is a common chelating agent used extensively in the analysis of

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            OF MOSSES



           DIRECTED BY

             MAY 19
               TABLE OF CONTENTS

SECTION                                 PAGE

Acknowledgements                          2

Abstract                                  3

Introduction                              4

Aims and objectives                       10

Experimental                              11




Recommendations for further study

Appendix 1

Appendix 2



               I would like to take this opportunity to extend my gratitude to all

               the people who helped me during my project work and over the

               last year in college , especially the following;

               Dr. who gave his time and help in abundance.

               The technical staff of Sligo I.T. science department.

               The library staff of Sligo I.T. .

               I would also like to thank the following ,my class for their

friendship and help, and for all the late nights out. To my housemates for all the

craic and more late nights. The wise man who shut Fureys down making us go

out even more to find a new pub. To the girls for having smooth legs even

though its a tough job. Of course anyone who had to put up with me .

               Finally I would like to thank my mother for all her help

encouragement and nagging over the last few years, it got me this far.

                  “think where mans glory begins and ends

                   and say my glory was I had such friends”

                              W.B. Yeats


               Using solutions which contain various different metal complexes

,the ability of mosses to bind the metals was examined. The ability of the moss

to bind metal was then examined when chelating agents were present in the

solutions, the effect of varying the chelating agents concentration was also


               All tests were carried out by monitoring the decrease in metal

concentration in solutions which were exposed to a quantity of moss. In the case

of metal complexes the samples were taken on varying dates after exposure, and

in the case of the chelating agents samples were taken hourly to examine effects

on the rate of metal uptake.

               A test was also carried out to examine if the moss would uptake

the actual chelating agents themselves.


               To understand why certain tests were carried out in this project

we must first understand the actual binding of metals to the moss surface. This

however is not a simple process in fact there is still much debate about the

process, some experts claim that the process involves cation exchange, however

there is evidence that this is not the case, this evidence does allow for the fact

that this process does play some part in the overall binding. Probably the most

likely process was put forward by Xue, Stumm and Sigg (1988) who conducted

tests on the metal binding ability of algae, they concluded that the process

involved surface complex formation between the algal surface and the metals.

               The above mentioned work indicated that the surface of a

biological molecule contains various functional groups such as carboxylic,

amino, hydroxo, thio, and many others which interact coordinately with the

heavy metals . This however still does not explain all the characteristics of metal

binding in mosses, the process is understood to involve an equilibrium, between

metal bound to the moss and metal in solution. This is thought to be a part of an

adsorption like process which closely follows the Freundlich adsorption


               Some of the experimental work conducted during this project

will attempt to clarify some of the theories involved in the metal binding of

mosses, one test will be aimed at seeing whether the moss can uptake the actual

complexing/chelating agents themselves.

               The first set of tests will be aimed at the ability of the moss to

uptake metals that are complexed by simple inorganic ligands. If the metal

which is complexed does not have a full coordination sphere it should then be

possible for the ligands on the moss surface to bind to the metal coordinately,

thereby binding the metal. If however the metal has a full or crowded

coordination sphere it should be difficult for the moss ligands o approach and

bind to the metal. Of course here the lability of the ligands bound to the metal

becomes important, if the ligands are labile then it is reasonable to expect them

to be ejected from the metal in favour of the moss ligands. Therefore we could

expect that metals which contain a full coordination sphere of strong (non

labile) ligands ,will not be bound to the moss by coordinate processes. If then

metals in this form are bound to the moss there must also be physical adsorption

processes occurring at the moss surface.

               A test will be carried out on one portion of moss to determine if

an equilibrium process is actually occurring. This will be achieved by

attempting to leach metals from the moss surface, if metal is leached from the

surface two conclusions can be drawn firstly that an equilibrium is present

between metal in solution and metal on the moss, proof of this is simple as in

clean water metal will leach from the moss surface to reestablish the

equilibrium. The second conclusion which could be drawn is that if an

equilibrium is present then metal must be physically adsorbed onto the moss

surface as well as metal bound to moss ligands, the equilibrium of course

would only effect physically adsorbed metal as the chemically adsorbed (bound)

metal would not be free to reenter the water as this would require bond breaking

which is highly unlikely to occur.

               The other important test conducted here was the examination of

the effect of chelating agents on the uptake of metal by the moss. The two

chelating agents used were EDTA (ethelenediamminetetraacetic acid) and

humic acid.

        EDTA is a common chelating agent used extensively in the analysis of

metals, the four oxygen and the two nitrogen atoms in the molecule bind to the

metal, the stability constants for metal EDTA complexes are high which

indicates that the EDTA chelate is non labile from most metals. This means that

the moss ligands should not be able to uptake the metal coordinately and once

more physical adsorption processes must be involved for uptake to occur.

       A variation on this test will also be used in that the concentration of the

EDTA will be varied. It is expected that the presence of the bulky EDTA

molecule will slow the rate of binding but may also ultimately reduce the total

uptake. From the test hourly monitoring of metal concentration should indicate

this. If the rate of uptake is slower and assuming that the bonds between metal

and chelate are not broken then the rate of adsorption of metal EDTA complex

is slower than the uptake of metal alone. Of course if the total uptake is lower

then this would indicate a physical adsorption whose equilibrium is more biased

towards metal EDTA in solution. In other words the moss would have a

preference to free metal.

        Humic acid was also used in a similar test . Studies conducted by

Aualitia and Pickering show that Lead and Copper sorbed onto humic acid are

non labile to anodic stripping voltametry however, there is an equilibrium

between metal in solution and metal sorbed to humic acid. In the context of this

test, it is known that the moss does not uptake the humic acid, therefore

depending on this equilibrium the rate of metal uptake will be affected. The

reason for this is that there is only a specific proportion of metal free in the

solution the amount being dependant on the bias of the equilibrium, as this is

taken up by the moss the concentration of free metal in solution is reduced and

the metal humic acid equilibrium is disturbed and metal is released from the

humic acid to reestablish the equilibrium this process continues until both

equilibria are in synch that is that the equilibria between the metal and the moss

and metal and humic acid are themselves in equilibrium, this should leave more

metal not bound to the moss, but also should slow the rate of uptake as the

equilibria balance each other.

        As the humic acid has different affinities for metals we should see

varying changes in the uptake across the metals, the metals to be used in this test

are Lead, Copper and zinc as these form water soluble complexes with humic

acid. These are uptaken by the moss to a considerable level these will also be

used in the tests involving EDTA so that a comparison of the two chelating

agents can be conducted.

        A test will be carried out using EDTA and metal leached moss to see

whether the moss is capable of uptaking the actual chelating agent itself and the

extent of the uptake of the EDTA. This test is to be used to examine if the

uptake of metals bound to EDTA is equivalent to the uptake of EDTA, this

could be used to determine if the metals made any difference to the uptake of

EDTA if of course EDTA itself is actually adsorbed. The term adsorbed is used

here as the EDTA molecule will be in anionic form, as the moss ligands

themselves are anions it should not be possible to bind coordinately with the

EDTA. This test should show if adsorption processes are occurring at the moss

surface. If this is the case then it would be reasonable to expect other anions to

be adsorbed on the moss surface. This can be reasonably expected as moss can

be best described as a “scavenger” taking up everything that is placed in solution

with it in order to survive. If this is occurring then the uptake of complexes may

not be governed by the uptake of metals but more by the uptake of the

ligand/chelating molecules.

        The pH of a number of solutions shall be measured the information

gained from this should be able to clarify the role of cation exchange in the

metal uptake process. As all the ligands mentioned contained labile hydrogen

atoms it is reasonable to assume that some of these protons will exchange for

metal atoms during the binding process thereby allowing the metals to bind

coordinately with the ligands. It is this that will be monitored in this test. If a

significant lowering in pH is monitored then it is reasonable to assume that this

cation exchange process is significant, however a slight lowering of solution pH

would indicate that cation exchange plays only a slight part in the metal binding

processes occurring at the surface of the moss.

        The above tests have been specifically chosen for two reasons firstly

they will show the effect that using complexed forms of metal will have on the

ability of moss to bind the metal. The tests will also aid in the understanding of

the processes involved in the metal binding of moss, the tests should indicate

some of the processes involved and prove that they are actually occurring.

                        AIMS AND OBJECTIVES

       The aim of this work is to examine the effect that ligands bound to

metals will have on the ability of moss to bind that metal, the study will also

encompass chelating agents and their effects.

       The object of this is to gain a further understanding of the processes

occurring at the moss surface, the work will elucidate which processes are

actually occurring and should be capable of indicating the extent and therefore

the importance of each of the theorised mechanisms.


        Much of the experimental portion of this project is fairly similar and to

avoid repetition a general outline of the tests will be given firstly.

        For the majority of tests a portion of moss is placed in a solution

containing either metal or metal complex. 10ml samples are then extracted from

the solution at predetermined or convenient times. The samples are then filtered

to remove any residual moss (to ensure that the instrument sample intake

capillary is not blocked). Once filtered the samples are analysed for the

appropriate metal using flame atomic absorption using a Perkin Elmer flame

atomic absorption spectrophotometer.


                Solutions containing 10 parts per million of metal are prepared,

500mls of these solutions are placed in a beaker which contained 600mls of

loose packed washed moss. Samples were then taken at days convenient to lab

usage. The metal complexes used were Zinc nitrate ( ), Copper chloride ( ),

Manganese chloride ( ) and


                For the purpose of these tests three metals were chosen those

were Lead, Copper and Zinc the chelating agents chosen were EDTA and

Humic acid. Solutions were then prepared from these, the working solutions

contained the following: each solution contained 10ppm of metal, 2,5 and

10ppm of chelating agent were then added to separate solutions. Roughly 30g of

washed moss were then placed into 600mls of each solution. Samples were then

extracted from each solution on an hourly basis for twelve hours, samples were

also taken at 24 and 30 hours after introduction.


               A portion of moss was placed into roughly 600mls of distilled

water at intervals samples were extracted. After each sample the moss was

washed and placed into fresh water and the process was repeated. The samples

were then analysed for a number of relevant metals.


               A portion of the moss from the above test was washed and placed

into contact with 500mls of a 0.1 molar EDTA solution after 24 hours four 20ml

samples were extracted and were analysed for EDTA content.

               The EDTA analysis involved the samples being titrated using a

0.1 molar Copper solution and an Erichrome Black T (EBT) indicator.



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