Toxic effects of environmental-relevant exposure to polyethylene terephthalate (PET) micro and nanoparticles in zebrafish early development.

Polyethylene terephthalate (PET) is a commonly used thermoplastic in industry due to its excellent malleability and thermal stability, making it extensively employed in packaging manufacturing. Inadequate disposal of PET packaging in the environment and natural physical-chemical processes leads to the formation of smaller particles known as PET micro and nanoplastics (MNPs). The reduced dimensions enhance particle bioavailability and, subsequently, their reactivity. This study involved chemical degradation of PET using trifluoroacetic acid to assess the impact of exposure to varying concentrations of PET MNPs (0.5, 1, 5, 10, and 20 mg/L) on morphological, functional, behavioral, and biochemical parameters during the early developmental stages of zebrafish (Danio rerio). Characterization of the degraded PET revealed the generated microplastics (MPs) ranged in size from 1305 to 2032 µm, and that the generated nanoplastics (NPs) ranged from 68.06 to 955 nm. These particles were then used for animal exposure. After a six-day exposure period, our findings indicate that PET MNPs can diminish spontaneous tail coiling (STC), elevate the heart rate, accumulate on the chorion surface, and reduce interocular distance. These results suggest that PET exposure induces primary toxic effects on zebrafish embryo-larval stage of development.

Açaí (Euterpe oleracea Mart.) green synthesis of silver nanoparticles: antimicrobial efficacy and ecotoxicological assessment.

The synthesis of silver nanoparticles (AgNPs) is usually based on expensive methods that use or generate chemicals that can negatively impact the environment. Our study presents a simple one-step synthesis process for obtaining AgNP using an aqueous extract of Amazonian fruit açai (Euterpe oleracea Mart.) as the reducing and stabilizing agents. The bio-synthesized AgNP (bio-AgNP) were comprehensively characterized by diverse techniques, and as a result, 20-nm spherical particles (transmission electron microscopy) were obtained. X-ray diffraction analysis (XRD) confirmed the presence of crystalline AgNP, and Fourier-transform infrared spectroscopy (FT-IR) suggested that polyphenolic compounds of açaí were present on the surface. The bio-AgNP showed antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii. In Caenorhabditis elegans exposed to 10 µg/L bio-AgNP for 96 h, there were no significant effects on growth, reproduction, or reactive oxygen species (ROS) concentration; however, there was an increase in superoxide dismutase (SOD) and glutathione-S-transferase (GST) enzymatic activity. In contrast, when worms were exposed to chemically synthesized AgNP (PVP-AgNP), an increase in ROS, SOD, and GST activity and a reduction in oxidative stress resistance were observed. In conclusion, our study not only showcased the potential of açaí in the simple and rapid production of AgNP but also highlighted the broad-spectrum antimicrobial activity of the synthesized nanoparticles using our protocol. Moreover, our findings revealed that these AgNPs exhibited reduced toxicity to C. elegans at environmentally realistic concentrations compared with PVP-AgNP.

Effects of including of Japanese Pumpkin Seeds and Pomace in the Diets of Pacific White Shrimp (Penaeus vannamei).

A 60-day feeding trial was conducted to evaluate the effects of including pumpkin seeds and pomace in the diets of Pacific white shrimp Penaeus vannamei, and the effects of these supplements on growth performance, body composition, and total polyphenol, flavonoid and carotenoid contents, as well as on total antioxidant activity, and body color parameters. Five diets were evaluated: pumpkin seeds (PS) at 50 and 100 g·kg-1, pumpkin pomace (PP) at 50 and 100 g·kg-1, and a control treatment. Pacific white shrimp (P. vannamei) juveniles (0.60 ± 0.01 g) were stocked in 15 tanks (310 L), containing 30 shrimps per tank, and the treatments were randomly distributed in triplicate. At the end of the experiment, shrimps were euthanized, weighed, and dissected for further analyses. The inclusion of PS in the diets impaired growth performance, reduced the total flavonoid content and had a pro-oxidative effect on muscle. The inclusion of PP in the diets did not affect growth performance, improved the feed conversion ratio, increased the total flavonoid content in the diets and hepatopancreas, and improved the antioxidant activity of the feeds and shrimp muscle. The total carotenoid content of the feeds increased with the inclusion of PS or PP in the diets; however, the total carotenoid content of shrimp increased only in those fed PP diets. Shrimp fed with PS diets showed a yellowish color and higher saturation when fresh and a reddish color and yellow hue angle after cooking. Shrimp fed PP diets turned reddish and yellowish, both when fresh and after cooking. The inclusion of PS in P. vannamei diets is not recommended; however, PP can be included at 100 g·kg-1 without affecting the growth parameters. Further studies evaluating the inclusion of higher PP levels in shrimp diets are recommended.

Polystyrene microplastic alters the redox state and arsenic metabolization in the freshwater bivalve Limnoperna fortunei.

Most organisms possess the capacity to metabolize arsenic (As) accumulating compounds to less toxic forms, thus minimizing the adverse effect induced by this metalloid. However, other contaminants may to interfere with As metabolism, contributing to the accumulation of more toxic compounds. Microplastics (MPs) are omnipresent in aquatic environment and may induce toxicological effects (alone or in combination with other contaminants) on living organisms. Therefore, the objective of the present study was to evaluate the effect of the exposure of the freshwater clam Limnoperna fortunei to a combination of MP (4 and 40 µg/L of polystyrene microbeads, 1.05 µm) and As (50 µg/L) for 48 h, evaluating the accumulation and metabolization of As and oxidative stress parameters, such as catalase (CAT), glutathione-S-transferase activities, total antioxidant competence, reduced glutathione (GSH), and lipid damage in the gills and digestive glands. Results revealed that low MP concentration disrupts the redox state of the digestive gland by a decrease in the antioxidant activity (CAT and total antioxidant capacity). GSH levels in the gills of animals exposed to MP (4 µg/L) alone and the combination of MP + As increased, concomitant with an increase in the percentage of toxic compounds, indicating the effect of MP on As metabolism. Although, few studies evaluated the effect of coexposure to MP + As by considering metabolization of metalloid in freshwater bivalve, our results revealed that exposure to MP reduced the metabolization capacity of As, favoring the accumulation of more toxic compounds besides the MP alone, which showed a pro-oxidant effect in L. fortunei.

Effects of Lyophilized Açaí (Euterpe oleracea) Supplementation on Oxidative Damage and Intestinal Histology in Juvenile Shrimp Penaeus vannamei Reared in Biofloc Systems.

The objective of this was to evaluate the ability of bioflocs to assimilate and transfer antioxidant compounds present in açaí Euterpe oleracea to juvenile Penaeus vannamei shrimp grown in a biofloc system. Juvenile shrimp were distributed into four treatment groups (control, 5, 20, and 80 mg açaí L-1), containing 31 shrimps/tank (90 L), and cultivated for 30 days. Every 24 h throughout the experimental period, the respective açaí concentrations were added directly to the cultivation water. The bioflocs and hepatopancreas lost their antioxidant capacity with increasing concentrations of açaí; however, lipid damage was mitigated after treatment with 20 mg of açaí L-1 (p < 0.05). The application of 20 mg açaí L-1 increased the mean height and area of the middle intestinal microvilli (p < 0.05). Mortality and protein and lipid damage in shrimp muscle increased with daily administration of 80 mg açaí L-1 (p < 0.05). It is concluded that the bioflocs were able to assimilate the antioxidants present in açaí and transfer them to the shrimp, and the administration of 20 mg açaí L-1 presented the best performance, demonstrating the possibility of its application in the cultivation of P. vannamei in a biofloc system.

The Effects of Lipoic Acid on Yolk Nutrient Utilization, Energy Metabolism, and Redox Balance over Time in Artemia sp.

Lipoic acid (LA) is a mitochondrial coenzyme that, depending on the concentration and exposure time, can behave as an antioxidant or pro-oxidant agent and has a proven ability to modulate metabolism by promoting lipid and glucose oxidation for energy production. To assess the effects of LA on energy metabolism and redox balance over time, Artemia sp. nauplii was used as an animal model. The administered concentrations of the antioxidant were 0.05, 0.1, 0.5, 1.0, 5.0, and 10.0 µM. Therefore, possible differences in protein, triglyceride, glucose, and lactate concentrations in the artemia samples and total ammoniacal nitrogen (TAN) in the culture water were evaluated. We also measured the effects of LA on in vivo activity of the electron transport system (ETS), antioxidant capacity, and production of reactive oxygen species (ROS) at 6, 12, 18, and 24 h post-hatching. There was a decrease in glucose concentration in the LA-treated animals, and a decrease in ammonia production was observed in the 0.5 µM LA treatment. ETS activity was positively regulated by the addition of LA, with the most significant effects at concentrations of 5.0 and 10.0 µM at 12 and 24 h. For ETS activity, treatments with LA presented the highest values at 24 h, a period when ROS production decreased significantly, for the treatment with 10.0 µM. LA showed positive regulation of energy metabolism together with a decrease in ROS and TAN excretion.

Multigenerational effects of co-exposure to dimethylarsinic acid and polystyrene microplastics on the nematode Caenorhabditis elegans.

In the current study, we assessed the impact of DMA (dimethylarsinic acid) and MPs (microplastics) interactions in C. elegans over the course of five generations. We found that the redox state of the organisms changed over generations as a result of exposure to both pollutants. From the third generation onward, exposure to MPs reduced GST activity, indicating reduced detoxifying abilities of these organisms. Additionally, dimethylarsinic exposure decreased the growth of organisms in the second, fourth, and fifth generations. In comparison to isolated pollutants, the cumulative effects of co-exposure to DMA and MPs seem to have been more harmful to the organisms, as demonstrated by correlation analysis. These findings demonstrate that DMA, despite being considered less hazardous than its inorganic equivalents, can still have toxic effects on species at low concentrations and the presence of MPs, can worsen these effects.

Silver nano/microparticle toxicity in the shrimp Litopenaeus vannamei (Boone, 1931).

The effects of silver nano/microparticles (AgP) on juvenile Litopenaeus vannamei shrimp were evaluated through several responses, aiming to use it as a prophylactic and therapeutic method. Shrimps (3.19 ± 0.13 g) were exposed to clear water for 3 h with increasing concentrations of nanosilver (0; 25; 100; and 400 µg/l). After 3 h of exposure, they were transferred to water without nanosilver for 30 days (recovery). The weight gain and weekly growth were not affected by AgNP. Total antioxidant capacity (ACAP) increased in the hepatopancreas (exposure period) and gills (recovery) in shrimp exposed to AgNP. In muscle, ACAP was induced in shrimp exposed to 100 µg/l AgNP (exposure). In the gills, there was an increase in TBARS in shrimp exposed to 100 µg/l AgNP (recovery). In the concentration of protein-associated sulfhydryl groups (P-SH), a decrease was observed in the hepatopancreas (recovery) in the 100 µg/l AgNP treatment. In chromaticity parameters, an increase in reddish tones was observed in shrimp exposed to 100 µg/l AgNP (recovery). An increase in granular hemocytes was verified in shrimp exposed to 25 and 400 µg/l AgNP during exposure. Tissues analyzed histologically showed normal patterns without apoptosis or necrosis processes, and after 30 d of recovery, only in one muscle sample of shrimp exposed to µg/l of AgNP was silver detected. It is concluded that a prophylactic action of short duration (3 h) mostly did not affected the welfare of shrimp L. vannamei and can be considered its use as a therapeutic strategy.

Comparison of the toxic effects of organic and inorganic arsenic in Caenorhabditis elegans using a multigenerational approach.

Although arsenic (As) is a persistent contaminant in the environment, few studies have assessed its effects over generations, as it requires an animal model with a short lifespan and rapid development, such as the nematode Caenorhabditis elegans. Furthermore, few studies have evaluated the effects of As metabolites such as dimethylarsinic acid (DMAV), and several authors have considered DMA as a moderately toxic intermediate of As, although recent studies have shown that this chemical form can be more toxic than inorganic arsenic (iAs) even at low concentrations. In the present study, we compared the toxic effects of arsenate (AsV) and DMAV in C. elegans over 5 subsequent generations. We evaluated biochemical parameters such as reactive oxygen species (ROS) concentration, the activity of antioxidant defense system (ADS) enzymes such as catalase (CAT) and glutathione-S-transferase (GST), and nonenzymatic components of ADS such as reduced glutathione (GSH) and protein-sulfhydryl groups (P-SH). Exposure to 50 µg L-1 of AsV led to an increase in ROS generation and GSH levels together with a decrease in GST activity, while exposure to DMAV led to an increase in ROS levels, with an increase in lipid peroxidation, CAT activity, and a decrease in GSH levels. In addition, both treatments reduced animal growth from the third generation onward and caused disturbances in their reproduction throughout all 5 generations. This study shows that the accumulated effects of DMA need to be considered; it highlights the importance of this type of multigenerational approach for evaluating the effects of organic contaminants considered low or nontoxic.

Interactive effects of freshwater acidification and selenium pollution on biochemical changes and neurotoxicity in Oreochromis mossambicus.

Effect of selenium and acidification in freshwater environment was assessed solitary but no reports are available on the impacts of both factors act together. In the present study, effects of combined simultaneous exposure to selenium (Se) and low pH were assessed in Mozambique tilapia, Oreochromis mossambicus. Responses were measured based on antioxidant defenses (enzymatic SOD, CAT, GPx and non-enzymatic GSH), biotransformation enzyme (GST), metallothionein levels (MT), oxidative damage (LPO, CP), Na+/K+-ATPase (NKA) activity in gills and liver tissues and neurotoxicity (acetylcholinesterase, AChE) response in brain tissue. Fish were exposed to combined treatment at different pH levels (7.5, control (optimum pH for tilapia growth); 5.5, low pH) and Se concentrations (0, 10, and 100 µg L-1). Toxicity levels of Se were not significantly different under control and low pH indicating that pH did not affect Se toxicity. Levels of GSH and MT were enhanced in Se-exposed fish at both pH. Combined effects of high Se concentration and low pH decreased SOD and CAT activities and increased those of GPx and GST. However, organisms were not able to prevent cellular damage (LPO and CP), indicating a condition of oxidative stress. Furthermore, inhibition of Na+/K+-ATPase activity was showed. Additionally, neurotoxicity effect was observed by inhibition of cholinesterase activity in organisms exposed to Se at both pH conditions. As a result, the combined stress of selenium and freshwater acidification has a slight impact on antioxidant defense mechanisms while significantly inhibiting cholinesterase and Na+/K + -ATPase activity in fish. The mechanisms of freshwater acidification mediating the toxic effects of trace non-metal element on freshwater fish need to investigate further.

Characterization, stability, and in vivo effects in Caenorhabditis elegans of microencapsulated protein hydrolysates from stripped weakfish (Cynoscion guatucupa) industrial byproducts.

This study aimed to microencapsulate protein hydrolysates from stripped weakfish (Cynoscion guatucupa) industrial byproducts produced by Alcalase (HA) and Protamex (HP) by spray drying, using maltodextrin as wall material. The physicochemical characteristics, and in vitro antioxidant and Angiotensin-I converting enzyme-inhibitory activities were evaluated during storage. Both microencapsulated hydrolysates showed spherical shape (~3.6 µm particle diameter), low water activity (<0.155) during storage and reduced hygroscopicity (~30%) compared to the free hydrolysate. Infrared spectroscopy evidenced the maltodextrin-hydrolysate interaction. Based on the in vitro results, nematoid C. elegans in L1 larval stage were treated with free and microencapsulated HP, which demonstrated a protective effect on nematoid exposed to oxidative stress (survival ~ 13% control, 77% free HP, and 85% microencapsulated HP) and improved their growth and reproduction rate. Thus, microencapsulation appears to be a good alternative to maintain hydrolysates stability during storage, showing bioactivity in C. elegans.

Co-exposure to nTiO2 impairs arsenic metabolism and affects antioxidant capacity in the marine shrimp Litopenaeus vannamei.

Aquatic animals are vulnerable to arsenic (As) toxicity. However, rarely does a contaminant occur alone in the aquatic environment. For this reason, this study was conducted to evaluate whether titanium dioxide nanoparticles (nTiO2) can interfere with the effects induced by As in Litopenaeus vannamei. Arsenic accumulation and metabolic capacity; expression and enzymatic activity of GSTΩ (glutathione-S-transferase omega isoform); antioxidant responses such as GSH, GR, and GST (reduced glutathione levels, glutathione reductase, and glutathione-S-transferase activity, respectively); and lipid peroxidation in the gills and hepatopancreas of shrimp were evaluated. The results are summarized as follows: (1) higher accumulation of As occurred in both tissues after exposure to As alone; (2) co-exposure to nTiO2 affected the capacity to metabolize As; (3) GSTΩ gene expression was not modified, but its activity was decreased by co-exposure to both contaminants; (4) As alone increased the GSH levels in the hepatopancreas, and co-exposure to nTiO2 reduced these levels in both tissues; (5) a decrease in the GST activity in the gills occurred with all treatments; (6) in the gills, GR activity was increased by As, and nTiO2 reversed this increase, whereas in the hepatopancreas co-exposure inhibited enzyme activity; (7) only in the hepatopancreas lipid damage was observed when animals were exposed to As or nTiO2 but not in co-exposure. The results showed that the As induces toxic effects in both tissues of shrimp and that co-exposure to nTiO2 can potentiate these effects and decrease the capacity to metabolize As, favoring the accumulation of more toxic compounds.

Impact of different crystalline forms of nTiO2 on metabolism and arsenic toxicity in Limnoperna fortunei.

Arsenic (As) is a ubiquitous contaminant in the environment and it is known to induce oxidative stress in aquatic organisms. In an attempt to remove As from water, some studies have suggested the titanium dioxide nanomaterial (nTiO2) as a promising alternative. However, it has been observed that nTiO2 can induce toxicity alone or in combination with metals, and this toxicity is dependent on its crystalline form of nanomaterial (mainly rutile as nTiO2R and anatase as nTiO2A, respectively). Considering that both (nTiO2 and As) can occur together, the objective of this study was to evaluate if co-exposure to rutile and anatase may influence accumulation, metabolisation, and toxicity of arsenite (As+3) in the golden mussel Limnoperna fortunei after 48 h of co-exposure to nTiO2 (1 mg/L) and As (50 µg/L). Accumulation and chemical speciation of As in organisms were determined. Also, biochemical analyses, such as the activity of the enzymes glutathione S-transferase omega (GSTΩ), catalase (CAT) and glutathione S-transferase (GST), as well as lipid peroxidation (LPO) were investigated. Results showed that co-exposure to nTiO2A + As changed accumulation pattern of metalloid in gills and digestive gland. Both crystalline forms of nTiO2 affected the metabolisation capacity favoring the accumulation of more toxic As compounds and nTiO2A alone or in combination with As showed induce oxidative stress in gills of L. fortunei. In this way, it has a high potential risk of the co-exposure of these contaminants to aquatic organisms, and it also needs to consider the nanomaterial (nTiO2) properties and their application in the environmental remediation, carefully and judiciously.

Modulation of nodularin toxicity in shrimp Litopenaeus vannamei (BOONE, 1931) fed with dietary açai (Euterpe oleracea) inclusion.

This study evaluated the effect of dietary inclusion of lyophilized açaí Euterpe oleracea (LEO) on redox status of shrimp Litopenaeus vannamei (initial weight 1.5 ± 0.39 g) upon exposure to cyanotoxin nodularin (NOD) in bioflocs system. Three hundred juvenile shrimps were randomly divided into two groups and fed twice a day with two diets: one containing 0.00 (control diet) and the other 10.0% LEO (w/w) for 30-days. After the feeding period, both shrimp groups were submitted to three treatments (14 L; 7 shrimp/tank) with different concentrations of cyanotoxin NOD (0.00; 0.25; and 1.00 µg/L) dissolved in water with 96 h of exposure. Then, the shrimps were sampled (n = 15/treatment) for the determination of reduced glutathione (GSH), the activity of glutathione-S-transferase (GST), sulfhydryl groups associated to proteins (P-SH), and lipid peroxidation (TBARS) in the hepatopancreas, gills and muscle. The NOD accumulation was measured in the muscle. The results revealed that dietary LEO significantly increased GSH levels in the hepatopancreas and gills of the shrimps exposed to NOD. Toxin exposure did not modify GST activity in all organs. Muscle TBARS levels were lower in the shrimp fed with the LEO diet and exposed to NOD. The NOD toxin did not accumulate in the muscle but notably was detected in the control groups fed or not with dietary LEO. Açaí was able to induce the antioxidant system of L. vannamei, as well as lowered the oxidative damage in shrimps exposed to NOD, suggesting its use as a chemoprotectant against cyanotoxins.

Graphene oxide and GST-omega enzyme: An interaction that affects arsenic metabolism in the shrimp Litopenaeus vannamei.

Arsenic (As) is one of the most widespread contaminants; it is found in almost every environment. Its toxic effects on living organisms have been studied for decades, but the interaction of this metalloid with other contaminants is still relatively unknown, mainly whether this interaction occurs with emerging contaminants such as nanomaterials. To examine this relationship, the marine shrimp Litopenaeus vannamei was exposed for 48 h to As, graphene oxide (GO; two different concentrations) or a combination of both, and gills, hepatopancreas and muscle tissues were sampled. Glutathione S-transferase (GST)-omega gene expression and activity were assessed. As accumulation and speciation (metabolisation capacity) were also examined. Finally, a molecular docking simulation was performed to verify the possible interaction between the nanomaterial and GST-omega. The main finding was that GO modulated the As toxic effect: it decreased GST-omega activity, a consequence related to altered As accumulation and metabolism. Besides, the molecular docking simulation confirmed the capacity of GO to interact with the enzyme structure, which also can be related to the decreased GST-omega activity and subsequently to the altered As accumulation and metabolisation pattern.

The impact of polar fraction of the fine particulate matter on redox responses in different rat tissues.

Particulate matter (PM) contains different chemical substances that have been associated with health effects and an increased risk of mortality due to their toxicity. In this study, fine particulate matter (PM2.5) samples were collected in a region with rural characteristics (Seropédica (Se)) and another with some industries (Duque de Caxias (DC)) (Brazil, RJ). Rats were exposed to PM2.5 extracts daily for 25 days at different dilutions: 10×, 5×, and a concentrated solution (CS). Biochemical analyses were investigated for total antioxidant capacity (ACAP), lipid peroxidation (LPO) levels, reduced glutathione (GSH) concentration, activity of glutamate cysteine ligase (GCL), and activity of glutathione S-transferase (GST). The liver showed a significant increase in GCL (DC-5×, DC-CS and Se-CS) and GST activities (DC-CS and Se-CS) in both regions when compared to the control group. In the renal cortex, GCL activity decreased in most of the tested groups while GST activity increased only in the 5× groups of both regions (DC and Se). In the renal medulla, GCL activity decreased for Se-10× and DC-CS but increased for Se-5×, and GST activity increased in the Se-10×, DC-5×, and DC-CS groups. Lung GCL increased in all groups for both regions. Moreover, this organ also showed an increase in GST activity when higher metal concentrations were present (5× and CS). TBARS levels were increased for all tissues in most tested concentrations. These data indicate that soluble compounds (e.g., metals) from PM2.5 sampled in areas with different pollution indexes can change the redox status and cause damage to different tissues.

Genotoxic effect of dimethylarsinic acid and the influence of co-exposure to titanium nanodioxide (nTiO2) in Laeonereis culveri (Annelida, Polychaeta).

Few data are available about the effect of dimethylated forms (DMA) on aquatic organisms. As rarely a contaminant occurs alone, studies evaluating the combined effect of different contaminants in aquatic organisms are needed. In fact, the presence of nanomaterials, such as titanium dioxide nanoparticles (nTiO2), in the aquatic environment is now a reality due to its intensive production and use. So, this study evaluated the toxicological effects of DMA in an acute exposure condition and considered the potential influence of nTiO2 on the effects induced by DMA in the polychaete, Laeonereis culveri. The animals were exposed over 48 h to DMA (50 and 500 µg/l) alone or in combination with nTiO2 (1 mg/l). Biochemical parameters such as concentration of reactive oxygen species (ROS), glutathione-S-transferase (GST) activity, levels of reduced glutathione levels (GSH) and macromolecular (lipid and DNA) damage were evaluated, as well the DNA repair system. In addition, the accumulation of total As and the chemical speciation of the metalloid in the organisms was determined. The results showed that: (1) only the group exposed to 500 µg of DMA/l accumulated As and when co-exposed to nTiO2, this accumulation was not observed. (2) The levels of ROS increased in the group exposed to 50 µg/l of DMA alone and the effect was reversed when this group was co-exposed to nTiO2 (3) None of the treatments showed altered GST activity or GSH levels. (4) All groups that received nTiO2 (alone or in combination with DMA) showed lipid peroxidation. (5) The exposure to DMA (both concentrations) alone or in combination with nTiO2 induced DNA damage in L. culveri. These results showed that DMA exhibits a genotoxic effect and that co-exposure to nTiO2 had an influence on its toxicity. So the occurrence of both contaminants simultaneously can represent a threat to aquatic biota.

Circular Estimate Method (CEM) - a Simple Method to Estimate Caenorhabditis elegans Culture Densities in Liquid Medium.

BACKGROUND: Nematodes are used in many different fields of science, including environmental and biomedical research. Counting and/or estimating nematode numbers is required during research. Although being one of the most common procedures, this apparently simple task is a time-consuming process, prone to errors and concerns regarding procedure, reliability, and accuracy. When an estimate is necessary, there is a traditional manual counting procedure that in this study it will be called as "drop method" (DM). This popular method that extrapolates an animal count from a small drop of fluid shows a high coefficient of variation. To solve this problem, the present study used the free-living nematode Caenorhabditis elegans to develop a new estimation procedure that was based on a relationship between area and volume of a larger sample. RESULTS: The new method showed a low coefficient of variation and a close relationship between estimated and real counts of the total number of nematodes in large C. elegans suspensions. Reactive oxygen concentration was measured as an example of method application and to allow comparison between methods. CONCLUSION: The proposed method is accurate, facile and reproducible, requiring simple, inexpensive materials that make it an excellent alternative to the DM manual counting procedure. Although the DM is faster, its estimates are not as accurate or as precise as those of the new proposed method.

Fish oil and meal replacement in mullet (Mugil liza) diet with Spirulina (Arthrospira platensis) and linseed oil.

This research evaluated the effect of the simultaneous substitution of fish meal and fish oil at 0, 30, 50, 70 and 100% (termed SS0, SS30, SS50, SS70 and SS100, respectively) with both Spirulina (Arthrospira platensis) and linseed oil. The effect on growth, colour, fatty acid (FA) profile, and liver and muscle antioxidant response in mullets (Mugil liza) were evaluated. A full substitution was not possible due to a final body weight reduction (p < 0.05). The colorimetric analyses showed an improvement in fillet colouration (p < 0.05). Also, we found a saturation of carotenoids in muscle at 30% substitution. The content of highly unsaturated fatty acids - HUFA (DHA, EPA and ARA) in muscle diminished as the substitution level increased and, at the same time, the level of these FAs in the diet diminished. We found interesting evidence of elongation from 18:3n-3 to 20:3n-3 when substitution with linseed oil levels was above 50% but no desaturation to 20:4 or 20:5n-3 was found, which could insinuate a certain n-3 HUFA requirement under the experimental conditions. Moreover, we noted an increase in the antioxidant capacity up to the treatment SS70; the SS100 treatment showed a diminution in the antioxidant capacity. It is concluded that a partial inclusion of Spirulina and linseed oil (50%) is possible, and that this replacement can increase antioxidant responses, improve its growth performance and can modulate the mullets fillet quality without affecting negatively fillet fatty acid content.