Bioethical principles applied to aquatic ecotoxicological studies

Aquatic ecotoxicology 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 ecotoxicology published from 2010 to 2014 were reviewed. The numbers of animals utilized in ecotoxicology 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.

Bioethical principles applied to aquatic ecotoxicological studies http://dx.doi.org/10.1590/1983-80422015232079 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 effluents 5 .
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 justice 6 .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 beings 7 .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 bioethicswhich 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 studies 10 .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 Bioethical principles applied to aquatic ecotoxicological studies http://dx.doi.org/10.1590/1983-80422015232079as the acceptance of self-determination before the situations of the environment 16 , 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 community 26 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 Bioethical principles applied to aquatic ecotoxicological studies http://dx.doi.org/10.1590/1983-80422015232079 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 situ or 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.

Research article
Bioethical principles applied to aquatic ecotoxicological studies http://dx.doi.org/10.1590/1983-80422015232079Regarding 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 program 50 .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 conditions 54 .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 out in 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

Research article
Bioethical principles applied to aquatic ecotoxicological studies http://dx.doi.org/10.1590/1983-80422015232079guidelines 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 bioethics 61 , 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 pub-lications 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.

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

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