Preparing for the future offspring: European perch (Perca fluviatilis) biosynthesis of physiologically required fatty acids for the gonads happens already in the autumn.

Long-chain polyunsaturated fatty acids (PUFA) are critical for reproduction and thermal adaptation. Year-round variability in the expression of fads2 (fatty acid desaturase 2) in the liver of European perch (Perca fluviatilis) in a boreal lake was tested in relation to individual variation in size, sex, and maturity, together with stable isotopes values as well as fatty acids (FA) content in different tissues and prey items. ARA and DHA primary production was restricted to the summer months, however, perch required larger amounts of these PUFA during winter, as their ARA and DHA muscle content was higher compared to summer. The expression of fads2 in perch liver increased during winter and was higher in mature females. Mature females stored DHA in their gonads already in late summer and autumn, long before the upcoming spring spawning period in May. Lower δ13CDHA values in the gonads in September suggest that these females actively synthesized DHA as part of this reproductive investment. Lower δ13CARA values in the liver of all individuals during winter suggest that perch were synthesizing essential FA to help cope with over-wintering conditions. Perch seem able to modulate its biosynthesis of physiologically required PUFA in situations of stress (fasting or cold temperatures) or in situations of high energetic demand (gonadal development). Biosynthesis of physiologically required PUFA may be an important part of survival and reproduction in aquatic food webs with long cold periods.

Eutrophication effect on production and transfer of omega-3 fatty acids in boreal lake food webs.

Eutrophication, i.e. increasing level of nutrients and primary production, is a central environmental change of lakes globally with wide effects on food webs. However, how eutrophication affects the synthesis of physiologically essential biomolecules (omega-3 fatty acids) and their transfer to higher trophic levels at the whole food web level is not well understood. We assessed food web (phytoplankton, zooplankton, and fish) biomass, community structure and fatty acid content (eicosapentaenoic acid [EPA], and docosahexaenoic acid [DHA]), together with fatty acid specific primary production in 12 Finnish boreal lakes covering the total nutrient gradient from oligotrophic to highly eutrophic lakes (4-140 µg TP l-1; 413-1814 µg TN l-1). Production was measured as the incorporation of 13C-NaHCO3 into phytoplankton fatty acids and differentiated into volumetric production (production per litre of water) and productivity (production per phytoplankton biomass). Increases in nutrients led to higher biomass of phytoplankton, zooplankton and fish communities while also affecting community composition. Eutrophication negatively influenced the contribution of phytoplankton biomass preferentially grazed by zooplankton (<35 µm). Total volumetric production saturated at high phytoplankton biomass while EPA volumetric production presented a logarithmic relationship with nutrient increase. Meanwhile, total and EPA productivity had unimodal responses to this change in nutrients. DHA volumetric production and productivity presented large variation with increases in total phosphorus, but a unimodal model best described DHA changes with eutrophication. Results showed that eutrophication impaired the transfer of EPA and DHA into zooplankton and fish, showing a clear negative impact in some species (e.g. perch) while having no effect in other species (e.g. roach, ruffe). Results show non-linear trends in fatty acid production and productivity peaking at nutrient concentrations 22-35 µg l-1 TP followed by a gradual decrease.

Endogenous AhR agonist FICZ accumulates in rainbow trout (Oncorhynchus mykiss) alevins exposed to a mixture of two PAHs, retene and fluoranthene.

Multiple studies have reported synergized toxicity of PAH mixtures in developing fish larvae relative to the additive effect of the components. From a toxicological perspective, multiple mechanisms are known to contribute to synergism, such as altered toxicodynamics and kinetics, as well as increased oxidative stress. An understudied contributor to synergism is the accumulation of endogenous metabolites, for example: the aryl hydrocarbon receptor 2 (AhR2) agonist and tryptophan metabolite 6-Formylindolo(3,2-b)carbazole (FICZ). Fish larvae exposed to FICZ, alongside knock-down of cytochrome p450 (cyp1a), has been reported to induced symptoms of toxicity similar to those observed following exposure to PAHs or the dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin. Here, we explored if FICZ accumulates in newly hatched rainbow trout alevins (Oncorhynchus mykiss) exposed to two PAHs with different properties: retene (potent AhR2 agonist) and fluoranthene (weak AhR2 agonist and Cyp1a inhibitor), either alone or as a binary mixture for 3 and 7 days. We found that exposure to the mixture resulted in accumulation of endogenous FICZ, synergized the blue sac disease index (BSD), and altered the body burden profiles of the PAHs, when compared to the alevins exposed to the individual components. It is thus very plausible that accumulation of endogenously derived FICZ contributed to the synergized BSD index and toxicity in exposed alevins. Accumulation of endogenously derived FICZ is a novel finding that extends our general understanding on PAHs toxicity in developing fish larvae, while at the same time highlighting why environmental risk assessment of PAHs should not be based solely results from the assessment of individual compounds.

Metabolic plasticity of mixotrophic algae is key for their persistence in browning environments.

Light availability is the main regulator of primary production, shaping photosynthetic communities and their production of ecologically important biomolecules. In freshwater ecosystems, increasing dissolved organic carbon (DOC) concentrations, commonly known as browning, leads to lower light availability and the proliferation of mixotrophic phytoplankton. Here, a mixotrophic algal species (Cryptomonas sp.) was grown under five increasing DOC concentrations to uncover the plastic responses behind the success of mixotrophs in browning environments and their effect in the availability of nutritionally important biomolecules. In addition to the browning treatments, phototrophic, heterotrophic and mixotrophic growth conditions were used as controls. Despite reduced light availability, browning did not impair algal growth compared to phototrophic conditions. Comparative transcriptomics showed that genes related to photosynthesis were down-regulated, whereas phagotrophy gene categories (phagosome, lysosome and endocytosis) were up-regulated along the browning gradient. Stable isotope analysis of phospholipid fractions validated these results, highlighting that the studied mixotroph increases its reliance on heterotrophic processes with browning. Metabolic pathway reconstruction using transcriptomic data suggests that organic carbon is acquired through phagotrophy and used to provide energy in conjunction with photosynthesis. Although metabolic responses to browning were observed, essential fatty acid content was similar between treatments while sterol content was slightly higher upon browning. Together, our results provide a mechanistic model of how a mixotrophic alga responds to browning and how such responses affect the availability of nutritionally essential biomolecules for higher trophic levels.

Changes in cardiac proteome and metabolome following exposure to the PAHs retene and fluoranthene and their mixture in developing rainbow trout alevins.

Exposure to polycyclic aromatic hydrocarbons (PAHs) is known to affect developing organisms. Utilization of different omics-based technologies and approaches could therefore provide a base for the discovery of novel mechanisms of PAH induced development of toxicity. To this aim, we investigated how exposure towards two PAHs with different toxicity mechanisms: retene (an aryl hydrocarbon receptor 2 (Ahr2) agonist), and fluoranthene (a weak Ahr2 agonist and cytochrome P450 inhibitor (Cyp1a)), either alone or as a mixture, affected the cardiac proteome and metabolome in newly hatched rainbow trout alevins (Oncorhynchus mykiss). In total, we identified 65 and 82 differently expressed proteins (DEPs) across all treatments compared to control (DMSO) after 7 and 14 days of exposure. Exposure to fluoranthene altered the expression of 11 and 19 proteins, retene 29 and 23, while the mixture affected 44 and 82 DEPs by Days 7 and 14, respectively. In contrast, only 5 significantly affected metabolites were identified. Pathway over-representation analysis identified exposure-specific activation of phase II metabolic processes, which were accompanied with exposure-specific body burden profiles. The proteomic data highlights that exposure to the mixture increased oxidative stress, altered iron metabolism and impaired coagulation capacity. Additionally, depletion of several mini-chromosome maintenance components, in combination with depletion of several intermediate filaments and microtubules, among alevins exposed to the mixture, suggests compromised cellular integrity and reduced rate of mitosis, whereby affecting heart growth and development. Furthermore, the combination of proteomic and metabolomic data indicates altered energy metabolism, as per amino acid catabolism among mixture exposed alevins; plausibly compensatory mechanisms as to counteract reduced absorption and consumption of yolk. When considered as a whole, proteomic and metabolomic data, in relation to apical effects on the whole organism, provides additional insight into PAH toxicity and the effects of exposure on heart structure and molecular processes.

Exposure to retene, fluoranthene, and their binary mixture causes distinct transcriptomic and apical outcomes in rainbow trout (Oncorhynchus mykiss) yolk sac alevins.

Polycyclic aromatic hydrocarbons (PAHs) are widely spread environmental contaminants which affect developing organisms. It is known that improper activation of the aryl hydrocarbon receptor (AhR) by some PAHs contributes to toxicity, while other PAHs can disrupt cellular membrane function. The exact downstream mechanisms of AhR activation remain unresolved, especially with regard to cardiotoxicity. By exposing newly hatched rainbow trout alevins (Oncorhynchus mykiss) semi-statically to retene (32 µg l-1; AhR agonist), fluoranthene (50 µg l-1; weak AhR agonist and CYP1a inhibitor) and their binary mixture for 1, 3, 7 and 14 days, we aimed to uncover novel mechanisms of cardiotoxicity using a targeted microarray approach. At the end of the exposure, standard length, yolk area, blue sac disease (BSD) index and PAH body burden were measured, while the hearts were prepared for microarray analysis. Each exposure produced a unique toxicity profile. We observed that retene and the mixture, but not fluoranthene, significantly reduced growth by Day 14 compared to the control, while exposure to the mixture increased the BSD-index significantly from Day 3 onward. Body burden profiles were PAH-specific and correlated well with the exposure-specific upregulations of genes encoding for phase I and II enzymes. Exposure to the mixture over-represented pathways related to growth, amino acid and xenobiotic metabolism and oxidative stress responses. Alevins exposed to the individual PAHs displayed over-represented pathways involved in receptor signaling: retene downregulated genes with a role in G-protein signaling, while fluoranthene upregulated those involved in GABA signaling. Furthermore, exposure to retene and fluoranthene altered the expression of genes encoding for proteins involved in calcium- and potassium ion channels, which suggests affected heart structure and function. This study provides deeper understanding of the complexity of PAH toxicity and the necessity of investigating PAHs as mixtures and not as individual components.

Salmo trutta is more sensitive than Oncorhynchus mykiss to early-life stage exposure to retene.

Salmonids are known to be among the most sensitive fish to dioxin-like compounds (DLCs), but very little is known about the sensitivity of the brown trout (Salmo trutta), which has declined and is endangered in several countries of Europe and Western Asia. We investigated the sensitivity of brown trout larvae to a widespread dioxin-like PAH, retene (3.2 to 320 µg.L-1), compared to the larvae of a salmonid commonly used in toxicology studies, the rainbow trout (Oncorhynchus mykiss). Mortality, growth, cyp1a induction and the occurrence of deformities were measured after 15 days of exposure. Brown trout larvae showed a significantly higher mortality at 320 µg.L-1 compared to rainbow trout larvae. While the occurrence of deformities was only significantly increased at 320 µg.L-1 for the rainbow trout, brown trout larvae displayed pericardial edemas and hemorrhages already at 10 or 100 µg.L-1. cyp1a induction was increased significantly already at ≥3.2 µg.L-1 for the brown trout, versus ≥32 µg.L-1 for the rainbow trout. Least square regression analysis of the concentration-response relationships suggested that S. trutta larvae were at least 2 times more sensitive than O. mykiss larvae for cyp1a induction. The present study suggests that S. trutta larvae are more sensitive than O. mykiss larvae to a potent DLC, retene. As it is possible that S. trutta populations have declined partly because of pollution by DLCs, we recommend generating more data regarding the sensitivity of threatened fish populations, in order to ensure better risk assessment.

Retene, pyrene and phenanthrene cause distinct molecular-level changes in the cardiac tissue of rainbow trout (Oncorhynchus mykiss) larvae, part 2 - Proteomics and metabolomics.

Polycyclic aromatic hydrocarbons (PAHs) are global contaminants of concern. Despite several decades of research, their mechanisms of toxicity are not very well understood. Early life stages of fish are particularly sensitive with the developing cardiac tissue being a main target of PAHs toxicity. The mechanisms of cardiotoxicity of the three widespread model polycyclic aromatic hydrocarbons (PAHs) retene, pyrene and phenanthrene were explored in rainbow trout (Oncorhynchus mykiss) early life stages. Newly hatched larvae were exposed to sublethal doses of each individual PAH causing no detectable morphometric alterations. Changes in the cardiac proteome and metabolome were assessed after 7 or 14 days of exposure to each PAH. Phase I and II enzymes regulated by the aryl hydrocarbon receptor were significantly induced by all PAHs, with retene being the most potent compound. Retene significantly altered the level of several proteins involved in key cardiac functions such as muscle contraction, cellular tight junctions or calcium homeostasis. Those findings were quite consistent with previous reports regarding the effects of retene on the cardiac transcriptome. Significant changes in proteins linked to iron and heme metabolism were observed following exposure to pyrene. While phenanthrene also altered the levels of several proteins in the cardiac tissue, no clear mechanisms or pathways could be highlighted. Due to high variability between samples, very few significant changes were detected in the cardiac metabolome overall. Slight but significant changes were still observed for pyrene and phenanthrene, suggesting possible effects on several energetic or signaling pathways. This study shows that early exposure to different PAHs can alter the expression of key proteins involved in the cardiac function, which could potentially affect negatively the fitness of the larvae and later of the juvenile fish.

Retene, pyrene and phenanthrene cause distinct molecular-level changes in the cardiac tissue of rainbow trout (Oncorhynchus mykiss) larvae, part 1 - Transcriptomics.

Polycyclic aromatic hydrocarbons (PAHs) are contaminants of concern that impact every sphere of the environment. Despite several decades of research, their mechanisms of toxicity are still poorly understood. This study explores the mechanisms of cardiotoxicity of the three widespread model PAHs retene, pyrene and phenanthrene in the rainbow trout (Oncorhynchus mykiss) early life stages. Newly hatched larvae were exposed to each individual compound at sublethal doses causing no significant increase in the prevalence of deformities. Changes in the cardiac transcriptome were assessed after 1, 3, 7 and 14 days of exposure using custom Salmo salar microarrays. The highest number of differentially expressed genes was observed after 1 or 3 days of exposure, and retene was the most potent compound in that regard. Over-representation analyses suggested that genes related to cardiac ion channels, calcium homeostasis and muscle contraction (actin binding, troponin and myosin complexes) were especially targeted by retene. Pyrene was also able to alter similar myosin-related genes, but at a different timing and in an opposite direction, suggesting compound-specific mechanisms of toxicity. Pyrene and to a lesser extent phenanthrene were altering key genes linked to the respiratory electron transport chain and to oxygen and iron metabolism. Overall, phenanthrene was not very potent in inducing changes in the cardiac transcriptome despite being apparently metabolized at a slower rate than retene and pyrene. The present study shows that exposure to different PAHs during the first few days of the swim-up stage can alter the expression of key genes involved into the cardiac development and function, which could potentially affect negatively the fitness of the larvae in the long term.

Temporal variations in embryotoxicity of Lake Ontario American eel (Anguilla rostrata) extracts to developing Fundulus heteroclitus.

The recruitment of American eel (Anguilla rostrata) juveniles to Lake Ontario (LO), Canada has declined significantly since the 1980s. To investigate the possible contribution of maternally-transferred persistent organic pollutants (POPs) to this decline, this study measured temporal variations in the toxicity of complex organic mixtures extracted from LO American eels captured in 1988, 1998 and 2008 to developing Fundulus heteroclitus exposed by intravitelline (IVi) injection. The 1988 and 1998 eel extracts were most toxic, causing a pattern of sublethal embryotoxic responses similar to those previously reported in F. heteroclitus embryos exposed to single dioxin-like compounds (DLCs): stunted growth, craniofacial deformities, EROD activity induction, and reduced predatory capacities. The potency of extracts declined over time; the only significant effect of the 2008 eel extracts was EROD induction. The chemically-derived TCDD-TEQs of eel extracts, calculated using measured concentrations of some DLCs and their relative potencies for F. heteroclitus, overestimated their potency to induce EROD activity possibly due to interactions among POPs. Other POPs measured in eel extracts (non-dioxin-like PCBs, PBDEs and organochlorinated pesticides) did not appear to be important agonistic contributors to the observed toxicity. The toxicity of the complex mixtures of POPs measured in LO eels may have been underestimated as a result of several factors, including the loss of POPs during extracts preparation and a focus only on short-term effects. Based on the model species examined, our results support the hypothesis that contamination of LO with DLCs may have represented a threat to the American eel population through ecologically-relevant effects such as altered larval prey capture ability. These results prioritize the need to assess early life stage (ELS) toxicity of DLCs in Anguilla species, to investigate long-term effects of complex eel extracts to ELS of fish, and to develop biomarkers for potential effects in eel ELS sampled in the field.

Applicability of the TCDD-TEQ approach to predict sublethal embryotoxicity in Fundulus heteroclitus.

The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxic equivalent quantity (TCDD-TEQ) approach was used successfully to predict lethal embryotoxicity in salmonids, but its applicability to sublethal effects of mixtures of organohalogenated compounds in other fish species is poorly known. The sublethal toxicity of two dioxin-like compounds (DLCs), 3,3',4,4'-tetrachlorobiphenyl (PCB77) and 2,3,4,7,8-pentachlorodibenzofuran (2,3,4,7,8-PnCDF), two non-dioxin-like (NDL) polychlorinated biphenyls (PCBs), 2,2',5,5'-tetrachlorobiphenyl (PCB52) and 2,3,3',4',6-pentachlorobiphenyl (PCB110), and of Aroclor 1254, a complex commercial mixture of PCBs, was assessed in Fundulus heteroclitus embryos exposed by intravitelline injection. At 16 days post-fertilization, the two DLCs and Aroclor 1254 altered prey capture ability in addition to inducing classical aryl hydrocarbon receptor-mediated responses: ethoxyresorufin-O-deethylase (EROD) induction, craniofacial deformities and reduction in body length. None of these responses was induced by the two NDL PCBs, at doses up to 5400 ng g(-1)wet weight. Dose-response curves for prey capture ability for the 2 DLCs tested were not parallel to that of TCDD, violating a fundamental assumption for relative potency (ReP) estimation. Dose-response curves for EROD induction were parallel for 2,3,4,7,8-PnCDF and TCDD, but the ReP of 2,3,4,7,8-PnCDF for F. heteroclitus was 5-fold higher than the World Health Organization (WHO) fish toxic equivalent factor (TEF) based on embryolethality in salmonids. The chemically derived TCDD-TEQs of Aroclor 1254, calculated using 3,3',4,4',5-pentachlorobiphenyl (PCB126) concentrations and it ReP for F. heteroclitus, overestimated its potency to induce EROD activity possibly due to antagonistic interactions among PCBs. This study highlights the limitations of using TEFs based on salmonid toxicity data alone for risk assessment to other fish species. There is a need to assess the variability of RePs of DLCs in different species for a variety of endpoints and to better understand interactions between DLCs and other toxic chemicals.

Relative potency of PCB126 to TCDD for sublethal embryotoxicity in the mummichog (Fundulus heteroclitus).

The relative potency (ReP) of 3,3',4,4',5-pentachlorobiphenyl (PCB126) to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for sublethal responses was assessed in Fundulus heteroclitus embryos. Eggs were treated with intravitelline injections of graded sublethal doses of PCB126 (312-5000 pg g(-1) wet weight, ww) or TCDD (5-1280 pg g(-1) ww). At 16 days post-fertilization (DPF), craniofacial deformities were observed in larvae hatched from eggs treated with the two highest doses of PCB126 (2500-5000 pg g(-1) ww). Both compounds caused a dose-responsive reduction of larval growth and prey capture ability (at ≥1250 pg g(-1) ww), and induction of ethoxyresorufin-O-deethylase (EROD) activity (at ≥80 pg g(-1) ww). The dose-response relationships for EROD activity for PCB126 and TCDD had similar slopes and the ReP of PCB126 to TCDD for EROD activity was estimated at 0.71. This is 140-fold higher than the World Health Organization (WHO) TCDD equivalency factor (TEF) of PCB126 for fish (0.005), which is based on rainbow trout (Oncorhynchus mykiss) embryolethality data. The slope of the dose-response relationship for prey capture ability for PCB126 was steeper than for TCDD, suggesting different mechanisms of action. Expression levels of several genes were also studied by quantitative real-time polymerase chain reaction (qPCR) following exposure to single doses of TCDD or PCB126 (1280 and 1250 pg g(-1) ww, respectively) causing similar EROD induction. A different pattern of responses was observed between PCB126 and TCDD: PCB126 appeared to induce antioxidant responses by inducing sod2 expression, while TCDD did not. These results suggest that relative potencies are species-specific and that the current ReP for PCB126 underestimates its toxicity for some fish species. It is recommended to develop species-specific RePs for a variety of sublethal endpoints and at environmentally relevant doses.

A simple and reliable in vivo EROD activity measurement in single Fundulus heteroclitus embryo and larva.

Dioxin-like compounds (DLC) induce toxic responses in early life stages of fish through activation of the aryl hydrocarbon receptor (AhR) which is frequently assessed by ethoxyresorufin-O-deethylase (EROD) activity. A novel spectrofluorimetric method was developed to quantitatively assess EROD activity in individual living embryos and prolarvae of a marine model fish species, the mummichog Fundulus heteroclitus. This in vivo method is based on the measurement of the production of resorufin by single live embryos or prolarvae after 5 h incubation with ethoxyresorufin. Freshly fertilized eggs were treated topically from 2.5 to 50 pg egg⁻¹, with 3,3',4,4',5-pentachlorobiphenyl (PCB126), a prototypical DLC. EROD activity was assessed in embryos (7 days post-fertilization) and prolarvae (16 days post-fertilization). Resorufin was measured both in the culture medium (25‰ seawater) and in whole fish homogenates, to assess the percentage retained in the body. Approximately 95% and 17% of the resorufin produced in vivo was retained in embryos and prolarvae respectively. EROD activity in homogenates of embryos and in the culture medium of prolarvae increased linearly with dose. EROD activity measured by the in vivo method was highly correlated to that measured by a traditional in vitro technique using S9 fractions for both embryos and prolarvae. Both in vivo and in vitro EROD activity were higher in prolarvae than in embryos pretreated with PCB126. EROD induction measured in prolarvae by the in vivo and in vitro methods were similar whereas higher induction was measured in vivo than in vitro in embryos. The in vivo method was more sensitive and as reliable as the in vitro technique, and required a lower number of fish (4 compared to 3 pools of 5). This in vivo method is useful to link EROD induction in individual embryos or prolarvae to other organism-level responses. Further studies with other categories of xenobiotics should be performed to assess potential toxic effects on resorufin absorption/excretion processes which could affect in vivo measurements.

Long-term food-exposure of zebrafish to PCB mixtures mimicking some environmental situations induces ovary pathology and impairs reproduction ability.

Although the use of polychlorinated biphenyls (PCBs) has been banned for several decades, they are still present in the environment and are occasionally mechanically released from sediment or transferred through the trophic chain. Field analyses have established correlations between exposure to PCBs and alterations in fish physiology including reproductive function. Experimental exposures have been mainly performed using dioxin-like PCBs or other congeners at very high concentrations. However, these studies are often difficult to relate to real-life conditions. In the present study, we performed a life-cycle exposure using zebrafish model and mixtures representative of some environmental situations in terms of doses, composition and containing mainly non dioxin-like congeners. Exposure was performed through diet which is the main contamination route in the field. We demonstrated a bioaccumulation of PCBs in males and females as well as a maternal transfer to the eggs. Survival, growth and organ size were similar for all conditions. Several reproductive traits were altered after exposure to a PCB-contaminated diet, including a reduction in the number of fertilized eggs per spawn as well as an increase of the number of poorly fertilized spawns. This latter observation was found irrespective of the sex of contaminated fish. This is related to modifications of ovary histology revealing a decrease of maturing follicles and an increase of atretic follicles in the ovaries of females exposed to PCBs. These results indicate that exposure to PCBs mixtures mimicking some environmental situations, including mainly non dioxin-like congeners, can lead to a dramatic reduction in the number of offspring produced by a female over a lifetime. This is of great concern for wild species living under natural conditions.