Pollution-related changes in nest microbiota: Implications for growth and fledging in three passerine birds.

Non-ferrous smelters emit toxic metals into the environment, posing a threat to wildlife health. Despite the acknowledged role of microbes in host health, the impact of such emissions on host-associated microbiota, especially in wild birds, remains largely unexplored. This study investigates the associations of metal pollution, fitness, and nest microbiota (serving as a proxy for early-life microbial environment) which may influence the nestling health and development. Our study focuses on three passerine birds, the great tit (Parus major), blue tit (Cyanistes caeruleus), and pied flycatcher (Ficedula hypoleuca), within control and metal-polluted sites around a Finnish copper-nickel smelter. The polluted sites had been contaminated with arsenic (As), cadmium (Cd), copper (Cu), nickel (Ni), and zinc (Zn). We performed bacterial 16S rRNA sequencing and metal analyses on 90 nests and monitored nestling body mass, fledging success, and various biotic and abiotic factors. Our findings revealed species-specific responses to metal exposure in terms of both fitness and nest microbiota. P. major and C. caeruleus showed sensitivity to pollution, with decreased nestling growth and fledging in the polluted zone. This was accompanied by a shift in the bacterial community composition, which was characterized by an increase in some pathogenic bacteria (in P. major and C. caeruleus nests) and by a decrease in plant-associated bacteria (within C. caeruleus nests). Conversely, F. hypoleuca and their nest microbiota showed limited responses to pollution, indicating greater tolerance to pollution-induced environmental changes. Although pollution did not correlate with nest alpha diversity or the most abundant bacterial taxa across all species, certain potential pathogens within the nests were enriched in polluted environments and negatively correlated with nestling fitness parameters. Our results suggest that metal pollution may alter the nest bacterial composition in some bird species, either directly or indirectly through environmental changes, promoting pathogenic bacteria and potentially impacting bird survival.

The effects of short-term glyphosate-based herbicide exposure on insect gene expression profiles.

Glyphosate-based herbicides (GBHs) are the most frequently used herbicides worldwide. The use of GBHs is intended to tackle weeds, but GBHs have been shown to affect the life-history traits and antioxidant defense system of invertebrates found in agroecosystems. Thus far, the effects of GBHs on detoxification pathways among invertebrates have not been sufficiently investigated. We performed two different experiments-1) the direct pure glyphosate and GBH treatment, and 2) the indirect GBH experiment via food-to examine the possible effects of environmentally relevant GBH levels on the survival of the Colorado potato beetle (Leptinotarsa decemlineata) and the expression profiles of their detoxification genes. As candidate genes, we selected four cytochrome P450 (CYP), three glutathione-S-transferase (GST), and two acetylcholinesterase (AChE) genes that are known to be related to metabolic or target-site resistances in insects. We showed that environmentally relevant levels of pure glyphosate and GBH increased the probability for higher mortality in the Colorado potato beetle larvae in the direct experiment, but not in the indirect experiment. The GBHs or glyphosate did not affect the expression profiles of the studied CYP, GST, or AChE genes; however, we found a large family-level variation in expression profiles in both the direct and indirect treatment experiments. These results suggest that the genes selected for this study may not be the ones expressed in response to glyphosate or GBHs. It is also possible that the relatively short exposure time did not affect gene expression profiles, or the response may have already occurred at a shorter exposure time. Our results show that glyphosate products may affect the survival of the herbivorous insect already at lower levels, depending on their sensitivity to pesticides.

Ecosystem consequences of herbicides: the role of microbiome.

Non-target organisms are globally exposed to herbicides. While many herbicides - for example, glyphosate - were initially considered safe, increasing evidence demonstrates that they have profound effects on ecosystem functions via altered microbial communities. We provide a comprehensive framework on how herbicide residues may modulate ecosystem-level outcomes via alteration of microbiomes. The changes in soil microbiome are likely to influence key nutrient cycling and plant-soil processes. Herbicide-altered microbiome affects plant and animal performance and can influence trophic interactions such as herbivory and pollination. These changes are expected to lead to ecosystem and even evolutionary consequences for both microbes and hosts. Tackling the threats caused by agrochemicals to ecosystem functions and services requires tools and solutions based on a comprehensive understanding of microbe-mediated risks.

Does Glyphosate Affect the Human Microbiota?

Glyphosate is the world's most widely used agrochemical. Its use in agriculture and gardening has been proclaimed safe because humans and other animals do not have the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). However, increasing numbers of studies have demonstrated risks to humans and animals because the shikimate metabolic pathway is present in many microbes. Here, we assess the potential effect of glyphosate on healthy human microbiota. Our results demonstrate that more than one-half of human microbiome are intrinsically sensitive to glyphosate. However, further empirical studies are needed to determine the effect of glyphosate on healthy human microbiota.

Adaptation of bacteria to glyphosate: a microevolutionary perspective of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase.

Glyphosate is the leading herbicide worldwide, but it also affects prokaryotes because it targets the central enzyme (5-enolpyruvylshikimate-3-phosphate, EPSP) of the shikimate pathway in the synthesis of the three essential aromatic amino acids in bacteria, fungi and plants. Our results reveal that bacteria may easily become resistant to glyphosate through changes in the 5-enolpyruvylshikimate-3-phosphate synthase active site. This indicates the importance of examining how glyphosate affects microbe-mediated ecosystem functions and human microbiomes.

Bird Feces as Indicators of Metal Pollution: Pitfalls and Solutions.

Bird feces are commonly used as a proxy for measuring dietary metal exposure levels in wild populations. Our study aims to improve the reliability and repeatability of fecal metal measurements and gives some recommendations for sampling. First, we studied levels of variation in metallic element (arsenic, calcium, cadmium, cobalt, copper, nickel, lead) concentrations: temporal variation within an individual, among siblings in a brood and among-brood/spatial variation. Second, we explored the variation caused by dual composition (urate vs. feces) of bird droppings. Two sets of fresh fecal samples were collected from pied flycatcher (Ficedula hypoleuca) nestlings living in a metal polluted area in summers 2017 (dataset 1) and 2018 (dataset 2). We found a great deal of temporal intra-individual variation in metal levels, suggesting that dietary exposure varied markedly in a short time scale (within a day). A sample from only one nestling per brood did not well describe the brood mean value, and we recommend that at least four siblings should be sampled. Brood level samples give relatively good temporal repeatability for most metals. For all the metals, the levels in the fecal portion were more than double to those in the urate portion. Since the mass proportion of urate in the bird droppings varied a great deal among samples, standardizing sampling, e.g., by collecting only the fecal part, would markedly reduce the variation due to composition. Alternatively, urate portion could be used for biomonitoring of internally circulated bioavailable metal.

Glyphosate-based herbicides influence antioxidants, reproductive hormones and gut microbiome but not reproduction: A long-term experiment in an avian model.

Controversial glyphosate-based herbicides (GBHs) are the most frequently used herbicides globally. GBH residues in the wild, in animal and human food may expose non-target organisms to health risks, yet the developmental and cumulative effects of GBHs on physiology and reproduction remain poorly understood. We present the first long-term study on the effects of subtoxic GBH exposure (160 mg/kg) on multiple key physiological biomarkers (cellular oxidative status and neurotransmitters), gut microbiome, reproductive hormones, and reproduction in an avian model. We experimentally exposed in Japanese quail females and males (Coturnix japonica) to GBHs and respective controls from the age of 10 days-52 weeks. GBH exposure decreased hepatic activity of an intracellular antioxidant enzyme (catalase), independent of sex, but did not influence other intracellular oxidative stress biomarkers or neurotransmitter enzyme (acetylcholinesterase). GBH exposure altered overall gut microbiome composition, especially at a younger age and in females, and suppressed potentially beneficial microbes at an early age. Many of the microbial groups increased in frequency from 12 to 28 weeks under GBH exposure. GBH exposure decreased male testosterone levels both at sexual maturity and at 52 weeks of exposure, but did not clearly influence reproduction in either sex (maturation, testis size or egg production). Future studies are needed to characterize the effects on reproductive physiology in more detail. Our results suggest that cumulative GBH exposure may influence health and reproduction-related traits, which is important in predicting their effects on wild populations and global poultry industry.

Glyphosate-based herbicide has soil-mediated effects on potato glycoalkaloids and oxidative status of a potato pest.

Glyphosate is the most used herbicide worldwide, targeting physiological pathways in plants. Recent studies have shown that glyphosate can also cause toxic effects in animals. We investigated the glyphosate-based herbicide (GBH)-induced changes in potato (Solanum tuberosum) plant chemistry and the effects of a GBH on the survival rate and oxidative status of the Colorado potato beetle (Leptinotarsa decemlineata). The beetles were reared on potato plants grown in pots containing soil treated with a GBH (Roundup Gold, 450 g/l) or untreated soil (water control). The 2nd instar larvae were introduced to the potato plants and then collected in 2 phases: as 4th instar larvae and as adults. The main glycoalkaloids of the potato plants, α-solanine and α-chaconine, were measured twice during the experiment. The α-solanine was reduced in potato plants grown in GBH-treated soil, which can be detrimental to plant defenses against herbivores. GBH treatment had no effect on the survival rate or body mass of the larvae or the adult beetles. In the larvae, total glutathione (tGSH) concentration and the enzyme activity of catalase (CAT), superoxide dismutase, and glutathione-S-transferase were increased in the GBH treatment group. In the adult beetles, CAT activity and tGSH levels were affected by the interactive effect of GBH treatment and the body mass. To conclude, environmentally relevant concentrations of a GBH can affect the potato plant's glycoalkaloid concentrations, but are not likely to directly affect the survival rate of the Colorado potato beetle, but instead, modify the antioxidant defense of the beetles via diet.

Weather effects on breeding parameters of two insectivorous passerines in a polluted area.

Direct and indirect effects of environmental pollution affect negatively to birds' breeding performance in both urban and industrial environments, but much less is known on how pollution and natural stress factors work together. In our long-term study (1991-2018), we explored whether industrial pollution and associated habitat changes increase the sensitivity of breeding parameters (hatching and fledging success, nestling growth) to temperature and precipitation in two insectivorous bird species, the great tit (Parus major) and the pied flycatcher (Ficedula hypoleuca). We found that both species are rather weather sensitive in terms of their fledging success, but especially in the F. hypoleuca, the negative effect was strengthened in a polluted environment. For both study species, all the breeding parameters, except growth of F. hypoleuca nestlings, were inferior in the polluted area and negatively affected by cold weather. Independent of pollution, the duration of rainy spells during the nestling period had an overall negative effect on fledging success of F. hypoleuca, and this effect became stronger at cold temperatures. The length of rainy spells was, however, positively associated with nestling wing length in both species, possibly because of better availability of some important food resources for wing growth in more humid conditions. The weather-pollution interactions in our study populations were not overwhelmingly strong, but those found in F. hypoleuca show that such interactions exist, they are species-specific and in our study system most likely associated to pollution-related resource (e.g. food) limitation. Higher sensitivity of F. hypoleuca to low temperatures is likely related to its less well-insulated nests and higher dependence on aerial prey, the availability of which is especially reduced during cold and rainy spells. Our study indicates that anthropogenic stress, such as pollution, has synergistic effects with natural stress factors affecting passerine birds' breeding performance.

Effects of parental exposure to glyphosate-based herbicides on embryonic development and oxidative status: a long-term experiment in a bird model.

Controversial glyphosate-based herbicides (GBHs) are the most frequently used herbicides globally. GBH residues are detected in soil, water, crops, and food products, potentially exposing non-target organisms to health risks; these organisms include wildlife, livestock, and humans. However, the potential for GBH-related parental effects are poorly understood. In the case of birds, GBHs may be transferred directly from mothers to eggs, or they may indirectly influence offspring performance by altered maternal resource allocation to eggs. We experimentally exposed a parental generation of Japanese quails (Coturnix japonica) to GBHs (200 mg/kg feed) or respective controls. Glyphosate residues were found in eggs (ca 0.76 kg/mg). Embryonic development tended to be poorer in the eggs of GBH-exposed parents (76% of eggs showed normal development) compared to control parents (89% normal eggs). Embryonic brain tissue from GBH-exposed parents tended to express more lipid damage (20% higher), yet other biomarkers showed no apparent differences. We detected no differences in egg quality (egg, yolk, or shell mass, egg hormone concentration) across the treatment groups. Given this is the first long-term study testing parental effects of GBHs with birds, more studies are needed characterizing GBH-associated changes in maternal allocation and for example epigenetic programming.

Glyphosate-based herbicide affects the composition of microbes associated with Colorado potato beetle (Leptinotarsa decemlineata).

Here, we examined whether glyphosate affects the microbiota of herbivores feeding on non-target plants. Colorado potato beetles (Leptinotarsa decemlineata) were reared on potato plants grown in pots containing untreated soil or soil treated with glyphosate-based herbicide (GBH). As per the manufacturer's safety recommendations, the GBH soil treatments were done 2 weeks prior to planting the potatoes. Later, 2-day-old larvae were introduced to the potato plants and then collected in two phases: fourth instar larvae and adults. The larvae's internal microbiota and the adults' intestinal microbiota were examined by 16S rRNA gene sequencing. The beetles' microbial composition was affected by the GBH treatment and the differences in microbial composition between the control and insects exposed to GBH were more pronounced in the adults. The GBH treatment increased the relative abundance of Agrobacterium in the larvae and the adults. This effect may be related to the tolerance of some Agrobacterium species to glyphosate or to glyphosate-mediated changes in potato plants. On the other hand, the relative abundances of Enterobacteriaceae, Rhodobacter, Rhizobium and Acidovorax in the adult beetles and Ochrobactrum in the larvae were reduced in GBH treatment. These results demonstrate that glyphosate can impact microbial communities associated with herbivores feeding on non-target crop plants.

Female Preference and Adverse Developmental Effects of Glyphosate-Based Herbicides on Ecologically Relevant Traits in Japanese Quails.

Controversial glyphosate-based herbicides (GBHs) are the most frequently used herbicides globally. An increasing number of studies have identified GBH residues in soil, water, and even human food that may expose nontarget organisms including wildlife, livestock, and humans to health risks. After a heated debate, the European Union allowed the use of GBHs to continue until 2022, after which their risks will be re-evaluated. Thus, decision makers urgently need scientific evidence on GBH residues and their possible effects on ecosystems. An important, yet neglected, aspect is to assess whether animals show preference or avoidance for GBH-contaminated food, as it can influence the likelihood of adverse health effects in wildlife. Here, using Japanese quails (Coturnix japonica) as our model, we show that females preferred GBH-contaminated food compared to control food. In females, exposure to GBHs caused delayed plumage development, and GBH residues were present in eggs, muscles, and liver. These results indicate that female preference is not adaptive, potentially exposing nontarget animals to greater risk of adverse effects of GBHs in natural and agricultural environments. Our results on tissue residues suggest that further studies are needed to understand the risks of such residues in the food chain.

Can Indirect Herbicide Exposure Modify the Response of the Colorado Potato Beetle to an Organophosphate Insecticide?

Organisms live in complex multivariate environments. In agroecosystems, this complexity is often human-induced as pest individuals can be exposed to many xenobiotics simultaneously. Predicting the effects of multiple stressors can be problematic, as two or more stressors can have interactive effects. Our objective was to investigate whether indirect glyphosate-based herbicide (GBH) exposure of the host plant has interactive effects in combination with an insecticide (azinphos-methyl) on an invasive pest Colorado potato beetle (Leptinotarsa decemlineata Say). We tested the effects of GBH and insecticide on the survival, insecticide target genes expression (acetylcholinesterase genes) and oxidative status biomarkers (glutathione S-transferase [GST], glucose-6-phosphate dehydrogenase [G6PDH], glutathione reductase homolog [GR], glutathione peroxidase homolog [GPx], total glutathione [totGSH], glutathione reduced-oxidized [GSH: GSSG], catalase [CAT], superoxide dismutase [SOD], lipid hydroperoxides). We found that exposure to indirect GBH has no single or interactive effects in combination with the insecticide on larval survival. However, prior exposure to GBH inhibits Ldace1 gene expression by 0.55-fold, which is the target site for the organophosphate and carbamate insecticides. This difference disappears when individuals are exposed to both GBH and insecticide, suggesting an antagonistic effect. On the other hand, oxidative status biomarker scores (PCAs of GPx, GR, and CAT) were decreased when exposed to both stressors, indicating a synergistic effect. Overall, we found that indirect GBH exposure can have both antagonistic and synergistic effects in combination with an insecticide, which should be considered when aiming for an ecologically relevant risk assessment of multiple human-induced stressors.

Effects of a glyphosate-based herbicide on survival and oxidative status of a non-target herbivore, the Colorado potato beetle (Leptinotarsa decemlineata).

Glyphosate is the globally most used herbicide against a wide range of weeds. Glyphosate has been considered safe to animals as it mainly targets physiological pathways in plants. However, recent toxicological studies have revealed that glyphosate can cause various toxic effects also on animals. In this study, we investigated the direct toxic effects of a glyphosate-based herbicide (GBH, Roundup® Bio) on 1) survival and 2) oxidative status of a non-target herbivore by using Colorado potato beetles (Leptinotarsa decemlineata), originating from Poland and USA, as model species. Larvae were randomly divided into three groups: 1) high concentration (100% Roundup Bio, 360 g/l), 2) low concentration (1.5% Roundup Bio) and 3) control group (water). Larvae were exposed to Roundup for different time periods: 2 h, 24 h, 48 h, 72 h and 96 h. Larval survival decreased in the group treated with high concentration of GBH compared to controls, whereas the low concentration group did not differ from the control group. GBH treatment had no association with oxidative status biomarkers (i.e. catalase, superoxide dismutase, glutathione-S-transferase, glutathione and glutathione related enzymes), but increased lipid hydroperoxide levels after 2 h exposure, suggesting increased oxidative damage soon after the exposure. Larvae of different origin also differed in their oxidative status, indicating population-dependent differences in antioxidant defence system. Environmentally relevant concentrations of GBH are not likely to affect larval survival, but high concentrations can reduce survival and increase oxidative damage of non-target herbivores. Also, populations of different origin and pesticide usage history can differ in their tolerance to GBH.

Antioxidant Enzyme Activities Vary with Predation Risk and Environmental Conditions in Free-Living Passerine Birds.

Prolonged physiological stress response may lead to an excessive production of reactive oxygen species (ROS) and ultimately to oxidative stress and severe fitness costs. We investigated whether natural variation in predation risk, induced by pygmy owls (Glaucidium passerinum), modifies the oxidative status of two free-living food-supplemented passerine bird species-the great tit (Parus major) and the willow tit (Poecile montanus)-in March 2012 and 2013. Predation risk significantly affected antioxidant enzyme activities of willow tits. Antioxidant enzyme activities (principal component factor 2 [PC2] representing glutathione-S-transferase and superoxide dismutase activities) were higher in high predation risk areas in 2013 than in low predation risk areas in the same year. Higher enzyme activities may suggest higher ROS production in birds living under high predation risk. In addition, antioxidant enzyme activities (PC2) were also higher in high predation risk areas in 2013 than in high predation risk areas in the previous year, 2012. This may represent variation in the risk represented by pygmy owls, which is probably inversely related to the natural fluctuations in the densities of their main prey, voles. In willow tits, PC1 (representing catalase, total glutathione, the ratio of reduced to oxidized glutathione, and protein carbonylation) was not affected by perceived predation risk, nor were antioxidant levels or enzyme activities in great tits. Higher enzyme activities observed in willow tits suggest that predator presence can modify the antioxidant status of avian prey, but the response also seem to be influenced by other environmental characteristics, like harsh winter conditions.

Telomere damage and redox status alterations in free-living passerines exposed to metals.

Telomere length may reflect the expected life span and possibly individual quality. Environmental stressors are known to increase oxidative stress and accelerate telomere attrition: however the interactions between redox status and telomere dynamics are not fully understood. We investigated whether exposure to heavy metal pollution is associated with oxidative stress and telomere damage in two insectivorous passerines, the Great tit (Parus major) and the Pied flycatcher (Ficedula hypoleuca). We were also interested to know whether within-brood competition could influence the nestling redox status or telomere length. Breeding females and nestlings were sampled near the point pollution source and compared to birds in non-polluted control zone. We measured heavy metal concentrations, calcium, metallothioneins, telomere lengths and redox status (oxidative damage, and enzymatic and non-enzymatic antioxidants) in liver samples. Great tit nestlings in the polluted zone had significantly shorter telomeres compared to those in the unpolluted control zone. In addition, those great tit nestlings that were lighter than their average siblings, had shorter telomeres compared to the heavier ones. In pied flycatchers neither pollution nor growth stress were associated with telomere length, but adult females had significantly shorter telomeres compared to the nestlings. All the results related to redox status varied remarkably among the species and the age groups. In both species antioxidants were related to pollution. There were no significant associations between redox status and telomere length. Our results suggest that wild birds at a young age are vulnerable to pollution and growth stress induced telomere damage. Redox status seems to interact with pollution and growth, but more studies are needed to clarify the underlying physiological mechanisms of telomere attrition. Our study highlights that all the observed associations and differences between the sampling zones varied depending on the species, age, and degree of exposure to pollution.

Effects of dietary lead exposure on vitamin levels in great tit nestlings - An experimental manipulation.

Exposure to metal pollution negatively affects animal physiology, including nutrient metabolism, but in the wild an effect can seldom be attributed to a single metal. Moreover, little is known about how the metabolism of vitamins, essential micronutrients for developing juveniles, is affected by toxic metals. Therefore we experimentally investigated the effects of lead (Pb), a widespread toxic metal, on four fat-soluble vitamins A (total and retinol), D3, E (total and α-tocopherol) and K and carotenoids (lutein, zeaxanthin and unidentified) in great tit (Parus major) nestlings. In addition to a control group where no Pb was provided, two Pb-dosed groups were compared to a metal exposed group in the vicinity of a Ni-Cu smelter. We examined whether Pb treatment affects vitamin homeostasis and how the response of Pb-treated birds relates to that of a population under industrial exposure of Pb and other metals. For this purpose, vitamin and carotenoid levels were quantified with UPLC-MS from plasma of 7 days-old nestlings. All metal exposed groups showed increased vitamin A and retinol levels. However, vitamin levels were not directly associated with fecal Pb levels, with the exception of retinol, which was positively correlated with fecal Pb. Alpha-tocopherol, lutein and zeaxanthin levels were positively associated with body mass and wing growth rate. To conclude, Pb exposure increased plasma vitamin A and retinol levels while the levels of other vitamins and carotenoids rather reflected secondary pollution effects via differences in habitat and diet quality at the smelter site. Our findings suggest Pb exposed nestlings may allocate the vitamins needed for growth and development to fight the physiological stress thus compromising their fitness.

Temporal trends in metal pollution: using bird excrement as indicator.

Past mining and smelting activities have resulted in metal polluted environments all over the world, but long-term monitoring data is often scarce, especially in higher trophic levels. In this study we used bird (Parus major and Ficedula hypoleuca) excrement to monitor metal pollution in the terrestrial environment following 16 years of continuously reduced emissions from a copper/nickel smelter in Finland. In the early 1990s, lead and cadmium concentrations dropped significantly in excrement, but the reduction did not directly reflect the changes in atmospheric emission from the smelter. This is likely due to a continuous contribution of metals also from the soil pool. We conclude that bird excrement can be used to assess changes in the environment as a whole but not specifically changes in atmospheric emission. Inter-annual variation in excrement concentration of especially copper and nickel demonstrates the importance of long-term monitoring to discern significant trends.

No delayed behavioral and phenotypic responses to experimental early-life lead exposure in great tits (Parus major).

Early-life exposure to pollutants, such as lead, may have long-lasting consequences on health, behavior, and cognition. However, experiments on delayed effects of specific pollutants are very rare in wild animals. We experimentally exposed wild nestling great tits (Parus major) to dietary lead (high, low, or control group) in levels relevant to exposure levels of wild populations in Europe and studied delayed effects on phenotypic and behavioral traits in captivity. We also included a group of birds from a vicinity of a copper smelter, exposed to a mixture of toxic metals and altered food supply during development. This experimental setup allowed us to compare the strength of direct (exposure to lead per se) and indirect (pollution-related changes in diet) effects of pollutants. Our experimental lead treatment significantly increased lead levels in bone and feces compared with controls. However, we found no carry-over effect of early-life dietary lead on morphology, plumage coloration, or heat shock proteins. Treatment did not affect activity, exploration, neophobia, or success in learning and spatial memory task. We conclude that with the exposure levels and relatively short exposure period used, delayed effects on the measured traits were not found. However, it is important to further study other types of behavioral traits and ultimately fitness effects.

Effects of early-life lead exposure on oxidative status and phagocytosis activity in great tits (Parus major).

Lead is a highly poisonous metal with a very long half-life, distributing throughout the body in blood and accumulating primarily in bones and kidney. We studied the short and long-term effects of early-life lead exposure on antioxidant defense and phagocytosis activity in a small passerine bird, the great tit (Parus major) by manipulating dietary lead levels of the nestlings. We had three experimental groups, exposed to environmentally relevant lead concentrations; high (4 µg/g body mass), low (1 µg/g body mass) and control (0 µg/g body mass) group. As a comparison, a great tit population breeding in the vicinity of a metal smelter was included to the experimental set-up. We measured glutathione, the ratio of reduced and oxidized glutathione, and the antioxidant enzymes: glutathione peroxidase, glutathione-S-transferase, catalase and superoxide dismutase together with protein carbonylation and phagocytosis activity to study the effects of lead on the oxidative status and immune function of birds. We found differences in enzyme activities between the study groups, but in most cases the smelter group differed from the other groups. Despite the differences observed in antioxidant enzymes, our results indicate only minor short-term effects of lead exposure on oxidative status, since either glutathione ratio or protein carbonylation were not affected by lead. Phagocytosis activity was not linked to higher lead concentrations either. Interestingly, protein carbonylation was positively associated with enzyme activities and glutathione level. Our results did not show major long-term effects of lead on the oxidative status of great tits.