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JEB
Journal Home page : www.jeb.co.in «E-mail : editor@jeb.co.in
Journal of Environmental Biology, Vol. Special issue, 73-84, January 201435© Triveni Enterprises, Lucknow (India)
Journal of Environmental Biology ISSN: 0254-8704
CODEN: JEBIDP
Effect of pesticides used in banana and pineapple plantations on
aquatic ecosystems in Costa Rica
noel.diepens@wur.nl
Noël J. Diepens *, Sascha Pfennig , Paul J. Van den Brink , Jonas S. Gunnarsson , Clemens Ruepert andLuisa E. Castillo
1,2 1 2,3 4 1 1
1
2
3
4
Central American Institute for Studies on Toxic Substances (IRET), Universidad Nacional, Heredia, 83-3000, Costa Rica
Department of Aquatic Ecology and Water Quality Management, Wageningen University, Wageningen University and Research Centre, P.O. Box
47, 6700 AA Wageningen, the Netherlands
Alterra, Wageningen University and Research Centre. P.O. Box 47, 6700 AA Wageningen, the Netherlands
Department of Ecology, Environment and Plant Sciences, Stockholm University, Stockholm, SE-106 91, Sweden
*Corresponding Author E-mail:
Abstract
Key words
Current knowledge on fate and effect of agricultural pesticides comes is mainly from temperate
ecosystems. More studies are needed in tropical systems in order to assess contamination risks to non-
target endemic tropical species from the extensive use of pesticides e.g. in banana and pineapple
plantations. In this study, acute laboratory toxicity tests with organophosphate pesticides ethoprophos and
chlorpyrifos were conducted on two Costa Rican species, cladoceran and fish
. Tests showed that chlorpyrifos was more toxic than ethoprophos to and
and that was also more sensitive than to both pesticides. Additionally, bioassays
were performed by exposing and to contaminated water collected from the field.
Chemical analyses of field water revealed that fungicides were generally the most frequent pesticide group
found, followed by insecticides/nematicides and herbicides. The bioassays and values obtained from the
literature confirmed that was more sensitive to pesticide contamination than and that
was more sensitive than , suggesting that the native cladoceran is a more suitable test
species than its temperate counterpart. Species sensitivity distributions showed no significant difference in
sensitivity between tropical and temperate fish and the arthropod species exposed to chlorpyrifos in this
study. Choline esterase activity (ChE) was measured in in laboratory tests in order to assess the
applicability of this biomarker. ChE inhibition in was observed in the laboratory at levels below the
LC of both ethoprophos and chlorpyrifos, confirming that ChE is an efficient biomarker of exposure. Both
indigenous Costa Rican species used in this study were found to be suitable standard tropical test species.
Further studies are needed to investigate how protective the safe environmental concentrations, derived
from LC of native tropical species, are for protecting tropical aquatic natural communities.
Acute toxicity, Bioassays, Chlorpyrifos, ChE inhibition, Ethoprophos, Tropical aquatic ecosystems
Daphnia ambigua
Parachromis dovii D. ambigua P.
dovii D. ambigua P. dovii
D. magna P. dovii
D. magna P. dovii D.
ambigua D. magna
P. dovii
P. dovii
10
50
Introduction
Costa Rica is the second largest banana producer in the
world. Commercially grown bananas are among the world's most
pesticide intensive crops. Approximately, 45 kg of active
ingredient (a.i.) per hectare per year are used in the Costa Rican
banana plantations (Castillo 1997; Ramírez, 2010;et al.,
REPCar, 2011; Castillo 2012; Bravo 2013). The most
important group of pesticides used are fungicides, followed by a
wide range of insecticides, nematicides and herbicides (see
(Castillo 1997; Ramírez, 2009) for a review). Currently,
pineapple and rice are increasing their production area.
Pineapple production uses an average of 30 kg of a.i. of
pesticides per hectare per year and currently the crop is grown
et al., et al.,
et al.,
Publication Info
Paper received:
Revised received:
Accepted:
27 April 2013
24 June 2013
05 September 2013
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over 45000 ha (Castillo 2012). This together with the
banana plantation, these crops potentially brings along more
environmental pollution and health risks (Barraza , 2011).
Two widely used organophosphate (OP) pesticides are
chlorpyrifos and ethoprophos. Plastic bags that are used to
protect banana bunches are usually impregnated with
chlorpyrifos (1% mass concentration equals 0.7 kg a.i. ha y
(Matlock Jr and de la Cruz, 2002). Ethoprophos is one of the most
commonly used nematicides to protect the root systems of
banana and pineapple plants. In banana plantations, it is applied
a maximum of three times a year (16-21 kg a.i. ha y ; (Matlock Jr
and de la Cruz, 2002)). Together with carbamate pesticides, OPs
are neurotoxins, which act by inhibiting the cholinesterase
enzymes (ChE) acetylcholinesterase (AChE) and butyryl cholin
esterase (BChE) in the nervous system. Inhibition of ChE has
been widely used to assess the effects of anticholinesterase
compounds on wildlife (Thompson, 1999). In fish, inhibition of
et al.,
et al.
-1 -1
-1 -1
20% or more is well accepted as indicator of exposure to OPs
(Varó ,2007).
The river Rio Madre de Dios (RMD) in the Atlantic
lowlands of Limon, Costa Rica (Fig. 1) receives drainage water
from banana plantations, pineapple and rice fields before it flows
into a coastal lagoon, which is part of a protected coastal wetland
with a large biodiversity. Due to the high rain precipitation in this
region, with an average of 3.6 m per year (Waylen 1996),
the intensive use of pesticides and fertilizers in these extensive
agricultural systems may reach the RMD river and its lagoon
trough run-off and endanger the non-target fauna such as fish and
invertebrates. Several massive fish kills have been observed in
these water courses, especially after heavy rains and are
believed to be caused by pesticide run-off. Generally, fish species
such as guapote ( ), black surfperch ( ),
tilapia ( ) and other cichlid species have
been found among the dead fish (Pais, 2009; Pais, 2010).
et al.
et al.,
P. dovii Embiotoca jacksoni
Oreochromis niloticus
N.J. Diepens et al.
74
Fig. 1 : Sampling sites in watershed Rio Madre de Dios (14758 ha), Limon, Costa Rica. RMD is Rio Madre de Dios and LMD is Laguna Madre de Dios.
RMD control was used as control site and together with Bridge RMD, Canal Pama, LMD X RMD they were used for the bioassays and ChE analyses.
Caño Azul, RMD X Freeman, LMD mid and LMD x Pacuare were used for water quality and pesticide residue analyses only
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A temperate and tropical species sensitivity distribution
calculation was used to evaluate to what extent these tropical
species are suitable for toxicity testing and environmental risk
assessment in tropical regions.
Two cladoceran species (Crustacea) tropical
(Scourfield, 1947) and
(Straus, 1820) and the guapote fish (Cyclidae)
(Günther, 1864) were used as test organisms.
The fish were cultivated and kept under a natural light : dark cycle
(approximately 12:12 hr) in the laboratory at the Biology
Department of the National University (UNA), Heredia, Costa
Rica. Aged tap water (24 hr) with a temperature of 18-25°C was
used in the fish tanks. The guapote fish were fed a mixture of fish,
soya, cornflour, shrimps and soya oil, supplemented with vitamins
and minerals. Fry were also fed with and cultivated
zooplankton. After 12 to 18 days, the fish were transferred for
acclimatization for at least 24hr to the laboratory of
ecotoxicological studies (ECOTOX) of IRET (Central American
Institute for Studies on Toxic Substances), UNA Universidad
Nacional, Heredia, Costa Rica. They had a mean length of 7.3 ±
0.5 mm and a weight of 8.8 ± 1.0 mg. and
were cultured in laboratory of ECOTOX. The crustaceans were
kept in glass beakers of 200 ml in a temperature controlled room
(18-22°C) with a light : dark cycle of 16:8 hr. They were fed yeast,
cereal leaves, tetramin (0.5 ml l ), Se (5.2 ml l ), B (0.095 ml l ),
sp. (0.275 ml l ) and sp. (0.57 ml l ). Only
neonates from adults that already had one or more clutches were
used for the tests. Neonates between 24 and 72 hr old were taken
for acclimatization (<24 hr) and used for testing.
The tested pesticides were chlorpyrifos (CAS nr.
2921-88-2, Dr. Ehrenstorfer, purity 98.4 ± 0.5%) and ethoprophos
(CAS nr. 13194-48-4, Dr. Ehrenstorfer, purity 91%). All tests were
conducted in the ECOTOX laboratory. Stock solutions of the
pesticides were made by diluting chlorpyrifos in acetone (1:1) and
ethoprophos in deionized water from a Milli-Q water purification
system (Millipore). Test concentrations for fish were prepared by
diluting the stock solution with carbon filtered, UV treated water.
For daphnids, hard reconstituted water was used, which was
prepared by adding KCl (0.008 g l ), MgSO (0.12 g l ),
CaSO .H O (0.12 gl ) andNaHCO (0.192 g l )todeionized water
(Dutka, 1989), with a conductivity of 3.96 ms cm and a pH of 8.
For control, same water was used as for the dilutions. The
maximum amount of acetone used in the concentration ranges
was also added to the acetone control.
A range finding test was performed with fish to determine
the concentration range of chlorpyrifos for the final test. The
concentration ranges for the other test were based on earlier
experiences at the ECOTOX laboratory or by using the geometric
mean of LC values present in the EPA database
(www.epa.gov/ecotox).
Materials and Methods
Test species :
Chemicals :
Daphnia ambigua Daphnia
magna
Parachromis dovii
Artemiasalina
D. ambigua D. magna
Chlorella Selenastrum
temperate
-1 2+ -1 -1
-1 -1
-1 -1
-1 -1
-1
12
4
4 2 3
50
75Effect of pesticides on aquatic ecosystems in Costa Rica
Besides acute mortality, pesticides may also cause reproduction
failure and other chronic effects, which could lead to a decline in
fish populations. Furthermore, pesticide pollution may result in
acute and chronic effects on macroinvertebrates (Castillo
2000), which could lead to changes in the benthic community
structure and species richness (Pringle and Ramírez, 1998;
Castillo 2006), and in turn change the food web structure.
Despite the high use of pesticides in Costa Rica and other
tropical countries and their possible environmental impact, there
is still relatively little knowledge about the fate and toxicity of
pesticides in tropical aquatic ecosystems compared to temperate
systems (Castillo 1997; Lacher and Goldstein, 1997;
Mortensen 1998; Pringle and Ramírez, 1998; Robinson
1999; Matlock Jr and de la Cruz, 2002; Kwok 2007;
Daam and Van den Brink, 2010). For instance, Maltby (2005)
collected toxicity data from different databases and only 11% of
the species originated from tropical regions, while more than 50%
were temperate species.There is an on-going debate on whether
species sensitive distributions (SSDs) derived from temperate
toxicity benchmark values (i.e. LC50 or NOEC values) can be
used to assess the contamination risks for tropical species as well
or reversely if it is necessary to calculate benchmark values only
based on native tropical species.
A few recent studies have found no significant difference
between the slope of SSD curves based on toxicity data for
tropical and temperate species, suggesting that SSDs obtained
based on temperate species could be applied to protect tropical
species (Dyer 1997; Maltby 2005; Kwok 2007;
Daam and Van den Brink, 2010; Rico 2010). In line with this,
Daam (2008) found no significant difference in species
sensitivity at the community level for chlorpyrifos between
temperate and tropical aquatic ecosystems. For some pesticides,
including chlorpyrifos, data are available for the comparison of
temperate and tropical species. For other pesticides such as
ethoprophos, however, this is not the case, and therefore
temperate data must be used to perform an ecological risk
assessment for tropical species.
The aim of present study was to understand the effects of
pesticides in tropical aquatic ecosystems by conducting
standardized laboratory acute toxicity tests with chlorpyrifos and
ethoprophos on two Costa Rican indigenous species,
and . Furthermore, water
samples were collected in the field to measure pesticide residue
levels in rivers and creeks receiving agricultural runoff. Standard
toxicity bioassays were performed under laboratory conditions
using these field collected water samples on and
. ChE inhibition assays were performed in the laboratory
on to evaluate the use this biomarker for exposure
assessment. Furthermore, we compared the differences in
sensitivity of these two tropical species (fish and crustacean) to
their temperate counterparts and .
et al.,
et al.,
et al.,
et al., et
al., et al.,
et al.
et al., et al., et al.,
et al.,
et al.
cladoceran
Daphnia ambigua Parachromis dovii
Daphnia magna
P. dovii
P. dovii
Oncorhynchus mykiss D. magna
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76
Acute toxicity test set up:
Environmental sampling :
Bioassays :
All tests were performed in triplicate
(n=3) with 10 individuals per treatment and performed according
to adapted standard protocols of OECD (1992) and conducted at
an ambient temperature (24.9 ± 1.2°C) and a light:dark cycle of
12:12 hr. Crustaceans were exposed in 150 ml medium water in a
glass beaker covered with parafilm, without aeration and food.
Ten fish were put in each beaker glass with 200 ml of aerated
water and were not fed during the tests. Each beaker was
continuously aerated during the tests using air pumps connected
to a glass Pasteur pipette. After two days, 150 ml of test water was
replaced by new stock solution. Oxygen concentration
(Wissenschaftlich-Technische Werkstätten (WTW) GmbH
Oxi325), pH (Corning pHmeter 220), conductivity (WTW cond
315i), water temperature (WTW cond315i) and air temperature
(Digital thermometer Cresta) were recorded at 24, 48, 72 and 96
hr. This was done for one randomly picked replicate of each
concentration. Water samples of the stock solutions and of one
random replicate per concentration at the end of each test were
taken for pesticide concentration analyses to the chemical
laboratory (LAREP) of IRET, Heredia, Costa Rica. For control and
acetone control, only one replicate of each was collected for
pesticide analysis.
Mortality was monitored every 24 hr and was defined for
fish as the lack of gill movement. A individual was
considered dead if the animal did not visually move after one
minute of stimulation, using a magnifying lens. Dead animals
were removed from the test immediately.
Eight sites within the watershed Rio
Madre de Dios located in the Caribbean lowlands of Costa Rica
downstream from large banana and pineapple plantations (Fig. 1)
were sampled during rainy season (8-10-2009). One sample of
surface water was collected from each site for pesticide analysis.
The water samples were collected in a pre-washed 4 l capacity
amber glass bottles, which were rinsed once with surface water.
At four sites (Fig. 1), additional water samples were taken for
laboratory bioassays. The bottles were kept on ice during their
transport to the laboratory (approx. half a day). A site situated
upstream of most of the plantations was assumed to be relatively
unpolluted and therefore chosen as a reference. The site near the
bridge over the "Rio Madre de Dios" (Bridge RMD) was chosen as
a relative uncontaminated site but was surrounded by plantations
and used as an experimental treatment for the bioassay. Another
site, the "Canal Pama" channel, collects drainage water from
various banana plantations and was chosen for the bioassay
because of high fish mortality that has been reported from this
site. The last site of four selected sites for the bioassays was
located at the entrance of the river into the lagoon (RMD X LMD).
At all sites oxygen concentration (WTW Oxi325), pH (WTW
pH320), conductivity (WTW cond 315i) and water temperature
(WTW cond 315i) were measured.
For the bioassays (n=3), i.e. laboratory toxicity tests
Daphnia
carried out with water from the four sites described above,
(72 hr, semi static with a 75% water exchange after every 24 hr)
and (48 hr, static) were used. was used
instead of due to availability at that moment. For both
species, ten individuals were exposed to 200 ml of field water
obtained from each site. Besides the field samples and the field
control, a negative control (n=3) with carbon-filtered, UV treated
water for fish and reconstituted water for (as described
above) was also included in the experimental setup. The tests
were conducted at ambient temperature (24.7 ± 0.9°C). Mortality
was recorded every 24 hr for and fish.
Surviving fish from toxicity tests and
bioassays were analysed for cholinesterase (ChE) inhibition. At
the end of each toxicity tests, ten fish were randomly chosen from
three replicates of each test concentration and from each of the
bioassay tests. When the number of surviving fish was less than
ten, remaining fish were used. The fish were rinsed with deionised
water and kept frozen individually in Eppendorf tubes at -18°C.
Whole fish samples were thawed and homogenized in 1 ml ice-
cold phosphate buffer (0.1 M, pH 7.2) using a Branson SLPt
sonifier. Samples were then centrifuged at room temperature
(Eppendorf 5415c, 10,000 rpm, 5 min). Supernatants were
transferred to new Eppendorf tubes and stored at -18°C until
further analysis. Protein concentration in the samples were
determined in triplicate by the Bradford method (Bradford, 1976)
adapted to microplate (Bio-Rad Laboratories 2005). Bovine -
globulin was used as standard. Dilutions were made to normalize
protein content to approximately 0.5 mg ml . Dye reagent and
protein standard were purchased from Bio-Rad. The ChE activity
was determined at room temperature (25°C) by the method of
Ellman (1961) adapted to a microplate (Lopes 1996;
Mena 2012). Acetylthiocholine (Sigma-Aldrich) hydrolysis
was measured as an increase of absorbance at 414 nm caused
by the reaction of thiocholine with DTNB (Sigma-Aldrich) to
produce yellow 5-thio-2-nitro-benzoic acid anion. Absorbance
was measured at an interval of 2.5 min for 15 min. For the
determination of ChE activity, change in absorbance between 5
and 10 min was calculated. A Thermo Multiskan Ascent
microplate reader was used for both enzymatic and protein
determinations. ChE activities were calculated as nmol of
hydrolyzed substrate per min and mg of protein. The results were
expressed as units (U) per mg of protein (U = n mol min ).
For determination of pesticides
concentration, water was extracted by solid phase extraction
(SPE) and pesticides were analyzed by gas chromatography with
mass detector (GC-MS) and by liquid chromatography with
photodiode array detector (LC-PDA). In the field samples, 16
pesticides were analyzed: ametryn, bromacil, carbofuran,
carbaryl, chlorothalonil, chlorpyrifos, diazinon, difenoconazole,
diuron, epoxiconazole, ethoprophos, fenamiphos, hexazinone,
metalaxyl, terbufos, triadimenol. They were arranged in five
pesticide groups (insecticides, nematicides, herbicides,
P. dovii
D. magna D. magna
D. ambigua
Daphnia
Daphnia
et al., et al.,
et al.,
Cholinesterase assay :
Pesticide analyses :
g
-1
-1
N.J. Diepens et al.
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77
fungicides and insecticides/ nematicides) based on Tomlin
(2003). The SPE cartridges (200 mg ENV+/6 ml Isolute or C18
500mg 3ml ) were previously conditioned with ethyl acetate (5 ml,
analytical grade of Fluka Pestanal), methanol (10 ml analytical
grade of J.T. Baker hplc solvent), deionized water (10 ml) and
methanol (5 ml). The water samples (1l) were passed through the
cartridges, than they were dried by centrifugation (2 min at 5000
rpm) and then by vacuum for 8 min. For GC-MS analyses, the
pesticides were eluted from the cartridges with two times 6 ml
ethyl acetate for 6 ml cartridges and 3 ml ethyl acetate for 3 ml
cartridges. These samples were either diluted with acetone :
cyclohexane (1:9) or concentrated to approximately 0.1 ml with
nitrogen gas and filled up with acetone : cyclohexane (1:9) to a
final volume of 1 ml. In a parallel extraction step for LC-PDA
analysis with the SPE cartridges (200 mg ENV+/6 ml Isolute),
elution was performed with 2 x 5 ml of methanol. This extracts
were evaporated at 30-350ºC under nitrogen current to 0.05 ml,
and reconstituted in 1 ml methanol / water (40:60) mixture.
Th e GC-MS extracts were ana ly ze d by gas
chromatography with mass spectrometer with an Agilent 7890A
GC and 5975C MS (Agilent Technologies, Palo Alto, USA), in
synchronous SIM Scan mode, a CTC Combipal autosampler
(CTC Analytics AG, Switzerland), and a capillary column BP35
(Agilent Technologies, Palo Alto, USA) (25 m x 0.25 mm x 0.25
µm). The data acquisition was carried out using Chemstation
software and NIST05 Mass Spectral Database. The temperature
program was 90ºC for (1 min) to 210ºC with 20ºC min , and then
up to 300ºC with 4ºC min , the interface was held on 280ºC, the
ion source on 230ºC, the injector on 230ºC. The sample (1 µl) was
injected in splitless mode. Pesticides concentrations were
quantified by external calibration.
LC-PDA analyses were performed using a Shimadzu
HPLC LC-10AD with an SPD-M10A diode array detector
(Shimadzu, Kyoto, Japan). The chromatographic column was a
LiChroCART HPLC RP-18e column (125 mm x 3 mm x 5 µm
particle size, Merck, Germany). 50 µl of extracts were analyzed.
The mobile phase consisted of 20 mM sodium acetate in ultra
pure water/methanol 56:44 (solvent A) and methanol (solvent B).
Mobile phase was delivered at a flow rate of 0.5 ml min . Gradient
elution program started from 100% of solvent A, decreased to
50% Ain 15 min and held for 5 min; decreased to 20% Ain 5 min
and held for 5 min and finally increased to 100% A in 5 min and
held 5 min. The total run time was 45 min. Identification was
performed using retention time and the UV-spectra of the
pesticides included in the analysis. Quantification was performed
by the external standard method. Good recovery yields were
obtained for all pesticides between 77 and 105%.
To calculate LC and LC values andtheir 95%
confidence limits, log-logistic analyses were performed using the
statistical software Genstat, 12 edition (Lawes Agricultural Trust,
VSN International Ltd.). The LC concentrations were converted
from µg l into molarity (mol l ) by using molecular weight
-1
-1
-1
-1
th
-1 -1
Data evaluation : 50 10
50
(chlorpyrifos: 350.6 g mol and ethoprophos: 242.3 g mol
(Tomlin, 2003)). All calculations were done using nominal
concentrations. Adjustments for control mortality were made by
the program. The following formula, modified from Schroer
(2004), was used to construct a dose response curve.
(1)
Where 'y' is the fraction of dead animals (dimensionless), (conc)
is the applied dose in µg l on basis of the nominal concentrations
at t= 0 and the parameters are: a(ln LC ), b (slope in l µg ) and c
(fraction of background effect or mortality in the control). The
parameters were provided by the Genstat program.
From the bioassay results, box plots were created using
statistical program PASW (SPSS version 18). To detect spatial
variation in effects among the different sampling sites, a one-way
ANOVAwith a LSD post-hoc test was conducted in PASW.
For comparison of sensitivity of temperate versus
tropical species to ethoprophos and chlorpyrifos, species
sensitivity distribution curves (SSDs) were plotted using
previously reported g eometric mean LC values of the t emperate
species rainbow trout and A
temperate and tropical SSD for fish for chlorpyrifos was plotted
using the ETX 2.0 software (Van Vlaardingen 2004) that uses the
method of Aldenberg and Jawarska (2000) to generate the SSD
distribution, the derived hazard concentrations HC (hazard
concentration that affec ts 5 % of the species), HC and their 95%
lower and upper confident limits. The SSD is described by a
statistical cumulative frequency distribution of either LC or
NOEC values, based on a log normal distribution. Acute toxicity
data (LC , 96 hr) from the EPA database (www.epa.gov/ecotox)
were used here to construct the SSDs. If multiple LC data were
available for one species, the geometric mean was taken. Since
only two species were available between 23.5N-23.5S
(classification for tropical species by (Kwok 2007)), all
species between 35N and 35S, with at least one distribution limit
within the tropical region, were used. Other species outside this
range were categorised as temperate. Overlap was allowed
outside the range of 23.5N-23.5S. Using these criteria, 12
tropical and 13 temperate fish species were available to
construct SSDs for chlorpyrifos. For ethoprophos, only seven
different species were ava ilable of which none could be classified
as tropical and only three as temperate according the criteria
above, therefore no SSD was made. The temperate and tropical
distributions were compared on significant slope differences
using a Kolmogorov-Smirnov test (sign. level 0.05) (PASW
statistics 18).
The analytical recovery for chlorpyrifos was 99.8% and
for ethoprophos 97.9%. In the tests performed with
hardly any dissipation of the pesticides was observed. This was in
-1 -1
-1
-1
et al.
Oncorhynchus mykiss D. magna.
et al.,
D. ambigua,
50
50
5
50
50
50
50
Comparison of temperate versus tropical species
sensitivities :
Results and Discussion
Effect of pesticides on aquatic ecosystems in Costa Rica
1 + e -b(lnconc-a)
1-c
y conc( ) =
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78
contrast to the tests performed with , where dissipation
was up to 99.3%.The fish systems were aerated for 96 hr, which
could have led to high volatilization of both chemicals during the
test since both chemicals have a relatively high Henry's law
constant (0.478 Pa m mol at 25ºC for chlorpyrifos and 0.0135 Pa
m mol at 25ºC for ethoprophos (Tomlin, 2003). For both species,
nominal values were used for further calculations.
All mortality levels in the controls were below 20%
(maximum acceptable level set by the (OECD, 1992) for )
except for the test with and ethoprophos,in which
33% mortality was observed in the control. These test results are
used for further calculations because they still yielded a clear
dose response relationship but the results should be interpreted
with caution.
Results of acute toxicity tests showed that
was more sensitive to both organophosphates (OPs) compared
to and that chlorpyrifos was more toxic to these two
species than ethoprophos, although they have a similar mode of
action (i.e. both are neurotoxins that cause inhibition of AChE)
(Table 1 and Fig. 2). The difference in toxicity of two pesticides
and in species sensitivity have been reported earlier for other OPs
P. dovii
Daphnia
D. ambigua
D. ambigua
P. dovii
3 -1
3 -1
(Persoone 1985, cited in (Varó 2000)). The difference
in sensitivity may be the result of differences in enzymatic
expression or metabolism (Boone and Chambers, 1997).
Furthermore, arthropods crustaceans are usually the most
sensitive taxonomic group to insecticides, i.e. compared to
vertebrates and other aquatic groups (Maltby 2005; Daam
and Van den Brink 2010). The variation in toxicity between two
compounds could also be explained by difference in chemical
structure leading to different physico-chemical properties such as
solubility and hydrophobicity and thus to difference in uptake and
elimination of the compound by the individuals.
For no toxicity data have previously been reported
for both OP compounds. The LC value of reported in this
paper for ethoprophos is similar to the earlier reported LC values
for samespecies and compound (96 hr LC : 54, 158 and 220 µg l ).
The fish used in these experiments were older and therefore
possibly less sensitive, which could explain the slightly higher
LC values. Compared to the standard temperate test species on
(geometric mean 96 hr LC was 19 µg l for
chlorpyrifos (n=13) and 3153 µg l for ethoprophos (n=6)),
was less sensitive for chlorpyrifos and more sensitive to
ethoprophos, both within an order of magnitude.
et al., et al.,
et al.,
P. dovii
P. dovii
O. mykiss
P. dovii
50
50
50
50
50
-1
-1
-1
Dose response curve ethoprophosParachromis dovii
Log concentration
Measured mortality
1 10 100 1000
Mortality (%)
100
80
60
40
20
0
(B)
Dose response curve chlorpyrifosParachromis dovii
Measured mortality
(A)
Mortality (%)
100
80
60
40
20
0
Log concentration
1 10 100 1000
Dose response curve Daphnia ambigua ethoprophos
Mortality (%)
100
80
60
40
20
0
Log concentration ( g l )m-1
1 10 100 1000
Measured mortality
(D)
Fig. 2 : Dose response curves for (A and B) and (C and D) and for chlorpyrifos (A and C) and ethoprophos (B and
D) (n=3 with 10 individuals per replicate)
Parachromis dovii Daphnia ambigua
Dose response curve chlorpyrifosDaphnia ambigua
Measured mortality
Mortality (%)
100
80
60
40
20
0
(C)
Log concentration ( g l )m-1
1 10 100 1000
N.J. Diepens et al.
Online Copy
The LC of for chlorpyrifos was higher than
the earlier published LC value of 0.035 µg l (95% CI 0.032-
0.037 µg l ) (Harmon 2003). Ethoprophos showed a steep
dose response curve, which indicates that a small change in
concentration can result in a large change in the mortality
response (Fig. 2D). This was illustrated by LC value that was
very close to the LC value (Table 1). was 10 to 100
times more sensitive than the standard temperate test species,
(geometric mean of LC : 0.8 µg l for chlorpyrifos (n=2)
and 63.9 µg l for ethoprophos (n=2)). LC values for
chlorpyrifos were found lowest among arthropods and fish
species in de EPAdatabase. Tropical HC for chlorpyrifos derived
by Maltby (2005) from an SSD with acute toxicity data for
arthropods had a value of 0.06 µg l (95% CI 0.002-0.16) and was
not statically different from the temperate value (0.13 µg l ). The
lower confidence level of HC derived from an SSD gives a
conservative estimate of the ecosystem threshold concentrations
(Maltby 2005). With a LC value below the tropical HC ,
is a sensitive species, therefore they could be used as a
tropical freshwater indicator species instead of in
tropical risk assessments. Furthermore, this species is easy to
culture and is suitable for routine toxicity tests (Harmon
2003). Moreover, sp. are taxonomic stable, sensitive to
wide range of chemicals and widely distributed (Sarma and
Nandini, 2006).
The physico-chemical parameters of the surface water
measured at the field sites showed natural fluctuations in pH and
conductivity; i.e. higher pH and conductivity levels towards the
sea. In the tropics, it should be taken into account that warmer
water can contain less oxygen. In Canal Pama, the lowest oxygen
concentration (1.84 mg l ) was measured, dead fish were
observed and a change in water colour (from brown to green) was
observed within few hours, indicating a rapid algal bloom. With five
different pesticides (difenoconazole, epoxiconazole, ethoprophos,
fenamiphos, triadimenol) detected, second highest number of
pesticides were detected in Canal Pama (Table 2). A few days
before the analysis it had rained heavily (local meteorological
data), probably causing runoff of pesticides and nutrients from
many banana plantations that drained into Canal Pama and at this
time fish mortality was observed. The highest concentration was
found for the nematicide fenamiphos (2 µg l )(Table 2), which was
lower than the acute 96 hr LC for fish, crustacean and algae but
slightly higher than 48 hr LC for aquatic invertebrates (EU
FOOTPRINT).The death of fish at the time of sampling (a few days
after the first fish mortality) was probably caused due to
50
50
10
50
50
50
5
5
50 5
50
50
D. ambigua
et al.,
D. ambigua
D.
magna
D. ambigua
et al.
et al., D.
ambigua
D. magna
et al.,
Daphnia
-1
-1
-1
-1
-1
-1
-1
-1
all the
The SSD (Fig. 3) shows that the LC of falls within
the confidence limits (95%) of tropical HC but outside the range
of the temperate. Based on this SSD, tropical fish species were
indicated as less sensitive to chlorpyrifos, yet no statistical
difference was detected between the temperate and tropical
distributions (p=0.975). This is consistent with a previous
comparison study for fish performed by Dyer (1997) who
found no difference between tropical and temperate sensitivity for
carbaryl, lindane, malathion, pentachlorophenol and phenol.
Moreover, Rico (2010) (methylparathion) and Rico
(2011) (malathion and carbendazim) showed that three
indigenous tropical fishes and freshwater invertebrates species
of the Amazon were not more sensitive than their temperate
counterparts.
Castillo (1997) recommended the guapote fish as a
tropical freshwater standard test species because of its economic
value and/or sensitivity. Its economic value is considerable since
it is popular in local fisheries and sport fishing (tourism) and is
used for human consumption. In addition, this fish species is easy
to cultivated, lay a substantial number of eggs (800-1000) and is
therefore suitable for toxicity testing. However, the results show
that the guapote fish is not particularly sensitive for the tested
compounds. Therefore, in order to protect 95% of tropical fish
species from chlorpyrifos, an uncertainty factor of 100 should be
applied to its LC to obtain a threshold value that lies within the
confident limits of tropical HC (Fig. 3). More toxicity tests need to
be conducted to reveal its sensitivity to other toxicants.
50
50
50
5
P. dovii
et al.
et al. et al.
et al.
79
Table 1 :
LC (µg l ) (95% CI) LC (µg l ) (95% CI) LC (mol l )
Results of acute toxicity tests for chlorpyrifos and ethoprophos with and
Chlorpyrifos 53 (29, 98) 117 (87, 158) 3.3*10
Ethoprophos 73 (43, 125) 242 (179, 328) 1.0*10
Chlorpyrifos 0.021(0.013, 0.034) 0.039 (0.029, 0.053) 1.1*10
Ethoprophos 7.01 (6.66, 7.37) 7.63 (7.30, 7.97) 3.1.10
Parachromis dovii Daphnia ambigua
Parachromis dovii
Daphnia ambigua
10 50 50
-1 -1 -1
-7
-6
-10
-8
Fig. 3 : Temperate and tropical species sensitivity distribution (SSD) for
chlorpyrifos and fish
0.0
0.2
0.4
0.6
0.8
1.0
0.01 0.1 1 10 100 1000 10000 100000
Potentially affected fraction
SSD fish chlorpyrifos
LC ( g l )
50 m-1
Temperate: HC =0.66
(0.10-2.09) HC =
16.29 (6.37-41,68)
5
50
Tropical: HC = 1.36
(0.11- 6.05) HC =
75.30 (22.00-257.77)
5
50
Effect of pesticides on aquatic ecosystems in Costa Rica
Online Copy
Castillo (1997). Earlier sampling in the catchment showed
that 67% of the samples taken from RMD, the following seven
pesticides were frequently found in water and/or sediment:
chlorothalonil, bromacil, difenoconazole, diuron, ethoprophos,
esfenvalerate and cypermethrin. In this study, bromacil,
esfenvalerate and cypermethrin were not found in any of the
samples. In general, epoxiconazole and ethoprophos were most
frequently detected at respectively six and five of the eight
samples, while only traces of chlorpyrifos were detected at Caño
Azul. This reflects the wide use of ethoprophos and its mobility
through the watershed, possible due to its high solubility (700 mg
l at 20ºC (Tomlin, 2003)). Robinson (1999) found that 30%
of the applied ethoprophos runs off into rivers while the runoff
potential of chlorpyrifos is low (Ware and Racke, 1993). Although
chlorpyrifos is widely used in the fruit protection bags, its leaching
potential due to rain events could be minimal because of its low
solubility (1.4 mg l at 25ºC (Tomlin, 2003)) and because the
product is the applied to the inside of the bag, thus with limited
exposure to rain. When reaching the water column, the
compound likely to bind to organic material in the sediment or
living animals and may bioaccumulate in the biota (Varó ,
2000; Varó , 2002). On the other hand, it is rapidly
metabolized into less toxic molecules and eliminated from the
body (Ware and Racke, 1993; Barron and Woodburn 1995). More
research is required to determine the fate of chlorpyrifos in the
plastic banana protection bags.
Ethoprophos concentrations measured in this study (max
concentration in water 1µg l ) were 10 fold higher than previous
reported concentration while chlorpyrifos concentrations were
under the detection limit in this study. Calculating the risk quotient
(maximal environmental concentration/predicted no effect
concentration for most sensitive species in this study (LC
et al.
et al.
et al.
et al.
-1
-1
-1
In Cent ra l Am er ic a, ma xi mu m e nv ir on me ntal
concentrations found in water, sediment and biota were higher for
chlorpyrifos (water 0.18 µg l ; sediment 34.2 µg kg ; biota 8 µg
kg (Castillo ., 1997)) than for ethoprophos (water 0.10 µg l
(Unpublished data); sediment 1.98 µg kg (Robinson .,
1999).
-1 -1
-1 -1
-1
et al
et al
50
combination of multiple stressors i.e.; low oxygen concentration
combined withpesticide exposure (Fig. 4).
Most pesticides (ametryn, carbofuran, carbaryl,
ch lo rpyrifos, diuron, difenoconazole, epoxico na zo le ,
ethoprophos, fenamiphos) were found in Caño Azul (Table 2) as
was hypothesized due to the extensive drainage of banana and
pineapple plantations but no dead fish or algae blooms were
observed here. Carbofuran (5.0 µg l ) and diuron (2.0 µg l ) were
found at high concentrations. At the control site of Rio Madre de
Dios (RDM control) no pesticides were found confirming its
suitability as use as a control (Table 2). From the Caño Azul to the
crossing of RMD with Freeman (RMD X Freeman), the
concentration and the number decrease from eight pesticides to
one. The decrease of pesticides can be explained by biological
and chemical degradation, such as photolytic degradation,
vaporization and sorption to organic matter. Compared to
temperate systems, these processes can be faster due to the
tropical conditions (see (Daam and Van den Brink 2010) for a
review), though many of the detected pesticides are known to be
persistent in water. In lagoon, the amount and concentration of
the pesticides increased probably due to entry of Canal Pama and
other side streams. Especially, ethoprophos (0.9 - 1.0 µg l ) and
epoxiconazole (0.2 - 0.3 µg l ) has been found consistently
throughout the whole lagoon.It should be noted that one single
pesticide and physico-chemical parameter analysis is not
sufficient to obtain a full overview of pesticide concentration
dynamics due to the spatial and temporal variation in applications
and rain events. Currently, a long-term monitoring program (by
the TROPICA project and IRET) is running, which aims at the
determination of pesticide concentrations in water and sediment
in this watershed and evaluates the overall risks of pesticides to
native species in this ecosystem. This will give important insight to
the dynamics in such systems.
In general, the most frequently found pesticide groups in
this study were fungicides followed by herbicides and insecticides
/ nematicides (Table 2), which is in accordance with the findings of
-1 -1
-1
-1
80
Table2 :
Pesticides RMD control Bridge RMD Caño Azul Canal Pama RMD X Freeman LMD X RMD LMD mid LMD X Pacuare
Concentrations (µg l ) of different pesticides per sample site found during one sampling time
metryna 0.5 traces 0.06 0.06
arbaryl 0.4
arbofuran 5.0
hlorpyrifos traces
hlorothalonil 0.05 traces 0.07 0.05
iazinon 0.05
ifenoconazol 0.8 0.3
iuron 2.0 0.1 0.1 0.2
poxiconazol 0.6 0.6 0.1 0.2 0.3 0.2
thoprofos 0.1 traces 0.9 0.9 1.0
enamiphos 2.0
etalaxyl traces
riadimenol 0.07
-1
A
C
C
C
C
D
D
D
E
E
F
M
T
N.J. Diepens et al.
Online Copy
Fig. 5 : Bioassays for (A) (72 hr) and (B) (48 hr) showing mortality per sample site in the watershed Rio Madre de Dios. Values are mean
of three replicates SD. Differences in letters indicate sites with significant difference ( =0.05)
P. dovii D. magna
+a
Bioassay 72 hrParachromis dovii
(A)
aa
a
aa
40
30
20
10
0
Mortality (%)
Control
RMD
Bridge
RMD
Canal
Pama
Lagoon
RMD
Negative
control
Bioassay 48 hrDaphnia magna
(B)
a
bba
cc
40
30
20
10
0
Mortality (%)
Control
RMD
Bridge
RMD
Canal
Pama
Lagoon
RMD
Negative
control
Therefore, it proved to be a good indicator of pollution and is
useful for rapid detection of environmental contamination. The
toxicity test, showed that was more sensitive than
for the evaluated compounds. Since it is an indigenous
tropical species, it might be even more suitable as an indicator
species in the tropics and replace in a tropical test
battery.
In the laboratory toxicity tests with different concentrations
of ethoprophos and chlorpyrifos, there was a significant treatment
effect on ChE activity analysed in whole fish tissue (Fig. 6Aand 6B)
(one-way ANOVA). Post-hoc comparisons showed that for
ethoprophos the three highest concentrations were not
significantly different, and for chlorpyrifos 10 and 30 µg l , and the
highest two concentrations were not significantly different (Fig. 6).
The unexpected high fish mortality rate (33%)at 3 µg l chlorpyrifos
concentration (Fig. 2A) is probably due to other factors, such as
biological effects or a fungal infection, than the pesticides toxicity
since high ChE activity does not reveal any inhibition. Fig. 6 shows
D. ambigua D.
magna
D. magna
-1
-1
divided by a safety factor 10) gives a rough estimate that there is a
substantial risk in tropical ecosystems for both compounds with
risk quotients of 16 and 46 for ethoprophos and chlorpyrifos
respectively. Besides acute effects, chronic effects may occur at
lower concentrations.
Fig. 5 shows the difference in response of the bioassays
performed with and among the sampled sites.
Mortality of both species was below 40% at all sampling sites. For
fish, no significant difference (p=0.482) in mortality between sites
could be detected. Significant differences in mortality among sites
(p=0.001) were found for . The LSD post-hoc test
showed that the observed mortality at Bridge RMD was
significantly different from Canal Pama (p=0.001) and lagoon
(p=0.007) but similar to both controls. RMD and Lagoon RMD
were not significantly different (p=0.290) but differed in observed
mortality with all other sites.
For a higher mortality in the most polluted sites
and difference among sample sites could be detected (Fig. 5).
P. dovii D. magna
D. magna
D. magna
81
Fig. 4 : Conceptual model of environmental processes possibly leading to fish kills after a rain event
Runoff
Nutrients
Pesticides Death fish
Death algea
grazers
Algae bloom
Rain event
Low dissolved oxygen
concentrations
Higher water
temperature
Decaying organic
material
Sun
Pesticide
application
Effect of pesticides on aquatic ecosystems in Costa Rica
Online Copy
Fig. 7 : ChE inhibition in after 72 hr exposure to surface water
taken from watershed Rio Madre de Dios. Values are mean of three
replicates SD. Differences in letters indicate that the treatments were
significantly different from each other (alpha =0.05).
P. dovii
+
ChE activity in bioassays
acd
dc
d
ba
80.00
60.00
40.00
20.00
0.00
Activity ( n mol mg protein min )
-1 -1
RMD
Control
Bridge
RMD
Canal
Pama
Lagoon
RMD
Negative
control
effects at individual level, such as changes in behaviour,
reproduction and feeding success, which could affect populations
in long term.
ChE activities measured in the bioassay (Fig. 7) shows
that ChE activity measured in the negative control of the toxicity
tests was significantly similar to ChE activity levels measured at
the field control site (RMD control). ChE activity in fish exposed to
water from Bridge RMD and Canal Pama did not differ from each
other and were both significantly lower than control. However,
Canal Pama was similar to Lagoon RMD. The strongest ChE
inhibition was found in the Lagoon RMD, where only one OP could
be detected. This was surprising since there was also a higher
dilution in the lagoon with incoming seawater from the Carribean
Sea and from other adjacent streams. These other streams,
however, could bring other OPs and carbamates that we did not
look for in our chemical analyses. During the bioassays, no fish
mortality was observed, the ChE inhibition however, showed a
clear response i.e. 37% inhibition in Canal Pama and 62%
inhibition in the Lagoon RMD. This again indicates the usability of
this biomarker as an early warning indicator of exposure to OPs
and carbamates. Yet, the majority of the pesticides found
consisted of fungicides and herbicides, which have a different
mode of action than OPs and carbamates. Therefore, a
combination with other biomarkers and/or toxicity tests is needed
in order to detect pesticide contamination and asses its
environmental risks in this watershed.
In conclusion, the proposed tropical species were similar
or more sensitive than their temperate counterparts. More toxicity
data are needed about to evaluate possible similarities or
respectively differences between temperate and tropical species
to chemical contamination. Further studies are also needed to
investigate how protective safe environmental concentrations,
derived with LC sof temperate species, are forprotecting tropical
50
that the amount of ChE decreased with increasing concentrations
with significant difference for ethoprophos between the control and
the first concentration and for chlorpyrifos between the first and
second concentration. This and the threshold value of 20%
inhibition (Varó 2007) indicates that inhibition begins for
ethoprophos at 20 µg l (32%) and for chlorpyrifos at 10 µg l
(38%). For both pesticides noimmobile or dead fish were observed
at these concentrations while ChE inhibition showed already sub
lethal effects. Therefore, this biomarker could be used as an early
warning indicator. Since ChE recovery was low or even assumed
to be irreversible it can provide long-term exposure information
instead of a snapshot by chemical analysis of water samples.
Moreover, inhibition of ChE might be linked to different indirect
et al.,
-1 -1
82
Fig. 6 : ChE inhibition in P. after 96 hr exposure to (A) chlorpyrifos and (B) ethoprophos. Values are mean of three replicates SD. Difference in
letters indicate that the treatments were significantly different from each other (alpha =0.05)
dovii +
(A)
a
c
c
d
d
b
150.00
100.00
50.00
0.00
ChE activity ( n mol mg protein min )
-1 -1
Concentration ( g l )m-1
0 3 10 30 100 300
(B)
a
c
dd
d
b
250.00
200.00
150.00
100.00
50.00
0.00
ChE activity ( n mol mg protein min )
-1 -1
Concentration ( g l )m-1
0 20 60 180 540 1620
N.J. Diepens et al.
Online Copy
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Integra. Environ. Asse.
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aquatic natural communities. Both recommended tropical
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This study was funded by the TROPICA project, Swedish
Research Council FORMAS, grant nr 2005-473-3035-21 and by
complementary funding from IRET. We are grateful for the
valuable help from all people from both the ECOTOX and LAREP
laboratories of IRET, Universidad Nacional, Heredia, Costa Rica.
We also want to thank Julio Knight and his family for assistance
during the fieldwork and important knowledge of the study area.
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