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.

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.

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.

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

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

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

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

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.

Metal Bioaccumulation by the Neotropical Clam Anomalocardia flexuosa to Estimate the Quality of Estuarine Sediments.

This investigation evaluated the bioaccumulation potential of the tropical estuarine bivalve Anomalocardia flexuosa for trace metals. To this aim, chemical and sedimentological analyses and bioaccumulation tests were performed. The sediments were mainly composed by fine-sands and mud, with variable levels of organic matter and CaCO3. Muddy sediments from a depositional site (P2) presented the highest concentrations of metals, despite SEM/AVS not indicating bioavailability. Bioaccumulation factors showed high ratios for Cd, Ni, and Zn, while associations between the contents of mud, organic matter, CaCO3 and metals in sediments and tissues of A. flexuosa were indicated by a principal component analysis. The SEM/AVS was not effective to predict the bioavailability through dissolved metals. The results showed that contaminants were bioavailable, while the performed bioaccumulation test proved to be a reliable technique for assessing sediment contamination in estuarine regions. Moreover, A. flexuosa was considered an adequate test organism for bioaccumulation studies.

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.

Levels of metals and toxicity in sediments from a Ramsar site influenced by former mining activities.

This study aimed to evaluate the sediment quality in a Ramsar site located in the Southeast Brazil, by using several lines of evidence (LOEs). Chemical and sedimentological analyses, ecotoxicological tests, and whole-sediment Toxicity Identification Evaluation (TIE) were performed. The sediments were predominantly composed of fine sands and muds. Higher concentrations of metals in the sediments collected close to Iguape were observed; however according to the SEM/AVS analyses, most sediments should not be toxic and the potentially toxic samples could occur along the whole channel. In fact, sediments of most stations were toxic in at least one survey. The PCA revealed associations between mud, metals and toxicity. Acutely toxic sediments presented Pb concentrations above the threshold effect levels, while chronic toxicity seemed to be influenced also by SEM. The most contaminated and toxic areas are not necessarily those located close to the primary sources, but are actually those with finer sediments. Toxicity Identification and Evaluation (TIE) showed that metals and ammonia were responsible for the toxicity. The results suggest that metals from former mining areas located at the upper Ribeira de Iguape River are transported to the Cananéia Iguape Peruíbe Protected Area and accumulate in the sediments at potentially toxic levels. The location of muddy and more contaminated sediments appears to move along the estuary, accompanying the displacement of the maximum turbidity zone, which, in turn, depends on seasonal freshwater inputs. The results also showed that using several lines of evidences to asses contamination in a protected area provides reliable information to subsidise further actions to control the sources of contaminants.

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.

Pollution status of marine protected areas worldwide and the consequent toxic effects are unknown.

Chemical pollution is considered a factor that may threaten marine protected areas (MPAs), and recent studies have found contamination and associated biological effects in some MPAs. However, organized data on this topic are lacking. This study reviewed the literature on pollution in MPAs in order to compile data, determine whether MPAs are influenced by pollution and, whenever possible, describe how they are being affected by contaminants. The results show that the pollution status is unknown in most MPAs worldwide. When any information is available, it is often insufficient to diagnose the threats to biodiversity or to support further actions. More robust and extensive information is available on a small number of MPAs, and much less information is available regarding the negative effects of pollution. More than 80% of the areas studied exhibited evidence of contamination at potentially toxic concentrations or were found to have a status that produced toxic effects on the biota. The scientific community is encouraged to study pollution in MPAs worldwide.