Lead Toxicity in Mute Swans

MATERIALS AND METHODS
Blood was obtained from an urban ftock of Mute Swans
Cygnus olor (Gmelin) which were known to have elevated
lead levels (O'Halloran and Duggan, 1984). In addition,
blood samples were collected from Chilean flamingos Pheonicopannus
andinus (Philippi); Black-necked swans Cygnus
melanocurphus (Molina); Emperor Geese Anser canagicus
(Sewastianov); a Canada Goose Branta canadensis (Linnaeus)
and Cape Barren Geese Cereopsis novaehollandiae
Latham, from Fota Wildlife Park, County Cork. Eight
Warren Studler chickens, Gallus domesticus (L), were
sampled from a private poultry fann in Cork City. In each
case, blood was collected from the brachia! vein using a 23
gauge l in. needle and placed in potassium EDTA tubes to
prevent clotting. Haemoglobin concentration was estimated
by the following methods:
(a) Measurement of total haemoglobin as cyanhaemiglobin
A sample of 20 µl blood was mixed thoroughly with 5 ml
of Drabkin's solution (i.e. 200 mg potassium ferricyanide,
50 mg potassium cyanide, 140 mg potassium dihydrogen
phosphate and distilled water made up to a litre) and spun
in a Mason centrifuge at 2500 g (determined stroboscopically).
The absorbance values of the decanted supernatant
were read immediately or within 12 hr using a flow-through
cuvette with 1 cm light path in a C.E.393 digital spectrophotometer
at 540 nm. The haemoglobin concentration of
the blood sample in g/100 ml blood was calculated from a
standard curve prepared from known standards (Merz and
Dade, Hi.Cn Standard, Hergestellt, Dundingen, Switzerland).
The pellet, following centrifugation, was retained for
later extraction with alkaline-haem detergent (ADH).
(b) Measurement of total haemoglobin as alkaline haematin
l. This method followed that ofBell et al. (1965): 0.06 ml
of well mixed blood was pipetted into 20 ml of 0.1 N Na OH
solution. After mixing, the solution was kept at 37°C for
1 hr, then cooled rapidly to room temperature and, within
30 min, the absorption at 705 nm measured and compared
with standards prepared from alkaline haematin (99% pure
from Serva, Heidelberg, FRG).
2. The procedure for haemoglobin determination by the
alkaline haematin D-575 method followed Zander et al.
(1984). Twenty microlitres of blood were mixed with 3 ml of
a solution of 25 g scintillation grade Triton X-100 in 11 of
0.1 mol/l NaOH (termed ADH). The absorbance values
were read, immediately or within 12 hr of preparation, at
575 nm. These results were then converted to haemoglobin
concentrations. The absorbance readings were compared
with those of known concentration prepared from purified
alkaline haematin (99% pure from Serva, Heidelberg, FRG)
and haemoglobin concentrations calculated.
(c) Treatment of pellet
Following centrifugation and reading of the supernatant,
ADH was added to the wet pellet and vortex mixed for
10 sec and the absorbance measured. Values of haemoglobin
were calculated from standards as above. All concentrations
of haemoglobin are presented in g/100 ml.
Statistics
Statistical analyses (calculation of means, standard errors
and linear regression analyses) was carried out using a
Minitab (Pennsylvania State University) statistical package.
RESULTS
A comparison of chicken haemoglobin values
using the three methods of estimation is given in
Table I. Each sample was analysed I 0 times and the
mean and standard error calculated. The quantity of
haemoglobin estimated from the pellet for all bird
species studied is presented (Tables 1 and 2) and is
added to the level of haemoglobin estimated from
reference method to yield the total haemoglobin
(Tables I and 2). It can be seen there was good
agreement between the level of haemoglobin estimated
using the alkaline haematin method of Zander
et al. (1984) and the sum of the soluble and sedimentable
cyanhaemiglobin by the reference method.
Note also that the precision of this modified reagent
is much better than that of Bell et al. ( 1965). Values
of haemoglobin using the reference method and
ADH-575 for different species of birds is presented in
Table 2, again each sample was analysed 10 times and
the mean and standard error calculated. Figure I
shows a scattergram of levels of haemoglobin from
mute swans. The linear relationship can be described
by the following regression equation y = l. l 9x +
0.686 (y = alkaline haematin, x = cyanhaemiglobin).
DISCUSSION
As a general test for health in animals an accurate
estimate of haemoglobin is very useful. In all animals
except mammals the presence of nuclei in red blood
cells poses problems for the cyanhaemiglobin procedure.
Because this method utilises a secondary
biological standard comparisons between laboratories
is difficult and so is not useful for external quality
control assessment schemes. While the cyanhaemiglobin
method is useful for obtaining reproducible
results in birds, this study has shown that
a modified alkaline haematin method (ADH-575
after Zander et al., I 984) is more appropriate for an
accurate and reproducible assessment of avian haemoglobin.
Table 1. Chicken haemoglobin levels estimated using three different methods (.f ± SE of 10
replicates, g/ I 00 ml)
Alkaline haematin
ADH-575 after
Specimen Pellet Total Zander et al. Bell et al.
no. Cyanhaemiglobin haemoglobin haemoglobin (1984) (1965)
1 7.74 ± 0.08 2.15±0.33 9.90 ± 0.20 9.79±0.13 9.30 ± 0.20
2 8.49±0.13 3.12±0.40 11.62 ± 0.40 10.85 ± 0.18 10.73 ± 0.68
3 7.33 ± 0.06 2.27 ± 0.08 9.61 ±0.12 9.63 ± 0.13 10.51 ± 0.60
4 8.20 ± 0.11 1.96 ± 0.12 10.15±0.17 9.67±0.15 9.87 ± 0.34
5 8.53 ± 0.13 2.40 ± 0.09 10.94 ±0.16 10.75±0.15 11.46 ± 0.83
6 9.90 ± 0.27 2.64±0.13 12.54 ± 0.24 12.50 ± 0.20 14.14 ± 0.60
7 9.12±0.25 2.66 ± 0.15 11.24 ± 0.22 11.24 ± 0.22 11.13 ± 1.00
8 8.23 ± 0.09 2.40 ± 0.11 10.63 ± 0.12 10.63 ± 0.12 13.50 ± 0.77
Total 8.44 ± 0.28 2.45±0.12 10.82 ± 0.34 10.63 ± 0.34 11.13 ±0.60
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