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Element losses following distilled water rinsing of leaves of the seagrass Posidonia oceanica (L.) Delile
Aquatic botany ELSEVIER
Aquatic Botany 52 (1995) 229-235
Technical communication
Element losses following distilled water rinsing of leaves of the seagrass Posidonia oceanica (L.) Delile G. L e d e n t a, M . A . M a t e o
b,
M. Warnau a, A. T e m a r a Ph. D u b o i s a
a, j.
R o m e r o b,.
~'Laboratoire de Biologie Marine (CP 160/15), Universiti Libre de Bruxelles, 50 av. F.D. Roosevelt, B-I050 Bruxelles, Belgium h Dpm Ecologga, Universidad de Barcelona. Diagonal 645, 08028 Barcelona, Spain
Accepted 11 August 1995
Abstract Rinsing of seagrasses with distilled water is often carried out prior to element analysis. The present study shows that such rinsing of the leaves of the Mediterranean seagrass Posidonia oceanica (L.) Delile results in significant element leakages, depending on leaf age. In adult leaves, only Fe concentrations are reduced. In younger leaves, significant concentration reductions occur for S, Mg, Na, Zn, Pb, Cd, Fe, Cr, and Ti, while concentrations of N, P, Ca, and Cu remain unaffected. C and K concentrations increase following distilled water rinsing owing to weight loss by leaching of Na and probably other major ions. The relative contribution of the different age classes to total shoot weight implies that distilled water rinsing also affects element analysis of whole shoots. The use of distilled water rinsing prior to element analysis should thus be avoided. Keywords: Carbon; Nitrogen; Phosphorus; Sulphur; Sodium; Potassium; Calcium; Magnesium; Heavy metals; Posidonia oceanica; Rinsing methodology
1. Introduction Element analysis in seagrasses (as in other aquatic plants) is a common method used to investigate a number o f aspects of plant biology, such as pollution (e.g. Ward, 1989) and mineral nutrition (e.g. Duarte, 1990). Rinsing the material prior to analysis is frequently performed in order to remove the epiphytes, sediment particles and detritus which are found * Corresponding author. Tel.: 34-3-4021511; Fax: 34-3-4111438: E-mail: [email protected]. 0304-3770/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved )00506-4
SSDI0304-3770(95
230
G. Ledent et aL / Aquatic Botany 52 (1995) 229--235
in abundance on seagrass leaves. This is carried out with distilled water (Harlin and ThorneMiller, 1981; Ward et al., 1984; Costantini et al., 1991; Carter and Eriksen, 1992; Catsiki and Panayotidis, 1993; among others), sea water (Brix and Lyngby, 1984; Nienhuis, 1986; Burkholder et al., 1994) or tap water (Fourqurean, 1992; Fourqurean et al., 1992). The removal of the surface seawater of the plant has been invoked as a reason for rinsing with distilled water (Birch, 1975); however, the effects of such a procedure on the element constituents of the plant other than sodium and chloride have apparently not been addressed until now. The aim of the present paper is to assess if the commonly used procedure of rinsing with distilled water prior to analysis biases the resulting leaf concentrations of heavy metals (Zn, Pb, Cd, Fe, Cr, Cu, Ti) and other mineral components (C, N, P, S, Na, K, Ca, Mg) in the Mediterranean seagrass Posidonia oceanica (L.) Delile.
2. Materials and methods 2.1. Sampling and rinsing treatment
Shoots ofP. oceanica were collected during two sampiings:( 1 ) in the meadow offLacco Ameno (Ischia Island, Italy) in April 1993 at 5 m depth; (2) in the meadow off the Medes Islands (NE Spain) in September 1994 at 5 m depth. Immediately after both samplings (i.e. within 2 h), shoots were randomly sorted into two groups, each of five shoots. The first group was rinsed with seawater from the sampling site and the second group was rinsed with distilled water. Before rinsing, rhizomes and juvenile leaves (length under 10 cm) were discarded, and epiphytes eliminated with a Plexiglas blade. Rinsing treatments were carried out actively for 1 min, the time during which maximum salt losses occur (Birch, 1975). Rinsing volumes were in the range 500-1000 ml, for about 5 g dry weight (DW) of leaves. After rinsing, adult leaves (leaves with differentiated petiole) and intermediate leaves (leaves without differentiated petiole, sensu Giraud, 1979) were separated, and part of the surface water was dried off with a paper tissue. Leaf samples were then dried ( 100°C, 48 h) and stored in polyethylene containers until analysis. C, N, P and heavy metals were analyzed in both adult and intermediate leaves from the first sampling. In the second sampling only intermediate leaves were kept for the analysis of the following elements: C, N, P, S, Na, K, Ca, and Mg. 2.2. Heavy metal analysis
A known amount of each sample (usually 0.2 g DW, finely chopped using stainless steel scissors) was digested in a clean test tube with 65% HNO3 (Merck, p.a., 10 ml g - 1 DW of sample). Acid digestions were carried out successively at 20°C, 40°C, 60°C, and 80°C for 24 h, 24 h, 12 h and 12 h, respectively. Digests were then diluted up to 20 mi with milli-Q water (Millipore) and filtered on Whatmann GF/A glass microfiber filters. The concentrations of Zn, Pb, Cd, Fe, Cr, Cu, and Ti were measured by atomic emission spectrometry using a Jobin-Yvon 38 + ICPS. Accuracy of the metal analysis methodology was checked
G. Ledent et al. /Aquatic Botany 52 (1995) 229-235
231
Table 1 Certified and measured metal concentrations (rag g - ~DW) in CRM no. 278 Metal
Certified ( mean + SD)
Measured (range) (n = 5 )
Zn Pb Cd Fe Cr Cu Ti
76 ,+ 2 1.91 _+0.04 0.34 ,+ 0.02 133 _+4 0.8 _+0.08 9.60_+0.16 2 ,+ 0.2"
77-82 1.91-1.95 0.31-0.37 126-161 0.87-1.04 9.60-10.71 2.8-3.8
"Uncertified values.
by processing a certified reference material (Mytilus edulis, CRM no. 278, Community Bureau of Reference) along with the experimental samples (Table 1).
2.3. Other elements Dried samples were finely ground using a Cyclotec mill. Subsamples were analyzed in a Carlo-Erba NA 1500 elemental analyzer for C and N concentrations. Additional subsamples, ground as above, were digested adding a mixture of 65% HNO3 and 60% HC104 (2:1 ) acids, and heated in a microwave oven (see Mateo and Sabat6, 1993 for more details). The concentrations of Ca, Na, Mg, K, P, and S were analyzed by atomic emission spectrometry using a Thermo Jarrel Ash Polyscan 61E.
2.4. Statistical analysis Data concerning the first sampling were analyzed using two-way analysis of variance (ANOVA), with a single degree of freedom for both effects (treatment and leaf age) and the interaction and 16 degrees of freedom for the error term. In the cases of significant interaction, a Tukey post-hoc test was used to assess the statistical differences between means. The data concerning the second sampling were analyzed using a one-way ANOVA, with a single degree of freedom for the effect of the treatment and eight degrees of freedom for the error term. The variability explained by each factor is derived from the sum of squares.
3. Results
The concentrations of Cu, N, P (Tables 2, 3 and 4) and Ca (Tables 3 and 4) were not affected by the rinsing method. Cu, N, and P concentrations showed a significant decrease with leafage (Tables 2 and 4). The concentrations of C and K increased following distilled water rinsing (Table 4), although this increase was small for C (relative changes of less than 2% in leaves from the first sampling and 7% in leaves from the second; see Tables 2 and 3 ) and moderate for K ( 11%; see Table 3). All the other elements showed an important
232
G. Ledent et a l . / Aquatic Botany 52 (1995) 229-235
Table 2 Results of element analysis in Posidonia oceanica according to rinsing method and leaf age (first sampling) Seawater
Adult Izg g - i D W Zn Pb Cd Fe Cr Cu Ti %DW C N P
Distilled water
Interm.
Adult
Interm.
207.0 (13.6) 246.5 (9.3) 211.5 (39.7) 187.5 (20.1) 13.3 (1.9) 14.3 (0.5) 12.2 (2.3) 3.7 (0.2) 1.97 (0.33) 2.57 (0.15) 2.10 (0.34) 1.88 (0.48) 84.8 (25.9) 64.0 (13.6) 1.42 (0.48) 1.48 (0.46) 1.58 (0.48) 0.29 (0.38) Adult 16.1 ( 1.6); Intermediate 23.8 (2.7) 2.18 (0.96) 1.51 (0.34) 2.12 (0.54) 0.13 (0.23) 36.5 (0.5)
38.9 (0.6) 37.7 (0.9) Adult 1.61 (0.16); Intermediate 2.02 (0.13) Adult 0.07 (0.02); Intermediate 0.11 ( 0.01 )
39.1 (0.8)
Mean values are given with standard errors in parentheses. For each group, n = 5. The number of cases increases if different groups are pooled owing to lack of significance among them (see Table 4). Interm., intermediate leaves; Adult, adult leaves.
loss after distilled water rinsing, ranging from 24% (Zn) to 91% (Ti) for heavy metals, and from 21% (Mg) to 44% (Na) for the other elements. In seawater-rinsed leaves, element concentrations tended to be higher in intermediate leaves than in adult leaves, except for Ti (Table 2) ; the decrease in element concentration caused by distilled water rinsing mainly affected intermediate leaves for most elements (Zn, Pb, Cd, Cr and Ti), as shown by the significant interaction between rinsing method and leaf age (Table 4, Tukey post-hoc test, P < 0.05 for all cases). Only Fe losses occurred independently of leaf age. 4. Discussion The mechanisms involved in element losses are probably diverse. Substitution or dilution of the surface water and water in the extracellular space by distilled water can explain losses Table 3 Results of element analysis in Posidonia oceanica according to rinsing method (second sampling, only intermediate leaves). Values are presented as % DW Seawater
C N P Na K Ca Mg S
Distilled water
34.4 (1.1)
37.0 (0.7) 1.44 (0.17) 0.12 (0.01)
5.48 (0.26) 2.25 (0.14)
3.08 (0.27) 2.51 (0.13) 2.45 (0.87)
0.89 (0.09) 0.96 (0.03)
0.70 (0.12) 0.60 (0.06)
G. Ledent et al. / Aquatic Botany 52 (1995) 229-235
233
Table 4 Variability in element concentrations measured in Posidonia oceanica as explained by rinsing method and leaf age Element
% variability Rinsing
Age
R x A
Error
28.0" *
49.8
First sampling Zn
20.6"
Pb
44.0" * *
Cd
12.0 ~
Fe
21.9"
Cr
14.8"
Cu
0.0
Ti
5.1 3.9 21.3" *
28.6" * *
9.5
25.0 *
57.8
5.7
68.5
25.8" *
76.9" * *
1.5
38.1 21.6
46.1" " *
11.3"
29.1
8.4"
5 9 . 4 " "*
4.3
279
N
1.6
69.6* * *
0.64
28.2
P
5.3
42.1" *
2.1
50.5
C
13.5" *
1.7 1 7 . 9 " *"
Second sampling C
-
-
45.5
N
54.5 * * 4
-
-
96
P
20
-
-
80
Na
92.8 * * *
-
-
7.2
K
34.7 * "
-
-
65.3
Ca
10.4
-
-
89.6
Mg
3 3 . 3 '~
-
-
66.7
S
91.2" "
-
-
8.8
Statistical significance:
"0.1 < P < 0 . 0 5 ; * P < 0 . 0 5 ; * " P < 0 . 0 1 ; *" * P < 0 . 0 0 1 . - ,
not tested.
of Na, and perhaps of Mg, whose concentrations in seawater are relatively high. Birch (1975) attributes losses of chloride after distilled water rinsing to surface water dilution and, partially, leaching from the extracelluIar space. In our case, part of the surface water was eliminated prior to the analysis, and the Na dissolved in the water of the extracellular space represents a small amount (0.37 g Na per 100 g DW: accepting the value of extracellular space given for Posidonia australis Hook. f. by Tyerman, 1989 of 0.071 cm 3 g fresh weight (FW), and assuming 0.18 g DW g - ~ FW for our P. oceanica leaves, personal observation). This implies that there must be some Na leaching from the intracellular pool to account for the total Na loss (2.40 g Na per 100 g DW). Indeed, Na and CI appear to circulate readily between intracellular (both cytoplasmic and vacuolar) and extracellular fluids (Tyerman, 1989). Heavy metal concentrations in seawater are extremely low, in the range of tens of nanograms per litre (Morley and Burton, 1991), and thus the losses observed must occur either through leaching from the intracellular pools or through washing of metals adsorbed to charged polysaccharides in the cell wall and intercellular matrix (as is the case in some algae: Morris and Bale, 1975; Eide et al., 1980). The different responses of intermediate and adult leaves may be due to several factors. In the adult leaves, part of the mineral content has been retranslocated (Pedersen and Borum,
234
G. Ledent et al./ Aquatic Botany 52 (1995) 229-235
1992) or leached, which can result in a lower sensitivity to osmotic shock. It is also possible that metals are associated with more insoluble compounds, and/or located in different sites than in the younger intermediate leaves. The observed increases in C and K can be considered an artifact resulting from the loss of Na and, probably, other major ions. Loss of CI resulting from surface and extracellular water dilution should presumably follow the loss of Na in a ratio of 18.8:10.8 by weight (derived from the seawater composition: Margalef, 1985) or lower (6.1:5.5) if differences between intracellular ion composition and seawater are taken into account (Birch, 1975). Thus, C1 losses of 2.7-4.2 g per 100 g DW seem a reasonable estimate. Hence, total losses (CI + Na + S + Mg) would amount to between 5.6 and 7.1 g per 100 g DW, which explains the observed increase in C and K. Moreover, during the treatment with distilled water, a leaching of N, P, Ca, and Cu occurred but was not detected owing to the decrease in leaf weight. Subsequently, the values of metal (and other elements) leaching derived from data in Tables 2 and 3 are underestimates. From a methodological point of view, it is clear that distilled water rinsing influences the results of element analysis. Intermediate leaves account for 35-50% of the whole shoot weight (see Buia et al., 1992), and thus the use of distilled water rinsing would result in an underestimation of the total element content of the plant. Moreover, if we address the question of metal content according to leaf age, the opposite conclusion would be drawn, depending on the water used for rinsing (Table 2). In summary, distilled water rinsing affects the results of element analysis of P. oceanica leaves, mainly concerning C, K, Na, Mg, S, Zn, Pb, Cd, Fe, Cr and Ti. In the case of major ions, such as K and Na, one should address the question of which mineral pools are the object of study; seawater rinsing will result in estimations of total element content (including surface water and water in the free space), while rinsing with distilled water will lead to underestimates of cellular content. In the case of heavy metal analysis, rinsing with distilled water should be avoided and replaced by seawater rinsing; or at least the question of the effect of such treatment should be addressed when working with aquatic plants other than P. oceanica.
Acknowledgements We thank Prof. L. Mazzella (Stazione Zoologica di Napoli, Italy) for providing laboratory and diving facilities and Prof. M. Hecq (Universit6 de Mons-Hainaut, Belgium) and Elionor Pelforth (ICP-AES Department of the Scientific and Technical Services of Barcelona University) for providing access facilities to ICPS. Research supported by an IRSIA grant to G. Ledent (ref. 930674) and M. Warnau (ref. 910528), and by an ECC STEP Program (ref. STEP-0063-C). Contribution of the Centre Interuniversitaire de Biologie Marine (CIBIM). Ph. Dubois is a Research Associate of the National Fund for Scientific Research (Belgium). References Birch, W.R., 1975. Some chemical and calorific properties of tropical marine angiosperms compared with those of other plants. J. Appl. Ecol., 12: 201-212.
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Brix, H. and Lyngby, J.E., 1984. A survey of the metallic composition of Zostera marina L. in the Limfjord, Denmark. Arch. Hydrobiol., 99: 347-359. Buia, M.C., Zupo, V. and Mazzella, L., 1992. Primary production and growth dynamics of Posidonia oceanica. P.S.Z.N.I: Mar. Ecol., 13: 1-15. Burkholder, J.M., Glasgow, Jr., H.B. and Cooke, J.E., 1994. Comparative effects of water-column nitrate enrichment on eelgrass Halodule wrightii, and widgeongrass Ruppia maritima. Mar. Ecol. Prog. Ser., 105:12 l - 138. Carter, R.J. and Eriksen, R.S., 1992. Investigation into the use ofZostera mueUeri lrmisch ex Aschers. as a sentinel accumulator for copper. Sci. Tot. Environ., 125: 185-192. Catsiki, V.A. and Panayotidis, P,, 1993. Copper, chromium and nickel in tissues of the Mediterranean seagrasses Posidonia oceanica and Cymodocea nodosa (Potamogetonaceae) from Greek coastal areas. Chemosphere, 26: 963-978. Costantini, S., Giordano, R., Ciaralli, L. and Beccaloni, E., 1991. Mercury, cadmium and lead evaluation in Posidonia oceanica and Codium tomentosum. Mar. Pollut. Bull., 22: 362-363. Duarte, C.M., 1990. Seagrass nutrient content. Mar. Ecol. Prog. Ser., 67: 201-207. Eide, I., Myklestad, S. and Melson, S., 1980. Long-term uptake and release of heavy metals by Ascophyllum nodosum (L.) Le Jol. (Phaeophyceae) in situ. Environ. Pollut., 23: 19-28. Fourqurean, LW., 1992. Relationships between porewater nutrient and seagrasses in a subtropical carbonate environment. Mar. Biol., 114: 57~55. Fourqurean, J.W., Zieman, J.C. and Powell, G.V.N., 1992. Phosphorus limitation of primary production in Florida Bay: evidence from C:N:P ratios of the dominant seagrass Thalassia testudinum. Limnol. Oceanogr., 37: 162171. Giraud, G., 1979. Sur une m6thode de mesure et de comptage des structures foliaires de Posidonia oceanica (L.) Delile. Bull. Mus. Hist. Nat. Marseille, 39: 33-39. Harlin, M.M. and Thorne-Miller, B., 1981. Nutrient enrichment of seagrass beds in a Rhode-Island coastal lagoon. Mar. Biol., 65: 221-229. Margalef, R. (Editor), 1985. The Western Mediterranean. Pergamon, Oxford, 374 pp. Mateo, M.A. and Sabat& S., 1993. Wet digestion of vegetable tissue using a domestic microwave oven. Anal. Chem., 279: 273-279. Morley, NH. and Burton, J.D., 1991. Dissolved trace metals in the Northern West Mediterranean sea: spatial and temporal variations. Water Pollut. Res. Rep., 13: 223-231. Morris, A.W. and Bale, A.J., 1975. The concentration of cadmium, copper, manganese and zinc by Fucus i,esiculosus in the Bristol Channel. Estuar. Coastal Mar. Sci., 3: 153-163. Nienhuis, P.H., 1986. Background levels of heavy metals in nine tropical seagrass species in Indonesia. Mar. Pollut. Bull., 17:508-511. Pedersen, M.F. and Borum, J., 1992. Nitrogen dynamics of eelgrass Zostera marina during a late summer period of high growth and low nutrient availability. Mar. Ecol. Prog. Ser., 80: 65-73. Tyerman, S.D., 1989. Solute and water relations of seagrasses. In: A.W.D. Larkum, A.J. McComb and S.A. Shepherd (Editors), Biology of Seagrasses. Elsevier, Amsterdam, pp. 723-759. Ward, T.J., 1989. The accumulation and effects of metals in seagrass habitats. In: A.W. Larkum, A.J. McComb and S.A. Shepherd (Editors), Biology of Seagrasses. Elsevier, Amsterdam, pp. 797-820. Ward, T.J., Warren, L.J. and Tiller, K.G., 1984. The distribution and effects of metals in the marine environment near a lead-zinc smelter, South Australia. In: J.O. Nriagu (Editors), Environmental Impact of Smelters. Wiley, New York, pp. [-73.
Aquatic Botany 52 (1995) 229-235
Technical communication
Element losses following distilled water rinsing of leaves of the seagrass Posidonia oceanica (L.) Delile G. L e d e n t a, M . A . M a t e o
b,
M. Warnau a, A. T e m a r a Ph. D u b o i s a
a, j.
R o m e r o b,.
~'Laboratoire de Biologie Marine (CP 160/15), Universiti Libre de Bruxelles, 50 av. F.D. Roosevelt, B-I050 Bruxelles, Belgium h Dpm Ecologga, Universidad de Barcelona. Diagonal 645, 08028 Barcelona, Spain
Accepted 11 August 1995
Abstract Rinsing of seagrasses with distilled water is often carried out prior to element analysis. The present study shows that such rinsing of the leaves of the Mediterranean seagrass Posidonia oceanica (L.) Delile results in significant element leakages, depending on leaf age. In adult leaves, only Fe concentrations are reduced. In younger leaves, significant concentration reductions occur for S, Mg, Na, Zn, Pb, Cd, Fe, Cr, and Ti, while concentrations of N, P, Ca, and Cu remain unaffected. C and K concentrations increase following distilled water rinsing owing to weight loss by leaching of Na and probably other major ions. The relative contribution of the different age classes to total shoot weight implies that distilled water rinsing also affects element analysis of whole shoots. The use of distilled water rinsing prior to element analysis should thus be avoided. Keywords: Carbon; Nitrogen; Phosphorus; Sulphur; Sodium; Potassium; Calcium; Magnesium; Heavy metals; Posidonia oceanica; Rinsing methodology
1. Introduction Element analysis in seagrasses (as in other aquatic plants) is a common method used to investigate a number o f aspects of plant biology, such as pollution (e.g. Ward, 1989) and mineral nutrition (e.g. Duarte, 1990). Rinsing the material prior to analysis is frequently performed in order to remove the epiphytes, sediment particles and detritus which are found * Corresponding author. Tel.: 34-3-4021511; Fax: 34-3-4111438: E-mail: [email protected]. 0304-3770/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved )00506-4
SSDI0304-3770(95
230
G. Ledent et aL / Aquatic Botany 52 (1995) 229--235
in abundance on seagrass leaves. This is carried out with distilled water (Harlin and ThorneMiller, 1981; Ward et al., 1984; Costantini et al., 1991; Carter and Eriksen, 1992; Catsiki and Panayotidis, 1993; among others), sea water (Brix and Lyngby, 1984; Nienhuis, 1986; Burkholder et al., 1994) or tap water (Fourqurean, 1992; Fourqurean et al., 1992). The removal of the surface seawater of the plant has been invoked as a reason for rinsing with distilled water (Birch, 1975); however, the effects of such a procedure on the element constituents of the plant other than sodium and chloride have apparently not been addressed until now. The aim of the present paper is to assess if the commonly used procedure of rinsing with distilled water prior to analysis biases the resulting leaf concentrations of heavy metals (Zn, Pb, Cd, Fe, Cr, Cu, Ti) and other mineral components (C, N, P, S, Na, K, Ca, Mg) in the Mediterranean seagrass Posidonia oceanica (L.) Delile.
2. Materials and methods 2.1. Sampling and rinsing treatment
Shoots ofP. oceanica were collected during two sampiings:( 1 ) in the meadow offLacco Ameno (Ischia Island, Italy) in April 1993 at 5 m depth; (2) in the meadow off the Medes Islands (NE Spain) in September 1994 at 5 m depth. Immediately after both samplings (i.e. within 2 h), shoots were randomly sorted into two groups, each of five shoots. The first group was rinsed with seawater from the sampling site and the second group was rinsed with distilled water. Before rinsing, rhizomes and juvenile leaves (length under 10 cm) were discarded, and epiphytes eliminated with a Plexiglas blade. Rinsing treatments were carried out actively for 1 min, the time during which maximum salt losses occur (Birch, 1975). Rinsing volumes were in the range 500-1000 ml, for about 5 g dry weight (DW) of leaves. After rinsing, adult leaves (leaves with differentiated petiole) and intermediate leaves (leaves without differentiated petiole, sensu Giraud, 1979) were separated, and part of the surface water was dried off with a paper tissue. Leaf samples were then dried ( 100°C, 48 h) and stored in polyethylene containers until analysis. C, N, P and heavy metals were analyzed in both adult and intermediate leaves from the first sampling. In the second sampling only intermediate leaves were kept for the analysis of the following elements: C, N, P, S, Na, K, Ca, and Mg. 2.2. Heavy metal analysis
A known amount of each sample (usually 0.2 g DW, finely chopped using stainless steel scissors) was digested in a clean test tube with 65% HNO3 (Merck, p.a., 10 ml g - 1 DW of sample). Acid digestions were carried out successively at 20°C, 40°C, 60°C, and 80°C for 24 h, 24 h, 12 h and 12 h, respectively. Digests were then diluted up to 20 mi with milli-Q water (Millipore) and filtered on Whatmann GF/A glass microfiber filters. The concentrations of Zn, Pb, Cd, Fe, Cr, Cu, and Ti were measured by atomic emission spectrometry using a Jobin-Yvon 38 + ICPS. Accuracy of the metal analysis methodology was checked
G. Ledent et al. /Aquatic Botany 52 (1995) 229-235
231
Table 1 Certified and measured metal concentrations (rag g - ~DW) in CRM no. 278 Metal
Certified ( mean + SD)
Measured (range) (n = 5 )
Zn Pb Cd Fe Cr Cu Ti
76 ,+ 2 1.91 _+0.04 0.34 ,+ 0.02 133 _+4 0.8 _+0.08 9.60_+0.16 2 ,+ 0.2"
77-82 1.91-1.95 0.31-0.37 126-161 0.87-1.04 9.60-10.71 2.8-3.8
"Uncertified values.
by processing a certified reference material (Mytilus edulis, CRM no. 278, Community Bureau of Reference) along with the experimental samples (Table 1).
2.3. Other elements Dried samples were finely ground using a Cyclotec mill. Subsamples were analyzed in a Carlo-Erba NA 1500 elemental analyzer for C and N concentrations. Additional subsamples, ground as above, were digested adding a mixture of 65% HNO3 and 60% HC104 (2:1 ) acids, and heated in a microwave oven (see Mateo and Sabat6, 1993 for more details). The concentrations of Ca, Na, Mg, K, P, and S were analyzed by atomic emission spectrometry using a Thermo Jarrel Ash Polyscan 61E.
2.4. Statistical analysis Data concerning the first sampling were analyzed using two-way analysis of variance (ANOVA), with a single degree of freedom for both effects (treatment and leaf age) and the interaction and 16 degrees of freedom for the error term. In the cases of significant interaction, a Tukey post-hoc test was used to assess the statistical differences between means. The data concerning the second sampling were analyzed using a one-way ANOVA, with a single degree of freedom for the effect of the treatment and eight degrees of freedom for the error term. The variability explained by each factor is derived from the sum of squares.
3. Results
The concentrations of Cu, N, P (Tables 2, 3 and 4) and Ca (Tables 3 and 4) were not affected by the rinsing method. Cu, N, and P concentrations showed a significant decrease with leafage (Tables 2 and 4). The concentrations of C and K increased following distilled water rinsing (Table 4), although this increase was small for C (relative changes of less than 2% in leaves from the first sampling and 7% in leaves from the second; see Tables 2 and 3 ) and moderate for K ( 11%; see Table 3). All the other elements showed an important
232
G. Ledent et a l . / Aquatic Botany 52 (1995) 229-235
Table 2 Results of element analysis in Posidonia oceanica according to rinsing method and leaf age (first sampling) Seawater
Adult Izg g - i D W Zn Pb Cd Fe Cr Cu Ti %DW C N P
Distilled water
Interm.
Adult
Interm.
207.0 (13.6) 246.5 (9.3) 211.5 (39.7) 187.5 (20.1) 13.3 (1.9) 14.3 (0.5) 12.2 (2.3) 3.7 (0.2) 1.97 (0.33) 2.57 (0.15) 2.10 (0.34) 1.88 (0.48) 84.8 (25.9) 64.0 (13.6) 1.42 (0.48) 1.48 (0.46) 1.58 (0.48) 0.29 (0.38) Adult 16.1 ( 1.6); Intermediate 23.8 (2.7) 2.18 (0.96) 1.51 (0.34) 2.12 (0.54) 0.13 (0.23) 36.5 (0.5)
38.9 (0.6) 37.7 (0.9) Adult 1.61 (0.16); Intermediate 2.02 (0.13) Adult 0.07 (0.02); Intermediate 0.11 ( 0.01 )
39.1 (0.8)
Mean values are given with standard errors in parentheses. For each group, n = 5. The number of cases increases if different groups are pooled owing to lack of significance among them (see Table 4). Interm., intermediate leaves; Adult, adult leaves.
loss after distilled water rinsing, ranging from 24% (Zn) to 91% (Ti) for heavy metals, and from 21% (Mg) to 44% (Na) for the other elements. In seawater-rinsed leaves, element concentrations tended to be higher in intermediate leaves than in adult leaves, except for Ti (Table 2) ; the decrease in element concentration caused by distilled water rinsing mainly affected intermediate leaves for most elements (Zn, Pb, Cd, Cr and Ti), as shown by the significant interaction between rinsing method and leaf age (Table 4, Tukey post-hoc test, P < 0.05 for all cases). Only Fe losses occurred independently of leaf age. 4. Discussion The mechanisms involved in element losses are probably diverse. Substitution or dilution of the surface water and water in the extracellular space by distilled water can explain losses Table 3 Results of element analysis in Posidonia oceanica according to rinsing method (second sampling, only intermediate leaves). Values are presented as % DW Seawater
C N P Na K Ca Mg S
Distilled water
34.4 (1.1)
37.0 (0.7) 1.44 (0.17) 0.12 (0.01)
5.48 (0.26) 2.25 (0.14)
3.08 (0.27) 2.51 (0.13) 2.45 (0.87)
0.89 (0.09) 0.96 (0.03)
0.70 (0.12) 0.60 (0.06)
G. Ledent et al. / Aquatic Botany 52 (1995) 229-235
233
Table 4 Variability in element concentrations measured in Posidonia oceanica as explained by rinsing method and leaf age Element
% variability Rinsing
Age
R x A
Error
28.0" *
49.8
First sampling Zn
20.6"
Pb
44.0" * *
Cd
12.0 ~
Fe
21.9"
Cr
14.8"
Cu
0.0
Ti
5.1 3.9 21.3" *
28.6" * *
9.5
25.0 *
57.8
5.7
68.5
25.8" *
76.9" * *
1.5
38.1 21.6
46.1" " *
11.3"
29.1
8.4"
5 9 . 4 " "*
4.3
279
N
1.6
69.6* * *
0.64
28.2
P
5.3
42.1" *
2.1
50.5
C
13.5" *
1.7 1 7 . 9 " *"
Second sampling C
-
-
45.5
N
54.5 * * 4
-
-
96
P
20
-
-
80
Na
92.8 * * *
-
-
7.2
K
34.7 * "
-
-
65.3
Ca
10.4
-
-
89.6
Mg
3 3 . 3 '~
-
-
66.7
S
91.2" "
-
-
8.8
Statistical significance:
"0.1 < P < 0 . 0 5 ; * P < 0 . 0 5 ; * " P < 0 . 0 1 ; *" * P < 0 . 0 0 1 . - ,
not tested.
of Na, and perhaps of Mg, whose concentrations in seawater are relatively high. Birch (1975) attributes losses of chloride after distilled water rinsing to surface water dilution and, partially, leaching from the extracelluIar space. In our case, part of the surface water was eliminated prior to the analysis, and the Na dissolved in the water of the extracellular space represents a small amount (0.37 g Na per 100 g DW: accepting the value of extracellular space given for Posidonia australis Hook. f. by Tyerman, 1989 of 0.071 cm 3 g fresh weight (FW), and assuming 0.18 g DW g - ~ FW for our P. oceanica leaves, personal observation). This implies that there must be some Na leaching from the intracellular pool to account for the total Na loss (2.40 g Na per 100 g DW). Indeed, Na and CI appear to circulate readily between intracellular (both cytoplasmic and vacuolar) and extracellular fluids (Tyerman, 1989). Heavy metal concentrations in seawater are extremely low, in the range of tens of nanograms per litre (Morley and Burton, 1991), and thus the losses observed must occur either through leaching from the intracellular pools or through washing of metals adsorbed to charged polysaccharides in the cell wall and intercellular matrix (as is the case in some algae: Morris and Bale, 1975; Eide et al., 1980). The different responses of intermediate and adult leaves may be due to several factors. In the adult leaves, part of the mineral content has been retranslocated (Pedersen and Borum,
234
G. Ledent et al./ Aquatic Botany 52 (1995) 229-235
1992) or leached, which can result in a lower sensitivity to osmotic shock. It is also possible that metals are associated with more insoluble compounds, and/or located in different sites than in the younger intermediate leaves. The observed increases in C and K can be considered an artifact resulting from the loss of Na and, probably, other major ions. Loss of CI resulting from surface and extracellular water dilution should presumably follow the loss of Na in a ratio of 18.8:10.8 by weight (derived from the seawater composition: Margalef, 1985) or lower (6.1:5.5) if differences between intracellular ion composition and seawater are taken into account (Birch, 1975). Thus, C1 losses of 2.7-4.2 g per 100 g DW seem a reasonable estimate. Hence, total losses (CI + Na + S + Mg) would amount to between 5.6 and 7.1 g per 100 g DW, which explains the observed increase in C and K. Moreover, during the treatment with distilled water, a leaching of N, P, Ca, and Cu occurred but was not detected owing to the decrease in leaf weight. Subsequently, the values of metal (and other elements) leaching derived from data in Tables 2 and 3 are underestimates. From a methodological point of view, it is clear that distilled water rinsing influences the results of element analysis. Intermediate leaves account for 35-50% of the whole shoot weight (see Buia et al., 1992), and thus the use of distilled water rinsing would result in an underestimation of the total element content of the plant. Moreover, if we address the question of metal content according to leaf age, the opposite conclusion would be drawn, depending on the water used for rinsing (Table 2). In summary, distilled water rinsing affects the results of element analysis of P. oceanica leaves, mainly concerning C, K, Na, Mg, S, Zn, Pb, Cd, Fe, Cr and Ti. In the case of major ions, such as K and Na, one should address the question of which mineral pools are the object of study; seawater rinsing will result in estimations of total element content (including surface water and water in the free space), while rinsing with distilled water will lead to underestimates of cellular content. In the case of heavy metal analysis, rinsing with distilled water should be avoided and replaced by seawater rinsing; or at least the question of the effect of such treatment should be addressed when working with aquatic plants other than P. oceanica.
Acknowledgements We thank Prof. L. Mazzella (Stazione Zoologica di Napoli, Italy) for providing laboratory and diving facilities and Prof. M. Hecq (Universit6 de Mons-Hainaut, Belgium) and Elionor Pelforth (ICP-AES Department of the Scientific and Technical Services of Barcelona University) for providing access facilities to ICPS. Research supported by an IRSIA grant to G. Ledent (ref. 930674) and M. Warnau (ref. 910528), and by an ECC STEP Program (ref. STEP-0063-C). Contribution of the Centre Interuniversitaire de Biologie Marine (CIBIM). Ph. Dubois is a Research Associate of the National Fund for Scientific Research (Belgium). References Birch, W.R., 1975. Some chemical and calorific properties of tropical marine angiosperms compared with those of other plants. J. Appl. Ecol., 12: 201-212.
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