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Behavioral and histological effects of rotenone in fish (Guppy, Poecilia

reticulata)

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DOI: 10.3922/j.psns.2014.4.22

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 Psychology & Neuroscience, 2014, 7, 4, 619-623
DOI: 10.3922/j.psns.2014.4.22

Behavioral and histological effects of rotenone in fish (Guppy,

Poecilia reticulata)
André Walsh-Monteiro1, Wylfredo Pragana2, Edmar Tavares Costa2, and Amauri Gouveia Jr2
1- Instituto Federal do Pará, Tucuruí, PA, Brazil
2- Universidade Federal do Pará, Belém, PA, Brazil

Abstract
Rotenone is a toxic compound that causes neuronal death in dopaminergic areas and is commonly used as a pesticide in
agriculture. The aim of the present study was to investigate the effects of acute rotenone poisoning on the telencephali ventralis
pars ventralis (Vv; i.e., an area of dopaminergic innervation) in adult Poecilia reticulata fish and its behavioral effects in the
open field test. Forty adult guppy fish (King Cobra) were divided into five groups: 0, 5, 10, and 12.5 µg/L rotenone exposure for
24 h. The fish were then tested in the open field for 10 min and then sacrificed. The encephalon was removed, and Nissl staining
was performed. The cell counts were performed in a 200 µm2 area of Vv tissue. Rotenone increased locomotor activity in the
open field, enhanced exploratory activity in the center of the open field, and reduced the number of cells in the Vv. Keywords:
behavior, rotenone, toxicology, animal model.

Received 19 April 2014; received in revised form 30 July 2014; accepted 11 October 2014. Available online 16 December 2014.

Introduction dopaminergic neurons and movement disorders (Sherer,

Rotenone is a secondary metabolite product Kim, Betarbet, & Greenamyre, 2003).
(Edwards & Wratte, 1981) with pesticide and ictiotoxic Rotenone promotes the systemic inhibition of
properties. It is found in plants and commonly used in mitochondrial complex I, in which the production of
artisanal fishing in South America and Southeastern Asia reactive oxygen species (ROS) blocks the electron
(Chacon, 1973). It is a lipophilic compound that easily transport chain and causes neurodegeneration (Miyoshi,
crosses biological barriers because of its hydrophobicity, 1998; Betarbet, Sherer, Mackenzie, Garcia-Osuna,
thus having high absorption and uptake in the brain. Panov, & Greenamyre, 2000). Meng et al. (2011)
Despite being widely distributed in the brain, rotenone studied the association between pesticides, such as
can cause selective neurodegeneration in specific rotenone, and gene products related to PD. They found
regions (Talpade, Greene, Higgins, & Greenamyre, that prolonged exposure to environmental toxins tends
2000) and has been directly associated with the onset of to increase oxidative stress in exposed subjects.
Parkinson’s disease (PD; Tanner et al., 2011). Heikkila, Although rotenone is used as a pesticide, its level
Nicklas, Vyas, and Duvoisin (1985) injected rotenone of exposure is small relative to other pesticides, making
in rat brains, which caused the death of dopaminergic epidemiological and toxicological studies of rotenone
neurons. Later studies showed that rotenone can be used quite scarce. One of the few epidemiological reports on
as a model for studying PD because its effects mimic rotenone in humans was published by Hancock et al.
the features of the disease (Schmidt & Alam, 2006), (2008). Toxicological studies on the lethal and sublethal
including the selective degeneration of nigrostriatal effects of this neurotoxin in fish are even rarer (Cheng &
Farrell, 2007). In one of these studies, Ling showed that
André Walsh-Monteiro, Laboratório de Biociências e fish are sensitive to acute doses of rotenone, indicating
Comportamento, Instituto Federal do Pará, Campus Tucuruí.
Wylfredo Pragana and Edmar Tavares Costa, Laboratório
that 1 ppm or lower concentrations are sufficient to
de Doenças Neurodegenerativas, Instituto de Ciências cause death within a few hours. The LC50 in 24 h for
Biológicas, Universidade Federal do Pará. Amauri Gouveia these animals was determined to be between 5 and 100
Jr., Laboratório de Neurociências e Comportamento, Núcleo µg/L (Ling, 2003). Considering ecological aspects,
de Teoria e Pesquisa do Comportamento, Universidade toxicological studies with fish may help clarify the role
Federal do Pará. Correspondence regarding this article of toxins in the environment, in which animals come
should be directed to: André Walsh-Monteiro, Laboratório
de Biociências e Comportamento, Instituto Federal do Pará, into contact with xenobiotic elements that are dissolved
Campus Tucuruí, Rua Salto Santiago s/nº, Vila Permanente, and incorporated into food chains (Ahmad, 1995).
Tucuruí, PA, 68464-000, Brazil. Phone: +55 (94) 8114-0236. The effect of neurotoxins can be studied through
Fax: +55 (94) 3778-4684. Email: andre.walsh@ifpa.edu.br behavioral tests, such as the open field test (Hall, 1934),
620 Walsh-Monteiro et al.

which assesses emotional behavior by measuring times per day with specific food for the species (Guppy
the time spent at the edge and center of the circular Mep 200, Alcon, Brazil). Feeding was interrupted only
apparatus and locomotor activity (Prut & Belzung, 2003) during the 24-h period of exposure to rotenone.
in fish when they explore new bright environments. A 1 week acclimation period was used to observe and
Another way to study the effects of neurotoxins is to control health conditions, recover from transportation
histologically analyze tissue to identify the brain areas stress, and adapt to the new environment, as recommended
that are affected by exposure to neurotoxins. by Murty (1988).
Rotenone is associated with PD, and we
neurohistologically analyzed the telencephali ventralis Test apparatus
pars ventralis (Vv) area. Several studies have indicated
During the 24 h of intoxication, the animals were
this area is rich in dopamine and homologous to the
individually placed in aquaria with a volume of 1 L.
substantia nigra in mammals (Wullmann & Vernier,
The aquaria were coated with black vinyl to protect the
2009). This area in mammals undergoes substantial
rotenone solution from photobleaching.
changes when exposed to drugs that are commonly used
For the open field test, a circular tank was used,
in experimental models of PD (e.g., 1-methyl-4-phenyl-
with matte walls and background (37.70 cm2) and a 2 L
1,2,3,6-tetrahydropyridine, paraquat, and rotenone;
capacity. The background had two perpendicular lines
Gorell, Johnson, Rybicki, Peterson, & Richardson, 1998).
that intersected at the center of the apparatus where they
Furthermore, comparative analyses of vertebrates have
met a circle (7.54 cm2). This structure resulted in the
strongly suggested that the Vv is a septal formation area.
formation of four peripheral quadrants and one central
In the present study, Poecilia reticulata, a
circle. Thus, the animal’s displacement in two directions
benthopelagic nonmigratory Amazonian fish that is
(center-periphery and periphery-periphery) could be
commonly known as Guppy or Lebiste, with a King
Cobra lineage (Whitney & Hahnel, 1980), was used. measured.
This teleost fish is widely used in toxicological studies
because it is quite sturdy and easy to handle. In the open Intoxication
field test, this fish presents a stereotyped behavioral Four groups were formed (n = 10). Three groups were
pattern, in which exposure to the new environment is exposed for 24 h to different sublethal concentrations of
followed by higher activity in the peripheral zone than rotenone (5, 10, and 12.5 µg/L) diluted in water. The
in the central zone. This pattern is only modified through fourth group was not exposed to rotenone (negative
habituation (Warren & Callaghan, 1976). control). Rotenone has limited solubility (0.2 mg/L
The purpose of the present study was to describe at 20°C), and successive dilution was performed to
the effects of acute rotenone exposure at different obtain the final concentrations. The concentrations were
concentrations on Poecilia reticulata using behavioral determined to be within the minimum sensitivity range
and neurohistological analyses. for the family Poecilidae, which is 17 μg/L (Ling, 2003).
All of the groups, including the negative control, were
Materials and methods exposed to their respective solutions for 24 h.
All of the experimental procedures were consistent
Open field test
with the standards of the International Guiding
Principles for Biomedical Research Involving Animals The open field test was conducted in a controlled
(National Research Council, Institute for Laboratory environment with low noise and fluorescent lamps
Animal Research, 2004) and received approval from positioned above the test tank to avoid shadow formation
the Ethics Committee for Animal Use of the Federal from the animal and walls of the apparatus.
University of Pará. One fish was placed in the center of the apparatus
at the beginning of each session, and the test duration
Subjects was 10 min. The experiment was recorded on a webcam
(LifeCam, Microsoft, USA) coupled to a notebook
In the current investigation, the experimental
computer (Vostro 3560, Dell, USA). Each animal was
subjects were adult male fish of the Poecilia reticulata
tested 10 times.
species, King Cobra lineage, weighing between 0.5
and 0.6 g, that were purchased from an Aquanorte
store (Belém, Pará, Brazil). The animals were housed Craniotomy and brain section
in 20 L aquaria (pH 7.0-7.6, 25.90ºC and 27.00°C After 24 h exposure to rotenone, the animals were
at room temperature), with constant aeration and anesthetized with 5% tricaine methamesulfonate and then
filtration with an hydraulic pump (HF-0400, Atman, subjected to craniotomy. After extraction, the brains were
Brazil). The photoperiod was 12 h light/12 h dark. fixed in 4% paraformaldehyde and diluted in a solution
The water was supplied by the public network, and of 0.1% phosphate-buffered saline (PBS) and 2.5%
the limnological parameters were adjusted according glutaraldehyde for 24 h at room temperature. The brains
to the limits recommended by the American Public were then transferred to a cryoprotectant solution that
Health Association (2012). The animals were fed three consisted of 30% sucrose in 0.1% PBS for 48 h at -4°C.
Effects of rotenone in fish 621

Coronal sections of the brains, as thin as 40 μm, first displayed. The nonparametric Kruskal-Wallis test
were cut with a cryostat (CM 1520, Leica, Germany). (H test) was then performed, with a significance level
The histological sections were then mounted on of p < .05.
gelatinized slides, and Nissl staining was performed.
Results
Nissl staining In the open field test, locomotor activity was
For the neurohistological study, the tissue was measured in the four peripheral quadrants and central
stained with Cresyl violet. The staining protocol was circle. Table 1 shows the values ​​obtained in the analysis
adapted from Klüver & Barrera (1953). The sections of overall crossings (total) and statistical comparisons
were mounted on gelatin-coated slides, dehydrated in between crossings in the center-periphery (central) and
ethanol, delipidated in xylene, rehydrated in ethanol, periphery-periphery (periphery).
and immersed in 0.25% Cresyl violet acetate for 2 min. Table 2 shows the p values of the comparisons
The sections were then dehydrated again, immersed between groups that were exposed to rotenone and the
in xylene, and covered with DPX mounting medium control group. Peripheral, central, and total locomotor
(BDH, Poole, United Kingdom). activity was analyzed. The comparison between the 5
μg/L and 10 μg/L groups indicated no differences in
Histological study any of the variables, suggesting that the subjects were
A Lucida optical microscope equipped with an equally affected by these doses.
image capture system (Labophot-2, Nikon, Japan) was Figure 1 presents central, peripheral, and total
used at 400× magnification. Cell counting was performed locomotor activity in the open field after 24 h exposure
in a previously defined area of approximately 200 μm2. to rotenone. All the groups preferred the periphery of
The limits of this area were defined by considering the open field over the center.
the natural border circumscribed by the group of cells The number of crossings in the peripheral region of
(cluster) and neuroanatomical features, such as bundles the apparatus was not significantly different between the
of nerves that cross the area. Cell counting was always 5 μg/L and 10 μg/L groups and the control group. The
performed with the fifth section of the telencephalon. number of crossings in the 5 μg/L and 10 μg/L groups
Five sections were manually counted for each animal. was higher than in the control group. No significant
difference was found between the 5 μg/L and 10 μg/L
Statistical analysis groups. Significantly differences were found in all of
The results were analyzed using BioEstat 5.0 the other comparisons. The 5 μg/L and 10 μg/L groups
software for Windows (Ayres, Ayres, Ayres, & Santos, had a very similar average number of crossings. Both
2007). The arithmetic mean and standard deviation were concentrations apparently had a similar anxiogenic

Table 1. Locomotor activity (number of crossings) in the open field.

Group Central Periphery Total p

Control 1.80 ± 1.16 11.44 ± .66 13.24 ± 1.15 .0005**

5 µg/L rotenone 2.89 ± .64 15.77 ± 2.48 18.66 ± 2.47 < .0001**

10 µg/L rotenone 3.26 ± .80 15.33 ± 2.45 18.59 ± 2.47 < .0001**

12.5 µg/L rotenone 4.63 ± 1.99 10.77 ± 2.15 15.40 ± 1.97 .0857

Table 2. Comparison of number of crossings in the open field between groups.

Group Central Periphery Total

H = 19.20 H = 36.47 H = 38.90
p < .0007 p < .0001 p < .0001

Control × 5 µg/L .0542 .0197* .0009**

Control × 10 µg/L .0102* .0384* .0008**

Control × 12.5 µg/L .0002** .5497 .0960

5 µg/L × 10 µg/L .5194 .7943 .9572

5 µg/L × 12.5 µg/L .0790 .0034** .0992

10 µg/L × 12.5 µg/L .2661 .0076** .0886

622 Walsh-Monteiro et al.

Table 3. Comparison of total number of neurons in Vv

between groups.

Group Neurons
H = 32.24
p < .0001

Control × 5 µg/L .0362*

Control × 10 µg/L .0018**

Control × 12.5 µg/L .0001**

5 µg/L × 10 µg/L .3017

5 µg/L × 12.5 µg/L .0005**

10 µg/L × 12.5 µg/L .0147*

Figure 1. Locomotor activity, presented as the number of crossings,
between groups.

effect. This anxiogenic effect was less noticeable in the effect occurs uniformly throughout the brain, only
12.5 μg/L group. dopaminergic nigrostriatal neurons appear to suffer
With regard to central ambulation, crossings were degeneration (Betarbet et al., 2000). In the present
less frequent than in the peripheral area. The 10 μg/L study, the neurodegenerative effects of rotenone on the
and 12.5 μg/L rotenone groups exhibited a significantly Vv were identified, and the cell count in this area was
greater number of crossings compared with the control inversely proportional to the pesticide concentration
group. Locomotor activity in the 10 μg/L and 12.5 μg/L with acute exposure.
groups was ​​equivalent to the control group. Even the lowest concentration of rotenone (5 μg/L)
Total ambulation in the 5 μg/L and 10 μg/L caused significant neuronal loss in the Vv compared
groups was significantly higher than in the control with the control group (p = .0362). The 12.5 μg/L
group, demonstrating the anxiogenic effect of these concentration caused an approximately 9% loss of
concentrations. Average ambulation ​​was significantly neurons in the Vv compared with the control group
higher In the groups exposed to rotenone. (control: 394.80 ± 1.55; 12.5 μg/L: 359.30 ± 5.29; p
The quantitative analysis of cells in the Vv showed < .0001). The 12.5 mg/L group also exhibited higher
that the number of neurons was inversely proportional locomotor activity in the central area of the apparatus in
to the concentration of rotenone (Figure 2). A significant the open field test. Therefore, the Vv in fish may have
reduction of the number of cells was observed in the 5 the same intrinsic sensitivity to defects in mitochondrial
μg/L (390.40 ± 2.59), 10 μg/L (387.60 ± 3.34), and 12.5 complex I as the neurons in the substantia nigra. In the
μg/L (359.30 ± 5.29) groups compared with the control present study, higher rotenone concentrations increased
group (394.80 ± 1.55; Table 3). The 12.5 μg/L group had locomotor activity, and rotenone also exerted an
significantly elevated neuronal loss compared with the anxiolytic effect, reflected by behavioral disinhibition
other rotenone groups. and an increase in central ambulation.
A significant increase in locomotor activity (total
ambulation) was found in the 5 μg/L (p = .0009) and
Discussion 10 μg/L (p = .0008) rotenone groups compared with
Rotenone is deleterious to mitochondria. It the control group. The lesions observed in the Vv are
binds to complex I proteins of the electron transport consistent with the hypothesis that rotenone directly
chain, blocks adenosine triphosphate production, and affects locomotion. Thus, although the low (5 μg/L)
promotes the formation of free radicals, which activate and moderate (10 μg/L) doses of rotenone enhanced
mechanisms of oxidative stress (Drechsel & Patel, total ambulation, preference for the periphery was
2008), resulting in neurodegeneration. Although this preserved (p < .0001) in both groups compared
with controls, even with equivalent percentages of
peripheral ambulation (84.51%, 82.46%, and 86.40%,
respectively). These results indicate an anxiogenic effect
of these concentrations, similar to the findings reported
by López-Patiño, Yu, Cabral, and Zhdanova (2008),
who studied the effects of cocaine in Danio rerio, and
Hallgren, Volkova, Reyhanian, Olse, and Hällström
(2011), who studied the effects of 17α-ethynylestradiol
in P. reticulata.
Locomotor alterations were also found in the 12.5
μg/L rotenone group, in which central ambulation
Figure 2. Total number of neurons in Vv. was significantly higher than in controls (p = .0002).
 Effects of rotenone in fish 623

Although preference for the peripheral areas of the Edwards, P. J. & Wratten, S. D. (1981). Ecologia das interações entre
insetos e plantas. São Paulo: EPU Ed.
apparatus occurred at the detriment to the central area, Gorell, J. M., Johnson, C. C., Rybicki, B. A., Peterson, E. L., &
which was observed in the control group and 5 and 10 Richardson, R. J. (1998). The risk of Parkinson’s disease with
μg/L groups, the difference in locomotor activity was not exposure to pesticides, farming, well water, and rural living.
Neurology, 50(5), 1346-1350.
significant, and the percentage of peripheral crossings Hall, C. S. (1934). Emotional behavior in the rat: I. Defecation and
decreased by 69.93%. Thus, the rate of locomotor urination as measures of individual differences in emotionality.
activity in the central area of the apparatus was two- Journal of Comparative Psychology, 18(3), 385-403.
times higher in the 12.5 mg/L group compared with Hallgren, S., Volkova, K., Reyhanian, N., Olse, K. H., & Hällström, I.
P. (2011). Anxiogenic behaviour induced by 17α-ethynylestradiol
the lower concentrations, suggesting an antagonistic in male guppies (Poecilia reticulata). Fish Physiology and
effect. The 12.5 μg/L concentration exerted a bimodal Biochemistry, 37(4), 911-918.
pharmacological effect, in which peripheral activity was Hancock, D. B., Martin, E. R., Mayhew, G. M., Stajich, J. M., Jewett,
R., Stacy, M. A., Scott, B. L., Vance, J. M., & Scott, W. K. (2008).
reduced (related to fear) and exploratory behavior was Pesticide exposure and risk of Parkinson’s disease: A family-based
increased (crossings toward the center of the apparatus). case-control study. BMC Neurology, 8, 6.
The anxiolytic effect induced by 12.5 μg/L rotenone, Heikkila, R. E., Nicklas, W. J., Vyas, I., & Duvoisin, R. C. (1985).
Dopaminergic toxicity of rotenone and the 1-methyl-4-
reflected by a significant increase in central ambulation phenylpyridinium ion after their stereotaxic administration to rats:
compared with the control group (p = .0002), appeared implication for the mechanism of MPTP toxicity. Neuroscience
to be associated with extensive damage in the Vv Letters, 62(3), 389-394.
Klüver, H. & Barrera, E. (1953). A method for the combined staining of
caused by this concentration. Neurodegeneration was cells and fibers in the nervous system. Journal of Neuropathology
significant, even when compared with the 10 μg/L and Experimental Neurology, 12(4), 400-403.
concentration (p = .0147). These results appear to Ling, N. (2003). Rotenone: a review of its toxicity and use for
fisheries management. Science for Conversation, no. 211.
indicate higher motivation to explore at the detriment of Wellington: Department of Conservation.
avoidance behavior (McNaughton & Corr, 2004). López-Patiño, M. A., Yu, L., Cabral, H., & Zhdanova, I. V. (2008).
Anxiogenic effects of cocaine withdrawal in zebrafish. Physiology
and Behavior, 93(1-2), 160-171.
Conclusions McNaughton, N. & Corr, P. J. (2004). A two-dimensional
neuropsychology of defense: fear/anxiety and defensive distance.
In the present study, the adverse effects of rotenone Neuroscience and Biobehavioral Reviews, 28(3), 285-305.
were observed at the lowest concentration (5 μg/L), Meng, F., Yao, D., Shi, Y., Kabakoff, J., Wu, W., Reicher, J., Ma,
causing significant neuronal loss in the Vv and resulting Y., Moosmann, B., Masliah, E., Lipton, S. A., & Gu, Z. (2011).
Oxidation of the cysteine-rich regions of parkin perturbs its E3
in progressive motor hyperstimulation (Viggiano, 2008) ligase activity and contributes to protein aggregation. Molecular
up to a concentration of 12.5 μg/L. This may have caused Neurodegeneration, 6, 34.
a progressive collapse of the inhibitory system, which Miyoshi, H. (1998). Structure-activity relationships of some complex
I inhibitors. Biochimica et Biophysica Acta, 1364(2), 236-244.
is equivalent to septal regions, resulting in an increase Murty, A. S. (1988). Toxicology of pesticides to fish. Boca Raton: CRC
in locomotor activity and behavioral disinhibition. Press.
However, more behavioral, biochemical, and cellular National Research Council, Institute for Laboratory Animal Research
studies are necessary to confirm the possibility of using (2004). The development of science-based guidelines for laboratory
animal care: Proceedings of the November 2003 International
rotenone as a model of PD in fish similarly to rats. Workshop. Washington DC: National Academies Press.
Prut, L. & Belzung, C. (2003). The open field as a paradigm to
measure the effects of drugs on anxiety-like behaviors: a review.
Acknowledgements European Journal of Pharmacology, 463(1-3), 3-33.
Schmidt, W. J. & Alam, M. (2006). Controversies on new animal
This work was supported by grants from CNPq models of Parkinson’s disease pro and con: the rotenone model
(AGJ and WP) and CAPES (AWM). of Parkinson’s disease (PD). Journal of Neural Transmission
Supplement, 70, 273-276.
Sherer, T. B., Kim, J. H., Betarbet, R., & Greenamyre, J. T.
References (2003). Subcutaneous rotenone exposure causes highly
Ahmad, S. (1995). Oxidative stress from environmental pollutants. selective dopaminergic degeneration and α-synuclein
Archives of Insect Biochemistry and Physiology, 29(2), 135-157. aggregation. Experimental Neurology, 179(1), 9-16.
American Public Health Association (2012). Standard methods for the Talpade, D. J., Greene, J. G., Higgins, D. S., Jr., & Greenamyre, J.
examination of water and wastewater, 22nd edition. Washington T. (2000). In vivo labeling of mitochondrial complex I (NADH:
DC: American Public Health Association, American Water Works ubiquinone oxidoreductase) in rat brain using [3H]dihydrorotenone.
Association, Water Environment Federation Publication. Journal of Neurochemistry, 75(6), 2611-2621.
Ayres, M., Ayres, M., Jr., Ayres, D. L., & Santos, A. A. S. (2007). Tanner, C. M., Kamel, F., Ross, G. W., Hoppin, J. A., Goldman, S.
BioEstat: aplicações estatísticas nas áreas das ciências M., Korell, M., … Langston, J. W. (2011). Rotenone, paraquat and
biomédicas. Belém: Instituto Mamirauá. Parkinson’s disease. Environmental Health Perspectives, 119(6),
Betarbet, R., Sherer, T. B., Mackenzie, G., Garcia-Osuna, M., Panov, 866-872.
A. V., & Greenamyre, T. J. (2000). Chronic systemic pesticide Viggiano, D. (2008). The hyperactive syndrome: Metaanalysis of
exposure reproduces features of Parkinson’s disease. Nature genetic alterations, pharmacological treatments and brain lesions
Neuroscience, 3(12), 1301-1306. which increase locomotor activity. Behavioural Brain Research,
Chacon, J. O. (1973). O Timbó (rotenona) usado como inseticida e 194, 1-14.
tóxico para peixes. Boletim Técnico DNOCS, 2, 123-129. Warren, E. W. & Callaghan, S. (1976). The response of male guppies
Cheng, W. W. & Farrell, A. P. (2007). Acute and sublethal toxicities (Poecilia reticulata, Peters) to repeated exposure to an open field.
of rotenone in juvenile rainbow trout (Oncorhynchus mykiss): Behavioral Biology, 18(4), 499-513.
Swimming performance and oxygen consumption. Archives of Whitney, F. L. & Hahnel, P. (1980). O livro dos guppies. Portugal:
Environmental Contamination and Toxicology, 52(3), 388-396. Presença Ed.
Drechsel, D. A. & Patel, M. (2008). Role of reactive oxygen species in Wullimann, M. F. & Vernier, P (2009). Evolution of the nervous
the neurotoxicity of environmental agents implicated in Parkinson’s system in fishes. In J. H. Kaas (Ed.), Evolutionary neuroscience
disease. Free Radical Biology and Medicine, 44(11), 1873-1886. (pp. 147-168). New York: Academic Press.

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