Cr and Hg Toxicity Assessed In Situ Using
the Structural and Functional
Characteristics of Algal Communities
A. K. SINGH and L. C. RAI
Laboratory of Algal Biology,
Centre of Advanced Study in Botany,
Banaras Hindu University,
Varanasi, 221005, India
ABSTRACT
The toxicity of mercury and chromium on algal community structure have been assessed
using in situ N,ase activity, pigment diversity, autotrophic index, and I4C uptake of
algae. The location was in the river Ganga and controlled ecosystem pollution experiment
enclosures were used. Maximum inhibition of algal number was observed a t 0.8 pg Hg
mL-' followed by 8.0 p g Cr mL-l. Unicellular forms, except for Anorthoneis excentrica,
were very sensitive to test metals used. The decline in algal number was concentration
dependent and metal specific at generic and species levels. Complete elimination of three
and six species was observed respectively at 8.0 p g Cr mL-' and 0.8 pg Hg mL-' after
12 days' exposure. Likewise, a concentration-dependent and metal-specific increase in
autotrophic index and pigment diversity of phytoplankton was recorded for Hg and Cr.
Inhibition of '"C uptake of phytoplankton in Ganga water was almost equal (79%) at 0.8
p g Hg mL-' and 8.0 pg Cr mL-' (78%). Although complete inhibition of in situ Nzase
was observed a t 0.8 pg Hg mL-I, it was only 80%with 8.0 p g Cr mL-'. Our study suggests
that heavy metals inhibit both structural and functional variables of phytoplankton in
field microcosms. Hence this technique seems to hold potential for the biomonitoring of
heavy metal toxicity in the field.
INTRODUCTION
Of the various freshwater bodies and rivers in India, the river Ganga
occupies an unique position. It is the life line for the Indian population
and drains an area of 861,404 km2, accounting for over 40% of the
irrigated land and sustains 37% of the country's population. Heavy
metals are among the more dangerous substances that deteriorate the
water quality (Rai et al., 1981a; Mathur et al., 1987).
Algae are a valuable tool for bioassay of metal toxicity. But most
Environmental Toxicology and Water Quality: An International Journal
Vol. 6, 97-107 (1991)
CCC 1053-4725/91/01097-011$04.00
0 1991 John Wiley & Sons, Inc.
98ISINGH AND RAI
studies dealing with heavy metal toxicity to algae are confined t o the
laboratory microcosm under a defined set of culture media and environmental conditions (Whitton, 1984; Rai and Raizada, 19891, using one
species of algae and a single metal or bimetallic combinations. Although laboratory test data do give valuable information about toxicity,
they cannot be used directly to monitor changes in the algal communities of aquatic system in the field.
Use of controlled ecosystem pollution experiment enclosures
(CEPEX) have proved to be a valuable advance in metal toxicity bioassays of ecosystems level (Cairns, 1983; Taub, 1984; Wong and Trevors,
1988). Some important advantages of multispecies tests are that they
can incorporate some of the emergent properties of communities or
ecosystems, and serve as an intermediate step between the simplicity
of single species toxicity tests and the unreproducible complexity of the
environment. Thus, it is believed hazard evaluation may be improved
significantly by the use of multispecies toxicity tests to compliment
single-species test data (ONeill and Waide, 1981; Cairns, 1983, 1985).
Surprisingly, this technique has primarily been used only in the oceanic
ecosystems of the United States, Canada, etc. (see Shubert, 1984). Recognizing the suitability of this method for toxicity monitoring, we have
used this technique t o assess Cr and Hg toxicity by employing structural and functional characteristics of the algae of the river Ganga in
India. The following parameters were measured: (a) changes in algal
community at different concentrations of Cr and Hg, (b) autotrophic
index, (c) pigment diversity, (d) in situ nitrogen fixation, and (e) 14C
uptake of phytoplankton.
MATERIALS AND METHODS
Water-tight glass CEPEX chambers (120 x 60 x 45 cm) with the
support of an iron angle framework were made. Such enclosures were
placed in the river water approximately 7 m away from the bank.
Chambers filled with Ganga water were kept submerged by erecting
parallel and horizontal bamboo poles in the flowing water in such a
way that the edge of the chambers was always 15 cm above the water
surface. Keeping one chamber as a control, the others were spiked with
different doses of Cr (4.0 and 8.0 pg mL-l) and Hg (0.4and 0.8 pg
mL-'). Algae and water samples were taken both from the control and
experimental chambers every 3 days, and were analyzed with respect
t o the following characteristics: qualitative and quantitative changes
in algae, pigment diversity, autotrophic index (AI), 14Cuptake, and in
situ N,ase activity.
Cr AND Hg TOXICITY ASSESSED IN SZTU/99
Structural Aspects
A sample of water (2 L) was passed through 0.45-pm filter paper (47-mm
diameter) by applying 30.3 kPa pressure. The algae so obtained were
counted with a hemocytometer and expressed as the number ofindividuals L-I. Systematic analysis was done using standard taxonomic keys
(e.g., Hustedt, 1930; Desikachary, 1959; and Randhawa, 1959).
Functional Aspects
For 14C uptake, a 500-mL water sample was filtered and algae transferred into scintillation vials containing 1.0 mL NaHI4CO, (specific
activity 18.5 x lo5 Bq, pH 9.5) and incubated for 2 h at 25 2°C and
12.0 Wm-' light intensity. The I4C uptake by the algal suspension was
stopped periodically by adding 0.2 mL 50% acetic acid. Then 5.0 mL
of scintillation cocktail was added and the resulting suspension was
bubbled with air for 5 min. Counting was done in a Beckman liquid
scintillation counter and the rate of I4C uptake was expressed in CPM
(counts min- '). Part of the phytoplankton sample collected by filtration
was used to determine pigment diversity. The latter was calculated as
the ratio of carotenoid to chlorophyll a (Margalef, 1958). The autotrophic index was calculated by measuring the planktonic biomass (dry
weight) as well as chlorophyll a from a known amount of sample (Weber
and McFarland, 1969).
*
(AI) =
Biomass (dry wt) organic matter
Chlorophyll a
In situ nitrogen fixation was measured using specially fabricated
equipment fitted with 6 bottles of 275 mL capacity each illuminated
with tube light (12.0 Wm-' light intensity) and cooled by continuously
flowing river water. This assembly was kept rotating continuously. Into
each of the 6 bottles containing 250 mL water sample, 10%of acetylene
was injected from an airtight syringe. Then each bottle was sealed by
a n airtight stopper and parafilm. After exposure for 1h, the reaction
was terminated by injection of 50% (w/v) trichloroacetic acid (Riddolls,
1985). The nitrogenase activity was measured by the acetyleneethylene assay method (Stewart et al., 1968) after bringing the bottles
to the laboratory.
RESULTS
Structural Aspects
A reduction in the number of algal genera and species following Cr and
Hg treatment (Tables I and 11) began after their addition to the CEPEX
100/SINGH AND RAI
TABLE I
Changes in algal genera and species after 12 days' exposure to different concentrations
of chromium in CEPEX chamber
Algal cells or filaments or colonies (L-')
4.0 wg mL-'
Name of algae
Chlorophyceae
Chlorella sp.
Coelastrum lanceolatum
Hormidium sp.
Microspora sp.
Pediastrum simplex
Scenedesmum bicaudatus
Cyanophyceae
Anabaena sp.
Merismopedia minima
Nostoc linckia
Oscillatoria formosa
Oscillatoria sp.
Spirulina sp.
Bacillariophyceae
Anorthoneis excentrica
Cylindrotheca sp.
Fragilaria sp.
Melosira granulata
Control
nos. lo4
23.6
0.6
0.1
4.4
13.1
2.6
2.7
27.9
5.7
4.1
0.1
12.8
5.0
0.2
24.5
1.1
11.2
10.1
2.1
Nos.
lo4
12.2
0.2
0.05
3.1
6.0
1.4
1.3
15.1
2.8
1.5
0.1
6.9
3.8
0.1
11.1
0.9
5.8
4.0
0.4
Percent
inhibition
48
63
57
29
54
45
51
46
51
63
57
46
23
43
54
19
48
60
79
8.0 fig mL-'
Nos.
lo4
6.8
0.1
0
1.7
3.8
0.8
0.3
10.1
2.1
0.7
00
5.8
2.2
0.1
9.1
0.7
4.8
3.5
0
Percent
inhibition
71
80
100
60
70
69
90
61
63
82
100
55
55
53
63
37
57
65
100
chambers. This inhibition was very pronounced until day 12. The decline in algal number was concentration dependent and metal specific.
Species-specific sensitivity to Hg and Cr was also observed during the
present investigation. Maximum inhibition of algal number occurred at
0.8 pg HgmL-I followed by 8.0 pg Cr mL-'. The sensitivity hierarchy of
algae for Cr is Chlorophyceae > Bacillariophyceae > Cyanophyceae,
and for Hg it is Cyanophyceae > Bacillariophyceae > Chlorophyceae.
Filamentous forms such as Microspora sp., Hormidium sp., Oscillatoria
formosa, Oscillatoria sp., Spirulina sp., and Zygnema sp. showed more
tolerance toward Hg and Cr than the unicellular forms such as Pediastrum simplex, Chlorella vulgaris, and Gyrosigma sp. A complete elimination of Anorthoneis excentrica at 0.4 pg Hg mL-l suggested its extreme sensitivity to Hg. Sensitivity increased as follows: P. simplex
followed by Pediastrum sp. and Gyrosigma. Nostoc linckia and Melosira
granulata were the most sensitive to Cr. However, algae resistant to
Cr showed the following order of increasing tolerance: A. excentrica
Cr AND Hg TOXICITY ASSESSED IN SZTUI101
TABLE I1
Changes in algal genera and species after 12 days' exposure to different concentrations
of mercury in CEPEX chamber
~~
Algal cells or filaments or colonies (L-')
0.4 p g mL-l
Name of algae
Chlorophyceae
Chlorella vulgaris
Coelastrum lanceolatum
Microspora sp.
Pediastrum simplex
Pediastrum sp.
Scenedesmum sp.
Spirogyra sp.
Ulothrix sp.
Zygnema sp.
Cyanophyceae
Anacystis sp.
Anabaena sp.
Lyngbya sp.
Merismopedin minima
Oscillatorin formosa
Phormidium sp.
Spirulina sp.
Bacillariophyceae
Anorthoneis excentrica
Cylindrotheca sp.
Gyrosigma sp.
Nitzschia sp.
Fragilaria sp.
Control
nos. lo4 Nos.
26.5
1.7
0.03
11.9
2.1
1.0
1.6
4.0
3.3
1.3
31.2
0.8
9.6
3.3
4.6
13.2
0.3
0.3
38.6
1.8
8.6
10.2
6.0
11.9
lo4
11.4
0.6
0.01
5.5
0.6
0.4
0.4
1.6
1.8
0.6
11.5
0.2
2.9
1.5
0.8
5.8
0.1
0.1
13.8
0
4.0
1.8
2.9
5.0
Percent
inhibition
57
63
77
54
70
59
76
60
45
51
63
69
70
55
82
56
60
76
64
100
54
82
51
58
0.8 p g mL-'
Percent
Nos. lo4 inhibition
4.5
0.3
00
2.8
0
0
0
0.4
0.8
0.2
4.2
0
0.4
0.9
0
2.8
0.07
0.02
5.3
0
1.6
0.4
1.2
2.0
83
83
100
76
100
100
100
89
75
84
86
100
96
73
100
79
75
94
86
100
81
95
79
83
followed by Spirulinu sp., Oscillatoria formosa and Hormidium. Lyngbya sp. followed by Ulothrix sp., and Microspora sp. were found most
resistant to Hg.
Although the number of individuals per species declined, no change
in the species number (16 originals) following Cr (4.0 pg mL-') treatment was noticed (Table I). A decrease of approximately 48 and 64%in
algal population following exposure to 4.0 and 8.0 pg Cr mL-' was
observed after 12 days. A time-dependent decrease in algal number
following exposure to Hg was observed (results not shown). Of 21 species
present in untreated control only 14 species could survive in the CEPEX
chambers treated with 0.8 pg Hg mL-' after 12 days of exposure (Table
11).
LOzISINGH AND RAI
TABLE I11
Effect of different concentrations of Cr and Hg on in situ 14C uptakea
Concentration
( p g mL-')
0h
Control
Chromium
4.0
8.0
Mercury
0.4
0.8
14C02uptake x lo3 counts min-'
0.5 h
% Inhibition
1h
% Inhibition
2h
% Inhibition
0.1
0.1
28.9
18.7
35.2
48.7
31.7
35.0
54.2
34.1
37.1
0.1
11.3
60.9
13.7
71.8
12.0
77.8
0.1
0.1
13.6
7.3
52.6
74.6
26.5
11.3
45.5
76.8
28.7
11.4
46.9
79.0
-
a Analysis of variance (ANOVA):Ftlrnez,8
= 8.24,p < 0.025; Ftreatrnent4,8 = 21.42, p <
0.001.
Functional Aspects
The analysis of variance of the results of I4C uptake by phytoplankton
of Ganga water showed that the variation was more significant in
respect t o treatment ( p < 0.001) than time (Table 3). A concentrationdependent inhibition of 14C uptake was recorded both for Hg and Cr.
Approximately 79, 47, 77, and 37% inhibition of 14C uptake was observed respectively with 0.8 and 0.4 p g Hg mL-' and 8.0 and 4.0 p g Cr
mL-l. A high value of the A1 was recorded with 0.8 pg Hg mL-l and
8.0 pg Cr mL-l after exposure for 15 days. A1 depicted a reverse trend
to that found with nitrogenase activity, i.e., a concentration-dependent
increase in the A1 value was found both for Cr and Hg. Although there
was increase in the A1 value from the beginning of the experiment
(Table IV), a statistically significant increase was observed only after
6 days when the values had become maximal for both of the test metals.
The statistical tests showed that the variation was highly significant
both for time and treatment ( p < 0.001).
The effect of Cr and Hg on pigment diversity of the algal plankton
of the river Ganga also followed the pattern of A1 for both of the metals
(Table V). An increase in pigment diversity showed a dependence on
metal concentration and duration of exposure, and the variation was
highly significant both for time and treatment. It did not show a sudden
increase as observed for A1 values. Highest pigment diversity was found
with 0.8 p g Hg mL-l and 8.0 p g Cr mL-'. A statistically significant
increase in pigment diversity was recorded in the control CEPEX chamber with increasing exposure time.
The effect of Cr and Hg on the in situ nitrogen-fixing potential of
Cr AND Hg TOXICITY ASSESSED IN SZTV/l03
TABLE IV
Effect of different concentrations of Cr and Hg on algal autotrophic index using
in situ enclosuresa
Number of days
Concentration
(pg mL-'1
Control
Chromium
4.0
8.0
Mercury
0.4
0.8
0
3
6
9
12
15
12.9
12.9
12.9
12.9
12.9
12.0
14.0
16.0
17.8
19.9
12.9
18.0
20.2
20.3
28.3
12.9
28.1
31.8
36.6
42.0
13.0
48.4
48.5
58.5
65.7
13.1
50.0
60.4
62.7
66.0
"ANOVA:Ftlme4,16 = 1 3 . 3 6 , <
~ 0.001; Ftlme4,16 = 1 0 . 3 1 , <
~ 0.001.
Ganga water demonstrated (Fig. 1)a concentration-dependent decrease
in the nitrogenase activity of the phytoplankton. N,ase activity was
completely inhibited at 0.8 pg Hg mL-l. Approximately an 80% reduction of N,ase activity was observed at 8.0 pg Cr mL-'. However, about
55 and 33% in situ N,ase activity was observed, respectively, at 4.0 pg
Cr mL-' and 0.4 pg Hg mL-'. Thus Hg proved more toxic than chromium against in situ N,ase activity of planktonic communities of Ganga
water.
0
0.8
2
4
6
CONCENTRATION ( p g rn1-l)
8
Fig. 1. Effect of different concentrations of chromium and mercury on nitrogenase
activity. Cr &-A) and Hg (0-0).
104/SINGHAND RAI
TABLE V
Effect of different concentrations of Cr and Hg on algal pigment diversitya
Carotenoid: chlorophyll a ratio
Number of days
Concentration
( y g mL-')
Control
Chromium
Mercury
0
4.0
8.0
0.4
0.8
3
6
9
12
15
0.51
0.50
0.51
0.51
0.51
0.51
0.64
0.94
0.98
0.88
0.51
0.78
0.98
0.92
1.20
0.54
0.85
1.06
1.06
1.26
0.52
0.92
1.20
1.20
1.48
0.53
1.02
1.40
1.40
1.50
DISCUSSION
Pollution generally brings about a reduction in species diversity and
induces changes in the physiology affecting such processes as cell division, growth and production of extracellular substances all of which
may bring about changes in community structure (Cairns, 1985). The
change in species composition is toward selection of more tolerant species. Sanders et aZ. (1981)postulated that tolerant species not dominant
in the natural assemblage are able to compete successfully with the
usual dominant species only when those species are stressed.
The data presented in Tables I and I1 on variation in response
of algal genera and species t o Cr and Hg are interesting and complex.
Since all the experimental conditions were similar, any difference in
the sensitivity of algae could possibly be due to differences in the
composition of their cells. A general tolerance of filamentous forms
like Microspora sp., Hormidium sp., 0. formosa, Oscillatoria sp.,
Spirulina sp., and Zygnema over unicellular forms, and the sensitivity
of Anorthoneis excentrica to Hg and resistance to Cr, suggest that
the responses were not only metal specific but species specific also.
Therefore, our study not only supports the laboratory-based findings
of Starodub et al. (1988) and Rai and Raizada (1989), but goes one
step further to demonstrate the applicability of such studies at the
field level.
The ratio of carotenoid to chlorophyll a has been used as a
reliable tool for monitoring pollution conditions in an aquatic ecosystem (Margalef, 1958). Thus an increase in pigment diversity (carotenoid/chlorophyll a ) at increasing concentrations of Hg and Cr may
be due to the inhibition of chlorophyll a synthesis and/or an increase
Cr AND Hg TOXICITY ASSESSED IN SITUI105
in carotenoid content, and therefore an overall increase in the ratio
(Rai et al., 1981b).
An increase in A1 as observed in the CEPEX enclosures with increasing concentrations of Hg and Cr suggests that these metals have
an inhibitory effect on photosynthetic pigments of planktonic algae. An
increase in A1 value may be due to inhibition of chlorophyll contents
(De Filippis and Pallaghy, 1976; Rai et al., 1981b) in Chlorella vulgaris
due to metal toxicity.
Metals may be toxic to nitrogenase in many different ways: (a)
there can be direct action on the enzyme complex, or (b) there may
be an effect on the supply of ATP or the reductant pool, which are
prerequisites for activity of the nitrogenase enzyme. Since photosynthesis is the main source of ATP and reductant, inhibition of this process
may reduce ATP content and reductant. Even so, a high-level inhibition
of 14Cuptake by test metals seems to have a direct effect on nitrogenase
activity. Although metal-induced inhibition of carbon fixation followed
the trend of nitrogenase, the level of inhibition was higher for carbon
fixation than nitrogenase. Our observations agree well with those of
Blinn et al. (1977), where inhibition of phytoplankton productivity
following Hg treatment in CEPEX chambers in Lake Arizona was
found. Inhibition of carbon fixation in marine algae as a result of metal
exposure is well known (see Rai et al., 1981a; Whitton, 1984). Thus the
results of structural and functional characteristics together not only
attest to the suitability of laboratory data, but extend and recommend
further that these characteristics can be used for toxicity assessment
in field microcosms. However, extensive studies involving different
freshwater habitats are required before recommending its use in toxicity assessment.
CONCLUSION
The toxicity of Cr and Hg on community structure, pigment diversity,
autotrophic index, in sztu 14Cuptake, and nitrogenase activity of phytoplankton of the river Ganga using CEPEX enclosures has been studied.
A concentration-dependent inhibition of all the variables was observed.
Filamentous algae were found to be more tolerant than unicellular
forms. The results of this study attest to the suitability of this approach
for field assessment of metal toxicity.
Our thanks are due to the Head and Programme Coordinator of CAS in Botany
for facilities. This work was supported by a grant from the Department of
106/SINGH AND RAI
Environment and Forests Ganga Project Directorate, Government of India and
the University Grants Commission, New Delhi, in the form of Career Award to
L.C. Rai.
References
Blinn, D.W., T. Tompkins, and L. Zaleski. 1977. Mercury inhibition on primary productivity using large volume plastic chambers in situ. J. Phycol. 13:58-61.
Cairns, J. Jr. 1983. Are single species toxicity tests alone adequate for estimating environmental hazard? Hydrobiol. 17:1363-1374.
Cairns, J. Jr. 1985. Multispecies Toxicity Testing. Pergamon Press, Oxford.
De Filippis, L.F., and C.K. Pallaghy. 1976. The effect of sublethal concentrations of
mercury and zinc on Chlorella. I. Growth characteristics and uptake of metals. Zeit.
Pflanzenphysiol. 78:197-207.
Desikachary, T.V. 1959. Cyanophyta. Indian Council of Agricultural Research, New
Delhi.
Hustedt, F. 1930. Bacillariophyta. Heft 10. In A. Pascher (ed.), Die Susswasser-flora
Mitteluropas, G. Fischer, Jena.
Margalef, R. 1958. Information theory. Ecol. Gen. System. 3:36-71.
Mathur, A., Y.C. Sharma, D.C. Rupainwar, B.C. Murthy, and S. Chandra. 1987. A study
of river Ganga a t Varanasi with special emphasis on heavy metal pollution. Pollut.
Res. 6:37-44.
ONeill, R.V., and J.B. Waide. 1981. Ecosystem theory and the unexpected implications
for environmental toxicology, P. 43-73. In B.W. Cornaby (Ed.), Management of Toxic
Substances in Our Ecosystems. Ann Arbor Science, Ann Arbor, MI.
Rai, L.C., and M. Raizada. 1989. Effect of bimetallic combinations of Cr, Ni and Pb on
growth, uptake of nitrate, ammonia. 14C02fixation and nitrogenase activity of Nostoc
muscorum. Ecotoxicol. Environ. Safety 17:75-85.
Rai, L.C., J.P. Gaur, and H.D. Kumar. 1981a. Phycology and heavy metal pollution. Biol.
Rev. 56:99-151.
Rai, L.C., J.P. Gaur, and H.D. Kumar. 1981b. Protective effects of certain environmental
factors on the toxicity of zinc, mercury and methylmercury to Chlorella vulgaris.
Environ. Res. 25:250-259.
Randhawa, M.S. 1959. Zygnemaceae. Indian Council of Agricultural Research, New
Delhi.
Riddolls, A. 1985. Aspect of nitrogen fixation in Lough Naugh. I. Acetylene reduction
and the frequency of Aphanizomenon flos-aquae. Freshwater Biol. 15:289297.
Sanders, J.G., J.H. Batchelder, and J.H. Ryther. 1981. Dominance of a stressed marine
phytoplankton assemblage by a copper tolerant pennate diatom. Bot. Mar. 24:
39-41.
Shubert, L.E. 1984. Algae as Ecological Indicators. Academic Press, London.
Starodub, M.E., P.T.S. Wong, C.I. Mayfield, and Y.K. Chau. 1988. Influence of complexation and pH on individual and combined heavy metal toxicity to a freshwater green
alga. Can. J. Fish. Aquat. Sci. 44:1173-1180.
Stewart, W.D.P., G.P. Fitzgerald, and R.H. Burris. 1968. Acetylene reduction by nitrogen
fixing blue-green algae. Arch. Mikrobiol. 62:336-348.
Taub, F.B. 1984. Measurement of pollution in synthesized aquatic microcosms, P.
159-192. In H.H. White (Ed.), Concepts in Marine Pollution Measurements. University
of Maryland, College Park, MD.
Weber, C.I., and B.H. McFarland. 1969. Periphyton biomass chlorophyll ratio as a n index
Cr AND Hg TOXICITY ASSESSED IN SITUI107
of water quality. Presented a t the 17th annual meeting of the Midwest Benthological
Society, Gilbertsville, KY.
Whitton, B.A. 1984. Algae as monitors of heavy metals in freshwaters, P. 257-280. In
L.E. Shubert (Ed.), Algae as Ecological Indicators. Academic Press, London.
Wong, P.T.S., and J.T. Trevors. 1988. Chromium toxicity to algae and bacteria, P.
305-315. In J.O. Nriagu and E. Nieboer (Eds.), Chromium in the Natural and Human
Environments, John Wiley & Sons, New York.