Determination of trace metals by voltamperometry in zebra mussel (Dreissena polymorpha) employed as environmental bio-indicator

Abstract

In the present work, metals (cadmium, lead, copper, nickel, tin, selenium, and mercury) have been estimated in the Ebro River (Spain) using the zebra mussel (Dreissena polymorpha) as an environmental bio-indicator. In two sequential studies, in 2006 and 2008, concentrations of metals were calculated in water as well as in the shells and fleshes of the zebra mussels. Samples were collected from assorted locations of the river. Metals were determined successfully at trace levels through voltamperometry, a sensitive technique. It has been noted that analysis of bioaccumulators like zebra mussels can be helpful in evaluating metal pollution in water.

Keywords:

• Dreissena polymorpha
• Ebro River
• metal determination
• voltamperometry
• zebra mussel

Introduction

Zebra mussels (Dreissena polymorpha) are widely used bio-indicators of persistent organic pollutants 1, trace metals 2, and radionuclides 3 in several worldwide ecosystems. They were applied successfully to assess the trace metal contamination in freshwaters. Dreissena polymorpha is a filter-feeding bivalve and is widely spread based on the planktonic larval stage. It is easily collectable species throughout the year having enough biomass for trace analysis. Because of these qualities, it is commonly employed as a bio-indicator for finding out metal contamination in water ecological systems 2. Several studies have been reported about using zebra mussel (Dreissena polymorpha) as a bio-indicator in different parts of the world, for instance, in subalpine lakes, Italy 2, Lake Erie and Ontario, USA 4, Danube River, Vienna 5, Bergen Harbor area, Norway 6, in Gulf of Olympia, Greece 7 and by us in River Ebro (Spain) 8.

Several spectroscopic techniques are being used to find out metals in mussel samples. Electrothermal atomic absorption spectroscopy was used for determination of Cd, Co, Cr, Cu, Hg, Ni, Pb, and Zn 2. Inductively coupled plasma emission spectroscopy has been used for determination of Ca, Cd, Cr, Cu, Fe, Hg, K, Mg, Mn, Mo, Na, Ni, Pb, Se, Sr, V, and Zn 4 9. Cold vapor atomic absorption spectroscopy was utilized for mercury 10 while electro thermal atomic absorption spectroscopy for cadmium by using slurries 11. Also, cadmium and lead were determined by atomic absorption spectroscopy coupled with graphite furnace 12 13. Automated ultrasonic slurry sampling and microwave-assisted digestion was employed to quantify arsenic with electro thermal atomic absorption spectroscopy 14. Flame atomic absorption spectrometry and flow injection analysis were employed for finding out cadmium in mussels 15. Apart from spectroscopic techniques, a rapid voltammetric method has been developed for the trace determination of copper in fish samples 16. In this study, a procedure based on anodic stripping voltamperometry is described and conditions were optimized for determination of Zn, Cd, Pb, Cu, Ni, Fe, Al, Se, Cr, and Sn in mussel samples. Anodic stripping voltammetry is a voltammetric method for quantitative determination of specific ions. The analyte is electroplated on the electrode during a deposition step, and oxidized from the electrode during the stripping step. The current is measured during the stripping step. The oxidation of ions is registered as a peak in the current signal at the potential at which the ions begin to oxidized. The method is simple, sensitive and less time-consuming compared with other techniques in use.

Results and discussion

Zebra mussel (Dreissena polymorpha) is a freshwater bivalve native to the drainage basins of Black and Caspian Sea, spreading along the coasts of eastern USA and Europe. For the first time, they were found in Spain during summer 2001 in Ebro River 8. Exterior of zebra mussel is similar to marine mussel. During the biological cycle, the larva produced is scattered with flowing water. It grows rapidly, and can tolerate temperature changes and salinity of water. It is a perfect invader in water ecosystems and reduces quantity of phytoplankton in water rapidly. Its presence increases the rate of deposition of organic matter in sea depths that increases transparency of water. As a bio-accumulator, they can deposit organic matter and pollutants 8. The sampling site chosen were entry and exit of Ribaroja dam at Ebro River. The reason for choosing the said site was the fact that in dam when water is stagnant, there will be more bioaccumulation and more metals will be collected by mussels than running water.

The method was optimized before analysis. The sensitivity of voltamperometry varies from metal to metal, as shown in Table 2. In 2007, similar work had been carried out for judging metals in water but in different times of the year (January, February, November, and December) 8. In the present work, metal concentrations in zebra mussel (Dreissena polymorpha), taken from Ebro River in January, March, July, and October, were measured by voltamperometry and the results are shown in Figure 1. Technique was found to be more sensitive for lead, zinc, and cadmium whereas rather less sensitivity was found for mercury, tin, and iron. Figure 2a, Figure 3a, and Figure 4a show results at entry point of Ribaroja dam in flesh, sludge, and water in that order while Figure 2b, Figure 3b, and Figure 4b show results at exit point of the dam.

Figure 1.  Metals in zebra mussels (Dreissena polymorpha) by voltamperometry technique in Ebro River in different times of year. 1=zinc (mg/kg), 2=cadmium (mg/kg), 3=lead (mg/kg), 4=copper (mg/kg), 5=nickel (mg/kg), 6=iron (g/kg), 7=aluminum (g/kg), 8=tin (mg/kg), 9=selenium (mg/kg), 10=mercury (mg/kg), 11=chromium (mg/kg).

Figure 2a.  Concentrations of metals in flesh samples of zebra mussels (Dreissena polymorpha) by voltamperometry technique taken from entry point of dam at Ebro River in different times of year; units=mg/kg, 1=cadmium, 2=lead, 3=copper, 4=nickel, 5=tin, 6=selenium, 7=mercury, 8=chromium.

Figure 2b.  Concentrations of metals in flesh samples of zebra mussels (Dreissena polymorpha) by voltamperometry technique taken from exit point of dam at Ebro River in different times of year; units=mg/kg, 1=cadmium, 2=lead, 3=copper, 4=nickel, 5=tin, 6=selenium, 7=mercury, 8=chromium.

Figure 3a.  Concentrations of metals in sludge samples taken from entry point of dam at Ebro River collected along zebra mussel colonies in different times of year; units=mg/kg, 1=cadmium, 2=lead, 3=copper, 4=nickel, 5=tin, 6=selenium, 7=mercury, 8=chromium.

Figure 3b.  Concentrations of metals in sludge samples taken from exit point of dam at Ebro River collected along zebra mussel colonies in different times of year; units=mg/kg, 1=cadmium, 2=lead, 3=copper, 4=nickel, 5=tin, 6=selenium, 7=mercury, 8=chromium.

Figure 4a.  Concentrations of metals in water samples taken from entry point of dam at Ebro River collected along zebra mussel colonies in different times of year; units=mg/kg, 1=cadmium, 2=lead, 3=copper, 4=nickel, 5=tin, 6=selenium, 7=mercury, 8=chromium.

Figure 4b.  Concentrations of metals in water samples taken from exit point of dam at Ebro River collected along zebra mussel colonies in different times of year; units=mg/kg, 1=cadmium, 2=lead, 3=copper, 4=nickel, 5=tin, 6=selenium, 7=mercury, 8=chromium.

Maximum accumulation of cadmium has taken place in March at exit point of dam. Same was the case with lead, nickel, and selenium. In general, concentration of metals, except copper, lessens as the year passes on, however, mercury has concentrated. The metal concentrations were higher in winter since these species accumulate metal for their physiological activities in cold. Afterwards, as the time goes on, metal concentration decreases through natural excretory processes. Maximum zinc concentration was found in December and January while cadmium in November and December. It supports the idea of metal build-up for physiological activities in cold but after winter, unload metals. Lead has the same tendency but not prominent. Metals like lead, mercury, tin, and chromium have wide range of applications in industry and in agriculture; therefore, they come to the sea in huge quantities. Study of analytes like zebra mussels will allow us to note metal contamination in sea, during specific times of year.

It was found that in the flesh samples taken from the dam exit point, metal concentration was high because of holding water in dam that made metal build-up suitable. Although, absorption and desorption went on simultaneously, but the samples got in March, have higher values because of high rates of absorption of metals. The sludge samples taken from the entry point of the dam have highest values in January. Metal concentration in water was highest in October because of activities taking place in summer. However, metal content in waters near to sediments was almost constant indicating an establishment of dynamic equilibrium (absorption–desorption).

Experimental

Reagents, standards, and instruments

All reagents used in this work were of high grade purity purchased from either Merck or Sigma-Aldrich. Double distilled water was used to prepare standards and samples. Stock solutions of metals were prepared by dissolving appropriate quantity of pure metal or anhydrous metal salt of high purity in distilled water. The standards were prepared by successive dilution of stock solutions. Anodic stripping voltametry was performed on a VA-Computrace (Model-757) equipped with GOES-646 processor. Mercury electrode was used for potential measurements, whereas silver/silver chloride electrode and platinum electrode were employed as reference electrode, respectively.

Sampling sites and timing

Samples were collected by different locations along the river (exit and entry points of Ribaroja dam), and at different depths, and in different times of year. Annual range of water parameters is shown in Table 1.

Table 1. Annual range of different water parameters collected by various depth levels.

Depth (m)	T^°C	SAT% O2	CE1	pH	Chlorophyll (mg/L)
0.1	6.3–24.9	84.0–155.1	713.0–1645.0	6.4–7.5	3.6–19.5
2	6.2–24.6	70.8–152.5	708.0–1645.0	6.4–9.5	3.4–24.5
5	6.1–24.6	67.7–152.5	708.0–1640.0	6.3–7.5	3.3–20.5
10	6.2–22.2	68.4–111.1	804.0–1639.0	6.3–7.5	3.8–13.9
20	6.2–18.8	42.3–110.9	874.0–1639.0	6.3–7.5	2.7–10.9
Note: d=(S–1)/ln N (Margalef's diversity index), where S is the number of species and N is the number of individuals.

Sample preparation

The zebra mussel colonies were drawn out alive (together with associated soil and water) from Ebro River. The three components (zebra mussels, sludge of moist soil, and water) were separated. Afterwards, mussels were soaked in distilled water for few hours and then in dilute HCl (1:100). They were spread over a filter paper and dried. Rind was separated with plastic tweezers. Comprehensive washing was carried out to remove all types of impurities like eggs and mussels in the embryonic process, since they can cause interruption during analysis. Flesh and shells were separated. Shells were crushed with mortar and flesh with turmix.

Shells were heated in china dish at 110^oC for 2 hours to remove moisture. Organic content was removed by calcining at 550^oC for 2 hours in controlled temperature furnace. Material gained after calcination was digested with 5 mL nitric acid (1:1) for 10–15 min and then diluted to 100 mL after filtration.

Flesh was also treated like shell sample, except that 1 g calcium carbonate was added to each 10 g of flesh sample to avoid metal loss during calcination. After digestion with 5 mL nitric acid (1:1), sample was preserved.

Analysis of metals

Electroanalytical system was calibrated for zinc, cadmium, mercury, lead, copper, nickel, selenium, iron, aluminum, chromium and tin, one by one, and then respective samples of each metal were run. Detection limits, linear range and percentage recovery of each metal were got by using synthetic samples (Table 2).

Table 2. Analytical parameters (detection limits, linear range, and percentage recovery) of voltamperometry.

Element	LODx10^−6 (mg/Kg)	Linear range (µg/Kg)	Recovery (%)
Zn	50	0.5–10.0	99.72
Cd	25	0.2–5.0	99.54
Pb	50	0.5–10.0	99.63
Cu	2500	250.0–5000.0	99.45
Ni	50	0.5–10.0	99.87
Fe	100	1.0–20.0	99.78
Al	2500	250.0–5000.0	99.62
Se	2500	250.0–5000.0	99.57
Cr	2500	250.0–5000.0	99.63
Sn	500	5.0–100.0	99.71
Hg	1000	10.0–200.0	99.87

Conclusions

Metals, nutritious as well as pollutants, were determined by voltamperometry in zebra mussel (Dreissena polymorpha), and collected at entry and exit points of Ribaroja dam located at Ebro River (Spain). The study has shown that metals like copper, cadmium, zinc, chromium, iron, selenium and mercury varies in water; in different parts of years depending on different factors like temperature, retention time in dam, and absorption capacity of zebra mussel. Voltamperometry is a good source to see variations in metal concentration in water that will help to explore outcomes of metal pollution in watery systems.

Acknowledgements

The authors would like to thank the members of “Grupo Especial de Actividades Subacuáticas de la Guardia Civil” for diving in the Ebro River and collecting the mussels for this study; the Spanish Electric Company “Endesa” for financial help for this project and also the Arágon Regional Government for financing our research group E 75.