Lipids play important roles in growth, reproduction, locomotion, and migration of fish. Municipal effluents, which are complex mixtures of biological and chemical compounds including flame retardants, have been shown to alter lipid metabletabolism in environmentally and experimentally exposed fish. Down-regulation of several genes coding for fatty acid metabolism enzymes has previously been reported in male northern pike (Esox lucius) collected in the St. Lawrence River (QC, Canada) downstream of a major primary wastewater treatment plant (WWTP) point of discharge. The main objective of this study was to investigate the effects of exposure to the Montreal’s WWTP effluent on the lipidomic profile (i.e., fatty acids, acylcarnitines, and phospholipids) as well as the transcription of genes related to lipid metabolism in the liver of northern pike collected upstream and downstream of this WWTP effluent. Halogenated flame retardant concentrations were also determined in pike liver and used as markers of exposure to this effluent. Greater concentrations of saturated and monounsaturated lysophosphatidylcholines (LPCs) and lower concentrations of polyunsaturated LPCs were determined in the liver of pike collected downstream of the WWTP compared to those collected upstream. Lower mRNA levels of peroxisome proliferator-activated receptor alpha (pparα), a major regulator of lipid metabolism, were also measured in pike exposed to Montreal’s WWTP effluent. In addition, the relative contributions (%) of LPC 18:2 and LPC14:0, compounds used as markers of obesity and inflammation, were significantly correlated with halogenated flame retardant concentrations and fish girth. Results of the present study suggest that chronic environmental exposure to a primary WWTP effluent can modulate the transcription of genes related to lipid metabolism, and hence affect the hepatic phospholipid composition of pike from the St. Lawrence River.

Many freshwater ecosystems are becoming saltier and/or warmer, but our understanding of how these factors interact and affect the physiology and life history outcomes of most aquatic species remain unknown. We hypothesize that temperature modulates ion transport rates. Since ion transport is energetically expensive, increases in salinity and/or temperature may influence ion flux rates and ultimately, organismal performance. Radiotracer (22Na+, 35SO4-2, and 45Ca2+) experiments with lab-reared mayflies (N. triangulifer) and other field-collected insects showed that increasing temperature generally increased ion transport rates. For example, increasing temperature from 15 °C to 25 °C, increased 22Na+ uptake rates by two-fold (p < 0.0001) and 35SO4-2 uptake rates by four-fold (p < 0.0001) in the caddisfly, Hydropsyche sparna. Smaller changes in 22Na+ and 35SO4-2 uptake rates were observed in the mayflies, Isonychia sayi and Maccaffertium sp., suggesting species-specific differences in the thermal sensitivity of ion transport. Finally, we demonstrated that the toxicity of SO4 was influenced by temperature profoundly in a 96 -h bioassay. Under the saltiest conditions (1500 mg/L SO4), mayfly survival was 78% at 15 °C, but only 44% at 25 °C (p < 0.0036). Conceivably, the energetic cost of osmoregulation in warmer, saltier environments may cause significant major ion toxicity in certain freshwater insects.

Canada’s oil sands industry continues to expand and the volume of diluted bitumen (dilbit) transported across North America is increasing, adding to spill risk and environmental contamination. Dilbit exposure is known to cause adverse effects in fish, but linking molecular and cellular changes with ecologically-relevant individual performance metrics is needed to better understand the potential consequences of a dilbit spill into the aquatic environment. Therefore, this study examined the effects of dilbit exposure on subcellular responses in cardiac and skeletal muscle in relation to swimming performance in a migratory fish species at risk of exposure, Atlantic salmon. Smolts were exposed subchronically to environmentally relevant concentrations of the water-soluble fraction of dilbit (WSFd) for 24 d, and then a subset of exposed fish underwent a depuration period of 7 or 14 d, for a total of 3 experimental time points. At each time point, repeat swimming performance was assessed using sequential critical swimming speed tests (Ucrit) separated by a 24 h rest period, and then several tissues were collected to determine biotransformation enzyme activation, energetic responses, and gene expression changes. Ucrit was unaffected in fish exposed to 67.9 μg/L total initial polycyclic aromatic compounds (PAC), but fish showed a decreased reliance on lipid metabolism for adenosine triphosphate (ATP) in the heart that was maintained through 7 d depuration. In contrast, Ucrit increased in fish exposed to 9.65 μg/L PAC, corresponding to an increased reliance on anaerobic metabolic pathways in cardiac and red skeletal muscle, with partial recovery after 7 d depuration. As expected, at both concentrations WSFd hepatic cyp 1A-mediated biotransformation reactions increased, as measured by EROD activity, which remained elevated for 7 d but not after 14 d depuration. Transcript abundance of cyp1a was also increased in muscle tissue and recovered by 14 d depuration. The expression of other stress-related genes increased in white muscle of dilbit-exposed fish, but were largely unchanged in cardiac and red muscle. The transcriptional profile of cardiac tissue was compared to that of sockeye salmon similarly exposed to WSFd in a previous experiment, and is provided in supplemental text. Combined, these results demonstrate that dilbit exposure alters gene expression and enzyme activities related to xenobiotic exposure, cellular stress, and muscle energetics in juvenile Atlantic salmon without impairing swimming performance, and that most of these changes are recoverable within 14 d depuration.

Petrogenic chemicals are common and widespread contaminants in the aquatic environment. In Canada, increased extraction of bitumen from the oil sands and transport of the major crude oil export product, diluted bitumen (dilbit), amplifies the risk of a spill and contamination of Canadian waterways. Fish exposed to sublethal concentrations of crude oil can experience a variety of adverse physiological effects including osmoregulatory dysfunction. As regulation of water and ion balance is crucial during the seawater transition of anadromous fish, the hypothesis that dilbit impairs seawater acclimation in Atlantic salmon smolts (a fish at risk of exposure in Canada) was tested. Smolts were exposed for 24 d to the water-soluble fraction of dilbit in freshwater, and then transferred directly to seawater or allowed a 1 wk depuration period in uncontaminated freshwater prior to seawater transfer. The seawater acclimation response was quantified at 1 and 7 d post-transfer using established hematological, tissue, and molecular endpoints including gill Na+/K+-ATPase gene expression (nka). All smolts, irrespective of dilbit exposure, increased serum Na+ concentrations and osmolality within 1 d of seawater transfer. The recovery of these parameters to freshwater values by 7 d post-transfer was likely driven by the increased expression and activity of Na+/K+-ATPase in the gill. Histopathological changes in the gill were not observed; however, CYP1A-like immunoreactivity was detected in the pillar cells of gill lamellae of fish exposed to 67.9 μg/L PAC. Concentration-specific changes in kidney expression of a transmembrane water channel, aquaporin 3, occurred during seawater acclimation, but were resolved with 1 wk of depuration and were not associated with histopathological changes. In conclusion, apart from a robust CYP response in the gill, dilbit exposure did not greatly impact common measures of seawater acclimation, suggesting that significant osmoregulatory dysfunction is unlikely to occur if Atlantic salmon smolts are exposed sub-chronically to dilbit.

Recently, research on the biological effects of nanoplastics has grown exponentially. However, studies on the effects of nanoplastics on freshwater organisms and the mechanisms of the biological effects of nanoplastics are limited. In this study, the content of reactive oxygen species (ROS), gene and protein expression in the MAPK-HIF-1/NFkB pathway, and antioxidant gene expressions and enzyme activities were measured in Daphnia pulex exposed to polystyrene nanoplastic. In addition, the full-length extracellular signal-regulated kinases (ERK) gene, which plays an important role in the MAPK pathway, was cloned in D. pulex, and the amino acid sequence, function domain, and phylogenetic tree were analyzed. The results show that nanoplastic caused the overproduction of ROS along with other dose-dependent effects. Low nanoplastic concentrations (0.1 and/or 0.5 mg/L) significantly increased the expressions of genes of the MAPK pathway (ERK; p38 mitogen-activated protein kinases, p38; c-Jun amino-terminal kinases, JNK; and protein kinase B, AKT), HIF-1 pathway ( prolyl hydroxylasedomain, PHD; vascular endothelial growth factor, VEGF; glucose transporter, GLUT; pyruvate kinase M, PKM; hypoxia-inducible factor 1, HIF1), and CuZn superoxide dismutase (SOD) along with the activity of glutathione-S-transferase. As the nanoplastic concentration increased, these indicators were significantly suppressed. The protein expression ratio of ERK, JNK, AKT, HIF1α, and NFkBp65 (nuclear transcription factor-kB p65) as well as the phosphorylation of ERK and NFkBp65 were increased in a dose-dependent manner. The activities of other antioxidant enzymes (catalase, total SOD, and CuZn SOD) were significantly decreased upon exposure to nanoplastic. Combined with our previous work, these results suggest that polystyrene nanoplastic causes the overproduction of ROS and activates the downstream pathway, resulting in inhibited growth, development, and reproduction. The present study fosters a better understanding of the biological effects of nanoplastics on zooplankton.

Pyraclostrobin is a fungicide used widely across the world. However, its photolysis pathway and toxic mechanism is unclear. In this study, photolysis and photo-induced toxicity of pyraclostrobin to Vibrio Fischeri were determined. The results showed that direct photolysis dominated the degradation of pyraclostrobin. Gas Chromatography-Mass spectrometry and quantum chemical calculation revealed that the pyraclostrobin was firstly photo-degraded into Methyl N-phenyl-carbamate and 1-(4-chlorophenyl)-3-hydroxy-1H-pyrzole, synthetic intermediates of pyraclostrobin, then into aniline, benzoquinone and acids. Toxicity assay showed that bioluminescent inhibition rate to V. fischeri fluctuated with radiation/illumination time and the toxicity curve can be classified into three phases (Phase I: 0 – 10 min, incline; Phase II: 10 – 60 min, decline; Phase III: 60 – 120 min, incline). The up-and-down curve indicates the change of parent compound during the photolysis. Simulation of molecular docking showed that the CDOCKER interaction energy of pyraclostrobin (-44.71) lower than other intermediate products (>-30.00), indicating that the parent compound is more toxic than its intermediates. An increased toxicity observed in the toxicity curve was attributed to the generation of benzoquinone with log1/EC50 of 6.73, which can greatly change structure of target luciferase in Vibrio Fischeri. In addition, the addition of radical scavengers can inhibit the bioluminescence of the tested solutions, indicating the involvement of radicals in the transformation of intermediates. This paper reveals that one of photochemical transformation products of pyraclostrobin can cause more toxic than its parent compound to bacteria. Environmental risk assessment should consider not only the parent compound, but also its metabolites.

ZnO nanoparticles (NPs) form binary mixtures of ZnO particles and released Zn2+ in the environment, and the quantitative contributions of these components to toxicity are still uncertain. Herein, quantitative contribution of ZnO particle and Zn2+ to cytotoxicity of ZnO NPs to Escherichia coli were determined during 48 h bioassay. The cytotoxicity and mechanisms of ZnO NPs were dynamic and affected by ionic strength, Fe3+, humic acid, and temperature due to the unfixed ZnO particle:Zn2+ ratio. ZnO NPs and ZnO bulk particles (BPs)had comparable cytotoxicity but distinct cytotoxic mechanisms. ZnO NPs cytotoxicity arises mainly from ZnO particles for 3 h and from Zn2+ afterwards (8 to 48 h). The cytotoxicity of ZnO BPs depends predominantly on ZnO particles for 12 h and on Zn2+ from 24 to 48 h. The cytotoxicity of ZnO NPs and BPs is partially attributable to Zn accumulation, and dependent on ZnO particle:Zn2+ ratio. The linear regressions of acute toxicity for ZnO NPs vs. BPs and Zn2+ yielded excellent r2 (0.9994 and 0.9998) from literature data and good r2 (≥ 0.714) under certain environmental factors, which can be applied to assess environmental risk of ZnO NPs. Furthermore, dynamic cytotoxicity and mechanisms should be seriously considered during the environmental risk assessment of ZnO NPs.

Tributyltin (TBT), an aromatase inhibitor, has been found to disrupt gametogenesis and reproductive behavior in several fish species. However, whether TBT is capable of affecting other behaviors such as aggressive behavior and fear response in fish and the underlying mode(s) of action remain unclear. To study aggressive behavior, adult zebrafish (Danio rerio) males were continuously exposed to two nominal concentrations of TBT (TBT-low, 100 ng/L and TBT-high, 500 ng/L) for 28 days. To study the fear response, the fish were divided into four groups (Blank and Control, 0 ng/L TBT; TBT-low, 100 ng/L; and TBT-high, 500 ng/L). The fish were then treated with DW (Blank) or with alarm substance (AS) (Control, TBT-low and TBT-high). After exposure, the aggressive behavior of the fish was tested using the mirror test (mirror-biting frequency, approaches to the mirror and duration in approach zone).and fighting test (fish-biting frequency) The mirror-biting frequency, approaches to the mirror, duration in approach zone and fish-biting frequency of the TBT-exposed fish increased significantly compared to those of the control fish, indicating enhanced aggressive behavior. The fear response parameters tested using the novel tank dive test (onset time to the higher half, total duration in the lower half and the frequency of turning) of the TBT-exposed fish were also significantly increased after AS administration, suggesting an enhanced fear response. Further investigation revealed that TBT treatment elevated the plasma level of 11-ketotestosterone (11-KT) and decreased the plasma level of estradiol (E2) in a concentration-dependent manner. Moreover, TBT up-regulated the mRNA levels of ar, c-fos and bdnf1, and suppressed the expression of btg-2 in fish. In addition, exposure to AS increased the plasma level of cortisol and down-regulated the mRNA expression levels of genes involved in 5-HT synthesis (such as tph1b and pet1) in both control and TBT-treated fish. AS significantly suppressed the mRNA level of tph1b, tph2, pet1 and npy in the TBT-high group compared to the control fish. The present study demonstrates that TBT enhances aggressive behavior and fear responses in male zebrafish probably through altering plasma levels of 11-KT, E2 and cortisol and altering the expression of genes involved in the regulation of aggressive behavior (ar, c-fos, bdnf1 and btg-2) and fear responses (tph1b, tph2, pet1 and npy). The present study greatly extends our understanding of the behavioral toxicity of TBT to fish.

Due to their widespread use, silver (Ag) and titanium dioxide (TiO2) nanoparticles (NPs) are commonly discharged into aquatic environments via wastewater treatment plants. The study was aimed to assess the effects of wastewater-borne AgNPs (NM-300 K; 15.5 ± 2.4 nm; 25-125 μg L-1) and TiO2NPs (NM-105; 23.1 ± 6.2 nm; 12.5-100 μg L-1), from a laboratory-scale wastewater treatment plant, on Daphnia magna, at individual and subcellular level. For effect comparison, animals were also exposed to ASTM-dispersed NPs at the same nominal concentrations. The behaviour of D. magna was evaluated through monitoring of swimming height and allocation time for preferred zones after 0 h and 96 h of exposure. Biochemical markers of neurotransmission, anaerobic metabolism, biotransformation, and oxidative stress were subsequently determined. No 96-h EC50 (immobilization ≤ 4%) could be obtained with wastewater-borne NPs and ASTM-dispersed TiO2NPs, whereas the ASTM-dispersed AgNPs resulted in an immobilization 96-h EC50 of 113.8 µg L-1. However, both wastewater-borne and ASTM-dispersed TiO2NPs, at 12.5 µg L-1, caused immediate (0 h) alterations on the swimming height. Allocation time analyses showed that animals exposed to ASTM-dispersed AgNPs spent more time on the surface and bottom at 0 h, and in the middle and bottom at 96 h. This pattern was observed neither with ASTM-dispersed TiO2NPs nor with wastewater-borne AgNPs or TiO2NPs. At the biochemical level, the more pronounced effects were observed with wastewater-borne AgNPs (e.g. induction of lactate dehydrogenase and glutathione S-transferase activities, and inhibition of catalase activity). This integrative approach showed that: (i) the behavioural and biochemical response-patterns were distinct in D. magna exposed to environmentally relevant concentrations of wastewater-borne and ASTM-dispersed NPs; (ii) the most pronounced effects on allocation time were induced by ASTM-dispersed AgNPs; and (iii) at the subcellular level, wastewater-borne AgNPs were more toxic than wastewater-borne TiO2NPs. This study highlights the need for the assessment of the effects of wastewater-borne NPs under realistic exposure scenarios, since processes in wastewater treatment plants may influence their toxicity.

Transgenic fish are powerful models that can provide mechanistic information regarding the endocrine activity of test chemicals. In this study, our objective was to use a newly developed transgenic zebrafish line expressing eGFP under the control of the cyp19a1a promoter in the OECD Fish Short Term Reproduction Assay (TG 229) to provide additional mechanistic information on tested substances. For this purpose, we exposed adult transgenic zebrafish to a reference substance of the TG 229, i.e. prochloraz (PCZ; 1.7, 17.2 and 172.6 µg/L). In addition to “classical” endpoints used in the TG 229 (reproductive outputs, vitellogenin), the fluorescence intensity of the ovaries was monitored at 4 different times of exposure using in vivo imaging. Our data revealed that 172.6 µg/L PCZ significantly decreased the number of eggs laid per female per day and the concentrations of vitellogenin in females, reflecting the decreasing E2 synthesis due to the inhibition of the ovarian aromatase activities. At 7 and 14 days, GFP intensities in ovaries were similar over the treatment groups but significantly increased after 21 days at 17.2 and 172.6 µg/L. A similar profile was observed for the endogenous cyp19a1a expression measured by qPCR thereby confirming the reliability of the GFP measurement for assessing aromatase gene expression. The overexpression of the cyp19a1a gene likely reflects a compensatory response to the inhibitory action of PCZ on aromatase enzymatic activities. Overall, this study illustrates the feasibility of using the cyp19a1a-eGFP transgenic line for assessing the effect of PCZ in an OECD test guideline while providing complementary information on the time- and concentration-dependent effects of the compound, without disturbing reproduction of fish. The acquisition of this additional mechanistic information on a key target gene through in vivo fluorescence imaging of the ovaries was realized without increasing the number of individuals.

The number of submarine power cables using either direct or alternating current is expected to increase drastically in coming decades. Data concerning the impact of magnetic fields generated by these cables on marine invertebrates are scarce. In this context, the aim of this study was to explore the potential impact of anthropogenic static and time-varying magnetic fields on the behavior of recently settled juvenile European lobsters (Homarus gammarus) using two different behavioral assays. Day-light conditions were used to stimulate the sheltering behavior and facilitate the video tracking. We showed that juvenile lobsters did not exhibit any change of behavior when submitted to an artificial magnetic field gradient (maximum intensity of 200 µT) compared to non-exposed lobsters in the ambient magnetic field. Additionally, no influence was noted on either the lobsters’ ability to find shelter or modified their exploratory behavior after one week of exposure to anthropogenic magnetic fields (225 ± 5 µT) which remained similar to those observed in control individuals. It appears that static and time-varying anthropogenic magnetic fields, at these intensities, do not significantly impact the behavior of juvenile European lobsters in daylight conditions. Nevertheless, to form a complete picture for this biological model, further studies are needed on the other life stages as they may respond differently.

We investigated the impacts of carbon dioxide (CO2)-induced acidification on energy metabolism in muscle tissue of rainbow trout Oncorhynchus mykiss fry. Fry were exposed to freshwater in equilibrium with air containing CO2 at volume concentrations of 380, 1000 and 2000 ppm for 30 days. At this critical and sensitive life stage, we showed dose-dependent reductions in weight gain and changes in energy metabolism under projected future CO2 levels. Rainbow trout fry exposed to elevated CO2 concentrations produced more energy in muscle tissue because ATP levels were significantly elevated. Fry exposed to 1000 ppm CO2 had a higher activity of pyruvate kinase (PK), higher concentrations of pyruvate, acetate, 2-oxoglutarate, phenylalanine, tyrosine, inosine, uracil and uridine, and lower concentrations of anserine and IMP in muscle tissue based on metabolomic analysis. We infer that the increased ATP production in fry exposed to 1000 ppm CO2 is produced through glycolysis. In the 2000 ppm group, the protein content of muscle was lower than in the control, and the activities of PK, cytochrome C oxidase (COX), Na+/K+-ATP and Ca2+/Mg2+-ATPase were significantly higher. Concentrations of pyruvate, 2-oxoglutarate, histidine, anserine and IMP were significantly lower, and alanine, methionine, leucine, isoleucine, valine, lysine, pantothenate, uracil and inosine were significantly higher. Accelerated tissue decomposition may have sustained the higher production of ATP in the 2000 ppm group.

There is little information in scientific literature as to how conditions created by a microcystin (MC) producing cyanobacterial bloom affect the oxidant/antioxidant, biotransformation and neurotoxicity parameters in adult frogs in situ. We investigated biochemical parameters in the skin and muscle of Pelophylax kl. esculentus from Lake Ludaš (Serbia) by comparing frogs that live on the northern bloom side (BS) of the lake with those that inhabit the southern no-bloom side (NBS). A higher protein carbonylation level and lower antioxidant defense system capability in the skin of frogs living in conditions of the cyanobacterial bloom were observed. Inhibition of glutathione-dependent machinery was the major mechanism responsible for the induction of cyanobacterial bloom-mediated oxidative stress in frog skin. On the other hand, the detected higher ability of muscle to overcome bloom prooxidant toxicity was linked to a higher efficiency of the biotransformation system through glutathione-S-transferase activity and/or was the consequence of indirect exposure of the tissue to the bloom. Our results have also revealed that the cyanobacterial bloom conditions induced the cholinergic neurotransmitter system in both tissues. This study provides a better understanding of the ecotoxicological impact of the MC producing cyanobacterial bloom on frogs in situ. However, further investigations of the complex mechanism involved in cyanobacterial bloom toxicity in real environmental conditions are required.

Recent studies suggest that 2,4-DABA, a neurotoxic excitatory amino acid present in virtually all environments, but predominantly in aquatic ecosystems may be a risk factor for development of neurodegenerative diseases in animals and humans. Despite its neurotoxicity and potential environmental importance, mechanisms underlying the excitatory and putative excitotoxic action of 2,4-DABA in neurons are still unexplored. We previously reported on extensive two-stage membrane depolarization and functional disturbances in leech Retzius neurons induced by 2,4-DABA. Current study presents the first detailed look into the electrophysiological processes leading to this depolarization. Intracellular recordings were performed on Retzius neurons of the leech Haemopis sanguisuga using glass microelectrodes and input membrane resistance (IMR) was measured by injecting hyperpolarizing current pulses through these electrodes. Results show that the excitatory effect 2,4-DABA elicits on neurons’ membrane potential is dependent on sodium ions. Depolarizing effect of 5·10-3 mol/L 2,4-DABA in sodium-free solution was significantly diminished by 91% reducing it to 3.26 ± 0.62 mV and its two-stage nature was abrogated. In addition to being sodium-dependent, the depolarization of membrane potential induced by this amino acid is coupled with an increase of membrane permeability, as 2,4-DABA decreases IMR by 8.27 ± 1.47 MΩ (67.60%). Since present results highlight the role of sodium ions, we investigated the role of two putative sodium-dependent mechanisms in 2,4-DABA-induced excitatory effect – activation of ionotropic glutamate receptors and the electrogenic transporter for neutral amino acids. Excitatory effect of 5·10-3 mol/L 2,4-DABA was partially blocked by 10-5 mol/L 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) a non-NMDA receptor antagonist as the first stage of membrane depolarization was significantly reduced by 2.59 ± 0.98 mV (40%), whilst second stage remained unaltered. Moreover, involvement of the sodium-dependent transport system for neutral amino acids was investigated by equimolar co-application of 5·10-3 mol/L 2,4-DABA and L-alanine, a competitive inhibitor of this transporter. Although L-alanine exhibited no effect on the first stage of membrane depolarization elicited by 2,4-DABA, it substantially reduced the second stage (the overall membrane depolarization) from 39.63 ± 2.22 mV to 16.28 ± 2.58 mV, by 58.92%. We therefore propose that the electrophysiological effect of 2,4-DABA on Retzius neurons is mediated by two distinct mechanisms, i.e. by activation of ionotropic glutamate receptor that initiates the first stage of membrane depolarization followed by the stimulation of an electrogenic sodium-dependent neutral amino acid transporter, leading to additional influx of positive charge into the cell and the second stage of depolarization.

Within monitoring frameworks, biomarkers provide several benefits because they serve as intermediates between pollutant exposure and effects, and integrate the responses of contaminants that operate through the same mechanism of action. This study was designed to verify the use of transcriptomic biomarkers developed in our prior work (i.e., Coastal Biosensor of Endocrine Disruption; C-BED assay) on Mytilus edulis and identify additional biomarkers for legacy pollutants. M. edulis were collected from a reference site in Pemaquid, ME, USA and deployed by the Massachusetts Water Resources Authority (MWRA) at locations in and outside Boston Harbor, MA, USA: including (1) Boston Inner Harbor (IH), (2) the current outfall (OS), (3) 1 km away from the current outfall (LNB), and (4) Deer Island (DI), the site where untreated wastewater was formally discharged into the bay. Differential gene expression was quantified with a high density microarray. Seven genes significantly correlated with whole tissue concentration of PAHs, and six genes significantly correlated with whole body concentrations of PCBs, two groups of legacy contaminants that were elevated at stations IH, OS, and DI. Enrichment analysis indicated that IH mussels had the highest induction of stress response genes, which correlated with the higher levels of contaminants measured at this site. Based on the C-BED assay gene analysis, stations IH and OS exhibited signs of endocrine disruption, which were further confirmed by incorporating the results for the C-BED assay within the Integrated Biomarker Response (IBR) approach. This study successfully demonstrated the potential use of transcriptomic biomarkers within a monitoring program to identify the presence and organismal responses to endocrine disrupting and legacy contaminant classes.

Fish studies report consumption of microplastics (MPs) in the field, and concern exists over associated risks. However, laboratory studies with adult fish are scarce. In this study, outbred and see-through Japanese medaka (Oryzias latipes) were fed diets amended with 500, 1000, or 2000 µg/g 10 µm fluorescent spherical polystyrene microplastics (MPs) for 10 weeks during their maturation from juveniles to spawning adults. No behavioral changes, growth differences, or mortalities occurred. In vivo examinations and histologic sections showed no evidence of translocation of MPs from the gut to other internal organs. Mature females experienced dose-dependent decreases in egg number. Scanning electron microscopic examination of gills and gut revealed MPs in both areas. Swollen enterocytes were observed on apices of gut folds only in exposed fish. These were particularly apparent in foreguts of the high exposure group. Enterocytes with eroded brush borders were found in foregut of high and medium exposure groups. Increased mucus production, in long strands and sheets, was seen over primary and secondary lamellae of gills. Histological analysis showed alteration in buccal cavity, kidney, and spleen. Thickening and roughening of epithelium in headgut and pharynx and cellular alterations in spleen occurred. Head kidney was the primary site of alteration. Glomerulopathy and nephrogenesis were observed in exposed fish, increasing in severity with exposure level.

Tritium (3H), a radioactive isotope of hydrogen, is ubiquitously present in the environment. In a previous study, we highlighted a mis-regulation of genes involved in muscle contraction, eye transparency and response to DNA damages after exposure of zebrafish embryo-larvae from 3 hpf to 96 hpf at 0.4 and 4 mGy/h of tritiated water (HTO). The present study aimed to link this gene mis-regulation to responses observed at higher biological levels. Analyses on spontaneous tail movement, locomotor activity and heart rate were performed. Histological sections of eyes were made to evaluate the impact of HTO on eye transparency and whole embryo immunostainings were realized to assess DNA double strand breaks repair using gamma-H2AX foci. We found a decrease of basal velocity as well as a decrease of response in 96 hpf larvae exposed at 0.4 mGy/h after a tactile stimulus as compared to controls. Histological sections of larvae eyes performed after the exposure to 4 mGy/h did not show obvious differences in lens transparency or retinal development between contaminated and control organisms. Gamma-H2AX foci detection revealed no differences in the number of foci between contaminated organisms and controls, for both dose rates. Overall, results highlighted more detrimental effects of HTO exposure on locomotor behavior in 96 hpf larvae exposed at the lowest dose rate. Those results could be linked to mis-regulation of gene involved in muscle contraction found in a previous study at the same dose rate. It appears that not all effects found at the molecular scale were confirmed using higher biological scales. These results could be due to a delay between gene expression modulation and the onset of physiological disruption or homeostatic mechanisms to deal with tritium effects. However, crossing data from different scales highlighted new pathways to explore, i.e. neurotoxic pathways, for better understanding HTO effects on organisms.

Fish reproduction in natural environments tends to occur in conditions, including extreme pH values, ideal for the survival of offspring. In this context, the purpose of this study was to assess the influence of pH on both survival and early development of two fish species, Astyanax lacustris (short-distance migrating fish) and Piaractus mesopotamicus (long-distance migrating fish), with different reproductive strategies,. Our experiments were divided into two tests (incubation and larviculture) at five pH levels. First was incubation through a water recirculation system using 2 L incubators followed by larviculture using a 2.5 L aquarium. pH had no influence on the hatching rate of A. lacustris eggs, but affected the yolk-sac diameter, while the newly-hatched larvae had some skeletal disorders. For P. mesopotamicus, pH influenced the hatching rate and perivitelline space, while the newly-hatched larvae had several skeletal disorders. The larviculture of A. lacustris revealed no effect on the survival rate, but the analyzed morphometric variables indicated significant differences between the treatments. An acidic pH negatively influenced the survival rate of P. mesopotamicus, and all morphometric variables also varied significantly between the treatments. We concluded that both the survival and development of A. lacustris and P. mesopotamicus revealed different responses at pH levels during incubation and larviculture tests.

Current and proposed transcontinental pipelines for the transport of diluted bitumen (dilbit) from the Canadian oil sands traverse the coastal watersheds of British Columbia, habitat essential to Pacific salmonids. To determine the potential risks posed to these keystone species, juvenile sockeye (Oncorhynchus nerka; 1+ parr) were acutely (24 - 96 h) or subchronically (21 - 42 d) exposed to 4 concentrations of the water-soluble fraction (WSF) of unweathered Cold Lake Blend dilbit (initial total PAC concentrations: 0, 13.7, 34.7 and 124.5 μg/L) in a flow-through system. Dilbit effects on iono-osmoregulation, the physiological stress response, and the immune system were assessed by both biochemical and functional assays. Hydrocarbon bioavailability was evidenced by a significant induction of liver ethoxyresorufin-O-deethylase (EROD) activity in exposed fish. Acute and subchronic exposure significantly reduced gill Na+-K+-ATPase activity and resulted in lower plasma osmolality, Cl-, and Na+ concentrations. Acute exposure to dilbit resulted in a classic physiological stress response, however at 21 d of exposure, plasma cortisol remained elevated while other measured parameters had returned to baseline values. A compromised immune system was demonstrated by a 29.5% higher mortality in fish challenged with Vibrio (Listonella) anguillarum following dilbit exposure compared to unexposed controls. Exposure of juvenile salmonids to the WSF of dilbit (at TPAC concentrations at the ppb level) resulted in sublethal effects that included a classic physiological stress response, and alterations in iono-osmoregulatory homeostasis and immunological performance.

Population control of invasive sea lamprey relies heavily on lampricide treatment of infested streams. The lampricide 3-trifluoromethyl-4-nitrophenol (TFM) is thought to impair mitochondrial ATP production through uncoupling oxidative phosphorylation. However, the effect of TFM on the entire electron transport chain (complexes I to V) in the mitochondria is not clear. In addition, TFM is reduced in phase I metabolism by sea lamprey at higher levels than in other fish species. The effects of these TFM reductive metabolites on mitochondria have not been explored. In this study, we sought to examine the effects of TFM and its reductive metabolite amino-TFM (TFMa) on cardiac mitochondrial oxygen consumption and membrane potential to delineate potential mechanisms for toxicity. To determine if molecules with similar structure also exhibit similar effects on mitochondria, we used 4-nitro-3-methylphenol (NMP) and its reductive metabolites 4-amino-3-methylphenol (NMPa) and 4-nitroso-3-methylphenol (NMPn) for comparisons. We found that mitochondrial bioenergetics was heavily affected with increasing concentrations of TFM, NMP, and NMPa when complexes I and II of the electron transport chain were examined, indicating that the toxic action of these compounds was exerted not only by uncoupling complex V, but also affecting complexes I and II.

Phytoremediation is an effective and environmentally friendly approach to treat antibiotic contaminated water, however, the mechanisms of migration and transformation of antibiotics in plant tissues are still far from clear. In this study, the floating macrophyte Eichhornia crassipes was exposed to a series of antibiotic ciprofloxacin (CIP) concentrations. The results showed that the CIP was taken up and accumulated in the roots, which were the major accumulative tissue. CIP content increased with lipid content. During cultivation, the root bioconcentration factor (RCF) gradually increased. The average CIP content detected in aerial parts was 12.80 μg g-1, an order of magnitude lower than in the roots. At low CIP concentrations, the highest leaf bioconcentration factor (LCF) and transfer factor (TF) indicated highly efficient translocation from roots to aerial parts. The soluble protein growth rate of leaves, which is associated with metabolic activity, increased following CIP exposure. Overall, eight major transformation products in E. crassipes tissues were identified, and three possible transformation pathways were proposed involving the processes of desethylation, dehydroxylation, oxidation, hydroxylation and cleavage of the piperazine and quinoline rings. These findings could prove beneficial for improving the management or amelioration methods used for treating water contaminated with antibiotics.

Aquatic ecosystems are subject to many anthropogenic disturbances, and understanding their possible impacts is a real challenge. Developing approaches based on the behaviour of bivalve mollusks, an integrating marker of the state of the organisms, and therefore of their environment, is relevant, whether within a natural ecosystem or an ecosystem subject to industrial activities. The main objective of this study was to identify by HFNI Valvometry a reliable and reproducible clam behavioural response in the presence of crude oil in a multistress context. To closely replicate actual field conditions, Corbicula fluminea was exposed in outdoor artificial streams that were subject to natural variations and were continuously fed by fresh water from the Gave de Pau (S.W. France). After a period of 26 days in these artificial streams, the clams (n = 14-16 per condition) were separately exposed for 10 days to crude oil alone, crude oil and barium, crude oil and noise pollution, crude oil and turbidity pulses, barium alone, noise pollution alone, turbidity pulses alone or natural changes alone. The secondary objective was to characterize the accumulation of polycyclic aromatic hydrocarbons (PAHs) in 3 tissues (gills, adductor muscles and foot) in clams exposed for 10 days to crude oil alone or under multistress conditions (n = 5 clams per condition) and then to compare the accumulation and behaviour of clams under these conditions. The response of clams to crude oil alone or under multistress conditions was visually and statistically significant and not confounded by the other disturbances tested, despite large variations in water temperature. In the presence of crude oil, the behaviour of clams was characterized by an increase in valve-closure duration, a decrease in valve-opening amplitude and an increase in valve agitation index. In the presence of crude oil, the clam behaviour showed no direct relationship with PAH accumulation in the gills, adductor muscles or foot, although hypothetical mechanisms are discussed. This work supports the growing interest in studying the behaviour of bivalve mollusks in the context of biomonitoring of the aquatic environment surrounding oil facilities.

Deltamethrin (DLM) is a synthetic pyrethroid used for agricultural purposes to control insects and has been found to pollute the aquatic environment and leads to serious health problems. Lactobacillus plantarum L-137 (L-137) has gained more popularity as functional supplement for its immunomodulatory effects and antioxidant potential. This study was designed to examine the potential of L-137 on liver function, histopathology, immune and antioxidant related gene expressions in Nile tilapia exposed to subacute DLM for 30 days. Fish (mean weight of 28.18 ± 1.34 g) was distributed into four groups (triplicates): the first and second groups fed the control diet, while the third and fourth groups fed L-137 at 50 mg/kg and the second and fourth groups were exposed to DLM (15 μg/L) in rearing water (control, DLM, L-137 and DLM + L-137, respectively). DLM-treated fish groups showed a significant increase in blood biochemical parameters (creatinine, urea and bilirubin) as well as hepatic enzymes (ALP, AST and ALT) (P < 0.05). Blood total protein, globulin, albumin, WBCs, RBCs, Hb, phagocytic index, phagocytic and lysozyme activities were significantly decreased in fish exposed to DLM (P < 0.05). Additionally, DLM toxicity downregulated the transcription of immune genes (IL-1β and IL-8), while upregulated the stress related genes (HSP70 and CASP3). The histopathological images of Nile tilapia exposed to DLM revealed damage in gills, intestine, spleen and liver which confirmed the toxic effects. Conversely, L-137 presented protective effects and restored the aforementioned parameters when fish exposed to DLM and fed L-137. Further, L-137 restored the antioxidative capacity (CAT and GPx). Thus, L-137 supplementation exhibited defensive effects against DLM toxicity in Nile tilapia through improving blood biochemical responses, immune, and antioxidant related gene expressions as well as histopathological effects.

Clarithromycin (CLA), a macrolide antibiotic, has been frequently detected in the global surface waters. Concerns have been raised over the potential impacts of CLA on the non-target aquatic species, particularly algae acting as the primary producers in the ecosystem. This study therefore evaluated the toxicological effects of CLA at a range of concentration levels (0, 5, 20, 40, 80 μg L-1) on two green algae, Raphidocelis subcapitata (R. subcapitata) and Chlorella vulgaris (C. vulgaris). The algal growth, photosynthetic pigment contents, lipid peroxidation biomarker malondialdehyde (MDA), responses of antioxidants including superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione peroxidase (GP), and glutathione S-transferase (GST) were measured. After 7 d exposure, the growth of R. subcapitata was inhibited with the CLA exposure levels higher than 20 μg L-1, whereas the inhibition in C. vulgaris was detected at the concentration level of 80 μg L-1. The MDA contents in both species were elevated. To cope with the increased levels of ROS, the activities of enzymatic antioxidants (SOD, CAT, GP, and GST) and the content of non-enzymatic antioxidant (GSH) in R. subcapitata were all enhanced. However, in C. vulgaris, enhancement was detected only in the activities of antioxidant enzymes (SOD, CAT, and GP). In addition, chlorophyll a, b, and carotenoid contents were all significantly increased in R. subcapitata but decreased in C. vulgaris. The results suggested that R. subcapitata is more sensitive to CLA exposure than C. vulgaris. This study provides insights into the CLA - oxidative stress process in two algae.

Manganese (Mn) is a hypoxic reactive metal commonly found in marine sediments. Under hypoxic conditions the metal becomes fully reduced to Mn2+ and is biologically available to the benthic community for uptake. Mn is also a potent neurotoxin and it may play a role in the etiology of idiopathic blindness that has been observed in American lobsters. An acute study was designed to expose American lobster, Homarus americanus, to 0, 20, 80, 150, and 300 mg L-1 (ppm) for 96 hrs to explore disparities in Mn accumulation among several tissues: optic nerve, brain, hepatopancreas, muscle, hemolymph, gill, and exoskeleton. These concentrations were based on realistic pore-water concentrations (20 mg L-1), high sediment concentrations (80 mg L-1), and unrealistically high concentrations to determine lethality (150 and 300 mg L-1). A positive correlation between Mn accumulation and exposure concentration was observed in all tissues examined. In the internal tissues, manganese concentrations showed a high affinity towards brain, optic nerve, and hemolymph. In the exoskeleton and gills, Mn concentrations were also high, possibly because of internal uptake as well as external adsorption. Concentrations of Mn in tissues from the acute exposure study followed the pattern: hemolymph > gill > exoskeleton > optic nerve > brain > hepatopancreas = muscle. A long-term exposure study lasting seven weeks was designed to investigate the potential link between high Mn exposure and idiopathic blindness, a condition that affects an estimated 50% of the adult American lobster population off Southern New England (SNE), USA. A comparison of these exposure studies showed evidence of time-dependent Mn accumulation in brain, muscle, exoskeleton, and gill tissue. Although the relationship between Mn exposure and blindness was not apparent, there was a modest trend in the development of blindness (Chi-square, p = 0.102) in animals exposed to a high concentration (150 mg L-1) of the metal. With no mortalities occurring in the acute study and only one mortality in the long-term study, it is highly unlikely that Mn is acutely toxic to American lobsters at environmentally relevant concentrations. Its potential role in idiopathic blindness remains to be determined.

Fipronil is a phenylpyrazole insecticide widely used to control pests in agriculture even though evidence of harmful side effects in non-target species has been reported. A comprehensive study on the effects of dietary administration of Regent®800WG (80% fipronil) in European sea bass juveniles was carried out under two temperature regimes: a) natural conditions, and b) 3 °C above the natural temperature (an increase predicted for the NW Mediterranean by the end of this century). Fipronil was added to the fish food (10 mg fipronil /Kg feed) and the effects were studied at several time points including right before administration, 7 and 14 days after daily fipronil feed and one-week after the insecticide withdrawal from the diet (depuration period). A wide array of physiological and metabolic biomarkers including feeding rate, general condition indices, plasma and epidermal mucus metabolites, immune response, osmoregulation, detoxification and oxidative-stress markers and digestive enzymes were assessed. General linear models and principal component analyses indicated that regardless of water temperature, fipronil resulted in a significant alteration of several of the above listed biomarkers. Among them, glucose and lactate levels increased in plasma and decreased in epidermal mucus as indicators of a stress response. Similarly, a depletion in catalase activity and higher lipid peroxidation in liver of fipronil-exposed fish were also indicative of an oxidative-stress condition. Fipronil induced a time dependent inhibition of Cytochrome P450-related activities and an inhibition of phase II glutathione-S-transferase. Moreover, fipronil administration was able to reduce the hypo-osmoregulatory capability as shown by the increase of plasmatic osmolality and altered several digestive enzymes including trypsin, lipase, alpha amylase and maltase. Finally, analyses in bile and muscle confirmed the rapid clearance of fipronil but the persistence of the metabolite fipronil-sulfone in bile even after the 7-day depuration period. Altogether, the results reveal a notable impact of this compound on the physiological condition of the European sea bass. The results should be considered in future environmental risk assessment studies since fipronil could be hazardous to fish species, particularly those inhabiting estuarine ecosystems exposed to the discharge of agriculture runoffs where this pesticide is mainly used.

Harmful algal bloom (HAB) toxins have severe negative impacts on marine mammals, particularly for Florida bottlenose dolphins (Tursiops truncatus) which frequently experience mass mortality events. Dolphins on the Florida Atlantic coast inhabit a region endemic to two HAB species, Karenia brevis and Pyrodinium bahamense, which produce the neurotoxins brevetoxin (PbTx) and saxitoxin (STX), respectively. Although toxic HABs and associated dolphin mortality events have been reported from this region, there is a lack of available data necessary for comparing toxin exposure levels between bloom (‘exposed’) conditions and non-bloom (‘baseline’) conditions. Here we present a 10-year dataset of PbTx and STX concentrations detected in dolphins stranding in this region, and compare the toxin loads from HAB-exposed dolphins to those detected in dolphins recovered in the absence of a HAB. We analyzed liver tissue samples from dead-stranded dolphins (n = 119) recovered and necropsied between 2002–2011, using an enzyme-linked immunosorbent assay (ELISA) modified for use with mammalian tissues. For dolphins recovered during baseline conditions, toxin-positive samples ranged in concentration from 0.27-1.2 ng/g for PbTx and from 0.41-1.9 ng/g for STX. For K. brevis-exposed dolphins, concentrations of up to 12.1 ng PbTx/g were detected, and for P. bahamense-exposed dolphins, concentrations of up to 9.9 ng STX/g were detected. Baseline PbTx values were similar to those reported in other regions where K. brevis blooms are more frequent and severe, but HAB-exposed PbTx values were considerably lower relative to these other regions. Since no baseline STX dolphin data exist for any region, our data serve as a first step towards establishing reference STX values for potential dolphin mortality events associated with STX-producing blooms in the future. This study demonstrates that although HABs in eastern Florida are only infrequently associated with dolphin mortalities, the presence of toxins in these animals may pose significant health risks in this region.

Ecotoxicological effects of glucocorticoids and steroid mixtures in the environment are not sufficiently known. Here we investigate effects of 11-14 days exposure of female zebrafish to the glucocorticoid clobetasol propionate (Clo), cortisol (Cs), their mixture and mixtures with five different class steroids (Clo + triamcinolone + estradiol + androstendione + progesterone) in liver, brain and gonads. Cs showed little activity, while Clo reduced the condition factor at 0.57 and 6.35 µg/L. Clo induced differential expression of genes in the liver at 0.07-6.35 µg/L, which were related to circadian rhythm (per1, nr1d2), glucose metabolism (g6pca, pepck1), immune system response (fkbp 5, soccs3, gilz), nuclear steroid receptors (pgr and pxr), steroidogeneses and steroid metabolism (hsd11b2, cyp2k22). Clo caused strong transcriptional down-regulation of vtg. Similar upregulations occurred in brain for pepck1, fkbp5, socs3, gilz, hsd11b2, and nr1d2a, while cyp19b was down-regulated. Effects of Clo + Cs mixtures were similar to potent Clo alone. Transcriptional alterations were different in five steroids mixtures with no alteration of vtg in the liver due to counteraction of Clo and estradiol. Induction of fkbp5 (brain) and sult2st3 (liver) and downregulation of cyp19a (gonads) occurred at 1 µg/L. Histological effects of the five steroids mixture in gonads were characterized by a decrease of mature oocytes. Our data indicate that effects of steroids of different classes sum up to an overall joint effect driven by the most potent steroid Clo.

Production of industrial effluents have led to increased copper (Cu) pollution of aquatic ecosystems, impacting the physiology of aquatic vertebrates. Past work has shown that Cu exerts its toxicity by disruption ion regulation and/ or increasing oxidative stress. However, it remains unclear how Cu may influence aerobic metabolism and hypoxia tolerance, two possible targets of its toxicity. To address this issue, we exposed freshwater acclimated killifish (F. heteroclitus) to a 96 h Cu treatment at a target concentration of 100 μg L-1. We determined resting oxygen consumption (MO2), MO2max after exhaustive exercise, and followed MO2 for 3 h in post-exercise recovery in water with either no Cu or 100 μg L-1 Cu. We assessed hypoxia tolerance by determining the critical oxygen tension (Pcrit). It was found that killifish exposed to combined 96 h Cu exposure and Cu present during metabolic measurements, showed a significant decrease in MO2max and in aerobic scope (MO2max - MO2rest), compared to control fish under either measurement condition. However, changes in blood and muscle lactate and muscle glycogen were not consistent with an upregulation of anaerobic metabolism as compensation for reduced aerobic performance in Cu exposed fish. Hypoxia tolerance was not influenced by the 96 h Cu exposure or by presence or absence of Cu during the pCrit test. This study suggests that Cu differentially influences responses to changes in oxygen demand and oxygen availability.

Mangroves are tidal wetlands that are often under strong anthropogenic pressures, despite the numerous ecosystem services they provide. Pollution from urban runoffs is one such threats, yet some mangroves are used as a bioremediation tool for wastewater (WW) treatment. This practice can impact mangrove crabs, which are key engineer species of the ecosystem. Using an experimental area with controlled WW releases, this study aimed to determine from an ecological and ecotoxicological perspective, the effects of WW on the red mangrove crab Neosarmatium africanum. Burrow density and salinity levels (used as a proxy of WW dispersion) were recorded, and a 3-week caging experiment was performed. Hemolymph osmolality, gill Na+/K+-ATPase (NKA) activity and gill redox balance were assessed in anterior and posterior gills of N. africanum. Burrow density decreased according to salinity decreases around the discharged area. Crabs from the impacted area had a lower osmoregulatory capacity despite gill NKA activity remaining undisturbed. The decrease of the superoxide dismutase activity indicates changes in redox metabolism. However, both catalase activity and oxidative damage remained unchanged in both areas but were higher in posterior gills. These results indicate that WW release may induce osmoregulatory and redox imbalances, potentially explaining the decrease in crab density. Based on these results we conclude that WW release should be carefully monitored as crabs are key players involved in the bioremediation process.

The aquatic environment is continuously under threat because it is the final receptor and sink of waste streams. The development of industry, mining activities and agriculture gave rise to an increase in metal pollution in the aquatic system. Thus a wide occurrence of metal mixtures exists in the aquatic environment. The assessment of mixture stress remains a challenge considering that we can not predict the toxicity of a mixture on the basis of single compounds. Therefore the analysis of the effects of environmentally relevant waterborne mixtures is needed to improve our understanding of the impact of metal pollution in aquatic ecosystems. Our aim was to assess whether 10% of the concentration of the 96 h LC50 (the concentration that is lethal to 50% of the population in 96 h) of individual metal exposures can be considered as a “safe” concentration when applied in a trinomial mixture. Therefore, common carp were exposed to a sublethal mixture of Cu 0.07 ± 0.001 µM (4.3 ± 0.6 μg/L), Zn 2.71 ± 0.81 µM (176.9 ± 52.8 μg/L) and Cd 0.03 ± 0.0004 µM (3.0 ± 0.4 μg/L) at 20 °C for a period of one week. Parameters assessed included survival rate, bioaccumulation and physiological biomarkers related to ionoregulation and defensive mechanisms such as MT induction. Our results showed a sharp increase in Cu and Cd concentration in gills within the first day of exposure while Zn levels remained stable. The accumulation of these metals led to a Na drop in gills, liver and muscle as well as a decreased K content in the liver. Biomarkers related to Na uptake were also affected: on the first day gene expression for H+-ATPase was transiently increased while a concomitant decreased gene expression of the Na+/H+ exchanger occurred. A fivefold induction of metallothionein gene expression was reported during the entire duration of the experiment. Despite the adverse effects on ionoregulation all fish survived, indicating that common carp are able to cope with these low metal concentrations, at least during a one week exposure.

Nitrite is a major environmental pollutant in aquatic environments that negatively affects aquatic species. In this study, we investigated the impact of nitrite exposure on plasma biochemical parameters and immune responses in Takifugu rubripes. Fish were exposed to various concentrations of nitrite (0, 0.5, 1, 3, and 6 mM) for 96 h. After 0, 12, 24, 48, and 96 h of exposure, fish blood samples were collected to assay the levels of total protein (TP), albumin (Alb), glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (ALT), complement C3 (C3), complement C4 (C4), immunoglobulin (IgM), and lysozyme activity (LZM). The gills were sampled to analyze the mRNA levels of heat shock protein 70 (hsp70), heat shock protein 90 (hsp90), tumor necrosis factor α (tnf-α), B-cell activating factor (baff), interleukin-6 (il-6), and interleukin-12 (il-12). Levels of GOT, ALT, C3, and C4 were significantly enhanced in the high nitrite concentration group (3 and 6 mM), whereas those of TP, Alb, LZM, and IgM decreased significantly with the same treatments. Nitrite significantly upregulated hsp70, hsp90, tnf-α, il-6, il-12, and baff mRNA levels after 96 h of exposure. These results indicated that nitrite exposure altered the blood physiological status and immune system response, resulting in dysfunction and immunotoxicity in T. rubripes. Furthermore, our results reveal the possible mechanism of aquatic-nitrite-induced toxicity in fish.

Labile dissolved organic carbon (DOC) is a major pollutant in coastal marine environments affected by anthropogenic impacts, and may significantly contribute to coral bleaching and subsequent mortality on coastal reefs. DOC can cause bleaching indirectly through the rapid proliferation of copiotrophic and pathogenic bacteria. Here we demonstrate that labile DOC compounds can also impair the coral-dinoflagellate symbiosis by directly affecting coral physiology on both the host and algal symbiont level. In a controlled aquarium experiment, we monitored over several weeks key physiological parameters of the tropical coral Stylophora pistillata exposed to ambient and elevated labile DOC levels (0.1 and 1.0 mM) in combination with low and high nitrogen (i.e. ammonium) conditions (0.2 and 4.0 μM). At the symbiont level, DOC exposure under low ammonium availability decreased the photosynthetic efficiency accompanied by ∼75% Chl a and ∼50% symbiont cell reduction. The photosynthetic functioning of the symbionts recovered once the DOC enrichment ceased indicating a reversible shift between autotrophic and heterotrophic metabolism. At the host level, the assimilation of exogenous DOC sustained the tissue carbon reserves, but induced a depletion of the nitrogen reserves, indicated by ∼35% decreased protein levels. This suggests an imbalanced exogenous carbon to nitrogen supply with nitrogen potentially limiting host metabolism on the long-term. We also demonstrate that increased ammonium availability delayed DOC-induced bleaching likely by keeping symbionts in a photosynthetically competent state, which is crucial for symbiosis maintenance and coral survival. Overall, the present study provides further insights into how coastal pollution can de-stabilize the coral-algal symbiosis and cause coral bleaching. Therefore, reducing coastal pollution and sustaining ecological integrity are critical to strengthen the resilience of coral reefs facing climate change.

The growing popularity of physical sunscreens will lead to an increased release of ingredients from zinc oxide (ZnO) sunscreens into marine environments. Though zinc (Zn) is a necessary micronutrient in the ocean, greater than natural Zn concentrations may be released into marine environments by use of sunscreens. The extent of the consequences of this addition of Zn to the ocean are not fully understood. We investigated the effects of materials released by ZnO- sunscreens on the development of California purple sea urchin, Strongylocentrotus purpuratus. Embryos incubated in various concentrations of Zn (0.01, 0.05, 0.1, 0.5, and 1 mg/L), the sources of which included zinc-containing compounds: ZnO and zinc sulfate (ZnSO4); and ZnO sunscreens: All Good, Badger, and Raw Elements brands. Based on EC50 values, ZnO-containing sunscreens were slightly, but not significantly, more toxic than ZnO and ZnSO4, suggesting that sunscreens may release additional unknown materials that are detrimental to sea urchin embryo development. All concentrations of Zn-exposure resulted in significant malformations (skeletal abnormality, stage arrest, axis determination disruption), which were identified using light and fluorescence confocal microscopy. The concentration of Zn2+ internalized by the developing embryos correlated positively with the concentration of Zn in seawater. Additionally, exposure to both ZnO sunscreens and ZnO and ZnSO4 at 1 mg/L Zn, significantly increased calcein-AM (CAM) accumulation, indicating decreased multidrug resistant (MDR) transporter activity. This is one of the first studies documenting ZnO-containing sunscreens release high concentrations of Zn that are internalized by and have detrimental effects on aquatic organisms.

Various strategies exist to control noxious cyanobacterial populations, although the application of a newly developed granular compound (sodium carbonate peroxyhydrate ‘SCP’, trade name ‘PAK® 27’ algaecide) containing hydrogen peroxide (H2O2) as the active ingredient, has been recently proven as an effective and ecofriendly treatment. However, in aquaculture settings the application of SCP to treat cynobacterial blooms may affect non-targeted biota, such as fish due to H2O2 being known to elicit toxic oxidative stress in fish. Consequently, a better understanding of the side effects as a function of dosing concentrations would help to improve treatment efficacy and fish welfare. Thus, the aim of the current study is to assess the potential risks of SCP to largemouth bass (Micropterus salmoides), a high priced fish in the U.S. To this end, fish were exposed to two recommended doses of SCP corresponding to either 2.5 or 4.0 mg/L H2O2 for 6 days, with a control group in parallel. After 6 days, the effect of SCP exposure on oxidative stress, histopathological changes and anti-oxidant potential in the brain, liver, gills and muscle were investigated. Results show that exposure to 4.0 mg/L H2O2 -SCP incited oxidative damage, evidenced by an over-accumulation of H2O2 and malondialdehyde (MDA) in the brain and liver, which were accompanied by an increment in xanthine oxidase activity. Unlike 4.0 mg/L H2O2, these oxidative stress biomarkers in the brain and liver tissue of 2.5 mg/L H2O2-SCP exposed fish were restrained within control levels and concomitant with an increase in superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR) and glutathione-s-transferase (GST) activity. In contrast, many of these anti-oxidants sentinels in the 4.0 mg/L H2O2 exposed fish were either unaffected or significantly inhibited, which resulted in over-accumulation of H2O2 and MDA. In addition, a series of histopathological alterations were observed, and the most severe brain injuries and liver inflammation were recorded in 4.0 mg/L H2O2-SCP exposed fish. In addition, based on oxidative parameters, both doses of SCP resulted in a relatively mild oxidative stress in gills but no effect in muscle, probably explaining the modest anti-oxidative responses in the former and almost complete lack of anti-oxidative responses in the latter. Overall, our findings suggests that application of SCP at 4.0 mg/L H2O2 for controlling cyanobacterial blooms in aquaculture settings can possess potential risk to the farmed fish.

Aquatic media are ultimate recipients of various contaminants including pesticides pervasively applied in agrosystems. Characterizing the ecotoxicity of pesticides and their mixtures to aquatic wildlife at field-realistic levels is thus crucial for environmental risk assessment. This study aims at assessing the effects of two current-use insecticides, imidacloprid and chlorpyrifos, on Gammarus fossarum using multi-level biomarkers. In microcosms, gammarids were exposed for 72 h to insecticides tested individually or in mixture at 0.01, 0.1 and 1 µg/L of each chemical. Multi-metric responses were assessed at the individual level (behavioural traits: locomotion, respiration and amplexus formation) and the cellular level (enzymes involved in growth, moulting, digestion and cell stress). The results showed insecticide-elicited behavioural and biochemical responses from the lowest concentration of 0.01 µg/L. Overall, single exposures stimulated behavioural traits and inhibited enzymatic activities, highlighting subtle impacts at different organizational levels but these were not dose related. For binary mixtures, antagonistic effects (i.e. less-than-additive) on biomarkers were mainly observed when compared with single exposures. Multi-variable analyses indicated the complementarity of behavioural and biochemical biomarkers in identifying sublethal biological alterations and dose-dependent multiple action sites of insecticides. Besides, the mortality observed only for the mixture at 1 µg/L demonstrated a high lethal potential of insecticides in a simple binary combination. To conclude, this study demonstrates disturbances in individual performances and cellular impairments occurring at environmentally realistic exposure levels in a non-target wild species. Since the sublethal effects, such as those identified with this multi-biomarker approach, could lead to long-term alterations in population dynamics of agricultural areas, they constitute promising early endpoints for risk assessment of insecticides.

Cadmium (Cd) affects plants and animal health seriously. Ca2+ signals in plant cells are important for adaptive responses to environmental stresses. Here we showed that 50 μM Cd shock stimulated the Ca2+ signal via modifying the instantaneous Ca2+ flux from influx of 17 pmol·cm-2·s-1 to the efflux of 240 pmol·cm-2·s-1 at 100 μm from rhizoid tip. And the Ca2+ signal transferred to the vein and mesophyll cell. The Ca addition decreased the accumulation of Cd. The gene expression of glutamate receptor–like (GLR) proteins, which is activated by Glu and triggers Ca2+ flux, was increased significantly by 24 h Cd stress. Glu content was increased under Cd stress and exogenous Glu triggered the Ca2+ signal in duckweed, while Ca2+ addition caused no influence to Glu content. GABA, which is synthesized from Glu and acts as an inhibitory neurotransmitter, has been decreased with 24 h Cd treatment. GABA addition increased the abscission rate and Glu addition decreased the abscission rate during Cd stress, suggesting that the Glu/GABA ratio is important for responding to Cd. This research shows the sight of the Glu, Ca2+, GABA signaling networks during Cd stress.

Tributyltin (TBT) and dioxin-like polychlorinated biphenyls (PCBs) are environmental contaminants that are highly toxic to fish and co-occur in New Bedford Harbor (NBH), an estuarine Superfund site located in Massachusetts, USA. Atlantic killifish (Fundulus heteroclitus) that reside in NBH (and other highly contaminated sites along the east coast of the United States) have developed resistance to activation of the aryl hydrocarbon receptor (AHR) pathway and the toxicity of dioxin-like chemicals, such as 3,3',4,4',5-pentachlorobiphenyl, PCB126. In many biological systems, TBT disregulates adipose and bone development via the PPARγ-RXR pathway; AHR activation also disrupts adipose and bone homeostasis, potentially through molecular crosstalk between AHR and PPARγ. However, little is known about how co-exposure and the interaction of these pathways modulate the toxicological effects of these contaminants. Here, we tested the hypotheses that TBT would induce teratogenesis in killifish via activation of PPARγ and that PCB126 co-exposure would suppress PPARγ pathway activation in PCB-sensitive killifish from a reference site (Scorton Creek, SC, PCB-sensitive) but not in PCB-tolerant NBH killifish. Killifish embryos from both populations exposed to TBT (50 and 100 nM) displayed caudal fin deformities. TBT did not change the expression of pparg or its target genes related to adipogenesis (fabp11a and fabp1b) in either population. However, expression of osx/sp7, an osteoblast marker gene, and col2a1b, a chondroblast marker gene, was significantly suppressed by TBT only in SC killifish. An RXR-specific agonist, but not a PPARγ-specific agonist, induced caudal fin deformities like those observed in TBT-treated embryos. PCB126 did not induce caudal fin deformities and did not exacerbate TBT-induced fin deformities. Further, PCB126 increased expression of pparg in SC embryos and not NBH embryos, but did not change the expression of fabp1b. Taken together, these results suggest that in killifish embryos the PPARγ pathway is regulated in part by AHR, but is minimally active at least in this early life stage. In killifish, RXR activation, rather than PPARγ activation, appears to be the mechanism by which TBT induces caudal fin teratogenicity, which is not modulated by AHR responsiveness.

Increasing microplastics pollution of marine and terrestrial water is a concerning issue for ecosystems and human health. Nevertheless, the interaction of microplastics with freshwater biota is still a poorly explored field. In order to achieve information concerning the uptake, distribution and effect of microplastics in planarians, Dugesia japonica specimens have been fed with mixtures of food and differently shaped and sized plastic particles. Feeding activity and food intake were non-altered by the presence of high concentrations of different types of plastic particles. However, the persistence of microplastic within the planarian body was a function of size/shape, being small spheres (<10 µm in diameter) and short fibers (14 µm large and 5/6 µm length) more persisting than larger spheres and longer fibers which were eliminated almost entirely by ejection in a few hours. Transmission electron microscopy analysis demonstrated that at least part of microplastics was phagocytized by the enterocytes. Chronic exposure to small plastic did not alter the regenerative ability but caused a significant reduction of the gut epithelium thickness and lipid content of enterocytes, together with the induction of apoptotic cell death, modulation of Djgata 4/5/6 expression and reduced growth rate. The ability of microplastic to perturb planarian homeostasis is concerning being them extremely resilient against mechanical and chemical insults and suggests possible harmful effects upon other more susceptible species in freshwater ecosystems.

Ocean acidification, due to increased levels of anthropogenic carbon dioxide, is known to affect the physiology and growth of marine phytoplankton, especially in polar regions. However, the effect of acidification or carbonation on cellular metabolism in polar marine phytoplankton still remains an open question. There is some evidence that small chlorophytes may benefit more than other taxa of phytoplankton. To understand further how green polar picoplankton could acclimate to high oceanic CO2, studies were conducted on an Antarctic Chlorella sp. Chlorella sp. maintained its growth rate (∼0.180 d-1), photosynthetic quantum yield (Fv/Fm = ∼0.69) and chlorophyll a (0.145 fg cell-1) and carotenoid (0.06 fg cell-1) contents under high CO2, while maximum rates of electron transport decreased and non-photochemical quenching increased under elevated CO2. GCMS-based metabolomic analysis reveal that this polar Chlorella strain modulated the levels of metabolites associated with energy, amino acid, fatty acid and carbohydrate production, which could favour its survival in an increasingly acidified ocean.

In the present study, the biochemical responses and antioxidant enzymes activity of the Dunaliella salina, a green microalga, to the interaction of silver nanoparticles (AgNPs) and salicylic acid (SA) were investigated. Algal suspensions in the phase of logarithmic growth were subjected to the concentrations of 0, 5, 15, and 25 pM AgNPs with or without 1 mM SA. AgNPs level of 25 pM declined cell division but highly accumulated levels of chlorophyll, β-carotene, proteins, free amino acid, carbohydrates, and hydrogen peroxide, which was associated with enhanced the activity of proteolysis, lipid peroxidation, and antioxidant enzymes. SA-treated cells at 25 pM AgNPs improved cell growth but declined the activities of antioxidant enzymes and proteolytic along with a lower accumulation of metabolites except β-carotene relative to untreated controls. These results suggest that AgNPs treatment induce oxidative stress in D. salina cells, which tolerated by alga through the metabolic modifications and accumulating β-carotene, while SA induces AgNPs tolerance by the mechanisms that direct carbon flux to growth and β-carotene biosynthesis rather than the antioxidant enzymes or osmoprotectant metabolites.

The present work aimed to investigate the effects of acute (12, 24, 48 and 96 h) and subchronic (168 and 336 h) exposure of seahorse, Hippocampus reidi to water-accommodated fraction (WAF) of diesel fuel on biotransformation parameters, antioxidant defenses and DNA integrity. In addition, a recovery experiment was performed, where the organisms remained in absence of the contaminant for 336 h, after WAF exposure for 168 h (totaling 504 h). At the end of each experimental protocol, the concentration of pyrene-, benzo(a)pyrene- and naphthalene-type metabolites in bile, hepatic activity of glutathione-S-transferase (GST), superoxide dismutase (SOD), and catalase (CAT), as well as lipid peroxidation (LPO) levels in hepatocytes, were analyzed, in addition to the DNA damage and the micronucleus (MN) test in the peripheral blood. It was observed that both acute and subchronic WAF exposure affected the investigated parameters in different ways. In general, the exposed groups presented higher mean values for the investigated parameters if compared with their respective controls. After the recovery experiment, the mean values of PAH metabolites, LPO, DNA damage and MN frequency were significantly lower than those of animals exposed for 168 h, indicating that the recovery period was appropriately long for the evaluated biomarkers return to the control levels. The results indicated that the selected H. reidi biomarkers proved to be adequate and complementary tools in determining the first impacts of acute and subchronic exposure caused by WAF of diesel fuel in fish, as well as their recovery in clean water.

Acidification of freshwater ecosystems is recognized as a global environmental problem. However, the influence of acidic water on the early stages of freshwater fish is still unclear. This study focused on the sublethal effects of acidic water on the lateral line system of zebrafish embryos. Zebrafish embryos were exposed to water at different pH values (pH 4, 5, 7, 9, and 10) for 96 (0∼96 h post-fertilization (hpf)) and 48 h (48∼96 hpf). The survival rate, body length, and heart rate significantly decreased in pH 4-exposed embryos during the 96-h incubation. The number of lateral-line neuromasts and the size of otic vesicles/otoliths also decreased in pH 4-exposed embryos subjected to 96- and 48-h incubations. The number of neuromasts decreased in pH 5-exposed embryos during the 96-h incubation. Alkaline water (pH 9 and 10) did not influence embryonic development but suppressed the hatching process. The mechanotransducer channel-mediated Ca2+ influx was measured to reveal the function of lateral line hair cells. The Ca2+ influx of hair cells decreased in pH 5-exposed embryos subjected to the 48-h incubation, and both the number and Ca2+ influx of hair cells had decreased in pH 5-exposed embryos after 96 h of incubation. In addition, the number and function of hair cells were suppressed in H+-ATPase- or GCM2-knockdown embryos, which partially lost the ability to secrete acid into the ambient water. In conclusion, this study suggests that lateral line hair cells are sensitive to an acidic environment, and freshwater acidification could be a threat to the early stages of fishes.

Cytochrome P450 (CYP) enzymes are one of the largest protein families, and they metabolise a wide range of lipophilic organic endogenous and exogenous compounds. Many cytochrome P450 genes have been cloned and characterised, and they are frequently used as biomarkers in environmental toxicology studies because of their sensitivity and inducibility. In the present study, the full-length cDNAs of DpCYP370B and DpCYP4 were cloned from Daphnia pulex for the first time. The sequence of DpCYP370B consisted of an ORF of 1515 bp that encoded a 504 amino acid polypeptide, while the sequence of DpCYP4 comprised an ORF of 1527 bp that encoded a 508 amino acid polypeptide. Homologous alignments revealed the presence of a conserved cysteine haeme-iron ligand signature, FxxGxxxCxG, located in the C-terminal portion. Both the proteins contained a sequence for a transmembrane region that was deduced to be located in the endoplasmic reticulum. Subsequently, the expression levels of DpCYP370B and DpCYP4, as well as those of CYP4AN1, CYP4C33, and CYP4C34, were investigated using quantitative real-time PCR after exposure to five polystyrene nanoplastic concentrations: 0 (control), 0.1, 0.5, 1, and 2 mg/L for 21 days. Except for DpCYP4, the highest mRNA expression was observed at 0.5 mg/L nanoplastics; next, the expression of three of the enzymes (DpCYP370B, CYP4AN1, CYP4C34,) decreased to that of the control level at 1 and 2 mg/L doses of nanoplastics. The expression of DpCYP4 did not significantly change compared with that of the control group. These results indicated that CYP genes might play an important role in protecting D. pulex against nanoplastic pollutants.

To understand effects of two widely used antidepressant on the antioxidant defense mechanism in the marine rotifer Brachionus koreanus, we assessed acute toxicity and measured population growth, reactive oxygen species (ROS) levels, glutathione (GSH) levels, and antioxidant enzymatic activities (GST, GR, and SOD) in response to fluoxetine hydrochloride (FLX) and sertraline hydrochloride (SER). The no observed effect concentration-24 h of fluoxetine and sertraline were 1000 μg/L and 450 μg/L, respectively, whereas the median lethal concentration (LC50)-24 h of fluoxetine and sertraline were 1560 μg/L and 507 μg/L, respectively. Both fluoxetine and sertraline caused significant reduction in the population growth rate indicating that both antidepressants have a potential adverse effect on life cycle parameters of B. koreanus. The intracellular ROS level and GSH level were significantly modulated (P < 0.05) in response to fluoxetine and sertraline. In addition, antioxidant enzymatic activities have shown significant (P < 0.05) modulation in response to FLX and SER in B. koreanus. Furthermore, transcriptional profiles of antioxidant genes (GSTs, SODs, and GR) have shown modulation in response to FLX compared to SER-exposed B. koreanus. Our results indicate that fluoxetine and sertraline induce oxidative stress, leading to reduction in the population density and modulation of antioxidant defense mechanism in the marine rotifer B. koreanus.

Lethal and sublethal effects of trinitrotoluene (TNT) and its degradation products 2-amino-4,6-dinitrotoluene (2-ADNT) and 4-amino-2,6-dinitrotoluene (4-ADNT) to zebrafish embryos (Danio rerio) were investigated in a 120 hours exposure scenario. Lethal concentrations (LC50) were 4.5 mg/l for TNT, 13.4 mg/l for 2-ADNT and 14.4 mg/l for 4-ADNT. Embryos exposed to 2-ADNT or 4-ADNT revealed a high proportion of chorda deformations among the surviving individuals. Genotoxicity of the nitroaromatic compounds in zebrafish embryos was investigated by comet assay isolating cells from whole embryos after 48 hours in vivo exposure. Significant genotoxicity was induced by all three compounds tested, in comparison to the corresponding controls at 0.1 mg/l and 1.0 mg/l as lowest tested concentrations. The genotoxicity caused by TNT was about three to four times higher than that of 2-ADNT and 4-ADNT. To our knowledge, this is the first study demonstrating the genotoxicity of TNT in fish embryos by in vivo exposure. The results are discussed in the context of dumped munition in the marine environment.

The simultaneous presence of natural and anthropogenic stressors in aquatic ecosystems can challenge the identification of factors causing decline in fish populations. These stressors include chemical mixtures and natural abiotic and biotic factors such as water temperature and parasitism. Effects of cumulative stressors may vary from antagonism to synergism at the organismal or population levels and may not be predicted from exposure to individual stressors. This study aimed to evaluate the combined effects of chronic exposure to cadmium (Cd) and elevated water temperature (23 °C) or parasite infection in juvenile rainbow trout (Oncorhynchus mykiss) using a multi-level biological approach, including RNA-sequencing. Fish were exposed to diet-borne Cd (6 μg Cd/g wet feed), individually and in combination with thermal (23 °C) or parasitic stressors, for 28 days. The parasite challenge consisted of a single exposure to glochidia (larvae) of the freshwater mussel (Strophitus undulatus), which encysts in fish gills, fins and skin. Results indicated lower fish length, weight, and relative growth rate in fish exposed to a higher water temperature (23 °C). Body condition and hepatosomatic index of trout were, however, higher in the 23 °C temperature treatment compared to the control fish kept at 15 °C. Exposure to thermal stress or parasitism did not influence tissue Cd bioaccumulation. More than 700 genes were differentially transcribed in fish exposed to the individual thermal stress treatment. However, neither Cd exposure nor parasite infection affected the number of differentially transcribed genes, compared to controls. The highest number of differentially transcribed genes (969 genes) was observed in trout exposed to combined Cd and high temperature stressors; these genes were mainly related to stress response, protein folding, calcium metabolism, bone growth, energy metabolism, immune system, and functions overlapped with responses found in fish solely exposed to higher water temperature. Only 40 genes were differentially transcribed when fish were exposed to Cd and glochidia and were related to the immune system, apoptosis process, energy metabolism and malignant tumor. These results suggest that dietary Cd may exacerbate the temperature stress and to a lesser extent, parasitic infection stress on trout transcriptomic responses. Changes in the concentrations of liver ethoxyresorufin-o-deethylase, heat shock protein 70 and thiobarbituric acid reactive substances coupled to changes in the activities of cellular glutathione S-transferase and glucose-6-phosphate dehydrogenase were also observed at the cellular level. This study may help understand effects of freshwater fish exposure to cumulative stressors in a changing environment.

Oxidative stress parameters were evaluated during the first 72 h of embryonic development of purple sea urchin Strongylocentrotus purpuratus continuously exposed to control conditions, to cadmium alone (Cd, 30 µg/L), to zinc alone (Zn, 9 µg/L) or to a Cd (28 µg/L) plus Zn (9 µg/L) mixture. These sublethal concentrations represent ∼ 10% of the acute EC50. Bioaccumulation, antioxidant capacity against peroxyl radicals (ACAP), total glutathione (GSH) level, glutathione-S-transferase (GST), glucose-6-phosphate dehydrogenase (G6PDH) and superoxide dismutase (SOD) activity, and lipid peroxidation (LPO) were analyzed at 24 h (blastula), 48 h (gastrula), and 72 h (pluteus) stages of development. Zinc (an essential metal) was well-regulated, whereas Cd (non-essential) bioaccumulated and whole-body [Cd] increased from blastula to pluteus stage in sea urchin larvae. In controls, ACAP progressively declined from 24 h to 72 h, while LPO reciprocally increased, but other parameters did not change. Cd alone was more potent than Zn alone as a pro-oxidant, with the major effects being decreases in SOD activity and parallel increases in LPO throughout development; GST activity also increased at 24 h. Zn alone caused only biphasic disturbances of ACAP. In all cases, the simultaneous presence of the other metal prevented the effects, and there was no instance where the oxidative stress response in the presence of the Cd/Zn mixture was greater than in the presence of either Cd or Zn alone. Therefore the sublethal effects of joint exposures were always less than additive or even protective, in agreement with classical toxicity data. Furthermore, our results indicate that SOD and Zn can play important roles in protecting sea urchin embryos against Cd-induced lipid peroxidation.

Endogenous estrogens play major roles in many aspects of female reproductive development in fish. In order to develop a relatively high-throughput assay to determine the potential impact on reproductive development, vitellogenic rainbow trout ovarian follicles were exposed to a suite of contaminants in vitro and then assessed for the ability to produce estradiol-17β (E2) after a 500 ng/ml salmon gonadotropin (sGTH) challenge. There was a positive correlation between ovarian follicle size and E2 production, but an inverse correlation between size and responsiveness to sGTH. Significant impacts on E2 levels were observed following treatment with different endocrine disrupting chemicals, such as 17α-ethinylestradiol (EE2), prochloraz, or trenbolone. EE2 was remarkably potent and significantly reduced ovarian follicle responsiveness to sGTH at concentrations as low as 0.1 nM. Of the other contaminants tested, only tamoxifen impacted E2 levels, and only at concentrations near the limits of solubility. Flutamide, fluoxetine, 4-hydroxy tamoxifen, hydroxyflutamide, and norfluoxetine had little or no impact. Quantitative PCR analyses of steroidogenesis-related genes were carried out on EE2 treated ovarian follicles, but significant transcriptional responses to EE2 were not observed. Overall, this study suggests that xenoestrogens and anti-estrogens are more likely to interfere with ovarian E2 synthesis than other classes of EDCs. This also provides a template for further testing of the effects of EDCs on ovarian function.

It is widely recognized that endocrine disrupting chemicals (EDCs) released into the environment through anthropogenic activities can have short-term impacts on physiological and behavioral processes and/or sustained or delayed long-term developmental effects on aquatic organisms. While numerous studies have characterized the effects of EDCs on temperate fishes, less is known on the effects of EDCs on the growth and reproductive physiology of tropical species. To determine the long-term effects of early-life exposure to common estrogenic chemicals, we exposed Mozambique tilapia (Oreochromis mossambicus) yolk-sac fry to 17β-estradiol (E2) and nonylphenol (NP) and subsequently characterized the expression of genes involved in growth and reproduction in adults. Fry were exposed to waterborne E2 (0.1 and 1.0 µg/L) and NP (10 and 100 µg/L) for 21 days. After the exposure period, juveniles were reared for an additional 112 days until males were sampled. Gonadosomatic index was elevated in fish exposed to E2 (0.1 µg/L) while hepatosomatic index was decreased by exposure to NP (100 µg/L). Exposure to E2 (0.1 µg/L) induced hepatic growth hormone receptor (ghr) mRNA expression. The high concentration of E2 (1.0 µg/L), and both concentrations of NP, increased hepatic insulin-like growth-factor 1 (igf1) expression; E2 and NP did not affect hepatic igf2 and pituitary growth hormone (gh) levels. Both E2 (1.0 µg/L) and NP (10 µg/L) induced hepatic igf binding protein 1b (igfbp1b) levels while only NP (100 µg/L) induced hepatic igfbp2b levels. By contrast, hepatic igfbp6b was reduced in fish exposed to E2 (1.0 µg/L). There were no effects of E2 or NP on hepatic igfbp4 and igfbp5a expression. Although the expression of three vitellogenin transcripts was not affected, E2 and NP stimulated hepatic estrogen receptor (erα and erβ) mRNA expression. We conclude that tilapia exposed to E2 and NP as yolk-sac fry exhibit subsequent changes in the endocrine systems that control growth and reproduction during later life stages.

Ocean acidification and marine biofouling, which may interact in the future, pose two major threats to global coastal ecosystems. Yet, the fate of highly invasive fouling species in a rapidly acidifying ocean remains poorly understood, due to lack of information on multigenerational consequences at different levels of biological organization. Here, we investigated antioxidant responses of the mussel, Musculista senhousia, a swiftly spreading invasive fouling species in global coastal waters, following transgenerational exposure to elevated pCO2. In the face of seawater acidification, M. senhousia without a prior history of transgenerational exposure to elevated pCO2 showed resistance to lipid peroxidation, but significantly increased activities of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), indicated oxidative stress responses. However, enhanced transgenerational immunity occurred, as exemplified by observations that mussels originating from parents exposed to elevated pCO2 exhibited significantly lower activities of SOD, CAT and GPx in comparison to those spawn from parents exposed to ambient pCO2. Rapid transgenerational acclimation of M. senhousia in terms of reduced oxidative stress responses can likely be linked to the enhanced capacity of maintaining acid-base homeostasis previously demonstrated. These findings provide the first evidence of transgenerational plasticity at the biochemical level in highly invasive fouling bivalve species, and represent a step forward in understanding how they respond and acclimate in an acidifying ocean.

One of the categories of environmental contaminants possibly contributing to declining sockeye salmon (Oncorhynchus nerka) in the Fraser River, British Columbia, Canada is pesticides. In this 4-month study, the effects of environmentally relevant concentrations of a waterborne neonicotinoid, clothianidin (0.15, 1.5, 15 and 150 μg/L), on embryonic, alevin and early swim-up fry sockeye salmon derived from four unique genetic crosses of the Pitt River, BC stock were investigated. There were no significant effects of clothianidin on survival, hatching, growth or deformities, although genetic variation significantly affected these endpoints. Clothianidin caused a significant 4.7-fold increase in whole body 17β-estradiol levels in swim-up fry after exposure to 0.15 µg/L, but no effects were observed on testosterone levels. In addition, hepatic expression of the gene encoding glucocorticoid receptor 2 was also impacted at the highest concentration of clothianidin tested, and was found to be ∼4-fold lower compared to the sockeye reared in control water. These results indicate additional examination of clothianidin and its effects on salmonid gonad development and the reproductive and stress endocrine axes in general, is warranted.

Calanus hyperboreus is the largest copepod and a key species in the Arctic food web. During the spring bloom, C. hyperboreus builds up large lipid reserves, which enable it to survive and produce eggs during overwintering. The ecological effects of oil exposure on overwintering C. hyperboreus are unknown. The present study empirically tested if exposure to the polycyclic aromatic hydrocarbon (PAH) pyrene from crude oil affects the survival, egg production, and hatching success of overwintering C. hyperboreus. We also tested the delayed effects on faecal pellet production and lipid recovery in clean seawater. Direct exposure did not reduce survival and egg production, but reduced hatching success 3-18 times by the end of the exposure period. Remarkably, we documented strong delayed effects of pyrene on faecal pellet production and the recovery of lipid reserves. The current study reveals a high vulnerability of this key species of Arctic zooplankton to oil exposure during winter. Together with our previous study on C. glacialis, we complete the picture of the impact of oil on the largest and most lipid-rich copepod C. hyperboreus, which potentially can have huge ecological consequences for the fragile Arctic marine food web.

Numerous anthropogenic sources, such as pulp mill and sewage treatment effluents, contain androgenic endocrine disrupting compounds that alter the reproductive status of aquatic organisms. The current study injected adult male mummichog (Fundulus heteroclitus) with 0 (control), 1 pg/g, 1 ng/g or 1 μg/g body weight of the model androgen 5α-dihydrotestosterone (DHT) with the intent to induce a period of plasma sex hormone depression, a previously-observed effect of DHT in fish. A suite of gonadal steroidogenic genes were assessed during sex hormone depression and recovery. Fish were sampled 6, 12, 16, 18, 24, 30 and 36 h post-injection, and sections of testis tissue were either snap frozen immediately or incubated for 24 h at 18 °C to determine in vitro gonadal hormone production and then frozen. Plasma testosterone (T) and 11-ketotestosterone (11KT) were depressed beginning 24 h post-injection. At 36 h post-injection plasma T remained depressed while plasma 11KT had recovered. In snap frozen tissue there was a correlation between plasma sex hormone depression and downregulation of key steroidogenic genes including steroidogenic acute regulatory protein (star), cytochrome P450 17a1 (cyp17a1), 3β-hydroxysteroid dehydrogenase (3βhsd), 11β-hydroxysteroid dehydrogenase (11βhsd) and 17β-hydroxysteroid dehydrogenase (17βhsd). Similar to previous studies, 3βhsd was the first and most responsive gene during DHT exposure. Gene responses from in vitro tissue were more variable and included the upregulation of 3βhsd, 11βhsd and star during the period of hormone depression. The differential expression of steroidogenic genes from the in vitro testes compared to the snap frozen tissues may be due to the lack of regulators from the hypothalamo-pituitary-gonadal axis present in whole-animal systems. Due to these findings it is recommended to use snap frozen tissue, not post-incubation tissue from in vitro analysis, for gonadal steroidogenic gene expression to more accurately reflect in vivo responses.