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Aquatic Toxicology 105 (2011) 312–320

Contents lists available at ScienceDirect

Aquatic Toxicology
journal homepage: www.elsevier.com/locate/aquatox

Cholinesterase inhibition and behavioral toxicity of carbofuran on Oreochromis
niloticus early life stages
P.C. Pessoa a , K.H. Luchmann b , A.B. Ribeiro a , M.M. Veras a , J.R.M.B. Correa c , A.J. Nogueira b ,
A.C.D. Bainy b , P.S.M. Carvalho a,∗
a
Universidade Federal de Pernambuco, Zoology Dept., Recife, Brazil
b
Universidade Federal Rural de Pernambuco, Estacão de Piscicultura Johei Koike, Recife, Brazil
¸
c
Universidade Federal de Santa Catarina, Biochemistry Dept., Florianopolis, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Nile tilapia Oreochromis niloticus at 9 days post-hatch were exposed in semi-static experiments to the car-
Received 15 December 2010 bamate insecticide carbofuran, which is applied in agricultural systems in Brazil. Although the molecular
Received in revised form 15 June 2011 mechanism of carbofuran toxicity is well known, a detailed understanding of the ecological mecha-
Accepted 21 June 2011
nisms through which carbofuran effects can propagate towards higher levels of biological organization
in fish is incomplete. Mortality rates were quantified for larvae exposed for 96 h to 8.3, 40.6, 69.9, 140,
Keywords:
297 and 397 g/L carbofuran, and the LC50 96 h was 214.7 g/L. In addition, the biochemical biomarker
Carbofuran
cholinesterase inhibition and behavioral biomarkers related to vision, swimming, prey capture and preda-
Oreochromis niloticus larvae
Cholinesterase
tor avoidance were quantified in individual larvae, as well as their growth in weight. The behavioral
Swimming parameters were quantified by analysis of digitally recorded videos of individual larvae within appropri-
Vision ate experimental setups. The activity of the enzyme cholinesterase decreased after exposure to carbofuran
Predator–prey with a lowest observed effects concentration (LOEC) of 69.9 g/L. Visual acuity deficits were detected
Growth after carbofuran exposure with a LOEC of 40.6 g/L. Swimming speed decreased with carbofuran expo-
sure, with a LOEC of 397.6 g/L. The number of attacks to prey (Daphnia magna nauplii) decreased in
larvae exposed to carbofuran, with a LOEC of 397.6 g/L. Growth in weight was significantly reduced
in a dose dependent manner, and all carbofuran groups exhibited a statistically significant decrease in
growth when compared to controls (p < 0.05). The number of predator attacks necessary to capture larvae
decreased after exposure to carbofuran, and the LOEC was 69.9 g/L. These results show that exposure
of sensitive early life stages of tilapia O. niloticus to sublethal concentrations of carbofuran can affect
fundamental aspects of fish larval ecology that are relevant to recruitment of fish populations, and that
can be better understood by the application of behavioral biomarkers.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction of these unwanted toxic effects in birds, fish, mammals, insects
and aquatic invertebrates (USEPA, 2006). Although carbofuran is
Insecticides are deemed necessary to control pests in several being reevaluated by Brazilian authorities, it is still legally applied
commercial crops, but they can cause toxic effects in non-target throughout the country. Sugar cane plantation is the main agri-
organisms in different terrestrial and aquatic ecosystems (Bretaud cultural practice in the state of Pernambuco, northeastern Brazil,
et al., 2000). Among them, carbofuran (2,3-dihydro-2,2-dimethyl- where carbofuran is used to control nematoids that can reduce
benzofuran-7-methylcarbamate) is a nematicide and acaricide that crop productivity (Barros et al., 2005). It is also used to control a
has been recently banned in the United States and Europe because coleopteran that damages irrigated rice crop in the southeast of the
Brazil (Plese, 2005). Aquatic environments are particularly prone
to carbofuran contamination as indicated by its groundwater ubiq-
∗ Corresponding author at: Universidade Federal de Pernambuco, Depto. de Zoolo- uity score (GUS) index of 4.5, characterizing a relatively high risk
gia, Centro de Ciencias Biologicas, Av. Prof. Moraes Rego, 1235 – Cidade Universitária, of being transported from the soil of application areas to adjacent
Recife – PE – CEP: 50670-901, Brazil. Tel.: +55 81 99584918; fax: +55 81 21268353. water bodies after rain events (Plese, 2005). Carbofuran is degraded
E-mail addresses: priscila contatos@hotmail.com (P.C. Pessoa), in the environment through hydrolysis, oxidation and reduction,
khluchmann@yahoo.com.br (K.H. Luchmann), andersonbritor@yahoo.com.br
(A.B. Ribeiro), marimaldi@gmail.com (M.M. Veras), majin renato@yahoo.com.br
as well as by microbial activity, and its main metabolites are 3-
(J.R.M.B. Correa), augustopesca@ig.com.br (A.J. Nogueira), bainy@mbox1.ufsc.br hydroxycarbofuran and 3-ketocarbofuran, equally toxic to target
(A.C.D. Bainy), pcarvalho@ufpe.br (P.S.M. Carvalho). and nontarget organisms (Otieno et al., 2010). Carbofuran 96 h

0166-445X/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquatox.2011.06.020


P.C. Pessoa et al. / Aquatic Toxicology 105 (2011) 312–320 313

LC50 to different freshwater fish vary from 88 to 1990 g/L (USEPA, 26 (initial juvenile) according to (Fujimura and Okada, 2007). Fish
2004), whereas crustaceans and insects are among the most sensi- were transported to the Ecotoxicology Laboratory at Universidade
tive organisms, with 96 h LC50 s varying from 1.6 to 500 g/L (Dutra Federal de Pernambuco (UFPE) in plastic containers, and were kept
et al., 2009). Environmental agencies in the state of Pernambuco in 70 L tanks until experiments begun. Water was renewed daily at a
do not monitor organic contaminants in water bodies, and there rate of 100%, after passing through 5 and 1 m pressure filters. Tem-
is no information available on carbofuran concentrations in water. perature was kept at 25.5 ± 0.5 ◦ C, pH was 7.5 ± 0.5, total hardness
It has been shown that carbofuran concentration in irrigated rice was 100 mg CaCO3 L−1 and oxygen was 6 ± 0.5 mg L−1 . Fish were
fields in the southeast of Brazil can reach maximum concentrations fed 45% protein commercial fish food (Alcon Artemia, Camboriu,
of 233 g/L in laminar water (Plese, 2005). Additionally, carbofu- Brazil), and total ammonia, nitrite and nitrate were undetectable.
ran concentrations of 26 g/L and 264 g/L have been detected Experiments have been carried out in accordance with The Code of
in a headwater stream, and in agricultural field drains that flow Ethics of the World Medical Association (Declaration of Helsinki)
to this headwater stream in UK, respectively (Matthiessen et al., for animal experiments.
1995). Estimated environmental concentrations of carbofuran in
surface water for selected use patterns in the United States have
been modeled and vary from 5.2 to 36 g/L using maximum rates of 2.2. Exposure procedures
application (USEPA, 2006). Adverse sublethal effects can result from
exposure to these concentrations of carbofuran. Goldfish exposed A stock solution of carbofuran at 8.75 g/L was prepared by adding
to carbofuran concentrations as low as 1 g/L showed reduced 2.5 mL of the commercial form of carbofuran as 350 g/L Furadan
attraction to a chironomid extract (Saglio et al., 1996), and when 350-SC (FMC Corporation, Philadelphia, USA) into a 100 mL volu-
the same species was exposed to 5 g/L, significant alterations in metric flask completed with distilled water. Different volumes of
sheltering and burst swimming were found (Bretaud et al., 2002). In this stock solution were added daily into mixing aquariums with
another study, the amplitude of the electro-olfactogram of pacific 20 L of clean water to create nominal exposure concentrations of
salmon (Oncorhynchus sp.) exposed to 10.4 g/L carbofuran was 10, 50, 100, 200, 300 and 400 g/L carbofuran plus a control group.
reduced (Jarrard et al., 2004). After homogenization of stock solutions added into aquariums with
Low concentrations of organophosphates and carbamates can 20 L volume, water flowed by gravity into exposure aquariums with
inhibit acetylcholinesterase, leading to accumulation of the neu- 15 L of water, providing a daily renewal rate of 133% for all treat-
rotransmitter acetylcholine in the synaptic gap of cholinergic ments, including the control.
synapses and neuromuscular junctions, effect that has become a A second stock solution was prepared from the dissolution of
classic biomarker of exposure to these chemicals (Sturm et al., 2.5 g of carbofuran 98% pure (Sigma–Aldrich, St. Louis, USA) into
1999). Acetylcholinesterase inhibition has been used as an indi- 100 mL of reagent grade ethanol in a volumetric flask. Different vol-
cator of potential fish exposure to these agrochemicals in Brazil umes of this stock solution were added daily into mixing aquariums
(Oliveira et al., 2007). However, information regarding whether this with 20 L to create exposure concentrations of 10, 100, 200, 300,
biomarker of exposure might be used to ultimately predict more 400 and 500 g carbofuran L−1 plus a control and a solvent control
ecologically relevant endpoints related to this exposure are lacking. group. This solution was used only for mortality experiments.
As pointed out by Scholz and Hopkins (2006), there are important Samples of water from these treatments were analyzed by liquid
data gaps that need to be addressed before these predictions can be chromatography for carbofuran after extraction of 1 L with methy-
made, and these include an understanding of sublethal effects that lene chloride, and the extract was dried and concentrated to a
can be linked to deficits in individual fitness. Acetylcholinesterase volume of 10 mL. The extract was cleaned up on a C-18 cartridge,
inhibition in fish sublethaly exposed to these agrochemicals has filtered, and eluted on a C-18 analytical column with a mobile
been related to several measures of behavioral toxicity, includ- phase of 50% acetonitrile in water at a flow rate of 2.0 mL/min,
ing neuromotor effects on swimming activity (Brewer et al., 2001; with a retention time of 3.5 min and ultra-violet (UV) detection
Little and Finger, 1990), effects on specific sensorial systems such as at 280 nm, according to (USEPA, 2010). Method detection limit
vision (Dutta et al., 1992) and smell (Tierney et al., 2008), as well as was 3.2 g carbofuran L−1 . Chemical analysis results indicated that
behavioral measures encompassing more complex situations like measured water concentrations were below nominal concentra-
predator–prey interactions (Scholz et al., 2000). Proper functioning tions by a factor varying from 5% to 30%, and measured carbofuran
of swimming skills and sensorial systems is essential for success- concentrations were subsequently used in all graphs and analysis.
ful detection, attack and capture of prey, as well as for predator The effect of carbofuran concentrations prepared from 98% car-
evasion (Fuiman et al., 2006). The joint assessment of traditional bofuran (Sigma–Aldrich, St. Louis, USA) and prepared from the
biomarkers like cholinesterase inhibition with more ecologically commercial form Furadan 350-SC on larval mortality was com-
relevant behavioral biomarkers will provide important knowledge pared. On this experiment groups freely swimming larvae were
that can help in the development of predictive models of popu- exposed to each of the carbofuran concentrations described above
lation level effects for fishery resources. Within this framework, in 15 L aquariums, a group of 100 fish for each concentration from
the present study was designed to evaluate the effects of exposing each stock solution preparation method.
larval tilapia Oreochromis niloticus to carbofuran on cholinesterase All behavioral parameters and cholinesterase activity measured
inhibition and behavioral parameters related to vision, swimming, were based on larvae exposed to carbofuran solutions prepared
prey capture, predator evasion and growth. from the commercial form of carbofuran, Furadan 350-SC, for
higher environmental realism. On these experiments 10 larvae
were kept in each one of four 250 mL volume floating beakers
2. Materials and methods adapted with 300 m windows to allow water flow in and out
while they floated in the 15 L exposure aquariums. A total of 40
2.1. Animals larvae (4 replicates of 10) were exposed to each concentration
described before. Each group of 10 larvae was fed once a day with
Nile tilapia (O. niloticus) larvae at 9 days post hatch, total length 0.3 g of 45% protein commercial fish food (Alcon Artemia, Cambo-
9 mm, were obtained from the Fish Culture Facility Professor Johei riu, Brazil). Dead larvae were counted and removed daily. Mortality
Koike at Universidade Federal Rural de Pernambuco (UFRPE). Fish rates were calculated by totaling all dead larvae from each exposure
used in experiments were between stages 23 (advanced larvae) and concentration at the end of a 96 h exposure period.


314 P.C. Pessoa et al. / Aquatic Toxicology 105 (2011) 312–320

2.3. Video recording system 2.7. Prey capture

Behavioral tests for swimming activity, feeding and predator During prey capture tests run on the same aquariums used for
evasion were based on digital video recordings obtained from a swimming tests an individual tilapia larva was used in each test.
closed circuit television (CFTV) system based on cameras with Model prey used were Daphnia magna neonates (24–48 h old), total
6–60 mm zoom lenses that allowed the capture of a full superior length between 3 mm and 5 mm. Each larval tilapia was acclimated
view of each experimental arena. A video encoder board (Geovision for 5 min inside a PVC tube in the test aquarium, while 5 D. magna
model GV-800, Irvine, CA) received the images from the cameras neonates were also enclosed in another PVC tube inside the test
monitoring one experimental arena from each exposure concen- aquarium, and the experimental arena setup was enclosed by a
tration (a total of 7 cameras) simultaneously, and videos were black curtain to avoid disturbance. All PVC tubes holding the preda-
recorded on a hard disk for later analysis. tor and prey were tied to a PVC rod that when lifted allowed the
simultaneous release of all predator and prey in each of the 7 are-
2.4. Cholinesterase activity nas monitored simultaneously by the video system. Predator–prey
interactions were recorded for approximately 10 min, and 15 fish
Each sample consisted of pools of 3 whole tilapia larvae homog- from each exposure concentration were individually tested. Pos-
enized after being euthanized, due to their small size. For each terior video analysis generated data on the number of attacks
carbofuran concentration, 5 samples were analyzed (n = 5 for each performed by each tilapia larva towards prey during the test.
concentration). Larval pools were homogenized in Tris buffer (HCl
50 mM, KCl 0.15 M, pH 7.4, PMSF 0.1 mM) in the proportion of 2.8. Predator evasion
1 g of sample to 4 mL of buffer (1:4), using a Potter homoge-
nizer (Glas-Col). Samples were then centrifuged at 9000 × g during Individual tilapia larvae originating from each of the different
20 min at 4 ◦ C. The supernatant (S9 fraction) was separated in exposure concentrations and a control predator (Parachromis man-
aliquots and stocked at a −80 ◦ C freezer for posterior measure- aguensis) were confined inside PVC tubes at opposing sides of an
ment of cholinesterase activity according to the method described aquarium (14 cm length × 10.5 cm width × 13.5 cm height). All PVC
by Ellman et al. (1961) adapted to 96 well microplates. Analy- tubes holding the prey (tilapia) and predator were tied to a PVC rod
ses were run on duplicates at 25 ◦ C using the microplate reader that when lifted allowed the simultaneous release of all prey and
Spectramax 250 (Molecular Devices, Sunnyvale, CA). Reagents were predator in each of the 7 arenas monitored simultaneously by the
purchased from Sigma (St. Louis, MO, EUA). Total protein was mea- video system (Fig. 1). Prey–predator interactions were recorded for
sured according to Peterson (1977), using bovine serum albumin approximately 10 min. A total of 15 fish from each treatment were
as standard. Cholinesterase activity was normalized to total pro- tested individually. Posterior video analysis generated data on the
tein content of samples. We used only acetylthiocholine iodide number of predator attacks needed to capture each prey (tilapia).
(ASCh) as substrate during our measurements in whole fish, but
cholinesterases present in liver and muscle of Nile tilapia have 2.9. Weight gain
properties that resemble both AChE and BChE (Rodríguez-Fuentes
and Gold-Bouchot, 2004). Therefore, our measurements might Before exposure initiated, 25 control tilapia larvae were sacri-
relate to total cholinesterase activity, and will be referred to as ChE. ficed, dried on a paper towel, and weighted on an analytical balance
with a precision of ±0.001 g (Toledo, Brazil, model AR1530). At
the end of the 96 h exposure to each carbofuran concentration, 15
2.5. Swimming activity
tilapia larvae from each treatment were weighted using the same
procedure to evaluate weight gain during this period.
Before the start of the experiment, larvae were individually put
in aquariums (8 cm length × 6 cm width × 8.5 cm depth) with a col-
2.10. Reaction distance to prey and application of Blaxter’s
umn of water 3 cm deep, and acclimated for 10 min inside the
feeding model
experimental arena setup enclosed by a black curtain to avoid dis-
turbance by people moving in the lab. Each video recording was
Reaction distance (RD) to an object of specific size is an alter-
created in segments of 3 min. Recordings were processed by the
native form of representation of the visual acuity of an animal, and
software Spyneurotracking (Bose, 2005), which identifies the ani-
is defined as the maximum distance at which an object of certain
mal coordinates x and y in each frame of the video recordings, and
dimensions can be away from the observer, and still be resolved.
calculates the average swimming speed of the animal in cm s−1 . A
RD can be calculated from the visual acuity angle and the size (D)
total of 15 fish were tested in each treatment.
of either a prey or predator, from equation
D
2.6. Visual acuity 2
× tan( ˛ ) ×
2 180
RD =
10
Visual acuity tests were based on the system and operational
where: RD = reaction distance (cm); D = prey dimension (length
procedures previously described by Carvalho et al., 2002, and is
in mm) ˛ = visual acuity angle (◦ ); = 3.14.
related to the capacity of discriminating detail. During this test indi-
RD and swimming speed can be used to calculate the volume
vidual larvae were kept inside a glass chamber surrounded by black
scanned by larval fish during their search for food. This informa-
and white stripes of varying widths. Briefly, both optomotor (swim-
tion relates the visual and motor capacity of the fish with its ability
ming) and optokinetic (eye movement) responses were monitored
to search and detect food in a three dimensional environment. RD
as fish were exposed to moving stripes of decreasing width until the
is an important parameter used to estimate the reaction area, the
optokinetic response ceased. An acuity angle was then calculated
transversal section of the visual field of fish in the vertical and hori-
based on the smallest stripe width to which the fish responded pos-
zontal directions, according to the formula proposed by Blaxter and
itively. Ten individual tilapia larvae from each exposure treatment
Staines (1971):
were tested for visual acuity after 4 and 5 days of exposure to the
different carbofuran concentrations. A total of 15 fish were tested 2
Reaction area (RA) (cm2 ) = × (RD)2
in each treatment. 3



Fig. 1. Design of the arenas for predator–prey interaction monitoring, showing superior and lateral views of the system.

where: = 3.14; RD = reaction distance in cm 1.0
From RA it is possible to estimate the search volume by multi- 0.9
plying RA times swimming speed: 0.8
0.7
Search volume (SV) (L s−1 )

Mortality rate
0.6
RA (cm2 ) × swimming speed (cm s−1 ) 0.5
=
1000 0.4

This simple modeling approach relates the visual and motor 0.3
capacity of the fish with its ability to search and detect food in a 0.2
three dimensional environment. 0.1
0.0
1 10 100 1000
2.11. Statistical analyses
Carbofuran concentration (µg.L-1)
Mortality rates were corrected for control responses using
Fig. 2. Mortality rates, probit adjusted lines and fiducial limits for Furadan (black
Abbott’s procedure and evaluated for fitness to probit model with circles) and carbofuran (white circles) exposed tilapia larvae Oreochromis niloticus.
Chi-square goodness of fit tests (p < 0.05), using the software Toxs-
tat version 3.5 (West, Inc. and Gulley, D.D., University of Wyoming,
3.2. Cholinesterase activity
USA). If the data fit the probit model, 96 h LC50 and 95% fiducial
limits were also calculated.
A dose dependent inhibition of cholinesterase activ-
For behavioral and cholinesterase parameters, tests for nor-
ity was detected (Fig. 3). Activity varied from 443.7 ±
mality (Kolmogorov–Smirnov test) and homoscedasticity (Levene
162.4 nmol min−1 mg protein−1 in control larvae (0% inhibi-
median test) of the data were applied. If the data were nor-
tion) to 143.7 ± 27.3 nmol min−1 mg protein−1 in larvae from
mal and homoscedastic, we used one-way ANOVA to compare
300 g/L carbofuran (68% inhibition). The lowest observed effect
means from larvae exposed to the different carbofuran treatment
groups, followed by a Dunnett’s test procedure to test for dif-
ferences between the control and carbofuran treatments. A LOEL 700
(lowest observable effect level) and NOEL (no observable effect
level) were estimated following Dunnett’s tests. In case data failed 600
normality or homoscedasticity a non-parametric Kruskal–Wallis
ANOVA was used, followed by Dunn’s test to estimate the LOEL and
(nmol.min-1 mg protein-1)

500
NOEL for each parameter. All statistical analysis for cholinesterase,
Cholinesterase activity

behavioral and growth data were based on the software Sigmaplot
version 11 (Jandel Scientific, Erkrath, Germany). 400

300
*
3. Results
* *
3.1. Mortality rates
200
*
Tilapia larval mortality rates exposed to both types of carbofuran 100
solutions increased in a dose dependent manner (Fig. 2). Mortality
rates calculated from both exposure types fit a probit model, and the 0
LC50 -96 h for the commercial form of carbofuran was 220.7 g/L, 0 8.3 40.6 69.9 140 297 397
with 95% fiducial limits of 191.6 and 254.3 g/L. The LC50 -96 h for Carbofuran concentration (µg.L-1)
carbofuran at 98% purity form was 214.7 g/L, with 95% fiducial
Fig. 3. Cholinesterase activity (nmol min−1 mg protein−1 ) of carbofuran exposed
limits of 200.1 and 229.2 g/L. tilapia larvae Oreochromis niloticus. (*): Different from control values (p < 0.05).



1,2

Number of tilapia larvae attacks on Daphnia
32
30
28
1,0
26
24
Swimming speed (cm.s-1)

0,8 22
20
18
0,6 16
* 14

0,4
12 *
10
8
0,2 6
4
2
0,0 0

0 8.3 40.6 69.9 140 297 397
0 8.3 40.6 69.9 140 297 397
Carbofuran water concentration (µg.L-1)
Fig. 6. Number of attacks on Daphnia performed by carbofuran exposed tilapia lar-
Fig. 4. Swimming speed (cm s−1 ) of carbofuran exposed tilapia larvae Oreochromis vae Oreochromis niloticus. N = 15 fish for all treatments, (*): different from control
niloticus. N = 15 fish for all treatments, (*): different from control values (p < 0.05). values (p < 0.05).

concentration (LOEC) was 69.9 g/L, and the no observed effect
concentration (NOEC) was 40.6 g/L. Cholinesterase inhibition dependent increase in median acuity angle was detected along
in larvae exposed to carbofuran concentrations of 69.9, 140, 297 exposure concentrations of 8.3, 40.6, 69.9, 140, 297 and 397 g/L,
and 397 g/L were 59%, 66%, 68% e 46%, respectively, and were with median visual acuities of 1.4◦ , 1.9◦ , 1.9◦ , 2.4◦ , 3.6◦ and 3.4◦ ,
significantly different than control values (ANOVA F6, 34 = 6.07, respectively (Fig. 5). The LOEC for visual acuity was 40.6 g/L, and
followed by Dunnet’s test, p < 0.05). the NOEC was 8.3 g/L (Kruskal–Wallis H6 = 56.8, p < 0.001, fol-
lowed by Dunn’s test, p < 0.05).
3.3. Swimming speed
3.5. Prey capture
A tendency of dose dependent decrease in swimming speed of
exposed larvae was detected (Fig. 4), although only at the 397 g/L A dose dependent decrease in the number of attacks per-
exposure group the swimming speed of 0.25 cm s−1 was signif- formed by carbofuran exposed tilapia towards 24 h old D. magna
icantly reduced when compared to the control larvae speed of was observed, although a statistically significant difference was
0.65 cm s−1 (ANOVA F6,55 = 3.61, followed by Dunnet’s test, p < 0.05). detected only at the 397 g/L exposure group. Median number
of attacks varied from 17 in the control to 5 attacks at 397 g/L,
3.4. Visual acuity the LOEC (Fig. 6) (Kruskal–Wallis, H6 = 14.3, p < 0.001, followed by
Dunn’s test, p < 0.05). The median reductions in number of attacks
Visual acuity was significantly affected by carbofuran exposure. compared to controls were 17.6%, 23.5%, 11.7%, 29.4%, 35.2% and
Median visual acuity angle was 0.4◦ in control larvae, and a dose 70.5% for the exposure groups of 8.3, 40.6, 69.9, 140, 297 and
397 g/L.
7
*
* 3.6. Predator evasion
6
Control tilapias were more successful in escaping the model
predator P. managuensis, which needed a median of 5 attacks to
5 capture each individual tilapia tested. Carbofuran exposed tilapia
Visual acuity (degrees)

larvae were captured by the predator after a median number of
4 attacks of 4, 3, 1, 1 and 1 for exposure groups at 8.3, 40.6, 69.9,
140 and 297 g/L, respectively (Fig. 7), and the latter three groups
were statistically different from control values (Kruskal–Wallis,
3 * H5 = 14.1, p = 0.015, followed by Dunn’s test, p < 0.05). The
* * NOEC for predator evasion was 40.6 g/L and the LOEC was
2
69.9 g/L.

3.7. Weight gain
1
Weight gain in carbofuran exposed larvae was significantly
0 reduced at all exposure concentrations (Fig. 8). Tilapia larvae had
0 8.3 40.6 69.9 140 297 397 an average initial weight at the beginning of the experiment of
10.7 ± 1.8 mg (mean ± standard deviation). Weight gain after the
exposure period was equal to 6.5 mg in the control group, and a
Fig. 5. Visual acuity (◦ ) of carbofuran exposed tilapia larvae Oreochromis niloticus. dose dependent decrease in weight gain was found. At the exposure
N = 15 fish for all treatments, (*): different from control values (p < 0.05). groups of 8.3, 40.6, 69.9, 140, 297 and 397 g/L carbofuran, weight



Table 1
Reaction distances to prey, reaction areas and volume searched by carbofuran exposed Nile tilapia larvae hunting Daphnia magna neonates based on visual acuity and
swimming speeds applied to Blaxter’s feeding model.

Carbofuran concentration Visual Swimming Prey total Reaction Reaction Search volume
( g/L) acuity (◦ ) speed (cm s−1 ) length (mm) distance (cm) area (cm2 ) (L s−1 )

Control 0.4 0.65 2.0 28.6 1718.9 1.109
8.3 1.3 0.81 2.0 8.9 165.3 0.134
40.6 2.1 0.66 2.0 5.6 65.4 0.043
69.9 2.1 0.69 2.0 5.6 65.4 0.045
140 2.6 0.56 2.0 4.4 41.3 0.023
297 4.7 0.31 2.0 2.5 12.7 0.004
397 4.5 0.25 2.0 2.5 13.3 0.003
Number of predator attacks to capture tilapia larvae

3.8. Visual acuity and swimming speed applied to Blaxter’s
feeding model
9
Control larval tilapia reaction distance, reaction area and search
8
volume towards prey represented by a 2 mm total length D. magna
7 neonate is equal to 28.6 cm, 1718 cm2 and 1.1 L s−1 , respectively
(Table 1). A reduction in these parameters related to the capac-
6 ity of the free swimming larvae to encounter prey was calculated
for carbofuran exposed larvae using measured data on visual acuity
5
and swimming speed applied to (Blaxter and Staines, 1971) feeding
4 model assumptions. Reaction distances were reduced from 31% to
9% of control RD from lowest (10 g/L) to highest (400 g/L) carbo-
3 furan exposure groups, respectively. Reaction areas were reduced

2 * from 10% to 0.8% of control RA from lowest (10 g/L) to high-
* * est (400 g/L) carbofuran exposure groups, respectively. Search
1
volumes were reduced from 12% to 0.3% of control SV from low-
est (10 g/L) to highest (400 g/L) carbofuran exposure groups,
0 respectively.
0 8.3 40.6 69.9 140 297
Carbofuran concentration (µg.L-1) 4. Discussion

Fig. 7. Number of attacks of the model predator Parachromis managuensis performed This study reveals sublethal effects of the exposure of larval
to capture each individually tested carbofuran exposed tilapia larvae Oreochromis
tilapia to waterborne carbofuran on cholinesterase activity and a
niloticus. N = 15 fish for all treatments, (*): different from control values (p < 0.05).
suite of behavioral endpoints that are directly related to impor-
tant ecological mechanisms relevant for their survival, growth and
gains of 3.5, 2.8, 2.7, 0.2, −1.3 and −1.1 mg were found, respec- recruitment to the adult population.
tively, all means statistically different than the control (ANOVA I,
F6,49 = 27.7, p < 0.001, followed by Dunnett’s test, p < 0.05). The LOEC 4.1. Mortality rates
for weight gain was 8.3 g/L.
Carbofuran 96 h LC50 in freshwater fishes range from 88 g/L in
bluegill sunfish Lepomis macrochirus to 1990 g/L in fathead min-
0,012 now Pimephales promelas, a 20 fold difference in sensitivity (USEPA,
Tilapia weight gain after exposure (g)

2004). A carbofuran 96 h LC50 of 480 g/L has been determined for
0,010 juvenile (0.6–3 g wet weight) O. niloticus (Stephenson et al., 1984).
Our results for carbofuran and Furadan 96 h LC50 of 214 g/L and
0,008 220 g/L, respectively, indicate that O. niloticus larvae (0.01 g wet
weight) are more sensitive than larger juveniles and also that the
0,006 * species is among the most sensitive fishes to carbofuran exposure.
This increased sensitivity of larval tilapia could be explained by
* * the smaller amount of AChE present in smaller fish that can be
0,004
rapidly affected by pesticides when compared to larger fish (Dutta
0,002 and Arends, 2003).
* Additionally, the similarity of the 96 h LC50 for carbofuran in
its pure form compared to its agricultural formulation Furadan
0,000
* * indicate that the toxic potency of the active ingredient in the agri-
cultural formulation is not affected by the inert substances added
-0,002
(Fig. 2).

0 8.3 40.6 69.9 140 297 397
4.2. Cholinesterase activity
The measurement of acetylcholinesterase inhibition in feral
Fig. 8. Weight gain (g) at the end of 96 h of carbofuran exposed tilapia larvae Ore-
ochromis niloticus. N = 15 fish for all treatments, (*): different from control values fish is widely established as a biomarker to diagnose exposure to
(p < 0.05). organophosphate and carbamate pesticides (Sturm et al., 1999).



Nevertheless, the potential use of this biomarker as part of an tion decline (Weis et al., 1999, 2001). Fish during early life stages
early warning system of impending ecologically relevant effects need to feed frequently to supply its energetic demands, and an effi-
is a central challenge in fish ecotoxicology, due to gaps in knowl- cient prey capture skill is essential for growth and survival (Zhou
edge about whether this inhibition might affect the fitness of the et al., 2001). Contaminants can affect the motivation to feed as well
exposed individuals (Scholz and Hopkins, 2006). This potential as the ability to capture prey. A decrease in feeding rates mea-
effect in the overall fitness can be assessed by the quantification sured as a reduction in attacks or capture of live prey as well as
of behavioral parameters relevant for the survival and growth of in reduced consumption of artificial food is a common and ecolog-
exposed fish. Furthermore, they can provide the basis for ecological ically relevant effect of contaminants (Sandheinrich and Atchison,
mechanisms involved in the propagation of these effects towards 1990; Weis et al., 2003). Examples related to organophosphates
the population level, where they are a matter of societal concern. include decreased ability to capture Artemia after exposure of
Recent advances in this direction have been made, and an elegant atlantic salmon Salmo salar to fenitrothion (Morgan and Kiceniuk,
modeling approach has been used to relate sublethal reductions 1990), and decreased ability to capture fathead minnows P. prome-
in acetylcholinesterase activity to reductions in wild salmon pop- las by hybrid striped bass exposed to diazinon (Gaworecki et al.,
ulations’ productivity and growth rates (Baldwin et al., 2009). A 2009). In this study prey capture skills of exposed tilapia were
20% AChE inhibition is commonly used as a threshold to deter- affected by carbofuran, as control larvae attacked Daphnia more
mine exposure to organophosphate and carbamates (Bretaud et al., frequently than exposed tilapia (Fig. 6), although the high vari-
2000). We found a LOEC of 69.9 g/L carbofuran for ChE inhibi- ability of the data limited statistically significant differences to
tion in tilapias exhibiting 59.4% inhibition relative to controls, and the 397 g/L treatment. The complexity of predator prey rela-
a non statistically significant ChE inhibition of 22.1% was detected tions might have influenced this variability. In spite of that, results
at the lowest concentration tested of 8.3 g/L (Fig. 3). In addition, of both endpoints (swimming and attacks on prey) indicate a
we found statistically significant differences in weight gain at this clear tendency of decrease with increasing carbofuran concentra-
lowest concentration of 8.3 g/L (Fig. 8), which opens up the pos- tion.
sibility that this threshold discussed above by Bretaud et al. (2000) Vision is essential for several important behavioral activities
might also be correlated with a threshold for more ecologically like prey detection, orientation towards prey, search for sexual
relevant effects. Growth of early life stages is considered an impor- partners, and detection and escape from predators. An inhibition
tant endpoint for risk assessment of effects of contaminants on fish of Ache activity in diazinon exposed Indian carp Labeo rohita has
recruitment. The lack of statistical significance in ChE inhibition at been correlated with deficits in the optomotor response (Dutta
8.3 g/L might also be related to a relatively large variability in the et al., 1992), which is considered a measure of the visual abil-
results (Fig. 3), as pools of 3 whole larvae had to be homogenized. ity of fish. However, Dutta et al. (1992) analyzed the optomotor
It was unfeasible to use tissues like brain where the variability of response using a method that does not quantify the actual visual
the results could have been lower. acuity of the fish being tested, but rather only whether they respond
Nevertheless, it is essential to better understand how sublethal or not to a certain fixed width of the black and white stripes within
dose-dependent ChE inhibition relates to concomitant alterations their field of view during testing. Using a different methodology,
in behavioral parameters, if we want to improve our capacity to (Carvalho and Tillitt, 2004) exposed rainbow trout to 2,3,7,8-TCDD
propose models of contaminant effects from the suborganismal to and quantified the visual acuity angle of the tested fish also ana-
the population level of biological organization. Deleterious effects lyzing optomotor and optokinetic responses. This difference in
of ChE inhibiting pesticides on fish exposed during early life stages methodology has important ecological implications because the
have been detected in individual performance behavioral parame- visual acuity angle can be expressed in terms of a reaction dis-
ters like spontaneous swimming speed (Brewer et al., 2001), swim- tance to prey, and further into reaction areas and search volumes
ming stamina (Van Dolah et al., 1997), vision dependent behaviors towards prey, important parameters used in fish foraging model-
like the optomotor response (Dutta et al., 1992), or olfactory depen- ing (Blaxter, 1986; Breck and Gitter, 1983). Deleterious effects on
dent behaviors like attraction to a food extract (Saglio et al., 1996). visual acuity of a fish involve a decrease in its reaction distance
Additionally, ChE inhibition has also been correlated with maladap- to other subjects, either prey or predators. In the first situation,
tive behavioral effects on more complex situations involving the deficient prey detection skills can lead to decreased energy intake,
interaction of exposed fish either with their potential prey (Morgan and potential growth deficits. The reaction distance of fenitroth-
and Kiceniuk, 1990) or predators (Sandahl et al., 2005). Our results ion exposed atlantic salmon towards adult Artemia quantified in
indicate a significant dose-dependent ChE inhibition clearly cor- experiments with live prey was a sensitive biomarker indicating
related with several measures of behavioral performance that are significant effects at 0.3% the LC50 (Morgan and Kiceniuk, 1990). In
usually evaluated separately in different studies. this study with larval tilapia, visual acuity was the second most
sensitive parameter, with a LOEC of 40.6 g/L (Fig. 5). Further-
4.3. Swimming more, combining the average swimming speeds and visual acuity
angles in a modeling approach, we predicted that the reaction area
A decrease in swimming speed after exposure to organophos-
and search volume towards prey for the exposed larvae would
phates and carbamates has been detected in several fish, as
be reduced to less than 12% of the prey search volume in con-
in carbamate exposed golfish Carassius auratus, rainbow trout
trol fish at the lowest carbofuran concentration tested, 8.3 g/L
Oncorhynchus mykiss and medaka Oryzias latipes (Brewer et al.,
(Table 1). According to this approach, control fish would be able
2001; Heath et al., 1993; Zinkl et al., 1991), and also in organophos-
to search 1.1 L s−1 of water for prey per second, and this search vol-
phate exposed seabass Dicentrarchus labrax (Almeida et al., 2010).
ume would be reduced to 0.13 L s−1 after exposure to the lowest
Our results with larval tilapia support this tendency as we found a
concentration mentioned above, and further reduced to 0.043 L s−1
significant tendency of hypo activity after a 96 h exposure period
at 40.6 g/L. These concentrations are within a range that can be
(Fig. 4), although a statistically significant effect was observed only
detected in various aquatic environments, as reported previously.
at 397 g/L.
Interestingly, this prediction of a modeled reduced prey detection
capability based on swimming speed and visual acuity is confirmed
4.4. Vision, feeding and weight gain
by a clear tendency of reduced attacks on prey (Fig. 6) and by a
It is recognized that alterations in feeding behavior by aquatic significantly decreased growth rate (Fig. 8), which was the most
contaminants can be related to deficits in growth and to popula- sensitive parameter, with a LOEC of 8.3 g/L.



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