Chemosphere, Vol.14, No.

5, Printed in Great Britain

pp 417-426,

1985

0045-6535/85 $3.00 + .OO 1985 Pergamon Press Ltd.

C A D M I U M UPTAKE BY THE W A T E R HYACINTH: EFFECTS OF ROOT MASS, SOLUTION VOLUME, COMPLEXERS AND OTHER METAL Department

IONS 1

James K. Hardy* of Chemistry and the Center for Environmental Studies The University of Akron, Akron, OH 44325 USA and David H. O'Keeffe* Department of G h e m i s t r y The University of Michigan-Flint, Flint, MI 48503 USA ABSTRACT

At a fixed Cd 2+ concentration water hyacinth (Eichhornia crassipes) plants with greater root mass (dry weight) take up more metal ions as a function of time, and more metal ions are taken up by a plant as the solution volume is increased. Experiments in which several different metal ion complexers were present suggest that (i) the roots possess sites which initially reversibly bind free Cd 2+ ,C ~ , ( ~ ) w ~ e t ~ e ~ d ~ m ~ r ~ o C ~ e C ~ o ~ o o ~ 3 e i ~ l ~ a ~ 3 a ~ t i ~ 3 e ~ h ~ O ~ o ~ e e tissues e f f e c t i v e l y removing it from the e q u i l i b r i u m processes in solution. Many metal ions are taken up by the plant but only the m i c r o n u t r i e n t Zn 2+ competes well wihh Cd 2+ for uptake. Thus, there may be binding sites on the roots for specific metal ions.

INTRODUCTION

A number of laboratories (Eichhornia crassipes) tiated a systematic

have examined

the ability of the water hyacinth from water. 2-9 to investigate We recently inifactors which affect factors of imporpollution ions competing metal

to remove heavy metals In continuing ability mass,

investigation

of a number of solution

cadmium uptake by the plant. 10 tance to the water hyacinth's control device, and c o m p l e x i n g

to be an effective biological solution volume,

the effects of root

agents have now been evaluated.

The results have also allowed us

to gain some insight

into the metal uptake process. EXPERIMENTAL

The tracer used for most studies tained as a carrier-free solution

to monitor cadmium uptake was 109Cd 2+, ob(New England Nuclear Co.).

in 0.i M HCI

417

418

Cadmium solutions were prepared tific).

from reagent grade C d ( N O 3 ) 2 . 4 H 2 0

(Fisher Scien-

All other c h e m i c a l s used were r e a g e n t grade w h e r e possible and deioniFor studies e m p l o y i n g i09Cd2+ as a tracer, a Nu-

zed w a t e r was used throughout.

cleus Model 2010 single channel a n a l y z e r / s c a l e r (TI) w e l l - t y p e s c i n t i l l a t i o n d e t e c t o r was used. Elmer M o d e l 4000 atomic a b s o r p t i o n spectrometer,

e q u i p p e d with a 2 x 2 inch NaI For the volume study a Perkine q u i p p e d w i t h a cadmium elecThis instrument was al-

trodeless d i s c h a r g e lamp, was used to d e t e r m i n e cadmium.

so e m p l o y e d in the assay of the metals u s e d in the c o m p e t i n g ions study. W a t e r h y a c i n t h s were grown in the l a b o r a t o r y in h a l f - s t r e n g t h Hoagland's i0 For most e x p e r i m e n t s mature plants of apin age were used. Plants

s o l u t i o n as p r e v i o u s l y described.

p r o x i m a t e l y one gram dry w e i g h t and about three weeks v a r y i n g in size and age w e r e as a f u n c t i o n of root mass. used in e x p e r i m e n t s

d e s i g n e d to test c a d m i u m uptake

E x p e r i m e n t s w e r e p e r f o r m e d in a laboratory m a i n t a i n -

ed at a b o u t 22C and at c o n s t a n t r e l a t i v e humidity. W i t h the e x c e p t i o n of the volume study, were variations in cadmium c o n c e n t r a t i o n

f o l l o w e d by d e t e r m i n i n g the s o l u t i o n 109Cd2+ a c t i v i t y at regular intervals. c o n d i t i o n investigated, i n d i v i d u a l plants were p l a c e d in

For each e x p e r i m e n t a l

bottles c o n t a i n i n g d e i o n i z e d water,

the d e s i r e d a m o u n t of c a d m i u m and s u f f i c i e n t For

tracer to a l l o w for an initial a c t i v i t y of at least 1500 cpm/5ml aliquot. m o s t studies 500 ml bottles (6 cm in diameter, 14 cm in height)

c o n t a i n i n g a to-

tal of 400 ml solution were used.

For the volume study glass containers of from

500 to 2500 ml total volume c o n t a i n i n g 400 to 2000 ml of s o l u t i o n were used. Each study was done at least in triplicate. was withdrawn, At regular intervals a 5 ml a l i q u o t solution. For those

a n a l y z e d and r e t u r n e d to the e x p e r i m e n t a l

samples a s s a y e d by atomic a b s o r p t i o n s p e c t r o s c o p y , a l i q u o t was returned. As p l a n t t r a n s p i r a t i o n

any r e m a i n i n g portion of the (about 50 ml/day), the so-

is h i g h

lution volume was r e g u l a r l y m o n i t o r e d d u r i n g the e x p o s u r e p e r i o d and a d d i t i o n a l d e i o n i z e d w a t e r a d d e d as n e e d e d to m a i n t a i n a c o n s t a n t volume. The effects of

various factors on c a d m i u m uptake were s t u d i e d by v a r y i n g a single factor in solution, then m o n i t o r i n g the s o l u t i o n c a d m i u m level. In all studies a c a d m i u m is rapid and no

plant damage

c o n c e n t r a t i o n of 1 ppm w a E used. i0 is observed.

Uptake at this c o n c e n t r a t i o n

RESULTS Uptake as a F u n c t i o n of R o o t Mass. A total of forty plants w e r e e x p o s e d to Cd 2+ in d e i o n i z e d water and monitored for their ability to take up the metal. e x a m i n e a w i d e range of r o o t masses. r e m o v e d from solution, grams Cd 2+ r e m o v e d versus root mass relationship, 24 hours while not linear, After The plants w e r e s e l e c t e d so as to

4 hours of e x p o s u r e the plants w e r e dried and weighed. (Figure la) A plot of m i c r o indicates that a At

their roots removed,

(mg dry weight)

exists between root mass and c a d m i u m uptake.

(Figure ib) a l e v e l i n g effect becomes a p p a r e n t as c a d m i u m is d e p l e t e d

419

from solution

(400 mg total).

Values at 48 hours

(not shown)

indicate that this

l e v e l i n g effect becomes even more pronounced.

4(]0
X X

400. g : x xx,~-' xXx~'~'" x ~D "'" x "'" x " " X ,X X X X 4X C X X

x x

300
X l

d
l

3BO.
x XXx

200

2BO. I

x /x x* 100-

x/
0

E
0

(3

10B

200

300

(3

1BO

200

:300

Roo%mass (mg dr W ~elgh%]

Roo%mass (mg d r g

~eigh%]

Fi@ure ~.

M i c r o g r a m s c a d m i u m removed at root mass.

(a) 4 h and

(b) 24 h as a function of

Uptake as a F u n c t i o n of S o l u t i o n Volume. Plants were e x p o s e d to c a d m i u m solutions of volumes and 2000 ml. 400, 800, 1200, 1600,

For this study a t o m i c a b s o r p t i o n s p e c t r o s c o p y was used to m o n i t o r As obser-

c a d m i u m uptake as the q u a n t i t i e s of tracer r e q u i r e d were prohibitive. ved in Figure 2, a p p a r e n t l y linear r e l a t i o n s h i p s

exist b e t w e e n solution volume As

and the total amount of c a d m i u m r e m o v e d at 4, 24 and 48 hours of exposure.

the e x p o s u r e time increases the slope of the line also increases w h i c h is consistent w i t h our earlier results showing i n c r e a s e d metal 10 posure times. ion uptake at longer ex o

Uptake in the Presence of Complexers. One p r o b l e m a s s o c i a t e d w i t h c o n v e n t i o n a l m o v a l arises t r e a t m e n t methods for o a d m i u m reII from the p r e s e n c e of c o m p l e x i n g agents. These species can act to m a k i n g it impossible to remove the metal from

mask the p r e s e n c e of the metal, solution.

A n u m b e r of c o m p l e x i n g agents were i n v e s t i g a t e d to d e t e r m i n e their ef. In i n c r e a s i n g a f f i n i t y for

fect on c a d m i u m removal by the w a t e r hyacinth.

420

/
1600g
X

I00-

80
t200 d 6,0

R
800 E M 0 v
p-

400

20
o

5'00

1000

1500

2000 0 0
I '2

50LUTIC;~ VOLUME (ml)

2'4
EXPOSURE rIME (h)

3'5

4'8

Figure ~. Uptake of c a d m i u m as a f u n c t i o n of s o l u t i o n volume (+=4h, o= 24h, x= 48 h)

Figure 3. C a d m i u m uptake in the p r e s e n c e of c o m p l e x i n g agents (+= control, ,=NTA, o= HEDTA, x= EDTA =CDTA). A v e r a g e of 6 plants per c o m p l e x e r studied.

cadmium,

the complexers used were: acid

acetate,

glycine,

histidine,

s a l i c y l i c acid, -triaand

nitrilotriacetic cetic a c i d

(NTA), N - ( 2 - h y d r o x y e t h y l ) e t h y l e n e d i n i t r i l o - N , N ' , N ' acid(EDTA),

(HEDTA), e t h y l e n e d i n i t r i l o - N , N , N ' , N ' - t e t r a a c e t i c acid

trans-l,2-cyclohexylenedinitrilotetraacetic 1.70, 4.22, 5.39, 5.55, 9.80, 13.1, 16.28,

(CDTA) with log K 1 values of Phos-

and 19.84 respectively. 12,13

phate was also included in this set. ble c a d m i u m complex, essential nutrient Initial

While this species does not form a solufor p r e c i p i t a t e f o r m a t i o n and is an

it has the p o t e n t i a l

for the plant.

studies were c o n d u c t e d by a d d i n g s u f f i c i e n t a m o u n t of c o m p l e x e r to In e a c h case, the c o m p l e x -

r e p r e s e n t a i:i molar ratio of c a d m i u m to complexer.

er was a d d e d to a 1 ppm c a d m i u m s o l u t i o n and the r e s u l t i n g solution a d j u s t e d to pH 7 w i t h either 6 M KOH or HNO 3. of acetate, glycine, histidine, As c o m p a r e d to a control plant the p r e s e n c e or p h o s p h a t e had no e f f e c t on

s a l i c y l i c acid, NTA, HEDTA,

c a d m i u m uptake

(data not shown).

E D T A and C D T A each acted to signiin Figure 3. As can be seen,

ficantly reduce the rate of c a d m i u m uptake as shown

the percent c a d m i u m removed at all time p o i n t s d e c r e a s e s as the log K 1 for the c o m p l e x e r increases, phase (0-4 h). w i t h the effect most a p p a r e n t on uptake in the fast uptake

As log K 1 increases there is a c o n c o m i t a n t r e d u c t i o n in the exIn all cases though, the rate of c a d m i u m uptake

tent of the fast uptake phase. in the s~ow phase

(> 4 h) is about the same.

421

The effect of c o m p l e x e r c o n c e n t r a t i o n on uptake was also briefly examined. N T A / C d 2+ molar plants) lative ratios of 0.i:i, i:i and i0:i y i e l d e d average (based upon six

p e r c e n t Cd 2+ r e m a i n i n g at 48 hrs of 2.9, to control plants not e x p o s e d to NTA.

28.9 and 64.4 r e s p e c t i v e l y rethe

These results help corroborate

idea that the roots only take up free Cd 2+ in solution.

C a d m i u m Release In our p r e v i o u s study we r e p o r t e d that plants initially exposed to c a d m i u m

for 48 ~ o u r s and then t r a n s f e r r e d to a Cd 2+ free solution e x h i b i t e d no significant release of the metal. I0 To d e t e r m i n e if the presence of complexing agents plants were initial-

could result in the release of p r e v i o u s l y taken up cadmium,

ly e x p o s e d to 1 ppm cadmium for 24 hours and then t r a n s f e r r e d to solution containing 9 x 10 -5 M CDTA, EDTA or HEDTA. m o l a r ratio of c o m p l e x e r to Cd 2+. nally removed ratio versus time This c o n c e n t r a t i o n r e p r e s e n t e d a i0:i

A plot of the solution c a d m i u m / c a d m i u m origi-

(Figure 4) shows that the larger is the log K 1

of the c o m p l e x e r used the greater is the amount of Cd 2+ r e l e a s e d in the 8-24 hour time frame up to a m a x i m u m of 60% of the c a d m i u m o r i g i n a l l y taken up by the plant w h e n C D T A was the c o m p l e x e r present in the solution. appears to be a mirror image of the fast uptake phase This initial release This

for the metal. I0

rapid release phase is then followed by a slow re-uptake of the metal at a rate comparable to the initial slow uptake phase. represents

Since CDTA, with a log K 1 of 19.84 for formation of Cd(CDTA), .8 -

.6

O I1

m ~

--I ~ X
~ e

/
.2

I /
I

,
I
24

Exposure Time [hI

Figure ~. C a d m i u m release in the p r e s e n c e of c o m p l e x i n g agents (+=control, =HEDTA, x=EDTA, I = C D T A ) . Ratio = micrograms Cd 2+ in solution per m i c r o g r a m s Cd 2+ o r i g i n a l l y removed. A v e r a g e of 6 plants per c o m p l e x e r studied.

422

a1
i

.i+I---- ~ _ _ ~

A~&

& .,...._

.2

I
24

I
48

I
72

Exposure Time [h]

C a d m i u m release in_~he p r e s e n c e of C D T A after various Cd 2+ e x p o s u r e Figure 5. R a t i o = m i c r o g r a m s Cd Z in s o l u t i o n per m i c r o g r a m Cd 2+ o r i g i n a l l y reperiods. A v e r a g e of 6 plants moved. (A=I day, I=2 days, = 3 days, X=4 days, + = 5 days). per initial e x p o s u r e period.

the strongest c o m p l e x e r i n v e s t i g a t e d and d e m o n s t r a t e d lease potential, its a b i l i t y to remove time. the metal

the .greatest c a d m i u m re-

from plants was m o n i t o r e d as a

function of c a d m i u m e x p o s u r e for from 1-5 days, f u n c t i o n of

Plants were i n i t i a l l y e x p o s e d to 1 ppm Cd 2+

s u b s e q u e n t l y e x p o s e d to 9x10 -5 M C D T A and then m o n i t o r e d as a As seen in Figure 5, there is a decrease in (as c o m p a r e d

time for c a d m i u m release.

the m a x i m u m a m o u n t of c a d m i u m r e l e a s e d in the 8-24 h o u r time frame to the a m o u n t i n i t i a l l y removed) with increasing

initial c a d m i u m e x p o s u r e time.

E f f e c t of Metal Ions on C a d m i u m Uptake. It has been r e p o r t e d that the p r e s e n c e of o t h e r heavy m e t a l s results d e c r e a s e in c a d m i u m uptake. 4'5 in a

We have a l r e a d y shown that the p r e s e n c e of an inIn or-

c r e a s i n g c o n c e n t r a t i o n of zinc ion results in d e c r e a s e d c a d m i u m uptake. 10 der to further i n v e s t i g a t e

this e f f e c t plants were e x p o s e d to cadmium solutions in-

c o n t a i n i n g v a r i o u s metal ions r e p r e s e n t i n g a range of ionic radii and charge, c l u d i n g m e t a l s o c c u r r i n g n a t u r a l l y in water.

S o l u t i o n s were p r e p a r e d which con-

tained 1 p p m cadmium and s u f f i c i e n t metal to r e s u l t in a 10:1 m o l a r ratio of metal ion to cadmium. This ratio w o u l d be e x p e c t e d to make any effect in cadEach metal was used as its n i t r a t e salt and the The lower pH value was used if p r e c i p i t a t e

m i u m uptake r e a d i l y apparent.

solutions w e r e a d j u s t e d to pH 6-7. f o r m a t i o n was evident.

E a r l i e r w o r k has shown that this d e c r e a s e in pH does not

423

significantly 24 hours removed in

alter c a d m i u m

uptake . 10
_

The p e r c e n t as well

decrease

in cadnLium u._aJ.e at ~ i, of these m e t a l s in Table I.

hhe p r e s e n c e

of these m e t a l s

as the p e r c e n t

from s o l u t i o n

by the w a t e r

hyacinth

at 24 hours

are r e p o r t e d ~ounts

As can be seenr the c o m p e t i n g decrease

the p l a n t was present, uptake

capable but only

of r e m o v i n g

sicnificant

of each of

metals

in the case of zinc ion was a d r a m a t i c

in c a d m i u m

observed.

TABLE E f f e c t of C o m p e t i n g Competing Ion ca2+ Fe 3+ Hg 2+ Co 2+ Cu 2+ Ni 2+ Mg 2+ Na + Zn 2+ Cd 2+b aRange for 3 plants Solution DISCUSSION Concentration (ppm) 3.6 5.0 17.9 5.3 5.7 5.2 2.2 2.0 5.8 1.0

I Uptake % C o m p e t i n g Ion Removed = 38.0 - 40.7 29.3 - 54.1

Ions on C a d m i u m

% D e c r e a s e In C a d m i u m Uptake a 2.1 - 5.5 3.7 - 5.9 6.0 - 7.0 6.9 - 7.1 7.0 -13.2 7.8-12.8 21.4 - 2 2 . 3 21.6 -25.4

36.5 - 59.8 32.0 - 58.0 24.0 13.5- 24.1 39.1

19.5 - 25.1 29.4 - 39.6

81.1 -82.5

30.7 - 31.8 71.2 - 73.8

bcontrol

The root mass ture

results remove

shown more

in F i g u r e cadmium

1 clearly

demonstrate per unit

that p l a n t s

w i t h greater the na-

from s o l u t i o n effect

time.

To d e t e r m i n e

of the a p p a r e n t

leveling

of c a d m i u m

uptake,

additional

experiments is var-

will n e e d to be p e r f o r m e d ied and time points tion of root surface these current results beyond

in w h i c h

the initial

concentration Monitoring

of c a d m i u m uptake

24-48 hours

examined.

as a funcHowever, sizes, and

area may also y i e l d more certainly indicate of taking

informative having

results. different

that plants

therefore tissues.

ages,

are all c a p a b l e

up and c o n c e n t r a t i n g

cadmium

in their

Increasing with increased are less

solution

volume

(fixed c d 2+ c o n c e n t r a t i o n ) in F i g u r e 2. However also

does the

correlate

well

Cd 2+ uptake

as shown

fact that that

the

slopes

than one at all

three

time p o i n t s

reveals

the uptake

424

efficiency decreases as the solution volume increases.

In terms of the overall

amount of Cd 2+ taken up it is noted that increasing the solution volume, which increases the amount of Cd 2+ to be taken up, results in increasing uptake per unit time. This demonstrates that the water hyacinth is able to effectively remove Cd 2+ from solution when the amount of metal small or quite large. The influence of metal ion complexers on the uptake process Cd 2+ bound to some type of site on the roots. tion. plant. (Figures 3 and 4) is consistent with an equilibrium existing between free Cd 2+ in solution and This equilibrium is being influin soluion present is either quite

enced by a competing equilibrium involving free Cd 2+ and that complexed

The results suggest that only free Cd 2+ is taken out of solution by the That is, as the amount of Cd 2+ in solution is steadily decreased by bethe amount of Cd 2+ re(Figure 3) that the i0 Notice however

ing bound to complexers having increasing log K 1 values, moved from solution by the roots decreases.

effect seems limited to what we have referred to as the fast uptake phase. ers.

The slow uptake phase does not seem to be altered by the presence of the complexThis seems logical as we interpret the slow uptake phase to be the process whereby the plant removes Cd 2+ from the equilibrium system into the interior of the root cells. Complexer effects appear to fall into three categories: have no apparent influence; (i) those which and (3) those

(2) those which partially eliminate;

which almost completely eliminate the fast uptake phase.

We believe the fast

phase to be due to the saturation of the metal ion binding sites on the roots. This idea is supported by our earlier finding that stirring the solution dramatically increases rate of uptake. 10 tate (1.70), glycine Complexers having small log K 1 values: ace(4.22), histidine (5.39), salicylic acid (5.55), as well as

phosphate,

do not effectively compete with the root binding sites for free Cd 2+ NTA, which has a log KI= 9.80, does compete somewhat with the root (Category 2). Finally, complexers hav(Ca(13.1), EDTA (16.28), and CDTA (19.84), appear to

(Category i).

binding sites for free Cd 2+ in solution ing large log K 1 values: HEDTA tegory 3).

compete much more effectively for free Cd 2+ than do the root binding sites is somewhat larger than the value for NTA HEDTA (13.1). Additional evidence

These data also suggest that the log K 1 for the root binding sites (9.80) and smaller than the value for

in support of the equilibrium involving free Cd 2+ and

that bound to the roots is based upon the results obtained in the Cd 2+ release experiments. As shown in Figure 4 the amount of Cd 2+ released from the roots This the With time, however,

is proportional to the binding strength of complexer present in solution. release also mirrors the original fast uptake phase. re-uptake even in the presence of the complexers.

Cd 2+ is removed from the equilibrium system by the plant as shown by the slow This conclusion is further strengthened by noting the results presented in Figure 5. The longer any given

425

plant is exposed to Cd 2+ prior to its being subsequently treated with a strong complexer the roots. (CDTA), the smaller is the amount of Cd 2+ that can be released from These observations are also consistent with the idea that while the eventually the metal

roots possess binding sites for free Cd 2+ in solution,

ions occupying those sites are translocated into the plant cells and thus removed from the equilibrium processes in solution. (Table I) only Zn 2+) ions compete The fact that of all the metal ions tested

well with Cd 2+ ions for uptake by the roots of the water hyacinth is consistent i0 with binding sites specific for these two metal ions as previously suggested. It is of course quite interesting to note that the water hyacinth took up sub2+ stantial amounts of every metal ion tested, including the heavy metal ion Hg Presumably the roots have binding sites for metal ions required for normal growth and maintenance of the plant, and some of those sites are prehaps utilized in the uptake of other metal ions ~e.g. Cd2+). The actual number of distinctly different metal ion binding sites on the roots remains unknown. Certain plants have 14 Consequently, the ability of any given plant

been found to be metal tolerant.

to function well as a biological pollution control device must also depend upon the ultimate fate of the metal ion(s) once they are tekan up by those plants.

ACKNOWLEDGEMENT

The assistance of A. Rao, R. Blanchard,

G. Hanley and M. Dearth in mainis greatly appreciated.

taining plants and in conducting some of the experiments

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Wolverton,

B.C.

(1975).

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Tokunaga,

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M.

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