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Boletín de la Sociedad Chilena de Química

versión impresa ISSN 0366-1644

Bol. Soc. Chil. Quím. v.45 n.3 Concepción set. 2000

http://dx.doi.org/10.4067/S0366-16442000000300016 

USE OF THE FRESH WATER PLANTS ZANNICHELLIA PALUSTRIS
AND MYRIOPHYLLUM ACUATIUM
FOR BIOMONITORING OF Cd,
Pb, AND Cu IN ANDEN RIVERS OF CHILE

S.Stegen1 *; F.Queirolo1, S.Cortés1; J.Pastenes2;
P. Ostapczuk3; F. Backhaus3, C. Mohl3

1 Department of Chemistry, Universidad Católica del Norte, Angamos 0610,
Antofagasta, Chile
2 Department of Biomedicine, Universidad de Antofagasta, Campus Coloso,
Antofagasta, Chile.
3 Institute of Applied Physical Chemistry, Research Center Jülich,
D-52425 Jülich, Germany
(Received: March 15, 2000 - Accepted: June 21, 2000)

In memorian of Dr. Guido S. Canessa C.

Abstract

The species Zannichellia palustris and Myriophyllum acuatium, collected along the Loa River basin, North Chile, were chosen as possible bioindicators for Cd, Pb, and Cu. These species have a cosmopolitan distribution and can survive very sudden and large fluctuations in salinity. They are widely spread in Chile and in spite of their enormous salt tolerance they seldom penetrate to the marine habit.

Quantification in water and algae samples was made by Differential Pulse Anodic Stripping Voltammetry (DPASV) after UV (water) and open wet digestion (algae). The cadmium concentrations found in algae samples ranged from 0.72±0.01 to 32±0.31 µg/g d.w. Values for lead were less and estimated 0.4±0.01 to 2.13±0.08 µg/g d.w.. Copper concentrations were between 13±0.2 and 119±0.5 µg/g d.w.. In comparison with the element concentrations in the water samples, a significant biomagnification, especially for cadmium in Myriophyllum acuatium was observed.

Keywords: river water plants, bioindicator, heavy metals, North Chile, Loa river

RESUMEN

Las especies Zannichellia palustris y Myriophyllum acuatium recolectadas a lo largo de la Cuenca del Río Loa, fueron escogidas como posibles bioindicadores de Cd, Pb y Cu. Estas especies tienen una distribución cosmopolita y pueden tolerar súbitamente grandes fluctuaciones de salinidad. Se encuentran ampliamente distribuídas a lo largo de Chile y a pesar de su enorme tolerancia a la sal, raramente penetran en el habitat marino.

La cuantificación en las aguas y algas se realizó mediante la técnica electroanalítica de Voltamperometría Diferencial de Pulso con Redisolución Anódica (DPSAV), tras una mineralización por UV para las aguas y húmeda de sistema abierto para las algas. La concentración encontrada para cadmio en las muestras de algas fluctúan entre 0.72±0.01 y 32±0.31 µg/g p.s. y para Cu, entre 13±0.2 y 119±0.5 µg/g p.s. En comparación a la concentración de los elementos encontradas en las aguas, se observó una significativa biomagnificación, especialmente para Cd, en la especie Myriophyllum acuatium.

Palabras claves: plantas acuáticas de río, bioindicador, metales pesados, norte de Chile, Río Loa.

INTRODUCTION

The concept of a bioindicator is applied to those organisms that are able to accumulate substances considered as contaminants, without being themselves greatly affected. The process through which the organism concentrates a substance to levels greater than the environment is known as bioaccumulation. 1, 2) Each organism has a different optimum life range. For example, those species with a wide tolerance range should be able to cope with environmental alterations more easily. These organisms are the bioindicators of an altered environment.

The factors considered for the bioindication process (via element accumulation) depend greatly on the bioindication method used. The two main bioindication methods are the "in situ" or passive method (the plant grows in the area studied) and the so-called indirect or transplantation method (plants are transplanted and exposed to a study area for a certain time span). This paper is based on the first bioindication method.

In general, a bioindicator must be sensitive to environmental changes. The quantification of a pollutant, indicated by the experimentally collected data, changes the bioindicator into a biomonitor, relative to the quantification of the parameter studied.

In the water, nutrients are always available for plants. Therefore, when the amount of mineral nutrients increases, water plant growth may be very fast. However, the toxic compounds contaminating the water are also available for plants and may cause death in some cases.

Physiologically, water plants are different from ground plants because they absorb nutrients through all the submerged area and, they can extract carbon dioxide from the bicarbonate dissolved in the water by photosynthesis. Many undergo a serious reduction of the vegetative body during unfavorable periods. Due to their relatively long life cycles, their permanent existence, and their tolerance to different environmental impacts, etc., vegetable species from water environment, both sea and river, have been commonly reported as the major contamination indicators. 3,4)

The chemical composition of river waters in North Chile is influenced by two main conditions: the saline environment, which explains the high concentration of major elements 5,6,7) and volcanism, which is related to the presence of several volatile trace elements. 8,9)

The metals studied here were lead, cadmium and copper. These elements show a great affinity with certain functional groups (particularly SH-groups of proteins) and act, directly or indirectly, interfering with different metabolic processes 10). These metal-thionines are found in human beings, animals, simple organisms and even in major plants 10,11). Copper is also an essential micronutrient for most plants and it takes part in several metabolic processes. It has been shown that the lack of copper may influence many physiological processes. On the other hand, copper becomes toxic for water plants at higher levels 12).

This research was aimed first at implementing a reliable analytical methodology and, in the second stage, the quantification of potentially ecotoxic heavy metals, especially Cd, Pb, and Cu in water and water plant samples from the Loa River basin in North Chile.

EXPERIMENTAL

Sampling

The Loa River basin (Fig. 1) is located in the northern third of the Antofagasta Region and contains one of the few rivers discharging into the Northern Chile Pacific Ocean. Together with the Atacama River (San Pedro de Atacama), they are the most important rivers of the province. The Loa river, 440 km long, originates at the northern base of the Miño volcano, almost at the border of Tarapacá and Antofagasta (21º 15’ S). The sampling area is located in the higher course of the river Loa from its origin in Calama, more precisely, a 13º-km span between Lequena and Calama. Here the Loa is the most important tributary from the Andes: San Pedro River (Inacaliri) which originates from the east, in the vicinity of San Pedro de Conchi, 76 km from Calama and, the river Salado, also originates from the east, 3 km down Chiu-Chiu.


From the very beginning, all used equipment and sample manipulation was aimed at a chemical analysis of Cd, Pb, and Cu present at trace level. Special care was taken to avoid any contamination.

The water samples were collected in precleaned 0.5 l polyethylene (PE) bottles. Samples were immediately filtered through a 0.45 µm cellulose acetate filter (Sartorius) into a 100 ml PE bottels (twice from each sampling area), acidified to pH 1 with nitric acid (65%, Merck, suprapur) and then transferred to polyethylene bags.

About 5 kg of Myriophyllum aquaticum and Zannichellia palustris were taken from each of the sampling areas of the river basin (Fig. 1), by applying a random sampling procedure 13). Aquatic plants were sampled by hand protected with vinyl gloves at the river bottom and transferred into polyethylene bags.

The samples were stored in a cold box and transported by car within Chile. In spite of the long distances and time required for a sampling exercise, this study was limited only to the medium and high area of the basin. Plant samples collected in the river San Salvador quickly decomposed and were not included into the study.

The sampling was made in July 1995, when the plants are subject to a higher "stress" due to the lowest water temperatures in the winter season. Also, there is a less ion mobility in the environment 14). In Table 1 some water parameters at the sampling time are presented.

Table I. Collection areas and conditions at the sampling time (July, 1995).

Species studied

The species were identified at the Museo Nacional de Historia Natural in Santiago, Chile.

Myriophyllum acuaticum (Fig.2)
Family: Haloragaceae
Common name in northern Chile: Frog herb, Little water pine tree (Hierva del sapo, Pinito de agua).
Characterization: rooted submersed water herb with a long branched stem and divided leaves. The stem emerges in summer to flourish and later givesrice to fruit bodies. It lives and grows in the water or muddy substrates at different depths. It is a cosmopolitan species existing worldwide.
Notation Assigned: M


Zannichellia palustris L. (Fig.3)
Family: Zannichelliaceas
Common name in northern Chile: lake horny herb (cachudita de las lagunas).
Characterization: a submersed water plant rooted in the muddy substrate, with a branched filamentous stem and linear leaves. It grows in brakish water of salt marshes, ditches and estuaries. It may occasionally grow into the open air without being damaged. It is a cosmopolitan plant growing throughout South America and can survive very sudden and large fluctuation in salinity.
Notation Assigned: Z


Equipment and procedures

Freeze-drying: For freeze-drying a Finn-Aqua, Hürth, Germany LYOVAC GT2 was used together with a Leybold trivac D8B vacuum pump. The freeze drying was performed at 22ºC to the final pressure of 6,2x10-2 mbar.

Homogenization: Homogenization was performed with a Fritsch, Idar-Oberstein, Germany, Planetary Mill Pulverisette 5 made of ZrO2 at 225 rpm for 60 min.

Digestion: Water samples were digested by UV (Kuerner, Rosenheim, Germany)

Residual water determination: The dry mass-correction with infrared was accomplished by a Mettler Toledo, Germany, LP16-M infrared balance. Sub-samples used for the determination of the correction factor cannot be used for elemental determination due to loss of volatile elements15).

Determination: DPASV curves for the simultaneous determination of cooper, cadmium and lead were recorded after a digestion procedure with the model 348B Polarographic Analyzer using the 303A Static Mercury Drop Electrode both from EG&G PAR (Princeton, NJ). Details of determination procedure are presented in Table II. Using the method of calibration curve at low concentration level 16) following detection limits for used parameter and recalculated to the dry mass were found: for cadmium 5 ng/g, for lead 5 ng/g and for copper 50 ng/g.

Table II. Experimental conditions for Cd, Pb, and Cu determination by Differential Pulse Anodic Stripping voltametry (DPASV)

* Supporting electrolyte: 10 ml de HCIO4 s.p. ( 79 % Merck) diluted to 10g with deionized water (Milli-Q-Water-Purification-System, Millipore, Germany).

Reagents and reference solutions

Deionized water was obtained from a Milli-Q-Water-Purification-System (Millipore, Germany). All reagents were Merck (Darmstadt, Germany). Acids used in the wet digestion procedures was HClO4 (70% s.p.) and HCl (30% s.p.) and HNO3 (65% p.a.) distilled under sub-boiling conditions prior to use. Stock standard solutions (1g/L) of Cu2+, Cd2+ and Pb2+ were prepared from Tritisol solutions and acidified at pH <2.

Sea Lettuce BCR-CRM 279 was employed as certified reference material for optimizing and setting up the analytical procedure.

Digestion procedure

About 0.200±0.001g of the dry sample was weighed into 50-ml quartz vessels (suprasil). 2 ml of a mixture of acids, HNO3/HCl04, at a ratio of 40:1 was added. The vessels were covered with quartz lids and put on a electric heater at 50ºC for 15 minutes. They were placed at room temperature until the originally foamy and dark solution became light yellow. Later, heat was applied and temperature was gradually increased up to 330ºC. The vessels were permanently covered so that, with a near reflux, acid evaporation could be slowed down and thus obtain a better mineralization of the sample17,18). The acids were evaporated and after cooling pure water and 0.1 ml of HClO4 was added. If the final residue is dark or colored, a new portion of the acid mixture must be added and mineralization be repeated. The silicon content must be quantitatively collected. It is important to note that the mineralized samples had a high SiO2 content.

The resulting solution was quantitatively transfered into an 10 ml polyethylene flask and diluted with water to the end volume of 10 ml. The solution was stored in refrigator at 4ºC until the analytical stage.

Quality Control

The analytical procedure was tested using the certified reference material Sea Lettuce BCR-CRM-279. The results obtained for all the metals are in an excellent agreement with the certified values for the method of open wet digestion (Fig. 4).


Fig. 4 Concentration of Cd (mg/g) by DPASV after open wet digestion in quartz vesseles in Sea Letuce samples (BCR-CRM 279) with differet weights.

For external quality control, some of samples were analysed by the German Environmental Specimen Bank at the Institute of Applied Physical Chemistry, Research Center of Jülich, Germany using ICP-MS. Table III present the comparison between the data obtained by ICP-MS and DPASV. No significant differences between both methods were observed.

Table III. Interlaboratory comparison of Cd, pb, and Cu determination in some fresh water plants.

RESULTS AND DISCUSION

From Table IV it is obvious that the Rio Loa in the water composition deviate from "normal" river water. Concentrations of alkaline and earth alkaline elements in river water from North Chile are significantly higher than those found in "normal" river waters, but below the concentration present in sea water.

Table IV. Water composition of Río Loa in comparison with other rivers

The water content of the plants was measured as the difference between fresh mass and mass after freeze-drying. Water content was generally 71% for both types of plants. Due to handling in the laboratory environment, after freeze-drying some residual water was present in the samples. The mass correction factor for dry weight was estimated by balance with an infrared oven at 105ºC. In all the samples the residual water content was about 4%.

Element pattern of Zannichellia palustris (Fig. 5a) and Myriophyllum acuatium (Fig. 5b) indicate that manganese concentration in both kind of water plants probably due an biological regulation mechanism is very similar and not depend on sampling area.

As, Cu, Pb, Se, Sr, Tl and Ba concentrations are similar in both types of plants (Fig. 5a and 5b), but depend strongly on sampling area. Very high As concentrations found in all sampling areas confirm the high pollution level of natural waters with arsenic in North Chile19,20). Very high Tl concentrations found in two sampling areas indicate some point sources of this element exist, where nature will be identified in the future.


Fig. 5a. Element pattern of Zannichellia palustris


Fig. 5 b. Element pattern of Myriophyllum acuaticum

Co, Ni, Zn and Cd concentrations found in Myriophyllum acuatium were significant higher than in Zannichellia palustris.

The results obtained by DPASV show, that the species studied have wide tolerance ranges for cadmium and copper (Table V). Observed range for cadmium is from 0.72 to 32 µg/g. These very high concentrations were not found in sea water algae 21). It seems that the tributary stream Rio Salado is highly polluted by cadmium. In a sampling area close to the sources (AT-M) the highest cadmium, lead and copper concentration was found in Myriophyllum acuatium. It is probable that the pollution is related to the volcano activity, which shows up in the geyser at the Tatio. Down stream (sampling area S-M) lower concentrations of cadmium, lead and copper were observed and there is the obvious dilution of elements with increasing distance from the sources. In the river Loa the situation is more complicated. In samples from sampling area Q-M cadmium values of about 10 µg/g were found. Up-stream (sampling area LEQ-M) less cadmium content was found. Down-stream (sampling area CL-M) the lowest cadmium concentration was found. These localized source of cadmium pollution is due human activities. There are important anthropogenic sources of heavy metals in the sampling areas: a sewerage discharge point at La Cascada (sampling area CAS-Z), west of Calama, and the use of fertilizers in agricultural areas (Quinchamale, Chiu-Chiu, Lasana, Ayquina, etc.). Besides, water rights management in the upper portion of the Loa basin influences directly the composition of its lower waters.

Table V. Element concentrations (d.w.) in fresh water plants

Lead concentrations found in both fresh water plants were comparable with concentrations observed also in other algae22). In the river Loa only in one sampling area (CL-M), an increased lead level was observed. At this sampling area also a high copper concentration was found. Probably the pollution by both elements is based on mining activities.

In the upper part of the Loa, the waters have a high copper concentration from the origin itself. According to the geological profile23), the Loa originates near to the Miño volcano, a sector where there is an outcrop of a non-exploited copper porphide rock. This influences directly the composition of water since this constantly leach the rocks in the deposit. Elevated copper concentrations were found in samples collected up-stream, which is in agree with the geological situation. In samples collected down-stream, less copper was found. In some sampling areas close to the mine Chuquicamata (CL-M, SL-M, SL-Z and CAS-Z), the influence of mining activity to the copper concentration in water can not be excluded.

CONCLUSIONS

Low concentrations of Cd, Pb and Cu in fresh water samples make the use of these matrix for monitor of heavy metals difficult, because the detection limits of that technique is in many cases very close to the determined concentrations. Also errors by sampling and processing before determination can falsify the results.

This preliminary study demonstrates that fresh water plants Zannichellia palustris and Myriophyllum acuatium bioaccumulate these elements at concentration levels which are easy to analyse by common analitical method with less probability on analytical errors.

In future work it is necessary to estimate the biological variabilty between individual plants at one sampling area and estimate the bioaccumulation factors for both plants based on adequate water analysis.

ACKNOWLEDEMENTS

Financial suport of the Universidad Católica del Norte, Chile, and of the International Bureau of Research Center Jülich, Germany, are greatfully acknowledged.

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