Emersion and recovery alter oxygen consumption, ammonia and urea excretion, and oxidative stress parameters, but not diffusive water exchange or transepithelial potential in the green crab (Carcinus maenas).

The green crab (Carcinus maenas) is an inshore species affected by intertidal zonation patterns, facing periods of emersion during low tide and submersion during high tide. During these periods of air and subsequent water exposure, these species can face physiological challenges. We examined changes in O2 consumption rate (MO2), and ammonia and urea excretion rates over sequential 14 h periods in seawater (32 ppt, control), in air and during recovery in seawater after air exposure (13°C throughout). At the end of each exposure, the anterior (5th) and posterior (8th) gills and the hepatopancreas were removed for measurements of oxidative stress parameters (TBARs and catalase in the gills and hepatopancreas, and protein carbonyls in the gills). MO2 remained unchanged during air exposure, but increased greatly (3.4-fold above control levels) during the recovery period. Ammonia and urea net fluxes were reduced by 98% during air exposure, but rebounded during recovery to >2-fold the control rates. Exchangeable water pools, rate constants of diffusive water exchange, unidirectional diffusive water flux rates (using tritiated water) and transepithelial potential were also measured during control and recovery treatments, but exhibited no significant changes. Damage to proteins was not observed in either gill. However, lipid damage occurred in the anterior (respiratory) gill after the air exposure but not in the posterior (ionoregulatory) gill or hepatopancreas. Catalase activity also decreased significantly in recovery relative to levels during air exposure in both the anterior gill and hepatopancreas, but not in the posterior gill. The crabs did not modify water metabolism or permeability. We conclude that MO2 was maintained but not enhanced during air exposure, while ammonia and urea-N excretion were impaired. As a result, all of these parameters increase greatly during re-immersion recovery, and oxidative stress also occurs. Clearly, emersion is not without physiological costs.

Exercise and emersion in air, and recovery in seawater in the green crab (Carcinus maenas): metabolic, acid-base, cardio-ventilatory and ionoregulatory responses.

In nature, the green crab exhibits emersion and terrestrial activity at low tide. Treadmill exercise in air (20-23°C) of crabs acclimated to 32 ppt seawater (13°C) revealed an inverse relationship between velocity and duration: 2.0 body lengths (BL) s-1 was sustainable for several minutes, and 0.25 BL s-1 was sustainable for long periods. Fatigue was not due to dehydration. Physiological responses over an 18 h recovery in seawater after near-exhaustive exercise (0.25 BL s-1, 1 h) in air were compared with responses after quiet emersion (1 h) in air. Exercising crabs exhibited transient scaphognathite slowing and progressive increases in heart rate, whereas emersed crabs exhibited persistent inhibition of ventilation and transient heart slowing. Upon return to seawater, all these rates increased above both control and treatment levels. Post-exercise disturbances were more marked and/or longer lasting (e.g. EPOC, hyperventilation, tachycardia, metabolic acidosis, lactate elevation, ionic disturbances) than those after simple air exposure. However, an increase in net acidic equivalent excretion to the environment occurred after emersion but not after exercise. Instead, post-exercise crabs relied on carapace buffering, signalled by elevated haemolymph Ca2+ and Mg2+. Prolonged lowering of haemolymph PCO2 associated with hyperventilation also played a key role in acid-base recovery. EPOC after exercise was 3-fold greater than after emersion, sufficient to support resting MO2 for >14 h. This reflected clearance of a large lactate load, likely by glycogen re-synthesis rather than oxidation. We conclude that the amphibious green crab uses a combination of aquatic and terrestrial strategies to support exercise in air, emersion in air and recovery in seawater.

Transepithelial potential remains indicative of major ion toxicity in rainbow trout (Oncorhynchus mykiss) after 4-day pre-exposure to major salts.

The Multi-Ion Toxicity (MIT) Model uses electrochemical theory to predict the transepithelial potential (TEP) across the gills as an index of major ion toxicity in freshwater animals. The goal is to determine environmental criteria that will be protective of aquatic organisms exposed to salt pollution. In recent studies, TEP disturbances above baseline (ΔTEP) during short-term exposures to major ions have been proven as indicative of their toxicity to fish, in accord with the MIT model. However, the acute 1-h exposures used in these previous studies might not be realistic relative to the 24 h or 96 h test periods used for toxicity assessment. To address this temporal inconsistency, the current study investigated both the TEP responses to serial concentrations of 10 major salts (NaCl, Na2SO4, NaHCO3, KCl, K2SO4, KHCO3, CaCl2, CaSO4, MgCl2, MgSO4) and plasma ion levels in juvenile rainbow trout after they had been pre-exposed to 50% of the 96h-LC50 levels of these same salts for 4 days. The pre-exposures caused no mortalities. In general, plasma ions (Na+, K+, Ca2+, Mg2+, Cl-) were well-regulated; however, pre-exposure to sulfate salts resulted in the greatest number of alterations in plasma ion levels. TEP responses remained largely similar to those of naïve trout (without salt pre-exposure). All salts caused hyperbolic concentration-dependent increases in TEP that were well-described by the Michaelis-Menten equation. In the pre-exposed trout, the variation of ∆TEP at the 96h-LC50 concentrations was only 2.2-fold, compared to nearly 28-fold variation among the molar concentrations of the various salts at the 96h-LC50s, identical to the conclusion for naïve trout. Overall, the results remove the temporal inconsistency of previous tests and remain supportive of the MIT model. In addition, the recorded alterations in certain plasma ions, baseline TEP, and Michaelis-Menten constants improve our knowledge on specific physiological responses after extended major ion exposure.

Trans-epithelial potential (TEP) response as an indicator of major ion toxicity in rainbow trout and goldfish exposed to 10 different salts in ion-poor water.

Freshwater ecosystems are facing increasing contamination by major ions. The Multi-Ion Toxicity (MIT) model, a new tool for risk assessment and regulation, predicts major ion toxicity to aquatic organisms by relating it to a critical disturbance of the trans-epithelial potential (TEP) across the gills, as predicted by electrochemical theory. The model is based on unproven assumptions. We tested some of these by directly measuring the acute TEP responses to a geometric series of 10 different single salts (NaCl, Na2SO4, KCl, K2SO4, CaCl2, CaSO4, MgCl2, MgSO4, NaHCO3, KHCO3) in the euryhaline rainbow trout (Oncorhynchus mykiss) and the stenohaline goldfish (Carassius auratus) acclimated to very soft, ion-poor water (hardness 10 mg CaCO3/L). Results were compared to 24-h and 96-h LC50 data from the literature, mainly from fathead minnow (Pimephales promelas). All salts caused concentration-dependent increases in TEP to less negative/more positive values, in patterns well-described by the Michaelis-Menten equation, or a modified version incorporating substrate inhibition. The ΔTEP above baseline became close to a maximum at the 96-h LC50, except for the HCO3- salts. Furthermore, the range of ΔTEP values at the LC50 within one species was much more consistent (1.6- to 2.1-fold variation) than the molar concentrations of the different salts at the LC50 (19- to 25-fold variation). ΔTEP responses were related to cation rather than anion concentrations. Overall patterns were qualitatively similar between trout and goldfish, with some quantitative differences, and also in general accord with recently published data on three other species in harder water where ΔTEP responses were much smaller. Blood plasma Na+ and K+ concentrations were minimally affected by the exposures. The results are in accord with most but not all of the assumptions of the MIT model and support its further development as a predictive tool.

The potential for salt toxicity: Can the trans-epithelial potential (TEP) across the gills serve as a metric for major ion toxicity in fish?

An emerging Multi-Ion Toxicity (MIT) model for assessment of environmental salt pollution is based on the premise that major ion toxicity to aquatic organisms is related to a critical disturbance of the trans-epithelial potential across the gills (ΔTEP), which can be predicted by electrochemical theory. However, the model has never been evaluated physiologically. We directly tested key assumptions by examining the individual effects of eight different salts (NaCl, Na2SO4, MgCl2, MgSO4, KCl, K2SO4, CaCl2, and CaSO4) on measured TEP in three different fish species (fathead minnow, Pimephales promelas = FHM; channel catfish, Ictalurus punctatus = CC; bluegill, Lepomis macrochirus = BG). A geometric concentration series based on previously reported 96-h LC50 values for FHM was used. All salts caused concentration-dependent increases in TEP to less negative/more positive values in a pattern well-described by the Michaelis-Menten equation. The ΔTEP responses for different salts were similar to one another within each species when concentrations were expressed as a percentage of the FHM LC50. A plateau was reached at or before 100 % of the LC50 where the ΔTEP values were remarkably consistent, with only 1.4 to 2.2-fold variation. This relative uniformity in the ΔTEP responses contrasts with 28-fold variation in salt concentration (in mmol L-1), 9.6-fold in total dissolved solids, and 7.9-fold in conductivity at the LC50. The Michaelis-Menten Km values (salt concentrations causing 50 % of the ΔTEPmax) were positively related to the 96-h LC50 values. ΔTEP responses were not a direct effect of osmolarity in all species and were related to specific cation rather than specific anion concentrations in FHM. These responses were stable for up to 24 h in CC. The results provide strong physiological support for the assumptions of the MIT model, are coherent with electrochemical theory, and point to areas for future research.

Contamination and risk implications of endocrine disrupting chemicals along the coastline of China: A systematic study using mussels and semipermeable membrane devices.

A systematic study has been carried out to assess the contamination of endocrine disrupting chemicals (EDCs) in five highly urbanized coastal cities spanning from temperate to subtropical environments along the coastline of China. In each of these cities, species of native mussels (Mytilus galloprovincialis, M. coruscus or Perna viridis) were deployed alongside with semipermeable membrane devices (SPMDs) for one month at a reference site and a polluted site. The level of 4-nonylphenol (4-NP), bisphenol A (BPA), 17ß-estradiol (E2) and 17α-ethynylestradiol (EE2) in SPMDs and transplanted mussels were determined and compared. The concentration of EDCs in mussels from polluted sites of Qingdao and Shenzhen ranged from 99.4±9.40 to 326.1±3.16ng/g dry wt. for 4-NP, Dalian and Shanghai from 170.3±4.00 to 437.2±36.8ng/g dry wt. for BPA, Dalian and Shenzhen from 82.9±3.03 to 315.6±6.50ng/g dry wt. for E2, and Shenzhen and Shanghai from 124.5±3.25 to 204.5±9.26ng/g dry wt. for EE2, respectively. These results demonstrate that concentrations of EDCs in mussels along the coastline of China are substantially higher than levels reported in mussels and seafood elsewhere. Despite high levels of EDCs and per capita seafood consumption in China, analysis indicated that 4-NP and BPA intake from mussels at polluted sites per se are still below the Tolerable Daily Intake (TDI). In contrast, the daily intake of E2 and EE2 (6.5 and 5.5µg/person/day, respectively) from mussel consumption exceeded the Acceptable Daily Intake (ADI) established by the WHO, USA and Australia by large margins, suggesting significant public health risks. A strong correlation was found between EDC concentrations in SPMDs and transplanted mussels, and the advantages of using mussels and SPMDs for monitoring EDCs in the aquatic environment are discussed.

Transgenerational impairments of reproduction and development of the marine invertebrate Crepidula onyx resulted from long-term dietary exposure of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47).

Polybrominated diphenyl ethers have become ubiquitous in the environment and elevated concentrations have often been found in marine organisms. Using the gastropod Crepidula onyx as a study model, this multigenerational study sets out to test the hypotheses that 1) parental dietary exposure to environmentally realistic levels of 2,2',4,4'-tetrabrominated diphenyl ether (BDE-47) would lead to transgenerational impairments on fitness traits of marine invertebrates, and 2) the organisms might develop adaptation/acclimation after exposure for one or more generations. F0 generation of C. onyx was fed with the dinoflagellate Isochrysis galbana encapsulated with two concentrations of BDE-47 (1.78 and 16.0 ng million cells-1, respectively), and half of the F1 offspring from the higher concentration treatment was returned to control condition (transgenerational group), while the other half received BDE-47 treatment continuously (continuous treatment group). Bioaccumulation and maternal transfer of BDE-47 were evident in all life stages of the F0 generation and in F1 eggs, respectively. Exposure to BDE-47 reduced fecundity, delayed sexual maturity, and impeded embryonic development in F0 to F2. In particular, developmental toxicity of F2 embryos was apparent in the transgenerational group, but not in the continuous treatment group, even when BDE-47 was not detected in the F2 embryos nor in their mothers and they have never been exposed to the chemical. This study also suggested that the offspring might have developed adaptation/acclimation to the exposure of BDE-47 within two generations of exposure, and that the physiological alterations associated with acclimation/adaptation might have hindered the normal larval development under a stress free condition. These findings highlighted the need for long-term multigenerational studies in the ecological risk assessment of chemicals alike.

Uptake and biotransformation of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) in four marine microalgae species.

Hydroxylated- and methoxylated- polybrominated diphenyl ethers (OH-PBDEs and MeO-PBDEs) are more toxic than PBDEs and occur widely in the marine environment, and yet their origins remain controversial. In this study, four species of microalgae (Isochrysis galbana, Prorocentrum minimum, Skeletonema grethae and Thalassiosira pseudonana) were exposed to BDE-47, which is synthetic and is the predominant congener of PBDEs in the environment. By chemical analysis after incubation of 2 to 6 days, the efficiency of uptake of BDE-47 and, more importantly, the potential of undergoing biotransformation to form OH-PBDEs and MeO-PBDEs by the microalgae were investigated. Growth rates of these axenic microalgae were not affected upon exposure to environmentally relevant concentrations (0.2-20 µg BDE-47 L-1), and accumulation ranged from 0.772 ± 0.092 µg BDE-47 g-1 lipid to 215 ± 54 µg BDE-47 g-1 lipid within 2 days. Debromination of BDE-47 and formation of BDE-28 occurred in all microalgae species (0.01 to 0.87%), but biotransformation to OH-PBDEs was only found in I. galbana upon exposure to extremely high concentration. The results of this study showed that biotransformation of microalgae species is unlikely an explanation for the OH-PBDEs and MeO-PBDEs found in the marine environment.

A novel approach for estimating the removal efficiencies of endocrine disrupting chemicals and heavy metals in wastewater treatment processes.

The wide occurrence of endocrine disrupting chemicals (EDCs) and heavy metals in coastal waters has drawn global concern, and thus their removal efficiencies in sewage treatment processes should be estimated. However, low concentrations coupled with high temporal fluctuations of these pollutants present a monitoring challenge. Using semi-permeable membrane devices (SPMDs) and Artificial Mussels (AMs), this study investigates a novel approach to evaluating the removal efficiency of five EDCs and six heavy metals in primary treatment, secondary treatment and chemically enhanced primary treatment (CEPT) processes. In general, the small difference between maximum and minimum values of individual EDCs and heavy metals measured from influents/effluents of the same sewage treatment plant suggests that passive sampling devices can smooth and integrate temporal fluctuations, and therefore have the potential to serve as cost-effective monitoring devices for the estimation of the removal efficiencies of EDCs and heavy metals in sewage treatment works.

Heavy metal contamination along the China coastline: A comprehensive study using Artificial Mussels and native mussels.

A comprehensive study was carried out to assess metal contamination in five cities spanning from temperate to tropical environment along the coastal line of China with different hydrographical conditions. At each of the five cities, Artificial Mussels (AM) were deployed together with a native species of mussel at a control site and a polluted site. High levels of Cr, Cu and Hg were found in Qingdao, high level of Cd, Hg and Pb was found in Shanghai, and high level of Zn was found in Dalian. Furthermore, level of Cu contamination in all the five cities was consistently much higher than those reported in similar studies in other countries (e.g., Australia, Portugal, Scotland, Iceland, Korea, South Africa and Bangladesh). Levels of individual metal species in the AM showed a highly significant correlation with that in the native mussels (except for Zn in Mytilus edulis and Cd in Perna viridis), while no significant difference can be found between the regression relationships of metal in the AM and each of the two native mussel species. The results demonstrated that AM can provide a reliable time-integrated estimate of metal concentration in contrasting environments over large biogeographic areas and different hydrographic conditions, and overcome the shortcomings of monitoring metals in water, sediment and the use of biomonitors.

Polybrominated diphenyl ethers do not affect metamorphosis but alter the proteome of the invasive slipper limpet Crepidula onyx.

Man-made polybrominated diphenyl ethers (PBDEs) used as flame retardants in various consumer products may be harmful to marine organisms. Larvae of some marine invertebrates, especially invasive species, can develop resistance to PBDEs through altered protein expression patterns or proteome plasticity. This is the first report of a proteomics approach to study BDE-47 induced molecular changes in the invasive limpet Crepidula onyx. Larvae of C. onyx were cultured for 5 days (hatching to metamorphosis) in the presence of BDE-47 (1 µg L(-1)). Using a 2-DE proteomics approach with triple quadrupole and high-resolution TOF-MS, we showed that BDE-47 altered the proteome structure but not the growth or metamorphosis of C. onyx larvae. We found eight significant differentially expressed proteins in response to BDE-47, deemed the protein expression signature, consisting of cytoskeletal, stress tolerance, metabolism and energy production related proteins. Our data suggest C. onyx larvae have adequate proteome plasticity to tolerate BDE-47 toxicity.

Polybrominated diphenyl ethers (PBDEs) alter larval settlement of marine intertidal organisms across three phyla via reducing bacterial abundance on the biofilms.

Polybrominated diphenyl ethers (PBDEs) have been widely used as flame retardants over the last three decades, and are now ubiquitous in the marine environment. While the harmful effects of PBDEs on the abnormal development and reproductive impairment in mammals and fish are well documented, the effects on marine invertebrates remain virtually unknown. Using three model intertidal species accross three phyla, including the polychaete Hydroides elegans (Phylum Annelida), the gastropod Crepidula onyx (Phylum Mollusca), and the barnacle Balanus amphitrite (Phylum Arthopoda), this study demonstrated that (a) chronic exposure to BDE-47 (at spiking concentrations up to 1000 ng L(-1)) throughout the entire larval stage did not affect settlement, development or growth of all three species per se, despite bioaccumulation was clearly evident (measured body burden ranging from approximately 7000 to 13 000 ng BDE-47 g(-1) lipid), and (b) BDE-47, at measured concentrations of 15 and 113 ng g(-1) lipid, reduced the bacterial abundance in biofilms and resulted in a concomitant change in larval settlement pattern of all the model intertidal species across three phyla.