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Environmental
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a n d Toxicology
2003 Springer-Verlag New York Inc.
400
E. Boncompagni et al.
Fig. 1. The three study areas in Pakistan and their presumed pollution
levels
is also exposed to ship wastes and oil spills. Therefore, we expected considerable urban and industrial pollution. Samples were
collected in May 2000 at a colony in the Ghas Bunder lagoon, with
115 nests of Little Egret, 20 nests of Intermediate Egret, and ten
nests of Indian Pond Heron Ardeola grayii.
Sample Collection
At each study area, we marked sample nests with eggs in one egret
colony. Priority was given to Little Egrets, but at Haleji and Taunsa,
Little Egret nests were few, therefore we sampled also nests of Intermediate or Cattle Egrets. All these egrets belong to the same genus,
breed in mixed colonies, and forage on the same habitats within 510
km around the colony. At Karachi, Little Egret belonged to the dark
morph (subspecies gularis), while at the other areas they belonged to
the white morph (subspecies garzetta). At each area, using uniform
techniques, we collected the following:
Fresh eggs: One per nest, and ten eggs per area whenever possible.
Feathers: 10 20 body feathers taken from one chick per nest, the
same nests where eggs had been collected, 10 15 days after
hatching. Newly grown feathers of chicks minimize the problem of
external attachment of contaminants to the feather.
Prey: Diet samples from the spontaneous regurgitation of the egret
chicks when handled. Prey items were sorted into main types; one
composite sample was then made of several items of the same
type, collected from one nest. At Haleji, the 19 samples were pools
of four fish species (Oreochromis niloticus, Puntius phuturio,
Colisa lalia, Glossogobius giuris). At Karachi, fish (Liza abu) were
included in each of three pooled samples. At Taunsa, the five prey
samples corresponded to pools of frogs and tadpoles, insets, and
mice.
Sediments: One surface layer, 5-cm deep from areas most frequently used by the egrets for foraging (assessed during repeated
surveys of the wetlands within 10 km of the colony). Each sample
consisted of a mixture of 4 12 individual sediments, collected at
different spots within the same habitat, in order to obtain more
representative samples.
401
Chemical Analysis
Results
AAS. Cd, Cr, and Pb were analyzed by a Perkin Elmer Analiyst 600
AA Spectrophotometer, equipped with transversely heated graphite
furnace, longitudinal AC Zeeman-effect background correction, and
autosampler AS-800. Hg was analyzed by a Perkin Elmer FIAS-100
equipment for cold vapor generation, and by a Perkin Elmer 3110 AA
Spectrophotometer. Instrument control and data calculation were performed on PC using the AA WinLab software. In order to eliminate
organic matter, the samples were mineralized, without previous washing, using a CEM-MDS 81D microwave oven, operating in teflon
bombs with Newtec Valves, and were dissolved in 30% nitric acid, as
described by Fasola et al. (1998). The mineralization technique was
tested for element recovery, on reference material (whole egg powder,
8415 Reference Material, National Institute of Standards and Tecnology). The tests for Cd, Cr, Hg, and Pb, gave recoveries from 98% and
101%. Preliminary tests were carried out using standard solutions of
pure single element, dissolved in nitric acid/water 30% vol/vol at three
concentrations. The calibration curves were linear for the following
ranges of concentrations: Cd, 2 4 6 ppb; Cr, 510 20 ppb; Hg,
2 4 8 ppb; Pb, 3 6 12 ppb; corresponding to detection limits of
0.020, 0.030, 0.010, and 0.030 ppm, respectively. Estimates were
made according to standard addition method (Bader 1980) supported
by PC software.
NAA. Samples irradiation was performed at the irradiation position of the
Triga Mark II Nuclear Reactor (Pavia University) at a flux of 1 1013 n
cm2 s2. Condition and irradiation time, counting, and delay, were
chosen according to the nuclear characteristics of each element. Gamma
ray spectrometry was performed using a high resolution intrinsic Ge
detector. Data were processed using a computer program from ORTEC.
Data Analysis
All concentrations are expressed in g/g (ppm) on dry weight (dry wt).
Concentrations below the detection limit of our techniques, were replaced
by imputed values obtained by quantile-quantile regression (Millard and
Neerchal 2001), after having checked the log-normal data distribution, for
each element and each sample group (i.e. the samples of one type and
from one study area). This procedure allows unbiased and robust descriptive statistics, but it is not applicable to the samples with many values
below detection (i.e. 50%). In these cases we replaced the nondetected
values with one-half the lowest detected concentration. Geometric means
were calculated from imputed values, whereas for the frequency and the
minimum, we used the original values with nondetected.
Differences among means were tested by nonparametric KruskallWallis ANOVA, in order to minimize problems caused by the inputed
values, and by abnormal data distribution. When a difference was
found among study areas, a posteriori pairwise comparisons were
performed using the Mann-Whitney U test and a Bonferroni procedure
to guarantee a tablewise significant 0.05 levels (Holm 1979). Since the
Little Egret was the only species to be found at all study areas, and
since element concentrations differed among egret species (see Results), the intersite differences were tested by comparing the Little
Egret samples only.
402
E. Boncompagni et al.
Little Egret
Number
Mean
Minmax
Intermediate Egret Number
Mean
Minmax
Karachi Little Egret
Number
Mean
Minmax
Taunsa Little Egret
Number
Mean
Minmax
Cattle Egret
Number
Mean
Minmax
Difference between species, Haleji (p )
Difference between species, Taunsa
(p )
Difference among areas, Little Egrets
only (p )
Manganese Mercury
8 (100%)
0.17
0.060.43
20 (14%)
0.01
ND0.044
19 (57%) 14 (59%)
0.211
0.07
ND1.32 ND1.026
4 (25%) 3 (0%)
0.11
ND0.34
9 (10%) 9 (0%)
0.087
ND0.22
0.0001
8 (100%)
0.25
0.180.44
20 (91%)
0.06
ND0.13
17 (100%)
0.08
0.011.2
4 (100%)
0.11
0.050.54
9 (100%)
0.03
0.010.08
0.0001
8 (100%)
0.45
0.290.58
15 (35%)
0.001
ND0.16
14 (93%)
0.06
ND0.10
3 (100%)
0.10
0.060.18
9 (100%)
0.06
0.020.12
0.0001
NS
NS
NS
NS
NS
0.004
NS
0.0001
14 (100%)
1.70
0.95.2
NS
Selenium
Zinc
8 (100%)
0.33
0.220.58
20 (100%)
0.34
0.141.19
16 (100%)
0.31
0.040.84
4 (100%)
0.36
0.200.48
9 (100%)
0.10
0.030.30
NS
8 (100%)
2.28
1.73.4
18 (100%)
2.16
1.03.5
16 (100%)
3.32
1.78.4
4 (100%)
2.91
1.24.1
9 (100%)
2.6
2.03.8
NS
8 (100%)
45.9
25.965.9
18 (100%)
47.6
29.890.8
16 (68%)
2.9
ND329.0
4 (100%)
6.4
1.1028.10
9 (90%)
11.9
ND63.0
NS
0.02
NS
NS
NS
0.06
0.06
The values are: Number of samples, percentage frequency of samples with concentrations above detection limit, geometric mean, maximum and
minimum values. Concentrations in ppm on dry wt. The difference among study areas was tested by Kruskal-Wallis ANOVA, and the difference
among species by Mann-Whitney test. ND not detected. NS nonsignificant.
Little Egret
Intermediate Egret
Karachi
Little Egret
Taunsa
Little Egret
Cattle Egret
Number
Mean
Minmax
Number
Mean
Minmax
Number
Mean
Minmax
Number
Mean
Minmax
Number
Mean
Minmax
12 (50%)
0.48
ND2.62
3 (33%)
0.10
ND1.12
12 (7.6%)
0.10
ND1.23
NS
NS
Chromium
Mercury
Manganese
Selenium
Zinc
4 (100%)
1.26
0.2499.0
3 (100%)
0.22
0.200.25
12 (100%)
0.94
0.148.75
3 (100%)
0.47
0.154.76
12 (100%)
0.72
0.156.55
NS
NS
NS
3 (100%)
0.21
0.160.25
3 (100%)
0.16
0.140.22
11 (100%)
0.89
0.432.65
2 (100%)
0.97
0.711.32
10 (100%)
0.41
0.0211.48
NS
NS
0.03
3 (100%)
2.71
1.64.9
3 (100%)
2.17
1.15.3
12 (0%)
3 (100%)
1.71
1.372.30
3 (100%)
1.15
0.871.36
12 (83%)
3.83
ND23.2
3 (100%)
9.02
3.3527.7
12 (100%)
7.74
3.1821.7
NS
NS
NS
3 (100%)
154.0
123184
3 (100%)
214.0
186264
12 (67%)
199.7
ND1710
3 (33%)
0.4
ND113
8 (11%)
1.8
ND428
NS
NS
NS
Values as in Table 1.
Discussion
Contamination Levels
For each element we discuss the concentrations found in Pakistan, in relation to the levels in other regions, to the threshold
that may affect wild birds, and to concentrations that may
indicate alarming environmental pollution.
For cadmium, the concentrations we found in eggs from Paki-
403
Intermediate
Egret
No.
Mean
Min-max
Karachi
Little Egret
No.
Mean
Min-max
Taunsa
Cattle Egret No.
Mean
Min-max
Difference among study area (p )
5 (80%)
0.55
ND1.35
Cadmium
Chromium
Lead
Manganese
Mercury
19 (100%) 19 (100%)
19 (100%)
19 (100%)
0.09
0.040.15
3 (100%)
0.10
0.070.13
5 (100%)
0.06
0.030.12
NS
0.41
0.190.78
3 (100%)
4.62
3.596.37
5 (100%)
0.73
0.481.05
0.002
0.10
0.060.14
3 (100%)
0.57
0.530.64
5 (80%)
0.03
0.020.13
0.004
0.29
0.230.40
3 (100%)
16.54
10.5524.91
5 (100%)
1.48
0.723.35
0.0001
6 (100%)
130.89
56.2321.2
Selenium
Zinc
5 (100%)
2.31
1.133.49
5 (100%)
69.3
28180
Values as in Table 1.
No.
Mean
Min-max
Karachi
No.
Mean
Min-max
Taunsa
No.
Mean
Min-max
Difference among areas (p )
Arsenic
Cadmium
Chromium
Lead
Mercury
Selenium
Zinc
3
8.63
4.5119.1
3
4.84
4.126.30
NS
10
0.22
0.190.26
9
0.23
0.120.40
10
0.14
0.0571.33
0.011
10
35.13
28.3744.27
9
56.44
18.77117.87
10
25.45
15.7755.98
0.002
10
11.73
9.0915.55
9
18.47
6.2447.72
10
7.72
3.8422.87
0.002
10
0.02
0.020.02
9
0.62
0.172.67
10
0.01
ND0.03
0.0001
3
3.10
2.913.43
3
4.85
3.248.47
NS
3
42.4
7119
3
31.8
1254
NS
Haleji Lake
Values as in Table 1. The percentage frequency of samples with concentrations above detection limit was 100 for all samples, except for Hg at
Taunsa (50%).
Table 5. Bioaccumulation ratios, as proportion of the concentration in eggs, in feathers, and in prey, in relation to the concentration in the
sediments
Arsenic
Cadmium
Chromium
Lead
Mercury
Selenium
Zinc
Egg
Feather
Prey
0.022 (0.003, 3)
0.237 (0.305, 5)
0.003 (0.003, 5)
0.012 (0.015, 5)
14.61 (10.66, 5)
0.737 (0.290, 3)
0.215 (0.154, 3)
0.039 (0.018, 3)
0.114 (1)
0.428 (0.040, 3)
0.120 (0.152, 3)
0.127 (0.111, 3)
2.910 (2.390, 3)
0.477 (1)
2.180 (1)
0.021 (0.011, 5)
26.09 (30.29, 5)
1.563 (0.313, 3)
1.593 (2.698, 3)
The values are mean ratio (standard deviation, sample size). The mean ratios shown here were calculated across all the three study areas and the
three egret species. An individual sample is one egret species at a single study area.
404
E. Boncompagni et al.
background level for water birds, and most of them were below
the threshold that may affect the survival or the reproductive
success of the birds. A similar situation of relatively low
contamination, was found in our three study areas for organochlorine pesticides and PCBs (Sampera et al. in press). However, somewhat high concentrations were found in fish from
Karachi, for Pb that was at levels that may harm fish reproduction, and for Hg that was at limit concentration for human
consumption. Other alarming concentrations were found for Cr
and Se in sediments, especially those from Karachi, that were
above, or about, the critical levels for contaminated soil. Moreover, eggs from Karachi and Taunsa had Se concentrations that
may affect egret reproduction.
feathers were similar because chicks had been fed prey collected in similar habitats around the colony.
Given these differences in element concentration among
egret species, the choice of an indicator species is critical, even
within a guild of similar species, and interareas comparisons
must be based on one single indicator species, particularly for
eggs. Indeed, when similar bird species were compared, their
contamination levels usually differed (Furness and Greenwood
1993; Burger and Gochfeld 1993, 1995; Goutner and Furness
1997; Mattig et al. 2000).
Bioaccumulation
The element that exhibited high bioaccumulation in our material was Hg. Uptake and accumulation of Hg by birds depends
on multiple factors, diet, age, and metabolism (Lewis et al.
1993; Wolfe et al. 1998). Birds deposit Hg and other elements
into growing feathers, where it no longer produces adverse
effects, since Hg bonded to feather keratin becomes physiologically isolated (Spalding et al. 1994). Therefore, feathers are
a very effective pathway to excrete Hg (Honda et al. 1986;
Furness et al. 1990). Hg in feathers reflects mainly the amount
derived from diet at the time when feather are developing,
therefore the level of Hg in chick feather indicates the local
pollution in the foraging area around colonies (Burger 1993).
Cadmium, Se, and Zn exhibited low accumulation in our
materials. Cd is believed not to be accumulated by birds
(Custer 2000) nor to be transferred into their eggs (White et al.
1978). Selenium occurs in many forms, but most of the laboratory data that were used to derive toxic threshold concentrations, were based on the highly toxic form, selenomethionine,
that is believed to be a major, but probably not the only,
chemical form in the food of birds in nature. Nothing is known
about bioaccumulation of Zn.
The other elements exhibited no accumulation in our materials. Inorganic Cr compounds are poorly absorbed by animals,
although natural complexes in diet may be more available and
more toxic (Gauglhofer and Bianchi 1991). Pb is mainly stored
in calcareous tissues (Scheuhammer 1987). Arsenic is widely
distributed in nature, but it has been rarely been determined in
animals, and because of its detoxification and rapid excretion,
chronic poisoning by As is infrequent in wildlife (Eisler 1988).
For the elements subject to bioaccumulation, both high and
low, the feathers of predatory birds, such as the egrets, may be
more sensitive indicators of environmental contamination, as
proposed by Burger (1993). Our study confirms that feathers
harbor higher concentrations of trace metals than eggs (Honda
et al. 1986). However, in our samples, more differences among
areas were significant for contaminants in eggs than in feathers.
But this may be an artifact of the larger sample size for eggs
than for feathers.
On the other hand, for the elements not accumulated in
organisms, the sediments or the organisms low in the food
chain, like fish or crustaceans, are better indicators of their
presence in the environment, because they harbor higher concentrations of these elements than predatory birds.
405
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