Analytical Methods: Total mercury (THg) concentrations were determined using a Nippon Instruments MA-3000 Direct Mercury Analyzer (DMA), which uses direct thermal decomposition, gold amalgamation, and cold vapor atomic absorption spectroscopy. For quality assurance and quality control, certified reference materials National Research Council Canada (NRCC) DORM-4 (fish protein) and NRCC TORT-3 (lobster hepatopancreas) were analyzed at the beginning and end of each analysis day. Around 20 mg of dried, homogenized tissues were weighed out for THg analysis, and replicate samples and DORM-4 were analyzed every seven samples. A blank boat was combusted after samples expected to have high THg concentrations (e.g., livers from large fish) to reduce any potential THg carryover to subsequent samples. The average variation of replicate measurements was 4.1%, and analyses of DORM-4 (average measured THg = 417 ± 26 ng Hg/g dry weight, n = 161) and TORT-3 (296 ± 15 ng Hg/g dry weight, n = 40) were within 1.2% and 1.5% of mean certified values, respectively. Only data collected between standard reference materials that were within the range of certified values were included in analyses.

We analyzed tissues known to play roles in methylmercury (MeHg) accumulation and excretion for MeHg. We selected sets of dorsal muscle, stomach lining, intestine, and liver samples from 10 lancetfish representing a range of masses (116.0 – 3920.8 g) for MeHg analysis (n = 40 samples total). We also analyzed brains from 33 whole lancetfish for MeHg. MeHg concentrations were determined using species-specific isotope dilution mass spectrometry following microwave-assisted acid digestion. This method quantifies the MeHg concentration of a sample based on the recovery of an isotopically labeled Me201Hg solution added during sample preparation. A stock Me201Hg solution was synthesized from 201HgII (96.3% purity) (Gilmour and Riedel, 1995; Hintelmann et al., 2000; Hintelmann and Ogrinc, 2002) and diluted to a 13 ng/mL working solution. The MeHg concentration of the Me201Hg working solution was periodically quantified using reverse isotope dilution with standards prepared from a certified 1000 ppm MeHg (II) chloride standard (Alfa Aesar). Exact concentrations were used to calculate MeHg concentrations in the samples after signal deconvolution of Me201Hg and Me202Hg peak areas.

To prepare samples for analysis, around 20 mg of tissue were weighed into acid-cleaned 50 mL PTFE-TFM pressure vessels with 10 mL of 5 M nitric acid and 85 µL of the 13 ng/mL Me201Hg working solution. The volume of Me201Hg added was selected to target a 202Hg/201Hg mixing ratio of 0.23 to minimize uncertainty in signal deconvolution (Heimbürger et al., 2015; Rousseau et al., 2013). Samples were digested using an Anton Paar Multiwave 5000, which heated the samples to 70˚C for 10 minutes after a 5-minute ramp-up period. After digestion, sample digestates were diluted with 30 mL of Milli-Q water and stored at 4˚C.

MeHg in each sample was derivatized for analysis using ascorbic acid-assisted direct ethylation (Munson et al., 2014). 100 µL of sample digestate were added to a clean 60 mL amber borosilicate glass vial with 45 mL of Milli-Q water. Sample pH was adjusted to 4.8 using 2 M acetate/glacial acetic acid buffer before ascorbic acid (2.5% w/v) was added. Samples were ethylated with sodium tetraethylborate (1% NaTEB in 2% potassium hydroxide), and ethylation was allowed to proceed for at least 10 minutes before analysis. All reagents were prepared using Milli-Q water as needed. Samples were purged with Hg-free ultra-high purity argon gas, volatile Hg species were separated via gas chromatography using a Tekran 2700 Methyl Mercury Auto-Analysis System, and Me201Hg and Me202Hg were detected using an Agilent 8900 triple quadrupole inductively coupled plasma mass spectrometer (ICP-MS). Replicate samples, DORM-4, and TORT-3 were analyzed in each batch prepared for microwave digestion. The average variation of replicate measurements was 6.7%, and analyses of DORM-4 (average measured MeHg = 363 ± 24 ng Hg/g dry weight, n = 9) and TORT-3 (128 ± 8 ng Hg/g dry weight, n = 9) were within 2.3% and 6.9% of mean certified values, respectively.

Data Analysis: Because MeHg analysis is costly and time-consuming, we analyzed a subset of tissues for MeHg to confirm the relationship between THg and MeHg concentrations. THg and MeHg concentrations were highly correlated in dorsal muscle, liver, stomach lining, and intestine (Spearman’s rank correlation, r = 1.00, 0.99, 0.98, and 0.83, respectively). However, we analyzed all brains for MeHg, so comparisons of measured MeHg concentrations between tissues were skewed by uneven sample size. To address this issue, we fit a generalized additive model (GAM) using restricted maximum likelihood estimation, a gaussian family distribution, and an “identity” link function using the mgcv package (version 1.9.0; Wood, 2011) to predict percent MeHg from lancetfish mass by tissue type using paired MeHg and THg measurements (Table S1). Percent MeHg was calculated as the MeHg concentration divided by the THg concentration of the same sample. We then used the GAM to estimate MeHg concentrations of dorsal muscle, stomach lining, intestine, and liver samples from fish with measured brain MeHg concentrations. We only measured MeHg in brain due to low sample mass, and thus do not report percent MeHg.

All mercury concentrations are reported in ng Hg/g on a wet-weight basis using percent moisture data obtained from the freeze-drying process. All data processing was performed in R Statistical Software (version 4.3.2; R Core Team, 2023).