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.

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.

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.

Chemical pollution impairs the health of fish species and fishery activities along the Algeria coastline, Mediterranean Sea.

Chronic exposure to multiple pollutants affects aquatic organisms, even at low concentrations, and can impair fishery activities along marine coastlines. The bioavailability of toxic metals and the presence of metals and polycyclic aromatic hydrocarbons (PAHs) in both water and sediment can explain the worst-case scenario of fish health and fishery production decline along the Algeria coastline. The hepatosomatic index (HIS), gonadosomatic index (GSI), and condition factor (K) in the studied species from the Algiers, Bou Ismail, and Zemmouri bays are the first indicators of the poor environmental health along the studied region. These findings could be explained by the bioavailability of Zn, Cu, Cr, Mn, Hg, and Ni and the detection of PAHs in the water and sediment of these bays. Additionally, histopathological damage in the liver is described in sardine (Sardina pilchardus), anchovy (Engraulis encrasicolus), and sardinelle (Sardinella aurita) highlights the current study in the investigation of the risk of exposure to biota or human populations. The occurrence of permanent lesions in the livers of fish impairs organ function and increases the incidence of diseases affecting the fish community. Furthermore, the factor analysis with principal component analysis (FA/PCA) dataset explains the physiological disturbances described in all studied species. These findings revealed that Zemmouri bay is the most affected by chemicals, suggesting that S. pilchardus is the most sensitive species. Finally, the results showed that the bioavailability of chemicals present in the studied bays confirms poor water quality, which can explain the decrease in fishery production along the Algerian Coastline.

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.

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.

Combined effects of temperature and copper on oxygen consumption and antioxidant responses in the mudflat fiddler crab Minuca rapax (Brachyura, Ocypodidae).

This study investigates the combined effects of waterborne copper exposure and acute temperature change on oxygen consumption and the oxidative stress biomarkers, glutathione S-transferase (GST) and glutathione peroxidase (GPx), in the gills and hepatopancreas of the fiddler crab Minuca rapax. Crabs held at 25 °C were acclimated to 0 (control), 50, 250 or 500 µg Cu L-1 for 21 days, and were then subjected to 15, 25 and 35 °C for 24 h. Aerial oxygen consumption rates of crabs in copper free media increased with increasing temperature from 15 to 35 °C, Q10 values reaching ≈3. Crabs exposed to increasing copper concentrations exhibited variable responses, Q10 values falling to ≈1.5. Copper had no effect on oxygen consumption at 25 °C. However, at 35 °C, rates decreased in a clear concentration-response manner in the copper exposed crabs, revealing impaired aerobic capability. At 15 °C, oxygen consumption rates increased with copper concentration, except for a decrease at 500 µg Cu L-1. Gill GST activity was ≈2-fold that of the hepatopancreas, while hepatopancreas GPx activity was 3-fold that of the gills. Gill GST activities were reduced by copper exposure only at 25 °C while hepatopancreas GST activities were altered by copper at all temperatures. Hepatopancreas GST and GPx activities increased in crabs exposed to copper at 35 °C, revealing oxidative stress induction. Hepatopancreas GST and GPx activities were reduced in copper exposed crabs at 15 °C, suggesting a diminished capability to mitigate the effects of copper exposure at low temperature. These findings reveal that copper exposure increases oxygen consumption at low temperatures but decreases consumption at high temperature. Hepatopancreas GPx activities decreased at low temperature and increased at high temperature. These novel findings demonstrate that the interaction between copper exposure and temperature should be considered when evaluating biomarker activities in semi-terrestrial crabs.

Seasonal environmental parameters influence biochemical responses of the fiddler crab Minuca rapax to contamination in situ.

The mudflat fiddler crab Minuca rapax, typical of mangroves and intertidal zones in the Western Atlantic Ocean, responds to fluctuations in environmental parameters by biochemical and physiological adjustments. Such biochemical effects are commonly employed in environmental studies as biomarkers of estuarine contamination. This study evaluates biochemical responses in the gills and hepatopancreas of M. rapax in situ from localities exhibiting different types and levels of contamination, against a backdrop of fluctuations in environmental parameters like salinity and temperature common to estuarine regions. The biochemical biomarkers metallothionein (MT)-like protein titers and glutathione S-transferase (GST), glutathione peroxidase (GPx) and acetylcholinesterase (AChE) activities were used to evaluate responses to environmental contamination and seasonal changes in environmental parameters. Crabs were collected during two seasons, the austral winter and summer, at three sites along the coast of the state of São Paulo, Brazil that present decreasing degrees of environmental contamination: Ilha Diana, Santos (ID) > Rio Itapanhaú, Bertioga (RI) > Picinguaba, Ubatuba (P), a pristine control site. Our findings show that MT were induced in crabs from the contaminated sites (ID and RI) mainly during winter, revealing the activation of detoxification mechanisms; however MT were also induced in P crabs during the summer rainy season. GPX, GST and AChE activities were altered in P crabs during summer and in ID and RI crabs in winter. While enzyme activities in summer crabs may reflect seasonal changes in precipitation and salinity, in winter these altered activities appear to reflect contamination, although an effect of environmental parameters cannot be excluded. These findings reveal a strong seasonal influence on biochemical biomarker responses in Minuca rapax, a relevant factor to consider when interpreting the impact of environmental contamination in estuaries.

Enteric nervous system analyses: New biomarkers for environmental quality assessment.

The gastrointestinal tract (GIT) of fish is a target of contaminants since it can absorb these substances. We evaluated the morphophysiological alterations in the GIT of Sphoeroides testudineus collected in two estuaries presenting differences in their environmental quality (NIA and IA). The intestine was analyzed for histological and neuronal changes; liver and gills for biochemical markers; muscle tissues for neurotoxicity and peripheral blood for genotoxic damage. The results showed alterations in the GIT of the animals collected in the IA, such as muscle tunica and goblet cell density reduction, increased intraepithelial lymphocytes density and changes in neuronal density. Furthermore, changes were observed in MTs and LPO in the gills. Thus, we suggest that TGI is functioning as a barrier that responds to ingested contaminants, in order to reduce their absorption and translocation. Thus, alterations in morphophysiological and enteric neurons in S. testudineus can be used as biomarkers of environmental contamination.

Implications on the Pb bioaccumulation and metallothionein levels due to dietary and waterborne exposures: The Callinectes danae case.

This study aimed to assess the bioaccumulation of Pb and induction of metallothionein-like proteins (MT) in Callinectes danae through single and combined dietary and waterborne exposures. Male C. danae individuals were collected in the south area of the Cananéia-Iguape-Peruíbe Protected Area (APA-CIP), in São Paulo State, Brazil. After an acclimatization period, exposure assays were performed during 7 and 14 days, at two Pb concentrations (0.5 e 2.0 µg/g) in 4 treatments: 1) control; 2) contaminated water only; 3) contaminated food only; 4) contaminated water and food. The results indicate that C. danae is highly tolerant to Pb exposure at the evaluated concentrations. In gills, Pb bioaccumulation is more dependent of water efflux and time of exposure (higher Pb values). However, pathways act simultaneously in the induction of MT expression in this tissue. The decreases in Pb accumulation in the combined treatments and MT increases after 14 days in gills suggests that these proteins play a detoxification function in the presence of Pb. In hepatopancreas, depending on the predominance of a certain pathway or combined pathways, accumulation occured at different times. For muscle tissue, bioaccumulation was observed due to contaminated water exposure, but not dietary exposure, probably because Pb concentrations were low.

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.