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Will leaded young mallards take wing? Effects of a single lead shot ingestion on growth of juvenile game-farm Mallard ducks Anas platyrhynchos
Science of the Total Environment 409 (2011) 2379–2383
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Science of the Total Environment j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / s c i t o t e n v
Will leaded young mallards take wing? Effects of a single lead shot ingestion on growth of juvenile game-farm Mallard ducks Anas platyrhynchos Eric Plouzeau a, Olivier Guillard b,⁎, Alain Pineau c, Philippe Billiald a, Philippe Berny d a
Muséum National d'Histoire Naturelle, USM505 Ecosystèmes et interactions toxiques, 12 rue Buffon, CP39, Paris Cedex 05, France CHU Poitiers, Laboratoire de biochimie 86021 Poitiers, France Université Nantes, Faculté de Pharmacie, Laboratoire de Toxicologie 44000 Nantes, France d Laboratoire de Toxicologie, Ecole Nationale Vétérinaire de Lyon, BP 83, 69280 Marcy L'Etoile, France b c
a r t i c l e
i n f o
Article history: Received 11 October 2010 Received in revised form 1 March 2011 Accepted 8 March 2011 Available online 7 April 2011 Keywords: Mallard (Anas platyrhynchos) Lead shot Blood lead Zinc protoporphyrin ZEAAS (Zeeman Electrothermal Atomic Absorption Spectrophotometry)
a b s t r a c t This study aims to monitor retention of a single ingested lead shot in young mallards, and to evaluate effect on growth in relation to lead shot size class during late wing growth and the first wing molt period (8 to 12 weeks old). Toxicological tests, radiography and biometric measurements were conducted on 51 juvenile Mallard ducks. Forty one of them were given per os a single lead shot in three different commercially available sizes: No. 2 (LS2), No. 4 (LS4) and No. 6 (LS6). Less than 20% of total lead shots were found on X-rays at Day 21 (D) and none remained at D28, with mean retention time in gizzard calculated 12.85 ± 1.34 days for all treated groups. Young ducks developed high blood lead levels for each LS treatment at D15 in males and females, the maximal values being for LS2 (297.00 ± 78.64 μg/100 mL and 483.14 ± 83.70 μg/100 mL, respectively (p b 0.001)). Zinc protoporphyrin (ZPP) levels increased at D15 with LS2 and LS4 in males and only with LS6 in females. Treated ducks developed no symptoms of plumbism except light diarrhea, and at D40, all mallards had survived. We found that LS2 pellets released more lead in gizzards and produced the highest levels of blood lead, suggesting that LS2 pellets are more likely to intoxicate mallards than smaller sizes. The biometric measurements performed showed no statistical difference in weight or bill and wing length between control and treated groups, a finding suggesting that absorption of a single lead shot by young Mallard ducks does not affect their development during the first wing molt period, and appears not to compromise the flight capacity of young (post-juvenile) mallards. However, younger mallards and/or effects on growth of exposure to more than a single-shot dose still need to be investigated. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Lead (Pb) intoxication due to lead shot ingestion is a widespread cause of mortality in water birds on wetlands worldwide, and affects a wide range of species: Cygnus, geese and ducks, flamingos, and river birds (Mateo et al., 2007; Mateo, 2009). It likewise affects rare and globally endangered species, such as the marbled teal (Marmaronetta angustirostris) (Mateo et al., 2001) and the white-headed duck (Oxyura leucocephala) (Svanberg et al., 2006). Poisoning of water fowl due to lead shot is not only a major environmental problem, yet it may also be a potential public health concern (Pain et al., 2010). Lead shots are massively present in wetland soils due to decades of waterfowl hunting practice, as more than 2 million lead shots per hectare were found in several studies in Spain (Mateo et al., 1998), France (Pain, 1991) and California (Rocke et al., 1997). These lead
⁎ Corresponding author. Tel./fax: + 33 549444967. E-mail addresses: [email protected] (E. Plouzeau), [email protected] (O. Guillard), [email protected] (A. Pineau), [email protected] (P. Billiald), [email protected] (P. Berny). 0048-9697/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2011.03.012
shots are ingested by waterbirds along with or instead of grit, which is known to produce mechanical degradation of feed in duck gizzards. The prevalence of lead shot ingestion has been correlated with grit composition in the local environment (Figuerola et al., 2005), and proportion of grit size N 1 mm (Mateo et al., 2000). The type of diet and soil ingestion affects both lead shot retention time in the gizzard and toxicity (Sanderson, 2002). Given their extensive geographical distribution, Mallard ducks (Anas platyrhyncos) are useful in the monitoring of plumbism in wetlands (Mateo et al., 1997). In Ebro delta, Spain, 25% (Guitart et al., 1994) up to 33% (Mateo et al., 1998) of mallards were once poisoned by lead shots. In French Camargue, where up to 45% of all Mallard ducks may have carried lead pellets in their gizzards (Pain, 1990), studies have shown a survival rate 19% lower in lead-affected than in non-affected mallards (Tavecchia et al., 2001). On the one hand, in adult mallards, clinical signs of intoxication and weight loss occur when numerous lead shots have been ingested, leading to the death of the animal (Brewer et al., 2003). On the other hand, a single shot administration with redosing after 5 weeks does not modify the weight of subclinical intoxicated adult mallards (Havera et al., 1992).
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Juvenile mallards gain flight capacity at 8 weeks (56 days) of age, a period during which the possible effects of ingested lead shot intoxication on the growth of body and wings still need to be investigated in order to determine whether or not a single lead-shot ingestion may alter flight potentialities. In the light of these observations, this study aimed to monitor retention of a single ingested lead shot by blood analysis, and to evaluate effect on growth (biometry measurements of beak and wings) in accordance with lead shot size class during the first wing molt period (8 to 12 weeks old). We produced a subclinical intoxication, a transitory state from which mallards should all recover within 1 month with minimal alteration of their health and wellbeing. We monitored lead-shot excretion directly by collection in feces, and indirectly by X-ray of the gizzard. 2. Material and methods 2.1. Lead shots Lead shots from a commercial hunting trademark were calibrated (weight-scale Ohaus, accuracy ± 0.001 g): Commercial lead shot (LS2) size class: 0.325 ± 0.03 g, n = 14 Commercial lead shot (LS4) size class: 0.177 ± 0.03 g, n = 14 Commercial lead shot (LS6) size class: 0.125 ± 0.03 g, n = 13 2.2. Mallard ducks 51 seven-week-old game-farmed Mallard ducks of both sexes were divided by random choice into four groups. Control group: 5 males, 5 females, n = 10. Lead shot group No. 2 (LS2): 7 males, 7 females, n = 14. Lead shot group No. 4 (LS4): 8 males, 6 females, n = 14. Lead shot group No. 6 (LS6): 7 males, 6 females, n = 13. All ducks from lead shot groups were force-fed with a single lead shot on Day 1 of experimentation (D1), and shortly afterwards X-rays were performed in order to monitor lead presence in the gizzard. Mallards were housed in 4 identical outdoor enclosures sized 2.5 m × 3 m and separated by a 1 m high fence. In order to monitor lead shot excretion in feces, all enclosures were built on a plastic tarpaulin connected to a steel tank, collecting feces on a daily basis while cleaning the enclosure by means of a garden hose. Collected lead shot was weighted in order to determine Mallard weight loss, and their morphology was described. Experiments were carried out in accordance with the European Communities Council Directive.
Blood lead level analysis was used as direct evidence witness of lead intoxication, and zinc protoporphyrin (ZPP) was chosen as an indirect marker of intoxication. For biochemical analysis, blood sampling was performed on the brachial vein at days D1, D15, and D30 (2.5 mL whole blood, stored in 3 mL EDTA, BD Vacutainer® tubes, Becton-Dickinson, USA). 2.5. Method for blood lead level analysis by Zeeman Electrothermal Atomic Absorption Spectrophotometry (ZEAAS) Perkin-Elmer® (CT, USA) 4110 ZL atomic absorption spectrometer, with a transversally heated graphite atomizer and longitudinal Zeeman background correction, was used along with AS72 autosampler (Perkin-Elmer®, CT, USA). The ZEAAS was set up in accordance with the instrument parameters recommended by the manufacturer. The graphite furnace program for lead determination in whole blood by ZEAAS is described in Table 1. All reagents and standards were from Merck® (Germany). The lead standards were stored in high-quality plastic polypropylene vials (Sarstedt®, Germany) and refrigerated when not in use. Blood lead concentrations were determined by the method of addition calibration after chemical defecations by HNO3 M according to the preparatory and analytic technique of Stoeppler et al. (1978), as modified and adapted to the Stabilized Temperature Platform Furnace (STPF) concept. Assessment of analytical performance of the method is described by Pineau et al. (2002). In this study, within-day (n = 15) and day-to-day (n = 10) precision data were collected on a pool of whole blood of ducks (11.80 ± 0.78 μg/100 mL) and the coefficients of variation (CV%) were respectively 2.05 and 3.95. In order to verify the accuracy of the method, a recovery study was carried out in triplicate by adding two known quantities of lead with a high (60 μg/100 mL) and a low (20 μg/100 mL) concentration to another pool of whole blood from ducks (10.71 ± 0.72 μg/100 mL). The results show an average recovery of 96.0 ± 1.2% and 95.7 ± 1.6%, respectively. Detection limits (LOD: μg/100 mL) and quantification limits (LOQ: μg/ 100 mL) were calculated as respectively 3 times and 10 times the standard deviation of 20 replicate measurements of the whole blood blank. The results for LOD were 0.02 μg/100 mL and for LOQ 0.06 μg/ 100 mL. In addition, analytical performance was monitored by participation in an interlaboratory survey, the Quebec Toxicology Center Interlaboratory Comparison Program (Weber, 1988). 2.6. Method used in zinc protoporphyrin analysis Zinc protoporphyrin (ZPP) was determined using the protofluor-Z hematofluorometer (Helena®, USA) that was calibrated daily, using high -and low-concentration calibrator solutions. Results are expressed in μg/g Hb (reference value: 1 μg/g Hb).
2.3. X-rays
2.7. Statistical analysis
Standard medical radiography was used to monitor lead shots in the digestive track of the animals (Atomscope® HF80 plus, X-ray generator Toshiba® D-102, adjustment 0.02 s, 54 Kv, films Kodak® Lanex Fast F), on days D1, D3, D4, D5, D6, D7, D11, D21, and D28.
Data are expressed as means ± SEM and analyzed by a Kruskal– Wallis test followed by a Dunn's multiple comparison test as a post hoc test according to the statistical program GraphPad Instat (GraphPad Software, San Diego, CA, USA). Correlations were
2.4. Biometry, blood sampling
Table 1 Furnace program for lead determination in whole blood by ZEAAS.
Biometrical measurements were taken on all Mallard ducks at days D − 7, D1, D7, D15, D30 and D39: Whole body weight (weight-scale OHAUS® LS5000, accuracy ± 4 g). Wing and beak measurement were made with a caliper. Left wing length, distance from the wrist (ulna– carpometacarpus joint) to the distal end of the 10th primary remix, and beak length (distance from first feathers of the front to the distal end of the premaxilla) were likewise measured.
Step
Temp (°C)
Ramp time (s)
Hold time (s)
Air flow (mL/min)
Drying Ashing Atomizationa Cleaning Cooling
120 450 1700 2400 20
20 10 0 1 3
30 20 5 2 5
250 250 0 250 250
a
Read.
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Table 2 Whole blood lead levels in males and females (μg/100 mL ± SEM). Control
LS6
LS4
LS2
Males/Females
M
F
M
F
M
F
M
F
D1 D15 D30
11.54 ± 2.54 11.06 ± 2.06 11.74 ± 2.49
15.96 ± 2.24 13.46 ± 2.06 29.48 ± 18
8.50 ± 0.90 76.61† ± 12.49 104.00†† ± 49.50
10.83 ± 2.05 321.00⁎⁎; ‡‡‡ ± 114.89 82.7 ± 12.53
12.30 ± 0.99 126.75††† ± 13.03 65.3† ± 8.93
8.97 ± 1.67 241.00‡‡‡ ± 52.59 69.17 ± 11.4
10.36 ± 2.05 297.00⁎;††† ± 78.64 100.7⁎⁎;† ± 16.6
10.96 ± 1.62 483.14⁎⁎⁎;‡‡‡ ± 83.70 107.6⁎ ± 8.18
⁎p b 0.05; ⁎⁎p b 0.01; ⁎⁎⁎p b 0.001, compared to respective gender control. † p b 0.05; ††p b 0.01; †††p b 0.001, compared to respective D1 control male for each LS treatment. ‡‡‡ p b 0.001, compared to respective D1 control female for each LS treatment.
evaluated by Spearman test, α = 5%. A p-value b 0.05 was considered significant. 3. Results 3.1. Clinical findings and blood lead analysis None of the animals showed any neurological or behavioral symptom that could be associated with known plumbism clinical findings. However, in all groups except the control group, light brown diarrhea was observed, that can be interpreted as a classical symptom of plumbism (Dumonceaux and Harrison, 1994). No Mallard died during the experiment. Initial blood lead levels are low and similar in all groups at D1 (Table 2). In the control group, whatever the day (D1, D15 or D30), blood lead levels remain low. At D15, blood lead levels in males were significantly higher than control for LS2 treatment (p b 0.05). In addition, at D15 blood lead levels in females had also increased compared to control for LS2 and LS6 treatments. At D30 in males and females, LS2 treatment only induced a significant increase of blood lead levels when compared to controls (Table 2). For males, blood lead levels were always higher at D15 and D30 versus D1 whatever the treatment (LS2, LS4 and LS6). However, for females significant differences were observed only between D15 and D1 whatever the treatment (LS2, LS4 and LS6). 3.2. Lead shot retention time and erosion Radiography showed lead shot disappearance from digestive content with high accuracy during the first 3 weeks. At D21, 34 lead shots (82.9%) were no longer visible, and no lead shot was found at D28. Based on these radiographic results, mean retention time was calculated for all treated groups: 12.85 ± 1.34 days (Table 3). 3.3. Effect of lead shot size class on blood lead levels Among the 41 administered lead shots, 14 were retrieved in feces and could be attributed to individual Mallard ducks through comparison with radioscopic results. Initially totally spherical, the lead shots retrieved in the first days after administration were ovoid, whereas those found around D15 were flat. Mean mass loss was 49.6% for all groups (n = 14), yet lead shots size 2 (n = 7) lost 64.5% of their initial mass, and each one may have released up to 210 mg lead in the
Mallard's digestive tract, whereas LS4 (n = 5) lost 46.3% (82 mg), and LS6 (n = 2) 38.0%, which is equivalent to only 47 mg (Table 3). 3.4. Effect of retention time on blood lead levels We found a positive correlation between retention time and blood lead concentration at D15 in the LS2 group (Spearman Rs = 0.570, p b 0.05, n = 14) but not in the other groups. A global correlation is also found if all treated groups are considered at D15 (Spearman Rs = 0.368, p b 0.05, n = 41). We did not find any correlation at D30. 3.5. Protoporphyrin-zinc blood levels Initial blood ZPP levels are low and similar in all groups at D1 (Fig. 1). For males at D15, LS2 and LS4 treatment induce significant increase for ZPP levels (p b 0.01) compared to D1 control (Fig. 1, panel A). At D30, ZPP levels were significantly higher than D1 control (p b 0.05) (Fig. 1, panel A). In females, only LS6 treatment at D15 increased ZPP levels compared to those obtained in D1 control (p b 0.05) (Fig. 2, panel B). Correlation between lead and ZPP levels are observed only at D15 in LS6 group (Spearman test: r = 0.608, p b 0.01, n = 13). These results confirm the lead intoxication, but are not demonstrative of a lead shot size class effect on blood ZPP levels. 3.6. Biometry While the young ducks' weight rose during the experiment, we did not find any statistical difference between groups (data not shown). Nor was any statistical difference found in the beak (data not shown) or wing length parameters within the four groups (Fig. 2, panels A and B), and all of the birds showed satisfactory macroscopic feather development. A subclinical lead intoxication was produced with 3 different lead shot size classes and growth was monitored during the first wing molt period (8 to 12 weeks old). Our results confirm that pellets of size class No. 2 are more likely to intoxicate mallards than smaller ones, as the biggest lead shots are more eroded and may release the most heavy metal in the digestive tract. 4. Discussion Our values remained in ranges consistent with previous studies involving a single pellet dose (Havera et al., 1992); these results constantly associate subclinical acute lead intoxication with blood lead decrease 1 month after lead shot ingestion. This probably is often
Table 3 Excretion time and mass alteration of lead shots.
Initial number of lead shots Mean day (D) of lead shot excretion based on radiographic results (D ± SEM) Number of lead shots retrieved in feces which could be linked with radiographic results Mean mass before transit (g ± SEM) Mean mass after transit (g ± SEM)
LS2
LS4
LS6
Total — mean ± SEM
14 11.11 ± 2.09 7 0.32 ± 0.05 0.11 ± 0.02
14 13.29 ± 2.51 5 0.17 ± 0.05 0.09 ± 0.025
13 14.27 ± 2.45 2 0.12 ± 0.05 0.08 ± 0.03
41 12.85 ± 1.34 14
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6
ZPP (µg/gHb)
5
Control LS2 LS4 LS6
A (males)
D15
500
D30
** ** *
Wing length in males (mm SEM)
A (males)
4 3 2
D1
400
Control LS2 LS4 LS6 D15
300
D30
D1
200 100
1 0 0
B (females)
ZPP (µg/gHb)
5
Control LS2 LS4 LS6
B (females) 500
D30
D15
*
4 3
D1 2
Wing length in females (mm SEM)
6
400
Control LS2 LS4 LS6 D15
300
D30
D1
200 100
1 0 Fig. 1. Blood ZPP levels in males (panel A) and females (panel B) following LS treatment *p b 0.05; **p b 0.01, compared to respective D1 controls.
the case under natural conditions: if the Mallard does not redose itself with a second (or more) collected lead shot from the sediments, it survives the single pellet ingestion. Green et al. (2008) provides models in California condors (Gymnogyps californianus) for a decrease of blood lead levels over time after absorption that is consistent with our observations. Our results are consistent with the conclusions drawn by Sanderson and Belrose (1986), who estimated that a single lead shot would disappear from the gizzard in about 20 days and by Kerr et al. (2010) on the bobwhite quail (Colinus virginianus), who found that all pellets were absorbed or excreted in less than 14 days. However, excretion was somewhat more rapid than described by Srebocan and Rattner (1988), who found 7 No. 4 lead shots among 8 still in the gizzard at D21 and more recently by Sanderson (2002), who found a mean of 2 out of 5 lead shots remaining in the gizzards of mallards fed on commercial pellets at D21, as well as Rodríguez et al. (2010) who evaluate the retention time in digestive track around 30 days. Differences in fiber composition of daily dietary given to captive mallards, as well as severity of lead-induced diarrhea may be responsible for these variations. The biggest lead shots (LS2) showed the greatest mass loss, suggesting that they released more soluble lead in the digestive tract, which was absorbed and found in blood lead analysis. Along with many American states, some 14 European countries have adopted specific regulations on hunting practices based on nontoxic ammunition; they effectively limit the amounts of lead shot available in wetlands (Mateo, 2009). Recently, Pokras and Kneeland (2008) advocated a transdisciplinary approach aimed at resolving the long-time problem of lead poisoning. Based upon our results, which confirm those of previous studies (Mateo et al., 2007; Brewer et al.,
0 Fig. 2. Wing length of males (panel A) and females (panel B) following LS treatment.
2003; Kelly et al., 1998), we suggest that in areas where no lead shot ban is applied, the No. 2 lead shot size class should no longer be used for hunting on wetlands. In all treated groups, the biometric measurements performed showed no significant impact of lead intoxication on weight, bill and wing growth, thereby suggesting that a single lead shot absorption by young Mallard ducks does not affect their development during the first wing molt period, and should not compromise the flight capacity of young (post-juvenile) mallards. However, these results do not demonstrate that lead shot poisoning has no effect at all on the growth of young mallards. Previous studies on freshly hatched Herring gulls (Burger and Gochfeld, 1988) and Common Terns (Gochfeld and Burger, 1988) have shown a dose-related effect of lead (lead nitrate solution) on weight and wing length growth. In our study, blood lead levels were significantly lower, and the birds were no longer chicks, but rather post-juvenile mallards having reached the final last wing growth stage. Under natural conditions, ducks may ingest more than one lead shot at the same time and/or at short intervals of time. Further investigations on younger mallards with more than a single lead shot dose consequently need to be conducted.
5. Conclusion In our study, a single lead shot ingestion by juveniles Mallard ducks during the 8th to 12th week did not modify their global or allometric growth, despite the high blood lead levels in circulation that we found. Nevertheless, the study mimics a sub-clinical intoxication due to a single lead shot ingestion, and further studies with higher numbers of lead shots and/or sequential redosing may quite possibly show different results.
E. Plouzeau et al. / Science of the Total Environment 409 (2011) 2379–2383
Acknowledgements We thank K. Tiphaneau, S. Devant and A. Marrauld for their excellent technical assistance. References Brewer L, Fairbrother A, Clark J, Amick D. Acute toxicity of lead, steel, and an iron– tungsten–nickel shot to Mallard ducks (Anas platyrhynchos). J Wildl Dis 2003;39: 638–48. Burger J, Gochfeld M. Effects of lead on growth in young herring gulls (Larus argentatus). J Toxicol Environ Health 1988;25:227–36. Dumonceaux G, Harrison GJ. Toxins. In: Ritchie BW, Harrison GJ, Harrison LR, editors. Avian medicine: principles and application. Lake Worth: Wingers Publishing; 1994. p. 1030–52. Figuerola J, Mateo R, Green AJ, Mondain-Monval JY, Lefranc H, Mentaberre G. Grit selection in waterfowl and how it determines exposure to ingested lead shot in Mediterranean wetlands. Environ Conserv 2005;32(3):226–34. Gochfeld M, Burger J. Effects of lead on growth and feeding behaviour of commons terms (Sterna hirundo). Arch Environ Contam Toxicol 1988;17:513–7. Guitart R, Figueras J, Mateo R, Bertolero A, Cerradelo S, Martinez-Vilalta A. Lead poisoning in waterfowl from the Ebro Delta, Spain: calculation of lead exposure thresholds for mallards. Arch Environ Contam Toxicol 1994;27:289–93. Green RE, Hunt WG, Parish CN, Newton I. Effectiveness of action to reduce exposure of free-ranging California condors in Arizona and Utah to lead from spent ammunition. PLoS ONE 2008;3(12):e4022. Havera SP, Wood SG, Georgi MM. Blood and tissue parameters in wild mallards redosed with lead shot. Bull Environ Contam Toxicol 1992;49:238–45. Kelly ME, Fitzgerald SD, Aulerich RJ, Balander RJ, Powell DC, Stickle RL, et al. Acute effects of lead, steel, tungsten–iron, and tungsten–polymer shot administered to game-farm mallard. J Wildl Dis 1998;34:673–87. Kerr R, Holladay S, Jarrett T, Selcer B, Meldrum B, Williams S, et al. Lead pellet retention time and associated toxicity in northern bobwhite quail (Colinus virginianus). Environ Toxicol Chem 2010;29(12):2869–74. Mateo R, Green AJ, Lefranc H, Baos R, Figuerola J. Lead poisoning in wild birds from southern Spain: a comparative study of wetland areas and species affected, and trends over time. Ecotoxicol Environ Saf 2007;66(1):119–26. Mateo R. Lead poisoning in wild birds in Europe and the regulations adopted by different countries. In: Watson RT, Fuller M, Pokras M, Hunt WG, editors. Ingestion of lead from spent ammunition: implications for wildlife and humans. Boise, Idaho, USA: The Peregrine Fund; 2009, doi:10.4080/ILSA.2009.0107. Mateo R, Green AJ, Jeske CW, Urios V, Gerique C. Lead poisoning in the globally threatened marbled teal and white-headed duck in Spain. Environ Toxicol Chem 2001;20(12):2860–8.
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Mateo R, Belliure J, Aguilar Serrano JM, Guitart R. High prevalences of lead poisoning in wintering waterfowl in Spain. Arch Environ Contam Toxicol 1998;35:342–7. Mateo R, Guitart R, Green AJ. Determinants of lead shot, rice, and grit ingestion in ducks and coots. J Wildl Manage 2000;64:939–47. Mateo R, Martinez-Vidalta A, Guitart R. Lead shot pellets in the Ebro delta, Spain: densities in sediments and prevalence of exposure in waterfowl. Environ Pollut 1997;96(3):335–41. Pain DJ. Lead shot densities and settlement rates in Camargue Marshes, France. Conserv Biol 1991;57:273–86. Pain DJ. Lead shot ingestion by waterbirds in the Camargue, France: an investigation of levels and interspecific differences. Environ Pollut 1990;66:273–85. Pain DJ, Cromie RL, Newth J, Brown MJ, Crutcher E, et al. Potential hazard to human health from exposure to fragments of lead bullets and shot in the tissues of game animals. PLoS ONE 2010;5(4):e10315. Pineau A, Fauconneau B, Rafael M, Viallefont A, Guillard O. Determination of lead in whole blood: comparison of the LeadCare blood lead testing system with Zeeman longitudinal electrothermal atomic absorption spectrometry. J Trace Elem Med Biol 2002;16:113–7. Pokras MA, Kneeland MR. Lead poisoning: using transdisciplinary approaches to solve an ancient problem. EcoHealth 2008;5(3):379–85. Rocke TE, Brand CJ, Mensik JG. Site-specific lead exposure from lead pellet ingestion in sentinel mallards. J Wildl Manage 1997;61(1):228–34. Rodríguez JJ, Oliveira PA, Fidalgo LE, Ginja MM, Silvestre AM, Ordoñez C, et al. Lead toxicity in captive and wild Mallards (Anas platyrhynchos) in Spain. J Wildl Dis 2010;46(3):854–63. Sanderson GC. Effects of diet and soil on the toxicity of lead in mallards. Ecotoxicology 2002;11(1):11–7. Sanderson GC, Belrose FC. A review of the problem of the lead poisoning in waterfowl. Illinois Natural History Survey Spec. Publ., 4. ; 1986. 34 pp. Srebocan E, Rattner BA. Heat exposure and the toxicity of one number four lead shot in mallards, Anas platyrhynchos. Bull Environ Contam Toxicol 1988;40:165–9. Stoeppler M, Brandt K, Rains TC. Contributions to automated trace analysis. Rapid method for the automated determination of lead in whole blood by electrothermal atomic-absorption spectrophotometry. Analyst 1978;103:714–22. Svanberg F, Mateo R, Hillström L, Green AJ, Taggart M, Raab A, et al. Lead isotopes and lead shot ingestion in the globally threatened marbled teal (Marmaronetta angustirostris) and white-headed duck (Oxyura leucocephala). Sci Total Environ 2006;370:416–24. Tavecchia G, Pradel R, Lebreton JD, Johnson AR, Mondain-Monval JY. The effect of lead exposure on survival of adult mallards in the Camargue, southern France. J Appl Ecol 2001;38:1197–207. Weber JP. An interlaboratory comparison program for several toxic substance in blood and urine. Sci Total Environ 1988;71:111–23.
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Will leaded young mallards take wing? Effects of a single lead shot ingestion on growth of juvenile game-farm Mallard ducks Anas platyrhynchos Eric Plouzeau a, Olivier Guillard b,⁎, Alain Pineau c, Philippe Billiald a, Philippe Berny d a
Muséum National d'Histoire Naturelle, USM505 Ecosystèmes et interactions toxiques, 12 rue Buffon, CP39, Paris Cedex 05, France CHU Poitiers, Laboratoire de biochimie 86021 Poitiers, France Université Nantes, Faculté de Pharmacie, Laboratoire de Toxicologie 44000 Nantes, France d Laboratoire de Toxicologie, Ecole Nationale Vétérinaire de Lyon, BP 83, 69280 Marcy L'Etoile, France b c
a r t i c l e
i n f o
Article history: Received 11 October 2010 Received in revised form 1 March 2011 Accepted 8 March 2011 Available online 7 April 2011 Keywords: Mallard (Anas platyrhynchos) Lead shot Blood lead Zinc protoporphyrin ZEAAS (Zeeman Electrothermal Atomic Absorption Spectrophotometry)
a b s t r a c t This study aims to monitor retention of a single ingested lead shot in young mallards, and to evaluate effect on growth in relation to lead shot size class during late wing growth and the first wing molt period (8 to 12 weeks old). Toxicological tests, radiography and biometric measurements were conducted on 51 juvenile Mallard ducks. Forty one of them were given per os a single lead shot in three different commercially available sizes: No. 2 (LS2), No. 4 (LS4) and No. 6 (LS6). Less than 20% of total lead shots were found on X-rays at Day 21 (D) and none remained at D28, with mean retention time in gizzard calculated 12.85 ± 1.34 days for all treated groups. Young ducks developed high blood lead levels for each LS treatment at D15 in males and females, the maximal values being for LS2 (297.00 ± 78.64 μg/100 mL and 483.14 ± 83.70 μg/100 mL, respectively (p b 0.001)). Zinc protoporphyrin (ZPP) levels increased at D15 with LS2 and LS4 in males and only with LS6 in females. Treated ducks developed no symptoms of plumbism except light diarrhea, and at D40, all mallards had survived. We found that LS2 pellets released more lead in gizzards and produced the highest levels of blood lead, suggesting that LS2 pellets are more likely to intoxicate mallards than smaller sizes. The biometric measurements performed showed no statistical difference in weight or bill and wing length between control and treated groups, a finding suggesting that absorption of a single lead shot by young Mallard ducks does not affect their development during the first wing molt period, and appears not to compromise the flight capacity of young (post-juvenile) mallards. However, younger mallards and/or effects on growth of exposure to more than a single-shot dose still need to be investigated. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Lead (Pb) intoxication due to lead shot ingestion is a widespread cause of mortality in water birds on wetlands worldwide, and affects a wide range of species: Cygnus, geese and ducks, flamingos, and river birds (Mateo et al., 2007; Mateo, 2009). It likewise affects rare and globally endangered species, such as the marbled teal (Marmaronetta angustirostris) (Mateo et al., 2001) and the white-headed duck (Oxyura leucocephala) (Svanberg et al., 2006). Poisoning of water fowl due to lead shot is not only a major environmental problem, yet it may also be a potential public health concern (Pain et al., 2010). Lead shots are massively present in wetland soils due to decades of waterfowl hunting practice, as more than 2 million lead shots per hectare were found in several studies in Spain (Mateo et al., 1998), France (Pain, 1991) and California (Rocke et al., 1997). These lead
⁎ Corresponding author. Tel./fax: + 33 549444967. E-mail addresses: [email protected] (E. Plouzeau), [email protected] (O. Guillard), [email protected] (A. Pineau), [email protected] (P. Billiald), [email protected] (P. Berny). 0048-9697/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2011.03.012
shots are ingested by waterbirds along with or instead of grit, which is known to produce mechanical degradation of feed in duck gizzards. The prevalence of lead shot ingestion has been correlated with grit composition in the local environment (Figuerola et al., 2005), and proportion of grit size N 1 mm (Mateo et al., 2000). The type of diet and soil ingestion affects both lead shot retention time in the gizzard and toxicity (Sanderson, 2002). Given their extensive geographical distribution, Mallard ducks (Anas platyrhyncos) are useful in the monitoring of plumbism in wetlands (Mateo et al., 1997). In Ebro delta, Spain, 25% (Guitart et al., 1994) up to 33% (Mateo et al., 1998) of mallards were once poisoned by lead shots. In French Camargue, where up to 45% of all Mallard ducks may have carried lead pellets in their gizzards (Pain, 1990), studies have shown a survival rate 19% lower in lead-affected than in non-affected mallards (Tavecchia et al., 2001). On the one hand, in adult mallards, clinical signs of intoxication and weight loss occur when numerous lead shots have been ingested, leading to the death of the animal (Brewer et al., 2003). On the other hand, a single shot administration with redosing after 5 weeks does not modify the weight of subclinical intoxicated adult mallards (Havera et al., 1992).
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Juvenile mallards gain flight capacity at 8 weeks (56 days) of age, a period during which the possible effects of ingested lead shot intoxication on the growth of body and wings still need to be investigated in order to determine whether or not a single lead-shot ingestion may alter flight potentialities. In the light of these observations, this study aimed to monitor retention of a single ingested lead shot by blood analysis, and to evaluate effect on growth (biometry measurements of beak and wings) in accordance with lead shot size class during the first wing molt period (8 to 12 weeks old). We produced a subclinical intoxication, a transitory state from which mallards should all recover within 1 month with minimal alteration of their health and wellbeing. We monitored lead-shot excretion directly by collection in feces, and indirectly by X-ray of the gizzard. 2. Material and methods 2.1. Lead shots Lead shots from a commercial hunting trademark were calibrated (weight-scale Ohaus, accuracy ± 0.001 g): Commercial lead shot (LS2) size class: 0.325 ± 0.03 g, n = 14 Commercial lead shot (LS4) size class: 0.177 ± 0.03 g, n = 14 Commercial lead shot (LS6) size class: 0.125 ± 0.03 g, n = 13 2.2. Mallard ducks 51 seven-week-old game-farmed Mallard ducks of both sexes were divided by random choice into four groups. Control group: 5 males, 5 females, n = 10. Lead shot group No. 2 (LS2): 7 males, 7 females, n = 14. Lead shot group No. 4 (LS4): 8 males, 6 females, n = 14. Lead shot group No. 6 (LS6): 7 males, 6 females, n = 13. All ducks from lead shot groups were force-fed with a single lead shot on Day 1 of experimentation (D1), and shortly afterwards X-rays were performed in order to monitor lead presence in the gizzard. Mallards were housed in 4 identical outdoor enclosures sized 2.5 m × 3 m and separated by a 1 m high fence. In order to monitor lead shot excretion in feces, all enclosures were built on a plastic tarpaulin connected to a steel tank, collecting feces on a daily basis while cleaning the enclosure by means of a garden hose. Collected lead shot was weighted in order to determine Mallard weight loss, and their morphology was described. Experiments were carried out in accordance with the European Communities Council Directive.
Blood lead level analysis was used as direct evidence witness of lead intoxication, and zinc protoporphyrin (ZPP) was chosen as an indirect marker of intoxication. For biochemical analysis, blood sampling was performed on the brachial vein at days D1, D15, and D30 (2.5 mL whole blood, stored in 3 mL EDTA, BD Vacutainer® tubes, Becton-Dickinson, USA). 2.5. Method for blood lead level analysis by Zeeman Electrothermal Atomic Absorption Spectrophotometry (ZEAAS) Perkin-Elmer® (CT, USA) 4110 ZL atomic absorption spectrometer, with a transversally heated graphite atomizer and longitudinal Zeeman background correction, was used along with AS72 autosampler (Perkin-Elmer®, CT, USA). The ZEAAS was set up in accordance with the instrument parameters recommended by the manufacturer. The graphite furnace program for lead determination in whole blood by ZEAAS is described in Table 1. All reagents and standards were from Merck® (Germany). The lead standards were stored in high-quality plastic polypropylene vials (Sarstedt®, Germany) and refrigerated when not in use. Blood lead concentrations were determined by the method of addition calibration after chemical defecations by HNO3 M according to the preparatory and analytic technique of Stoeppler et al. (1978), as modified and adapted to the Stabilized Temperature Platform Furnace (STPF) concept. Assessment of analytical performance of the method is described by Pineau et al. (2002). In this study, within-day (n = 15) and day-to-day (n = 10) precision data were collected on a pool of whole blood of ducks (11.80 ± 0.78 μg/100 mL) and the coefficients of variation (CV%) were respectively 2.05 and 3.95. In order to verify the accuracy of the method, a recovery study was carried out in triplicate by adding two known quantities of lead with a high (60 μg/100 mL) and a low (20 μg/100 mL) concentration to another pool of whole blood from ducks (10.71 ± 0.72 μg/100 mL). The results show an average recovery of 96.0 ± 1.2% and 95.7 ± 1.6%, respectively. Detection limits (LOD: μg/100 mL) and quantification limits (LOQ: μg/ 100 mL) were calculated as respectively 3 times and 10 times the standard deviation of 20 replicate measurements of the whole blood blank. The results for LOD were 0.02 μg/100 mL and for LOQ 0.06 μg/ 100 mL. In addition, analytical performance was monitored by participation in an interlaboratory survey, the Quebec Toxicology Center Interlaboratory Comparison Program (Weber, 1988). 2.6. Method used in zinc protoporphyrin analysis Zinc protoporphyrin (ZPP) was determined using the protofluor-Z hematofluorometer (Helena®, USA) that was calibrated daily, using high -and low-concentration calibrator solutions. Results are expressed in μg/g Hb (reference value: 1 μg/g Hb).
2.3. X-rays
2.7. Statistical analysis
Standard medical radiography was used to monitor lead shots in the digestive track of the animals (Atomscope® HF80 plus, X-ray generator Toshiba® D-102, adjustment 0.02 s, 54 Kv, films Kodak® Lanex Fast F), on days D1, D3, D4, D5, D6, D7, D11, D21, and D28.
Data are expressed as means ± SEM and analyzed by a Kruskal– Wallis test followed by a Dunn's multiple comparison test as a post hoc test according to the statistical program GraphPad Instat (GraphPad Software, San Diego, CA, USA). Correlations were
2.4. Biometry, blood sampling
Table 1 Furnace program for lead determination in whole blood by ZEAAS.
Biometrical measurements were taken on all Mallard ducks at days D − 7, D1, D7, D15, D30 and D39: Whole body weight (weight-scale OHAUS® LS5000, accuracy ± 4 g). Wing and beak measurement were made with a caliper. Left wing length, distance from the wrist (ulna– carpometacarpus joint) to the distal end of the 10th primary remix, and beak length (distance from first feathers of the front to the distal end of the premaxilla) were likewise measured.
Step
Temp (°C)
Ramp time (s)
Hold time (s)
Air flow (mL/min)
Drying Ashing Atomizationa Cleaning Cooling
120 450 1700 2400 20
20 10 0 1 3
30 20 5 2 5
250 250 0 250 250
a
Read.
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Table 2 Whole blood lead levels in males and females (μg/100 mL ± SEM). Control
LS6
LS4
LS2
Males/Females
M
F
M
F
M
F
M
F
D1 D15 D30
11.54 ± 2.54 11.06 ± 2.06 11.74 ± 2.49
15.96 ± 2.24 13.46 ± 2.06 29.48 ± 18
8.50 ± 0.90 76.61† ± 12.49 104.00†† ± 49.50
10.83 ± 2.05 321.00⁎⁎; ‡‡‡ ± 114.89 82.7 ± 12.53
12.30 ± 0.99 126.75††† ± 13.03 65.3† ± 8.93
8.97 ± 1.67 241.00‡‡‡ ± 52.59 69.17 ± 11.4
10.36 ± 2.05 297.00⁎;††† ± 78.64 100.7⁎⁎;† ± 16.6
10.96 ± 1.62 483.14⁎⁎⁎;‡‡‡ ± 83.70 107.6⁎ ± 8.18
⁎p b 0.05; ⁎⁎p b 0.01; ⁎⁎⁎p b 0.001, compared to respective gender control. † p b 0.05; ††p b 0.01; †††p b 0.001, compared to respective D1 control male for each LS treatment. ‡‡‡ p b 0.001, compared to respective D1 control female for each LS treatment.
evaluated by Spearman test, α = 5%. A p-value b 0.05 was considered significant. 3. Results 3.1. Clinical findings and blood lead analysis None of the animals showed any neurological or behavioral symptom that could be associated with known plumbism clinical findings. However, in all groups except the control group, light brown diarrhea was observed, that can be interpreted as a classical symptom of plumbism (Dumonceaux and Harrison, 1994). No Mallard died during the experiment. Initial blood lead levels are low and similar in all groups at D1 (Table 2). In the control group, whatever the day (D1, D15 or D30), blood lead levels remain low. At D15, blood lead levels in males were significantly higher than control for LS2 treatment (p b 0.05). In addition, at D15 blood lead levels in females had also increased compared to control for LS2 and LS6 treatments. At D30 in males and females, LS2 treatment only induced a significant increase of blood lead levels when compared to controls (Table 2). For males, blood lead levels were always higher at D15 and D30 versus D1 whatever the treatment (LS2, LS4 and LS6). However, for females significant differences were observed only between D15 and D1 whatever the treatment (LS2, LS4 and LS6). 3.2. Lead shot retention time and erosion Radiography showed lead shot disappearance from digestive content with high accuracy during the first 3 weeks. At D21, 34 lead shots (82.9%) were no longer visible, and no lead shot was found at D28. Based on these radiographic results, mean retention time was calculated for all treated groups: 12.85 ± 1.34 days (Table 3). 3.3. Effect of lead shot size class on blood lead levels Among the 41 administered lead shots, 14 were retrieved in feces and could be attributed to individual Mallard ducks through comparison with radioscopic results. Initially totally spherical, the lead shots retrieved in the first days after administration were ovoid, whereas those found around D15 were flat. Mean mass loss was 49.6% for all groups (n = 14), yet lead shots size 2 (n = 7) lost 64.5% of their initial mass, and each one may have released up to 210 mg lead in the
Mallard's digestive tract, whereas LS4 (n = 5) lost 46.3% (82 mg), and LS6 (n = 2) 38.0%, which is equivalent to only 47 mg (Table 3). 3.4. Effect of retention time on blood lead levels We found a positive correlation between retention time and blood lead concentration at D15 in the LS2 group (Spearman Rs = 0.570, p b 0.05, n = 14) but not in the other groups. A global correlation is also found if all treated groups are considered at D15 (Spearman Rs = 0.368, p b 0.05, n = 41). We did not find any correlation at D30. 3.5. Protoporphyrin-zinc blood levels Initial blood ZPP levels are low and similar in all groups at D1 (Fig. 1). For males at D15, LS2 and LS4 treatment induce significant increase for ZPP levels (p b 0.01) compared to D1 control (Fig. 1, panel A). At D30, ZPP levels were significantly higher than D1 control (p b 0.05) (Fig. 1, panel A). In females, only LS6 treatment at D15 increased ZPP levels compared to those obtained in D1 control (p b 0.05) (Fig. 2, panel B). Correlation between lead and ZPP levels are observed only at D15 in LS6 group (Spearman test: r = 0.608, p b 0.01, n = 13). These results confirm the lead intoxication, but are not demonstrative of a lead shot size class effect on blood ZPP levels. 3.6. Biometry While the young ducks' weight rose during the experiment, we did not find any statistical difference between groups (data not shown). Nor was any statistical difference found in the beak (data not shown) or wing length parameters within the four groups (Fig. 2, panels A and B), and all of the birds showed satisfactory macroscopic feather development. A subclinical lead intoxication was produced with 3 different lead shot size classes and growth was monitored during the first wing molt period (8 to 12 weeks old). Our results confirm that pellets of size class No. 2 are more likely to intoxicate mallards than smaller ones, as the biggest lead shots are more eroded and may release the most heavy metal in the digestive tract. 4. Discussion Our values remained in ranges consistent with previous studies involving a single pellet dose (Havera et al., 1992); these results constantly associate subclinical acute lead intoxication with blood lead decrease 1 month after lead shot ingestion. This probably is often
Table 3 Excretion time and mass alteration of lead shots.
Initial number of lead shots Mean day (D) of lead shot excretion based on radiographic results (D ± SEM) Number of lead shots retrieved in feces which could be linked with radiographic results Mean mass before transit (g ± SEM) Mean mass after transit (g ± SEM)
LS2
LS4
LS6
Total — mean ± SEM
14 11.11 ± 2.09 7 0.32 ± 0.05 0.11 ± 0.02
14 13.29 ± 2.51 5 0.17 ± 0.05 0.09 ± 0.025
13 14.27 ± 2.45 2 0.12 ± 0.05 0.08 ± 0.03
41 12.85 ± 1.34 14
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6
ZPP (µg/gHb)
5
Control LS2 LS4 LS6
A (males)
D15
500
D30
** ** *
Wing length in males (mm SEM)
A (males)
4 3 2
D1
400
Control LS2 LS4 LS6 D15
300
D30
D1
200 100
1 0 0
B (females)
ZPP (µg/gHb)
5
Control LS2 LS4 LS6
B (females) 500
D30
D15
*
4 3
D1 2
Wing length in females (mm SEM)
6
400
Control LS2 LS4 LS6 D15
300
D30
D1
200 100
1 0 Fig. 1. Blood ZPP levels in males (panel A) and females (panel B) following LS treatment *p b 0.05; **p b 0.01, compared to respective D1 controls.
the case under natural conditions: if the Mallard does not redose itself with a second (or more) collected lead shot from the sediments, it survives the single pellet ingestion. Green et al. (2008) provides models in California condors (Gymnogyps californianus) for a decrease of blood lead levels over time after absorption that is consistent with our observations. Our results are consistent with the conclusions drawn by Sanderson and Belrose (1986), who estimated that a single lead shot would disappear from the gizzard in about 20 days and by Kerr et al. (2010) on the bobwhite quail (Colinus virginianus), who found that all pellets were absorbed or excreted in less than 14 days. However, excretion was somewhat more rapid than described by Srebocan and Rattner (1988), who found 7 No. 4 lead shots among 8 still in the gizzard at D21 and more recently by Sanderson (2002), who found a mean of 2 out of 5 lead shots remaining in the gizzards of mallards fed on commercial pellets at D21, as well as Rodríguez et al. (2010) who evaluate the retention time in digestive track around 30 days. Differences in fiber composition of daily dietary given to captive mallards, as well as severity of lead-induced diarrhea may be responsible for these variations. The biggest lead shots (LS2) showed the greatest mass loss, suggesting that they released more soluble lead in the digestive tract, which was absorbed and found in blood lead analysis. Along with many American states, some 14 European countries have adopted specific regulations on hunting practices based on nontoxic ammunition; they effectively limit the amounts of lead shot available in wetlands (Mateo, 2009). Recently, Pokras and Kneeland (2008) advocated a transdisciplinary approach aimed at resolving the long-time problem of lead poisoning. Based upon our results, which confirm those of previous studies (Mateo et al., 2007; Brewer et al.,
0 Fig. 2. Wing length of males (panel A) and females (panel B) following LS treatment.
2003; Kelly et al., 1998), we suggest that in areas where no lead shot ban is applied, the No. 2 lead shot size class should no longer be used for hunting on wetlands. In all treated groups, the biometric measurements performed showed no significant impact of lead intoxication on weight, bill and wing growth, thereby suggesting that a single lead shot absorption by young Mallard ducks does not affect their development during the first wing molt period, and should not compromise the flight capacity of young (post-juvenile) mallards. However, these results do not demonstrate that lead shot poisoning has no effect at all on the growth of young mallards. Previous studies on freshly hatched Herring gulls (Burger and Gochfeld, 1988) and Common Terns (Gochfeld and Burger, 1988) have shown a dose-related effect of lead (lead nitrate solution) on weight and wing length growth. In our study, blood lead levels were significantly lower, and the birds were no longer chicks, but rather post-juvenile mallards having reached the final last wing growth stage. Under natural conditions, ducks may ingest more than one lead shot at the same time and/or at short intervals of time. Further investigations on younger mallards with more than a single lead shot dose consequently need to be conducted.
5. Conclusion In our study, a single lead shot ingestion by juveniles Mallard ducks during the 8th to 12th week did not modify their global or allometric growth, despite the high blood lead levels in circulation that we found. Nevertheless, the study mimics a sub-clinical intoxication due to a single lead shot ingestion, and further studies with higher numbers of lead shots and/or sequential redosing may quite possibly show different results.
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Acknowledgements We thank K. Tiphaneau, S. Devant and A. Marrauld for their excellent technical assistance. References Brewer L, Fairbrother A, Clark J, Amick D. Acute toxicity of lead, steel, and an iron– tungsten–nickel shot to Mallard ducks (Anas platyrhynchos). J Wildl Dis 2003;39: 638–48. Burger J, Gochfeld M. Effects of lead on growth in young herring gulls (Larus argentatus). J Toxicol Environ Health 1988;25:227–36. Dumonceaux G, Harrison GJ. Toxins. In: Ritchie BW, Harrison GJ, Harrison LR, editors. Avian medicine: principles and application. Lake Worth: Wingers Publishing; 1994. p. 1030–52. Figuerola J, Mateo R, Green AJ, Mondain-Monval JY, Lefranc H, Mentaberre G. Grit selection in waterfowl and how it determines exposure to ingested lead shot in Mediterranean wetlands. Environ Conserv 2005;32(3):226–34. Gochfeld M, Burger J. Effects of lead on growth and feeding behaviour of commons terms (Sterna hirundo). Arch Environ Contam Toxicol 1988;17:513–7. Guitart R, Figueras J, Mateo R, Bertolero A, Cerradelo S, Martinez-Vilalta A. Lead poisoning in waterfowl from the Ebro Delta, Spain: calculation of lead exposure thresholds for mallards. Arch Environ Contam Toxicol 1994;27:289–93. Green RE, Hunt WG, Parish CN, Newton I. Effectiveness of action to reduce exposure of free-ranging California condors in Arizona and Utah to lead from spent ammunition. PLoS ONE 2008;3(12):e4022. Havera SP, Wood SG, Georgi MM. Blood and tissue parameters in wild mallards redosed with lead shot. Bull Environ Contam Toxicol 1992;49:238–45. Kelly ME, Fitzgerald SD, Aulerich RJ, Balander RJ, Powell DC, Stickle RL, et al. Acute effects of lead, steel, tungsten–iron, and tungsten–polymer shot administered to game-farm mallard. J Wildl Dis 1998;34:673–87. Kerr R, Holladay S, Jarrett T, Selcer B, Meldrum B, Williams S, et al. Lead pellet retention time and associated toxicity in northern bobwhite quail (Colinus virginianus). Environ Toxicol Chem 2010;29(12):2869–74. Mateo R, Green AJ, Lefranc H, Baos R, Figuerola J. Lead poisoning in wild birds from southern Spain: a comparative study of wetland areas and species affected, and trends over time. Ecotoxicol Environ Saf 2007;66(1):119–26. Mateo R. Lead poisoning in wild birds in Europe and the regulations adopted by different countries. In: Watson RT, Fuller M, Pokras M, Hunt WG, editors. Ingestion of lead from spent ammunition: implications for wildlife and humans. Boise, Idaho, USA: The Peregrine Fund; 2009, doi:10.4080/ILSA.2009.0107. Mateo R, Green AJ, Jeske CW, Urios V, Gerique C. Lead poisoning in the globally threatened marbled teal and white-headed duck in Spain. Environ Toxicol Chem 2001;20(12):2860–8.
2383
Mateo R, Belliure J, Aguilar Serrano JM, Guitart R. High prevalences of lead poisoning in wintering waterfowl in Spain. Arch Environ Contam Toxicol 1998;35:342–7. Mateo R, Guitart R, Green AJ. Determinants of lead shot, rice, and grit ingestion in ducks and coots. J Wildl Manage 2000;64:939–47. Mateo R, Martinez-Vidalta A, Guitart R. Lead shot pellets in the Ebro delta, Spain: densities in sediments and prevalence of exposure in waterfowl. Environ Pollut 1997;96(3):335–41. Pain DJ. Lead shot densities and settlement rates in Camargue Marshes, France. Conserv Biol 1991;57:273–86. Pain DJ. Lead shot ingestion by waterbirds in the Camargue, France: an investigation of levels and interspecific differences. Environ Pollut 1990;66:273–85. Pain DJ, Cromie RL, Newth J, Brown MJ, Crutcher E, et al. Potential hazard to human health from exposure to fragments of lead bullets and shot in the tissues of game animals. PLoS ONE 2010;5(4):e10315. Pineau A, Fauconneau B, Rafael M, Viallefont A, Guillard O. Determination of lead in whole blood: comparison of the LeadCare blood lead testing system with Zeeman longitudinal electrothermal atomic absorption spectrometry. J Trace Elem Med Biol 2002;16:113–7. Pokras MA, Kneeland MR. Lead poisoning: using transdisciplinary approaches to solve an ancient problem. EcoHealth 2008;5(3):379–85. Rocke TE, Brand CJ, Mensik JG. Site-specific lead exposure from lead pellet ingestion in sentinel mallards. J Wildl Manage 1997;61(1):228–34. Rodríguez JJ, Oliveira PA, Fidalgo LE, Ginja MM, Silvestre AM, Ordoñez C, et al. Lead toxicity in captive and wild Mallards (Anas platyrhynchos) in Spain. J Wildl Dis 2010;46(3):854–63. Sanderson GC. Effects of diet and soil on the toxicity of lead in mallards. Ecotoxicology 2002;11(1):11–7. Sanderson GC, Belrose FC. A review of the problem of the lead poisoning in waterfowl. Illinois Natural History Survey Spec. Publ., 4. ; 1986. 34 pp. Srebocan E, Rattner BA. Heat exposure and the toxicity of one number four lead shot in mallards, Anas platyrhynchos. Bull Environ Contam Toxicol 1988;40:165–9. Stoeppler M, Brandt K, Rains TC. Contributions to automated trace analysis. Rapid method for the automated determination of lead in whole blood by electrothermal atomic-absorption spectrophotometry. Analyst 1978;103:714–22. Svanberg F, Mateo R, Hillström L, Green AJ, Taggart M, Raab A, et al. Lead isotopes and lead shot ingestion in the globally threatened marbled teal (Marmaronetta angustirostris) and white-headed duck (Oxyura leucocephala). Sci Total Environ 2006;370:416–24. Tavecchia G, Pradel R, Lebreton JD, Johnson AR, Mondain-Monval JY. The effect of lead exposure on survival of adult mallards in the Camargue, southern France. J Appl Ecol 2001;38:1197–207. Weber JP. An interlaboratory comparison program for several toxic substance in blood and urine. Sci Total Environ 1988;71:111–23.