Anuran tadpole assemblages in riparian areas of the Middle Paraná River, Argentina (original) (raw)

Abstract

Acute toxicity and genotoxicity of the S-metolachlor-based herbicide Dual Gold ® on Leptodactylus luctator (Hudson, 1892) tadpoles (Anura: Leptodactylidae). Herbicides used in agriculture and their metabolites are frequently detected in surface water bodies, where they can persist and cause adverse effects on aquatic organisms. The aim of this study was to evaluate the acute toxicity and genotoxic effects of the S-metolachlor (SM)-based herbicide Dual Gold ® (DG ®), on Leptodactylus luctator tadpoles (Anura: Leptodactylidae). To assess the toxicity of the herbicide, including the median lethal concentration (LC50) at 24h, the no-observed-effect concentration (NOEC), and the lowest-observed-effect concentration (LOEC), tadpoles were exposed to five nominal concentrations of DG ® (5.0, 6.2, 7.8, 9.8, and 12.2 mg/L), and to dechlorinated water as a negative control (NC). The LC50 24h of DG ® was 7.0 mg/L, the NOEC was 5.0 mg/L and the LOEC=6.2 mg/L. L. luctator tadpoles were sensitive to the herbicide, reaching 100% mortality after 24 h of exposure to the highest concentration tested (12.2 mg/L). To evaluate the potential genotoxicity of the herbicide, the frequencies of micronuclei (MN) and other erythrocyte nuclear abnormalities (ENA) were determined in larvae exposed to three nominal concentrations of DG ® (1.0, 5.0, and 6.2 mg/L) for 48 and 96 h. The frequencies of MN and ENA were compared with a positive control (40 mg/L of Cyclophosphamide) and a negative control. The frequencies of MN and ENA in the erythrocytes of tadpoles exposed to the test concentrations of DG ® and Cyclophosphamide were significantly higher than in the negative control group at both 48 and 96 h (with the only exception of MN at 1.0 mg/L at 48 h). Our results confirm the genotoxic and cytotoxic effects of this widely used herbicide in agriculture, a fact that represents a potential risk to amphibians that develop in ponds associated with or immersed in agroecosystems.

Figures (3)

Figure 1. Details of erythrocytes observed in Leptodactylus luctator tadpoles exposed to the SM-based herbicide DG®. (A) Normal erythrocytes; (B) Binucleated erythrocyte (BE); (C) Erythroplastid or anucleated erythrocyte (EP); (D) Notched nucleus (NN); (E) Kidney-shaped nucleus (K); (F) Pyknotic nucleus (PN); (G) Micronucleus (MN); (H) Lobed nucleus (LN); (1) Budded nucleus (BN); (J) Blebbed nucleus (BbN); (K) Apoptotic cell (AP); (L) Erythrocyte in mitosis (M). May Griinwald-Giemsa staining, 100x. Black bar in image D represents a scale of 10 um (for images A-D). Black bar in image L represents a scale of 5 wm (for images E-L). Detalle de eritrocitos observados en larvas de Leptodactylus luctator expuestas al herbicida DG®, a base de SM. (A) eritrocitos normales, (B) eri- trocito binucleado (BE); (C) eritoplastido o eritrocito anucleado (EP); (D) nucleo con muesca (NN); (E) nucleo en forma de rifion (K); (F) nucleo picnotico (PN); (G) Microntcleo (MN); (H) nucleo lobado (LN); (1) nicleo con brote (BN); (J); nucleo ampollado (BbN); (K) célula apoptotica (AP); (L) eritrocito en mitosis (M). May Griinwald-Giemsa, 100x. La barra negra en la imagen D representa una escala de 10 um (imagenes A-D). La barra negra en la imagen L representa una escala de 5 um (imagenes E-L).

Figure 1. Details of erythrocytes observed in Leptodactylus luctator tadpoles exposed to the SM-based herbicide DG®. (A) Normal erythrocytes; (B) Binucleated erythrocyte (BE); (C) Erythroplastid or anucleated erythrocyte (EP); (D) Notched nucleus (NN); (E) Kidney-shaped nucleus (K); (F) Pyknotic nucleus (PN); (G) Micronucleus (MN); (H) Lobed nucleus (LN); (1) Budded nucleus (BN); (J) Blebbed nucleus (BbN); (K) Apoptotic cell (AP); (L) Erythrocyte in mitosis (M). May Griinwald-Giemsa staining, 100x. Black bar in image D represents a scale of 10 um (for images A-D). Black bar in image L represents a scale of 5 wm (for images E-L). Detalle de eritrocitos observados en larvas de Leptodactylus luctator expuestas al herbicida DG®, a base de SM. (A) eritrocitos normales, (B) eri- trocito binucleado (BE); (C) eritoplastido o eritrocito anucleado (EP); (D) nucleo con muesca (NN); (E) nucleo en forma de rifion (K); (F) nucleo picnotico (PN); (G) Microntcleo (MN); (H) nucleo lobado (LN); (1) nicleo con brote (BN); (J); nucleo ampollado (BbN); (K) célula apoptotica (AP); (L) eritrocito en mitosis (M). May Griinwald-Giemsa, 100x. La barra negra en la imagen D representa una escala de 10 um (imagenes A-D). La barra negra en la imagen L representa una escala de 5 um (imagenes E-L).

Figure 2. Frequency of micronuclei (MN) (per-1000 cells) in Leptodactylus luctator tadpoles exposed to different concentra- tions of the SM-based herbicide DG®. NC: negative control (0 mg/L of SM); PC: positive control (40 mg/L of cyclophospha- mide); Ta=1 mg/L of SM; Tb=5.0 of mg/L SM; Tc=6.2 mg/L of SM. RBCs= red blood cells. * p<0.05: significant differences compared to the negative control (binomial proportion test). Frecuencia de micronticleos (MN) (por 1000 células) en larvas de Leptodactylus luctator expuestas a diferentes concentracio- nes del herbicida DG®, a base de SM. NC: control negativo (0 mg/L de SM); CP: control positivo (40 mg/L de ciclofosfamida); Ta=1 mg/L de SM; Tb=5.0 de mg/L SM; Tc=6.2 mg/L de SM. *p<0.05: diferencias significativas con respecto al control ne- gativo (Test de proporcion binomial). Figure 3. Frequency of erythrocytes nuclear abnormalities (ENA) (per-1000 cells) in Leptodactylus luctator tadpoles ex- posed to different concentrations of SM-based herbicide DG®. NC: negative control (0 mg/L of SM); PC: positive control (40 mg/L of cyclophosphamide); Ta=1 mg/L of SM; Tb=5.0 mg/L of SM; Tc=6.2 mg/L of SM. RBCs= red blood cells. * p<0.05: sig- nificant differences compared to the negative control (binomial proportion test). Frecuencia de Aberraciones nucleares en los ertirocitos (ANE) (por 1000 células) en larvas de Leptodactylus luctator expuestas a diferentes concentraciones del herbicida DG®, a base de SM. NC: control negativo (0 mg/L de SM); CP: control positivo (40 mg/L de ciclofosfamida); Ta=1 mg/L de SM; Tb=5.0 mg/L de SM; Tce=6.2 mg/L de SM. *p<0.05: diferencias significativas con respecto al control negativo (Test de propor- cion binomial). Ecotoxicity of the herbicide Dual Gold® on Leptodactylus luctator tadpoles

Figure 2. Frequency of micronuclei (MN) (per-1000 cells) in Leptodactylus luctator tadpoles exposed to different concentra- tions of the SM-based herbicide DG®. NC: negative control (0 mg/L of SM); PC: positive control (40 mg/L of cyclophospha- mide); Ta=1 mg/L of SM; Tb=5.0 of mg/L SM; Tc=6.2 mg/L of SM. RBCs= red blood cells. * p<0.05: significant differences compared to the negative control (binomial proportion test). Frecuencia de micronticleos (MN) (por 1000 células) en larvas de Leptodactylus luctator expuestas a diferentes concentracio- nes del herbicida DG®, a base de SM. NC: control negativo (0 mg/L de SM); CP: control positivo (40 mg/L de ciclofosfamida); Ta=1 mg/L de SM; Tb=5.0 de mg/L SM; Tc=6.2 mg/L de SM. *p<0.05: diferencias significativas con respecto al control ne- gativo (Test de proporcion binomial). Figure 3. Frequency of erythrocytes nuclear abnormalities (ENA) (per-1000 cells) in Leptodactylus luctator tadpoles ex- posed to different concentrations of SM-based herbicide DG®. NC: negative control (0 mg/L of SM); PC: positive control (40 mg/L of cyclophosphamide); Ta=1 mg/L of SM; Tb=5.0 mg/L of SM; Tc=6.2 mg/L of SM. RBCs= red blood cells. * p<0.05: sig- nificant differences compared to the negative control (binomial proportion test). Frecuencia de Aberraciones nucleares en los ertirocitos (ANE) (por 1000 células) en larvas de Leptodactylus luctator expuestas a diferentes concentraciones del herbicida DG®, a base de SM. NC: control negativo (0 mg/L de SM); CP: control positivo (40 mg/L de ciclofosfamida); Ta=1 mg/L de SM; Tb=5.0 mg/L de SM; Tce=6.2 mg/L de SM. *p<0.05: diferencias significativas con respecto al control negativo (Test de propor- cion binomial). Ecotoxicity of the herbicide Dual Gold® on Leptodactylus luctator tadpoles

Table 1. Frequency (%o) of different types of erythrocytes nuclear abnormalities (ENA) in L. /uctator tadpoles exposed to different concentrations of SM (mean + SE). NC: negative control (0 mg/L of SM); PC: positive control (40 mg/L of cyclo- phosphamide); Ta=1 mg/L of SM; Tb=5.0 mg/L of SM; Tc=6.2 mg/L of SM; BE: binucleated erythrocytes; K: kidney-shaped nuclei; EP: erythroplastids or anucleated erythrocytes; BN: budded nuclei; LN: lobed nuclei; BbN: blebbed nuclei; NN: notched nuclei; PN: pyknotic nuclei; AP: apoptotic cells. *p<0.05: significant differences compared to the negative control (binomial proportion test). Frecuencia (%o) de distintos tipos de aberraciones nucleares en eritrocitos (ANE) de larvas de L. luctator expuestas a diferentes concentraciones de SM (media + SE). NC: control negativo (0 mg/L de SM); CP: control posi- tivo (40 mg/L de ciclofosfamida); Ta=1 mg/L de SM; Tb=5.0 mg/L de SM; Tc=6.2 mg/L de SM; BE: eritrocitos binucleados; K: nucleos en forma de rion; EP: eritoplastidos o eritrocitos anucleados; BN: nicleos con brotes; LN: ntcleos lobados; BBN: nucleos ampollados; NN: nucleos con muescas; PN: nucleos picnoticos; AP: célula apoptotica. *p<0.05: diferencias significativas con respecto al control negativo (Test de proporcion binomial). mg/L has been reported for SM on embryos of the common frog Pelophylax perezi (Quintaneiro et al., 2018). Our results suggest that L. /uctator tad- poles are less tolerant to SM than P. perezi embry- os. Differences in sensitivity could be attributed to intrinsic differences between species, as well as to the different developmental stages evaluated, and to the commercial formulation used in this study, which, in addition to the active ingredient contains excipients such as surfactants and solvents, which may influence its toxicity. Previous studies have shown that a commercial formulation of various agrochemicals may be more toxic than the active ingredient itself (K6nen & Cava, 2008; Nikoloff et al., 2013; Lajmanovich et al., 2014; Bach et al., 2016). While DG® contains more than twice the amount of SM in its formulation than Primextra® II, it is not possible to directly compare the sensi-

Table 1. Frequency (%o) of different types of erythrocytes nuclear abnormalities (ENA) in L. /uctator tadpoles exposed to different concentrations of SM (mean + SE). NC: negative control (0 mg/L of SM); PC: positive control (40 mg/L of cyclo- phosphamide); Ta=1 mg/L of SM; Tb=5.0 mg/L of SM; Tc=6.2 mg/L of SM; BE: binucleated erythrocytes; K: kidney-shaped nuclei; EP: erythroplastids or anucleated erythrocytes; BN: budded nuclei; LN: lobed nuclei; BbN: blebbed nuclei; NN: notched nuclei; PN: pyknotic nuclei; AP: apoptotic cells. *p<0.05: significant differences compared to the negative control (binomial proportion test). Frecuencia (%o) de distintos tipos de aberraciones nucleares en eritrocitos (ANE) de larvas de L. luctator expuestas a diferentes concentraciones de SM (media + SE). NC: control negativo (0 mg/L de SM); CP: control posi- tivo (40 mg/L de ciclofosfamida); Ta=1 mg/L de SM; Tb=5.0 mg/L de SM; Tc=6.2 mg/L de SM; BE: eritrocitos binucleados; K: nucleos en forma de rion; EP: eritoplastidos o eritrocitos anucleados; BN: nicleos con brotes; LN: ntcleos lobados; BBN: nucleos ampollados; NN: nucleos con muescas; PN: nucleos picnoticos; AP: célula apoptotica. *p<0.05: diferencias significativas con respecto al control negativo (Test de proporcion binomial). mg/L has been reported for SM on embryos of the common frog Pelophylax perezi (Quintaneiro et al., 2018). Our results suggest that L. /uctator tad- poles are less tolerant to SM than P. perezi embry- os. Differences in sensitivity could be attributed to intrinsic differences between species, as well as to the different developmental stages evaluated, and to the commercial formulation used in this study, which, in addition to the active ingredient contains excipients such as surfactants and solvents, which may influence its toxicity. Previous studies have shown that a commercial formulation of various agrochemicals may be more toxic than the active ingredient itself (K6nen & Cava, 2008; Nikoloff et al., 2013; Lajmanovich et al., 2014; Bach et al., 2016). While DG® contains more than twice the amount of SM in its formulation than Primextra® II, it is not possible to directly compare the sensi-

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