INTRODUCTION

Lead (Pb) is a common environmental pollutant that has physiological effects on both humans and animals (Cromie et al. 2014, Masindi and Muedi 2018). In the document “Risk Communication on Chemical Substances” published by the Ministry of the Environment of the Government of Japan, an impact dose is defined as a dose that does not cause any clinical symptoms, a toxic dose causes clinical symptoms and potentially death, and a lethal dose has a high probability of death (Ministry of the Environment 2008). If a bird ingests a lethal dose, death occurs immediately (Pain et al. 2009). Toxic doses cause non-specific neural and gastric symptoms, a decline in the reproductive success rate, and immune system suppression (Kendall et al. 1996, Williams et al. 2018).

Impact doses also cause non-specific symptoms, but these generally have no effect on birds. The symptoms of impact doses are increased foraging distance from their own population (Pain et al. 2019), suppression of humoral immunity (Snoeijs et al. 2004), and increased production of immunoglobulin E (IgE) and cytokines in response to an allergen that causes inflammation (Tepper et al. 1990, Heo et al. 1997, Miller et al. 1998, Wood et al. 2004), but these generally have no effect on birds. Nevertheless, of these symptoms, immunity suppression can lead to mass mortality in the event of highly pathogenic infections in the habitat of Pb-contaminated birds (Ushine et al. 2020a). It is difficult to point out the effects of contaminants in wildlife based on impact dose, given the likely absence of noticeable symptoms (Johnson et al. 2007). However, impact doses are considered to have significant effects on the long-term survival of species. For example, Eurasian Blackbirds (Turdus merula) have a reduced lifetime breeding success rate because of chronic Pb contamination (Fritsch et al. 2019).

As anthropogenic effects on the conservation of bird species are increasing annually, it is necessary to properly evaluate how these effects threaten the survival of bird species (Richard et al. 2021). In several marine mammals, polychlorinated biphenyl (PCB) contamination causes immunosuppression, leading to mass mortality during viral infections (Ross et al. 1995). In Eurasian Sparrowhawks (Accipiter nisus), it has been reported that even below the pollution threshold for cadmium (Cd) their growth rate is reduced (Grúz et al. 2019). In fact, mass mortality of wild birds has been reported in multiple places globally (Camphuysen et al. 2002, Fey et al. 2015), and it is possible that low-level contamination from environmental pollutants was responsible for these deaths.

Black-headed Gulls (Chroicocephalus ridibundus, Laridae) belong to the gull family and are widely distributed across Eurasia (BirdLife International 2022). They are flocking birds that form breeding colonies (Andersson et al. 1981), and those that breed in high latitudes also migrate (Burger et al. 2020). They spend the non-breeding season in Japan (BirdLife International 2022). Black-headed Gulls often select urban areas as their breeding and wintering habitats, thus, they are a species familiar to humans (Feng and Liang 2020), but also a wild bird easily affected by human society (Kitowski et al. 2018).

Previous research has shown that Black-headed Gulls are contaminated with Pb, which affects the percentages of heterophils and lymphocytes in the peripheral blood (Ushine et al. 2020b). In addition to Pb contamination, oil spills (Newman et al. 2000, Seiser et al. 2000), plastic ingestion (Lavers et al. 2019), methylmercury exposure (Sonne et al. 2020), and seabird-specific infections (Kirkwood et al. 1995) have all been reported to affect the blood chemistry of seabirds. However, among these potentially pathogenic factors, Pb is rather unique, as it is a heavy metal widely used by humans and is one of the most common environmental pollutants (Assi et al. 2016).

The purpose of the present study was to determine the minimum Pb level that affects six types of cells and molecules in the peripheral blood with immunity function, i.e., the total number of white blood cells (WBC; cell/µL), proportion of lymphocytes (Lym; %), proportion of heterophils (Het; %), ratio of heterophils to lymphocytes (H/L ratio), copy number of CD4 messenger RNA (CD4; number of copies/µL), and copy number of CD8α messenger RNA (CD8α; number of copies/µL) in Black-headed Gulls during the winter season. We sought to clarify the relationship between the blood Pb level (BLL) and the ratio of immune cells and molecules in peripheral blood, and to determine the minimum Pb contamination level with a significant effect on the ratio of immune cells and molecules. We hypothesize that the impact dose of BLL causes a significant change in the six types of cells and molecules in peripheral blood with immunity function. This study therefore aims to contribute to the risk assessment of wild bird pathogens by clarifying the effects of Pb contamination on wild birds and their immune function on cells and molecules.

Among wild birds, seabirds are more susceptible than others to the effects of environmental pollutants (Walsh 1990). For instance, male Black-legged Kittiwakes (Rissa tridactyla), individuals with high mercury levels, are more likely to neglect their breeding behavior (Tartu et al. 2015). Dichloro-diphenyl-trichloroethane (DDT) is known to cause population declines and behavioral disorders in some sea bird species (Burger and Gochfield 2004). Furthermore, it was suggested that bird species that select urban areas as their habitat tend to be more susceptible to artificial environmental pollutants than those that live in the suburbs (Lebedeva 1997). Thus, our report can also serve as an appropriate case study of the effects of environmental pollution for seabirds and other wild birds living in urban areas.

METHODS

Study area

This study was conducted in Tokyo Bay, Chiba Prefecture (35°N, 139°E), and Mikawa Bay, Aichi Prefecture (34°N, 137°E; Fig. 1). These areas were reported as wintering areas for the Black-headed Gull arriving from Russia and China (Yamashina Institute for Ornithology 2002).

Sample animals

Black-headed Gulls were captured by hand, noose trap, or whoosh net from November 2018 to April 2021 at Tokyo Bay and from January 2019 to April 2021 at Mikawa Bay. Peripheral blood (less than 0.5% of body mass) was collected using a heparin-coated syringe (Nipro, Osaka, Japan) as previously reported (Fair et al. 2010). After hemostasis was confirmed, the gull was released with a color ring and a metal ring for identification in the left and right tarsus of the gulls, respectively, in the Tokyo Bay population. For the Mikawa Bay gulls, only a metal ring was attached to the right tarsus. Blood smears were prepared immediately from the collected blood by the pulled glass method. The smears were fixed with 100% methanol and stained with Wright-Giemsa stain (Fujifilm Wako Chemicals, Osaka, Japan). After encapsulation in the mounting medium (Fujifilm Wako Chemicals, Osaka, Japan), the differential count of leukocytes was calculated under an optical microscope at 1000× resolution.

Identification of sex and age

Age was assessed based on the plumage and birds were classified into two groups: yearlings (with first-year plumage) and adults (not in first-year plumage; Baker 2016). Sex was determined by targeting a gene on the sex chromosome that codes for a chromodomain helicase DNA-binding protein (Çakmak et al. 2017). The protocols for the polymerase chain reaction (PCR) were carried with reference to Ushine et al. (2016).

Evaluation of immunity

The following six indices of peripheral blood were used to evaluate immunity in Black-headed Gulls (Ushine et al. 2020b): WBC count (cell number per deciliter), total Lym per 100 leukocytes and total Het per 100 leukocytes (both determined using a blood smear), Het/Lym (H/L) ratio, and copy number of messenger RNA both CD4 and CD8α, which were calculated by performing a real-time PCR with primers specific to Black-headed Gulls (GenBank® accession numbers of: LC533369 for CD4, and LC533370 and LC533371 for CD8α).

Measurement of Pb levels

Clots in the peripheral blood were autoclaved and transported to Hokkaido University (Kita 8, Nishi 5, Kita-ku, Sapporo, Hokkaido, Japan). BLL was measured using the method described by Ushine et al. (2020a), that is, blood samples were digested with 5 mL of 30% nitric acid (Kanto Chemical Corp., Tokyo, Japan) and 1 mL of 30% hydrogen peroxide (Kanto Chemical Corporation) in a microwave digestion system (Berghof, Eningen, Germany). Pb concentrations were measured with an inductively coupled plasma-mass spectrometer (Agilent Technology, Santa Clara, California, USA; Toyomaki et al. 2020). Analytical quality control was performed using the certified reference material Seronorm™ Trace Elements Whole Blood L-2 (Sero, Billingstad, Norway). Replicate analysis of reference material showed good accuracy (relative standard deviation less than 3%) and recoveries (95–105%). Finally, the measurements were converted to the amount of Pb per µL of blood.

BLL classification

Pb toxicity symptoms depend on the pollutant concentration in each species (Burger and Gochfeld 2004). A commonly used reference value is a Pb contamination level of 20 µg/dL, reported in Anseriformes, Falconiformes, and Accipitriformes (Pain et al. 2019). Reference values for BLL have been determined in some avian groups that are known to be affected by severe poisoning or pollution (Swaileh and Sansur 2006). For example, the reference concentration of Pb in the order Anseriformes is 20 µg/dL (Martinez-Haro et al. 2011) and in the genus Haliaeetus is 40 µg/dL (Bedrosian and Craighead 2009). However, establishing the threshold levels as reference values for heavy metal pollution requires exposure experiments using animals (Ministry of the Economy, Trade and Industry 2007). It is difficult to set a threshold for Black-headed Gulls because no captive exposure experiments have been performed for this species. Thus, herein, the BLL was classified into four groups according to quartile values (< 1.0, 1.0–2.0, 2.0–3.5, and > 3.5 µg/dL) following the methodology described by Krishnan et al. (2012).

Statistical analysis

Differences in Pb levels with respect to age, sex, and population were confirmed using the Wilcoxon rank-sum test. If the indices had any significant differences, the gull populations were classified according to them. Next, the Shapiro-Wilk test was used to evaluate if the variables WBC, Het, Lym, H/L ratio, CD4, and CD8α were normally distributed. To obtain normal distributions, CD4 and CD8α copy counts were logarithmically transformed before analysis. Finally, linear regression analysis was performed with BLL as the explanatory variable. The lowest BLL (< 1.0 µg/dL) group was used as reference. Statistical analyses were performed using R software (ver. 3.5.0; R Foundation for Statistical Computing, Vienna, Austria) and Stata (ver. 14.0; StataCorp LLC, College Station, Texas, USA). The significance level was set at 0.05 for all statistical analyses.

RESULTS

A total of 170 gulls were captured and their blood samples were analyzed (Table 1). Differences in Pb contamination by age, sex, and population were not significant, and block structures for these variables were not included. The results of the six blood indices and BLL are shown in Table 2. Linear regression analysis revealed significant trends in five indices (Table 3). First, the group with BLL < 1.0 µg/dL had lower Het than that with BLL > 3.5 µg/dL (P > 0.05). Second, the group with BLL < 1.0 µg/dL had higher Lym than that with BLL > 3.5 µg/dL (P < 0.05; Fig. 2). The H/L ratio was significantly higher in the group with BLL > 3.5 µg/dL than in animals with BLL < 1.0 µg/dL (Fig. 3). WBC (Fig. 4) and CD8α (Fig. 5) counts were significantly higher in the groups with BLL 1.0–2.0 and 2.0–3.5 µg/dL than in those with BLL < 1.0 µg/dL (P < 0.05).

DISCUSSION

Noteworthily, not all six of the cells and molecules showed significant changes in minimal BLL. Herein, the proportion of Het increased for BLL > 3.5 µg/dL, whereas Lym decreased at the same concentration. An increase in the proportion of Het affects the immune response suppression caused by the inflammatory response (Harmon 1998) and a decrease in that of Lym has a negative effect on the regulation of immune function (Sharma 1991). Therefore, when BLL > 3.5 µg/dL, the Het and Lym levels change substantially, possibly leading to negative effects on immune function. Similarly, the H/L ratio showed a significant increase when BLL > 3.5 µg/dL as compared with animals with BLL < 1.0 µg/dL. The H/L ratio is maintained at a certain value for each avian species (Minias 2019) and deviations in the H/L ratio indicate abnormal conditions, such as stress and starvation (Totzke et al. 1999). Our results suggest that the proportions of Het and Lym were affected at BLL > 3.5 µg/dL as a result of fluctuations in their levels. The H/L ratio may possibly be altered by other environmental contaminants besides Pb. For example, in some passerine species, exposure to carbon monoxide generated in urban areas significantly alters their H/L ratio (Ribeiro et al. 2022). Therefore, factors other than Pb may have existed for the change in this ratio.

WBC and CD8α levels were significantly higher in the BLL 1.0–3.5 µg/dL group than in the BLL < 1.0 µg/dL group, unlike the results for Het and Lym. The biological effect of Pb pollution is dependent on the Pb level and dose per unit time (Franson and Pain 2011). Herring Gulls (Larus argentatus) showed a 16% reduction in equilibrium capacity after exposure to 100 mg/kg Pb in comparison with 50 mg/kg Pb exposure (Burger and Gochfeld 2010). Fritsch et al. (2019) reported that the reproductive success rate of the Blackbird decreased with less than 10 µg Pb/g dry weight of contamination. In some avian species, low Pb pollution has been reported to reduce bodily strength (Kendall et al. 1996, Haig et al. 2014). Notably, this study is the first to report changes in WBC and CD8α in response to Pb and they appear to depend on the level of the pollutant.

In the current study, only CD4 was found to be not significantly affected by BLL. However, CD4+ cells are known to be the most affected immune cell population by Pb pollution (Fenga et al. 2017). Cao (2016) reported that the number of CD4 memory cells increases upon Pb contamination, whereas naive CD4 cells decrease. This reported effect is suspected to be one of the reasons why this study did not show a significant relationship between BLL and CD4; it is also possible that the Pb level in this study was too low to affect CD4. Pb contamination in adult humans is reported to contribute to the activation of T helper 2 (Th2) cells and to be involved in the proliferation of T helper 1 (Th1) cells, which are CD4+ T cells (Arstila et al. 1994). In contrast, children with high BLL have a significantly lower proportion of CD4+ cells than those with low BLL (Cao 2016). In the present study, the Black-headed Gulls did not develop any toxicity symptoms, implying that they were chronically polluted with Pb at an impact dose level rather than at toxic or lethal levels. Additionally, Pb effects differ depending on the type of cells expressing CD4 (Arstila et al. 1994); it is therefore possible that no significant change could be observed in the CD4 levels. In a 30,000 µg/dL Pb exposure experiment in rats, the CD4 level was significantly decreased, whereas those of CD8 remained unchanged (Fang et al. 2012). Therefore, there seems to be no clear relationship between CD4 and BLL in gulls.

Although the mechanism of Pb contamination on avian immune suppression has not been elucidated, some case studies reported immunosuppression after Pb contamination. For example, the immune activity of Th1 cells is significantly suppressed at a blood value ≥ 10 µg/dL in the juvenile Mallard (Anas platyrhynchos; Vallverdú-Coll et al. 2019). In a Pb exposure experiment in Japanese Quail (Coturnix japonica), the expression of Toll-like receptor-3, that has an antiviral effect, was significantly decreased in animals with 5000 µg/dL BLL as compared those with 500 µg/dL BLL (Nain and Smits 2011). For some bird taxa, reference values for pollution have been set based on the concentration at which symptoms of poisoning are observed, such as in Anseriformes (Martinez-Haro et al. 2011) and Haliaeetus species (Bedrosian and Craighead 2009). For humans, the biological effects of low Pb pollution have been investigated in detail in children; thus, in 2012, the Center for Disease Control and Prevention (CDC) set a standard value of 5.0 µg for Pb contamination (CDC 2012). However, in 2021, the CDC revised this standard to 3.5 µg because of the impact that Pb has on the intellectual development of infants (CDC 2021). In avian species, low-level Pb pollution has potential health effects. For example, chronic Pb contamination was shown to significantly increase the infection rate and number of intestinal parasites species in Mallards (Prüter et al. 2018), and a positive relationship between the parasitism rate of Plasmodium relictum and Pb levels in the feathers of House Sparrow (Passer domesticus; Bichet et al. 2013) was reported; none of these birds showed any sign of poisoning, suggesting that an impact dose of Pb increases the risk of infection. Therefore, it is reasonable to consider that the BLL revealed in this research may increase the risk of infection by pathogens.

The abundance of birds has been declining globally since the 1970s and there are concerns for future declines (Lees et al. 2022). Various anthropogenic causes have been considered (Richard et al. 2021). Considering our findings, it is possible that the wild birds affected by Pb contamination do not only suffer direct effects, such as lowered reproductive success (Fritsch et al. 2019), but also indirect negative effects on immune function, possibly making them more susceptible to infectious diseases. Several surveys aimed at understanding the levels of pollution affecting individual birds or avian groups have been conducted in the past (Mateo et al. 2003, Ayaş 2007, Martinez-Haro et al. 2011). Herein, we estimated the degree of Pb contamination and its impact on a single species, the Black-headed Gull, in Japanese populations. Our findings confirmed that there were many individuals in the field with low-concentration contamination. Black-headed Gulls are reported to have a high risk of transmitting avian influenza to European poultry (Feare 2010) and to be carriers of multidrug-resistant Salmonella (Čižek et al. 2006). Thus, low-level contamination with Pb may be enabling these infections. Pb is a major environmental pollutant, against which measures must be taken. There are two ways to minimize the impacts of Pb on wild birds: reducing the amount of Pb we consume in the future and removing Pb that is already in the environment. Active substitution of Pb will reduce Pb emission into the environment. On the other hand, removal methods are currently trialed with regard to Pb that has already accumulated in bird sanctuaries, such as removing sludge from wetlands and pond bottoms (Jurries 2003, von Sperling 2007). In addition, waterfowl that ingest Pb as a grit can benefit from the addition of gravel to the bottom of the water body. These methods can decrease the harmful effects of anthropogenic environmental pollutants on wild birds and aid long-term bird conservation.

CONCLUSIONS

This study revealed that even the migratory Black-headed Gulls that inhabit Japan only for the winter season are contaminated with Pb, and that having BLL > 3.5 µg/dL affects the proportions of heterophils and lymphocytes in their peripheral blood. Moreover, it was confirmed that the number of WBCs and the copy number of CD8α messenger RNA, which are also related to immune function, increase significantly at BLL 1.0–3.5 µg/dL. Altogether, the results of this study suggest that Pb contamination at a low, non-toxic impact dose significantly affects the proportion of some blood cells and molecules with immune function. Hence, if a gull continues to inhabit polluted areas, it is more likely to be infected by pathogens or further contaminated with Pb, a concept that should support the development of measures against Pb pollution for the conservation of wild bird species.

LITERATURE CITED

Andersson, M., F. Götmark, and C. G. Wiklund. 1981. Food information in the Black-headed Gull, Larus ridibundus. Behavioral Ecology and Sociobiology 9(3):199-202. https://doi.org/10.1007/BF00302938

Arstila, T. P., O. Vainio, and O. Lassila. 1994. Central role of CD4⁺ T cells in avian immune response. Poultry Science 73(7):1019-1026. https://doi.org/10.3382/ps.0731019

Assi, M. A., M. N. M. Hezmee, A. W. Haron, M. Y. M. Sabri, and M. A. Rajion. 2016. The detrimental effects of lead on human and animal health. Veterinary World 9(6):660-671. https://doi.org/10.14202/vetworld.2016.660-671

Ayaş, Z. 2007. Trace element residues in eggshells of Grey Heron (Ardea cinerea) and Black-crowned Night Heron (Nycticorax nycticorax) from Nallihan Bird Paradise, Ankara-Turkey. Ecotoxicology 16:347-352. https://doi.org/10.1007/s10646-007-0132-6

Baker, K. 2016. Black-headed Gull. Pages 337-339 in Identification of European Non-Passerines: a BTO guide, second revised edition. British Trust for Ornithology, Norfolk, UK.

Bedrosian, B., and D. Craighead. 2009. Blood lead levels of Bald and Golden Eagles sampled during and after hunting seasons in the Greater Yellowstone Ecosystem. Pages 219-220 in R. T., Watson, M. Pokras, M. Fuller, and G. Hunt, editors. Ingestion of spent lead ammunition: implications for wildlife and humans. The Peregrine Fund, Boise, Idaho, USA. https://doi.org/10.4080/ilsa.2009.0209

Bichet, C., R. Scheifler, M. Cœurdassier, R. Julliard, G. Sorci, and C. Loisea. 2013. Urbanization, trace metal pollution, and malaria prevalence in the House Sparrow. PLoS ONE 8:e53866. https://doi.org/10.1371/journal.pone.0053866

BirdLife International. 2022. Species factsheet: Larus ridibundus. BirdLife International, Cambridge, UK. http://datazone.birdlife.org/species/factsheet/Black-headed-gull-larus-ridibundus

Burger, J., and M. Gochfeld. 2004. Marine birds as sentinels of environmental pollution. EcoHealth 1:263-274. https://doi.org/10.1007/s10393-004-0096-4

Burger, J., and M. Gochfeld. 2010. Effects of lead on learning in Herring Gulls: an avian wildlife model for neurobehavioral deficits. Neurotoxicology 26(4):615-624. https://doi.org/10.1016/j.neuro.2005.01.005

Burger, J., M. Gochfeld, G. M. Kirwan, D. Christie, and E. Garcia. 2020. Black-headed Gull (Chroicocephalus ridibundus), version 1.0. In J. del Hoyo, A. Elliott, J. Sargatal, D. A. Christie, and E. de Juana, editors. Birds of the world. Cornell Lab of Ornithology, Ithaca, New York, USA. https://doi.org/10.2173/bow.bkhgul.01

Çakmak, E., C. A. Pekşen, and C. C. Bilgin. 2017. Comparison of three different primer sets for sexing birds. Journal of Veterinary Diagnostic Investigation 29(1):59-63. https://doi.org/10.1177/1040638716675197

Camphuysen, C. J., C. M. Berrevoets, H. J. W. M. Cremers, A. Dekinga, R. Dekker, B. J. Ens, T. M. van der Have, R. K. H. Kats, T. Kuiken, M. F. Leopold, J. van der Meer, and T. Piersma. 2002. Mass mortality of Common Eiders (Somateria mollissima) in the Dutch Wadden Sea, winter 1999/2000: starvation in a commercially exploited wetland of international importance. Biological Conservation 106(3):303-317. https://doi.org/10.1016/S0006-3207(01)00256-7

Cao, J. J. 2016. Early life exposure to toxic environments: effects on lung and immune cell development in mice and men. Thesis. University of Groningen, Groningen, Netherlands. https://www.semanticscholar.org/paper/Early-life-exposure-to-toxic-environments-%3A-effects-Cao-Song/4df8f5642da321ff6ed41554b1f696828cde5f33

Centers for Disease Control and Prevention (CDC). 2012. Adult blood lead epidemiology and surveillance (ABLES). National Institute for Occupational Safety and Health (NIOSH), Atlanta, Georgia, USA. http://www.cdc.gov/niosh/topics/ables/default.html.

Centers for Disease Control and Prevention (CDC). 2021. Childhood lead poisoning prevention. Blood lead reference value. CDC, Atlanta, Georgia, USA. https://www.cdc.gov/nceh/lead/data/blood-lead-reference-value.htm

Čížek, A., M. Dolejská, R. Karpíšková, D. Dědičová, and I. Literák. 2006. Wild Black-headed Gulls (Larus ridibundus) as an environmental reservoir of Salmonella strains resistant to antimicrobial drugs. European Journal of Wildlife Research 53:55-60. https://doi.org/10.1007/s10344-006-0054-2

Cromie, R., J. Newth, J. Reeves, M. O’Brien, K. Beckmann, and M. Brown. 2014. The sociological and political aspects of reducing lead poisoning from ammunition in the UK: why the transition to non-toxic ammunition is so difficult. Pages 104-124. Proceedings of the Oxford lead symposium, lead ammunication: understanding and minimising the risks to human and environment health. Edward Grey Institute, The University of Oxford, Oxford, UK. http://www.oxfordleadsymposium.info/wp-content/uploads/OLS_proceedings/papers/OLS_proceedings_cromie_newth_reeves_obrien_beckman_brown.pdf

Fair, J. M., E. Paul, J. Jones, A. B. Clark, C. Davie, and G. Kaiser. 2010. Minor manipulative procedures. Pages 131-167 in J. M. Fair, E. Paul, and J. Jones, editors. Guidelines to the use of wild birds in research. The Ornithological Council, Washington, D.C., USA. https://www.ndsu.edu/fileadmin/research/documents/IACUC/Ornithology_Guidelines_August2010.pdf

Fang, L., F. Zhao, X. Shen, W. Ouyang, X. Liu, Y. Xu, T. Yu, B. Jin, J. Chen, and W. Luo. 2012. Pb exposure attenuates hypersensitivity in vivo by increasing regulatory T cells. Toxicology and Applied Pharmacology 265(2):272-278. https://doi.org/10.1016/J.TAAP.2012.10.001

Feare, C. J. 2010. Role of wild birds in the spread of highly pathogenic avian influenza virus H5N1 and implications for global surveillance. Avian Diseases 54(s1):201-212. https://doi.org/10.1637/8766-033109-ResNote.1

Feng, C., and W. Liang. 2020. Behavioral responses of Black-headed Gulls (Chroicocephalus ridibundus) to artificial provisioning in China. Global Ecology and Conservation 21:e00873. https://doi.org/10.1016/j.gecco.2019.e00873

Fenga, C., S. Gangemi, V. Di Salvatore, L. Falzone, and M. Libra. 2017. Immunological effects of occupational exposure to lead (review). Molecular Medicine Reports 15:3355-3360. https://doi.org/10.3892/mmr.2017.6381

Fey, S. B., A. M. Siepielski, S. Nusslé, K. Cervantes-Yoshida, J. L. Hwan, E. R. Huber, M. J. Fey, A. Catenazzi, and S. M. Carlson. 2015. Recent shifts in the occurrence, cause, and magnitude of animal mass mortality events. Proceedings of the National Academy of Sciences 112(4):1083-1088. https://doi.org/10.1073/pnas.1414894112

Franson, J. C., and D. J. Pain. 2011. Lead in birds. Pages 563-594 in W. N. Beyer, and J. P. Meador, editors. Environmental contaminants in biota, second edition. CRC, Boca Rotan, Florida, USA. https://doi.org/10.1201/b10598

Fritsch, C., L. Jankowiak, and D. Wysocki. 2019. Exposure to Pb impairs breeding success and is associated with longer lifespan in urban European Blackbirds. Scientific Reports 9:486. https://doi.org/10.1038/s41598-018-36463-4

Grúz, A., O. Mackle, A. Bartha, R. Szabó, J. Déri, P. Budai, and J. Lehel. 2019. Biomonitoring of toxic metals in feathers of predatory birds from eastern regions of Hungary. Environmental Science and Pollution Research 26:26324-26331. https://doi.org/10.1007/s11356-019-05723-9

Haig, S. M., J. D’Elia, C. Eagles-Smith, J. M. Fair, J. Gervais, G. Herring, J. W. Rivers, and J. H. Schulz. 2014. The persistent problem of lead poisoning in birds from ammunition and fishing tackle. Condor 116(3):408-428. https://doi.org/10.1650/CONDOR-14-36.1

Harmon, B. G. 1998. Avian heterophils in inflammation and disease resistance. Poultry Science 77(7):972-977. https://doi.org/10.1093/ps/77.7.972

Heo, Y., W. T. Lee, and D. A. Lawrence. 1997. In vivo the environmental pollutants lead and mercury induce oligoclonal T cell responses skewed toward Type-2 reactivities. Cellular Immunology 179:185-195. https://doi.org/10.1006/cimm.1997.1160

Johnson, C. K., T. Vodovoz, W. M. Boyce, and J. A. Mazet. 2007. Lead exposure in California condors and sentinel species in California. Report prepared for the California Department of Fish and Game (CDFW). CDFW, Sacramento, California, USA.

Jurries, D. 2003. Biofilters (bioswales, vegetative buffers, & constructed wetlands) for storm water discharge pollution removal. State of Oregon Department of Environmental Quality (DEQ) Northwest region document. DEQ, Portland, Oregon, USA. https://nacto.org/docs/usdg/biofilters_bioswales_vegetative_buffers_constructed_wetlands_jurries.pdf

Kendall, R. J., T. E. Lacher Jr., C. Bunck, B. Daniel, C. Driver, C. E. Grue, F. Leighton, W. Stansley, P. G. Watanabe, and M. Whitworth. 1996. An ecological risk assessment of lead shot exposure in non-waterfowl avian species: upland game birds and raptors. Environmental Toxicology and Chemistry 15(1):4-20. https://doi.org/10.1002/etc.5620150103

Kirkwood, J. K., A. A. Cunningham, C. Hawkey, J. Howlett, and C. M. Perrins. 1995. Hematology of fledgling Manx Shearwaters (Puffinus puffinus) with and without ‘Puffinosis’. Journal of Wildlife Diseases 31(1):96-98. https://doi.org/10.7589/0090-3558-31.1.96

Kitowski, I., D. Jakubas, P. Indykiewicz, and D. Wiącek. 2018. Factors affecting element concentrations in eggshells of three sympatrically nesting waterbirds in Northern Poland. Archives of Environmental Contamination and Toxicology 74:318-329. https://doi.org/10.1007/s00244-017-0481-y

Krishnan, E., B. Lingala, and V. Bhalla. 2012. Low-level lead exposure and the prevalence of gout: an observational study. Annals of Internal Medicine 157:233-241. http://dx.doi.org/10.7326/0003-4819-157-4-201208210-00003

Lavers, J. L., I. Hutton, and A. L. Bond. 2019. Clinical pathology of plastic ingestion in marine birds and relationships with blood chemistry. Environmental Science & Technology 53(15):9224-9231. https://doi.org/10.1021/acs.est.9b02098

Lebedeva, N. V. 1997. Accumulation of heavy metals by birds in the Southwest of Russia. Russian Journal of Ecology 28(1):41-46.

Lees, A. C., L. Haskell, T. Allinson, S. B. Bezeng, I. J. Burfield, L. M. Renjifo, K. V. Rosenberg, A. Viswanathan, and S. H. M. Butchart. 2022. State of the world’s birds. Annual Review of Environment and Resources 47:231-260. https://doi.org/10.1146/annurev-environ-112420-014642

Martinez-Haro, M., A. J. Green, and R. Mateo. 2011. Effects of lead exposure on oxidative stress biomarkers and plasma biochemistry in waterbirds in the field. Environmental Research 111(4):530-538. http://doi.org/10.1016/j.envres.2011.02.012

Masindi, V., and K. L. Muedi. 2018. Environmental contamination by heavy metals. Pages 115-133 in H. E. M. Saleh, and R. F. Aglan, editors. Heavy Metals. IntechOpen, London, UK. http://doi.org/10.5772/intechopen.76082

Mateo, R., M. Taggart, and A. A. Meharg. 2003. Lead and arsenic in bones of birds of prey from Spain. Environmental Pollution 126(1):107-114. https://doi.org/10.1016/s0269-7491(03)00055-1

Miller, T. E., K. A. Golemboski, R. S. Ha, T. Bunn, F. S. Sanders, and R. R. Dietert. 1998. Developmental exposure to lead causes persistent immunotoxicity in Fischer 344 rats. Toxicological Sciences 42(2):129-135. http://doi.org/10.1006/toxs.1998.2424

Minias, P. 2019. Evolution of heterophil/lymphocyte ratios in response to ecological and life-history traits: a comparative analysis across the avian tree of life. Journal of Animal Ecology 88(4):554-565. https://doi.org/10.1111/1365-2656.12941

Ministry of the Economy, Trade and Industry. 2007. Guidebook for risk assessment of chemicals [title translated from the Japanese]. Ministry of the Economy, Trade and Industry, Tokyo, Japan. https://www.meti.go.jp/policy/chemical_management/law/prtr/pdf/guidebook_jissen.pdf

Ministry of the Environment. 2008. Chemicals Advisor Certification Examination Text [title translated from the Japanese]. Ministry of the Environment, Tokyo, Japan. http://www.env.go.jp/chemi/communication/taiwa/text/3s.pdf

Nain, S., and J. E. G. Smits. 2011. Subchronic lead exposure, immunotoxicology and increased disease resistance in Japanese quail (Corturnix coturnix japonica). Ecotoxicology and Environmental Safety 74(4):787-792. https://doi.org/10.1016/j.ecoenv.2010.10.045

Newman, S. H., D. W. Anderson, M. H. Ziccardi, J. G. Trupkiewicz, F. S. Tseng, M. M. Christopher, and J. G. Zinkl. 2000. An experimental soft-release of oil-spill rehabilitated American Coots (Fulica americana): II. effects on health and blood parameters. Environmental Pollution 107(3):295-304. https://doi.org/10.1016/S0269-7491(99)00171-2

Pain, D. J., I. J. Fisher, and V. G. Thomas. 2009. A global update of lead poisoning in terrestrial birds from ammunition sources. Pages 99-118 in R. T. Watson, M. Fuller, M. Pokras, and W. G. Hunt, editors. Proceedings of the conference: ingestion of lead from spent ammunition: implications for wildlife and humans. The Peregrine Fund, Boise, Idaho, USA. https://science.peregrinefund.org/legacy-sites/conference-lead/2008PbConf_Proceedings.htm https://doi.org/10.4080/ilsa.2009.0108

Pain, D. J., R. Mateo, and R. E. Green. 2019. Effects of lead from ammunition on birds and other wildlife: a review and update. Ambio 48:935-953. https://doi.org/10.1007/s13280-019-01159-0

Prüter, H., M. Franz, S. Auls, G. A. Czirják, O. Greben, A. D. Greenwood, O. Lisitsyna, Y. Syrota, J. Sitko, and O. Krone. 2018. Chronic lead intoxication decreases intestinal helminth species richness and infection intensity in Mallards (Anas platyrhynchos). Science of The Total Environment 644:151-160. https://doi.org/10.1016/j.scitotenv.2018.06.297

Ribeiro, P. V. A., V. F. Gonçalves, V. C. de Magalhães Tolentino, C. Q. Baesse, L. P. Pires, L. P. Mendes Paniago, and C. de Melo. 2022. Effects of urbanisation and pollution on the heterophil/lymphocyte ratio in birds from Brazilian Cerrado. Environmental Science and Pollution Research 29:40204-40240. https://doi.org/10.1007/s11356-022-19037-w

Richard, F. -J., I. Southern, M. Gigauri, G. Bellini, O. Rojas, and A. Runde. 2021. Warning on nine pollutants and their effects on avian communities. Global Ecology and Conservation 32:e01898. https://doi.org/10.1016/j.gecco.2021.e01898

Ross, P. S., R. L. De Swart, P. J. Reijnders, H. Van Loveren, J. G. Vos, and A. D. Osterhaus. 1995. Contaminant-related suppression of delayed-type hypersensitivity and antibody responses in harbor seals fed herring from the Baltic Sea. Environmental Health Perspectives 103(2):162-167. https://dx.doi.org/10.1289/ehp.95103162

Seiser, P. E., L. K. Duffy, A. D. McGuire, D. D. Roby, G. H. Golet, and M. A. Litzow. 2000. Comparison of Pigeon Guillemot, Cepphus columba, blood parameters from oiled and unoiled areas of Alaska eight years after the Exxon Valdez oil spill. Marine Pollution Bulletin 40(2):152-164. https://doi.org/10.1016/S0025-326X(99)00194-0

Sharma, J. M. 1991. Overview of the avian immune system. Veterinary Immunology and Immunopathology 30(1):13-17. https://doi.org/10.1016/0165-2427(91)90004-v

Snoeijs, T., T. Dauwe, R. Pinxten, F. Vandesande, and M. Eens. 2004. Heavy metal exposure affects the humoral immune response in a free-living small songbird, the Great Tit (Parus major). Archives of Environmental Contamination and Toxicology 46:399-404. https://doi.org/10.1007/s00244-003-2195-6

Sonne, C., U. Siebert, K. Gonnsen, J. -P. Desforges, I. Eulaers, S. Persson, A. Roos, B. -M. Bäcklin, K. Kauhala, M. T. Olsen, K. C. Harding, G. Treu, A. Galatius, E. Andersen-Ranberg, S. Gross, J. Lakemeyer, K. Lehnert, S. S. Lam, W. Peng, and R. Dietz. 2020. Health effects from contaminant exposure in Baltic Sea birds and marine mammals: a review. Environment International 139:105725. https://doi.org/10.1016/j.envint.2020.105725

Swaileh, K. M., and R. Sansur. 2006. Monitoring urban heavy metal pollution using the House Sparrow (Passer domesticus). Journal of Environmental Monitoring 8(1):209-213. https://doi.org/10.1039/b510635d

Tartu, S., F. Angelier, J. C. Wingfield, P. Bustamante, P. Labadie, H. Budzinski, H. Weimerskirch, J. O. Bustnes, and O. Chastel. 2015. Corticosterone, prolactin and egg neglect behavior in relation to mercury and legacy POPs in a long-lived Antarctic bird. Science of The Total Environment 505:180-188. https://doi.org/10.1016/j.scitotenv.2014.10.008

Tepper, R. I., D. A. Levinson, B. Z. Stanger, J. Campos-Torres, A. K. Abbas, and P. Leder. 1990. IL-4 induces allergic-like inflammatory disease and alters T cell development in transgenic mice. Cell 62(3):457-467. https://doi.org/10.1016/0092-8674(90)90011-3

Totzke, U., M. Fenske, O. Hüppop, H. Raabe, and N. Schach. 1999. The influence of fasting on blood and plasma composition of Herring Gulls (Larus argentatus). Physiological and Biochemical Zoology 72(4):426-437. https://doi.org/10.1086/316675

Toyomaki, H., J. Yabe, S. M. M. Nakayama, Y. B. Yohannes, K. Muzandu, A. Liazambi, Y. Ikenaka, T. Kuritani, M. Nakagawa, and M. Ishizuka. 2020. Factors associated with lead (Pb) exposure on dogs around a Pb mining area, Kabwe, Zambia. Chemosphere 247:125884. https://doi.org/10.1016/j.chemosphere.2020.125884

Ushine, N., T. Kato, and S. Hayama. 2016. Sex identification in Japanese birds using droppings as a source of DNA [title translated from the Japanese]. Bulletin of the Japanese Bird Banding Association 28(2):51-70. https://doi.org/10.14491/jbba.MS082

Ushine, N., O. Kurata, Y. Tanaka, T. Sato, Y. Kurahashi, and S. Hayama. 2020b. The effects of migration on the immunity of Black-headed Gulls (Chroicocephalus ridibundus: Laridae). Journal of Veterinary Medical Science 82(11):1619-1626. https://doi.org/10.1292/jvms.20-0339

Ushine, N., S. M. M. Nakayama, M. Ishizuka, T. Sato, Y. Kurahashi, E. Wakayama, N. Sugiura, and S. Hayama. 2020a. Relationship between blood test values and blood lead (Pb) levels in Black-headed Gull (Chroicocephalus ridibundus: Laridae). Journal of Veterinary Medical Science 82(8):1124-1129. https://doi.org/10.1292/jvms.20-0246

Vallverdú-Coll, N., R. Mateo, F. Mougeot, and M. E. Ortiz-Santaliestra. 2019. Immunotoxic effects of lead on birds. Science of The Total Environment 689:505-515. https://doi.org/10.1016/j.scitotenv.2019.06.251

von Sperling, M. 2007. Waste stabilisation ponds. IWA Publishing, London, UK. https://doi.org/10.2166/9781780402109

Walsh, P. M. 1990. The use of seabirds as monitors of heavy metals in the marine environment. Pages 183-204 in R. W. Furness, R. W. and P. S. Rainbow, editors. Heavy metals in the marine environment. CRC, Boca Raton, Florida, USA. https://doi.org/10.1201/9781351073158

Williams, R. J., S. D. Holladay, S. M. Williams, and R. M. Gogal Jr. 2018. Environmental lead and wild birds: a review. Reviews of Environmental Contamination and Toxicology 245:157-180. https://doi.org/10.1007/398_2017_9

Wood, N., K. Bourque, D. D. Donaldson, M. Collins, D. Vercelli, S. J. Goldman, and M. T. Kasaian. 2004. IL-21 effects on human IgE production in response to IL-4 or IL-13. Cellular Immunology 231(1-2):133-145. https://doi.org/10.1016/j.cellimm.2005.01.001

Yamashina Institute for Ornithology. 2002. Atlas of Japanese Migratory Birds from 1961 to 1995 [title translated from the Japanese]. Yamashina Institute for Ornithology, Chiba, Japan. https://www.biodic.go.jp/banding/pdf/atlas_low_1.pdf