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.

Persisting Effects in Daphnia magna Following an Acute Exposure to Flowback and Produced Waters from the Montney Formation.

Hydraulic fracturing extracts oil and gas through the injection of water and proppants into subterranean formations. These injected fluids mix with the host rock formation and return to the surface as a complex wastewater containing salts, metals, and organic compounds, termed flowback and produced water (FPW). Previous research indicates that FPW is toxic to Daphnia magna (D. magna), impairing reproduction, molting, and maturation time; however, recovery from FPW has not been extensively studied. Species unable to recover have drastic impacts on populations on the ecological scale; thus, this study sought to understand if recovery from an acute 48 h FPW exposure was possible in the freshwater invertebrate, D. magna by using a combination of physiological and molecular analyses. FPW (0.75%) reduced reproduction by 30% and survivorship to 32% compared to controls. System-level quantitative proteomic analyses demonstrate extensive perturbation of metabolism and protein transport in both 0.25 and 0.75% FPW treatments after a 48 h FPW exposure. Collectively, our data indicate that D. magna are unable to recover from acute 48 h exposures to ≥0.25% FPW, as evidence of toxicity persists for at least 19 days post-exposure. This study highlights the importance of considering persisting effects following FPW remediation when modeling potential spill scenarios.

Context-dependent relationships between swimming, terrestrial jumping and body composition in the amphibious fish Kryptolebias marmoratus.

Understanding the mechanisms that create phenotypic variation within and among populations is a major goal of physiological ecology. Variation may be a consequence of functional trade-offs (i.e. improvement in one trait comes at the expense of another trait) or alternatively may reflect the intrinsic quality of an organism (i.e. some individuals are simply better overall performers than others). There is evidence for both ideas in the literature, suggesting that environmental context may mediate whether variation results from trade-offs or differences in individual quality. We tested this overarching 'context dependence' hypothesis by comparing the aquatic and terrestrial athletic performance of the amphibious fish Kryptolebias marmoratus captured from two contrasting habitats, a large pond and small burrows. Overall, pond fish were superior terrestrial athletes but burrow fish were better burst swimmers, suggestive of a performance trade-off at the population level. Within each population, however, there was no evidence of a performance trade-off. In burrow fish, athletic performance was positively correlated with muscle content and body condition, consistent with the individual quality hypothesis. In pond fish, there was only a relationship between glycolytic white muscle and aquatic burst performance. Notably, pond fish were in better body condition, which may mask relationships between condition and athletic performance. Overall, our data highlight that population-level trends are insufficient evidence for the existence of phenotypic trade-offs in the absence of similar within-population patterns. Furthermore, we only found evidence for the individual quality hypothesis in one population, suggesting that patterns of phenotypic covariance are context dependent.

Physiological and behavioural strategies of aquatic animals living in fluctuating environments.

Shallow or near-shore environments, such as ponds, estuaries and intertidal zones, are among the most physiologically challenging of all aquatic settings. Animals inhabiting these environments experience conditions that fluctuate markedly over relatively short temporal and spatial scales. Living in these habitats requires the ability to tolerate the physiological disturbances incurred by these environmental fluctuations. This tolerance is achieved through a suite of physiological and behavioural responses that allow animals to maintain homeostasis, including the ability to dynamically modulate their physiology through reversible phenotypic plasticity. However, maintaining the plasticity to adjust to some stresses in a dynamic environment may trade off with the capacity to deal with other stressors. This paper will explore studies on select fishes and invertebrates exposed to fluctuations in dissolved oxygen, salinity and pH. We assess the physiological mechanisms these species employ to achieve homeostasis, with a focus on the plasticity of their responses, and consider the resulting physiological trade-offs in function. Finally, we discuss additional factors that may influence organismal responses to fluctuating environments, such as the presence of multiple stressors, including parasites. We echo recent calls from experimental biologists to consider physiological responses to life in naturally fluctuating environments, not only because they are interesting in their own right but also because they can reveal mechanisms that may be crucial for living with increasing environmental instability as a consequence of climate change.

Investigating the Potential Toxicity of Hydraulic Fracturing Flowback and Produced Water Spills to Aquatic Animals in Freshwater Environments: A North American Perspective.

Unconventional methods of oil and natural gas extraction have been a growing part of North America's energy sector for the past 20-30 years. Technologies such as horizontal hydraulic fracturing have facilitated the exploitation of geologic reserves that were previously resistant to standard drilling approaches. However, the environmental risks associated with hydraulic fracturing are relatively understudied. One such hazard is the wastewater by-product of hydraulic fracturing processes: flowback and produced water (FPW). During FPW production, transport, and storage, there are many potential pathways for environmental exposure. In the current review, toxicological hazards associated with FPW surface water contamination events and potential effects on freshwater biota are assessed. This review contains an extensive survey of chemicals commonly associated with FPW samples from shale formations across North America and median 50% lethal concentration values (LC50) of corresponding chemicals for many freshwater organisms. We identify the characteristics of FPW which may have the greatest potential to be drivers of toxicity to freshwater organisms. Notably, components associated with salinity, the organic fraction, and metal species are reviewed. Additionally, we examine the current state of FPW production in North America and identify the most significant obstacles impeding proper risk assessment development when environmental contamination events of this wastewater occur. Findings within this study will serve to catalyze further work on areas currently lacking in FPW research, including expanded whole effluent testing, repeated and chronic FPW exposure studies, and toxicity identification evaluations.

Toxicity in aquatic model species exposed to a temporal series of three different flowback and produced water samples collected from a horizontal hydraulically fractured well.

In the present study, we compared the toxicity and associated chemical characterizations of flowback and produced water (FPW) collected from a single horizontal hydraulically fractured well at different time points during FPW production. Since few studies on whole mixture toxicity related to FPW exist, our aims were to determine both overall toxicity of the FPW mixture in a suite of organisms (Daphnia magna, Lumbriculus variegatus, Danio rerio, and Oncorhynchus mykiss) and also determine if toxicity changes depending on variation in FPW chemical properties as a function of time sampled (1.33, 72, and 228 h FPW samples collected immediately post-well production onset were analyzed in current study). FPW chemical composition was determined via quadra-pole inductively coupled plasma - mass spectrometry/mass spectrometry (ICP-MS/MS), full-scan high performance liquid chromatography/Orbitrap mass spectrometry (HPLC/Orbitrap-MS), and gas chromatography-mass spectrometry (GC-MS). We observed that FPW sampled later in the production process contained higher ion and total dissolved solids concentrations, whereas the highest concentrations of dissolved organic compounds were observed in the earliest FPW sample analyzed. Toxicity associated with FPW exposure was deemed to be species-specific to a certain extent, but general trends revealed the earliest FPW sampled contained highest toxic potential. Accordingly, we theorize that although the saline conditions of FPW are the foremost toxicological drivers to freshwater organisms, dissolved organics associated with FPW significantly contribute to the overall toxicity of exposed organisms.

Acquisition of alanyl-alanine in an Agnathan: Characteristics of dipeptide transport across the hindgut of the Pacific hagfish Eptatretus stoutii.

This study used 3 H-L -alanyl-L -alanine to demonstrate dipeptide uptake using in vitro gut sacs prepared from the hindgut of the Pacific hagfish Eptatretus stoutii. Concentration-dependent kinetic analysis resulted in a sigmoidal distribution with a maximal (± SE) uptake rate (Jmax -like) of 70 ± 3 nmol cm-2 h-1 and an affinity constant (Km -like) of 1072 ± 81 µM. Addition of high alanine concentrations to transport assays did not change dipeptide transport rates, indicating that hydrolysis of the dipeptide in mucosal solutions and subsequent uptake via apical amino acid transporters was not occurring, which was further supported by a Km distinct from that of amino acid transport. Transport occurred independent of mucosal pH, but uptake was reduced by 42% in low mucosal sodium. This may implicate cooperation between peptide transporters and sodium-proton exchangers, previously demonstrated in several mammalian and teleost species. Finally, apical L -alanyl-L -alanine uptake rates (i.e., mucosal disappearance) were significantly increased following a meal, demonstrating regulation of uptake. Overall, this examination of dipeptide acquisition in the earliest extant Agnathan suggests evolutionarily conserved mechanisms of transport between hagfish and later-diverging vertebrates such as teleosts and mammals.

The role of dissolved organic carbon concentration and composition on nickel toxicity to early life-stages of the blue mussel Mytilus edulis and purple sea urchin Strongylocentrotus purpuratus.

Nickel (Ni) emissions resulting from production and transportation raise concerns about the impact of Ni exposure to marine ecosystems. Ni bioavailability models are established for FW systems, but the influence of chemical parameters (e.g. dissolved organic carbon (DOC)) on Ni toxicity within marine systems is less well understood. To examine the effects of DOC concentration and composition on Ni toxicity, acute toxicity tests were conducted on early life-stages of blue mussels (Mytilus edulis) and sea urchin embryos (Strongylocentrotus purpuratus) in full strength sea water (32 ppt). Nine different field collected samples of water with varying concentration (up to 4.5 mg C/L) and composition of DOC were collected from the east coast of the United States. Organic matter compositional analysis included molecular fluorescence and absorbance spectroscopy. The different DOC sources had different protective effects against embryo toxicity. The control (no DOC) Ni 48 h-EC50 for Mytilus embryos was 133 µg/L (95% confidence interval (C.I.) of 123-144 µg/L), while Strongylocentrotus embryos displayed control 96-h EC50 values of 207 µg/L (167-247 µg/L). The most significantly protective sample had high humic acid concentrations (as determined from fluorescence spectroscopy), which yielded an EC50 of 195 µg/L (169-222 µg/L) for Mytilus, and an EC50 of 394 µg/L (369-419 µg/L) for S. purpuratus. Among all samples, protection was related to both DOC quantity and quality, with fluorescence-resolved humic and fulvic acid concentrations showing the strongest correlations with protection for both species. These data suggest that DOC is protective against Ni toxicity in M. edulis and S. purpuratus, and that accounting for a DOC quality factor will improve predictive toxicity models such as the biotic ligand model.

A novel pathway of nutrient absorption in crustaceans: branchial amino acid uptake in the green shore crab (Carcinus maenas).

Estuaries are environments enriched with dissolved nutrients such as amino acids. To date, marine arthropods are the only invertebrate group that have not been demonstrated to access this potentially important nutrient resource. Using in vitro gill perfusion techniques, we sought to investigate the ability of the green shore crab (Carcinus maenas) to take up the amino acid l-leucine directly from the water. Investigation of the concentration-dependent transport kinetics of radiolabelled l-leucine showed that there are two specific transport pathways across Carcinus gills, one with high affinity and low capacity, and the other with high capacity and low affinity. Using putative competitive substrates and reduced sodium preparations, we were able to identify the putative amino acid transport system associated with high-affinity uptake. This is the first study to demonstrate the absorption of dissolved organic nutrients across the gill epithelium of a marine arthropod.

Sublethal and Reproductive Effects of Acute and Chronic Exposure to Flowback and Produced Water from Hydraulic Fracturing on the Water Flea Daphnia magna.

Hydraulic fracturing is an industrial process allowing for the extraction of gas or oil. To fracture the rocks, a proprietary mix of chemicals is injected under high pressure, which later returns to the surface as flowback and produced water (FPW). FPW is a complex chemical mixture consisting of trace metals, organic compounds, and often, high levels of salts. FPW toxicity to the model freshwater crustacean Daphnia magna was characterized utilizing acute (48 h median lethal concentrations; LC50) and chronic (21 day) exposures. A decrease in reproduction was observed, with a mean value of 18.5 neonates produced per replicate over a 21 day chronic exposure to 0.04% FPW, which was a significant decrease from the average of 64 neonates produced in the controls. The time to first brood was delayed in the highest FPW (0.04%) treatment. Neonates exhibited an LC50 of 0.19% of full-strength FPW, making them more sensitive than adults, which displayed an LC50 value of 0.75%. Quantitative PCR highlighted significant changes in expression of genes encoding xenobiotic metabolism (cyp4) and moulting (cut). This study is the first to characterize chronic FPW toxicity and will help with the development of environmental monitoring and risk assessment of FPW spills.

Effect of environmental salinity manipulation on uptake rates and distribution patterns of waterborne amino acids in the Pacific hagfish.

Among vertebrates, hagfish are the only known iono- and osmoconformers, and the only species thus far documented to absorb amino acids directly across the skin. In the current study, short-term (6h) manipulations of exposure salinities (75-125% seawater) were conducted to determine whether changes in osmotic demands influenced the uptake and tissue distribution of waterborne amino acids (alanine, glycine and phenylalanine), in the Pacific hagfish, Eptatretus stoutii. No changes in erythrocyte or muscle amino acid accumulation rates were noted, but the patterns of plasma amino acid accumulation were suggestive of regulation. Contrary to expectations, glycine transport across the skin in vitro was enhanced in the lowest exposure salinity, but no other salinity-dependent changes were demonstrated. Overall, this study indicates that uptake and distribution of amino acids varies with salinity, but not in a manner that is consistent with a role for the studied amino acids in maintaining osmotic balance in hagfish.

Mechanisms of Nickel Toxicity in the Highly Sensitive Embryos of the Sea Urchin Evechinus chloroticus, and the Modifying Effects of Natural Organic Matter.

A 96 h toxicity test showed that the embryos of the New Zealand sea urchin (Evechinus chloroticus) are the most sensitive of all studied marine species to waterborne nickel (Ni), with the EC50 for the development of fully formed pluteus larvae found to be 14 µg L(-1). Failure to develop a standard larval shape suggested skeletal impairment. Whole body ions (Na, Mg) increased with Ni exposure and calcium influx was depressed. The effects of natural organic matter (NOM) on Ni accumulation and toxicity were also examined in three different seawater sources (nearshore, offshore, and near the outlet of a "brown water" stream). At low dissolved organic carbon (DOC) concentrations the brown water NOM was protective against Ni toxicity, however at higher DOC concentrations it exacerbated developmental toxicity in the presence of Ni. These results show that sea urchin development is highly sensitive to Ni via a mechanism that involves ionoregulatory disturbance, and that Ni toxicity is influenced by environmental factors such as NOM. These data will be critical for the development of water quality guidelines for Ni in the marine environment.

Iron transport across the skin and gut epithelia of Pacific hagfish: Kinetic characterisation and effect of hypoxia.

In most animals, the acquisition of the essential trace metal iron (Fe) is achieved by the gut, but in hagfishes, the skin is a nutrient absorbing epithelium, and thus may also play a role in Fe uptake. In the current study, the absorption of Fe, as Fe(II), across the intestinal and cutaneous epithelia of Pacific hagfish (Eptatretus cirrhatus) was investigated. Both epithelia absorbed Fe, with saturation at lower tested concentrations, superseded by a diffusive component at higher Fe exposure concentrations. Affinity constants (Km) of 9.4 and 137µM, and maximal Fe transport rates (Jmax) of 0.81 and 0.57nmolcm(-2)h(-1) were determined for the skin and the gut, respectively. This characterises the skin as a relatively high-affinity Fe transport epithelium. The majority of the absorbed Fe in the skin remained in the tissue, whereas in the gut, most absorbed Fe was found in the serosal fluid, suggesting distinct mechanisms of Fe handling between the two epithelia. To determine if reduced dissolved oxygen altered Fe transport, hagfish were subjected to hypoxia for 24h, before Fe transport was again assessed. Hypoxia had no effect on Fe transport across gut or skin, likely owing to the relative lack of change in haematological variables, and thus an unaltered Fe demand under such conditions. These data are the first to kinetically characterise the absorption of a nutritive trace metal across the epithelia of hagfish and add to the growing understanding of the role of the skin in nutritive transport in this group.

Novel route of toxicant exposure in an ancient extant vertebrate: nickel uptake by hagfish skin and the modifying effects of slime.

Utilizing an in vitro technique, the skin of Pacific hagfish (Eptatretus stouti) was shown to take up nickel from the water via a high affinity, low capacity transport pathway. Uptake was biphasic, with saturation occurring at low nickel exposure concentrations, superseded by linear, diffusive uptake at levels greater than 50 µM. In vivo exposures showed that nickel accumulated mainly in the gill, heart, and brain, representing a tissue distribution distinct from that found in teleosts. Slime on the epidermal surface was shown to significantly reduce the uptake of low concentrations (10 µM) of the metals zinc and nickel, but slime had no effect on organic nutrient (the amino acid l-alanine) absorption. At a higher metal exposure concentration (1 mM), slime was no longer protective, indicating saturation of metal-binding sites. This is the first study to show that metals can be taken up by the integument of hagfish. The ability of the skin to act as a transport epithelium may be of particular importance for a burrowing, benthic scavenger, such as hagfish, which are likely to be exposed to relatively enriched levels of metal toxicants through their habitat and lifestyle, and this may have consequences for human health through hagfish consumption.

Low salinity enhances NI-mediated oxidative stress and sub-lethal toxicity to the green shore crab (Carcinus maenas).

Nickel (Ni) is a metal of environmental concern, known to cause toxicity to freshwater organisms by impairing ionoregulation and/or respiratory gas exchange, and by inducing oxidative stress. However, little is known regarding how nickel toxicity is influenced by salinity. In the current study we investigated the salinity-dependence and mechanisms of sub-lethal Ni toxicity in a euryhaline crab (Carcinus maenas). Crabs were acclimated to three experimental salinities--20, 60 and 100% seawater (SW)--and exposed to 3mg/L Ni for 24h or 96 h. Tissues were dissected for analysis of Ni accumulation, gills were taken for oxidative stress analysis (catalase activity and protein carbonyl content), haemolymph ions were analysed for ionoregulatory disturbance, and oxygen consumption was determined in exercised crabs after 96 h of Ni exposure. Total Ni accumulation was strongly dependant on salinity, with crabs from 20% SW displaying the highest tissue Ni burdens after both 24 and 96-h exposures. After 96 h of exposure, the highest accumulation of Ni occurred in the posterior (ionoregulatory) gills at the lowest salinity, 20% SW. Posterior gill 8 exhibited elevated protein carbonyl levels and decreased catalase activity after Ni exposure, but only in 20% SW. Similarly, decreased levels of haemolymph Mg and K and an increased level of Ca were recorded but only in crabs exposed to Ni for 96 h in 20% SW. Oxygen consumption after exercise was also inhibited in crabs exposed to Ni in 20% SW. These data show for the first time the simultaneous presence of all three modes of sub-lethal Ni toxicity in exposed animals, and indicate a strong salinity dependence of sub-lethal Ni toxicity to the euryhaline crab, C. maenas, a pattern that corresponded to tissue Ni accumulation.

Salinity-dependent nickel accumulation and oxidative stress responses in the euryhaline killifish (Fundulus heteroclitus).

The mechanisms of nickel (Ni) toxicity in marine fish remain unclear, although evidence from freshwater (FW) fish suggests that Ni can act as a pro-oxidant. This study investigated the oxidative stress effects of Ni on the euryhaline killifish (Fundulus heteroclitus) as a function of salinity. Killifish were exposed to sublethal levels (5, 10, and 20 mg L(-1)) of waterborne Ni for 96 h in FW (0 ppt) and 100 % saltwater (SW) (35 ppt). In general, SW was protective against both Ni accumulation and indicators of oxidative stress [protein carbonyl formation and catalase (CAT) activity]. This effect was most pronounced at the highest Ni exposure level. For example, FW intestine showed increased Ni accumulation relative to SW intestine at 20 mg Ni L(-1), and this was accompanied by significantly greater protein carbonylation and CAT activity in this tissue. There were exceptions, however, in that although liver of FW killifish at the highest exposure concentration showed greater Ni accumulation relative to SW liver, levels of CAT activity were greatly decreased. This may relate to tissue- and salinity-specific differences in oxidative stress responses. The results of the present study suggest (1) that there was Ni-induced oxidative stress in killifish, (2) that the effects of salinity depend on differences in the physiology of the fish in FW versus SW, and (3) that increased levels of cations (sodium, calcium, potassium, and magnesium) and anions (SO4 and Cl) in SW are likely protective against Ni accumulation in tissues exposed to the aquatic environment.

Evidence for transporter-mediated uptake of environmental L-glutamate in a freshwater sponge, Ephydatia muelleri.

The freshwater sponge, Ephydatia muelleri, lacks a nervous or endocrine system and yet it exhibits a coordinated whole-body action known as a "sneeze" that can be triggered by exposure to L-glutamate. It is not known how L-glutamate is obtained by E. muelleri in sufficient quantities (i.e., 70 µM) to mediate this response endogenously. The present study tested the hypothesis that L-glutamate can be directly acquired from the environment across the body surface of E. muelleri. We demonstrate carrier mediated uptake of two distinct saturable systems with maximal transport rates (Jmax) of 64.27 ± 4.98 and 25.12 ± 1.87 pmols mg-1 min-1, respectively. The latter system has a higher calculated substrate affinity (Km) of 2.87 ± 0.38 µM compared to the former (8.75 ± 1.00 µM), indicative of distinct systems that can acquire L-glutamate at variable environmental concentrations. Further characterization revealed potential shared pathways of L-glutamate uptake with other negatively charged amino acids, namely D-glutamate and L-aspartate, as well as the neutral amino acid L-alanine. We demonstrate that L-glutamate uptake does not appear to rely on exogenous sodium or proton concentrations as removal of these ions from the bathing media did not significantly alter uptake. Likewise, L-glutamate uptake does not seem to rely on internal proton motive forces driven by VHA as application of 100 nM of the VHA inhibitor bafilomycin did not alter uptake rates within E. muelleri tissues. Whether the acquired amino acid is used to supplement feeding or is stored and accumulated to mediate the sneeze response remains to be determined.

Invasive investigation: uptake and transport of l-leucine in the gill epithelium of crustaceans.

Many aquatic species are well known as extremely successful invaders. The green crab (Carcinus maenas) is an arthropod native to European waters; however, it is now known to be a globally invasive species. Recently, it was discovered that the C. maenas could transport nutrients in the form of amino acids across their gill from the surrounding environment, a feat previously thought to be impossible in arthropods. We compared the ability for branchial amino acid transport of crustacean's native to Canadian Pacific waters to that of the invasive C. maenas, determining if this was a novel pathway in an extremely successful invasive species, or a shared trait among crustaceans. Active transport of l-leucine was exhibited in C. maenas, Metacarcinus gracilis, Metacarcinus magister, and Cancer productus across their gill epithelia. Carcinus maenas exhibited the highest maximum rate of branchial l-leucine transport at 53.7 ± 6.24 nmolg-1 h-1, over twice the rate of two native Canadian crustaceans. We also examined the influence of feeding, gill specificity, and organ accumulation of l-leucine. Feeding events displayed a heavy influence on the branchial transport rate of amino acids, increasing l-leucine transport rates by up to 10-fold in C. maenas. l-leucine displayed a significantly higher accumulation rate in the gills of C. maenas compared to the rest of the body at 4.15 ± 0.78 nmolg-1 h-1, with the stomach, hepatopancreas, eyestalks, muscle tissue, carapace and heart muscle exhibiting accumulation under 0.15 nmolg-1 h-1. For the first time, the novel transport of amino acids in Canadian native arthropods is described, suggesting that branchial amino acid transport is a shared trait among arthropods, contrary to existing literature. Further investigation is required to determine the influence of environmental temperature and salinity on transport in each species to outline any competitive advantages of the invasive C. maenas in a fluctuating estuarine environment.

The effects of winter cold acclimation on acute and chronic cadmium bioaccumulation and toxicity in the banded killifish (Fundulus diaphanus).

Temperate freshwater fishes can experience large seasonal temperature fluctuations that could affect their exposure and sensitivity to trace metals. Yet, temperature effects are overlooked in ecotoxicology studies, especially for cold temperatures typical of the winter. In the present study, the effects of long-term cold acclimation on Cd bioaccumulation and toxicity were investigated in a freshwater fish, the banded killifish (Fundulus diaphanus). Killifish were acclimated to 14 °C or gradually cooled (2 °C/week) to 4 °C and cold acclimated for 6 weeks. Then, both acclimation groups were exposed to environmentally realistic waterborne Cd concentrations (0, 0.5 or 5 µg Cd L-1) for a further 28 d at their respective acclimation temperatures. Tissue metal bioaccumulation, fish survival, condition, and markers of oxidative and ionoregulation stress, were measured after 0, 2, 5 and 28 days of Cd exposure. Cadmium tissue accumulation increased over the exposure duration and was typically lower in cold-acclimated fish. In agreement with this lower bioaccumulation, fewer Cd toxic effects were observed in cold-acclimated fish. There was little evidence of a difference in intrinsic Cd sensitivity between 4 °C- and 14 °C-acclimated fish, as Cd toxicity appeared to closely follow Cd bioaccumulation. Our study suggests that current environmental water quality guidelines would be protective in the winter for the abundant and ecologically-important banded killifish.

Copper toxicity does not affect low tide emersion tolerance of Mytilus galloprovincialis.

Intertidal mussels are well adapted to withstand emersion from water during low tide, but they may be intermittently exposed to waterborne toxicants such as copper, which targets physiological processes including metabolism, ammonia excretion, and osmoregulation. To determine if copper exposure damages intertidal organisms' ability to tolerate tidal emersion, Mediterranean mussels (Mytilus galloprovincialis) were exposed to copper for 96 h followed by 6 h of emersion. Oxygen uptake increased after copper exposure which suggests that copper accumulation caused moderate stress in the mussels, but ammonia excretion and anaerobic metabolism were unaffected by mixed copper and emersion exposures. Shell composition analyses indicate that cycles of copper exposure and tidal emersion may affect bivalve shell growth, but copper deposition into shells may decrease the metal's overall toxicity. Results suggest that copper does not damage M. galloprovincialis's tolerance to tidal emersion, and insight is provided into the mussel's ability to overcome mixed stressor exposures.