Bioenergetic Shifts in Humpback Whale Fibroblasts Upon Chemical Exposure.

Southern Hemisphere humpback whales accumulate persistent and toxic chemicals, which are transported to Antarctica through distant sources and in situ usage. The extreme seasonal migration-associated fast of humpback whales results in the remobilization of persistent and lipophilic environmental contaminants from liberated fat stores. Mitochondria play a key role in lipid metabolism, and any disruption to mitochondrial function is expected to influence whole-organism bioenergetics. It is therefore of interest to advance understanding of the impact of known contaminants of the Antarctic sea-ice ecosystem upon humpback whale cellular bioenergetics. Using cell line-based in vitro testing, this study employed the Seahorse Extracellular Flux Analyzer to study cellular metabolic activity in live humpback whale fibroblast cells. The assay, based on oxygen consumption rate, provides insights into the cause of cellular bioenergetic disruption. Immortalized skin fibroblasts were exposed to four priority environmental chemicals found in the Antarctic sea-ice ecosystem. Our findings reveal chemical-dependent functional alterations and varying bioenergetic profile responses. Chlorpyrifos was observed to decrease mitochondrial basal oxygen consumption; dieldrin increased basal oxygen consumption; trifluralin's impact was dose-specific, and endosulfan displayed no effect. Our results provide unique insights into environmental chemical mechanisms of action on cellular bioenergetics, generating much-needed taxa-specific chemical effect data in support of evidence-based conservation policy and management.

Novel Use of Cell Profiling Technology to Visualize Mitochondrial Responses of Humpback Whale Fibroblasts to Chemical Exposure.

Cetaceans are at elevated risk of accumulating persistent and lipophilic environmental contaminants due to their longevity and high proportion of body fat. Despite this, there is a paucity of taxa-specific chemical effect data, in part due to the ethical and logistical constraints in working with highly mobile aquatic species. Advances in cetacean cell culture have opened the door to the application of mainstream in vitro toxicological effect assessment approaches. Image-based cell profiling is a high-throughput, microscopy-based system commonly applied in drug development. It permits the analysis of the xenobiotic effect on multiple cell organelles simultaneously, hereby flagging its potential utility in the evaluation of chemical toxicodynamics. Here we exposed immortalized humpback whale skin fibroblasts (HuWaTERT) to six priority environmental contaminants known to accumulate in the Southern Ocean food web, in order to explore their subcellular organelle responses. Results revealed chemical-dependent modulation of mitochondrial texture, with the lowest observed effect concentrations for chlorpyrifos, dieldrin, trifluralin, and p,p'-dichlorodiphenyldichloroethane of 0.3, 4.1, 9.3, and 19.8 nM, respectively. By contrast, no significant changes were observed upon exposure to endosulfan and lindane. This study contributes the first fixed mitochondrial images of HuWaTERT and constitutes novel, taxa-specific chemical effect data in support of evidence-based conservation policy and management.

Antarctic sea-ice low resonates in the ecophysiology of humpback whales.

The past six years have been marked by some of the most dramatic climatic events observed in the Antarctic region in recent history, commencing with the 2017 sea-ice extreme low. The Humpback Whale Sentinel Programme is a circum-polar biomonitoring program for long term surveillance of the Antarctic sea-ice ecosystem. It has previously signalled the extreme La Niña event of 2010/11, and it was therefore of interest to assess the capacity of existing biomonitoring measures under the program to detect the impacts of 2017 anomalous climatic events. Six ecophysiological markers of population adiposity, diet, and fecundity were targeted, as well as calf and juvenile mortality via stranding records. All indicators, with the exception of bulk stable isotope dietary tracers, indicated a negative trend in 2017, whilst C and N bulk stable isotopes appeared to indicate a lag phase resulting from the anomalous year. The collation of multiple biochemical, chemical, and observational lines of evidence via a single biomonitoring platform provides comprehensive information for evidence-led policy in the Antarctic and Southern Ocean region.

Mercury levels in humpback whales, and other Southern Ocean marine megafauna.

Mercury is a known potent neurotoxin. The biogeochemical cycle of mercury in the remote Antarctic region is still poorly understood, with Polar climate change contributing added complexity. Longitudinal biomonitoring of mercury accumulation in Antarctic marine megafauna can contribute top-down insight into the bio-physical drivers of wildlife exposure. The bioaccumulative nature of organic mercury renders high trophic predators at the greatest risk of elevated exposure. Humpback whales represent secondary consumers of the Antarctic sea-ice ecosystem and an ideal biomonitoring species for persistent and bioaccumulative compounds due to their extended life-spans. This study provides the first results of mercury accumulation in humpback whales, and places findings within the context of mercury accumulation in both prey, as well as six other species of Antarctic marine megafauna. Combined, these findings contribute new baseline information regarding mercury exposure to Antarctic wildlife, and highlights methodological prerequisites for routine mercury biomonitoring in wildlife via non-lethally biopsied superficial tissues.

Sex ratios of migrating southern hemisphere humpback whales: A new sentinel parameter of ecosystem health.

Southern hemisphere humpback whales have evolved energetically demanding capital breeding and migratory life-history behaviours. It has been hypothesised that not all individuals of a population participate in the seasonal migration each year, or only undertake partial migrations. Given the cost of migration and reproduction, we explored the possibility that specifically, not all mature females participate in the seasonal migration every year, or significantly delay or shorten their migration, in response to poor feeding conditions. That is, females must attain a minimum threshold of accumulated energy reserves to commit to a reproductive event that likely occurs as a product of mating during migration. With a 1:1 male to female birth ratio, yet a male bias observed along the main migratory corridor; this study utilised inter-annual migratory cohort sex ratios to explore their potential to serve as measures of population fecundity, as a function of ecosystem health. The sex ratios of randomly biopsied adult humpback whales, sampled at a defined location and set time-points along the main migratory corridor from 2008 to 2016 were investigated. Northward migration sex ratios in 2009, 2014 and 2016 revealed a lower male bias suggesting good female participation in the migration and therefore apparent optimal provisioning during the two preceding summers. By contrast, the 2011 southward migration, revealed the highest male bias recorded of 5.75:1. Southward migration sex ratios were found to oscillate closely with measures of population adiposity, a sentinel parameter employed for long-term surveillance of the Antarctic sea-ice ecosystems under the Southern Ocean Observing System-endorsed Humpback Whale Sentinel Program. Anomalously poor humpback whale body condition recorded in 2011 was attributed to poor Antarctic feeding conditions during the extreme La Niña event of 2010/11. These findings lend support for the application of migratory cohort sex ratios, standardised by time and location, as a measure of relative inter-annual population fecundity. This work therefore contributes a new non-lethal tool for the study of population health, as a function of ecosystem productivity, and facilitates the inclusion of fecundity as a sentinel parameter into long-term Antarctic ecosystem surveillance under the Humpback Whale Sentinel Program.

Turning microplastics into nanoplastics through digestive fragmentation by Antarctic krill.

Microplastics (plastics <5 mm diameter) are at the forefront of current environmental pollution research, however, little is known about the degradation of microplastics through ingestion. Here, by exposing Antarctic krill (Euphausia superba) to microplastics under acute static renewal conditions, we present evidence of physical size alteration of microplastics ingested by a planktonic crustacean. Ingested microplastics (31.5 µm) are fragmented into pieces less than 1 µm in diameter. Previous feeding studies have shown spherical microplastics either; pass unaffected through an organism and are excreted, or are sufficiently small for translocation to occur. We identify a new pathway; microplastics are fragmented into sizes small enough to cross physical barriers, or are egested as a mixture of triturated particles. These findings suggest that current laboratory-based feeding studies may be oversimplifying interactions between zooplankton and microplastics but also introduces a new role of Antarctic krill, and potentially other species, in the biogeochemical cycling and fate of plastic.

Signals from the south; humpback whales carry messages of Antarctic sea-ice ecosystem variability.

Southern hemisphere humpback whales (Megaptera novaeangliae) rely on summer prey abundance of Antarctic krill (Euphausia superba) to fuel one of the longest-known mammalian migrations on the planet. It is hypothesized that this species, already adapted to endure metabolic extremes, will be one of the first Antarctic consumers to show measurable physiological change in response to fluctuating prey availability in a changing climate; and as such, a powerful sentinel candidate for the Antarctic sea-ice ecosystem. Here, we targeted the sentinel parameters of humpback whale adiposity and diet, using novel, as well as established, chemical and biochemical markers, and assembled a time trend spanning 8 years. We show the synchronous, inter-annual oscillation of two measures of humpback whale adiposity with Southern Ocean environmental variables and climate indices. Furthermore, bulk stable isotope signatures provide clear indication of dietary compensation strategies, or a lower trophic level isotopic change, following years indicated as leaner years for the whales. The observed synchronicity of humpback whale adiposity and dietary markers, with climate patterns in the Southern Ocean, lends strength to the role of humpback whales as powerful Antarctic sea-ice ecosystem sentinels. The work carries significant potential to reform current ecosystem surveillance in the Antarctic region.

Persistent Organic Pollutants in the East Antarctic Atmosphere: Inter-Annual Observations from 2010 to 2015 Using High-Flow-Through Passive Sampling.

In the first multiyear sampling effort for POPs in the eastern Antarctic atmosphere, 32 PCBs and 38 organochlorine pesticides were targeted in air collected with a high-flow-through passive sampler. Agricultural chemicals were found to dominate atmospheric profiles, in particular HCB and endosulfan-I, with average concentrations of 12 600 and 550 fg/m3, respectively. HCB showed higher concentrations in the austral summer, indicative of local, temperature-dependent volatilisation, while endosulfan-I appeared to show fresh, late-austral-summer input followed by temporally decreasing levels throughout the year. The current-use herbicide, trifluralin, and the legacy pesticides mirex and toxaphene, were detected in Antarctic air for the first time. Trifluralin was observed at low but increasing levels over the five-year period. Its detection in the Antarctic atmosphere provides evidence of its persistence and long-range environmental transport capability. While a time frame of five years exceeds the duration of most Antarctic air monitoring efforts, it is projected that continuous monitoring at the decadal scale is required to detect an annual 10% change in atmospheric concentrations of key analytes. This finding emphasizes the importance of continuous, long-term monitoring efforts in polar regions, that serve a special role as sentinel environments of hemispheric chemical usage trends.

Spring Melt and the Redistribution of Organochlorine Pesticides in the Sea-Ice Environment: A Comparative Study between Arctic and Antarctic Regions.

Complementary sampling of air, snow, sea-ice, and seawater for a range of organochlorine pesticides (OCPs) was undertaken through the early stages of respective spring sea-ice melting at coastal sites in northeast Greenland and eastern Antarctica to investigate OCP concentrations and redistribution during this time. Mean concentrations in seawater, sea-ice and snow were generally greater at the Arctic site. For example, α-HCH was found to have the largest concentrations of all analytes in Arctic seawater and sea-ice meltwater samples (224-253 and 34.7-48.2 pg·L-1 respectively compared to 1.0-1.3 and <0.63 pg·L-1 respectively for Antarctic samples). Differences in atmospheric samples were generally not as pronounced however. Findings suggest that sea-ice OCP burdens originate from both snow and seawater. The distribution profile between seawater and sea-ice showed a compound-dependency for Arctic samples not evident with those from the Antarctic, possibly due to full submersion of sea-ice at the former. Seasonal sea-ice melt processes may alter the exchange rates of selected OCPs between air and seawater, but are not expected to reverse their direction, which fugacity modeling indicates is volatilisation in the Arctic and net deposition in the Antarctic. These predictions are consistent with the limited current observations.

A nutritional-toxicological assessment of Antarctic krill oil versus fish oil dietary supplements.

Fish oil dietary supplements and complementary medicines are pitched to play a role of increasing strategic importance in meeting daily requirements of essential nutrients, such as long-chain (≥ C20, LC) omega-3 polyunsaturated fatty acids and vitamin D. Recently a new product category, derived from Antarctic krill, has been launched on the omega-3 nutriceutical market. Antarctic krill oil is marketed as demonstrating a greater ease of absorption due to higher phospholipid content, as being sourced through sustainable fisheries and being free of toxins and pollutants; however, limited data is available on the latter component. Persistent Organic Pollutants (POP) encompass a range of toxic, man-made contaminants that accumulate preferentially in marine ecosystems and in the lipid reserves of organisms. Extraction and concentration of fish oils therefore represents an inherent nutritional-toxicological conflict. This study aimed to provide the first quantitative comparison of the nutritional (EPA and DHA) versus the toxicological profiles of Antarctic krill oil products, relative to various fish oil categories available on the Australian market. Krill oil products were found to adhere closely to EPA and DHA manufacturer specifications and overall were ranked as containing intermediate levels of POP contaminants when compared to the other products analysed. Monitoring of the pollutant content of fish and krill oil products will become increasingly important with expanding regulatory specifications for chemical thresholds.

Metabolic concentration of lipid soluble organochlorine burdens in the blubber of southern hemisphere humpback whales through migration and fasting.

Southern hemisphere humpback whales undertake the longest migrations and associated periods of fasting of any mammal. Fluctuations in lipid energy stores are known to profoundly affect the toxicokinetics of lipophilic organochlorine compound (OC) burdens. Results from blubber biopsy sampling of adult, male humpback whales at two time points of the annual migration journey revealed dramatic concentration effects for the majority of OC compounds. The observed concentration effect was, however, not linear with measured average blubber lipid loss indicating significant redistribution of OCs and hence the importance of alternate lipid depots for meeting the energetic demands of the migration journey. Applying lipophilic OC burdens as novel tracers of whole-body lipid dynamics, the observed average concentration index suggests an average individual weight loss of 13% over 4 months of the migration journey. This value is based upon lipid derived energy and is in good agreement with previous weight prediction formulas. Notably, however, these estimates may greatly underestimate individual weight loss if significant protein catabolism occurs. Biomagnification factors between migrating southern hemisphere humpback whales and their principal prey item, Antarctic krill, closely resembled those of baleen whales feeding on herbivorous zooplankton in the Arctic. This study emphasizes the importance of considering prolonged periods of food deprivation when assessing chemical risks posed to wildlife. This is of particular importance for Polar biota adapted to extremes in ecosystem productivity.

Dietary exposure of Antarctic krill to p,p'-DDE: uptake kinetics and toxicological sensitivity in a key polar species.

This study evaluated the dietary uptake kinetics and sublethal toxicity of p,p'-dichlorodiphenyl dichloroethylene (p,p'-DDE) in Antarctic krill. The uptake rate constant (characterised by the seawater volume stripped of contaminant sorbed to algae) of 200 ± 0.32 mL g(-1) wet weight h(-1), average absorption efficiency of 86 ± 13% and very low elimination rate constant of 5 × 10(-6) ± 0.0031 h(-1) demonstrate the importance of feeding for p,p'-DDE bioaccumulation in Antarctic krill. Faecal egestion of unabsorbed p,p'-DDE of 8.1 ± 2.7% indicates that this pathway contributes considerably to p,p'-DDE sinking fluxes. A median internal effective concentration (IEC50) of 15 mmol/kg lipid weight for complete immobility indicates baseline toxicity and that Antarctic krill exhibit comparable toxicological sensitivity as temperate species under similar 10 d exposure conditions. These findings support the critical body residue approach and provide insight to the role of Antarctic krill in the biogeochemical cycling of p,p'-DDE in the Southern Ocean.

Behavioural sensitivity of a key Southern Ocean species (Antarctic krill, Euphausia superba) to p,p'-DDE exposure.

Persistent organic pollutants (POPs) have been frequently measured throughout the Southern Ocean food web for which little information is available to assess the potential risks of POP exposure. The current study evaluated the toxicological sensitivity of a key Southern Ocean species, Antarctic krill, to aqueous exposure of p,p'-dichlorodiphenyl dichloroethylene (p,p'-DDE). Behavioural endpoints were used as indicators of sublethal toxicity. Immediate behavioural responses (partial immobility and tail flicking) most likely reflect neurotoxicity, while the p,p'-DDE body residue causing a median level of sublethal toxicity in Antarctic krill following 96h exposure (IEC50(sublethal toxicity)=3.9±0.21mmol/kg lipid weight) is comparable to those known to cause sublethal narcosis in temperate aquatic species. Critical body residues (CBRs) were more reproducible across tests than effective seawater concentrations. These findings support the concept of the CBR approach, that effective tissue residues are comparable across species and geographical ranges despite differences in environmental factors.

Aqueous uptake and sublethal toxicity of p,p'-DDE in non-feeding larval stages of Antarctic krill (Euphausia superba).

This study evaluated the toxicological sensitivity of non-feeding larval stages of a key Antarctic species (Antarctic krill, Euphausia superba) to p,p'-dichlorodiphenyl dichloroethylene (p,p'-DDE) exposure. The aqueous uptake clearance rate of 84 mL g(-1) preserved weight (p.w.) h(-1) determined for p,p'-DDE in Antarctic krill larvae is comparable to previous findings for small cold water crustaceans and five times slower than the rates reported for an amphipod inhabiting warmer waters. Natural variations in larval physiology appear to influence contaminant uptake and larval krill behavioural responses, strongly highlighting the importance of time of measurement for ecotoxicological testing. Sublethal narcosis (immobility) was observed in larval Antarctic krill from p,p'-DDE body residues of 0.2 mmol/kg p.w., which is in agreement with findings for adult krill and temperate aquatic species. The finding of comparable body residue-based toxicity of p,p'-DDE between polar and temperate species supports the tissue residue approach for environmental risk assessment of polar ecosystems.

Altered developmental timing in early life stages of Antarctic krill (Euphausia superba) exposed to p,p'-DDE.

Persistent organic pollutants (POPs) are persistent, toxic and bioaccumulative anthropogenic organic chemicals, capable of undergoing long range environmental transport to remote areas including the Antarctic. p,p'-dichlorodiphenyl dichloroethylene (p,p'-DDE) has been identified as a dominant POP accumulating in Antarctic krill (Euphausia superba), which is a key Southern Ocean species. This study examined the developmental toxicity of p,p'-DDE via aqueous exposure to Antarctic krill larvae. p,p'-DDE exposure was found to stimulate developmental timing in the first three larval stages of Antarctic krill, while extended monitoring of larvae after a five day exposure period had ended, revealed delayed inhibitory responses during development to the fourth larval stage. Stimulatory responses were observed from the lowest p,p'-DDE body residue tested of 10.1±3.0 µmol/kg (3.2±0.95 mg/kg) preserved wet weight, which is comparable to findings for temperate species and an order of magnitude lower than the exposure level found to cause sublethal behavioural effects in Antarctic krill. The delayed responses included increased mortality, which had doubled in the highest p,p'-DDE treatment (95±8.9% mortality at 20 µg/L p,p'-DDE) compared to the solvent control (44±11% mortality) 2 weeks after end of exposure. Development of surviving metanauplius larvae to calyptopis 1 larvae was delayed by 2 days in p,p'-DDE exposed larvae compared with untreated larvae. Finally, the developmental success of surviving p,p'-DDE exposed larvae was reduced by 50 to 75% compared to the solvent control (100% developmental success). The lowest observed effect concentration for all delayed effects was 1 µg/L, the lowest exposure concentration tested. These findings demonstrate the importance of delayed and indirect effects of toxicant exposure. Further, the findings of this study are important for environmental risk assessment of POPs in the Southern Ocean ecosystem and strongly highlight the significance of developmental endpoints for ecotoxicological testing.

Rapid exposure assessment of PSII herbicides in surface water using a novel chlorophyll a fluorescence imaging assay.

Recently a new Maxi-Imaging-PAM (Max-I-PAM) instrument for phytotoxicity assessment via chlorophyll fluorescence imaging was introduced. This new instrument allows rapid detection of the effects of PS II inhibiting herbicides which are high use agricultural chemicals frequently detected in surface waters in Australia and elsewhere. Several studies have applied the new instrument for detection of phytotoxicants in water using microalgae suspensions; however, these use preliminary protocols and to date no validated method is available for high throughput testing of environmental samples in 96-well plates. Here we developed and applied a new protocol allowing dose-response assessment of four samples within 2 h (8 dilutions in duplicate). The technique was found to be sensitive, with a detection limit of 2.3 ng l(-1) for the herbicide diuron when testing solid phase extracts (SPE) of 1000 ml water samples, and reproducible both between experiments (coefficient of variation (CV)=0.30) and within the 96-well plate (CV=0.06). Relative potencies were determined for four reference PS II impacting herbicides (diuron>hexazinone>atrazine>simazine). Extracts from 1000 ml environmental samples and diuron spiked ultrapure water as well as passive sampler extracts were evaluated and good agreement was found between diuron equivalent concentrations calculated from bioassay results (DEQ(IPAM)) and DEQ(CHEM) values calculated from LCMS chemical analysis of the four reference compounds in the same samples. Overall, the technique provides a valuable bioanalytical tool for rapid and inexpensive effects-based assessment of PS II impacting herbicides in environmental mixtures.