Avian eggshell thickness in relation to egg morphometrics, embryonic development, and mercury contamination

Abstract Eggshell thickness is important for physiological, ecological, and ecotoxicological studies on birds; however, empirical eggshell thickness measurements for many species and regions are limited. We measured eggshell thickness at the equator and the egg poles for 12 avian species and related eggshell thickness to egg morphometrics, embryonic development, egg status, and mercury contamination. Within an egg, eggshells were approximately 5.1% thicker at the equator than the sharp pole of the egg, although this difference varied among species (0.6%–9.8%). Within Forster's tern (Sterna forsteri), where eggshell thickness was measured at 5 equally spaced positions along the longitude of the egg, eggshell thickness changed more rapidly near the sharp pole of the egg compared to near the blunt pole of the egg. Within species, eggshell thickness was related to egg width and egg volume for six of the 12 species but was not related to egg length for any species. Among species, mean eggshell thickness was strongly related to species mean egg width, egg length, egg volume, and bird body mass, although species mean body mass was the strongest predictor of species mean eggshell thickness. Using three species (American avocet [Recurvirostra americana], black‐necked stilt [Himantopus mexicanus], and Forster's tern), whose nests were carefully monitored, eggshell thickness (including the eggshell membrane) did not differ among viable, naturally abandoned, dead, or failed‐to‐hatch eggs; was not related to total mercury concentrations of the egg content; and did not decrease with embryonic age. Our study also provides a review of all existing eggshell thickness data for these 12 species.

species and regions. For example, eggshell thickness can influence egg physiology, specifically gas exchange, because the diffusive properties of gas through pores in the eggshell relate to the ratio of pore length (eggshell thickness) to pore radius (Rahn, Paganelli, & Ar, 1987). Ecologically, eggshell thickness may vary as a result of factors including maternal age (Massaro & Davis, 2006), egg-laying order (Castilla, Herrel, Robles, Malone, & Negro, 2010), egg mass , and egg pigmentation (Gosler, Higham, & Reynolds, 2005). Some of these factors vary enough within and among clutches that they could cause marked differences in eggshell thicknesses among eggs; however, it is unclear whether this variation would result in an eggshell thickness that could influence whether or not an egg would hatch. Eggshell thickness also is an important egg measurement for ecotoxicological studies because it can be directly influenced by contaminant exposure (Cooke, 1973;Hickey & Anderson, 1968) and eggshell thickness is a necessary component in the accurate calculation of egg contaminant concentrations .
In ecotoxicological studies, eggshell thickness influences the estimation of an egg's contaminant concentration . The ideal reporting metric of contaminant concentrations in avian eggs is the calculation of fresh wet weight (fww) of the egg (Ackerman, Herzog, & Schwarzbach, 2013), a calculation that typically uses estimates of egg density, egg volume, and fresh egg mass. Estimating these measurements without removing the eggshell can result in a 6%-13% underestimate of egg contaminant concentrations . Consequently, the calculation of contaminant concentrations in egg contents can be improved by estimating and subsequently excluding the thickness of the eggshell . The common allometric equations to estimate eggshell thickness use egg length, egg width, eggshell mass, or whole egg mass (Ar et al., 1974;Khurshid, Farooq, Durrani, Sarbiland, & Chand, 2003;Maurer et al., 2010;Morrison & Kiff, 1979;Osborne & Winters, 1977;Ratcliffe, 1970) and are derived from large, multispecies datasets, although their accuracy has not been well validated for individual eggs (Ancel & Girard, 1992;Maurer, Portugal, & Cassey, 2012). Additionally, eggshell thickness may be estimated using bird body mass (Birchard & Deeming, 2009). The main equation to predict eggshell thickness from egg mass was derived from Schönwetter (1960Schönwetter ( -1992; this equation can be misused because it was derived using estimates of eggshell thickness from other equations and was not empirically based (Maurer et al., 2012). Furthermore, egg mass decreases by as much as 15% during embryonic development (Brown, 1976;Drent, 1970;Westerskov, 1950) and egg mass can also decrease as a result of desiccation from environmental exposure. Therefore, predictive equations based on egg mass (Ar & Rahn, 1985;Osborne & Winters, 1977;Rahn & Paganelli, 1989) will only be accurate for freshly laid eggs because the relationship between eggshell thickness and egg mass changes after the time point when the egg was laid. Thus, eggshell measurements are needed within and among species to test and improve upon allometric relationships for estimating eggshell thickness (using egg morphometrics or bird mass) that are not based on egg mass and are accurate for individual species.
We used 12 avian species to provide empirical eggshell thickness measurements in relation to egg morphometrics, embryonic development, egg status at the time of collection, and mercury contamination. The methodology we used provided more precise and repeatable eggshell thickness measurements than prior studies that used analog micrometers (Santolo, 2018), and these eggshell thicknesses can be applied in other ecological, physiological, and toxicological studies. Specifically, we examined the following: (a) eggshell thickness at multiple positions on the egg; (b) the relationship between eggshell thickness and egg morphometrics (length, width, and volume) both within and among species, as well as the relationship between species mean eggshell thickness and species mean bird body mass; (c) whether eggshell thickness decreases with embryonic development; (d) whether there are differences in eggshell thickness related to the egg status at the time of collection (normally developing eggs, eggs naturally abandoned by parents, dead embryos in eggs from nests where no sibling eggs hatched, and dead embryos in eggs from nests where sibling eggs hatched); and (e) whether eggshell thickness is related to egg content mercury concentrations.
Eggshell thickness in relationship to mercury contamination was chosen because few studies have examined the effects of mercury on eggshell thickness and mercury concentrations were analyzed for related contaminant studies.

| Sample collection
We salvaged and collected eggs from 12 avian species, representing 6 families from 4 orders (Table 1), as part of related contaminant studies during 2014-2018 (Peterson & Ackerman, 2020). Eggs of 11 species were from multiple sites within San Francisco Bay and the Central Valley in California (USA), some Caspian tern (Hydroprogne caspia) eggs were from the Potholes Reservoir in Washington State (USA), and wood duck (Aix sponsa) eggs were from Fallon, Nevada (USA). In the field, eggs were placed in egg cartons and kept in small coolers with wet ice until they were transported back to the laboratory. Eggs were stored in a refrigerator (2°C) until processing.

| Eggshell processing
First, the exterior of each egg was cleaned with deionized water, swabbed with isopropyl alcohol, rinsed with deionized water, and allowed to dry. Before egg dissection, length (±0.01 mm) and width (±0.01 mm) were measured using digital calipers (Mitutoyo, Aurora, Illinois, USA) and whole egg mass (±0.01 g) was obtained with a digital balance (Ohaus Adventurer™ Pro AV212, Ohaus Corporation).
We then cut an approximately 15 mm diameter circle at the blunt end of each egg using stainless-steel scissors, removed the blunt end of the eggshell, and transferred the egg contents into a sterile polypropylene jar. The blunt pole of the eggshell was removed and discarded during egg processing for some eggs, prior to the development of this specific study. Embryos were aged to the nearest whole day , and the egg contents were prepared for determination of mercury . Most eggs (68.8%) were identified as fertile and aged to at least 1 day in incubation (mean 7.8 ± 5.2 days; interquartile range 4-11 days; range 1-27 days). Additionally, 3.2% were identified as fresh and fertile (day 0 of incubation). The remaining eggs were either infertile or embryonic age could not be determined. After egg dissection, eggshells were stored in a freezer at −20°C.
Prior to processing of eggshells and measurement of eggshell thickness, eggshells were removed from the freezer and allowed to warm to room temperature. The outside of the eggshell was reexamined to determine whether there was any remaining exogenous material that needed to be removed. Then, we rinsed the inside of eggshells with a mild detergent (Alconox) and used a cotton swab to wipe out the inside. If necessary, a small stainless-steel spatula was gently used to dislodge any contents adhered to the inside of the eggshell that could not be dislodged with a cotton swab. After any remaining egg contents were dislodged, the inside was rinsed multiple times with deionized water. Eggshell membranes were not removed. We recorded the condition of the ultrathin outermost eggshell membrane, closest to the egg contents (Simkiss, 1961), because that membrane occasionally becomes detached from the rest of the eggshell during dissection and it is almost always absent from the blunt pole as it peels away from the eggshell in the blunt pole region as the air cell expands during embryonic development. The main TA B L E 1 Eggshell thickness was measured for 12 avian species from 4 orders and 6 families Note: Mean female body mass was obtained from published studies (Ackerman, Hartman, et al., 2013;Ackerman et al., 2008;Bluso et al., 2006;Delnicki & Reinecke, 1986;Dunning, 2008;Herring et al., 2008Herring et al., , 2010bPage et al., 2009;Robinson et al., 1999). a Available body mass measurements were for the subspecies Sternula antillarum athalassos.
Once cleaned, eggshells were placed in a drying oven for 24 hr at 40°C and stored in a desiccator until they were measured.

| Eggshell thickness measurements
We measured eggshell thickness at 3 positions on each eggshell when possible: equator, sharp pole, and blunt pole ( Figure 1). We measured eggshell thickness using a Magna-Mike ® 8600 Hall effect thickness gauge (Olympus Scientific Solutions Americas Corporation) with a 1.58 mm magnetic measurement ball. We measured the minimum thickness of the eggshell and membrane as the ball was rolled across the inside of the eggshell at three measurement positions: the equator, the sharp pole, and the blunt pole, following the methods of Santolo (2018). At the equator measuring position, we slowly rotated the egg 3-5 times over the measurement ball to make sure the entire equator was sampled. At the sharp and blunt pole measuring positions, the measurement ball was rolled around in a small circle to capture the entire end of the eggshell. Our method measured across maculated (pigment spots) and plain sections of eggshells. Because some studies showed differences in eggshell thickness between pigmented and unpigmented sections (Gosler et al., 2005), our method captured the thinnest spot at that measurement position on the eggshell, which may have represented a pigmented section. If only a portion of the eggshell was intact at the equator, we measured as much of the eggshell as possible and recorded the percent of the eggshell area that was sampled. We excluded measurements from sections of eggshell that had mold on them and any eggshells where the main inner eggshell membrane was removed or was visibly separating from the calcite portion of the eggshell. The thickness gauge was calibrated at the start of every day of measurement and any time when the machine was inactive for more than 1 hr.
To examine more specifically whether and how eggshell thickness changed longitudinally from the sharp to the blunt pole of the eggshell, we conducted a separate experiment where we measured eggshell thickness at 5 positions ( Figure 1) on a subset of Forster's tern (Sterna forsteri) eggshells (n = 40) where the blunt pole eggshell piece was retained after egg dissection. The sharp pole and blunt pole were measured in the same way as described above, but we also measured eggshell thickness at 3 additional and equally spaced regions on the egg at the ¼ egg (i.e., halfway between the sharp pole and the midpoint of the egg), the ½ egg (i.e., the midpoint of the egg), and the ¾ egg (i.e., halfway between the midpoint of the egg and the blunt pole). The ½ egg was measured at the midpoint of the egg and not the egg equator, which is defined as the widest part of the egg and typically the equator is closer to the blunt pole than the sharp pole of the egg (Figure 1). Each of the 5 positions on an individual eggshell was measured within the same calibration period (<15 min). We used the same protocol described above, turning the eggshell 3-5 times over the measurement ball around each longitudinal section.

| Mercury determination
Eggs were analyzed for total mercury (THg) using a Nippon at 5 different equally spaced positions on Forster's tern eggshells (n = 40). Percentages represent the percent thicker or thinner each location is relative to the measurement location directly to the left, moving from the sharp pole toward the blunt pole. Note that the ½ egg was measured at the midpoint of the egg and not the widest part of the egg (which is called the egg equator; shown with a dashed line) that is typically closer to the blunt pole than the sharp pole certified reference materials, 99.9 ± 2.1% (n = 328) for calibration verifications, and 100.6 ± 3.5% (n = 322) for matrix spikes.

| Statistical analyses
We examined the influence of species, measurement position, egg morphometrics (egg length, egg width, and egg volume) and bird body mass, embryo age, egg status at the time of collection, and egg THg content on eggshell thickness using a combination of weighted regression, mixed effects linear models, and general linear models.
All analyses were performed in the statistical program R (R Core Team, 2019).

| Eggshell thickness among species and within individual eggs
We examined whether there were differences in eggshell thickness within and among species at specific egg measurement positions within individual eggshells. First, we compared the eggshell thickness values from 5 positions in the detailed study of Forster's tern eggs with a linear mixed effects model using the lme4 package (Bates et al., 2015). In the model, we included measurement position as a fixed effect and eggshell identification as a random effect. We used the Kenward-Roger approximation for degrees of freedom and tested for significance with F tests generated from the afex package (Singmann, Bolker, & Westfall, 2015). We examined differences in model-generated least squares mean eggshell thicknesses among the five eggshell measurement positions with a Tukey honest significant difference adjustment.
Using data from multiple species (n = 12), we ran two linear mixed effects models with species, measurement position on the eggshell, and a species × measurement position interaction as fixed effects and eggshell identification nested within nest identification as random effects. The first model included eggshells with paired equator and sharp pole measurements, and the second model included eggshells that had an equator, sharp pole, and blunt pole measurement.
We examined whether there was a consistent difference in eggshell thickness between the sharp pole and the equator within and among species using two approaches. First, we examined the relationship between the sharp pole and the equator using a general linear model with individual eggshell thickness measurements and an equator eggshell thickness × species interaction. Second, we examined the linear relationship among species, using a weighted regression on species mean values of each measurement. We calculated weights as the natural log of the sample size to reduce the weight on higher sample sizes such that species with more samples were weighted only slightly more than species with fewer samples. We then calculated residuals for all individual eggshell measurements from the regression equation generated using the species means.
Finally, we used the mean and 95% CI of the residuals to determine whether the residuals for each species fell above, included, or were below zero. If the 95% CI of the residuals for a species included zero, that would suggest that the mean eggshell thickness at the sharp pole for that species was within the range of what would be expected based on the eggshell thickness at the equator. Conversely, if the 95% CI of the residuals for a species was entirely above zero, that would suggest that the eggshell thicknesses at the sharp pole for that species were thicker than would be expected based on the eggshell thickness at the equator. If the 95% CI of the residuals for a species was entirely below zero, that would suggest that the eggshell thicknesses at the sharp pole for that species were thinner than would be expected based on the eggshell thickness at the equator.

| Eggshell thickness versus egg morphometrics and bird body mass
We quantified the relationship between eggshell thickness at the equator and egg morphometrics (egg length, width, and volume) within and among species. First, we examined the relationship between the eggshell thickness at the equator and either the egg length, egg width, or egg volume (in separate models), using a general linear model with equator eggshell thickness measurements for individual eggs and an equator eggshell thickness × egg morphometric measurement interaction. We then ran individual models for each species with more than 10 samples. Egg volume was calculated using an egg shape coefficient (K v ), egg length, and egg width (egg vol- Hoyt, 1979). Second, to qualitatively compare the relationship between the eggshell thickness at the equator and egg morphometrics within versus among species, we quantified the linear relationship among species (length, width, and volume in separate models), using a weighted regression on species mean values, with weights calculated as the natural log of the sample size to reduce the weight on higher sample sizes such that species with more samples were weighted only slightly more than species with fewer samples. We then calculated residuals for all individual eggshells from the regression equation generated using species means and used these residuals to determine whether the mean residual value for each species fell above, included, or was below zero, using the mean and 95% CI for each species. If the 95% CI of the residuals for a species included zero, that would suggest that the mean eggshell thickness at the equator pole for that species was within the range of what would be expected based on the egg length or width. Conversely, if the 95% CI of the residuals for a species was entirely above zero, that would suggest that the eggshell thickness at the equator for that species was thicker than would be expected based on the egg length or width. If the 95% CI of the residuals for a species was entirely below zero, that would suggest that the eggshell thickness at the equator for that species was thinner than would be expected based on the egg morphometric measurements.
We also quantified the relationship between species mean eggshell thickness at the equator and species mean bird body mass.
For bird masses, we used published mean female body masses prior to analysis because we did not expect bird body mass to scale linearly with eggshell thickness (Birchard & Deeming, 2009). We used AICc (corrected for small sample sizes) to compare regression models with different predictors of eggshell thickness.

| Eggshell thickness versus embryo age
We examined whether eggshell thickness measured at the equator and sharp pole decreased with embryonic development, using a subset of normally developing eggs of American avocet (Recurvirostra americana), black-necked stilt (Himantopus mexicanus), and Forster's tern that were collected during weekly nest monitoring. We used a general linear model with fixed effects for species, embryo age (in days), and a species × embryo age interaction. We did not include nest identification as a random effect because we had only 1 normally developing egg from each nest.

| Eggshell thickness versus egg status
For a subset of American avocet, black-necked stilt, and Forster's tern eggs that were sampled during weekly nest monitoring visits, the status of each egg was categorized upon collection as active, abandoned, dead, or failed to hatch . Active eggs were normally progressing in nests that were actively being incubated, whereas abandoned eggs were from nests where the parents naturally had abandoned the nest. Eggs classified as dead contained dead embryos and had stopped progressing normally in nests while they were still being incubated and no sibling eggs in the clutch hatched. Failed-to-hatch eggs contained dead embryos and also did not hatch but were from nests where other sibling eggs in the clutch successfully hatched. We ran two separate mixed effects linear models to compare eggshell thickness at either the equator or the sharp pole with egg status, species, embryo age, an egg status × species interaction, and nest identification as a random effect. Nest identification was included as a random effect, because some nests with dead, abandoned, or failed-to-hatch eggs had multiple eggs salvaged.

| Eggshell thickness versus egg content mercury
To examine whether eggshell thickness was related to MeHg exposure, we used the subset of normally progressing American avocet, black-necked stilt, and Forster's tern eggs. We determined THg concentration in the egg as a proxy for MeHg since 96% of the Hg in bird eggs is in the MeHg form . We used two general linear models, one for the equator eggshell thickness and one for the sharp pole eggshell thickness, with fixed effects for the egg content THg concentration (fww), species, embryo age, and an egg content THg concentration × species interaction. We did not include nest identification as a random effect because only 1 normally developing egg in this dataset was from each nest.

| Eggshell thickness among species
We observed a 190% difference in mean eggshell thickness at the equator between the species with the thinnest eggshells (California

| Eggshell thickness at 5 equally spaced positions on the egg
We began with a detailed study of eggshell thickness among 5 equally spaced positions on Forster's tern eggs (n = 40). Eggshells were thickest at the ¼ egg and ½ egg and thinnest at the poles (F 4,156.0 = 31.96, p < .001; Figure 1). The increase in eggshell thickness was greater between the sharp pole and the ¼ egg than between the blunt pole and the ¾ egg ( Figure 1). Furthermore, average eggshell thickness was similar at the ¼ egg and the ½ egg (t = 1.29, p = .70), whereas eggshell thickness at the ¾ egg was 3.2% thinner than the ½ egg (t = 2.70, p = .059). For this set of eggshells, the ½ egg was 10.8% thicker than the blunt pole (t = 8.06, p < .001) and 8.5% thicker than the sharp pole (t = 6.32, p < .001), and the sharp pole was similar in thickness to the blunt pole (t = 1.74, p = .42).

| Eggshell thickness at the equator and poles
When we considered all 12 species, avian eggshells generally were thickest at the equator and thinner at the sharp and blunt poles. On average, eggshells were 5.1% thicker at the equator than the sharp F I G U R E 2 Arithmetic mean eggshell thickness (±SD) at the egg equator, sharp pole, and blunt pole for 12 different avian species (raw data). Asterisks indicate species with <10 eggs measured. Refer to Table 2 for sample sizes and egg length and egg width measurements TA B L E 2 Sample size (n) for egg morphometric data (egg length and egg width) salvaged and collected from 12 avian species between 2014 and 2018 throughout western North America Note: Sample size and raw data on minimum eggshell thickness (including the main inner eggshell membrane) measurements at the equator, sharp pole, and blunt pole. Sample sizes differ because egg morphometric measurements were not obtained for all eggs and some eggshells were not measured at all three egg positions. NA indicates when no measurements were taken at that measurement position for that species.
pole when there were at least 10 eggshells measured from a species (n = 10 species; Table 2), although mean differences between eggshell thickness at the equator and sharp pole within a species varied from 0.6% to 9.8% (F 11,2500.0 = 19.46, p < .001; Figure 2

| Relationship between sharp pole and equator eggshell thickness within species
When individual eggshell thickness measurements ( When models were run individually, eggshell thickness at the sharp pole was significantly related to eggshell thickness at the equator for all 10 species (all F ≥ 7.05, all p ≤ .015, all R 2 ≥ .11; Figure 3a; Table 3).

| Relationship between sharp pole and equator eggshell thickness among species
When mean values were used for each species, eggshell thickness at the sharp pole was strongly related to eggshell thickness at the equator (R 2 = .99; Figure 3b). The slope of the relationship was 0.95, indicating that eggshell thickness at the sharp pole was consistently 95% of the thickness at the equator (sharp pole eggshell thickness = eggshell thickness at the equator × 0.95300 + 0.00029).
The 95% CI of the residuals for all species except mallard (Anas platyrhynchos), where only 2 eggshells were measured, included zero, indicating that there were no species where the mean relationship between eggshell thickness at the sharp pole and the equator differed from expected.

| Eggshell thickness versus egg morphometrics and bird body mass
The relationship between eggshell thickness at the equator and egg length, egg width, or egg volume was stronger among species than within species. Among species, mean species body mass was the best predictor of species mean eggshell thickness, better than species mean egg length, egg width, or egg volume.

| Individual species comparisons
The slope of the relationship between eggshell thickness and egg morphometrics within each species did not differ among species for length (F 11,2844 = 0.67, p = .77; Figure 4) but varied among individual species for width (F 11,2844 = 1.84, p = .042; Figure 4) and for volume

| Among species
Among species, the mean eggshell thickness at the equator in- Double-crested cormorant had thicker eggshells at the equator than would be predicted based on the length or width of the egg, and wood duck eggshells also were thicker at the equator than would be predicted based on the length, but not the width, of the egg. Great egret had thinner eggshells than would be predicted based on the length, but not the width, of the egg. The estimated species mean eggshell thickness, using the equation with species mean egg length, ranged from 17.7% lower (double-crested cormorant) to 13.2% higher (great egret) than the mean measured eggshell thickness. Wood duck (16.9% lower), snowy plover (15.4% lower), Forster's tern (10.3% higher), American avocet (9.5% higher), Caspian tern (9.4% higher), and mallard (6.8% lower) had a predicted mean eggshell thickness that was more than 6.0% higher or lower than the mean measured eggshell thickness. The remaining four species had a predicted mean eggshell thickness that was within 6% of the mean measured eggshell thickness.   to 15.5% higher (great egret) than the mean measured eggshell thicknesses. American avocet (14.0% higher), California gull (10.5% lower), California least tern (6.3% lower), and double-crested cormorant (6.2% higher) had a predicted mean eggshell thickness that was more than 6.0% higher or lower than the mean measured eggshell thickness. The remaining six species had a predicted mean eggshell thickness that was within 6% of the mean measured eggshell thickness.
We compared the four different models to predict species mean eggshell thickness from species mean egg morphometric measurements (egg length, width, or volume) or bird body mass, and log 10 (bird body mass) was the best predictor. The AIC c value of −42.1 for log 10 (bird body mass) was more than a ΔAIC c of 2 from the models using egg length (ΔAIC c = 5.0), egg width (ΔAIC c = 8.3), or egg volume (ΔAIC c = 8.8).

| Eggshell thickness versus embryo age
We did not find support for a decrease in the eggshell thickness

| Eggshell thickness versus egg content THg
We did not detect a relationship between eggshell thickness and egg content THg concentrations. At the equator, after removing the non-
For the 10 species that had been previously measured, the mean eggshell thicknesses were similar to previous studies in North America with some slight differences among studies (Table 5). For example, mean American avocet and black-necked stilt eggshells were 10.0% and 3.8% thicker, respectively, in the present study than previous mean eggshell thickness measurements for the same species in Utah (Henny, Anderson, & Crayon, 2008), although the ranges of eggshell thicknesses observed in the two studies were similar.
Black skimmer eggshells were, on average, 5.6% to 10.3% thicker in the present study than in southern California (Roberts, 1997;Santolo, 2018), but were ≥42.3% thinner than the mean of the most recently sampled eggshells (1990s) of black skimmer from the east coast of the United States (Burger et al., 1995). Forster's tern eggshell thickness in the present study was similar to Forster's tern eggshell thickness from other regions in California (Grant, 1982;Roberts, 1997;Santolo, 2018 Eggs classified as dead contained dead embryos and had stopped progressing normally in nests while they were still being incubated and no sibling eggs in the clutch hatched. Failed-to-hatch eggs contained dead embryos and also did not hatch but were from nests where other sibling eggs in the clutch successfully hatched thickness in addition to local contamination by DDT and other chlorinated pesticides that might decrease eggshell thickness (Anderson & Hickey, 1972). Many previous studies that examined eggshell thickness in birds were conducted specifically to look for eggshell thinning as a result of environmental contamination. Furthermore, our methodology of using newer technology (a Hall effect thickness gauge) provides a more precise and repeatable thickness measurement than analog micrometers that have been used in many previous studies (Santolo, 2018). Additionally, the Hall effect thickness gauge reduces the potential for the eggshell membrane to be compressed while obtaining the eggshell thickness measurement.
The slope of 0.95 we observed for the relationship between eggshell thickness at the sharp pole and the equator indicates that the eggshell thickness at the sharp pole is consistently 95% of the eggshell thickness at the equator. Therefore, when a whole egg is not available and only eggshell fragments can be collected, if the sharp pole can be identified and measured, this relationship may be used to estimate eggshell thickness at the equator.
Physical characteristics of eggs and biological attributes of the clutch may explain variability in eggshell thickness within species.
Our use of a minimum eggshell thickness measurement would likely capture the more pigmented and sometimes thinner sections of an eggshell (Gosler et al., 2005), although variation in eggshell thickness with pigmentation has not been supported in all studies (Maurer, Portugal, Boomer, & Cassey, 2011), suggesting that variation in pigmentation within and among some species may complicate interpretations of eggshell thickness. Additionally, the laying order of a clutch may influence the length and width of an egg (Ackerman, Eagles-Smith, Herzog, Yee, & Hartman, 2016;Penland, 1981); consequently, this may explain some of the observed variability in eggshell thickness within species. Laying order influenced eggshell thickness in falcons (Falco sp.), with the thickest eggshells observed for the first egg laid , but a study on passerines (Passeriformes) found no such relationship (Orłowski et al., 2016). In addition, other studies indicate that the overall clutch size influences an individual egg's eggshell thickness, with bigger clutches associated with thinner individual eggshells in passerine birds (Orłowski et al., 2016).
We did not detect a decrease in the average eggshell thickness at the equator or sharp pole of the egg with embryonic development, and we found some support for a small increase in thickness of the eggshell membrane and eggshell when all embryo ages were included. Most studies observed eggshell thinning as a result of advances in embryonic development, when the eggshell membrane was removed from the calcite eggshell (Ancel & Girard, 1992;Finnlund et al., 1985;Orłowski & Hałupka, 2015;Santolo, 2018). However, removal of the eggshell membrane can remove some of the mammillary core and result in an underestimate of the eggshell thickness (Simkiss, 1961). Furthermore, eggshell membranes may increase in thickness during embryonic development (Castilla, Van Dongen, et al., 2010;Finnlund et al., 1985). However, when we examined eggshells with embryos older than one week of age, we found no evidence of eggshell thinning with embryonic F I G U R E 6 Eggshell thickness at the egg equator was not related to total mercury (THg) concentrations in the egg contents, quantified using fresh wet weight ( Heinz, Erdman, Haseltine, and Stafford (1985) Wisconsin 1977 3 0.373

Yes
Not specified Heinz et al. (1985) TA B L E 5 (Continued) Note: Means are assumed to be arithmetic unless otherwise indicated. Standard deviation (SD) standard error (SE) or confidence limit (CL) is indicated using a superscript after the value. Eggs for multiple studies were salvaged and collected from locations known to contain contaminants that could influence eggshell thickness. Most studies that included museum specimens from prior to 1947 state that the researchers compared eggshell thickness between pre-DDT eggshells from museums and post-DDT eggshells that were measured at the egg equator. Although most blow-out holes in museum eggshells are at or near the equator, if eggs from museum collections were measured closer to the blunt end of the egg than the true egg equator it would result in a thinner eggshell measurement (Santolo, 2018

TA B L E 5 (Continued)
development and we no longer observed an increase in eggshell and membrane with embryonic age. Of note, only 3.5% of embryos were in the final quarter of development. Detectable eggshell thinning due to embryonic development may not occur until the final quarter of the incubation period, similar to observations in capercaillie (Tetrao urogallus) eggs (Orłowski, Merta, et al., 2019).
Organochlorine compounds, such as DDT (dichlorodiphenyltrichloroethane), were widely used beginning in the 1940s and were found to decrease eggshell thickness and affect egg survival (Cooke, 1973;Hickey & Anderson, 1968). It is possible that Hg within the egg itself, which also indicates Hg within the female developing the egg , may also influence eggshell thickness, but studies are less conclusive for this contaminant. We found that eggshell thickness, measured either at the equator or the sharp pole, was not correlated with egg content THg concentrations. In contrast, eggshells of Japanese quail thinned significantly when fed diets containing known quantities of mercuric chloride (Stoewsand et al., 1971). Additionally, research on mallard eggshells (Heinz, 1979) showed that eggs of game farm mallards fed a diet of 0.5 ppm Hg had slightly thinner eggshells at the equator (6.6%) than mallards fed on a control diet, but this occurred only in the third generation of mallards fed Hg and not in the first two generations fed Hg (Heinz, 1979). Other field studies have indicated more ambiguous results. Previous research on clapper rail (Rallus longitrostris) indicated that eggshells were thinner at a contaminated site than a control site, with Hg the only metal that significantly differed by site (Rodriguez-Navarro et al., 2002). However, when data were analyzed by site there was no detectable relationship between eggshell thickness and Hg concentrations; consequently, the observed variation in eggshell thickness may have been driven by site characteristics rather than Hg (Rodriguez-Navarro et al., 2002). Additionally, eggshell thickness and mercury concentrations were not related in eggs of Forster's tern (n = 79), black skimmer (n = 41) (King et al., 1991), or several shorebird species (Hargreaves et al., 2011).
Eggshell thickness was not related to egg status in the subset of the three avian species where we monitored nests weekly (American avocet, black-necked stilt, and Forster's tern), similar to previous research on least tern (Koenen & Leslie, 1996). Specifically, eggshells from eggs that did not hatch, either from clutches where no eggs hatched (dead egg status) or from clutches where other eggs in the clutch hatched (failed-to-hatch egg status), were not thicker or thinner, on average, than eggs that were progressing normally.
Accounting for eggshell thickness can be important in the accurate calculation of contaminant concentrations in avian eggs.
Specifically, using estimates of eggshell thickness results in more accurate (6%-13%) fresh wet weight egg contaminant concentrations than when the eggshell is ignored . Using measured eggshell thickness will provide the most accurate calculation of fresh wet weight contaminant concentrations for an individual egg. However, measuring eggshell thickness for every egg can be time consuming and costly. If eggshell thickness cannot be measured for individual eggs, we suggest using mean eggshell thickness for the species (  (Birchard & Deeming, 2009;Maurer et al., 2012). However, individual species can deviate substantially from expected eggshell thicknesses. For example, in the present study, the mean equator eggshell thickness of snowy plover was 26.0% thicker than would be predicted based on the among-species equation for body mass.
Therefore, using multispecies equations to estimate eggshell thick- ness is a tool that should be employed with caution only in the absence of empirically measured eggshell thickness data for the specific species being studied.

ACK N OWLED G M ENTS
We comments that improved the manuscript; and two anonymous reviewers for comments that improved the quality of the manuscript.
The use of trade, product, or firm names in the publication is for descriptive purposes and does not imply endorsement by the U. S. Government.

CO N FLI C T O F I NTE R E S T S
The authors declare no conflicts of interest.

E TH I C S S TATEM ENT
Research was conducted with the approval of the U.S. Geological

Survey Western Ecological Research Center's Animal Care and Use
Committee.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data in this article are in ScienceBase: https://doi.org/10.5066/ P981OW6T.