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Toxic effect of metal ions on biochemical oxygen demand

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DOI: 10.1016/S0043-1354(99)00104-9

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 Wat. Res. Vol. 34, No. 1, pp. 147±152, 2000
# 1999 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
PII: S0043-1354(99)00104-9 0043-1354/99/$ - see front matter

www.elsevier.com/locate/watres

TOXIC EFFECT OF METAL IONS ON BIOCHEMICAL

OXYGEN DEMAND
S. K. MITTAL* and R. K. RATRA
Department of Chemical Engineering, Thapar Institute of Engineering & Technology (Deemed
University), Patiala 147 004, India

(First received 1 June 1998; accepted in revised form 1 February 1999)

AbstractÐToxic e€ect of metal ions like Al, Co, Ni, Cu, Zn, Pb and Hg on the Biochemical Oxygen
Demand (BOD) exertion has been studied at various concentrations of the metal ions. Presence of these
metal ions in the e‚uent samples signi®cantly a€ects the observed BOD values. The magnitude of their
dependence varies with the nature and concentration of the metals working as a toxicant. The
detrimental e€ect of metal ions on the observed BOD is a resultant of the biological as well as chemical
processes taking place in the sample. Addition of metal ions like Al, Zn, Cu and Pb results in the
inhibition as well as increase in BOD. Mechanism for the e€ect of metal ion on BOD has been
proposed and is correlated with their electrochemical potentials. # 1999 Elsevier Science Ltd. All rights
reserved

Key wordsÐbiochemical oxygen demand, toxic e€ect, metal ion, suppression

INTRODUCTION heavy metals on BOD test. BOD5 (BOD measured

Trace quantities of heavy metals such as nickel,
manganese, lead, chromium, cadmium, zinc, copper,
iron and mercury are constituents of most waste
waters. Some of these metals are necessary for
growth of biological life, and absence of sucient
quantities of them could limit growth of algae, for
example. The presence of any of these metals in
excessive quantities will interfere with many ben-
e®cial uses of the water because of their toxicity.
Therefore, it is desirable to measure and control the
concentration of these substances. Since both the
biological treatment process and BOD measuring
technique are based on same principle of biochemi-
cal degradation of organic matter by microorgan-
isms in presence of oxygen, the presence of toxic
metals like copper, zinc, lead and other heavy
metals will have an in¯uence on both the processes.
Because of the limited solubility of oxygen in the
aquatic medium contained in the BOD bottle, the
toxic e€ect of heavy metal will be suciently large
compared to that in the treatment plant producing
e‚uent being tested for BOD. This di€erence may
be due to the reason that on continuous aeration,
the e‚uent of the treatment plant gets a regular
supply of oxygen.
Little work (Medine et al., 1981; Franke et al.,
1981; Boston and Hure, 1983; Berthouex and
Szewczyk, 1984) has been done on the e€ect of
*Author to whom all correspondence should be addressed.
Fax: +175-214498; e-mail: mittal@tietp.ren.nic.in
after incubation period of 5 days) is signi®cantly
a€ected by even small concentrations (1±2 mglÿ1)
of Cu or Cr. Stones (1981) measured the percentage
suppression caused by 1 mglÿ1 of selected heavy
metals dosed in the form of inorganic salts, on the
BOD of domestic sewage. Kansal and Mehrotra
(1982) studied the e€ect of di€erent concentrations
of cadmium ions on two wastes of di€erent nature.
Schubert (1980) showed that even for easily biode-
gradable material sub-toxic concentrations of toxic
material can inhibit biodegradation. Copper inhi-
bition of treatment process is found to show a
synergetic e€ect (Admeroti, 1988) with nickel and
zinc. Since on the addition of certain heavy metals,
BOD of certain industrial waste water and domestic
waste water may be suppressed, hence a systematic
study on the e€ect of heavy metal ions on BOD of
waste water is needed to understand the mechanism
involved in the process of BOD.
In our investigations, e€ects of metals like alu-
minium, cobalt, nickel, copper, zinc, mercury and
lead on BOD5 have been studied and a new mech-
anism for BOD exertion in presence of metal ions is
proposed.
MATERIALS AND METHODS
Methodology and test set-up
BOD determination is an empirical test in which stan-
dardised laboratory procedures (APHA, 1989) are used to
determine the relative oxygen requirements of waste water,
e‚uents, and polluted waters. The test measures the oxy-
gen utilised during a speci®ed incubation period for bio-

147
148 S. K. Mittal and R. K. Ratra
chemical degradation of organic material (carbonaceous
demand) and the oxygen used to oxidise inorganic ma-
terial such as sulphide and ferrous ions. Although only 5-
day BOD (BOD5) is described here, many variations of
oxygen demand measurement exist. These include using
shorter and longer incubation periods, tests to determine
rate of oxygen uptake, and continuous oxygen uptake
measurement techniques.
Sampling and storage
Samples were prepared synthetically in the laboratory
and then stored in the refrigerator. Samples were not col-
lected from any waste stream since the study was of rela-
tive nature. At the time of analysis, samples were allowed
to come at a temperature of 208C.
Apparatus
Incubation bottles. 300 ml capacity amber glass bottles
were thoroughly cleaned with chromic acid followed by
distilled water and drained before use. As a precaution
against drawing air in the dilution bottle during incu-
bation, a water seal (space between mouth of the BOD
bottle and glass stopper ®lled with water) was used. A foil
cap was placed over the ¯ared mouth of the bottle to
reduce evaporation of the water seal during incubation.
BOD incubation. BOD incubator (Indian Equipment
Co., Mumbai) thermostatically controlled at 20 20.18C
was used for the tests.
Reagents. Appropriate amounts of the chemicals (A R
grade) were used to prepare solutions required for BOD
measurements.
Procedure
Preparation of dilution water. The desired volume of
water was put into a suitable container and 1 ml each of
phosphate bu€er, MgSO4, CaCl2 and FeCl3 solutions per
litre of seeded dilution water was added as described
below. Dilution water was brought to 208C before use and
saturated with dissolved oxygen (DO) by shaking in two
big containers for 15 min.
Seeding. Synthetic milk waste was used in the test. A
compatible seed from a treatment plant of a local milk
plant was collected. Seed water was taken from the corner
of an aeration tank before the settling tank. Seeded water
is then allowed to rest for settling and supernatant liquid
was then used as a seed for the BOD test.
Seed control. BOD of seeding material was measured
and dilution was made so that its DO uptake comes
between 0.6 to 1.25 mg lÿ1.
Synthetic waste sample preparation. A synthetic waste
sample was prepared from raw milk in the laboratory. A
synthetic sample was preferred to the actual waste sample
from industrial e‚uents because the investigations are of a
relative nature and the industrial sample may contain con-
taminants other than organic material, such as detergents,
heavy metals and other foreign materials, which may inter-
fere with the metal under test. Raw milk has BOD of
about 70,000±100,000 mglÿ1 and was diluted with distilled
water so that it comes in the range of 700±1000 mglÿ1.
Preparation of metal ion solutions. Metals selected for
the study were aluminium, cobalt, nickel, copper, zinc,
mercury and lead. Appropriate amounts of nitrate salts of
these metals were dissolved in distilled water and dilutions
{(0.5% sample was taken, because the expected BOD was
between 700±1000 mglÿ1. Therefore, maximum
expected DO depletion would have been between 3.5±
5.0 mglÿ1. As saturated DO in summer days is usually
around 7.0 mglÿ1, 2 mgl ÿ1 of DO would have
remained in the test solution in any case, which is an
essential norm for BOD test.)
were done to prepare a 1000 ppm solution of each metal
ion.
Estimation of BOD. Separate tests were performed for
each metal ion at di€erent concentrations in the range
0.1±100 mglÿ1.
The required number of duly washed bottles were
arranged in sets of three bottles each. 1.5 ml (0.5%){ of
synthetic waste water sample was pipetted into each bottle
of all the sets. These bottles were then ®lled with seeded
dilution water. A known quantity of metal ion solution
was added in each set of bottles ranging from 0.1±100
mglÿ1.
All bottles were carefully ®lled with seeded dilution
water to the top of the brim, not being allowed to over-
¯ow, because over¯owing would disturb the concentration
of metal ion in the bottle. The initial DO of ®rst bottle of
each set was taken and the remaining two were capped,
watersealed and incubated for 5 days at 208C in BOD
Incubator. After 5 days of incubation, the ®nal DO of the
samples were determined.
Estimation of dissolved oxygen. Initial and ®nal DO
were determined by the titration method. The Alsterberg
modi®cation of Winkler's method (APHA, 1989) was
adopted. To ®nd the DO (of each bottle), 1.0 ml of
manganese sulphate solution was pipetted by dipping the
end of pipette below the surface of liquid; 1.0 ml of alka-
line potassium iodide solution was added, the stopper was
inserted and the contents were mixed thoroughly. The pre-
cipitation formed was allowed to settle. Then 2.0 ml of
sulphuric acid solution was added to the bottle; the pre-
cipitates were dissolved by vigorous shaking. The calcu-
lated amount of sample solution in a titration ¯ask was
taken and titrated against sodium thiosulphate solution
(0.025N) using starch as indicator. The volume in ml of
the titrant used was recorded, which gave directly the
mglÿ1 of dissolved oxygen.
Calculations. Initial and ®nal DO values are calculated
using the following equation and results are used to ®nd
change in BOD (increase/inhibition) given in Table 1.
…D1 ÿ D2 † ÿ …B1 ÿ B2 † f
BOD5 …mglÿ1 † ˆ
P
where
D1=DO of diluted sample immediately after prep-
aration, mglÿ1; D2=DO of diluted sample after 5-day in-
cubation at 208C, mglÿ1; P = Decimal volumetric fraction
of sample used; B1=DO of seed control before incu-
bation, mglÿ1; B2=DO of seed control after incubation,
mglÿ1; and f = ratio of seed in diluted sample to seed in
seed control.
Here f = 1, because the same seeded dilution water is
used in each case. Seed corrections were applied to all the
observations.
RESULTS AND DISCUSSION
Addition of metal ions in the sample solutions
resulted in the change in BOD5. Results of BOD5
depletion/increase with the addition of di€erent
concentration of metallic ions are presented in
Table 1. In case of cobalt, nickel and mercury there
is a regular decrease in observed BOD5 with an
increase in the amount of metal ion. The percentage
inhibition increases from 3.7 to 114% for nickel,
9.8 to 78.3% for cobalt and 76.1 to 118.4% for
mercury ions. Toxicity of mercury is large, even at
low concentrations. BOD5 inhibition of more than
100% may be due to the reason that the ``seed''
 Toxic e€ect of metal ions on biochemical oxygen demand 149

Table 1. BOD5 exertion in presence of metal ions at 208C

Sample No. Type of sample Change in BOD (inhibition/increase), % age

Nickel Cobalt Mercury Aluminium Zinc Copper Lead

1 1 ml/l seed+1 ml/l each of nutriant ± ± ± ± ± ±

2 W.W+0.0 mg/l metal ions ± ± ± ± ± ± ±
3 (+)0.1 mg/l 3.70(ÿ) 9.80(ÿ) 76.1(ÿ) 1.30(+) 7.80(+) 0.60(ÿ) 1.75(+)
4 (+)0.25 mg/l 11.1(ÿ) 13.0(ÿ) 90.8(ÿ) 7.90(+) 15.6(+) 3.00(ÿ) 12.3(+)
5 (+)0.50 mg/l 24.1(ÿ) 18.5(ÿ) 110(ÿ) 11.3(+) 23.4(+) 3.50(ÿ) 1.75(+)
6 (+)0.75 mg/l 38.1(ÿ) 22.5(ÿ) 117(ÿ) 17.1(+) 10.4(+) 11.9(ÿ) 3.50(ÿ)
7 (+)1.0 mg/l 50.0(ÿ) 30.7(ÿ) 118(ÿ) Nil 6.30(ÿ) 16.4(ÿ) 8.77(ÿ)
8 (+)2.0 mg/l ± 37.5(ÿ) 117(ÿ) 11.8(ÿ) 10.9(ÿ) 20.9(ÿ) 12.3(ÿ)
9 (+)5.0 mg/l 64.8(ÿ) 41.6(ÿ) 118(ÿ) 52.6(ÿ) 18.8(ÿ) 31.3(ÿ) 20.0(ÿ)
10 (+)10 mg/l 81.5(ÿ) 45.6(ÿ) 118(ÿ) 61.8(ÿ) 41.8(ÿ) 29.8(ÿ)
11 (+)20 mg/l 90.7(ÿ) 55.2(ÿ) 118(ÿ) 67.1(ÿ) 28.7(ÿ) 31.3(ÿ) 14.0(ÿ)
12 (+)50 mg/l 109(ÿ) 67.4(ÿ) 118(ÿ) 80.3(ÿ) 37.5(ÿ) 9.90(ÿ) 7.00(ÿ)
13 (+)75 mg/l ± 117(ÿ) ± 7.5(+) 1.75(ÿ)
14 (+)100 mg/l 114(ÿ) 78.3(ÿ) 118(ÿ) 90.8(ÿ) 46.9(ÿ) 20.9(+) Ð

acts as a source of BOD5 in addition to the sample
BOD5.
In case of aluminium and zinc, there is ®rst an
increase and then a sudden fall in BOD5 values fol-
lowed by gradual decrease. Percentage change in
BOD5 ranges from 1.3%(+) to 90.3%(ÿ) for alu-
minium, and for zinc it is 7.8%(+) to 46.9%(ÿ).
The magnitude of percentage change for aluminium
is about twice that of zinc at 100 mglÿ1 concen-
tration.
In presence of copper ions, inhibition of BOD5
®rst increases from 0.6 to 41.8% and then decreases
almost at the same rate resulting in an overall
increase of 21% at 100 mglÿ1. However, in the case
of lead, ®rst there is an increase in the oxygen
demand and then the inhibition starts which
increases up to 29.8%Ðand then reversal of the
trend is noticed. Subsequently, no overall change in
BOD5 is observed at 100 mglÿ1 metal ion concen-
tration.
During the process of BOD measurement there
are microbial organisms (added as seed) present
along with the ®nite amount of metal ion concen-
trations. In general, the microbial organism oxidises
the organic matter using oxygen from the aqueous
medium and exerts BOD. Presence of metal ion
might a€ect the growth of microbial seed. Metal
ion has an anity to combine with the microorgan-
ism (Chan and Bhargava, 1992). This process
involves the penetration of metal ion through the
cell membrane, resulting in the metal complex with
the organism, thereby either inhibiting or stimulat-
ing the catalytic function of the enzymes of the
microorganism.
During the process of BOD measurement a small
amount (1 ml) of the seed (which normally grows
with time) and sucient amount of nutrients are
added to favour the process of their development.
However, the presence of metal ions a€ects the
growth of microbial seed as well as inhibits their
population, and a prolonged lag period develops
which persists until resistant organisms have
evolved. The presence of metal ions may prolong
this lag period even beyond 5 days, thus a€ecting
the BOD5 results.
Toxicity of the metal ions to microorganisms
may be lethal or sublethal depending upon the
nature and concentration of the ion added. As
observed in case of mercury, even very small con-
centrations proved lethal and result in complete in-
hibition (Figs 1±10) whereas in case of aluminium,
the lethal dose is at very high concentration.
Similarly, a sublethal dose was observed for other
metals which do not show complete inhibition. The
toxic e€ect may be chronic or acute. These terms
are more relevant for BODs other than BOD5, e.g.,
BOD3 or BODu (BOD with very large incubation
time). Similar observations are reported by Kansal
et al. (1982).
From the above results it may be said that there
is competition between the microbial species (seed)
and the metal ions to combine with the dissolved
oxygen present in the medium. Our observation
supports the hypothesis that there are two competi-
tive pathways, represented as Scheme 1 and Scheme
2, in which microbial population of bacteria can
react, either with organic matter (Scheme 1) or with
metal ion (Scheme 2).
If the microbial seed combines with metal ion in
the medium, then it will not react with the organic
Fig. 1. BOD exertion in presence of metal ions (0.1)
mg lÿ1.
150 S. K. Mittal and R. K. Ratra

Fig. 2. BOD exertion in presence of metal ions

(0.25 mg lÿ1). Fig. 5. BOD exertion in presence of metal ions
(1.0 mg lÿ1).

Fig. 3. BOD exertion in presence of metal ions Fig. 6. BOD exertion in presence of metal ions
(0.50 mg lÿ1). (2.0 mg lÿ1).

Fig. 7. BOD exertion in presence of metal ions

(5.0 mg lÿ1).

Fig. 4. BOD exertion in presence of metal ions

(0.75 mg lÿ1).

Scheme 1.

Fig. 8. BOD exertion in presence of metal ions

Scheme 2. (10.0 mg lÿ1).
 Toxic e€ect of metal ions on biochemical oxygen demand
Fig. 9. BOD exertion in presence of metal ions
(20.0 mg lÿ1).
Fig. 10. BOD exertion in presence of metal ions
(50.0 mg lÿ1).
matter and hence the uptake of oxygen will be less,
so, inhibition of BOD exertion results. Otherwise,
the microbial seed consumes the organic matter,
resulting in depletion of oxygen, leading to exertion
of BOD.
Dissolved oxygen in the aqueous medium can be
consumed in two ways. Either it may be consumed
during the oxidation of organic matter or it will be
taken up by the metal ions, resulting in their oxide
formation. The latter phenomenon was observed in
our study and we noticed that a number of times
BOD was found to increase even larger than the
original BOD values, probably due to the oxidation
of metal ions.
In the cases of aluminium and zinc, apparently
there is an oxidation of the metal in addition to
consumption of oxygen by the microbial matter,
since the oxygen demand increases (Table 1) on the
introduction of metal ions in the sample solution.
This hypothesis is supported by the standard oxi-
dation potential values for all the metal ions under
consideration. Metal ions with larger oxidation po-
tential are easily oxidised, while those with smaller
values have relatively less tendency to undergo oxi-
dation. Aluminium and zinc have the largest oxi-
dation potentials (1.66 and 0.763 V, respectively),
so, they are easily oxidised and consume oxygen in
addition to its requirement for the normal BOD
process. This results in the overall increase of BOD
151
in sharp contrast to the expected fall in its value
(due to the presence of metal ion). Oxidation of
copper (indicated from the increase in BOD) is
observed only at a high concentration because of its
lower oxidation potential as shown in the series
below:
Al (1.66 V) > Zn (0.763 V) >> Co(0.277 V) >
Ni(0.246 V) > Pb(0.126 V) >> Cu(ÿ0.337 V) >>
Hg(0.79 V).
The behaviour shown by lead is irregular. No
increase in the oxygen demand is apparent for
cobalt, nickel and mercury, as expected in the series
shown above.
Histograms of the study showing the e€ect of
metal ions at selected concentrations on BOD (inhi-
bition or increase) are presented in Figs 1± 10. A
general look at the diagrams shows that at low con-
centrations of 0.1±0.25 mglÿ1, a distinctly large in-
hibition in BOD5 is observed for mercury ions. No
signi®cant inhibition or increase is observed for all
other metal ions in this concentration range.
A typical trend is noticed at a concentration of 1
mglÿ1 for all the metal ions. Aluminium shows no
inhibition, while all other metal ions inhibit BOD
exertion due to their presence. It is beyond this con-
centration when the presence of all the metal ions
in¯uence BOD, resulting in lowering its values
(with the exception of copperÐshowing an increase
at 100 mglÿ1, probably due to metal oxide for-
mation along with normal process of BOD exer-
tion). More than 100% inhibition is noticed for
nickel and mercury, which may be due to the fact
that the `seed' itself consumes oxygen.
Toxicity controlling techniques. Li and Tan
(1994a,b) have suggested that the most e€ective
way to overcome the presence of inhibitory or toxic
compounds in waste water is the use of acclimated
seed. Toxicity can also be overcome by pretreat-
ment. For example, toxic metals can be complexed
with chelating agents such as EDTA, a strong com-
plexing agent which can form stable complexes with
almost all metal ions, causing toxicity to BOD
measurement. Dilution is the other technique which
can be applied without using extra chemicals or
time. The toxic e€ect is diluted out because dilution
reduces the concentration of toxins below threshold
toxic levels. Stirring the contents of the bottle con-
tinuously can also help to overcome the toxic e€ect.
Microbial toxicity may also be reduced by increas-
ing the organism concentration.
AcknowledgementsÐThe authors are thankful to Dr M. P.
Kapoor, Director, Thapar Institute of Engineering &
Technology, Patiala, for providing research facilities.
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