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.

Five years after the collapse of the Fundão Dam: lessons from temporal monitoring of chemistry and acute toxicity.

In November 2015, the Fundão Dam break released millions of tons of metal-rich tailings into the Doce River Basin (DRB), causing catastrophic damage and potential ecological effects that reached the Atlantic Ocean. This study aimed to evaluate the geochemistry and toxicity of water and sediments collected in the DRB from 2015 to 2019 and to determine the spatial and temporal trends. Water and sediment samples were analyzed for metals and As by inductively coupled plasma optical emission spectrometry (ICP-OES), and acute toxicity for Daphnia similis or D. magna. Results were explored using geochemical indices and correlation analyzes. Overall, higher concentrations of metals and As in water and sediments were observed immediately after dam breakage, but the levels exhibited a decreasing trend over time, although the levels of some elements such as As and Mn remained high in the upper DRB. The geochemical indices indicated mostly low to moderate contamination, and the enrichment factor (EF) demonstrated a higher enrichment of Mn in the upper DRB. Acute toxicity to water fleas (D. similis and D. magna) was occasionally observed in waters and sediments, but the reference samples were toxic, and the short-term effects were not correlated with metals and As. Overall, the results showed limited bioavailability of metals and As and a decreasing trend in their concentrations, indicating an ongoing recovery process in DRB. These results are important to decision-making regarding the disaster and actions for environmental restoration.

PAH residues and toxicity levels two years after an extensive oil spill on the northeast Brazilian coast.

The most extensive oil spill ever recorded in tropical oceans occurred between August 2019 and March 2020, affecting approximately 3000 km of the Brazilian coast. This study assessed the chemical contamination and toxicity of sediments collected from affected reef areas during two sampling surveys conducted 17 and 24 months after the peak of oil slick inputs. Our results indicated that neither PAH levels nor measured toxicity showed a significant contribution from the spilled oil, with concentrations and biological effects indistinguishable from those in unaffected areas. Similarly, no differences were observed between seasons. Furthermore, there was no discernible relationship between sediment toxicity results and the measured PAH concentrations. Therefore, while biological responses indicated toxicity in most assessed areas, these responses are likely related to other local sources. This evidence suggests a natural oil attenuation process contributing to local environmental recovery. Nonetheless, further investigation is needed for other areas affected by oil spills.

Adults of Sun Coral Tubastraea coccinea (Lesson 1829) Are Resistant to New Antifouling Biocides.

Biocides used in antifouling (AF) paints, such as 4,5-dichlorine-2-n-octyl-4-isothiazole-3-one (DCOIT), can gradually leach into the environment. Some AF compounds can persist in the marine environment and cause harmful effects to non-target organisms. Nanoengineered materials, such as mesoporous silica nanocapsules (SiNCs) containing AF compounds, have been developed to control their release rate and reduce their toxicity to aquatic organisms. This study aimed to evaluate the acute toxicity of new nanoengineered materials, SiNC-DCOIT and a silver-coated form (SiNC-DCOIT-Ag), as well as the free form of DCOIT and empty nanocapsules (SiNCs), on the sun coral Tubastraea coccinea. T. coccinea is an invasive species and can be an alternative test organism for evaluating the risks to native species, as most native corals are currently threatened. The colonies were collected from the Alcatrazes Archipelago, SP, Brazil, and acclimatized to laboratory conditions. They were exposed for 96 h to different concentrations of the tested substances: 3.33, 10, 33, and 100 µg L-1 of free DCOIT; 500, 1000, 2000, and 4000 µg L-1 of SiNC; and 74.1, 222.2, 666.7, and 2000 µg L-1 of SiNC-DCOIT and SiNC-DCOIT-Ag. The test chambers consisted of 500 mL flasks containing the test solutions, and the tests were maintained under constant aeration, a constant temperature of 23 ± 2 °C, and photoperiod of 12 h:12 h (light/dark). At the end of the experiments, no lethal effect was observed; however, some sublethal effects were noticeable, such as the exposure of the skeleton in most of the concentrations and replicates, except for the controls, and embrittlement at higher concentrations. Adults of T. coccinea were considered slightly sensitive to the tested substances. This resistance may indicate a greater capacity for proliferation in the species, which is favored in substrates containing antifouling paints, to the detriment of the native species.

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.

Unraveling sources of hydrocarbons in subtropical estuaries with distinct degrees of protection in the Southwestern Atlantic Ocean, Brazil.

Sedimentary aliphatic hydrocarbons and polycyclic aromatic hydrocarbons (PAHs) were studied in subtropical estuaries with distinct degrees of legal protection, located in the Southwestern Atlantic Ocean, São Paulo State, Brazil. A multivariate approach was adopted, using the Hierarchical cluster analysis followed by the Principal Matrix Factorization analysis to support the hydrocarbons sources findings using diagnostic ratios. In general, the sites with the highest values of hydrocarbons were in the vicinity of well-urbanized cities, where sewage discharge, harbor and industrial activities take place. Pyrolitic PAHs were the predominant source of PAHs in the sites. The region can be considered not highly contaminated by hydrocarbons; however, specific sites under local anthropogenic impact from sewage and/or urban drainage, presented relatively high hydrocarbons levels. These findings highlight the importance of sources identification as reliable approach to be included in the management plan of protected areas under the inputs of several vectors of contamination.

Linking MS1 and MS2 signals in positive and negative modes of LC-HRMS in untargeted metabolomics using the ROIMCR approach.

Data-independent acquisition (DIA) mode in liquid chromatography (LC) high-resolution mass spectrometry (HRMS) has emerged as a powerful strategy in untargeted metabolomics for detecting a broad range of metabolites. However, the use of this approach also represents a challenge in the analysis of the large datasets generated. The regions of interest (ROI) multivariate curve resolution (MCR) approach can help in the identification and characterization of unknown metabolites in their mixtures by linking their MS1 and MS2 DIA spectral signals. In this study, it is proposed for the first time the analysis of MS1 and MS2 DIA signals in positive and negative electrospray ionization modes simultaneously to increase the coverage of possible metabolites present in biological systems. In this work, this approach has been tested for the detection and identification of the amino acids present in a standard mixture solution and in fish embryo samples. The ROIMCR analysis allowed for the identification of all amino acids present in the analyzed mixtures in both positive and negative modes. The methodology allowed for the direct linking and correspondence between the MS signals in their different acquisition modes. Overall, this approach confirmed the advantages and possibilities of performing the proposed ROIMCR simultaneous analysis of mass spectrometry signals in their differing acquisition modes in untargeted metabolomics studies.

A Glow before Darkness: Toxicity of Glitter Particles to Marine Invertebrates.

Glitter particles are considered a model of microplastics, which are used in a wide range of products. In this study, we evaluated the toxicity of two types of glitter (green and white, with distinct chemical compositions) dispersions on the embryonic development of the sea urchins Echinometra lucunte, Arbacia lixula, and the mussel Perna perna. The Toxicity Identification and Evaluation (TIE) approach was used to identify possible chemicals related to toxicity. Glitter dispersions were prepared using 0.05% ethanol. The tested dispersions ranged from 50 to 500 mg/L. The white glitter was composed of a vinyl chloride-methyl acrylate copolymer. The effective concentrations of green glitter to 50% embryos (EC50) were 246.1 (235.8-256.4) mg/L to A. lixula, 23.0 (20.2-25.8) mg/L to P. perna and 105.9 (61.2-150.2) mg/L, whereas the EC50 of white glitter to E. lucunter was 272.2 (261.5-282.9) mg/L. The EC50 for P. perna could not be calculated; however, the lowest effect concentration was 10 mg/L-that was the lowest concentration tested. The filtered suspension of green glitter had Ag levels exceeding the legal standards for marine waters. TIE showed that metals, volatiles, and oxidant compounds contribute to toxicity. The results showed that glitter may adversely affect marine organisms; however, further studies are necessary to determine its environmental risks.

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.

Toxicity of antifouling biocides on planktonic and benthic neotropical species.

Organotin-based (OTs: TBT and TPT) antifouling paints have been banned worldwide, but recent inputs have been detected in tropical coastal areas. However, there is a lack of studies evaluating the toxicity of both legacy and their substitute antifouling booster biocides (e.g., Irgarol and diuron) on neotropical species. Therefore, the acute toxicity of four antifouling biocides (TBT, TPT, Irgarol, and diuron) was investigated using the marine planktonic organisms Acartia tonsa and Mysidopsis juniae, the estuarine tanaid Monokalliapseudes schubarti (water exposure), and the burrowing amphipod Tiburonella viscana (spiked sediment exposure). Results confirmed the high toxicity of the OTs, especially to planktonic species, being about two orders of magnitude higher than Irgarol and diuron. Toxic effects of antifouling compounds were observed at levels currently found in tropical coastal zones, representing a threat to planktonic and benthic invertebrates. Furthermore, deterministic PNECmarine sediment values suggest that environmental hazards in tropical regions may be higher due to the higher sensitivity of tropical organisms. Since regulations on antifouling biocides are still restricted to a few countries, more ecotoxicological studies are needed to derivate environmental quality standards based on realistic scenarios. The present study brings essential contributions regarding the ecological risks of these substances in tropical and subtropical zones.

Multilevel assessment of chlorothalonil sediment toxicity to Latin American estuarine biota: Effects on biomarkers, reproduction and survival in different benthic organisms.

Chlorothalonil is an organochlorine compound that has long been used in agriculture. In recent years, this compound has been used as an antifouling booster biocide and its presence has been reported in marine coastal environments, especially in navigational areas. Although sediment can be a sink for chlorothalonil due to high affinity to fine particulate matter, toxicity studies with non-target marine and/or estuarine organisms is focused on waterborne exposure only. This study aimed to determine sediment-borne ecotoxicological effects of chlorothalonil on different benthic organisms of the Latin American biota using a integrative multilevel approach. Marine/estuarine organisms were exposed to sediments spiked with chlorothalonil (from 0 to 10.0 µg g-1) and effects at sub-individual (biochemical biomarkers in Anomalocardia flexuosa), individual (lethal effects to Tiburonella viscana and Artemia salina) and subpopulation levels (Nitokra sp. reproduction) were assessed. Increasing chlorothalonil concentrations in sediment caused increasing ecotoxicological effects in different levels of biological organisation, from biochemical to subpopulation levels. The highest exposure concentrations showed increased biomarkers of effects (lipid peroxidation and DNA damage in gills and/or digestive gland of A. flexuosa), lower fecundity and lower survival of the test organisms. GPx activity associated with LPO levels in the digestive gland suggested a response to the oxidant challenge provided by the biocide. At the lowest concentration (0.001 µg g-1), chlorothalonil detoxification mechanisms and defense against its oxidising action, involving GSH and glutathione-dependent enzymes (GST and GPx) were induced. At intermediate concentrations, there was a tendency of decreasing GSH levels, probably due to conjugation with chlorothalonil, which also affected the activities of the glutathione-dependent enzymes. At the highest tested concentration (10.0 µg g-1), chlorothalonil may have restimulated GSH synthesis in the gills of A. flexuosa, although the prooxidant activity has induced effects. This study contributes to assessing the environmental risk of chlorothalonil in sediment for non-target marine and estuarine organisms.

Plastic pellets make Excirolana armata more aggressive: Intraspecific interactions and mortality in field and laboratory ecotoxicological assays.

Microplastics, including plastic pellets, get stranded on sandy beaches. They persist in the oceans for long periods and frequently carry contaminants. Acute and chronic toxicity has been observed when marine organisms are exposed to high densities of plastic pellets in laboratory assays. We investigated the toxicity of beach-stranded plastic pellets on macrobenthic populations (Excirolana armata; Crustacea; Isopoda) under natural conditions (in situ). We simulated different pellets densities on a beach not contaminated by pellets, exposing isopods for 6 h and testing possible behavioral responses (i.e., vertical displacement) and mortality effects. No effect was observed on vertical displacement, but higher mortality was reported for organisms exposed to plastic pellets. The lowest pellet density tested commonly found in coastal areas was sufficient to trigger mortality. We also observed that lethargic individuals (near-death) were preyed on by the healthy individuals remaining in the test chambers.

From molecular endpoints to modeling longer-term effects in fish embryos exposed to the elutriate from Doce River.

Sediments represent a major sink and also a main source of contaminants to aquatic environments. An environmental disaster from a mining dam breakage in 2015 in South-East Brazil re-suspended complex mixtures of chemicals deposited in the sediment, spreading contaminants along the Doce River Basin (DRB) major river course. While high levels of contaminants in sediment were well described, toxicological effects in aquatic organisms were poorly investigated. Thus, the effects of these potentially toxic chemicals were assessed in the present study through different endpoints (biochemical to populational levels) in fish embryos of the South-American silver catfish exposed to elutriates from different sites of the DRB. Despite no significant mortality observed, our results showed that exposure to the elutriates, especially those from the closest site to the dam collapse, caused higher deformities rates and DNA damage in the fish embryos than in the control group. Multivariate analysis showed that these sublethal effects may be related to the high levels of metals introduced by mining activities, compromising long-term survival and reproduction success. In addition, it was possible to observe the influence of other sources of pollutants along the river. According to our data, the mathematical model simulated a significant impact on the population density at longer-term exposure, for the sites that showed the most prominent toxicity responses. The fish embryo toxicity test proved to be an effective assay to assess the ecotoxicological effects of the pollutants from a major river contaminated by a mining dam collapse and showed that the survival rate per se was not a suitable endpoint to assess the toxicity of the pollutants. As a consequence, we contributed to shed a light on a potential underestimated impact of pollutants in sediments of the DRB on the native organisms at distinct biological levels of organizations.

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).

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.

Univariate or multivariate approaches for histopathological biomarkers in the context of environmental quality assessments?

Although the simplification of multivariate histopathological data into univariate indices can be useful for the assessment of environmental quality, this implies a great loss of information. The objective of the present study was to evaluate the effectiveness, in the context of environmental quality assessment, of an approach that integrates individual histopathological responses in a discriminated manner with the results of contaminants by means of multivariate analyses. This analysis was compared to the diagnosis of environmental quality provided by the use of the univariate Bernet histopathological index. Contaminant loads (sediments and fish) and the liver histopathology of Cathorops spixii were integrated through multivariate analysis. Integrated individual histopathological responses allowed classifying environmental quality from more to less impacted sites, while the univariate index showed some inconsistencies with chemical loads and allowed identifying only the most impacted site.

Integrated analysis of fish intestine biomarkers: Complementary tools for pollution assessment.

The gastrointestinal tract and its enteric nervous system are the first routes of food and xenobiotics uptake. Considering the importance of this organ, this study evaluated intestinal biomarkers of Sphoeroides testudineus integrating the data to generate tools for pollution assessment. The fish were collected in three sites of São Paulo Coast and their intestines were analyzed for biochemical, histology, and neuronal density and morphometry biomarkers. To evaluate the differences among the data, a PERMANOVA was applied, followed by a FA/PCA. The PERMANOVA indicated differences (P < 0.001) between the regions (RA, A1, and A2). Four factors were extracted from the FA/PCA (62% cumulative), showing that the animals from A2 presented severe alterations, mainly in intestinal morphometry and neuronal density. A1 alterations refer mainly to the increase of neuronal metabolism. Our results also evidence a gradient of environmental quality related to the protection level (AR > A1 > A2).

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.

Toxicity of innovative antifouling additives on an early life stage of the oyster Crassostrea gigas: short- and long-term exposure effects.

Recent advances in nanotechnology have allowed the encapsulation of hazardous antifouling (AF) biocides in silica mesoporous nanocapsules (SiNC) reducing their short-term toxicity. However, the chronic effects of such novel nanoadditives remain understudied. The present study aimed to assess short- and long-term sub-lethal effects of soluble forms (DCOIT and Ag) and nanostructured forms (SiNC-DCOIT and SiNC-DCOIT-Ag) of two AF biocides and the "empty" nanocapsule (SiNC) on juveniles of Crassostrea gigas after 96 h and 14 days of exposure. Juvenile oysters exposed for a short period to free DCOIT and AgNO3 presented worse physiological status comparing with those exposed to the nanostructured forms. The long-term exposure to DCOIT and Ag+ caused an extensive biochemical impairment comparing with the tested nanomaterials, which included oxidative damage, activation of the antioxidant defense system, and neurotransmission impairment. Despite the negative effects mostly observed on the health condition index and AChE, the encapsulation of the abovementioned AF biocides into SiNC seems to be a technological advantage towards the development of AF nanoadditives with lower long-term toxicity comparing with the soluble forms of such biocides.