Ecotoxicology (2012) 21:2143–2152 DOI 10.1007/s10646-012-0967-3
Toxicity of methylmercury injected into eggs of thick-billed murres and arctic terns Birgit M. Braune • Anton M. Scheuhammer • Douglas Crump • Stephanie Jones • Emily Porter Della Bond
•
Accepted: 13 June 2012 / Published online: 4 July 2012 Ó Her Majesty the Queen in Right of Canada 2012
Abstract Mercury (Hg) has been increasing in some marine birds in the Canadian Arctic over the past several decades. To evaluate the potential reproductive impact of Hg exposure, eggs of two species of arctic-breeding seabirds, the thick-billed murre and arctic tern, were dosed with graded concentrations of methylmercury (MeHg) and artificially incubated in the laboratory to determine species differences in sensitivity. Based on the dose–response curves, the median lethal concentrations (LC50) for thickbilled murre and arctic tern embryos were 0.48 and 0.95 lg g-1 Hg on a wet-weight (ww) basis, respectively. Compared with published LC50 values for other avian species, the murres and terns had a medium sensitivity to MeHg exposure. LC50 values were also calculated for the actual Hg concentration measured in the embryos, that is, the maternally-deposited Hg plus the injected MeHg dose. This increased the LC50 values to 0.56 lg g-1 Hg ww in the thick-billed murre and to 1.10 lg g-1 Hg ww in the arctic tern. Although muscarinic acetylcholine and N-methyl-D-aspartic acid glutamate receptor levels have been correlated with increasing Hg concentrations in brains of adult birds, no significant associations were found in brain tissue of the murre or tern embryos. The incidence of gross external anatomical deformities was 4.3 % in the murre embryos and 3.6 % in the tern embryos. However, given that the eggs were taken from wild populations, it is B. M. Braune (&) A. M. Scheuhammer D. Crump S. Jones D. Bond Environment Canada, National Wildlife Research Centre, Carleton University, Raven Road, Ottawa, ON K1A 0H3, Canada e-mail:
[email protected] E. Porter 473F Moodie Drive, Ottawa, ON K2H 8T7, Canada
unlikely that the deformities observed in this study were due to MeHg exposure alone. Keywords Seabird
Methylmercury Toxicity Egg injection
Introduction Mercury (Hg) has been increasing in marine birds and mammals in some regions of the Canadian Arctic over the past several decades (Braune 2007; Braune et al. 2005; Rige´t et al. 2011). Methylmercury (MeHg) biomagnifies through the food chain (Atwell et al. 1998; Campbell et al. 2005) making those species feeding at high trophic positions more vulnerable to dietary Hg exposure. Marine bird species which feed at high trophic levels in the Canadian Arctic include the thick-billed murre (Uria lomvia) (Campbell et al. 2005) and the arctic tern (Sterna paradisaea) (Akearok et al. 2010). Hg concentrations have been steadily increasing in eggs of thick-billed murres from the Canadian high Arctic (Braune 2007). Arctic terns, which also breed in the Canadian Arctic, are experiencing population declines (Gilchrist and Robertson 1999; Hatch 2002). Although arctic terns share a comparable trophic position with other seabird species, their egg Hg concentrations are higher than those measured in eggs of other high Arctic marine birds (Akearok et al. 2010). The most bioavailable and toxic form of Hg is MeHg and dietary MeHg is efficiently transferred to avian eggs in a dose-dependent manner, making reproduction one of the most sensitive endpoints of Hg toxicity in birds (Wolfe et al. 1998). Nearly 100 % of the total Hg (THg) transferred to eggs is in the form of MeHg with the majority (about 85–95 %) being deposited into the albumen (Wiener
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et al. 2003). Therefore, the Hg concentration in bird eggs is a good indicator of risk for Hg-associated reproductive impairment, such as reduced hatchability and increased rates of embryonic deformity and mortality (Thompson 1996; Wolfe et al. 1998). Studies have shown that neurochemical parameters, such as muscarinic acetylcholine (mACh) and N-methylD-aspartic acid (NMDA) glutamate receptor levels, are also affected by low-level dietary exposure to MeHg in adult birds and mammals (Basu et al. 2006, 2007; Scheuhammer et al. 2008). These two neurotransmitter receptors (mACh, NMDA) play important roles in the regulation and control of reproductive hormones and, therefore, their perturbation may signify early impacts to reproductive potential. Although several recent studies have reported neurochemical changes in association with increasing Hg concentrations in brains of adult birds (e.g. Scheuhammer et al. 2008), the measurement of these neurochemical endpoints in embryonic birds and the possible effects of in ovo exposure to MeHg on developing avian neurochemical pathways, have not been previously studied. Embryotoxicity thresholds for Hg, determined for a limited number of species, primarily from captive breeding studies, are often applied generically to all avian species. However, based on a study of 26 avian species, Heinz et al. (2009) recently showed that there are significant interspecies differences in sensitivity to the embryotoxic effects of MeHg. Given that the embryo is the life stage at which birds are most sensitive to MeHg, our objectives were: (i) to determine the relative sensitivity of two arctic marine bird species, the thick-billed murre and arctic tern, to MeHg exposure using an egg injection protocol similar to the one developed by Heinz et al. (2006), and (ii) to determine the effects of MeHg exposure on the neurosignaling pathways of the developing avian brain using changes in neurochemical parameters (e.g. neuroreceptor concentrations) in response to in ovo MeHg exposure.
B. M. Braune et al.
first few days of incubation is a useful attribute for an experimental protocol that requires temporary storage of eggs in the field after collection before transportation to the laboratory. Storage of unincubated eggs for several days at cool temperatures prior to placement in an incubator is an accepted practice in the poultry industry (Fasenko 2007). The natural incubation period for arctic tern eggs ranges from 21 to 23 days (Hatch 2002). Thick-billed murre eggs from Coats Island have a natural incubation period that ranges from 31 to 37 days with a mean of 33 days (Gaston and Hipfner 2000). Sample collection The study spanned 2 years with one species tested per year. In June 2009, 120 fresh, unincubated thick-billed murre eggs were collected from a colony on Coats Island (62°300 N, 83°000 W) in northern Hudson Bay, Canada. Eggs were collected within 18 h of being laid and stored up to 4 days in foam-lined coolers in the field at 8–15 °C until they could be transported to the National Wildlife Research Centre (NWRC) laboratories in Ottawa. In early July 2010, 125 fresh, unincubated arctic tern eggs were collected from a colony of 300–350 arctic tern nests on Nasaruvaalik Island (75°490 N, 96°180 W) just north of Cornwallis Island in the Canadian high Arctic. Nests were marked and visited daily. Only the first-laid egg from each clutch/nest was collected. Eggs were collected within 24 h of being laid and were stored up to 7 days in foam-lined coolers in the field at 6–16 °C until they could be transported to the NWRC laboratories in Ottawa. Eggs of both species were gently wiped clean with water in the field immediately after collection. All work was carried out under the appropriate research, collection and export permits, and all procedures involving the handling of animals were conducted according to protocols approved by the Animal Care Committee at the NWRC.
Methods
Experimental design
Species information
At the NWRC laboratories, eggs were randomly assigned to artificial incubators (Brinsea Contaq Z6). However, due to incubator difficulties in 2010, the arctic tern eggs were transferred to a Petersime incubator on incubation day 2. Our experimental protocol was based on that of Heinz et al. (2006) with a few minor variations (e.g. solvent, solvent volume, sealant). Heinz et al. (2006, 2009) chose corn oil as their solvent whereas we used safflower oil. Properties of various plant-derived oils are very similar with respect to their function as delivery vehicles. For example, Heinz et al. (2006) found no difference between the use of corn oil or soybean oil as a solvent. In their tests of solvent
The thick-billed murre lays a single egg which is incubated continuously from the time it is laid (Gaston and Hipfner 2000) whereas the arctic tern lays one to three eggs per clutch and incubation is initially irregular until the full clutch has been laid (Hatch 2002). Even when regularly deserted at night, most arctic tern eggs will hatch (Hatch 2002) and thick-billed murre eggs removed 5 days after laying and kept at ambient air temperature for 48 h exhibited normal hatching success (Gaston and Powell 1989). This apparent insensitivity to temperature fluctuations during the
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volumes, Heinz et al. (2006) found no statistically significant difference in embryo survival between the use of 0.5 and 1 lL but chose 1 lL for their final protocol. We chose 0.5 lL as our solvent volume in order to minimize injection volume. We also used AirPoreTM Tape instead of a hot glue gun to seal the hole in the egg because it provides a more natural barrier, allowing air exchange but not bacterial transfer, and it did not adhere to the incubator’s contact membrane as did the glue. Given that the protocol determined that eggs should be injected when the embryos achieved a similar stage of development as a 3-day-old chicken embryo, it was necessary to pro-rate the incubation periods of the tern and murre eggs with the 21-day incubation period of domestic poultry. Therefore, on incubation day 5 for the murres and incubation day 4 for the terns, eggs were weighed, candled, carefully examined for hairline cracks or other physical aberrations, and randomly assigned to eight dose groups consisting of 12 eggs each plus a vehicle-control group of 12 eggs for each species. Uninjected control groups of 12 murre eggs and 17 tern eggs were also included. Unsuitable eggs were removed from the experiment but viability was difficult to ascertain by candling due to the shell pigmentation in both species and, therefore, infertile eggs were not removed from the sample groups until the end of the experiment. Eggs were dosed with environmentally-relevant graded concentrations of methylmercury (II) chloride (MeHg chloride, PESTANALÒ, analytical standard from Sigma-Aldrich) dissolved in safflower oil as follows: control group (not injected but same handling as other eggs), vehicle-control group [injected with safflower oil (vehicle)], and dose groups of 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 lg g-1 MeHg on a wet-weight (ww) basis in the egg. Heinz et al. (2006) demonstrated that the MeHg dose introduced into the air cell of an egg passes through the inner shell membrane and into the albumen, and they concluded that air cell injections provided the best dose–response results. Following the same protocol, the vehicle control and dosing solutions of MeHg were injected into the air cell in our study. In order to deliver the correct dose, the mass of the egg contents was calculated by multiplying the mean total egg weight for the eggs sampled by 0.891 for the murre eggs and 0.944 for the tern eggs to account for the average eggshell mass as determined by previously collected data for the respective species at the colonies sampled. The location of the air cell was marked during candling. The eggshell in the area of the air cell was swabbed with 70 % reagent grade alcohol and a hole was drilled in the shell at the air cell using a hand-held Dremel tool with a 3/32-inch diameter diamond Dremel bit. Each egg was then injected with the prescribed dose of MeHg chloride dissolved in 0.5 lL of safflower oil per gram of egg contents delivered using a Biohit E-pet
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5–120 lL electronic pipette. The hole was subsequently sealed with AirPoreTM Tape (Qiagen, Mississauga, ON). After dosing, eggs were allowed to sit upright for *30 min prior to being returned to the incubators. The murre eggs were incubated using Brinsea Contaq Z6 incubators (Sandford, England) held at 39.5 °C and 60 ± 5 % relative humidity with a turning frequency of 1–1.75 h and a cooling interval of 10 min every 24 h. Approximately 1 week prior to hatch, the incubators were changed from contact to forced draft mode (contact membranes removed) and the temperature reduced to 37.5 °C. About 3 days prior to peak hatch, the eggs were no longer turned. The tern eggs were incubated using a Petersime Model XI incubator (Zulte, Belgium) held at 37.5 °C and 60 ± 5 % relative humidity with a turning frequency of 2 h. Approximately 3 days prior to peak hatch, the eggs were no longer turned (tilting of trays stopped). For both species, egg viability was assessed periodically during incubation using a digital egg monitor (Avitronics Buddy Mk2; Truro, England). Survival to 90 % of development was used as the endpoint measurement for embryo survival. Embryos that survived to pipping (starring of eggshell indicating beginning of hatch) were euthanized by decapitation. The stage of development of all embryos was classified according to Hamburger and Hamilton (1951) and all embryos at stage 35 or higher (at least 76 % development) were examined for any gross anatomical anomalies. Embryos (including the yolk sac, fecal sac and chorioallantoic membrane) and remaining egg contents (if any) were then weighed. The brains were removed from 68 thick-billed murre embryos and 56 arctic tern embryos, weighed and stored in liquid nitrogen for analysis of NMDA and mACh receptor density as well as THg. The embryo and any egg contents were then homogenized and frozen at -40 °C prior to being analyzed for THg. Although it was expected that THg would be almost 100 % MeHg in the embryos as it is in eggs, three samples from each dose group were analyzed for MeHg in order to confirm the ratio of MeHg:THg. Mercury analyses Embryos and egg contents were homogenized and freezedried for THg analysis, whereas brain samples, due to the small sample mass, were freeze-dried and then pulverized with a stainless steel spatula. All 68 murre embryo brain samples were analyzed for THg but only 55 of the 56 tern embryos yielded enough brain sample for confident THg analysis. THg in embryo/egg content samples was analyzed using an advanced Hg analyzer (AMA-254; Altec Ltd., Canalytical, Burlington, ON) according to NWRC Method No. MET-CHEM-AA-03H [see also EPA Method 7473; Salvato and Pirola (1996)] and the brain samples were
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analyzed using a direct Hg analyzer (DMA-80; Milestone, Sorisole, Italy). Both methods employ thermal decomposition, catalytic reduction, gold–mercury amalgamation, desorption and atomic absorption spectroscopy in an oxygen-rich atmosphere. Organic Hg (primarily present as MeHg in biological tissues) was quantified according to NWRC Method MET-CHEM-AA-04E by extraction of organomercurials into toluene followed by back-extraction into sodium thiosulphate and measurement of THg in the final extract as described in Scheuhammer et al. (1998). THg in the final extract was analyzed using the AMA-254. Analytical accuracy for THg and organic Hg measured using the AMA-254 was determined by analyzing blanks with each sample set, as well as certified reference materials (CRMs): oyster tissue 1566b from the National Institute of Standards and Technology (NIST), TORT-2 (lobster hepatopancreas) and DOLT-3 (dogfish liver) from the National Research Council of Canada (NRCC) for both THg and organic Hg and, for THg only, BCR-463 (tuna fish) and ERM CE278 (mussel tissue) from the Institute for Reference Materials and Measurements (IRMM). CRM recoveries were within the acceptable limits averaging 103.4 ± 3.4 % for THg and 97.3 ± 11.7 % for organic Hg in the murre samples, and 106.1 ± 8.6 % for THg and 103.5 ± 16.6 % for organic Hg in the tern samples. Analytical precision was checked by analyzing replicate samples for both THg and organic Hg. The relative standard deviation (% RSD) among replicate samples was 3.1 ± 3.4 % (n = 19) for THg and 2.0 ± 0.6 % (n = 2) for organic Hg in the murre samples, and 3.7 ± 2.9 % (n = 22) for THg and 8.9 % (n = 1) for organic Hg in the tern samples. The practical detection limit for THg measured by AMA-254 was 0.006 lg g-1 dry weight (dw) and for organic Hg, it was 0.0375 lg g-1 dw. Similar quality assurance procedures were employed for the brain samples analyzed for THg using the DMA-80. CRM recoveries for THg were 106.7 ± 9.3 % for the murre samples, and 95.6 ± 8.2 % for the tern samples. The % RSD among replicate samples was 3.2 ± 3.9 % (n = 14) for the murre samples, and 3.0 ± 1.9 % (n = 10) for the tern samples. The practical detection limit for THg was 0.0024 lg g-1 dw for the murre samples, and 0.010 lg g-1 dw for the tern samples. THg in the safflower oil used as the solvent/vehicle for the dose solutions was also measured using the DMA-80 and found to be well below the practical detection limits. Although the MeHg chloride (PESTANALÒ, SigmaAldrich) used to prepare the dose solutions was certified as 99.2 % pure MeHg, two of the dose solutions (1.6 and 6.4 lg g-1 MeHg) were analyzed by Flett Research Limited (Winnipeg, MB) to confirm the composition and were found to contain 100.3 and 99.8 % MeHg, respectively.
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Neurotransmitter receptor assays Only brains of embryos which had reached at least 90 % of development were included for neurotransmitter receptor assays. Concentrations of NMDA glutamate receptors and mACh receptors were assayed in embryonic brain tissue homogenates using methods described previously for adult birds and mammals (Basu et al. 2006, 2007; Scheuhammer et al. 2008). In brief, tritiated ligands with high specific affinities for these receptors were incubated with tissue homogenates in the presence and absence of an unlabelled ligand of similar affinity. For mACh receptor assays, a Na/ K buffer was used (50 mm NaH2PO4, 5 mm KCl, 120 mm NaCl, pH 7.4) and for NMDA receptors, a Tris buffer was employed (50 mm Tris, 100 lm glycine, 100 lm L-glutamic acid, pH 7.4). 30 lg of prepared membrane was re-suspended in the appropriate buffer and added to microplate wells containing a 1.0 lm GF/B glass filter (Millipore, Boston, MA, USA). For mACh receptor binding, samples were incubated with 1 nm [3H]-QNB (42 Ci mmol-1; NEN/PerkinElmer, Boston, MA, USA) for 60 min. For NMDA receptor binding, samples were incubated with 5 nm [3H]-MK-801 (22 Ci mmol-1; NEN/ PerkinElmer) for 120 min. All assays were carried out on a shaking platform at room temperature. Binding reactions were terminated by vacuum filtration. The filters were rinsed three times with buffer and then allowed to soak for 48 h in 25 lL of OptiPhase Supermix Cocktail (PerkinElmer). Radioactivity retained by the filter was quantified by liquid scintillation counting in a microplate detector (Wallac Microbeta, PerkinElmer) having a counting efficiency of approximately 35 %. Specific binding to both receptors was defined as the difference in radioligand bound in the presence and absence of 100 lM unlabelled atropine (for mACh receptors) and MK-801 (for NMDA receptors). Binding was reported as fmol of radioisotope bound per mg of membrane protein (fmol mg-1). All samples were assayed in quadruplicate for total and non-specific binding. Intra- and inter-plate variation in binding was less than 10 % as determined by use of internal, pooled controls. Data handling Since brains were removed for the neuroreceptor assays, the THg concentration in the embryo was calculated based on the THg concentration and weight measurements for the brain and carcass for each embryo which generated the THg content for the carcass and brain. The summed THg content for carcass and brain was then divided by the total weight (sum of carcass and brain) to generate the THg concentration for the whole embryo. Statistica version 7 (StatSoft Inc., Tulsa, OK) was used to carry out analysis of variance (ANOVA) and linear correlation analyses. ANOVA was
Toxicity of MeHg injected into eggs
a
100
Thick-billed Murre
12
Percent Survival
80 12
11
12
60
11
11
40
LC50 = 0.48 µg g-1 (95% CI: 0.26-0.99)
20
11
8 7
0
0
0.05
0.1
0.2
0.4
0.8
1.6
3.2
6.4
-1
Mercury Injected (µg g ww)
b 100 Thick-billed Murre
12
80 12
Percent Survival
used to test for differences in percent organic Hg to THg concentrations in embryos among dose groups followed by Tukey’s test for mean separation to distinguish statistically significant differences. Levene’s test was used to test for homogeneity of variances. Linear correlation analyses (Pearson r) were used to test for associations between THg and neurochemical variables in the brain, and between whole embryo THg and brain THg. The critical level of significance for all statistical analyses was set at a = 0.05. Following the statistical methods utilized by Heinz et al. (2009), the probit procedure in SAS version 9.2 (SAS Institute, Cary, NC) was used to calculate the median lethal concentration (LC50) and 95 % confidence interval (CI) for each species. Survival data were corrected for control mortality using Abbott’s formula (Abbott 1925) as done by Heinz et al. (2009). The same probit procedure was used to calculate the LC50 and 95 % CI for each species based on the measured THg concentrations, that is, maternallydeposited THg plus the injected MeHg dose. For this analysis, samples were grouped according to measured concentrations which paralleled the sample distribution by dose group for the murres, but required post hoc re-distribution of some tern samples, particularly at the higher concentrations. This changed some of the group sample sizes for the arctic terns. As well, measured THg concentrations were not corrected for control mortality since the control group had a concentration value from the maternally-deposited THg and could no longer be considered as a control.
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11
12
11
60
11
40
20
LC50 = 0.56 µg g (95% CI: 0.38-0.85) -1
11
8 7
0
0.25
0.22
0.31
0.40
0.49
0.86
1.59
3.29
6.55
-1
Mercury Measured (µg g ww)
Results Of the 108 thick-billed murre eggs assigned to the eight dose groups plus the vehicle-control, six eggs were removed from the experiment because of bacterial infections and seven eggs were deemed infertile. Of the remaining 95 eggs, including those dosed with MeHg, 46 (48 %) reached at least 90 % development with variable percent survival among dose groups (see Fig. 1a). Of 70 murre embryos examined for gross anatomical deformities, three (4.3 %) exhibited deformities (Table 1). Two embryos (from dose groups 0.05 and 0.8 lg g-1) exhibited hydrocephaly (swelling of the head due to an accumulation of cerebrospinal fluid in the brain), and a third embryo (from dose group 0.4 lg g-1) had its intestines exposed through an opening in the stomach (gastroschisis). Of the 108 arctic tern eggs assigned to the eight dose groups plus the vehicle-control, 34 eggs were removed from the experiment. One egg had a hairline crack in the shell leaking contents and 33 eggs were deemed infertile. Of the remaining 74 eggs, including those dosed with MeHg, 46 (62 %) reached at least 90 % development with
Fig. 1 Survival of thick-billed murre embryos through 90 % of development when eggs were injected with MeHg. a Percent survival plotted by dose group. b Percent survival plotted by groups based on measured Hg (injected MeHg ? maternally-deposited Hg). Sample sizes are given above each point on the graph. The LC50 and 95 % CI are also shown
variable percent survival among dose groups (see Fig. 2a). Of 56 tern embryos examined for gross anatomical deformities, two (3.6 %) exhibited one or more deformities (Table 1). One embryo (from dose group 0.05 lg g-1) exhibited hydrocephaly, had no eyes and an abnormally short upper bill, and another embryo (from dose group 0.2 lg g-1) was missing its lower bill and right eye. The calculated LC50 for the murre embryos was 0.48 lg g-1 ww based on MeHg injected into the eggs uncorrected for maternally-deposited THg (Fig. 1a), and for the tern embryos, the LC50 was 0.95 lg g-1 ww (Fig. 2a). When calculations were based on the actual Hg measured in the embryos, that is, maternally-deposited THg plus the injected MeHg dose, the LC50 values increased to 0.56 lg g-1 ww in the murres (Fig. 1b) and 1.10 lg g-1 ww in the terns (Fig. 2b). Organic Hg in the murre egg contents/ embryos averaged 98.3 ± 1.9 % of THg. Analysis of
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Table 1 Deformities observed in controls and MeHg dosed embryos Dose group
n examined
n with deformities
Hydrocephaly
Gastroschisis
Eyes
Bill
1
1
1
1
Thick-billed murre Control without injected safflower oil Control with injected safflower oil
9
0
12
0
0.05 lg g-1 Hg
9
1
0.1 lg g-1 Hg
10
0
0.2 lg g-1 Hg
9
0
0.4 lg g Hg 0.8 lg g-1 Hg
9 8
1 1
1.6 lg g-1 Hg
3
0
3.2 lg g-1 Hg
1
0
6.4 lg g-1 Hg
0
0
-1
1
1 1
Arctic tern Control without injected safflower oil Control with injected safflower oil
10
0
8
0
0.05 lg g-1 Hg
11
1
0.1 lg g-1 Hg
7
0
0.2 lg g-1 Hg
8
1
-1
0.4 lg g
Hg
4
0
0.8 lg g-1 Hg
4
0
1.6 lg g-1 Hg
4
0
3.2 lg g-1 Hg
0
0
6.4 lg g-1 Hg
0
0
1
Only embryos which were at least 76 % developed according to the developmental stages as defined by Hamburger and Hamilton (1951) were examined
variance indicated a significant difference among dose groups (ANOVA: n = 27, F(8,18) = 3.62, p = 0.01) but Tukey’s post hoc test for means found a significant difference only between the 0.05 and 0.8 lg g-1 dose groups suggesting no meaningful pattern to the difference. In the tern samples, organic Hg averaged 94.5 ± 1.3 % of THg with no significant difference among dose groups (ANOVA: n = 27, F(8,18) = 0.701, p = 0.69). Of the 68 brain samples harvested from the thick-billed murre embryos, only 54 provided sufficient tissue for measurements of THg as well as NMDA and mACh receptor density. Likewise, only 55 of the 56 arctic tern brains sampled were analyzed for THg and NMDA, and only 54 had sufficient tissue for measurement of mACh binding activity. Measured brain THg concentrations ranged from 0.34 to 10.8 lg g-1 dw (0.047–1.07 lg g-1 ww) in the murre embryos and 0.63–14.8 lg g-1 dw (0.062–1.66 lg g-1 ww) in the tern embryos. No significant correlations between THg concentrations and densities of either the NMDA or mACh neuroreceptor were found in either the murre embryos (NMDA: r2 = 0.027, p = 0.24; mACh: r2 = 0.001, p = 0.81) or the tern embryos (NMDA: r2 = 0.026, p = 0.24; mACh: r2 = 0.001, p = 0.78).
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Discussion Toxicity of MeHg to avian embryos The LC50 values calculated for the thick-billed murre and arctic tern embryos were higher when injected MeHg plus maternally-deposited THg were taken into account than when only injected MeHg was considered. However, the difference in the measured THg across dose groups is not as consistent as one might expect if one were to simply sum the average THg found in the control eggs with the injected MeHg dose (see Figs. 1, 2). The discrepancies in the MeHg dose administered and THg values measured may be due to a variety of factors including incomplete delivery of the dose to the egg, variation in the accuracy of dose solution preparation, and variability in the maternally-deposited THg. For example, variability in maternally-deposited THg likely accounts for the slightly higher first measured THg value (0.25 lg g-1) on the X-axis in Fig. 1b compared with the second value (0.22 lg g-1). Therefore, it is extremely important to include actual measured concentration values with any egg dosing studies in order to verify the results.
Toxicity of MeHg injected into eggs
a
11
100
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7
Arctic Tern
9 10
Percent Survival
80 6
60
8 9
40
LC50 = 0.95 µg g (95% CI: 0.59-1.58) -1
20
6
8
3.2
6.4
0
0
0.05
0.1
0.2
0.4
0.8
1.6
Mercury Injected (µg g-1 ww)
b
11
100
7
Arctic Tern
9 10
Percent Survival
80
9
60 8
40
7
LC50 = 1.10 µg g (95% CI: 0.86-1.61) -1
20
7
6
4.06
7.07
0
0.41
0.42
0.47
0.51
0.72
1.13
1.99 -1
Mercury Measured (µg g ww)
Fig. 2 Survival of arctic tern embryos through 90 % of development when eggs were injected with MeHg. a Percent survival plotted by dose group. b Percent survival plotted by groups based on measured Hg (injected MeHg ? maternally-deposited Hg). Sample sizes are given above each point on the graph. The LC50 and 95 % CI are also shown
Using estimated median LC50 for 26 tested species dosed with MeHg, Heinz et al. (2009) demonstrated that the sensitivity of avian embryos to MeHg can vary dramatically among species with median LC50 values ranging from 1 lg g-1 ww or higher in eggs of the low sensitivity group (e.g. Canada goose Branta canadensis, mallard Anas platyrhynchos, hooded merganser Lophodytes cucullatus, lesser scaup Aythya affinis, laughing gull Larus atricilla) to \0.25 lg g-1 ww in eggs of those species exhibiting high sensitivity (e.g. American kestrel Falco sparverius, osprey Pandion haliaetus, white ibis Eudocimus albus, snowy egret Egretta thula). Species, such as the common tern (Sterna hirundo), royal tern (S. maxima), and Caspian tern (S. caspia) as well as the herring gull (Larus argentatus) were categorized as having medium sensitivity to MeHg based on LC50 values ranging between 0.25 and 1 lg g-1 ww of Hg. Since Heinz et al. (2009) carried out their
studies using eggs collected from areas believed to be free of Hg contamination, they did not account for maternallydeposited MeHg in their dose–response calculations, and their dose–response graphs show only the injected MeHg concentrations. However, previous studies have shown that thick-billed murre and common tern eggs at the colonies sampled in this study are not free from Hg contamination (see Akearok et al. 2010; Braune 2007) and therefore, dose–response curves were calculated in two ways: (i) following the same methods as Heinz et al. (2009) for comparative purposes, and (ii) using actual measured THg values which included both the injected MeHg dose plus the maternally-deposited THg. Applying the relative sensitivity categories suggested by Heinz et al. (2009), both the thick-billed murres (LC50 = 0.48 lg g-1 ww) and arctic terns (LC50 = 0.95 lg g-1 ww) were categorized as having medium sensitivity to MeHg based on the calculations using only the injected MeHg dose groups. This placed the arctic tern in the same sensitivity category as the three other tern species (common, royal, Caspian) tested by Heinz et al. (2009). When measured THg values were used, that is, maternally-deposited THg plus the injected MeHg dose, estimated LC50 values for both species increased (Figs. 1, 2) and the sensitivity category for the arctic tern changed from medium to low sensitivity. However, the 95 % CI for the LC50 estimates for both species (see Figs. 1, 2) suggest some latitude in these values. Kenow et al. (2011) estimated LC50 values for common loon (Gavia immer) embryos based on measured THg values (LC50 = 1.78 lg g-1 ww) and suggested that common loons also have a low sensitivity to injected MeHg compared with those species tested by Heinz et al. (2009). To put the LC50 estimates for the murre and terns into a realistic environmental context, the average THg concentration in thick-billed murre eggs collected from the Coats Island colony in 2009 was 0.16 lg g-1 ww, and for a high Arctic murre colony at Prince Leopold Island on Lancaster Sound, it was 0.40 lg g-1 ww (Braune, unpublished data). Concentrations of THg measured in first-laid (early) arctic tern eggs collected in 2008 from Nasaruvaalik Island averaged 0.49 lg g-1 ww (Akearok et al. 2010). Although the average colony THg concentrations do not exceed the estimated LC50 values for either species, they are within the same order of magnitude. Thompson (1996) noted that, although there is a degree of agreement between dosing experiments in determining threshold levels of Hg toxicity in eggs, similar Hg concentrations measured in eggs of wild populations do not always show effects upon hatching. Further, Heinz et al. (2009) cautioned that MeHg injected into the egg may be two to four times more embryotoxic than maternally-deposited MeHg although the sensitivity relative to other species should be the same whether the MeHg was injected or deposited naturally by
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Neurochemical receptors Although neurochemical changes in association with increasing Hg concentrations have been reported in brains of adult birds (e.g. Scheuhammer et al. 2008), the effects of in ovo exposure to MeHg on developing avian neurochemical pathways have not previously been investigated. A study using artificially-incubated herring gull eggs from the Great Lakes confirmed that the muscarinic (mACh) and NMDA receptors could be detected and confidently measured in brain tissue dissected from pipping herring gull embryos (Scheuhammer, unpublished data). Both NMDA and mACh receptors were detected with no tissue-specific issues confirming that changes in these two neurochemical receptors could potentially be used as biomarkers of MeHg exposure in the developing avian brain. However, brain tissue from thick-billed murre and arctic tern embryos in our study showed no significant correlations between THg concentrations and densities of either the NMDA or mACh neuroreceptors. Reasons for this apparent lack of response of embryonic neuroreceptors to Hg exposure in ovo are currently unknown. THg concentrations in the embryonic brain were significantly correlated with increasing THg concentrations in the whole embryo in both species
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a
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the mother. Taking these factors into account, our dose– response data have most likely overestimated embryonic sensitivity of thick-billed murres and arctic terns to MeHg, resulting in more conservative (lower) LC50 estimates. It has been shown that reproductive success in birds can decrease by 35–50 % due to dietary MeHg exposure insufficient to cause obvious signs of toxicity in adults (Wolfe et al. 1998). For example, documented effects of MeHg exposure not addressed in our study include aberrant reproductive behaviour and reduced clutch sizes (Thompson 1996; Wolfe et al. 1998). Based on a recent evaluation of published field and laboratory studies for nonmarine birds, the range of egg Hg concentrations associated with no adverse effects is 0.7–1.6 lg g-1 ww while the range of egg Hg concentrations associated with adverse effects is 0.8–5.1 lg g-1 ww (Shore et al. 2011). Shore et al. (2011) also used the data generated by Heinz et al. (2009) for species sensitivity to the embryotoxic effects of Hg to propose an indicative Hg value of 0.6 lg g-1 ww in eggs as being protective for most (95 %) species. The estimated LC50 values for the thick-billed murre border on the proposed indicative value whereas those of the arctic tern exceed it, although the average colony THg concentrations for murre and tern eggs reported from the Canadian Arctic currently fall below the range of concentrations associated with adverse effects. However, there is evidence that Hg concentrations are increasing in some biota in the Canadian Arctic (Braune 2007; Rige´t et al. 2011).
B. M. Braune et al.
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Fig. 3 Relationship of THg concentrations (lg g-1 ww) in embryo versus brain of a thick-billed murres (n = 55, r2 = 0.93, p \ 0.0001) and b arctic terns (n = 55, r2 = 0.92, p \ 0.0001), which are [90 % developed
(Fig. 3). However, it is possible that the brain THg levels were too low to be associated with significant neurochemical change in these species as has been postulated for adults of other avian species which also showed no significant correlations (Hamilton et al. 2011; Rutkiewicz et al. 2010). Alternatively, neuroreceptors in the developing avian brain may respond differently to chemical stressors such as MeHg compared with the adult brain. Anatomical deformities In any animal population, there will be an incidence of embryos that die or are unable to hatch due to deformities (Butcher and Nilipour 2002). In commercial domestic poultry hatching operations, the occurrence of deformities is low, ranging from 0.22 to 0.30 % of the total hatch (Butcher and Nilipour 2002). Expected rates of deformities are also low in uncontaminated populations of wild birds, usually less than 1 % (Ohlendorf et al. 1986). Laboratory studies have shown that exposure to MeHg can induce teratogenic
Toxicity of MeHg injected into eggs
effects in developing embryos (Heinz and Hoffman 1998, 2003; Heinz et al. 2011). However, the extent to which these teratogenic effects occur in wild birds exposed to MeHg is unknown (Heinz et al. 2011). In a study of 25 species dosed with MeHg, Heinz et al. (2011) examined 2,292 embryos and found a total of 8.2 % individuals with one or more deformities across all dose groups. In our study, 4.3 % of thick-billed murre embryos and 3.6 % of arctic tern embryos examined exhibited some form of anatomical deformity. In a field study examining malposition as a potential mechanism for Hg-induced hatching failure, Herring et al. (2010) also found some type of deformity in 2 % of embryos of American avocets (Recurvirostra americana), black-necked stilts (Himantopus mexicanus) and Forster’s terns (Sterna forsteri) examined (total n = 470). In our study of murre and tern embryos, abnormalities included hydrocephaly, missing eyes and deformed/missing bills. These types of major external deformities have also been observed in other MeHg dosing studies (Birge and Roberts 1976; Heinz and Hoffman 1998, 2003; Heinz et al. 2011; Hoffman and Moore 1979). However, given that wild birds, such as terns and murres, are exposed to a wide variety of environmental contaminants, and the occurrence of the observed deformities occurred across a number of dose groups including the lower dose groups, it is unlikely that the deformities observed in this study were due to exposure to MeHg alone. Acknowledgments Thanks to A. Gaston and M. Mallory of Environment Canada for their help in coordinating the field work, and S. Kennedy of Environment Canada for making his staff and laboratory available for the egg injections and subsequent egg incubation. Thanks to K. Elliott (University of Manitoba), K. Woo (Environment Canada) and J. Nakoolak (Coral Harbour) for their help in collecting the murre eggs in 2009, and K. and J. Boadway (University of New Brunswick), C. Vallerand (Environment Canada), V. Amarualik (Resolute Bay) and T. Noah (Grise Fiord) for their help in collecting the tern eggs in 2010. We also thank F. St-Louis (Environment Canada), J. Rutkiewicz (University of Michigan) and G. Braune for their help with the egg incubation and embryo dissections, and E. Neugebauer for carrying out the Hg analyses. Finally, we would like to extend our gratitude to G. Heinz of the U.S. Geological Survey for extensive discussions regarding the protocol used in this study, and to J. Klimstra of the U.S. Fish and Wildlife Service for his statistical advice. N. Burgess of Environment Canada also provided valuable insights regarding the study protocol and data analysis. Funding was provided by the Northern Contaminants Program of Aboriginal Affairs and Northern Development Canada. Conflict of interest of interest.
The authors declare that they have no conflict
References Abbott WS (1925) A method of computing the effectiveness of an insecticide. J Econ Entomol 18:265–267
2151 Akearok JA, Hebert CE, Braune BM, Mallory ML (2010) Inter- and intraclutch variation in egg mercury levels in marine bird species from the Canadian Arctic. Sci Total Environ 408:836–840 Atwell L, Hobson KA, Welch HE (1998) Biomagnification and bioaccumulation of mercury in an arctic marine food web: insights from stable nitrogen isotope analysis. Can J Fish Aquat Sci 55:1114–1121 Basu N, Scheuhammer AM, Rouvinen-Watt K, Grochowina N, Klenavic K, Evans RD, Chan HM (2006) Methylmercury impairs components of the cholinergic system in captive mink (Mustela vison). Toxicol Sci 91:202–209 Basu N, Scheuhammer AM, Rouvinen-Watt K, Grochowina N, Evans RD, O’Brien M, Chan HM (2007) Decreased N-methyl-Daspartic acid (NMDA) receptor levels are associated with mercury exposure in wild and captive mink. Neurotoxicology 28:593–597 Birge WJ, Roberts OW (1976) Toxicity of metals to chick embryos. Bull Environ Contam Toxicol 16:319–324 Braune BM (2007) Temporal trends of organochlorines and mercury in seabird eggs from the Canadian Arctic, 1975 to 2003. Environ Pollut 148:599–613 Braune BM, Outridge PM, Fisk AT, Muir DCG, Helm PA, Hobbs K, Hoekstra PF, Kuzyk ZA, Kwan M, Letcher RJ, Lockhart WL, Norstrom RJ, Stern GA, Stirling I (2005) Persistent organic pollutants and mercury in marine biota of the Canadian Arctic: an overview of spatial and temporal trends. Sci Total Environ 351–352:4–56 Butcher GD, Nilipour AH (2002) Chicken embryo malpositions and deformities. VM129. Gainesville: University of Florida Institute of Food and Agricultural Sciences. Accessed 20 January 2012: http://edis.ifas.ufl.edu/vm095 Campbell LM, Norstrom RJ, Hobson KA, Muir DCG, Backus S, Fisk AT (2005) Mercury and other trace elements in a pelagic Arctic marine food web (Northwater Polynya, Baffin Bay). Sci Total Environ 351–352:247–263 Fasenko GM (2007) Egg storage and the embryo. Poult Sci 86: 1020–1024 Gaston AJ, Hipfner JM (2000) Thick-billed murre (Uria lomvia). In: Poole A (ed) The birds of North America. Cornell Lab of Ornithology, Ithaca. Retrieved from the Birds of North America. http://bna.birds.cornell.edu/bna/species/497 Gaston AJ, Powell DW (1989) Natural incubation, egg neglect, and hatchability in the Ancient Murrelet. Auk 106:433–438 Gilchrist HG, Robertson GJ (1999) Population trends of gulls and Arctic terns nesting in the Belcher Islands, Nunavut. Arctic 52:325–331 Hamburger V, Hamilton HL (1951) A series of normal stages in the development of the chick embryo. J Morphol 88:49–92 Hamilton M, Scheuhammer A, Basu N (2011) Mercury, selenium and neurochemical biomarkers in different brain regions of migrating common loons from Lake Erie, Canada. Ecotoxicology 20:1677–1683 Hatch JJ (2002) Arctic tern (Sterna paradisaea). In: Poole A (ed) The birds of North America. Cornell Lab of Ornithology, Ithaca. Retrieved from the Birds of North America. http://bna.birds. cornell.edu/bna/species/707 Heinz GH, Hoffman DJ (1998) Methylmercury chloride and selenomethionine interactions on health and reproduction in mallards. Environ Toxicol Chem 17:139–145 Heinz GH, Hoffman DJ (2003) Embryotoxic thresholds of mercury: estimates from individual mallard eggs. Arch Environ Contam Toxicol 44:257–264 Heinz GH, Hoffman DJ, Kondrad SL, Erwin CA (2006) Factors affecting the toxicity of methylmercury injected into eggs. Arch Environ Contam Toxicol 50:264–279
123
2152 Heinz GH, Hoffman DJ, Klimstra JD, Stebbins KR, Kondrad SL, Erwin CA (2009) Species differences in the sensitivity of avian embryos to methylmercury. Arch Environ Contam Toxicol 56:129–138 Heinz GH, Hoffman DJ, Klimstra JD, Stebbins KR, Kondrad SL, Erwin CA (2011) Teratogenic effects of injected methylmercury on avian embryos. Environ Toxicol Chem 30:1593–1598 Herring G, Ackerman JT, Eagles-Smith CA (2010) Embryo malposition as a potential mechanism for mercury-induced hatching failure in bird eggs. Environ Toxicol Chem 29:1788–1794 Hoffman DJ, Moore JM (1979) Teratogenic effects of external egg applications of methyl mercury in the mallard, Anas platyrhynchos. Teratology 20:453–461 Kenow KP, Meyer MW, Rossman R, Gendron-Fitzpatrick A, Gray BR (2011) Effects of injected methylmercury on the hatching of common loon (Gavia immer) eggs. Ecotoxicology 20:1684–1693 Ohlendorf HM, Hoffman DJ, Saiki MK, Aldrich TW (1986) Embryonic mortality and abnormalities of aquatic birds: apparent impacts of selenium from irrigation drainwater. Sci Total Environ 52:49–63 Rige´t F, Braune B, Bignert A, Wilson S, Aars J, Andersen M, Asmund G, Aubail A, Basu N, Born E, Dam M, Dietz R, Evans M, Evans T, Gamberg M, Gantner N, Green N, Gunnlaugsdo´ttir ´ lafsdo´ttir K, H, Kannan K, Kirkegaard M, Letcher R, Muir D, O Olsen M, Renzoni A, Roach P, Skaare JU, Sonne C, Stern G, Wiig Ø (2011) Temporal trends of Hg in Arctic biota, an update. Sci Total Environ 409:3520–3526 Rutkiewicz J, Scheuhammer A, Crump D, Jagla M, Basu N (2010) Investigation of spatial trends and neurochemical impacts of
123
B. M. Braune et al. mercury in herring gulls across the Laurentian Great Lakes. Environ Pollut 158:2733–2737 Salvato N, Pirola C (1996) Analysis of mercury traces by means of solid sample atomic absorption spectrometry. Mikrochim Acta 123:63–71 Scheuhammer AM, Atchison CM, Wong AHK, Evers DC (1998) Mercury exposure in breeding common loons (Gavia immer) in central Ontario, Canada. Environ Toxicol Chem 17:191–196 Scheuhammer AM, Basu N, Burgess NM, Elliott JE, Campbell GD, Wayland M, Champoux L, Rodrigue J (2008) Relationships among mercury, selenium, and neurochemical parameters in common loons (Gavia immer) and bald eagles (Haliaeetus leucocephalus). Ecotoxicology 17:93–101 Shore RF, Pereira G, Walker LA, Thompson DR (2011) Mercury in nonmarine birds and mammals. In: Beyer WN, Meador J (eds) Environmental contaminants in biota: interpreting tissue concentrations, 2nd edn. CRC Press, Boca Raton, pp 603–618 Thompson DR (1996) Mercury in birds and terrestrial mammals. In: Beyer WN, Heinz GH, Redmon-Norwood AW (eds) Environmental contaminants in wildlife: interpreting tissue concentrations. SETAC Special Publication Series. Lewis Publishers, Boca Raton, pp 341–356 Wiener JG, Krabbenhoft DP, Heinz GH, Scheuhammer AM (2003) Ecotoxicology of mercury. In: Hoffman DJ, Rattner BA, Burton GA, Cairns J (eds) Handbook of ecotoxicology, 2nd edn. Lewis Publishers, Boca Raton, pp 409–463 Wolfe MF, Schwarzbach S, Sulaiman RA (1998) Effects of mercury on wildlife: a comprehensive review. Environ Toxicol Chem 17:146–160
