OEDOGONIUM cardiacum exposed to varying concentrations of sodium cyanide for 15 day periods exhibited both morphological and physiological alterations. Organisms were exposed to the pollutant in concentrations of 1, 10, 25, 50, and 100 parts per million. Exposure period for organisms in each concentration was 15 days. As the concentration of the pollutant increased fragmentation also increased. Exposure also caused organisms to lose chlorophyll. The third morphological alteration was the incidence of rupture. Physiological effects altered by exposure included: reduced oxygen evolution, retardation of starch production and death. Death occurs when organisms are exposed to high concentrations over the total 15 day period

Sparks, E.

English

NASA Technical Reports Server

N79-19028
s
Paper No. 19
THE EFFECTS OF THE POLLUTANT, SODIUM CYANIHE, ON THE
MORPHOLOGY AND PHYSIOLOGY OF OEDOGONIUM CARDIACUM
Elbert Sparks, St/I/man College, Tuscaloosa, Alabama
ABSTRACT
OEDOGONIUM cardiacum exposed to varying concentrations
of sodium cyanide for 15 day periods exhibited both
morphological and physiological alterations. Organisms
were exposed to the pollutant in concentrations of i,
lO, 25, 50, and I00 parts per million. Exposure period
for organisms in each concentration was 15 days. The 15
day period permitted assays of effects caused by the
pollutant.
As the concentration of the pollutant increased frag-
mentation also increased• Exposure also caused organisms
to lose chlorophyll. There is a direct proportion of
chlorophyll loss to increased pollutant concentrations.
This is true for chlorophyll '_" and "b". The third
morphological alteration is the incidence of rupture.
Cells in the higher concentrations rupture and organelles
are lost.
Physiological effects altered by exposure include:
reduced oxygen evolution, retardation of sexual and
asexual reproduction, retardation of starch production
and death. Death occurs when organisms are exposed to
high concentrations over the total 15 day period.
INTRODUCTION
Cyanide has a long history of being a poison• (Betty 1976).
Many people have heard of cyanide as being the substance used in
gas chamber executions. However, ._anyare not a,Jareof the fact
that cyanide is a common water pollutant. (Berry 1974). Small
quantities of the pollutant occt.rnaturally in water, such as
that produced by millipedes _s a defense mechanisms against
predators. (Berry 1974). Much of the cyanide in industrial use
is commercially synthesi_ed. Cyahi_e consists of carbon and
_itro_en atoms joined by a triple bonC. The joining of these
atoms form the cyanide radical. The compound, referred to as the
pollutant. Sodium cyanide is used in this study. Compounds
containing the cyanide radical form a group of versat/la reagents
with many chemical aztd industrial applications. The cyanide
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https://ntrs.nasa.gov/search.jsp?R=19790010857 2020-03-20T18:12:35+00:00Z
pollutant enter industrlal waste streams from a variety of ,
processing industries such as extracting gold and silver ores,
synthetics manufacturing, coal cooking furnaces and electro-
plating.
The effect of cyanide on the morphology and physiology of
the green algae Oedogonium cardiacum may give some indications
of the effects on organis_ dependent on the algal plant for
food and oxyge11. The effects could be traced to fish and
ultimately man. Early studies indicate that concentrations of
cyanide as low as 0.1 ppm in water can kill fish. (Doudoroff 56).
This study will assess the degree to which diseases rather
than death will be produced in organisms utilizing the plant as
a source of food. In addition, problems will be defined,
hypotheses will be established and studies will be designed to
essess the effects experienced by food chain organisms.
Naterlals and Hethods
Oedo_onium cardlacum, a filamentous freshwater green algae
is the organism for study. Tt consists of several cells - per
filament. Each cell has a nucleus and single large chloroplast
of irregular net-like shape with many pyrenoids. The basal cell
is modified as a holdfast. Growth of the filament occurs as
various cells divide-mitotically.
Reproducciol. oc:urs both asexually and sexually. Asexual
reproduction is by tragmentation or by the production of zoo-
spores. Sexual reproduction is o_ the oogamous type. A filament
may coutain a large swollen cell, the oogoeium, and small
structures resembling zoospores in the antheridium. A sperm
(zoospore) from the antheridi_m swims to the enlarged oogonium
and enters through a pore In the wall of this cell and fuse with
the single egg contained therein. The diploid zy$ote germinates
producing four haploid zoospores each of which develops into a
new fila_nt.
The organism is grown in soil wa_er medium or Bold's basal
medium. Sodium cyanide is added to the medium in various con-
centratioms. The control contains only the medium while con-
centratlons of I ppm, IO ppm. 25 ppm, and I00 ppm constituted the
experimental groups, ppm dilutions indicate a weight colume
ratio of milligrams of sodium cyanide per liter of solution e.g.
mg/l.
Each concentration Is grown in conlcal.flasks for a period
of fifteen (15) days, The growth period is conducted in twelve
(12) hour light and dark periods. The light period coincide with
the daylight period. The temperature for the growth period is
2 3°C + 2.
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1979010842-204
Pa+ly observations are conducted wl_:, +._;_-'light microscopa.
The_ o_s,_rvations permit essessment o( ft..., _.4ation, cellular
rupture, _otmation of reproductive _t. ,,+:+,;r,. and l_ss of
cellu_a_ co,+,st ltuents.
Re_piratic_ r,_sures are cer.:. __,_ t_y the use of Warburg
apparatus. Each _oncentration is _t:._ _,;+_'ed to five (5) fifteen
(15) minute respiratory measures _,_il_ ior the fifteen (15) day
study period.
Chlorophyll extracclon ttch_qoes enable masures of
chlorophyll loss for the fifteen (I_) day perio<1. Chlorophyll
"a", '_" and total chlorophyll is measured by the spectrophoto-
met :r. Calculations based on spectrophoto_tric readinss pro-
vide data indicating the amount of chlorophyll loss during the
fifteen (I..5) day period.
Chromatographic assays are employed to determine the
presence of carbohydrates produced and stored in tt_ filaments
at the end of the experiment..ion period. Carbohydrates are
extracted and subjected to -,per chromatography for analysis of
the carbohydrates.
Discuss ion
Oedoxoniur,_ c is grown in either soll water me.dlum er Bold's
basal -_._iumcontaining sodium cyanide in concentration_ of 0 Pl_.,
l ppm, iO ppm, 50 ppm, an_ lOO ppm for a period of fifteen (15)
days. The growth period consists of twelve (12) hour dark and
t_ive (12) hour lil_htperiods. The temperature for the gros_th
perio_ is 23°C _+ 2 de_rees centrigra_Ie. Daily ob_ervations with
the light microscope indicates that as the concentrations of
pollutant increased fr,_mentation o_ the fila_nts increased.
Observation_ indicate that 1% of the tilaments in the control
(ppm) sttowed some _ra_mentation _hile _7. of the filaments
fragrr_ntedin SO0 pp,_concentration. Figure One (1) indicates
the percentage of _ragmcntation in the concentrations _tudled.
Cell fre£ments occurred in nll concentra,.lon_. However, the
frequency of _ragments increased with the increase of the
polluta_'.. Fra.,_mentatlonInclutlesbrea_Ir_ol _ingle cells o_"
fil_nts, breat_in_ of lil,::_e_ts, or atmorm_l L,_istin3of cells
causing the walls to ruptt_re and hre,d_. The incidence o_
fragmentation is n_re apparent in the cell:' _:ith the gre_ltest
expost, re.
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1979010842-205
f',
.9
Filament Fragmentation increased
as sodium cyanide concentrations
increased.
o
"_ .7
q j.
_ s
"4 .3
0
Oppm lppm LOppr, 25ppm 50 ppm lOOppm
Concentration of Pollu,:ant
Fig. I- Filament Frag_cntation
In :tr:_ _tion to fila_tcnt '. cd_t_ntation tire nt_la_>er ot cetL_
void o, p. oLoi_I_st in_r-_ase<_ ,_ _lle conceL_tt'ations illci'eA_c, ,
Few cells in the conLrot (:) p?_,_)f_a:-I,ace void of ceLLula."
cvns_ittent.,. However, an i,n_',.'ease in celts _q_tho_L protopl.'Yt.':
wexe vksi.',_le in cnncentldtiOtxc O_ )'J _pn; cmd 103 ppr_.
:tepL'odu,.tive ._3_l't'ctt|reL 1 _.eveLopLll_ on tiiaraents in t,ie
control _.,ere nt._:_ero_;s. Bot_ o.),onia rind .lntheri_ia are vI_L ,le
in the conLt'oL. .Se::rat tepro_'t,,:_ion £s apparent in the appe,.,_--
ance o_ antheri&a an_: oo,,oni.:_ on _i_u_:went_. Anthe_'idta .ti-e
in,'.ic,:ted L,'., _i_ort _:atLs formec in the cell pro£ucin.; sperra
ceE_J. _lte nkl,R_er Of CYO,_S _:2_l:. incYe_tbe ._ r,_Lu,,_t£on o. L,ze
anthe_idit;m L,:"_-s p_ace.
AI,jo t'eproduction cart ',e hotel! b,. the ._ppearance of L"e
oojoniu_. _ncreasea in the _ei)ru,!,,ctLon :;tructurcs indicate a
tendency for increase in off sp,in_;.
A_ cor,.:entrations increase the number o[ ou_._nia an,
anthertdta decreaaed, :' 7. of the filaments in the control ,:on-
tatn':d an antheridta or oogonia. In some instances ftta_enL_
contained !,o_.h antheridium an,: an oo;:onium. As th- concentra io.s
increased the ipcidence of reproductive _tructure_ hecrea_ed. The
100 ppm concentration shoed 17. of the [llamnts _ith reprod_.ct£v¢
2O4
1979010842-206
J i
structures. As sodium cyanide concentrations
are increased reproductive st_ctures
.9 on filaments decrease ,
.9
.8
U
o
Oppm ippm lOppm 25ppm 50ppm lOOppm
Concentrations of Sodium Cyanide
Fig. 2 - _productlve Structures
The percentage of reproductive structures decreased from
8 % to i % in concentrations of 0 ppm to i00 ppm.
Respiration of oxygen evolution is a measure of the
physiological activities of the cells. Measures were conducted
for five (5) fifteen (15) minute periods per day. The measures
are averaged to provide a measure of the oxygen evolved for that
day. The fifteen (15) day measures for 0 to i00 ppm are recorded
in Figure 3. Tne data indicate that the e_,ntrol organisms were
producing six (6) micco liters of oxygen on the tenth (i0) day
and the I ppn_ and the i0 )pm had ceased to evolve any measurable
amounts of oxygen on the tenth (I0) day.
205
P
1979010842-207
1.5 •
1.4 • d
1.3
i 6,1
1.1 , • Key Oppm lppm lOppm
I
-9 +
g .8 ,
0 06 d
m
•_ .4 + ///_ eg .3 " '/'_ • /U •
# e 41 -e • o • • --_
1 2 3 4 5 6 7 8
Time in Days
t
; ' Fig. 3 - Respiration Measures
Figure 4 contains data for 25 ppm, 50 ppm, and 100 ppm. The
50 ppm concentration indicates practically no oxygen evolved about
nine (9) days. On the nineth and tenth days almost five (5)
mlcrolitex-s of oxygen are evolved. This measure is not indi-
cative of the 25 ppm and I00 ppm. Oxygen is produced on two (2)
days and then decreases through the tenth (i0) days measures.
I,
.9=
.8 • Key 25ppm 50 ppm 100 ppm
• _ .6* I " j,
o ! I
> .5 *
.4e 1 %
/I:,..
• _2=__ .
W • • • 0 ILl • In •1.4
I 2 3 4 5 6 7
,,4
o Fig. 4- Time in Days
•
•+ {J
206
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1
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1979010842-208
The dsta indicate that oxygen production decreases as the
concentrations of the pollutant increase.
Chlorophyll is extracted at the close of ten (_0) day study
_riod. The extraction procedure provides data for chlorophyll
'_" and '_" and total chlorophyll. The _uure does not indicate
the amount of chlorophyll lost per day. The _asure is the
amount lost o_r ten (10) day study _ri_. Visible observations
indicate that chlorophyll is lost to so_ degree. The control
organisms are quite green wh£1e the organisms in the 100ppm con-
cen_ration are a white to m£1_ white color. Indicating chloro-
phyll has been lost during the expertJnentat£on period.
The chlorophyll is extracted aud _asures are _de using
the B & L spectrophoto_ter. Calculat£ons show that the control
organis_ contain .4 _ uf chlorophyll "a" per _r_ of tissue,
.5 mg of chlorophyll *_" per gram of tissue, and 8 mg of total
chlorophyll per gram of tissue. Organisms in the 10_pm lost
1.6 mg of chlorophyll '_" per gram of tissue, 1.9 mg of
chlorophyll *_" per gram of tissue and 2.1 mg total chlorophyll
per gram of tissue. Figure 5 include calculations for each
concentration for the study period.
• 2.2
2.1 •
2•
1.9 ,
1.8 • _ _
Z.7 •
1.6 •
i._ •
1.4
*
1.3 ,
1.2 •
I.I •
.9,
_ .8• --
°3Q
°2•
Oppm lppm lOppm 25ppm 50ppm lOOppm
Fig. 5 - Chlorophyll Lose
207
t
p
t
s 1 ' J,
1979010842-209
Spectrophometr_c measures are based on the pgicentage
transmittance. Therefore, the higher the bar graph the smaller
the amount of _hlorophyll present. As a result more chlorophyll
was lost from the organisms exposed to higher concentrations of
sodium cyanide. Chlorophyll extraction did noL occur until the
study was completed. However, visible observatlous on a daily
basis indicated that the I00 ppm concentration organ%sms were
void of much of their chlorophyll two (2) to three (3) days
before the other concentrations.
Findings
O. cardiacum exposed to sodium cyanide concentrations of
0 ppm, 1 ppm, l0 ppmp 50 ppm, and 100 ppm for fifteen (15) days
showed a varieLy of morphological and physiological changes.
Morphological changes that occurred include an increase in the
number of cells fragmenting as the concentration of the
pollutant is increased. Along with increased fregmentation, a
larger number of cells rupture and all cytoplasmic material and
organells are lost. Rupturing increases as the pollutant con-
centration decreases.
e
Reproductive structures occur in large numbers in the
growth medium containing 0 ppm of sodium cyanide. However, in-
crease in concentration causes a decrease in the number of re-
productive structures forming.
Physiologically, chlorophyll is lost from organisms ex-
posed to concentration of pollutants from I ppm to lO0 ppm. The
largest amount of chlorophyll is lost by the organisms in the
100 ppm concentration.
Carbohydrates are in smaller quantities as measured by
paper chromatography. The extraction procedure and analysis
technique yielded only small traces carbonhydrate material in
the control organisms. In concentrations of i0 to 100 ppm no
assessment of carbohydrate was apparent in chromatographis
analysis
Sodium cyanide in i ppm to 100 ppm cause a wide variety of
morphological and physiological effects on O. cardlacum.
Supported by NIH Grant No. RR-08021-04
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1979010842-210
BIBLIOGRAPHY
;-_ Berry, James _., David Osgood and Philip A. St. John. 1974.
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OF ISOPROPGL N-PHENYI CARBAI_%TE ON THE. GREEN ALGAE
OEDOGONIUM O%RDIACUM. J. Cell Biology. 63: 84.
Dodge, J. D., 1973. THE FINE STFJJCT_RE OF ALGAE CELLS. Aca(emic
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Dou3oroff, P., 1956. SO_E EXPERI_ENT:_ ON THE TOXICITY OF COMPLEX
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Ennis, '_1.B., Jr. 1948. SO_IE CYTOLOGICAL EFFECTS OF O-ISOPIIOYL
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Leedale, G. F., 1970. PHYLOGENETIC ASPECTS OF NUCLEAR CYTOLOGY
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Pickett - Heaps, J.D., K.L. McDonald and D. H. Tippit. 1975.
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The effects of the pollutant, sodium cyanide, on the morphology and physiology of oedogonium cardiacum

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