INTERACTIVE EEFECTS OF CADMIUM, COPPER AND NICKEL ON THE GROWTH OF SITKA SPRUCE AND STUDIES OF METAL UPTAKE FROM NUTRIENT SOLUTIONS

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1 New Phytol. (1986) 103, INTERACTIVE EEFECTS OF CADMIUM, COPPER AND NICKEL ON THE GROWTH OF SITKA SPRUCE AND STUDIES OF METAL UPTAKE FROM NUTRIENT SOLUTIONS BY K. W. BURTON', E. MORGAN' AND A. ROIG^ ^Department of Science, The Polytechnic of Wales, Treforest, Mid-Glam, S. Wales, CF37 ldl, UK and ^Department of Organic Resources, Centre for Edaphology and Applied Biology of the Segura Valley, CSIC, Murcia, Spain (Accepted 3 March 1986) SUMMARY The interactive effects of,, and were studied in Sitka spruce [Picea sitchensis (Bong.) Carr] seedlings grown in nutrient solutions supplemented with combinations of these metals over three experiments of 3 x 3 x 3 factorial design. The results indicated that the toxic effects of and may be additive when both metals are present at concentrations which individually reduce yields, and that tree growth at several forestry sites in South Wales may be more severely affected than previously reported. Analysis of variance of the concentrations of the added metals in the plants showed that concentrations of in both roots and shoots were significantly reduced by treatments {P < 001) and that this effect was especially pronounced at the highest concentrations of and (significant x interaction, P < 0 01). Analysis of variance of the concentrations of the nutrients Zn, Mg, Mn, and Ca in roots and shoots showed that both and significantly (P < 0 01) reduced the concentrations of Mn, and also interactively affected these {P < 0-01). Concentrations of Mg in the shoots were also significantly reduced by treatments. Key words: Sitka spruce, interactive effects, cadmium, copper, nickel. INTRODUCTION Most studies of the impacts of heavy metals on higher plants have tended to concentrate on effects of individual metals added to the rooting medium, and have generally neglected possible interactive effects. Tbese interactive effects migbt be antagonistic, independent, additive, or synergistic in nature (Davis & Beckett 1978). Such effects are widespread in animals (Underwood 1977), and from the little work that has been carried out in plants, and on trees specifically (Carlson & Bazzaz, 1977), there would seem to be no reason to suggest that they are less widespread or complex in tbe plant kingdom. Studies of the impacts of individual heavy metals may lead to a better understanding of the ways in which plants are affected by tbose individual metals, but may not be very relevant if more than one contaminating metal is present which can influence plant function, growth, and the pattern of uptake of other metals. This may occur frequently because of natural associations between contaminating metals. For example, tbe soil and vegetation around non-ferrous smelters often have enhanced concentrations cf and (Hutchinson & Whitby, 1974). Tberefore, the combined impact of contaminating metals should be determined by examining tbe individual and interactive effects of these metals X/86/ $03.00/ The New Phytologist

2 55O K. W. BURTON, E. MORGAN AND A. ROIG In many areas of South Wales there are enhanced levels of several heavy metals (Goodman & Roberts, 1971; Burton & John, 1977). It has been indicated that the growth of Sitka spruce [Picea sitchensis (Bong.) Carr], a commonly grown forestry tree in upland S. Wales, may be affected as a result of contamination by,,, and Pb (Burton, Morgan & Roig, 1983, 1984). These studies were based on assessments of the metal concentrations at which growth is impaired, and related to the foliar metal concentrations at several forestry sites, but interactive effects were not considered. The present study examines interactions between metals which might affect the growth of sitka spruce and the uptake of metals. MATERIALS AND METHODS Sitka spruce seeds (P. sitchensis) of known provenance and origin, and of uniform seed size were obtained from the Forestry Commission. Seeds were germinated at 25 C over 10 to 14 d in trays of peat. The seedlings were transferred to supports suspended over 250 ml polypropylene pots covered in black polythene (15 seedlings per pot) and containing quarter strength Ingestad nutrient solution (Ingestad, 1959). The composition of this solution was increased to half strength after a further 5 d and to full strength 5 d later. The ph of the solution was 4-5 (not adjusted) and its full strength composition is as given in Table 1. The pots were kept in an electrically heated propagator (minimum temperature 20 C) with a day/ night ratio of 16/8 h (Morgan, 1983). The nutrient solutions were changed every 5 d to prevent depletion of oxygen and nutrients. Table 1. Nutrient solution composition used in growing the Sitka spruce seedlings (Ingestad, 1959) Elemental composition Compound Concentration (mgl-') Element Concentration (mg 1-') NH^NO^ KH2PO4.2H2O KCl CaCla. 6H2O MgSO,. 7H2O EeCI.,. 6H2O MnCla. 4H2O H3BO3 CUCI2.2H2O ZnClj. 2H2O Na^MoO,. 2H2O N P K Ca Mg, S Fe Mn B Zn Mo , ' When the seedlings were 31 d old,,, and were added (as chloride salts) to the nutrient solutions following a 3 x 3 x 3 factorial design. Three experiments were conducted. In exp 1,,, and were each added at three concentrations (Table 2). The lowest concentrations of,, and were those in the full strength nutrient solution. The intermediate concentrations were set at the concentrations which just reduce plant growth (Morgan, 1983), and the highest concentrations at double the intermediate concentrations. Experiment 2 was conducted subsequently at the same solution concentrations in the hope of

3 Metal and growth of Sitka spruce 551 Table 2. Concentrations {mg l~^) of the metals, and added to the nutrient solutions in expts 1, 2 and 3 Experiments 1 and treatment level 2; Experiment 3: treatment level Metal confirming the results of expt 1. In expt 3 the treatments were duplicated and the solution concentrations of,, and were adjusted slightly (Table 2) to increase the toxic effects of and and to reduce tbe rather large eftects of found in the first two experiments. The replication of the treatments in expt 3 allowed tbe calculation of tbe three-factor interactions (Davies, 1979). When 73 d old the 15 plants from each treatment were harvested, divided into sboots and roots, dried overnight at 105 C and tbe dry weigbt yields measured. Total concentrations of metals in shoots and roots were determined by atomic absorption spectropbotometry after digestion of tbe dried material in 3 :1 nitricperchloric acid mixture. Concentrations of,, and were determined in expt 1 and Ca, Mg, Mn, and Zn concentrations were also measured in expts 2 and 3. RESULTS The results of tbe measurements of yields and metal concentrations in the three experiments, wbich are too many to report in full here, were subjected to analysis of variance. The analyses of variance of the average yield responses of the Sitka spruce seedlings, based on tbe three experiments, are summarized in Table 3 as the i<" ratios of the different treatments and tbe directions of tbe responses. In expts 1 and 2 there were significant reductions in the yields due to treatments (P < 0-01) though the effect was not quite significant in expt 3, probably the result of decreasing the added concentrations of (Table 2). In expt 2 there were significant reductions in yields due to treatments (P < 0-01 total and sboot yields, P < 0-05 root yields), though no effects by were demonstrated in any of the experiments (Table 3). There were no significant interactive effects upon yield even where and individually affected yields (expt 2). Had there been significant interactive effects between and, tbis would have indicated non-additivity of tbe individual effects, i.e. either synergism or antagonism (Anderson & McLean, 1974). To examine tbe effect of treatments upon the responses produced by the treatments, tbe average total yields (expt 2) at the different and solution concentrations are plotted in Figure 1, and it appears tbat tbe responses to tbe two metals are approximately additive, but not synergistic. Tbe results of the analysis of variance of the concentrations of,, and in roots and shoots in the three experiments are summarized in Table 4 as E ratios of the different treatments and the directions of the responses. Generally, it can be seen tbat when tbe solution concentrations of the metals were enhanced their

4 552 K. W. BURTON, E. MORGAN AND A. ROIG Table 3. F ratios from Anovars of treatment effects upon the total yields of dry matter in the seedlings and those of shoots and roots in expts 1, 2 and 3 Treatment Total yield Shoot yield Root yield Expt 1 x x x Expt 2 x x X Expt 3 x x X x x ** ** ** ** ** ** ** * ** Significant, P < 0-05; ** Highly significant, P < 001. ^ 90 - B Copper solution concentrotion (mg L" ) Eig. 1. Effect of and treatments on average total seedling yields (expt 2). SE, standard error of means from error mean square (Anderson & McLean, 1974). Cadmium solution concentration; ( ) 0 mg 1-'; ( ) 0-25 mg I"'; (A) 0-5 mg I"'. concentrations in tbe shoots and roots were also significantly increased. It can be also noted that when an effect by a particular treatment was significant in the roots it was usually significant in the shoots. This would seem to be logical since, depending on the mobility of the metal, any large increase or decrease in metal

5 Metal and growth of Sitka spruce 553 Table 4. F ratios from Anovars of treatment effects upon the shoot and root concentrations of, and in expts 1, 2 and 3 Shoot Root Treatment Expt 1 X x x 38-80** ** ** ** 7-15* * ** 47-36** ** ** ** ** ** ** Expt 2 X x x 34-24** ** * ** ** ** ** ** ** ** * Expt 3 x x x x x 17-68** ** ** 76-82** ** ** ** ** ** ** ** 24-67** ** ** 12-25** Significant, P < 005; ** Highly significant, P < concentrations in tbe roots would affect tbe availability of that metal for uptake to tbe shoots. Analyses of variance of the concentrations of in the plants show tbat tbere were large effects of the treatments (Table 4). Increasing solution concentrations significantly affected concentrations of in tbe roots in all tbe experiments (P < 0-01) and in the shoots in expts 1 and 2 (P < 0-01). In expt 3 the effect was not quite significant in the shoots, which again was probably the result of reducing the solution concentrations in this experiment. There were significant interactive effects between and upon shoot and root concentrations. This effect is sbown in Figure 2, wbicb is a plot of tbe average root concentrations at the different and solution concentrations in expt 3. It was found that treatments witb reduced tbe concentrations of (P < 0-01), and tbat tbis effect was most pronounced at the highest solution concentrations of (significant x interaction at P < 0-01). Concentrations of were significantly aifected in expts 1 and 3 by treatments (Table 4). However, in expt 1 there was a very significant increase in concentrations in tbe sboots over the tbree treatments (P < 0-01), whereas in expt 3, the treatments significantly reduced concentrations in both roots and sboots (P < 001). This was probably tbe result of doubling tbe concentrations of in the solutions and halving those of in expt 3 compared with expt 1, and may indicate that the effect is dependent upon the relative concentrations of the two elements. Tbere were also several otber significant effects upon and

6 554 K. W. BURTON, E. MORGAN AND A. ROIG Cadn'iium solution concentrotion (mg I Eig. 2. Effect of and treatments on root concentrations (expt 3)..SE, standard error of means from error mean square (Anderson & McLean, 1974). Copper solution concentration: (#) 0-02 mg 1-'; (A) 2-5 mg I"'; ( ) 5 mg I*'. Table 5. F ratios from Anovars of treatment effects upon the shoot and root concentrations of Zn, Mg, Mn and Ga in expts 2 and 3 Shoot Root Treatment Zn Mg Mn Ca Zn Mg Mn Ca Expt 2 x x X Expt 3 x x X X X ** ** -5-57* ** ** ** -6-36' ft* 31 "91 ** ** ** "? ** ** ** ** ** ** -8-87** 34-55** 6-70** 9-42** 8-68** Significant, P < 0-05; ** Highly significant, P < concentrations in tbe plants, although these were present in only one experiment. Analyses of variance of the effects of the different treatments upon tbe concentrations of Ca, Mg, Mn and Zn in tbe plants were conducted for expts 2 and 3, and are again summarized as F ratios witb the directions of the responses in Table 5. Mn concentrations in tbe shoots and roots of the seedling were significantly affected in both experiments by and treatments individually (P < 0-01) and interactively (P < 0-01). Figure 3 is a plot of the average Mn concentrations in the

7 Metal and growth of Sitka spruce E Copper solution concentration (mg L ) Eig. 3. Eflect of and treatments on root Mn concentrations (expt 3). SE, standard error of means from error mean square (Anderson & McLean, 1974). Cadmium solution concentration: (A)0-5mgl-';(«)0mgl-';(O) 1 mgl"'. roots at tbe different concentrations of and in tbe solutions as found in expt 3, and sbows that both and individually reduced Mn root concentrations to approximately 25 % of tbeir control values. However, Figure 3 shows that this effect was no greater when both metals were present. Tbis is refiected by tbe significant positive interactions upon the Mn concentrations in each case (P < 0-01) and indicates tbat and interact antagonistically in tbeir effects upon Mn concentrations. There were also significant effects upon the concentrations of Mn in tbe roots in expt 3 by treatments, and interactive effects between and, and, and a complex three factor interaction involving the three added metals. Table 5 also shows that the treatments with significantly reduced the Mg concentrations in the shoots (P < 0-05 expt 2, P < 0-01 expt 3) and roots (P < 0-01 expt 3), though the effects upon Mg concentrations in expt 1 are not known since they were not measured. The only significant effects upon Zn and Ca uptake were also produced by treatments in the seedling sboots (expt 2). Here again the effects may not bave been present in expt 3 as the concentrations in the solution had been decreased. DISCUSSION One of the main aims of this work was to discover in what way the three metals interacted to affect tbe growth of Sitka spruce. Tbe effects may be additive as assumed in the zinc equivalent concept (Chumbley, 1977) or, alternatively, they may be independent, antagonistic or synergistic (Maccol & Beckett, 1985). Tbe effects of and upon tbe yields were individually significant in expt 2 and an examination of the data (Fig. 1) seems to indicate that the overall effects were additive since increases in tbe solution concentrations led to greater reductions in total yields tban caused by alone and vice versa. Davis & Carlton-Smith (1984) reported that the phytotoxic effects of mixtures of elements were only additive when the concentrations of one or more of the elements exceeded its upper critical concentration, defined as the lowest tissue concentration

8 556 K. W. BURTON, E. MORGAN AND A. ROIG of an element at which its presence leads to a reduction in yield. Therefore the results presented in tbis study are in broad agreement with tbis concept. Carlson & Bazzaz (1977) studied tbe interactive effects of Pb and in two-year-old American sycamore {Platanus occidentalis L.) and reported synergistic reductions in several growth parameters, indicating tbat more mature trees may also be affected by contaminating metals. Tbe foliar concentrations of,,, and Pb were measured in young needles taken from the upper crowns of Sitka spruce growing at several forestry sites in South Wales (Burton et al., 1983). A comparison of these concentrations with their respective upper critical tissue concentrations, measured in Sitka spruce seedlings, showed tbat these critical concentrations were exceeded at several forestry sites, and it was concluded that tree growth at tbese sites may be at risk (Burton et al., 1983). As the toxic effects of and upon yields would seem to be additive (Fig. 1) and since it has been shown that more mature trees may be affected by interactions between contaminating metals, it seems reasonable to conclude tbat tree growth is more at risk than previously reported. The effects on the uptake of the added metals and nutrients by treatments in Sitka spruce seedlings are given in Tables 4 and 5 respectively. Some individual effects by,, and upon uptake of otber metals into plants have been reported previously in other species, but little attention has been paid until recently to the more complex interactive effects involving two or more elements. Beckett & Davis (1978), in a non-factorial experiment in which young barley plants were grown in sand culture witb Zn, and added to the rooting medium, found that effects of one element on the uptake of another from the same medium were not large and usually occurred at solution concentrations whicb reduced the yields of the barley plants. However, tbis was not found to be the case in these experiments where the magnitudes of effects were often very large and occurred where there were no significant effects upon yield (expt 3). One reason for this could be that sitka spruce is more sensitive to such effects, which are thought to vary widely from species to species (Webber, 1981). The reduction in the uptake of into sboots and roots in response to treatments with was consistently significant over the three experiments. This effect has been reported for other plant species (Saitoh, Hanada & Mochizuki, 1973 ; Cataldo & Wilding, 1978). Figure 2 shows tbe effect of treatments upon concentrations in the roots (expt 3), and would seem to indicate that can block the uptake of since the effect of treatments was greatest at the highest solution concentrations of both and (significant x interaction, P < 0-01). uptake was also significantly affected by treatments, though the actual effect seemed to depend on the relative concentrations of the two metals in the rooting medium. Other workers have also reported such contradictory results in rye plants grown on sludge-amended soil (nningham, Keeney & Ryan, 1975). Analyses of variance of the effects of the different treatments upon the nutrients Ca, Mg, Mn, and Zn in expts 2 and 3 showed that, of these nutrients, only Mn levels in tbe plants were highly sensitive to the presence of,, and in the nutrient solution (Table 5). Figure 3 sbows tbat both and individually induced large decreases in the levels of Mn in tbe roots. It has been reported previously that Mn uptake by bushbeans was reduced in the presence of (Wallace et at., 1977), and the results obtained bere therefore agree with these findings. Copper treatments also decreased Mn uptake, and interacted with in such a way that Mn uptake was not reduced any further wben botb and

9 Metal and growth of Sitka spruce 557 were present. These effects were quite large and affected the uptake of Mn into shoots and roots in expts 2 and 3 (P < 0-01, Table 5). Another consistent though smaller significant effect was that of treatments upon Mg uptake into shoots and roots. However, studies of the infiuence of metals upon concentrations of Mg in plants have not been reported in tbe literature, making any appraisal of tbe results difficult. ACKNOWLEDGEMENTS Tbe authors wish to thank Dr Binns of the Forestry Commission for his help and advice. They are also grateful to the Science and Engineering Research Council and the British Council for financial support to F. Morgan and A. Roig respectively. REFERENCES ANDKH.SON, V. L. & MCLEAN, R. A. (1974). Design of E.vperiments, A Reatistic Approach. Marcel Decker, New York. BECKKTT, P. H. T. & DAVIS, R. D. (1978). The additiv-ity of the toxic effects of, and Zn in young barley. A^CK)/^/iyro/»^;.?r, BURTON, K. W. & JOHN, E. D. (1977). A study of heavy metal contamination on the Rhondda Fawr, South Wale.s. Water, Air and Soil Poltution, BURTON, K. W., MORGAN, E. & ROIG,.\. (1983). The influence of heavy metals upon the growth of Sitka spruce in south Wales forests. I. Upper critical and foliar concentrations. Plant and Soil, 73, BURTON, K. W., MORGAN, E. & RotG, A. (1984). The influence of heavy metals upon the growth of Sitka spruce in South Wales forests. II. Greenhouse experiments. Plant and Soil, 78, CARLSON, R. W. & BAZZAZ, F. A. (1977). (Jrowth reduetion in american sycamore (Platanus occidentalis L.) cau.sed by Pb- interaction. Environmental Pollution, 12, CATAI.DO, D. A. & WILDING, R. F. (1978). Soil and plant factors influencing the accumulation of heavy metals by plants. Environmental Health Perspectives. 27, CHtiMiiLRV, C. CJ. (1977). Permissible levels of lo.xic metals in sewage used on agricultural land. Agricultural Development and Advisory Service Advisory Paper, No 10., Ministry of.agriculture, Food and r'isheries. Pinner. CUNNINGHAM, J. D., KI-;I;NEY, D. R. & RYAN, J. A. (1975). Yield and metal composition of rye grown on sludge amended soil. Journal of Environmental Quality. 4, 448^54. DAVII;S, O. L. (1979). The Design and Analysis of Industrial Experiments, 2nd edn. Longman, London. DAVIS, R. D. & HiiCKii-rT, P. H. T. (1978). Upper critical levels of toxic elements in plants. 2. Critical levels of copper in young barley, wheat, rape, lettuce and ryegrass, and of nickel and zinc in young barley and ryegrass. Neiv Phytologist, 80, DAVLS, R. D. & CARL-ION-SMITH, C. H. (1984). An investigation into the phytotoxicity of zinc, copper and nickel using sewage sludge of eon trolled n-ietal eon tent. Eni'ironwental Pollution (Series B), 8, GOODMAN, G. T. & ROFIRKT.S, T. M. (1971). Plants and soil as indicators of metals in the air. Nature, London, 231, HUTCHINSON, T. C. & WHITBY, L. M. (1974). Heavy metals pollution in the Sudbury region of Canada, 1. Soil and vegetation contamination by nickel, copper and other metals. Environmental Conservation, 1, 123' 132. INGUS-IAD, T. (1959). Studies on the nutrition of forest tree seedlings, 2. Mineral nutrition of spruce. Physiologia Phwtarnm, 12, 56S 593. MAI-NUOL, R. I). & Bi-fKETT, P. 11. T. (1985). Critical tissue concentrations of potentially toxic elements. Phmt and Soil, 85, MoDGAN, E. (1983). The influence of heavy metals upon tree growth in South Wales forests. Ph. D. Thesis. The Polytechnic of Wales, Pontypridd, S. Wales. SAITO, H., HANADA, S. & MofHizuKi, T. (1973). Kffect of cadmium on the uptake of zinc. Bakujutsii Kotiudu. 21, UNDHRWOOD, \l. J. (1977). Trace luements in tlunian and.4nimat Nutrition, 4th edn. Academic Press, New York. WALLACE, A., ROMNEY, K. M., ALEXANDER, C;. V. SOUM, S. M. & PATHL, P. M. (1977). Some interactions in plants, other heavy metals and chelating agents. Journal of Agronomy, 69, Wi:imiiR, J. (1981). Trace metals in agrieulture. In: t'lifect of tieavy Metal Pollution on Plants, vol. 2, (\Ld. by N. W. Lepp), pp Applied Science Publishers, London.

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