Zoltán Nyiri

Effects of breeding habitat (woodland versus urban) and metal pollution on the egg characteristics of great tits (Parus major)

In an urban environment, birds are exposed to metals, which may accumulate in their tissues and cause oxidative stress. Female birds may eliminate these pollutants through depositing them into eggs, thus eggs become suitable bioindicators of pollution. In this study, we aimed to analyse whether eggshell spotting pattern, egg volume, eggshell thickness and egg yolk antioxidant (lutein, tocopherol, retinol and selenium) levels were related to the breeding area (woodland versus urban) and the metal levels in the eggshell of a small passerine species, the great tit (Parus major). In the urban habitat, soil and eggshells contained higher concentrations of metals, and soil calcium level was also higher than that in the woodland. Eggshell spotting intensity and egg volume did not differ between eggs laid in the woodland and the urban park, and these traits were not related to the metal levels of the eggshell, suggesting that these egg characteristics are not sensitive indicators of metal pollution. A more aggregated eggshell spotting distribution indicated a higher Cu concentration of the eggshell. We found that eggshells were thinner in the less polluted woodland habitat, which is likely due to the limited Ca availability of the woodland area. Great tit eggs laid in the urban environment had lower yolk lutein, retinol and selenium concentrations, however, as a possible compensation for these lower antioxidant levels, urban females deposited more tocopherol into the egg yolk. It appears that females from different breeding habitats may provide similar antioxidant protection for their offspring against oxidative damage by depositing different specific dietary antioxidants. Egg yolk lutein and retinol levels showed a negative relationship with lead concentration of the eggshell, which may suggest that lead had a negative impact on the amount of antioxidants available for embryos during development in great tits.

Analysis of Potential Migrants from Plastic Materials in Milk by Liquid Chromatography–Mass Spectrometry with Liquid–Liquid Extraction and Low-Temperature Purification

A simple and fast analytical method was developed for the determination of six UV stabilizers (Cyasorb UV-1164, Tinuvin P, Tinuvin 234, Tinuvin 326, Tinuvin 327, and Tinuvin 1577) and five antioxidants (Irgafos 168, Irganox 1010, Irganox 3114, Irganox 3790, and Irganox 565) in milk. For sample preparation liquid-liquid extraction with low-temperature purification combined with centrifugation was used to remove fats, proteins, and sugars. After the cleanup step, the sample was analyzed with high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). External standard and matrix calibrations were tested. External calibration proved to be acceptable for Tinuvin P, Tinuvin 234, Tinuvin 326, Tinuvin 327, Irganox 3114, and Irganox 3790. The method was successfully validated with matrix calibration for all compounds. Method detection limits were between 0.25 and 10 μg/kg. Accuracies ranged from 93 to 109%, and intraday precisions were <13%.

Darker eggshell spotting indicates lower yolk antioxidant level and poorer female quality in the Eurasian Great Tit (Parus major)

ABSTRACT Protoporphyrin pigment causes the red-brown eggshell colors; however, for many species, the function of this pigment is unknown. It has been proposed that eggshell pigmentation may strengthen the shell or that it may be a sexually selected signal, which advertises the quality of the female and that of her offspring to the male parent. In this study, we aimed to discover whether protoporphyrin-based eggshell pigmentation patterns of Eurasian Great Tits (Parus major) were related to female or egg quality. Additionally, we tested whether different methods of eggshell pigmentation estimation could be reliable predictors of eggshell protoporphyrin levels. We found that spot intensity, spot size, spotting coverage, and brown spot chroma indicated the protoporphyrin pigment concentration of the eggshell. Our results revealed that Eurasian Great Tit females that laid eggs with darker pigmentation had more lymphocytes in their circulation and had paler yellow breast and lower UV plumage reflectance, possibly indicating poorer health and individual quality. However, we did not find evidence that eggshell pigmentation patterns indicated the body condition, body size, or plasma oxidative status of females. Furthermore, we found that eggs with darker spots contained lower concentrations of antioxidants in the yolk. High protoporphyrin levels may be detrimental to females as they may cause oxidative damage, and this may be why birds that laid eggs with darker spots deposited lower amounts of antioxidants into the egg yolk. Shell spot darkness may also indicate territory quality, as females that laid smaller clutches also laid eggs with higher eggshell pigmentation levels. Thus, our results suggest that shell spot darkness may indicate the state of health of the female, egg yolk antioxidant level, and possibly also the quality of the territory.

Effects of environmental conditions on the egg mass, yolk antioxidant level, eggshell thickness and eggshell spotting patterns of Great Tits (Parus major)

AbstractAvian mothers can influence the fitness of their offspring by resource investment into the egg. Allocation of macro- and micronutrients into the eggs may be costly for the female, therefore, we expect that resource investment may be affected by the environmental and social conditions the mother experiences during egg formation. Here, we investigated whether environmental circumstances experienced by the reproducing female exert an influence on egg mass, yolk antioxidant (lutein and tocopherol) concentration, eggshell thickness, and the eggshell spotting patterns of Great Tits (Parus major). Our study showed that when caterpillars were less abundant, in colder weather and when breeding in an area of higher local breeding density, female Great Tits laid eggs of lower mass, suggesting that adverse environmental circumstances constrain the macronutrient investment into the eggs. However, we found no evidence that yolk antioxidant concentration and eggshell thickness were affected by environmental factors. Female Great Tits may use their endogenous stores or spend more time and energy in finding sufficient amounts of dietary antioxidants and calcium under unfavourable environmental conditions, which may have a cost effect on their own conditions. We found that birds that bred in the beginning of the season laid eggs with darker eggshell spots. Moreover, females laid more spotted eggs in colder weather and when breeding in higher density areas. Our results suggest that Great Tits deposit more of the potentially harmful pro-oxidant protoporphyrin pigment into the eggshell under unfavourable environmental conditions.ZusammenfassungAuswirkungen von Umweltbedingungen auf Eimasse, Antioxidantiengehalt des Dotters, Schalendicke und Fleckung der Eischale bei Kohlmeisen (Parus major) Vogelmütter können die Fitness ihrer Nachkommenschaft durch die Ressourcen beeinflussen, die sie in ein Ei investieren. Die Abgabe von Makro- und Mikronährstoffen an die Eier kann für das Weibchen kostspielig sein, daher ist zu erwarten, dass die Investition von Ressourcen durch Umweltbedingungen und soziale Faktoren beeinflusst wird, denen die Mutter während der Eibildung ausgesetzt ist. Hier untersuchten wir, inwieweit die Umweltbedingungen, denen das Weibchen während der Fortpflanzung ausgesetzt war, einen Einfluss auf die Eimasse, die Konzentration von Antioxidantien (Lutein und Tocopherol) im Dotter, die Schalendicke und die Fleckung der Eischalen von Kohlmeisen (Parusmajor) ausübten. Unsere Studie zeigte, dass Kohlmeisen-Weibchen in Zeiten geringeren Raupenangebots, bei kälterem Wetter und in Brutgebieten mit höherer lokaler Brutdichte Eier von geringerer Masse legten, was darauf hindeutet, dass ungünstige Umweltbedingungen die Investition von Makronährstoffen in die Eier beschränken können. Allerdings fanden wir keine Hinweise darauf, dass die Konzentration von Antioxidantien im Dotter und die Schalendicke durch Umweltfaktoren beeinflusst wurden. Unter ungünstigen Umweltbedingungen können Kohlmeisen-Weibchen körpereigene Reserven angreifen oder mehr Zeit und Energie auf die Suche nach ausreichenden Mengen nahrungsgebundener Antioxidantien und Kalzium aufwenden, was zu Lasten ihrer eigenen Kondition gehen kann. Wir stellten fest, dass früh in der Saison brütende Vögel Eier mit dunklerer Schalenfleckung legten. Außerdem legten die Weibchen bei kälterem Wetter und in dichter besetzten Brutgebieten mehr gefleckte Eier. Unsere Ergebnisse deuten an, dass Kohlmeisen unter ungünstigen Umweltbedingungen mehr des potenziell schädlichen prooxidativen Pigments Protoporphyrin in der Eischale ablagern.

Solubility determination as an alternative to migration measurements

ABSTRACT Solubility values for six UV stabilisers (Cyasorb UV-1164, Tinuvin P, Tinuvin 234, Tinuvin 326, Tinuvin 327 and Tinuvin 1577) and five antioxidants (Irgafos 168, Irganox 1010, Irganox 3114, Irganox 3790 and Irganox 565) were determined in all the liquid food simulants (3% (m/V) acetic acid–water mixture, 10% (V/V), 20% (V/V), 50% (V/V) ethanol–water mixture and vegetable oil) proposed in European Union Regulation No. 10/2011/EC, as well as in fruit juice and cola drink. The applied method was obtained by modification of the method for the determination of water solubility as described in OECD guideline Test No. 105. By using ultrasonication and shorter equilibration time, the time demand of the solubility determinations were decreased notably. Solubility values proved to be lower than the specific migration limits (as specified in 10/2011/EC) at 25°C for almost all target compounds in food simulants A, B, C and D1 as well as in fruit juice and cola drink. The exceptions were Tinuvin P and Irganox 3790 in simulant D1. The solubility in food simulant D2 was higher than 1000 µg ml–1 for all target compounds. These results show that the solubility of some additives in food simulants can be so low that it makes migration studies for certain additive–food simulant pairs dispensable.

Biliverdin- and protoporphyrin-based eggshell pigmentation in relation to antioxidant supplementation, female characteristics and egg traits in the canary (Serinus canaria)

Avian eggs exhibit a large variability in coloration and patterns, which are produced by blue-green biliverdin and red-brown protoporphyrin pigments. Several hypotheses have been proposed to explain the function of eggshell coloration. In this experimental study, we tested two hypotheses (signalling-function hypothesis and structural-function hypothesis) on both eggshell pigment types in an open-nesting songbird, the canary (Serinus canaria). Also, we aimed to examine whether deposition of pigments into the eggshell has any cost in terms of the plasma oxidative status of the female. We found that eggshell average blue-green chroma was increased by antioxidant supplementation, although we note that there had already been a pre-existing bias in plasma antioxidant capacity between the supplemented and control groups. Eggshell average blue-green chroma was positively related to female body condition during egg laying. However, blue-green eggshell colour was not related to female oxidative status during or after the laying period, and blue-green chroma increased with laying order. Accordingly, we found some support for that eggshell blue-green colour could reflect maternal antioxidant availability and body condition, but did not find evidence that it has a cost for the female’s oxidative status. By contrast, eggshell spot brightness was positively related to body condition, suggesting that darker spotting reflected poorer nutritional condition in the canary. Eggshell blue-green pigmentation was not significantly connected to the egg volume or yolk antioxidant level, but we found that eggs with lower yolk antioxidant concentration had higher average eggshell brown chroma. In sum, our results suggest that eggshell colour reflected female antioxidant and nutrient availability. Finally, we found that eggs with thinner eggshells had a more aggregated spot distribution, supporting the view that aggregated spots may help to strengthen eggshells.Significance statementAvian eggs have a large variability in colours and patterns, which are due to two pigments: the blue-green biliverdin and the red-brown protoporphyrin. In this study, we tested whether eggshell pigmentation, measured by a spectrophotometer, could reflect female and egg quality in the canary, an open nesting songbird. We found that females supplemented with antioxidants before and during egg-laying laid more intense blue-green eggs. Females in better body condition laid eggs that had a more intense blue-green coloration, but lower intensity of brown coloration. Egg yolk antioxidant level was lower in eggs with more intense brown eggshell coloration. These results suggest that eggshell coloration could reflect the antioxidant and nutrient availability of females. Moreover, we found that eggs with thinner eggshells had a more aggregated spot distribution on the eggshell, supporting the view that aggregated brown pigment spots may help to strengthen eggshells.

Optimisation of solid-state urea clathrate formation as a chemical separation method coupled to compound-specific stable carbon isotope analysis

ABSTRACT Solid-state urea clathrate formation (SSUCF) as a chemical separation method prior to stable carbon isotope fingerprinting of diesel fuel contaminations was studied. The stable carbon isotope ratios (δ13C) of n-alkanes in diesel fuel can be used to trace the origin of a contamination. The accurate measurement of the stable isotopic composition of individual compounds requires baseline separation from any other co-eluting compounds. For this purpose silica gel column chromatography (SGCC) and SSUCF were applied. Detailed optimisation of SSUCF was performed: different activators, clathrate formation temperatures, activator volumes, clathrate formation times and sample capacity were investigated. The main benefits of the developed method are reduced clathrate formation time and increased recoveries for lower molecular weight n-alkanes. The recoveries of the developed SSUCF method ranged between 63 and 100% for C10–C24 n-alkanes with relative standard deviation no more than 7%. The precision of the gas chromatography-isotope ratio mass spectrometry measurement was acceptable with a standard deviation of the δ13C values ranging between 0.08 and 0.15‰. The absence of isotopic fractionation was also investigated. The robustness of the method was tested within a model experiment. Nine different water samples including distilled water, tap water, river water, industrial wastewaters and groundwater samples were spiked with the same diesel fuel. The water samples were extracted with n-hexane and after purification with both SGCC and SSUCF n-alkanes were measured. The δ13C values of n-alkanes were found to be similar for all samples. The importance of sample purification prior to compound-specific isotope analysis (CSIA) was also demonstrated within this model experiment by analysing samples from different stages of the sample preparation. Our results show that the proposed method can remarkably improve the precision of compound-specific stable carbon isotope analysis of n-alkanes originating from diesel contamination of the aquatic environment.

Determination of particulate phase polycyclic aromatic hydrocarbons and their nitrated and oxygenated derivatives using gas chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry

An analytical method has been developed for the quantitative determination of polycyclic aromatic hydrocarbons (PAHs) and their nitrated and oxygenated derivatives (nitro- and oxy-PAHs respectively) in particulate matter (PM) samples. The sample preparation procedure included only a simple and quick sonication-assisted extraction step, clean-up based on addition of water and centrifugation as well as pre-concentration under N2 stream. The determination of 16 PAHs and 4 oxy-PAHs was carried out by gas chromatography-mass spectrometry, while liquid chromatography-tandem mass spectrometry was used in the case of the 11 investigated nitro-PAHs. The optimized method was fully evaluated in terms of trueness, precision (repeatability), limit of detection (LOD), limit of quantification (LOQ), sensitivity and linearity. The LOQ values ranged at pgm-3 level for the investigated PAHs (42pgm-3), oxy-PAHs (either 42 or 83pgm-3) and nitro-PAHs (either 83 or 167pgm-3) as well. The developed method was applied for the quantitative determination of PAHs, nitro- and oxy-PAHs in urban PM2.5 (particles with aerodynamic diameter smaller than 2.5μm) samples (n=36) collected in Budapest, Hungary. Almost 100% of the PM2.5 samples contained the investigated PAHs and oxy-PAHs in detectable and quantifiable amounts; however, the concentration of the nitro-PAHs was generally lower than the corresponding LOD/LOQ values. According to our results, during the 3-year long sampling campaign the concentration of benzo(a)pyrene never exceeded the limit value (1ngm-3) set by the European Commission.

Eggshell Biliverdin and Protoporphyrin Pigments in a Songbird: Are They Derived from Erythrocytes, Blood Plasma, or the Shell Gland?

Biliverdin and protoporphyrin pigments are deposited into the eggshell when the developing egg is in the shell gland. However, the site of synthesis of eggshell pigments is still uncertain, although it may influence the possible costs and potential functions of eggshell coloration in avian species. Eggshell pigments may be derived from red blood cells or be produced in other organs and then transferred to the shell gland, or they may be synthesized de novo in the shell gland. We studied in the canary (Serinus canaria) whether eggshell blue-green and brown pigmentations are associated with experimentally elevated anemia, female hematocrit level, immature erythrocyte percentage, and feces and plasma pigment levels during egg laying to find out the possible origin of eggshell pigments. We found no significant effects of hematocrit level or experimentally elevated anemia on intensity of eggshell blue-green and brown pigmentations; therefore, we consider it less likely that eggshell pigments are derived from erythrocytes. In addition, we found no significant associations between female feces biliverdin concentration during egg laying and intensity of eggshell blue-green pigmentation, suggesting that eggshell biliverdin may not originate from the spleen or liver. We found a negative association between plasma and feces protoporphyrin concentrations during egg laying and eggshell brown chroma. This result suggests that an increased production of protoporphyrin in the liver, which could have elevated plasma and feces protoporphyrin concentrations, could inhibit eggshell protoporphyrin pigmentation, probably through affecting enzymatic activities. We suggest that both pigments are produced de novo in the shell gland in the canary, but circulating pigment levels may influence shell gland pigment synthesis, thus connecting the physiological status of the female to eggshell coloration.

Effects of Temperature and Duration of Storage on the Stability of Antioxidant Compounds in Egg Yolk and Plasma

Antioxidants help protect tissues from oxidative damage caused by reactive oxygen species. In view of the widespread interest in egg yolk and plasma antioxidants in relation to environmental and life-history variables, there is a need for knowledge on the necessary conditions for sample storage, which is currently lacking. In this study, our purpose was to examine the kinetics of the degradation of lutein, retinol, and tocopherol in egg yolk samples and the total antioxidant capacity in plasma samples stored at three different temperatures (−20°, −50°, and −80°C) for 24 mo. We found that yolk lutein was stable during the study period. Yolk retinol and tocopherol showed a steep early decline and then remained relatively stable, but retinol showed significant losses at the end of the study period too. In contrast to our expectations, there was no difference in the stability of antioxidant compounds of egg yolk samples stored at different temperatures. Plasma OXY level was stable during the first 6 mo, showed a slight decline between 6 and 12 mo, and declined more intensely after 12 mo of storage. We suggest that studies focusing on the analysis of egg yolk retinol or tocopherol concentrations and total plasma antioxidant capacity should analyze the samples in the first 6–7 mo after collection. For the analysis of yolk lutein, samples can be stored for 24 mo without significant degradation. The storage temperature of −20°C seemed to be sufficient, as a lower temperature did not significantly affect the slope of degradation of the samples.