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- Materials and methods
- Supporting Information
All organisms currently face significant challenges imposed by global climate change (Root et al. 2003) and other anthropogenic changes (Rosenzweig et al. 2008; Johnston & Roberts 2009); not even organisms living in the deep ocean can escape exposure to environmental contaminants (de Boer et al. 1998). Therefore, it is becoming increasingly important to develop new monitoring tools to assess habitat quality and manage sensitive areas and wildlife. Bird eggs may provide an efficient means of assessing the environmental quality of avian breeding habitats, particularly with respect to industrial processes and agricultural pesticides. Many persistent organic compounds associated with these activities are known to bio-accumulate in animal tissues as they are transferred from low trophic levels to higher ones. The influence of the bio-accumulation of these contaminants on avian reproduction became readily apparent during the late 1960s (Ratcliff 1967; Hickey & Anderson 1968), particularly through eggshell thinning induced by exposure to dichlorodiphenyldichloroethylene (DDE), a metabolite of the persistent insecticide dichlorodiphenyltrichloroethane (DDT) (Gilbertson 1974). Within the Great Lakes, the levels of persistent organic particulates have decreased dramatically over the last half-century (Hebert, Norstrom & Weseloh 1999); however, significant quantities of organic compounds are still prevalent in colonially nesting waterbirds (Lavoie et al. 2010). Recent models also suggest that cycles of contamination may be linked to oscillating currents and global patterns of climate change (Bustnes, Gabrelsen & Verreault 2010). Such studies reveal the importance of continued monitoring to track long-term patterns and evaluate potential risks to plants, animals and humans from environmental contamination.
Despite dramatic variation in avian egg coloration within and among species (Collias 1993; Kilner 2006), only two related pigments are primarily responsible for this variation: biliverdin (blue-green in colour) and porphyrin (brown in colour) (Kennedy & Vevers 1976), both of which are derived from haem biosynthesis (McGraw 2006). A variety of porphyrins are created endogenously through enzymatic interactions and these nonmetallic porphyrins create haem through the addition of an Fe+ ion (McGraw 2006), which can be oxidized to create biliverdin (McDonagh 2001). The concentration of one of these pigments, porphyrin, has been recommended as a bioindicator in faecal samples (Akins et al. 1993; Casini et al. 2003). However, as both porphyrin and biliverdin occur along the same biochemical pathway, arguments for the use of porphyrin may also be relevant for biliverdin (Mateo et al. 2004; Jagannath et al. 2008).
A number of factors support the possible utility of avian pigments, and particularly avian egg pigments, as bioindicators of environmental stress. Proximity to urbanization (Horak et al. 2000) and exposure to polychlorinated biphenyls (PCBs) (McCarthy & Secord 2000; Bortolotti, Fernie & Smits 2003; Bortolotti, Smits & Bird 2003) are known to influence avian plumage and soft part coloration. Egg coloration in birds may be similarly influenced by environmental quality as it has been linked with female body condition (Morales, Sanz & Moreno 2006; Soler et al. 2008), rainfall (Avilés et al. 2007), soil calcium availability (Gosler, Higham & Reynolds 2005) and health (Moreno et al. 2005; Martínez-de la Puente et al. 2007), yet this possibility has received surprisingly limited attention.
The relationship between environmental contaminants and eggshell pigmentation was recently examined in the Eurasian sparrowhawk Accipiter nisus Linnaeus (Jagannath et al. 2008). In a sample of eggs collected across the United Kingdom in a single year, Jagannath et al. (2008) found that blue hue was positively correlated with DDE concentration, while chroma decreased with DDE concentration. An experimental study has also shown that the contamination by lead caused a 53-fold increase in protoporphyrin and a 66-fold increase in biliverdin in faecal samples of mallards Anas platyrhynchos Linnaeus (Mateo et al. 2004). These findings are consistent with the observation that organochlorines, halogenated hydrocarbons and heavy metals influence the haem biosynthesis pathway (Kennedy et al. 1998; Casini et al. 2003; Mateo et al. 2003b, 2004).
Our objective was to determine whether eggshell coloration could serve as a nondestructive bioindicator of environmental stress, using herring gulls Larus argentatus Pontoppidan as an indicator species. Herring gulls can provide insight about changes in the health or quality of their ecosystem, and early research on Great Lakes herring gulls documented that DDT decreased egg hatchability (Keith 1966). Herring gulls have therefore been the focus of the long-term Great Lakes Herring Gull Monitoring Programme, spanning multiple colonies along the shores of the Great Lakes in Canada and the United States, with the objective of examining the concentrations and effects of environmental contaminants in herring gulls and their eggs. This programme has documented the levels of various organochlorines and metal contaminants in this species for 40 years (Hebert, Norstrom & Weseloh 1999). Fortunately, the levels of most legacy contaminants in Great Lakes herring gull eggs have declined significantly because the use of DDT was banned in 1972 and 1974 in the United States and Canada, respectively (Pekarik & Weseloh 1998; Jermyn-Gee et al. 2005). Herring gull eggshells contain both biliverdin and porphyrin (Kennedy & Vevers 1976), which allows for substantial variation in colour. This variability in eggshell coloration and dramatic temporal variation in contaminant load, as well as the herring gull's susceptibility to organochlorines (Neimi et al. 1986; Breton, Fox & Chardine 2008), makes this system ideal for examining the possible influence of contaminants on egg coloration. The eggs used in this long-term monitoring programme have been stored in a national archive and are available for continued research projects.
In this study, we investigated the relationship between egg coloration and environmental contaminants measured through the Great Lakes Herring Gull Monitoring Programme. Our objective was to examine whether environmental contaminants influence eggshell coloration, and to determine whether eggshell coloration could serve as a useful proxy for the degree of environmental contamination. First, we examined the possible influence of a suite of contaminants on eggshell coloration. On the basis of previous research on the effects of DDE concentration on egg colour (Jagannath et al. 2008), we predicted that contaminant load would be negatively related to blue-green chroma. We then used a cross-validated partial least squares linear discriminant function to determine whether raw spectral data obtained from field-portable spectrometers could be used to classify the contaminant load in the field.
- Top of page
- Materials and methods
- Supporting Information
On the basis of the analyses of a long-term data set, our findings reveal significant associations between a principal component summarizing contaminant load, representing a suite of persistent organic contaminants and egg coloration in herring gulls. We found that as contaminant load increased, blue-green chroma decreased while ultraviolet chroma increased, when controlling for colony and time since collection. Our findings support the only other study to examine the relationship between egg colour and contaminant load (Jagannath et al. 2008) and provide the first evidence that this pattern holds across a suite of contaminants within a large, multi-year data set. We also provide a discriminant function that can effectively classify herring gull eggs into high and low contaminant classes in the field using egg reflectance spectra. Our findings suggest that egg coloration could be used as a bioindicator of contaminant load and provide further support for the use of colonial waterbirds for monitoring environmental quality (Kushlan 1993).
The only other study to examine the relationship between environmental contaminants and egg coloration found a negative association between DDE levels and chroma of Eurasian sparrowhawk eggs, and a positive association between DDE levels and blue hue (Jagannath et al. 2008). Although we did not consider the impact of contaminant load on hue, we did find that chroma, in our case blue-green chroma, was negatively associated with overall contaminant load, suggesting the potential for broader applicability of both studies, particularly as the eggs of many species contain the blue-green pigment biliverdin (Kennedy & Vevers 1976). Our findings build on those of Jagannath et al. (2008) by showing that this pattern was conserved across a long-term data set. Our findings also reveal that blue-green chroma was negatively related to a suite of contaminants rather than a single contaminant, which is important because wild birds will experience multiple environmental contaminants.
This relationship between environmental contaminants and eggshell coloration may relate to the physiological mechanisms of pigment production. Previous work has shown that the contaminants can influence the haem biosynthesis pathway by either enhancing or inhibiting the production of porphyrin or biliverdin (Casini et al. 2003). For instance, upstream degradation of haem through induced haem oxygenase activity has been proposed as a mechanism to explain increases in biliverdin associated with dioxin toxicity (Niittynen et al. 2002). In addition, some species exposed to lead poisoning produce excess biliverdin (Mateo et al. 2003a, 2004), whereas others produce excess haemoglobin (Styles & Phalen 1998; Pollock 2006). Based on this previous research, the negative relationship between contaminant load and blue-green chroma in herring gull eggs could be caused by the combined influence of those contaminants on the haem biosynthesis pathway. However, the herring gulls in this study were exposed to a suite of contaminants. Because the concentrations of these contaminants are correlated (Peakall & Fox 1987; Wiemeyer, Bunck & Stafford 1993), this type of data set cannot be used to assess the individual effects of contaminants on egg coloration.
Another nonmutually exclusive possibility is that demographical factors may mediate a relationship between egg coloration and exposure to contaminants. For example, egg coloration varies with age in some species (Moreno et al. 2005; but see, Hargitai, Herenyi & Torok 2008). If environmental contaminants have a differential influence on survival between age classes, variation in egg coloration may be mediated through indirect age-specific effects because of altered population level demographics, rather than a direct effect of contaminants on pigment production. Long-term population studies across a gradient of contaminant exposure would be required to assess such a hypothesis. Future studies involving the experimental manipulation of individual contaminants have the potential to greatly enhance our understanding of the direct and indirect effects of contaminants on the haem biosythesis pathway and, consequently, on avian eggshell pigment deposition.
By tracking decades of decline in contaminants on the Great Lakes, the Herring Gull Monitoring Programme provided a natural experiment to examine the long-term effects of environmental contaminants on eggshell coloration. In addition to demonstrating the capacity for contaminant load to predict blue-green and ultraviolet chroma, we also developed a discriminant function that allows raw spectral data to classify eggs into high and low contaminant levels with greater than 80% success. One might assume that the function would work best on the freshest eggs; however, the function performed similarly well across years (1989 = 87%, 1993 = 79%, 1997 = 82%). We chose to use raw spectral data rather than chroma values in our discriminant analyses to develop an approach that would minimize data processing so that eggs could quickly be classified in the field, and to provide consistency across studies, as researchers often use different chroma metrics (reviewed in, Cherry & Gosler 2010). Because most environmental data, including spectral data, violate the multivariate normality and heterogeneity of variance assumptions (e.g. Alden, Dauer & Rule 1982), we used a PLS-LDA which is not constrained by the same assumptions as a normal linear discriminant analysis (Barker & Rayens 2003). However, previous research using LDA for environmental assessment (Christman & Dauer 2003) has rightly pointed out that discriminant analysis is robust against violations of their assumptions (Lachenbruch 1975; Everitt & Dunn 1991; Sharma 1996) and when prediction is the goal, the percentage of correctly assigned values from an independent validation data set, which in our case was 84%, is the best means for assessing the influence of these violations. Our findings therefore suggest that egg coloration has the potential to be used to approximate contaminant load in the field, and the availability of numerous handheld, battery-operated spectrophotometers make the application of such a bioindicator very feasible.
While large variation in herring gull egg coloration was useful for an initial test of the potential utility of using egg colour as a bioindicator, large inter-clutch variation in egg colour may pose a challenge to assessing contaminant loads. Moreover, many other factors are known to influence egg coloration in birds including genetic determination (e.g., Punnett 1933; Hardiman, Collins & Urban 1975) physiological condition (Soler et al. 2008; Morales, Velando & Torres 2011), life-history traits such as age (Moreno et al. 2005; but see, Hargitai, Herenyi & Torok 2008) and environmental factors (Gosler, Higham & Reynolds 2005; Avilés et al. 2007). We therefore encourage future examinations of other potential ecological correlates of egg colour, such as age, temperature, rainfall and calcium availability, using geographically and temporally broad data sets.
As egg colour is likely to vary between herring gull colonies for reasons other than the influence of contaminants, it will be critical to determine the baseline variation in colour and examine the influence of contaminant load on this variation within target colonies. Similarly, such baseline data would need to be determined in any new species or population before reflectance data are used to predict contaminant load. We used eggs that were emptied and stored for several years, so future research should calibrate our discriminant function on fresh egg measurements, which will account for fading and potentially different eggshell transmission properties. The application of these findings as a monitoring tool would be most beneficial if future investigations target broadly distributed species with pigmented eggs, particularly those species that are already subject to widespread monitoring, such as herring gulls, bluebirds Sialia sialis Linnaeus, European starlings Sturnus vulgaris Linnaeus, or great tits Parus major Linnaeus.
The evaluation of contaminant levels in biota is important for the conservation of natural resources and for monitoring long-term health risks to humans. Long-term monitoring programmes provide a means to examine the progress of environmental remediation and for forecasting potential health risks. Recent research on great tits and blue tits Cyanistes caeruleus Linnaeus suggests that avian eggs may make valuable bioindicators in general, because these birds appear to be exposed to contaminants in the same way and exhibit similar mechanisms of accumulation and maternal transfer to their eggs (Van den Steen et al. 2010). Together with previous research, our findings suggest that the colour of avian eggs may serve as valuable bioindicator of contaminant load. Moreover, there are only two pigment classes controlling egg coloration in birds (Gorchein, Lim & Cassey 2009), and it is therefore possible that these patterns are conserved across all birds. Egg colour may provide a simple, inexpensive and nondestructive indicator of contaminant concentration, while numerous long-term monitoring programmes of colonial and semi-colonial birds worldwide should facilitate the global application of using avian egg coloration as a bioindicator of environmental contamination.