cyclohexane: Fate, Fertility, and Reproductive Success in American

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The Flame Retardant β‑1,2-Dibromo-4-(1,2dibromoethyl)cyclohexane: Fate, Fertility, and Reproductive Success in American Kestrels (Falco sparverius) Sarah C. Marteinson,¶ Robert J. Letcher,§ Laura Graham,† Sarah Kimmins,‡ Gregg Tomy,∥ Vince P. Palace,∥ Ian J. Ritchie,# Lewis T. Gauthier,§ David M. Bird,# and Kim J. Fernie*,¶ ¶

Ecotoxicology & Wildlife Health Division, Science & Technology Branch, Environment Canada, Burlington, Ontario, Canada Ecotoxicology & Wildlife Health Division, Science & Technology Branch, Environment Canada, National Wildlife Research Centre, Ottawa, Ontario, Canada † Department of Animal and Poultry Science, Guelph University, Guelph Ontario, Canada ‡ Department of Animal Science, McGill University, Ste-Anne-de-Bellevue, Quebec, Canada ∥ Department of Fisheries and Oceans Canada, Winnipeg, MB, Canada # Avian Science and Conservation Centre, Department of Natural Resource Sciences, McGill University, Ste-Anne-de-Bellevue, Quebec, Canada §

S Supporting Information *

ABSTRACT: Captive American kestrels (Falco sparverius) were exposed via diet during reproduction to an environmentally relevant concentration of β1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane (β-TBECH). The β-TBECH isomer was injected into the food source at a daily dosing concentration of 0.239 ng/g kestrel/day (22 pairs); control birds were exposed via diet to the safflower oil vehicle only (24 pairs). Eight pairs in each group were exposed for four weeks and sacrificed for tissue analysis; the remaining pairs completed their breeding cycle, with exposure ceasing at the end of incubation (82 days). α- and β-TBECH appeared to be rapidly metabolized and/or eliminated from fat, liver, and plasma; both isomers and potential hydroxylated metabolites of β-TBECH (plasma) were undetected. Notwithstanding, compared to controls, pairs exposed to β-TBECH laid fewer eggs (p = 0.019) and laid lighter eggs (successful eggs: p = 0.009). Exposed pairs also demonstrated poorer egg fertility (p = 0.035) although testis mass and histology were similar among males. Reductions in egg production and fertility resulted in decreased hatchling success (p = 0.023). The β-TBECH-exposed pairs also produced fewer males overall (p = 0.009), which occurred concurrently with increased estradiols maternally deposited in eggs (p = 0.039). These findings demonstrate that β-TBECH may be detrimental for breeding in wild birds receiving similar exposure levels.

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INTRODUCTION Brominated flame retardants (BFRs) have been in use since the 1970s. Currently there is a wide variety of BFRs, several of which are environmentally persistent, bioaccumulate, and have toxic effects. Many BFRs are additive meaning that they are mixed but not bound to polymers, allowing them to leach out into the environment. The additive BFRs, that have been used in the highest quantities over the last few decades, polybrominated diphenyl ethers (PBDEs) and hexabromocyclododecane (HBCD), have become the most prominent global pollutants among the BFRs (e.g., ref 1). These compounds are lipophilic and capable of disrupting steroid hormone pathways by binding to receptors, for example,2 and can cause various endocrine, behavioral, reproductive, metabolic, and neurological outcomes in exposed animals.3−11 Consequently, some PBDEs were voluntarily removed by the © 2012 American Chemical Society

industry and, along with HBCD, all are now banned, regulated, or under review in many countries including Canada, the United States, and China, as well as by the European Union and the Stockholm Convention (summarized at www.bsef. com/regulation). A number of other less prominent replacement BFRs have been identified as having the potential to be persistent and bioaccumulative.12 Several of these newer BFRs are now detected in wildlife sentinel species, particularly in birds, such as in the eggs of herring gulls (Larus argentatus)13 and peregrine falcons (Falco peregrinus)14 from the Laurentian Received: Revised: Accepted: Published: 8440

March 21, 2012 June 22, 2012 July 9, 2012 July 9, 2012 dx.doi.org/10.1021/es301032a | Environ. Sci. Technol. 2012, 46, 8440−8447

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Table 1. Summary of the Kestrel Study Subjects, Sample Sizes, and Analyses Used in the Current β-TBECH Exposure Experiments group

a

experiment

exposure period (d)

sample size (pairs)

sex male female male female

1

tissue analysis

28

16 (8 control; 8 βTBECH)

2

reproductive assessment

mean = 82

30 (14 control; 16 β-TBECH)

agea (years)

analyses

1 1−3 2−4 1 (93%) 2 (7%)

tissue concentrations of β-TBECH and metabolites (liver, fat, plasma); testicular histology reproductive success and timing; egg fertility, size, and shell thickness; egg concentrations of β-TBECH and hormones

When ages varied, they were evenly distributed between the exposed and control subgroups.

disrupting effects on several reproductive measurements. In females, clutch size,25,26 egg mass, shell thickness, and egg fertility25 were reduced and delays in breeding were also noted.27,28 In males, exposure to 17α-methyltestosterone reduced the number of spermatids in the testes, which was associated with the reduced fertility of the eggs laid by their mates.25 The objective of the present study was to determine the effects of dietary exposure to the β-TBECH isomer on the reproductive success of American kestrels (Falco sparverius), a species that is sensitive to other BFRs in terms of reproduction, endocrine function, and behavior.7,29−32 Specifically, β-TBECH was examined because it is the dominant TBECH isomer in the tissues of gulls and beluga whales.13,19 If β-TBECH acts as an androgen agonist in vivo in kestrels, similar detrimental effects on fertility and reproductive success demonstrated by birds exposed to androgen agonists may be predicted.

Great Lakes of North America. Very little to no toxicological information exists for any of these emerging BFR contaminants. A new replacement BFR that is increasing in commercial use is 1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane (TBECH) which is also lipophilic with a log Kow of 5.2515 and has been identified by the United States Environmental Protection Agency as a compound of environmental concern.16 The commercial product is a mixture of two isomers, α-TBECH and β-TBECH; however, these can be thermally isomerized into γTBECH and δ-TBECH.17 TBECH has been shown to accumulate in earthworms living in spiked soil (10, 100, 10,000 ng/g soil), identifying its potential for biomagnification.18 Furthermore, all 4 isomers were detected (γ- and δisomers) or quantified (α- and β-isomers) in the eggs of herring gulls from across the Great Lakes of North America at similar levels each year between 1997 and 2006; sum α- and β-TBECH concentrations in 2006 ranged from an average of 0.95−2.29 ng/g lw with the latter isomer dominant.13 Only β-TBECH was quantifiable in the blubber of arctic beluga whales (Delphinapterus leucas) at levels ranging from 1.1−9.3 ng/g lw.19 These studies comprise the only information available to date on the presence of TBECH in animals. In in vitro assays, all four TBECH isomers have been identified as androgen agonists and are capable of binding to the active site of the human androgen receptor (AR), causing activation.20,21 These in vitro assays identified TBECH as the first confirmed single environmental pollutant that acts as a potent androgen agonist.20,21 In comparison, HBCD and many PBDE congeners are antiandrogenic as well as antiestrogenic and antiprogesteronic and are capable of disrupting the thyroid axis.2 It is not yet known whether TBECH may affect other endocrine pathways in vitro. There is little information on the potential effects of TBECH exposure in animals. and to date reports have been limited to three papers detailing the effects of exposure in fish.18,22,23 These studies demonstrated that TBECH can be maternally transferred to eggs in zebrafish (Danio rerio) exposed by diet to 10 or 100 nmols per g of their food to a chemical mixture that included technical TBECH for 42 days.24 Additionally, some endocrine effects were noted in brown trout (Salmo trutta) exposed to β-TBECH at levels of 0.5, 5.4, or 54 μg/20 mL corn oil. Circulating estradiol or testosterone concentrations were reduced at certain time points in males and females, although no clear trend was identified.22 The thyroid axis of trout was also sensitive to β-TBECH where thyroid cell height was increased at all exposure levels (∼215, 118 pmol/g), and total thyroxine was transiently reduced at the high exposure level.23 The potential effects of TBECH exposure on other reproductive parameters remain unknown for any animal species. However, in birds, exposure to other androgen agonists, testosterone or methyltestosterone, has demonstrated

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MATERIALS AND METHODS As in previous experiments,7,11 this study used captive-bred American kestrels with documented individual histories at the Avian Science and Conservation Centre of McGill University. In April 2010, 92 kestrels divided into 46 pairs were selected and separated into two groups. The first group, used for tissue analysis, consisted of 8 control pairs and 8 pairs exposed to βTBECH. The second group consisted of 30 pairs, 14 control and 16 exposed, that were used for reproductive assessment (Table 1). Paired birds were placed in breeding chambers (1.0 m × 2.4 m × 2.4 m) with a nest-box. Birds were subject to natural climatic conditions and were fed treated, day-old frozen−thawed cockerels (Gallus domesticus) from a local hatchery. Birds had access to food ad libitum, which was refreshed daily. Kestrel husbandry was conducted in accordance with the Canadian Council on Animal Care Guidelines,33 and the research received approval from the Animal Care Committee of McGill University. Chemicals and Reagents. For preparation of the exposure material, standards and solutions of α- and β-TBECH, BDE-30, and ultra high-purity β-TBECH (neat; > 97% purity) were purchased from Wellington Laboratories (Guelph, ON, Canada). OmniSolve solvents, suitable for gas chromatography and pesticide residue analysis, were used for extraction and clean up. An amount of 0.205 mg of pure β-TBECH was dissolved in 100 uL of high grade toluene (to ensure complete dissolution prior to transfer to safflower oil). After dissolution in the toluene, this solution was quantitatively transferred with 3 × 250 μL of high grade n-hexanes to a 1 L amber glass bottle; previously the bottle had been chemically cleaned with 3 washes of acetone followed by 3 washes with hexanes and allowed to dry. Precisely 700 mL (0.7 L) of organic safflower oil was added to the amber bottle. The mouth of the bottle was lightly covered with (chemically washed) aluminum foil and 8441

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integrity. Eggshell thickness was measured at five places around the egg’s equator, and a mean was calculated following Fernie et al.7,11 and referenced work therein. Reproductive measures of the pairs were calculated on a perpair basis following Fernie et al.7,11 and included the following: Julian lay date (number of days from pairing to egg laying), clutch size (number of eggs in the first clutch), fertility (the total number of fertile eggs in the clutch and proportion of eggs that were fertile at candling), hatching and fledging production (the total number of hatchlings or fledglings produced for successful pairs), the proportion of fertile eggs that hatched, the proportion of hatchlings that fledged, the number of male and female young produced (a male to female ratio was calculated), and overall reproductive success (the number of eggs that produced fledglings). The first egg was not included in analyses of fertility, hatching, or fledging success, because it was removed immediately from the nest. Egg Hormone Analysis. Yolks from the first eggs of 26 of the pairs used for reproductive assessment were successfully analyzed for concentrations of testosterone, estrone, and estradiol (henceforth referred to as estradiols), corticosterone, thyroxine (T4), triiodothyronine (T3), and β-TBECH. Egg yolks were extracted using a modification of a previously published simplified method of extraction of steroid hormones from avian egg yolks.36 In brief, 0.5 mL of assay buffer (0.02 M Trizma, 0.300 M NaCl; pH 7.5) was added to 0.1 g of homogenized yolk sample in a microcentrifuge tube, vortexed for 3 min, and incubated for 1 h at 37 °C. Following incubation, 0.5 mL of ETOH was added, and samples were vortexed for 3 min. Samples were then centrifuged for 15 min at 15,000 g. Extracts containing the hormones were decanted and stored at −20 °C until enzyme-immunoassay (EIA). Extracts were centrifuged at 4000 g to further remove lipids prior to evaporation and reconstitution in assay buffer for the EIA. All of the EIAs employed polyclonal antibodies made in rabbits, the details of which can be found in the Supporting Information. Determination of TBECH Isomers and Quality Control. The concentrations of β-TBECH were determined in the safflower dose solutions as well as the egg (control n = 2, exposed n = 4), fat, liver, and plasma (control n = 3, exposed n = 7) samples from a random subset of individuals. A sub-subset of five plasma samples (control n = 1, exposed n = 4) was also screened for potential α-TBECH, debrominated metabolites, and hydroxyl- (OH-) containing TBECH metabolites. The determination of α- and β-TBECH in the eggs, tissues, and plasma was similar to methods reported in Gauthier et al.13 with modifications. The β-TBECH method limits of detection (MLODs) for liver, fat, plasma, and egg were 0.04, 0.4, 0.07, and 0.4 ng/g wet weight (ww), respectively. The β-TBECH method limits of quantification (MLOQs) for liver, fat, plasma, and egg were 0.11, 1.2, 0.2, and 1.2 ng/g ww, respectively. The overall mean average percent recovery of the internal standard (IS) for all samples (egg, liver, fat, and plasma) was 74 ± 25%. Blank samples contained low levels of β-TBECH, and thus background ‘noise’ was minor and generally unnecessary to correct for in the samples. To assess the recovery and reproducibility of β-TBECH in each batch of samples, pork liver homogenate from an Ottawa market (for liver, lung, brain, and diet), olive oil (for fat), and bovine serum (for plasma) were spiked with a known concentration of α- and β-TBECH. Recovery was highly quantitative and precise with a %RSD of

allowed to stir (with a magnetic stir bar in the solution) overnight (∼12 h). This was to ensure virtually complete outgassing of the very small volume of toluene and n-hexanes and for a completely homogeneous distribution of the betaTBECH in the safflower oil. Diatomaceous earth was heated at 600 °C overnight in a muffle furnace prior to use. Silica was activated at 120 °C overnight in an oven and then treated with sulfuric acid (equal weight). The mixture was homogenized for >24 h prior to use. Dosing solutions were prepared using neat β-TBECH at Environment Canada’s National Wildlife Research Centre in Ottawa (Letcher Lab). Exposure Protocol. A safflower oil solution of β-TBECH was prepared at an estimated concentration of 293 ng of βTBECH/mL safflower oil (or 0.293 μg/mL), although analysis showed the actual concentration to be 0.301 μg of β-TBECH/ mL oil. A volume of 0.05 mL solution was injected into individual (dead) cockerel brains after they were thawed and immediately prior to daily feeding of the birds. Kestrels were each fed 1.75 cockerels per day; kestrels in the treatment exposure group received a dose of 26.3 ng of β-TBECH/day (0.239 ng/g kestrel/day), and the control kestrels were fed cockerels containing safflower oil only. Exposure concentrations were environmentally relevant and based on those reported in the eggs (collected in 2006) of wild herring gulls in the Great Lakes.13 Testicular Evaluation. The testes of the one-year-old males from the first group of kestrel pairs (n = 8 control and 8 β-TBECH-exposed, Table 1) were used to examine the possible effects of β-TBECH on male fertility. These males were paired with females in March and were exposed to β-TBECH for two weeks prior to pairing and two weeks while paired. The males were sacrificed during the onset of spermatogenesis. The method of testicular analysis followed previous BFR studies in our laboratory.31,32 Briefly, the right and left testes were removed within 30 min of death, weighed, and fixed in Bouin’s solution for ≥6 h. Sections were stained with eosin and hematoxylin. All seminiferous tubules were examined at a magnification of 670×; the number of tubules containing final spermatids was counted, and a proportion relative to the total number of tubules was calculated. Two cross sections from the first quarter and middle sections of the right and left testis of each individual were examined, and the mean number of seminiferous tubules was calculated. The gonadosomatic index was calculated as total testes mass ×100/body − testes mass.34 Reproductive Assessment. The second group of kestrels involving 30 pairs (n = 14 control and 16 TBECH-exposed, Table 1) were observed for an entire reproductive cycle. Following previous BFR kestrel exposure protocols established by Fernie et al.,7,11 exposure began four weeks prior to pair formation and continued through courtship and incubation (average of 82 days). In these pairs, most females were one year of age (two were two years old), and all males were two to four years old and separated equally by age between the two groups (Table 1). Reproductive parameters included the dates of egg laying, hatching, and fledging for each chick. All eggs were weighed when freshly laid, and length and width measurements were recorded with digital calipers for volume calculation35 as per our previous BFR kestrel research.9,24 At mid-incubation, eggs were reweighed and candled to determine their fertility by the presence of an embryo. The first egg laid by each pair of kestrels was removed from the nest immediately and directly frozen at −20 °C until analyzed. These eggs were used for chemical and hormone analysis as well as to measure eggshell 8442

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60%) in zebrafish, and when exposure ceased, TBECH was eliminated more rapidly than BDE-28, -128, -209, HBCD, and tetrabromobisphenol A (TBBP-A).37 In juvenile brown trout, elimination of β-TBECH did not occur as quickly; however, at 17 days, its half-life was 10 times lower than that of γ-HBCD. Metabolites were not detected in muscle or liver of the trout, and there was no evidence of β-TBECH bioisomerization to α-TBECH.22 Those authors proposed that the elimination rate of β-TBECH exceeded either metabolism or storage rates or that β-TBECH may not have been metabolized at all. However, uptake and storage of βTBECH in wild birds is possible as evidenced by its detection in the eggs of Great Lakes herring gulls,13 which could indicate that exposure levels in wild birds may be higher than those in the present study. Egg Production and Quality. Although β-TBECH did not accumulate in the kestrels at detectible levels, and likely was metabolized and/or eliminated rapidly by these birds, their reproductive success was adversely affected by their exposure to β-TBECH. The kestrel pairs exposed to β-TBECH laid fewer eggs than control pairs (U14,16 = 56.0, p = 0.019) with a mode of four eggs in the treated group compared to five eggs for control pairs (excluding the first egg removed for analysis) (for 8443

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high-exposure DE-71 group being fertile, compared to 67% of all eggs being fertile for the entire β-TBECH kestrel group in the present study. This suggests that for birds there may be a greater impact on fertility for β-TBECH exposure than for DE71. There were no differences in testes mass, gonadosomatic index, nor the proportion of seminiferous tubules containing final spermatids, between control and β-TBECH-exposed males. These results contrast with the effects seen in kestrels exposed to HBCD or DE-71 where increased testicular mass and altered histology were reported.31,32 The lack of testicular changes suggests that sperm production in males may not have been a factor in the reduced fertility of the eggs at the present exposure level. The testes of Japanese quail exposed to 17αmethyltestosterone demonstrated reduced testicular mass and spermatid numbers at the highest exposure levels (6, 13 mg/kg bw/d)25 which were much higher than the levels in the present study. Furthermore, pairs exposed to β-TBECH actually demonstrated increased copulation frequency compared to controls (Fernie, J. and Marteinson S., Environment Canada, unpublished data), eliminating reduced sperm delivery as a likely cause for decreased egg. Delays in egg-laying can reduce egg fertility in birds;42 however, exposed pairs demonstrated a trend in advanced laying in the present study. Together, these results suggest that the reduced fertility of eggs in the present pairs exposed to β-TBECH may have been a result of other factors, including sperm morphology, mobility, ability for capacitation, and/or fertilization. Additionally, changes in the female may also have been a contributing factor, and the alterations in egg mass and hormone deposition reported herein suggest that the female reproductive tract is affected. In contrast to the present results, though pairs exposed to the PBDE mixture DE-71, also had reduced egg fertility, in contrast they demonstrated delayed egg-laying and reduced copulation frequency.7,30 Additionally, males exposed in ovo to DE-71 had increased testicular mass and histological changes, suggesting different modes of action on fertility for these two chemicals. Kestrel pairs exposed to β-TBECH had a larger number of fertile eggs that failed to hatch than control pairs. In the kestrel group exposed to β-TBECH, 47% of nests had at least one failed egg (mean of 1.5 eggs) compared to 23% of control nests (mean of 2.6 eggs). In addition, only the β-TBECH pairs demonstrated cracking of their eggs or a lack of incubation (n = 3). Though this happened in a small proportion of kestrel pairs, it demonstrates a possible lack of appropriate parental attention toward their eggs, which occurs rarely in control birds in this laboratory. Similarly, kestrels exposed to PCBs by diet also demonstrated an increased incidence of cracked eggs without eggshell thinning, likely as a result of parental inattentiveness.43 For Japanese quail exposed to 17α-methyltestosterone, there was a dose dependent increase in the incidence of cracked eggs in comparison with controls; the quail eggs were artificially incubated, and the authors concluded that it may have been related to the reduced eggshell thickness observed at the highest exposure level only.25 Reproductive Success. Ultimately, the reproductive success of a pair depends on the number of offspring that pairs successfully raise. For the kestrels exposed to β-TBECH, fewer hatchlings were produced per pair in total (t20 = 2.45, p = 0.023) (Figure 1) but not in proportion to the number of fertile eggs laid per pair. The reduced hatching production in βTBECH-exposed pairs was thus mainly the result of the smaller number of eggs laid as a whole as well as the reduced egg

Figure 2. Comparison of the mean mass for hatched eggs producing either 1. male or 2. female young as well as 3. infertile and 4. failed fertile eggs between American kestrels exposed by diet to β-TBECH and control kestrels exposed to the safflower oil vehicle (interaction plot from a 2-factor ANOVA).

No significant alterations (p > 0.05) in eggshell thickness were noted in the group exposed to β-TBECH compared to controls. This is consistent with previous work on androgen exposure in female Japanese quail, where only the high exposure level (13 mg/kg bw/d) of androgens reduced eggshell integrity.25 In comparison to other BFR studies that also used environmentally relevant exposure levels, this result is parallel to kestrels exposed to HBCD (0.51 μg HBCD/g kestrel/d) where eggshell thickness was not altered.11 However, eggs laid by female kestrels exposed by diet to DE-71 (0.98 ug DE-71/g kestrel/day) demonstrated eggshell thinning by 6%.7 Timing of Breeding. The timing of egg laying is a useful measure of the changes in seasonal breeding and may have effects on subsequent reproductive measures. Exposure to βTBECH at the present level resulted in an advance of the initiation of egg laying in the exposed pairs compared to controls, by an average of 6 days, though this trend did not reach statistical significance. These results are similar to other kestrel pairs exposed by diet to HBCD, which also displayed advanced egg laying.11,41 In contrast, exposure to testosterone in other avian species caused delays in egg laying for female homing pigeons (Columba livia domestica)27 and dark-eyed juncos (Junco hyemalis),28 and the timing of egg laying was delayed in kestrels exposed to PBDEs by diet to the pair, or developmentally to the male only.7,30 Egg laying in birds is in part regulated by thyroid hormones, as well as steroidal hormones and behavior, and the timing of breeding in these pairs may be related to one of these factors. Egg Fertility and Viability. The kestrels exposed to βTBECH produced fewer fertile eggs per pair compared to controls, overall (U14,15 = 52.5, p = 0.035) (Figure 1), similar to Japanese quail exposed to 17α-methyltestosterone.25 On average, 88% of the eggs laid by control pairs were fertile, compared to 73% fertility in β-TBECH clutches; however, this proportional fertility did not reach statistical significance. Reductions in fertility were also reported in kestrels exposed to the DE-71 PBDE mixture by diet7 or developmentally to males.30 Although exposure concentrations in the PBDE study were higher than the present study, the magnitude of effect was similar between the two studies with 65% of all eggs laid in the 8444

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example, increased maternal estrogen,47 progesterone,48 or corticosteroids49 can bias the sex ratio toward females, and increased maternal testosterone can bias the sex ratio toward males.50 Of all of the hormones measured in the first egg laid (estradiols, testosterone, corticosteroids, T3, and T4), only in ovo concentrations of estradiols significantly differed, and they were higher in the first egg of the β-TBECH exposed pairs (3.03 ± 0.32 ng/g) compared to control eggs (2.29 ± 0.18 ng/ g) (t test for unequal variances: t = −2.20, p = 0.039). This suggests that an endocrine explanation for the reduced number of male chicks in the β-TBECH group may be a possibility. However, levels of estradiols in the first egg were not statistically correlated with the number of males or females in the brood. Another hypothesis that may explain the hatchling sex ratios is selective embryo mortality. Indeed, a larger proportion of nests in the β-TBECH-exposed group had at least one failed egg. Additionally, the candling technique was used to assess fertility at mid-incubation. This technique is accurate and can detect embryos that have died; however, it cannot reveal embryos that were not viable or died before any visible growth occurred. As a result, any embryos dying at a very early stage may not have been detected. If a disproportionate number of male embryos died in early incubation, this could result in a skewed sex ratio. Further research is warranted to determine how exposure to β-TBECH may be affecting the sex allocation and embryo toxicity in birds. However, regardless of the mechanism, any deviation from a normal sex ratio may have important implications for the reproductive success and stability of wild populations. Environmental Implications. That TBECH isomers have been detected in the eggs of wild herring gulls13 and the blubber of beluga whales19 demonstrates that uptake and storage of these isomers is possible in wild animals, which has been confirmed in earthworms18 and fish24 in the laboratory. Despite the lack of detectable levels of β-TBECH in the kestrels, the accumulation of TBECH in other animals in conjunction with the definitive reproductive effects observed in the present kestrels suggests that environmental exposure may be greater for wild birds than the low β-TBECH exposure in the present study. Critical reproductive parameters including clutch size, egg size, and fertility and hatching success were reduced, and egg deposition of estradiol was increased in the present β-TBECH-exposed kestrel pairs compared to controls. These findings corroborate the results of previous studies in fish18,22,23 where TBECH was found to be bioactive with the potential for endocrine disruption at environmentally relevant exposure concentrations. The pattern of reproductive effects relating to β-TBECH exposure in the present kestrels is largely consistent with its androgenic endocrine disrupting potential20,21 and demonstrates reproductive effects similarly observed in birds exposed to historically prominent BFRs: PBDEs and HBCD.7,11,30 Exposure to β-TBECH may be a potential concern for wild birds experiencing similar or greater exposure levels to β-TBECH. Additional research is warranted with birds and other animal models to further determine the potential effects of exposure to TBECH.

fertility of this group (Figure 1). However, the increased incidence of failed eggs may also have been a contributing factor. Despite the reduced number of viable eggs in the βTBECH group, by the end of the breeding season, the two groups had produced a similar number of fledglings per pair (Figure 1). The results of the current study suggest that βTBECH affects reproduction early in the reproductive period, specifically prior to hatching through fewer eggs being laid and the laying of eggs with poorer fertility; effects on similar stages were reported in captive kestrels exposed to HBCD.11 For the control kestrels in the current study, mortality of some nestlings after hatching resulted in fledging success that was comparable to the kestrels exposed to β-TBECH, which can occur with inexperienced one-year-old females. Thus overall reproductive success was not greatly affected by exposure to β-TBECH in these ideal laboratory conditions. Kestrels receiving exposure to DE-71 by diet produced fewer fledglings compared to controls,7 while conversely exposure to HBCD did not affect reproductive success at environmentally relevant concentrations.11 Sex Ratio and Egg Hormones. The reduced number of viable eggs in the β-TBECH group appeared to be at the expense of male nestlings being successfully produced. Pairs exposed to β-TBECH had fewer male offspring per brood than control pairs (t13 = 3.05, p = 0.009) (Figure 3). The sex ratio of

Figure 3. The mean total number of male or female kestrel offspring per brood produced by control pairs compared to pairs exposed to βTBECH.

control pairs was biased toward male nestlings where the mean for control pairs was 2.71 males to females compared to 1.43 in the exposed group. However, in kestrels, young females, such as those in both groups in the present study, naturally tend to produce more male offspring, the smaller of the two sexes.44 This phenomenon is thought to be related to their inexperience and to have evolved so that first-time breeders will have increased success with lower parental investment.44 In birds, sex allocation is dependent on genetics; however, unlike mammals, other mechanisms are also involved. These include several factors such as food availability, which was controlled for in this study, as well as the body condition of the female,44,45 which were not affected in the present study. Females, being the heterogametic sex in birds, additionally have physiological control of sex allocation, which may be primarily linked to changes in circulating hormone concentrations.46 For

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ASSOCIATED CONTENT

S Supporting Information *

Further details on the analytical methods for both chemical analysis and hormone analysis. This material is available free of charge via the Internet at http://pubs.acs.org. 8445

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AUTHOR INFORMATION

Corresponding Author

*Phone: 905-336-4843. Fax: 905-336-6430. E-mail: kim. [email protected]. Corresponding author address: Ecotoxicology & Wildlife Health Division, Science & Technology Branch, Environment Canada, 867 Lakeshore Rd., Burlington, Ontario, Canada, L7R 4A6. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank L. Bardo and M. Gagnon (McGill University). Funding was provided by the Chemicals Management Plan and the Ecotoxicology and Wildlife Health Division, both of Environment Canada (K. J. Fernie, R. J. Letcher), and the Natural Sciences and Engineering Research Council of Canada (239250-07 to R. J. Letcher and 327004-06 to S. Kimmins). The authors thank G. Barrett (Environment Canada), photographer of the TOC art.

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