Microplastics and 17α Ethinylestradiol: How Do Different Aquatic Invertebrates Respond to This Combination of Contaminants?

The synthetic hormone 17α ethinyl estradiol (EE2) is a molecule widely used in female contraceptives and recognized as a contaminant of attention (Watch List) in the European Union due to its high consumption, endocrine effects and occurrence in aquatic environments. Its main source of introduction is domestic sewage where it can be associated with other contaminants such as microplastics (MPs). Due to their characteristics, they can combine with each other and exacerbate their isolated effects on biota. This study evaluated the combined effects of microplastics (MPs) and 17α ethinylestradiol (EE2) on two tropical estuarine invertebrate species: Crassostrea gasar and Ucides cordatus. Polyethylene particles were spiked with EE2 and organisms were exposed to three treatments, categorized into three groups: control group (C), virgin microplastics (MPs), and spiked microplastics with EE2 (MPEs). All treatments were evaluated after 3 and 7 days of exposure. Oysters exhibited changes in phase 2 enzymes and the antioxidant system, oxidative stress in the gills, and reduced lysosomal membrane stability after exposure to MPs and MPEs. Crabs exposed to MPs and MPEs after seven days showed changes in phase 1 enzymes in the gills and changes in phases 1 and 2 enzymes in the hepatopancreas, such as disturbed cellular health. The combined effects of microplastics and EE2 increased the toxicity experienced by organisms, which may trigger effects at higher levels of biological organization, leading to ecological disturbances in tropical coastal ecosystems.

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.

Using large amounts of firefighting foams releases per- and polyfluoroalkyl substances (PFAS) into estuarine environments: A baseline study in Latin America.

We analyzed per- and polyfluoroalkyl substances (PFAS) in aqueous film-forming foams (AFFFs) used to extinguish a major fire in a petrochemical terminal from the Port of Santos (Brazil). Eight AFFFs from seven known commercial brands and one unknown sample (AFFF-1 to AFFF-8) were evaluated. 17 PFAS were identified and quantified using high performance liquid chromatography (LC/MS). The concentrations of Σ17 PFAS in the AFFFs ranged from 500 to 9000 ng/g, with prevalence of short chain PFAS (~85 %), followed by long chain PFAS. Perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA), included in the global treaty of the Stockholm Convention, were also detected. We estimated that at least 635.96 g of PFAS were introduced in the estuary, representing a massive input of these substances. This investigation reports the PFAS composition of AFFFs used in firefighting in the GRULAC Region (Group of Latin American and Caribbean countries).

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.

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.

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.

Marine contamination and cytogenotoxic effects of fluoxetine in the tropical brown mussel Perna perna.

Concerns are growing about the presence of fluoxetine (FLX) in environmental matrices, as well as its harmful effects on non-target organisms. FLX in aquatic ecosystems has been detected in a range varying from pg/L to ng/L, while adverse effects have been reported in several organisms inhabiting freshwater and marine environments. The present study quantifies FLX concentrations in seawater samples from Santos Bay, Brazil and assesses metabolic responses and sublethal effects on the tropical brown mussel Perna perna. Levels of ethoxyresorufin­O­deethylase, dibenzylfluorescein dealkylase, glutathione S-transferase, glutathione peroxidase, cholinesterase, lipoperoxidation, and DNA damage were assessed in the gills and digestive gland of these animals, and lysosomal membrane stability was also assessed in hemocytes. FLX altered phase I and II enzyme activities, caused cytogenotoxic effects, and negatively impacted the overall health of mussels exposed to environmentally relevant concentrations. These findings contribute to characterize the risks of introducing this drug into the marine environment.

Detoxification, oxidative stress, and cytogenotoxicity of crack cocaine in the brown mussel Perna perna.

The presence of cocaine and its metabolites and by-products has been identified in different aquatic matrices, making crack cocaine the target of recent studies. The aim of this study was to evaluate the sublethal effects of crack on the brown mussel Perna perna. Mussels were exposed to three concentrations of crack cocaine (0.5, 5.0, and 50.0 µg L-1) for 168 h. Gills, digestive glands, and hemolymph were extracted and analyzed after three different exposure times using a suite of biomarkers (EROD, DBF, GST, GPX, LPO, DNA damage, ChE, and lysosomal membrane stability [LMS]). After 48 and 96 h of exposure, EROD, DBF, GST, GPX activities and DNA strand breaks in the gills increased significantly after 48 and 96 h of exposure. Alterations in LMS were also observed in the mussels exposed to all crack concentrations after 96 and 168 h. Our results demonstrated that crack cocaine is metabolized by CYP-like and GST activities in the gills. GPX was not able to prevent primary genetic damage, and cytotoxic effects in the hemocytes were also observed in a dose- and time-dependent response. Our study shows that the introduction of illicit drugs into coastal ecosystems must be considered a threat to marine organisms.

Could male reproductive system be the main target of subchronic exposure to manganese in adult animals?

Manganese (Mn) is one of the most common chemical elements on Earth and an essential micronutrient in animal organism. However, in supraphysiological levels and long-term exposures, it is a potential toxicant. Although nervous system is the most studied in relation to Mn toxicity, other tissues can have their function impaired by Mn in high doses. The present study investigated the possible adverse effects of subchronic exposure to supraphysiologic level of Mn (5 mg/kg or 15 mg/kg, intraperitoneally) on reproductive, neurobehavioral, renal and hepatic parameters of male rats. For the first time, the vulnerability of these parameters to Mn was concomitantly investigated. While our results demonstrate that Mn treatments were not sufficient to produce a marked effect of neurotoxic, hepatotoxic or renal toxicity in adult rats, we found typical indicators of reproductive toxicity such as histopathological changes (major in testes and epididymis) and impaired sperm concentration and quality. Mn, under these experimental conditions, seems to exert reproductive toxicity by different testicular mechanisms, i.e. direct and indirect action on germ cells. On the other hand, exposure to Mn did not change the pattern of cognitive and emotional behaviors and the histological organization of kidneys of experimental rats. The liver showed a weight increasement and hidropic degeneration, probable due to the detoxification overload. In summary, for the first time it was demonstrated that adult male reproductive system was more sensitive to Mn toxicity than nervous, hepatic and renal systems, although nervous system is known as the main target tissue of this metal.

Ecotoxicological effects of losartan on the brown mussel Perna perna and its occurrence in seawater from Santos Bay (Brazil).

The antihypertensive losartan (LOS) has been detected in wastewater and environmental matrices, however further studies focused on assessing the ecotoxicological effects on aquatic ecosystems are necessary. Considering the intensive use of this pharmaceutical and its discharges into coastal zones, our study aimed to determine the environmental concentrations of LOS in seawater, as well as to assess the biological effects of LOS on the marine bivalve Perna perna. For this purpose, fertilization rate and embryolarval development were evaluated through standardized assays. Phase I (ethoxyresorufin O­deethylase EROD and dibenzylfluorescein dealkylase DBF) and II (glutathione S-transferase GST) enzymes, glutathione peroxidase (GPx), Cholinesterase (ChE), lipoperoxidation (LPO) and DNA damage were used to analyze sublethal responses in gills and digestive gland of adult individuals. Lysosomal membrane stability was also assessed in hemocytes. Our results showed the occurrence of LOS in 100% of the analyzed water samples located in Santos Bay, Sao Paulo, Brazil, in a range of 0.2 ng/L-8.7 ng/L. Effects on reproductive endpoints were observed after short-term exposure to concentrations up to 75 mg/L. Biomarker responses demonstrated the induction of CYP450 like activity and GST in mussel gills exposed to 300 and 3000 ng/L of LOS, respectively. GPx activity was also increased in concentration of exposure to 3000 ng/L of LOS. Cyto-genotoxic effects were found in gills and hemocytes exposed in concentrations up to 300 ng/L. These results highlighted the concern of introducing this class of contaminants into marine environments, and pointed out the need to include antihypertensive compounds in environmental monitoring programs.

A tiered approach to assess effects of diclofenac on the brown mussel Perna perna: A contribution to characterize the hazard.

Pharmaceutical discharges into the aquatic ecosystem are of environmental concern and sewage treatment plants (STPs) have been pointed out as the major source of these compounds to coastal zones, where oceanic disposal of sewage occurs through submarine outfalls. Diclofenac (DCF) is one of the most frequently detected pharmaceuticals in water, but little is known about the effects on marine organisms. In this study, we employed a tiered approach involving the determination of environmental concentrations of DCF in marine water and the adverse biological effects for fertilization, embryo-larval development and biomarker responses of the mussel Perna perna. Results indicate that effects in fertilization rate and embryo-larval development were found in the order of mg·L-1. However, low concentrations of DCF (ng·L-1) significantly decreased the lysosomal membrane stability and COX activity, as well as triggered DNA damage, oxidative stress and changes in antioxidant defenses. Our results point to an environmental hazard at coastal ecosystems and suggest the need for improvements in the treatment of domestic wastewater aiming to reduce DCF concentrations, as well as regulation on current environmental legislation and monitoring of aquatic ecosystems.

Shell alterations in limpets as putative biomarkers for multi-impacted coastal areas.

During the last years, shell alterations in gastropods have been proposed as tools to be used in monitoring programs. However, no studies were so far performed investigating the relationships among shell parameters and classical biomarkers of damage. The relationship between shell alterations (biometrics, shape and elemental composition) and biomarkers (LPO and DNA strand break) was evaluated in the limpet L. subrugosa sampled along a contamination gradient in a multi-impacted coastal zone from southeastern Brazil. Statistically significant differences were detected among sites under different pollution levels. The occurrence of shell malformations was consistent with environmental levels of several hazardous substances reported for the studied area and related to lipid peroxidation and DNA damage. In addition, considering the low mobility, wide geographic distribution, ease of collection and abundance of limpets in coastal zones, this putative tool may be a cost-effective alternative to traditional biomarkers. Thus, shell alterations in limpets seem to be good proxies for assessing biological adverse effects in multi-impacted coastal zones.

Effects of novobiocin and methotrexate on the benthic amphipod Ampelisca brevicornis exposed to spiked sediments.

The marine amphipod Ampelisca brevicornis was used as model organism of benthic macrofauna to assess the possible adverse effects of pharmaceuticals bound to sediments. Organisms were exposed to sediment spiked with novobiocin (NOV) and methotrexate (MTX) for 10 days in order to estimate the acute toxicity (lethal effects) produced by the two compounds. The surviving organisms were pooled and analyzed to determine their sublethal responses associated with different phases of metabolism (enzyme activities in phases I and II), oxidative stress (antioxidant enzyme activities and lipid peroxidation), and genotoxicity (DNA damage in the form of strand breaks). No lethal or sublethal effects were observed in the amphipods exposed to NOV. For organisms exposed to sediments spiked with MTX the results were found to calculate the concentration that was lethal to 50% of the organisms exposed in the toxicity tests (LC50 of 30.36 ng/g). MTX also induced the metabolism of enzyme detoxification activities in phases I and II. Oxidative stress and DNA damage in particular were also observed, indicating responses associated with MTX's mechanism of action. Both mortality and the set of applied biomarkers allowed for the assessment of bioavailability, oxidative stress, and genotoxicity of NOV and MTX. The information obtained in this investigation can assist in ecological risk assessment of marine sediments contaminated by pharmaceuticals.

Ecological risk evaluation of sediment metals in a tropical Euthrophic Bay, Guanabara Bay, Southeast Atlantic.

Surface sediments were collected from Guanabara Bay, at 14 stations distributed in five sectors, over three sampling campaigns. Analyses of metals, grain size fractions and total organic carbon analyses were performed. The geo-accumulation index and the enrichment factor were estimated to assess contamination status based on background values. Additionally, the sediment quality guidelines were applied to evaluate the adverse biological effects. Results show that there was no seasonal variation in sediment quality based on any methodology, and all methods utilized showed that NW sector and HRJ sector were the worst affected and that the NE sector had the best conditions. The sediments of GB are polluted mainly by Cr, Pb and Zn. According to ΣSEM/AVS, these metals are not available to the biota, although toxicity tests dispute this. Among the various methods employed, those using background values for the area seem to best reflect the local historical contamination.

The application of biochemical responses to assess environmental quality of tropical estuaries: field surveys.

A battery of biomarkers of exposure (EROD, DBF, GST and GPx) and effect (lipid peroxidation and DNA damage - strand breaks) were analyzed in gill tissues from caged and native oysters Crassostrea rhizophorae exposed to two tropical estuarine systems in SW Brazil: Santos (S1, S2, S3, S4) and Paranaguá (P1 - control, P2, P3, P4). The exposure lasted 28 days. Native oysters were sampled in the same areas where caged systems were exposed. Significant induction of biomarkers of exposure to organic compounds and oxidative stress (p < 0.05) were observed in all transplanted individuals from Santos resulting in DNA damage and lipid peroxidation. Biological adverse effects were more evident in oysters transplanted in the Santos Estuarine System, a recognized contaminated area, than in the Paranaguá Estuarine System, surrounded by urban areas and Environmental Protected Areas. Native specimens from both estuaries showed adaption to the impacts of several contamination sources affecting the ecosystem. The use of transplanted C. rhizophorae proved to be a suitable tool for assessing and monitoring the environmental quality in mangrove ecosystems. This integrated approach employing multi-biomarker responses under field conditions could be incorporated as a descriptor of health status in tropical estuarine systems.