Artículos

 

Effects of cadmium and zinc on Procambarus clarkii: Simulation of the Aznalcóllar mining spill

 

Efectos de cadmio y zinc en Procambarus clarkii: Simulación del accidente minero de Aznalcóllar

 

M.L. Martín-Díaz¹*, S.R. Tuberty², C.L. McKenney Jr.³, D. Sales¹ and T.A. Del Valls¹

 

¹ Facultad de Ciencias del Mar y Ambientales Campus Río San Pedro s/n 11510 Puerto Real, Cádiz, Spain. * E-mail: laura.martin@uca.es

² University of West Florida Center for Environmental Diagnostics and Bioremediation One Sabine Island Dr. Gulf Breeze, FL 32561, USA.

³ US Environmental Protection Agency NHEERL Gulf Ecology Division One Sabine Island Dr. Gulf Breeze, FL 32561, USA.

 

Recibido en junio de 2003;
aceptado en mayo de 2004.

 

Abstract

Female red swamp crayfish, Procambarus clarkii, were exposed for 21 days in the laboratory to different dissolved concentrations of zinc (1000 µg L^-1 and 3000 µg L^-1) and cadmium (10 µg L^-1 and 30 µg L^-1), determined in the Guadiamar River after the Aznalcóllar mining spill (SW Spain). Female gonadosomatic and hepatosomatic indexes were analyzed at the end of the bioassay, and a general decrease in the gonadosomatic index and increase in the hepatosomatic index were observed in individuals at the same maturation stage, exposed to increasing heavy metal concentrations. Only the decrease in the gonadosomatic index values was significant at the highest zinc concentration. Decreases in gonadosomatic indexes were associated with reduced fecundity.

Key words: Aznalcóllar mining spill, heavy metals, crustaceans, gonadosomatic index, hepatosomatic index.

 

Resumen

Hembras de cangrejo rojo, Procambarus clarkii, se expusieron en el laboratorio durante 21 días a diferentes concentraciones de zinc (1000 µg L^-1 y 3000 µg L^-1) y de cadmio (10 µg L^-1 y 30 µg L^-1), determinadas en el Río Guadiamar tras el accidente minero de Aznalcóllar (SO de España). Se analizaron los índices gonadosomáticos y hepatosomáticos de las hembras al final del bioensayo, observándose una disminución del índice gonadosomático y un aumento del hepatosomático en los individuos que se encontraban en el mismo estado de maduración, expuestos a un incremento de concentraciones de metales. Únicamente se observó una disminución significativa del índice gonadosomático a la mayor concentración de zinc. La disminución en los índices gonadosomáticos se ha relacionado con una disminución de la fecundidad.

Palabras clave: accidente minero de Aznacóllar, metales pesados, crustáceos, índice gonadosomático, índice hepatosomático.

 

Introduction

The Guadalquivir River is the main river of the southern Iberian Peninsula and shelters different kinds of ecologically important species. The Aznalcóllar mining spill poured into the nearby Guadiamar River, which flows into the Guadalquivir estuary, almost 6 Hm³ of mud and acidic waters, with high concentrations of metals in solution, including Cd, Cu, Mn, As, Pb and especially Zn. Consequently, the sediment and water quality of the river and estuary was negatively affected (Gutiérrez-Yurrita and Montes, 1998; Riba et al., 2002).

Environmental contamination represents a threat to aquatic species world-wide (Guillette et al., 1998). The red swamp crayfish, Procambarus clarkii (Girard, 1852), a native of the southern United States and introduced into the southern Iberian Peninsula, has been intensely studied as a bioindicator of the effects of pollutants and because of its economic importance. Extensive literature is available on heavy metals and this crayfish: effects in ovarian development on this species have been studied by Naqvi and Howell (1993) and Reddy et al. (1997). Ovarian development, a hormonally-regulated process, can serve as an indicator of the condition of the gonads (Fingerman et al., 1998). It can be determined through the variation of the gonadosomatic index that represents the percentage of the weight of the animal due to the gonad, and has been studied in vivo in the effect of Cd on the gonadosomatic index of P. clarkii (Reddy et al., 1997). The relationship between ovarian maturation and development of the hepato-pancreas, and its dependence on hormonally-regulated processes, made the hepatopancreas an object of study at the same time as the ovary. The hepatosomatic index, the percentage of the weight of the animal due to the hepatopancreas, is also important because of its role in ovarian maturation, and it could be an indicator of the condition of this organ.

The hepatopancreas synthesizes and releases serum vitello-genin, which is immunologically identical to lipovitellin, the major high-density lipoprotein in the fully-developed ovary (Paulus and Laufer, 1987). Reproductive processes in the brachyurans, including vitellogenesis, are governed by neuralhormonal mechanisms (Adiyodi and Adiyodi, 1970). Therefore, in addition to the oocytes, the cells in the hepatopancreas that synthesize vitellogenin are potential targets for hormonal factors regulating reproduction (Paulus and Laufer, 1987).

This study was designed as a preliminary evaluation of the effect of different concentrations of Cd and Zn, using real dissolved concentrations from a mining spill episode, on the development of the ovaries and its relationship with changes in the contribution of the hepatopancreas to the total weight of P. clarkii. At the same time, the gonadosomatic and hepatoso-matic indexes were tested as biomarkers of environmental heavy metal contamination.

 

Material and methods

Approach

The present study was performed taking into account heavy metal concentrations of dissolved Cd and Zn, previously determined the first months after the spill during the monitoring of the impact on the Guadiamar River and surrounding areas by Junta de Andalucía (www.cma.junta-andalucia.es). Intermoult female P. clarkii were obtained from the Department of Wildlife and Fisheries, Mississippi State University, USA. The animals were placed in a laminar-flow holding tank with filtered (6 mm) and dechlorinated fresh water and allowed to acclimate for two weeks prior to exposures. The tank conditions were maintained at 25°C and a 12:12 h light:dark cycle. The material used for the present experiment was previously washed with nitric acid (10%) and cleaned with distilled water (Milli-Q) to avoid metal contamination. Crayfish were exposed for 21 days to 1000 µg L^-1 and 3000 µg L^-1 of Zn (zinc chloride), and 10 µg L^-1 and 30 µg L^-1 of Cd (cadmium chloride) (Sigma) in filtered and dechlorinated fresh water. A single set of reference females was used. Eight female P. clarkii were randomly placed in 30-L tanks with 5-L volume of exposure solutions (filtered dechlorinated fresh water and metal stock solution). Chelipeds were removed in order to reduce antagonistic behavior and cannibalism. The exposure solutions were replaced every three days and the animals were fed on a diet of frozen brine shrimp just prior to the water change to reduce fouling of the exposure solutions. Exposure conditions were as described for the acclimation period.

Determination of the gonadosomatic and hepatosomatic indexes

At the end of the experiment, the crayfish were weighed and dissected, and their ovaries and hepatopancreas were removed and weighed. The gonadosomatic index or GSI (ratio of wet weight of ovaries to whole body wet weight x 100) and the hepatosomatic index or HSI (ratio of weight of the midgut gland to whole body weight x100) were calculated. Ovarian maturation in crustaceans includes a color change and an enlargement in size of the ovaries as the oocytes proliferate and increase in diameter during yolk incorporation (Charniaux-Cotton and Payen, 1998). In order to determine the ovarian maturation stage, a sample of ovarian tissue was examined under a light microscope to estimate the oocyte diameter by ocular measurements. Although eight female crayfish were chosen for each treatment in the present bioassay, only females in the same maturation stage (stage VI) were selected for analysis of the results.

The GSI and HSI data obtained for individuals exposed to Cd and Zn were compared to those belonging to the control treatment by Student's t-test with significance set at the 95% and 99% confidence intervals.

 

Results

Effect of Cd and Zn on the gonadosomatic index

The ovarian stage of all the females used for the data analysis (control and metal treatments) was the same, stage VI, and the diameter of the oocytes was 10 µm. This makes it possible to compare GSI means of all female crayfish treated in this experiment at the same ovarian maturation stage. The GSI means belonging to the different heavy metal exposures and control are given in table 1. Increasing Zn concentration exposure resulted in a decrease in the GSI values. The values obtained were lower than those determined for control crayfish, although only the GSI mean of the individuals exposed to the highest Zn concentration (3000 µg L^-1) was significantly different to the control (P < 0.01). This response showed that exposure to the highest Zn concentration significantly affects the development of the oocytes.

The GSI means of the crayfish exposed to Cd did not show a clear relationship with the Cd concentration values, like that observed in crayfish exposed to Zn. The GSI values for animals exposed to Cd were lower, but not significantly different to the control.

Effect of Cd and Zn on the hepatosomatic index

The HSI was determined for the same animals used in the GSI determination. The HSI means of the individuals exposed to the different heavy metal concentrations and control are given in table 2. Results show higher HSI means in animals exposed to Zn and Cd than those means obtained in the negative toxicity control, although they were not significantly different. Increasing Zn concentration was related to an increase in HSI values; nevertheless, crayfish exposed to Cd did not show a clear relationship with Cd concentration values.

 

Discussion

Studies of the effects of contaminants on gonad development of crustaceans have been relatively few in number (Fingerman et al., 1998). These parameters, in particular GSI, can serve as indicators of the condition of the gonads (Fingerman et al., 1998). They not only provide the information that an effect biomarker can give, but also provide information about the effect in future generations because of the role of gonad maturation in reproduction. The GSI has been used in studies developed to determine the effect of the injection of methyl farnesoate (Laufer et al., 1998), the effect of temperature and photoperiod (Daniels et al., 1994), and the effect of the injection of Cd (Reddy et al., 1997) on P clarkii. The stimulation or inhibition of this index has been directly related to stimulation or inhibition of ovarian maturation. Only in the study developed by Laufer et al. (1998) were both criteria (size of the ovary relative to individual body weight (GSI) and color to assess maturation) taken into account together to assess the effect of the injection of methyl farnesoate on ovarian maturation in females of red swamp crayfish P. clarkii, resulting in differences of GSI values and oocytes with similar diameters.

The reduction in GSI values observed in the present study, for crayfish exposed to Cd and Zn (only significant at the highest concentration) compared with the control, could be due to an increase or decrease in total body weights; however, no differences in body weight were found between animals exposed to Cd or Zn and the control. The decrease in GSI values obtained for crayfish exposed to both metals and concentrations, in individuals at the same stage of development and similar oocyte diameter, may be due to a fewer number of oocytes maturing in response to the heavy metal treatment, resulting in reduced fecundity. Although the number of developing oocytes was not counted in this study, this speculation is not wholly improbable, because Cd was found to reduce fecundity and hatching success of P clarkii at 0.5 mg of Cd per gram body weight (Naqvi and Howell, 1993), and all broods of the cladoceran Moina irrasa had a reduced number of progeny at concentrations of 200 L^-1 of Zn (Zou, 1997).

Increases in HSI values were related to decreases in GSI values in every treatment. Deposition of energy reserves in the hepatopancreas (increase of hepatopancreas weight) may have been required before yolk deposition in the eggs (increased ovary weight).

Only individuals at the same stage of ovarian development and with similar oocyte diameter were used in this study. This fact and the mortality registered in all treatments and control (25%) due to cannibalism between individuals, did not allow greater analysis of the results because of the small number of replicates. Nevertheless, the data obtained can be taken into account as a first approach.

The concentrations of heavy metals released during the first days of the Aznalcóllar mining spill produced adverse biological effects (Riba et al., 2003). The effects produced on the reproductive efficiency of native species could not be measured at that time, but the concentrations of heavy metals measured could have affected the organisms during the first months of the accidental spill. In the present study, hormon-ally-regulated processes were used as indicators of the health of the environment, and tested in the laboratory with real environmental concentrations. Nevertheless, more studies regarding the use of GSI and HSI values as indicators of toxic-ity in the environment are necessary, because although a direct relationship was shown between these indexes and dissolved Zn concentrations, they did not show a clear trend in animals exposed to Cd.

 

Acknowledgements

This research was supported by a grant from the PICOVER program (Consejería de Medio Ambiente, Junta de Andalucía, Spain) and performed at the US Environmental Protection Agency, Pensacola, Florida. The authors would like to thank the members of the EPA for their help and support, and especially Kathy, Tere and Andy for their tireless efforts and friendship during the course of this study.

 

References

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