California condors and DDT: Examining the effects of endocrine disrupting chemicals in a critically endangered species

ABSTRACT

In 1987, the last free-flying California condor (Gymnogyps californianus) was captured and brought into captivity, rendering the species extinct in the wild. At the time, only 27 condors remained. Today, the population numbers approximately 430 individuals and though condors continue their remarkable recovery, they still face numerous challenges. One challenge, specific to condors inhabiting coastal regions, is exposure to the endocrine disrupting chemical (EDC) p,p'-DDE, through the scavenging of marine mammal carcasses. The exposure levels these birds currently experience appears to be causing eggshell thinning, reminiscent of the phenomenon that nearly collapsed populations of several avian species decades ago. We were interested in further exploring the potential effects of EDCs on California condors. Investigating EDC effects on a critically endangered species like the condor can be difficult, with limited options for studies that can be feasibly conducted. Therefore, we conducted non-invasive, in vitro estrogen receptor (ESR) activation assays to characterize activation by EDCs that coastal condors encounter. Here, I give a brief history of EDCs effects on birds, and in particular the California condor. Additionally, our ESR data are summarized and mechanisms of eggshell thinning are reviewed, highlighting the potential implications of EDC exposure on the continued recovery of the California condor.

KEYWORDS:

• California condor
• DDT
• estrogen receptor
• p,p'-DDE

Introduction

In 1962, Rachel Carson's Silent Spring brought the potential hazards of widespread pesticide use into the public conscience. At the time of its publication, the effects of pesticides, especially DDT (dichlorodiphenyltrichloroethane), on wildlife species were becoming clear. As the title of the book suggests – a reference to the disappearance of springtime migration of songbirds – the detrimental effects of DDT use on avian species was of particular concern. Initial indications that DDT exposure compromises reproduction in birds predate Silent Spring by a few years. Ratcliffe¹ was one of the first to identify what is now the most well known effect of DDT and its metabolite p,p'-DDE (dichlorodiphenyldichloroethylene) on birds, by associating the use of the pesticide with the laying of thin-shelled eggs by peregrine falcons and other birds of prey.² Since then, DDT use and elevated levels of its metabolite p,p'-DDE have been associated with eggshell thinning and other reproductive problems in many bird species.^3-5 Furthermore, DDT and its metabolites produced through chemical and biological degradation (DDXs; see Table 1) are suggested to function as endocrine disrupting chemicals (EDCs) in numerous vertebrate taxa.^6,7

Table 1. Half maximal activation (EC₅₀) and percent activation relative to maximal activation by estradiol (% Max E₂) of California condor estrogen receptors (ESRs) 1 and 2 by DDT and its metabolites. Adapted from Felton et al., 2015.

Structure	Name	ESR1 EC50	ESR1 % Max E2	ESR2 EC50	ESR2 % Max E2
	p,p'-DDT	6.6 × 10^−6 M	82.3 ± 5.3	5.3 × 10^−6 M	74.6 ± 9.6
	o,p'-DDT	3.6 × 10^−6 M	72.9 ± 4.0	2.9 × 10^−6 M	98.1 ± 12.7
	p,p'-DDD	2.6 × 10^−5 M	16.1 ± 1.9	2.5 × 10^−5 M	39.7 ± 1.3
	o,p'-DDD	2.7 × 10^−6 M	68.2 ± 7.0	3.8 × 10^−6 M	60.0 ± 6.6
	p,p'-DDE	1.4 × 10^−5 M	16.1 ± 2.4	5.9 × 10^−6 M	28.1 ± 3.0
	o,p'-DDE	3.2 × 10^−6 M	47.0 ± 5.8	2.9 × 10^−6 M	55.8 ± 5.2

Bird species residing near the coast of California were particularly hard hit by DDXs, to the extent that entire breeding colonies of some species produced few, if any, chicks.^8-10 The major source of contamination that affected bird species residing within the Channel Islands was the discharge of millions of pounds of DDT by the Montrose Chemical Company into the Pacific Ocean and onto the Palos Verdes Shelf in the 1940's–1970's. Since dumping has stopped and the use of DDT was banned in 1972, environmental levels of DDXs have decreased, but continue to persist within the food web at concentrations potentially sufficient to harm wildlife.¹¹ One such species that appears to be impacted by DDT, more than 4 decades after its use was banned, is the critically endangered California condor (Gymnogyps californianus)¹², which was reintroduced to coastal regions of central California in 1997.

The decline and resurgence of the California condor

Like other bird species in California, the increased use of DDT also coincided with a decrease in the California condor population. However, the extent to which DDXs contributed to the historical decline of the California condor is uncertain. DDT use became widespread in the United States in the mid-1940's.¹³ In 1960, when DDT use was near its greatest, fewer than 60 condors remained in California. That number was down from 100 birds in 1940 and an estimated 600 birds in the 1890's.¹⁴ Clearly, the condor population was in decline well before the widespread use of DDT. In his seminal publication on California condor life history, Carl Koford attributed condor mortalities primarily to hunting, accidental poisoning and trapping by carnivore control efforts and other anthropogenic factors.¹⁵ He did not suggest that pesticide use was contributing to the condor's decline, nor would he necessarily have reason to, as it would be years before the first reports^1,2 were published linking DDT use with thin eggshell productions in a raptor species. Nevertheless, an analysis of condor eggshell fragments collected before and after the widespread use of DDT suggests the pesticide was a contributing factor to the condor's decline. Eggs collected after 1963 (n = 4; mean shell thickness = .485 mm) exhibited excessive thinning (∼30%) compared to those collected before 1944 (n = 2; mean shell thickness = .717 mm), prior to the widespread use of DDT.¹⁶ Others have suggested these findings are not evidence of DDT-associated eggshell thinning, but instead are an artifact of measuring fragments from regions of eggs with thinner shells and/or measuring shells from smaller eggs compared to those collected before 1944.¹⁷ Regardless of the relative magnitude of specific contributing factors to their mid-century decline, the condor population continued to dwindle, reaching a low of 22 birds in 1982, and it became clear the species would disappear without human intervention.¹⁴ In 1987, the last wild California condor was captured¹⁸, making the species extinct in the wild with a total captive population of 27 individuals.

Today there are more than 430 California condors with over half the population living in the wild. As the population grew, primarily due to the success of captive breeding programs, reintroduction sites were established and condors now reside in California, Arizona, Utah and Baja California, Mexico. The largest threat to the condor's continued recovery is lethal lead poisoning from scavenging fragments of lead-based ammunition present in the carrion of terrestrial mammals.^19,20 Without a reduction in the current levels of lead poisoning, the condor population cannot become self-sustaining¹⁹ and as a result, extensive efforts are made to reduce lead exposure. However, condors continue to be exposed to harmful levels of lead, to the extent that attempts are made to capture each wild bird at least once a year to perform lead testing and lead chelation therapy if needed.²¹

In California, condors inhabit both inland and coastal environments. Coastal reintroduction sites represent the condor's historical range¹² and have the benefit of providing opportunities to scavenge marine mammal carrion that should provide lower risk of lead exposure since marine mammals are not legally hunted. However, by scavenging marine mammals, and in particular carcasses of top marine predators such as California sea lions (Zalophus californianus), condors are at risk of increased exposure to bioaccumulated organochlorine chemcials, like DDT and p,p'-DDE. This risk was realized prior to reintroductions, but since DDT levels were in decline the decision was made to release birds in Big Sur, CA.¹²

While the role that DDT played in the condor's decline is uncertain, its effect on Big Sur, CA birds appears clear. In that population, 16 pairs of condors nested from 2006–2010 and eggshells from 12 of the nests were collected for analysis.¹² Compared to eggs from condors inhabiting inland regions of southern California, shells from coastal eggs were 34% thinner (0.46 mm vs 0.70 mm), which is a similar decrease in thickness of eggshells collected before and after the widespread use of DDT.¹⁶ Moreover, compared to their inland counterparts coastal condors have plasma p,p'-DDE concentrations that are 20-fold higher, with mean circulating levels reaching low micromolar concentrations.²² While lethal exposure to lead in coastal environments might be lower than inland habitats²³, sub-lethal exposure to EDCs appears to be affecting condor reproduction, presenting yet another challenge to those managing the recovery of the species.

Studying EDC effects on critically endangered species

We were interested in further investigating how p,p'-DDE and other EDCs might affect reproduction in California condors, but definitively addressing such questions in a species with fewer than 500 total individuals presents unique challenges. Namely, opportunities to conduct in vivo studies are limited, if not impossible.²⁴ We therefore used an in vitro approach by identifying and cloning condor estrogen receptors (ESRs) 1 and 2, which are both established EDCs targets, from reproductive tissues collected opportunistically during condor necropsies.²⁹ We then expressed recombinant condor ESRs in human embryonic kidney cells (HEK293) and characterized activation of condor ESRs by DDXs (Table 1) and other EDCs to which condors are actually or potentially exposed to using a luciferase reporter plasmid system.²⁹ We have had previous success using this approach in identifying dietary phytoestrogens as a potential cause of poor fertility in the southern white rhinoceros (Ceratotherium simum simum)²⁵, demonstrating the utility of non-invasive methods in studying EDC effects on species of conservation concern.

Activation of condor ESRs by EDCs

Nearly all of the EDCs we tested activated both condor ESRs, but most were only capable of doing so at micromolar concentrations.²⁶ Of the 2 receptors, ESR2 was generally more sensitive, with 8 of the 12 EDCs stimulating greater maximal activation of ESR2 compared to ESR1 relative to the maximal activation of the receptors by estradiol (E₂). Of the DDXs studied, o,p'-DDT and p,p'-DDT isomers were the most potent ESR agonists, with each stimulating similar levels of receptor activation. These findings are consistent with in vitro chicken ESR studies,²⁷ but are not in agreement with in vivo studies in chickens or other avian species,^28,29 in which p,p'-DDT is not estrogenic. While interesting, this discrepancy highlights an obvious limitation in relying solely on in vitro systems, which is that they cannot always predict in vivo responses to EDCs. Nevertheless, analyzing in vitro data within the context of other available data, such as EDC exposure levels or EDC effects on other closely related species, allows for speculation on the physiological effects of EDCs on species in which extensive in vivo work cannot be conducted.

To date, the only DDX for which actual condor exposure levels are available is p,p'-DDE,²² which is widely considered to be anti-androgenic and not to have significant estrogenic activity, based on both in vitro and in vivo studies.^5,30 Although it was one of the least estrogenic EDCs we tested, we did find that low micromolar concentrations of p,p'-DDE stimulated ESR1 and ESR2 activation to approximately 10 and 20%, respectively, of the maximal activation by E₂. Though high, these concentrations are similar to those found in vivo in coastal-scavenging condors. Recent analyses of plasma from 22 coastal-dwelling condors revealed mean p,p'-DDE concentrations of nearly 3 μM, with levels reaching as high as 8.9 μM.²² Given that p,p'-DDE is the dominant DDX in condor plasma, which is reported to account for nearly 96% of all DDXs found²², it is possible that other DDXs are not present in sufficient concentrations to function as EDCs through activation of condor ESRs.

Mechanisms of eggshell thinning

There is still no certain mechanistic explanation of p,p'-DDE–induced eggshell thinning in adult birds, despite a decades-old link between exposure to the contaminant and the phenomenon in numerous avian species. The most likely mechanism, based on evidence from multiple studies across different species, is that p,p'-DDE interferes with the synthesis of prostaglandins, which subsequently reduces the amount of calcium transported across the eggshell gland resulting in the production of thin-shelled eggs.⁵ There is little evidence to suggest that p,p'-DDE alters estrogen signaling, as has been previously proposed, either by increasing the metabolism of circulating estrogens or by directly interfering with estrogen receptors (ESRs).⁵ However, an alternative mechanism of eggshell thinning has been described that is likely ESR-dependent. In the chicken, in ovo exposure to ESR agonist, ethinyl estradiol (EE₂), or the estrogenic DDT isomer, o,p'-DDT, results in alterations to the development of the shell gland, including reduced carbonic anhydrase activity that causes the production of thin eggshells as adults.^31,32 While these 2 mechanisms ultimately result in a similar outcome – the production of eggs with compromised shells – they differ in terms of their duration, highlighting the importance of the timing of exposure in predicting the severity of the resulting effect.^6,33 As evidenced by the rebound of many wild bird populations soon after DDT use was banned,¹⁰ the effects of adult exposure can often be reversed if exposure levels are reduced. In contrast, when developmental EDC exposure occurs, the functional and structural changes to the eggshell gland that result in thin shell production persist for longer periods of time and perhaps indefinitely.³²

Potential implications of EDC exposure to the continued recovery of the California condor

Although our study does not provide additional insight into current levels of condor eggshell thinning, since p,p'-DDE-induced eggshell thinning in adult birds does not appear to involve ESRs, it does raise the possibility that in ovo exposures to this EDC may be sufficient to alter eggshell gland development. How well circulating contaminant levels reflect levels found in the yolk of condor eggs is currently unknown, but in laying hens, blood p,p'-DDE levels are similar to those found in the yolk and other tissues.³⁴ Thus, it is plausible that maternal transfer into the yolk exposes developing condor embryos to high levels of p,p'-DDE in ovo. If this is the case, the question becomes whether p,p'-DDE in ovo activate ESRs like it does in vitro and if so, is the degree of activation sufficient to alter eggshell gland development as EE₂ and o,p'-DDT do in other species?

Studies specifically looking at the effects of p,p'-DDE on eggshell gland development are lacking, however, there is evidence that high p,p'-DDE concentrations can exhibit estrogenic activity in ovo. In their landmark investigation into the reproductive collapse of the southern California western gull population, well after the discontinued use of DDT, Fry and Toone²⁸ examined the effects of environmentally relevant DDXs exposure on the development of gull embryos. Their study primarily focused on the estrogenic effects of o,p'-DDT, which caused gonadal feminization in males and development of the typically rudimentary right oviduct in female embryos. These effects were observed at both the high and the low concentrations of o,p'-DDT and E₂ tested. In eggs treated with p,p'-DDE, gonadal feminization was observed in male embryos, but only at the higher concentrations tested. Although this effects on males corroborates our ESR activation data showing p,p'-DDE exhibits weak estrogenic activity, it should be noted that no effects of p,p'-DDE on female embryos were detected. However, this study looked only at development of the right oviduct as a measure of estrogenicity and assessments of the left oviduct and its associated shell gland were not made. As the authors of the study recognize, it is therefore possible that developmental exposure to the DDXs tested may be responsible for other abnormalities such as the production of thin eggshells.²⁸ We of course now know this to be the case for chicken embryos exposed to o,p'-DDT, but whether in ovo p,p'-DDE exposure can do the same, particularly in California condors, is unclear and warrants further investigation.

The reintroduction of California condors to coastal habitats and the subsequent p,p'-DDE-induced eggshell thinning they have experienced is an obstacle to the species' recovery, yet the problem is currently manageable. For instance, wild-laid eggs can be weighed regularly to check for excessive water loss, which is indicative of thin shells, and removed for artificial incubation to ensure successful hatching if necessary.¹² With time, and without any significant new inputs of DDT into the coastal ecosystem, it is anticipated that levels of p,p'-DDE exposure will drop below the threshold where eggshell thinning occurs and will no longer present as issue for coastal condors.¹² Although we do not know what that timeline or threshold are, the scenario is possible given the recovery of other species following reductions in DDT use and p,p'-DDE exposure. However, with the potential for p,p'-DDE (or other EDCs) to cause developmental effects resulting in long-term reproductive problems such as thin eggshell production, as speculated here, this problem could persist well into the future and provide further challenges for the condor. One such challenge is that production of thin eggshells due to in ovo EDC exposure would not be recognized until birds reach the breeding age of 5–6 y. Furthermore, it is common for condors to be moved between reintroduction sites as part of the species' management and as the population grows, condors are expanding their range on their own.²¹ Therefore, it is possible that chicks exposed to EDCs in ovo could be moved or naturally relocate to habitats with low EDC exposure risk, but still exhibit reproductive issues consistent with EDC exposure as adults. Most importantly, however, is that problems resulting from developmental EDC exposure could be persistent and particularly detrimental to condors, especially given their life span of 50–60 y and slow reproductive output of usually one egg every 2 y. In raising awareness for the potential of these effects to occur, condors can be monitored and if evidence suggests that a problem is transpiring, the population can be managed accordingly.

Our study has provided some insight into the possible effects of EDCs on California condors, but more work is certainly needed to better evaluate the risks facing this species. One aspect of EDC exposure not yet addressed is how chemical mixtures affect condor ESR activation. Even though contaminant loads in condors are dominated by p,p'-DDE, it is possible that others, including both legacy and emerging chemicals, are present in levels sufficient to have combinatorial effect that differ from activation by p,p'-DDE alone. Additional studies examining EDC interactions with other hormone receptors are also warranted and are currently underway. Together, findings from such in vitro, non-invasive approaches can provide a more comprehensive picture of how EDCs interact with condor endocrine systems, potentially compromising reproduction. This information can hopefully be used to assist managers in ensuring that birds are released in suitable habitats, while also raising awareness about potential challenges that condors may face in the future.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

Thank you to Dr. Barbara Durrant, Dr. Thomas Jensen and Rachel Felton (San Diego Zoo) and Dr. Myra Finkelstein (UC-Santa Cruz) for providing valuable feedback on this manuscript.