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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
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
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.
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Universidade Federal do Tocantins
Câmpus de Gurupi Programa de Pós-Graduação em Produção Vegetal
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
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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
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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!
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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
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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.
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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
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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
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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
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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
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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
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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
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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).
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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
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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
Samples hydrographic region:Tocantins Araguaia
******
***
******
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).
3.2.2 Planarian Locomotor velocity (pLMV)
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).
3.2.3 Fecundity rate
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
**
***
***
***
***
Samples hydrographic region:Tocantins Araguaia
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.
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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
3.2.1 Planarian Locomotor velocity (pLMV)
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
3.3.1 Fecundity rate
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.
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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)
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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
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Annex 3. Analysis of organic and inorganic compounds in water samples of point 3
of hydrographic region Tocantins Araguaia.
77
78
Annex 4. Live cycle of Dugesia tigrina, adults of 3 weeks (a), cocoons (b), hatchlings
(c).
1 mm
c
b
a
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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
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Annex 8. Blastema regeneration, control treatment
Blastema
0 hours
72 hours
24 hours
48 hours
96 hours