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Universidade Federal do Tocantins

Câmpus de Gurupi Programa de Pós-Graduação em Produção Vegetal

ANA MARIA CÓRDOVA LÓPEZ

BACIA ARAGUAIA-TOCANTINS: AVALIAÇÃO DO GRAU DE CONTAMINAÇÃO USANDO Dugesia tigrina COMO MODELO

GURUPI - TO

2015



ANA MARIA CÓRDOVA LÓPEZ

BACIA ARAGUAIA-TOCANTINS: AVALIAÇÃO DO GRAU DE CONTAMINAÇÃO USANDO Dugesia tigrina COMO MODELO

Dissertação apresentada ao Programa de Pós-graduação em Produção Vegetal da Universidade Federal do Tocantins como parte dos requisitos para a obtenção do título de Mestre em Produção Vegetal.

Orientador: Prof. Dr. Renato de Almeida Sarmento, UFT.

Co-Orientador: Prof. Dr. Amadeu Soares, UA.

GURUPI - TO 2015

Dados Internacionais de Catalogação na Publicação (CIP) Biblioteca da Universidade Federal do Tocantins

Campus Universitário de Gurupi

L864b López, Ana Maria Córdova Bacia Araguaia-Tocantins: avaliação do grau de contaminação

usando Dugesia tigrina como modelo. Ana Maria Córdova López – Gurupi, TO, 2015.

80f.

Dissertação de Mestrado – Universidade Federal do Tocantins, Câmpus Universitário de Gurupi – Programa de Pós-Graduação em Produção Vegetal, 2015. Orientador: Prof. Dr. Renato de Almeida Sarmento Coorientador: Prof. Dr. Amadeu M. V. M. Soares

1. Efeitos sub-letais. 2. Metais. 3. Planárias. I. Sarmento, Renato de Almeida II. Universidade Federal do Tocantins. III. Título.

CDD 635

Elaborado pelo sistema de geração automática de ficha catalográfica da UFT com os dados fornecidos pela autora.

TODOS OS DIREITOS RESERVADOS – A reprodução total ou parcial, de qualquer forma ou por qualquer meio deste documento é autorizado desde que citada a fonte. A violação dos direitos

do autor (Lei nº 9.610/98) é crime estabelecido pelo artigo 184 do Código Penal.

iv



ATA nº 17/2015

ATA DA DEFESA PÚBLICA DA DISSERTAÇÃO DE MESTRADO DE ANA MARIA CÓRDOVA LÓPEZ, DISCENTE DO PROGRAMA DE PÓS-GRADUAÇÃO EM PRODUÇÃO VEGETAL DA

UNIVERSIDADE FEDERAL DO TOCANTINS

Aos 9 dias do mês de dezembro do ano de 2015, às 8:15 horas, na Sala 15 do Bala II, reuniu-se a Comissão Examinadora da Defesa Pública, composta pelos seguintes membros: Prof. Orientador Dr. Renato de Almeida Sarmento do Campus Universitário de Gurupi/Universidade Federal do Tocantins, Prof. Dr. Amadeu M.V.M. Soares da Universidade de Aveiro/Aveiro-Portugal, Prof. Dr. Gil Rodrigues dos Santos/Campus Universitário de Gurupi/Universidade Federal do Tocantins e Prof. Dr. Marçal Pedro Neto /Campus Universitário de Gurupi/Universidade Federal do Tocantins, sob a presidência do primeiro, a fim de proceder a arguição pública da dissertação de mestrado de Ana Maria Córdova López, intitulada " Bacia Araguaia-Tocantins: avaliação do grau de contaminação usando Dugesia tigrina como modelo". Após a exposição, a discente foi arguida oralmente pelos membros da Comissão Examinadora, tendo parecer favorável à aprovação, habilitando-a ao título de Mestre em Produção Vegetal.

Nada mais havendo, foi lavrada a presente ata, que, após lida e aprovada, foi assinada pelos membros da Comissão Examinadora.

Dr. Amadeu M.V.M. Soares Primeiro examinador

Dr. Gil Rodrigues dos Santos Segundo examinador

Dr. Marçal Pedro Neto Terceiro examinador

Dr. Renato de Almeida Sarmento Universidade Federal do Tocantins

Orientador e presidente da banca examinadora

Gurupi, 9 de Dezembro de 2015.

Dr. Rodrigo Ribeiro Fidelis Coordenador do Programa de Pós-graduação em Produção Vegetal

v

A Deus por ter enchido

meu coração e minha mente de paz

em todos os momentos que precisei.

A minha família, em especial

a minha amada filha Annie Nicole.

DEDICO

vi

AGRADECIMENTOS

A Deus por ter reconfortado e fortalecido minha fé ante todas as dificuldades, por ter

colocado as pessoas certas no momento certo, meu Deus é maior. A minha mãe Maria López, por ter batalhado e vencido em muitos problemas sendo

um exemplo de vida a seguir. Minhas vitórias seguem o que me foi ensinado. A minha amada filha Annie Nicole Cienfuegos López, meu maior estímulo para avançar na pesquisa e chegar com êxito ao final. Aos meus queridos irmãos Rosa, Luis, Dante, Victor e Patricia e a tia Nora Lopez

pelos conselhos a mim concedidos nos momentos mais difíceis. Aos meus amigos fornecidos por Deus Ronice, Althieries, Prinscilla, Irais, Danilo, Raquel, Gleicielly, Diogenis e Diana, vocês fizeram a diferença em minha vida.

Ao professor Gil Rodrigues e sua esposa Rita, que com muita gentileza acolheram-

me e brindaram-me a ajuda necessária para iniciar o mestrado. Aos meus Orientadores Renato Sarmento e Amadeu Soares, que contribuíram para a culminação da pesquisa. Ao João Pestana, pela atenção e por estar sempre disponível em todos os

momentos, contribuído com seus conhecimentos para o enriquecimento e conclusão deste trabalho. Ao grupo de pesquisa de ecotoxicologia do laboratório de ecologia funcional e aplicada por ter colaborado no desenvolvimento da pesquisa. Ao Programa de Pós-Graduação em Produção Vegetal da UFT, por ter-me dado a oportunidade de cumprir uma das minhas metas de vida. À Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES pela

concessão da bolsa de mestrado.

Muito obrigada!

vii

RESUMO GERAL

O aumento das fronteiras agrícolas, devido à demanda no mercado trouxe consigo o

desenvolvimento de cultivos geneticamente modificados e uso massivo de pesticidas

como ferramentas para melhora da produção agrícola. Consequentemente, muitos

ecossistemas podem ser afetados pela presença destes produtos tóxicos,

prejudicando espécies dos diferentes níveis das cadeias tróficas. Organismos

bentônicos como planárias são uns dos principais prejudicados pelo uso de tais

contaminantes, estes organismos desempenham um papel fundamental dentro das

cadeias tróficas, uma vez, que são predadores controladores de espécies de insetos

e outros organismos. Nesse sentido, este estudo teve como objetivo avaliar a

sensibilidade de Dugesia tigrina ante exposições de glifosato e amostras de água

recolhidas na Região hidrográfica Tocantins-Araguaia (RHTA), avaliar parâmetros

letais e sub-letais como alimentação, velocidade de locomoção da planária (VLMp),

regeneração e reprodução, ante a exposição de glifosato e amostras de água

recolhidas em áreas de intensa produção agrícola da RHTA. Para isso planárias

adultas foram expostas à diversas concentrações de glifosato e amostras de água

de RHTA. Avaliou-se a taxa de alimentação (larvas de Chironomus xantus

consumidas por hora), VLMp (linhas percorrida por minuto), Regeneração do

blastema (mm regenerados e tempo de regeneração) após 96 h de exposição e a

reprodução (número de casulos e crias produzidos por planária) durante cinco

semanas de exposição. Determinou-se a concentração letal para metade dos

organismos expostos (CL50) após 48 h de exposição de 37,06 mg·L-1 de glifosato.

Em exposições sub-letais de 96 h determinou-se a menor concentração com efeito

observável (MCEO) de 3,39 mg·L-1 de glifosato para VLMp e regeneração do

blastema. Em exposições sub-letais de 5 semanas, atingiu-se um MCEO de 1,71

mg·L-1 glifosato para a reprodução. Nas exposições das amostras de água de RHTA,

na época chuvosa a máxima diminuição da taxa de alimentação ocorreu no ponto 2

com 37,6% e a máxima diminuição VLMp ocorreu no ponto 5 com 36,44%. Na

estação seca, a taxa de alimentação foi afetada, com uma redução de 26,6% no

ponto 2 e na taxa fecundidade com uma diminuição de 79,77% no ponto R2. Nos

pontos mencionados foram detectados a presença de metais nas amostras de água.

Conclui-se que as respostas do comportamento, fisiológicas e reprodução de D.

tigrina foram sensíveis a exposições de glifosato e amostras de água contaminadas

da RHTA, e consequentemente, esta planária pode ser considerada um organismo

adequado para a realização de monitoramentos de tais ecossistemas, demonstrando

sensibilidade quando submetida a exposições por períodos curtos e longos de

poluentes.

Palavras-chave: efeitos sub-letais, metais, planárias, glifosato

viii

ABSTRACT

The increase in agricultural frontiers, due to demand in the market has brought with it

the development of genetically modified crops and massive use of pesticides as tools

for improvement of agricultural production. Consequently, many ecosystems are

damaging by the presence of these toxic products, harming species at different levels

of trophic chains. Benthic organisms such as flatworms are one of the main harmed

by the use of these contaminants, these organisms play a key role in food chains

because they are predators controlling species of insects and other organisms. In

this sense, this study aimed, to evaluate the sensitivity of Dugesia tigrina against

glyphosate exposure and water samples collected in the TAHR, evaluating the lethal

and sub-lethal parameters such as planarian locomotor velocity (pLMV), feeding rate,

regeneration and reproduction, at exposure of the herbicide glyphosate and water

samples collected in areas of intensive agricultural production of Tocantins-Araguaia

hydrographic region (TAHR). For these adult planarians were exposed to various

concentrations of glyphosate and TAHR water samples. We evaluated the feed rate

(Chironomus xanthus larvae consumed per hour), pLMV (lines crossed per minute),

regeneration blastema (mm regenerated and time to regenerate) after 96 hours

exposure and reproduction (number of cocoons and hatchlings produced by

planarian) after 5 weeks of exposure. I determined lethal concentration that kills half

of the organisms (LC50), after 48 hours of exposure: 37.06 mg·L-1 of glyphosate. In

sub-lethal exposures of 96 hours, I also determined the lowest observed effect

concentration (LOEC) 3.39 mg·L-1 glyphosate in the pLMV and regeneration of

blastema. In five weeks of sub-lethal exposures the LOEC is 1.71 mg·L-1 glyphosate

for reproduction. Exposures to water samples from the TAHR, in the rainy season the

maximum decrease in feed rate occurred at the point 2 with 37.6% and the maximum

decrease pLMV occurred at the point 5 with a 36.44%. In the dry season, the feed

rate was affected with a decrease 26.6% in point 2 and fecundity rate with a

decrease of 79.77% in the point R2. Points where the presence of metals in water

samples have been detected. The conclusion is that behavior responses,

physiological and reproduction of Dugesia tigrina were sensitive to glyphosate

exposure and water samples polluted of TAHR, and consequently, Dugesia tigrina

can be considered an adequate organism for performing monitoring of such

ecosystems, demonstrating sensitivity when subjected to exposures for short and

long periods of pollutants.

Keywords: sub-lethal effects, metals, planarians, glyphosate.

ix

SUMÁRIO

SUMÁRIO.................................................................................................................................. ix

LISTA DE ABREVIATURAS SÍMBOLOS E UNIDADES ............................................ xi

LISTA DE FIGURAS.................................................................................................. xii

CAPÍTULO I ............................................................................................................................. xii CAPÍTULO II ........................................................................................................................... xiii

LISTA DE TABELAS ................................................................................................. xv

CAPÍTULO I ............................................................................................................................. xv

ANEXOS ................................................................................................................... xv

INTRODUÇÃO GERAL ............................................................................................. 16

REFERÊNCIAS BIBLIOGRÁFICAS ..........................................................................................19

CAPITULO I .............................................................................................................. 22

Agricultural pollution in freshwater ecosystems and sub-lethal effects in Dugesia tigrina......................................................................................................................... 22

Abstract ....................................................................................................................................23

1. INTRODUCTION ................................................................................................ 25

2. MATERIAL AND METHODS .............................................................................. 27

2.1 Study área ........................................................................................................... 27

2.3 Animals ................................................................................................................ 30

2.3.1 Development and design test of behavioral responses .....................................................30 2.3.2 Feed rate .........................................................................................................................30 2.3.3 Locomotion velocity (pLMV) .............................................................................................30 2.3.4 Fecundity rate ..................................................................................................................31

2.4 Statistical analysis ............................................................................................... 31

3. RESULTS ........................................................................................................... 31

3.1 Wet season ....................................................................................................... 31

3.1.1 Feed rate .....................................................................................................................31 3.1.2 Planarian Locomotor velocity (pLMV) ...........................................................................32

3.2 Dry season ........................................................................................................ 33

3.2.1 Feeding rate .................................................................................................................33 3.2.2 Planarian Locomotor velocity (pLMV) ...........................................................................33 3.2.3 Fecundity rate ..............................................................................................................34

4. DISCUSSION ..................................................................................................... 35

5. CONCLUSIONS ................................................................................................. 39

6. REFERENCES ................................................................................................... 39

CAPÍTULO II ............................................................................................................. 46

Acute and chronic effects of glyphosate on the freshwater planarian Dugesia tigrina 46

Abstract ....................................................................................................................................47 Resumo ....................................................................................................................................48

1. INTRODUCTION ................................................................................................ 49

2.1 Test organism ................................................................................................. 50

2.2 Original Roundup ® preparation ..................................................................... 50

x

2.3 Experimental animals ..................................................................................... 51

2.3.1 Acute Test ...................................................................................................... 51

2.3.2 Sub-lethal effects ............................................................................................ 51

2.3.2.1 Planarian Locomotor velocity (pLMV) ......................................................... 51

2.3.2.2 Feeding rate ................................................................................................ 52

2.3.2.3 Regeneration............................................................................................... 52

2.3.3 Reproduction test............................................................................................ 52

2.3.3.1 Fecundity rate (Fc) and Fertility rate (Fr) ..................................................... 52

2.4 Chemical analysis ........................................................................................... 53

2.4.1 Chemical analysis of Glyphosate in water ................................................ 53

2.5 Statistical analysis ......................................................................................... 54

3. RESULTS ........................................................................................................... 54

3.1 Acute toxicity .................................................................................................. 54

3.2 Sub-Lethal exposures ..................................................................................... 55

3.2.1 Planarian Locomotor velocity (pLMV) ............................................................. 55

3.2.2 Feeding rate ................................................................................................... 56

3.2.3 Regeneration .................................................................................................. 56

3.2.3.1 Regeneration blastema ............................................................................... 56

3.2.3.2 Formation of photoreceptors ....................................................................... 57

3.2.3.3 Regeneration auricles ................................................................................. 58

3.3 Reproduction .................................................................................................. 59

3.3.1 Fecundity rate ................................................................................................. 59

3.3.2 Fertility rate ..................................................................................................... 59

4. Discussion .......................................................................................................... 61

5. CONCLUSIONS ................................................................................................. 63

6. REFERENCES ................................................................................................... 64

CONSIDERAÇÕES FINAIS ......................................................................................................73 Annexes ...................................................................................................................................74 Annex 1. Glyphosate concentrations measured in samples used in lethal and sub-lethal expositions (mean ± SD). ..........................................................................................................74

xi

LISTA DE ABREVIATURAS SÍMBOLOS E UNIDADES

SEM : Erro padrão da média

RD : Roundup

pLMV : Velocidade de locomoção da planaria

LC 50 : Concentração letal que produz a morte do 50% da população

FC : Fecundidade

Fr : Fertilidade

RHTA : Região hidrográfica Tocantins-Araguaia

NOEC: Concentração onde não se observam efeitos

LOEC: menor concentração com efeito observável

xii

LISTA DE FIGURAS

CAPÍTULO I

Fig. 1 - Study area, (a) reference R2, (b) rice fields in Formoso do Araguaia, (c) spraying of pesticides on soybeans in Lagoa da Confusão, (d) Pesticides bottles abandoned within soy plantations in Formoso of Araguaia, (e ) map of hydrographic region Tocantins Araguaia, adapted (AGÊNCIA NACIONAL DE ÁGUAS [ANA], 2009) The number denote the water collection points. _______ 29 Fig. 2 - Feeding rate (Number of larvae Chironomus xantus consumed for hour) of Dugesia tigrina, mean (±SEM), n = 10, after 96 hours of exposure to sample water of HRTA. Asterisks denote significant differences in comparisons with the control treatment (ASTM), ** p < 0.01, *** p < 0.001 (Dunnett's test). ______ 32 Fig. 3 - Planarian locomotor velocity (pLMV) of Dugesia tigrina (gridlines crossed /min), mean (±SEM), n = 10, after 96 hours of exposure to sample water of HRTA. Asterisks denote significant differences in comparisons with the control treatment (ASTM), *** p < 0.001 (Dunnett's test). ________________ 32 Fig. 4 - Feeding rate (Number of larvae Chironomus xantus consumed for hour) of Dugesia tigrina, mean (±SEM), n = 10, after 96 hours of exposure to sample water of HRTA. Asterisks denote significant differences in comparisons with the control treatment (ASTM), *** p < 0.001 (Dunnett's test). ________________ 33 Fig. 5 - Planarian locomotor velocity (pLMV) of Dugesia tigrina (gridlines crossed /min), mean (±SEM), n = 10, after 96 hours of exposure to sample water of HRTA. (Dunnett's test). __________________________________________ 34 Fig. 6 - Cumulative fecundity rate in Dugesia tigrina, mean (±SEM), n = 3, in five weeks exposure to sample water of HRTA. Asterisks denote significant differences in comparisons with the control treatment (ASTM), * p < 0.05 ** p < 0.01 *** p < 0.001 (Dunnett's test). __________________________________ 34

xiii

CAPÍTULO II

Fig. 1 - Lethal effects of glyphosate in Dugesia tigrina, after 48 hours of exposure. Expressed mean (±SEM), n =5. Asterisks denote significant differences in comparisons with the control treatment (ASTM), * p <0.05, *** p <0.001 (Dunnett's test). _________________________________________________ 55 Fig. 2 - Planarian locomotor velocity (pLMV) of Dugesia tigrina (gridlines crossed /min), mean (±SEM), n =15, after 96 h of exposure to sub-lethal glyphosate concentrations. Asterisks denote significant differences in comparisons with the control treatment (ASTM), *** p <0.001 (Dunnett's test). ______________________________________________________________ 55 Fig. 3 - Feeding rate (Number of larvae Chironomus xantus consumed for hour) of Dugesia tigrina, mean (±SEM), n =10, after 96 h of exposure to sub-lethal glyphosate concentrations. Asterisks denote significant differences in comparisons with the control treatment (ASTM), * p <0.05, *** p <0.001 (Dunnett's test). _________________________________________________ 56 Fig. 4 - Regeneration Blastema (length in mm) after 36h decapitation of Dugesia tigrina, mean (±SEM), n =10, after 96h exposure to sub-lethal glyphosate concentrations. Asterisks denote significant differences in comparisons with the control treatment (ASTM), ** p <0.01, *** p <0.001 (Dunnett's test). _________________________________________________ 57 Fig. 5 - Time (h) of regeneration of photoreceptors in D. tigrina, mean (±SEM), n =10, after 96h exposure to sub-lethal glyphosate concentrations. Asterisks denote significant differences in comparisons with the control treatment (ASTM), * p <0.05, *** p <0.001 (Dunnett's test). _______________________ 58 Fig. 6 - Time (h) of regeneration of auricles in D. tigrina, mean (±SEM), n =10, after exposure 96h to sub-lethal glyphosate concentrations. Asterisks denote significant differences in comparisons with the control treatment (ASTM), * p <0.05 (Dunnett's test). ____________________________________________ 58 Fig. 7 - Cumulative fecundity rate, in D. tigrina, mean (±SEM), n =3, in five week exposure to sub-lethal RD concentrations. Asterisks denote significant differences in comparisons with the control treatment (ASTM), *** p <0.001 (Dunnett's test). _________________________________________________ 59

xiv

Fig. 8 - Cumulative Fertility rate, in D. tigrina, mean (±SEM), n =3, in five week exposure to sub-lethal RD concentrations. Asterisks denote significant differences in comparisons with the control treatment (ASTM), *** p <0.001 (Dunnett's test). _________________________________________________ 60 Fig. 9 -Deformations in the photoreceptors and injuries, produced in first week of exposure to exposure to sub-lethal RD concentrations. Images show, (a) control without effects, (b) deformations of the photoreceptors (c) head injurie in D. tigrina a 14.91mg/L concentration. Expressed mean (±SEM), n =3. Stars denote significant differences from control treatment, ASTM, ** p <0.01, *** p <0.001 (Dunnett's test). ___________________________________________ 61

xv

LISTA DE TABELAS

CAPÍTULO I

Table 1. Parameters in rainy season, numbers represent sample water collected in different site of hydrographic region Tocantins Araguaia: Lagoa da Confusão (1-2), Formoso do Araguaia (3-5) ..................................................................... 36

Table 2. Parameters of dry season, numbers represent sample water collected in different site of hydrographic region Tocantins Araguaia: Lagoa da Confusão (1-2), Formoso do Araguaia (3-5), Reference site (R1, R2). ............................... 38

ANEXOS

Annex 1: Glyphosate concentrations measured in samples used in lethal and sub-lethal expositions (mean±SD)..................................................................... 74

Annex 2: Median lethal concentration (LC50) in 24, 48 and 96 hours of exposed D. tigrina to RD original, calculated as measured concentrations of active ingredient glyphosate. Probit Analysis and 95% CI. .......................................... 75

Annex 3: Analysis of organic and inorganic compounds in water samples of point 3 of hydrographic region Tocantins Araguaia. ......................................... 76

Annex 4: Live cycle of Dugesia tigrina, adults of three weeks (a), cocoons (b), hatchlings (c). ................................................................................................... 78

Annex 5: Feed rate ........................................................................................... 79

Annex 6: Planarian locomotor velocity (pLMV)................................................ 80

Annex 7: Fecundity (Fc) and fertility (Fr) rate ................................................... 81

Annex 8: Blastema regeneration, control treatment ........................................ 82

16

INTRODUÇÃO GERAL

O Brasil é um país com amplo desenvolvimento na agricultura, a qual outorga

um produto interno bruto de 5,2% (THE WORLD BANK, 2014). As grandes

monoculturas e interrupções dos habitats estão facilitando a propagação de pragas e

doenças nas culturas. Para o combate e controle destas pragas utilizam-se os

chamados pesticidas (TANG et al., 2013; LUPI et al., 2015), como por exemplo, os

herbicidas, inseticidas, fungicidas, nematicidas e raticidas (ONGLEY, 1996). Os

pesticidas são produtos de processos físicos, químicos ou biológicos destinados ao

uso em diversos setores de produção agrícola, proteção de ambientes urbanos,

industriais ou ecossistemas cuja finalidade seja alterar a composição da flora ou da

fauna, a fim de preservá-las da ação danosa de seres vivos considerados nocivos

(PRESIDÊNCIA DA REPÚBLICA, 2002).

No Brasil, foi analisado o impacto da legislação no registro de pesticidas,

onde se faz evidente o incremento de 584 (1990) a 863 (2000) registros de novos

produtos comercias de pesticidas, dos quais 58% admitem doses letais 50 (DL50) em

ratos que variam entre 200 mg·Kg-1 a mais de 2000 mg·kg-1 para formulações

líquidas e 50 mg·Kg-1 a mais de 500 mg·Kg-1 para formulações sólidas (GARCIA

GARCIA et al., 2005). Conforme o Estatuto da Legislação e Controle regulamentar

de pesticidas de saúde pública, no nível de países endêmicos a vetores, conclui-se

que existem deficiências nos quadros legislativos e regulamentares para os

pesticidas de saúde pública entre os estados membros da OMS. Esta situação

prejudica a utilização eficaz destes pesticidas para controlar os vetores que

transmitem doenças, os quais representam riscos desnecessários para a saúde

humana e para o ambiente (MATTHEWS et al., 2011).

Os pesticidas, além de cumprirem o papel de proteger as culturas agrícolas

das pragas, doenças e plantas daninhas, podem oferecer riscos à saúde humana e

ao ambiente ( FEOLA et al., 2011; AKOTO et al., 2013; ZHAO et al., 2014). O uso

incorreto de pesticidas pode causar a contaminação dos solos, da atmosfera, das

águas superficiais e subterrâneas, dos alimentos, apresentando consequentemente

efeitos negativos em organismos terrestres, aquáticos e intoxicação humana pelo

consumo de água e alimentos contaminados, assim como o risco de intoxicação

ocupacional de trabalhadores e produtores rurais (BEMPAH et al., 2011; ALAMDAR

et al., 2014; FREIRE et al., 2015).

17

Em vista da grande utilização de pesticidas aliada à falta de conhecimento

sobre seu comportamento, se dispõe como ferramenta básica da ecotoxicologia,

ciência que estuda os efeitos tóxicos nos organismos vivos, particularmente sobre as

populações e comunidades dentro de ecossistemas definidos; esses estudos

incluem as vias de entrada e transporte dos agentes em causa e a sua interação

com o ambiente. Muitos bioensaios “in situ” foram desenvolvidos para determinar a

sensibilidade das respostas de diversas espécies a poluentes ambientais, como por

exemplo: Daphnia magana, Chironomus riparius, Pseudokirchneriella subcapitata,

Danio rerio, Dugesia japonica (LIU et al., 2008; RODRIGUES et al., 2015; RIBEIRO

et al., 2014; BROCK, VAN WIJNGAARDEN, 2012). A maioria dos estudos utilizam

respostas biológicas (sobrevivência, crescimento, reprodução e desenvolvimento)

como critérios de determinação de toxicidade. Estas respostas são ecologicamente

relevantes, uma vez que, são componentes importantes do desenvolvimento, saúde,

estrutura e a dinâmica das populações (SIBLEY et al.,1997;CUHRA et al., 2013;

PATHIRATNE, KROON, 2015).

Os organismos que habitam os ecossistemas aquáticos são uns dos

principais afetados pela contaminação de tóxicos (AHMAD et al., 2015; CANUEL,

HARDISON, 2015). Estes organismos aquáticos, como por exemplo, as planárias

são predadores de mosquitos, larvas e outros organismos. (BLAUSTEIN, 1990).

Consequentemente, quando se fornecem mosquitos para um grupo de planárias,

estas começaram a mover-se para capturara-lo, e após inserção de sua faringe no

corpo do inseto, o alimento é sugado e digerido (SHEIMAN et al., 2002).

Nos últimos anos as planárias têm sido amplamente estudadas na

neurobiologia devido estes organismos possuírem um sistema nervoso bilobulado e

estruturas sensoriais, tais como, fotorreceptores e quimiorreceptores, assim, como a

facilidade de regeneração das partes amputadas, além de tratar-se de organismos

de fácil manutenção em laboratório ( MACRAE, 1964; MACRAE, 1967; REDDIEN,

SÁNCHEZ, 2004; OVIEDO et al., 2008). Em estudos biogeográficos e ecológicos,

foram cariotipadas um total de 140 planárias de água doce das espécies Girardia

schubarti, Girardia tigrina e Girardia anderlani, de 16 áreas do Rio Grande do Sul-

Brasil. Girardia tigrina foi detectada entre a vegetação nos corpos de água lênticos,

sendo organismos diplóides (2n = 16) e triplóides (3n = 24) (KNAKIEVICZ et al.,

2007). Diante disso, Dugesia (Girardia) tigrina surge como excelente espécie nativa

18

candidata para testes ecotoxicológicos de modo a avaliar o estado ecológico dos

ecossistemas aquáticos brasileiros. Contudo, para uma efetiva e regular utilização

de planárias como organismos modelo para avaliação ecotoxicológica torna-se

necessário o levantamento de informação referente à toxicidade de determinados

contaminantes nomeadamente pesticidas.

Este estudo ecotoxicológico tem como objetivo principal avaliar a

sensibilidade de Dugesia tigrina ante exposições do herbicida glifosato e amostras

de água recolhidas na Região hidrográfica Tocantins-Araguaia, avaliar parâmetros

letais (mortalidade), e também parâmetros sub-letais como alimentação, locomoção,

regeneração e reprodução ante a exposição ao herbicida Glifosato e amostras de

água recolhidas em áreas de intensa produção agrícola da Região Hidrográfica

Tocantins Araguaia.

19

REFERÊNCIAS BIBLIOGRÁFICAS

AHMAD, H.; YOUSAFZAI, A.M.; SIRAJ, M.; AHMAD, R.; AHMAD, I.; NADEEM, M.S.; AHMAD, W.; AKBAR, N.; MUHAMMAD, K. Pollution Problem in River Kabul : Accumulation Estimates of Heavy Metals in Native Fish Species. BioMed Research International, v. 2015, n. 537368, p. 7, 2015.

AKOTO, O.; ANDOH, H.; DARKO, G.; ESHUN, K.; OSEI-FOSU, P. Health risk assessment of pesticides residue in maize and cowpea from Ejura, Ghana. Chemosphere, v. 92, n. 1, p. 67–73, 2013.

ALAMDAR, A.; SYED, J.; MALIK, R.; KATSOYIANNIS, A.; LIU, J.; LI, JUN.; ZHANG,

G.; JONES, K. Organochlorine pesticides in surface soils from obsolete pesticide

dumping ground in hyderabad city, pakistan: Contamination levels and their potential

for air-soil exchange. Science of the Total Environment, v. 470-471, p. 733–741,

2014.

BEMPAH, C.K.; DONKOR, A.;YEBOAH, P.; DUBEY, B.; OSEI-FOSU, P. A. A

preliminary assessment of consumer’s exposure to organochlorine pesticides in fruits

and vegetables and the potential health risk in Accra Metropolis, Ghana. Food

Chemistry, v. 128, n. 4, p. 1058–1065, 2011.

BLAUSTEIN, L. Evidence for predatory flatworms as organizers of zooplankton and

mosquito community structure in rice fields. Hydrobiologia, v. 199, p. 179–191,

1990.

BROCK, T. C. M.; VAN WIJNGAARDEN, R. P. A. Acute toxicity tests with Daphnia

magna, Americamysis bahia, Chironomus riparius and Gammarus pulex and

implications of new EU requirements for the aquatic effect assessment of

insecticides. Environmental Science and Pollution Research, v. 19, n. 8, p. 3610–

3618, 2012.

CANUEL, E. A.; HARDISON, A. K. Sources, Ages, and Alteration of Organic Matter in Estuaries. Annual Review of Marine Science, v. 8, n. 1, p. annurev–marine–

122414–034058, 2015.

CUHRA, M.; TRAAVIK, T.; BØHN, T. Clone- and age-dependent toxicity of a

glyphosate commercial formulation and its active ingredient in Daphnia magna.

Ecotoxicology, v. 22, n. 2, p. 251–262, 2013.

FEOLA, G.; RAHN, E.; BINDER, C. R. Suitability of pesticide risk indicators for Less

Developed Countries: A comparison. Agriculture, Ecosystems & Environment, v.

142, n. 3-4, p. 238–245, 2011.

FREIRE, C.; KOIFMAN, R.; KOIFMAN, S. Hematological and hepatic alterations in

Brazilian population heavily exposed to organochlorine pesticides . J Toxicol

20

Environ Health A, v. 78, n. 8, p. 534-48, 2015.

GARCIA GARCIA, E.; BUSSACOS, M. A.; FISCHER, F. M. Impact of legislation on

registration of acutely toxic pesticides in Brasil. Revista de Saúde Pública, v. 39, n.

5, p. 832–839, 2005.

KNAKIEVICZ, T.; LAU, A. H.; PRÁ, D.; ERDTMANN, B. Biogeography and

karyotypes of freshwater planarians (Platyhelminthes, Tricladida, Paludicola) in

southern Brazil. Zoological science, v. 24, n. 2, p. 123–129, 2007.

LIU CL , XI YL , HUANG L, W. J. Impact of glyphosate and acetochlor on Dugesia

japonica ingestion and regeneration. Chinese journal of Applied Ecology, v. 19, n.

11, p. 2509–2514, 2008.

LUPI, L.; MIGLIORANZA, K.S.B.; APARICIO, V.C., MARINO, D.; BEDMAR, F.;

WUNDERLIN, D. A. Occurrence of glyphosate and AMPA in an agricultural

watershed from the southeastern region of Argentina. Science of The Total

Environment, v. 536, p. 687–694, 2015.

MACRAE, E. K. Observations on the fine structure of photoreceptor cells in the

planarian Dugesia tigrina. Journal of ultrastructure research, v. 10, n. 3, p. 334–

349, 1964.

MACRAE, E. K. The fine structure of sensory receptor processes in the auricular

epithelium of the planarian, Dugesia tigrina. Zeitschrift für Zellforschung, v. 82, n.

4, p. 479–494, 1967.

MATTHEWS, G.; ZAIM, M.; YADAV, R.S.; SOARES, A.; HII, J.; AMENESHEWA, B.;

MNZAVA, A.; DASH, A. P.; EJOV, M.; TAN, S.H.; VAN DEN BERG, H. Status of

legislation and regulatory control of public health pesticides in countries endemic with

or at risk of major vector-borne diseases. Environmental Health Perspectives, v.

119, n. 11, p. 1517–1522, 2011.

ONGLEY, E. D. (FAO) Irrigation and Drainage Paper 55: Control of Water

Pollution from Agriculture. [s.l: s.n.]. Disponível em:

<http://www.fao.org/docrep/W2598E/W2598E00.htm>.

OVIEDO, N. J.; NICOLAS, C. L.; ADAMS, D. S.; LEVIN, M. Establishing and

maintaining a colony of planarians. CSH protocols, v. 2008, p. pdb.prot5053, 2008.

PATHIRATNE, A.; KROON, F. J. Using species sensitivity distribution approach to

assess the risks of commonly detected agricultural pesticides to Australia’s tropical

freshwater ecosystems. Environmental Toxicology and Chemistry, v. Accepted A,

2015.

21

PRESIDÊNCIA DA REPÚBLICA. DECRETO No 4.074, DE 4 DE JANEIRO DE 2002

Regulamenta. Brasil-Casa Civil , Subchefia para Assuntos Jurídicos, , 2002.

Disponível em: <http://www.mma.gov.br/port/conama/legiabre.cfm?codlegi=515>

REDDIEN, P. W.; SÁNCHEZ ALVARADO, A. Fundamentals of Planarian

Regeneration. Annual Review of Cell and Developmental Biology, v. 20, n. 1, p.

725–757, 2004.

RIBEIRO, F.; GALLEGO-URREA, J. A.; JURKSCHAT, K.; CROSSLEY, A.;

HASSELLÖV, M.; TAYLOR, C.; SOARES, A M V M.; LOUREIRO, S. Silver

nanoparticles and silver nitrate induce high toxicity to Pseudokirchneriella

subcapitata, Daphnia magna and Danio rerio Fabianne. Science of the Total

Environment, v. 466-467, p. 232–241, 2014.

RODRIGUES, A.C.M.; GRAVATO, C.; QUINTANEIRO, C.; GOLOVKO, O.; ŽLÁBEK,

V., BARATA, C.; SOARES, A M.V.M.; PESTANA, J. L. T. Life history and

biochemical effects of chlorantraniliprole on Chironomus riparius. Science of The

Total Environment, v. 508, p. 506–513, 2015.

SHEIMAN, I. M.; ZUBINA, E. V; KRESHCHENKO, N. D. Regulation of the Feeding

Behavior of the Planarian Dugesia ( Girardia ) tigrina. Journal of Evolutionary

Biochemistry and Physiology, v. 38, n. 4, p. 414–418, 2002.

SIBLEY, P. K.; BENOIT, D. A.; ANKLEY, G. T. The significance of growth in

Chironomus tentans sediment toxicity tests: Relationship to reproduction and

demographic endpoints. Environmental Toxicology and Chemistry, v. 16, n. 2, p.

336–345, 1997.

TANG, S.; LIANG, J.; TAN, Y.; CHEKE, R.A. Threshold conditions for integrated pest

management mdoels with pesticides that have residual effects. Journal of

mathematical biology, v. 66, p. 1–35, 2013.

THE WORLD BANK. Agriculture, Value added (% of GDP). Disponível em:

<http://data.worldbank.org/indicator/NV.AGR.TOTL.ZS/countries/BR?display=graph>

ZHAO, M.; ZHANG, Y.; ZHUANG, S.; ZHANG, Q.; LU, C.; LIU, W. Disruption of the

hormonal network and the enantioselectivity of bifenthrin in trophoblast: maternal-

fetal health risk of chiral pesticides. Environmental science & technology, v. 48, n.

14, p. 8109–16, 2014.

22

CAPITULO I

Agricultural pollution in freshwater ecosystems and sub-lethal

effects in Dugesia tigrina

23

Agricultural pollution in freshwater ecosystems and sub-lethal effects in

Dugesia tigrina

Abstract

The increasing agricultural production in consequence of the market demand has

generated the development of crop improvements and increased production

techniques. The genetically modified crops (GMC) and physical-chemical products

are some of the tools used in agriculture for production improvements. The present

study suggests the use of the sp. Dugesia tigrina (Girard) for biomonitoring the

aquatic ecosystem of the watershed Araguaia-Tocantins, area of intense agricultural

production. It was used adult planarians to assess behavioral responses to the

exposure of water samples from different parts of the Tocantins-Araguaia region,

areas of intensive agricultural production in two seasons (wet and dry). We evaluated

the feed rate (number of larvae Chironomus xantus consumed per hour), planarian

locomotor velocity (pLMV) (crossed lines per minute) and the fertility rate (number of

cocoons produced per week). In the wet season, feeding rate and pLMV were

affected by the presence (above permitted levels) of Cl and any of these metals as

Al, Fe, Zn were found in points 1, 2, 4, 5 respectively, the maximum decrease in feed

rate occurred in point 2 (37.6%) and pLMV with 36.40% in the point 4. In the dry

season, feeding rate and fertility rate were affected, with a decrease in the maximum

point 2 (26.6%) and in point 5 (91.7%), respectively. Our results showed that the

behavioral responses of D. tigrina to short and long exposures can be used for

freshwater ecosystems monitoring.

Key-words: Biomonitoring; Metals; Locomotion; Feed rate; Fecundity rate.

24

Poluição agrícola em ecossistemas de água doce e efeitos sub-letais em

Dugesia tigrina

Resumo

O aumento da produção agrícola, em consequência da demanda do mercado tem

gerado o desenvolvimento de técnicas de melhoramento das culturas e aumento da

produção. As culturas geneticamente modificadas (GMC) e produtos físico-químicos

são algumas das ferramentas utilizadas na agricultura para a melhoria da produção.

O presente estudo sugere a espécie Dugesia tigrina (Girard) como organismo

modelo para o biomonitoramento do ecossistema aquático da bacia Araguaia-

Tocantins, áreas de intensa produção agrícola. Utilizou-se planárias adultas para

avaliar respostas comportamentais para a exposição de amostras de água de

diferentes partes da região do Tocantins-Araguaia, áreas de produção agrícola

intensiva em duas épocas (chuva e seca). Foram avaliadas a taxa de alimentação

(número de larvas de Chironomus xantus consumido por hora), a velocidade de

locomoção das planárias (pLMV) (linhas cruzadas por minuto) e a taxa de

fecundidade (número de casulos produzido por semana). Na estação chuvosa, a

taxa de alimentação e pLMV foram afetados pela presença (acima dos níveis

permitidos) de Cl e alguns metais como Al, Fe, Zn que foram encontrados nos

pontos 1, 2, 4, 5, respectivamente. A queda máxima na taxa de alimentação ocorreu

no ponto 2 (37,6%) e pLMV com 36,40% no ponto 4. Na estação seca, a taxa de

alimentação e taxa de fecundidade foi afetada, com uma redução máxima no ponto

2 (26,6%) e no ponto 5 (91,7%), respectivamente. Os resultados mostraram que as

respostas comportamentais e reprodutivas de D. tigrina a exposições curtas e longas

podem ser usadas para monitoramento de ecossistemas de água doce.

Palavras-chave: biomonitoramento; locomoção; metais; taxa de alimentação; taxa

de fecundidade.

25

1. INTRODUCTION

Brazil is a country rich in the availability of land and water in abundance, a

factor that contributes to the success of the agricultural holding, giving 5.2% of GDP.

(THE WORLD BANK, 2014). In this scenario, the Tocantins state is consolidated as

the new agricultural frontier of the country, strategically located by environmental

conditions and available water resources. Agricultural holding on a large scale refers

to opening new areas for the implementation of monocultures (SAWYER, 2008), for

example of soybeans, corn, watermelon, rice, sugar cane, eucalyptus, etc.

(CAMPANHA et al., 2005; CONAB, 2014; MARINHO et al., 2014). This fact has

facilitated the spread of diseases, pests and weeds in crops (SOUZA et al., 2014;

SOARES et al., 2015 KOWALCZUCK et al., 2012; GUERRA et al., 2012; OLIVEIRA ,

FREITAS, 2008). Thus for achieving high productivity, pesticides have been the most

efficient manner in short-term control adopted (TANG et al., 2013; GREEN, 2014;

EDWARDS et al., 2014; TABASHNIK et al., 2014).

In the context of high production rates, the addition of pesticides such as

agricultural fertilizers have been used in various steps of the production cycle of

cultures, whereas such compounds become subject to environmental contamination

(BURNEY et al., 2010; KAYSER et al., 2015). However, the use of these compounds

in agriculture is not unanimous, although its impacts can be considered as positive by

some segments of society, for others its impacts are seen as negative, mainly

through the environmental point of view (SIMBERLOFF et al., 2013).

In order to overcome these problems, genetically modified crops (GMC) were

developed - e.g. GCM resistant to glyphosate (soybeans, corn, cotton) (SOUZA et

al., 2014; MEYER, CEDERBERG, 2010; MONSANTO, 2015a; MONSANTO, 2015b).

Scientific research showed that environmental stress can promote changes in

the biology and ecology of weeds (FRIED et al., 2009; BÜRGER et al., 2015; SMITH

et al., 2016), herbivores, and their interactions (HARMON, BARTON, 2013;

WHALEN, HARMON, 2015). Among the midst of ecological interactions, altered by

environmental pressure, non-target organisms are significantly affected, as in the

case of pollinating bees (VAN DER SLUIJS et al., 2013). It is noteworthy that even

those compounds considered environmentally-friendly are likely to cause

environmental imbalances (XAVIER et al., 2015).

26

On the other hand, the environmental imbalance can cause the appearance of

other pests (SZCZEPANIEC et al., 2011), and these in turn can be controlled, they

are subjected to abusive applications of pesticides which generate residual effects of

varying times (TANG et al., 2013b). Thus, in an agricultural area, size of water

resources as in the state of Tocantins, the aquatic ecosystem ultimately be affected,

either by evaporation, leaching (with subsequent contamination of groundwater), or

runoff of these compounds by the action of rain (AMARAL et al. 2008; SÁNCHEZ-

BAYO et al., 2013; ANDERSON et al., 2015).

It is also known that the various global warming resulting from the

environmental pressure has caused changes in aquatic ecosystems, with regard to

the physical, chemical and biological water, such as nitrogen deposition,

eutrophication, increased acidification (carbonates, sulphates and nitrates) as well as

reduction of dissolved oxygen caused by the continuous rise in temperature (EISSA,

ZAKI, 2011). Such factors may adversely affect aquatic organisms as well as having

influence in combined action with pesticides and other waste from the production of

crops process.

At the level of the trophic chain, the most sensitive organisms are those most

affected, and in fact, contamination of the aquatic ecosystem, whether for

bioaccumulation, or direct exposure, will expose humans to various contaminants

(COSTANTINI, 2015). Thus, it is noted that contamination of aquatic ecosystems has

been monitored by testing on bioindicators, which reflects the state of that specific

environment (BROCK, VAN WIJNGAARDEN, 2012; CONNON et al., 2012; FOLMAR

et al., 1979; JESUS et al., 2013).

There are a plethora of studies on the ecotoxicological biomonitoring

parameters in order to predict future implications of higher levels of organization in

the ecosystem (BROCK, VAN WIJNGAARDEN, 2012; CUHRA et al., 2013; SIBLEY

et al., 1997). Model organisms in ecotoxicology as Daphnia magna, Chironomus

riparius, Danio rerio, etc. are widely used to assess behavioral responses such as

feeding, breeding and growing (LIU et al., 2015; RODRIGUES et al., 2015;

VEHNIÄINEN; KUKKONEN, 2015). These responses are important parameters in

monitoring changes and impact on ecosystems (FLEEGER et al., 2003; WEIS et al.,

2001) .

27

Planarians (phylum Platyhelminthes, class Turbellaria, order tricladida) are

living organisms, that play a major key role in the trophic chain, since they are

primarily predators of insects, larvae and other freshwater invertebrates (BOLL et al.,

2015). These organisms are made up of a nervous system lobed, with sensory

structures such as photoreceptors and auricles (MACRAE, 1964, 1967). These

organisms are easy to maintain and manipulate in experimental tests at laboratory

level. These characteristics make the planarians a model organism in

ecotoxicological studies for evaluation and monitoring of freshwater ecosystems

(OVIEDO et al., 2008; ELLIOTT, SÁNCHEZ, 2013).

This study pretends to evaluate the behavioral response (feed rate, planarian

locomotion mobility) and reproduction of D. tigrina against the exposure of water

coming from watershed Araguaia-Tocantins, in areas subject to agricultural pressure

in the dry and wet season.

2. MATERIAL AND METHODS

2.1 Study área

Water samples Tocantins-Araguaia Hydrographic region were collected in four

municipalities, Formoso do Araguaia (S 11º 48' 02,8" and W 049º 37' 26,3"), Lagoa

da Confusão (S 10º 50' 64,3" and W 049º 42' 51,0"). Water samples were collected

in areas of intense agricultural production. The local reference is located far from

sources of pollution (S 10º 48' 38, 00" and W 49º 38' 52,00") Samplings were

conducted at two seasons, wet season (Janeiro 2014) and dry season (September

2015).

28

Description of the sampling points (Fig 1):

ASTM: Basic mineral salts medium, considered Control.

Reference 1: lake with crystal clear water, located near the city and far away from

the crops.

Reference 2: Output of water between rocks fountain, about 5 km from the extraction

of calcareous and away from crops.

Point 1: water flows out by irrigation canals within the Lagoa da Confusão crops.

Point 2: water flows out by the Urubu river, water that feeds some of the plantations

in Lagoa da Confusão.

Point 3: water dammed "Taboca" captured the river Formoso, Formoso with which

the project is irrigated.

Point 4: water flows out by small irrigation canals crop in Formoso do Araguaia.

Point 5: water flows out of large irrigation canals crop in Formoso do Araguaia.

The collection of water in wet season was conducted in areas with

developmental soybean and rice, the point 5 has high dissolution power. The

collection of water in dry season was conducted in areas with crop soybean. Water

collection was carried out in plastic PET bottles, as specified by ANA (2011). After

transported to the laboratory of Ecotoxicology at the Federal University of Tocantins

and stored at -20°C for later analysis.

29

R1

R2

12

54

3

a b c d

e

Fig. 1 - Study area, (a) reference R2, (b) rice fields in Formoso do Araguaia, (c) spraying of pesticides on soybeans in Lagoa da Confusão, (d) Pesticides bottles abandoned within soy plantations in Formoso of Araguaia, (e ) map of hydrographic region Tocantins Araguaia, adapted (AGÊNCIA NACIONAL DE ÁGUAS [ANA], 2009) The number denote the water collection points.

30

2.2 Water analysis

In the wet season, the analysis of water samples were performed by the

laboratory CONÁGUA AMBIENTAL laboratory. This study revealed the organic and

inorganic compounds. (Annex 3). In the dry season, the analysis of water sample is

performed in Federal University of Viçosa, this study identify the presence of

pesticides.

2.3 Animals

Dugesia tigrina was cultivated with ASTM hard water (ASTM, 1980) medium,

at 22ºC ± 1ºC, dark, and constant aeration . The population was fed twice a week

with bovine liver. Except 96 hours prior to and during each test, when they were not

fed (Annex 4).

2.3.1 Development and design test of behavioral responses

Dugesia tigrina (0.862 ± 0,098 cm, total length) were exposed for four days to

water sample, collected in different site in hydrographic region Tocantins Araguaia.

Exposure 10 organism per treatment in crystal dishes with 20 ml of water sample.

2.3.2 Feed rate

After 96 hours of exposure, transfer of individual planarian to crystallizing

dishes with 20 ml ASTM medium. Freed is for twenty larvae of C. xantus (six days’

ages) per planarian. Feed rate per hour is determined with number of larvae

consumed per planarian for 12 hours (Annex 5).

2.3.3 Locomotion velocity (pLMV)

Aluminum plate (35 cm of diameter) is adhered millimeter paper (grid lines

spaced 0.1 cm apart). Additional 300 ml of ASTM medium, calculated the pLMV, with

grid lines prerecorded of minute (Annex 6).

31

2.3.4 Fecundity rate

Dugesia tigrina (1.25 ± 0.088 cm, total length) were exposed for 5 weeks to

water sample, collected in different site in hydrographic region Tocantins Araguaia.

Exposure 12 organisms and three replicates in glass beakers, containing 100 mL of

water sample Concentration was replaced each week, after feeding with bovine liver.

Conditions test, dark, temperature (22°C ± 1°C). Evaluated fecundity rate for week,

number of cocoons produced by sexual reproducing animals, methodology described

by KNAKIEVICZ et al. (2006). Control treatment (ASTM) is maintained in the same

conditions (Annex 7).

2.4 Statistical analysis

The behavioral responses of D. tigrina (pLMV, feeding rate and fecundity rate)

when they exposed to various water samples of hydrographic region Tocantins

Araguaia, compared with the Control (ASTM), using one-way ANOVA with Dunnett's

Multiple Comparison test, was performed using GraphPad Prism version 5.03 for

Windows, GraphPad Software, San Diego California USA, www.graphpad.com.

Verification existence of linear association was assessed using D'Agostino &

Pearson omnibus normality test and homoscedasticity was verified by Brown–

Forsythe test.

3. RESULTS

3.1 Wet season

3.1.1 Feed rate

Feeding rate per hour of D. tigrina was significantly (F(6,63) = 6.25, p < 0.001,

Fig. 2) with compare of control. Planarian when exposed to the sample water 1, 2

and 5 decreased by 27.5, 37.6 and 26.4% respectively, when compared to control

treatment (ASTM).

32

ASTM R1 1 2 3 4 50.0

0.5

1.0

1.5

2.0

***

****

Samples hydrographic region:Tocantins Araguaia

Fee

din

g r

ate

Fig. 2 - Feeding rate (Number of larvae Chironomus xantus consumed for hour) of Dugesia tigrina, mean (±SEM), n = 10, after 96 hours of exposure to sample water of HRTA. Asterisks denote significant differences in comparisons with the control treatment (ASTM), ** p < 0.01, *** p < 0.001 (Dunnett's test).

3.1.2 Planarian Locomotor velocity (pLMV)

Locomotion velocity (pLMV) of D. tigrina, measured lines crossed per min, was

significantly (F(6,63) = 11.05, p < 0.0001, Fig. 3), decreases after 96 hours exposure to

water samples, with a decreases of 34.52, 26.88, 31.97, 34.69 and 36.40% in the

sample R1, 1,2,3,4, when compared to control treatment(ASTM).

ASTM R1 1 2 3 4 5

0

5

10

15

20

25


******

***

******

pL

MV

(g

rid

lin

es c

ross

ed /

min

)

Fig. 3 - Planarian locomotor velocity (pLMV) of Dugesia tigrina (gridlines crossed /min), mean (±SEM), n = 10, after 96 hours of exposure to sample water of HRTA. Asterisks denote significant differences in comparisons with the control treatment (ASTM), *** p < 0.001 (Dunnett's test).

33

3.2 Dry season

3.2.1 Feeding rate

Feeding rate of D. tigrina was significantly (F(7, 72) = 5.588, p < 0.001, Fig. 4).

When exposed to the sample water 2 (Lagoa da Confusão) decreased by 26.26%

the feeding rate, when compared to control treatment (ASTM).

ASTM R1 R2 1 2 3 4 50.0

0.5

1.0

1.5

*

Endopoints hydrographic region:Tocantins Araguaia

Fee

din

g r

ate

Fig. 4 - Feeding rate (Number of larvae Chironomus xantus consumed for hour) of Dugesia tigrina, mean (±SEM), n = 10, after 96 hours of exposure to sample water of HRTA. Asterisks denote significant differences in comparisons with the control treatment (ASTM), *** p < 0.001 (Dunnett's test).


Locomotion velocity of Dugesia tigrina was not significantly (p > 0.05), when

compared to control treatment (ASTM) (Figure 5).

34

ASTM R1 R2 1 2 3 4 50

10

20

30

Endopoints hydrographic region:Tocantins Araguaia

pL

MV

(g

rid

lin

es c

ross

ed /

min

)

Fig. 5 - Planarian locomotor velocity (pLMV) of Dugesia tigrina (gridlines crossed /min), mean (±SEM), n = 10, after 96 hours of exposure to sample water of HRTA. (Dunnett's test).


During the five weeks of assessment, D. tigrina showed different fecundity

rates. Fecundity rate (Fc) of D. tigrina was significantly (F(7,16) =26.68 p < 0.001 Fig.

6), when compared with the control (ASTM), decreases in 30.34, 79.77, 24.72, 30.34,

44.94 and 76.40% when exposed to water samples R1, R2,1, 3, 4.5 respectively.

ASTM R1 R2 1 2 3 4 50

1

2

3

**

***

***

***

***


Cu

mu

lati

ve

fec

un

dit

y r

ate

Fig. 6 - Cumulative fecundity rate in Dugesia tigrina, mean (±SEM), n = 3, in five weeks exposure to sample water of HRTA. Asterisks denote significant differences in comparisons with the control treatment (ASTM), * p < 0.05 ** p < 0.01 *** p < 0.001 (Dunnett's test).

35

4. DISCUSSION

The results of this study showed behavioral responses of D. tigrina exposure

to water samples from different parts of the Tocantins Araguaia region. Behavioral

responses and reproduction are the result of synergistic or antagonistic interactions

of the physicochemical parameters of each sample. The alteration of some of the

parameters can influence positively or negatively the organisms. For better

understanding and interpretation of these results the detection of contaminants in the

water was achieved. The behavioral responses of D. tigrina changed when exposed

to different water samples from the two seasons (wet season and dry season) (Table

1).

In the wet season, the water collected was performed in canals for irrigation of

soybean and rice, the point 5 has a high dissolving power. The feeding rate and

pLMV were affected by the presence of metals in the water samples of TAHR. These

effects produced in the behavior D. tigrina were caused by the presence of some

metals in water samples (Table 1). The maximum decrease in feed rate was given in

the sample 2, where a high concentration of zinc in water with 0.438 mg·L-1 (2.4

levels more than allowed). In the water samples did not report the presence of any

type of pesticide. Analysis of water samples in the reference point, any contaminate

was not found. Analyses of water samples revealed the presence of contaminants in

some collection points. In point 1 it was detected the presence of 0.04 mg·L-1 of total

chlorine (allowable levels: 0.01 mg·L-1), which caused a decrease of 27.5 and 26.8%

in the feed rate and pLMV respectively in D. tigrina. In point 4 they reported 0.105

mg·L-1 of aluminum and dissolved iron 0.5358 mg·L-1 (1.79 levels of maximum

allowable concentration), which caused a decrease in 36.4% pLMV. In point 5

feeding rate decreased by 26.4% was caused by the presence of 0.335 mg·L-1

dissolved iron (slightly above allowable concentration). This level of Al affects

neurotoxicity, cytotoxicity and causes oxidative stress (GARCÍA-MEDINA et al., 2011;

FERNÁNDEZ-DÁVILA et al., 2012; KUMAR, GILL, 2014).

The level of metal in the water of the reservoir was lower than the maximum

set forth in the legislation, except for that of Cd and Fe. In sediments, Cu, Cd, Cr, and

Ni presented concentrations above the threshold effect level (TEL). Pb and Cr were

above the limits for the Geophagus brasiliensis. The tendency of metals present in

the muscles is Al > Cu > Zn > Fe > Co > Mn > Cr > Ag > Ni > Pb > Cd > As. In the gills, it

36

was Al > Fe > Zn > Mn > Co > Ag > Cr > Ni > Cu > As > Pb > Cd, and the liver presented

Al > Cu > Zn > Co > Fe > Mn > Pb > Ag > Ni > Cr > As > Cd. The bioconcentration and

bioaccumulation of metal in the tissues follow the global tendency

liver > gills > muscle (VOIGT et al., 2015).

Table 1. Parameters in wet season, numbers represent sample water collected in different site of hydrographic region Tocantins Araguaia: Lagoa da Confusão (1-2), Formoso do Araguaia (3-5)

Parameters

Allowable

limit

Sample of hydrographic region Tocantins Araguaia

R 1 2 3 4 5

Total dissolved solids (TDS) 500 mg·L-1 8.17 32.06 66.22 62.81 11.82 11.82

Turbidity 100 1.84 4.8 3.35 2.39 3.8 3.36

Chlorophyll a µg·L-1 500 µg·L-1 2.16 12.02 5.29 1.68 1.77 1.2

pH 6-9 7.06 6.69 6.49 6.61 5.44 7.1

DO mg·L-1 > 5 5.4 4.7 3.9 6 6.2 7

Cyanobacterias cel·ml-1 50 000 cel·ml-1 0 1430 1258 6977 0 3603

Dissolved aluminum mg·L-1 0.1 mg·L-1 0 0 0 0 0.105 0

Total chlorine mg·L-1 0.01 mg·L-1 0 0.04 0 0 0 0

Dissolved iron mg·L-1 0.3 mg·L-1 0 0 0 0 0.5358 0.335

Total Zinc mg·L-1 0.18 mg·L-1 0 0 0.438 0 0 0

Surfactants mg·L-1 U 0.74 0.76 0.64 0.63 0.61 0.68

Glyphosate 60 ug·L-1 LQ LQ LQ LQ LQ LQ

Thermotolerant coliforms MPN

1000/100 ml <180 <180 <180 <180 < 180 <180

U- unregulated ; MPN- Most Probable Number

In experiments with algae, Zinc it was shown to affect both the rate of growth

of Chlamydomonas reinhardtii and Cyanidium caldarium. showed the IC50 of free

Zn2+ for C. caldarium was 4.87 mg·L-1 and 0.038 mg·L-1 for C. reinhardtii (MIKULIC;

BEARDALL, 2014). Exposures of earth worms (Eisenia foetida) in soil contaminated

with metals such as concentrations As (280 ± 30), Zn (136 ± 3), Cu (11.3 ± 0.7)

mg·kg-1 for 21 days caused significant mortality and differences in body weight

(GARCÍA-GÓMEZ et al., 2014). Above 3 x 10-5 M CuSO4 concentrations caused

significant damage in DNA in cells Dugesia (Girardia) schubarti (GUECHEVA et al.,

2001). Enrichment factor of heavy metals indicate Cd > Pb > Zn > Cu > Cr > Co > Mn

> Fe pattern of enrichment from higher to lower impact structure. Scale of pollution

indicates most of the sampling points are practically uncontaminated to moderately

contaminated by Fe and Mn along with moderately contaminated by Cu, Co, Cr, and

37

Zn in most of the cases. It can be said that the non-conservative nature of these

elements is governed by several geochemical removal processes like flocculation of

dissolved organic matter, adsorption onto suspended matter, and freshly precipitated

oxy-hydroxides of Fe and Mn. The distribution, mobility, and bio-availability of these

elements can be different for equal total concentrations, given the different nature

and properties of the materials build up the concerned aquifer unit. In addition, trace

elements are unevenly distributed in a regional scale (spatial CA) that readily

influences their availability to water environment. Rather in a local perimeter, actual

contamination behavior of heavy metals may be controlled not only by its different

fractions but changes in ground water pH and salinity, CO3, and HCO3 profile along

with other nutrient contents that also control the impact structure (HARICHANDAN et

al., 2013).

In the dry season, water samples were collected during the soybean crop, so

the presence of water was low, with little power of dissolution. The effects observed

in the feed rate in point 2, is probably due to the presence of metals found in the

analysis of the wet season. At this time the analysis of physical-chemical parameters

were not performed, only quantified the presence of three pesticides (Glyphosate,

Thiamethoxam and cyproconazole), but were not found traces of these contaminants

in the water. Fecundity rate decreases significantly (p < 0.001) in the points R1, R2,

3, 4 and 5, the decreased in R2 (spring water), is likely Groundwater contamination

bay salt produced by one of the calcareous which it is 5 km where there extraction of

Ca, Mg, the conductivity in this point is superior to the other points evaluated (Table

2). The fecundity rate decline in the rest of the points is probably due to the presence

of metals and other compounds.

In research was calculated LC50: 42 ± 0.08 mg·L-1 for adult D. tigrina, after 96

hours exposure to Cu+2. Behavior planarian was influenced to exhibit Cu+2, mL OAEC

observed adult intact, with values of 0.40 mg·L-1 (24 hours and 72 hours) and 0.20

mg·L-1 of Cu+2. (48 and 96 hours). Chronic exposure (5 weeks) of Cu+2 showed a

detectable effect on reproductive performance after exposure to Cu+2, low

concentrations as 0.05 mg·L-1 makes fertility and fecundity good biomarkers for

evaluating Cu+2 effects of chronic exposure (KNAKIEVICZ; FERREIRA, 2008). The

48 median lethal concentration that killed 50% of individuals (LC50) were calculated

as 6.31 mg·L-1 Cu2+ of Dugesia japonica (ZHANG et al., 2014).

38

Table 2. Parameters of dry season, numbers represent sample water collected in different site of hydrographic region Tocantins Araguaia: Lagoa da Confusão (1-2), Formoso do Araguaia (3-5), Reference site (R1, R2).

Sample of hydrographic

region Tocantins Araguaia

Parameters: Mean (±SD)

pH Condutivity (µs/cma)

OD (mg·L-1)

Temperature (°C)

ASTM 7.5 (±0.015) 560 (±0.35) 5.5 (±0.01) 21 (±0.1)

R1-Reference 7.14 (±0.03) 13.9 (±0.2) 4.9 (±0.01) 21 (±0.1)

R2-reference 7.72 (±0.01) 566 (±3) 4.9 (±0.08) 21 (±0.5) 1 7.05 (±0.02) 37.4 (±0.015) 5 (±0.06) 21.2 (±0.1) 2 6.57 (±0.02) 27.5 (±0.1) 4.2 (±0.01) 21.8 (±0.05)

3 7.88 (±0.01) 53.1 (±1.95) 5.4 (±0.01) 21.2 (±0.29)

4 7.94 (±0.02) 115.9 (±1) 4.9 (±0.02) 21.3 (±0.26) 5 7.91 (±0.01) 124.8 (±0.95) 5.1 (±0.02) 21.3 (±0.30)

Study indicates that both the nano-size and ionic dissolution play a significant

role in the cytotoxicity of ANPs towards freshwater algae, and the exposure period

largely determines the prevalent mode of nano-toxicity (PAKRASHI et al., 2013).

Metals bioconcentration in Chironomus javanus increases with exposure to

increasing concentrations and Cd was the most toxic to C. javanus, followed by Cu,

Fe, Pb, Al, Mn, Zn and Ni (Cd > Cu > Fe > Pb > Al > Mn > Zn > Ni) (SHUHAIMI-

OTHMAN et al., 2011). Research shows the impact of iron oxide, nanocomposite of

cadmium sulfide and silver sulfide, cadmium sulfide and silver sulfide nanoparticles

(NPs) on a fresh water alga Mougeotia sp. On day five all the NPs showed toxicity

except iron oxide NPs which enhanced the growth in lower concentrations (0.1 and 1

mg·L-1), but it induced the toxicity in higher concentration of iron oxide NPs (in 5, 10

and 25 mg·L-1 NPs). Compared to the other NPs in this study, the iron oxide NPs

showed the least toxic effect. Lipid peroxidation and ROS generation were increased

upon NPs treatment. NPs exposures suppressed the antioxidant defense system,

thereby increasing the oxidative stress, leading to the death of the cells

(JAGADEESH et al., 2015).

39

5. CONCLUSIONS

The tributaries of the hydrographic region Araguaia-Tocantins comprising the

municipalities of Formoso do Araguaia and Lagoa da confusão exhibit contamination

by heavy metals and other pollutants, which caused chronic effects on freshwater

planarian D. tigrina.

Dugesia tigrina can be used as an organism for biomonitoring ecosystems

lotic.

Acknowledgements

We thank to Coordination for the Improvement of Higher Education Personnel

(CAPES), and the Federal University of Tocantins for financial support.

6. REFERENCES

AGÊNCIA NACIONAL DE ÁGUAS (ANA). Plano estratégico de recursos hídricos da bacia hidrográfica dos rios Tocantins e Araguaia. Brasília: [s.n.].

AGÊNCIA NACIONAL DE ÁGUAS (ANA). Guia nacional de coleta e preservação

de amostras: água, sedimento, comunidades aquáticas e efluentes líquidos.

São Paulo, 2011.

AMARAL N.R.; DE OLIVEIRA, R.L.; DE LIMA, J.M.; GUERREIRO, M.C. Lixiviação

do inseticida thiamethoxam em macrolisímetros de duas classes de solo. Ciencia e

Agrotecnologia, v. 32, n. 6, p. 1818–1823, 2008.

ANDERSON, J. C.; DUBETZ, C.; PALACE, V. P. Neonicotinoids in the Canadian

aquatic environment: A literature review on current use products with a focus on fate,

exposure, and biological effects. Science of The Total Environment, v. 505, p.

409–422, 2015.

ASTM. Standard practice for conducting acute toxicity tests with fishes,

macroinvertebrates and amphibians. American Standards for Testing and Materials.

Philadelphia, P.A. . 1980.

BOLL, PK.; ROSSI, I.; AMARAL, SV.; LEAL-ZANCHET, A. A taste for exotic food:

Neotropical land planarians feeding on an invasive flatworm. PeerJ, p. 8, 2015.

BROCK, T. C. M.; VAN WIJNGAARDEN, R. P. A. Acute toxicity tests with Daphnia

40

magna, Americamysis bahia, Chironomus riparius and Gammarus pulex and

implications of new EU requirements for the aquatic effect assessment of

insecticides. Environmental Science and Pollution Research, v. 19, n. 8, p. 3610–

3618, 2012.

BÜRGER, J.; DARMENCY, H.; GRANGER, S.; GUYOT, S. H.M.; MESSÉAN, A.;

COLBACH, N. Simulation study of the impact of changed cropping practices in

conventional and GM maize on weeds and associated biodiversity. Agricultural

Systems, v. 137, p. 51–63, 2015.

BURNEY, J. A; DAVIS, S. J.; LOBELL, D. B. Greenhouse gas mitigation by

agricultural intensification. Pnas, v. 107, n. 26, p. 12052–12057, 2010.

CAMPANHA, M. M.; SILVA SANTOS, R.H.; DE FREITAS, G. B.; PRIETO

MARTINEZ, H. E.; RIBEIRO GARCIA, S.L.; FINGER, F.L. Growth and yield of coffee

plants in agroforestry and monoculture systems in Minas Gerais, Brazil.

Agroforestry Systems, v. 63, n. 1, p. 75–82, 2005.

Companhia Nacional de Abastecimento (CONAB). Perspectivas para a

agropecuária. Perspec. agropec, v. 2, p. 1–155, 2014.

CONNON, R. E.; GEIST, J.; WERNER, I. Effect-Based Tools for Monitoring and

Predicting the Ecotoxicological Effects of Chemicals in the Aquatic Environment.

Sensors, v. 12, n. 12, p. 12741–12771, 2012.

COSTANTINI, M. L. Effect of multiple disturbances on food web vulnerability to

biodiversity loss in detritus-based systems. Ecosphere, v. 6, n. July, p. 1–20, 2015.

CUHRA, M.; TRAAVIK, T.; BØHN, T. Clone- and age-dependent toxicity of a

glyphosate commercial formulation and its active ingredient in Daphnia magna.

Ecotoxicology, v. 22, n. 2, p. 251–262, 2013.

DIAS GUERRA, W.; DE OLIVEIRA, P. C.; PUJOL-LUZ, J. R. Gafanhotos

(Orthoptera, Acridoidea) em áreas de cerrados e lavouras na Chapada dos Parecis,

Estado de Mato Grosso, Brasil. Revista Brasileira de Entomologia, v. 56, n. 2, p.

228–239, 2012.

EDWARDS, C. B.; JORDAN, D. L.; DIXON, P. M.; YOUNG, B. G.; WILSON, R. G.;

WELLER, S. Benchmark study on glyphosate-resistant crop systems in the United

States . Economics of herbicide resistance management practices in a 5 year field-

scale study . Pest Manag Sci, v. 70, n. 12, p. 3759, 2014.

EISSA, A. E.; ZAKI, M. M. The impact of global climatic changes on the aquatic

environment. Procedia Environmental Sciences, v. 4, p. 251–259, 2011.

41

ELLIOTT, S. A.; SÁNCHEZ ALVARADO, A. The history and enduring contributions of

planarians to the study of animal regeneration. Wiley Interdisciplinary Reviews:

Developmental Biology, v. 2, n. 3, p. 301–326, 2013.

FERNÁNDEZ-DÁVILA, M. L.; RAZO-ESTRADA, A. C.; GARCÍA-MEDINA, S.;

GÓMEZ-OLIVÁN, L. M.; PIÑÓN-LÓPEZ, M. J.; IBARRA, R. G.; GALAR-MARTÍNEZ,

M. Aluminum-induced oxidative stress and neurotoxicity in grass carp (Cyprinidae-

Ctenopharingodon idella). Ecotoxicology and Environmental Safety, v. 76, p. 87–

92, 2012.

FLEEGER, J. W.; CARMAN, K. R.; NISBET, R. M. Indirect effects of contaminants in

aquatic ecosystems. Science of The Total Environment, v. 317, n. 1-3, p. 207–233,

2003.

FOLMAR, L. C.; SANDERS, H. O.; JULIN, A M. Toxicity of the herbicide

glyphosphate and several of its formulations to fish and aquatic invertebrates.

Archives of environmental contamination and toxicology, v. 8, n. 3, p. 269–278,

1979.

FRIED, G.; PETIT, S.; DESSAINT, F.; REBOUD, X. Arable weed decline in Northern

France: Crop edges as refugia for weed conservation? .Biological Conservation, v.

142, n. 1, p. 238–243, 2009.

GARCÍA-GÓMEZ, C.; ESTEBAN, E.; SÁNCHEZ-PARDO, B.; FERNÁNDEZ, M. D.

Assessing the ecotoxicological effects of long-term contaminated mine soils on plants

and earthworms: relevance of soil (total and available) and body concentrations.

Ecotoxicology, p. 1195–1209, 2014.

GARCÍA-MEDINA, S.; RAZO-ESTRADA, C.; GALAR-MARTINEZ, M.; CORTÉZ-

BARBERENA, E.; GÓMEZ-OLIVÁN, L. M.; ÁLVAREZ-GONZÁLEZ, I.; MADRIGAL-

BUJAIDAR, E. Genotoxic and cytotoxic effects induced by aluminum in the

lymphocytes of the common carp (Cyprinus carpio). Comparative Biochemistry

and Physiology Part C, v. 153, n. 1, p. 113–118, 2011.

GREEN, J. M. Current state of herbicides in herbicide-resistant crops. Pest

Management Science, v. 70, n. 9, p. 1351–1357, 2014.

GUECHEVA, T.; HENRIQUES, J. A P.; ERDTMANN, B. Genotoxic effects of copper

sulphate in freshwater planarian in vivo, studied with the single-cell gel test (comet

assay). Mutation Research - Genetic Toxicology and Environmental

Mutagenesis, v. 497, n. 1-2, p. 19–27, 2001.

HARICHANDAN, R.; ROUTROY, S.; MOHANTY, J. K.; PANDA, C. R. An

assessment of heavy metal contamination in soils of fresh water aquifer system and

42

evaluation of eco-toxicity by lithogenic implications. Environmental Monitoring and

Assessment, v. 185, p. 3503–3516, 2013.

HARMON, J. P.; BARTON, B. T. On their best behavior: how animal behavior can

help determine the combined effects of species interactions and climate change.

Annals of the New York Academy of Sciences, v. 1297, n. 1, p. 139–147, 2013.

JAGADEESH, E.; KHAN, B.; CHANDRAN, P.; KHAN, S. S. Toxic potential of iron

oxide, CdS/Ag2S composite, CdS and Ag2S NPs on a fresh water alga Mougeotia

sp. Colloids and Surfaces B: Biointerfaces, v. 125, p. 284–290, 2015.

JESUS, F. T.; OLIVEIRA, R.; SILVA, A.; CATARINO, A. L.;SOARES, A.M.V.M.;

NOGUEIRA, A. J. A; DOMINGUES, I. Lethal and sub lethal effects of the biocide

chlorhexidine on aquatic organisms. Ecotoxicology, v. 22, n. 9, p. 1348–1358, 2013.

KAYSER, C.; LARKIN, T.; SINGHAL, N. Amendment of biosolids with waste

materials and lime: Effect on geoenvironmental properties and leachate production.

Waste Management, p. 2015, 2015.

KNAKIEVICZ, T.; VIEIRA, S. M.; ERDTMANN, B.; FERREIRA, H. B. Reproductive

modes and life cycles of freshwater planarians. Invertebrate Biology, v. 125, n. 3, p.

212–221, 2006.

KNAKIEVICZ, T.; FERREIRA, H. B. Evaluation of copper effects upon Girardia tigrina

freshwater planarians based on a set of biomarkers. Chemosphere, v. 71, n. 3, p.

419–428, 2008.

KOWALCZUCK, M.; CARNEIRO, E.; CASAGRANDE, M. M.; MIELKE, O. H. H. The

Lepidoptera Associated with Forestry Crop Species in Brazil: A Historical Approach.

Neotropical Entomology, v. 41, n. 5, p. 345–354, 2012.

KUMAR, V.; GILL, K. D. Oxidative stress and mitochondrial dysfunction in aluminium

neurotoxicity and its amelioration: A review. NeuroToxicology, v. 41, p. 154–166,

2014.

LIU, C.L; XI, Y.L; HUANG, W. J. Toxic effects of perfluorononanoic acid on the

development of Zebrafish (Danio rerio) embryos. Journal of Environmental

Sciences, v. 32, p. 26–34, 2015.

MACRAE, E. K. Observations on the fine structure of photoreceptor cells in the

planarian Dugesia tigrina. Journal of ultrastructure research, v. 10, n. 3, p. 334–

349, 1964.

MACRAE, E. K. The fine structure of sensory receptor processes in the auricular

epithelium of the planarian, Dugesia tigrina. Zeitschrift für Zellforschung und

43

Mikroskopische Anatomie, v. 82, n. 4, p. 479–494, 1967.

MARINHO, C. D; MARTINS, F. J O; AMARAL, A. T.; GONÇALVES, L. S.; DOS

SANTOS, O. J A P.; ALVES, D. P.; BRASILEIRO, B. P.; PETERNELLI, L. A.

Genetically modified crops: Brazilian law and overview. Genetics and Molecular

Research, v. 13, n. 3, p. 5221–5240, 2014.

MEYER, D. E.; CEDERBERG, C. SIK-Rapport Nr 809: Pesticide use and

glyphosate- resistant weeds – a case study of Brazilian soybean production.

[s.l: s.n.]. Disponível em: <http://commodityplatform.org/wp/wp-

content/uploads/2011/03/s>.

MIKULIC, P.; BEARDALL, J. Contrasting ecotoxicity effects of zinc on growth and

photosynthesis in a neutrophilic alga (Chlamydomonas reinhardtii) and an

extremophilic alga (Cyanidium caldarium). Chemosphere, v. 112, p. 402–11, 2014.

MONSANTO. Monsanto ’ s Strong Third Quarter Performance Reinforces

Confidence in Full-Year Guidance ; Global Portfolio , Leading Innovation for

Farmers and Financial Discipline Fuel Growth in Challenging Agriculture

Environment Monsanto Reaffirms Vision for Ag and. Disponível em:

<http://news.monsanto.com>. Acesso em: 23 ago. 2015.

MONSANTO COMPANY © 2002-2015. Monsanto’s Intacta RR2 ProTM Poised To

Deliver A New Wave Of Benefits For South American Countries. Disponível em:

<http://www.monsanto.com/newsviews/pages/intacta-rr2-pro-benefits- for-south-

american-countries.aspx>. Acesso em: 23 ago. 2015.

OLIVEIRA, A. R.; FREITAS, S. P. Levantamento fitossociológico de plantas

daninhas em áreas de produção de cana-de-açúcar. Planta Daninha, v. 26, n. 1, p.

33–46, 2008.

OVIEDO, N. J. et al. Establishing and maintaining a colony of planarians. CSH

protocols, v. 2008, p. pdb.prot5053, 2008.

PAKRASHI, S.; DALAI, S.; PRATHNA T.C.; TRIVEDI, S.; MYNENI, R.; RAICHUR, A.

M.; CHANDRASEKARAN, N.; MUKHERJEE, A Cytotoxicity of aluminium oxide

nanoparticles towards fresh water algal isolate at low exposure concentrations.

Aquatic Toxicology, v. 132-133, p. 34–45, 2013.

RODRIGUES, A.C.M.; GRAVATO, C.; QUINTANEIRO, C.; GOLOVKO, O.; ŽLÁBEK,

V., BARATA, C.; SOARES, A M.V.M.; PESTANA, J. L. T. Life history and

biochemical effects of chlorantraniliprole on Chironomus riparius. Science of The

Total Environment, v. 508, p. 506–513, 2015.

44

SÁNCHEZ-BAYO, F.; TENNEKES, H. A. Impact of Systemic Insecticides on

Organisms and Ecosystems. Additional information is available at the end of the

chapter, v. Chapter 13, p. 365–414, 2013.

SAWYER, D. Climate change, biofuels and eco-social impacts in the Brazilian

Amazon and Cerrado. Philosophical transactions of the Royal Society of

London. Series B, Biological sciences, v. 363, n. 1498, p. 1747–1752, 2008.

SHUHAIMI-OTHMAN, M.; YAKUB, N.; UMIRAH, N. S.; ABAS, A. Toxicity of eight

metals to Malaysian freshwater midge larvae Chironomus javanus (Diptera,

Chironomidae). Toxicology and Industrial Health, v. 27, n. 10, p. 879–886, 2011.

SIBLEY, P. K.; BENOIT, D. A.; ANKLEY, G. T. The significance of growth in

Chironomus tentans sediment toxicity tests: Relationship to reproduction and


336–345, 1997.

SIMBERLOFF, D.; MARTIN, J. L.; GENOVESI, P.; MARIS, V.; WARDLE, D. A.;

ARONSON, J.; COURCHAMP, F.; GALIL, B.; GARCÍA-BERTHOU, E.; PASCAL, M.;

PYŠEK, P.; SOUSA, R.; TABACCHI, E.; VILÀ, M. Impacts of biological invasions:

What’s what and the way forward. Trends in Ecology and Evolution, v. 28, n. 1, p.

58–66, 2013.

SMITH, R. G.; ATWOOD, L.W.; MORRIS, M.B.; MORTENSEN, D.A.; KOIDE, R.T.

Evidence for indirect effects of pesticide seed treatments on weed seed banks in

maize and soybean. Agriculture, Ecosystems & Environment, v. 216, p. 269–273,

2016.

SOARES, A.; GUILLIN, E.; BORGES, L.; SILVA, A.; ALMEIDA, Á.; GRIJALBA, P.;

GOTTLIEB, A.M.; BLUHM, B.H.; OLIVEIRA, L. More Cercospora Species Infect

Soybeans across the Americas than Meets the Eye . PLoS One., v. 10, n. 8, p.

133495, 2015.

SOUZA, T.L.P.O.; DESSAUNE, S.N.; MOREIRA, M.A.; BARROS, E.G. Soybean rust

resistance sources and inheritance in the common bean ( Phaseolus vulgaris L .).

Genetics and Molecular Research, v. 13, n. 3, p. 5626–5636, 2014.

SZCZEPANIEC, A.; CREARY, S.F.; LASKOWSKI, K.L.; NYROP, J.P.; RAUPP, M.J.

Neonicotinoid Insecticide Imidacloprid Causes Outbreaks of Spider Mites on Elm

Trees in Urban Landscapes. PLoS ONE, v. 6, n. 5, p. e20018, 2011.

TABASHNIK, B.E.; MOTA-SANCHEZ, D.; WHALON, M.E.; HOLLINGWORTH, R. M.;

CARRIÈRE, Y. Defining terms for proactive management of resistance to Bt crops

and pesticides. Journal of economic entomology, v. 107, n. 2, p. 496–507, 2014.

45

TANG, S.; LIANG, J.; TAN, Y.; CHEKE, R.A. Threshold conditions for integrated pest

management mdoels with pesticides that have residual effects. Journal of

Mathematical biology, v. 66, p. 1–35, 2013.


<http://data.worldbank.org/indicator/NV.AGR.TOTL.ZS/countries/BR?display=graph>

.

VAN DER SLUIJS, J.P.; SIMON-DELSO, N.; GOULSON, D.; MAXIM, L.;

BONMATIN, J.M.; BELZUNCES, L. P. Neonicotinoids, bee disorders and the

sustainability of pollinator services. Current Opinion in Environmental

Sustainability, v. 5, n. 3-4, p. 293–305, 2013.

VEHNIÄINEN, E.-R.; KUKKONEN, J. V. K. Multixenobiotic resistance efflux activity in

Daphnia magna and Lumbriculus variegatus. Chemosphere, v. 124, p. 143–9, 2015.

VOIGT, C. L.; DA SILVA, C. P.; DORIA, H. B.; FERREIRA, R.M. A.; DE OLIVEIRA

RIBEIRO, C.A.; DE CAMPOS, S.X.. Bioconcentration and bioaccumulation of metal

in freshwater Neotropical fish Geophagus brasiliensis. Environmental Science and

Pollution Research, v. 22, p. 8242–8252, 2015.

WEIS, J. S.; SMITH, G.; ZHOU, T.; SANTIAGO-BASS, C.; WEIS, P. Effects of

Contaminants on Behavior: Biochemical Mechanisms and Ecological Consequences.

BioScience, v. 51, n. 3, p. 209, 2001.

WHALEN, R.; HARMON, J. P. Temperature alters the interaction between a

herbivore and a resistant host plant. Arthropod-Plant Interactions, v. 9, p. 233–240,

2015.

XAVIER, M.; MESSAGE, D.; PICANC, M. C.; CHEDIAK, M.; JU, P.A.S.; RAMOS, R.

S. Acute Toxicity and Sublethal Effects of Botanical Insecticides to Honey Bees.

JOURNAL OF INSECT SCIENCE, v. 15, p. 1–6, 2015.

ZHANG, X.; ZHANG, B.; YI, H.; ZHAO, B. Mortality and antioxidant responses in the

planarian (Dugesia japonica) after exposure to copper. Toxicology and industrial

health, v. 30, n. 2, p. 123–31, 2014.

46

CAPÍTULO II

Acute and chronic effects of glyphosate on the freshwater planarian

Dugesia tigrina

47

Acute and chronic effects of glyphosate on the freshwater planarian Dugesia

tigrina

Abstract

Demand of glyphosate based herbicides including Roundup® is rising due to the

increase in transgenic crops that are resistant to glyphosate. Consequently, there is

now an indiscriminate use of glyphosate with potential adverse effects in the

environment. This study aimed at determining the sensitivity of the freshwater

planarian Dugesia tigrina to acute and chronic exposures of Roundup®. The

organisms were exposed to a range of lethal concentration of glyphosate to

determine the LC50 and the sub-lethal bioassays assessed the effects of sub-lethal

concentrations of glyphosate on the planarian locomotor velocity (pLMV), feeding

rate, regeneration endpoints and fecundity/fertility rate. Regeneration endpoints

included the formation of blastema, photoreceptors and auricles after decapitation as

well as deformaties in photoreceptors observed under exposure to glyphosate. The

estimated 48 hours LC50 was 37.06 mg·L-1. The LOEC values of 3.39 mg·L-1

glyphosate for pLMV, regeneration endpoints, and 1.71 mg·L-1 glyphosate for

reproduction, concentration 14.91 mg·L-1 of glyphosate fertility rate was completely

inhibited. Our results chronic exposures to glyphosate based herbicides can impair

reproduction of aquatic planarians. Moreover, the sensitivity and suitability of

freshwater planarian as ecotoxicological model species is discussed.

Keywords: Glyphosate toxicity, behavior, regeneration, reproduction.

48

Efeitos agudos e crônicos de glifosato na planária de água doce Dugesia tigrina

Resumo

A utilização de cultivares transgênicas resistentes à molécula de glifosato tem

intensificado a demanda por herbicidas, como por exemplo, o Roundup®. O uso

indiscriminado de glifosato tem gerado potenciais efeitos adversos ao meio

ambiente. Assim, este estudo objetivou determinar a sensibilidade da planária de

água doce Dugesia tigrina a exposições agudas e crônicas ao Roundup®. As

planárias foram expostas a diversas concentrações letais de glifosato para a

determinação da CL50 e em bioensaios utilizando-se concentrações subletais,

avaliou-se os efeitos do glifosato sobre a velocidade de locomoção, taxa de

alimentação, parâmetros de regeneração e taxa de fecundidade/fertilidade das

planárias. Os parâmetros da regeneração incluíram a formação de blastema,

fotorreceptores e aurículas após a decapitação, bem como a presença de

deformidades em fotorreceptores dos organismos sob exposições ao glifosato. A

CL50 foi estimada em 37,06 mg·L-1 à 48 horas. Os valores LOEC de 3,39 mg·L-1 de

glifosato para pLMV, os terminais da regeneração, e 1,71 mg·L-1 de glifosato para a

reprodução, a taxa de fertilidade foi completamente inibida a concentração de 14,91

mg·L-1 de glifosato. Os resultados de exposições crônicas ao herbicidas com base

na molécula de glifosato pode prejudicar a reprodução de planárias aquáticas. Além

disso, discute-se a sensibilidade e adequação das planárias de água doce como

espécies modelo ecotoxicológicas.

Palavras-chave: Dugesia tigrina; comportamento; regeneração, reprodução;

toxicidade do glifosato.

49

1. INTRODUCTION

Responses of organisms to short and long term exposures to various

substances are essential, which could predict the behavioural responses of

populations in ecosystems. Evaluation of these responses may provide greater

sensitivity in predicting beneficial or detrimental biological, chemical or physical

effects in ecosystems (CONNON et al., 2012; SIH et al., 2011). Behavioral responses

to short-term exposures such as feeding, locomotor velocity and regeneration were

tested in species (LOPES et al., 2014; RAMAKRISHNAN, DESAER, 2011; ZHANG

et al., 2015; SHEIMAN, KRESHCHENKO, 2015). Also, biological responses such as

reproduction and life cycle to long periods of exposure have been reported (NYMAN

et al., 2013; RODRIGUES et al., 2015; CUHRA et al., 2013; PRESTES et al., 2013).

These responses are ecologically relevant, as they are important components of

developmental, health, structure and dynamics of populations (GROH et al., 2015).

Planarians are aquatic invertebrates commonly found in freshwater streams

and ponds that prey predominantly upon insects, insect larvae, and other

invertebrates (REDDIEN; ALVARADO, 2004). Recently, these organism have been

widely used for the development of studies in several areas, because of their ease of

maintaining and manipulating these organisms in laboratory settings (OVIEDO et al.,

2008), and have been used in regenerative, neurotoxicology research to monitor

drug responsiveness at the organismal level (BALESTRINI et al., 2014; ELLIOTT,

ALVARADO, 2013; HAGSTROM et al., 2015; KITAMURA et al., 2003; REDDIEN,

ALVARADO, 2004). Roundup original, liquid herbicide based on glyphosate active

ingredient is one of the products widely used in agriculture. Demand for herbicide

glyphosate based products is increasing worldwide, this demand is favored by

increased transgenic crops such as soybeans, corn, cotton, etc. (MONSANTO,

2015a; CHAUZAT, FAUCON, 2007; GRUBE et al., 2011; GREEN, OWEN, 2011).

The development of transgenic crops resistance to glyphosate (GR) generally leads

to increasing the amount of dosage for control (GIBSON et al., 2015). Brazil is one of

the largest producers of transgenic crops such as soybean, corn, cotton, etc

(MONSANTO COMPANY © 2002-2015), ranking second worldwide with 40.3 million

hectares after USA biotech crop (JAMES, 2014). Consequently, it is also one of the

major consumers of glyphosate (MEYER, CEDERBERG, 2010). Besides fulfilling the

role of protecting agricultural crops from pests, pesticides can pose risks to the

50

environment resulting in the pollution of soil, air, surface water, ground water and

food (HUSSAIN et al., 2009; LUPI et al., 2015; ALAMDAR et al., 2014;

PATHIRATNE, KROON, 2015 ; CHATTERJEE et al., 2015). Furthermore, these

negative effects can lead to human intoxication by consuming contaminated food and

water, plus the risk of occupational poisoning of workers and farmers (BRAZ-MOTA

et al., 2015; LONDON et al., 2010; HU et al., 2015; DELIRRAD et al., 2015; FREIRE

et al., 2015; ARRUDA et al., 2011).

The main objective of this study was to evaluate the lethal and sub-lethal

effects in Dugesia tigrina to exposures of glyphosate. First, we determined the lethal

concentration for 50% of the population (LC50). Later the sub-lethal effects, we

evaluated short-term exposure, planarian locomotor velocity (pLMV), feeding rate,

regeneration (blastema, photoreceptors and auricles) and long-term exposure tests

effects on reproduction.

2. MATERIAL AND METHODS

2.1 Test organism

Dugesia tigrina was a generous gift from University of Sao Paulo (Sao Paulo

SP-Brazil). We maintained the organism in medium ASTM hard water in the Federal

University of Tocantins (Gurupi – TO – Brazil), with 22°C ± 1°C, constant aeration

and dark. The population was fed twice a week with bovine liver, except 96 hours

prior to and during each test, when they were not fed, adapting culture conditions of

OVIEDO et al., (2008) (Annex 4).

2.2 Original Roundup ® preparation

Herbicide liquid original ® (RD) composed with N-(phosphonomethyl) glycine

isopropylamine salt (480g·L-1), acid equivalent (Glyphosate) 360 g·L-1, and other

ingredients 684 g·L-1. Lot: RRO01/1307-00, were manufactured by Monsanto

Company (Av. Nations Unidas 12.901, São Paulo – Brazil). Stock solution of 5000

mg·L-1 (pH = 4.50) was dissolved with distilled water, which was protected from light

to avoid degradation of RD and stored at -20°C. Experimental solutions were

prepared by dilution of the stock solution in ASTM medium

51

2.3 Experimental animals

2.3.1 Acute Test

We determined the median lethal concentration for Dugesia tigrina

(0.86±0.098 cm, total length) after 24, 48 and 96h to a gradient of glyphosate

concentrations (refer to Annex 1). Conditions were maintained at 22°C ± 1 under

dark conditions and no food. This exposure contained 5 groups of organisms with 5

replicates per concentration in crystallizing dishes containing 20 mL of experimental

solution. All test dilutions were prepared using artificial medium (ASTM).Control

treatments (ASTM) were maintained in the same conditions as glyphosate

treatments.

2.3.2 Sub-lethal effects

Dugesia tigrina were exposed (0.83 ± 0.11 cm total length) for 96 hours to

different concentration of RD: 1.71(± 0.16), 3.39 (± 0.41), 8.32 (± 0.36) and 14.91 (±

0.83) mg·L-1 (measured concentrations, mean ± SD). Conditions were maintained at

22°C ± 1 under dark light conditions and no food. Exposure was performed with

groups of 15 organisms with 3 replicates per treatment, in borosilicate glass beakers

containing 100 mL of experimental solution. Control treatments (ASTM) were

maintained in the same conditions as RD treatments.

2.3.2.1 Planarian Locomotor velocity (pLMV)

We evaluated pLMV in aluminum plates (diameter: 35.0 cm), previously

covered with millimeter paper (grid lines spaced 0.5 cm apart) and adhesive plastic

film. Fifteen organisms per treatment were individually placed in the center of an

aluminum plate, with a layer of ASTM covering the bottom. Post- exposure pLMV

was thus measured as the number of lines crossed (grid lines spaced 0.5 cm apart)

each planarian crossed or re-crossed per minute, for 2 minutes of observation, after

an adaptation period of 30 seconds (Annex 6).

52

2.3.2.2 Feeding rate

After-exposure feeding rate was evaluated with 10 experimental units per

treatment into crystallizing dishes with 20 mL of ASTM medium. Live Chironomus

xantus larvae (25 units) of 6 days old and total length of 0.59 ± 0.065 cm were added

as food in each replicate. Feeding rate per hour was measured as the number of

larvae ingested per planarian for 12 hours (Annex 5).

2.3.2.3 Regeneration

After-exposure, ten planarians per treatment were decapitated with a single

cut behind the auricles in microscope slides with a drop of ASTM. Decapiteated

planarians were then transferred to crystallizing dishes with 20 mL of ASTM medium.

We followed the regeneration of blastema (length in mm), and the formation of

photoreceptors and auricles every 6 hours, with a MIKROS magnifying glass

equipped with a calibrated eye-piece micrometer (Annex 8 ).

2.3.3 Reproduction test

Adult planarians at the onset of reproductive age (1.39 ± 0.08 cm total length),

were exposed for five weeks to different concentration of RD: 1.71, 3.39, 8.32 and

14.91 mg·L-1. During exposure fecundity and fertility rate was evaluated for weeks.

Control treatments (ASTM) were maintained in the same conditions as RD

treatments. Exposure treatments were performed with 12 organisms, and 3 replicates

in borosilicate glass beakers containing 100 mL of experimental solution that was

replaced each week, after feeding with bovine liver. Conditions were maintained at

22°C ± 1°C under dark light conditions.

2.3.3.1 Fecundity rate (Fc) and Fertility rate (Fr)

During exposure we evaluated the number of cocoons and hatchlings

produced by sexual planarians as described by KNAKIEVICZ et al., (2006).

Fecundity rate was determined by the number of cocoons produced by week, divided

53

by the number of planarians exposed. Fertility rate was determined by the number of

hatchlings produced per individuals or pairs per week divided by the number of

planarians exposed (Annex 7).

2.4 Chemical analysis

Nominal RD concentrations in experimental solutions used were verified by

chemical analysis. Three water samples (20 mL per sample) per treatment were

analyzed. All samples were frozen at −20°C.

2.4.1 Chemical analysis of Glyphosate in water

Nominal RD concentrations in experimental solutions used were verified by

chemical analysis. Three water samples (20 mL per sample) per treatment were

analyzed. All samples kept in the dark and frozen at −20°C.

N-(phosphonomethyl) glycine-monoisopropylamine salt (glyphosate-IPA) 96%

(CAS: 1071-83-6, Lot: MKBJ9130V), was obtained from Sigma–Aldrich, Brasil Ltda.

Av. Nations Unidas.23.043 (São Paulo - Brazil). Solutions of glyphosate were

prepared in distilled water at a concentration of 0, 100, 200, 400 and 1000mg·L-1 as

determined by a calibration curve. The concentrations tested exhibited good linearity

in the concentration range between 100mg·L-1 to 1000 mg·L-1 glyphosate; R2 =

0.9992.

Analyses of glyphosate in water samples were performed on a Gas

Chromatograph Model GC Solution brand SHIMADZU, equipped with a FID sensor.

For registration and analysis of chromatograms, the equipment is connected to a

microcomputer, using the GC Solution Program. The compounds were separated

and identified on a DB5 capillary column (30 m x 0.25 mm). For chromatographic

separation, 1 mL of sample was injected with the aid of Hamilton® syringe (10 ml) in

splitless injection system. Nitrogen gas was used as a carrier with scheduled linear

speed to 43.2 cm·s-1 and the gases hydrogen and synthetic air formed in the flame

detector.

Temperatures of injector and detector were controlled isothermal at 300ºC and

320ºC. The initial column temperature was 150°C (maintained for 5 minutes),

54

increasing 10ºC per minute up to 280ºC (maintained for 20 minutes). The flow of

carrier gas in the column was 1.8 ml·min-1.

2.5 Statistical analysis

Acute test was evaluated after 24, 48 and 96 hours and Probit analyses used

to estimate the LC50 values, and confidence intervals using in Minitab software.

We evaluated the effects of glyphosate exposure in the sub lethal parameters

(locomotion, feeding, regeneration and reproduction) in comparison with controls

treatments using one-way ANOVA with Dunnett's Multiple Comparison test. All

analyses for sub-lethal parameters were performed in GraphPad Prism version 5.03

for Windows, GraphPad Software, San Diego California USA, www.graphpad.com”.

The normality of data was verified using D'Agostino & Pearson omnibus normality

test and homoscedasticity was verified by Brown–Forsythe test.

3. RESULTS

3.1 Acute toxicity

High concentrations of glyphosate initially induced injuries in exposed

organisms followed by mortality. Acute test analysis shows that mortality increases

with increasing concentrations of glyphosate (F(18, 76) = 24.12, p < 0.001, Fig 1). In

annex 2 shows a summary of results for LC50 in Dugesia tigrina after 24, 48 and 96

hours and 95% confidence intervals values. Mortality was not observed in the control

treatment.

55

0 28 31.7 33 34.3 35.1 38 40.9 42 44 46 48.3 49.6 51 540

20

40

60

80

100R2 = 0.8511

LC50 = 37.06 mg/L (CI 95% : 35.93 - 38.17)

*

*

******

******

******

************

Glyphosate(mg/L)

% M

ort

alit

y

Fig. 1. Lethal effects of glyphosate in Dugesia tigrina, after 48 hours of exposure.

Expressed mean (± SEM), n = 5. Asterisks denote significant differences in comparisons with the control treatment (ASTM), *p < 0.05, ***p < 0.001 (Dunnett's test).

3.2 Sub-Lethal exposures


Locomotor velocity (pLMV) of D. tigrina, measured as lines crossed per min,

was significantly (F(4, 70) = 34.03, p < 0.001, Fig. 1), decreased by 22.14, 29.56 and

42.43% after glyphosate exposure with 3.39, 8.32 and 14.91 mg·L-1 respectively,

when compared to control treatment (ASTM).

Control 1.71 3.39 8.32 14.910

5

10

15

20

25

***

Glyphosate(mg/L)

pL

MV

(g

rid

lin

es c

ross

ed/m

in)

******

Fig. 2. Planarian locomotor velocity (pLMV) of Dugesia tigrina (gridlines crossed/min), mean (± SEM), n = 15, after 96 hours of exposure to sub-lethal

56

glyphosate concentrations. Asterisks denote significant differences in comparisons with the control treatment (ASTM), *** p < 0.001 (Dunnett's test).

3.2.2 Feeding rate

Feeding rate of D. tigrina was significantly reduced after glyphosate exposure

(F(4, 45) = 6.9, p < 0.001, Fig. 2). Over a period of 12 hours, feeding rate of planarians

was reduced by 22.31 and 43.50% in the 8.32 and 14.91 mg·L-1 respectively

glyphosate treatment, when compared to the control treatment (ASTM).

Control 1.71 3.39 8.32 14.910.0

0.2

0.4

0.6

0.8

1.0

***

*

Glyphosate(mg/L)

Fee

din

g r

ate

Fig. 3. Feeding rate (Number of larvae Chironomus xantus consumed for hour) of Dugesia tigrina, mean (± SEM), n = 10, after 96 hours of exposure to sub-lethal glyphosate concentrations. Asterisks denote significant differences in comparisons with the control treatment (ASTM), * p < 0.05, *** p < 0.001 (Dunnett's test).

3.2.3 Regeneration

3.2.3.1 Regeneration blastema

The regeneration of the blastema (length in mm) of D. tigrina was significantly

(F(4, 45) = 10.02, p < 0.001, Fig. 3), with a decreased by 22.47, 25.37 and 39.86% in

the 3.39, 8.32 and 14.91 mg·L-1 glyphosate concentration, respectively, when

compared to control treatment (ASTM).

57

Control 1.71 3.39 8.32 14.910.0

0.3

0.6

0.9

1.2

1.5

**

***

**

Glyphosate(mg/L)

Bla

stem

a (l

eng

th i

n m

m)

Fig. 4. Regeneration Blastema (length in mm) after 36 hours decapitation of Dugesia tigrina, mean (± SEM), n = 10, after 96 hours exposure to sub-lethal glyphosate concentrations. Asterisks denote significant differences in comparisons with the control treatment (ASTM), ** p < 0.01, *** p < 0.001 (Dunnett's test).

3.2.3.2 Formation of photoreceptors

To evaluate glyphosate affects on regeneration of we checked the time D.

tigrina take for photoreceptors formation after decapitation. New Photoreceptors

appear 74 ± 4.19 hours after decapitation in the control treatments and the

photoreceptors formation was significantly delayed after 96h glyphosate exposure

(F(4, 45) = 8.15, p < 0.001, Fig. 4), with a delay of 5.4 and 9 hours at concentrations of

8.32 and 14.91 mg·L-1 glyphosate respectively, when compared to control treatment

(ASTM).

58

Control 1.71 3.39 8.32 14.9160

70

80

90

100

***

*

Glyphosate(mg/L)

Tim

e (h

)

Fig. 5 Time (h) of regeneration of photoreceptors in D. tigrina, mean (± SEM), n = 10, after 96 hours exposure to sub-lethal glyphosate concentrations. Asterisks denote significant differences in comparisons with the control treatment (ASTM), * p < 0.05, *** p < 0.001 (Dunnett's test).

3.2.3.3 Regeneration auricles

Regeneration auricles of D. tigrina in control treatment (ASTM) started 81 ±

3.16 hours after decapitation. Time for regeneration of auricles was significantly (F(4,

45) =2.78 p < 0.001, Fig. 5), delayed after 96h RD exposure, with a delay of 4 hours

for auricles formation in the 14.91 mg·L-1 glyphosate concentration, when compared

to the control treatment.

Control 1.71 3.39 8.32 14.9170

75

80

85

90

95

*

Glyphosate(mg/L)

Tim

e (h

)

Fig. 6. Time (h) of regeneration of auricles in D. tigrina, mean (± SEM), n = 10, after

exposure 96 hours to sub-lethal glyphosate concentrations. Asterisks denote

59

significant differences in comparisons with the control treatment (ASTM), * p < 0.05 (Dunnett's test).

3.3 Reproduction


During the exposure period (Five weeks), planarians in the control group

showed the highest fecundity rate, increasing during successive weeks. Fecundity

rate (Fc) of D. tigrina was significantly decreased after glyphosate exposure (F(4, 10) =

36.32, p < 0.001), Fig. 6). After five-week exposure, Fc decreased of 56.25, 77.49,

and 94.44% in the 1.71, 3.39, 8.32 mg·L-1 glyphosate concentration, while those

treated with 14.91 mg·L-1 glyphosate inhibit the fecundity rate when compared to

control treatment (ASTM).

control 1.71 3.39 8.32 14.910

1

2

3

***

***

***

***

Glyphosate(mg/L)

Cu

mu

lati

ve

fec

un

dit

y r

ate

Fig. 7 Cumulative fecundity rate, in D. tigrina, mean (± SEM), n = 3, in five week exposure to sub-lethal RD concentrations. Asterisks denote significant differences in comparisons with the control treatment (ASTM), *** p < 0.001 (Dunnett's test).

3.3.2 Fertility rate

During the exposure time (Five week), the number of hatchlings produced by

each planarian (Fertility rate) changed according to Roundup glyphosate treatment.

Fertility rate (Fr) of D. tigrina was significantly (F(4, 10) = 58.95, p < 0.001, Fig. 7),

60

decreases of 56.58, 78.50 and 98.25 in the 1.71, 3.39 and 8.32 mg·L-1 glyphosate

concentration respectively. While those treated with 14.91 mg·L-1 glyphosate inhibit

the fertility rate when compared to control treatment (ASTM).

control 1.71 3.39 8.32 14.910

2

4

6

8

***

***

***

***

Glyphosate(mg/L)

Cu

mu

lati

ve

fert

ility

rat

e

Fig. 8. Cumulative Fertility rate, in D. tigrina, mean (± SEM), n = 3, in five week exposure to sub-lethal RD concentrations. Asterisks denote significant differences in comparisons with the control treatment (ASTM), *** p < 0.001 (Dunnett's test).

Additionally, during the reproduction is observed the deformation and injuries

of planarians. In the first week of exposure to glyphosate concentration, is produced

deformations photoreceptors and injuries in the organisms. The deformations

photoreceptors and injuries of Dugesia tigrina was significantly (F(4, 14) =23.25, p <

0.001), and (F(4, 14) =16.68 p < 0.001) (Fig 8) respectively, increased after first week

glyphosate exposure. In the following weeks, organisms adapt and do not exhibit

these behaviors. The control did not show deformities or injuries.

61

Fig. 9. Deformations in the photoreceptors and injuries, produced in first week of exposure to exposure to sub-lethal RD concentrations. Images show, (a) control without effects, (b) deformations of the photoreceptors (c) head injurie in D. tigrina a 14.91 mg·L-1 concentration. Expressed mean (± SEM), n = 3. Stars denote significant differences from control treatment, ASTM, ** p < 0.01, *** p < 0.001 (Dunnett's test).

4. Discussion

Our study reveals the importance and sensitivity of testing the behavior of

short and long exposure in the systemic, neurotoxicity and cytotoxicity effects of RD

in freshwater planarians.

The experiments with D. tigrina indicated that RD induce the LC50 = 47.17

mg·L-1 (12 h) and 37.06 mg·L-1 (48 h). Similar results were obtained by exposing

Dugesia japonica to glyphosate, reporting LC50 41,78 mg·L-1 (12 h) and 35.48 mg·L-1

(48 h) (LIU et al., 2008). Other species such as Caridina nilotica reported LC50

values of 2.5 mg·L-1 (neonates), 7.0 mg·L-1 (juveniles) and 25.3 mg·L-1 (adults) of RD

(MENSAH et al., 2011). In Ceriodaphnia dubia and Acartia tonsa LC50 (48 h) was

5.39 and 1.77 mg·L-1, respectively ( TSUI; CHU, 2003).

The sub-lethal effects on the behavior pLMV and feeding rate in D. tigrina to

glyphosate exposures are likely due to neurotoxicity, RD affects glutamate uptake,

release and metabolism within neural cells leading to Ca2+ influx through NMDA

receptors and L-VDCC (CATTANI et al., 2014). Concentrations above 1 mg / L of RD

inhibits AChE enzyme activity, which is responsible for the acetylcholine degrading at

synaptic level in fishes (MENÉNDEZ-HELMAN et al., 2012; MODESTO, MARTINEZ,

62

2010; GHOLAMI-SEYEDKOLAEI et al., 2013). Above concentrations of 6.20 mg·L-1

of glyphosate significant differences in ingestion of Dugesia japonica are exhibited

(LIU et al., 2008).

The sub-lethal effects on the regeneration of blastema, photoreceptors and

auricles in D. tigrina is significant, presented a lowest observed effect concentration

(LOEC) of 3.39, 8.32 and 14.91 mg·L-1 of glyphosate respectively. Chronic effects in

the deformations of photoreceptors (a week of exposure), presented a lowest

observed effect concentration (LOEC) of 3.39 mg·L-1 glyphosate. The effects on

regeneration and deformation the photoreceptors in D. tigrina to expositions

glyphosate is likely the capacity cytotoxic and genotoxic (POLETTA et al., 2009,

BENACHOUR et al., 2007). Above concentrations of 6.20 mg·L-1 of glyphosate show

significant differences in regeneration of Dugesia Japonica (Liu et al., 2008).

Research shows, the presence of micronucleated erythrocytes (DNA damage) in fish

at concentrations above 10 mg·L-1 (CAVAS; KONEN, 2007). Concentration of 1 mM

(Omega, Cosmic and Cargly) and 8 -12 mM (RD formulations) inducing cell cycle

disorder (MARC; MULNER-LORILLON; BELLÉ, 2004).

In chronic test with long term exposure, glyphosate causes serious

reproduction (Fecundity rate) damage and presented a lowest observed effect

concentration (LOEC) of 1.71 mg·L-1 glyphosate, and inhibit with 14.91 mg·L-1 RD

concentration. Unlike the other pesticides, RD inhibits steroidogenesis in mouse cells

by disrupting expression of StAR protein, consequently progesterone levels decrease

at higher concentrations of 24.4 ± 0.67 mg·L-1 without inducing a parallel decrease in

total protein synthesis, indicating that this herbicide did not cause acute cellular

toxicity or a general disruption in translation (WALSH et al., 2000). It's not determined

the presence of sex steroids: androgens, estrogens, progesterone in D. tigrina, but in

freshwater planarians as Bdellocephala brunnea the presence of testosterone levels

was determined during spermatogenesis (FUKUSHIMA et al., 2008). Research

shows that glyphosate caused a reduction in the number of eggs spawned by female

zebrafish exposed to high concentrations (10 mg·L-1) of glyphosate. However, this

concentration is well above concentrations measured to date in the environment and

unlikely to occur in aquatic systems (UREN WEBSTER et al., 2014). Reproduction of

the soil dwellers (Lumbricus terrestris) was reduced by 56% within three months after

herbicide application (176.12 ml·m−2 of herbicide was applied) (GAUPP-

63

BERGHAUSEN et al., 2015). In Daphnia magna chronic exposure, particularly to

formulated Roundup, causes serious reproduction damage at levels close to (1.35

mg·L-1) or even below (0.45 mg·L-1)(CUHRA; TRAAVIK; BØHN, 2013).

In this study LOEC lowest (1.71 mg·L-1) was obtained in the fertility rate of D.

tigrina, it is slightly higher compared to the accepted threshold values for glyphosate

in surface waters in the United States in general (0.7 mg·L-1) and in the state of

California specifically (1.0 mg·L-1) (EPA, Washington, 1992).

Behavioral responses such as locomotor activity, feeding, regeneration and

reproduction planarian at exposure of various substances are used to determine the

toxicity (LOMBARDO et al., 2011; RAFFA et al., 2001; RAFFA, MARTLEY, 2005).

Altering these patterns of responses in the presence of toxic are ecologically

important, since these organisms are predators in aquatic ecosystems, consuming

larvae and aquatic insects, controlling the proliferation of many harmful insects to

human health (PRASNISKI, LEAL-ZANCHET, 2009) (BLAUSTEIN, 1990).

Modification of planarians responses can lead to significant changes in ecosystems,

natural and anthropogenic disturbance modified food web topological properties in

the river invertebrate community, with such disturbance-induced changes being

reflected in substantial variations in the structural stability of detritus-based food

webs in the face of species loss (COSTANTINI, 2015).

5. CONCLUSIONS

Commercial products based on the herbicide glyphosate (including Roundup),

are classified as moderately toxic to aquatic organisms, but in our studies exposures

of 5 weeks in the concentrations 1.71 mg·L-1 glyphosate cause significant damage to

the reproduction, consequently this damage can threaten populations in aquatic

ecosystems flatworms, which would result in an increase in other organisms as

harmful to human health insects. Should be considered to lower concentrations of

1.71 mg·L-1 glyphosate and longer exposure these effects could also be observed.

64

Acknowledgements

We thank to Coordination for the Improvement of Higher Education Personnel (CAPES),

and the Federal University of Tocantins for financial support.

6. REFERENCES

ALAMDAR, A.; SYED, J.; MALIK, R.; KATSOYIANNIS, A.; LIU, J.; LI, JUN.; ZHANG, G.; JONES, K. Organochlorine pesticides in surface soils from obsolete pesticide dumping ground in hyderabad city, pakistan: Contamination levels and their potential for air-soil exchange. Science of the Total Environment, v. 470-471, p. 733–741, 2014.

ARUNDA, J.F.; DE MELLO, J.; JANDOTTI, A.C.; DE PAULA E.R. Prevalência dos

casos de intoxicação por pesticidas (2007-2010) em São Miguel do Oeste – SC.

Prevalence of cases of pesticide poisoning (2007-2010) in São Miguel do Oeste –

SC. Publicatio UEPG: Ciencias Biologicas e da Saude, v. 17, n. 2, p. 123–131,

2011.

AGÊNCIA NACIONAL DE ÁGUAS (ANA). Plano estratégico de recursos hídricos da bacia hidrográfica dos rios Tocantins e Araguaia. Brasília: [s.n.].

AGÊNCIA NACIONAL DE ÁGUAS (ANA). Guia nacional de coleta e preservação de amostras: água, sedimento, comunidades aquáticas e efluentes líquidos São Paulo, 2011.

AHMAD, H. et al. Pollution Problem in River Kabul : Accumulation Estimates of Heavy Metals in Native Fish Species. BioMed Research International, v. 2015, n.

537368, p. 7, 2015.

ALAMDAR, A. et al. Organochlorine pesticides in surface soils from obsolete pesticide dumping ground in hyderabad city, pakistan: Contamination levels and their potential for air-soil exchange. Science of the Total Environment, v. 470-471, p.

733–741, 2014.

AMARAL CASTRO, N. R. et al. Lixiviação do inseticida thiamethoxam em macrolisímetros de duas classes de solo. Ciencia e Agrotecnologia, v. 32, n. 6, p. 1818–1823, 2008.

ANDERSON, J. C.; DUBETZ, C.; PALACE, V. P. Neonicotinoids in the Canadian aquatic environment: A literature review on current use products with a focus on fate, exposure, and biological effects. Science of The Total Environment, v. 505, p. 409–422, 2015.

ASTM. Standard practice for conducting acute toxicity tests with fishes, macroinvertebrates and amphibians. American Standards for Testing and Materials. Philadelphia, P.A. . 1980, p. 1980.

65

BEMPAH, C. K. et al. A preliminary assessment of consumer’s exposure to organochlorine pesticides in fruits and vegetables and the potential health risk in Accra Metropolis, Ghana. Food Chemistry, v. 128, n. 4, p. 1058–1065, 2011.

BENACHOUR, N. et al. Time- and dose-dependent effects of roundup on human embryonic and placental cells. Archives of Environmental Contamination and Toxicology, v. 53, n. 1, p. 126–133, 2007.

BLAUSTEIN, L. Evidence for predatory flatworms as organizers of zooplankton and mosquito community structure in rice fields. Hydrobiologia, v. 199, p. 179–191, 1990.

BOLL, P. et al. A taste for exotic food: Neotropical land planarians feeding on an invasive flatworm. PeerJ, p. 8, 2015.

BROCK, T. C. M.; VAN WIJNGAARDEN, R. P. A. Acute toxicity tests with Daphnia magna, Americamysis bahia, Chironomus riparius and Gammarus pulex and implications of new EU requirements for the aquatic effect assessment of insecticides. Environmental Science and Pollution Research, v. 19, n. 8, p. 3610–

3618, 2012.

BÜRGER, J. et al. Simulation study of the impact of changed cropping practices in conventional and GM maize on weeds and associated biodiversity. Agricultural Systems, v. 137, p. 51–63, 2015.

BURNEY, J. A; DAVIS, S. J.; LOBELL, D. B. Greenhouse gas mitigation by agricultural intensification. Pnas, v. 107, n. 26, p. 12052–12057, 2010.

CAMPANHA, M. M. et al. Growth and yield of coffee plants in agroforestry and monoculture systems in Minas Gerais, Brazil. Agroforestry Systems, v. 63, n. 1, p.

75–82, 2005.

CATTANI, D. et al. Mechanisms underlying the neurotoxicity induced by glyphosate-based herbicide in immature rat hippocampus: Involvement of glutamate excitotoxicity. Toxicology, v. 320, p. 34–45, 2014.

CAVAS, T.; KONEN, S. Detection of cytogenetic and DNA damage in peripheral erythrocytes of goldfish (Carassius auratus) exposed to a glyphosate formulation using the micronucleus test and the comet assay. Mutagenesis, v. 22, n. 4, p. 263–268, 2007.

COMPANHIA NACIONAL DE ABASTECIMENTO (CONAB). Perspectivas para a agropecuária. Perspec. agropec, v. 2, p. 1–155, 2014.

CONNON, R. E.; GEIST, J.; WERNER, I. Effect-Based Tools for Monitoring and Predicting the Ecotoxicological Effects of Chemicals in the Aquatic Environment. Sensors, v. 12, n. 12, p. 12741–12771, 2012.

COSTANTINI, M. L. Effect of multiple disturbances on food web vulnerability to biodiversity loss in detritus-based systems. Ecosphere, v. 6, n. July, p. 1–20, 2015.

66

CUHRA, M.; TRAAVIK, T.; BØHN, T. Clone- and age-dependent toxicity of a glyphosate commercial formulation and its active ingredient in Daphnia magna. Ecotoxicology, v. 22, n. 2, p. 251–262, 2013.

DIAS GUERRA, W.; DE OLIVEIRA, P. C.; PUJOL-LUZ, J. R. Gafanhotos (Orthoptera, Acridoidea) em áreas de cerrados e lavouras na Chapada dos Parecis, Estado de Mato Grosso, Brasil. Revista Brasileira de Entomologia, v. 56, n. 2, p. 228–239, 2012.

EDWARDS, C. B. et al. Benchmark study on glyphosate-resistant crop systems in the United States . Economics of herbicide resistance management practices in a 5 year field-scale study . Pest Manag Sci, v. 70, n. 12, p. 3759, 2014.

EISSA, A. E.; ZAKI, M. M. The impact of global climatic changes on the aquatic environment. Procedia Environmental Sciences, v. 4, p. 251–259, 2011.

ELLIOTT, S. A.; SÁNCHEZ ALVARADO, A. The history and enduring contributions of planarians to the study of animal regeneration. Wiley Interdisciplinary Reviews: Developmental Biology, v. 2, n. 3, p. 301–326, 2013.

ENVIRONMENTAL PROTECTION AGENCY, WASHINGTON, D. O. OF THE A. A. FOR W. Drinking Water Criteria Document for Glyphosate. [s.l: s.n.].

FERNÁNDEZ-DÁVILA, M. L. et al. Aluminum-induced oxidative stress and neurotoxicity in grass carp (Cyprinidae-Ctenopharingodon idella). Ecotoxicology and Environmental Safety, v. 76, p. 87–92, 2012.

FLEEGER, J. W.; CARMAN, K. R.; NISBET, R. M. Indirect effects of contaminants in aquatic ecosystems. Science of The Total Environment, v. 317, n. 1-3, p. 207–233, 2003.

FOLMAR, L. C.; SANDERS, H. O.; JULIN, A M. Toxicity of the herbicide glyphosphate and several of its formulations to fish and aquatic invertebrates. Archives of environmental contamination and toxicology, v. 8, n. 3, p. 269–278, 1979.

FREIRE, C.; KOIFMAN, R.; KOIFMAN, S. Hematological and hepatic alterations in Brazilian population heavily exposed to organochlorine pesticides . J Toxicol Environ Health A, v. 78, n. 8, p. 15287394, 2015.

FRIED, G. et al. Arable weed decline in Northern France: Crop edges as refugia for weed conservation? Biological Conservation, v. 142, n. 1, p. 238–243, 2009.

FUKUSHIMA, M. et al. Detection and changes in levels of testosterone during spermatogenesis in the freshwater planarian Bdellocephala brunnea. Zoological science, v. 25, n. 7, p. 760–765, 2008.

GARCIA GARCIA, E.; BUSSACOS, M. A.; FISCHER, F. M. Impact of legislation on registration of acutely toxic pesticides in Brasil. Revista de Saude Publica, v. 39, n.

5, p. 832–839, 2005.

67

GARCÍA-GÓMEZ, C. et al. Assessing the ecotoxicological effects of long-term contaminated mine soils on plants and earthworms: relevance of soil (total and available) and body concentrations. Ecotoxicology, p. 1195–1209, 2014.

GARCÍA-MEDINA, S. et al. Genotoxic and cytotoxic effects induced by aluminum in the lymphocytes of the common carp (Cyprinus carpio). Comparative Biochemistry and Physiology Part C, v. 153, n. 1, p. 113–118, 2011.

GAUPP-BERGHAUSEN, M. et al. Glyphosate-based herbicides reduce the activity and reproduction of earthworms and lead to increased soil nutrient concentrations. Nature Publishing Group, n. August, p. 1–9, 2015.

GHOLAMI-SEYEDKOLAEI, S. J. et al. Optimization of recovery patterns in common carp exposed to roundup using response surface methodology: Evaluation of neurotoxicity and genotoxicity effects and biochemical parameters. Ecotoxicology and Environmental Safety, v. 98, p. 152–161, 2013.

GREEN, J. M. Current state of herbicides in herbicide-resistant crops. Pest Management Science, v. 70, n. 9, p. 1351–1357, 2014.

GUECHEVA, T.; HENRIQUES, J. A P.; ERDTMANN, B. Genotoxic effects of copper sulphate in freshwater planarian in vivo, studied with the single-cell gel test (comet assay). Mutation Research - Genetic Toxicology and Environmental Mutagenesis, v. 497, n. 1-2, p. 19–27, 2001.

GUEDES-ALONSO, R. et al. Molecularly imprinted solid-phase extraction coupled with ultra high performance liquid chromatography and fluorescence detection for the determination of estrogens and their metabolites in wastewater. Journal of Separation Science, p. 1–23, 2014.

HARICHANDAN, R. et al. An assessment of heavy metal contamination in soils of fresh water aquifer system and evaluation of eco-toxicity by lithogenic implications. Environmental Monitoring and Assessment, v. 185, p. 3503–3516, 2013.

HARMON, J. P.; BARTON, B. T. On their best behavior: how animal behavior can help determine the combined effects of species interactions and climate change. Annals of the New York Academy of Sciences, v. 1297, n. 1, p. 139–147, 2013.

JAGADEESH, E. et al. Toxic potential of iron oxide, CdS/Ag2S composite, CdS and Ag2S NPs on a fresh water alga Mougeotia sp. Colloids and Surfaces B: Biointerfaces, v. 125, p. 284–290, 2015.

JESUS, F. T. et al. Lethal and sub lethal effects of the biocide chlorhexidine on aquatic organisms. Ecotoxicology, v. 22, n. 9, p. 1348–1358, 2013.

KAYSER, C.; LARKIN, T.; SINGHAL, N. Amendment of biosolids with waste materials and lime: Effect on geoenvironmental properties and leachate production. Waste Management, p. 2015, 2015.

KNAKIEVICZ, T. et al. Reproductive modes and life cycles of freshwater planarians. Invertebrate Biology, v. 125, n. 3, p. 212–221, 2006.

68

KNAKIEVICZ, T. et al. Biogeography and karyotypes of freshwater planarians (Platyhelminthes, Tricladida, Paludicola) in southern Brazil. Zoological science, v. 24, n. 2, p. 123–129, 2007.

KNAKIEVICZ, T.; FERREIRA, H. B. Evaluation of copper effects upon Girardia tigrina freshwater planarians based on a set of biomarkers. Chemosphere, v. 71, n. 3, p.

419–428, 2008.

KOWALCZUCK, M. et al. The Lepidoptera Associated with Forestry Crop Species in Brazil: A Historical Approach. Neotropical Entomology, v. 41, n. 5, p. 345–354, 2012.

KUMAR, V.; GILL, K. D. Oxidative stress and mitochondrial dysfunction in aluminium neurotoxicity and its amelioration: A review. NeuroToxicology, v. 41, p. 154–166,

2014.

LIU CL , XI YL , HUANG L, W. J. Impact of glyphosate and acetochlor on Dugesia japonica ingestion and regeneration. Chinese journal of Applied Ecology, v. 19, n. 11, p. 2509–2514, 2008.

LIU, H. et al. Toxic effects of perfluorononanoic acid on the development of Zebrafish (Danio rerio) embryos. Journal of Environmental Sciences, v. 32, p. 26–34, 2015.

LOMBARDO, P. et al. Fine-scale differences in diel activity among nocturnal freshwater planarias (Platyhelminthes: Tricladida). Journal of circadian rhythms, v.

9, n. 1, p. 2, 2011.

LUPI, L. et al. Occurrence of glyphosate and AMPA in an agricultural watershed from the southeastern region of Argentina. Science of The Total Environment, v. 536, p. 687–694, 2015.

MACRAE, E. K. Observations on the fine structure of photoreceptor cells in the planarian Dugesia tigrina. Journal of ultrastructure research, v. 10, n. 3, p. 334–

349, 1964.

MACRAE, E. K. The fine structure of sensory receptor processes in the auricular epithelium of the planarian, Dugesia tigrina. Zeitschrift für Zellforschung, v. 82, n. 4, p. 479–494, 1967.

MARC, J.; MULNER-LORILLON, O.; BELLÉ, R. Glyphosate-based pesticides affect cell cycle regulation. Biology of the Cell, v. 96, n. 3, p. 245–249, 2004.

MARINHO, C. D. et al. Genetically modified crops: Brazilian law and overview. Genetics and Molecular Research, v. 13, n. 3, p. 5221–5240, 2014.

MATTHEWS, G. et al. Status of legislation and regulatory control of public health pesticides in countries endemic with or at risk of major vector-borne diseases. Environmental Health Perspectives, v. 119, n. 11, p. 1517–1522, 2011.

MENÉNDEZ-HELMAN, R. J. et al. Glyphosate as an acetylcholinesterase inhibitor in cnesterodon decemmaculatus. Bulletin of Environmental Contamination and

69

Toxicology, v. 88, n. 1, p. 6–9, 2012.

MENSAH, P. K.; MULLER, W. J.; PALMER, C. G. Acute toxicity of Roundup? herbicide to three life stages of the freshwater shrimp Caridina nilotica (Decapoda: Atyidae). Physics and Chemistry of the Earth, v. 36, n. 14-15, p. 905–909, 2011.

MEYER, D. E.; CEDERBERG, C. SIK-Rapport Nr 809: Pesticide use and glyphosate- resistant weeds – a case study of Brazilian soybean production. [s.l: s.n.]. Disponível em: <http://commodityplatform.org/wp/wp-content/uploads/2011/03/s>.

MIKULIC, P.; BEARDALL, J. Contrasting ecotoxicity effects of zinc on growth and photosynthesis in a neutrophilic alga (Chlamydomonas reinhardtii) and an extremophilic alga (Cyanidium caldarium). Chemosphere, v. 112, p. 402–11, 2014.

MODESTO, K. A.; MARTINEZ, C. B. R. Roundup® causes oxidative stress in liver and inhibits acetylcholinesterase in muscle and brain of the fish Prochilodus lineatus. Chemosphere, v. 78, n. 3, p. 294–299, 2010.

MONSANTO. Monsanto ’ s Strong Third Quarter Performance Reinforces Confidence in Full-Year Guidance ; Global Portfolio , Leading Innovation for Farmers and Financial Discipline Fuel Growth in Challenging Agriculture Environment Monsanto Reaffirms Vision for Ag and. Disponível em: <http://news.monsanto.com>. Acesso em: 23 ago. 2015.

MONSANTO COMPANY © 2002-2015. Monsanto’s Intacta RR2 ProTM Poised To Deliver A New Wave Of Benefits For South American Countries. Disponível em:

<http://www.monsanto.com/newsviews/pages/intacta-rr2-pro-benefits- for-south-american-countries.aspx>. Acesso em: 23 ago. 2015.

OLIVEIRA, A. R.; FREITAS, S. P. Levantamento fitossociológico de plantas daninhas em áreas de produção de cana-de-açúcar. Planta Daninha, v. 26, n. 1, p.

33–46, 2008.

ONGLEY, E. D. FAO Irrigation and Drainage Paper 55: Control of Water Pollution from Agriculture. [s.l: s.n.]. Disponível em: <http://www.fao.org/docrep/W2598E/W2598E00.htm>.

OVIEDO, N. J. et al. Establishing and maintaining a colony of planarians. CSH protocols, v. 2008, p. pdb.prot5053, 2008.

PAKRASHI, S. et al. Cytotoxicity of aluminium oxide nanoparticles towards fresh water algal isolate at low exposure concentrations. Aquatic Toxicology, v. 132-133,

p. 34–45, 2013.

PATHIRATNE, A.; KROON, F. J. Using species sensitivity distribution approach to assess the risks of commonly detected agricultural pesticides to Australia’s tropical freshwater ecosystems. Environmental Toxicology and Chemistry, v. Accepted A,

2015.

POLETTA, G. L. et al. Genotoxicity of the herbicide formulation Roundup

70

(glyphosate) in broad-snouted caiman (Caiman latirostris) evidenced by the Comet assay and the Micronucleus test. Mutation Research - Genetic Toxicology and Environmental Mutagenesis, v. 672, n. 2, p. 95–102, 2009.

PRASNISKI, M. E. T.; LEAL-ZANCHET, A. M. Predatory behavior of the land flatworm Notogynaphallia abundans (Platyhelminthes: Tricladida). Zoologia (Curitiba), v. 26, n. 4, p. 606–612, 2009.

PRESIDÊNCIA DA REPÚBLICA. DECRETO No 4.074, DE 4 DE JANEIRO DE 2002 Regulamenta Brasil. Casa Civil , Subchefia para Assuntos Jurídicos, , 2002. Disponível em: <http://www.mma.gov.br/port/conama/legiabre.cfm?codlegi=515>

RAFFA, R. B.; HOLLAND, L. J.; SCHULINGKAMP, R. J. Quantitative assessment of dopamine D2 antagonist activity using invertebrate (Planaria) locomotion as a functional endpoint. Journal of Pharmacological and Toxicological Methods, v. 45, n. 3, p. 223–226, 2001.

RAFFA, R. B.; MARTLEY, A. F. Amphetamine-induced increase in planarian locomotor activity and block by UV light. Brain Research, v. 1031, n. 1, p. 138–140,

2005.

REDDIEN, P. W.; SÁNCHEZ ALVARADO, A. Fundamentals of Planarian Regeneration. Annual Review of Cell and Developmental Biology, v. 20, n. 1, p. 725–757, 2004.

RIBEIRO, F. et al. Silver nanoparticles and silver nitrate induce high toxicity to Pseudokirchneriella subcapitata, Daphnia magna and Danio rerio Fabianne. Science of the Total Environment, v. 466-467, p. 232–241, 2014.

RODRIGUES, A. C. M. et al. Life history and biochemical effects of chlorantraniliprole on Chironomus riparius. Science of The Total Environment, v. 508, p. 506–513, 2015.

SÁNCHEZ-BAYO, F.; TENNEKES, H. A. Impact of Systemic Insecticides on Organisms and Ecosystems. Additional information is available at the end of the chapter, v. Chapter 13, p. 365–414, 2013.

SAWYER, D. Climate change, biofuels and eco-social impacts in the Brazilian Amazon and Cerrado. Philosophical transactions of the Royal Society of London. Series B, Biological sciences, v. 363, n. 1498, p. 1747–1752, 2008.

SHEIMAN, I. M.; ZUBINA, E. V; KRESHCHENKO, N. D. Regulation of the Feeding Behavior of the Planarian Dugesia ( Girardia ) tigrina. Journal of Evolutionary Biochemistry and Physiology, v. 38, n. 4, p. 414–418, 2002.

SHUHAIMI-OTHMAN, M. et al. Toxicity of eight metals to Malaysian freshwater midge larvae Chironomus javanus (Diptera, Chironomidae). Toxicology and Industrial Health, v. 27, n. 10, p. 879–886, 2011.

SIBLEY, P. K.; BENOIT, D. A.; ANKLEY, G. T. The significance of growth in Chironomus tentans sediment toxicity tests: Relationship to reproduction and

71


336–345, 1997.

SIMBERLOFF, D. et al. Impacts of biological invasions: What’s what and the way forward. Trends in Ecology and Evolution, v. 28, n. 1, p. 58–66, 2013.

SMITH, R. G. et al. Evidence for indirect effects of pesticide seed treatments on weed seed banks in maize and soybean. Agriculture, Ecosystems & Environment, v. 216, p. 269–273, 2016.

SOARES, A. et al. More Cercospora Species Infect Soybeans across the Americas than Meets the Eye . PLoS One., v. 10, n. 8, p. 133495, 2015.

SOUZA, T. L. P. O. et al. Soybean rust resistance sources and inheritance in the common bean ( Phaseolus vulgaris L .). Genetics and Molecular Research, v. 13,

n. 3, p. 5626–5636, 2014.

SZCZEPANIEC, A. et al. Neonicotinoid Insecticide Imidacloprid Causes Outbreaks of Spider Mites on Elm Trees in Urban Landscapes. PLoS ONE, v. 6, n. 5, p. e20018, 2011.

TABASHNIK, B. E. et al. Defining terms for proactive management of resistance to Bt crops and pesticides. Journal of economic entomology, v. 107, n. 2, p. 496–507,

2014.

TANG, S. et al. Threshold conditions for integrated pest management mdoels with pesticides that have residual effects. Journal of mathematical biology, v. 66, p. 1–35, 2013a.

TANG, S. et al. Threshold conditions for integrated pest management models with pesticides that have residual effects. Journal of Mathematical Biology, v. 66, n. 1-

2, p. 1–35, 2013b.


<http://data.worldbank.org/indicator/NV.AGR.TOTL.ZS/countries/BR?display=graph>.

TSUI, M. T. K.; CHU, L. M. Aquatic toxicity of glyphosate-based formulations: Comparison between different organisms and the effects of environmental factors. Chemosphere, v. 52, n. 7, p. 1189–1197, 2003.

UREN WEBSTER, T. M. et al. E ff ects of Glyphosate and its Formulation, Roundup, on Reproduction in Zebra fi sh ( Danio rerio ). Environmental science & technology, v. 48, p. 1271–1279, 2014.

VAN DER SLUIJS, J. P. et al. Neonicotinoids, bee disorders and the sustainability of pollinator services. Current Opinion in Environmental Sustainability, v. 5, n. 3-4,

p. 293–305, 2013.

VEHNIÄINEN, E.-R.; KUKKONEN, J. V. K. Multixenobiotic resistance efflux activity in Daphnia magna and Lumbriculus variegatus. Chemosphere, v. 124, p. 143–9, 2015.

72

VOIGT, C. L. et al. Bioconcentration and bioaccumulation of metal in freshwater Neotropical fish Geophagus brasiliensis. Environmental Science and Pollution Research, v. 22, p. 8242–8252, 2015.

WALSH, L. P. et al. Roundup inhibits steroidogenesis by disrupting steroidogenic acute regulatory (StAR) protein expression. Environmental Health Perspectives, v.

108, n. 8, p. 769–776, 2000.

WEIS, J. S. et al. Effects of Contaminants on Behavior: Biochemical Mechanisms and Ecological Consequences. BioScience, v. 51, n. 3, p. 209, 2001.

WHALEN, R.; HARMON, J. P. Temperature alters the interaction between a herbivore and a resistant host plant. Arthropod-Plant Interactions, v. 9, p. 233–240, 2015.

XAVIER, M. et al. Acute Toxicity and Sublethal Effects of Botanical Insecticides to Honey Bees. JOURNAL OF INSECT SCIENCE, v. 15, p. 1–6, 2015.

ZHANG, X. et al. Mortality and antioxidant responses in the planarian (Dugesia japonica) after exposure to copper. Toxicology and industrial health, v. 30, n. 2, p.

123–31, 2014.

ZHAO, M. et al. Disruption of the hormonal network and the enantioselectivity of bifenthrin in trophoblast: maternal-fetal health risk of chiral pesticides. Environmental science & technology, v. 48, n. 14, p. 8109–16, 2014.

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CONSIDERAÇÕES FINAIS

A agricultura intensiva desenvolvida nas diferentes partes da RHTA tem

contribuído para a poluição dos ecossistemas aquáticos, prejudicando organismos

bentônicos, como Dugesia tigrina e, consequentemente, aos organismos dos

diferentes níveis da cadeia alimentar.

Nas práticas agrícolas utilizam-se vários tipos de agroquímicos, tais como o

herbicida glifosato. Este herbicida é usado em diferentes etapas na condução das

lavouras. Diante disso, no presente estudo não foi detectada a presença do

herbicida glifosato nas amostras de água analisadas, o que provavelmente, pode ter

ocorrido devido à degradação de sua molécula na presença de luz, ou até mesmo,

pelo elevado poder de dissolução das águas nos canais de irrigação das áreas de

cultivo e a presença de metais.

Este trabalho demostrou que a planária de água doce Dugesia tigrina pode

ser usada como ferramenta para avaliar os ecossistemas aquáticos. Produtos

tóxicos como Glifosato e metais provocaram alterações na fisiologia, na reprodução

e nas repostas comportamentais das planarias. Exposições de 5 semanas à

concentrações de 1.71 mg·L-¹ de glifosato e a presença de metais provocaram

sérios danos na reprodução das planarias. Estes resultados são de relevância

ecológica, uma vez que, menores concentrações com maior tempo de exposição

representam elevados riscos.

As alterações do comportamento das planárias e da reprodução pela

presença de tóxicos, está colocando em risco a manutenção da estrutura dos

ecossistemas aquáticos, já que as planárias tem um papel fundamental como

predador dentro das cadeias tróficas.

Embora não se tenha detectado a presença de glifosato nas amostras de

água, isso não garante que este herbicida esteja ausente durante toda a época do

ano ou que o herbicida não seja tóxico aos ecossistemas, uma vez que, foi

demostrado neste estudo que mediante exposições curtas de 96 h promoveram

alterações no comportamento das planárias.

74

Annexes

Annex 1. Glyphosate concentrations measured in samples used in lethal and sub-

lethal expositions (mean ± SD).

Experiment

Roundup Nominal concentrations (mg·L-¹)

Glyphosate (mg·L-¹)

Lethal

20 20.72 22 22.60 24 24.84 26 28 30 31.69 32 34 34.34 36 35.19 38 40 40.91 42 44 46 48 48.29 50 49.57 52 50.95 54

Sub-lethal

1.87 1.71 (± 0.165) 3.75 3.39 (± 0.41) 7.5 8.32 (± 0.37) 15 14.91 (± 0.83)

75

Annex 2. Median lethal concentration (LC50) in 24, 48 and 96 hours of exposed D.

tigrina to RD original, calculated as measured concentrations of active ingredient

glyphosate. Probit Analysis and 95% CI.

Time (h) LC50 (mg·L-¹) 95% confidence limits (mg·L-¹)

Lower Upper

24 47.17 45.17 49.81

48 37.06 35.93 38.17

96 29.32 28.38 30.23

76

Annex 3. Analysis of organic and inorganic compounds in water samples of point 3

of hydrographic region Tocantins Araguaia.

78

Annex 4. Live cycle of Dugesia tigrina, adults of 3 weeks (a), cocoons (b), hatchlings

(c).

1 mm

c

b

a

79

Annex 5: Feed rate

80

Annex 6. Planarian locomotor velocity (pLMV)

81

÷

Fc: 12/14

Fc: 0.85 12 14

12 9 ÷

Fr: 12/9

Fr: 1.33

Annex 7. Fecundity (Fc) and fertility (Fr) rate

82

Annex 8. Blastema regeneration, control treatment

Blastema

0 hours

72 hours

24 hours

48 hours

96 hours

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