The distribution of cadmium, copper, lead, and zinc in eelgrass (Zostera marina L.)

The Science of the Total Environment, 24 (1982) 51---63

51

Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

THE DISTRIBUTION OF CADMIUM, COPPER, LEAD, AND ZINC IN EELGRASS (Zostera marina L.)

H A N S BRIX and JENS ERIK LYNGBY

Botanical Institute, University of Aarhus, 68 Nordlandsvej, DK-8240 Risskov (Denmark) (Received October 30th, 1981; accepted November 20th, 1981)

ABSTRACT The distribution of Cd, Cu, Pb, and Zn in eelgrass (Zostera marina L.) was studied at three locations with different heavy metal loads in the Limfjord, Denmark. The eelgrass was fractionated into roots, rhizome, stem, and leaves according to age, and the heavy metal concentrations in each fraction were determined. The distribution patterns of the four heavy metals in eelgrass were independent of the heavy metal loads at the sampling stations. The concentrations of all metals were greater in the roots than in the rhizomes. In the aerial* parts two different age-dependent distribution patterns were observed. The concentrations of Cd, Pb, and Zn increased with age while the opposite was true for Cu. The distribution of lead correlated with the distribution of ash content. These age-dependent distribution patterns were maintained throughout the observation period and were most pronounced for Cu and Zn in winter. The heavy metal distribution in eelgrass is discussed in relation to gross morphology, especially age-structure. It is suggested that the accumulation of Cd, Pb, and Zn is due to a slow irreversible uptake or to the existence of more binding sites in old leaves. The distribution of Cu can be explained by translocation within the plant, dilution due to growth or leakage from the older leaves.

INTRODUCTION

In recent years (e.g. Jacques et al., 1975; Garcia et al., 1979) the distribution of heavy metals in terrestrial plants, especially species of economic value, has been studied extensively. The heavy metal concentrations in different parts of plants have been investigated in only a few aquatic macrophytes. The c o m m o n use of brown algae as an indicator of heavy metal contamination has provided some knowledge for the distribution of heavy metals in these non-rooted macrophytes. In general, the heavy metal concentrations are lowest in the youngest parts of the thallus and increase with age (e.g. Bryan and Hummerstone, 1973; Myklestad et al., 1978). Although several investigations have described heavy metal concentrations in submerged** angiosperms (e.g. Boyd, 1970; Adams et al., 1973; Cowgill, 1974), the information on heavy metal distributions in these is sparse. aerial = above the sediment/water interface. * * submerged = below the sediment/water interface. 0048-9697/82/0000--0000/$02.75 © 1982 Elsevier Scientific Publishing Company

52

Eelgrass (Zostera marina L.) is the most widely spread seagrass in the northern hemisphere (den Hartog, 1970) and the most extensively studied species of seagrass (Zieman and Wetzel, 1980). Some information on the distribution of nutrients in eelgrass, such as nitrogen, is available (e.g. Harrison and Mann, 1975), but no data concerning the heavy metal distribution are, to the authors knowledge, available. The aim of this investigation was to study the distribution of cadmium, copper, lead, and zinc in eelgrass through the seasons at three locations with different heavy metal loads. Results will be discussed in relation to gross morphology, especially age-structure of eelgrass.

MATERIAL AND METHODS

The investigation was carried out at three locations in the Limfjord, Denmark, viz. Aalborg, Nibe, and R~bnbjerg (Fig. 1). The Aalborg area is the most eutrophic part of the Limfjord (Limfjordskomiteen, 1976) and elevated concentrations of heavy metals in sediment and biota have been reported (Vandkvalitetsinstituttet, 1976; Brix et al., 1982). The salinity averaged 24, 12, and 22~00, at Aalborg, Nibe, and RCnbjerg, respectively. A more detailed description of the three sampling stations, e.g. heavy metal concentrations in sediment and water, is presented by Lyngby and Brix (1982).

Fig. 1. Map of the Limfjord showing the location of the three sampling stations: Aalborg, Nibe, and R{bnbjerg. (From Lyngby and Brix, 1982).

53 Eelgrass was collected from September 1979 until July 1980. However, bad weather made sampling impossible at R~bnbjerg in September and January. Sufficiently large samples of eelgrass (approx. 2 kg wet weight) were taken to provide a representative sample from each area. The eelgrass was thoroughly rinsed in seawater at the sampling stations and then transported to the laboratory in plastic bags. The eelgrass was divided into aerial and submerged parts. The aerial parts were further fractionated into stem and, according to age, leaves 1 to 5 (Fig. 2). Epiphytes were removed by scraping and washing. The fractions were dried at 105°C to constant weight and then ground in a porcelain mortar. T w o or five replicates of the homogenates were digested in concentrated nitric acid and hydrogen peroxide (AR-grade) as described by Brix et al. (1982). Blanks were run concurrently. Cadmium, copper, and lead concentrations were determined by flameless AAS (Perkin-Elmer 305B, HGA-74) and zinc concentrations by flame AAS (Perkin-Elmer 503). Deuterium background correction was used in the determination of cadmium and lead. On one occasion, 7--9 July 1980, the aerial and submerged biomass of eelgrass was harvested at the three sampling stations. Ten samples of 1/36 m 2 were taken at random inside eelgrass stands using a corer as described b y

Leaf

Leaf 1 3

Leaf 2

Leaf z.

Leaf 5

Stem

i:!.i.:. Rhi;'ome ....... ;...q ~

i

" ; !i. :

i" I

Roois " " ' .,.. .,-~.,-.:, ::,::.-..-, . . . . -.-. (- :-..-: - . - , . . . . . . . . . "-.

•

Fig. 2. Drawing of an eelgrass plant, showing the eight fractions into which eelgrass was divided. The age of the leaves increases from leaf 1 to leaf 5. The stem fraction is the plant portion from the rhizome to the leaf base.

54 Schierup (1978). The corer was emplaced into the sediment to a depth of 30 cm, filled with water, and then a plastic ball was pressed into the upper opening to create a vacuum, and the corer withdrawn. Care was taken to ensure that no parts of the plans were lost. Most of the sediment was washed from the root-rhiz0mes through a 2-mm plastic sieve .at the sampling stations, and the samples were transported to the laboratory in separate plastic bags. In the laboratory, the number of shoots in each sample was counted. Black rhizomes were discarded together with non-rooted upper plant parts, which were considered to belong to plants from outside the sample. Epiphytes were carefully removed by scraping. The samples were factionated into roots, rhizomes, stems, leaves 1 to 5, and flowering shoots. Each fraction was carefully rinsed in running water to remove sediment particles and other adherent material. The dry weight and heavy metal concentrations of each fraction were determined as previously described. The ash contents of the homogenates were determined by ashing in a muffle furnace at 550°C for 24 h. In the determination of ash content, the coefficients of variation were less than 5%. Either t-tests or analyses of variance were carried out on data where samples were collected at all stations. Heavy metal concentrations were logtransformed in order to obtain additivity between the effects and to stabilize the variances. RESULTS Biomass

The mean shoot density, biomass, and average shoot weight of eelgrass at the three sampling stations are presented in Table 1. The mean shoot density at Nibe and R~bnbjerg was significantly (P < 0.001) higher than for samples obtained at Aalborg (approx. 4 times). However, the average weight of the shoots at Nibe and R~bnbjerg was 30--50% lower than at Aalborg. The total biomass was significantly (P ~ 0.001) higher at R~bnbjerg than at the two other stations (Table 1). The ratio between aerial and submerged biomass was approx. 1 at Nibe and RCnbjerg; at Aalborg the aerial biomass constituted a greater proportion. The distribution of biomass is shown in Fig. 3. Flowering shoots were sampled only at Nibe and R~bnbjerg and constituted 17.6 and 24.4% of the above-ground biomass at these stations, respectively. The distribution of biomass in the non-reproductive shoots was nearly identical at the three stations. The greatest biomass was associated with the stem-fraction. At Nibe and R~bnbjerg the stem-fraction constituted ~ 4 0 % of the aerial biomass, while it was only " 3 0 % at Aalborg. At Aalborg, Nibe, and RCnbjerg the roots constituted 24.2, 19.7, and 32.1%, respectively, of the total submerged biomass. The observed differences might be explained by the existence of different

55 TABLE 1 M E A N S H O O T DENSITY, B I O M A S S A N D A V E R A G E S H O O T T H R E E S A M P L I N G S T A T I O N S IN JULY, 1980 (mean +-s.d.)

Aalborg Nibe R~bnbjerg

WEIGHT

AT THE

Shoot density (shoots/m 2 )

aerial biomass (g/m 2 )

submerged biomass (g/m 2 )

Total biomass (g/m 2 )

Average shoot weight (g/shoot)

295 +157 1089 +-360 1348 -+362

87 +49 150 +51 262 +-58

55 +-27 136 +-56 245 +-50

143 +-62 286 +60 507 -+66

0.30 0.14 0.19

Biomass (g d.w. m -2) 0

Flowering

100

200

0

100

200

0

100

200

C

Leaf 5 Leaf 4 Leaf 3 Leaf 2 Leaf 1 Stem Rhizome Root

Fig. 3. The distribution of biomass (g dry weight/m2 ) in eelgrass (Zostera marina L.) at Aalborg (A), Nibe (B), and R~bnhjerg (C) in July, 1980. Bars indicate standard deviation of ten samples. ecological t ypes o f eelgrass at the three sampling stations or different environmental factors, such as salinity and exposure (McRoy, 1970; Bak, 1980). Ash content A significant (P < 0.001) difference in ash c o n t e n t of the aerial fractions was r e c o r d e d (Fig. 4). T he stem had t he greatest ash c o n t e n t and the leaves showed an increase in ash c o n t e n t with age. Similar results have been rep o r t e d by Harrison and Mann (1975), who f o u n d t hat the ash c o n t e n t of y o u n g leaves o f eelgrass was generally lower than in older leaves. In submerged parts the ash c o n t e n t was greatest in the roots. The highest value was r e c or ded at Aalborg where t he roots were brownish and seemed less viable t han those at Nibe and R~bnbjerg. However, difficulties in removing all a d h e r e n t sediment particles from t he roots might have influenced these results.

56

Ash content 10

20

30

40

50

0

10

20

30

40

50

0

10

20

30

40

50

Bm

A

Leaf 5

(%)

C

Leaf 4 Leaf 3 Leaf 2 Leaf 1 Stem

m

m

Rhizome Root

Fig. 4. The distribution of ash content (% dry weight) in eelgrass (Zostera marina L.) at Aalborg (A), Nibe (B), and R~$nbjerg (C) in July, 1980.

Cadmium The cadmium concentrations in eelgrass were significantly different at the three stations (P ~ 0.001) and highest at R~nbjerg (Fig. 5) which reflected the high cadmium concentration in the water at this station, as reported by Lyngby and Brix (1982). In aerial parts of eelgrass, cadmium concentration showed a significant (P ~ 0.001) increase with age of leaves, and this pattern was maintained throughout the observation period (Fig. 9A). Generally, the cadmium concentrations increased about two-fold from leaf 1 to leaf 5. The lowest concentrations in the aerial parts were recorded in the stems {Fig. 5).

Cd (ppm) 0

0.5 "'

''''

1.0 ....

1.5

'

0

A

0.5

1.0

1.5

0

1

~J''''I'''AILAL~I S ~ ' ~ ~

2

3 C

Leaf 5

Leaf, Lea,3 Lea,2 Lea,,

~ ~ ~ ~

Rhizome ~ "

~ ~ ~ im

ibm

Root

Fig. 5. The distribution of cadmium (ppm dry weight) in eelgrass (Zostera marina L.) at Aalborg (A), Nibe (B), and R~bnbjerg (C) in July, 1980. Bars indicate standard deviation of 5 replicates. Note different scale at R~bnbjerg.

57

In the submerged parts, the concentrations of cadmium were highest in the roots at all stations.

Copper The concentrations of copper in eelgrass at the three stations were significantly different (P ~ 0.001). The highest concentrations were recorded at Aalborg, being 2 to 5 times as great as the concentrations at the other stations {Fig. 6). The copper concentrations were significantly (P ~ 0.05) different in the leaf-fractions. In contrast to the other metals the copper concentration was highest in leaf 1 and decreased with age. This pattern was most pronounced in winter, where the concentration in leaf 1 was at least twice as great as the concentration in leaf 4 (Fig. 9B). Furthermore, a significant (P ~ 0.01) difference in copper concentrations throughout the observation period was observed. In the submerged parts the copper concentrations were highest in the roots. The high concentration recorded at Aalborg reflected the elevated copper levels in the sediment at this station (Lyngby and Brix, 1982). Lead

The concentrations of lead in eelgrass were significantly different (P 0.001) at the three stations. The lead level at Aalborg was 15--20 times higher than the lead level at the two other stations (Fig. 7). The concentrations of lead in the leaves showed a significant increase with increasing leaf age (P ~ 0.001). This pattern was similar on all sampling dates (Fig. 9C). The lead concentrations were 3--10 times higher in leaf 5 than in leaf 1. In the stem-fraction the lead concentrations were higher than in leaf 1. This pattern was similar to the distribution pattern Of ash content in eelgrass, indicating a positive inter-relationship between lead and ash content.

Cu (ppm) 0

5

10

15

20

25

0

5

A m

Leaf 5

10

15

0 S

5 m

10

15

C

;o-o;o;o-, ° ° 64..,

Leaf 4 Leaf 3 Leaf 2

i

Leaf 1 Stem Rhizome Root

I

27.4

I I

I

Fig. 6. The distribution of copper (ppm dry weight) in eetgrass (Zostera marina L.) at Aalborg (A), Nibe (B), and R~nbjerg (C) in July, 1980. Bars indicate standard deviation of 5 replicates. Note different scale and broken column in the root fraction at Aalborg.

58

Pb (ppm) 0

20

10 ll..I

....

30

I ....

I....I

40 ....

50

0 ....

I

1

2

I ....

| ....

3 I ....

4 ~L,,

5

0

, .J

Leaf 5

1 ....

| ....

2

3

I ....

| ....

4 I ....

5 |

B

A _ _

C

Leaf 4 Leaf 3 Leaf 2 Leaf 1

91

m

Stem Rhizome

~ I

I

z3

146

I

Root

!

Fig. 7. The distribution of lead (ppm dry weight) in eelgrass (Zostera marina L . ) a t Aalborg (A), Nibe (B), and R~nbjerg (C) in July, 1980. Bars indicate standard deviation of 5 replicates. Note different scale at Aalborg and broken columns in the root fractions at Aalborg and Nibe.

In the roots, the lead concentrations were 6--12 times higher than in the rhizomes. Zinc

The zinc concentrations in eelgrass differed significantly ( P ( 0 . 0 0 1 ) at the three sampling stations but were generally highest at Aalborg (Fig. 8). A significant difference between zinc concentrations in leaf-fractions was observed (P ( 0 . 0 0 1 ) . Generally, the zinc concentrations increased with age of the leaves; this distribution pattern was maintained throughout the investigation period (Fig. 9D). At Nibe and R~bnbjerg the distribution pattern was most pronounced during the winter, while the zinc concentrations in the different aerial fractions were nearly identical in July (Fig. 8).

Zn (ppm) 0 ± [ , , I

100 ....

I ....

200 ! ....

0

I

100 , , , l l

....

I ....

200 IAJ~LJ

A

0 ,

100 , , , , I , , , , L

B

....

200 I ....

I

C

Leaf 5 Leaf 4 Leaf 3 Leaf 2

Leaf 1 Stem Rhizome Root

i . F'

| , i I

liBD~

Fig. 8. The distribution of zinc (ppm dry weight) in eelgrass (Zostera marina L.) at Aalborg (A), Nibe (B), and R~nbjerg (C) in July, 1980. Bars indicate standard deviation of 5 replicates.

59

-50

1.0-

A

"'g

p

• .-.40

........... "............. ....+.+ -.

--3O z5

.............

05.

."

•

•

iL

--20

o_

..........

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......

~ . " l t .

_-:=~: ..... ~ - - ~ "

3

0..C

I

I

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//.,,,,,,,,

20-

il

~ _

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..r~

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i

i

i

i

a.

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i

ii

.

-

c° "o

--10

i

-0

- ~

_- 2 0 0 ~

, - -

--150 ~ .

E

..Q o._

10-

._

-•-.

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-

5-

..........

....

-.lh ....

". ""e

Sap. I O c t

•

-

-

.+ ,o

+.,..

"~

0-

- ..•

",0

.........

• • ............

-I~""

• .........

I Nov. I Dec. I Jan. I

' " .

.

.

". " .

.

.

.

. " - - I. I

. ........ .

o +"

I Jul~,

• ..... • Leaf 1

• ....

• Leaf 3

• ..... • Leaf 2

•

• L e a f /.

100

.~

-

--

IF . . . . . . . . 4,

U

"/

-50

.-r

;ep. I Oct. I Nov. I Dec. I Jan. I

II

I July

i-0

Fig. 9. The concentrations of cadmium (A), copper (B), lead (C), and zinc (D) in leaf 1 (dotted line), leaf 2 (dotted-dashed line), leaf 3 (dashed line), and leaf 4 (solid line) of eelgrass (Zostera marina L.) at Aalborg through the research period. Note broken time axis.

The concentrations of zinc in the roots were twice as great as the concentrations in the rhizomes at Aalborg and Nibe, but no significant difference was observed at R~bnbjerg. TABLE 2 THE TOTAL CONTENT OF Cd, Cu, Pb, AND Zn (/~g/m 2 ) IN THE BIOMASS O F EELGRASS (Zostera marina L.) A T THE THREE SAMPLING STATIONS IN JULY, 1980 Cd(~g/m 2 )

Cu(~g/m 2 )

Pb(~g/m 2 )

Zn(pg/m 2 )

Aalborg

aerial submerged to~l

39 20 59

969 792 1761

990 1853 2843

12095 4635 16730

Nibe

aerial submerged total

55 53 108

682 455 1137

87 201 288

10110 11316 21426

R~bnbjerg

ae~al submerged toml

356 310 666

1499 1079 2578

264 221 485

15854 15160 31014

60

Heavy metal content in biomass Table 2 shows the total amount of cadmium, copper, lead, and zinc in eelgrass per unit area. The greatest amount of cadmium, copper, and zinc was recorded at ROnbjerg. The total cadmium content was 6--11 times greater at this station compared to the other stations. The total amount of lead associated with the eelgrass biomass was greatest at Aalborg being 10 and 6 times the amount at Nibe and R~bnbjerg, respectively. The major portion of cadmium and copper occurred in the aerial parts of the biomass at the three stations, whereas lead occurred mainly in the submerged parts, except at R~bnbjerg. Zinc was uniformly distributed between the two parts, except at Aalborg, where levels in the aerial biomass were approx. 3 times those found in the submerged biomass.

DISCUSSION

In the submerged parts of eelgrass the greatest concentrations of heavy metals were recorded in the root-fraction. This could be explained by the greater surface area (Smith et al., 1979) and thereby greater heavy metal adsorption capacity of the roots compared to the rhizomes and/or deposition of the heavy metals in the roots. Koeppe {1977) reported that lead might be bound on the outside of plant roots as crystalline or amorphous deposits and, could also be sequestrated in the cell walls or deposited in vesicles inside the cells. These factors may be responsible for the relatively high root--rhizome ratio observed for lead compared to that of the other heavy metals. The concentrations of several heavy metals in submerged parts of freshwater macrophytes are reported to be greater than the concentrations in aerial parts (e.g. Welsh and Denny, 1980). However, in seagrasses, the opposite pattern has been reported (Pulich, 1980; Brix et al., 1982; Lyngby and Brix, 1982) which might be explained by the fact that the rhizomes of seagrasses constitute a great proportion of the submerged biomass, and that the heavy metal concentrations in the rhizomes are generally lower than in the roots. Comparing the heavy metal concentrations only in the root fraction, with the concentrations in the aerial parts, a similar pattern to that reported for freshwater macrophytes is obtained. The concentrations of cadmium, lead, and zinc in leaves of eelgrass increase with age. Acropetal translocation of cadmium, lead, and zinc is reported to be insignificant in submerged aquatic plants (Faraday and Churchill, 1979; Welsh and Denny, 1979; Lyngby et al., 1982). Translocation is therefore probably a negligible factor in the age-dependent distribution of these heavy metals in the leaves of eelgrass. In brown algae the highest concentrations of heavy metals are associated with the older parts of the thallus, and this has been ascribed to a slow accumulation of heavy metals and/or to a synthesis of more binding sites with time (Bryan and Hummerstone, 1973; Skipnes et al., 1975; Myklestad et al., 1978). Pectic substances

61 constitute a significant fraction of cell wall material in eelgrass and are believed to play an important role in the absorption of ions (Maeda et al., 1966). These results suggest that the age-dependent distribution of cadmium, lead, and zinc in eelgrass might be due partly to a greater amount of adsorption sites e.g. pectates in older leaves, and partly to a slow irreversible uptake of these metals. An electron microscopy study (Sharpe and Denny, 1976) showed that lead in Potamogeton pectinatus was b o u n d in cell walls according to Donnan equilibria and that pinocytotic invaginations might convey some heavy metal into the protoplast. These mechanisms are perhaps features c o m m o n to all submerged angiosperms (Sharpe and Denny, 1976), and might also play a significant role in the accumulation of some heavy metals in eelgrass. The age-dependent distribution of copper in eelgrass was different to that of the other heavy metals. Copper concentrations were greatest in the younger leaves and decreased with age. Both copper and zinc are micronutrients, b u t the mobility of copper is reported to be greater than that of zinc (Tiffin, 1972). The carrier of copper in plant fluids is probably amino acids, based on the affinity of copper for the N-atom in the amino group (Tiffin, 1972). In recent investigations, Welsh and Denny (1979; 1980) found that copper was translocated from the roots to the stem apices and youngest leaves of Potamogeton spp., and that a considerable quantity of copper in the plant was in a soluble fraction. These results indicate that translocation cannot be neglected in the evaluation of the distribution pattern of copper in eelgrass. Harrison and Mann (1975) found the highest nitrogen content in the y o u n g plant parts of eelgrass and ascribed the decrease with age to be due to either leaching to the water column or translocation to the rhizome. In Potamogeton spp. the lower copper concentration recorded in the older tissue was explained by re-translocation to the younger plant parts or by leakage of copper from the older leaves. These results suggest that the observed age-dependent distribution of copper in eelgrass might be explained in several ways: (1) translocation of copper from the submerged parts to the youngest leaf and a dilution with growth, (2) leakage of copper from the older leaves to the surrounding water, and (3) re-translocation of copper from the older leaves to the youngest leaf. However, a combination of these processes might also explain the age-dependent distribution pattern, b u t needs to be clarified b y further investigations. Large quantities of heavy metals, even in greater amounts than are available in the sediment, may be tied up in the seagrass biomass (Pulich et al., 1976). The heavy metal concentrations in eelgrass are several orders of magnitude greater than the concentrations in the ambient and interstitial water from which it is accumulated (Lyngby et al., 1982). The majority of eelgrass enters detritus f o o d chains (Fenchel, 1977) and the heavy metals b o u n d in the eelgrass biomass may thereby be made available to higher trophic levels. The highest concentrations of copper, lead, and zinc in eelgrass were recorded at the contaminated sampling station Aalborg. However, the minor biomass at this station caused the amount of heavy metals in eelgrass per

62

unit area to be relatively small compared to the a m o u n t per unit area at Nibe and R~bnbjerg. This implies that the a m o u n t of heavy metals available to the decomposer food chains per unit area in uncontaminated areas may be of the same magnitude or even greater than in contaminated areas. However, considering the accumulation of heavy metals in the single organisms of the f o o d chains, the concentrations in eelgrass rather than the a m o u n t per unit area are of interest. In conclusion, two age-dependent distribution patterns of heavy metal concentrations in eelgrass were observed in this investigation. The concentrations of cadmium, lead, and zinc increased with age, and the concentration of copper decreased with age of the leaves.

ACKNOWLEDGEMENTS

We wish to t h a n k Drs. Lennart Rasmussen and Hans-Henrik Schierup for critical comments on the manuscript and B.Sc. Marianne Hansen for linguistic improvements.

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