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.

Multiple biomarkers in pufferfish as a proxy of environmental health in brazilian marine protected areas.

Marine Protected Areas (MPAs) are designed to conserve biodiversity and vulnerable ecosystems. This study aimed to assess the environmental quality of three Brazilian MPAs, based on the integrated analysis of biomarkers in pufferfish. The MPAs are differentiated by the degree of anthropogenic influences. The Barra do Una Estuary sustainable reserve (JUR) is a reference area due to its low levels of contamination and species diversity. The Cananéia Estuarine System (CAN) has been recognized as a biosphere reserve by UNESCO, as well as a Ramsar wetland. This MPA was influenced by upstream mining activities, resulting in the introduction of metals in the estuarine waters and the discharge of untreated urban sewage. The São Vicente estuary (SSV) lacks proper sanitation infrastructure. All collections were conducted during winter season, and, after collection, the animals were euthanized, their soft tissues were removed, and multiple biomarkers were analyzed in the gills and liver, as biometric, genotoxic, biochemical, and morphological. A one-factor multivariate analysis was applied to evaluate the differences between the data sets, and the matrices were analyzed using PERMANOVA to evaluate the "estuary" factor. The results were integrated using PCA with a 0.4 cut value and an Enhanced Integrated Biomarker Response (EIBR) was calculated. PCA was correlated with biochemical, genotoxic, and morphological biomarkers. In general, SVV differed from CAN and JUR as shown by both univariate and multivariate analyses. SVV also showed the highest EIBR, followed by CAN. Organisms from SSV showed greater gill pathology, elevated AChE activity and lipoperoxidation (LPO), and micronuclei frequency. CAN present intermediate EIBR, with severe pathologies in the liver. CAN seems to present an intermediate environmental quality between SSV and JUR indicating the importance of the existence of MPAs for environmental conservation and the need to monitor such areas, to maintain their suitable environmental quality.

Interactive effects of microplastics and benzo[a]pyrene on two species of marine invertebrates.

This study aimed to evaluate B[a]P and low-density polyethylene microplastics (MPs) toxicty, alone and in mixture (0.03 to 30 µg L-1 of B[a]P; and 5, 50 and 500 mg L-1 for MPs). Five mg L-1 of MPs is considerably higher than commonly reported environmental concentrations, although it has been reported for marine environments. Individual (sea urchin embryo-larval development and mortality of mysids) and sub-individual responses (LPO and DNA damage in mysids) were assessed. The toxicity increased as the B[a]P concentration increased, and microplastics alone did not cause toxicity. B[a]P toxicity was not modified by the lowest concentration of MPs (5 mg L-1), but at higher MPs concentrations (50 and 500 mg L-1), the effects of B[a]P on sea urchin development and in biomarkers in mysids were diminished. Microplastics interacted with B[a]P in seawater, reducing its toxicity, probably due to adsorption of B[a]P to the surface of microplastics.

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.

A preliminary study on multi-level biomarkers response of the tropical oyster Crassostrea brasiliana to exposure to the antifouling biocide DCOIT.

This study investigated the sublethal effects of environmentally relevant concentrations of DCOIT on the neotropical oyster Crassostrea brasiliana. Gills and digestive glands of animals exposed to increasing concentrations of DCOIT were analyzed for biochemical, cellular, and histopathological responses. Exposure to DCOIT (0.2 to 151 µg L-1) for 120 h triggered oxidative stress in both tissues (through the modulation of GPX, GST, GSH and GR), which led to damage of membrane lipids (increase of LPO and reduction of the NRRT). DCOIT increased histopathological pathologies in gills, such as necrosis, lymphocyte infiltration and epithelial desquamation. This study showed that short term exposure to environmental concentrations of DCOIT causes negative effects on C. brasiliana at biochemical, physiological, and histological levels. Therefore, the use of DCOIT as a booster biocide in antifouling paints should be further assessed, as it may cause environmental hazards to marine organisms.

Biochemical and histopathological responses in peripubertal male rats exposed to agrochemicals isolated or in combination: A multivariate data analysis study.

The widespread use of agrochemicals results in the exposure of the general human population, including children, to several of these chemicals simultaneously. In the present preclinical study, it was investigated the hepatic damages caused by exposure to acephate, carbendazim and mancozeb when administered alone or in different combinations (binary and ternary). Juvenile male Wistar rats were exposed to agrochemicals from post-natal day 53, by gavage. The doses of agrochemicals applied here were determined from previous studies whose results showed no signs of systemic toxicity. All exposures provoked a significant increase in DNA damage (except for acephate alone) and activation of the xenobiotic biotransformation system (except for the ternary mixture). Interestingly, the ternary mixture did not exhibit an exacerbation in adverse effects caused by agrochemicals isolated or in binary combination, even though they are sharing genotoxicity damage induction as a common toxicity pathway. Conversely, some effects observed for isolated or binary combinations of agrochemicals were not observed for ternary combination, suggesting a chemical interaction that could imply antagonism character. Using a multivariate data analysis approach, exposure to isolated agrochemicals were related to a group of adverse effects characterized by hepatic lesion and the attempt of the tissue to mobilize defense cells and increase mitotic rates to minimize damages. Binary mixtures also share similarities in relation to the effects they exhibited, mainly a moderate to high increase in the GST activity and in histopathological alterations suggesting that binary combinations trigger an increased response of the mechanism of xenobiotics biotransformation. Together, obtained results bring important insights regarding adverse effects and possible interaction of the three agrochemicals whose residues are commonly detected in agro-food products.

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.

Heavy Metals in Tissues of Blue Crabs Callinectes danae from a Subtropical Protected Estuary Influenced by Mining Residues.

This short note aims to report in detail a preliminary assessment of the concentrations of Cd, Hg and Pb in tissues of blue crabs Callinectes danae collected from the Cananéia-Iguape-Peruíbe estuarine complex (CIP), in the South of São Paulo State coast, Brazil. In October 2014, blue crabs were collected from CIP. Tissues were removed by dissection and metal determination was performed by GF-AAS and CV-AAS. According to statistical analysis, Pb and Cd concentrations in gills were significantly higher than those found in muscles and hepatopancreas, respectively. There were no significant differences in Hg concentrations between samples. Cd, Hg and Pb concentrations in gills and hepatopancreas were lower than those reported in a previous study performed at CIP. However, Cd concentration in hepatopancreas was higher than the Brazilian limit for consumption and new efforts to monitor Cd concentrations in C. danae tissues must be performed.

Embryo toxicity assay in the fish species Rhamdia quelen (Teleostei, Heptaridae) to assess water quality in the Upper Iguaçu basin (Parana, Brazil).

The Iguaçu River is one of the largest and most important rivers in the Southern of Brazil. The Upper Iguaçu Basin is responsible for water supply (80%) of the Metropolitan Region of Curitiba (MRC). After crossing a large urban region, the river is polluted by domestic and industrial sewage, but despite of that few ecotoxicological studies have been performed in order to evaluate the water quality from Iguaçu River. The aim of the present study was to investigate the risk of exposure of Iguaçu water to biota and also human population. In this terms, was utilized the survival effect and the morphological deformities in larval embryos of Rhamdia quelen, a native South America species. The results showed a high level of pollution in all studied sites along the Upper Iguaçu River including PAHs and toxic metals such as lead. The lethal and non-lethal effects described in earlier stages of development suggest an elevated risk to biota. This data was corroborated by the theoretical model, showing that the pollutants present in water from Iguaçu River may further reduce the fish population density including risk of local extinction. The present study reflect the needs to conduct in-depth research to evaluate the real impact of human activities on the endemic fish biota of Iguaçu River including the risk for human populations.

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.

Sediment toxicity identification evaluation (TIE phases I and II) based on microscale bioassays for diagnosing causes of toxicity in coastal areas affected by domestic sewage.

Domestic sewage is a major problem in highly urbanized coastal areas worldwide. In the present study, toxicity identification evaluation (TIE) phases I and II were applied to sediment interstitial water from 2 locations along the São Paulo coast in southeastern Brazil: the sewage outfalls from the city of Santos, a densely urbanized area, and the city of Bertioga, a less urbanized area. An adapted microscale sea urchin embryo-larval development bioassay was employed. Phase TIE-I manipulations were 1) ethylenediamine tetraacetic acid (EDTA) addition, 2) aeration at modified pH, 3) C18 solid-phase extraction (SPE), and 4) addition of Na2 S2 O3 . The results of the Santos TIE-I indicated toxicity by sulfides and substances with affinity to C18 resin. In Bertioga, toxicity was the result of NH3 , metals, and arsenic, as well as substances with affinity to C18 resin. Phase TIE-II aimed to specify the causes of toxicity by testing the toxicity of eluates of the sublation and C18 SPE manipulation. The results reinforced the role of both surfactants and nonpolar compounds as causative agents of toxicity in both Santos and Bertioga. Chemical analyses of sediment interstitial water or whole sediment ruled out the influence of polycyclic aromatic hydrocarbons (PAHs) or polychlorinated biphenyls (PCBs) in the toxicity of both sampling sites. Other hydrophobic substances may play a role in the toxicity of Santos and Bertioga effluents. Efforts to remove such substances from sanitary effluents must be prioritized. Environ Toxicol Chem 2017;36:1820-1832. © 2017 SETAC.

Assessing genotoxic effects in fish from a marine protected area influenced by former mining activities and other stressors.

The goal of the current study was to evaluate different genotoxicity tools in order to assess a marine protected area (MPA) affected by former mining activities and urban settlements. A catfish (Cathorops spixii) was analyzed for genotoxic effects at the (i) molecular and at the (ii) chromosomal levels. Through factor analysis, genotoxicity was found to be linked to levels of metals bioaccumulated and PAH metabolites in the bile. Micronucleus and nuclear alteration were less vulnerable to the effects of confounding factors in mildly contaminated areas since they were more frequently associated with bioaccumulated metals than the DNA analysis. The different genotoxicity responses allowed for the identification of sources of pollution in the MPA. This approach was important for detecting environmental risks related to genotoxic contaminants in a mildly contaminated MPA.

Assessing pollution in marine protected areas: the role of a multi-biomarker and multi-organ approach.

Marine protected areas (MPAs) are vulnerable to many pressures, including pollution. However, environmental quality monitoring in these areas traditionally relies on only water chemistry and microbiological parameters. The goal of the current study was to investigate the role of a set of biomarkers in different target organs (liver, kidney, and gills) of fish in order to assess the environmental quality of an MPA (MTs, GPx, GST, GSH, DNA damage, LPO, AChE, and condition index). Chemical analyses were also performed on liver and muscle tissues to evaluate metal body burdens, and PAHs were identified in bile. A demersal fish (Cathorops spixii) that is widely consumed by the local population was used as bioindicator species, and the results were integrated using multivariate analysis. The use of the biomarker approach allowed for the identification of both seasonal and spatial variations in pollution sources around the Environmental Protected Area of Cananéia-Iguape-Peruíbe (APA-CIP). Higher metal body burdens associated with biological responses were found in the sites under the influence of urban areas during the dry season, and they were found in the sites under the influence of the Ribeira de Iguape River (RIR) during the rainy season. The liver was found to be more responsive in terms of its antioxidant responses, whereas gills were found to be more responsive to biomarkers of effect. These results show that this set of biomarker analyses in different organs of fish is a useful tool for assessing chemical pollution in an MPA.

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.