Multi-level biological responses of Daphnia magna exposed to settleable atmospheric particulate matter from metallurgical industries.

Metallurgical industries are a continuous source of air pollution due to the amount of settleable particulate matter (SePM) they release. This SePM is a complex mixture formed by metallic nanoparticles and metals, which reach terrestrial and aquatic ecosystems and can be a significant source of contamination. The aim of this study was to evaluate the adverse effects of SePM at different levels of biological organization in order to estimate its ecological impacts on aquatic ecosystems. For this purpose, the crustacean Daphnia magna was exposed to different concentrations of SePM (0.01, 0.1, 1, 5, 10 g/L) using a multi-level response approach. The endpoints studied were: avoidance throughout 24 h in a non-forced exposure system, reproduction (total number of neonates per female after 21 days of exposure), acetylcholinesterase activity (AChE) after 48 h, and finally, the feeding rates during a short-term exposure (48 h) and a long-term exposure (21 day + 48 h). There was a negative effect of SePM on all responses measured at high concentrations. The avoidance was concentration-dependent and represented 88 % and 100 % at the two highest concentrations. The AChE activity was significantly inhibited at 5 and 10 g/L. The total number of neonates increased from 1 g/L of SePM and the first brood occurred earlier as of 5 g/L compared to control. The post-exposure feeding rates were lower during long-term exposure at the highest concentration. Chemical analyses were performed to characterize the metals present in this SePM, but this study did not report any direct relationship with toxicity, due to the chemical heterogeneity of the particles. The emission of compounds caused by anthropogenic activity may have significant ecological consequences, so it is important to consider these possible effects on aquatic biota generated by the mixture of metals present in SePM originated from metallurgical activities. Environmental and sectorial regulations are needed to prevent contamination and ecological disturbances.

Combined effects of diesel and air exposure on oxidative stress parameters of mussels Perna perna (Mytilidae, Bivalvia).

This study aimed to assess the combined effect of hypoxia and exposure to diesel on biochemical parameters of Perna perna mussels. Mussels previously kept for 48 h in clean seawater were submitted to hypoxia for 24 h followed by reoxygenation in clean seawater for 48 h. The same procedure was done but using seawater containing 0.01 mL/L of diesel, before and after hypoxia. Antioxidant enzymes as well as levels of glutathione and lipid peroxidation were measured in gills and digestive glands. The neutral red retention time assay was also evaluated in hemocytes. Results showed that cycles of air exposure and reoxygenation caused oxidative stress and antioxidant modulation in both the gills and digestive glands. The presence of diesel in water triggered additional modulation of antioxidants under hypoxia and reoxygenation stress, apparently enhancing the capacity of mussels to avoid lipid peroxidation.

Settleable atmospheric particulate matter harms a marine invertebrate: Integrating chemical and biological damage in a bivalve model.

Some atmospheric pollutants may affect aquatic ecosystems after settling, generating contamination, bioaccumulation, and threats to aquatic species. Metallurgical processes result in the emission of settleable atmospheric particulate matter (SePM), including metals and metalloids, along with rare earth elements (REE) that are considered emerging contaminants. We report the 30-day exposure of brown mussels (Perna perna) to SePM collected in a metallurgical area of southeast Brazil close to estuarine ecosystems, followed by a 30-day clearance period, to evaluate the toxic potential of SePM to this model mollusk. The bioaccumulation of 28 elements identified in SePM and the sublethal effects were evaluated. REEs were found in SePM (Ce, Y, and La). Significant bioaccumulation of eight metals (Fe, Ni, Cu, Zn, Rb, Sr, Cd, and Ba) was found in the bivalves and correlates with the cytotoxicity and genotoxicity, showing a dose-dependent mode and suggesting a pre-pathological condition that could lead to ecological disturbances over time. Conversely, the unchanged lipid-peroxidation level after SePM exposure could indicate the effectiveness of the antioxidant system in protecting gills and digestive glands. The clearance period was not enough to successfully reverse the negative effects observed. So far, the current results enhance the comprehension of the negative role of SePM on metal bioaccumulation and metal-induced toxicity to aquatic biota. Thus, this report adds innovative findings on the role of SePM in aquatic pollution in coastal areas affected by atmospheric pollution, which should be relevant for future public policies to verify and control the environmental pollution.

Aquatic Pollution and Risks to Biodiversity: The Example of Cocaine Effects on the Ovaries of Anguilla anguilla.

Pollution is one of the main causes of the loss of biodiversity, currently one of the most important environmental problems. Important sources of aquatic pollution are illicit drugs, whose presence in waters is closely related to human consumption; their psychoactive properties and biological activity suggest potential adverse effects on non-target organisms, such as aquatic biota. In this study, we evaluated the effect of an environmentally relevant concentration of cocaine (20 ng L−1), an illicit drug widely found in surface waters, on the ovaries of Anguilla anguilla, a species critically endangered and able to accumulate cocaine in its tissues following chronic exposure. The following parameters were evaluated: (1) the morphology of the ovaries; (2) the presence and distribution of enzymes involved in oogenesis; (3) serum cortisol, FSH, and LH levels. The eels exposed to cocaine showed a smaller follicular area and a higher percentage of connective tissue than controls (p < 0.05), as well as many previtellogenic oocytes compared with controls having numerous fully vitellogenic and early vitellogenic oocytes. In addition, the presence and location of 3ß-hydroxysteroid dehydrogenase, 17ß-hydroxysteroid dehydrogenase, and P450 aromatase differed in the two groups. Finally, cocaine exposure decreased FSH and LH levels, while it increased cortisol levels. These findings show that even a low environmental concentration of cocaine affects the ovarian morphology and activity of A. anguilla, suggesting a potential impact on reproduction in this species.

Combined effects of polyethylene spiked with the antimicrobial triclosan on the swamp ghost crab (Ucides cordatus; Linnaeus, 1763).

Domestic sewage is an important source of pollutants in aquatic ecosystems and includes both microplastics (MPs) and pharmaceuticals and personal care products (PPCPs). This study sought to assess the biological effects of the interaction between plastic particles and the antibacterial agent triclosan (TCS). The study relied on the swamp ghost crab Ucides cordatus as a model. Herein polyethylene particles were contaminated with triclosan solution. Triclosan concentrations in the particles were then chemically analyzed. Swamp ghost crab specimens were exposed to experimental compounds (a control, microplastics, and microplastics with triclosan) for 7 days. Samplings were performed on days 3 (T3) and 7 (T7). Gill, hepatopancreas, muscle and hemolymph tissue samples were collected from the animals to evaluate the biomarkers ethoxyresorufin O-deethylase (EROD), dibenzylfluorescein dealkylase (DBF), glutathione S-transferase (GST), glutathione peroxidase (GPx), reduced glutathione (GSH), lipid peroxidation (LPO), DNA strands break (DNA damage), cholinesterase (ChE) through protein levels and neutral red retention time (NRRT). Water, organism, and microplastic samples were collected at the end of the assay for post-exposure chemical analyses. Triclosan was detected in the water and crab tissue samples, results which indicate that microplastics serve as triclosan carriers. Effects on the gills of organisms exposed to triclosan-spiked microplastics were observed as altered biomarker results (EROD, GST, GPx, GSH, LPO, DNA damage and NRRT). The effects were more closely associated with microplastic contaminated with triclosan exposure than with microplastic exposure, since animals exposed only to microplastics did not experience significant effects. Our results show that microplastics may be important carriers of substances of emerging interest in marine environments in that they contaminate environmental matrices and have adverse effects on organisms exposed to these stressors.

Non-destructive biomarkers can reveal effects of the association of microplastics and pharmaceuticals or personal care products.

Methods to assess the effects of contaminants on marine organisms typically involve euthanasia to obtain samples, but less invasive techniques may be more appropriate for working with threatened species. In this study, were assessed the biological responses of crabs exposed to microplastics and contaminants of emerging concern. Biochemical and cellular effects (lipid peroxidation, DNA damage, cholinesterase activity, and lysosomal membrane stability) in hemolymph were analyzed in a kinetic study, at 3 and 7 days, in U. cordatus exposed to microplastics spiked with Triclosan (TCS) or 17α-Ethynylestradiol (EE2). The results showed that the contaminants were produced toxic effects in the crabs exposed either to the microplastics alone (oxidative stress, genotoxicity, and neurotoxicity), or to microplastics with TCS or EE2 adsorbed (neurotoxic and cytotoxic). The present study showed the responsiveness of non-lethal analyzes to understanding the biological effects of combined exposure to microplastics and chemical pollution.

Environmentally realistic concentrations of cocaine in seawater disturbed neuroendrocrine parameters and energy status in the marine mussel Perna perna.

Cocaine (COC) is a powerful illicit drug frequently detected in the aquatic environment. COC acts by inhibiting the reuptake of dopamine (DOPA) and 5-hydroxytryptamine (5-HT - serotonin) and causes endocrine disturbances in mammals. This study investigated similar effects from cocaine exposure in the marine mussel Perna perna, as well as neurotoxicity and energy imbalances. Mussels were exposed to COC (0.2 µg.L-1 and 2 µg.L-1) for periods of 48, 96, and 168 h. Acetylcholinesterase (AChE) was measured in adductor muscle tissue to determine neurotoxicity, and neurotransmitter levels (DOPA and 5-HT), monoamine oxidase (MAO) and cyclooxygenase (COX) activity, and energy status (mitrochondrial electron transport, MET, and total lipids, TLP) were evaluated in the mussels' gonads. COC decreased AChE activity in the mussels exposed to 0.2 µg.L-1 and 2 µg.L -1 after 168 h, and all concentrations of COC increased neurotransmitter levels. Increases in MET (0.2 µg.L -1, for all exposure periods) and TLP (0.2 µg.L 1 after 48 h, and 2 µg.L -1 after 96 h and 168 h) were also observed. No significant change was detected in MAO activity. COC also decreased COX activity in the mussels exposed to 0.2 µg.L -1 (48 h and 96 h) and 2 µg.L -1 (96 h). These results suggest that COC may compromise neurotransmitter levels and COX activity. Furthermore, the changes in MET and LPT suggest that COC affects the energy balance of the mussels, and could negatively affect physiological processes such as metabolism, hormone production, and embryonic development.

Sub-lethal combined effects of illicit drug and decreased pH on marine mussels: A short-time exposure to crack cocaine in CO2 enrichment scenarios.

The increasing CO2-concentrations in the atmosphere promote ocean acidification. Seawater chemistry changes interact with contaminants, such as illicit drugs in the coastal zones. This work evaluates impacts of pH decrease and crack-cocaine exposure on the commercial mussel Perna perna through biomarker responses (lysosomal membrane stability, lipid peroxidation, and DNA strand breaks). The organisms were exposed to different crack-cocaine concentrations (0.5, 5.0, and 50 µg L-1) combined with different pH values (8.3, 8.0, 7.5, 7.0, 6.5, and 6.0) for 96 h. Crack-cocaine in the different acidification scenarios triggered cyto-genotoxicity, which affected the overall health of mussels exposed to cocaine environmentally relevant concentration. This study produced the first data on biomarker responses associated with CO2-induced acidification and illicit drugs (crack-cocaine) in marine organisms.

Effects of substances released from a coal tar-based coating used to protect harbor structures on oysters.

Products coal tar-based are largely used as concrete structures as protective coatings but some questions about leaching and potential toxic effects remain unclear. A laboratory experiment exposing oysters to miniaturized concrete pillars painted with Lackpoxi N1761 over time was performed and trace elements and 17 PAH were monitored in seawater exposure media, and oyster tissues. The original paint composition was also analyzed, and high concentrations of trace elements and PAH were detected. Sharp increases in PAH concentrations were observed after 6-96 h in exposure media and oyster tissues, suggesting that these compounds were leached from the painted structures. In parallel, the integrity of the hemocytes lysosomal membranes of exposed organisms has been damaged. Based on the response of this biomarker, the use of Lackpoxi N1761 is potentially harmful to the environment and it is required that coal tar-based paints be evaluated according to the environmental risk assessment protocols.

Repeated crack cocaine administration alters panic-related responses and delta FosB immunoreactivity in panic-modulating brain regions.

Crack cocaine is the crystal form of cocaine, produced by adding sodium bicarbonate to cocaine base paste. Brazil is the largest consumer of crack cocaine in the world. Users of crack cocaine show important physiological and behavioral alterations, including neuropsychiatric symptoms, such as anxiety-related symptoms. Nevertheless, few pre-clinical studies have been previously performed to understand the neurobiological effects of crack cocaine. The purpose of the present study was to investigate effects of the subchronic treatment (5 days, IP) of rats with crack cocaine in an animal model of anxiety/panic, the elevated T-maze (ETM). The ETM model allows the measurement of two behavioral defensive responses, avoidance and escape, in clinical terms, respectively, associated to generalized anxiety and panic disorder, the two main psychiatric conditions that accompany substance use disorders. Immediately after the ETM model, animals were tested in an open field for locomotor activity assessment. Analysis of delta FosB protein immunoreactivity was used to map areas activated by crack cocaine exposure. Results showed that crack treatment selectively altered escape displayed by rats in the ETM test, inducing either a panicolytic (18 mg/kg IP) or a panicogenic-like effect (25 and 36 mg/kg IP). These effects were followed by the altered functioning of panic-modulating brain regions, i.e., the periaqueductal gray and the dorsal region and lateral wings of the dorsal raphe nucleus. Treatment with 36 mg/kg of crack cocaine also increased locomotor activity. These are the first observations performed with crack cocaine in a rodent model of anxiety/panic and contribute to a better understanding of the behavioral and neurobiological effects of crack cocaine.

Genotoxicity, oxidative stress, and inflammatory response induced by crack-cocaine: relevance to carcinogenesis.

Crack-cocaine is a cocaine by-product widely consumed by general population in developing countries. The drug is low cost and is associated with more intense effects when compared to other illicit drugs. Genotoxicity, oxidative stress, and inflammatory response are considered crucial events in carcinogenesis, since they actively participate in the multistep process. The purpose of this paper was to provide a mini review regarding the relationship between carcinogenesis and genotoxicity, oxidative stress, and inflammation induced by crack-cocaine. The present study was conducted on search of the scientific literature from the published studies available in PubMed, MEDLINE, Scopus, and Google Scholar for all kind of articles (all publications to November 2020) using the following key words: crack-cocaine, DNA damage, genotoxicity, cellular death, cytotoxicity, mutation, oxidative stress, inflammation, and mutagenicity. The results showed that published papers available were almost all in vivo test system being conducted in humans or rodents. Crack-cocaine was able to induce genotoxicity and oxidative stress in mammalian cells. However, the role of inflammatory response after exposure to crack-cocaine was not conclusive so far. In summary, this study is consistent with the notion that crack-cocaine is a chemical carcinogen as a result of genotoxicity and oxidative stress induced in mammalian and non-mammalian cells.

Mussels get higher: A study on the occurrence of cocaine and benzoylecgonine in seawater, sediment and mussels from a subtropical ecosystem (Santos Bay, Brazil).

Data on the occurrence of cocaine (COC) and benzoylecgonine (BE) in marine environmental compartments are still limited, with few studies reporting superficial water contamination, mainly in tropical zones. In this sense, environmental data of these substances are essential to identify potential polluting sources, as well as their impact in costal ecosystems. The aim of this study was to evaluate the occurrence of COC and BE in seawater, sediment and mussels from a subtropical coastal zone (Santos Bay, São Paulo, Brazil), as well as to determine a field measured Bioaccumulation Factor (BAF). COC and BE were detected in all water samples in concentrations ranging from 1.91 ng·L-1 to 12.52 ng·L-1 and 9.88 ng·L-1 to 28.53 ng·L-1, respectively. In sediments, only COC was quantified in concentrations ranging from 0.94 ng·g-1 to 46.85 ng·g-1. Similarly, only COC was detected in tissues of mussels 0.914 µg·kg-1 to 4.58 µg·kg-1 (ww). The field-measured BAF ranged from 163 to 1454 (L·kg-1). Our results pointed out a widespread contamination by cocaine and its main human metabolite benzoylecgonine in Santos Bay. Mussels were able to accumulate COC in areas used by residents and tourists for bathing, fishing, and harvest, denoting concern to human health. Therefore, our data can be considered a preliminary assessment, which indicates the need to evaluate drugs (including illicit as COC) in environmental and seafood monitoring programs, in order to understand their risks on the ecosystem and human health.

Review on the occurrence and biological effects of illicit drugs in aquatic ecosystems.

Illicit drugs (IDs) and their metabolites are recognized as contaminants of emerging concern. After consumption, illicit drugs are partially metabolized and excreted unchanged in urine and feces or as active metabolites reaching wastewater treatment plants (WWTPs). Furthermore, most WWTPs are insufficient in the treatment of effluents containing IDs, which may be released into aquatic ecosystems. Once in the water or sediment, these substances may interact and affect non-target organisms and some evidences suggest that illicit drugs may exhibit pseudo-persistence because of a continuous environmental input, resulting in long-term exposure to aquatic organisms that may be negatively affected by these biologically active compounds. We reviewed the literature on origin and consumption, human metabolism after consumption, aquatic occurrences, and toxicity of the major groups of illicit drugs (opioids, cannabis, synthetic drugs, and cocaine). As a result, it could be concluded that illicit drugs and their metabolites are widespread in diverse aquatic ecosystems in levels able to trigger sublethal effects to non-target organisms, besides to concentrate in seafood. This class of emerging contaminants represents a new environmental concern to academics, managers, and policymakers, whose would be able to assess risks and identify proper responses to reduce environmental impacts.

Effects of Microplastics Associated with Triclosan on the Oyster Crassostrea brasiliana: An Integrated Biomarker Approach.

Urban waste is a complex mixture of different substances, including microplastics and pharmaceuticals and personal care products. Microplastics have a high affinity for hydrophobic substances. One of these substances is triclosan, a bactericide used in a variety of hygiene products. Therefore, microplastics (MPs) may serve as a vector between triclosan and aquatic organisms. The current study sought to evaluate the effects of the interaction between microplastics and triclosan based on a mechanistic approach in which the oyster Crassostrea brasiliana was used as a model. The organisms were exposed to three conditions: the control, microplastic (MP), and microplastic contaminated with triclosan (MPT). The organisms were exposed for 3 or 7 days. After the exposure time, hemolymph was sampled for performing the neutral red retention time assay and, subsequently, the gills, digestive glands, and adductor muscles were dissected for measuring biomarkers responses (EROD, DBF, GST, GPx, GSH, lipid peroxidation, DNA strand breaks, and AChE). Our results demonstrate combined effects of MPs associated with triclosan on oyster physiology and biochemistry, as well as on lysosomal membrane stability. These results contribute to understanding the effects of contaminants of emerging concern and microplastics on aquatic organisms.

Lead toxicity on a sentinel species subpopulation inhabiting mangroves with different status conservation.

Lead is a priority pollutant introduced in the aquatic environment by different sources commonly located in estuarine regions, such as ports, marinas and industries. Environmental agencies around the world set the maximum allowable concentration of lead in effluents, surface water and sediment, but few studies reported its accumulation and chronic toxicity in mangrove benthic invertebrates using concentrations believed to be safe. In the case of Brazilian mangrove environments, Ucides cordatus is a crab species of choice to be used in bioaccumulation studies. We have assessed biomarkers' responses (DNA strand breaks, micronucleated cells, metallothioneins, enzymatic activity of aminolevulinic acid dehydratase and neutral red retention time) and the total bioaccumulation in six tissues of U. cordatus crabs resident to mangrove areas under different conservation status during a 28-day period bioassay. We also investigated Pb subcellular partition and biomarkers' responses using a supposedly safe concentration (10 µg L-1). During the Pb exposure, the highest concentration of Pb was observed in crab gills. Crabs also showed a high ability to allocate Pb in detoxified forms. Multivariate analysis pointed out that bioaccumulation (total, active and detoxified) is linked to biomarkers. Even in supposedly safe dosage, U. cordatus triggered its defense mechanisms expressing more metallothioneins and presented relevant cyto-genotoxic damage. Our data suggest the development of biological tolerance to Pb in crabs from polluted areas. Our results provided a new insight about lead toxicity even at concentrations considered environmentally safe, which could support new strategies to manage estuarine areas considering their respective conservation status.

In vivo and in vitro analysis of cytogenotoxicity in populations living in abnormal conditions from Santos-Sao Vicente estuary.

The aim of the study was to evaluate cyto- and genotoxic effects in populations living in subnormal clusters in Santos São Vicente estuary. For in vivo study, samples of buccal mucosa and peripheral blood cells were collected. Micronucleus assay and single-cell gel (comet) assay were performed. For in vitro study, Chinese ovary hamster (CHO) cells were exposed to contaminated water. The results showed that people living in the contaminated estuary have increased DNA damage in oral mucosa and peripheral blood cells, as detected in the micronucleus and comet assays respectively. In addition, estuarine water was able to promote cytotoxicity at the highest concentrations, as well as decrease the number of cells in the G1 phase. In summary, our results indicate that water from the Santos-São Vicente estuary is capable of inducing cytogenotoxicity in mammalian cells in vivo and in vitro.

Seasonal monitoring of cocaine and benzoylecgonine in a subtropical coastal zone (Santos Bay, Brazil).

Illicit drugs and their metabolites represent a new class of emerging contaminants. These substances are continuously discharged into wastewater which have been detected in the aquatic environment in concentrations ranging from ng.L-1 to µg.L-1. Our study detected the occurrence of cocaine (COC) and benzoylecgonine (BE) in a subtropical coastal zone (Santos Bay, SP, Brazil) within one year. Water samples (surface and bottom) were collected from the Santos Submarine Sewage Outfall (SSOS) area. COC and BE were measured in the samples using ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-ESI-MS/MS). Concentrations ranged from 12.18 to 203.6 ng.L-1 (COC) and 8.20 to 38.59 ng.L-1 (BE). Higher concentrations of COC were observed during the end of spring, following the population increase at summer season. COC and its metabolite occurrence in this coastal zone represent a threat to coastal organisms.

Common snook juveniles, Centropomus undecimalis, as biomonitor organisms to evaluate cytogenotoxicity effects of surface estuarine water from Southern Brazil.

Centropomus undecimalis fish inhabit the highly contaminated Santos-São Vicente Estuarine System (SESS) and could be a good biomonitor of contaminants. This study aimed to investigate the cytogenotoxic potential of superficial water from SESS using C. undecimalis as a biomonitoring model and to validate the use of farmed fish as controls. Using biochemical (DNA damage and Lipid Peroxidation - LPO), cellular (erythrocyte nuclear abnormality - ENA) and tissue (8-OHdG immunoexpression) biomarkers, our results showed fish from SEES had higher LPO concentration in gills and higher frequency of reniform, lobed and total ENA in erythrocytes when compared with control farmed fish. Thus, SESS surface water are cytogenotoxic for blood and gills cells of fishes. C. undecimalis has shown to be a good biomonitor model and farmed fish can be used as control only if livers were not the target organs of study since the dietary food from farmed fish causes steatosis.

Mangrove metal pollution induces biological tolerance to Cd on a crab sentinel species subpopulation.

Metals are persistent pollutants, able to accumulate in the biota and magnify in trophic web. In the specific case of cadmium contamination, it has been the subject of considerable interest in recent years because of its biological effects and it is one of major pollutant in estuarine areas. Ucides cordatus is considered a mangrove local sentinel crab species in Brazil and there are previous studies reporting crab subpopulations living from pristine to heavily metal impacted areas in São Paulo coast (Southeastern Brazil). Taking into account the background knowledge about these subpopulations, we proposed the hypothesis that crabs from a highly polluted mangrove (Cubatão - CUB) have developed biological tolerance to cadmium compared to animals from an Environmental Protected Area (Jureia - JUR). Aiming to verify this hypothesis, we have investigated total bioaccumulation and subcellular partition of Cd, besides biomarkers' responses during a long-term exposure bioassay (28 days, with weekly sampling) using a supposedly safe Cd concentration (0.0022 mg L-1). Specimens from the pristine area (JUR) accumulated higher total Cd, as such as in its biologically active form in gills. Animals living in the polluted site (CUB) presented higher amounts of Cd in the mainly detoxifying tissue (hepatopancreas), which could be considered a pathway leading to tolerance for this metal. Multivariate analysis indicated that bioaccumulation (active, detoxified and total Cd) is linked to geno-cytotoxic damages. CUB subpopulation was considered more tolerant since it presented proportionally less damage and more capacity to allocate Cd in the main detoxifying forms and tissues.