Integrated Biological Response Research Articles

Tracing toxic path of antimony: From bioaccumulation to DNA hypomethylation in zebrafish (Danio rerio)

The increasing concentration of Antimony (Sb) in ecological environments has raised serious concerns about its potential biotoxicological impact. This study investigated the toxicokinetics, Global DNA Methylation (GDM), biomarker expression, and Integrated Biological Response (IBR) of Sb at different concentrations in zebrafish. The toxic mechanism of Sb exposure was simulated using molecular dynamics (MD). The results showed that significant differences effect existed (BCFk: liver > ovary > gut > brain) and uptake saturation phenomenon of Sb among zebrafish tissues. Over a 54-day exposure period, the liver emerged as the main target site for Sb-induced GDM, and the restoration was slower than in other tissues during the 54-day recovery period. Moreover, the concentration of Sb had a significant impact on the normally expression of biomarkers, with GSTM1 inhibited and MTF2, MT1, TET3, and p53 showing varying degrees of activation at different Sb concentrations. This could be attributed to Sb3+ potentially occupying the active site or tightly binding to the deep cavity of these genes. The IBR and MD results highlighted DNMT1 as the most sensitive biomarker among those assessed. This heightened sensitivity can be attributed to the stable binding of Sb3+ to DNMT1, resulting in alterations in the conformation of DNMT1's catalytic domain and inhibition of its activity. Consequently, this disruption leads to damage to the integrity of GDM. The study suggests that DNA methylation could serve as a valuable biomarker for assessing the ecotoxicological impact of Sb exposure. It contributes to a better understanding of the toxicity mechanisms in aquatic environments caused potential pollutants.

Butyl benzyl phthalate exposure impact on the gut health of Metaphire guillelmi

Agricultural films are extensively utilized in high-intensity agriculture, with China's annual usage reaching 1.5 million tons. Unfortunately, the recovery rate is less than 60%, leading to an inevitable accumulation of plastic mulch in agricultural soils. This accumulation primarily introduces butyl benzyl phthalate (BBP) into soil ecosystems, whose specific effects remain largely unclear, thereby posing potential risks. The present study focuses on the exposure impact of BBP on earthworms, Metaphire guillelmi, a commonly found endogenic earthworm within real farmland, as it provides insight into the direct interaction between biota gut health and contaminants. Specifically, we studied the biomarkers related to oxidative stress, the digestive system, and neurotoxicity within the gut of Metaphire guillelmi, and the integrated biological response (IBR) index was utilized to track these markers at different timeframes after BBP exposures. Our findings indicate that BBP exposures lead to oxidative damage, digestive system inhibition, and neurotoxicity, with IBR indexes of 14.6 and 17.3 on the 14th and 28th days, respectively. Further, the underlying mechanisms at a molecular level through molecular docking were investigated. The results showed that the most unstable interaction was with the Na+K+-ATPase (binding energy: −2.25 kcal−1), while BBP displayed stable bonds with superoxide dismutase and 8-hydroxydeoxyguanosine via hydrogen bonds and hydrophobic interaction. These interactions resulted in changes in protein conformation and their normal physiological functions, offering new insights into the molecular mechanism underlying enzymatic activity changes. This study has significant implications for the prediction of toxicity, environmental risk assessment, and the establishment of regulations related to BBP.

Randomly-shaped nanoplastics induced stronger biotoxicity targeted to earthworm Eisenia fetida species: Differential effects and the underlying mechanisms of realistic and commercial polystyrene nanoplastics

Nanoplastics (NPs) are widely distributed in various environments, including soil, and have been known to adversely affect soil organisms. Currently, most of the obtained studies were principally focused on the ecological risks of commercial sphere-type microbeads (SNPs), while ignoring that they might be different from randomly-shaped nanoplastics (RNPs) in a real environment. Thus, this study was undertaken to probe the shape-dependent effects of NPs on the earthworm Eisenia fetida and the corresponding poisoning mechanisms, and discriminate the toxicity differences between SNPs and RNPs at the molecule, cell, tissue, and animal levels. The results showed SNPs and RNPs exhibited lethal effects to earthworms with the LC50 determined to be 27.42 g/kg and 21.69 g/kg, respectively after a 28-day exposure. SNPs and RNPs exposure can cause ROS-induced ROS release in worm, inducing oxidative stress through mitochondria-mediated pathway, leading to lipid peroxidation, DNA damage, and histopathological changes, thereby contributing to decreased stress resistance against exogenous stressors. To reduce ROS-mediated oxidative damage, the antioxidant defense system in E. fetida can be activated, which scavenges unwanted ROS. High doses of SNPs and RNPs inhibited the AChE activity in worms, causing excess acetylcholine accumulation in the synaptic space, which finally lead to neurotoxicity. Also, two kinds of NPs can induce the abnormal expression of genes relevant to oxidative stress, reproduction, growth, and tight junction protein in E. fetida, which ultimately contribute to various detrimental effects, tissue damage and dysfunction, reproductive and developmental toxicity. The results obtained from the Integrated Biological Response (IBR) suggested that long-term exposure to high-dose SNPs and RNPs can induce the stronger toxicity effects to E. fetida worms, and RNPs-induced toxicity can be different and stronger than that of SNPs. Our results provide insights for revealing the environmental effects posed by randomly-shaped NPs-contaminated soil, and are of importance for assessing the contribution of NPs with different physical characteristics to soil eco-safety.

Disturbances in growth, oxidative stress, energy reserves and the expressions of related genes in Daphnia magna after exposure to ZnO under thermal stress

The toxicological effects of metal contamination are influenced by the ambient temperature. Therefore, global warming affects the toxicity of metal contamination in aquatic ecosystems. ZnO is widely used as a catalyst in many industries, and causes contamination in aquatic ecosystems. Here, we investigated the effects of ZnO concentration under elevated temperature by observing growth, oxidative stress, energy reserves and related gene expression in exposed Daphnia magna. Body length and growth rate increased in neonates exposed to ZnO for 2 days but decreased at 9 and 21 days under elevated temperature. ZnO concentration and elevated temperature induced oxidative stress in mature D. magna by reducing superoxide dismutase (SOD) activity and increasing malondialdehyde (MDA) levels. In contrast, juveniles were unaffected. Carbohydrate, protein and caloric contents were reduced throughout development in D. magna treated with ZnO and elevated temperature in all exposure periods (2, 9 and 21 days). However, lipid content also decreased in mature D. magna treated with ZnO cultured under elevated temperature, while that of juveniles showed an increase in lipid content. Therefore, energy was perhaps allocated to physiological processes for detoxification and homeostasis. Moreover, expression patterns of genes related to physiological processes changed under elevated temperature and ZnO exposure. Taken together, our results highlight that the combination of temperature and ZnO concentration induced toxicity in D. magna. This conclusion was confirmed by the Integrated Biological Response (IBR) index. This study shows that changes in biological levels of organization could be used to monitor environmental change using D. magna as a bioindicator.

Extreme environmental doses of diisobutyl phthalate exposure induce oxidative stress and DNA damage in earthworms (Eisenia fetida): Evidence at the biochemical and molecular levels

Diisobutyl phthalate (DIBP), as a plasticizer, is widely used and has caused many extreme soil contamination scenarios, posing potential risks to soil fauna. However, the toxic effects and mechanisms of DIBP on soil fauna remain unclear. In this study, earthworms (Eisenia fetida) were used as model animals to explore the subchronic toxicity of extreme DIBP soil exposure (300, 600, and 1200 mg/kg) for 28 days. The results showed that the level of reactive oxygen species (ROS) and the contents of malondialdehyde (MDA) and 8-hydroxydeoxyguanosine (8-OHdG) in E. fetida were significantly increased during continuous DIBP exposure. In addition, the activities of superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) were significantly inhibited while glutathione S-transferase (GST) activity was activated during continuous exposure. Integrated biological response (IBR) analysis showed that DIBP had positive dose-dependent toxicity and negative time-dependent toxicity to E. fetida, and SOD/CAT were selected as sensitive biomarkers. The molecular docking study found that DIBP could stably bind to SOD/CAT through hydrogen bonding, which further proved its sensitivity. This study provides primary data for ecological and environmental risk assessment of extreme dose DIBP soil pollution.

The response of bacterial community to UVB was significantly different between immature periphyton and mature periphyton, but not for physiological indicators

Periphyton has important ecological functions. It can even exist in environments with strong ultraviolet radiation. However, knowledge of periphyton under ultraviolet is limited, which limits the understanding and application of periphyton in environments with high ultraviolet radiation. In this study, immature periphyton (IMP) and mature periphyton (MP) under ultraviolet B (UVB) irradiation were investigated and compared in terms of physiological characteristics and bacterial community. Analysis of the physiological characteristics showed that the response patterns of IMP and MP to UVB were similar. IMP and MP could adapt to UVB of 1 W/m2 well. However, high-intensity UVB (2 and 3 W/m2) reduced the periphyton biomass, inhibited photosynthesis and antioxidant enzyme activity and caused severe lipid peroxidation in both IMP and MP. Integrated Biological Response (IBR) analysis and toxicological model fitting showed that the ED50 values of UVB for IMP and MP were 1.25 and 1.50 W/m2, respectively. 16 S rRNA gene analysis showed that in both IMP and MP, bacterial community composition, assembly and function were affected by UVB. In addition, the response of the bacterial community in IMP to UVB was stronger than that in MP. The diversity of the IMP community was inhibited by UVB, but that of the MP community was not. Proteobacteria and Deinococcus-Thermus are key microorganisms responsible for tolerance to UVB stress. Neutral community model fitting showed that both UVB and the development process caused the determinism of bacterial succession. However, UVB may weaken the deterministic process caused by development. Functional prediction showed that many metabolic functions of periphyton were inhibited by UVB in IMP and MP. However, UVB caused different changes (enhancement or inhibition) of some ecological functions in them. This study provides valuable information for understanding periphyton in environments with UVB radiation, which may be used to improve the application of periphyton in these environments.

Evaluation of fluorene-caused ecotoxicological responses and the mechanism underlying its toxicity in Eisenia fetida: Multi-level analysis of biological organization

Fluorene is an important toxic chemical that exists ubiquitously in the environment, and it has also been suggested to exert potential deleterious effects on soil invertebrates. However, knowledge about the toxic effects of fluorene and its underlying mechanisms of the effects on key soil organism earthworms remains limited. From this view point, this study was undertaken to explore the potential effects of fluorene and its underlying mechanisms in Eisenia fetida at the level of experimental animals, tissue, cell, and molecule. It was concluded that fluorene exerted lethal activity to adult E. fetida on day 14 with the LC50 determined to be 88.61 mg/kg. Fluorene-induced ROS caused oxidative stress in E. fetida, resulting in DNA damage, protein carbonylation, and lipid peroxidation. Moreover, changed antioxidative enzymatic activities, non-enzymatic antioxidative activities, and total antioxidative capacity in E. fetida by fluorene stress are associated with antioxidative and protective effects. High-dose fluorene (> 2.5 mg/kg) exposure significantly caused histopathological lesions including the microstructure of body wall, intestine, and seminal vesicle of earthworms. Also, the reproductive system of E. fetida was clearly disrupted by fluorene stress, leading to poor reproduction ability (decreased cocoon and juvenile production) in earthworms. It is found that E. fetida growth was significantly inhibited when treated with high-dose fluorene, thereby causing normal growth disorders. Additionally, fluorene stress triggered the abnormal mRNA expression related to oxidative stress (e.g., metallothionein and heat shock protein 70), growth (translationally controlled tumour protein), reproduction (annetocin precursor) in E. fetida. Together, both high-dose and long-term exposure elicited more severe poisoning effects on earthworms using the Integrated Biological Response (IBR) index, and E. fetida coelomocyte DNA was the most negatively affected by fluorene stress. This study comprehensively evaluated fluorene-induced toxicity in E. fetida, and its underlying molecular mechanisms mediating the toxic responses have been elucidated. These findings provide valuable data for assessing potential ecological risks posed by fluorene-contaminated soil.

Effects of a novel fungicide benzovindiflupyr in Eisenia fetida: Evaluation through different levels of biological organization.

Although benzovindiflupyr (BZF), which is a novel succinate dehydrogenase inhibitor fungicide, has considerable application potential worldwide, its extensive use is toxic to non-targeted soil organisms. Therefore, this study aimed to evaluate the acute and subchronic toxicity of BZF to earthworms (Eisenia fetida). The acute toxicity of BZF to adult and larval earthworms was measured, as indicated by the following LC50 values obtained after 14 days of exposure: 416mg/kg for adult earthworms and 341mg/kg for juveniles. Subchronic toxicity tests were conducted using only adult earthworms. The earthworms' weight gain was slower on days 14 and 28 after commencing the BZF T100 treatment (50mg/kg of soil). Following 14 days of BZF exposure, enzymes and gene expressions associated with the mitochondrial respiratory chain and energy metabolism were activated to some extent, and the reactive oxygen species level and malondialdehyde content also increased. Antioxidant and detoxifying enzymes and metallothionein gene, Heat shock protein 70 gene associated with resistance to oxidative damage were also activated to varying degrees. Increased BZF concentrations corresponded to increased genotoxicity. Integrated biological response (IBR) values were calculated at the biochemical and molecular levels to show increased toxicity with increased BZF concentration. Although a series of biomarkers changes occurred after initiating BZF treatment, these changes were all likely to have been resisted by the earthworms' own antioxidant defense system and only showed phenotypic (weight-related) changes with treatments of 50mg/kg. In conclusion, reasonable levels of BZF application may have little impact on earthworms. Our findings provide insights on the toxic effects of BZF on earthworms and may prove useful for risk assessments relating to BZF's impacts on soil ecosystems.

Combined effects of mulch film-derived microplastics and atrazine on oxidative stress and gene expression in earthworm (Eisenia fetida)

With the wide use of mulch film and pesticides, mulch film-derived microplastics are very likely to produce combined effects with pesticides in agricultural soil. However, little is known about their combined toxicity on terrestrial organisms. This study aimed to investigate the combined toxicity of unused or farmland residual transparent low-density polyethylene mulch film-derived microplastics (MPs and MPs-aged, respectively) (550–1000 μm) and atrazine (ATZ; 0.02 and 2.0 mg/kg) on the earthworm (Eisenia fetida). After single and combined exposure to ATZ and microplastics for 28 d, the results showed an accumulation of reactive oxygen species, a decrease in superoxide dismutase, catalase, and glutathione-S-transferase activities, an increase in the malondialdehyde and 8-hydroxydeoxyguanosine levels, and abnormal expression of annetocin, heat shock protein 70, translationally controlled tumor protein and calreticulin genes. Integrated biological response (IBR) values calculated at the biochemical level indicated that the combined exposure to ATZ and microplastics, particularly to high concentrations of ATZ, induced greater oxidative stress in E. fetida compared with that of exposure to ATZ or microplastics alone. In addition, the IBR values calculated at the gene level did not show regular changes after combined exposure to ATZ and microplastics compared with those of a single exposure. The oxidative stress and abnormal expression of genes in E. fetida induced by MPs-aged were higher than those induced by MPs; a similar trend was observed for oxidative stress induced by MPs/MPs-aged + ATZ2.0, whereas an opposite trend was observed for the abnormal expression of genes in E. fetida induced by MPs/MPs-aged + ATZ0.02/ATZ2.0. Our results suggest that mulch film-derived microplastics have the potential to enhance the toxicity of ATZ within the soil environment.

Physiological and biochemistry responses of graphene combined with triphenyl phosphate (TPP) to <italic>Mytilus galloprovincialis</italic>

After released into the environment, graphene nanomaterials can interact with other pollutants, due to their excellent adsorption properties. This brings changes to the environmental behavior and toxicological effects of graphene nanomaterials and other pollutants. Therefore, it is of great significance to further investigate the joint toxic effects of graphene and other pollutants. Triphenyl phosphate (TPP) is an organophosphate flame retardant and widely distributed in the Bohai sea. TPP can interact with graphene easily, as it contains three benzene rings in the molecule structure. Previous studies showed that graphene could increase the accumulation of TPP because of its hydrophobicity and durability. Due to the increasing applications of graphene and TPP, it is of great significance to study the combined toxicological effects of graphene and TPP. Marine bivalves are important model organisms to assess the effects of nanoparticles on aquatic biota, and may play a significant role in nanoparticles uptake, biotransformation and trophic transfer through food chains. The physiological response index can be used to evaluate the effects of environmental stressors on marine bivalves. These physiological parameters not only present the key processes related to energy budget, but also reflect the physiological plasticity of animals to stress environments. As a species with high tolerance and efficient accumulation of pollutants, mussel Mytilus galloprovincialis has been widely used as the bioindicator for environmental monitoring and ecotoxicological research. In this study, the physiological responses of graphene and TPP on M. galloprovincialis were explored based on the analyses of five groups, including seawater control, DMSO control, graphene (0.5 mg/L), TPP (0.5 mg/L) and graphene (0.5 mg/L) + TPP (0.5 mg/L). The antioxidant status, physiological responses and histological changes of gill and hepatopancreas tissues were measured after 14-d exposure to graphene and TPP. The oxidative stress effects of graphene and TPP on M. galloprovincialis were examined by obtaining a series of enzyme activity indicators, such as the activities of catalase (CAT), superoxide dismutase (SOD), glutathione-s-transferase (GST), glutathione peroxidase (GPx) and the content of glutathione (GSH). The key physiological parameters that characterized changes in physiological response were measured, including clearance rate (CR), ammonia excretion rate (NR), oxygen consumption rate (OR) and oxygen to nitrogen (O:N). In addition, the integrated biological response (IBR) index was also calculated to compare the toxic effects of graphene, TPP and graphene + TPP on M. galloprovincialis . The results showed that the exposure of graphene + TPP mitigated oxidative stress and tissue damage in mussels induced by graphene. Graphene had significant effects on the oxygen consumption and ammonia excretion ( P M. galloprovincialis was increased significantly after TPP exposure. Compared with the single graphene exposure, respiratory inhibition induced by graphene decreased, while oxygen-nitrogen ratio (O:N) increased after the combined exposure. It was evidenced that the combined exposure resulted in less environmental pressure on mussels compared with graphene exposure. The M. galloprovincialis was in a sub-health state after graphene and TPP exposure, and long-term continuous exposure could pose a potential threat to its population maintenance. In addition, IBR index showed that the combined exposure of graphene and TPP exhibited significantly lower stress on mussels, compared with the exposure of graphene and TPP, respectively.

An integrative biological effects assessment of a mine discharge into a Norwegian fjord using field transplanted mussels

The blue mussel (Mytilus sp.) has been used to assess the potential biological effects of the discharge effluent from the Omya Hustadmarmor mine, which releases its tailings into the Frænfjord near Molde, Norway. Chemical body burden and a suite of biological effects markers were measured in mussels positioned for 8 weeks at known distances from the discharge outlet. The biomarkers used included: condition index (CI); stress on stress (SoS); micronuclei formation (MN); acetylcholine esterase (AChE) inhibition, lipid peroxidation (LPO) and Neutral lipid (NL) accumulation. Methyl triethanol ammonium (MTA), a chemical marker for the esterquat based flotation chemical (FLOT2015), known to be used at the mine, was detected in mussels positioned 1500 m and 2000 m downstream from the discharge outlet. Overall the biological responses indicated an increased level of stress in mussels located closest to the discharge outlet. The same biomarkers (MN, SoS, NL) were responsible for the integrated biological response (IBR/n) of the two closest stations and indicates a response to a common point source. The integrated biological response index (IBR/n) reflected the expected level of exposure to the mine effluent, with the highest IBR/n calculated in mussels positioned closest to the discharge. Principal component analysis (PCA) also showed a clear separation between the mussel groups, with the most stressed mussels located closest to the mine tailing outlet. Although not one chemical factor could explain the increased stress on the mussels, highest metal (As, Co, Ni, Cd, Zn, Ag, Cu, Fe) and MTA concentrations were detected in the mussel group located closest to the mine discharge.

The CRF Family of Neuropeptides and their Receptors - Mediators of the Central Stress Response.

Background: Dysregulated stress neurocircuits, caused by genetic and/or environmental changes, underlie the development of many neuropsychiatric disorders. Corticotropin-releasing factor (CRF) is the major physiological activator of the hypothalamic-pituitary-adrenal (HPA) axis and conse-quently a primary regulator of the mammalian stress response. Together with its three family members, urocortins (UCNs) 1, 2, and 3, CRF integrates the neuroendocrine, autonomic, metabolic and behavioral responses to stress by activating its cognate receptors CRFR1 and CRFR2.Objective: Here we review the past and current state of the CRF/CRFR field, ranging from pharmacologi-cal studies to genetic mouse models and virus-mediated manipulations.Results: Although it is well established that CRF/CRFR1 signaling mediates aversive responses, includ-ing anxiety and depression-like behaviors, a number of recent studies have challenged this viewpoint by revealing anxiolytic and appetitive properties of specific CRF/CRFR1 circuits. In contrast, the UCN/CRFR2 system is less well understood and may possibly also exert divergent functions on physiol-ogy and behavior depending on the brain region, underlying circuit, and/or experienced stress conditions.Conclusion: A plethora of available genetic tools, including conventional and conditional mouse mutants targeting CRF system components, has greatly advanced our understanding about the endogenous mecha-nisms underlying HPA system regulation and CRF/UCN-related neuronal circuits involved in stress-related behaviors. Yet, the detailed pathways and molecular mechanisms by which the CRF/UCN-system translates negative or positive stimuli into the final, integrated biological response are not completely un-derstood. The utilization of future complementary methodologies, such as cell-type specific Cre-driver lines, viral and optogenetic tools will help to further dissect the function of genetically defined CRF/UCN neurocircuits in the context of adaptive and maladaptive stress responses.

Assessing developmental toxicity of caffeine and sweeteners in medaka (Oryzias latipes).

The use of artificial sweeteners (ASWs) has increased and become more widespread, and consequently ASWs have appeared in aquatic environments around the world. However, their safety to the health of humans and wildlife remains inconclusive. In this study, using medaka embryos (Oryzias latipes), we investigated developmental toxicity of aspartame (ASP) and saccharin (SAC). Since ASWs are often consumed with caffeine (CAF) and CAF with sucrose (SUC), we tested biological activities of these four substances and the mixtures of CAF with each sweetener. The embryos were exposed to ASP at 0.2 and 1.0 mM, SAC at 0.005 and 0.050 mM, CAF at 0.05 and 0.5 mM, or SUC at 29 and 146 mM, starting from less than 5 h post fertilization until hatch. Control embryos were treated with embryo solution only. Several endpoints were used to evaluate embryonic development. Some of the hatchlings were also tested for anxiety-like behavior with the white preference test. The results showed that all four substances and the mixtures of CAF with the sweeteners affected development. The most sensitive endpoints were the heart rate, eye density, and hatchling body length. The hatchlings of several treatment groups also exhibited anxiety-like behavior. We then used the Integrated Biological Response (IBR) as an index to evaluate the overall developmental toxicity of the substances. We found that the ranking of developmental toxicity was SAC > CAF > ASP > SUC, and there was a cumulative effect when CAF was combined with the sweeteners.Electronic supplementary materialThe online version of this article (doi:10.1186/s40064-015-1284-0) contains supplementary material, which is available to authorized users.

Integrated biomarker assessment of the effects of tailing discharges from an iron ore mine using blue mussels (Mytilus spp.)

The blue mussel (Mytilus spp.) has been used to assess the potential biological effects of the discharge effluent from the Sydvaranger mine, which releases its tailings into Bøk fjord at Kirkenes in the north of Norway. Metal bioaccumulation and a suite of biomarkers were measured in mussels positioned for 6weeks at varying distances from the discharge outlet. The biomarkers used included: stress on stress (SS); condition index (CI); cellular energy allocation (CEA); micronuclei formation (MN); lysosomal membrane stability (LMS), basophilic cell volume (VvBAS); and neutral lipid (NL) accumulation. The individual biomarkers were integrated using the integrated biological response (IBR/n) index. The accumulation of Fe was significantly higher in mussels located closer to the discharge outlet, indicating that these mussels had been exposed to the suspended mine effluent. The IBR/n results were in good agreement with the location of the mussels in relation to the distance from the discharge outlet and expected exposure to the mine effluent. Several biomarkers showed responses resulting in higher IBR/n values of analysed mussels within a 3km distance from the tailing discharge.

Effects of increasing temperatures on biomarker responses and accumulation of hazardous substances in rope mussels (Mytilus galloprovincialis) from Bizerte lagoon

This study examined the influence of increasing temperatures in spring and summer on biochemical biomarkers in Mytilus galloprovincialis mussels sampled from Bizerte lagoon (northern Tunisia). Spatial and seasonal variations in a battery of seven biomarkers were analyzed in relation to environmental parameters (temperature, salinity, and pH), physiological status (condition and gonad indexes), stress on stress (SoS), and chemical contaminant levels (heavy metals, polycyclic aromatic hydrocarbons (PAHs), and PCBs) in digestive glands. Integrated biological response (IBR) was calculated using seven biomarkers (acetylcholinesterase (AChE), benzo[a]pyrene hydroxylase (BPH), multixenobiotic resistance (MXR), glutathione S-transferase (GST), catalase (CAT), malondialdehyde (MDA), and metallothioneins (MT). Seasonal variations in biological response were determined during a critical period between spring and summer at two sites, where chemical contamination varies by a factor of 2 for heavy metals and a factor 2.5 for PAHs. The analysis of a battery of biomarkers was combined with the measurement of physiological parameters at both sites, in order to quantify a maximum range of metabolic regulation with a temperature increase of 11 °C between May and August. According to our results, the MT, MDA, CAT, and AChE biomarkers showed the highest amplitude during the 11°C rise, while the BPH, GST, and MXR biomarkers showed the lowest amplitude. Metabolic amplitude measured with the IBR at Menzel Abdelrahmen-the most severely contaminated station-revealed the highest metabolic stress in Bizerte lagoon in August, when temperatures were highest 29.1°C. This high metabolic rate was quantified for each biomarker in the North African lagoon area and confirmed in August, when the highest IBR index values were obtained at the least contaminated site 2 (IBR = 9.6) and the most contaminated site 1 (IBR = 19.6). The combined effects of chemical contamination and increased salinity and temperatures in summer appear to induce a highest metabolic adaptation response and can therefore be used to determine thresholds of effectiveness and facilitate the interpretation of monitoring biomarkers. This approach, applied during substantial temperature increases at two sites with differing chemical contamination, is a first step toward determining an environmental assessment criteria (EAC) threshold in a North African lagoon.

Pro-oxidant and antioxidant responses in the liver and kidney of wild Goodea gracilis and their relation with halomethanes bioactivation

In mammals, it has been shown that halomethanes (HM) are bioactivated by enzymes such as CYP 2E1 and the theta isoform of GST to produce reactive metabolites. However, in fish, little information is available, although HM can form autochthonously in aquatic environments. This study assessed the effect of HM in dusky splitfin (Goodea gracilis) from three lakes of the Valley of Mexico by analysing specific HM biomarkers as well as a broad range of biomarkers. The concentration of HM was a function of its half-life (higher in deep waters), while its precursors and solar radiation are secondary factors that determine its concentration. The kidney showed higher basal metabolism than the liver, probably because of its function as a haematopoietic and filtration organ. Using integrated biological response version 2 (IBRv2), it was found that the hepatic and renal O₂· content is a pro-oxidant force capable of inducing oxidative stress (ROOH, TBARS and RC=O). Early damage was found to be dependent on low concentrations of HM in Major Lake, whereas late damage was observed in fish exposed to higher concentrations of HM in Zumpango Lake and Ancient Lake. The activities of enzymes involved in antioxidant defence seemed to be inefficient. The quantitative assessment of biomarkers (ANOVA) and the estimate of parameter A obtained from IBRv2 provided different information. However, the data support the greater predictive power of IBRv2, but it requires a series of interrelated biomarkers to infer these possibilities. G. gracilis presents marked patterns of adaptation, which are dependant on the HM concentrations in environmental mixtures, although the response is complex and many toxicants could induce similar responses.

Marine ecosystem health status assessment through integrative biomarker indices: a comparative study after the Prestige oil spill “Mussel Watch”

Five integrative biomarker indices are compared: Bioeffects Assessment Index (BAI), Health Status Index (HSI), integrated biological response (IBR), ecosystem health condition chart (EHCC) and Integrative Biomarker Index (IBI). They were calculated on the basis of selected biomarker data collected in the framework of the Prestige oil spill (POS) Mussel Watch monitoring (2003–2006) carried out in Galicia and the Bay of Biscay. According to the BAI, the health status of mussels was severely affected by POS and signals of recovery were evidenced in Galicia after April-04 and in Biscay Bay after April-05. The HSI (computed by an expert system) revealed high levels of environmental stress in 2003 and a recovery trend from April-04 to April-05. In July-05, the health status of mussels worsened but in October-05 and April-06 healthy condition was again recorded in almost all localities. IBR/n and IBI indicated that mussel health was severely affected in 2003 and improved from 2004 onwards. EHCC reflected a deleterious environmental condition in 2003 and a recovery trend after April-04, although a healthy ecosystem condition was not achieved in April-06 yet. Whereas BAI and HSI provide a basic indication of the ecosystem health status, star plots accompanying IBR/n and IBI provide complementary information concerning the mechanisms of biological response to environmental insult. Overall, although the integrative indices based on biomarkers show different sensitivity, resolution and informative output, all of them provide coherent information, useful to simplify the interpretation of biological effects of pollution in marine pollution monitoring. Each others’ advantages, disadvantages and applicability for ecosystem health assessment are discussed.

High-Resolution Mass Spectrometric Identification and Quantification of Glucocorticoid Compounds in Various Wastewaters in The Netherlands

In the past two decades much research effort has focused on the occurrence, effects, and risks of estrogenic compounds. However, increasing emissions of new emerging compounds may also affect the action of hormonal pathways other than the estrogenic hormonal axis. Recently, a suite of novel CALUX bioassays has become available that enables looking further than estrogenic effects only. By employing these bioassays, we recently showed high glucocorticogenic activity in wastewaters collected at various sites in The Netherlands. However, since bioassays provide an integrated biological response, the identity of the responsible biological compounds remained unknown. Therefore, our current objective was to elucidate the chemical composition of the wastewater extracts used in our previous study by means of LC-high-resolution Orbitrap MS/MS and to determine if the compounds quantified could account for the observed glucocorticoid responsive (GR) CALUX bioassay response. The mass spectrometric analysis revealed the presence of various glucocorticoids in the range of 13-1900 ng/L. In extracts of hospital wastewater-collected prior to sewage treatment-several glucocorticoids were identified (cortisol 275-301 ng/L, cortisone 381-472 ng/L, prednisone 117-545 ng/L, prednisolone 315-1918 ng/L, and triamcinolone acetonide 14-41 ng/L) which are used to treat a great number of human pathologies. A potency balance calculation based on the instrumental analyses and relative potencies (REPs) of the individual glucocorticoids supports the conclusion that triamcinolone acetonide (REP = 1.3), dexamethasone (REP = 1), and prednisolone (REP = 0.2) are the main contributors to the glucocorticogenic activity in the investigated wastewater extracts. The action of these compounds is concentration additive and the overall glucocorticogenic activity can be explained to a fairly large extent by their contribution.