Bioethical principles applied to aquatic ecotoxicological studies

Abstracts

Aquatic eco-toxicology analyzes the adverse effects of chemical pollutants in the biota. The aim of this study was to analyze the adequacy of the laboratory trials with aquatic organisms, based on the principles of the 3Rs. Twenty articles on aquatic eco-toxicology published from 2010 to 2014 were reviewed. The numbers of animals utilized in eco-toxicological trials, acclimatization, research approved by the committee on ethics and utilization of anesthetics were verified. The findings indicate that the principles of refinement and replacement, of the 3Rs program, were the most encountered in the research; while most did not cite approval of experimental protocols by an ethics committee, nor the use of anesthetics on the organisms during the experiments. The research published indicates a need to reduce the number of in the bio-trials, and to apply principles of bioethics in experimentation with aquatic organisms.

Ecotoxicology; Animal experimentation; Bioethics; Ethics-Animals

A ecotoxicologia aquática analisa os efeitos adversos dos poluentes químicos na biota, utilizando-se de animais nos ensaios laboratoriais. Neste trabalho, objetivou-se analisar a adequação dos ensaios laboratoriais realizados com organismos aquáticos com base nos princípios dos 3 R. Foram realizadas revisões de 20 artigos sobre ecotoxicologia aquática, publicados no período de 2010 a 2014. A análise verificou o número de animais utilizados nos ensaios ecotoxicológicos, aclimatação, aprovação da pesquisa por comitê de ética e uso de anestésicos. Os resultados indicam que os princípios de refinamento e substituição, do programa dos 3R, foram os que mais ocorreram nas pesquisas, porém a maioria delas não citou aprovação dos protocolos experimentais por um comitê de ética nem o uso de anestésicos nos organismos durante os experimentos. As pesquisas publicadas indicam a necessidade de redução do número de organismos nos bioensaios, bem como a aplicação dos princípios bioéticos na experimentação realizada com organismos aquáticos.

Ecotoxicologia; Experimentação animal; Bioética; Ética-Animais

La ecotoxicología acuática analiza los efectos adversos de los contaminantes químicos en la biota, utilizando animales en las pruebas de laboratorio. En este trabajo se tuvo como objetivo analizar la adecuación de las pruebas de laboratorio realizados con organismos acuáticos basadas en los principios de las 3Rs. Se realizaron revisiones en 20 artículos sobre Ecotoxicología acuática, publicados en el periodo de 2010 a 2014. Se verificó el número de animales utilizados en las pruebas ecotoxicológicas y en la aclimatación. La investigación fue aprobada por el comité de ética y para la utilización de anestésicos. Los resultados indican que los principios Refinamiento y Sustitución, del programa de las 3Rs, fueron los más encontrados en los estudios, pero que la mayoría no citó aprobación de los protocolos experimentales por un comité de ética, ni para el uso de anestésicos en los organismos durante los experimentos. Las investigaciones publicadas indican la necesidad de reducir el número de organismos en las biopruebas, así como la aplicación de los principios de la bioética en la experimentación realizada con los organismos acuáticos.

Ecotoxicología; Experimentación animal; Discusiones bioéticas; Ética animal

Ecotoxicology describes the relationship between chemical pollutants, the environment in which they are released and organisms that live there. Further studies of these substances are necessary in order to minimise their potential risks and to put in practice cleansing measures to attenuate their likely impacts ¹. So, ecotoxicology is presented as a tool to analyse the exposure of various xenobiotics to the environment where they were inserted ² and also the adverse effects of exogenous pollutants in the environment and aquatic organisms ³.

The ecotoxicological assessment is extremely important in the control, regulation and classification of toxic substances regarding their potential environmental risk. The identification of the effects and the evaluation of the dose-response relationship are initial steps in the process of environmental risk analysis ⁴. Many tests are also performed with the aim to evaluate both the relative sensitivity of aquatic organisms in relation to a specific toxic agent as well as the efficiency of different methods of treatment of industrial effluents⁵.

Bioethics requires the combination of various disciplines, aimed at the proper use of the knowledge acquired by science as well as ethical values ​​and biological facts. The first and best-known theoretical line of thought, the principlist bioethics , is guided by the principles of beneficence, nonmaleficence, autonomy and justice⁶. The beneficence is defined as the analysis of the benefits to the individual participant of the research and, subsequently, the benefits to society; nonmaleficence is characterised by the prevention of damage during the course of the study, which could affect the subject of the research ; autonomy is defined as respect for the individual's capacity for choice ; and justice is related to an equitable distribution of benefits among the beings⁷. In relation to the principles proposed by the Universal Declaration on Bioethics and Human Rights

(abbreviated as DUBDH in Brazil)⁸, benefit and harm refer to the maximisation of benefits and minimisation of harm to individuals of the study and others involved; and protection of the environment, the biosphere and the biodiversity, characterised by an understanding of the interdependence between human beings and other life forms.

In many cases, the principles of bioethics - which include, in addition to the principlism theory and the Universal Declaration on Bioethics and Human Rights 8, the ideas of the 3 R, proposed by scholars Russell and - are not observed when working with most organisms used in procedures, or the information is not cited in the scientific articles published. In addition, the bioethical discussions propose several alternative methodologies, which development and implementation in institutions which use animals in scientific studies are a complex and extensive process ¹⁰.

The use of animals in research has been the cause of various bioethical discussions focused on the large number of animals and the suffering caused to them. The program of the 3 Rs - reduction, refinement and replacement - aims to decrease the number of animals, minimise their pain, discomfort, and provide alternatives for the replacement of in vivo studies¹⁰. And, based on the 3 Rs principles, this work aimed to analyse the adequacy of laboratory tests performed with aquatic organisms in articles published between 2010 and 2014.

Bioethics and its principles

The term "bioethics" was disseminated by the American biologist and oncologist Van Rensselaer Potter in his book "Bioethics: Bridge to the Future" ¹¹. This new field of study was created with the objective of enabling biological sciences to guarantee and improve the quality of life of future generations ¹². The use of animals in scientific experiments increased with the advancement of technologies, making it necessary to expand the ethical discussions about these researches. As already mentioned, the zoologist William M. S. Russell, together with the microbiologist Rex L. Burch, proposed the 3 Rs principles in 1959, known as replacement , reduction and refinement ¹³. These principles redirected the scientific work involving animal research to the use of alternative models in order to reduce not only the use of animals in experiments, but also the pain and suffering that the tests caused to them ¹⁴.

Historically, bioethics discussion was more closely linked to medical settings,so much that, initially, the formulation of the guiding ethical principles of scientific activity occurred more in function of studies involving human beings. This conception, later known as principlism theory, is based on four principles: nonmaleficence, beneficence, justice and respect for autonomy ¹⁵. The nonmaleficence means not to harm or to prevent harm to living beings; beneficence refers to the realisation of good and maximum reduction of harm that one can cause to another, recognising their moral values; justice has equity as the main axis, that is, all must be attended in the same way, and respect for autonomy is described as the acceptance of self-determination before the situations of the environment¹⁶, understanding autonomy as the capacity for choice of the individual ⁷.

Another milestone in connection with the organisation and guidance of bioethical matters was the Universal Declaration on Bioethics and Human Rights, adopted in October 2005 by the United Nations Educational, Scientific and Cultural Organisation (UNESCO) ⁸. There are principles, in the document, such as human dignity and human rights, autonomy and individual responsibility, consent, privacy and confidentiality, social responsibility and health. In addition, it is worth noting another two norms provided by the Universal Declaration on Bioethics and Human Rights, which are of particular interest for scientific research: benefit and harm, referring to the maximisation of benefits and minimisation of harm to the research participants and others involved; protection of the environment, the biosphere and the biodiversity, which assumes the interdependence of human subjects with other forms of life.

These principles are much more often applied to studies related to human beings than to animals of different taxonomic categories, which is why there are still many ethical issues to be discussed, among them is included one that is quite relevant to this article: Can these principles of bioethics be applied to all groups of organisms?

Aquatic ecotoxicology studies and bioethics

With the high demand for new technological products and the continued destruction of natural resources by human activities, many xenobiotics have been incorporated into the environment through direct or indirect ways, be them point or diffuse sources ¹⁷. Studies on these beings can be conducted directly in the field or through tests in laboratory (experimental tests with distinct methodologies), regulated by national and international standards on the use of bioindicators organisms in such tests ¹⁸. The aquatic organisms are usually used in ecotoxicological tests for various reasons ¹⁹, among them their physiological processes, such as nutrition and breathing, as many of them breathe through gills in direct contact with water, which can carry many contaminants into the interior of these organisms, causing morphophysiological changes in them ²⁰.

To identify these morphophysiological changes (biomarkers) in aquatic species , the use of laboratory techniques able to assess the degree of contamination on an area for a certain pollutant is necessary ²¹. . In these bioassays, one can check damage to organisms (that die or keep still), estimating the extent of their impact on the environment and human beings ²². . The organisms used in these studies are fish and benthic macroinvertebrates, such as, for example, crustaceans from the gender Daphnia, that are easily grown in situ and exhibit reproductive characteristics that meet the purpose of these studies ²³. In the case of fish, among the species considered an international standard of reference is the Danio rerio (zebrafish), mainly because its genome sequence is similar to the genome sequence of other vertebrates, including human beings ²⁴.

Currently, the scientific community has turned to the ethical aspects of research with animals of the phylum Chordata (subphylum Vertebrata), for which there is specific Brazilian legislation ²⁵. There are also specific ethics committees that evaluate studies with insects and cephalopods, but its acting is still limited, since the current legislation gives priority to sentient organisms, which means vertebrate animals, whose sentience is widely recognised in the scientific community²⁶ Now, insentient organisms, among which are included the invertebrates, do not receive the same protection intended to sentient beings ²⁷.

Most of the time, researches involving ecotoxicology tests (with invertebrates or with vertebrates) occur without approval of ethics committees. Moreover, many researchers of the aquatic ecotoxicological field still have little contact with bioethics, either in their initial training or in their laboratory work. This situation needs to be addressed and changed so we can have a positive influence on the results of researches that get published and which serve as reference to new works in the area.

Given what was shown here, the aim of this study was to analyse the adequacy of laboratory tests performed with aquatic organisms on articles published from 2010 to 2014, based on the principles of the 3 Rs. In this sense, 20 scientific articles published in 16 different journals were analysed, which address ecotoxicological studies with animals of different taxonomic categories, aimed at the identification of the most widespread bioethical principles applied to animal testing among researchers.

Methodological path

This study is a literature review which data had been gathered from scientific articles about the procedures performed in aquatic ecotoxicological tests realised between the years 2010 and 2014. We analysed 20 articles, which were systematically searched in databases registered at the Coordination of Training of Higher Education Personnel ( abbreviated as “Capes” in Brazil) and from the Scientific Electronic Library Online (SciELO), containing the following descriptors: "ecotoxicology"; "Ecotoxicological studies"; "Bioethics"; "Ethics".

First, for the organisation of data, we read the summaries of 20 selected scientific publications in order to identify the objects of study of the works, prioritising ecotoxicological tests ^28,29. At least one of the bioethic principles was observed in all 20 studies. The technique used by the authors for the analysis of content was based on the reading of scientific papers chosen by examining the key information such as number of animals used in ecotoxicological testing, development phase and acclimatisation, origin of organisms (in situor ex situ) and research approved by an ethics committee. According to the content analysis of each work, we checked the adequacy of such data to the principles of the 3Rs (Replacement, reduction and refinement) ¹³. The refinement was analysed taking as base the acclimatisation, the research approved by an ethics committee, methodological protocols of the ABNT (NBR 12713/2009 ³⁰ e 13373/2010 ³¹), (ABNT- abbreviation in portuguese of Brazilian Association of Technical Standards), the use of anaesthetics. The identification technique of the bioethical principles of William M. S. Russell and Rex L. Burch 9 was the analysis of content “ thematic-categorial”, involving the following phases: pre-analysis; exploitation of material or codification; treatment of results; inference and interpretation. Statistical softwares were used in order to obtain charts and percentages for organisation of data related to scientific production. The 20 works are listed in Table 1, below.

Table 1
Articles published between 2010 and 2014 and used in the bibliographic revision of this study

Nº	Authors	Name of the Article	Magaazine	Year
1	Rechenmacher C, Siebel AM, Goldoni A, Klauck CR, Sartori T, Rodrigues MT, Rodrigues MAS, Gehlen G, Ardenghi PG, Silva LB 28	A multibiomarker approach in rats to assess the impact of pollution on Sinos River, Southern Brazil	Brazilian Journal of Biology	2010
2	Nepomuceno JC, Silva AC 32	Assessment of the frequency of peripheral micronuclei in erythrocytes of the mandi-amarelo (Pimelodus maculatus) in the Paranaíba river	Perquirere: Revista do Núcleo Interdisciplinar de Pesquisa e Extensão do Unipam	2010
3	Brandolt TD, Lobo E 33	Ecotoxicological assessment using bioassays with Daphnia magna in rice paddy’s water in the county of Mostardas, RS, Brazil	Tecno-Lógica	2010
4	Barbola IF, Moraes MFPG, Anazawa TM, Nascimento EA, Sepka ER, Polegatto CM, Milléo J, Schühli GS34	Assessment of aquatic macroinvertebrate communities as a tool for the monitoring of a reservoir in the Pitangui river basin, Paraná, Brazil	Iheringia, Série Zoologia	2011
5	Morais AV, Cortez FS, Bretas ES, Cesar E, Pereira CDS, Santos AR 35	Assessment of the environmental quality of the Itaguaré river, Bertioga-SP, based on toxicity tests and microbiologic indicators of current water quality	O Mundo da Saude	2011
6	Mendes BG, Budziak D, Stolberg J, Peixer ZI, Dalmarco JB, Simionatto EL, Pedrosa RC, Felipe KB, Ogawa J, Pegoraro C, Scheffer L, Besen MR, Oliveira LJGG, Geremias R36	Study of the water quality of the Marombas river (SC/Brasil), using physicochemical parameters and bioassays	Revista de Ciências Ambientais	2011
7	Holanda JN, Maciel AP, Santos RL37	Ecotoxicological assessment, using Danio rerio, of wash water from the purification of methyl soybean biodiesel	Boletim do Laboratório de Hidrobiologia	2012
8	Damato M, Barbieri E 38	Study of the acute toxicity and metabolic alterations caused by the exposition of the fishHyphessobrycon callistus, used as an indicator of environmental healthto cadmium	O Mundo da Saúde	2012
9	Mannarino CF, Moreira JC, Ferreira JA, Arias ARL 39	Assessment of the impact , on the aquatic biota, of the combined effluent of the treatment of landfill leachate from municipal solid waste and sewage	Ciência & Saúde Coletiva	2013
10	Mohr G, Lobo EA 40	Assessment, using bioassays of the efficiency of a water treatment system in a small rural property	Revista Jovens Pesquisadores	2013
11	Gaspar N, Gago J, Repolho T, Luís O41	Assessment of the toxicity of lime mud on the sea urchin Paracentrotus lividus (Lamarck, 1816). Perspectives for new applications of this industrial waste	Revista da Unidade de Investigação do Instituto Politécnico de Santarém	2013
12	Hernandez OD, Castro F, Paez M42	Bioacumulación de mercurio en larvas de anuros en la zona afectada por la minería de oro en el río Dagua, Buenaventura, Valle del Cauca, Colombia	Acta Biológica Colombiana	2013
13	Canto T, Pich CT, Geremias R 43	Bioassays of toxicity in leachate in the landfill of the county of de Araranguá (Santa Catarina, Brasil)	Revista Biociências	2013
14	Sousa DBP, Almeida ZS, Carvalho-Neta RNF44	histological biomarkers in two species of estuary catfish from the maranhense litoral ,Brazil	Arquivo Brasileiro de Medicina Veterinária e Zootecnia	2013
15	Jacobowski AC, Zobiole NN, Padilha PM, Moreno SE, Arruda EJ 45	Mutagenic effect of copper edetate ([Cu (EDTA)] - 2) ,free and nano encapsulated, in mice and fish	Ecotoxicology and Environmental Contamination – EEC	2013
16	Rezende KFO, Santos RM, Santos RM, Shimada JCB, Silva JRMC, Muniz CASD 46	Histopathology of the gills of Nile tilapiaOreochromis niloticus, from the Billings dam in the Area of environmental protection Bororé-Colônia	Atlas de Saúde Ambiental – ASA	2013
17	Pereira DP, Santos DMS, Carvalho Neta AV, Cruz CF, Carvalho Neta RNF 47	Morpholocigal alterations in the gills ofOreochromis niloticus(Pisces,Cichlidae) as biomarkers of aquatic pollution in the Jansen Lagoon, São Luís, MA (Brazil)	Bioscience Journal	2014
18	Benites LM, Doncato KB, Minho TS, Perazzo GX48	Assessment of the mutagenic potential of copper compounds in the water of the Uruguay river	Ciência e Natura	2014
19	Franco-Bernardes MF, Maschio LR, Azeredo-Oliveira MTV, Almeida EA 49	Biochemical and genotoxic effects of a commercial formulation of the herbicide tebuthiuron inOreochromis niloticus of different sizes	Ecotoxicology and Environmental Contamination – EEC	2014
20	Nour EAA, Candello FP, Santos EMR, Barretto AS, Domingues LM 29	Biologic treatment of formaldehyde: residual toxicity monitored by bioassays with Daphnia similis	Ecotoxicology and Environmental Contamination – EEC	2014

Use of principles of bioethics in ecotoxicological test

The data obtained in the analysis of the scientific papers listed above show that there is some concern of the authors with the program of the 3 Rs , since most of the work had some kind of information related to the care of animals used in ecotoxicological studies. According to this program, the concepts found were refinement, reduction and replacement (Figure 1), of which the first two appeared more frequently.

Figure 1
Distribution of the 3 Rs in relation to analysed scientific articles

Regarding the number of animals used in the ecotoxicological procedures, 4 articles did not mention the amount of animals, but other studies (16) presented that information. Most of the studies analysed (14) used between 11 and 50 individuals in the researches; however, four studies used between 300 and 18,473 specimens. Only one scientific paper used up to 10 individuals, adapting to the "reduction" principle of the 3Rs program⁵⁰. The large number of organisms used in a scientific experiment can be explained by the absence of legislation that limits the use of invertebrates in scientific studies ²⁴, given that these studies were conducted with aquatic and semi-aquatic macroinvertebrates. These kinds of study using thousands of individuals do not yet include the program of the 3 R ^29,50.

To date, the concern with ethical aspects focuses on tests with animals in the phylum Chordata, subphylum Vertebrata. There are some groups and ethics committees which also evaluate studies on insects and cephalopods. This fact, however, is still the exception. As a result, organisms lower in the zoological scale, which are classified as unprotected, can also be used instead of vertebrates, characterising the "replacement" principle. One example is the use of Daphnia pulgans (water flea), Artemia salina (brine shrimp)and bees in studies, mainly of ecotoxicology ⁵¹. Thus, 30% of the studies analysed used species such as Daphnia similis (water flea), Daphnia magna (freshwater water flea), Ceriodaphnia dubia (freshwater water flea) and Artemia sp. (brine shrimp) and these were assessed as important in the ”replacement" principle of the 3 R. program.

Just over half of the analysed studies (53%) reported that the acclimatisation of organisms was performed before the ecotoxicological procedures , indicating that the principle of refinement was observed, since the animals were kept in conditions that minimised their pain and stress ⁵². This type of procedure is, moreover, provided by Brazilian law ⁵³. The acclimatisation is characterised by long-lasting adaptive physiological adjustments of organisms that result in increased tolerance to continuous or repeated exposure to multiple stressors, usually realised under field conditions⁵⁴. The acclimatisation and the reduction or alleviation of pain are considered improvements in animal care, since researchers must know how to evaluate and monitor the pain in order to provide ethical treatment to animals ⁵⁵. In addition to ethical concerns, the pain and stress of the animals lead to undesirable variables in the study, as they can significantly interfere with the interpretation of the study’s results ⁵⁶.

The use of anaesthesia was reported in 15% of the analysed articles, indicating that the principle of refinement was used, since the technique provides decreased suffering of organisms used in experiments ⁵². . During the practice of some of the laboratory procedures, the animals are subjected to various types of pain and suffering; therefore, they must be sedated and anaesthetised properly ^57,58.

It is important to highlight that 20% of the scientific work carried outin situ collected peripheral blood to be smeared on microscope slides, and then the animals were returned to their habitat. This kind of research - which return all organisms to the environment , benefiting both researchers and the animals researched - are desirable because they decrease the impacts on the local wildlife and produce more reliable information on the real situation of the natural habitat of the species studied, which makes it possible to work even with mammals and endangered species ⁵⁹.

Very few articles (20%) mentioned the approval of their experimental protocol by an institutional ethics committee. The vast majority of the studies (80%) did not report such approval, and did not even point out that the research involved larvae of species that are classified in the category of "least concern" by the International Union for Conservation of Nature (IUCN), and only 25% mentioned the use of protocols standardised by the ABNT (NBR 12713/2009 ³⁰ e 13373/2010 ³¹). Scientific studies involving animals should be analysed by an ethics committee, whose role is to guide and review protocols, and increase knowledge about proper moral conduct towards animals ⁶⁰. It is important that the experimental protocol is analysed regarding the aspect of the methodology used, in order to identify the impacts caused to animals regarding pain, suffering, confinement, stress and death ¹⁴.

Researchers have the responsibility to submit their research protocols to an Ethics Committee that guarantees the possibility to adapt best practice guidelines which ensure the welfare of organisms subjected to a study. The use of animal species in different contexts (from ecotoxicological tests to animal production and population management within the scope of conservation programs) deserves the observation of the principles of bioethics⁶¹, so that science can evolve and respect all forms of life on the planet.

Considerações finais

The discussion on methodologies and innovative concepts introduced in scientific research with animals has been increasing in scientific publications in recent years. However, in published works containing results of ecotoxicological tests on aquatic environments, there are still several issues that need to be debated, such as the concrete need for approval of experimental protocols by ethics committees; assurance that bioethical principles are coupled up to laboratory methodologies; use of anaesthesia to minimise animal suffering; reduction of the number of organisms in laboratory tests, and adoption of alternative methods in the use of animals, without necessarily affecting the result of the research. To discuss bioethical ideas is fundamental, and the principles of the 3 Rs should be considered in the preparation of projects and in current scientific research practices in the aquatic ecotoxicology area.

Referências

• ¹
  Stützer G, Guimarães G. Aspectos toxicológicos e ambientais relacionados com o uso de produtos fitossanitários. In: Zambolim L, Conceição MZ, Santiago T, organizadores. O que os engenheiros agrônomos devem saber para orientar o uso de produtos fitossanitários. Viçosa: UFV; 2003. p. 69-84.
• ²
  Carvalho NL, Pivoto TS. Ecotoxicologia: conceitos, abrangência e importância agronômica. Monografias Ambientais. [Internet]. 2011 [acesso 6 fev 2015];2(2):176-92. Disponível: http://cascavel.ufsm.br/revistas/ojs-2.2.2/index.php/remoa/article/view/2315/1598
  » http://cascavel.ufsm.br/revistas/ojs-2.2.2/index.php/remoa/article/view/2315/1598
• ³
  Lombardi JV, Ferreira CM, Rodrigues EL. Toxicologia aquática. In: Ranzani-Paiva MJT, Takemoto RM, Lizama MAP, organizadores . Sanidade de organismos aquáticos. São Paulo: Varela; 2004. p. 262-97.
• ⁴
  United States Environmental Protection Agency. Short-term methods for estimating the toxicity of effluents and receiving waters to freshwater and marine organisms. [Internet]. 4ª ed. Washington: EPA; 2002 [acesso 6 fev 2015]. Disponível: http://water.epa.gov/scitech/methods/cwa/wet/upload/2007_07_10_methods_wet_disk3_ctf1-6.pdf
  » http://water.epa.gov/scitech/methods/cwa/wet/upload/2007_07_10_methods_wet_disk3_ctf1-6.pdf
• ⁵
  Metcalf L, Eddy HP. Wastewater engineering: treatment in reuse. [Internet]. 4ª ed. Boston: McGraw Hill; 2003 [acesso 6 fev 2015]. Disponível: http://www.mumbaidp24seven.in/reference/Ch_1WastewaterEngineering4thed_byMetcalfandEddy.pdf
  » http://www.mumbaidp24seven.in/reference/Ch_1WastewaterEngineering4thed_byMetcalfandEddy.pdf
• ⁶
  Schatzmayr HG, Müller CA. As interfaces da bioética nas pesquisas com seres humanos e animais com a biossegurança. Ciênc Vet Tróp. [Internet]. 2008 [acesso 6 fev 2015];11(Supl 1):130-4. Disponível: http://www.rcvt.org.br/suplemento11/130-134.pdf
  » http://www.rcvt.org.br/suplemento11/130-134.pdf
• ⁷
  Silva JACD, Teixeira RKC, Monma CA, Neotti T. Perfil bioético dos anteprojetos enviados ao comitê de ética em pesquisa da Universidade do Estado do Pará. Rev. bioét. (Impr.). [Internet]. 2011 [acesso 6 fev 2015];19(2):563-75. Disponível: http://revistabioetica.cfm.org.br/index.php/revista_bioetica/article/view/645/673
  » http://revistabioetica.cfm.org.br/index.php/revista_bioetica/article/view/645/673
• ⁸
  Organização das Nações Unidas para a Educação, Ciência e Cultura (Unesco). Declaração Universal sobre Bioética e Direitos Humanos. [Internet]. Paris: Unesco; 2006 [acesso 17 mar 2015]. Disponível: http://unesdoc.unesco.org/images/0014/001461/146180por.pdf
  » http://unesdoc.unesco.org/images/0014/001461/146180por.pdf
• ⁹
  Russell WMS, Burch RL. The principles of humane experimental technique. Londres: Ufaw ; 1992.
• ¹⁰
  Cazarin KCC, Corrêa CL, Zambrone FAD. Redução, refinamento e substituição do uso de animais em estudos toxicológicos: uma abordagem atual. Revista Brasileira de Ciências Farmacêuticas – RBCF. [Internet]. 2004 [acesso 6 fev 2015];40(3):289-99. Disponível: http://www.scielo.br/pdf/rbcf/v40n3/04.pdf
  » http://www.scielo.br/pdf/rbcf/v40n3/04.pdf
• ¹¹
  Potter VR. Bioethics: bridge to the future. Upper Saddle River: Prentice Hall; 1971.
• ¹²
  Clotet J. Por que bioética? Revista Bioética. [Internet]. 1993 [acesso 6 fev 2015];1(1):13-9. Disponível: http://www.revistabioetica.cfm.org.br/index.php/revista_bioetica/article/view/474/291
  » http://www.revistabioetica.cfm.org.br/index.php/revista_bioetica/article/view/474/291
• ¹³
  Franco AL, Nogueira MNM, Guimarães KS, Frota MF, Fernandes CMS, Sena MC. Animal research: a bioethical reflection. Acta bioeth. [Internet]. 2014 [acesso 6 fev 2015];20(2):247-53. Disponível: http://www.scielo.cl/scielo.php?script=sci_abstract&pid=S1726-569X2014000200012&lng=es&nrm=iso&tlng=en
  » http://www.scielo.cl/scielo.php?script=sci_abstract&pid=S1726-569X2014000200012&lng=es&nrm=iso&tlng=en
• ¹⁴
  Feijó AGS, Sanders A, Centurião AD, Rodrigues GS, Schwanke CHA. Análise de indicadores éticos do uso de animais na investigação científica e no ensino em uma amostra universitária da área da saúde e das ciências biológicas. Sci Med. [Internet]. 2008 [acesso 6 fev 2015];18(1):10-9: Disponível: http://revistaseletronicas.pucrs.br/ojs/index.php/scientiamedica/article/viewFile/2234/2807
  » http://revistaseletronicas.pucrs.br/ojs/index.php/scientiamedica/article/viewFile/2234/2807
• ¹⁵
  Hossne WS. Bioética: princípios ou referenciais. Mundo Saúde. [Internet]. 2006 [acesso 6 fev 2015];30(4):673-6. Disponível: http://www.saocamilo-sp.br/pdf/mundo_saude/41/20_bioetica_principio.pdf
  » http://www.saocamilo-sp.br/pdf/mundo_saude/41/20_bioetica_principio.pdf
• ¹⁶
  Koerich MS, Machado RR, Costa E. Ética e bioética: para dar início à reflexão. Texto Contexto Enferm. [Internet]. 2005 [acesso 6 fev 2015];14(1):106-10. Disponível: http://www.scielo.br/pdf/tce/v14n1/a14v14n1.pdf
  » http://www.scielo.br/pdf/tce/v14n1/a14v14n1.pdf
• ¹⁷
  Andréa MM. O uso de minhocas como bioindicadores de contaminação de solos. Acta Zoológica Mexicana. [Internet]. 2010 [acesso 6 fev 2015];26(número especial 2):95-107. Disponível: http://www.scielo.org.mx/pdf/azm/v26nspe2/v26nspe2a7.pdf
  » http://www.scielo.org.mx/pdf/azm/v26nspe2/v26nspe2a7.pdf
• ¹⁸
  Torres GA, González SP, Peña ES. Descripción anatomica, histológica y ultraestructural de la branquia e hígado de tilapia (Oreochromis niloticus). Int J Morphol. [Internet]. 2010 [acesso 6 fev 2015];28(3):703-12. Disponível: http://www.scielo.cl/scielo.php?pid=S0717-95022010000300008&script=sci_arttext
  » http://www.scielo.cl/scielo.php?pid=S0717-95022010000300008&script=sci_arttext
• ¹⁹
  Montes CS, Ferreira MAP, Santos SSD, Von Ledebur EICF, Rocha RM. Branchial histopathological study of Brachyplatystoma rousseauxii (Castelnau, 1855) in the Guajará bay, Belém, Pará State, Brazil. Acta Scientiarum: Biological Sciences. [Internet]. 2010 [acesso 6 fev 2015];32(1):93-99. Disponível: http://eduem.uem.br/ojs/index.php/ActaSciBiolSci/article/viewFile/4800/5085
  » http://eduem.uem.br/ojs/index.php/ActaSciBiolSci/article/viewFile/4800/5085
• ²⁰
  Cantanhêde SM, Medeiros AM, Ferreira FS, Ferreira JRC, Alves LMC, Cutrim MVJ et al. Uso de biomarcador histopatológico em brânquias deCentropomus undecimalis (Bloch, 1972) na avaliação da qualidade da água do Parque Ecológico Laguna da Jansen, São Luís-MA. Arq Bras Med Vet Zootec. [Internet]. 2014 [acesso 6 fev 2015];66(2):593-601. Disponível: http://www.scielo.br/scielo.php?pid=S0102-09352014000200037&script=sci_arttext
  » http://www.scielo.br/scielo.php?pid=S0102-09352014000200037&script=sci_arttext
• ²¹
  Costa CR, Olivi P, Botta CMR, Espíndola ELG. A toxicidade em ambientes aquáticos: discussão e métodos de avaliação. Quim Nova. [Internet]. 2008 [acesso 6 fev 2015];31(7):1820-30. Disponível: http://www.scielo.br/pdf/qn/v31n7/v31n7a38.pdf
  » http://www.scielo.br/pdf/qn/v31n7/v31n7a38.pdf
• ²²
  Linde-Arias AR, Inácio AF, Novo LA, Albuquerque C, Moreira JC. Multibiomarker approach in fish to assess the impact of pollution in a large brazilian river, Paraiba do Sul. Environmental Pollution. [Internet]. 2008 [acesso 6 fev 2015];156(3):974-9. Disponível: http://ac.els-cdn.com/S0269749108002522/1-s2.0-S0269749108002522-main.pdf?_tid=5807f0ee-ae40-11e4-ac4e-00000aacb360&acdnat=1423255390_213d451feb2b30287c588875e3bdd976
  » http://ac.els-cdn.com/S0269749108002522/1-s2.0-S0269749108002522-main.pdf?_tid=5807f0ee-ae40-11e4-ac4e-00000aacb360&acdnat=1423255390_213d451feb2b30287c588875e3bdd976
• ²³
  Domingos, FXV. Biomarcadores de contaminação ambiental em peixes e ostras de três estuários brasileiros e cinética de derivados solúveis do petróleo em peixes [tese]. Curitiba: Universidade Federal do Paraná; 2006.
• ²⁴
  Oliveira EM, Goldim JR. Legislação de proteção animal para fins científicos e a não inclusão dos invertebrados: análise bioética. Rev. bioét. (Impr.). [Internet]. 2014 [acesso 6 fev 2015];22(1):45-56. Disponível: http://www.scielo.br/pdf/bioet/v22n1/a06v22n1.pdf
  » http://www.scielo.br/pdf/bioet/v22n1/a06v22n1.pdf
• ²⁵
  Brasil. Lei nº 11.794, de 8 de outubro de 2008. Procedimentos para o uso científico de animais. Diário Oficial da União. 10 set 2008;145(196): Seção 1, p. 1-2.
• ²⁶
  Presgrave OAF. The need for the establishment of a Brazilian Centre for the Validation of Alternative Methods (BraCVAM). Altern Lab Anim. [Internet]. 2008 [acesso 6 fev 2015];36(6):705-8. Disponível: http://www.incqs.fiocruz.br/images/stories/incqs/bracvam/ATLA_BraCVAM_2008.pdf
  » http://www.incqs.fiocruz.br/images/stories/incqs/bracvam/ATLA_BraCVAM_2008.pdf
• ²⁷
  Luna SPL. Dor, senciência e bem-estar em animais: senciência e dor. Revista Ciência Veterinária nos Trópicos. [Internet]. 2008 [acesso 6 fev 2015];11(Supl 1):17-21. Disponível: http://www.rcvt.org.br/suplemento11/17-21.pdf
  » http://www.rcvt.org.br/suplemento11/17-21.pdf
• ²⁸
  Rechenmacher C, Siebel AM, Goldoni A, Klauck CR, Sartori T, Rodrigues M T et al. A multibiomarker approach in rats to assess the impact of pollution on Sinos River, Southern Brazil. Braz J Biol. [Internet]. 2010 [acesso 6 fev 2015];70(4 Supl):1223-30. Disponível: http://www.scielo.br/scielo.php?pid=S1519-69842010000600012&script=sci_arttext
  » http://www.scielo.br/scielo.php?pid=S1519-69842010000600012&script=sci_arttext
• ²⁹
  Nour EAA, Candello FP, Santos EMR, Barretto, AS, Domingues LM. Tratamento biológico de formaldeído: toxicidade residual monitorada por bioensaios com Daphnia similis Ecotoxicology and Environmental Contamination. [Internet]. 2014 [acesso 6 fev 2015];9(1):77-85. Disponível: http://siaiweb06.univali.br/seer/index.php/eec/article/view/4557/3647
  » http://siaiweb06.univali.br/seer/index.php/eec/article/view/4557/3647
• ³⁰
  Associação Brasileira de Normas Técnicas. NBR 12713: ecotoxicologia aquática, toxicidade aguda: método de ensaio com Daphnia spp (Cladocera, Crustacea). Rio de Janeiro: ABNT; 2009.
• ³¹
  Associação Brasileira de Normas Técnicas. NBR 13373: ecotoxicologia aquática, toxicidade crônica: método de ensaio comCeriodaphnia spp(Crustacea,Cladocera). Rio de Janeiro: ABNT; 2010.
• ³²
  Nepomuceno JC, Silva AC. Avaliação da frequência de micronúcleos em eritrócitos periféricos de mandi-amarelo (Pimelodus maculatus) do rio Paranaíba. Perquirere. [Internet]. 2010 [acesso 6 fev 2015];7(1):167-79. Disponível: http://perquirere.unipam.edu.br/documents/23456/36602/Avaliacao_da_frequencia_de_micronucleos_em_e_ritrocitos.pdf
  » http://perquirere.unipam.edu.br/documents/23456/36602/Avaliacao_da_frequencia_de_micronucleos_em_e_ritrocitos.pdf
• ³³
  Brandolt TD, Lobo EA. Avaliação ecotoxicológica utilizando bioensaios com Daphnia magna em água de lavoura de arroz no município de Mostardas, RS, Brasil. Tecno-Lógica. [Internet]. 2010 [acesso 6 fev 2015];14(2):47-51. Disponível: https://online.unisc.br/seer/index.php/tecnologica/article/view/1580/1147
  » https://online.unisc.br/seer/index.php/tecnologica/article/view/1580/1147
• ³⁴
  Barbola IF, Moraes MFPG, Anazawa TM, Nascimento EA, Sepka ER, Polegatto CM et al. Avaliação da comunidade de macroinvertebrados aquáticos como ferramenta para o monitoramento de um reservatório na bacia do rio Pitangui, Paraná, Brasil. Iheringia, Sér Zool. [Internet]. 2011 [acesso 6 fev 2015];101(1-2):15-23. Disponível: http://www.scielo.br/pdf/isz/v101n1-2/v101n1-2a02.pdf
  » http://www.scielo.br/pdf/isz/v101n1-2/v101n1-2a02.pdf
• ³⁵
  Morais AV, Cortez FS, Bretas ES, Cesar A, Pereira CDS, Santos AR. Avaliação da qualidade ambiental do rio Itaguaré, Bertioga-SP, com base em testes de toxicidade e indicadores microbiológicos de balneabilidade. Mundo Saúde (Impr.). [Internet]. 2011 [acesso 6 fev 2015];35(1):55-63. Disponível: http://saocamilo-sp.br/pdf/mundo_saude/83/55a63.pdf
  » http://saocamilo-sp.br/pdf/mundo_saude/83/55a63.pdf
• ³⁶
  Mendes BG, Budziak D, Stolberg J, Peixer ZI, Dalmarco JB, Simionatto EL et al. Estudo da qualidade das águas do rio Marombas (SC/Brasil), utilizando parâmetros físico-químicos e bioensaios. Revista de Ciências Ambientais. [Internet]. 2011 [acesso 7 fev 2015];5(2):43-58. Disponível: http://revistas.unilasalle.edu.br/index.php/Rbca/article/view/260/252
  » http://revistas.unilasalle.edu.br/index.php/Rbca/article/view/260/252
• ³⁷
  Holanda JN, Maciel AP, Santos RL. Avaliação ecotoxicológica da água de lavagem da purificação de biodiesel de soja metílico utilizandoDanio rerio como organismo-teste. Boletim do Laboratório de Hidrobiologia. [Internet]. 2012 [acesso 6 fev 2015];25(1):13-20. Disponível: http://www.periodicoseletronicos.ufma.br/index.php/blabohidro/article/viewFile/1924/73
  » http://www.periodicoseletronicos.ufma.br/index.php/blabohidro/article/viewFile/1924/73
• ³⁸
  Damato M, Barbieri E. Estudo da toxicidade aguda e alterações metabólicas provocadas pela exposição do cádmio sobre o peixeHyphessobrycon callistus utilizado como indicador de saúde ambiental. Mundo Saúde (Impr.). [Internet]. 2012 [acesso 6 fev 2015];36(4):574-81. Disponível: http://bvsms.saude.gov.br/bvs/artigos/mundo_saude/estudo_toxicidade_aguda_alteracoes_metabolicas.pdf
  » http://bvsms.saude.gov.br/bvs/artigos/mundo_saude/estudo_toxicidade_aguda_alteracoes_metabolicas.pdf
• ³⁹
  Mannarino CF, Moreira JC, Ferreira JA, Arias ARL. Avaliação de impactos do efluente do tratamento combinado de lixiviado de aterro de resíduos sólidos urbanos e esgoto doméstico sobre a biota aquática. Ciênc Saúde Coletiva. [Internet]. 2013 [acesso 6 fev 2015];18(11):3235-43. Disponível: http://www.scielo.br/pdf/csc/v18n11/14.pdf
  » http://www.scielo.br/pdf/csc/v18n11/14.pdf
• ⁴⁰
  Mohr G, Lobo EA. Avaliação da eficiência de um sistema de tratamento da água em pequena propriedade rural, utilizando bioensaios. Revista Jovens Pesquisadores. [Internet]. 2013 [acesso 6 fev 2015];3(1):12-22. Disponível: http://online.unisc.br/seer/index.php/jovenspesquisadores/article/view/3752/2660
  » http://online.unisc.br/seer/index.php/jovenspesquisadores/article/view/3752/2660
• ⁴¹
  Gaspar N, Gago J, Repolho T, Luís O. Avaliação da toxicidade das lamas de cal sobre o ouriço-do-mar Paracentrotus lividus (Lamark, 1816). Perspectivas para novas aplicações destes resíduos industriais. Revista da UIIPS. [Internet]. 2013 [acesso 9 fev 2015];1(2):82-102. Disponível: http://www.ipsantarem.pt/wp-content/uploads/2013/06/Revista-da-UIIPS_N2_Vol1_2013_ISSN-2182-9608.pdf
  » http://www.ipsantarem.pt/wp-content/uploads/2013/06/Revista-da-UIIPS_N2_Vol1_2013_ISSN-2182-9608.pdf
• ⁴²
  Hernandez OD, Castro F, Paez M. Bioacumulación de mercurio en larvas de anuros en la zona afectada por la minería de oro en el río Dagua, Buenaventura, Valle del Cauca, Colombia. Acta Biol Colomb. [Internet]. 2013 [acesso 6 fev 2015];18(2):341-8. Disponível: http://www.redalyc.org/pdf/3190/319028011010.pdf
  » http://www.redalyc.org/pdf/3190/319028011010.pdf
• ⁴³
  Canto T, Pich CT, Geremias R. Bioensaio de toxicidade em percolados no aterro sanitário do município de Araranguá (Santa Catarina, Brasil). Revista Biociências. [Internet]. 2013 [acesso 6 fev 2015];19(2):53-60. Disponível: http://revistas.unitau.br/ojs-2.2/index.php/biociencias/article/viewFile/1650/1263
  » http://revistas.unitau.br/ojs-2.2/index.php/biociencias/article/viewFile/1650/1263
• ⁴⁴
  Sousa DBP, Almeida ZS, Carvalho-Neta RNF. Biomarcadores histológicos em duas espécies de bagres estuarinos da costa maranhense, Brasil. Arq Bras Med Vet e Zootec. [Internet]. 2013 [acesso 6 fev 2015];65(2):369-76. Disponível: http://www.scielo.br/pdf/abmvz/v65n2/11.pdf
  » http://www.scielo.br/pdf/abmvz/v65n2/11.pdf
• ⁴⁵
  Jacobowski AC, Zobiole NN, Padilha PM, Moreno SE, Arruda EJ. Efeito mutagênico do edetato de cobre ([Cu (EDTA)]-2) livre e nanoencapsulado em camundongos e peixes. Ecotoxicol Environ Contam. [Internet]. 2013 [acesso 6 fev 2015];8(1):13-9. Disponível: http://siaiweb06.univali.br/seer/index.php/eec/article/view/4620/2544
  » http://siaiweb06.univali.br/seer/index.php/eec/article/view/4620/2544
• ⁴⁶
  Rezende KFO, Santos RM, Santos RM, Shimada JCB, Silva JRMC, Muniz CASD. Histopatologia das brânquias de tilápia do Nilo Oreochromis niloticus, provenientes da represa Billings, área de proteção ambiental Bororé-Colônia. Atas de Saúde Ambiental - ASA. [Internet]. 2013 [acesso 6 fev 2015];1(1):57-68. Disponível: http://revistaseletronicas.fmu.br/index.php/ASA/article/view/308/486
  » http://revistaseletronicas.fmu.br/index.php/ASA/article/view/308/486
• ⁴⁷
  Pereira DP, Santos DMS, Carvalho Neta AV, Cruz CF, Carvalho Neta RNF. Alterações morfológicas em brânquias de Oreochromis niloticus (Pisces, Cichlidae) como biomarcadores de poluição aquática na laguna da Jansen, São Luís, MA (Brasil). Biosc J. [Internet]. 2014 [acesso 6 fev 2015];30(4):1213-21. Disponível: http://www.seer.ufu.br/index.php/biosciencejournal/article/view/21852/14937
  » http://www.seer.ufu.br/index.php/biosciencejournal/article/view/21852/14937
• ⁴⁸
  Benites LM, Doncato KB, Minho TS, Perazzo GX. Avaliação do potencial mutagênico de cobre da água do rio Uruguai. Ciência e Natura. [Internet]. 2014 [acesso 6 fev 2015];36(2):107-13. Disponível: http://cascavel.ufsm.br/revistas/ojs-2.2.2/index.php/cienciaenatura/article/view/13610/pdf
  » http://cascavel.ufsm.br/revistas/ojs-2.2.2/index.php/cienciaenatura/article/view/13610/pdf
• ⁴⁹
  Bernardes MFF, Maschio LR, Azeredo-Oliveira MTV, Almeida EA. Biochemical and genotoxic effects of a commercial formulation of the herbicide tebuthiuron in Oreochromis niloticus of different sizes. Ecotoxicological Environmental Contamination. [Internet]. 2014 [acesso 6 fev 2015];9(1):59-67. Disponível: http://www6.univali.br/seer/index.php/eec/article/view/5779/3440
  » http://www6.univali.br/seer/index.php/eec/article/view/5779/3440
• ⁵⁰
  Muñoz DR. Bioética: a mudança da postura ética [editorial]. Rev Bras Otorrinolaringol. [Internet]. 2004 [acesso 6 fev 2015];70(5):578-9. Disponível: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0034-72992004000500001
  » http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0034-72992004000500001
• ⁵¹
  Presgrave OAF. Métodos alternativos. In: Lapchik VBV, Mattaraia VGM, Ko GM, organizadores. Cuidados e manejo de animais de laboratório. São Paulo: Atheneu; 2009. p. 619-34.
• ⁵²
  Balls M. The three Rs and the humanity criterion. Nottingham: FRAME; 2009.
• ⁵³
  Alvim MS. A experimentação animal na nova Lei 11.794/08 à luz da interpretação conforme a Constituição. Revista Brasileira de Direito Animal. [Internet]. 2010 [acesso 6 fev 2015];7(5):221-49. Disponível: http://www.portalseer.ufba.br/index.php/RBDA/article/viewFile/11045/7966
  » http://www.portalseer.ufba.br/index.php/RBDA/article/viewFile/11045/7966
• ⁵⁴
  Bridi AM. Adaptação e aclimatação animal. [Internet]. s.d. [acesso 7 fev 2015]. Disponível: http://www.uel.br/pessoal/ambridi/Bioclimatologia_arquivos/AdaptacaoeAclimatacaoAnimal.pdf
  » http://www.uel.br/pessoal/ambridi/Bioclimatologia_arquivos/AdaptacaoeAclimatacaoAnimal.pdf
• ⁵⁵
  Flecknell PA. Refinement of animal use: assessment and alleviation of pain and distress. Laboratory Animals. [Internet]. 1994 [acesso 6 fev 2015];28(3):222-31. Disponível: http://lan.sagepub.com/content/28/3/222.full.pdf+html
  » http://lan.sagepub.com/content/28/3/222.full.pdf+html
• ⁵⁶
  Broom DM, Molento CFM. Bem-estar animal: conceito e questões relacionadas: revisão. Archives of Veterinary Science. [Internet]. 2004 [acesso 6 fev 2015];9(2):1-11. Disponível: http://www.unb.br/posgraduacao/docs/fav/BEMESTARANIMALCONCEITOQUESTOESRELACIONADAS.pdf
  » http://www.unb.br/posgraduacao/docs/fav/BEMESTARANIMALCONCEITOQUESTOESRELACIONADAS.pdf
• ⁵⁷
  Pereira CEM, Silva JDM, Romeiro VR. Aspectos éticos da experimentação animal. Acta Cir Bras. [Internet]. 1998 [acesso 15 abr 2015];13(2). Disponível: http://dx.doi.org/10.1590/S0102-86501998000200010
  » http://dx.doi.org/10.1590/S0102-86501998000200010
• ⁵⁸
  Schnaider TB, Souza C. Aspectos éticos da experimentação animal. Rev Bras Anestesiol. [Internet]. 2003 [acesso 15 abr 2015];53(2):278-85. Disponível: http://www.scielo.br/pdf/rba/v53n2/v53n2a14
  » http://www.scielo.br/pdf/rba/v53n2/v53n2a14
• ⁵⁹
  Rocha-Mendes F, Napoli RP, Mikich SB. Manejo, reabilitação e soltura de mamíferos selvagens. Arq Ciên Vet Zool Unipar. [Internet]. 2006 [acesso 6 fev 2015];9(2):105-9. Disponível: http://revistas.unipar.br/veterinaria/article/download/351/318
  » http://revistas.unipar.br/veterinaria/article/download/351/318
• ⁶⁰
  Feijó AGS. A função dos comitês de ética institucionais ao uso de animais na investigação científica e docência. Bioética. [Internet]. 2004 [acesso 6 fev 2015];12(2):11-22. Disponível: http://www.revistabioetica.cfm.org.br/index.php/revista_bioetica/article/view/131/136
  » http://www.revistabioetica.cfm.org.br/index.php/revista_bioetica/article/view/131/136
• ⁶¹
  Oliveira RF, Galhardo L. Sobre a aplicação do conceito de bem-estar a peixes teleósteos e implicações para a piscicultura. Rev Bras Zootec. [Internet]. 2007 [acesso 7 fev 2015];36(Suppl):77-86. Disponível: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1516-35982007001000009&lng=en&nrm=iso&tlng=pt
  » http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1516-35982007001000009&lng=en&nrm=iso&tlng=pt

• Work produced within the framework of the Studies and Research Studies in Gender and Sexualities of the State University of the Southwest of Bahia, Jequié/BA, Brazil. (Universidade Estadual do Sudoeste da Bahia – UESB).

Publication Dates

• Publication in this collection
  May-Aug 2015

History

• Received
  13 Jan 2015
• Reviewed
  24 Mar 2015
• Accepted
  7 May 2015