Comparing Toxicity Endpoints on Lecane quadridentata (Rotifera: Monogononta) Exposed to Two Anticholinesterases Pesticides Ignacio Alejandro Pe´rez-Legaspi,1 J. Luis Quintanar,2 Roberto Rico-Martı´nez3 1
Divisio´n de Estudios de Posgrado e Investigacio´n, Instituto Tecnolo´gico de Boca del Rı´o, Km 12 Carr, Veracruz-Co´rdoba, Boca del Rio, Veracruz 94290, Me´xico 2
Departamento de Fisiologı´a y Farmacologı´a, Universidad Auto´noma de Aguascalientes, Ave. Universidad 940, Aguascalientes, Ags. 20100, Me´xico
3
Departamento de Quı´mica, Universidad Auto´noma de Aguascalientes, Ave. Universidad 940, Aguascalientes, Ags. 20100, Me´xico
Received 15 April 2010; revised 13 September 2010; accepted 21 September 2010 ABSTRACT: Toxicity tests were performed on the freshwater rotifer Lecane quadridentata exposed to the pesticides carbaryl and methyl parathion (lethal, sublethal, and chronic) to compare the sensitivity between different endpoints: (a) 48-h mortality; (b) 30-min in vivo inhibition of esterase activity; (c) 5-day inhibition of the instantaneous growth rate. The emphasis of this work was to find the most appropriate endpoint to evaluate the toxicity of these pesticides in view of their sensitivity, duration, and ecological relevance. The comparison between the three toxicity tests show that the 5-day chronic tests have the lowest EC50 (2.22 and 6.6 mg/L), lowest-observed-effect concentration (2.5 and 2.5 mg/L), and noobserved-effect concentration (1.0 and 1.2 mg/L) values for carbaryl and methyl parathion, respectively. This indicates that the estimate of the instantaneous rate of natural increase r is the most sensitive endpoint regarding the toxicity of these pesticides. This sensitivity might be due to the effect on reducing the growth potential form the first generation on. Lethal and sublethal tests are closely related, suggesting that the immediate effect after inhibition of esterases is death. In general, the sensitivity of L. quadridentata is similar to other species of rotifers exposed to methyl parathion. Therefore, the 5-day chronic toxicity test with the freshwater rotifer L. quadridentata should be considered a good candidate to evaluate the effect of anticholinesterase pesticides, due to its high sensitivity and ecological relevance. # 2010 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2010.
Keywords: organophosphorous; carbamates; toxicity tests; endpoint; rotifer
INTRODUCTION Organophosphate and carbamate pesticides are widely used in agricultural crops, forests, rangelands, and wetlands. Correspondence to: I. A. Pe´rez-Legaspi; e-mail:
[email protected] Contract grant sponsor: Consejo Nacional de Ciencia y Tecnologı´a (CONACyT). Contract grant number: 136453. Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/tox.20668
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However, although these pesticides degrade quickly, they have nonspecific toxicity and share the same mode of action: they inhibit the activity of the enzyme acetylcholinesterase (AChE) causing accumulation of the neurotransmitter acetylcholine and thus altering the normal functioning of the nervous system. These toxic effects result in behavioral and physiological changes and death and depend on the degree of exposure and toxicity of the pesticide (Nimmo and McEwen, 1994; Newman and Unger, 2003). In the zooplankton, there are reports of effects of these
2010 Wiley Periodicals, Inc.
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pesticides on population dynamics, survival, reproduction, sex ratio, morphology, and swimming behavior, resulting in some populations being more vulnerable to predation. Therefore, these pesticides are capable of adversely affecting freshwater zooplankton at different levels of ecological organization, thereby altering community structure and functioning of aquatic ecosystems (Hanazato, 2001). Toxicity tests are valuable tools to evaluate effects of chemicals. Some endpoints should be considered biomarkers with rapid response and ecological relevance (Burbank and Snell, 1994). In rotifers, many endpoints have been assessed: mortality, reproduction, amictic female production, resting egg production, probability of extinction, behavior, feeding, swimming activity, in vivo enzyme activity, and gene expression (Snell and Janssen, 1998; Wallace et al., 2006). The use of rotifers in ecotoxicological assays offers several advantages: small size, short generation time, ease of handling and culture, great ecological relevance, sensitivity, and availability of neonates (Janssen et al., 1993; Snell and Janssen, 1998; Marcial et al., 2005). The LC50 (lethal concentration where 50% of individuals exposed die) value is a sensitive parameter, which under standard conditions is considered the basis for developing other tests, as it allows good estimates of the toxicity of a substance, which can be applied to monitor water quality (Wallace et al., 2006). However, other significant parameters like growth, reproduction, and physiological and biochemical aspects should be considered (Simon et al., 1998). Most toxicity tests that have been performed with rotifers are lethal tests (acute), which are standardized, simple, and reproducible; the mortality or immobility is the endpoint used to estimate the LC50 value of a population exposed to a certain toxicant for 24 or 48 h without food. Most of these studies with rotifers focus on the genus Brachionus (Ferrando and Andreu-Moliner, 1992; Snell and Janssen, 1998). The lethal effects of the organophosphate methyl parathion have been evaluated in the freshwater rotifers B. calyciflorus (Fernandez-Casalderrey et al., 1993; Sarma et al., 1998), B. angularis, (Gama-Flores et al., 1999), B. patulus (Sarma et al., 2001), and Asplachna sieboldi (Gama-Flores et al., 2004). Chronic toxicity tests allow assessment of sublethal effects resulting of prolonged exposure rather than death (Nimmo and McEwen, 1994). Chronic tests have shown that methyl parathion and carbaryl are capable of altering the aquatic community structure by reducing the intrinsic rate of natural increase r and other population parameters in the cladocerans Daphnia pulex (Dodson et al., 1995; Hanazato and Hirokawa, 2004) and D. magna; (Hanazato and Yasuno, 1987; Ferna´ndez-Casalderrey et al., 1995). In the rotifers Brachionus calyciflorus, B. angularis, and B. patulus, methyl parathion affects population dynamics and decreases r (Ferna´ndez-Casalderrey et al., 1993; GamaFlores et al., 1999; Sarma et al., 2001; Gama-Flores et al.,
Environmental Toxicology DOI 10.1002/tox
2004). Methyl parathion also affects the predator–prey interaction between two rotifer species (Sarma et al., 1998). In general, chronic tests assessing r are more sensitive than acute tests even when compared with other reproductive tests and other aquatic species (Snell and Moffat, 1992; Marcial et al., 2005). Also, the 5-day chronic test with the rotifer Lecane quadridentata has similar sensitivity than the 2-day B. calyciflorus test (Hernandez-Flores and Rico-Martinez, 2006). These chronic tests are very sensitive and with great ecological relevance, because they estimate the growth potential of the exposed population and include much of the organism’s life cycle (Snell and Moffat, 1992). Moreover, its sensitivity and easy performance have been proposed as good substitutes of the 21-day test with D. magna (Radix et al., 1999). The assessment of sublethal effects should focus in the first responses to toxic substances. Such responses include stress, biochemical and physiological changes, reproductive failure, and behavioral changes (Burbank and Snell, 1994; Nimmo and McEwen, 1994). Among enzymes, the activity of AChE has been the most investigated in organisms as a biomarker for anticholinesterase-type pesticides (Grue et al., 1983). This biomarker is useful, because it indicates the site of action and degree of inhibition (Walker et al., 2006). AChE is considered a good biomarker for many species of vertebrates and invertebrates (Mayer et al., 1992). In rotifers several enzymatic systems (esterases, phospholipases A2, and glycosidases) have been used as in vivo biomarkers in 30-min sublethal tests. The species used are B. calyciflorus (Burbank and Snell, 1994), L. quadridentata, L. luna, and L. hamata for esterases (Pe´rezLegaspi et al., 2002), and phospholipases A2 (Pe´rezLegaspi and Rico-Martinez, 2003) and glycosidases with B. plicatilis and B. rotundiformis (Araujo et al., 2000, 2001). The in vivo enzyme inhibition tests with rotifers are in the range of sensitivity of standardized tests such as 5-min Microtox test, 24-h D. magna 24-h galactosidases activity, and the 7-day Ceriodaphnia dubia test. Therefore, the rotifer tests have been proposed as good assessment tools for monitoring toxic substances. An integral evaluation of toxic effects indicates that the sensitivity depends on the species and type of test being performed (McDaniel and Snell, 1999). Therefore, a single species cannot be considered as representative of freshwater rotifers (Wallace et al., 2006). Thus, it is desirable to have a diverse battery of toxicity tests and species to put emphasis on the ecological relevance of the integral results to monitor toxic substances in aquatic ecosystems. Therefore, the aim of this study is to evaluate the sensitivity between three endpoints: (a) mortality, (b) in vivo inhibition of esterase activity, and (c) intrinsic rate of natural increase r; through the development of acute, sublethal, and chronic toxicity tests using the freshwater rotifer L. quadridentata exposed to the pesticides methyl parathion and carbaryl.
COMPARING PESTICIDES TOXICITY ENDPOINTS IN L. quadridentata
MATERIALS AND METHODS We used amictic neonate females of the freshwater rotifer L. quadridentata (EHRENBERG, 1832) less than 24-h old, hatched from parthenogenetic eggs. This rotifer was originally collected from Lake Chapala, Jalisco, Mexico. The geographic coordinates of this location have already been published (Rico-Martı´nez and Silva-Briano, 1992; SilvaBriano et al., 2003). L. quadridentata has been maintained during more than 10 years in laboratory conditions (Pe´rezLegaspi and Rico-Martı´nez, 1998). Rotifers were cultured in Petri dishes with synthetic freshwater EPA medium (96 mg NaHCO3, 60 mg CaSO4.2H2O, 60 mg MgSO4, and 4 mg KCl per liter) at pH 7.5 in deionized water (U.S. EPA, 1985) at 258C 6 28C and 16:8 l:d (light: dark) period in a bioclimatic chamber (Revco Scientific, Asheville, NC). Rotifer cultures were fed with the green algae Nannochloris oculata (UTEX strain LB2194), which is grown in Bold’s Medium (Nichols, 1973). Toxicants of the highest purity available were always used. The carbamate pesticide carbaryl was obtained from Sigma Co., USA, and the organophosphate pesticide methyl parathion from Supelco Co., USA. We dissolved carbaryl into acetone (JT Baker Co., USA) due to its low toxicity (LC50 [ 7 g/L) reported previously for L. quadridentata (Pe´rez-Legaspi and Rico-Martı´nez, 2001) and considered a solvent control of the highest acetone concentration used for each pesticide concentration tested to monitor the toxicity due to solvent. All media and experiments used deionized water obtained from a Labconco Water Pro PS System (Labconco, Kansas City, MO). The pesticides were tested at their nominal concentrations, and no analytical chemistry was performed. Each definitive test included a negative control (EPA medium without toxicant), a solvent control (acetone), and five pesticide concentrations. All statistical analyses were performed using the software Statistica 6 (StatSoft Inc., 2001) with P \ 0.05.
Acute Toxicity Tests Asexual eggs of Lecane quadridentata were collected and incubated at 258C in Petri dishes with EPA medium. Toxicity range finding tests were conducted previous to the definitive tests for each pesticide. Acute toxicity tests were conducted in 24-well polystyrene plates (Costar Co, USA), following the protocol of Pe´rez-Legaspi and Rico-Martı´nez (2001). Briefly, neonates (n 5 10, five replicates) were placed in each well containing a final test volume of 1.0 mL and incubated for 48 h at 258C in darkness. For the definitive tests, we tested five concentrations for both methyl parathion (2.5, 5, 7.5, 10, and 20 mg/L) and carbaryl (5, 10, 25, 50, and 100 mg/L). The test was concluded after 48 h; after that time, the number of immobilized rotifers was recorded, and the LC50, no-observed-effect concentration (NOEC), and the lowest-observed-effect concentration
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(LOEC) values were determined. The independent variable was the toxicant concentration (which, in the case of the negative control, is zero), and the dependent variable was mortality. Mortality data were analyzed by means of the DL50 software (S.B.I.-I.R.C.T. Montpellier, Fevrier, 1987), which estimates the LC50 values and achieves a chisquare to test the validity of the linearity of the regression (P \ 0.05). The statistical analysis was performed by simple regression to obtain LC50 estimates of concentration curves and the regression coefficient (R2) values. Then, we performed one-way analysis of variance (ANOVA) to obtain NOEC and LOEC values using Tukey tests.
Sublethal Toxicity Tests We performed the in vivo esterase inhibition test according to Burbank and Snell (1994) with slight modifications (Pe´rez-Legaspi et al., 2002). Test concentrations for the definitive test were a series of five concentrations for both methyl parathion (0.625, 1.25, 2.5, 5, and 10 mg/L) and carbaryl (1, 2.5, 5, 10, and 20 mg/L). The neonates (n 5 15, three replicates) were added to each well of the plate to expose into each pesticide concentration, negative, and solvent control, respectively, during 30-min period. After exposure, we added 5 lM of the fluorogenic substrate cFDAam (5-carboxifluorescein diacetate acetoxymethylester from Molecular Probes, OR) for 15 min to assess activity of esterases in vivo as the end-point. Enzymatic activity was stopped by adding 10% formalin solution 200 M per well. Rotifers were observed at 203 magnification under a Leica fluorescence microscope with an excitation spectrum of 450–490 nm and an emission barrier of 515 nm. The intensity of fluorescence in the gut was measured to obtain a mean fluorescence value. Background fluorescence was measured and taken from the surroundings of the rotifer and subtracted from the gut fluorescence value. These actions were performed by image analysis using a Motic images plus 2.0 camera to capture images and Kodak Digital Science 1D (to analyze digital data). The statistical analysis was performed using ANOVA’s and post hoc comparison Tukey tests to establish the NOEC and LOEC points. The independent variable was the toxicant concentration or negative control, and the dependent variable was the fluorescence intensity measured as an indirect evaluation of enzyme activity. Simple linear regression was used to obtain the EC50 (effect concentration where a 50% reduction in esterase activity is observed) and the confidence limits.
Chronic Toxicity Tests These tests measure inhibition of the instantaneous growth rate (r) in the rotifer Lecane quadridentata according to the protocol of Herna´ndez-Flores and Rico-Martı´nez (2006). Briefly, in the definitive test, five neonates per well (five replicates) were exposed to series of five concentrations for
Environmental Toxicology DOI 10.1002/tox
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TABLE I. Results of the acute test, the chronic 5-day test, and the sublethal in vivo esterase inhibition tests with the freshwater rotifer Lecane quadridentata exposed to carbaryl and methyl parathion Lethal 48-h Endpoint
LC50
Carbaryl toxicity test mg/L 13.72 CL 6.071–31.01 CV % 81.13 R2 0.6332 Methyl parathion toxicity test mg/L 9.495 CL 8.28–10.89 CV % 125 R2 0.9416
LOEC
Sublethal 30-m NOEC
EC50
Chronic 5-d
LOEC
NOEC
EC50
LOEC
NOEC
10
5.0
17.19 10.54–23.84 48.41 0.8729
5.0
2.5
2.22 1.35–3.09 183.8 0.8639
2.5
1.0
10.0
7.5
9.4 6.05–12.75 39.38 0.8930
5.0
2.5
6.62 2.63–10.6 31.53 0.7062
2.5
1.25
Abbreviations: LC50, lethal concentration where 50% of individuals exposed die; EC50, effect concentration where a 50% decrease in sublethal (esterase activity) or in chronic (reproduction) test is observed; LOEC, lowest observed effect concentration; NOEC, no observed effect concentration; CL, 95% confidence limits for LC50 or EC50 values; CV, coefficients of variation; R2, correlation degree among variables.
both carbaryl (0.5, 1, 2.5, 5, and 7.5 mg/L) and methyl parathion (0.625, 1.25, 2.5, 5, and 10 mg/L), including negative and solvent control. At the beginning of experiment, 1 3 106 cells/mL of Nannochloris oculata per well were added as food during the 5-day test without renewal. After exposure, all individuals were counted for each treatment, and then r was obtained by the following equation: r 5 (ln Nt 2 ln No)/t, where r is the instantaneous growth rate, Nt the number of individuals after 5 days, No the initial number of individuals at the beginning, ln the natural logarithm, and t the time at 5 days. Statistical analysis was performed using ANOVA and post hoc comparison Tukey tests to determine the NOEC and LOEC values. The independent variable was the toxicant concentration or the negative control as zero, and the dependent variable was the individual number recorded after 5 days of exposure to assess the instantaneous growth rate. Simple linear regression was used to obtain the EC50 value, and the confidence limits.
RESULTS The results of the three toxicity tests (mortality, In vivo esterase inhibition, and growth inhibition) for L. quadridentata are shown in Table I. The lowest LC50/EC50, LOEC and NOEC values were obtained in the chronic reproductive, which suggests that this test is the most sensitive. On the other hand, both the lethal and sublethal toxicity tests have similar values. The LC50 values from 48-h lethal toxicity tests showed that methyl parathion (9.495 mg/L) is more toxic than carbaryl (13.72 mg/L). Similarly, the EC50 values obtained from 30-m sublethal toxicity test show that methyl parathion (9.4 mg/L) is more toxic than carbaryl (17.19 mg/L). In contrast, the EC50 values recorded from 5-day chronic toxicity test showed that carbaryl (2.22 mg/L) is more toxic than methyl parathion (6.62 mg/L).
Environmental Toxicology DOI 10.1002/tox
We determined actual values for carbaryl using high performance liquid chromatography (HPLC) with a HPLC Perkin Elmer Series 200 with a column RPC8 and detected by mass spectrometer from Agilent Technologies. For carbaryl, we have a recovery efficiency of 98.9% 6 10.7% (mean 6 one SD; n 5 4). Methyl parathion was determined by HPLC using a VARIAN chromatographer model CP3800 with a CP-Sil5B column and detected using mass spectrometer from Agilent Technologies. The recovery efficiency for methyl parathion was 141.9% 6 22.5% (n 5 10). Concentration/response curves (Fig. 1) are shown for the three toxicity tests performed for each pesticide. In general, according to the R2 values, the correlation coefficient showed high degrees between the endpoints (mortality, esterase inhibition, and reproduction), and the pesticides evaluated in an exposure concentration-response dependent manner. The comparisons among lethal, sublethal, and chronic toxicity endpoint ratios for both pesticides assessed are shown in Table II. These small ratios showed that the EC50 values for sublethal endpoint for carbaryl (0.79) and methyl parathion (1.01) were close to the LC50 values. These results suggest that mortality might be an immediate effect produced by both pesticides. On the other hand, the nearest ratios between lethal and sublethal endpoints showed a similar sensitivity between them, which suggest that the 30 min in vivo esterase inhibition test compares well with the lethal toxicity test for both carbaryl and methyl parathion pesticides.
DISCUSSION The results of our investigation clearly show that the chronic 5-day test with Lecane quadridentata was the most sensitive of all three tests used for both carbaryl and methyl parathion. The results of the LC50 48-h and EC50 30-m
COMPARING PESTICIDES TOXICITY ENDPOINTS IN L. quadridentata
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Fig. 1. Dynamics of concentration curves for L. quadridentata exposed to the pesticides assessed by three different toxicity tests.
values indicate that methyl parathion is more toxic than carbaryl (Table I). This result might be related to the irreversible inhibition of AChE active site typical of an organophosphate such as methyl parathion. The enzyme remained phosphorylated and inactive for a long period of time. In contrast, the lesser adverse effect of carbaryl might be the
formation of an enzyme-carbaryl bond that is readily hydrolyzed, and the carbamylated enzyme is able to recover in a short time. Therefore, inhibition is considered reversible (Walker et al., 2006). Our 48-h LC50 value obtained for methyl parathion was 9.50 mg/L, which is lesser than that of B. calyciflorus 24-h
Environmental Toxicology DOI 10.1002/tox
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TABLE II. Acute–subacute–chronic-ratios found among the different tests performed with the freshwater rotifer Lecane quadridentata Lethal 48-h
Sublethal 30-m
Chronic 5-d
Carbaryl LC50/LOECL 1.37 LC50/EC50e 0.79 LC50/EC50r 6.18 LC50/NOECL 2.74 LC50/LOECe 5.48 LC50/LOECr 5.48 LC50/NOECe 13.72 LC50/NOECr 13.72 Methyl parathion LC50/LOECL 1.18 LC50/EC50e 1.01 LC50/EC50r 1.43 LC50/NOECL 1.26 LC50/LOECe 1.89 LC50/LOECr 3.79 LC50/NOECe 3.79 LC50/NOECr 7.59 Abbreviations: LC50, lethal concentration where 50% of individuals exposed die; LOECL, LOEC values of lethal test; NOECL, NOEC values of lethal test; EC50e, EC50 values of esterases activity test; LOECe, LOEC values of esterases activity test; NOECe, NOEC values of esterases activity test; EC50r, EC50 values of instantaneous growth rate test; LOECr, LOEC values of instantaneous growth rate test; NOECr, NOEC values of instantaneous growth rate test.
LC50 (29.19 mg/L). However, this difference may be due to the methyl parathion used for B. calyciflorus was technical grade 80% purity (Ferna´ndez-Casalderrey et al., 1993), whereas methyl parathion used for L.quadridentata was of analytical grade (100% or so). Similarly, there are reported values of 24-h LC50 for B. calyciflorus (9 mg/L) and A. sieboldi (13 mg/L), but these authors do not mention the conditions of the test (presence of algal food and quality of methyl parathion evaluated) (Sarma et al., 1998). There are several 24-h LC50 values for B. angularis (0.636, 2.783, and 6.52 mg/L) and B. patulus (8.8 and 10.66 mg/L) with different concentrations of the algae Chlorella vulgaris as food reported (which influences the standardization of the method and the toxicity of the pesticide) (Sarma et al., 2001; Gama-Flores et al., 2004). Therefore, it appears that under controlled conditions in acute tests L. quadridentata offers good sensitivity to methyl parathion in relation to other species of rotifers used. However, rotifer are far less sensitive than cladocerans (D. magna 24-h LC50 5 0.31 ng/L). The EC50, LOEC, and NOEC values of the chronic toxicity tests showed greater sensitivity than the same values of the lethal and sublethal tests, suggesting that the increase in exposure time increases the sensitivity of the population to the toxicity of both pesticides evaluated (Table I and Fig. 1). The chronic tests measures the inhibition of the potential population growth, which considers all stages of life (embryos, juveniles and adults) during the trial period, becoming an important ecological parameter (Snell and Moffat, 1992; Ferrando et al., 1996). The 5-day chronic reproductive test with L. quadridentata considers the longevity of 68.57% during the time of exposure. In contrast, the 2-day B. calyciflorus chronic test comprises 33% of their longevity during the test period (Snell and Moffat, 1992).
Environmental Toxicology DOI 10.1002/tox
Values for EC50 methyl parathion (6.6 mg/L) and LOEC (2.5 mg/L) from reproductive test of L. quadridentata (Table I) are greater than the EC50 values reported for B. angularis on the peak of population density (0.545 and 1.031 mg/L), and the rate of natural increase (0.335 and 1.488 mg/L) in two food concentrations (C. vulgaris), respectively (Gama-Flores et al., 2004). On the other hand, Ferna´ndez-Casalderrey et al. (1993) reported that concentrations of methyl parathion of 5 mg/L and up are capable of modifying the intrinsic rate of natural increase and other demographic parameters of B. calyciflorus throughout their entire life cycle. Also Gama-Flores et al. (1999) reported that food concentration affects the toxicity of methyl parathion and population density of B. calyciflorus, but did not determine parameters such as EC50, LOEC, and NOEC. Sarma et al. (2001) mentioned that the sensitivity of the intrinsic rate of natural increase r depends on the density of food and has a significant effect in reducing the toxicity of methyl parathion in acute and chronic conditions for the rotifer Brachionus patulus. In general, L. quadridentata shows similar sensitivity to the other species of rotifers exposed to methyl parathion on reproductive tests. However, once again, cladocerans are far more sensitive in this parameter than rotifers (21-day D. magna MATC for methyl parathion 5 0.25 ng/L) (Fernandez-Casalderrey et al., 1995). However, Radix et al. (1999) reported that toxicity values have similar response from 2-day B. calyciflorus and 21-day D. magna tests for 25 chemicals, with a good correlation for 80% of the substances evaluated. Therefore, the 2-day B. calyciflorus test can be a good substitute of the 21-day D. magna test. On the other hand, Hanazato and Yasuno (1987) mention that carbaryl does not affect the population dynamics of rotifers and that rotifers are less sensitive than cladocerans, because they use to their advantage the decrease in population density of their competitors and exploit the available resources rapidly increasing its population size. However, the values of LC50/EC50, LOEC, and NOEC obtained in this study demonstrate that carbaryl has adverse effects on freshwater rotifers and probably influenced by the reversible effect of carbamates to recover quickly turn combined with the great reproductive potential, and short generation time rotifers are able to recover their population size quickly with respect to the cladocerans. Sublethal tests esterase inhibition show that the EC50 value (9.4 mg/L) is similar to the LC50 (9.50 mg/L) of the lethal tests for methyl parathion, whereas, for carbaryl, the EC50 (17.19 mg/L) is greater than the LC50 (13.72 mg/L). This indicates that sublethal esterase tests for 30 min offer an approximate sensitivity to the lethal tests for methyl parathion, and it is less sensitive for carbaryl. However, this type of sublethal tests compares well with the acute test, provide answers in shorter times, has good coefficient of variation, and have good accuracy (Table I). The ratio EC50/CL50 for both tests suggests that they are closely
COMPARING PESTICIDES TOXICITY ENDPOINTS IN L. quadridentata
related (Table II), which means that the anticholinesterase pesticide immediate effect after inhibition of esterases is death. Comparison between the LOEC and NOEC values of both sublethal tests shows that the sensitivity is half of the values for mortality. In general, good sensitivity is reported when esterases-NOEC values are compared against the 24-h LC50 values in the case of B. calyciflorus 2-day or when compared with other tests like D. magna 24-h, Microtox, and Ceriodaphnia dubia 7-day (Burbank and Snell, 1994). Also, there are reports for three species of Lecane that the testing of esterase inhibition is a lot more sensitive than lethal tests (LC50 48-h) for metals and organic compounds (Pe´rez-Legaspi et al., 2002). Therefore, in vivo inhibition of esterase activity test is considered a suitable biomarker to assess the impact in a short time of different substances (Burbank and Snell, 1994). However, in the past, this test has not been used to assess organophosphate and/or carbamate pesticides. Our results suggest that these tests are probably not are the most appropriated to assess carbaryl or methyl parathion. It is important to compare different endpoints with welldefined criteria and to compare characteristics of the tests like standardization, sensitivity, durability, and ecological significance of the test organisms (Preston and Snell, 2001). This analysis will reveal the species and the most appropriate endpoint to assess toxic substances in aquatic environments. Also, this analysis should include a set of relationships like EC50/LC50, NOEC, and LOEC comparisons for each toxicity test. All these analysis would contribute to establish criteria for water quality that helps maintain the structure and functioning of the receiving water ecosystem. In conclusion, the 5-day L. quadridentata chronic toxicity test, which evaluates the reproduction using the instantaneous rate of natural increase, should be considered a good candidate for use as a biomarker to assess the effect of anticholinesterase pesticides because of its good sensitivity and ecological significance. We thank Dr. Samantha Ramos for her comments, and M. Sc. Jose´ Luis Herna´ndez Duque for help in the determination of carbaryl and methyl parathion concentrations. We appreciate the valuable comments of the anonymous referees for improved the quality of this manuscript.
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