ABSTRACT This study reports on accumulation of Cd, Co, Cu, Fe, Mn, Ni and Pb in insect species (grasshoppers

E Markova

I Karadjova

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XI ANNIVERSARY SCIENTIFIC CONFERENCE                                BIOTECHNOL. & BIOTECHNOL. EQ. 23/2009/SE 
120 YEARS OF ACADEMIC EDUCATION IN BIOLOGY                                                   SPECIAL EDITION/ON-LINE 
45 YEARS FACULTY OF BIOLOGY 
204
METAL ACCUMULATION IN INSECTS (ORTHOPTERA, ACRIDIDAE) NEAR 
A COPPER SMELTER AND COPPER-FLOTATION FACTORY (PIRDOP, 
BULGARIA) 
I. Karadjova1, E. Markova2 
1 Faculty of Chemistry, University of Sofia 
2 Faculty of Biology, University of Sofia “St. Kliment Ohridski”, Department of Ecology and Environmental Protection, blvd. 
Dragan Tzankov 8, Sofia–1164, Bulgaria 
Correspondence to: Emilia Markova 
E-mail: markovae@abv.bg 
ABSTRACT 
This study reports on accumulation of Cd, Co, Cu, Fe, Mn, Ni and Pb in insect  species (grasshoppers) from different sites in 
the vicinity of a copper smelter and copper-flotation factory in mining region Pirdop, Bulgaria. Higher concentrations of Pb 
and Cd were found in the summer and autumn captured insects from the sample sites closer to the flotation factory than those 
from the control site, indicating, that elevated levels of these metals in this mining region is an important factor influencing 
body burdens. Observed differences were associated with higher metal concentrations in the plants from these sites because 
positive correlation between Cd and Pb concentrations in grasshoppers and plants for summer and autumn samples was 
found. It should be pointed that, for spring captured insects differences in Pb and Cd content for all sample saites under 
investigation were less significant. The concentrations of Cu, Fe, and Mn are more dependent on the feeding properties of the 
insects than on the distance from the emitter. In general metal body burdens for Cd, Cu, Mn, Pb and Ni were higher in summer 
captured insects than in spring irrespective of site or metal. 
 
Keywords: Acrididae, grasshoppers, Pirdop, pollution, trace 
metals  
Introduction 
The area around copper smelter and copper-flotation factory 
near copper mine provide an opportunity to study the effects 
of geological and antropogenic factors on plants and animal 
communities. Several studies have been undertaken on the 
accumulations of metals in soils, plants and vegetables from 
this region and, as  one would expect, concentrations of 
metals are usually highest at the sites closest to the smelter 
(2). However there is a lack of information for the metal 
accumulation in herbivorous insects. Data presented in the 
literature for the ecophysiological response of invertebrates to 
chronic and acute metal stress and their potential use as 
bioindicators are ambiguous (6; 3; 7). Generally higher metal 
concentrations  have been reported in invertebrates collected 
from more contaminated sites and considerable differences 
have been observed between the metal concentrations of the 
various group of invertebrates (1; 4; 5). There is a growing  
evidence of a connection between air pollution and the degree 
of damage inflicted by herbivorous insects; however,  the 
ecological significance of these relationships is still unclear 
(8). This paper aims to describe the patterns of metal  (Cd, 
Cu, Co, Fe, Mn, Ni and Pb) accumulation in herbivorous 
insects - grasshoppers, (Orthoptera, Acrididae)  at selected 
sites in the vicinity of a copper flotation factory and copper 
smelter in mining region Pirdop, Bulgaria. The samples are 
collected during spring, summer and autumn. 
Materials and methods 
Grasshoppers (Orthoptera, Acrididae) have one generation 
per year and their life cycle is characterized by four distinct 
stages. Single egg masses are laid in the soil at the end of 
October, where they remain throughout the winter. Larvae 
hatch in early spring, feed from may to June and pass through 
4-5 instars.  Adults insects were collected seasonally in the 
middle of  June (spring), at the end of august (summer) and in 
the middle of October (autumn). Five sample sites were 
chosen with respect to prevailing winds and based on 
 
BIOTECHNOL. & BIOTECHNOL. EQ. 23/2009/SE              XI ANNIVERSARY SCIENTIFIC CONFERENCE 
SPECIAL EDITION/ON-LINE                   120 YEARS OF ACADEMIC EDUCATION IN BIOLOGY 
               45 YEARS FACULTY OF BIOLOGY 
205
previous investigations for soil and plant pollution.  Site 1 is 
located far from the cooper flotation factory and copper 
smelter and is control, unpolluted point for this region.  Sites 
4 and 2 are located at distances of 0.5 and 3 km north 
respectively from copper flotation factory, site 5 is between 
copper flotation factory and copper smelter and site 3 is 2 km 
west from copper flotation factory. Five parallel samples 
were analyzed from each sample site. 
Ultrasonic bath was used to clean invertebrates of surface 
deposits. After drying to constant weight  (60-70 °C  for 22-
24 h)  samples were digested using nitric acid.  
Metal concentrations of all samples were analyzed by 
flame atomic absorption spectrometry, Perkin Elmer 1100B 
(Cu, Fe and Mn) and electrothermal atomic absorption 
spectrometry, Perkin Ellmer Zeeman 3030 (Cd, Ni and Pb). 
Calibration standards were prepared from Titrisol 
concentrates (Merck) and standard addition method was used 
for calibration. Analytical blanks and standard reference 
material (TORT -1 Lobster Hepatopankreas, National 
Research Council Canada) were treated simultaneously in the 
same manner to confirm accuracy of processes. All samples 
consist of individual animals, i.e. no samples were pooled. 
Statistical analysis of the data was carried out using 
Kruskal-Wallis H-test and Mann-Whitney U-test, differences 
were considered significant at P<0.05 (9). These 
nonparametric comparison tests were considered after all 
values obtained were examined for normality. The 
Kolmogorov-Smirnov test was used as an estimate of 
goodness-of-fit to normality and only a very limited number 
of data sets revealed a good fit to normal distribution. 
Results and Discussion 
Analytical results obtained are presented at Fig. 1 for all 
studied elements, seasons and sites. As can be expected in 
spring insignificant differences were observed in metal 
concentration for all investigated sample sites. Only for Fe 
relatively higher contents were found for site 4 and site 5, 
which are closer to the emission source.  
However grasshoppers clearly showed sites-dependent 
metal accumulation patterns for Cd and Pb in summer 
samples.  Taking into account, that the main pollutants 
emitted in this region are Cu, Cd, Fe and Pb,  significantly 
elevated concentrations  found for Cd and Pb in order site 
5>site 4>site 3 confirm the expectation that the animals' body 
burden reflects site pollution (p<0.05, U-test). Less 
contrasting differences were found for Cu,  while Fe,  Mn and 
Ni ranges were not significantly different between all sites 
(p<0.05, H-test). It might be assumed that because Cu, Fe and 
Mn are essential ions  in insect metabolism there are 
physiological mechanism, which aids in its regulations and 
prevent toxic levels. One explanation for site independent 
nickel distribution is that the volatility of this element is 
much lower in comparison with Cd and Pb. Taking into 
account that grasshoppers are herbivorous  insects, pooled 
plant samples were analyzed from the same sample sites. An 
analysis of the correlation coefficient (linear, r2) between 
metal concentrations in pooled plant samples and insects 
revealed significant (P<0.05) relationship for Cd (r2=0.95) 
and Pb (r2=0.93).  
It should be pointed, that Cu, Cd Mn, Ni and Pb 
concentrations differ significantly with seasons. In general 
for all sample sites summer captured individuals accumulated 
more Cd, Cu, Mn, Ni and Pb then spring captured animals. 
This accumulation is more pronounced for Cd and Pb and 
less expressed for Cu, Mn and Ni. Season accumulation 
pattern (spring-summer) is well established for insects 
captured from more polluted sites e.g. site 4 and site 5. 
Season accumulation (summer-autumn) was observed only 
for Pb. 
In autumn site-specific metal accumulation  patterns were 
found only for Pb. For all other investigated elements same 
levels of content were analyzed for five sample sites.  
 Site-independent and season independent accumulation 
may explain the rather tangled picture of Fe levels in 
grasshoppers. Probably some specific feeding and life habits 
of these insects reflect on Fe accumulation. 
Conclusion 
- Cadmium and lead  the main pollutants of the region, 
became heavily accumulated in grasshoppers at the 
most contaminated sites.  
- Possitive correlation was found between Cd and Pb 
concentrations in pooled plant samples and in insects 
from the same sample site. 
- Iron and manganese appeared to be under homeostatic 
control. 
- The expression of site-specificity of copper another 
main pollutant for this region and Ni is not  clear. 
 
 
 
XI ANNIVERSARY SCIENTIFIC CONFERENCE                                BIOTECHNOL. & BIOTECHNOL. EQ. 23/2009/SE 
120 YEARS OF ACADEMIC EDUCATION IN BIOLOGY                                                   SPECIAL EDITION/ON-LINE 
45 YEARS FACULTY OF BIOLOGY 
206
1 2 3 4 5
0.0
0.2
0.4
0.6
0.8
1.0
Ca
dm
iu
m
 c
on
c.
 ( µ
g/
g 
dw
)
Sites
 spring
 summer
 autumn
1 2 3 4 5
0
2
4
6
8
10
12
14
16
Le
ad
 c
on
c.
 ( µ
g/
g 
dw
)
Sites
 spring
 summer
 autumn
 
 
1 2 3 4 5
0
20
40
60
80
C
op
pe
r c
on
c.
 ( µ
g/
g 
dw
)
Sites
 spring
 summer
 autumn
1 2 3 4 5
0
40
80
120
160
200
240
 spring
 summer
 autumn
Iro
n 
co
nc
. (
µg
/g
 d
w
)
Sites
 
 
1 2 3 4 5
0
20
40
60
80
M
an
ga
ne
se
 c
on
c.
 ( µ
g/
g 
dw
)
Sites
 spring
 summer
 autumn
1 2 3 4 5
0.0
0.2
0.4
0.6
0.8
N
ic
ke
l c
on
c.
 ( µ
g/
g 
dw
)
Sites
 spring
 summer
 autumn
 
Fig. 1. Median and quartiles of metal concentrations in grasshoppers at different sites for spring, summer and autumn 
 
 
BIOTECHNOL. & BIOTECHNOL. EQ. 23/2009/SE              XI ANNIVERSARY SCIENTIFIC CONFERENCE 
SPECIAL EDITION/ON-LINE                   120 YEARS OF ACADEMIC EDUCATION IN BIOLOGY 
               45 YEARS FACULTY OF BIOLOGY 
207
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3. Hopkin S. P. (1989). Ecophysiology of Metals in 
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6. Martin M. H., Coughtrey P. J. (1982).  Biological 
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20 YEARS OF ACADEMIC EDUCATION IN BIOLOGY SPECIAL EDITION/ON-LINE 45 YEARS FACULTY OF BIOLOGY METAL ACCUMULATION IN INSECTS (ORTHOPTERA, ACRIDIDAE) NEAR A COPPER SMELTER AND COPPER-FLOTATION FACTORY (PIRDOP, BULGARIA)

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20 YEARS OF ACADEMIC EDUCATION IN BIOLOGY SPECIAL EDITION/ON-LINE 45 YEARS FACULTY OF BIOLOGY METAL ACCUMULATION IN INSECTS (ORTHOPTERA, ACRIDIDAE) NEAR A COPPER SMELTER AND COPPER-FLOTATION FACTORY (PIRDOP, BULGARIA)

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