Effect of Cadmium, Zinc, Copper and Fluoranthene on Soil Bacteria
Nilufer Cevik2, Ayten Karaca1
Ankara University, Faculty of Agriculture, Department of Soil Science, 06110, Ankara,
Turkey
Abstract
The influence of Cd, Cu, Zn, and fluoranthene (FLA), separately applied, and combinations of
one of these heavy metals with FLA on the growth of soil bacteria were examined through a
90 day incubation period and compared with the behavior of no treatment (cntrol). In the soils
amended with all doses of Cd, Cu and Zn alone and combination with FLA, total bacterial
population was always significantly lower than those of the control soil. Significant
reductions of bacterial counts were observed for both doses of heavy metals combined with
FLA. Low concentration of heavy metals (50 mg kg-1 ) which was not affective when added
separately was found to reduce bacterial growth when applied in combination with FLA. At
higher levels of heavy metals (150 mg kg-1 ) addition of FLA also increased the toxicity of the
metals.
Comparisons of whole treatments revealed that total bacterial growth was more inhibited by
150 mg kg-1 Cd alone (%85), and 50 mg kg-1 Cd + 150 mg kg-1 FLA and 150 mg kg-1 Zn +
150 mg kg-1 FLA treatments (%82,5). Based on these results, it could be concluded that FLA
may enhance the toxicity of low concentrations of heavy metals (50 mg kg-1) to bacteria in
soils.
Keywords: flouranthene, heavy metals, bacteria, soil
1
Corresponding Author: Tel:+903125961758, Fax:+903123178465, akaraca@agri.ankara.edu.tr, Ankara
University, Faculty of Agriculture, Soil Science Department, 06110, Ankara, Turkey
2
Ankara University, Faculty of Agriculture, Soil Science Department, 06110, Ankara, Turkey
Introduction
Hazardous organic and metallic residues or by-products can enter into plants, soils, and
sediments from processes associated with domestic, municipal, agricultural, industrial, and
military activities. Heavy metal contamination of soils originating from agricultural (e.g.,
fertilizers and sewage sludge) or industrial activities (e.g., metal mining and smelting) is one
of the major environmental problems in many parts of the world. From agricultural, home,
and industrial usage, pesticides can enter into crop residues, municipal sludges, farm manures,
and soils. Organic contaminants (e.g., polychlorinated biphenyls [PCBs], polycyclic aromatic
hydrocarbons [PAHs]) other than pesticides can enter into the soil from fuel combustion or
from sewage sludge and other feed stocks. Spillage of fuel oil hydrocarbons can contaminate
soils. These hydrocarbons will inhibit seed germination and plant growth, but plants do not
appear to accumulate the hydrocarbons (1).
The soil microbial community should be a sensitive indicator of metal contamination effects
on bioavailability and biogeochemical processes. In recent years, several reports have
documented the harmful effects on soil microorganisms and microbial activity of the longterm heavy metal contamination of agricultural soils (2, 3). However, very few publications
on the effects of non-pesticide organic pollutants (4) are available.
Hydrophobic organic pollutants such as polyaromatic hydrocarbons (PAHs) may have
negative side-effects on microorganisms (5, 6, 7, 8, 9) and microbial-mediated processes in
soils (10). According to the review by Sikkema et al. (11), lipophilic cyclic hydrocarbons such
as PAHs interact with membrane microorganisms including bacteria and yeasts. These
interactions lead to changes in the structure and function of the membranes. Increases in
permeability to protons and ions can be observed (11, 12, 13). PAHs may enhance the toxicity
of the metals because they can penetrate into the perforated microbial cells more easily (14).
We assume that presence of PAHs in contaminated soils will be enhanced the toxicity of the
heavy metals due to low solubility of PAHs and their adsorption by soil generally organic
matter. Until now, no studies have been published about the combination effect of heavy
metals and polyaromatic hydrocarbons in soil. Only one study have been published about
combination affect of heavy metals and hydrocarbons on soil bacteria which containing pure
growth media (agar-plate) by Gogolev and Wilke (14). Anthropogenic pollutants hardly
occur in the environment as single constituents. Usually, industrial and domestic activity
simultaneously emits numerous pollutants to soil and water. Thus, combination effects rather
than separate effects of pollutants are expected to occur in the environment.
The purpose of this study was to evaluate the effects of heavy metals alone and in
combination with FLA on soil bacterial number.
Materials and Methods
Experimental design
The pH of the soil was 7.35, particle ratio was 59:26:15 (clay:silt:sand, respectively) and the
SOM content was 1.8% by weight. Electrical conductivity (EC) was 0.16 dSm-1, total N
content 0.11 %, available P content 20%, CEC 42.16 meq 100 g-1, extractable Cd, Cu and Zn
were 0.179, 0.080 and 0.390 mg kg-1, respectively. The soil can be classified as unpolluted.
An incubation experiment was conducted in plastic pots, each containing 250 g coarsely
sieved soil with various treatments. Each treatment was replicated three times and the
experiment was carried out in a randomized complete block design. These were the
treatments:
1. The control pots were unamended.
2. Soils were supplemented with analytical reagent grade CdSO4, CuSO4, and ZnSO4 to
yield 50 and 150 mg kg-.
3. Soils were supplemented with analytical reagent grade flouranthene (Avocado, 20644-0) to yield 75 and 150 mg FLA kg-1 soil.
4. Heavy metals amended soils (50 and 150 mg kg-1) were supplemented with 75 and
150 mg kg-1 FLA.
Soil moisture was adjusted to 65% of water holding capacity. The incubation was performed
in a growth chamber at 28 0C. Water losses were compensated by the addition of distilled
water during incubation.
Sampling and analysis
Soil bacterial numbers were enumerated after 1, 7, 30, 45 and 90 days. Soil extractable heavy
metal contents were measured after 1st and 90 days of incubation. Soil pH and EC were
measured in a 1:2.5 soil: water mixture (15); SOM by a modified Walkley-Black Method
(16); particle size distribution according to Bouyoucos (1951); total N by the Kjeldahl method
(17). Extractable Cd, Cu and Zn in soils were extracted with a DTPA solution (0.005 M
DTPA + 0.005 M CaCl2 + 0.1 M TEA, pH 7.3) (18). The DTPA-extractable heavy metals in
solution were determined by inductively coupled plasma (ICP- VISTA AX CCD
Simultaneous model).
The total culturable bacteria population determined by the plate count agar technique (19).
Total bacterial number were counted by plating dilutions of soil on plate count agar medium
which contained 20,5 g agar in 1 l distilled water. Soil suspensions were derived by extraction
of soil (0,5 g moist wt.) with ringer solutions (2 tablets 1 l-1) in test pots. Agar was steam
sterilized at 121 °C for 15 minutes. Approximately 15 ml warm agar (~50 °C) was mixed 0,1
ml of the diluted soil extracts. The number of colonies was determined from three replicate
plates after 5 days of incubation at 28 °C darkness in an incubator.
Subsequent statistical analysis was performed using Minitab for Windows (version 2.14).
Results and Discussion
Heavy Metal Availability
The changes in the amount of DTPA extractable Cd, Zn and Cu in the first and the last
incubation time are shown in Table 1. Higher metal input resulted in consistently higher metal
concentration than did lower metal input. At the beginning of the incubation, the DTPAextractable Cd, Zn and Cu were higher in heavy metal + FLA combination treatments than
metal alone treatments. However, at the end of the incubation, the extractable heavy metal
concentration was higher in metal alone treatments than heavy metal + FLA combination
treatments (P<0.05). In general, organic contaminants added with FLA had a negative effect
on the bacterial numbers of soil, which, in some cases, counteracted the positive effect that a
low level of heavy metal contamination might have had on them.
Effect of Cadmium on Total Count of Soil Bacteria
Changes in total soil bacteria number treated with 50 and 150 mg kg-1 Cd alone and
combination with 75 and 150 mg kg-1 FLA during incubation are shown in Figure 1a and 1b,
respectively. In the soils amended with both doses of Cd alone and combination with FLA,
total bacterial population was significantly lower than those of the control soil. However,
bacterial numbers were higher in 75 mg kg-1 FLA alone added soils than control soils in 45
and 60 days of incubation period. The total culturable bacteria population was significantly
lower in all treatments with Cd+FLA combination than their respective non-FLA-amended
controls (P<0.05). We assume that the presence of fluoranthene with Cd in soils will be
enhanced the toxicity to soil bacteria. Increasing rates of FLA also enhanced negative effect
of Cd to soil bacteria. Our results are in agreement with results of Gogolev and Wilke (14).
Moreover 150 mg kg-1 Cd treatments reduced the bacteria to a larger extent than 50 mg kg-1
Cd treatments.
The supposition that 50 and 150 mg kg-1 Cd addition had a detrimental effect on the bacterial
community is supported by the fact that a significant negative correlation was found between
the total culturable bacterial population and extractable Cd (r=-0.518 and r= -0,657,
respectively P<0.001).
Effect of Zinc on Total Count of Soil Bacteria
Changes in total soil bacteria number treated with 50 and 150 mg kg-1 Zn alone and
combination with 75 and 150 mg kg-1 FLA during incubation are shown in Figure 2a and 2b,
respectively. At the beginning of the incubation, the addition of Zn with and without FLA
increased the bacterial numbers with respect to FLA alone treatments. However, by the end of
the incubation period, FLA alone treatments had significantly higher bacterial numbers than
Zn alone and Zn+FLA combination treatments. The total bacterial population was
significantly reduced with the addition of Zn+FLA treatments than Zn alone treatments during
the incubation period (P<0.05). Increasing rates of Zn and FLA also enhanced negative effect
of Zn to soil bacteria, whereas, 150 mg kg-1 Zn which combined with 150 mg kg-1 FLA
inhibited soil bacterial growth to a larger extent than all other treatments. Gogolev and Wilke
(13) who studying with liquid nutrient medium also reported that bacterial growth was more
affected from combinations than separate Zn treatment.
Bacterial numbers obtained from soils which adding 50 and 150 mg kg-1 Zn separately and
combining with FLA were not correlated the levels of extractable Zn.
Effect of Copper on Total Count of Soil Bacteria
Changes in total soil bacteria number treated with 50 and 150 mg kg-1 Cu alone and
combination with 75 and 150 mg kg-1 FLA during incubation are shown in Figure 3a and 3b,
respectively. In control treatment, the total culturable bacteria population was significantly
greater than in all other treatments, at the first and the last months of incubation, whereas the
addition of FLA alone resulted in significantly greater bacterial populations at 45 and 60 th
days of incubation. The bacteria population decreased by increasing the Cu and FLA rate. All
combination treatments had significantly lower bacteria population than alone treatments
(P<0.05). However, this decrease was much greater in the treatments which combinated with
150 mg kg-1 FLA than 75 mg kg-1 FLA. Moreover, less bacteria were counted in soil samples
that containing 150 mg kg-1 Cu in comparison to 50 mg kg-1 Cu (P<0.05).
The correlation coefficient between the number of bacteria and extractable Cu in this study
were -0.601 and r= -0,549, respectively (P<0.01).
Andrade 2004 (20) indicated that there was a high correlation (r > 0.90) between the
concentrations of Cu, Ni and Pb metals and hydrocarbon content of the polluted soils, which
shows the combined addition of these metals through the fuel oil.
Conclusion
According to our findings, both heavy metals separately and combining with FLA reduced
bacterial growth significantly considering to control depended on dose and time. When
compared the effects of heavy metal alone and combinations with FLA on bacterial number
considering to 90 days of incubation period, the most effective inhibitor metal to bacteria was
Cd in soils treated with 50 mg kg-1 alone and combinations with FLA (Cd>Zn>Cu).
Although soil bacteria was more inhibited by Cd when 150 mg kg-1 heavy metals doses were
applied separately (Cd>Zn>Cu), 150 mg kg-1 heavy metals doses combined with 75 and 150
mg kg-1 FLA bacterial growth was inhibited by Zn (Zn>Cd>Cu).
Comparisons of whole treatments revealed that total bacterial growth was more inhibited by
separate 150 mg kg-1 Cd treatment (%85), and 50 mg kg-1 Cd + 150 mg kg-1 FLA and 150 mg
kg-1 Zn + 150 mg kg-1 FLA treatments (%82,5).
This incubation experiment suggested that FLA enhance the metal toxicity to bacteria in soils.
Considering the low solubility of FLA and its strong adsorption to organic material, it seems
that deleterious effects of heavy metals enhanced by the presence of FLA in contaminated
soils with heavy metals such as Cd, Zn and Cu. This is also supported by Gogolev and Wilke
(14).
Sikkema et al. (11) stated that hydrophobic pollutants such as PAHs interact with lipophilic
compounds of cytoplasmatic membranes of microorganisms. This could cause changes in the
membrane structure and might alter the permeability of the membranes. Therefore, we assume
that PAHs may enhance the toxicity of the metals because they can penetrate into the
perforated microbial cells more easily in soil.
References
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Table1. Concentrations of DTPA-extractable Cd, Zn and Cu (mg kg-1) in treated soils (1st day and 90th
day)
DTPA Extractable Cd mg kg-1
Control
50 Cd
50Cd+75 FLA
50Cd+150 FLA
1st day
0.179 C
4.070 B
4.156 A
4.149 A
90th day
0.432 D
5,888 A
4.716 C
4.599 B
Control
150 Cd
150Cd+75 FLA
150Cd+150 FLA
1st day
0.179 C
5.261 B
7.158 A
7.214 A
90th day
0.432 D
6.715 A
6.156 B
6.014 C
DTPA Extractable Zn mg kg-1
Control
50 Zn
50Zn+75 FLA
50Zn+150 FLA
1st day
0.08 C
14.91 B
17.66 A
17.16 A
90th day
0.093 D
16.00 A
11.88 B
10.97 C
Control
150 Zn
150Zn+75 FLA
150Zn+150 FLA
1st day
0.08 D
49.5 A
19.5 B
18.20 C
90th day
0.093 D
84.75 A
31.50 B
20.10 C
DTPA Extractable Cu mg kg-1
Control
50 Cu
50Cu+75 FLA
50Cu+150 FLA
1st day
0.39 D
42.48 C
44.00 B
43.00 A
90th day
0.23 D
19.38 A
10.63 B
11.95 C
Control
150 Cu
150Cu+75 FLA
150Cu+150 FLA
1st day
0.39 D
102.00 C
137.00 A
129.00 B
90th day
0.23 D
98.00 A
64.68 B
62.80 C
Least significant difference (LSD, P<0.05)= 49.59
50 Cd
A
1600
B
A
1000
C
1200
B
D
A
A
C
F
E
E
E
D
B
D
E
B
D
C
F
C
E
D
B
A
200
F
F
F
D
400
B
B
C
C
600
D
800
A
Total soil bacteria
(CFU g-1 dry soil)
1400
0
Control
50 Cd
50 Cd+75 FLA 50 Cd +150 FLA
75 FLA
150 FLA
-1
Treatm ents (m g kg )
1
7
30
45
60
90 (day)
LSD: 7,2791
Fig. 1a Changes of total soil bacteria population in 50 mg kg-1 Cd treatments during 90 days
of incubation period, CFU: Colony-forming units, LSD: Least significant difference.
Significant differences between treatments at each time point indicated by different letters,
(P<0,05 level).
Control
150 Cd
1
7
150 Cd +75
150 Cd +150
FLA
FLA
Treatments (mg kg-1)
30
45
B
C
A
A
B
D
60
C
E
D
75 FLA
E
C
B
F
B
B
F
F
D
E
D
C
0
D
F
F
C
B
B
A
300
C
600
E
E
900
C
A
Ba
1200
A
Total soil bacteria
(CFU g-1 dry soil)
1500
A
150 Cd
150 FLA
90 (day)
LSD: 6,0901
Fig. 1b Changes of total soil bacteria population in 150 mg kg-1 Cd treatments during 90 days
of incubation period. For abbreviations, see Fig. 1a.
50 Zn
A
2400
B
1500
D
C
1800
50 Zn
7
30
45
60
B
D
F
E
75 FLA
E
D
C
C
D
50 Zn+75 FLA 50 Zn+150 FLA
Treatments (mg kg-1)
1
A
A
E
E
E
D
E
C
F
F
B
0
Control
C
B
C
B
A
300
D
C
E
600
C
900
B
A
1200
A
Total soil bacteria
(CFU g-1 dry soil)
2100
150 FLA
90 (day)
LSD: 4,3063
Fig. 2a Changes of total soil bacteria population in 50 mg kg-1 Zn treatments during 90 days
of incubation period. For abbreviations, see Fig. 1a.
150 Zn
A
2100
C
D
150 Zn
C
B
A
A
B
150 Zn+150
FLA
75 FLA
D
E
B
D
D
C
150 Zn+75 FLA
F
D
E
D
E
C
F
D
C
0
Control
B
B
F
D
E
C
A
300
B
600
C
900
E
A
1200
A
(CFU g-1 dry soil)
Total soil bacteria
1500
B
1800
150 FLA
Treatments (mg kg-1)
1
7
30
45
60
90 (day)
LSD: 5,6383
Fig. 2b Changes of total soil bacteria population in 150 mg kg-1 Zn treatments during 90 days
of incubation period. For abbreviations, see Fig. 1a.
50 Cu
D
C
D
C
C
E
F
E
F
E
B
F
D
F
E
C
E
C
D
A
300
F
F
C
B
D
B
B
B
600
E
900
A
A
A
1200
A
(CFU g-1 dry soil)
Total soil bacteria
1500
C
A
A
1800
0
Control
50 Cu
50 Cu+75 FLA
50 Cu+150 FLA
75 FLA
150 FLA
-1
Treatments (mg kg )
1
7
30
45
60
90 (day)
LSD: 4,3063
Fig. 3a Changes of total soil bacteria population in 50 mg kg-1 Cu treatments during 90 days
of incubation period. For abbreviations, see Fig. 1a.
1600
A
150 Cu
B
1200
B
C
D
Control
150 Cu
150 Cu+150
FLA
D
F
D
75 FLA
F
C
C
B
F
D
E
B
150 Cu+75 FLA
E
C
F
F
C
0
E
E
E
D
C
B
B
A
200
D
400
C
600
C
800
A
A
A
1000
A
Total soil bacteria
(CFU g-1 dry soil)
1400
150 FLA
Treatments (mg kg-1)
1
7
30
45
60
90 (day)
LSD: 3,9869
Fig. 3b Changes of total soil bacteria population in 150 mg kg-1 Cu treatments during 90 days
of incubation period. For abbreviations, see Fig. 1a.