UNIVERSIDADE ESTADUAL DO MARANHÃO
CENTRO DE EDUCAÇÃO, CIÊNCIAS EXTAS E NATURAIS
DEPARTAMENTO DE QUÍMICA E BIOLOGIA
PROGRAMA DE PÓS GRADUAÇÃO EM RECURSOS AQUÁTICOS E PESCA
HELLON CUNHA VIANA
MONITORAMENTO DA QUALIDADE AMBIENTAL COM USO DE BIOMARCADORES HISTOLÓGICOS EM Sciades herzbergii (Pisces, Ariidae) E Bagre bagre (Pisces, Ariidae) EM UM SISTEMA ESTUARINO NO NORDESTE AMAZÔNICO
São Luís – MA
2018
HELLON CUNHA VIANA
MONITORAMENTO DA QUALIDADE AMBIENTAL COM USO DE BIOMARCADORES HISTOLÓGICOS EM Sciades herzbergii (Pisces, Ariidae) E Bagre bagre (Pisces, Ariidae) EM UM SISTEMA ESTUARINO NO NORDESTE AMAZÔNICO
Documento de dissertação apresentado em cumprimento às exigências do Programa de Pós-Graduação em Recursos Aquáticos e Pesca da Universidade Estadual do Maranhão.
Orientadora: Prof.ª Dr.ª Raimunda Nonata Fortes Carvalho Neta
São Luís – MA
2018
HELLON CUNHA VIANA
MONITORAMENTO DA QUALIDADE AMBIENTAL COM USO DE BIOMARCADORES HISTOLÓGICOS EM Sciades herzbergii (Pisces, Ariidae) E Bagre bagre (Pisces, Ariidae) EM UM SISTEMA ESTUARINO NO NORDESTE AMAZÔNICO
Documento de dissertação apresentado em cumprimento às exigências do Programa de Pós-Graduação em Recursos Aquáticos e Pesca da Universidade Estadual do Maranhão.
Aprovado em: _____/_____/_____
Banca examinadora
______
Prof.ª Dr.ª Raimunda Nonata Fortes Carvalho Neta (Orientadora)
Universidade Estadual do Maranhão (UEMA)
______
Prof.ª Dr.ª Débora Martins Silva Santos
Universidade Estadual do Maranhão (UEMA)
2° Examinador
______
Prof.ª Dr.ª Marianna Jorge Basso
Universidade Federal do Maranhão (UFMA)
3° Examinador
“Dedico este trabalho aos amigos que estiveram ao meu lado e que sempre torceram e torcem pelo meu sucesso, e em especial a toda minha família que é de onde tiro a coragem, a força e o suporte necessário para alcançar os meus objetivos.”
“Quando temos sede parece-nos que poderíamos beber todo um oceano: é a fé; e quando bebemos, bebemos um copo ou dois: é a ciência.” Anton Tchekhov
Agradecimentos Primeiramente gostaria de agradecer à toda minha família, sempre ao meu lado.
Ao meu pai, Sr. Neemias Viana, pelo apoio incondicional para minha formação superior e ampliação dos meus conhecimentos, por ser esse cara justo e bom educador.
À minha mãe, Dona Eliane Viana, essa professora guerreira, com seu espirito de educador, por sempre investir tudo o que pôde, sem mesquinhez e com a melhor das intenções. Sempre com uma lição de moral e bondade para passar, trabalhando turno dobrado e que ainda assim cuida da casa, dos filhos, mãe dedicada e maior incentivadora do sucesso pelo conhecimento.
Agradecer à minha noiva, Simone Silva, pela companhia, carinho, amor, pelas horas de sono mal dormidas, pelas discussões, por toda essa jornada, praticamente juntos, em busca de um mesmo ideal.
Às minhas irmãs, Myrna e Milca, pelo amor fraternal e por também fazerem parte desse sonho, crescemos, desejamos, almejamos e alcançamos nossos objetivos.
À Professora Dr.ª Raimunda Nonata Fortes Carvalho Neta, que é uma mulher de cabeça aberta e perspicaz. Sempre atenciosa e incentivadora da busca pelo conhecimento de forma honesta e verdadeira. Obrigado pelo acolhimento, incentivo e orientação na hora que mais precisei.
À professora Dr.ª Débora Martins Silva Santos pela atenção prestada, pela sua disposição para tirar dúvidas e propor soluções e por se dirigir aos alunos de maneira honesta e sempre solícita.
À professora Dr.ª Marianna Basso Jorge por ter aceitado o convite para compor à banca avaliadora e por ter sanado algumas dúvidas e sugerido boas ideias.
Aos demais Professores do DQB que fizeram e fazem parte de toda essa compilação de conhecimentos.
Aos colegas LABOAQ e GPEMAAQ, em especial a Wanda Batista, que ajudou nas tarefas de laboratório e na organização, e os demais colegas, como a Simone, Andressa e Jorge que ajudaram nas coletas.
Ao pescador Denércio (Deco), pela paciência e determinação nas coletas, sempre colocando nosso trabalho em primeiro lugar.
A todos os colegas da turma PPGRAP 2017 com os quais tivemos muito bons momentos e pelo suporte nas horas mais difíceis e de maior concentração.
Fica aqui meu muito obrigado e desejo de sucesso a todos!
Resumo
Os ambientes aquáticos sofrem constantemente com a poluição através de diversos tipos de efluentes lançados sem o devido tratamento. O objetivo deste estudo foi comparar biomarcadores histológicos (lesões branquiais e hepáticas) em Sciades herzbergii e Bagre bagre indicativos de impactos ambientais na Baía de São José, Maranhão. Foram realizadas quatro coletas trimestralmente em quatro pontos na Baía de São José, Maranhão (A1 – referência; A2 – impactada, A3 – contaminada e A4 - referência). Foi realizado a biometria e aferição do peso total. As amostras de fígado e brânquia passaram pelo processo de histologia básica. Foram realizados cortes de 5µm, colorado com hematoxylina-eosina e examinado sob microscópio óptico (ZEISS). A análise histopatológica mostrou uma elevado número de alterações hepáticas como respostas inflamatórias (centros de melanomacrófagos - MMCs), hiperplasia e vacuolização citoplasmática em S. herzbergii da área impactada. Também foi possível identificar uma diferença significativa entre o índice do fígado (Iorg.) em peixes que vivem na área A2 em relação às demais áreas. Valores do índice hepatossomático (HSI) e o fator de condição (FC) mostraram-se similares nas áreas A1, A2 e A3, no entanto diferiu da área A4. Os peixes das áreas A3 e A2 tiveram um maior número de anormalidade no fígado. Resultados indicaram que os fígados dos indivíduos adultos da área A2 (260.50 ± 161.50 MMCs/mm²) apresentaram um número maior de MMCs quando comparados à indivíduos adultos da área A3 (60.00 ± 30.10 MMCs/mm²). Indivíduos jovens dos pontos A1 (100.00 ± 0.00) e A2 (95.33 ± 33.00) apresentaram valores consideráveis quando comparados à jovens do ponto A3 (49.00 ± 0.00). O maior percentual de lesões branquiais, tais como, deformação da lamela secundária, fusão parcial, fusão total, necrose, hipertrofia e hiperplasia foram constatadas em S. herzbergii que habitam a área A2 em relação as demais áreas estudadas. O presente estudo indica que o S. herzbergii é mais apropriado do que o Bagre bagre para estudos com biomarcadores histológicos, tanto para lesões hepáticas (efeitos crônicos) e em especial levando-se em consideração as lesões branquiais (efeitos agudos) e se mostrou útil para o monitoramento de regiões estuarinas, por causa da grande sensibilidade demonstrada para a área potencialmente impactada (A2) comparada às áreas de referencia (A1 e A4) e área contaminada (A3). Palavras- chave: Biomonitoramento, biomarcador, histopatologia, bagre, poluição.
Abstract
The aquatic environments constantly suffer from pollution through several types of effluent released without proper treatment. The aim of this study was to compare histological biomarkers (gill and hepatic lesions) in S. herbergii and B. bagre indicative of impacts from fishery and aquaculture systems in São José Bay, Maranhão. Four samplings were carried out quarterly at four different sites in the São José Bay, Maranhão. (A1 - reference, A2 - impacted, A3 - contaminated and A4 - reference). Biometric data and total weight were assessed. Liver and gill samples underwent basic histology. Sections of 5um were stained with hematoxylin-eosin and examined under an optic microscope (ZEISS). Histopathological analysis showed a high number of hepatic alterations such as inflammatory responses (melanomacrophagous centers - MMCs), hyperplasia and cytoplasmic vacuolization in S. herzbergii of the impacted area. It was also possible to identify a significant difference between the liver indexes (Iorg.) in fishes that live in the impacted area in relation to the other areas. The values of the hepatosomatic index (HSI) and the condition factor (K) were similar in areas A1, A2 and A3 but differed from area A4. Fish from areas A3 and A2 had a greater number of abnormalities in the liver. Results indicated that the adults‟ livers of the A2 area (260.50 ± 161.50 MMCs / mm²) had a higher number of MMCs when compared to the adults A3 area (60.00 ± 30.10 MMCs / mm²). Young individuals of A1 (100.00 ± 0.00) and A2 (95.33 ± 33.00) presented considerable values when compared to young A3 (49.00 ± 0.00). The highest rate of branchial lesions, such as secondary lamella deformity, partial fusion, total fusion, necrosis, hypertrophy and hyperplasia were observed in S. herzbergii that inhabit the potentially contaminated area in relation to the other areas. The present study indicates that S. herzbergii is more appropriate than Bagre bagre for histological biomarkers studies both for hepatic lesions (chronic effects) and especially taking into account the branchial lesions (acute effects). It has proved useful for monitoring of estuarine regions because of the great sensitivity shown for the potentially impacted area (A2) compared to the reference areas (A1 and A4) and contaminated area (A3). Keywords: Biomonitoring, biomarker, histopathology, catfish, pollution.
LISTA DE FIGURAS
TEXTO INTEGRADOR
Figura 1. Mapa de localização de captura do S. herzbergii e B. bagre na baía de São José, MA...... 25 Figura 2. Mapa de cobertura do solo da bacia hidrográfica do Rio Santo Antônio...... 26 Figura 3. Exemplar de S. herzbergii capturado na Baía de São José, Maranhão ...... 27 Figura 4. Exemplar de B. bagre capturado na Baía de São José, Maranhão...... 27
CAPÍTULO I
Figure 1. Site map of S. herzbergii and B. bagre catches at the Santo Antonio River basin and Curupu Bay estuary complex, Maranhão...... 36 Figure 2. Boxplots showing hepatosomatic index (HSI) variation (a) and condition factor (K) (b) in the different sampled areas...... 44 Figure 3. Boxplots showing melanomacrophage centers (MMCs) (c) for S. herzbergii and B. bagre in the different areas sampled...... 455 Figure 4. Photomicrography (H&E) of histopathological liver changes. (a) Macrovesicular steatosis (lipid cells). (b) Inflammatory response (leukocyte infiltrate). (c) Atrophy and tumor (psamomatous body). (d) Aneurysms and edema. (e) Necrosis. (f) Granulomatosis. (g) Melanomacrophage centers (MMCs). (h) Hyperplasia. The arrow () indicates the changes found...... 49 Figure 5. Photomicrography (H&E) of histological gill changes. (a) Normal gills. (b) Hyperplasia. (c) Necrosis and partial fusion. (d) Secondary lamellae disorganization. (e) Hypertrophy. (f) Epithelium raising. (g) Venous sinus dilation, total fusion and aneurysms. (h) Parasites. The arrow () indicates the changes found...... 51
CAPÍTULO II
Figure 1. Geographic location of sampling points. A1 – reference, A2 – impacted and A3 – contaminated...... 742
Figure 2. Photomicrograph of the liver of S. herzbergii sampled at three different points of the São José bay - MA. (A) H&E staining of a section of the liver showing lipid components. (B) Melanomacrophage center presence. (C) Lipid cells, psamomatous body in hepatopancreas tissue and MMCs. (D) antibodies surrounding the hepatocyte cytoplasm as well as the bile duct epithelium. (E, F) The histological appearance of normal liver. Asterisk: melanomacrophage center; m: macrophages; lp: lipid cells; s: sinusoids; pb: psamomatous body; bd: bile duct...... 796 Figure 3. Number of melanomacrophage centers (MMCs/mm²) (standard error) in young and adult S. herzbergii livers at the three capture sites. Asterisk (*) indicates a significant statistic difference comparing the reference and impacted areas...... 797 Figure 4. Number of melanomacrophage centers (MMCs/mm²) during the sample period. ... 79
LISTA DE TABELAS
CAPÍTULO I
Table 1. Biometric data and gonadosomatic index of S. herzbergii and B. bagre captured at Curupu bay...... 411 Table 2. Physical parameters of water (mean) recorded in dry and rainy periods at the fish collection sites...... 422 Table 3. Histopathological alterations identified in the liver of S. herzbergii and B. bagre of each sampling point...... 466 Table 4. Histopathological alterations identified in the gill of S. herzbergii and B. bagre of each sampling point...... 499 Table 5. Liver index of S. herzbergii and B. bagre for each site and sampled period...... 52 Table 6. Gill index of S. herzbergii and B. bagre to each site and period of collection...... 53
CAPÍTULO II
Table 1. Average length and percentage of young and adults of S. herzbergii from different sampled locations...... 775
SUMÁRIO 1 INTRODUÇÃO ...... 15
2 OBJETIVOS ...... 16
2.1 Objetivo geral ...... 16
2.2 Objetivos específicos ...... 16
3 FUNDAMENTAÇÃO TEÓRICA ...... 16
3.1 Monitoramento ambiental em sistemas estuarinos ...... 16
3.2 Biomonitoramento em ecossistemas aquáticos com uso de peixes ...... 18
3.3 Biomarcadores em organismos aquáticos ...... 20
3.4 Sciades herzbergii (Bloch, 1974) ...... 24
3.5 Bagre bagre (Linnaeus, 1766) ...... 24
4 METODOLOGIA ...... 25
4.1 Área de estudo ...... 25
4.2 Licença e comitê de ética ...... 26
4.3 Coleta das espécies, biometria e dados abióticos ...... 26
4.4 Histologia dos tecidos ...... 28
4.5 Quantificação dos Centros de Melanomacrófagos (MMCs) ...... 29
4.6 Fator de condição (K) ...... 29
4.7 Índice hepatossomático (HSI) ...... 29
4.8 Análises estatísticas ...... 30
5 RESULTADOS ...... 31
Article 1 – Histopathology on Sciades herzbergii (Pisces, Ariidae) and Bagre bagre (Pisces, Ariidae) gills to assess environmental pollution ...... 31
Abstract ...... 32
1. Introduction ...... 33
2. Material and Methods ...... 35
2.1 Study area ...... 35
2.2 Licensing and ethics declaration ...... 36
2.3 Collection of organisms ...... 37
2.4 Abiotic water parameter analysis ...... 37
2.5 Tissue processing and histopathology ...... 38
2.6 Microscopic analysis ...... 38
2.7 Statistical analysis ...... 40
3. Results ...... 40
3.1 Fish and abiotic water parameter data ...... 40
3.2 Biometric indexes ...... 42
3.3 Macroscopic observations ...... 44
3.4 Prevalence of histological alterations ...... 44
3.5 Quantitative analysis and comparison of fish from each studied area ...... 52
4. Discussion ...... 54
Conclusions ...... 60
Acknowledgements ...... 61
Funding ...... 61
References ...... 61
Article 2: Aggregation of hepatic melanomacrophage centers in S. herzbergii (Pisces, Ariidae) as indicators of environmental change and well-being ...... 70
Abstract ...... 71
1. Introduction ...... 72
2. Materials and Methods ...... 73
2.1 Study area ...... 73
2.2 Licensing and ethics declaration ...... 74
2.3 Tissue Processing and Histopathology ...... 74
2.4 Melanomacrophage Center Quantification (MMCs) ...... 75
2.5 Condition factor ...... 75
2.6 Statistical analysis ...... 76
3. Results ...... 76
3.1 Histopathology ...... 77
3.2 MMC quantitative analysis ...... 79
3.3 Condition factor and melanomacrophage centers ...... 80
4. Discussion ...... 81
Conclusions ...... 84
Acknowledgements ...... 84
Funding ...... 85
References ...... 85
6 CRONOGRAMA ...... 110
7 CONSIDERAÇÕES FINAIS ...... 111
REFERÊNCIAS ...... 111
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1 INTRODUÇÃO O Golfão Maranhense é constituído pelas baías de São Marcos e São José, separadas pela Ilha de São Luís. A Baía de São José insere-se no contexto das regiões tropicais úmidas e tem grande importância ambiental pela caracterização marcada por estuários e reentrâncias (TEIXEIRA; SOUZA FILHO, 2009). Os sistemas estuarinos apresentam extensas florestas de mangue e fornecem os principais nutrientes para a região costeira através da concentração de material originado na sua bacia de drenagem e aportes de origem antropogênica (AZEVEDO et al., 2008).
O crescente processo de urbanização e o desenvolvimento econômico próximo às áreas costeiras, sob influência de ações antropogênicas, como uso e ocupação do solo, uso de fertilizantes na agricultura, pecuária, piscicultura e o aporte de efluentes domésticos e industriais sem tratamento adequado, tem causados danos irreparáveis em todos os ecossistemas aquáticos (SIMÕES et al., 2007; DOMINGOS et al., 2009; LINS et al., 2010; SANTOS et al., 2012; CANTANHÊDE et al., 2014). Os peixes têm sido utilizados como bioindicadores e alguns de seus órgãos (brânquia e fígado) têm funcionado para análises de biomarcadores através de alterações histológicas, cuja principal finalidade é a identificação de contaminação em ambientes aquáticos através da avaliação das lesões causadas por potenciais poluentes (CARVALHO-NETA; ABREU-SILVA, 2010). Os modernos programas de biomonitoramento ambiental tem utilizado essa técnica por apresentarem respostas mais rápidas e mais concisas às possíveis contaminações (CARVALHO-NETA; TORRES; ABREU-SILVA, 2012; CARVALHO-NETA; ABREU-SILVA, 2013; CARVALHO NETA et al., 2014; SALES et al., 2017).
Dentre as espécies de peixes, destaca-se o Sciades herzbergii (guribu), uma das poucas espécies que possui todo o seu ciclo de vida nos estuários maranhenses, sendo considerado como uma espécie “estuarino-residente” (CARVALHO-NETA; ABREU-SILVA, 2010). E o Bagre bagre (bandeirado), peixe marinho que ocorre comumente próximo à desembocadura de rios (FIGUEIREDO; MENEZES, 1978; CARVALHO-NETA; CASTRO, 2008) e que fazem migração na época de reprodução (ABSOLON; ANDREATA, 2009). Esses táxons são considerados bons bioindicadores, por serem topo de cadeia alimentar, importante fonte de proteína e renda para comunidades de pesca artesanal e por sua ampla distribuição geográfica (CARVALHO-NETA; CASTRO, 2008). São bem relatadas na literatura em estudos de Biomonitoramento de contaminação aquática no Complexo Estuarino de São Marcos
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(SOUSA; ALMEIDA; CARVALHO-NETA, 2013a; SOUSA; ALMEIDA; CARVALHO- NETA, 2013b; CARVALHO-NETA; TORRES; ABREU-SILVA, 2012; CARVALHO NETA et al., 2014) porém são escassos os trabalhos na Baía de São José, apesar da complexidade de ecossistemas e de sua importância ecológica.
Este trabalho será apresentado em dois capítulos em forma de artigos científicos que serão posteriormente enviados a revistas para sua publicação. O primeiro capítulo aborda a avaliação de alterações encontradas em fígados e brânquias de S. herzbergii e B. bagre, através de observações de necropsia, prevalência de alterações histológicas e índices biométricos para avaliar poluição ambiental. O segundo capítulo consiste na utilização de centros de melanomacrófagos hepáticos de S. herzbergii como biomarcador de exposição a poluentes ambientais, em particular o material oriundo de sistemas de piscicultura.
2 OBJETIVOS
2.1 Objetivo geral Avaliar o uso de biomarcadores histológicos (lesões branquiais e hepáticas) em Sciades herzbergii e Bagre bagre indicativos de impactos ambientais na Baía de São José, Maranhão.
2.2 Objetivos específicos Identificar e quantificar as lesões branquiais e as lesões hepáticas nas espécies Sciades herzbergii e Bagre bagre na baía de São José. Correlacionar dados biométricos das espécies Sciades herzbergii e Bagre bagre com os biomarcadores histológicos.
3 FUNDAMENTAÇÃO TEÓRICA
3.1 Monitoramento ambiental em sistemas estuarinos A Baía de São José está situada no litoral do Estado do Maranhão, limitada a oeste pela Ilha de São Luís, que a separa da Baía de São Marcos, e a leste pelas ilhas que formam a Baía de Tubarão, formando assim o Golfão Maranhense (COUTINHO, P. N; MORAIS, 1976). As amplitudes de marés estão entre as maiores do litoral brasileiro, com até 6,5 metros nas marés equinociais (CASTRO; MIRANDA, 1998) e correntes com velocidade superior a 7,5 nós registradas pela DHN (1972). De um modo geral, os padrões de circulação e mistura das águas conferem ao ambiente como um sistema dinâmico (CARVALHO-NETA; CASTRO, 2008). A Baía de São José possui um espelho d‟água de aproximadamente 665
17 km², recebendo na sua formação o aporte fluvial dos rios Itapecuru e Munim (PNRH, 2006). O clima é tropical úmido, com estações seca (julho a dezembro) e chuvosa (janeiro a junho) bem definidas, com temperatura média em torno de 26ºC (NUGEO, 2011). A vegetação predominante são os manguezais, considerados os mais estruturalmente complexos do Brasil (MOCHEL, 1997). Este aspecto é atribuído em parte pelas diversas características da linha de costa, assim como, o imenso volume de água doce aportada pelos rios, as altas taxas de precipitação, bem como as altas amplitudes de maré (TEIXEIRA; SOUZA FILHO, 2009).
Nesse contexto, alguns sistemas estuarinos de rios perenes destacam-se por sua importância social e econômica na região. Um desses sistemas é o estuário do Rio Santo Antônio, localizado na porção nordeste da Ilha do Maranhão, compreendendo o município de Paço do Lumiar, parte de São José de Ribamar e de São Luís. O comprimento total da bacia do rio Santo Antônio é de aproximadamente 21,0 km (FERREIRA, 2003).
Os principais processos erosivos observados na bacia do rio Santo Antônio são causados por agentes oceanográficos, tais como, ondas, marés, correntes e por agentes climáticos, tais como, ventos, temperatura, pluviosidade e umidade (FEITOSA, 1996). O clima é tropical úmido, caracterizado por dois períodos: um chuvoso, com grandes índices pluviométricos, de janeiro a junho e outro de estiagem, com poucas chuvas, de julho a dezembro (KOPPEN, 1948). A temperatura média é de 26°C, com temperaturas mais elevadas durante os meses de outubro e dezembro e inferiores, em abril e maio. A precipitação média anual oscila em torno de 2000 mm (NUGEO, 2011).
A ocupação desenfreada e a rápida expansão urbana vêm gerando alguns problemas ambientais e sociais ao longo da área da bacia hidrográfica do rio Santo Antônio, destacando- se a sustentabilidade do sistema de aproveitamento da água para consumo humano e a destruição da flora e da fauna, principalmente porque compreende áreas de manguezais e matas de galeria (FERREIRA, 2003).
A mata ciliar está completamente devastada no curso superior do rio Santo Antônio e a sua destruição está relacionada ao acelerado processo de ocupação sem planejamento urbano, do município de São Luís, particularmente com a construção dos conjuntos habitacionais, Cidade Operária e Paranã (FERREIRA, 2003). O médio e o baixo curso do rio Santo Antônio estão razoavelmente conservados, dada as características do município de Paço do Lumiar, com ocupações de pequenos povoados, como, Pau deitado, Mojó, Salina, Pindoba, Iguaíba e Timbuba, onde ainda é considerado preservada a vegetação das margens do rio (FERREIRA,
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2003). No baixo curso do rio, o desmatamento nas áreas de manguezais é pouco evidenciado, sendo um dos ecossistemas mais conservados de toda a Ilha de São Luís, servindo de subsistência para populações ribeirinhas, que fazem utilização da área para pesca, extração de mariscos e moluscos.
Os processos erosivos ocorrem principalmente no período chuvoso, com a ação das chuvas e aumento do volume do rio e a ação antrópica está relacionada à intensa extração de barro e areia para a construção civil. Ocorre também a lixiviação de todo o material que fica solto, sendo transportado para o rio, ocasionando processos de assoreamento e de alteração da morfologia do canal. Dar-se ainda o lançamento de esgoto in natura e lixo diretamente nas nascentes e nos canais do rio (FERREIRA, 2003).
As principais atividades registradas nessa região são a pesca artesanal, agricultura familiar e piscicultura. Nesse contexto, o monitoramento ambiental do rio Santo Antônio e baía de São José de Ribamar são de extrema importância para a conservação dos principais corpos hídricos da região.
Recentemente, esta região têm apresentado um crescente desenvolvimento de empreendimentos aquícolas, trazendo uma grande preocupação quanto aos lançamentos de efluentes de piscicultura de forma inapropriada. Esses efluentes podem conter amônia e nitrito, que são compostos tóxicos para organismos aquáticos. Estes poluentes estão presentes no ambiente natural pela excreção dos organismos aquáticos, no entanto podem aumentar suas concentrações pelo processo de degradação do alimento não consumido e pelo processo incompleto de nitrificação (DOS SANTOS SILVA et al., 2018). Assim, a utilização de peixes estuarinos, principalmente os bagres, por sua importância ecológica e econômica, em estudos de biomonitoramento, pode torná-los um potencial modelo biológico para indicar contaminação ambiental nos ecossistemas aquáticos.
3.2 Biomonitoramento em ecossistemas aquáticos com uso de peixes O desenvolvimento de metodologias de diagnósticos eficientes é o primeiro passo para a resolução de problemas relacionados à má gestão dos recursos hídricos. Nas últimas quatro décadas, pesquisadores do mundo inteiro argumentam que as metodologias tradicionais de classificação das águas baseadas tão somente nas características físico-químicas e bacteriológicas são insuficientes para atender aos usos múltiplos da água. Dessa forma, as respostas biológicas foram incorporadas na avaliação dos sistemas aquáticos (BUSS; BAPTISTA; NESSIMIAN, 2003).
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Duas formas de metodologias são utilizadas para a avaliação, a primeira , utiliza testes toxicológicos, que são as respostas de organismos aquáticos a estressores específicos, tais como, alterações bioquímicas, fisiológicas, comportamentais, metabólicas e do ciclo de vida (VAN DER OOST; BEYER; VERMEULEN, 2003). Neste contexto, destacam-se os bioensaios de reação aguda, especializados na determinação de poluição letal e subletal de substâncias sintéticas ou naturais de origem mineral, animal ou vegetal (EWELL et al., 1986). No entanto, a avaliação em laboratório envolve ainda a análise da exposição crônica, conferindo os efeitos deletérios quanto a genotoxicidade, carcinogenicidade e mutagenicidade (REYNOLDSON; METCALFE-SMITH, 1992). A segunda metodologia avalia, em nível macro, os impactos ambientais por meio da medição da alteração da organização estrutural e funcional das comunidades biológicas ou dos ecossistemas (BUSS; BAPTISTA; NESSIMIAN, 2003).
Segundo Metcalfe (1989), o uso das respostas biológicas como indicadores de degradação ambiental é vantajoso em relação às medidas físicas e químicas da água, pois estas registram apenas o momento em que foram coletadas, como uma fotografia do rio, necessitando assim de um grande número de análises para a realização de um monitoramento temporal eficiente. Outra desvantagem é que, se forem feitas longe da fonte poluente, as medições químicas não serão capazes de detectar perturbações sutis sobre o ecossistema (PRATT; COLER, 1976).
Os ecossistemas aquáticos estão frequentemente sob exposição de substâncias tóxicas lançadas no ambiente. A poluição em ambientes aquáticos é bastante difusa, porém as principais fontes de contaminação são os efluentes industriais, processos de drenagem agrícola (fertilizantes, agrotóxicos), derrames acidentais e não acidentais de lixos químicos (metais pesados, compostos orgânicos e inorgânicos) e efluentes domésticos (LINS et al., 2010). O biomonitoramento ambiental através da avaliação de bioindicadores acrescenta informações a respeito da resposta biológica que aquele táxon apresenta na presença desses poluentes (WHO, 1996). O termo bioindicador pode ser reconhecido como sendo espécies sentinelas que são utilizadas como primeiros indicadores de efeito da contaminação de seu habitat (ADAMS et al., 2002). Em se tratando de ambiente aquático, plantas aquáticas, algas, crustáceos, moluscos, peixes, mamíferos, aves, entre outros, podem ser alvos de estudos, portanto são considerados bioindicadores. Um bom biomonitor deve sobreviver em ambientes saudáveis, mas também apresentar resistência relativa ao contaminante que está exposto, ser taxonomicamente bem definido e facilmente reconhecível por não-especialistas, apresentar
20 distribuição geográfica ampla, ter baixa variabilidade genética e ecológica, preferencialmente possuir tamanho grande, apresentar baixa mobilidade e longo ciclo de vida e dispor de características ecológicas bem conhecidas (JOHNSON et al., 1993). Outros aspectos que podem facilitar o desenvolvimento de um estudo são a abundância dessa espécie no ambiente e facilidades de coleta e de se adaptar a ensaios laboratoriais (AKAISHI et al., 2004).
Nesse contexto, a seleção de espécies que possam refletir a situação ambiental do baixo curso do rio Santo Antônio e baía de Curupu, torna-se relevante para monitoramento das interferências que essa região vêm sofrendo ao longo dos anos. Os peixes por serem organismos pertencentes ao topo da cadeia alimentar são comumente utilizados por possuírem intrínseca relação com toda a cadeia inferior, indicando respostas de efeitos crônicos, acumulativos e persistentes no nível de cadeia, além de efeitos diretos à nível individual (CARVALHO NETA et al., 2014).
Três categorias aplicáveis de bioindicadores para estudos de biomonitoramento de ambientes aquáticos e terrestres são considerados (GERHARDT, 2009). O primeiro, é um bioindicador a nível ambiental, no qual a espécie ou grupo de espécies respondem previsivelmente à perturbação ambiental ou alteração ambiental, tais como organismos sentinelas, acumuladores e os que são utilizados em bioensaios. O segundo, é um bioindicador a nível ecológico, onde os indivíduos são conhecidos pela sensibilidade aos locais poluídos ou contaminados e pode ocasionar a fragmentação do habitat. O terceiro, é um bioindicador a nível de biodiversidade, é levado em consideração a riqueza de um táxon que é utilizado como indicador da riqueza de espécies de uma comunidade.
No presente trabalho iremos enfocar os peixes como bioindicadores a nível ambiental, por se tratar de um estudo que utiliza biomarcadores de contaminação aquática e outros parâmetros ambientais para compreender a saúde dos indivíduos provenientes de ecossistemas naturais do rio Santo Antônio e baía de Curupu, Maranhão.
3.3 Biomarcadores em organismos aquáticos O uso de parâmetros biológicos para medir a qualidade da água se baseia nas respostas dos organismos em relação ao meio onde vivem. Como os rios estão sujeitos a inúmeras perturbações, a biota aquática reage a esses estímulos, sejam eles naturais ou antropogênicos. A habilidade de proteger os ecossistemas depende da capacidade de distinguir os efeitos das ações humanas das variações naturais, buscando categorizar a influência das ações humanas sobre os sistemas biológicos (CAIRNS; VAN DER SCHALIE, 1980). Nesse contexto, a
21 definição de biomonitoramento mais aceita é o uso sistemático das respostas de organismos vivos para avaliar as mudanças ocorridas no ambiente, geralmente causadas por ações antropogênicas (MATTHEWS et al., 1982).
Por sua vez, os organismos integram as condições ambientais durante toda a sua vida, permitindo que a avaliação biológica seja utilizada com bastante eficiência na detecção tanto de ondas tóxicas intermitentes agudas quanto de lançamentos crônicos contínuos (DE PAUW; VANHOOREN, 1983). Além disso, as metodologias biológicas são bastante eficazes na avaliação de poluição não pontual (difusa), tendo, portanto, grande valor para avaliações em escala regional (PRATT; COLER, 1976).
A identificação dos biomarcadores mais apropriados para um melhor diagnóstico do ecossistema em que se pretende investigar, deve-se levar em consideração o poluente a ser estudado e o melhor modelo biológico para a avaliação de impacto ambiental (JESUS; CARVALHO, 2008). Para tanto, existem várias classificações acerca dos biomarcadores e sua aplicabilidade (AELION et al., 2009).
A divisão clássica da ecotoxicologia aquática e as mais utilizadas pelos pesquisadores, subdivide os biomarcadores em nível de exposição, efeito e de suscetibilidade (GOLDSTEIN et al., 1987; WORLD HEALTH ORGANIZATION, 2001; AMORIM, 2003; JESUS; CARVALHO, 2008). Por outro lado, os biomarcadores são categorizados de acordo com indicadores e/ou marcadores específicos presentes nos indivíduos, tais como: enzimas de biotransformação, estresse oxidativo, proteínas reguladoras, parâmetros hematológicos, imunológicos, reprodutivos, genotóxicos, neuromusculares, fisiológicos e a nível morfológico ou histológico (VAN DER OOST; BEYER; VERMEULEN, 2003).
Neste trabalho, iremos utilizar a classificação de Van der Oost et al. (2003) que leva em consideração a utilização de marcadores biológicos histológicos. A histologia é uma ferramenta sensível para se diagnosticar efeitos tóxicos diretos e indiretos que afetem tecidos animais. Por isso é considerada um excelente método de avaliação de impacto ambiental causado por agentes tóxicos sobre os animais constituintes de uma determinada fauna e, portanto, é amplamente utilizada (PÉREZ et al., 2018). Exclusivamente, o estudo de patologias nos tecidos resulta dados sobre lesões, mas não especifica a causa pontual dessa lesão, ou seja, não diagnostica o poluente em questão, somente pontua a resposta biológica aos estressores ambientais. Entretanto, a associação de métodos de análises e estudos histológicos pode auxiliar o entendimento mais detalhado de diversas situações (LINS et al.,
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2010). A escolha de um órgão é crucial para a relevância dos dados obtidos. Alguns órgãos, como as brânquias, que apresenta contato direto com o poluente, o fígado e o rim, que metabolizam e excretam os xenobióticos, podem indicar alterações de ações agudas ou crônicas desses poluentes em tecidos animais (SANTOS et al., 2012; CARVALHO-NETA et al., 2014).
Em bagres, como na maioria dos teleósteos, a brânquia é a principal superfície corporal envolvida nas trocas gasosas (HUGHES, 1982). Desempenha um papel crucial na regulação ácido-base, na osmoregulação e na excreção de substâncias azotadas (GOSS et al., 1998). As consequências da ação de poluentes neste órgão-alvo podem desencadear processos degenerativos, necróticos e transtornos do crescimento celular (HIBIYA, 1982). Dessa maneira, as alterações histológicas encontradas nas brânquias são facilmente reconhecidas, tornando-se importante para determinar os danos causados aos níveis de exposição dos peixes aos poluentes (ARELLANO; STORCH; SARASQUETE, 1999).
O epitélio da brânquia é a principal superfície de contato com o ambiente e na presença de agentes estressores, as brânquias podem exibir modificações que são consideradas respostas de defesa. Agentes estressores químicos, físicos ou biológicos induzem reações nos tecidos causando distúrbios circulatórios, como congestão, telangectasia, edema e a proliferação de células como a hiperplasia epitelial e de células mucosas, além de processos inflamatórios que podem levar a necrose dos tecidos (GARCIA-SANTOS et al., 2007; FONTAÍNHAS-FERNANDES et al., 2008; ROQUES et al., 2015).
Telangectasia lamelar ou aneurisma é uma alteração característica dos sinusóides branquiais e está associada a traumas físicos ou químicos. Apresenta-se após manejos mais severos e pode estar associada com lesões parasitárias, resíduos metabólicos ou contaminantes químicos. Quando muitas lamelas são afetadas, a função respiratória pode diminuir especialmente em temperaturas altas, quando os níveis de oxigênio são baixos e a demanda metabólica é alta (SANTOS et al., 2012).
Alguns estudos realizados com peixes têm demonstrado que a exposição das brânquias a poluentes ambientais produzem alterações nos filamentos branquiais como o espessamento do epitélio filamentar, que podem advir da proliferação de células de cloro e de células indiferenciadas do epitélio. A proliferação celular pode conduzir à fusão parcial ou total das lamelas branquiais (REIS et al., 2009).
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As alterações histológicas, por exemplo, a fusão lamelar, podem funcionar como mecanismos de defesa, diminuindo a área de superfície da brânquia ou, também, aumentando a barreira de difusão ao agente poluente ou estressor; entretanto, a maior dificuldade respiratória pode induzir à vasodilatação, podendo levar ao aparecimento de edemas e descolamento do epitélio branquial (ERKMEN; KOLANKAYA, 2000).
O fígado é considerado o principal órgão responsável pelo metabolismo das substâncias químicas, por meio de diferentes reações e processos enzimáticos, sendo o mais importante sítio anatômico de armazenamento, biotransformação e excreção de pesticidas (CENGIZ; UNLU, 2006). Também é um dos órgãos que tendem a acumular as substâncias tóxicas em concentrações superiores aos da corrente sanguínea, em função do alto grau de vascularização e importância fisiológica e toxicocinética (PEDROZO et al., 2002). Além do mais, estudos intensivos e detalhados de lesões hepáticas representam os exemplos mais convincentes de relação causal entre doenças em peixes e poluição do ambiente aquático (VAN DER OOST; BEYER; VERMEULEN, 2003).
O componente hepático é constituído pelos hepatócitos e um sistema de condutos que drenam a bile. Os hepatócitos são células poliédricas, grandes, geralmente com um núcleo central e nucléolos característicos, espalhados como cordões dispostos em duas camadas celulares, e rodeado por sinusóides. Os ductos biliares são geralmente encontrados perto da veia porta e eles são revestidos por epitélio cuboide simples (VICENTINI et al., 2005; WEBB, 2009).
Os hepatócitos podem ser considerados o primeiro alvo de toxicidade de uma substância, o que caracteriza o fígado como um órgão biomarcador da poluição ambiental (ZELIKOFF, 1998). Alterações como vacuolização dos hepatócitos, redução do estoque de glicogênio, inflamação, alteração no formato dos capilares sinusóides e neoplasmas podem ser interpretados como respostas ao estresse ambiental, sendo, considerados indicadores histopatológicos da qualidade do ambiente (TEH; ADAMS; HINTON, 1997).
A histopatologia, integrada com outros métodos, como a bioquímica, a imunologia, avaliação de crescimento e diagnóstico de doenças, é utilizada para a avaliação dos efeitos ambientais como a qualidade da água e as condições de internas de peixes. Observam-se também as alterações histológicas no fígado, sendo comumente reconhecidos vacuolização dos hepatócitos, degeneração gordurosa do fígado, alterações na atividade metabólica, alterações no parênquima hepático e necrose (RASKOVIC et al., 2011).
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3.4 Sciades herzbergii (Bloch, 1974) A espécie S. herzbergii pertence a Ordem Siluriformes, família Ariidae. Os peixes dessa espécie são popularmente conhecidos como bagre guribu, ocupam ambientes de água marinha, doce e estuarina demersal em climas tropicais. A sua distribuição ocorre no continente sul americano, no Caribe, rios do atlântico e estuários da Colômbia ao Brasil. Os adultos dessa espécie ocorrem em estuários com águas túrbidas, mangues e baixos cursos de rios e toleram mudanças de salinidade. São encontrados em regiões costeiras rasas (CERVIGÓN et al., 1992), alimentam-se de uma grande variedade de animais pelágicos, crustáceos, vermes e animais encontrados entre as raízes dos manguezais e sua reprodução ocorre entre setembro e dezembro. Uma fêmea coloca 20-30 ovos, com um diâmetro de 10-12 mm e os machos os incubam em sua boca (FROESE; PAULLY, 2018). A incubação demora 10 a 12 dias. Após 50-60 dias, jovens totalmente funcionais são libertados. As fêmeas atingem a maturidade em idade mais jovem que os machos e crescem mais rapidamente (MARCENIUK, 2005).
3.5 Bagre bagre (Linnaeus, 1766) O Bagre bagre, conhecido popularmente como bandeirado, é uma espécie demersal com comportamento restrito de migração. Essa espécie ocupa ambientes de águas marinhas costeiras, e faz migrações para ambientes estuarinos ou planícies costeiras em busca de alimento e para desovar (DA SILVA et al., 2016). Alimenta-se principalmente de pequenos peixes e invertebrados, como crustáceos. Os machos mantém os ovos fertilizados na sua cavidade oral por aproximadamente três meses, sem se alimentar, quando então os jovens estão prontos e permanecem no ambiente estuarino até ficarem maduros, não apresenta estágios pelágicos larvais (RIMMER, 1985; ARAÚJO, 1988).
O Bagre bagre é amplamente conhecido por sua distribuição ao longo da costa do continente Sul Americano, da Colômbia à boca do Rio Amazonas, Brasil. É mais comum em profundidades até 50 m, em áreas Tropicais (MARCENIUK, 2005). Em contraste com outros arídeos, que são tipicamente endêmicos de uma área geográfica restrita, espécies do gênero Bagre são amplamente distribuídos (MARCENIUK, 2007), sendo considerados como característica de espécie invasora (LODGE, 1993). Em algumas partes do Brasil, como no nordeste amazônico, essa espécie é alvo de intensivas pescarias industriais e algumas capturas artesanais (MARCENIUK, 2015).
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4 METODOLOGIA
4.1 Área de estudo Os pontos amostrais localizam-se nos municípios de Paço do Lumiar e São José de Ribamar – MA (figura 1). Os embarques foram realizados no Porto do Pau Deitado, utilizou- se uma embarcação motorizada conhecida popularmente como biana. As coletas do S. herzbergii foram realizadas em um ponto no baixo curso do Rio Santo Antônio 2°29'36.54"S 44° 3'34.38"W (A2 - Pecoapara), um na foz, já na Baía de Curupu 2°27'47.58"S 44° 2'45.42"W (A1 - Panaquatira) e um terceiro ponto 2°33'18.36"S 44° 3'10.02"W (A3 - Porto do Vieira). Um único ponto foi amostrado para o Bagre bagre 2°28'56.64"S44° 1'44.40"W (A4 - Panaquatira).
Figura 1. Mapa de localização de captura do S. herzbergii e B. bagre na baía de São José, MA.
Foi realizado um mapeamento de uso e ocupação do solo para uma melhor compreensão das unidades paisagísticas da bacia do rio Santo Antônio (Figura 2). Através desse levantamento, a área caracterizou-se como sendo bem preservada por causa das extensas áreas verdes (11.435,43 km³) que representam quase 73,80% de toda a área
26 mapeada. Os corpos hídricos ocupam uma área de aproximadamente 4.057,02 km², correspondendo à 26% da região. Somente 1,50 km² da bacia hidrográfica da bacia do rio Santo Antônio correspondem a áreas ocupadas.
Figura 2. Mapa de cobertura do solo da bacia hidrográfica do Rio Santo Antônio, MA.
4.2 Licença e comitê de ética O protocolo para captura dos peixes foi aprovado pelo Comitê de ética da Universidade Estadual do Maranhão (número 064/2018 CRMV-MA).
4.3 Coleta das espécies, biometria e dados abióticos Quatro coletas foram realizadas, (1ª - setembro de 2017; 2ª – dezembro de 2017; 3ª – março de 2018 e 4ª – maio de 2018). A coleta do S. herbergii (figura 3) foi realizada com o uso de covos, conhecidos popularmente como manzuá. Sardinhas foram usadas como iscas, espetadas em cordões de até 3kg, presos na parte interna dos corvos. Os covos foram instalados no baixo curso do rio Santo Antônio, durante a maré baixa, em baixas profundidades e deixados de um dia para o outro, quando então ocorreu a despesca. A coleta do Bagre bagre (figura 4) foi realizada com o uso de redes de emalhar flutuantes, com uma malha de 15 a 40 mm entre nós adjacentes, 4 a 5 metros de altura e 200 metros de
27 comprimento para as capturas. As amostragens ocorreram durante a maré de quadratura com esforço de pesca padronizado de 6 horas, correspondendo a todo o ciclo da vazante.
Figura 3. Exemplar de S. herzbergii capturado na Baía de São José, Maranhão.
Figura 4. Exemplar de B. bagre capturado na Baía de São José, Maranhão.
Os peixes foram identificados segundo Cervigon et al. (1992). Após a identificação, a atualização taxonômica foi realizada acessando o Projeto Fishbase (FROESE; PAULY, 2018), em seguida foram colocados em caixas térmicas com gelo e encaminhados ao Laboratório de Biomarcadores em Organismos Aquáticos da Universidade Estadual do Maranhão – LABOaq. Foi aferido alguns dados de biometria, como peso total (Wt), comprimento total (Lt) e comprimento padrão (Lp). Posteriormente, uma incisão na parte ventral dos peixes foi realizada para a retirada dos órgãos (brânquias, fígados e gônadas) e aferição do peso das gônadas (Wg) e peso do fígado (Wl). A classificação dos estádios de maturação gonadal foi realizada segundo Brown-Peterson et al. (2011), I – Imaturo, II – Em desenvolvimento, III – Capaz de desovar, IV – Em Regressão e V – Em Regeneração.
Foram coletados 73 indivíduos da espécie S. herzbergii (24 machos e 49 fêmeas) e 42 B. bagre (27 machos e 15 fêmeas). São mostrados os dados biométricos (Lt – comprimento total, Lp – comprimento padrão, Wt – peso total, Wg – peso das gônadas e GSI – índice gonadossomático) dos peixes coletados na baía de Curupu.
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Os dados abióticos foram aferidos com auxílio do multiparâmetro da marca AK88, sondas de temperatura (ºC), salinidade, pH e Oxigênio dissolvidos (OD) auxiliaram na análise da qualidade da água. Os parâmetros físicos da água aferidos, pH, temperatura e oxigênio dissolvido apresentaram-se uniformes em todas as áreas e períodos analisados.
4.4 Histologia dos tecidos O protocolo de avaliação de poluição aquática proposta por Bernet et al. (1999) serviu para determinar o grau de severidade das lesões branquiais e hepáticas encontradas nos indivíduos dessa pesquisa. De acordo com o protocolo foi determinado um fator de importância (w) da lesão, que divide-se em três níveis: 1 – mínima importância patológica ou reversível; 2 – moderada importância patológica ou reversível na maioria dos casos e 3 – marcada importância patológica ou irreversível. O alcance da alteração histológica também foi registrado, classificando o grau e a extensão da lesão (a): 0 (sem mudanças), 2 (ocorrência leve), 4 (ocorrência moderada), e 6 (ocorrência severa), com valores intermediários também sendo considerados.
O uso do fator de importância e dos valores das alterações permitiu o cálculo de dois índices. Como as lesões de cada órgão foram estudadas separadamente, aplicou-se o cálculo de dois índices, o índice do órgão (Iorg.) e o índice de reação do órgão (Iorg rp), conforme proposto por Bernet et al. (1999):
Iorg. = Σrp Σ alt (a.w)
Onde: org = órgão (constante); rp = padrão de reação; alt = alteração encontrada no órgão; a = grau de extensão da alteração; w = fator de importância.
O índice representa o grau de dano para um órgão. Ele é a soma da multiplicação dos fatores de importância pelos valores de todas as mudanças encontradas no órgão examinado. Um elevado índice indica um alto grau de dano.
Iorg rp = Σ alt (a.w)
Onde: org e rp = constante.
A qualidade da lesão em um órgão é expressada pelo índice da reação. Ele é calculado pelo somatório da multiplicação do fator de importância pelo valor das alterações do padrão de reação correspondente. A soma dos cinco índices de reação do órgão é equivalente ao
índice do órgão (Iorg.).
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Onde: rp1 – distúrbios circulatórios (hemorragia, hiperemia, aneurisma e edema intercelular); rp2 – mudanças regressivas (alterações estruturais, atrofia, depósitos, necrose, alterações no plasma e alterações nucleares); rp3 – mudanças progressivas (hiperplasia e hipertrofia); rp4 – inflamação (exsudase, ativação do sistema reticuloendotelial e infiltração) e rp5 – tumores (benigno e maligno).
4.5 Quantificação dos Centros de Melanomacrófagos (MMCs) Primeiramente, os centros de melanomacrófagos consistem em agregação de macrófagos, como células e fragmentos de células fagocitadas, principalmente eritrócitos e pigmentos, como, melanina, hemosiderina e lipofuscina, localizados no tecido reticuloendotelial do fígado, rim e pâncreas (AGIUS; ROBERTS, 2003). Uma análise quantitativa da agregação de MMCs foi realizada em seções de tecido hepático dos peixes. Cada campo digital foi fotografado utilizando Zen Imaging software Axiovision 4.8 montado no microscópio óptico Zeiss com as objetivas de 4X, 10X e 40X. A agregação de MMCs foi medida usando análise de imagem no software ImageJ e foi medida aleatoriamente como número de MMCs por mm2. Cada seção foi retirada da região central do fígado, sendo possível mensurar todo o órgão.
4.6 Fator de condição (K) O fator de condição é um indicador quantitativo do grau de higidez ou de bem-estar de um indivíduo. Ele é usado para indicar variações na densidade populacional e nas condições alimentares. O valor reflete as condições nutricionais recentes e/ou gastos de reservas em atividades metabólicas, possibilitando relações com condições ambientais e aspectos comportamentais das espécies (VAZZOLER, 1996).
O fator de condição (K) foi estimado para cada indivíduo e suas médias obtidas para cada período, segundo a expressão (VAZZOLER,1996): K = Wt/Ltb, onde: Wt = peso total, Lt = comprimento e b = coeficiente angular da relação peso/comprimento.
4.7 Índice hepatossomático (HSI) O fígado de cada peixe foi pesado para o cálculo do índice hepatossomático (HSI) (massa do fígado/massa corporal x 100). O índice hepatossomático pode estar relacionado com a mobilização das reservas energéticas necessária para o processo de vitelogênese, reprodução ou preparação para o período mais seco (AZEVEDO; DE CASTRO; SILVA, 2017).
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4.8 Análises estatísticas Estatísticas descritivas são apresentadas na forma de médias, desvios-padrão, medianas e prevalência percentual. Os resultados histopatológicos de HSI, CF, e quantitativos foram comparados entre os locais utilizando o teste de variância ANOVA e teste Tukey. As Análises estatísticas foram realizadas utilizando-se o software PAlaeontological STatistics (PAST), version 1.0.0.0 (RYAN; HAMMER; HARPER, 2001) e significância estatística foi aceita para p ≤ 0.05. Os resultados da quantidade de MMCs foram comparados entre os locais utilizando o teste de Kruskal-Wallis. Para as variáveis com um resultado de teste significativo, comparações entre pares entre os diferentes locais foram feitas usando o teste U não-paramétrico de Mann-Whitney, controlando o erro do Tipo I entre os testes por meio do ajuste de Bonferroni. As diferenças estatísticas foram consideradas significativas quando p <0,05. Os dados foram verificados quanto à normalidade usando os testes de Kolmogorov- Smirnov ou Shapiro-Wilk.
31
1 5 RESULTADOS
2 Article 1 – Histopathology on Sciades herzbergii (Pisces, Ariidae) and Bagre bagre (Pisces,
3 Ariidae) gills to assess environmental pollution
4 Este artigo será submetido à revista Chemosphere. ISSN: 0045-6535. Fator de impacto: 4.427.
5 Qualis A1 (2018) para a área Zoologia/Recursos pesqueiros.
6 Hellon Cunha Viana a, Simone Karla Lima e Silva b, Marianna Basso Jorge c, Débora Martins
7 Silva Santos d, Raimunda Nonata Fortes Carvalho Neta d.
8 a Master Student in Aquatic Resources and Fisheries (PPGRAP) by State University of
9 Maranhão (UEMA), Campus Paulo VI, São Luis, Maranhão, Brazil.
10 b Master in Biodiversity and Conservation by Federal University of Maranhão (UFMA), São
11 Luis, Maranhão, Brazil.
12 c Department of Oceanography and Limnology, Federal University of Maranhão (UFMA),
13 São Luis, Maranhão, Brazil.
14 d Department of Chemistry and Biology, State University of Maranhão (UEMA), Campus
15 Paulo VI, São Luis, Maranhão, Brazil.
16 Corresponding author: Hellon Cunha Viana¹. Email: [email protected], phonenumber:
17 +55 98 983109904
18 „Declarations of interests: none‟
19
20
21
32
22 Abstract
23 Fish have been used as biomonitors, and hepatic and gill alterations as biomarkers for
24 contamination identification in aquatic environments. The objective of this research was to
25 evaluate data corresponding to alterations found in S. herzbergii and B. bagre gills and livers,
26 including necropsy observations, prevalence of histological alterations and biometrical
27 indexes. Biological samples were collected at four different sample sites (A1 – reference; A2
28 – impacted A3 – contaminated and A4 - reference) at Curupu Bay, Maranhão. The greatest
29 percentage of gill injuries, such as secondary flap deformity, partial fusion, total fusion,
30 hypertrophy and hyperplasia were recorded in S. herzbergii, which live in area A2 in relation
31 to the rest of the studied areas. These injuries remained between the end of the dry period and
32 start of the rainy period. Histopathological analysis showed an elevated number of hepatic
33 alterations such as inflammatory responses, melanomacrophage centers, hyperplasia and
34 cytoplasmtic vacuolization (lipid cells) in S. herzbergii from area A2. It was also possible to
35 identify a significant difference in the liver index (Iorg.) of fish, which live in area A2 in
36 relation to the other areas. Hepatosomatic index values (HSI) and condition factor (K) were
37 similar in areas A1, A2 and A3, however differed from area A4. Fish from areas A3 and A2
38 had the largest number of liver abnormalities (fat nodes). Results suggest that histological
39 alterations in S. herzbergii liver and gills might be used to assess areas with different levels of
40 water pollution in estuary environments.
41 Keywords: Biomonitoring, catfish, Pollution.
42
43
44
45
33
46 1. Introduction
47 Anthropogenic changes such as rapid population growth and urbanization of large areas have
48 led to a decline in the quality of aquatic ecosystems (Pérez et al., 2018). In addition, water
49 quality directly relates to the sanitary conditions of humans and animals that inhabit these
50 environments (Ghisi et al., 2016). Uncontrolled domestic and industrial effluent discharges,
51 agrochemical dumps, heavy metals, organic compounds among other xenobiotics (Van der
52 Oost et al., 2003) directly affect fish, among aquatic organisms. In this sense, fish are sentinel
53 organisms that can be used as bioindicators of environmental stress, both of abiotic and biotic
54 changes caused by pollutants (Débora Batista Pinheiro Sousa et al., 2013; DBP Sousa et al.,
55 2013, Carvalho Neta et al., 2014). The first responses of organisms to stress are changes at the
56 cellular and tissue levels (Facey et al., 2005).
57 Fish gills have a large surface in direct contact with water, gill changes have been widely used
58 as environmental contamination indicators (Zhang et al., 2014; Olivares-Rubio and Vega-
59 López, 2016). Experimental studies have assessed gill damage induced by xenobiotics, such
60 as heavy metals and pesticides (Garcia-Santos et al., 2007; Carvalho-Neta and Abreu-Silva,
61 2013; Olurin et al., 2016) and gill structure after a period of exposure to contaminants and
62 being relocated to good quality environments (Sales et al., 2017; Choi et al., 2018).
63 Studies on species from the South American continent, USA, Europe, Africa and Asia have
64 shown that liver histopathology, through alterations found in liver are advantageous exposure
65 biomarkers to contaminants (Au, 2004; Triebskorn et al., 2008; Van Dyk et al., 2012, Zhang
66 et al., 2014, Sardi et al., 2016, Pérez et al., 2018). Some of these studies were incorporated
67 into biomonitoring programs and programs of this nature require a level of histopathological
68 data and biomonitoring fish species from the region in question (Carvalho-Neta and Abreu-
69 Silva, 2010).
34
70 The northern region of Maranhão is well developed for its proximity to port complexes and
71 accelerated urban and industrial development (Carvalho-Neta et al., 2012). As a result, many
72 water bodies have suffered negative impacts related to releases of industrial effluents,
73 untreated sewage, among others (Cantanhêde et al., 2014).
74 Among pollutant mixtures, fishery effluents stand out the most, containing ammonia and
75 nitrite, which are toxic compounds for aquatic organisms. These pollutants are present in the
76 natural environment through aquatic organism excretion; however, they can increase their
77 concentrations through the processes of unconsumed food degradation and incomplete
78 nitrification (dos Santos Silva et al., 2018). Aquaculture has been responsible for continued
79 growth in fish supplying for human consumption (47% of 171 million tons produced in 2016),
80 because since the 1980s, fish production has been relatively static (FAO, 2018). Aquaculture
81 in Brazil produced more than 586 thousand tons of fish (70.9% referring to fish farming
82 followed by 19.3% to shrimp farming) (IBGE, 2016). Maranhão currently occupies the
83 seventh position in fish production, climbing two positions in comparisons to bulletins
84 published in previous years (IBGE, 2016).
85 Exposure regimes for pollutants experienced by fish are difficult to determine. Often, there is
86 fluctuation of chemicals in the river water due to varying rates of biodegradation as well as
87 physical-chemical conditions and water flow rates. These factors may alter the levels of
88 effluent dilution and fish movements between habitats (Vrana et al., 2005). However, some
89 histological changes, such as hyperplasia, cell hypertrophy and vascular damage depending
90 on the number of lesions assessed may be indicators of acute environmental contamination by
91 pollutants (Carvalho Neta et al., 2014). The taxon Sciades herzbergii is a typical benthic fish
92 and maintains a close relationship with the environment; it is common to coastal areas and has
93 been used as a model for the effect of contaminants in estuarine systems. Bagre bagre is a
94 migratory species which resides in estuarine environments in the Gulf of Maranhão
35
95 (CARVALHO-NETA; TORRES; ABREU-SILVA, 2012). These catfish species are widely
96 distributed and are used for research because of their sedentary habit and economic
97 importance for river front communities and small-scale fisheries which are located throughout
98 the Santo Antônio River basin (CARVALHO NETA et al., 2014).
99 This study sought to incorporate all data related to liver and gill abnormalities found in S.
100 herzbergii and B. bagre, including necropsy observations, prevalence of histological changes
101 and biometric indexes (hepatosomatic index-HSI and K-condition factor). It was determined
102 whether these measures differed and/or reflected the state of pollution of different aquatic
103 ecosystems on a regional scale. The approach divided four different sampling sites (A1 -
104 reference, A2 - impacted, A3 - contaminated and A4 - reference) in terms of site and pollution
105 status. Quantitative histopathological scoring systems were integrated to assess histological
106 changes in fish from each site in terms of severity of occurrence and pathological importance.
107
108 2. Material and Methods
109 2.1 Study area
110 The four selected collection sites are located between the mouth of the Santo Antônio River
111 and Curupu Bay, in the eastern region of São Luís Island, Maranhão (Fig. 1). It is
112 characterized as a rural region, marked by extensive mangrove areas. However, there is an
113 accelerated urbanization process. A region at the mouth of the river, and therefore further
114 away from the banks of the river, was selected as reference area for S. herzbergii (A1 - 2 °
115 27'47.58 "S 44 ° 2'45.42" W). A closer and more accessible location to the channel, where
116 fishery effluents are theoretically released, was characterized as an impacted area (A2 - 2°
117 29'36.54"S 44° 3'34.38" W). A third point, within an urban center, which constantly suffers
118 from domestic effluent releases was selected as a contaminated area (A3 - 2° 33'18.36 "S 44°
36
119 3'10.02" W). In addition, a fourth, further away from the coast, was selected as reference area
120 for the B. bagre A4 (2° 28'56.64 "S44° 1'44.40" W). Shipments left the Port of Pau Deitado,
121 municipality of Paço do Lumiar and they took a motorized vessel called biana.
122
123 Figure 1 Geographic location of S. herzbergii and B. bagre catches at the Santo Antonio River
124 basin and Curupu Bay estuary complex, Maranhão.
125
126 2.2 Licensing and ethics declaration
127 The capture of fish was authorized by the Ethics Committee of Maranhão State University
128 (number 064/2017-2018 CRMV-MA) and met the guidelines of the Brazilian College for
129 Animal Experimentation (SBCAL/COBEA, 2018).
130
37
131 2.3 Collection of organisms
132 The collection of S. herzbergii was carried out with the use of cages, known as manzuá.
133 Sardines were used as bait, arranged on cords up to 3kg and tied inside of the cages. The
134 cages were installed during the low tide, in low depths and left overnight, when the harvesting
135 occurred. The collection of B. bagre was carried out using floating gill nets with a mesh of 15
136 to 40 mm between adjacent nots, 4 to 5 meters in height and 200 meters in length for catches.
137 Samplings occurred during the quadrature tide with standard fishing effort of 6 hours,
138 corresponding to the entire ebb. The fish were acclimatized in thermal iceboxes to the Aquatic
139 Organisms Biomarker Laboratory at the State University of Maranhão - LABOaq.
140 Measurement of total length (cm) and total weight (g) were carried out to calculate condition
141 factor according to Vazzoler (1996). Evisceration was performed for macroscopic
142 identification of maturational stages, where: I - Immature, II - In development, III - Able to
143 spawn, IV - Regression and V - Regeneration (Brown-Peterson et al., 2011). The liver of each
144 fish was weighed to calculate the hepatosomatic index (HSI) (liver mass/body mass x 100).
145 The gonadosomatic index (GSI) was also evaluated (gonad mass/body mass x 100), it is the
146 value that expresses body weight allocated in the gonads.
147
148 2.4 Abiotic water parameter analysis
149 Abiotic data measured with assistance from the AK88 multi-parameter, temperature (ºC)
150 probes, salinity, pH and dissolved oxygen (DO) helped in the water quality analysis.
151
38
152 2.5 Tissue processing and histopathology
153 The same LABOaq team collected fish and tissue samples from each sampling point where
154 the same methodological process was applied. A basic histology procedure was applied, fish
155 were dissected and liver and gill samples were set in 10% formaldehyde, gills were
156 decalcified with nitric acid and later the organs were dehydrated in increasing alcohol
157 concentrations, they were diaphanized in xylol and went through paraffin inclusion. Sections
158 of liver and gills (5 μm) were stained with hematoxylin-eosin (H & E) (Merck) for hepatic
159 and branchial structure description, respectively.
160
161 2.6 Microscopic analysis
162 The optic microscope assisted in the quantitative assessment of histological changes. The
163 liver and gill slides were inspected with 4X, 10X and 40X lenses, depending on the lesion. To
164 determine the degree of hepatic and branchial lesion severity, the water pollution assessment
165 protocol proposed by Bernet et al. 1999 was used. In short, histological changes were
166 assessed according to identification of changes in five reaction patterns (rp). Where: rp1 -
167 circulatory disorders (hemorrhage, hyperemia, aneurysm and intercellular edema); rp2 -
168 regressive changes (structural changes, atrophy, deposits, necrosis, changes in plasma and
169 nuclear alterations); rp3 - progressive changes (hyperplasia and hypertrophy); rp4 -
170 inflammation (exsudase, reticuloendothelial system activation and infiltration) and rp5 -
171 tumors (benign and malignant).
172 When the lesion was identified, the importance factor (w) was evaluated at three levels: 1 -
173 minimal pathological or reversible importance; 2 - moderate pathological or reversible
174 importance in most cases and 3 - marked pathological or irreversible importance. We also
175 recorded the extent of the histological alteration, which classified degree and extent of the
39
176 lesion (a): 0 (no changes), 2 (mild occurrence), 4 (moderate occurrence), and 6 (severe
177 occurrence), with intermediate values also being considered.
178 Since lesions of each organ were studied separately, the calculation of two indices were
179 applied, the organ index (Iorg.) and organ reaction index (Iorg rp), as proposed by Bernet et al.
180 1999:
181
182 Iorg. = ΣrpΣalt (a.w)
183
184 Where: org = organ (constant); rp = reaction standard; alt = alteration found in the organ; a =
185 alteration extension degree; w = importance factor.
186 The index represents a degree of organ damage. It is the result of the sum of multiplying
187 importance factors with the values from all changes found in the examined organ. A high
188 index indicates a high degree of damage.
189
190 Iorgrp = Σ alt (a.w)
191
192 Where: org and rp = constant.
193 Lesion quality in an organ is expressed by the reaction index. It is calculated by the sum of
194 multiplying the importance factor with the value of the corresponding standard reaction
195 alterations. The sum of the organ reaction‟s five indices (Irp1; Irp2; Irp3; Irp4 e Irp5) is
196 equivalent to the organ index (Iorg.).
197
40
198
199 2.7 Statistical analysis
200 Descriptive statistics are presented in the form of averages, standard deviations, medians and
201 percentage prevalence. Histological results, hepatosomatic and condition factor variation, and
202 quantitative were compared between sites using the ANOVA variance test and Tukey test.
203 Statistical analyses were performed using the software PAlaeontological STatistics (PAST),
204 version 1.0.0.0 (Ryan et al., 2001) and statistical significance was accepted for p ≤ 0.05.
205 MMC quantity results were compared between sites using the Kruskal-Wallis test. For
206 variables with a significant test result, comparisons between pairs between different sites were
207 made using the Mann-Whitney non-parametric U test, controlling the Type I error between
208 the tests by Bonferroni adjustment. Data was checked for normality using the Kolmogorov-
209 Smirnov or Shapiro-Wilk tests.
210
211 3. Results
212 3.1 Fish and abiotic water parameter data
213 In the first collection (September/2017), four fish were caught at point A1, 10 fish at point A2
214 and 11 fish at point A4. In the second collection (December/2017), six fish were collected at
215 point A1, 3 fish at point A2, 12 fish at point A3 and 11 fish at point A4. In the third collection
216 (March/2018), 6 fish from point A1, 2 fish from point A2, 11 fish from point A3 and 9 fish
217 from point A4 were captured. In the fourth and final collection (May/2018), six fish were
218 caught at point A1, 5 fish at A2 points, 8 fish at point A3 and 11 fish at point A4. In total, 73
219 individuals of S. herzbergii (24 males and 49 females) and 42 B. bagre (27 males and 15
220 females) were collected (Table 1).
221
41
222
223
224 Table 1. Biometric data and gonadosomatic index of S. herzbergii and B. bagre captured at
225 Curupu bay.
Sampling Gender Lt (cm) Ls (cm) Wt (g) Wg (g) GSI point Females 34.50 ± 4.60 28.57 ± 3.88 399.77 ± 153.75 6.3415 ± 5.8604 1.3151 ± 1.0912 (A1) Males 28.57 ± 4.00 22.12 ± 4.81 237.89 ± 115.85 0.2433 ± 0.3364 0.0787 ± 0.0688 Females 30.72 ± 4.31 25.47 ± 3.94 286.85 ± 113.66 3.0635 ± 3.9105 0.9941 ± 1.1658 (A2) Males 20.27 ± 9.67 16.99 ± 8.27 281.71 ± 65.52 0.2872 ± 0.2401 0.1112 ± 0.1085 Females 30.25 ± 3.68 25.03 ± 2.88 269.22 ± 100.55 5.3420 ± 5.8499 1.6152 ± 1.5846 (A3) Males 26.25 ± 3.03 21.69 ± 2.69 177.25 ± 60.12 0.2107 ± 0.1031 0.1286 ± 0.0710 Females 29.02 ± 2.95 23.03 ± 2.44 165.13 ± 55.56 2.9654 ± 10.0217 1.2378 ± 4.0645 (A4) Males 28.02 ± 3.42 22.12 ± 2.86 144.96 ± 46.30 0.1912 ± 0.2037 0.1415 ± 0.1488 226
227 Values given as mean ( ) and standard deviation (± SD). Lt: Total length; Ls: Standard length;
228 Wg: Weight of gonad; GSI: Gonadosomatic index.
229
230 At point A1, it was divided among 12 young individuals and 10 adults; at point A2, 16 young
231 fish and 4 adults; at point A3, specimens were divided into 7 young and 24 adults and finally,
232 at point A4, 24 young individuals and 18 adults were identified. Biometric data (Lt - total
233 length, Lp - standard length, Wt - total weight, Wg - weight of gonads and GSI -
234 gonadosomatic index) of fish collected in Curupu Bay are shown. Females from the A1 area
235 showed the highest length and weight averages, whereas males from the A3 area had the
236 smallest average size, and males and females from the A4 area showed lower average weight.
237 The fish in the A2 area were mostly young and presented lower average GSI. In relation to the
238 physical parameters of the water measured, pH, temperature and dissolved oxygen were
239 uniform in all analyzed areas and periods. Salinity showed greater variation between drought
42
240 and rainy periods. All parameters sampled were in compliance with Brazilian legislation
241 (Conama 357/2005) (table 2).
242 Table 2. Physical parameters of water (mean) recorded in dry and rainy periods at the fish
243 collection sites.
244
(A1) (A2) (A3) a Recommended Parameters Dry Rainning Dry Rainning Dry Rainning values season season season season season season pH 7.57 7.40 7.42 7.41 7.46 7.70 from 6.5 to 8.5 Temp. (°C) 30.20 29.65 33.30 30.40 31.60 31.20 < 40°C DO (mg/L) 8.76 11.19 9.14 9.91 8.69 11.16 > 5mg/L Salinity 37.50 27.05 37.45 26.65 38.70 26.60
245
246 Values given as mean ( ). pH: Hydrogenic potential; Temp.: Temperature; DO: Dissolved
247 oxygen. a Recommended values based on resolution nos. 357 and 430 of Brazil‟s National
248 Environmental Council, CONAMA 2005. Reference area (A1); impacted area (A2);
249 contaminated area (A3).
250
251 3.2 Biometric indexes
252 The HSI and K variation from four different research areas were investigated. The highest
253 HSI averages were found in area A1 (1.77 ± 0.44) and A3 (1.98 ± 0.42) and the lowest
254 averages were found in areas A2 (1.73 ± 0.37) and A4 (1.07 ± 0.38) (Fig. 2A). The greatest
255 HSI variations occurred at points A3 and A4, as demonstrated in the boxplot quartiles
256 compared with the smallest variations at areas A1 and A2. A statistical difference was noted
257 in relation to point A4 and the other collection sites (p<0,05).
43
258 The average K of all fish sampled from areas A1, A2, A3 and A4 was 0.98 ± 0.08, 1.03 ±
259 0.10, 1.00 ± 0.08 and 1.52 ± 0.14, respectively. The highest variation in K value occurred
260 with individuals from point A4. The variation of the other groups was similar, with the A1
261 group indicating a lower K than expected (K = 1). The condition factor of S. herzbergii
262 ranged from 1.0387 to 0.9672 for area A1. K ranged from 1.0432 to 0.9700 in area A2 and
263 decreased from 1.0433 to 0.9800 in area A3. The ANOVA did not show significant values of
264 K in fish from points A1, A2 and A3, but there was a significant difference in relation to the
265 A4 point (p <0.05). The condition factor of each fish was examined and the monthly average
266 indicated that, in at least two different periods of the year, fish were in good condition (Fig.
267 2B).
3,5 A 3,0
2,5
2,0
1,5
1,0
Hepatosomatic (HSI) indexHepatosomatic 0,5
0,0 Area 1 Area 2 Area 3 Area 4 268
44
2,0 B 1,8 1,6 1,4 1,2 1,0
Condition factor (K) factor Condition 0,8 0,6 0,4 Area 1 Area 2 Area 3 Area 4 269
270 Figure 2 Boxplots showing hepatosomatic index (HSI) variation (a) and condition factor (K)
271 (b) in the different sampled areas.
272 3.3 Macroscopic observations
273 Fish livers from the sampled points did not show many noticeable macroscopic abnormalities.
274 Abnormalities, such as fat nodes, were identified in fish from sites A2, A3 and A4 and were
275 more prevalent (31%) in area A2 fish. These livers were characterized by the presence of fatty
276 nodes with regions of tissue coloring ranging from yellow, green, black and red. Only 6%,
277 12% and 9% of the fish presented similar macroscopic abnormalities in areas A1, A3 and A4,
278 respectively.
279
280 3.4 Prevalence of histological alterations
281 At all points sampled, MMCs prevailed in livers compared to any other type of lesion
282 identified, at more than 90%. Changes such as hyperplasia and lipid cells were also widely
283 observed (> 80%) in areas A1, A2 and A3, with a lower frequency in the A4 area (<60%).
45
284 The average number of MMCs/mm² was higher in areas A2 (72.00 ± 113.36) and A3 (70 ±
285 50.59), showing similarities between both (Fig. 3). Areas A1 (41 ± 21.52) and A4 (22 ±
286 16.50) had the lowest average MMCs/mm². The greatest variation occurred in area A2. The
287 number of MMCs was significantly lower in individuals' livers from areas A1 and A4 (p <
288 0.05) compared to individuals from areas A2 and A3. A statistical difference (p = 0.01) was
289 observed in hepatic MMCs among groups of individuals from the four areas observed.
450,0 400,0 350,0
300,0
250,0
200,0 (MMCs) 150,0 100,0
Melanomacrophage centers Melanomacrophage 50,0 0,0 Area 1 Area 2 Area 3 Area 4 290
291 Fig.3. Boxplots showing melanomacrophage centers (MMCs) (c) for S. herzbergii and B.
292 bagre in the different areas sampled.
293
294 The most frequent hepatic lesions found in this study (> 70%) were lipid cells, hyperplasia,
295 hypertrophy, necrosis, atrophy, intercellular edema and melanomacrophage centers.
296 Hemorrhages, aneurysms, structural and nuclear alterations showed frequencies of
297 intermediate lesions (40-70%). While the other lesions, deposits, granuloma, neutrophilic
298 infiltration and leukocyte infiltrate showed the lowest frequencies (0 - 40%) (Table 3).
299
46
300
301
302
303
304
305 Table 3. Histopathological alterations identified in the liver of S. herzbergii and B. bagre of
306 each sampling point.
Reaction Sampling sites Alteration w pattern A1 A2 A3 A4
Circulatory Haemorrhage/Hiperaemia/Aneurysm 1 26.32 66.67 66.67 52.63 disturbances Intercellular edema 1 73.68 60.00 86.67 68.42 Strctural alerations 1 31.58 33.33 66.67 47.37 Lipidosis 1 78.95 86.67 93.33 57.89 Regressive Deposits 1 15.79 20.00 0.00 15.79 changes Nuclear alterations 2 21.05 13.33 66.67 36.84 Atrophy 2 36.84 60.00 80.00 57.89 Necrosis 3 68.42 66.67 93.33 73.68 Hipertrophy 1 63.16 73.33 86.67 52.63 Progressive Hiperplasia 2 84.21 100.00 93.33 57.89 changes Granulomatosis 2 36.84 13.33 26.67 15.79 Leukocyte infiltrate 2 10.53 13.33 20.00 26.32 Neutrophilic infiltration 1 10.53 20.00 0.00 0.00 Inflammation Melanomacrophages centres 1 94.74 93.33 93.33 89.47
307
308 Values givens as percentage (%). The importance factor (w) of each alteration. Reference area
309 for S. herzbergii (A1); impacted area (A2); contaminated area (A3). Reference area for B.
310 bagre (A4).
311
47
312 A large number of circulatory disorders, such as hemorrhaging and edema (Fig. 4D) were
313 identified in the four study areas; the A3 area demonstrated a greater amount of these lesions
314 while the A2 area found the lowest incidence.
315 Regarding regressive changes, changes including cell necrosis (Fig. 4E), autolysis (Fig. 4C),
316 and massive lipid cell presence (Fig. 4A) prevailed in the A3 area. Structural and nuclear
317 changes also prevailed in the same region.
318 With regard to progressive changes, the presence of hyperplasia lesions (Fig. 4H) was more
319 constant in the A2 region. A huge hypertrophy process in area A3 fish was also noted. The
320 presence of granuloma (Fig. 4F) prevailed in fish of area A1 and was less constant in fish of
321 area A2.
322 Among inflammatory processes, hepatic melanomacrophagous centers (Fig. 4G) were the
323 most frequent lesions compared to any other type of alteration identified. In all studied areas
324 (n = 115), hepatic MMCs (60%) were characterized as multiple structures dispersed through
325 the liver parenchyma. An increase in the number and size of these structures (> 100) was
326 identified in 11% of assessed fish, while the presence of some of these structures (< 30) was
327 observed in 21% of the investigated livers. Liver samples without MMCs were the case in
328 only 5% of the fish. A similar MMCs percentage distribution was noted for all the studied
329 areas. However, at specific sites where a large increase in the number of MMCs was observed
330 in a single liver sample, it was found in fish from areas A2 and A3.
331 An inflammatory response characterized by mononuclear leucocyte infiltration (Fig. 4B) was
332 identified in 26.32% of the fish in the A4 area. Similar responses were identified in the other
333 study areas, 20%, 13.33%, 10.53%, in areas A3, A2 and A1, respectively.
48
334 Tumors characterized as psamomatous bodies (Fig. 4C) were more common in area A2 and
335 A3 fish. As there was no mitosis or necrosis, it was not possible to indicate benignity. No
336 tumors were found in fish livers from areas A1 and A4.
337
49
338 Figure 4 Photomicrography (H&E) of histopathological liver changes. (a) Macrovesicular
339 steatosis (lipid cells). (b) Inflammatory response (leukocyte infiltrate). (c) Atrophy and tumor
340 (psamomatous body). (d) Aneurysms and edema. (e) Necrosis. (f) Granulomatosis. (g)
341 Melanomacrophage centers (MMCs). (h) Hyperplasia. The arrow () indicates the changes
342 found.
343 In relation to the gills, the main histological changes consisted of secondary lamella
344 deformity, partial fusion, total fusion, necrosis (regressive changes), hypertrophy and
345 hyperplasia (progressive changes) (table 4). It was also possible to observe parasite presence e
346 in more than 1/4 of the fish studied in area A2.
347 Table 4. Histopathological alterations identified in the gill of S. herzbergii and B. bagre of
348 each sampling point.
Reaction Sampling sites Alteration w pattern A1 A2 A3 A4
Haemorrhage/Hiperaemia/Aneurysm 1 47.37 66.67 46.67 45.00
Circulatory Profusion and dilation of blood 1 52.63 73.33 46.67 40.00 disturbances vessels Intercellular edema 1 42.11 26.67 33.33 20.00
Epithelial lifting 1 26.32 80.00 40.00 25.00
Regressive Deformation of lamellae 1 73.68 100.00 66.67 50.00 changes Partial lamellar fusion 1 78.95 100.00 73.33 60.00 Necrosis 3 5.26 100.00 53.33 40.00 Total lamellar fusion 1 63.16 80.00 66.67 55.00 Progressive Hipertrophy 1 78.95 93.33 66.67 55.00 changes Hiperplasia 2 73.68 100.00 66.67 55.00 Parasite 5.26 26.67 0.00 5.00
349
350 Values givens as percentage (%). The importance factor (w) of each alteration. Reference area
351 for S. herzbergii (A1); impacted area (A2); contaminated area (A3). Reference area for B.
352 bagre (A4).
50
353
354 Despite the large number of gill lesions of the studied fish, the normal gill structure was also
355 observed (Fig. 5A). The hyperplasia lesion was recorded in 100% of the specimens studied in
356 area A2 (Fig. 5B), as well as necrosis, partial fusion (Fig. 5C) and lamella disorganization
357 (Fig. 5D). Hypertrophy (Fig. 5E) also showed great representativeness in the four areas
358 studied. In relation to circulatory disorders, the epithelial lining (Fig. 5F) was more prevalent,
359 followed by venous sinus dilation and aneurysms (Fig. 5G). Parasite presence was observed
360 in some cases (fig 5H).
51
361
362 Figure 5 Photomicrography (H&E) of histological gill changes. (a) Normal gills. (b)
363 Hyperplasia. (c) Necrosis and partial fusion. (d) Secondary lamellae disorganization. (e)
364 Hypertrophy. (f) Epithelium raising. (g) Venous sinus dilation, total fusion and aneurysms.
365 (h) Parasites. The arrow () indicates the changes found.
366
52
367 3.5 Quantitative analysis and comparison of fish from each studied area
368 For hepatic reaction patterns, Irp1 (circulatory disorders) reaction had the highest average in
369 individuals in area A2 (126.50 ± 40.44), in March 2018 (rainy period). For this same reaction
370 pattern, individuals from the A3 area showed values of 71.60 ± 48.90, in May, 2018 (rainy
371 period). The lowest averages were found in the areas A4 (15.00 ± 0.00) and A1 (2.00 ± 0.71),
372 respectively. In relation to Irp2 (regression changes) pattern (104.50 ± 104.52), Irp 3
373 (progressive changes) (60.86 ± 63.32) and Irp4 (inflammation) (95.17 ± 149.58), the
374 specimens from area A2 also showed higher values than with other study areas.
375 Consequently, the liver index was significantly higher (91.48 ± 103.46) in A2 individuals than
376 the other specimens of areas A1, A4 and A3. However, the lowest organ index was also
377 measured in the same area (19.5 ± 15.87). These higher averages correspond to individuals
378 captured in March 2018 (rainy period) and the lowest ones in December 2017 (dry period).
379 The lowest average in the Irp2 reaction pattern (9.13 ± 7.32) was observed in individuals from
380 area A1, in December 2017 (drought period). The lowest average values of the Irp3 (14.69 ±
381 8.26) and Irp4 (13.25 ± 5.19) were analyzed in individuals from the A4 area, in March and
382 May 2018 (rainy period), respectively (Table 5).
383
384
385
386
387
388 Table 5. Liver index of S. herzbergii and B. bagre for each site and sampled period.
Sampling sites Month Reaction pattern of the Alteration Organ index
Reference site S. Irp1 Irp2 Irp3 Irp4 I Liver herzbergii (A1)
53
Dec/17 2.00 ± 0.71 9.13 ± 7.32 19.17 ± 13.96 56.60 ± 13.43 20.48 ± 20.86 Mar/18 16.80 ± 8.66 45.64 ± 47.88 26.29 ± 21.71 64.78 ± 67.27 38.48 ± 45.25 May/18 9.80 ± 18.38 20.89 ± 18.38 16.65 ± 7.40 102.2 ± 54.41 25.36 ± 34.02
Impacted site (A2)
Dec /17 28.00 ± 5.10 9.43 ± 4.95 19.89 ± 20.47 33.33 ± 2.62 19.5 ± 15.87 Mar/18 126.50 ± 40.44 104.50 ± 104.52 60.86 ± 63.32 95.17 ± 149.58 91.48 ± 103.46 May/18 68 ± 43.54 59.9 ± 58.25 40.19 ± 22.66 142.4 ± 108.19 63.39 ± 61.65 Cotaminated site (A3) Dec /17 2.00 ± 1.00 29.58 ± 28.15 19.43 ± 16.98 57.8 ± 27.26 27.88 ± 27.03 Mar/18 18.00 ± 10.00 20.53 ± 19.54 23.47 ± 13.49 32.6 ± 16.57 22.14 ± 16.04 May/18 71.60 ± 48.90 44.59 ± 41.96 30.12 ± 19.90 24.43 ± 17.11 42.29 ± 38.62 Reference site B. bagre (A4) Dec /17 15.00 ± 0.00 58.50 ± 36.47 22.44 ± 16.19 38.17 ± 17.60 38.08 ± 29.53 Mar/18 20.10 ± 12.26 55.71 ± 59.84 14.69 ± 8.26 18.8 ± 15.73 30.77 ± 41.15 May/18 26.12 ± 5.73 40.23 ± 41.37 22.58 ± 10.10 13.25 ± 5.19 27.05 ± 26.12 389
390 Values given as mean ( ) and standard deviation (± SD). Reaction pattern: Irp1: circulatory
391 disturbances; Irp2: regressive changes; Irp3: progressive changes; Irp4: Inflammation.
392
393 The reaction patterns of the most representative branchial lesions were circulatory disorders in
394 the A1 area. Fish collected in this area showed a higher index of the gill in September 2017
395 (dry period). The main lesions found in area A2 were progressive changes (Irp3) followed by
396 circulatory disorders (Irp1). Fish collected in December 2017 (drought period) showed a
397 higher index of the organ compared to the other specimens of all the research. For fish of area
398 A3, the progressive changes were more representative and the organ index was higher in the
399 rainy period (May 2018) in this region. Individuals in the A4 area showed a higher index of
400 lesions related to progressive changes and the organ index was higher in September 2017. The
401 gill index was significantly higher in individuals from area A2 than the other specimens from
402 areas A1 and A3 (Table 6).
403 Table 6. Gill index of S. herzbergii and B. bagre to each site and period of collection.
54
Sampling sites Month Reaction pattern of the alteration Organ index Reference site for rp1 rp2 rp3 I Gill S. herzbergii (A1) Sep /17 149.14 ± 297.57 59.00 ± 29.81 73.10 ± 86.92 86.57 ± 165.08 Dec/17 25.00 ± 32.47 12.71 ± 17.33 12.80 ± 11.14 14.24 ± 18.10 Mar/18 17.00 ± 3.00 12.93 ± 18.52 18.50 ± 12.80 15.04 ± 16.30 May/18 7.30 ± 3.29 13.00 ± 6.18 15.33 ± 17.46 12.40 ± 12.23 Impacted site (A2) Sep /17 9.75 ± 13.53 77.05 ± 202.14 34.64 ± 35.91 49.44 ± 141.94 Dec/17 277.83 ± 556.69 59.67 ± 72.01 632.66 ± 1080.34 299.15 ± 723.11 Mar/18 9.50 ± 8.50 16.86 ± 11.04 31.20 ± 35.20 20.93 ± 24.05 May/18 17.33 ± 10.09 11.42 ± 8.09 32.31 ± 28.12 19.34 ± 19.66 Contaminated site (A3) Sep /17
Dec/17 28.67 ± 21.72 81.29 ± 113.02 16.64 ± 11.93 36.07 ± 65.03 Mar/18 68.00 ± 76.52 15.93 ± 76.52 17.14 ± 11.28 26.27 ± 42.76 May/18 33.67 ± 67.24 12.60 ± 8.76 183.62 ± 477.66 81.71 ± 307.50 Reference site for B. bagre (A4) Sep /17 3.50 ± 1.50 9.00 ± 10.58 131.75 ± 100.01 73.73 ± 96.08 Dec/17 7.86 ± 4.64 23.71 ± 17.32 45.57 ± 36.00 28.84 ± 28.49 Mar/18 8.67 ± 5.79 7.33 ± 4.64 9.91 ± 4.68 9.24 ± 4.99 May/18 33.82 ± 90.53 9.21 ± 8.31 32.13 ± 31.86 22.97 ± 56.10 404
405 Values given as mean ( ) and standard deviation (± SD). Reaction pattern: Irp1: circulatory
406 disturbances; Irp2: regressive changes; Irp3: progressive changes; Irp4: Inflammation.
407
408 4. Discussion
409 In this study, the most frequent histological alterations in the liver were inflammatory
410 responses (MMCs), hyperplasia and cytoplasmatic vacuolization (lipid cells), which are
411 common responses of fish exposed to degraded aquatic environments. The macroscopic
412 appearance of nodules in tissue regions and the difference in staining between different organs
413 examined show that the liver holds a large amount of fat and this was evidenced by
55
414 microscopic and hepatocellular vacuolization results, characteristic of steatosis. The condition
415 factor of the fish from areas A1, A2 and A3 obtained similar average values.
416 A study carried out on the kidneys of Prochilodus lineatus showed positive correlation
417 between condition factor, water temperature at different periods and the number of
418 melanomacrotic centers (Balamurugan et al., 2012). Monitoring of fish hygiene conditions
419 through fluctuation of the condition factor due to environmental changes has already been
420 reported for feeding (Yan et al., 2015) and breeding cycles (Azevedo et al., 2017).
421 As with the condition factor, hepatosomatic index variation was similar between areas A1, A2
422 and A3 and differed from area A4. Comparing different area samples, the highest values of
423 HSI were measured in areas A1, A2 and A3. The association between effluent release and fish
424 liver size increase has been reported (Galloway et al., 2003) (Saravanan et al., 2011).
425 Phosphatic substances from sanitary effluents and fisheries can cause toxin production, such
426 as microcystins, which can accumulate in fish liver (Deblois et al., 2011) and cause cellular
427 changes. The high number of alterations such as hepatocyte hypertrophy may be related to the
428 toxin presence as a result of the eutrophication process, which is potentiated by the release of
429 high phosphororus levels in water bodies (Van Dyk et al., 2012). The presence of heavy
430 metals in the water, in divergence with the amount permitted under Brazilian legislation
431 (resolution 357/2005) (Conama, Brazil, 2005), has already been reported in the investigated
432 area, that is, in the lower course of the Santo Antônio river (Cantanhêde et al., 2016). A
433 methodology through microbiological analyses and enzymatic responses in bivalves also
434 indicated high levels of contamination in the same region and indicated a risk to human
435 consumption (Ribeiro et al., 2016).
436 Abiotic factors such as temperature, pH, conductivity and dissolved oxygen can change
437 species richness and fish assemblage, which can also be attributed to anthropogenic impacts
56
438 (Carvalho Neta et al., 2014). Similar studies in areas with the same characteristics as in this
439 study have shown lower GSI averages in catfish from potentially contaminated areas and
440 suggest that this decrease may result in abnormal gonad development, late maturation, high
441 levels of atresia and fish intersexuality (Carvalho- Neta and Abreu-Silva, 2010; Carvalho
442 Neta et al., 2014).
443 Similar to the biometric indices, results from microscopic analyses also showed differences
444 between the period and the different sites. Liver index was significantly high in area A2 and
445 high in area A3 compared to fish in areas A1 and A4. This pattern was identified by HSI
446 results as well as microscopy and macroscopic analyses' results. Fish livers from area A2 and
447 A3 were severely affected in terms of liver health, as evaluated in this study.
448 Histopathological results showed a clear and specific difference between the sites accessed,
449 reflecting the pollution level in these systems. Histological change variation associated with
450 the reaction pattern was identified, however, high prevalence of regressive changes in fish
451 suggests that most histological changes in fish from natural environments are regenerative
452 (Van Dyk et al., 2012).
453 The liver plays a number of essential functions for organisms, which increases the concern
454 with bioaccumulation and public health. Its functions include carbohydrate metabolism, lipid
455 storage, synthesis and fatty acid oxidation and glycogen storage, as well as being the main
456 detoxification center (Choi et al., 2018).
457 Inflammatory responses were constituted by macrophage infiltration of hepatic tissue, defense
458 cells with an important role in the immune response to foreign agents, whose function is to
459 remove, by phagocytosis, foreign particles derived from cellular degradation (Passantino et
460 al., 2014; Basilone et al., 2018). MMCs may increase in number and size depending on
461 environmental stress conditions and have been used as a water quality biomarker (Agius and
57
462 Roberts, 2003). A study with Micropterus salmoides showed a significant increase in the
463 number of MMCs in fish from an impacted area when compared to an uninfluenced area
464 (Blazer et al., 1987). In this study, hepatic melanomacrophages of S. herzbergii were observed
465 primarily close to blood vessels and involved in storage, relocation and recycling of iron
466 compounds from sterile or damaged venous red blood cells. The highest number of quantified
467 MMCs/mm² was detected in area A2 organisms and corresponded to the youngest subjects in
468 the study.
469 The number of lipid cells found in the liver of B. bagre was lower in relation to the other three
470 areas evaluated and the values for this same alteration were growing according to the level of
471 impact of the area, A1 472 anthropic pollution gradient observed at the collection sites (Cantanhêde et al., 2016), since 473 the concentration of xenobiotics in the environment increases the incidence of lesions suffered 474 by the liver (Ghisi et al., 2016). 475 Cellular vacuolization is harmful from the moment that cellular metabolism is altered as a 476 result of chemical stress, and may interfere with normal cellular functioning. These 477 substances can cause major damage, such as necrosis. In addition, the accumulation of lipids 478 in the vesicles constitutes a mechanism of cellular response when in the presence of lipophilic 479 chemical agents. In an attempt to immobilize these substances, it prevents their interaction 480 with the other cellular components and, in this case, minimizes its toxic effect (Olivares- 481 Rubio and Vega-López, 2016). These results indicate prolonged physiological disturbances 482 leading to glycogen depletion and lipid accumulation. The association of lipid cells with 483 MMCs has been observed in fish from regions contaminated with xenobiotic mixtures 484 (Triebskorn et al., 2008). Vacuolization and hepatocyte swelling are the most common lesions 485 for nitrogen compounds (dos Santos Silva et al., 2018). 58 486 Histological parameters are generally indicative of irreversible damage as they are involved in 487 high-level biological organization changes. An important impact of effluent exposure at the 488 histological level was observed in this study. The main histological gill changes were 489 hyperplasia, hypertrophy, partial fusion, total fusion and aneurysms. These lesions gradually 490 affect gill functions and are responses for acute and/or chronic exposure to a wide variety of 491 contaminants (Pérez et al., 2018). 492 Hyperplasia was characterized by increased cell proliferation, which may lead to melting 493 lamellae and, more rarely, filaments (Hinton et al., 1992). Lamellar fusions are results of 494 excessive epithelial cell proliferation in filaments and a natural defense mechanism to protect 495 lamellar epithelium from direct contact with toxic agents (Vigário and Sabóia-Morais, 2014). 496 Epithelial elevation, lamellar hyperplasia and fusion are changes resulting from a variety of 497 stressors, such as metals, ammonia, phenolic infections by microorganisms and even 498 ectoparasites (Hinton et al., 1992). 499 Aneurysms are usual results of pillar cell collapse and impairs vascularization integrity with 500 the high volume blood release that pulls the lamellar epithelium out (Hinton and Lauren, 501 1990). The high rate of apoptosis or necrosis found in area A2 fish may be related to potential 502 DNA damage caused by direct exposure to contaminants present in river water (Sales et al., 503 2017). In teleosteal fish, a significant apoptosis increase has been used as an aquatic 504 environment biomarker after exposure to different types of xenobiotics known to damage 505 aquatic animals' liver and gills (Piechotta et al., 1999). 506 Cellular hypertrophy usually indicates cellular activity increase in and is a very common 507 morphological alteration related to gill damage, leading to epithelium thickening (Almeida 508 and de Oliveira Ribeiro, 2014). Cells respond to stress by adaptive changes that limit or repair 509 damage, preventing cell death (Mosser et al., 1997). Parasite presence, as verified in fish in 59 510 this research, is also dangerous for these animals' respiratory quality (Raine et al., 2017). This 511 may be related to immunodeficiency defense, acid-base problems and even osmoregulation 512 problems (Akaishi et al., 2004). 513 High histopathological index for the gill tissue in individuals from area A2 can be attributed 514 to animals' high exposure to xenobiotics that reach this place, suggesting that these substances 515 undergo an accumulation process there. In addition to the fishery effluents, this site receives 516 agrochemical waste and sewage effluents in natura from riverside communities. In addition, 517 no filtration processes were found for residues resulting from these anthropic activities, so 518 water quality is compromised. This is reflected in health conditions in animals' from this area. 519 In the lower course of the Santo Antônio River there is intense fishing activity (a large 520 number of boats that use fuel) followed by the extraction of shellfish (constantly revolving the 521 mud), in addition to the occupational process along the riverbanks and unrestrained 522 population growth, which seriously interferes with natural systems. There are also erosive 523 processes and leaching, which carry toxic materials into the waterways. The effects of 524 contaminants in the aquatic environment cause serious problems in gill tissues considering the 525 important functionalities of this organ in respiratory processes and osmotic and ionic 526 regulation, which could lead to individuals' deaths (Ghisi et al., 2016). 527 Despite histopathological assessment results, other factors such as pollution level, fish age 528 and sample size should be considered. In this study, fish were collected during the dry and 529 rainy periods. Histopathological differences were identified with seasonal variations. Age 530 determination samples were not performed. However, a macroscopic gonadal analysis was 531 performed and it was found that a large quantity of young fish from the area close to the 532 fishery (A2) showed a high quantity of MMCs in comparison to adult individuals from the A3 60 533 area. However, age determination is strongly recommended as part of a histological protocol 534 standard for assessing fish sanity levels. 535 Results of this study will be useful for monitoring and managing the health of fish 536 populations. It will allow specific liver and branchial lesion identification at a site and infer 537 regional similarities and/or patterns in terms of histological changes of the liver and gill. This 538 study provides a better understanding of health effects related to fish pollution by comparing 539 polluted and relatively natural systems and advances our understanding of the normal range of 540 liver and gill histology for S. herzbergii and B. bagre. 541 Santo Antônio River Basin administrators when making decisions about this body of water 542 should consider these results. Biomonitoring studies are fundamental for the development of 543 public policies that prioritize conservation. There is also a need for more effective inspections 544 of xenobiotic components entering the site and decreasing their environmental quality, which 545 compromises directly and indirectly dependent organisms' quality of life. 546 547 Conclusions 548 This study concluded that between the analyzed catfish species, the taxon Sciades herzbergii 549 is the most suitable for monitoring environmental impacts in transitional areas and estuarine 550 environments through histopathological gill assessment. This was confirmed by acute effects 551 suffered by gills and the high number of lesions demonstrated on individuals inhabiting the 552 potentially impacted area in comparison to the other areas studied. 553 High HSI, the low condition factor of fish in areas A2 and A3, and the high number of lesions 554 caused in the liver of S. herzbergii and their chronic effects prove that the species is a good 555 biomonitor for monitoring environmental impacts in areas of transition and estuarine 556 environments. 61 557 558 Acknowledgements 559 I would like to thank the employees of LABOAQ. 560 561 Funding 562 This work was supported by Maranhão State Research Foundation - FAPEMA (Fundação de 563 Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Estado do Maranhão) 564 (Grant number: BM-02175/17). 565 566 References 567 Agius, C., Roberts, R.J., 2003. Melano-macrophage centres and their role in fish pathology. 568 J. 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Fator de impacto: 4.427. 4 Qualis A1 (2018) para a área Zoologia/Recursos pesqueiros. 5 Hellon Cunha Viana a, Simone Karla Lima e Silva b, Marianna Basso Jorge c, Débora Martins 6 Silva Santos d, Raimunda Nonata Fortes Carvalho Neta d. 7 a Master Student in Aquatic Resources and Fisheries (PPGRAP) by State University of 8 Maranhão (UEMA), Campus Paulo VI, São Luis, Maranhão, Brazil. 9 b Master in Biodiversity and Conservation by Federal University of Maranhão (UFMA), São 10 Luis, Maranhão, Brazil. 11 c Department of Oceanography and Limnology, Federal University of Maranhão (UFMA), 12 São Luis, Maranhão, Brazil. 13 d Department of Chemistry and Biology, State University of Maranhão (UEMA), Campus 14 Paulo VI, São Luis, Maranhão, Brazil. 15 Corresponding author: Hellon Cunha Viana¹. Email: [email protected], phonenumber: 16 +55 98 983109904 17 „Declarations of interests: none‟ 18 19 20 21 71 22 Abstract 23 Melanomacrophage centers (MMCs) in fish liver is an indicator of environmental and stress 24 condition because they are involved in xenobiotic biotransformation. This project aimed to 25 assess the MMC number in the liver of young and adult Sciades herzbergii individuals from 26 areas with different contamination levels. Fish were captures at three points (reference point - 27 A1, potentially impacted point - A2 and contaminated point - A3), using cages (manzuá) 28 during receding tide in São José bay (Maranhão, Brazil), in four samplings. Livers were 29 immediately dissected and the samples underwent the basic histology procedure. 5μm 30 sections were stained with hematoxylin-eosin. Results indicated that the livers from adult 31 individuals in area A2 (260.50 ± 161.50 MMCs/mm²) had a higher number of MMCs when 32 compared to adult individuals in the A3 area (60.00 ± 30.10 MMCs/mm²). Young individuals 33 showed considerable values at points A1 (100.00 ± 0.00) and A2 (95.33 ± 33.00), compared 34 to young at point A3 (49.00 ± 0.00). These high values are unexpected for young individuals. 35 The average MMC number showed a correlation with the more intense rainy period. The 36 variation in the number of melanomacrophage centers for monitoring fish healthiness 37 conditions was shown to be a good environmental change indicator. The use of hepatic 38 MMCs as a biomarker for exposure to environmental pollutants, in particular material from 39 fishery systems, such as ammonia and nitrite, is confirmed by this study. 40 Keywords: Biomonitoring, Biomarker, Sciades herzbergii, melanomacrophage center. 41 42 43 44 72 45 1. Introduction 46 Melanomacrophage centers (MMCs) consist of macrophages, such as phagocytic cells and 47 fragments, mainly erythrocytes and pigments, such as melanin, hemosiderin and lipofuscin, 48 located in the reticuloendothelial liver tissue, kidney and pancreas (Agius and Roberts, 2003). 49 MMCs are found in several vertebrates such as reptiles (Johnson et al., 1999; Domiciano et 50 al., 2017) amphibians (Franco-Belussi et al., 2013, Franco-Belussi and de Oliveira, 2016, Wu 51 et al., 2017) and fish (Mela et al., 2013; Sales et al., 2017). The number of melanomacrophage 52 centers, their size and distribution varies according to species, organ, age, nutritional level and 53 stress conditions (Fishelson, 2006). The function of the MMCs is to destroy, detoxify or 54 recycle foreign materials (Van der Oost et al., 2003). 55 Fish liver is considered a good indicator for environmental condition, stress condition, since it 56 controls several vital functions in these animals' physiology, both in anabolism and 57 catabolism, and xenobiotic metabolism (Passantino et al., 2014). In this context, liver 58 histopathology can be used as an appropriate methodology for the detection of fish exposure 59 to various environmental organic pollutants (ammonia, nitrite) (Faccioli et al., 2014). 60 Sciades herzbergii is a species of catfish distributed on the South American continent, the 61 Caribbean, Atlantic rivers and estuaries from Colombia to Brazil (Marceniuk, 2005). Adults 62 of this species occur in estuaries with turbid waters, mangroves, low river courses, tolerate 63 salinity changes and are found in shallow coastal regions (da Silva et al., 2016). Its fishing is 64 carried out mainly by small-scale and subsistence fishing communities. Due to low cost in 65 markets and fairs, its fishing is intense and occurs all year round at São Luis Island river 66 mouths. 67 Next to these areas is the low course of the Santo Antônio River, where a massive fishery 68 network is settling up and launching effluents without any previous treatment, in areas where 73 69 heavy fishing takes place. Concern increases in the rainy period when fishery water is 70 constantly drained, preventing collapse or exceeding the acceptable water level. There are 71 some studies with this species in aquatic contamination biomonitoring programs in nearby 72 and similar regions (Carvalho-Neta et al., 2012; Débora Batista Pinheiro Sousa et al., 2013; 73 DBP Sousa et al., 2013; Carvalho-Neta et al., 2014). However, the approach based on MMCs 74 in S. herzbergii liver to evaluate anthropic impact is unknown in the state of Maranhão. This 75 work aimed to validate hepatic MMCs in S. herzbergii as biomarkers environmental pollutant 76 exposure, in particular fishery effluents in São José Bay (Maranhão, Brazil). 77 78 2. Materials and Methods 79 2.1 Study area 80 Four collections were carried out, (1st - September 2017, 2nd - December 2017, 3rd - March 81 2018 and 4th - May 2018). S. herzbergii samples were collected at three sampling points 82 between the Santo Antônio River mouth and São José Bay. The most distant area of the coast 83 was considered as a reference point (A1 - 2°27'47.58 "S 44° 2'45.42"W), because it has 84 theoretically less contact with domestic effluents discharged into the water bodies. A second 85 area was considered the impacted point (A2 - 2° 29'36.54 "S 44° 3'34.38" W), located in the 86 channel of the Santo Antônio river, near the stream that receives fishery effluents. In addition, 87 a third point, located at Port of Viera, in São José de Ribamar, was considered a contaminated 88 point (A3 - 2° 33'18.36 "S 44° 3'10.02" W), as domestic effluent discharge without any 89 treatment in their waters has been verified over years (Fig. 1). Shipments left the Port of Pau 90 Deitado and the boat used was a motorized biana. 74 91 92 Figure 1 Geographic location of sampling points. A1 – reference, A2 – impacted and A3 – 93 contaminated. 94 95 2.2 Licensing and ethics declaration 96 The capture of fish was authorized by the Ethics Committee of Maranhão State University 97 (number 064/2017-2018 CRMV-MA) and met the guidelines of the Brazilian College for 98 Animal Experimentation (SBCAL/COBEA, 2018). 99 100 2.3 Tissue Processing and Histopathology 101 For basic histological analysis and MMC identification, the fish were dissected and liver 102 samples were set 10% formaldehyde, subsequently dehydrated in increasing alcohol 103 concentrations and embedded in paraffin. Liver sections of liver (5 μm) were stained with 75 104 hematoxylin-eosin (H&E) (Merck) for liver structure description and slide reading was done 105 with the aid of light microscopy (ZEISS). Evisceration was performed for macroscopic 106 identification of maturational stages, where: I - Immature, II - In development, III - Able to 107 spawn, IV - Regression and V - Regeneration (Brown-Peterson et al., 2011). 108 109 2.4 Melanomacrophage Center Quantification (MMCs) 110 A quantitative analysis of MMC aggregation was performed on hepatic tissue sections of the 111 fish. Each digital field was photographed using Zen Imaging Axiovision 4.8 software 112 mounted on the optical microscope with the 4X, 10X and 40X lenses. MMC aggregation was 113 identified through image analysis in the ImageJ software and randomly quantified the number 114 of MMCs per mm2. Each section was removed from the liver's central region and it was 115 possible to measure the whole organ. 116 117 2.5 Condition factor 118 Condition factor (K) is a quantitative indicator of the individual‟s degree of healthiness or 119 well-being. It is used to indicate variations in population density and food conditions. The 120 value reflects recent nutritional conditions and/or reserve expenditures on metabolic activities, 121 allowing relationships with environmental conditions and species' behavioral aspects 122 (Vazzoler, 1996). 123 K was estimated for each individual and their average obtained for each period, according to 124 the expression: K = Wt/Ltb x 100, where: Wt = total weight, Lt = total length and b = 125 weight/length angular coefficient. 126 76 127 2.6 Statistical analysis 128 The average number of MMC/mm2 was compared between sites using the Kruskal-Wallis 129 test. For variables with a significant test result, comparisons between pairs among different 130 sites were made using the non-parametric Mann-Whitney U test, controlling Type I error 131 between tests by Bonferroni adjustment. Statistical differences were considered significant 132 when p <0.05. Data was checked for normality using the Kolmogorov-Smirnov or Shapiro- 133 Wilk tests. Statistical analyses were performed using the software PAlaeontological STatistics 134 (PAST), version 1.0.0.0 (Ryan et al., 2001) and statistical significance was accepted for p ≤ 135 0.05. Results were expressed by average and standard error. An ANOVA and Tukey test were 136 performed for different sampled sites and condition factor. 137 138 3. Results 139 A total of 73 individuals (12 young and 10 adults at point A1, 16 young and 4 adults at point 140 A2 and 7 young and 24 adults at point A3) were collected. Young and adults in the A1 area 141 showed a similar percentage in relation to the number of specimens, with a slight numerical 142 advantage compared to the young. In relation to area A2, the difference reached 80% in 143 relation to young and 20% of adults. In the A3 area, the adult presence (77%) predominated in 144 relation to young people (23%) (Table 1). Adult fish at point A1 were the largest on average 145 total length, both young (28.01 ± 3.05) and adults (36.95 ± 2.43). The adults at point A2 had 146 an average length of 31.28 ± 2.72 and young presented the lowest total length averages (26.01 147 ± 8.92) of the entire collection. At point A3, average adult length was 29.94 ± 4.02 and young 148 26.76 ± 2.27. 149 150 77 151 Table 1. Average length and percentage of young and adults of S. herzbergii from different 152 sampled locations. MATURATIONAL STAGES (%) TOTAL GROUP SITES T. LENGTH (cm) I II III IV V (%) YOUNG A1 28.01 ± 3.05 41 14 55 ADULT A1 36.95 ± 2.43 36 9 - 45 YOUNG A2 26.01 ± 8.92 45 35 80 ADULT A2 31.28 ± 2.72 20 20 YOUNG A3 26.76 ± 2.27 16 7 23 ADULT A3 29.94 ± 4.02 35 32 10 77 153 154 Values givens as mean ( ) and standard deviation (±SD). Reference area (A1); impacted area 155 (A2); contaminated area (A3). Maturational stages: I - Immature, II - In development, III - 156 Able to spawn, IV - Regression and V – Regeneration. 157 158 3.1 Histopathology 159 Histological appearance of the normal liver of S. herzbergii is shown in Fig. 2E, Fig. 2F. 160 Hepatic parenchyma is composed of the central lobular vein (CLV) surrounded by hepatocyte 161 cords and sinusoid capillaries. The hepatocyte presented a polygonal shape with a spherical 162 and centralized nucleus (Passantino et al., 2014). The hepatic parenchyma was homogenous 163 with hepatocytes of polygonal forms containing a spherical nucleus; hepatic cytoplasm 164 showed to be weakly basophilic and with presence of lipid cells in small and large vacuoles 165 (Fig. 2A). Hepatocytes were organized as branched and anastomosed cords arrayed by 166 sinusoid networks. The melanomacrophage centers in the hepatic parenchyma, which are 167 composed of cell and pigment aggregation, contain iron ions, lipofuscin and ceroids (Fig. 2B). 168 Aadipose metaplasia and tumor presence was also observed, as there was no sign of mitosis or 169 necrosis, it was not possible to indicate benignity, this psamomatous body was found in 170 hepatopancreas tissue (Fig. 2C). The hepatopancreas are the main digestive enzyme supply 78 171 sites, which in carnivorous fish, have higher lipase activity when compared to omnivorous 172 and herbivorous species. On the other hand, bile, secreted by hepatocytes, can enter the 173 proximal part of the intestine or be stored in the gallbladder, with the function of facilitating 174 digestion and absorption of lipids and lipophilic substances, such as fat soluble vitamins (A, 175 D, E and K) (Arantes et al., 2016). Hepatocytes were thus observed in the bile duct 176 epithelium, both in young and adult individuals (Fig. 2D). 177 79 178 Figure 2 Photomicrograph of the liver of S. herzbergii sampled at three different points of the 179 São José bay - MA. (A) H&E staining of a section of the liver showing lipid components. (B) 180 Melanomacrophage center presence. (C) Lipid cells, psamomatous body in hepatopancreas 181 tissue and MMCs. (D) antibodies surrounding the hepatocyte cytoplasm as well as the bile 182 duct epithelium. (E, F) The histological appearance of normal liver. Asterisk: 183 melanomacrophage center; m: macrophages; lp: lipid cells; s: sinusoids; pb: psamomatous 184 body; bd: bile duct. 185 186 3.2 MMC quantitative analysis 187 The number of melanomacrophage centers (MMCs/mm²) in young and adult S. herzbergii 188 livers at three capture sites (Fig. 3). The MMC number was significantly low in livers of area 189 youth A3 (P< 0.05) compared to youth from areas A2 and A1. 1200 1000 800 600 (µm²mm²) 400 200 Melanomacrophage centers Melanomacrophage 0 Reference Impacted Contaminated Young Adult 190 191 Figure 3 Number of melanomacrophage centers (MMCs/mm²) (standard error) in young and 192 adult S. herzbergii livers at the three capture sites. Asterisk (*) indicates a significant statistic 193 difference comparing the reference and impacted areas. 194 80 195 3.3 Condition factor and melanomacrophage centers 196 The S. herzbergii condition factor ranged from 1.0387 to 0.9672 for area A1 (Fig. 4B). K 197 ranged from 1.0432 to 0.9700 in area A2 (Fig. 4D) and decreased from 1.0433 to 0.9800 in 198 area A3 (Fig. 4F). The ANOVA did not show significant values for the different sampling 199 points and the fish condition factor. The condition factor of each fish was examined and the 200 monthly average indicated that in at least two different times of the year, fish were in good 201 condition. A total of 5-8 fish were randomly selected for histological analyses at each point 202 and each season. The melanomacrophage center aggregation was lower in adults from area 203 A3 (30.50 ± 12.60 MMCs/mm²) in May 2018 (Fig. 4E) and higher in area A2 adults (260.50 204 ± 161.50 MMCs/ mm²) in March 2018 (Fig. 4C). In young individuals, a greater 205 quantification of melanomacrophage centers was observed in areas A1 (100.00 ± 0.00) (Fig. 206 4A) and A2 (95.33 ± 33.00) (Fig. 4C), in March and May 2018, respectively. Observations of 207 the average number of MMC aggregates showed a difference in the periods sampled for areas 208 A1 and A2, showing correlation with the period of more intense rains. For area A3, the 209 average number of MMCs showed constancy throughout the sampled period. 81 210 211 Figure 4 Number of melanomacrophage centers (MMCs/mm²) during the sample period. 212 213 4. Discussion 214 The large amount of MMCs between young and adult individuals indicates that the 215 aggregation of hepatic melanomacrophage centers in S. herzbergii can be used as an indicator 216 for environmental change and well-being. This strong proliferation of MMCs in young 217 individuals caught in areas close to fisheries (A2) may indicate damage to organisms' health 218 status because of a higher degree of environmental contamination. 219 Histological analysis of the liver of S. herzbergii allowed the identification of both free and 220 grouped melanomacrophages, mainly near blood vessels. The data found in this study 82 221 confirms and amplifies available information about fish MMCs of fish that are considered 222 catabolism residues of several compounds (Ribeiro et al., 2011). Lipofuscin and ceroid are 223 consecutive products of the same oxidation process, derived from a cell membrane rupture or 224 lipid metabolism rupture, and increase with age. These pigments can accumulate in fish 225 exhibiting a wide variety of pathological conditions, including nutritional deficiencies, 226 bacterial and viral diseases, and disorders caused by toxic material (Agius, 1979). Stress itself 227 can increase macrophage-like cells and increase red cell degradation (Peters and Schwarzer, 228 1985). Therefore, in the presence of environmental pollution, which may be responsible for 229 increased cell damage, increased phagocytic activity could be expected, with consequent 230 increase in MMCs. 231 Hepatic S. herzbergii melanomacrophages are involved in the activity of storing, relocating, 232 and recycling iron compounds from sterile or damaged venous red blood cells. In this study, 233 the highest number of MMCs was detected in organisms from area A2 (1833) and correspond 234 to the youngest individuals in the study. This fish group also exhibited the highest average 235 MMCs/mm² compared to other areas. It presented a similar number (1630) to area A3 and 236 three times more than area A1 (617). These results are in conflict with literature data because 237 a strong correlation between fish aging - hence tissue degeneration - and increase in the 238 number and size of MMCs have been demonstrated in different species‟ liver and kidney 239 (Agius, 1980). In this case, it is likely that the environment in question is undergoing a 240 contamination process and mainly young individuals. In addition to these physiological 241 changes related to aging, an increase in the MMC number was also demonstrated in liver, 242 spleen and kidney of fish stressed due to a polluted environment (Agius and Roberts, 2003). 243 As a result, the role of these aggregates as biomarkers for the effects due to environmental 244 exposure to polluting chemical substances has been extensively documented in freshwater and 245 marine teleosts (Passantino et al., 2005; Mela et al., 2007; Pascoli et al., 2011; Van Dyk et al., 83 246 2012; Passantino et al., 2014; Zhang et al., 2014; Arantes et al., 2016; Basilone et al., 2018) 247 and in the case of the fish from the environments evaluated in this study. 248 There is little research on the environmental situation of the Santo Antônio River basin, where 249 its low course is the target of our investigation. It is known that there is irregular occupational 250 movement, followed by accelerated urban occupation in mangroves and gallery forest areas, 251 favoring the destruction of flora and fauna, and consequently diminishing the region's water 252 system quality. The upper and middle course is totally impacted by disorderly occupation and 253 leaching processes added to the release of in natura sewage. 254 The presence of heavy metals (iron, mercury, aluminum, lead and cadmium), not complying 255 Brazilian legislation (resolution 357/2005) (Conama, Brazil 2005), has already been observed 256 in the area investigated in recent studies (Cantanhêde et al., 2016). In addition, microbial 257 bivalve contamination was associated with high levels of microbiological contamination, 258 especially in the period of the most intense rains and high risk for human consumption 259 (Ribeiro et al., 2016). 260 The low course is considered preserved, however, it is possible to observe similar 261 occupational characteristics in the middle and upper reaches of the river. Growing fishery 262 activity and also the scarcity of documented material on contamination by organic material 263 coming from fishery systems in the region studied, makes this work an unprecedented 264 investigation in this context. In similar regions, where there is already an intense and well- 265 documented fishery system, it is possible to affirm that effluents from these systems, when 266 released without previous treatment, increase phosphorus and nitrogen levels present in fish 267 feed. There is also a greater introduction of organic matter in the periods of expenditure, 268 which causes a decrease of dissolved oxygen in the water, essential for aquatic life 269 maintenance (Simões et al., 2007). 84 270 The variation in the number of melanomacrophage centers to monitor fish healthiness 271 conditions has been shown to be a good indicator of environmental changes (Balamurugan et 272 al., 2012). MMC aggregation was high in March and May of 2018 (rainy period). It is likely 273 due to environmental changes in that particular area. These results show the effectiveness of 274 MMC use as an indicator of fish exposure to environmental pollution and confirm, through 275 the high number of MMCs found in young S. herzbergii individuals, that areas close to 276 fisheries are suffering from exposure to organic compounds of anthropic origin higher than 277 the other groups of fish studied. 278 Observations showed that the liver of young S. herzbergii individuals possessed unexpectedly 279 large amount of melanomagnetic centers compared to adult fish, being a potential indicator of 280 environmental changes indicative of fishery system impacts in the northeastern Amazon area. 281 Through these observations, in-depth studies on young and adults of S. herzbergii are 282 suggested to obtain useful information for the evaluation of fish welfare and/or risk to human 283 health. 284 285 Conclusions 286 In conclusion, this study confirms the importance of the use of hepatic MMCs as exposure 287 biomarkers to environmental pollutants, in particular fishery system effluents. MMCs in S. 288 herzbergii showed differences in the individuals from analyzed environments, being able to 289 differentiate areas with different environmental impact levels. 290 291 Acknowledgements 292 I would like to thank the employees of LABOAQ. 293 85 294 Funding 295 This work was supported by Maranhão State Research Foundation - FAPEMA (Fundação de 296 Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Estado do Maranhão) 297 (Grant number: BM-02175/17). 298 299 References 300 Agius, C., 1979. The role of melano‐ macrophage centres in iron storage in normal and 301 diseased fish. J. Fish Dis. https://doi.org/10.1111/j.1365-2761.1979.tb00175.x 302 Agius, C., 1980. Phylogenetic development of melano–macrophage centres in fish. J. 303 Zool. https://doi.org/10.1111/j.1469-7998.1980.tb01446.x 304 Agius, C., Roberts, R.J., 2003. Melano-macrophage centres and their role in fish 305 pathology. J. 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Toxicity assessment of environmental pollutant 435 phenanthrene in clam Venerupis philippinarum using oxidative stress biomarkers. 436 Environ. Toxicol. Pharmacol. https://doi.org/10.1016/j.etap.2014.01.018 110 6 CRONOGRAMA Meses de 2017 Meses de 2018 Meses de 2019 Atividade mar abr Mai jun jul ago set out nov dez jan fev mar abr mai jun jul ago set out nov dez jan fev mar Levantamento X X X X X X X X X X X X X X X X X X bibliográfico Coleta das X X X X espécies Análise do X X X X X X X X material Redaçãobiológico dos X X X X X X X X X X X X X X resultados Qualificação X Seminário II X Defesa da X Dissertação Relatório X parcial Relatório X final X - Executado X - Em execução 111 7 CONSIDERAÇÕES FINAIS Foi confirmada a importância do uso de MMCs hepáticas como biomarcadores de exposição a poluentes ambientais, em particular efluentes oriundos de sistemas de piscicultura. O número de MMCs em S. herbergii mostraram diferenças nos ambientes analisados, indicando que a espécie pode acumular altas concentrações de poluentes, que podem comprometer suas funções biológicas, representando também uma fonte de alimento inseguro para os seres humanos. O elevado HSI, o baixo fator de condição dos peixes nas áreas contaminada e impactada e o elevado número de lesões causadas no fígado e seus efeitos crônicos em indivíduos que habitam a área potencialmente impactada em comparação as demais áreas estudadas, comprova que o S. herzbergii é uma espécie biomonitora de impactos ambientais em áreas de transição e ambientes estuarinos. As principais histopatologias em brânquias foram hiperplasia, hipertrofia, fusão parcial, fusão total e aneurismas. O presente estudo concluiu que a espécie S. herzbergii é boa para o monitoramento de impactos ambientais em áreas de transição e ambientes estuarinos através da avaliação histopatológica das brânquias. 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