Long-term trends in contaminant exposure were examined in eggs of two aquatic-feeding species, the Herring Gull (Larus argentatus) and the Snapping Turtle (Chelydra serpentina), collected from locations within the Hamilton Harbour Area of Concern, an industrialized area historically associated with high concentrations of legacy persistent organic pollutants, metals and other compounds. Significant declines in concentrations of sum polychlorinated biphenyls, four organochlorine pesticides and 2,3,7,8-tetrachlorodibenzo-p-dioxin were evident in Herring Gull eggs collected from nesting colonies within the harbour (range in % declines = 86%–94%) and in Snapping Turtle eggs from two area of concern wetland/creek locations (range in % declines = 36%–89%) from the 1980s to 2012. Temporal trends in polychlorinated biphenyl congener profiles in eggs were examined by grouping polychlorinated biphenyl congeners according to structure-activity relationships based on their susceptibility to be metabolized by the cytochrome P450 system. Significant temporal changes in the percentages of non-metabolizable congeners (i.e. increase) and congeners metabolized by P450 1A enzymes (i.e. decrease) were evident in eggs of both species. It is not clear if these distinct temporal patterns are related to changes in metabolism following reductions in polychlorinated biphenyl burdens or changes in bioavailability of congeners in the environment. Large decreases in contaminant burdens in eggs of these two species at two different geographic scales within the area of concern (i.e. local scale for Turtles and regional scale for Gulls) are reflective of improved environmental conditions within the harbour and support their utility as top-predator wildlife bio-indicator species.
In 1987, Hamilton Harbour was identified as one of 43 Areas of Concern (AOCs) on the Great Lakes where local environmental degradation had severely impacted the area's ability to support aquatic life. Although numerous factors, including poor water quality, eutrophication, loss of habitat, and invasive species have contributed to the degradation of the harbour, the presence of high concentrations of persistent organic pollutants (POPs) was one of the reasons for the listing of Hamilton Harbour as an AOC (Hamilton Harbour Remedial Action Plan [RAP], 1992). Heavy industry, such as steel mills and associated industry, and municipal activities have contributed to loadings of polychlorinated biphenyls (PCBs) and metals into the harbour while upstream agricultural activity draining into tributaries were likely the main sources of organochlorine (OC) pesticides. The historical discharge of these compounds resulted in numerous exceedences of water quality objectives, sediment screening criteria and fish consumption guidelines in the 1970s and 1980s (Hamilton Harbour RAP, 1992).
Hamilton Harbour has undergone, and is continuing to undergo, remediation activities to limit the availability of POPs to biota. Efforts to remediate Hamilton Harbour contaminant sources include improvements to sewage treatment plants to reduce loadings of organics and capping of sediment severely contaminated with heavy metals and PAHs at Randle Reef (Hamilton Harbour RAP, 2002; Graham et al., 2013). Concentrations of PCBs were notably elevated in surficial sediment in Windermere Arm (Milani and Grapentine, 2002). PCBs in sediment are naturally degraded slowly over time by microbial processes and profiles can change as certain PCB congeners are subject to reductive dechlorination which has been shown to occur primarily at the meta and para positions (Pakdeesusuk et al., 2003; Xu et al., 2012). Differences in the extent of dechlorination influence the bioavailability of these congeners to foraging benthic invertebrates, fish and wildlife. PCB congener structure, biological properties (e.g. hydrophobicity) and other factors are important in determining which congeners are metabolized or accumulated in an organism. Higher chlorinated PCB congeners tend to increase with increasing trophic level in the food web (Antoniadou et al., 2007).
There are no ideal environmental media for monitoring temporal (or spatial) trends of POPs. The use of wildlife has the advantage that the rates of elimination of POPs are generally much faster in orders of days and weeks than the rates of environmental degradation where half-lives in sediment, for example, can be several years and decades (Norstrom et al., 1986; Clark et al., 1987; Sinkkonen and Paasivirta 2000); hence changes in body burdens reflect changes in the bioavailability of POPs. Since most POPs are sequestered in lipid rich tissues, eggs make a convenient and temporally predictable medium for monitoring (Gewurtz et al., 2011). High levels of contamination have been reported in eggs of Herring Gulls (Larus argentatus) and Snapping Turtles (Chelydra serpentina) from Hamilton Harbour relative to other Great Lakes locations (Weseloh et al., 2006; de Solla et al., 2007). There is direct evidence that POPs in the Hamilton Harbour area are bioavailable to wildlife feeding there. Adult and juvenile farm-raised Mallards (Anas platyrhynchos) that were released and subsequently collected 10 days later at the Hamilton Harbour Confined Disposal Facility had sum PCB concentrations in breast muscle that were 5300 times greater than at release on day 0 (Gebauer and Weseloh, 1993).
The main objective of this study was to assess temporal trends in burdens of legacy contaminants in eggs of two sentinel species, Herring Gulls and Snapping Turtles, in the Hamilton Harbour AOC since the early 1980s. Historical high concentrations of PCBs in the AOC provide a unique opportunity to examine relative differences among individual PCB congeners in both species over time based on their propensity for metabolism by the cytochrome P450 system.
Herring Gull eggs were collected annually from one of several colonies within Hamilton Harbour (see Figure 1 in Hall and O'Connor, 2016) from 1981 to 2012, usually in late April. Each year, a single egg was collected from one of 9–13 freshly-laid clutches and then pooled for a single chemical analysis (Turle and Collins, 1992). Exceptions included 1981 in which ten individual eggs were chemically analyzed and in years 1983, 1985, 1988 and 1990 when no egg collections were conducted. Egg contents were frozen at −40°C or lower prior to chemical analysis. Egg collections in Hamilton Harbour have been performed as part of the Great Lakes Herring Gull Monitoring Program which has been monitoring POPs annually at 15 Herring Gull colonies throughout the Great Lakes since 1974 (Weseloh et al., 2011).
Freshly-laid Snapping Turtle eggs were collected in June from Cootes Paradise within the Hamilton Harbour AOC (see Figure 1 in Hall and O'Connor, 2016, this issue). Eggs were collected annually from sites in Cootes Paradise in years 1986–1991, 1993–1995, 1998, 1999, 2002, and 2012. Five eggs were selected from each clutch, although occasionally fewer eggs were selected from smaller clutches (Bishop et al., 1995). Eggs were selected in a pseudo-random but stratified manner such that eggs were selected throughout the entire clutch (de Solla et al., 2007). Egg contents from each clutch were pooled and frozen in hexane-cleaned amber glass jars. Numbers of clutches analyzed ranged from 6–15 per year, with the exception of collections in 1987, 1991, and 1993 where analyses were performed on one or two clutches.
From 1981 to 1985, egg samples were analyzed for PCBs and OC pesticides at the Ontario Research Foundation (ORF)–currently Process Research ORTECH Inc.–in Mississauga, Ontario using high resolution gas chromatography-electron capture detection (GC-ECD; Reynolds and Cooper, 1975; Norstrom et al., 1980). PCBs were analyzed as Aroclor 1254:1260 (1:1) and estimated by a single peak corresponding to PCB 138 (Turle et al., 1991). Starting in 1986, egg samples were analyzed at the National Wildlife Research Centre (NWRC) in Ottawa, with the exception of one analysis which was performed at the Great Lakes Institute for Environmental Research (GLIER) in Windsor, Ontario (a subset of 2002 Turtle eggs). PCBs and other OCs were quantified by GC-ECD for egg samples collected from 1986–1996 and then by GC with mass selective detection (GC-MSD) for egg samples collected from 1997–2012 (see details of chemical methodology in de Solla et al., 2007, 2010). Concentrations of PCBs were determined as individual congeners. For temporal trend analysis, sum PCBs are based on the sum concentration of 36 congeners common to both GC-ECD and GCD-MSD methods and both Turtle and Gull egg analyses; these include: PCB #28, 31, 42, 44, 49, 52, 56/60, 64/41, 66/95, 70/76, 87, 97, 99, 101/90, 105, 110, 118, 138, 141, 146, 151, 153, 156/171, 158, 170/190, 172, 174, 178, 180, 183, 187/182, 194, 195, 196/203, 200, and 206. These 36 PCB congeners comprised on average 89.1% and 91.8% of total sum PCBs quantified for Gulls and Turtles, respectively. Concentrations of the four most abundant OC pesticides found in eggs from Hamilton Harbour are reported which include: bis(4-chlorophenyl)-dichloroethylene (p,p'-DDE), dieldrin, mirex and sum chlordane (as the sum concentration of cis, trans, oxy-chlordane, cis- and trans-nonachlor). In general, samples were run with duplicate injections and extractions, method blanks, one or two in-house reference materials (RM), and certified reference material to meet quality control standards. Method Detection Limits (MDLs) for organochlorines ranged between 0.0001 µg g−1 to 0.005 µg g−1. For observations that were below MDLs, replacement values were calculated for individual PCB congeners and OC pesticides using maximum likelihood (de Solla et al., 2012). Since changes in GC methodology used to quantify compounds could influence reported trends (de Solla et al., 2010), GC-ECD concentrations for individual PCB congeners and OC pesticides were adjusted to allow for direct comparability to GC-MSD concentrations. This was examined using in-house RM consisting of 1:9 dilution of Herring Gull eggs and large chicken eggs (see de Solla et al., 2010 for details) run for 61 analyses at NWRC using both GC methods. For compounds where a significant difference was found between mean concentrations of the RM using the two methods, GC-ECD concentrations were adjusted by multiplying by a constant, i.e. the ratio of GC-MSD to GC-ECD concentrations. Mean percent lipids in eggs of both species did not change over the study period with overall means (±SD) of 8.6 (±0.8)% and 6.3 (±1.3)%, for Gulls and Turtles, respectively.
Eggs were also analyzed for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by gas chromatography/mass spectrometry at NWRC beginning in 1984 (Norstrom and Simon, 1991; Simon and Wakeford, 2000). Snapping Turtle eggs were analyzed as a single pooled sample for collections at Cootes Paradise in 1989, 1990, 1998, 1999 and 2002. All samples were above MDLs for TCDD which ranged from 0.1–2 pg g−1.
Simple linear regression analysis was used to examine temporal trends in contaminant concentrations in Gull eggs from Hamilton Harbour and in Turtle eggs from Cootes Paradise. For early years when individual PCB congeners were not quantified or fewer than 36 individual PCB congeners were quantified, sum 36 PCB concentrations were estimated by calculating the average ratio of concentrations of sum 36 PCBs and the Aroclor 1254:1260 (1:1) equivalents (Turle et al., 1991); these ratios were equal to 0.47 and 0.42 in eggs of Gulls and Turtles, respectively. Regressions were performed on means, where individual analyses of eggs or clutches were performed, or using a single pooled egg sample. Contaminant data were natural logarithm (ln) transformed for statistical analysis.
Temporal trends in PCB congener profiles in eggs of the two species were also examined. We classified each of the 36 PCB congeners into one of four groups by the presence/absence and position of vicinal H-atoms (i.e. meta-para (m,p), ortho-meta (o,m)), which can be used to indicate the propensity of a congener to be metabolized (Kannan et al., 1995). PCB congeners were classified as: Group I: non-metabolizable (lacking both m,p and o,m vicinal H-atoms); Group II: those metabolized by P450 2B enzymes (having m,p vicinal H-atoms); Group III: those metabolized by P450 1A enzymes (having o,m vicinal H-atoms), and Group IV: those metabolized by both P450 1A and 2B enzymes (having both m,p and o,m). For each group, PCB congener concentrations were summed and the percent contribution of each group to the sum 36 PCB concentration was determined for all eggs. While the sum 36 PCB concentration in Gull eggs in 2006 was consistent with concentrations in bracketing years, the relative contributions of each group to the sum were not consistent and so this year was not included in analyses. Linear regression analysis was performed to examine temporal trends observed for each PCB group for both species. Inter-species comparisons of temporal trends for PCB groups were performed by comparing the slopes of the regression lines using a Student's t-test. Analyses were completed using STATISTICA 6.0 software and assessed using a significance level of p ≤ 0.05.
Concentrations of sum PCBs, OC pesticides and TCDD declined in Herring Gull eggs from Hamilton Harbour from 1981 (or 1984 for TCDD) to 2012 (Figure 1; range in r2 = 0.47–0.82; p < 0.0001 in all cases). Between the first year and last year of analysis, declines in contaminant concentrations in Gull eggs ranged from 86% for sum 36 PCBs to 94% for mirex. Concentrations of sum PCBs and four OC pesticides also declined in Snapping Turtle eggs from Cootes Paradise from 1986 to 2012 (Figure 2; range in r2 = 0.30 – 0.77; p < 0.05 in all cases). Declines in mean contaminant concentrations between the first and last year of analysis ranged from 36% for mirex to 89% for p,p'-DDE. TCDD also declined in Turtle eggs from 1989 to 2002 (r2 = 0.79; p = 0.046) with an overall decline of 71% in concentration (Figure 2). In eggs of both species, exponential curves show rapid declines in concentrations followed by relatively slower rates of decrease in recent years.
There were temporal changes in the relative contribution of some groups of PCBs to sum PCB concentrations in eggs of both species. The percentage of Group I non-metabolizable PCBs increased over time in eggs of Gulls (r2 = 0.44, p = 0.0008) and Turtles from Cootes Paradise (r2 = 0.83, p = 0.00003) from 1986/7 to 2012 (Figure 3a). PCB congeners 153 and 180 were the most prevalent congeners in Group I, together comprising on average 32.7% and 32.6% of sum PCB concentrations in eggs of Gulls and Turtles, respectively. In contrast, the percentage of Group III PCBs (metabolized by P450 1A enzymes) decreased over time in eggs of Gulls (r2 = 0.70, p = 0.000007) and Turtles (r2 = 0.88, p = 0.000006, Figure 3b). PCB congeners 138 and 118 were the most prevalent congeners in Group III, together comprising on average 22.3% and 30.6% of sum PCB concentrations in eggs of Gulls and Turtles, respectively. Concentrations of groups I and III PCB congeners together comprised, on average, 94.5% and 97.5% to sum PCB concentrations in eggs of Gulls and Turtles, respectively. A significant difference in the temporal trends (i.e. slopes) was found between the two species for the Group I (t30 = 3.55, p = 0.0013) and Group III (t30 = 3.68, p = 0.0009) PCBs. Relative to Gulls, faster rates of change were found in Turtles for Group I (i.e. increase) and Group III (i.e. decrease) PCBs. Groups II and IV PCBs together contributed less than 6% to sum PCB concentrations in both species. No significant temporal changes in the relative contributions of either of these groups were found with one exception, i.e. for Group II PCBs in Turtles where a significant decrease was found (r2 = 0.40, p = 0.03; data not shown).
Large and significant declines in POP burdens in eggs of both Snapping Turtles and Herring Gulls indicate consistent changes in exposure to contaminants in the Hamilton Harbour AOC from the 1980s to 2012. The magnitude of these changes outweighs any differences in longevity, metabolism and/or diet (see below) between the two species. Declines in the bioavailability of these compounds can be attributed to severe restrictions on their use, improved industrial practices and the effectiveness of remedial activities in reducing chemical inputs into Hamilton Harbour (Hamilton Harbour RAP, 2002). Long-term declines in PCBs have been reported since the 1970/80s in suspended sediment and various fish species caught in Hamilton Harbour (Bhavsar et al., 2016; Jia et al., 2016). Despite these declines, the persistent nature of these compounds means that they continue to be available and accumulate in biota.
Similar temporal patterns in these two species in the AOC, as a reflection of their surrounding environment over a 30-year study period, provide additional support that maternal contaminant burdens deposited into eggs are largely reflective of the female's diet just prior to egg production rather than long term accumulation in body tissue. While concentrations of PCBs in body tissues of Snapping Turtles have been shown to increase with increasing size and age, no such pattern of increasing burdens with age is evident in eggs (Hebert et al., 1993; Bishop et al., 1994). Furthermore, Bishop et al. (1994) contend that since clutch mass accounts for a small percentage (7–13%) of female body mass and the period of egg production in Snapping Turtles occurs over several months, it is probable that lipids required for egg production (and hence POP burdens) are derived from dietary intake rather than utilization of fat stores. Similarly, resources used for egg production in Herring Gulls (which are year-round residents) are derived from dietary intake over a several week period (Hobson, 2006). Half-lives for p,p'-DDE, oxychlordane and dieldrin were in the range of 100–300 days in experimentally-dosed Herring Gulls (Norstrom et al., 1986; Clark et al., 1987) which is short relative to the time frame here. Depuration rates for organochlorines in Turtle tissues are not known. Evidence suggesting that Turtles are not accumulating POPs indefinitely is inferred by the lack of difference in the rates of decline between Hamilton Harbour Gull and Turtle eggs for both sum PCBs (t34 = 0.32, p = 0.75) and p,p'-DDE (t34 = 1.42, p = 0.17) from 1986–2012 (results not reported).
Dietary shifts in the trophic levels will also influence the interpretation of contaminant temporal trends (Hebert and Weseloh, 2006). This was likely this was not an issue in Gull eggs from Hamilton Harbour where no significant change in stable nitrogen isotope values (as an indicator of Gull trophic position) was evident between 1981–2012 (CE Hebert, ECCC unpublished). Significant temporal declines for PCBs and OCs are consistent with that found for mercury in eggs from this colony from 1974–2009 by Weseloh et al. (2011) who examined changes in Gull diet using stable carbon isotopes, trophic position and fatty acids. Year-to-year fluctuations in contaminant concentrations could be due to factors including variation in local feeding conditions, availability of forage fish, and weather conditions (Hebert et al., 1997).
Differences in diet and metabolism may account for reported differences in POP burdens between Gulls and Turtles. Snapping Turtles are generalist omnivores and their diet consists of fish, aquatic plants, frogs, insects, juvenile birds, mollusks, and other food items (Lagler, 1943). Herring Gulls are facultative fish-eating birds whose diet consists primarily of small fish and invertebrates, although other food items may be taken. While Turtles and Gulls likely occupy a similar trophic level, Gulls have higher metabolic rates and thus greater energetic demands, and in turn have a greater daily food requirement (Nagy et al., 1999) and thus dietary exposure to POPs. This difference in accumulation of POPs between the two species was reflected in consistently higher contaminant burdens in Gull eggs versus Turtle eggs. Since Gulls tend to forage within 15 km of their colony (DVCW, pers. obs.), contaminants in eggs tend to reflect regional contamination around the harbour and surrounding area. Snapping Turtles, with a much smaller home range, are useful as indicators of localized geographic contamination (de Solla et al., 2007). While Turtle egg collection sites were not within Hamilton Harbour proper, Cootes Paradise is directly connected to the harbour where fish would feed (and thereby accumulate POPs) prior to their migration into wetlands and creeks to spawn. The contribution of burdens acquired from sources outside of the AOC, such as through consumption of migratory fish or for Gulls which foraged outside of the AOC, may have also contributed to POP burdens. Given the similarities in temporal contaminant patterns for these indicator species evident at two geographic scales, these influences are likely minor relative to conditions within the harbour.
Cytochrome P450 enzymes (CYP) are important for metabolizing endogenous and exogenous substrates including organic contaminants and are found in birds, reptiles and mammals. Structural differences in individual PCB congeners can influence the activity of these enzymes and hence biotransformation to more hydrophilic forms and subsequent elimination (Borlakoglu et al., 1990). In this study, similar temporal patterns were found between two top predator fish-eating species using PCB congeners categorized into groups based on their structural similarities. Group I PCB congeners 153 and 180 with no vicinal H-atoms are difficult to metabolize, persistent and known to accumulate in wildlife (Antoniadou et al., 2007). Despite large temporal declines in sum PCB concentrations, the combined contributions of these two congeners remained high (nearly 33%) over time in both species. Also persistent, Group III PCB congeners such as 138 and 118 have o,m vicinal H-atoms and are metabolized by P450 1A enzymes. It is unclear if the distinct temporal patterns shown by these groups are related to changes in metabolic ability following decreases in maternal burdens or if PCB profiles in prey and the environment have changed due to removal-type biotic and abiotic processes (e.g. volatilization, sedimentation, natural degradation) following significant reductions in PCB inputs. It is important to note that not all congeners necessarily responded in the same way within a group. For example in both species, no significant temporal decline (i.e. change) was found for percent contribution of PCB 138 while a significant temporal decline was found for percent contribution of PCB 118. Structural differences relating to the number of ortho-substituted chlorines present (i.e. two for PCB 138 vs one for PCB 118) and hence greater recalcitrance of PCB 138 likely contribute to this difference in trends. Nonetheless, the remaining Group III PCBs contributed to the overall significant decline found for this group in both species. Groups II and IV PCBs contributed relatively smaller proportions to sum PCBs in eggs. Similar to the pattern for the highly chlorinated Group I PCBs (with a minimum of six chlorines), Braune et al. (2001) reported significant temporal increases in the contributions of the hexachlorobiphenyl PCBs to sum PCBs (as the most abundant homolog group) in eggs of three species of seabirds over a 23-year period. These trends were concomitant with overall temporal decreases in sum PCB burdens in these species.
High contributions of groups I and III PCBs relative to the groups II and IV PCBs have also been found in eggs of Osprey (Pandion haleaetus) and liver, fat and blood of four species of Sea Turtles from the Atlantic and southwest Pacific Ocean (Keller et al., 2004; de Solla and Martin, 2009; Richardson et al., 2010). Richardson et al. (2010) suggest that accumulation of the Group III PCBs metabolized by P450 1A enzymes in Sea Turtles might indicate poor biotransformation which is consistent with the limited CYP 1A expression reported previously in Sea Turtle species. In this study, it is difficult to speculate on the differences in metabolic efficiencies of the two classes of enzymes in eggs within and between the two species which would also be directly related to the PCB congener profiles found in their prey. In contrast to eggs, higher relative contributions of the groups II and IV PCBs were found in Osprey chick plasma compared to groups I and III PCBs which may have been due to differences in consumption of local prey items and/or differences in metabolism between adults and juveniles (de Solla and Martin, 2009). The relative contributions of metabolizable PCBs (which approximated sum concentrations of groups II and III PCBs in this study) in blood of Antarctic Adélie Penguins (Pygoscelis adeliae) also differed significantly within one breeding season (van den Brink et al., 2000).
For a species to be an effective biomonitor of environmental conditions, it is vital that acquired contaminant burdens are reflective of these conditions both geographically and temporally. Large and significant temporal declines in concentrations of legacy contaminants were found in eggs of two species which are representative of environmental conditions at two different geographic scales within the AOC (i.e. at local scale for Turtles within tributaries/wetlands and at regional scale for Gulls which forage over a larger range). These findings are consistent with temporal contaminant patterns in other media in Hamilton Harbour and are further evidence of improved environmental conditions in the AOC. Significant temporal changes in the percentages of non-metabolizable congeners (i.e. increase) and congeners metabolized by P450 1A enzymes (i.e. decrease) were evident in eggs of both species. It is not clear if these distinct temporal patterns are related to changes in metabolism following reductions in PCB burdens or changes in bioavailability of congeners in the environment. Analyses of long-term PCB trends at a site with a history of PCB contamination provide a unique opportunity to examine temporal patterns in PCB profiles in two aquatic species. Similarities in temporal patterns between the two species support their utility as top-predator wildlife bio-indicator species.
We would like to thank Christine Bishop, John Struger, and Kim Fernie for their early work studying POPs in Snapping Turtles in Hamilton Harbour, as well as the numerous field assistants, contractors and staff who participated in Gull and Turtle egg collections over the years.