Arch. Environ. Contam. Toxicol.

45, 399 406 (2003)

DOI: 10.1007/s00244-003-0198-y

A R C H I V E S O F

Environmental
Contamination
a n d Toxicology
2003 Springer-Verlag New York Inc.

Egrets as Monitors of Trace-Metal Contamination in Wetlands of Pakistan

E. Boncompagni,1 A. Muhammad,2 R. Jabeen,3 E. Orvini,4 C. Gandini,5 C. Sanpera,6 X. Ruiz,6 M. Fasola1
1
2
3
4
5
6

Dipartimento Biologia Animale, Universita`, Piazza Botta 9, I-27100 Pavia, Italy

Ecotoxicology Research Program, National Agricultural Research Center, Park Road, Islamabad 45500, Pakistan
World Wildlife Fund-Pakistan, P.E.C.H.S., 606-607 Fortune Center, Shahra-e-Faisal, Karachi 75400, Pakistan
Dipartimento Chimica Generale, Universita`, Viale Taramelli, I-27100 Pavia, Italy
Dipartimento Chimica Farmaceutica, Universita`, Viale Tarmelli, I-27100 Pavia, Italy
Departamento Biologia Animal, Universidad de Barcelona, Avenida Diagonal 645, 08028 Barcelona, Spain

Received: 3 September 2002 /Accepted: 16 April 2003

Abstract. Environments in Pakistan are subject to increasing

pollution, but previous studies were very scanty. During 1999
and 2000, we assessed trace element contamination at three
wetlands, Karachi Harbour (with presumed industrial-urban
pollution), Taunsa Barrage (agricultural pollution), and Haleji
Lake (relatively unpolluted), using as indicators the eggs and
the feathers of colonial waterbirds, particularly Little Egrets,
their prey, and the sediments collected within their foraging
areas. The concentrations of As, Cd, Cr, Pb, Hg, Mn, Se, and
Zn were generally within the normal background level, and
mostly below the threshold that may affect bird survival or
reproduction. 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. Alarming concentrations were
found for Cr and Se in sediments from Karachi, that were
above the critical levels for contaminated soil, and Se in eggs,
that may affect egret reproduction. The differences among the
three wetlands were less marked than hypothesized. The egret
species within the same area differed in the concentration of
certain elements in their eggs, possibly because females may
have foraged in different habitats before breeding, whereas no
interspecies difference was found in chick feathers, presumably
because their food had been collected in similar habitats around
the colony. High bioaccumulation from sediments to organic
samples occurred for Hg, while Cd, Se, and Zn exhibited low
accumulation; for all these elements, feathers of predatory
birds such as the egrets are the best indicators of environmental
contamination. On the other hand, As and Cr did not bioaccumulate, and the sediments, or the organisms low in the food
chain, like fish or crustaceans, are better indicators of their
presence in the environment than predatory birds.

As the environments in Pakistan are subject to increasing

Correspondence to: M. Fasola; email: fasola@unipv.it

pollution from industrial, urban, and agricultural sources, there

is a growing urgency to monitor contaminant levels, and to
assess their effects. However, previous studies on contamination in Pakistan were limited to a few reports of contaminants
in water and in aquatic organisms (Tariq et al. 1994; Akhtar et
al. 1997; Khan and Khan 1997), and in the laggar falcon
(Movalli 2000).
We assessed the persistent pollutants in selected wetlands of
Pakistan and of China during 1999 and 2000, using as indicator
the Little Egret (Egretta garzetta), a widespread colonial
waterbird (Ciconiiformes: Ardeidae). The results on organic
contaminants in Pakistan were reported by Sanpera et al.
(2003). In this paper, we assess contamination in Pakistan by
trace elements of environmental concern, Arsenic (As), Cadmium (Cd), Chromium (Cr), Lead (Pb), Manganese (Mn),
Mercury (Hg), Selenium (Se), and Zinc (Zn). We studied three
wetlands, presumably subject to different pollution regimes,
Karachi Harbour (industrial-urban pollution), Taunsa Barrage
(agricultural pollution), and Haleji Lake (relatively unpolluted). In order to assess contamination at different levels through
the wetland ecosystem, we used as indicators the eggs and the
chick feathers of predatory egrets, their prey, and the sediments
collected within their foraging areas. When Little Egrets were
unavailable, we sampled similar and syntopic species, the
Intermediate Egret Egretta intermedia, and the Cattle Egret
Bubulcus ibis. Predatory birds such as egrets have been advocated as bioindicators of pollution since they are at the top of
their food webs, are particularly susceptible to bioaccumulation, and integrate contaminants over time (Custer et al. 1990;
Furness and Greenwood 1993). Collateral aims of our research
were: to assess the bioaccumulation of the trace elements from
sediments through various organic materials, eggs, feathers,
and prey; to check the use of these sample materials for
assessing environmental pollution by trace elements; and to
tests the variability among similar and sintopic species in
reflecting the pollution of the same environment.
Bioaccumulation of metals in birds is less documented than
for organic contaminants (Clarkson 1995). The few existing
studies compared feathers to body tissues or eggs (Honda et al.
1986; Spahn and Sherry 1999), and the intake through the food

400

E. Boncompagni et al.

chain (Pascoe et al. 1996). It is generally believed that eggs

indicate the general contamination of the environment where
the female bird was feeding during egg formation, usually the
wetlands around the colony, although the birds prior to breeding may derive their body burden from a larger area. Several
authors advocated the use of eggs of predatory birds such as
egrets, as effective materials for monitoring exposure to contaminants (Kushlan 1993; Erwin and Custer 2000; Custer et al.
1990; Furness and Greenwood 1993). Eggs are easy to collect,
their homogeneous composition facilitates data interpretation,
and collecting eggs poses low risks to the study species. Feathers reflect local contamination better than eggs, since birds
sequester and excrete heavy metals in their feathers during the
short period of plumage growth (Burger 1993). Therefore, trace
elements in chick feathers derive mostly from the prey collected by their parents around the colony during chick development, with a minimal derivation from the egg (Burger et al.
1992). The prey and the sediment may indicate contamination
of particular habitats. For sediments, the main source of heavy
metals is the soil material, but surface contamination with trace
metals is evident in sites with a history of pollution (Pendias
and Pendias 1992).
On the applicative side, our results on pollution level in three
of the main wetlands of Pakistan will (1) provide baseline data
for comparison with future conditions in Pakistan and elsewhere in Asia, where contamination studies are still scanty; (2)
highlight potential threats to human health; (3) provide information for the management of the study wetlands, and for the
strengthening of pollution control regulations.

Materials and Methods

Study Areas
During the egret breeding season 1999 and 2000, we sampled three
study areas, selected for their presumed differing exposure to contamination, for the presence of breeding egret colonies, and for their
national and global importance (Figure 1):

Haleji Lake, Sindh (2548N, 6747E), a freshwater reservoir

(10.5 km2) with associated marshes and lagoons. There are no
agricultural nor industrial activities in the surroundings, therefore
contaminants may reach the lake only through water inflow and the
atmosphere, and the area was presumed relatively unpolluted.
Samples were collected in May 1999 at colonies, located on reed
beds on the lake, with 60 nests of Little Egrets, 300 of Intermediate
Egrets, 200 of Night Herons Nycticorax nycticorax, and 500 of
Little Cormorants Phalacrocorax niger.
Taunsa Barrage, Punjab (3042N, 7050E), a storage reservoir
behind a barrage on the River Indus. Adjacent areas are cultivated
with cotton, sugar cane, wheat, and the barrage is used also for
power generation. Therefore, we expected contamination from
agricultural, and to a lesser degree from industrial sources. Samples
were collected in MayJune 2000 at a colony 5 km west of the
barrage, with 26 nests of Little Egret, 22 of Intermediate Egret, 305
of Cattle Egret, and 15 of Night Heron.
Karachi Harbor (2447N, 6711E), an area of tidal creeks, mangroves, and intertidal mudflats, around the harbor of Karachi (8
million people). About 45% of Pakistani industries are located near
Karachi, and all their effluents plus the city sewage find their way,
untreated, into the sea (A. Khan, Unpublished report). The harbor

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.

For transport in difficult logistic conditions, eggs, prey, and sediments

were homogenized, dried at 50C to constant weight, and stored in
polyethylene vials clean from metals.

Egrets as Trace-Metal Monitors

401

Chemical Analysis

Results

The samples were analyzed at Pavia University (Dipartimento

Chimica and Dipartimento Chimica Farmaceutica), using two techniques: Atomic Adsorption Spectrophotometry (AAS) for Cd, Cr, Hg,
and Pb; and Neutronic Activation Analysis (NAA) for As, Mn, Se, Zn.
Unfortunately, some feather samples were too small (mass 1 g) for
both techniques, so we devoted this limited material only to one
analysis, and feather concentration data were not available for some
elements. Similarly, some sediment samples could only be analyzed by
AAS, and concentrations for some elements were not available.

In the eggs, most elements were almost ubiquitous (Table 1):

Cr, Pb, Hg, Se, and Zn were detected in all or in the majority
of the samples from each study area. Cd was absent from
Taunsa but widespread elsewhere. Arsenic was present in few
samples. Between the pairs of egret species sampled at each
area, significant differences were found for Cd, Cr, and Pb,
with Little Egret showing higher concentrations than Intermediate Egrets at Haleji. Given these differences between egret
species, we based all the comparisons among areas on the Little
Egret samples only. The concentrations were similar for the
three areas, for most elements in eggs. Significant differences
were found only for Cd and for Pb, with higher levels at Haleji
(see p values in Table 1). The pairwise tests showed significant
differences for both Cd and Pb between Haleji and Karachi, but
not between Haleji and Taunsa, probably because of the low
number of samples from Taunsa. Differences close to significance were also found for Se (higher at Karachi) and for Cr and
Zn (higher at Haleji).
Feather samples were limited in amount, and consequently
the analyses were not possible for some elements and areas
(Table 2). The pattern of element presence in feathers duplicate
the one in the eggs: most elements were ubiquitous, while As
was less frequent. No difference emerged between egret species for element concentration in feathers, neither at Haleji nor
at Taunsa. Among areas, significant differences in concentration were found only for Hg, with lower values at Haleji (but
the pairwise tests with the other two areas were nonsignificant).
For prey, all the elements were almost ubiquitous (Table 3),
as found for eggs and feathers. Significant differences in concentration were found for Cr, Hg, and Pb, with highest levels at
Karachi; the pairwise comparisons were significant only between Karachi and Haleji. We recall that the egret diet composition differed among study areas. Prey were only fish at
Karachi and at Haleji, while frogs, tadpoles, insects, and mice
were included in the diet at Taunsa. Therefore, the comparison
of concentrations was not homogenous for prey type.
Unfortunately, sediment concentrations (Table 4) were not
available for some elements at Haleji. Different concentrations
emerged for Cd (significantly lower at Taunsa than at both
other two areas), for Cr (significantly higher at Karachi than at
both other two areas), for Pb (at Karachi significantly higher
than at Taunsa, and higher but not significantly so than at
Haleji), and for Hg (significantly higher at Karachi than at both
other two areas).
Bioaccumulation ratios from sediment through prey, feathers,
and eggs, are detailed for each element in Table 5. The elements
can be classified in relation to the magnitude of their bioaccumulation ratios from sediments to organisms. Hg exhibited a high
accumulation, ratios from 3 to 26, and increased by one order of
magnitude from prey to their predatory birds. Cd, Se, and Zn had
low accumulation, ratios between 0.2 and 2, and usually decreasing slightly from prey to predator. All the other elements exhibited
no accumulation, ratios lower than 0.2, and decreasing from prey
to predators. Unfortunately, data are lacking for Cd and for Pb in
feathers. The variability in accumulation ratios among areas, however, was high (see the standard deviations in Table 5), and the
variation of the elements with low or no accumulation, was often
larger than their mean values.

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.

Table 1. Concentration of elements of environmental concern in eggs

Arsenic
Haleji

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 )

Cadmium Chromium Lead

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.

Table 2. Concentration of elements of environmental concern in feathers

Arsenic
Haleji Lake

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

Difference between species, Haleji (p )

Difference between species, Taunsa (p )
Difference among areas, Little Egrets only (p )

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-

stan were similar to those in water birds from Europe (Renzoni et

al. 1986; Becker and Sperveslage 1989), but they were much
lower than those from US (Gochfeld 1997). The concentrations
we found in prey were similar to those found in heron food from
USA (Blus et al. 1985; Spahn and Sherry 1999).
Chromium is an essential element for animals, but tissue
concentrations 4 ppm dry wt indicate alarming contamination
(Outridge and Scheuhammer 1993). Levels of Cr in eggs from
Pakistan were lower than in eggs of herons (Rattner et al. 2000)
and of gulls (Gochfeld 1997) in the United States. The levels

Egrets as Trace-Metal Monitors

403

Table 3. Concentration of elements of environmental concern in prey

Arsenic
Haleji Lake

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.

Table 4. Concentration of elements of environmental concern in sediments

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.

we found in feathers were far lower than those in egrets and

storks (Burger et al. 1992; Burger and Gochfeld 1993), and
were similar to those in feathers of Little Egrets from Hong
Kong (Connel et al. 2002). No toxic hazards are expected at the
levels we found.
For lead, no thresholds levels in feathers or eggs have been
proposed. The levels we found in Pakistan were lower than in
eggs of gulls (Gochfeld 1997), and in feathers of Little Egrets
(Fasola et al. 1998), and similar to those found in seabirds and
in herons from Europe (Renzoni et al. 1986; Becker and
Sperveslage 1989). Much higher Pb levels were found in egret
feathers near Hong Kong (Burger and Gochfeld 1993). The

levels in prey from Haleji Lake and Taunsa were similar to

those found in food of herons from the United States (Spahn
and Sherry 1999), while the level of Pb in fish from Karachi
were much higher, by about one order of magnitude.
Mercury is a pervasive contaminant. Normal, background
concentration for egrets could be 0.25 ppm wet weight (wet
wt) in eggs (King et al. 1991), and 4 ppm in feathers (Doi et al.
1984). Several studies indicate that levels of 1.5 ppm in eggs,
and 5 40 ppm in feathers, are associated with adverse effects
(Scheuhammer 1987; Burger and Gochfeld 1997). Concentrations of Hg we found in Pakistan were lower, or within the
range found in eggs of egrets and seabirds (Renzoni et al. 1986;

404

Burger and Gochfeld 1997; Gochfeld 1997; Rattner et al.

2000). In feathers, the levels we found were well below the
concentration that may affect birds, and were lower than those
found elsewhere in egrets and in other water birds (Honda et al.
1986; Goutner and Furness 1997; Fasola et al. 1998; Burger et
al. 1992; Burger and Gochfeld 1993, 1997; Goutner et al.
2000; Connel et al. 2002; Frederick et al. 2002). The values we
found in prey fish from Pakistan (maximum 0.097 0.57 ppm
dry wt from Karachi) were similar to those found in Mediterranean fish (Goutner et al. 2000), and they were below the
concentration associated with toxic effects in freshwater fish
(510 ppm wet wt; Beyer et al. 1996). However, the admitted
limit for seafood consumption by humans in most countries is
0.5 ppm, and the Hg level we found in fish from Karachi were
around this concentration.
Arsenic, a toxic, nonessential element, is readily bioconcentrated. Levels of As similar to ours were found in eggs of
herons (Blus et al. 1985).
Manganese, an essential element, may become toxic at high
doses. Relatively little is known about Mn levels in birds. For
eggs, the levels found in Pakistan were within the range of
values found elsewhere in water birds (Gochfeld 1997; Gochfeld and Burger 1998; Rattner et al. 2000). For feathers, the
concentrations in our samples were higher than in egrets from
Korea (Honda et al. 1986), similar to those in egrets from the
United States and Egypt (Burger et al. 1992), and lower than in
egrets from Hong Kong (Burger and Gochfeld 1993; Connel et
al. 2002).
Selenium is an essential element, but may become toxic at
concentrations only twice those required (Lemly 1997). In
water birds, background concentration in eggs average 0.32.1
ppm wet wt (Gochfeld and Burger 1998), but higher levels,
3 6 ppm dry wt, were also found (Renzoni et al. 1986; Custer
et al. 1998; Rattner et al. 2000). The concentrations we found
in eggs from Pakistan were similar to these relatively high
levels, and were in some cases higher than the threshold, 35
ppm dry wt, that should affect avian reproduction (Beyer et al.
1996; Ohlendorf et al. 1986). About feathers, the concentrations of Se we found in Pakistan were similar to those found for
water birds (Burger et al. 1992; Burger and Gochfeld 1993).
The threshold that seems to affect fish is 4 ppm dry wt (Beyer
et al. 1996), and the values we found in fish from Pakistan were
below this concentration.
The levels of zinc in eggs of wild birds have rarely been
documented. In the egg of some Mediterranean seabirds, Zn
ranged from 37 to 86 ppm dw (Renzoni et al. 1986). The values
we found in eggs and feathers from Pakistan were far lower
than those found in egret feathers from Korea (Honda et al.
1986), and in eggs of egrets and of seabirds (Renzoni et al.
1986; Morera et al. 1997; Connel et al. 2002), therefore these
concentrations were probably not detrimental. The Zn levels
we found in prey (shrimps) were below those causing death in
crustaceans (200 ppm; White and Raimbow 1982).
Regarding sediments, the concentrations of Hg, As, Zn, Cd,
Cr, Pb we found in Pakistan were lower than the critical levels
assumed as threshold for contaminated soil (Netherlands Ministry of Housing, and Canadian Council of Minister, in Alloway and Ayres 1997), with the only exception of Cr from
Haleji sediments that was above the critical level.
In summary, the concentrations of the various elements in
the samples from Pakistan were generally within the known

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.

Differences Among Areas in Pakistan

Overall, the differences among the three study areas were less
marked than we had hypothesized, and only partially confirmed
our initial expectation that Haleji was clean, Taunsa intermediate, and Karachi highly polluted. Indeed, Karachi had higher
concentrations of the heavy metals Cd, Cr, Pb, and Hg, especially in sediments and prey, but the concentrations differed
from the other two areas only by 50 100% for most elements
and sample types, a difference that can be considered relatively
limited. Only for Hg, the concentrations at Karachi differed
considerably, by one order of magnitude, from the other two
areas. On the other hand, in some samples the concentrations
were higher at Taunsa (e.g., Hg in feathers) or at Haleji (e.g.,
Cd and Pb in eggs). In summary, Karachi had the highest
concentrations of elements of environmental concern, but at
levels only moderately higher than the other two areas: Taunsa
and Haleji were similar, except for surprisingly high levels of
Cd in eggs at Haleji, and for Hg in feathers at Taunsa. In
general agreement with these overall evaluations of contamination levels, Sanpera et al. (2002, 2003) found decreasing
contamination with organochlorines and PCBs, from Karachi,
to Taunsa, to Haleji, and relatively limited differences.

Differences between Egret Species

The three egret species we sampled within the same area
differed significantly in the concentration of certain elements in
their eggs, while no interspecies difference was found in chick
feathers. Eggs reflect the exposure of the laying female to
contaminants in the environment, both prior and during egg
formation. On the other hand, chick feathers reflect the intake
during the relatively short period of feather growth, and from a
foraging area close to the colony. In each of our three study
areas of Pakistan, we observed the different species of egrets
foraging on the same habitats around their colony during chick
development. At Taunsa, both Little and Cattle egrets foraged
on small lakes, channels, ponds, other wetlands, rice paddies,
flooded or dry fields; at Karachi, both Little and Intermediate
egrets foraged on channels, mangroves, and intertidal mud
surfaces. We conclude that in our samples from Pakistan, the
egg content differed between egret species because females
may have foraged in different habitats before breeding, while

Egrets as Trace-Metal Monitors

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.

Acknowledgments. This study was part of the research on colonial

waterbirds as bioindicators of pollutant levels and effects in China and

405

Pakistan, funded by the European Union (IC18-CT98-0294). We wish

to thank U. K. Baloch (Islamabad), the staff of WWF-Karachi, and G.
Gaudenzi for their help with fieldwork; M. Coppola and S. Comizzoli
for the AAS analysis; R. A. Khan (Haleji Lake) and the Sindh and
Punjab Wildlife Departments for logistic support.

References
Akhtar W, Ali I, Zaidi SSH, Jilani S (1997) The state of pollution
levels of Karachi Harbour and adjoining coastal water. Water Air
Soil Poll 94:99 107
Alloway BJ, Ayres DC (1997) Chemical principles of environmental
pollution. Blackie, London
Bader M (1980) A systematic approach to Standard Addition Methods
in instrumental analysis. Chem Edu 57:703
Becker PH, Sperveslage H (1989) Organochlorines and heavy metals
in herring gull (Larus argentatus) eggs and chicks from the same
clutch. Bull Environ Contam Toxicol 42:721727
Beyer WN, Heinz GH, Redmon-Norwood AW (1996) Environmental
contaminants in wildlife. Interpreting tissue concentrations.
Lewis, Boca Raton, Florida
Blus LJ, Henny CJ, Anderson A, Fitzner RE (1985) Reproduction,
mortality, and heavy metal concentrations in Great Blue Herons
from three colonies in Washington and Idaho. Colonial Waterbirds 8:110 116
Burger J (1993) Metals in avian feathers: Bioindicators of environmental pollution. Rev Environ Toxicol 5:203311
Burger J, Gochfeld M (1993) Heavy metal and selenium levels in
feathers of young egrets and herons from Honk Kong and
Szechuam, China. Arch Environ Contam Toxicol 25:322327
Burger J, Gochfeld M (1995) Biomonitoring of heavy metals in the
Pacific basin using avian feathers. Environ Toxicol Chem 14:
12331239
Burger J, Gochfeld M (1997) Risk, Mercury levels, and birds: Relating
adverse laboratory effects to field biomonitoring. Environ Res
75:160 172
Burger J, Parsons K, Benson T, Shukla T, Rothsyein D, Gochfeld M
(1992) Heavy metal and selenium levels in young Cattle Egrets
from nesting colonies in the Northeastern United States, Puerto
Rico and Egypt. Arch Environ Contam Toxicol 23:435 439
Clarkson TW (1995) Environmental contaminants in the food chain.
Am J Clin Nutr 61 (Suppl. 3):682 686
Connel DW, Wong BSF, Lam PKS, Poon KF, Lam MHW, Wu RSS,
et al. (2002) Risk to breeding success of Ardeids by contaminants
in Hong Kong: Evidence from trace metals in feathers. Ecotoxicology 11:49 59
Custer TW (2000) Environmental contaminants. In: Kushlan JA,
Hafner H (eds) Heron conservation. Academic Press, London, pp
251268
Custer TW, Pendleton G, Ohlendorf HM (1990) Within-clutch and
among-clutch variation of organochlorine residues in eggs of
black-crowned night-herons. Environ Monitor Assess 15:8390
Custer TW, Hines RK, Stewart PM, Melancon MJ, Henshel DS,
Sparks DW (1998) Organochlorine, mercury and selenium in
great blue heron eggs from Indiana Dunes National Lakeshore,
Indiana. J Great Lakes Res 24:311
Doi R, Ohno H, Harada M (1984) Mercury in feathers of wild birds
from the mercury-polluted area along the shore of the Shiranui
Sea, Japan. Sci Total Environ 40:155167
Eisler R (1988) Arsenic hazards to fish, wildlife, and invertebrates: A
synoptic review. US Fish and Wildlife Service Biological Report
85 (1.12)
Erwin M, Custer TW (2000) Herons as indicators. In: Kushlan JA,
Hafner H (eds) Heron conservation. Academic Press, San Diego,
California, pp 310 330

406

Fasola M, Movalli PA, Gandini C (1998) Heavy metal, organochlorine

pesticide, and PCB residues in eggs and feathers of herons breeding in northern Italy. Arch Environ Contam Toxicol 34:8793
Frederick PC, Spalding MG, Dusek R (2002) Wading birds as bioindicators of mercury contamination in Florida, USA: Annual and
geographic variation. Environ Toxicol Chem 21:163167
Furness RW, Greenwood JJD (1993) Birds as monitors of environmental change. Chapman and Hall, London
Furness RW, Lewis SA, Mills JA (1990) Mercury levels in the
plumage of red-billed gulls (Larus novaehollandie scopulinus) of
known sex and age. Environ Poll 63:3339
Gauglhofer J, Bianchi V (1991) Chromium. In: Merian E (ed) Metals
and their compounds in the environment. VCH Publisher, Weinheim, Germany
Gochfeld M (1997) Spatial patterns in bioindicator: Heavy metal and
selenium concentration in eggs of Herrin gulls (Larus argentatus)
in the New York bight. Arch Environ Contam Toxicol 33:6370
Gochfeld M, Burger J (1998) Temporal trends in metal levels in eggs
of the endangered Roseate Tern (Sterna dougallii) in New York.
Environ Res 77:36 42
Goutner V, Furness RW (1997) Mercury in feathers of little egret
Egretta garzetta and night heron Nycticorax nycticorax chicks and
in their prey in the Axios Delta, Greece. Arch Environ Contam
Toxicol 32:211216
Goutner V, Furness RW, Papakonstantinou K (2000) Mercury in
feathers of Audouins gull (Larus audouinii) chicks from northeastern mediterranean colonies. Arch Environ Contam Toxicol
39:200 204
Holm S (1979) A simple sequentially rejective multiple test procedure.
Scan J Stat 6:6570
Honda K, Min BY, Tatsukawa R (1986) Distribution of heavy metals
and their age-related changes in the eastern great white egret,
Egretta alba modesta, in Korea. Arch Environ Contam Toxicol
15:185197
Khan SA, Khan M (1997) Monitoring of water pollution in the Kabul
River (Pakistan) under low flow conditions. J Chem Soc Pakistan
19:126 132
King KA, Custer TW, Quinn JS (1991) Effects of mercury, selenium
and organochlorine contaminants on reproduction of Forsters
terns and Black skimmers nesting in a contaminated Texas bay.
Arch Environ Contam Toxicol 20:32 40
Kushlan JA (1993) Colonial waterbirds as bioindicators of environmental change. Colonial Waterbirds 16:223251
Lemly AD (1997) Environmental implications of excessive selenium:
A review. Biomed Environ Sci 10:415 435
Lewis SA, Becker PH, Furness RW (1993) Mercury levels in eggs,
tissues, and feathers of herring gulls Larus argentatus from the
German Wadden Sea coast. Environ Poll 80:293299
Mattig FR, Ro sner H-U, Giessing K, Becker PH (2000) Environmental
chemicals in eggs of Dunlins (Calidris alpina) from Northern
Norway compared to eggs of coastal bird species breeding in the
Wadden Sea. J Ornithol 141:361369
Millard SP, Neerchal NK (2001) Environmental Statistics with S-Plus.
CRC Press, Boca Raton, Florida
Morera, M, Sanpera C, Crespo S, Jover L, Ruiz X (1997) Inter and
intraclutch variability in heavy metals and selenium levels in
Audouins Gull eggs from the Ebro Delta, Spain. Arch Environ
Contam Toxicol 33:7175

E. Boncompagni et al.

Movalli PA (2000) Heavy metal and other residues in feathers of

Laggar Falcon Falco biarmicus jugger and the use of this species
as a bioindicator of environmental pollution in Pakistan. Environ
Poll 109:267275
Ohlendorf HM, Hothem RL, Bunck CM, Aldrich TW, Moore JF
(1986) Relationships between selenium concentrations and avian
reproduction. Trans N Am Wildl Nat Res Conf 51:330 342
Outridge PM, Scheuhammer AM (1993) Bioaccumulation and toxicology of chromium: Implications for wildlife. Rev Environ Contam Toxicol 130:3177
Pascoe GA, Blasnchet RJ, Linder G (1996) Food chain analysis of
exposure and risks to wildlife at a metal-contaminated wetland.
Arch Environ Contam Toxicol 30:306 318
Pendias AK, Pendias H (1992) Trace elements in soils and plants,
second edition. CRC Press, London
Rattner BA, Hoffman DJ, Melancon MJ, Olsen GH, Schmidt SR,
Parsons KC (2000) Organochlorine and metal contaminant exposure and effects in hatching Black-crowned night herons (Nycticorax nycticorax) in Delaware Bay. Arch Environ Contam Toxicol 39: 38 45
Renzoni A, Focardi S, Fossi C, Leonzio C, Mayol J (1986) Comparison between concentrations of mercury and other contaminants in
eggs and tissues of corys shearwater Calonectris diomedea collected on Atlantic and Mediterranean islands. Environ Poll 40:
1735
Sanpera C, Ruiz X, Llorente GA, Jover L, Jabeen R (2002) Persistent
organochlorine compounds in sediment and biota from the Haleji
Lake: A wildlife sanctuary in south Pakistan. Bull Environ Contam Toxicol 68:237244
Sanpera C, Ruiz X, Jover L, Llorente G, Jabeen R, Muhammad A, et
al. (2003) Persistent organic pollutants in wetlands from Pakistan:
Using egrets eggs as monitoring tools. Arch Environ Contam
Toxicol 44:360 368
Scheuhammer AM (1987) The chronic toxicity of aluminum, cadmium, mercury and lead in birds: A review. Environ Poll 46:263
295
Spahn SA, Sherry TW (1999) Cadmium and lead exposure associated
with reduced growth rates, poorer fledging success of little blue
heron chicks (Egretta caerulea) in South Louisiana wetlands.
Arch Environ Contam Toxicol 37:377384
Spalding MG, Bjork RD, Powell GVN, Sundlof SF (1994) Mercury
and cause of death in Great White Herons. J Wildl Manage
58:735739
Spalding MG, Frederick PC, McGill HC, Bouton SN, McDowell LR
(2000) Methylmercury accumulation in tissues and its effects on
growth and appetite in captive great egrets. J Wildl Dis 36:41122
Tariq J, Jaffar M, Ashraf, M, Moazzam M (1994) Trace metal concentration, distribution and correlation in water, sediment and fish
from the Ravi River, Pakistan. Fisheries Research 19:131139
White DH, Finley MF, Ferrel JF (1978) Histopathologic effects of
dietary cadmium on kidneys and testes of Mallard ducks. J Toxicol Environ Health 4:551558
White SL, Rainbow PS (1982) Regulation and accumulation of copper, zinc and cadmium by the shrimp Palaemon elegans. Mar Ecol
Prog Ser 8:95101
Wolfe MF, Schwarzbach S, Sulaiman RA (1998) Effects of mercury
on wildlife: A comprehensive review. Environ Toxicol Chem
17:146 160