PB83-149377
Factors Influencing Metal Accumulation by Algae
Syracuse Univ., NY
Prepared for
Industrial Environmental Research Lab,
Cincinnati, OH
Dec 82
U.S. DEPARTMENT OF COMMERCE
National Technical Information Service
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EPA-600/2-82-100
December 1982
PBi3-1 4*377
FACTORS INFLUENCING
METAL ACCUMULATION BY ALGAE
by
J. Charles Jennett,
J. E. Smith and J. M. Hassett
Syracuse University
Syracuse, New York 13210
Grant No. R804734
Project Officer
Hugh B. Durham
Industrial Pollution Control Division
Industrial Environmental Research Laboratory
Cincinnati, Ohio 45268
INDUSTRIAL ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/2-82-100
2.
3. RECIPIENT'S ACCESSION NO.
gyg 3 1 i93Y
4. TITLE AND SUBTITLE
FACTORS INFLUENCING METAL ACCUMULATION BY ALGAE
5. REPORT DATE
December 1982
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
J.Charles Jennett, J.E. Smith and J.M. Hassett
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Syracuse University
149 Hinds Hall
Syracuse, NY 13210
10. PROGRAM ELEMENT NO.
1BB610
11. CONTRACT/GRANT NO.
R804734
12. SPONSORING AGENCY NAME AND ADDRESS
Industrial Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, OH 45268
13. TYPE OF REPORT AND PERIOD COVERED
Final Report. 9/76-1/79
14. SPONSORING AGENCY CODE
EPA/600/12
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Shallow beds of algae (algal meanders) have proved to be highly effective at re-
moving heavy metals and organometallics from lead-zinc mine and mill wastes. A research
program was initiated (1) to determine conditions under which algae were most effective
at concentrating significant quantities of As, Cd, Cu, Hg, Ni, Pb, or Zn, and (2) to
apply the meander technology to new types of wastewater-metal problems.
Studies were performed on 20 species of algae to determine interactions of experi-
mental variables affecting metal adsorption; species and strains of algae; type, form,
and concentrations of metal; pH; culture age; micronutrient composition of culture
medium; exposure to metal; and light intensity and exposure period. These numerous
variables were studies by means of a rapid analytical technique, the Titertek™ super-
natant collection system, to determine the adsorption of metal radionuclides.
Neither Zn nor As were bound significantly at any pH. Experimental and literature
data indicate that algae remove certain metals economically from a variety of waters
and that the meander system can be used to recover these metals for processing.
17.
KEY WORDS AND DOCUMENT ANALYSIS
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b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
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Release to Public
19. SECURITY CLASS (ThisReport)
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21. NO. OF PAGES
133
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLI
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NOTICE
This document has been reviewed in accordance with
U.S. Environmental Protection Agency policy and
approved for publication. Mention of trade names
or commercial products does not constitute endorse-
ment or recommendation for use.
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FOREWORD
When energy and material resources are extracted, processed, converted,
and used, the related pollutional impacts on our environment and even on our
health often require that new and increasingly more efficient pollution
control methods be used. The Industrial Environmental Research Laboratory-
Cincinnati (lERL-Ci) assists in developing and demonstrating new and improved
methodologies that will meet these needs both efficiently and economically.
This report describes a detailed laboratory study of factors influencing
heavy metal accumulation by 20 species of algae. Laboratory conditions under
which particular algae were most effective at concentrating significant
quantities of arsenic, cadmium, copper, mercury, nickel, lead or zinc were
determined. These data can be utilized further to evaluate the feasibility
of using algal meanders to remove a variety of heavy metals from industrial
wastewaters. The Energy Pollution Control Division is to be contacted for
further information.
David G. Stephan
Industrial Environmental Research Laboratory
Cincinnati
111
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ABSTRACT
Shallow beds of algae (algal meanders) have proved to be highly effec-
tive at removing heavy metals and organometallics from lead-zinc mine and
mill wastes. A lab-scale research program was initiated to determine conditions
under which algae were most effective at concentrating significant quantities of
As, Cd, Cu, Hg, Ni, Pb, or Zn, for the purpose of potential application of the
meander technology to new types of wastewater-metal problems.
Studies were performed on 20 species of algae to determine interactions
of experimental variables affecting metal adsorption, species and strains of
algae, type, form, and concentrations of metal, pH, culture age, micronutrient
composition of culture medium, exposure to metal, and light intensity and
exposure period. These numerous variables were studied by means of a rapid
analytical technique, the Titertek supernatant collection system, to deter-
mine the adsorption of metal radionuclides.
Metal removal was observed to be fast (3 hrs), young growing cultures
were seen to be most effective, and concentration factors > 1 x 10 were
observed. The pH had little effect on accumulation of metals except lead.
Heavy metals could be stripped from algal mats with 0.01 M EDTA or 0.1 N HNCL.-
Ca and Mg were not effective competitors for the binding sites of Hg, Pb and
Cd. Neither Zn nor As were bound significantly at any pH. Experimental and
literature data indicate that algae remove certain metals economically from a
variety of waters and that the meander system can be used to recover these
metals for processing.
This report is submitted in fulfillment of Grant No. R804734 by Syracuse
University under the sponsorship of the U. S. Environmental Protection Agency.
This report covers the period from September 13, 1976 through-January 1, 1979
and work was completed as of June 30, 1979.
iv
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CONTENTS
Foreword . . ill
Abstract iv
Figures.. vi
Tables vii
1. Introduction 1
2. Conclusions 3
3. Recommendations 5
4. Literature Review
Morphology 7
Metal Concentration by Algae 8
Interactions Between Metals and Algae
Arsenic 11
Cadmium 11
Copper 12
Lead 13
Mercury 14
Nickel 16
Zinc 17
5. Materials and Methods
Algae i 20
Metal Uptake Studies
Whole Culture Studies 24
Microtiter Assay Techniques 27
6. Results
Algal Growth 36
Batch Studies 36
Microtiter Assays 48
7. Discussion of Project Results 95
References 101
Appendix
Tables of Concentration Factors Arranged by Metal 108
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FIGURES
Number Page
1 Details of Microtiter Technique (Protocol I) 30
2 Details of Improved Microtiter Technique (Protocol II) 32
3 Experimental Procedures (Protocol II) 35
4 Uptake of Arsenic (III)by various algae 40
5 Uptake of Arsenic (V) by various algae 41
6 Uptake of Cadmium by various algae 42
7 Uptake of Copper by various algae 43
8 Uptake of Lead by various algae 44
9 Uptake of Mercury by various algae 45
10 Uptake of Nickel by various algae 46
11 Uptake of Zinc by various algae 47
12 Uptake of Lead-210 at pH 6 89
13 Uptake of Lead-210 at pH 7 90
14 Uptake of Lead-210 at pH 8 91
15 Uptake of Mercury-203 at pH 5 92
16 Uptake of Mercury-203 at pH 6 93
17 Uptake of Mercury-203 at pH 7 94
vi
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TABLES
Number ?age
1 Algal Cell Coverings 9
2 Sources and Cultivation of Algae 21
3 Culture Media Used 25
4 Algal-Metal Combinations Used in Batch Studies 27
5 Radionuclide Data 29
6 Metal Uptake - Batch Studies 37
7 Microdiluter Reliability 49
8 Effect of Biomass on Lead-210 Removal 50
9 Influence of Culture Algae on Lead-210 Removal 50
10 Removal of Lead-210 and Cadmium-109 from Water Samples 51
11 Buffers Used in Protocol II 52
12 Mercury-203 Counts for a Control Plate 53
13 Algae Used in Protocol II 54
14 Cadmium - Young Algae Data 55
15 Cadmium Removals - Young Algae 56
16 Lead - Young Algae Data 62
17 Lead Removals - Young Algae 63
18 Mercury - Young Algae Data 66
19 Mercury Removals - Young Algae 67
20 Zinc - Young Algae Data 73
21 Cadmium - Old Algae Data 74
22 Cadmium Removals - Old Algae 75
23 Lead - Old Algae Data 77
24 Lead Removal - Old Algae 78
25 Mercury - Old Algae Data 81
26 Mercury Removals - Old Algae 83
27 Zinc - Old Algae Data 85
28 Lead-210 Removal in Various Buffers 86
29 Concentration Factors for Cadmium 96
30 Concentration Factors for Lead 97
31 Concentration Factors for Mercury 98
vii
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t
SECTION I
INTRODUCTION
Effluents from secondary lead smelters are a major source of heavy
metals in the aquatic environment. These effluents are frequently low in pH
and contain a wide range of heavy metals. The largest single heavy metal
constituent is, of course, lead. Relatively large amounts of zinc, cadmium,
arsenic, copper, and other toxic cations may also be found, along with a wide
variety of anions often associated with these metals (chloride, sulfate,
nitrate and chromate for example). Lead smelter effluents also contain toxic
trace organics generated when batteries and other materials are crushed and
the heavy metals are removed from their plastic encasements. The fact that
heavy metals are dangerous in the aquatic environment has been well documented
in the literature. The problem is a major one and prevalent
throughout the United States and the rest of the world.
One possible solution is to apply the type of algal-meander -treatment
system currently in use for treating mine and mine-mill wastes at one site in
the new lead belt region in southeast Missouri—the world's largest lead
mining region (Jennett and Wixson 1975a). A detailed laboratory study of the
factors influencing heavy metal, accumulation by algae is the subject of this
investigation and an attempt has been made to evaluate the feasibility of
using algal meanders to remove a variety of heavy metals.
DESCRIPTION OF THE MEANDER PROCESS
491
In the Missouri algal-meander system, mine and mill wastes were treated
in a standard tailings pond followed by a series of shallow meanders in which
the growth of algae is encouraged. These algae grow, utilize the waste mill-
ings reagents and trap heavy metals, both particulate and dissolved, on their
surface. When the algal mat breaks loose, it is then trapped in a settling
pond at the end of the,meander system. The pond is equipped with baffled
weirs which prevent algal overflow into the receiving stream. Based on total
heavy metals removed, the system is more than 99 percent effective. Dissolved
heavy metals are also removed to levels well below U.S. Public Health Service
drinking water standards. Work by N.L. Gale at the University of Missouri-
Rolla (Wixson and Jennett 1974) has shown also that lead in its highly soluble
acetate form is removed effectively by the algae.
The process has been shown to be an adsorptive one and temperature is
not a major factor in removal (Jennett and Wixson 1975b). The algae do not
become a solid waste problem (Jennett and Wixson 1975a, b; Wixson and Jennett
1
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1974, Ryck and Whitely 1974). Solids do build up in the final sedimentation
pond for 1 to 2 years, but then this buildup stops or slows down in a manner
analogous to sewage lagoons. Furthermore, the algae with their coating of
heavy metals become relatively dense and settle well. No problems have been
reported with floating material. The leaching of heavy metals from the
entrapment through the soil to the groundwater table must be checked at each
individual site but most normal soils (except sand) have such' high cation
exchange capacities that such leaching is a highly unlikely event (Linneman
1975).
To date the algal-meander system has been used to treat only one waste,
a- combined lead-zinc mine and mill waste in the new lead belt mining region.
Since 1971, this region has been the site of a large interdisciplinary inves-
tigation, supported by the National Science Foundation, of environmental pollut-
ion by lead and other heavy metals. Wixson and Jennett in 1974 confirmed that
below the AMAX-Homestake Lead Tollers, Inc. mining mills_, severe stream degra-
dation and heavy metal buildup_was occurring. During the course of their
^study, the meandering system wasbuilt to a full operating scale and as a re-
suit the stream was returned to essentially pre-mining conditions (Jennett
and Wixson"l975a,b; Ryck and Whitely 1974). The history of this treatment
system and the quality of the water in receiving streams has been well docu- .
mented for lead and zinc (Jennett and Wixson 1975a,b; Wixson and Jennett 1974).
This technology, however, has not been applied to wastes containing other
toxic metals. This has been largely due to the lack of information on the
system's range of application and the newness of the technology. The ability
of algae to concentrate heavy metals from their aqueous environment has been
reported by radioecologists examining debris from, nuclear tests (Polikarpoy
1966), chemical geologists attempting to explain low-grade deposits of metal
(Ferguson and Bubella 1974), and ecologists concerned with the self-purifi-
cation of streams below mining operations (Trollope and Evans 1976). While
these observations are clearly valuable,, a more detailed knowledge of the
interaction between algae and heavy metals could aid in understanding these
processes. A systematic study of these'interactions has been difficult because
a wide range of variables affects metal accumulation by algae. These variables
include the length of the exposure period, the type of metal, oxidation states,
pH,,, salinity, and presence of organic pollutants. To understand the factors
influencing metal accumulation by algae in the meander system, laboratory
studies were initiated with pure cultures of representative species from other
laboratories and from the algal culture collection at the University of Texas
(Starr 196.4).
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SECTION 2
CONCLUSIONS
The ability of representative algae to concentrate large quantities of
specific heavy metals was demonstrated by adapting the Titertek supernatant
collection system (Flow Laboratories, Rockville, Maryland) for assaying radio-
nuclides of heavy metals. This technique permitted the simultaneous study of
several factors affecting heavy metal accumulation and provided a sufficient
number of replicates for statistical analysis.
Calculation of concentration factors for heavy metals revealed signifi-
cant differences between species of the same genus and between strains of the
same species (i.e. Nostoc). Some species regularly had concentration factors
of 1 x 10 . In general, young cultures exhibited very much higher concen-
tration factors than old cultures. Most of the metal removed by algae can be
removed by a rinse with 0.01 M ethylenediaminetetraacetic acid (EDTA) or 0.1 N_
nitric acid (HN00). This information suggests that surface adsorption sites'
are the principaf repository for the metallic ions or particles.
The ability of algae to remove metals is generally in this order:
mercury > lead > cadmium. Neither zinc nor arsenic was removed significantly
at any pH during a 3~hour exposure. The presence of chelating agents in the
medium (such as EDTA) inhibited the removal of the heavy metals. Calcium and
magnesium were not effective competitors for the binding sites of mercury,lead,
and cadmium. There was remarkably little effect of pH on metal accumulation
in the range of pH 5 to 8 when young cells were employed. Lead was the prin-
cipal exception because it was removed more efficiently at pH 4 to 5 than at
other pH values.
The removals of lead and mercury from nutrient solution by algae are
found to be rapid phenomena, usually accomplished in three hours or less at
room temperature. If the cells were placed in buffer after the initial adsorp-
tion of lead, a limited, slow release of lead occurred for a few hours, perhaps
while some surface polymer-lead complexes were released from dead or dying
cells. Mercury was removed from solution in nearly identical fashion by two
strains of Nostoc and showed little evidence of being released. In the control
wells without algae there were reproducible losses of Hg that occurred
within the first 24 hours at pH 6. This may be related to reports in the
literature claiming volatilization of mercury by algae.
Chlamydomonas (a green flagellated form) proved to be dramatically
superior to all other species in its,ability to remove lead. Concentration
factors for the organism of 1.9 x 10 were noted. This organism showed essent-
ially the same concentration factor for lead in the pH range of 4 to 9.
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In other experiments, Ulothrix and Chlorella had concentration factors for
cadmium greater than 10 .
The empirical successes of the algal-meander system for removing lead
from surface waters can be explained in part by the concentration and seques-
tering of lead and cadmium on the surface of algal cells. Very little heavy
metal was leached from these cells at pH 5 to 9 in the presence of divalent
cations and other inorganic constituents of algal growth media.
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SECTION 3
RECOMMENDATIONS
The remarkable ability of selected algae to concentrate heavy metals on
their cell surfaces suggests that the use of intensive algal culture may be a
very practical way to reclaim wastewater and/or recover useful quantities of
metals. Several directions for future research and development are clearly
indicated before this potential can be realized:
A. The glycocalyx—i.e., the mucopolysaccharide and protein capsule
surrounding all algae must be isolated from metal-avid species and studied
for its biochemical structure, exchange capacities, and biphasic physical
properties. With these data in hand, it is practical to search for organisms
with glycocalyces having desirable properties for the adsorption of metals.
B. If algal beds are to be maintained on a continuing basis, it will be
necessary to follow the reproductive capacity of the organism(s) in the pres-
ence of excessive levels of metal. In fact, it may be necessary to devise
schedules for metal adsorption that are followed by regeneration at loxj metal
levels.
The present meander technology depends entirely on the presence of
naturally occurring wild algae, probably growing as a mixed flora. Since some
of the species may have concentration factors in excess of 1 x 10 under cer-
tain conditions, and others may not concentrate metal at all, it appears profi-
table to encourage the former by selective algal farming. Simple enrichment
culture techniques are feasible for the meander manager.
C. A screening operation should be devised with the objective of finding
natural or induced mutants that a) have accumulation coefficients one or more
orders or magnitude greater than wild types, b) exhibit greater selectivity
for a particular metal ion than the wild types, and c) tend to remain in their
benthic form rather than in their free floating form. The blue-green algae
may be the better choice for this work because they are usually more metal-
avid than green algae, and because they are haploid. The latter characteris-
tic makes it easier to induce mutations and to select for variants in blue-
green algae diploid organisms.
D. Almost no data are to be found concerning the maintenance of beds of
benthic algae on a continuous basis. Experiments are needed to learn the long-
term consequences of heavy metal accumulation in such beds, to monitor deten-
tion and release of metals during chronic exposures, and to study the detach-
ment of benthic cells coated with heavy metals.
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E. Methods must be devised for systematically discharging metals, or
for otherwise recovering them, from meander beds. Simultaneously, it must be
determined whether or not it is feasible to recycle the algal cells as inocu-
lum or feedstock after they have been stripped of metal by chemical washing.
F. Investigation of practical questions regarding the suitability of
metal-stripped secondary sewage effluent for agricultural purposes must be
examined. 'Conversely, the efficacy of sewage as a dependable nutrient source
for algae has not been established. Certainly the presence of oil, cyanates,
solvents, etc., could be expected to interfere with the productive synthesis
of the glycocalyx adsorptive surface.
G. The effects of light, temperature, and salt concentration have not
been evaluated on a continuously operating meander. Quantitative data have
not been collected on the optimum output of glycocalyx because the factors
which stimulate it are essentially unknown.
H. It should be determined whether or not an appropriate substitute
can be found for the algal cells in the meander (aggregates or other surfaces
coated with semi-purified glycocalyx, for example). This search might provide
a renewable and/or strippable surface that would eliminate some of the
vagaries associated with the maintenance of open algal cultures.
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SECTION 4
LITERATURE REVIEW
INTRODUCTION
The literature contains numerous studies concerning the effects of heavy
metals on algae. The topics studied include toxic and mutagenic effects, sup-
pression of growth, effect on photosynthetic rates, assessment of certain algae
as indicators of metallic pollution, development of resistance to heavy metals,
and concentration of heavy metals through food chains and biotransformation
studies. The review presented here will concern itself with these studies as
well as the phenomenon of algal concentration of heavy metals, and the effects
of heavy metals on the morphology of algae.
MORPHOLOGY
Gertz and Suffet (1977) state that the first structure of an algal cell
encountered by a heavy metal is the algal cell wall. Though this is true in
many cases, the great morphological diversity of algae makes such a state-
ment overly simple. Table I summarizes the various categories of cell cover-
ings found in some algal groups. These categories include naked membrane
stage, normal, pellicle, silica frustule, silica scales, sheath or capsule.
In this table, membrane refers, to the plasmalemma or cell niemhrane,
usually 7 to 8 nm thick with an external "fuzz" of proteinaceous material.
Stage is the term for zoospores or gametes that are reproductive structures
present in the life cycles of some algae. Normal refers to organisms possess-
ing a cell membrane for their entire life cycle. Pellicle is a structure with
an outer plasma membrane, under which lie flat strips of proteinaceous material,
often with muciferous bodies at various points on the pellicle. A silica
frustule is a complex silicaceous structure found exclusively in diatoms. The
plasmalemma remains outside while the frustule is forming; after formation,
the outer membrane is abandoned, and a new plasmalemma is formed inside the
cell.. Silica scales refer to silacaceous coverings found outside the cell
membrane (Dodge 1973).
The sheath or capsule is the covering external to the cell wall found in
the blue-green algae. It is either a thick and solid structure or a thin and
dissolving slime layer (Drews, 1973). These sheaths are polysaccharide in
nature (Fuhs 1973). In the blue-green algae, the cell walls are multilayered
and composed of murein (the rigid, formative component of the cell wall)
carbohydrates (mannose, galactose, xylose, and glucose polymers), amino acids,
fatty acids, and possibly murein-associated proteins (Drews 1973).
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Other components found include rhamnose, arabinose, ribose, and giucuronic
acid (Fogg, et al., 1973).
In algae other than the blue-greens, the cell wall, when present, is
either incomplete or complete. In the former case, the portion of the cell
wall present (called a lorica) is constructed of microf ibrils thought to be
cellulosic in nature. On the other hand, algae with complete cell walls
show a wide variety of structures. One red alga studied had a cell wall
constructed of microf ibrils of cellulose. The yellow-green algae show ran-
domly arranged cellulosic microf ibrils (Dodge 1973).
Green algae in general show cell walls made up mostly of cellulose
walls which consist mainly of cellulose microf ibrils . An example of this
is Cladophora. Sometimes the wall is an outer protein-rich cuticular reg-
ion with several alternating microf ibrillar and amorphous layers. Ulva ex-
hibits a less distinct organization in its cell wall with a larger amount
of incorporated amorphous materials. Spirogyra has an outer layer of mucila-
ginous materials, and Chlorella is coated with a layer of sporopollenin. In
Chlamydomonas , the wall is composed, at least in part, of proteinaceous ma-
terials (including glycoproteins) . Some green algae have cell walls based
on xylan and mannan rather than cellulose (Dodge 1973) .
OBSERVATIONS CONCERNING METAL CONCENTRATION BY ALGAE
General
The removal and concentration of heavy metals from their aqueous environ
ment. by algae was first studied in detail in the 1950's and 1960's as part of
the problem of the release of nuclear debris from weapons testing and from
reactor cooling water effluent. Scientists took advantage of the released
isotopes, as well as newly developed analytical techniques, to study pathways
of various elements in the environment. One early result of this work was the
observation that certain isotopes were concentrated by biota to a much greater
extent than the concentration of that isotope in water. From these observat-
ions came the concept of the concentration factor (CF) such that
where C and C are respectively the concentration of the radionuclide in the
aquatic organism and in the aqueous medium. The concept is also applicable
to stable isotopes (Polikarpov, 1966).
This, rather new branch of science (radioecology) was necessarily limited
to isotopes with long half-lives that were produced under conditions mentioned
above. Furthermore, field observations were limited to areas close to test
sites and/or reactors and laboratory work emphasized the accumulation of
fission, products. -/The early data were, therefore, rich in information on
isotopes of cerium, yttrium, zirconium, niobium, molybdenun, iodine, strontium,
germanium, and praseodymium, as well as the induced radioisotopes of zinc,
phosphorous, chromium, sulfur, manganese, iron, and cobalt (Polikarpov, 1966).
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TABLE 1. SUMMARY OF CELL COVERINGS FOUND IN SOME ALGAL GROUPS
Naked Membrane
Cell Wall
Algal Type Stage Normal Pellicle Silica Silica Sheath or Incomplete Complete Reference
Only Frustule Scales Capsule (Lorica)
Bacillariophytes
(Diatoms)
Chlorophytes
(Green algae)
Chrysophytes
(Golden algae)
Cyanophytes
(Blue-green algae)
Euglenophytes
(Brown algae)
Phaeophytes
(Brown algae)
Rhodophytes
(Red algae)
Xanthophytes
(Yellow-green
algae)
Dodge (1973)
-H-
Drews (1973)
Dodge (1973)
After Dodge (1973)
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More recently, however, emphasis has shifted to the study of elements of in-
dustrial and ecological importance—principally arsenic, cadmium, copper,
lead, mercury, nickel, and zinc.
Data available from the literature have been assembled into Tables A-l
to A-7, which describe the information available about a specific metal.
The data were produced by the following types of observation: radioecological
(field observations involving measurements of radioisotope contaminants),
ecological (field studies involving stable isotopes of the metal in question),
radioisotope (laboratory studies involving radionuclides as tracers), and
stable isotope (small scale laboratory experiments using stable isotopes).
Data presented in Tables A-l to A-7 require some further explanation.
Biomass was determined in some cases on a wet (fresh) weight basis and in
other cases on a dry weight basis. Data from Booth and Knauer (1972) indi-
cate that, for algae, wet weight is greater than dry weight by a factor of
approximately 57, a point to be considered when comparisons are made from
the tables.
A third point to consider in data interpretation has to do with epi-
phytic organisms. One recent study (Patrick and Loutit, 1977) noted that a
biological slime consisting of protozoans, diatoms, and bacteria of the genus
Sphaerotilus coated the macrophyte Alisma plantago-aquatica growing down-
stream from an industrial discharge. Removing these epiphytes from leaf sur-
faces removed some iron and lead, 15-50% of the chromium, 30-35% of the copper
and 10-50% of the zinc. The authors attribute most of the reduction in ad-
sorbed metals to the removal of the bacteria..
Some interesting observations are presented about metal concentration
by algae in Tables A-l to A-7. First, all the metals are concentrated to some
degree. Second, there are differences in the degree of concentration among
metals for a given alga. For example, the marine brown alga Fucus vesiculosis
has a cadmium concentration factor XCF) of 2.7 x 10 , copper.CFs of 2.5 x-10
to 2.7 x 10"4, a lead2CF of 2.4 x £0 , nickel CFs of 2.8 x 10 to 6.8 x 10 and
zinc CFs of 4.2 x 10 to 6.4 x 10 , all values calculated on a dry weight basis,
Using the maximum CF reported for each metal gives an order of Zn > Cu > Ni >
Cd > Pb. If the minimum CF is chosen for each metal, however, the order
changes to Ni > Cd > Cu > Pb > Zn. Some of the reasons for the variations of
CFs for a metal could include differences in ages among specimens collected,
•seasonal variations, temperature differences.
A wide range of CFs was observed for particular metals among several
different algae. For example, Zygnema had a nickel CF of 6 x 10 while
Microspora, collected from the same stream, had a nickel CF of only 36.
Both are filamentous green algae. It therefore appears that some algae
scavenge metals more efficiently than others and it is not due to bacterial
epiphytes growing on their surfaces.
10
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OBSERVATIONS ON THE INTERACTIONS BETWEEN METALS AND ALGAE
Arsenic
Radioecological studies of arsenic and algae are made difficult by the
fact that the most important radionuclide of arsenic, the neutron-induced
As, has a half-life of only 26.8 hours (Polikarpov 1966). Therefore, little
work has been done on this subject.
Arsenic is found in water in various anionic forms, depending on pH and
Eh: H-AsO , H AsO ~ HAsO ~, AsO_ ~, H AsO^ , and HAsO, ~ (Ferguson and
Gavis 1972;. Other forms are possible under'extreme conditions as a result of
industrial discharges (National Research Council, 1977a).
Lunde (1972) studied the accumulation of arsenic organic compounds in
algae derived from the inorganic arsenic present in culture media. The five
species,studied,with the concentration factor being the ratio between7grganic-
bound As in either a lipid or an aqueous-extract and the inorganic As in
the medium,had CFs of 2.4 x 10 to 2.9 x 10 . The concentrations were deter-
mined by neutron activation analysis. The lipid phase invariably contained
more arsenic than the aqueous extract; Phaeodactylum tricornutum had the high-
est concentrations of arsenic.
The same worker (Lunde, 1970) measured the arsenic contents of known
algae at various points along the European coast. Using neutron activation
analysis, he determined that brown algae of the family Laminariaceae had
higher affinities for arsenic than members of the family Fucaceae. Other
experiments done on a laboratory scale with labelled sodium arsenite showed
that the test algae achieved high concentrations in as short a time as 2 hours.
A rinse of dilute hydrochloric acid removed the arsenic, indicating surface
adsorption rather than transport into the tissues (National Research Council,
1977a).
Cadmium
As with arsenic, the relationship between cadmium and algae has not been
studied extensively because no important cadmium radionuclides are included in
fallout or reactor cooling water effluent. Furthermore, the relationship be-
tween human health and cadmium exposure has only recently been firmly estab-
lished (Friberg, et al., 1971).
2+ +
Cadmium exists in natural waters as Cd , CdOH , and Cd(OH)9 depending on
pH and Eh (Weber and Posselt 1974). In sea water, cadmium is probably in the
form CdCl . One study of a pilot plant tertiary treatment/aquaculture system
showed that the green algae studied concentrated cadmium to a greater extent
than the diatom mixture employed (Table A-2). Furthermore, resuspension of algae
contaminated with cadmium (600 ppm on a dry weight basis) into fresh sea water
revealed that, after 2 hours, only 10% of cadmium had been released (Kerfoot
and Jacobs, 1976). A study done on the freshwater alga Nitella showed that the
rate and extent of cadmium uptake depended upon the amount of calcium and mag-
nesium present. Uptake occurred faster and to a greater extent in hard water
(Kinade and Erdman 1975).
11
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Although the nature of the interaction between cadmium and algae is
unknown, there is evidence that it is associated with protein. Proteins known
to complex cadmium, the tnetallothioneins, are rich in cysteine (one out of every
four amino acids) and the complex results from an interaction between cadmium
and sulfhydryl groups (Kagi and Vallee 1961). These proteins are widely
distributed in nature including bacteria, yeast, and marine plants.
Copper
Copper is a nutrient necessary for algal growth. In higher concentrations
it is a widely used algicide (National Research Council 1977b). A fairly exten-
sive body of literature is available on its interactions with algae. There are
no radioecologic data, however, since the principal radioisotope of copper, the
induced Cu, has a half-life of only 12.8 hours. (Polikarpov 1966).
Copper is present in sea water in both particulate forms and as organic
complexes (Stiff, 1971). In fresh water, copper is found as Cu , various pro-
'ducts of hydrolysis, CuCO., (aq) , and organic complexes (Stiff. 1971; Sylva, 1976)
The specific fate of copper taken up by algae is unknown even though high
concentration factors have been reported (Table A-3). Bryan (1971), working
with the brown seaweed Laminaria digitata, speculated that alginates (uronic
acid polymers) in cell walls and intercellular spaces act as ion exchange
materials. He noted that alginates extracted from the weed exhibited affinities
for divalent metals in the same decreasing order Pb> Cu> Cd> Ba> Sr> Ca>
Co> Ni> Zn> Mn> Mg as the whole cells.
Laboratory studies on the effects of copper on freshwater algae have pro-
duced a variety of conclusions. Ferguson and Bubela (1974) used algal cell
matter broken up by freeze drying, chopping, and/or pressure extrustion. They
noted concentration factors (in terms of mass of total organic carbon) of
3.3 x 10 for Ulothrix, 4.4 x 10J for Chlamydomonas. and 9.7 x 10 for Chlorella.
They speculated that metals recovered from solution by biosorption might pro-
duce sediments rich in netals. Steeman Nielsen and Wium-Andersen (1971),
working with the diatom Nitzschia palea, noted that the photosynthetic rate
decreased even in short-term experiments and concluded that copper probably
penetrates the cells. The growth rate of this alga was not appreciably affected
by copper,'apparently because some of the organic matter excreted by the alga
into the medium was able to bind copper and thus remove any toxic effect.
An earlier paper (Hassall 1963) compared the uptake of copper by living
and by scalded cells. Cells were scalded by placing the centrifuge tubes con-
taining them in a boiling water bath for three minutes. Copper uptake by
scalded cells was found to be more rapid than uptake by living cells even though
the ultimate concentration of copper is the same in both cases. Two thirds of
the copper was firmly retained, even by dead cells, and neither rinses with
distilled water nor potassium sulphate solution removed it.
Steeman Nielsen and Wium-Andersen (1969) reached a similar conclusion
while working with Chlorella pyrenoidosa. They observed that very little of the
copper bound by the alga entered the cytoplasm. They concluded that the major
portion of the copper was bound in the cell walls and the slime envelopes.
12
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They substantiated their argument by noting that, in this organism, the cell
wall and slime envelope constitute 6% of the total carbon of the cell. A
later study from the same laboratory supported this view (Steeman Nielsen and
Kamp-Nielsen, 1970).
That different species of algae respond differently to copper was illus-
trated by Button and Hostetter (1977) who shox^ed that the diatom Cyclotella
meneghiniara was copper sensitive while the green alga Chlamydomonas rein-
hard til was copper resistant. The diatom removed approximately five times
more copper on a cell-to-cell basis than the green alga. Cell walls were
cleaned by boiling the diatom in hot nitric acid and the green alga in hot
4.5% KOH. It was found that cleaned cell walls of Chlamydomonas removed more
copper from solution than living Chlamydomonas while the cleaned cell walls of
Cyclotella adsorbed little copper compared with the living cells. The authors
concluded that the cell walls of Chlamydomonas impart copper resistance by
adsorbing copper, possibly to glycoproteins, whereas in the diatom most of the
copper was associated with the plasmalemma or entered into the cell. A partial
desorption of copper after 6 hours, as compared to the 3 hour values, was noted
in several experiments.
Gibson (1972) noted that the blue-green alga Anabaena flos-aquae accumu-
lated four to five times more copper than the green alga Scenedesmus quadricauda
and also observed that copper (II) sulfate was more toxic to young cultures of
A., f los-aquae than to older cultures.
Russell and Morris (1970) presented data that indicated algae can become
copper tolerant. The organism under study was the marine-fouling alga Ectocar-
pus siliculosis. Foster (1977) studied Chlorella vulgaris and noted a non-
tolerant strain accumulated five to ten times more copper than a tolerant strain,
even at comparable external copper concentrations.
Stokes (1975) studied copper tolerant Scenedesmus isolated from a polluted
lake and found specific copper-containing complexes in the nuclei. Copper was
not, however, found in the cell walls or the cell membrane. A later paper
(Silverberg, et al., 1976) gave data obtained from electron microscopic tech-
niques. It was found that the intranuclear inclusions were in the form of cen-
tral dense-core complexes. Copper was the only heavy metal found in the in-
clusion. More recent work (Stokes, et al., 1977) revealed that the inclusions
were composed of copper-binding proteins with molecular weights of about 10,000
daltons. Apparently the alga detoxified copper by this method.
Lead
The relationship between lead and algae has not been extensively studied.
Lead is not a fission product of nuclear explosives and is not found in reactor
cooling water effluents (Polikarpov, 1966). In sea water, lead may be in the
form PbC03 (aq), PbCl , PbCl2 (aq) , Pb , PbCl" and perhaps PbOH as well as
complexed and chelated forms (Zirino and Yamamoto, 19721, In fresh water, the
forms PbOH , Pb(OH)2 (aq), and Pb (OH)~ and possibly Pb are expected to
predominate (Leckie and James, 1974).
As noted before, Bryan (1971) reported that lead was bound more strongly
than any other bivalent metal to alginates extracted from brown algae.
13
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The high affinity of.algae for lead is noteworthy and concentration
factors, as high as 7 x 10 have been reported (Table A-4) . That different
species of algae respond differently to lead was noted by Malanchuk and
Gruendling (1973) who subjected five algal-species to lead nitrate at 10.,20
and 30 ppm. Response was measured by light dependent fixation rates of CCL.
The blue-green Anabaena sp and the green desmid Cosmarium botrytis were
affected by the lowest levels of lead while CO,., fixation fell off sharply only
above 10 ppm Pb for Chlamydpmpnas reinhardtii and the diatom Navicula pellic-
ulosa. However, in the flagellated chrysophyte Ochromonas malhamensis, C09
fixation was found to increase with increasing lead concentration. The authors
speculate that lead might act by physically blocking movement across mem-
branes. They justify this statement by noting the intricate cell morphology
and the high surface area-to-volume ratio in the lead sensitive Cosmarium.
Hessler (1974) studied the green marine form Platymonas subcordiformis
in various concentrations of lead chloride. Noting that normal, flagellated
cells survived less well than flagella-free mutants, she suggested that the
flagella may be on sites of lead uptake. The mutant's resistance to lead could
not be explained solely by the lack of flagella, however.
Schulze-Baldes and Lewin (1976) studied the course of lead uptake in
batch assays with the marine diatom Phaeodactylum tricornutum as well as
Platymonas subcordiformis. They noted a rapid increase in the lead content
of cells exposed to lead and attributed the initial uptake to its adsorption to
the cell surface. Cultures which had different concentrations of EDTA .added
immediately prior to lead treatment took up less lead than cultures without
EDTA treatment, indicating that only uncomplexed lead is available for ad-
sorption. Exposing the algae to lead for various lengths of time and then
to EDTA showed that the bound lead fraction increased considerably with time
of exposure, a fact explained by the translocation of lead into the cells.
Silverberg (1975) studied the ultrastructural localization of lead in
the tolerant filamentous green alga Stigeoclonium tenue. The algae were ex-
posed to various concentrations of lead nitrate for 9 days and then subjected
to X-ray microanalysis and a staining procedure utilizing sodium rhodizonate.
Two strategies for coping with lead exposure were noted. At low concentra-
tions of lead, electron dense precipitates on the surface of the cell wall
were noted. At exposures to higher concentrations of lead, the metal was
found in the internal vacuoles of the algae. Invaginations on the cell mem-
brane indicated that movement inwards was by pinocytosis.
Mercury
Mercury is not found in large amount's in either' radioactive debris' from
weapons testing or reactor cooling water effluent (Polikarpov, 1966). Interest
in mercury in the environment has increased dramatically since the awareness of
the dangers of mercury contamination became apparent in the late 1960's and
early 1970's (D'ltri and D'ltri 1977). Most of the studies involving the re-
lationship between mercury and algae have been made since 1970.
14
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Gavis and Ferguson (1972) reviewed the cycling of mercury through the
environment. Mercury can exist in water as the native metal, in the +1 state
and in the +2 state. Under various conditions of Eh and pH, mercury may exist
as HgS , Hg(OH)2 (aq), Hg° (aq), HgCl(OH) (aq), and HgCl (aq). The chemistry
of mercury is further complicated by the affinity of the metal for sulfhydryl
groups as well as its ability to be methylated. In sea water, inorganic mer-
cury is predominantly in the form HgCl , (Glooschenko 1969).
Glooschenko (1969) studied the marine diatom Chaetoceras costatum under
conditions of light and dark with dividing, nondividing, and dead cell popu-
lations to determine the relative importance of surface adsorption versus ac-
tive uptake of mercury. He found that formalin killed cells accumulated more
mercury than any other population, including the dividing cell population
(figured on a per cell basis). Cells in the dark were found to take up slight-
ly more mercury than cells in the light. Dividing cells in the light showed a
longer period of mercury accumulation than nondividing cells. He concluded
that, while some active uptake may be present, the most important process for
mercury uptake by this diatom was by surface adsorption.
Kamp-Nielsen (1971) studied Chlorella pyrenoidosa with mercury and con-
cluded that a smaller number of sites bound mercury than copper, and mercury
was bound more specifically on the cytoplasmic membrane. The bound metal ions
apparently increased the permeability of the membrane since potassium and possi-
bly phosphate leaked out of the cells. Hannan and Patoiullet (1972), while
studying the effect of inorganic mercuric salts on the growth rates of four
different algae, concluded that the uptake of mercury could not be accounted
for by surface adsorption alone. A later paper by this,group (Hannan, et al,
1973) reported exposing Phaeodactylum tricornutum to HgCl_ and then ex-
tracting pigments and other cellular debris with methanol. About 2% of the
bound mercury was extracted by this method, a fact attributed to intracellular
transport.
Shieh and Barber (1973) studied Chlorella and noted that mercury uptake
was unaffected by light. Lowering the temperature to 1°C revealed that the in-
flux of mercury consisted of two phases, a rapid phase completed within sev-
eral minutes and a slower phase with no saturation within sixty minutes. A
breakdown in cell membrane permeability was also noted. Fujita, et al. (1976)
studied the effects of light, magnesium, and cyanide on the accumulation of
mercury by the freshwater diatom Synedra ulna. Observing that mercury accumu-
lation was stimulated by light as well as magnesium ions, they concluded that
uptake of mercury was related to energy pathways. Cyanide at O.OlmM was found
to reduce uptake to 50% of the control value, possibly by complexation with
mercuric ion.
That the silica shell of the green colonial Pediastrum boryanum was re-
sponsible for mercury accumulation was suggested by Richardson, et al, (1975)
who observed that skeletonized colonies took up mercury rapidly for two hours.
Living cells continued to remove mercury after two hours, a fact attributed to
adsorption by other cell components. Similar results were reported by Fujita
and Hashizume (1975) with the freshwater diatom Synedra ulna. They noted that
acid-digested silicate frustules took up mercury more rapidly than dividing cells,
reaching equilibrium in a matter of minutes. Living cells took up more mercury
15
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but took it up more slowly, attaining a maximum level only after 24 hours.
Davies (1974) reported experiments with the marine alga Isochryses galbana
that indicated some mercury was translocated to cell interiors.
Burkett (1975) worked with the green filamentous alga Cladophora glom-
erata which he radiolabelled with methyl mercuric chloride. He found that
live Cladophora adsorbed more methylmercury than the formalin-killed algal
speciments. It was found that release of methylmercury from Cladophora
occurred slowly, if at all. DeFilippis and Pallaghy (1976a), utilizing
Chlorella, calculated that 14% of the total cell content of mercury was situ-
ated in the cell walls. Mercury-treated cultures experienced a greater in-
crease of biomass than control cultures. This was explained by hypothesizing
that mercury prevented the export of carbon compounds such as glycollates into
the medium.
Since algae can recover from initial contact with mercury, several workers
have searched for the mechanism of metal tolerance. Ben-Bassat, et al. (1972),
working with aerated cultures of Chlorella pyrenoidosa, noted that the ability
to recover from exposure to mercury salts was directly proportional to the cell
density of the algal inoculum. Mercury taken up by the cells was apparently
not readily available for the conversion to a more volatile form, as indicated
by a minimum mercury concentration of 2 to 4 femtomoles of mercury per cell.
DeFilippis and Pallaghy (1976b) developed Chlorella strains which were resis-
tant to mercury. Resistant cells were found to volatilize much more mercury
than sensitive cells when at 25C; at 2C neither strain volatilized mercury.
Both strains, however, accumulated the same amounts of mercury—an amount cal-
culated to be very close to the sulfhydryl concentration in the cells—which
suggests that sulfhydryl groups have been the binding sites. No differences
in sulfhydryl concentration between resistant and sensitive strains were noted.
Ben-Bassat and Mayer (1977) determined that the reducing factor or factors were
extracellular agents and that the volatile mercury species released was Hg, as
indicated by its entrapment on PdCl paper, a specific test for mercury. The
reducing factor or factors were found to have a molecular weight of about 1200,
to be heat stable at 85C for short periods, and to give a positive reaction for
reducing sugars.
Nickel
There has been very little research on the relationship between nickel and
algae, due in part to its low concentrations in natural waters (National Research
Council, 1975). Furthermore, the principal radioisotope of nickel ( Ni) though
possessing a long half-life, emits a beta-particle of such low energy that it is
of no great ecological concern (Polikarpov, 1966).
Equilibrium forms, for nickel have been calculated for sea water and typical
fresh water by Sibley and Morgan (1975). In sea water, nickel was found to be
principally in the ionic state, while in fresh water the carbonate species were
the most important, followed by the free ion and then the hydroxide. Some of the
nickel (40%) was computed to be adsorbed on particulate matter.
16
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Alginates extracted from the brown seaweed Laminaria digitate were found
to have a lower affinity for nickel than for any other divalent ion except
magnesium (Bryan 1971) . Concentration factors for nickel can be found in
Table A-6.
Stokes (1975) studied algae isolated from lakes polluted with both copper
and nickel and compared them with related algae from laboratory stocks. She
was able to obtain concentration factors of up to about 1 x 10 for the isolated
strain of Scenedesmus studied. Concentration of nickel was found to occur over
a narrow pH range.A number of other factors, including calcium concentration,
were found to influence the uptake of nickel. No information as to ultra-
structural localization was given, however.
Zinc
Zinc, like copper, has been widely studied for its effects on algae.
Early work emphasized the algal concentration of Zn because of its long half
life (245 days) and energetic gamma ray. Concentration factors established
for. zinc are among the highest for the metals reported here and all species
studied have been able to concentrate zinc to a considerable extent (Table A-7).
Like copper, zinc is a required micronutrient for algae, while in higher con-
centrations it becomes toxic (Polikarpov, 1966).
Equilibrium calculations reveal that in surface sea water zinc is most
likely present in the form of a chlorocomplex (Zirino and Yamamoto, 1972;
Sibley and Morgan, 1975). In fresh water the principal form of zinc was calcu-
lated to be the hydrated ion, but carbonate, sulfate, and hydroxyl species are
present to some extent (Sibley and Morgan, 1975).
Early studies on the uptake of zinc by the marine diatom Nitzschia
closterium were conducted by Chipman, et al. (1958). Their work .indicated
that this species accumulated zinc far in excess of metabolic needs. A culture
with an initial concentration of 43 x 10 cells per liter removed all but a
trace of the zinc (added as the sulfate) within 24 hours, with 80% removal ,
being attained within the first hour. A concentration factor of about 5 x 10
was calculated using isotope concentration in equal cell and medium volumes as
the basis for comparison. EDTA, when added to the culture, prevented the uptake
of zinc. Distilled water rinses removed little of the zinc, although repeated
rinses with increasingly concentrated zinc solutions removed up to 43% of the
cell bound zinc isotope.
Bachman and Odum (1960) used Zn to trace the uptake of zinc by brown algae
under conditions of light and dark. Noting that no uptake was observed in the
samples incubated in the dark and that light-incubated samples showed a corre-
lation between uptake and oxygen production, the authors concluded that a meta-
. bolic process was involved in the uptake process. In fact, Zn uptake was
suggested as a possible assay for primary production.
A series of three papers by Gutknecht (1961, 1963, 1965) provided an alter-
native explanation for the correlation between zinc uptake and oxygen production.
The first paper in this series reported that freshly killed thalli (undifferen-
tiated plant bodies) of the green macroalga Ulva lactuca sorbed zinc more
17
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rapidly than live thalli. Furthermore, a pronounced pH dependence of both
light and dark zinc sorption, with six times as much zinc adsorbed from media
buffered at pH 9 as at pH 7, led to the conclusion that the apparent corre-
lation between uptake and photosynthesis was due to the changes in pH caused
by metabolic activity. The second paper in this series extended the investi-
gation to the red alga Porphyra unibilicalis and the brown alga Laminaria
agardi, with essentially the same results. Studies with metabolic inhibitors
(phenylurethane and uranyl nitrate) showed that they had a relatively slight
effect on uptake rates. The last paper utilized several more species of algae.
Results from this study are included as a part of Table A-7.
Bryan (1971), when discussing the affinity series of divalent cations for
alginates and heavy metals, noted that the position of zinc in the series
(higher only than magnesium) seemed anomalous since zinc was highly concentrated
by the brown algae from which the alginates were extracted. Bryan therefore
postulated that other materials such as cell proteins probably governed the
distribution of zinc. ..
A series of papers by Austrian workers (Schuster and Broda, 1970; Matzu
and Broda, 1970; Findenegg et al, 1971) reported on investigations utilizing
Chlorella pyrenqidosa. In the first paper, experiments utilizing cell walls
of the algae were described. Characterization of the chemical nature of the
cell wall by a pH titration and the determination of adsorption isotherms re-
vealed that two kinds of sites were available for zinc binding. The authors,
using information available on the biochemical composition of the cell wall,
concluded that phosphate groups, perhaps those associated with lipids, were
primarily responsible for zinc adsorption. The second paper described the
short-term uptake of zinc under culture conditions of light and air (representing
an energy rich situation) or dark and nitrogen-(an energy poor situation). Under
ideal conditions (pH 5, 5 x 10~ M Zn, 5 x 10 M Ca), about 20% of the zinc was
removed from solution after 20 hours but under minimal energy conditions only 5%
was removed. The authors concluded that the short-term uptake was a physico-
chemical mechanism, with the zinc remaining in the cell walls. The last paper
in this series described a chemostat for determining uptake, something that the
authors felt was advantageous since extracellular metabolites could not accumu-
late in the medium. Here it was found that at 30C the uptake of a 1.5 x 10~
zinc solution (buffered at pH 6.25) showed a strong light dependency, with up-
take about doubling within an hour after the light was turned on. At 5C, the
light-dependent uptake of zinc was practically nil.
Kempner and Miller (1972) , while they were determining the minimal medium
for Euglena gracilis, observed that the initial concentration of zinc(10 M)
was depleted completely within a few hours. Even though no further zinc was
available, cell growth continued at the same rate until the population had
increased a hundred fold. Falchuk, et al. (1975) reported that in zinc-
deprived Euglena gracilis, cell size increased, rate of cell division decreased,
and amount of intracellular paraiaylon increased.
Parry and Howard (1973) studied the marine alga Dunaliella tertiolecta
and observed the same pH dependence on uptake that Gutnecht noted. The pH of
the culture medium varied over 1.4 pH units as a result of photosynthesis or
respiration, a factor used to explain the pH dependence of zinc uptake. The
authors noted that this alga does not possess a cell wall; proteins (specific-
ally the amino groups) were suggested as binding sites.
18
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DeFilippis and Pallaghhy (1976a, b) utilized Chlorella in studying up-
take of zinc and the mechanisms of resistance to the metal. In the first
report, it was noted that zinc was taken up at the same initial rate at 2C
and 25C, even though the higher temperatures resulted in about one-third
more zinc being taken up after 2 hours. The zinc (1 mM as the chloride) was
postulated to be bound to the free spaces in the cell wall. A long-term
slower uptake of zinc was also noted. The second paper in the series showed
that resistance to zinc was induced by Chlorella after a long-term (50 days)
exposure to a 1 mM ZnCl solution. The resistance seemed to be due to a zinc
exclusion phenomenon since sensitive cells removed far more zinc than resis-
tant cells.
Styron, et al^ (1976) used a factorial design to determine the effects
of 8 temperatures (6 to 40°C) and 10 salinities (3.5 to 44.0 g/1) on the up-
take and concentration of zinc,for 6 marine algae. Concentration factors for
zinc ranged from 1 to 2.2 x 10 for one organism (Achnanthes, Table A-7) and
showed clearly the importance of environmental factors in determining uptake
rates.
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SECTION 5
MATERIALS AND METHODS
ALGAE
Sources and Types of Algae
The selection of representative algae to screen for heavy metal uptake
was dictated by several factors. First, it was desired to have species of
algae from the major taxonomic groups (cyanophytes, chlorophytes, etc.).
Second, it was necessary to select algae exhibiting various morphological
features (colonial, filamentous, unicellular, etc.). Third, the species
selected had to be amenable to laboratory culture in one of a relatively few
culture media. Fourth, the algae had to grow to a sufficient biomass_ in a ._.
relatively short period of time. Fifth, the algae selected had to grow in a
strict inorganic medium because such a medium would result in a relatively
low risk of bacterial contamination of the cultures.
Table 2 lists the algae selected for possible use, the sources from which
the algae were obtained, information about their taxonomic and morphological
features, and a qualitative assessment of their growth in the three media em-
ployed in the study. It should be noted that not every media-algal combination
was attempted. If satisfactory growth was achieved in Bristol's medium, no
other medium was employed.
All algal cultures were unialgal, a fact checked occasionally by micro-
scopic examination. A.ny culture found contaminated by other algae was dis-
carded and, when possible, a new culture was started from agar slants. The
only exceptions were cultures derived from samples taken from Cazenovia and
Onondaga Lakes. One-mi subsamples of each lake were cultured in both Bristol's
and the diatom medium. No attempt was made to quantify or identify the algae
in these mixed cultures.
Culture Media
If has been said that there are perhaps as many media and modifications
used for algal growth as there are active phycologis.ts today (Nichols 1973) .
Certainly the beginning worker in phycology is apt to be confused by the wide
variety of recipes available for culture media. Therefore, it was decided to
use a limited number of media and accept the fact that not all algae could be
cultured successfully.
20
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TABLE 2. SOURCES AND CULTIVATION OF ALGAE
Algae
Source *
Type
Growth on media'
B D SW
ro
A. Bacclllariophytes (diatoms)
Navicula pelliculosa
B. Chlorophytes (green algae)
Chlamydomonas sp.
Chlorella pyrenoidosa
ChlorotyIlium sp.
Kirchnerella sp.
Mougeotia sp.
Scenedesmus obliquus
Spirogyra sp.
Ulothrix fimbrinata
Zygnema
UTEX 668
Dr. C. Kuehnert
Syracuse University
Dr. N. Lazaroff
SUNY Binghamton
UTEX 758
UTEX 2016
Dr. N. Lazaroff
Dr. N. Lazaroff
UTEX 923
Pennate diatom
Unicellular,2 flagella +
Unicellular +
Filamentous +
Colonial, enclosed in +
gelatinous matrix
Filamentous, planktonic +
Colonial, 4-8 cells, +
planktonic
Filamentous, planktonic +
Filamentous, benthic +
Filamentous, enclosed by +
mucilaginous sheath
(continued)
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TABLE 2. (continued)
Algae
Source*
Type
Growth on media t
B D SW
to
ro
C. Cyanophytes (blue-green algae)
Gleotrichia sp.
Nostoc muscprum A
Nostoc F
Nostoc muscorum II
Nostoc L
Nostoc muscorum W
Nostoc 31
Osciliatoria sp.
Nostoc 586
Schizothrix calcicola
D. Mixed Cultures
Cazenovia Lake
Onondaga Lake
Dr. Lazaroff
Colonial
*University of Texas Culture Collection and Identification.
tB=Bold's medium; D=diatom medium; SW=soil water medium.
+ = success
- = failure
-------
Three media were selected for use: Bold's modification of Bristol's med-
ium (Nichols 1973), a freshwater diatom medium (Darley and Volcani 1971) and a
soil-water medium. Table 3 displays information about the first two media.
The soil-water medium was prepared by placing approximately 1/2 in. of
commercial potting soil and 300 ml of distilled water in a 500-ml Ehrlenmeyer
flask, adding a pinch of CaC03, plugging the flask with cotton and cheesecloth,
and then steaming for approximately an hour on two successive days.
All chemical reagents used were analytical grade. The water was distilled
water. Final pHs x^ere 6.6 for the Bristol's medium, 8.3 for the diatom medium,
and variable alkaline pHs (depending on the amount of CaCCL added) for the soil-
water medium.
Culturing Techniques
Agar Slants—
Cultures obtained were either axenic or bacterized unialgal. Those that
were axenic were maintained on agar slants composed of Bristol's medium solidi-
fied with 1.5% agar. Transfers were accomplished about every 4 months using a
loop and streaking onto fresh slants. Those algae which could be maintained in
this manner were: Chlorella, Chlamydomonas, Scenedesmus, Gleotrichia, all of
the Nostoc strains, and Schizothrix calcicola. These slants were maintained at
20°C.
Liquid Cultures—
Seventy-five ml of culture medium were dispensed into 125ml Erlenmeyer
flasks that were then plugged with cotton and cheesecloth and sterilized by
autoclaving. After cooling, 1 ml of an existing liquid culture was pipetted
into the fresh medium. Transfers were made every 10 to 20 days. Cultures were
sometimes inoculated from agar slants by transferring a loop of cells from
the solid to the liquid medium.
The flasks were maintained on a gyrorotatory table (New Brunswick Scientific
Co.) at 120 oscillations per minute. Light, at 560 lux, was supplied by cool-
white fluorescent tubes on a 16 hour light, 8 hour dark basis. Temperature was
maintained at 20°C.
Certain strains selected for further study were grown on a larger scale.
Thirteen one-liter glass bottles were filled with 10 liters of nutrient medium and
sterilized by .autoclaving. Laboratory compressed air was first filtered through
cheesecloth and then passed through glass tubing into the nutrient medium at a
rate sufficient to cause the suspension of the algae. Illumination (at 430 lux)
was supplied by a bank of Life Line fluorescent tubes on the same light-dark
regime.
Determination of Dry Weights
Algal dry weights were obtained by using two techniques. The first method
employed involved suction filtration of a known volume (usually 5 or 10 nil)
through a 4.25 cm diameter Whatman #2 filter paper disc. The filter paper and
23
-------
a corresponding disposable aluminum weighing dish had previously been dried at
103 °C and weighed to the nearest o.1 mg on an analytical balance. The
filter disc, with the filtered algae, was then placed in its corresponding weigh-
ing dish and redried, again at 103 UC) to constant weight. The difference
between final and initial weights divided by the volume filtered yielded dry
weight on a mg/ml basis.
There were several problems with this technique.. First, the suction appar-
ently lysed some samples as the filtrate was sometimes green. This was particu-
larly true for samples of unicellular algae such as Chlorella and Chlamydomonas.
Second, those algae with mucilaginous sheaths blocked the filter, leading to fil-
tration time of over an hour for a 5 ml sample. This was especially true for the
blue-green algae.
Because of these problems, precision was poor. For example, filtering 10 ml
of a 10 day culture of Schizothrix calcicola yielded a dry weight of 0.15 mg/ml,
while 20 ml of the same culture yielded a value of 0.28 mg/ml. Twenty ml of a 10
day culture of Oscillatoria yielded a value of 0.22 mg/ml, while 10 ml of the same
culture showed a value of 0.15 mg/ml.
The above lack of precision led to a modification that was both more rapid
and more precise. Aluminum weighing pans were marked and dried at 103 degrees C,
cooled in a desiccator and weighed. A sample of the algal culture, usually 5 or
10 ml, was then pipetted directly into the pan which was then returned to 103
degrees C and dried to constant weight. An example of the improved precision is
demonstrated by the following data. Four replicate samples (5 ml) of a Scenedesmus
culture were determined, with the dry weights (after correction) found to be
1.38 mg/ml, 1.26 mg/ml, 1.32 mg/ml and 1.38 mg/ml. Equal volumes of sterile media
were, handled in the same manner to provide a correction for the salt content of
the media. All dry weights after January 1978 were determined by the improved
technique.
Cell Counting Techniques
Cell counts, when done, were made using the Utermohl technique, as described
by Schwoerbel (1970). One ml of the culture was.diluted with 19 ml of distilled
water. Lugol's solution (10 g KI, 5 g l£ and 5 g sodium acetate in 70 ml of water)
was added dropwise until the color resembled that of a fine cognac. The resulting
mixture was then allowed to stand for 1 day, after which the vessel.was shaken and
10 ml-poured into a 10 cc tubular chamber, mounted on a plate chamber covered with
a plexiglass cap and allowed to sediment for about 4 hours. The tube was then slid
off the plate chamber, replaced by a cover glass and the plate chamber, with the
sedimented algae, placed on an inverted microscope (Unitron BU-13) and counted
using a Whipple disc of 1.3 x 10 nm . Five fields were counted and the average
used to calculate the number of organisms per ml.
METAL UPTAKE STUDIES
Whole Culture Studies
In an attempt to learn something of the time scale of metal uptake for use in
designing the microtiter assay technique described later, several preliminary stud-
ies were performed using various algal-heavy metal combinations. Table 4 lists the
algal culture used, their age in days, and the cation(s) with which they were tested.
24
-------
TABLE 3. CULTURE MEDIA USED
N>
A. Hold's Bristol Solution
Stock Solution
Macronutrients
1. K2HP04'3H20
2. KH2P04
3 . NaN03
4. NaCl
5. MgS04- 7H20
6. CaCl2. 2H20
Micronutrients
7 . EDTA
KOH
8. FeS04- 7H20
H2S04
9. H3B03
-10. ZnS04« 7H20
MnCl2. 4H20
Na2Mo04- 211^
CuS04> 5H20
Co(N03) 2' 6H20
3 g/400 ml
7 g/400 ml
10 g/400 ml
1 g/400 ml
3 g/400 ml
1 g/400 ml
50 g/1
31 g/1
4.8 g/1
1.0 ml/1
11.42 g/1
8.82 g/1
1.44 g/1
1.00 g/1
1.57 g/1
0.49 g/1
Used for one liter
of medium Final Concentration
mg/1
10 ml 75
10 ml 175
10 ml 250
10 ml 25
10 ml 75
10 ml 25
ug/1
1 ml 50
31
1 ml 4.98
1 ml 11.42
1 ml 8.82
1.44
1.00
1.57
0.49
mM
0.43
1.29"
2.94
0.43
0.30
0.17
JJM
171.09
553.00
17.9
184.7
30.7
7.3
4.9
62.9
16.8
(continued)
-------
TABLE 3. (continued)
B. diatom medium
Stock solution
Macronutrients
1. Ca(N03)2 . 4H20
2V ITD/t QU C\
m ix^rllrU . , jtl U
3. MgSO .7H 0
4. Na2Si03 9H20
5. Na2C03
Micronutrient mixture
6. H3B03
ZnCl,
CuCl2. H20
NaMoO.- 2H00
24 2
CoCl • 6H 0
<— £•
MnCl2- 4H 0
FeSo. • 7H.O
4 2
Sodium tartrate 2H 0
Used for one liter
of medium Final concentration
4 g/400 ml 10 ml
3 g/400 ml 1.8 ml
3 g/400 ml 3.5 ml
4 g/400 ml 10 ml
0.8 g/400 ml 10 ml
0.568 g/1 1 ml
0.624 g/1
0.268 g/1
0.252 g/1
0.42 g/1
0.36 g/1
2.50 g/1
1.76 g/1
mg/1
100
13.5
25
100
20
yg/1
0.568
0.624
0.268
0.252
0.42
0.36
2.50
1.76
mM
0.42
0.059
0.10
0.47
0.18
oE
9.16
4.58
1.58
1.04
1.76
1.82
8.99
7.65
-------
TABLE 4. ALGAL-METAL COMBINATIONS USED IN BATCH STUDIES
Cations
Alga Age As(III) As(V) Cd Cu(II) Hg(II) Ni(II) Pb Zn
(days)
Chlamydomonas
Chlorella
Spirogyra
Ulothrix
Ulothrix
Gleotrichia
Gleotrichia
Gleotrichia
No s toe 31
Nostoc 586
Nostoc 586
Oscillatoria
Oscillatoria
12
12
12
12
20
12
20
40
40
12
20
12
20
+ +++ + + + +
+ + + +
+ + + + + + +
+ + + +
+
+
+ +
+ + + + + + +
+
+ + + + + + + +
+
The basic procedure was to take approximately 75 ml of algal culture and
transfer it to centrifuge tubes. After centrifugation, the supernatant was
discarded and the cell mass resuspended in 50 ml of any one of the following
solutions: lead nitrate, cadmium nitrate, copper sulfate, nickel nitrate,
sodium arsenite, sodium arsenate, zinc chloride, or mercuric nitrate. All
solutions except the mercuric nitrate were at a concentration of 20 yg/ml with
respect to the metal. Mercury was present at 50 ug/ml. Sodium nitrate was
added to all heavy metal solutions to attain a final cation concentration of
0.04M.
The cultures were left unagitated in the dark. No attempt was made to
monitor or control the pH in the flasks except for a final pK reading. Three
milliliter samples were withdrawn at 3, 12, 18 and 24 hours, after which the
cultures were filtered by gentle suction and washed with 5 ml of distilled water
and 5 ml of 0.1 N HNO_. Dry weights of the algal mass were determined. The four
samples and the two washes were analyzed for heavy metal concentration on a
Perkin-Elmer model 603 atomic absorption spectrophotometer using the appropriate
hollow cathode lamps. All analyses were performed according to the manufacturers
recommendations (Perkin-Elmer 1976).Results were compared to control solutions
(no algae) subjected to the same sampling and analytical regime.
Microtiter Assay Techniques
Introduction—
The large array of variables affecting metal uptake by algae made a syste-
matic study of removal mechanisms difficult. Therefore, an important first step
in this study was the adaptation of microtitration equipment and techniques, for
use with radionuclide tracer methods. What follows is the description of the
original technique as it was employed (Protocol I) and then the improved method-
ology (Protocol II) developed during the study. First, however, information
common to both techniques will be given.
27
-------
Radionuclides—
Table 5 lists information about the radionuclides used in this phase of
the study. Isotopes were diluted to the necessary volume with distilled water,
vigorously mixed to ensure a uniform nuclide concentration and dispensed to, the
plates as described below. After sampling, the nuclide to be counted was trans-
ferred to a 15 x 115 mm glass counting tube and counted on a Nuclear Chicago
gamma scintillation counter (Model 4216) equipped with a Nal crystal detector.
Preparation of Algae for Microtiter Assay—
Algal cultures were centrifuged for five minutes at 2000 x g. The super-
natant then was discarded and the algal cells were resuspended in distilled
water to one-fourth the original volume. The four-fold concentrate was then
dispensed into the designated wells by a micropipette. Filamentous algae as
well as some colonial forms could not be dispensed through the micropipetters.
These cultures were placed in an Oster blender (John Oster Manufacturing Co.,
Milwaukee, Wisconsin) and blended at high speed for 90 seconds. Robinson (1968)
has shown that .-90 seconds produced filaments of reasonably homogeneous lengths
with a minimum of cell damage. Cultures subjected to this regime included
:Chlorotyllium, Spirogyra, Ulothrix, Mougeotia, Zygnema, Gleotrichia and
"Nostoc 31.
Microtiter Assay Technique (Protocol I)—
Microtitration equipment—Microtitration equipment used included inicro-
titration plates, micropipettes, and a multi-mic.rodiluter. The first series
of experiments utilized 96 wells (8 rows by 12 columns), disposable, V-bottom,
acrylic plates (Flow Laboratories, Rockville, Maryland, #76-364-05). Each well
had a capacity of about 0.2 ml. Micropipettes used were either reusable poly-"
propylene pipettes with stainless steel tips, or disposable- pipettes made from
plastic tubing with plastic tips. Each delivered drops of 0.025 nil at a claimed
precision of -2%. The reusable pipettes were used to dispense inert solutions;
the disposable pipettes were used for radionuclide solutions. The multi-micro-
diluter (Cooke Engineering Co., Alexandria, Virginia) allowed the simultaneous
use of six 0.025 ml microdiluters that had been trimmed to 90 mm in length. The
microdiluters allowed the transfer of 0.025 ml samples from the wells to the
counting tubes.
Procedure— The initial microtitration assay technique (Protocol I) is illus-
trated in Figure 1. Each well in a plate was filled with 4 drops from a micro-.
pipette. Every well received a drop of Bristol's solution (diluted 1/10 with
distilled water), a drop of buffer, and a drop of radionuclide solution. Experi-
mental wells received a drop of algal cells and control wells received a drop of
distilled water-. Dilution of the algal cells in the wells was therefore 1:4, re-
storing the algae to the same concentration as the culture from which they were
derived. The plates were then incubated in a dark, humidified environment at
room temperature (21°C) for three hours. After this labelling period the'plates"
were centrifuged for 20 minutes at 2000 x g in a floor model centrifuge using
carriers designed for the purpose. The six-place multi-microdiluter was used to
transfer 0.025 ml from the supernatant of individual wells to 5 ml of 20 mM EDTA
in scintillation tubes. The microdiluters were prewetted and blotted to improve
their uniformity of sample uptake; occasionally they were flamed with alcohol
(Conrath, 1972). After the supernatant was transferred, the diluters were rinsed
in a flowing stream of water. After the plate was sampled, the tubes were
counted.
-------
TABLE 5. RADIONUCLIDE DATA
ro
VD
Nuclide
2°3Hg
109Cd
21°Pb
" Zn
76 As
Source* Anion Solvent Half-life f
NEN N0~ 5 ml of 46.6 d
J 0.5N HNO
NEN Cl~ 5 ml of 450 d
0.5N HC1
A N0~ 5 ml of 21 y
3N HNO
UMRR Zn~ 7 ml of 243.6 d
2N HC1
UMRR As?°i 5 ml of 26.5 h
0.5N HNO
Mass tt Activity
0.84 mg 6 MC/mg
5 MC total
0.59 mg 3.37 MC/mg
2 MC total
0.03 mg 2 MC total
47 mg 3 MC total
28.2 mg 20 MC
* NEN = New England Nuclear Corp.; A = Amersham Corp.; UMRR = University of Missouri Research Reactor,
t
tt
Handbook of Chemistry and Physics.
Information supplied by isotope manufacturer.
-------
Q.025 ml
PIPETTE DROPPER
I/10 BRISTOL SOLUTION
20 mM BUFFER +
HEAVY METAL
INOCULUM
RADIOISOTOPE .
CENTRIFUGATION
2,000 x G
20-30 Mln
ADSORPTION
3 HR, 96-WELL
V-PLATE
AUTOMATED
GAMMA WELL
COUNTER
u u u u u~u
SCINTILLATION
TUBES
DECONTAMINATION
OF MICRODILUTORS
m
m
ITI
ft!
rfl
m
0.020 M EDTA 5 ml
Figure 1. Details of microtiter technique (Protocol I).
MULTIMICRODILUTOR
-------
Statistical methods—The information derived from the gamma counter (i e.,
the counts per minute remaining in the supernatant) was used as the basis for
the data reduction and statistical analysis employed during this phase of the
study. The mean and standard deviation of the control and experimental wells
were determined. For a given experiment there were usually 12 control wells
and several groups of 6 experimental wells. The means were used to compute
the percentage removal by use of the formula
% Removal = X control - X experimental
X" control x 100
where X control is the control mean and X experimental the test mean. Signifi-
cance of the differences between experimental and control means was determined
by use of Student's _t test (Steel and Torrie 1960).
Microtiter Assay Technique (Protocol II)—
Equipment—Though the micropipettes used were the same as those used in
the original technique, different plates and supernatant collection device were
used for improved methodology. The plates were 96~well (8 rows by 12 columns),
disposable U-bottom, polystyrene plates (Flow Laboratories, Rockville, Mary-
land, #76-311-05). Besides having U-bottom wells, these plates were consider-
ably stiffer than those used previously. Each well had a capacity of approxi-
mately 0.25 ml.
TM
The Titertek supernatant collection apparatus used included harvesting
frames, a press, an alignment rack, transfer tube frames, and a transfer fork
(Flow Laboratories, Rockville, Maryland). The harvesting frames were dispos-
able plastic holders containing 48 cellulose acetate absorption cartridges,
each with a glass-fiber filter disc. The adsorption cartridges were spaced in
4 rows and 12 columns corresponding to wells in the plates.
The press was used to push the 48 absorbing cartridges simultaneously into
the microtitration plates to a uniform depth and the alignment rack aligned the
transfer tube frames to the same spacing as the harvesting frame. The transfer
tubes (strips of twelve plastic tubes) were designed to receive the absorbing
cartridges. The transfer fork was used to push the cartridges from the frame
into the transfer tubes.
Procedure—The technique was developed originally for clinical laboratories
which use immunoassays based on the release of Cr from erythrocytes (Herschberg
et al., 1977). In this work it was used to assay the unadsorbed radionuclide re-
maining in the incubation mixture. The technique is detailed in Figure 2.
After the labelling period, the plastic holder containing the 48 cellulose ace-
tate adsorption cartridges was placed over 48 wells of a plate. The cartridges
were pressed down into the wells' and held for about 2 seconds, a time sufficient
to absorb 0.080-0.085 ml of supernatant. The glass-fiber filter at the end of
the cartridge had a diameter slightly larger than the microplate well and there-
fore formed a seal between itself and the well wall. After the cartridge had
been lifted off the plate, the filter discs were left behind, preventing the
adsorption of any algal cells. The cellulose acetate cartridges (still in the
31
-------
u>
to
O.O25ml
Pipette Dropper
I/IO Bristol
Solution
Buffer
Inoculum
Radioisotope
Automated
Gamma well
Counter
\\\\\\\\\\\\\\\\\\\\\\\\\\\\\
Adsorption
3hr, 96 Well
u-notch-Plate
Scintillation
Tubes
Absorption Cartridge
Frame
Micro Plate well
Class Fiber Filter
—: Supernatant
Press
Cartridge Containing
Supernatant
Cells
Filter
Figure 2. Details of improved microtiter technique (Protocol II).
-------
harvesting frame) were then placed over a rack of 48 transfer tubes and were
pushed down, one cartridge to a tube, with the plastic transfer fork designed
for the purpose. The plastic transfer tubes were broken off and dropped into
the glass scintillation tubes for gamma counting. The process was then re-
peated for the remaining 48 wells on the plate. The plastic tubes inside the
scintillation vials were nearly vertical and showed only minute differences in
counting geometry. Some initial problems with reproducibility of counts were
corrected by drying the cartridges (either in their transfer rack or scintilla-
tion tubes) for about 3 hours at 60 degrees C.
Statistical methods—The increased capacity of the Improved system
(Protocol II) called for an improved method of data collection, reduction and
analysis. These ends were achieved by means of a computer program developed
specifically for this project.
Data developed from Protocol I showed the desirability of conducting the
metal uptake studies over a wider pH range than first employed. Therefore, a
control plate (8 rows of 12 wells each) was used with rows maintained at pH 3,
pH 4 and pH 10 by various buffers. There were no algae in the control plate
wells; as shown in Figure 3, this control plate was used in conjunction with
several experimental plates. Each experimental plate was divided into two reg-
ions each with 8 rows and 6 columns. The rows were maintained at pH 3 to pH 10
by buffers. A droplet of algae was placed in each of the 6 wells in the pH 4
to 10 rows, while distilled water rather than algae was used in the 6 wells at
a buffered pH 3. The pH 3 wells thus served as an interplate control (IPC).
Data obtained from the gamma counter were used in conjunction with the
algal dry mass and concentration of the isotope to calculate the following:
i) Removal efficiency:
X control -X experimental
X control
ii) Accumulation co-efficient
yg metal removed
g algal dry weight
iii) (ug metal removed)/(g algal dry weight)
(yg metal)/(ml total medium volume)
Significant differences between residual activity in the experimental and
control wells were determined by use of Dunnett's t_ criterion, which is designed
to test several treatment means against a common control mean. This technique
utilizes an estimate of the pooled variance and provides one t value by which
significance is determined for each control-experiment mean pa"ir. The calcu-
lated value of t_ was determined by use of the formula:
t = d '
sd
33
-------
where d' is the difference between 1 experimental mean and the control mean and
sd is the joint standard error of the difference, determined by use of the
formula
sd=
where n1 and n,. are the number of replicates in the experimental and control
groups (6 and 12 respectively, as shox,m in Figure 3) and s~ is the error mean
square for the entire experiment (Steel and Torrie, 1960).
Data were collected and stored in an IBM 370 computer. Upon completion of
data entry, the statistical program was initiated and the results printed. It
is important to note that comparisons between control and experimental means
were all done at the same pH; there were then, in effect, eight experiments
being analyzed simultaneously, one at each pH.
Experimental controls were maintained in two ways. First, the mean counts
from each experimental plate were compared to the control mean at the same pH.
The pH 3 rows contained no algae and were utilized as interplate controls (IPC,
see Figure 3). The IPC data were used to test the hypothesis.that all the plates
had been handled similarly. If the IPC data from an experimental plate were not
significantly different from the control mean at pH 3, it was assumed that the
experimental plate had been handled in the same manner as the control plate and
the data at each pH were then accepted for analysis. If the IPC data for an
experimental plate were different from the control mean at pH 3, all the data on
that plate were rejected.
The second control procedure was to use one-half of an experimental plate
with distilled water rather than algae at all pHs. The means for each row from
the half plate were compared to each row from the entire control plate. In this
manner, unusual trends across the entire pH range could be detected.
Summary—
The improved technique had several advantages over the original technique.
The newer technique was faster because the plates did not have to be centri-
fuged, and sampling a plate took 30 seconds compared to about 10 minutes for
the original method. In addition, there was no danger of the cells being taken
up (as occasionally happened when the microdiluters disturbed the algal pellet
in the original method).
Most importantly, as the results sections will show, precision of counts
between sample wells was increased dramatically. This was probably due, in
part at least, to the elimination of any cross contamination between wells. The
improved method was superior also in radiological safety, because all the liquid
to be counted was held in the absorbing elements, minimizing chances for contami-
nation. The use of disposable absorbing elements also reduced the volume of
radioactive wastes that had to be disposed of as a result of decontamination
rinsing and washing.
34
-------
CO
en
ALGAL TEST PLATE
ALGA I ALGA 2
pH CONTROL PLATE
pH\
oooooooooooo
10 10
90
y
Q o
•7 -j
( <
6C
D
5C
D
4/1
't
3 (IPC) 3
oooo oooooooo
OOOO OOr\ r^» OOOO
OOOO OOOO OO OO
ooo oooooooo
00.00 ooo oonoo
OnOo ooo ooo oo
oooooooo oooo
Figure 3. Experimental procedure as used in improved microtiter assay technique (Protocol II)
for comparing the removal of heavy metal radionuclides by 2 algae at different pi-1.
+ wells with algal inoculum; o wells without algae; IPC = interplate control.
-------
SECTION 6
RESULTS
ALGAL GROWTH
Algal growth was sufficient for 20 species to provide a representative
selection of algae for the experiments. Some species proved impossible to
culture and others exhibited unusual rates of growth. Table 2 reveals that no
growth was obtained from any form in soil-water media. At first this seems
surprising because some workers considered this a general medium, suitable for
many forms (Nichols 1973; Starr 1964). The choice of African violet potting
'soil was probably unfortunate because this soil is high in humus. Starr (1964)
states that success with this medium depends on the selection of a garden soil
with a medium humus content.
A few algal forms were characterized by unusual growth rates. Mougeotia
and Zygnema grew only slowly and to a low final biomass. Nostoc 31, Nostoc W,
and Gleotrichia exhibited a long lag phase. For each of these forms, inocu-
lation was followed by about 15 days of apparent inactivity. After this period,
growth to a useful final biomass was rapid. For these forms, then, no young
culture data are available.
BATCH STUDIES
General Discussion
The results of the batch studies are presented in Figures 4 through 11
and Table 6.
The figures show the concentration of metals in the sample as a function
of the time the samples were withdrawn from the culture. It should be noted
that, for reasons of clarity, not all data points are shown on the figure.
The table includes the dry weight determined for each algal culture
used, the age of the culture (in days), the concentration of metal found in
the four, timed samples, and the concentrations in both the distilled water and
acid rinse. It will be recalled that all metals had an initial concentration
of 20 jag/ml except mercury, which, was present at 50 pg/ml.
Arsenic III—
The data show no obvious trends in the removal of arsenic III. The
highest removal (about 35%) was accomplished by Chlamydomonas, which also had
the lowest biomass (0.12 mg/ml).
36
-------
TABLE 6. METAL UPTAKE - BATCH STUDIES
Metal Culture (age)
Arsenic III Chlamydomonas
Ulothrix
Ulothrix
Gleotrichia
Gleotrichla
Nostoc 31
Nostoc 586
Nostoc 586
Oscillatorla
Oscillatoria
Control
to Arsenic V Chlamydomonas
"^ Ulothrix
Nostoc 31
Nostoc 586
Oscillatoria
Control
Cadmium Chlamydomonas
Chlorella
Spirogyra
Ulothrix
Gleotrichia
Nostoc 586
Oscillatoria
Control
(12)
(12)
(20)
(20)
(40)
(40)
(12)
(20)
(12)
(20)
(12)
(12)
(40)
(12)
(12)
(12)
(12)
(12)
(12)
(12)
(12)
(12)
Biomass
(mg/ml)
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
.12
.50
.18
.58
.92
.58
.34
.56
.22
.44
.10
.24
.44
.40
.20
.06
.04
.12
.34
.06
.22
.14
3 hr
17
18
17
18
19
15
17
16
18
21
20
20
18
18
18
20
22
12
12.8
13.2
11.6
12.4
9.6
13.2
12 hr
19
18
18
18
19
15
17
17
18
18
20
21
18
17
19
22
22
11.2
11.2
13.2
10.0
12.8
12.4
11.6
Samples
18 hr
15
18
17
18
20
16
15
18
19
17
20
21
18
18
17
19
21
13.2
12.0
12.8
10.4
13.2
12.4
12.8
15.2
(yg metal/ml),.
24 hr DW"
13
20
15
19
18
18
16
16
15
14
20
18
20
18
16
18
20
13.6
12.8
13.6
10.0
10.8
10.8
12.4
16.0
1
1
3
6
6
4
1
1
4
3
2
2
4
4
3
3.4
3.2
2.9
2.5
3.6
2.9
3.6
ARf
2
2
1
3
3
0
1
1
0
0
3
2
0
2
0
1.
1.
0.
4.
4.
1.
2.
7
6
5
0
8
7
2
(continued)
-------
TABLE 6. (continued)
LO
oo
Metal
Copper
Lead
Mercury
Culture
Chlamydomonas
Chlorella
Spii .•'•gyra
Ulothrix
Gleotrichia
Nostoc 586
Oscillatoria
Control
Chlamydomonas
Chlorella
Spirogyra
Ulothrix
Nostoc 586
Oscillatoria
Control
Clamydomonas
Spriogyra
Nostoc 586
Oscillatoria
Control
(age)
(12)
(12)
(12)
(12)
(12)
(12)
(12)
(12)
(12)
(12)
(12)
(12)
(12)
(12)
(12)
(12)
(12)
Biomass
(mg/ml)
0.08
0.08
0.26
0.20
0.14
0.20
0.26
0.08
0.08
0.20
0.22
0.28
0.32
0.16
0.36
0.36
0.24
3 hr
13.6
16.0
16.8
12.0
13.2
7.2
17.2
2.2
11.9
14.4
1.1
6.0
4.8
14
29
6
26
34
12 hr
16.8
16.0
16.4
19.2
10.8
9.2
18.4
18.8
1.6
1.3
15.4
4.3
3.7
3.1
14.0
8
14
4
20
32
Samples (yg metal
18 hr 24 hr
19.2
16.0
15.2
15.2
14.0
8.4
17.6
17.2
3.3
2.9
14.7
3.4
4.8
2.2
17.2
8
11
6
19
34
14.4
15.2
14.4
14.8
11.6
16.0
16.8
16.8
4.2
9.4
14 . 1
1 . 6
4.0
1.1
12.0
9
12
9
19
36
/ml)
DW
5.4
1.2
3.6
2.9
5.2
3.7
3.2
1.7
4.1
2.8
0.7
0.8
0.5
0.8
4
4
2
AR
0.3
1.9
2.0
2.3
3.6
1.6
2.0
16.0
13.7
4.0
30.1
28.3
21.7
4.0
23
20
50
31
(continued)
-------
TABLE 6. (continued)
u>
Metal Culture
Nickel Chlamydomonas
Chlorella
Spirogyra
Ulothrix
Nostoc 586
Oscillatoria
Control
Zinc Chlamydomonas
Spirogyra
Ulothrix
Nostoc 586
Oscillatoria
Control
(age)
(12)
(12)
(12)
(12)
(12)
(12)
(12)
(12)
(12)
(12)
(12)
Biomass
(mg/ml)
0
0
0
0
0
0
0
0
0
0
0
.04
.08
.32
.08
.10
.20
.12
.20
.20
.38
.28
3
19
19
16
16
17
18
22
17
14
16
15
17
19
hr
.6
.6
.4
.0
.6
.0
.4
.2
.0
.0
.6
.6
.6
12 hr
18.8
16.8
16.8
18.8
18.4
19.2
23.2
14.0
13.6
25.2
12.8
9.2
22.8
Samples (yg metal/ml)
18 hr 24 hr DW
19.2
19.2
18.0
19.2
20.8
18.8
21.6
14.0
10.8
16.0
11.6
16.8
18.4
18.4
20.0
18.4
18.4
19.6
15.2
24.0
16.8
15.6
20.4
15.6
15.6
16.8
4.5
2.8
4.5
4.5
4.9
4.5
5.0
2.8
2.8
2.8
2.8
3.2
AR
2
1
2
2
1
2
1
2
8
2
10
0
.5
.3
.5
.5
.4
.5
.1
.8
.0
.4
.4
.8
* Concentration in distilled water rinse.
t Concentration in HNO., rinse.
-------
30H
£ 20-
\
O>
o
UJ
on 10
<
• • • • CONTROL
A • • • NOSTOC 586 ( 12 day)
O • • • CHLAMYDOMONAS (12 day)
A • • • OSCILLATORIA (20 day)
• • • • NOSTOC 31 (40 day )
0
9 12 15
TIME (hours)
18
21
24
Figure 4. Uptake of arsenic (III) by various algae.
-------
3CH
20-
o
z
LJ
CO
QL
10-
T~
6
D CONTROL
A NOSTOC 586 (12 day)
O CHLAMYDOMONAS (12 day)
A OSCILLATORIA (12 day)
O NOSTOC 31 (40 day)
0
12
TIME (hours)
15
nr~
18
24
Figure 5. Uptake of arsenic (V) by various algae.
-------
NJ
30H
20-
E
V.
o>
Q
3 10-
0
• • • - CONTROL
A • • • NOSTOC 586 (12 day)
O • • • CHLAMYDOMONAS (12 day
A • • • OSCILLATORIA (12 day)
• • • • ULOTHRIX (12 day)
i
12
15
18
21
i
24
TIME (hqurs)
Mgure 6. Uptake of cadmium by various algae.
-------
CO
20-
E
X.
en
o:
LJ
o_
Q_
o
0 10-
0
• • • • CONTROL
A • • • NOSTOC 586 (12 day)
O • • • CHLAMYDOMONAS (12 day)
A • • • OSCILLATORIA (12 day)
• • • -GLEOTRICHIA (12 day)
i
3
I
6
9 12 15
TIME (hours)
8
24
Figure 7. Uptake of copper by various algae.
-------
20-
10-
Q
<
LJ
• • CONTROL
• • CHLAMYDOMONAS (12 day)
' ' NOSTOC 586 (12 day)
A . . . OSCILLATORIA (12 day)
«... ULOTHRIX (12 day)
O • ' 'SPIROGYRA (12 day)
-O
0
9 12
TIME (hours)
15
18
21
24
Figure 3. Uptake of lead by various algae.
-------
40-
30-
20-
or
Z>
o
cc
LJ
ioH
0
0
• • • • CONTROL
A • • • NOSTOC 586 (12 day)
O • • • CHLAMYDOMONAS (12 day)
A- • • OSCILLATORIA (12 day)
• • • • SPIROGYRA (12 day )
r
6
9 12 15
TIME (hours)
18
21
:6
2A
Figure 9. Uptake of mercury by various algae.
-------
30-
25-
e
\
D»
20-
LJ
O
2
• • • • CONTROL
A • • • NOSTOC 586 (12 day)
O • • • CHLAMYDOMONAS (12 day)
A ' • • OSCILLATORIA (12 day)
• • • • ULOTHRIX (12 day)
0
I
3
i
12
i
15
i
18
I
21
24
TIME (hours)
Figure 10. Uptake of nickel by various algae.
-------
25-
_ 20H
e
\
0>
O 15-
10-
• • - - CONTROL
A- • • NOSTOC 586 (12 day)
O • • • CHLAMYDOMONAS (12 day)
A- • • OSCILLATORIA (12 day)
• • • • SPIROGYRA (12 day )
D
D
0
9 12 15
TIME (hours)
18
24
Figure IT. Uptake of zinc by various algae.
-------
Arsenic "v —
There are no obvious trends in .he removal of arsenic V. Most experimental
values are coo close to the control value to be considered significantly differ-
ent. The lowest experimental value (16 yg/ml, compared to a control value of
20 mg/ial) was attained bv '.Costoc 586, which had among the highest biomass
(0.40 mg/ml).
Cadmium—
Cadmium was likewise not dramatically removed by the algae used in this part
of the study. The lowest experimental values (about 10 yg/ml) were achieved by
Ulothrix, which had the highest biorr.ass (0.34 mg/ml). That the acid rinse for
both Ulothrix and Xostoc 556 (with the next lowest experimental value) had high
concentrations of cadmium indicates a surface adsorption mechanism for cadmium
removal.
Copper—
The highest removal of copper (about 30% compared to the controls) was at-
tained by Gleotrichia, which had a biomass of about 0.14 mg/ml. This alga, though
usually a dark brown mass of filaments, turned greenish on exposure to copper.
Lead—
Lead appeared to be removed by all the forms studied in this experiment,
with removal (compared to the 18 hour control) of from 15% for Spirogyra to about
87% for Osciliatoria. The green filamentous Ulothrix showed high values of lead
removal, and the morphologically similar Spirogyra showed values little different
from the controls, even though both algae had nearly identical biomasses. The
high values of lead in the acid rinses suggests again a surface adsorption.
Mercury—
The most dramatic removal of mercury was accomplished by Nostoc 586, which
had an experimental value of 6 jag/ml as compared to the control.value of 34 yg/ml
after only three hours. Again the values of mercury in the acid rinses were high.
Nickel-
Though all algae seemed to exhibit some removal of nickel (17 to 37% after
24 hours), no trends could be discovered from the data.
Zinc—
The algae that seemed to accumulate zinc were Spirogyra (with 41% removal
after 18 hours) and Nostoc 586 (with 37% removal during the same length of time).
Again, the acid rinses of these two algae had high values of zinc, suggesting
surface adsorption sites.
RESULTS OF MICROTITES. ASSAYS
Initial Techniques - Protocol I
General Discussion—
Several variables were studied by means of the original technique utilized
irt this study. These variables will be discussed in turn. First, however,
information common to all experiments will be presented.
48
-------
.-,,,-,
"
, . . . . , . 76 . 109 „ . 203 .,
raaion.ucj.iGes used in cms part or tne stuay were .-is, Co., rig
?b, and,03 Zn. Dilutions of the stock solution were mace to provide radio-
active counts appreciably above the background. The pri was maintained, at 6.5 and
8.0 by the use of Tricine buffer (:I-tris (Hydroxymechyl) rr.ethylglycine) at 0.020 :!
£or pel 6.5 and 3.0; pK 5 was attained by addition of KC1 to maintenance medium.
Determination of Error —
An important first step in the development of any technique is the determi-
nation of, the error associated with the technique. To this end, a pla-te was made
up with ~" ?b, water, and buffer to 3 different pHs, and the plate was sampled by
two operators. The results are shown in Table 7.
TABLE 7. RELIABILITY OF MICRODILITERS FOR SAMPLING
RADIONUCLIDE (LEAD 210) IN MICROCULTURE PLATES
Operator
A
B
DH
5.0
6.5
8.0
5.0
6.5
8.0
n
14
13
14
12
12
12
X
1442
1480
1393
1401
1402
1444
a
X
162
333
135
195
236
201
CV*
0.11
0.23
0.10
0.14
0.17
0.14
Coefficient of Variation
Candidate Organisms—
The eight candidate organisms selected for the initial work were the green
Chlamydomonas, Chlorella, Chlorotyllium, and Ulothrix and the blue-green Gleo-
trichia, Nostoc 586, Nostoc 31 and Oscillatoria. Each alga was exposed to the
nuclides for three hours in a dark environment.
Effect of Biomass—
The effect of biomass was studied by use of Nostoc 586 and Pb. The cells
were centrifuged, washed, and resuspended in various amounts of distilled water
to achieve the desired relative concentration. Results of this experiment are
given in Table 8.
49
-------
TABLE 8. EFFECT OF BIOMASS ON LEAD 210 REMOVAL BY NOSTOC 586
Relative concentration
of Nostoc 586
0.24
0.5
1
2
4
8
Control
Dry wt.
(mg/ml)
0.21
0.43
0.86
1.72
3.44
6.88
0
Residual CPM in .
supernatant"
4210 - 520
3930 - 500
2990 - 720
2080 - 220
2980 - 660
1930 - 170
3240 - 350
* Mean of 6 wells + 1 standard deviation; control is mean of 10 wells.
TABLE 9. INFLUENCE OF CULTURE AGE ON REMOVAL OF LEAD 210
Organism
Nostoc 586
Chlamydomonas
Oscirllatoria
Control
Age (days)
15
55
15
55
15
55
—
Dry wt.
(mg/ml)
0.71
1.40
0.15
0.13
0.31
0.71
0
Residual CPM in
supernatant*
2000 - 520
1790 - 210
1210 i 180
2270 - 320
2600 - 600
1520 - 130
3340 t 400
*Mean of 6 wells - standard deviation.
50
-------
210 109
TABLE 10. REMOVAL OF Pb and Cd BY WATER SAMPLES*
SEEDED WITH NOSTOC 586 and ULOTHRIX
-f—
Residual CPM' ' in Suuernatants
1)
2)
3)
4)
5)
6)
Water Source'
Onondaga Lake
Nine Mile Creek
(State Fair Blvd)
Nine Mile Creek
(Amboy)
Geddes Brook
Meadowbrook Crk.
Allied effluent
1/10 Bristol's
nitrate solution
Distilled H20
No inoculum
Nostoc 586
PH
7.4
7.3
7.7
7.5
7.1
7.6
6.8
6.7
210Pb
2430
2860
2740
2570
1840
2900
2140
2310
2530 - 280
109Cd
25800
29200
21200
24900
21900
24000
18100
20700
Ulothrix
21°Pb
2370
3100
2380
2570
2640
2590
1980
2050
109Cd
24600
27100
20500
21000
23900
23700
21600
22500
24400 - 2600
* All samples 0.5 ppm Pb; 0.025 ppm Cd.
' Water Quality:
1) = Saline lake receiving metro Treatment Plant effluent,
2) = Sterile with chlorine, sodium carbonate, sodium chloride
3) = Rural farm stream, downstream from Camillus village,
4) = Suburban stream, surface runoff; trout, pan fish,
5) = Urban stream, storm overflow channel, and
ij.6) = Unnamed brook contaminated with chemical plant cooling water.
1' Average of 6 wells; control = average of 12 wells - 1 standard deviation.
' 51
-------
Influence of culture Age—
The influence of culture age on lead removal was studied by comparing the
uptake of lead 210 by 15-and 55-day old cultures of Nostoe 586, Chlamydomonas,
and Oscillacoria. Resulcs of this study are presented in Table 9.
Heavy Metal Accumulation from Natural Waters—
Samples fro-, various sources of water were gathered for the experiment des-
cr^bec in Table 10. Each sample was seeded with radionuclide (either" Pb or
Cd), algae (either Nostoe 586 or Ulothrix), and the uptake of the metal deter-
mined. The pH of the various water samples ranged from 7.1 to 7.7. Distilled
water and the normal maintenance medium (Bristol's solution diluted 1/10) were
also used to compare with uptake from the various natural water samples.
Irnnroved Technique-Protocol II
General Discussion—
The results of the initial screenings, coupled with the increased capacity
of the improved technique, led to the decision to screen for heavy metal removal
over a broader range of pHs. Therefore, results were obtained using the buffers
and pHs described in Table 11.
TABLE 11. BUFFERS* USED IN IMPROVED MICROTITER ASSAY
(Protocol II)
PH
3
4
5
6
7
8
9
10
Component A
0.0 5M KHC H 0A
0.05M KHC H 0,
844
0.05M KHC8H404
0.05M KH2P04
0.05M KH2P04
0.05M KH^O/^
0.013M Na2B407'10H20
0.13M Na2B,07'10H20
Component B
0.22M HC1
0.0001M HC1
0.023M NaOH
0.0006M NaOH
0.29M NaOH
0.047M NaOH
0.005M HC1
0.02M NaOH
* Handbook of Chemistry and Physics, 57th Ed., 1977.
52
-------
/•The radionuclicies used in this part of the study were Cd, Hg, Pb,
and Zn. Arsenic was excluded because of the negative results obtained during
the first phase of the study and also because, with a half-life of only 26.8
hrs . the original activity rapidly diminished and required large, corrections
for decay within the experimental period.
Precision—
The precision of the new technique, as well as the necessity for a change
in procedure, is demonstrated bv the following data. A plate was prepared with
3 drops of water and 1 drop of Hg solution, sampled, and the glass tubes
counted immediately in the gamma counter. The same tubes were counted 18, 66,
and 90 hours after the initial counts. The data obtained are presented in
Table 12.
The striking downward trend of the mean counts led to the hypothesis that,
as the liquid medium evaporated from the top of cellulose acetate cartridges,
the radionuclide was redistributed along the length of the filter element,
thereby changing the counting geometry and the mean counts. This hypothesis
was tested in two ways. First, some dye was used to monitor visually any up-
ward transport of dissolved matter as the solvent evaporated. It was observed
that movement of the dye ceased after a period of about 3 days. Second, a
plate was prepared and sampled and the tubes placed in an oven at 60 C for
about 12 hours before counting. The tubes were recounted after 12 hours.
TABLE 12. MERCURY 203 COUNTS FOR A CONTROL PLATE
Time (hrs) n* X x CV+
0
18
66
90
8
8
8
8
7253
6007
5162
5056
628
509
338
269
0.087
0.085
0.065
0.053
Notes: *Data from one column.
•fCoefficient of variation.
Though the first counts (with data analyzed for the 12 wells of the first row)
produced a mean of 5786, a standard deviation of 583, and a coefficient of
variation of 0.10, the second series of counts of the same tubes produced a
mean of 5749, a standard deviation of 567, and a coefficient of variation
(again) of 0.10.
While the above observations certanly do not prove the hypothesis as
stated, the goaloof reproducible: counts[was .obtained. _ All _tubes were there-
fore dried at 60 C for at least 6 hours prior to countingT" ~
53
-------
Results—
The results from the screening experiment -,;ill be presented in tabular
fors. For this series of experiments all algae were exposed to the nuclide
for 3 hours in the dark. Table 13 shows the algal cultures and ages used with
the various nuclides.
TABLE 13. ALGAE USED IN IMPROVED MICROTITER ASSAY
(PROTOCOL II)
Alga
Young Cultures'
Old Culturest
,. 210_. 203,,
Ca ?D As,
5_ 109_ , 210_, 203H 65_
Zn Cd Pb Hg Zn
Navicula pelliculosa
Chlamvdomo na s
Chlorella
Mougeotia
Scenedesmus
Ulothrix
Zygnema
Gleotrichia
Nostoc 586
Nostcc muscorum A
Nostoc H
Nostoc L
Nostoc W
Oscillatoria
Schizothrix
Cazenovia (Bristol's)
Cazenovia (Diatom)
Onondaga (Bristol's)
Onondaga (Diatom)
4-
4-
4-
4-
4-
4-
4*
4-
4-
4-
4-
4-
4-
4-
4-
4-
4-
4-
4-
4-
4-
4-
4-
4-
4-
4-
4-
4-
4-
4-
4- 4-
4-
4-
4-
4- 4-
4-
4- 4-
4- +
4-
4-
4-
4-
4-
4-4-4-
O_ 4^.
4- 4-
4- 4-
4- 4-
4-
4- 4-
* Cultures were 11 days old
•f Cultures were 44 days old
11-Day algae and cadmium—The objective of the statistical program was to
identify those algal-pH combinations that revealed significant removals of cad-
mium. This was accomplished by identifying experimental means lower (at the 95%
confidence level) than the corresponding control mean. Table 14 shows the
analysis of variance data used to determine significant differences between
experimental and control means, while Table 15 shows those algal-pH combinations
that revealed significant removals of cadmium.
The F-test is used. in. a sample calculation to show how the data were used
to determine significant removals. Figure 3.(page 35) depicts the experimental
design used in these assays. The interplate control (IPC) data were used to
test the hypothesis that all experimental plates were handled similarly to the
control plate. The calculation uses the treatment, me.an square (IMS) and. the..
error mean square (EMS) to calculate an F value. Using the IPC (pH 3) data from
Table 14 gives:
54
-------
TABLE 14. STATISTICAL DATA FOR CADMIUM - YOUNG ALGAE EXPERIMENT
Ul
Ui
Source of Variability
Treatment SS*
Treatment df
*
Treatment MS
Error SS
Error df
Error MS
Total SS
Total df
Control data
n
X
o
X
pll 3
28.9
18
1.60
1.59
101
1.57
188
119
12
8960
1130
pH 4
55.0
18
3.06
192
101
1.90
247
119
12
7570
890
pH 5
34.4
18
1.91
133
101
1.32
168
119
12
7960
660
pll 6
57.0
18
3.17
88.0
101
0.871
145
119
12
7530
830
pll 7
127
18
7.04
119
101
1.18
246
119
12
6540
870
pll 8 pll 9
69.5 52.3
18 18
3.86 2.91
89.3 139
101 JO.I
0.884 1.38
159 I'H
119 119
12 12
6080 5(>40
930 JO'JO
pll 10
75.5
18
4.19
136
101
1.35
212
119
12
7380
1 330
*Al.l sums of squares (SS) and mean squares (MS) are x 10~ .
'('Degrees of freedom. ,
-------
TABLE 15 . SIGNIFICANT REMOVALS OF CADMIUM" BY YOUNG CULTURES OF ALGAE
ALGA (biomass rag/ml) pH 4
Navicula pelliculosa (0.085)
t
t-value ns
Mean
Ox
Removal efficiency
Accumulation coefficient
Concentration factor
Chlamydomona s (0.04)
t-value ns
Mean
Ox
Removal efficiency
Accumulation coefficient
Concentration factor
Chlorella pyrenoidosa (0.015)
t-value • ns
Mean
Ox
Removal efficiency
Accumulation coefficient
Concentration factor
pH 5 pll 6
•l-l-
ns 4.99!
5200
830
0.31
1330
3640
ns 3 . 54
5880
730
0.22
2010
5480
3.78 4.04
5790 5640
810 1020
0.27 0.25
6660 6100
18,200 16,700
pll 7
4.47
4110
380
0.37
1600
4370
ns
5.78
3400
510
0.48
11,700
32,000
pll 8 pll 9 pH 10
5.38 ns ns
3550
1050
0.42
1790
4900
ns ns ns
3.84 ns 3.28
4270 5490
550 600
0.30 0.26
7520 6250
19,800 17,100
(continued)
-------
TABLE 15. (continued)
ALGA (biomass mg/ml)
pH 4
pH 5
pll 6
pli 7 pH 8
pll 9
pll 10
Scenedesmus obliquus
t-value
Mean
°x
Removal efficiency
Accumulation coefficient
Concentration factor
ns ns 3.70
5800
780
0.23
990
2700
3.69
4540
610
0.31
1320
3600
3.40 ns ns
4480
670
0.26
1130
3100
Ui
Ulothrix fimbrinata (0.017)
t-value
Mean
ax
Removal efficiency
Accumulation coefficient
Concentration factor
Zygnema (0.03)
t-value
Mean
ax
Removal efficiency
Accumulation coefficient
Concentration factor
ns
ns
5590
670
0.26
3130
8560
3.33
6050
890
0.24
5170
ns
3.13
6070
790
0.19
4180
14,100 11,400
4.14
ns
ns
ns
ns
ns
ns
ns
ns
(continued)
-------
TABLE 15. (continued)
' - ' II II .1 - ._..-•-•:- _. .- - l_ -I-m "I. _ _ - U _ -1- T -_--!. --JT- _ J— --mi J,--, .TUU-- _. «.-_— 11 - -1----L Tl. -_ _ _ ^~ _ - — .-J.. . _..».... _. -. _ _ Tl _-
ALGA (biomass nig /ml)
Nostoc 586 (0.17)
t-value
Mean
0X
Removal efficiency
Accumulation coefficient
Concentration factor
Oscillatoria (0.19)
t-value
^ Mean
00 Ox
Removal efficiency
Accumulation coefficient
Concentration factor
Onondaga - Bristol's (0.23)
t-value
Mean
ax
Removal efficiency
Accumulation coefficient
Concentration factor
pH 4 pH 5 pll 6
ns 2.977 3.12
6250 6070
990 820
0.21 0.19
460 420
1260 1140
ns ns 3.55
5870
1270
0.22
420
1.160
ns ns us
f
pH 7 pit 8 pll 9 pll 10
ns 3.22 ns us
4560
730
0.25
540
1470
3.00 ns ns ns
4910
460
0.25
480
1310
3.17 n s 1 1 s us
4820
7C.O
0.26
420
1140
(corit i niu'd )
-------
TABLE 15. (continued)
--__- _ _ ..
ALGA (biomass mg/ml)
Onondaga - Diatom (0.175)
t- value
Mean
°x
Removal efficiency
Accumulation coefficient
Concentration factor
pH 4 pH 5 pH 6 pll 7 pll 8 pH 9 pll 10
ns ns ns ns 2.96 ns us
4680
410
0.23
480
1310
Cadmium concentration = 0.36 Mg/ml; activity = 1.24 |)C/ml.
u, t
*° Not significant
= 2.90 at the 95% confidence level.
crit
-------
. _
1.57(£35) - 1.02
This value is to be compared with a tabulated F value (Steel and Torrie, 1960,
p. 436-441) having 18 numerator and 101 denominator degrees of freedom (treat-
ment and error degrees of freedom respectively). The interpolated value is
1.50. Since the calculated F value is lower than the tabulated F value, the
hypothesis that all plates were treated similarly is accepted. A calculated F
value higher from the tabulated value would indicate that at least one IPC mean
was different from the other control means and would be evidence of some syste-
matic error. The data in the following tables showed no evidence of systematic
error.
The next step is to identify specific algal-pH combinations revealing sig-
nificant cadmium removals. This was done by use of Dunnett's _t_ criterion. The
calculation uses a control and experimental mean at a given pH, the error mean
square (EMS) for that pH, and the number of experimental and control replicates.
Using Navicula pelliculosa at pH 6 for an example, one finds the experimental
mean of 5200 (Table 15), control mean and EMS of 7530 and 8.7 x 10 , respectively
(Table 14) and 6 and 12 experimental and control replicates, respectively (Fig-
ure 3) .
The calculated _t - value is therefore
7530 - 5200 = 4.99
t=
71 x 10J (1/12 + 1/6)
The calculated _t_ value is compared with tabulated values of Dunnett's _t (Steel
and Torrie, 1960, p.447) using 18 treatment means and 101 error degrees of free--
dom. The extrapolated value is 2.90. The larger calculated _t value indicates
this experimental mean is significantly different from the pH 6 control mean and
is taken as evidence of a significant removal of cadmium by Navicula pelliculosa
at this pH.
The accumulation coefficient (ug metal accumulated per gram of algal dry
weight) can be calculated,from the data in Table 15. Again using the data from
Navicula pelliculosa at pH 6, the calculation is:
0.31 x 0.366 ug/ml
0.085 mg/ml x lg/1000 mg
This assumes that all the metal removed is concentrated by the alga. Since
80 to 85% of the supernatant is collected by the cartridges, most of the remain-
ing volume is the algal cells. The assumption is therefore reasonable.
The concentration factor is simply the accumulation, coefficient divided by
the initial metal concentration. For the same data, this is:
1330 ug/g _
0.366 ug/ml ~ 363°
Slight discrepancies (such as the tabular value of 3640 versus 3630 calculated
here) are caused by rounding errors. The data in the tables were calculated and
then rounded to 3 significant digits but the calculations used values alreadv
rounded. -"-j-eauy
60
-------
.,-a-^arioa of che data in Table 15 reveals some interesting trends.
-iV3-""r--e -^ efficient accumulator of met a}, is clearly Chlorella pyrenoidosa,
"..trC'concencracicn factors on the order of 10"* over the range of pHs from 5 to 8.
••locWi-Tfi-brinaca also shows high concentration factors but only at pH 5 and
g-~~ -_ C5r,erai, the blue-green algae were not efficient accumulators of cadmium.
i1-Day alsae and lead—The experimental results for young algae and lead
are presencec'in Tables 16 and 17. The most noteworthy feature of these results
is che dramatic superiority of Chlamydomonas to accumulate lead. Between 70 and
30% of che lead was removed at each pH from 4 to 9. Concentration factors were
on the order cf 10"*. No other algae removed as much lead over as wide a pH range,
11-Dav aliae and mer.curv—Tables 18 and 19 show the results of the young
algae-mercury experiment. The highest percentage of removals were achieved by
che blue-green algae, especially Nostoc H and Nostoc 586. The acidic pHs
favored removal of mercury by these organisms. The highest concentration fac-
tors were exhibited by Chlorella pyrenoidosa, with values on the order of 10 for
all ?Hs from 4 to 10. The mixed cultures of algae (i.e., Cazenovia and Onondaga
Lake algae) removed mercury from solution but not as efficiently as the most
successful pure cultures.
11-Day algae and zinc—The data for the zinc-young algae experiment are
shown in Table"20.The application of the F-test to the data at each pH shows
chat at least one mean was significantly different from the others at pHs 7 to
10. This is in fact the case in as much as the means for Mougeotia at these
pHs were significantly higher than the control mean. Assuming a systematic
error and re-analyzing the data without the Mougeotia data, one would reach the
conclusion that no organism removed zinc to a statistically significant degree
under the conditions employed in this study. The lower counts on the control
plate at pH 9 and 10 were undoubtedly due to a pH effect because they were dupli-
cated on the other two control plates.
44-Day algae and cadmium—Tables 21 and 22 show the data from the cadmium-
old algae experiment. As in the earlier experiment with young algae and cadmium,
no single organism is superior to any other. In fact, removals are no better
than 30% (for Navicula pelliculosa at pH 8). In general, it appears that an
acidic pH favors cadmium accumulation by algae. As in all experiments with
older cultures of algae, the accumulation coefficients and concentration factors
are dramatically lower than younger cultures of the same species.
44-Day algae and lead—Data from the lead-old algae experiment are presented
in Tables 23 and 24. As in the young algae lead experiment, the single organism
that accumulates lead most effectively is Chlamydomonas. which removed between
60 and 70% of the lead at pH 4 to 9. As in the young culture experiment, there
was, no significant removal at pH 10. Concentration factors were on the order of
10 . The next most competent accumulators of lead were the blue-green Nostoc H
and Nostoc W which removed between 30 to 40% and 10 to 20% of the lead, respect-
ively. Concentration factors again were on the order of 10 . Removal was fairly
consistent from pH 4 to 9.
61
-------
TABLE 16. STATISTICAL DATA FOR LEAD - YOUNG ALGAE EXPERIMENTS
N>
Source of Variability
Treatment SS
Treatment df
*
Treatment MS
Error SS
Error df
Error MS
Total SS
Total df
Control data
n
X
0
X
pH 3
7,40
18
0.411
34.5
101
0.344
42.2
119
12
3780
520
pll 4
41.8
18
2.33
55.1
101
0.546
97.0
119
12
3010
540
pll 5
35.9
18
1.99
48.2
101
0.477
84.0
119
12
3100
390
pH 6
38.7
18
2.15
27.2
101
0.269
65.9
119
12
3210
500
pll 7
38.5
18
2.14
29.8
101
0.295
68.3
119
12
3390
730
.
pll 8
36.1
18
2.00
33.9
101
0.335
69.9
119
12
3 1 00
620
pli 9
52.9
18
1 . 94
21.2
101
0.2 JO
74 . 1
119
12
3260
380
pll 10
21.8
18
1.21
32.8
101
0.325
54 . 7
J 19
1.2
328D
330
•Jc f
All sums of squares (SS) and mean squares (MS) are x 10 .
Degrees of freedom.
-------
TABLE 17. SIGNIFICANT REMOVALS OF LEAD BY YOUNG CULTURES OF ALGAE
er>
ALGA (biomass mg/ml) pH 4 pH 5 pH 6 pH 7 pll 8
Chlamydomonas (0.04)
tt
t-value 6.46" 6.40 9.12 8.45 7.12
Mean 670 880 830 1130 950
ox 190 200 112 190 130
Removal efficiency 0.78 0.72 0.74 0.69 0.69
Accumulation coefficient 490 450 460 420 430
Concentration factor 19,500 17,900 18,600 16,700 17,400
Mougeotia (0.035)
t-value ns1t ns ns ns ns
Mean
ax
Removal efficiency
Accumulation coefficient
Concentration factor
Ulothrix fimbrinata (0.017)
t-value ns ns ns ns
Mean
°x
Removal efficiency
Accumulation coefficient
Concentration factor
pll 9 pll 10
.L.I.
11.0 ns "
640
0.80
500
20,100
3.93 ns
2320
1020
0.29
210
8180
4.65
21.50
350
0.34
500
1 y , 900
(continued)
-------
TABLE 17. (continued)
ALGA (biomass rag/ml) pH 4
Nostoc muscorum A (0.005)
t-value ns
Mean
Ox
Removal efficiency
Accumulation coefficient
Concentration factor
Nostoc 586 (0.017)
t-value ns
Mean
°K
Removal efficiency
Accumulation coefficient
Concentration factor
Nostoc H (0.23)
t-value ns
Mean
°x
Removal efficiency
Accumulation coefficient
Concentration factor
pH 5 pll 6 pH 7 pH 8
ns ns ns ns
ns 3.26 4.13 ns
2360 2290
1400 200
0.27 0.33
39 48
1560 1920
ns 4.48 ns ns
2040
200
0.36
40
1580
pH 9 pll 10
3.01 ns
2540
470
0.22
1090
43,800
5 . 64 ns
1920
330
0.41
60
2400
4.80 ns
2120
370
0.35
38
1520
(continued)
-------
TABLE 17. (continued)
Cn
ALGA (biomass mg/ml) pll 4
Oscillatoria (0.19)
t-value ns
Mean
°x
Removal efficiency
Accumulation coefficient
Concentration factor
Schizothrix calcicola (0.23)
t-value ns
Mean
a
x
Removal efficiency
Accumulation coefficient
.Concentration factor
pH 5 pH 6 pH 7 pll 8
ns ns ns ns
ns ns 3.91 ns
2340
210
0.31
35
1410
pll 9
4 . 10
2320
230
0.29
38
1520
4.00
2310
220
0.24
33
1320
pll 10
ns
4.32
2060
310
0.37
42
1690
Lead concentration 0.025 ug/ml; 1.67 pC/ral.
tt
t .-.= 2.90 at 95% confidence level.
crit.
Not significant.
-------
TABLE 18. STATISTICAL DATA FOR MERCURY - YOUNG ALGAE EXPERIMENTS
Cft
Treatment SS
Treatment df^
Treatment MS
Error SS
Error df
Error MS
Total SS
Total df
Control data
n
X
0
X
pH 3
43.1
18
2.39
136
101
1.35
179
119
12
5900
830
pH 4
122
18
6.79
54.4
101
0.539
176
119
12
4000
1230
pH 5
71.1
18
3.95
36.7
101
0.363
107
119
12
3080
1160
pH 6
87.0
18
4.83
45.9
101
0.454
133
119
12
4150
750
pH 7
33.1
18
1.84
13.5
101
0.134
46.6
119
12
2980
250
pll 8
36.9
18
2.05
34.7
101
0.344
71.7
119
12
2680
580
pll 9
40.1
18
2.23
30.0
101
0.297
70.1
119
12
2880
410
pH 10
101
18
5.60
48.5
101
0.480
149
119
12
3530
530
* -6
All sums of squares (SS) and mean squares (MS) are x 10
Degrees of freedom.
-------
TABLE 19. SIGNIFICANT REMOVALS OF MERCURY BY YOUNG CULTURES OF ALGAE
ALGA (biomass ing /ml)
Navicula pelliculosa (0.085)
t-value
Mean
°K
Removal efficiency
Accumulation coefficient
Concentration factor
Chlamydomonas (0.04)
t-value
Mean
ax
Removal efficiency
Accumulation coefficient
Concentration factor
Chlorella pyrenoidosa (0.015)
t-value
Mean
Ox
Removal efficiency
Accumulation coefficient
Concentration factor
pH 4
4.371'
2400
480
0.40
190
4700
6.90
1470
210
0.63
650
15,810
5.74
1900
260
0.53
1440
35,100
pH 5
3.73
1950
430
0.37
180
4290
4.39
1750
870
0.43
440
10,700
3.67
1970
230
0.36
980
24,000
pH 6
7.09
1760
410
0.58
280
6780
4.46
2640
310
0.36
370
9060
8.15
1400
220
0.66
1810
44,100
pH 7
4.79
2100
300
0.29
140
3460
nsft
11.5
890
110
0.70
1920
46,700
pH 8
3.15
1760
220
0.34
170
4040
ns
6.50
780
210
0.71
1940
47,300
pH 9
4.87
1550
210
0.46
220
5420
4.63
1620
420
0.44
450
10,900
7.10
950
130
0.67
1830
44,800
pH 10
3.57
2290
250
0.35
170
4130
ns
4.85
1850
340
0.48
1300
31,700
(continued)
-------
TABLE 19. (continued)
ALGA (biomass mg/ml)
Mougeotia (0.035)
t-value
Mean
°x
Removal efficiency
Accumulation coefficient
Concentration factor
Scenedesmus obliquus (0.085)
t-value
gj Mean
°x
Removal efficiency
Accumulation coefficient
Concentration factor
Ulothrix fimbrinata (0.017)
t-value
Mean
ax
Removal efficiency
Accumulation coefficient
Concentration factor
pH 4 pH 5 pll 6
ns ns 5.53
2280
270
0.45
530
ns 3.07 6.84
2150 1840
420 290
0.30 0.56
150 270
3540 6540
4.77 ns 3.81
2250 2860
730 600
0.44 0.31
1060 750
25,740 18,220
pH 7 pH 8
6.47 ns
1790
240
0.40
470
6.97 4.05
1700 1500
180 340
0.43 0.44
210 210
5040 5190
ns ns
pll 9
4.06
1780
430
0.38
450
5.70
J330
250
0.54
260
6340
3.79
1850
550
0.36
860
21,070
pll 10
4.14
2090
360
0.4.1.
480
4 . 4 7
1980
230
0.44
210
5160
4.18
2080
260
0.41
990
24,13,0
(continued)
-------
TABLE 19. (continued)
ALGA (biomass mg/ml)
Zygnema (0.030)
t-value
Mean
a
Removal efficiency
Accumulation coefficient
Concentration factor
Nostoc 586 (0.17)
t-value
Mean
ax
Removal efficiency
Accumulation coefficient
Concentration factor
Nostoc muscorum A (0.005)
t-value
Mean
0x
Removal efficiency
Accumulation coefficient
Concentration factor
pH 4 pH 5
3.34 ns
2780
560
0.31
420
10,200
9.50 8.12
520 630
130 170
0.87 0.80
210 190
5120 4680
ns ns
pH 6
6.31
2020
210
0.51
700
17,100
8.64
1240
790
0.70
170
4130
4.18
2740
490
0.34
2790
68,000
pH 7
4.50
2150
370
0.28
380
9,220
5.41
1970
360
0.33
80
1950
3.54
2330
320
0.22
1780
43,500
pli 8
3.29
1720
270
0.36
490
12,000
5.14
1180
550
0.56
140
3290
3.06
1790
220
0.33
2740
66,900
pH 9
3.74
1860
350
0.35
480
11,800
7.34
880
390
0.69
170
4080
4.62
1620
420
0.44
3590
87,500
pH 10
3.45
2330
500
0.34
460
11,300
5.67
1570
760
0.56
130
3270
3.41
2350
250
0.33
2740
66,900
(continued)
-------
TABLE 19. (continued)
ALGA (biomass mg/ml)
Nostoc H (0.23)
t-value
Mean
ax
Removal efficiency
Accumulation coefficient
Concentration factor
Oscillatoria (0.19)
t-value
Mean
°x
Removal efficiency
Accumulation coefficient
Concentration factor
Schizothrix calcicola (0.22)
t-value
Mean
°x
Removal efficiency
Accumulation coefficient
Concentration factor
pll 4
8.36
940
90
0.77
140
3330
7.01
1430
460
0.64
140
3380
6.21
1720
520
0,57
110
2590
pH 5
7.19
910
60
0.70
130
3060
6.63
1080
620
0.65
140
3420
6.19
1210
180
0.61
110
2760
pH 6
7.43
1640
190
0.60
110
2630
7.88
1490
670
0.64
140
3370
6.47
1970
200
0.53
100
2390
pH 7
6.81
1730
200
0.42
70
1820
IIS
7.69
1570
150
0.47
90
2150
pH 8
3.31
1720
730
0.36
60
1570
3.93
1530
890
0.43
90
2260
6.12
890
130
0.67
120
3040
pH 9
5.46
1390
1120
0.52
90
2240
5.75
1310
810
0.54
120
2860
7.15
930
30
0.68
130
3070
pH 10
4.14
2100
620
0.41
70
1770
ns
6.29
1350
370
0.62
110
2800
(continued)
-------
TABLE 19. (continued)
ALGA (biomass mg/ml)
Cazenovia - Bristols (0.25)
t-value
Mean
°x
Removal efficiency
Accumulation coefficient
Concentration factor
Cazenovia - Diatom (0.005)
t-value
Mean
°x
Removal efficiency
Accumulation coefficient
Concentration factor
Ononda'ga - Bristols (0.23)
t-value
Mean
°x
Removal efficiency
Accumulation coefficient
Concentration factor
pH 4 pH 5 pll 6
ns ns 6.82
1850
280
0.55
90
2220
ns ns 8.22
1380
180
0.69
5480
13,000
4.20 3.35 3.83
2460 2060 2850
430 420 2170
0.38 0.33 0.31
70 60 60
1670 1430 1350
pH 7
3.94
2260
500
0.24
40
970
5.86
1900
330
0.36
2950
71,900
5.46
1980
210
0.33
60
1460
pll 8
4.16
1470
310
0.45
70
1820
4.20
1460
130
0.46
3760
91,600
ns
pll 9
4.84
1560
250
0.46
80
1830
5 . 23
1.370
240
0 . ')2
4290
10'j, <>()<)
n:.;
pll 10
us
'( 6(,
22(>0
;no
0.3(>
2950
71 ,»()()
IKS
(continued)
-------
TABLE 19. (continued)
ALGA (biomass rag/ml)
pH 4
pll 5
pH 6
pH 7
pH 8
pH 9
pH 10
Onondaga - Diatom (0.175)
t-value
Mean /
°x
Removal efficiency
Accumulation coefficient
Concentration factor
4.24
2450
630
0.39
90
2220
4.24
1800
500
0.42
100
2370
8.94
1130
200
0.73
170
4150
7.90
1530
320
0.49
110
2770
5.16
1170
250
0.56
130
3220
5.87
1280
270
0.56
130
3170
4.72
1895
320
0 . 4 6
1.10
2640
Mercury concentration 0.041 yg/ml; 0.243 jjC/ml.
crit = 2<9° at 95* confidence level.
-Not significant.
-------
TABLE 20. STATISTICAL DATA FOR ZINC - YOUNG ALGAE EXPERIMENT
Source of Variability
Treatment SS
Treatment df
Treatment MS
Error SS
Error df
Error MS
Total SS
Total df
Control data
n
X
°x
pH 3
5.11
18
0.284
38.8
101
0.384
43.9
119
12
4500
320
pll 4
7.70
18
0.428
39.0
101
0.386
46.7
119
12
3680
740
pH 5
6.41
18
0.356
28.8
101
0.285
35.2
119
12
3710
780
pH 6
6.72
18
0.373
48.2
101
0.478
55.0
119
12
3660
560
pH 7
56.7
18
3.15
47.1
101
0.466
104
119
12
2250
750
pll 8
44.7
18
2.48
77.4
101
0.766
122
119
12
2200
750
pll 9
25.0
18
1.39
34.9
101
0.345
59.9
119
12
1190
690
pll 10
18.9
18
1.05
25.6
101
0.253
44.4
119
12
560
2.10
All sums of squares (SS) and mean squares (MS) are x 10
Degrees of freedom.
-------
TABLE 21. STATISTICAL DATA FOR CADMIUM - OLD ALGAE EXPERIMENT
Source of Variability
Treatment SS*
Treatment df "*"
Treatment MS
Error SS
Error df
Error MS
Total SS
Total df
Control data
n
X
a
X
pH 3
16.7
8
2.09
167
51
3.27
183
59
12
7020
330
pH 4
66.7
8
8.33
59.0
51
1.16
126
59
12
7010
230
pH 5
48.6
8
6.08
56.0
51
1.10
105
59
12
7140
300
pH 6
390
8
48.7
363
51
7.13
753
59
12
7570
500
pH 7
209
8
26.1
509
51
9.98
718
59
12
5190
780
pH 8
284
8
35.5
473
51
9.28
757
--59
12
4570
810
pH 9
897
8
112
471
51
9.24
1369
59
12
4290
930
pi I 10
552
8
69.1
505
51
9 . 90
1057
59
12
4860
1060
* -S
All sums of squares (SS) and mean squares (MS) are x 10 .
Degrees of freedom.
-------
TABLE 22. SIGNIFICANT REMOVALS OF CADMIUM BY OLD CULTURES OF ALGAE
ALGA (biomass mg/ml)
Navicula pelliculosa (0.95)
t-value
Mean
-------
TABLE 22. (continued)
ALGA (biomass mg/ml)
Nostoc 586 (2.03)
t-value
Mean
°x
Removal efficiency
Accumulation coefficient
Concentration factor
Nostoc muscorum A (1.88)
t-value
Mean
Ox
Removal efficiency
Accumulation coefficient
Concentration factor
Nostoc H (1.76)
t-value
Mean
ax
Removal efficiency
Accumulation coefficient
Concentration factor
pH 4
ns
4.47
6250
350
0.11
14
60
4.39
6260
300
0.11
14
60
pH 5
4.19
6450
210
0.10
11
50
4.15
6450
250
0.10
12
50
2.89
6660
510
0.07
9
40
pH 6 pit 7 pH 8 pH 9 pli 10
3.37 ns ns ns ns
6150
330
0.19
22
90
ns ns ns na ns
3.97 ns ns ns ns
5900
350
0.22
30
130
Cadmium concentration 0.238 yg/ml; 0.802 yC/ml.
t crlfc = 2.87 at 95% confidence level.
Not significant
-------
TABLE 23. STATISTICAL DATA FOR LEAD - OLD ALGAE EXPERIMENT
Source of Variability
Treatment SS
Treatment df '
Treatment MS
Error SS
Error df
Error MS
Total SS
Total df
Control data
n
X
°x
pH 3
1.16
10
0.116
14.8
61
0.243
16.0
71
12
6470
790
pll 4
134
10
13.4
8.17
61
0.134
143
71
12
6290
280
pll 5
101
10
10.1
8.13
61
0.133
109
71
12
6290
340
pH 6
138
10
13.8
7.75
61
0.127
146
71
12
6560
470
pH 7
101
10
10.1
9.70
61
0.159
111
71
12
6660
310
pll 8
99.6
10
9.96
11.0
61
0.181
111
71
12
6680
310
pll 9
114
10
1.1.4
7.58
61
0.124
122
71
12
6380
380
pH 10
18.9
10
1.89
1.3.0
61
0.213
:n .9
71
1.2
5080
270
*
All sums of squares (SS) and mean squares (MS) are x 10.
Degrees of freedom.
-------
TABLE 24. SIGNIFICANT REMOVALS OF LEAD BY OLD CULTURES OF ALGAE
00
ALGA (biomass mg/ml)
Chlamydomonas (1.96)
t -value
Mean
°x
Removal efficiency
'Accumulation coefficient
Concentration factor
Gleotrichia (1.80)
U— value
Mean
°x
Removal efficiency
Accumulation coefficient
Concentration factor
Nostoc 586 (2.03)
t-value
Mean
°x
Removal efficiency
Accumulation coefficient
Concentration factor
pH 4
,
23.9"
1910
210
0.70
13.0
350
8.94
4660
450
0.26
5.40
140
4.02
5560
360
0.12
2.20
60
pH 5
20.4
2570
400
0.59
11.0
300
8.40
4680
430
0.26
5.20
140
ns
pll 6
26.0
1930
110
0.71
13.0
360
9 . 6.1
4850
390
0.26
5.40
150
6.83
5340
280
0.19
3.40
90
pll 1
19.4
2780
380
0.58
11.0
300
ns
5.79
5510
150
0.17
3 . 20
90
pH 8
18.5
2750
250
0.59
11 . 0
300
ns
3.88
5860
320
0.12
2.30
60
pll 9 pll 10
it
23.9 ns
2170
120
0.66
13.0
340
3.79 ns
57.1.0
760
0..1.0
2.20
58.0
8.46
4890
.190
0.23
4.30
1.20
(continued)
-------
TABLE 24. (continued)
11 ' • _.._.. - ..._.-..-.._.. -
ALGA (biomass mg/ml)
Nostoc H (1.76)
t-value
Mean
Ox
Removal efficiency
Accumulation coefficient
Concentration factor
Nostoc L_ (1.56)
t-value
Mean
°x
Removal efficiency
Accumulation coefficient
Concentration factor
Nostoc W (1.29)
t-value
Mean
°x
Removal efficiency
Accumulation coefficient
Concentration factor
pH 4
10.7
4340
500
0.31
6.6
180
2.92
5760
260
0.09
2.00
50
5.20
5340
740
0.15
4.40
120
pll 5
8.50
4740
500
0.25
5.3
140
ns
5.61
5270
420
0.16
4.70
130
pll 6
14.6
3950
210
0.40
8.50
230
ns
6.65
5370
220
0.18
5.30
140
pH 7
11.1
4450
320
0.33
7.00
190
ns
5.68
5530
250
0.17
4.90
130
pll 8 pll 9 pll 10
8.61 13.9 ns
4850 3930
270 310
0.27 0.38
5.80 8.20
160 220
ns 3.63 ns
5740
200
0.10
2.40
60
ns 7 . 00 ns
5150
390
0.19
5.60
150
(continued)
-------
TABLE 24. (continued)
oo
o
ALGA (blomass mg/ral)
Schizothrlx calcicola (1.95)
t-value
Mean
0X
Removal efficiency
Accumulation coefficient
Concentration factor
Oscillatoria (1.73)
t-value
Mean
Ox
Removal efficiency
Accumulation coefficient
Concentration factor
pH 4
3.83
5590
210
0.11
2.10
60
15.6
3440
170
0.45
9.80
260
pll 5
ns
4.41
5490
350
0.13
2.80
70
pH 6
6.52
5400
200
0.18
3.40
90
12.2
4390
320
0.33
7.10
190
pH 7
5.39
5590
360
0.16
3.10
80
5.45
5570
590
0.16
3.50
90
pH 8
3.63
5910
160
0.12
2.20
60
5.91
5430
460
0.19
4.10
110
pH 9 pH 10
4 . 94 ns
5510
210
0.14
2.60
70
12.0 ns
4260
320
0.37
7.20
190
Lead concentration 0.0375 yg/ml; 0.25 yC/ml.
tt,
t . = 2.89 at 95% confidence level.
crit
Not significant.
-------
TABLE 25. STATISTICAL DATA FOR MERCURY - OLD ALGAE EXPERIMENT
Source of Variability
Treatment SS *
Treatment df '
Treatment MS *
Error SS
Error df
Error MS
Total SS
Total df
Control data
n
X
. %
pll 3
5.85
5
1.17
14.6
30
0.487
20.5
35
6
3080
110
„ ,
pll 4
139
5
27.8
2.74
30
0.0913
142
35
6
2440
110
pll 5
88.1
5
17.6
3.02
30
1 . 01
91 . 1
35
6
.1870
90
~
pll 6
51.1
5
10.2
6.36
30
2.12
57.4
35
6
1650
80
pll 7
29.1
5
5.82
14 . 9
30
0.499
44.1
35
6
1440
130
pll 8 pll 9 pll 10
18.2 10 J .17.1.
5 5 5
3.64 32.2 34.1'
4.67 3.63 JO. 4
30 30 30
0.156 0.1.21 0.347
22.9 1.65 1-1.1
35 35 3.')
6 (. <>
1.1.90 .1450 .1.580
180 1.40 160
All sums of squares (SS) and mean squares (MS) are x 10''.
Degrees of freedom.
-------
44-Day algae and mercury—All five of the organisms screened in the nercury-
old algae experiments showed significant removals of netal, as revealed by die
data in Tables 25 and 26. Indeed, all^the algae showed remarkably similar con-
centration factors (on the order of 10") and removal efficiencies (generally 50%
to 70%). Once again, as in the young culture experiments, the acidic pHs favored
mercury removal by the algae.
44-Day algae and zinc—The data for the zinc-old algae experiments are pre-
'sented in Table 27. F-tests reveal significant effeccs at pH 9 and 10. However,
as in the earlier experiments with zinc, the effects are probably related to
changes in pH rather than to actual metal accumulation.. The raw data at pH 10
reveal that most treatment means are significantly higher than the control means,
indicating that the experimental wells with algae apparently prevented the for-
mation of insoluble zinc compounds like those that occurred in the control wells
at pH 10.
Results from other Microtiter Assays (Protocol II)
Experiments Using Various Buffers at pH 6—
The ease with which data could be obtained with the new technique (Protocol
II) led to the design of several experiments to answer more specific questions
raised by the earlier results. One such experiment concerned the choice of buf-
fers.
Chlamydomonas was chosen as the test organism and lead as the test inetal.
Several buffers, all at pH 6, were made up as described in Table 28. For this
experiment, plates were made up in the normal manner with each group of 6 experi-
mental wells having 6 control wells with the same buffer. Samples were obtained
after, the cells had been exposed to the buffer-metal combination for both 3 and 6
hours. A row of wells at pH 3 and without algae was utilized as an interplate
control (IPC) as in previous experiments.
The results of this experiment (Table 28) show that the choice of buffer can
affect the accumulation of lead by Chlamydomona s. One buffer, the citric acid-
sodium citrate buffer, effectively prevented the uptake of lead by Chlamydomonas,
probably because of the formation of a soluble complex. Citrate is well known as
a chelating agent for certain metals (e.g. iron) and is often added to culture
media to make the metallic micronutrients available for the organisms (Nichols
1973).
Except for the citrate buffer, all other buffers showed surprisingly similar-
results of removal efficiencies, accumulation coefficients, and concentration
factors.
Experiments Over a 24-Hour Period
The improved technique (Protocol II) was used to gather data on metal accumu-
lation by algae over a longer period of time. Algae found to accumulate metals
were selected from the data of the screening experiments and representative pHs
chosen. Plates were prepared in two duplicate 48-well (4 row by 12 column) sect-
ions, with each section having 5 groups of 6 experimental wells, two algae at
each of two pHs, one alga at a third pH, and 3 rows of 6 control wells (one row
for each pH). Four plates were prepared for each radionuclide used in this
82
-------
TABLE 26. SIGNIFICANT REMOVALS OF MERCURY BY OLD CULTURES OF ALGAE
ALGA (biomass mg/ml)
pH 4 pH 5
pll 6
PH 7
pH 8 pH 9
pll 10
Scenedesmus obliquus (1.88)
oo
t-value
Mean
°x
Removal efficiency
Accumulation coefficient
Concentration factor
Gleotrichia (1.80)
t-value
Mean
°x
Removal efficiency
Accumulation coefficient
Concentration factor
Nostoe 586 (2.03)
t-value
Mean
ox
Removal efficiency
Accumulation coefficient
Concentration factor
22.1'
1210
100
0.50
3.84
270
19.3
750
140
0.60
4.58
320
11.8
660
150
0.60
4.60
320
35.3
490
70
0.80
6.40
22.7
560
130
0.70
5.60
9.49
1080
180
0.48
3.90
440
24.7
1070
90
0.56
4.00
280
390
23.7
500
50
0.73
5.20
360
270
12.7
590
240
0.65
4.60
320
5.95
670
80
0.53
4 10
280
8.11
600
110
0.49
3.80
260
10.9
750
150
0.48
3.70
250
2.77
1080
520
0.25
2.00
140
5.89
680
20
0.53
3.80
260
5.39
800
210
0.33
2.60
180
8.52
580
40
0.51
3.70
250
ns
ns
9.54
550
60
0.65
5.00
350
ns
4.23
1120
300
0.29
2.10
140
(continued)
-------
TABLE 26. (continued)
oo
-P.
ALGA (biomass rag/ml)
Nostoc H (1.76)
t-value
Mean
Removal efficiency
Accumulation coefficient
Concentration factor
Schizothrix calcicola (1.96)
t-value
Mean
Removal efficiency
Accumulation coefficient
Concentration factor
pH 4
27.1
940
80
0.61
5.00
350
30.9
730
120
0.70
5.20
360
pll 5
23.4
520
80
0.72
5.90
410
23.6
510
70
0.73
5.40
370
pi! 6
11.5
680
50
0.59
4.80
330
12.8
580
60
0.65
4.80
330
pll 7
4.60
840
70
0.41
3.40
240
6.05
600
30
0.54
4.00
280
pn 8
6.28
740
50
0.38
3. 10
220
9.41
510
70
0.57
4.20
290
pll ')
iiri
14 . 5
530
40
0.64
4.70
330
pll 1(1
3.91
1.1 SO
81)
0.27
2.20
.1.30
8 . 3 '.>
670
130
0.57
4.20
290
t
Mercury concentration = 0.0144 yg/ml; activity = 0.086 uC/ml.
Tcrit = 2°72 at the 95% confidence level.
Not significant.
-------
TABLE 27. STATISTICAL IJA'I'A FOR XING - OLD ALGAE EXPKKIMKNT
Source of Variability
*
Treatment SS
Treatment df
*
Treatment MS
Error SS
Error df
Error MS
1
Total SS
DO
01
Total df
Control data
n
X
**
pH 3-
9.96
8
1.24
104
51
2.04
114
59
12
6410
510
pll 4
16.4
8
2.05
69.1
51
1.35
85.5
59
12
6220
360
. ,
pll 5
12.8
8
1 . 60
67.9
51
1.33
80.7
59
12
6350
380
pll 6
29.9
8
3.74
94 . 0
51
1.84
1.24
59
12
6410
360
pll 7
36.5
8
4 . 56
121
51
2.37
158
59
12
6760
530
pll 8 pll 9 pll 10
.1.2.2 .114 3(.o
8 8 8
1 . 53 .1.4 .2 45.8
73.6 296 176
51 5.1 51
1.44 5.80 3.46
85.8 4.10 '343
59 59 59
12 12 .12
6520 6460 720
1.90 340 540
All sums of squares (SS) and mean squares (MS) are x 10 ".
Degrees of freedom.
-------
TABLE 28. RESULTS* OF BUFFER COMPARISON EXPERIMENT...
(pH 6) USING CHLAMYDOMONAS; AND LEAD 210'
Buffer Time
3 hr 6 hr
s
pH 6 Borax-phosphate (Borax and potassium dihydrogen phosphate)"
Control mean 24,830 24,300
Control ax 1030 850
Experimental mean 11,500 10,400
Experimental a 520 230
Removal efficiency 0.54 0.57
Accumulation coefficient 115 123
Concentration factor 1920 2040
pH 6 Citrate (sodium citrate and citric acid) s
Control mean 24,800 24,000
Control ax 1270 600
Experimental mean 22,900 22,400
Experimental ax 1970 1660
Removal efficiency 0.08 0.07
Accumulation coefficient 16 14
Concentration factor 270 240
pH 6 Succinate (succinic acid and sodium hydroxide) '
Control mean 23,600 22,600
Control ax 1060 960
Experimental mean 10,500 9600
Experimental a 200 470
Removal efficiency 0.56 0.58
Accumulation coefficient 119 123
Concentration factor 1980 2050
pH 6 Citrate-phosphate (citric acid and sodium monohydrogen
phosphate)
Control mean 23,260 22,600
Control a 800 960
Experimental mean 12,100 9600
Experimental a^ 500 470
Removal efficiency 0.48 0.58
Accumulation coefficient 103 123
Concentration factor 1710 2050
(continued)
86
-------
TABLE 28. (continued)
Time
Buffer
3 hr
6 hr
pH 6 Phthalate (potassium hydrogen phthalate and sodium hydroxide)
Control mean
Control ax
Experimental mean
Experimental a
Removal efficient
Accumulation coefficient
Concentration factor
21,700
700
10,500
350
0.52
110
1840
pH 6 Tris-maleate (tris, maleic anhydride and sodium hydroxide)
§
For sample calculations see Section II. B. 2. of Chapter 4.
Twelve day culture of algae; dry weight = 0.28 mg/ml.
21,300
570
9,600
230
0.55
118
1960
Control mean
Control a
Experimental mean
Experimental a
Removal efficiency
Accumulation coefficient
Concentration factor
21,700
700
9,400
390
0.57
121
2,020
20,200
800
7,700
220
0.62
133
2,210
§
Lead concentration 0.06 yg/ml.
Buffers made up as in Handbook of Biochemistry and Molecular Biology (3rd ed.)
87
-------
study—Cd, Pb, He, ?b and Zn. Forcy-eight wells at a time were sampled by use of
one rack containing the adsorption cartridges. Samples were withdrawn at 0, 0.5,
1, 3, 6, 9, 12, and 24 hours. The plates were kept in the dark at 22°C.
Data fro:?, these experiments are shown as Figures 12 to 17.
Cadmium—Test species utilized in the cadmium uptake experiment were
Navicula pelliculosa (at pH 6, 7 and 8) and Scenedesmus obliquus (at pH 6 and 7).
Neither alga was found to accumulate cadmium, a. finding inconsistent with earlier
results. The control values for cadmium dropped inexplicably over the 24 hour
period to the sane extent as the experimental wells masking any uptake by either
of the algae.
Lead—The results for the lead experiment are shown as Figures 12 through 14.
For this experiment, the cultures were 15-day cultures; Chlamydomo nas had a dry
weight of 0.35 ng/nl while Nostoe 586 produced 0.27 mg/ml of dry weight. Once
again, the efficiency of Chlamydomonas in removing lead is striking, with initial
removal being most rapid at pH 6, where after only 30 minutes, experimental value
was less than 50% of the control value. After 24 hours, experimental values at
all pHs had risen to about 70% of the control values, perhaps as a result of a
slow leaching of lead-organic complexes from the cell surface. Nostoc 586 was
somewhat less efficient at removing lead in this experiment.
Mercury—The dramatic and rapid removal of mercury by the blue-green Nostoc
H (15-day culture, 0.36 mg/ml dry weight) and Nostoc 586 (15-day culture, 0.27
rag/al dry weight) was once again noted and is shown in Figures 15 to 17. A slow
downward trend of control values at every pH may be explained by physical adsorp-
tion of the mercury on the plastic surfaces of the individual wells. Final ex-
perimental values were less than 20% of the control value at pH 6 for both strains
of Nostoc at pH 6.
Zinc—No uptake was noted for zinc, a finding that was consistent with
earlier results. Test species were Nostoc 586 (at pH 6 and 7) and Chlamydomona s
at pH 6, 7, and 8).
88
-------
CO
12,000-
Z
OL
UJ
Q.
ID
V)
10,000
8,000
o:
UJ
a.
H-
2
13
O
O
6,000-
4,000
O
CHLAMYDOMONAS
0
TIME (hours)
Figure 12. Uptake of lead - 210 at pH 6.
-------
14,000-J
h-
<
2
£E
LU
Q.
=5
HI
h-
2
5
on
LJ
Q.
I-
2
O
O
12,000-
CONTROL
NOSTOC 586
CHLAMYDOMONAS
TIME (hours)
Figure 13. Uptake of lead - 210 at pH 7.
-------
>o
Z
o:
UJ
o.
UJ
Z
2
DC
UJ
Q.
ID
O
O
14,000
12,000-
± 10,000-
8,000-
6,000-4-
0
l
3
O •
A -
• CONTROL
• NOSTOC 586
• CHLAMYDOMONAS
12
15
18
21
i
24
TIME ( hours )
Figure 14. Uptake of lead - 210 at pH 8.
-------
5,000-
vo
N
a:
Ld
o_
Z>
O)
LU
h-
o:
UJ
o.
o
o
4,000
3,000-
2,000-
1,000-
TIME (hours)
Figure 15. Uptake of mercury - 203 at pH 5.
-------
VO
OJ
4,000
Z
DC.
UJ
O.
3
CO
3,000
2,000-
o:
UJ
a. /
CO
o
o
1,000-
CONTROL
NOSTOC 586
NOSTOC H
0
TIME (hours)
Figure 16. Uptake of mercury - 203 at pH 6.
-------
O • • • CONTROL
a ... NOSTOC H
0
TIME (hours )
Figure 17. Uptake of mercury - 203 at pi I 7.
-------
SECTION 7
DISCUSSION OF PROJECT RESULTS
The results of previous work, summarized in Section 4, were organized
using the concentration factor (CF) as the means by which che ability of
various algae to accumulate heavy metals was evaluated. Since chis parameter
was calculated for the algae used in the second phase of this study, a comparison
seems .appropriate and is provided in Tables 29 through 31.
Several comments about the data in these tables can be made. First pub-
lished works by several investigators have shown zinc to be accumulated by a
wide variety of algae, while this study has not yet found zinc to be amassed
to any significant degree. However, previously cited work by Broda et al.
(1971) indicated that zinc uptake by a species of Chlorella was light dependent.
Since the present study represents an initial screening effort in which all cul-
tures were kept in the dark, it is possible that this arbitrary selection of
initial conditions prevented zinc uptake by the test algae employed in this
study. An alternative hypothesis is that test algae did not accumulate zinc
under any conditions.
With the exception of zinc, our results show reasonably good agreement
with those reported in the literature, with all concentration factors between
studies being within a factor of 2 to 3. As Cope land and Aver (1972) point
out, a concentration factor cannot be considered a fixed number, but is rather
a variable dependent on environmental conditions. The agreement between these
and previous studies seems reasonable.
Previous studies that have also examined the kinetics of heavy metal accumu-
lation by algae,in general,report that uptake was an extremely rapid phenomenon
(Schulze, Baldes and Lewin, 1976; Richardson, Millington and Miles, 1975; Kemp-
ner and Miller, 1972). The results of the 24 hour experiments in this study
clearly confirmed the earlier observations in this regard. The negative results
for the cadmium experiments cannot be explained at this time.
One obvious manner in which the present study extends previous work is
the calculation of concentration factors for species of algae previously un-
studied. These data (Tables 29, 30, & 31) were obtained under identical con-
ditions and record both intraspecific (in the case of Nostoc) and interspecific
differences in heavy metal accumulation by algae. New data are provided on the
effect of culture age and initial pH during the labelling period on the accumu-
lation of heavy metals by algae (Tables 29, 30, & 31) and deserve some comment.
The present study about doubles the number of algae for which concentrat-
ion factors for cadmium have been determined (Tables 14 & 15). This is not only
95
-------
TABLE 29. CONCENTRATION FACTORS FOR CADMIUM DETERMINED DURING THIS STUDY
VO
Organism
Navicula pelliculosa
Chlamydomonas
Chlorella
Scenedesmus obliquus
Ulothrix flmbrinata
Zygnema
Gleotrichia
Nostoc 586
Oscillatoria
Nostoc H
Type
Diatom
Green, flagellated
Green, colonial
Green, colonial
Green, filamentous
Green, filamentous
Blue-green, filamentous
Blue-green, colonial
Blue-green, filamentous
Blue-green, colonial
1 2
Culture Age Concentration Factor
Young
Old
Young
Young
Young
Old
Young
Young
Old
Young
Old
Young
Old
3.
70
5.
1.
2.
40
1.
8.
70
1.
50
1.
40
6 - 4.9 x
- 320
5 x 103
7 - 3.2 x
7 - 3.6 x
- 110
1 - 1.4 x
6 x 10
- 160
1 - 1.5 x
- 90
2 - 1.3 x
130
10J
io4
io3
io4
io3
io3
Notes: 1. Young = 11 day; Old
2, Concentration Factor
= 44 day.
= concentration of metal in
algae
concentration of metal in medium
-------
TABLE 30. CONCENTRATION FACTORS FOR LliAI.) AS DKTEKMl.NF.I) UUKINC THIS STUDY
VO
__________ .,..,.,. .... . _,-,..- — . - , - , - .
Organism
Clilamydomonas
Mougeotia
Ulothrix flmbrinata
Nostoc 586
Nostoc muscorum A
Nostoc H
Nostoc L
Nostoc W
Oscillatoria
Schizothrix calcicola
Type
Green, flagellated
Green, filamentous
Green, filamentous
Blue-green, colonial
Blue-green, colonial
Blue-green, colonial
Blue-green, colonial
Blue-green, colonial
Blue-green, filamentous
Blue-green, colonial
1.
Culture Ayu
Young
Old
Young
Young
Young
Old
Young
Young
Old
Old
Old
Young
Old
Young
Old
(.'(') net'
1.7 -
3.0 -
8.2 x
2.0 x
1.6 -
1.4 x
4.4 x
1.5 -
1.4 -
50 -
1.2 -
1.5 x
90 -
1.3 -
60 -
•)
n(*. rat i on Ivic tor
.- . . . A . -
2 .0 x 10
•;
3.6 x 1.0"
10 3
io4
2.4 x 10^
1.5 x 10
10*
1.6 x 10^
2.2 x 10.
60
1.5 x IO2
io3
260
1.7 x IO3
90
Notes: 1. Young = 11 day; Old = 44 day.
2. Concentration Factor = concentration of
metal in algae
concentration of metal in medium
-------
TABLE 31. CONCENTRATION FACTORS FOR MERCURY AS DETERMINED DURING THIS STUDY
00
Organism
Navicula pelliculosa
Chlamydomonas
Clilorella
Mougeotia
Scenedesmus obliquus
Ulothrix fimbrinata
Zygnema
Gleotrichia
Nostoc 586
Nostoc muscorum A
Nostoc 11
Oscillatoria
Schizothrix calcicola
Type
Diatom
Green, flagellated
Green, unicellular
Green, filamentous
Green, colonial
Green, filamentous
Green, filamentous
Blue-green, colonial
Blue-green, colonial
Blue-green, colonial
Blue-green, colonial
Blue-green, filamentous
Blue-green, colonial
Culture Age
Young
Young
Young
Young
Young
Old
Young
Young
Old
Young
Old
Young
Young
Old
Young
Young
Old
Concent. rat : i.on Factor
3.5 - 6.8 x
9.1 x JO"3- 1.6 x IO4
2.4 - 4.7 x 10
1.1 - 1.3 x 10*
3.5 - 6.5 x 10~
2.5 - 3.5 x 10
1.8 - 2.6 x .IO4
9.2 x 1.03- 1.7 x IO4
1.4 - 4.4 x IO2
1.9 - 5.1 x 1.0^
1.4 - 3.6 x .1.0
4.4 - 8.8 x l.O4
1.6 - 3.3 x I0:j
J.5 - 4.J x 101-
2.3 - 3.4 x I.()J
2.2 - 3.7 x I0;j
2.9 - 3.7 x 10
Notes: 1. Young = 11 day old; Old = 44 day.
2. Concentration Factor = concentration of metal in algae
concentration of metal in medium
-------
important to waste treatment applications, but also to environmental impact
studies. Also noteworthy is the close agreement of concentration factors from
the literature with those of this study, with CFs on the order of 10 common to
both. What is not immediately apparent is the generally lower CFs for the blue-
green algae. In fact, three 10-day old cultures of blue-green algae, Nostoc^
mu sco rum A, Nostoc H and Schizothrix calcicola did not remove cadmium signifi-
cantly at any pH, while only one green algae, Mougeotia, showed such negative
results. The absence of blue-green algae in Table 29 leads one to wonder xrtiether
they were examined by other workers and found not to accumulate cadmium or
whether the present study represents the first effort to study the uptake of
cadmium by blue-green algae. Although the total number of species examined is
small, the present study suggests that green algae are somewhat more efficient
at accumulating cadmium than the blue-green algae.
3 4
Concentration factors for lead are generally on the order of 10 to 10
for all algae studied. All the blue-green algae screened were successful at
removing lead at least at one pH while the diatom Navicula pelliculosa and the
green Chlorella and Scenedesmus obliquus were not successful at accumulating
lead at any pH. As will be discussed below, Chlamydomonas proved to be the
most efficient at removing lead from solution.
Mercury was demonstrated again (Tables 18 and 19) to be concentrated avidly
by most species of algae. Every organism screened in this study removed mercury
at least at one pH, with concentration factors on the order of iQ'l to 101*. Algae
may be one of the most effective means for removing this element. Ben-Bassat
and Mayer (1975) and DeFillipis and Pallaghy (1976) found that algae can reduce
mercuric ion to elemental mercury which is subsequently volatilized to the at-
mosphere. One might well ask whether the same phenomenon occurred in our ex-
periments, giving artificially high concentration factors. Although the possi-
bility cannot be denied categorically, the short time scale of our experiments
(3 hours) and the quiescent conditions under which the plates were held would
argue against loss of very much mercury to the atmosphere. This is especially
true when one recalls that most other studies employed aerated cultures and a
time scale of several days. Ben-Bassat and Mayer (1977) also indicated that
the reducing f actor (s) were extracellular. Since the algal cells in the present
study were washed and resuspended in distilled water before introduction to the
assay plates, the concentration of any reducing f actor (s) would be diminished,
at least initially. However, the phenomenon of mercury reduction and volatili-
zation by algae clearly deserves further study.
Another contribution of this work was the systematic study of the effect
of pH on heavy metal accumulation. The studies cited from the literature were
generally field observations during which the pH of the surroundings varied
naturally, or were laboratory studies where the culture media characteristically
determined the pH of metal uptake, and therefore, uptake across a wide variety
of pH conditions could not be determined. In general, those algae which are
most proficient at removing metals will remove them over a . wide pH range .
For example, Chlamydomonas removes lead across a pH range from 4 to 9 at about
the same efficiency (0.69-0.80) while the less effective alga Mougeotia removes
lead only at pH 9 and then only at an efficiency of 0.29. Clearly Chlamydomonas
is more competent at removing lead from solution. nuamyaomonas
99
-------
Other observations made during this study tend to confirm and extend sug-
gestions made by earlier workers. For example, Hessler (19/4) hvpothesized that
the flagella of Platvmonas subcordiforais provided the. sites for a significant
fraction of the lead taken up by this algae. U'-iile this hypothesis was noc spe-
cifically tested in the laboratory, the dramatic superiority shown by Chlamydom-
onas in removing lead, as compared with the other algae evaluated combined with
the fact that Chlainydomonas was the only flagellated fora used, argues for che
importance of the flagella sites of lead accumulation.
Kerfoot and Jacobs (1976) noted that cadmium was accumulated to a greater
extent by a green playtomonad than by a mixture of diatoms. As noted above, the
green algae (especially Chlorella)screened in this study seemed to be more effic-
ient accumulators of cadmium than either the diatom (Navicula pelliculosa) or the
blue-green algae employed.
Perhaps one of the most important contributions of this work, however, was
the adaption and development of microtiter equipment, especially the litertek
supernatant collection apparatus, to the study of the problem of algal uptake
of heavy metals. The technique and procedures developed during the course of
this study have already provided usable data on a relatively limited number of
variables. Future work will no doubt utilize the rapidity and sensitivity of
the technique to accumulate more data covering a wider range of variables.
100
-------
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107
-------
APPENDIX
Tables of Concentration Factors Arranged by Metal
TABLE A-l. ARSENIC CONCENTRATION BY ALGAE
I-*
0
oo
Organism
Benthic
algae
Brown
algae
Phytoplankton
Type Habitat
Assorted Marine
it it
Lake
Michigan
Type of
Observation
Radioecological
Ecological
ii
Concentration
Factor
2xl03
2xl02 -6xl03
1.5xl()3
Basis References
Wet Lowman, et al.
11 liowen 1.966
" Copeland and
Ayers 1972
-------
TABLE A-2 CADMIUM CONCENTRATION BY ALGAE
Organism
Benthic algae
Plankton
Brown algae
Fucus
vesiculosis
Porphyra
umbilical is
Prasinecladus
subsala
Phaeodactylum
tricornutum and
Chaetoceras sp-
Cladophora
fracta
C. glomerata
Mougeotia sp-
Spirogyra crassa
Type
Assorted
n
n
Brown,
macroscopic
Red
macroscopic
Green,
flagellated
Diatoms
Green ,
filamentous
..
n
n
Habitat
Marine
n
n
U.K.
Coast
M
Lab culture
(marine)
n
Fresh
Water
n
n
n
Type of
Observation
Radioecological
Ecological
ii
n
n
Stable isotope
n
Radioecological
n
n
M
Concentration Basis Reference
Factor
2
2x10 Wet Lowman, et al.1971
9.1xl02 " Bowen 1966
8.9xl02 " "
3
2.7x10 Dry Preston, et al.
1972
6.6xl02 "
6.7x10 " Kerfoot and Jacobs
1976
AxlO3
1.6xl04" Polikarpov 1966
1.7xl04 »
2.8xl03 "
3.3xl03 "
-------
TABLE A-3 COPPER CONCENTRATION BY ALGAE
Organism
Phytoplankton
Phytoplankton
Benthic algae
Phytoplankton
Plankton
Brown algae
Fucus
vesiculosis
Fucus
vesiculosis
Pprphyra
umbilicalis
Fucus
vesiculosis
Ascophyllum
nodosum
Chondrus
Type Habitat TyPe °f Concentration Basis Reference
Observation factor
Assorted Lake Michigan
" Monterey Bay
" Marine
ii ii
ii ii
ii it
Brown, U.K.
macroscopic Coast
ti ii
Red , "
macroscopic
Brown, Menai
macroscopic Straights, U.K.
n ii
Red, Sea of Japan
Ecological 1.2xl03 Dry Copeiund & Ayers 1972
3
" 2.8x10 Dry Martin & Knauer 1972
2
Radioecological 1x10 Wet Lowman, et al. 1971
3xl04
Ecological 1.7x10* " Bowen 1966
" 1.0-9.2x10*
" 2.5-2.7x10 Dry Bryan & Hummer stone
" 1975
" 4.5-xlO3 " Preston, et al. 1972
6.3xl03
" 2.5-6.4xl03 " Foster 1976
8.6xl03
2.3xl03 " Saenko, et a.l . 1976
yendoi
macroscopic
(continued)
-------
TABLE A-3.(continued)
Organism TyPe Habitat
Ptilota Red, Sea of
filicina macroscopic Japan
Polysiphonia " "
japonica
Rhodomela " "
latrix
Heterochordaria Brown, "
abietina macroscopic
Chorda filum " "
Laminaria " "
chichoriodes
JL. japonica " "
Agarum " "
cribrosum
Pelvetia " .•
wrightii
Sargassum " "
pallidum
Enteromorpha Green, "
prolifera macroscopic
Ulva " "
fenestrata
Type of Concentration Basis Reference
observation factor
Ecological 5.2xl03 Dry Saenko, et a.l . 1976
2.8xl03
l.SxlO3
8xl02
5.6xl02
1.6xl03
3.6xl02
•• 9xlQ2 „ „
1. 4.4xl02 " "
11 1.7xl03 "
2.8xl03
11 8.4xl03 "
(continued)
-------
TABLE A-3.(continued)
Organism
Ulvaria
splendens
Cod ium
yessoensis
Chlamydomonas
nivalis
Mougeotia
Zygnema
Ulothrix
Cladophora
Spirogyra
Oedogonium
Triboneraia
Type
Green,
macroscopic
it
Green,
flagellated
Green,
filamentous
it
it
n
Green,
filamentous
n
Yellow-
Habitat Type of
Sea of Ecological
Japan
ii n
Greenland "
snow
Polluted "
Stream
n n
n ii
n n
Polluted "
stream
n n
Polluted "
Concentration Basis Reference
5.7xl03 Dry Saenko, et al. 1976
5xl02 " "
25 " Fjerdingstad 1973
A
1.2x10 " Trollope & Kvans .1976
3.5xl04
l.OxlO4
1.8-3.5xl04 "
3.3xl03-1.4xl04"
6.6xl03
1.3-8.3xl04
Coccomyxa
green, fil-
amentous
Green,
colonial
5x10
Oscillatoria
Blue-green "
filamentous
2.10
(continued)
-------
TABLE A-3. (continued)
Organism Type
Scenedesmus Green,
colonial
Scenedesmus "
tt
Scenedesmus "
Habitat Type of
observation
Lab Stable isotope
culture
n n
n ii
Concentration Basis Reference
factor
3.7-4.0xl03 Dry Stokes 1975
2.5-3.0xl03
7.6xl03-4.1xl(/'" "
* Laboratory strain.
t Strain isolated from metal polluted lake.
tt Low value in absence of nickel: high value in presence of 3.0 pp. Ni.
-------
TABLE A-4, LEAD CONCENTRATION BY ALGAE
- " " - ..:.-.;.—- T- , - - *,-.,- : . - - - - - - -
Organism
Benthic algae
Phytoplankton
Plankton
Brown algae
Fucus
vesiculosa
Porphyra
umbilicalis
Phaeodactylum
tricornutum
Platymonas
subcordif orm i s
Mougeotia sp
Tribonema
Coccpmyxa
Zygnema
Type Habitat
Assorted Marine
ii ii
Brown, U.K.
macroscopic Coast
Red , "
macroscopic
Diatom Lab culture
(marine)
Green,
flagellated
Green, Polluted
filamentous
Yellow-green, "
filamentous
Green, "
colonial
Green, "
filamentous
Type of observation Concentration Basis Reference
factor
2
Radioecological 7x10 wet Lowman, et al.
1971
" Axl 0 " lf
Ecological 4.1xl04 " Bowen 1966
7xl04
3
" 2.4x10 Dry Preston, et al
.1.972
" 2.103
Stable isotope 2x10 " Scliu.l./.-dia Ides
S, Lewi n .ll)7i'>
IxlO3
Ecological 2x10 " Tiro 1.1 ope and
I'jvans 1 '.i7d
3.0xlOJ-1.8xl.04"
tl •) i I , j ' kl H
" 2.5xl04
(con I" i in
-------
TABLE A-4. (continued)
in.
Organism
Ulothrix
Oscillatoria
Microapora
Cladophora
Spirogyra
Oedogonium
Type Habitat
Green, Polluted
filamentous stream
Blue-green "
filamentous
Green, "
filamentous
n n
n n
n n
Type of observation Concentration Basis
factor
3
Ecological 7.6x10 Dry
5.6xl03
" 7.4xl02
" 2.2-9.0xl02 "
4.0xl02-3.8xl03"
" 5.7xl02
Reference
Trollope and
Evans 1976
"
"
: n
n
II
-------
TABLE A-5. MERCURY CONCENTRATION BY ALGA)::
Organism
Phytoplankton
Phytoplankton
Brown algae
Cladojjhora fracta
C. glomerata
C. glomerata
Pediastrum
boryanum
Synedra ulna
Chlorella+
ft
Chlorella
Phaeodactylum
tricornutum
Type
Type Habitat Observation
Assorted Lake Ecological
Michigan
" Monterey Bay "
" Marine "
Green, Fresh water Radioecological
filamentous
ii it ii
" Lab culture Radioisotope
Green, "
colonial
Diatom " "
Green, " "
unicellular
ii n ii
Diatom Lab culture "
(marine)
Concentration
factor
5 . 9xl03
2.2xl02
2.5xl02
5.9xl03
5.4xl03
6.5xl01-5.6xl03
1.8x10^
1.3-1.7xl.04
7.101xAx8x.l02
? 1
1.6x10-1.2x10
1.3xl04
Basis Reference
Wet Copelaiul and
Ayers, 19/2
" Knauer and
Martin, 1972
" Bowen, 1966
Dry Polikarpov,
1966
It II
" Burkett, 1975
Wet Richardson
et al., 1975
" Fujita and
llashizume,1975
" Del'illppis and
Pallaghy,1976a
" Hannan, et al.
1973
* 203
UJ
HgCl
TT 203U
Phenyl Hg acetate-
-------
TABLE A~6. NT.CKEL CONCENTRATION BY ALCAIC
Organism
Benthic algae
Phytoplankton
Plankton
Brown algae
Pliytoplankton
Fticus veslculosis
Porphyra
umbilicalis
Ascophyllum
nodosum
Fucus vesiculosi.s
Cliondrus yendoi.
Ptilota filicina
Rliodowela larix
Heterochordaria
abietina
Type
Assorted
it
ii
H
M
Brown,
macroscopic
Red,
macroscopic
Brown,
macroscopic
ii
Red,
macroscopic
ii
ii
Brown ,
macroscopic
.
Type Concentration
Habitat observation factor liasi.s Reference:
Marine Radioecologicai 1x10 Wet l,owman,el' L\ 1 .
.1971
5xl<)3
" Ecological. 1.7xl03 " Bowen, 1'Jf.f.
1.4-5.0xl02
Monterey " 5.7x10" Dry Martin .-.mil
Bay Knaner, 1.972
U.K.. Coast " 2.8x10 " Preston, el ;\\ . ,
1972
" " l.lxlO3
Menai " 4.6x10 " Foster, .!'//(>
U.K.
" " 2.8-6.8xl.()J
Sea of Japan " 20 20 Saenko, c:L a I . ,
.1976
ii 2xl()^
11 " 3x5xl()2
40
(cont iniiod )
-------
TABLE A-6.
(continued)
Organism
Chorda filum
Laminar ia
ch ichor io ides
L. japonica
Agarum
cribrosum
Pelvetia
P wrigiitii
00
Sargassum
pa 11 id um
Enteromorpha
prolif era
Ulva
f enestrata
Lllvaria
s p. lend ens
Cod ium
yessoensis
Chlamydomonas
nival is
Type
Brown,
macroscopic
it
it
it
n
ii
Green,
macroscopic
ii
n
n
Green,
flagellated
Habitat Type of observation Concentration Basis Reference
factor
Sea of Ecological 50 Dry Saenko et al
Japan 1976
n n 6Q M n
n .. 3.2xl02 " "
" " 3.2xl02 " "
" " 4.4xl()2 " "
n .. 1.7xl03
II ll ->Q n n
2xl02
" " 7.8xl()-
II II "l/\ M tl
Greenland " 1.4x10" " !•' ;jei:«.l in-sltul
snow !<)7'.i
(continued)
-------
TABLE A-6. (continued)
1111 - •"• • • • ' ' - - •
Organism
Mougeotia
Zygnema
Ulothrix
Cladophora
Spirogyra
Oedogonium
Tribonema
Coccomyxa
Osc ilia tor ia
Microspora
Scenedesmus*
Type Habitat
Green, Polluted
filamentous stream
n n
n n
ii n
n n
n M
Yellow-green "
filamentous
Green, "
colonial
Blue-green "
filamentous
Green, "
filamentous
Green, Lab
colonial culture
Type of observation Concentration Basis
factor
3
Ecological 1.6x10 Dry
6xl03
l.AxlO2
3.9-8.9xl02
" 2.5xl02-1.7xl03"
" 1.2xl03
I.l-3.0xl03
" 1.2xl03 "
9.1xl03
36
Stable isotope 1.2xl03-8.2xl03"
Reference
Trollope and
Evans 1976
11
tl
II
tl
II
II
It
11
II
Stokes 1975
* Low value in absence of copper, high value in presence of 1.0 ppm Cu.
-------
TABLE A-7. CONCENTRATION 0V ZINC BY ALCAK
^ . * ," , 1 . fc • » - T - - - - - - - - - - - • ' • - ••- - * .. ~ - - - -
Organism
Benthic algae
Phytoplankton
Plankton
Brown algae
Phytoplankton
•-* Fucus
o vesiculosis
F. serratus
Ascophyllum
nodosum
Pelvetia
canaliculate
Laminaria
saccharina
L. digitata
Ascophyllum
nodosum
Fucus
Type Habitat
Assorted Marine
ii n
n n
n n
" Monterey
Bay
Brown, Marine
macroscopic
ii it
ii ii
n ii
n n
n n
" Norway
Fjord
" Marine
Type of observation Concentnit. ion
factor
2
Radioecological 4.1x10
1.5xl()4
Ecological 10 -6.5x10
102-1.3xJ()4
5.5xl03
Radioecological 4 . 2x10
" 6xl02-l.lxl03
" 1.4xl02
IxlO3
4.2xl02
4xl02-lxl03
Ecological 50
Radioecological l.lxlO3
Basil-
Wet
ii
n
Dry
Wet.
M
n
n
Lowmun, el ;i 1.
197.1
Marti.n and
Knauer .1972
Polikarpov
1966
Skipnes, et al,
1975
Rice 1961
-------
TABLE A-7. (continued)
Organism
Pelvetia
canaliculata
Ascophyllum
nodosum
Fucus
vesiculosis
n
Ascophyllum
nodosum
Fucus
vesiculosis
Dictyota
dichotoma
Porphyra
umbilicalis
Cystoseira
barbata
Heterochordaria
abietina
Type
Brown,
macroscopic
n
Brown
macroscopic
Golden
macroscopic
Red
macroscopic
Brown
macroscopic
Habitat Type of observation
Marine Radioecological
n n
U.K. Ecological
Coast
n n
Menal
Straights
U.K.
n n
Lab Radioisotope
culture
(marine)
n ii
Marine Radioecological
Sea of Ecological
Japan
Concentration lias is Keferenco
factor:
1x1 03 Wet: Ki.ee .!%.!.
1.4x.U)3
1.1—6.4x10 Dry Hryan and
Hummers Love l.')7!l
4
2x10 " I'irestori, et a 1 .
1.0-6. Ax !.(/' " Foster
1.3xlf/'
3.3x10" Wet Uitluieclit .\l)h'j
2.8xl()2
2.6xl02
1.9xl02 " Polikarpov 1%6
l.SxlO2 " Saenko, et. al
1976
(corit inued )
-------
TABLE A-7. (continued)
Is)
N>
Organism Type
Chorda filum Brown,
macroscopic
Laminarla "
chichorioidea
L. japonica "
Agarum crlbrosum "
Costaria costata "
Pelvetia wrightii "
Sargassum pallidum "
Ulva nigida Green „
macroscopic
U. pertusa "
U. fenestrata "
Ulvaria splendens "
Codium yessoensis "
Enteromorpha "
prolifera
Ulva lactua "
Codium decorticatum "
Habitat Type of observation Concentration Basis Reference
factor
Sea of Ecological 60 Wet Saenko, et al
Japan 1976
ii M l.SxlO2
ii ii 70 •• ••
ii ii 2.102 " "
H « 60 i.
" " 20 " "
" " 40 Dry "
Marine Radioecological 1.3xl02 " Polikarpov 1966
" " 2.9x10 " "
Sea of Ecological 30 " Saenko, et al .
Japan 1976
M •• lxlQ2
ii 5.4xl02
•• .. 5xlQ2
Lab Radioisotope 2.9x10 Wet Gutknecht 1965
culture
(marine)
" " 30 " "
(continued)
-------
TABLE A-7. (continued)
Ni
Organism
Euglena
viridis
Chlorella
vulgaris
Scenedesmus
quadricauda
Cladophora
fracta
C. glomerata
Mougeotia
Spirogyra
crassa
Chlorella
. Scenedesmus
quadricauda
Cladophora
glomerata
Spirogyra
Type
Unicellular,
euglenophyte
Green,
unicellular
Green,
colonial
Green,
filamentous
Green,
unicellular
Green,
colonial
Green,
filamentous
n
Habitat Type of observation Concentration Basis Reference
factor
3 5
Lab Stable isotope 2.4x10 -1.1x10 Dry Coleman, et al.
culture 1971
11 " 4.2xl03-9.6xl04 "
11 Radioisotope 7.8xl02 " Polikarpov 1966
Fresh " 6.1xl03
water
" " 3.9xl03
" " 3.7xl04
ii ii 1.9xl03 "
n 1.4xl02
Lab " 7.8xl02 " Gileva 1960
culture
" 3.9xl03
" " 3.2xl04 "
Polluted Ecological 6.6-9.0x10 " Trollops and
stream Kvans J976
(continued)
-------
TABLE A-7. (continued)
'""'"""" - - - - - - - 1 - - - I • I - II • - - 1 • - > - • •
Organism
Mougeotia
Zygnema
Ulothrix
Cladophora
Oedogonium
Tribonema
to Coccomyxa
Oscillatoria
Spirogyra
Chara
Co asjpera
Co ftagalis
"^_i
Type
Green,
filamentous
it
ii
ti
Yellow-green,
filamentous
Green,
colonial
Blue-green ,
filamentous
Green,
filamentous
n
ID
II
Habitat Type of observation Concentration
factor
Polluted Ecological 1.3xl()3
stream
" " A.OxlO3
7.3xl02
" " 4.0xl03-1.2xl04
11 " l.SxlO3
11 . 1.7xl03
" " 1.7xl02
" " 9.6xl02 "
A
Fresh Radioisotope 3.2x10
water
„
" " 2.3xl03
" " 2.9xl03
Basis Reference
Dry Trollope and
Kvans 1.97 h
n n
n n
ii n
n n
II M
II
" Pollikarpov
1966
II tt
II tl
* Values obtained from factorial experiment involving 8 temperatures and 6 salinities.
+ Minimal medium experiment.
------- |