Ecotoxicological studies on the pejerrey (Odontesthes bonariensis, Pisces Atherinopsidae)
Pedro Carriquiriborde and Alicia Ronco
Centro de Investigaciones del Medio Ambiente, Facultad de Ciencias Exactas, UNLP - CONICET. Calle 47 y 115, (1900) La Plata, Bs. As., Argentina.
Address correspondence to: Dr. Pedro Carriquiriborde. Centro de Investigaciones del Medio Ambiente, Facultad de Ciencias Exactas, UNLP, Calle 47 y 115, (1900) La Plata, Bs. As., ARGENTINA. Tel/Fax: (+54-221) 4229329. E-mail: pcarriqu@quimica.unlp.edu.ar
Key words: Odontesthes bonariensis; Ecotoxicology; Environmental pollution; Heavy metals; Pesticides.
The pejerrey (Odontesthes bonariensis, Cuvier and
Valenciennes, 1835) is an Atherinid fish characteristic
of the Southern sector of the ‘del Plata' basin (Bonetto
and Castello, 1985). This species not only possess ecological
relevance (it could represent the largest fish biomass,
especially in low diversity euryhaline lakes;
Baigún and Delfino, 2001), but also has regional socioeconomic
relevance due to commercial and sport fishing,
the last representing an annual economic movement
of $72 million for the Buenos Aires Province alone
(Lopez et al., 2001). In addition, pejerrey is a promissory
species for the aquaculture as a result of the high
quality of its flesh, appreciated by local and foreign
consumers; a fact that in recent years has driven important
advances related to the larvae-culture (Miranda and
Somoza, 2003), and caged culture (Colautti and Remes
Lenicov, 2001) of the species.
On the other hand, pejerrey inhabit rivers and lakes
located in the most densely populated and therefore most
highly modified regions of Argentina due to human activities.
These aquatic systems are usually natural receptors
of pollutants released to the environment from
point (industry, sewage) and non point (agriculture, urban
runoff) sources. Previous studies testify to differing
degrees of organic and inorganic contamination of
these water bodies and their biota (Ringuelet, 1967;
Catoggio, 1990; AGOSBA-OSN-SIHN, 1992; Ronco et
al., 1995; Kreimer et al., 1996; Colombo et al., 2000;
Menone et al., 2000; Villar et al., 2001).
Despite the ecological and socioeconomical relevance
of the pejerrey, and the evidence of differing
degrees of pollution in the water bodies that the species
inhabit, no published studies on the potential effects of
environmental pollutants on the wellbeing of the
pejerrey were found at the beginning of our investigations
in 1997, and little information on this subject is
yet available. As a consequence, the aim of this publication
is to review the advances in ecotoxicological aspects
of O. bonariensis obtained up to the present in
our laboratory (Carriquiriborde, 2004). The studies covered
aspects such as lethal and sublethal responses from
exposure to some organic and inorganic pollutants of
environmental relevance, the influence of some physicochemical
characteristics of the water on the toxicity
of metals, the bioconcentration pattern of metals according
to chemical speciation, and the vulnerability of the
species in representative areas of the Southern sector of
the del Plata basin.
Methodological Approach
A summary of the ecotoxicological studies conducted during the last seven years by the working group of CIMA to assess adverse effects of pollutants of environmental relevance on O. bonariensis is shown in Table 1. The general conceptual approach for studies included the following aspects: 1) Evaluation of acute lethal effects of the selected toxicants on the species, in order to understand the relative toxicity of each contaminant, and the comparative sensitivity of O. bonariensis in relation to other fish species and aquatic organisms. Additionally, it provided information on the concentration/ response and time/response functions of the selected metals and pesticides. Acute lethal toxicity tests studies were also used to assess the dependence of the adverse effects of metals in relation to the physicochemical characteristics of the water and the source and age of the fish, 2) Evaluation of sublethal effects related to the toxicokinetics and toxicodynamics of the contaminants in order to quantify the bioconcentration of chemicals and their effects at the cellular levels in two main target organs after waterborne exposure to chemicals; the gills and the liver. Effects of chemicals at the cellular levels were investigated through the assessment of the response of the nucleolar activity and the DNA integrity. Selected biological responses were also evaluated as potential biomarkers of exposure and/or effect, 3) Evaluation of the vulnerability of O. bonariensis in the natural environment in order to identify the potential risk of lethal and sublethal exposure to the species in its main natural area of distribution. For this purpose, the toxic exposure ratio (TER) (Harrass, 1996) was calculated on the basis of the information reported in previous studies about the physicochemical characteristics and chemical concentrations in representative water bodies comprising the main distribution area of the species, and the sensitivity levels obtained in our studies.
TABLE 1.
Ecotoxicological studies conducted with O. bonariensis
Study of Lethal Effects
The study of the acute lethal effects of the metals (Cd+2, Cu+2, Cr+6 and Hg+2) and the pesticides (the herbicide glyphosate and the insecticide cypermethrin)1 indicate that O. bonariensis sensitivity lies in the following order: cypermethrin > Hg+2 > Cd+2 > Cu+2 > Cr+6> glyphosate (Table 2). Sensitivity to cypermethrin was two orders of magnitude greater than Hg(II) and six orders of magnitude greater than glyphosate, the least toxic of the tested chemicals.
TABLE 2.
Relative sensitivity of O. bonariensis respect to a selected group
of chemicals with environmental relevance, and in comparison
with other well studied fish species and aquatic organisms.
The LC50 values indicate that no general rule can
be made relating to the nature of the chemical (organic
or metal) and the relative toxicity on O. bonariensis. The
observed relative order of toxicity for the tested chemicals
was equivalent to that reported for Oncorhynchus
mykiss and also for the mean available LC50 data for
fish species. However, it was slightly different to that
observed for Cyprinus carpio, which showed an inversion
of the sensitivity to Cd and Cu. The lower sensitivity
to Cd and higher to Cu was also a pattern reported
for crustaceans, and distinctively different
from that observed for O. bonariensis (Table 2).
The relative sensitivity of O. bonariensis in comparison
with other fish species was, with the exception
of glyphosate, always higher than that reported as the
average value for all available fish species. In particular,
when O. bonariensis was compared with the sensitivity
of two well known species it was found to be closer
to that reported for O. mykiss (a sensitive species) than
for C. carpio (tolerant species). In relation to other Neotropical
fish species, the sensitivity of O. bonariensis to all the studied metals ranged between 2.5 and 11 times
higher than that reported for the 15 day old Cichlasoma
facetum exposed in hard water (Bulus Rossini and
Ronco, 2004). In addition, the sensitivity of O.
bonariensis to cypermethrin was also higher than that
reported for Cichlasoma dimerus (Domitrovic, 2000),
for Aequidens portalegrensis to Hg (Hirt and Domitrovic,
1999) and Cd (Hirt and Domitrovic, 2002), and for Cnesterodon decemmaculatus to Cu (Villar et al., 2000).
In comparison with another relevant group in the
aquatic environment such as the crustacean, the sensitivity
observed for O. bonariensis was one and two orders
of magnitude higher for cypermethrin and Cd, respectively
and of the same order of magnitude for Cu,
Cr, and glyphosate (Table 2). However, in comparison
with D. magna, a micro-crustacean broadly used in toxicity
bioassays, O. bonariensis presented higher sensitivity
only to cypermethrin, a comparable sensitivity to
Cd and glyphosate, and a markedly lower sensitivity to
Cu and Cr.
The Figure 1 shows an example of the dependence
of the LC50 with the time of exposure2 . The dependence
of Cr+6 LC50 values with time show a good agreement
with the model derived from the critical body residue
concept (McCarty and Mackay, 1993), and was
consistent with the fact observed for Cu and Ni, that
binding of the metals to fish gills predicts acute toxicity
better than free-ion activity (Meyer, 1999). In the
figure, it is also possible to observe that the age of the
organism and water hardness influence the values of
the LC50(t) (a slight but significant increase of Cr+6 toxicity was observed when organisms were younger and
the water hardness lower). However, the effect induced
by these two variables was accurately interpreted by the
same time-response model. A similar behavior was also
seen for Cd+2 exposed organisms (data not shown). In
contrast with the model presented for Cu+2 by Villar et
al. (2000), the developed model would provide a mechanistically
based description of the relationship between
exposure time and the LC50 for the studied metals.
FIGURE 1. Time dependence of LC50 values for O. bonariensis exposed to Cr(VI). Cr 30 H: 30d old organism exposed to Cr(VI) in hard water (255 mg CaCO3/L); Cr 30 S: 30d old organism exposed to Cr(VI) in soft water (30 mg CaCO3/L); Cr 15 H: 15d old organism exposed to Cr(VI) in hard water (255 mg CaCO3/L); black geometric figures: indicate mean LC50 values; bars: 95% confidence limits; curves: adjusted model to the LC50 values; r2: coefficient of determination; horizontal lines: incipient values (LC50(¥)).
The dependence of the estimated LC50 for O. bonariensis with water hardness was more evident for Cd+2 than for Cr+6, and the relationship between the logarithms of these two variables was satisfactorily described by an empirical model proposed for cadmium by USEPA (2001), showing a positive linear correlation (Fig. 2). The figure also shows that O. bonariensis is within the group of sensitive species to Cd detaching from the more tolerant group (regression lines with higher y-intercept). Also the application of a model developed by Welsh et al. (1996) for Pimephales promelas to describe copper toxicity dependence with the water chemistry was used here to explain the behavior of this metal for the case of pejerrey. This model accurately predicted acute lethal effects of Cu on O. bonariensis exposed at different water hardness (data not shown, see Carriquiriborde and Ronco, 2002). Therefore, these models could be used to assess the risk of metal exposure for the species in the field as a function of physicochemical characteristics of the water bodies.
FIGURE 2. Dependence of Cd(II) LC50 for O. bonariensis with water hardness, and its comparison
with other fish species (EPA, 2001).
Symbols: LC50 for different fish species (see insert); solid line and equation: linear regression
function fitted for O. bonariensis at different hardness values; dotted lines: linear regression
function fitted to Cd(II) LC50(96) data published for other species at different hardness values.
Sublethal effects3
Studied sublethal effects in laboratory exposure experiments
covered the assessment of the bioconcentration
and the response of nucleolar activity to chromium and
cadmium; and the genotoxicity of cadmium, copper and
chromium in gill and liver tissues.
Bioconcentration depends on the relation between
the uptake and elimination rate of chemicals. The study
of the bioconcentration4 of the Cd+2 and Cr+6 in the gills
and liver of O. bonariensis showed that both metals
bioconcentrate in the studied organs, each one following
a characteristic pattern (Fig. 3). Small concentrations
of waterborne Cd (» 0.1 mM) were highly accumulated
in the gill (1000 fold), but only little Cd was
accumulated in the liver (60 fold). On the other hand,
relatively high concentrations of waterborne Cr (»20
mM) were moderately bioconcentrated in the gill (only
2.5 fold), but presented low accumulation in the liver.
FIGURE 3. Chromium and cadmium
bioconcentration patterns for O. bonariensis after 384h exposure.
Values: represent approximate micromolar
concentrations; shades: symbolize concentration
levels.
Under the assayed conditions, cadmium is mainly
present as the free cation, Cd+2, that quickly binds to
gill surface to be later slowly up-taken (Pagenkopf, 1983;
Playle 1998) passively through Ca+2 channels (Verbost et al., 1989; Wicklund Glynn et al., 1994). In a different
way, chromium is mainly present as the anion CrO4-2/Cr2O7-2 (Kotás and Stasicka, 2000) and it is believed
that the metal is up-taken through transporters for SO4 -2 and PO4 -3 (Wetterhahn, 1979; Ottenwälder and
Wiegand, 1988). As a consequence, these properties and
mechanisms characteristic of each metal could be responsible
for the differences observed in the pattern and
the magnitude of the bioconcentration of Cd+2 and Cr+6 in the gills and liver of O. bonariensis. The higher affinity
of the Cd+2 for the gill surface described by other
authors agrees with the higher accumulation of the metal
observed in this tissue. Furthermore, this could explain
the observed correlation between the model derived from
the CBR concept and the LC50(t), and may also explain
the higher lethal acute toxicity of Cd+2 in comparison
with Cr+6. Moreover, according to Norey et al.
(1990), the bioconcentration pattern of Cd observed in O. bonariensis, higher in the gill than in the liver, places
the pejerrey within the group of sensitive species such
as the salmonids to this metal.
The nucleolar activity is mainly given by the product
of ribosome biogenesis, which is directly linked with
the protein synthesis of the cell. In the yeast, 75% of the
most highly transcribed genes are ribosomal protein
genes, representing an important energetic cost for the
cell (Jelinsky and Samson, 1999). Consequently, it is
tightly regulated (Leary and Huang, 2001; Jorgensen et
al., 2002) and is very sensitive to factors that induce cellular
stress and DNA damage (Rubbi and Milner, 2003).
Our experiments demonstrate that the nucleolar activity5 in the gill and liver of O. bonariensis was affected by
sublethal concentration of Cr+6 and Cd+2, also showing a
characteristic response depending on the metal and the
organ. Whereas a general reduction of the nucleolar activity
(assessed by the mean nucleolar area) was induced
by Cr+6 both in the gills and the liver, a decrease of this
parameter was induced by Cd+2 only in the gill. Conversely,
a significant increase of the nucleolar activity
was caused by this metal in the liver. The nucleolar activity assessment end points NOEC and LOEC found for
each metal and tissue are shown in Table 3.
TABLE 3.
Summary of sublethal effects induced by Cd, Cr, and Cu in O. bonariensis
DNA integrity6 , assessed by means of the comet
assay identifies double and simple strand breaks and
labile alkaline sites induced in the DNA by chemicals.
Results showed that all the tested metals (Cd+2, Cr+6,
Cu+2) were able to induce genotoxic effects on the studied
tissues of O. bonariensis. It was evidenced as a significant
increase in the frequency of DNA damaged
nucleus presented in the gill and liver of metal exposed
fish with respect to those in the control group. As was
expected from a waterborne exposure, the three metals
induced higher DNA damage in the gill than in the liver
in a time dependent manner (Carriquiriborde et al.,
2002a, 2003). The NOEC and LOEC found for each
metal and tissue is shown in Table 3.
The observed sublethal effects can also be linked
to the bioconcentration pattern of the Cd and Cr, through
the CBR concept. In the gill, organ with the highest concentration
of both metals, the nucleolar activity was significantly
reduced and was the only organ where DNA
damage was observed. Reduction of the nucleolar activity
can be related to the demonstrated capacity of Cd+2 at cytotoxic concentrations to induce "in vitro" and "in
vivo" general inhibition of cellular proliferation, gene
expression, and protein synthesis (Beyersmann and Hechtenberg, 1997). In addition, it was observed that
Cd induced DNA damage only in the gill, a reported
effect only at cytotoxic concentrations of this metal
(Beyersmann and Hechtenberg, 1997). The reduction
caused by Cr+6 can be linked to the capability of this metal
to induce cross links with proteins and DNA during its
intracellular reduction from Cr+6 to Cr+3 (Stearns and
Wettterhahn, 1994). In addition, both metals are able to
generate general cellular injury by means of oxidative
stress (Stohs and Bagchi, 1995). On the other hand, in
the liver, while chromium (the higher concentrated of both
metals, Fig. 3) still decreased the nucleolar activity and
induced DNA damage, cadmium only increased nucleolar
activity. The observed increase of the nucleolar activity
induced by cadmium in the liver is consistent with
the observation that small quantities of the metal induce
protein synthesis, gene expression and cell proliferation
(Beyersmann and Hechtenberg, 1997). The same authors
state that the stimulation induced by cadmium on cellular
signals at various stages of mitogenic cascades, protooncogene
expression, DNA synthesis, and cell proliferation
may be a clue for the interpretation of the
carcinogenic action of this metal.
Vulnerability of O. bonariensis in the natural environment
Vulnerability of the species to be adversely affected by the Cd, Cu, Cr, and cypermethrin was assessed considering
the information generated on the lethal and
sublethal effects of these metals and information on
water physical-chemistry and metal concentration reported
for water bodies located within the main distribution
area of the species.
It was shown in the previous sections that physicochemical
characteristics of the water affect metal toxicity
on O. bonariensis. The effect of the hardness, pH
and DOC on Cd+2 and Cu+2 lethal acute toxicity can be
easily assessed by means of empirical models, but no
model is yet available for Cr. Consequently, in order to
predict more accurately the vulnerability of O.
bonariensis to these metals within the main area of distribution
of the species, values of hardness, pH, DOC,
and metal concentration were collected from the available
literature and analyzed.
The reported pH range for the Lower Paraná River
was 6.1-7.9 (Villar et al., 1999), for the Río de la Plata
5.8-9.6 (AA-AGOSBA-ILPLA-SHN, 1997), for
Samborombón River 8.3-8.4 (Mercado, 2000), for
Chascomús Lake 8.0-9.0 (Conzonno and Claverie,
1990), and for San Miguel del Monte pond 8.23-9.20
(Gabellone et al., 2001). In addition, mean hardness
values, estimated as the sum of Ca+2 and Mg+2 concentrations
reported in the literature, were expressed as
calcium carbonate. Hardness interval values found for
the Lower Paraná River were 22.5-32.0 mg CaCO3/L
(estimated from Villar et al., 1999), and for the Río de
la Plata, 22.5-217.5 mg CaCO3/L (estimated from AAAGOSBA-
ILPLA-SHN, 1997). Mean hardness values
calculated for the lower, middle and upper sectors of
the Samborombón River were 86, 87, 155 mg CaCO3/
L (estimated from Mercado, 2000). Hardness ranges
obtained for Vitel and Chascomús ponds were 94.9-
205.4 mgCaCO3/L (estimated from Olivier, 1961), and
61.5-161.3 mgCaCO3/L (estimated from Conzonno and
Claverie, 1990), respectively. Hardness reported for
Salado River (Guerrero's Bridge) was 262 mg CaCO3/
L. Finally, the mean and minimum DOC values reported
were 2.8 - 4.2 for the Lower Paraná River and
4.0-7.9 mgC/L for the Río de la Plata (Villar et al.,
1999). Minimum and maximum values found for the
same parameter in some representative water bodies
from the Salado River Basin were: Vitel pond 12.0-
57.0 (Olivier, 1961) and Chascomús pond 7.7-18.7
(Conzonno and Claverie, 1990).
The maximum concentration of Cd, reported for
the lower Paraná River, was 1 mg/L (Gómez et al., 1998).
For the Río de la Plata, the range of maximum values
reported for this metal was 1.0-3.7 mg Cd/L (AGOSBAOSN-
SIHN, 1992; Villar et al., 1998), with the highest
value found in front of Berazategui. For the Salado River
Basin, the observed range in Cd concentration was from
1.0-5.0 mg Cd/L, with higher concentrations found in
the upper sector of the basin (Ronco and Argemí, 1998;
Carriquiriborde et al., 2002b). In addition, the range of
maximum values of Cr reported for the Lower Paraná River and Río de la Plata were 19.7 - 44.2 and 5 - 4,100
mg Cr/L, respectively (Villar et al., 1998; AGOSBAOSN-
SIHN, 1992; AA-AGOSBA-ILPLA-SHN, 1997).
Extremely high values were observed in the Riachuelo
River mouth with concentrations of 72,200 mg Cr/L
(Kreimer et al., 1996). The values reported for the Salado
River Basin ranged between 35 and 71 mg Cr/L, and
again, the higher levels were found in the upper sector
of the basin (Ronco and Argemí, 1998; Carriquiriborde et al., 2002b). Moreover, the range of copper concentrations
reported within the distribution area of the species
were 3-8 mg/L in the lower Paraná River, 4-12 mg/L
in Río de la Plata (Villar et al., 1999), and between 12
to 16 in the Salado River Basin, with the highest value
observed in del Monte pond (Carriquiriborde et al., 2002b). Finally, the interval of cypermethrin concentrations
reported for streams and the mouths of affluent
rivers of the lower Paraná River was 1 - 190 μg /L
(Marino et al., 2004).
A summary of the physicochemical parameter values
corresponding to the worst case scenario (lowest
values of hardness, pH, and DOC, and highest metal
concentrations) was constructed from the published field
data of water bodies and placed within the main distribution
area of the species. The LC50(¥) values corrected
as a function of these physicochemical parameters and
the toxic effect ratio (TER) values according to Harrass
(1996), represented as a quotient between the adjusted
lethal acute toxicity value and the reported field metal
concentration, is displayed in Table 4.
TABLE 4.
Vulnerability of O. bonariensis to heavy metal acute exposure
in the southern sector of the del Plata basin.
According with the data presented above it is possible
to assume that: a) the physicochemical characteristics
reported for the water bodies of the southern sector
of the del Plata basin showed a decrease of hardness
and pH values from the South to the North and from the
West to the East, showing the lower Paraná as the place
of maximum vulnerability for the species to metal exposure,
and b) the concentration of metal and
cypermethrin near or greater than those that induced
biological effects in O. bonariensis were reported for
metals in the Río de la Plata and upper Salado River Basin, and for cypermethrin in the lower Paraná River.
The TER, shows the Río de la Plata as the place
where risk of lethal acute exposures to the three studied
metals for O. bonariensis is the highest, not only as a
consequence of the elevated metal level contents reported
for its waters, but also resulting from the relatively
low values of hardness and pH, and DOC that
characterize this water body. In addition, risk of O.
bonariensis to Cd and Cu lethal acute exposure should
be considered in the lower Paraná River, as well as to
Cd in the Salado River Basin. From another point of
view, a high risk of acute lethal effects (TER<10) was
found for Cd in the whole distribution area, for Cu in
Río de la Plata and the Lower Paraná, and for Cr only
in Río de la Plata. On the other hand, the lowest TER
was obtained for cypermethrin in the mouth of affluent
rivers of the lower Paraná River which drain from ‘Pampa Ondulada', one of the most intensely cultivated
areas of Argentina, indicating a high risk of exposure
for O. bonariensis to this compound related to
agricultural activities.
Concluding Remarks and Perspectives
In the present paper, ecotoxicological studies obtained
during the last seven years in our laboratory on O. bonariensis, were reviewed. On the basis of the current
knowledge, it is possible to state that:
• O. bonariensis is a very sensitive species to environmental
pollution, and also presents a relative
response pattern to chemicals comparable to most
fish species.
• Acute lethal effects of waterborne Cd+2 and Cr+6 (and probably other metals) on O. bonariensis is
mainly related to the ability of each metal to bind
to the gill and accumulate there. The magnitude
of the metal accumulation in the gills not only
depends on its chemical nature but also on its
bioavailability, which is conditioned by physicochemical
characteristics of the water such as its
hardness. The lethal acute toxicity of these metals
also markedly depends on the age of the organism
and the time of exposure up to 48h but,
after this time variation of lethality is much lower.
• Sublethal effects of Cd(II) and Cr(VI) (and probably
other metals) are also directly related to the
distribution and concentration of the metal in the
specific tissues. Reduction of the nucleolar activity
induced by both metals is in good accordance
with the general cellular damage proposed
for these metals at cytotoxic concentrations, while
the increase of the nucleolar activity induced by
Cd may be related with its ability to stimulate
protein synthesis, gene expression and cell proliferation
at very low concentrations. As a consequence,
nucleolar activity seems to be a good indicator
of cell arrest or proliferation induced by
metals in O. bonariensis. Genotoxic effects of Cd
and Cr assessed by the COMET assay are in good
correspondence with the general response observed
for the nucleolar activity, and with the
capacity to induce DNA damage as previously
reported for these metals.
• According to the physicochemical characteristics
and chemical concentrations reported for the
water bodies included in the natural area of distribution
of O. bonariensis, and the high sensitivity
of the species to the studied environmental
pollutants, it is possible to affirm that vulnerability
to metal exposure is at a maximum in the soft
and slightly acidic waters of the lower Paraná River, and that potential risk of exposure to this
class of chemicals exist near urban areas, such as
the occidental margin of the Río de la Plata. On
the other hand, high potential risk of exposure to
cypermethrin exists in rivers draining into agricultura areas.
Although unprecedented information on ecotoxicological
aspects of O. bonariensis was summarized
in the present paper, it only represents the beginning in
understanding the effects of pollutants on this important
natural resource and emblematic fish of the shallow
ponds of the ‘pampas' region. It is clear that a great
amount and diversity of studies will still be necessary
to have a clear insight on the impacts of toxicants on
the species in its natural environment.
From the results obtained in the reviewed studies
it is possible to establish a number of futures lines of
research, however, many others would still be relevant.
For example, the presented methodology could be used
to complete the scheme presented in table 1, or to asses
the adverse effects of other pesticides and chemicals
of environmental concern. In addition, new experiments
could be conducted to asses the more recently
proposed Biotic Ligand Model as a tool to evaluate
the effect of the physicochemical characteristics on the
toxicity of metal. Furthermore, the observed response
of the nucleolar activity to metals opens a new field to
study possible mechanisms involved in such responses.
On the other hand, it would be interesting to conduct field studies in selected regions to assess the potential
risk areas and possible effects on O. bonariensis natural
populations.
According to current challenges in ecotoxicology,
studies with O. bonariensis could also be oriented to
assess: i) chronic effects of pollutants, such as endocrine
disruption, DNA damage/mutagenesis, deficiencies
in the immune system, and neurological effects; ii)
multiple effects by single pollutants, as for example
multiple target sites and multiple modes of toxic action,
and time- and organ-dependent effects; iii) effects
of complex mixtures of pollutants, assessing the effects
of wastewater treatment plant effluents, field runoff,
pollutants and their degradation products, and complexes
of chemical compounds; iv) multiple stressors, as for
example assessing the join effect of UV, temperature
and pathogens on pollutants toxicity; and v) ecosystem
complexity, such as variations in species sensitivities,
effects of propagation from organisms to populations
and ecosystems, and identification of the stressor-effect
relationship.
The challenges in ecotoxicology also call for novel
bioanalytical methodologies such as modern molecular
and genetic tools or sophisticated test systems for
routine screening. However, it will be necessary to understand
how subtle changes at the molecular level or
laboratory scale affect O. bonariensis in its environment.
It is evident that further studies will have to be
undertaken to provide better tools in the species management
and protection.
Acknowledgements
Financial support for studies was from the National Research Council of Argentina (CONICET) and the University of La Plata. Authors wish to acknowledge F Dulout (one of the PhD Thesis Directors), JC Deluca, S Picco, MP Heras from CIGEBA-UNLP; D Colautti, G Berasain from the Fisheries Office; and F Argemi, JP Streitenberger from CIMA-UNLP for their specific collaboration in different aspects of the studies summarized in this review. Also authors wish to thank the Fisheries Office of the Ministry of Production of the Buenos Aires Province for supplying test organisms, and Mr Roberto Cantarini for the supply of fish food. Cytological studies were performed in the Centre for Basic and Applied Studies on Genetics (CIGEBA), Faculty of Veterinary, UNLP.
1. Lethal acute toxicity tests were conducted following recommendations of the USEPA (1993). Static-renewal tests were employed to evaluate the 50%
lethal concentration (LC50) for Cd+2, Cr+6, Cu+2, Hg+2, cypermethrin, and glyphosate. Fifteen or 30-day old organisms were used as test organisms. Assay
were performed in hard water, (La Plata tap water; 255.3 mg CaCO3/L) and reconstituted soft water (31.4 mg CaCO3/L). Two hundred and fifty ml
polyethylene or glass test chambers filled with 200ml of test solution were utilized for acute lethal toxicity assessment of metals and pesticide, respectively.
Toxicity tests were conducted by triplicate using 5 or 10 organisms per replica in preliminary or final tests, respectively. Temperature was kept at 22 ± 1ºC
and and pH 7.2. Larvae were fed with 24h Artemia sp. nauplii once at 48h exposure. Dead fish were removed and recorded every 24h during 96h. LC50
were estimated at each recording time by means of the Probit method (Finney, 1971) using a specific software (Probit; USEPA version 1.5).
2. Time dependency of the LC50 values was explained by the following model: LC50(t) = LC50(¥) / [1- e (-ke ¥ t)]2, derived from the Critical Body Residue
(CBR) concept and more specifically the Lethal Body Burden (LBB) approach (McCarty, 1986; McCarty and Mackay, 1993).
Effects of physicochemical characteristics of the water on Cd+2 and Cu+2 toxicity were assessed by means of empirical equations obtained from
USEPA (2001) and Welsh et al. (1996), respectively. The equation used to explain the relationship between water hardness and Cd+2 LC50 was: Ln(LC50Cd)=
1.205[Ln(hardness)]-3.949. The equation used to explain the relationship between pH, DOC, and hardness and Cu+2 LC50 was: Log(LC50Cu)= 0.192(pH)
+ 0.136(pH ¥ log DOC) + 0.166(Ca) -0.981, replacing Ca by the hardness, expressed as mg CaCO3/L, as the product of 0.4 ¥ [Ca], then the equation is
Log(LC50Cu)= 0.192(pH) + 0.136(pH ¥ log DOC) + 0.0664(Hardness) -0.981. The parameters of these equations were recalculated for O. bonariensis using the LC50 values obtained in the laboratory at different hardness values.
3. Sublethal exposure experiments were performed using six-month old
juveniles exposed in 20 L polyethylene test chambers to three sublethal
concentrations of Cd+2, Cr+6 or Cu+2 for a maximum period of 384 h. Assays
were performed by triplicate using 2 organisms per replica per treatment
(time/concentration). Fish were sampled at 6, 48, 192 and 384 h.
Static renewal experiments were conducted using La Plata tap water, temperature
was kept at 22 ± 1ºC, pH was 7.2, and juveniles were fed with
adults of Daphnia sp. every 48h.
4. Bioconcentration of Cd and Cr in gill and liver of O. bonariensis juveniles
sampled at 48 and 384h were measured by means of electrothermic
absorption spectroscopy using a GBC Scientific Equipment PTY. LTD.
Model: AA902 equipped with a graphite furnace atomizer GF2000 and an
autosampler, and following the operative manual guidelines. Tissues were
previously digested following the method proposed by Handy and Depledge
(1999).
5. Nucleolar activity was evaluated in gill and liver cells sampled from juveniles exposed to Cd+2 and Cr+6 during 6, 48, 192, and 384h. Histological
preparations and nucleolar area and number assessment were conducted following the method proposed by Arkhipchuk and Palamarchuk (1997) and
adapted by Carriquiriborde et al. (2000).
6. DNA integrity in the gill and the liver cells of O. bonariensis juveniles exposed to Cd+2, Cr+6 and Cu+2 for 48 and 348h was assessed using the single cell
gel electrophoresis assay (COMET assay) according to Singh et al. (1988). Tissue samples were pretreated following the methodology proposed by
Deventer (1996) but using only trypsin to isolate cell from tissue. Qualitative analysis of DNA damage was performed scoring the observed nuclei in 5
levels, 0, I, II, III, IV according to the degree of DNA migration (tail).
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Received on April 18, 2005.
Accepted on May 31, 2005.