Philippa Kaur

To the Editor — In an insightful Comment Bliege Bird and Codding1 highlight a number of important issues to consider in the analysis of cross-cultural anthropological data. However, a casual reader of the Comment could be forgiven for taking away the message that cross-cultural data in anthropology is inherently flawed, and so is of limited use. We want to emphasize that comparative analysis plays an essential role in all non-experimental sciences, including anthropology and archaeology. This is because when systems cannot be manipulated due to scales of time and space, or issues of logistics or ethics, the only way to evaluate alternative outcomes is by analysing the results of natural experiments. More

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AffiliationsDepartment of Integrative Biology, Oregon State University, Corvallis, OR, USABenjamin D. Dalziel & Mark NovakDepartment of Mathematics, Oregon State University, Corvallis, OR, USABenjamin D. DalzielCollege of Earth, Ocean and Atmospheric Sciences, Oregon State University, Corvallis, OR, USAJames R. WatsonDepartment of Ecology and Evolutionary Biology, Cornell University, Ithaca, NY, USAStephen P. EllnerAuthorsBenjamin D. DalzielMark NovakJames R. WatsonStephen P. EllnerCorresponding authorCorrespondence to
Benjamin D. Dalziel. More

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Mercury additions to the study catchmentMETAALICUS was conducted on the Lake 658 catchment at the Experimental Lakes Area (ELA; now IISD-ELA), a remote area in the Precambrian Shield of northwestern Ontario, Canada (49° 43′ 95″ N, 93° 44′ 20″ W) set aside for whole-ecosystem research31. The Lake 658 catchment includes upland (41.2 ha), wetland (1.7 ha) and lake surface (8.4 ha) areas. Lake 658 is a double basin (13 m depth), circumneutral, headwater lake, with a fish community consisting of forage (yellow perch (P. flavescens) and blacknose shiner (Notropis heterolepis)), benthivorous (lake whitefish (C. clupeaformis) and white sucker (Catostomus commersonii)), and piscivorous (northern pike (E. lucius)) fishes. The lake is closed to fishing.Hg addition methods used in METAALICUS have been described in detail elsewhere19,32,33. In brief, three Hg spikes, each enriched with a different stable Hg isotope, were applied separately to the lake surface, upland and wetland areas. Upland and wetland spikes were applied once per year (when possible; Fig. 1a) by fixed-wing aircraft (Cessna 188 AGtruck). Mercury spikes (as HgNO3) were diluted in acidified water (pH 4) in a 500 l fiberglass tank and sprayed with a stainless-steel boom on upland (approximately 79.9% 200Hg) and wetland (approximately 90.1% 198Hg) areas. Spraying was completed during or immediately before a rain event, with wind speeds less than 15 km h−1 to minimize drift of spike Hg outside of target areas. Aerial spraying of upland and wetland areas left a 20-m buffer to the shoreline, which was sprayed by hand with a gas-powered pump and fire hose to within about 5 m of the lake32. Average net application rates of isotopically labelled Hg to the upland and wetland areas were 18.5 μg m−2 yr−1 and 17.8 μg m−2 yr−1, respectively.The average net application rate for lake spike Hg was 22.0 μg m−2 yr−1. For each lake addition, inorganic Hg enriched with approximately 89.7% 202Hg was added as HgNO3 from four 20-l carboys filled with acidified lake water (pH 4). Nine lake additions were conducted bi-weekly at dusk over an 18-week (wk) period during the open-water season of each year (2001–2007) by injecting at 70-cm depth into the propeller wash of trolling electric motors of two boats crisscrossing each basin of the lake32,33. It was previously demonstrated with 14C additions to an ELA lake that this approach evenly distributed spike added in the evening by the next morning34.We did not attempt to simulate Hg in rainfall for isotopic lake additions because it is impossible to simulate natural rainfall concentrations (about 10 ng l−1) in the 20-l carboys used for additions. Instead, our starting point for the experiment was to ensure that the spike was behaving as closely as possible to ambient surface water Hg very soon after it entered the lake. Several factors support this assertion. By the next morning each spike addition had increased epilimnetic Hg concentrations by only 1 ng l−1 202Hg. Average ambient concentrations were 2 ng l−1. Thus, while the Hg concentrations in the carboys were high (2.6 mg l−1), the receiving waters were soon at trace levels. Furthermore, we investigated if the additions altered the degree of bioavailability or photoreactivity of Hg(ii) in the receiving surface water. We examined the bioavailability of spike Hg(ii) as compared to ambient Hg in the lake itself using a genetically engineered bioreporter bacterium35. On seven occasions, epilimnetic samples were collected on the day before and within 12 h of spike additions. The spike was added to the lake as Hg(NO3)2, which is bioavailable to the bioreporter bacterium (detection limit = 0.1 ng Hg(ii) l−1), but we never saw bioavailable ambient or spike Hg(ii) in the lake, presumably because it was quickly bound to dissolved organic carbon (DOC). This indicates that, in terms of bioavailability, the spike Hg was behaving like ambient Hg soon after additions. Photoreactivity in the surface water was examined on seven occasions, by measuring the % of total Hg(ii) that was dissolved gaseous Hg for spike and ambient Hg, either 24 h or 48 h after the lake was spiked36. There was no significant difference (paired t-test, P > 0.05), demonstrating that by then the lake spike was behaving in the same way as ambient Hg during gaseous Hg production.Lake, food web and fish samplingWater samples were collected from May to October every four weeks at the deepest point of Lake 658. Water was pumped from six depths through acid-cleaned Teflon tubing into acid-cleaned Teflon or glass bottles. Water samples were filtered in-line using pre-ashed quartz fibre filters (Whatman GFQ, 0.7 µm). Subsequently, Hg species were measured in the filtered water samples (dissolved Hg and MeHg) and in particles collected on the quartz fibre filter (particulate Hg and MeHg).From 2001 to 2012, Lake 658 sediments were sampled at 4 fixed sites up to 5 times per year. Sampling frequency was highest in 2001, with monthly sampling from May to September, and declined over the course of the study. Fixed sites were located at depths of 0.5, 2, 3 and 7 m. A sediment survey of up to 12 additional sites was also conducted once or twice each year. Survey sites were selected to represent the full range of water depths in both basins. Cores were collected by hand by divers, or by subsampling sediments collected using a small box corer. Cores were capped and returned to the field station for processing within a few hours. For each site, three separate cores were sectioned and composited in zipper lock bags for a 0- to 2-cm depth sampling horizon, and then frozen at −20 °C.Bulk zooplankton and Chaoborus samples were collected from Lake 658 for MeHg analysis. Zooplankton were collected during the day from May to October (bi-weekly: 2001–2007; monthly: 2008–2015). A plankton net (150 μm, 0.5 m diameter) was towed vertically through the water column from 1 m above the lake bottom at the deepest point to the surface of the lake. Samples were frozen in plastic Whirl-Pak bags after removal of any Chaoborus using acid-washed tweezers. Dominant zooplankton taxa in Lake 658 included calanoid copepods (Diaptomus oregonensis) and Cladocera (Holopedium glacialis, Daphnia pulicaria and Daphnia mendotae). Chaoborus samples were collected monthly in the same manner at least 1 h after sunset. After collection, Chaoborus were picked from the sample using forceps and frozen in Whirl-Pak bags. Chaoborus were not separated by species for MeHg analyses, but both C. flavicans and C. punctipennis occur in the lake. Profundal chironomids were sampled at the deepest part of the lake using a standard Ekman grab sampler. Grab material was washed using water from a nearby lake and individual chironomids were picked by hand.All work with vertebrate animals was approved by Animal Care Committees (ACC) through the Canadian Council on Animal Care (Freshwater Institute ACC for Fisheries and Oceans Canada, 2001–2013; University of Manitoba ACC for IISD-ELA, 2014–2015). Licenses to Collect Fish for Scientific Purposes were granted annually by the Ontario Ministry of Natural Resources and Forestry. Prior to any Hg additions, a small-mesh fence was installed at the outlet of Lake 658 to the downstream lake to prevent movement of fish between lakes. Sampling for determination of MeHg concentrations (measured as total mercury (THg), see below) occurred each autumn (August–October; that is, the end of the growing season in north temperate lakes) for all fish species in Lake 658, and for northern pike and yellow perch in nearby reference Lake 240 (Extended Data Tables 2, 3). Fish collections occurred randomly throughout the lakes. Forage fish (YOY and 1+ yellow perch, and blacknose shiner) were captured using small mesh gillnets (6–10 mm) set for 90% of the Hg in muscle tissue from yellow perch in Lake 658 is MeHg40,41, here we report fish mercury data as MeHg.THg concentrations (ambient, lake spike, upland spike and wetland spike) in fish muscle samples were quantified by ICP-MS39. Samples were digested with HNO3/H2SO4 (7:3 v/v) and heated at 80 °C until brown NOx gases no longer formed. The THg in sample digests was reduced by SnCl2 to Hg0 which was then quantified by ICP-MS (Thermo-Finnigan Element2) using a continuous flow cold vapour generation technique41. To correct for procedural recoveries, all samples were spiked with 201HgCl2 prior to sample analysis. Samples of CRMs (DORM2 (2001–2011), DORM3 (2012–2013), DORM4 (2014–2015); National Research Council of Canada) were submitted to the same procedures; measured THg concentrations in the reference materials were not statistically different from certified values (P > 0.05). Detection limit for each of the spikes was 0.5% of ambient Hg.Calculations and statistical methodsAnalyses were completed with Statistica (6.1, Statsoft) and Sigmaplot (11.0, Systat Software). We present wet weight (w.w.) MeHg concentrations for all samples, except sediments which are dry weight (d.w.) concentrations. For zooplankton, Chaoborus, and profundal chironomids, d.w. MeHg concentrations were multiplied by a standard proportion (0.15) to yield w.w. concentrations for each sample42. The resulting w.w. concentrations were averaged over each open water season to determine annual means. For fish muscle biopsies, d.w. MeHg concentrations were multiplied by individual d.w. proportions to yield w.w. MeHg concentrations for each sample. To avoid any size-related biases, we calculated standardized annual MeHg concentrations (ambient and lake spike) for northern pike and lake whitefish by determining best-fit relationships between FL and MeHg concentrations for each year (quadratic polynomial, except for a linear fit for lake whitefish in 2004), and using the resulting regression equations to estimate MeHg concentrations at a standard FL43 (the mean FL of all fish sampled for each species: northern pike, 475 mm; lake whitefish, 530 mm). Square root transformation of raw northern pike data was required to satisfy assumptions of normality and homoscedasticity prior to standardization. The resulting data represent standardized concentrations of lake spike and ambient MeHg for each species each year.We used the ratio of lake spike and ambient Hg in each sample as a measure of the amount by which Hg concentrations were changed with the addition of isotopically enriched Hg:$${rm{P}}{rm{e}}{rm{r}}{rm{c}}{rm{e}}{rm{n}}{rm{t}},{rm{i}}{rm{n}}{rm{c}}{rm{r}}{rm{e}}{rm{a}}{rm{s}}{rm{e}}={[{rm{l}}{rm{a}}{rm{k}}{rm{e}}{rm{s}}{rm{p}}{rm{i}}{rm{k}}{rm{e}}{rm{H}}{rm{g}}]}_{i}/{[{rm{a}}{rm{m}}{rm{b}}{rm{i}}{rm{e}}{rm{n}}{rm{t}}{rm{H}}{rm{g}}]}_{i}times 100$$
(1)
where [lake spike Hg]i is the concentration of lake spike MeHg in sample i, and [ambient Hg]i is the concentration of ambient MeHg in sample i. For northern pike and lake whitefish, we calculated the mean annual relative increase from all individuals (not the size-standardized concentration data).Biomagnification factors (BMF) were calculated to describe differences in Hg concentrations between predator and prey5:$${rm{BMF}}={log }_{10}({[{rm{MeHg}}]}_{{rm{p}}{rm{r}}{rm{e}}{rm{d}}{rm{a}}{rm{t}}{rm{o}}{rm{r}}}/{[{rm{MeHg}}]}_{{rm{p}}{rm{r}}{rm{e}}{rm{y}}})$$
(2)
where [MeHg]predator is the mean (forage fish) or standardized (large-bodied fish) concentration of MeHg in the predator (ng g−1 w.w.) and [MeHg]prey is the mean concentration of MeHg in the prey (ng g−1 w.w.). MeHg concentration of prey items were averaged from samples collected throughout the open-water season immediately prior to autumn sampling of fish species to represent an integrated exposure for calculation of BMF. We used a dominant prey item to represent the diet of each fish species. For age 1+ yellow perch, northern pike, and lake whitefish, dominant prey items were zooplankton, forage fishes (YOY and 1+ yellow perch, and blacknose shiner) and Chaoborus, respectively.To assess loss of lake spike MeHg by northern pike during the recovery period (2008–2015), we calculated28 whole body burdens (in μg) of lake spike MeHg for the standardized population and for individuals that had been sampled in autumn 2007 (t0 is the final time spike Hg was added to the lake) and again in at least one subsequent year during annual autumn sampling (n = 16 fish, of which 1–9 individuals were recaptured annually from 2008–2015). This calculation of MeHg burden is a relative measure of whole fish Hg content because MeHg is higher in muscle tissue than in other tissue types28,40. For the standardized population data, we used best-fit relationships between FL (in mm) and body weight (in g; quadratic polynomial) to determine body weight at the standard FL. We multiplied this body weight by standard ambient and spike MeHg concentrations (in ng g−1 w.w.) in muscle tissue for each year to determine body burdens over time (in ng). For individual fish, we multiplied spike MeHg concentration (in ng g−1 w.w.) by body weight (in g) to yield individual body burdens (in ng). To account for differences among individuals and between individuals and the population, we normalized the data to examine the mean proportion of original (t0) lake spike MeHg burden present in northern pike each year of the recovery period (2008–2015).$${rm{change}},{rm{in}},{rm{burden}},{rm{from}},{t}_{0}={{rm{burden}}}_{{rm{tx}}}/{{rm{burden}}}_{{rm{t}}0}$$
(3)
We used a best fit regression (exponential decay, beginning in the second year of recovery) to estimate the half-life (50% of original burden) of lake spike MeHg for the population.Northern pike and lake whitefish ages were determined by cleithra and otoliths, respectively, if mortality had occurred, but most ages were quantified using fin rays collected from live fish44 (K. H. Mills, DFO or North/South Consultants). Northern pike of the sizes selected for biopsy sampling had a median age of 3 years (range: 2–12 years; n = 305); the median age of lake whitefish was 17 years (range: 3–38 years; n = 86).Reporting summaryFurther information on research design is available in the Nature Research Reporting Summary linked to this paper. More