HEAVY METAL (CD, CU, PB AND ZN) DETERMINATION IN WOODLOUSE (ONISCUS ASELLUS)

Filip M. G. Tack^a*, Nicolas Bogaert^a, Marc G. Verloo^a, Frederik Hendrickx^b, Jean-Pierre Maelfait^b, Johan Mertens^b

^aChemistry and Applied Ecochemistry, Ghent University, Coupure Links 653, B-9000 Gent, Belgium

^bLaboratory of Animal Ecology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium

^*Corresponding author: filip.tack@rug.ac.be

Abstract

The performance of selected destruction methods for the determination of Cd, Cu, Pb and Zn in woodlouse (Oniscus asellus) was assessed. Total analysis involving microwave destruction with HF, HCl and HNO₃ was used as a reference method. Low values for the dry ashing method indicated incomplete dissolution of the elements and/or losses through volatilization. The method involving concentrated HNO₃ produced erroneous values for Cd, Cu and Pb. Results were consistent with the reference method provided H₂O₂ was used during digestion. The method involving HNO₃/HClO₄ in one destruction step yielded low recoveries. This was corrected provided that two destruction steps were applied, but was at the expense of reproducibility and rendered the method more laborious. Because of the good performance combined with speed and simplicity, destruction with HNO₃/H₂O₂ (method 4) was most appropriate.

Introduction

There is currently a great interest for the analytical determination of heavy metals in arthropods. Some arthropods are known to be strong heavy metal accumulators (Butovsky & Van Straalen, 1995). They may therefore play an important role in the transfer of these elements from soils into the food chain and could be useful indicators for metal pollution (Dallinger, 1992).

Matrix destruction for the determination of heavy metals in arthropods is usually accomplished using wet destruction methods. Details of the procedures followed unfortunately are rarely provided. Most procedures applied involve a wet digestion using nitric acid, alone (Rabitsch, 1995; Read et al, 1998), or in combination with perchloric acid (van Straalen & van Wensem, 1986; Wilczek & Migula, 1996) or hydrogen peroxide (Marinussen & van der Zee, 1997). The aim of this study was to assess the performance of selected destruction methods for the determination of Cd, Cu, Pb and Zn in woodlouse (Oniscus asellus).

METHODS

Woodlice (Oniscus asellus) were collected in September 1998 by hand picking in the field. Animals were dried at 70ºC for 48 hrs and thoroughly crushed using a mortar and a pestle to assure good homogeneity. To mimic the situation where individual animals are analyzed, aliquots between 20 - 30 mg were weighed.

A US-EPA method for the determination of total metal contents in environmental matrices (US-EPA, 1996) was used as a reference method (method 1). Samples were placed into 45 ml teflon destruction bombs along with 3 ml 65% HNO₃ and 1 ml 37% HCl. The recipients were placed during 15 minutes in an ultrasonic bath to evacuate nitrous vapors. Closed bombs were heated in a microwave (250 Watt during 5 minutes, 400 Watt during 5 minutes, 600 Watt during 4 minutes). The destruate was filtered over a 0.45 µm membrane filter which was transferred again into the destruction vessel along with 1 ml 48% HF, 1 ml 37% HCl and 1 ml HNO₃ 65%. This mixture was subjected to the same heating program. The resulting solution was combined with the filtrate and diluted to 50 ml.

A method of dry ashing was adopted from a procedure commonly used for plant analysis (Tack et al, 1997) (method 2). Samples were weighed into porcelain crucibles and ashed during 3 hours at 450°C. The residue was digested with 5 ml of 6 mol/L HNO₃. The mixture was evaporated to a low volume, which was dissolved in 5 ml of 3 mol/L HNO₃, filtered and diluted to 50 ml.

Method 4 was according to Marinussen & Van Der Zee (1997)(Marinussen & van der Zee, 1997). Samples were treated with 5 ml ultra-pure 65% HNO₃. The suspension was heated up to 130ºC for 1 h under reflux. A total amount of 4 ml 20% H₂O₂ was added in aliquots of 0.5 ml. For the destruction involving only HNO₃, the same procedure was followed without addition of H₂O₂ (method 3).

Methods 5 and 6 are adapted from van Straalen & van Wensem (1986). Digestion was performed by means of 5 ml 7:1 mixture of ultra-pure 65% nitric acid and 70% perchloric acid. The suspension was heated at 85ºC and then at 160ºC, each during 45 minutes under reflux. Then, the mixture was evaporated at 170-180ºC until no more white fumes evolved. If the color of the residue is not white, indicating incomplete destruction, the digestion is repeated on the residue. The residue is finally dissolved in 0.1 mol/L HNO₃ and heated during 30 minutes before dilution to 50 ml. Our samples were subjected to either one destruction (method 5) or two destructions (method 6).

Flame atomic absorption equipped with deuterium background correction was used for the determination of Zn in the extracts. Cd, Cu and Pb were analyzed by graphite furnace atomic absorption equipped with Zeeman background correction.

Results and discussion

Metal contents obtained using different destruction procedures were compared with reference to method 1 (Figure 1). This is a vigorous destruction method proposed by US-EPA for the determination of total metal contents in a variety of matrices (US-EPA, 1996).

Except for Zn, recoveries of the ashing method (method 2) with respect to the microwave destruction method consistently were too low. Low recoveries can be due to incomplete dissolution of the element from the ash and/or volatilisation of the element.

Figure 1. Concentrations of heavy metals as determined using different destruction procedures (method 1: Microwave destruction with HNO₃/HCl/HF; 2: dry ashing; 3: HNO₃; 4: HNO₃/H₂O₂; 5: HNO₃/HClO₄, 1 destruction; 6: HNO₃/HClO₄, 2 destructions).

The method involving HNO₃ was performed without (method 3) and with addition of H₂O₂ (method 4). When no H₂O₂ was used, only 66% of Pb was recovered compared to microwave digestion. In contrast, values obtained for Cu and Zn were too high, approx. 110% of the microwave destruction method. The brown color of the solutions suggested that destruction of organic matter was incomplete when H₂O₂ was not used. This may result in incomplete dissolution of elements such as Pb and/or give rise to interference during analytical determination. This fast and convenient method is frequently used. Current results indicate that it is unsuited for the determination of metals in arthropods and likely other biota. Use of H₂O₂ during digestion was effective and yielded good results for all elements.

When a single destruction step was applied in the HNO₃/HClO₄ method (method 5), Cu and Pb were much lower than for the other methods and Pb exhibited a much higher variability (Table 1). Application of two consecutive destructions (method 6) yielded values consistent with the reference method. In our case, the color of the residue remaining after one destruction was a very pale yellow, which was easily mistaken as indicating complete destruction.

Analysis of variance with nested design was used to partition the sum of squares into different sources of variability (Table 2). Methods 3 and 5 were not further considered.

Table 1. Variation coefficients associated with the use of the different destruction methods

	1	2	3	4	5	6
Cd	8.2%	15.1%	6.3%	9.6%	5.8%	18.4%
Cu	5.3%	3.9%	5.8%	5.2%	6.6%	11.0%
Pb	9.5%	17.4%	52.7%	12.8%	24.5%	12.6%
Zn	4.4%	5.3%	9.3%	6.4%	1.1%	6.0%

The variation coefficients associated with analytical determination were at or below 5% for Cd, Cu and Zn (Table 2). This is acceptable for analytical determinations in biological matrices. For Pb, the variation coefficient was 9%, which is related to concentrations being in the lower range for determination with graphite furnace atomic absorption.

Table 2. Estimated variation coefficient associated with each source of variability (methods 1, 2, 4 and 6)

Source of variation (factor)	Cd	Cu	Pb	Zn
Method	11.7	4.3	12.6	2.4
Destruction/sample	6.6	0.0	7.5	4.6
Analytical detmn.	5.5	3.8	9.0	2.3

In conclusion, three of the tested methods were appropriate for the determination of Cd, Cu, Pb and Zn in woodlouse. Of these, the method involving HNO₃/H₂O₂ is by far the easiest and fastest to carry out in the laboratory. At the same time, it allows to obtain recoveries and reproducibilities comparable with the two other more laborious approaches.

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