Plant and Soil 66, 181-193 (1982). 0032-079X/82/02/0181-13500.20/0. Ms. 4859 �9 1982 Martinus Nijhoff/Dr W. Junk Publishers, The Hague. Printed in The Netherlands.

Seasonal and species variation in the content of cadmium and associated metals in pasture plants at Shipham

H1LARY M A T T H E W S and IAIN T H O R N T O N Applied Geochemistry Research Group, Imperial College, London, U K

Key words Cd Metal contaminated soil Pasture plants Seasonal variation Tolerance Translocation Uptake Zn

Summary Very high Cd and Zn concentrations exist in soils reclaimed from old mine workings at Shipham but little of the metal is transferred into the pasture herbage. The amount of metal in the soil therefore only influences the amount in the aerial part of the plant to a small degree. It would appear to be the plant species which to a large extent governs its metal burden: grasses accumulating the least Cd and members of the Compositae the most.

Within this species variation exists a change in metal content that corresponds to the time of year, metal levels in plants peaking in the winter between January and March. This increase in the metal content of the shoot reflects the redistribution of metal previously bound within the root. The movement of Pb may be associated with changes in the phosphate status of the plant. In grasses it

would appear that the root possesses the highest metal burden, and for Holcus lanatus, tolerance to both Cd and Zn has been established. The ammonium-aceta te and DTPA-extractable Zn/Cd ratio in soils corresponds closely to that found in the roots of both ryegrass and Yorkshire fog. A much higher ratio was observed in the shoots of these plants.

Introduction

Agricultural soils around the village of Shipham in Somerset have very high concentrations of CD and Zn as a result of mining activity during the late 16th to

mid 19th centuries 6. The ore, calamine (ZnCO3), was worked from the dolomitic conglomerate host rock by means of a series of shallow bell-pits, which remained undisturbed until recently. Increased demand for grazing however has led to the reclamation of some mined land for pasture. After levelling the area, reseeding was carried out using a commercial grass mixture. Seed was sown at the beginning of the very dry summer in 1976, and failed. In its place, however, a self- seeded sward became established, providing good but rather patchy ground cover. Many weed species are present, and include Thlaspi alpestre, a plant characteristic of metal mine wastes 26, and sometimes used as a metal indicator species z. For most of the year the sward appears healthy but some chlorosis in both grass and weed species becomes apparent during late winter and early spring.

The soil, which still contains many large boulders and stones as a legacy of its mining past, is a freely draining brown earth of the Wrington series 5 with a neutral pH and CEC of 20 meq/100 g. Elevated soil metal values are found quite widely in the dolomitic conglomerate of this region. Some 8 km 2 of agricultural

181

182 M A T T H E W S A N D T H O R N T O N

l a n d h a s so i l s w i t h C d c o n c e n t r a t i o n s in e x c e s s o f 100 ixg/g 5, w i t h m a x i m u m

v a l u e s o f 6 0 0 ~ g / g C d a n d 6~o Z n f o u n d in t o p s o i l s in o n e o f t h e r e c l a i m e d

f i e lds 15.

I n o r d e r t o e x a m i n e t h e e x t e n t t o w h i c h t h e s e m e t a l s a r e b e i n g t r a n s f e r r e d f r o m

soi l t o p l a n t a n d so m a d e a v a i l a b l e t o g r a z i n g a n i m a l s t h r o u g h t h e f o o d c h a i n it

h a s b e e n n e c e s s a r y to:

(a) d e t e r m i n e t h e r e l a t i o n s h i p b e t w e e n so i l a n d p l a n t m e t a l c o n t e n t o n so i l s

w i t h v a r y i n g d e g r e e s o f c o n t a m i n a t i o n ,

(b) e x a m i n e t h e d i f f e r e n c e s in p l a n t m e t a l c o n t e n t b e t w e e n t h e m a i n s p e c i e s

p r e s e n t in t h e s w a r d , a n d

(c) a s s e s s t h e p r e s e n c e o f a n y s e a s o n a l v a r i a t i o n s in m e t a l c o n t e n t in t h e s e

s pec i e s .

T h e p r e s e n t w o r k is n o t c o n c e r n e d d i r e c t l y w i t h t h e m o b i l i t y o f m e t a l s in

p l a n t s , b u t w i t h c h a n g e s in t h e i r c o n c e n t r a t i o n a n d d i s t r i b u t i o n a t d i f f e r e n t t i m e s

o f t h e y e a r .

Methods

Herbage was sampled randomly from four, 5 m 2 plots. Sampling was carried out on 14 occasions over a 22-month period (April 1979~anuary 1981) for plot 1, and on 8 occasions over a 12 month period (January 1980-January 1981) for the remaining 3 plots. Plot 1 was located at Shipham on previously mined land that has recently been levelled and reclaimed as pasture. Plot 2 was located in an adjacent field where the grass forms an established ley, with no known underlying mineralisation. Plot 3 was also on an established ley with a lower soil Cd value (only grass and clover were available for sampling at this site). Plot 4 formed the control site and was located nearby at Banwell. The soils from Plots 1 and 2 have been classified as belonging to the Wrington series, and overly Dolomitic Conglomerate parent material, whilst those from plots 3 and 4 lie on the Worcester/Tickenham boundary, and are developed from Keuper Marl 5. All 4 sites were subject to intermittent grazing by cattle and sheep during the course of this research.

Of the 15 plant species sampled from plot 1, 7 were selected for examination on the remaining plots. Fresh herbage samples were cut I cm above the soil and thoroughly washed in 3 rinses of deionised water prior to drying at c. 80~ After milling in a Christy and Norris mill, designed to minimise trace metal contamination, subsamples were wet ashed using a 4 : 1 mixture of nitric/perchloric acid.

Entire plant systems were collected by digging up turfs about 30 cm 2 and 45 cm deep using a spade. Soil was gently shaken from the roots and the individual plants were then divided into root, shoot or root, stem, leaf and seed-head components, as applicable, using stainless steel scissors. The plant parts were carefully and thoroughly washed three times in deionised water. Great attention was given to washing the root, until thorough visual inspection, using a hand lens, revealed that no soil particles remained adhering to the roots, and the deionised water used for washing was clear. Because it was impossible to clean soil from the root-shoot junction, this was discarded. Titanium has been proposed as an indicator element for soil contamination of herbage samples 1 s. The soils at Shipham contain c. 6001ag/g Ti and the carefully washed roots were found to contain < 101ag/g Ti, reflecting soil contamination levels of < 2~o. These samples were dried and analysed using the method outlined above.

Composite soil samples, comprising 9 subsamples, were collected from each of the 4 plots at depth intervals of 0-15, 15 30 and 30-45 cm with a hand auger. After oven drying the less than 200 pm fraction was digested in concentrated HNO 3 for I hour prior to dilution and analysis for 'total' metal content.

CADMIUM IN PASTURE PLANTS AT SHIPHAM 183

All samples were analysed using a flame atomic absorption spectrophotometer (Perkin Elmer model 403), employing Ca correction as necessary.

Heavy metal tolerance was investigated in Holcus lanatus samples using the method outlined by Wilkins 3o and subsequently modified by Jowett 12 and McNeilly and Bradshaw 16. Root extension was measured for grass tillers grown in a Ca(NO3) 2 control (Ca 0.5 g/l), and corresponding tillers grown in metal amended solutions: Cd 2 lag/g, Zn-7.5 ~tg/g. At the end of a 3-week period the Tolerance Index was determined. Solutions were changed every other day.

Results and discussion

The soils of the 4 plots sampled provided a gradient of metal contamination (Table 1) for the metals Cd, Zn and Pb; amounts of Cu were similar at 3 of the 4 plots sampled and greater at plot 1. Plant metal contents were found broadly to reflect those of the soil, however the difference in Cd content between grass sampled from the site with the highest degree of contamination and that from the unmineralised control was small (Fig. 1).

Work by Page e t al . 20 has established that for plants grown by solution culture techniques the Cd content in the leaves increases as solution Cd concentrations increase, but this does not occur in a linear fashion. Further research, based on making Cd additions to soil 17, has shown corresponding increases in plant Cd but again, uptake exhibits a non-linear, upper-limit type of response. This 'tailing-off" in plant response to increased Cd levels within the substrate is clearly shown amongst samples of mixed grasses collected from a range of sites at and around Shipham (Fig. 2). The percentage of soil Cd taken up by grass decreases as soil Cd levels rise (Table 2), keeping the overall metal burden low.

Table 1. Soil metal content and pH for the four plots examined

Plot Depth Cd Cu Pb Zn Ca pH CEC Loss on igni-

No. (cm) (lag/g) (lag/g) (lag/g) (lag/g) (%) (H20) (meq/ tion as esti- 100g) mate of OM%

1 0-15 440 49 7800 50000 7.24 7.2 18 5.3

15-30 432 53 10640 54000 6.68 30~5 373 95 17066 62400 6.72

2 0-15 53 26 893 3613 1.41 6.9 25 5.1

15-30 52 23 660 2940 2.20

30-45 63 25 555 3187 2.62

3 0-15 24 22 475 2040 0.21 6.1 19 10.2

15-30 19 20 367 1560 0.20 30-45 16 19 236 1180 0.20

4 0-15 3 23 135 453 0.27 6.3 17 8.1

15-30 3 24 134 308 0.30 30~5 2 22 88 208 0.24

184 MATTHEWS AND THORNTON

_

Fig. 1.

_

_

_

0

CLOV._~___ER

~ M I X E D G R A S S

I,,ol s312, I 3 I Site I Site 2 Site 3 Site 4 (Soil Cd value)

Mean concentrations of Cd in clover and mixed grass at the four Shipham sites.

3 . 0 -

2.5.

0

0

2.0, r

r 1.5.

o~ r J~ 1.0. I . B ,I-

0.5-

=$

i i i i i i i i

2 0 0 4 0 0 6 0 0 8 0 0

S o i l C d M g / g

i i

1 0 0 0

Fig. 2. The relationship between soil Cd concentration and grass metal content.

C A D M I U M IN P A S T U R E PLANTS AT S HIP HAM 185

Chaney and Hornick 3, reviewing results of several greenhouse and field trials, found pH to be the main soil factor governing Cd uptake. At the near neutral pH of the Shipham soils Cd would be strongly bound to soil constituents and consequently of limited availability to plants. The presence of very large amounts of Zn and Ca at these sites may also depress Cd uptake through competition for exchange sites on the root surface. This effect has been previously established experimentally in ryegrass by Jarvis et al.9, using a solution culture technique. The amount of Cd present within the plant however would appear to depend very much upon the individual species concerned (Fig. 3). Grasses were found to have relatively low metal contents; this is in agreement with work by Simon z4 who noted that monocotolyedonous plants take up less Cd than dicotolyedons; consequently weed species acquire a greater burden of Cd than grasses. Members of the daisy family (Compositae) were found able to accumulate the largest amounts of metals: these include, in addition to daisy, yarrow and dandelion. Examining the literature provided further instances of unusually high levels of trace elements in members of this family and yielded data for chicory 23, black- eyed susan and rough blazing star 17. These species differences are thought by some researchers to reflect a genetic basis for Cd uptake and translocation 4 which does not depend on any gross difference in plant morphology or anatomy. Even on the low metal control site the relative patterns for metal uptake by the different plant species remain (Fig. 4). It would appear however that plants capable of taking up and retaining most Cd on the control site also have a greater capacity for accumulating this metal on the contaminated sites. Consequently Cd levels in daisy showed an eleven-fold increase between plots 1 and 4, whilst grass Cd values increased by a factor of only three between the same sites.

In addition to species differences, plant metal content changes with season, with maximum metal values occurring over the period from January to March. This variation is especially pronounced for Pb, but also occurs with Cd and Zn. Increases from the low summer values to the winter peak are around 500% for Pb and 300% for Cd and Zn (in Hotcus lanatus); similar increases were not apparent for Cu (Fig. 5). This difference in relative increase between the metals and in particular the small changes in concentration of Cu throughout the year suggest that the process is not simply a growth effect as proposed by some researchers 22, whereby changes in the amount of plant dry matter due to senescence bring

Table 2. The relationship between Cd concentrations in grass and topsoil (0-15 cm)

Plot Topsoil Cd Grass Cd Soil/grass No. (p.g/g) (lag/g d.m.) Cd ratio

1 440 2.8 157.1 2 53 2.7 19.6 3 24 2.1 11.4 4 3 0.9 3.3

186 MATTHEWS AND THORNTON

RYEGRASS I i Lollum perenne

FESCUE Festuca rubra

YORKSHIRE FOG Holcus lanatus

DAISY Bellls perennls

DANDELION Taraxacum spp

YARROW Achnlea mlllefollum

BUTTERCUP Ranunculus spp

CLOVER Trlfollum repens

J

i iiiiiiiiiiiiiiii iil

0 10 20 3o 40 50 60 70 80 ug / g C a d m i u m

Fig. 3. Variations in Cd content of common pasture species at Shipham.

about corresponding changes in plant metal contents. Large differences in Pb concentration in grasses between summer and winter months were first reported for uncontaminated sites in Scotland 17, and then for old Pb mining areas in Derbyshire 2s. These seasonal changes may reflect either a change in the total metal burden within the plant, or merely a redistribution of metals from root to shoot. In solution culture trials senescence in plants has been shown to result in an increased uptake of Cd into both root and shoot 21. In addition, plants which are less metabolically active exhibit a greater Pb and Cd burden 8. This may be because actively growing roots are capable of restricting the movement of Pb to aerial plant parts 1~. In the 1980/81 season it would appear that the winter increase in metals is less marked than for the previous year: this may be due to milder weather placing the plants under less physiological stress.

The seasonal change in plant metal level could also result from a seasonal change or redistribution of some essential element such as phosphorus within the plant. Pb and Cd in most plants are found in a precipitated form associated with the cell walls 8'9. In corn this precipitate has been identified as a Pb-phosphate complex 13. It has been suggested that the transport and mobility of Pb and Cd within the plant relates to the solubilities of their phosphates 7. Consequently seasonal variation in the phosphate level could have a profound effect on the distribution of metals. Work carried out at Long Ashton Research Station 29 has shown a three-fold decline in the phosphate content of apple leaves between summer and winter months, and initial observations in this research show a similar decline within the grass species examined. Thus falling phosphate

CADMIUM IN PASTURE PLANTS AT SHIPHAM 187

PLANT SPECIES GRASS ~ ]

(mixed 9raell epp)

c.ow. I N I (Tr Ifollum repens)

(Plantago lan�9

BUTTERCUP ~ 1 (Ranun�9 spp)

co-.- i ] MOUSE EAR IC~tlau f ~

~176 I I (Taraxacum spp)

DAISY (Bellis perennis)

6 ,i ~ 1~ 1~ 2'0 ='4 Cd (IJg/g)

Soil Cd (pg/g)

~ 4 4 0

18 ~2 ~8 go g.

Fig. 4. Mean Cd values in the herbage at the Shipham study sites (sampled on 6 occasions between January 1980 and September 1980).

concentrations within the plant would appear to correlate with increasing metal burdens.

Further studies included an examination of changes in the metal content of grass roots. From these it would appear that the increased metal burden in the aerial part of the plant corresponds to a decrease in root metal content (Table 3), thus helping to establish that it is remobilisation of the metal, and not increased uptake that is responsible for these seasonal variations in the plant shoots.

Whilst the form(s) of the metals within these soils are not known, preliminary work carried out using selective extractant solutions, including ammonium acetate (1 M pH 7) and DTPA, showed that in each instance a far greater proport ion of Cd could be extracted from these soils than Zn. This unequal. extraction is reflected by the low Zn/Cd ratios obtained using selective extractant solutions, compared with the much higher ones obtained from a ' total ' nitric attack (Table 4).

Analysis of whole plants has shown that for the two grass species ryegrass and yorkshire fog, the roots provide the site of highest metal burden (Fig. 6). This finding corresponds with those from research based on solution culture techniques when 88~o Cd and 57-80~o Pb were localised within the root tissue. It was not possible to differentiate in this research between surface bound metal, and the metal in the root cells. However, the seasonal redistribution of Cd and Pb as measured by a change in the root/shoot metal ratios appeared to indicate that a significant portion of the metal is present within the plant. It is of interest that the Zn : Cd ratio in the plant roots was found to correspond to the Zn : Cd ratio in the soil; as determined by the selective extractants (Table 5). This was very much lower than the Zn : Cd ratio determined in the shoots, so it would appear than Zn,

188 MATTHEWS AND THORNTON

o~ \ o~ =L 'ID 0

1 C A D M I U M

ol =L J~ O.

60-

50-

40 .

30-

20-

10-

0

L E A D

350-

300-

250-

200- o~

=" 150- c N 100-

50 -

0

ZINC

0

10-

8-

6-

4-

2-

0

C O P P E R

APF MAY JUL AUG SEP NOV DEC JAN FEB MAF APIq JUl~

Fig. 5. Seasonal variation in metal levels in Holcus lanatus (Yorkshire fog) sampled at Shipham from Plot 1 (smoothed data based on rolling mean over a 3-month period).

upon enter ing the roots , is t r ans loca ted to the shoots whilst Cd does not become

re loca ted in the same way. This may reflect the essential na ture of Zn to p lants

and its subsequent active uptake. Zn : Cd ra t ios in the aerial par ts of the p lants

s tudied were found to be species specific. W o r k at Sh ipham has shown tha t Cd tolerance, as measured by relat ive roo t

extension 24. relates clearly to the a m o u n t of Cd present in the soils from which the

p lan t was collected (Fig. 7). This l inear re la t ionship indicates tha t metal to lerance

CADMIUM IN PASTURE PLANTS AT SHIPHAM

Table 3. Seasonal changes in the contents of Cd and Pb in plant shoots and roots

189

Sampling month

Root Shoot Root/shoot ratio

Cd Pb Cd Pb Cd Pb

July 51 288 5.3 77 8.1 3.7 September 79 366 3.4 29 23.2 12.6 November 67 207 6.6 64 10.2 3.2 January 86 210 9.3 86 9.2 2.4 March 60 125 10.3 114 5.8 1.1

is a mult i -gene characteristic 1. Cd tolerance has been found to exceed 100~o in

Holcus lanatus plants, collected from a highly con tamina ted site at Shipham 29.

The roots grew better in solut ions con ta in ing 2 ~g/g Cd, than in solut ions

without Cd. However this was not found to be the case in the current research, a

difference which may reflect the fact that full nut r ien t solut ion was not used in

this latter study. The degree of Zn tolerance appeared to be lower than that for Cd

but the two exhibit a direct correlat ion with each other (Fig. 8). Experiments

using radio-zinc have shown that tolerant clones ofAgrostis tenuis Sibth are able

to bind greater amoun t s of metal within the root cell walls than non- to le ran t

clones27: this suggests that the tolerance mechanism is a modificat ion of the

normal process within the plant which prevents metals being t ranslocated from

the root to the shoot.

Acknowledgements The research was funded by a grant from RTZ Services Ltd., Bristol, including a bursary to Hilary Matthews. We are grateful to our colleagues in the Applied Geochemistry Research Group for analytical support.

Received 31 August 1981. Revised March 1982

Table 4. Comparison of the Zn/Cd ratios obtained using total and partial extractants on Shipham soils

Extractant Zn/Cd ratios

Mean Range

Nitric acid 88 68-112

Ammonium acetate 18 9 27 DTPA 22 13- 30

190 M A T T H E W S AND T H O R N T O N

300

I I I I

2 0 0 -

1 8 0 -

1 6 0 -

A

m 1 4 0 - \

=L , - - 120-

100 -

1:3

r 8 0

6 0

4 0 -

2 0 -

o

Cd distribution in an example of the soil/plant system at Shipham for Holcus lanatus. Fig. 6.

References

1 AntonovicsJ, B r a d s h a w A D a n d T u r n e r R G 1971 Heavy metal tolerance in plants. Adv. Ecol. Res. 7, 1-85.

2 Bradshaw A D and Chadwick M J 1980 The ecology and reclamation of derelict and degraded land. Blackwell Scientific Publications, Oxford.

C A D M I U M IN PASTURE PLANTS AT SHIPHAM

8 0 -

191

7 0 -

60 -

LIJ (J z 50 - <r n "

IL l .--I 0 40 - I - -

3 0 -

Q ,,r u 20

10, '+

0 0

-I-

+

4"

I I ' I " I " I " I " I

20 4 0 60 80 100 120 140

SOIL CADMIUM ( H g / g )

Fig. 7. The influence of soil Cd content on the degree of Cd tolerance exhibited by Holcus lanatus

collected from Shipham pastures (each point represents the mean of data obtained for 10 tillers).

Table 5. Zn/Cd ratios in soils and grasses ~om the four study sites around Shipham

Plant species Sample Soil Soil Root Shoot site Cd ~g/g Zn/Cd Zn/Cd Zn/Cd

Holcus lanatus 1 304 101 43 92 2 94 77 31 90 3 25 78 23 111 4 3 77 29 42

Mean 83 32 84

Lolium perenne 1 324 96 26 119 2 57 86 36 79 3 25 78 19 101 4 3 77 23 79

Mean 84 26 95

192 M A T T H E W S AND T H O R N T O N

A

LU

Z

LU /

O

0 Z m

N

2 0 -

15-

10 -

5

§

+

+

0 I I I I I I I 0 10 20 30 40 50 60 70

CADMIUM TOLERANCE ( % )

+

Fig. 8. The relationship between Cd and Zn tolerance indices in Holcus lanatus taken from Shipham

pastures.

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C A D M I U M IN PASTURE PLANTS AT SHIPHAM 193

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