 2019 argus body (SOD, CAT, LDH, weight AChE enzymes and MDA) (after 96 h)

1 Insecticides

2 Organophosphates and carbamates

3 Organophosphates and carbamates are pesticides that act via cholinesterase inhibition.

4 Cholinestarases are enzymes that cause the hydrolysis of acetylcholine, an excitatory

5 neurotransmitter that is abundant in the nervous system and present in neuromuscular junctions.

6 AChE inhibitors are either reversible or irreversible. Carbamates killing action is based on

7 reversible AChE inactivation and are considered safer than organophosphates insecticides,

8 which irreversibly inhibit AChE causing more severe cholinergic poisoning.17

9 Exposure to AChE inhibitory pesticides may result in a buildup of acetylcholine in

10 neuromuscular junctions and disruption of neural function, possibly affecting locomotor

11 performance and other activities such as food consumption, which could influence growth. The

12 typical symptoms of AChE inhibitory pesticides acute poisoning are agitation, muscle

13 weakness, muscle fasciculation, body/limb tremors, twitching, miosis, hypersalivation,

14 sweating. Severe poisonings may cause respiratory failure, unconsciousness, confusion,

15 convulsions and/or death.17,18

16 Malathion is an organophosphate insecticide that has been studied in the lizard species

17 Calotes versicolor (Oriental garden lizard) and Sceloporus occidentalis (Western fence

18 lizard).18-20 Khan19 measured the cholinesterase activity in the liver and kidney of adult

19 specimens of C. versicolor injected with 0.1% and 1% malathion and detected a decrease up to

20 30% and 65.09%, respectively, in its activity.

21 The terrestrial and arboreal locomotor performances of juvenile S. occidentalis were

22 evaluated after exposure to malathion. Doses of 0.2, 2, 20, 100 and 200 mg/kg body weight

23 were applied, the doses of 0.2 and 200 mg/kg were administered once while the others were

24 applied three times, weekly. Mass, length, growth and food consumption were not significantly

1 different from control. Mortality was similar among all groups, though the higher doses seemed

2 to have more deaths, but it was not statistically significant. 18,20

3 Clinical symptoms of poisoning were exhibited by 70% of lizards that received a dose

4 of 200 mg/kg malathion and 85% in the lizards that received three doses of 100 mg/kg (the

5 effects subsided within 24 h). Terrestrial sprint velocity was increased in the both groups,

6 although not significantly. The mean maximum arboreal velocity was reduced in the high dose

7 group after the second dose was administered and half of the lizards refused to transverse in the

8 arboreal setting, suggesting the lizards were less likely to engage in coordination-dependent

9 activities.18,20

10 Similar methods were used to study the terrestrial and arboreal sprint velocity of S.

11 occidentalis after administration of the carbamate carbaryl. Single doses of 2.5, 25 or 250 mg/g

12 were used in adult specimens. No deaths were observed, however 58% the lizards of the high

13 dose group exhibited clinical signs of its exposure, which persisted up to 48 h after exposure.

14 Carbaryl was found to have a stimulatory effect in lower doses (2.5 and 25 mg/g), but at the

15 highest dose the sprint speed was impaired. Endurance was also found to be reduced, as the

16 speed diminished over time, especially in the highest dose group. The medium and high dose

17 groups also exhibited symptoms of impairment in arboreal tests, as it was more challenging and

18 this data can be more relevant to the species, as it uses trees to capture preys and evade

19 predators.21

20 Another species, Ophisops elegans (Snake-eyed lizard), was used to evaluate the

21 histopathological effects of carbaryl on the digestive system and testes. Adult specimens were

22 administered 2.5, 25 or 250 mg/kg in a single oral dose. No deaths were observed, though in

23 the high dose groups the animals had slower movements.22,23 Carbaryl potentially affect germ

24 cell development and possibly have adverse effects on spermatogenesis and male fertility. It

25 induced seminiferous tubule degeneration, increase of diameters of tubules, the disarrangement 24

1 of spermatogenic cell lines, disrupted germ cell association, vacuolization, sloughing and

2 hemorrhage in a dose-related trend.22

3 The most important histological defects in the digestive system were observed in the

4 stomach. Hemorrhage in the esophagus and vacuolization of gastric gland cells were the

5 alterations observed in the low dose group. Hemorrhage intensified in the esophagus and

6 appeared in the large intestine, degeneration in the epithelial layer was observed in the stomach

7 and small intestine in the medium dose. At high dose, epithelial cells were scattered and gastric

8 glands disappeared in the stomach, in the small intestine, collapse of villi and hemorrhage were

9 prominent an in the large intestine, scattered secretory granules of goblet cells were observed.23

10 Thus, carbaryl has the potential to disrupt both male fertility and the digestive system of

11 lizards.22,23

13 Pyrethroids

14 Pyrethroids are synthetic insecticides derived from natural pyrethrins that target

15 voltage-dependent sodium channels, which are present in axons of neurons leading nerve

16 impulses through the organism. Closure of such channels is prevented by pyrethroids, keeping

17 the axonal membrane depolarized, which leads to repetitive movement, paralysis and ultimately

18 death.24,25

19 Alexander et al.26 tested the effect of deltamethrin diluted at 17,5 g (the recommended

20 dose at the time) and 25 g in two lizard species, Meroles suborbitalis (Spotted sand lizard) and

21 Pedioplanis namaquensis (Namaqua sand lizard). Specimens were contaminated either directly

22 via spray or indirectly via sprayed soil. Within an hour of treatment symptoms of poisoning

23 were observed a loss of coordination, loss of righting response, sensitivity to bright light and

24 muscle spasms and panic. Animals with indirect contact took twice as long to manifest

25 symptoms. Though the lizards appeared to recover by the next day, they all died within two 25

1 months of treatments. The authors recommend cautions when spraying the pesticide to try and

2 diminish the effects it has on non-target lizard species.26

3 The toxicity of a natural pyrethrin insecticide was tested in adult Anolis carolinensis

4 (Green anole lizards) through percutaneous exposition (18.7, 37.5, 75, 150 and 300 mg/L) in

5 different temperatures (15, 20, 25, 30, 35, and 38ºC). The animals were dipped in the solution,

6 except the head, for two seconds. Mortality of lizards maintained at 15 and 20ºC and pyrethrin

7 concentration levels of 75, 150 and 300 mg/L were significantly higher than of lizards

8 maintained at 35 and 38ºC and exposed to lower doses. At 35ºC, no mortality occurred when

9 lizards were exposed to the 75 mg/L of pyrethrin mixture; however, at this same concentration,

10 70% of the lizards maintained at 20ºC died. These results suggest that temperature is a factor

11 to be considered when evaluating the toxicity of pesticides.27

12 Cholinesterase activity in the liver and kidney of C. versicolor was evaluated under

13 the effect of 0.1% and 1% cypermethrin, administered by injection. Cypermethrin caused up to

14 35 and 54% decrease in the cholinesterase activity.19

15 Beta-cypermethrin was tested in E. argus to check oxidative stress and test whether

16 saliva is a good diagnostic tool for oxidative stress in lizards. A single dose of 20 mg/kg body

17 weight was administered through oral gavage. Antioxidant enzymes (SOD and CAT), Lactate

18 dehydrogenase (LDH), AChE and MDA were measured from serum, saliva, brain, liver, kidney

19 and testes. The tested biomarkers increased or decreased in different manners in different

20 tissues. Saliva was more correlated with kidney and gonad and LDH was more relevant to

21 predict oxidative stress and could be a testing manner for toxicity that did not evolve the death

22 of the animal.28

23 Reproductive toxicity was tested in E. argus after treated food ingestion of alpha-

24 cypermethrin (ACP). Mealworms were treated with 2 and 20 mg/kg of its wet weight and fed

25 to the lizards for eight weeks. Antioxidant enzyme levels, SOD, CAT and glutathione-S- 26

1 transferase (GST), MDA levels, hormone levels from serum, gonads, histopathology and

2 reproductive output was evaluated in adult male and female. Food consumption in both groups

3 were decreased and decreases in body mass index and mortality was observed in a dose-

4 dependent trend, having a stronger effect in females. Significant variations in GST and CAT

5 activities and MDA levels in gonads, suggest that lizards were under oxidative stress.29

6 No histopathological changes were observed in ovaries, although testis presented

7 seminiferous tubule damage, germ cells sloughing and degeneration, and giant cells. ACP

8 exposure also increased testosterone levels in males and reduced egg production of females.

9 Females were in the breeding phase and may have been more susceptive to the pesticide. These

10 negative effects highlight that ACP dietary exposure is a potential threat to lizards’

11 reproduction.19

12 Bioaccumulation, thyroid gland lesions, thyroid hormone levels, and gene expression

13 related to the hypothalamus-pituitary-thyroid axis (HTP) was evaluated after administration of

14 two lambda-cyhalothrin (LCT) enantiomers in juvenile male E. argus at three oral doses of 10

15 mg/kg body weight, for three weeks. Samples from plasma, feces, thyroid, liver and brain were

16 analyzed. The body and liver weight and were lower in the (+)-LCT enantiomer and it was more

17 concentrated in the feces than the (-)-LCT. HPT axis-related genes expression were altered in

18 both enantiomers.30

19 The thyroid gland treated with the (+)-LCT enantiomer showed an irregular shape of

20 the follicles and there were clear signs of reabsorption in the follicle lumen. In the (-)-LCT

21 enantiomer exposure group, the follicular area was enlarged and the number of follicles was

22 decreased and the colloids were also decreased and no reabsorbing vacuoles were observed.

23 Enantiomer (-)-LCT appeared to accumulate higher levels in the liver, however, its toxic effects

24 on lizard growth, T3 level and thyroid activity were relatively lower than those of (+)-LCT.

25 Suggesting that (-) enantiomer caused less disruption on lizard thyroid than (+) enantiomer.30 27

2 Neonicotinoids

3 Neonicotinoids are systemic pesticides with selective toxicity for insects that are

4 agonists with irreversible binding at nicotinic acetylcholine receptors (nAChRs), which present

5 in the nervous system and are activated by acetylcholine. Contrary to acetylcholine,

6 acetylcholinesterase does not act on neonicotinoids, leading to their prolonged action on the

7 nAChRs which may cause paralysis and death.31-33

8 Distribution, metabolism and hepatotoxicity of neonicotinoids dinotefuran (DIN),

9 thiamethoxam (TMX) and imidacloprid (IMI) in E. argus was assessed at a dose of 20 mg/kg

10 body weight through oral gavage, twice a week for 35 days. The accumulation of neonicotinoids

11 increases the risk to lizards. Residual concentration of DIN was the highest among the three

12 neonicotinoids selected, followed by TMX. IMI was most easily metabolized in lizards, and its

13 terminal metabolite CPA was the main form of existence in lizards. Results indicated that all

14 three neonicotinoids are easily excreted by excretion, and the order of accumulation in lizards

15 was DIN > TMX > IMI. Bodyweight decrease during the experiment, being significantly

16 different only in the DIN group by the end of the experiment. Significant decrease in the number

17 of nuclei was observed in the liver tissues of all groups. The number of hepatocytes in the DIN,

18 TMX and IMI exposure groups decreased by 42 ± 11%, 30 ± 5% and 56 ± 8%, respectively.

19 Mild fibrosis occurred in all exposure groups, which was a precursor to cirrhosis. Accumulation

20 of cells occurred in the DIN and IMI exposure groups.34

21 In the continuous exposure of 35 days, exposure to DIN caused a decrease in plasma

22 GH concentration, down-regulation of ghr, igf1 and igfbp2 gene expression, accompanied by

23 oxidative stress damage in the liver, showing significant growth inhibition. Although IMI

24 caused severe liver oxidative stress damage, the effect of IMI on the GH/IGF pathway was not

25 obvious. Compared to DIN and IMI, TMX was the least toxic to lizards. In general, 28

1 neonicotinoids can significantly damage the liver of lizards and cause growth inhibition, being

2 worthy of attention in the conservation of farmland lizards. Although concentrations of

3 neonicotinoids in environmental soils usually do not reach the laboratory exposure

4 concentration, lizards are exposed to a high concentration of pesticides during the pesticide

5 application season. 34

7 Benzoylureas

8 The hypothalamus-pituitary-thyroid axis is important to the development of lizards

9 and the toxic effect of the insecticide flufenoxuron was evaluated on this axis through plasma

10 thyroid hormone levels, thyroid gland histopathology and expression profiles of thyroid

11 hormone receptors, deiodinases, and transthyretin. E. argus was tested in the two phases of skin

12 shedding, resting and proliferating phase, adult male and female were used. Three doses of 50

13 mg/kg were used, administered once a week via oral gavage.35

14 The results indicated that the sensibility of the specimens of E. argus to the pesticide

15 varied according to the stages. The proliferation phase suggested down-regulation on the T4

16 levels, whereas the T4 level was significantly increased at the resting phase after exposure.

17 There was more serious damage to the thyroid gland in the proliferation phase. The expression

18 of HPT axis-related genes correlates with the gender and tissue. It was tested in the liver, gonad,

19 brain, kidney, skin and thyroid gland, but there was a high expression only in the kidney, brain

20 and liver. In the female liver, the expression of tra, trb, dio1, dio2 and ttr genes was more

21 seriously affected at the proliferation phase than that at the resting stage after treatment and in

22 the brain, it was similar between both phases. In summary, flufenoxuron affect the thyroid

23 endocrine system of lizards, having different effects in females and males.35

25 29

1 Fungicides

2 Myclobutanil, a triazole fungicide, was evaluated in adult E argus. A single dose of 20

3 mg/kg body weight through oral gavage was used and SOD, CAT, LDH and AChE enzymes,

4 as well as MDA, were measured from serum, saliva, brain, liver, kidney and testes for oxidative

5 stress. SOD activities were failed to be evaluated in serum and saliva. Myclobutanil did not

6 affect SOD activities. CAT activities increased in kidney and decreased in brain and gonad.

7 MDA levels decreased in gonad, serum and saliva, which indicates alteration in cell

8 metabolism. LDH activity was increased in the brain and kidney, indicating cellular damage

9 and anaerobic metabolism, while in the gonads, serum and saliva it decreased, suggesting

10 reduced metabolism. Activity of AChE in the kidney and gonad decreased, while the activity

11 in saliva increased, indicating different metabolism between different tissues and organs.

12 Oxidative stress was less severe than beta-cypermethrin, tested in the same experiment.28

14 Conclusion

15 Lizards can be contaminated with pesticides through several routes, mainly oral or

16 dermal exposure, however, most works preferred oral administration, thus lacking data about

17 dermal exposure. Younger animals are more sensitive to external pollutants and should get more

18 attention in research, as eggs can be easily contaminated and this review showed that only one

19 report has been published which investigates neonates exposed in ovo.

20 Some pesticides are neurotoxic, targeting enzymes or receptors form the nervous

21 system, what reflects in behavior. It has been shown that locomotor velocity and even

22 impairment of more coordinated movements may be a result of pesticide contamination.

23 Oxidative stress assessment is also a common tool being used to assess toxicology of pesticides,

24 as gene expression and bioaccumulation. 30

1 Pesticide effects cannot be generalized from a single group, therefore lizard data needs

2 to be considered in toxicology assessments, meaning more research needs to be done to acquire

3 these data, as only 13 pesticides have been studied in lizards so far.

5 Funding

6 The author(s) disclosed receipt of the following financial support for the research,

7 authorship, and/or publication of this article: This work was supported by the Fundação de

8 Amparo à Pesquisa do Estado de Goiás (FAPEG) [scholarship number 201810267000852].

10 Declaration of Conflicting Interests

11 The author(s) declared no potential conflicts of interest with respect to the research,

12 authorship, and/or publication of this article.

25 31

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25 35

1 MACRO- AND MICROSCOPIC ANATOMY OF THE BRAIN OF Tropidurus

2 torquatus

4 Running title: NEUROANATOMY OF Tropidurus torquatus

6 SUMMARY

7 Amniotes gained reproductive independence of water and reptiles have a key role in 8 understanding this revolutionary change. It allowed many adaptations to arise, from locomotor 9 patterns to behaviors and the nervous system was adapted, accommodating new habits. We 10 described the macroscopic anatomy and cytoarchitecture of the brain of Tropidurus torquatus, 11 an abundant lizard in South America. Fifteen specimens were captured, euthanized and their 12 brains were dissected. From these, eight brains were included in paraffin, stained in 13 hematoxylin-eosin and sectioned at transversal, sagittal and frontal planes. Their brain is 14 composed of forebrain (telencephalon and diencephalon), midbrain (tectum and tegmentum) 15 and hindbrain (medulla oblongata and cerebellum). The main and accessory olfactory bulbs are 16 the most rostral structure of the brain and are composed of six layers and connected to the brain 17 through the olfactory peduncles. Brain hemispheres are composed of pallium and subpallium. 18 Medial, dorsomedial, lateral and dorsal cortices, as well as the dorsal ventricular ridge, are part 19 of the pallium. Striatum, pallidum and septum compose the subpallium. The diencephalon is 20 composed of thalamus, epithalamus and hypothalamus, with structures of the pineal complex 21 projecting from the epithalamus. The midbrain has a ventral tegmentum and a dorsal tectum 22 composed of torus semicircularis and a 14 layered optic tectum. The midbrain tegmentum is 23 continuous with the medulla oblongata tegmentum. Their cerebellum arises from the medulla 24 oblongata, forming a three-layered plate like structure. In general, the brain of Tropidurus 25 torquatus resembles those of other lizards, with its own adaptations.

26 27 KEYWORDS: Histology, Lizard, Neuroanatomy, Morphology, Reptile.

28 Number of figures: 10

29 Number of tables: 0

30 36

1 INTRODUCTION

2 Reptiles’ embryos present amniotic membrane, an adaptation that arose with water

3 independence in reproduction. But this feature is not the only one associated with the transition

4 from water to land that happened to tetrapods. Adaptations to the head to accommodate the

5 differences in feeding, hearing and other behaviors and specialized limbs to support the body

6 off the ground were some of the changes that happened with equivalent changes in the nervous

7 system (Shedlock & Edwards, 2009).

8 Survival and reproduction of organisms in the environment in which they live is

9 important to a species’ success and the nervous system coordinate activities towards these goals.

10 Mammalian and reptile brain share ancestry and a number of functional attributes and since the

11 reptile brain is simpler, it may provide invaluable help in deciphering modern neuroscience

12 questions (Naumann et al. 2015). Anatomically, the Central Neural System (CNS) is subdivided

13 into the brain and spinal cord. The brain has three major divisions in development,

14 prosencephalon or forebrain, which gives rise to the diencephalon and telencephalon.;

15 mesencephalon or midbrain; and rhombencephalon or hindbrain, which gives rise to medulla

16 oblongata and cerebellum (Senn, 1979; Wyneken, 2007).

17 Pallium and subpallium comprises the telencephalon. The pallium has a cortical

18 region, composed of medial, dorsomedial, dorsal and lateral components and a large subcortical

19 region, the dorsal ventricular ridge. Striatopallidal complexes and septum are part of the

20 subpallium (Ebbesson & Voneida, 1969; Wright et al., 2009). The main and accessory olfactory

21 bulbs compose the most rostral part of the telencephalon (Llahi & García-Verdugo, 1989)

22 Epithalamus, dorsal and ventral thalamus and hypothalamus comprise the

23 diencephalon and an area called pretectum marks the transition between the diencephalon and

24 midbrain. (Butler & Northcutt, 1973; Cruce, 1974). The epithalamus is the most dorsal part of 37

1 the diencephalon while the hypothalamus is the most ventral part of the diencephalon

2 (Engbretson, Reiner & Brecha, 1981; Bruce & Neary, 1995).

3 Tectum and tegmentum are the main parts of the midbrain. A large optic tectum and a

4 torus semicircularis comprise the tectum. (Huber & Crosby, 1933). The tegmentum itself

5 contains several continuous structures in both midbrain and hindbrain. A transitional area called

6 the isthmus separates the midbrain tegmentum from the hindbrain (Díaz, Yanes, Trujillo &

7 Puelles, 2000).

8 Two major divisions comprise the hindbrain, medulla oblongata and cerebellum. The

9 ventral part of the medulla oblongata is composed of tegmentum which contains the reticular

10 formation. Additionally, there is major passage of axons between the spinal cord, midbrain and

11 forebrain in the medulla oblongata (Hoogland, 1982; Newman & Cruce, 1982). A curved plate

12 is the characteristic shape of lizards’ cerebellum (Ten Donkelar & Bangma, 1992).

13 Lizards have been identified as model organisms for various types of studies due to

14 their easy observation, capture and handling. One of these species, Tropidurus torquatus (Wied,

15 1820) has been explored in several studies, including about temperature (Kiefer, Van Sluys, &

16 Rocha, 2007), diet (Siqueira, Kiefer, Sluys, & Rocha, 2011) reproduction (Ortiz, Boretto,

17 Piantoni, Álvarez & Ibargüengoytía, 2014) and embryonic development (Py-Daniel et al.,

18 2017). Its specimens are extremely abundant, being distributed from Brazil to Argentina, with

19 a seasonal reproductive cycle in the rainy season. The species is diurnal and preferentially

20 inhabit open environments, feeding on invertebrates, flowers and fruits (Rodrigues, 1987;

21 Kiefer, Van Sluys, & Rocha, 2005; Ortiz et al., 2014).

22 We aimed to describe the macroscopic anatomic and cytoarchitecture of the brain of

23 T. torquatus, highlighting its main regions and structures.

25 38

1 MATERIAL AND METHODS

2 Macroscopic analysis

3 Fifteen juvenile and adult specimens of T. torquatus were used. Animals of both sexes

4 were collected with a noose at the Universidade Federal de Goiás - Regional Jataí. They were

5 euthanized with an intraperitoneal lethal dose of bupivacaine hydrochloride (100 mg/kg) and

6 dissected with the help of tweezers, scissors and a dissecting microscope (Hoops, 2015). The

7 skin of the head was removed, followed by removal of eyes and muscles around the brain. Next

8 the bones protecting the brain and the dura mater were extracted, exposing the structure. Finally,

9 the brain was carefully removed from the remaining brain case and severed at the spinal cord.

10 The external morphology and topography of the brain were described and documented with a

11 dissecting microscope (Leica ICC50 HD®). This research is supported through the capture

12 permit (SISBIO 61909-1) and ethics committee (UFG/REJ - CEUA 013/18).

14 Histologic analysis

15 Eight brains were stained with hematoxylin-eosin (HE). The material was dehydrated

16 in a series of alcohol 100% (5 baths, 50 min each), followed by submersion in xylol (2 baths /

17 50 min each) and paraffin inclusion (3 baths / 50 min each). The brains were included in three

18 sectioning planes (sagittal, frontal and transversal) and then sectioned with microtome at 5 μm.

19 For the staining protocol the paraffin was melted in an incubator (1 hour) and the remaining

20 paraffin was removed with xylene (2 baths, 20 min each). Then it was passed through a series

21 of alcohol solutions (100, 90, 70 and 50%, 5 min each) and bathed in distilled water (10 min)

22 before the hematoxylin staining (5 min). Next it was submitted to running tap water (10 min)

23 and counter-stained with eosin (4 min). It was dehydrated through alcohol 70% (5 quick

24 immersions), alcohol 80% (1 min), alcohol 90% (2 min), alcohol 100% (5 min) and finally

25 submersed in xylene (2 baths, 5 min each) and mounted with entellan. 39

1 Histological images were analyzed and photographed using an optic microscope

2 (LEICA DM750®) with an embedded camera (LEICA ICC50 HD®), with objective lens 4x

3 (0.10), 10x (0.22), 20x (0.40) and 40x (0.65). After capture, macro- and microscopic images

4 were processed using the software Affinity Photo® v1.5.2.69 to merge continuous pictures of

5 the same region, Adobe Photoshop CS6® v13.0 for background processing and adjusting tone

6 and light and CorelDRAW X7® v17.1.0.572 to assemble images and point structures.

8 RESULTS AND DISCUSSION

9 The brain of T. torquatus extends from the medulla oblongata to the olfactory bulbs.

10 It is limited caudally from the spinal cord by the foramen magnum and rostrally by olfactory

11 capsules (Figure 1A). The olfactory bulbs are located rostrally to the eyes, being connected to

12 the brain by the olfactory peduncles. It is composed of forebrain (telencephalon and

13 diencephalon), midbrain (tectum and tegmentum) and hindbrain (medulla oblongata and

14 cerebellum) (Figures 1B, 2-5). A system of ventricles is associated with every region of the

15 brain, and its ependyma form an inner-most layer in all regions containing ventricles (Figures

16 6, 7). The choroid plexus is located inside the ventricles (Figure 7D). Pia and dura mater are

17 meninges that cover the brain and are closely associated with the ventricular system.

19 Olfactory bulbs

20 Olfactory nerves enter the ventromedial surfaces of the olfactory bulbs of T. torquatus,

21 coming from the nasal capsule and vomeronasal organ, respectively, which is a lizard feature.

22 In general, the olfactory bulbs are described as small and oval or pear-shaped, as in Anolis

23 garmani, Anolis grahami, Anolis lineatopus. Chameleon Vulgaris, Tupinambis teguixin (=

24 Tupinambis nigropunctatus) and Salvator merianae (= Tupinambis teguixin) (Shanklin, 1930; 40

1 Curwen, 1937; Armstrong, Gamble, & Goldby, 1953; Cruce, 1974; Reis, 2017) and in Iguana

2 iguana iguana, they are more triangular-shaped (Northcutt, 1967).

3 Main (rostral) and accessory (caudomedial) olfactory bulbs comprise the olfactory

4 bulbs (as in T. torquatus, A. garmani, A. grahami, A. lineatopus, Gekko gecko, I. iguana iguana

5 and Podarcis hispanica) (Armstrong et al., 1953; Northcutt, 1967; Smeets, Hoogland &

6 Lohman, 1986; Llahi & García-Verdugo, 1989). The accessory olfactory bulbs in G. gecko are

7 thinner and more distinct from the main bulbs than in T. torquatus, A. garmani, A. grahami, A.

8 lineatopus, Gekko gecko, I. iguana iguana and Podarcis hispanica (Smeets et al., 1986).

9 In lizards, the olfactory fibers are directed to the brain hemispheres through thin, long

10 and cylindrical olfactory peduncles, which enter the brain hemispheres near their rostral ends,

11 with some of its fibers being directed to the olfactory tubercles, as in T. torquatus, A. garmani,

12 A. grahami, A. lineatopus, C. Vulgaris, G. gecko, I. iguana iguana, P. hispanica and T. teguixin

13 (Shanklin, 1930; Curwen, 1937; Armstrong et al., 1953; Northcutt, 1967; Cruce, 1974; Smeets

14 et al., 1986; Llahi & García-Verdugo, 1989.

15 In T. torquatus the olfactory peduncles become thicker as they reach the cerebral

16 hemispheres (Figure 2), which is also observed in G. gecko, I. iguana iguana and T. teguixin

17 (Northcutt, 1967; Cruce, 1974; Smeets et al., 1986) and this structure appears to be significantly

18 thinner in A. garmani (Armstrong et al., 1953). Olfactory ventricles are present and are

19 connected to lateral ventricles in T. torquatus, G gecko and I. iguana iguana (Northcutt, 1967;

20 Smeets et al., 1986). While there is no information for most species, Shanklin (1930) noted the

21 lack of an olfactory ventricle in C. Vulgaris.

22 Microscopically in T. torquatus, both olfactory bulbs present 6 layers: olfactory nerve

23 fibers, glomerular, external plexiform, mitral, internal plexiform and granular layers. In the

24 main bulb the layers are located concentrically around the ventricle, while in the accessory one

25 it is mainly on the medial wall because the ventricle is located laterally (Figure 6). This is 41

1 similar to A. garmani, A. grahami, A. lineatopus and P. hispanica (Armstrong et al., 1953;

2 Llahi & García-Verdugo, 1989) and distinct from the findings in I. iguana iguana, for which

3 only three cell layers are described: external granular, mitral and internal granular layers

4 (Northcutt, 1967) and C. Vulgaris, describing that the cells appear granular cells (Shanklin,

5 1930). This divergence could be due to different staining methods, which may not have detailed

6 the cytoarchitecture of the olfactory bulb in these species.

8 Telencephalon

9 Macroscopically the brain hemispheres present cordiform shape (T. torquatus, A.

10 garmani, G. gecko, I. iguana iguana, P. hispanica, T. teguixin and S. merianae) and are visually

11 larger than the oval shaped optic tectum (Curwen, 1937; Armstrong et al., 1953; Northcutt,

12 1967; Lohman & Van Woerden-Verkley, 1976; Smeets et al., 1986; Llahi & García-Verdugo,

13 1989; Reis, 2017). Brain hemispheres are composed of a superficial pallium and a subpallium.

14 The pallium component is comprised of medial, dorsomedial, lateral and dorsal cortex, and the

15 ventrolateral dorsal ventricular ridge. The dorsomedial cortex is continuous with the medial

16 cortex, but its cells are larger and less densely packed.

17 Three layers are present in the cortices, external and internal plexiform layers with

18 scarce cells and an organized cell layer in between. The dorsal cortex presents a less organized

19 cell layer and it is partially overlapped by the dorsomedial and lateral cortices (Figure 7). This

20 disposition was observed in T. torquatus, A. garmani, A. grahami, A. lineatopus, C. Vulgaris,

21 G. gecko, I. iguana iguana, T. teguixin, S. merianae (Shanklin, 1930; Curwen, 1937; Armstrong

22 et al., 1953; Northcutt, 1967; Lohman & Mentink, 1972; Lohman & Van Woerden-Verkley,

23 1976; Bruce & Butler, 1984; Smeets et al., 1986; Butler, 1976). In G. gecko and T. teguixin the

24 internal plexiform layer was described as a subcortical layer of scattered cells and a fiber layer, 42

1 we did not notice any distinct fiber layer in T. torquatus (Lohman & Mentink, 1972; Smeets et

2 al., 1986)

3 A clear distinction between anterior and posterior parts of the dorsal ventricular ridge

4 is not visible with H.E. staining, and it presents as uniform distributed cells in T. torquatus

5 (Figures 3-5). Some parts of the ventricular ridge are covered by the cortices but lateral parts

6 of the ventricular ridge are covered by a layer of pia mater and ependyma, the pallial membrane

7 (Figure 3A), which was described in A. garmani, A. grahami and A. lineatopus (Armstrong et

8 al., 1953).

9 Subpallium is composed of septum, striatum and pallidum, from which the first is

10 located medially between medial cortex and striatum. The amygdaloid complex is also present

11 in the subpallium (G. Gecko), but it was not possible to distinguish it in T. torquatus (Smeets

12 et al., 1986). The lateral ventricle is associated with the telencephalon and is located between

13 the cortices and dorsal ventricular ridge (Figure 3). The hippocampal and anterior commissures

14 cross the hemispheres and were identified in T. torquatus (Figure 1C).

16 Diencephalon

17 In T. torquatus, C. Vulgaris, G. gecko and T. teguixin, four regions compose the

18 diencephalon: epithalamus, thalamus (dorsal and ventral), and hypothalamus (Shanklin, 1930;

19 Cruce, 1974; Bruce & Butler, 1984) The diencephalon is associated with it is the third ventricle

20 (Figures 3-5). It is almost completely covered by the hemispheres on the dorsal surface, only

21 part of the epithalamus being exposed (Figure 2A).

22 Most part of the epithalamus is composed of the habenula, from which some fibers

23 crosses at the habenula commissure. Structures of the pineal complex project from the

24 epithalamus, the pineal organ, dorsal sac, paraphysis and parietal eye (Figure 8) (Shanklin,

25 1930; Cruce, 1974; Smeets et al., 1986). The pineal organ is oval shaped in T. torquatus and 43

1 triangular shaped in S. merianae (Reis, 2017). The pretectum lies at the transition of the

2 diencephalon and the mesencephalon and could not be clearly distinct in T. torquatus Shanklin,

3 1930; Cruce, 1974; Smeets et al., 1986).

4 The thalamus is only visible in the sagittal section. In its dorsal part the posterior

5 commissure was identified in T. torquatus (Figures 1C, 3C). The largest part of the

6 diencephalon is the hypothalamus, part of which is visible on the ventral surface and the

7 protruding infundibulum is medially located in this region. Also, ventrally, thick optic nerves

8 intersect at the optic chiasm and enter the brain through optic tracts, surrounding the

9 infundibulum, where the hypophysis is connected to the brain (Figure 2). A supraoptical

10 commissure is present ventrally in the hypothalamus, caudal to the optic chiasm (Figure 1B)

11 (Shanklin, 1930; Cruce, 1974; Smeets et al., 1986).

13 Mesencephalon

14 Mesencephalic tectum and tegmentum comprise the mesencephalon. The tegmentum

15 is located ventrally, continuous with the hindbrain. Its tectum has an optic tectum and torus

16 semicircularis (Figures 3-5). Macroscopically, the optic tectum is oval shaped and noticeably

17 smaller than the cerebral hemispheres and partially covered by the cerebellum (also described

18 in S. merianae), while the torus semicircularis is a small median structure located caudoventral

19 to the optic tectum, completely covered by the cerebellum (Figures 1B, 2) (Reis, 2017).

20 The torus semicircularis is funnel shaped and it is larger in its medial part, which both

21 antimeres are partly fused at the midline. It thins out gradually as it extends laterally, this is

22 seen in T. torquatus, Gallotia galloti and T. teguixin (Browner & Rubinson 1977; Díaz et al.,

23 2000). A band of fibers crosses both parts of the tectum forming the tectal commissure (Figures

24 1C, 3C). The cerebral aqueduct passes through the midbrain toward the fourth ventricle (Figure

25 1B). 44

1 The optic tectum has 14 layers starting from the ventricle and can be organized into 6

2 strata in T, torquatus, I. iguana iguana and T. teguixin. (Figure 9) (Butler & Ebbesson, 1975;

3 Foster & Hall, 1975). This organization was first described by Ramón (1896 apud Huber &

4 Crosby, 1933): stratum fibrosum periventriculare [ependyma / epithelial zone (1); molecular

5 zone (2)], stratum griseum periventriculare [cellular zone (3); molecular zone (4); cellular zone

6 (5)], stratum album centrale [central fiber zone (6)], statum griseum centrale [central cellular

7 zone (7)], stratum fibrosum and griseum superficiale [cellular zone (8); molecular zone (9);

8 cellular zone (10); molecular zone (11); cellular and optic fiber zone (12); molecular zone (13)]

9 and stratum opticum [optic fiber zone (14)]. Layer 7 is thicker than the other cell layers and

10 layer 6 is the largest fiber layer. In T. torquatus, layer 14 is thicker rostrally and layers 7-11 are

11 sparser, with a less distinct organization than in I. iguana iguana, (Foster & Hall, 1975).

13 Hindbrain

14 Medulla oblongata and cerebellum compose the hindbrain. The ventral part of the

15 medulla oblongata is composed of tegmentum, which is continuous with the tegmentum of the

16 midbrain. One structure of the tegmentum is the reticular formation, distinct for its appearance

17 of loose fibers (Figure 4). The medulla oblongata is large lateral-laterally and it caudally tappers

18 toward its division with the spinal cord, which also appears larger rostrally. Laterally the

19 medulla oblongata has a curved appearance and several roots of nerves protrude from its

20 surface, both laterally and ventrally.

21 Dorsally in the medulla oblongata, part of the rhomboid fossa is visible, being covered

22 rostrally by the cerebellum, it is formed by the fourth ventricle and the choroid plexus lies over

23 it (Figure 2). The rhomboid fossa in A. garmani, G. gecko and, T. teguixin is longer (caudo-

24 caudally) than in T. torquatus, and present a more cylindrical shape, while in the latter it is

25 triangular shaped (Curwen, 1937; Armstrong et al., 1953; Smeets et al., 1986). It appers to be 45

1 more similar between I. iguana iguana, S. merianae and T. torquatus (Northcutt, 1967; Reis,

2 2017). It is oblique in Anniella nigra, mainly covered by nerve roots, with only a vertical slit

3 being visible in dorsal view (Larsell, 1926).

4 The cerebellum protrudes from the dorsorostral part of the medulla oblongata,

5 connected to it through the cerebellar peduncle (Figure 10B). It has a plate shape and it curves

6 rostrally (Figure 2), divided into two lateral flocculi (also observed in S. merianae) and

7 hemispheres with a median part and two lateral parts (Figure 10A) (Reis, 2017).

8 The curved plate shape of the cerebellum, which covers the optic tectum, is shared

9 among lizards, including A. garmani, I. iguana iguana, Phrynosoma douglasii, T. teguixin, S.

10 merianae, Sceloporus biseriatus and Sceloporus graciosus (Larsell, 1926; Curwen, 1937;

11 Armstrong et al., 1953; Northcutt, 1967; Cruce, 1974; Foster & Hall, 1975; Reis, 2017).

12 However, in the lizards G. gecko and P. hispanica, it doesn’t cover the optic tectum and it

13 appers to be slightly curved backwards in the former and smaller in the latter (Shanklin, 1930;

14 Smeets 1986).

15 In the lizard A. nigra, a legless lizard, the cerebellum is small, in size and compared to

16 the rest of the brain. It is almost hidden from view, lying in a depression formed by the midbrain,

17 medulla oblongata and nerve roots. The cerebellum of Gerrhonotus principis presents

18 interesting features. In dorsal view it presents a triangular shape, its median part has a tongue-

19 like structure projecting backwards following the contour of the rhomboid fossa. The lateral

20 parts are curved forward, similar to the cerebellum of other lizards mention above (Larsell,

21 1926).

22 Its cortex is formed by three layers: granular layer, Purkinje cell layer and molecular

23 layer. The most external and dorsocaudal layer is the granular layer, composed of small and

24 densely packed granule cells and larger Golgi cells. The molecular layer is the most

25 ventrorostral and inner layer, with the presence of many dendrites from the adjacent Purkinje 46

1 cells and axons from granule cells, and few basket and stellate cells. The Purkinje cell layer is

2 located between the other two layers, formed by a single line of cells (Figure 10C). These layers

3 and cells are very similar to the ones found in C. vulgaris, P. douglasii, S. biseriatus and S.

4 graciosus (Larsell, 1926; Shanklin, 1930).

5 In A. nigra, the layers are more similar to that of anurans, with the granular layer located

6 ventrocaudally and the molecular layer dorsorostrally. The Purkinje cell layer is also formed by

7 a single line of cells. The median part of the cerebellum of G. principis presents the granular

8 layer ventrocaudally, similar to A. nigra, while it bends rostrally as it extends laterally, like that

9 of most lizards (e.g. T. torquatus). The author proposed that this lateral part is what

10 predominated in other lizards with the common conformation to the cerebellum (Larsell, 1926).

12 ACKNOWLEDGEMENTS

13 This work was supported by the Fundação de Amparo à Pesquisa do Estado de Goiás

14 (FAPEG) [scholarship number 201810267000852]. We would like to thank Finep for the aid

15 under MCT/Finep/Ação Transversal - Novos Campi – 05/2016. We thank the Laboratories of

16 Human and Comparative Anatomy, Morphophysiology and Medical Research of the

17 Universidade Federal de Goiás.

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22 51

1 FIGURE LEGENDS

3 Figure 1: A – Topography of the brain of Tropidurus torquatus. B – Macroscopic anatomy of

4 the brain of Tropidurus torquatus. C – detail of picture B. A – dorsal view; B, C - medial view.

5 INDEX of structures: ac – anterior commissure; ce – cerebellum; di – diencephalon; ep –

6 epithalamus; hc – hippocampal commissure; hy – hypothalamus; II – optic nerve; iv – fourth

7 ventricle; ma – mesencephalic aqueduct; mb – midbrain; mc – medial cortex; mo – medulla

8 oblongata; ob – olfactory bulb; oc – optic chiasm; op – olfactory penducle; pc – posterior

9 commissure; sc – spinal cord; sep – septum; soc – supraoptical commissure; sp – subpallium;

10 tcm – tectal commissure; tg – tegmentum; ts – torus semicircularis. Scale Bar (A): 2 cm; Scale

11 Bar (B, C): 5 mm.

12 52

2 Figure 2: Macroscopic anatomy of the brain of Tropidurus torquatus. A – dorsal view; B –

3 ventral view; C – lateral view. INDEX of structures: aob – acessory olfactory bulb; ch –

4 cerebellar hemisphere; cp – cerebellar penducle; dc – dorsal cortex; dvr – dorsal ventricular

5 ridge; ep – epithalamus; fl – flocculus; hy – hypothalamus; I – olfactory nerve; II – optic nerve;

6 in – infundibulum; iv – fourth ventricle; lp – lateral part of cerebellar hemisphere; mo – medulla

7 oblongata; mob – main olfactory bulb; mp – median part of cerebelar hemisphere; oc – optic 53

1 chiasm; op – olfactory penducle; ot – optic tectum; otr – optic tractum; out – olfactory tubercle;

2 sc – spinal cord; tg – tegmentum; V – trigeminal nerve. Scale Bar: 5 mm.

5 Figure 3: Transversal sections and scheme of the brain of Tropidurus torquatus. HE staining.

6 INDEX of structures: ce – cerebellum; cp – cerebellar penducle; dc – dorsal cortex; di –

7 diencephalon; dmc – dorsal medial cortex; dvr – dorsal ventricular ridge; ep – epithalamus; hy

8 – hypothalamus; lc – lateral cortex; lv – lateral ventricle; mb – midbrain; mc – medial cortex;

9 mo – medulla oblongata; oc – optic chiasm; ot – optic tectum; pa – pallidum; pm – pallial

10 membrane; rf – reticular formation; sep – septum; sp – subpallium; st – striatum; ta – thalamus;

11 tg – tegmentum; ts – torus semicircularis;. Scale Bar: 1 mm. 54

2 Figure 4: Sagittal sections and scheme of the brain of Tropidurus torquatus. HE staining.

3 INDEX of structures: ce – cerebellum; dc – dorsal cortex; dmc – dorsal medial cortex; dvr –

4 dorsal ventricular ridge; hy – hypothalamus; lc – lateral cortex; mb – midbrain; mc – medial

5 cortex; mo – medulla oblongata; ot – optic tectum; rf – reticular formation; sep – septum; ta –

6 thalamus; tcm – tectal commissure. Scale Bar: 1 mm. 55

2 Figure 5: Frontal sections and scheme of the brain of Tropidurus torquatus. HE staining.

3 INDEX of structures: ce – cerebellum; cp – cerebellar penducle; dc – dorsal cortex; di –

4 diencephalon; dmc – dorsal medial cortex; dvr – dorsal ventricular ridge; ha – habenula; iii –

5 third ventricle; iv – fourth ventricle; lc – lateral cortex; lv – lateral ventricle; mb – midbrain;

6 mc – medial cortex; mo – medulla oblongata; ot – optic tectum; ov – optic ventricle; po – pineal

7 organ; rf – reticular formation; sep – septum; sp – subpallium; tg – tegmentum; ts – torus

8 semicircularis. Scale Bar: 1 mm. 56

2 Figure 6: Olfactory bulb of Tropidurus torquatus. Frontal sections, HE staining. INDEX of

3 structures: aob – acessory olfactory bulb; em – ependyma; epl – external plexiform layer; gll –

4 glomerular layer; grl – glanular layer; ipl – internal plexiform layer; mcl – mitral cell layer;

5 mob – main olfactory bulb; olv – olfactory ventricle; onl – olfactory nerve layer; op – olfactory

6 penducle. Scale Bar (A): 500 µm; Scale Bar (B): 100 µm.

9 57

2 Figure 7: Cortices of Tropidurus torquatus. Sagittal sections. A – dorsal cortex; B – medial

3 cortex; C – lateral córtex; D – dorsomedial cortex. INDEX of structures: chp – choroid plexus;

4 cl – cell layer; dc – dorsal cortex; em – ependyma; epl – external plexiform layer; ipl – internal

5 plexiform layer; lv – lateral ventricle. Scale Bar: 100 µm.

8 Figure 8: Epithalamus of Tropidurus torquatus and associated structures. A – dorsal view of

9 the brain; B – frontal section of pineal complex -; C – sagittal section of habenula. INDEX of

10 structures: bh – brain hemisphere; ce – cerebellum; dsa – dorsal sac; ha – habenula; hac – 58

1 habebular commissure; ot – optic tectum; par – paraphysis; pe – parietal eye; po – pineal organ.

2 Scale Bar (A): 5 mm; Scale Bar (B): 200 µm; Scale Bar (C): 100 µm.

5 Figure 9: Optic tectum of Tropidurus torquatus. Sagittal section. Numbers represents layers.

6 INDEX of structures: 1-14 – layers; sac – stratum album centrale; sfgs – stratum fibrosum and

7 griseum superficiale; sfp – stratum fibrosum periventriculare; sgc – statum griseum centrale;

8 sgp – stratum griseum periventriculare; so – stratum opticum; Scale Bar: 100 µm.

11 Figure 10: Cerebellum of Tropidurus torquatus. A – frontal section; B, C – sagittal sections.

12 Scale Bar (A): 500 µm; INDEX of structures: cp – cerebellar penducle; dms – dorsal median

13 sulcus; fl – flocculus; fs – floccular sulcus; grl – glanular layer; lp – lateral part of cerebellar 59

1 hemisphere; ml – molecular layer; mp – median part of cerebelar hemisphere; pcl – purkinje

2 cell layer. Scale Bar (B, C): 100 µm.

25 60

1 DEVELOPMENTAL NEUROTOXICOLOGY OF 2,4-D IN Tropidurus torquatus 2 3 ABSTRACT 4 2,4-D is a selective herbicide that acts as a synthetic auxin. It is usually eliminated through 5 urine in animals, but when the renal clearance rate is exceeded, adverse effects appear. Few 6 studies evaluate the effect of 2,4-D on embryonic development and no work has been done with 7 reptiles. We aimed to evaluate the effect of 2,4-D on the embryonic development of the brain 8 of Tropidurus torquatus. This species is abundant and by inhabiting crops it can naturally 9 contaminate itself with pesticides, making it a fitting animal model. Embryos were collected 10 post-oviposition and randomly assigned to two groups, control and 2,4-D. The 2,4-D group 11 substrate was contaminated shortly after collection with a dose corresponding to 1.5L/ha diluted 12 in water, and the control’s substrate was moistened with the same amount of water. Embryos 13 were collected on days 15, 30 and 60 after oviposition and their brains were submitted to 14 paraffin inclusion for hematoxylin-eosin staining and the volume of the optical tectum was be 15 estimated using stereology to compare both groups. A decrease in volume was observed in the 16 optic tectum of the embryos in the 2,4-D group on day 15, which suggests that at the 17 recommended dose there is a mild neurotoxic effect to this structure. No differences were found 18 on day 30, suggesting that the 2,4-D effect may be reversible, due to repair mechanisms in the 19 brain. The volume decrease might have been caused by decrease in cell and fibers density, 20 which has been an observed effect of 2,4-D in other species.

21 Key words: lizard, reptile, embryo, pesticide, stereology.

23 Acknowledgments: 24 This work was supported by the Fundação de Amparo à Pesquisa do Estado de Goiás 25 (FAPEG) [scholarship number 201810267000852]. We would like to thank Finep for the aid 26 under MCT/Finep/Ação Transversal - Novos Campi – 05/2016, the Laboratories of Human and 27 Comparative Anatomy, Morphophysiology and Medical Research of the Universidade Federal 28 de Goiás. 29 30 INTRODUCTION 31 2,4-D is a selective herbicide, first synthesized in 1941, which acts as a plant growth 32 regulating hormone. It maintains high levels of auxin, which causes excessive growth 33 stimulation until death (Aronzon et al. 2014; Song 2014; Ertug et al. 2014; Peterson et al. 2016). 61

1 There are several commercial formulations of this herbicide and the most commonly are amine 2 salts and esters, with dimethylamine being the most used (Ertug et al. 2014; Peterson et al. 3 2016). 4 In animals, this herbicide is distributed throughout the body, without accumulation in 5 any specific organ. It is eliminated in the urine without being metabolized unless it exceeds the 6 renal clearance rate (Loomis et al. 2015; USEPA 2016; Pannu et al. 2018; Sharma and Rajesh 7 2018), and its toxicity is dose-dependent (Pereira and Stabille 2017; Sharma and Rajesh 2018). 8 It is classified as extremely toxic (Class I) and is highly soluble in water and soil, being a 9 hazardous product for the environment and a risk to humans (Song 2014; ANVISA [2018]; Cai 10 et al. 2018). 11 Exposure of the organism to toxicants during development may cause problems, 12 especially in the brain. In adult individuals, the brain-blood barrier provides a well-controlled 13 stable environment for the brain. However, in embryos this barrier is immature or even absent, 14 which may render the developing brain more vulnerable to toxins and this may cause 15 intellectual and behavioral problems (Lanphear et al. 2005; Ek et al. 2012; Saunders et al. 2012). 16 There are many studies evaluating the effect of 2,4-D on wildlife and humans, but few 17 studies investigate its effects on embryonic development, only been studied in zebrafish 18 embryos, chick eggs, and rats’ mothers and neonates. The main results of the neurotoxicity of 19 2,4-D were alterations in behavioral patterns, disrupted locomotor patterns, changes in the 20 levels of lipids, proteins, DNA and neurotransmitters. Histopathological changes were found in 21 zebrafish, including increased apoptosis, disrupted motor neuron growth and reduction of axon 22 projections to the optic tectum (Kim et al. 1988; Duffard et al. 1996; Charles et al., 2001; De 23 Moro et al. 2003; Ton et al. 2006; Sturtz et al. 2008; Uyanıkgil et al. 2009; Arozon et al. 2011; 24 Diamante et al. 2014; Ertug et al. 2014; Li et al. 2017; USEPA 2016). 25 The brain of reptiles presents forebrain, midbrain (composed of optic tectum and torus 26 semicircularis) and hindbrain. Although the optic tectum is usually associated with the optical 27 system, it receives visual, somatosensory and auditory inputs, as well as non-sensory inputs 28 from other centers in the brain. Its pathways are related to tactile and motor orientation to 29 stimuli, capturing prey and escaping predators (Butler and Hodos 2005; Guirado and Dávila 30 2009). 31 There has been no research evaluating 2,4-D toxicity on lizards or any other species of 32 reptiles. In pesticides toxicological assessments in general, umbrella species are used to assess 33 risks to herpetofauna, but since not all organisms react the same way to pollutants, further 62

1 studies are needed to actually evaluate the effect of environmental pollutants on these species 2 (Campbell and Campbell 2002; Weir et al. 2010; Ortiz-Santaliestra et al. 2018). 3 Tropidurus is a very abundant genus of lizards distributed throughout South America, 4 comprising 30 species (Rodrigues 1987; Uetz and Hošek 2016). The species Tropidurus 5 torquatus (Wied 1820), known as amazon lava lizard, has one of the largest distributions of the 6 genus, being distributed from Brazil to Argentina, comprising three large population groups 7 (Rodrigues, 1987). The specimens are diurnal and preferentially inhabit open environments, 8 (Rodrigues, 1987), feeding on invertebrates, flowers and fruits. They adopt a hunting strategy, 9 but Teixeira and Giovanelli (1999) report other strategies for this species, such as area patrol 10 and prey attack in large quantities. 11 Regions of high agricultural productivity are inhabited by the species, being possibly 12 chosen for the availability of insects, an abundant source of food for the species. Their terrestrial 13 habitat makes the species T. torquatus susceptible to pesticide contamination through walking 14 and standing on contaminated soil and by ingesting food with the presence of chemical residues. 15 Similarly, their eggs may be contaminated upon contact with the soil that has contaminants or 16 through the contaminated maternal organism (Amaral 2012). Consequently, T. torquatus 17 presents itself as an animal model for toxicological studies on lizards. 18 Therefore, we aimed to analyze the neurotoxicity of the herbicide 2,4-D DMA® 806 BR 19 on the development of T. torquatus brain, using stereological technique for the volume analysis 20 of the optic tectum, reporting any changes found. 21 22 MATERIALS AND METHODS 23 Collection and maintenance of lizards 24 Thirty-six pregnant T. torquatus females were collected at the Federal University of 25 Goiás - Jataí. The animals were captured with a noose and maintained in terrariums until 26 oviposition, after which they were released. The collection took place during the reproductive 27 season (October 2018 to January 2019). 28 The animals were randomly divided into four terrariums to minimize stress during the 29 captive period. Each terrarium had a 10 cm layer of moistened vermiculite substrate. The 30 light/dark cycle (12/12 hours) was maintained with the use of 60W incandescent lamps, for 31 thermoregulation of individuals, as well as pieces of clay tiles for environmental enrichment. 32 The animals were fed with cockroaches and mealworms ad libitum and a source of clean water 33 was always present in each terrarium. 63

1 The research is supported by the collection permission (SISBIO 61909-1) and ethics 2 committee of the Federal University of Goiás - UFG / REJ (CEUA 013/18). 3 4 Egg collection, maintenance and herbicide exposure 5 Two groups were created, control (Con) and 2,4-D treatment (2,4-D). After 6 oviposition, individualized litters were removed from terrariums and eggs randomly allocated 7 into the groups, and maintained in separate incubators. The incubators were programmed to 8 maintain a constant temperature of 30ºC in the presence of water. 9 Eggs were placed in plastic cups containing 20 mg of vermiculite mixed with 40 ml of 10 distilled water. The herbicide was diluted according to the recommended dose for soybeans 11 (1.5L p.c./ha) for the area of the plastics cups, resulting in 0.075 ml of the commercial product 12 2,4-D DMA® 806 BR DMA diluted in 40 ml of distilled water and 60.25 mg/ml of 2,4-D 13 dimethylamine salt per cup. 14 Embryos were collected on days 15, 30 and 60 after oviposition, forming the groups 15 Con/D15, 2,4-D/D15, Con/D30, 2,4-D/D30, Con/D60 and 2,4-D/D60. There were 32 viable 16 embryos collected in day 15, 16 being for control and 16 for 2,4-D treatment. For day 30 there 17 were 26 viable embryos, being 9 for control and 17 for 2,4-D treatment. For day 60 there were 18 only three viable embryos, on the 2,4-D group. The embryos were removed from their eggs and 19 quickly anesthetized by five drops of bupivacaine hydrochloride, euthanized by five drops of 20 dose of potassium chloride, fixed in 2% Karnovsky solution and posteriorly preserved in 70% 21 alcohol. The specimens were photographed with a dissecting microscope (LEICA ICC50 HD®) 22 and then classified according to the developmental stages determined by Py-Daniel et al. 23 (2017). The images were processed using Adobe Photoshop CS6® v13.0 and CorelDRAW X7® 24 v17.1.0.572. 25 26 Histology 27 One embryo of each stage and group was kept unprocessed, with the remaining having 28 their heads removed, embedded in paraffin in the transversal plane and stained with 29 hematoxylin-eosin (HE). For this, the samples were dehydrated in a series of alcohol 100% (5 30 baths, 50 min each), followed by submersion in xylol (2 baths / 50 min each) and paraffin 31 inclusion (3 baths / 50 min each) and then sectioned with microtome at 5 μm. 32 For the staining protocol the paraffin was melted in an incubator (1 hour) and the 33 remaining paraffin was removed with xylene (2 baths, 20 min each). Then the samples passed 34 through a series of alcohol solutions (100, 90, 70 and 50%, 5 min each) and bathed in distilled 64

1 water (10 min) before the hematoxylin staining (5 min). Following, they were submitted to 2 running tap water (10 min) and counter-stained with eosin (4 min). It was dehydrated through 3 alcohol 70% (5 quick immersions), alcohol 80% (1 min), alcohol 90% (2 min), alcohol 100% 4 (5 min) and finally submersed in xylene (2 baths, 5 min each) and mounted with methacrylate 5 resin. 6 7 Stereology 8 For the stereology technique it was used 13 embryos for Con/D15, 6 embryos for 2,4- 9 D/D15, 7 embryos for Con/D30 and 13 embryos for 2,4-D/D30. 10 Sampled sections were collected by means of systematic random sampling process 11 since it gives an equal chance to all part of tissue to be sample. By means of this approach, 12 systematic deviation from real value would be eliminated, i.e. this result would be unbiased. 13 For samples from day 15 the section sampling fraction (ssf) was 1/10, meaning that 1 in every 14 10 sections through the optic tectum was analyzed, while for day 30 it was used a ssf of 1/17. 15 Images of these sections were captured with the aid of a microscope (LEICA DM750®) with 16 image capture system and specialized software (LEICA ICC50 HD®). The software Stepanizer 17 v1 was used to overlap a grid over the images. A grid of 8x8 (64) points was used for day 15 18 (a(p)= 93811.4956 µm²), while a grid of 9x7 (63) points was used for samples of day 30 (a(p)= 19 133657.427µm²) (Figure 1). A point was marked every time it was over the region of interest. 20 21 22 23 24 25 26 A B 27 28 Fig. 1 Software Stepanizer, used to apply stereology to estimate the volume of the optic tectum 29 of Tropidurus torquatus. A: Sample of day 15; B: Sample of day 30 30 31 Volume estimations were done using the Cavalieri principle, whereby a volume is 32 uniformly sectioned into a series of two-dimensional slices, the position of the first slice being 33 random with respect to the volume. The Cavalieri principle states that the volume of an 34 arbitrarily shaped object which is cut into parallel slices of equal thickness (t) can be estimated 65

1 by multiplying the sum of all ground areas of each particular slice by the slice thickness. Optic 1 2 tectum volumes were estimated by the following formula: [푉] = 푡 ∗ 푎(푝) ∗ ∑ 푝; [V] = 푠푠푓 3 volume; t = section thickness; a(p) = area associated with one test point; p= total number of 4 points hitting the optic tectum (Gundersen et al. 1988). 5 The coefficient of error (CE) for each embryo and the coefficient of variance (CV) for 6 each group were calculated. 7 8 Statistical analysis 9 Statistical analysis was performed using BioStat v5.0®. Lilliefors test was used to 10 check the normality of the data, which was normally distributed. Student’s T-test was used to 11 determine if the mean volume of the optic tectum of two sets of data other (Con/15 and 2,4- 12 D/15; Con/D30 and 2,4-D/D30) were significantly different from each other. ANOVA one-way 13 and LSD test were used to determine if the mean volume of the optic tectum in the stages of the 14 embryos were significantly different from each other. Pearson correlation’s test was used to 15 determine if the volume of the brain increased as the stages increased. Microsoft excel 2016 16 was used to generate graphics. 17 18 RESULTS 19 From 193 embryos collected, 65 survived, from which 38 was exposed to 2,4-D and 20 27 was in the control group. Most of the collected embryos did not survive / were not viable 21 due to unknown causes. The number of embryos collected and embryo survival rate are exposed

22 in Table 1. Most embryos of day 15 were in stage 32 and most embryos of day 30 were at stage 23 36 and most experimental embryos at stage 37 (Table 2). There were no macroscopic 24 differences between the stages of the embryos from control and 2,4-D groups (Figure 2). Day 25 60 and stage 38 removed from their respective analyses due to insufficient number of embryos. 26 27 Table 1 Number of embryos of Tropidurus torquatus collected in the control and 2,4-D 28 groups. Control 2,4-D Total Alive 27 26.5% 38 41.8% 65 33.7% Dead 75 73.5% 53 58.2% 128 66.3% Total 102 91 193 29 66

1 Table 2 Stage of collected embryos of Tropidurus torquatus in control and 2,4-D groups, 2 according to Py-Daniel (2017). Control 2,4-D Total Day 15 Stage 32 11 11 22 Stage 33 5 5 10 Total 16 16 32 Day 30 Stage 36 6 5 11 Stage 37 3 9 12 Stage 38 0 3 3 Total 9 17 26 Day 60 Stage 40 0 1 1 Stage 41 0 2 2 Total 0 3 3 3

4 5 Fig. 2 Embryos of Tropidurus torquatus. A-D: Control group; E-H: 2,4-D group. A, E – Stage 6 32; B, F – Stage 33; C, G – Stage 36; D, H – Stage 37. te – telencephalon; di – diencephalon; 7 hb – hindbrain. Bar: 5 mm 8 9 The estimated volume of the optic tectum of T. torquatus exposed to control and 2,4-D, 10 as well the coefficient of error and coefficient of variation of the estimations are shown in Table 11 3. There was a significant decrease in the volume of the optic tectum of embryos exposed to 67

1 2,4-D that were collected in day 15 post-oviposition. For day 30, no statistical difference was 2 found (Figure 3). There were no significant differences regarding the volume of the optic tectum 3 in stages of the embryos between treatments (Figure 4). The correlation analyses indicate that 4 the optic tectum increases its volume according to the advancement of the embryos’ stages 5 (Figure 5). 6 7 Table 3 Estimated volume of optic tectum of 2,4-D treated embryos of Tropidurus torquatus 8 by Cavalieri. Groups Treatment day Volume of optic tectum ± SD CE CV Control 15 0.91 ± 0.17 0.017 0.18 30 1.87 ± 0.38 0.018 0.19 2,4-D 15 0.72 ± 0.18 0.019 0.23 30 1.84 ± 0.30 0.018 0.15 9 SD: standard deviation; CE: coefficient of error; CV: coefficient of variation. 10

2.5 Ab Ab 2

1.5 Control Aa

1 Ba 2,4-D Volume mm³

0.5

0 D15 D30 11 12 Fig. 3 Volume comparison of the optic tectum of Tropidurus torquatus from embryos exposed 13 to water (control) and 2,4-D. D15: day 15 post oviposition; D30: day 30 post oviposition. Bars 14 mean standard deviation. A,B Indicate difference within the same day evaluated (P < 0.05). a,b 15 Indicate difference between days within the same treatment (P < 0.05) 16 68

3 Ab 2.5 Ab Ab Ab 2

1.5 Control Aa

Volume mm³ Volume Aa Aa 1 2,4-D Aa

0.5

0 32 33 36 37 Stages 1 2 Fig. 4 Volume comparison of the optic tectum of Tropidurus torquatus from embryos exposed 3 to water (control) and 2,4-D within the identified stages. Bars mean standard deviation. A,B 4 Indicate difference within the same day evaluated (P < 0.05). a,b Indicate difference between 5 days within the same treatment (P < 0.05) 6

2.5

1.5

1 Volume Volume mm³

0.5

0 31 32 33 34 35 36 37 38 Stages 7 8 Fig. 5 Correlation analysis between the estimated brain volume and the developmental stages 9 of the embryos of Tropidurus torquatus collected at days 15 (stages 32 and 33) and 30 (stages 10 36 and 37), indicating a positive correlation between volume and stage advancement (r = 0,86; 11 P < 0,001) 12 13 14 69

1 DISCUSSION 2 Since there is a lack of research towards the effect of 2,4-D in lizards, our study aimed to 3 use stereology to evaluate the neurotoxicity of a pesticide in the developing brain of T. 4 torquatus. Our results suggest that 2,4-D may cause damage to the optic tectum of T. torquatus 5 and these data may be extrapolated to other lizards. We observed a volume reduction 15 days 6 after exposition to the herbicide. However, there was no significant difference on day 30. This 7 could suggest that the embryos from day 30 suffered neurotoxic damage in their early post- 8 oviposition development, but the repair mechanisms of the brain reversed this damage. Or the 9 embryos analyzed in day 30 never suffered a significant neurotoxic damage, which suggests 10 some embryos, which were analyzed in day 15, were more susceptible to volume decrease due 11 to unknown causes. 12 The only other study using eggs (chicken), evaluated myelination and found alterations 13 in the levels of lipids, indicating hypomyelination. No changes were found past the phase of 14 proliferation and development of the oligodendrocytes (De Moro et al. 1993). The same could 15 happen to lizards, even though we did not evaluate myelination. This may also be related to the 16 reduction of fibers and axon projections reported in zebrafish (Ton et al. 2006), suggestion that 17 axonal fibers are one of the targets of 2,4-D in the brain. 18 Fish are apparently more sensitive to 2,4-D than rats, perhaps due to the water 19 environment, and zebrafish and guppy presented severed changes in behavior and neurotoxic 20 effects in doses ranging 15-100mg/L. Increased apoptosis and loss of neurons, disrupted motor 21 neuron growth, and reduction of axon projections to the optic tectum was observed in these fish 22 (Ton et al. 2006; Uyanıkgil et al. 2009; Ertug et al. 2014). Either reduced cell size, reduced cell 23 density or reduction in fibers, could explain the volume decrease we observed in T. torquatus. 24 Further studies are necessary to evaluate these parameters. 25 2,4-D can be detected in the brain even at a low dose (10mg/kg), which was better 26 explained by the competitive inhibition of 2,4-D elimination via active transport in the anionic 27 system of the choroid plexus (Kim et al. 1988; Oliveira and Palermo-Neto 1993). In pregnant 28 mice it was shown that 2,4-D accumulated more in the fetal brain than in the mother’s and the 29 areas of the brain with the greatest accumulation were the brainstem, cerebellum and frontal 30 cortex (Kim et al. 1988). This suggests that the infiltration of 2,4-D in the brain of embryos is 31 higher, possible because the mechanisms of defense are not fully developed. Since that in 32 eutherian animals the toxicant needs to penetrate in the mother’s organism first, this may 33 provide some protection for the embryos. Oviparous animals, such as lizards, lack this barrier 34 and are more possibly more vulnerable to external damage. 70

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ANEXO 1 – Extrato da permissão de coleta do SISBIO

Ministério do Meio Ambiente - MMA Instituto Chico Mendes de Conservação da Biodiversidade - ICMBio Sistema de Autorização e Informação em Biodiversidade - SISBIO

Extrato da solicitação Nº 61909 em PDF - Gerado em: 19/01/2018 as 11:01:58 horas

Dados básicos da Solicitação Nº da solicitação: 61909 Situação atual: Submetida para análise Data da situação atual: 19/01/2018

Tipo da solicitação: Autorização para atividades com finalidade científica

Título do Projeto: MORFOLOGIA, ONTOGENIA E ECOTOXICOLOGIA DE Tropidurus torquatus ASSOCIADA AO USO DE HERBICIDAS NO DESENVOLVIMENTO EMBRIONÁRIO

Dados do pesquisador Nome: Fabiano Campos Lima Nacionalidade: Brasileira CPF: 07520553647 E-mail: [email protected] Identidade: 14713361 SSP MG

Endereço: Rua Joaquim Caetano 1948

Bairro: Samuel Graham CEP: 75804-058 Município: JATAI UF: GO Fone: (0xx64) 3636-8138 Fax:

Profissão: Biólogo Nível escolar: Pós-Doutorado

Dados do vínculo institucional Instituição: UNIVERSIDADE FEDERAL DE GOIAS CNPJ: 01.567.601/0001-43 Fone: (0xx64) 3606-8210

Tipo de vínculo: Servidor público Email: [email protected] Observação:

Atividades da solicitação Descrição das atividades/substrato Tipo do item Coleta/transporte de amostras biológicas in situ Atividades Coleta/transporte de espécimes da fauna silvestre in situ Atividades Manutenção temporária (até 24 meses) de vertebrados silvestres em cativeiro Atividades Captura de animais silvestres in situ Atividades

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Locais onde as atividades serão executadas Descrição do local Bioma Município UF Tipo do local Abrange caverna? Universidade Federal de Goiás - Regional Jataí Cerrado JATAI GO Fora de UC Federal Não

Táxon(s) Táxon Grupo taxonômico Gênero Tropidurus Répteis

Táxon(s) X Atividades Grupo Táxon Envolve espécie ameaçada? Qtd prevista Atividade Répteis Gênero Tropidurus NÃO 20 Coleta/transporte de espécimes da fauna silvestre in situ Répteis Gênero Tropidurus NÃO Manutenção temporária (até 24 meses) de vertebrados silvestres em cativeiro Répteis Gênero Tropidurus NÃO Captura de animais silvestres in situ Répteis Gênero Tropidurus NÃO Coleta/transporte de amostras biológicas in situ

Táxon(s) X Materiais, métodos e amostras biológicas Grupo taxonômico Descrição Tipo Répteis Captura manual Método de captura/coleta Répteis Fragmento de tecido/órgão Amostras biológicas Répteis Laço de Lutz Método de captura/coleta

Destino(s) do(s) material(is) biológico(s) coletado(s) Descrição do destino Tipo do destino UNIVERSIDADE FEDERAL DE GOIAS LaboratÃ³rio de Anatomia Humana e Comparativa - UFG

Cronograma de atividades Descrição da atividade Data início Data Fim Coleta de animais (Tropidurus sp.) no Campus da UFG - Regional Jataí 01/03/2018 31/03/2019 Exposição ao herbicida - Ovos de animais da primeira coleta serão submetidos ao protocolo de exposiç 20/03/2018 31/05/2018 Exposição ao herbicida - Ovos de animais da segunda coleta serão submetidos ao protocolo de exposiç 01/12/2018 31/05/2019 Técnicas anatômicas diversas - Dissecação, preparo histológico, microscopia de varredura, diafanizaç 01/04/2018 31/12/2020 Analise estatística 01/12/2018 31/08/2020 Analise dos dados 31/05/2018 31/08/2020

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Áreas do conhecimento Descrição da atividade Embriologia Morfologia Saúde Anatomia

Dados básicos Nome do campo Descrição Atualmente é utilizado o termo Ecotoxicologia para relacionar os efeitos tóxicos de determinadas substâncias em organismos vivos ou mesmo no ecossistema (IBAMA, 2009). Grande parte das substâncias que podem causar danos a estrutura dos seres vivos são produzidas sinteticamente pelo homem, e muitas vezes utilizadas de maneira indiscriminada. A economia brasileira tem como base a agricultura. Setores diversos se esforçam para manter cada vez mais alto a produção agrícola com intuito de alavancar as reservas do país e aumentar o lucro dos produtores. Por muito tempo têm sido utilizado produtos diversos objetivando eliminar organismos indesejados em diferentes áreas, dentre elas a agricultura, onde destacam-se os agrotóxicos. Esses produtos possuem fontes, estruturas e meios de ação variados. No último século as indústrias passaram a produzir agrotóxicos sintéticos orgânicos em grande escala, principalmente da classe organoclorados, sendo o mais importante e conhecido deles o DDT (1,1,1-tricloro-2,2-bis(4-clorofenil)etano) (JARDIM et al., 2009; MANSANO, 2016). O Brasil é um grande produtor e exportador de produtos agrícolas, sendo este um dos principais pilares da economia. O clima tropical é propício ao surgimento e propagação de organismos que afetam a qualidade das lavouras, sendo necessário o uso de agrotóxicos. As primeiras unidades produtivas destes datam de meados da década de 1940. Anualmente, são utilizados mais de um milhão de toneladas, o que corresponde ao consumo de mais de um bilhão de litros (MANSANO, 2016). É importante entender que embora um agrotóxico seja aplicado para ter efeito contra uma ou poucas espécies, sua ação não é limitada, podendo afetar todo o ecossistema (MOORE, 1967). O uso de agrotóxicos causa forte pressão seletiva ao seus organismos alvo, levando ao uso de doses maiores para exterminar os indivíduos resistentes e seus descendentes, elevando sua quantidade no meio ambiente (MOORE, 1967; D?AMATO et al., 2002). Introdução/Justificativa De acordo com a Lei Federal 7.802, de 11 de julho de 1989, agrotóxicos são produtos e agentes de processos físicos, químicos ou biológicos, destinados ao uso nos setores do agronegócio em diversos ecossistemas, cuja finalidade seja alterar a composição da flora ou da fauna, objetivando preservá-las da ação danosa de seres vivos considerados nocivos (BRASIL, 1989). O herbicida glifosato é amplamente utilizado, principalmente no Brasil, onde existem relatam sobre algumas alterações nos organismos quando expostos. Devido à sua classificação de toxicidade (classe IV), considerado pouco perigoso, espera-se que não prejudique o ambiente, os animais e o ser humano. Porém, quando em grandes quantidades e por exposição continua, podem ocorrer alterações diversas, sendo necessários estudos que objetivam avaliar os impactos diretos ao meio ambiente, ao desenvolvimento dos organismos e suas possíveis manifestações. O lagarto T. torquatus é um organismo modelo para estudos devido seu conjunto de características, o que facilita o estudo com uma amostra significativa de embriões a cada ciclo reprodutivo e que não afete sua população, devido a sua abundância. No ambiente ele pode se contaminar devido a seu hábito terrestre, caminhando e permanecendo sobre solo contaminado, e também por ingestão de alimento com a presença de resíduos químicos. Em quantidades suficientes o glifosato, como qualquer agrotóxico, se acumula no tecido adiposo, o que é magnificado em organismos de nível trófico superior, processo conhecido com biomagnificação. Analisar o desenvolvimento cerebral mostrará se o herbicida possui efeito deletério significativo sobre esse lagarto, pois embriões e juvenis são mais sensíveis as alterações. Caso o resultado seja positivo para alterações por exposição ao glifosato, estes dados podem ser extrapolados para outros animais, incluindo o ser humano, deixando um alerta sobre o uso indiscriminado deste agrotóxico.

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Nome do campo Descrição Objetivo geral Descrever a ontogenia de T. torquatus em condições normais e analisar o efeito do herbicida Glifosato no desenvolvimento embrionário. Descrever a anatomia de T. torquatus: Descrever o desenvolvimento embrionário normal de T. torquatus; Objetivos específicos Descrever o desenvolvimento embrionário de T. torquatus sobre efeito do herbicida Glifosato; Comparar o desenvolvimento embrionário normal sob o efeito do herbicida Glifosato, relatando as possíveis alterações encontradas. Serão coletadas fêmeas grávidas de T. torquatus na Universidade Federal de Goiás ? Regional Jataí. Os animais serão enlaçadas e transportadas para um local onde ficarão mantidos até a ovipostura, após a qual serão devolvidos ao local de origem.Os animais serão divididos aleatoriamente em dois grupos iguais que serão mantidos em terrários. O terrário contendo o grupo controle, bem como aquele contendo o grupo de estudo estarão sob as mesmas condições ? exceto pela exposição ao agente herbicida no grupo de estudo. Após a postura, os ovos serão retirados dos terrários, e as ninhadas individualizadas em potes de plásticos com 1:1 de vermiculita e água, sendo então transferidos para uma incubadora, programada para manter temperatura constante de 30ºC, na presença de água para manter a umidade. De ambos os grupos serão coletados diariamente dois ovos. Os embriões serão removidos de seus ovos e imediatamente será realizada a eutanásia por meio de uma solução 1:1 de 0,5% de cloridrato de bupivacaína e 2% de cloridrato de lidocaína. O grupo de estudo será composto por ovos recém ovipostos os quais serão mantidos em terrário separado do grupo controle. O substrato em que os embriões serão artificialmente incubados será contaminado com o herbicida Glifosato, que será diluindo em concentrações de 4,2, 42, 420 partes por bilhão (ppb) em água destilada, com um valor equivalente e valores acima e abaixo ao valor de ingestão diária aceitável no Brasil (0,042 mg/kg), determinado pela Agência Nacional de Vigilância Sanitária (ANVISA). Cada tratamento será baseado nos mesmos volumes para os espécimes determinados. A coleta ocorrerá conforme protocolo anteriormente informado. Macroscopia e Mesoscopia Material e métodos Uma coleção de embriões constando toda sequência ontogenética de ambos os grupos será diafanizada e suas cartilagens e ossos corados com Alcian Blue e Alizarina red S, respectivamente. Análise do desenvolvimento e dos espécimes adultos Para fins de comparação a anatomia da espécie adulta também será descrita, sendo utilizados 10 espécimes no total, cinco fixados e cinco post mortem. Os espécimes fixados serão descritos conforme métodos usuais e os demais serão eutanaziados com uma dose letal de pentobarbital sódico (100 mg/kg) intraperitoneal e dissecados a fresco submersos em solução fisiológica (pH 7,2) (SEBBEN, 2012). Este protocolo permite a documentação fotográfica das estruturas de interesse sem o inconveniente da fixação prévia. Microscopia Fragmentos de órgãos diversos (fígado, encéfalo, gônadas, tubo digestório, pulmões, coração) de ambos os grupos serão submetidos ao protocolo histológico com coloração de Hematoxilina e Eosina (H.E.), P.A.S., Tricômico de Masson, Nissl, Golgi-Cox. Embriões de cada estágio serão submetidos a microscopia eletrônica de varredura. A análise estatística será realizada utilizando-se o programa SPSS (Statistical Package for Social Science), versão 22.0. Para criação e validação do banco de dados, será utilizado o programa Microsoft Excel versão 2007 ou superior. Será executada análise descritiva dos dados, através da avaliação das medidas de tendência central (média, moda e mediana); medidas de dispersão (desvio padrão, variância, mínima, máxima, erro padrão e amplitude), além da realização da distribuição (assimetria, curtose) e histogramas na busca de possíveis erros de entrada e validação dos dados.

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Nome do campo Descrição AMARANTE JUNIOR, P. O.; SANTOS, T. C. R.; BRITO, N. M.; RIBEIRO, M. L. Glifosato: Propriedades, Toxicidade, Usos E Legislação. Quimica Nova, v. 25, n. 4, p. 589?593, 2002. ARRUDA, J. L. S. Ecologia de Tropidurus torquatus (Squamata: Tropiduridae) no bioma pampa, extremo Sul do Brasil. 2009. 76 f. Dissertação (Mestrado em Biodiversidade Animal). Universidade Federal de Santa Maria, Santa Maria. BALON, E. K. Early ontogeny of the lake chaar, Salvelinus (Cristivomer) namaycush. Chaars: Salmonid fishes of the genus Salvelinus. pp. 475-685. Dr. W. Junk Publishers, Netherlands. 1980 BOUGHNER, J. C.; BUCHTOVÁ, M.; FU, K.; DEWERT, V.; HALLGRÍMSSON, B.; RICHMAN, J. M. Embryonic development in Python sebae ? I: Staging criteria and macroscopic skeletal morphogenesis of the head and limbs. Zoology. 110:212-230. 2007. BRASIL. Ministério da Agricultura. Portaria no. 329, de 02 de setembro de 1985. http://bvsms.saude.gov.br/bvs/saudelegis/mapa_gm/1985/prt0329_02_09_1985.html, acessada em Outubro, 2017. BRASIL. Lei Federal 7.802, de 11 de julho de 1989. http://www.planalto.gov.br/CCIVIL_03/decreto/2002/D4074.htm, acessada em Outubro, 2017. D?AMATO, C.; TORRES, J. P. M.; MALM, O. DDT (Dicloro difenil tricloroetano): Toxicidade e contaminação ambiental - Uma revisão. Quimica Nova, v. 25, n. 6 A, p. 995?1002, 2002. DARUICH, J.; ZIRULNIK, F.; SOFÍA GIMENEZ, M. Effect of the Herbicide Glyphosate on Enzymatic Activity in Pregnant Rats and Their Fetuses. Environmental Research, v. 85, n. 3, p. 226?231, 2001. FABREZI, M., V. ABDALA AND M. I. M. OLIVER, Developmental basis of limb homology in lizards. The Anatomical Record, 290:900-912. 2007. GILBERT, S. F. Developmental Biology. Sunderland: Mass. Sinauer Associates. 2006. GILBERT, S. F.; LOREDO, G. A.; BRUKMAN, A.; BURKE, A. C. Morphogenesis of the turtle shell: the development of novel structure in tetrapod evolution. Evo Devo. 3:47?58. 2001. GRIER, J. W. Ban of DDT and subsequent recovery of reproduction in bald eagles. Science, 1982. Referências bibliográficas HALLBERG, G. R. Pesticides pollution of groundwater in the humid United States. Agriculture, Ecosystems and Environment, v. 26, n. 3?4, p. 299?367, 1989. HARRISON, L.; LARSSON, H. Estimating evolution of temporal sequence changes: a practical approach to inferring ancestral developmental sequences and sequence heterochrony. Systematic Biology. 57: 378?387. 2008. HOLMGREN, N. On the origin of the tetrapod limb. Acta Zoologica (Stockholm), v. 14, p. 185?295. 1933. IBAMA, Instituto Brasileiro de Meio Ambiente e Recursos Naturais Renováveis. Produtos agrotóxicos e afins comercializados em 2009 no Brasil: uma abordagem ambiental. RABELO, R. M. 2009. Brasília. 84p.

JARDIM, I. C. S. F.; ALMEIDA ANDRADE, J.; QUEIROZ, S. C. D. N. Resíduos de agrotóxicos em alimentos: uma preocupação ambiental global - um enfoque às maçãs. Quimica Nova, v. 32, n. 4, p. 996?1012, 2009. JEFFERY, J. E.; BININDA-EMONDS, O. R. P.; COATES, M. I. & RICHARDSON, M. K. A new technique for identifying sequence heterochrony. Systematic Biology. 54: 230?240. 2005. JEFFERY, J. E.; BININDA-EMONDS, O. R. P.; COATES, M. I.; RICHARDSON, M. K. Analyzing evolutionary patterns in amniote embryonic development. Evo Devo 4: 292?302. 2002. KURAKU, S.; USUDA, R.; KURATANI, S. Comprehensive survey of carapacial ridge-specific genes in turtle implies cooption of some regulatory genes in carapace evolution. Evo Devo 7:3?17. 2005. KIEFER, M. C.; SLUYS, M. VAN; ROCHA, C. F. D. Body temperatures of Tropidurus torquatus (Squamata, Tropiduridae) from coastal populations: Do body temperatures vary along their geographic range? Journal of Thermal Biology, v. 30, n. 6, p. 449?456, 2005.

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KOHLSDORF, T.; RIBEIRO, J. M.; NAVAS, C. A. Territory quality and male dominance in Tropidurus torquatus (Squamata, Tropiduridae). Phyllomedusa, v. 5, n. 2, p. 109?118, 2006. MABEE, P. M.; OTMSTEAD, K. L.; CUBBAGE, C. C. An experimental study of intraspecific variation, developmental, timing

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Nome do campo Descrição Os animais serão divididos aleatoriamente em dois grupos iguais que serão mantidos em terrários com uma camada de 10 cm com substrato de vermiculita de grão médio, na proporção 1:1 com água. Esses serão colocados em local que minimize o estresse durante o período em que os espécimes serão mantidos em cativeiro. Condições do mantenedouro Lâmpadas incandescentes de 60W, pedaços de concreto e troncos serão utilizados dentro dos terrários para a termorregulação dos espécimes. O ciclo claro/escuro (12/12 horas) será mantido controlado por um temporizador. Os animais serão alimentados com insetos ad libitum e uma fonte de água limpa deve ser mantida sempre em cada terrário.

Histórico da Solicitação 19/01/2018 11:17 Submetida para análise 19/01/2018 10:54 Em elaboração

Histórico da distribuição Tipo da Distribuição Data Unidade Fone da Unidade Lim. Receber Lim. Parecer Descrição da situação Emissão de Parecer (análise obrigatória) 19/01/2018 RAN (0xx62) 3225-9968 30/01/2018 09/02/2018 Aguardando recebimento

ANEXO 2 – Aprovação no Comitê de Ética

Ministério da Educação Universidade Federal de Goiás Coordenação de Pesquisa e Inovação Comissão de Ética no Uso de Animais/CEUA-Jataí

Jataí, 08 de maio de 2018.

PARECER CONSUBSTANCIADO REFERENTE AO ATENDIMENTO DE PENDÊNCIA DO PROTOCOLO Nº. 013/18

I - Finalidade do projeto de pesquisa: Iniciação Científica e Mestrado II - Identificação:  Data de apresentação a CEUA: 17/04/2018  Título do projeto: Morfologia, ontogenia e ecotoxicologia de Tropidurus torquatus associada ao uso de herbicidas no desenvolvimento embrionário.  Pesquisador Coordenador no SAP: Fabiano Campos Lima  Pesquisador Responsável/ Unidade: Fabiano Campos Lima/Unidade Acadêmica Especial de Ciências Biológicas.  Pesquisadores Participantes: Leticia Menezes Freitas, Mônica Rodrigues Ferreira Machado, Kleber Fernando Pereira, Lilia Cristina de Souza Barbosa, Odeony Paulo dos Santos, Juliana Flávia Ferreira e Silva Paranaíba, Maira Rocha Amorim dos Santos, Raphaela Lorrayne de Jesus Costa, Karoline Cardoso Silva Santos.  Médico Veterinário/CRMV: Mônica Rodrigues Ferreira Machado/CRMV 3558  Unidade onde será realizado: Unidade Acadêmica Especial de Ciências Biológicas

III - Objetivos e justificativa do projeto: O objetivo geral do projeto é: Descrever a ontogenia de T. torquatus em condições normais e analisar o efeito do herbicida Glifosato no desenvolvimento embrionário.

Os objetivos específicos são:  Descrever a anatomia de T. torquatus:  Descrever o desenvolvimento embrionário normal de T. torquatus;  Descrever o desenvolvimento embrionário de T. torquatus sobre efeito do herbicida Glifosato;

Comissão de Ética no Uso de Animais/CEUA-JATAÍ Bloco 5 – sala 23 B, Campus Jatobá. Br 364 Km 192, nº. 3.800 Parque Industrial, Jataí (GO) - 75801-615 Email: [email protected] MINISTÉRIO DA EDUCAÇÃO UNIVERSIDADE FEDERAL DE GOIÁS PRÓ-REITORIA DE PESQUISA E INOVAÇÃO COMISSÃO DE ÉTICA NO USO DE ANIMAIS/CEUA

 Comparar o desenvolvimento embrionário normal sob o efeito do herbicida Glifosato, relatando as possíveis alterações encontradas.

Justificativa do projeto: O herbicida glifosato e amplamente utilizado, principalmente no Brasil, onde existem relatam sobre algumas alterações nos organismos quando expostos. Devido a sua classificação de toxicidade (classe IV), considerado pouco perigoso, espera-se que não prejudique o ambiente, os animais e o ser humano. Porém, quando em grandes quantidades e por exposição continua, podem ocorrer alterações diversas, sendo necessários estudos que objetivam avaliar os impactos diretos ao meio ambiente, ao desenvolvimento dos organismos e suas possíveis manifestações. O lagarto T. torquatus é um organismo modelo para estudos devido seu conjunto de características, o que facilita o estudo com uma amostra significativa de embriões a cada ciclo reprodutivo e que não afete sua população, devido a sua abundância. No ambiente ele pode se contaminar devido a seu habito terrestre, caminhando e permanecendo sobre solo contaminado, e também por ingestão de alimento com a presença de resíduos químicos. Em quantidades suficientes o glifosato, como qualquer agrot6xico, se acumula no tecido adiposo, o que e magnificado em organismos de nível tr6fico superior, processo conhecido com biomagnificação. Analisar o desenvolvimento cerebral mostrará se o herbicida possui efeito deletério significativo sobre esse lagarto, pois embriões e juvenis são mais sensíveis as alterações. Caso o resultado seja positivo para alterações por exposição ao glifosato, estes dados podem ser extrapolados para outros animais, incluindo o ser humano, deixando um alerta sobre o uso indiscriminado deste agrotóxico. Também procura-se saber se o limite de ingestão diária permitido para o glifosato e o suficiente para impedir danos ao organismo, especialmente durante o período de desenvolvimento.

IV - Sumário do projeto:  Discussão sobre a possibilidade de métodos alternativos e necessidade do número de animais: Não consta  Prevê Projeto Piloto: Não

 Espécie animal utilizado/ número total de animais/ Número de animais por tratamento ou grupo experimental: Tropidurus torquatus/ 188 animais/10 fêmeas no grupo controle e 10, expostas ao herbicida. Diariamente serão retirados 02 ovos por grupo.  Descrição do animal utilizado (Explicitar: espécie/ linhagem/ sexo (informar número por sexo)/ peso e/ou idade etc): Tropidurus torquatus/ 20 fêmeas e 168 embriões, sendo 84 machos e 84 fêmeas.  Fonte de obtenção do animal: Coleta à campo das fêmeas na UFJ, de acordo com as normas do SISBIO 61909, e coleta dos ovos para a obtenção dos embriões no laboratório de Anatomia Humana e Comparada da UFJ.  Descrição das instalações utilizadas e número de animais/área/qualidade do ambiente (ar, temperatura, umidade), alimentação/hidratação: As fêmeas serão entrelaçadas, com auxílio de laço, e transportadas para um laborarório de Anatomia da UFJ, onde ficarão mantidas até a ovipostura, após a qual serão devolvidos ao local de origem. A coleta ocorrerá durante época reprodutiva (agosto a fevereiro) do ano de 2018. As fêmeas serão alimentadas com insetos ad libitum e uma fonte de água limpa estará disponível a todo momento. 04 fêmeas serão mantidas no mesmo terrário de vidro com dimensões de 60x40x50cm com lâmpadas incandescentes de 60W, pedaços de concreto e troncos serão utilizados dentro dos terrários para a termorregulação dos espécimes. O ciclo claro/escuro (12/12horas) será controlado por um temporizador. O terrário será forrado com substrato de vermiculita de grão médio na proporção 1:1 com água. Uma grade será usada para manter os animais no terrário, providenciando ventilação. Após a ovipostura, os ovos serão retirados dos terrários, e as ninhadas individualizadas em potes de plásticos com 1:1 de vermiculita e água, sendo então transferidos para uma incubadora, programada para manter temperatura constante de 30ºC, na presença de água para manter a umidade. De ambos os grupos serão coletados diariamente dois ovos. O terrário contendo o grupo controle, bem como aquele contendo o grupo de estudo estarão sob as mesmas condições, exceto pela exposição ao agente herbicida no grupo de estudo.  Utilização de agente infeccioso/gravidade da infecção a ser observada e análise dos riscos aos pesquisadores/alunos: Não se aplica  Procedimentos experimentais do projeto de pesquisa: O grupo de estudo será composto por ovos recém ovipostos os quais serão mantidos em terrário separado do grupo controle. O substrato em que os embriões serão artificialmente incubados será contaminado com o herbicida Glifosato, que será diluindo

em concentraçõesde4,2, 42, 420 partes por bilhão (ppb) em água destilada, com um valor equivalente e valores acima e abaixo ao valor de ingestão diária aceitável no Brasil (0,042 mg/kg), determinado pela Agência Nacional de Vigilância Sanitária (ANVISA). Cada tratamento será baseado nos mesmos volumes para os espécimes determinados. Uma coleção de embriões constando toda sequência ontogenética de ambos os grupos será diafanizada e suas cartilagens e ossos corados com Alcian Blue e Alizarina Red S, respectivamente. Outra coleção de embriões constando toda sequência ontogenética de ambos os grupos será dissecada e todos os órgãos fotografados com lupa estereosc6pica com sistema de captura de imagem. A partir da análise de características morfológicas embrionárias o desenvolvimento normal de T. torquatus será descrito. Os embriões expostos ao herbicida serão analisados segundo o mesmo protocolo, comparando-se com o grupo controle e relatando as possíveis alterações no desenvolvimento. Fragmentos de órgãos diversos (fígado, encéfalo, gônadas, tubo digestório, pulmões, coração) de ambos os grupos serão submetidos ao protocolo histológico com coloração de Hematoxilina e Eosina (H.E.), P.A.S., Tricômico de Masson, Nissl, Golgi-Cox. Em ademais, embriões de cada estágio serão submetidos a microscopia eletrônica de varredura.  Métodos utilizados para minimizar o sofrimento e aumentar o bem-estar dos animais antes, durante e após a pesquisa. Pontos Finais Humanitários: As fêmeas capturadas serão mantidas em terrários com enriquecimento ambiental e sob condições de temperatura, umidade e ciclo circadiano controlados. Alimento e água serão oferecidos sem restrições. Os terrários serão inspecionados diariamente apenas para coletar ovos, não sendo necessário a manipulação dos animais adultos. Os ovos, após protocolo experimental, serão injetados com superdose de solução l:l de 0,5%decloridrato de bupivacaina e 2% de cloridrato de lidocaína objetivando eutanásia dos embriões antes mesmo da manipulação.  Grau de invasividade: GI1 para as fêmeas e GI3 para os embriões.  Material utilizado em outros projetos: Não se aplica.  Método de eutanásia: Diariamente serão aleatoriamente selecionados e os embriões serão removidos e, imediatamente será realizada a eutanásia por meio de uma solução 1:1 de 0,5% de cloridrato de bupivacaína e 2% de cloridrato de lidocaína.  Destino do animal: As fêmeas capturadas grávidas serão devolvidas ao local de coleta após a oviposição. Os embriões serão fixados e ap6s a pesquisa serão armazenados no Laborat6rio de Anatomia Humana e Comparada da UFJ.

V – Comentários do relator frente às orientações da CEUA:  Quanto aos documentos exigidos pela CEUA/UFG: Todos os documentos foram entregues e estão de acordo com a exigência.  Quanto aos cuidados e manejo dos animais e riscos aos pesquisadores: Para evitar a exposição da equipe ao herbicida, serão utilizadas luvas de látex e máscaras FPA l 0. Para os demais procedimentos, a equipe e possui experiência no desenvolvimento das atividades. VI - Parecer da CEUA: De acordo com a documentação apresentada à CEUA, consideramos o projeto APROVADO, smj desta Comissão.

Solicitamos aos pesquisadores: A aprovação do SISBIO foi anexada ao projeto. O biotério localizado no laboratório de Anatomia Humana e Comparada da UFJ deve ser cadastrado no CIUCA (Cadastro das Instituições de Uso Científicos de Animais).

Informação aos pesquisadores: Reiteramos a importância deste Parecer Consubstanciado, e lembramos que a pesquisadora responsável deverá encaminhar à CEUA-Jataí o Relatório Final baseado na conclusão do estudo e na incidência de publicações decorrentes deste, de acordo com o disposto na Lei nº. 11.794 de 08/10/2008, e Resolução Normativa nº. 01, de 09/07/2010 do Conselho Nacional de Controle de Experimentação Animal-CONCEA. O prazo para entrega do Relatório é de até 30 dias após o encerramento da pesquisa, a qual está prevista para finalizar suas ações até 31 de dezembro de 2021.

VII - Data da reunião: 08/06/2018.

Dra. Mirian Machado Mendes Coordenadora da CEUA-Jataí

Comissão de Ética no Uso de Animais/CEUA-JATAÍ Bloco 5 – sala 23 B, Campus Jatobá. Br 364 Km 192, nº. 3.800 Parque Industrial, Jataí (GO) - 75801-615 Email: [email protected]