Timothy D. Jardine

Lipid corrections in carbon and nitrogen stable isotope analyses: comparison of chemical extraction and modelling methods

1. Lipids have more negative delta(13)C values relative to other major biochemical compounds in plant and animal tissues. Although variable lipid content in biological tissues alters results and conclusions of delta(13)C analyses in aquatic food web and migration studies, no standard correction protocol exists. 2. We compared chemical extraction and mathematical correction methods for freshwater and marine fishes and aquatic invertebrates to better understand impacts of correction approaches on carbon (delta(13)C) and nitrogen (delta(15)N) stable isotope data. 3. Fish and aquatic invertebrate tissue delta(13)C values increased significantly following extraction for almost all species and tissue types relative to nonextracted samples. In contrast, delta(15)N was affected for muscle and whole body samples from only a few freshwater and marine species and had a limited effect for the entire data set. 4. Lipid normalization models, using C : N as a proxy for lipid content, predicted lipid-corrected delta(13)C for paired data sets more closely with parameters specific to the tissue type and species to which they were applied. 5. We present species- and tissue-specific models based on bulk C : N as a reliable alternative to chemical extraction corrections. By analysing a subset of samples before and after lipid extraction, models can be applied to the species and tissues of interest that will improve estimates of dietary sources using stable isotopes.

Biomagnification of Mercury in Aquatic Food Webs: A Worldwide Meta-Analysis

The slope of the simple linear regression between log10 transformed mercury (Hg) concentration and stable nitrogen isotope values (δ(15)N), hereafter called trophic magnification slope (TMS), from several trophic levels in a food web can represent the overall degree of Hg biomagnification. We compiled data from 69 studies that determined total Hg (THg) or methyl Hg (MeHg) TMS values in 205 aquatic food webs worldwide. Hg TMS values were compared against physicochemical and biological factors hypothesized to affect Hg biomagnification in aquatic systems. Food webs ranged across 1.7 ± 0.7 (mean ± SD) and 1.8 ± 0.8 trophic levels (calculated using δ(15)N from baseline to top predator) for THg and MeHg, respectively. The average trophic level (based on δ(15)N) of the upper-trophic-level organisms in the food web was 3.7 ± 0.8 and 3.8 ± 0.8 for THg and MeHg food webs, respectively. For MeHg, the mean TMS value was 0.24 ± 0.08 but varied from 0.08 to 0.53 and was, on average, 1.5 times higher than that for THg with a mean of 0.16 ± 0.11 (range: -0.19 to 0.48). Both THg and MeHg TMS values were significantly and positively correlated with latitude. TMS values in freshwater sites increased with dissolved organic carbon and decreased with total phosphorus and atmospheric Hg deposition. Results suggest that Hg biomagnification through food webs is highest in cold and low productivity systems; however, much of the among-system variability in TMS values remains unexplained. We identify critical data gaps and provide recommendations for future studies that would improve our understanding of global Hg biomagnification.

Analytical error in stable isotope ecology.

The increasing popularity of stable isotope analysis (SIA) as an ecological research tool and the ease of automated analysis have created a knowledge gap between ecologists using SIA and the operators of isotope ratio mass spectrometry (IRMS) equipment. This has led to deterioration in the understanding of IRMS methodology and its proper dissemination in the ecological literature. Of 330 ecological research papers surveyed, 63 (19%) failed to report any form of analytical error associated with IRMS. Of the 267 papers that reported analytical error, there was considerable variation both in the terminology and approach used to quantify and describe error. Internal laboratory standards were often used to determine the analytical error associated with IRMS, so chosen because they are homogenous and have isotopic signatures that do not vary over time. We argue that true ecological samples collected in the field are complex bulk mixtures and often fail to adhere to these two criteria. Hence the analytical error associated with samples is potentially greater than that of standards. A set of standard data run over time with a precision typically reported in the ecological literature (1 standard deviation: 1SD=0.26‰) was simulated to determine the likelihood of spurious treatment effects depending on timing of analysis. There was a 90% likelihood of detecting a significant difference in the stable nitrogen ratio of a single sample (homogenized bovine liver) run in two time periods when n>30. Minor protocol adjustments, including the submission of blind replicates by researchers, random assignment of sample repeats within a run by analytical labs, and reporting 1SD of a single sample analyzed both within and between runs, will only serve to strengthen the interpretation of true ecological processes by both researchers and reviewers.

Nonlethal Sampling of Sunfish and Slimy Sculpin for Stable Isotope Analysis: How Scale and Fin Tissue Compare with Muscle Tissue

Abstract We found that the sampling of tissues that do not result in the death of the fish, such as scale and fin tissue, may be substituted for muscle tissue in stable isotope analysis (SIA) of fishes. Comparisons were made between the values of δ13C and δ15N found in muscle tissue with the corresponding scale tissue of three sunfish species (bluegill Lepomis macrochirus, pumpkinseed L. gibbosus, and redbreast sunfish L. auritus) and with caudal fin tissue of slimy sculpin Cottus cognatus. The fish showed strong linear correlation in δ13C values between their nonlethally sampled scale or fin tissue and their muscle tissue (combined sunfish: r = 0.97; slimy sculpin: r = 0.84). Sunfish δ13C values were higher in scale tissue than in muscle tissue and required a correction factor for converting the scale values to the muscle values (regression equation: y = 1.1673x + 1.0531). Slimy sculpin δ13C fin and muscle values were similar and did not require a correction factor. The correlation of δ15N values between...

High fidelity: isotopic relationship between stream invertebrates and their gut contents

Abstract Benthic macroinvertebrate families were sampled along 3 rivers in New Brunswick, Canada. Stable isotopes of C and N were compared between body tissue and gut contents of individuals. δ13C and δ15N of body tissue and gut contents were strongly correlated (r = 0.94 and 0.93, respectively) over a wide range of δ values. In nonpredators, only minor fractionation of δ13C and δ15N was observed. In predators, diet–tissue fractionation of 13C was minor, but 15N fractionation that may have been related to diet quality (N content) was observed. The influence of diet quality on N-isotope fractionation was inconsistent in direction and strength among families. Our results suggest that subjecting primary consumers to gut clearance prior to processing for stable-isotope analysis is unnecessary, but the guts of predators should be removed before processing.

A non-lethal sampling method for stable carbon and nitrogen isotope studies of tropical fishes

Despite prior studies showing good agreement between fin and muscle isotope ratios in temperate fishes, the non-lethal method of fin sampling has yet to become a standard technique in isotopic food-web studies, and the relationship between the two tissues has never been tested in the tropics. We hypothesised that fin and muscle δ13C and δ15N would be strongly correlated in tropical fishes, thus allowing non-lethal sampling of these species. To test this hypothesis, we analysed fin and muscle tissues from 174 tropical fishes representing 27 species from the Mitchell River, Queensland, Australia. Fin tissue was a strong predictor of muscle-tissue δ13C (r2 = 0.91 for all species) and was slightly enriched in 13C (0.9‰), consistent with the results of studies on temperate species. Fin tissue was a poorer predictor of muscle-tissue δ15N (r2 = 0.56 for all species) although the mean difference between the tissues was small (<0.1‰). Differences were smallest in the largest fish, possibly because the elemental composition (%N) of fin more closely resembled that of muscle. These measurements provide more impetus for increased use of fin tissue as a non-destructive means of testing hypotheses about fish food webs in the tropics and elsewhere.

Consumer - resource coupling in wet-dry tropical rivers

1. Despite implications for top-down and bottom-up control and the stability of food webs, understanding the links between consumers and their diets remains difficult, particularly in remote tropical locations where food resources are usually abundant and variable and seasonal hydrology produces alternating patterns of connectivity and isolation. 2. We used a large scale survey of freshwater biota from 67 sites in three catchments (Daly River, Northern Territory; Fitzroy River, Western Australia; and the Mitchell River, Queensland) in Australias wet-dry tropics and analysed stable isotopes of carbon (δ(13) C) to search for broad patterns in resource use by consumers in conjunction with known and measured indices of connectivity, the duration of floodplain inundation, and dietary choices (i.e. stomach contents of fish). 3. Regression analysis of biofilm δ(13) C against consumer δ(13) C, as an indicator of reliance on local food sources (periphyton and detritus), varied depending on taxa and catchment. 4. The carbon isotope ratios of benthic invertebrates were tightly coupled to those of biofilm in all three catchments, suggesting assimilation of local resources by these largely nonmobile taxa. 5. Stable C isotope ratios of fish, however, were less well-linked to those of biofilm and varied by catchment according to hydrological connectivity; the perennially flowing Daly River with a long duration of floodplain inundation showed the least degree of coupling, the seasonally flowing Fitzroy River with an extremely short flood period showed the strongest coupling, and the Mitchell River was intermediate in connectivity, flood duration and consumer-resource coupling. 6. These findings highlight the high mobility of the fish community in these rivers, and how hydrological connectivity between habitats drives patterns of consumer-resource coupling.

Aquatic and terrestrial organic matter in the diet of stream consumers: implications for mercury bioaccumulation

The relative contribution of aquatic vs. terrestrial organic matter to the diet of consumers in fluvial environments and its effects on bioaccumulation of contaminants such as mercury (Hg) remain poorly understood. We used stable isotopes of carbon and nitrogen in a gradient approach (consumer isotope ratio vs. periphyton isotope ratio) across temperate streams that range in their pH to assess consumer reliance on aquatic (periphyton) vs. terrestrial (riparian vegetation) organic matter, and whether Hg concentrations in fish and their prey were related to these energy sources. Taxa varied in their use of the two sources, with grazing mayflies (Heptageniidae), predatory stoneflies (Perlidae), one species of water strider (Metrobates hesperius), and the fish blacknose dace (Rhinichthys atratulus) showing strong connections to aquatic sources, while Aquarius remigis water striders and brook trout (Salvelinus fontinalis) showed a weak link to in-stream production. The aquatic food source for consumers, periphyton, had higher Hg concentrations in low-pH waters, and pH was a much better predictor of Hg in predatory invertebrates that relied mainly on this food source vs. those that used terrestrial C. These findings suggest that stream biota relying mainly on dietary inputs from the riparian zone will be partially insulated from the effects of water chemistry on Hg availability. This has implications for the development of a whole-system understanding of nutrient and material cycling in streams, the choice of taxa in contaminant monitoring studies, and in understanding the fate of Hg in stream food webs.

Rapid carbon turnover during growth of Atlantic salmon (Salmo salar) smolts in sea water, and evidence for reduced food consumption by growth-stunts

Wild Atlantic salmon smolts were captured during spring out-migration in the Northwest Miramichi River, New Brunswick, Canada, and placed on an isotopically distinct hatchery diet to determine the relative contributions of growth and metabolic turnover to isotopic change. As expected for an ectothermic species, growth explained a large amount of isotopic variation in changing stable carbon ratios of muscle tissue (average r2= 0.46 ) for the 3 years of study. Turnover rates of muscle carbon in all 3 years in growing fish (0.24–0.66 month−1) were higher than previously reported values for other ectothermic species, but there was little evidence for isotopic change in non-growers (average r2= 0.041, p > 0.1). It is unlikely that non-growers had consumed any of the hatchery diet over a 2-month period, thus preventing them from acquiring the new carbon isotopic signature. This period of food deprivation resulted in nitrogen-15 enrichment in liver relative to muscle (p= 0.003). It is advised that future isotope studies of metabolic turnover rates in ectotherms be conducted on slow-growing animals over a long time period. This would serve to avoid the obscuring effects of growth on isotopic change, and provide stronger comparisons to endothermic tissue turnover rates.

Productivity, Disturbance and Ecosystem Size Have No Influence on Food Chain Length in Seasonally Connected Rivers

The food web is one of the oldest and most central organising concepts in ecology and for decades, food chain length has been hypothesised to be controlled by productivity, disturbance, and/or ecosystem size; each of which may be mediated by the functional trophic role of the top predator. We characterised aquatic food webs using carbon and nitrogen stable isotopes from 66 river and floodplain sites across the wet-dry tropics of northern Australia to determine the relative importance of productivity (indicated by nutrient concentrations), disturbance (indicated by hydrological isolation) and ecosystem size, and how they may be affected by food web architecture. We show that variation in food chain length was unrelated to these classic environmental determinants, and unrelated to the trophic role of the top predator. This finding is a striking exception to the literature and is the first published example of food chain length being unaffected by any of these determinants. We suggest the distinctive seasonal hydrology of northern Australia allows the movement of fish predators, linking isolated food webs and potentially creating a regional food web that overrides local effects of productivity, disturbance and ecosystem size. This finding supports ecological theory suggesting that mobile consumers promote more stable food webs. It also illustrates how food webs, and energy transfer, may function in the absence of the human modifications to landscape hydrological connectivity that are ubiquitous in more populated regions.