Sampling
Killer whale biopsy samples of skin and blubber from 38 individuals were collected year-round from August 2017 to July 2018 in northern Norway. All whales were sampled according to relevant guidelines and regulations, and conducted under the permit FOTS-ID 10176 issued by Mattilsynet (the Norwegian Food Safety Authority, report nr. 2016/179856). Details of seasonal sampling locations, stable isotope dietary descriptors and classification of sampled individuals are described in a previous study14. In the current study, total Hg was analysed in skin from all individuals (n = 38), whereas organohalogen contaminants (OHC) was analysed in blubber of 31 individuals due to insufficient blubber for the remaining 7 individuals.
OHC analysis
OHC analysis was conducted at the Laboratory of Environmental Toxicology at the Norwegian University of Life Sciences, Oslo, Norway. We analysed a total of 83 OHCs: 49 organochlorines (OCs), including 34 PCBs and 15 organochlorine pesticides (OCPs), 18 brominated flame retardants (BFRs), including newer and unregulated compounds, and 16 hydroxylated metabolites (OH-metabolites) of PCBs and polybrominated diphenylethers (PBDEs). A full list of analysed compounds can be found in Supplementary Table S1.
We analysed OCs and BFRs using a multicomponent method, first described in 197842, and since modified for a range of compounds and biological matrices43,44,45,46. The analysis of the OH-metabolites was conducted according to previously published methods47,48. An outline of the method is described in the Supplementary Information. Reported concentrations were blank corrected based on the average concentration detected within blank samples. The limit of detection (LOD) was defined as three times the average noise in chromatograms, and ranged from 0.40 to 11.10 ng/g w.w. for OCs, 0.012 to 0.362 ng/g w.w. for BFRs and 0.013 to 0.040 ng/g w.w. for OH-metabolites (see Supplementary Table S2). Internal reference materials for OCs and BFRs (contaminated seal blubber, MTref01) and OH-metabolites (contaminated seal blood, MTref03) were also extracted in conjunction with sample material to assess method performance. Internal standard recoveries are listed in Supplementary Table S2.
Hg analysis
We analysed total Hg by atomic absorption spectrometry at the University of Oslo, using a Direct Mercury Analyser (DMA-80, Milestone Srl, Soirsole, Italy). Killer whale skin samples were freeze dried in a Leybold-Heraeus GT2 freeze dryer with a Leybold Vakuum GmbH vacuum pump (Leybold, Cologne, Germany) and then homogenised to a fine powder using an agate pestle and mortar. Approximately 0.002 g of killer whale skin were analysed in parallel with sample blanks and certified reference material (DORM-4, fish protein; DOLT-5, dogfish liver, National Research Council, Ottawa, Canada). If enough material, samples were analysed in duplicates to ensure precision of measurements and the arithmetic mean value used. Average recoveries of the certified reference materials were within 10% of the reported values. The detection limit of the instrument was 0.05 ng mercury.
Data treatment
We included OHC compounds found in levels above the instrument’s LOD in a minimum of 65% of the individual whale samples for statistical analysis (see Supplementary Table S1, Supporting Information for pollutants excluded). For individual concentrations below the LOD, we imputed left-censored data by replacing missing values with a random number between 0 and the LOD assuming a beta distribution (α = 5, β = 1) to retain the pattern of the dataset. In total, 95 values below the LOD were replaced, representing 6.52% of the OHC dataset. All total Hg samples were above the LOD.
We defined the ΣPCBs as the sum of all 28 PCB congeners detected in more than 65% of the whale samples (PCB-28, -66, -74, -87, -99, -101, 105, -110, -114, -118, -128, -137, -138, -141, -149, -151, -153, -156-, 157, 170, -180, -183, -187, -189, 194, -196, -206, -209). The definition for ΣPCBs varies within killer whale literature, with some studies analysing only a few core PCB congeners35, some all 209 of the possible congeners36, and others not providing a definition (e.g. for thresholds for possible health effects7). There will therefore inevitably be some errors in comparisons. However, since the ΣPCBs in killer whales is dominated by a few commonly reported congeners, typically PCB-153 and -13816,37, it is unlikely that the inclusion of other minor constituents will have a major influence on the total load. PCBs were further grouped according to the number of chlorine substitutions per molecule, i.e. homologue group to compare the pattern of PCBs. ΣDDTs was defined as the sum of p,p′-DDT, p,p′-DDD and p,p′-DDE, the ΣPBDEs as the sum of BDE-28, -47, -99, -100, -153 and -154 and the sum of chlordanes (ΣCHLs) as the sum of oxychlordane, trans-chlordane, cis-chlordane, trans-nonachlor and cis-nonachlor.
Statistical analyses
Statistical analyses were performed using R v. 3.4.149. The significance level was to set to α = 0.05, except in cases where the value was adjusted due to multiple testing, and was two-tailed. In addition to visual inspection, normality was tested using the Shapiro–Wilk’s test50 and homogeneity of variance by Levene’s test51 using the R package car52.
Whale dietary groups
The dietary groups used in this study are based on a previous study, which used stable isotope values inputted into a Gaussian mixture model to assign sampled individuals to two fish-eating groups: Herring-eaters and Lumpfish-eaters and one mammal-eating group Seal-eaters14. The three dietary groups were characterised by disparate, non-overlapping isotopic niches that were consistent with predatory field observations. The seal-eating group was defined by higher δ15N values than the two fish-eating groups.
We found that the herring and lumpfish-eating killer whales did not differ in either their OHC levels (Tukey’s HSD: p = 0.49) or total Hg levels (pairwise Welch’s t-test: p = 0.67). In this study, we thus combined the dietary groups Herring-eaters and Lumpfish-eaters into the group Fish-eaters, to enable easier comparison to the seal-eating killer whales.
We then used Welch’s t-test to compare the ΣPCB levels in the seal-eating and fish-eating dietary groups (using a log10 transformation), and to compare the total Hg levels in the skin between the two dietary groups.
OHC dataset
We used multivariate analysis to compare and visualise the differences in all the OHCs between the dietary groups, age and sex classes using the vegan package in R53. Principle Component Analysis (PCA) was used to visualise the main structure of the data: reducing the dimensions to two new, uncorrelated, latent variables termed principle components 1 and 2 (PC1 and PC2). We log-10 transformed contaminant levels to ensure normality and homogeneity of variance, and the presence of any influential outliers were checked by the Cook’s distance test. Redundancy Analysis (RDA) was used to extract and summarise the variation in the OHC levels constrained, and thereby explained, by a set of explanatory variables54. Significant associations between response variables and the explanatory variables were identified by an RDA based forward model selection, followed by a Monte Carlo forward permutation test (1,000 unrestricted permutations). The samples’ scores along PC1 were subject to one-way Analysis of Variance (ANOVA) followed by Tukey’s honestly significant difference post hoc test (Tukey’s HSD) to analyse differences between the three dietary groups. PC1 scores were also used to evaluate correlation to total Hg levels in the skin using a Spearman’s rank correlation test. Absolute concentrations were subject to PCA with lipid % as a covariate, after checking its significance using RDA, as lipid normalising data in inferential statistics can often lead to misleading conclusions55.
We lipid-normalised OHC values when comparing levels to threshold values for toxicity or other killer whale populations, and used the geometric mean as the average for each dietary group to reflect the log normal distribution of the data. In accordance with convention, efforts were made to only compare adult males with other worldwide populations, as reproductive female whales are known to transfer a substantial portion of their OHC burden to their calves35,36,38. In any case of comparison, similar metrics were compared (i.e. arithmetic mean, geometric mean, median) and all variables kept similar (i.e. sex, age, biopsy/stranded animals). We make the assumption in this study that the killer whales sampled in 2002 in Norway were fish-eaters for the following reasons: firstly, the whales were sampled on herring overwintering grounds, feeding on herring, and photographs were taken of five of the eight adults sampled and were identified as herring-eaters from previous field observations16. Secondly, the PCB pattern in the sampled whales showed 76% of ΣPCBs higher chlorinated congeners (hexaCBs or higher), which is more similar to the fish-eaters from our study (80% higher chlorinated congeners) than the seal-eaters (87% higher chlorinated congeners). Thirdly, the upper 95% confidence range of all pollutants reported in the 2002 killer whales falls below both the geometric and arithmetic mean values for seal-eaters from this study.
Total Hg dataset
The normal distribution of the data within each dietary group meant we used the arithmetic mean as an average. The three dietary groups (Herring-eaters, Lumpfish-eaters and Seal-eaters) were compared using a pairwise Welch’s t-test with a Benjamini–Hochberg False Discovery Rate correction to adjust for multiple testing. Because we found no difference between the Herring-eaters and Lumpfish-eaters (p = 0.67), we combined these two groups to a group called “Fish-eaters” for easier comparison with the seal-eaters. The total Hg levels in the skin of the two groups, Fish-eaters and Seal-eaters were compared using Welch’s t-test.
There is a strong positive correlation between Hg levels in the skin and liver in toothed whales, and this can be used to compare Hg levels measured in skin with hepatic toxicity threshold values56,57,58. To extrapolate to liver from skin in our samples, we chose an equation based on a model using concentrations in the liver (Hgliver μg/g w.w) and skin (Hgskin μg/g w.w) of bottlenose dolphins (Tursiops truncatus) (Eq. 1)58. We converted dry weight to wet weight using the water content for each individual whale measured during freeze drying.
$$ln left( {Hg_{liver} } right) = 1.6124 times ln left( {Hg_{skin} } right) + 2.0346$$
(1)
When comparing Hg concentrations to other worldwide populations, both male and female whales were included. This was due to a lack of information of sex in one of the populations for comparisons and because killer whales are unlikely to pass on Hg burdens to calves5,59.
Source: Ecology - nature.com
