Mechanistic characterization of waterborne selenite uptake in the water flea, Daphnia magna, indicates water chemistry affects toxicity in coal mine-impacted waters.

Concentrations of selenium that exceed regulatory guidelines have been associated with coal mining activities and have been linked to detrimental effects on aquatic ecosystems and the organisms therein. Although the major route of selenium uptake in macroinvertebrates is via the diet, the uptake of waterborne selenite (HSeO3-), the prominent form at circumneutral pH, can be an important contributor to selenium body burden and thus selenium toxicity. In the current study, radiolabelled selenite (Se75) was used to characterize the mechanism of selenite uptake in the water flea, Daphnia magna. The concentration dependence (1-32 µM) of selenite uptake was determined in 1-hour uptake assays in artificial waters that independently varied in bicarbonate, chloride, sulphate, phosphate and selenate concentrations. At concentrations representative of those found in highly contaminated waters, selenite uptake was phosphate-dependent and inhibited by foscarnet, a phosphate transport inhibitor. At higher concentrations, selenite uptake was dependent on waterborne bicarbonate concentration and inhibited by the bicarbonate transporter inhibitor DIDS (4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid). These findings suggest that concentrations of phosphate in coal mining-affected waters could alter selenite uptake in aquatic organisms and could ultimately affect the toxic impacts of selenium in such waters.

Effects of Acute and Subchronic Waterborne Thallium Exposure on Ionoregulatory Enzyme Activity and Oxidative Stress in Rainbow Trout (Oncorhynchus mykiss).

The mechanisms of acute (96-hour) and subchronic (28-day) toxicity of the waterborne trace metal thallium (Tl) to rainbow trout (Oncorhynchus mykiss) were investigated. Specifically, effects on branchial and renal ionoregulatory enzymes (sodium/potassium adenosine triphosphatase [ATPase; NKA] and proton ATPase) and hepatic oxidative stress endpoints (protein carbonylation, glutathione content, and activities of catalase and glutathione peroxidase) were examined. Fish (19-55 g) were acutely exposed to 0 (control), 0.9 (regulatory limit), 2004 (half the acute median lethal concentration), or 4200 (acute median lethal concentration) µg Tl L-1 or subchronically exposed to 0, 0.9, or 141 (an elevated environmental concentration) µg Tl L-1 . The only effect following acute exposure was a stimulation of renal H+ -ATPase activity at the highest Tl exposure concentration. Similarly, the only significant effect of subchronic Tl exposure was an inhibition of branchial NKA activity at 141 µg Tl L-1 , an effect that may reflect the interaction of Tl with potassium ion handling. Despite significant literature evidence for effects of Tl on oxidative stress, there were no effects of Tl on any such endpoint in rainbow trout, regardless of exposure duration or exposure concentration. Elevated basal levels of antioxidant defenses may explain this finding. These data suggest that ionoregulatory perturbance is a more likely mechanism of Tl toxicity than oxidative stress in rainbow trout but is an endpoint of relevance only at elevated environmental Tl concentrations. Environ Toxicol Chem 2024;43:87-96. © 2023 SETAC.

Waterborne amino acids: uptake and functional roles in aquatic animals.

Dissolved organic matter is a ubiquitous component of freshwater and marine environments, and includes small nutrient molecules, such as amino acids, which may be available for uptake by aquatic biota. Epithelial transporters, including cotransporters, uniporters and antiporters, facilitate the absorption of dissolved amino acids (often against concentration gradients). Although there is a lack of mechanistic and molecular characterization of such transporters, pathways for the direct uptake of amino acids from the water appear to exist in a wide range of marine phyla, including Porifera, Cnidaria, Platyhelminthes, Brachiopoda, Mollusca, Nemertea, Annelida, Echinodermata, Arthropoda and Chordata. In these animals, absorbed amino acids have several putative roles, including osmoregulation, hypoxia tolerance, shell formation and metabolism. Therefore, amino acids dissolved in the water may play an important, but overlooked, role in aquatic animal nutrition.

Mechanistic examination of thallium and potassium interactions in Daphnia magna.

The trace element thallium (Tl) exerts its toxic effects, at least in part, through its mimicry of potassium (K+) and subsequent impairment of K+ homeostasis. However, the specific nature of this effect remains poorly understood, especially in aquatic biota that are threatened by elevated concentrations of Tl associated with mining and refining effluents. In this study experiments were conducted to mechanistically examine the relationship between Tl and K+ in terms of uptake and toxicity in the regulatory model species Daphnia magna. In one set of experiments the effects of K+, the K+ analog rubidium (Rb+), and generalized K+ channel blocker cesium (Cs+) on Tl-induced acute toxicity were examined. The presence of increasing concentrations of K+ and Rb+ in exposure water reduced waterborne Tl toxicity, indicating that the actions of Tl were mediated at least in part through interactions with K+. However, in the presence of elevated water Cs+, the toxicity of Tl paradoxically increased. Pharmaceuticals with putative blocking actions on K+ channels failed to alter whole-body K+ of control organisms, but in the case of clozapine and chlorpropamide, whole-body K+ status was significantly elevated relative to exposures with Tl alone, which tended to reduce this metric. These data identify inwardly rectifying and voltage gated K+ channels as potential loci of Tl toxicity. Experiments using rubidium (Rb+) as a tracer of K+, showed that waterborne Tl affects the uptake of K+, but the magnitude of inhibition by Tl was not sufficient to explain the effect on whole-body K+. While these data indicate interactions between Tl and K occur at K+ transporters in D magna, they also indicate that environmental levels of K+ are likely to ameliorate toxicity in most natural waters.

Accumulation of Thallium in Rainbow Trout (Oncorhynchus mykiss) Following Acute and Subchronic Waterborne Exposure.

The accumulation and tissue distribution of toxicants in aquatic biota can be determinative of their toxic impact to both exposed organisms and their potential human consumers. In the present study, accumulation of the trace metal thallium (Tl) in gill, muscle, plasma, and otoliths of rainbow trout (Oncorhynchus mykiss) following acute (96-h) and subchronic (28-day) waterborne exposures was investigated. Owing to known interactions between Tl and potassium ions (K+ ), plasma and muscle K+ concentrations were also determined. Branchial Tl accumulated in a dose-dependent manner in both acute and subchronic exposures, while plasma Tl was rapidly mobilized to tissues and accumulated only at exposure concentrations of 141 µg L-1 or higher. For muscle tissue, Tl concentrations at 28 days were markedly lower than those at 96 h at comparable exposure concentrations (0.9 µg L-1 ), indicating the presence of mechanisms that act to reduce Tl accumulation over time. However, after acute exposure, muscle Tl reached concentrations that, if consumed, would exceed acceptable daily intake values for this element, indicating some risk to human health from the consumption of fish from waters heavily contaminated with Tl. Otoliths showed Tl concentrations that reflected exposure concentration and length, confirming their capacity to provide insight into fish exposure history. No changes in tissue K+ concentrations were observed, suggesting that accumulation of Tl in rainbow trout plasma and muscle does not occur at the expense of K+ homeostasis. In addition to highlighting the capacity of rainbow trout to accumulate Tl to levels that exceed recommended dietary doses to human consumers, the present study provides the first data of tissue-specific Tl accumulation in an important regulatory species. Environ Toxicol Chem 2023;42:1553-1563. © 2023 SETAC.

Physiological and biochemical responses of the estuarine pulmonate mud snail, Amphibola crenata, sub-chronically exposed to waterborne cadmium.

Physiological and biochemical responses of the pulmonate mud snail, Amphibola crenata, to waterborne cadmium (Cd) were investigated to determine the mechanisms of toxicity and impacts of a 21-d Cd exposure. Mud snails were exposed to nominal Cd concentrations of 0, 0.2, 4 and 8 mg L - 1 and bioaccumulation, whole animal physiological (oxygen consumption, ammonia excretion and oxygen:nitrogen), and tissue level biochemical (catalase activity, lipid peroxidation, glycogen, glucose and protein) endpoints were measured every 7 days. At the two highest Cd exposure concentrations complete mortality was observed over 21-d. In surviving animals, oxygen consumption declined and ammonia excretion rate increased with Cd exposure concentration and duration. The increased ammonia excretion likely reflected enhanced protein metabolism as suggested by a reduced oxygen:nitrogen (O:N). Increasing waterborne Cd concentration and exposure time led to increasing metal accumulation in all tissues. The snail viscera showed the highest Cd accumulation. Both catalase activity and lipid peroxidation in the viscera significantly increased with Cd exposure concentration and time, whereas, the foot muscle and remaining tissues (kidney, mantle, remaining digestive tissues and heart) showed increased catalase activity and lipid peroxidation at higher Cd concentrations (4 and 8 mg L - 1), suggestive of an effect of Cd on oxidative stress. Over the course of 21 days, Cd exposure resulted in significantly lower levels of glycogen in viscera relative to Cd-free controls, reflecting an increased energy demand. Haemolymph glucose rose initially and then fell with increased exposure duration, while haemolymph protein generally exhibited an increased concentration in Cd-exposure groups, reflecting the changes in energy substrates noted for somatic tissues. These results suggest that the physiological and biochemical responses of A. crenata to Cd are conserved relative to other aquatic animals, and were tissue-specific, dose- and time-dependant.

Sub-chronic exposure to waterborne extracellular microcystin-LR impairs calcium homeostasis in rainbow trout.

Fish mortality is associated with harmful algal blooms, although whether toxicity is related directly to the presence of cyanotoxins or the prevailing water chemistry remains unclear. Similarly, while planktivorous fish may be exposed to toxin through the diet, the hazard posed by waterborne extracellular toxin to carnivorous fish is less well understood. In this study rainbow trout (Oncorhynchus mykiss) were exposed for up to 28 d to waterborne microcystin-LR at nominal concentrations of 1.5 and 50 µg L-1 (measured values 2 and 49 µg L-1, respectively). The former represents the Canadian drinking water guideline, and the latter an elevated environmental level. This study hypothesised that waterborne toxin exposure would specifically impact gill function, and given the importance of this tissue in freshwater fish ion regulation, effects on plasma ions and branchial ion transporter activity would be observed. Microcystin-LR exposure resulted in a significant and persistent hypocalcaemia at the higher exposure concentration, but plasma sodium and branchial activities of the sodium/potassium ATPase, proton ATPase and calcium ATPase enzymes remained unaffected. An in vitro assessment failed to show any effect of microcystin-LR on branchial calcium ATPase activity even at exposure concentrations as high as 1000 µg L-1. A transient increase in hepatic alkaline phosphatase activity was also observed at 49 µg L-1, but there were no effects of toxin exposure on branchial or hepatic lactate dehydrogenase activity. These results suggest that microcystin-LR exposure does not have a general effect on ion regulation, but instead produces a novel and specific impact on calcium metabolism in rainbow trout, although the mechanism underlying this effect remains unknown.

Factors Affecting the Binding of Diltiazem to Rainbow Trout Plasma: Implications for the Risk Assessment of Pharmaceuticals in Aquatic Systems.

The accumulation of organic toxicants in fish plasma, and how they partition between the bound and unbound fraction once absorbed, are important metrics in models that seek to predict the risk of such contaminants in aquatic settings. Rapid equilibrium dialysis of diltiazem, an ionizable weak base and important human pharmaceutical contaminant of freshwaters, was conducted with rainbow trout (Oncorhynchus mykiss) plasma. The effect of fed state, fish sex, fish strain/size, and dialysis buffer pH on the binding of radiolabeled diltiazem (9 ng ml-1 ) was assessed. In fed fish, 24.6%-29.5% of diltiazem was free, unbound to plasma proteins. Although starvation of fish resulted in a decrease in plasma protein, the bound fraction of diltiazem remained relatively constant. Consequently, the protein-bound concentration of diltiazem increased with length of starvation. In general, rainbow trout strain was a significant factor affecting plasma binding, although the two strains tested also differed markedly in size. Dialysis buffer pH significantly influenced plasma binding, with a higher unbound diltiazem fraction at pH 6.8 than pH 8.0. These data indicate that empirical measures of plasma binding in fish are important for accurate risk assessment and that the physiological status of a fish is likely to impact its sensitivity to toxicants such as diltiazem. Environ Toxicol Chem 2022;41:3125-3133. © 2022 SETAC.

Effect of thallium on phototactic behaviour in Daphnia magna.

Thallium (Tl) is a trace metal enriched in wastewaters associated with mining and smelting of base metals. The toxicity of Tl to aquatic biota is poorly understood, particularly with respect to its sublethal effects. In this study, phototactic behavioural responses of naïve (i.e. no previous exposure to Tl) Daphnia magna, a key regulatory freshwater crustacean species, were examined in waters containing Tl. Fed and fasted neonate daphnids (< 24 h old) and fed adults (10-15 days old) showed no significant response at any tested water Tl concentration. However, in fasted adults, an increase in the positive phototactic response (measured as a greater number of daphnids closer to the light source after a 5-min exposure) was seen at Tl concentrations of 917 and 2099 µg L-1, values representative of extreme environmental Tl concentrations. The presence of Tl also decreased the swimming speed of adult Daphnia towards a light source. In the presence of cimetidine, a histamine receptor blocker, the increase in positive phototaxis induced by Tl disappeared, suggesting that Tl acts to perturb the phototaxis response through sensory inhibition. Conversely, although there was a trend towards enhanced activity, Tl had no significant effect on acetylcholinesterase, a marker of locomotor capacity.

The relationship between population attributes of the mud snail Amphibola crenata and sediment contamination: A multi-estuary assessment.

This study assessed the potential of the New Zealand mud snail Amphibola crenata to act as a bioindicator of contaminated estuarine sediment. Seventeen sites with varying contaminant burdens were identified within six New Zealand regions. Attributes (population density, individual length distribution and individual dry weight condition index) were measured for field-collected A. crenata, and related to measurements of sediment trace metals and nutrients. Population density of the mud snail was relatively high in sites with elevated nutrients and organic matter. The length distribution of A. crenata showed significant regional and site-specific variations. Minimum, mean, and median shell length of A. crenata were positively correlated with sediment cadmium and zinc concentration. Overall, the sites were able to be distinguished by A. crenata population attributes and the sediment metal and nutrient content. These results suggest that A. crenata population information has potential value for assessing estuarine sediment metal and nutrient contamination.

Acute waterborne strontium exposure to rainbow trout: Tissue accumulation, ionoregulatory effects, and the modifying influence of waterborne calcium.

Flowback and produced water (FPW) is an end-product of the hydraulic fracturing method of oil and gas extraction that is highly enriched in alkaline earth metals such as strontium (Sr). While Sr concentrations in FPW can exceed toxic thresholds for fish, the accompanying high concentrations of calcium (Ca) in FPW may ameliorate any toxicity. In this study, Sr bioaccumulation and molecular, biochemical, and physiological changes in ionoregulatory endpoints were investigated in rainbow trout (Oncorhynchus mykiss). Exposures were conducted over a 96-h period at Sr concentrations ranging from 1.7 to 1948 µM, with effects at the highest Sr exposure concentration also separately examined in waters of varying Ca concentration (10 to 958 µM). Plasma and gill Sr burdens increased as a function of increasing waterborne Sr, and accumulation increased further as water Ca concentrations were lowered. Despite this, there was no consistent, dose-dependent effect of Sr on plasma or gill Ca concentrations, although impacts on plasma and branchial sodium (Na) concentrations were observed. Waterborne Sr significantly inhibited branchial Ca2+-ATPase activity, albeit only at the highest tested Sr concentration (1948 µM). In exposure treatments where Sr was highly elevated and water Ca was reduced, the hepatic gene expression of Ca signaling receptors ß-2 adrenergic receptor (Adrb2) and inositol-1,4,5-triphosphate receptor-2 (Itpr2) were inhibited, highlighting novel potential pathways of Sr toxicity in rainbow trout. Overall, these data indicate that water Ca has a strong effect on Sr bioavailability, but over an acute exposure period there is limited evidence for an effect of Sr on Ca homeostasis. Although Sr is elevated in effluents associated with the oil and gas industry, the co-occurrence of high Ca concentrations might protect freshwater fish against acute effects related to Sr exposure.

Radium in New Zealand agricultural soils: Crop uptake and estimation of current and future ionising radiation dose.

Crop uptake of 226Ra over a range of key New Zealand agricultural and horticultural growing areas was analysed to establish the dietary implications of an increase in soil 226Ra activity concentrations. Thirty crop samples, covering both feed and food commodities, were quantified for 226Ra activity concentrations, and concentration ratio (CRs) from the soil activity were calculated. The calculated CRs correlated with international default values for estimating crop uptake. Variation in CRs established that there was no increase in the crop activity concentration, relative to soil 226Ra from pasture foliage at a fertiliser impacted site, with a gradient of soil 226Ra activity concentrations. Based on the calculated CRs, the upper bound of the theoretical range of dietary exposures to 226Ra was 78.1 µSv/yr for teenage boys. Future forecasting of the increased dietary dose of 226Ra that might occur at the current soil loading rate, based on current fertiliser activity concentrations, confirmed that long-term loading of soil with 226Ra is unlikely to present a dietary risk. The forecast model calculated that the increase in dietary ionising radiation burden is unlikely to reach thresholds requiring regulatory intervention for two millennia.

Chemical niches and ionoregulatory traits: applying ionoregulatory physiology to the conservation management of freshwater fishes.

Alterations in water chemistry can challenge resident fish species. More specifically, chemical changes that disrupt ion balance will negatively affect fish health and impact physiological and ecological performance. However, our understanding of which species and populations are at risk from ionoregulatory disturbances in response to changing freshwater environments is currently unclear. Therefore, we propose a novel framework for incorporating ionoregulatory physiology into conservation management of inland fishes. This framework introduces the concepts of fundamental chemical niche, which is the tolerable range of chemical conditions for a given species based on laboratory experiments, and realized chemical niche, which is the range of chemical conditions in which a species resides based on distribution surveys. By comparing these two niches, populations that may be at risk from ionoregulatory disturbances and thus require additional conservation considerations can be identified. We highlight the potential for commonly measured ionoregulatory traits to predict fundamental and realized chemical niches but caution that some traits may not serve as accurate predictors despite being important for understanding ionoregulatory mechanisms. As a sample application of our framework, the minimum pH distribution (realized niche) and survival limit pH (fundamental niche) of several North American fishes were determined by systematic review and were compared. We demonstrate that ionoregulatory capacity is significantly correlated with a realized niche for many species, highlighting the influence of ionoregulatory physiology on fish distribution patterns along chemical gradients. Our aim is that this framework will stimulate further research in this field and result in a broader integration of physiological data into conservation management decisions for inland waters.

The effect of marine dissolved organic carbon on nickel accumulation in early life-stages of the sea urchin, Strongylocentrotus purpuratus.

Dissolved organic carbon (DOC) is known to ameliorate the toxicity of the trace metal nickel (Ni) to aquatic animals. In theory, this effect is mediated by the capacity of DOC to bind Ni, rendering it less bioavailable, with the resulting reduction in accumulation limiting toxicological effects. However, there is a lack of experimental data examining Ni accumulation in marine settings with natural sources of DOC. In the current study, radiolabelled Ni was used to examine the time- and concentration-dependence of Ni accumulation, using naturally sourced DOC, on developing larvae of the sea urchin Strongylocentrotus purpuratus. Contrary to prediction, the two tested natural DOC samples (collected from the eastern United States, DOC 2 (Seaview park, Rhode Island (SVP)) and DOC 7 (Aubudon Coastal Center, Connecticut)) which had previously been shown to protect against Ni toxicity, did not limit accumulation. The control (artificial seawater with no added DOC), and the DOC 2 sample could mostly be described as having saturable Ni uptake, whereas Ni uptake in the presence of DOC 7 was mostly linear. These data provide evidence that DOC modifies the bioavailability of Ni, through either indirect effects (e.g. membrane permeability) or by the absorption of DOC-Ni complexes. There was some evidence for regulation of Ni accumulation in later-stage embryos (96-h) where the bioconcentration factor for Ni declined with increasing Ni exposure concentration. These data have implications for predictive modelling approaches that rely on known relationships between Ni speciation, bioavailability and bioreactivity, by suggesting that these relationships may not hold for natural marine DOC samples in the developing sea urchin model system.

Hypoxia modifies calcium handling in the Pacific hagfish, Eptatretus stoutii.

Hagfishes may encounter low dissolved oxygen in their natural habitats, a consequence of association with hypoxic sediments and their feeding behaviour. In teleost fish, hypoxia exposure decreases ion uptake, speculated to be a mechanism for energy conservation. Although hagfishes osmoconform, they do regulate extracellular fluid concentrations of divalent cations such as calcium. The current study hypothesised that exposure of hagfish to hypoxia (0.4 kPA, 24 h) would reduce calcium uptake (determined via in vitro isolated skin and gut epithelial transport assays) and calcium accumulation (determined by in vivo whole animal exposures, using radiolabelled calcium (45Ca) to assess newly acquired calcium). A decrease in in vitro epidermal uptake was observed at sub-environmental calcium levels (10 µM), but not at environmental calcium levels (10 mM). No changes in gut calcium uptake were determined. Conversely, hypoxia led to a more rapid in vivo accumulation of calcium in tissues (skin, muscle, liver, heart, plasma, brain), mediated mostly by a significant increase in accumulation at the gill. These differences were only apparent after 1-h of exposure to the radiolabel (i.e., the last hour of the 24-h hypoxia exposure) and were not observed after 3 and 24 h periods of radiolabel exposure. This outcome was the opposite of the hypothesised effect. The reasons for a more rapid accumulation of calcium in hypoxic hagfish are unknown but may relate to roles for calcium in enhancing hypoxia tolerance in hagfishes or could be a consequence of changes in ventilatory frequency.

The skin of adult rainbow trout is not a significant site of ammonia clearance from the blood.

We hypothesized that the skin acts as an extrabranchial route for ammonia excretion in adult rainbow trout (Oncorhynchus mykiss) following high environmental ammonia (HEA) exposure. Trunks of control or HEA-exposed trout were perfused with saline containing 0 or 1 mmol l-1 NH4 + . Cutaneous ammonia excretion rates increased 2.5-fold following HEA exposure, however there was no difference in rates between trunks perfused with 0 or 1 mmol l-1 NH4 + . The skin is therefore capable of excreting its own ammonia load, but it does not clear circulating ammonia from the plasma.

Environmental DNA and environmental RNA: Current and prospective applications for biological monitoring.

Traditional environmental biomonitoring approaches have limitations in terms of species detectability and their capacity to account for spatial and temporal variation. Furthermore, as invasive techniques they can be harmful to individual organisms, populations and habitats. The application of non-invasive sampling methods that extract, isolate and identify nucleic acid sequences (i.e. DNA, RNA) from environmental matrices have significant potential for complementing, or even ultimately replacing, current methods of biological environmental assessment. These environmental DNA (eDNA) and environmental RNA (eRNA) techniques increase spatial and temporal acuity of monitoring, and in the case of the latter, may provide functional information regarding the health of individuals, and thus ecosystems. However, these assessments require robust analysis of factors such as the detectability and specificity of the developed assays. The presented work highlights the current and future uses of nucleic acid-based biomonitoring regimes, with a focus on fish and aquatic invertebrates and their utility for water quality, biodiversity and species-specific monitoring. These techniques are compared to traditional approaches, with a particular emphasis on the potential insights that could be provided by eRNA analysis, including the benefits of microRNAs as assay targets.

Reductionist approaches to the study of ionoregulation in fishes.

The mechanisms underlying ionoregulation in fishes have been studied for nearly a century, and reductionist methods have been applied at all levels of biological organization in this field of research. The complex nature of ionoregulatory systems in fishes makes them ideally suited to reductionist methods and our collective understanding has been dramatically shaped by their use. This review provides an overview of the broad suite of techniques used to elucidate ionoregulatory mechanisms in fishes, from the whole-animal level down to the gene, discussing some of the advantages and disadvantages of these methods. We provide a roadmap for understanding and appreciating the work that has formed the current models of organismal, endocrine, cellular, molecular, and genetic regulation of ion balance in fishes and highlight the contribution that reductionist techniques have made to some of the fundamental leaps forward in the field throughout its history.

Spatial and temporal change in trace element profiles in seawater, sediment and mussels associated with an earthquake rubble sea-fill.

Elemental profiles in seawater, sediment and green-lipped mussels (Perna canaliculus) were determined, following the deposition of earthquake demolition rubble into a newly developed sea-fill located adjacent to a busy commercial port with a history of diverse contaminant inputs. Zinc and lead displayed environmental concentrations that varied over time during sea-fill construction, and which declined with distance from the sea-fill, indicating that the sea-fill activity was the source of these two contaminants. A transplantation study using reference site mussels caged near the sea-fill, supported this finding. However, none of the trace metal burdens in resident or transplanted mussels were of regulatory concern. An integrated approach comprising the monitoring of multiple environmental matrices, examination of contaminant burdens as a function of time and distance from a putative point source, and the use of active biomonitoring techniques, is necessary to robustly identify novel contamination inputs in historically-polluted marine settings.