Journal of the Indian Fisheries Association

20, 1990, 43-50

TOXICITY OF HEAVY METALS TO BRINE SHRIMP ARTEMIA

S.N. GAJBHIYE AND R. HIROTA*

National Institute of Oceanography, Regional Centre, Seven Bungalows,
Versova, Bombay- 400 061

ABSTRACT
Bioassay were carried out on 48 h cultured nauplii of brine shrimp Artemiti by exposing
them to seven trace elements viz. copper (Cu), zinc (Zn), cadmium (Cd), nickel (Ni), lead (Pb),
iron (Fe) and manganese (Mn). Synergistic effects of all these elements and additive effects
of Cu and Zn, Cd and Pb, and Ni and Fe were also investigated. Comparatively, the degree of
toxicity for compound bioassays was higher than individual simple tests. The values
were averaged and the expected median lethal cor.centration (LC 5J of tested heavy metals
was obtained by probit analysis on the basis of cumulative numbers of dead organisms
after 24 and 48 h. The order of toxicity of the metals to Artemia was Pb > Cd > Cu > Ni
> Zn > Fe > Mn. Potency ratios of the seven metals were also calculated. The 24 and 48
h variations obtained in LC50 values were significantly different and relative implications of
these are discussed.

INTRODUCTION
Due to the persistence and toxic nature of
the metals, knowledge on the toxicity of
different metals is important for evaluating
tolerable levels of the metal to various
types of biological components. The discharge
of these elements into the rivers, estuaries
and coastal waters poses serious pollution
problems and consequently affects the flora
and fauna. To predict the ecological effects
of heavy metals, various kinds of organisms
have been recommended and among them
fishes are widely considered as suitable .for
bioassay. However, studies by Bryan (1971),
Connor (1972), Saliba & Ahsanullah (1973),
Ahsanullah & Arnot (1978), and Ahsanullah
(1982) reveal that invertebrate larvae are
usually more sensitive to heavy metal
contamination. These larva~ and other juveniles
have an important role in the dietary regime
of cultivable fishes and shellfishes. Existing
information (Hudinaga & Miyamura, 1962;
Riley, 1966; Wickins, 1972 and Sorgeloos, 1980)
suggest that only a few species are utilized
as good live feed organisms andamong them
the brine shrimp Artemia comes in for prime
consideration. Like other organisms, Artemia
also accumulate the trace elements and
subsequently transfer them to higher levels.
*Aitsu Marine Biological station, Kumamoto University Amakusa-Matsushima, Kumamoto 861.6l'Japan.
Therefore, it is necessary to know the
relationship between Artemia and its tolerance
range for various heavy metals, so that the
cultures can be used more safely and
effectively. In recent years, a variety (?f
Arteniia culture methods have been
developed. However, informations on the
toxicity level of metals on Artemia are limited
(Corner & Sparrow, 1956; Brown &
Ahsanullah, 1971; Saliba & Ahsanullah, 1973;
Trieft 1980 and Kissa et al., 1984). Therefore,
an attempt has been made to investigate the
impact of Cu; Zn, Ni, Cd, Pb, Fe and Mn on
this animal. Tests were also conducted to
determine the synergistic effects of Cu and
Zn, Cd and Pb, Ni and Fe, and combined
toxicity of all these metals on Artemia.
MATERIALAND METHODS
The cysts of Artemia (San Francisco Bay
Strain) were incubated in filtered sea water.
The water temperature was maintained at
28 1C throughout the incubation period.
Instant nauplii were used for bioassay.
Bioassay. for 50% survival of Artemia
nauplii for . 24 and 48 h exposures were
conducted using standard methods of static

43
44 GAJBHIYE AND HIROTA

bioassay tests (APHA, 1971; FAO, 1977). 4.0 ppm Cd concentrations within 24 h. The
Batches of 20 Artemia nauplii were placed in concentration range selected for Ni was 1.0 to
a beaker of 250 ml capacity. Before the 40.0 ppm and the observed mortality rate
introduction of the animals, each beaker was varied from 0 to 100%. Experiments with Pb
dosed with toxicant to get the desired were conducted in the range of 0.5 to 4.0 ppm
concentrations. Observations on the state of concentrations and at both 24 and 48 h tests,
specimens in the beaker were made at regular 100% mortality was observed in 2.5 ppm
intervals of3 h. The organism was considered _concentration. The toxicity experiments with
dead when a nauplius failed to respond to Fe conducted in the range of 1.0 to 60.0 ppm.
mechanical stimulation. Each experiment was 100% mortality for Fe was noticed at and
run in triplicate with control to monitor the above 30 ppm solutions. Experiments with Mn
death of animals. Experiments were were also carried out within a very wide
repeated twice with regular aeration. Mean range of 4.0 to 100 ppm concentrations and
values of the experiments (triplicate repeated highest concentration that did not result in
twice) were considered for statistical mortality was 8 ppm.
analysis (FAO, 1977).
While conducting compound tests equal
The inorganic metal salts used were proportions of metals were added. The
cupric chloride (Cuclz), zinc nitrate (ZnN0 3), synergestic effect of Cd and Pb indicated
cadmium chloride (CdClz), nickel chloride that mortality rate increased significantly at
(NiCiz), lead nitrate (Pb(No3)z), ferric chloride and above 1 ppm (Table I; Fig, 2). For Cu and
(FeC13) and manganese chloride (MnCiz)~ Zn no mortality was observed upto 12 h of
exposure at concentrations below 5 ppm.
The chemical properties of test water
The com pound toxicity of Fe and Ni showed
were as follows. The pH levels in all test
at 10 ppm and above that nauplius could not
containers with metal salts remained between
survive for more than an hour. The combined
7.5 to 8.4. Ranges for salinity and DO were
effect of all seven metals indicated 100%
from 32.94 to 33.53%o and 4.66 to 5.10 mVl
mortality at and beyond 1 ppm within an hour.
respectively.
There was marginal difference in mortality
Statistical analysis of the data was done rate between 24and48hexperimental period
by taking into consideration the mean values. for the additive effect of all the metals.
On the basis of the cumulative number of
dead specimens after 24 and 48 h exposures, The established order of toxicity of
the expected median lethal concentration common trace elements based on the
(LC5J of tested heavy metals was obtained available information (GMP/UNESCO, 1977)
by probit analysis. w-ith respect to the most sensitive life stages
RESULTS AND DISCUSSION is as follows, Cu > Zn > Ni > Pb > Cd >Fe> Mn.
However, in the present investigation the
Cu was tested in the range of 0.1 to 20 degree of toxicity of the metals to Artemia
ppm ( Table I; Fig. 1). The highest was in the order ofPb > Cd > Cu > Ni > Zn >
concentration of Cu that did not result in Fe > Mn. It also differs significantly with
mortality was 0.5 ppm and the 100% rnortality already reported values for Artemia (Brown
was observed at 20 1Jor.n concentration. and Ahsanullah, 1971) namely Hg > Cu > Cd >
" '
Experiments with Zn were conducted -at Fe > Zn > Pb. The striking difference was
concentrations ranging from 0.5 to 30 ppm. found for lead as it was reported less toxic,
Exposure at 0.5 and 1.0 ppm Zn concentrations whereas in the present study it was found to
did not show any mortality during 24 h. be the most toxic except cadmium. The
Toxicity experiment with Cd was conducted difference in the result could be due to
in a narrow range of 0.5 to 4.0 ppm. Even different types of experimental conditions,
100% mortality was observed in 2.5, 3.0 and strain of eggs and objective of the bioassay
 TOXICITY OF HEAVY METALS TO BRINE SHRIMP ARTEM!A 45
COPPER

~~~a
10[
E
1/)
;;:
~6
0
0

10 15
CADMIUM NICKEL

LEAD IRON

20 30
Concentration
MANGANESE

I
I 0
I
I 0 0
I
I
~
I

20 40 60 80 100
Concentration (ppm)
Fig. 1. LC50 of different heavy metals toArtemia nauplius. A-LC50 for 24 hours exposure, B-LC50 for 48 h
exposure, 0-mortality rate for 24 h exposure and X- mortality rate for 48 h exposure.
46 GAJBHIYE AND HIROTA

Zinc, LII!(Jd, Cadmium, Nickel, }ron, MoogoMS~e

O)(

Ill
E A 8
Ill ll
c
0

----~-------------~
0'>
....
0

0
~ ,,
~:
0
z )(
I
I I
0)(0 0
I I
___ __j
I I I 1 II
3.0 ~5 lO 1.5 2.0 2.5

Copper, Zinc Iron, Nickel

A
A
101 Ol(

oil

~
".
.....
0
"0
..,0
o
0
0
z
0)(
,. I

5 10 15 20 2S
Concentration (ppm) Con<eotration (ppm)

Fig. 2. LC50 of different metals (compound bioassay test) to Artemia nauplius. A-LC50 for 24 h exposure,
B-LC50 for 48 h exposure, 0-mortality rate for 24 h exposure and X- mortality rate for 48 h exposure.

studies. The pres~nt investigation was
carried out at slightly higher temperature
(maintained 28 1C) compared to earlier
studies ( Saliba & Ansanullah, 1973
and Ahsanullah & Arnott, 1978).
In general, the results obtained in the
present study reflect that the nauplii of Artemia
were relativdy resistant to heavy metals and
could tolerate wider ranges. Comer & Sparrow
(1956) and Kissa et al. (1984) found that the
larvae of the brine shrimp Artemia were very
resistant to heavy metal toxicity which is in
agreement with the present observation.
However, all tested metals exhibit toxicity to
Artemia at varying degrees, a few were relatively
more toxic and some others moderately toxic.
Earlier studies reveal that the toxicity of Cd to
marine animals varies with compounds, species
and stages of development. Reported values
(GESAMP, 1976) were in .the range of
0.1-100 j.!g/1. The estimated 24 and 48 h LC 50
values ofCd (1.7 and 1.5 ppm) and Pb (1.7 and
1.4 ppm) show that these elements substantially
affect the survival of Artemia nauplii. Potency
ratios of the seven metals tested (Table II)
reflect the intensity of the toxic nature of one
metal with other metals. The ratios ofPb and Cd
 TOXICITY OF III:AVY Ml:TALS TO URI!\1'. SIIRIMP ARTEMIA 47

Table I The LC50 l'alues of heavy metals (ppm) to brine shrimp estimated from prohit analysis

Metal 24 h LCSO (SO) 48 h LCSO (SO)

Copper 9.5 0.006 4.0 0.042

Zinc 17.8 0.007 12.3 0.020
Cadmium 1.7 0.009 1.5 0.021
Nickel 15.6 0.036 11.2 0.033
Lead 1.7 0.011 1.4 0.015
Iron 18.2 0.022 13.9 0.020
Manganese 75.0 0.023 51.8 0.015
Cadmium & Lead 0.5 0.036 0.3 0.069
Copper & Zinc 5.6 0.033 3.4 0.074
Iron & Nickel 5.6 0.035 3.4 0.074
All seven metals 0.6 0.036 0.5 0.029
SO : Standard deviation

with Cu, Zn, Ni, Fe and Mn vary from 2. 7 to 44.1
indicating that they were several times toxic
in nature than the rest. Cd and Pb are cumulative
poisonous to organisms and their susceptibility
vary greatly with individuals. As reported by
Ahsanullah (1976) Cd was found to be more
toxic to the adults of all marine invertabrates
than Zn. In the case of Pb the toxicity to
marine organisms was not known precisely
(Portmann, 1972). I Iowever, acute toxicity levels
(LC50 values upto 96 h) to marine animals
have been reported (GESAMP, 1976) to be
between 1 and 10 !lg/1.
In the present study the toxicity of Cu
and Zn was estimated as 9.5 and 4.0, and 17.8

Table II : Potency rates of heal')' metal based on 24 and 48 h LC50mlues

Name of metal Exposure time (h)

Ratio of Ratio with 24 48

Lead Cadmium 1.0 1.1 1

Copper 5.6 2.9 1
Nickel 9.2 1 8.0 1
Zinc 10.5 1 8.8 1
Iron 10.7 1 9.9 1
Manganese 44.1 37.0 1
Cadmium Copper 5.6 1 2.7 1
Nickel 9.2 1 7.5 1
Zinc 10.5 1 8.2 1
Iron 10.7 1 9.3 1
Manganese 44.1 1 34.5 1
Copper Nickel 1.6 1 2.8 1
Zinc 1.9 1 3.1 1
Iron 1.9 1 3.5 1
Manganese 7.9 1 13.0 1
Nickel Zinc 1.1 1.1 1
Iron 1.2 y1 1.2 1
Manganese 4.8 1 4.6 1
Zinc Iron 1.0 1 1.1 1
Manganese 4.2 1 4.2 1
Iron Manganese 4.1 1 3.7 1
48 GAJBHIYE
and 12.3 ppm respectively for 24 and 48 h
exposure (Table 1). They were likely to
increase against the exposure time. Cu and
Zn toxicity in the marine environment have
been studied by number of workers.
Accordingly they vary significantly between
species to species. But in most of the cases
(Raymont & Shields, 1964; Ahsanullah &
Arnott, 1978 and Arnott & Ahsanullah, 1979) it
was observed that Cu was more toxic than Zn.
Acute toxicity of Cu to marine animals was
usually reported to be the order of a few
ppm for adults of numerous species. During
the.present observation, the potency ratio
(Table II) of Cu with other metals shows
that it was 1.9 - 3.1 times more toxic than Zn,
1.6- 2.8 times toxic than Ni, 1.9- 3.5 times toxic
than Fe, and 7.9- 13.0 times toxic than Mn. For
Zn toxicity, as reported by Negilski (1976) in
static test with juvenile mullets, the acute
incipient lethal level of Zn was 9.0 mg/1. In
another study with Cu and Zn (Ahsanullah
& Arnott, 1978) lethal levels of 0.17 and 1.23
mg/1 were obtained respectively for larvae of
crab Paragrapsus guadridentalus.
The calculated 24 and 48 h LC50 levels for
Ni and Fe were 15.6 and 11.2, and 18.2 and
13.9ppm respectively (Tablell). However, in
the order of toxicity, Ni ranked fourth
and Fe ranked sixth. It was reported earlier
for Zooplankton (Gajbhiye, et al., 1985) that
Ni was less toxic than Cu. Fe was less toxic
thanPb, Cd, Cu,ZnandNi, butitwas4.1 and3.7
times more toxic than Mn. The acute toxicity
~f Ni to the am phi pod Allorchestes compressa
was found to be 5.56 mg/1 for 96 h
(Ahsanullah, 1982).
Regarding Mn, very little information was
available on the toxicity to marine organisms.
Mn toxicity to Artemia nauplii does not
appear to be high. The LC 50 values for 24
and 48 h exposure were 75.0 and 51.8 ppm
(Table I) reflecting that it was relatively a
nontoxic element. Both Fe and Mn were
considered comparatively innocuous to the
aquatic organisms (GMP/UNESCO, 1977). The
reported toxicity of Mn to the American
oyster Crassostrea virginica was 16.0 ppm for
AND HIROTA
48 h duration (Calabrese et al. 1973).
As compared to simple individual toxicity
of the metals, the compound toxicity of Cu,
Zn, Cd, Pb, Ni, Fe and Mn appear to be very
acute. The estimated LC50 values for 24 and 48
h exposure were 0.6 and 0.5 ppm (Table 1). This
indicates that all these metals act on Artemia
nauplii in a different manner. It may be
due to the synergistic effect. This occurs
when the additive effect of two or more
metals act together which is greater than the
expected individual effect. In the case of
addilive effects of Hg and Cu, the observations
(Corner and Sparrow, 1956) have led to the
view that Cu and Hg act differently and the
changes induced by one poison are to
reinforce those caused by other.
Even for the other experiments, the
differences between combined toxicities of
Cu and Zn, Cd and Pb, and Fe and Ni were far
greater than those observed during simple
individual bioassays. The LC50 values for Cd
andPb,CuandZn, andNi and Fewere0.5and
0.3, 5.6 and 3.4, and 5.6 and 3.4 ppm
respectively for 24 and 48 h duration (Table I).
Such variability in toxicity of the same
metals indicate the additive effect of the
compounds enhancing the rate of toxicity.
Artemia is a filter feeder and has the
capability to control filteration (Reeve, 1963).
It is very likely that the heavy metals in
question may combine with sulfhydryl groups
on enzymes connected with the filtcration
system. The mechanism of toxicity
manifestation involves SH binding (Trieff,
1980). The binding probably occurs in the
filtering system or possibly on the pleopod or
external gills.
It is concluded that the nauplii of Artemia
possess a considerable resistance to heavy
metal toxicity. The estimated LC50 values for
24 and 48 h suggest that Pb and Cd were
relatively more toxic than Cu, Zn, Ni, Fe and
Mn. The order of toxicity takes the following
sequence : Pb > Cd > Cu > Ni > ~n > Fe > Mn.
The synergistic effect of these metals
 TOXICITI' OF HEAVY METALS TO BRINE SIIRIMP ARTEMIA
enhanced the rate ot toxicity which was
evident by the increased mortality in Artemia.
However, further detailed studies on the
compound toxicity and intake capacity are
needed for concrete predictions.
ACKNOWLEDGEMENTS
The authors are grateful to the Ministry of
Education of Japan and India for awarding
the fellowship and financial assistance for
this project. We are grateful to the Director,
National Institute of Oceanography, Goa for
allowing us to undertake this study. Thanks
are due to Mr. & Mrs. Shimasaki and Mr.
Uranaka of Aitsu Marine Biological Station
for assistance in the laboratory.
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