C:HAPTE~: V

D I SC:USS I ON

The response of algae to. heavy metal toxicants varies with

the metal and also with the algal species. Different

pa~ameters investigated in the present study also show varying

sensitivities of algae to heavy metals. Vocke (1978) observed

that the order of toxicity based on EC50 calc~lated from the

growth data was Cd > Se > Hg > As for ~nk!.§t.r-QQ~§!!!!:!§ Spa at1d Cd >

~!'£.~Q£.Q!~!:!§ Spa another order was Qbserved. Rachlin et ala

(1982a) reported the toxi.:ity in the o.rder Cu > Cd > Zn > Pb f·or

EricksQn et ala (1970) observed

different s~nsiti~ities of differe~t species of algae to. Cu.

Christensen and Nyholm (1984) documented higher toxicity of Cu to.

In the present study the

order of toxicity based on EC50s was found to be Ni > Cd > Cu >

Cr (Fig. 3) for ~n~£~§t.i§ Spa and Cu > Ni > Cd > Cr for §Qi~y!in~

Sp • (Fig. 11).

The EC50 values repo.rted by differe!1t autho.rs on different

species of algae vary widely. For example, Canto.n and 8looff

(1982) reported an EC50 48 hrs val ue f';)r !;b!.Q~~!.!.if! as 5. 1 ppm Cd.

In the present study an EC50 value of 33.5 ± 2.1 ppm Cd was

for the same time interval. For

for the same time interval EC50 obtained was

292

extremely high. The high EC50 values obtained can be due to the

high ionic strength and hardness of the medium (Brown 1968;

Sinley ~1 ~l·, 1974; Bellavere and Gorbi 1981; Canton and Slooff

1982; Stephenson, 1983) used in the present study (Table 2) a~d

also the high tolerance of cynophytes to the ,metal concerned.

Vocke (1978) also observed higher EC50 values for the cynophytes

than chlorophytes. The relatively more tolerance of cyanophytes

to high temp~rature than other planktonic group has been

demonstrated by Sharma (1985). The EC56 values of ~n~£~~ti~ for

Cu decreased from 52.77 ~ 1.78 ppm at 48 hrs to 1.90 ~ 1.01 ppm

at 120 hrs (Fig. 3). In case of Cd the decrease was from 33.5 + v

2.1 ppm (at 48 hrs) to 1.30 + 1.66 ppm <120 hrs). The higher

sensi:t;ivity of the unicellular species is indicated by the lower

EC50 value obtained for them than the filamentous

For exampl e, the EC50 for Ni at 1.20 hrs fort!ne£~~ti.2 was « 0.1

ppm while it was 16.52 ~ 1.39 ppm for §Qit:.!:!!.lnl!. This much

variation was not~obtained in case of eu for the two species.

For eu the values were 1.'30 ~ 1.01 ppm fetr 8.!:1e£~§ti.2 and 5.08 +

1.'33 ppm for §Qir.!:!lin§; at 120 hrs. In case of Cd the values were

1.30 + 1.66 ppm and 21.94 t 1.88 ppm respectively for the two

species. .Wi-th increase in exposure time there was a fall in EC50

valu~s during the first 24 hrs interval. For example EC50 48 hrs

for Cu for ~n~£Y§1!§ was 52.27 t 1.78 ppm while EC50 72 hrs was

6.03 + 1.24 ·ppm. With further increase in the duration of

exposure the EC50 values did not show a proportional decrease.

293

Similar was the case for Cd and Ni. §Qir.!:!.!.ine also behaved the

same way with all the metals. A number of workers have reported

a similar decrease in EC50 and LC50s within a short duration of

time (e.g., 24 hrs, 48 hrs), followed by the rate of change

de<:reasing sharply, thus the curve becoming asymptot,ic. Brown

(1973) has discussed this feature when she elaborated the

distinction between ~he quantitative response and the quantal

re-ponse in environmental toxicological studies. Khangarot

(1981) observed similar attaining of an asymptotic character by

the EC50 values for tel costs. During the fir~t 12-24 hrs there

was a fall in EC50 for Ni and Zn for all the four species of

telcosts studied by him. This feature, i.e. the toxicity curve

(EC50 vs. time) becoming asymptotic indicates an incipient lethal

level whi.:h is defined by Sprague {1969) as "that level of the

environmental entity beyond which 50X of the population cannot

live for an indefinite time".

ane£~§~i§ . grows faster than §eir.yline, the 'r' values for

the former during the present study being ~ 8.3 ~ 10-3 (Table 4)

and for the latter, ~ 5.1 ~ 10-3 (Tabl~ 13). The corresponding

doubling time was 76.8 hrs and 125.3 hrs respective-lye Growth

rate was found to vary with slight changes in growth conditions.

Compared to the survival ratio the growth rate is found to be

more sensitive to heavy metal contamination. For exampl e, with

2.0 ppm eu .the survival ratio of ene£Y§~i§ got reduced by 54.8X

within 120 hrs (Table 5), while the growth rate reduction for the

/

29~

same concentration was 85.51. (Table 4). Almost similar was the

situation in case of Cd, Ni and Cr as evident from Tables 4 and

5. In case of Ni (2.0 ppm) at 120 hrs the survival ratio got

reduced by 841., ~hile the growth rate was reduced by 184.31.. For

§Qir~!in~ also the situation was not different. Fisher and Frood

(1980) , DeFilippis (1 '381) Christensen (1984) ,

Christensen and Nyholm (1984), have also documented growth rate

to be a more sensitive parameter. Maestrini et ala (1984) noted

that -many of the bia-chemical reactions leading to growth are

very sensitive to alterations in light, temperature etc. So the

scope of comparison of growth rate is very much confined.

During ,the prsent st':ldy in most of the cases, when low

concentration of metal was administered to alg~e, an enhanc ement-

in grcMJth and net biomass' was observed. For e);ample, with metal

concentration less than 1.00 ppm an increased biomass and

survival ratio was obtained at varying intervals from O'hr

onwards. Only in case of §gir~!in~ treated with Cu and Cr this ;

situation persisted upt;? the end of the experiment (168 hrs)

(Table 14; Fig. 10). In case of Bn~£~§!i§ the enhanced growth

could be observed only with Cu and Cd (Table 5; Fig. 4). Itl all

the cases, after a short duration of higher growth, the process

gradually re.:eded and at the end of the experiment no ,

concentration regime resulted in higher biomass and growth rate.

The phenomenon of enhanced biological activity was ,observed in

, .

295

case of carbon assimilation and also chi a content in many of the

concentration regimes used in the study (Tables 24, 31, 38 and

45),

The stimulatory effect of subinhibitory level of toxicants

is known as Hormesis. Hormet i c actions were observedi n animal

and plants of wide phylogenetic origin exposed to different

'chemicals (Luckey .! ~!. 1'375; 1'380; Stebbing

1981 ; 1'381 ; Deviprasad and Deviprasad 1982;).

The increase in biomass with the lower concentrations may be due

to over reaction of the test organism to stress applied or an

adaptive mechanism which results in additional resi st anc e to

further exposure to toxicants. When the concent~ation of

toxicant reaches above the level at which the organism can

counter act a steady inhibition of this ability of the organism

resul ts CStebbi ng and P!?mroy -1978).

Alteratio~ in pH modifies the toxicity of. metals. The

wodifications vary with the species. and also with the metals.

" For example, Babich and Stozky (1983) noted enhanceroent in Cd

toxicity to different fungal species with increase in pH.

Michnowicz and Weaks (1984) observed decrease in toxicity with

increasing pH. pH affects Ca) the physiological activities of

organisms and Cb) the chemical fractionation of the toxic metal.

Decrease in pH from the optimum interferes with the metab':llic

a.::tivities, thus leading to inhibition of growth. When §Q!.r;.!:!!.i!1~

was inoculated at 6.4 pH growth was completely stopped upto a

c~rtain duration.

was -1.5 x 10-3 •

296

Growth rate for the time interval 0-144 hrs

With increasing time the algae regai'ned growth

(Fig. 13). During the time interval when there was no explicit

growth, the pH showed a gradual increase. With the addition of

hydrochloric acid to the culture medium a super saturatibn of the

medium with free COz c~n be expected, due to the reaction

occurring between NaHC03 (main carbon source in the medium) and

the acid. It also may lead to many oiher chemical reactions

lead~ng to deleterious effect on the algae.. The increase in pH

brought about in the medium by the alga (Fig. 13) may be due b:J

the release of organic exudates and also due to the dead cells.

The enchanced toxicity of metals at low pH in case of Cu and

Cd <Table 15) indicates the importance of the ioni c f':Jrm of the

metal (which will be predominant i~ acidic conditions) rather

than the hydroxide or carbonate form which are more dominant in

the alkaline conditions prevailing in the normal medium (pH 8.3).

Sylva (1976) reports that CuC03 , CuOH+ forms are the predominant

species at this high pH ranges in artificial sea water medium.

As the concentration of different anions and cations are more

in the case of medium used in the present study .carbonate and

hydroxide fractions of the metals are expfi

concentrations. Toxicity of different chemical forms of metals

are found to vary with target organis£fl. For example, Sunda ·and

Gui.llard (1976) documented that for Cu the· to'xi c i ty_ to

297

phyt,oplankton is highly r~lated wi th ionic Cu than total Cu.

Shaw and Brown (974) reported that both ionic Cu (Cu2 +) as well

as CuCO:s (dissolved) are important for Cu toxicity to rainbow

trout. In, case of other metals, vi·z. , Ni and Cr no drastic

changes like those in presence of Cu and Cd .:oul d be noted.

Babich and Stozky (1983) noted that in case of Ni while the

increase in pH reduced its toxicity, for Cu no clear trend could

be as.:ertained. All metals ~xcept Cu when administered at pH 6.4

resulted in higher survival ratio than pH 7.2. This s.ituatic)n

result~ because of the the alga being incapable of growing in

lc)wer pH regimes. But from the higher survival ratio observed

(Tabl e 14 and 15) it. is qui te reasonable to conclude that the

metals other than Cu show ,an antagonistic response to the pH

ef fect. The decreased toxicity of the metals at the lower pH

range is due to the hig~er competitiori b~tween the metalic

cations and protons which will be predominant at the lower pH

ranges. for the binding si tes ,on the cell wall. In case of Cu

this situation is not encountered du~ to the higher affinity of

the- metal 'ion to the organic binding si tes.

Decrease in metal toxicity in the presence of chelators is

widely observed.

toxicity of metal

Chelators like EDTA, NTA etc. decrease the

by way df binding with the metal ions and

decreasing their availability (Allen, 1980) • Algae themselves

make use of chelators in order to capture the nutrierit metalic

ions (Huntsman and Sunda 1980). During the present study the

298

survival ratio obtained when §QiLY!iD~ Spa was treat~d with 10.0

ppm of Cu, 9d, Ni and Cr were 55.1 ~ 5.9; 88.7 + 4.7; 73.2 ± 6.4

and 99.8 + 3.8% at 120 hrs (Table 16) in presence of EDTA, three

times in molar concentration that of each ITletal. T~lese- values

were quite- higne-r than when the- alga was tre-ated with 8.0 ppm of

tbe same- metal s wi thout EDTA (Tabl e- 14,). Binding of metal ions

by chelators depends on the co-ordination characte-ristics of the

metal specie-so The stabil~ty of complexes with cbelators, liKe-

EDTA increases with incre-ase- in .atomic radius and de-creases with

the charge incre-ase of cations with an oute-r she-II of 18

electrons (d 10 cations In case- of

transition metal cations (0-10 d e-Iectrons - Cu2 +; N°2+ 1 , etc. )

tbe stahi Ii ty may be independent on the nature of chel at i ngo agent

(Mellor 1979). Irving and Williams (1'348) and Mellor and Maley

(1948) reported higher stability of Cu with chelators than Ni and

Cd. Polydentate chel ators impart more stabi I i ty thatlfl'lOnode-ntate

ones. Jackson and Morgan (1978) examined the influence of

chelators on cbe-mical speciation, their influence on the- chemical

interaction between metals, and also the tr'ansport of the metal

ions to the surface and obse-rved none Df the proce-sses accounted

for the role of chelators. The ameliorating effect of chelator~

on metal toxicity is a well noted feature (Morris and Russel,

1973; 1982; Me Leese- and Ray, 1984) and many of the

cbelator compounds are employe-d in the-rapeutic uses (Catsch and

Harmuth-Hoe-ne- 1979). Hung (1982) states that efficiency of

, "

299

ch~lators to reduce toxicity depends on their strength in forming

stabl~ compl~xes.

-When an organism is treated with mor~ than one toxicant at a

time, the, response in many cases is different quantitatively or

qualitatively from that caused by a single toxicant (Finney 1971;

Anderson and d'Apollonia 1978; Babich and Stotzky 1983). Based

on the quantitative alterations 'induced by mixtures of to:dcants,

Finney (1971) distinguished the joint acti9ns into independent

joint action, similar joint action and synergistic action. It has

been reported that different pollutants applied together modify

each othet's toxicity and result in synergistic CBraek ~i ~!.

19i6; Say and Whitton 1977; Christensen et ala 1979; Babich and

stotz ky 1 '983b; Sell ers and Ram 1984), antagoni st,i c (Chr i st ensen

~i ~!. 1979; Finlayson and Verrue 1982; Babich and Stotzky 1983;

Sellers and Ram 1984; Roales and Perlmutter, 1974) or additive

(Th.::>mpson ~i ~!.

1984) actions.

1980; Shehata and Whitton 1982; Sellers and Ram

Potentiated ~ctions similar to synergist~c

actions of chemical pollutants wit~ non-chemical stress like

o:.-;ygen depletion (Neu-hoff, 1983) and starvati.:;)n CBaghdiguian and

Ri va 1985) were also reported for di fferent .::>rganisms. The

interaction betwe~n the metals added in combination are

concentration dependent, the ratio between the components

deciding the interaction (Finlayson and Verrue 1982). Babich and

Stotzky (1983) report that the mechanism of synergism is an area

in which very little study has been conducted. Brown (1968)

300

proposed the use~f toxicity units to predict the combined effect

mixtures. According to Colby (1967)'s method if the expected

survival ratio is higher than the observed, the interaction is

synergistic, if it is lesser, then antagonistic. In ,:ase of Cu x

Ni combination for ~Q~£~§li§ Spa in almost all the concentration

ranges. studiedCTable 6) the expected ratio was always lesser

than the observed ones indicating an ameliorating effect of the

metals ~hen treated together. In case of §Qi~YlinA sp~ the

observation was opposite except in combination of 4.0 ppm Cu x

0.1 to 4.0 ppm Ni after 72 hrs of contamination (Table 19). For

&.J~£~§~i§ Spa the expected ratio was higher than the observed for

Cd x Cu combination (Table 8). For this combination, as Cu

concentration incresaed, the observed ratio gradually became more

than the expected ones. For §Qi~YliQ§ sp. in the case of Cu x Cd

combination (Table 18) in all the concentration ranges studied

the expected valu~ was higher than observed values. While for

for Cu x Cr combinations the expected and observed

survival ratios ~ere quite closer to each other,

Spa the former was much higher than the latter (Table 9 and 20).

When An2£l!"§ti§ Spa was treated wi th Cu x Cr mi :dures, except for ,

the first 24 hrs and also for 0.1 ppm Cu x Cr combinations in all

other cases results indicating a slight potentiation of toxicity

were obtained. With Cd x Ni combiMation for~n~£~§ti§ except for

the first 24 hrs, always e~;pected values were lesser than the

observed ones (Table 10). For §Qi~Y!.in~ with Cd x Ni ccimbination

3Ul

in case of higher concentration only higher values were obtained

f'Jr the ey;pected 'survival ratios thati the 'Jbserved ones. With

Cr y; Cd comb ina t i on s f or ~tlS\.£.Y.§ti.§' sp • an ameleorating effect

during the first 24 hrs was observed. With ~igher concentration

c'Jmbinat ions (4.0 ppm' Cr x Ni) of these metal s the expected

survival ratio was higher than observed ones indicating a

potenti~ting effect on toxicity of the metals (Table 11). The

same ~ombination showed uniform higher expected survival rat i 0

than observed ones in case of §Q.i.r.h!ii.tlS\. Spa (Tabl e 23). Lesser

expected values than observed ones were obtained in case of

with Ni x Cr combination (Table 12). In case of

§~itY!itl~ sp.' however the situ~tion was reverse (Table 22).

Antagonistic interactions of toxicants are the results of

the competitive interaction or some other physiological phenQmena

effected by the toxicant. For example .. Stratton (1985) observed

antaqon~stic action between a py~ethroid insecticide, - , pi::-rmithrin

'and S-triazine herbicide, atrazine and Hg and concluded that this

result was due to the membrane effects of Hg. Hg ion shows high

capacity to solubilize cell membrane, and this may interfere

with the uptake processes of the insecticide resulting in a

reduction of the toxic effects Qf the insecticides. The

mechanism of synergistic actions'are little known (Stratton 1985;

Babich and St6zby 1983b). Eisler and Gardiner (1973) supposed

that the alteration ,induced by one toxicant on the solubility 'Jf

302

the other, resul ting in .::hange of uptake pro.:ess may be

responsible for the interaction observed' between pollutants.

This may be possible mainly in the environmental phase of metal

interact ion. It is reported that many of the neutrient compounds

help in making binding sites available to the metal toxi cants.

For example, Skaar ~t. 2!.. (1974) have reported the relation

between phosphate content and Ni uptake. The synergistic action

can be affected by different possibilities like the affinity to

different binding sites and differenc~ in toxic action,

enhancement of permeability to ohe toxicant by the presence of

another, the si tuation of stress and strain (as observed in ca'se

of oxygen depletion or starvation and metal toxicity - Neu~hoff,

1983; Baghdiguian and Riva, 1985; Pascoe and Woodworth, 1980) on

the physiological system etc. If there are different binding

sites for each toxicant so that there is no cc.rrapetition between .

the toxicants for sites, ~nd the effect of toxic action due to

each toxicant is similar, then the result may be similar

response addition. Toxic effects of synergistic nature also can

be due to interactions between the toxicants at the sites of

detoxification (Ande.rson and d'Apollonia 1978).

principle photosynthetic pigment present in

cyanobacteria is chlorophyll a. The accessoTY pigments reported

from the, group are phycocyanins and phycocythrins (Evans ~t. ~!._

1983). Cyanobacterial. photosynthetic systems are identical to C3

pI ant s. They also show some characteristics of C4 plants like

3ua

reduced rates of photorespiration, low carbonic anhydrase

activity and higher level of phosphoenol pyruvate carboxylase

activity than Ca plants (Coleulan and Coleman 1981). The main

differences between the photosynthetic systern of cyanobacterium

and that of eukaryots is the absence of lamellar stacking. The

lamella is found irregularly throughout the cytoplasm or around

the edge- of the cells in concentric shells, and the light

harvesting antenna of photo-system II has no ChI b. The

phycobilisomes are attached to the outer surfaces of thylakoid

-membranes (Bower and Bendall 1983). The unicellular species

~!J§f.:i§!.i§ contain more ChI a than filamentous species. In case

of ~n~f.:i§!.i§-the ChI a cont~nt observed in the ~tudy was: D.74%

of total dry weight and for §QirY!in~ it was: 0.42%. A slightly

higher ratio of carbon assimilation to ChI a content

(assimilation efficiency) was also seen in case of a~~£~§ti§

(Table 38 and 45). ~n~£:i§ti§ showed higher instantaneous growth

coefficient and lower doubling ,time thatl §Q!..r:..~l.!..nfa (Table- 4 atld

13). Piorreck and Pohl (1984) reported gradual decrease with

time in ChI' a content

In case

of blue green alga they reported ~ 5 fold-lower ChI a cont tmt

than green algae. Piorreck ~t fa1... (1984)

observed alteration in ChI a content with the ni trogen

concentration of the media. In media with 0.10% nitrogen content

they reported ~ 0.92% and ~ 0.5% of total dry weight of ChI a

30~

conte-nt for !!tl~£..:t.§t!.§ arid §Q!.r:.!:!!.!.tl~ re-spe-ctive-Iy. ChI a conte-nts

observed in the present study was significantly lowe-r than that

report ed by Pi orr ed~ ~t ~!.. (1'384), eventhough the- nitrogen

conce-ntration prese-nt in the medium use-d in the study was highe-r

(0.18Y.; Table 2 - med~um composition) than that re-porte-d (0.10%)

by Piorreck ~t ~!.. (1984).

A numbe-r of studies has been re-porte-d on the- alterations

induced in ChI a conte-nt and photosynthe-tic activities of alga by

the pre-sence of he-avy me-tals. Harr is et al. (1970) reported

inhibition of photosynthetic activity of algal specie-s by Hg in

ppb levels. Woolery and Le-win (1976) observed 75% re-duction in

photosynthesis of phaeodyctylum in presence of 10.0 ppm Pb.

(1983) reported Cd induced inhibition of ChI a

content and carbon assimilation of gbl§m~~2mQn§§ r§inb~r~!!. 1

uM (0.1 ppm~ Cd reduced ChI a c'J,.,tent by 3% wi thi n 3 hrs

exposure and 33% by 48 hrs exposure. Same concentrations of Cd

reduced the photosynthetic oxygen production by 13% by 3 hrs

exposure- and 23% by 24 hrs exposure. 80% reduction of

photosynthetic activity was observed by Kallquist and Meadows

(1978)· by 0.1 ppm Cu,. They reporte-d that the growth rate was more

sensitive to Cu than the photosynthetic oxygen production.

Steeman-Nielsen and WiumrAnderse-n (1971) observed higher

sensitivity to Cu of photosynthesis than growth rate in case of

gb12L~1!..§· Wium-Andersen (1974) observed lower effectiveness of Cr

305

in inhibiting photosynth~sis than Cu . In th~ pres~nt study .

photosynthetic activity (carb'on assimilation) was" found to be

more sensitive to metal than ChI a content. This is demonstrated

by the decreasing ratio of carbon assimilation to ChI a content

with increasing metal concentration (Table 38 and 45). In case

of ~n~£~§1i§ and §gi~Y1in~ inhibition of carbon assimilation was

• affected in the order Cu ) Ni > Cd > Cr. With low concentration

of metal an enhancement in carbon assimilation was observed in

case of certain metals us~d in this study (Table 38 and 45).

Thripathy ~1 91. (1 '381) reported that Ni inhibited the

electrone transport, and photosysterrl II is more sensitiv~ .than

photosystem I. Alteratiori in ChI a emission characteristics were

also observed by the same authors. Thripathy and Mohanty (1980)

and Thripathy ~t. 91. (1'383) observed the higher sensitivity of

photosyste~ II to Co and In. Disorganisation of grana on Cd

treatment and non-conmpetitive inhibition of photosynthetic

Cl402 fixation by isolated chloroplasts of Spinach by Cd and In

were also reported by Berry and Downton 1982). They reported

that Cd was almost do~ble toxic than In. Metal ions act at the

oxfdi zing si t es of illuminated chloroplasts. Increase in the

permeability of thylakoid membranes towards protons, thereby

inhibition of ATP formation also has been reported (Hipki ns

1'383). Rachlin ~f 91. (1982c) observed that hevy metals like Cd

and Ni altered the microstructure of thylakoids, which are "the ~

si~es where the cellular chlo~ophyll and carotienoid pigments are

localized. Wu and Lor~nzen (1984) have discussed about th~

higher s~nsitivity of photosystem II than photosystem I of

chIarella towards Cu. Unlik~ most of th~ studies distussed above

Wu and LOretlZen (984) conducted study on intact cells. Th~y

suggested that the inhibition of photosynthesis can be due to the

accumulation of photosynthates which in turn, is because of the

disturbance of the transfo~mation of thephotosynthates to other

cellular substrates, by Cu •. A possibility of displacement of Mg

atoms from the chlorophyll molecule by metal, resulting in

compounds like Cu-porphyr in,' and also the Cu:'-med i at ed

peroxidation of photosynthetic membranes or the inhibition of

energy cot1versi.::)n is, also discussed by Wu and Lorenzen (1984) •

When 8u~£~§ii§ and §Qi[y!iu~ were treated with heavy metals

under both illuminated and dark conditions higher inhibition .of

Chi a content was observed under illuminated conditions (Table 24

and 31). This was the situation when the algae were treated with

metals in combination also (T,,!ble 25 te:. 30; 32-37) • E:-:cept ions

to this general observation was noted only in case of the

inhibition of ChI a content by one metal in presence of 10.0 ppm

of the other metal. In the!:;e cases the slopes of metal

concentration vs ChI a content plots o.f th,~ illuminated samples

were equal or lesser to those of samples kept under qark

condi ti.::)ns (Fig. 21 to 34). Steeman-Nielsen and Wium-Andersen

(1971) noted high inhibitory action of Cu on photosynthesis of

307

diatomes at higher illuminations. Cedano-Maldonado and Swader

-(1972) observed pot~ntiation of Cu inhibition of photosynthetic

electrone' transport by light inten1.5ity. Baker (1982)

noted the light dependent itlhibitiot1 of electron transport with

Zn. They concluded that the light independent inhibitory action

of zinc was at the water splitting site (oxidizing side of

photosystem II) and the light dependent itlhibttory site was

between photbsystem I and II. They proposed that the light

dependent inhibition is related to the primary charge separation

across the thylakoid membranes resulting from the primary

photochemic~l activities of photosystem I and II. A ·possible

alteration in the metabolic turnover of ChI a can be affected by

metal ·exposure. Some processes involved in the synthesis as well

as degradations of the chldrophyll molecule i~ to a great exteht

light dependent. Enzymes like protochlorophyllide reductase

which are involved in the conversion of protochlorophyllide to

chlorophyll ide are light dependent enzymes (Baker 1'384) • The

conversi ()n of de novo phytochrome (Pr) to- the act i ve form CPfr)

is also a light dependent process (Colbert ~t ~l. 1'383) • Active

phytochrclme· increases the steady state levels of the mRNAs

speci fic for the Ribulose phosphate carboxylase and the light

dependent chlorophyll alb binding protein' by enhancing the

transcriptional rates (Steiumuller ~t ~l. 1'385). Itl case of

algae the expression of distinct genes can be coupled with the

blue-light receptor in the photosyntheti~ system (Kloppstech ~t

308

~!. 1984). As basic photosynthetic mechanism in blue-greens are

similar to that of higher plants (Fay 1983) the above cited

mechanisms can shed light on results obtained in ,the present

study. The possible reason for the lower inhibition of Chi a by

heavy metals under dark may be the light dependent inhibition o~

enzymes and other factors which gets activated by illuminati~n.

The displacement of Mg from the chlorophyll molecule by the metal

molecule leading to a change in the functional characteri sti cs

may also be a possible reason. These processes lead to an

alteration in the metabolic turn over of ChI a in the preSet1Ce of

taxi c metal s.

The ,slopes 'fl' of most of the best fitting curves between

metal concentration and ChI a content or carbon assimilation were

altered when the al~ae were treated with more than one metal ih

combination. Generally 'the slope with metal A in presence of

0.01 ppm of metal B was more than in absence of it except mostly

when the metal B happened to be .cr. This increase in slope can

be taken as indicative of the potentiative action of metal B (at

low concentration) on A. But as metal B concentration increases

the in~ercept of the equation (which is indicative of only metal

B toxicity) gets reduced along with the slope also getting

reduced. This si tuation was more prominent for carbon

assimilation in case of §Qi~yliD§ Spa (Table 45 to 51). This

reduction in slopes indicates that as the metal B concentration

is increased the inhibition due to A only is getting reduced and

309

I

lJith further increase in B it can reach a stage where reduction

in carbon assimilation is independent of the metal A

concentration.

Al teratiotT in the protei!l cont~nt was observed in presence

of di fferent metals in case of both 8n2£.i::§£i.§ 2m! :2Qi.r..!:!!.i.!:l~

during the present study. Bu·t the protein content was found to

be far

studi ed.

less sensitive to heavy metals than any other parameter

For example, in case of 8U§.h.i::§£i.§. Spa with 1.0 ppm Cu

the survival ratio was reduced to 68.3% within a duration of 120

hrs .... hile pro"tein content was only reduced to 85.6% by 144 hrs

(Tabl e 5 and 52). The order of inhibition of protein content for

~.l2£.i::§!:.i§sp.' was Ni > Cu > Cd > Cr'. The survival ratio was also

inhibited by the same order. In case of §Qir..!:!!.in2 sp, the

difference between sensitivity to metals of protein content and

survival ratio was much less than in case of 8n§.£~§£i.§. Spa (Table

52 atld 59). When the alga was treated with 1.0 ppm Cu for a 144

hr interval the survival ratio was 89% and ~roteincontent 91.1%

of control, at the end of this period. Carbon assimilation was a

more sensitive parameter than either protein content or survival

ratio to judge toxicity of metals to both the algae. The

potentiation or amelioration of toxicity in case of metal

combinations was

.:ontent reduction.

not cl~arly indicated in terms of protein

310

Heavy metal toxicants are found to interfere with many

biosynthetic pathways of protein. Gallaghar and Grey ( 1982)

reported on the Cd, interference with RNA metabolism in murine

lymphocytes. The inhibition of RNA synthesis by Ni was found to

be through its effect on RNA polymerase (Mushak, 1980) • In many

cases the exposure of organisms to hevy metals is fQund to

release some specific protein synthetic processes.

(1972) had observed synthesis of metal binding protein .when

~E£~1i2 Spa was exposed to Cd. They observed that this newly

synthesized protein was more or less similar to the

metallothionen proteins observed under similar condition in

higher organisms. Silverberg ~~ El. (1976) reported that on

exposure to metal (Cu) green algae develop intranuclear metal

protein complexes. Synthesis of metallothionen like proteins in

~b!.Qr.~!l~ J2:ir.~!J.QiQ9§~ is also discussed 'by Hart and Scai fe (1977)

and Hart ~~ E!. (197'3) • Many heavy metals have high affinity

towards the -SH group present in the protein moity. So high

concentration of metals may lead to the denaturation of many

enzymes and other proteins. Many metals show the capacity to

displace metallic prosthetic groups of the metalloenzymes (Mitra

and Bernstein 1980; Jacobson ~~ E!. 1983) thus resulting in

inactivation of the enzymes. Hence eventhough low concet1tration

of metals may lead to the release of some detoxification

mechanisms, c..

at higher concentration the denaturation capaity and A

-displacement capacity prevail thus resulting in confoYinational

311

changes, and·functional alterations, resulting in a net reduction

in protein content.

Algae show high capacity for metal uptak~f their surface

area per unit we~ght being one of the main reasons. The

cl::)ncentration factor varie~ with metals and algal species

involved. Concentration factors varying in the order of thousand

have been reported by a number of workers (Jennette ~i ~l. 1982).

Les and Walker (1984) noted concentration factors in the range of

3-4000 for Cu and Cd Dongmant1 and

Nert1berg ( 1'382) observed comparable range forI!:l~!.~§§i.Q§i.r.2.

!:'QtU!2 and Cd. In the present study the concentration factors

shown by both species for different metals were in the range of

An inverse correlation between concentration factors

and concentration of metal in the medium was observed (Table 66

and 81). Dongmann and Nurnberg (1982) and Geisweid and Urbach

(983) , and Les and Walker (1984) also observed a decrease in

concentration factor with increase in metal ~oncentration in the

medium. This indicates a saturation in metal uptake by algae.

In the present study metal absorption by algae followed' a

common relationship m = KCn em = metal conca in algae mol/gm;

C = metal conca in the medium mol/l; K and n constants) and n

was always <1.0 (e.g., Table 67 and 82). Rebhum,and Ben-Amotz

(984) and Geisweid and Urbach (1983) observe-d a similar relatil::>n

for Cd uptake for various species of algae. In case of ~!:llQr.~!l~

sp. Rebhum. and Ben-Amotz (1984) observed n > 1.0 indicating

312 _.

increase in metal uptake with metal concentration. l3eisweid and

Urbach (1983) observed 'n' values <1.0 and concluded that the

metal uptake by alg~e follows-a non-Lang~uir type of sorption.

In the present study the values of 'n' were always found lesser

thatl that observed by the above .:ited workers. The ionic strength

of the medium has been reported to influence the metal uptake by

algae (Jennett ~:t ~l. 1984; l3eisweid and Urbach 1983). The

medium employed in tHe present study has a very high ionic

strength (1 = 0.287).

Les and Walker (1984) and Davies (1973) reported that the

metal uptake by algae followed the Langmuir sorption isotherm.

Dongrtlann and Nurnberg (1982) fi tted metal uptake resul ts to the

relation K = Km_xC/(Kc + C) (K = metal concentration in the

alga.l biomass; K_x = maximum molality in the algal biomass;

= the saturation constant; C = metal concentration mol/I).

Geisweid and Urbach (1983) fitted similar relation to the three

species of algae they studied and Cd; and observed K ..........

(mol/cell) in the range of 1.0 x 10-1~ to 5.0 X 10-6 and binding

constant (satur~tion constant) in the range of 1.0 x 10-~ and 2.0

X 10-6 (mol/l). When the same equation was fited (without

assuming that metal uptake process during the long tiule exposure,

is exactly similar to the short term uptake process) to the

lresul t of the present study, Ni showed the hi ghest val ues of K~,..a ...

and K, whi Ie Cr showed the lowest values at 12 hrs of . e:t;posure

313

(Table 68). An increase in the K __ x and Kc values with exposure

time has been observed in the study for Cr and Ni. Cr+6 unlike

Cr+3 has only low capacity to get sorbed on particles I

(Pfeiffer

1980, 1982) • A clear trend of decrease was observed in

case of Cd. In case- of §~i~YliD~ Spa also the highest Km_K and

Kc: was obtained for Ni (Table 68).

The uptake process included the primary stage of sorption of

the metals to the algal cell wall and this process is basically a

passive process. Bentley-Mowat and Reid (1977) Geisweid and

Urbach (1983), Tobin g~ 91. (1984) , Bollag and Duszota (1984)

reported the ac~umulation of more metal by dead cells than living

ones. Davies (1978) suggested that the algal cell wall consists

of a mosaic of cationic and anionic exchange sites and the net

charge on the cell surface is rela~ed to the pH and other

properties of the medium. Starry and Kratzer (1984) concluded

that the algal cell wall behaves predominantly as polyfunctional

weakly acidic cation exchanger. After the preliminary binding

of the metal with the cell wall material, the transport of the

sorbed metal to di fferent metal binding si tes occur. This

involves many metabolic processes like synthesis of spe.:ial type

of proteins like metellothionein and polyphosphate bodies

(Peverly ~~ §1. 1978; Sickogoad and Stormer 1979; Baxter and

Jensen 1980; Jensen g~ .5!1. 1'382; 1984; Rachlin g~ .5!1. 1982,

1984) • Ih the present study metal accumulation with increasing

exposure periods did not show a clear time dependent alteration.

314

Even though the maximum absorption process occurs within very

short duration (Les and Walker- 1984; Davi es '1973; Geisweid and

Urbach 1'383; Hawkins and Griffith 1982) followed by a stagnation

in the process, long time exposure may lead to a gradual increase

in the accumulated metalCStarry ~i ~!. 1983) • This process may

be also related to the possible interference 01 the exclusion

mechani smeffecti ve in living or'gani sms.