Interactive Toxic Effect and

Bioconcentration between Cadmium and

Chromium Using Continuous Algal Culture

ISAO AOYAMA and HIDE0 OKAMURA

Laboratory of Ecological Chemistry,
Research Institute for Bioresources,
Okayama University,
2-20-1, Chuo, Kurashiki, Okayama 710, Japan

ABSTRACT
Numerous studies have been conducted on the effects of toxic chemicals using algal
batch culture. On the other hand, continuous culture research has the advantage of
allowing optimal cell growth. Few studies on the interactive toxic effect between chemi-
cals have been carried out either with batch culture or with continuous culture. The
purpose of this study is to find the relationship between interactive toxic effects and the
bioconcentration of heavy metals using continuous algal culture. The green alga Chlorella
ellipsoidea was used as a test organism. As test substances cadmium and chromium
were used.
The amount of heavy metals both in algae and in the medium was determined after
the cells had been exposed to heavy metals for 10 days. The amount of Cd taken up in
the cells and the growth inhibition rates caused by Cd increased as the concentration
of Cd in the medium became higher. The inhibition rates rose drastically but the amount
of Cd in the cells remained a t almost the same level even if Cr was added. The bioconcen-
tration factors of Cd were ca. 10,000 in all the cases tested, regardless of the presence
of Cr. The amount of Cr in the cell exposed to Cr was low, while that amount and the
growth inhibition rates for all the concentrations tested increased when Cd was added
to the medium containing Cr. The bioconcentration factors of Cr reached 156,000 as the
concentration of Cd in the medium increased. It seems that the toxicity of Cr was
enhanced because the presence of Cd in the medium raised the amount of Cr in the
cell. 0 1993 John Wiley & Sons, Inc.

INTRODUCTION
The evaluation of the toxicity of chemicals released into the aquatic
environment has become an increasingly important issue in ecotoxico-
Environmental Toxicology and Water Quality: An International Journal
Vol. 8, 255-269 (1993)
0 1993 John Wiley & Sons, Inc. CCC 1053-47251931030255-15
256/AOYAMA AND OKAMURA

logical studies. In almost all natural waters, metals are present to-
gether in solution. When mixed together, they have supplementary
synergistic or antagonistic effects on organisms living in the aquatic
environment. For this reason, the research on the interactive toxic
effects between combined chemicals is extremely important. Most of
the studies on interactive effects have been conducted in batch culture,
that is, in a closed system (Aoyama et al., 1987; Bartlett et al., 1974;
Stratton 1983; Wong et al., 19781,but there has been very little research
concerning continuous culture. In comparison with batch culture, con-
tinuous culture is more analogous to a natural environment like a
lake, which has an inflow from one side and an outflow in the other
side. The continuous culture system involves a continuous supply of
fresh medium allowing optimal cell growth and a continuous removal
of catabolic waste.
As algae are primary producers and are at the bottom of the aquatic
tropic level, their inhibition by metals would, in turn, affect the well-
being of their aquatic communities. Some research using continuous
algal culture has been reported in ecotoxicological studies.
The chemostat was used in a study of the toxicity of Cd on algae
(DeNoyelles et al., 1980). There have been some studies in which
the toxicity of chemicals in continuous culture was compared with
that in batch culture. Continuous cultures have proven to be superior
t o batch cultures for studying competitive interactions, and poly-
chlorinated biphenyls caused greater alteration of species composi-
tion in continuous cultures than in batch cultures (Fisher et al.,
1974).
The higher sensitivity of the turbidostatic continuous culture
method was reported in the case of an experiment dealing with the
effect of Cd (Premazzi et al., 1978). In contrast with this, the difference
in the effect of mercuric acetate on several marine algae incubated in
batch and turbidostat culturing conditions was found not t o be signifi-
cant (Kayser, 1976). Wong et al. (1978, 1983) evaluated the toxicity of
the metal mixture using both batch and continuous culture techniques,
and summarized the advantages and disadvantages of the use of algal
batch and continuous culture techniques in a study on metal toxicity.
As for the bioconcentration of chemicals by algae grown in batch and
continuous culture, there appears to be a dose-response relationship
between cellular haxachlorobiphenyls and relative growth rate (Leder-
man and Rhee, 1982a, 198213).
One of the purposes of this research is to find out whether the
dilution rate influences the intensity of toxicity of a chemical acting
 INTERACTIVE TOXIC EFFECT OF CD AND CR/257

Fig. 1. Culture system for continuous algal culture. 1: Culture flask; 2: reservoir
flask; 3: drain flask; 4a and 4b: peristaltic pump; 5 : fluorescent lamp; 6 : magnetic stirrer;
7: aquarium; 8: sampling syringe.

on an organism grown in continuous culture. Another is to find out

the relationship between interactive toxic effects and bioconcentration
of combined chemicals, using continuous algal culture. As a test organ-
ism, green alga Chlorella ellipsoidea was used, and as test chemicals
Cd and Cr were used because their toxicities are well known.

MATERIALS AND METHODS

Culture of Algae
The freshwater green algae, C . ellipsoidea Gernec (IAM.C-27),provided
by the Institute of Applied Microbiology,Tokyo University, was used as
a test organism. The green algae Chlorella was grown in the chemostat
shown in Fig. 1. As a culture medium, Myers 1N-A, solution (Aoyama
et al., 19871, whose composition was modified by using Fe-citrate in-
stead of FeSO,, was diluted 10 times with water before using (Okamura
et al., 1989). The cultures were maintained at 25°C with continuous
light of 8000 lux at the surface of the vessel. Algal density was expressed
as cells per milliliter of algal suspension and was evaluated using a
Cell Counter appparatus (Nippon Koden Co. Ltd).
In a series of experiments, 4 vessels, each containing 100 mL of
the medium without toxicants, were prepared, and Chlorella was inocu-
lated from the precultured algae. The algal suspension was mixed with
a magnetic stirrer to supply C 0 2 gas actively. The excess volume of
258iAOYAMA AND OKAMURA

the culture solution was discharged with the peristaltic pump. The
algae first grew in a test medium free of toxicant. Once they reached
a steady state, the reservoir medium was replaced with the one contain-
ing test heavy metals.

Determination of the Amount of Heavy Metals

As test chemicals, Cd and Cr were used. The chemical formulas of the
salts used were Cd(NOJ, and K,CrO, . The heavy metal ions dissolved
in deionized-distilled water were stored a t 4°C until the time they were
used. The algal suspension containing test heavy metals was filtrated
using a membrane filter (pore size: 0.45 pm, 4 : 47 mm, Advantec Toyo
Co., Ltd). The algal cells containing the metals on the membrane filter
were digested with nitric acid : perchloric acid (1: 3) a t 200°C overnight,
and the medium containing the metals was not pretreated. The amount
of heavy metals both in the algae and in the medium was determined
using a flameless atomic absorption spectrophotometer (AA-8200, Nip-
pon Jarrel-Ash Co., Ltd).

Analysis of the Experimental Results

The method used to calculate the Relative Growth Rate (RGR) is shown
in the following equation (Lederman and Rhee, 1982b):
RGR (per hour) = DR + l / ( t , - t,).ln (x,/x,)(1)
where DR is the dilution rate (per hour), which is the ratio of the inflow
rate to the flask volume. The terms xl and x, are the cell densities
(cells/mL) a t time, (tl, hours) and time, (t,, hours), respectively. If no
toxicants are added, RGR is theoretically equal to DR because the cell
densities at time t, and time t, are the same a t a steady state. If a subtle
disturbance in cell density is observed in the early stages following
the administration of a chemical, RGR is drastically influenced. The
inhibition rate was calculated with the following equation using the
RGR obtained from Eq. (1):
Inhibition Rate (5%) = (1 - RGR,,,/RGR,,,,)~ 100
(2)
where RGR,,,, is the RGR in the control and RGR,,, is the RGR of the
sample with a toxicant.

RESULTS AND DISCUSSION

Effect of RGR on the Toxicity of a Chemical
In a chemostat, it is known that the RGR is equal to the dilution rate,
which is defined as the inflow rate divided by the flask volume. The
 INTERACTIVE TOXIC EFFECT OF CD AND C ~ i 2 5 9

concenlraliori
( lleqll)
I e Cd 0
! x--- Cd 7
I A- td I0

0 10 20
tirne(days)

Fig. 2. Effect of Cd on the growth of ChloreZZa a t 0.02 h-' RGR.

higher the RGR is, the lower the cell density is. Since the standing
crop of Chlorella is dependent on the RGR,the toxic effect of a chemical
on Chlorella may be altered according t o the RGR.
At first, we conducted the experiment in order to find out whether
the toxicity of a chemical was influenced by the RGR or not. RGR was
adjusted to three different levels and Cd was used as a test heavy
metal. Figure 2 shows the changes in cell density when RGR was
0.02ih. The 10-day Cd exposure experiment was followed by a recovery
experiment for another 10 days.
The Cd toxicity is clearly shown in this figure. Cell density de-
creased logarithmically at the highest concentration tested. The
changes in cell density at the lower Cd concentration were not different
from those of the control. In the recovery experiment, the cell density at
the highest concentration tested increased logarithmically and reached
the initial cell density.
Figures 3 and 4 show the changes in cell density when the RGR
was 0.05 and O.l/h, respectively. The toxic effect of Cd was observed

.-
cuncerilralion
( IJeqil 1
Cd 0
x-- cd 7
t I h- Cd 10

-
a' I
260IAOYAMA AND OKAMURA

in both cases when the Cd concentration was more than 10 peq/L.

When RCR was O.l/h, the change in cell density in the control was
not different from that when RGR was 0.05ih. This is likely due to
experimental error because some Chlorella grew on the wall of the
vessel. Such growth on the wall is frequently reported and it is the
main problem to be solved when using this continuous culture tech-
nique (Wong et al., 1983).
For the quantitative evaluation of the toxicity intensity, the rela-
tionship between inhibition rate and the Cd concentration is shown in
Fig. 5, over several exposure periods. The dilution rate (per hour) was
0.02 for A, 0.05 for B, and 0.1 for C. After exposure, a small change in
cell density has a significant effect on inhibition rates, calculated using
Eqs. (1)and (2). It is not advisable to evaluate the toxicity of chemicals
for a short exposure period, because it is too difficult to determine
whether the effect was caused by a n experimental error or by the
chemicals. So it seems that the toxicity should be evaluted using a n
inhibition rate obtained from a relatively long period of exposure. The
range in the inhibition rate for the control was within about t 2 0 %
of the average value. At the highest concentration tested, complete
inhibition was found after 3 days when the lowest RGR of 0.02/h was
used. At the same concentration, 60% inhibition was obtained after 5
days when RGR was 0.051h. At the highest RGR of O.l/h, only 20%
inhibition was shown after 24 h exposure. Thus, a stronger toxic effect
of Cd was shown when the RGR was lower, that is, when the standing
crop of Chlorella was higher. In contrast with this, it is sometimes
reported in the research using batch culture that chemicals cause
stronger toxicity when cell density is lower. It seems that the physiolog-
ical activity of Chlorella might be more depressed when cell density
was higher in the continuous algal culture.
 INTERACTIVE TOXIC EFFECT OF CD AND C R i 2 6 1

concentration
( IJeqll)
E C d 0
eZaCd 7
I C d 10
OCd 70

(C 1
r m - l o d or e v a l u a t i o n ( d a y e )

Fig. 5 . Percent inhibition of Cd over several exposure periods (A) 0.02 h-' RGR,
(B) 0.05 h-' RGR, and (C) 0.1 h-' RGR.

Interaction Effect of Both Metals on Chlorella

It was found that RGR had to be low in order to achieve a high sensitiv-
ity to the chemicals. The following experiments were conducted to
evaluate the interactive toxic effect of heavy metals, and RGR was
adjusted to 0.02/h. In these experiments, the doubling time of Chlorella
was about 2 days. Therefore, Chlorella in the control was assumed to
have 5 generations every 10 days. At first, only Cd was used, and the
concentrations were adjusted to 0, 2, 10, and 20 peq/L. Figure 6 shows
the changes in cell density when Cr was administered independently.
The Cr concentrations used were 0, 10, 20, and 50 peq/L. Cell density
decreased with the increase of the Cr concentration in the medium,
262iAOYAMA AND OKAMURA

Fig. 6. Effect of C r on the growth of Chlorella a t 0.02 h - ' RGR.

but the extent of the decrease in the rate was less than that obtained
when Cd was used alone. At the end of the exposure experiment, when
the medium was changed to a fresh solution, the cell density increased,
and recovered something between 60 and 80%of the initial cell density.
Figure 7 shows the results when both Cd and Cr were added to
the medium simultaneously. In this case, the Cd concentrations used
were the same as those when Cd was used on its own, but the Cr
concentration was fixed at 50 peqiL. The cell density decreased loga-
rithmically from the second day till the end of the exposure at all
concentration levels tested, and the decreasing rates were dependent
on the Cd concentration.
After the end of the exposure, the cell density did not increase for
several days and the length of this period was also dependent on the
Cd concentration tested. After these periods, the cell density began to
increase but reached only 50-60% of the initial cell density at the end

- lo?[- l e
Cd,Cr
0 . 50
2 , 50
10. 5 0
20, 5 0

! A~ ,in

time( duys)

Fig. 7. Interaction effect between Cd and C r on the growth of Chlorella a t 0.02 h - '
RGR.
 INTERACTIVE TOXIC EFFECT OF CD AND CRi263

concenlralion
- 100 veqll)
-3
>c B C d
eZaCd
0
2
L
1 C d 10
nw 20
5
)1
.a
$ 0
- 20 (A)
Z 4 F B 1 0
-5 100 Deriod o r evaluatlon(daye)

P) B Cr 0
$ lY2 C r 10
ICr 20
C
0Cr 50
)1
n
$ 0

(B)
.Lo 2 L F 8 10

-5 100 - Pel-lod o f ; evaiuatlonldaye)

u -
Cd, Cr
1: 0 , 50
2 . 50
10, 5 0
20, 5 0

.
- 1 , O " ' I
2 L
" " "
6 0 1 0
(C 1
pel-Iod o r evaluatlon[daye)

Fig. 8. Percent inhibition of Cd and Cr over several exposure periods. (A) Cd only,
(B) Cr only, and (C) Cd and Cr.

of the recovery experiment. It was clearly shown that the toxicity of

one metal was enhanced by the presence of the other metal during
both exposure and recovery experiments.
The relationships between inhibition rates and the concentrations
of both Cd and Cr over several exposure periods are shown in Fig. 8,
with a quantitative evaluation of the toxicity of both metals. Fig. 8(A)
shows the case in which Cd was administered to its own. Complete
inhibition was found after 3 days when Cd concentration was 20 peql
L. Figure 8(B) shows the results for Cr. The inhibition rate was the
highest when the exposure period was 1day. It decreased as the expo-
sure period became longer, except for one case involving the highest
concentration tested. In Fig. 8(C), the interactive effect of Cd and Cr
on the growth inhibition is shown. The inhibition rates were higher
than those obtained when each heavy metal was used alone. It is likely
264iAOYAMA AND OKAMURA

Cd 0
Cd 2
Cd I0
Cd 20

Fig. 9. Changes in the concentration of heavy metals present alone in the medium
(A) Cd and (B) Cr.

that the interaction mode of Cd and Cr is synergistic or at least additive.

But it is difficult to evaluate the interaction mode quantitatively, be-
cause we do not yet have the means to judge these modes under present
experimental conditions.

Bioconcentration of Metals by Chlorella

The changes in the metal concentration in the medium when each
heavy metal was administered alone are shown in Fig. 9. Figure 9(A)
and (B) show the concentration of Cd and Cr, respectively. The arrows
marked beside the Y axis show the nominal concentrations.
The Cd concentration decreased for the first 24 h after exposure.
It was thought that Cd was absorbed or adsorbed by Chlorella, or
adsorbed on the wall of the culture vessel. At the highest concentration
tested, the Cd concentration gradually increased after 24 h until the
end of exposure. It was assumed that Chlorella took Cd up to the
saturation point in the first 24 h, so cell density decreased during that
time and after that Cd concentration tended to increase a little. When
the initial Cd concentration was 10 peqiL, the concentration in the
medium tended to decrease and fell to about 70% of the initial concen-
tration at the end of the exposure experiment. At this concentration
 INTERACTIVE TOXIC EFFECT OF CD AND C R / 2 6 5

c o i i c e n l r u li o n
( IJeull 1
Cd, C r
I 0----0 , 50

w., -
g20 A-- 2 , 50
x--
2a’
~
I D . 50
2 0 , 50

I >>,

0 10 20
“ m e ( days)

iirne(duys)

Fig. 10. Changes in the concentration of heavy metals in the medium under the
presence of both metals (A) Cd and (B) Cr.

level, it was assumed that Chlorella did not take Cd to saturation from
the fact that cell density did not decrease.
In the recovery experiment, at the highest concentration tested,
Cd in the medium remained at 50% of the initial concentration even
on the 5th day of the experiment. At the end of the recovery experiment,
the Cd concentrations were almost 0.
In Fig. 9(B), the changes in Cr concentration in the medium are
shown when Cr was used alone. The Cr concentration was relatively
stable in the exposure experiment in comparison with Cd. The changes
in the concentrations of both heavy metals in the medium are shown
in Fig. 10. The decreasing rate of the Cd concentration in Fig. 10(A),
which was found for several days after exposure, was larger than that
when Cd was used alone. This was independent of the initial Cd concen-
tration tested. From Fig. 10(B),we can see that the Cr concentration
tended to decrease for 10 days except for one case. As the concentrations
of both metals continued to decrease until the second day, one metal
seemed to increase in taking up of the other metal by Chtorella at the
beginning of the exposure experiment.
The amounts of each heavy metal in the algal cells at the end of
the exposure experiment were determined and are shown in Table I.
The amount of Cd taken up in the cells increased as the Cd concentra-
266iAOYAMA AND OKAMURA

TABLE I
Concentrations of Cd and Cr in a cell a t the end
of exposure
Cd (fgicell)
Concentration (peq/L) in medium
Combination
of chemicals 0 2 10 20
Cd alone ii
51.1 192 518
Cd + Cr d
35.8 187 428
Cr (fgicell)
Concentration (peqiL) in medium
Combination
of chemicals 0 10 20 50
Cr alone 26.5 42.6 74.6 80.0
Cd + Cr 65.8 310 1810 2900

* Less than 10 fgicell.

tion in the medium increased, but even if Cr was added to the medium
containing Cd, the amount of Cd in the cell remained almost the same.
The amount of Cr taken up in the cell increased with the Cr concentra-
tion in the medium, but it was a t most 80 fg/cell(l fg = 10 l5 g), while
~

that amount increased and became 35 times higher when Cd was added
to the medium containing Cr.
Bioconcentration factors (BCFs) were calculated using these data.
These are shown in Table 11. The BCFs of Cd were about 10,000 in all
the cases tested in these experiments, regardless of the presence or
absence of Cr. The BCFs of Cr decreased with the Cr concentration in
the medium. However, in the presence of Cd, the BCFs of Cr increased
significantly, reaching about 156,000 a t the point of the highest concen-
tration of Cd in the medium. It was clearly shown that the bioconcentra-
tion of Cr by Chlorella was stimulated by the presence of Cd.

Relationships Between G r o w t h Inhibition

and Bioconcentration
The relationships between growth inhibition and the bioconcentration
of each heavy metal by Chlorella are shown in Fig. 11.Both the amount
of Cd taken up in the cell and the inhibition rates caused by Cd used
alone increased with increases in the amount of Cd in the medium.
When Cr was added to the medium containing Cd, the inhibition rates
rose drastically, but the amount of Cd in the cell remained almost the
 INTERACTIVE TOXIC EFFECT OF CD AND C ~ / 2 6 7

TABLE I1
Bioconcentration factors of Cd and Cr a t the end
of exposure
Cd
Concentration (peq/L) in medium
Combination
of chemicals 0 2 10 20
Cd alone NC" 11900 9090 9000
Cd + Cr NC 8960 7900 8700
Cr
Concentration (peqiL) in medium
Combination
of chemicals 0 10 20 50
Cr alone NC 17500 12000 2860
Cd + Cr 2460 13800 73500 156000
a NC: Not calculated

same. The amount of Cr in the cells exposed only to Cr was low, while
the inhibition rates were about 20%. When Cd was added to the medium
containing Cr, both the amount of Cr and the growth inhibition rates
increased for all the concentrations tested.
It was found that growth inhibition was caused by the fact that
the bioconcentration of Cr by Chlorella was stimulated by the presence
of Cd. On the other hand, the bioconcentrat,ion of Cd by Chlorella was
not affected by the presence of Cr.

1
C
0
.-
.-
c

.-n medium

10 100 1000 10000

amount of h e a v y metal ( f g l c e l l )
Fig, 11. Relationship between percent inhibition and the concentration of heavy
metals in a cell.
268iAOYAMA AND OKAMURA

CONCLUSIONS
1. The toxicity of Cd was detected with greater sensitivity when
the dilution rate, that is, the relative growth rate was lower in continu-
ous algal culture.
2. Both the amount of Cd taken up in the cell and the inhibition
rates caused by Cd increased as the Cd concentration in the medium
became higher.
3. The inhibition rates caused by Cd also increased with the Cd
concentration in the medium but the amount of Cd in the cell remained
at almost the same level even if Cr was added.
4. The amount of Cr taken up in the cell was relatively small.
When Cd was added, both the Cr uptake and the inhibition rates in-
creased for all the concentrations tested.
5 . It seems that the toxicity of Cr was enhanced because the pres-
ence of Cd in the medium raised the amount of Cr in the cell.
6. In environmental monitoring, it is assumed that Cd and Cr
would affect each other in terms of bioconcentration and toxicity, and
that the intensity of toxicity would be affected more by the concentra-
tion in the cells than by that in the water. Therefore, the Cr concentra-
tion as well as the Cd concentration in the biota should be determined
in order to evaluate the toxic effect.

This study was partly supported by a grant provided by Nissan Science Founda-
tion, which we wish to gratefully acknowledge.

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