United States
Environmental Protection
Agency
Environmental Research
Laboratory
Narragansett Rl 02882
EPA-600 •3-30-025
February 1980
Research and Development
6EFA
Toxicity of Metals to
Marine Phytoplankton
Cultures
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal spe-
cies, and materials. Problems are assessed for their long- and short-term influ-
ences. Investigations include formation, transport, and pathway studies to deter-
mine the fate of pollutants and their effects. This work provides the technical basis
for setting standards to minimize undesirable changes in living organisms in the
aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-80-025
February 1980
TOXICITY OF METALS TO MARINE PHYTOPLANKTON CULTURES
by
.William B. Wilson and Larry R. Freeberg
Texas A&M University at Calveston
Galveston, Texas 77550
Grant No. R801511
Project Officer
C. S. Hegre
Environmental Research Laboratory
Narragansett, Rhode Island 02882
This study was conducted
in cooperation with
Marine Research Laboratory
Texas A&M University at Galveston
Galveston, Texas 77550
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
NARRAGANSETT, RHODE ISLAND 02882
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DISCLAIMER
This report has been reviewed by the Environmental Research Laboratory,
U.S. Environmental Protection Agency, and approved for publication. Approval
does not signify that the contents necessarily reflect the views and policies
of the U.S. Environmental Protection Agency, nor does mention pf trade names
or commercial products constitute endorsement or recommendation for use.
ii
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FOREWORD
The Environmental Research Laboratory of the U.S. Environmental
Protection Agency is located on the shore of Narragansett Bay, Rhode Island.
In order to assure the protection of marine resources, the laboratory is
charged with providing a scientifically sound basis for Agency decisions on
the environmental safety of various uses of marine systems. To a great
extent, this requires research on the tolerance of marine organisms and their
life stages as well as of ecosystems to many forms of pollution stress.
In addition, a knowledge of pollutant transport and fate is needed.
This report describes a five-year study of the relationship between
growth conditions of marine phytoplankton and the toxicity of metals to
them. It illustrates that toxicity is governed not only by the composition
of seawater but also by the duration of exposure. An approach to toxicity
testing which minimizes the uncertanties caused by these variations has been
devised.
Tudor Davies
Director, ERL-Narragansett
iii
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PREFACE
As more knowledge is developed regarding various features of the marine
environment, the role of the trace metals and their activities emerge as a
more and more prominent feature. Not only do they act as toxicants, but also
as stimulants. As more of their interactions with other metals and trace
amounts of other substances are recognized, the apparent importance of metals
activity in marine productivity becomes more prominent. The source, avail-
ability and chemical form of metals become more important and the activity
of chemical and physical conditions demands consideration in the environment,
especially in regard to productivity. Also, as more data become available,
the rewards of sophisticated research procedures become obvious and the
values of long-used techniques and methods show the merit they deserve as
standards.
Perhaps this work will emerge as a more sensitive assay procedure that
will reduce the work per analysis in its simplest form and that will increase
the sensitivity of phytoplankton bioassays, so that it can be employed as a
standard technique.
The turbidostat and chemostat procedures show sufficient capabilities
to merit additional efforts to perfect these procedures for regular use.
Although these assays were time-consuming and were subject to operational
failures, the possibility of detecting long-term effects of toxicants on
organisms merits additional evaluation. Results indicated that population
reductions not detectable by other means may be discerned in a continuous-
flow system.
iv
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ABSTRACT
The objectives of this program were to evaluate the toxicity of nine
metals to cultures of four species of marine phytoplankton. The relationships
of acute, instantaneous and chronic toxicity were evaluated using growth rates
in continuous-flow culture systems. The latter methods employed both the
chemostat and the turbidostat techniques. The instantaneous procedure mea-
sures short-term changes of metabolic activity indicated by 14-C uptake that
result from metal addition within a relatively short time period after cul-
tures are exposed to metal additions. Four levels were determined for the
acute toxicity of metals to each organism. The use of fluorometric measure-
ments of relative chlorophyll a of actively growing cultures was a fast,
accurate assay method that facilitated the Minimum Toxicity Level calcula-
tion, increased the sensitivity of the method, and reduced variability.
Refinement should result in this method being more useful for giving more
uniform assay results. Also, the procedures are necessary for this method
to succeed.
The medium composition in which they live was apparently the main factor
that protected phytoplankton from the toxic effects of metallic ions. Sea
water that is "more pure", i.e., contains none or low amounts of dissolved
organic substances not usually found in sea water, probably offers less
protection to the phytoplankton populations than the so-called polluted
water. On the other hand, polluted waters impose more variable conditions
which usually result in the survival of fewer species. That is, the sea
water components that protect estuarine organisms from metals toxicity may
limit the growth of many species directly.
Results indicated that the nine metals tested could be grouped based on
their acute toxicity. The toxicity levels of the groups differed by approx-
imately one order of magnitude from 0.05 mg/L, 0.5 mg/L and 5.0 mg/L. Cu,
Hg and Ag comprise the most toxic group and Mo, V and Ni were the least
toxic group. This group arrangement did not apparently apply to conditions
of continuous-flow studies. At least the differences in toxicity between
members of the groups were not an order of magnitude different in concentra-
tion.
Differences in salinity had more effect on metals toxicity than did
differences in temperature. The use of salinity levels that were near the
lower salinity tolerance levels of an assay organism may have had a major
influence on this result. There was no significant effect within the range
of temperatures tested, on the toxicity of metals to the cultures. Metals
were more toxic in low salinity medium than in high salinity medium or in
the combined conditions of low salinity and high temperature. High salinity
(>30 ppt) reduced the Cu and Hg toxicity to G. splendens. Also, there was
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an indirect effect of salinity on the toxicity of Cu and Ag to G. splendens.
The major effects of salinity on toxicity was in medium that had salinity
levels similar to the low level tolerated by the organism. For example,
T. pseudonana cultures were more susceptible to metals in low salinity medium
(3-7 ppt) than in high salinity medium (28 ppt). Also, nine metals were
less toxic in medium with the maximum salinity levels employed (28 ppt) than
in media with lower salinity levels.
The four organisms employed in the toxicity assays were not equally sen-
sitive to the nine metals tested. The two dinoflagellates were more suscept-
ible to metals toxicity. G. splendens was possibly more sensitive collect-
ively than G. halli and the metals had the least effect on I. galbana. Also,
the toxicity levels were different between metals. For example, the Hg LMTL
varied from 0.005 mg Hg/L for G. splendens to 0.1 mg/L for J. galbana. The
basic analysis employed in the toxicity evaluations was similar to that
employed to assay the "natural" chelation level of sea water (Davey
1973; as described by Hannan and Palouillet, 1972, for fresh water pollutants
and algae growth). A good feature of this assay is that each test portion
can be measured repeatedly without opening the container portion. The most
toxic metal group (Hg"1""1", Cu and Ag) was much more toxic than the least toxic
group (Ni, V and Mo). The toxic levels of Ni, V, and Mo were 6 to 10 times
less toxic than Hg, Cu and Ag. Individually, Ag was the most toxic metal
tested.
Differences in the low (LMTL) and high (HMTL) results indicated a 10-
fold or more range between the two levels and these differences increased as
the toxicity of the metals decreased. For example, the LMTL of Cu to
G. splendens was one-tenth the HMTL of Cu but the LMTL and HMTL of vanadium
was one-twentieth.
Results of residual, instantaneous and chronic toxicity experiments
indicated that these four organisms could be subjected to high concentrations
of the nine metals for periods of 24 to 144 hours and subsequently grow after
being transferred to fresh medium. Some reports indicated that phytoplankton
"take-up" metals rapidly. Thus, the lack of continued mortality of cultures
after transfer to fresh medium must indicate that some short-term growth
effects are not permanent. Indeed, some of the effects observed could rep-
resent that part of culture population development commonly referred to as
"lag phase." These effects were exemplified by the results of the instan-
taneous and residual toxicity results. Cultures were tolerant of relatively
high metals concentrations and had a high degree of recovery from toxic
levels. The order of toxicity (i.e., most toxic to least toxic metals) in
chemostat tests was similar to that of the acute toxicity results.
Instantaneous toxicity experiments showed that the metals produced a
rapid, short-term reduction of l^C uptake, but the effects were transient
and recovery occurred, in some cases, within 1 hour. Low amounts of some
metals (as low as 10 yg Cu/L) reduced both the growth rate and ^C uptake
of J. galbana, but concentrations as high as 1250 yg Cu/L did not stop cell
division. The growth of I. galbana in instantaneous experiments and turbid-
ostat cultures was stimulated by Hg additions of 10 yg Hg/L or less and by
11.2 yg Cr/L.
vi
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The optimum temperature for the growth of j. galbana in batch culture
was from 20 to 25C (Kain and Fogg, 1958). The growth was less at tempera-
tures below 15C and above 25C. The optimum temperatures for growth in con-
tinuous cultures was from 17 to 26C (Freeberg, 1971). Growth was signifi-
cantly less at 17C or below and above 26C. Data on the combined effects of
temperature and salinity on growth often indicated that the optimum temp-
erature level was between 16 and 24C, but not as high as 28 or as low as 12C.
Turbidostat results were similar to those from the chemostat, but dif-
ferences did occur. Strickland (1965) hypothesized that there was possibly
a temperature range of IOC within which the division time of an algal cell
was essentially constant. On the other hand, Freeberg (1971) found the Kc
changed with each change (3C) in temperature. The equation for the linear
increase in the Kc of J. galbana from 14 to 26C was y = 0.0645(x)-0.281
(Freeberg, 1971). In this study it was y = 0.065(x)-0.295 with a correla-
tion coefficient of 0.99.
Cells in a nutrient-rich culture may become light-limited by way of
mutual shading associated with high population levels and growth may be
limited in a manner similar to cells in a nutrient-limited culture. The Kc
of I. galbana did not change significantly in cultures with cell concentra-
tions from 1.0 x 105 to 1.75 x 105 cells/ml, but the KC decreased (21%) with
2.0 x 10^ cells/ml and 5 raw/cm^ irradiation. A light-limited culture regime
was indicated by results with 1.75 x 105 and 2.0 x 105 cells/ml and 5 or 10
raw/cm^ irradiation.
The variability of KC values may be partially attributed to the rela-
tively low concentration of cells in the cultures. Batch cultures contained
from 2 to 14 x 106 cells/ml (Kain and Fogg, 1958). Two-liter chemostats
contained from 1.6 to 3.0 x 10^ cells/ml (Caperon, 1968). In batch cultures
the population level is continuously changing and the growth rate is inte-
grated over a range of cell concentrations. The minimum population level
(2 x 10^ cells/ml) (Kain and Fogg, 1958) was an order of magnitude greater
than the concentration indicated as light-limited in this study. The maximum
growth rate of 0.55 was similar to the Kc of light-limited cultures in this
study. Light intensity, culture volume, and culture vessel width may also
control the concentration at which the cells become light-limited.
The variation in the growth of G. halli at 17 different combinations of
light and temperature supported the previously described hypothesis of con-
stant algal growth within wide ranges of light and temperature (Strickland,
1965). The optimum temperature for the growth of G. halli was from 23 to
32C, however, the Kc values changed with each 3C change from 23 to 29C.
Light intensities had little effect upon the KC of G. halli, yet the cell
concentration was directly related to the light intensity. The KC of G.
halli with 10 mw/cm2 irradiation declined significantly only after the cell
number exceeded approximately 2.4 x 10^ cells/ml. A 50% increase in irradia-
tion from 10 to 15 raw/cm^ increased the cell number 2.5 times to 6.1 x 10^
cells/ml. In contrast to J. galbana, the Chi a/cell of G. halli was not
related to the cell number. These data further supported the hypothesis that
algal growth may become limited at moderate cell numbers in cultures, and
vii
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algal bioassay tests may be insensitive to subtle changes due to prior sup-
pression (light-limited) of algal growth.
The action of a heavy metal on living cells has been shown to be a two-
step process. The first action site occurs at the surface boundaries of the
cell. As the metal diffuses into the cell, the second action occurs intra-
cellularly (Rothstein, 1959) . The first reactions are characterized by
adsorption of the metal on the cell membrane, followed by a physiological
disturbance, if any, resulting from the chemical alterations of the diffusion
barrier by the membrane-bound metal. These reactions are relatively rapid
and are usually reversible, while the interactions within the cell, the sec-
ond action, exhibit a time lag and are not essentially reversible (Rothstein,
1959).
The first actions have been demonstrated for copper and mercury with a
number of phytoplankton species. Mandelli (1969) reported a rapid uptake of
copper by nine species of marine phytoplankton for the first 15 to 30 minutes
of exposure. Continued Cu uptake, at a reduced rate, was dependent upon cell
division. Glooschenko (1969) found that dead, live non-dividing and live
dividing cells of the marine diatom chaetoceros costatum initially took up
203ng rapidly, while only live dividing cells in the light continued to take
up Hg. The initial Hg uptake was not dependent on photosynthesis and
Glooschenko suggested that, although some active uptake may occur in actively
metabolizing cells, the most important process in Hg uptake by Chaetoceros
was passive surface adsorption. The primary effect of cytoplasmic membrane
bound Hg in chlorella was light-independent excretion of potassium through
the diffusion barrier (Ramp-Nielsen, 1971). Cell division was inhibited by
the alteration in membrane permeability, yet the light processes of photo-
synthesis were not affected until Hg ions penetrated the cells. Lag period
in cell division, indicating the first type of reaction, were extended in
relation to the initial Hg concentration, yet the extended lag periods could
be reduced or eliminated by complexing the Hg with the addition of citric
acid. Nuzzi (1972) noted that inhibition in the growth of Phaeodactylum
tricornutum by Hg (as phenylmercurie acetate) was reversed with the addition
of monothiol compounds, e.g., glutathione. Ben-Bassat et al (1972) reported
the lag period prior to the growth of Chlamydomonas reinhardi was directly
related to the initial Hg concentration in the medium, and suggested that
recovery to a normal growth rate was due to loss of Hg from not only the
cells but also from the total culture system. He determined that mercury
was reduced by 40% in the culture system (medium and cells) after 8 days
whereas there was a 5% reduction in the controls system. They theorized the
Hg was biologically converted to a volatile and less permeable form, which
had less affinity to the cells. In other words, a single sublethal dose of
Hg inhibits algal cell division, apparently via alteration in cell membrane
permeability, and upon natural or induced removal of the membrane-bound Hg,
normal cell division resumed.
The cytoplasmic concentration of Hg in Isochrysis galbana cultures was
ca 10^ times that in the medium, and intracellular uptake was controlled by
membrane diffusions (Davies 1974, 1976). A greater driving concentration of
Hg than initially added to the medium was required for the calculated diffu-
sion coefficients, and Davies suggested that membrane adsorption was the
viii
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mechanism that enabled such a buildup of Hg at the cell surfaces. Davies
reported the specific growth rate of I. galbana was inversely related to the
intracellular concentration of Hg. He was not able to distinguish a differ-
ence in toxicity of surface adsorbed or intracellular Hg, thus the site or
mode of Hg toxicity has yet to be demonstrated. Davies (1974) reported an
increase in the mean cell volume of J. galbana when initially exposed to Hg
and suggested the enlarged cells were probably due to inhibition of cell
division by the metal forming complexes with sulphur-containing compounds
known to be important for cell division, e.g., methionine. Moreover, Davies
(1976) was not able to determine any significant excretion of intracellular
potassium by I. galbana or Dunaliella tertiolecta after exposure to Hg. He
concluded that, "The primary effect of mercury upon phytoplankton was not
disruption of cell membranes causing loss of intracellular cations, but the
dislocation (probably intracellular) of metabolic processes governing growth
and division."
Experimental data derived from continuous exposure of algal cells to
steady-state concentrations of Hg in turbidostats indicated there were Type I
reactions over a wide range of Hg concentrations. First, the Kc of both
J. galbana and G. halli were affected when the cultures were exposed to Hg,
yet intracellular metabolic parameters (l^C uptake and Chi a) were not
affected until the lethal concentration was approached. Second, the Hg in-
duced effect upon the KC of each species, either enhancement or inhibition,
was rapidly reversed and the KC was equivalent to that of the control when
the addition of Hg was discontinued. Third, metabolic functions are usually
temperature-dependent, yet the Kc of G. halli grown in a wide range of Hg
concentrates at 23 and 30C was temperature-independent. Unfortunately, anal-
ysis of membrane and intracellular Hg concentrations similar to those of
Davies (1976) were not made in this study, and intracellular metabolic param-
eters, as reported in this study were not determined by Davies. A study
employing continuous culture techniques and analysis of a variety of meta-
bolic parameters in 'conjunction with isolating the cellular location of Hg
concentrations would contribute to the elucidation of mercury's mode of
action. Also, it is suggested that the effect of Hg upon membrane perme-
ability and/or uptake of ions or molecules known to be important for cell
division should be included in future studies, e.g., sulfur, usually taken up
as S04, is required for cell division and the inhibition of sulfur uptake
could be a passive mechanism which reduced cell division without an effect
upon metabolic parameters, such as ^C uptake.
J. galbana and G. halli were not only more sensitive to chronic expo-
sures of Cr than to Hg, but the mode of action of Cr appeared to be differ-
ent. Although a separation of Type I and Type II reactions were not dis-
cernible, internal metabolic parameters of both species were affected by Cr,
and the KC was directly related to the changes in l^C uptake or Chi a/cell.
The Chi a/cell of J. galbana was not significantly affected by Cr while the
l^c uptake was inversely related to the Cr concentration over the total
range of Cr tested. The ^C uptake of G. halli was not significantly affect-
ed by Cr but the Chi a/cell was greater than that of the control culture if
the Cr concentrations were less than 12 ug/L. The Chi a/cell of G. halli
was inversely related to the medium Cr concentration from approximately 7.9
yg Cr/L to the maximum concentration tested (35.6 yg Cr/L).
ix
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A model for species succession and/or community dominance by one species
was illustrated not only by the difference in the sensitivity of the species
tested, but also by the dissimilar patterns of response exhibited by each
species exposed to Hg and Cr. For example, Hg was stimulatory, inhibitory,
and lethal to the growth of G. halli within a range that did not significantly
affect the growth of I, galbana.
Although some results indicated that metals were more toxic in chemostat
cultures than in turbidostat cultures, the high sensitivity in the former is
believed to result from the condition of chemostat populationst Physio-
logically a stressed population would be most likely to be more sensitive to
metals than a non-stressed population. Although we have no measure of the
stress that exists in a phytoplankton culture, a declining population is
probably more subject to metals toxicity than a fast-growing population.
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CONTENTS
Foreword ill
Preface iv
Abstract v
Figures xii
Tables xiii
Abbreviations and Symbols xvi
Acknowledgments xvii
SECTION 1. Introduction I
SECTION 2. Acute Toxicity of Nine Metals to Marine
Phytoplankton Cultures 5
Materials and Methods 5
Results 12
SECTION 3. Residual Toxicity of Nine Metals to G. splendens,
I. galbana, T. pseudonana and G. halli 19
Results 20
SECTION 4. The Effects of Temperature and Salinity on the
Acute Toxicity of Metals to G. splendens,
I. galbana and T. pseudonana 24
Materials and Methods 24
Results 25
SECTION 5. Instantaneous Effects of Metals on Photosynthesis
Rates of G. splendens, 21. pseudonana and
J. galbana. 36
Materials and Methods 36
Results 37
SECTION 6. The Effects of Light Intensity, Temperature,
Cell Density and Chronic Exposure of Mercury
and Chromium on Two Marine Phytoplankters in
Continuous Culture 38
Materials and Methods 40
Results 46
SECTION 7. The Chronic Toxicity of Mercury, Chromium and
Nickel to G. halli and J. galbana (Chemostat) ... 61
Materials and Methods 61
Results 63
SECTION 8. References 81
xi
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FIGURES
Number Page
1 Variability of Cu toxicity to G. splendens (values in mg/L) ... 9
2 The relationship of MIL, NIL, NGL and TML to the toxicity of
Cu to G. splendens (results from 11 experiments) 10
3 Diagrammatic sketch of the basic turbodistat design 41
4 Diagrammatic sketch of the basic chemostat design 62
5 Response of G. halli culture to Hg concentration of 50 mvig/L
(one experiment) 65
6 Response of G. halli culture to Ni concentration of 0.2 yg/L
(one experiment) 66
7 Response of G. halli culture to Hg concentration of 2.0 myg/L
(one experiment) 67
8 Response of G. halli culture to Ni concentration of 0.15 pg/L
(one experiment) , . 68
xii
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TABLES
Number Page
1 Composition of Chelate-Free Artificial Seawater Medium which
Supported the Best Growth of I. galbana, T. pseudonana,
C. nana, G. splendens and G. halli 6
2 Acute Toxicity of Nine Metals to Cultures of Four Marine
Phytoplankton Organisms (values in mg/L) 13
3 The Low Minimum Toxicity Level (LMTL-1), Low No Growth Level
(LNGL-2) and Low Total Mortality Level (LTML-3) of Nine Metals
to Four Phytoplankton Cultures (values in mg/L) 15
4 Group List of Nine Metals Based on Their Gravimetric (mg/L)
and Molecular (pg at/1) Weights of Combined LMTL and LNGL
Acute Toxicity Values to G. halli, G. splendens, I. galbana
and T. pseudonana 17
5 High Minimum Toxicity Level (HMTL-1), High No Growth Level
(HNGL-2) and High Total Mortality Level (HTML-3) of Nine
Metals to Four Marine Phytoplankton Cultures 18
6 Residual Toxicity (A) of Nine Metals to J. galbana, T. pseudo-
nana and G. splendens (values in mg/L) 21
7 Residual Toxicity (A) of Nine Metals to G. halli After
Exposures of 1, 24, 48, 72 and 144 Hours 22
8 Effects of Temperature and Salinity on the Growth Rates (K)
of G. splendens, I. galbana and T. pseudonana 26
9 The Effects of Temperature on the Toxicity of Metals to
G. splendens (values in mg/L) 28
10 Effects of Salinity on the Toxicity of Metals to G. splendens
and J. galbana (values in mg/L) 29
11 Combined Effects of Temperature and Salinity on the Toxicity
of Metals to G. splendens (values in mg/L) 31
xiii
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TABLES (continued)
Number Page
12 Effects of Temperature-Salinity Combinations on the Toxicity
of Zn, Hg, Ag and Cu to J. galbana 32
13 Combined Effects of Temperature and Salinity on the Toxicity
of Metals to T. pseudonana (clone 3H) (values in mg/L) 33
14 Specific Growth Rate, Flux, Chi a/Cell and 14C Uptake for
Various Population Levels of I. galbana Grown at 23C and with
5 and 10 mw/cm^ Irradiation 47
15 Specific Growth Rate, Chi a/Cell, and 14C Uptake of J. galbana
Exposed to Various Light-Temperature Combinations 48
16 Specific Growth Rate, Flux, Chi a/Cell and ^C Uptake for
Various Population Levels of G. halli Grown at 23C and with
5, 10 raw/cm^ Irradiation 50
17 Specific Growth Rate, Chi a/Cell and C Uptake of G. halli
Exposed to Various Temperature-Irradiation Combinations 52
18 Specific Growth Rate, Chi a/Cell, and C Uptake of I. galbana
Grown at 23C with 10 raw/cm^ Irradiation and Continuous Exposure
to Various Concentrations of HgCl2 55
19 Specific Growth Rate, Chi a/Cell, and C Uptake of G. halli
Grown at 30C with 10 mw/cm^ Irradiation and Continuous Exposure
to Various Concentrations of Kg*"1" 56
20 Specific Growth Rate, Chi a/Cell, and C Uptake of G. halli
Grown at 23C with 10 mw/cm^ Irradiation and Continuous Exposure
to Various Concentrations of Kg"*"1" 58
21 Specific Growth Rate, Chi a/Cell, and C Uptake of I. galbana
at 23C with 10 raw/cm^ Irradiation and Continuous Exposure to
Various Concentrations of CrCl3 59
22 Specific Growth Rate, Chi a/Cell and C Uptake of G. halli
at 30C with 10 mw/cm^ Irradiation and Continuous Exposure to
Various Concentrations of CrCl3 60
xiv
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TABLES (continued)
Number Page
23 Chronic Toxicity of Mercury to G. halli in a Chemostat 70
24 Chronic Toxicity of Chromium to G. halli in a Chemostat .... 72
25 Chronic Toxicity of Nickel to G. halli in a Chemostat 74
26 Chronic Toxicity of Mercury to I. galbana in a Chemostat .... 75
27 Chronic Toxicity of Chromium to J. galbana in a Chemostat ... 77
28 Chronic Toxicity of Nickel to J. galbana in a Chemostat .... 79
xv
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LIST OF ABBREVIATIONS AND SYMBOLS
ABBREVIATIONS AND SYMBOLS
Cu
Zn
Ag
Co
Pb
Ni
Se
Ba
Cd
mg.
Fe
Mo
V
Cr
20C
ppt
L.
ml.
p.s.i.
B
Mn
Ti
Si
Zr
NH4C1
copper
zinc
silver
cobalt
lead
nickel
selenium
barium
cadmium
milligram
iron
molybdenum
vanadium
chromium
twenty degrees
centigrade
parts per thousand
liter
milliliter
pounds per square inch
boron
manganese
titanium
silica
zirconium
ammonium chloride
NaAC03
pH
EDTA
chl a
in vitro
<
>
yg
Carbon-14
or 14C
x
No
0
var
magnesium chloride
sodium bicarbonate
symbol for hydrogen
ion concentration
ethylene diamine
tetra-acetic acid
chlorophyll a
in glass, transfers
were made without
transferring the
culture or any portion
thereof from the glass
container
percent or percentage
less than
more than
microgram
carbon-14 isotope
mean numerical value
number
standard deviation
variation
XVI
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ACKNOWLEDGMENTS
The cooperation and suggestions of personnel of the National Marine
Water Quality Laboratory, Office of Research and Development, U.S. Environ-
mental Protection Agency are gratefully acknowledged. Specifically the
efforts and consideration of C. S. Hegre, project officer, and S. Erickson,
M. Malcomb and E. Davey in the planning and evaluation of this project were
most helpful and appreciated. Many problems that occurred during various
aspects of this work were made less difficult by the efforts and coopera-
tion of M. Watson, G. Marlatt and A. Aldrich.
During various stages of this project, as many as three independent
investigators were concerned with different phases of the program. One
principal contributor, Larry R. Freeberg, planned, conducted research and
prepared reports on the material included as Section 6, pages 38-59,
titled "The Effects of Light, Temperature, Cell Density and Chronic Exposure
of Mercury and Chromium on Two Marine Phytoplankters in Continuous Culture."
Another contributor of the work reported herein as Section 5, "Instantaneous
Effect of Metals on Photosynthesis Rate of G. splendens, T. pseudonana and
J. galbana" was Emory A. Sutton. Much of the work reported here was devel-
oped and conducted independently, yet in many ways, the results were similar
from one section to another.
xvii
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SECTION 1
INTRODUCTION
That heavy metals are toxic to aquatic organisms has probably been
known to some extent since the Phoenicians used pitch and possibly copper
sheathing on their ships to prevent the attachment of marine organisms
(Masseille, 1933). Also, lead sheathing, sheathing of other metals and
sheathing composed of mixtures of metals were used for this purpose, but
Sir Humphrey Davy was possibly the first to conduct experimental studies on
the prevention of the attachment of marine organisms to vessels by use of
copper sheathing (Woods Hole Oceanographic Institution, 1952). Subsequently,
and at present, copper, mercury and to a lesser extent other metals are
employed as the active component of anti-fouling and anti-boring paints and
coatings on marine vessels, buoys and structures. Much of the research on
metals toxicity to aquatic organisms involved freshwater species, especially
fish. One of the earliest reports (Richet, 1881) compared the toxicity of
various metals to fish. Other studies on metals toxicity to fish such as
those by Thomas (1915), Carpenter (1927), Jones (1938), Jones (1939) and
Domogalla (1935) are reviewed by Doudoroff and Katz (1950).
Most of the early studies on the effects of metals on marine organisms
have examined copper and mercury toxicity. Miller (1946) determined the
effects of copper on Bugrula. Other investigations on the effects of copper
and/or mercury on marine organisms are as follows: Hunter (1950) on the
amphipod I&rinogananarus; Pyefinch and Mott (1948) barnacles and their larvae;
and Waksman et al (1942) and Starr and Jones (1957) on marine bacteria.
Corner and Sparrow (1956) presented evidence that copper affects the respira-
tion of crustaceans, whereas the mode of action of mercury was different.
Barnes and Stanbury (1948); Pyefinch and Mott (1948), Hunter (1950);
Doudoroff and Katz (1953) and Corner and Sparrow (1956) indicated that copper
and mercury were synergistically toxic to various aquatic organisms.
In 1904, Moore and Kellerman used copper sulfate to control the growth
of freshwater algae, especially phytoplankton, in reservoirs. Subsequently,
the use of copper sulfate to control freshwater algae has been a standard
practice (Whipple, 1947; Hale, 1950; Monie, 1956; and Fisher, 1956). Green-
wald (1956, 1957) lists over 200 publications on the use of copper and other
metals to control aquatic plants, especially algae. Palmer and Maloney (1955)
screened 76 compounds (including Cu, Zn, Ag and Hg compounds) for their
toxicity to six cultures of freshwater planktonic algae. Maloney and Palmer
(1956) determined the toxicity of six of the more toxic of these compounds
(including Cu and Zn compounds) to cultures of 30 freshwater species of
Chlorophyta, Cyanophyta and Bacillariophyceae. In tests employing nine
metals (Cd, Cu, Co, Pb, Hg, Ag, Se, Ba and Ni) and four freshwater phyto-
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plankton organisms—Chlorella, Scenedesmus, Haematococcus, and Chlamydomonas,
Hutchinson, 1973; and Stokes, et al, 1973, determined that (1) silver inhib-
ited the growth of batch cultures of the most sensitive organism—Chlorella,
at levels of 0.005 ppm; (2) all metals except barium and lead were toxic at
concentration of 0.5 ppm or less; and (3) some combinations of metals, e.g.
Cu and Ni were synergistically toxic and other combinations, e.g. Cd and Se
were less toxic. The relative toxicity of the metals to Chlorella were as
follows:
Ag>Cd>Cu>Se>Ni>Co>Ba>Pb
and to Scenedesmus were as follows:
Ag>Cd>Ni>Se>Cu>Ba>Pb
Whitton's (1970) review of heavy metal toxicity to freshwater algae
emphasized the effects of environmental factors on metals toxicity and dis-
cussed such factors as chelation, salinity, sediments and adaption as possi-
ble causes of observed variabilities of metal toxicity. Recent studies of
metals toxicity evaluated long-term effects on growth and other physiological
parameters (Harriss etal/1970; Matson et al, 1972; Tomkins and Blinn, 1976;
Blinn et al, 1977.
Although metallic compounds have been used to control freshwater phyto-
plankton and many studies have been conducted on their relative toxicity to
the forms and factors that cause variabilities of toxicity, the toxicity of
metals to marine plants has not been as thoroughly investigated. Miyaki
(1935) used copper sulfate in an attempt to control a dinoflagellate bloom
in a Japanese estuary. Shilo et al, 1955, determined the values of pyridyl-
mecuric acetate and copper sulfate in the control of Prymnesium paruum in
brackish water ponds in Israel. Copper, silver, mercury and gold were the
most toxic elements tested to Gymnodinium breve cultures (Wilson 1958).
Rounsetell and Evans (1958) stated that "20 pounds of CuSO^ crystals per acre
will destroy the red tide organism, but will give only temporary relief for
a period of 10-14 days —". Marvin and Proctor (1964) listed some metallic
and organo-metallic compounds to be among the most toxic of 4,000 plus com-
pounds screened for their toxicity to Gymnodinium breve. Ukeles (1962)
determined that urea and ethyl mercury compounds were the most effective of
17 compounds tested in the inhibition of growth of axenic cultures of Mono-
chrysis lutheri, Dunaliella euchlora, Chlorella sp., Protococcus sp. and
Phaeodactylum tricornutum.
Many of the recent studies on metals toxicity to marine phytoplankton
have emphasized physiological effects, such as growth and carbon-14 uptake,
rather than acute toxicity. Mandelli (1969) determined that between 0.03
and 0.26 mg. cu. per liter inhibited the growth of nine marine phytoplankton
species and that the uptake of copper by these cultures occurred mostly
within the first 30 minutes after exposure. Phytoplankton were inhibited
by copper concentration progressively more as the exposure time was increased
from 20 to 60 minutes (Steeman-Nielsen et al, 1970) and growth depression of
a diatom by copper varied from one natural seawater sample to another
(Erickson, 1970). This variable attribute of different seawater samples was
employed by Davey et al (1973) to estimate the copper complexation capacity
of seawater. Mercury (as phenylmercuric acetate) concentrations as low as
0.06 mg. per liter was inhibitory to three phytoplankton species (Nuzzi, 1972).
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These results emphasize the possible deleterious effects of very low metal
concentrations and some of the factors that effect metal toxicity.
On the other hand, certain metals are known to be essential for the
growth of marine phytoplankton and their availability often determines the
level of primary productivity. Harvey (1955) listed Fe, Mn, Cu, Zn, Mo and
Co as probable essential elements for phytoplankton growth. Hutner et al
(1950) discussed the roles of metals in protisan metabolism. The role of
metals in phytoplankton growth and the availability of metals in seawater,
as nutrients or as toxicants is emphasized by the studies of Thomas, 1959;
Menzel and Ryther, 1961; Johnston, 1963, 1964; Tranter and Newell, 1963;
Wilson, 1966 and Barber, 1973. Davies (1970) considered that all phytoplank-
ton require organo-iron complexes to synthesize chlorophyll. The potential
toxicity of very low concentrations of metals and the essentiality and/or
uptake of these metals indicates that one or more systems make metals avail-
able and either that system or another system reduces the ionic concentration
to which the organisms are exposed in the medium.
Physical and chemical factors alter the toxicity of metals according to
several investigators. Moore and Kellerman (1905) proposed that the amount
of copper required to control freshwater algae should be increased 2.5% for
each degree below 15C and decreased the same amount for each degree above
15C, and the amount of copper employed for control should be increased in
water with high alkalinity and/or high organic content. Whipple (1947)
states that the toxicity of copper in freshwater depends on the temperature,
organic matter and suspended material of the water. Shilo (Shelubsky, 1954)
emphasized that temperature changes do not alter copper toxicity, but that
organic matter resulting from phytoplankton growth reduces toxicity. Oxygen
content, pH and alkalinity affect the solubility of metals in seawater (Wood
Hole Oceanographic Institution, 1952) and thus affect metals toxicity.
Barnes and Stanbury (1948) discussed the formation of copper complexes by
chlorides, sulfates and carbonates. Since dissolved 02, pH, alkalinity,
chlorides, sulfates and carbonates of seawater are related to salinity, the
toxicity of metals may be salinity-dependent. On the other hand, the com-
position of oceanic and estuarine water may be markedly different in that
estuarine waters may exhibit variable major ionic ratios as well as variable
total dissolved solids. Although the effects of differences in ionic ratios
on estuarine organisms is considered to be of less significance than the
total salt content (Kinne, 1964)' little information is available on the com-
bined effects of heavy metals and variable ionic ratios Provasoli et al (1954)
The program included six major investigation areas titled in the sec-
tions as follows:
2. The acute toxicity of nine metals (Ag, Cr, Cu, Fe, Hg, Mo, Ni, V
and Zn) to cultures of four marine phytoplankton organisms
(GLenodinium halli Frudenthal and Lee, Gymnodinium splendens
Labour, Isochrysis galbana Parke and Thalassiosira pseudonana (3H)
Hasle and Heimdal; = (Cyclotella pseudonana Husfedt).
3. The residual toxicity of nine metals to G. splendens, J. gaJbana.,
T. pseudonana and G. halli.
-------
4. The effects of temperature and salinity on the acute toxicity
of metals to G. splendens, I. galbana and T. pseudonana,
5. Some instantaneous effects of Hg, Cu and Cr on the photosynthetic
rate of G. splendens, T. pseudonana and J. galbana.
6. The effects of light intensity, temperature, cell density and
chronic exposure to mercury and chromium on two marine phytoplank-
ters in continuous culture (Turbidostat tests).
7. The chronic toxicity of Hg'H' Cr+++and Ni++to G. halli and I. gal-
bana (Chemostat tests).
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SECTION 2
ACUTE TOXICITY OF NINE METALS TO
MARINE PHYTOPLANKTON CULTURES
MATERIALS AND METHODS
Bacteria-free cultures of G. splendens Lebour, G. halli Frudenthal
and Lee, I. galbana Parke and T. pseudonana Hasle and Heimdal were grown in
a chelate-free artificial seawater medium (TABLE 1) after it had passed
through a Chelex 100 column (Davey et al, 1970). Glassware used in toxicity
tests was silicone-coated. T. pseudonana grew better if the medium received
additional silica (TABLE 1). Although it was not employed for medium of
these tests, G. splendens and G. halli grew better if the medium contained
Tris (hydroxy) methyl aminomethane (0.4 grams/L; pH 8.1; Matheson Coleman
and Bell).
A standard temperature and salinity of 20C and 28 ppt were employed in
all tests with I. galbana and T. pseudonana and a temperature of 28C and
28 ppt was employed as control levels for G. splendens, I. galbana (Freeberg,
1976), T. pseudonana (3H, Guillard and Ryther, 1962 and G. halli (Wilson,
unpublished data). Screw-capped 125-ml microfernbach and 16 x 125mm Corning
disposable test tubes with polypropylene thimble closures were employed in
all tests. Preliminary results indicated that metals were more toxic in
culture portions of 50-ml or more than in 10-ml portions. These effects were
apparently a surface-volume relationship and the use of new test tubes and
other glassware used in toxicity tests were rinsed and filled with triple
glass-distilled water and autoclaved at 20 psi for 20 minutes before and
after being silicone-coated.
Triplicate 10-ml culture portions were employed for each concentration
of the test metal. Dilutions of metal solutions, prepared with distilled
water, were added in 0.1-ml amounts to the test tubes prior to adding the
culture portions. Cultures were enumerated on a Model B Coulter Counter and
examined microscopically prior to use in tests.
Test portions were examined after exposure periods of 24 and 48 hours
and relative chlorophyll a measurements were made, in vitro, with a Turner
Model 111 Fluorometer (Lorenzen, 1966) at these times. The door on the
Fluorometer was modified to accommodate the 125mm-long test tubes. All test
portions were arranged in a single row which was a. uniform distance from the
uniform bright portion of 40-watt, cool-white, fluorescent light. Light
intensity measurements were monitored with a Gossen Luna-Pro Light Meter.
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TABLE 1. COMPOSITION OF CHELATE-FREE ARTIFICIAL SEAWATER MEDIUM WHICH
SUPPORTED THE BEST GROWTH OF I. galbana, T. pseudonana, C, nana,
G. splendens and G. halli
Component Amount per liter
NaCl
KC1
MgCl2-6H20
MgS04-7H20
CaCl2-2H20
K2HP04
KN03
Metals T
Vitamin 6^2
Thiamine HC1
Biotin
Sulfides
Adenine Sulfate
NaHCOs
24.0 g
0.6 g
4.5 g
6.0 g
0.7 g
10.0 mg
10.0 rag
1.0 ml (1)
T.O yg
10.0 mg
0.5 yg
5.0 ml (2)
1.0 mg
0.2 g (3)
(1) Metals T added after passing medium through Chelex 100 column
Metal T mg per milliliter
Fe (as FeCl3«6H20) 0.05
B (as H3B03) 0.05
Se (as H2Se03) 0.01
V (as NH4V03) 0.005
Cr (as K2Cr04) 0.002
Mn (as MnCl2'6H20) 0.01
Mb (as (NH4)6Mo7024'4H2b) 0.01
Cu (as CuCl2-2H20) 10.001
Zn (as ZnCl2) 0.005
Ti (as Ti02) 0.05
Si (as Na2Si03-9H20) 0.05
Zr (as ZrOCl2-8H20) 0.02
Ba (as BaCl2) 0.01
(2) Sulfides: NH4C1 0.2 g; KH2P04 0.1 g; MgCl2-6H20 0.04 g;
NaHC03 0.2 g; Na2S-9H20 0.15 g. Raise to one liter with
distilled water.
(3) NaHCOs was added prior to autoclaving. Occasionally this
medium turned cloudy (precipitate) on autoclaving (15 Ibs.,
15 min.) but cleared after 3 or 4 days equilibration with
atmospheric C02- The pH also increased to 8.4-8.8 during
autoclaving but returned to 8.0-8.2 as the medium cleared.
Aseptic filtration was employed after the medium cleared.
(continued)
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TABLE 1 (continued)
Two modifications of artificial seawater medium employed in this study.
Constituent
EDTA*
TRISt
Fe (as FeCl3-6H20)
B (as H3B03)
Se (as H2Se03)
V (as NH4V03)
Cr (as K2Cr04)
Mn (as MnCl2-6H20)
Ti (as TiC>2)
Si (as Na2Si03-9H20)
Zr (as ZrOCl3-8H20)
Ba (as BaCl2>
Sulfides Mix
NaHC03
Gates & Wilson
(per liter)
10.0 mg
100.0 mg
250.0 iag
250.0 Mg
50.0 ug
25.0 ug
10.0 ug
50.0 ug
250.0 ug
250.0 ug
100.0 ug
50.0 ug
1.0 ml
_
NH-15
(per liter)
10.0 mg
400.0 mg
289.4 ug
262.2 yg
30.6 ug
26.1 ug
14.8 ug
51.4 ug
249.4 ug
247.2 ug
56.6 ug
49.5 ug
5.0 ml
~*
NH-15 CF
(per liter)
_
-
57.9 ug
52.4 ug
6.1 ug
5.2 ug
3.0 ug
10.3 ug
49.9 ug
49.4 ug
11.3 ug
9.9 ug
5.0 ml
200.0 mg
*2-Amino-2-hydroxymethyl-l, 3-propanediol
tDisodium ethylene diaminotetraacetate
The modification designated NH-15 was employed for the assessment of physi-
cal factors on growth. Chelate-free medium (NH-15 CF) was used to determine
the chronic effects of Hg, Ni and Cr. The media were prepared (8 L amounts)
in 12-liter Pyrex bottles, filtered through Whatman's GF/A glass fiber
filters, and autoclaved at 15 psi for 1 hour. A sterile NaHC03 solution was
added to NH-15 CF after autoclaving when the temperature of the medium had
decreased below 35C. This solution was prepared by placing a 250 ml screw
cap Erlenmeyer flask containing 13.5 grams of NaHC03 in an oven at ± 30
minutes, followed by an aseptic addition of 150 ml of sterile water to the
flask.
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Rigid control of light quality and intensity was required to avoid light
elicited variations of the chlorophyll a measurements. This system is an
excellent method of evaluating growth without disturbing the culture popula-
tion and measurements can easily be made often, if necessary. Examination
of the tubed test-portions were made also with an American Optical inclined
stage stereoscopic microscope with 3x and lOx magnification.
Experiments for the determination of the toxicity of various materials
to phytoplankton cultures had a major advantage over toxicity experiments
involving most organisms in that a large number of experimental containers
each with approximately 10^ to 101 organisms/L can be utilized for each
test condition. On the other hand, it was frequently difficult to detect
toxic effects on phytoplankton culture populations. This was especially
true with organisms that are non-motile and do not lyse readily after expo-
sure to what proved to be a lethal concentration of a material. This is the
case with the diatom, T. pseudonana and this is one reason we adopted the
fluorometric method to detect in vitro change in chlorophyll a. In addition,
we extended the total period of observation from 24 hours to 48 hours.
Although these methods were employed and care was exercised to prevent vari-
able results, variations in the toxicity of a metal did occur between experi-
ments (Figure 1). This variability involved experiments with G. splendens
in which motility was used to evaluate toxicity effects. Primarily because
of such variations, the minimum toxic concentration, the maximum non-toxic
concentrations and the minimum concentration that appeared to cause the lysis
of most cells in a test portion are included as levels of toxicity in this
report. The term "toxic concentration" as used in this report on acute
toxicity is in the following account.
In theory, the addition of a specific amount of substance to a phyto-
plankton culture portion may result in one of eight conditions, or more,
after a relatively short period of exposure (e.g., 48 hours) as follows:
1. Effective culture growth exceeds that of a control portion
(one that received no added test material).
2. Effective culture growth was similar to that of the control
portion.
3. Growth greatly exceeds mortality but effective growth is less
than the control.
4. Growth slightly exceeds mortality or effective growth is much
less than the control.
5. Growth is similar to mortality, or there is no effective growth.
6. Growth is less than mortality or there is slight effective
mortality.
7. Growth is much less than mortality or there is high effective
mortality.
8. Total or almost total mortality of the test population.
It is possible for all of these events to occur in different test
portions of a single bioassay (Figure 2).
In experiments conducted during this program in which the population
levels of test portions of a culture were followed for 48 hours or longer
8
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>50
100
J
1000
HOURS
Figure 1. Variability of Cu toxicity to G. splendens (.values in rog/L).
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70
• 50
x
a.
O
£ 40
30
3 20
ui
oc.
10
I
10
20
30
40
TIME OF EXPOSURE (HOURS>
---NGL
--TML
i
50
Figure 2. The relationship of MTL, NTL, NGL and TML to the toxicity of Cu
to G. splendens (results from 11 experiments).
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on a daily schedule by determining the relative chlorophyll a, in vitro, the
following pattern of results occurred if the test culture was growing at the
time the experiment was initiated.
1. Populations in control portions continued to grow, i.e., the chlor-
ophyll a content was higher after 48 hours than at the time the
test was started.
2. Populations in certain test portions with low metal addition con-
tinued to grow. In some cases, the culture portion grew faster or
the same as the control portion.
3. Populations in test portions that received increasingly higher
metal additions than the control had progressively lower final
populations.
4. The population levels of some test portions did not change greatly
during the test period.
5. Populations that received progressively higher metal additions than
those mentioned in Items 1 through 4 decreased significantly and in
some cases they decreased to the level of "0" relative chlorophyll
a value with the most sensitive Fluorometer window setting (30X).
Although the population level of a culture may have been high, if the
culture was not growing at the time an experiment was initiated, the popula-
tion level of all test portions and the controls usually decreased. Since
the population of the culture portion that received no addition of the test
material (control portion) may decline more than those of test portions that
received growth inhibiting levels of a substance, and this often occurs, the
assay results are not a valid measure of toxicity. Indeed, such assays con-
ducted with natural populations could be misleading.
As a result of these developments, only actively growing cultures were
used in the acute toxicity experiments. Cultures were established by trans-
ferring large inocula into fresh medium 3 to 5 days (2*. pseudonana and J. gal-
bana), 4 to 6 days (G. .halli) or 14-18 days (G. splendens) before a culture
was to be used in an experiment. Based on this procedure and the previously
described observations, the toxicity terms used in this report are as
follows:
Minimum Toxic Level (MTL) was that level of minimum metal addition in which
the increase of the chlorophyll a content of the test population was
approximately 65 percent of the increase of the control population
during the 48-hour test period.
Maximum Non-Toxic Level (NTL) was that level of maximum metal addition at
which the increase of relative chlorophyll a of the test population
was significantly greater than 65 percent of the increase in the
control population during the 48-hour test period. Since results of
more than one experiment are included in these determinations this
value can be either higher, lower or the same as the MTL.
11
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No Growth Level (NGL) that level of metal addition in which the relative
chlorophyll a of the test population changes less than 5 percent during
the 48-hour test period.
Minimum Total Mortality Level (TML) that level of metal addition at which the
chlorophyll a of the test population decreased during the 48-hour test
period to a fluorometric reading of less than 5 (on scale of 0 to 100)
with the most sensitive (SOX) window of the Fluorometer.
These definitions are not entirely correct. Most results indicate that
there are progressively lesser degrees of metal inhibition associated with
progressively lower metal additions (Figure 2). The end-point of inhibition
is difficult to establish. Often test portions that receive small specific
amounts of two metals in one experiment may grow to a lower terminal popula-
tion level than the control portion, but in another comparable experiment the
terminal population level may be higher than that of the control. Also, the
four toxicity levels (Figure 2) will obviously be progressively higher
(NTL>MTL>NGL>TML), if only results of a single experiment are considered, but
combining results of three or more experiments often resulted in a lower de-
fined level having the same toxic effects as the next even higher toxicity
level. Although these levels were arbitrarily selected, they represent toxi-
city levels that are basic to evaluating the effects of a substance on a
phytoplankton culture.
RESULTS
The four organisms were not equally inhibited by any one of the nine
metals (TABLE 2). The toxicity levels of some metals were similar for two
organisms (e.g., the LMTL of the nine metals to G. splendens and G. halli),
but not as many as three organisms. The lower MTL of Ni were nearer the
same for all four organisms (0.1 to 0.5 mg/L) than were those of any other
metals (TABLE 2). On the other hand, the toxicity levels of some metals
differed by an order of magnitude or more between organisms. Also, based on
the LMTL and LNGL, with the exception of Ni and Mo, the metals were much more
toxic to the two dinoflagellates than to I. galbana and T. pseudonana cul-
tures.
The nine metals were more toxic to G. splendens and G. halli than to
T. pseudonana and least toxic to I. galbana. Although 13 of the 18 LMTL and
LNGL of the nine metals were higher for I. galbana than for T. pseudonana,
all nine of the LTML for I. galbana were higher than those for T. pseudonana.
The toxicity levels of the nine metals to the two dinoflagellates were simi-
lar. If there was a difference in their sensitivity to these metals,
G. splendens was possibly more susceptable to lower additions of more of the
metals than was G. halli.
Despite these observed differences in the toxicity levels between
organisms, there were broad patterns of toxicity levels of groups of the
metals to all four organisms. Based on the LMTL and LNGL of the nine metals
to the four organisms, three groups of metals can be arranged (TABLE 3).
Although there are exceptions and there are overlapping values, the means of
12
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TABLE 2. ACUTE TOXICITY OF NINE METALS TO CULTURES OF FOUR MARINE PHYTO-
PLANKTON ORGANISMS (VALUES IN MG/L)
G. splendens
Toxicity Level
Metal
Hg
Cu
Ag
Zn
Cr
Fe
Ni
V
Mo
L*
.005
.01
.005
.05
.05
.2
.2
1
2
MTL
H
.09
.1
.04
1.0
.8
5
6
20
35
L
.002
.005
.002
.02
.02
.1
.1
.5
1
NTL
H
.08
.09
.03
.8
.5
4
5
18
30
NGL
L
.02
.01
.01
1
.1
.5
1
2
5
H
.1
.1
.08
5
7
25
15
25
70
L
.04
.07
.02
2
.5
1
15
10
10
TML
H
.3
.2
.1
10
25
30
20
25
200
Number of
Experiments
9
18
14
9
9
8
10
10
9
X. galbana
Toxicity Level
Metal
Hg
Cu
Ag
Zn
Cr
Fe
Ni
V
Mo
L
0.1
.05
.02
.5
.5
1
.5
20
10
MTL
H
.9
.2
.1
4
10
25
3
30
>300
L
.08
.02
.01
.2
.4
.5
.2
18
20
NTL
H
.8
.3
.2
3
5
20
2
40
>300
NGL
L
.2
.1
.05
1
.6
6
.5
50
20
H
1
1
1
7
>40
30
>50
>50
>300
L
.3
1
.7
7
>40
>35
>50
50
>300
TML
H
5
>2
*5
>10
>40
>35
>50
>50
>300
Number of
Experiments
19
28
28
14
12
17
12
12
7
*Two levels of toxicity (low-L) and (high-H) are designated for each
toxicity level. The low level (L) is the minimum addition of the metal
in the total number of experiments (right column) that had the designated
toxicity to occur with the metal addition shown and the high level (H) is
the maximum metal addition that produced this toxicity effect.
(continued)
13
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TABLE 2 (continued)
T. pseudonana
Toxicity Level
Metal
Hg
Cu
Ag
Zn
Cr
Fe
Ni
V
Mo
L
.06
.06
.04
.1
.02
8
.1
10
1
H
.5
.08
.08
.5
.08
>20
.4
30
100
L
.05
.05
.03
.08
.01
7
.05
5
.5
H
.1
.07
.06
.4
.05
>20
.2
30
80
L
.08
.08
.08
.5
.08
20
.2
10
H
1
.5
.2
2
.6
>20
10
40
10 >100
L
.08
.1
.08
1
.1
>20
.2
20
60
H
1
.5
.5
20
>10
>20
>100
40
>100
mimuei. UL
Experiments
10
10
14
10
9
10
10
10
10
G. halli
Toxicity Level
MTL
Metal
Hg
Cu
Ag
Zn
Cr
Fe
Ni
V
Mo
L
.02
.005
.005
.02
.02
.5
.2
2
2
H
.05
.14
.04
.2
.5
5
5
20
25
L
.01
.002
.002
.01
.01
.2
.1
1
1
NTL
H
.02
.12
.02
.1
.2
2
2
10
20
NGL
L
.03
.01
.01
.2
.1
2
.5
10
20
TML
H
.1
.16
.07
2
.5
>10
20
25
50
L
.05
.05
.05
5
.2
10
2
50
50
H
>.l
>5
2
50
>20
100
50
100
>100
Number of
Experiments
8
10
6
5
10
5
6
4
4
14
-------
TABLE 3. THE LOW MINIMUM TOXICITY LEVEL (LMTL-1), LOW NO GROWTH LEVEL
(LNGL-2) AND LOW TOTAL MORTALITY LEVEL (LTML-3) OF NINE METALS
TO FOUR PHYTOPLANKTON CULTURES (VALUES IN MG/L)
ORGANISM
Metal
Hg 1
2
3
Cu 1
2
3
Ag 1
2
3
Zn 1
2
3
Cr 1
2
3
Ni 1
2
3
Fe 1
2
3
V 1
2
3
Mo 1
2
3
T. pseudonana
0.06
0.08
0.08
0.06
0.08
0.1
0.04
0.08
0.08
0.1
0.5
1.0
0.2
0.08
0.1
0.1
0.2
0.2
8.0
20.0
>20.0
10.0
10.0
20.0
1.0
10.0
60.0
J . ga 2b ana
0.1
0.2
0.3
0.05
0.1
1.0
0.02
0.05
0.7
0.5
1.0
7.0
0.5
0.6
>40.0
0.5
0.5
>50.0
1.0
6.0
>35.0
20.0
50.0
50.0
10.0
20.0
>300.0
G. splendens
0.005
0.02
0.04
0.01
0.01
0.07
0.005
0.01
0.02
0.05
1.0
2.0
0.05
0.1
0.5
0.2
1.0
15.0
0.2
0.5
1.0
1.0
2.0
10.0
2.0
5.0
10.0
G. halli
0.02
0.03
0.05
0.005
0.01
0.05
0.005
0.01
0.05
0.02
0.2
5.0
0.02
0.1
2.0
0.2
0.5
2.0
0.5
2.0
10.0
2.0
10.0
50.0
2.0
20.0
50.0
15
-------
the LMTL of the three groups progressively differed by a six-fold factor as
follows: Group I—0.03 mg/L, Group II—0.18 mg/L, Group III—1.3 mg/L and
Group IV—6.0 mg/L. A questionable feature of this group arrangement involves
Fe and Ni. As mentioned previously, the variation of the LMTL of Ni for the
four organisms was 0.1 to 0.5 mg/L (Mean-0.2 mg/L), the smallest range of the
nine metals. On the other hand, the Fe LMTL varied from 0.2 to 8.0 mg/L
(Mean-1.2 mg/L); the largest toxicity range of these metals. The high toler-
ance of T. pseudonana to Fe additions was the major contributor to this vari-
ability. If either the LMTL of G. splendens and G. halli or the LMTL values
for J. galbana and T. pseudonana are combined, the proposed groups are less
variable. If the molecular concentration of the toxicity levels of the nine
metals are compared, the same group arrangement resulted (TABLE 4). The
results presented above pertained to the low levels of toxicity observed in
the numerous tests conducted. Although these lower values represent levels
of toxicity that must be of primary consideration, there are other results,
(e.g., the high levels of toxicity),that should be considered for a more
complete evaluation of the tests.
The high levels of toxicity (HMTL, HNGL and HTML) were approximately
one order of magnitude higher than the low values previously presented
(TABLE 5). For example, Group I metals had HMTL from 0,04 to 0.9. mg/L where-
as the LMTL values were 0.005 to 0.1 mg/L and Group I mean HMTL and LMTL were
0.19 and 0.03 mg/L, respectively. The greatest differences in the low and
high levels of toxicity that occurred in the tests were in the Group II
metals—Zn and Cr. For example, values for the four organisms were from 0.08
to 10.0 mg/L compared to the LMTL of 0.02 to 0.5 mg/L. Although some tests
were repeated as many as five times in attempts to determine the cause.Cs) or
reduce this variability, the variability continued and the causes, remained
unknown. In addition, although the general trend of increased toxicity
according to group number was observed with the high toxicity levels, the
HMTL, HNGL and HTML of the Group II and Group III metals overlapped more than
did the low toxicity level values. The higher variability and more ill-
defined groups should probably be expected, as this was usually the case as
the toxicity of a substance was less.
16
-------
TABLE 4. GROUP LIST OF NINE METALS BASED ON THEIR GRAVIMETRIC (MG/L) AND
MOLECULAR yg AT/L WEIGHTS OF COMBINED LMTL AND LNGL ACUTE TOXICITY
VALUES TO G. halli, G. splendens, I. galbana and T. pseudonana
Toxicity Level
LMTL
Group
Group
Group
Metal
I Ag
Cu
Hg
II Cr
Zn
Ni
III Fe
V
Mo
MG/L
0.005-0,04
0.005-0.06
0.005-0.1
0.02-0.5
0.02-0.5
0.1-0.5
0.2-0.5
1.0-20.0
1.0-10.0
Ug AT/L
0.04-0,37
0.08-0.94
0,03-0.50
0.39-9.6
0,31-7.6
1.7-8.5
1,7-8.5
19.6-392
10.4-104
LNGL
MG/L
0.01-0,08
0.01-0.08
0.02-0.08
0.08-0,6
0.2-1,0
0.2-1.0
0.5-20.0
2.0-50,0
5.0-20.0
yg AT/L
0.09-0.74
0.16-1.27
0,10-0.40
1,5-11,5
0.31-15.3
3.4-16.9
8,9-357
39.2-980
52.0-208
17
-------
TABLE 5. HIGH MINIMUM TOXICITY LEVEL (HMTL-1), HIGH NO GROWTH LEVEL (HNGL-2)
AND HIGH TOTAL MORTALITY LEVEL (HTML-3) OF NINE METALS TO FOUR
MARINE PHYTOPLANKTON CULTURES
Hg
Cu
Ag
Zn
Cr
Ni
Fe
V
Mo
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
i
2
3
1
2
3
1
2
3
1
2
3
Tp
0.5
1.0
1.0
0.08
0.5
0.5
0.08
0.2
0.5
0.5
2.0
20.0
0.08
>10.0
>10.0
0.4
10.0
>100.0
>20.0
>20.0
>20.0
30.0
40.0
40.0
100.0
>100.0
>100.0
19
0.9
1.0
5.0
0.2
1.0
>2.0
0.1
1.0
>5.0
4.0
7.0
>10.0
10.0
>40.0
>40.0
3.0
>50.0
>50.0
25.0
30.0
>35.0
30.0
>50.0
>50.0
>300.0
>300.0
>300.0
Gs
0.09
0.1
0.3
0.1
0.1
0.2
0.04
0.08
0.1
1.0
5.0
10.0
0.8 -
7,0
25.0
6.0
15.0
20.0
5.0
25.0
30.0
20.0
25.0
25.0
35.0
70.0
200.0
Gh
0.05
0.1
>0.1
0.14
0.16
>5.0
0.04
0.07
2.0
0.2
2.0
50.0
5.0
5.0
>200.0
5.0
20.0
50.0
5.0
>10.0
100.0
20.0
25.0
100.0
25.0
50.0
>100.0
Toxicity
Level
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
1
2
3
18
-------
SECTION 3
RESIDUAL TOXICITY OF NINE METALS TO
G. splendens, I. galbana, T. pseudonana and G. halli
Toxicological reports give accounts of the uptake of sufficient quanti-
ties of a substance in a short time period to produce subsequent mortality or
metabolic inhibition. In the evaluation of the toxicity of metals to phyto-
plankton, a consideration which had not received attention was whether or not
a phytoplankton population could be exposed to a toxic metal concentration
and subsequently grow at a normal rate and if so, what was the highest con-
centration that would not permanently restrict subsequent growth and how did
this value compare to the acute toxicity level. Mandelli (1969) showed that
the uptake of Cu by four phytoplankton organisms occurred in the first hour
of exposure. Button and Hostetter (1977) showed that the uptake of copper by
two freshwater phytoplankton organisms was fast during the first five minutes
of exposure and continued at a reduced rate for a period of two hours, but
there were small, if any, amounts removed from the medium afterwards. Both
reports showed that the organisms released Cu after they were placed in fresh
medium and the latter publication indicated that most of the copper (46% and
59%) was released within the first two hours. Thus, organisms that survived
the period of exposure to•a toxic metal concentration may subsequently
release the absorbed material and experience no residual effect. On the
other hand, a population may survive the exposure period but sustain perma-
nent physiological damage and eventually die-off. Residual toxicity tests
were conducted in an attempt to evaluate the possibility of permanent impair-
ment to cultures of four phytoplankton organisms.
In the residual toxicity experiments with G. splendens, I. galbana and
T. pseudonana, culture portions of the organisms were exposed to various
concentrations of each metal for a period of 48 hours following the same pro-
cedure as described for the acute toxicity tests. After this 48-hour expo-
sure, either 0.1 ml (J. galbana and T. pseudonana) or 0.3 ml (G. splendens)
from each test portion was transferred to 10 ml portions of fresh culture
medium (TABLE 1) and incubated for 14 days in the standard culture condition
described in the previous section. If growth occurred after the transfer,
the growth rates were determined from the relative chlorophyll a measurement
that were made every 1 to 3 days. If growth rates were, below normal, sub-
cultures were made to determine if the growth of that specific population was
permanently impaired. If no growth occurred in a test portion, after the
transfer to fresh culture medium, it was assumed that there were no viable
cells or that their growth had been permanently inhibited. The highest level
in which growth occurred was recorded as the level of residual toxicity. This
was done because cultures grew from many of the test portions that received
the maximum additions of the metals. Residual toxicity tests with G. halli
19
-------
were conducted in this same manner except that 0.1 ml portion of the test
portions were transferred to fresh culture medium after exposure to the
various metal addition for period of 1, 24, 48, 72 and 144 hours.
RESULTS
In some instances cultures exposed to high metals concentrations sub-
sequently grew at a reduced rate during the 14-day incubation period, but
their normal growth after subsequent subcultures were made indicate that
there was no permanent metabolic impairment. Although it could not be deter-
mined with certainty, results indicated that if some organisms survived in
a test portion during the exposure period, they resumed normal growth follow-
ing one or two transfers in fresh medium. Indeed, cells transferred from
test portions in which no surviving cells were detected, by microscopic exam-
ination, subsequently grew. Yet, it was frequently observed in cultures of
G. splendens, G, halli and J. galbana that most of the cells in a test por-
tion lysed during the exposure period, but a few individuals survived as
indicated by their motility. Other test portions that appeared by micro-
scopic examination to contain living cells did not grow after being trans-
ferred. Fluorometric measurements often were better indicators of the pres-
ence of live cells. A relative chlorophyll a value of "0" with the most
sensitive (SOX) window setting was usually a reliable indicator that a test
portion contained no living cells, but test portions that gave a measurable
fluorometric reading did not always grow after being transferred.
Although metal additions were increased to relatively high levels, the
organisms remained viable in over 50% of the test portions that contained the
maximum concentration tested.
Higher addition than those listed (TABLES 6 and 7) were not tested
because the saturation level of the metal in seawater had been greatly ex-
ceeded and precipitation of the various components of the medium could have
inhibitory effects that were not directly related to the metal concentrations.
Special systems are needed for each metal if concentrations as high or higher
than those employed here are to be evaluated realistically.
In most cases, the residual toxicity level of each metal was much
higher than the acute toxicity level (TABLES 6 and 7). The residual toxicity
levels for V with I. galbana and Fe with T. pseudonana were between 10 and
over 1000 times that of the LMTL values. On the other hand, the residual
toxic levels were 10 times the HNGL or less in 28 of 36 cases and in 17 of
36 cases, they were 2 times the HNGL, or less. This latter consideration
may indicate that the no growth level was slightly higher than the residual ,
toxicity level or perhaps these were two different assessments of the same
basic condition.
The residual toxicity levels of Ag, Cu and Hg to G. splendens were less
(more toxic) in all cases and those of Fe, Ni, V and Mo were less in 7 of 9
.cases than those of the other three organisms. This comparison may be indi-
cative of a higher sensitivity of G. splendens to these metals which, except
for results with G. halli, was indicated by the acute toxicity tests. On the
20
-------
TABLE 6. RESIDUAL TOXICITY (A) OF NINE METALS TO I. galbana, T. pseudonana
AND G. splendens (VALUES ARE IN MG/L)
Metal
Ag
Cu
Hg
Zn
Cr
Fe
Ni
V
Mo
J. gaJb ana
ABC
7(8) 0.02 1
10(9) 0.05 1
6(5) 0.1 1
10* (D) 0.5 7
40* 0.5 >40
35* 1.0 30
50* 0.5 >50
100* 20 >50
300* 10 >300
T. pseudonana
ABC
1(2) 0.04 1
20(30) 0.06 0.5
5(6) 0.06 0.2
100* 0.1 2
10* 0.2 >10
20* 8.0 >20
100* 0.1 10
100* 10 40
100* 1 >100
G. splendens
ABC
0.1(0.2) 0.005 0.08
0.1(0.2) 0.01 0.1
0.2(0.3) 0.005 0.1
20* 0.05 5
60* 0.05 7
30(35) 0,2 25
80(100) 0.2 15
30(35) 1.0 25
100(200) 2.0 70
(A) The highest concentration (mg/L) of the metal to which the organisms
were exposed for a period of 48 hours (i.e., the equivalent amount
added to the test portion) and subsequently grew after being trans-
ferred to fresh medium. Numbers in parentheses are the lowest levels
tested to which the organisms were exposed for 48 hours and did not
grow after being transferred.
(B) The LMTL values of the metals from Table 2.
(C) The HNGL values of the metals from Table 2.
(D) Asterisk (*) indicates that this addition was the highest concentra-
tion tested.
21
-------
TABLE 7. RESIDUAL TOXICITY (A) OF NINE METALS TO G. halli AFTER EXPOSURES
OF 1, 24, 48, 72 AND 144 HOURS.
Metal
Ag
Cu
Hg
Zn
Cr
Fe
Ni
V
Mo
Hours of Exposure
1
10* (B)
0.2(0.5)
10*
50*
200*
100*
100(200)
500*
500*
24
10*
0.2(0.5)
10*
50*
100(200)
100*
100(200)
500*
500*
48
10*
0.2(0.5
10*
50*
100(200)
100*
100(200)
500*
500*
72
5(10)
0.5(1)
0.2(0.5)
50*
50(100)
100*
100(200)
500*
500*
144
5(10)
0.5(1)
0.5(1.0)
50*
50(100)
100*
10(20)
500*
500*
(A) The highest concentration (mg/L) of the metal to which G. halli was
exposed for periods of 1, 24, 48, 72 or 144 hours (i.e., the equiva-
lent amount added to the test portion) and subsequently grew after
being transferred to fresh medium. Numbers in parentheses are the
lowest levels tested to which G. halli was exposed for the indicated
time and did not grow after being transferred.
(B) Asterisk (*) indicates that this addition was the highest amount
tested.
22
-------
other hand, the G. halli 48-hour residual toxicity results were similar to
those of X. galbana and T. pseudonana.
The results of the G. halli residual toxicity tests with exposure
periods of 1 to 144 hours indicated that a 48-hour exposure period does not
always give representative toxicity results and that the uptake and release
of the different metals may be different. The residual toxicity of some
metals did not increase as the period of exposure increased. In fact in the
case of Cu, the 1, 24 and 48 hour residual toxicity levels (0.2 mg/L) were
less than the 72 and 144 hour exposures (0.5 mg/L). Considering the fast
uptake of Cu (Mandelli 1969 and Button and Hostetter 1977), an exposure of
one hour would be expected to be as effective as a longer exposure. A possi-
ble explanation is that the ionic concentration of Cu in the medium decreased
during the 72 hour period and the cells released Cu.
The uptake of the other metals of the nine tested for which a residual
toxicity level was determined (Ag, Hg, Cr, Ni) did not appear to be as fast
as that of copper. There was no effect of exposures of 1, 24 and 48 hours'
to the maximum Ag and Hg additions (10 mg/L). A 1-hour exposure to 200 mg Ni
resulted in no growth after transfer but longer exposures did not increase
the toxicity, except that after 144 hours the toxicity level was 20 mg/L.
Also, there were increases in the toxicity of Cr after exposures of 24 and
72 hours. These differences in the time of exposures required to elicit a
toxicity level involve many features of the system such as the various possi-
ble reactions of the metal with the organism and the combined reactions of
the metal, the medium and the organism.
23
-------
SECTION 4
THE EFFECTS OF TEMPERATURE AND SALINITY ON THE ACUTE TOXICITY OF
METALS TO G. splendens, I. galbana AND T. pseudonana
If the limits of the salinity or temperature ranges in which these
three organisms grew are disregarded, the broad features of their growth
response to temperature and salinity are predictable. The temperature
response is such that as the temperature increases from some level below
which there is no growth, growth rates increase gradually to an optimum level
which extended upward approximately 6 to 12C. Further thermal increases of
4 to 6C caused a sharp decline of the growth rate. The salinity response
was somewhat similar, but reversed. Growth rates increased gradually as the
salinity decreased from some level above which no growth occurred. The sharp
decline in growth rates in response to salinity occurred near the low end of
the growth range and the optimum salinity level for growth was relatively
broad. If these two general patterns are considered together, the levels of
temperature and salinity near which the sharpest declines in growth occurred,
high temperature and low salinity, would be the primary suspected combination
of conditions that may interact detrimentally to the growth of these organ-
isms. Secondarily, the combined effects of low temperature and low salinity
may have reduced the growth rates. No attempt was made to determine the
upper salinity levels that the three organisms tolerated, therefore the
effects of high salinity, i.e. near the upper tolerance limits, in combina-
tion with different temperatures were not determined.
MATERIALS AND METHODS
Five walk-in constant temperature culture rooms were employed in this
work. Usually the temperature in these rooms was maintained at 12C, 16C,
20C, 24C and 28C ± 1. Temperatures below 12C were established by lowering
the room usually maintained at 12 and temperatures above 28 were estab-
lished by altering the concentrations of the major salts (NaCl, KC1, MgCl2*
6H£0, MgS04'7H20 and CaCl2'H20) without altering their relative ratios and
without changing the concentrations of the other medium components (TABLE 1)
Cultures for each temperature, salinity or temperature-salinity combi-
nations were established by step-wise subculturing into progressively lower
or higher temperatures, salinity levels and combination temperature and
salinity levels. The growth rate of each of the three organisms was deter-
mined for each condition and only those combinations of temperature and
salinity in which the organisms grew were employed in toxicity tests.
All other procedures and conditions employed in these experiments were
24
-------
the same as described previously for acute toxicity experiments except that
the growth of the test portions were determined by measuring the relative
chlorophyll a, in vitro, before the portions were delivered to the culture
tubes and after 1, 24 and 48 hours exposure to the test concentrations of
the metals. Counts were not made of the test portions during these experi-
ments .
The evaluations of the effects of the metals were made by calculating
the MTL values for each combination of metal concentrations and temperature,
salinity or salinity-temperature combination. Also, the 50% mortality (M-50)
values were determined for each of these conditions or combinations of condi-
tions. The 50% mortality (M-50) values were determined by plotting the
48-hour fluorometer readings (arithmetic scale) for the various concentrations
of the test metal (log scale) for temperature-salinity combinations on semi-
log paper. The 50% mortality values was that concentration on the long-scale
that corresponded to a population level on the curve that was 50% of the
initial fluorometer reading (T=0) for the population. The MTL value was that
concentration on the same curve that represented the 65% growth level of the
population based on the initial (T=0) and final (T=48 hours) readings of the
controls.
RESULTS
The temperature and salinity requirements of G. splendens (Sweeney,
1951; Thomas, et al, 1973; unpublished data); Isochrysis galbana (Kain and
Fogg, 1958; Freeberg, 1976, unpublished) and T. pseudonana (Guillard and
Ryther, 1972; Ryther and Guillard, 1962; Guillard and Mykelstad, 1970) have
been treated in previous reports. However, the cultures we employed may have
had or have developed slightly different requirements from the isolates used
by the above investigators. For this reason the general features of the
effects of temperature and salinity were examined and results are included
in this report.
G. splendens grew within a temperature range of 16C to 33C and a salin-
ity range of 14 ppt to 37 ppt. It did not grow at a temperature of 14 C; and
it grew only slightly at 33C in combination with 28 ppt salinity but not
14 ppt salinity (TABLE 8) No growth occurred in media with salinity levels
of 12 ppt or less, but G. splendens grew well in media with 37 ppt salinity—
the highest tested. Based on our results, G. splendens is a eurythermal,
euryhaline organism that grew best at temperatures between 20C and 30C and
salinity levels between 14 and 37 ppt. The upper limit of the temperature
range was approximately 3C higher than that reported for a California iso-
late of G. splendens (Thomas et al, 1971).
J. galbana grew within a temperature range of 12C and 28C (the lowest
and highest tested) and within the salinity range of 6 and 37 ppt, the lowest
and highest tested. It's growth rate was low at 28C with salinity levels
between 14 ppt and 37 ppt, but if the salinity was less than 12 ppt, it did
not grow. J. galbana did not survive at 30C at any salinity level. The
highest division rates occurred at temperatures from 16 to 24C and with
salinity levels between 14 ppt and 24 ppt. This organism showed both
25
-------
eurythermal and euryhaline characteristics and it was tolerant of short-term
temperature and salinity changes. Of the conditions tested, temperature
levels of 28C and higher were the most inhibitory.
T. pseudonana (clone 3H) adjusted to progressively lower salinity
levels down to the lowest tested (3 ppt) and grew well at all temperatures
and salinity combinations employed. This organism grew best within broader
ranges of temperature and salinity than did G. splendens and J. galbana. It's
maximum growth rates were with salinity levels between 10 ppt and 28 ppt
and with temperatures between 12C and 28C. The minimum growth occurred with
high temperatures (24-28C) and low salinity (3 ppt-5 ppt) combinations.
This isolate had euryhaline and eurythermal attributes,
TABLE 8. EFFECTS OF TEMPERATURE AND SALINITY ON THE GROWTH RATES (K) OF
G. splendens, I. galbana and 21. pseudonana
G. splendens
T C 12 16 20 24 28 30 32 33
S ppt
10
12
14
16
18
20
24
28
37
CD*
CD
CD
CD
CD
CD
CD
CD
CD
NGt
NG
.33*
.38
.35
.27
.25
.29
.2
NG
NG
.41
.44
.41
.49
,42
.28
.43
CD
CD
.43
,48
.40
,5
,45
.29
.47
.31
.42
.37
,48
.51
.38
.31
.29
.35
.43
,42
.53
.48
.51
,2
.18
.20
.23
.20
,25
CD
.16
* CD = controls died.
t NG = controls lived, but there was no growth.
f K values = numerical values representing growth/cell/day.
(continued)
26
-------
TABLE 8 (continued)
T C 12
S ppt
6
9
12
14
16
20
24
28
37
.53
.50
.61
.68
.61
.75
.73
.71
.68
16
,46
.58
.57
1.0
.75
.84
0.83
0.87
.79
I.
20
0.57
.74
.87
1.0
.94
1.1
.92
1.0
.8
galbana
24
.7
1.0
.99
1.1
1.0
1.1
0.94
1.1
1.1
28
CD
CD
NG
0.36
0.34
0.36
0.36
0.23
0.17
30
CD
CD
CD
CD
CD
CD
CD
CD
3
5
7
10
14
21
28
0.71
.62
.69
.60
.81
1.0
.65
0.72
.68
.70
.68
.75
.92
.75
r.
0.69
0.72
.66
i .79
.78
.91
.98
pseudonana
0.58
.67
.72
.77
.94
1.2
.77
0.45
0.49.
.42
.88
.99
1.4
.95
27
-------
Temperature Effects on Acute Metals Toxicity to G. splendens.
Results of toxicity tests of the nine metals to G. splendens grown in
medium with 28 ppt salinity and temperatures of 16C, 20C, 24C and 28C did not
show that there was a general effect of these temperatures on metals toxicity.
The toxicity of Ni and Zn increased as the temperature was increased (TABLE 9)
but temperatures of 16C to 28C had no effect on the toxicity of V, Cu, Mo, Hg
and Ag.
There were no effects of temperature on the toxicity of Fe and Cr to
G. splendens. Concentrations between 0.1 and 1.0 mg/L of these metals
apparently stimulated the growth of this organism. It was not possible to
assign the possible effect of this stimulatory activity to the final experi-
mental results. G. splendens Cr-LMTL was at 28C but the Cr-HMTL was at 24C.
Perhaps this organism was less susceptible to metals at temperatures that
were optimum for growth. Ni, V, Fe, Cr, Cu and Hg were less toxic at 24C
than at 28C, but the M-50 value for these metals did not show similar results.
On the other hand, the MTL values for 7 of the 9 metals were higher (less
toxic) at 24C than at 16C. The two exceptions were Ni and Zn.
TABLE 9. THE EFFECTS OF TEMPERATURE ON THE TOXICITY OF METALS TO
G. splendens (VALUES IN MG/L)
Metal
Ag
Cu
Hg
Temperature
16C
20C
24C
28C
M-50
0.032
0.054
0.035
0.041
MTL
0.0075
0.018
0.013
0.013
M-50
0.095
0.086
0.094
0.045
MTL
0.01
0.017
0.047
0.032
M-50
0.029
0.038
>0.05
>0.05
MTL
0.012
0.026
0.023
0.019
Ni
Cr
Zn
16C
20C
24C
28C
16C
20C
24C
28C
>20
>20
5
3
17
>30
26
>30
.0
.0
.9
.5
.5
.0
.0
.0
1.
0.
0.
0.
Fe
2.
1.
3.
1.
0
95
56
13
0
7
6
4
>1.
>1.
>1.
>1.
>20.
>20.
>20.
>20.
0
0
0
0
0
0
0
0
0
0
0
<0
V
5
9
15
13
.012
.012
.016
.01
.8
.5
.5
.5
8.0
4.5
2.8
1.5
17.0
50.0
45.0
>100.0
0
0
0
0
Mo
1
6
2
5
.22
.24
.11
.12
.0
.8
.0
.8
Salinity Effects on Acute Metals Toxicity.
There was a possible effect of salinity on the toxicity of Cu and Ag to
G. splendens, but I. galbana did not respond similarily to Cu and Ag or Fe,
28
-------
Zn and Hg.
Salinity had an indirect effect on the toxicity of Cu and Ag to G. splen-
dens. Toxic effects were progressively less in media with progressively
higher salinity levels from 14 ppt to 28 ppt (TABLE 10).
Unlike the toxicity of Cu and Ag at different salinity levels to
G. Splendens, the results of tests with I. galbana did not indicate that
salinity altered the toxicity of these metals. Also, salinity had no effect
on the toxicity of Fe, Zn and Hg to I. galbana (TABLE 10). Tests with Cu in
media with seven different salinity levels from 7 ppt to 37 ppt had MTL
values from 0.13 to 0.15 mg Cu/L except in medium with 37 ppt salinity in
which the Cu was less toxic (MTL=0.3 mg Cu/L).
TABLE 10. EFFECTS OF SALINITY ON THE TOXICITY OF METALS TO G. splendens
AND I. galbana. (VALUES IN MG/L)
G. splendens
M-50 MTL
14 ppt
16 ppt
20 ppt
24 ppt
28 ppt
7 ppt
10 ppt
14 ppt
16 ppt
20 ppt
28 ppt
37 ppt
12 ppt
20 ppt
28 ppt
Ag-20C
.021
.024
.030
.052
.047
Cu-20C
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
>1.0
Hg-20C
.18
.16
.17
0013
0065
0085
0060
0094
M-50
Cu-30C
.043 <0
.062 <0
>0 . 10 0
>0.10 0
J.
MTL M-50 MTL
.005
.005
.01
.034
galbana
Ag-20C Fe-20C
.13
.15
.14
.14
.15
.13
.30
.11
.10
.10
.09
.08
.086
.081
9.0 1.55
025
025
024 8.5 1.6
026
9.8 1.45
M-50 MTL
Zn-20C
6.8 4.4
3.2 2.7
3.8 2.3
29
-------
Combined Effects of Temperature and Salinity on Metals Toxicity.
The effects of temperatures near the tolerance limits (14 and 30C) in
combination with salinity levels of 14 ppt and 28 ppt were employed to eval-
uate the effects of temperature and salinity combined on the acute toxicity
of metals to G. splendens. In addition, tests with Ag, Cu and Hg were con-
ducted at 32C and 33C at 14 and 28 ppt salinity.
The interacting effects of temperature and salinity on metals toxicity
did not follow a specific toxicity trend. With the exception of Fe, either
the M-50 values, the MTL values or both indicated that the toxic levels of
the metals were lower (the toxicity was higher) in media with 14 ppt than
with 28 ppt salinity (TABLE 11). Also, values for Mo, Ni, Zn and Cr indi-
cated that these metals were more toxic in medium with low salinity and high
temperature (14 ppt and 30C) than in media with other temperature and salin-
ity combinations. Although the M-50 and MTL values did not show that V, Ag,
Cu and Hg were more toxic in low salinity medium at high temperatures, com-
pared to the levels with high salinity and low temperature conditions, the
toxicity levels of these metals with these conditions were low. The control
test populations in conditions of 33C and 14 ppt salinity died within 48
hours whereas those in portions subjected to 33C and 28 ppt grew at a slow
rate. The Fe-MTL values were higher at 30C than at 16C in both 14 ppt and
28 ppt salinity media. Also, this was true for the M-50 values. The toxi-
city of Fe to G. splendens in media with these four temperature and salinity
combinations were the only results that indicated that temperature had in-
creased toxicity more than salinity. The lowest toxicity levels (highest MTL
and M-50 values) were at 30C rather than at 16C.
The effects of different combinations of temperature and salinity on the
toxicity of Zn, Hg, Ag and Cu to J, galbana were not significantly different.
High and low MTL and M-50 values associated with some T-S combinations were
not significantly different from other results with the same or similar tem-
perature-salinity, metal and organism combinations (TABLE 12), These combi-
nations were either similar or within range of the LMTL and HMTL for I. gal"
bana and these four metals (TABLES 2, 3 and 5). Some organisms (<50%) sur-
vived for 48 hours at 30C, but not if the portion had .02 yg Cu/L or more
added at this temperature in 14 ppt salinity medium.
Results of tests on the combined effects of temperature and salinity on
metals toxicity to T. pseudonana (Clone 3H) indicated that the metals were
more toxic at 14 ppt than at 28 ppt. The MTL and M-50 values for Cr and Ni
and the Mo-MTL values did not necessarily follow this trend (TABLE 13). Tests
results with the other six metals ususally indicated that the metals were
more toxic with low salinity-high temperature conditions.
The M-50 value of Mo in 14 ppt salinity medium was less than 100 mg/L at
the 5 temperatures, employed and more than 100 mg/L with 28 ppt salinity
medium (TABLE 13), This difference did not occur in the MTL value, but 4 of
5 MTL values in 28 ppt medium were more than 10.0 mgMo/L, whereas 3 of 5
values at 14 ppt were less than 10.0 mgMo/L, The mean M-50 value for Mo at
14 ppt and 28C was the lowest M-50 value for this metal.
30
-------
TABLE 11. COMBINED EFFECTS OF TEMPERATURE AND SALINITY ON THE TOXICITY OF
METALS TO G. splendens (VALUES IN MG/L)
14 ppt
28 ppt
14 ppt
28 ppt
Ag
M-50
16C 30C 32C 33C
.018 .021 .020 CD1
.033 .044 .025 .015
MTL
.007 .011 .002 CD
.008 .010 .002 .01
Cu Hg
M-50 M-50
16C 30C 32C 33C 16C 30C 32C 33C
.030 .028 .045 CD .018 .016 .025 CD
.087 .057 .050 .008 .06 .074 .09 .015
MTL MTL
.019 .010 NG2 CD .008 .004 .005 CD
.038 .018 .028 .004 .011 .014 .010 .005
Cr Ni Mo Fe
M-50 M-50 M-50 M-50
14 ppt
28 ppt
14 ppt
28 ppt
16C 30C
.3 .25
.22 .24
MTL
.026 .012
.028 .022
16 C
8 '
20
30C
6.5
38
MTL
1.8
2.5
0.4
1.8
16C 30C 16C 30C
4.5 6 1.0 1.1
16 13 0.9 3.7
MTL MTL
1.6 1.3 .15 .28
2.4 5.2 .16 .28
Zn V
M-50 M-50
14 ppt
28 ppt
14 ppt
28 ppt
16 C 30C
2.3 6.0
6.1 3.5
MTL
.25 1.4
.7 .3
16 C
1.8
24
30C
7
42
MTL
.64
11
2.2
26
CD = Controls died
2NG = No growth
31
-------
TABLE 12. EFFECTS OF TEMPERATURE-SALINITY COMBINATIONS ON THE TOXICITY OF
Zn, Hg, Ag and Cu TO J. galbana
Zn Hg Ag Cu
M-50 M-50 M-50 M-50
Temperature 16C 28C 16C 28C 16C 28C 16C 28C
C
Salinity ppt
12 3.4 1.0 0.28 0.20 0.08- 0.096 0.68 0.11
16 2.8 3.0 0.33 0.18 0.011 0.11 0.76 0.22
20 2.4 0.8 0.31 0.28 0.21 0.14 0.85 0.35
28 2.4 3.0 0.3 0.09 0.33 0.55 0.55 0.54
MTL MTL MTL MTL
12 2.0 1.0 1.3 .10 0.022 0.015 0.035 0.01
14
16 0.43 0.36 0.074 0.058 0.024 0.038 0.02 0.07
20 0.81 0.23 0.1 0.12 0.064 0.060 0.07 0.05
24
28 1.2 0.43 0.1 0.11 0.024 0.11 0.018 0.025
32
-------
TABLE 13. COMBINED EFFECTS OF TEMPERATURE AND SALINITY ON THE TOXICITY OF
METALS TO T. pseudonana (CLONE 3H) (VALUES IN MG/L)
Temperature
C 12C
Salinity
PPt
14 ppt
28 ppt
14 ppt
28 ppt
14 ppt
28 ppt
14 ppt
28 ppt
14 ppt
28 ppt
14 ppt
28 ppt
14 ppt
28 ppt
14 ppt
28 ppt
3 ppt
5 ppt
•* f f »-
7 ppt
90
>100
60
15
>20
19
12
12.5
26
35
12
19
>10
>10
.10
.072
.025
.074
.046
16C 20C
Mo
M-50
64 66
>100 >100
MTL
3.5 35
89 80
.Fe
M-50
15 15
>20 >20
MTL
11.5 11
9.7 >20
V
M-50
18 13
27 35
MTL
9.8 8.4
19 23
Ni
M-50
>10 0.4
>10 0.9
MTL
.031 .028
.14 .030
M-5Q
.20
.16
.13
24C
70
>100
8.6
<1.0
15
>20
12
>20
12
33
7.9
20.0
0.1
.18
.017
.021
28C
52
>100
4.0
30
15
>20
12
>20
21
37
8.6
16.0
>10
>10
.08
.18
AS.
.028
.047
.027
12C
0.5
>20
<0.1
.35
>1
>1
.065
.062
.048
.088
.027
.039
.06
.64
.012
.082
X
.014
.03
.023
16C 20C
Zn
M-50
2.8 1.8
>20 >20
MTL
.17 <.l
.28 .1
Cr
M-50
>1 .35
>1 >1
MTL
.058 .028
.037 .046
Hg
M-50
.043 .056
>.l .095
MTL
.031 .032
.053 .062
Cu
M-50
.12 .19
.71 .56
MTL
.046 .048
.068 .052
MTL
.084
.07
.068
24C 28C
,75 2.0
>10 >20
<.l .2.
<.l .17
.48 .60
>1 >1
.017 >.01
.082 .15
.049 .030
.075 .073
.034 .018
. 047 . 043
.09 .07
.19 .71
.031 .017
.072 .034
.013
.016
.014
(continued)
33
-------
TABLE 13 (continued)
Temperature
C 12C 16C 20C 24C 28C 12C 16C 20C 24C 28C
Salinity
PPt Ag.
M-50 MTL
10 ppt .071 .14 .19 .075 .038 .031 .074 .076 .044 .021
14 ppt .095 .16 .056 .037 .056 .032
21 ppt .052 .12 .063 .023 .068 .040
28 ppt .07 .19 .15 .080 .088 ' .025 .031 .042 .048 .052
34
-------
There were no significant effects of temperature on the toxicity of Fe
and Zn at 14 and 27 ppt. As previously noted, Fe additions as high as 10 mg
Fe/L stimulated the growth of T. pseudonana.
Although 7 of 10 Cr M-50 values were more than 1.0 mg/L (the maximum
added) , the three Cr M-50 values that were less than this concentration were
in 14 ppt salinity media. On the other hand, Cr-MTL values indicated that
Cr was more toxic at 14 ppt than at 28 ppt salinity in combination with 24C
and 28C.
Vanadium was more toxic to T. pseudonana at 14 ppt than in medium with
28 ppt salinity. Although portions of T. pseudonana cultures that were in
14 ppt medium had lower MTL values than those in 28 ppt, high concentrations
of V were tolerated by T. pseudonana and there were no combinations of tem-
perature and salinity that uniquely elicited a higher or lower toxic level
of this metal.
The toxicity of Hg and Cu to T. pseudonana was less at 28 ppt than at
14 ppt salinity. Also, with the exception of comparable M-50 and MTL values
at 12C and 14 ppt salinity, these toxicity levels were less in conditions of
28C and 14 ppt than with any other combination. High and low temperatures
combined with 14 ppt salinity had significant effects on the toxicity of Hg
and Cu to T. pseudonana, Hg and Cu were more toxic at 12C and 28C than at
16, 20 and 24C.
The toxicity of Ag to T. pseudonana was tested at seven salinity levels
between 3 ppt and 28 ppt in combination with either three or five temperature
levels (TABLE 13). The Ag M-50 and MTL values were higher at 20C than at
either 12C or 28C at all salinity levels. The M-50 values for Ag at 3 ppt
were among the lowest of the values determined.
The Ag MTL values were higher at 20C than at 12C or 28C. Also, except
for those values in combination with 21 ppt and 28 ppt salinity the MTL
values at 12C were higher than those at 28C. The Ag MTL value (0.013 mg
Ag/L) that occurred with the highest temperature (28C) and lowest salinity
(3 ppt) was the lowest value (highest toxicity) recorded in assays with
T. pseudonana, but it was comparable to the Ag MTL (0.014 mg Ag/L) recorded
for two other T-S combinations (3 ppt, 12C; 7 ppt, 28C). The Ag MTL values
with 28C increased as the salinity was increased. The Ag MTL values in
medium with 28 ppt increased (less toxic) as the temperature level was in-
creased from 12C to 28C, but this apparent combined effect of temperature on
toxicity at the other salinity levels employed did not occur.
35
-------
SECTION 5
INSTANTANEOUS EFFECTS OF METALS ON PHOTOSYNTHESIS
RATES OF G. splendens, T. pseudonana AND J. galbana
Much of the work pertaining to the toxicity of metals to phytoplankton
has been conducted in freshwater and at concentration levels aimed at con-
trolling or eliminating the algal populations. Results indicated that the
toxicity of copper varied considerably from one time to another and depended
on temperature and alkalinity (Bartsch, 1954). The composition of saltwater
would probably make a more complex condition with respect to reactions with
metals. Erickson, Lackie and Maloney (1970) determined that there was a wide
variation in the tolerance between different members of the estuarine phyto-
plankton community to metals.
MATERIALS AND METHODS
Bacteria-free, unialgal cultures of J. galbana, G. splendens and
T. pseudonana were grown in a chelate-free artificial seawater medium
(TABLE 1) with the major salts (NaCl, KC1, MgS04, MgCl£ and CaCl2) adjusted
to result in water with a salinity level of 20 ppt. Medium was prepared with
triple glass distilled water. Glassware was cleaned as previously described
(Aldrich and Wilson, 1960). Photosynthesis was measured by using a modified
method of Steeman-Nielson (1952) and Strickland and Parsons (1968). A
Coulter Counter Model B was used for cell counts and radioactivity was
counted with a Nuclear Chicago Model 4338B counting system. The organism to
be used in an experiment was growing in a batch culture. Triplicate, 100 ml
portions of the culture were placed in 125 ml Fernbach Flasks which received
1 yc 14c, incubated for 1 to 24 hours at 21 to 25C in 800-1000 ft. C of
"cool white" fluorescent light. After incubation, the photosyntheses rate
was measured by withdrawing a culture portion and carry out the described
procedure.
The remaining culture portion of each flask received a specific volume
of a metal solution to give the desired concentration of the metal to be
tested. Triplicate 10 ml amounts of each metal concentration were dispensed
into 16 x 125 mm test tubes furnished with a polypropylene closure. The cul-
ture portions were incubated for 24 hours and filtered on a 0,45 y membrane
and the 14c uptake was counted. All test tubes were maintained at a uniform
distance from the light during incubation.
36
-------
RESULTS
Although Cu concentrations as low as 10 yg Cu/L depressed both the
short-term photosynthesis and growth rate of I. galbana, concentrations as
high as 1250 yg Cu/L did not stop cell division or photosynthesis. The
doubling time increased from 32 hours in the control to 62 hours in medium
with 62.5 pg Cu/L and up to 224 hours in medium that received 1250 yg Cu/L.
Culture poritons that received 1250 yg Cu/L had a photosynthesis rate that
was 24% of the control (no metal added) after 120 hours exposure.
G. splendens grew less in medium that received 70 or 80 yg Cu/L after
incubation periods up to 24 hours than in the control medium, but recovered
and had increased growth after periods of 48 hours or more. Addition of
20 yg Cu/L had no effect on the growth of this organism.
Culture portions of I. galbana were exposed to medium with Cr additions
of 1 to 8 mg Cr/L. Additions within this range reduced the photosynthetic
activity compared to the control (no metal added), but after 96 hours expo-
sure the culture portions recovered and organism from all portions grew after
being transferred to fresh NH-15-CF medium. Also, the color of organisms in
portions that received 8 mg Cr/L was green rather than the normal yellow-
brown.
J. galbana culture portions that received additions of 20 yg Hg/L had a
mean photosynthesis rate that was 58% higher than the mean control portion.
The former (20 yg Hg/L) portion had a mean cell number after 48 hours incuba-
tion that was 33% higher than the control portions. Hg reduced photosynthe-
sis of J. galbana more than comparable Cu additions. Also there was a 16%
increase of cell numbers after 48 hours in portions that received 160 yg Cu/L.
The growth rate of T. pseudonana was modified by Hg additions. Growth
was not uniform in respect to Hg amounts added. Culture portions exposed to
40 yg Hg/L increased in cell numbers less than 10% after 96 hours, but cul-
ture portions that received 10 yg Hg/L or less (control portions) increased
approximately 400%. Medium portions that received 20 and 40 yg Hg/L grew
much less than the control.
Additions of 1.0 mg Hg/L stopped cell division of J. galbana and it did
not resume within 72 hours. Culture portions that received additions of 70
yg Hg/L had a growth rate that was reduced by 47% after 1 hour compared to
the controls. With additions of this level the photosynthesis rate of
I. galbana recovered to its pre-test condition 4 hours after the addition
was made. The l^C uptake rate was reduced after 2 hours exposure to medium
with an addition of 80 yg Hg/L. In this same experiment, additions of
80 yg Hg/L reduced -^C uptake within the same 2 hour period, but after 72
hours, the lowest addition tested (5 yg Hg/L) reduced the l^C rate to levels
as low as 89% of the control. Carbon-14 uptake was less in medium with addi-
tions of 40 and 80 yg Hg/L, but the cells continued to divide in these por-
tions. Lower addition of Hg (5 and 10 ygHg/L) had higher 14C uptake rates
than the controls after 96 hours.
37
-------
SECTION 6
THE EFFECTS OF LIGHT INTENSITY, TEMPERATURE, CELL DENSITY
AND CHRONIC EXPOSURE OF MERCURY AND CHROMIUM ON
TWO MARINE PHYTOPLANKTERS IN CONTINUOUS CULTURE
The concentration at which some metals promote growth is usually only
slightly less than the concentration at which they are toxic. Copper has
been reported to be essential for the synthesis of cytochromes, peroxides,
catalases and it has been demonstrated that Cu is necessary for the develop-
ment of chlorophyll (Corcoran and Alexander 1964), yet it has been reported
to be an algal toxicant, e.g. Marvin et al, 1960; Steemann-Nielsen and Wium-
Anderson 1970; Niemi 1972. Sverdrup et al, (1942) reported an average of
10 wg Cu per liter in natural seawater and Guillard and Ryther (1962) added
5 yg Cu per liter when enriching seawater. Wilson (1958) found 30 yg Cu per
liter was lethal to Gymnodinium breve and Mandelli (1969) found the growth
of three species of marine flagellates was inhibited by copper concentrations
as low as 30-50 yg per liter. These combined results indicate that the inhi-
bitory concentration of copper is only two or three times the concentration
that supports phytoplankton growth.
Johnston (1964) suggested that the species composition of a phytoplank-
ton community as well as overall phytoplankton growth was associated with
the physical and/or chemical availability of trace metals. A phytoplankton
population may be limited by the availability of a trace metal as follows:
(1) standing crop—the total biomass attainable by a population of algae may
be limited by the total amount of a metal present (biologically non-active
reservoir); and (2) productivity—the growth rate may be limited by the flow
rate of a metal (replenishment rate). The effect of a trace metal upon
phytoplankton growth must be related in some manner to the capability for
uptake and retention of the metal. For example, Spencer (1957) demonstrated
the growth-promoting activity of manganese is related to the concentration
of the ionic form, and is independent of the non-ionic concentration. Thus,
the chemical form in which a metal is present may control its biological
availability, and the flow rate of a metal may be a consequence of the rate
at which the biologically active form is replenished from the non-active
reservoir. Provasoli (1958) suggested: "By balancing chelators and trace
metal concentrations one can create a non-toxic reservoir of metals, thus
allowing prolonged division of fast-growing organisms".
Wilson and Collier (1955) and subsequently, Johnston (1964), Barber
and Ryther (1969), and Prakash and Rashid (1968) have shown that the addition
of known chelators or natural humic substances will enhance the growth of
phytoplankton in seawater. Traditional interpretation has suggested that
chelation makes trace elements available for biological uptake, but Steeman-
38
-------
Nielsen and Wiurn-Anderson (1970) argue that an inhibiting concentration
(ionic) of a metal, principally copper, could be rendered non-toxic by chela-
tion. Except for the study of Wilson (1966) there are few studies on com-
plexed metal ions accompanying growth promotion studies of phytoplankton.
Slowey et al (1967) and Alexander and Corcoran (1967) have demonstrated that
up to 50% of the copper in seawater may be complexed into an organic form.
Fitzgerald and Lyons (1973) and Knauer and Martin (1972) indicated a similar
percentage of Hg in the complexed form. Thus, organic complexes of both
mercury and copper may constitute at least 50% of their total concentration,
if a chelator is present.
Data obtained from acute bioassays may vary depending on the time lapse
between the addition of a metal and analysis of parameters to evaluate the
metal's effects. Steemann-Nielsen and Wium-Anderson (1970) found that 20
minutes after adding a toxic concentration of copper to static cultures of a
diatom the photosynthesis was reduced 10%, while after 40 minutes photosyn-
thesis was reduced 50% and after 60 minutes there was a 60% reduction in
photosynthesis. Studies allowing longer exposure times prior to determining
the effects on growth rates may be further complicated by an extension of the
lag phase before cell division starts. Ben-Bassat et al (1972) showed the
lag phase of Chlamydomonas was extended when exposed to increasing concentra-
tions of mercuric chloride. After cell division started, the growth rate was
similar to that of the control and the final population level approached that
of the control. Thus, the time factor is of great importance in a biossay of
this type, and comparison of different studies are complicated by a variety of
exposure times considered appropriate by investigators (e.g., Erickson et al
1970; Harriss et al 1970; Niemi 1972; Nuzzi 1972).
Time related factors to be considered with biologically active metals
in seawater are removal of the metal from the medium by surface deposition on
container walls or particulate matter, precipitation, and phytoplankton uptake.
Duursma and Sevenhuysen (1966) found that soluble concentrations of metals
are normally reduced in seawater by precipitation into insoluble compounds,
usually hydroxides or carbonates, which have low solubility constants. They
determined that mercury (added as HgCl2) in Pyrex flasks of filtered seawater,
decreased 50% in 2 hours and after 6 hours the initial concentration was re-
duced by 90%. The loss of Hg from solution was attributed to surface deposi-
tion, in that the Hg was recoverable with acid treatment of the flask walls
(Rice et al 1973). Mandelli (1969) reported a rapid uptake of copper by five
species of marine phytoplankton during the first 15-30 minutes of exposure to
various concentrations of copper and the total uptake was related to the ini-
tial concentration of the metal. A similar rapid uptake of Hg203 by a diatom
was reported by Gloosehenko (1969) and subsequent uptake depended upon con-
tinued cell division.
The dynamic (continuous culture) bioassay is best suited for determin-
ing the chronic effects of a test material at acutely sub-lethal concentra-
tions. In a dynamic culture the time factor, both in relation to the popula-
tion in the culture and in relation to the metal concentration, is reduced or
eliminated. After steady-state growth of the test organism is established,
the culture is time independent and its functions are not functions of time;
thus, one need not be concerned with the age of the culture (Malek 1966).
39
-------
The essential feature of this technique is that growth in a dynamic culture
takes place under steady-state conditions; that is, growth occurred in a con-
stant environment and at a constant rate.
The distinct advantage of a continuous culture system is the capacity
to continuously renew a predetermined concentration of the test material in
the culture regime. As previously discussed, metals in solution are normally
reduced in seawater by precipitation, absorption and phytoplankton uptake.
In a continuous culture system the ionic form of a metal and the medium are
added to the culture vessel through separate inflow tubes simultaneously.
The metal flow rate is maintained as a constant ratio of the medium flow rate.
Thus, specific concentrations of a metal ion can be maintained in steady-
state equilibrium within the culture, relatively independent of other cations
in the medium.
The principal objectives of this study were as follows:
1. The first objective .was to determine the effects of Hg and Cr on
the growth of J. galbana and G. halli. Up to 24 concentrations of each metal
were established in steady-state cultures ranging from that concentration at
which an effect on the growth rate of J. galbana and G. halli was not detect-
able to a concentration at which cell division was completely inhibited.
2. The effects of Hg on the growth of G. halli in cultures that were
growing with a combination of sub-optimal irradiance and temperature was
evaluated.
MATERIALS AND METHODS
Continuous Culture Apparatus
The continuous culture apparatus employed for this study was of the
turbidometric type (Freeberg 1971). The basic design (Figure 3) consisted
of a 1-liter round bottom culture flask housed in a waterbath box, in which
water was continuously circulated. The waterbath temperature was maintained
(± 0.6C) by a Dapper fie Company Model A4406B Mobile force flow unit which
resulted in a constant culture temperature (± 0.2C). A silicon (P-N junction
type) photocell was housed in each water-jacketed box on opposite sides of
the waterbath box, and a monitoring lamp was positioned to maintain equal
illumination on both photocells. The photocells were connected'in series
and the microampere output difference between the two photocells was ampli-
fied and metered in a control center. If the metered microampere signal
exceeded a preset value, a solenoid was activated which allowed the inflow
of fresh medium into the culture vessel until the signal decreased below the
preset value. Thus, the population density, represented by turbidity between
the photocells,.was held constant (± 5%).
The control center amplification unit was modified by replacing the 1. 5
volt battery, of the low voltage circuit, with a variable microampere DC
power supply. The power supply could be regulated to provide the amperage
necessary to nullify any difference, within limits, between the microampere
40
-------
AIR--:
METALS SOLUTION
THERMOMETER
WELL
PHOTOCELL
- AIR
MEDIUM
RESERVOIR
SOLENOID
VALVES
b: AIR
NFRARED
FILTER
;>_._ OVERFLOW
INFRARED
FIITER MONITORING
LIGHT
Photocell
Figure 3. Diagrammatic sketch of the basic turbidostat
design.
41
-------
output of the two photocells. Thus, a range of population levels (turbidity)
could be selected, at which a culture was maintained in the turbidostat.
A delivery system capable of supplying metallic ions in solution to the
culture vessel simultaneously with the medium, yet independent of the medium,
was developed for the turbidostat (Figure 3). A 100 milliliter graduated
cylinder was connected to the culture vessel by small diameter silicone tubing.
The addition of metal solution was controlled by a solenoid, which operated
in a similar manner to that of the medium supply system. The two solenoid
pinch valves were activated simultaneously by the control center, and the
metal solutions flow rate was 1, 2 or 3% of the medium flow rate. The rela-
tive flow rates of the solution and medium were established by adjusting the
orifices at the tips of the glass inlet tubes. The amount of metal solution
(My) delivered to the culture vessel was determined by subtracting the volume
prior to the test and the theoretical metal concentration (Cm) in the culture
vessel during steady-state conditions was calculated as follows:
MA = My x M£
MA is the total amount of metal ion added to the culture vessel, My is the
volume of metal solution delivered to the culture vessel, M£ is the concentra-
tion of the metal ion in the metal solution; and
dV
where Cm is the theoretical metal concentration in the culture vessel during
steady-state conditions, and dV is the volume of culture overflow.
The energy necessary for photosynthesis was supplied by irradiance from
two 150-watt incandescent flood lamps—one on each side of the waterbath.
The spectral quality of the irradiance was controlled by a chemical filter
which was an integral part of the waterbath box. Jerlov (1951) classified
various coastal and oceanic seawater types according to their transparency
to daylight. The spectrum of the filtered tungsten light more nearly simu-
lated that of Jerlov's coastal seawater No. 2 than either of the two commonly
used fluorescent light sources (cool-white and white daylight). Light inten-
sity was varied by changing the distance of the flood lamps from the culture
vessel and the irradiance was determined in energy units (mw/cm^) with a YSI-
Kettering Model 65 Radiometer.
Culture Methods and Materials
The two species of marine phytoplankton employed in this study were
Jsochrysis galbana Parke (Chrysophyta), a small (5-6 urn diameter) naked fla-
gellate; and Glenodimium halli Freudenthal and Lee (Pyrrophyta), a lightly
armored dinoflagellate. Axenic cultures of these organisms were maintained
at the Texas ASM University, Department of Marine Sciences Laboratory in
Galveston, Texas. The culture of I. galbana was isolated by M. W. Parke from
the saltwater fish ponds of Port Erin in 1938 and obtained by the Texas A&M
laboratory from Dr. R. Ukeles, National Oceanic and Atmospheric Administra-
tion (National Marine Fisheries Services) Laboratory, Milford, Connecticut.
42
-------
The G. halli culture was isolated from Offats Bayou, Galveston in 1955 by
W. B. Wilson (personal communication). These two species were employed in
this study because each species could tolerate the mechanical stirring re-
quired to maintain homogeneous distribution without cell damage or attach-
ment to the culture vessel, and both species have a specific growth rate of
approximately 1.0 div/day. The growth requirements of I. galbana have been
studied and it has been commonly employed in phytoplankton investigations
(e.g., Kain and Fogg, 1958; Mclaughlin, 1958; Caperon, 1968; Davies, 1974).
The growth requirements of G. halli have not been determined and culture
studies employing G. halli at this time, other than in this laboratory, are
unknown.
The artificial seawater media employed was basic formulation from Gates
and Wilson (1960) and was modified by W. B. Wilson (personal communication)
as shown in TABLE 1 for this study.
Experimental metals solutions were made by dissolving the chloride salts
of Hg+2 and Cr+3 in glass distilled water. Stock solutions of 100 ug Hg or
Cr per ml were prepared in 500 ml glass stoppered volumetric flasks (67.7 mg
HgCl2 or 128.2 mg CrCl3-6H20 per 500 ml water). Solutions were made by dis-
tilled water dilution of the stock solution. The metal stock solutions and
all dilutions were autoclaved at 15 psi for 15 minutes. Aseptic manipulation
of stock solutions decreased the possibility of contamination.
All glassware and silicone tubing that was in contact with the medium,
metal solutions or the culture were cleaned using the procedure of Aldrich
and Wilson (1960). Glassware and tubing were placed in, or filled with, a
detergent (Alconox—approximately 5 g/liter) and heated to near boiling (90C)
for 15 minutes. The detergent was removed by rinsing with distilled water
and brushing the glassware surfaces. The glassware and tubing were .placed in
or refilled with distilled water, heated to 100C, and then rinsed with dis-
tilled water. The glassware was filled with 10% (V/V) HN03, heated to 50-60C,
emptied, and rinsed with distilled water eight times. The culture vessel was
filled with glass distilled water and pretreated by autoclaving at 15 psi for
a minimum of 20 minutes.
Upon completion of the cleaning procedure, the culture vessel assembly,
tubing, overflow catch vessel, and two cotton air filters were autoclaved at
15 psi for a minimum of 15 minutes. The culture system was assembled while
positive sterile air pressure was maintained in all containers that required
aseptic conditions. This procedure decreased the probability of bacterial
contamination during assembly, inoculation, and normal operation. The air
for aeration and positive pressure was supplied by an Aquarium Pump Supply Co.
Silent Giant, Model 120 air pump. The air was filtered by a Koby Inc. Air
Purifier and Flow Equalizer, Model, Junior, a sterile cotton filter, and
connected to the culture air inflow with Tygon tubing. All joints were flamed
during assembly. After the culture apparatus was assembled, the culture ves-
sel was filled with medium and the inoculum was introduced aseptically into
the vessel through the thermometer well port.
A magnetic stirrer with a teflon-coated stirring rod in the culture
vessel was employed to facilitate homogeneous mixing. Failure to maintain
43
-------
a homogeneous population can lead to erratic oscillations in the population
density (Powell, 1955). If the magnetic stirrer was stopped, such oscilla-
tions were detected by significant differences in the cell counts of samples
within a period of less than 24 hours.
Bacterial sterility tests were conducted biweekly, or if contamination
was suspected. An unexpected decrease in growth rate or the attachment of
cells to the vessel walls usually indicated bacterial contamination. Steril-
ity tests were made by aseptically transferring 1.0 ml of culture into each
of two media. One medium contained 10 ml of peptone seawater broth, the other
a 10 ml peptone seawater agar slant (Spencer 1952). Tests were incubated at
37C for 10 days.
The cell numbers of a continuous culture was considered to be a vari-
able factor affecting the physiology of the organism in culture. The flow of
medium or test material per cell (flux) may change with different cell num-
bers. This hypothesis was investigated by determining the Kc, Chi a, and
uptake/cell of each species over a wide range of population levels in steady-
state growth at 23C with irradiance levels of 5, 10 and 15 mw/cm^. A stan-
dard cell concentration for each species was employed in all tests. The
standard population level selection was the minimum level at which the turbi-
dostat control center was able to maintain the population level constant with-
in a ± 5% range; and the concentration at which the least change in Kc and
Chi a/cell occurred within that range.
Irradiation intensities of 5, 10 and 15 milliwatts per square centi-
meter (mw/cm2), which represented approximately 14, 29 and 43% of the energy
available in full sunlight, were supplied to the cultures. Eppley et al
(1969) reported full sunlight measured in energy units was equal to approxi-
mately 35 mw/cm2 (1 langly/min = 70 mw/cm2). The three intensities simulated
the energy that would be expected at. depths of 8.2, 4.7 and 2.3 m, respec-
tively, in water classified by Jerlov as coastal seawater No. 2 (Jerlov 1954).
Ryther (1956) suggested the factor, 70 mw/cm2, equals 15,500 ft-c, for con-
verting the photosynthetically active irradiation of full sunlight (400-700
mw/m2) to units of illumination. Therefore the irradiation levels employed
gave theoretical illumination values of approximately 1100, 2200 and 3300
ft-c which were adequate for light saturation. Illumination values of 700,
1400 and 2100 ft-c were determined with a Gossen Luna Pro exposure meter.
The Luna Pro meter could be read only to the nearest 100 ft-c in the range
of 1000 to 5000 ft-c.
The interrelationship of light and temperature on the growth of each
species was investigated by varying the temperature of three turbidostats
simultaneously in 3C increments or, in a few instances, 2C increments. The
temperature was varied from 14 to 28C with I. galbana and from 20 to 35C with;
G. halli.
The Kc, Chi a/cell and 14C uptake/cell were determined for cultures of
each organism with steady-state concentrations of HgCl2 and CrCl3- The
metal concentrations in the culture were changed by varying the concentration
of the metallic salt solution. Concentrations of each metal were varied
within a range from a minimum, at which the Kc was not significantly affected,
44
-------
either beneficially or deleteriously, to a maximum at which cell division was
inhibited and the Kc approached zero. The Kc, Chi a/cell and l^c/cell uptake
were determined in control cultures of each organism, in which 1, 2 or 3%
distilled water were delivered in place of the metals solution.
Chlorophyll a was determined daily using a modification of Strickland
and Parson (1968). Ten-mi duplicate portions were removed from a 40-ml cul-
ture sample. The remaining 20-ml portion was used for cell enumeration with
a Model B Coulter Counter. Each of the two chlorophyll a samples were fil-
tered on a Whatman GF/C type filter pad that had received 0,5 ml of a dis-
tilled water 1% MgCOs solution (Yentsch and Menzel, 1963; Long and Cooke,
1971). The filter pad was extracted with 90% acetone, ground with a Thomas
Tissue Grinder //B51590, centrifuged and the fluorescence of the supernatent
was measured with a Turner Model 111 Fluorometer as modified by Lorenzen
(1966). Cells enumerated with a Model B Coulter Counter are correct to with-
in 2% (Mattern et al 1957; Caperon 1968).
The specific growth rates of the populations were calculated as follows:
v = 1 dn
Kn n 3T>
n is the number of organisms per unit volume and t is time (Novick and Szilard
1950). Assuming the number of organisms per unit volume was constant, the
specific growth rate may be determined by
„ _ 1 dv
KV " V dt'
if V is the volume of the culture. The turbidostats employed were capable of
maintaining the population number constant within ± 2% with ideal conditions.
This capability did not normally occur because of (1) sampling errors and
accuracy of cell enumeration; (2) the position of the monitoring light was not
stable (mounting of lamp housing), thus the light path changed; and
(3) changes in optical density between the two photocells which was not due
to the culture, e.g., condensation on windows, air bubbles on the side of the
culture vessel, and waterbath turbidity. The population level was normally
maintained within ± 5%. The assumption of constant population number was
validated by mathematically correcting the overflow volume by the percent
population level change as follows:
V = !^ x V,
where V is the total volume of the culture. The volume correction (Vc) was
added to the overflow volume and Kc was calculated as follows:
dv+Vc
v = ±.
c V dt
The culture overflow was measured and K<. calculated twice each day. The KC
45
-------
values are volume of constant culture overflow per volume of the culture
vessel per day which, if the chlorophyll a/cell remains, are directly related
to divisions per cell per day.
The photosynthetic response of each species to changes in physical
environment and various concentrations of mercury and chromium were evaluated
from daily radioactive carbon/cell uptake. A 50 ml portion was obtained from
the culture vessel and triplicate 10 ml portions were placed into screw-cap
test tubes. The remaining portion was used for cell enumeration. One ml of
14c-labeled NaHC03 solution containing 1 microcurie of activity was added to
each of the 3 test samples. The samples were incubated in the corresponding
turbidostat waterbath box for 1 hour at the same temperature and irradiation
as the steady-state culture. The cells were concentrated on glass fiber
filters (type GF/A) dried for a minimum of 12 hours and the radio activity
was counted 3 times for 1 minute.
RESULTS
Culture Conditions—Isochrysis galbana
The specific growth rate (Kc) of I. galbana was inversely related to
the cell concentration (TABLE 14). In cultures with the lowest population
level tested, 50,000 cells/ml, there was a maximum Kc of 1.57 and the KC
decreased with population levels greater than 200,000 cells/ml to a minimum
Kc of 0.45 with 400,000 cells/ml. The Kc was constant (1.16 to 1.17 ± 0.13)
with cell numbers of approximately 75,000 to 200,000 cells/ml. The KC deter-
mined in two different turbidostats, one exposed to 5 mw/cm2 and the other
exposed to 10 mw/cm2 irradiation, were similar at each of the population
levels tested, with one exception. With cell numbers of 200,000 cells/ml,
the KC of the culture irradiated with 10 mw/cm2 (1.17) was 27% greater than
the culture with 5 mw/cm2 (0.92). The medium flow rate/cell/day (flux) de-
creased as the cell concentration increased. There was a maximum flux of
31.4 x 10~6 ml/cell with 400,000 cells/ml.
The chlorophyll a per cell (10~8 ug Chi a/cell) of J. galbana grown at
23C with 5 mw/cm2 irradiation was higher if cell concentrations were greater
than 135,000 cells/ml than in cultures with lesser population levels
(TABLE 14). Excluding the apparent anomalous value with population levels
of 75,000 cells/ml, the Chi a/cell did not change (mean value of 6.54) from
the minimum population level tested to approximately 135,000 cells/ml and
increased to a maximum of 13.00 x 10~8 yg Chi a/cell with 300,000 cells/ml
(TABLE 14).
The optimum temperature range for the growth of I. galbana was from 20
to 25C with the three light intensities employed (TABLE 15). The KC in-
creased linearly with a slope of 0.065 from 17 to 25C, remained constant at
25C and decreased at temperatures greater than 25C (TABLE 15). J. galbana
did not grow and the cell concentration of the culture decreased rapidly at
28C with each of the three light intensities tested. The Chi a/cell was 26%
less at temperatures of 17C than at 20 to 26C and the Chi a/cell values were
similar from 20 to 26C for each of the three light intensities.
46
-------
TABLE 14. SPECIFIC GROWTH RATE, FLUX, CHL a/CELL AND 14C UPTAKE FOR VARIOUS
POPULATION LEVELS OF I. galbana GROWN AT 23C AND WITH 5 AND 10
mw/cm2 IRRADIATION
Irradiation
mw/cm
5
5
5
5
5
5
5
5
5
5
10
10
10
10
10
10
10
Population level
cells /ml (X105)
.50
.75
1.00
1.25
1.35
1.35
1.75
2.00
3.00
4.00
.50
1.00
1.30
1.30
2.00
3.00
4.25
Flux1 •
(X10~6)
27.4
15.5
12.0
9.3
8.7
8.8
6.7
4.6
2.6
1.1
31.4
12.2
9,5
9.3
6.2
2,3
1.2
KC2.
1,37
1.16
1,20
1.16
1.18
1.19
1,17
0.92
0.79
0.45
1.57
1,20
1.21
1.21
1,17
0.70
0,50
Chi a /cell
yg (X10-8)
6.54
7.76
6.55
6.24
6.45
6,57
7.96
9.29
13.00
11.22
—
5.78
5.82
6.30
5.82
-
"
14c Uptake3
(X104)
2,56
2.29
2.91
-
-
4.76
3,79
3.59
1.81
2.37
-
-
-
6.40
2.92
-
"
Notes :
1. Flux: The medium flow rate is calculated as follows: F=Kc 5 where PL
PIT10 '
is the population level in cells/ml and is given in ml/cell/day,
2. The specific growth rate is calculated as follows: Kc=1 dv+Vc ana is
v dt
given in divisions per cell per day.
3. The l^C uptake is given as:
Counts/min
•jig Chi a
47
-------
TABLE 15. SPECIFIC GROWTH RATE, CHL a/CELL, AND !^C UPTAKE OF I. galbana
EXPOSED TO VARIOUS LIGHT-TEMPERATURE COMBINATIONS
Irradiation
mw/ cm2
5
5
5
5
5
5
5
5
5
Mean 3 •
10
10
10
10
10
10
10
Mean3 •
15
15
15
15
15
15
15
15
Mean3 •
Temperature
°C
17
20
23
23
23
23
25
25
28
-
17
20
23
23
23
25
28
-
17
20
23
23
23
25
25
28
—
M-
0.80
1.07
1.16
1.18
1.19
1.44
1.21
1.31
0.0
-
0.79
1.20
1.21
1.21
1.20
1.22
0.0
-
0.77
1.13
1.19
1.23
1.23
1.14
1.38
0.0
—
Chi a/cell
yg (X10-8)
4.69
5.44
6.24
6.45
6.57
5.52
6.37
8.24
_
6.10
3.98
4.75
5.82
6.30
5.78
6.51
-
5.66
3.09
3.73
4.72
4.95
4.29
5.25
5.98
—
4.59
l^C Uptake2-
(X1Q4)
3.72
3.23
-
-
4.76
7.15
2.02
3.89
_
3.90
4.05
8.76
-
6.40
8.99
3.09
-
6.40
6.25
12.22
-
6.94
-
5.55
-
-
6.60
Notes :
1. The specific growth rate is calculated as follows:
given in divisions per cell per day.
2. The 14C uptake is given as: cnmn X10*.
1 dv+Vc
Kc = — —^ —
and is
3. The mean value was determined for all temperatures in which cell division
occurred, excluding the highest and lowest values.
48
-------
The Chi a/cell was inversely related to the light intensity (TABLE 15).
Analysis of variance indicated a reduction of Chi a/cell as the light inten-
sity was increased. The mean Chi a/cell value for the temperatures with 10
mw/cm2 irradiation (5.66) was not significantly different from those values
with either 5 or 15 mw/cm2 irradiation (6.10 and 4.59, respectively), but the
mean Chi a/cell with 15 mw/cm2 irradiation was 23% less than the mean value
with 5 mw/cm2 irradiation.
The culture conditions designated as standard for I. galbana cultures
in the remainder of this report are as follows :
1) Population level - 1.2 ± .10 x 10$ cells/ml
2) Irradiation - 10 mw/cm2
3) Temperature - 23C
Culture Conditions — Glenodinium halli
The Kc of G. halli was inversely related to the population level of the
culture, if the cell numbers were greater than 25 x 10-* cells/ml (TABLE 16) .
The KC was constant at 0.98, with a standard deviation of 0.08, from the
lowest cell concentration tested (6.8 x 10^ cells /ml) to approximately 2.3 x
10^ cells/ml with 10 mw/cm2 irradiation. With 15 mw/cm2 irradiation, the
population level at which the Kc remained essentially constant (0.97 ± 0.09)
extended to approximately 6.2 x 10^ cells/ml (TABLE 16). The Kc's were
similar (0.49 ± 0.04) with 1.2 x 105 cells/ml and 15 raw/cm2 irradiation.
The medium flux per cell decreased parabolically with increased cell
numbers (TABLE 16) . Rapid decreases in flux at low cell concentrations cor-
responded with the plateau region of the Kc curve and as the flux asymtoti-
cally approached zero, the Kc leveled off at approximately 0.45. The
greatest change of flux corresponded to the maximum slope of the Kc curves.
The Chi a/cell did not change significantly with cell numbers at the
three light intensities employed. The mean Chi a/cell values for 5, 10 and
15 mw/cm2 irradiation were similar (15.84, 16.49 and 15.68, respectively)
(TABLE 16).
Results did not indicate a change in -C uptake cell with increased
cell numbers. The l^C uptake by G. halli was higher with higher irradiation
levels (TABLE 17). Mean 14c uptake values at 10 and 15 mw/cm2 (3.38 and 5. 06,
respectively) were higher than the value with 5 mw/cm2 irradiation (3.03).
The optimum temperatures for the growth of G. halli were from 23 to 32C,
with the three light intensities employed in this study (TABLE 17) . Mean KC
values increased linearly with a slope of 0.147 from 20 to 29C, remained
relatively constant from 29 to 32C, and at 35C, there was no cell division.
Light intensity of 5, 10 and 15 mw/cm2 had no effect on the mean KC of
G. halli. The mean Kc values (0.95, 1.03, 1.24) for all temperatures and
each light intensity were not significantly different at the 95% confidence
level, although KC values at some specific temperatures were significantly
49
-------
TABLE 16. SPECIFIC GROWTH RATE, FLUX, CHL a/CELL AND 14c UPTAKE FOR VARIOUS
POPULATION LEVELS OF G. halli GROWN AT 23C AND WITH 5, 10 mw/cm2
IRRADIATION
Irradiation
mw/cm^
5
5
5
5
5
5
5
5
5
Mean6-
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
Mean6 •
15
15
15
15
15
15
15
15
15
15
15
Mean6-
Population level Flux-1--
cells/ml (X103) (X10~6)
7.4
9.3
13.4
22.7
24.4
28.4
38.0
57.4
68.0
6.8
7.9
9.9
11.9
15.9
21.9
23.5
23.8
32.0
32.8
33.4
36.4
45.5
57.2
61.3
71.6
75.5
75.9
6.5
13.0
20.7
21.7
22.5
31.0
47.4
61.8
119.7
151.4
196.9
118.9
98.9
64.9
39.2
31.2
27.1
17.9
11.7
8.7
147.1
144.3
98.9
82.4
38.8
44.8
39.6
35.3
24.4
23.2
23.4
20.9
9.0
10.0
6.9
5.9
6.0
5.9
173.9
76.9
49.3
41.0
42.2
29.4
19.8
14.2
4.4
3.2
2.3
KC2- Chi a/cell
Pg (X10-8)
0.88
0.92
0.87
0.89
0.76
0.77
0.68
0.67
0.59
1.00
1.14
0.98
0.98
0.95
0.98
0.93
0.84
0.78
0.76
0.78
0.76
0.66
0.57
0.42
0.42
0.45
0.45
1.13
1.00
1.02
0.89
0.95
0.91
0.94
0.88
0.53
0.48
0.46
14.74
16.93
16.60
19.16
15.93
15.88
14.84
15.99
14.37
15.84
17.19
18.33
15.13
15.03
14.69
16.89
15.50
14.93
14.93
15.22
21.88
-
14.26
15.04
18.19
17.38
19.04
16.91
16.49
15.78
14.19
21.22
18.59
13.89
14.78
15.64
15.40
15.10
16.08
15.60
15.68
l^C up take3- Medium4 •
(X104)
—
—
3.80
—
2.30
-
2.99
3.43
3.32
3.25
—
-
3.55
-
-
_
3.70
3.46
4.90
4.96
5.50
-
3.87
6.59
4.02
3.68
4.82
5.08
4.41
-
-
5.50
4.00
-
5.25
5.41
4.54
5.58
4.74
4.16
4.93
NH-15Si5'
NH-15Si
NH-15Si
NH-15Si
NH-15CF
NH-15
NH-15
NH-15
NH-15
NH-15Si
NH-15 Si
NH-15Si
NH-15 Si
NH-15Si
NH-15 Si
NH-15CF
NH-15Si
NH-lSSi
NH-15S1
NH-150F
NH-15Si
NH-15Si
NH-15
NH-15Si
NH-15Si
NH-15
NH-15
NH-15Si
NH-15Si
NH-15CF
NH-15CF
NH-15Si
NH-15S1
NH-15
NH-15
NH-15
NH-15
NH-15
(continued)
50
-------
TABLE 16 (continued)
Notes:
1. Flux: The medium flow rate per cell is calculated as follows:
F = _£. XIO^ where PL is the population level in cells /ml and F is
PL
given in ml/ cell/day.
1 dv
2. The specific growth rate is calculated as follows: Kc = — -rr an
-------
TABLE 17. SPECIFIC GROWTH RATE, CHL a/CELL AND 14c UPTAKE OF C. halli
EXPOSED TO VARIOUS TEMPERATURE-IRRADIATION COMBINATIONS
Irradiation
mw/cm2
5
5
5
5
5
5
5
5
5
Mean3 •
10
10
10
10
10
10
10
10
10
10
10
10
Mean •
15
15
15
15
15
15
15
15
15
15
15
Mean3 •
Temperature
°C
20
23
23
23
26
26
29
29
32
20
23
23
23
23
26
26
29
29
32
32
35
-
20
23
23
23
26
26
29
29
32
32
35
Kc1'
0,16
0.50
0.27
0.76
1.24
1.18
1.60
1.38
1.34
0.95
0.42
0.47
0.50
0.46
0.93
1.18
1.37
1.42
1.50
1.54
1,45
0.0
1.03
0,41
1.02
0.65
0.89
1,17
1.32
1.65
1,76
1.66
1.58
0.0
1.24
Chi a/cell
Ug (X10-8)
24.90
19.73
18.57
15,93
20.54
16.40
24.76
18.28
19.71
23.30
19.36
16,57
17.49
15,29
19.39
16.64
21.50
18.32
_
21.70
-
18.87
19.42
21,22
15.22
18.59
20.12
16.34
20,10
16.70
-
20.97
18.89
14c Uptake2-
(X104)
2.20
-
3.20
2.30
3,60
3. .30
2.60
3.20
3,03
2.80
3.40
4.40
2.70
3.70
3.90
3.40
3.30
3.40
—
3,10
-
3.38
3.00
5.50
7.00
4.00
5.00
5,50
4.20
6.70
-
4.20
5.06
Notes:
1. The specific growth rate is calculated as follows:
given in divisions per cell per day.
K =
1 dv+Vc
, .
and ls
(continued)
52
-------
TABLE 17 (continued)
counts/min
- — - -
yg Chi a
o mi. ^ ^1 • •
2. The J-^C uptake is given as:
3. The mean values were calculated for all temperatures in which cell divi-
sion occurred, excluding the highest and lowest values.
53
-------
different with certain light intensities. For example, at 20 and 32C the Kc
(0.16 and 1.34) of the cultures receiving 5 mw/cm2 irradiation were less than
those that received 10 (0.42 and 1.54) and 15 mw/cm2 (0.41 and 1.66) irradia-
tion. The Kc values at 26C were not significantly different at the 95% con-
fidence level at the three irradiation levels (TABLE 17).
The Chi a/cell was constant at each of the fifteen temperature-irra-
diation combinations tested. Results from analysis of variance indicated the
Chi a/cell was similar (19.71, 18.87, 18.89) for cultures, irrespective of
the light intensity-temperatures combination.
The 14c uptake values did not vary significantly within the temperature
range employed, but there was a significant difference of 14c uptake values
with increased light intensity.
Standard culture conditions for G. halli cultures for the remainder of
this report are as follows:
1) Population level - 2.0 + 0.5 x 10* cells/ml
2) Irradiation - 10 mw/cm2
3) Temperature - 30C
The Chronic Effect of Mercury CHg+2) on the Growth, of r, ga.lba.na and G. halli
The chronic effects of mercury on J, galbana were apparent only after the
Hg concentration in the turbidostat culture exceeded 200 yg Hg/1, KC values
for I. galbana exposed to mercury, at concentrations less than 200 yg Hg/1,
were similar to those of the control in which distilled water was continu-
ously added in the place of the Hg solution. The Kc values decreased with
a slope of -0.002 with mercury concentrations greater than 200 yg Hg/1, The
Chi a/cell and 14c uptake values did not change significantly throughout the
range of mercury concentrations tested, in which cell division occurred
(TABLE 18). A steady-state culture was not established with medium contain-
ing 602 yg Hg/1. After three days, cell division ceased and the cell popu-
lation was not maintained. The Chi a/cell (2.17) after three days incubation
in medium with 602 yg Hg/1 was 47% less than the mean Chi a/cell value (4.09)
for all other Hg concentrations tested. The l^C uptake (1.34) after three
days in medium with 602 yg Hg/1 was 36% less than the mean 14c uptake value
(2.09) for all other Hg concentrations tested (TABLE 18),
The growth of G. halli was enhanced by the addition of less than 90 yg
Hg/1 (TABLE 19). The KC was higher than that of the control (1,05) with
additions of 11.9 yg Hg/1 in which the maximum KC was 1.99. The KC was less
in medium with higher Hg concentrations. Cell division ceased in medium with
121 yg Hg/1 after three days. The maximum Kc of 1.99 with 11.9 yg Hg/1 rep-
resented an 86% increase above that of the control value of 1.05.
The Chi a/cell and 14C uptake of G. halli grown at 30C decreased signi-
ficantly (95% confidence level) only after the concentration of mercury was
more than 100 yg Hg/1 (TABLE 19). The Chi a/cell (11.66) in medium with
107.3 yg Hg/1 was 31% less than the mean Chi a/cell value (16.96) for all
54
-------
TABLE 18. SPECIFIC GROWTH RATE, CHL a/CELL, AND 14C UPTAKE OF I. galbana
GROWN AT 23C WITH 10 MW/CM2 IRRADIATION AND CONTINUOUS EXPOSURE
TO VARIOUS CONCENTRATIONS OF HgCl2
Hg+2
yg/L
2.8% H20
1.20
29.6
50.0
103.9
134.1
165.0
221.0
301.7
335.3
485.4
538.0
601.8
Mean
Notes :
1:
c
1.24
1.26
1.33
1.36
1.25
1.29
1.32
1.02
1.06
0.78
0.58
0.33
0.00
™
Chi a /cell
yg(X10-8)
4.82
4.81
4.82
4.43
3.88
4.67
3.94
3.44
4.19
3.31
4.12
3.31
2.17
4.09
14C Uptake2'
(X10*)
1.47
1.79
1.73
2.07
2.54
2.31
2.16
2.12
2.33
2.58
1.73
1.71
1.34
2.09
1 dv+Vc
V = — anH
is given in divisions per cell per day.
14,, , . . counts/min
2. The C uptake is given as: Chi a
55
-------
TABLE 19. SPECIFIC GROWTH RATE, CHL a/CELLS, AM) C UPTAKE OF G. halli
GROWN AT 30C WITH 10 MW/CM2 IRRADIATION AND CONTINUOUS EXPOSURE
TO VARIOUS CONCENTRATIONS OF Kg4"1"
Hg^
wg/L
3.4% H20
0.079
0.097
0.12
0.30
1.13
1.34
3.75
4.21
11.9
28.2
37.2
64.2
92.6
89.7
107.3
121.0
Mean4 •
K^
1.05
1.08
1.19
1.28
1.23
1.35
1.24
1.62
1.61
1.99
1.91
1.79
1.50
1.04
0.91
0.83
0.00
Chi a/cell
yg(X10-8)
15.91
18.41
14.12
14.25
16.17
19.85
15.55
18.79
16.26
18.62
16.04
19.14
22.94
13.22
16.22
11.66
7.323-
16.96
14C Uptake2'
(X104)
5.02
3.08
4.73
4.03
3.82
3.91
3.65
3.90
3.71
3.56
2.74
3.73
2.69
1.76
2.78
1.78
1.493-
3.46
Notes:
1 dv+Vc
1. The specific growth rate is calculated as follows: K^ = -r— and
is given in divisions per cell per day.
„ „„ 14,, ^ , . . counts/min
2. The C uptake is given as: —r^
* 6 jig Chi a
3. The values reported were from the 5th day of exposure at the indicated
Hg concentration.
4. The mean values were determined for all Hg concentrations less than
100 yg/L, excluding the highest and lowest values.
56
-------
test portions. The 1*C uptake in medium with 121 yg Hg/1 of 1.49 was sig-
nificantly less (95% confidence level) than the value of 1.78 in medium with
107.3 yg Hg/1, l*c uptake value of 3.46.
Results of turbidostat experiments on effects of mercury on the growth
of G. halli at 23C were similar to those observed at 30C (TABLE 19). The Kc
rates were higher (1.27) in medium with Hg concentrations of 6.32 yg Hg/1
but were less with 109 yg Hg/1. Cell division stopped in medium with 109
and 118 yg Hg/1. The maximum Kc value of 1.27 in medium with 6.32 yg Hg/1
represented a 154% increase above the maximum control value of 0.50.
The Chi a/cell and 14C uptake of G. halli grown at 23C with various con-
centrations of HgCl2 changed significantly only after the mercury concentra-
tion exceeded 100 yg Hg/1. The Chi a/cell in medium with 109.1 and 118.0
yg Hg/1 were 47 and 58% less than the mean value of 17.33 for values of test
portions that contained less than 100 yg Hg/1 (TABLE 20). The 14C uptake
value in medium with 118.0 yg Hg/1 was 76% less than the mean l^C uptake of
3.76 for cultures .with,100 yg Hg/1 or less.
An intensive study of the chronic effect of mercury on the growth of
G. halli at 23C in medium with concentrations of 1.0 yg Hg/1 or less, indi-
cated an unusual pattern of growth response for the 13 concentrations of Hg
tested (TABLE 20). The Kc (0.67) of G. halli grown in medium with the lowest
mercury concentration tested (0.012 yg Hg/1) was 34% greater than the KC of
the control culture (0.50). The KC increased linearly to a maximum of 0.79
with additions at 0.052 yg Hg/1. Subsequently, the Kc were less and contin-
ued to be less to a minimum of 0.51 at 0.462 and 0.652 yg Hg/1. The Kc in-
creased to 0.72 in medium with 0.99 yg Hg/1 (TABLE 20). The KC was not less
than the control (0.50) in medium with any of the 13 Hg concentrations tested.
The chronic effect of chromium on the growth of J. galbana at 23C with
10 mw/cm2 irradiation was observed by determining changes in the KC and l^C
uptake as the steady-state concentration of Cr was increased from 0.72 to
167.8 yg Cr/1. The Kc with Cr concentrations in medium of 2.21 yg Cr/1 or
less were not significantly (95% confidence level) different from the KC
(1.24) of the control culture. With Cr concentrations from 9.8 to 32.3 yg/1
the growth of J. galJbana was stimulated and the Kc was significantly (95%
confidence level) higher than the KC of the control culture (TABLE 21). The
Kc maximum of 1.57 developed in medium with 11.2 yg Cr/1 and the minimum KC
of 0.34 occurred in medium with the highest Cr concentration (167.8 yg/1)
tested. The ^C uptake of 1.47 (control culture) was less than that of 1.96,
the lowest concentration of Cr (0.72 yg/1) tested. The KC was less in the
concentration of Cr ( 92.8 yg Cr/1). The Chi a/cell values did not change
significantly (95% confidence level) from that of the control culture over
the total range of Cr concentrations tested.
The growth of G. halli was stimulated at the lowest Cr concentration
(0.04 yg Cr/1) tested and the KC (1.16) was significantly (95% confidence
level) higher than the KC (1.01) of the control culture (TABLE 22). The KC
values were higher in medium with Cr concentrations of 0.2, 0.38, 3.8, 7.88,
12.0 and 12.8 yg Cr/1 than in the control. The minimum KC of 0.25 developed in
57
-------
TABLE 20. SPECIFIC GROWTH RATE, CHL a/CELL, AND C UPTAKE OF G. halli
GROWN AT 23C WITH 10 MW/CM2 IRRADIATION AND CONTINUOUS EXPOSURE
TO VARIOUS CONCENTRATIONS OF Kg**
Kg44
2.7% H20
2.8
0.012
0.021
0.052
0.078
0.100
0.031
0.166
0.316
0.438
0.462
0.652
0.890
0.990
1.83
2.67
3.50
4.95
6.32
13.1
24.7
51.0
80.9
109.1
118.0
Mean3-
Notes :
1. The specific
_# 9 • __
K^
0.50
0.50
0.67
0.75
0.79
0.75
0.75
0.72
0.65
0.57
0.54
0.51
0.51
0.60
0.72
0.73
0.73
0.69
0.80
1.27
1.22
1.20
1.02
0.75
0.16
0.0
—
growth rate is
i • » •
Chl a/cell
yg(X10-8)
16.57
19.73
19.56
17.12
15.54
19.23
16.78
20.24
21.55
16.50
15.14
16.68
19.76
19.78
16.93
17.49
19.05
18.57
16.87
15.14
16.66
14.99
14.61
14.17
9.24
7.21
17.33
calculated as follows:
i ^ n
14C Uptake2'
(X104)
4.36
2.87
4.50
4.17
3.42
4.80
3.72
2.90
2.41
3.15
-
-
-
-
-
1.92
-
2.42
4.36
3.54
4.96
5.38
-
-
0.92
3.76
jr _ i dv+Vc and
v dt
_
2.
3.
is given in divisions per cell per day.
count s/min
— — ^ -
Vg l>nl a
The mean value was determined for all Hg concentrations less than
100 vg/L excluding the lowest and highest values.
_, 14,, .. , .
The C uptake is gxven as:
58
-------
TABLE 21. SPECIFIC GROWTH RATE, CHL a/CELL, AND 14C UPTAKE OF I. galbana
AT 23C WITH 10 MW/CM? IRRADIATION AND CONTINUOUS EXPOSURE TO
VARIOUS CONCENTRATIONS OF CrCl3
Cr+3
2.8% H20
0.72
2.21
9.8
11.2 i
23.6
32.3
51.8
56.3
92.8
113.7
167.8
KC1-
1.24
1.24
1.23
1.40
1.57
1.43
1.32
1.27
1.04
0.82
0.75
0.34
Chi a/cell
yg(X10-8)
4.82
5.20
5.33
5.14
4.37
4.68
4.66
4.91
4.56
5.07
5.10
5.19
14C Uptake2'
(X104)
1.47
1.96
1.71
1.46
1.37
1.29
1.26
0.94
0.99
1.05
0.90
0.47
4.95
Notes:
1. The specific growth rate is calculated as follows:
2.
3.
given in divisions per cell per day.
The 14C uptake is given as: coun/min
1 dv+Vc
-- -j- —
v dt
, .
and is
pg UnX 3.
The mean values were calculated for all chromium concentrations tested,
excluding the highest and lowest values.
59
-------
medium with the highest concentration (35.6 pg Cr/1) tested. The Chi a/cell
of G. halli in media with Cr concentrations of 7.88 or less (19.57) was sig-
nificantly higher than the Chi a/cell of the control culture (14.27) (95%
confidence level) . The minimum Chi a/cell was in medium with Cr concentra-
tions of 35.06 yg Cr/L, the highest Cr concentration tested. The Chi a/cell
maximum was 20.88 yg Chi a x 10~8 in medium that contained 0.20 yg Cr/L
(TABLE 22). The 14c uptake was not significantly changed (mean 2.80) by Cr
concentrations of 25.3 yg Cr/L or less. With media concentrations of 28.4
and 35.6 yg/Chl a the l^C uptake was significantly less (1.52 and 1.36, re-
spectively) than the ^C uptake (2.61) in medium with 25.3 yg Cr/1.
(TABLE 22).
TABLE 22. SPECIFIC GROWTH RATE, CHL a/CELL AND ^UPTAKE OF G. halli AT 30C
WITH 10 MW/CM2 IRRADIATION AND CONTINUOUS EXPOSURE TO VARIOUS
CONCENTRATIONS OF CrCl3
Cr+3
yg/L
N/A
0.04
0.20
0.38
3.8
7.88
12.0
12.8
14.3
19.7
25.3
28.4
35.6
Mean^ •
v JL •
1.01
1.16
1.21
1.40
1.32
1.23
1.17
1.02
1.00
0.74
0.76
0.66
0.25
-
Chi a/cell
yg(X10-8)
14.27
18.84
20.88
19.57
20.03
18.38
15.75
12.60
11.90
8.38
9.29
9.10
7.42
-
14C Uptake2-
(X104)
3.67
2.58
2.85
2.01
1.80
3.80
2.43
3.42
3.51
3.00
2.61
1.52
1.36
2.80
Notes:
1 dv+Vc
1. The specific growth rate is calculated as follows: Kc = -i-— and is
given in divisions per cell per day,
o _, i A _ . . . . counts/min
2, The •L4C uptake is given as: =^
e ug Chi a
3. The mean value was calculated for all concentrations of chromium up to
and including 25,3 yg Cr/L, excluding the lowest and highest values.
60
-------
SECTION 7
THE CHRONIC TOXICITY OF MERCURY, CHROMIUM AND NICKEL
TO G. halli AND J. galbana (CHEMOSTAT)
The addition of a metal to a fixed volume of seawater may result in an
instantaneous concentration of the metal in this medium that is near the
amount added per unit volume. But, heavy metals react with components of
seawater and form low solubility (approaching zero) products (Duursma, 1966)
and the concentration of a specific metal that results from a given addition
decreases with time. Thus, the effects of a specific concentration of a
specific metal on a specific organism cannot be determined in this manner.
The ionic concentration of metals can be increased by the use of chelators
but this decreases the activity of the metal ions. A steady-state concentra-
tion of the metal that approaches the desired concentration can be established
in a continuous-flow culture system. Continuous flow culture systems are
principally of the chemostat (Novick and Sailard, 1950; Monad, 1950) or the
turbidostat (e.g., Maddox and Jones, 1964; Freeberg, this report) types. But
this steady-state condition can only be accomplished if the medium and metals
solution, which is in a non-reacting solvent, are delivered in the proper
amounts from separate reservoirs. The turbidostat differs from the chemostat
in that the flow of the turbidostat decreases, increases or stops according
to the growth of the population whereas the flow of a chemostat continues at
the set rate independent of the growth of the population. The population
controls the flow of the turbidostat and the flow of the chemostat is mechani-
cally controlled at a specific rate. Studies were conducted on the toxicity
of Hg and Cr to cultures of Glenodinium halli and Isochrysis galbana in a
turbidostat and in a chemostat. Also, the toxicity of Ni to G. halli was
studied in a chemostat. Results of turbidostat experiments were included in
Section 6. of this report.
MATERIALS AND METHODS
The chemostat that was developed for metals toxicity tests consisted of
two peristaltic pumps controlled by a Monroe Electronics Timer (Model 231
with Model 510-3 accessory) that synchronized the delivery of the test
material and the medium from separate reservoirs through tubing to a 3-way,
Y-type connector located above the culture vessel (Figure 4). We used Mano-
stat, Harvard, Sigmamotor and Virtis peristaltic pumps. None of these were
completely satisfactory, mainly because the rotor pinched or ruptured the
tubing. The air was supplied from a Silent Giant Pump Model 120 (Aquarium
Pump Supply, Inc.) through the third arm of the connector. The air supply
was passed through a coil in a heated 6" x 6" chamber and filtered through
a sterile fiberglass column and a Koby, Inc. Junior Air Purifier and Flow
Equalizer and delivered through the third arm of the 3-way connector. The
61
-------
TEST MATERIAL RESERVOIR
PUMP H-
PUMP
Of
-TIMER
AIR
PUMP
MEDIUM RESERVOIR
OVERFLOW
'3-WAY "Y" CONNECTOR
SAMPLING PORT
CCATCH VESSEL
Figure 4.
MAGNETIC STIRRER
Diagrammatic sketch- of the basic chemostat design.
62
-------
air pump could be operated continuously or activated at the same time that
the pumps were activated by the timer for medium and test material delivery.
Since our off-on cycle on the timer was 99 sec:5-12 sec., the air, medium
and test material were supplied synchronously. This off-on cycle was more
appropriate for the growth of cultures. It stirred the cultures adequately;
facilitated the medium delivery; dissolved medium gas balance and overflow
uniformity. The 3-way connector was mounted on a glass delivery tube that
extended to the bottom of the culture vessel. Thus, the culture medium and
test material came together immediately before delivery to the bottom of the
culture vessel and was mixed into the culture volume by the turbulent air
flow. This intermittent agitation was beneficial to the chemostat growth of
dinoflagellates. The culture vessel was air-tight except through the over-
flow port at the surface of the culture volume. The overflow port could be
accurately calibrated, with the dimension of our culture vessel, for any
volume from 100 to 1200 ml by adjusting the overflow tube length. We employed
a 1-liter volume. The aeration and a magnetic stirrer were used to maintain
a homogeneous distribution of the culture, however, the stirrer was not re-
quired. The 1000 ft. c. light intensity for the culture was supplied by
three 15-watt daylight fluorescent lights placed four inches from the culture
vessel on each of three sides. The entire apparatus was housed in. a walk-in
constant temperature room. As many as six chemostats were in operation at
one time with a single set of pumps and timers.
Initially the flow of the medium was 500 ml ± 50 ml per day and the flow
of the test material (distilled water solution of HgCl2, CrCl3, or NiCl2) was
5 ml ± 0.5 ml per day. Rates were established by the timer, the pump rate
and the size of the tubing. Subsequently, the flow rate of both materials
were doubled so that there was one complete volume exchange per day.
Sampling for population counts and Chlorophyll a measurements were made
at the same time daily by catching the overflow during a one-half to one hour
period. The overflow vessel was an integral part of the system and was main-
tained aseptically. Cell counts were made with a Model B Coulter Counter and
Chlorophyll a was measured with a modification of the fluorometric method of
Strickland and Parsons (1968) as modified (p. 43, Section 6 of this report).
The test solutions of Hg, Ni or Cr were prepared according to the concentra-
tion desired in the culture vessel so that 10 ml of this solution mixed with
one liter of culture medium resulted in the desired concentration. This metal
test solution, prepared with distilled water was contained in a graduated
cylinder. The cylinder was closed with a two-hole silicone stopper. The
volume delivered each day from this reservoir was subtracted from the total
overflow volume from the culture to determine the amount of medium and test
material solution delivered.
The medium was prepared in 8-liter amounts in 2.5 gallon Pyrex carboys
closed with a two-holed, shirted stopper (Baltimore Biological Laboratory).
Both thick-wall silicone and viton tubing was used for all connections be-
tween reservoirs, culture vessels, air filters and catch-vessels. The medium
or test solutions as well as components of the system that were in contact
with them were autoclaved at either 15 psi for 15 minutes or for 30 minutes
for the 8-liters of medium. All connections were made prior to autoclaving,
if possible. Otherwise all connecting terminals were wrapped with gauze and
63
-------
masking tape and each connection made aseptically, in the constant tempera-
ture room after autoclaving. Glassware cleaning procedures were described on
p. 43, Section 6.
All experiments with G. halli were conducted in medium with 28 ppt
salinity at 28C and those with I. galbana were conducted at 28 ppt salinity
and 12C as controls. Sterility tests were made with Spencers (1952) seawater
peptone and peptone seawater agar every third day of operation, at the time
of a change in the concentration of the metal tested, at the finish of a run
or at other times that sterility was questionable.
RESULTS
Results of some of the first experiments conducted with G. halli indi-
cated that Hg, Cr and Ni were much more toxic in a chemostat than in the
static acute toxicity assays (Figures 5 and 6). Concentrations as low as 50
myg Hg/L, 0.1 yg Cr/L and 0.2 yg Ni/L reduced population levels and the Chlo-
rophyll a content by an order of magnitude or more within 16 days or less.
Although these tests were repeated either three or four times, these metal
concentrations did not again elicit these high toxic effects. Also, lower
concentrations had no effects (Figures 7 and 8). Indeed, the concentration
of the metals that were toxic subsequently were similar to or less than an
order of magnitude less than the LMTL values of the acute toxicity tests with
both G. halli and J. galbana (TABLE 2).
Other than these initial experiments conducted to determine the coxicity
of Hg to G. halli (Run A and B, TABLE 23), population levels of G. halli, as
indicated by cell counts and Chlorophyll a, were maintained in media with
concentrations from 0.01 to 10.0 yg Hg/L (Run 1 and 2, TABLE 23). Collec-
tively, these results indicate that the minimum concentration that was toxic
to G. halli in this system was from 20 to 100 yg Hg/L. The mean population
levels for the periods that the system contained 0.08 to 10 yg Hg/L were
significantly higher than during the periods that the medium contained 0.02
yg Hg/L or less (Run 1, TABLE 23). These results may indicate that these
concentrations of Hg were stimulatory, but this effect did not occur with 5
and 10 yg Hg/L in another test (Run 2, TABLE 23).
The toxic levels of Cr to G. halli were between 10 and 100 yg Cr/L.
Exposures of 5 to 12 days to media that contained from 0.01 to 100 yg Cr/L
did not reduce the cell number or Chlorophyll a content significantly (Run 1
and 2, TABLE 24) and noticeable population changes occurred only after the Cr
level was raised to 1000 yg Cr/L. On the other hand, in subsequent tests,
the toxic level was 100 yg Cr/L or less (Runs 3 and 4, TABLE 24). Less than
100 yg Cr/L reduced the number of organisms in another series (Run A,
TABLE 24), but the population levels and Chlorophyll a content declined
throughout the 35-days test, possibly caused by something other than the Cr
concentration. The toxicity level of Ni to G. halli was approximately 50 yg
Ni/L. Concentration of 0.05 to 40 yg Ni/L did not cause a significant change
in the population numbers or Chlorophyll a concentrations. On the other hand,
concentrations of 50 yg Ni/L caused a significant decrease in both measure-
ments (Run 1, 2 and A, TABLE 25) and a concentration of 100 yg Ni/L caused a
64
-------
Ui
E
-».
CO
u >1
o 4
QC
UJ
to
z
I
o o
2-
• CHLOROPHYLL a
o--NUMBER OF CELLS /ml -
o o
o
UJ
h-
_So
24
Figure 5. Response of G. halli culture to Hg concentration of 50 myg/L
(one experiment).
-------
8
•? 6
E
V.
(A
j
UJ
O
£E
U)
m 4
3
z
i
o
_j
2
0
> i i i i i ii
• • -CHLOROPHYLL a
* o -NUMBER OF CELLS /ml _
• ^ t
f
o p
ooooooo
a *
UJ
l—
o: o
< 0
h-
co
o
z 0 O
Ni-0.2#g/L •
, . . i i , • • 9 fr
2 4 68 10 12 14 16 1
16
14
12
o>
,0*-
o
_J
_J
8 i
Q.
o
DC.
6 3
I
0
4
2
B
TIME (DAYS)
Figure 6. Response of G. halli culture to Ni concentration of
0.2 vg/L (one experiment).
66
-------
D
nnnnn
E
CO
_j 4
UJ
o
oc
UJ
CD
~
7 2
o
o
-J
1 1 III 1 1 1 1 1 1 1
1
*
_ • o o
o o
* o o * * ® o ° o oc
°o° ° • ° • o°
_ 9 . _
•
• * * • *
9
• • • • -
•
-
•-CHLOROPHYLL a
o- NUMBER OF CELLS /ml _
Hg-2.0m/
8 =1
o
_l
6 >
I
a.
O
oc
4 0
I
o
2
TIME (DAYS)
Figure 7. Response of G. halli culture to Hg concentration of 2 myg/L
(one experiment).
-------
00
8
E
-s.
UJ
o
-------
more rapid decline in the number of organisms and Chlorophyll a.
Chronic effects of Hg to r, galbana indicated that the toxicity level
was 100 yg Hg/L, or possibly less. The first test series with J. galbana
and Hg with concentrations from 0.02 to 800 pg Hg/L indicated that the toxi-
city level was between 80 and 800 yg Hg/L (Run 1, TABLE 26), Although the
mean population number and Chlorophyll a of the culture that received 80 yg
Hg/L was less than the one that received the next lower amount (8 yg Hg/Ll,
the difference was not significant. In the second test series, a concentra-
tion of 100 pg Hg/L caused a significant population decrease within 12 days
an^ the population decreased more rapidly in medium with 200 pg Hg/L (Run 2.
TABLE 26).
The minimum concentration of Cr that produced a decline of J, galbana
populations in a chemostat was between 5 and 100 pg Cr/L, Populations were
reduced by concentrations of 100 pg Cr/L (Runs 2 and 3, TABLE 271. The mean
population numbers and Chlorophyll a levels were significantly less in media
with 5 to 40 pg Cr/L than during periods that the media contained 0,5 pg Cr/L
or less. These differences may be meaningless as the population continued to
grow at a reduced rate, but there was a general downward trend of both cell
numbers and Chlorophyll a of populations in media that contained progressively-
higher concentrations of Cr from 5 to 40 pg Cr/L. Both experiments.indicate
that 100 pg Cr/L was toxic to J. galbana (Runs 2 and 3, TABLE 271,
Nickel was toxic to chemostat cultures of J. galbana at concentrations
between 40 and 100 pg Ni/L. Concentrations of 40 pg Ni/L or less had no
effect on the growth of I. galbana as indicated by the cell counts or Chloro-
phyll a concentrations but the populations decreased in media that received
80 pg Ni/L or more (Run 2 and 3, TABLE 28), Another test (Run 1, TABLE 28)
indicated that 0.2 to 0.4 pg Ni/L may have been toxic to J, galbana but sub-
sequent experiments did not show that these concentrations reduced the popu-
lations of the system.
At the termination of each run of a chemostat experiment, cultures of
G. halli and I. galbana usually grew if allowed to remain in the culture
vessel in a static system. At the end of the incubation periods, the cultures
were examined to determine whether or not the population level had increased.
After most initial inhibitory, effects were observed, the air, medium and
metal solution flows were stopped and the culture remained in a static incu-
bation state for 8 to 15 days. Although the cells were enumerated and the
relative Chlorophyll a was determined, no attempts were made to determine
growth rates during this period. The results of these examinations show that
I. galbana populations increased in all systems after the flow was stopped
and that G. halli often, but not always, increased during this period. For
the most part, G. halli increased in all cases except those in which the
media contained metal concentrations that were much higher than the minimum
toxicity level (e.g. 500 pg Hg/L, TABLE 23 and 1000 yg Cr/L, TABLE 24). On
the other hand, G. halli populations died in media with relatively low metal
concentrations, e.g., .05 pg Hg and 2 pg Hg (Runs A and B, TABLE 23, 30 ug
Cr/L (Run A, TABLE 24) and 50 pg Ni/L (Run A, TABLE 25).
69
-------
TABLE 23. CHRONIC TOXICITY OF MERCURY TO G, halli IN A CHEMOSTAT
Run 1
Hg added
Ug/L
Days of
operation
1st day
Last day
X Ch a
a Ch a
Var.Ch a
No. x 107
1st day
No. x 107
Last day
X" No. cells
a No. cells
Var. No.
cells
0
7
6.15
6.68
7.23
0.73
0.44
2.9
3.8
3.6
0.56
0.27
.01
10
8.26
8.31
7.46
0.95
0.80
3.9
3.0
2.79
0.75
0.52
02
10
6.94
8.21
5.94
1.68
2.70
2.4
3.4
2.70
0.70
0.44
04
10
7.85
10.32
8.43
1.00
0.89
3.7
5.0
2.92
0.56
0.28
08
11
10.24
12.45
12.32
1.31
1.15
6.1
7.1
7.31
0.70
0.44
.1
7
12.70
11.15
11.83
1.10
1.04
7.1
6.0
6.60
0.77
0.51
l.'O
8
12.06
12.99
11.88
0.74
0.48
6.1
8.9
7.61
0.84
0.62
*
2.0
6
12.20
12.35
12.51
0.73
0.46
9.0
6.5
7.57
0.84
0.58
4.0
4
13.45
13.52
13.32
0.28
0.06
7.3
9.3
8.12
0.84
0.53
10
5
13.96
12.52
13.45
1.05
0.88
9.7
9.9
9.36
0.42
0.14
100
6
9.46
4.89
7.23
2.22
3.70
6.8
4.3 -
6.10
1.22
1.12
500
4
0.48
0.01
0.25
0.33
0.06
0.50
0.30
0.40
0.14
0.01
(continued)
-------
TABLE 23. (continued)
Hg added
Days of
operation
1st day
Last day
X~Ch a
cr Ch a
Var.Ch a
No. x 107
1st day
No. x 107
Last day
X~ No . cells
a No. cells
Var. No,
cells
0
6
13.60
11.35
12.65
0.94
0.73
6.9
6.3
6.52
0.37
0.11
Run
5
11
11.13
10.02
10.06
1.53
2.14
6.9
4.4
4.72
1.26
1.45
2
10
10
9.57
7.75
8.77
1.10
1.08
5.1
4.2
4.85
0.52
0.24
20
4
4.90
0.03
2.51
2.47
4.59
3.3
0.38
2.27
1.35
1.38
0
8
26.43
24.02
23.42
3.10
8.17
20.0
18.0
16.8
0.28
0.07
Run A
.05
16
24,41
0.02
6.31
11.71
128.5
18
0.13
5.19
5.60
28.93
0
9
3.2
7.8
5.65
1.45
1.85
1.7
4.2
2.86
0.87
0.70
Run B
1.0
10
9.8
15.2
14.67
2.57
5.94
5.2
14.0
9.93
2.90
7.59
2.0
15
14.
0.
6.
6.
35.
13.
0.
6.
4.
17.
9
1
66
18
69
0
8
26
37
85
-------
TABLE 24. CHRONIC TOXICITY OF CHROMIUM TO G. halli IN A CHEMOSTAT
to
Cr added
yg/L
Days of
operation
1st day
Last day
X~Ch a
a Ch a
Var.Ch a
No. x 107
1st day
No. x 107
Last day
X No. cells
a No. cells
Var. No.
cells
0
9
9.56
13.31
11.38
2.03
3.67
5.2
5.1
5.11
0.65
0.38
.01
10
15.02
13.51
11.89
1.91
3.25
9.0
4.9
4.8
1.70
2.59
Run 1
02
11
12.40
15.92
12.66
2.73
6.74
5.1
7.1
5.90
1.55
2.19
04
9
15.49
13.30
13.91
2.55
5.79
6.9
9.1
7.74
0.80
0.58
08
12
14.40
11.79
13.76
1.80
2.91
7.8
8.4
9.26
0.76
0.53
.2
5
11.84
13.82
13.98
1.59
2.02
9.1
8.6
9.04
0.47
0.18
0.1
11
17.12
14.56
14.67
1.16
1.22
8.6
7.9
8.25
0.53
0.25
Run
1.0
5
14.55
12.35
13.28
0.92
0.72
6.5
5.8
6.28
0.38
0.12
2
2.0
4
12.79
14.05
13.55
1.18
1.04
5.8
5.6
5.98
0.33
0.08
10
5
15.29
13.43
14.05
0.79
0.53
7.2
6.0
6.18
0.72
0.04
100
5
11.61
13.43
12.75
0.99
0.65
4.6
5.0
4.87
0.23
0.04
1000
3
4.21
0.53
2.07
1.91
2.44
4.8
3.5
4.0
0.7
0.32
(continued)
-------
TABLE 24. (continued)
Cr added
Ug/L
Days of
operation
1st day
Last day
x"Ch a
a Ch a
Var.Ch a
No. x 107
1st day
No. x 107
Last day
x" No. cells
a No . cells
Var. No.
cells
Run
0
14
24.93
15.14
21.67
2.35
3.41
11.0
8.7
12.0
1.42
1.31
3
100
8
16.03
0.24
7.47
5.96
28.61
8.1
1.3
5.8
2.48
7.80
0
9
21.
18.
18.
2.
4.
11.
11.
9.
1.
1.
71
13
73
21
36
0
0
97
17
22
Run 4
10
8
20.19
18.00
19.15
0.8
0.56
10.0
13.0
12.5
1.6
2.25
20
7
15.48
0.45
5.47
6.03
31.2
11.0
3.5
6.14
3.22
8.90
16.
11.
14.
2.
7.
14.
8.
10.
2.
3.
0
8
03
80
38
96
3
0
1
5
16
89
1
11
13.13
4.40
11.1
2.90
7.7
8.1
2.7
6.92
1.80
2.87
Run A
15
14
2.67
0.20
1.36
0.82
0.61
2.7
0.80
1.68
1.10
1.16
30
2
0.14
0.16
0.15
0.09
0.10
0.61
0.69
0.65
0.06
0.00
-------
TABLE 25, CHRONIC TOXICITY OF NICKEL TO G. halli IN A CHEMOSTAT
Ni added
Pg/L
Days of
operation
1st day
Last day
X~.Ch a
a Ch a
Var.Ch a
No. x 107
1st day
No. x 107
Last day
X No. cells
a No. cells
Var. No.
cells
0
7
13.67
11.38
12.58
1.03
0.91
6.4
6.9
6.8
0.50
0.21
0.05
11
10.86
11.01
9.61
1.00
0.89
5.6
5.0
4.65
0.61
0.33
.1
12
12.32
12.63
9.84
2.14
4.19
5.5
5.7
3.92
1.16
1.23
.2
9
12.18
12.67
12.18
0.79
0.55
5.4
8.1
5.94
1.05
0.99
Run
.4
12
11.38
12.52
13.9
1.41
1.82
7.4
8.2
8.84
1.10
1.11
1
1
7
13.88
12.06
12.88
0.71
0.43
9.7
8.4
8.89
0.66
0.37
5
10
13.92
20.51
17.92
2.36
5.00
8.7
12.0
10.47
1.22
1.35
20
6
20.69
13.67
15.60
3.43
9.82
11.0
7.3
S.65
1.30
1.41
40
6
12.35
15.74
14.20
1.45
1.57
7.5
9.2
8.30
0.79
0.47
50
5
11.21
2.01
5.26
3.40
12.79
5.9
2.6
3.82
1.38
1.53
100
3
0.35
0.18
0.21
0.12
0.01
1.5
1.5
1.4
0.17
0.02
(continued)
-------
TABLE 25. (continued)
Ni added
Pg/L
Days of
operation
1st day
Last day
X Ch a
a Ch a
•••i
•-" Var.Ch a
No. x 107
1st day
No. x 107
Last day
X No. cells
a No. cells
Var. No.
cells
0
5
27.63
20.58
23.11
3.07
7-52
16.0
13.0
14.0
1.40
1.60
Run
10
13
19.58
13.52
16.04
1.99
3.65
12.0
6.7
8.77
1.90
3.32
2
20
12
16.03
16.56
13.42
3.22
9.53
8.6
9.5
6.97
2.03
3.78
50
15
16.44
6.94
11.3
3.61
12.51
9.8
4.7
5.95
1.67
2.59
0
8
12.69
9.79
11.52
2.48
5.15
6.1
6.2
6.4
1.40
1.63
Run A
10
12
'9.57
3.07
12.48
7.84
56.39
5.3
4.0
8.35
4.14
15.74
50
8
2.36
0.06
0.37
0.80
0.57
2.3
0.6
1.02
0.77
0.51
-------
TABLE 26. CHRONIC TOXICITY OF MERCURY TO J. galbana IN A CHEMOSTAT
Hg added
Ug/L
Days of
operation
1st day
Last day
X Ch a
a Ch a
ol Var.Ch a
No. x 107
1st day
No. x 107
Last day
X~ No. cells
a No. cells
Var. No.
cells
0
6
6.03
9.22
8.60
1.91
3.05
1.5
2.7
2.7
1.4
1.64
.02
8
16.17
19.52
21.43
4.86
20.70
4.1
3.9
4.54
0.83
0.62
.04
9
18.32
19.82
20.24
1.90
3.20
2.7
5.4.
3.66
1.03
0.95
Run
.08
10
15.21
17.32
17.50
1.17
1.12
3.0
2.8
3.32
0.46
0.19
1
.2
21
16.52
4.78
6.64
3.40
10.97
2.4
0.99
1.36
0.64
0.40
.5
7
6.37
7.45
6.14
1.37
1.61
1.6
2.0
1.44
0.49
0.21
1
9
7.17
12.61
11.51
2.59
5.95
1.2
2.0
1.88
0.47
0.19
2 4 8 80 800
55 46 9
10.24 13.67 11.94 9.42 8.65
11.47 11.47 10.32 7.81 0.77
12.03 13.48 10.90 8.47 3.12
1.46 0.39 0.80 0.55 2.62
1.59 0.11 0.49 0.25 6.00
1.5 2.1 1.6 1.4 1.3
1.8 1.7 1.7 1.4 0.27
1.83 1.86 1.63 1.33 0.61
0.25 0-17 0.05 0.08 0.33
0.05 0.02 0.00 0.00 0.10
Run 2
0
6
24.69
24.70
23.18
1.20
1.20
10.0
5.0
I
6.37
1.95
3.17
100
12
21.37
11.76
16.13
4.74
20.6
3.1
1.6
3.17
1.19
1.30
200
9
6.12
0.86
3.20
1.91
3.26
1.2
0.3
0.75
0.36
0.11
-------
TABLE 27. CHRONIC TOXICITY OF CHROMIUM TO T, galbana IN A CHEMOSTAT
Cr added
yg/L
Days of
operation
1st day
Last day
X~Ch a
a Ch a
Var. Ch a
No. x 107
1st day
No. x 107
Last day
X No. cells
cr No. cells
Var. No.
cells
0
6
14.66
17.64
16.26
2.10
3.66
4.6
4.0
3.35
1.15
1.10
Run 1
005
13
17.07
14.32
22.03
6.36
37.3
4.8
2.5
4.3
1.60
2.30
01
10
13.81
12.52
14.21
1.10
1.10
2.3
2.5
2.5
0.40
0.18
0
10
17.07
19.92
18.48
1.81
2.94
7.0
3.2
4.85
1.54
2.13
02
11
16.76
13.81
18.04
2.65
6.30
4.0
2.2
3.82
0.98
0.88
04
9
16.50
20.21
22.23
3.23
9.13
3.8
3.9
4.75
0.93
0.75
Run 2
08
10
21.86
21.69
21.34
1.51
2.08
3.6
2.7
2.99
0.48
0.21
.5
15
18.38.
17.72
20.05
2.50
5.85
2.1
2.2
2.51
0.37
0.13
5
11
17.42
7.62
8-27
4.52
18.64
2.3
1.2
1.32
0.48
0.21
10
7
6.60
4.12
4.28
1.14
1.11
1.3
1.1
0.91
0.23
0.05
20
8
5.01
1.37
3.73
1.31
1.50
1.0
0.41
0.74
0.22
0.04
40 100
8 7
2.69 6.38
5.89 1.63
3.54 3-96
1.42 2.26
1.76 4.37
0.90 1.0
0.91 0.32
0.72 0.68
0.15 0.38
0.02 0.13
1
mg
5
2.65
1.07
1.55
0.64
0.32
0.42
0.42
0.42
0.04
0.00
(continued)
-------
TABLE 27 (continued)
00
Cr added
ug/L
Days of
operation
1st day
Last day
X Ch a
a Ch a
Var. Ch a
No. x 107
1st day
No. x 107
Last day
X No. cells
a No. cells
Var. No.
cells
0
6
20.26
26.25
24.29
2.86
6.80
5.7
9.1
6.63
1.46
1.79
Run 3
100
22
26.47
0.31
7.39
10.17
98.75
6.5
0.12
1.73
2.17
4.50
-------
TABLE 28. CHRONIC TOXICITY OF NICKEL TO I. galbana IN A CHEMOSTAT
Nl added
Wg/L
Days of
operation
1st day
Last day
X~Ch a
a Ch a
Var.Ch a
No. x 107
1st day
No. x 107
Last day
X~ No. cells
0 No. cells
Var. NO,
cells
Run 1
0 .2
9 15
42.04 25.93
35.85 10.21
38.43 17.48
4.50 5.30
18.88 23.88
6.0 5.2
8.1 2.1
6.49 3.48
1.18 1.15
1.14 1.23
.4
11
12.01
5.23
8.77
2.32
4.90
2.3
2.1
2.06
.38
.13
0
11
9.70
10.24
8.41
1.99
3.59
2.6
2.5
2.49
.65
.38
.1
11
9.26
7.74
8.55
1.79
2.88
2.1
1.7
2.74
.78
.56
.2
9
6.37
14.62
11.82
3.78
12.74
1.6
4.3
3.63
1.37
1.68
.5
12
13.45
14.07
13.91
1.70
2.66
3.2
3.2
3.33
.64
.38
Run 2
1
11
13.18
11.15
12.27
0.98
0.87
3.0
2.1
3.04
.58
.31
5
8
5.23
5.69
5.24
.85
.64
0.88
1.4
1.37
.38
.12
10
8
6.05
11.23
8.30
2.16
4.08
1.2
2.7
1.86
.61
.33
20
8
12.41
9.9
9.48
1.82
2.9
2.8
2.1
2.0
.46
.18
40
8
10.01
12.35
11.10
1.07
1.00
2.0
2.2
2.18
.36
.11
80
9
10.26
2.61
4.91
3.98
14.08
1.8
.74
1.12
.57
.29
160
9
2.06
2.73
2.40
.28
.06
.83
.74
.71
.1
.01
(continued)
-------
TABLE 28 (continued)
00
o
Ni added
yg/L
Days of
operation
1st day
Last day
X Ch a
o Ch a
Var.Ch a
No. x 107
1st day
No. x 107
Last day
X No. cells
-------
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The typists for this Report were Mrs. Margie Watson and Ms. Nora McKenna
91
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
i. REPORT NO.
EPA-600/3-80-025
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
i. REPORT DATE
February 1980 issuing date
TOXICITY OF METALS TO MARINE PHYTOPLANKTON CULTURES
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
William B. Wilson and Larry R. Freeberg
8. PERFORMING ORG>
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Texas A&M University at Galveston
Galveston, Texas 77550
10. PROGRAM ELEMENT NO.
1BA819
11. CONTRACT/GRANT NO.
R801511
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory - Narragansett, RI
Office of Research and Development
U.S. Environmental Protection Agency
Narragansetf, Rhode Island 02882
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA/600/05
15. SUPPLEMENTARY NOTES
16. ABSTRACT
The objectives of this program were to evaluate the toxicity of nine
metals to cultures of four species of marine phytoplankton. The relationships
of acute, instantaneous and chronic toxicity were evaluated using growth rates
in continuous-flow culture systems. The latter methods employed both the
ehemostat and the turbidostat techniques. The instantaneous procedure measures
short-term changes of metabolic activity indicated by 14-C uptake that result
from metal addition within a relatively short time period after cultures
are exposed to metal additions. Four levels were determined for the acute
toxicity of metals to each organism. The use of fluorometric measurements
of relative chlorophyll-a of actively growing cultures was a fast, accurate
assay method that facilitated the Minimum Toxicity Level calculation, increased
the sensitivity of the method, and reduced variability. Refinement should
result in this method being more useful for giving more uniform assay results.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
selenium
Copper
Mercury
Silver
Nickel
cadmium
lead
barium.
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Marine Biology 0801
Phytoplankton 0603
Salinity 0704
Toxicity 0620
cobalt
0603
Continuous culture
G. Splendens
I. Galbana
T. pseudonana
G. Halli
06F
06T
08A
13. DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
110
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 <9-73)
92
* U.S. GOVEBNMENT PfllHTING OFFICE: 1990-657-146/5572
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