Variations in tree sparrow (Passer montanus) egg characteristics under environmental metal pollution

Science of the Total Environment 687 (2019) 946–955

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Variations in tree sparrow (Passer montanus) egg characteristics under environmental metal pollution Jian Ding a, Wenzhi Yang a, Ying Yang a, Shiwei Ai b, Xiaojuan Bai a, Yingmei Zhang a,⁎ a b

Gansu Key Laboratory of Biomonitoring and Bioremediation for Environmental Pollution, School of Life Sciences, Lanzhou University, Lanzhou 730000, China School of Public Health, Lanzhou University, Lanzhou 730000, China

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Tree sparrow egg characteristics can be used as bioindicators of metal pollution. • Bigger clutch size was accompanied by higher within-clutch CV for egg volume. • Eggshells were significantly thinner and darker in metal polluted area. • More aggregated spotting eggshells were associated with higher Zn and Pb levels.

a r t i c l e

i n f o

Article history: Received 9 April 2019 Received in revised form 8 June 2019 Accepted 8 June 2019 Available online 10 June 2019 Editor: Jay Gan Keywords: Environmental metal pollution Egg characteristics Passer montanus

⁎ Corresponding author. E-mail address: [email protected] (Y. Zhang).

https://doi.org/10.1016/j.scitotenv.2019.06.140 0048-9697/© 2019 Elsevier B.V. All rights reserved.

a b s t r a c t Environmental metal pollution is known to adversely affect bird reproduction, for which the variations of egg characteristics are considered very important. Our study explored whether variations in egg characteristics, such as egg volume, eggshell spotting pattern, eggshell coloration, and eggshell thickness, were correlated with heavy metal levels (Cu, Zn, Pb, and Cd) and Ca levels in the eggshells of tree sparrows (Passer montanus), a widespread passerine species. Eggs were collected from a long-term heavy metal polluted area (Baiyin, BY, northwest China) and a relatively unpolluted area (Liujiaxia, LJX, northwest China). Our results showed that the embryonated (eggshell: Cu: p = 0.003, Pb: p = 0.002) and non-embryonated (egg contents: Pb: p = 0.044, Ca: p = 0.045) eggs collected from BY contained relatively higher metal concentrations than those from LJX. Eggs from BY were smaller in volume (p b 0.01) and thinner in eggshell thickness (p b 0.01) than those from LJX. Mean egg volume increased with clutch size in BY (p = 0.017), which was also accompanied by an increase in the within-clutch coefficient of variation (CV) for egg volume (p = 0.045). Clutches with a higher CV for egg volume tended to contain higher concentrations of Zn and Pb (Zn: p = 0.084; Pb: p = 0.081) in the eggshells from BY. No differences were found in the eggshell spotting coverage ratio of eggs; however, eggshells were much darker in BY than in LJX. A more aggregated eggshell spotting distribution indicated higher eggshell Zn and Pb levels (BY: Zn: p = 0.040, Pb: p = 0.076; LJX: Pb: p = 0.066). The results demonstrate that the egg characteristics of tree sparrows can be used as indicators of metal pollution, especially for the within-clutch CV for egg volume, eggshell spotting pattern and eggshell coloration. © 2019 Elsevier B.V. All rights reserved.

J. Ding et al. / Science of the Total Environment 687 (2019) 946–955

1. Introduction Wild birds exposed to metals and other anthropogenic substances may accumulate pollutants in their tissues (Spalding et al., 2000; Sakellarides et al., 2006; Gasparik et al., 2010; Yohannes et al., 2017) and suffer negative reproductive effects (Eeva and Lehikoinen, 1995; Nam and Lee, 2006; Eeva et al., 2009). The use of bird eggs as bioindicators of pollution has been repeatedly emphasized (Burger, 1994; Dauwe et al., 2005; Ruuskanen et al., 2014; Hargitai et al., 2016). Egg characteristics such as size and eggshell thickness are vital life history traits for breeding under maternal control and are commonly considered to be important indicators of egg quality. These traits have been used as early indicators of changes in reproduction, growth, and nestling survival of birds (Tuan et al., 1991; Christians, 2002). The balance between egg size and clutch size is significant because it is important for determining the number of surviving offspring and overall fitness. Variability in egg size within clutches is an important reproductive strategy of birds under changing environmental conditions (Krist, 2011). Egg size and eggshell thickness may be subject to both constraints and adaptive adjustments depending on environmental conditions (Tryjanowski et al., 2004; Pollentier et al., 2007; Kvalnes et al., 2013). Ca availability is very important for the formation of eggshells, the strength of eggshells; however, heavy metals can disturb intestinal absorption of Ca and the metabolic pathway of Ca during egg laying (Graveland, 1998; Dauwe et al., 2005), and result in smaller eggs and thinner eggshells (Eeva and Lehikoinen, 1995; Nam and Lee, 2006). Eggshell spotting pattern and coloration may also be affected by environmental pollution (Jagannath et al., 2008; Hanley and Doucet, 2012; Hargitai et al., 2016). The red-brownish spotting of the eggs that are laid by many passerine species is caused by the protoporphyrin pigment, which is produced by the metabolism of red blood cells (Baird et al., 1975). Some significant roles for this pigment include a structural function as a cushion, making the eggshell more resistant when it becomes thinner (Gosler et al., 2005; Hargitai et al., 2013); alternatively, the pigment may act as a signal of a female's condition because of its effect as a pro-oxidant (Moreno and Osorno, 2003). Heavy metal exposure can lead to the production of thinner eggshells (Grandjean, 1976; Eeva and Lehikoinen, 1995) and can alter the synthesis and excretion of protoporphyrin pigment (Casini et al., 2003; Mateo et al., 2006), which may influence the eggshell spotting pattern and coloration. Therefore, birds exposed to heavy metal pollution may produce more spotted, darker eggs or may produce spotted eggs with more aggregated pigment (Espín et al., 2016; Hargitai et al., 2016); thus, spottiness and coloration of eggshell can be used as non-destructive bio-assays of egg health. Generally, egg production in passerine birds represents a short-term maternal transfer of resources, including the substances and contaminants derived from recently assimilated food and possibly micronutrients from long-term storage reserves (Ward and Bryant, 2006; Van Dyke et al., 2013). Additionally, the high lipid content of bird eggs can concentrate hydrophobic contaminants (Van den Steen et al., 2010). Many resident passerine species, such as great tits (Parus major) and blue tits (Cyanistes caeruleus), are particularly effective species for monitoring local environmental contamination because of their small territories and foraging areas (Dauwe et al., 2002; Eeva et al., 2009). We selected as study species the tree sparrow (Passer montanus), a widely distributed, sedentary, omnivorous species, which forages in urban environments (Chu and Zheng, 1982; Pan et al., 2008) and could therefore be a suitable bioindicator of local contamination. Several studies have suggested the effects of metal pollution on the sparrows (Pinowski et al., 1993; Swaileh and Sansur, 2006; Kekkonen et al., 2012; Baker et al., 2017) and there are several studies on the variation in egg size of tree sparrow under normal environmental conditions (Lebedeva, 1999; Pinowski et al., 2001). However, we found that there is a lack of understanding of the variation in egg size within clutches of tree sparrows exposed to environmental metal pollution and of how varying degrees of metal pollution contribute to variations

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in the within-clutch coefficient of variation (CV) for egg size. Thus, our study aimed to (i) determine overall variation in egg size within clutches, (ii) identify differences in eggshell thickness, eggshell spotting pattern and coloration between two study sites differing in contaminant levels, (iii) evaluate concentrations of metals (Cu, Zn, Pb, Cd, and Ca) in eggs from two study sites, and (iv) determine the relationship between egg characteristics and metal concentrations. 2. Materials and methods 2.1. Ethics statement The experiments complied with the ethics committee of the School of Life Sciences, Lanzhou University. 2.2. Study species and study site The tree sparrow is a resident bird with a habitat range of approximately 2–3 km (Pan and Zheng, 2003). According to 2 years of continuous observation, the breeding season of the tree sparrows in our study sites lasts from the first week of April until the end of July, with the peak reproduction period occurring from mid-May to early July. Females lay two clutches of eggs per year, with an average of 4.52 ± 0.90 (mean ± SD) eggs. Two study sites, 110 km apart, were selected. These included: 1) Silong town in Baiyin (BY) city (E104°23′, N36°25′), a moderately polluted area that has been contaminated by industrial wastewater from metal smelting for a long time, with heavy metals (Cu, Zn, Pb, and Cd) being the main environmental pollutants (Nan et al., 2002; Liu, 2003; Wang et al., 2012); and 2) Weijiachuan village in Liujiaxia (LJX) town (E103°15′, N35°56′), an upstream site of the Yellow River with similar fauna and natural characteristics to BY but a relatively unpolluted area. At these two sites, in 2017, we measured the concentrations of heavy metals in soils and water (Table S1), and in the tissues of adult birds (Table S2). A previous study demonstrated that sparrow food sources from BY contained higher heavy metal concentrations than those from LJX (Ai, 2018), which might indicate that tree sparrows in BY are at risk of consuming higher amount of heavy metals. Moreover, the concentrations of Pb and Cd in all food sources from the two sites exceeded the limits set by the national standard (GB 2762-2017) (Ai, 2018). 2.3. Field work Our study was carried out on nest box breeding populations of tree sparrows from 2017 to 2018 (BY: 38 nests; LJX: 45 nests). These nests commonly contained clutches of three to six eggs. Females laid one egg per day until the clutch was complete. On rare occasions, a day of laying would be skipped because of severe weather. Starting from the beginning of April, all nest boxes were checked weekly. Occupied nest boxes (defined as those with nest material) were then checked daily to determine the clutch initiation date and the exact egg laying order. Eggs were numbered with a waterproof marker according to their laying order. Egg length and breadth were measured with digital calipers (to the nearest 0.01 mm) and the volume was estimated using the following formula: volume = 0.51 × length × breadth2 (Hoyt, 1979). Shape (elongation) was calculated according to the following formula: elongation = length/breadth (Hoyt, 1976). To obtain a more detailed understanding of egg size for the two sites, we also calculated the within-clutch coefficient of variation (CV) for egg volume, using a formula that adjusted the CV for small sample sizes (Sokal and Rohlf, 1995). From 2017 to 2018, we collected eggs at different hatching stages, the eggs were opened along their equator and the contents were placed in glass dishes to examine the presence of the embryos (Orłowski et al., 2016). The development of the embryo was classified into three stages

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(early, 0–4 d; midterm, 5–8 d; late, 9–11 d), it was determined that an obvious embryo appears on roughly the fifth day of the incubation period. We collected 7 clutches (3–5 clutch size, 34 eggs) from BY and 13 clutches (4–6 clutch size, 68 eggs) from LJX for analyses during the early incubation period (circa days 2–3), in which the embryos were not visible. In order to reduce influence on the reproduction of the tree sparrows, we only collected 10 eggs from BY and 30 eggs from LJX during the later incubation period (circa days 7–8), during which the eggs contained visible embryos. Based on our video observation and relevant research (Seel, 1968), the hatching of all eggs, except for the last to be laid in each clutch, is synchronized in tree sparrows; therefore, the age of embryo is roughly the same as the hatching time. All eggs were stored in the freezer (−20 °C) until further analysis.

2.4. Eggshell spotting pattern and coloration Pictures of the eggs were taken every morning with a digital camera (Canon EOS M; Canon Inc., Tokyo, Japan) to ensure that the newly laid eggs were photographed and measured in a timely fashion. To stabilize the eggs before photographing, each egg was placed on the lid of a freezing tube and was photographed against a white panel background next to a ruler. Tree sparrows are known to lay intensely colored eggs and have interclutch variability (Poláček et al., 2013); therefore, we took four separate images capturing four egg regions for each egg (sharp pole, blunt pole, and two middle sections of the two opposite sides). Pictures were saved as JPG files and were processed using Adobe Photoshop (PS) (Adobe Systems, Mountain View, CA). Most studies of eggshell spottiness have graded the spotting pattern by artificial visual scoring of photographs (Sanz and Garcia-Navas, 2009; Brulez et al., 2014; Hargitai et al., 2016). However, subjectivity may affect results. The eggshell spots in the aforementioned studies can be clearly defined, whereas the spots of tree sparrow eggs overlap and do not have clear boundaries. To avoid subjectivity, we only calculated the proportion of the speckled region (eggshell spotting coverage ratio). First, we used the automatic lasso tool in Photoshop to buckle down the eggshell image. Owing to the curvature of eggs and unequal shading, we collected images from the central areas of the four different egg regions for subsequent spottiness analyses according to the methodology described by Šulc et al. (2016). Because the images were highly dependent on luminosity conditions, we could not directly analyze the whole gray threshold when the images were split up to obtain threshold images. Second, the threshold images were imported into ImageJ for pixel analysis (Rasband, 2012). We obtained the proportion of the image that was speckled as the number of total pixels in the image occupied by spots (black) divided by the measured total area of the images (black white). The spotting coverage ratio for each egg was calculated as the mean of the four egg regions. All images were processed in the same manner to avoid systematic error (Fig. 1). The surface color of the eggshells was measured with a portable battery-driven spectrophotometer NS810 (3nh Scientific Inc., Shenzhen, China). Eggs were placed directly on the measuring port of the spectrophotometer with a diameter of 8 mm. The eggshells could completely fill the space covered by the specimen measuring port. Due to overlapping of the spots on the eggshells, the precision of our color measure was not limited to the color of the spots, but also included the relatively non-spotted areas. Reference calibrations against zero and a white standard tablet associated with the apparatus were performed periodically according to the apparatus instructions. For each egg, reflectance spectra were produced as the mean of three egg regions (sharp pole, middle section, and blunt pole), with three random measurements taken from each region. The CIE LCh color scale was used for color measurements. Lightness (L*) refers to position on a grayscale between black and white, chroma (C*) refers to spectral variance, and hue (h°) corresponds to wavelength of light (CIE color space, 1976, USA).

Fig. 1. Four egg regions were used to analyze eggshell pigment spotting of tree sparrow eggs (A, B = middle section of the egg, C = sharp pole, D = blunt pole).

2.5. Eggshell thickness measurement After thawing, we opened the eggs along the equator with a surgical blade previously rinsed with acetone. Egg contents were transferred into pre-weighted 2 mL centrifuge tubes, which were placed in a refrigerator at 4 °C for 24 h before analysis. Eggshells were sponged using medical cotton swabs soaked in 96% ethanol and were then dried at room temperature. Eggshells may become thinner due to the development of embryos, which is an intrinsic feature of oviparous vertebrates (Orłowski and Hałupka, 2015). Assessments of the change in eggshell thickness or of the concentration of metal within an eggshell for ecotoxicological studies should consider this embryo-induced eggshell thinning. In our previous work, we collected the eggs during the early, middle and late stages of hatching, compared the eggshell thickness, and then found that the development of the embryo would lead to a decrease of the eggshell thickness. Eggshell pieces were taken from the sharp, blunt and middle regions of the eggs to measure eggshell thickness. Eggshell thickness was measured three times from each region with a digital micro-caliper with a precision of 0.001 mm to calculate the average eggshell thickness for each egg. Eggshell membranes were not removed from eggshells.

2.6. Element analysis After measuring eggshell thickness, eggshells were washed vigorously in deionized water followed by an acetone solution (1 mol/L) to

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remove internal and external contamination. Eggshells were then transferred into pre-weighted 15 mL centrifuge tubes. All eggshell samples were dried in an oven at 60 °C for 12 h. The wet weights of the egg contents and dry weights of the eggshell samples were recorded using an analytical balance to the nearest 0.001 g. After their weights were determined, samples were digested using an intelligent microwave digestion instrument (PreeKem TOPEX ; PreeKem Scientific Instruments Co., Ltd., Shanghai, China). The microwaved samples were diluted with deionized water and stored at 4 °C until subsequent analysis. Metal element concentrations (Cu, Zn, Pb, Cd and Ca) in all digested solutions were determined using a flame atomic absorption spectrophotometer (FAAS, Analytik Jena, ZEEnit 700P, Germany). Before the Ca concentration was analyzed, solutions were diluted (eggshell solution: 100 times; egg content solution: 20 times). The measurement process was checked using reference material, DORM-3 (fish protein), provided by the National Research Council of Canada Institute for National Measurement. The precision of the method was determined, i.e., the difference between the mean value obtained by analyzing the certified reference material (six measurements on 0.9 g samples) and its certified value for Cu, Zn, Pb, and Cd performed on the same sample. This was within 10% of the concentrations stated for the reference material. 2.7. Statistical analysis Samples collected for the analysis of eggshell thickness and metal concentrations were collected in 2017 and 2018; samples for analyzing eggshell spottiness and coloration were collected in 2018. Statistical analyses were performed using the SPSS 22.0 statistical software package (IBM SPSS Inc., USA) and OriginPro 9.0 (OriginLab, USA). The normal distribution of the data was checked using Kolmogorov-Smirnov test. Eggshell Cu and Cd concentrations for BY were log-transformed to achieve normality. The significance of the data was assessed by oneway analyses of variance (ANOVA) combined with Duncan's test, when the variance of data was homogeneous, or the Dunnett test, when the variance was not homogeneous. The Mann-Whitney U test was used where appropriate. Metal concentrations in the eggshells for each egg were used for the correlation. Mean metal concentrations in the eggshells for the entire clutch were used only when evaluating the correlation between within-clutch CV for egg volume with metal concentrations. Correlation analysis was used by two-tailed Pearson analysis. A significance level of p b 0.05 was chosen for all statistical tests. All values were expressed as the mean ± SD. 3. Results

Fig. 2. Egg volume (A) and coefficient of variation (CV) for egg volume within a tree sparrow clutch (B) in relation to clutch size.

3.1.3. Eggshell thickness Eggshells were thicker in LJX than in BY for both non-embryonated (F1,69 = 22.932, p b 0.01) and embryonated eggs (F1,25 = 10.398, p b 0.01). Shells from non-embryonated eggs were thicker than from embryonated ones at both sites (all data p b 0.001) (Fig. 3).

3.1. Comparison of egg traits between study sites 3.2. Comparison of the element concentration between two sites 3.1.1. Egg size Mean egg volume increased with clutch size in BY clutches (r = 0.384, p = 0.017), but not in LJX clutches (r = 0.026, p = 0.866) (Fig. 2A). Mean egg volume per clutch size were compared between the two sites, but only clutches containing four eggs had significantly higher mean egg size in LJX compared to BY (Table 1). No differences were found in the CV for egg volume among the different clutch size classes in both sites (Table 1). The within-clutch CV for egg volume increased with clutch size in clutches from BY (r = 0.327, p = 0.045), but this relationship was not observed in clutches from LJX (r = 0.058, p = 0.705) (Fig. 2B). 3.1.2. Eggshell spotting pattern and coloration No differences were found in eggshell spotting coverage ratio between the two sites, but there were significant differences in eggshell coloration between the two sites (L* of sharp and blunt region: LJX N BY, p b 0.05; h°: LJX N BY, p b 0.01) (Table 2).

3.2.1. Concentrations of elements in eggshells There were significant differences in the concentrations of Zn (LJX = 33.75 ± 12.98 N BY = 23.68 ± 9.18, F1,38 = 4.686, p = 0.037) and Pb (BY = 49.15 ± 15.39 N LJX = 39.94 ± 2.76, F1,38 = 10.575, p = 0.002) in the embryonated eggshells between the two sites. There were significant differences in the concentrations of Cu (BY = 5.16 ± 2.45 N LJX = 3.74 ± 2.05, F1,100 = 9.531, p = 0.003) and Zn (LJX = 28.55 ± 14.29 N BY = 22.09 ± 10.61, F1,100 = 5.435, p = 0.022) in the non-embryonated eggshells between the two sites. The levels of the other two elements in the eggshells (Cd, Ca) showed no differences between two sites (Fig. 4A). 3.2.2. Concentrations of elements in egg contents The contents of embryonated eggs had significantly different concentrations of Pb and Ca between the two sites (Pb: BY = 1.67 ± 1.01 N LJX = 1.16 ± 0.43, F1,30 = 4.387, p = 0.044; Ca: BY = 4.52 ± 2.69

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Table 1 Comparison of egg size of tree sparrow between Baiyin (BY) and Liujiaxia (LJX). Clutch size 3 4 5 6 F p

N

Length (cm)

Width (cm)

Volume (cm3)

Shape

CV for volume

BY LJX BY

LJX

BY

LJX

BY

LJX

BY

LJX

BY

LJX

3 20 13 2

2.01 ± 0.10 2.00 ± 0.06⁎⁎ 1.99 ± 0.09 2.01 ± 0.10 0.768 0.513

1.42 ± 0.05 1.42 ± 0.04 1.46 ± 0.05 1.47 ± 0.05 11.561 b0.001

1.42 ± 0.07 1.46 ± 0.05⁎⁎ 1.44 ± 0.05⁎ 1.45 ± 0.04 2.023 0.112

1.39 ± 0.05 1.37 ± 0.08 1.34 ± 0.05 1.35 ± 0.06 2.818 0.041

1.41 ± 0.09 1.39 ± 0.10 1.38 ± 0.07⁎⁎ 1.38 ± 0.07 0.442 0.723

2.012 ± 0.240 2.007 ± 0.165 2.101 ± 0.331 2.194 ± 0.180 8.033 b0.001

2.084 ± 0.260 2.160 ± 0.172⁎⁎ 2.105 ± 1.192 2.160 ± 0.182 0.623 0.601

0.023 ± 0.007 0.043 ± 0.028 0.061 ± 0.044 0.066 ± 0.015 0.791 0.507

0.038 ± 0.021 0.050 ± 0.021 0.053 ± 0.023 0.047 ± 0.014 0.644 0.591

3 16 16 10

1.96 ± 1.11 1.95 ± 0.11 1.96 ± 0.08 1.98 ± 0.07 0.554 0.646

⁎⁎ p b 0.01 compared with BY population. ⁎ p b 0.05 compared with BY population.

coloration in BY samples (L*: r = −0.791, p b 0.001; h°: r = −0.537, p = 0.015) and LJX samples (L*: r = −0.545, p = 0.001; h°: r = −0.416, p = 0.016). For BY samples, the eggshells with higher h° had higher concentrations of Zn, Pb and Cd in the shells (Zn: r = 0.629, p = 0.003; Pb: r = 0.695, p = 0.001; Cd: r = 0.501, p = 0.025) (Fig. 5C and D). The blunt poles of the eggshells with higher spotting coverage ratios had higher concentrations of Zn and Pb in BY samples (Zn: r = 0.397, p = 0.040, Pb: r = 0.348, p = 0.076) (Fig. 5F), and there was a higher concentrations of Pb in LJX samples (Pb: r = 0.289, p = 0.066).

N LJX = 2.88 ± 1.68, F1,36 = 4.324, p = 0.045) (Fig. 4B). No differences were found in the non-embryonated eggs. 3.2.3. Differences in element concentrations between non-embryonated and embryonated eggs The shells of embryonated eggs had significantly higher concentrations of Pb and Cd than non-embryonated eggs in BY (Pb: F1,27 = 10.255, p = 0.003; Cd: p = 0.008). Similar differences were found for Cu, Pb and Cd in LJX (Cu: F1,97 = 19.302, p b 0.001; Pb: F1,65 = 7.352, p = 0.009; Cd: F1,97 = 6.517, p = 0.012). Only the Ca concentration in the embryonated egg contents was significantly higher than that in non-embryonated eggs for both sites (BY: F1,29 = 30.722, p b 0.001; LJX: F1,84 = 15.619, p b 0.001) (Fig. 4).

4. Discussion Egg size is very consistent within an avian species, only rarely do environmental factors have strong effects on egg size (Christians, 2002). However, fluctuating and changing environments have been critical to the evolution of bird life histories (Tuljapurkar et al., 2009; Chevin et al., 2010). To maximize breeding and surviving fitness, a balance must exist between reproductive effort and self-maintenance in individual birds owing to limited environmental resources and changing environmental pressures. The variation in egg size among different clutch sizes is an important mechanism by which females can adjust in their reproductive patterns according to environmental conditions and their own body conditions (Smith and Bruun, 1998; Yosef and Zduniak, 2008). Additionally, offspring survival has been shown to be related to changes in egg size owing to environmental pressures (Krist, 2011). Therefore, optimum egg size is extremely important for subsequent development. We found that the mean egg volume from BY was smaller than that from LJX only for four-egg clutches, and this difference was not found for larger clutch sizes. A trade-off between the number propagules and size is one of the most general patterns

3.3. Relationship between egg traits and egg element concentrations Owing to the embryo-induced eggshell thinning, we only analyzed the non-embryonated eggs. Egg volume and eggshell thickness were significantly correlated with Pb (egg volume: r = −0.264, p = 0.040; eggshell thickness: r = −0.272, p = 0.029) and Cd (egg volume: r = −0.337, p = 0.008; eggshell thickness: r = −0.335, p = 0.006) concentrations in LJX samples (Fig. 5A and B) but not found in BY samples (all data p N 0.159). Higher within-clutch CV for egg volume tended to be associated with higher concentrations of Zn and Pb in the eggshells collected from BY (Zn: r = 0.544, p = 0.084; Pb: r = 0.548, p = 0.081) (Fig. 5E). Average eggshell spotting coverage ratio was significantly correlated with eggshell thickness in LJX samples (r = −0.392, p = 0.011); however, this was not found in BY samples (r = 0.216, p = 0.279). The average egg spotting coverage ratio was significantly related to eggshell

Table 2 Comparison of eggshell spotting pattern and coloration of tree sparrow between Baiyin (BY) and Liujiaxia (LJX). Traits

Spotting coverage ratio

L*

C*

h°

L*: lightness, C*: chroma; h°: hue.

BY

Sharp region Middle region Blunt region Average Sharp region Middle region Blunt region Average Sharp region Middle region Blunt region Average Sharp region Middle region Blunt region Average

LJX

Mean ± SD

N

Mean ± SD

N

0.44 ± 0.14 0.51 ± 0.11 0.68 ± 0.08 0.53 ± 0.11 55.14 ± 6.23 50.93 ± 6.71 41.99 ± 6.45 49.48 ± 5.64 8.11 ± 2.17 9.06 ± 2.23 8.48 ± 1.85 8.55 ± 1.93 80.44 ± 10.77 75.78 ± 11.50 68.14 ± 7.16 75.44 ± 9.22

27 27 27 27 27 20 27 20 27 20 27 20 27 20 27 20

0.40 ± 0.17 0.49 ± 0.14 0.65 ± 0.10 0.53 ± 0.10 58.66 ± 6.66 52.94 ± 5.57 46.51 ± 5.61 50.55 ± 4.46 7.94 ± 2.69 8.18 ± 2.04 7.86 ± 1.92 8.15 ± 1.83 93.68 ± 19.52 86.02 ± 12.73 78.86 ± 9.44 83.05 ± 9.76

41 41 41 41 37 33 37 33 37 33 37 33 37 33 37 33

F

df

p

1.077 0.380 1.999 0.066 4.609 1.391 8.912 0.586 0.066 2.202 1.700 0.562 10.133 8.645 24.505 7.883

66 66 66 66 62 51 62 51 62 51 62 51 62 51 62 51

0.303 0.540 0.162 0.798 0.036 0.244 0.004 0.448 0.798 0.144 0.197 0.457 0.002 0.005 b0.001 0.007

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Fig. 3. Comparison of mean eggshell thickness between non-embryonated and embryonated eggs of tree sparrows collected from BY and LJX. The sample sizes of non-embryonated/ embryonated eggs were 34/10 from BY and 68/30 from LJX. Different symbols on bars representing the same site indicate statistical differences between two groups of eggs (a, b: p b 0.05; aa, bb: p b 0.01).

among life-history traits in most taxa (Fox and Czesak, 2000; Roff, 2002). However, even though the present study did not detect any difference in larger clutch sizes, it is possible that, in the median clutch size, the females in better condition were able to lay larger eggs in the small broods, and there was only a trade-off between size and number for clutch sizes above the median (Fox and Czesak, 2000; Kvalnes et al., 2013). Although no differences were found in the within-clutch CV for egg volume between the two sites, this was increased in BY. Higher withinclutch CV for egg volume tended to be associated with higher concentrations of Zn and Pb in the eggshells from BY, but not in those from LJX. We speculated that this may reflect the different reproductive strategies adopted by parents under environmental pressures. First, they might try to maximize the fledging of young from a larger clutch, or they might adopt a bet-hedging strategy that divides within-clutch resources unequally to avoid investing in eggs with poor survival prospects (Crean and Marshall, 2009). Second, the within-clutch CV for egg volume may contribute to the development of an offspring size hierarchy, where females could select the best condition that is dependent on sibling competition (Smith and Bruun, 1998). Third, less variable eggs in small clutch sizes at both sites may suggest that the parents were more confident in their ability to fledge young successfully. The eggshell coloration of tree sparrow is mainly brownish due to the presence of protoporphyrin pigment. This allows the use of a classification system for eggs because darker eggs will contain more pigment (Poláček et al., 2017). In BY, eggshells were much darker, and the hue tended to be more red-brown than in LJX, thus these eggs contained higher concentrations of protoporphyrin. However, there was no difference in the eggshell spotting coverage ratio between the two sites. Porphyrins have been reported as pro-oxidants that can induce oxidative stress, and their accumulation can rapidly increase the activity of the antioxidant enzymes (Afonso et al., 1999). Females in poorer health condition and with Ca deficiencies are expected to lay thinner-shelled eggs, thus increasing the concentration of the pigment on the eggshell matrix (Martinez-de la Puente et al., 2007). Thinner eggshells have a more aggregated spotting distribution, possibly because pigment spots can strengthen the blunt end of the eggshell (García-Navas et al., 2011; Hargitai et al., 2013).

Spotting may play a functional role in the chemistry of avian eggs by enabling females to variously distribute micronutrients and trace elements into different regions of eggshells. Cu and Cd levels were significantly higher in speckled eggshell regions of the quail eggs, and the levels of Cu, Pb and Cd were higher in darker eggs than in bright ones (Orłowski et al., 2017). It also has been shown that thinner eggshells of great tits with higher concentrations of Cu had a more aggregated and uneven spotting distribution (Hargitai et al., 2013; Hargitai et al., 2016). Here, we found that eggshells with a higher spotting coverage ratio on the blunt poles contained higher concentrations of Zn and Pb in samples from BY, with a similar relationship found between eggshell Pb levels and spotting coverage ratio in samples from LJX. Higher levels of Pb in eggshells might alter the crystal structure or porosity of the eggshells, thus making the shell thickness less uniform (Grandjean, 1976; Eeva and Lehikoinen, 1995) and leading to an increase in spottiness to mechanistically strengthen the eggshell by increasing protoporphyrin levels. Decreasing avian shell thickness is normal when induced by a developing embryo, which absorbs Ca and other elements from the eggshell (Karlsson and Lilja, 2008). The discrepancy between the two groups of egg samples (embryonated and non-embryonated eggs) from both sites could be due to physiological processes mobilizing elements from the shells because of the embryos developing. Our study provided evidence that the presence of an embryo can produce significant differences in the concentration of metal elements in eggshells and egg contents. These two groups of eggs differed in shell thickness; embryonated eggs from both sites had 7% thinner shells on average compared to nonembryonated eggs. Eggshells were thinner in BY than in LJX for both embryonated and non-embryonated eggs, with eggshell thickness measured at the blunt pole showing the smallest differences (Table 1). The apparent embryo-induced eggshell thinning occurs along the length of an egg; starting from the sharp pole through the equator (the middle part) and shoulders (the widest part) to the blunt end, where the decline is least pronounced (Maurer et al., 2011). Eggshells were thinner when the concentrations of Pb and Cd in the eggshells were higher in LJX; however, a similar result was not found in BY. We also found a higher average eggshell spotting coverage ratio for thinner eggshells in LJX, but not in BY. One possible explanation is that the eggshells

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Fig. 4. Comparison of mean concentrations of elements measured in shells (A) and contents (B) of non-embryonated and embryonated eggs of tree sparrows from BY and LJX. The sample sizes of eggshells from non-embryonated/embryonated eggs were 34/10 from BY and 68/30 from LJX, and the sample sizes of egg contents were 28/10 from BY and 60/30 from LJX.

from the contaminated area are thinner because of an inability to change shell thickness. However, eggshells from the unpolluted area may be more sensitive to metal pollution and may therefore become thinner even when exposed to small quantities of pollutants. Eggs from BY contained higher mean concentrations of metals (Cu: eggshell of non-embryonated egg, Pb: eggshell of embryonated egg, and Ca: egg content of embryonated egg) than those from LJX. This was not found for Zn, which might be because Zn is an essential element in the quaternary structure of proteins such as ovalbumin and conalbumin, making it a necessary requirement for the normal embryonic development of birds (Morera et al., 1997). Our study indicated that the concentration of Cu, Pb, and Cd in the shells of embryonated eggs were significantly higher than in the shells of nonembryonated eggs. The deposition of some elements in some shell layers may be not subject to embryonic erosion (i.e. the palisade

layer or cuticle) (Orłowski et al., 2016), or that they may be contained in the eggshell membrane, which has been shown to have a high binding affinity with a variety of heavy metals (Suyama et al., 1994). We found that the concentration of Ca in the contents of embryonated eggs was significantly higher than that in the nonembryonated eggs from both sites, which suggests that Ca resorbs from the mammillary layer of the shell into the egg contents during embryonic growth (Hincke et al., 2012). Low Ca availability can cause thinner eggshells that break easily or lose excessive embryo water, resulting in egg desiccation, which is the main cause of hatching failure (Eeva and Lehikoinen, 1995; Graveland and Drent, 1997). We found no differences in the concentration of Ca in eggshells between the two sites. Although there was a difference in the concentration of Ca in embryonated egg contents between the two sites (BY N LJX), this was accompanied by an increase in Pb

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Fig. 5. Relationship between egg traits and eggshell element concentrations. LJX: egg volume (A), eggshell thickness (B); BY: hue (C, D), within-clutch CV for egg volume (E), and coverage ratio (F).

concentration (BY N LJX). Some metals such as Pb interact with the Ca metabolism, which may result in the metals becoming incorporated more readily into the egg contents and affecting embryo development (Scheuhammer, 1987). 5. Conclusions Our findings suggest that tree sparrow egg characteristics can be used as bioindicators of metal pollution. In our metal-polluted study site, increased clutch sizes had higher within-clutch CV for egg volume. Eggshell color measurements and spotting pattern appeared to reflect egg quality. A more aggregated eggshell spotting distribution was associated with higher Zn and Pb concentrations in the eggshell. We also

emphasized the need to consider embryo-induced changes in eggshell thickness and metal elements of eggs in ecotoxicology studies. Declaration of Competing Interest The authors declare no competing financial interests. Acknowledgements Thank for the help in the field work and element analyses from Rui Guo, Huijie Zhang, Shengnan Wang and Hao Sun. This work was supported by the National Natural Science Foundation of China (Grant No. 31572216).

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Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2019.06.140.

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