Characterization of AhR agonists reveals antagonistic activity in European herring gull (Larus argentatus) eggs

Science of the Total Environment 514 (2015) 211–218

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Characterization of AhR agonists reveals antagonistic activity in European herring gull (Larus argentatus) eggs Martine Muusse a,b,⁎, Guttorm Christensen c, Tânia Gomes a, Anton Kočan d, Katherine Langford a, Knut Erik Tollefsen a, Lenka Vaňková d, Kevin V. Thomas a a

Norwegian Institute for Water Research (NIVA), Gaustadalleen 21, N-0349 Oslo, Norway University of Oslo, Dept. of Bioscience, Postboks N-0316 Blindern, Oslo, Norway Akvaplan NIVA, Fram Centre, N-9296 Tromsø, Norway d Research Centre for Toxic Compounds in the Environment, Faculty of Science, Masaryk University, Kamenice 753/5, 625 00 Brno, Czech Republic b c

H I G H L I G H T S • • • • •

European herring gull eggs were analyzed for dioxins, furans and PCBs. TEQs between 55 and 292 pg/g lw were determined using CALUX specific REPs. REPs are in 16 of the 23 samples higher than the biological AhR agonist activity. This suggests the presence of AhR antagonists, but none could be identified. These levels could pose a risk for the developing embryo or the (human) consumer.

a r t i c l e

i n f o

Article history: Received 3 October 2014 Received in revised form 28 January 2015 Accepted 28 January 2015 Available online xxxx Editor: Adrian Covaci Keywords: European herring gull eggs Aryl hydrocarbon receptor agonists Dioxins Furans PCBs

a b s t r a c t European herring gull (Larus argentatus) eggs from two Norwegian islands, Musvær in the south east and Reiaren in Northern Norway, were screened for dioxins, furans, and dioxin-like and selected non-dioxin-like polychlorinated biphenyls (PCBs), and subjected to non-target analysis to try to identify the aryl hydrocarbon receptor (AhR) agonists, responsible for elevated levels measured using the dioxin responsive chemically activated luciferase expression (DR-CALUX) assay. Eggs from Musvær contained chemically calculated toxic equivalent (WHO TEQ) levels of between 109 and 483 pg TEQ/g lw, and between 82 and 337 pg TEQ/g lw was determined in eggs from Reiaren. In particular PCB126 contributed highly to the total TEQ (69–82%). In 19 of the 23 samples the calculated WHO TEQ was higher than the TEQCALUX. Using CALUX specific relative effect potencies (REPs), the levels were lower at between 77 and 292 pg/g lw in eggs from Musvær and between 55 and 223 pg/g lw in eggs from Reiaren, which was higher than the TEQCALUX in 16 of the 23 samples. However, the means of the REP values and the TEQCALUX were not significantly different. This suggests the presence of compounds that can elicit antagonist effects, with a low binding affinity to the AhR. Non-target analysis identified the presence of hexachlorobenzene (HCB) (quantified at 9.6–185 pg/g lw) but neither this compound nor high concentrations of PCB126 and non-dioxin-like PCBs could explain the differences between the calculated TEQ or REP values and the TEQCALUX. Even though, for most AhR agonists, the sensitivity of herring gulls is not known, the reported levels can be considered to represent a risk for biological effects in the developing embryo, compared to LC50 values in chicken embryos. For human consumers of herring gull eggs, these eggs contain TEQ levels up to four times higher than the maximum tolerable weekly intake. © 2015 Elsevier B.V. All rights reserved.

1. Introduction European herring gulls (Larus argentatus) are omnivorous sea birds that live and breed on the coast of Northern Europe and Scandinavia. In ⁎ Corresponding author at: Norwegian Institute for Water Research (NIVA), Gaustadalleen 21, N-0349 Oslo, Norway. E-mail address: [email protected] (M. Muusse).

http://dx.doi.org/10.1016/j.scitotenv.2015.01.101 0048-9697/© 2015 Elsevier B.V. All rights reserved.

Northern Norway the eggs of the European herring gull are seen as a delicacy and traditionally eaten in spring. Previous studies have raised concern about the concentration of contaminants, such as polychlorinated biphenyls (PCBs), brominated flame retardants (BFRs) and dichlorodiphenyltrichloroethane (DDT) in herring gull eggs from Norway (Helgason et al., 2008; Knutsen et al., 2008). In 2002 the Norwegian government advised children and women, whom were pregnant, breastfeeding, or of a reproductive age, not to consume herring gull

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eggs due to high levels of organic contaminants present (Mattilsynet, 2013). In addition to concerns for the egg-consuming public, high contaminant concentrations pose a risk for the herring gull populations. There have been reports of lower hatchling survival and skewed hatchling sex ratios in arctic glaucous gulls (Larus hyperboreus) with high levels of contamination (Erikstad et al., 2011) and a study on survival rate in glaucous gulls on Bjørnøya in Norway reports that females are more sensitive to high levels of organochlorines (OCs) than males (Erikstad et al., 2013). In herring gull eggs from the Great Lakes (USA), a negative correlation with total PCB levels (45 congeners) and plasma retinol (vitamin A) and T-cell mediated immunity was seen (Grasman et al., 1996). Elevated levels of PCBs were found in deceased glaucous gulls analyzed in 1989, and the authors concluded that it cannot be excluded that these levels have been at least partly responsible for the deaths of these gulls (Gabrielsen et al., 1995). One group of compounds that are particularly hazardous for humans and wildlife are those compounds that bind to the aryl hydrocarbon receptor (AhR), for example polychlorinated dibenzo-p-dioxins (PCDDs or dioxins), polychlorinated dibenzofurans (PCDFs or furans) and PCBs. After binding to the AhR, these compounds move into the nucleus and activate cytochrome P-450 1A1 (CYP1A1), which modulates the expression of many other genes and gene products. A number of adverse outcomes have been reported for polychlorinated compounds, including hepatoxic responses such as porphyria, immunotoxicity, developmental and reproductive toxicity, disruption of endocrine pathways, chloracne, and carcinogenesis in humans and wildlife (Safe, 2001). In the mid-1980s, the World Health Organization (WHO) developed the concept of toxic equivalently factors (TEFs) as a measurement of how strongly a compound binds to the AhR (Van den Berg et al., 2006). The dioxin-like toxicities of all documented AhR agonists have been ranked relative to 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD), the most potent AhR agonist, and have been assigned TEF values. By definition, the TEF of TCDD is 1 and for example PCB126, which has a ten times lower affinity for the AhR, has a TEF of 0.1 (Table 1). This allows the total amount of AhR

agonists to be expressed as TCDD toxic equivalents (TEQs). Twentynine compounds have been assigned TEF values. These are other dioxins, furans, non-ortho (no) PCBs and some mono-ortho (mo) PCBs (Van den Berg et al., 2006). In addition to using a target analytical approach to quantify the concentrations of individual known AhR agonists, AhR agonist activity can be measured by using the in vitro dioxin responsive chemically activated luciferase expression (DRCALUX) assay. Most compounds with TEF values have been tested using this assay, giving a slightly different response than the WHO TEF values would predict: CALUX specific relative effect potencies (REPs, Behnisch et al., 2003). Other compounds, such as certain polycyclic aromatic hydrocarbons (PAHs) and BFRs, and pesticides such as hexachlorobenzene (HCB) have been demonstrated to bind to the AhR, but due to the lack of experimental data these compounds have as yet not been given official TEF values. Their AhR agonist activity is instead documented by using induction equivalently factors (IEFs), based on in vitro assays (van Birgelen, 1998; Machala et al., 2001; Behnisch et al., 2003). Most previously published studies on contaminants in different gull species or their eggs have quantitatively analyzed specific contaminants (Herbert et al., 1999; Kannan et al., 2001; Helgason et al., 2008). Although this is an effective way of analyzing environmental samples, contaminants that are not specifically targeted could easily be overlooked. This applies in particular to compounds with low concentration (dose)-high toxicity, compounds with unknown toxicity and compounds that otherwise have not been identified as priority pollutants, or compounds of emerging concern in monitoring approaches. In addition, the fact that chemicals co-exist in the environment as complex mixtures (cocktails) with the potential to cause combined effects that are not predicted by the effect of the individual compounds, warrants a more holistic and non-biased chemical monitoring strategy. One way to accommodate this is by combining biological assays (such as the DR-CALUX assay measuring AhR agonist activity) and targeted and non-targeted screening. Such an integrated approach has been implemented with success, including studies by Hurst et al. (2004), Houtman et al. (2006) and Muusse et al. (2012).

Table 1 WHO TEF values for humans/mammals (van den Berg et al., 2006) versus the bird TEF (van den berg et al., 1998) and herring gull IEF values (Kennedy et al. 1996a,b, Hervé et al., 2010), and the CALUX specific REP values (Behnisch et al., 2003).

Dioxins

Furans

no-PCBs

mo-PCBs

Compounds with TEF value

Human/mammal TEF

Bird TEF

Herring gull IEF

CALUX specific REP

2,3,7,8-TCDD 1,2,3,7,8-PeCDD 1,2,3,4,7,8-HxCDD 1,2,3,6,7,8-HxCDD 1,2,3,7,8,9-HxCDD 1,2,3,4,6,7,8-HpCDD OCDD 2,3,7,8-TCDF 1,2,3,7,8-PeCDF 2,3,4,7,8-PeCDF 1,2,3,4,7,8-HxCDF 1,2,3,6,7,8-HxCDF 2,3,4,6,7,8-HxCDF 1,2,3,7,8,9-HxCDF 1,2,3,4,6,7,8-HpCDF 1,2,3,4,7,8,9-HpCDF OCDF 3,3',4,4'-TCB (PCB 77) 3,4,4',5-TCB (PCB 81) 3,3',4,4',5-PeCB (PCB 126) 3,3',4,4',5,5'-HxCB (PCB 169) 2,3,3',4,4'-PeCB (PCB 105) 2,3,4,4',5-PeCB (PCB 114) 2,3',4,4',5-PeCB (PCB 118) 2,3',4,4',5'-PeCB (PCB 123) 2,3,3',4,4',5-HxCB (PCB 156) 2,3,3',4,4',5'-HxCB (PCB 157) 2,3',4,4',5,5'-HxCB(PCB 167) 2,3,3',4,4',5,5'-HeCB(PCB 189)

1 1 0.1 0.1 0.1 0.01 0.0003 0.1 0.03 0.3 0.1 0.1 0.1 0.1 0.01 0.01 0.0003 0.0001 0.0003 0.1 0.03 0.00003 0.00003 0.00003 0.00003 0.00003 0.00003 0.00003 0.00003

1 1 0.05 0.01 0.1 b0.001 0.0001 1 0.1 1 0.1 0.1 0.1 0.1 0.01 0.01 0.0001 0.1 0.05 0.1 0.001 0.0001 0.0001 0.00001 0.00001 0.00001 0.0001 0.00001 0.00001

1

1 0.54 0.3 0.14 0.066 0.046 0.0005 0.32 0.21 0.5 0.13 0.039 0.18 0.11 0.029 0.041 0.0065 0.0013 0.0042 0.067 0.0034 0.000012 0.000048

0.9 21

b0.0003 0.06 0.07 b0.00009 b0.00009

0.000024 0.00021 0.00008 0.000008 0.000007

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Previously we have analyzed European herring gull eggs from two locations in Norway for the presence of all AhR agonists using the DRCALUX assay (Muusse et al., 2014). Elevated AhR agonist levels of between 15 and 401 pg TEQCALUX/g lw were detected in total egg extracts (i.e. non-acid treated) from Musvær, Northern Norway, and from 6 to 360 pg TEQCALUX/g lw in eggs from Reiaren, Southern Norway. The objective of the study reported here is to attempt to identify all of the AhR agonists present. Since these samples represent total egg extracts, no acid clean-up was performed prior to the DR-CALUX assay since this would remove labile AhR agonists that are also of interest. This work aims to advance the knowledge of which compounds give rise to this bioactivity, using targeted analysis for priority persistent organic pollutants (POPs) with known AhR agonist activity (dioxins, furans and dioxin-like PCBs), in addition to some non-dioxin-like PCBs. A non-target approach was also deployed in an attempt to identify unknown or poorly documented AhR-agonists present in the samples. 2. Materials and methods 2.1. Sampling Twenty-three European herring gull (L. argentatus) eggs were collected in May 2012: eleven eggs from Musvær island (69.88°N, 18.55°E), and twelve eggs from Reiaren island (59.15°N, 10.46°E) in Norway. Details of the sampling procedure are provided in Muusse et al. (2014). 2.2. Extraction, clean-up and chemical analysis Extraction of organic compounds from the eggs was performed by liquid–liquid extraction as described previously (Muusse et al., 2014). In brief, the egg yolk was carefully separated from the white and embryo (if present) after thawing, and extracted with acetone:cyclohexane (3:2, v/v) in an ultrasonic bath for 15 min and subsequently shaken for an hour. Samples were centrifuged (10 min, 1300 g) and the supernatant was used for further clean-up. Two different clean-up methods and chemical analyses were used: 1) Targeted analysis for dioxins, furans and dioxin-like and nondioxin-like PCBs, and 2) non-target analysis and target analysis for PAHs and the quantification of HCB, DDT and its metabolite dichlorodiphenyldichloroethylene (DDE). The latter extraction method was also used for generating the extracts exposed to bioassay screening by the DR-CALUX assay. 2.2.1. Target analysis The extract (3 mL) was cleaned and subjected to the targeted analysis of PCDDs, PCDFs and dioxin-like PCBs using the following methods: The samples were evaporated and lipid amount was determined gravimetrically, before an internal standard was added (50 pg each of 2,3,7,8substituted tetra- to hepta-CDD/Fs, 100 pg OCDD, 100 pg OCDF 13C12labeled, 1000 pg each of 12 dioxin-like PCBs and 2500 pg each of 6 indicator non-dioxin-like PCBs). Subsequently, the samples were dissolved in 4 mL hexane and cleaned-up using a multilayer column packed with (from bottom): Silanized glass wool, silica, potassium silicate, silica, 44% H2SO4/silica, silica, and anhydrous Na2SO4. Hexane (120 mL) was used for the elution of analytes from the column. The eluant was fractioned into 3 fractions using an active carbon AX-21/Celite 545 (1:19) column. The 1st fraction, eluted with 12 mL cyclohexane– dichloromethane–methanol (2:2:1, v/v) containing non-dioxin-like PCBs, the 2nd fraction (F2) containing dioxin-like PCBs (6.5 mL toluene) and the 3rd fraction (F3) containing PCDD/Fs (80 mL toluene, reverse flow) were collected. The F1, F2 and F3 were up-concentrated to near dryness before 1000 pg of PCB162 (13C12-labeled), 600 pg of PCB162 (13C12-labeled) and 15 pg of 1,2,3,4-TCDD (13C12-labeled) recovery standards dissolved in n-nonane were added.

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A gas chromatograph/high resolution mass spectrometer (GC/HRMS) (Thermo Scientific dual GC Trace 1310 and HRMS DFS) was used for the separation and quantification of the analytes. PCDD/Fs in the F3 were separated on a 60 m × 0.25 mm i.d. × 0.25 μm Restek Dioxin 2 capillary column and dioxin-like PCBs in the F2 and non-dioxin-like PCBs in the F1 by a 60 m × 0.25 mm i.d. × 0.25 μm SGE HT8 column. Mass spectrometric resolution was set at 10,000 (10% valley). GC/HRMS chromatograms were evaluated using the Thermo Scientific TargetQuan software and the levels of PCDD/Fs and dioxin-like-PCBs were determined by applying the isotope dilution approach, using the U.S. EPA 1613 (USEPA, 1994) and 1668 method (USEPA, 1999); method performance data and quality control is given in SI1. 2.2.2. Non-target analysis Lipids were removed from an aliquot (1.5 mL) by gel permeation chromatography (GPC; Waters 2695 separations module coupled to a Waters 486 absorbance detector at 254 nm) fitted with Envirogel columns (19 mm × 150 mm + 19 mm × 300 mm; Waters). The mobile phase was dichloromethane (DCM) with a flow rate of 5 mL/min and the extract was collected between 14.40 and 21.00 min. 1.5 mL of the sample extracts was injected. The system performance was checked by injecting a test solution containing corn oil, bis(2-ethylhexyl)phthalate, methoxychlor, perylene and sulfur. Ten percent of the cleaned-up yolk extract was transferred to dimethyl sulfoxide (DMSO) and used for the DR-CALUX assay. The remaining extract was up-concentrated and used for non-targeted analysis on a gas chromatography coupled to high-resolution time of flight mass spectrometry (GC–HR-ToF-MS) (GCT Premier, Waters, USA). The source temperature was 180 °C with a resolution over 8000, and the injector was at 240 °C. A BD-5 column was used (60 m × 0.25 μm × 0.25 mm, Agilent). The oven program had an initial temperature of 60 °C which was held for 3 min before ramping at 4 °C/min to 280 °C which was held for 10 min. This aliquot was used for targeted analysis for PAHs, performed with the TargetLynx® software (Waters, Manchester, UK) using a 5-point calibration. The calibration was linear between 25 and 4000 ng/mL with a limit of detection of 3 ng/mL. In addition all peaks were integrated manually and compounds were tentatively identified using the National Institute of Standards and Technology (NIST) library. The concentration of HCB, DDE and p-p′-DDT was quantified by adding 50 ng of internal standard (PCB30, 53 and 204) to the extracted samples (1 μL). Following treatment with concentrated sulfuric acid the compounds were analyzed by gas chromatography with a micro-electron capture detector (GC/μECD). The injector temperature was 255 °C and the detector temperature was 285 °C. A DB-5 column was used (60 m × 0.25 mm, Agilent). The extracts were injected in a splitless mode at 90 °C and held for 2 min. The oven temperature was raised as follows: 20 °C/min to 180 °C, 2 °C/min to 270 °C and 20 °C/min to 310 °C held for 5 min. Hydrogen was used as a carrier gas at a flow of 1 mL/min. The quantification was performed using a 7 point calibration curve in the concentration range 2 to 1000 ng/mL using PCB30 as an internal standard. 2.2.3. DR-CALUX assay The DR-CALUX uses a rat hepatoma cell-line, HL16.Lc2, stably transfected with an AhR-regulated luciferase reporter gene from the firefly (Photinus pyralis). This cell-line was kindly provided by Mike Denison (University of California, Davis, USA) and the analyses were performed as described in detail by Muusse et al. (2014). 2.3. Statistics Statistical analysis was performed with Excel and GraphPad Prism 5 (GraphPad software, La Jolla California, USA) and XLSTAT2014© (Addinsoft, NY, USA). Analytical data was tested for normality using a D'Agostino and Pearson omnibus normality test. Data that was not normally distributed was log transformed to meet the criteria for Gaussian

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distribution. Pearson correlation coefficients between the bioassay derived TEQ (TEQCALUX) and the TEQ derived by GC/HRMS (WHO TEQ and CALUX specific REP values) were calculated. Principal component analysis (PCA) was performed to evaluate the influence of the different AhR agonist groups on the TEQCALUX and visualized by Excel, and differences between populations or analyze methods were investigated using a paired t-test. The statistical significance levels were set at p b 0.05. 3. Results and discussion 3.1. Targeted analysis for dioxins, furans and dioxin-like PCBs No PAHs were detected in the egg extracts; however, elevated concentrations of dioxins, furans and PCBs were detected. The total TEQ values (using the internationally accepted TEF values for humans, WHO TEF (Van den Berg et al., 2006)), were between 109 and 438 pg WHO TEQ/g lw (10–41 pg WHO TEQ/g ww) for the eggs from Musvær and between 82 and 337 pg WHO TEQ/g lw (6–26 pg WHO TEQ/g ww) for the eggs from Reiaren (Table 2, see SI2 for details on the individual congeners). With the exception of the mo-PCBs, no statistical differences were identified between the two bird populations. The concentration of PCDFs was low in both populations, however, the contribution of PCB126 (3,3′,4,4′,5-PeCB) was high, being the most potent AhR agonist among the PCBs. With a WHO TEF value of 0.1, PCB126 contributed to the total TEQ value in all samples with 69–82% (Table 1). It is common for PCB126 to contribute significantly to the total TEQ in herring gull eggs. For example, 57–72% of the TEQ was accounted for by PCB126 in herring gull eggs from the

Table 2 Summary of the lipid percentage, the WHO TEQ values and CALUX REP values, as well as the TEQCALUX in pg/g lw, the HCB concentration in pg/g lw and the sum of indicator PCBs in ng/g lw, in herring gull eggs from two locations. WHO TEF values from van den Berg et al. (2006), REP values from Behnisch et al. (2003), and CALUX data from Muusse et al. (2014).

Lipid % TEQCALUX (pg/g CALUX lw) WHO Total TEQ (pg/g PCDD/F lw) PCDD PCB no PCB mo PCB PCB 126 CALUX Total REP (pg/g PCDD/F lw) PCDD PCB no PCB mo PCB PCB126 (ng/g Ind. PCB lw) (pg/g HCB lw) (pg/g p,p'-DDE lw) (pg/g DDT lw)

Pooled(n=23) Musvær(n=11) (min-max)

Reiaren(n=12) (min-max)

24

29 (24-32)

19 (11-25)

135

104 (15-401)

163 (6.2-360)

249

262 (109-438)

236 ( 82-337)

24

21 (18-36)

26.6 (9.2-35)

21 225 201 23 190 167

16 (9.0-30) 241 (96-402) 222 (86-378) 19 (9.5-27) 207 (81-351) 178 (77-292)

24.6 (8.7-33) 209 (73-306) 183 (64-248) 27 (9.1-44)a 174 (59-237) 157 (55-223)

23

23 (14-45)

23 (8.2-32)

17 144 129 15 127 2033

14 (8.9-25) 155 (63-258) 140 (56-237) 14 (7.4-21) 139 (54-235) 1807 (1055-2399)

20 (7.5-27)a 134 (47-195) 119 (40-171) 15 (7.5-24) 117 (39-168) 2269 (1102-3703)

71

123a (29-185)

23 (9.6-52)

446

446 (155- 905)

431 (225- 679)

11.8

bLOD

10 (1.7-28)

No-PCB: non-ortho PCBs: sum of PCB77, 81, 126, and 169. Mo-PCB: mono-ortho PCBs: sum of PCB105, 114, 118, 123, 156, 157, and 167, 189. Ind. PCB: Indicator PCBs: sum of PCB28, 52, 101, 153, 138, and 180. a Value is significantly higher than in the other population.

Great Lakes in the USA and the total WHO TEQ was between 1082 and 2540 pg/g lw (Kannan et al., 2001). A study on predatory birds from the UK in 2014 however, reported concentrations of PCB levels below the limit of detection to 248 pg WHO TEQ/g lw, while not detecting PCB126 in any of the samples (Pereira et al., 2014). In herring gull eggs from the north of Norway, average levels of 65.5 pg WHO TEQ/ g ww were measured (Pusch et al., 2005), and on Northern Norwegian islands, four PCB congeners with WHO TEF values (PCB105, 118, 156, 157), were measured in herring gull eggs, with values of between 45 and 83 pg WHO TEQ/g lw (Helgason et al., 2008), while in the current study the WHO TEQ (sum of PCB105, 118, 156, 157) accounts for 9– 36 pg/g lw. To summarize, the TEQ levels in the current study are up to an order of magnitude lower than eggs from the USA and a factor of between 2 and 5 times lower than herring gull eggs from Norway in studies from 2002 and 2005. An additional seven non-dioxin-like PCBs, the indicator PCBs, were also analyzed and quantified. These were PCB28, 52, 101, 138, 153 and 180. Concentrations of sum indicator PCBs were between 1055 and 3703 (mean: 2033) ng/g lw (Table 2, see SI1 for details on the individual congeners). These levels are comparable to PCB levels found in herring gull eggs from Northern Norwegian islands (Helgason et al., 2008). GC-HR-ToF-MS screening of the eggs revealed the presence of two compounds of interest, namely HCB and p,p′-DDT. Concentrations and the (possible) AhR agonist properties of HCB will be discussed later. p,p′-DDE was present at concentrations of between 155 and 905 (mean: 446) pg/g lw (Table 2). Concentrations of DDT were all close to the limit of detection (LOD), due to chromatogram interference with high PCB concentrations. DDT and its breakdown product p,p′DDE are not known to bind to the AhR. An attempt was made to estimate the contribution of known AhR agonists to the total AhR agonist signal by comparing CALUX-derived TEQs and chemically derived WHO TEQ and REP values. For the majority of samples (19 out of 23), the chemically measured WHO TEQ was higher than the biologically measured TEQCALUX values determined for the same eggs (Muusse et al., 2014), (Fig. 1, SI1). In the five samples with the lowest TEQCALUX, the chemically analyzed WHO TEQ was more than 10 times higher and for one sample even 16 times higher. To assess if they correlate better with the TEQCALUX, CALUX specific REPs were calculated. The average REP values are significantly lower than the average WHO TEQ values (p b 0.0001), which is mainly due to lower values for PCB126 and PCB118. For 16 out of 23 samples, the CALUX specific REP values were higher than the biologically measured TEQCALUX, however, the means between the REP and the TEQCALUX are not significantly different (p b 0.05), hence the REPs correlate better with the DR-CALUX results than the WHO TEF values (Fig. 1). Also other studies have found that, especially for PCBs, the CALUX specific REPs correlate better with the TEQCALUX (Carbonnelle et al., 2004). Few other studies have reported a higher chemical TEQ than TEQCALUX; however, in the blood serum of Flemish women, the chemical WHO TEQ was twice as high as the TEQCALUX (Koppen et al., 2001), and in two out of five sediment samples from UK estuaries, TEQCALUX was lower than the chemically measured WHO TEQ (Hurst et al., 2004). These studies investigated the use of DR-CALUX as a screening tool for measurement of dioxin-like compounds and samples were therefore cleaned with acidified silica prior to DR-CALUX analysis, removing any unstable compounds. The aims of the current study, however, are not to validate the use of the DR-CALUX assay, but to measure all compounds with an affinity for the AhR. For this reason the DR-CALUX assay as well as the non-target analysis was performed on non-acid cleaned extracts, to measure the biological effect of the mixture of all compounds available, and in addition trying to identify which compounds are responsible for these effects. The low TEQCALUX values in comparison to the REP values, in combination with relatively low, though mostly significant, Pearson correlation coefficients between the TEQCALUX and the REP values for both populations pooled (Table 3), indicate that the determination of the

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Fig. 1. Correlation between TEQCALUX and total WHO TEQ values of A) Musvær and B) Reiaren and the correlation between TEQCALUX and REP values of C) Musvær and D) Reiaren, in pg/g lw in herring gull eggs. The solid line is the trend line, the dotted black lines are the 95% confidence bands, the gray dashed lines are the lines of equality and the numbers in the graphs correspond to the sample numbers.

chosen analytes (known AhR agonists) alone is not able to account for all the bioactivity, and that the effects of other compounds are also measured on the DR-CALUX assay. One of the possible causes for this underestimation of the concentration of AhR agonists by the DR-CALUX assay might be the presence of compounds that can elicit antagonist effects, with a low binding affinity to the AhR. This can result in a competition with stronger AhR agonists, resulting in a net antagonistic effect in the DR-CALUX assay when in concentrations close to saturation. Another possibility is the presence of partial agonists, compounds with a lower EC50 than TCDD, resulting in a lower than expected biological response. PCB52 and 153 as well as other non-planar PCBs, are partial agonists to the AhR, and have been reported to be able to behave as antagonists (Garrison et al., 1996; O'Kane et al., 2014) and also PCB126 is thought to act as an AhR antagonist when it co-occurs with TCDD (Sanctorum et al., 2007). PCB126 has a positive Pearson correlation coefficient with the Table 3 Pearson correlation coefficients between TEQCALUX and the chemical data of the different contaminant groups represented by REPCALUX (REP values by Behnisch et al., 2003) for both populations together and the populations separately. TEQCALUX CALUX REP

Both populations (n= 23)

Musvær (n=11)

Reiaren (n=12)

Total REP PCDD/F PCDD PCB NO PCB MO PCB PCB 126

0.54a 0.29 0.76a 0.55a 0.56a 0.42 0.56a

0.72a -0.03 0.77a 0.76a 0.76a 0.73a 0.76a

0.51 0.56 0.48 0.49 0.52 0.22 0.53

a

significant correlation (pN0.05)

TEQCALUX in the present study, and the non-dioxin-like PCB52, 138, 153 and 180, had positive, albeit insignificant, correlations. PCB101 had a significant correlation of 0.55 and PCB28 had a negative, but insignificant, correlation of −0.11. Another possible partial agonist is HCB, which is present in the current samples in concentrations of 29–185 pg/g lw (Musvær eggs) and 9.6–52 pg/g lw (Reiaren eggs, Table 2). Schroijen et al. (2004) reported antagonistic activities for HCB, while van Birgelen (1998) has suggested that HCB be included in the TEF concept with a TEF value of 0.0001. Nevertheless, the contribution of HCB as an AhR agonist to the overall TEQ will likely be negligible (0.001–0.019 pg TEQ/g lw) if using a TEF of 0.0001, and additionally, the correlation coefficient between HCB and the TEQCALUX was insignificant (p = 0.11) and the 7 samples whose TEQCALUX was higher than the chemically measured REP values did not have HCB concentrations that were significantly different from the other samples, thus the contribution of HCB to the overall TEQ/REP still remains unresolved. As far as we are aware, there is only one other study where contaminants in herring gull eggs have been characterized in such a manner, namely a study by Kennedy et al. (1996b), where chicken hepatocyte cultures were used to measure ethoxyresorufin-O-deethylase (EROD) inducing potencies of several dioxins, furans and dioxin-like PCBs. Using herring gull egg extracts (containing all PCDD/Fs, PCBs, structurally related non-polar halogenated aromatic hydrocarbons (HAHs) and chlorinated pesticides) as an example, the EROD activity, in chicken hepatocyte specific relative effect potencies (REPs), was compared with chemical TEQ levels. 99% of the activity could be explained and 98% of the active compounds were PCBs. The results from the current study indicate that seagull eggs contain a complex mixture of dioxins, furans and PCBs, in combination with other

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compounds such as HCB. These compounds have the capacity to interact in different ways with the AhR, making the interpretation of the results extremely difficult. Despite the fact that several compounds with reported partial agonist/antagonist properties were measured in these samples, a significant correlation could not be detected and therefore, while the combination of the CALUX data and the chemical analysis suggests antagonistic activity, the presence of antagonists could not be verified.

3.3. Principal component analysis To identify how contaminants were associated with the populations and how they influence the TEQCALUX in both populations, a PCA was applied to the data (Fig. 2). The CALUX specific REP values for the different groups of contaminants, including the TEQCALUX, were used, along with the concentrations of HCB and the indicator PCBs. The overall PCA clearly shows distinct responses between seagull populations. PC1 displays the TEQCALUX to be more closely associated with the population from Reiaren (sample numbers 12–23). The Musvær eggs (sample numbers 1–11) had higher HCB concentrations and also the PCDF, no-PCB and PCB126 REPs are more closely related to Musvær eggs, while the eggs from Reiaren exhibited higher PCDD and mo-PCB REPs, as well as higher concentrations of indicator PCBs. Furthermore, even though no direct association of TEQCALUX with a single contaminant group was detected, the PCA analysis confirms that HCB does not appear to influence the TEQCALUX much.

3.2. Differences between the two populations Differences in contaminant levels between the two populations were not significant, however, when the correlation coefficients were calculated for the two populations separately, the correlation coefficients between the TEQCALUX and the total CALUX specific REP values for the south-eastern population (Musvær) increased to 0.72, while the correlation coefficients of the northern population (Reiaren) decreased to 0.50 (Table 3). This indicates that, regardless of insignificant differences between the measured REP values of the two populations, there is probably a difference in contamination of other compounds that could interact with the AhR. These differences in correlation coefficients between the herring gull populations could be an effect of different feeding behaviors; herring gulls in Reiaren live in a pristine environment, while Musvær herring gulls live in a more populated area and probably have a higher access to food of anthropogenic sources. This could potentially also explain the higher concentration of the herbicide HCB in Musvær eggs. Glaucous gulls (L. hyperboreus) individuals are shown to feed on different trophic levels, some feed predominantly on eggs and young from other birds while others are specialize in catching fish, which means that they can have different contaminant patterns (Bustnes et al., 2003; Erikstad and Reiertsen, 2007), which can explain the differences in contaminant pattern between individual eggs.

3.4. Risks to herring gulls and humans Different TEF values are defined for birds as opposed to the human/ mammal WHO TEFs (Van den Berg et al., 1998), because birds seem to be less sensitive to PCDDs but more sensitive to PCBs than humans/ mammals. However also the bird TEF values are a (necessary) generalization if single species are to be considered, and therefore Kennedy et al. (1996a,b) and Hervé et al. (2010) have calculated specific IEFs for herring gulls for some AhR agonists (Table 1). Contrary to birds in general, PCB126 has a lower induction of cytochrome P4501A in herring gull hepatocyte cultures compared to those isolated from mammals (IEF of 0.06 against a TEF of 0.1 for mammals or birds (Kennedy et al., 1996a, b)). In addition, the furan 1,2,3,7,8-PeCDF has a higher induction in herring gull hepatocytes (IEF of 21 compared to a TEF of 0.1 for birds or 0.03

3

1

2

3

PCDF REP HCB

5

PC2 (17.70 %)

1

10

4

6

9

8

2

11 no-PCB REP

19 0

PCB126 REP

7 17

13 12

14

21

23

-1

22 18

20

16 15

mo-PCB REP TEQCALUX Ind. PCBs PCDD REP

-2

-3 -3

-2

-1

0 PC1 (61.71 %)

1

2

3

Fig. 2. PCA analysis of the eggs of both populations (Musvær: nrs 1–11, indicated with circles and Reiaren: nrs 12–23, indicated with squares) together with the contaminant groups: PCDD REP, PCDF REP, no- and mo-PCB REPs and PCB126 REP, and the concentrations of HCB, the indicator PCBs and the TEQCALUX values. The samples with REP values lower than the TEQCALUX values are indicated by open symbols.

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for mammals (Hervé et al., 2010)). Using these IEF values, and the TEFs for birds for the congeners that have not been tested on herring gulls specifically, the IEQ in the current study would be between 45 and 393 pg/g lw, which is quite similar to the mammal WHO TEQ values calculated for these samples, since, due to low concentrations of 1,2,3,7,8PeCDF, the high IEF for this compound in herring gulls only marginally contributes to the total herring gull specific IEQ. For the sake of comparison, the WHO TEF values for mammals were used when other studies are discussed. Several studies on contaminants in different species of seagulls have indicated that eggs from Norwegian birds contain levels of organochlorine contaminants that can pose a risk for the populations. A recent report has measured among others eight PCB congeners with TEF values in herring gull eggs. The WHO TEQ for these congeners (using the human/mammal TEF values) was 32–80 pg/g lw in eggs from Northern Norwegian islands (Huber et al., 2014), compared to a WHO TEQ of 8– 27 pg/g lw when the same congeners are considered in the current study. These concentrations are considered ‘with risk for biological effects’ by the Norwegian Environmental Agency (Andersen et al., 2014), where a LD50 of 52 pg TEQ/g ww (LD50 for domestic chicken embryos (Carro et al., 2013)) was used as a risk reference. To compare, in the current study the total WHO TEQ (all congeners) in ww is 6– 41 pg/g, which is lower than the LD50 for domestic chicken embryos, but in the same order of magnitude. Even though it is not completely clear how sensitive herring gulls are to dioxin-like compounds in comparison to mammals or other birds, for consumers of the eggs the levels of dioxins and PCBs are relatively high. The European Union (EU) Scientific Committee on Food has set the tolerable weekly intake (TWI) of dioxins and PCBs on 14 pg TEQ/kg body weight (EU Legislation, 2001), which would be an intake of 840 pg TEQ for a person weighing 60 kg. In the current study, with an average TEQ/egg of 2057 pg (range of between 511 and 3454 pg TEQ/egg), only two of the eggs are below this limit. For chicken eggs to be sold on the market a maximum limit of 6 pg TEQ/g lw (sum PCDD/Fs and DL-PCBs) is proposed by the EU (EU Commision Regulation, 2006), a concentration all of the eggs in the current study exceeded by up to an order of magnitude. Before the Norwegian government advised people to eat less herring gull eggs in 2002, 36% of the coastal Norwegian inhabitants ate one or more eggs per year (Pusch et al., 2005). This number has now gone down to 12%, but some individuals still consume up to 40 eggs per year (Birgisdottir et al., 2012). The present data clearly demonstrate that human consumption of herring gull eggs is still an issue to consider when assessing the overall impact of environmental pollution by halogenated compounds. 4. Conclusions The chemically calculated WHO TEQ in herring gull eggs measured up to 16 times higher than the TEQCALUX, and only in 4 out of 23 samples the TEQCALUX was higher than the WHO TEQ. When the TEQCALUX was compared to CALUX specific REP values, the TEQCALUX is higher in 7 out of 23 samples, but the average REP is still higher than the average TEQCALUX, however, the mean REP value is not significantly different from the TEQCALUX mean. This suggests the presence of AhR antagonists in the samples, but even though some documented antagonists, such as PCB126, non-dioxin-like PCBs and HCB, have been identified, a clear negative correlation with the TEQCALUX could not be found and for PCB126 the correlation was even positive. Therefore the presence of antagonists could not be proven. This might be due to low concentrations of some of the individual antagonists/partial agonists and effects of this complex cocktail of compounds, possibly in combination with other factors, for example metabolism of compounds in the DR-CALUX cell, which to date, are poorly understood. DR-CALUX and chemical analysis are based on completely different principles. Where a cell based assay measures biological activity of the

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mixture of all compounds available in the sample in vitro, chemical analysis detects and quantifies a determined amount of compounds from the same mixture. These results show that both bioassays and chemical analysis are necessary to understand the toxicity of complex samples such as these herring gull eggs. If only DR-CALUX would have been used as a screening method, some of the eggs in the lower range of AhR agonist activity would have been falsely regarded as negative samples, while, if only chemical analysis would have been used, the effect of all these compounds in a mixture would have been overseen. The chemically calculated WHO TEQ levels were similar to those found in other studies of herring gull eggs and even though little is known about the sensitivity of herring gulls for AhR agonists, the levels are high enough to pose a possible threat to the developing embryo when the EC50 for domestic chickens is used as a risk reference. In addition, for all eggs but two, the TEQ concentrations per egg are higher than the TWI for human consumers and all eggs have a higher TEQ in pg/g lw than that excepted for chicken eggs on the market.

Acknowledgments The authors thank the Research Council of Norway for funding this project through the Miljø2015 program (project 183762). The contribution of Andreas Sven Høgfeldt and Alfhild Kringstad in assisting with the GPC extraction and quantification on the GC/ECD, respectively, is acknowledged as well as the assistance of Inger Lise Nerland with statistics. This work was carried out with the support of core facilities of Research Centre for Toxic Compounds in the Environment (RECETOX) — National Infrastructure for Research of Toxic Compounds in the Environment; project number LM2011028, funded by the Ministry of Education, Youth and Sports of the Czech Republic under the activity “Projects of major infrastructures for research, development and innovations”.

Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2015.01.101.

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