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Chronic Toxicity of Ethyl Parathion and Isobutoxyethanol Ester of. 2,4-Dichlorophenoxyacetic Acid to Estuarine Juvenile and Adult Crabs. Enrique M. Rodrfguez* ...
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Arch. Environ. Contam. Toxicol. 22, 140-145 (1992)

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© 1992 Springer-Verlag New York Inc.

Chronic Toxicity of Ethyl Parathion and Isobutoxyethanol Ester of 2,4-Dichlorophenoxyacetic Acid to Estuarine Juvenile and Adult Crabs Enrique M. Rodrfguez*, Jos6 M. Monserrat*, and Oscar A. Amin** *Animal Physiology Laboratory and **Arthropoda Laboratory, Department of Biological Sciences, University of Buenos Aires, Ciudad Universitaria, (1428) Buenos Aires, Argentina Abstract. The chronic toxicity of ethyl parathion and isobutoxyethanol ester of 2,4-dichlorophenoxyacetic acid was determined for the crabs Uca uruguayensis and Chasmagnathus granulata collected in Argentina in 1989. Parathion toxicity was more time-dependent than 2,4-D. Four-week LC50 values for parathion were size and species independent, as expected under chronic exposure conditions. As shown in previous studies, the opposite was noted when crabs were exposed to acute exposure. Parathion was much more toxic than 2,4-D, in all cases. Also, parathion exerted a very much lower chronic lethal effect than other pesticides at a fraction of the respective acute LC50. A different susceptibility between sexes was found in C. granulata exposed to 2,4-D.

The pesticides ethyl parathion (0, O-Diethyl O-p-nitrophenyl phosphorothionate) and 2,4-D (isobutoxyethanol ester of 2, 4-dichlorophenoxyacetic acid) have been widely employed in Argentina for the last thirty years, with an increasing trend to use them in several crops. The continued application of these substances (Kuhnemann 1977; Lenardon et al. 1987; Comisi6n Administradora del Rio de la Plata 1989) may adversely affect a great variety of species living in the Rio de la Plata estuary, including the ones in its outer section (the Samboromb6n Bay) that comprises an extensive coastal zone. The littoral zones of these areas are densely inhabited by the estuarine crabs Uca uruguayensis (Ocypodidae), and Chasmagnathus granulata (Grapsidae), which serve as food for many fish species of high commercial value. Since both juveniles and adults live permanently near shore, they are susceptible to receive the influence of aquatic pollutants contained in the adjacent estuarine waters. This study continues previous results (Rodrfguez and L o m b a r d o 1991; Rodriguez and Amfn 1991; Monserrat et al. 1991) aimed at evaluating the lethal effects of parathion and 2,4-D to the species mentioned above. The specific purpose of this paper is to establish the chronic lethal toxicity of ethyl parathion and 2,4-D to: 1) adult males and females and between sexes of the two crab species, and 2) juveniles of C. granulata.

Materials and Methods

Adult males and females of U. uruguayensis and C. granulata, were collected in October 1988 at Faro San Antonio beach, near Punta Rasa, southern limit of Samboromb6n Bay (36°18'S and 56°48'W). C. granulatajuveniles were collected in September 1989 at the same site. Crabs were acclimatized for two weeks in the laboratory under experimental conditions selected for the bioassays that would be carried out later. Weights and carapace widths of tested crabs used are given in Table 1. Since crabs were randomly assigned to different treatments, only the dilution water control individuals were weighed and measured. Chronic toxicity of parathion and 2,4-D was assessed following the procedures recommended by the American Public Health Association (1976). Ethyl parathion (purity 99%), Compafiia Qu~mica, Buenos Aires, Argentina) and isobutoxyethanol ester of 2,4-D (purity 95%, Sfntesis Qufmica, Buenos Aires, Argentina) were employed. To prepare the stock solutions, pentaethylene oxide nonyl phenolate was used as solvent, in equal proportions to the pesticides, and distilled water was added. Artificial marine salts used in a previous work (Monserrat et al. 1991) were added to dechlorinated tap water (80 mg/L total hardness as CaCO 3) to obtain the dilution water used both for acclimatization and assays, at the desired salinity of 12 +_ 1%o. (12 g/L), pH 7 -+ 0.5. A constant temperature (23 + I°C) and a controlled photoperiod (12L:12D, fluorescent light) were maintained during the experiments. Assays with U. uruguayensis were carried out in 1-L glass jars containing 150 mL of saline water, and assays with C. granulata adults were performed using 24-L glass containers with 3 L of media, while the same jars above mentioned, containing 500 mL, were used in the assays with juveniles. In all cases, each concentration was run in duplicate, and eight to ten crabs were placed in each container. Table 2 details the tested concentrations of each pesticide for each experiment. Toxic ranges were established on the basis of 96-h LC50 values previously estimated for adults (Rodriguez and Lombardo 1991), being the highest concentrations of the chronic series 4.3 to 6.7 times as low as the respective 96-h LC50 value. For C. granulatajuveniles, similar series to those used for U. uruguayensis adults were run, taking into account their size similarity. Both dilution water and solvent controls were run, the latter with solvent concentration in the highest or in the two highest pesticide concentrations in each series (see Table 2). Twice a week, crabs were fed with chicken liver and rabbit food, and all test solutions were renewed. Mortality was recorded every 24 h, and dead animals were

Toxicity of Ethyl Parathion and 2,4-D Ester to Crabs

141

Table 1. Mean weight (g) and mean caraspace width (ram) of tested crabs

Species/ stage--sex

Mean weight - SD

Mean carapace width - SD

N

15.21 -+ 2.12 8.81 ± 1.61 0.54 +- 0.22

28.45 ± 1.68 24.83 +- 1.90 10.58 ± 1.05

16 16 20

0.72 ± 0.19 0.46 + 0.14

11.38 ± 1.01 11.09 ± 1.15

20 20

C. granulata Adults--males Adults--females Juveniles

U. uruguayensis Adults--males Adults--females

Table 2. Concentration series and total number of tested animals (N) Species/ pesticide--stage

Pesticide concentrations

Solvent control concentrations

N

1-10-100ixg/L 5-50-500 mg/L 0.1-1-10i~g/L 0.5-5-50mg/L

0.0790p~l/L 0.0405-0.4049ml/L 0.0079~I/L 0.0405ml/L

150 150 100 100

0.1-1-10Ixg/L 0.3-3-30 mg/L

0.0079ul/L 0.0243ml/L

180 180

C. granulata Parathion--adult 2,4-I)--adult Parathion-juvenile 2,4-D--juvenile

U. uruguayensis Parathion--adult 2,4-D---adult

removed. The criterion for death used was absence of movement after gently touching the animals with a glass rod, confirmed by observation of cheliped laxity. Duncan multiple range test was employed for comparing survival mean values (Bliss 1967), previously estimating the residual variance by Repeated Measures ANOVA (Winner 1971)for arcsin transformed survival data (live individuals/initial number of individuals ratio). Time of exposure was the repeated measure factor while treatment (concentrations and controls) was taken as second factor; moreover, only in the case of adults, sex was taken as a third factor. LC50 values and 95% confidence limits were estimated by probit analysis (Finney 1971). When no convergence occurred, the alternative Lichtfield and Wilcoxon graphic method (Granmo and Larsstuvold 1976)was used. LC50 values were compared according to the procedure outlined by the American Public Health Association (1976).

Results

In the assays with C. granulata adults, on the 15th day of exposure, the animals of one replicate were used for an additional experiment designed to measure their oxygen consumption. Although this procedure did not affect the crabs, a high mortality occurred due to the tagging method employed for individual identification of animals of the experiment, and the replicate had to be discarded. Figure 1 summarizes survival percentage data for all assays. Mortality both in dilution water and solvent controls, as well as in all concentrations used, is shown at the end of each week, for the 4-wk exposure period. Table 3 details the results of survival mean multiple comparisons, with respect to the factors considered in the ex-

periments. Comparing concentrations, the highest concentrations of each series differ significantly (p < 0.01) from controls and/or respective lowest concentrations, causing the highest mortality. Only in the assay of 2,4-D in adult U. uruguayensis were no significant differences encountered (p > 0.05). Comparing the effect of parathion in different weeks of exposure, a significant reduction in survival was observed, at least when comparing first and last weeks in all cases (Table 3, Figure l) (p < 0.01). For 2,4-D, a significant effect was found only on C. granulata adults, though more gradual compared to C. granulata adults exposed to parathion (Table 3, Figure 1). Concerning sex, the ANOVA analysis showed that the only detectable difference between sexes consisted in a significantly higher survival (p < 0.01) of C. granulata females exposed to 2,4-D. LC50 values at the end of the exposure period, with their respective 95% confidence intervals, are listed in Table 4. Values for 2,4-D in adults of both species could not be estimated because of the low mortality recorded at 4 wk (less than 50%), taking the highest concentration as a minimum reference value. Even though for adults of C. granulata, the highest concentration employed (500 mg/L) caused more than 50% mortality, it was not considered in the analysis, due to the high mortality recorded in the respective solvent control (Figure 2). Comparing 4-wk LC50 values among all species and stages, no significant differences (p > 0.05) were found for parathion in any case. Although no direct comparison of LC50 values could be made between pesticides for adults of each species, because--as mentioned above--LC50 values could not be accurately estimated for 2,4-D, obtained results indicate that parathion was more toxic than 2,4-D, for both species. In fact, minimum values for 2,4-D-LC50 were not included within the confidence intervals of the corresponding parathion-LC50 values; the values between pesticides differing at least in four orders of magnitude (Table 4). Direct comparison between parathion and 2,4-D LC50 values for juveniles show the same pattern, with a significantly higher toxicity of parathion (p < 0.01).

Discussion

Survival in highest concentration of each series decreased significantly in almost all cases. Concentrations equal to or lower than 1/55 of the 96-h LC50 (corresponding to a mean of second highest concentrations of adult series of both species) did not affect survival of adults under a 4-wk chronic exposure. On the other hand, parathion showed a lethal toxicity significantly higher (p < 0.01) than 2,4-D, in all cases (3,036-fold for juveniles of C. granulata, and more than 8, 300-fold and than 11,500-fold for adults of U. uruguayensis and C. granulata respectively). Concerning the effect of time of exposure, two different trends were observed for both pesticides. Chronic lethal toxicity of parathion seems to be more time-dependent than that of 2,4-D. Despite the incipient lethal level that parathion reached at 96 h for adults of both species (Rodriguez and

142

E.M. Rodriguez et al. U. u r u g u a y e n s i s

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% o f z,Jrvival

% of survival 100,

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WC

SV(1O)

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1

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C. g r a n u l a t a

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20 10 0

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2,4-D CONCENTRATION

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~WK1 ~WK2 ~WKZ tool W~K 4 Fig. 1. Percentage survival of crabs exposed to each pesticide, as a function of concentrations and time of exposure (weeks). WC: dilution water control. SV: solvent control (corresponding to the pesticide concentration specified between brackets)

Lombardo 1991) and for juveniles of C. granulata (Rodr~guez and Amin 1991), the increase of mortality during the 4-wk period indicates that no lethal threshold concentration exists when the exposure to parathion becomes chronic. This fact suggests an absence of acclimation to the insecticide; similar results have been obtained in other species exposed to parathion (Banas and Sprague 1986) and other organophosphate pesticides (Palawski et al. 1983). This effect may be related to the slow compensation of the cholinesterase inhibition caused by the above-mentioned pesticides (O'Brien 1966; Palawski et al. 1983).

The only significant difference encountered between sexes (females of C. granulata more resistant to 2,4-D than males) is not indicative of a clear pattern respect to sex-differential toxicity for the species under study, also taking into account the absence of significant differences between sexes of U. uruguayensis u n d e r acute e x p o s u r e to both p e s t i c i d e s (Rodriguez and Lombardo 1991). No clear trend is evident from other studies that consider the sex-differential toxic effects of pesticides and other stressors on aquatic fauna. Females more resistant than males were reported in: crabs (Uca pugilator) under saline and

Toxicity of Ethyl Parathion and 2,4-D Ester to Crabs

143

Table 3. Results of mean survival comparison data. Treatments ordered by decreasing survival means. Horizontal lines indicate no significant differences (p > 0.05). WC: dilution water control. SV: solvent control (corresponding to the pesticide concentration specified between brackets) Pesticide/species--stage

Between concentrations

Between weeks

Parathion (~g/L) C. granulata adults

WC / SV(100) / 1 / 10 / 100

1 2/3

4

C. granulata--juveniles

1/0.I/WC

SV(10)/10

1 2/3

4

U. ur~guayensis--adults

1/0.1/WC

SV(10)/10

1 2/3

4

4

2,4-D (mg/L)

C. granulata--adults

SV(50)

WC 5 / 50 / SV(500) / 500

1 2/3

C. granulata--juveniles

SV(50)

5/0.5/WC/50

1 2/3/4

U. uruguayensis adults

SV(30) / 3 / 0.3 / 30 / WC

1 2/3/4

Table 4. Four-week LC50 values and comparisons with acute ones. p: probability level for differences between 96-h and 4-wk LC50, 96-h LC50 were taken from previous works (see text) Pesticide

Species/stage

Parathion (~g/L)

C. granulata adult juvenile

4-wk LC50

95% Conf. Limits

Slope

R2

96-h LC50

96h/4wk ratio

p

4.34 10.00

0.09- 18.61 3.05- 32.82

0.72 4.08

0.94 0.99

560 360

129 36