UNIVERSIDADE FEDERAL DE SANTA MARIA
CENTRO DE CIÊNCIAS NATURAIS E EXATAS
PROGRAMA DE PÓS-GRADUAÇÃO EM BIODIVERSIDADE ANIMAL
TOXICIDADE DO CÁDMIO EM EMBRIÕES E LARVAS
DE Rhamdia quelen (HEPTAPTERIDAE) EM
DIFERENTES ALCALINIDADES DA ÁGUA
DISSERTAÇÃO DE MESTRADO
Ana Paula da Silva Benaduce
TOXICIDADE DO CÁDMIO EM EMBRIÕES E LARVAS DE
Rhamdia quelen (HEPTAPTERIDAE) EM DIFERENTES
ALCALINIDADES DA ÁGUA
por
Ana Paula da Silva Benaduce
Dissertação apresentada ao Curso de Mestrado do Programa de
Pós-Graduação em Biodiversidade Animal da Universidade Federal de
Santa Maria (UFSM, RS), como requisito parcial para obtenção do grau
de
Mestre em Ciências Biológicas.
Orientador: Prof. Bernardo Baldisserotto
© 2005
Todos os direitos autorais reservados a Ana Paula da Silva Benaduce. A reprodução de partes ou do todo deste trabalho só poderá ser com autorização por escrito do autor.
Universidade Federal de Santa Maria
Centro de Ciências Naturais e Exatas
Programa de Pós-Graduação em Biodiversidade Animal
A Comissão Examinadora, abaixo assinada,
aprova a Dissertação de Mestrado
TOXICIDADE DO CÁDMIO EM EMBRIÕES E LARVAS DE Rhamdia
quelen (HEPTAPTERIDAE) EM DIFERENTES ALCALINIDADES DA
ÁGUA
elaborada por
Ana Paula da Silva Benaduce
como requisito parcial para obtenção do grau de
Mestre em Ciências Biológicas
COMISSÃO EXAMINADORA:
Bernardo Baldisserotto, Dr.
(Presidente/Orientador)
Cláudia Bueno dos Reis Martinez, Dr. (UEL)
Vânia Lucia Pimentel Vieira, Dr. (UFSM)
AGRADECIMENTOS
À Deus pela oportunidade e por me dar força e persistência para seguir este
caminho.
Ao meu orientador, Prof. Dr. Bernardo Baldisserotto, que por sua incalculável
ajuda e paciência dei meus primeiros passos na pesquisa científica e, com sua
confiança e incentivo, tive a oportunidade de amadurecer pessoalmente e
profissionalmente.
Aos meus pais, Paulo e Cledia, por abdicarem de seus sonhos, para
realizarem os meus e por serem sempre exemplos de quem vive seus princípios e
valores. Obrigada por acreditarem que o maior legado que se pode deixar a um filho
é a educação e o conhecimento.
À minha irmã Ana Laura pelo amor, carinho, companheirismo e por sua
disposição em me ajudar em momentos difíceis.
À minha avó Hélia e ao meu avô Ângelo por serem exemplos de vida e por
todas as orações realizadas.
À minha avó Laurentina por todo carinho e expectativa por ter a primeira neta
mestre.
Ao meu avô Raymundo que, apesar de não estar fisicamente presente, com
certeza deu sua ajudinha “lá de cima” para a realização desse trabalho.
Aos familiares que foram apenas expectadores desta caminhada, agradeço o
carinho e as muitas vezes que me trouxeram de volta realidade.
À “Léiadir” por estar sempre disposta a me ajudar, não me deixar esmorecer
frente às dificuldades e por ser, acima de tudo, uma grande amiga.
Ao Milton pelo amor, amizade, incentivo e pelas frases: “Vai dar certo!”, “Eu
sei que tu consegue!” que, sem dúvida, foram de grande importância durante toda
essa caminhada.
À amiga Ana Paula Fabian, minha irmã de coração, pelo carinho e amizade
sincera, nunca medindo esforços para me ajudar.
À Maria Amália que, com sua grande simplicidade, sempre compartilhou
comigo sua amizade e seus conhecimentos.
parceria nas infindáveis horas de dedicação no laboratório, pela amizade e
companheirismo.
Aos Profs. Érico e Valderi do Departamento de Química pela ajuda prestada
na análise das concentrações de cádmio.
À Profª. Silvia e aos estagiários do Laboratório de Parasitologia Veterinária
por permitirem a realização de um super “book” de fotos de ovos e larvas de jundiá.
Aos meus amigos que souberam me entender e ajudar nos momentos em
que precisei.
Três coisas
“De tudo, ficaram três coisas:
A certeza de que estamos sempre começando...
A certeza de que é preciso continuar...
A certeza de que seremos interrompidos antes de terminar...
Portanto, devemos:
Fazer da interrupção, um caminho novo...
Da queda, um passo da dança...
Do medo, uma escada...
Do sonho, uma ponte...
Da procura, um encontro..."
RESUMO
Dissertação de Mestrado
Programa de Pós-Graduação em Biodiversidade Animal
Universidade Federal de Santa Maria
TOXICIDADE DO CÁDMIO EM EMBRIÕES E LARVAS DE Rhamdia
quelen (HEPTAPTERIDAE) EM DIFERENTES ALCALINIDADES DA
ÁGUA
Autora: Ana Paula da Silva Benaduce
Orientador: Bernardo Baldisserotto
Data e Local da Defesa: Santa Maria,16 de dezembro de 2005.
O presente estudo avaliou os efeitos do cádmio (Cd) em embriões e larvas de
jundiá, Rhamdia quelen, em duas alcalinidades da água. Ovos e larvas foram
expostos a diferentes concentrações de Cd na água em dois níveis de alcalinidade
(63 e 92 mg.L
-1CaCO
3): 0,5 - controle; 4,5; 8; 18 µg Cd.L
-1. A taxa de fertilização foi
similar para todos os tratamentos, mas as deformações na superfície dos ovos foram
significativamente maiores nos expostos a 18 µg Cd.L
-1e 63 mg.L
-1CaCO
3. A
sobrevivência aos 3 dias após a eclosão foi significativamente maior nas larvas
controle do que nas expostas a 4,5 e 8 µg Cd.L
-1e nas expostas a 18 µg Cd.L
-1do
que nas expostas a 4,5 µg Cd.L
-1na alcalinidade de 63 mg.L
-1CaCO
3, o que não foi
observado na alcalinidade de 92 mg.L
-1CaCO
3. Além disso, na menor alcalinidade
foram observadas maiores porcentagens de deformações nos barbilhões e na
coluna espinhal nas maiores concentrações de Cd na água. Ao final do período
experimental, 21 dias após a eclosão, o aumento de Cd na água diminuiu a
sobrevivência na menor alcalinidade, e uma alta porcentagem de deformações na
coluna espinhal foi observada na maior concentração de Cd na água. Na maioria
das vezes, em 63 mg.L
-1CaCO
3, o peso, a altura da cabeça, a espessura da
membrana caudal, o comprimento da nadadeira caudal diminuíram com o aumento
da concentração de Cd na água. Esse fato não foi observado em 92 mg.L
-1CaCO
3.
A taxa de crescimento específico foi menor nas maiores concentrações de Cd em 63
mg.L
-1CaCO
3. Esses resultados sugerem que a alcalinidade de 92 mg.L
-1CaCO
3reduz a toxicidade do cádmio em larvas de jundiá.
ABSTRACT
Master Dissertation
Post-Graduation in Animal Biodiversity
Universidade Federal de Santa Maria
TOXICITY OF CADMIUM IN THE SILVER CATFISH Rhamdia quelen
(HEPTAPTERIDAE) EMBRYOS AND LARVAE AT DIFFERENT
ALKALINITIES
Author: Ana Paula da Silva Benaduce
Adviser: Bernardo Baldisserotto
Place and Date of Defence: Santa Maria, December 16
th, 2005.
The present study evaluated the effect of waterborne cadmium (Cd) in
embryos and larvae of silver catfish, Rhamdia quelen, in two water alkalinities. Eggs
and larvae were exposed to different waterborne Cd concentrations at two alkalinities
levels (63 and 92 mg.L
-1CaCO
3): 0.5 - control, 4.5, 8, 18 µg Cd.L
-1. The fertilization
rate was similar in all treatments, but the number of eggs with irregular surface was
significantly higher in those exposed to 18 µg Cd.L
-1and 63 mg.L
-1CaCO
3. Survival
rate registered 3 days after hatching was significantly higher in control larvae
compared to those exposed to 4.5 and 8 µg Cd.L
-1and in those exposed to 18 µg
Cd.L
-1compared to larvae exposed to 4.5 µg Cd.L
-1in the alkalinity of 63 mg.L
-1CaCO
3, while these significant difference were not observed at the water alkalinity of
92 mg.L
-1CaCO
3. Furthermore, in the lower alkalinity, higher incidence of barbells
and spinal column deformities was observed in the highest waterborne Cd
concentration. At the end of experimental period, 21 days after hatching, it was
observed in the lower alkalinity that with the increase of waterborne Cd the survival
rate decreased, and also that the percentage of spinal column deformity was high in
the highest waterborne Cd concentration. Usually, at 63 mg.L
-1CaCO
3weight, head
height, membranous layer thickness, and tail fin length decreased as waterborne Cd
increased. However, these relationships were observed only in some parameters at
92 mg.L
-1CaCO
3. As expected, specific growth rate was lower in the highest
waterborne Cd concentration at 63 mg.L
-1CaCO
3. These results suggest that
LISTA DE TABELAS
TABELA 1 - Deformações das larvas de jundiá expostas a diferentes concentrações
de Cd na água em dois níveis de alcalinidade...39
LISTA DE FIGURAS
FIGURA 1 - Acúmulo total de Cd nos os ovos e larvas de jundiá expostos a
diferentes concentrações de Cd na água em dois níveis de alcalinidade...44
FIGURA 2 - Relação entre a sobrevivência larval e a concentração de Cd na água e
nas larvas aos 3 e 21 dias após a eclosão...45
FIGURA 3 - Larvas de jundiá aos 3 dias após a eclosão...46
FIGURA 4 - Larvas de jundiá aos 7 dias após a eclosão...47
FIGURA 5 - Larvas de jundiá aos 21 dias após a eclosão...48
FIGURA 6 - Volume do ovo e do saco vitelínico nos jundiás expostos a diferentes
concentrações de Cd na água em dois níveis de alcalinidade...49
FIGURA 7 - Comprimento total e peso das larvas de jundiá expostas a diferentes
concentrações de Cd na água em dois níveis de alcalinidade...50
FIGURA 8 - Altura da cabeça das larvas de jundiá expostas a diferentes
concentrações de Cd na água em dois níveis de alcalinidade...51
SUMÁRIO
1 INTRODUÇÃO...13
2 REVISÃO BIBLIOGRÁFICA...15
2.1
Considerações sobre Rhamdia quelen (Quoy e Gaimard,
1824)...15
2.2
Cádmio...16
2.3
Qualidade da água: alcalinidade...18
3 ARTIGO CIENTÍFICO ...20
Title, names of authors...21
Abstract...22
Introduction...23
Materials and methods...24
Results...27
Discussion...30
References...32
Tables...39
Figure captions...41
4 CONCLUSÃO...53
5 REFERÊNCIAS BIBLIOGRÁFICAS...54
6 ANEXOS...62
1 INTRODUÇÃO
A qualidade dos parâmetros físicos e químicos da água são requisitos indispensáveis para o bom desenvolvimento e sobrevivência de animais aquáticos. A poluição aquática interfere na qualidade da água e, consequentemente, nos seres vivos presentes naquele ambiente. Os efeitos tóxicos dos poluentes compreendem efeitos letais e sub-letais. Os organismos que sobrevivem em ambientes contaminados sofrem alterações fisiológicas, bioquímicas e histológicas (Larsson et al., 1976; Pratap & Wendelaar Bonga, 1993), o que pode levar à diminuição na taxa de crescimento e na capacidade reprodutiva, alteração no comportamento, alterações morfológicas, entre outros.
O cádmio é um subproduto da mineração de zinco e um dos metais que
apresenta elevada toxicidade para os peixes. Experimentos realizados mostram que
sua toxicidade varia de acordo com a espécie de peixe, tempo de exposição e
concentração. A qualidade dos parâmetros físicos e químicos da água também
interfere na toxicidade deste metal, por exemplo, quanto maior a temperatura maior
é a toxicidade do Cd (Alabaster & Lloyd, 1982) e quanto maior a dureza da água
menor a toxicidade (Sprague, 1987).
A alcalinidade é um parâmetro químico da água relacionado com a capacidade de neutralizar ácidos. Freqüentemente o aumento da alcalinidade deve-se a um processo de calagem da água. A calagem neutraliza a acidez, estimula o ciclo de nutrientes, promove a reorganização biológica, influencia fatores físicos e químicos da água, resultando em respostas favoráveis da biota em curto período de tempo (Rojas et al., 2001).
Embriões e larvas de peixes em desenvolvimento são altamente sensíveis a agentes tóxicos, e por isso são freqüentemente usados em testes de toxicidade (Rand, 1995; Connell et al., 1999). Como a toxicidade dos metais, inclusive do cádmio, é alterada pelos parâmetros químicos da água é provável que uma variação na alcalinidade da água influencie na toxicidade do cádmio.
O jundiá, Rhamdia quelen, espécie utilizada neste trabalho, apresenta distribuição geográfica neotropical e é adaptada a diferentes qualidades da água. Possui hábito alimentar omnívoro com tendência piscívora, crescimento rápido, é euritérmico e estenoalino, entre outras características (Gomes et al., 2000; Chippari-Gomes et al., 1999; Marchioro & Baldisserotto, 1999). É uma espécie nativa que apresenta excelente aceitação no mercado consumidor, e na pesca esportiva.
2 REVISÃO BIBLIOGRÁFICA
2.1 Considerações sobre Rhamdia quelen (Quoy e Gaimard, 1824)
O jundiá Rhamdia quelen (Siluriformes, Heptapteridae) é um peixe de água doce que vive em lagos e poços fundos dos rios, preferindo os ambientes de águas mais calmas com fundo de areia e lama. É encontrado do sudeste do México ao centro da Argentina (Silfvergrip, 1996).
Essa espécie é adaptada ao clima da região, e o seu manejo é relativamente fácil, apresentando excelente aceitação no mercado consumidor. Seu hábito alimentar é omnívoro com tendência a piscívoro, mas também se alimenta de crustáceos, insetos, restos vegetais, e detritos orgânicos. Sua coloração varia de marrom-avermelhado claro a cinza ardósia (Gomes et al., 2000).
A maturidade sexual é atingida no primeiro ano de vida (Narahara et al., 1985), os machos a partir de 16,5 cm e as fêmeas a partir dos 17,5 cm. É uma espécie ovulípara (Godinho et al., 1978; Andreatta, 1979) e na natureza desova em ambientes com água limpa e fundo pedregoso, não apresentando cuidado parental (Gomes et al., 2000). As fêmeas de jundiá apresentam fase reprodutiva de agosto a março e podem produzir cerca de 216.000 ovos.kg-1 de peso vivo (Gomes et
al., 2000).
O jundiá é uma espécie com desenvolvimento oocitário do tipo assincrônico, desova múltipla e fecundidade baixa (Narahara et al., 1989). Variações na fecundidade podem estar relacionadas ao suprimento alimentar, densidade populacional, estresse, temperatura, entre outros fatores ambientais (Bagental, 1978). Entretanto, Gomes et al. (2000) afirmam que a fecundidade está relacionada com as condições alimentares às quais os reprodutores estão submetidos no período de um ano antes da reprodução.
A reprodução pode ser induzida em jundiás com grande facilidade, e essa espécie responde bem à indução com hipófise de carpa (Lopes et al., 2001) e a gonadotrofina coriônica humana (Radünz Neto, 1981). Em sistema artificial a taxa de fertilização e eclosão dos ovos é em torno de 95 e 90% respectivamente (Andreatta, 1979).
A desova e o desenvolvimento embrionário de jundiá são bastante rápidos e o desenvolvimento larval ocorre entre 3-5 dias (Gomes et al., 2000). Segundo Godinho et al. (1978), esse desenvolvimento é influenciado por fatores ambientais como a temperatura da água. O tempo de eclosão dos ovos depende da temperatura da água, por exemplo, com a temperatura da água em torno de 24oC a eclosão demora entre 24 e 36 horas (Andreatta, 1979). Para um melhor desenvolvimento dos ovos Ferreira et al. (2001) recomenda pH próximo a 9,0, já as larvas apresentam melhor crescimento e sobrevivência em pH entre 8,0 e 8,5 (Lopes et al., 2001). Segundo Townsend et al. (2003) e Silva et al. (2003) a faixa de dureza entre 30 e 70 mg.L-1 CaCO3 é a melhor
para a eclosão e larvicultura dessa espécie.
Resíduos industriais e agrícolas são alguns dos principais fatores de contaminação das águas por metais pesados, tais como: cobre (Cu2+), chumbo (Pb), zinco (Zn2+), cádmio (Cd2+) e mercúrio (Hg2+), e representam um sério problema de poluição na água devido às propriedades tóxicas desses metais e seus efeitos adversos na qualidade da água (Xiaorong et al, 1997).
Segundo Plachy (1999), o Cd é um dos 11 metais na lista de produtos tóxicos, persistentes e bioacumulativos (PBT, Persistent and Bioaccumulative Toxic) da Agência de Proteção Ambiental Americana (EPA, Environment Protection Agency). Ainda segundo este autor, o Cd faz parte da lista de substâncias e processos consideravelmente perigosos ao planeta do Registro Internacional de Produtos Químicos Potencialmente Tóxicos (IRPTC, International Register of Potentially Toxic Chemical) do Programa Ambiental das Nações Unidas.
O Cd, originado principalmente das minas de zinco, é um dos metais pesados mais tóxicos (Bryan, 1984) e é uma das maiores preocupações ambientais e de saúde pública (Sprague, 1987; WHO, 1992; Rainbow, 1995). Algumas fontes humanas de liberação de Cd são baterias, pigmentos, equipamentos eletrônicos, acessórios fotográficos, defensivos químicos, entre outros (ILO, 1998). Vários trabalhos (Hiatt & Huff, 1975; Kaviraj & Das, 1994) têm salientado o potencial tóxico deste metal e sua capacidade de produzir mudanças bióticas nos ecossistemas aquáticos. Como um poluente cumulativo não-degradável, o cádmio pode permanecer nos níveis tróficos aquáticos por muitos séculos (Sorensen, 1991). Seus efeitos adversos têm sido estudados em muitas espécies animais e em vários órgãos alvos, tais como fígado e rim e sistemas reprodutivo e respiratório.
Peixes de água doce são particularmente vulneráveis à exposição ao Cd (Sorensen, 1991) que promove efeitos adversos no crescimento, reprodução e osmorregulação (Sangalang & O´Halloran, 1972; Pratap & Wendelaar Bonga, 1990; Cheng et al., 2001, Hansen et al., 2002). As atividades de enzimas metabólicas no fígado, rim, músculos, e outros tecidos são também perturbadas (Sastry & Subhadra, 1982). Cd é conhecido por aumentar a permeabilidade das membranas celulares e então causar prejuízos ao metabolismo celular (Grose et al., 1987) e mesmo em baixas concentrações também perturba as funções centrais do peixe por afetar muitos processos bioquímicos e fisiológicos (Larsson et al., 1976).
A toxicidade do Cd é geralmente atribuída ao cátion divalente livre (Pagenkopf, 1983). Quando a água para o abastecimento contém resíduos industriais, o Cd é amplamente distribuído no ambiente aquático e prejudicial aos organismos presentes, principalmente para os peixes, já que interfere no equilíbrio iônico. Sua toxicidade pode ser afetada por parâmetros físicos e químicos da água. Por exemplo, Playle et al. (1993a) observou que a concentração de 42,1 mg Ca2+.L-1 na água evita a ligação de Cd nas brânquias de “fathead minnows” (Pimephales promelas) expostos a 5,6 g Cd.L-1 por 2-3 horas. Segundo Meinelt et al. (2001), o aumento da concentração de Ca2+ melhorou a sobrevivência dos embriões de “zebrafish” (Danio rerio) em água contaminada com Cd. Carbono orgânico dissolvido e matéria orgânica dissolvida também oferecem proteção contra a toxicidade do Cd (Playle et al., 1993b, Richards et al., 1999).
para peixes de água doce é alta e similar à do Cu2+, com patofisiologia substancial em concentrações muito abaixo de 100 g.L-1 (Wood, 2001).
O Cd também provoca diminuição na taxa de crescimento e
indução de desenvolvimento de anormalidades, tais como lordose
e ciclopia (Larsson et al., 1976; McKim, 1977; Weis & Weis, 1977).
Recentes avanços na pesquisa com Cd têm demonstrado que este
metal possui uma capacidade endócrina disruptiva em teleósteos
(Sangalang & O’Halloran 1972; Thomas, 1990; Hontela et al., 1996;
Gupta et al., 1997, Jones et al., 2001).
O Conselho Nacional do Meio Ambiente (CONAMA) é o orgão brasileiro responsável pelo estabelecimento de concentrações limites de substâncias na água. Segundo a resolução n°357 de 17/03/2005 o nível máximo de Cd permitido em águas doces brasileiras nas classes I e II é de 1 µg Cd.L-1. Entretanto, pesquisas feitas na Baía de Santos e Estuário de Santos e São Vicente mostraram que águas superficiais apresentavam entre 0,6-1,1 µg Cd.L-1, águas profundas entre 0,4-1,6 µg Cd.L-1 e o sedimento entre 0,17-0,20 µg Cd.g-1 (Eysink et al., 1985). Análise dos peixes que habitavam este local mostrou que nas vísceras havia entre 0,05-0,22 µg Cd.g-1 e nos músculos entre 0,05-0,06 µg Cd.g-1 (Boldrine & Pereira, 1985). Segundo Flores (1990) as Sangas da Carvoeira e Quebra-Jugo, localizadas na região de Candiota no estado do RS, possuiam cerca de 8 e 7 µg Cd.L-1, respectivamente. Estudos realizados no litoral do estado do Rio de Janeiro comprovaram a contaminação do sedimento (Kehrig et al., 2003) e também de alguns peixes carnívoros (Pfeiffer et
al., 1985) por Cd, o que mostra que a contaminação por esse metal também ocorre no Brasil.
2.3 Qualidade de água: alcalinidade
Alcalinidade reflete a capacidade que um ecossistema apresenta em neutralizar ácidos, referindo-se à concentração total de sais na água que reagem neutralizando íons H+ (Alabaster & Lloyd, 1982). Segundo Arana (2004) muitos materiais podem contribuir com a alcalinidade da água, sendo a maior parte os hidróxidos, carbonatos e bicabonatos.
Águas naturais com alcalinidade igual ou acima de 40 mg.L-1 CaCO3 são consideradas mais
produtivas do que aquelas com baixa alcalinidade (Mairs, 1996). Entretanto, Mairs (1996) ressalta que a alta produtividade não se deve diretamente a elevação da alcalinidade, mas sim ao aumento da disponibilidade de fósforo e outros elementos essenciais causados pela alcalinidade elevada. Rojas
et al. (2001) mostraram que alcalinidades entre 15 - 56 mg.L-1 CaCO3 não interferiram na
sobrevivência das larvas de curimbatá (Prochilodus lineatus). Entretanto, nesse experimento foi utilizado carbonato de cálcio para elevar a alcalinidade e isso provocou um aumento também na dureza da água, o que pode ter interferido nos resultados.
A alcalinidade também influencia na especiação de metais na água pela formação de metal-carbonato ou espécies de bimetal-carbonatos. Muitos cátions na água, especialmente o Ca2+, competem com os metais pelos sítios de ligação nas brânquias (Pagenkopf, 1983; McDonald et al., 1989). Welsh
et al. (2000) observou que a toxicidade do cobre em salmonídeos diminui com o aumento da
alcalinidade de 97 para 194 mg.L-1 CaCO3. Entretanto, os níveis de alcalinidade na água devem ser
Toxicity of cadmium in the silver catfish Rhamdia quelen (Heptapteridae) embryos and larvae at different alkalinities
Ana Paula S. Benaduce a, Daiani Kochhann a, Érico M.M. Flores b, Valderi L. Dressler b, Bernardo Baldisserotto a, *
Departamento de Fisiologia e Farmacologia (a) e Departamento de Química (b), Universidade Federal de Santa Maria, 97105-900, Santa Maria, RS, Brazil
* Corresponding author:
Departamento de Fisiologia e Farmacologia Universidade Federal de Santa Maria 97105-900, Santa Maria, RS, Brazil
Tel.: + 55 55 3220-9382; fax: + 55 55 3220-8241 E-mail: bernardo@smail.ufsm.br
Abstract
The present study evaluated the effect of waterborne cadmium (Cd) in embryos and larvae of silver catfish, Rhamdia quelen, in two water alkalinities. Eggs and larvae were exposed to different waterborne Cd concentrations at two alkalinities levels (63 and 92 mg.L-1 CaCO3): 0.5 - control, 4.5, 8,
surface was significantly higher in those exposed to 18 µg Cd.L-1 and 63 mg.L-1 CaCO3. Survival rate
registered 3 days after hatching was significantly higher in control larvae compared to those exposed to 4.5 and 8 µg Cd.L-1 and in those exposed to 18 µg Cd.L-1 compared to larvae exposed to 4.5 µg Cd.L-1 in the alkalinity of 63 mg.L-1 CaCO3, while these significant difference were not observed at the
water alkalinity of 92 mg.L-1 CaCO3. Furthermore, in the lower alkalinity, higher incidence of barbells
and spinal column deformities was observed in the highest waterborne Cd concentration. At the end of experimental period, 21 days after hatching, it was observed in the lower alkalinity that with the increase of waterborne Cd the survival rate decreased, and also that the percentage of spinal column deformity was high in the highest waterborne Cd concentration. Usually, at 63 mg.L-1 CaCO3 weight,
head height, membranous layer thickness, and tail fin length decreased as waterborne Cd increased. However, these relationships were observed only in some parameters at 92 mg.L-1 CaCO3. As
expected, specific growth rate was lower in the highest waterborne Cd concentration at 63 mg.L-1 CaCO3. These results suggest that alkalinity of 92 mg.L
-1
CaCO3 reduces waterborne Cd toxicity in
silver catfish larvae.
Key words: fish, eggs, sodium bicarbonate, heavy metal.
Introduction
Cadmium (Cd) is an important xenobiotic in aquatic ecosystems. It is a non-essential metal, but competes with other essential metallic ions when accumulated in organisms and produces unpredictable changes (Dutta and Kaviraj, 2001). As a nondegradable cumulative pollutant, Cd can remain in aquatic trophic levels for centuries, and freshwater fish are particularly vulnerable to Cd exposure (Sorensen, 1991) which it interferes with ion and water balance in fish (Hwang et al., 1995).
Cd enters through Ca2+ channels in chloride cells and subsequently blocks Ca2+ extrusion to the blood across the basolateral membrane through the inhibition of Ca2+ -ATPase (Verbost et al., 1989; Wicklund Glynn et al., 1994). Cd accumulates mainly in three organs: liver, kidney and gills (Wicklund Glynn, 1990; McGeer et al.,2000). Significant Cd influx can also occur through the skin, fins, and gills (Wicklund Glynn, 2001). For small fishes, the skin may serve as a particularly important site because of a high surface area to body ratio (Hayton and Barron, 1990).
digestive efficiency (Sastry and Gupta, 1979), affects olfaction sensitivity (Scott et al., 2003), hypocalcemia, hypokalemia, and hyperglycemia (Sorensen, 1991). Cd has also adverse effects on reproduction, ion regulation, growth (Sangalang and O’Halloran, 1972; Pratap and Wendelaar Bonga, 1990, 1993; Jones et al., 2001), and might provoke erratic swimming (Peterson et al., 1983) and mortality (Cheng et. al., 2001).
Effects of Cd on early life stages of fish have been well recorded (Cheng et al., 2000). Toxicity of this metal has also been demonstrated in a variety of species ranging from mice, birds, amphibia and fishes in which developmental abnormalities, growth retardation and prenatal deaths were reported (Webster, 1990). Teleosts are highly sensitive to Cd and markers of toxicity include premature hatching, decreased growth rates and the induction of developmental abnormalities such as cyclopia and spinal lordosis (Larsson et al., 1975; McKim, 1977; Weis and Weis, 1977).
Waterborne Cd toxicity can be affected by several water physico chemical parameters as alkalinity, pH, water hardness, and mainly Ca2+ and humic substances (Meinelt et al., 2001). There is abundant evidence that Cd is less toxic in hard water than in soft water, but most studies have failed to separate the effects of alkalinity and pH from those of the two hardness cations (reviewed by Davies et al., 1993). Nevertheless, there are studies which demonstrated that specifically Ca2+ and not carbonate alkalinity, was the dominant protective factor against Cd toxicity to rainbow trout (Oncorhynchus mykiss) (Davies et al., 1993; Richards and Playle, 1999). Furthermore, geochemical modeling indicates that Ca2+ competition rather than carbonate complexation is the main factor keeping Cd off the gills in hard water (Hollis et al., 1997, 1999).
The silver catfish, Rhamdia quelen, is a freshwater fish that can be found in Central and South America. This fish reaches sexual maturity in nature at the end of the first year of life, and gonadal development starts with males having at least 13.4 cm and females 16.5 cm. The reproductive period of silver catfish is from August to March in South Brazil with multiple spawning, and females can produce around 216.000 eggs.kg-1 live weight. There is no parental care of the eggs or larvae (Gomes et al., 2000). The embryological development is fast, and the larval development occurs in 3 –5 days (Narahara et al., 1985).
As fishes are important food resource and a major ecosystem component, it is important to assess the deleterious effects of Cd at an early stage. In addition, the effect of water alkalinity on larval development and Cd toxicity was rarely studied. Therefore, the objective of the present study was to evaluate the effect of waterborne Cd on survival and embryonic development of silver catfish in two different water alkalinities.
Materials and methods
kg) 40 mL of milt. The milt was added to oocyte mass to provide fertilization and divided into 8 equal parts and placed in 8 plastic containers. After fertilization, eggs were hydrated and incubated in the same water to be used for treatment (to be described). Twelve minutes later dead eggs were manually separated on Petri dishes and the viable fertilized eggs (determined by observation of the initial cell division on a microscope) present in 3 mL were counted. Eggs were then placed in incubators (4 L polyethylene bottles – 800 eggs.bottle-1
) at 25°C. Each bottle received continuous aeration with a 20W air pump (air flow of 3.2 L.min-1) that also promoted water movement. Hatching occurred 36 hours after fertilization. Three days after hatching, larvae were fed in excess six times a day (08:00, 10:00, 12:00, 14:00, 16:00, and 18:00 h) with ground dry pellets according to Lopes et al. (2001). The incubators were siphoned everyday, and consequently at least 20% of the water in the bottles was replaced with water previously adjusted to the appropriate Cd concentration and alkalinity. Three days after hatching the remaining larvae were transferred to 40 L polyethylene boxes until to the end of the experiment (21 days).
Samples of eggs were collected 12 hours after fertilization and larvae at days 3, 7, 14, and 21 after hatching (6 specimens from each replicate). Three eggs and larvae were fixed for 24 h in the Karnovisky’s solution and stored in 0.5% glutaraldehyde. Other 3 eggs and larvae were frozen at -20°C to posterior analysis of total Cd concentration.
The fertilization rate (FR) and the post-hatch survival (PH) were calculated by:
FR= (number of incubated eggs – number of dead eggs)/(number of incubated eggs) X 100. Post-hatch survival (PH) was calculated by adaptation of the method of Geertz Hansen and Rasmussen (1994) three days after hatching, when larvae were transferred to the 40 L boxes.
PH= (number of incubated eggs – number of dead eggs – dead larvae)/(number of incubated eggs) X 100.
Morphometric characteristics associated with swimming abilities, vision and feeding were measured in each fixed larvae using a binocular stereomicroscope fitted with an ocular graticule. These characteristics were: (1) total length, (2) head height measured at the level of the operculum, (3) eye diameter, (4) membranous layer thickness, (5) digestive tract length (6) tail fin length, (7) anal fin length. In the yolk sac larvae, the maximum and minimum diameters of the ellipsoidal yolk sac were also measured and the volume calculated using the following formula: V=0.1667 LH2; were H is the minimum diameter and L is the maximum diameter of the yolk sac mass (Heming and Budding, 1988). All measurements were taken along lines parallel or perpendicular to the horizontal axis of the body (Gisbert, 1999). Maximum and minimum diameters of the eggs were measured to calculate the volume. Eggs with irregular surface were classified as deformed eggs, and larvae with a rudimentary barbells and a axial curvature greater than 10° (Cheng et al., 2000) were classified as barbells and spinal deformity.
Specific growth rate (SGR) was calculated according to Jørgensen and Jobling (1993). On day 21, all surviving larvae were collected to determined survival (%) and biomass (individual mean weight X number of surviving larvae).
Eggs and larvae were exposed to different waterborne Cd concentrations (as CdCl2) at two
alkalinities levels of 63 and 92 mg.L-1 CaCO3 (three replicates per treatment) (in µg Cd.L -1
18. Actual measured waterborne Cd concentrations were (mean ± SEM): 0.59 0.09 and 0.53 0.13, 4.18 0.28 and 5.05 0.45, 7.95 0.74 and 7.58 0.7, 18.4 0.68 and 11.06 0.39 µg Cd.L-1 for alkalinity of 63 and 92 mg.L-1 CaCO3, respectively. Alkalinity was increased by the addition of
sodium bicarbonate (NaHCO3) and was estabilized two weeks before starting the experimental period.
The hightest waterborne Cd concentrations were different between the alkalinities because at the alkalinity of 92 mg.L-1 CaCO3 that was the formation of Cd oxides and hydroxides which might
precipitate.
Waterborne Cd levels were measured once a week using a graphite furnace atomic absorption spectrometer (Analytik Jena Model EA5; Jena, Germany) equipped with a deuterium lamp background corrector and Cd hollow cathode lamp, operated at 5 mA (wavelength 228.8 nm, spectral bandwidth 0.5 nm). Palladium was used as chemical modifier and the pyrolysis and atomization temperatures were 700 and 1800oC, repectively. All measurements were made in integrated absorbance. Eggs and larvae were previously digested with concentrated nitric acid (HNO3) for
subsequent determination of total Cd concentration. Water alkalinity was determined daily by the sulfuric acid (H2SO4) method (Greenberg et al., 1976) and adjusted when necessary. Dissolved
oxygen (YSI model Y5512 oxygen meter) and water pH (Quimix 400A pH meter) were also measured daily, and the last adjusted to around pH 8.0 (for both alkalinities) with sodium hydroxide, because this value had been identified as the best for survival and growth of silver catfish larvae (Lopes et al., 2001). The following physicochemical parameters of the water were analyzed at 3-day intervals: total ammonia, total hardness and nitrite. Water hardness was determined by the EDTA titrimetric method, nitrite by Boyd and Tucker (1992), total ammonia nitrogen (NH3 + NH4+) by direct Nesslerization
method (Greenberg et al., 1976), and non-ionized ammonia nitrogen (N-NH3) was calculated as
described by Piper et al. (1982). Ca2+ level was determined by flame atomic absortion spectrometry (Perkin-Elmer Model 3030, Germany) equipped with Ca2+ hollow cathode lamp, operated at 10 mA (wavelength 422.7 nm, slitwidth of 0.7 nm). Waterborne Na+ and K+ concentrations were measured with a flame photometer Micronal B286. Through experimental period overall mean of water parameters were: dissolved oxygen (5.61 0.02 mg.L-1), temperature (22.59 0.03 ºC), pH (8.21 0.03), non-ionized ammonia (0.071 0.004 mg.L-1), total ammonia (0.970 0.047 mg.L-1), nitrite (0.142 0.01 mg.L-1), water hardness (22.9 1.3 mg.L-1 CaCO3), Ca
2+
(5.2 mg.L-1), Na+ (5.64 0.12 mg.L-1), and K+ (1.49 0.02 mg.L-1).
Data have been reported as mean S.E.M. (N). Homogeneity of variances among groups was tested with the Levene test. Most data presented homogeneous variances, and comparisons among different treatments were made by two-way analysis of variance and Tukey test. However, data of percentage of deformation were analyzed by ANOVA Kruskall-Walis and Mann-Whitney test. Analysis was performed using the software Statistica (version 5.1), and the minimum significance level was set at P < 0.05. The relationship between different waterborne Cd concentrations at both alkalinities, in some variables, was calculated with the aid of the same software.
Throughout the experiment all water parameters did not show any significant difference among treatments and remained within the appropriate range for silver catfish development.
Total cadmium accumulation in eggs and larvae maintained at 63 mg.L-1 CaCO3 increased as
waterborne Cd concentration increased, showing a significant relationship 12 hours after fertilization, 3, 7, and 21 days after hatching (Figure 1). This relationship was also observed at 3, 7 and 21 days at 92 mg.L-1 CaCO3, but not at 12 hours after fertilization. Nevertheless, in this period eggs exposed to
8.0 µg Cd.L-1 and 92 mg.L-1 CaCO3 showed significantly higher Cd accumulation than those exposed
to control water and same alkalinity(Figure 1).
The fertilization rate was similar to all treatments (overall mean 99.55% 0.26 (N = 3 incubators from four treatments). Three days after hatching larvae survival did not present significant difference between the control treatments at both alkalinities. Larvae survival of the control treatment at 63 mg.L-1 CaCO3 was significantly higher than those exposed to 4.5 and 8 µg Cd.L
-1
. Although survival of larvae exposed to the highest waterborne Cd level at 63 mg.L-1 CaCO3 did not show
significant difference when compared to control larvae, it was significantly higher than of those maintained at 4.5 µg Cd.L-1 (Figure 2A). However, no relationship between survival and total Cd accumulation in the larvae was observed at this moment (Figure 2B). Twenty one days after hatching, at 63 mg.L-1 CaCO3 larvae survival decreased with the increase of waterborne Cd levels and total
cadmium accumulation in the larvae, reaching almost zero (there was only one larva left) in those submitted to 18 µg Cd.L-1. These relationships were not observed in larvae maintained at 92 mg.L-1 CaCO3 (Figures 2C and 2D). However, it was observed that larvae exposed to 4.5 and 8 µg Cd.L
-1
presented significantly lower survival than control larvae at this alkalinity.
Throughout all experimental period the percentage of visible abnormalities on the surface egg, barbells and spinal column of larvae were small and without significant difference between the control treatments at two alkalinity levels.
Twelve hours after fertilization the percentage of eggs exposed to 63 mg.L-1 CaCO3 alkalinity
and 18 µg Cd.L-1 with irregular surface (36.11% 7.35 (N = 3 incubators from four treatments) ) was significant higher than those exposed to 0.5, 4.5 and 8 µg Cd.L-1 (0% (N = 3), 5.56% 5.56 (N = 3), 0% (N = 3), respectively). However, eggs exposed to 92 mg.L-1 CaCO3 did not show significant
difference among treatments regarding irregular surface (overall mean = 0% (N = 3)).
Larvae exposed to the two highest waterborne Cd concentrations studied for 3-7 days after hatching showed significantly higher percentage of barbells deformity compared to control larvae, and the increase of alkalinity to 92 mg.L-1 CaCO3 did not protect against these deformities. However, at 3
days in alkalinity of 92 mg.L-1 CaCO3 the percentage of this deformity was higher in larvae exposed to
At 12 hours after fertilization the egg volume did not show any significant difference between both control treatments. In the alkalinity of 92 mg.L-1 CaCO3 the egg volume was significantly higher in
the control than in those exposed to other waterborne Cd levels in the same alkalinity, although at 63 mg.L-1 CaCO3 there was no significant difference among treatments (Figure 6). At 3 days after
hatching the yolk sac volume did not show any difference between the control treatments, but at 63 mg.L-1 CaCO3 the yolk sac volume increased with the increase of waterborne Cd levels. Nevertheless,
at 92 mg.L-1 alkalinity this relationship was not observed
Throughout the experimental period length, weigth, head height, tail fin length and membranous layer thickness of larvae were not significantly affected by water alkalinity in the control treatments, except weight that was significantly higher in larvae exposed to 63 mg.L-1 CaCO3 than
those exposed to 92 mg.L-1 CaCO3 7 days after hatching (Figures 7, 8 and 9). In addition, length and
membranous layer thickness did not show any significant difference among treatments up to 7 days after hatching, except larvae exposed to 18 µg Cd.L-1 and 63 mg.L-1 CaCO3, that showed significantly
lower length than those exposed to 0.5 (control) and 8 µg Cd.L-1 (Figures 7 and 9). However, three days after hatching head height was higher in control larvae than those exposed to some waterborne Cd concentrations at both alkalinities (Figure 8). Usually length, weight, head height, tail fin length and membranous layer thickness decreased as waterborne Cd increased 7, 14 and 21 days after hatching in larvae maintained at 63 mg CaCO3. These significant relationships occurred only with some
parameters in a few days in larvae maintained at 92 mg CaCO3 (Figures 7, 8, 9).
Throughout all experiment SGR did not show any significant difference between the control treatments. Seven days after hatching larvae of the control treatment and 63 mg.L-1 CaCO3 showed
significantly higher SGR than those exposed to 18 µg Cd.L-1 in the same alkalinity. At 21 days, in this same alkalinity, SGR was significantly higher in control larvae and those exposed to 4.5 µg Cd.L-1 than those exposed to 8 and 18 µg Cd.L-1. It was also observed that larvae maintained at 8 µg Cd.L-1 showed higher SGR than those maintained at 18 µg Cd.L-1. The SGR of larvae exposed to 92 mg.L-1 CaCO3 did not present significant difference among treatments (Table 2).
The biomass at the end of 21 days at the 63 mg.L-1 CaCO3 decreased with the increase of
waterborne Cd concentration (from 4.86g 0.98 (N = 3 boxes from four treatments) in the control treatment to 0.02g 0 (N = 1 larvae) in the highest cadmium concentration), and this relationship is expressed by y = -0.2475 + 3.946 X (r2= 0.759), where y – biomass (g) and x – µg Cd.L-1
. Larvae maintained at 92 mg.L-1 CaCO3 water alkalinity did not show significant difference of biomass among
treatments.
Discussion
This study demonstrated that both studied alkalinity (63 and 92 mg.L-1 CaCO3) did not
in this experiment cited before the water hardness also changed because calcium carbonate was used to increase alkalinity, which could have changed results.
Water hardness in the 30-70 mg.L-1 CaCO3 range is supposed to be the best for silver catfish
hatching and larviculture (Silva et al., 2003, 2005; Townsend et al., 2003). However, in the present study, water hardness of 22.9 1.3 mg.L-1 CaCO3, larvae showed a higher SGR than those exposed
to the same water hardness (20-30 mg.L-1 CaCO3) in the studies of Townsend et al. (2003) and Silva
et al. (2005).
Water pH and hardness are both closely related to alkalinity in natural waters. The effect of hardness (Calamari et al., 1980; Hollis et al. 1997, 2000b), pH (Peterson et al., 1985), DOM (Richards et al., 1999), humic substances (Playle et al., 1993a, b) and other parameters on Cd toxicity were extensively reported, while alkalinity has seldom been treated as a relevant factor.
Twenty one days after hatching it was observed that survival of larvae kept at 63 mg.L-1 CaCO3 decreased as waterborne Cd concentration increased, and this tendency was not observed in
the highest alkalinity. It is assumed that low alkalinity may interfere with ionic transport across epithelia of freshwater fish resulting in the net accumulation of unwanted trace elements, like Cd, in their tissues (Haines, 1981; Spry and Wiener, 1991). Increase on water alkalinity within a 7-8 pH range decreased Zn toxicity (Gómez et al., 1998). Spry and Wiener (1991) related that low waterborne Cd and high Ca2+ resulted in lower Cd uptake across the gills of the fish than that expected in low-alkalinity waters. Moreover, Hg concentrations in fishes from low-alkalinity lakes often exceed those in fishes from Hg-contaminated waters with higher alkalinities, indicating that Hg bioavailability is somehow enhanced in low-alkalinity lakes (Spry and Wiener, 1991). Copper toxicity is inversely correlated with water hardness and alkalinity (Chakoumakos, 1979).
Throughout all experimental period, except at 12 hours after fertilization and 92 mg.L-1 CaCO3,
the increase of waterborne Cd increased larval body Cd accumulation. This was expected because accumulation of Cd by fish typically increases with increasing waterborne Cd concentration (Eaton, 1974). Juvenile rainbow trout exposed for 30 days to 3 g Cd.L-1 showed an increased on whole body Cd accumulation in a time-dependent fashion (Hollis et. al., 2001). In juvenile common carp, Cyprinus
carpio, the increase of waterborne Cd concentration also increased Cd concentration in different
organs as a function of time of exposure and concentration (Smet et al., 2001). Rainbow trout (Onchorhyncus mykiss) exposed to waterborne Cd showed Cd accumulation primarily in kidney, liver and gills (Hollis et al., 1999, 2000a, b; McGeer et al., 2000), but dietary exposure led to higher Cd accumulation in the kidney (Szebedinsky et al., 2001).
Webster (1990) reported that waterborne Cd can cause developmental abnormalities, growth retardation and prenatal deaths in fishes and others animal classes. In the present study eggs exposed to 18 g Cd.L-1 and 63 mg.L-1 CaCO3 presented significantly higher deformation 12 h after
fertilization than the other treatments in the same alkalinity. However, eggs maintained at 92 mg.L-1 CaCO3 did not show deformation. Cheng et al. (2000) noted that the chorion of zebrafish embryos
Silver catfish larvae exposed to the two highest waterborne Cd concentrations at 63 mg.L-1 CaCO3 usually presented the highest percentage of barbells and spinal column deformities. At 92
mg.L-1 CaCO3 alkalinity, the same situation was observed, except at 3 days where high percentage of
deformities was observed in larvae maintained at 4.5 g Cd.L-1. These morphological deformities were expected because spinal deformities are commonly identified as being one of the major pathological traits among fish dwelling in areas polluted with toxic chemicals, including metals (Kocan, 1996). In addition, zebrafish embryos exposed for 48 h to 11,200 g Cd.L-1 displayed abnormalities in the craniofacial region, altered axial curvature and defective somites (Cheng et al., 2001). As Cd inhibits Ca2+ transport (Verbost et al., 1989), probably these spinal deformities are related to decrease on internal Ca2+ for bone growth. Supporting this hypothesis, Chang et al. (1997) related that larvae of tilapia (Oreochromis mossambicus) exposed to 20 g Cd .L-1 showed a reduction of 32-45% of Ca influx. In addition, hypocalcemia is a classic symptom of Cd exposure in freshwater fish and can be direct cause of death (Giles, 1984).
At 3 days after hatching in the lower alkalinity (63 mg.L-1 CaCO3) the yolk sac volume
increased as the Cd concentration increased. It knows that the major route of ion uptake are thought to occur via rudimentary “chlorides cells” which occur on the yolk-sac epithelium and skin, as well as on the developing gills (Hwang and Hirano, 1985), so this increased of yolk-sac volume may be related with the edema development caused by Cd interference on larval ion regulation. This change on yolk sac volume was not observed at water alkalinity of 92 mg.L-1 CaCO3, indicating a protective
effect of a higher alkalinity level to silver catfish larvae.
Throughout the experimental period growth decreased (as observed by
weight, length, SGR, and biomass decrease) as waterborne Cd increased in the
lower alkalinity (63 mg.L
-1CaCO
3). This relationship was not observed at the higher
alkalinity (92 mg.L
-1CaCO
3). Therefore, the present study demonstrated that water
alkalinity within the 63-92 mg.L
-1CaCO
3range did not cause adverse effects in silver
catfish eggs and larvae. Furthermore, alkalinity of 92 mg.L
-1CaCO
3decreased
waterborne Cd toxicity (levels higher than 8 g Cd.L
-1) compared to 63 mg.L
-1CaCO
3probably because alkalinity influences metal complexation by inorganic compounds
such as carbonates and hydroxides (Bradley and Sprague, 1985).
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Table 1. Deformities of silver catfish larvae (% of total larvae) as a function of water alkalinity and waterborne Cd ( g.L-1).
Days after hatching
0.5 - control 44.44 29.39ab 5.56 5.56a 11.11 11.11 0a 4.5 83.33 9.62a 22.22 14.69a 16.67 9.62 22.22 14.69ab 8.0 20.83 4.16b 66.67 0b 33.33 16.67 44.45 14.69b 11.0 83.33 16.66ab 91.66 8.33c 0 41.66 41.66ab Means S.E.M. (N = 3) identified by different letters in the columns (in the same alkalinity) were significantly different (P< 0.05) as determined by Anova Kruskall-Wallis and Mann-Whitney test. *only one larvae
Table 2. Specific growth rate (SGR) of silver catfish larvae as a function of water alkalinity and waterborne Cd ( g.L-1).
Days after hatching
Cd ( g.L-1) 7 14 21 63 mg.L-1 CaCO3 0.5 g.L-1 (control) 26.48 2.61a 10.52 0.89 17.06 0.83a 4.5 g.L-1 15.68 5.59ab 9.23 1.81 18.08 0.33a 8.0 g.L-1 14.91 2.48ab 9.18 0.25 12.99 0.42b 18 g.L-1 7.35 3.38b 7.53 0 6.46 0c* 92 mg.L-1 CaCO3 0.5 g.L-1 (control) 13.37 0.27 13.44 0.48 17.80 2.86 4.5 g.L-1 17.79 2.55 8.74 0.28 20.31 0.70 8.0 g.L-1 17.84 3.75 9.77 1.88 13.16 2.93 11 g.L-1 20.10 0.99 12.47 0.52 14.64 1.75
Figure captions
Figure 1. Total Cd accumulation in eggs and larvae of silver catfish exposed to different waterborne Cd concentrations at 63 and 92 mg.L-1 CaCO3 alkalinities.
Means S.E.M. (N = 3) identified by different letters indicate significant difference among waterborne Cd levels in the same alkalinity by ANOVA and Tukey test (p<0.05).
Data were fitted to the following equations, where y - µg Cd.g-1 and x - µg Cd.L-1: 12 hours after fertilization and 63 mg.L-1 CaCO3 : y= 0.006 + 0.019x ( r
2
= 0.986).
3 days after hatching: 63 mg.L-1 CaCO3: y= 0.033 + 0.012x ( r2 = 0.774); 92 mg.L-1 CaCO3: y= -0.022 +
0.023x ( r2 = 0.984).
7 days after hatching: 63 mg.L-1 CaCO3: y= 0.074 + 0.070x ( r2 = 0.978); 92 mg.L-1 CaCO3: y= 0.095 +
0.077x ( r2 = 0.998).
21 days after hatching: 63 mg.L-1 CaCO3: y= -0.133 + 0.098x ( r 2
= 0.872); 92 mg.L-1 CaCO3: y=
-0.009 + 0.049x ( r2 = 0.837).
Figure 2. Relationships between silver catfish larvae survival and waterborne Cd concentration or total Cd accumulation at 3 (A and B) and 21 (C and D) days after hatching, respectively. In D, total Cd accumulation values of larvae exposed to 18 µg Cd.L-1 and 63 mg.L-1 CaCO3 were from 7 days
because at 21 days there were no surviving larvae.
Means S.E.M. (N = 3) identified by different letters indicate significant difference among waterborne Cd levels in the same alkalinity by ANOVA and Tukey test (P<0.05).
Data were fitted to the following equations:
C - 63 mg.L-1 CaCO3 : y = 0.820 + 0.801/(1+((x + 0.196)/0.95) 2 ) ( r2 = 0.979), where y - survival (%) and x - µg Cd.L-1. D - 63 mg.L-1 CaCO3 : y = -0.0031x 3
+ 0.15x2 -2.5x + 15.3 ( r2 = 1.00), where y - survival (%) and x - µg Cd.g-1.
Figure 3. Silver catfish larvae 3 days after hatching (10 X). 3a - control and 63 mg.L-1 CaCO3; 3b -
control and 92 mg.L-1 CaCO3; 3c - 18 µg Cd.L -1
and 63 mg.L-1 CaCO3; 3d - 11 µg Cd.L -1
and 92 mg.L-1 CaCO3. B- barbells, ML- membranous layer, TF- tail fin, SC- spinal column, YS- yolk sac. Arrow in 3c
Figure 4. Silver catfish larvae 7 days after hatching (7 X). 4a - control and 63 mg.L-1 CaCO3; 4b -
control and 92 mg.L-1 CaCO3; 4c - 18 µg Cd.L-1 and 63 mg.L-1 CaCO3; 4d - 11 µg Cd.L-1 and 92 mg.L-1
CaCO3. . B- barbells, ML- membranous layer, TF- tail fin, SC- spinal column. Arrows in 4c and 4d
indicate deformities of the spinal column.
Figure 5. Silver catfish larvae 21 days after hatching. 5a - control and 63 mg.L-1 CaCO3 (10 X); 5b -
control and 92 mg.L-1 CaCO3 (10 X); 5c - 8 µg Cd.L -1
and 63 mg.L-1 CaCO3 (7 X); 5d - 11 µg Cd.L -1
and 92 mg.L-1 CaCO3 (7 X). B- barbells. Arrows in 5c and 5d indicate deformities of the spinal column.
Figure 6. Egg and yolk sac volume of silver catfish exposed to different waterborne Cd concentration at 63 and 92 mg.L-1 CaCO3 alkalinities.
Means S.E.M. (N = 3) identified by different letters indicate significant difference among waterborne Cd levels in the same alkalinity by ANOVA and Tukey test (p<0.05).
Data were fitted to the following equation, where y - yolk sac (mm3) and x - µg Cd.L-1: 3 days after hatching and 63 mg.L-1 CaCO3 : y= 0.030 + 0.002x ( r
2
= 0.797).
Figure 7. Total length and weight of silver catfish larvae exposed to different waterborne Cd concentration at 63 and 92 mg.L-1 CaCO3 alkalinities.
* significantly different between the alkalinities at the same waterborne Cd level.
Means S.E.M. (N = 3) identified by different letters indicate significant difference among waterborne Cd levels in the same alkalinity by ANOVA and Tukey test (p<0.05).
Data were fitted to the following equations:
Total length, where y - lenght (mm) and x - µg Cd.L-1:
14 days after hatching and 63 mg.L-1 CaCO3 : y= 8.74 - 0.136x ( r 2
= 0.924). 21 days after hatching and 63 mg.L-1 CaCO3 : y= 12.51 - 0.204x ( r
2
= 0.929). Weight, where y - weight (mg) and x - µg Cd.L-1:
3 days after hatching and 92 mg.L-1 CaCO3 : y= 2.34 - 0.044x ( r2 = 0.966).
7 days after hatching and 63 mg.L-1 CaCO3 : y= 5.192 - 0.125x ( r 2
= 0.854). 14 days after hatching and 63 mg.L-1 CaCO3: y= 10.42 - 0.274x ( r
2
= 0.761). 21 days after hatching and 63 mg.L-1 CaCO3 : y= 36.04 – 1.55x ( r
2
= 0.894).
Figure 8. Head height of silver catfish larvae exposed to different waterborne Cd concentration at 63 and 92 mg.L-1 CaCO3 alkalinities.
Means S.E.M. (N = 3) identified by different letters indicate significant difference among waterborne Cd levels in the same alkalinity by ANOVA and Tukey test (p<0.05).
Data were fitted to the following equations, where y - head height (mm) and x - µg Cd.L-1: 7 days after hatching and 92 mg.L-1 CaCO3 : y= 0.669 + 0.021x ( r
2
= 0.818). 14 days after hatching and 63 mg.L-1 CaCO3 : y= 1.38 - 0.016x ( r
2
= 0.899). 14 days after hatching and 92 mg.L-1 CaCO3 : y= 1.40 - 0.022x ( r2 = 0.740).
