Some heterocyclic aromatic compounds are Ah receptor agonists in the DR-CALUX assay and the EROD assay with RTL-W1 cells

Purpose  

Heterocyclic aromatic compounds containing nitrogen, sulfur, or oxygen heteroatoms (NSO-HET) have been detected in air, soil, marine, and freshwater systems. However, only few publications are available investigating NSO-HET using in vitro bioassays. To support better characterization of environmental samples, selected NSO-HET were screened for dioxin-like activity in two bioassays.     

Use of polyvinyl chloride pipes (PVC) as potential artificial shelters for amphibians in a coastal plain forest of southeastern Brazil

We studied the use of polyvinil chloride pipes (PVC pipes) by frogs as artificial shelters in a Coastal Plain forest in the Vale Natural Reserve, north of Espírito Santo state, southeastern Brazil from June 2015 to May 2016. We used 14 plots distributed in the Coastal Plain Forest following the RAPELD methodology of sampling. The frogs were sampled in transects along the plots, using the active search method, with visual and auditory sampling. We recorded two species of frogs (Aparasphenodon brunoi and Scinax argyreornatus) using PVC pipes as shelters in the plots in the Forest. Of the 468 PVC pipes established along plots, 38 of them (8.1%) were used as shelter by the frogs. For both frog species, about 33% of microhabitats used in the forest were the interior of PVC pipes. We concluded that the PVC pipes might constitute artificial shelters for at least two of the frog species living in the community of the Coastal Plain Forest, potentially being of relevance in conservation programs. In altered areas and those in which there has been the loss of types of microhabitats, the addition of these PVC pipes as artificial shelters may represent additional and complementary microhabitats to keep or increase the population stocks of arboreal and semi-arboreal frog species and having a size that allow them entering the shelter. These artificial shelters can play an important role in conservation of these frogs in fragmented areas where the availability of the microhabitats of these frog species were locally reduced.     

The use of sedimentary %C, %N, delta(15)N, and Pb concentrations to assess historical changes in anthropogenic influence on Portuguese estuaries

Vertical profiles of C, N, delta(15)N and Pb were measured in the Mondego and Mira estuaries as markers that conveyed notions as to the relative influence of anthropogenic influence over the past decades. Recent carbon changes in both estuaries may reflect changes in estuarine productivity, probably as a consequence of sediment reworking and erosion, and also of losses in salt marsh area and dwarf eelgrass beds. delta(15)N values began to diverge considerably before the %C and %N and were higher in Mondego. delta(15)N signatures detected N enrichment at relatively low rates, and indicated that Mondego received more enriched N than Mira. Lead concentrations differed between estuaries, with higher concentrations in Mondego. The secular increase in %N, Pb, and delta(15)N signatures was significantly related to human density in the watersheds of the estuaries and were sensitive indicators of anthropogenic activity.     

Fractionation and Determination of Ah Receptor (AhR) Agonists in Organic Waste After Anaerobic Biodegradation and in Batch Experiments with PCB and decaBDE (8 pp)

Goals, Scope and Background

Anaerobic digestion of organic household waste can lead to an increase in dioxin-like content, as determined by dioxin-specific bioassays. This may be a result of bioactivation of Ah receptor (AhR) agonists into more potent congeners. Work towards identifying the contributing compound groups is important in order to understand the mechanisms and to assess the relevance behind this increase in dioxin-like toxicity, since the residue can be used as a soil fertilising agent. The aim with the present work was to identify compound groups with AhR agonistic properties that caused the previously reported increase in dioxin-like activity after anaerobic biodegradation

Methods

Firstly, chemical fractionation combined with dioxin bioassay testing was used to find bioactive classes of compounds. Secondly, batch digestion experiments with an externally added polychlorinated biphenyl (PCB) mixture (Clophen A50) and with decabrominated diphenyl ether (decaBDE), respectively, were studied as a possible process for transformation of precursors into more potent, dioxin-like compounds. Mesophilic (37ºC) and thermophilic (55ºC) anaerobic digestion were studied. Two different dioxin-specific bioassays were used to analyse AhR agonists in the biodegraded material, the CELCAD and the DR-CALUX.

Results and Discussion

AhR agonist activity was detected in both di- and polyaromatic fractions of digestate extracts, which indicated that a diverse mixture of compounds contributed to the bioassay responses. No quantifiable activities were induced by the monoaromatic fractions. Further fractionation based on planarity revealed higher concentrations of AhR agonists than what was detected after the first fractionation, probably due to non-additive biological interactions of compounds in the extract that were removed in the second fractionation. These results showed significant activity in the non-planar diaromatic fractions and in the co-planar fractions of both diaromates and polyaromates. In the batch experiment with externally added PCB, an increase in dioxin-like activity was seen after 21 days of digestion at mesophilic conditions. After completed digestion, the content of AhR agonists was equal to the start concentration. PCB analysis with GC-MS indicated that dehalogenation of PCBs occurred in the digestors. The batch experiment with decaBDE showed no significant changes in TEQ-concentrations over time.

Conclusions

The results show that the previously reported increase of AhR agonists during mesophilic anaerobic digestion is probably due to an accumulation of several different groups of AhR agonists, both diaromatic and polyaromatic, and both co-planar and non-planar. Batch experiments with externally added PCBs and decaBDE, respectively, did not result in any accumulation of AhR agonist activity after completed digestion, even though chemical analysis indicate a dechlorination of PCBs. Complex, unfractionated extracts were difficult to test using the bioassay approach. Removal of AhR antagonists or otherwise interacting compounds during fractionation may yield bio-TEQ values that are much higher than in the original extract.

Recommendations and Perspective

. Our results indicate that the environmental risk that AhR agonists may pose concerning large-scale anaerobic digestion of organic household waste probably depends on the efficiency of the digester and the sludge residence time. In order to obtain reliable results with the bioassays, an extensive cleanup and fractionation procedure is necessary. Without clean up and fractionation, there is a risk for false negatives and misleading conclusions. DR-CALUX and CELCAD were both suitable for these kinds of studies, provided that suitable fractionation methods are used.

     

Potential risks of natural mercury levels to wild predator fish in an Amazon reservoir

Mercury (Hg) is a toxic metal that bioaccumulates in aquatic organisms and along food chain. Many studies have reported the problem of mercury exposure in aquatic systems from Amazon basin, but very few have focused on the potential risks to wild fish. The present study reports the bioaccumulation of mercury and alterations in target organs of the predator fish Hoplias malabaricus (traíra) from Samuel reservoir, Amazon basin, Northern Brazil. About 18% of fish had mercury levels in muscle exceeding the safe limit for ingestion through food, established by WHO (0.5 μg Hg g(-1)). Fish were separated in two groups according to mercury bioaccumulation in liver (<0.2 μg Hg g(-1)-group I and >0.2 μg Hg g(-1)-group II) for biomarker comparisons. Catalase activity and number of macrophage centers were statistically higher in group II, confirming the potential of Hg to interfere with redox balance and to recruit defense cells to the liver. Conversely, erythrocyte nuclear alterations were less frequent in group II, indicating a more rigorous selection of erythrocytes or hormesis pattern of response. Glutathione S-transferase activity, lipid peroxidation, and histopathological analyses were not statistically different in the liver and gills of both groups. Comparison of lipid peroxidation levels of these fish with others captured in Southern Brazil during another study and the high incidence of morphological alterations in the liver and gills suggest that the bioaccumulation of mercury during continuous exposure is posing potential risks to the species.     

Chronic genetic damages in <Emphasis Type="Italic">Geophagus brasiliensis</Emphasis> exposed to anthropic impact in Estuarine Lakes at Santa Catarina Coast–Southern of Brazil

Biological monitoring through animals exposed to pollutants using biomarkers provides a promising tool for the identification of pollutants that may cause damage to human health and/or to sustainability of ecosystems. The effects of pollutants in fish tissues are important tools to understand the impact of human activities in natural ecosystems. The aim of this work was to study the water quality of two estuarine lakes in Santa Catarina, Brazil (Camacho Lake and Santa Marta Lake). Geophagus brasiliensis is a species widely distributed in Brazil and was used in this work. Comet assays in peripheral red blood and kidney cells, micronucleus tests in peripheral red blood cells, measurements of acetylcholinesterase activity in axial muscle and histopathological analysis of liver were used as biomarkers. Three sampling campaigns were undertaken in November 2004, June 2005 and November 2005. Thirty adult animals were sampled from each of three different sites (P1—Santa Marta Lake, P2 and P3—Camacho Lake). A negative control was sampled in a non-polluted site at Costa Ecological Park, Paraná. The positive control for genotoxicity was obtained by treating animals with copper sulphate. The results showed that both studied lakes are impacted by potential genotoxic substances. Severe lesions in liver of G. brasiliensis were also observed. The inhibition of acetylcholinesterase activity suggests the presence of pesticides or metals in the studied sites. This work shows that the water quality of Santa Marta and Camacho Lakes have been compromised and further control source of pollutants into these ecosystems is required.     

Phytomanagement of Cd-contaminated soils using maize (Zea mays L.) assisted by plant growth-promoting rhizobacteria

Zea mays (L.) is a crop widely cultivated throughout the world and can be considered suitable for phytomanagement due to its metal resistance and energetic value. In this study, the effect of two plant growth-promoting rhizobacteria, Ralstonia eutropha and Chryseobacterium humi, on growth and metal uptake of Z. mays plants in soils contaminated with up to 30 mg Cd kg^?1 was evaluated. Bacterial inoculation increased plant biomass up to 63 % and led to a decrease of up to 81 % in Cd shoot levels (4–88 mg Cd kg^?1) and to an increase of up to 186 % in accumulation in the roots (52–134 mg Cd kg^?1). The rhizosphere community structure changed throughout the experiment and varied with different levels of Cd soil contamination, as revealed by molecular biology techniques. Z. mays plants inoculated with either of the tested strains may have potential application in a strategy of soil remediation, in particular short-term phytostabilization, coupled with biomass production for energy purposes.     

Toxicity assessment of 2,4-D and MCPA herbicides in primary culture of fish hepatic cells

In this study, we used primary cultures of fish hepatic cells as a tool for evaluating the effects of environmental contamination. Primary hepatic cell cultures derived from the subtropical fish Metynnis roosevelti were exposed to different concentrations (0.275, 2.75 and 27.5 μg L^?1) of the herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 4-chloro-2-methylphenoxyacetic acid (MCPA). Cellular respiratory activity was evaluated by polarography using three substrates: 0.5 M glucose, 0.5 M succinate and 0.5 M α-ketoglutarate. Significant changes were observed in cellular oxygen consumption with 0.5 M α-ketoglutarate. Even at low concentrations, 2,4-D and MCPA were potent uncouplers of oxidative phosphorylation. Primary cultures of M. roosevelti liver cells may provide a useful tool for the evaluation of environmental contaminant effects. A review of regulations regarding permitted concentrations of these herbicides is needed.     

Method validation for control determination of mercury in fresh fish and shrimp samples by solid sampling thermal decomposition/amalgamation atomic absorption spectrometry

A method for the determination of total mercury in fresh fish and shrimp samples by solid sampling thermal decomposition/amalgamation atomic absorption spectrometry (TDA AAS) has been validated following international foodstuff protocols in order to fulfill the Brazilian National Residue Control Plan. The experimental parameters have been previously studied and optimized according to specific legislation on validation and inorganic contaminants in foodstuff. Linearity, sensitivity, specificity, detection and quantification limits, precision (repeatability and within-laboratory reproducibility), robustness as well as accuracy of the method have been evaluated. Linearity of response was satisfactory for the two range concentrations available on the TDA AAS equipment, between approximately 25.0 and 200.0 μg kg^?1 (square regression) and 250.0 and 2000.0 μg kg^?1 (linear regression) of mercury. The residues for both ranges were homoscedastic and independent, with normal distribution. Correlation coefficients obtained for these ranges were higher than 0.995. Limits of quantification (LOQ) and of detection of the method (LDM), based on signal standard deviation (SD) for a low-in-mercury sample, were 3.0 and 1.0 μg kg^?1, respectively. Repeatability of the method was better than 4%. Within-laboratory reproducibility achieved a relative SD better than 6%. Robustness of the current method was evaluated and pointed sample mass as a significant factor. Accuracy (assessed as the analyte recovery) was calculated on basis of the repeatability, and ranged from 89% to 99%. The obtained results showed the suitability of the present method for direct mercury measurement in fresh fish and shrimp samples and the importance of monitoring the analysis conditions for food control purposes. Additionally, the competence of this method was recognized by accreditation under the standard ISO/IEC 17025.